桑脉带相关病毒RT-LAMP检测方法的建立

Development of reverse transcription loop-mediated isothermal amplification for the detection of Mulberry vein banding-associated virus

桑脉带相关病毒Mulberry vein banding-associated virus (MVBaV)是广西桑树病毒病的主要病原病毒。本研究根据MVBaV基因组的核外壳蛋白(nucleocapsid protein, N)基因序列设计了5组引物,筛选获得了1组有效引物,建立了该病毒的反转录环介导等温扩增技术(reverse transcription loop-mediated isothermal amplification, RT-LAMP)检测方法。该方法能在恒温条件(63℃)下1 h内检测MVBaV,其灵敏度是RT-PCR检测方法的10倍。对6个田间样品进行RT-LAMP检测,其结果与RT-PCR的结果一致。该方法可直接在反应管中加入SYBR GreenⅠ,通过颜色变化即可判定结果,无需经过琼脂糖电泳或专门仪器,具有灵敏、快速和特异性好的优点,可应用于MVBaV的快速诊断及其田间监测。

Mulberry vein banding-associated virus (MVBaV) is one of the main pathogenic viruses infecting mulberry in Guangxi. Five sets of specific primers were designed based on MVBaV nucleocapsid protein (N) gene for reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay, and one feasible set of primers was selected. The RT-LAMP method was developed for the detection of MVBaV from mulberry. In the RT-LAMP method, MVBaV genomic RNA could be amplified under isothermal conditions (63℃) within 1 h, and was 10 times more sensitive than RT-PCR. Consistent results of six mulberry samples from the field were obtained by the RT-LAMP and RT-PCR assay, suggesting the reliability of RT-LAMP in MVBaV detection.The results were validated by the measurement or observation of the color of the reaction mixture after adding SYBR GreenⅠwithout gel electrophoresis or special equipments. This RT-LAMP method enabled sensitive, rapid and specific detection of MVBaV and could be applied for the rapid diagnosis and surveillance of MVBaV in the field.