玉米褪绿斑驳病毒RT-LAMP快速检测方法的建立(英文)

Reverse Transcription-loop-mediated Isothermal Amplification for Detection of Maize Chlorotic Mottle Virus

玉米褪绿斑驳病毒(Maize chlorotic mottle virus,MCMV)是我国对外公布的检疫性有害生物,采用逆转录环介导等温扩增技术(Reverse transcription-loop-mediated isothermal amplification,RT-LAMP),建立了一种快速、灵敏和特异的MCMV检测方法。根据MCMV外壳蛋白基因序列的保守区设计了3组引物,通过特异性试验筛选确定了第二组引物。灵敏度试验显示该方法的灵敏度与RT-PCR方法相当,而检测时间明显缩短,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。研究组还对玉米种子中携带的玉米褪绿斑驳病毒病毒进行了RT-LAMP检测,结果表明,裸眼判定与仪器监测结果一致。该研究所建立的MCMV 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MCMV的现场快速检测。

Maize chlorotic mottle virus （MCMV） is a quarantine pest as approved by Chinese government： A rapid, sensitive and specific MCMV detection method using reverse transcription-loop-mediated isothermal amplification （RT-LAMP） was established in this study. Based on the sequence of MCMV coat protein coding gene, 3 sets of primers were designed and specificity test showed that the second set of primers was specific to MCMV, Similar sensitivities were observed on RT-LAMP and RT-PCR, except that RT-LAMP was quicker, and the reaction could be finished within 1 h. In addition, the presence or absence of the fluorescence under daylight allows naked easy detection of the amplification of MCMV genomic RNA using calcein. The RT-LAMP assay was applied successfully to detect MCMV in maize 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 without the need for complicated equipment, and is suitable for rapid field detection of MCMV.