牛呼吸道合胞体病毒一步法实时荧光定量PCR检测方法的建立及应用

Establishment and application of a one-step real-time fluorescence quantitative PCR assay for bovine respiratory syncytial disease

为建立一种牛呼吸道合胞体病毒(Bovine respiratory syncytial virus, BRSV)快速简便的检测方法,本研究基于BRSV M基因保守区序列,设计特异性引物及探针,通过优化反应条件,建立用于BRSV检测的一步法实时荧光定量PCR,并验证了该方法的敏感性、特异性和重复性;同时利用建立的检测方法对采集的临床样本进行检测。结果表明:本研究所建立的BRSV荧光定量检测方法,其特异性好,仅对BRSV存在特异性扩增;敏感性高,最低可达10 copies/μL;稳定性好,组内变异系数和组间变异系数小。使用所建立的BRSV一步法实时荧光定量PCR对宁夏地区94份临床样品进行检测,阳性率为5.3%(5/94)。上述结果表明,本研究建立的检测方法可为BRSV的快速诊断提供有力的技术支持。

To establish a rapid and simple method for the detection of bovine respiratory syncytial virus(BRSV),we designed specific primers and probes based on the sequence of the conserved region of the BRSV M gene,and established a one-step real-time fluorescence quantitative PCR assay for BRSV detection by optimizing the reaction conditions,and validated the sensitivity,specificity and stability of the assay.Finally,the method was also used to test clinical samples.The results showed that this study successfully established a BRSV fluores-cence quantitative assay with good specificity and specific amplification only for BRSV,with high sensitivity at a minimum of 10 copies/μL,with good stability with low intra-group and inter-group coefficients of variation.The detection rate of 94 clinical samples collected in the Ningxia region using the established BRSV one-step real-time fluorescence quantitative PCR assay,the positive rate was 53%(5/94).The above results indicated that the method established in this study might serve as a powerful technical tool for rapid diagnosis of BRSV.