茄匍柄霉LAMP快速检测体系的建立及应用  

Establishment and Application of LAMP Rapid Detection Assay for Stephylium solani

作　　者：谢学文[1] 刘世程 石延霞[1] 张胜丰 陈利达 柴阿丽[1] 范腾飞 李磊 李宝聚[1] XIE Xue-Wen;LIU Shi-Cheng;SHI Yan-Xia;ZHANG Sheng-Feng;CHEN Li-Da;CHAI A-Li;FAN Teng-Fei;LI Lei;LI Bao-Ju(Institute of Vegetables and Flowers,Chinese Academy of Agricultural Sciences,Beijing 100081,China;Xuzhou Tairui Agricultural Technology Co.,Ltd.,Xuzhou 221133,China)

机构地区：[1]中国农业科学院蔬菜花卉研究所,北京100081 [2]徐州市贾汪区泰瑞农业科技有限公司,徐州221133

出　　处：《农业生物技术学报》2022年第10期2045-2052,共8页Journal of Agricultural Biotechnology

基　　金：国家重点研发计划(2020YFD1000300);宁夏回族自治区重点研发计划(2021BBF03002);国家大宗蔬菜产业技术体系(CRS-25);中国农业科学院创新工程项目(CAAS-ASTIP-IVFCAAS);农业部园艺作物生物学与种质创制重点实验室项目。

摘　　要：匍柄霉(Stemphylium solani)是一种重要的植物病原真菌,可侵染多种作物。环介导等温扩增(loopmediated isothermal amplification,LAMP)技术具有简便、准确、快速等突出优点,在病原菌检测领域有广泛应用。本研究以SYBR GreenⅠ为指示剂,以甘油醛-3-磷酸脱氢酶基因(glyceraldehyde-2-phosphate dehydrogenase,gpd)为检测靶标基因设计6条引物(2条内引物FIP/BIP,2条外引物F3/B3,环引物LF25/LB25)进行LAMP扩增,同时通过环引物加速反应,建立了一套茄匍柄霉LAMP快速检测体系。特异性验证结果显示,仅16株茄匍柄霉有阳性扩增,具有较高的特异性。灵敏度检测结果显示,该检测体系灵敏度达2.4 pg/μL,显著高于普通PCR检测技术。环引物加速LAMP反应结果表明,加入环引物能使反应时间缩短至27 min,可有效加速LAMP反应。通过钙黄绿素进行显色反应,在自然光下仅模板为茄匍柄霉的反应液变为绿色,其余反应液均为橙色。采用该体系检测茄匍柄霉接种后番茄(Lycopersicon esculentum)叶片病原菌含量动态变化,接种病原菌12 h时,该体系即有阳性显色反应,接种后颜色随时间推移逐渐加深,与接种病原菌后叶片发病情况的规律相同。该反应结果不借助其他仪器即可观察,环引物加速反应使检测时间进一步缩短至27 min,能够快速、准确反应病原菌含量。该体系在匍柄霉快速检测方面具有较强的优势,可用于病害潜伏、未显症期的快速检测,对及时、科学防治茄匍柄霉叶斑具有重要意义。Stephylium solani is an important plant pathogenic fungus with a wide range of hosts.Loop-mediated isothermal amplification(LAMP)technology has been widely used in many fields such as plant protection because of its strong specificity,high sensitivity,rapid response and low cost.To establish a rapid,efficient and accurate detection technology for Lycopersicon esculentum petiole leaf spot,SYBR GreenⅠwas used as the indicator and the glyceraldehyde-2-phosphate dehydrogenase(gpd)gene was used as the target.6 primers(2inner primers FIP/BIP,2 outer primers F3/B3 and loop primer LB25/LF25)were designed for LAMP amplification.At the same time,a set of rapid LAMP detection system for S.solani was established by accelerating the reaction with loop primers.The results of specificity verification showed that only 16 strains of S.solani had positive amplification and had high specificity.The sensitivity results showed that the LAMP detection sensitivity of the system was 2.4×103 fg/μL,significantly higher than the PCR detection technology.The results showed that the addition of ring primers could shorten the reaction time to 27 min,and effectively accelerate the LAMP reaction.Through the color reaction of calcein,under natural light,only the reaction solution with the template of S.solani turned green,and the other reaction solutions were orange.The system was used to detect the dynamic changes of pathogen content in tomato leaves after inoculation with S.solani.12 h after inoculation,the LAMP detection had a positive color reaction,and the color gradually became darker with time,which was consistent with the severity of tomato leaves after artificial inoculation.The reaction results can be directly observed.The loop primer accelerates the reaction to further shorten the detection time to 27min,which can quickly and accurately reflect the pathogen content.The LAMP detection system established in this study has strong advantages in the rapid detection of S.solani.It can be used for the rapid detection of latent and non sy

关 键 词：茄匍柄霉 环介导等温扩增(LAMP) 快速分子检测 

分 类 号：S431.9[农业科学—农业昆虫与害虫防治]