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Background: Recombinase polymerase amplification (RPA) is a novel nucleic acid isothermal amplification technique that can achieve rapid detection of the target, under 37 to 42°C conditions, within 30 minutes. It has the advantage of extreme sensitivity, strong specificity, and low instrument dependency and is particularly suitable for real-time detection in the field. It can be widely used in fields such as in vitro diagnostics, biosafety, and agriculture. This study was based on RPA technology, targeting the gyrA gene of Neisseria gonorrhoeae (N. gonorrhoeae), to establish a quick, accurate, and easy to operate method for detecting N. gonorrhoeae and to evaluate its specificity, sensitivity, and clinical, practical value.
Methods: Specific primers and probes suitable for RPA and qPCR methods based on the specific conserved region of the gyrA gene of N. gonorrhoeae on GenBank (no. U08817.1) were designed An RPA method was developed and N. gonorrhoeae ATCC49226 and a number of clinical isolates were used as study subjects to validate the specificity and sensitivity of the RPA method for the detection of N. gonorrhoeae. A real-time fluorescence quantitative polymerase chain reaction (qPCR) method, with N. gonorrhoeae ATCC49226 as the research object, was established to verify the sensitivity of qPCR method for detecting N. gonorrhoeae. Finally, clinical samples were tested by using RPA and qPCR methods as performance validation experiments to determine the clinical utility of the RPA technique in detecting N. gonorrhoeae.
Results: The established RPA detection method showed excellent specificity, with a specific amplification curve for N. gonorrhoeae alone, no cross-reactivity with other bacteria, and excellent reproducibility. The detection results could be obtained within 30 minutes, under the condition of 39°C, which was significantly lower than the detection time of traditional methods. The sensitivity of the RPA method for detecting pathogenic bacteria samples was 4 × 102 CFU/mL, which is consistent with the detection limit of qPCR methods. RPA and qPCR methods were used to detect 121 clinical isolates, out of which 30 strains of N. gonorrhoeae showed a specific amplification curve, while the remaining 91 strains of non-N. gonorrhoeae did not. Both methods had 100% accuracy and specificity in detecting N. gonorrhoeae.
Conclusions: The RPA method developed in this study has the characteristics of being quick, accurate, and easy to operate, which was of great value for the rapid detection of N. gonorrhoeae in clinical samples.
DOI: 10.7754/Clin.Lab.2024.240424
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