关键词: 菜豆荚斑驳病毒, 大豆花叶病毒, 多重RT-PCR, 双链RNA

Abstract: 【Objective】 A multiplex RT-PCR method was developed for simultaneous and rapid detection of Bean pod mottle virus (BPMV) and Soybean mosaic virus (SMV), both of which could cause fairly similar symptom in imported soybean seeds. 【Method】 Two sets of specific primer for the conserved coat protein genes in viral genome regions were designed according to the sequences from GenBank. BPMV and SMV infected healthy soybean seeds were selected as experimental materials with double strand RNA (dsRNA) used as template for multiplex RT-PCR. A series of assays (involving concentrations of primer pairs, Tm value and the number of cycling) were conducted to optimize the multiplex RT-PCR method, of which the specificity was analyzed by detecting BPMV and SMV from the extract of infected and healthy soybean seeds. In order to determine the sensitivity of the established multiplex RT-PCR, a ten-fold serial dilution (from 10^-1 to 10^-6) of infected soybean seed extract was obtained by adding corresponding volume of healthy soybean extract. For the reliability analysis, the amplified DNA fragments of multiplex RT-PCR were recovered, purified, inserted into pMD18-T vector and sequenced. Suspected virus-infected seeds from the United States, Argentina, China and Brazil were detected using the multiplex RT-PCR and single RT-PCR. In addition, multiplex one-step immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR) and multiplex one-step tubecapture reverse transcription-polymerase chain reaction (TC-RT-PCR) were conducted by transferring extract of virus-infected soybean seeds to PCR tubes with or without anti-BPMV/SMV antibodies coated, followed by RT-PCR amplification. 【Result】 The optimal parameters for the multiplex RT-PCR were 0.4 μmol·L^-1 for each of the primers, 52℃ for the Tm value and 35 for the number of cycling. Results of specificity analysis showed two amplified DNA fragment bands (542 and 221 bp, respectively) from BPMV/SMV infected soybean seeds, a 542 bp band from BPMV infected soybean seeds and a 221 bp band from soybean seeds infected with SMV only. No amplified DNA bands were observed from the healthy soybean seeds. Results of sensitivity determination revealed that neither multiplex RT-PCR nor single RT-PCR could amplify any DNA fragments from the infected soybean seeds at a concentration of 10^-3 dilution of extract of infected soybean seeds, suggesting they shared same sensitivity with a limit of 10^-2 dilution of the extract of infected seeds. PCR products, with lengths of 542 bp for BPMV and 221 bp for SMV, respectively, were demonstrated to share high homology with corresponding genes published before by sequencing and analyzing, validating the reliability of the multiplex RT-PCR method. The method was also employed to detect BPMV and SMV from soybean seeds originating from different countries. Three out of four samples of soybean seeds from the US were infected with BPMV and one with SMV, one sample from Argentina and two samples from China with SMV, which was in line with results of single RT-PCR method. Furthermore, two specifically amplified DNA bands were observed from BPMV/SMV infected seeds when performing one-step multiplex IC-RT-PCR and one-step multiplex TC-RT-PCR, whereas no bands showed in the healthy seeds. 【Conclusion】 The multiplex RT-PCR method in this paper could be used for rapid detection of BPMV and SMV in imported soybean seeds.

Key words: Bean pod mottle virus (BPMV), Soybean mosaic virus (SMV), multiplex RT-PCR, dsRNA