Simultaneous Detection Of Three Quarantine Viruses And One Endogenous Gene From Soybean Seeds By Multiplex RT-PCR

Soybean, as the most important economic crops in the world, has a large trade in all countries every year, but it can be infected by many fungi, bacteria, viruses, worms, insects and pathogens during its process of growth. Bean pod mottle virus(BPMV), Tobacco ringspot virus(TRSV) and Tomato ringspot virus(ToRSV) are all quarantine viruses that infect soybean seeds, therefore, they are our main targets of detection. The establishment of the method for multiple PCR detection is very important for improving the detection efficiency and serving the trade transportation.In this study, the soybean seeds which had been infected by BPMV, TRSV, ToRSV were chosen as experimental materials. Several pairs of primers were synthesised according to the CP gene of BPMV, TRSV, ToRSV, then the specificity of the single gene PCR reaction was verified, at the same time, the soybean endogenous gene (BD30K) was chosen as a control to ensure the extraction of RNA, the synthesis of the first chain of cDNA and that the PCR process is correctly operated, as well as preventing the occurrence of the false negative result. By properly assembling the four pairs of virus-specific primers and regulating the conditions of the multiple PCR reactions, four pairs of virus-specific primers were chosen and the conditions of the multiple PCR reactions was determined. A multiple RT-PCR system was established to detect the four viruses in soybean at the same time by adjusting the concentration of buffer to 1.4×and concentration of the BD30K primers was increased to twice of others primers. The chosen primers could amplify a 275bp fragment of BPMV, a 580bp of TRSV, a 794bp of ToRSV and 104bp of BD30K simultaneously in the samples. The results indicated that good specificity and sensitivity were obtained in simultaneous detection. The minimum content of RNA detected in BPMV, TRSV, ToRSV and BD30K were 0.9ng, 0.186ng, 8.0pg, 3.8ng respectively; When the soybeans with three viruses were mixed together, the minimum content of RNA detected was 1.16ng. The data indicated that the multiple PCR method is advantageous in that it is time-efficient, with high specificity, sensitivity and easy in operation. It can be used by the port for the rapid detection of the three viruses in the soybean seeds entering our country and has a broad application prospect in the entry-exit inspection and quarantine.In addition, an one-tube duplex Real-time PCR approach for detection BPMV and TRSV was developed in this study. The same concentration of plasmids carring the CP gene of BPMV or that of TRSV was used as posivitive control, and the soybean seeds mix-infected by BPMV and TRSV were chosen as experimental material for Real-time PCR detection. The results showed that both viruses could be detected in the same tube without cross-reaction. Although the detection limitations for these two viruses were equivalent and up to 35 pg/ml in the positive samples, but in sample, the detection limitations for these two viruses varied for the concentration of these two viruses in the soybean seeds was different. By comparing the sensitivity of the real-time PCR with those of DAS-ELISA and RT-PCR, the results showed that the real-time PCR was 103 and 106 times more sensitive than DAS-ELISA in detecting BPMV and TRSV in soybean seeds, and 100 times more sensitive than normal RT-PCR. So the duplex real-time PCR technology has a wide application prospect in the detection of viruses, and the establishment of this method plays a significant role in agricultural production, as well as the circulation, inspection and quarantine of seeds.