利用聚合酶連鎖反應-限制酶片段長度多型性快速鑑定變形桿菌屬菌種

Polymerase chain reaction-restriction fragment length polymorphism for rapid Identification of Proteus species

本研究我們以recombinase A gene (recA)為目標基因，開發出PCR-RFLP快速鑑定變形桿菌屬菌種之分子鑑定的方法。首先我們以GeneBank中Proteus mirab ilis recA基因(accession no. X14870)全長1348 bp的DNA進行BLASTN比對，BLASTN比對結果顯示第328核苷酸位置的G至第937核苷酸位置的 A共610個核苷酸區域與許多細菌之recA基因有很高的相同性(identity)。接著我們在此一區域設計了對變形桿菌屬recA基因的退化性引子(degenerate pri mers) recA5F與recA3R，以此二條退化性引子進行PCR反應可將四種變形桿菌屬菌種之441 bp recA基因部分片段增幅。然後我們根據限制酶圖譜分析之結果，將四種變形桿菌屬菌種之441 bp recA DNA利用TaqⅠ剪切，電泳結果顯示四種變形桿菌屬菌種之441 bp 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441 bp recA DNA當作目標基因之PCR-RFLP方法可快速且正確的區別鑑定四種變形桿菌屬菌種。

In this study, we developed a PCR-RFLP method for rapid molecular identification of bacteria within the Proteus genus, targeting the recombinase A gene (recA). Initially, we performed a BLASTN comparison using the full-length 1348 bp DNA sequence of the Proteus mirabilis recA gene (accession no. X14870) obtained from GeneBank. The BLASTN results indicated a high degree of identity between a region spanning from the 328th nucleotide position (G) to the 937th nucleotide position (A), encompassing 610 nucleotides, and the recA genes of many other bacteria. Next, we designed a pair of degenerate primers, recA5F and recA3R, targeting the recA gene region within the Proteus genus. Using these two degenerate primers in a PCR reaction allowed us to amplify a 441 bp partial segment of the recA gene in four different Proteus species. We digested the amplified 441 bp recA PCR products with the restriction enzymes Taq I. The analysis showed that the RFLP profiles of PCR products from each species of Proteus were quite distinguishable. In conclusion, the results of this study demonstrate that the PCR-RFLP method targeting the 441 bp recA DNA can effectively and accurately differentiate and identify the four Proteus species.