Rapid analysis of Escherichia coli O157:H7 using isothermal recombinase polymerase amplification combined with triple-labeled nucleotide probes

• Published in: Molecular and cellular probes 2020-04, Vol.50, p.101501, Article 101501
• Main Authors: Hu, Jinqiang, Wang, Yi, Su, Haijian, Ding, Huimin, Sun, Xincheng, Gao, Hui, Geng, Yao, Wang, Zhangcun
• Format: Article
• Language: English
• Subjects: Analysis; Animals; Cattle; DNA Primers - genetics; DNA Probes - genetics; Escherichia coli; Escherichia coli O157 - genetics; Escherichia coli O157 - isolation & purification; Food Contamination - analysis; Milk - microbiology; Nucleotides - genetics; Polymerase Chain Reaction - methods; Recombinase polymerase amplification; Recombinases - metabolism; Reproducibility of Results; Sensitivity and Specificity
• ISSN: 0890-8508; 1096-1194
• DOI: 10.1016/j.mcp.2019.101501

Rapid analytical methods are urgently needed to evaluate Escherichia coli (E. coli) O157:H7 in food. In this work, a novel recombinase polymerase amplification (RPA)-based lateral flow dipstick (LFD) method was developed to detect E. coli. Briefly, suitable primers and probes were designed and screened. Then, RPA reaction parameters, including volume, time, and temperature, were optimized. The specificity and sensitivity of RPA-LFD were analyzed, and a contaminated milk sample was used to test the detection performance of the proposed method. The optimal RPA reaction conditions included a minimum volume of 10 μL, incubation time of 10 min, temperature range of 39–42 °C, the primer pair EOF4/EOR3, and the probe EOProb. RPA-LFD was highly sensitive, it could detect as little as 1 fg of the genomic DNA of E. coli O157:H7, and 19 nontarget DNA of foodborne bacteria did not yield amplification products. Finally, the limit of detection of RPA-LFD for E. coli O157:H7 in artificially contaminated raw milk was 4.4 CFU/mL. In summary, the RPA-LFD assay developed in this study is an effective tool for the rapid investigation of E. coli O157:H7 contamination in raw milk samples. •Minimal volume, optimal temperature and shortest time of RPA reaction was 10 μL, 39–42 °C and 10 min, respectively.•LOD of RPA-LFD were 1 fg and 4.4 CFU/mL for genomic DNA of E. coli O157: H7 and for E. coli O157: H7, respectively.•RPA was highly specific for E. coli O157: H7 using 19 non-target foodborne bacteria as controls.•Experimental results can be directly judged by naked eye with free equipment.