双重RT-PCR快速检测水仙普通潜隐病毒和尼润潜隐病毒

Rapid Detection of Narcissus Common Latent Virus and Nerine Latent Virus by Duplex RT-PCR

目的:针对水仙上水仙普通潜隐病毒(Narcissus common latent virus, NCLV)和尼润潜隐病毒(Nerine latent virus, NeLV)建立同时检测两种病毒的双重RT-PCR方法。方法:参照GenBank上已报道的NCLV和NeLV CP基因序列设计特异性引物,在单一RT-PCR检测方法的基础上,通过对PCR反应体系和反应条件的优化,建立了可以同时检测NCLV和NeLV的双重RT-PCR方法,并对其特异性、灵敏度进行了测定,同时应用于水仙实际样品的检测。结果:建立的双重RT-PCR检测方法具有良好的特异性和灵敏度,能够有效区分水仙上NCLV、NeLV两种病毒,并且检测灵敏度不低于单一RT-PCR,水仙实际样品检测结果与单一RT-PCR检测结果相符。结论:建立的NCLV、NeLV双重RT-PCR检测方法特异性强、灵敏度高、准确性好,适用于口岸及农业生产上NCLV、NeLV的快速检测。

This paper aims to develop a rapid, sensitive and specific duplex RT-PCR method to simultaneously detect Narcissus common latent virus(NCLV) and Nerine latent virus(NeLV). Two pairs of specific primers were designed from the conserved regions of CP gene in the published NCLV and NeLV sequences from GenBank. A duplex RT-PCR assay was established and optimized for simultaneous detection of these two viruses based on single RT-PCR. The specificity and sensitivity were also determined. In addition, this assay was applied to detect narcissus samples. The established duplex RT-PCR method can effectively distinguish NCLV and NeLV on narcissus which showed high specificity and sensitivity. The method demonstrating sensitivity is not lower than that of single RTPCR. The result obtained from the duplex RT-PCR assay is consistent with those of single PCR assays. It is concluded that the duplex RT-PCR assay reported here is highly specific, sensitive and accurate, which could be applied to the rapid detection of both NCLV and NeLV at port and agricultural production.