Conventional PCR and real-time quantitative PCR detection of Helminthosporium solani in soil and on potato tubers
Conventional PCR and real-time quantitative PCR detection of Helminthosporium solani in soil and on potato tubers
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DOI：
10.1023/a:1011247826231
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发表时间：
2001-01-01
影响因子：
1.8
通讯作者：
Duncan, JM
中科院分区：
文献类型：
--
作者：
Cullen, DW;Lees, AK;Duncan, JM
马铃薯银皮病是由茄长蠕孢菌（Helminthosporiumsolani）引起的一种重要经济病害。设计了两套PCR引物Hs 1F 1/Hs 2 R1（外引物）和Hs 1 NF 1/Hs 2NR 1（巢式引物），分别扩增H.索拉尼采用巢式PCR提高单轮PCR的特异性和敏感性。用71个欧洲和北美分离株的DNA分别扩增出447 bp和371 bp的产物。solani，引物的特异性通过不存在来自其它真菌和细菌植物病原体的DNA的扩增产物来证实。本文改进了一种从土壤和马铃薯块茎中直接提取DNA的简便快速方法，在3 h内即可得到纯度和质量均符合PCR要求的DNA。特异性检测H.测定播种土壤中的茄病菌为1.5孢子g（-1）土壤。H.通过PCR还在自然侵染的土壤中以及从感染的和表面上健康的块茎的果皮和果皮提取物中检测到茄病。使用原始引物序列设计特异性引物和TaqManTM荧光探针以进行实时定量（TaqManTM）PCR。特异性检测H.与常规巢式PCR和凝胶电泳一样，在第一轮基于mboxTaqMan的PCR期间获得土壤和块茎中的solani。这种快速和定量PCR检测方法可以准确估计块茎和土壤中的H。solani，从而提供了一种研究生物生态学的工具，并作为疾病风险评估的一个重要组成部分。
Silver scurf is an economically important blemish disease of potato caused by the fungus Helminthosporium solani. Two sets of PCR primers, Hs1F1/Hs2R1 (outer) and Hs1NF1/Hs2NR1 (nested) were designed to unique sequences of the nuclear ribosomal internal transcribed spacer (ITS1 and ITS2) regions of H. solani. Nested PCR was used to increase the specificity and sensitivity of single round PCR. Each primer set amplified a single product of 447 bp and 371 bp respectively, with DNA from 71 European and North American isolates of H. solani, and the specificity of primers was confirmed by the absence of amplified product with DNA from other fungal and bacterial plant pathogens. A simple and rapid procedure for direct extraction of DNA from soils and potato tubers was modified and developed to yield DNA of a purity and quality suitable for PCR within 3 h. The sensitivity of PCR for the specific detection of H. solani in seeded soils was determined to be 1.5 spores g(-1) of soil. H. solani was also detected by PCR in naturally infested soil and from peel and peel extract from infected and apparently healthy tubers. Specific primers and a TaqMan(TM) fluorogenic probe were designed using the original primer sequences to perform real-time quantitative (TaqMan(TM)) PCR. The same levels of sensitivity for specific detection of H. solani in soil and tubers were obtained during first round mboxTaqMan-based PCR as with conventional nested PCR and gel electrophoresis. This rapid and quantitative PCR assay allows an accurate estimation of tuber and soil contamination by H. solani, thus providing a tool to study the ecology of the organism and to serve as a crucial component for disease risk assessments.