BVDV SYBR GreenⅠ荧光定量PCR方法与RT-PCR方法的建立与应用

摘要

利用SYBR GreenⅠ荧光定量PCR方法和RT-PCR方法建立2种有效地实验室检测牛病毒性腹泻病毒的方法.根据GenBank中登录的牛病毒性腹泻病毒基因序列,选取14株BVDVⅠ型和7株BVDVⅡ型,经序列比对后,设计合成1对特异性引物,建立了检测BVDV的RT-PCR方法和SYBR GreenⅠ荧光定量PCR方法.通过对RT-PCR方法和SYBR GreenⅠ荧光定量PCR方法的特异性、灵敏性和重复性进行检测.结果显示,该方法可从BVDV标准毒株Oregon C24V中扩增出250 bp的特异性片段,对牛细小病毒、牛副流感病毒、牛腺病毒、呼肠孤病毒的扩增结果均为阴性,对BVDV C24V株、GSTZ株、Av69株、S183株和BVDVⅡ型扩增结果为阳性.经对标准毒株的细胞毒进行检测,RT-PCR的灵敏性为10 -1TCID50/mL,SYBR Green Ⅰ荧光定量PCR方法的灵敏性为3.4×102copies/μL.应用2种方法对临床血清样本进行检测,结果比病毒分离方法更为敏感,操作简便.表明建立的RT-PCR方法和SYBR Green Ⅰ荧光定量PCR方法具有特异、灵敏、高效、快速的特点,可用于BVDV的流行病学监测、实验室检测以及诊断.%Rapid,sensitive and specific diagnostic method is necessary to confirm infection of Bovine viral diar-rhea virus(BVDV).RT-PCR and Real-time PCR with SYBR Green Ⅰ was used as a diagnostic method to detect BVDV.According to genomes of Bovine viral diarrhea virus(BVDV) in GenBank,a pair of primers had been de-signed to develop conventional RT-PCR method and Real-time PCR with SYBR Green Ⅰ for detection of BVDV. Depending on estimations of specificity and sensitivity and stability of the two methods,RT-PCR method could amplify the specific PCR product with 250 bp in length from BVDV standard strain (Oregon C24),and Real-time PCR with SYBR GreenⅠcould also obtain positive signal.The two methods didn′t obtain any signals from BPV,BPIV3,BAV-3 and REO which are main pathogens for cattle industry,but could obtain positive signals from some BVDV strains (C24V, GSTZ,Av69,S183 and typeⅡBVDV strain).As for the limit of detection of per method,the limit of detection of RT-PCR could reach to 10-1TCID50/mL;the limit of detection of Real-time PCR with SYBR GreenⅠcould reach to 3.4×102 copies/μL.The two methods were used to detect clinical serum samples efficiently,suggesting that the current develop-ments of detection methods can take application for epidemiology of BVDV and clinical assay of BVDV.