广东省蒲瓜病毒种类鉴定及多重RT-PCR检测方法的建立  

Identification of Viruses Infecting Bottle Gourd in Guangdong Province and Establishment of Multiplex RT-PCR Detection Method

作　　者：苏琦 汤亚飞[2] 佘小漫[2] 蓝国兵[2] 于琳 吴正伟 李正刚 何自福[2] SU Qi;TANG YaFei;SHE XiaoMan;LAN GuoBing;YU Lin;WU ZhengWei;LI ZhengGang;HE ZiFu(College of Coastal Agricultural Sciences,Guangdong Ocean University,Zhanjiang 524088,Guangdong;Plant Protection Research Institute,Guangdong Academy of Agricultural Sciences/Guangdong Provincial Key Laboratory of High Technology for Plant Protection,Guangzhou 510640)

机构地区：[1]广东海洋大学滨海农业学院,广东湛江524088 [2]广东省农业科学院植物保护研究所/广东省植物保护新技术重点实验室,广州510640

出　　处：《中国农业科学》2023年第23期4684-4695,共12页Scientia Agricultura Sinica

基　　金：国家自然科学基金(32272509);广东省农业科学院农业优势产业学科团队建设项目(202103TD,202105TD);广州市科技计划(202102020504);广州市青年科技人才托举项目(QT-2023-040);广东省农业科学院科技人才培养专项(R2023PY-JX011);广东省农业科学院协同创新中心项目(XT202210)。

摘　　要：【目的】探明侵染广东省蒲瓜的病毒种类,建立一种可以同时检测多种蒲瓜病毒的RT-PCR检测方法,提高蒲瓜病毒种类鉴定效率,为蒲瓜病毒病的精准监测与防控提供依据。【方法】2018—2022年间,从广东省广州市、惠州市、清远市、汕头市和湛江市的蒲瓜主要种植区采集蒲瓜病毒病疑似样品78份,对采集的样品按照地区和症状进行分类,提取每份样品的总RNA,将一个地区具有相同症状样品的RNA等量混合后进行小RNA深度测序分析。根据小RNA深度测序分析结果,设计特异引物进行RT-PCR验证,从而明确侵染广东省蒲瓜的病毒种类。挑选6种检出率高、危害严重的蒲瓜病毒,根据GenBank数据库设计多重PCR检测引物,通过优化反应体系和反应条件,建立同时检测6种蒲瓜病毒的多重RT-PCR方法。【结果】从采集的78份蒲瓜疑似病毒病样品中,共检测到12种病毒,按照检出率从高到低分别为葫芦内源RNA病毒(Lagenaria sicerariaalphaendornavirus,LSEV)(64.1%)、黄瓜绿斑驳花叶病毒(cucumber green mottle mosaic virus,CGMMV)(62.8%)、小西葫芦虎纹花叶病毒(zucchini tigre mosaic virus,ZTMV)(51.3%)、西瓜绿斑驳花叶病毒(watermelon green mottle mosaic virus,WGMMV)(43.6%)、西瓜病毒A(watermelon virus A,WVA)(32.1%)、番木瓜环斑病毒(papaya ringspot virus,PRSV)(19.2%)、小西葫芦黄花叶病毒(zucchini yellow mosaic virus,ZYMV)(9.0%)、瓜类蚜传黄化病毒(cucurbit aphid-borne yellows virus,CABYV)(7.7%)、中国南瓜曲叶病毒(squash leaf curl China virus,SLCCNV)(7.7%)、瓜类褪绿黄化病毒(cucurbit chlorotic yellows virus,CCYV)(5.1%)、瓜类黄矮失调病毒(cucurbit yellow stunting disorder virus,CYSDV)(3.8%)、甜瓜黄斑病毒(melon yellow spot virus,MYSV)(1.3%)。78份样品中,复合侵染率高达80.8%,其中2、3、4、5、6和7种病毒复合侵染的检出率分别为14.1%、16.7%、23.1%、17.9%、7.7%和1.3%。在多重RT-PCR体系中先加入WVA+CCYV+ZTMV+PRSV�【Objective】The objective of this research is to determine the various viruses that infect bottle gourd,establish a multiplex RT-PCR detection method that can simultaneously detect multiple bottle gourd viruses,so as to enhance the efficiency of identifying bottle gourd virus species,and provide a foundation for accurate prevention and control of bottle gourd viral diseases.【Method】From 2018 to 2022,78 bottle gourd samples of suspected virus infection were collected from the main planting areas of bottle gourd in Guangdong Province,including Guangzhou,Huizhou,Qingyuan,Shantou,and Zhanjiang.The collected samples were sorted by region and symptom,and the total RNA of each sample was extracted.The RNA samples with similar symptoms from each region were mixed for small RNA deep sequencing analysis.According to the results of small RNA deep sequencing analysis,specific primers were designed for RT-PCR verification to identify the specific virus species infecting bottle gourd in Guangdong Province.Six viruses with higher incidence and more losses were selected,and multiplex PCR detection primers based on the GenBank database were designed.After optimizing the reaction system and conditions,a method for simultaneous detection of all six viruses via multiplex RT-PCR was established.【Result】A total of 12 viruses were detected in 78 bottle gourd samples of suspected virus infection.These viruses were ranked in order of infection rate,from highest to lowest:Lagenaria siceraria alphaendornavirus(LSEV)(64.1%),cucumber green mottle mosaic virus(CGMMV)(62.8%),zucchini tigre mosaic virus(ZTMV)(51.3%),watermelon green mottle mosaic virus(WGMMV)(43.6%),watermelon virus A(WVA)(32.1%),papaya ringspot virus(PRSV)(19.2%),zucchini yellow mosaic virus(ZYMV)(9.0%),cucurbit aphid-borne yellows virus(CABYV)(7.7%),squash leaf curl China virus(SLCCNV)(7.7%),cucurbit chlorotic yellows virus(CCYV)(5.1%),cucurbit yellow stunting disorder virus(CYSDV)(3.8%),melon yellow spot virus(MYSV)(1.3%).It was found that 80.8%of the 78 samples w

关 键 词：蒲瓜 病毒病 小RNA深度测序 RT-PCR检测 多重PCR方法 

分 类 号：S436.429[农业科学—农业昆虫与害虫防治]