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苹果轮纹病菌LAMP快速检测方法的建立
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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 NameAffiliationE-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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