Establishment And Application Of A Multiplex PCR To Determine Virulence-associated Genes In Avian Pathogenic Escherichia.coli

With the rapid development of poultry industry,various bacterial diseases become serious,especially avian colibacillosis,which is one of the most important dieases restricting the growth of poultry industry.In order to establish a special method for colibacillosis diagnoses,a multiplex PCR has been established.This method could amplify five virulence-asscoiated genes in APEC,conclouding papC and tsh of adhesion genes,iucD and irp2 of iron-acqusition genes,and iss of increased serum survival protein.Then this method was used to detect five virulence genes in the APEC strains isolated from diseased poultry in Anhui province.The results were as follows.1.The Amplifications for papC,iucD,irp2,tsh and iss of E.coli standard strainsAccording to pathogenic-mechanism of APEC and the function of baterial adhesin,iron-acquisition and increased serum survival,five pairs of oligonucleotide primers were designed basing on the high conservative regions of the gene sequence in GeneBank,concluding the papC gene of type P fimbriae,iucD gene of aerobaetin, tsh gene of temperature-sensitive hemagglutinin,irp2 gene of iron-repressible protein, and iss gene of increased serum survival protein.To select the positive control of multiplex PCR,sole gene amplifications were performed by PCR respectively,using DNA templates of three E.coli standard strains of typical serogroups as O₁ (CVCC249),O₂(CVCC1565)and O₇₈(CVCC1555).The conditions of sole gene PCR amplification were as follows:2μL of each DNA extract of three kinds of serogroup APEC strains were added to reaction mixture(25μL)containing 0.5μL of each primer pairs(10pmol),1μL of the four deoxynucleoside triphosphates(10mM),2.5μL of PCR buffer(10x),3μL of25mM magnesium chloride,and 2 units of Taq DNA ploymerase. Amplifications were performed and the cycling conditions were the following: pre-denaturation,3 min at 94℃;denaturation,30 sec at 94℃;annealing,30 sec at 58℃;extension,3 min at 68℃;repeated for 30 cycles totally,then extension 10min at 72℃.The result showed that the positive rates of papC,iucD,irp2,tsh and iss genes were all 100%.Therefore,the DNA templates of these APEC strains all can be used for the positive control of multiplex PCR.One of the serogroups of APEC O₂(CVCC1565)was selected to amplified the five genes respectively.And the nucleotide acid sequences were detected,the homology were analyzed in NCBI BLAST 2 SEQUENCES,comparing with the sequences logging in GeneBank.The results showed that the sequences of segments of each gene were consistent with the expected,and the homologies were 98%～99%. 2.Establishment of Multiplex PCR for papC,iucD,irp2,tsh and iss of APECBasing on the conditions of sole gene PCR amplification,the reactive conditions of multiplex PCR were optimized step by step for the combination of primers concentrations,magnesium ion concentration,the volume of Taq DNA polymerase, anneal temperature,extension time and cycle times.The optimum conditions of multiplex PCR were as follows:2μL of DNA template was added to reaction mixture (25μL)containing 0.5μL of each primer pair(10pmol),1μL of the four deoxynucleoside triphosphates(10mM),2.5μL of PeR buffer(10x),2.5μL of 25mM magnesium chloride,and 2.5 units of Taq ploymerase.The PCR amplification parameters were as follows:pre-denaturate at 94℃for 3min,denatureate 94℃30s, annealing 58℃30s,extend 3min at 68℃,repeate for 30 cycles totally,then extend 9min at 72℃finally.The detecting limit of this method was 10² cfu·mL^-1,and the established multiplex PCR assay is specific.3.Virulence Genes Detection ofAPEC Anhui strains by Multiplex PCRTo make sure the practicability of the multiplex PCR established,nine APEC Anhui strains were detected for five virulence genes,and,the virulence genotypes were defined.In this study,genome DNA templates of the nine APEC strains were extracted respectively to amplify papC,iucD,irp2,tsh and iss genes.The results showed that one Escherichia coli strain isolated from duck and three strains from chiken carried four virulence genes,one Escherichia coli strain isolated from goose and four strains from chiken carded five virulence genes.And the positive rates of papC,iucD,irp2,,tsh and iss were 100%,100%,100%,55.6%and 100%respectively. The nine avian pathogenic Escherichia coli strains were distributed into two virulence genotypes,of which 5 strains were iss⁺irp2⁺papC⁺iucD⁺tsh⁺(55.6%);4 strains were iss⁺irp2⁺papC⁺iucD⁺tsh^-(44.4%).The results demonstrated that papC,iucD,irp2,tsh and iss genes existed widely in APEC Anhui strains.Above all,the established multiplex PCR assay is specific,accurate and sensitive (10² cfu·mL^-1).It could be used for detecting any kind or any combination of the five virulence genes,and also could be used in the colibacillosis fast diagnoses,molecular epidemiology survey and the detection for APEC empoisoned poultry product. Moreover,it might provide a basis for the further study about the molecular pathogenic mechanism of APEC.