一步RT-PCR检测水仙黄条病毒

Detection of Narcissus Yellow stripe virus by One- step RT- PCR

目的:建立用于快速检测水仙黄条病毒(Narcissus yellow stripe virus,NYSV)的一步RT-PCR方法。方法:根据已报道的NYSV基因序列设计特异性引物,采用一步RT-PCR建立了快速检测水仙黄条病毒的方法。结果:该方法具有良好的特异性,能够从感染水仙黄条病毒的水仙样品上扩增出预期大小的特异性目的片段,而与其他病毒无交叉反应;一步RT-PCR产物序列测定结果表明,该产物的序列与NYSV序列高度同源,同源性达99%;灵敏度测定结果显示,一步法RT-PCR与两步法RT-PCR的灵敏度相当,可以检测稀释到10-4倍的RNA。结论:应用建立的一步RT-PCR对一批水仙样品进行NYSV检测,结果与两步法RT-PCR完全相同,说明该方法可准确用于NYSV的检测。

Objective：To establish one -step RT- PCR method for rapid detection of Narcissus yellow stripe virus （NYSV）. Method：One -step RT- PCR was developed to detect NYSV in narcissus sample by using a pair of specific primers designed according to the pub- lished nucleotide sequence of NYSV. Result：The method showed good specificity, which could successfully amplify target fragment from NYSV -infected sample,while not from other virus infected sample. The sequence analysis showed that nucleotide sequence of the ampli- fied product was 99% identical to the sequences of NYSV in GenBank. The sensitivity of one - step RT - PCR for detection of NYSV was equal to that of two -step RT- PCR. The two methods could detect NYSV when tested RNA was diluted to 10^-4. Conclusion： NYSV in narcissus samples was determined by one -step RT -PCR, and the result was consistent with that of two -step RT -PCR, which suggested that one - step RT - PCR could be accurately used for detection of NYSV.