Development of propidium monoazide-recombinase polymerase amplification (PMA-RPA) assay for rapid detection of Streptococcus pyogenes and Streptococcus agalactiae.

Streptococcus pyogenes (Group A Streptococcus, GAS) and Streptococcus agalactiae (Group B Streptococcus, GBS) are common pathogens that threaten public health. In this study, a double recombinase polymerase (RPA) amplification assay was developed to rapidly detect these pathogens. Specificity tests revealed that the GAS and GBS strains were positive for speB and SIP genes, respectively. In clinical samples, the double assay performed similarly to the traditional biochemical method. The limits of detection were both ≤100 copies per reaction. In tests for simulant-contaminated samples, bacterial-culture media containing 103  CFU/mL original concentrations of S. pyogenes and S. agalactiae were positive in RPA assays after incubating for 4 h. Results can be obtained at 37 °C in 20 min. To determine whether propidium monoazide (PMA) can eliminate the influence of DNA extracted from dead cells, a bacterial suspension was treated with PMA before DNA extraction. Findings of RPA assay showed that DNA extracted from dead cells had no fluorescence signal. Therefore, the PMA-RPA assay is a promising technology for field tests and rapid point-of-care diagnosis.