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

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。

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.