Establishment of Duplex PCR Assay of Eimeria tenella and Enteropathogenic Escherichia coli and Application of Epidemiological Investigation
[Objective]A duplex PCR detection assay that could for simultaneous detection of Eimeria tenella(E.tenella)and Enteropathogenic Escherichia coli(EPEC)was established,which provided technical support for clinical detection of E.tenella and secondary EPEC.[Method]Two specific primers were designed based on E.tenella and EPEC(escV),and the reaction conditions of multiple PCR were optimized by annealing temperature,template concentration and primer concentration.A duplex PCR detection method for the two pathogens was established,and 490 fecal samples of meat and laying hens in Guizhou Province were detected by this method.Epidemiological analysis was performed.[Result]The minimum concentration combination of template DNA required for this method was 3.5 ng/μL,the optimal annealing temperature was 62℃,and the optimal primer concentration combination was 0.1 nmol/μL.The results of epidemiological investigation showed that the positive rates of E.tenella and EPEC in Bijie samples were both 36.36%,and the positive rate of mixed infection was 9.09%.Sample from Liping of Guizhou Province had only EPEC infection,and the positive rate of EPEC was 23%.The Guizhou Xishui sample did not test positive,and the positive rate of E.tenella and EPEC of Jinping samples were 11.11%and 22.22%,respectively,and there was no co-infection.E.tenella,EPEC and mixed infection positive rate of total samples was respectively 10.20%,18.37%and 2.04%.[Conclusion]The established duplex PCR assay for E.tenella and EPEC has the advantages of high specificity,high sensitivity and good repeatability.
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