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番茄煤污假尾孢叶斑病菌实时荧光定量PCR检测技术的建立及应用
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引用本文:康华军,柴阿丽,石延霞,谢学文,郭建光,李宝聚.番茄煤污假尾孢叶斑病菌实时荧光定量PCR检测技术的建立及应用.植物保护学报,2019,46(6):1214-1221
DOI:10.13802/j.cnki.zwbhxb.2019.2018216
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作者单位E-mail
康华军 中国农业科学院蔬菜花卉研究所, 北京 100081  
柴阿丽 中国农业科学院蔬菜花卉研究所, 北京 100081 chaiali@caas.cn 
石延霞 中国农业科学院蔬菜花卉研究所, 北京 100081  
谢学文 中国农业科学院蔬菜花卉研究所, 北京 100081  
郭建光 山西省运城市盐湖区蔬菜发展中心, 运城 044000  
李宝聚 中国农业科学院蔬菜花卉研究所, 北京 100081 libaoju@caas.cn 
中文摘要:为快速、准确地对番茄煤污假尾孢Pseudocercospora fuligena进行检测与定量分析,基于其Avr4基因设计特异性引物JWB-9F/JWB-7R,建立实时荧光定量PCR检测技术,分析该检测技术的特异性和灵敏度,并利用采集自重庆市、河北省和广西壮族自治区的14份材料对该检测技术的应用效果进行验证。结果表明,引物JWB-9F/JWB-7R仅可从番茄煤污假尾孢基因组DNA中扩增出232 bp的目的片段,特异性良好;实时荧光定量PCR检测技术的灵敏度为67.09 copies/μL,是普通PCR检测技术的1 000倍。且该实时荧光定量PCR检测技术可以实现未显症样本中番茄煤污假尾孢的定量检测,检测限为6.02×102 copies/μL,实际应用效果较好。表明所建立的实时荧光定量PCR检测技术可用于番茄煤污假尾孢叶斑病的早期诊断和预测预报。
中文关键词:煤污假尾孢  Avr4  实时荧光定量PCR  检测
 
Establishment and application of real-time quantitative PCR assay for detection of fungal pathogen Pseudocercospora fuligena in tomato
Author NameAffiliationE-mail
Kang Huajun Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, China  
Chai Ali Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, China chaiali@caas.cn 
Shi Yanxia Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, China  
Xie Xuewen Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, China  
Guo Jianguang Vegetable Development Center of Yanhu District, Yuncheng 044000, Shanxi Province, China  
Li Baoju Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, China libaoju@caas.cn 
Abstract:To quickly and accurately detect and quantify Pseudocercospora fuligena of tomato, a realtime fluorescent quantitative PCR (qPCR) method was established. The specific primer set JWB-9F/JWB-7R for qPCR was designed based on Avr4 gene of P. fuligena, the specificity and sensitivity of qPCR method were analyzed, and a total of 14 materials from Chongqing City, Hebei Province, and Guangxi Zhuang Autonomous Region were used to test the practical application of this qPCR method. The results showed that the primer set JWB-9F/JWB-7R could only amplify a specific fragment of 232 bp from the genomic DNA of P. fuligena and the limit of detection was 67.09 copies/μL, which was 1 000 times that of conventional PCR. The pathogen could be quantitatively detected from asymptomatic tomato materials by this qPCR method and the detection limit was 6.02×102 copies/μL. The results indicated that the qPCR method in this study can be used for early diagnosis and forecast of tomato black leaf spot.
keywords:Pseudocercospora fuligena  Avr4  real-time fluorescent quantitative PCR (qPCR)  detection
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