Establishment Of Rapid Detection Method Of Food-borne Pathogens And Development And Application Of The Detection Kit

Foodborne pathogens are the main factors that endanger food security and human health.The problem of food safety is becoming more awared and more serious in many countries. It’sassociated closely with the national development and people’s health and happiness. With theimprovement of people’s life level, more and more attention was paid to food safety. Foodsafety problems has increasingly become a focus of social concern. Microbial food poisoningis in first place among the food borne diseases prevalent in our country. Thus, foodbornepathogens detection is an important aspect in food hygiene and safety testing. Commonfoodborne pathogens, including Salmonella, pathogenic E.coli, Campylobacter, Vibrioparahaemolyticus, Staphylococcus aureus, Bacillus cereus, Clostridium perfringens, Proteusgenus, Enterobacter sakazakii, Listeria monocytogenes, Yersinia enterocolitica, Shigella. Faceof increasing multiple mixed pollution. The current detection method appears to lag.Therefore the establishment of efficient, rapid and accurate detection of foodborne pathogenicmicroorganisms methods become more and more urgent.Multiplex PCR has the same basic principle with conventional PCR, a number of primersare added to same PCR reaction system, achieve more than one target genes amplification inthe same system at the same time. It overcomes the drawback that conventional PCR reactioncan only amplify one target gene. Multiplex PCR provides a wider space for development, soas to achieve the fast detection of a variety of foodborne pathogens at the same time. Savingcost and time.In this study, six kinds of food-borne pathogen Campylobacter jejuni, Vibrioparahaemolyticus, Yersinia enterocolitica, Enterobacter sakazakii and Clostridiumperfringens, Proteus bacillus to establish a rapid detection method for multiplex PCR.According to Characteristic genes sequences of the hipO gene of Campylobacter jejuni, thetlh gene of Vibrio parahaemolyticus, the Ail gene of Yersinia enterocolitica, the16SrRNA gene of Enterobacter sakazakii, the cpa gene of Clostridium perfringens and the tuf gene ofProteus mirabilis to design six special primers. The target gene fragment amplified by PCRrespectively was1028bp,450bp,274bp,282bp,120bp,541bp. The two triplex PCR methodswere developed for the simultaneous detection of the six pathogens from meat.In the PCR process, DNA concentration, Mg2+concentration, template volume,annealing temperature and PCR circles are optimized to determine the optimal PCR. Thereaction mixture consisted of50uL,10×PCR buffer5uL, Mg2+(25mM)3μL, mixture ofdNTPs(25mM)4μL, Taq enzyme（5U）1μL,15pmol hipO,5pmol tlh and7.5pmol Ail;5pmol16SrRNA,15pmol cpa and5pmol tuf, ddH2O to50μL. The reaction was run under thefollowing conditions: Cool start; DNA pre-denaturation at94℃for5min, DNA denaturationat94℃for30s, primer annealing at57℃for40s, and DNA extention at72℃for70s, run35cycles; the final extention was performed at72℃for15min.The PCR products were examined by2%agarose gel electrophoresis. The MultiplexPCR products of plastic recycling to connect to the pMD18-T vector and transformed intoE.coli DH5α, positive clones were confirmed by DNA sequencing. The result of sequencingcompared with the target gene sequence at Genebank showed that PCR amplified productswere certified.The sensitivity of simultaneous detection of the six kinds of food borne pathogens withm-PCR, using artificially contaminated meat and dairy products in the trial. Among Vibrioparahaemolyticus, Enterobacter sakazakii and Clostridium perfringens, the sensitivity ofmultiplex PCR compared with the single PCR, low a gradient; While the other three foodborne pathogens in the same. The detection limits were3.0×101cfu/ml for Campylobacterjejuni,6.2×101cfu/ml for Vibrio parahaemolyticus and1.5×101cfu/ml for Yersiniaenterocolitica;1.9×101cfu/ml for Enterobacter sakazakii,5.8×101cfu/ml for Clostridiumperfringens and2.4×101cfu/ml for Proteus mirabilis.In our study, we developed the two triplex PCR methods test the actual sample, theprevalence of Tai’an area of food borne pathogens. In various samples collected from chickenabattoirs, processing plants and retail vendors in Tai’an, Shandong Province of China. A totalof409samples including chicken feces and feathers, chicken washes, chicken carcasses fromprocessing plants, fresh raw chickens, and frozen chickens were collected.392samples were collected from Tai’an a dairy farm and supermarkets including fresh milk and powdered milkand used for the validation of the triplex PCR method. Campylobacter jejuni was detected in20.8%of the samples, whereas Vibrio parahaemolyticus, Yersinia enterocolitica, Enterobactersakazakii, Clostridium perfringens, Proteus mirabilis were identified in1.5%,13.2%,0.5%,6.0%,6.0%respectively. In this study, real detection was made. The result indicates: thesensitivity of the m-PCR assay is100%.By the experimental results showed that: The results of the experiments demonstratedthat the multiplex PCR assay was rapid, simple, sensitive and specific. The whole test can becompleted in5h. The detection kit establish important foundation for simultaneous detectionfor these six pathogens in polluted sample. It can be used for food and raw materials’bio-safety monitoring and also can be used in clinical diagnosis.