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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

International Microbiology (2023)Cite this article

Abstract

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 Tms of 75.5–87.5 °C. The LoD was < 15.548 fg/μL, and nine modular units successfully detected the target pathogens. The MRT-NAAT AMR set included 24 modular units 65–785 bp in product size with a Tms of 75.5–87.5 °C; it showed high performance for detecting GPP target genes and variants.

Conclusions

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.

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Fig. 1
Fig. 2

Data availability

The datasets used and/or analyzed in this study are available from the corresponding author upon reasonable request.

Abbreviations

AMR:

Antimicrobial resistance

GPP:

Global priority pathogen

MRT-NAAT:

Modular real-time nucleic acid amplification test

GLASS:

Global Antimicrobial Resistance Surveillance System

MALDI-TOF MS:

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry

Ct :

Threshold cycle

Tm :

Melting temperature

WGS:

Whole-genome sequencing

NGS:

Next-generation sequencing

ATCC:

American Type Culture Collection

LoD:

Limit of detection

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Funding

This work was supported by research funds 2019-ER5402-01 and 2020-ER5403-00 of the Korea Disease Control and Prevention Agency.

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Authors and Affiliations

  1. Seoul National University College of Medicine, Seoul, Republic of Korea

    Eunsik Bang & Hee Won Cho

  2. Department of Laboratory Medicine, Seoul National University College of Medicine, Seoul, Republic of Korea

    Sujin Oh, Jeong Su Park & Kyoung Un Park

  3. Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul, Republic of Korea

    Da-ha Park & Young Ho Suh

  4. PHiCS Institute, Seoul, Republic of Korea

    Ho Eun Chang

  5. Department of Laboratory Medicine, Seoul National University Bundang Hospital, Seongnam, Republic of Korea

    Jeong Su Park & Kyoung Un Park

  6. Department of Pediatrics, Seoul National University Bundang Hospital and Seoul National University College of Medicine, Seoul, Republic of Korea

    Hyunju Lee

  7. Department of Internal Medicine, Seoul National University Bundang Hospital and Seoul National University College of Medicine, Seoul, Republic of Korea

    Kyoung-Ho Song, Eu Suk Kim & Hong Bin Kim

Authors
  1. Eunsik Bang
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  2. Sujin Oh
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  11. Young Ho Suh
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Contributions

Conceptualization: EB, HWC, DP, YHS, and KUP; data collection: EB, SO, HWC, DP, HEC, JSP, HL, KS, ESK, HBK, and KUP; formal analysis: EB, SO, HWC, HEC, and KUP; writing, original draft: EB; writing, review, and editing: EB, SO, HEC, and KUP. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Kyoung Un Park.

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Ethics approval and consent to participate

The study was conducted according to the guidelines of the Declaration of Helsinki and was reviewed by the Institutional Review Board of Seoul National University Bundang Hospital (No. X-2112–729-901).

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Not applicable.

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The authors declare no competing interests.

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Bang, E., Oh, S., Cho, H.W. et al. 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. Int Microbiol (2023). https://doi.org/10.1007/s10123-023-00321-9

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  • DOI: https://doi.org/10.1007/s10123-023-00321-9

Keywords

  • WHO global priority pathogens
  • Pathogen identification
  • Antimicrobial resistance
  • Diagnosis
  • Modularity
  • Real-time
  • Nucleic acid amplification test