Rapid analysis of Escherichia coli O157:H7 using isothermal recombinase polymerase amplification combined with triple-labeled nucleotide probes. (April 2020)

• Record Type: Journal Article
• Authors: Hu, Jinqiang; Wang, Yi; Su, Haijian; Ding, Huimin; Sun, Xincheng; Gao, Hui; Geng, Yao; Wang, Zhangcun
• Is Part Of: Molecular and cellular probes. Volume 50(2020)
• Publication Date: 2020-04
• Subjects: Escherichia coli -- Recombinase polymerase amplification -- Analysis; Molecular probes -- Diagnostic use -- Periodicals; Pathology, Cellular -- Technique -- Periodicals; Cell Biology -- Periodicals; Molecular Biology -- Periodicals; Sondes moléculaires -- Utilisation diagnostique -- Périodiques; Cytopathologie -- Technique -- Périodiques; 572
• DOI: 10.1016/j.mcp.2019.101501
• Languages: English
• ISSNs: 0890-8508

Abstract

Abstract: 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. Highlights: 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.