Journal of Textile Research ›› 2021, Vol. 42 ›› Issue (10): 92-98.doi: 10.13475/j.fzxb.20201101207

• Dyeing and Finishing & Chemicals • Previous Articles     Next Articles

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. 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 Online:2021-10-15 Published:2021-10-29
  • Contact: GUO Huiqing E-mail:guohq65@aliyun.com

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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

CLC Number: 

  • TSO7

Tab.1

Parameter of two sets of primers"

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

Tab.2

Corresponding amount of primers and probes at each final concentration in system"

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

Fig.1

Amplification curve of two sets of primers"

Fig.2

Amplification curve of primers and probes at different final concentrations 1—0.2 μmol/L; 2—0.4 μmol/L; 3—0.6 μmol/L;4—0.8 μmol/L; 5—1.0 μmol/L。"

Fig.3

Amplification results of different templates 1—1 μL; 2—2 μL; 3—3 μL; 4—4 μL; 5—5 μL。"

Fig.4

Amplification results at 60 ℃"

Fig.5

Specific amplification curve"

Tab.3

Test results of two methods"

编号 添加水平/
(CFU·mL-1)		 传统方法 PCR方法
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

Fig.6

Amplification curves of 7 dilutions"

Fig.7

Amplification curve of some positive samples added with target bacteria"

Tab.4

Parallel test of 20 samples of 5 samples"

编
号		 添加
水平/
(CFU/mL)		 传统方法 PCR方法
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

Fig.8

Amplification results of natural pollution sample"

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