應用多目標即時聚合酶鏈反應同時檢驗金黃色葡萄球菌、沙門氏菌、仙人掌桿菌及腸炎弧菌   全文下載

Simultaneous Detection of Food Pathogens, Staphylococcus aureus, Salmonella Enterica, Bacillus cereus and Vibrio parahaemolyticus by Multiplex Real-Time Polymerase Chain Reaction

使用即時定量多目標PCR同時檢測金黃色葡萄球菌之SAOUHSC_02297基因、沙門氏菌之invA基因、仙人掌桿菌之hbl基因、腸炎弧菌之tlhA基因，分別設計4組引子及探針，皆可檢測出且無偽陽性，即表示專一性佳且彼此無干擾情形。即時定量多目標PCR同時檢測金黃色葡萄球菌、沙門氏菌、仙人掌桿菌及腸炎弧菌，除仙人掌桿菌靈敏度為103.5 CFU/mL，其餘皆可達102.5 CFU/mL，而即時定量多目標PCR檢測單一菌株與混合菌株之線性相關係數R2皆大於0.98，表示即時定量多目標PCR定量檢測病原菌之誤差在可接受範圍，且檢驗流程於12小時內可完成。

The study established a multiplex real-time polymerase chain reaction (PCR) for simultaneous detection and quantitation of Staphylococcus aureus, Salmonella Enterica, Bacillus cereus and Vibrio parahaemolyticus in the same reaction at one time, thus reducing the amounts of reagents and cutting down on the labor and time. Four sets of genetic marker specific primers corresponding to SAOUHSC_02297, invA, hbl and tlhA gene, and probes were labeled with FAM, HEX, TEXAS RED and Cy5 respectively. Multiplex real-time PCR was carried out with an iQTM Multiplex Powermix Kit (BIO-RAD) Sequence Detection System. The result showed that the correlation coefficient between multiplex real-time PCR estimates and plate counts of 10-serial dilutions in specific bacteria was above 0.98, independent of 101-4-fold numbers of three other pathogenic strains. With optimized conditions, the lowest detection concen-trations of four pathogens were 102.5 CFU/mL for Sta. aureus, S. Enterica and V. parahaemolyticus and 103.5 CFU/mL for B. cereus. The duration of the entire experiment from DNA isolation and purification to PCR amplification was less than 12 h.