Work

Establishment of Recombinase-Aided Amplification Combined with Lateral Flow Dipstick for Rapid Detection of Yersinia pestis Based on the pla Gene

Year: 2026

Type: article

Abstract: This study establishes a rapid detection method for Yersinia pestis that combines recombinase-aided amplification with a lateral flow dipstick (RAA-LFD) and targets the pla gene. Based on the pla gene sequences of six representative strains from NCBI, three primer pairs (P-F1/R1, P-F2/R2, and P-F3/R3) and one RAA-nfo probe were designed. The RAA-LFD assay was constructed and evaluated for sensitivity, specificity, repeatability, and clinical applicability. Initial screening via polymerase chain reaction followed by agarose gel electrophoresis (PCR-AGE) showed P-F3/R3 produced a faint band, while P-F1/R1 and P-F2/R2 yielded bright, specific bands. RAA-LFD condition optimization demonstrated that: clear test lines (T lines) were observable at primer concentrations of 250–2000 nM, with the strongest signal at 2000 nM; the T line signal of P-F1/R1 remained stable at 35–41 °C, peaking slightly at 39 °C, whereas P-F2/R2 decreased markedly at 35 °C (p < 0.01); and the integrated optical density (IOD) of the T line increased with reaction time from 5 to 30 min, with a significant difference between 5 and 30 min (p < 0.001). The optimal reaction conditions were consequently determined as follows: primer pair P-F1/R1, a final primer concentration of 2000 nM, and incubation at 39 °C for 30 min. All five Y. pestis strains tested positive, while seven non-target strains and blank controls were negative, consistent with PCR results. Regarding sensitivity, the limit of detection (LOD) of RAA-LFD was 3.8 × 102 copies/μL, approximately 10-fold lower than conventional PCR (3.8 × 103 copies/μL). In the repeatability evaluation, intra-assay CVs were 1.79%, 0.59%, and 7.76%; inter-assay CVs were 1.75%, 2.35%, and 1.70%, all within 10%. Results for 130 clinical samples showed 100% agreement with PCR and qPCR. RAA-LFD established in this study provides a reliable, simple approach for on-site plague screening and primary laboratory diagnosis.

Source: Veterinary Sciences

DOI: 10.3390/vetsci13101091