Development of a rapid and sensitive one-pot recombinase polymerase amplification assay for the simultaneous detection of Bacillus anthracis and Yersinia pestis with naked eye result read-out.

Bacillus anthracis and Yersinia pestis are zoonotic bacteria serving as the etiological agents for anthrax and plague, respectively. Due to the rapidly progressing and lethal nature of anthrax and plague, fast and reliable detection is essential for effective disease control and management. Nucleic acid amplification-based approaches offer fast and sensitive pathogen detection. Isothermal amplification techniques (IAT) are being developed to address the limitations of the PCR-based nucleic acid amplification technology. Recombinase polymerase amplification (RPA) is an IAT with low operating temperatures and fast turnaround time while maintaining specificity and sensitivity in easily accessible formats. This study focuses on developing a novel isothermal recombinase polymerase amplification assay combined with lateral-flow result read-out (RPA-LFD) for rapid and simultaneous detection of B. anthracis and Y. pestis under 25 min at 42 °C. The outcomes of the feasibility studies establish its real-world applicability for clinical diagnosis (blood) and also for environmental detection (soil and water) with detection limits comparable to real-time PCR. The absence of cross-reactivity and high sensitivities in different matrices demonstrates its significant potential for in-field, rapid, and reliable detection of biothreat agents in both biothreat and natural outbreak scenarios.