Rapid detection of carbapenemase genes by multiplex real-time PCR

J Antimicrob Chemother. 2012 Apr;67(4):906-9. doi: 10.1093/jac/dkr563. Epub 2012 Jan 9.

Abstract

Objectives: To develop a single multiplex real-time PCR assay to detect six different genetic types of carbapenemases already identified in Enterobacteriaceae (KPC, GES, NDM, IMP, VIM and OXA-48).

Methods: A total of 58 bacterial isolates were tested. Thirty were previously characterized as resistant to carbapenems and documented by PCR and sequencing analysis to carry the following genes: bla(KPC) type, bla(GES) type, bla(IMP) type, bla(VIM) type, bla(OXA-48) and bla(NDM-1). These positive strains included 21 Enterobacteriaceae, 1 Acinetobacter baumannii and 8 Pseudomonas aeruginosa isolates. The remaining 28 isolates previously tested susceptible to carbapenems and were negative for these genes. Bacterial DNA was extracted using the easyMag extractor (bioMérieux, France). The real-time PCR was performed using the Rotor-Gene 6000 instrument (Corbett Life Science, Australia) and specific primers for each carbapenemase target were designed using the DNAStar software (Madison, WI, USA).

Results: Each one of the six carbapenemase genes tested presented a different melting curve after PCR amplification. The melting temperature (T(m)) analysis of the amplicons identified was as follows: bla(IMP) type (T(m) 80.1°C), bla(OXA-48) (T(m) 81.6°C), bla(NDM-1) (T(m) 84°C), bla(GES) type (T(m) 88.6°C), bla(VIM) type (T(m) 90.3°C) and bla(KPC) type (T(m) 91.6°C). No amplification was detected among the negative samples. The results showed 100% concordance with the genotypes previously identified.

Conclusions: The new assay was able to detect the presence of six different carbapenemase gene types in a single 3 h PCR.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acinetobacter baumannii / enzymology*
  • Acinetobacter baumannii / genetics
  • Bacterial Proteins / analysis*
  • Bacterial Proteins / classification
  • Bacterial Proteins / genetics*
  • Bacteriological Techniques / methods
  • Enterobacteriaceae / enzymology*
  • Enterobacteriaceae / genetics
  • Genotype
  • Humans
  • Multiplex Polymerase Chain Reaction / methods*
  • Pseudomonas aeruginosa / enzymology*
  • Pseudomonas aeruginosa / genetics
  • Real-Time Polymerase Chain Reaction / methods*
  • Sensitivity and Specificity
  • Time Factors
  • Transition Temperature
  • beta-Lactamases / analysis*
  • beta-Lactamases / classification
  • beta-Lactamases / genetics*

Substances

  • Bacterial Proteins
  • beta-Lactamases
  • carbapenemase