Simultaneous rapid identification of blaKPC-coding carbapenemase- producing Klebsiella pneumoniae by duplex chip digital PCR

Abstract:

[Background] Carbapenem-resistant Klebsiella pneumoniae strains have been on the rise with the extensive use and abuse of carbapenems. The production of carbapenemase is a common mechanism by which these strains resist killing by the carbapenems. The available detection methods for carbapenemase-producing K. pneumoniae are time- and labor-intensive, with poor specificity and sensitivity. [Objective] To develop an assay for the detection of K. pneumoniae with bla_KPC gene based on duplex chip digital PCR (duplex-cdPCR). [Methods] The specific primers and probes for the detection of the characteristic gene yhaI of K. pneumoniae and the carbapenem resistance gene bla_KPC were designed. The range of absolute quantification of nucleic acids, detection limit and optimal reaction conditions of the duplex-cdPCR for yhaI and bla_KPC were determined simultaneously. The specificity, sensitivity, and precision of the method were analyzed and the clinical strains were detected. [Results] Compared with duplex-qPCR, duplex-cdPCR had the sensitivity of about 1.5 orders of magnitude higher and the detection limit was 3.74 copies/μL (yhaI) and 1.93 copies/μL (bla_KPC), respectively. The optimized duplex-cdPCR showed the specificity consistent with that of duplex-qPCR. In this report, a total of 58 clinical strains were detected by the optimized duplex-cdPCR, of which 43 strains were detected as K. pneumoniae and 13 were K. pneumoniae harboring bla_KPC gene. This demonstrated 100% concordance with the results of mass spectrometry and drug resistance profile.[Conclusion] A duplex-cdPCR assay capable of simultaneously detecting the characteristic gene yhaI of K. pneumoniae and the carbapenem resistance gene bla_KPC in the same target was established. This assay appears to be highly specific, sensitive, and accurate. The duplex assay is suitable for nucleic acid detection and quantitative analysis of K. pneumoniae with bla_KPC gene. Moreover, it also provides a new technical reference for the detection of carbapenem-resistant pathogenic bacteria carrying other carbapenemase gene types.