Establishment And Application Of A Multlplex-PCR Assay For Pathogenicity Island Of Pathogenic E.coli And Salmonella

Two important pathogenic bacterias are pathogenic Escherichia coli （pathogenicE.coli）and Salmonella which cause food-borne illness. The result of epidemiology showsthat the virulence of pathogenic E.coli and Salmonella having relevance with thepathogenicity island. The discovery and study of the pathogenicity island have significantinfluence on profound understanding of the occurrence and development of bacterialdisease and how to prevent and control.In this study,Pairs of primers were designed with the pathogenity island which inpathogenic E.coli and Sallmonella. and specific amplify the pathogenity island for eachother by PCR to find the best amplification conditions.Through the comparison to find thepais of primers to establish the multiplex-PCR. Method was used to detect E.coli andsalmonella which isolate from the farmer and slaughterhouse of Hefei.In order to supplytheoretic for prevent and control the colibacillosis and solmonellosis.The results as followed:1. PCR for pathogenicity island on pathogenic E.coli and SalmonellaIn this study, Pathogenicity island is regarded as the point,According to theGenBank,three pairs of primers were designed for each other that irp1,irp2,fyuA ofpathogenic E.coli and mgtC,sopB,sseL of Salmonella，extract the DNA from the E.coli（CVCC1565） and Salmonella（ATCC9150），and then did PCR to optimized amplicationconditions.The result shows that the primers irp1,irp2,fyuA can amplified the799bp,414bp,948bp specific fragments of E.coli（CVCC1565） and the primersmgtC,sopB,sseL can amplified the500bp,269bp,1000bp specific fragments ofSalmonella（ATCC9150）. The optimization system of PCR include as follows:0.5μL primerF（100um），0.5μL primer R（100um），0.5μL TaqDNA polymerase（5U），2.5μL10×PCRbuffer（Mg2+），2.5μL DNApellet,1.0μL dNTP（10mM），add the sterilize water to25μL.2. Multiplex-PCR for pathogenicity island on pathogenic E.coli and SalmonellaAccording to the result of optimization system of PCR，specific laboratory tests wasset up for each primers.The result shows that primers irp1,irp2,fyuA can only amplified thespecific fragments from DNA samples of E.coli（CVCC1565） and the primersmgtC,sopB,sseL can only amplified the specific fragments from DNA samples ofSalmonella（ATCC9150）. Two groups of primers were designed in order to find the bestprimer combination for multiplex-PCR:1.fyuA of pathogenic E.coli and mgtC ofSalmonella;2.fyuA of pathogenic E.coli and sseL of Salmonella,and choose the pairs1:fyuAof pathogenic E.coli and mgtC of Salmonella to establish the multiplex-PCR for pathogenicity island on pathogenic E.coli and Salmonella. The sensitivity test showed thatDNA used for the mμLtiplex-PCR might be as low as2.2×10¹cfu·mL^-1forE.coli（CVCC1565） and2.0×10¹cfu·mL^-1for Salmonella（ATCC9150）,respectively. Theresults indicated that this multiplex-PCR assay was rapid,sensitive and specific.3.The application of the Multiplex-PCR for pathogenicity island on E.coli andSalmonella in Animals.In this study,The isolation,culture and biochemical identification of the24samples whichcollected from the farmer and slaughterhouse of Hefei,Anhui Province.Seleted24stains ofeach E.coli and Salmonella,And use the multiplex-PCR to detecte it.The result shows: inthe isolated strains of the E.coli has20strains contain the gene of fyuA that the positiverate is83.3%,and the Salmonella has23strains contain gene of mgtC which the positiverate is95.8%. The results indicated that this multiplex-PCR assay is useful for rapiddiagnosis and epidemiology survey for E.coli and Salmonella in Animals.The study shows that the Multiplex-PCR for pathogenicity island of pathogenic E.coliand Salmonella is rapid,sensitive and specific for simultaneous diagnosis for E.coli andSalmonella in Animals. And it can provide theoretical basis for prevent and control theColibacilosis and the Salmonellosis of zoonosis.