Biotechnology Bulletin ›› 2022, Vol. 38 ›› Issue (3): 264-275.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0768

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Development of Fluorescence Quantitative Lyophilized Detection Kit Based on Escherichia coli O157∶H7

SU Yuan1(), ZHU Long-jiao1, CAO Ji-juan2, LIU Jian-long3, XU Wen-tao1()   

  1. 1. Key Laboratory of Safety Assessment of Genetically Modified Organism(Food Safety),Ministry of Agriculture and Rural Affairs, College of Food Science and Nutritional Engineering,China Agricultural University,Beijing 100083
    2. Dalian Minzu University,Dalian 116600
    3. Hebei Elisa Biotechnology Co.,Ltd.,Zhuozhou 072750
  • Received:2021-06-17 Online:2022-03-26 Published:2022-04-06
  • Contact: XU Wen-tao E-mail:sy18223736@163.com;xuwentao@cau.edu.cn

Abstract:

Escherichia coli O157∶H7,as a food-borne pathogen,is widely distributed and causes great harms. To develop a kit based on E. coli O157∶H7 that can not only achieve rapid,simple and sensitive detection,but also achieve room temperature storage and transportation,this study utilized real-time fluorescence quantitative polymerase chain reaction(qPCR),combinig with vacuum lyophilizing technology,and developed an E. coli O157∶H7 fluorescence quantitative lyophilizing detection kit. This kit retains nucleic acid detection performance,and is easy to store and transport at room temperature,and reduces aerosol pollution. The developed kit adopts lyophilizing technology to retain the detection performance of nucleic acid amplification reagents. After rehydration,the accumulation of fluorescent signals is monitored in real time through cyclic amplification under the action of Taq DNA polymerase to achieve quantitative fluorescence detection. By the proposed method 2.1 copies/μL of eaeA gene can be detected within 40 min. This technology provides a fine research foundation and technical reference for the detection of E. coli O157∶H7,and fills the lack of detection kits with high sensitivity and easy storage in the market.

Key words: E. coli O157∶H7, nucleic acid, fluorescent quantitative PCR, lyophilized protective agent, kits