双重RT-PCR法同步检测单头异沙叶蝉携带的两种小麦病毒  

Simultaneous detection of two wheat viruses harbored by a single leafhopper (Psammotettix alienus) by duplex RT-PCR

作　　者：杜真真 刘艳[1] 王锡锋[1] DU Zhenzhen;LIU Yan;WANG Xifeng(State Key Laboratory for Biology of Plant Diseases and Insect Pests,Institute of Plant Protection,Chinese Academy of Agricultural Sciences,Beijing 100193,China)Abstract It was found that the leafhopper(Psammotettix alienus)

机构地区：[1]中国农业科学院植物保护研究所,植物病虫害生物学国家重点实验室,北京100193

出　　处：《植物保护》2020年第3期175-179,187,共6页Plant Protection

基　　金：国家重点研发计划(2016YFD0300705);国家自然科学基金(31871938)。

摘　　要：异沙叶蝉可传播小麦矮缩病毒Wheat dwarf virus(WDV)和小麦黄条纹病毒Wheat yellow striate virus(WYSV)两种病毒。本文根据WDV和WYSV的基因序列分别设计两种病毒的特异性引物对,以含有上述两种病毒的异沙叶蝉样品总RNA为模板,以随机引物为3′端通用引物反转录获得cDNA,然后在同一个PCR反应体系加入两对引物,分别得到与预期相符的773 bp和322 bp扩增产物条带。通过对引物浓度、dNTP和rTaq用量以及退火温度等条件进行优化,建立了能在同一异沙叶蝉体内检测WDV与WYSV两种小麦病毒的双重RT-PCR方法。该双重PCR方法特异性强、敏感性高,可以快速准确地在一个体系里同步检测介体昆虫体内的两种病毒,有效地检测虫体内病毒带毒率,这些结果可为病毒预测预报和病害防治提供参考。It was found that the leafhopper(Psammotettix alienus) can transmit both Wheat dwarf virus(WDV) and wheat-infecting virus-Wheat yellow striate virus(WYSV). Here, the specific primer sets for WDV and WYSV were designed according to viral genomic sequences, respectively. Total RNA of leafhopper, which was positive for both WYSV and WDV, was used as a template for the first strand cDNA synthesis. Reverse-transcription was performed using random primer as 3′ primer for each virus. WDV and WYSV specific primer sets was then added into the PCR system producing two distinct fragments of 773 and 322 bp, respectively. A method of duplex reverse-transcription polymerase chain reaction(RT-PCR) was developed for the simultaneous detection of WDV and WYSV in single leafhopper. The specific reagents and enzymes in the duplex RT-PCR reaction system, primers, dNTPs, and rTaq were first optimized by testing different concentrations and volumes of these components and the annealing temperature was then optimized. Thus, the duplex RT-PCR method optimized in this study was to develop and validate a highly specific and simple RT-PCR method for accurate detection of two viruliferous rates of leafhopper population, which could facilitate better forecasting and control of the virus diseases.

关 键 词：异沙叶蝉 小麦矮缩病毒 小麦黄条纹病毒 双重RT-PCR 

分 类 号：S435.12[农业科学—农业昆虫与害虫防治]