江苏省葫芦科作物6种病毒的多重RT-PCR方法及应用

Establishment and application of multiplex RT-PCR assays for simultaneous detection of six viruses on Cucurbit crops in Jiangsu province

江苏省葫芦科作物上的病毒主要有小西葫芦黄花叶病毒（ZYMV）、西瓜花叶病毒（WMV）、黄瓜花叶病毒（CMV）、黄瓜绿斑驳花叶病毒（CGMMV）、烟草花叶病毒（TMV）和番木瓜环斑病毒（PRSV），通常发生复合侵染。基于GenBank中这6种病毒的核苷酸序列保守区设计特异性引物，在单重RT-PCR技术的基础上，通过对影响2种多重RT-PCR扩增的DNA聚合酶浓度、dNTPs及镁离子浓度、退火温度、延伸时间、循环次数等因素进行优化，对检测灵敏度进行测定，建立了2种PCR反应同时检测葫芦科作物上6种病毒的多重RT-PCR方法。结果显示：2种多重RT-PCR体系均能同时扩增出各自特异性片段，测序分析结果表明序列同源性在97％以上：优化的2种多重RT-PC体系检测灵敏度为总RNA稀释10^2～10^3倍，应用于江苏省210份样品的检测结果与单重RT-PCR一致．体现了检测的可靠性；检测结果表明近年来江苏省葫芦科作物以种子传播的ZYMV和CGMMV侵染为主，病毒的复合侵染率达75．24％。

Cucurbit crops are frequently co-infected by zucchini yellow mosaic virus ( ZYMV) , watermelon mosaic virus (WMV), cucumber mosaic virus (CMV), cucumber green mottle mosaic virus (CGMMV), tobacco mosaic virus ( TMV) and papaya ringspot virus ( PRSV) in Jiangsu province. Based on the RT-PCR for the detection of single virus, the two multiplex RT-PCR ( mRT-PCR) systems were established for simultaneous detection of the six viruses by using six sets of specific primers designed on the basis of conserved sequences of the viruses in GenBank. The main factors suches con-centrations of DNA polymerase, Mg2+ and dNTPs, PCR conditions of annealing temperature, extension time and amplifica-tion cycles, sensitivity of the detection were examined and optimized. The results showed that expected fragments of the six viruses were amplified in the two mRT-PCR systems and the sequences shared above 97 percent similarities with those of other relative viruses. The optimized mRT-PCR could detect diluted 100 to 1000 times. The results of the mRT-PCR were consistent with sRT-PCR on detecting 210 sam-ples of cucurbit crops in Jiangsu province which reflects the reliability of the protocol. The results also revealed that in recent years the seed transmission viruses ZYMV and CGMMV were the dominant infectious viruses on cucurbit <br> crops in Jiangsu province, with the co-infection rate of 75. 24%.