柔嫩艾美耳球虫和肠致病性大肠杆菌双重PCR检测方法的建立及流行病学调查应用

Establishment of Duplex PCR Assay of Eimeria tenella and Enteropathogenic Escherichia coli and Application of Epidemiological Investigation

【目的】建立可同时检测柔嫩艾美耳球虫(Eimeria tenella,E.tenella)和肠致病性大肠杆菌(Enteropathogenic Escherichia coli,EPEC)的双重PCR检测方法,为临床E.tenella继发EPEC病的检测提供技术支持。【方法】基于E.tenella和EPEC(escV)分别设计2条特异性引物,并通过退火温度、模板浓度和引物浓度等优化多重PCR的反应条件,建立2种病原体的双重PCR检测方法,并运用该方法检测贵州地区490份肉鸡、蛋鸡粪便样品,进行流行病学分析。【结果】该方法所需模板DNA的最低浓度组合为3.5ng/μL,最佳退火温度为62℃,最佳引物浓度组合为0.1 nmol/μL。流行病学调查显示,贵州毕节样本中E.tenella和EPEC的阳性率均为36.36%,混合感染阳性率9.09%;贵州黎平仅有EPEC感染,阳性率为23%;贵州习水样本中未检测出阳性,锦屏样本中E.tenella阳性率为11.11%,EPEC阳性率为22.22%,无混合感染。总样本中E.tenella、EPEC及2种混合感染的阳性率分别为10.20%、18.37%和2.04%。【结论】建立的柔嫩艾美耳球虫(Eimeria tenella,E.tenella)和肠致病性大肠杆菌(Enteropathogenic Escherichia coli,EPEC)双重PCR检测方法具有特异性强、敏感性高和重复性好的优点。

【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.