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Background <p><i>Vibrio cholerae</i> O139 is a significant waterborne pathogen responsible for severe cholera outbreaks, particularly in regions with poor sanitation. Early and accurate detection of this strain is necessary to limit rapid disease spread and reduce mortality rates. Conventional detection methods are hindered by long processing times, high costs, and dependency on specialized equipment, thus limiting their usability in resource-poor settings.</p> Methods and results <p>This study employed a whole-cell Systematic Evolution of Ligands by EXponential enrichment (SELEX) process to isolate high-affinity DNA aptamers for <i>V. cholerae</i> O139. Twelve aptamer sequences were isolated after 12 SELEX rounds, two counter-selections against <i>E. coli</i>, and the best affinity and specificity were noted with the aptamer VcA7 by a low dissociation constant (Kd = 14.72 ± 0.89 pM) by flow cytometry protocols. In silico docking analysis indicated a high affinity of VcA7 to bind with the Outer membrane protein U (OmpU), a significant surface receptor of <i>V. cholerae</i> O139. Fluorescence microscopy and specificity assays identified the selective binding to the target pathogen without cross-reactivity with closely related bacterial pathogens, such as <i>V. parahaemolyticus</i>, <i>E. coli</i>, and <i>S. aureus</i>.</p> Conclusion <p>The aptamer VcA7 displayed high affinity and specificity for <i>V. cholerae</i> O139, surpassing other contenders in both computational and experimental screening. Its high binding activity to OmpU, combined with its high selectivity, render VcA7 a valuable biorecognition element for aptamer-based diagnostic tools. This research helps in the design of rapid, affordable, and field-deployable diagnostic kits for cholera surveillance in resource-poor countries and endemic environments.</p>

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A novel DNA aptamer for Vibrio cholerae O139 detection: a whole-cell SELEX approach

  • 期刊论文

  • 发表日期：

    2025年6月9日

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  • Masilamani Karthikeyan,
  • Mohandass Ramya,
  • Pasupathi Rathinasabapathi

摘要

Background

Vibrio cholerae O139 is a significant waterborne pathogen responsible for severe cholera outbreaks, particularly in regions with poor sanitation. Early and accurate detection of this strain is necessary to limit rapid disease spread and reduce mortality rates. Conventional detection methods are hindered by long processing times, high costs, and dependency on specialized equipment, thus limiting their usability in resource-poor settings.

Methods and results

This study employed a whole-cell Systematic Evolution of Ligands by EXponential enrichment (SELEX) process to isolate high-affinity DNA aptamers for V. cholerae O139. Twelve aptamer sequences were isolated after 12 SELEX rounds, two counter-selections against E. coli, and the best affinity and specificity were noted with the aptamer VcA7 by a low dissociation constant (Kd = 14.72 ± 0.89 pM) by flow cytometry protocols. In silico docking analysis indicated a high affinity of VcA7 to bind with the Outer membrane protein U (OmpU), a significant surface receptor of V. cholerae O139. Fluorescence microscopy and specificity assays identified the selective binding to the target pathogen without cross-reactivity with closely related bacterial pathogens, such as V. parahaemolyticus, E. coli, and S. aureus.

Conclusion

The aptamer VcA7 displayed high affinity and specificity for V. cholerae O139, surpassing other contenders in both computational and experimental screening. Its high binding activity to OmpU, combined with its high selectivity, render VcA7 a valuable biorecognition element for aptamer-based diagnostic tools. This research helps in the design of rapid, affordable, and field-deployable diagnostic kits for cholera surveillance in resource-poor countries and endemic environments.