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苹果轮纹病菌LAMP快速检测方法的建立

引用本文：汪少丽,曲恒华,王英姿,王培松,栾炳辉,王冠华.苹果轮纹病菌LAMP快速检测方法的建立.植物保护学报,2020,47(1):127-133

DOI：10.13802/j.cnki.zwbhxb.2020.2019031

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作者	单位	E-mail
汪少丽	山东省烟台市农业科学研究院, 烟台 265500
曲恒华	烟台市果茶工作站, 山东烟台 264001
王英姿	山东省烟台市农业科学研究院, 烟台 265500 烟台大学生命科学学院, 山东烟台 264005	ytnkyzbs@yt.shandong.cn
王培松	山东省烟台市农业科学研究院, 烟台 265500
栾炳辉	山东省烟台市农业科学研究院, 烟台 265500
王冠华	烟台大学生命科学学院, 山东烟台 264005

中文摘要:为建立快捷、灵敏检测苹果轮纹病菌Botryosphaeria dothidea的环介导等温扩增（loop-medi-ated isothermal amplification，LAMP）检测方法，以其内转录间隔区ITS序列为靶标，设计6条LAMP引物，对其特异性进行检测，优化反应条件并建立苹果轮纹病菌的LAMP检测方法。引物特异性检测结果表明，2株苹果轮纹病菌反应结果呈绿色为阳性，而其它3株对照菌株反应结果呈橙色为阴性，表明了LAMP检测引物的高特异性。优化后的LAMP最佳反应条件：温度65℃、扩增时间60 min、FIP/BIP、F3/B3、LF/LB引物终浓度分别为1.0、0.25、0.5 μmol/L。LAMP检测方法对苹果轮纹病菌DNA的检测灵敏度达到了100 ag/μL，是常规PCR检测灵敏度的100倍。田间疑似轮纹病组织检测结果发现LAMP方法对苹果轮纹病菌的检出率高达68%，而传统分离鉴定方法的检出率仅为24%。表明所建立的苹果轮纹病菌LAMP快速检测方法简便快捷、特异性好、灵敏度高，尤其适用于基层植保机构对于苹果轮纹病菌的田间快速检测。

中文关键词:苹果轮纹病菌内转录间隔区(ITS) 环介导等温扩增(LAMP) 快速检测

Development of a loop-mediated isothermal amplification assay for rapid detection of apple ring rot pathogen Botryosphaeria dothidea

Author Name	Affiliation	E-mail
WANG Shaoli	Yantai Academy of Agricultural Sciences, Yantai 265500, Shandong Province, China
QU Henghua	Yantai Fruit and Tea Workstation, Yantai 264001, Shandong Province, China
WANG Yingzi	Yantai Academy of Agricultural Sciences, Yantai 265500, Shandong Province, China College of Agronomy, Yantai University, Yantai 264005, Shandong Province, China	ytnkyzbs@yt.shandong.cn
WANG Peisong	Yantai Academy of Agricultural Sciences, Yantai 265500, Shandong Province, China
LUAN Binghui	Yantai Academy of Agricultural Sciences, Yantai 265500, Shandong Province, China
WANG Guanhua	College of Agronomy, Yantai University, Yantai 264005, Shandong Province, China

Abstract:To establish a convenient and quick detection method for Botryosphaeria dothidea, based on internal transcribed spacer sequence (ITS) of B. dothidea and using six LAMP primers, the loop-mediated isothermal amplification (LAMP) method was successfully established after optimizing reaction conditions. The specificity assay of LAMP showed that the products of two B. dothidea strains displayed green and positive reaction, and three other control strains displayed orange and negative reaction, which suggested that the LAMP detection was specific for B. dothidea. The optimized conditions of LAMP reaction were constant temperature 65℃, 60 min of extension and 1.0 μmol/L FIP/BIP, 0.25 μmol/L F3/B3、0.5 μmol/L LF/LB. The sensitivity of LAMP detection was 100 ag/μL for B. dothidea DNA, which was 100 times more sensitive than that of regular PCR detection. Finally, LAMP was applied to detect suspected apple ring rot samples collected from the field. The positive rate of LAMP detection was 68%, while the positive rate by using traditional identification methods was only 24%. The results indicated that the LAMP established in the study could rapidly and easily detect B. dothidea, and had the potential for detection of B. dothidea specifically for grassroots plant protection agencies.

keywords:Botryosphaeria dothidea internal transcribed spacer loop-mediated isothermal amplification rapid detection

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