猪塞尼卡病毒TaqMan探针荧光RT-PCR方法的建立和应用

Development and application of reverse real time PCR for Senecavirus A in porcine

塞尼卡病毒(Senecavirus A,SVA)为小核糖RNA病毒科塞尼卡病毒属的单股正链RNA病毒,可引起猪的水泡样病变、跛行和新生仔猪突然死亡等,我国已经有多个省市检测到了本病毒。为建立一种快速高效、适应范围广的SVA检测方法,本研究以分离到的1株SVA流行病毒株GXT91作为参考毒株,设计合成了TaqMan探针和1对引物建立了SVA的荧光RT-PCR方法,并分别进行该方法的敏感性、特异性和重复性试验。结果显示:该方法的敏感性达到了17.4个分子拷贝/μL,与口蹄疫病毒、猪瘟病毒等病原无交叉反应,表明它具有较高的特异性,组间和组内重复性试验的变异系数均小于2%,表明其重复性较好。进一步运用该方法对48份临床样本进行检测,从中发现了12份阳性样品。这表明本研究建立的荧光RT-PCR是一种良好的诊断SVA的方法,可用于待测样本中SVA的诊断和流行病学调查监测等。

Senecavirus A(SVA) is a single strand positive RNA virus belonging to the Senecavirus genus of family Picornaviridae,which can cause porcine idiopathic vesicular lesions on the snout and coronary bands,lameness and acute death of newborn piglets.SVA has been reported in several provinces in China.In order to develop a high sensitive and applicable method to diagnose SVA,a new reverse real time PCR method was constructed on the base of new TaqMan probe and a pair of primers which was designed by the reference sequence of a SVA isolate in our laboratory.The sensitivity,specificity and repeatability were further versified.The sensitivity of this method could reach 17.4 copies virus/μL,with high specificity to differentiate any other viruses of foot and mouse disease virus,classical swine fever virus,porcine respiratory and reproductive virus.The intro-and inter-assay repeatability was acceptable with less than 2% coefficient variation.Some applications were verified successfully with detection of 12 positive from 48 clinical samples.This research indicated the real time RT-PCR of SVA diagnosis is a preferential assay for clinical samples and cell culture in epidemiological surveillance.