Flanders 病毒 TaqMan RT-PCR 检测方法的建立

摘要

目的：应用TaqMan PCR技术建立针对Flanders病毒（Flanders virus ，FLAV）的实时荧光定量PCR检测方法。方法下载GenBank中FLAV基因序列资料并进行多序列比对分析，选择其L基因中的高保守序列片段进行FLAV特异引物与探针的设计，使用来自于不同科属的15株病毒验证方法特异性，通过重复／平行实验验证方法稳定性，并利用体外转录的L基因RNA标准品建立基因拷贝数绝对定量分析模型。结果引物与探针工作特异性良好，同一样品重复检测Ct值的变异系数均小于1．7％，定量分析模型灵敏度达到100 copies／PCR。结论建立完成针对FLAV的TaqMan PCR检测方法。实验结果显示，本方法具有高特异性、高敏感性、高稳定性、简便、易操作的特点，为今后对 FL A V的检测、监测及相关研究提供技术手段。%The Flanders virus (FLAV) is a number of family Rhabdoviridae ,contains a single‐stranded ,negative‐sense vi‐ral RNA .Here we describe a molecular detection method developed for fast measurement of FLAV based on Taqman RT‐PCR method .In this study ,FLAV specific primers and probe were designed based on the FLAV L gene sequences published in GeneBank .Quantitative standard curve of FLAV TaqMan PCR was also successfully established .The specificity and stability test showed that the system is specific and the coefficient variables were all less than 1 .7% .Quantitative standard curve based on the genomic copy was drawn ,and the lowest detectable limit (LOD) of system was 100 copies/PCR ,with higher sensitivity and stability than that of the conventional RT‐PCR assay targeting the same gene .