建兰花叶病素荧光PCR检测技术研究.docVIP

荧光RT-PCR法快速检测微量建兰花叶病毒的研究 柳爱春 刘超 李峰 (杭州市农业科学研究院，浙江 杭州 310024) 摘要： 根据基因库中建兰花叶病毒(Cymbidium mosaic virus)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（Arachnis sp．）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，实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，且操作快速,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。 关键词：建兰花叶病毒；荧光RT-PCR；快速检测；蜘蛛兰 Rapid detection of trace amounts of Cymbidium mosaic virus by real time RT-PCR LIU Ai-chun ,LIU Chao ,Li Feng ( Hangzhou Academy of Agricultural Sciences Zhejiang , Hangzhou 310024 , China) Abstract According to the conserved sequence of the coat protein gene of Cymbidium mosaic virus (CymMV) in the gene bank, 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 (Arachnis 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 gene bank 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 . Keywords Cymbidium mosaic virus，real-time RT-PCR，Rapid detection，Arachnis sp. 建兰花叶病毒(Cymbidium mosaic virus，简称CymMV)和齿兰环斑病毒(OdontogLossum ringspot virus，简称ORSV)是发生最为普遍的兰花病毒，广泛分布于全世界栽培兰花的地区［1-6］，这两种病毒传染性强，其中CymMV相对ORSV更易传染，在市场上蝴蝶兰、文心兰和卡特兰CymMV的感染率可高达66%以上［7］，病毒侵染导致植株生长不良甚至死亡，有时病毒感染后还会在植株的营养生长期出现较长时间的隐症现象，但带毒植株开花小而少，花期缩短，发病兰株观赏价值和经济价值大为下降，还会导致种质退化，对兰花产业造成严重危害。 多年来，国内学者对不断深入地对CymMV的检测技术开展研究工作：1997年，潘俊松等从石桷兰中分离出CymMV用于制备抗血清［8］；郑平等用组织涂抹酶联免疫吸附技术检测CymMV和ORSV［9］；周国辉等用RT-PCR方法对病毒分子进行鉴定［10］；2007年施农农等对用