4种番茄病毒多重RT-PCR检测及应用

Establishment and application of multiplex RT-PCR for simultaneous detection of four viruses infecting tomato

番茄是全世界栽培最为普遍的果菜之一,2011年世界番茄栽培面积约805万亩,年产量约3 773万t,我国是世界番茄种植大国之一,2011年面积96667公顷,产量约679万t,随着番茄种植面积不断扩大,番茄病毒病的危害逐年加重。世界范围内番茄除了已有的TMV抗病资源与育成的抗病品种外,缺少CMV等抗病种质与抗病品种。

Tomato virus diseases happens all the year round, causing serious yield losses. For quick detection of these viruses and mading foundation to disease control and breeding for disease resistance, a multiplex reverse transcription polymerse chain reaction （mRT-PCR） system was established for simultaneous detection on Tobacco mosaic virus （ TMV）, Cucumber mosaic virus （ CMV） , Tomato spotted wilt virus （ TSWV ） and Potato virus X （PVX） in tomato. Four sets of specific primers were designed according to the coat protein （CP） genes of the four viruses, respectively. The concentrations of the primers, Mg2＋, Taq DNA polymerase and dNTPs were examined and the PCR conditions including annealing temperature and amplification cycles were optimized. Expected fragments of 156 bp （PVX）, 257 bp （TMV）, 314 bp （TSWV） and 448 bp （CMV） were amplified by this mRT-PCR system, which verified the mRT-PCR system was successfully established. The multiplex RT-PCR system can accurately and quickly determine the infection of the four virus with a similar detection sensitivity to each single RT-PCR.