苹果潜隐性病毒一步三重检测体系的建立与优化

Establishment and optimization of one-step triple RT-PCR detection system of apple latent viruses

【目的】在两步单重检测体系基础上,建立能同时检测苹果茎痘病毒(ASPV)、苹果茎沟病毒(ASGV)和苹果褪绿叶斑病毒(ACLSV)的一步三重检测体系,以简化苹果潜隐性病毒检测方法。【方法】以携带ASPV、ASGV、ACLSV的苹果优系‘浓果-25’组培苗为试材,比较一步单重、一步两重和一步三重RT-PCR 3种检测体系的检测效果,对一步三重检测法中的退火温度、反转录时间、延伸时间、循环数和引物加量进行优化,并用带毒情况不同的苹果田间根茎叶、组培苗、脱毒苗对优化后的RT-PCR一步三重检测体系进行验证。【结果】一步单重RT-PCR体系一次只能检测1种病毒,一步两重RT-PCR体系一次能同时检测2种病毒,一步三重RT-PCR体系一次能同时检测3种病毒。优化后的一步三重RT-PCR检测体系为:50℃反转录20 min;94℃预变性20 min;94℃变性30 s,53~56℃退火30 s,72℃延伸30 s,30个循环,引物加量为各病毒上、下游引物(10μmol/L)各1.0μL,无需进行终延伸。优化后的一步三重检测体系验证结果与两步单重检测结果一致,初步证明该检测体系合理。【结论】建立了苹果潜隐性病毒一步三重检测体系,该体系能同时对苹果样品中ASPV、ASGV和ACLSV 3种病毒进行检测。

【Objective】Based on the two-step single detection system,a one-step triple detection system capable of simultaneously detecting apple stem pox virus(ASPV),apple stem groove virus(ASGV)and apple chlorotic leaf spot virus(ACLSV)was established and optimized to simplify apple virus detection.【Method】The apple seedlings of‘Nongguo-25’carrying ASPV,ASGV and ACLSV were used and the detection results of single-step,one-step double and one-step triple RT-PCR detection systems were compared.The annealing temperature,reverse transcription time,extension time,number of cycles and amount of primers in the one-step triple detection method were optimized.Apple roots,tissue cultured seedlings and virus-free seedlings with different toxic conditions were used to verify the established system.【Result】One step single RT-PCR system can detect only one virus at a time,one step double RT-PCR system can detect two viruses at one time,while one step triple RT-PCR system can detect three viruses simultaneously.The optimized conditions of one-step triple RT-PCR system were:reversing transcription at 50℃for 20 min,predenaturation at 94℃for 20 min,denaturation at 94℃for 30 s,annealing at 53-56℃for 30 s,extension at 72℃for 30 s,30 cycles,10μmol/L upstream primer 1.0μL,10μmol/L downstream primer 1.0μL,and no final extension.The verification results were consistent with the two-step single method,showing that the established detection system was reasonable.【Conclusion】One-step triple detection system for apple latent viruses was established to simultaneously detect ASPV,ASGV and ACLSV viruses.