Rapid real-time PCR for the detection of IMP, NDM, VIM, KPC and OXA-48 carbapenemase genes in isolates and spiked stool samples

Abstract

An in-house real-time PCR was developed to detect the carbapenemase genes IMP, NDM, VIM, KPC and OXA-48 from both bacterial colonies and directly from fecal material. The assay is a multiplex PCR performed in two tubes with NDM, VIM and IMP genes detected in one tube and OXA-48 and KPC genes in the other. Despite the large amount of primers and probes necessary to cover all variants of especially the VIM and IMP genes, the efficiency of the PCR reactions was from 95 to 100%. When tested on 170 clinical strains and compared to culture results from a reference laboratory, 100% correspondence was found. Fecal samples were spiked with carbapenemase producing strains in different concentrations and analyzed by both PCR and conventional culture. Sensitivity of the PCR analysis varied from 100 to 10,000 cfu/ml fecal material and was comparable to culture on ChromID® CARBA plates. In conclusion, our in-house PCR assay may be able to detect all published variants of IMP, NDM, VIM, KPC and OXA-48 within 2.5 hours and can be performed directly on fecal samples.