The Distribution Regularity And Genetic Diversity Of Campylobacter Jejuni In Food

Campylobacter jejuni was the normal living bacteria in the intestinal tract of poultry, domestic animals and some birds. Humans would be infected by eating contaminated foods. This paper investigated the prevalence and level of C. jejuni contamination in national foods. Then we constructed the fingerprint by ERIC-PCR (Enterobacteia Repetitive Intergenic Consensus-Polymerase Chain Reaction) typing, with an attempt to find out its correlation, which serves as a reference in C. jejuni contamination prevention and control.For the moment, the detection of C jejuni was always confined to qualitative research of single city or districts, there was no total C. jejuni contamination level study of several provinces. In order to know the state of presence of Campylobacter jejuni of foods in representative cities in China, including Shenzhen, Shaoguang, Shantou, Zhanjiang, Heyuan, Hainan, Fujian, Guangxi, Lanzhou, Harbin, Xi’an, Taiyuan, Bejing, Jinan, Hefei, Nanchang, Chengdu, Wuhan, Kunming and Shanghai. According to the national standard GB4789.9-2008with a little modification, to detect the contamination of C.jejuni from seven types of food samples including vegetables, meat product, cooked food, seafood, frozen food, dairy product and edible fungi.19C. jejuni-positive meat product samples in1161food samples were detected, the total positive rate wasl.64%, the positive rate of meat products was6.75%, the MPN was850/100g, and19strains were isolated. Fresh poultry meat was the main food of C. jejuni contamination in the16provinces of China, more control behaviors must be taken to avoid C. jejuni contamination in fresh poultry meat.Many research showed that the resolution of ERIC-PCR was not lower than the other typing methods, including RAPD, RFLP, PFGE, MLST, and it had the advantages of good stability, simplicity of operator and low-cost. Exploration of conditions was in a wide range so that we could get a preliminary reaction systems. Then the three factors(except the primers), template concentration and annealing temperature were further optimized in a small range so that we could got the final optimized systems. In the end, we used the optimized ERIC-PCR method to type the24C. jejuni isolates. According to the results of biochemical reactions to biochemical type the strains, and we also compared the ERIC-PCR molecular typing method with biochemical typing method. The optimal concentrations of Mg2+, dNTPs, TaqDNA polymerase, template were3.0mmol/L,2.5mmol/L and5U/25μL,24～120ng/25μL, respectively, and the annealing temperature was35℃.24C. jejuni isolates were classified to19types belong to4clusters. The resolution ratio of ERIC-PCR method was0.920and showed good resolution. The24C. jejuni isolates were classified to19biochemical types belonging to4clusters by biochemical typing method, which showed the diversity of genes in the strains. The reaction system and procedures of ERIC-PCR subtyping method have good stability and high resolution, and shows the genetic diversity of the strains better than biochemical typing method, which are applicable for the tracking and typing of C. jejuni strains.This paper investigated the C. jejuni contamination in national food, then established the ERIC-PCR typing method of C. jejuni and genotyped the isolates. Now, we preliminary know the distribution regularity of C. jejuni contamination in food, lay a good foundation on the establishment of fingerprinting database tracing to the source of C. jejuni.On the basis of previous investigation,12pathogenic genes were detected by PCR methods and ERIC-PCR fingerprints were constructed of C. jejuni isolates respectively. In pathogenic genes PCR results was showed that the positive rates of cadF, racR, flaA, cdtA, cdtB, cdtC, dnaJ, ciaB, imaA in all C. jejuni isolates were more than50%respectively, while pldA and wlaN were10%. Interestingly, virB11gene was not found in all isolates. Total of19C. jejuni isolates could be divided to10genotypes belonging to3clusters by ERIC-PCR fingerprints. The majority (15strains) were in cluster A with61%～100%similarity. The cluster B and cluster C had2strains respectively, the similarity was between67%and80%showed that the genetic relationship of each strain was farther.