基于逆转录重组酶聚合酶扩增技术的苹果病毒新型快速检测方法  

A novel rapid detection method for apple viruses based on reverse transcriptase polymerase amplification

作　　者：焦健 夏炎 武雪莉 王苗苗 宋春晖 庞宏光 白团辉[1] 宋尚伟[1] 黄松 郑先波[1] JIAO Jian;XIA Yan;WU Xueli;WANG Miaomiao;SONG Chunhui;PANG Hongguang;BAI Tuanhui;SONG Shangwei;HUANG Song;ZHENG Xianbo(College of Horticulture,Henan Agricultural University,Zhengzhou 450002,China;XinYang Agriculture And Forestry University,Xinyang 464006,China)

机构地区：[1]河南农业大学园艺学院,河南郑州450002 [2]信阳农林学院,河南信阳464006

出　　处：《河南农业大学学报》2021年第6期1097-1103,共7页Journal of Henan Agricultural University

基　　金：国家自然科学基金项目项目(31801818);河南省现代农业产业体系项目(S2014-11-G02,Z2014-11-03);河南省高等学校科技创新团队项目(19IRTSTHN009);河南省科技攻关项目(202102110188)。

摘　　要：利用逆转录重组酶聚合酶扩增技术(Reverse transcription-recombinase polymerase amplification,RT-RPA),为苹果病毒的快速检测提供技术支持。以苹果茎沟病毒(apple stem grooving virus,ASGV)、苹果茎痘病毒(apple stem pitting virus,ASPV)、苹果褪绿叶斑病毒(apple chlorotic leaf spot virus,ACLSV)和苹果坏死花叶病毒(apple necrotic mosaic virus,ApNMV)为检测对象,通过特异性RPA引物设计以及反应条件优化,对检测方法的灵敏度、特异性进行评价,并与传统RT-PCR方法进行比较。结果表明,所设计的RPA引物特异性高,反应温度为37℃,反应时间为20 min,RT-RPA对4种苹果病毒的检测灵敏度为9.63×10^(1) copies,是普通RT-PCR的10^(2)~10^(3)倍。所建立的RT-RPA检测方法具有等温反应、检测速度快、灵敏度高等优点,可以用于快速检测苹果病毒病。To provide technical support for detecting apple virus diseases,this study established a rapid detection method for 4 apple virus diseases using reverse transcription-recombinase polymerase amplification(RT-RPA).The specific RPA primers for apple stem grooving virus(ASGV),apple stem pitting virus(ASPV),apple chlorotic leaf spot virus(ACLSV)and apple necrotic mosaic virus(ApNMV)were designed and the reaction conditions were optimized.Moreover,the sensitivity and specificity of the developed RT-RPA method were evaluated and compared with the traditional RT-PCR method.The designed primers had high specificity,and the sensitivity of RT-RPA was 9.63×10^(1) copies/reaction,which was 10^(2) to 10^(3) times higher than that of RT-PCR,when the reaction temperature was 37℃and the reaction time was 20 min.The established RT-RPA detection method had the advantages of isothermal reaction,rapid detection and high sensitivity,which can be used for rapid detection of apple virus diseases.

关 键 词：逆转录重组酶聚合酶扩增 苹果病毒病 快速检测 等温扩增 

分 类 号：S661.1[农业科学—果树学]