应用多重RT-PCR检测百合无症病毒和百合斑驳病毒

Detection of Lily symptomless virus and Lily mottle virus by Multiplex RT-PCR

根据病毒外壳蛋白基因序列,设计了2对检测百合无症病毒(LSV)、百合斑驳病毒(LMoV)的引物,对扩增条件进行优化,建立了同时检测LSV和LMoV的多重RTPCR检测方法。此方法可特异地从带有LSV和LMoV的样品中扩增出2条带LSV(876bp)、LMoV(662bp)。灵敏性测定结果表明,该双重PCR可从稀释104组织中检测出病毒,具有与单一PCR相同的灵敏性。扩增产物测序表明,LSV扩增产物与其它分离物核苷酸同源性为87.8%～99.3%,LMoV扩增产物与其它分离物的同源性为90.1%～99.5%。

Two sets of specific primers were designed according to LSV and LMoV CP gene sequence, and the conditions for PCR were optimized. The multiplex PCR method which can simultaneously detect the two lily viruses was developed. It was showed that all samples which infected LSV and LMoV could be amplified by multiplex PCR, yielding tow specific bands of LSV(876 bp) and LMoV(662 bp). The two viruses were detected from dilutions of 10 4 by multiplex RT-PCR and RT-PCR, respectively. Sequence analysis on the amplified products show that the nucleotide sequences homology of LSV was 87.8%~99.3% compared with sequences of other isolates, and LMoV was 90.1%~99.5%.