| 辣椒土传病原菌三重PCR检测体系的建立与应用 |
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| 引用本文： | 邹芬,何烈干,李湘民,黄瑞荣,马辉刚.辣椒土传病原菌三重PCR检测体系的建立与应用[J].南方农业学报,2022,53(12):3453-3459. |
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| 作者姓名： | 邹芬 何烈干 李湘民 黄瑞荣 马辉刚 |
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| 作者单位： | 江西省农业科学院植物保护研究所, 江西南昌 330200 |
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| 基金项目： | 江西省蔬菜产业技术体系项目（JXARS-06）；江西省现代农业科研协同创新专项（JXXTCX201901-03） |
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| 摘 要： | 【目的】建立简便、快速、准确同时检测辣椒疫霉（Phytophthora capsici）、白绢病菌（Sclerotium rolfsii）和青枯病菌（Ralstonia solanacearum）的多重PCR检测体系，为辣椒多种土传病害同时早期诊断提供技术支撑。【方法】以3种辣椒土传病害病原菌辣椒疫霉、白绢病菌和青枯病菌为研究对象，参考相关文献筛选出3种病原菌的3对特异性引物PCA1F/PCA1R、SCR-F/SCR-R和RalfliC-F/RalfliC-R，以引物浓度、退火温度、循环次数及延伸时间为变异因子，探索最佳反应体系，建立三重PCR检测方法，并基于田间实际发病样品对方法进行验证。【结果】优化的三重PCR反应体系25.0 μL: 3对引物PCA1F/PCA1R、SCR-F/SCR-R和RalfliC-F/RalfliC-R浓度分别为0.24、0.24和0.28 μmol/L，DNA模板各10 ng，2×Multiplex PCR Mix 12.5 μL，ddH2O补足至25.0 μL。最佳扩增程序: 94℃预变性1 min； 94℃ 30 s，55.8℃ 30 s，72℃ 45 s，进行35个循环； 72℃延伸10 min。建立的三重PCR体系可分别扩增出352、540和724 bp的特异性目的片段，体系灵敏度达10-1 ng/μL。利用建立的反应体系，对来自江西省不同市（县）的54份辣椒和土壤样品进行检测，样品检出率为100%。【结论】建立的三重PCR检测体系可对辣椒田间发病植株和土壤中辣椒疫霉、白绢病菌和青枯病菌进行快速、准确检测，可应用于辣椒多种土传病害并发的早期预防和流行监测。
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| 关 键 词： | 辣椒 三重PCR 辣椒疫霉 白绢病菌 青枯病菌 |
| 收稿时间： | 2022-02-22 |
Development and application of triplex PCR detection system for pepper soil-borne pathogens |
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| ZOU Fen,HE Lie-gan,LI Xiang-min,HUANG Rui-rong,MA Hui-gang.Development and application of triplex PCR detection system for pepper soil-borne pathogens[J].Journal of Southern Agriculture,2022,53(12):3453-3459. |
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| Authors: | ZOU Fen HE Lie-gan LI Xiang-min HUANG Rui-rong MA Hui-gang |
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| Affiliation: | Institute of Plant Protection, Jiangxi Academy of Agricultural Sciences, Nanchang, Jiangxi 330200, China |
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| Abstract: | 【Objective】 In this study, a multiplex PCR method was developed for early and rapid detection of Phytophthora capsici, Sclerotium rolfsii and Ralstonia solanacearum at the same time, and provide technical support for early diagnosis of soil-borne disease pathogens in pepper field.【Method】 Three soil-borne disease pathogens of pepper, P. capsici, S. rolfsii and R. solanacearum, were selected, and three pairs of specific primers, PCA1F/PCA1R, SCR-F/SCR-R and RalfliC-F/RalfliC-R, were screened with reference to relevant papers. The optimal reaction system was explored by setting different primer concentrations, annealing temperatures, cycle numbers and extension times to establish a triplex PCR detection method, and that was validated based on the actual diseased samples in the field.【Result】 In the optimized triplex PCR system 25.0 μL, the concentrations of the primer pairs of PCA1F/PCA1R, SCR-F/SCR-R and RalfliC-F/RalfliC-R were 0.24, 0.24 and 0.28 μmol/L, respectively;DNAtemplates were 10 ng each;2×Multiplex PCR Mix 12.5 μL, added ddH2O to make up to 25.0 μL. The optimal amplification procedure was as follows:pre-denaturation at 94℃ for 1 min followed by 35 cycles of denaturation at 94℃ for 30 s, annealing at 55.8℃ for 30 s and extension at 72℃ for 45 s; finally extension at 72℃ for 10 min. Established triplex PCR system could amplify specific fragments of P. capsici, S. rolfsii and R. solanacearum with the length of 352, 540 and 724 bp, respectively, and with the sensitivity of 10-1 ng/μL. Using the above reaction system, 54 pepper and soil samples collected from different counties and cities in Jiangxi were detected, and the detection rate of the samples was 100%.【Conclusion】 The established triplex PCR is able to rapidly and accurately detect P. capsici, S. rolfsii and R. solanacearum in diseased plants and soil in the field. It can be used for early prevention and epidemic monitoring of various soil borne diseases of pepper. |
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