| 三重PCR检测茄子青枯病菌、黄萎病菌和白绢病菌 |
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| 引用本文：邹芬,何烈干,李湘民,黄瑞荣,马辉刚.三重PCR检测茄子青枯病菌、黄萎病菌和白绢病菌.植物保护学报,2023,50(1):206-213 |
| DOI：10.13802/j.cnki.zwbhxb.2023.2021044 |
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| 中文摘要:为快速、准确对田间茄子发病植株和土壤中3种土传病害的病原菌青枯病菌Ralstonia solanacearum、黄萎病菌Verticillium dahliae和白绢病菌Sclerotium rolfsii进行检测,筛选这3种病菌的特异性引物,建立这3种病菌的三重PCR检测体系,对其退火温度、引物浓度、10×PCR Buffer(Mg2+plus)、dNTP和Taq DNA聚合酶进行优化,对优化后体系的特异性和灵敏度进行检测,并利用优化体系对田间采集的病害样品和土壤样品进行检测。结果表明,在优化后的50μL三重PCR检测体系中,RS-1-F/RS-3-R、dllz1/dllz2和SRITSF/SRITSR引物的最佳浓度分别为0.16、0.16和0.28μmol/L,10×PCR Buffer(Mg2+plus)、dNTP和Taq DNA聚合酶体积分别为6、5和1μL,检测体系的最佳退火温度为54℃。按照优化后的反应条件能分别扩增出青枯病菌、黄萎病菌和白绢病菌3种病菌的特异条带,分别为716、350和500bp,其他对照病菌无条带。该体系对病菌DNA检测灵敏度达到0.1ng/μL。该检测体系对田间采集的病害样品和土壤样品中病菌的检出率达95%以上。 |
| 中文关键词:茄子 土传病害 三重PCR 青枯病菌 黄萎病菌 白绢病菌 |
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| Triplex PCR detection of three soil-borne pathogens Ralstonia solanacearum,Verticillium dahliae and Sclerotium rolfsii in eggplants |
| Author Name | Affiliation | E-mail | | ZOU Fen | Institute of Plant Protection, Jiangxi Academy of Agricultural Sciences, Nanchang 330200, Jiangxi Province, China | | | HE Lie-gan | Institute of Plant Protection, Jiangxi Academy of Agricultural Sciences, Nanchang 330200, Jiangxi Province, China | | | LI Xiang-min | Institute of Plant Protection, Jiangxi Academy of Agricultural Sciences, Nanchang 330200, Jiangxi Province, China | | | HUANG Rui-rong | Institute of Plant Protection, Jiangxi Academy of Agricultural Sciences, Nanchang 330200, Jiangxi Province, China | | | MA Hui-gang | Institute of Plant Protection, Jiangxi Academy of Agricultural Sciences, Nanchang 330200, Jiangxi Province, China | mahg1997@sina.com |
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| Abstract:In order to rapidly and accurately detect three soil-borne pathogens, Ralstonia solanacearum,Verticillium dahliae and Sclerotium rolfsii, in diseased eggplants and in the field soil, three sets of specific primers were screened to establish a triplex PCR detection system. The important factors affecting the multiplex PCR reaction, including annealing temperature, primer concentration, and volumes of 10×PCR Buffer(Mg2+plus), dNTP, and Taq DNA polymerase were optimized, and the specificity and sensitivity were determined. Besides, the optimized system was tested in detection of the pathogens from diseased tissues and field soil samples. The results showed that in the 50 μL triplex PCR system, the optimal annealing temperature was 54℃, the optimal primer concentrations of RS-1-F/RS-3-R, dllz1/dllz2and SRITSF/SRITSR was 0.16, 0.16 and 0.28 μmol/L, respectively, and 10×PCR Buffer(Mg2+ plus),dNTP, Taq DNA polymerase was 6, 5, 1 μL, respectively. Using the optimized reaction system, the specific fragments of the three pathogens with the length of 716, 350, 500 bp could be amplified, respectively,while no band was amplified from the other control pathogens. The detection limitation was 0.1 ng/μL.The detection rate from diseased tissues and soil samples was over 95% using this detection system. |
| keywords:eggplant soil-borne disease triplex PCR Ralstonia solanacearum Verticillium dahliae Sclerotium rolfsii |
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