基于TaqMan探针非洲猪瘟病毒E301R基因荧光定量PCR检测方法的建立及验证  

Development and verification of a qPCR method based on TaqMan probe targeting E301R gene of African swine fever virus

作　　者：葛海亮 张柯慧 于少雄 曹宏伟 李素[2] 杨玉莹[1] 仇华吉 GE Hailiang;ZHANG Kehui;YU Shaoxiong;CAO Hongwei;LI Su;YANG Yuying;QIU Huaji(College of Animal Sciences,Yangtze University,Jingzhou 434023,Hubei Province,China;不详)

机构地区：[1]长江大学动物科学学院,湖北荆州434023 [2]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室,黑龙江哈尔滨150069

出　　处：《中国生物制品学杂志》2024年第9期1133-1139,共7页Chinese Journal of Biologicals

基　　金：国家自然科学基金面上项目(32072866);黑龙江省自然科学杰出青年基金项目(JQ2020C002)。

摘　　要：目的建立基于TaqMan探针的非洲猪瘟病毒(African swine fever virus,ASFV)E301R基因荧光定量PCR(fluorescent quantitative polymerase chain reaction,qPCR)检测方法,并进行验证,以期应用于临床样本中ASFV的检测。方法通过对ASFV E301R基因序列进行分析,选择基因保守区设计特异性引物和TaqMan探针。PCR扩增E301R基因片段,克隆至pCAGGS载体,构建质粒标准品,建立基于TaqMan探针的qPCR检测方法,并验证方法的线性范围、精密性、特异性、敏感性及准确性。采用建立的方法检测92份临床样本,并与商品化ASFV荧光PCR试剂盒检测结果进行比较。另采用建立的方法对E301R基因转录动力学进行分析。结果质粒标准品在1.6×(10^(1)~108)拷贝/μL范围内,与Ct呈良好的线性关系,线性回归方程为:y=-3.239 x+40.774,R2=0.994;重复性及中间精密性验证CV均<2%;除ASFV外,猪瘟病毒(classical swine fever virus,CSFV)、猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)、伪狂犬病病毒(pseudorabies virus,PRV)及猪圆环病毒2型(porcine circovirus type 2,PCV2)均未出现扩增曲线;最低可稳定检出1.6×10^(1)拷贝/μL的质粒标准品;浓度为1.6×10^(2)和1.6×10^(1)拷贝/μL质粒标准品的加标回收率在90%~110%之间。建立的方法与商品化ASFV荧光PCR试剂盒对92份临床样本检测结果的符合率为96.7%(Kappa=0.932,P=1.000)。E301R基因可能为ASFV感染的中期转录基因。结论建立的基于TaqMan探针的qPCR检测方法具有良好的精密性、特异性、敏感性及准确性,可用于临床样本中ASFV的检测。Objective To develop and verify a fluorescent quantitative polymerase chain reaction(qPCR)method based on TaqMan probe for the detection of E301R gene of African swine fever virus(ASFV),so as to apply the method to the detection of ASFV in clinical samples.Methods By analyzing the E301R gene sequence of ASFV,specific primers and TaqMan probes were designed for the conserved region of the gene.The fragment of E301R gene amplified by PCR was cloned into pCAGGS vector,and the standard plasmid was constructed.The qPCR detection method based on TaqMan probe was developed and verified for the linear range,precision,specificity,sensitivity and accuracy.The developed method was used to detect 92 clinical samples,and the results were compared with those detected by commercial real-time PCR diagnostic kit.In addition,the transcriptional dynamics of E301R gene was analyzed by using the developed method.Results In the range of 1.6×(10^(1)-108)copies/μL,the standard plasmid showed a good linear relationship with Ct values,and the linear regression equation was:y=-3.239 x+40.774,with the correlation coefficient of 0.994.The CVs of repeatability and intermediate precision verification were both less than 2%.Except for ASFV,there was no amplification curve of classical swine fever virus(CSFV),porcine reproductive and respiratory syndrome virus(PRRSV),pseudorabies virus(PRV),and porcine circovirus type 2(PCV2).The minimum detection limit was 1.6×10^(1)copies/μL of standard plasmid.The spike recoveries of 1.6×10^(2)and 1.6×10^(1)copies/μL standard plasmid were between 90%and 110%.The coincidence rate of the detection results between the developed method and commercial real-time PCR diagnostic kit for 92 clinical samples was 96.7%(Kappa=0.932,P=1.000).E301R gene may be the transcription gene in the middle stage of ASFV infection.Conclusion The developed detection method of qPCR based on TaqMan probe has good precision,specificity,sensitivity and accuracy which can be used for the detection of ASFV in clinical samples.

关 键 词：非洲猪瘟病毒 E301R基因 TAQMAN探针 荧光定量PCR法 转录动力学 

分 类 号：S852.651[农业科学—基础兽医学]