Rapid detection assays for Bacillus anthracis, Yersinia pestis, and Brucella spp. via triplex-recombinase polymerase amplification.

BACKGROUND

Bacillus anthracis (B. anthracis), Yersinia pestis (Y. pestis), and Brucella spp. are zoonotic bacteria that cause anthrax, plague, and brucellosis, respectively. Outbreaks typically occur in remote regions with poor transportation and limited laboratory testing. Therefore, a simple, sensitive, multiplex nucleic acid detection method is essential for effective disease management and control.

METHODS

Primers and probes for the three pathogens were designed to reduce interference from related strains. Three recombinase polymerase amplification (RPA) reactions were conducted at 39 °C for 10 min to produce species-specific fluorescence signals for the three pathogens. These were integrated, and conditions were optimized for rapid, sensitive triplex-RPA assays without cross-reactivity. A triplex-RPA reaction with lateral flow dipsticks (LFDs) was developed and applied to blood samples, newly isolated strains, and simulated samples.

RESULTS

Highly sensitive and specific primers and probes were developed, achieving a maximum sensitivity of 1 copy/µL in single-reaction RPA. The optimized triplex RPA detection technique, combined with fluorescence, effectively identified B. anthracis, Y. pestis, and Brucella spp. within 20 min, whereas LFDs achieved detection in 10 min. The assay also performed comparably to conventional polymerase chain reaction techniques when tested on blood samples, newly isolated strains, and simulated samples.

CONCLUSIONS

This study offers reliable methods for detecting B. anthracis, Y. pestis, and Brucella spp. in rural hospitals and public health initiatives.