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<p><i>Es</i><i>cherichia coli</i> O157 (<i>E. coli</i> O157) is a well-known foodborne pathogen that significantly threatens to human health and public safety and causes economic loss globally. Given this severe threat, there is an urgent need to develop accurate and timely detection methods for <i>E. coli</i> O157. In this study, a new method was developed for detecting <i>E. coli</i> O157, which combined recombinase polymerase amplification (RPA) with polymer flocculation sedimentation (PFS). Firstly, four upstream primers and 12 downstream primers were designed, and 12 pairs of primers were combined. Then, the optimal primer combinations were screened by using both PCR and RPA methods. Meanwhile, the RPA reaction conditions and the concentration range of PEG8000/NaCl were optimized. Subsequently, the specificity and sensitivity analyses of RPA-PFS assay were further conducted. Finally, artificially contaminated raw milk samples with <i>E. coli</i> O157 and real raw milk samples used to evaluate the potential application of the RPA-PFS method. The results showed that EO3 (F1/R3) was the best primer combination with the optimal reaction temperature, shortest reaction time, and minimum reaction volume of 39/40&#xa0;°C, 15&#xa0;min and 10 μL, respectively. The optimal concentration of PEG8000/NaCl binding solution was (0.25&#xa0;g/mL)/(2.5&#xa0;M). RPA-PFS assay was specific for 11 non-target foodborne bacteria and could detect <i>E. coli</i> O157 genomic DNA at a detection limit of 2.7&#xa0;fg. Furthermore, RPA-PFS assay could successfully detect <i>E. coli</i> O157: H7 with 43&#xa0;CFU/mL in artificially contaminated raw milk within 20&#xa0;min and was negative for real raw milk sample regardless of bacteria enrichment. In summary, RPA-PFS assay established in this study is a rapid, sensitive, specific, and visual detection tool for <i>E. coli</i> O157, and might be used in resource-limited areas.</p>

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Simple, Rapid, and Visual Detection of Escherichia coli O157 in Raw Milk by Recombinase Polymerase Amplification with Polymer Flocculation Sedimentation

  • 期刊论文

  • 发表日期：

    2025年9月1日

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  • Jinqiang Hu,
  • Shunxin Xie,
  • Jiahui Fan,
  • Yayue He,
  • An Guo,
  • Hui Gao,
  • Yao Geng

摘要

Escherichia coli O157 (E. coli O157) is a well-known foodborne pathogen that significantly threatens to human health and public safety and causes economic loss globally. Given this severe threat, there is an urgent need to develop accurate and timely detection methods for E. coli O157. In this study, a new method was developed for detecting E. coli O157, which combined recombinase polymerase amplification (RPA) with polymer flocculation sedimentation (PFS). Firstly, four upstream primers and 12 downstream primers were designed, and 12 pairs of primers were combined. Then, the optimal primer combinations were screened by using both PCR and RPA methods. Meanwhile, the RPA reaction conditions and the concentration range of PEG8000/NaCl were optimized. Subsequently, the specificity and sensitivity analyses of RPA-PFS assay were further conducted. Finally, artificially contaminated raw milk samples with E. coli O157 and real raw milk samples used to evaluate the potential application of the RPA-PFS method. The results showed that EO3 (F1/R3) was the best primer combination with the optimal reaction temperature, shortest reaction time, and minimum reaction volume of 39/40 °C, 15 min and 10 μL, respectively. The optimal concentration of PEG8000/NaCl binding solution was (0.25 g/mL)/(2.5 M). RPA-PFS assay was specific for 11 non-target foodborne bacteria and could detect E. coli O157 genomic DNA at a detection limit of 2.7 fg. Furthermore, RPA-PFS assay could successfully detect E. coli O157: H7 with 43 CFU/mL in artificially contaminated raw milk within 20 min and was negative for real raw milk sample regardless of bacteria enrichment. In summary, RPA-PFS assay established in this study is a rapid, sensitive, specific, and visual detection tool for E. coli O157, and might be used in resource-limited areas.