CAJ | 학술논문

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102~103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%。
강소성호호과작물상적병독주요유소서호호황화협병독( ZYMV)、서과화협병독( WMV)、황과화협병독( CMV)、황과록반박화협병독( CGMMV)、연초화협병독( TMV)화번목과배반병독( PRSV)，통상발생복합침염。기우GenBank중저6충병독적핵감산서렬보수구설계특이성인물，재단중RT-PCR기술적기출상，통과대영향2충다중RT-PCR확증적DNA취합매농도、dNTPs급미리자농도、퇴화온도、연신시간、순배차수등인소진행우화，대검측령민도진행측정，건립료2충PCR반응동시검측호호과작물상6충병독적다중RT-PCR방법。결과현시：2충다중RT-PCR체계균능동시확증출각자특이성편단，측서분석결과표명서렬동원성재97%이상；우화적2충다중RT-PC체계검측령민도위총RNA희석102~103배，응용우강소성210빈양품적검측결과여단중RT-PCR일치，체현료검측적가고성；검측결과표명근년래강소성호호과작물이충자전파적ZYMV화CGMMV침염위주，병독적복합침염솔체75.24%。
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%.