猪繁殖与呼吸综合征病毒TaqMan-MGB实时荧光定量RT-PCR方法的建立及应用

摘要

In order to quickly and accurately detect porcine reproductive and respiratory syndrome (PRRSV) , one TaqMan-MGB Real-time RT-PCR assay for detection of PRRSV was established using specific primers and probe based on PRRSV gene.The developed Real-time RT-PCR method could detect PRRSV classic strain, highly pathogenic mutant strain, as well as the new NADC30-like strain at the same time.The specificity, sensitivity and repeatability of the established method were detected.120 clinical samples were detected using the developed Real-time RT-PCR method and the conventional PCR method.The results showed that TaqMan-MGB Realtime RT-PCR method was specific for PRRSV classic strain, highly pathogenic mutant strain, aswell as the NADC30-like strain, and the test results were negative with CSFV, PEDV, TGEV, RV, PRV, PPV and PCV2.The method showed a good linear relationship within the template ranges from 101 to 108 copies/μL, and its sensitivity was 100 times that of routine PCR assay.The correlation of the standard curve was 0.9999, and the efficiency was 93％.The limit of detection concentration was 101 copies/μL.The coefficient of variation in the intra-and inter-assays were0.17％to 0.90％ and 0.65％ to 2.34％, respectively.The positive detection rate of PRRSV in clinical samples using the developed Real-time RT-PCR method (59.2％) was higher than that using the conventional PCR method (44.2％) .The establishment of Real-time RT-PCR method provided a rapid and accurate detection means for early diagnosis and epidemiological investigation of the disease.%为了快速、准确地检测猪繁殖与呼吸综合征病毒 (porcine reproductive and respiratory syndrome, PRRSV), 本研究根据PRRSV基因序列设计特异性引物和探针, 建立了一种可同时检测PRRSV经典毒株、高致病性变异毒株以及近几年中国新出现的NADC30-like毒株的TaqMan-MGB实时荧光定量方法, 并对该方法的特异性、敏感性和重复性进行验证, 同时用建立的实时荧光定量方法与常规PCR方法对临床收集的120份疑似PRRSV样品进行检测.结果表明, 该方法特异性良好, 对PRRSV的经典毒株、高致病性变异毒株及NADC30-like毒株均有良好的扩增, 但对猪瘟病毒 (CSFV) 、猪流行性腹泻病毒 (PEDV) 、猪传染性胃肠炎病毒 (TGEV) 、猪轮状病毒 (RV) 、猪伪狂犬病病毒 (PRV) 、猪细小病毒 (PPV) 和猪圆环病毒2型 (PCV2) 的检测结果均为阴性, 无交叉反应;模板浓度在101～108拷贝/μL范围内具有良好的线性关系, 标准曲线结果显示其扩增的相关系数为0.9999, 扩增效率为93％;敏感性高, 约是常规PCR方法的100倍, 最低可以检测到101拷贝/μL的模板;重复性好, 批内和批间重复性试验变异系数分别为0.17％～0.90％和0.65％～2.34％;用本研究所建立的实时荧光定量检测方法对120份临床样品进行检测, PRRSV阳性检出率为59.2％ (71/120), 而常规PCR方法的PRRSV阳性检出率为44.2％ (53/120) .该方法的建立为PRRSV的实验室诊断、流行病学调查, 以及预防和控制中国PRRSV的流行提供了快速、准确的检测手段.