| 利用常规PCR和实时荧光定量PCR检测杨梅凋萎病菌 |
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| 引用本文： | 任海英,戚行江,梁森苗,郑锡良.利用常规PCR和实时荧光定量PCR检测杨梅凋萎病菌[J].植物病理学报,2016,46(1):1-10. |
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| 作者姓名： | 任海英 戚行江 梁森苗 郑锡良 |
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| 作者单位： | 浙江省农业科学院园艺研究所, 杭州 310021 |
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| 基金项目： | 浙江省重大科技专项重点农业项目(2012C12009-5);国家公益性行业(农业)科研专项(201203089) |
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| 摘 要： | 凋萎病是近年来危害杨梅的主要病害。为了快速灵敏的检测杨梅凋萎病菌(Pestalotiopsis versicolor和P.microspora),本研究开发了常规PCR和SYBR Green实时荧光定量PCR技术各一套。利用P.versicolor(JN861773)和P.microspora(JN861776)的ITS1-5.8S rDNA-ITS2序列的相同部分设计引物对(Pvm1L/Pvm1R)。该引物对利用常规PCR技术能特异性扩增出杨梅凋萎病菌188 bp的目标产物而对照菌株则呈阴性。该常规PCR体系能够检测人工接种后21 d和田间自然发病的有病症杨梅组织中的凋萎病菌,检测下限是0.6×10~5拷贝数。利用Pvm1L/Pvm1R进行SYBR Green实时荧光定量PCR,检测灵敏度是常规PCR的100倍,检测下限是0.6×10~3拷贝数,能够检测出人工接种及田间已经感染但尚未表现症状的杨梅组织中的凋萎病菌。这两项技术简单、快速、灵敏特异性强,可以应用于杨梅凋萎病的诊断和苗木检疫。
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| 关 键 词： | 杨梅 凋萎病菌 常规PCR SYBR Green实时荧光定量PCR 分子检测 |
Use of conventional and real-time quantitative PCR to detect Pestalotiopsis,the cause of bayberry twig blight |
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| REN Hai-ying,QI Xing-jiang,LIANG Sen-miao,ZHENG Xi-liang.Use of conventional and real-time quantitative PCR to detect Pestalotiopsis,the cause of bayberry twig blight[J].Acta Phytopathologica Sinica,2016,46(1):1-10. |
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| Authors: | REN Hai-ying QI Xing-jiang LIANG Sen-miao ZHENG Xi-liang |
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| Institution: | Institute of Horticulture, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China |
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| Abstract: | Twig blight disease is one of the main diseases on bayberry (Myrica rubra Sieb.& Zucc). We deve-loped an effective method to detect and quantify the pathogens Pestalotiopsis versicolor and P. microspora by using conventional PCR and SYBR Green real-time PCR. A primer set, Pvm1L/Pvm1R, was designed based on a conserved sequence of the internal transcribed spacer (ITS1-5.8S rDNA-ITS2) region of the ribosomal DNA gene of P. versicolor (JN861773) and P. microspora (JN861776). The 188 bp DNA fragments were amplified from 30 isolates of P. versicolor and 30 isolates of P. microspora, and no product was amplified from isolates of 11 other fungal genera. Conventional PCR was able to detect the pathogens on symptomatic and artificially infected bayberry plants at 21 days after inoculation, and the detection limit was 0.6×105 copies of the Pestalotiopsis DNA. In addition, the Pvm1L/Pvm1R primer set were successfully adapted to SYBR Green real-time PCR, which had a limit 100 times lower (0.6×103) than that by conventional PCR and was able to detect the pathogens in symptomless, artificially inoculated, and naturally infected plants. The conventional and SYBR Green real-time PCR developed in this study were simple, fast, sensitive, and specific, and can be used to detect Pestalotiopsis spp. from infected bayberry in the field. |
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| Keywords: | Myrica rubra pathogen of twig blight disease conventional PCR SYBR Green real-time quantitative PCR molecular detection |
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