Detection of human papilloma viruses

Detection of human papillomavirus

The present invention relates to a method for the detection of human papillomavirus (HPV) in biological material, as well as nucleotide sequences used in the process.

The method thereby comprises the steps of:

a) extraction / isolation of nucleic acids from biological material, and

b) detecting HPV-specific DNA from the isolated nucleic acids. Such a method is known from various publications.

Infections with HPV associated with a variety of diseases of the skin and mucosa. Mostly it involves benign tumors that present themselves as condylomata, warts or papillomas, which heal usually after months to years spontaneously. With the help of molecular biology and genetic engineering also the context of various cancers could be detected with Papillomvirusinfektionen. These tumors are primarily to carcinomas of the anogenital region, in particular cervical cancers, but also skin carcinomas in immunosuppressed patients or patients with rare disease Epidermophyton dysplasia verruciformis (EV). The causal involvement of HPV in cancers in the area of ​​the anogenital tract is now regarded as generally accepted. Globally, in virtually all cervical carcinomas and. Precursor lesions DNA of HPV detected.

Meanwhile more than 100 HPV genotypes have been identified, the clinical syndromes that can be caused by infection, are often characteristic of a particular HPV type. Common warts of the extremities and plantar warts are caused primarily by HPV types 1, 2, 4, 10, 27, 28, 57 and 65 thereof. The HPV that are associated with cancers in the anogenital region, are based on their oncogenic potential in so-called "low risk" - (eg 6, 11, 40) and "high risk" types of HPV (eg 16, 18, 45 and 56 ) divided. Low risk HPV types cause hautsächlich benign tumors of the outer genital area (condylomata) and low-grade intraepithelial neoplasia and are rarely associated with cancer. The presence of high-risk virus in epithelial cells of the anogenital tract, however represents a high risk factor for the development of carcinoma. In the development of skin carcinomas in EV patients the involvement of HPV types 5 and 8 is considered reasonably assured.

Cervical cancer and also in certain forms of skin cancer is preventable diseases if early detection and treatment are ensured. A reliable method for detecting an existing HPV infection can be detected by all HPV types and moreover enables typing is therefore a prerequisite for a targeted therapy.

Detection of precancerous lesions and early invasive carcinoma occurs already since the 50s by histological methods (cytology screening). The application of these methods had the consequence that the incidence of cervical cancer decreased. Nevertheless cytology is methodologically inadequate, resulting in both false negative (invasive carcinomas 15% to 55%, early invasive carcinomas 20% to 70%) and false positive results (5% to 15% with the result unnecessarily scaring patients). These shortcomings are:

• Subjective errors - cytology is complex, it requires intensive visual inspection of cells. This technique is standardized neither objective nor geographically. • Incorrect or inadequate specimen collection may lead to false negative or equivocal results.

• Histological method is generally only the current state of a tissue detected so that the prognostic value of this method is missing.

Improved detection methods, it is now managed directly detect the presence of papilloma rus in infected tissues:

In so-called HCM method (Hybrid Capture Micro Plate), one of the company Digene Corp., Gaithersburg, MD, USA for commercial beziehendem test, it is a signal-enhancing hybridization method. To succeed in the qualitative detection of 18 HPV types in tissue samples of uterine shark. As hybridization probes HPV-specific RNA sequences are used, covering the full viral genome.

After incubation, these probes and denatured HPV DNA from the infected tissue to RNA / DNA hybrids can be detected by a specific antibody form. Via the enzyme alkaline phosphatase, conjugated with the antibody is carried out by adding an appropriate substrate of the detection by chemiluminescence.

The HCM method allows differentiation between two HPV DNA groups: the HPV types 6, 11, 42, 43, 44 carcinogenic low potential and the HPV types 16, 18, 31, 33, 35, 39, 45 , 51, 52, 56, 58, 59, 68 with a medium to high carcinogenic potential. Accurate typing of papillomaviruses is not possible with this method. The disadvantage of this test method further, that there are often cross-reactions between two HPV classes due to the use of an RNA mixture. This can lead to false negative or false positive results. Another disadvantage is that the papillomaviruses HPV 5 and 8 are not detected by this method.

. Suren Theran et al, "Detection and typing of Human Papilloma Viruses in mucosal and cutaneous biopsis from immunosuppressed and in unocompetent patients and patients with Epidermodyspla- sia verruciformis: A unified diagnostic approach", Journal of Clinical Pathology "1998; 51: 606-610 , describe a polymerase se chain reaction (PCR) -based method for the detection of HPV DNA in skin and mucosal biopsies from immunosuppressed and immunocompetent and EV patients. the authors do this using different PCR primer pairs, all of which open within the reading frame of the viral gene for the structural protein Ll lie.

A typing of papillomaviruses is carried out by subsequent sequence analysis of the amplified gene fragment. Recent studies have shown that of all known HPV types less conserved the Ll gene is than originally thought. Therefore, with this method succeeds only possess a limited number of HPV types (HPV 2-6, 8, 10, 11, 16, 31, 41, 57). Whether the detection of high-risk HPV types 18, 45 and 56 is possible with this method is not described.

Jacobs also et al., "Group-Specific Differentiation between high- and low-risk human papillomavirus genotypes by General Primer-Mediated PCR and Two cocktails of oligonucleotides pro- bes ", Journal of Clinical Microbiology 1995, 33, 901-905, using a GP5 + / GP6 + designated primer pair, which is within the viral gene for the structural protein Ll. The in PCR amplified with this primer pair region of the Ll gene is 150 base pairs (bp). by means of specific probes, the amplified products by gel electrophoresis separation the high risk HPV types 16, 18, 31, 33, 35, 39, 45, 51 be 52, 54, 56 and 58 and the low risk HPV types 6, 11, 34, 40, 42, 43 and 44 associated. the specific hybridization probes each having a length of 30 bases.

. Tieben et al, "Deteσtion of Epidermodysplasia verruciformis- Like Human papillomavirus types in malignant and premalignant skin lesions of renal transplant recipients", British Journal of Dermatology 1994; 131, 226-230, describe a well PCR-based detection method of HPV DNA in skin tumors of patients after renal transplantation. The PCR primer pairs used here, various lie within the conserved virus-specific El gene, which encodes an ATP-dependent for DNA helicase. By using four different PCR primer pairs of the authors succeed in sensing the following HPV types: HPV 1-5, 6b, 7-9, 10a, 11, 12, 14a, 15, 16-22, 24, 25, 31, 33, 36-38, 46, 49, 50, 65. the inventor of the present application has found, however, that the primer pair CP4 / CP5 is not suitable for amplification.

A typing is in this publication on the sequencing of the amplified product. It is worth noting here that only three of the above primer pairs can be used, when such a typing should be connected to the PCR. can not use the above-mentioned primer pair CP4 / CP5. This method Tieben et al succeed. by typing the specific detection of HPV types la, 2a, 3 to 5, 6b, 8, 10a, 11-13, 14a, 15-18, 20-22, 24, 25, 31, 33, 36, 39, 41 , 42, 46, 47, 49, 50, 57, 58, 63 and 65 thereof.

The advantage over the Suren Theran et al. Described method is that here a greater number can be determined at different HPV types with a primer pair. Despite this improved specificity the typing of many HPV, especially the high-risk papillomaviruses HPV 45 and 56, and the HPV types with medium oncogenic potential 35, 51 and 52 with this method is not possible.

To the knowledge of the inventor of the present application the primer pair CP4 / CP5 is not used in clinical practice, here find currently rather only primer pairs application that are within the Ll gene, such as that of Jacobs et al. , supra described primer pair GP5 + / GP6 +.

Against this background, the object of the present invention to provide a method of the aforementioned type which enables reliable detection of all currently associated with tumor induction candidate HPV viruses, and provide hybridization sequences for selbiges process.

In the method mentioned initially, this object is achieved by the fact that the evidence of specific HPV DNA segments using at least one of the following the nucleotide sequences is performed: SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 from the attached sequence protocol, or sequences that bind to sequences (hybridize), to which at least one of the sequences SEQ ID No. 1, SEQ ID.... bind nr. 2 or SEQ ID NO. 3 or sequences which are complementary to the above sequences.

The object underlying the invention is solved completely in this way. The inventors of the present application has recognized that manages the detection of all known HPV types through the use of the nucleotide sequences for the first time described herein SEQ ID NO. 1, 2 and 3 due to their very high specificity and sensitivity. All three sequences are derived from the highly conserved viral El gene. Therefore the use of the sequences SEQ ID Nos. 1, 2 and 3, for example, as hybridization probes (DNA marker) or in a DNA amplification method is particularly suitable for the detection of a general HPV infection.

The described method leads to particularly good results when the detection of HPV infection by a specific amplification of the HPV-DNA segments using polymerase chain reaction (PCR) is carried out.

For this purpose, for example, the nucleotide sequences of SEQ ID NO. 1 and 2 are used as the PCR primer pair. Here an amplification arises onsprodukt of approximately 220 bp, which may be by means of simple standard methods, such as ethidium bromide staining after gel electrophoresis separation, demonstrated. A preferred embodiment of the present invention is that the amplification of specific HPV-DNA segments is carried out by polymerase chain reaction (PCR), which is characterized by the following temperature profile:

Heating: 5-15 min, 90-100 ° C;. Heat denaturation: 30 - 90 seconds, from 90 to 100 ° C; Hybridization: 30 - 90 seconds, 50 - 60 ° C; Polymerization: 30 - 180 seconds, 67 - 77 ° C; Cooling:. 2 - 10 min, 67 - 77 ° C.

The inventor of the present application has recognized that this temperature profile that differs from other published profiles for an efficient amplification reaction and therefore reliable typing is essential. A particularly efficient PCR reaction is achieved, according to findings of the inventor with the following conditions:

Heating: 10 min, 95 ° C;.

Hi zedenaturierung: 30 sec, 95 ° C;

Hybridization: 30 sec, 55 ° C;

Polymerization: 1 min. , 72 ° C;

Cooling: 5 min. , 72 ° C;

Heating rate 1 ° C / sec;

Cycles: 40th

The use of the sequence SEQ ID NO. 1, together with the sequence SEQ ID NO. 3 as a PCR primer pair provides an amplification product of the viral El gene of approximately 280 bp in length. This allows, due to the length of the amplicon (in comparison to the 220 bp amplicon when using SEQ ID NO. 1 and SEQ ID NO. 2 as PCR primer pair), an even more secure typing, for example by subsequent sequencing or Temperaturgradientengelelektrophorese, than when using the primer pair SEQ ID NO. 1 and 2.

Another advantage is the fact that fikationsproduktes is facilitated by the nature of the nucleotide sequence SEQ ID NO. 1, which is degenerate in any position, a possible sequencing of the amplicon. Of Tieben et al. Primers used are degenerate hand in two or three positions. This has the consequence that in the PCR very high Primermen- must be used gen and at any subsequent sequencing increased problems.

Against this background, the invention further relates to the use of the sequences SEQ ID Nos. 1 and 3 as a PCR primer pair in the method described.

It is also possible to use the described primer pairs in a method for quantitative PCR, which can be carried out on the so-called LightCycler ™ from Roche Molecular Biochemicals. Detection of the amplified DNA can be effected both via the used for the detection of double-stranded DNA dye CYBER ™ Green I as well as through sequence-specific DNA probes which are labeled with different dyes.

A particularly reliable typing of HPV is achieved by sequencing of the amplified HPV-DNA segments. The amplification is namely due to low Sequenzvaria- tions between the various HPV types characterizes what enables the mapping of the type.

The described method is preferably used as part of a method for diagnosis and / or early detection, particularly in connection with cancer, such as cervical cancer.

The inventor herein has found that this method, the ingredients have been used in other contexts mainly in the research area, is a reliable diagnostic method.

The new method is also suitable to be used as part of an early detection of cancers. Only an assured positive findings in terms of HPV infection can namely be the starting point for a targeted therapy.

The invention relates to the nucleotide sequences used in the process SEQ ID Nos. 1, 2 and 3 according to the enclosed sequence listing, as well as nucleotide sequences that hybridize to sequences to which the sequences SEQ ID Nos. 1, SEQ ID NO.: 2 or SEQ ID no. 3 tie.

The fact is that the nucleotide sequences of the invention may be modified by shortening, lengthening, substitution, insertion and / or deletion, however, are still functionally comparable. Against this background, the invention also relates to such modified nucleotide sequences. The same is true for nucleotide sequences, the sequences are at least one of the above complementary.

The inventor has recognized that the use of complementary nucleotide sequences which hybridize according to the complementary strand of HPV DNA, in the method described above, lead to the same results. Therefore, such sequences are also part of the present invention.

A special effort by the inventor is the realization that a nucleotide sequence of the open reading frames El of HPV to detect and even for the typing of HPV in Anogenitalbe- rich, particularly in the context of cervical disease, can be used because the El-region compared to the Ll region of all known HPV types shows a higher conservation.

The high conservation of the viral El gene relative to the viral len Ll gene ensures the largest possible amount of HPV types. Despite existing, minor sequence variations between the different HPV types ensure unexpectedly also a reliable typing.

It is understood that the features mentioned above can be used not only in the specified combination but also individually or in other combinations, without departing from the scope of the present invention.

The invention is illustrated by way of examples, from which further features and advantages will be apparent. Example 1: DNA extraction

For the detection of HPV, it is first necessary to remove biopsy or smear material. This is in 500 ul mes- ting buffer (0.1 M EDTA, 0.05 M Tris pH 8.0; 0.5% SDS) was added. These 2.5 ul Proteinase K are added (20 mg / ml) and the solution was incubated for 24 to 48 hours at 55 ° C.

This is followed by two-time phenol extraction (phenol includes: CHC1₃: Isoamyl alcohol = 25: 24: 1). The batch is mixed, not vortexed for 2 - 5 min. incubated (rotated upside down), then at 14,000 rpm for 5 min. centrifuged. There follows an extraction with CHCl₃/ Isoamyl alcohol (24: 1), incubation for 2 - 5 min, then 5 min.. at 14000 rpm centrifuge for ren.

This is followed by addition of 1/10 of the lot volume of 7.5 M ammonium acetate, the mixture is mixed and 2 to 4 hrs. At 55 ° C.

The mixture is then ethanol precipitated overnight at -20 ° C.

The thus extracted DNA is then used in a PCR reaction.

Example 2: PCR reaction

The PCR serves the targeted amplification of viral DNA segments. For this purpose, 0.25 uL, 1 uL, 2.5 ul and 5 ul of the extracted amount of DNA are used (without concentration determination); Additional primer 1: 100 ng, primers 2: 100 and 200 ng, dNTPs: 10 mM each; 5 ul Taq polymerase buffer (10x, without MgCl₂); 7 ul MgCl₂ (25 mM stock solution corresponding to 3.6 mM); 0.4 ul Taq Gold polymerase (equivalent to 2 units). The batch is made up to a total volume of 50 ul. The PCR was carried out in an MJ Research thermal cycler (PTC 200); set heating rate: 1 ° C / sec The PCR amplification runs for 40 cycles [10 min. 95 ° C (heating, only in the first cycle); 30 sec. 95 ° C; 30 sec. 55 ° C; 1 min. 72 ° C; 5 min. 72 ° C (cooling, only in the last cycle)].

For non-specific detection of HPV infection (screening) are preferably the sequences SEQ ID NO. 1 (forward) and SEQ ID NO. 2 (reverse) used as primer pair (100 ng). The nucleotide sequences of these primers and the situation in the HPV genome (HPV16 as an example) are:

SEQ ID NO. 1 5^.- (Nt2082) -AAC AAT GTG TAG ACA TTA TAA

ACG AGC (nt2108) -3 '

SEQ ID NO: 2 5 '-. (Nt2336) -ATT AAA CTC ATT CCA AAA TAT

GA (nt2314) -3 '

Through this screening process all known HPV types are currently detected.

Believed to be a typing, DNA sequencing, connect eg, one preferably uses the sequences SEQ ID NO. 1 (Forward) and SEQ ID NO: 3 (reverse; 200 ng) as a primer pair.:

SEQ ID NO. 3: 5 '- (nt2401) gag-GYT-GCA-ACC-AAA AMT GRC

T- (nt2380) -3 '

wherein Y is a pyrimidine (T or C) M Amino (A or C) and R is a purine (G or A).

For the reverse primers SEQ ID Nos. 2 and 3 the positions of the anticoding DNA strand are given to the reverse these primers and are complementary.

Example 3: DNA sequencing (typing)

The sequencing of the amplified product from Example 2 for the determination of the HPV type is loaded with the Big Dye Terminator Cycle Sequencing Ready Reaction Kit with Ampli Taq from ABI using 200 ng of primer SEQ ID NO. 1 or 3 and the PCR amplification preparation performed. This is previously concentrated and purified using Microcon 100 columns (Millipore). From the purified approach 50% for sequencing are used.

The sequencing PCR reaction is performed according to the following conditions: 10 sec. 96 ° C, 5 sec 55 ° C, 4 min.. 60 ° C, 30 cycles, with a heating rate of 1 ° C / sec.

Like then with 0.3 M sodium acetate and 2.5 times the volume of ethanol. The pellet is taken up in 25 .mu.l TSR (template suppression reagent from ABI), for 2 minutes. incubated at 90 ° C, then for min. 2 brought to 4 ° C, and then kept at room temperature.

The capillary electrophoresis using the ABI Prism 310 automated sequencer is a 47 cm capillary (diameter 50 microns) performed and evaluated with the 310 Genetic Analyser program of ABI. As the polymer, the polymer is POP 6 (Performance Optimize polymer 6) from ABI used.

By this typing following three HPV types can be identified when using the primers SEQ ID No. 1 and SEQ ID NO..:

HPV1 to HPV8 HPV51 and HPV54

HPV10 and HPV19 HPV56 HPV60 up

HPV21 and HPV28 HPV65 HPV67 up

HPV30 and HPV38 HPV70

HPV40 HPV72 HPV73 up

HPV42 HPV82 HPV83 HPV44 and HPV47 up

When using the PCR primer pairs SEQ ID NO. 1 and SEQ ID NO. 2, analogous results are obtained. Due to the smaller amplification following HPV types are not differentiating: HPV51 / HPV31

HPV31 / HPV70

HPV18 / HPV6

HPV18 / HPV2a

HPV33 HPV1

HPV31 / HPV83

HPV31 / HPV57

Example 4: Use of a nucleotide sequence of the open reading frame of HPV El

The genome of HPV is shown in FIG. 1 by way of example with reference to the 7905 bp HPV 16 genome. The genome is divided into three sections: a non-coding region (LCR, Long Control Region), an E- (early-) region for the "early genes" coded and an L (lateralis) region for "late genes" coded. The position of the open reading frame can be seen with reference to the position information.

From the open reading frames El, which encodes an ATP-dependent DNA helicase, one or more nucleotide sequences for the detection of HPV are used in the manner according to the invention that hereby all HPV types are detected. The use of such sequences is possible in the form of hybridization probes or PCR primers, etc.. Here comes a particularly HPV infection in the anogenital region, particularly in the context of cervical disease, into consideration.

The El region is characterized by a high degree of conservation within the known HPV types. Therefore, the El region is particularly suitable for detecting a general HPV infection. It has been found that there may be false negative results due to greater sequence variations between the different HPV types when using the open reading frame Ll for detecting HPV. This risk of the erroneous detection is minimized by the use of the El-region.

Nevertheless, the El-region can also be used for the typing of HPV that^' small sequence differences rich then by knowledge of the inventor of surprisingly.

Example 5: Quantitative PCR with the primer pair SEQ ID NO.

1 and SEQ ID NO. 3

With the aid of the LightCycler ™ the genome of HPV 33 using the primer pair SEQ ID NO. 1 and SEQ ID NO. 3 is detected. For this purpose, according to the manufacturer is the tenfold LC DNA Master Hybridization fast start sample mix (without MgCl₂) Is used and the MgCl₂-Entkonzentration Adjusted to 4 mM.

The used amount of DNA of HPV33 was 30 ng, the two primers could be employed to 0.9 uM each in the range of 0.45.

The PCR amplification was carried out according to the following protocol:

Initial denaturation at 95 ° C, 10 minutes

Amplification (50 cycles), each 95 ° C, 10 seconds, 55 ° C, 15 seconds, and 72 ° C, 15 seconds. The cooling was carried out at 40 ° C, 1 minute. The heat-up rates were in all but one step at a 20 ° C / sec, in the 72 ° C. step from the amplification each with 5 ° C / second.

At the end of the annealing, the fluorescence measurement was carried out.

With the aid of the binding to double-stranded DNA dye CYBER ™ GREEN I could be detected in comparison with an HPV Plasmidstandard a sensitivity of this method, which still allows detection of only ten copies of HPV genome per sample.

In a further test, two sequence-specific DNA probes were used, which were labeled with different dyes. The sequences of the two probes are selected so that they are hybridized to the target sequence of the amplified DNA segment that the 3 'end of a probe close to the 5' end of the other probe, said the two dyes almost another to be brought. Of a dye is a donor dye and is excited by a short wavelength light source, whereupon it emits fluorescent light at a slightly longer wavelength. When the two dyes are located close next to each other, stimulates the emitted energy of the donor dye to the acceptor dye, which sits at the second hybridization probe and emits a fluorescent light at a different wavelength.

For the detection of HPV33 both of the following probes were used: 3 'probe: CGTAAAATGT CAATAGGACA ATGGATACAA

5 'probe: AGATGTGAAA AAACAAATGA TGGAGGAAA

The 3 'probe was the dye LC Red640 and 5' labeled probe with the dye FITC. Both probes were used in the range of 0.15 to 0.25 micron.

The PCR amplification was carried out according to the manufacturer's protocol, and here was an achievable sensitivity of 10 copies per genome inserted sample are shown.

Example 6: Comparison between the primer pairs GP5 + / GP6 + and SEQ ID NO: 1 / SEQ ID NO: 3 and the Hybrid Capture method..

With the Hybrid Capture II HPV DNA test of the company Digene Corporation, Gaithersburg, MD, USA, different strokes Zervikalab- were HPV examined, there arose 15 High Risk HPV positive and 24 High Risk HPV-negative samples.

These samples were also tested using the primer pair SEQ ID NO. 1 and SEQ ID Nos. 3 and with the PCR reaction shown in Example 2 to HPV. In parallel, these samples were also with the GP5 + / GP6 + primer pair and in the publication by Jacobs et al. ibid indicated reaction conditions checked.

Of the 15 HCII High Risk HPV-positive samples were with GP5 + / GP6 + eleven samples were positive, with SEQ ID NO. 1 / SEQ ID NO. 3, however, all 15 samples tested positive. Of the 24 high-risk HPV HCII-negative samples were positive with GP5 + / GP6 + two samples, with SEQ ID NO. 1 / SEQ ID NO. 3 even seven samples positive.

In all cases that have tested positive for SEQ ID NO. 1 / SEQ ID NO. 3 could be determined by direct sequencing of the amplified products to one of the present HPV type. In the total of 13 positive GP5 + / GP6 + samples this succeeded only in seven cases, as revealed to "patient samples in the gel multiple bands, which seems to point to non-specific amplification products of cellular genes. Of these seven cases, more the result in two cases of the result with SEQ ID NO. 1 / SEQ ID NO. 3 in.

.. The here found by SEQ ID NO: 1 / SEQ ID NO: 3 HPV types were: 2, 16, 18, 31, 32, 33, 35, 42, 45, 51, 53, 72.

These comparative experiments show that the primer pair SEQ ID NO. 1 / SEQ ID NO. 3 is significantly more sensitive than the Hybrid Capture method or a method in which the probes are GP5 + / GP6 + employed. compared to SEQ ID NO. 1 / SEQ ID NO. GP5 + / GP6 + showed 3 nine false negative samples, while the Hybrid Capture method compared with SEQ ID NO. 1 / SEQ ID NO. 3 seven false negative samples showed.