Study On The Detection Of Pathogenic Bacteria In Chicken By Triplex PCR

The food security is a major public health problem in the world. In recent years,global food safety incidents are in high frequency and the food security attracts more andmore attention. Bacterial food poisoning is the main factor which causes the food-bornedisease.Strengthen the food inspection efforts is the most effective way to solve theproblem of food safety. At present, detection of food-borne pathogens mainly uses thetraditional isolation, culture and biochemical identification method in our country. It isincapable of detecting pathogens quickly and sensitively because of the complicatedoperation, time consuming and low sensitivity. With the advent of PCR，Individual PCRassays have been developed for detection and ideniifieation of the bacterial pathogens. Buta large number of individual PCR assays would be necessary if single Primer sets are usedin separate reactions on a large number of food samples，which can be a relatively costlyand time–consuming Process. Multiplex PCR has a broad prospect of application inpathogenic bacteria testing field and the simultaneous detection of several pathogens with amultiplex PCR approach which has the same basic principle as conventional PCR wouldsave cost and time.Triplex PCR is an amplification method that can amplify three target sequencesthrough adding to three pairs of specific primers. In this study, three pairs of specificprimers were designed according to the invA gene of Salmonella, the ECs3032sequencingof E.coli O157:H7and the ail gene of Yersinia enterocolitica, thus the multiplex PCR wasestablished to simultaneous diagnose three kinds of food-borne pathogenic bacteria.According to the influence factors of multiplex PCR, the orthogonal experimental designL16（44） table, were used to optimize multiple PCR amplification system of four factors(Taq DNA polymerase, Mg²⁺, dNTPs and primers) at four levels. Then annealingtemperature and the concentration of the buffer were optimized. As a result, the multiplexPCR reaction system was determined to be25μL:2.5μL10×PCR buffer,2.0μL Mg²⁺（50mmol/L）,2.0μL dNTPs（each2.5mmol/L）, emplate1.5μL，0.8μL reversse prime（reach），0.4μL Taq DNA polymerase （5U/μL）, ddH20to25μL. The cycle parameters：Pre-degenerated at94℃for5min, degenerated at94℃for1min, annealing at54.1℃for1min, extention at72℃for2min, run30cycles, the final extention at72℃for10min. The PCR Products were examined by2%agarose gel electrophoresis.It is showed that this method is of better specificity through the primer specificexperiment and the reaction specific experiment. Multiplex PCR products were confirmedby DNA sequencing. The results of sequencing were compared with the sequence of the target gene at GenBank and three homologies were99.15%,99.71%and97.61%respectively. Under the above optimum experimental condition, the sensitivity of thesimultaneous detection of the three bacterial pathogens with multiplex PCR were103CFU/mL. Extracting genomic DNA with kit extraction, the detection limit of artificiallycontaminated pork was103CFU/g for Salmonella,104CFU/g for Listeria monocytogenesand Yersinia enterocolitica. The sensitivity and detection limit of the multiplex PCR werebelow that of the simplex PCR. The enrichmented culture was incubated for9hours afterartificially contaminating chicken, and the detection limit of the multiplex PCR assay forchicken salmonella and e. coli O157: H7is103CFU/mL, enterocolitis and coulson’spopulation could reach102CFU/mL, the sensitivity and detection limit were slightly lowerthan single PCR detection.To test the actual samples, and compared with national standard method, proved thatthe method testing three kinds of foodborne pathogenic bacteria has higher specificity andsensitivity.