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.

Abstract

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). 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). The MRT-NAAT pathogen identification set was made up of 15 modular units 109-199bp in product size and with a Tms of 75.5-87.5°C. The LoD was < 15.548fg/μL, and nine modular units successfully detected the target pathogens. The MRT-NAAT AMR set included 24 modular units 65-785bp in product size with a Tms of 75.5-87.5°C; it showed high performance for detecting GPP target genes and variants. MRT-NAAT enables pathogen identification and AMR gene detection and is time-effective. By unifying the reaction settings of each modular unit, the modularity where combinations of primers can be used according to need could be achieved. This would greatly help in reflecting the researcher's need and the AMR status of a certain region while successfully detecting pathogens and AMR genes.