Detection Of Five Tobacco Viruses With Multiplex RT-PCR

Tobacco is one of the most important cash crops worldwide, China’s planting area ranks first in the world. Since the 1970s, tobacco virus disease increase year by year. It was reported that more than 16 viruses have infected tobacco in china,the main viruses contains: Tobacco mosaic virus (TMV), Cucumber mosaic virus (CMV), Potato virus X (PVX), Potato virus Y(PVY),Tobacco etch virus (TEV) ,Tobacco vein banding mosaic virus (TVBMV). Tobacco viruses present the tendency of compound infection. Against the harm of tobacco on the effective and rapid virus detection, prevention and control of tobacco virus has great practical significance.Multiplex RT-PCR by adding more than two pairs of specific primers, and amplified DNA fragments by PCR reactions in the same reaction system, with effective, systematic, economical efficiency and simplicity.In this study, designed primers according to five tobacco viruses TMV, CMV, TEV, PVY and TVBMV coat protein gene sequences , screened the primers at the same annealing temperature, optimized the concentration of templates and primers ,PCR reaction and amplification conditions, Five pairs of optimized primer ratio between TMV: CMV: TEVV: PVY: TVBMV = 1.4:1:1:1:1.5, the ratio of the five templates TMV: CMV: TEV: PVY: TVBMV = 1.4:1: 1:1:1.6, the concentration of Taq DNA polymerase to 0.10 U/μL, Mg2+ concentration to 2.4 mmol/L, dNTPs concentration to 0.28 mmol/L, annealing at 51℃, extending for 60 s and cycling 35 times succeed amplification five kinds of compound infection viruses of tobacco material by multiplex PT-PCR in the same system, obtained five specific fragments of 237bp(TMV), 273bp(CMV), 347bp(PVY), 456bp(TEV), 547bp(TVBMV),set up simultaneous detection of five viruses in multiplex RT-PCR system. Sequencing and homology analysis shows that multiplex RT-PCR results are reliable.Repeat experiments multiplex RT-PCR system for rapid detection of five kinds of tobacco viruses in the same time, and has a high sensitivity.The results show that the extraction of high quality RNA is the key factor for RT-PCR, the ratio of primer and template can directly affect the PCR results. Multiplex RT-PCR can detection tobacco viruses rapidly, establish the foundation of tobacco virus control and the tobacco virus gene function research.