禽偏肺病毒通用型EvaGreen荧光定量RT-PCR检测方法的建立

Development of a universal EvaGreen-based reverse transcription quantitative PCR method for detection of avian metapneumovirus

为建立各亚型禽偏肺病毒(aMPV)的快速检测方法,本研究对aMPV 4个亚型的核苷酸序列比对分析,针对aMPV N基因的保守区域设计了一对特异性引物,通过条件优化,建立了检测aMPV各亚型的通用型EvaGreen荧光定量RT-PCR方法。结果显示,该方法在模板为10~3拷贝/μL～10~8拷贝/μL范围内具有良好的线性关系;检测下限为10~3拷贝/μL,敏感性为常规RT-PCR的10倍。与其它常见禽类病毒无交叉反应;组内重复试验和组间重复试验的变异系数分别为0.89%～1.34%和2.01%～3.29%,具有较好的重复性。利用所建立的方法对河南省部分地区鸡场送检病鸡样品进行检测,结果显示被检地区鸡场的aMPV的平均阳性率为46.66%,与普通RT-PCR的符合率为100%。本研究建立的EvaGreen荧光定量RT-PCR方法可以用于aMPV的检测,为aMPV感染的流行病学调查提供了可行方法。

In order to establish a rapid assay for detect all subtypes of avian metapneumovirus （aMPV）, a universal EvaGreen-based reverse transcription quantitative PCR （RT-qPCR） assay was developed with the primers targeting the conserved region of N genes of aMPV subtypes. The standard curve for each subtype of aMPV showed a linear relationship between cycle threshold （Ct） and template concentration ranging from 103copies/μL to 108copies/μL. The detection limit of the developed EvaGreen RT-qPCR was 103copies/μL, which was ten-fold more sensitive than that of conventional RT-PCR. The developed EvaGreen RT-qPCR had no cross reactivity with other common avian pathogens and had high reproducibility with coefficient variation of 0.89% to 1.34% and 2.01% to 3.29% in intra- and inter-assay, respectively. Moreover, the developed EvaGreen RT-qPCR was used to aMPV surveillance in some poultry farms in Henan province, and the average positive rate was 46.6%. The coincidence rate of the developed EvaGreen RT-qPCR with conventional RT-PCR was 94.7%. The developed universal EvaGreen qRT-PCR could be used for detecting all subtypes of aMPV, which provided a technical support for aMPV epidemiological investigation.