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| 題 名 | 利用聚合酶連鎖反應-限制酶片段長度多型性檢測與鑑定李斯特氏菌種=Detection and Differentiation of Listeria Species by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism |
|---|---|
| 作 者 | 廖朝財; 許俐伶; 李憲明; 林春福; | 書刊名 | Journal of Biomedical & Laboratory Sciences |
| 卷 期 | 33:2 2021.06[民110.06] |
| 頁 次 | 頁79-94 |
| 分類號 | 415.123 |
| 關鍵詞 | 李斯特菌; recA基因; 聚合酶連鎖反應; 限制酶片段長度多型性; Listeria; recA genes; PCR; Restriction fragment length polymorphism; |
| 語 文 | 中文(Chinese) |
| 中文摘要 | 傳統鑑定李斯特氏菌種的過程通常都是耗事費時的,本研究我們以 recombinase A gene (recA) 為目標基 因,開發出 PCR-RFLP 快速鑑定李斯特氏菌種之分子鑑定的方法。首先我們利用 GenBank 資料庫中其它 李斯特氏菌種之 recA 基因序列相似性較高的區域製作探針 (probe),用 LA-PCR 增幅出 Listeria grayi 之 recA 基因,並將此增幅之基因進行定序。定序結果顯示 L. grayi 之 recA 基因序列全長為 1044 bp,較其它 五種李斯特氏菌種少三個核苷酸。接著我們根據六種李斯特氏菌種之 recA 基因設計三條引子 L-recA1047-5F、L-recA1047-3R 及 Lg-recA1044-3R,以此三條引子進行 PCR 反應可將六種李斯特氏菌種 之 recA 基因增幅,然後我們根據限制酶圖譜分析之結果,將六種李斯特氏菌種之 recA DNA 利用 TaqⅠ 剪 切,電泳結果顯示六種李斯特氏菌種之 recA DNA 利用 TaqⅠ剪切後呈現出不同長度的 DNA 片段的多型性。 總而言之,本研究結果顯示以 recA 基因當作目標基因之 PCR-RFLP 方法可快速且正確的區別鑑定六種李 斯特氏菌種。 |
| 英文摘要 | The traditional process of identifying Listeria species is usually labor-intensive and time-consuming. In this study, we reported a molecular method based on restriction fragment length polymorphism (RFLP) of PCR-amplified fragments of the recA gene that was designed to rapidly identify Listeria strains to the species level. First, to obtain the entire recA gene sequences of Listeria grayi, we comparison of recA gene sequences of Listeria species available in the GenBank database to select a conserved region as a probe for detecting the recA gene of L. grayi. By using LA-PCR, the entire region of the recA of L. grayi was amplified and sequenced. The sequencing results showed that the recA gene sequence of L. grayi had a total length of 1044 bp, which was three nucleotides less than the other five Listeria species. Then, according to all of the recA genes of Listeria species, primers L-recA1047-5F, L-recA1047-3R and Lg-recA1044-3R were derived to amplify the entire recA region of DNA from Listeria species by PCR. The amplified PCR products were subsequently digested with the restriction enzymes Taq I. The analysis showed that the RFLP profiles of PCR products from each species of Listera were quite distinguishable. Taken together, our results suggest that the present PCR-RFLP method allows rapid and accurate identification of the six species of the Listeria genus. |
本系統中英文摘要資訊取自各篇刊載內容。