TaqMan探针法实时荧光定量PCR检测西瓜潜隐病毒  

The Detection of Citrullus lanatus Cryptic Virus Using TaqMan-qPCR Method

作　　者：赵立群[1] 邱艳红 张晓飞 刘慧[2] 杨静静[2] 张建 张海军 徐秀兰[2,3] 温常龙 ZHAO LiQun;QIU YanHong;ZHANG XiaoFei;LIU Hui;YANG JingJing;ZHANG Jian;ZHANG HaiJun;XU XiuLan;WEN ChangLong(Beijing Agricultural Extension Station,Beijing 100029;Beijing Vegetable Research Center(BVRC),Beijing Academy of Agricultural and Forestry Sciences/National Engineering Research Center for Vegetables,Beijing 100097;Supervision,Inspection and Test Center of Vegetable Seed Quality of Ministry of Agriculture and Rural Affairs,Beijing 100097)

机构地区：[1]北京市农业技术推广站,北京100029 [2]北京市农林科学院蔬菜研究中心/国家蔬菜工程技术研究中心,北京100097 [3]农业农村部蔬菜种子质量监督检验测试中心,北京100097

出　　处：《中国农业科学》2021年第20期4337-4347,共11页Scientia Agricultura Sinica

基　　金：科技助力经济2020重点专项(202006)。

摘　　要：【目的】西瓜潜隐病毒(Citrullus lanatus cryptic virus,CiLCV)是近年来新发生的一种重要种传病害,研究旨在建立基于TaqMan探针法的实时荧光定量PCR(real-time fluorescent quantitative PCR with TaqMan probes,TaqMan-qPCR)检测技术,为开展种子、种苗带毒鉴定和病害防控提供技术支撑。【方法】通过基于小RNA高通量测序技术在北京西瓜产地发现了CiLCV,并克隆CiLCV的dsRNA1和dsRNA2全长序列,设计特异性引物建立RT-PCR和TaqMan-qPCR检测方法。以黄瓜绿斑驳花叶病毒(cucumber green mottled mosaic virus)、黄瓜花叶病毒(cucumber mosaic virus)、甜瓜内源病毒(Cucumis melo endornavirus)、瓜类褪绿病毒(cucurbit chlorotic yellows virus)、南瓜花叶病毒(squash mosaic virus)、番茄斑萎病毒(tomato spotted wilt virus)、西瓜花叶病毒(watermelon mosaic virus)、小西葫芦黄花叶病毒(zucchini yellow mosaic virus)8种常见病毒为对照进行检测方法特异性分析;利用建立的实时荧光定量标准曲线对方法灵敏度进行评价;进一步利用TaqMan-qPCR和RT-PCR技术监测我国葫芦科作物主产区的西瓜、黄瓜、甜瓜和南瓜(砧木)种苗带毒情况。【结果】通过分析获得的高质量小RNA数据,发现17条组装的contig与CiLCV基因组有较好同源性。进一步克隆获得CiLCV的dsRNA1和dsRNA2序列全长(分别为1603和1466 nt),发现其与河南省鉴定出的CiLCV同源性高达99.4%和99.8%(GenBank number KY081285、KY081284)。建立的RT-PCR检测技术对CiLCV有单一扩增条带;建立的TaqMan-qPCR检测技术具有较好特异性和灵敏度,且能够检测到最低拷贝数为2×10^(3)的病毒,灵敏度是RT-PCR的100倍。同时,发现有1份来自北京的西瓜种苗检出了CiLCV,而其余地区的西瓜、黄瓜、甜瓜和南瓜(砧木)种苗均未检出该病毒,表明我国葫芦科作物主产区种苗带毒情况总体不高。【结论】建立的TaqMan-qPCR检测CiLCV技术特异性强、灵敏度高,适用于口岸和实【Objective】Citrullus lanatus cryptic virus(CiLCV)is an important seed-transmitted virus that newly-emerging in watermelon in recent years.The objective of this study is to develop a detection method for CiLCV with real-time fluorescent quantitative PCR with TaqMan probes(TaqMan-qPCR),and to provide technical supports for the CiLCV detection from seeds and seedlings,and also for disease controlling in the future.【Method】The CiLCV was found in Beijing watermelon producing area with high-throughput sequencing based on small RNAs.The full sequence of CiLCV-dsRNA1 and CiLCV-dsRNA2 was cloned and specific amplifying primers were designed to set up the detection method of the RT-PCR and TaqMan-qPCR.The other eight common viruses(cucumber green mottled mosaic virus,cucumber mosaic virus,Cucumis melo endornavirus,cucurbit chlorotic yellows virus,squash mosaic virus,tomato spotted wilt virus,watermelon mosaic virus,zucchini yellow mosaic virus)were used as control to analyze the method specificity.The standard curve of CiLCV was performed to evaluate the method sensitivity.Furthermore,the novel detection methods of TaqMan-qPCR and RT-PCR were used to inspect CiLCV in watermelon,cucumber,melon,and rootstock pumpkin seedlings that randomly collected from the main producing areas of cucurbits crop in North China.【Result】The small RNAs data with high-quality were obtained,and 17 assembled contigs were found to share homology with CiLCV genomes after data analysis.The full-length of CiLCV-dsRNA1 and CiLCV-dsRNA2 was cloned with 1603 and 1466 nt in length,respectively,sharing the highest nt sequence identity(about 99.4%and 99.8%,respectively)with CiLCV that isolated from Henan Province(GenBank number KY081285,KY081284).The established RT-PCR for CiLCV showed a single amplified band,while the novel TaqMan-qPCR for detecting CiLCV showed good sensitivity and specificity,and could detect about 2×10^(3) copies of CiLCV.The detection sensitivity of TaqMan-qPCR was about 100 times higher than that of RT-PCR.In addition,on

关 键 词：西瓜潜隐病毒 实时荧光定量PCR TaqMan探针法 葫芦科作物 种传病害 

分 类 号：S432.41[农业科学—植物病理学]