ATP生物荧光增幅法在控制菌检测中的验证研究——应用于化妆品领域

Validation study on amplified ATP bioluminescence assay in the detectionof control bacteria-Application in cosmetics field

参照《中国药典》(2020版)药品微生物检验替代方法验证指导原则中定性检验方法的验证要求,选取了清洁类、护理类、美容修饰类等6种化妆品,从专属性、检测限、重现性和耐用性4个方面开展了ATP生物荧光增幅法与《化妆品安全技术规范》(2015版)中耐热大肠菌群、铜绿假单胞菌和金黄色葡萄球菌检验标准方法的部分等效性研究。结果显示,ATP生物荧光增幅法的专属性和耐用性良好;采用该方法检测3种控制菌代表菌株的LOD 50分别为0.20、0.35和0.14 CFU/g,经Fisher exact test统计分析,ATP生物荧光增幅法检测限均显著优于相应标准方法(P<0.05);3家不同实验室使用2种方法对相同人工污染样品的检测结果无统计学差异(P>0.05),说明ATP生物荧光增幅法重现性良好。研究结果表明,ATP生物荧光增幅法与《化妆品安全技术规范》(2015版)中耐热大肠菌群、铜绿假单胞菌和金黄色葡萄球菌检验的标准方法具有部分等效性,可实现化妆品中各类微生物在48 h的同步检测。

According to the verification requirements of qualitative test methods in the guiding principles for validation of alternative methods for microbiological testing in Chinese Pharmacopoeia(2020),six kinds of cosmetics,including cleanser,care cosmetics and makeup categories,were selected to conduct the partial equivalence study between amplified ATP bioluminescence assay and the standard detection method for the detection of thermotolerant coliform bacteria,Staphylococcus aureus and Pseudomonas aeruginosa in Safety and Technical Standards for Cosmetics(2015)in terms of specificity,detection limit,reproducibility and durability.The results showed that the amplified ATP bioluminescence assay had good specificity and durability.The LOD 50 of the representative strains of the three control bacteria detected by this method were 0.20,0.35 and 0.14 CFU/g,respectively.According to the statistical analysis of Fisher exact test,the detection limit of the amplified ATP bioluminescence assay was significantly better than that of the corresponding standard method(P<0.05).There was no significant difference in the detection results of the same artificially contaminated samples by these two methods in three different laboratories(P>0.05),indicating that the amplified ATP bioluminescence assay had good reproducibility.In general,the amplified ATP bioluminescence assay is partially equivalent to the standard methods for the detection of thermotolerant coliform bacteria,Staphylococcus aureus and P.aeruginosa in Safety and Technical Standards for Cosmetics(2015),which can realize the simultaneous detection of various microorganisms in cosmetics within 48 h.