# Patent

## Title
Cyclic nucleotide phosphodiesterases

## Abstract

This application is a continuation of U.S. application Ser. No. 08/604,991, filed on Feb. 29, 1996 now U.S. Pat. No. 6,001,565, filed as PCT/GB94/01967, Sep. 9, 1994.

## Background
This invention relates to the detection and speciation of Campylobacter bacteria, for example in clinical, environmental and food samples. In particular, this invention relates to a method of detecting whether a sample contains Campylobacter and to a method of differentiating between the main Campylobacter species jejune, coli, upsaliensis and lari.

Campylobacter species are recognised as the most frequent cause of bacterial gastroenteritis in the United Kingdom and many other countries throughout the world. In the U.K. approximately 90% and 10% of case isolates are identified as Campylobacter jejuni and Campylobacter coli respectively, plus a small number of other species such as Campylobacter upsaliensis and lari. The majority of the infections are sporadic the source of which remains largely unknown although the importance of several vehicles is now recognised.

There is a known desire to be able to detect and differentiate species of Campylobacter. However, it is also known that present Campylobacter enrichment culture techniques lack sensitivity, making detection difficult. Campylobacter jejuni does not multiply in foodstuffs and low numbers may be present together with a high background of indigenous microflora. Also, surface viable counts of Campylobacter can decrease rapidly and cells that are potentially culturable are often lost before samples reach a laboratory for analysis. Another factor making detection problematic is that antibiotics used in culture enrichment media may damage already weakened Campylobacter.

There are currently available assays for detection of a variety of food and water-borne pathogens; L. pneumophila, V. vulnificus, enteroinvasive E. coli, Shigella; but no satisfactory method of detecting Campylobacter or distinguishing between the four main Campylobacter species is known.

A method of detecting Campylobacter has been published by Giesendorf, B A J, et al in Applied and Environmental Microbiology, December 1992, pages 3804-3808. The method detects the species jejuni, coli and lari, and produces similar results to conventional methods but in a reduced time. The method suffers from a number of drawbacks. It does not enable detection of the species upsaliensis. Further, the method employs polymerase chain reaction (PCR) techniques but nevertheless requires a short enrichment culture before the PCR can be employed. Further still, the primer used for the PCR does not have the precise homology with DNA sequences in the three Campylobacter species that can be detected using the method.

Another method for detecting Campylobacter jejune and Campylobacter coli is known from Wegmuller, B E et al, Applied Environmental Microbiology, vol. 59, part 7, 1993 pages 2161-2165. The described method detects only the species jejuni and coli.

In addition to the above-identified problems with detection and speciation of Campylobacter, recent work on Campylobacter jejuni suggests that in certain circumstances it enters a "non-culturable, viable form" when subjected to environmental stresses, such as pH or temperature extremes, increased oxygen tension or nutrient depletion. In this form, Campylobacter infectivity is maintained but the organisms cannot be cultured. Thus there exists a need for the improvement of methods of detection of non-culturable forms of Campylobacter.

It is an object of this invention to provide a method of testing for the presence of Campylobacter that enables more efficient detection and eliminates or mitigates the problems with existing techniques. It is a further object to provide a method of distinguishing the Campylobacter species jejuni, coli, upsaliensis and lari.

Accordingly, in a first aspect the present invention provides a method of testing for the presence of Campylobacter, e.g. in a clinical, environmental or food sample, comprising the steps of performing polymerase chain reaction (PCR) using primers adapted to amplify a region selected from (a) a sequence of at least 72 base pairs from SEQ ID NO: 1 and (b) a sequence having sufficient homology with (a) such that formation of PCR product is correlated with presence of Campylobacter; and determining if any PCR product is formed.

It is preferred that sequence (b) has at least 75% homology with (a), preferably at least 90% homology and more preferably at least 95% homology. It is also preferred that the primers are at least 12 nucleotides in length, preferably between 19-22 nucleotides in length. In particularly preferred embodiments of the invention the primers consist of at least 12 contiguous nucleotides selected from (1) SEQ ID NO:2 and SEQ ID NO:3, (2) SEQ ID NO:4 and SEQ ID NO:5 and (3) sequences having sufficient homology with (1) or (2) such that formation of PCR product is correlated with presence of Campylobacter

PCR has become a well known and established tool for DNA analysis. A single gene sequence can be marketed from a large amount of other DNA and amplified to provide a suitable quantity for analysis. The basis of today's PCR was first published in 1971 be Kleppe, E et al, J. Mol. Biol., 1971, 56, 341. Further significant details and improvements on the PCR method have been added by Saiki, R K et al, Science, 1985, 230, 1350 and Mullis, K B, Sci. Am. 1990, 262, 36.

As will be appreciated by a person of skill in the art familiar with the PCR, it is important to operate at a temperature suitable to ensure that the primers used are specific for the sequence desired to be identified and amplified. To this end it is convenient to carry out the PCR reaction using the method of the invention at temperatures of at least 40.degree. C. preferably at least 45.degree. C. and in a particularly preferred embodiment at 48-52.degree. C.

The 1.9 kilobase fragment identified in SEQ ID NO:1 is an underlying feature of this invention and has been found to be highly conserved between Campylobacter isolates. The method of the invention confers the advantage that PCR product will only be detected when a Campylobacter strain is found in the sample tested. The method also confers the advantage that it will detect non-culturable viable forms of Campylobacter as well as viable cells. Thus the method is effective where other methods have not been able to detect any Campylobacter.

It is preferable to use a primer sequence that will only bind to one specific region of SEQ ID NO:1 and which will not engage in formation of primer dimers and thus contaminate the PCR. Examples of preferred primers for use in the method of the invention are shown in the SEQ ID NOs 2 and 3 and SEQ ID NOs 4 and 5. These primers form further aspects of the invention.

In a second aspect the invention provides a method of distinguishing between Campylobacter species jejuni, coli, upsaliensis and lari in a DNA containing sample by performing PCR utilizing primers capable of amplifying a selected Campylobacter DNA sequence, said sequence having restriction endonuclease sites specifically associated with different Campylobacter species and then testing for digestion of the PCR product by the specific restriction endonucleases.

Thus, Campylobacter DNA that is differentially cleared by restriction endonucleases is amplified, subject to digestion by the endonucleases and identified as from a particular species.

SEQ ID NO:1 was isolated from Campylobacter jejuni and is known to have a particular characteristic pattern of cleavage by restriction endonucleases. Campylobacter coli, upsaliensis and lari contain sequences corresponding to SEQ ID NO:1 that have altered patterns of cleavage characteristic of each species.

In an embodiment of the second aspect there is provided a method of distinguishing between Campylobacter species jejuni, coli, upsaliensis and lari, e.g. in a clinical, environmental or food sample containing Campylobacter, comprising the steps of: adapted to amplify a region of DNA SEQ ID NO:1 that includes nucleotides 124-196, or using primers adapted to amplify a DNA region corresponding thereto; and Dra I and Dde I.

The method of the second aspect is advantageous because it enables accurate speciation between the four clinically most significant species. In particular, when amplifying region 124-196 of SEQ ID NO:1, the PCR product from Campylobacter jejuni is cleaved by all three restriction endonucleases, whereas the PCR product from species coli is not cleaved by Dra I, the PCR product from species upsaliensis is only cleaved by Dde I and PCR product from lari is only cleaved by Alu I. It is a straightforward matter for a person skilled in the art to identify whether the PCR product is cleaved by one or more of the above endonucleases and thus the method enables simple speciation of Campylobacter into jejuni, coli, upsaliensis or lari.

The embodiments of the first aspect of the invention described above form embodiments of the second aspect of the invention also, provided that primers are selected so as to be adapted to amplify at least nucleotides 124-196 of SEQ ID NO:1, or a Campylobacter sequence corresponding thereto.

In a preferred embodiment of the second aspect the primers consist of at least 12 contiguous nucleotides from SEQ ID NOs: 4 and 5. Where the primers are SEQ ID NOs: 4 and 5 the PCR product is 256 bp and the respective products of cleavage by Alu I, Dra I and Dde I differentiate between jejuni, coli, upsaliensis and lari.

In a further embodiment of the invention, increased sensitivity and specificity for the detection of the presence of Campylobacter DNA, e.g. in food and liquid samples, is provided by the following additional methodologies:

1. A nested PCR has been developed, and is performed by an additional round of amplification using primer sequences international primer SEQ ID NOs:4 and 5. Two exemplary primer sequences are identified as Cru 0476 (SEQ ID NO:6) and Cru 0474 (SEQ ID NO:7). Following the second round of amplification, an amplicon of approximately 173 pb is obtained in the presence of Campylobacter DNA. This DNA fragment retains the sequences for the restriction endonucleases Alu 1, Dde 1, and Dra 1, thus still enabling the speciation of the contaminating Campylobacter.

2. Additional increased sensitivity and specificity is optionally achieved by southern transfer of the amplified PCR products obtained using oligonucleotide primers SEQ ID NOs:4 and 5, followed by hybridisation with an internal probe (e.g. SEQ ID NO:8 probe sequence). The probe sequence spans the restriction sites for speciation of the contaminating Campylobacter and therefore restriction digest analysis can be used in conjunction with the probe hybridisation to confer additional specificity. The probe can be labelled, for example with digoxigenin, or radiolabelled.

The extraction procedures for food and environmental samples preferably use an internal standard to enable qualitative estimation of extraction efficiency and the effects of non-specific inhibition. The PCR "MIMIC" (Clontech Laboratories, Palo Alto, Calif.) is a form of competitive PCR in which a non-homologous neutral DNA fragment is engineered containing the same primer templates as the target DNA. The amplimer produced from this construct is a fragment either smaller or larger than the target product. Known amounts of construct are added to the PCR reaction, and compete for the same primers, acting as an internal standard. Where a mimic is used, the mimic sequence is capable of being amplified by the same primers that amplify, under PCR conditions, the Campylobacter sequence. The mimic, if cleaved by restriction endonucleases, does not form fragments that interfere with detection and/or speciation of Campylobacter--the mimic is said to be "neutral".

It is preferred to carry out the PCR steps of the invention also using a mimic. In an example, mimic DNA is added to the sample and PCR is performed according to the invention. The PCR product is analyzed. If mimic DNA has been amplified, this indicates that the PCR reaction has occurred properly. The product can then be tested for products that indicate presence of Campylobacter. If no mimic DNA is amplified then this indicates PCR has not fully been carried out, or has been inhibited in some way.

It is further preferred to carry out PCR using mimic DNA of known and varying quantities. After amplification, the various results are compared and it is observed which of the results has comparable amounts of amplified mimic and (if present) Campylobacter DNA. Thus, an estimate of the quantity of Campylobacter DNA in the original sample is obtained.

The methods of the invention are further illustrated by the further embodiments of the invention described in the following Examples:

The PCR assay was developed by the following steps:

1> Identification of a highly conserved, species specific clone from a random library of Campylobacter jejuni insert fragments, cloned in the vector pBlueScript KS.

2> Chain termination sequencing of the 1.9 kilobase fragment in both directions.

3> Selection of presumptive primer pairs based on regions of equivalent G+C/A+T content, and low identity (prevention of `primer-dimer`).

4> Optimisation of reaction parameters: Mg.sup.++  concentration, Taq enzyme source, buffer composition, annealing temperature, cycling parameters.

Using a single amplification (35 cycles, annealing temperature 50.degree. C.) we detected approx. 10 CFU/ml of Campylobacter jejuni.

At this stringency, the assay was specific for Campylobacter jejuni, Campylobacter coli and Campylobacter upsaliensis. Using a lower annealing temperature (42.degree. C.), Campylobacter fetus and Campylobacter lari were also amplified.

The following procedures were used for PCR amplification of Campylobacter jejuni from milk and water samples.

1> cell lysis by boiling or freeze/thaw cycles, centrifuge, PCR supernatant directly.

2> Cell lysis by boiling, nucleic acid purification by phenol/chloroform extraction.

3> cell lysis by guanidine isothiocyanate, nucleic acid purification using nuclease binding matrix ("isoquick").

4> Cell concentration using magnetic particles coated with anti-Campylobacter IgG, cell lysis by boiling.

5> concentration and immobilisation of cells on 0.2 .mu.m nitrocellulose filters (`solid-phase` PCR).

6> Cell concentration using affinity column purification.

7> Guanidium isothiocyanate nucleic acid extraction. ##STR1## ##STR2##

We observed the following differentiation of Campylobacter species using PCR primers SEQ ID NO:4 and SEQ ID NO:5 and restriction endonucleases Alu I, Dra I and Dde I. PCR product digested with: Species    Alu I        Dra I  Dde I ______________________________________ C. jejuni  +            +      + C. coli    +            -      + C.         -            -      + upsaliensis C. lari    +            -      - ______________________________________

We further observed the following fragment sizes for different species. Restriction enzyme digests of PCR amplimers Species   Alu I      Dde I        Dra I Thermophilic/ Fragment        Fragment    Fragment enteropathogenic sizes (bp)      sizes (bp)  sizes (bp) ______________________________________ C. jejuni 2     108, 148 2    83, 173 2   123, 133 C. jejuni 2     108, 148 2    83, 173 2   123, 133 (hippurate + ve) C. coli   2     108, 148 2    83, 173 1   256 C. lari   2     108, 148 1    256     1   256 C. upsaliensis 1     256      3    30, 83, 143 1   256 ______________________________________

The results are also illustrated in FIG. 3; where when bands were not visible by eye they were detected by use of radiolabels.

To test the specificity of Campylobacter detection we used PCR primers on laboratory samples containing a wide range of organisms. The primers were SEQ ID NOs: 4 and 5, PCR product size in brackets: Annealing temperature of primers Species      37.degree. C. 42.degree. C. 50.degree. C. ______________________________________ C. jejuni    +           +       + (256) C. coli      +           +       + C. upsaliensis +           +       + C. fetus     +           .+-.    + C. lari      +           .+-.    + C. mucosalis .+-.        -       - C. sputorum  .+-.        -       - Achromobacter sp. -           -       - Acinetobacter .+-.        -       - calcoac.     (multiple) Acinetobacter sp. .+-.        -       - (multiple) Aeromonas    -           -       - hydrophila Citrobacter  -           -       - freundii Enterobact.  -           -       - aerogenes Enterobact.  .+-.        -       - agglomerans   (500) Enterobacter -           -       - cloacae Escherichia coli -           -       - Flavobacterium -           -       - Klebsiella   -           -       - aerogenes Klebsiella oxytoca .+-.        -       - (500) Proteus mirabilis -           -       - Proteus morganii -           -       - Providencia  -           -       - stuartii Pseudomonas  -           -       - aeroginosa Pseudomonas  -           -       - maltophilia Pseudomonas  -           -       - pickettii Salmonella   -           -       - enteritidis Salmonella   -           -       - typhimurium Serratia     -           -       - marcescens Serratia     -           -       - liquefaciens Shigella     -           -       - dysenteriae Shigella sonnei -           -       - Vibrio cholera -           -       - Vibrio furnassii .+-.        -       - (1000) Vibrio       .+-.        .+-.    - parahaemolyticus (180) Yersinia     -           -       - enterocolitica Oxford       .+-.        .+-.    - Staphlococcus (300) ______________________________________

Using standard culture techniques (published by Bolton F J, et al, J. Appl. Bacteriol., 1983, vol. 54, pages 115-125) we compared the detection of Campylobacter jejuni by culture with detection by the method of the invention (using primers SEQ ID NOs: 4 and 5) against time.

The success of culture detection declined over the time of the comparison, no culturable organisms being found remaining in the sample after 26 days--thus at this point detection by culture indicated no Campylobacter present.

By contrast, using the PCR method of the of the invention we were still able to detect Campylobacter DNA in a sample 42 days old. The results are illustrated in FIG. 1.

To confirm the accuracy of the PCR method of the invention we tested many samples that contained known species of Campylobacter. The results, illustrated in FIGS. 4-11, confirm the method is completely accurate for all samples tested, and correctly identified each one by species.

It allows rapid and sensitive detection of Campylobacter jejuni from environmental samples, provides a semi-quantitative indication of the bacterial load, and determines whether samples are contaminated with Campylobacter jejuni, coli, upsaliensis or lari.

The method is of use for examining epidemiology of Campylobacter infection such as a) seasonal peak, b) inverse correlation of surface water viable counts with human disease, c) role of water supply in (re)infection of broiler flocks with Campylobacter jejuni, d) contamination of foodstuffs at the point of sale, and e) determine origin of sporadic human infections.

Thus, a novel method incorporating polymerase chain reaction assay has been developed for the detection of Campylobacter in clinical, environmental and food samples, such as milk and water samples. The assay is rapid, highly sensitive, and specific for Campylobacter sp. Simple restriction analysis of the PCR product allows speciation between Campylobacter jejuni, coli, upsaliensis and lari.

FIG. 1 shows a comparison of culturability of Campylobacter jejuni against time with detection of Campylobacter jejuni using PCR of the invention.

FIG. 2 shows the sequence (SEQ ID NOs: 12, 14-19) of open reading frame "C" from insert fragment pBSKSCJ19B with primer/nested primer locations, and restriction sites.

FIG. 3 shows restriction enzyme analysis of PCR products amplified from C. jejuni, coli and upsaliensis.

FIGS. 4-11 shows the results of carrying out the PCR method of the invention on samples containing a wide ranges of known isolates. "P"=Penner Serotype Reference strains. "L"=Lior Serotype Reference Strains. Others are laboratory isolates. Standard size markers are on the gel ends. FIG. 4      C. Jejuni.   Alu I digest. FIG. 5      C. Jejuni.   Dde I digest. FIG. 6      C. Jejuni.   Dra I digest. FIG. 7      C. upsaliensis. Alu I and Dde I digest. FIG. 8      C. upsaliensis. Dra I digest. FIG. 9      C. Coli.     Alu I digest FIG. 10     C. Coli.     Dde I digest FIG. 11.    C. Coli.     Dra I digest ______________________________________ 1 accaacagcc attaaaaatc ttgactcagc catactcact ttaagaacac tggttgtcgg taatttttag aactgagtcg gtatgagtga aattcttgtg 51 gcggacctat ataataccgt tgcccaaatc cctgaaagca taaaaccaaa cgcctggata tattatggca acgggtttag ggactttcgt attttggttt 101 aatcacacct gaagtatgaa gtggtctaag tcttgaaaaa gtggcatatt ttagtgtgga cttcatactt caccagattc agaacttttt caccgtataa 151 gtcctggtaa ataatttaaa ttaggatatg ccatttgaaa agctataaga caggaccatt tattaaattt aatcctatac ggtaaacttt tcgatattct 201 gttcctatag ccataccaac aatgccaaac aatatggtcg caaacataaa caaggatatc ggtatggttg ttacggtttg ttataccagc gtttgtattt 251 atatcttgca accgtatagt cgtaatttaa tacattacct ggatgcatcg tatagaacgt tggcatatca gcattaaatt atgtaatgga cctacgtagc 301 actttctcct taaaattttt gataacaaga gaagattata gaatattaat tgaaagagga attttaaaaa ctattgttct cttctaatat cttataatta 351 tatacatttt ttcttaaaaa tgat/aatttt gttaatcatt tgttatgttt atatgtaaaa aagaattttt acta ttaaaa caattagtaa acaatacaaa 401 tatattttaa ggctaaatca gtcttattta ttgatattta tcttataacc atataaaatt ccgatttagt cagaataaat aattataaat agaatattgg 451 taaacttgtc acatttttta taaaatcttc acccacttta tctcttactc atttgaacag tgtaaaaaat attttagaag tgggtgaaat agagaatgag 501 tttttataaa agttcta{aca gcagtatcgc tcacatgt}c acctatccaa aaaaatattt tcaagat tgt cgtcatagcg agtgtaca g tggataggtt 551 acatttttct taatatcttc atgcaaaacc aaagctccag gttgctttaa tgtaaaaaga attatagaag tacgttttgg tttcgaggtc caacgaaatt 601 aagcaaagaa ataaaagcca attctttttt agttaaaaca atttctccac ttcgtttctt tattttcggt taagaaaaaa tcaattttgt taaagaggtg 651 cactgtaaat taaagttcgt ttatttttgt taaattgata ttcttcagaa gtgacattta atttcaagca aataaaaaca atttaactat aagaagtctt 701 atttttacaa gcatatttgc ttcaattttt tcacctatca gataatctaa taaaaatgtt cgtataaacg aagttaaaaa agtggatagt ctattagatt 751 aactttaaac aactcttcta tatcaacagg tttaatcaaa tatttatcta ttgaaatttg ttgagaagat atagttgtcc aaattagttt ataaatagat 801 taccaatatc aatagaacgc aaaagtctct ctt tctctga atacgcacta atggttatag ttatcttgcg ttttcagaga gaa{agagact tatgcgtgat 851 aga acaacaa ttgggacatc atctgaaatt tctttaatct ctcttgccat tct tgttgaa aaccctgtag tagactttaa agaaattaga gagaacggta 901 atccagtcca tccataatag gcatagcaat atctgtgata actaaatctg taggtcaggt aggtattatc cgtatcgtta tagacactat tgatttagac 951 gcttaaattt tttaaatttt ttaagcccct catctccatt ttgagctccg cgaatttaaa aaatttaaaa aattcggggt gtagaggtaa aactcgaggc 1001 attactttac taaagcgttc gcttaatata ttaatcattg attctctagc taatgaaatg atttcgcaag cgaattatat aattagtaac taagagatcg 1051 cttaacctca tcttcaacta ctaatattat taattcttta cattcttgtg gaattggagt agaagttgat gattataata attaagaaat gtaagaacac 1101 acat/ttctac tctaccctct cttttagttt taaaaatatc tcaaaacaag tgta aagatg agatgggaga gaaaatcaaa atttttatag agttttgttc 1151 ccccgtcttt tccattttta acttttattt ttccttggaa actttcgata ggggcagaaa aggtaaaaat tgaaaataaa aaggaacctt tgaaagctat 1201 atttgtctac ttatataaag tcctactcct ataccttgac taggatgttt taaacagatg aatatatttc aggatgagga tatggaactg atcctacaaa 1251 tgttgtaaaa taaggttgaa aaattttatc taaattttct ttatcaatcc acaacatttt attccaactt tttaaaatag atttaaaaga aatagttagg 1301 caccagcatt atcttttatt gtaattttca gataattttt tccaaatttt gtggtcgtaa tagaaaataa cattaaaagt ctattaaaaa aggtttaaaa 1351 gaaaaattta ttgttatgat tttccttttt ttgtttttaa atgcttctat ctttttaaat aacaatacta aaaggaaaaa aacaaaaatt tacgaagata 1401 tgaatttaaa atcaaattaa gaaaaactct tattaaacca ttctcatatg acttaaattt tagtttaatt ctttttgaga ataatttggt aagagtatac 1451 ccaaaacttc ataatcactt ttcgaaacaa tattaatatt tacatgattt ggttttgaag tattagtgaa aagctttgtt ataattataa atgtactaaa 1501 ttttctatag tttcaaaagc aatttccaag gctttattta aagtctcttt aaaagatatc aaagttttcg ttaaaggttc cgaaataaat ttcagagaaa 1551 tataaataca cactgctcta ctcctttgtt aaacaaagtt ctaaacacat 1601 caattgtttc tgacatattt ttaatcatat cttttgattg tgagtaaatt gttaacaaag actgtataaa aattagtata gaaaactaac actcatttaa 1651 tcagcaaatc ctttttcatc tttaagattt tgcttcattt gaaacatggc agacgtttag gaaaaagtag aaattctaaa acgaagtaaa ctttgtaccg 1701 aataccgagc tcatttaacg gttgtctcca ttgatgtgct atatcactaa ttatggctcg agtaaattgc caacagaggt aactacacga tatagtgatt 1751 tcatttgttc taatgaagat ttcaaaatct cttcatatgc tattttaata agtaaacaag attacttcta aagttttaga gaagtatacg ataaaattat 1801 tctttttcat ttttttccaa ggcaatttgc atttttttct caaatttttt agaaaaagta aaaaaaggtt ccgttaaacg taaaaaaaga gtttaaaaaa 1851 acctaactgt ataaattctt gttggtgatt tttaactgta ttttcaagat tggattgaca tatttaagaa caaccactaa aaattgacat aaaagaacta 1901 taatacttaa ttctcttaat ttagcgtgat ttagagcaag ctcttcat attatgaatt aagagaatta aatcgcacta aatctcgttc gagaagta ##STR3## Primer 1 Cru 0477  (SEQ ID NO: 9) 5'   agaacacgcggacctatatacgcaagtgaaatctcctccg 3' 40 mer Primer 2 Cru 0660  (SEQ ID NO: 10) 5'   cgatgcatccaggtaatgtattctgtcaatgcagtttgtag 3' 41 mer agaacacgcg gacctatata cgcaagtgaa atctcctccg tcttggagaa gggagagcgt ttgccccagc taccattgat gtgtacatga tcatggtcaa atgctggatg attgatgcag acagccgtcc caagtttcgt gagctgattg cagagttctc caaaatggct cgtgaccctc cccgctatct tgttatacag ggagatgaaa ggatgcactt gcctagccct acagattcca agttttatcg caccctgatg gaggaggagg acatggaaga cattgtggat gcagatgagt atcttgtccc acaccagggc tttttcaaca tgccctctac atctcggact cctcttctga gttcattgag cgctactagc aacaattctg ctacaaactg cattgacaga       3' #             SEQUENCE LISTING - (1) GENERAL INFORMATION: -    (iii) NUMBER OF SEQUENCES: 19 - (2) INFORMATION FOR SEQ ID NO:1: -      (i) SEQUENCE CHARACTERISTICS: #pairs    (A) LENGTH: 1947 base (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear -     (ii) MOLECULE TYPE: DNA (genomic) -     (vi) ORIGINAL SOURCE: (A) ORGANISM: Campylobacte - #r coli #1:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - ACCAACAGCC ATTAAAAATC TTGACTCAGC CATACTCACT TTAAGAACAC GC - #GGACCTAT 60 - ATAATACCGT TGCCCAAATC CCTGAAAGCA TAAAACCAAA AATCACACCT GA - #AGTATGAA 120 - GTGGTCTAAG TCTTGAAAAA GTGGCATATT GTCCTGGTAA ATAATTTAAA TT - #AGGATATG 180 - CCATTTGAAA AGCTATAAGA GTTCCTATAG CCATACCAAC AATGCCAAAC AA - #TATGGTCG 240 - CAAACATAAA ATATCTTGCA ACCGTATAGT CGTAATTTAA TACATTACCT GG - #ATGCATCG 300 - ACTTTCTCCT TAAAATTTTT GATAACAAGA GAAGATTATA GAATATTAAT TA - #TACATTTT 360 - TTCTTAAAAA TGATAATTTT GTTAATCATT TGTTATGTTT TATATTTTAA GG - #CTAAATCA 420 - GTCTTATTTA TTGATATTTA TCTTATAACC TAAACTTGTC ACATTTTTTA TA - #AAATCTTC 480 - ACCCACTTTA TCTCTTACTC TTTTTATAAA AGTTCTAACA GCAGTATCGC TC - #ACATGTCA 540 - CCTATCCAAA CATTTTTCTT AATATCTTCA TGCAAAACCA AAGCTCCAGG TT - #GCTTTAAA 600 - AGCAAAGAAA TAAAAGCCAA TTCTTTTTTA GTTAAAACAA TTTCTCCACC AC - #TGTAAATT 660 - AAAGTTCGTT TATTTTTGTT AAATTGATAT TCTTCAGAAA TTTTTACAAG CA - #TATTTGCT 720 - TCAATTTTTT CACCTATCAG ATAATCTAAA ACTTTAAACA ACTCTTCTAT AT - #CAACAGGT 780 - TTAATCAAAT ATTTATCTAT ACCAATATCA ATAGAACGCA AAAGTCTCTC TT - #TCTCTGAA 840 - TACGCACTAA GAACAACAAT TGGGACATCA TCTGAAATTT CTTTAATCTC TC - #TTGCCATA 900 - TCCAGTCCAT CCATAATAGG CATAGCAATA TCTGTGATAA CTAAATCTGG CT - #TAAATTTT 960 - TTAAATTTTT TAAGCCCCTC ATCTCCATTT TGAGCTCCGA TTACTTTACT AA - #AGCGTTCG 1020 - CTTAATATAT TAATCATTGA TTCTCTAGCC TTAACCTCAT CTTCAACTAC TA - #ATATTATT 1080 - AATTCTTTAC ATTCTTGTGA CATTTCTACT CTACCCTCTC TTTTAGTTTT AA - #AAATATCT 1140 - CAAAACAAGC CCCGTCTTTT CCATTTTTAA CTTTTATTTT TCCTTGGAAA CT - #TTCGATAA 1200 - TTTGTCTACT TATATAAAGT CCTACTCCTA TACCTTGACT AGGATGTTTT GT - #TGTAAAAT 1260 - AAGGTTGAAA AATTTTATCT AAATTTTCTT TATCAATCCC ACCAGCATTA TC - #TTTTATTG 1320 - TAATTTTCAG ATAATTTTTT CCAAATTTTG AAAAATTTAT TGTTATGATT TT - #CCTTTTTT 1380 - TGTTTTTAAA TGCTTCTATT GAATTTAAAA TCAAATTAAG AAAAACTCTT AT - #TAAACCAT 1440 - TCTCATATGC CAAAACTTCA TAATCACTTT TCGAAACAAT ATTAATATTT AC - #ATGATTTT 1500 - TTTCTATAGT TTCAAAAGCA ATTTCCAAGG CTTTATTTAA AGTCTCTTTT AT - #AAATACAC 1560 - ACTGCTCTAC TCCTTTGTTA AACAAAGTTC TAAACACATC AATTGTTTCT GA - #CATATTTT 1620 - TAATCATATC TTTTGATTGT GAGTAAATTT CAGCAAATCC TTTTTCATCT TT - #AAGATTTT 1680 - GCTTCATTTG AAACATGGCA ATACCGAGCT CATTTAACGG TTGTCTCCAT TG - #ATGTGCTA 1740 - TATCACTAAT CATTTGTTCT AATGAAGATT TCAAAATCTC TTCATATGCT AT - #TTTAATAT 1800 - CTTTTTCATT TTTTTCCAAG GCAATTTGCA TTTTTTTCTC AAATTTTTTA CC - #TAACTGTA 1860 - TAAATTCTTG TTGGTGATTT TTAACTGTAT TTTCAAGATT AATACTTAAT TC - #TCTTAATT 1920 #           1947   AAGC TCTTCAT - (2) INFORMATION FOR SEQ ID NO: 2: -      (i) SEQUENCE CHARACTERISTICS: #pairs    (A) LENGTH: 20 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear -     (ii) MOLECULE TYPE: DNA (genomic) #2:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: # 20               GAGA - (2) INFORMATION FOR SEQ ID NO: 3: -      (i) SEQUENCE CHARACTERISTICS: #pairs    (A) LENGTH: 21 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear -     (ii) MOLECULE TYPE: DNA (genomic) #3:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: #21                CATG T - (2) INFORMATION FOR SEQ ID NO: 4: -      (i) SEQUENCE CHARACTERISTICS: #pairs    (A) LENGTH: 20 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear -     (ii) MOLECULE TYPE: DNA (genomic) #4:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: # 20               TATA - (2) INFORMATION FOR SEQ ID NO: 5: -      (i) SEQUENCE CHARACTERISTICS: #pairs    (A) LENGTH: 21 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear -     (ii) MOLECULE TYPE: DNA (genomic) #5:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: #21                TGTA T - (2) INFORMATION FOR SEQ ID NO: 6: -      (i) SEQUENCE CHARACTERISTICS: #pairs    (A) LENGTH: 18 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear -     (ii) MOLECULE TYPE: DNA (genomic) #6:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: #  18              GA - (2) INFORMATION FOR SEQ ID NO: 7: -      (i) SEQUENCE CHARACTERISTICS: #pairs    (A) LENGTH: 18 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear -     (ii) MOLECULE TYPE: DNA (genomic) #7:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: #  18              CA - (2) INFORMATION FOR SEQ ID NO: 8: -      (i) SEQUENCE CHARACTERISTICS: #pairs    (A) LENGTH: 173 base (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear -     (ii) MOLECULE TYPE: DNA (genomic) #8:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - TACGACTATA CGGTTGCAAG ATATTTTATG TTTGCGACCA TATTGTTTGG CA - #TTGTTGGT 60 - ATGGCTATAG GAACTCTTAT AGCTTTTCAA ATGGCATATC CTAATTTAAA TT - #ATTTACCA 120 - GGACAATATG CCACTTTTTC AAGACTTAGA CCACTTCATA CTTCAGGTGT GA - #T 173 - (2) INFORMATION FOR SEQ ID NO: 9: -      (i) SEQUENCE CHARACTERISTICS: #pairs    (A) LENGTH: 40 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear -     (ii) MOLECULE TYPE: DNA (genomic) #9:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: #    40            TATA CGCAAGTGAA ATCTCCTCCG - (2) INFORMATION FOR SEQ ID NO: 10: -      (i) SEQUENCE CHARACTERISTICS: #pairs    (A) LENGTH: 41 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear -     (ii) MOLECULE TYPE: DNA (genomic) #10:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO: #   41             TGTA TTCTGTCAAT GCAGTTTGTA G - (2) INFORMATION FOR SEQ ID NO: 11: -      (i) SEQUENCE CHARACTERISTICS: #pairs    (A) LENGTH: 410 base (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear -     (ii) MOLECULE TYPE: DNA (genomic) #11:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - AGAACACGCG GACCTATATA CGCAAGTGAA ATCTCCTCCG TCTTGGAGAA GG - #GAGAGCGT 60 - TTGCCCCAGC TACCATTGAT GTGTACATGA TCATGGTCAA ATGCTGGATG AT - #TGATGCAG 120 - ACAGCCGTCC CAAGTTTCGT GAGCTGATTG CAGAGTTCTC CAAAATGGCT CG - #TGACCCTC 180 - CCCGCTATCT TGTTATACAG GGAGATGAAA GGATGCACTT GCCTAGCCCT AC - #AGATTCCA 240 - AGTTTTATCG CACCCTGATG GAGGAGGAGG ACATGGAAGA CATTGTGGAT GC - #AGATGAGT 300 - ATCTTGTCCC ACACCAGGGC TTTTTCAACA TGCCCTCTAC ATCTCGGACT CC - #TCTTCTGA 360 #             410ACTAGC AACAATTCTG CTACAAACTG CATTGACAGA - (2) INFORMATION FOR SEQ ID NO: 12: -      (i) SEQUENCE CHARACTERISTICS: #pairs    (A) LENGTH: 388 base (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear -     (ii) MOLECULE TYPE: DNA (genomic) #12:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - TGATTAACAA AATTATCATT TTTAAGAAAA AATGTATAAT TAATATTCTA TA - #ATCTTCTC 60 - TTGTTATCAA AAATTTTAAG GAGAAAGTCG ATGCATCCAG GTAATGTATT AA - #ATTACGAC 120 - TATACGGTTG CAAGATATTT TATGTTTGCG ACCATATTGT TTGGCATTGT TG - #GTATGGCT 180 - ATAGGAACTC TTATAGCTTT TCAAATGGCA TATCCTAATT TAAATTATTT AC - #CAGGACAA 240 - TATGCCACTT TTTCAAGACT TAGACCACTT CATACTTCAG GTGTGATTTT TG - #GTTTTATG 300 - CTTTCAGGGA TTTGGGCAAC GGTATTATAT AGGTCCGCGT GTTCTTAAAG TG - #AGTATGGC 360 #            388   ATGG CTGTTGGT - (2) INFORMATION FOR SEQ ID NO: 13: -      (i) SEQUENCE CHARACTERISTICS: #acids    (A) LENGTH: 58 amino (B) TYPE: amino acid (D) TOPOLOGY: linear -     (ii) MOLECULE TYPE: protein #13:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO: -      Tyr Asp Tyr Thr Val Ala Arg Tyr - # Phe Met Phe Ala Thr Ile Leu Phe #   15 -      Gly Ile Val Gly Met Ala Ile Gly - # Thr Leu Ile Ala Phe Gln Met Ala #                 30 -      Tyr Pro Asn Leu Asn Tyr Leu Pro - # Gly Gln Tyr Ala Thr Phe Ser Arg #             45 -      Leu Arg Pro Leu His Thr Ser Gly - # Val Ile #         55 - (2) INFORMATION FOR SEQ ID NO: 14: -      (i) SEQUENCE CHARACTERISTICS: #acids    (A) LENGTH: 5 amino (B) TYPE: amino acid (D) TOPOLOGY: linear -     (ii) MOLECULE TYPE: peptide #14:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO: -      Gln Asn Tyr His Phe #  5 1 - (2) INFORMATION FOR SEQ ID NO: 15: -      (i) SEQUENCE CHARACTERISTICS: #acids    (A) LENGTH: 5 amino (B) TYPE: amino acid (D) TOPOLOGY: linear -     (ii) MOLECULE TYPE: peptide #15:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO: -      Glu Lys Met Tyr Asn #  5 1 - (2) INFORMATION FOR SEQ ID NO: 16: -      (i) SEQUENCE CHARACTERISTICS: #acids    (A) LENGTH: 11 amino (B) TYPE: amino acid (D) TOPOLOGY: linear -     (ii) MOLECULE TYPE: peptide #16:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO: -      Leu Thr Lys Leu Ser Phe Leu Arg - # Lys Asn Val #   10 - (2) INFORMATION FOR SEQ ID NO: 17: -      (i) SEQUENCE CHARACTERISTICS: #acids    (A) LENGTH: 102 amino (B) TYPE: amino acid (D) TOPOLOGY: linear -     (ii) MOLECULE TYPE: peptide #17:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO: -      Leu Ile Phe Tyr Asn Leu Leu Leu - # Leu Ser Lys Ile Leu Arg Arg Lys #   15 -      Ser Met His Pro Gly Asn Val Leu - # Asn Tyr Asp Tyr Thr Val Ala Arg #                 30 -      Tyr Phe Met Phe Ala Thr Ile Leu - # Phe Gly Ile Val Gly Met Ala Ile #             45 -      Gly Thr Leu Ile Ala Phe Gln Met - # Ala Tyr Pro Asn Leu Asn Tyr Leu #         60 -      Pro Gly Gln Tyr Ala Thr Phe Ser - # Arg Leu Arg Pro Leu His Thr Ser #     80 -      Gly Val Ile Phe Gly Phe Met Leu - # Ser Gly Ile Trp Ala Thr Val Leu #   95 -      Tyr Arg Ser Ala Cys Ser 100 - (2) INFORMATION FOR SEQ ID NO: 18: -      (i) SEQUENCE CHARACTERISTICS: #acids    (A) LENGTH: 4 amino (B) TYPE: amino acid (D) TOPOLOGY: linear -     (ii) MOLECULE TYPE: peptide #18:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO: -     Ser Glu Tyr Gly 1 - (2) INFORMATION FOR SEQ ID NO: 19: -      (i) SEQUENCE CHARACTERISTICS: #acids    (A) LENGTH: 8 amino (B) TYPE: amino acid (D) TOPOLOGY: linear -     (ii) MOLECULE TYPE: peptide #19:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO: -      Val Lys Ile Phe Asn Gly Cys Trp #  5 1 __________________________________________________________________________

## Claims

1. A method of distinguishing between Campylobacter species, comprising: (a) identifying a selected Campylobacter DNA sequence, said sequence comprising one or more restriction endonuclease sites specifically associated with different Campylobacter species; and of said restriction endonuclease sites, thereby distinguishing between Campylobacter species C. jejuni, C. coli, C. upsaliensis and C. lari, wherein said DNA sequence comprises at least 72 nucleotides from SEQ ID NO: 1.

2. The method of claim 1, wherein a hybridization probe is used to distinguish between said Campylobacter species.

3. The method of claim 2, wherein said hybridization probe spans a restriction endonuclease site.

4. The method of claim 1, further comprising amplifying said selected Campylobacter sequence.

5. A method of distinguishing between Campylobacter species, comprising contacting a selected Campylobacter DNA sequence with a nucleic acid probe, wherein said selected Campylobacter DNA sequence comprises at least 72 nucleotides from SEQ ID NO:1 and contains one or more restriction endonuclease sites specifically associated with different Campylobacter species, and wherein said nucleic acid probe hybridizes with said selected Campylobacter DNA sequence.

6. The method of claim 5, wherein said nucleic, acid probe spans a restriction endonuclease site contained within said selected Campylobacter DNA sequence.

7. The method of claim 5, wherein said method distinguishes between Campylobacter species C. jejuni, C. coli, C. upsaliensis and C. lari.

8. The method of claim 5, wherein said selected Campylobacter sequence comprises nucleotides 124-196 of SEQ ID NO:1.

9. A nucleic acid probe for use in distinguishing between Campylobacter species, wherein said nucleic acid probe hybridizes to a restriction endonuclease site located within nucleotides 124-196 SEQ ID NO:1.

10. The method of claim 1, wherein Campylobacter jejuni is characterized by the presence of sites for endonucleases Alu I, Dde I and Dra I, C. coli is characterized by the absence of a site for endonuclease Dra I, C. upsaliensis is characterized by the absence of sites for endonucleases Alu I and Dra I, and C. lari is characterized by the presence of a site for endonuclease Alu I only.

11. The method of claim 7, wherein Campylobacter jejuni is characterized by the presence of sites for endonucleases Alu I, Dde I and Dra I, C. coli is characterized by the absence of a site for endonuclease Dra I, C. upsaliensis is characterized by the absence of sites for endonucleases Alu I and Dra I, and C. lari is characterized by the presence of a site for endonuclease Alu I only.