由tuf基因序列發展PCR-變性梯度膠體電泳方法以區分及鑑定葡萄球菌菌種   全文下載

Development of a PCR-denaturing Gradient Gel Electrophoresis Method Targeting the tuf Gene to Differentiate and Identify Staphylococcus Species

以往金黃色葡萄球菌常被認為是造成食品葡萄球菌毒素中毒的主要病原菌，但是最近一些研究指出其他的葡萄球菌也有產生腸毒素的能力，因此，造成葡萄球菌食品中毒的病原有可能不僅只是金黃色葡萄球菌，而是其他種類的葡萄球菌或是多種葡萄球菌同時污染食物所致。本研究中我們以細菌轉譯延長因子Tu 之基因（elongation factor Tu gene, tuf gene）為標的基因，使用由該基因序列所設計的引子進行葡萄球菌屬之專一性的聚合?鏈鎖反應（polymerase chain reaction, PCR），並結合變性梯度膠體電泳（denaturing gradient gel electrophoresis, DGGE）技術，對常見之葡萄球菌菌種進行測試，預期在發生葡萄球菌食品中毒時，可以應用於食物樣品之檢測，並快速鑑定造成中毒之菌種。使用本方法測試之結果， 11 個常見的葡萄球菌菌種可以區分為10 種不同的圖譜，另外分析9 株金黃色葡萄球菌菌株、2 株沃氏葡萄球菌、2 株頭葡萄球菌、2 株表皮葡萄球菌以及2 株腐生葡萄球菌，結果指出同種之菌株皆產生相同的圖譜。隨機混合數種菌種並添加至牛乳後再進行測試，結果可以偵測出所有添加入牛乳之菌種，其中受測的菌種中包括了具有產毒素潛力之菌種。"

Staphylococcal intoxications are often associated with staphylococcal enterotoxins produced by Staphylococcus aureus. However, several studies revealed that other Staphylococcus species, such as S. intermedius and S. warneri, could produce enterotoxins as well. To facilitate the identification of a mixed culture while tracing the causative staphylococci of food poison, a polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) method was developed to differentiate Staphylococcus species using Staphylococcus-specific primers targeting the elongation factor Tu gene (tuf gene). Eleven tested species were differentiated to 10 separated patterns. Variance of the patterns between strains within a species was analyzed using 9 strains of S. aureus and several strains of other species. It was shown that strains within a species migrated the same distance in the DGGE assay. When mixed cultures of different Staphylococcus species in milk were subjected to DNA extraction and PCR-DGGE, the resulted patterns faithfully corresponded to the species of the mixed cultures, including those of potential to secret enterotoxins.