摘要
为了建立一种快速检测猪伪狂犬病病毒(PRV)经典毒株和变异毒株的双重实时荧光定量PCR方法,试验根据GenBank上收录的30株PRV设计gE基因和gC基因的特异性引物和探针,构建含有gE基因和gC基因的标准质粒,优化扩增体系建立双重实时荧光定量PCR方法,并检验该方法的特异性、敏感性和重复性,同时绘制标准曲线,最后采用该方法对临床样品进行检测。结果表明:试验建立的双重实时荧光定量PCR方法的最佳退火温度是60℃,最佳引物体积为0.8μL,最佳探针体积为0.4μL。该方法对猪场常见疫病病原猪蓝耳病病毒(PRRSV)、猪瘟病毒(CSFV)、猪圆环病毒2型(PCV-2)、猪圆环病毒3型(PCV-3)、猪细小病毒(PPV)、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,特异性强;对两种标准质粒pUC57-gE和pUC57-gC的最低检测限均为1×10^(1)copies/μL,gE基因和gC基因对应的标准曲线分别为y=-3.446x+41.920(R^(2)=0.998)和y=-3.255x+39.185(R^(2)=0.998);组内和组间变异系数均小于2%。临床样品检测结果的阳性率为10.53%,与《伪狂犬病诊断方法》(GB/T 18641—2018)的检测结果相同;变异毒株的阳性率为100%,扩增gC基因后的测序结果与GenBank中收录的PRV变异毒株一致。说明试验成功建立了可同时快速、准确鉴定PRV经典毒株和变异毒株的双重实时荧光定量PCR方法。
In order to establish a dual real-time PCR method for the rapid detection of classical and variant strains of porcine Pseudorabies virus(PRV),in this experiment,according to the 30 PRV strains included in GenBank,specific primers and probes for gE gene and gC gene were designed,and standard plasmids containing gE gene and gC gene were constructed.The amplification system was optimized to establish a dual real-time PCR method,and the specificity,sensitivity and repeatability of the method were detected,and the standard curve was constructed.Finally,this method was used to detect clinical samples.The results showed that the optimal annealing temperature of the double real-time PCR method established in the experiment was 60 C;the optimal primer volume was 0.8μL,and the optimal probe volume was O0.4μL.This method did not have cross-reaction to common epidemic viruses in pig farms,Porcine reproductive and respiratory syndrome virus(PRRSV),Classic swine fever virus(CSFV),Porcine circovirus type 2(PCV-2),Porcine circovirus type 3(PCV-3),Porcine parvovirus(PPV),Porcine epidemic diarrhea virus(PEDV),and Transmissible gastroenteritis virus(TGEV),and had strong specificity.The minimum detection limits for the two standard plasmids pUC57-gE and pUC57-gC were 1×10^(1)copies/μL;the standard curves coresponding to gE gene and gC gene were y=-3.446x+41.920(R^(2)=0.998)andy=-3.255x+39.185(R^(2)=0.998),respectively.The coeffcients of variation were less than 2%both within and between groups.The positive rate of clinical sample test results was 10.53%,which was the same as that of the Diagnostic Method for Pseudorabies(GB/T 18641-2018);the positive rate of the variant was 100%,and the sequencing results after amplification of the gC gene were consistent with the PRV variants included in GenBank.The results suggested that the test had successfully established a dual real-time PCR method that could quickly and accurately identify the classic and variant strains of PRV at the same time.
作者
苏金辉
邓云贵
夏冰
SU Jinhui;DENG Yungui;XIA Bing(Beijing Dabeinong Technology Group Co.,Ltd.,Beijing 100086,China;Hebei Dabeinong Farming and Animal Husbandry Food Co.,Ltd.,Hengshui 053900,China;Institute of Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing 100700,China)
出处
《黑龙江畜牧兽医》
北大核心
2023年第21期84-89,共6页
Heilongjiang Animal Science And veterinary Medicine
基金
北京市博士后工作经费资助项目(2022-22-130)。
关键词
伪狂犬病病毒
变异毒株
鉴别诊断
特异性
敏感性
Pseudorabies virus
variant strain
differential diagnosis
specificity
sensibility(011)
