香石竹斑驳病毒属简并引物RT-PCR检测方法的建立  

Development of a Degenerate Primer RT-PCR Assay for Detection of Carmovirus

作　　者：廖富荣 陈红运 沈建国 方志鹏 黄蓬英 陈青 林玲玲 洪钧 LIAO FuRong;CHEN HongYun;SHEN JianGuo;FANG ZhiPeng;HUANG PengYing;CHEN Qing;LIN LingLing;HONG Jun(Xiamen Customs Technology Center,Xiamen 361026,Fujian;Technology Center of Fuzhou Customs District,Fuzhou 350001)

机构地区：[1]厦门海关技术中心,福建厦门361026 [2]福州海关技术中心,福州350001

出　　处：《中国农业科学》2023年第12期2288-2301,共14页Scientia Agricultura Sinica

基　　金：福建省农业引导性(重点)项目(2019N0029);厦门海关科研项目(2020XK03)。

摘　　要：【目的】使用简并引物RT-PCR方法并结合序列测定与分析,为甲型香石竹斑驳病毒属(Alphacarmovirus)、乙型香石竹斑驳病毒属(Betacarmovirus)和丙型香石竹斑驳病毒属(Gammacarmovirus)病毒建立一种快速、灵敏、广谱的检测鉴定方法。【方法】通过基因组序列的多重比对分析,在依赖于RNA的RNA聚合酶(RdRp)基因的保守区域设计1对简并引物Carmo-F2/Carmo-R2,在引物5′端分别加入非互补的富含AT序列(AATAAATCATAA)形成引物Carmo-F2a/Carmo-R2a,测试简并引物RT-PCR方法的广谱性、特异性和灵敏度,并对PCR产物进行测序、序列分析和系统发育分析。利用该方法对来自厦门的扶桑(Hibiscusrosa-sinensis)样品进行病毒检测。【结果】利用Carmo-F2/Carmo-R2和Carmo-F2a/Carmo-R2a两对引物建立的RT-PCR方法,扩增甲型、乙型和丙型香石竹斑驳病毒属病毒的部分RdRp基因,扩增片段分别约为500和550 bp。该方法成功用于检测甲型香石竹斑驳病毒属的香彩雀花碎色病毒(angelonia flower break virus,AnFBV)、小花矮牵牛斑驳病毒(calibrachoa mottle virus,CbMV)、香石竹斑驳病毒(carnation mottle virus,CarMV)、天竺葵花碎色病毒(pelargonium flower break virus,PFBV),乙型香石竹斑驳病毒属的木槿褪绿环斑病毒(hibiscus chlorotic ringspot virus,HCRSV),丙型香石竹斑驳病毒属的甜瓜坏死斑点病毒(melon necrotic spot virus,MNSV),显示出良好的广谱性。特异性检测表明,简并引物Carmo-F2/Carmo-R2和Carmo-F2a/Carmo-R2a在玉米褪绿斑驳病毒(maize chlorotic mottle virus,MCMV;Machlomovirus)、天竺葵线纹病毒(pelargonium line pattern virus,PLPV;Pelarspovirus)、香石竹环斑病毒(carnation ringspot virus,CRSV;Dianthovirus),健康的西瓜、甜瓜、南瓜、大豆、豌豆植物未扩增到预期大小的条带。灵敏度检测表明,简并引物Carmo-F2/Carmo-R2最低可检测到10-2稀释液,简并引物Carmo-F2a/Carmo-R2a可检测到10-3稀释液,在5′端加入非互补的富【Objective】The objective of this study is to establish a rapid,sensitive,and broad-spectrum screening method for simultaneous detection and identification of the genera Alphacarmovirus,Betacarmovirus,and Gammacarmovirus using degenerate primer RT-PCR combined with sequence analysis.【Method】Multiplexed analysis of genome sequences was aligned to search for suitable conserved regions for the design of the degenerate primers.One pair of degenerate primer Carmo-F2/Carmo-R2 was designed based on the RNA-dependent RNA polymerase(RdRp)gene sequences,and another pair primer Carmo-F2a/Carmo-R2a was formed by adding the non-complementary AT-rich sequences(AATAAATCATAA)to the 5′end of the degenerate primers.The broad-spectrum,specificity,and sensitivity of RT-PCR method were analyzed.The sequencing,BLASTn analysis and phylogenetic analysis of PCR products were performed.The method was used to screen and detect viruses on Chinese hibiscus(Hibiscus rosa-sinensis)samples from Xiamen,China.【Result】Degenerate primers Carmo-F2/Carmo-R2 and Carmo-F2a/Carmo-R2a were used to amplify partial RdRp gene of the members of genera Alphacarmovirus,Betacarmovirus,and Gammacarmovirus by RT-PCR.The fragment of approximately 500 and 550 bp was amplified,respectively.The developed RT-PCR assay was successfully used to detect angelonia flower break virus(AnFBV;Alphacarmovirus),calibrachoa mottle virus(CbMV;Alphacarmovirus),carnation mottle virus(CarMV;Alphacarmovirus),and pelargonium flower break virus(PFBV;Alphacarmovirus),hibiscus chlorotic ringspot virus(HCRSV;Betacarmovirus),melon necrotic spot virus(MNSV;Gammacarmovirus).The specificity test showed that no specific band could be obtained from maize chlorotic mottle virus(MCMV;Machlomovirus),pelargonium line pattern virus(PLPV;Pelarspovirus),carnation ringspot virus(CRSV;Dianthovirus),and healthy plants which including watermelon,melon,pumpkin,soybean,and pea.The sensitivity results showed that the primers Carmo-F2/Carmo-R2 could be detected up to 10-2 dilution and the primers

关 键 词：甲型香石竹斑驳病毒属 乙型香石竹斑驳病毒属 丙型香石竹斑驳病毒属 简并引物 RT-PCR 扶桑 木槿褪绿环斑病毒 

分 类 号：S432.41[农业科学—植物病理学]