草莓黄单胞菌RPA-LF快速检测方法的建立与应用
Development and evaluation of rapid visualization detection method for Xanthomonas fragariae based on RPA-LF
史春佳 1李刚 1刘林川 1秦梦瑶 1刘丽锋 1宋艳红 1赵霞 1周厚成1
作者信息
- 1. 中国农业科学院郑州果树研究所,河南 450009
- 折叠
摘要
建立一种适用于田间稳定、快速、准确地检测草莓黄单胞菌的技术方法,有助于该病原菌的早期诊断和有效控制.将重组酶聚合酶扩增(recombinase polymerase amplification,RPA)和胶体金侧流层析试纸条技术(lateral flow strip,LF)相结合.根据草莓黄单胞菌的限制性核酸内切酶亚基S基因序列保守区设计RPA特异引物及探针,通过对反应条件优化及对灵敏性、特异性与疑似样本的分析,成功建立了草莓黄单胞菌RPA-LF检测方法.该方法最佳反应条件为39℃恒温反应10 min,最低检测限为1.0×101 copies/μL,能够特异地检测出草莓黄单胞菌,从采集疑似症状植株到检测可在30 min内实现,而且用RPA-LF方法检测疑似样本结果与常规PCR检测方法一致.综上,建立的草莓黄单胞菌RPA-LF检测方法快速、简单、准确、可视化,可用于该病原菌的早期诊断.
Abstract
The objective of this study was to establish a stable,rapid,and accurate technical method for the detection of Xanthomonas fragariae in the field.Achieving early diagnosis and effective control of Xanthomonas fragariae.This study established an RPA-LF assay for Xanthomonas fragariae.We designed RPA-specific primers and probes in the conserved region of the restriction endonuclease subunit S gene sequence of Xanthomonas fragariae.And we also optimized the reaction conditions of the method,analyzed its sensitivity and specificity.Finally,this study detected suspicious samples performed.The optimal reaction conditions were 39℃for 10 min.The minimum detection limit was 1.0 × 101 copies/μL.The method was able to detect Xanthomonas fragariae specifically.It took only 30 min from the collection of the suspect plant to the detection of Xanthomonas fragariae.The RPA-LF assay for Xanthomonas fragariae established in this study is rapid,simple,accurate.Eventually,we can visualize this pathogen for early diagnosis.
关键词
草莓黄单胞菌/RPA-LF检测/田间/早期诊断Key words
Xanthomonas fragariae/RPA-LF assay/field/early diagnosis引用本文复制引用
基金项目
国家重点研发计划(2022YFD1600700)
河南省科技重大专项(221100110400)
河南省大宗水果产业技术体系建设项目(HARS-22-09-G2)
中国农科院科技创新工程项目(CAAS-ASTIP-2022-ZFRI)
出版年
2024
国家科技期刊平台
NSTL
万方数据