荧光RT-PCR法快速检测微量建兰花叶病毒研究

Rapid Detection of Trace Amounts of Cymbidium Mosaic Virus by Real-Time RT-PCR

根据基因库中建兰花叶病毒外壳蛋白基因的保守序列,设计并合成3对特异性引物,利用荧光RT-PCR方法在蜘蛛兰中检测到普通RT-PCR-琼脂糖电泳法检测不到的微量病毒。熔解曲线分析表明,每一扩增产物为单一产物形态。通过测序和同源性分析,试验证实扩增产物序列的实际长度与预期完全相符,蜘蛛兰样品的扩增产物基因序列与基因库中建兰花叶病毒相应序列的同源性为94%～96%。与普通RT-PCR的相比,荧光RT-PCR方法具有灵敏度高、操作快速、结果直观准确的特点,可应用于种苗中微量建兰花叶病毒的早期检测。

According to the conserved sequence of the coat protein gene of Cymbidium mosaic virus （CymMV）in the GeneBank, three pairs of primers used in real-time RT-PCR methods were designed and synthesized for detecting traces of the virus which could not be detected by ordinary RT-PCR-Agarose gel electrophoresis in spider orchid （A rachnis sp. ）. Melting curve analysis showed that each amplified product was a single product form. Sequencing and homology analysis indicated that the actual sequence lengths of the amplified products were entirely consistent with expectations, and the amplified gene had between 94% to 96% homology with the corresponding gene of CymMV in GeneBank in spider orchid. Compared to ordinary normal RT-PCR,the real-time RT-PCR method characterized by high sensitivity,fast operation,intuitive and accurate,is suitable for detection of trace amounts of CymMV and early diagnosis in seedlings.