title: Real-time PCR for detection of NDM-1 carbapenemase genes from spiked stool samples.
abstract: 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 of bla(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 × 10(1) to 3 × 10(1) CFU/100 mg feces with ChromID ESBL medium. The CHROMagar KPC culture medium had higher limits of detection (1 × 10(1) to 4 × 10(3) CFU/ml), especially with bacterial isolates having low carbapenem MICs. The limits of detection with the qPCR assay were reproducibly below 1 × 10(1) CFU/100 mg of feces by qPCR assay. Samples spiked with NDM-1-negative bacteria were negative by qPCR. The sensitivity and specificity of the bla(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 for bla(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.
authors.affiliation: Service de Bactériologie-Virologie, INSERM U914, Hôpital de Bicêtre, 94275 Le Kremlin-Bicêtre, France. thierry.naas@bct.aphp.fr
authors.forename: Thierry
authors.initials: T
authors.lastname: Naas
authors.forename: Ayla
authors.initials: A
authors.lastname: Ergani
authors.forename: Amélie
authors.initials: A
authors.lastname: Carrër
authors.forename: Patrice
authors.initials: P
authors.lastname: Nordmann
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics*; D005243:Feces / Q000382:microbiology*; D006801:Humans; D010957:Plasmids / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D001618:beta-Lactamases / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D001618:beta-Lactamases; C063912:carbapenemase