化妆品中耐热大肠菌群快速检测方法的研究

doi:10.3969/j.issn.1001-1803.2019.06.011

摘要/Abstract

摘要：

研究化妆品中耐热大肠菌群快速检测方法。分别采用3M Petrifilm ^TM大肠菌群测试片法和环介导等温扩增（Loop-mediated isothermal amplification,LAMP）技术检测人工污染化妆品中耐热大肠菌群,并与传统平板检测法进行了比较。结果表明,3M Petrifilm ^TM大肠菌群测试片法与传统平板检测法比较结果无统计学差异,且操作步骤简便;LAMP技术特异性强,灵敏度可达到2.03 μg/L,且耗时短。3M Petrifilm ^TM大肠菌群测试片法和LAMP技术均可用于化妆品中耐热大肠菌群的快速检测。

关键词: 化妆品, 大肠菌群, 测试片, 环介导等温扩增技术

Abstract:

The study aimed to investigate the rapid detection method of thermotolerant coliforms in cosmetics. The 3M Petrifilm ^TM coliform count plate method and Loop-mediated isothermal amplification（LAMP）technique were used to rapidly detect thermotolerant coliform bacteria in artificially contaminated cosmetics, and compared with the traditional flat plate detection method. The results show that the 3M Petrifilm ^TM coliform test method and the traditional detection method have no statistical difference, and the operation procedure is simple. The LAMP technique has strong specificity, sensitivity of 2.03 μg/L, and is time-consuming. 3M Petrifilm ^TM coliform count plate method and LAMP technique can be used for rapid detection of thermotolerant coliforms in cosmetics.

Key words: cosmetics, coliforms, count plate, Loop-mediated isothermal amplification technique

中图分类号:

TQ658

文霞,李彩玲,刘静霞,杨秀茳,张淑瑶,谢小保. 化妆品中耐热大肠菌群快速检测方法的研究[J]. 日用化学工业, 2019, 49(6): 410-414.

WEN Xia,LI Cai-ling,LIU Jing-xia,YANG Xiu-jiang,ZHANG Shu-yao,XIE Xiao-bao. Research on rapid detection method of thermotolerant coliforms in cosmetics[J]. China Surfactant Detergent & Cosmetics, 2019, 49(6): 410-414.

图/表 7

表 1

表 2

表 3

表 4

表 5

图 1

图 2

参考文献 13

[1]	Li Zhiming. Food hygiene microbiological examination[M]. Beijing: Chemical Industry Press, 2009.
[2]	State Food and Drug Administration . Safety and technical standards for cosmetics[S]. Beijing:State Food and Drug Administration, 2015.
[3]	Sun Hui, Li Tiantian . Comparison of Petrifilm and GB methods in the food coliform bacteria detection[J]. Liquor Making, 2011,38(6) : 54-56.
[4]	Bo Lichao, Li Fen, Zhang Jing . Research on the application of Petrifilm colony count test piece in disinfection quality monitoring of primary medical institutions[J]. Chinese Journal of Disinfection, 2018 ( 3) : 180-185.
[5]	Yang Lanhua, Wang Xuqiang, Lu Li , et al. Comparison of GB standard with 3M Petrifilm method to test aerobic bacterial count in cosmetics[J]. Chinese Journal of Health Laboratory Technology, 2008 ( 1) : 2297-2298.
[6]	Zhu Shizhen, Wang Lifang, Wang Zunwen . Application of Petrifilm colony count plate in microbial detection for cosmetics[J]. China Pharmacist, 2014 ( 9) : 1601-1602.
[7]	Notomi T, Okayama H, Masubuchi H , et al. Loop-mediated isothermal amplification of DNA[J]. Nucleic Acids Research, 2000,28(12) : 63. doi: 10.1093/nar/28.12.e63
[8]	Kumar S, Mondal K K . Visual detection of Escherichia coli contamination in milk and fruit juice using loop-mediated isothermal amplification[J]. Journal of Food Science and Technology, 2015,52(11) : 7417-7424. doi: 10.1007/s13197-015-1779-2
[9]	Jiang Yong, Liu Zhentao, Ma Shumian , et al. Comparative study of different inoculation methods for detection of heat-injured coliforms in food[J]. Journal of Food Safety and Quality, 2018,9(14) : 3745-3748.
[10]	Notomi T, Mori Y, Tomita N , et al. Loop-mediated isothermal amplification (LAMP) :principle, features, and future prospects[J]. Journal of Microbiology, 2015,53(1) : 1-5. doi: 10.1007/s12275-015-4656-9
[11]	Yan M, Xu L, Jiang H , et al. PMA-LAMP for rapid detection of Escherichia coli and shiga toxins from viable but non-culturable state[J]. Microbial Pathogenesis, 2017,105:245-250. doi: 10.1016/j.micpath.2017.02.001
[12]	Liu W, Huang S, Liu N , et al. Establishment of an accurate and fast detection method using molecular beacons in loop-mediated isothermal amplification assay[J] . Scientific Reports, 2017,7:40125.
[13]	Yang Luxi, Li Xiaoyan . LAMP quantitative method and process based on a fluorescence quantitative system: 201810265404.1[P]. 2018 -08-10.

来源	菌株名称	编号
标准菌株	大肠杆菌（Escherichia coli）（ATCC No.8039）	A
	铜绿假单胞菌（Pseudomonas aeruginosa）（ATCC No.9027）	B
	金黄色葡萄球菌（Staphylococcus aureus）（ATCC No.6538）	C
从污染的化妆品中分离的菌株	洋葱伯克霍尔德菌（Burkholderia cepacia）	D
	阴沟肠杆菌（Enterobacter cloacae）	E
	肺炎克雷伯氏菌（Klebsiella pneumonia）	F
	粘质沙雷氏菌（Serratia marcescens）	G
	沃氏葡萄球菌（Staphylococcus warneri）	H
	弗氏柠檬酸杆菌（Citrobacter freundii）	I

目的基因	序列（5’~3’）	大小	产物/ bp
F3	CGTTGAATACGACACCCCG	19	229
B3	CGTCATCAACCATGTCGCA	19
FIP	CTGAGCAGCACCGGTGATCATGTT- TTCACTACGCACACGTAGACTG	46	82
BIP	GATGCCGCAGACTCGTGAGCTTTT- ATGATGTACGGAACGCCTAC	34
LF	TTTAACATAGTCGGCGTGCCC	21	125
LB	ATCCTGCTGGGTCGTCAG	18

	细菌编号	A	B	C	D	E	F	G	H	I	H₂O
爽肤水①	平板法	2.1×10⁶	0	0	0	2.5×10⁶	2.8×10⁶	1.7×10⁶	0	2.5×10⁶	0
	Petrifilm法	2.3×10⁶	0	0	0	2.7×10⁶	2.6×10⁶	1.5×10⁶	0	2.2×10⁶	0
	LAMP法	+	-	-	-	-	+	+	-	+	-
爽肤水②	平板法	2.3×10⁶	0	0	0	2.5×10⁶	1.4×10⁶	4.7×10⁶	0	2.6×10⁶	0
	Petrifilm法	4.3×10⁶	0	0	0	2.5×10⁶	1.6×10⁶	5.5×10⁶	0	4.2×10⁶	0
	LAMP法	+	-	-	-	-	+	+	-	+	-
爽肤水③	平板法	5.1×10⁶	0	0	0	4.5×10⁶	1.8×10⁶	3.7×10⁶	0	6.5×10⁶	0
	Petrifilm法	2.9×10⁶	0	0	0	3.7×10⁶	2.4×10⁶	3.5×10⁶	0	7.2×10⁶	0
	LAMP法	+	-	-	-	-	+	+	-	+	-

	细菌编号	A	B	C	D	E	F	G	H	I	H₂O
乳液①	平板法	1.8×10⁶	0	0	0	2.0×10⁶	2.1×10⁶	1.7×10⁶	0	1.3×10⁶	0
	Petrifilm法	1.5×10⁶	0	0	0	2.1×10⁶	2.2×10⁶	1.8×10⁶	0	1.5×10⁶	0
	LAMP法	+	-	-	-	-	+	+	-	+	-
乳液②	平板法	2.8×10⁵	0	0	0	6.5×10⁵	2.8×10⁵	6.7×10⁵	0	8.5×10⁵	0
	Petrifilm法	4.3×10⁵	0	0	0	5.7×10⁵	2.6×10⁵	5.4×10⁵	0	7.2×10⁵	0
	LAMP法	+	-	-	-	-	+	+	-	+	-
乳液③	平板法	6.1×10⁵	0	0	0	5.6×10⁵	2.8×10⁵	3.7×10⁵	0	6.5×10⁵	0
	Petrifilm法	7.3×10⁵	0	0	0	4.8×10⁵	2.6×10⁵	4.5×10⁵	0	4.7×10⁵	0
	LAMP法	+	-	-	-	-	+	+	-	+	-

	细菌编号	A	B	C	D	E	F	G	H	I	H₂O
面霜①	平板法	2.4×10⁶	0	0	0	2.1×10⁶	2.1×10⁶	1.3×10⁶	0	2.4×10⁶	0
	Petrifilm法	2.1×10⁶	0	0	0	1.7×10⁶	1.8×10⁶	1.6×10⁶	0	2.1×10⁶	0
	LAMP法	+	-	-	-	-	+	+	-	+	-
面霜②	平板法	1.7×10⁶	0	0	0	1.5×10⁶	2.0×10⁶	2.0×10⁶	0	2.6×10⁶	0
	Petrifilm法	1.3×10⁶	0	0	0	1.7×10⁶	1.6×10⁶	1.8×10⁶	0	2.2×10⁶	0
	LAMP法	+	-	-	-	-	+	+	-	+	-
面霜③	平板法	2.1×10⁵	0	0	0	5.5×10⁵	2.8×10⁵	6.7×10⁵	0	2.4×10⁵	0
	Petrifilm法	2.3×10⁵	0	0	0	5.7×10⁵	2.6×10⁵	7.5×10⁵	0	5.2×10⁵	0
	LAMP法	+	-	-	-	-	+	+	-	+	-