一种基于M基因的尼帕病毒TaqMan-MGB荧光RT-PCR检测方法的建立

Development of a TaqMan-MGB probe real-time RT-PCR assay for detection of Nipah virus based on M gene

为建立一种快速、敏感和特异地检测尼帕病毒（Nipah virus,NiV）的方法,根据NiV M基因序列保守区设计了1对特异性引物和探针,通过优化反应条件,建立了一种检测NiV的TaqMan-MGB实时荧光RT-PCR方法,并对该方法进行特异性、定量线性范围、敏感性和重复性等试验。结果显示,该方法仅对NiV M基因的RNA标准对照（NiV-M-RNA）发生特异性扩增反应,对猪的其他常见病毒,包括经典猪瘟病毒、猪繁殖与呼吸综合征病毒、猪流行性腹泻病毒、猪流感病毒、伪狂犬病病毒、猪细小病毒和猪圆环病毒2型均不发生交叉反应。该方法对NiV-M-RNA的最适线性检测范围为4.6×101~4.6×107 copies/μL,标准曲线方程为y=-3.418x＋37.57,线性相关系数（r2）为0.999,检测下限为4.6copies/μL。对5个不同浓度（4.6×103~4.6×107 copies/μL）的NiV-M-RNA进行双份样品的4次重复检测,每个浓度重复试验的Ct值的变异系数均小于1.5%,具有良好的重现性。用该方法对368份临床样品进行NiV检测,结果均为阴性。本方法的建立为活猪临床样品中NiV检测提供了一种快速、敏感和特异的技术手段。

To establish a rapid,sensitive and specific assay for the detection of Nipah virus（NiV）,a TaqMan-MGB probe real-time RT-PCR was developed by optimization of reacting conditions.A pair of primers and TaqMan-MGB probe were designed according to the conserved regions of M gene sequences of NiV.There was no cross-reaction with classical swine fever virus,porcine reproductive and respiratory syndrome virus and porcine epidemic diarrhea virus,swine influenza virus,pseudorabies virus,porcine parvovirus,porcine circovirus type 2,respectively.The assay was linear for the NiV-M-RNA,in the range from4.6×101 to 4.6×107 copies/μL[standard equation：y=-3.418x＋37.57,correlation coefficient（r2）=0.999],and detection limit was up to 4.6copies/μL.Tested four times in duplicate by the assay,coefficient of variation（CV）of five diluted concentration（4.6×103 to 4.6×107 copies/μL）of NiV-M-RNA were less than 1.5%.A total of 368 samples from imported pigs and pork were tested by this assay,and all samples were negative.In conclusion,the development of this assay provides a rapid,sensitive and reliable molecular tool for NiV detection in the samples from the pigs and pork.