同时检测豇豆花叶病毒属与蚕豆病毒属病毒的通用RT-PCR检测方法

A Universal RT-PCR Method for the Simultaneous Detection of the Viruses in Genera Comovirus and Fabavirus

【目的】建立一个可同时检测豇豆花叶病毒属(Comovirus)和蚕豆病毒属(Fabavirus)病毒的通用RT-PCR检测方法。【方法】通过基因组序列的多重比对分析,在其保守区域设计简并引物,用于这2个病毒属病毒的通用RT-PCR检测,并进行灵敏性、特异性试验。为便于PCR产物直接测序,在简并引物中引入通用测序引物(RV-M和M13-47),并通过序列分析对病毒种类鉴定。利用该方法对来自福建柘荣的太子参病毒进行检测验证。【结果】设计一对简并引物,建立了一个用于扩增豇豆花叶病毒属和蚕豆病毒属病毒部分依赖于RNA的RNA聚合酶(Rd Rp)基因的通用RT-PCR方法。该方法成功用于检测安第斯马铃薯斑驳病毒(Ap Mo V)、蚕豆染色病毒(BBSV)、蚕豆真花叶病毒(BBTMV)、菜豆荚斑驳病毒(BPMV)、豇豆花叶病毒(CPMV)、豇豆重花叶病毒(CPSMV)、南瓜花叶病毒(Sq MV)、红三叶草斑驳病毒(RCMV)和萝卜花叶病毒(Ra MV)9种豇豆花叶病毒属病毒;蚕豆萎蔫病毒1号(BBWV1)、蚕豆萎蔫病毒2号(BBWV2)2种蚕豆病毒属病毒共计17个分离物,而不能与同亚科的线虫传多面体病毒属病毒及健康寄主植物反应,显示出良好的广谱性和特异性。在简并引物的5′端分别加入一段非互补的通用测序引物序列(RV-M和M13-47),不仅可以利用该通用测序引物进行PCR产物直接测序,而且检测灵敏度可以提高10—100倍。序列及系统发育分析表明,所扩增的序列可以把豇豆花叶病毒属和蚕豆病毒属病毒区分到种的水平。利用该方法测定了BBSV的部分Rd Rp基因序列,显示出与RCMV具有最近的亲缘关系。利用该方法在太子参中检出BBWV2。【结论】建立的通用RT-PCR方法可用于豇豆花叶病毒属和蚕豆病毒属病毒的广谱性检测,结合序列可进行病毒种类的快速鉴定,并且可能用于新病毒种类的检测鉴定。

[Objective] The objective of the study is to develop a universal RT-PCR method for the simultaneous detection of the viruses from Comovirus and Fabavirus.[Method]Analysis of the complete nucleotide sequence of comoviruses and fabaviruses was used to design a pair of degenerate primers for specific detection of members of the two genera. The sensitivity and specificity were evaluated, respectively. In order to direct the sequence, the non-complementary universal sequencing primer sequence RV-M and M13-47 were added, respectively, to the 5′termini of the primers. Amplicons were directly sequenced to verify their identity. Finally, the method was used to detect viruses in Pseudostellaria heterophylla coming from Zherong, Fujian Province. [Result]A generic PCR protocols was developed to detect the two virus genera in Comovirus and Fabavirus using degenerate primers designed to amplify part of the RNA-dependent RNA polymerase （RdRp） gene. An expected size product about 350 bp was amplified using the optimized PCR protocols from all 17 isolates of the 9 comovirus species and 2 fabavirus species tested including Andean potato mottle virus （APMoV）, Broad bean stain virus （BBSV）, Broad bean true mosaic virus （BBTMV）, Bean pod mottle virus （BPMV）, Cowpea mosaic virus （CPMV）, Cowpea severe mosaic virus （CPSMV）, Radish mosaic virus （RaMV）, Red clover mottle virus （RCMV）, Squash mosaic virus （SqMV）, Broad bean wilt virus 1 （BBWV1） and Broad bean wilt virus 2 （BBWV2）. When the RV-M and M13-47 primer sequences were added, respectively, to the 5′termini of the primers, it was not only observed that the PCR’s amplicons could be directly sequenced by the universal sequencing primer, but also it could improve the detection sensitivity by 10-100 times. No cross-reaction was observed with either healthy plants or from isolates in the genus Nepovirus of the Comovirinae. Phylogenetic analysis using the generic PCR’s amplicons sequence showed that it could differentiate comoviruses and fabaviruses at the species level. The partial sequence of the RdRp gene of BBSV was firstly determined by this method, and was shown to have the closest relationship with RCMV. Using this method, BBWV2 was detected in P. heterophylla.[Conclusion]The described generic assay could be applied for the broad spectrum detection of members of the genus Comovirus and Fabavirus and, in combination with the PCR’s amplicons sequence, for the identification of species in the two genera. The assay may also be useful for the detection of new or uncharacterized species within the two genera.