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Updated: May 14, 2025
Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus
Published on: September 5, 2013
Development and Validation of a Multiplex Real-Time PCR Assay for Rapid Screening of Main Carbapenemase Genes in
Francisco Javier Chamizo-López1,2, José Gutiérrez-Fernández2, María Dolores Rojo-Martín1
1Servicio de Microbiología, Hospital Regional Universitario de Málaga, Carlos Haya, s/n. 29010 Málaga, Spain.
Background/Objectives:
Carbapenem-resistant Enterobacterales, largely due to carbapenemase production, are significant public health threats, which compromise treatment with key β-lactam antibiotics. Early detection is essential for guiding therapy and controlling spread. This study describes the design, optimisation and validation of a multiplex real-time PCR for the screening of the most frequent carbapenemases in our area.
Methods:
Primers and probes targeted at genes encoding carbapenemases blaKPC, blaIMP, blaVIM, blaNDM and blaOXA-48-group were designed and adapted for the development, and in silico and experimental validation of a single-tube real-time PCR.
Results:
A good linear correlation between the fluorescence values in the real-time PCR and the log10 of bacterial concentration of each carbapenemase-containing bacterial suspension was observed (R2 > 0.98). The limit of detection was 2-15, 16-256, 42-184, 4-42, 42-226 CFU/reaction of VIM-, IMP-, NDM-, KPC- and OXA-48-carbapenemase-containing bacteria, respectively. Intra-assay coefficient of variation for the mean Ct values ranged from 0.99% for OXA-48 to 3.34% for KPC. Inter-assay variability remained below 7%. Real-time PCR tested on bacterial isolates yielded 100% sensitivity and specificity. Analysis of rectal swabs using extracted DNA and a DNA extraction-free protocol showed good concordance with culture-based phenotypic methods. Additionally, the molecular method could detect all targets, except for one sample where only the DNA extraction-free protocol detected NDM.
Conclusions:
The assay offers a rapid, sensitive and specific method for the screening of major carbapenemase genes, providing an effective tool for surveillance and infection control in clinical settings. The DNA extraction-free protocol converts this method into a good alternative for screening in 24/7 clinical laboratories. Further multiplexing to target other resistance genes, on demand, could add potential benefits to this laboratory-developed method.
