利用多重RT-PCR检测水仙黄条病毒、水仙退化病毒和水仙花叶病毒  

Simultaneous detection of Narcissus yellow stripe virus,Narcissus degeneration virus and Narcissus mosaic virus by multiplex RT-PCR

作　　者：金晶[1,2] 付翔[1] 沈建国[2] 蔡伟[2] 梅晨[2] 高芳銮[1] 吴祖建[1] 

机构地区：[1]福建农林大学植物病毒研究所,福建福州350002 [2]福建出入境检验检疫局检验检疫技术中心/福建省检验检疫技术研究重点实验室,福建福州350001

出　　处：《福建农林大学学报（自然科学版）》2015年第5期462-467,共6页Journal of Fujian Agriculture and Forestry University:Natural Science Edition

基　　金：福建省杰出青年科学基金资助项目(2014J06008);国家质检总局科技计划项目(2013IK299;2013IK094);质检公益性行业科研专项(201410076);国家级星火计划项目(2012GA720018);福州市科技计划项目(2013-N-54)

摘　　要：根据水仙黄条病毒、水仙退化病毒和水仙花叶病毒外壳蛋白(CP)基因的保守区序列设计3对特异性引物,通过优化包括Mg2+浓度、c DNA浓度、Taq DNA聚合酶浓度等在内的反应条件,建立了能同时检测这3种病毒的多重RT-PCR检测体系,并与单一RT-PCR做了比较.应用该体系能成功对3种病毒混合侵染的水仙样品进行多重RT-PCR扩增,获得751、623和483 bp 3条与预期大小相符的特异性条带,且RNA稀释至10-4倍时仍能被检测到.建立的多重RT-PCR检测体系具有良好的特异性和较高的灵敏度,比单一RT-PCR扩增更加方便、快速.To develop a multiplex RT-PCR assay to detect single or mixed infections with Narcissus yellow stripe virus( NYSV),Narcissus degeneration virus( NDV) and Narcissus mosaic virus( NMV),three compatible pairs of virus specific primers were designed based on the conserved regions of the partial coat protein( CP) gene sequences of NYSV,NDV and NMV. The system of multiplex RT-PCR was established after optimizing the parameters including the concentration of Mg2+,template c DNA,Taq DNA polymerase and annealing temperature. Expected fragments of 751,623 and 483 bp in size were amplified successively by the assay.The samples infected with the viruses could be detected after the total RNA was diluted to 10-4. The results showed that the multiplex RT-PCR assay could be an effective method to detect viruses in narcissus with high specificity and sensitivity.

关 键 词：水仙黄条病毒 水仙退化病毒 水仙花叶病毒 多重RT-PCR 

分 类 号：S41-32[农业科学—植物保护]