侵染西瓜的5种病毒ZYMV、WMV、TMV、SqMV和CMV的多重RT-PCR检测体系的建立与检测应用

Establishment and application of multiplex RT-PCR for simultaneous detection of five watermelon viruses ZYMV,WMV,TMV,SqMV and CMV

根据5种病毒小西葫芦黄花叶病毒(Zucchini yellow mosaic virus,ZYMV)、西瓜花叶病毒(Watermelon mosaic virus,WMV)、烟草花叶病毒(Tobacco mosaic virus,TMV)、南瓜花叶病毒(Squash mosaic virus,SqMV)和黄瓜花叶病毒(Cucumber mosai cvirus,CMV)的核苷酸保守区序列,设计特异性引物对,从影响多重RT-PCR(mRT-PCR)扩增的引物浓度、Mg2+浓度、TaqDNA聚合酶浓度、dNTPs浓度、退火温度等方面进行反应体系的优化,建立了一种能够同时检测ZYMV、WMV、TMV、SqMV和CMV的多重RT-PCR技术体系,并进行了实际应用。在一个体系中对上述5种病毒复合侵染的西瓜材料进行多重RT-PCR扩增,得到与试验设计相符的5条特异性条带,依次是542、485、410、354和293bp。该体系实现了对侵染西瓜的5种病毒的同时检测,极大地提高了检测效率,降低了检测成本,体现了多重RT-PCR的优越性。

A multiplex RT-PCR（mRT-PCR） system was established for simultaneous detection on Zucchini yellow mosaic virus（ZYMV）,Watermelon mosaic virus（WMV）,Tobacco mosaic virus（TMV）,Squash mo-saic virus（SqMV）and Cucumber mosaic virus（CMV）infected watermelon by using five sets of specific primers designed on the basis of conserved sequences of the 5 viruses. 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. The results showed that expected fragments of 542 bp（ZYMV）,485 bp（WMV）,410 bp（TMV）, 354 bp（SqMV） and 293 bp（CMV）were amplified and the mRT-PCR system was successfully established and the multiplex RT-PCR had been applied in practice, provided a simple, rapid and economic method for simultaneous detection of the five watermelon viruses.