应用逆转录环介导等温扩增技术检测大豆种子中烟草环斑病毒

Detection of Tobacco Ringspot Virus in Soybean Seeds by the Reverse Transcription Loop-mediated Isothermal Amplification Method

根据烟草环斑病毒(TRSV)外壳蛋白基因序列设计并合成了1组RT-LAMP引物,利用实时浊度仪监测反应过程,初步建立了烟草环斑病毒的RT-LAMP检测方法,并进行了特异性与灵敏度检测。结果显示,RT-LAMP灵敏度与普通RT-PCR法相当,但检测时间明显缩短,1 h即可完成反应。此外,体系中加入钙黄绿素,反应结束后,可通过裸眼观察荧光的有无来判断是否有扩增。对大豆种子中携带的烟草环斑病毒进行了RT-LAMP检测,裸眼判定与仪器监测结果一致。结果证明所建立的TRSV RT-LAMP方法具有快速、特异、灵敏,操作简单的特点,不需复杂仪器设备,适合于TRSV的现场快速检测。

A sets of primers were designed and synthesized based on the coat protein coding region of tobacco ringspot virus（TRSV）for reverse transcription loop-mediated isothermal amplification（RT-LAMP）assay.The amplification was detected using the real time turbidimeter.An accelerated RT-LAMP procedure with one step was developed for the detection of TRSV.The specificity and sensitivity of the RT-LAMP assay were tested.RT-PCR compared to RT-LAMP by serial dilutions of total RNA extracts,they had the same sensitivities,but RT-LAMP was quicker,and the reaction could be finished within 1 hour.In addition,the presence or absence of the fluorescent display in daylight allowed naked easy detection of the amplification of TRSV genomic RNA using Calcein.The RT-LAMP assay was applied successfully to detect TRSV in soybean seeds,and the result by the addition of Calcein was consistent with the result detected by the real time turbidimeter.The method is rapid,specific,sensitive and suitable for rapid field detection of TRSV.