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With the rapid development of the poultry industry, the various types of bacterial diseases is growing, especially E. coli disease has been one of the most important diseases that restrict the development of the poultry industry. For the establishment of a specific avian colibacillosis detection methods, the experiment based on the APEC virulence genes of papC, GeneBank included, iucD, tsh, irp2, iss sequence of the conserved region, the design and synthesis of five pairs of specific primers to establish a simultaneous detection of virulence genes APEC 5 multiplex PCR method, and apply this method to detect the virulence genes of APEC Anhui isolates. The results are as follows: 1.APEC middle of papC iucD irp2 tsh, iss single gene PCR amplification of the pathogenic mechanism of the APEC, settlers from the adhesion of bacteria involved in iron uptake and enhanced serum resistance aspects to consider, with reference to the GeneBank included poultry the pathogenic E. coli P type pili gene of papC, aerogenes gene iucD, temperature-sensitive hemagglutinin gene tsh, iron inhibitory protein gene irp2, serum resistance protein gene iss sequence, designed and synthesized 5 specific primers. E. coli O 1 (CVCC249), the O 2 (CVCC1565) the O 78 (CVCC1555) standard strain of the DNA as a template, The five genes that were single gene PCR amplification, screening the positive control of the multiplex PCR. After preliminary experiments obtained PCR reaction system comprising: 10 × PCR buffer, 2.5μL, MgCl 2 (25mM) 3.0 μl, dNTP (10mM) 1.0μL, primers each 0.5μL (10pmol), Taq DNA polymerase (5U μL of -1 ) 0.4 μL DNA template 2.0μL, plus sterile ultrapure water to 25 μL. PCR reaction parameters: 94 ° C denaturation 3min, 94 ℃ denaturation 30s, 58 ℃ annealing 30s, 68 ° C for 3min, 30 cycles of 72 ℃ for 10min. The 3 strains of the serotypes in the detection rate of the 5 genes was 100%, thus can be used to do a positive control template. Select one the the one serotype strains E.coliO 2 (CVCC1565), five genes from which the amplified nucleotide sequence was determined and presented in NCBI BLAST 2 the SEQUENCES, and published in GeneBank the gene homology analysis. The results show that the resulting fragment with the expected basic consistent, and at 98% to 99% homology. 2.APEC in of papC, iucD, irp2, tsh, iss establishment of a multiplex PCR method based on single-gene PCR amplification conditions, by gradually optimizing the combination of primer concentration, the concentration of magnesium ions the, TaqDNA polymerase, the annealing temperature, extension time, the number of cycles and DNA template extraction and other conditions, the establishment of a multiplex PCR method for simultaneous detection of virulence genes. Multiplex PCR reaction system of 25 μl, including 2.5μL 10 × PCR buffer, 2.5μL mM MgCl 2 (25mM), 1.0μL dNTP (10mM), 0.5μL primer (10pmol), 0.5μL Taq DNA polymerase (μL of , 5U -1 ), 2.0μL DNA template, and adding sterilized ultrapure water to 25μL. PCR reaction parameters: 94 ° C denaturation 3min, 94 ℃ denaturation 30s, 58 ℃ annealing 30s, 68 ° C for 3min, 30 cycles of 72 ℃ for 10min. Broth minimum detectable concentration was 10 2 cfu · mL -1 , and specificity. Anhui isolate virulence genes in 3.APEC of multiple PCR detection applications created multiplex PCR method to detect the nine APEC Anhui isolates, in order to verify the usefulness of the multiplex PCR method, and to determine the the nine APEC Anhui isolates virulence genotype. Multiplex PCR amplification of isolates were extracted genomic DNA as a template, of the papC, tsh, iucD, irp2 and iss gene. The results of a duck source and three chicken source APEC in amplified 4 virulence gene bands, the a goose source and four chicken source APEC were amplified five virulence gene bands. In the 9 APEC Anhui isolates in iss irp2, of papC and iucD gene carrying rate are 100%, tsh gene carrying rate of 55.6%. Nine APEC Anhui isolates distributed in two virulence genotype, the of which five iss irp2 iucD the of papC tsh , 55.6%; The four the iss irp2 of papC iucD tsh - , accounting for 44.4%. That the iss, irp2, papC and iucD gene widely exist in APEC in. These studies have shown that the multiplex PCR method can be established from the APEC isolates detected by any one or any combination of virulence genes, their specificity, good accuracy, sensitivity can reach 10 2 cfu · mL -1 . Therefore, this method has important applications in poultry colibacillosis early diagnosis, molecular epidemiology investigation and the of poultry products APEC pollution detection research foundation for the further from the molecular level to reveal the pathogenic mechanism of APEC.
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