纺织品中大肠杆菌O157:H7的检测方法

doi:10.13475/j.fzxb.20201101207

纺织学报 ›› 2021, Vol. 42 ›› Issue (10): 92-98.doi: 10.13475/j.fzxb.20201101207

纺织品中大肠杆菌O157:H7的检测方法

李轲¹, 张子宏¹, 禹建鹰¹, 连素梅², 丁友超³, 谢堂堂⁴, 傅科杰⁵, 郭会清¹()

1.郑州海关, 河南郑州 450003
2.石家庄海关, 河北石家庄 050051
3.南京海关工业产品检测中心, 江苏南京 210001
4.深圳海关工业品检测技术中心, 广东深圳 518067
5.宁波检验检疫科学技术研究院, 浙江宁波 315000

收稿日期:2020-11-26 修回日期:2021-07-19 出版日期:2021-10-15 发布日期:2021-10-29
通讯作者: 郭会清
作者简介:李轲(1976—),女,高级兽医师,硕士。主要研究方向为消费品生物安全。
基金资助:
河南省科技攻关项目(182102210226);国家认监委认证认可科技支撑计划项目(2017RJWKJ35);河南省科技攻关计划项目(162102210370)

Method to detect virulence gene rfbE of Escherichia coli O157:H7 in textiles

LI Ke¹, ZHANG Zihong¹, YU Jianying¹, LIAN Sumei², DING Youchao³, XIE Tangtang⁴, FU Kejie⁵, GUO Huiqing¹()

1. Zhengzhou Customs, Zhengzhou, Henan 450003, China
2. Shijiazhuang Customs, Shijiazhuang, Hebei 050051, China
3. Nanjing Customs Industrial Products Testing Center, Nanjing, Jiangsu 210001, China
4. Shenzhen Customs Industrial Products Testing Technology Center, Shenzhen, Guangdong 518067, China
5. Ningbo Institute of Inspection Science and Technology, Ningbo, Zhejiang 315000, China

Received:2020-11-26 Revised:2021-07-19 Published:2021-10-15 Online:2021-10-29
Contact: GUO Huiqing

摘要/Abstract

摘要：

为建立一种灵敏、特异、快速、高效地鉴定纺织品基质中大肠杆菌O157:H7的方法,筛选出大肠杆菌O157:H7特有的rfbE基因保守序列,设计PCR特异性引物和荧光双标记探针,结合免疫磁珠技术,集成创新开发一种目标菌低浓度、不可培养情况下的样品中高效、快速富集分离大肠杆菌O157:H7的技术,建立了一种针对纺织品基质的免疫磁珠富集-实时荧光定量PCR方法,其检测下限可达8 CFU/mL。利用该方法对收集到的30份阳性样品进行鉴定,鉴定结果与传统方法结果100%吻合。结果表明,新建方法稳定性强,实现了纺织品中大肠杆菌O157:H7的快速灵敏鉴定。

关键词: 纺织品, 大肠杆菌O157:H7, 毒力基因, 免疫磁珠, 实时荧光PCR

Abstract:

In order to establish a sensitive, specific, rapid and efficient method to evaluate E. coli O157:H7 in textiles, the conservative rfb E sequence of E. coli O157:H7 was screened for the design of PCR specific primers and fluorescent double labeled probes. Combined with immunomagnetic beads technology, a new technology was developed for quick and efficient isolation of E. coli O157:H7 under the condition of low concentration and non-culturable target bacteria. A real-time fluorescent quantitative PCR method was developed, and the detection limit could reach 8 CFU/g. 30 positive samples were identified using this method, and the results were 100% consistent with the results from using the traditional methods. The results show that the new method is stable, rapid and sensitive in identifying E. coli O157:H7 in textiles.

Key words: textile, Escherichia coli O157:H7, virulence gene, immunomagnetic bead, real-time PCR

中图分类号:

TSO7

李轲, 张子宏, 禹建鹰, 连素梅, 丁友超, 谢堂堂, 傅科杰, 郭会清. 纺织品中大肠杆菌O157:H7的检测方法[J]. 纺织学报, 2021, 42(10): 92-98.

LI Ke, ZHANG Zihong, YU Jianying, LIAN Sumei, DING Youchao, XIE Tangtang, FU Kejie, GUO Huiqing. Method to detect virulence gene rfbE of Escherichia coli O157:H7 in textiles[J]. Journal of Textile Research, 2021, 42(10): 92-98.

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引物名	引物序列5’-3’	扩增长度	Genebank编号	引物所在位置
1-rfbE-F	TTCCTCTCTTTCCTCTGCGG	197	CP035366.1	2724342-2724361
1-rfbE-R	TGACAAATATCTGCGCTGCT			2724538-2724519
1-rfbE-P	FAM-TCAGCTTGTTCTAACTGGGCT- TAMRA			2707901-2707921
2-rfbE-F	CTCATTCGATAGGCTGGGGA	181	CP035366.1	2724180-2724199
2-rfbE-R	CGCAGAGGAAAGAGAGGAAT			2724360-2724341
2-rfbE-P	FAM-CCGAGTACATTGGCATCGTG-TAMRA			2707674-2707693

序号	引物、探针终浓度/ (μmol·L^-1)	体系中引物、探针加入量/μL
①	0.2	0.5
②	0.4	1.0
③	0.6	1.5
④	0.8	2.0
⑤	1.0	2.5

编号	添加水平/ (CFU·mL^-1)	传统方法		PCR方法
编号	添加水平/ (CFU·mL^-1)	测试结果	概率/%	测试结果	概率/%
1	80(中)	1~10个平行实验全部阳性		1~10个平行实验全部阳性
2		1~10个平行实验全部阳性		1~10个平行实验全部阳性
3		10个平行有1个阴性	96	1~10个平行实验全部阳性	100
4		10个平行有1个阴性		1~10个平行实验全部阳性
5		1~10个平行实验全部阳性		1~10个平行实验全部阳性
6	8(低)	10个平行有1个阴性		10个平行有2个阴性
7		10个平行有2个阴性		10个平行有1个阴性
8		10个平行有3个阴性	76	1~10个平行实验全部阳性	100
9		10个平行有2个阴性		10个平行有2个阴性
10		10个平行有4个阴性		1~10个平行实验全部阳性
11	8×10⁵(高)	1~10个平行实验全部阳性	100	1~10个平行实验全部阳性	100
12	未添加	阴性	0	阴性	0

编号	添加水平/ (CFU/mL)	传统方法		PCR方法
编号	添加水平/ (CFU/mL)	测试结果	概率 /%	测试结果	概率 /%
1	80	20个平行全部阳性		20个平行全部阳性
2		20个平行全部阳性		20个平行全部阳性
3		20个平行全部阳性	100	20个平行全部阳性	100
4		20个平行全部阳性		20个平行全部阳性
5		20个平行全部阳性		20个平行全部阳性
12	未添加	阴性		阴性

纺织品中大肠杆菌O157:H7的检测方法

Method to detect virulence gene rfbE of Escherichia coli O157:H7 in textiles

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