Establishment and application of Real-Time Fluorescence Quantitative PCR Assay for Chicken Escherichia Coli
Aimto establish a sensitive,precise and rapid Real-time fluorescence quantitative PCR means for the detection of chicken escherichia coli,we designed primers based on a specific conserved sequence of uidA gene,which is chicken escherichia coli's specific gene.The uidA gene of Avian Pathogenic Escherichia Coli was constructed on 19T vector.We utilized the recombinant plasmid as a standard positive template to set up means for the detection of chicken escherichia coli and evaluated the specificity,Sensitiveness and repeatability of the method.The results showed that the Ct values of the established qPCR method showed a good linear relationship with those of the standard products in the range of 4.69 x 102～4.69 × 109 copies/μ L,the linear correlation coefficient was R2=0.993 and the standard curve equation was y=-3.2786x+39.032.At the same time,the melting curve is unimodal distribution and non-specific amplification.The results of Sensitivity test showed that the method possessed high sensitivities and the minimum detection limit was 4.69 × 102 copies/μ L,which was 1000 times that of the conventional PCR method.We also found that the positive rate of this method was 45％higher than ordinary PCR through the analysis of 60 clinical samples.The SYBR Green I real-time quantitative PCR method for the detection of chicken Escherichia coli established in this study is able to be used for the rapid diagnosis of chicken Escherichia coli,which is of great significance for the detec-tion of colibacillosis.
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