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

Authors

Yunhan Zhou, Xue Yang, Ziling Ding, Qiuyu Fan, Xiaofang Yan, Haifeng Liu, Ziyao Zhou, Zhijun Zhong, Jialiang Xin, Guangneng Peng

Published in

Veterinary sciences. Volume 13. Issue 10. Oct 14, 2026. Epub Oct 14, 2026.

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.

PMID:
42944185
Bibliographic data and abstract were imported from PubMed on 27 Oct 2026.

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