CA1314248C - Dna probes for identification and detection of campylobacter jejuni and campylobacter coli based on major outer membrane protein gene - Google Patents

Abstract

Abstract DNA probes to detect Campylobacter jejuni and Campylobacter coli were prepared from a bacteriophage .lambda.gtll library by screening using an antiserum directed against a C. jejuni 46 kilodalton major outer membrane protein (MOMP). Two DNA fragments (MOMP DNA probes) were identified, cloned into pUC13 and characterized by restriction endonuclease digestion (Figure 1). One MOMP DNA probe (pDT1720) hybridized only to C. jejuni strains, whereas the other (pDT1719) hybridized to both C. jejuni and C. coli strains. The invention DNA probes can be used to specifically identify the aforementioned bacterial species when the species designation is in doubt and can also be used to identify the microorganisms directly in stool specimens, as disclosed herein.
28 Claims, 1 Drawing

Description

~ ~314248 3 DNA Probe~ æor Identification and Detection of Campyobacter jejuni and Campylo~acter coli ba-~ed on Major OutQr Membrane Protein Gene.

The invention described herein was made in the course of work under a grant from the Natural Sciences and Engineering Research Council of Canada (Strategic Grant - Biotechnology).

BACKGROUND OF THE INVENTION
The bacterium Campylobacter jejuni is a major cause of bacterial gastroenteritis in both developed and developing countries (Blaser, M. J. and Reller, L. B. 1981. New Eng. J. Med. 305:1444-1452;
Skirrow, M. B. 1982. J. Hyg. Camb. 89:175-184). In contrast Campylobacter coli is responsible for 2-5% of cases of Campylobacter diarrhea in developed countries such as Canada (Karmali, M. A., Penner, J. L., Fleming, P. C., Williams, A. and Hennessy, J. N. 1983. ~. Infect. Dis. 147:243-246), although it is responsible for a larger proportion of cases in developing nations such as Africa (Georges-Courbot, M. C., Baya, C., Beraud, A. M., Meunier, D. M. and Georges, A. J. 1986. J. Clin. Microbiol.
23:592-594).

C. jejuni and C. coli should be differentiated from one another in the hospital clinical microbiology laboratory and particularly in the reference laboratory. However, C. jejuni and C. coli are differentiated only on the basis of a single biochemical test, the ability of C. jejuni to hydrolyse hippurate, in contrast to C. coli which does not (Hwang, M. A. and Ederer, G. M. 1975. J. Clin.
Microbiol. 1:114-115). Moreover, some variants of C. jejuni are hippurate negative, making differentiation based on hippurate hydrolysis difficult and not necessarily reliable (Penner, J. L.
1988. Clin. Microbiol. Rev. 1:157-172). The Campylobacter genus contains not only C. jejuni and C. coli but numerous other Campylobacter species (14 at last count). Some of these organisms are found in human stool specimens where they occasionally cause disease. In addition, many others are found in animal feces where ,~
~i 131~2~8 4 they apparently cause no harm. DNA hybridi~ation has been used in research laboratories to identify and classify strains of Campylobacter species (Belland, R. J. and Trust, T. J. 1982. J.
Gen. Microbiol. 128:2515-2522; Hebert, G. A., Edmonds, P. and Brenner, D. J. 1984. J. Clin. Microbiol. 20:138-140; Ng, L.-K., Stiles, M. E. and Taylor, D. E. 1987. Molec. Cell. Probes :233-243). DNA probes have usually consisted of total chromosomal DNA isolated from a known species often tagged with a 32P-labelled nucleotide. It would be useful to obtain specific DNA probes for identification of C. jejuni and C. coli, since the two species show a significant amount of DNA homology with one another which results in cross hybridization. Moreover, it is preferable to use a probe for a known and stable gene for identification of a particular species. Therefore, specific DNA probes for identification and differentiation of C. jejuni and C. coli would be useful in microbiological laboratories for testing these organisms directly.
The other major reason for developing DNA probes to detect C.
je juni and C. coli depends on the biology of these microorganisms.
Both species are microaerophilic and require a low concentration of oxygen for growth, 7% compared with the 21% 2 normally found in air. Therefore, special conditions are required for their isolation from stool specimens and their growth in the laboratory.
Anaerobic jars containing a special gas mixture or dedicated incubators are required. A number of selective media, which contain various antibiotics to kill other bacteria present in feces, have been devised for growth and selection of C. jejuni and C. coli (Hutchinson, D. N. and Bolton, F. J. 1984. J. Clin. Path.
37:956-957; Skirrow, M. B. 1977. Br. Med. J. 2:9-11). Although the procedures are not technically difficult for the trained microbiologist, they are expensive. Therefore DNA probes which could be used to detect C. jejuni and C. coli directly in stool specimens, without need to culture the organisms first, would be useful and could have commercially significant implications.

13142~8 5 DESCRIPTION OF THE PRIOR ART
Present methods for identifying and detecting the presence of C.
je juni may be found in: Claus P., Moseley, S. L., and Falkow, S.
1982. Prog. Food Nutr. Sci. 7:139-142; Korolik, V., Coloe, P. J., and Krishnapillai, V. 1988. J. Gen. Microbiol. 134:521-529, and Picken, R. N., Wang, Z. and Yang, H. L. 1987. Molec. Cell. Probes 1:245-259.
The United States Patent 4, 358, 535. November 9, 1982. Falkow, S. and Moseley, S. L. concerns specific DNA probes in Diagnostic Microbiology.

BRIEF SUMMARY OF THE INVENTION
The subject invention concerns two novel DNA probes, one specifically for identification of C. je juni (pDT1720) and the other for identification of both C. jejuni and C. coli (pDT1719).
These probes will enable workers to identify C. jejuni and/or C.
coli and differentiate them after microorganisms have been cultured and identified as Campylobacter species. The same probes may be used to identify C. jejuni and C. coli directly in stool specimens without the need to culture these organisms. Both probes were selected from a C. jejuni Agtll library as genes which encoded proteins that reacted with antiserum to a C. jejuni 46 kilodalton major outer membrane protein.

DESCRIPTION OF THE DRAWING
The Drawing (Figure 1) depicts the endonuclease restriction map of C. jejuni chromosomal DNA fragments inserted into the plasmid pUC13 to produce recombinant plasmids pDT1719 and pDT1720. Only the map of the inserted fragments is shown. The size of the inserts is in base pairs.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS
The subject invention, which is in the field of molecular biology, concerns DNA probes useful to identify and differentiate C. jejuni and C. coli, both with cultured organisms or directly in stool specimens.

,~ ~
~ , 131~248 These organisms are an important cause of bacterial gastroenteritis and are difficult to differentiate in the clinical laboratory with certainty. It is important to differentiate C.
jejuni and C. coli from one another for epidemiologicaI purposes and to differentiate them from other Campylohacter species as well as other bacterial species for epidemiological studies and for the information of the consulting physician. The current high cost of isolation and growth of C. jejuni and C. coli from stool specimens is due to the microaerophilic nature of these organisms. This invention when used to detect these microorganisms directly in stool specimens will circumvent the requirement for special jars or a dedicated incubator, expensive media containing antibiotics and other expensive supplements. It may take up to 72 hours to detect C. jejuni and C. col i in stool specimens by culture methods. This time period can be reduced to 18 hours by use of the DNA probes directly with technology available today. This time could be reduced further with future developments in DNA hybridization technology.
These DNA probes of the subject invention could be used to detect campylobacters in other specimens besides stool specimens, such as blood and serum samples. Other useful applications include detection of these organisms in food samples (e.g. chicken, turkey and game birds), milk and water samples. Since humans become infected with campylobacter by consuming these items, the DNA
probes would be useful to screen these products at the level of the food industry to determine the degree of contamination of food by Campylobacters.

Before detailing the construction and identity of the novel DNA probes of the subject invention, there are disclosed the materials and methods employed.

(1) MEDIA
C. jejuni and C. col i were grown from pure culture on Mueller Hinton agar (Oxoid Ltd., Basingstoke, U.K . ) . Escherichia col i Y10 was grown on L agar (Difco Laboratories, Detroit, Michigan, U.S.A.).

131424s~ 7 Medium for isolation of C. jejuni and C. coli from feces was the blood-free charcoal based medium of Hutchinson, D. N. and Bolton, F. J. (1984. J. Clin. Path. 37:956-957) which contain 35~g cephaperazone/ml and was supplied by Oxoid Ltd., Basingstoke, U.K.

(2) BACTERIAL STRAINS
The Escherichia coli strains used for the construction of the ~gtll library was Y1090 (Young, R. A., Bloom, B. R., Grosskinsky, C. M., Ivanyi, J., Thomas, D. and Davis, R. W. 1985. Proc. Nat. Acad.
Sci. U.S.A. 82:2583-2587) and for subsequent plasmid cloning E.
coli JM83 (Vieira, J. and Messing, J. 1982. Gene. 19:259-268).
The series of strains of different Campylobacter species (C.
jejuni, C. coli, C. fetus subps. fetus, C. fetus subsp. venerealis, C. laridis, C. hyointestinalis, C. sputorum, C. pylori) are maintained in the Diane E. Taylor culture collection at the University of Alberta. Other species were obtained from the Department of Medical Microbiology and Infectious Diseases, University of Alberta and were Pseudomonas aeruginosa, Salmonella typhimurium, Shigella sonnei, Bacillus subtilis and Staphylococcus aureus.

(3) PREPARATION OF ANTISERUM
A polyclonal antibody was prepared in rabbits against a 46 kilodalton (kDa) major outer membrane protein (MOMP) of C. jejuni UA580. The MOMP was prepared from C. jejuni UA580 by extraction with the detergent Triton X-100~ as described previously (Huyer, M., Parr, T. R., Jr., Hancock, R. E. and Page, W. J. 1986. FEMS
Microbiol. Lett. 37:247-250). The anti-MOMP antibody was adsorbed three times against E. coli JM83 and was tested for its activity against a range of Campylobacter species (Taylor, D. E. and Chang, N. 1987. Molec. Cell. Probes 1:261-274)-(4) CONSTRUCTION OF THE C. JEJUNI ~GT11 LIBRARYC. jejuni DNA from strain UA580 was partially digested with the restriction endonuclease Sau3A1. The resulting DNA fragments were 13l~2~8 "filled-in" using DNA polymerase I holoenzyme supplied by Boehringer-Mannheim Ltd., Montreal, Quebec to produce blunt-ended DNA fragments. EcoR1 linkers (supplied by Alberta Regional DNA
Synthesis Laboratory, Calgary, Alberta) were added to the fragments. The DNA fragments were ligated to dephosphorylated, EcoR1 cleaved, arms of the cloning vector ~gtll. The DNA was packaged into bacteriophage lambda heads using the methods described by the manufacturer of the packaging extracts (Promega Biotech. Inc., California).

(5) SCREENING OF C. JEJUNI LIBRARY
The ~gtll recombinant library was screened using the anti-MOMP
antiserum by a previously published procedure (Young, R. A., Bloom, B. P., Grosskinsky, C. M., Ivanyi, J., Thomas, D. and Davis, R. W.
1985. Proc. Nat. Acad. Sci. U.S.A. 82:2583-2587). An alkaline phosphatase conjugated goat anti-rabbit IgG was used to detect positive plaques using 5-bromo-4-chloro-3-indoyl phosphate (BCIP)/nitroblue tetrazolium (NBT) as the colour substrate.

(6) RESTRICTION ENDONUCLEASE DIGESTION AND ISOLATION OF DESIRED
FRAGMENTS
Restriction endonucleases were purchased from Boehringer Mannheim Canada or Bethesda Research Laboratories Ltd. Digestions were carried out according to supplier~s instructions. Separation of fragments was achieved by agarose gel electrophoresis in TBE buffer (9OmM TRIS, 0.89 M borate, 2mM EDTA). Isolation of the desired fragment was achieved using low melting point agarose (Biorad Laboratories Inc.) and the resulting DNA was then purified by phenol and chloroform extractions ~Maniatis, T., Fritsch, E. F. and Sambrook, J. 1982. Molecular Cloning: a laboratory manual. Cold Spring Harbor Laboratory, N. Y.).

(7) PLASMID DNA ISOLATION
Plasmid DNA was isolated by ethidium bromide-cesium chloride density gradient centrifugation using standard DNA methodology ~`

131~2~

(Birnboim, H. C. and Doly, J. 1979. Nucleic Acids Res.
7:1513-1523).

(8) ISOLATION AND CLONING OF MOMP DNA
DNA from recombinant lambda phage identified as positive using the anti~MOMP serum was isolated and purified using Lambda Sorb~ phage absorbent (Promega Biotech Inc. CA.), as described by the manufacturer. The cloned DNA fragment were isolated from EcoRI
digests of the recombinant phage DNA using low melting point agarose. The purified fragments were cloned into the EcoRI site of the cloning vector pUC13.

(9) PREPARATION OF MOMP PROBE DNA
MOMP DNA was isolated for use as probe from EcoRI digests of pUC13 subclones using low melting point agarose. DNA was labelled with both radioactive and nonradioactive labelling methods. The radioactive label used was [~-32P]-dATP and the probe DNA was labelled by nick translation. The nonradioactive probe DNA was labelled using the nonradioactive DNA labelling kit obtained from Boehringer Mannheim Ltd. This kit labels DNA using digoxigenin-labelled dUTP, which was incorporated into the probe and was detected using alkaline phosphatase conjugated anti-digoxigenin antiserum and 5-bromo-4-chloro-3-indolyl phosphate (BCIP)/nitroblue tetrazolium (NBT) as the color substrate.

(10) HYBRIDIZATIONS OF PROBES TO CAMPYLOBACTER DNA
Total DNA was extracted from various Campyl obacter sp. using a method described previously (Ezaki, T., Takeuchi, N., Liu, S., Kai, A., Yamamoto, H. and Yabuuchi, E. 1988. Microbiol. Immunol.
32:141-150). The DNA samples were then digested using Sau3AI, fractionated by agarose gel electrophoresis, and transferred to nitrocellulose membranes as described previously (Southern, E. M.
1975. J. Mol. Biol. 93:502-517). Hybridizations were carried out in the presence of 50% formamide at 37C or 42C using radioactively labelled probe DNA. The membranes were washed twice in 2xSSC and 0.1% SDS at 65C for 15 min. The membranes were ~' exposed to X-ray film (Kodak~ XAR 5) at -70C with an intensifying screen (DuPont Cronex~ Lightning Plus).

(11) DIRECT PROBING OF STOOL SAMPLES
A total of 140 stool samples were obtained from the University of Alberta Hospital and the Enteric Section of the Provincial Laboratory of Northern Alberta. Seventy of the stool samples had previously been reported to be culture positive for Campylobacter species using the blood-free charcoal based medium of Hutchinson and Bolton (10) which contains 35 ~g/ml cefaperazone. All except one Campylobacte~ spp. (69 samples) were identified as C. jejuni by hippurate hydrolysis (9) the other was identified as C. coli.
Seventy stool samples which were culture negative for Campylobacter spp. were also tested. The negative stool samples were stored at 5C prior to testing. Some culture positive stool samples were tested after storage at 5C, others were frozen at -20C for several weeks. Freezing did not appear to affect the test results.
Samples were tested in batches of 10. Pure cultures of C. jejuni and C. coli were tested concurrently with each batch of stool specimens as positive controls as well as a fecal specimen containing Salmonella spp. as a negative control. Approximately 200 ~l of each stool sample was suspended in 1.0 ml of Mueller-Hinton broth and vortexed until suspensions had an even consistency. Following this, 0.5 ml of the sample was transferred to 2.0 ml of Mueller-Hinton broth and mixed thoroughly. This suspension was passed through a Whatman~ #3 filter and a nitrocellulose membrane filter contained in ~ Millipore Swinex~ 25 filter unit under vacuum. In some cases pressure was applied using a 10 ml syringe to force the samples through the filter units. The Whatman #3 filter effectively removed some of the particulate matter in the stool samples while allowing any bacteria in the suspension to pass onto the nitrocellulose filter. The filter units were then filled with a lysing solution of 0.5M NaOH and 1.5M
NaCl and the filters were soaked in this solution for 10 minutes.
The lysing solution was removed by vacuum. The nitrocellulose filters were then removed from the filter units and transferred to ~' 131~2~8 a neutralizing solution (0.5 M Tris-HCl pH 7.5, 1.5 M NaCl) for 10 minutes. This was followed by a 10 minute wash in 2 X SSC. At this point the filters could be dried for screening later or screened immediately. The filters were sealed in plastic bags with approximately 1.0 ml/filter hybridization solution (50~ formamide, 5 X SSC, 0.02% SDS, 0.1% Sarkosine, 5% blocking reagent) as described in the protocol given by the manufacturer of the non-radioactive labelling kit (Boehringer Manneheim Ltd.) and were pre-hybridized in this solution for a minimum of 1 hr at 37C.
Fresh hybridization solution was added containing freshly heat denatured non-radioactively labelled probe (approximately 0.1 ~g of probe DNA/filter). The filters were incubated at 37C for 1 to 2 hrs. The filters were then rinsed briefly in 2 x SSC and placed in 20 mls of 0.5% blocking reagent solution (150mM NaCl, 100mM
Tris-HCl pH 7.5, 0.5% w/v Boehringer Mannheim blocking reagent) at room temperature for 30 minutes. The filters were then incubated in a 1:5000 dilution of alkaline phosphatase conjugated anti-digoxigenin antiserum in TBS (150mM NaCl, 100mM Tris-HCl pH
7.5) for 1 hr at room temperature. The filters were washed three times for 5 mins in TBS at room temperature and placed in the color developing solution. Color was allowed to develop for at least one hour or to a maximum of 24 hours in reduced light. Filters were air dried and stored in reduced light. Most filters were examined after 18 hours. A strong positive hybridization signal was denoted by a deep purple color, a weak one by a pale mauve. Negative hybridization was denoted by a filter the color of the fecal material itself, which ranged from white, yellow, brown or even pink, if the stool specimen contained blood.

EXAMPLE 1 - IDENTIFICATION AND DIFFERENTIATION OF C. JEJUNI AND C.
CO~I USING MOMP DNA PROBES
From the ~gtll library prepared with DNA from C. jejuni UA580, positive plaques were detected with a polyclonal antiserum to a 46 kDa MOMP from C. jejuni UA580. Bacteriophage lambda was isolated from the plaques and propagated in E. coli Y1090. DNA fragments . ~ , .~, 1 3 1 ~ 2 4 8 12 from C. jejuni UA5800 were then cloned into pUC13 using the EcoRI
site within the polylinker to give recombinant plasmids pDtl719 and pDT1720 containing DNA fragments of 1845-bp and 1475-bp, respectively. The restriction endonuclease maps of the two fragments are shown in the Drawing. Although the restriction endonuclease maps of the two fragments indicate that the two fragments are not closely related, hybridization experiments with dot blots and Southern blots demonstrated some cross-hybridization under low to moderate stringency conditions (37C), whereas at 42C
(high stringency condition) very little cross-hybridization was observed.
Although E. coli containing the ~gtll vector into which the C.
jejuni DNA fragments had been ligated, expressed a polypeptide that was detected in immunoblots using anti-MOMP antibody, the pUC13 derivatives pDT1719 and pDT1720 did not specify any MOMP related polypeptides. This is expected since ~gtll is an expression vector whereas pUC13 is not.
Initial studies to determine probe specificity employed probes labelled by nick translation with 32P-dATP. The probe prepared from pDT1720 hybridized only with C. jejuni DNA under conditions of both high stringency (42C) and moderate stringency (37C). The probe prepared from pDT1719 hybridized with C. jejuni and C. coli DNA and weakly with DNA from two out Gf five strains of C. laridis when hybridizations were performed at 37C. Raising the temperature of hybridization to 42C to give conditions of higher stringency reduced the degree of hybridization of pDT1719 with C.
coli and eliminated hybridization with C. laridis. Neither of the probes hybridized with other Campylobacter species including C.
fetus subsp fetus, C. fetus subsp venerealis, C. hyointestinalis, C. sputorum, C. upsaliensis or C. pylori. Neither did they hybridize with other unrelated bacteria, including E. coli, Salmonella typhimurium, S. typhi, Klebsiella spp., Shigella sonnei, Bacil l us subtilis nor Staphylococcus aureus.
Southern blot analysis was used to detect probe sequences present in C. jejuni and C. coli DNA. DNA isolated from eight C.
jejuni strains, and four C. coli strains obtained from different ; ~ ^ ' i ~"

geographic locations. DNA isolated from these strains was digested with Sau3Al and hybridized with the two probe insert DNAs. The pDT1719 probe hybridized with two major Sau3A1 DNA fragments of 675-bp and 530-bp in all C. jejuni strains tested. In contrast in the C. coli Sau3A1 digest this DNA probe hybridized predominantly to the 530-bp fragment. Hybridizations carried out with the probe from pDT1720 with Sau3A1 digests of DNA from C. jejuni strains showed one strongly hybridizing band of 1475-bp in the majority of the strains. This fragment is the same size as the probe itself.
In two C. jejuni strains (UAl and UA560) another slightly smaller band (1400-bp) was also found to hybridize with the pDT1720 probe.
~herefore these probes may be useful to compare strain differences based on restriction fragment length polymorphisms.

EXAMPLE 2 - DETECTION OF C. JEJUNI AND C. COII IN STOO~ SPECIMENS
USING THE MOMP DNA PROBES
The detection limits of pDT1719 and pDT1720 insert DNA were determined using pure cultures of C. jejuni tUA580) and C. coli (UA33). The pDT1720 probe was tested with Gnly the C. jejuni culture whereas the pDT1719 probe was tested with both C. jejuni and C. coli cultures. Detection limits were tested with both radiolabelled and non-radiolabelled probes. Cultures were usually diluted and lysed in situ on nitrocellulose disks. The detection limits of the pDT1720 probe for C. jejuni was determined to be approximately 5 x 105 organisms with the 32P-labelled probe and 1-2 x 105 with the Boehringer Mannheim Ltd. non-radioactive probe.
Similar results were obtained using the pDT1719 probe with C.
jejuni. However, for C. coli the detection limit of the pDT1719 probe was approximately 8 x 107 organisms with the radiolabelled probe and 3 x 107 with the non-radiolabelled probe.
The two probes were used to detect C. jejuni and C. col i directly in stool specimens. The non-radioactive labelling method was chosen for this work, as non-radioactive reagents are more acceptable in clinical laboratories, whereas radioactive methods are more suited to research environments. A total of 140 stool specimens were tested with both probes. Seventy of the stools were culture positive for Campylobacter species ( 69 C. jejuni and 1 C
coli). With the pDT1719 probe 55/70 of the samples gave strong positive reactions and 7 gave weak positive reactions. A strong positive hybridization signal was denoted by a deep purple colour, a weak one by a pale mauve colour. The results correspond to a sensitivity of 89%. With the pDT1720 probe 58/70 of the samples gave a strong positive reaction and 6 give weak positive reaction.
The C. coli was among those positively identified with the pDT1719 probe but not with the pDT1720 probe. Therefore the pDT1720 probe demonstrated a sensitivity of 93% based on 69 culture positive C.
jejuni samples.
Of the seventy stool specimens which were culture negative for C. jejuni, 11 gave a false-positive reaction. The same negative stool samples were identified as positive no matter which probe was used. The approximately 15% rate of false positives could be explained by the presence of enzymes in the feces, either alkaline phosphatase or related esterases, which would act on the substrate directly or otherwise affect the dye and give a false positive reaction. Other non-radioactive labelling systems may give a lower proportion of false positives.

The two examples given demonstrate some of the uses of the C.
jejuni and C. coli gene probes based on major outer membrane protein encoding sequences. It is likely that certain changes and modifications may be practiced within the scope of the appended claims.
The embodiments of the invention in which the exclusive property or privilege is claimed are defined as follows:

Claims (28)

1. A DNA probe fragment of C. jejuni UA580 (insert fragment of pDT1720) of unique restriction sites shown in the drawing (Figure 1) which can be used to identify C. jejuni sequences.

2. A DNA probe fragment of C. jejuni UA580 (insert fragment of pDT1719) of unique restriction sites shown in the drawing (Figure 1) which can be used to identify C. coli and C. jejuni sequences.

3. DNA fragment of claim 1 transferred to and replicated in a gram-negative procaryotic microorganism.

4. DNA fragment of claim 2 transferred to and replicated in a gram-negative procaryotic microorganism.

5. Plasmid pDT1720 comprising the entire genome of pUC13 with the C. jejuni UA580 fragment having the restriction endonuclease pattern as shown in the drawing (Figure 1) cloned into the polylinker.

6. Plasmid pDT1719 comprising the entire genome of pUC13 with the C. jejuni UA580 fragment having the restriction endonuclease pattern as shown in the drawing (Figure 1) cloned into the polylinker.

7. A procaryotic microorganism transformed by the pDT1720 plasmid of claim 5.

8. A procaryotic microorganism transformed by the pDT1719 plasmid of claim 6.

9. E. coli JM103 (pDT1720), a microorganism according to claim 7.

10. E. coli JM103 (pDT1719), a microorganism according to claim 8.

11. Plasmid pDT1720 comprising the entire genome of pUC13 and having the restriction endonuclease pattern shown in Figure 1, and DNA encoding a major outer membrane protein (46 kilodalton) determinant.

12. Plasmid pDT1719 comprising the entire genome of pUC13 and having the restriction endonuclease pattern shown in Figure 1, and DNA encoding a major outer membrane protein (46 kilodalton) determinant.

13. A procaryotic microorganism transformed by the plasmid pDT1720 of Claim 11.

14. A procaryotic microorganism transformed by the plasmid pDT1719 of Claim 12.

15. A process for identification of C. jejuni from cultured microorganisms which comprises (a) Cutting p]asmid pDT1720 with restriction endonuclease EcoRI to obtain a 1475-bp DNA fragment; and (b) Hybridization at 37°C-42°C to C. jejuni DNA using a DNA
dot blot method.

16. A process for identification of C. jejuni from cultured microorganism which comprises (a) Cutting plasmid pDT1720 with restriction endonuclease EcoRI to obtain a 1475-bp DNA fragment and (b) Hybridized at 37°C-42°C to C. jejuni DNA using the Southern transfer hybridization method.

17 17. A process for identification of C. jejuni and C. coli from cultured microorganism which compromises (a) Cutting plasmid pDT1719 with restriction endonuclease EcoRI to obtain an 1845-bp DNA fragment and (b) Hybridization at 37°C or 42°C to C. coli or C. jejuni DNA using a DNA dot blot method.

18. A process for identification of C. jejuni and C. coli from cultural microorganisms which comprises (a) Cutting plasmid pDT1719 with restriction endonucleases EcoRI to obtain an 1845-bp DNA fragment and (b) Hybridization at 37°C or 42°C to C. coli or C. jejuni DNA using the Souther Transfer Hybridization method.

19. A process to compare different strains of C. jejuni and C. coli for epidemiological and evolutionary studies which comprises (a) Cutting plasmid pDT1720 with restriction endonuclease EcoRI to obtain a 1475-bp DNA fragment and (b) Hybridization to DNA from C. jejuni and C. coli strain to compare restriction fragment length polymorphism.

20. A process to compare different strains of C. jejuni and C. coli for epidemiological and evolutionary studies which comprises (a) Cutting plasmid pDT1719 with restriction endonuclease EcoRI to obtain 1845-bp DNA fragment and (b) Hybridization with DNA from C. jejuni and C. coli strain to compare restriction fragment length polymorphism.

21. A process to detect (pathogen) C. jejuni in clinical specimens using pDT1720 DNA fragment of claim 1 comprising:
(a) depositing said specimen on filter (b) treating said specimen to affix DNA of said pathogen present in said specimen to said filter in substantially single stranded form (c) contacting said fixed single stranded DNA with pDT1720 DNA fragment labelled by non-radioactive method (d) Hybridization at moderate to high stringency (37° to 42°C) (e) Detection of duplex formation on said filter by means of a colour reaction.

22. A process to detect (pathogens) C. jejuni and C. coli in stool specimens using pDT1719 DNA fragment of claim 2 comprising:
(a) depositing said specimen on filter (b) treating said specimen to affix DNA of said pathogens present in said specimen to said filter in substantially single stranded form (c) contacting said fixed single stranded DNA with pDT1719 DNA fragment labelled by non-radioactive method (d) hybridization at moderate to high stringency (37°C-42°C) (e) detection of duplex formation on said filter by means of a colour reaction.

23. A process according to claims 21 or 22 using probes described in claims 1 and 2 wherein said specimen is a fecal sample.

24. A process according to claims 21 or 22 using probes described in claims 1 and 2 wherein said specimen is a food sample.

25. A process according to claims 21 or 22 using probes described in claims 1 and 2 wherein said specimen is a milk sample.

26. A process according to claims 21 or 22 using probes described in claims 1 and 2 where said specimen is a water sample.

27. A process according to claims 21 or 22 using probes described in claims 1 and 2 where said specimen is a blood specimen.

28. A process according to claims 21 or 22 using probes described in claims 1 and 2 where said specimen is a serum sample.