- 期刊
Simultaneous Detection of Food Pathogens, Staphylococcus aureus, Salmonella Enterica, Bacillus cereus and Vibrio parahaemolyticus by Multiplex Real-Time Polymerase Chain Reaction
應用多目標即時聚合酶鏈反應同時檢驗金黃色葡萄球菌、沙門氏茵、仙人掌桿菌及腸炎弧菌
摘要
使用即時定量多目標PCR同時檢測金黃色葡萄球菌之SAOUHSC_02297基囚、沙門氏菌之invA基因、仙人掌桿苗之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，即表示單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、仙人掌桿菌及腸先弧菌，除仙人掌桿菌靈敏度為10^3.5 CFU/mL，具餘皆可達10^2.5 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R^2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。
並列摘要
The study established a multiplex real-time polymerase chain reaction (PCR) for simultaneous detection and quantitation of Staphvlococcus 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, imvA, 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 iQ(superscript TM) Multiplex Powermix Kit (BIO-RAD) Sequence Detection System. The result showed that the correlation coefficient between multiplex real-time PCR estimate s and plate counts of 10-serial dilutions in specific bacteria was above 0.98, independent of 10^(1-4) -fold numbers of three other pathogenic strains. With optimized conditions, the lowest detection concentrations of four pathogens were 10^2.5 CFU/mL for Sta.attreus, S. Enterica and V. parahaemolyticus and 10^3.5 CFU/mL for B. cereus. The duration of the entire experiment from DNA isolation and purification to PCR amplification was less than 12 h.
延伸閱讀
- 傅筱婷、李佳音（2000）。多重引子聚合酶連鎖反應方法快速檢測致病與非致病性腸炎孤菌。中華民國獸醫學會雜誌，26(3)，223-232。https://doi.org/10.7009/JTCSVS.200009.0223
- Wong, H. C., You, W. Y., & Chen, S. Y. (2012). 應用具有內增幅控制之多套式聚合酵素鏈鎖反應於牡蠣中偵測致病性之霍亂弧菌、腸炎弧菌與創傷弧菌. 藥物食品分析, 20(1), 50-60+169. https://www.airitilibrary.com/Article/Detail?DocID=10219498-201203-201204110011-201204110011-50-60+169
- 呂昀陞、鄧文玲、楊雯馨、吳雅芳、鄭安秀、徐世典、曾國欽（2010）。應用多引子聚合酶連鎖反應技術同時檢測十字花科黑腐病菌細菌性葉斑病菌。植物病理學會刊，19(2)，137-147。https://doi.org/10.6649/PPB.201006_19(2).0003
- 曾浩洋、陳桐榮、虞積凱（1994）。應用聚合酶鏈反應於數種不同食品中腸毒素型金黃色葡萄球菌之檢測。藥物食品分析，2(3)，217-224。https://www.airitilibrary.com/Article/Detail?DocID=10219498-199409-2-3-217-224-a
- 莊其蓁（2007）。光動力殺菌對於抗Methicillin金黃葡萄球菌與表皮葡萄球菌之懸浮菌體與生物膜之研究〔碩士論文，國立臺灣大學〕。華藝線上圖書館。https://doi.org/10.6342/NTU.2007.00997