单管实时荧光RT-PCR方法同时检测大豆种子中的菜豆荚斑驳病毒和烟草环斑病毒

One-tube real-time PCR assay for simultaneous detection of Bean pod mottle virus and Tobacco ringspot virus in soybean seeds

本研究建立了大豆种子中菜豆荚斑驳病毒(BPMV)和烟草环斑病毒(TRSV)单管双重实时荧光PCR检测方法。将含有相同浓度的分别带有BPMV和TRSVCP基因的质粒溶液作为阳性对照,以受两种病毒侵染的大豆种子作为待测样品进行实时荧光PCR检测,结果表明能从同一管中同时检测出这两种病毒而不发生交叉反应。尽管在阳性对照中,二者的检测限相当,均可达到35 pg/mL,但在实际应用中,两种病毒由于在大豆种子中的浓度不一致而存在一定的差别。该方法快速、灵敏、简便,同时特异性更强,在出入境检验检疫中具有广泛的应用前景。

An one-tube duplex real-time PCR approach for detecting Bean pod mottle virus （BPMV） and Tobacco ringspot virus （TRSV） was developed in this study. The solution of the same concentration of plasmids carrying either the CP gene of BPMV or that of TRSV was used as posivitive controls and the soybean seeds mix-infected by BPMV and TRSV were as experimental samples for real-time PCR detection. The results showed that both viruses could be detected in the same tube without cross-reaction. Although the detection limits for these two viruses were equivalent and up to 35 pg/mL in the positive samples, there were some differences in practical detection due to the uneven concentration of these two viruses in the soybean seeds. This approach suggested a simple, sensitive, rapid and more specific detection of BPMV and TRSV for the entry-exit inspection and quarantine.