Isolation And Identification Of The Campylobacter Jejuni And Rapid Detection By Mutiplex PCR In Animal Food

Campylobacter jejuni,as an important intestinal pathogen,have upward trend in infection rates.Livestock polluted by C.jejuni,such as chickens and pigs,can lead to pollution of food and water.So detection and control of pollution of C.jejuni in food raw material and food is very important.There is a series of recommended standards for biochemical detection of C.jejuni. But it has risk for undetection to apply arbitrary method.A combined method, according to GB/T4789.9-2003,SN0175-92,ISO10272-1:2006,FDA/BAM:2001, USDA / FSIS:1998,was effective used for isolation and identification of Campylobacter jejuni in animal food.According this method,36 strains of suspected C.jejuni was isolated firstly from chicken meat samples in yaan.21 strains of C.jejuni were identified by biochemical and PCR method of 16S rDNA.The 16S rDNA sequence alignment of C.jejuni ATCC33560 and randomly selected isolated strain SCCJ-08-5 show that the result of detection is correct.The rate of pollution of chicken from yaan is 11.5%(21/183),applying combined biochemical detection.21 strains of C.jejuni tested to 22 antibiotics by Minimal inhibitory concentrations(MICs) for antibiotic susceptibility.The result indicated that 14.5%C.jejuni demonstrated resistance to Ciprofloxacin,all of C.jejuni were sensitive to Ciprofloxacin, Tetracycline,Erythromycin,get the MIC₅₀ and MIC₉₀ of these strains to 22 antibiotics.In this study,we have designed two pairs specific primers according to the hipO and 16S rRNA,and established a Multiplex polymerase chain reaction(M-PCR) to sensitively detect and identify C.jejuni in food.When the assay was applied,two specific fragments(699bp and 366bp) were amplified for C.jejuni,and not amplified for all other bacteria(11 species tested).The 16S rDNA and hipO sequence alignment of C.jejuni ATCC33560 and randomly selected isolated strain SCCJ-08-5 show that the result of detection is correct.The M-PCR method is simpler,rapider,and more sensitive than biological detection method,and can be provided as a new method to detect/monitor C.jejuni in food for the grass-roots health inspection and quarantine institutions.Detection by the M-PCR can be finished in 27h,and its sensitivity is 2.4-16 CFU/mL.Investigate the pollution of C.jejuni in animal food from market in yaan with M-PCR method and combined biological detection method at the same time. As a results of M-PCR method,38.0%(19/50) of chicken meat samples,28.3% (15/53)of pork meat samples,17.1%(6/35) of beef meat samples and 8.6%(4/46) of milk samples were found to be positive for C.jejuni;however,16%(8/50) of chicken meat samples,9.4%(5/53)of pork meat samples,5.7%(2/35)of beef meat samples and 2.2%(1/46) of milk samples were found to be positive for C.jejuni with culture method.These results indicated that M-PCR assay provides a specific, sensitive,and rapid method for quantitative detection of C.jejuni.Moreover,it is concluded that retail chinken meat,pork meat,beef meat and raw milk were commonly contaminated with C.jejuni in Sichuan yaan market,and could serve as a potential risk for the consumers in eastern China especially if proper hygienic and cooking considerations are amiss.