芋疫霉菌的巢式PCR检测

doi:10.19303/j.issn.1008-0384.2019.01.012

福建农业学报 ›› 2019, Vol. 34 ›› Issue (1): 76-82.doi: 10.19303/j.issn.1008-0384.2019.01.012

• 植物保护 • 上一篇

芋疫霉菌的巢式PCR检测

兰成忠^1,2, 卢学松¹, 姚锦爱¹, 丁雪玲¹, 蒋军喜²

1. 福建省作物有害生物监测与治理重点实验室/福建省农业科学院植物保护研究所, 福建福州 350013;
2. 江西农业大学农学院, 江西南昌 330045

收稿日期:2017-12-05 修回日期:2018-11-25 发布日期:2019-03-27
通讯作者: 蒋军喜(1964-),男,博士,教授,博士生导师,主要从事植物病害综合治理研究(E-mail:jxau2011@126.com) E-mail:jxau2011@126.com
作者简介:兰成忠(1979-),男,硕士,副研究员,主要从事植物病害防治技术研究(E-mail:lczhong7911@126.com)
基金资助:
国家自然科学基金项目（31400025）；福建省农业科学院青年科技英才百人计划项目（YC2015-4）；福建省农业科学院植物保护创新团队建设项目（STIT2017-1-8）

Nested-PCR Detection of Taro Leaf Blight Pathogen Phytophthora colocasiae

LAN Cheng-zhong^1,2, LU Xue-song¹, YAO Jin-ai¹, DING Xue-ling¹, JIANG Jun-xi²

1. Fujian Key Laboratory for Monitoring and Integrated Management of Crop Pest, Institute of Plant Protection/Fujian Academy of Agricultural Sciences, Fuzhou, Fujian 350013, China;
2. College of Agronomy, Jiangxi Agricultural University, Nanchang, Jiangxi 330045, China

Received:2017-12-05 Revised:2018-11-25 Published:2019-03-27

摘要/Abstract

摘要： [目的]建立芋疫霉菌快速、准确的PCR检测技术，为芋疫病流行规律监测和综合防控提供科学依据。[方法]根据芋疫霉菌与其他疫霉菌种类Ypt1基因序列差异，设计了1对芋疫霉菌PCR检测特异引物PCOF/PCOR，并对该引物的特异性、灵敏性和应用性进行了验证。[结果]在优化的反应体系与扩增条件下，PCOF/PCOR引物能特异性地从芋疫霉菌基因组DNA中扩增出1条172 bp的条带，而其他供试病原菌均无扩增条带。在25 μL PCR反应体系中，PCOF/PCOR引物对芋疫霉菌基因组DNA的检测灵敏度为100 pg，而以疫霉菌Ypt1基因通用引物ph1F/Yph2R为第一轮引物，PCOF/PCOR为第二轮引物，进行巢式PCR扩增，能检测到10 fg芋疫霉菌基因组DNA，检测灵敏度提高了10 000倍。采用巢式PCR，可从芋疫病发病的叶片和未显症叶片组织中检测到芋疫霉菌，检出率分别为100%和57.5%。[结论]所建立的巢式PCR可应用于芋疫霉菌的快速、特异和高灵敏度检测。

关键词: 芋疫霉菌, 特异引物, 巢式PCR, 分子检测

Abstract: [Objective]To develop a PCR assay for rapid and accurate detection, epidemiology information, and integrated disease management on Phytophthora colocasiae, the pathogen of taro phytophthora blight.[Method]A pair of species-specific primers, PCOF/PCOR, for P. colocasiae was designed based on the differences in Ras-related protein (Ypt1) gene sequence between P. colocasiae and other species in the same genus. The specificity, sensitivity and applicability of the primers were evaluated.[Result] With the optimized reaction conditions and amplification,PCOF/PCOR amplified only a single band of 172 bp with genomic DNA extracted from all P.colocasiae strains, while the other tested pathogens had no corresponding band. The sensitivity of conventional PCR method using PCOF/PCOR as primers was 100 pg of genomic DNA in a 25 μL reaction solution. Whereas, the newly developed nested-PCR performed using Ypt1 gene universal primers ph1F/Yph2R for the first-round and PCOF/PCOR for the second-round increased 10 000-fold on the sensitivity to 10 fg. The nested-PCR methodology could positively detected P. colocasiae 100% in diseased leaves or 57.5% in symptom-free infected tissues.[Conclusion] The newly established nested-PCR assay could be used for rapid, specific and sensitive detection of P. colocasiae.

Key words: Phytophthora colocasiae, specific primer, nested-PCR, molecular detection

中图分类号:

S435.72

兰成忠, 卢学松, 姚锦爱, 丁雪玲, 蒋军喜. 芋疫霉菌的巢式PCR检测[J]. 福建农业学报, 2019, 34(1): 76-82.

LAN Cheng-zhong, LU Xue-song, YAO Jin-ai, DING Xue-ling, JIANG Jun-xi. Nested-PCR Detection of Taro Leaf Blight Pathogen Phytophthora colocasiae[J]. Fujian Journal of Agricultural Sciences, 2019, 34(1): 76-82.

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芋疫霉菌的巢式PCR检测

Nested-PCR Detection of Taro Leaf Blight Pathogen Phytophthora colocasiae

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