逆转录环介导等温扩增技术检测南方菜豆花叶病毒

Detection of Southern bean mosaic virus by the reverse transcription loop-mediated isothermal amplification method

根据南方菜豆花叶病毒外壳蛋白基因序列设计并合成了4组RT-LAMP引物,通过引物筛选试验,确定SB1组引物为最佳引物,并进行了引物特异性与灵敏度检测试验,最终建立了南方菜豆花叶病毒的RT-LAMP检测方法。灵敏度检测试验显示,RT-LAMP方法比普通RT-PCR法灵敏度高10倍,而检测时间明显缩短,整个反应过程只需40 min。此外,体系中加入钙黄绿素,反应结束后,可裸眼观察颜色变化来判定结果。本研究所建立的SBMV RT-LAMP方法具有快速、稳定、灵敏、特异、操作简单的特点,适合于SBMV的现场快速检测。

Four sets of primers were designed and synthesized using the coat protein coding region of Southern bean mosaic virus （SBMV） for reverse transcription loop-mediated isothermal amplification （RT-LAMP） assay. Based on the initial experiments the set SB1 was selected for further evaluation. The specificity and sensitivity of the RT-LAMP assay were tested. An one step, accelerated RT-LAMP procedure was developed for the detection of SBMV. The sensitivity test showed that the RT-LAMP reaction could be finished within 40 minutes, and it was ten times more sensitive than RT-PCR assay. Moreover, adding calcein to the reaction tube, after the amplification reaction was completed, the alteration in color of reacted solution under daylight allowed visual detection of SBMV. The method is rapid, stable, sensitive, specific and simple, which is suitable for rapid field detection of SBMV.