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

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.