Establishment And Application Of A Multiplex PCR Method To Detect Avian Pathogenic Escherichia Coli O1,O2 And O78

Avian pathogenic Escherichia coli is one of the most important pathogen in poultry industry.The O-antigen serotype of APEC is highly complex.Poultry farms in different areas have its own dominant serotypes.And there is no cross protection between vaccines which produced by different serotypes.But for the same farm,the serotypes of pathogenic E.coli are relatively stable in a few years.Therefore,investigating the serotype of pathogenic Escherichia coli in poultry farm and producing the predominant serotype vaccine is a effective measure to prevent and control avian colibacillosis.According to the specificity of O-antigen gene clusters,we established a multiple PCR methods to detect APEC O1,O2 and O78 by using the flippase gene?wzx?as target.The key factors,such as primer concentration?Taq polymerase concentration?10×Buffer dosage?annealing temperature?annealing time?extension temperature and extension time,were optimized.The optimum condition of multiplex PCR were as follows:DNA,1?L;10×Ex Taq Buffer(Mg²⁺Plus 20mM),2.5?L;Ex Taq polymerase?5U/?L?,0.3?L;primers?10pmol?,each 0.5?L,and finally add sterilized double distilled water to fill up to 25?L.The PCR amplification parameters were as follow:pre-denaturate at 95?f or 5min,denaturate at 94?for 30s,annealing at 59?for 1min,extend at 72?for 1min,repeate for 30 cycles totally,and finally extend at 72?for 10min.The detecting limit of this method was 100pg/?L,and the established multiplex PCR assay is specific.To verify the stability and practicability of the this test method,54 APEC Changchun isolates were detected.The results showed that the positive detection rate of O1 group in 54 strains of APEC Changchun isolates was 9.26%?5/54?,O2 group was 7.41%?4/54?,O78 group was 40.74%,and the total positive detection rate of O1,O2 and O78 was 57.41%?31/54?.In order to evaluate the effect of multiple PCR methods,the traditional serum agglutination method was used to detect the serotypes of 54 APEC Changchun isolates.The results showed that the coincidence rate of the two methods was 85.19%?46/54?.It proved that the multiple PCR detection method had a considerable efficiency.