基于PMA-LAMP技术可视化检测消毒产品消毒效果

doi:10.3969/j.issn.2097-2806.2024.11.014

摘要/Abstract

摘要：

探讨建立一种氮溴化丙锭（Propidium monoazide，PMA）染料与环介导等温扩增法（Loop-mediated isothermal amplification，LAMP）联用的技术，实现可视化判断消毒产品消毒效果的目的。随机选取5种成分不同的市售消毒产品，回收消毒产品消毒处理后的菌液并进行PMA处理，并对处理后的菌液进行细菌基因组DNA提取。将大肠杆菌作为消毒效果检测的指示菌，针对大肠杆菌的fecA基因以及针对存在于所有细菌的16S rRNA基因进行LAMP扩增，并在反应体系中加入钙黄素染料。通过肉眼观察反应管颜色变化即可对消毒产品的消毒效果进行判断。将PMA-LAMP方法与PMA-qPCR方法进行比较后发现PMA-LAMP方法在操作步骤、检测时长、灵敏度方面均优于PMA-qPCR方法。在对消毒产品消毒效果的实际检测中，PMA-LAMP方法与《消毒技术规范（2002年版）》规定的标准方法的结果基本一致，两者杀灭对数值偏差在±0.5以内。该方法不依赖大型精密温控仪器，检测过程为45 min（不包括前处理步骤），实现了对消毒产品消毒效果现场快速可视化检测，具有良好的基层应用前景，将为消毒产品的性能评价提供参考、为行业市场监管提供技术支持。

关键词: 叠氮溴化丙锭, 环介导等温扩增法, 可视化, 消毒产品, 消毒效果

Abstract:

For visual detection of the disinfection effect of disinfectant products, a new method combining propidium monoazide （PMA, a dye） with loop-mediated isothermal amplification （LAMP） was established. Five commercial disinfection products with different ingredients were randomly selected in this research. Glass slides covered with bacteria were freshly prepared before use. After disinfection treatment by the disinfection products, the bacteria solutions were collected and then treated with PMA separately. Bacterial genomic DNA was extracted from the bacterial solutions. Subsequently, gDNA was used as template for LAMP and real-time PCR for amplification of fecA gene of Escherichia coli and 16S rRNA which universally existed in bacteria. Meanwhile, by adding calcein to the reaction system, the disinfection effect could be determined. The color change from fluorescent green to light orange in reaction tube could be observed by naked eyes. Excluding the step of disinfection treatment, the detection procedure could be completed within 45 min. Compared with PMA-qPCR, PMA-LAMP had the advantages of easy to operate, more sensitive, faster in detection, and no need of precise temperature-controlling instruments. For testing the disinfection effects of actual disinfection products, the results of PMA-LAMP were basically consistent with those of the standard method （refer to Technical Standard for Disinfection （2002 edition））, with a deviation within±0.5 between the two methods. The newly established PMA-LAMP method had good application prospect in field experiments. It would provide technical support for performance evaluation of disinfection products and market supervision.

Key words: propidium monoazide, loop-mediated isothermal amplification, visual detection, disinfection product, disinfection effect

中图分类号:

R187

梁媛媛, 夏琪琪, 杨启立, 徐新怡, 周志南, 刘鹏鹏. 基于PMA-LAMP技术可视化检测消毒产品消毒效果[J]. 日用化学工业（中英文）, 2024, 54(11): 1391-1396.

Yuanyuan Liang, Qiqi Xia, Qili Yang, Xinyi Xu, Zhinan Zhou, Pengpeng Liu. Visual detection of the disinfection effect of disinfectants by loop-mediated isothermal amplification combined with propidium monoazide[J]. China Surfactant Detergent & Cosmetics, 2024, 54(11): 1391-1396.

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引物名称		LAMP引物序列（5’-3’）
大肠埃希氏菌fecA基因	E-F3	ACGACGTATTTGAACATGCTGG
	E-B3	AAGGGGACGGGGATGCC
	E-FIP	GACGCCAGGGATGCGGTTATTTTCCGCCGTGAGGATTTCG
	E-BIP	CCACGACCTGGCGATGAACTTTTTCCATCAGGACGGTCGAGC
	E-LF	ACCTCACGCATGGTGGTTG
	E-LB	TTGGCATCCGGGGCCT
16S rRNA	16S-4-F3	CCTTTGTTGCCAGCGGTC
	16S-4-B3	ACTTCATGGAGTCGAGTTGC
	16S-4-FIP	AGGGCCATGATGACTTGACGTCGCCGGGAACTCAAAGGAG
	16S-4-BIP	TGGCGCATACAAAGAGAAGCGAAGACTCCAATCCGGACTACG

消毒剂	试验菌株	标准方法	PMA-LAMP方法			两方法对数值绝对差值
消毒剂	试验菌株	杀灭对数值	试验组对照标准曲线所得对数值	阳性对照组对照标准曲线所得对数值	结果	两方法对数值绝对差值
A	大肠埃希氏菌、金黄色葡萄球菌、铜绿假单胞菌混合菌液	5.57	<1 （Ct值>40.00）	6.17 （Ct值=20.62）	5.17	0.40
B		2.12	4.30 （Ct值=28.88）	6.06 （Ct值=21.10）	1.76	0.36
C		4.97	<1 （Ct值>40.00）	5.94 （Ct值=21.62）	4.94	0.03
D		5.50	<1 （Ct值>40.00）	6.21 （Ct值=20.42）	5.21	0.29
E		5.47	<1 （Ct值>40.00）	6.23 （Ct值=20.33）	5.23	0.24