小麦3种病毒病BSMV、BYDV-PAV、WYMV及WBD植原体病害的多重PCR同步检测

Simultaneous Detection of Three Wheat Virus BSMV, BYDV-PAV, WYMV and WBD Phytoplasma by Multiplex PCR

【目的】通过对检测小麦病毒病和植原体病害的多重PCR体系各成分和循环参数进行摸索和优化,建立了一种同时检测大麦条纹花叶病毒(barley stripe mosaic virus,BSMV)、大麦黄矮病毒PAV株系(barley yellow dwarfvirus,BYDV-PAV)、小麦黄花叶病毒(wheat yellow mosaicvirus,WYMV)和小麦蓝矮植原体(wheatbluedwarf,WBD)的多重PCR技术体系。【方法】运用根据3种病毒核苷酸保守区序列设计的特异性引物对、根据WBD植原体核糖体蛋白基因序列设计特异性引物对rpF1/R1,从影响多重PCR(M-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多重PCR体系的优化。【结果】成功的在一个体系中对BSMV、BYDV-PAV、WYMV和WBD复合侵染的小麦材料进行多重PCR扩增,得到503、600、898、1240 bp 4条特异性大小与试验设计相符的条带,建立了同时检测4种病原的多重PCR体系。【结论】该体系实现了植原体DNA病原和病毒RNA病原的同时检测,体现了多重PCR的优越性。

[Objective] Reaction components and reaction cycling parameters were optimized for establishing a multiplex PCR （M-PCR） system to simultaneous detect barley stripe mosaic virus, barley yellow dwarf virus-PAV, wheat yellow mosaic virus and wheat blue dwarf phytoplasma. [Method] Using three sets of specific primers designed according to the conserved sequences of the three virus and primers rpF1/R1 designed based upon the ribosomal protein region of WBD, the concentrations of the main ingredients, such as primers, Mg^2＋, Taq DNA polymerase, dNTPs and PCR conditions including annealing temperature and amplification cycles were examined and optimized. [Result] Expected fragments of 503 bp （BSMV）, 600 bp （BYDV-PAV）, 898 bp （WYMV） and 1 240 bp （WBD） were successfully amplified by this M-PCR system, which verified the M-PCR system was successfully established for simultaneous detection of these four pathogens. [Conclusion] The multiplex PCR provides a simple and rapid method for simultaneous detection of DNA phytoplasma pathogen （WBD） and RNA virus pathogen which showed the convenience of this protocol.