Establishment And Application Of Nucleic Acid-based Detection Methods For Campylobacter Jejuni

Campylobacter jejuni (C. jejuni) is recognized as a leading human food-borne pathogen. Traditional biochemical identification for C. jejuni is not reliable due to special growth requirements and the possibility that this bacterium can enter a viable but nonculturable (VNC) state. Nucleic acid -based tests have emerged as a useful alternative to traditional testing. The rapid and sensitive detection of C. jejuni is necessary for the maintenance of a safe food/water supply.In the present study, we have designed a pair primer (VS-F and VS-R) according to the species-specific sequence of VS1 and established a polymerase chain reaction (PCR) to sensitively detect and identify C. jejuni in food and water. In addition, two real-time PCR methods to quantitatively detect C. jejuni in food and water using a LightCycler instrument were developed. One is to quantitatively detect C. jejuni using the fluorescent double-stranded DNA binding dye SYBR Green I; the other is to quantitatively detect C. jejunii using a TaqMan probe and the 5'-3' exonuclease activity of the Taq DNA polymerase. The TaqMan probe was designed according to nucleotide sequence between two primers and the principle of fluorescene resonance energy transfer (FRET). When these assay were applied, the assay was positive for all of the isolates of C. jejuni tested (11 isolates, including type strain ATCC33560) and negative for all other Campylobacter spp (3 isolates) and several other bacteria (5species tested). The detection limits of three PCR assays were 8 CFU/ml, 5CFU/ml and 5CFU/ml respectively. Moreover, the two real-tune PCR methods can be completed within 60 min. A set of 300 frozen chicken meat samples, 300 milk samples and 300 water samples were screened for the presence of C. jejuni by culture, PCR, and real-time PCR methods. As a result of PCR method, 30.6% (92/300) of chicken meat samples, 27.3% (82/300) of milk samples and 13.6% (41/300) of water samples are found to be positive for C.jejuni. The apparently high number of C. jejuni was detected by the real-time PCR method in the three substrates examined. A part'of C. jejuni probably detected by real-time PCR were either not viable or VNC cells. These results indicated that the real-time PCR assay provides a specific, sensitive, and rapid method for quantitative detection of C. jejuni. Moreover, it is concluded that retail chicken meat, rawmilk, and environmental water were commonly contaminated with C. jejuni, and could serve as a potential risk for the consumers in eastern China especially if proper hygienic and cooking considerations are amiss.