Open AccessReal-Time PCR for Detection of NDM-1 Carbapenemase Genes from Spiked Stool Samples
Author(s) -
Thierry Naas,
Ayla Ergani,
Amélie Carrër,
Patrice Nordmann
Publication year - 2011
Publication title -
antimicrobial agents and chemotherapy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.07
H-Index - 259
eISSN - 1070-6283
pISSN - 0066-4804
DOI - 10.1128/aac.01734-10
Subject(s) - microbiology and biotechnology , taqman , detection limit , biology , real time polymerase chain reaction , feces , serial dilution , bacteria , dna extraction , polymerase chain reaction , plasmid , dna , gene , chemistry , chromatography , medicine , genetics , alternative medicine , pathology
An in-house quantitative real-time PCR (qPCR) assay using TaqMan chemistry has been developed to detect NDM-1 carbapenemase genes from bacterial isolates and directly from stool samples. The qPCR amplification ofbla NDM-1 DNA was linear over 10 log dilutions (r 2 = 0.99), and the amplification efficiency was 1.03. The qPCR detection limit was reproducibly 1 CFU, or 10 plasmid molecules, and there was no cross-reaction with DNA extracted from several multidrug-resistant bacteria harboring other β-lactam resistance genes. Feces spiked with decreasing amounts of enterobacterial isolates producing NDM-1 were spread on ChromID ESBL and on CHROMagar KPC media and were subjected to the qPCR. The limits of carbapenem-resistant bacterial detection from stools was reproducibly 1 × 101 to 3 × 101 CFU/100 mg feces with ChromID ESBL medium. The CHROMagar KPC culture medium had higher limits of detection (1 × 101 to 4 × 103 CFU/ml), especially with bacterial isolates having low carbapenem MICs. The limits of detection with the qPCR assay were reproducibly below 1 × 101 CFU/100 mg of feces by qPCR assay. Samples spiked with NDM-1-negative bacteria were negative by qPCR. The sensitivity and specificity of thebla NDM-1 qPCR assay on spiked samples were 100% in both cases. Using an automated DNA extraction system (QIAcube system), the qPCR assay was reproducible. The use of qPCR is likely to shorten the time forbla NDM-1 detection from 48 h to 4 h and will be a valuable tool for outbreak follow-up in order to rapidly isolate colonized patients and assign them to cohorts.