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Rapid Detection of bla KPC Carbapenemase Genes by Real-Time PCR
- Source :
- Journal of Clinical Microbiology. 46:2879-2883
- Publication Year :
- 2008
- Publisher :
- American Society for Microbiology, 2008.
-
Abstract
- Carbapenem resistance among Enterobacteriaceae is an emerging problem worldwide. Klebsiella pneumoniae carbapenemase ( bla KPC ) enzymes are among the most common β-lactamases described. In this study, we report the development and validation of a real-time PCR (q-PCR) assay for the detection of bla KPC genes using TaqMan chemistry. The q-PCR amplification of bla KPC DNA was linear over 7 log dilutions ( r 2 = 0.999; slope, 3.54), and the amplification efficiency was 91.6%. The q-PCR detection limit was 1 CFU, and there was no cross-reaction with DNA extracted from several multidrug-resistant bacteria. Perianal/rectal swabs ( n = 187) collected in duplicate from 128 patients admitted to Sheba Medical Center surgical intensive care units were evaluated for the presence of carbapenem-resistant bacteria by culturing on MacConkey agar-plus-carbapenem disks and for bla KPC genes by q-PCR. Carbapenem-resistant organisms, all K. pneumoniae , were isolated from 47 (25.1%) of the 187 samples collected, while bla KPC genes were detected in 54 (28.9%) of the patient samples extracted by the NucliSENS easyMAG system. Of these, seven samples were positive for bla KPC genes by q-PCR but negative for carbapenem resistance by culture, while all samples in which no carbapenem-resistant bacteria were detected by culture also tested negative by q-PCR. Thus, the sensitivity and specificity of the q-PCR assay after extraction by the NucliSENS easyMAG system were 100% and 95%, respectively. Similar values were obtained after DNA extraction by the Roche MagNA Pure LC instrument: 97.9% sensitivity and 96.4% specificity. Overall, the bla KPC q-PCR assay appears to be highly sensitive and specific. The utilization of q-PCR will shorten the time to bla KPC detection from 24 h to 4 h and will help in rapidly isolating colonized or infected patients and assigning them to cohorts.
- Subjects :
- Microbiology (medical)
Serial dilution
Klebsiella pneumoniae
Polymerase Chain Reaction
Sensitivity and Specificity
beta-Lactam Resistance
beta-Lactamases
law.invention
Microbiology
Bacterial Proteins
Enterobacteriaceae
law
TaqMan
Humans
Polymerase chain reaction
biology
Enterobacteriaceae Infections
Bacteriology
biochemical phenomena, metabolism, and nutrition
bacterial infections and mycoses
biology.organism_classification
DNA extraction
Real-time polymerase chain reaction
Genes, Bacterial
Bacteria
Subjects
Details
- ISSN :
- 1098660X and 00951137
- Volume :
- 46
- Database :
- OpenAIRE
- Journal :
- Journal of Clinical Microbiology
- Accession number :
- edsair.doi.dedup.....18b1834d69abea7370fc151b4e8ef730