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題名 | The Design of Specific Primers for the Detection of Ralstonia Solanacearum in Soil Samples by Polymerase Chain Reaction=以聚合酵素連鎖反應檢測土壤內青枯病菌的專一性引子的設計 |
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作者姓名(中文) | 李永安; 王啟仲; | 書刊名 | Botanical Bulletin of Academia Sinica |
卷期 | 41:2 2000.04[民89.04] |
頁次 | 頁121-128 |
分類號 | 433.4 |
關鍵詞 | 軟腐病菌; 聚合酵素連鎖反應; 假單胞菌; 青枯病菌; 黃原桿菌; Erwinia; Polymerase chain reaction; Pseudomonas; Ralstonia solanacearum; Xanthomonas; |
語文 | 英文(English) |
中文摘要 | 對青枯病菌的基因組核酸 (genomic DNA) 以任意放大多型性核酸 (RAPD) 技術,進行聚合酵素連鎖反應 (PCR) 的分析,發現可擴增出一段 0.7-kb 的 PCR 產物。以此 PCR 產物做為探針,與經 EcoRI 酵素處理的青枯病菌的基因組核酸,進行南方氏雜合反應,可在 2.7-kb 處有雜合訊息。將此 2.7-kb EcoRI 核酸片段選殖出後,再以此片段做為探針進行雜合反應,發現只有青枯病菌的基因組核酸可產生雜合訊息,而其他植物病原細菌如 Pseudomonas spp.、Xanthomonas campestris 病原小種、Erwinia spp. 等均無訊息產生。將此 2.7-kb EcoRI 核酸片段經核酸定序,並依該序列設計出一組 PCR 引子,此組引子對青枯病菌具有專一性,可擴增出 1.1-kb 的 PCR 產物,且 PCR 反應的敏感度可以達到 20 細胞。此 PCR 反應亦用於檢測土壤內的青枯病菌,若直接以含有青枯病菌的土壤萃取液進行 PCR 反應,發現無任何 PCR 產物。若從含有青枯病菌的土壤抽取核酸後再進行 PCR 反應,則可擴增出 1.1-kb 的 PCR 產物。為加速對土壤內青枯病菌的檢測工作,已研發出一個從土壤抽取核酸以進行 PCR 反應的簡易方法。 |
英文摘要 | A 0.7-kb DNA fragment, amplified by the randomly amplified polymorphic DNA (RAPD) method from Ralstonia solanacearum total DNA, was cloned and evaluated as a specific DNA probe. This 0.7-kb DNA fragment hybridized to a 2.7-kb EcoRI fragment in the EcoRI-digested total DNA of R. solanacearum. The 2.7-kb EcoRI fragment was also cloned and hybridized only to R. solanacearum but not to other Pseudomonas spp., pathovars of Xanthomonas campestris, and Erwinia spp. tested. The DNA sequence of this 2.7-kb fragment was obtained and used to design specific oligonucleotide primers for polymerase chain reaction (PCR) amplification. The primers amplified the same 1.1-kb PCR product from all R. solanacearum strains tested and failed to amplify DNA from any other plant pathogenic bacterial strains tested. DNA isolated from several saprophytic bacteria did not produce any PCR products with these primers. This specific PCR for R. solanacearum was also performed from colonies grown on BG medium with similar results. The sensitivity of the PCR assay using the specific primers was about 20 cells. The PCR assay was used to detect R. solanacearum in soil using these primer sets. No PCR product could be found when soil extract containing R. solanacearum was used directly in the assay. DNA extraction from soil was needed for the success of PCR assay. A simple method for DNA extraction from soil for PCR assay was developed and can hasten the detection of R. solanacearum in soil. |
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