逆转录环介导等温扩增技术检测烟草环斑病毒的研究

Research on detection of Tobacco ringspot virus by RT-LAMP

本研究采用环介导等温扩增技术(Loop-mediated isothermal amplification,LAMP),建立了一种快速、简便的烟草环斑病毒检测方法。根据TRSV外壳蛋白编码基因上的8个位点,共设计了6条引物,通过RT-LAMP扩增得到特征性的梯度条带。特异性试验表明,引物对TRSV的检测具有良好的特异性;灵敏度试验显示RT-LAMP比普通RT-PCR高10倍。通过反应温度和时间的优化,该方法只需在水浴锅中60℃等温扩增60 min,整个检测周期约1.5 h,结果采用SYBR green I染色显示,易于观察和判定。

The study established a rapid and simple Tobacco ringspot virus （TRSV）detection method using Loop-mediated isothermal amplification （LAMP）. According to the eight sites on TRSV coat protein coding genes, six primers were designed and characteristic ladder-like bands were obtained by RT-LAMP. Specificity tests showed that the primers were specific for detection of TRSV and sensitivity test showed that RT-LAMP was 10 times higher than RT-PCR. By the optimizing of temperature and time condition, the method just need the pot with a water bath 60℃ isothermal amplification for 60 min, the entire testing cycle is about 1.5 h, and it is very convenient to observe the results by using SYBR green I.