福建蝴蝶兰病毒小RNA测序及RT-PCR检测  

Detection and Identification of Virus Infecting Phalaenopsis Based on Small RNA Deep Sequencing Technology and Multiple RT-PCR Identification

作　　者：樊荣辉[1] 吴建设[1] 冯子楠 钟声远 钟淮钦[1] FAN Ronghui;WU Jianshe;FENG Zinan;ZHONG Shengyuan;ZHONG Huaiqin(Institute of Crop Sciences(Fujian Provincial Germplasm Resources Center),Fujian Academy of Agricultural Science,Fujian Engineering Research Center for Characteristic Floriculture,Fuzhou 350013,China)

机构地区：[1]福建省农业科学院作物研究所(福建省种质资源中心),福建省特色花卉工程技术研究中心,福州350013

出　　处：《园艺学报》2025年第2期503-512,共10页Acta Horticulturae Sinica

基　　金：省属公益类科研院所基本科研专项(2024R1030009);福建省林业科技种苗攻关项目(ZMGG-0710);福建省人民政府与中国农业科学院高质量发展超越“5511”协同创新工程项目(XTCXGC2021003);福建省农业科学院科技创新团队建设项目(CXTD2021010-2)。

摘　　要：从福建省福州、厦门、漳州、泉州采集56个蝴蝶兰(Phalaenopsis)疑似病毒样品,按侵染症状分为褪绿、黄化褐斑、黄化皱缩等3类样品,采用小RNA测序技术和RT-PCR进行检测,共发现8种病毒,按照检出率从高到低分别为建兰花叶病毒(Cymbidium mosaic virus,Cy MV)85.71%,齿兰环斑病毒(Odontoglossum ringspot virus,ORSV)35.71%,白三叶草花叶病毒(white clover mosaic virus,WCMV)32.14%,淮山药X病毒(yam virus X,Ya VX)21.43%,水仙花叶病毒(Narcissus mosaic virus,Na MV)10.71%,凤果花叶病毒(pepino mosaic virus,Pe MV)8.93%,马铃薯X病毒(potato virus X,Po VX)7.14%和仙人指X病毒(Schlumbergera virus X,Sc VX)7.14%。其中,Cy MV、ORSV和WCMV的检出率在30%以上,多为复合侵染,侵染率达75.51%。以小RNA测序序列为模板,设计出特异引物,建立了同时检测5种病毒的多重RT-PCR体系;ORSV、Cy MV、WCMV、Ya VX和Po VX的引物对浓度分别为0.30,0.06,0.20,0.50和0.40μmol·L^(-1),退火温度56℃时,可同时扩增出片段大小分别为1156、908、561、292和162 bp的目的条带,特异性良好。灵敏度检测结果显示,可从≥0.0001 mg的感病植物组织中检测到这5种病毒。This study collected 56 suspected virus samples of Phalaenopsis from Fuzhou,Xiamen,Zhangzhou,and Quanzhou in Fujian Province,which were divided into three types of mixed samplesbased on infection symptoms:chlorosis,yellowing brown spots,and yellowing wrinkles.Small RNA sequencing technology and RT-PCR were used for detection,and a total of 8 viruses were found.According to the detection rate,they were Cymbidium mosaic virus(CyMV)(85.71%),Odontoglossum ringspot virus(ORSV)(35.71%),white clover mosaic virus(WCMV)(32.14%),yam virus X(YaVX)(21.43%),Narcissus mosaic virus(NaMV)(10.71%),pepino mosaic virus(PeMV)(8.93%),potato virus X(PoVX)(7.14%),and Schlumbergera virus X(ScVX)(7.14%)from high to low.Among them,detection rates of CyMV,ORSV and WCMV are over 30%,and most of them are compound infections,accounting for 75.51%of the total infections.Using small RNA sequencing sequences as templates,specific primers were designed and a multiplex RT-PCR system was established for simultaneous detection of five viruses.The primer concentrations for ORSV,CyMV,WCMV,YaVX,and PoVX were 0.30,0.06,0.20,0.50 and 0.40μmol·L^(-1),respectively.At an annealing temperature of 56℃,target bands with fragment sizes of 1156,908,561,292 and 162 bp,respectively,can be simultaneously amplified with good specificity.The sensitivity test results show that these 5 viruses can be detected from infected plant tissues equivalent to or greater than 0.0001 mg.

关 键 词：蝴蝶兰 小RNA深度测序 多重RT-PCR 病毒 

分 类 号：S682.31[农业科学—观赏园艺]