坦布苏病毒荧光定量RT-PCR检测方法的建立及初步应用

Development and application of SYBR GreenⅠfluorescence quantitative RT-PCR for detection of Tembusu virus

为建立检测坦布苏病毒的SYBR GreenⅠ绝对荧光定量RT-PCR,针对坦布苏病毒E基因设计1对特异性引物,用PCR扩增E基因后将其连接到pMD19-T载体上构建重组质粒。重组质粒经PCR及测序鉴定后,作为阳性模板绘制了SYBR GreenⅠ荧光定量PCR标准曲线,并进行特异性、敏感性和重复性试验。结果显示,经反应条件优化后,绘制的SYBR GreenⅠ绝对荧光定量RT-PCR的标准曲线的线性关系显著(r2>0.999),平均试验间变异系数为0.26%;检测敏感性可达到2×101 copies/μL。应用该方法对人工感染坦布苏病毒的鸭组织进行的检测结果显示,从36份病料组织中检出35份为阳性,检出率为97%。结果表明,成功建立了检测坦布苏病毒的SYBR GreenⅠ荧光定量PCR,该方法较常规PCR更快捷、敏感、准确,适用于坦布苏病毒临床样品的检测。

In order to develop SYBR GreenⅠ absolute fluorescent RT-PCR for detecting Tembusu virus in duck,one pair of primers based on Tembusu virus E gene was designed and synthesized for amplification of E gene by RT-PCR,and the amplified product was identified and cloned into pMD19-T vector to construct a recombinant plasmid.The positive recombinant plasmid was extracted as the standard plasmid of fluorescent quantitative PCR.The standard plasmid was used as a quantitative template to establish the SYBR GreenⅠreal-time RT-PCR for Tembusu virus in duck,the specificity,sensitivity and repeatability of the real-time RT-PCR were evaluated respectively.In result,the SYBR GreenⅠ real-time RT-PCR was successfully established for detection of Tembusu virus in duck by optimizing the reaction conditions.The correlation coefficient of r2 was above 0.999,the mean repeatability tests coefficient of variation was0.26%,and the sensitivity of the assay was 2×101 copies/μL.Total 35 positive samples were detected out of 36 tissue samples from the infected-artificially ducks with the Tembusu virus AH-F10 strain,and the detection rate of 97%(35/36)was observed.In conclusion,a SYBR GreenⅠ fluorescent quantitative RT-PCR was established for detection of Tembusu virus in duck,which was more rapid,sensitive and accurate than the conventional RT-PCR,and might be used to detect the clinical samples of Tembusu virus infection.