中国兽医科学2017，Vol.47Issue(4)：466-470,5.DOI:10.16656/j.issn.1673-4696.2017.04.010
猪流行性腹泻病毒恒温隔绝式荧光PCR检测方法的建立
Development and application of on-site insulated isothermal PCR (iiPCR) for detecting porcine epidemic diarrhea virus
摘要
Abstract
The purpose of this study was to establish a method for the detaction of porcine epidemic diarrhea virus(PEDV) based on iiPCR.This study adopted and optimized the primers and probes for targeted PEDV S gene as well as Taq polymerase and reverse transcriptase.In order to compare with RT-qPCR,its specificity,sensitivity and stability were evaluated by detecting 15 PEDV positive samples and 7 negative samples as well as 115 diarrhea piglets samples.According to the results of iiPCR,optimal reaction system was described as follows:each of forward and reverse primers 3.5 μL,probe 0.25 μL,Taq polymerase 1 μL,reverse transcriptase 1.25 μL,Buffer A 25 μL,templates 2 μL DEPC water added up to 50 μL.Meanwhile,it took only 1h for the whole process.The sensitivity of detection limit was up to 17.5 copies/μL without cross reactions with TGEV,PPV and CSFV.Total coincidence rate with RT-qPCR was 90.9％.The sample detection rate in diarrhea piglets via iiPCR was 22.6％(26/115).In conclusion,this study successfully established iiPCR method for detecting PEDV,which provides a convenient and reliable method for on-site diagnosis of PED.关键词
猪流行性腹泻病毒/恒温隔绝式PCR/快速现场诊断Key words
porcine epidemic diarrhea virus/iiPCR/rapid on-site diagnostics分类
农业科技引用本文复制引用
沈思思,汤承,岳华,黄建德..猪流行性腹泻病毒恒温隔绝式荧光PCR检测方法的建立[J].中国兽医科学,2017,47(4):466-470,5.基金项目
十三五国家重点研发计划项目(2016YFD0500705) （2016YFD0500705）
