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Background <p><i>Neisseria gonorrhoeae</i> is a global public health threat, exacerbated by the rapid dissemination of ceftriaxone-resistant strains harboring <i>penA</i> allele 60.001. Current diagnostics are limited by long turnaround times, reliance on thermal cycling equipment, and poor suitability for resource-limited settings.</p> Methods <p>We developed an isothermal recombinase polymerase amplification assay coupled with a lateral flow strip (RPA-LFS) for separate identification of <i>N. gonorrhoeae</i> and the ceftriaxone resistance-associated SNP C932T (A311V) in <i>penA</i> 60.001 and limited closely related alleles. Species detection employed an RPA-Nfo probe targeting the conserved <i>porA</i> pseudogene. Resistance detection employed an allele-specific amplification refractory mutation system RPA (ARMS-RPA) strategy, incorporating artificial mismatches to specifically discriminate the C932T SNP. Assay performance was evaluated using genomic DNA of a panel of gonococcal and non-gonococcal strains and validated directly on unprocessed clinical urine samples.</p> Results <p>The <i>porA</i> RPA-LFS assay demonstrated a detection limit of 30 fg of genomic DNA with no cross-reactivity against closely related <i>Neisseria</i> species or other bacterial pathogens. The mismatch <i>penA</i> 60.001 RPA-LFS assay achieved a detection limit of 300 fg and exhibited absolute analytical specificity, with no amplification observed from wild-type or other mosaic <i>penA</i> alleles. Validation using 50 clinical urine samples yielded 100% concordance (95% CI: 92.9–100%) with gonorrhoea identification by reference nucleic acid amplification tests, with 100% sensitivity (95% CI: 87.1–100%) and 100% specificity (95% CI: 85.7–100%), and 100% concordance (95% CI: 85.7–100%) with resistance allele detection, with 100% sensitivity (95% CI: 67.6–100%) and 100% specificity (95% CI: 79.6–100%).</p> Conclusions <p>This integrated RPA-LFS platform provides rapid detection of gonococcal infections and ceftriaxone resistance without requiring a thermal cycler, delivering visual results within 20 minutes. The assay represents a promising point-of-care solution for timely diagnosis, targeted antibiotic therapy, and resistance surveillance in resource-limited settings, though further validation on diverse specimen types and integration of an internal amplification control are needed.</p>

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Development of a Point-of-Care Recombinase Polymerase Amplification Assay for Detection of Neisseria gonorrhoeae and Ceftriaxone-Resistance Associated penA Allele 60.001

  • 期刊论文

  • 发表日期：

    2026年7月24日

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  • Xia Sun,
  • Ying Fu,
  • Yi Liu,
  • Qiongyao Liu,
  • Luxia Chen,
  • Jiang Zhu,
  • Xu’ai Lin,
  • Hao Cheng,
  • Stijn van der Veen

摘要

Background

Neisseria gonorrhoeae is a global public health threat, exacerbated by the rapid dissemination of ceftriaxone-resistant strains harboring penA allele 60.001. Current diagnostics are limited by long turnaround times, reliance on thermal cycling equipment, and poor suitability for resource-limited settings.

Methods

We developed an isothermal recombinase polymerase amplification assay coupled with a lateral flow strip (RPA-LFS) for separate identification of N. gonorrhoeae and the ceftriaxone resistance-associated SNP C932T (A311V) in penA 60.001 and limited closely related alleles. Species detection employed an RPA-Nfo probe targeting the conserved porA pseudogene. Resistance detection employed an allele-specific amplification refractory mutation system RPA (ARMS-RPA) strategy, incorporating artificial mismatches to specifically discriminate the C932T SNP. Assay performance was evaluated using genomic DNA of a panel of gonococcal and non-gonococcal strains and validated directly on unprocessed clinical urine samples.

Results

The porA RPA-LFS assay demonstrated a detection limit of 30 fg of genomic DNA with no cross-reactivity against closely related Neisseria species or other bacterial pathogens. The mismatch penA 60.001 RPA-LFS assay achieved a detection limit of 300 fg and exhibited absolute analytical specificity, with no amplification observed from wild-type or other mosaic penA alleles. Validation using 50 clinical urine samples yielded 100% concordance (95% CI: 92.9–100%) with gonorrhoea identification by reference nucleic acid amplification tests, with 100% sensitivity (95% CI: 87.1–100%) and 100% specificity (95% CI: 85.7–100%), and 100% concordance (95% CI: 85.7–100%) with resistance allele detection, with 100% sensitivity (95% CI: 67.6–100%) and 100% specificity (95% CI: 79.6–100%).

Conclusions

This integrated RPA-LFS platform provides rapid detection of gonococcal infections and ceftriaxone resistance without requiring a thermal cycler, delivering visual results within 20 minutes. The assay represents a promising point-of-care solution for timely diagnosis, targeted antibiotic therapy, and resistance surveillance in resource-limited settings, though further validation on diverse specimen types and integration of an internal amplification control are needed.