猪流行性腹泻病毒实时荧光定量PCR检测方法的建立

Establishment of an EvaGreen real-time fluorescence quantitative PCR assay for porcine epidemic diarrhea virus

为开发一种快速、准确检测猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)的实时荧光定量PCR方法,根据引物设计原则在病毒基因组序列保守区设计了一对PEDV特异性引物,通过系统优化建立了EvaGreen实时荧光定量PCR方法,对其特异性、灵敏度和重复性进行了分析,并将该方法应用于90份临床样品检测。结果表明:该实时荧光定量PCR可以特异性地检测PEDV,对其他非靶标病毒无交叉反应,最低检测限为5 copies/μL;使用3个不同浓度的标准品进行批内和批间重复,Ct值的变异系数在2.8%以下,表明该方法重复性好;临床样品中PEDV的阳性率为48.9%。建立的实时荧光定量PCR是一种快速、准确、高度灵敏且特异的检测方法,可用于PEDV流行病学调查和临床诊断。

The purpose of this study is to develop an EvaGreen-based real-time fluorescence quantitative PCR assay to detect porcine epidemic diarrhea virus(PEDV)rapidly and accurately.A pair of specific primers for PEDV was designed in the conserved region of virus genome sequence according to the primer design principle.After system optimization,an EvaGreen real-time fluorescence quantitative PCR method was established,and its specificity,sensitivity and repeatability were analyzed.Subsequently,the established assay was applied to the detection of 90 clinical samples.The result revealed that the assay can specifically detect PEDV,and no cross-reaction was observed with other non-target viruses.The limit of detection of this assay was 5 copies/μL.Standards with 3 different concentrations were used for within-run and between-run repetition.The coefficient of variation(CV)of Ct value was less than 2.8%,which indicated that the proposed method had good repeatability.The positive rate of PEDV in the clinical samples was 48.9%.In summary,the real-time fluorescence quantitative PCR assay established in this work is a rapid,accurate,highly sensitive and specific detection method,which can be used for epidemiological investigation and clinical diagnosis of PEDV.