鉴别检测野生株和基因缺失株非洲猪瘟病毒多重荧光定量PCR方法的建立

Establishment of a multiplex real-time PCR method for differential detection of wild strains and gene-deleted strains of African swine fever virus

为建立一种鉴别检测野生型和基因缺失株非洲猪瘟病毒(ASFV)的方法,本研究根据GenBank中登录的ASFV P72、CD2v、MGF360-14L基因保守序列设计引物和探针,通过方阵法优化体系后,建立了一种可以同时检测P72、CD2v、MGF360-14L基因的多重荧光定量PCR方法。该方法与伪狂犬病病毒(PRV)、猪繁殖与呼吸综合征病毒(PRRSV)、猪瘟病毒(CSFV)、猪流行性腹泻病毒(PEDV)、猪传染性胃肠炎病毒(TGEV)均无交叉反应,可鉴别野生型和基因缺失株ASFV,特异性强;敏感性试验结果显示,该方法对P72、CD2v和MGF360-14L重组质粒标准品的最低检测限均为100 copies/m L,敏感性高;组内和组间变异系数均小于2%,表明该方法重复性较好。利用ASFV核酸检测试剂盒与本实验室建立的方法分别对35份临床样品进行检测,两种方法的检出率均为68.57%,阳性符合率为100%。上述结果表明,本研究建立的多重荧光定量PCR方法对ASFV的鉴别检测、病原流行病学调查等研究具有重要意义。

To develop a multiplex real-time PCR method for differential detecting wild strains and gene-deleted strains of African swine fever virus(ASFV),primers and probes were designed based on the P72,CD2 v and MGF360-14 L genes of ASFV in Gen Bank.After optimizing the system by the square matrix method,a multiplex real-time PCR method for simultaneous detection of P72,CD2 v and MGF360-14 L was successfully established.This method has no cross-reaction with PRV,PRRSV,CSFV,PEDV and TGEV,which indicated that it has strong specificity.The sensitivity results showed that the minimum detection limit of P72,CD2 v and MGF360-14 L were 100 copies/m L respectively.The intra-and inter-group coefficients of variation were both less than 2%,indicating that the method was reproducible.The multiplex real-time PCR assay and ASFV nucleic acid test kit were used to detect 35 clinical samples,both of the positive rate of ASFV were68.57%(24/35),the positive coincidence rate was 100%.The established multiplex real-time PCR assay has important implications for differential detection and epidemiological investigation of ASFV.