Development and Validation of a Novel Multiplex Real-Time PCR Assay for Rapid Detection of Carbapenemase Genes in Carbapenem-Resistant Isolates and Clinical Samples.

PURPOSE

Carbapenem-resistant (CRE) infection is an urgent threat to human health. This study aimed to develop and validate a novel multiplex real-time PCR (multi-qPCR) assay for the detection of the KPC, NDM, IMP, OXA-48-like, and VIM genes in CRE isolates and clinical samples, as well as to compare it with three phenotypic methods.

METHODS

The reliability and limit of detection (LOD) of the multi-qPCR assay were evaluated. PCR and DNA sequencing were used as the reference methods to identify carbapenemase genes in CRE isolates and clinical samples. The accuracy of the multi-qPCR assay, modified carbapenem inactivation and EDTA-modified carbapenem inactivation method (mCIMandeCIM), carbapenemase inhibitor-based combined disk test (CDT), and colloidal gold-based immunochromatographic test was compared with the reference methods with 182 isolates of CRE. Furthermore, 112 clinical samples were collected to validate the efficacy of this multi-qPCR assay.

RESULTS

The standard deviations (CVs) of intra-assay and inter-assay of the multi-qPCR assay were ≤ 0.53% and ≤ 2.04% for detecting the five major carbapenemase genes, respectively; while the LOD ranged from 2×10 copies/mL to 8×10 copies/mL. PCR and DNA sequencing confirmed 168 out of 182 CRE isolates producing carbapenemase(s): KPC (n = 93), NDM (n = 46), IMP (n = 8), OXA-48-like (n = 14), VIM (n = 1), KPC&NDM (n = 5), and KPC&NDM&IMP (n = 1). The accuracy of mCIMandeCIM, CDT, Colloidal Gold, and the multi-qPCR assay was 96.2%, 89.6%, 100%, and 100% respectively for detecting carbapenemase(s) producers. Moreover, the sensitivity and specificity of the multi-qPCR assay were all 100% for the detection of each carbapenemase gene in clinical samples, compared with PCR and sequencing.

CONCLUSION

For clinical isolate detection, the multi-qPCR assay is comparable to Colloidal Gold, and superior to mCIMandeCIM and CDT; while for clinical samples detection, it also shows excellent performance. Therefore, the multi-qPCR assay has great potential for clinical diagnosis.