ES2392445T3

Compositions and methods for detection of Hepatitis A virus nucleic acid

Abstract

A combination of at least two oligomers for amplification of a HAV target region comprising: oligomers of 23 to 26 nt contained in the sequence of SEQ ID NO: 138 that include at least the sequence of SEQ ID NO: 139 or SEQ ID NO : 140, or oligomers with a size within a range of 19 to 25 nt contained in the sequence of SEQ ID NO: 141, which contain at least one sequence of SEQ ID NOs 142 to 146, or promoter oligomers with a size within a range of 50 to 53 nt, which include specific portions of a HAV target of any one between SEQ ID NOs 21 to 27 .

Term

Term ended

Projected expiry passed 13 July 2025, 1.2 years ago.

24 claims: 2 independent, 22 dependent

1
REIVINDICACIONES 1. Una combinación de al menos dos oligómeros para la amplificación de una región diana de VHA que comprende:oligómeros de 23 a 26 nt contenidos en la secuencia de SEQ ID NO: 138 que incluyen por lo menos la secuencia de SEQ ID NO: 139 o SEQ ID NO: 140, u oligómeros con un tamaño dentro de un intervalo de 19 a 25 nt contenidos en la secuencia de SEQ ID NO: 141, que contienen al menos una secuencia de SEQ ID NOs 142 a 146, u oligómeros cebadores promotores con un tamaño dentro de un intervalo de 50 a 53 nt, que incluyen porciones específicas de una diana de VHA de una cualquiera entre SEQ ID NOs 21 a 27.
2
La combinación de al menos dos oligómeros de acuerdo con la reivindicación 1, seleccionados entre el grupo que consiste en:SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 143, SEQ ID NO: 144 y SEQ ID NO: 145.
3
La combinación de al menos dos oligómeros de acuerdo con la reivindicación 1, que comprende adicionalmente al menos un oligómero de una sonda de captura seleccionado entre el grupo consistente en SEQ ID NOs 1 a 7 o una secuencia específica de una diana fijada covalentemente a una secuencia o a un resto que se une a una sonda inmovilizada, seleccionándose dicha secuencia específica de una diana entre el grupo que consiste en SEQ ID NOs 8 a 14.
4
La combinación de al menos dos oligómeros de acuerdo con la reivindicación 3, en donde dicho al menos un oligómero de captura se selecciona entre el grupo consistente en SEQ ID NOs 2, 3 y 4 o una secuencia específica de una diana ligada covalentemente a una secuencia o a un resto que se une a una sonda inmovilizada, seleccionándose dicha secuencia específica de una diana entre el grupo consistente en SEQ ID NOs 9, 10 y 11.
5
La combinación de al menos dos oligómeros de acuerdo con la reivindicación 1, que comprende adicionalmente al menos un oligómero de una sonda de detección seleccionado entre el grupo consistente en SEQ ID NO 109 y SEQ ID NO:111.
6
La combinación de al menos dos oligómeros de acuerdo con la reivindicación 2, en donde dichos al menos dos oligómeros comprenden un primer oligómero de amplificación seleccionado entre el grupo consistente en SEQ ID NOs 21 a 27 y un segundo oligómero de amplificación seleccionado entre el grupo consistente en SEQ ID NOs 15 a 18 y80 a 85.
7
La combinación de al menos dos oligómeros de acuerdo con la reivindicación 6, en donde uno de dichos al menos dos oligómeros es SEQ ID NO:16 y el otro de dichos al menos dos oligómeros es SEQ ID NO: 22.
8
La combinación de al menos dos oligómeros de acuerdo con la reivindicación 7, en donde dicha composición comprende adicionalmente al menos un oligómero de captura seleccionado entre el grupo consistente en SEQ ID NOs 2, 3 y 4.
9
La combinación de al menos dos oligómeros de acuerdo con la reivindicación 7, en donde dicha combinación comprende adicionalmente al menos una sonda de detección seleccionada entre el grupo consistente en SEQ ID NOs 109 y 111.
10
La combinación de al menos dos oligómeros de acuerdo con la reivindicación 6, en donde dicha combinación comprende adicionalmente al menos un oligómero de captura seleccionado entre el grupo consistente en SEQ ID NOs 2 a 4.
11
La combinación de al menos dos oligómeros de acuerdo con la reivindicación 6, en donde dicha combinación comprende adicionalmente al menos una sonda de detección seleccionada entre el grupo consistente en SEQ ID NOs 109 y 111.
12
Un kit que comprende una combinación de al menos dos oligómeros de acuerdo con la reivindicación 1.
13
Un método para detectar la presencia de VHA en una muestra que comprende las etapas de:purificar un ácido nucleico de VHA a partir de otros componentes en una muestra que contiene VHA;amplificar una secuencia diana de VHA en el ácido nucleico purificado de VHA, o un ADNc obtenido a partir del mismo, empleando una reacción de amplificación in vitro que incluye al menos dos oligómeros de la amplificación, específicos de una región diana de VHA seleccionada, que incluyen: oligómeros de 23 a 26 nt contenidos en la secuencia de SEQ ID NO: 138 que incluyen por lo menos la secuencia de SEQ ID NO: 139 o SEQ ID NO: 140, u oligómeros con un tamaño dentro de un intervalo de 19 a 25 nt contenidos en la secuencia de SEQ ID NO: 141, que contienen al menos una secuencia de SEQ ID NOs 142 a 146, u oligómeros cebadores promotores con un tamaño dentro de un intervalo de 50 a 53 nt, que incluyen porciones específicas de una diana de VHA de una cualquiera entre SEQ ID NOs 21 a 27;para producir un producto amplificado de una región diana de VHA seleccionada;y detectar el producto amplificado empleando una sonda de detección que se hibrida específicamente con al menos una porción del producto amplificado.
14
El método de acuerdo con la reivindicación 13, en donde la etapa de purificación pone en contacto la muestra con al menos un oligómero de una sonda de captura que comprende una secuencia contenida en una cualquiera entre SEQ ID NOs 1 a 7 o una secuencia específica de una diana fijada covalentemente a una secuencia o a un resto que se une a una sonda inmovilizada, seleccionándose dicha secuencia específica de una diana entre el grupo que consiste en SEQ ID NOs 8 a 14, en donde dicho oligómero de una sonda de captura se hibrida específicamente con una secuencia en el ARN de VHA para formar un complejo de hibridación con el ARN de VHA, y se separa el complejo de hibridación que contiene el ARN de VHA de otros componentes de la muestra.
15
El método de acuerdo con la reivindicación 13, en donde la etapa de amplificación amplifica una secuencia empleando al menos dos oligómeros específicos de la primera región diana de VHA seleccionados entre el grupo consistente en:SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 143, SEQ ID NO: 144 y SEQ ID NO: 145;y en donde la etapa de detección emplea al menos una sonda de detección que se hibrida específicamente con el producto amplificado.
16
El método de acuerdo con la reivindicación 13, en donde en dicha etapa de detección dicho oligómero de detección se selecciona entre el grupo consistente en SEQ ID NOs 109 y 111.
17
El método de acuerdo con la reivindicación 13, en donde dichos al menos dos oligómeros de amplificación comprenden un primer oligómero de amplificación seleccionado entre el grupo consistente en SEQ ID NOs 21 a 27 y un segundo oligómero de amplificación seleccionado entre el grupo consistente en SEQ ID NOs 15 a 18 y 80 a 85.
18
El método de acuerdo con la reivindicación 17, en donde uno de dichos al menos dos oligómeros de amplificación es SEQ ID NO:16 y otro de dichos al menos dos oligómeros es SEQ ID NO: 22.
19
El método de acuerdo con la reivindicación 17, en donde en dicha etapa de purificación se pone en contacto la muestra con al menos un oligómero de captura seleccionado entre el grupo consistente en SEQ ID NOs 2, 3 y 4.
20
El método de acuerdo con la reivindicación 17, en donde dicha etapa de purificación comprende la etapa de capturar un ácido nucleico de VHA introduciendo al menos un oligómero de captura dentro de dicha muestra.
21
El método de acuerdo con la reivindicación 20, en donde dicho al menos un oligómeros de captura se selecciona entre el grupo consistente en SEQ ID NOs 2, 3 y 4 o una secuencia específica de una diana unida covalentemente a una secuencia o a un resto que se une a una sonda inmovilizada, en donde dicha secuencia específica de una diana se selecciona entre el grupo consiste en SEQ ID NOs 9 y 10 y 11.
22
El método de acuerdo con la reivindicación 21, en donde dicha sonda de detección se selecciona entre el grupo consistente en SEQ ID NOs 109 y 111.
23
El método de acuerdo con la reivindicación 17, en donde dicha etapa de purificación pone en contacto la muestra con al menos un oligómero de captura seleccionado entre el grupo consistente en SEQ ID NOs 2, 3 y 4.
24
El método de acuerdo con la reivindicación 17, en donde dicha sonda de detección se selecciona entre el grupo consistente en SEQ ID NOs 109 y 111.

Independent claims24

958 paragraphs in 1 section, as filed

Compositions and methods for the detection of hepatitis A virus nucleic acid

Field of the Invention

This invention relates to the diagnostic detection of a human virus and specifically relates to assays for detecting sequences of the human hepatitis A virus, using in vitro nucleic acid amplification and the detection of amplified sequences.

Background of the invention

Hepatitis A virus (HAV) is the causative agent of a form of hepatitis that can produce symptoms that include fever, fatigue, nausea, abdominal pain, diarrhea, loss of appetite and jaundice, for less than two months. Of people infected with HAV, approximately 10% to 15% have prolonged or recurrent symptoms for a period of six to nine months after infection. An immunity against HAV, based on the individual production of anti-HAV immunoglobulin G (IgG), is the consequence of symptomatic and asymptomatic infections.

Although the incidence of HAV infections has been drastically reduced in some parts of the world where vaccination against HAV (for example, through the use of inactivated HAV) has generally been used since the late 1990s, epidemic HAV infections may occur ( more than 700 cases per 100,000 inhabitants) in non-immune populations, where there are poor sanitary conditions, even if they are temporarily, for example, after an earthquake. HAV is excreted in the feces of infected people and is usually transmitted by fecal-oral route. Outbreaks spread in a population may be a consequence of food transmission that occurs when a food handler infected with HAV contaminates food during its preparation, or when food materials become contaminated during cultivation, harvesting, packaging or the treatment in the distribution system. Transmission can also result from contact with serum, blood products contaminated with VAH or contaminated needles, for example, by transfusion or by the use of injected drugs. People at risk of infection with HAV include those who have family or sexual contact with a person infected with HAV, people who have disorders of coagulation factors (for example, hemophilia) or a chronic liver disease, people who travel to countries where hepatitis A is common, men who have sex with men, illegal drug users and children living in areas with high rates of hepatitis A (for example, > 20 cases per 100,000 inhabitants).

HAV is a 27-nm RNA virus (picornavirus) that contains a positive single-stranded RNA genome of approximately 7.5 kb, for which a single serotype has been found worldwide. HAV replicates in the liver, is excreted in the bile, and is eliminated in the feces (up to 108 viruses per ml) during the acute phase of an infection. The incubation period is usually two to six weeks before symptoms appear. The diagnosis of hepatitis A cannot be differentiated from other types of viral hepatitis, by symptoms or other clinical features (for example, elevated serum aminotransferases). Typically, the diagnosis of hepatitis A is confirmed by serological tests that provide positive results by the presence of anti-HAV immunoglobulins (Ig). Anti-HAV IgM usually occurs five to ten days before symptoms appear and is undetectable in most patients up to six months later, while anti-HAV IgG appears early during infection and remains detectable. throughout the life of the individual. HAV RNA can be detected in the blood and feces of most people during the acute phase of the infection, by using methods to test nucleic acids, for example, amplification by chain reaction of polymerase (PCR), and nucleic acid sequencing, which has been used to identify the genetic relationship of HAV after extended infections in a population (Dato et al., Morbidity Mortality Wkly. Rpt., 2003, 52 (47): 1155-57; LaPorte et al., Morbidity Mortality Wkly. Rpt., 2003, 52 (24): 565-67). These methods, however, are not generally used for diagnostic purposes.

WO 03/106641 A (Chiron Corp.,) describes combinations of primers located in the 5'-UTR of the HAV genome. WO 91/11534 A (US Health, 8-8-1991) discloses primers located at 32-60 and 240-266 (p. 9) and a probe located at 161-187 (p. 11). Fujiwara

K. et al. (Digestive Diseases and Sciences, vol. 45, No. 12, pages 2422-2427) describe primers that amplify a fragment located at 277-551. Costa-Mattioli M. et al. (Journal of Viral Hepatitis, vol. 9, No. 2, pages 101-106) describe primers located at position 22 (direct) and 85-107 (reverse) and a probe located at position

58.

In the US, approximately 100 people die each year from acute liver failure due to hepatitis A (mortality rate of approximately 0.015%). Even in non-fatal cases of hepatitis A, there are substantial costs associated with HAV infections, including patient hospitalization costs, outpatient visits and lost work days. Public health costs associated with hepatitis A outbreaks include the location and administration of immunoglobulin to people exposed to an infected individual or an infectious source (for example, contaminated food or water) up to two weeks after exposure. The potential risk of infection, especially for widespread outbreaks in a population can cause significant psychological costs and economic losses. Due to the relative ease of transmitting HAV in water and contaminated food, and the morbidity associated with hepatitis A, HAV is a potential agent for use in biological terrorism.

There is a need to accurately detect the presence of HAV in biological and environmental samples. There is a need to quickly diagnose individuals infected with HAV. For example, since immunoglobulin must be administered to a person up to two weeks after exposure to HAV, there is a need for a rapid and accurate trial to promptly assess food handlers with hepatitis symptoms and inform Public health agencies from positive sources for HAV. There is a need to detect HAV present in contaminated materials, such as water and food, to avoid widespread outbreaks in populations or epidemics, as a result of the use or consumption of these materials. There is also a need to detect contamination with HAV, in products that can be used in medical treatment, for example, blood or serum used for transfusions or for the preparation of factors obtained from human fluids.

The present invention responds to these needs, by disclosing oligonucleotide sequences used in methods to test the nucleic acid to detect the presence of HAV nucleic acid in a sample.

Summary

The invention includes nucleic acid oligomers, useful for purification, amplification and detection of HAV target sequences. Such oligomers or combinations of oligomers may be contained in a kit configuration, whose embodiments may include additional oligomers and / or other reagents for amplification and / or detection of a HAV sequence. The invention also includes methods for the detection of HAV in a sample, which employ the steps of purifying HAV nucleic acid from other components in the sample, amplifying a target sequence of HAV RNA or cDNA obtained therefrom, by using an in vitro nucleic acid polymerase and any combination of oligomers for amplification, as described herein, to produce an amplified product, and the detection of the amplified product using a detection probe that specifically hybridizes with at least a portion of the amplified product. In one embodiment, the HAV nucleic acid is purified using at least one capture oligomer that includes a sequence that specifically hybridizes to a target region of the HAV RNA, to form a hybridization complex that includes the HAV RNA that separates. of other components of the sample.

One aspect described is a combination of at least two specific oligomers to amplify a HAV target region, which includes: for a first HAV target region, oligomers of about 23 to 26 nt, contained in the sequence of SEQ ID NO: 138, which include at least the sequence of SEQ ID NO: 139 or SEQ ID NO: 140, or oligomers with a size within a range from about 19 to 25 nt contained in the sequence of SEQ ID NO: 141, which contain at least one sequence of SEQ ID NOs 142 to 146, or promoter oligomer primers with a size within a range of from about 50 to 53 nt, which include specific portions of a HAV target of any one between SEQ ID NOs 21 to 27; for a second HAV target region, oligomers from about 21 to 27 nt, contained in the sequence of SEQ ID NO: 60 or contained in the sequence of SEQ ID NO: 86, which include at least the sequence of SEQ ID NO: 156 , or promoter oligomer primers with a size in a range from about 48 to 54 nt, which include specific portions of a HAV target of any one between SEQ ID NOs 29 to 32; for a third HAV target region, oligomers of approximately 24 to 30 nt, contained in the sequence of SEQ ID NO: 147, which include at least the sequence of SEQ ID NO: 148, or contained in the sequence of SEQ ID NO: 157 that include at least the sequence of SEQ ID NO: 158, or promoter priming oligomers that include specific portions of a HAV target of SEQ ID NO: 31 or SEQ ID NO: 32; for a fourth HAV target region, oligomers from about 18 to 27 nt, contained in the sequence of SEQ ID NO: 93 or SEQ ID NO: 95, which contain at least the sequence of SEQ ID NO: 97, SEQ ID NO: 159 or SEQ ID NO: 160, or a promoter primer oligomer that includes a specific portion of a HAV target of SEQ ID NO: 33; for a fifth HAV target region, oligomers from about 19 to 31 nt, contained in the sequence of SEQ ID NO: 149 that include at least the sequence of SEQ ID NO: 150, or promoter primer oligomers with a size within a range from about 51 to 56 nt, which include specific portions of a HAV target of any one between SEQ ID NOs 34 to 40; for a sixth HAV target region, oligomers from about 24 to 28 nt, contained in the sequence of SEQ ID NO: 161 that include at least the sequence of SEQ ID NO: 162, or promoter primer oligomers are embodiments of promoter primers that include specific portions of a HAV target of SEQ ID NO: 41 or SEQ ID NO: 42; and for a seventh HAV target region, oligomers from about 20 to 30 nt, contained in the sequence of SEQ ID NO: 151 that include at least any one among the sequences SEQ ID NO: 152 to SEQ ID NO: 155, or contents in SEQ ID NO: 163 which include at least the sequence of SEQ ID NO: 164, or contained in SEQ ID NO : 165 which include at least any one among the sequences SEQ ID NOs 166 to 168, or promoter priming oligomers with a size within a range from about 51 to 56 nt, which include specific portions of a HAV target of any one between SEQ ID NOs 43 to 49. Preferred embodiments of combinations of at least two specific oligomers of the first HAV target region are selected from SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 143, SEQ ID NO: 144 and SEQ ID NO: 145. Preferred embodiments of combinations of at least two specific oligomers of the second HAV target region are selected from SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60 , SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88 and SEQ ID NO: 156. Preferred embodiments of combinations of at least two specific oligomers of the third HAV target region are selected from SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 89 , SEQ ID NO: 90, SEQ ID NO: 91 and SEQ ID NO: 148. Preferred embodiments of combinations of at least two specific oligomers of the fourth HAV target region are selected from SEQ ID NO: 33, SEQ ID NO: 63, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96 and SEQ ID NO: 97. Preferred embodiments of combinations of at least two specific oligomers of the fifth HAV target region are selected from SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38 , SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 97, SEQ ID NO: 149 and SEQ ID NO: 150. Preferred combinations of at least two specific oligomers of the sixth HAV target region are selected from SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 161 and SEQ ID NO:

162. Preferred combinations of at least two specific oligomers of the seventh HAV target region are selected from SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167 and SEQ ID NO: 168. Other preferred embodiments further include at least one oligomer of a capture probe, selected from SEQ ID NOs 1 to 14. Still other embodiments further include at least one oligomer of a detection probe selected from SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121 to SEQ ID NO: 124, and SEQ ID NO: 126 to SEQ ID NO: 130. Preferred embodiments of oligomer combinations include at least two specific oligomers to amplify a selected HAV target region and at least one oligomer of a detection probe that is specific to a sequence contained in the HAV genomic sequence, located between the two. Selected oligomers, specific to amplify the selected HAV target region. Preferred embodiments of such combinations of oligomers can be packaged together in a kit, which may also contain other reagents, such as reagents used in the purification of HAV RNA from a sample, and / or reagents used in in vitro amplification of nucleic acid, and / or reagents used to produce a detectable signal from an oligomer of a detection probe.

Another aspect is a method of detecting the presence of HAV in a sample that includes the steps of purifying HAV nucleic acid from other components in a sample containing HAV; amplifying a HAV target sequence in purified HAV nucleic acid, or a cDNA obtained therefrom, using an in vitro amplification reaction that includes at least two amplification oligomers, specific to a selected HAV target region, which include: for a first HAV target region, oligomers from about 23 to 26 nt contained in the sequence of SEQ ID NO: 138, which include at least the sequence of SEQ ID NO: 139 or SEQ ID NO: 140, or oligomers with a size within a range from about 19 to 25 nt, contained in the sequence of SEQ ID NO: 141, which contain at least one sequence between SEQ ID NOs 142 to 146, or promoter priming oligomers with a size within a range from about 50 to 53 nt that include specific portions of a HAV target of any one between SEQ ID NOs 21 to 27; for a second HAV target region, oligomers of approximately 21 to 27 nt contained in the sequence of SEQ ID NO: 60 or contained in the sequence of SEQ ID NO: 86, which include at least the sequence of SEQ ID NO: 156, or promoter oligomer primers with a size in a range from about 48 to 54 nt, which include specific portions of a HAV target of any one between SEQ ID NOs 29 to 32; for a third HAV target region, oligomers from about 24 to 30 nt, contained in the sequence of SEQ ID NO: 147 that include at least the sequence of SEQ ID NO: 148, or contained in the sequence of SEQ ID NO: 157 which include at least the sequence of SEQ ID NO: 158, or promoter priming oligomers that include specific portions of a HAV target of SEQ ID NO: 31 or SEQ ID NO: 32; for a fourth HAV target region, oligomers from about 18 to 27 nt, contained in the sequence of SEQ ID NO: 93 or SEQ ID NO: 95, which contain at least the sequence of SEQ ID NO: 97, SEQ ID NO: 159 or SEQ ID NO: 160, or a promoter primer oligomer that includes a specific portion of a HAV target of SEQ ID NO: 33; for a fifth HAV target region, oligomers from about 19 to 31 nt, contained in the sequence of SEQ ID NO: 149 that include at least the sequence of SEQ ID NO: 150, or promoter primer oligomers with a size within a range from about 51 to 56 nt including specific portions of a HAV target of any between SEQ ID NOs 34 to 40; for a sixth HAV target region, oligomers from about 24 to 28 nt, contained in the sequence of SEQ ID NO: 161 that include at least the sequence of SEQ ID NO: 162, or promoter primer oligomers are embodiments of promoter primers that include specific portions of a HAV target of SEQ ID NO: 41 or SEQ ID NO: 42; and for a seventh HAV target region, oligomers from about 20 to 30 nt, contained in the sequence of SEQ ID NO: 151, which include at least any one among the sequences SEQ ID NO: 152 to SEQ ID NO: 155, or contained in SEQ ID NO: 163 that include at least the sequence of SEQ ID NO: 164, or contained in SEQ ID NO: 165 which include at least any one among the sequences SEQ ID NOs 166 to 168, or promoter oligomer primers with a size within a range from about 51 to 56 nt that include specific portions of a HAV target of any one between SEQ ID NOs 43 at 49, to produce an amplified product of the selected HAV target region; and to detect the amplified product using a detection probe that specifically hybridizes with at least a portion of the amplified product. In a preferred embodiment in the purification step, the sample is contacted with at least one oligomer of a capture probe comprising a sequence contained in any one between SEQ ID NOs 1 to 14, which specifically hybridizes to a sequence in HAV RNA to form a hybridization complex with HAV RNA, and the hybridization complex containing HAV RNA is separated from other components of the sample. In preferred embodiments in which a sequence is amplified in the first HAV target region, at least two specific oligomers of the first HAV target region are used, selected from SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO : 17, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 , SEQ ID NO: 27, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 143, SEQ ID NO: 144 and SEQ ID NO: 145; and then at least one detection probe that specifically hybridizes with the amplified product of the first HAV target region is employed. In preferred embodiments where a sequence is amplified in the second HAV target region, at least two specific oligomers of the second HAV target region are used, selected from SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88 and SEQ ID NO: 156; and then at least one detection probe that specifically hybridizes with the amplified product of the second HAV target region is employed. In preferred embodiments in which a sequence is amplified in the third HAV target region, at least two specific oligomers of the third HAV target region are used, selected from SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO : 61, SEQ ID NO: 62, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91 and SEQ ID NO: 148; and then at least one detection probe that specifically hybridizes with the amplified product of the HAV target region is employed. In preferred embodiments in which a sequence is amplified in the fourth HAV target region, at least two specific oligomers of the fourth HAV target region selected from SEQ ID NO: 33, SEQ ID NO: 63, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96 and SEQ ID NO: 97; and then at least one detection probe that specifically hybridizes with the amplified product of the fourth HAV target region is employed. In preferred embodiments in which a sequence in the fifth HAV target region is amplified, at least two specific oligomers of the fifth HAV target region are used, selected from SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO : 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67 , SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 97, SEQ ID NO: 149 and SEQ ID NO: 150; and subsequently at least one detection probe is used that specifically hybridizes with the amplified product of the fifth HAV target region. In preferred embodiments in which a sequence is amplified in the sixth HAV target region, at least two specific oligomers of the HAV target region are used, selected from SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO : 71, SEQ ID NO: 72, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 161 and SEQ ID NO: 162; and then at least one detection probe that specifically hybridizes with the amplified product of the sixth HAV target region is used. In preferred embodiments in which a sequence is amplified in the seventh HAV target region, at least two specific oligomers of the seventh HAV region are used, selected from SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO : 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76 , SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167 and SEQ ID NO: 168; and then at least one detection probe that specifically hybridizes with the amplified product of the seventh HAV target region is employed.

Detailed description

The present invention includes methods of detecting HAV present in samples that can be biological samples obtained from humans (for example, feces, blood, saliva, serum or urine), environmental samples (for example, water, soil) or others materials (for example, food) that are potentially contaminated with HAV. The methods are based on the detection of the presence of HAV nucleic acid sequences, by in vitro amplification of a region of the HAV genome and the detection of amplified nucleic acid by the use of a probe that specifically binds to a sequence. in the amplified nucleic acid. An embodiment of the method includes a step of isolating or purifying the HAV nucleic acid from a sample, before the step of amplifying a region of the HAV genome. In this embodiment, genomic HAV RNA is isolated by the use of a capture oligomer that specifically binds to a sequence in the HAV genome, preferably outside the region of the HAV genome that is amplified, and the separation of the complex formed by the capture oligomer and the attached HAV RNA of other components of the sample using a capture support, such as a particle to which the capture oligomer also binds. In the amplification of a portion of the HAV genomic sequence, one or more amplification oligomers that specifically bind to the HAV RNA or a complementary sequence, and in vitro enzymatic synthesis are used to make additional copies of a portion of the sequence. HAV genomics or a complementary sequence, through the use of amplification oligomers as primers for the synthesis of additional copies. A preferred embodiment uses an isothermal amplification reaction to make additional copies of a portion of the HAV genomic sequence. The amplified HAV sequence is then detected by the specific binding of one or more oligomers of the probe with the amplified nucleic acid, and the detection of a signal that is the result of the oligomer of a probe bound to the amplified sequence. The detection of a signal resulting from the oligomer of a probe bound to the amplified HAV sequence indicates the presence of HAV in the sample. These methods are useful for detecting the presence of HAV in a variety of samples, such as biological samples used to diagnose an infection with HAV in a human being, or environmental samples contaminated with HAV, to prevent the spread of HAV as a result of the use or Contaminated source consumption. These methods are also useful for the analysis of human fluid samples to study the presence of HAV, such as serum or plasma, to prevent subsequent infections with HAV as a result of the use of human fluid in a transfusion or for the preparation of therapeutic factors. The methods of the present invention are also useful for detecting the presence of HAV in human tissues or organs, to prevent their use in transplant therapy. Therefore, these methods are especially important for the detection of HAV contamination in human samples or in products obtained from human tissue.

The present invention encompasses nucleic acid compositions, such as oligomers that specifically hybridize with HAV RNA or nucleic acids obtained from HAV RNA, for example, cDNA or amplified sequences prepared from HAV RNA. One of these compositions is a capture oligomer used to purify HAV RNA from a complex mixture, such as a sample, by specific hybridization with HAV RNA and by fixing the hybridized HAV RNA to a capture support that allows separation of captured VAH RNA from other components of the sample. The purification method that uses such a capture oligomer is generally called a target capture, wherein the HAV RNA is the specific target nucleic acid. Another oligomer of the invention is an oligomer of nucleic acid amplification (sometimes referred to as a primer). Additional embodiments include probe oligomers that specifically hybridize with HAV RNA or amplified HAV nucleic acid sequences to provide a signal that detects the presence of a specific HAV sequence. These nucleic acid sequences are useful for the capture, amplification and detection of specific HAV sequences and, therefore, act together to detect the presence of HAV in a sample.

A sample includes any liquid that may contain HAV or any solid that may contain or have HAV on the surface. Samples include, for example, those from environmental sources such as water, biological sources such as human fluids or wastes, and food, packaging materials, or other components used in food processing. A biological sample includes any tissue or material obtained from a living or dead human being, which may contain HAV or HAV nucleic acid, including, for example, saliva, blood, plasma, serum, biopsy tissue, gastrointestinal tissue, urine. , feces or other fluids, tissues or body materials. A sample can be treated to physically or mechanically destroy its physical state to release HAV particles or HAV RNA, in an aqueous solution or a solvent, using conventional methods.

Nucleic acids include DNA or an analog thereof, RNA or an analog thereof, or mixed RNA-DNA polymers or oligomers, formed by at least two, and preferably ten or more bases linked by a main chain structure. The DNA and RNA may be composed of the common bases (A, T, G and C for the DNA, and A, G, C and U for the RNA), although the base analogs (for example, inosine) and Abbasic positions (i.e., a phosphodiester main chain that lacks a nucleotide in one or more positions, see US Patent No. 5,585,481) are also included in these terms. The polymers can have a length of many hundreds or thousands of nucleotides, while the oligomers generally refer to nucleic acids of 1000 or less bound nucleotides, and often comprise from two to about 100 bound nucleotides. The oligomers generally have a size within a range that has a lower limit of about 10 bases and an upper limit of about 150 bases, preferably a size within a range of about 15 to about 70 bases. The oligomers can be purified from naturally occurring biological sources, but preferably they are synthesized in vitro using any of a variety of well-known enzymatic or chemical methods (eg, Caruthers et al., 1987, Methods in Enzymol., 154: 287).

A main nucleic acid chain refers to groups or bonds known in the art (Eschenmoser, 1999, Science 284: 2118-2124), for example, sugar-phosphodiester bonds, 2'-O-methyl bonds, guanidine linkers in the DNA ("DNG"), S-methylthiourea linkers, methyl phosphonate bonds, phosphoramidate bonds, modifications in the main chain amide, such as in polyamide or peptide nucleic acids (PNA), phosphorothioate bonds, nucleic acid phosphonic ester bonds, pyranosyl oligonucleotide bonds, bicyclo and tricycle nucleic acid bonds, formacetal and 3'-thioformacetal bonds, morpholino bonds, or other modifications of the natural internucleoside phosphodiester bond, or combinations thereof (Majlessi et al., 1998, Nucl. Acids Res. 26 (9): 2224-2229; Dempcy et al., 1995, Proc. Natl. Acad. Sci. USA. 92: 6097-6101; Browne et al., 1995, Proc. Natl. Acad. Sci. USA. 92: 7051-7055; Arya and Bruice, 1998, J. Am. Chem. Soc. 120: 6619-6620; Reynolds et al., 1996, Nucl. Acids Res. 24 (22): 4584-4591; Gryaznov and Chen, 1994, Am. Chem. Soc. 116: 3143-3144; Chaturvedi et al., 1996, Nucl. Acids Res. 24 (12): 2318-2323; Hyrup and Nielsen, 1996, Bioorg. & Med. Chem. 4: 5-23; Hydig-Hielsen et al., PCT Patent Application Document WO 95/32305; Mesmaeker et al., Syn. Lett., November 1997: 12871290; Peyman et al., 1996, Angew. Chem. Int. Ed. Engl. 35 (22): 2636-2638; Aerschot et al., 1995, Angew. Chem. Int. Ed. Engl. 34 (12): 1338-1339; Koshkin et al., 1998, J. Am. Chem. Soc. 120: 13252-13253; Steffens and Leumann, 1997,

J. Am. Chem. Soc. 119: 11548-11549; Jones et al., 1993, J. Org. Chem. 58: 2983-2991; Summerton and Weller, 1997, Antisense & Nucl. Acid Drug Dev. 7: 187-195; Stirchak et al., 1989, Nucl. Acids Res. 17 (15): 6129-6141). A main nucleic acid chain can include a mixture of bonds in the same oligomer or polymer (for example, one

or several sugar-phosphodiester bonds and one or several 2'-O-methyl bonds in the strand) or it may have the same bonds along the strand (for example, all 2'-O-methyl bonds or all bonds of amide modification).

A target, a target sequence or a target nucleic acid may refer to a large sequence (for example, greater than 1000 nt) or to a smaller sequence within a larger nucleic acid, to which another sequence is attached, for example, using conventional pairing of complementary bases. A target nucleic acid can be RNA or DNA, of natural or synthetic origin. For example, a target can be a relatively large nucleic acid, such as the HAV genome, or a target can be a minor sub-sequence contained in the HAV RNA, its complement, or an amplification product prepared from it. , which specifically binds to another sequence in an oligomer. Those skilled in the art will appreciate that a target nucleic acid can exist in any form, for example, a coding or non-coding (+ or -) strand.

Complementary nucleic acids (or complementarity of nucleic acid) refers to a sequence of bases in a strand of nucleic acid that, due to the orientation of its functional groups, binds to a sequence of bases in an opposite strand, for example, by hydrogen bonds between bases A and T or U, and between bases C and G. Substantially complementary means that a base sequence of a strand is not completely or perfectly complementary to a sequence of bases in an opposite strand, but there are sufficient links between the bases of the two strands to form a stable hybridized complex with a set of conditions ( for example, the salt concentration in an aqueous solution, or a temperature). Such conditions can be predicted by using the base sequences and conventional mathematical calculations, known to those skilled in the art, to determine the melting temperature (Tm) at which 50% of the hybridized strands are denatured, or by the empirical determination of Tm using routine methods (for example, see Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed., (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989), at 9.50-51, 11.46-49, 11.55-57).

A hybridization condition refers to the combined environment in which one strand of nucleic acid binds to a second strand of nucleic acid through interactions of the complementary strand, to produce a hybridization complex. Such conditions include, for example, the temperature, the chemical components and the concentrations of the compounds (for example, salts, buffers, chelating agents, organic compounds) in aqueous and / or organic solutions containing the nucleic acids. Other factors, such as incubation time or reaction chamber dimensions, may contribute to hybridization conditions, which are well known in the art (for example, see Sambrook et al., Id., In 1.90-1.91 , 9.47-9.51, 11.47-11.57).

A marker refers to a molecular moiety that is detectable or produces a directly or indirectly detectable response, for example, by catalyzing a reaction that produces a signal. Markers include luminescent moieties (e.g., fluorescent, bioluminescent or chemiluminescent compounds), radioisotopes, members of binding partners (e.g., biotin and avidin or streptavidin), enzymes or enzyme substrates, reactive groups, or chromophores, for example, a dye or a particle that produces a detectable color. A detectable response or a signal is any perceivable or measurable data indicating the presence of a marker, for example, light, color, emission of radioactive decay, electrical signal, magnetic field or signal blocking, such as extinction or turbidity.

An immobilized oligomer or probe refers to an oligomer that is connected or fixed, covalently or non-covalently, to a capture support matrix, which provides a means to bind a capture hybrid containing a target nucleic acid, to the capture support. A preferred immobilized probe is an oligomer that binds, directly or indirectly, to a target nucleic acid to facilitate separation of the bound target nucleic acid, from unbound sample materials. In one embodiment, the target indirectly binds to the immobilized probe through a capture probe that binds the target and the immobilized probe, in a hybridization complex (see US Patent Nos. 6,110,678 and 6,280,952, Weisburg et al.). Any one of a variety of supports can be used, such as matrices or particles made of, for example, nitrocellulose, nylon, glass, polyacrylate, mixed polymers, polystyrene, polypropylene silane and magnetic materials. Monodisperse magnetic particles of relatively uniform size that can be easily recovered from the solution, applying a magnetic field, are a preferred embodiment of a support.

An oligomer or capture probe binds a target nucleic acid and an immobilized probe, that is, by using a specific moiety of a target that binds to the target sequence and a moiety that fixes the capture probe to an immobilized probe . In one embodiment, both fixations are the result of hybridization of complementary base sequences, that is, hybridization of a target sequence with a sequence of a capture probe that is complementary to a target, and hybridization of another portion of the target. capture probe with a complementary sequence of the immobilized probe. In other embodiments, one or more fixations can be produced by using the members of a specific binding partner (eg, biotin and avidin or streptavidin), which are well known in the art. Compositions and methods using capture probes are known (US Patent No. 6,110,678).

Separation or purification refers to the removal of one or more components from a sample, from other components of the sample. Sample components include nucleic acids in a phase in generally aqueous solution that may also include materials such as proteins, carbohydrates, lipids and other compounds. Preferably, the separation or purification of a nucleic acid removes at least about 70%, more preferably at least about 90% and, even more preferably, at least about 95% of the nucleic acid from other components of the sample.

An oligonucleotide or amplification oligomer refers to an oligomer that hybridizes with a target nucleic acid, or its complementary sequence, and that participates in a nucleic acid amplification reaction, by serving as a primer for in vitro nucleic acid synthesis. . The amplification oligomers may contain other functional sequences, such as a promoter sequence that binds to an RNA polymerase in an oligomer called the promoter primer. An amplification oligonucleotide generally contains at least about 10 contiguous bases, preferably at least about 12 contiguous bases, which are complementary to a target sequence (or a complementary strand thereof). The contiguous bases are preferably at least about 80%, more preferably at least about 90%, and more preferably about 100% complementary to the sequence that binds to the amplification oligomer. An amplification oligomer may be mixed RNA, DNA or RNA-DNA, and may optionally include modified nucleotides or linkages with the backbone.

A primer refers to an oligonucleotide that hybridizes with a template nucleic acid and has an end (generally 3 ') that can be extended in a polymerization reaction, catalyzed by an enzyme. The 5 'region of the primer may not be complementary to the target nucleic acid, for example, as in a promoter primer that includes a 5' promoter sequence that is not present in the target sequence. Those skilled in the art will appreciate that a promoter primer can act as a primer regardless of its promoter sequence (i.e., with or without the promoter sequence) and that any amplification oligomer can be modified to include a 5 'promoter sequence, and therefore it acts as a promoter primer.

Amplification refers to any known procedure for obtaining multiple copies of a target sequence, its complement or fragments thereof. Fragment amplification refers to the production of an amplified nucleic acid that contains less than the complete target nucleic acid sequence or its complement, for example, the amplification of a portion of the entire HAV genome. The amplification of a fragment or a portion of the entire target may result from the use of an amplification oligomer that hybridizes and initiates polymerization from an internal position of the target nucleic acid. Known methods for amplification include, for example, transcription-mediated amplification (TMA), replicase-mediated amplification, polymerase chain reaction (PCR), ligase chain reaction (LCR) and amplification by chain shift (SDA). Replicase-mediated amplification uses self-replicating RNA molecules and a replicase, such as QB replicase (for example, US Patent No. 4,786,600 to Kramer et al.). PCR uses a DNA polymerase, multiple primers and thermal cyclization to synthesize many copies of two complementary strands of DNA or cDNA (for example, US Patent Nos. 4,683,195, 4,683,202 and 4,800,159 Mullis et al.). The CSF uses at least four separate oligomers to amplify a target and its complementary chain through the use of multiple hybridization, ligation and denaturation cycles (eg, US Pat. No. 5,427,930 to Biekenmeyer et al., and 5,494,810 to Barany et al.). The SDA uses a primer that contains a recognition site for a restriction endonuclease and the endonuclease notches a strand of a semi-modified DNA duplex that includes the target sequence, followed by a series of primer extension and displacement steps. the chain (for example, US Patent No. 5,422,252 to Walker et al.). Transcription-mediated or transcription-associated amplification reactions use a polymerase to synthesize a double stranded complementary strand to the target, which contains a functional promoter for a specific RNA polymerase that produces transcripts that can be cyclized isothermally, to produce additional copies of transcripts that are detectable amplification products.

Transcription-mediated or transcription-associated amplification uses an RNA polymerase to produce multiple RNA transcripts from a nucleic acid template in isothermal reactions using an RNA polymerase, a DNA polymerase, deoxyribonucleoside triphosphates, triphosphates of ribonucleosides and a primer-promoter, and may optionally include one or more additional oligonucleotides. These amplification methods and reaction conditions have been described in detail previously (for example, see US Patent Nos. 5,399,491 and 5,554,516 to Kacian et al., No. 5,437,990 to Burg et al., PCT patents WO 88/01302 and WO 88/10315 of Gingeras, U.S. Patent No. 5,130,238 to Malek et al., and U.S. Patent Nos. 4,868,105 and 5,124 .246 from Urdea et al.).

Preferred embodiments of the present invention employ transcription mediated amplification (TMA, described in US Patent Nos. 5,399,491 and 5,554,516). It will be apparent to one skilled in the art, however, that the oligonucleotide primer methods and sequences described herein are readily applicable for use with any nucleic acid amplification method that synthetically extends the primers through the use of a polymerase. .

A detection probe is an oligomer that binds to a specific target sequence and, by binding, directly or indirectly produces a detectable signal that indicates the presence of the target sequence. A detection probe does not have to be labeled to produce a detectable signal, such as an electrical pulse that is produced by the attachment of the probe to the target. A labeled probe is composed of an oligomer that is directly or indirectly attached to a label. Methods for preparing and / or using labeled probes are well known (for example, Sambrook et al., Id., Chap. 10; US Pat. Nos. 6,361,945 to Becker et al., 5,658. 737 of Nelson et al., 5,656,207 of Woodhead et al., 5,547,842 of Hogan et al., 5,283,174 of Arnold et al., 4,581,333 of Kourilsky et al. And 5,731,148 of Becker et al. .). Detection probes may include a synthetic linker (US Pat. No. 5,585,481 and 5,639,604 to Arnold et al.), and a chemiluminescent label, such as an acridinium ester compound (EA) (US Patent Nos. 5,185,439, 5,656,207 and 5,658 .737).

A homogeneous detectable marker is a marker that can be detected in a homogeneous manner depending on whether or not the marker is bound to a target. That is, the detection of a marker in a homogeneous reaction does not require the physical separation of the unbound forms of the marker, from the mixture in which the signal is detected. Those skilled in the art will appreciate that a homogeneous reaction can take place in solution or on a support, for example, a matrix, a biochip or a gene chip. Homogeneous detectable markers and conditions for their detection are well known (for example, US Patent Nos. 5,283,174,

5,656,207 and 5,658,737).

"It consists essentially of" means that the component (s), composition (s) or stage (s) of the additional method (s), which do not materially change the basic and novel characteristics of The present invention can be included in the compositions, kits or methods of the present invention. Such characteristics include the ability to specifically detect the presence of HAV nucleic acid in a sample with a sensitivity of at least 80%, for samples containing 25 to 30 copies of HAV per ml, by using a probe combination of capture, amplification primers and oligomers of the detection probe, as described herein. Any component (s), composition (s) or step (s) of the method that has a material effect on the specificity and / or sensitivity of the detection of HAV present in a sample, using nucleic acid oligomers and in vitro methods described in this document, would be excluded from this expression.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as that commonly understood by experts in the relevant art. Definitions of many of the terms used herein are provided, for example, in Dictionary of Microbiology and Molecular Biology, 2nd ed. (Singleton et al., 1994, John Wiley & Sons, New York, NY), The Encyclopedia of Molecular Biology (Kendrew, compiler, 1994, Blackwell Science Ltd., Cambridge, MA), or The Harper Collins Dictionary of Biology (Hale & Marham, 1991, Harper Perennial, New York, NY). Unless otherwise mentioned, the techniques employed or contemplated herein are conventional methodologies well known to a person skilled in the art. Examples are included to illustrate some embodiments of the invention.

The present invention includes compositions (nucleic acid amplification oligomers, detection probes and optionally capture oligomers) and methods for detecting HAV nucleic acid in a sample. To select suitable sequences for use as the oligomers described herein, known genomic sequences of HAV (Beneduce et al., 1995, Virus Res. 36 (2-3): 299-309, Fujiwara et al., 2001, J Hepatol 35 (1): 112-119, Hu et al., 2002, Acta Virol. 46 (3): 153-157), which include those of different isolated materials, partial sequences and the complementary sequences available in a public database (for example, GenBank, entry no. AB020564 to AB020569) were aligned, matching the regions of identical or similar sequences and the aligned sequences were compared using well known techniques. Although sequence comparisons can be facilitated by the use of algorithms, those skilled in the art can make such comparisons manually and visually. Portions of HAV sequences containing relatively sparse sequence variants, among the sequences compared, were chosen as the basis for the design of synthetic oligomers suitable for use in the capture, amplification and detection stages, described herein. Other well-known characteristics of the sequence, such as the GC content and the relative abundance of expected secondary structures (eg, hairpin turns or intramolecular pairing), were also considered in the selection of oligomer sequences.

Based on these analyzes, the HAV genome regions around nucleotides 200, 3700, 4700, 5700, 5800, 6000 and 7000, were chosen as potential target regions for the detection of amplified HAV sequences. For each region, oligomers were designed for use in capturing HAV RNA from a sample, to purify it from other components of the sample, such as amplification oligomers and probe sequences. Preferred embodiments of the target regions are in portions of nt 0 to 305, nt 4714 to 4765, nt 5495 to 5788, nt 5788 to 6069 and nt 6952 to 7413 of the HAV genome.

Capture oligomer sequences generally include a sequence that specifically binds to a sequence close to the target region to be amplified and a "tail" region used in the fixation of the hybridization complex that includes the target to a solid support. , for example, by hybridization with an immobilized oligomer (for example, US Patent No. 6,110,678). Preferred capture oligomers include a specific sequence of a target that specifically binds to a HAV RNA sequence and a covalently fixed tail sequence (eg, dT3dA30), as shown in SEQ ID NOs 1 to 7 . Those skilled in the art will understand that the specific portion of a target of a capture oligomer (SEQ ID NOs 8-14), or its RNA equivalent, can be linked to any moiety that allows it to bind to an immobilized probe (for example , a different tail sequence or a member of a binding partner, such as biotin

or avidin). Any main chain can bind the base sequence of a capture oligomer. Some embodiments use 2'-O-methyl bonds in the specific portion of a target of a capture oligomer and conventional DNA bonds in the tail portion. A tail polynucleotide sequence may be any sequence complementary to a sequence of an immobilized probe, and generally has a sequence length of about 5 to 50 residues, and is preferably a substantially homopolymer sequence in a range of about 10 to about 40 residues (for example, C10 to C40) which is complementary to an immobilized homopolymer sequence (for example, G15).

The primer sequences specifically bind a HAV RNA target sequence or a complementary strand and flank an amplifying target sequence, although the primer sequences may contain additional sequences that do not bind to the target or its complementary sequence. A primer can be a promoter primer and include a 5 'promoter sequence, such as a T7 RNA polymerase promoter (SEQ ID NO: 19). Promoter primer embodiments include those of SEQ ID NOs 20 to 49. Other embodiments of HAV specific primers may include auxiliary sequences, such as restriction endonuclease recognition sequences (SEQ ID NOs 132 to 135). Those skilled in the art will appreciate that a specific sequence of a target of a primer, with or without a promoter or auxiliary sequence attached, can serve as a primer under a variety of in vitro amplification conditions. The amplification oligomers were designed for sequences in the HAV genome target regions (for example, around nucleotide positions 200, 3700, 4700, 5700, 5800, 6000 and 7000). Those skilled in the art will appreciate that these numbers refer to HAV target regions that are only approximate and that those oligomers can act in one assay for more than one target region. That is, the oligomers are not functionally limited by the numbers of identified target regions that are provided as an abbreviated reference to group the preferred embodiments of the invention. The amplification oligomers can be synthesized as complementary DNA, RNA, DNA or RNA sequences, or mixed RNA and DNA sequences, and can include one or more unconventional linkages with the nucleic acid backbone. For example, an oligomer of SEQ ID NO: 106 was synthesized with RNA bases and 2'-O-methyl bonds in residues 1 to 4 and DNA bases and conventional bonds in the other residues.

For a first HAV target region (around position 200), the amplification oligomers include those with a size in a range from about 23 to 26 nt that are contained in the sequence of SEQ ID NO: 138, and include at least the sequence of SEQ ID NO: 139 or SEQ ID NO: 140. Embodiments of such oligomers include those of SEQ ID NO: 51 to SEQ ID NO: 57. The embodiments of promoter primers for this region, with a size in a range from about 50 to 53 nt, are those that include specific portions of a target of SEQ ID NOs 21 to 27. The amplification oligomers for this target region, they also include those with a size within a range from about 19 to 25 nt contained in SEQ ID NO: 141, and which contain at least the sequence of any one between SEQ ID NOs 142 to 146. Embodiments of the amplification oligomers for this target region include those of SEQ ID NOs 15 to 18, 20 to 27, 50 to 57 and 80 to 85.

For a second HAV target region (around position 3700), the amplification oligomers include those with a size in a range from about 21 to 27 nt, contained in the sequence of SEQ ID NO: 60 or in SEQ ID NO: 86, and include at least SEQ ID NO: 156. Embodiment promoter embodiments that include such specific portions of a target for this region and have a size within a range of from about 48 to 54 nt, include those of SEQ ID NOs 29 to 32. The amplification oligomer embodiments for this region Target region include those of SEQ ID NOs 28 to 30, 58 to 60 and 86 to 88.

For a third HAV target region (around position 4700), the amplification oligomers include those with a size within a range from about 24 to 30 nt that are contained in SEQ ID NO: 147 and include at least the sequence of SEQ ID NO: 148, or are contained in SEQ ID NO: 157 and include at least the sequence of SEQ ID NO: 158. Embodiment oligomer embodiments for this target region include those of SEQ ID NOs 31, 32, 61, 62, 89, 90 and 91, of which, SEQ ID NO: 31 and SEQ ID NO: 32 are primer embodiments promoters that include a 5 'promoter sequence linked to the specific sequence of a target.

For a fourth HAV target region (around position 5700), the amplification oligomers include those with a size in a range from about 18 to 27 nt that are contained in the sequence SEQ ID NO: 93 or SEQ ID NO : 95. Embodiments of such oligomers include those containing at least any one between SEQ ID NO: 97, SEQ ID NO: 159 and SEQ ID NO: 160. Embodiment oligomer embodiments for this target region include those of SEQ ID NOs 33, 63 and 92 to 97, of which SEQ ID NO: 33 is a promoter primer embodiment that includes a 5 'promoter sequence linked to the specific sequence of a target.

For a fifth HAV target region (around position 5800), the amplification oligomers include those with a size in a range from about 19 to 31 nt that are contained in SEQ ID NO: 149 and include at least the sequence of SEQ ID NO: 150. The embodiments of promoter primers, with a size within a range from about 51 to 56 nt, which include such specific portions of a target are those of SEQ ID NOs 34 to 40. Other embodiments of amplification oligomers for this target region, include those of SEQ ID NOs 64 to 70, and 97.

For a sixth HAV target region (around position 6000), the amplification oligomers include those of approximately 24 to 28 nt, contained in the sequence of SEQ ID NO: 161 and include the sequence of SEQ ID NO: 162. Embodiment oligomer embodiments for this target region include those of SEQ ID NOs 41, 42, 71, 72, 98, 99 and 101, of which SEQ ID NOs 41 and 42 are embodiments of promoter primers that include a promoter sequence at 5 ', linked to the specific sequence of a target.

For a seventh HAV target region (around position 7000), the amplification oligomers include those with a size within a range from about 20 to 30 nt, contained in SEQ ID NO: 151 and that include at least one among the sequences SEQ ID NO: 152 to SEQ ID NO: 155. Other embodiments of amplification oligomers for this target region are contained in SEQ ID NO: 163 and include at least the sequence of SEQ ID NO: 164. Additional embodiments are amplification oligomers that are contained in SEQ ID NO: 165 and include at least one of the sequences SEQ ID NOs 166 to 168. The promoter primer embodiments, with a size in a range from about 51 to 56 nt, which include specific portions of a HAV target for this region, are SEQ ID NOs 43 to 49. Other embodiments of amplification oligomers for this region include those of SEQ ID NOs 73 to 79 and 102 to 108.

The oligomers were designed to hybridize and detect amplified HAV sequences, including detection probes of SEQ ID Nos. 109, 111, 113, 115, 117, 119, 121, 122, 123, 124, and 126 to 130. The experts in the art they will appreciate that a detection probe will be chosen to hybridize with a sequence contained within an amplified sequence that is determined by the combination of amplification oligomers that are used. The oligomers of the detection probe can be synthesized as DNA, RNA or mixed DNA and RNA polymers, and can include alternative linkages with the backbone, such as 2'-O-methyl bonds. For example, oligomers of SEQ ID NOs 109, 111, 117, 119, 121, 122, 128 and 130 were synthesized with 2'-O-methyl bonds, and oligomers of SEQ ID NOs 124 and 127 were synthesized as mixed nucleotides of DNA and RNA with 2'-Omethyl bonds from the second residue to the 3 'terminal residue. Preferred embodiments of the detection probes have a fixed chemiluminescent label, preferably an acridinium ester compound (EA) (US Pat. No. 5,185,439, 5,639,604, 5,585,481, and 5,656,744), which in preferred embodiments is attached to the probe by a non-nucleotide linker (see US Pat. Nos. 5,585,481, 5,656. 744 and 5,639,604, in particular from column 10, line 6 to column 11, line 3, and in Example 8). Embodiments of the probe oligomers were labeled using known methods, with an AE compound between residues 9 and 10 for SEQ ID NOs 119, 121 and 124, between residues 10 and 11 for SEQ ID NOs 115, 117, 126 , 127 and 128, between waste 11 and 12 for SEQ ID NOs 109, 111, 123, 124 and 130, between waste 12 and 13 for SEQ ID NOs 113, 122 and 129, and between waste 13 and 14 for SEQ ID NO: 122. The oligomers of the probe were tested and characterized by hybridization with complementary oligomer sequences, using conventional methods for the determination of the Tm and / or differential hydrolysis of the acridinium ester in a hybridization complex (described in detail in US Patent No. 5,283,174). For example, hybridizations were performed by using complementary pairs of the sequences SEQ ID NO: 109 and SEQ ID NO: 110, SEQ ID NO: 111 and SEQ ID NO: 112, SEQ ID NO: 113 and SEQ ID NO: 114, SEQ ID NO: 115 and SEQ ID NO: 116, SEQ ID NO: 117 and SEQ ID NO: 118, SEQ ID NO: 119 and SEQ ID NO: 120, SEQ ID NO: 124 and SEQ ID NO: 125, SEQ ID NO: 128 and SEQ ID NO: 100 and SEQ ID NO: 130 and SEQ ID NO: 131. Hybridization assays can be performed using other complementary sequences, such as SEQ ID NO: 124 with SEQ ID NO: 137 and SEQ ID NO: 129 with SEQ ID NO: 136.

The compositions of the present invention include kits for the detection of HAV nucleic acid sequences. Such kits include amplification oligomers as described herein that act as primers to amplify HAV nucleic acid sequences in vitro. Exemplary kits include a first amplification oligomer that specifically hybridizes with a sequence in a target region of the HAV RNA genome or its complementary sequence, and a second amplification oligomer that specifically hybridizes with another sequence of HAV in the target region, preferably complementary to the HAV genomic RNA sequence. Embodiments of the kits include amplification oligomers that are combinations of primers and promoter primers, as described herein. The kits may also contain one or more oligomers that serve as detection probes to detect amplified HAV sequences from the target region of the primers selected for the kit. The kits of the embodiments that include the probe oligomers use one or more of the sequences of the detection probe, as described herein, which may include a marker directly or indirectly attached to the oligomer of a probe. The kits may also contain oligomers that serve as capture oligomers to purify a HAV target RNA, from a sample. Embodiments of such capture oligomers, as described herein may contain a covalently fixed tail sequence or other binding moiety used in the capture of a target. The kits useful for practicing the methods described herein are also included in the invention, and preferred embodiments include at least two amplification oligomers, as described herein, and may also include reagents. to perform in vitro amplification, for example, enzymes, saline solutions and substrate compounds for nucleic acid synthesis. The oligomers described herein can be packaged in a variety of different embodiments, and therefore, those skilled in the art will appreciate that the invention encompasses many different kit configurations. For example, a kit may include amplification oligomers for only one target region of the HAV genome, or it may include amplification oligomers for multiple HAV target regions. Those skilled in the art will appreciate that a kit that includes a detection probe will include a probe that binds to a sequence amplified by the kit amplification oligomers. That is, the selection of the amplification oligomers and the detection probe oligomers for a kit will be linked by their desired target regions.

An embodiment of the assay to detect the HAV nucleic acid in a sample includes the steps of capturing the HAV target nucleic acid from a sample, by using a capture oligomer, amplifying a region of the HAV nucleic acid captured by the use of a combination of at least two primers, and detecting the amplified sequence of HAV by specifically hybridizing it with an oligomer of a detection probe and detecting a signal that is produced in the probe attached to the amplified HAV sequence. Preferred embodiments utilize an amplification reaction associated with transcription or mediated by transcription. The amplified nucleic acid or probe can be labeled, or both can be unlabeled and a detectable signal is produced from an indirect marker or a response associated with the hybridization complex, such as an electrical pulse resulting from the hybridization of the probe and amplified nucleic acid.

The capture step preferably uses a capture oligomer that includes a specific sequence of a target (for example, SEQ ID NOs 8 to 14) that specifically hybridizes with a HAV target sequence and a moiety that allows the hybridized target nucleic acid separate from other components of the sample. The capture step may use a capture oligomer that also includes a tail portion, for example, as in SEQ ID NOs 1-7, which serve as the remainder that allows the target nucleic acid to separate from other sample components. by hybridizing the tail portion with an immobilized probe, as previously described (US Patent No. 6,110,678). Preferred embodiments utilize supports that are magnetic spheres that are monodisperse (i.e., of uniform size ± approximately 5%) with covalently fixed or immobilized poly-dT oligomers that hybridize with a complementary sequence of the tail of the capture oligomer. The hybridization complex that includes at least the target nucleic acid and the capture oligomer, and preferably also includes the immobilized probe, is separated from other components of the sample, using conventional methods of physical separation (for example, application of a force magnetic, filtration or centrifugation) and the captured target nucleic acid can be washed once or several times to further purify the target nucleic acid from other components of the sample. For example, the particles with the target nucleic acid fixed in a hybridization complex are suspended once or several times in a wash solution that maintains the complex, and then the particles with the fixed complex are recovered from the wash solution, as and as described above.

The amplification of the captured HAV target sequence utilizes an in vitro amplification reaction that employs at least two primers flanking the sequence to be amplified, for example, a HAV sequence flanked by SEQ ID NO: 66 and SEQ ID NO : 95, or its complementary sequences. One embodiment employs a transcription-associated amplification reaction that produces many copies of RNA from a sequence, under substantially isothermal conditions (as described above in US Patent Nos. 5,399,491 and 5,554. 516). Transcription-associated amplification uses two types of primers (one a promoter primer that contains a promoter sequence for an RNA polymerase), enzymes (a reverse transcriptase and an RNA polymerase), substrates (deoxyribonucleoside triphosphates, ribonucleoside triphosphates) and appropriate salts and buffers in solution to produce multiple RNA transcripts from a nucleic acid template. Simply put, a promoter primer hybridizes specifically with a target RNA sequence and the reverse transcriptase creates a first strand of cDNA by extension from the 3 'end of the promoter primer and degrades the template strand in the resulting DNA duplex: RNA, using RNase H. activity A second primer binds to the cDNA and another strand of DNA is synthesized with the reverse transcriptase from the end of the second primer, to create a double stranded DNA with a functional promoter sequence to which the RNA polymerase binds. Multiple RNA transcripts ("amplicons") are transcribed and each can be a template in a new round of replication, as described above, thereby generating large amounts of the single stranded amplified sequence (eg, approximately 100 to 3,000 transcribed from a single mold). Embodiments using a transcription-associated amplification reaction may use promoter primers (SEQ ID NOs 20 to 49) with other primers (SEQ ID NOs 15 to 18, 80 to 99 and 101 to 108) to amplify HAV sequences selected for the detection.

The detection stage employs at least one probe that specifically binds to amplified HAV sequences. The embodiments may use any known detection method (for example, the detection of a radioactive, fluorescent, enzymatic, colorimetric, electrical or luminescent signal) to detect the binding of the detection probe with the amplified HAV sequences, and the detected signal indicates the presence of HAV in the sample. Embodiments of the probe oligomers (SEQ ID NOs 109, 111, 113, 115, 117, 119, 121, 122 to 124, 126 to 130) may be unlabeled or labeled, using any of a variety of known markers. In preferred embodiments, the detection step is carried out in a homogeneous detection reaction without removing the unbound detection probe from the mixture. Embodiments of the probe oligomers for use in homogeneous detection reactions are preferably marked with one among a variety of AE compounds that produce a chemiluminescent signal that is detected as previously described in detail (US Pat. U.S. 5,283,174, 5,656,744 and 5,658,737).

A preferred embodiment of the assay generally includes the following steps. A sample containing HAV is provided, which can be prepared by the use of conventional laboratory methods to prepare a substantially aqueous solution or a suspension containing HAV. An aliquot (0.5 ml) of the solution or suspension of the sample is mixed with approximately an equal volume (0.4 to 0.5 ml) of a target capture reagent, that is, a solution containing one or more capture oligomers (4 pmol / reaction), magnetic particles with fixed immobilized probes, complementary to a portion of the capture oligomers, and saline compounds to provide a hybridization condition. The target capture reagent preferably includes a detergent or other chaotropic agent that breaks the HAV particles and releases the HAV RNA to hybridize with the capture oligomers. The mixture is incubated 20-30 min at 60 ° C to allow hybridization of the specific portion of a target of the capture oligomer, with the VHA target sequence, and then at room temperature for 20-30 min to allow binding of the oligomer of capture and immobilized probe. A magnetic field is applied to the outside of the reaction vessel for approximately 10 min, to separate the particles with the fixed hybridization complexes that include HAV RNA, and the solution phase containing other components of the sample is removed. by aspiration. To wash the particles with the fixed hybridization complexes, they are suspended in 1 ml of wash buffer, separated from the solution substantially as described above, and the solution is removed. Particles with fixed hybridization complexes that include purified HAV RNA are mixed with a solution containing amplification reagents (buffers, salts, dXTP and XTP substrates), and a combination of amplification oligomers (a promoter primer and a combination of primers, each with 3 to 30 pmol, usually 15 pmol each), and covered with oil (0.2 ml of filtered silicone oil) to prevent evaporation, and incubated for 10 min at 60 ° C , then for 10 min at 42 ° C, and then the enzymes (reverse transcriptase and RNA polymerase) are added, and the mixture is incubated for 60 min at 42 ° C. For detection, the amplification reaction mixture is incubated with at least one oligomer of an acridinium labeled detection probe, to provide a maximum detectable signal (relative units of light or URL) of 2 million or less, as detected by using conventional methods in a luminometer (for example, Gen-Probe Leader®, Gen-Probe Incorporated, San Diego, CA). The detection probe is mixed with a diluted or undiluted aliquot of the amplification reaction mixture, in a hybridization solution, incubated for 20 min at 60 ° C to allow hybridization of the oligomer of a probe with the amplified target sequence . Next, the label on the unbound probes is hydrolyzed using a selection reagent (for example, a base) and incubated for 10 min at 60 ° C, followed by the addition of a detection reagent (for example, H2O2) to produce chemiluminescence, followed by neutralization of the pH (for example, by the addition of acid), and the detection of the chemiluminescent signal (URL) in a luminometer (for example, 1-5 seconds).

For use in the methods described above, capture oligomers, amplification oligomers and detection probes can be synthesized using conventional methods to produce mixed DNA, RNA or DNA and RNA polymers. Such oligomers may include conventional or modified bonds and / or nucleosides of natural origin (A, T or U, G, C), analogs (for example, inosine) or synthetic purine and pyrimidine derivatives (for example, P or K bases) (Lin & Brown, 1989, Nucl. Acids Res. 17: 10373-83; Lin & Brown, 1992, Nucl. Acids Res. 20: 5149-52).

The general principles of the present invention can be further appreciated by referring to the following examples, where some embodiments of the present invention are described. In addition to the specific components described in the examples, in general, the following reagents were used in the experiments described below. The target capture reagent consisted of 790 mM HEPES, 680 mM LiOH, 10% (v / v) lithium lauryl sulfate (LLS), 230 mM succinic acid, 0.03% (v / v) agent antifoam, 100 Ig / ml of magnetic particles (1 micron of SERA-MAG® particles, Seradyn, Inc. Indianapolis, Ind.) with covalently fixed poly-dT14 and one or more capture oligomers, each with 4 pmol per 400 Il . The wash buffer consisted of 150 mM NaCl, 10 mM HEPES, 6.5 mM NaOH, 1 mM EDTA, 0.3% (v / v) ethanol, 0.1% SDS, 0.02% (p / v) methyl paraben, 0.01% (w / v) propyl paraben, at pH 7.5. The amplification reagent consisted of 11.6 mM Tris base, 15 mM Tris-HCl, 22.7 mM MgCl2, 23.3 mM KCl, 3.33% glycerol, 0.05 mM Zn acetate, 0.665 mM dATP , 0.665 mM dCTP, 0.665 mM dGTP, 0.665 mM dTTP, 5.32 mM ATP, 5.32 mM CTP, 5.32 mM GTP and 5.32 mM UTP, at pH 7. The enzyme reagent consisted of 140 U / Il T7 RNA polymerase, 224 RTU / Il reverse transcriptase from Moloney murine leukemia virus (MMLV-RT), 16 mM HEPES, N-acetyl-L -70 mM cysteine, 3 mM EDTA, 0.05% (w / v) sodium azide, 20 mM Tris, 50 mM KCl, 20% (v / v) glycerol, 10% (v / v) Triton® X-102, trehalose 150 mM, at pH 7. (The enzymatic units are typically 1 U of T7 RNA polymerase incorporates 1 nmol of ATP into the RNA at 1 h at 37 ° C, using a DNA template containing a T7 promoter, and 1 U of MMLV-RT incorporates 1 nmol of dTTP in 10 min at 37 ° C, using oligo-dT 200-400 IM primer and poly-A template). The probe reagent consisted of 100 mM succinic acid, 2% (w / v) of LLS, 230 mM LiOH, 15 mM aldritiol-2, 1.2 M LiCl, 20 mM EDTA, 20 mM EGTA, 3% ( v / v) ethanol, adjusted to pH 4.7 with LiOH. The selection reagent consisted of 600 mM boric acid, 182 mM NaOH, 1% (v / v) octoxynol (TRITON® X-100), at pH 8.5. The detection reagents were the detection reagent I, which contained 1 mM nitric acid and 32 nM H2O2, and the detection reagent II (to neutralize the pH) which was 1.5 M NaOH (see US Pat. No. 5,283,174 for more details).

Example 1: Characterization of the detection probe

The oligomers of SEQ ID NOs 109, 111, 113, 119, 123, 126 and 130 were synthesized using conventional phosphoramidite chemistry (Caruthers et al., 1987, Methods in Enzymol, 154: 287) and an acridinium ester marker ( AE) was fixed through a linker through the use of well-known methods (US Patent No.

5,185,439 and 5,283,174), and the probes were purified using routine chromatographic methods (eg, HPLC). The probes were marked with AE between residues 11 and 12 of SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 123 and SEQ ID NO: 130, between residues 12 and 13 of SEQ ID NO: 113, between residues 10 and 11 of SEQ ID NO: 126, and between residues 9 and 10 of SEQ ID NO: 119. For characterization of the probe oligomers, each was hybridized with a complementary DNA and / or RNA oligomer (for example, SEQ ID NO: 109 with SEQ ID NO: 110, SEQ ID NO: 111 with SEQ ID NO: 112, SEQ ID NO: 113 with SEQ ID NO: 114, SEQ ID NO: 119 with SEQ ID NO: 120 and SEQ ID NO: 130 with SEQ ID NO: 131), at temperatures below the expected Tm of the probe, and then, Tm was determined experimentally using conventional methods. Differential hydrolysis of the AE marker in the probes hybridized with a complementary oligomer, as compared to AE in the unbound probe, was also determined experimentally by the use of conventional methods (see US Patent No. 5,283,174 ). Simply put, the relationship between the time needed to lose half of the signal due to the hydrolysis of AE in the hybrid was determined, compared to the time required for hydrolysis of the marker half in the unbound probe. The Tm were in the range of 59 ° C to 66 ° C for the oligomers of SEQ ID NOs 109, 111, 113, 119 and 130 when hybridized with a complementary DNA, and the Tm were in the range of 76 ° C to 81 ° C for the oligomers of SEQ ID NOs 109, 111, 123, 126 and 130, when hybridized with a complementary RNA. The differential hydrolysis ratios were in the range of 12 to 25 for the probes of SEQ ID NOs 109, 111, 113, 119 and 130 when hybridized with a complementary DNA, and the differential hydrolysis ratios were in the range of 18 to 104 for probes of SEQ ID NOs 109, 111, 123, 126 and 130 when hybridized with a complementary RNA. On the other hand, similar tests of hybridization and differential hydrolysis were carried out for the probes of SEQ ID NO: 121 marked between residues 9 and 10, SEQ ID NO: 122 marked between residues 13 and 14, SEQ ID NO: 124 marked between residues 9 and 10 and SEQ ID NO: 130 marked between residues 11 and 12, and the differential hydrolysis ratios were in the range of 43 to 190 when the probes hybridized with a complementary RNA. These results showed that all these synthetic oligomers of the probe hybridized specifically with their complementary target sequences and produced detectable signals, useful for specifically detecting amplified HAV sequences.

Example 2: Purification of HAV RNA from samples

The capture oligomers of SEQ ID NOs 1 to 7, synthesized using conventional phosphoramidite chemistry and purified using conventional methods, were tested for their ability to capture the HAV RNA released from the virus in human plasma samples. Samples were prepared by adding HAV particles with known concentrations, to normal human plasma (0.5 ml) and samples containing HAV (for example, 500 to 1000 per reaction) were mixed with an equal volume of capture capture reagent. a target containing each capture oligomer individually (4 pmol / reaction) and polidT magnetic particles. The mixtures were incubated for 30 min at 60 ° C, and then for 30 min at room temperature to form the hybridization complexes that captured HAV RNA in the particles. The magnetic particles with captured and fixed HAV RNA were separated by applying a magnetic field for 10 min to the outside of the vessel, then the solution phase was removed by aspiration to remove other components from the sample, and the particles with fixed hybridization complexes were washed twice sequentially, using 1 ml of the wash buffer at room temperature each time and removing the washing solution from the particles by aspiration. The particles with fixed hybridization complexes were then suspended in the probe reagent (0.1 ml) containing a labeled detection probe, as described in Example 1, and incubated for 20 min at 60 ° C , followed by the addition of the selection reagent (0.2 ml), mixing and incubating for 10 min at 60 ° C. Production and detection of the chemiluminescent signal was performed by adding 200 µl of detection reagent I, incubating and neutralizing the pH of the mixture by adding 200 µl of detection reagent II, and measuring the URL using a luminometer, substantially such and as described above. For all

5 For capture oligomers tested, the presence of HAV RNA in the sample was detected by detecting a positive signal significantly higher than background noise (URL for a similar sample that did not contain HAV). The tests showed small significant differences in yield between capture oligomers.

Example 3: Amplification and detection of HAV sequences

10 Samples of HAV in normal human plasma were prepared substantially as described in Example 2 and HAV RNA was captured by using various combinations of capture oligomers for assays, to amplify and detect selected target regions of the HAV genome. For a target region of residues 0-305 of the genome, SEQ ID NOs 2, 3 and 4 were used in the capture stage. For a target region in residues 4714-4765 of the genome, SEQ ID NOS 4, 5, 6 and 7 were used in the capture stage. For one

fifteen target region in residues 5495-5788 of the genome, SEQ ID NOS 1 and 6 were used in the capture stage. For a target region in residues 5788-6069 of the genome, SEQ ID NO: 2 was used in the capture stage. For a target region in residues 6952-7413 of the genome, SEQ ID NOS 1, 4, 5 and 7 were used in the capture stage. The capture steps were performed substantially as described in Example 2.

The captured HAV RNA was amplified in reactions substantially as described above, which

twenty they contained different combinations of amplification oligomers to serve as primers for different target regions in the HAV genome. The primers used to amplify the target regions were the following: SEQ ID NO: 16 and SEQ ID NO: 22 for the waste region 0-305, SEQ ID NO: 89 and SEQ ID NO: 32 for the waste region 4714-4765, SEQ ID NO: 92 and SEQ ID NO: 33 for the waste region 54955788, SEQ ID NO: 94 and SEQ ID NO: 37 for the waste region of 5788-6069, and SEQ ID NO: 108 and SEQ ID

25 NO: 46 for the waste region 6952-7413. The amplification reactions were all performed substantially the same as described above. That is, particles with the fixed HAV RNA, from the capture stage of a target, were mixed with the amplification reagent and the individual combination of amplification oligomers described above (generally 15 pmol of each), and they were covered with silicone oil (0.2 ml) to prevent evaporation, and incubated for 10 min at 60 ° C and then

30 for 10 min at 42 ° C. The enzyme reagent was added (reverse transcriptase and RNA polymerase), and the amplification reactions were incubated for 60 min at 42 ° C.

For detection, the amplification mixture was incubated with an oligomer of a labeled detection probe that specifically hybridizes with sequences contained in the amplified region. These included SEQ ID NO: 109 or SEQ ID NO: 111 for waste region 0-305, SEQ ID NO: 115 for waste region 4714-4765, SEQ ID 35 NO: 117 for waste region 5495-5788 , SEQ ID NO: 121 and / or SEQ ID NO: 122 for the waste region 5788-6069, and SEQ ID NO: 129 or SEQ ID NO: 130 for the waste region 6952-7413. The probes are provided in the probe reagent in a previously determined amount, based on the specific activity of the labeled probe to produce a maximum detectable signal of 2 million URLs or less from the labeled hybridized probe. The probes and the amplified sequences were incubated in the probe reagent at 55

40 60 ° C, and the chemiluminescent signal was produced from the hybridized probes and was substantially detected as described in Examples 1 and 2. For all combinations of primers tested with RNA captured from HAV, the sensitivity of the amplification assay detected between 400 and 1000 copies of HAV RNA present in the samples.

The combinations of capture oligomers, amplification oligomers and detection probes used for these assays are summarized in Table 1.

Table 1: Combinations of oligomers for HAV assays in samples

<dl><dt>Target region (Waste) </dt><dd>Oligomer (s) of capture Amplification oligomers Detection probe (s) </dd></dl>

Similar experiments were performed using the different capture oligomers (SEQ ID NOs 1, 2, 3, 4, 5, 6 and 7) separately with samples containing HAV, in which the capture stage of a target was substantially performed. as described above, in nine replicates for each test condition. For all these tests, the target region was residues 5788 to 6069 of HAV, for which they were used

amplification oligomers SEQ ID NO: 36 and SEQ ID NO: 96 in the amplification reactions with the HAV-captured RNA, as described above, and the amplified products were detected by measuring the chemiluminescence from the hybridized detection probe (SEQ ID NO: 123 or 124, marked with AE between residues 11 and 12), as described above. The results of these tests are shown in Table 2 (average URL for nine replicates).

Table 2: Amplification and detection of the target region in residues 5788-6069

<dl><dt>Purification by capture oligomer </dt><dd>Signal detected (average URL) </dd></dl>

These results show that all capture oligomers captured HAV RNA sufficiently purified from samples that were to be amplified and detected, to indicate the presence of HAV in the samples.

Example 4: Detection of HAV in plasma samples

In this example, an assay that detected HAV nucleic acid in plasma samples positive for

VHA To prepare the samples, a commercially available batch of HAV in human plasma was diluted in

negative plasma for HAV, to obtain samples with 25, 30, 100, 300 and 500 copies of HAV / ml; a negative control 15 was plasma without HAV. For each test, performed on samples of 20 replicates, samples of 0.5 were mixed

ml with 0.4 ml of the capture reagent of a target containing capture oligomers of SEQ ID NO: 4 (6.5

pmol / reaction) and SEQ ID NO: 5 (1.3 pmol / reaction) and the capture stage of a target was carried out

substantially as described in Example 3, except that it was incubated for 20 min at 60 ° C. For each

Assay, magnetic particles washed with the fixed hybridization complexes that included capture oligomers of SEQ ID NOs 4 and 5 bound to the HAV RNA, were employed in amplification reactions containing 75 Il

of amplification reagent containing amplification oligomers (SEQ ID NO: 36, with 13 pmol / reaction and SEQ

ID NO: 96 with 20 pmol / reaction). As described above, each mixture was covered with a layer of

oil, incubated 10 min at 60 ° C, then the enzyme reagent (25 Il) was added, and the mixture was incubated for

60 min at 41.5 ° C to allow amplification of the HAV target sequence. The amplified sequences were detected by the use of detection probes labeled with 2-methyl-AE (SEQ ID NOs 121 and 122, each with

0.007-0.13 pmol / reaction in 25 Il of a probe reagent), which were incubated for 15 min at 60 ° C for

hybridization of the probes with the amplified sequences of HAV. Next, 250 Il of reagent was added

selection and the mixture was incubated 10 min at 60 ° C to hydrolyze the label in unbound probes, and detection

it was performed as described above using detection reagents I and II to produce the chemiluminescent signal (URL) measured in a luminometer (LEADER ® HC Plus, Gen-Probe Inc.). The results

showed that the assay has a sensitivity of approximately 80% to 100% for samples containing 25

copies of HAV per ml, approximately 90% to 100% for samples containing 30 copies of HAV per ml,

approximately 98% to 100% for samples containing 100 copies of HAV per ml, and 100% for samples that

contain 300 and 500 copies of HAV per ml. No positive results were detected for negative controls (which did not contain HAV). These results show that the trial detects HAV in clinical samples with a

sensitivity of approximately 25 copies of HAV per ml of sample.

Example 5: Detection of HAV RNA and another viral target in the same sample

This assay includes the steps of capturing a target, amplification and detection, substantially as it is

described in Examples 1 to 4 to detect HAV and use additional oligomers to capture, amplify and detect another target, human parvovirus B19, in the same sample. To detect HAV, capture oligomers

are SEQ ID NOs 4 and 5, the amplification oligomers are SEQ ID NOs 36 and 96 and the detection probes are SEQ

ID NOs 121 and 122, used substantially as described in Example 4. To detect the acid

B19 parvovirus nucleic, a capture probe of SEQ ID NO: 169, amplification oligomers of

SEQ ID NOs 170 and 171 and detection probes of SEQ ID NO: 173 marked between residues 5 and 6 and SEQ ID NO: 45 174 marked between residues 9 and 10, similar to an assay previously described to detect parvovirus

B19 (US Patent No. US-2003-0124578-A1). 0.5 ml samples of human plasma

Normally they are prepared to contain known amounts of HAV and parvovirus B19, and then mixed with 0.4 ml of

target capture reagent containing capture oligomers SEQ ID NOs 4, 5 and 169). The stages of

capture of a target is performed substantially as described in Example 2 and the purified viral targets 50 are amplified in the same amplification reaction mixture, substantially as described.

described in Examples 3 and 4, but using the VHA specific and parvovirus specific amplification oligomers, described above. After amplification of the viral target sequences, the detection stage uses specific HAV probes and parvovirus specific probes, as described above, but the probes for the different viral targets are labeled with different 5 acridinium ester compounds. , to allow the detection of different signals in the detection stage by using differential kinetics (as described in US Patent No. 5,658,737). In these assays, both HAV and parvovirus B19 nucleic acids are detected in samples containing both targets. The sensitivity of the assay for the detection of HAV is approximately 25 copies / ml, that is, positive signals are detected in 80% to 100% of samples containing 25, 30, 100, 300 and 500 copies / ml of VHA The sensitivity of the assay for the detection of parvovirus B19 in the sample is as low as 150 international units / ml (IU / ml), that is, positive signals are detected in 20% to 40% of samples containing 150 IU / ml. The assay reliably detects 400 or more IU of parvovirus B19 / ml, that is, 70% to 100% positive detection for samples containing 400, 600, 800, 1600 and 3000 IU / ml. These results show that HAV nucleic acid is specifically detected when the sample includes HAV and another virus, parvovirus.

fifteen human B19, which is also specifically detected.

SEQUENCE LIST

<110> Gen-Probe Incorporated

<120> Compositions and methods for the detection of hepatitis A virus nucleic acid

<130> GP135-PCT

<140> Will be assigned

<141>

<150> US 60 / 587,734

<151>

<160> 174

<170> PatentIn version 3.1

<210> 1

<211> 54

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic oligomer, capture probe

<400> 1 ggacuuccaa gaggggcucc gtttaaaaaa aaaaaaaaaa aaaaaaaaaa aaaa 54

<210> 2

<211> 60

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic oligomer, capture probe

<400> 2 uuuagacucc uacagcucca ugcuaauttt aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 60

<210> 3

<211> 61

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic oligomer, capture probe

<400> 3

<210> 4

<211> 58

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic oligomer, capture probe

<400> 4 gaaauugaau aguaaguucc accuctttaa aaaaaaaaaa aaaaaaaaaa aaaaaaaa 58

<dl><dt><210> 5 <211> 60 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, capture probe </dt><dd /></dl>

<dl><dt><400> 5 gcauagcugc aggaaaauua aucauggttt aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa </dt><dd> 60 </dd></dl>

<dl><dt><210> 6 <211> 56 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, capture probe </dt><dd /></dl>

<dl><dt><400> 6 gcauagcugc aggaaaauua auctttaaaa aaaaaaaaaa aaaaaaaaaa aaaaaa </dt><dd> 56 </dd></dl>

<dl><dt><210> 7 <211> 58 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, capture probe </dt><dd /></dl>

<dl><dt><400> 7 gacaaaagaa aacuggagac uuucctttaa aaaaaaaaaa aaaaaaaaaa aaaaaaaa </dt><dd> 58 </dd></dl>

<dl><dt><210> 8 <211> 21 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer </dt><dd /></dl>

<dl><dt><400> 8 ggacttccaa gaggggctcc g </dt><dd> 21 </dd></dl>

<dl><dt><210> 9 <211> 27 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer </dt><dd /></dl>

<dl><dt><400> 9 tttagactcc tacagctcca tgctaat </dt><dd> 27 </dd></dl>

<dl><dt><210> 10 <211> 28 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer </dt><dd /></dl>

<dl><dt><400> 10 ttcatttctg tccatttctc atcattca </dt><dd> 28 </dd></dl>

<dl><dt><210> 11 <211> 25 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer </dt><dd /></dl>

<dl><dt><400> 11 gaaattgaat agtaagttcc acctc </dt><dd> 25 </dd></dl>

<dl><dt><210> 12 <211> 27 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer </dt><dd /></dl>

<dl><dt><400> 12 gcatagctgc aggaaaatta atcatgg </dt><dd> 27 </dd></dl>

<dl><dt><210> 13 <211> 23 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer </dt><dd /></dl>

<dl><dt><400> 13 gcatagctgc aggaaaatta atc </dt><dd> 23 </dd></dl>

<dl><dt><210> 14 <211> 25 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer </dt><dd /></dl>

<dl><dt><400> 14 gacaaaagaa aactggagac tttcc </dt><dd> 25 </dd></dl>

<dl><dt><210> 15 <211> 23 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer </dt><dd /></dl>

<dl><dt><400> 15 ccatggtgag gggacttgat acc </dt><dd> 23 </dd></dl>

<dl><dt><210> 16 <211> 22 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer </dt><dd /></dl>

<400> 16 cttgatacct caccgccgtt tg 22

<210> 17

<211> 23

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic oligomer

<400> 17 ttgatacctc accgccgttt gcc 23

<210> 18

<211> 25

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic oligomer

<400> 18 gatacctcac cgccgtttgc ctagg 25

<210> 19

<211> 27

<212> DNA

<213> Artificial Sequence

<220>

<223> T7 bacteriophage promoter

<400> 19 aatttaatac gactcactat agggaga 27

<210> 20

<211> 49

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic oligomer, promoter primer

<220>

<221> promoter

<222> (1)..(27)

<223>

<400> 20 aatttaatac gactcactat agggagaaga gaaacagatt aaagaaccc

<210> 21

<211> 53

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic oligomer, promoter primer

<220>

<221> promoter

<222> (1)..(27)

<223> <400> 21 aatttaatac gactcactat agggagagga agaaagaaga cagaaagcgt gaa 53

<210> 22

<211> 51

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic oligomer, promoter primer

<220>

<221> promoter

<222> (1)..(27)

<223>

<400> 22 aatttaatac gactcactat agggagagga agaaagaaga cagaaagcgt g 51

<210> 23

<211> 50

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic oligomer, promoter primer

<220>

<221> promoter

<222> (1)..(27)

<223>

<400> 23 aatttaatac gactcactat agggagagaa gaaagaagac agaaagcgtg 50

<210> 24

<211> 52

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic oligomer, promoter primer

<220>

<221> promoter

<222> (1)..(27)

<223>

<400> 24 aatttaatac gactcactat agggagatgg aagaaagaag acagaaagcg tg 52

<210> 25

<211> 51

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic oligomer, promoter primer

<220>

<221> promoter

<222> (1)..(27)

<223> <400> 25 aatttaatac gactcactat agggagatgg aagaaagaag acagaaagcg t 51

<210> 26

<211> 51

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic oligomer, promoter primer

<220>

<221> promoter

<222> (1)..(27)

<223>

<400> 26 aatttaatac gactcactat agggagactg gaagaaagaa gacagaaagc g 51

<210> 27

<211> 52

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic oligomer, promoter primer

<220>

<221> promoter

<222> (1)..(27)

<223>

<400> 27 aatttaatac gactcactat agggagagca aggggagagc cctggaagaa ag 52

<210> 28

<211> 54

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic oligomer, promoter primer

<220>

<221> promoter

<222> (1)..(27)

<223>

<400> 28 aatttaatac gactcactat agggagacag tatttataat ttcaacagtc acag 54

<210> 29

<211> 48

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic oligomer, promoter primer

<220>

<221> promoter

<222> (1)..(27)

<223> <400> 29 aatttaatac gactcactat agggagatct caacaaacca attatgtg 48

<210> 30

<211> 54

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic oligomer, promoter primer

<220>

<221> promoter

<222> (1)..(27)

<223>

<400> 30 aatttaatac gactcactat agggagacat gactctcaac aaaccaatta tgtg 54

<210> 31

<211> 51

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic oligomer, promoter primer

<220>

<221> promoter

<222> (1)..(27)

<223>

<400> 31 aatttaatac gactcactat agggagacaa ttgcttcctt aacataaact g 51

<210> 32

<211> 53

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic oligomer, promoter primer

<220>

<221> promoter

<222> (1)..(27)

<223>

<400> 32 aatttaatac gactcactat agggagacga tcaattgctt ccttaacata aac 53

<210> 33

<211> 47

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic oligomer, promoter primer

<220>

<221> promoter

<222> (1)..(27)

<223> <400> 33 aatttaatac gactcactat agggagacct tttcctctcc atgcctg 47

<210> 34

<211> 52

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic oligomer, promoter primer

<220>

<221> promoter

<222> (1)..(27)

<223>

<400> 34 aatttaatac gactcactat agggagagaa ttgaatttcc tccagcaaca tg 52

<210> 35

<211> 56

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic oligomer, promoter primer

<220>

<221> promoter

<222> (1)..(27)

<223>

<400> 35 aatttaatac gactcactat agggagaaca agaattgaat ttcctccagc aacatg 56

<210> 36

<211> 54

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic oligomer, promoter primer

<220>

<221> promoter

<222> (1)..(27)

<223>

<400> 36 aatttaatac gactcactat agggagaaca agaattgaat ttcctccagc aaca 54

<210> 37

<211> 52

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic oligomer, promoter primer

<220>

<221> promoter

<222> (1)..(27)

<223> <400> 37 aatttaatac gactcactat agggagacaa gaattgaatt tcctccagca ac 52

<210> 38

<211> 51

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic oligomer, promoter primer

<220>

<221> promoter

<222> (1)..(27)

<223>

<400> 38 aatttaatac gactcactat agggagaaag aattgaattt cctccagcaa c 51

<210> 39

<211> 53

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic oligomer, promoter primer

<220>

<221> promoter

<222> (1)..(27)

<223>

<400> 39 aatttaatac gactcactat agggagaaca agaattgaat ttcctccagc aac 53

<210> 40

<211> 52

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic oligomer, promoter primer

<220>

<221> promoter

<222> (1)..(27)

<223>

<400> 40 aatttaatac gactcactat agggagaccacaagaattga atttcctcca gc 52

<210> 41

<211> 52

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic oligomer, promoter primer

<220>

<221> promoter

<222> (1)..(27)

<223> <400> 41 aatttaatac gactcactat agggagactc 52 tgagccaatc ttggatgaac tc

<210> 42

<211> 52

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic oligomer, promoter primer

<220>

<221> promoter

<222> (1)..(27)

<223>

<400> 42 aatttaatac gactcactat agggagacag aacaattttc catcatgaca gt 52

<210> 43

<211> 55

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic oligomer, promoter primer

<220>

<221> promoter

<222> (1)..(27)

<223>

<400> 43 aatttaatac gactcactat agggagaggt cataaaatct cattctccac caatc 55

<210> 44

<211> 56

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic oligomer, promoter primer

<220>

<221> promoter

<222> (1)..(27)

<223>

<400> 44 aatttaatac gactcactat agggagagaa acactggtca taaaatctca ttctcc 56

<210> 45

<211> 52

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic oligomer, promoter primer

<220>

<221> promoter

<222> (1)..(27)

<223> <400> 45 aatttaatac gactcactat agggagaggt cacaaatgaa acactggtca ta 52

<210> 46

<211> 53

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic oligomer, promoter primer

<220>

<221> promoter

<222> (1)..(27)

<223>

<400> 46 aatttaatac gactcactat agggagagaa aggtcacaaa tgaaacactg gtc 53

<210> 47

<211> 52

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic oligomer, promoter primer

<220>

<221> promoter

<222> (1)..(27)

<223>

<400> 47 aatttaatac gactcactat agggagaaaa ggtcacaaat gaaacactgg tc 52

<210> 48

<211> 51

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic oligomer, promoter primer

<220>

<221> promoter

<222> (1)..(27)

<223>

<400> 48 aatttaatac gactcactat agggagagaa aggtcacaaa tgaaacactg g 51

<210> 49

<211> 53

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic oligomer, promoter primer

<220>

<221> promoter

<222> (1)..(27)

<223>

<dl><dt><400> 49 aatttaatac gactcactat agggagacaa atcatgaaag gtcacaaatg aaa </dt><dd> 53 </dd></dl>

<dl><dt><210> 50 <211> 22 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, primer </dt><dd /></dl>

<dl><dt><400> 50 agagaaacag attaaagaac cc </dt><dd> 22 </dd></dl>

<dl><dt><210> 51 <211> 26 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, primer </dt><dd /></dl>

<dl><dt><400> 51 ggaagaaaga agacagaaag cgtgaa </dt><dd> 26 </dd></dl>

<dl><dt><210> 52 <211> 24 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, primer </dt><dd /></dl>

<dl><dt><400> 52 ggaagaaaga agacagaaag cgtg </dt><dd> 24 </dd></dl>

<dl><dt><210> 53 <211> 23 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, primer </dt><dd /></dl>

<dl><dt><400> 53 gaagaaagaa gacagaaagc gtg</dt><dd> 23 </dd></dl>

<dl><dt><210> 54 <211> 25 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, primer </dt><dd /></dl>

<dl><dt><400> 54 tggaagaaag aagacagaaa gcgtg </dt><dd> 25 </dd></dl>

<dl><dt><210> 55 <211> 24 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, primer </dt><dd /></dl>

<dl><dt><400> 55 tggaagaaag aagacagaaa gcgt</dt><dd> 24 </dd></dl>

<dl><dt><210> 56 <211> 24 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, primer </dt><dd /></dl>

<dl><dt><400> 56 ctggaagaaa gaagacagaa agcg </dt><dd> 24 </dd></dl>

<dl><dt><210> 57 <211> 25 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, primer </dt><dd /></dl>

<dl><dt><400> 57 gcaaggggag agccctggaa gaaag </dt><dd> 25 </dd></dl>

<dl><dt><210> 58 <211> 27 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, primer </dt><dd /></dl>

<dl><dt><400> 58 cagtatttat aatttcaaca gtcacag </dt><dd> 27 </dd></dl>

<dl><dt><210> 59 <211> 21 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, primer </dt><dd /></dl>

<dl><dt><400> 59 tctcaacaaa ccaattatgt g </dt><dd> 21 </dd></dl>

<dl><dt><210> 60 <211> 27 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, primer </dt><dd /></dl>

<dl><dt><400> 60 catgactctc aacaaaccaa ttatgtg </dt><dd> 27 </dd></dl>

<dl><dt><210> 61 <211> 24 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, primer </dt><dd /></dl>

<dl><dt><400> 61 caattgcttc cttaacataa actg </dt><dd> 24 </dd></dl>

<dl><dt><210> 62 <211> 26 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, primer </dt><dd /></dl>

<dl><dt><400> 62 cgatcaattg cttccttaac ataaac </dt><dd> 26 </dd></dl>

<dl><dt><210> 63 <211> 20 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, primer </dt><dd /></dl>

<dl><dt><400> 63 ccttttcctc tccatgcctg </dt><dd> 20 </dd></dl>

<dl><dt><210> 64 <211> 25 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, primer </dt><dd /></dl>

<dl><dt><400> 64 gaattgaatt tcctccagca acatg </dt><dd> 25 </dd></dl>

<dl><dt><210> 65 <211> 29 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, primer </dt><dd /></dl>

<dl><dt><400> 65 acaagaattg aatttcctcc agcaacatg </dt><dd> 29 </dd></dl>

<dl><dt><210> 66 <211> 27 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, primer </dt><dd /></dl>

<dl><dt><400> 66 acaagaattg aatttcctcc agcaaca </dt><dd> 27 </dd></dl>

<dl><dt><212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, primer </dt><dd /></dl>

<dl><dt><400> 67 caagaattga atttcctcca gcaac </dt><dd> 25 </dd></dl>

<dl><dt><210> 68 <211> 24 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, primer </dt><dd /></dl>

<dl><dt><400> 68 aagaattgaa tttcctccag caac </dt><dd> 24 </dd></dl>

<dl><dt><210> 69 <211> 26 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, primer </dt><dd /></dl>

<dl><dt><400> 69 acaagaattg aatttcctcc agcaac </dt><dd> 26 </dd></dl>

<dl><dt><210> 70 <211> 25 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, primer </dt><dd /></dl>

<dl><dt><400> 70 ccacaagaat tgaatttcct ccagc </dt><dd> 25 </dd></dl>

<dl><dt><210> 71 <211> 25 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, primer </dt><dd /></dl>

<dl><dt><400> 71 ctctgagcca atcttggatg aactc </dt><dd> 25 </dd></dl>

<dl><dt><210> 72 <211> 25 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, primer </dt><dd /></dl>

<dl><dt><400> 72 cagaacaatt ttccatcatg acagt </dt><dd> 25 </dd></dl>

<dl><dt><210> 73 <211> 28 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, primer </dt><dd /></dl>

<dl><dt><400> 73 ggtcataaaa tctcattctc caccaatc </dt><dd> 28 </dd></dl>

<dl><dt><210> 74 <211> 29 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, primer </dt><dd /></dl>

<dl><dt><400> 74 gaaacactgg tcataaaatc tcattctcc </dt><dd> 29 </dd></dl>

<dl><dt><210> 75 <211> 25 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, primer </dt><dd /></dl>

<dl><dt><400> 75 ggtcacaaat gaaacactgg tcata </dt><dd> 25 </dd></dl>

<dl><dt><210> 76 <211> 26 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, primer </dt><dd /></dl>

<dl><dt><400> 76 gaaaggtcac aaatgaaaca ctggtc </dt><dd> 26 </dd></dl>

<dl><dt><210> 77 <211> 25 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, primer </dt><dd /></dl>

<dl><dt><400> 77 aaaggtcaca aatgaaacac tggtc </dt><dd> 25 </dd></dl>

<dl><dt><210> 78 <211> 24 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, primer </dt><dd /></dl>

<dl><dt><400> 78 gaaaggtcac aaatgaaaca ctgg</dt><dd> 24 </dd></dl>

<dl><dt><210> 79 <211> 26 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, primer </dt><dd /></dl>

<dl><dt><400> 79 caaatcatga aaggtcacaa atgaaa </dt><dd> 26 </dd></dl>

<dl><dt><210> 80 <211> 19 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, primer </dt><dd /></dl>

<dl><dt><400> 80 gacttgatac ctcaccgcc </dt><dd> 19 </dd></dl>

<dl><dt><210> 81 <211> 23 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, primer </dt><dd /></dl>

<dl><dt><400> 81 acttgatacc tcaccgccgt ttg </dt><dd> 23 </dd></dl>

<dl><dt><210> 82 <211> 23 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, primer </dt><dd /></dl>

<dl><dt><400> 82 cttgatacct caccgccgtt tgc </dt><dd> 23 </dd></dl>

<dl><dt><210> 83 <211> 24 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, primer </dt><dd /></dl>

<dl><dt><400> 83 cttgatacct caccgccgtt tgcc </dt><dd> 24 </dd></dl>

<dl><dt><210> 84 <211> 22 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, primer </dt><dd /></dl>

<dl><dt><400> 84 cuugatacct caccgccgtt tg </dt><dd> 22 </dd></dl>

<dl><dt><210> 85 <211> 23 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, primer </dt><dd /></dl>

<dl><dt><400> 85 cuugatacct caccgccgtt tgc </dt><dd> 23 </dd></dl>

<dl><dt><210> 86 <211> 26 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, primer </dt><dd /></dl>

<dl><dt><400> 86 gcagatagaa tgcttggatt gtctgg </dt><dd> 26 </dd></dl>

<dl><dt><210> 87 <211> 25 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, primer </dt><dd /></dl>

<dl><dt><400> 87 gcagatagaa tgcttggatt gtctg </dt><dd> 25 </dd></dl>

<dl><dt><210> 88 <211> 25 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, primer </dt><dd /></dl>

<dl><dt><400> 88 cagatagaat gcttggattg tctgg </dt><dd> 25 </dd></dl>

<dl><dt><210> 89 <211> 30 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, primer </dt><dd /></dl>

<dl><dt><400> 89 tctcctttta taatagcaac ttcaaattgg </dt><dd> 30 </dd></dl>

<dl><dt><210> 90 <211> 29 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, primer </dt><dd /></dl>

<dl><dt><400> 90 ctccttttat aatagcaact tcaaattgg </dt><dd> 29 </dd></dl>

<dl><dt><210> 91 <211> 25 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, primer </dt><dd /></dl>

<dl><dt><400> 91 ttataatagc aacttcaaat tggtc </dt><dd> 25 </dd></dl>

<dl><dt><210> 92 <211> 20 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, primer </dt><dd /></dl>

<dl><dt><400> 92 ggcaacatta gtgacaactg </dt><dd> 20 </dd></dl>

<dl><dt><210> 93 <211> 27 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, primer </dt><dd /></dl>

<dl><dt><400> 93 ggcaacatta gtgacaactg ttaatgg </dt><dd> 27 </dd></dl>

<dl><dt><210> 94 <211> 18 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, primer </dt><dd /></dl>

<dl><dt><400> 94 caggcatgga gaggaaaa </dt><dd> 18 </dd></dl>

<dl><dt><210> 95 <211> 20 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, primer </dt><dd /></dl>

<dl><dt><400> 95 caggcatgga gaggaaaagg </dt><dd> 20 </dd></dl>

<dl><dt><210> 96 <211> 19 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, primer </dt><dd /></dl>

<dl><dt><400> 96 caggcatgga gaggaaaag </dt><dd> 19 </dd></dl>

<dl><dt><210> 97 <211> 18 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, primer </dt><dd /></dl>

<dl><dt><400> 97 ggcatggaga ggaaaagg </dt><dd> 18 </dd></dl>

<dl><dt><210> 98 <211> 23 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, primer </dt><dd /></dl>

<dl><dt><400> 98 taagaaaatt gaaatgcaga gaa </dt><dd> 23 </dd></dl>

<dl><dt><210> 99 <211> 26 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, primer </dt><dd /></dl>

<dl><dt><400> 99 ctccaaaacg ctttttagaa agagtc </dt><dd> 26 </dd></dl>

<dl><dt><210> 100 <211> 22 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer </dt><dd /></dl>

<dl><dt><400> 100 ccatgattaa ttttcctgca gc </dt><dd> 22 </dd></dl>

<dl><dt><210> 101 <211> 26 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, primer </dt><dd /></dl>

<dl><dt><400> 101 ccaaaacgct ttttagaaag agtccc </dt><dd> 26 </dd></dl>

<dl><dt><210> 102 <211> 25 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, primer </dt><dd /></dl>

<dl><dt><400> 102 cgctgagttt gagcagaatt tagaa </dt><dd> 25 </dd></dl>

<dl><dt><210> 103 <211> 28 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, primer </dt><dd /></dl>

<dl><dt><400> 103 ctgagtttga gcagaattta gaaaatgc </dt><dd> 28 </dd></dl>

<dl><dt><210> 104 <211> 24 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, primer </dt><dd /></dl>

<dl><dt><400> 104 atgcatggct atgagtttta tcag </dt><dd> 24 </dd></dl>

<dl><dt><210> 105 <211> 26 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, primer </dt><dd /></dl>

<dl><dt><400> 105 gcatggctat gagttttatc agaaat </dt><dd> 26 </dd></dl>

<dl><dt><210> 106 <211> 27 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, primer </dt><dd /></dl>

<dl><dt><400> 106 gcauggctat gagttttatc agaaatt </dt><dd> 27 </dd></dl>

<dl><dt><210> 107 <211> 26 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, primer </dt><dd /></dl>

<dl><dt><400> 107 catggctatg agttttatca gaaatt </dt><dd> 26 </dd></dl>

<dl><dt><212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, primer </dt><dd /></dl>

<dl><dt><400> 108 gcatggctat gagttttatc agaaatt </dt><dd> 27 </dd></dl>

<dl><dt><210> 109 <211> 20 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer probe </dt><dd /></dl>

<dl><dt><400> 109 gccgtttgcc taggctatag </dt><dd> 20 </dd></dl>

<dl><dt><210> 110 <211> 20 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer </dt><dd /></dl>

<dl><dt><400> 110 ctatagccta ggcaaacggc </dt><dd> 20 </dd></dl>

<dl><dt><210> 111 <211> 25 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer probe </dt><dd /></dl>

<dl><dt><400> 111 cagggttctt taatctgttt ctcta </dt><dd> 25 </dd></dl>

<dl><dt><210> 112 <211> 25 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer </dt><dd /></dl>

<dl><dt><400> 112 tagagaaaca gattaaagaa ccctg </dt><dd> 25 </dd></dl>

<dl><dt><210> 113 <211> 27 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, probe </dt><dd /></dl>

<dl><dt><400> 113 atgatgtttg gatttcatca ttctgtg </dt><dd> 27 </dd></dl>

<dl><dt><210> 114 <211> 27 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer </dt><dd /></dl>

<dl><dt><400> 114 cacagaatga tgaaatccaa acatcat </dt><dd> 27 </dd></dl>

<dl><dt><210> 115 <211> 22 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, probe </dt><dd /></dl>

<dl><dt><400> 115 ggtcaaatcc aagtccaaaa ac </dt><dd> 22 </dd></dl>

<dl><dt><210> 116 <211> 22 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer </dt><dd /></dl>

<dl><dt><400> 116 gtttttggac ttggatttga cc </dt><dd> 22 </dd></dl>

<dl><dt><210> 117 <211> 20 <212> RNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, probe </dt><dd /></dl>

<dl><dt><400> 117 ccuauguuaa uuucugaggg </dt><dd> 20 </dd></dl>

<dl><dt><210> 118 <211> 20 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer </dt><dd /></dl>

<dl><dt><400> 118 ccctcagaaa ttaacatagg</dt><dd> 20 </dd></dl>

<dl><dt><210> 119 <211> 20 <212> RNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, probe </dt><dd /></dl>

<dl><dt><400> 119 caggcaugga gaggaaaagg </dt><dd> 20 </dd></dl>

<dl><dt><210> 120 <211> 20 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer </dt><dd /></dl>

<dl><dt><400> 120 ccttttcctc tccatgcctg </dt><dd> 20 </dd></dl>

<dl><dt><210> 121 <211> 23 <212> RNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, probe </dt><dd /></dl>

<dl><dt><400> 121 ggucuuccug gaaugugugg ugg </dt><dd> 23 </dd></dl>

<dl><dt><210> 122 <211> 23 <212> RNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, probe </dt><dd /></dl>

<dl><dt><400> 122 gucuuccugg aauguguggu ggg </dt><dd> 23 </dd></dl>

<dl><dt><210> 123 <211> 21 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, probe </dt><dd /></dl>

<dl><dt><400> 123 tcttcctgga atgtgtggtg g </dt><dd> 21 </dd></dl>

<dl><dt><210> 124 <211> 21 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, probe </dt><dd /></dl>

<dl><dt><400> 124 tcuuccugga auguguggug g </dt><dd> 21 </dd></dl>

<dl><dt><210> 125 <211> 20 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer </dt><dd /></dl>

<dl><dt><400> 125 caccacacat.tccaggaaga </dt><dd> 20 </dd></dl>

<dl><dt><210> 126 <211> 20 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, probe </dt><dd /></dl>

<dl><dt><400> 126 cttcctggaa tgtgtggtgg </dt><dd> 20 </dd></dl>

<dl><dt><210> 127 <211> 20 <212> RNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, probe </dt><dd /></dl>

<dl><dt><400> 127 cuuccuggaa uguguggugg </dt><dd> 20 </dd></dl>

<dl><dt><210> 128 <211> 22 <212> RNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, probe </dt><dd /></dl>

<dl><dt><400> 128 gcugcaggaa aauuaaucau gg </dt><dd> 22 </dd></dl>

<dl><dt><210> 129 <211> 26 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, probe </dt><dd /></dl>

<dl><dt><400> 129 tggagaaaga gaugauagaa uauagg </dt><dd> 26 </dd></dl>

<dl><dt><210> 130 <211> 24 <212> RNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, probe </dt><dd /></dl>

<dl><dt><400> 130 ggagaaugag auuuuaugac cagu </dt><dd> 24 </dd></dl>

<dl><dt><210> 131 <211> 24 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer </dt><dd /></dl>

<dl><dt><400> 131 actggtcata aaatctcatt ctcc </dt><dd> 24 </dd></dl>

<dl><dt><210> 132 <211> 28 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, primer </dt><dd /></dl>

<dl><dt><220> <221> Cla I recognition site <222> (3) .. (8) <223> </dt><dd /></dl>

<dl><dt><400> 132 ccatcgatgc gttttggaga ctacattc </dt><dd> 28 </dd></dl>

<dl><dt><210> 133 <211> 29 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, primer </dt><dd /></dl>

<dl><dt><220> <221> Pst I recognition site <222> (4) .. (9) <223> </dt><dd /></dl>

<dl><dt><400> 133 aaactgcaga tgaaggttcc tacaattcc </dt><dd> 29 </dd></dl>

<dl><dt><210> 134 <211> 29 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, primer </dt><dd /></dl>

<dl><dt><220> <221> Kpn I recognition site <222> (4) .. (9) <223> </dt><dd /></dl>

<dl><dt><400> 134 cggggtaccg cgttttggag actacattc </dt><dd> 29 </dd></dl>

<dl><dt><210> 135 <211> 29 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, primer </dt><dd /></dl>

<dl><dt><220> <221> Pst I recognition site <222> (4) .. (9) <223> </dt><dd /></dl>

<dl><dt><400> 135 aaactgcaga gaggtggaac ttactattc </dt><dd> 29 </dd></dl>

<dl><dt><210> 136 <211> 38 <212> RNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer </dt><dd /></dl>

<dl><dt><400> 136 uuaagccuau auucuaucau cucuuucucc aaacagga 38 </dt><dd /></dl>

<dl><dt><210> 137 <211> 27 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer </dt><dd /></dl>

<dl><dt><400> 137 gccccaccac acauuccagg aagacct </dt><dd> 27 </dd></dl>

<dl><dt><210> 138 <211> 42 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer </dt><dd /></dl>

<dl><dt><400> 138 gcaaggggag agccctggaa gaaagaagac agaaagcgtg aa </dt><dd> 42 </dd></dl>

<dl><dt><210> 139 <211> 8 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer </dt><dd /></dl>

<dl><dt><400> 139 gaagaaag </dt><dd> 8 </dd></dl>

<dl><dt><210> 140 <211> 21 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer </dt><dd /></dl>

<dl><dt><400> 140 gaagaaagaa gacagaaagc g </dt><dd> 21 </dd></dl>

<dl><dt><212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer </dt><dd /></dl>

<dl><dt><400> 141 ccatggtgag gggacttgat acctcaccgc cgtttgccta gg </dt><dd> 42 </dd></dl>

<dl><dt><210> 142 <211> 9 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer </dt><dd /></dl>

<dl><dt><400> 142 cttgatacc </dt><dd> 9 </dd></dl>

<dl><dt><210> 143 <211> 11 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer </dt><dd /></dl>

<dl><dt><400> 143 gacttgatac c </dt><dd> 11 </dd></dl>

<dl><dt><210> 144 <211> 22 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer </dt><dd /></dl>

<dl><dt><400> 144 22 cttgatacct caccgccgtt tg </dt><dd> 22 </dd></dl>

<dl><dt><210> 145 <211> 19 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer </dt><dd /></dl>

<dl><dt><400> 145 19 gatacctcac cgccgtttg </dt><dd> 19 </dd></dl>

<dl><dt><210> 146 <211> 17 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer </dt><dd /></dl>

<dl><dt><400> 146 17 cttgatacct caccgcc </dt><dd> 17 </dd></dl>

<210> 147

<211> 28

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic oligomer

<400> 147 cgatcaattg cttccttaac ataaactg 28

<210> 148

<211> 22

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic oligomer

<400> 148 caattgcttc cttaacataa ac 22

<210> 149

<211> 31

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic oligomer

<400> 149 ccacaagaat tgaatttcct ccagcaacat g 31

<210> 150

<211> 19

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic oligomer

<400> 150 gaattgaatt tcctccagc 19

<210> 151

<211> 58

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic oligomer

<400> 151 caaatcatga aaggtcacaa atgaaacact ggtcataaaa tctcattctc caccaatc

<210> 152

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic oligomer

<400> 152 ggtcacaaat gaaacactgg 20

<dl><dt><210> 153 <211> 23 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer </dt><dd /></dl>

<dl><dt><400> 153 aaaggtcaca aatgaaacac tgg </dt><dd> 23 </dd></dl>

<dl><dt><210> 154 <211> 12 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer </dt><dd /></dl>

<dl><dt><400> 154 gaaacactgg tc </dt><dd> 12 </dd></dl>

<dl><dt><210> 155 <211> 21 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer </dt><dd /></dl>

<dl><dt><400> 155 ggtcataaaa tctcattctc c </dt><dd> 21 </dd></dl>

<dl><dt><210> 156 <211> 24 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer </dt><dd /></dl>

<dl><dt><400> 156 cagatagaat gcttggattg tctg </dt><dd> 24 </dd></dl>

<dl><dt><210> 157 <211> 32 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer </dt><dd /></dl>

<dl><dt><400> 157 tctcctttta taatagcaac ttcaaattgg tc </dt><dd> 32 </dd></dl>

<dl><dt><210> 158 <211> 23 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer </dt><dd /></dl>

<dl><dt><400> 158 ttataatagc aacttcaaat tgg </dt><dd> 23 </dd></dl>

<dl><dt><210> 159 <211> 16 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer </dt><dd /></dl>

<dl><dt><400> 159 ggcatggaga ggaaaa </dt><dd> 16 </dd></dl>

<dl><dt><210> 160 <211> 17 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer </dt><dd /></dl>

<dl><dt><400> 160 ggcatggaga ggaaaag </dt><dd> 17 </dd></dl>

<dl><dt><210> 161 <211> 28 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer </dt><dd /></dl>

<dl><dt><400> 161 ctccaaaacg ctttttagaa agagtccc </dt><dd> 28 </dd></dl>

<dl><dt><210> 162 <211> 24 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer </dt><dd /></dl>

<dl><dt><400> 162 ccaaaacgct ttttagaaag agtc </dt><dd> 24 </dd></dl>

<dl><dt><210> 163 <211> 30 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer </dt><dd /></dl>

<dl><dt><400> 163 cgctgagttt gagcagaatt tagaaaatgc </dt><dd> 30 </dd></dl>

<dl><dt><210> 164 <211> 23 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer </dt><dd /></dl>

<dl><dt><400> 164 ctgagtttga gcagaattta gaa </dt><dd> 23 </dd></dl>

<dl><dt><210> 165 <211> 29 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer </dt><dd /></dl>

<dl><dt><400> 165 atgcatggct atgagtttta tcagaaatt </dt><dd> 29 </dd></dl>

<dl><dt><210> 166 <211> 21 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer </dt><dd /></dl>

<dl><dt><400> 166 catggctatg agttttatca g </dt><dd> 21 </dd></dl>

<dl><dt><210> 167 <211> 22 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer </dt><dd /></dl>

<dl><dt><400> 167 gcatggctat gagttttatc ag </dt><dd> 22 </dd></dl>

<dl><dt><210> 168 <211> 25 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer </dt><dd /></dl>

<dl><dt><400> 168 catggctatg agttttatca gaaat </dt><dd> 25 </dd></dl>

<dl><dt><210> 169 <211> 58 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, capture probe </dt><dd /></dl>

<dl><dt><400> 169 gttggctata cctaaagtca tgaatcctaa aaaaaaaaaa aaaaaaaaaa aaaaaaaa </dt><dd> 58 </dd></dl>

<dl><dt><210> 170 <211> 21 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, primer </dt><dd /></dl>

<dl><dt><400> 170 cccctagaaa acccatcctc t </dt><dd> 21 </dd></dl>

<dl><dt><210> 171 <211> 52 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, promoter primer </dt><dd /></dl>

<dl><dt><220> <221> promoter <222> (1) .. (27) <223> </dt><dd /></dl>

<dl><dt><400> 171 aatttaatac gactcactat agggagaagt accgggtagt tgtacgctaa ct </dt><dd> 52 </dd></dl>

<dl><dt><210> 172 <211> 25 <212> DNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, primer </dt><dd /></dl>

<dl><dt><400> 172 agtaccgggt agttgtacgc taact </dt><dd> 25 </dd></dl>

<dl><dt><210> 173 <211> 20 <212> RNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, probe </dt><dd /></dl>

<dl><dt><400> 173 gucauggaca guuaucugac </dt><dd> 20 </dd></dl>

<dl><dt><210> 174 <211> 22 <212> RNA <213> Artificial Sequence </dt><dd /></dl>

<dl><dt><220> <223> synthetic oligomer, probe </dt><dd /></dl>

<dl><dt><400> 174 guauuaucua gugaagacuu ac</dt><dd> 22 </dd></dl>

40 members in 7 offices

This recordMember, by publication datePriority claim

Priority claims9

Priority claims
Document	Office	Kind	Date
587734P	United States of America	–	2004-07-13
58773404	United States of America	P	2004-07-13
58773404	United States of America	P	2004-07-13
2005024952	United States of America	W	2005-07-13
2005024952	United States of America	W	2005-07-13
587734P	–	–	–
PCTUS2005024952	–	–	–
US20040587734P	–	–	–
WO2005US24952	–	–	–

Members40

Family members, oldest first
Document	Office	Kind
AU2005262317A1	Australia	A1	2006-01-19
CA2573532A1	Canada	A1	2006-01-19
CA2838428A1	Canada	A1	2006-01-19
US2006014142A1	United States of America	A1	2006-01-19
WO2006007603A2	World Intellectual Property Organization (WIPO)	A2	2006-01-19
WO2006007603A2	World Intellectual Property Organization (WIPO)	A2	2006-01-19
WO2006007603A3	World Intellectual Property Organization (WIPO)	A3	2006-09-14
WO2006007603A3	World Intellectual Property Organization (WIPO)	A3	2006-09-14
EP1771585A2	European Patent Office (EPO)	A2	2007-04-11
JP2008506392A	Japan	A	2008-03-06
EP1771585A4	European Patent Office (EPO)	A4	2009-03-04
US7544792B2	United States of America	B2	2009-06-09
AU2005262317B2	Australia	B2	2009-07-16
US2009208968A1	United States of America	A1	2009-08-20
JP4753943B2	Japan	B2	2011-08-24
JP2011182795A	Japan	A	2011-09-22
US8063197B2	United States of America	B2	2011-11-22
EP2402465A2	European Patent Office (EPO)	A2	2012-01-04
US2012009565A1	United States of America	A1	2012-01-12
US2012015348A1	United States of America	A1	2012-01-19
EP2412830A2	European Patent Office (EPO)	A2	2012-02-01
EP2402465A3	European Patent Office (EPO)	A3	2012-04-18
EP2412830A3	European Patent Office (EPO)	A3	2012-05-30
EP1771585B1	European Patent Office (EPO)	B1	2012-09-26
ES2392445T3This record	Spain	T3	2012-12-10
US8461324B2	United States of America	B2	2013-06-11
JP2013172750A	Japan	A	2013-09-05
US8563707B2	United States of America	B2	2013-10-22
JP5362761B2	Japan	B2	2013-12-11
CA2573532C	Canada	C	2014-01-07
EP2402465B1	European Patent Office (EPO)	B1	2014-11-19
EP2412830B1	European Patent Office (EPO)	B1	2014-11-19
US2015051092A1	United States of America	A1	2015-02-19
CA2838428C	Canada	C	2015-09-29
JP5886792B2	Japan	B2	2016-03-16
US9469881B2	United States of America	B2	2016-10-18
US2017009284A1	United States of America	A1	2017-01-12
US10392656B2	United States of America	B2	2019-08-27
US2019376131A1	United States of America	A1	2019-12-12
US11136622B2	United States of America	B2	2021-10-05

Compositions and methods for detection of Hepatitis A virus nucleic acid

Abstract

Term

SEQUENCE LIST

Priority claims9

Members40

Numbers

Titles2

Classification