Difficile Specific Oligonucleotides

This invention relates to oligonucleotides specific to bacteria of the species Clostridium difficile and useful as C. difficile DNA probes or in the amplification of C. difficile DNA.

Clostridium difficile, a Gram positive anaerobic spore-forming organism, is recognized as the major cause of pseudomembranous colitis and is implicated in antibiotic-associated colitis and diarrhoea. The pathogenicity of the organism is related to the production of an enterotoxin, toxin A, and a potent cytotoxin, toxin B. The diagnosis of C. difficile-associated disease depends on the isolation and identification of the organism and/or the demonstration of toxins in faecal specimens of patients and mono-specific antibodies for that purpose are disclosed in EP-A-0153519 and EP-A-0154064. Such procedures are time consuming, and a more rapid diagnosis is essential to enable the initiation of prompt therapy.

The present invention aims to avoid these difficulties by providing oligonucleotides specific to C. difficile DNA and is a development of the work reported in J. Clin. Microbiol. 24, 3, pp 384-387 (Sep. 1986). In that paper, the results of immunoblot studies on various strains of C. difficile were reported to various immunogenic proteins were identified common to all nine C. difficile strains tested. In particular, three common antigens were potentially identified with molecular weights in the range 50 to 70 kDa.

Following on from those studies, further antigens have now been identified common to all C. difficile strains tested, and the genes encoding those antigens have been identified, cloned and, in one case, sequenced giving rise to the present invention, namely the construction, for the first time, of DNA probes and oligonucleotide primers specific to C. difficile DNA.

FIG. 1 is a photograph showing the bands obtained by subjecting genetic DNA from C. difficile to PCR amplification using oligonucleotides according to the present invention as primers and subjecting the product of the CR reaction to separation on an agarose gel.

FIG. 2 is a similar photograph but showing the results obtained using a different pair of primers for the PCR reaction.

More specifically, in accordance with a first aspect of the present invention a 1947 bp DNA fragment encoding part of the A toxin gene of C. difficile has been identified, cloned and sequenced, the sequence being as set out in the Appendix I hereto as SEQ ID No.1 and which is to be taken as forming part of the present specification.

Analyzing that 1947 bp sequence in more detail, a continuous open reading frame is found containing four distinct groups of repeat nucleotide sequence with 88 to 100% identity within each group. These groups, identified herein as A, B, C, D (A=81 bp; B, C, D=63 bp) appear in the following arrangement:

The repeating sequence of the A, B, C and D blocks given above account for 1935 bp of the 1947 bp fragment. The remaining 12 are accounted for two linking areas each of 6 bp which occur between the A and D groups underlined.

Utilizing that information oligonucleotides and DNA probes capable of hybridizing to the C. difficile A toxin gene and potentially useful in the early detection of C. difficile infection may now be constructed. The probes may range in size from virtually the complete 1947 nucleotide sequence given in the appendix hereto, SEQ ID No 1 down to sequences of a more appropriate length for oligonucleotide synthesis, for example, sequence of from 15 to 300 nucleotides, more usually of from 15 to 50 nucleotides and especially from 15 to 30. The final selection of a particular oligonucleotide, or pair of oligonucleotides will depend upon a number of factors, well understood in the art, and including amongst others the stringency requirements, i.e. the ability or otherwise of the probe to tolerate local mismatching with the complementary sequence in the target DNA. Obviously the longer the probe the better the ability to withstand local mismatching without adversely affecting the hybridization of the oligonucleotide to the target DNA. However, the length of the oligonucleotide always has to be balanced against other factors such as ease of synthesis. The factors affecting that choice are, however, well recognized and well within the capabilities of the person skilled in the art.

Also, as the person skilled in the art will recognize, references herein to particular oligonucleotides and sequences in single stranded form and including the complete 1947 bp sequence, and which are written, as is required, reading from left to right, i.e. from the 5' terminus to the 3' terminus, automatically include the complementary sequence. Not only that, but oligonucleotide sequences given herein as DNA sequences can equally well be constructed as RNA sequences with uracil (U) replacing thymine (T).

Whilst, as indicated, the generality of the present invention extends to DNA (and RNA) probes complementary to any sequence of nucleotide bases to be found in the 1947 bp sequence set out hereinafter, certain sequences and pairs of sequences can be identified as being particularly preferred. For example, sequences selected from the repeat sequences already given are especially preferred as being complementary to two spaced but identical sections of the C. difficile A toxin gene. One such sequence is the 25 unit oligonucleotide:

(BW71 is simply an identification code) which sequence is to be found in the Toxin A Repeat C sequence given above.

A more particular aspect of the present invention relates to selected pairs of C. difficile oligonucleotide probes which can be used to amplify the C. difficile A toxin gene or sections thereof by polymerase chain reaction (PCR) technology and using selected pairs of oligonucleotides as primers.

In recent years PCR has developed into a major tool in DNA probe technology enabling highly sensitive detection of minute amounts of genetic material present in a sample by first amplifying the gene or gene fragment by PCR to replicate a multiplicity of identical gene or gene fragments. Not only does that enable better detection of that gene or gene fragment, but it provides a substantial reservoir of genetic material for subsequent research and analysis, and for subsequent cloning and expression in a suitable vector. However, PCR presupposes at least some knowledge of the nucleotide sequence in the gene or gene fragment to be amplified, in view of the need to provide an oligonucleotide primer for the PCR, which primer hybridized to the complementary sequence in the gene or gene fragment to be amplified and which initiates the PCR replication of the gene or gene fragment from that point. The use of a pair of primers which hybridize to the target DNA at different spaced apart locations enable precise lengths of the target gene or gene fragment to be amplified. Once again, it is theoretically possible to use any pair of oligonucleotides hybridizable at different points of the C. difficile A toxin gene as primers for the PCR amplification and subsequent detection of that gene, and consequently the detection of C. difficile in a sample, e.g. a clinical sample. However, it is preferable to use as primers for the PCR oligonucleotide which hybridize to spaced locations on one of the repeat sequences now identified as appearing in the C. difficile A toxin gene. Thus, in the amplification of the gene by PCR replication of the repeat sequence occurs in parallel at each location of that sequence in the gene, thus substantially increasing the replication rate, and increasing not only the sensitivity of the C. difficile detection, but also dramatically reducing that time required to carry out a diagnosis of C. difficile infection in a patient. In tests carried out by the present inventors, positive diagnoses of C. difficile infection have been confirmed by PCR within the space of five hours.

Based on the repeat sequences given above, various oligonucleotide pairings can be suggested of various different lengths and comprising a sequence of nucleotides complementary to the sequences at the 5'- and 3'- ends of each repeat sequence. Especially preferred are pairs of oligonucleotides complementary to the 5' and 3' termini of the tandem C repeat sequence. Two particular pairings which have been synthesized and used in accordance with this invention are

complementary to the opposite ends of the C. difficile A toxin gene repeat sequence C,

and

complementary to the opposite ends of the C. difficile A toxin gene repeat sequence A.

In general terms, the preferential pairings for PCR amplification of the C. difficile A toxin gene fragments may be identified as oligonucleotide sequences of from 15-25 nucleotides complementary to the 15 to 25 nucleotide sequences at the 5' to 3' ends of the A, B, C and D repeat sequences identified above, and especially of the A and C sequences.

In accordance, with a second aspect of the present invention a 31 kDa antigen has been identified as common to all C. difficile strains tested, and has been found to be encoded by a 1.8 kb HindIII digestion fragment of C. difficile DNA. Thus, in accordance with a second aspect of the present invention, C. difficile probes are provided derived from or complementary to 1.8 kb HindIII digestion fragment of C. difficile or to individual sequences of that fragment and comprising oligonucleotides again ranging in size from 15 units to substantially the complete sequence, but more usually ranging in size from 15 to 300 units, most usually 15 to 50 units, more preferably 15 to 30 units.

Although the correct reading frame has yet to be established, the complete sequence of the 1.8 kb HindIII digestion fragment encoding the 31 kDa C. difficile antigen is as set out in Appendix II as SEQ ID No.2 and which again is to be taken as form a part of the present specification.

Particular oligonucleotide probes complementary to selected sequences of the 1.8 kb HindIII fragment are identified as follows, the numbers in parentheses are simply identification codes.

In a broad aspect therefore, the present invention provides C. difficile specific oligonucleotides comprising oligonucleotides of at least 15 nucleotides complementary to, and capable to hybridization with, any sequence of 15 or more nucleotide bases of the DNA sequences hereinbefore set forth and identified respectively as the 1.8 kb HindIII DNA fragment of C. difficile and the 1947 bp A Toxin DNA fragment, said probes having a length upto and including the complete sequence.

Within that broad class, two broad sub-categories can be identified as constituting or including the preferred C. difficile specific oligonucleotides according to this invention. The first sub-category is constituted by sub-restriction fragments of either complete sequence, and as such sub-restriction fragments there may be especially identified the following:

An Eco RI/PstI 1448 bp sub-restriction fragment of the Toxin A gene.

Such restriction fragments may, of course, be obtained in known manner, for example by digestion of C. difficile genomic DNA with the appropriate restriction enzyme or enzymes and subsequent separation by gel electrophoresis, chromatography or in any other suitable manner known for the separation of individual DNA restriction fragments according to size.

The second principal sub-category of DNA probes according to the invention are synthetic oligonucleotide sequences, e.g. those already mentioned. Such oligonucleotide sequences are readily assembled using known oligonucleotide synthesis techniques.

Utilising selected complementary pairs of the above oligonucleotides as primers, polymerase chain reactions (PCR) have been carried out according to manufacturers specifications (Cetus Ltd.) to amplify known fragments of C. difficile DNA, both purified total C. difficile DNA and crude C. difficile DNA released from C. difficile bacterial cells. In these experiments:

oligonucleotides 031 and 032 have been shown to amplify a 0.303 kb fragment of C. difficile DNA;

oligonucleotides 028 and 029 have been shown to amplify a 1.2 kb fragment of C. difficile DNA;

oligonucleotides 031 and 029 have been shown to amplify a 0.7 kb fragment of C. difficile DNA;

oligonucleotides 037 and 029 have been shown to amplify a 0.35 kb fragment;

oligonucleotides 029 and 032 have been shown to amplify a 0.8 kb fragment of C. difficile DNA; and

Although PCR amplification appears to be the presently preferred method of C. difficile detection using the C. difficile specific oligonucleotides of this invention, other detection procedures are available and are well known in the art. To this end the C. difficile specific oligonucleotides of this invention may be provided with a variety of different labels: radioactive, fluorescent or enzyme, to provide a variety of DNA probes specific to C. difficile DNA and all permitting the detection of hybridized (bound) DNA probe to the unidentified DNA sample under investigation. Alternatively, of course, for sandwich hybridization techniques the DNA probes of this invention may be immobilized in any known appropriate fashion, e.g. by covalent bonding to a variety of different solid substrates, both particulate, e.g. glass, Sephadex, Sephacryl beads etc., and continuous surface substrates. The techniques of DNA labelling and immobilization are well known in the art, as are methods for the detection of microorganisms, in general, utilizing DNA probes and which will be equally suitable in accordance with the present invention for the specific detection of C. difficile. Such procedures and methods are not part of the present invention as such and need not be further described here.

However, it is to be understood that references herein to an oligonucleotide for use as DNA (or RNA) probe in accordance with this invention are to be taken as including also labelled, tailed and immobilized versions thereof as well as the oligonucleotide sequence per se, it also being understood that oligonucleotide lengths given herein do not include additional non-complementary sequences, such as poly(DA) or poly(DT) tails added for a specific purpose such as immobilizing the probe on a poly(DT) or poly(DA) coated surface.

The C. difficile specific oligonucleotides covered by this invention include both single- and double-stranded versions, it being understood that in any subsequent hybridization procedures such as the detection of C. difficile in clinical, faecal or other samples, such double-stranded oligonucleotide will require denaturing to provide the probes in single-stranded form.

Also included within the scope of this invention are a method of detecting the presence of C. difficile in a sample, for example a faecal sample, which comprises treating the sample to release the DNA or mRNA and detecting the presence of C. difficile DNA or mRNA using a C. difficile specific oligonucleotide according to the invention, and especially a pair of such oligonucleotides as primers for a PCR reaction and diagnostic kits for the diagnosis of C. difficile infection in a patient comprising a C. difficile specific oligonucleotide according to the invention, and especially PCR kits containing a pair of such oligonucleotides for use as primers in the PCR reaction.

The invention is illustrated by the following Examples:

PCR amplification of C. difficile A toxin gene using oligonucleotides BW69 and BW70

Nine stranded C. difficile strains, labelled A to E and W to Z were grown anaerobically at 37.degree. C. for 36 hours on selective media (cycloserine, cefoxitin and fructose sugar). Strains B, D, E, W, X and Z were specifically selected as being known to be toxigenic. Strains A, C and Y selected as controls were known to be non-toxigenic.

Single colonies of each strain were scraped with an inoculating loop into a 1.5 ml polypropylene tube containing 200 .mu.l sterile water and boiled for 10 minutes. The samples were then centrifuged for 5 minutes at 14000.times.g to obtain a supernatent C. difficile cell extract.

Oligonucleotides BW69 and BW70 (see above) were synthesized on an Applied Biosystems synthesizer using the automated phosphoramidite coupling method.

Two microliters of each C. difficile extract were added to a 100 .mu.l reaction volume with 10 mM tris-HCl (pH 8.3), 0.01% gelatin, 200 .mu.M of each of the four deoxytribonucleotides: A, C, T, G, 100.sub.D M of each of the two primers and 250 Taq polymerase (Perkin-Elmer Cetus, Calif.). The reaction mixtures were overlain with paraffin oil (100 .mu.l) placed in a thermal (Hybaid Ltd.) and amplified for 30 cycles. A rapid two step cycle of 94.degree. C. and 46.degree. C. for 30 seconds each was used except that the first 94.degree. C. step was extended to 3 minutes to ensure denaturation of the initial sample.

20 .mu.l aliquots of the amplified samples were electrophoresed in a horizontal 4% Nusieve GTG agarose gel (ICN Biochemicals Ltd.) containing 0.5 .mu.l ethidium bromide per ml, and the bands visualized under UV light.

In a parallel series of experiments, the cultured C. difficile strain W was serially diluted to provide samples containing 300, 30 and 3 bacteria (as well as 10.sup.7 other anaerobic enteric bacteria from the original faecal sample) which were likewise subjected to PCR with primers BW69 and BW70 (samples 1, 2 and 3).

The results are shown in FIG. 1 of the accompanying drawings which shows the band separation obtained with each of the samples A to E, W to Z and 1, 2 and 3. Lane M is that of a parallel DNA size marker giving bands at 67 80 110 147 190 242 and 267 bp.

Lanes B, D, E, W, X, Z, 1 and 2 (toxigenic C. difficile strains) each sow a characteristic five bands at 63 126 189 252 and 315 bp, which are not present in samples from the non-toxigenic strains (A, C and T), but which are apparent with samples containing as few as 300 or even 30 bacteria.

These results have been confirmed on a total of 45 C. difficile strains by PCR amplification using the primer pair BW69 and BW70. Of the 45 strains tasted, all 33 strains known to be toxigenic proved positive, whilst the 12 non-toxigenic strain proved negative.

Of 16 strains from 13 different species of Clostridium other than C. difficile, only three, all C. sordelii, have given positive results.

These results are tabulated in Table 1 and clearly show the substantial specificity of the present oligonucleotides towards toxigenic C. difficile, and the fact that positive diagnosis can be made on the basis of as few as 30 bacteria establishes its sensitivity. One of the reasons for this degree of sensitivity is conjectured to be the specific selection of a pair of primers complementary to the 5' and 3' ends of the C. difficile A toxin gene repeat sequence C. Virtually equivalent results can, however, be predicted for similar pairs of primers complementary to the 5' and 3' ends of the repeat sequences A, B and D.

PCR amplification of C. difficile DNA using oligonucleotide probes 028 and 029

Oligonucleotides 028 and 029 were synthesised as before on an Applied Siosystems synthesiser.

The two primers were then used at a concentration of 1 .mu.M to amplify C. difficile DNA obtained by boiling C. difficile cells in 50 .mu.l of H.sub.2 O for 10 minutes. The DNA was amplified using a cycle profile of 94.degree. C. 1 min., 31.degree. C. 1 min., and 72.degree. C. 3 min. After the last cycle the polymerization step was extended from 3 to 10 minutes. Twenty six cycles of amplification were performed in total. The reactions were carried out in 100 .mu.l volumes, 20 .mu.l samples were run on agarose gels and amplified DNA detected by ethidium bromide staining and comparison with mol. wt. standards. By this method C. difficile DNA has been amplified from cell population as low as 10.sup.2 C. difficile cells, thus indicating the extreme sensitivity of C. difficile specific oligonucleotides according to this invention. Simultaneous controls with other Clostridia spp. have failed to produce any evidence of amplification, indicating the specificity of probes according to the present invention to C. difficile.

Confirmation that the amplified fragments are C. difficile DNA is obtained both by hybridization with internal oligonucleotide probes and by using two internal primers for PCR after initial amplification, e.g. by using probes 028 and 029, to give amplified fragments of 1.2 kb, and then using 2% of this reaction product to perform further PCR with probes 037 and 029 to give a 0.35 kb fragment.

PCR amplification of C. difficile DNA using primers BW65 and BW66.

Following the same protocol as in Example 1, DNA was amplified from toxigenic and non-toxigenic C. difficile strains using primers BW65 and BW66.

The results are shown in FIG. 2. Lanes B D E W X and Z show the PCR amplified products from pure colonies of toxigenic C. difficile and Lanes A C and Y the non-toxigenic strains. Lanes 1, 2 and 3 serial dilutions of G toxigenic C. difficile strain W at 300 30 and 3 bacteria mixed with 10.sup.7 other anerobic enteric bacteria. Lane M is a molecular weight size marker showing DNA lanes at 67 80 110 147 190 242 and 267 bp. (BshI and MspI digests of plasmid pHC 314).

The results obtained in Examples 1 and 3 are summerized in Table 1 below.

These results clearly establish the specifity and sensitivity of the preferred oligonucleotides of the present invention i.e. those complementary to the terminal sequences of the A and C A-toxin repeat sequences, and indicate their utility as a tool in the detection and diagnosis of infections by C. difficile.