Kit and method for the detection of bacteria in a biological test sample by nucleic acid hybridization

BACKGROUND OF THE INVENTION

1. FIELD OF THE INVENTION

This invention concerns analytical methods and test kit for detecting the presence of bacteria in a test sample, particularly, in areas related to health care, such as in the analysis of human body fluids, e.g., urine and blood, for the purpose of aiding diagnosis, or in the testing of foods to detect contamination.

2. DESCHIPTION OF THE PRIOR ART

The classical method for detecting bacteria in a test sample involves culturing of the sample in order to expand the number of bacterial cells present into observable colony growths which can be enumerated. If desired, the cultures can also be subjected to additional testing in order to determine antimicrobial susceptibility and identification of particular bacterial species. Bacteriological culture methods are particularly labor intensive and require highly skilled technicians. Normally, 24 to 48 hours of incubation are required before a positive or negative result can be reliably determined. Other limitations of the culture method include the need to maintain viability of bacteria in the test sample during transport to and processing at the laboratory.

A wide variety of alternative techniques have been investigated and developed in the continuing attempts to devise methods which overcome the numerous drawbacks of the conventional culture approach. Light microscopy is frequently used to detect bacteria in clinical specimens. Usually the specimen is stained to improve the detection limit and help characterize the organisms present. The method is laborious and does not offer good detection limits. Immunological methods have been successfully developed to detect specific species and genera which have surface antigens which are distinguish able by specific antibody binding. Such an approach is not practical, however, for quantitating bacteria in a test sample which may contain bacteria from a variety of genera which do not share a common surface antigen from such sources as in body fluids, e.g., blood or urine.

Other tests have been developed based on detection of metabolic products of bacteria such as nitrites, nucleotides such as adenosine triphosphate, CO₂ released froni ¹⁴C-labeled glucose, and specific enzymes. The disadvantages of these techniques are many and include: ATP can be degraded by enzymes in the test sample, ATP can originate from nonbacterial sources such as tissues of the host, radioactive materials present a biohazard, and enzymes with similar activity can arise from the host.

Particle counting instrumentation has also been applied to detection of bacteria. Such instrumentstion measures perturbations in an electrical current across a small orifice caused by the presence of partiMS-1309 -CTP

־ cles in a,fluid flowing through the orifice. Besides requiring the use of complex and expensive apparatus, this method is highly nonspecific and requires a high level of care and particle־free conditions.

Other types of instrumentation measure the growth of bacteria in special liquid media. The instruments involved make periodic turbidimetric measurements with increases in turbidity indicating the presence of growing bacteria. . . ״

Therefore, there is a continuing, long-felt need for a rapid, accurate, and economical technique and means for quantitating bacterial presence. Despite the rapid advance of analytical techniques in closely related fields, as represented by the developments of the radioimmunoassay, spectrophotometry, fluorometry, microcalorimetry, and electrochemical techniques, the predominant technique used in bacteriological testing remains the plate culture method, which today is still much the same as the techniques used by Pasteur and the other early microbiologists of the 19th century. A recent analysis of the state-of-the-art in microbiology concluded that the needs of modern medicine could best be served by developing alternatives to culturing foi detecting microorganisms [Nature 302(1983) p. XXVII].

In 1964, Nygaard and Hall [«/י Mol. Biol. 0:125-142 (1964)] described a method for quantitative detection of DNA/RNA hybrids by filtration of samples through nitrocellulose membranes. They discovered that single stranded DNA adsorbs onto the membrane, whereas double30 stranded (duplexed) DNA and single-.or double-stranded RNAs pass through. Sample DNA in single-stranded form and radiolabeled RNA were allowed to incubate in solution resulting in hybridization binding between the radiolabeled RNA and complementary DNA strands in the sample. The reaction mixture was then passed through the nitrocellulose filter. The unhybridized labeled

MS-1309 -CIP labeled RNA labeled RNA washing. 1. filters was Res. Commun sample DNA was bound to ni------with a prehybridization solution

RNA passed through while the DNA-RNA hybrids had sufficient single-stranded DNA regions to adhere to the filter. Measurement of radioactivity bound to filter provided an estimate of the amount ofhybrid formed. .

The birth of this nucleic acid hybridization technique was followed by further refinements and applications to specific analytical situations. Gillespie an Spiegelman [d. Mol. Biol. 1965)2:829-843־)] fixed single-stranded sample DNA onto nitrocellulose filters and then submerged the filters into a solution of r to allow hybridization to occur. Excess . was removed by treatment with RNase and A DNA/DNA hybridization method on membrane developed by Denhardt [Biochem. Biophys.

(1966)]. Single-stranded ־ itrocellulose and treated ־ ‘ i to minimize nonspecific binding'of labeled probe DNA during the hybridization reaction. Grunstein and Hogness [Fro־־. »atl. Acad. Soi. USA 72:3961-3965(1975)] developed a solid phase colony hybridization technique for rapid screening of E. eoU colonies to determine which ones contained an inserte DNA sequence. Immobilization of nucleic acids to solid supports has also short DNA fragments, included using paper [Alwine et al, Proa.

Seed, Nualeia Aoids Bea Bioch. Biophys. Acta tof the nucleic acid prior [Thomas, Proc Nucleic been improved to include RNA and The techniques developed have with chemically reactive groups Natl. Acad. Soi. USA .74:4350(1977), ־ r. 10.1979(1982), and Hunger, 653:344(1981)] and derivatization to fixation on nitrocellulose . Natl. Aoad. Sol. USA 77 :5201(1980)I. acid hybridization assays have been developed for a number of diseases. These inclu the genetic diseases a-thalassemia [Kan et al, New Eng. J. Med. 295:1165(1976)], (!-thalassemias (Ortm

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75:5631

Wilson et al, 80:278 ; for the have also been reported.

et al. nature 292:627(1982)] and sickle cellahemia [Kan and Dozy, Proc. Bail. Acad. Sei. USA (1978), Geever et al, ibid 72:5081(1981), ibid 79:3628(1982) and Conner et al, ibid (1983)). Nucleic acid hybridization tests presence of infectious agents 1-.

These include a test for enterotoxin-producing bacteria in fecal specimens (Moseley et al, Dis. 52:892(1980), ibid 155:563(1982), and U.S. Pat NO. 4,358,535). See also Institut Pasteur, British Pat. 2,019.408B. Hepatitis virus in serum has also been detected by hybridization [Schafritz et al, Proc, a Acad. Sai. USA 79: 5675(1982) and Berninger et al, . .

Virol. 9:57(1982)).

Some studies have been undertaken of the generic relatedness of various bacteria based on nucleic act hybridization between denatured and fragmented single-stranded DNA from different bacteria [see Brenner, The Public Health Laboratory , Thompson lu . Inc., vol. 32 (1974) pp. 118130־: and the review by Brenner and Falkow, Ado. in Genetics 15:81-119(. .).

. ״f bacterial genomic DNA coding for various Conservation of bacterial genu!״v products such as ribosomal RNA, ribosomal proteins transfer RNAs, and the like has been the subject 0. some study [«7 ך; 1968)2:1105-1118 .'־C 381-385(1979); J. Bast. 133:1089(1978); J. Biol. Ches, 225:728-734(1980); and J. Bant. 129:1435(1977)). I 1 er St al [«״. J. Bloches,. 122:79-77(1981)] have reported that DNA sequences homologous to certain pro aryo 1c genes, B. soli tuf ^es. are present in some taxonomically unrelated genera.

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SUMMARY OF THE INVENTION

It has now been found that nucleic acid hybridization techniques can be applied to the task of quantifying bacteria in a variety of test samples, particularly in media of diagnostic significance such as body fluids. The present method therefore provides a highly convenient screening test for determining if there is significant bacterial presence in a test medium of interest, partidularly media which are normally sterile such as blood or urine.

The test sample is first subjected to conditions to release and denature (render single stranded) nucleic acids from bacteria present in the sample. The resulting single stranded nucleic acids are then contacted in an appropriate manner with a polynucleotide probe having a base sequence which is homologous with at least one base sequence in the nucleic acid content of substantially all of the various respective bacteria possibly present in the test sample. Contact between the probe and the denatured sample nucleic acids is performed under conditions favorable to hybridization between the probe and the aforesaid respective bacterial base sequences. Resuiting hybridization is then determined as a function of the extent of bacterial presence in the test sample.

The homologous base sequence of the probe is preferably at least about 10, and normally at least about 20, bases in length. The polynucleotide probe can be selected in several manners including empirical screening of the bacteria expected to be present in the test sample for a homologous sequence or sequences or predetermined selection of a conserved or substantially conserved gene.

It has most particularly been found that a most advantageous nucleic acid hybridization assay for de tecting bacteria is provided by the use of a .probe

MS-1309-C1P’ comprising at least a portion of one of the strands of a gene which codes for the synthesis of a nucleK acid or protein comprised in the mechanism of protein synthesis Important examples of such genes include those coding for transfer RNAs, ribosomal RNAs, ribosomal proteins ,. . aminoacyl-tRNA synthetases, initiation factors, clongation factors, and releasing factors.

Detection of hybridized probe can be preferably accomplished by solid phase hybridization involving use of a labeled form of the probe. The label can be any convenient detectable moiety such as radioactive isotopes or nonradioactive elements such as fluorescers, chemiluminescers, or molecules which form specific bind ing sites (e.g., haptens) for binding partners (e.g., antibodies) incorporated with a directly detectable species (e.g., enzymes, enzyme substrates, cofactors, or modulators, fluorescers, and chemiluminescers).

The present method for detecting bacteria numerous advantages over the prior art techniques, particularly the classical culturing methods. In the present method it is not necessary to isolate or pr.opagate the bacteria to be detected, which in some cases can be a highly infectious agent. Furthermore, it is not necessary to rely on the expression of certain metabolic products or surface antigens in order to make a determination. I״ addition, the present method does not require that test samples be handled in a manner necessary to guarantee viability of any bacteria present. The prior art methods in general will detect only viable cells in the sample. Often, microbes die during transport of specimens to the laboratory for testing. Also, a specimen can contain different typos of microorganisms having a variety of growth limitatio and requirements. Thus, when using the prior art methods, the specimen must be cultured under several different conditions in order to ensure detection of

MS-1309 -CIP any possible bacteria present. Such a serious Innit.ation is absolutely not required by the present method since the object of the assay is the stable nucleic acid composition of the bacteria present in a sample.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The use of nucleic acid hybridization as an analytical tool is based fundamentally on. the double - stranded, duplex structure of DNA. The hydrogen bonds between the purine and pyrimidine bases of the respective strands in double-stranded DNA can be reversibly broken. The two complementary single strands of DNA resulting from this melting״ or ״denaturation of DNA will associdte (sometimes referred to as reannealing or hybridization) reforming the duplexed structure. As is now well known in the art, contact of a first single stranded nucleic acid, either DNA or RNA, which comprises a base sequence sufficiently complementary to (i.e., homologous with) a second single stranded nucleic acid under appropriate solution conditions, will result in the formation of DNA/DNA, RNA/DNA, or RNA/RNA hybrids, as the case may be.

In accordance with the present invention, a polynucleotide (normally single stranded DNA or RNA) is selected as;an analytical reagent on the basis that it 25 comprises a base sequence which is homologous, i.e., sufficiently complementary, with base sequences found in substantially all bacteria of interest. Thus, hybridization between this polynucleotide probe and nucleic acids released in single stranded form from the 30 test sample indicates the presence of bacteria in the sample.

The bacteria of interest will vary from one analytical situation to another. Through experimentation or experience in the field it will be known which bacterial 3S species could be present in a particular test, sample.

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One then can select from a variety of methods to isolate one or more probes containing homologous base sequences from the nucleic acid content of such bacteria species. The homologous base sequence need not be a single continuous polynucleotide segment, but may be comprised of two or more individual segments interrupted by nonhomologous sequences. These nonhomologous sequences may be linear, or they may be self-complementary and form hairpin loops. In addition, the homologous region of the probe may be flanked at the 3' and 5 terminii by nonhomologous sequences, such as those comprising the DNA or RNA of a vector into which the homologous sequence had been inserted for propagation. In either instance, the probe as presented as an analyti cal reagent will exhibit detectable hybridization at one or more points with sample nucleic acids of interest. Linear or circular single stranded polynucleotides can be Used as the probe element, with major or minor portions being duplexed with a complementary polynucleotide 20 strand or strands, provided that the critical homologous segment or segments are in single stranded form and available for hybridization with sample DNA or RNA. Particularly preferred will be linear or circular probes wherein the homologous probe sequence is in essentially only single stranded form.

Homologous base sequences for the bacteria of interest ar6 obtainable in several manners. A particularly important method for obtaining a homologous sequence involves selection of a gene or segment there30 of which is known or experimentally determined to be conserved across substantially all bacterial species of interest. Such selected gene can be structural or regulatory, or perform some other function. If a structural gene, it need not be expressed in some or all 35 of the bacterial species of interest since the present method is based not on expression but rather on the

MS-1309-CIP presence of hybridizable nucleic acids, e.g., genomic DNA or RNA or extra-chromosomal nucleic acids.¹

The selected gene will usually comprise a structural gene for an expressed protein or nucleic acid. It has been particularly found that broad spectrum bacterial detection capability is provided where the homologous base sequence comprises at least a portion of one of the strands, i.e., either the sense or antisense strand, of a gene which codes for the synthesis of a nucleic acid or protein comprised in the mechanism of protein synthesis in the cell. Fundamentally, such mechanism involves the processes of transcription and translation. Transcription will be understood to define the process by which the genetic code in DNA is converted to messenger RNA (mRNA). Translation will be understood to define the process by which the nucleotide sequence of mRNA is translated into the amino acid sequence of a synthesized protein.

Therefore, in a preferred embodiment the homologous base sequence is obtained from a gene which codes for a nucleic acid or protein involved in the mechanisms of transcription or translation. In the case of transcrip tion, such nucleic acids and proteins include the RNA polymerases, protein initiator factors (sigma factor), and protein termination factors (rho factor). In the case of translation, included are those nucleic acids and proteins involved in the three fundamental stages of protein synthesis - initiation, elongation, and termination. Thus, such materials include transfer RNAs (tRNA- comprising the functionally distinct classes for each of the 20 fundamental amino acid building blocks), ribosomal RNAs (rRNA - comprising the various known fragments and subunits such as the 5S, 16S, 23S, and 40S elements), ribosomal proteins, aminoacyl-tRNA synthetases, initiation factors (including IF-1, .112־, and IF-3), elongation factors (including E‘F-1, 112־

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EF-G, and EF-Tu), and releasing factors. It should be understood that mRNA is not included in the abovedescribed preferred class since the genes which code for mRNA synthesis will vary according to the trait for which the gene codes. Thus, for the purposes of this invention, mRNA as such is not part of the mechanism of protein synthesis but rather can be considered as the variable informational input to an otherwise essentially constant chemical system.

In addition to the above preferred class of genes from which to select the probe of the present'invention, one can also choose from genes coding for other proteins and nucleic acids. Some examples are genes coding for such materials as various RNAs other . thafi those involved in protein synthesis, such as 5S-RNA and priming RNAs for DNA replication; genes coding for bacterial membrane proteins such as membrane lipoproteins from Gram-negative bacteria; genes coding for insertion sequences; transposons; and the like.

One strategy for development of a probe is to begin with cloned DNA which codes for ribosomal RNA. Ribosomal DNA has been cloned and sequenced for several species of plants, animals and bacteria including E. coli.

The 5S RNAs are 115 to 120 nucleotides long and have a high degree of sequence identity between prokaryotic and eucaryotic organisms. Probes based on 5S RNA would be useful for detection of bacteria in samples which do not contain significant eucaryotic cellular products.

16S Ribosomal RNA from E. coli is 1542 bases long and has a sequence nearly identical to those of other gram negative bacteria such as Proteus vulgaris.

There is a high degree of homology between 16S ribo35 somal RNAs from E. coli and Bacillus brevis. Therefore, the E. coli sequence could also be used to detect gram positive bacteria.

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In analyzing clinical samples for bacteria, it is desirable to differentiate between 16S bacterial RNA and mammalian 18S RNA and mitochondrial RNAs. This goal can be realized because the RNAs differ substan5 tially in length and the identity among sequences from various species of bacteria is much greater than between bacterial and mammalian sequences. If necessary, a probe based on 16S RNA can be made more specific for bacteria by selecting certain portions of these10 quence. For example, the 3'-hydroxyl terminus of mouse 18S RNA has segments which are very homologous with the 3״-terminus of E. coli 16S RNA. Elimination of a segment, for example, 230 bases, at the 3'terminus of a 16S RNA probe could be used to improve 15 the selectivity for bacteria over mammalian cells.

The 23S ribosomal RNA from E. coli is 2904 nucleotides long and the corresponding 28S RNA from the rat is larger, 4718 nucleotides. The rat sequence has a high degree of identity with sequences from 20 other higher organisms such as the frog (75¾ identity) and less identity with sequences from lower organisms such as yeast (about 50¾ identity). A probe which codes for 23S RNA from E. coli will thus be useful for detection of bacteria in clinical specimens which may 25 contain mammalian cells.

Another useful method for obtaining homologous base sequences for bacteria of analytical interest involves experimental screening of the nucleic acid content of such bacteria. This empirical method can be 30 accomplished by a variety of schemes.

In one such scheme, ribosomal RNA is isolated from one of the n bacterial strains of interest designated strain A and separated into size classes of SS, 16S and 23S RNA by sucrose gradient centrifugation. 35 The RNA is labeled with «³²P-ATP by using E. coli Poly A Polymerase. The DNA from all the bacterial strains

MS-1309-CIP strains of interest is isolated and fragmented by restriction endonuclease digestion. These DNA fragments are separated according to size by agarose gel electrophosesis and the DNA fragments are transferred to nitrocellulose by blotting. These DNA

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5S, 16S or 23S RNA hybridize to sonic of the DNA fragments from all the strains tested this indicates that there exists a common DNA sequence among all the strains. In order to enrich for and isolate this DNA׳sequence, the DNA restriction fragments of either strain B, C, D or E are separated by chromatography on an ion exchange resin (such as RPC-5). This procedure separates DNA restriction fragments on the basis of size, AT content and structural features. . The fractions from ion exchange chromatography are analyzed by agarose gel electrophoresis and blotted onto nitrocellulose. The nitrocellulose sheet is exposed to the ribosomal RNA probe to determine which fraction of DNA from the ion exchange chromatography is enriched for the common sequence shared with strain A. The DNA fractions enriched for ribosomal DNA are cloned and the clones screened for the presence of the ribosomal DNA. This DNA can then be processed by conventional techniques to yield polynucleotide probes useful in the present invention.

In another scheme, RNA is isolated from one of the n bacterial strains of interest and appropriately

דן labeled (e.g., with a P-ATP using E. ooli Poly A polymerase). The labeled RNA from strain A are then mixed with a sample of heat denatured DNA from strain B and resulting DNA/labeled RNA hybrids are separated from remaining nucleic acids. The separated hybrids, representing homologous sequences in the mRNA and tRNA from strain A to the DNA from strain B, are heat denatured and mixed with a sample of heat denatured DNA from strain C. This' is repeated for all n strains and the final DNA/labeled RNA hybrids will contain RNA sequences that are enriched for homology to a sequence of DNA that is present in all of the strains tested. Such hybrids can then be processed by conventional techniques to yield polynucleotide probes ,useful in the present invention.

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Having selected or experimentally obtained a suitable polynucleotide probe, reagent quantities can be prepared by conventional cloning techniques׳ in suitable vectors such as pBR.322, pllV33, pllC9, Lambda 5 and derivatives thereof, and pYIPlZ. A particularly preferred vector is M13 bacteriophage (and derivatives) since only single stranded DNA probe will be generated, thereby eliminating the need to denature the probe prior to use in the assay and preventing self hybridization [Molecular Cloning, A Laboratory'Manual, T. Maniatis et al, Cold Spring Harbor Laboratory (1982) pp. 51 et seq].

The extent and specificity of nucleic acid reassociation is affected by:

¹ 1. The purity of the nucleic acid preparation.

2. Base composition of the probe - G-C base pairs will exhibit greater thermal stability than . Λ-Τ base pairs. Thus, hybridizations involving higher G-C content will be stable at higher temperatures.

3. Length of homologous base sequence ־ Any short sequence of bases (e.g., less than 6 bases), has a high degree of probability of being present in many nucleic acids. Thus, little or no specificity can be attained in hybridizations involving such short sequences. The present homologous probe sequence will be at least 10 bases, usually 20 bases or more, and preferably greater than 100 bases. From a practical standpoint, the homologous probe sequence will often be between 300-1000 nucleotides.

4. Ionic strength ־ The rate of reannealing increases as the ionic strength of the incubation solution increases. Thermal stability of hybrids also increases.

5. Incubation temperature - Optimal reannealing occurs at a temperature about 25-30°C below the melting temperature (Tm) for a given duplex. Incubation at temperatures significantly below the optimum allows less related base sequences to hybridize.

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6. Nucleic acid concentration and incubation time ־ Normally, to drive the reaction towards hybrid!zation, one of the hybridizable sample nucleic acid or probe nucleic acid will be present in excess, usually

5. 100 fold excess or greater.

7. Denaturing reagents - The presence of hydrogen bond disrupting agents such as formamide and urea increases the stringency of hybridization.

8. Incubation time - The longer the incubation 10 time the more complete will be the hybridization.

Normally, incubation times are between 6 and 24 hours.

9. Volume exclusion agents The presence of these agents, as exemplified by dextran and dextran sulfate, are thought to effectively increase the con- centration of the hybridizing elements thereby increasing the rate of resulting hybridization.

Based on the above criteria and related factors known in the art, contact between the probe and denatured sample nucleic acids will be accomplished under conditions 20 favorable to hybridization. As conditions are made more stringent, greater complementarity is required for forma־ tion of probe hybrids. Hybridization conditions of low salt and high temperature increase the stringency of annealing. One of the most popular conditions for hybridization is in 2XSSC (where 1XSSC 0.15 ־M sodium chloride and 0.015M sodium citrate, pH 7.0) at 65°C. Under such conditions, hybridization generally will require at least about 1520־ contiguous, exactly matched base pairs in the probe.

In some applications, it may be desirable to allow hybridization between less complementary sequences, e.g., to allow for evolutionary mutations affecting nucleic acid sequence but not overall cellular function and structure. The ability of related but not identical complementary sequences to reanneal can be controlled by the conditions of stringency. High stringency, e.g., elevated temperatures, requires formation of greater

MS-1309-CIP numbers of base pairs in a given length of duplex since reassociation is restricted to closely, related sequences. At lower temperatures, i.e., less stringency, more distantly related sequences can reanneal. Addi5 tionally, increasing incubation time also permits less related sequences to anneal.

Practice of the present analytical method is not limited to any particular hybridization format. fhe manner in which hybridization resulting between the 10 probe and sample nucleic acids is determined is primarily a matter of convenience. Any conventional hybridization technique can be used. As improvements are made and as conceptually new formats are developed, such can be readily applied to carrying out the present 15 method.

Conventional hybridization formats which are particularly useful include those wherein the sample nucleic acids or the polynucleotide probe is immobilized on’a solid support (solid-phase hybridization) 20 and those wherein the polynucleotide species are all in solution (solution hybridization).

A. Solid-phase Hybridization

In this approach, one of the polynucleotide species participating in hybridization is fixed in an appro25 priate manner in its single stranded form to a solid support. Useful solid supports are well known in the art and include those which bind nucleic acids either covalently or noncovalently. Noncovalent supports which are generally understood to involve hydrophobic 30 bonding include naturally occurring and synthetic polymeric materials, such as nitrocellulose, derivatized nylon, and fluorinated polyhydrocarbons, in a variety of forms such as filters or solid sheets.

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־ »1 ־ .

Covalent binding supports are also useful and comprise materials having chemically reactive groups or groups, such as dichlorotriazinc, diazobenzyloxymethyl, and the like, Which can be activated for binding to polyuucleo tides.

A typical solid-phase hybridization technique begins with immobilization of sample nucleic acids : onto the support in single stranded form. This initial step essentially prevents reannealing of complementary 10 strands from the sample and caii be used as a means for concentrating sample material on the support for on hanced detectability. The polynucleotide probe >s then contacted, in a single stranded, labeled form, wnth the support. Appropriate labels are available by wljich to 15 detect resulting hybridization on the support, lyp* cally, a solid-phase hybridization technique will proceed as follows:

(1) the test sample is subjected to conditions to release and denature bacterial nucleic acids and resulting single stranded nucleic acids are nnmobllized on a solid support, e.g., a liquid sample such as a body fluid is applied to a nitrocellulose membrane, the deposited cells are lysed and released ONA denatured and the membrane is baked in vacuo at 80°C for 2 hours 25 to fix single stranded DNA to the membrane; alternatively, the cells are first lysed and released DNA s denatured and then applied to the nitrocellulose membrane; _l (2) the support is contacted with the labeled probe in excess under favorable hybridization conditions e g after saturating all nonspecific DNA binding sites on the membrane by treatment at 40-60’C with a prehybridization solution comprising buffer (^e.״ 2XSSC), protein such as bovine serum albumin, Ficoll 35 (a trademark identifying a copolymer of sucrose epichlorohydrin sold by Pharmacia Fine Chemicals,

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Piscataway, NJ), polyvinylpyrrolidone, and a den’atured foreign DNA such as from calf thymus or salmon sperm; typically the hybridization conditions will be the same as the prehybridizatic” condition except the time of incubation will usually be longer;

(3) removing labeled probe which has not become associated with the support by hybridization, e.g., by simple washing of the membrane; and (4) measuring the label on the support in accordance with the detectable characteristic of the label.

Traditionally, the label will comprise a radioisotope such as ³²P and will be detected by scintilla. tion counting or autoradiography, however, as will be more fully described below, nonradioisotopic detection schemes can also be used.

Additional steps may also be included in the above typical protocol. For example, where particularly short DNA fragments (e.g., less than about 1000 bases) or RNAs are to be immobilized, such polynucleotides can be first derivatized with glyoxal and t’en applied to the support. Alternatively, reactive cellulose can be used to covalently bind the polynucleotides, usually after an initial purification of the sample to isolate nucleic acids according to standard methods.

An example of a hybridization format using covalent binding, chemically derivatized paper, such as diazobenzyloxymethyl cellulose, is as follows.

(1) release and purify nucleic acids from bacteria present in the sample, (2) denature the sample nucleic acids by heating at 100’C for several minutes, (3) spot the denatured mixture onto the chemically activated paper, (4) allow to air dry, then store for 1216־ hours in a closed container, (5) wash the paper with water, 0.4N sodium hydroxide, and again with water,

MS-1309-CIP (6) rinse the paper with prehybridizatiori buffer, (7) incubate the paper for .1 hour in an elution , buffer [e.g., 99% (v/v) deionized formamide in 10 mM

Tris. HC1 buffer (pH 7.8)], (8) wash the paper again with prehybridization solution, (9) add labeled probe in hybridization solution, (10) wash the paper to remove excess, unhybridized probe, and (11) measure the label on the paper.

Instead of immobilizing sample nucleic acids and contact with labeled probe, it is also possible to label sample nucleic acids in situ by known methods and thereafter add the probe in immobilized form. The end measurement is the same, detection of the label associated with the support.

Another method of interest is the sandwich hybrid!zation technique wherein one of two mutually exclusive fragments of the homologous sequence of the probe is immobilized and the other is labeled. The presence of bacterial nucleic acids results in dual hybridization to the immobilized and labeled probe segments, again with the same ultimate measurement of support-associated label. See Methods in Enzymology 65:468(1980) and Gene 27:77-85(1983) for further details.

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An additional assay method would use a DNA probe which is complementary to a conserved sequence of an RNA such as 5S, 16S, or 23S RNA. The probe could be immobilized on a solid support and hybridized with the RNA from the sample, and the RNA/DNA duplex would be detected in any convenient manner, such as with labeled antibody specific for RNA/DNA hybrids [see Rudkin and Stollar, Nature 265:472 f 473 (1977), Stuart et al, PNAS 75:3751 (1981), Reddy and Safer,

BBRC 1(73:9 59-967 (1981), and Nakazato, Biochem. 19: 2835-2840 (1980)]. The use of immobilized DNA probes for detecting the presence of RNA sequences is possible because of the ability to prepare antibodies which are selective for DNA’RNA hybrids over the single stranded polynucleotides as is known in the art. Specific binding antibody fragments such as Fab and Fab’ can also be used. Determination of binding of antibody to hybridization duplexes can be accomplished in any convenient manner, preferably by using an anti-.

body or fragment thereof which is labeled with a detectable chemical group such as an enzymatically active group (e.g., an enzyme, an enzyme substrate, a coenzyme, or an enzyme inhibitor), a luminescer, a specifically bindable ligand (e.g. a hapten or biotin), a fluorescer, a chromophore, or a radioactive isotope, as is more fully described below. Alternatively, the antibody can be detected based on a native property such as its own antigenicity. A labeled anti(antibody) antibody will bind to the primary antibody rea30 gent where the label for the second antibody is a conventional label as above. Further, antibody can be detected by complement fixation or the use of labeled protein A, as well as other techniques known in the art for detecting antibodies.

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This assay method has the advantage that the probe can be immobilized, thus the analyst does not have to immobilize the sample RNA prior to the hybridization step. Sometimes immobilization of sample 5 nucleic acids can be problematical due to interferences by proteins and other materials in the sample. A further advantage is that each cell contains thousands of ribosomes. As a result, the detection .limit is greatly improved over methods which detect genomic 10 sequences. During hybridization of cellular RNAs with probes, it may be necessary in certain instances to protect the RNAs from degradation by ribonucleases. These enzymes can be effectively inhibited by a variety of substances such as heparin, sodium dodecyl 15 sulfate, ribonucleoside-vanadyl complexes, aurintricarboxylic acid and dextran sulfate.

B. Solution Hybridization

The present method can also be used for detection of bacterial nucleic acids in a solution format. This 20 normally requires that the homologous sequence be in single stranded form, be it RNA or DNA. This will be . referred to as the probe polynucleotide.

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In a solution format, the specimen nucleic acids are first released from bacterial cells in the sample by lysis, and then denatured. These steps may be combined by heating the sample, to .10b°C or by exposing it to base. After adding a solution containing a large excess of the probe, hybridization is allowed to occur under conditions of ionic strength and temperature empirically determined to give the desired probe specificity and sensitivity.

Hybrids can be detected and quantitated using a number of methods. For example, after hybridization the remaining single stranded nucleic acid can be hydrolyzed into small fragments with the single-strand specific nuclease Sj, Acid precipitation followed by centrifugation or filtration can be used to concentrate the hybrids and separate them from the hydrolyzed single-stranded polynucleotides. The amount of precipitate collected is then quantitated. In another approach, hybridized and single-stranded pdlynucleo20 tides can be separated by chromatography on hydroxyapatite. Other solution methods arc known and will be developed.

C. Labels

Preferably, when used as a labeled reagent, the 25 probe polynucleotide is single stranded DNA or RNA thereby obviating the need for denaturation of the probe prior to hybridization. This can be done by inserting the probe sequence into a single-stranded DNA bacteriophage (e.g., M13) or by isolating a labeled probe after an in vitro polymerization reaction.

The probe or other material to be labeled can be incorporated with a variety of labels. Useful labels include radioisotopes as well as nonradioisotopic

MS-1309-CIP labels. Isotopic labels include, but are hot limited tn 35ς 3 2 ״14 | -ן 5 2 1 ק _T. . ן S' ’ .

to, n, b, P, I, and C. The method of radio. actively labeling the probe or other material will depend upon its particular nature (e.g., RNA vs. ΠΝΛ, 5 single-stranded vs. double-stranded). Most labeling methods are enzymatic. These include, butare not limited to, the known methods of nick translation, end labeling, second strand synthesis, reverse transcription and transcription. All of these methods generally re10 quire the isotopically labeled nucleotides to function as enzyme substrates.

Alternatively, a radiolabel can be incorporated into the polynucleotide probe by chemical modification of.the polynucleotide probe. This method is used most 15 commonly with I labels.

With a radiolabeled polynucleotide probe, hybridization can be detected by autoradiography., scintillation counting or gamma-counting. The method used would depend upon the hybridization format, the type of test 20 (qualitative or quantitative), and the radioisotope used as a label.

Nonradioisotopic materials can also be used as labels. Such labels can be incorporated into the polynucleotide probe or other material to be labeled 25 by enzymatically incorporating modified nucleotides using one of the enzymatic procedures outlined above where the labeled nucleotides serve as enzyme substrates for the appropriate enzymes. Alternatively, a label could be introduced into a polynucleotide 30 probe by conventional chemical modifications of the probe.

Useful labels include, but are not limited to, haptens or other ligands, fluorescers, chemiluijiines cers, chromophores, and participants in enzymic reac35 tions (e.g,, enzymes, enzyme cofactors, enzyme substrates, and enzyme modulators, e.g. ,.inhibitors). These labels

MS-1309 -CIP are detected on the basis of their own physical properties (e.g., fluorescers and chromophores)׳or their reactive properties (e.g., the others listed) . For example, a cofactor-labeled probe can be detected 5 by adding the enzyme for which the label is a tof.ictoi and a substrate for the enzyme. Λ hapten and ligand labeled polynucleotide probe can be detected by adding an antibody to the hapten or a protein which binds the ligand, tagged with a detectable molecule. Such detect10 able molecule can be some molecule with a measurable physical property (e.g., fluorescence or absorbance) or a participant in an enzyme reaction (e.g., see above list). For example, one can use an enzyme which acts upon a substrate to generate a product with,a measurable 15 physical property. Examples of the latter include, but are not limited to, β-galactosidase, alkaline phosphatase and peroxidase. For in situ hybridization studies, ideally the final product is water insoluble.

A particularly useful labeling approach is a van²⁰ ation of second strand synthesis which can be applied to a single stranded DNA vector containing a region of defined sequence on the 5’ side of the probe sequence [Hu and Messing , Gene 17:271 - 277 (1982)]. In thi o approach, an oligonucleotide primer complementary to 25 the sequence on the 5’ side of the probe is used to initiate second strand synthesis with DNA polymerase. An appropriate label (which may be radioactive or nonradioactive as described above) can be incorporated into the second DNA strand. This approach.gives a 30 polynucleotide which is single stranded in the probe region, but which will contain a multiply labeled, double stranded region on the 5' side of the probe sequence.

MS-1309 - CIP

A technique not requiring incorporation of a label into a polynucleotide probe has been described by Rudkin and Stollar [Nature 265:472-473(1977)] and used by cytochemists for detection of hybridization in xitu. In this approach, single stranded RNA containing the desired sequence is hybridized to DNA from the sample. Hybridization is detected using labeled antibodies specific for RNA/DNA hybrids (these anti bodies do. not bind to double stranded DNA or single stranded RNA or DNA). This approach is applicable to the present method. Preferred antibody labels would be those described above.

In general, labels and labeling approaches which have been developed for use in immunoassays will be applicable to the present hybridization technique with modifications evident to the ordinary skilled worker. See U.S. Pat. Nos. 4,380,580; 4,279,992; 4,238,565; 4,134,792; 4,273,866; 3, 817,837; 4,043,872; 4 , 238,195; 3’,935,074; 3,998,943; 3,654,090; 3,992,631; .4,160,016; and 3,996,345; British Pat. Specs. 1,552,607 and 3,019,408; and European Pat. Applns. 70,.6.87 , 70,685, and 63,879; the pertinent parts of which relating to labeling schemes are incorporated herein by reference.

MS-1309-CIP

It will be clearly recognized that the present invention is not limited to any particular hybridjzation format or formats or particular labels. As new techniques and labels are developed, they will be applicable to the present method. Reference to Moleautar Cloning, A Laboratory Manual (oupra) will provide additional details concerning nucleic acid hybridization.

The test sample to be assayed for bacterial presence can be any medium of interest, and will usually be a liquid sample of medical, veterinary, environmental, nutritional, or industrial signi.ficance. Human and animal specimens and body fluids particularly can be assayed by the present method, including urine, blood (serum or . plasma), milk, cere־ brospinal fluid, sputum, fecal matter, lung aspirates, throat swabs, genital swabs and exudates, rectal swabs, and nasopharyngeal aspirates. The present invention has particular advantages for the detection of bacteria in urine (bacteriuria). The detection of,10^Jor more bacterial colony forming units per milliliter in a urine sample is presumptive of a urinary tract infection. In uncomplicated infections, the etiologi cal agents comprise two general categories according to incidence:

MS-1309-CIP

8

Category 1 (90% incidence)

Family: Enterobacteriaceae (Gram-negative facultatively anaerobic rods)

Q Eeoherichia (E. coli)

Klebsiella (K. pneumoniae) Enterobacter (E. cloacae, E. aerogene Proteus (P, vulgaris, P. mirabilts, Morganella morganti)

Category 2 (5-10% incidence)

Family: Pseudomonadaceae (Gram-negative aerobic rods and cocci) Peeudomonas (P. aeruginosa)

Family: Micrococcaceae (Gram-positive cocci) Staphylococcus (S. aureus)

Family: Streptococcaceae (Gram-positive cocci) Streptococcus Group D (Enterococci) in complicated infections, strain־ of Citvobact^. Serratia, and Providencia also may appear. ^p™ ^eshomologous for this entire group or subsets t ereo can be obtained according to the present invention depending on the degree of specificity and sensitivity desired for a particular bacteria test. . _

While the present invention has been particularly described with refer־־״־ to, and is most advantageously applied to, the detection of bacteria, it will be understood that this method can be used to detect other types of microorganisms such as protozoa, yeasts, viruses, and so forth. .. r

The test sample can be treated in a variety known manners in order to release bacterial nucleic acids in single stranded form. R־l״ ^£׳> ־־־־^{UCU C}acids can, for example, be obtained by mechanical d ruption (freeze/thaw, abrasion, sonication), p ysic chemical disruption (detergents such as Trit״־. Tween, sodium dodecylsulfate, alkali treatment, osmotic

MS-1309-CIP shock, heat-boiling water), or enzymatic lysis (lysozyme, proteinase K, pepsin). Denaturation of released nucleic acids is preferably accomplished by heating in boiling water or alkali treatment (e.g., 0.1 N sodium 5 hydroxide), which, if desired, can simultaneously be used to lyse the cells.

The present invention additionally provides a reagent system, i.e., reagent combination or means, comprising all of the essential elements required to conduct a desired assay method.־ The reagent system is presented in a commercially packaged form, as a composition or admixture where the comparability of the reagents will allow, in a test device configuration, or as a test kit, i.e., a packaged combination of one or more containers, devices, or the like holding the necessary reagents, and usually including written instructions for the performance of assays. Reagent systems of the present invention include all configurations and compositions for performing the various hybridization formats described herein. Particularly preferred is a test kit for performing a solid-phase hybridization protocol comprising (1) a solid support capable of immobilizing single stranded nucleic acid resulting from treatment of a test sample to release and denature bacterial nucleic acids, and (2) a labeled polynucleotide probe selected from the various types described herein. Preferably, such kit will additionally comprise foreign nucleic acid for use in substantially saturating nucleic acid adsorption sites on the support after immobilization of sample nucleic acid, along with, optionally, other desired ingredients for a prehybridization solution. Also, the kit will preferably include a chemical lysing and denaturing agent, e.g., alkali, for treating the sample to release single stranded nucleic acid therefrom. Ingredients for the hybridization reaction, if different from the prehybridization solution, will also be preferably

MS-1309-CIP ־30included in the kil . The reagent system can, of course, include other materials and solutions as arc known in the art and which may be desirable from a commercial and user standpoint, such as buffers, diluents, standards, and so forth.

The present invention will now be illustrated, but is not intended to be limited, by the following examples.

MS-1309-CIP

EXAMPLE 1

A. Growth of'miaroorganiams used as test strains.

The organism Escherichia coli No. 063 (University of Rochester Medical Center, Rochester, NY) wa.s obtained 5 from the urine of a patient that tested positive lor urinary tract infection. As a control non-urinary tract infection isolate, the microorganism Bacillus Bubtilis (No. 1A289 from the Bacillus Genetic Stock Center, Ohio State University, Columbus, OH) was used.

Each organism was grown in a liquid nutrient medium in the following manner.

Each shake flask was inoculated with either

E. aoli or B. subtilis. The shake flasks contained fifty milliliter (ml) of 2XYT broth. 2ΧΎΤ consists of 15 1.61 Bactotryptone, 1.0¾ yeast extract and 0.5¾ sodium chloride. Bactotryptone and yeast extract are available from Difco Laboratories, Detroit, MI. Both cultures were grown at 37°C in a rotary shaker for 6 hours.

In order to determine the bacterial count per unit 20 volume of each 6 hour broth culture, the culture was serially diluted by ten-fold increments into 2XYT broth. Each of the dilutions were plated on sterile agar medium containing tryptose blood agar base (TBAB) and 0.1¾ casamino acids (both components available from

Difco). Those plates were incubated at 37°C for 12 hours to allow colonies to form. The plates containing 30300־ colonies were counted and the number of cells per unit volume in the original broth cultures was determined by calculation.

MS-1309-CIP

B. Application of cells from broth culture to a solid support for immobilization of nuclei(. acids.

The six-hour broth cultures and freshly made dilutions of these cultures were applied to a solid support consisting of nitrocellulose (Bio-Rad TransTM blot transfer medium available from Bio-Rad Laboratories, Richmond, CA). The addition of a.precise number of cells to a constant surface area of the nitrocellulose was accompli shed by the use of an apparatus called a Minifold¹¹ which is commercially available from Schleicher 6 Schuell, Inc., Keene, NH. This apparatus is a filter manifold consisting of three sections which are clamped together into a sandwich during the filtration or immobilization TM process. The Minifold apparatus was fitted with one 4 inch () x 5 1/4 sheet of the nitrocellulose membrane, backed by two 4 x 5 1/4 sheets of Whatman

MM chromatography paper (available from Whatman Ltd., 20 England). The nitrocellulose and Whatman paper were presoaked in five ml of 1XSSC (1XSSC = 0.15M NaCl, 0.015M trisodium citrate). The membrane and backing papers were clamped into the apparatus. The apparatus was hooked up to a vacuum line and the vacuum applied.

To each well was added 0.10 ml of the 6 hour E. Coli culture and to successive wells, 0.10 ml the serial dilutions of this culture. The control 6 hour culture of B, subtilis was also collected on the filter in the same fashion as described for the E.coli culture. As an additional control, nutrient media was added to another set of wells.

MS-1309-CIP

3

C. Lysis of cells and denaturation of DNA on membrane containing the cells.

The nitrocellulose membrane containing the TM cells was removed from the Minifold apparatus and placed on a Whatman 3MM paper which had been saturated with a 0.5M sodium hydroxide solution. The membrane was placed on paper so the cells were always on the upper surface. The nitrocellulose membrane remained 10 in contact with the 3MM paper for 10 minutes at 65°C.

The nitrocellulose membrane was then placed or. a Whatman 3MM paper that had been presoaked with IM Tris, pH 7.4. The nitrocellulose remained in contact with the paper for 10 minutes at 65°C. The nitrocellulose 15 membrane was removed and placed on a second 3MM paper saturated with IM Tris, pH. 7.4, for 10 minutes at 65°C. The nitrocellulose was removed and placed on a Whatman 3MM paper saturated with a solution of 0.5M. Tris, 1.5M NaCl, pH 7.4, for 10 minutes at 65°C. The 20 nitrocellulose membrane was then air dried and baked at 80°C for 2 hours in a vacuum oven.

In a typical experiment, samples containing x 10⁸, 1 x 10⁷, 1 x 10⁶, 1 x 10⁵, 1 x 10⁴, 1 x 10³, 2 1 x 10 , 1 x 10 cells were applied to each well.

MS-1309 -CIP

D. Isolation and purification of M13-10 replica 1.1.ve form DNA.

The bacteriophage M13-10 (A'ICC. 39403-B1) was used as the source of the tuf A gene fragment. The tuf A gene of E. coli encodes for the protein Lf Lu. EF־Tu is a single polypeptide chain with a molecular weight of 43,225. The EF-Tu protein is an elongation factor in protein synthesis and־mediates the entry 01 an aminoacyl-tRNA into the A site of the ribosome. The EF-Tu protein carries a guanine nucleotide (GIF). The EF-Tu-GTP binds aminoacyl-tRNA. The size of the tuf A gene is 1,190 base pairs. The portion of the tuf A gene contained in M13-10 is an 800 base fragment. The tuf A gene is cloned between the Hine II and LcoRI restriction endonuclease sites of the vector M13mp9 (New England BioLabs, Beverly, MA).

The Escherichia coli host organism used for growth of M13-10 phage was JM103 (Mac, pro), supE, tin, strA, endA, sbcBlS, hsdH4-_t traD36, lac IqZMJ3. This strain is commercially available from Bethesda Research Laboratories, Gaithersburg, MD.

Fifteen ml of an overnight culture of the above

E. coli was inoculated into one liter of 2ΧΥΓ broth in a two liter Fernbach flask. This culture was grown for 3 hours at 37°C on a New Brunswick Gyrotary shaker (200 rpm). To the 3 hour culture 2 x 10¹¹ PFU (plaque forming units) of Μ13-Ϊ0 bacteriophage were added. This infected culture was allowed to incubate for 1 hour under the same conditions as described above. At this time, 150 mg of chloramphenicol was added to the culture Incubation was continued for an additional 45 minutes. Cells were harvested by centrifugation (GSA rotor, 5,000 rpm, 10 minutes) and washed once in cold 2XYT broth. The washed cells were suspended in 40 ml cold 10% sucrose solution. Lysis was accomplished by the

MS-1309-CIP •'» ‘f י « » addition of 20 mg of lysozyme and 16 ml of 0.25M ethylenediaminetetraacetic acid (liDTA) followed by incubation in an ice bath for 10 minutes. Lysis was completed by the addition of 4 ml 20% sodium dodecyl 5 sulfate (SDS). The lysis mixture was mixed gently.

The concentration of sodium chloride in the lysate solution was adjusted to IM with the addition of 2.90 gm of sodium chloride. The lysate mixture was incubated at 40°C overnight. The lysate was cleared by 4°C 10 centrifugation in a Beckman ultracentrifuge at 60,000 xg for 30 minutes. The supernatant was extracted three times with equal volumes of a phenolchloroform mixture composed of 4 parts of phenol 1 part of chloroform. The aqueous phase from the ex15 traction was added to two volumes of cold 95% ethyl alcohol. This mixture was incubated at -70°C for thirty minutes and then centrifuged 1,800 xg for 15 minutes at 4°C. The pellet that resulted was washed with 70% ethyl alcohol and then air dried. The DNA pellet was dis20 solved in 15 ml of a solution containing 0.01M Tris, P^{11 8}י 0.001M EDTA. This DNA solution was subjected to isopycnic cesium chloride density gradient centrifugation in the presence of ethidium bromide to separate covalently closed circular replicative form DNA from 25 linear DNA. Cesium chloride was added to the DNA solution at ambient temperature to a final refractive index of 1.3930. After centrifugation for 40 hours at 130,000 xg, the lower band containing the replicative form DNA was removed and extracted 6 times with 30 water-saturated sec-butanol to remove ethidium bromide. The extract was dialyzed extensively against TE buffer (0.01M Tris, pH 8.0, 0.001M EDTA) at 4°C. To remove contaminating RNA, 250 mg of ribonuclease (RNase) was added per ml of dialysate and the solution 35 incubated for 1 hour at 37°C. To remove the RNase, the

MS-1309-CIP solution was extracted with an equal volume of phenol saturated with 0.1M Tris, pH 8, buffer. The upper phase from the phenol extraction was made 0,.3M in sodium citrate and precipitated with 2 volumes of cold ethanol at -70°C for 30 minutes. The phage DNA was dissolved in TE buffer.

The M13-10 replicative form DNA was then cleaved by a double digest with Sal I, feolil under appropriate conditions such that the 800 bp (base pair) insert of the tuf A gene fragment was cut out of the vector.

E. Isolation and radioactive labeling of the tuj A fragment.

The double digested Sal I> EcoRI Μ13-1Ό DNA was adjusted to a concentration of 40 pg per 100 pl.

Horizontal agarose gel electrophoresis was performed in a Bio-Rad Sub-Cell™ electrophoresis chamber . (avail able from Bio-Rad Laboratories), Λ 1 percent low gelling temperature (LGT) agarose gel (available from Miles Research Products, Naperville, IL) in 0.Ό89Μ

Tris borate, 0.089M boric acid, 0.002M EDTA (TBE)> P^{11 8}י buffer containing 0.5 pg/ml ethidium bromide was formed in the Sub-Cell apparatus. The M13-10 double digested DNA solution was loaded on this gel at a concentration of 7 pg per well. Electrophoresis was performed at 50 volts for 3 hours at ambient temperature. The insert tuf A fragment DNA was cut out of the gel. These gel pieces were placed in a 50 ml polypropylene tube and heated to 70°C until the gel melted. Th(? molten gel was adjusted to a total volume of 10 ml with 0.2M NaCl, 0.02M Tris, pH 7.0, 0 001M EDTA. The DNA was isolated from this gel soluTM tion through the use of the Elutip d columns commerci ally available from Schleicher fi Schuell, Keene, NH.

MS-1309 -CIP ־ .37 The procedure used was t hat recommended by the supplier. The Elutip^TMd column was washed with 2 ml of a solution of 1.0M NaCl, 0.02M Tris-HCl, pH 7.3, 0.001M EDTA. The column was washed with 0.5 ml of a solution comprised of 0.2M NaCl, 0.Ό2Μ Tris-HCl, pH 6.3, 0.001M EDTA. .A 0.45 pm cellulose acetate disposable filter was attached to the syringe (available from Schleicher f! Schuell) and the DNA sample loaded onto the column. The DNA adsorbs to the matrix of the column. The column was washed with 3 ml of a solution containing 0. 2M NaCl, 0.02M fris-IICl, pH 7.3, 0.001M EDTA. Elution of the DNA sample from the column was accomplished by removing the cellulose acetate filter and adding 0.4 ml of the solution containing IM NaCl, 0.02M Tris-HCl, pH 6.3, 0.001M EDTA. The DNA was precipitated with cold ethanol and dissolved in 40 yl of water. 32

The tuf A gene fragment was labeled with P by nick translation as described below. A reaction mixture was prepared consisting of 0.94 pg of tuf A DNA fragment, 20 micromoles each of deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP) and deoxythymidine triphosphate (dTTP), 0.05M Tris-HCl, pH 7.8, 0.005M magnesium chloride, 0.01M 2mercaptoethanol, 0.50 pg of nuclease free bovine serum albumin (BSA), 2 units of DNA polymerase I (available from BRL, Gaithersburg, MD) 200 picograms DNase I, 0.0005M magnesium acetate, 250 pg glycerol, and 4.88 pmoles of [ά³²Ρ] deoxyadenosine triphosphate (dATP) with a specific activity of 410 Ci/mmol. The total reaction volume was adjusted to 50 microliters with distilled water and incubated at 15°C for one hour. The reaction was stopped by the addition of 5 microliters of 0.3M Na₂EDTA (pH 8.0) and extracted with an equal volume of Tris-saturated phenol. The extracted mix־ ture was chromatographed on a Sephadex G-50 column (Pharmacia Fine Chemicals, Piscataway, NJ) to remove

MS-1309 -CIP

2 the unreacted deoxytriphosphates. The ‘ P labeled DNA fragments were contained in a total volume of 400 microliters with a specific activity of 8 x 10 cpm per microgram of DNA.

. F. Hybridization protocol

The nitrocellulose membrane described in part B of this example was pretreated before hybridization by placing the membrane in a heat sealable bag (comincr cially available as Boil-A-Meal^^ bags from Sears,

Roebuck § Co., Chicago, IL) and adding 10 ml of a solntion consisting of 50% formamide (Matheson,¹ Coleman G Bell), 5XSSPE (1XSSPE -= 0.18M NaCl , 0.01M sodium phosphate, 0.001M Na₂ EDTA, pH 7.0), 5XBFP (1XBFP = 0.02¾ w/v of bovine serum albumin, Ficoll [MW. 400,000] and polyvinyl pyrrolidone), 1% glycine, and 100 pg/ml of denatured, sonicated carrier salmon sperm DNA. Ihe membrane was incubated in this solution for one hour at 42°C. The prehybridization solution was then removed. Four milliliters of a solution containing 50¾ v/v forma20 mide, 5XSSPE, 1XBFP, 100 ug/ml sonicated salmon sperm DNA, 10% w/v sodium dextran sulfate 500 and 0.3% SDS was prepared. Half of this solution (2 ml) was added to the bag containing the membrane and mixed.

The labeled probe DNA was denatured by heating the DNA in a solution of lOmM Tris (pH 7.5), ImM EDTA for minutes dt 95°C and cooling it quickly in an ice bath. The denatured probe DNA was added to the remaining 2 ml of solution at a quantity sufficient to provide 5 x 10⁶ cpm per membrane. The 2 ml of solution containing the probe was then added to the bag and the contents were mixed. The bag was sealed by using a heat-sealer.

The hybridization mixture was incubated for 20 hours at 42°C. The nitrocellulose membrane was removed 35 from the hybridization solution and washed in a solution

MS-1309-CIP of 2XSSPE and 0.2¾ SDS. This wash was repeated four times at 45°C. The membranes were air dried .and placed in contact with X-ray film (Dupont Cronex 4) and placed in a film cassette fitted with a Lightning Plus intensifying screen (available from Dupont). The film was exposed to the X-ray film for an appropriate time (this varies depending on the specificity and specific activity of the probe DNA and rate of the hybridization reaction).

G. Detection of the presence of a bacteria that had been isolated from urine of a human patient rni.t'1 a urinary tract infection.

In a typical experiment as in this example, the presence of 1X10⁶ bacteria comprised of Escherichia ooli No. 063 was detected using the tuf A gene fragment as a probe. The presence of a Gram positive organism Bacillus subtilis, a non-urinary tract isolate, was not detected even when as many as 1X10 cells were present.

EXAMPLE 2

A. Isolation and purification of DNA from bacterial strains that have been isolated from patients with urinary tract infections.

When the test organism is of the family Enterobacteriaceae, genus and species Escherichia ooh,. Proteus mirabilis, Enterobacter cloacae, Enterobacter aerogenes, Citrobacter freundii, Hafnia alvei, Klebsiella pneumoniae, Klebsiella oxytoca, Morganella morganni, Serratia rubideae, Providencia stuartii, and Klebsiella ozaenae, and the Families Streptococcaceae, Enterococcus, Micr'ococcaceae, and Staphylococcus aureus, the procedure for the isolation of genomic DNA is

MS-1309 ־CIP essentially the same with the exception of Staphylococcus aureus.

The general procedure for isolation of genomic DNA will be described for all the organisms named in the proceeding paragraph. Each of the aforesaid organisms was grown in 1 liter of TBB+CAA medium which is comprised of 1¾ tryptose, 0.3¾ beef extract, 0.1¾ casamino acids.(available from Difco Laboratories) and 0.5¾ sodium chloride for 18 hours with aeration.

Cells were harvested by centrifugation and washed in 75 ml of a solution of 0.15M NaCl and 0.015M trisodium citrate (1XSSC). The cells were suspended in a mixture of 25 ml of 1XSSC and 1.5 ml of 0. 2.5M EDTA. Lysis and RNA degradation was accomplished by the addition of 12.5 mg of lysozyme (egg white crystalline lysozyme, commercially available from Miles Laboratories, Naperville, IL) and 7.5 mg RNAse followed by incubation for 1 hour at 37°C. A 0.7 ml volume of 25¾ SDS was added to the mixture and incubation was continued for 1 hour followed by the addition of 10 ml of distilled water and 5 ml of 5M sodium perchlorate. Protein was removed by extracting the lysate with 50 ml of chloroformisoamyl alcohol in a ratio of 24:1. The upper phase was removed, 10 mg of pronase added, and the mixture incu25 bated for 2 hours at 37°C with gentle agitation. This mixture was extracted with 50 ml of chloroform-isoamyl alcohol The aqueous phase was extracted once more with chloroform-isoamyl alcohol. The DNA in the upper phase was precipitated with two volumes Of cold 95¾ ethanol. The DNA was dissolved in sterile distilled water or 0.1 XSSC.

In the case of Staphylococcus aureus, lysis was accomplished by using Lysostaphin (available commercially from Sigma Chemical Co., St. Louis, M0), adding 35 375 pg of this enzyme to lyse biomass recovered from liter of culture.

MS1309־-CIP

4.1

The DNA concentrations were determined using the fluorescence assay as described by A.R.Morgan et al, Nualeia Acids Research 7:547-594(1979).

B. Immobilization of genomic DNA isolated from organisms onto a solid support.

Samples of the DNA from each of the organisms named in Part A of this example and DNA from Bacillus subtilis and human placenta (human placental DNA is commercially available from Sigma Chemical Co., St. Louis, MO) were adjusted to DNA concentrations of 0.2 Mg/pl, 0.02 pg/pl, 0.002 yg/pl, 0.20 ng/pl, 0.02 ng/!.11 and 2.0 pg/μΐ. These six DNA concentrations were applied to a solid support consisting of a Gene-Screen¹ membrane (New England Nuclear, Boston, MA). The application of a precise volume of DNA solution to a constant surface area of the membrane was accomplished by the use of the Minifold filtration apparatus from Schleicher 8 Schuell, Inc., (see part B example 1). The Minifold apparatus was fitted with one 4 x 5 1/4 sheet, of nitrocellulose membrane that had been prewetted in 1XSSC. Vacuum was applied to the Minifold apparatus and 5 microliters of eacli concentration of DNA from each of the organisms named above was added. After the membrane was air dried, the membrane was immersed in a denaturing solution consisting of 2.5M NaCl and 0.5M NaOH for 2 minutes. Subsequently, the membrane was immersed in neutralizing solution which consisted of 3M sodium acetate (pH 5.5). Excess solution was , blotted from membrane with Whatman 3MM paper. The membrane was baked at 80°C in a vacuum oven for 1 hour.

MS-1309 -CIP

2

C. Isolation and purification of M13-10 replicative form DNA.

See part D, Example 1.

D. Isolation and radioactive labeling of the tuf A gene fragment.

See part E, Example 1.

E. Hybridization protocol.

See part F, Example 1.

F. Detection of the DNA of bacteria that comprise ninety-five percent of urinary tract infections, in humans.

In a typical experiment as in this example, the results were as shown in Table A.

MS-1309 -CIP

Table A tn 1' - ' . . .

<td> 0</td><td> Family</td><td> Genomic DNA-Organism</td><td> &gt; of Strains</td><td> Amount of DNA Detected</td>

<td></td><td></td><td></td><td></td><td></td>

<td> H</td><td> Enterobacteriaceae</td><td> Escherichia coli</td><td> 6</td><td> 1.0 nanogram</td>

<td> גר</td><td> II</td><td> Proteus mirabilis</td><td> 2</td><td> 10.0 </td>

<td></td><td> II</td><td> Enterobacter cloacae</td><td> 2</td><td> 10.0 </td>

<td></td><td> II</td><td> Enterobacter aerogenes</td><td> 1</td><td> 1.0 </td>

<td></td><td> II</td><td> Citrobacter freundii</td><td> 1</td><td> 1.0 </td>

<td></td><td> II</td><td> Hafnia alvei </td><td> 1 ..</td><td> 1.0 </td>

<td></td><td> II</td><td> Klebsiella pneumoniae</td><td> 5</td><td> 10,0 </td>

<td></td><td> II</td><td> Klebsiella oxytoca</td><td> ו</td><td> 1.0 : </td>

<td></td><td> '11 ,</td><td> Morganella morganii</td><td> 2</td><td> 10.0' ”</td>

<td></td><td> II</td><td> Serratia rubideae</td><td> 1</td><td> 10.0' </td>

<td></td><td> • II</td><td> 1 . Providencia stuartii</td><td> 2</td><td> 10.0 </td>

<td></td><td> II</td><td> Klebsiella ozaenae</td><td> 1 </td><td> 10.0 </td>

<td></td><td> Micrococcaceae</td><td> Staphgiococcus aureus</td><td> 1</td><td> *</td>

<td></td><td> Pseudomonadaceae .</td><td> Pseudomonas aeruginosa</td><td> 1</td><td> 1.0 </td>

<td></td><td> Streptoooccaceae</td><td> Enterococcus</td><td> 1</td><td> 4,00.0 </td>

<td></td><td> bacillaceae</td><td> bacillus subtilis</td><td> / , 1</td><td> Λ</td>

<td></td><td></td><td> Human placental DNA</td><td> ־</td><td> זו</td>

* = not detectable

The results indicate that the tuf A gene probe is able to hybridize with essentially all of the organisms that are etiologic agents of urinary tract infections. The control DNA samples, Bacillis sub5 tills, a soil microorganism, and human DNA does not hybridize with the tuf A gene.

EXAMPLE 3

A. Hybridization of DNA from urinary tract isolates lysed in situ on a nitrocellulose membrane filter

The following microorganisms were grown on TBAB (Tryptose Blood Agar Base) - Difco agar plates + casamino acids (CA) overnight, to obtain single colony isolates:

Escherichia coli AS

Escherichia coli A6

Klebsiella pneumoniae B4 Klebsiella pneumoniae B5 . Proteus mirabilis C2 . ׳ .

Enterobacter cloacae 1)4

MorganeIla morganii C7

A single colony isolate from each type of microorganism was used as inoculum for ten ml of TBAB + CA broth. These cultures were grown overnight at 37°C.

. . ״ ך - 7Each culture was diluted 10 to 10 in TBAB + CA media at 4°C to stop all growth. Each dilution was plated on TBAB + CA agar plates to determine the number of viable organisms per ml of media. Also, one ml of each dilution was transferred to a siliconized tube. To each tube was added: 50 micro30 liters lysozyme (10 mg/ml) dissolved in 10 mM Tris, 1 mM EDTA buffer, pH 8, 100 microliters of 1¾ SDS dissolved in 0.2 N NaOH (sodium hydroxide)., and 0.5 ml modified 20x SSPE which consists of 3.6 M NaCl,

MS-1309-CIP ־ 5 4 0.2 M NaH₂PO₄>H₂O, 0.16 M NaOH and 0.020 M Na_?EDTA. The SSPE is modified by adding sufficient 0.2 N NaOH solution to adjust the pH of the 20x SSPE to 7.4. The purpose of this mixture is. to disrupt and lyse 5 the bacterial cells and denature the DNA that is released from the cells.

The entire lysed cell mixture samples from each cell type were applied to a solid nitrocellulose support following the method of part B, Example 1. To 10 each well of the Minifold apparatus. was added the entire volume of cell lysate (which contains :1 ml of broth containing the cells and dilutions plus lysis reagents) of each sample.

A control culture of mammalian cells at a con7 15 centration of 1 x 10 cells per ml (determined by hemo cytometer count) and serial dilutions of these cells was also lysed and collected on the filter in the same manner as described for the cultures of microorganisms. Each sample and control was done in 20 duplicate.

The nitrocellulose membrane described above in this example was pretreated before hybridization by placing the membrane in the heat sealable bag commercially available from Sears Roebuck ξ Co . , and 25 adding 10 ml of a solution consisting of 5XBFP (1XBFP = 0.02% w/v bovine serum albumin, Ficoll of

M.W. 400,000, and polyvinylpyrrolidone), 5XSSPE, 0.3VSDS, and 100 yg/ml salmon sperm DNA. The membrane was incubated in this solution for one hour at 30 70“C and the prehybridization solution removed. Ten milliliters of a solution containing 5XSSPE, 1XBFP, 100 yg/ml salmon sperm DNA and 0.3% SDS was prepared. Half of this solution (5 ml) was added to the bag containing the membrane and mixed. The labeled probe 35 DNA- (tuf fragment from part E, Example 1) was denatured by heating the DNA in a solution of 10 mM Tris (pH 7.5),

MS-1300-CIP

.. ! (. 1 mM EDTA for 10 min. at 95°C and cooling it quickly in an ice bath. The denatured probe DNA was added to the remaining 5 ml. of solution at a quantity sufficient to provide 5 x 10^ cpm per membrane. The 5 solution containing the probe was added to the bag and the bag was heat sealed.

The hybridization mixture was incubated for 16 hours at 70°C. The nitrocellulose membrane was removed from the hybridization solution and washed in a 10 solution of 5XSSPE and 0.5¾ SDS. This wash was re־, peated four times at 70°C. The membranes were air dried; subsequently, the membranes, were cut up in such a manner as to cut out the filter area encompassed by each well of the Minifold as well as an equivalent 15 border area for each well. These filter squares were placed into individual scintillation vials containing

TM ml of Aquasol fluor (New England Nuclear, Villerica, MA). Duplicate samples of each cell type and its' serial, dilutions were counted on a Beckman 20 scintillation counter.

The results of this experiment are reported in Table B.

MS-130-’-CIP

MS-13Og-CIP

<td> Cell Type</td><td> Number of Viable Cells Applied to Each Well of Minifold¹¹^</td>

<td> 'seheriehia . coli 1057׳</td><td> 1.5x10? 1.5x10' 1.5x10? l.5x!0^b</td>

<td> Escherichia coli *165</td><td> 1'.7χ10ϊ ' 1.7x10: MxlO? 1.7x10'</td>

<td> Klebsiella pneumoniae</td><td> 1.1x102 1.1x10; 1.1x10? 1.1x10'</td>

<td> Klebsiella pneumoniae</td><td> 1.0x10? 1.0x10: 1.0x10? 1.0x10'</td>

<td> -roseus mirabilis</td><td> 2.0x10? .2.0x10: 2.0x10? 2.0x10°</td>

<td> ir.terobaoser cloacae</td><td> 5.0x10? 5.0x101' 5.0x10'</td>

<td> !.!orcanella mo'raanii k׳</td><td> 1.0x10? 1.0x10; 1.0x10'</td>

<td> Mammalian Cells</td><td> ' 1.0x10: ' 1.0x10'</td>

Table II

<td> P-CPM-x Counts Per Minute-Measure of Extent of Hybridization . of tuf Gene to DNA of Organism________</td><td> Standard ' Deviation ־</td>

<td> 2,539</td><td> 235</td>

<td> 699</td><td> .Ί22</td>

<td> 648</td><td> 64</td>

<td> 114</td><td> 11</td>

<td> 4,481</td><td> 076</td>

<td> 1,262 </td><td> 504</td>

<td> 676</td><td> 106</td>

<td> 118 .</td><td> 14</td>

<td> 3,062</td><td> 36ג .</td>

<td> 670 :</td><td> 111</td>

<td> 494</td><td> 114</td>

<td> 102</td><td></td>

<td> 1,509</td><td> 160</td>

<td> 430</td><td> . ’ 69</td>

<td> 111</td><td> 12</td>

<td> 98</td><td> 5</td>

<td> 2,677</td><td> 655</td>

<td> 599</td><td> יק</td>

<td> . 647</td><td> 1 to w 64</td>

<td> !14</td><td> 11</td>

<td> 1,341</td><td> 71.</td>

<td> 515</td><td> 1$</td>

<td> 194</td><td> / 26 '</td>

<td> 1,251 '</td><td> 267</td>

<td> 217</td><td> 52</td>

<td> . 161</td><td> /</td>

<td> •90 .</td><td> 18</td>

<td> 69.</td><td> '8</td>

8

EXAMPLE 4

A. Use of B. subtilis ribosomal RNA gene as a .probe for Escherichia coli.

A fragment of the ribosomal RNA genes from B.

subtilis cloned into pBR313 was used as the probe sequence for hybridization. This ribosomal gene clone is designated pl4Bl and is described in Stewart, G. C. , Wilson, F.E. and K. F. Bott', Detailed Physical Mapping of the Ribosomal RNA Genes of Bacillus sub10 tilis [Gene (1982) 19:153-162].

An 18 hour broth-culture (L-Broth which consists of 10g Bacto tryptone, 5g Bacto yeast extract, 0.5 g NaCl and 2 ml of IM NaOH per liter) of Escherichia coli strain MC4100 (Genotype F , ara D139, (lac) U169, 15 relA, rpsL, thi) Ito, K., Bassford, P. J., Jr. and .

Beckwith, J., Cell (1981) 24_:707- 717 . The culture was plated on L-agar plates to determine viable count to estimate the number of cells present per sample volume. The culture was also diluted serially to 10 . Λ 100 20 microliter sample of these dilutions was added to 50 microliters of lysis buffer. (Gegenheimer, P., Watson,

N. and D. Apirion. Journal Biological Chemistry, 252:3064-3073) and the sample tube immersed in boiling water bath for two minutes. A five microliter volume 25 of each sample was spotted onto a nitrocellulose membrane (Schleicher 5 Scheull B85) that had been presoaked in 20XSSC. The filter was dried in a vacuum oven at 80^eC for two hours.

The E. coli strain which contains plasmid pl4Bl was cultured using standard methods and the plasmid DNA isolated. (Maniatis, T., Fritsch, E. F., J. Sambrook. Molecular Cloning - A Laboratory Manual. CSH - Cold Spring Harbor, New York, pp. 88-93).

MS-1309-CIP

The pl4Bl DNA was labeled with by nick translation as described below. A reaction mixture was prepared consisting of 1.0 pg of pl4Bl DNA, 20 micromoles each of deoxycytidine triphosphate (dCTP), ' 5׳ deoxyguanosine triphosphate (dGTP) and deoxythymidine triphosphate (dTTP), 0.05 M Tris-HCl, pH 7.8, 0.005 M magnesium chloride, 0.01 M 2-mercaptoethanol, 0.50 pg of nuclease free bovine serum albumin (BSA), 2 units of DNA polymerase I (available from BRL, Gaithersburg, 10 MD) 20.0 picograms DNase I, 0.005'M magnesium acetaite,

דך

250 pg glycerol, and 4,88 pmoles of [a P] deoxyadenosine triphosphate (dATP) with a specific activity of 410 Ci/mmol. The total reaction volume was adjusted to 50 microliters with distilled water and incubated at 15°C for one hour. The reaction was stopped by the addition of 5 microliters of 0.3 M Na^EDTA (pH 8.0) and extracted with an equal volume of Tris - saturated phenol. The extracted mixture was chromatographed on a Sephadex G-50 column (Pharmacia Fine Chemicals,

Piscataway, N.J.) to remove the unreacted deosytriphosphates. The P-labeled DNA fragments were con&lt; tained in a total volume of 400 microliters with a 7 specific activity of 8X10 cpm per microgram of DNA. The nitrocellulose membrane described above in 25 this example was pretreated before hybridization by placing the membrane in the heat sealable bag commercially available from Sears Roebuck 5 Co. and adding 10 ml of a solution consisting of 5XBFP, 5XSSPE,

0.3? SDS and 100 pg/ml salmon sperm DNA. The membrane 30 was incubated in solution for one hour at 50%C and the prehybridization solution removed. Ten milliliters of a solution containing 5XSSPE, 1XBFP, 100 pg/ml salmon sperm DNA and 0.3? SDS was prepared. Half of this solution (5 ml) was added to the bag containing 35 the membrane and mixed. The labeled probe DNA was

MS-1309-CIP denatured by heating in a solution of 10 mM Tris (pH .7. 5), 1 mM EDTA for 10 min, at 95°C and cooling it quickly in an ice bath. The denatured probe DNA was added to the remaining 5 ml of solution at a quantity sufficient to provide 5X10^ cpm per membrane. The solution containing the probe was added to the bag and the bag was heat sealed.

The hybridization mixture was incubated for hours at 50°C. The nitrocellulose membrane was removed from the hybridization solution and washed in a solution of 5XSSPE and 0.5¾ SDS. This wash was repeated four times at 50°C. The membranes were air dried; subsequently, the membranes were cut up in such a manner as to cut out the filter area encompassed by the spotted sample as well as an equivalent border area. These filter squares were placed into individual scintillation vials containing 5 ml of Aquasol™ fluor. Duplicate samples of each cell type and its serial dilutions were counted on a Beckman scintillation counter. The results are shown in Table C.

MS-13Q9-CIP

Table C

Number of E. coli Cells in Lysis BuTfer Spotted on Membrane ³²P-CPM-Counts Per

Minutes - Measure of Extent of Hybridization of subtilis rRNA- Gene Fragment to ΈΤ coli

<td></td><td> 2.5x10?</td><td> Cells</td><td> 13,358</td>

<td></td><td> 2.5x10?</td><td> Cells</td><td> 1,649</td>

<td></td><td> 2.5x10״</td><td> Cells</td><td> 2 50 η Λ</td>

<td> 10</td><td> 2.5x10</td><td> Cells</td><td></td>

<td></td><td> Blank -</td><td> No Cells</td><td> 6 5</td>

In this experiment, the Bj_ subtilis ribosomal gene fragment probe was able to detect The presence 0 2.5x10$ Escherichia coli cells by hybridization.

The present invention has been particularly des cribed and exemplified above. Obviously, many other variations and modifications of the invention may be made without departing from the spirit and scope hereof