Multiplex real-time pcr assay for six major carbapenemase genes

Nori Yoshioka, Hideharu Hagiya, Matsuo Deguchi, Shigeto Hamaguchi, Masanori Kagita, Isao Nishi, Yukihiro Akeda, Kazunori Tomono

Research output: Contribution to journalArticlepeer-review

Abstract

The global dissemination of carbapenemase-producing Enterobacteriaceae (CPE) is a major concern in public health. Due to the existence of the diversity of carbapenemases, development of an easily available, cost-effective multiplex detection assay for CPE is required worldwide. Using clinically available and reliable equipment, COBAS® z480 (Roche Diagnostics K.K., Tokyo, Japan), we developed a multiplex real-time PCR assay for the detection of two combinations of carbapenemases; first, blaNDM, blaKPC, and blaIMP (Set 1), and second, blaGES, blaOXA-48, and blaVIM (Set 2). We constructed standard curves for each carbapenemase gene using serial dilutions of DNA standards, then applied reference or clinical isolates with each carbapenemase gene to this assay. The multiplex assay showed satisfactory accuracy to detect CPE genes, with the correlation coefficients of greater than 0.99 for all genotypes. The assay appropriately differentiated the reference or clinical strains harboring each carbapenemase gene without cross reactivity. Lastly, the assay successfully detected multiple genes without false-positive reactions by applying six clinical isolates carrying both NDM and OXA-48-like carbapenemase genes. Major advantages of our assay include multiplicity, simple operation, robustness, and speed (1 h). We believe that the multiplex assay potentially contributes to early diagnosis of CPE with a diverse genetic background.

Original languageEnglish
Article number276
Pages (from-to)1-6
Number of pages6
JournalPathogens
Volume10
Issue number3
DOIs
Publication statusPublished - Mar 2021

Keywords

  • Carbapenem-resistant Enterobacteriaceae
  • Carbapenemase-producing Enterobacteriaceae
  • Infection control
  • Multiplex detection assay

ASJC Scopus subject areas

  • Immunology and Allergy
  • Molecular Biology
  • Immunology and Microbiology(all)
  • Microbiology (medical)
  • Infectious Diseases

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