鉴别非洲猪瘟野毒株与基因缺失疫苗株的三重TaqMan探针荧光定量PCR检测方法的建立

The establishment of a triple TaqMan probe fluorescence quantitative PCR detection method to distinguish between wild African swine fever strains and gene-deleted vaccine strains

为建立一种鉴别非洲猪瘟野毒株与基因缺失疫苗株的TaqMan探针荧光定量PCR检测方法,根据非洲猪瘟病毒(ASFV)的B646L、EP402R、MGF360-13L基因序列,分别设计PCR引物和TaqMan探针,绘制标准曲线,并进行重复性试验、特异性试验、敏感性试验与临床样品检测,建立三重TaqMan探针荧光定量PCR检测方法。结果显示,以B646L、EP402R和MGF360-13L重组质粒为标准品绘制的标准曲线具有良好的线性关系,线性相关系数(R^(2))分别为0.995、0.997和0.997;建立的方法与多种猪常见病原不存在交叉反应,特异性良好;对B646L、MGF 360-13L与EP402R基因的检测下限均为10^(2.5) copies/μL,变异系数均<2%,该方法灵敏度高;当临床样品稀释至10^(-5)时,即滴度为10^(2.5)TCID_(50)/mL时仍能检测到病毒粒子,具有较高的临床使用价值。本研究建立了一种高效、灵敏、特异的ASFV分子检测方法,对ASF风险预警具有重要意义。

To establish a Taq Man probe fluorescence quantitative PCR detection method to identify African swine fever wild virus strains and gene deletion vaccine strains.Based on the gene sequences of B646L,EP402R,and MGF360-13L of ASFV,we designed PCR primers and Taq Man probes respectively,established a standard curve,and established triple Taq Man probes through repeatability experiments,specificity experiments,sensitivity experiments and clinical sample detection.Needle fluorescence quantitative PCR detection method.Experimental results show:With B646L,EP402R and MGF360-13L recombinant plasmids as the standard products,there is a good linear relationship,and the linear correlation coefficient R^(2)is 0.995,0.997 and 0.997,respectively.There is no cross reaction with many common swine pathogens,this method has good specificity.The lower limit of sensitivity detection of B646L,MGF360-13L and EP402R genes is all 10^(2.5) copies/μL,and the coefficient of variation is less than 2%.This method has high sensitivity.T Viral particles can still be detected when the clinical sample is diluted to 10,that is,the titer is 10^(2.5)TCID_(50)/m L,which has high clinical value.This study established an efficient,sensitive and specific molecular detection method for ASFV,which is of great significance for ASF risk early warning.