以oprI为靶基因实时荧光PCR法检测化妆品中绿脓杆菌.pdf

2014 年第 4 期 Vol.24 No. 4 检验检疫学刊 JOURNAL OF INSPECTION AND QUARANTINE 以oprI为靶基因实时荧光PCR法检测化妆品中绿脓杆菌 苏建晖 蔡 颖 许如苏 周广彪 曾梅锦 （汕头出入境检验检疫局 广东汕头 515031 ） 摘要 建立快速检测化妆品中绿脓杆菌的实时荧光PCR 方法。选取绿脓杆菌oprI基因中相对保守且高度 特异的核苷酸片段作为荧光PCR扩增的靶序列，设计引物和TaqMan探针，并研究了DNA提取方法，优化扩增 反应体系和仪器条件。与GB方法进行比对，检测结果一致，但检测时间均只有GB方法的1/10；方法的检测 灵敏度为2CFU ／荧光PCR反应体系，染菌样品经6 h增菌培养后，检测低限均达到2CFU/g ，证明该方法高度 敏感；通过对36株标准／参考菌株和51株非目的菌的检测，证实该方法高度特异；批内CP值的变异系数小于 2% ，说明该方法具有良好的可重复性。 关键词 化妆品；绿脓杆菌；检验；PCR 中图分类号 TQ658 Taking oprI as Target Gene to Test Pseudomonas aeruginosa in Cosmetics by RT-PCR Su Jianhui, Cai Ying, Xu Rusu, Zhou Guangbiao, Zeng Meijin (Shantou Entry-Exit Inspection and Quarantine Bureau, Shantou, Guangdong, 515031) Abstract: It established a method by real-time fluorescent PCR for rapid detection of Pseudomonas aeruginosa in cosmetics. The target sequences selected nucleotide fragment that is relatively conservative and highly specific for Pseudomonas aeruginosa oprI gene. It can be used as a fl uorescent PCR amplifi cation. Primers and a TaqMan probe was designed. The amplification reaction system, instrument conditions, the extraction method of DNA and the actual detection application in cosmetics was optimized. And compared with GB method, the detection results were consistent, but the detection time was only 1/10 by GB method. Detection sensitivity meet the 2 CFU per Each fl uorescence PCR reaction system. For bacterial infection samples after 6 hours of enrichment culture, lowest limit of detection meet 2CFU/g. It proved that the method is highly sensitive. Through against 36 strains of standard / reference strains and 51 strains of non objective bacterium detection, it confirmed that the method is highly specific and repeatable with the batch CP values less than 2% . The method can detect, not only qualitativ