摘要
为建立一种快速、高效检测猪繁殖与呼吸综合征病毒(PRRSV)的实时荧光定量PCR方法,本研究根据GenBank公布的中国流行PRRSV病毒株基因序列,制备包含PRRSV ORF7基因的重组质粒标准品,同时,针对PRRSV保守区域ORF7基因序列设计特异性引物和探针,并优化引物和探针终浓度,建立PRRSV Taq Man实时荧光定量PCR检测方法。以质粒标准品为模板构建检测方法线性模型,评估灵敏度、重复性和特异性,并在疑似临床样品中初步应用。本研究建立的PRRSV Taq Man荧光定量PCR检测方法线性关系良好,线性决定系数为0.9975;病毒载量检测限度为1μL 3.32×10^(1)拷贝;变异系数在组内和组间重复中均小于1.500%;仅与PRRSV发生特异性反应,未与其他病毒发生交叉反应。临床样品qPCR检测阳性率为36.92%,高于PCR检测阳性率(26.15%),阳性符合率为100%。基于PRRSV ORF7基因构建的Taq Man荧光定量PCR检测方法线性关系良好、灵敏度高、重复性好、特异性强,可快速、高效检测临床样品PRRSV,为PRRSV早期诊断、及时防控和流行病学调查提供了科学的技术支撑。
To establish a rapid and efficient qPCR method for the detection of porcine reproductive and respiratory syndrome virus(PRRSV),standard recombinant plasmid samples containing the ORF7 gene of PRRSV were prepared,according to the gene sequences of popular PRRSV strains in China annunciated by GenBank.Meanwhile,specific primers and probes were designed according to the ORF7 gene sequence of the conserved region of PRRSV,and the final concentrations of primers and probes were optimized to construct a real-time fluorescence quantitative PCR detection method for PRRSV Taq Man.The linear model of the assay was constructed by using the plasmid standard sample as the template to evaluate the sensitivity,repeatability and specificity,and the preliminary application was made in suspected clinical samples.The linear determination coefficient of the established Taq Man qPCR detection method for PRRSV was 0.9975 and the linear relationship was good.The minimum detection limit of viral load was 3.32×10^(1) copies/μL.The coefficient of variation was less than 1.500%in both intra-group and inter-group replicates.It only reacted specifically with PRRSV,and did not cross-react with other viruses.The positive detection rate of qPCR in clinical samples was 36.92%,which was higher than that of PCR(26.15%),and the positive coincidence rate was 100%.The Taq Man fluorescence quantitative PCR method based on PRRSV ORF7 gene had good linear relationship,high sensitivity,good repeatability and strong specificity.It can quickly and efficiently detect PRRSV in clinical samples,which provides scientific technical support for PRRSV early diagnosis,timely prevention and control as well as epidemiological investigation.
作者
安乐乐
罗迅
杨宣叶
胡欣妍
赵永清
AN Lele;LUO Xun;YANG Xuanye;HU Xinyan;ZHAO Yongqing(Biomedical Research Center,Northwest Minzu University,Lanzhou 730030,China;College of Life Science and Engineering,Northwest Minzu University,Lanzhou 730100,China)
出处
《江苏农业学报》
CSCD
北大核心
2024年第9期1681-1688,共8页
Jiangsu Journal of Agricultural Sciences
基金
甘肃省自然科学基金项目(21JR1RA221)
兰州市科技计划项目(2023-1-31)
中央高校基本科研项目(31920220050)。
