马铃薯5种病毒多重PCR检测技术的建立及应用

Establishment and Application of Multiplex PCR Rapid Detection of Potato Viruses

采用多重RT-PCR方法,建立可同时检测马铃薯X病毒(PVX)、马铃薯M病毒(PVM)、马铃薯Y病毒(PVY)、马铃薯A病毒(PVA)和马铃薯S病毒(PVS)的方法。根据Gen Bank中PVX、PVM、PVY、PVA及PVS(CP)基因序列设计特异引物,对多重RT-PCR退火温度、循环次数、延伸温度、引物组合浓度进行优化,建立了能同时检测5种马铃薯病毒的多重RT-PCR方法。该方法能同时扩增出PVX、PVM、PVY、PVA及PVS特异片段,其大小分别是138、213、369、468和657 bp。测序结果表明,5种病毒的序列与相应参考序列相似性达到97%以上。灵敏度分析结果表明,多重RT-PCR方法能够检测植物组织量为10-3 mg。应用建立的多重RT-PCR检测方法对田间样品和组培苗进行检测,结果显示,该方法可以准确、快速、灵敏地同时检测单一或复合侵染的5种马铃薯病毒。

A multiplex reverse transcription polymerase chain reaction(multiplex RT-PCR)method was developed for the simultaneous detection of five potato viruses,Potato virus X,Potato virus M,Potato virus Y,Potato virus A and Potato virus S. Five compatible sets of primers specific for each virus were designed based on conserved sequences of coat protein(CP)gene for multiplex RT-PCR assay. The crucial factors of multiplex RT-PCR including annealing temperature,number of the PCR cycles,extension temperature and primers concentration were optimized for the highest sensitivity and specificity. Five specific fragments were simultaneously amplified in uniplex PCR reaction. Their molecular weights were determined to be 138,213,369,468 and 657 bp. The sequence analysis indicated that the five viruses shared at least 97% homology with other related viruses. The sensitivity was shown to be capable of positively identifying the five viruses with ≥ 10-3 mg of freesia tissue sample. This multiplex RT-PCR protocol can simultaneously detect five potato viruses from field samples and plantlets with accurately,rapidity,sensitivity.