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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 a one-step RT-PCR method for rapid detection of Narcissus yellow stripe virus (NYSV). Methods: According to the reported sequence of NYSV gene, specific primers were designed and a rapid RT-PCR method was established to detect Nile virus. Results: The method was of good specificity and could amplify the target fragment of the expected size from the narcissus sample infected with narcissus sibiricum without cross-reaction with other viruses. The sequencing results of one-step RT-PCR products showed that, The sequence of the product was highly homologous to the NYSV sequence with a homology of 99%. The sensitivity assay showed that one-step RT-PCR was equivalent to the two-step RT-PCR and could detect 10-4-fold diluted RNA. Conclusion: NYSV was performed on a batch of Narcissus samples by one-step RT-PCR. The results were identical with those of two-step RT-PCR, which showed that the method was accurate for the detection of NYSV.