Contamination Of Clostridium Perfringens During Milk Production And Establishment And Application Of Multiplex Quantitative PCR Method For Gene Cpe/cpb2

Clostridium perfringens is a gram-positive anaerobic sporogene which is a zoonotic pathogen widely distributed in the natural environment,it can cause animal enterotoxemia,sudden death,necrotic enteritis,human gas gangrene and food poisoning as well as antibiotic-related diarrhea,seriously threaten the sustainable development of animal husbandry and human public health.Milk is an important animal-derived food in people’s daily life and to guarantee the consumers’health based on its safety and sanitation.In this study,samples of dairy cattle breeding and milking process were collected from three dairy farms in Shaanxi Province.C.perfringens were isolated and following serotyped by multiplex PCR,the contamination rate of bacterial and the propagation pattern was analyzed.All isolates were detected by using the established Taq Man real-time multiple fluorescent quantitative PCR for detecting toxin genes of C.perfringens atyp.cpb2,cons.cpb2 and cpe,and results as the following:1.The relevant samples involved in the milk production process including breeding and milking were collected from three large-scale dairy farms in Shaanxi Province,C.perfringens were isolated after enriched in the medium and gram staining microscopy then streaked onto BA plates,then following serotyped by multiplex PCR.Results showed 84 C.perfringens were isolated from 465 samples,the isolation rate is 18.1%.All 83 isolates identified as type A except 1 is type D.2.According to the atyp.cpb2,cons.cpb2 and cpe gene sequences of C.perfringens published in Gen Bank,the synthetic primers and probes were designed and synthesized,respectively.The Taq Man multiple fluorescence quantitative PCR method was established by optimizing the reaction conditions.The minimum detection of this method to recombinant plasmid atyp.cpb2-p GEM-T easy was 1×10~2 copy/μL,cons.cpb2-p GEM-T easy and cpe–p MD19-T were 1×10~3 copy/μL.The minimum detection to bacterial culture was 1.51-2.77×10~3 CFU/m L.Salmonella,Staphylococcus aureus,Streptococcus,E.coli,C.septicum failed to amplicate the corresponding curve.The CV values of intragroup and intergroup repeatability tests were all less than 5%.All 84 isolates were detected by using the established method,among this,25 isolates carried atyp.cpb2 gene with 29.8%positive rate,none of cons.cpb2 and cpe genes were detected.In conclusion,the prevalent serotype of C.perfringens in the milk production is type A,air and feces may be the main source of C.perfringens contamination,however,it may not cause human food-borne poisoning from milk.This study initially understands the contamination pattern of C.perfringens and further provides a scientific basis for the prevention and control of C.perfringens in the milk production.