微量植物组织直接RT-PCR反应检测4种植物病毒的方法建立与优化

Establishment and Optimization of Plant Microtissue Direct Reverse Transcription-Polymerase Chain Reaction(MD RT-PCR)for Detection of Four Types of Plant Viruses

建立并优化微量植物组织直接RT-PCR反应检测病毒的方法:在立体显微镜下,用26 G无菌注射器针头刺入植物茎、叶柄或根中,将沾有组织液的针头直接浸入RT反应液中,通过PCR反应产生病毒特异性DNA条带.该方法在葡萄、苹果、马铃薯和百合中有效检测了葡萄卷叶相关病毒3(GLRav-3)、苹果茎沟病毒(ASGV)、马铃薯卷叶病毒(PLRV)和黄瓜花叶病毒(CMV);消除了传统RT-PCR制备RNA模板复杂耗时的缺点.

A quick detection of viruses is required in virus-free plant tissue culture and plant quarantine inspection by institutions.Hence a microtissue direct reverse transcription-polymerase chain reaction(MD RT-PCR)method is established and optimized in this study.Under a stereoscopic microscope,a sterile syringe needle(26 G)is used to pierce plant stem,petioles or roots,and the needle with plant tissue liquid is directly transferred to RT mixture,followed by RT-PCR procedures to produce specific DNA band of virus.MD RT-PCR efficiently detectes four plant viruses belonging to four different genera that are Grapevine leafroll-associated virus-3(GLRa V-3),Apple stem grooving virus(ASGV),Potato leafroll virus(PLRV)and Cucumber mosaic virus(CMV)respectively in grapevines,apples,potatoes and lilies.MD RT-PCR eliminates the complicated,time-consuming procedure of RNA template preparation required in the traditional RT-PCR.Thus,MD RT-PCR has potential applications for quick detection of viruses in virus-free plant tissue culture and plant quarantine inspection by institutions.