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Background <p>Gonorrhea, caused by the human obligate pathogen <i>Neisseria gonorrhoeae</i>, is the second most common bacterial sexually transmitted infection worldwide. In the absence of an effective gonococcal vaccine, convenient and accurate diagnosis of <i>N. gonorrhoeae</i> infection is essential for optimal patient management and for interrupting ongoing transmission.</p> Methods <p>A custom-designed gold nanoparticle-based lateral flow biosensor (AuNP-LFB) was employed. The unique LAMP primers and a specific single-guide RNA (sgRNA) were designed to target the <i>orf1</i> gene of <i>N. gonorrhoeae</i>. The key diagnostic conditions, including detection temperature and time, were optimized. The sensitivity and specificity of the assay were validated, and its clinical feasibility was validated using seventy-eight genital secretion samples from patients suspected of <i>N. gonorrhoeae</i> infection.</p> Results <p>The custom-designed AuNP-LFB was successfully fabricated. The Ng-CRISPR-One-Pot assay achieved a limit of detection (LoD) as low as 7 copies per test and exhibits no cross-reactivity with other tested microbial species. The entire detection workflow, including nucleic acid extraction (10&#xa0;min), LAMP-CRISPR/Cas12b detection (56&#xa0;°C, 50&#xa0;min), and AuNP-LFB visual readout (&lt; 2&#xa0;min), can be completed within 65&#xa0;min without requiring specialized instrumentation. Compared with a reference <i>N. gonorrhoeae</i>-qPCR method, our assay achieved 100% sensitivity (95% CI: 93.94–100.00%) and 100% specificity (95% CI: 82.35–100.00%).</p> Conclusions <p>Collectively, these preliminary results indicate that the Ng-CRISPR-One-Pot assay holds promise as a practical point-of-care (POC) diagnostic tool, enabling accurate and convenient detection of <i>N. gonorrhoeae</i>, particularly in resource-limited settings.</p>

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One-pot LAMP-CRISPR/Cas12b coupled with nanoparticle-based biosensor for point-of-care test of Neisseria gonorrhoeae

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年7月16日

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  • Ling Xie,
  • Yan Tan,
  • Yanyan Qin,
  • Cencen Jia,
  • Ya Li,
  • Yanan Liu,
  • Xu Chen

摘要

Background

Gonorrhea, caused by the human obligate pathogen Neisseria gonorrhoeae, is the second most common bacterial sexually transmitted infection worldwide. In the absence of an effective gonococcal vaccine, convenient and accurate diagnosis of N. gonorrhoeae infection is essential for optimal patient management and for interrupting ongoing transmission.

Methods

A custom-designed gold nanoparticle-based lateral flow biosensor (AuNP-LFB) was employed. The unique LAMP primers and a specific single-guide RNA (sgRNA) were designed to target the orf1 gene of N. gonorrhoeae. The key diagnostic conditions, including detection temperature and time, were optimized. The sensitivity and specificity of the assay were validated, and its clinical feasibility was validated using seventy-eight genital secretion samples from patients suspected of N. gonorrhoeae infection.

Results

The custom-designed AuNP-LFB was successfully fabricated. The Ng-CRISPR-One-Pot assay achieved a limit of detection (LoD) as low as 7 copies per test and exhibits no cross-reactivity with other tested microbial species. The entire detection workflow, including nucleic acid extraction (10 min), LAMP-CRISPR/Cas12b detection (56 °C, 50 min), and AuNP-LFB visual readout (< 2 min), can be completed within 65 min without requiring specialized instrumentation. Compared with a reference N. gonorrhoeae-qPCR method, our assay achieved 100% sensitivity (95% CI: 93.94–100.00%) and 100% specificity (95% CI: 82.35–100.00%).

Conclusions

Collectively, these preliminary results indicate that the Ng-CRISPR-One-Pot assay holds promise as a practical point-of-care (POC) diagnostic tool, enabling accurate and convenient detection of N. gonorrhoeae, particularly in resource-limited settings.