复合侵染甘蔗的病原病毒RT-PCR检测

Detection of Viruses Co-infected Sugarcane by RT-PCR

根据甘蔗花叶病毒(sugarcane mosaic virus,SCMV)、高粱花叶病毒(sorghum mosaic virus,SrMV)和甘蔗黄叶病毒(sugarcane yellow leaf virus,SCYLV)外壳蛋白(CP)基因序列分别设计合成3对特异引物SCMV-F/R、SrMV-F/R和SCYLV-F/R,以表现花叶和黄叶复合症状的甘蔗叶片总RNA为模板,分别进行3种病毒单一RT-PCR检测,在SrMV和SCYLV特异引物RT-PCR反应体系中分别扩增到约850bp和630bp特异性片段。序列测定及同源性比对结果显示,850bp片段测序所得序列与浙江象山、临平和余杭SrMV分离物(GenBank登录号分别为AJ310194、AJ310195和AJ310198)对应序列同源性95%,630bp片段测序所得序列与广东、广西、福建SCYLV分离物(GenBank登录号分别为GU190165、GU190162、GU190159)对应序列同源性99%,表明该样品同时感染SrMV和SCYLV。在此基础上建立同时检测SrMV和SCYLV的一步双重RT-PCR体系,可检测1×10-6g病叶组织中的病毒,田间样品检测效果良好。

Three sets of specific primers （SCMV-F/R,SrMV-F/R,SCYLV-F/R） for each virus based on the CP gene sequence of sugarcane mosaic virus （SCMV）,sorghum mosaic virus （SrMV） and sugarcane yellow leaf virus （SCYLV）.Total RNA was extracted from sugarcane leaf with mosaic and yellow leaf symptom.The expected 850 bp and 630 bp fragment was amplified by RT-PCR with SrMV-F/R and SCYLV-F/R,respectively.Sequence analysis indicated 850 bp segment shared identities of 95% homologus with SrMV isolates from Xiangshan,Linping and Yuhang in Zhejiang Province （GenBank accession numbers AJ310194,AJ310195 and AJ310198）.630 bp segment shared identities of 99% homologus with SCYLV isolates from Guangdong,Guangxi and Fujian （GenBank accession numbers GU190165,GU190162,GU190159）.Sequence analysis confirmed the sugarcane was co-infected by SrMV and SCYLV.A double RT-PCR was applied to detecting SrMV and SCYLV base on single RT-PCR.Sensibility test showed it could simultaneously detect out SrMV and SCYLV from 10 -6 g sugarcane leafs infected by virus.It was proved simple,rapid and sensitive for detection of SrMV and SCYLV.