梨火疫病菌的RPA快速检测技术的研发  

Recombinase polymerase amplification assay for rapid on-site diagnosis of pear fire blight pathogen Erwinia amylovora

作　　者：蒲姝旸 张瑾逸 暴晓阳 孟青青 田茜[1] 蔡璐璐 赵文军[1,2] PU Shuyang;ZHANG Jinyi;BAO Xiaoyang;MENG Qingqing;TIAN Qian;CAI Lulu;ZHAO Wenjun(Institute of Plant Quarantine,Chinese Academy of Inspection and Quarantine,Beijing 100176,China;Chinese Academy of Inspection and Quarantine Sanya Center for Biosafety,Sanya 572024,China;Southern Breeding Administrate Office of Hainan Province,Sanya 572000,China;National Nanfan Research Institute(Sanya),Chinese Academy of Agricultural Sciences,Sanya 572024,China)

机构地区：[1]中国检验检疫科学研究院,北京100176 [2]三亚中国检验检疫科学研究院生物安全中心,三亚572024 [3]海南省南繁管理局,三亚572000 [4]三亚中国农业科学院国家南繁研究院,三亚572024

出　　处：《植物保护》2024年第4期242-249,282,共9页Plant Protection

基　　金：中国检验检疫科学研究院基本科研业务费(2022JK10)。

摘　　要：梨火疫病菌Erwinia amylovora是我国进境植物检疫性有害生物,主要侵染梨、苹果、山楂、海棠等多种蔷薇科植物引起梨火疫病。该病自1780年在美国被发现以来,已经传播到世界多个国家,对我国的果树生产也构成一定的威胁。严格植物检疫是全球防控梨火疫病菌扩散危害的重要方法之一,目前我国植物检疫部门主要从实验室分离鉴定、分子检测和田间症状识别及快速检测方面开展工作。本研究基于重组酶聚合酶扩增(RPA)技术和E.amylovora 16S-23S ITS区间的保守序列设计的引物和探针,建立了实时荧光RPA和侧流层析试纸条RPA两种检测方法。经测试,在39℃条件下,这两种检测方法都具有良好的特异性,分别能在20 min和10 min内完成扩增。实时荧光RPA对E.amylovora菌株DNA的检测阈值为1.38×10^(-2)ng/μL;侧流层析试纸条RPA对E.amylovora菌悬液和DNA的检测阈值分别为10^(4)cfu/mL和1.38×10^(-3)ng/μL。侧流层析试纸条RPA体系因反应快速、读取结果方便、不需要复杂仪器操作可以满足现场快速检测的需求。Erwinia amylovora is an imported quarantine plant pathogen in China,which infects Rosaceae plants such as pear,apple,hawthorn,causing pear fire blight disease.This disease,firstly discovered in the United States in 1780,has spread to many countries in the world and posed a threat to domestic pomefruits production.Strict quarantine is one of the important methods to control pear fire blight in the world.Chinese plant quarantine departments basically conducted Koch’s postulate,molecular detection and rapid detection on-site following the typical symptoms to detect the pathogen.In this study,a recombinase polymerase amplification with real-time fluorescent(real-time RPA)and with lateral flow dipstick(RPA-LFD)detection methods for E.amylovora were established,which provided technical support for rapid on-site diagnosis both in field and laboratory.Based on E.amylovora 16S-23S ITS region,we designed specific primers and probes and established real-time RPA and RPA-LFD detection methods.At 39℃,both methods showed good specificity and can give results in 20 mins and 10 mins,respectively.Real-time RPA detection limit of E.amylovora strain gDNA can reach 1.38×10^(-2)ng/μL,while RPA-LFD detection limits of E.amylovora suspension and its gDNA can reach 10^(4)cfu/mL and 1.38×10^(-3)ng/μL,respectively.RPA-LFD is accurate and can easily perform the test at the point of need.

关 键 词：梨火疫病 快速检测 重组酶聚合酶扩增(RPA) 实时荧光型RPA 侧流层析试纸条型RPA 

分 类 号：S436.612.1[农业科学—农业昆虫与害虫防治]