日勾维多细菌源菌双重PCR快速检测方法建立

doi:10.3969/j.issn.2097-2806.2024.01.006

摘要/Abstract

摘要：

建立日勾维多细菌源菌快速检测方法，快速有效地检出样品中的日勾维多细菌源菌。利用日勾维多细菌源菌的看家基因gyrB和ropB，设计2对特异性的扩增引物，以培养液作为DNA扩增模板，建立双重PCR快速检测方法，并通过优化扩增反应的退火温度，提高双重PCR的特异性;利用引物gyrB-F/gyrB-R和ropB-F/ropB-R（insert）扩增4株日勾维多细菌源菌，可得到2条特异性的目标DNA条带，但非目标菌会出现非特异性DNA条带。优化后的退火温度为65 ℃，以4株日勾维多细菌源菌为DNA模板时，可扩增出特异性的目标DNA条带;以大肠埃希氏菌、金黄色葡萄球菌、铜绿假单胞菌、阴沟肠杆菌和洋葱伯克霍尔德菌为DNA模板时，均检测不到DNA条带。当样品中的日勾维多细菌源菌达到4.4 CFU/mL时，双重PCR即可高灵敏检出样品中的日勾维多细菌源菌。可用双重PCR方法快速检出沐浴露、洗发水等淋洗类化妆品中的日勾维多细菌源菌。

关键词: 化妆品, 日勾维多细菌源菌, 双重PCR, 快速检测

Abstract:

A rapid detection method for Pluralibacter gergoviae was established, and this method was used to quickly and effectively detect Pluralibacter gergoviae in samples. Four house-keeping genes （gyrB, infB, atpD and ropB） were selected as target genes, and 4 pairs of primers were designed. The bacterial suspension was used as amplification, and the 4 pairs of primers could specifically bind with the Pluralibacter gergoviae to produce bright DNA bands. Among the four pairs of primers, only gyrB-F/gyrB-R and ropB-F/ropB-R obtained a small amount of non-specific DNA bands when Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Enterobacter cloacae and Burkholderia cepacia were used as DNA templates. Two pairs of specific primers gyrB-F/gyrB-R and ropB-F/ropB-R（insert） were used to establish the rapid detection method of duplex-PCR for Pluralibacter gergoviae in rinsing cosmetics. The specificity of duplex-PCR was improved by optimizing the annealing temperature of amplification. When the annealing temperature was 60 ℃, using the primer pairs gyrB-F/gyrB-R and ropB-F/ropB-R （insert） to amplify 4 strains of Pluralibacter gergoviae, two specific target DNA bands were obtained, but Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Enterobacter cloacae and Burkholderia cepacia would obtain non-specific DNA bands. When the annealing temperature was optimized and set at 65 ℃, the specific target DNA bands could be amplified when 4 strains of Pluralibacter gergoviae were used as DNA templates. When Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Enterobacter cloacae and Burkholderia cepacia were used as DNA templates, no DNA bands were detected. When the concentration of Pluralibacter gergoviae in the sample reached 4.4 CFU/mL, duplex-PCR was able to detect Pluralibacter gergoviae in the sample with high sensitivity. Duplex-PCR method could be used to quickly detect the Pluralibacter gergoviae in the rinsing cosmetics such as shower gel and shampoo.

Key words: cosmetics, Pluralibacter gergoviae, duplex-PCR, rapid detection

中图分类号:

TQ658

刘丰, 邓源昌, 应国红, 王晓炜. 日勾维多细菌源菌双重PCR快速检测方法建立[J]. 日用化学工业（中英文）, 2024, 54(1): 45-50.

Feng Liu, Yuanchang Deng, Guohong Ying, Xiaowei Wang. Establishment of duplex-PCR method for rapid detection of Pluralibacter gergoviae[J]. China Surfactant Detergent & Cosmetics, 2024, 54(1): 45-50.

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引物名称	长度/bp	引物序列（5'-3'）
gyrB-F	18	GAGCCTGGAAACCTTCAC
gyrB-R	18	CTTCTTGCTGTAGCCCTC
infB-F	17	CCTGCTGAACGCTATCC
infB-R	18	CACCTCTTTGCCGAGTTC
ropB-F	18	CGCAGGACATGATCAACG
ropB-R	18	ATCGTCTACGAAACGGCC
ropB-R（insert）	18	TGCCGAGATACGACGCTT
atpD-F	18	ACCCTCGGCCGTATTATG
atpD-R	18	GTTCATCTGGCCGTACAC
27f*	21	GAGAGTTTGATCCTGGCTCAG
1495r	20	CTACGGCTACCTTGTTACGA

样品编号	样品名称
1	烟酰胺香水沐浴露
2	海洋主义氨基酸海盐洗发水
3	儒意头皮净澈洗发水
4	极控古龙香氛沐浴露
5	香水滋润沐浴露
6	菲丝宜侧柏叶防脱洗发露

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