A real-time PCR assay was developed for the quantitative detection of Campylobacter jejuni in foods after enrichment culture. The specificity of the assay for C. jejuni was demonstrated with a diverse range of Campylobacter species, related organisms, and unrelated genera. The assay had a linear range of quantification over six orders of magnitude, and the limit of detection was approximately 12 genome equivalents. The assay was used to detect C. jejuni in both naturally and artificially contaminated food samples. Ninety-seven foods, including raw poultry meat, offal, raw shellfish, and milk samples, were enriched in blood-free Campylobacter enrichment broth at 37 degrees C for 24 h, followed by 42 degrees C for 24 h. Enrichment cultures were subcultured to Campylobacter charcoal-cefoperazone-deoxycholate blood-free selective agar, and presumptive Campylobacter isolates were identified with phenotypic methods. DNA was extracted from enrichment cultures with a rapid lysis method and used as the template in the real-time PCR assay. A total of 66 samples were positive for C. jejuni by either method, with 57 samples positive for C. jejuni by subculture to selective agar medium and 63 samples positive in the real-time PCR assay. The results of both methods were concordant for 84 of the samples. The total time taken for detection from enrichment broth samples was approximately 3 h for the real-time PCR assay, with the results being available immediately at the end of PCR cycling, compared to 48 h for subculture to selective agar. This assay significantly reduces the total time taken for the detection of C. jejuni in foods and is an important model for other food-borne pathogens.

A polymerase chain reaction (PCR) assay based on a solution hybridization format with colorimetric end-point detection (PCR ELISA) was investigated for the specific detection of Campylobacter jejuni and Campylobacter coli in food samples following enrichment culture. One hundred fifteen samples of raw meat and offal (poultry, porcine, ovine, and bovine), raw shellfish, and artificially contaminated milk were enriched in blood-free Campylobacter Enrichment Broth for 48 h. Enrichment cultures were subcultured to Campylobacter blood-free selective agar plates, and presumptive isolates were identified by phenotypic methods. DNA was extracted from 1-ml aliquots of the enrichment cultures using a rapid extraction method, and the DNA was used as the template in a PCR ELISA. A comparison of the PCR ELISA with the enrichment culture and subculture to selective agar method showed that the results of 112 of the 115 samples tested were in agreement by both methods. Seventy-one of the various food samples were positive in the PCR ELISA, and 70 samples were positive by culture. The PCR ELISA had a sensitivity of 99% and a specificity of 96%, with a positive predictive value of 97% and a negative predictive value of 98%. The PCR ELISA is a rapid, sensitive, and specific method for the detection of C. jejuni and C. coli in foods following enrichment culture and significantly reduces the time required for their detection.

ABSTRACT A PCR enzyme-linked immunosorbent assay (ELISA) assay was applied to the detection of Campylobacter jejuni and Campylobacter coli in environmental water samples after enrichment culture. Bacterial cells were concentrated from 69 environmental water samples by using filtration, and the filtrates were cultured in Campylobacter blood-free broth. After enrichment culture, DNA was extracted from the samples by using a rapid-boiling method, and the DNA extracts were used as a template in a PCR ELISA assay. A total of 51 samples were positive by either PCR ELISA or culture; of these, 43 were found to be positive by PCR ELISA and 43 were found to be positive by culture. Overall, including positive and negative results, 59 samples were concordant in both methods. Several samples were positive in the PCR ELISA assay but were culture negative; therefore, this assay may be able to detect sublethally damaged or viable nonculturable forms of campylobacters. The method is rapid and sensitive, and it significantly reduces the time needed for the detection of these important pathogens by 2 to 3 days.

A polymerase chain reaction (PCR) assay was developed based on a solution-hybridization colorimetric end-point detection format (PCR ELISA) for the identification of Campylobacter jejuni and Campylobacter coli. PCR primers were designed to target a gene sequence with species-specific motifs. Five biotin-labelled probes targeted to the species-specific motifs were investigated for the detection of digoxygenin-labelled PCR products from C. jejuni and C. coli using the PCR ELISA format. Two probes were identified, one which reacts with both the C. jejuni and C. coli target sequences (probe CC2) and one probe which reacts with the C. jejuni target sequence only (probe CJ2). The specificity of the assay with the CJ2 and CC2 probes was investigated with a range of Campylobacter spp., Arcobacter spp., Helicobacter spp. and a range of unrelated organisms. The PCR ELISA assay and probes were demonstrated to be specific for C. jejuni and C. coli. The sensitivity of the PCR ELISA assay was demonstrated to be 10-100-fold more sensitive than a gel-based PCR method using the same primers. This PCR ELISA assay is sensitive, specific and significantly reduces the time needed for the identification of C. jejuni and C. coli and has the potential to facilitate early detection of these important gastro-intestinal pathogens.

Meningitis caused by Campylobacter jejuni is rare, we describe a case following neurosurgery for intra-cranial haematoma in a chronic alcoholic patient. Conventional culture of CSF and blood was supplemented by polymerase chain reaction (PCR) detection of Campylobacter jejuni.

Taxonomic classification of bacteriophages specific for Campylobacter jejuni and C. coli has not been reported previously. A set of 16 virulent phages, distinguishable by their lytic spectra, has been used extensively for epidemiological typing of C. jejuni and C. coli at Preston Public Health Laboratory. These phages were investigated by electron microscopy, pulsed-field gel electrophoresis and restriction endonuclease analysis. All phages had icosahedral heads and long contractile tails. Accordingly, they were classified as members of the Myoviridae family. These phages could be subdivided into three groups according to genome size and head diameter: group I, two phages with head diameters of 140.6 and 143.8 nm and genome sizes of 320 kb; group II, five phages with average head diameters of 99 nm and average genome sizes of 184 kb; and group III, nine phages with average head sizes of 100 nm and average genome sizes of 138 kb. Phages NCTC12676 and NCTC12677 of group I had unusually large genomes of c. 320 kb which are two of the largest phage genomes to be described. Restriction endonuclease analysis demonstrated that DNA from the 16 phages was refractory to digestion by a number of restriction enzymes.

A novel method was developed for the detection of thermophilic enteropathogenic campylobacters based on the detection of mRNA using the reverse transcriptase polymerase chain reaction (RT-PCR). The RNA extraction method, DNase treatment and RT-PCR assay were shown to be specific for mRNA. The assay is specific for the thermophilic campylobactersCampylobacter jejuni,Campylobacter coliandCampylobacter upsaliensisand restriction fragment length polymorphism (RFLP) analysis of the 256 bp amplified product with the restriction endonucleasesAluI,DdeI andDraI revealed distinct species specific patterns. The assay was applied to the detection ofC. jejunicells killed by heating at 72°C for 5 min and mRNA was detected by RT-PCR immediately after heat killing but became undetectable within 4 h when the cells were held at 37°C. The assay therefore can differentiate between viable and dead cells ofC. jejuni.

Three commercial gas-generating systems — CampyGen (Oxoid, UK), Oxoid BR56 (Oxoid, UK), and CampyPak Plus (Becton Dickinson, USA)—and the evacuation replacement technique were compared for the recovery ofCampylobacter spp. from 500 human faecal samples collected from patients with gastroenteritis. Four hundred fifty faecal samples were tested upon receipt in the laboratory. Fifty faecal samples that had been previously found to be positive forCampylobacter spp. were tested retrospectively; these had been stored at 4°C for more than 48 h. A total of 41 (9.1%) of the fresh faecal samples and 41 of 50 (82%) of the stored faecal samples were positive for thermophilic campylobacters. The CampyGen, the Oxoid BR56, the CampyPak Plus, and the evacuation replacement system detectedCampylobacter spp. in 40 (97.6%), 39 (95.1%), 41 (100%), and 41 (100%) of the positive fresh faecal samples and in 37 (90.2%), 40 (97.6%), 39 (95.1%), and 40 (97.6%) of the stored samples, respectively. There was no statistical difference in performance of any of the four gas systems used (p=0.98; chi-square test). Eighty-six percent of the isolates wereCampylobacter jejuni and 14% wereCampylobacter coll. Biotyping and phage typing of the isolates demonstrated that they were of a diverse range of subtypes. This study demonstrates that thermophilic campylobacters can be isolated from human diarrhoeal faecal samples using any of the four microaerobic-atmosphere-generating systems.