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

doi:10.3864/j.issn.0578-1752.2015.08.07

Abstract

Abstract: 【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.

Key words: Comovirus, Fabavirus, degenerate primers, RT-PCR, detection and identification

YE Zhi-hong, LIAO Fu-rong, GUO Mu-jin, FANG Zhi-peng, CHEN Qing, CHEN Hong-yun, LIN Shi-ming, LIN Yi. A Universal RT-PCR Method for the Simultaneous Detection of the Viruses in Genera Comovirus and Fabavirus[J].Scientia Agricultura Sinica, 2015, 48(8): 1527-1537.

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

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