Development of diagnostic tests for pathogen identification and detection of antimicrobial resistance on WHO global priority pathogens using modular real-time nucleic acid amplification test

Background Concerns regarding antimicrobial resistance (AMR) have resulted in the World Health Organization (WHO) designating so-called global priority pathogens (GPPs). However, little discussion has focused on the diagnosis of GPPs. To enable the simultaneous identification of pathogens and AMR, we developed a modular real-time nucleic acid amplification test (MRT-NAAT). Methods Sequence-specific primers for each modular unit for MRT-NAAT pathogen identification and AMR sets were designed. The composition of the reaction mixture and the real-time PCR program were unified irrespective of primer type so to give MRT-NAAT modularity. Standard strains and clinical isolates were used to evaluate the performance of MRT-NAAT by real-time PCR and melting curve analysis. Probit analysis for the MRT-NAAT pathogen identification set was used to assess the limit of detection (LoD). Results The MRT-NAAT pathogen identification set was made up of 15 modular units 109–199 bp in product size and with a T m s of 75.5–87.5 °C. The LoD was