Rapid Detection of Carbapenemase-producing Genes by Multiplex Real-Time PCR with Melting Curve Analysis using a BD MAX™ Open System.

Abstract

Recently, the global spread of carbapenemase-producing Enterobacterales has become a concern, and rapid detection methods are required. We have developed a rapid and inexpensive multiplex real-time PCR with melting curve analysis using the BD MAX™ system and evaluated it. We used 31 carbapenemase-producing Gram-negative bacteria (bla_IMP group, 12; bla_GES group, 6; bla_NDM group, 5; bla_VIM group, 3; bla_KPC group, 3; bla_OXA-48-like group, 1 strain; and bla_IMP group + bla_GES, 1 strain), 10 AmpC-producing Gram-negative bacteria, and 10 ESBLproducing Gram-negative bacteria. A BD MAX™ open platform system was used. Carbapenemase-positive and carbapenemase-negative strains were correctly identified 30 of 31 (excluding a bla_IMP group and bla_GES group co-coding strain) and all 20 of 20 isolates, respectively. Melting temperature (Tm) values of the various genes were as follows: bla_IMP group, 81.2±0.5°C; bla_VIM group, 91.8±0.4°C; bla_NDM group, 85.4±0.3°C; bla_GES group, 90.5±0.3°C; bla_KPC group, 94.1±0.5°C; and bla_OXA-48-like group, 82.1°C. Identification of the various genotypes was possible from the Tm values. However, only a peak derived from the bla_GES group could be detected in the strains producing both bla_IMP group and bla_GES group simultaneously, suggesting that only the genotype with the highest expression level could be captured in cases of simultaneous production. In the carbapenemase-negative strains, no obvious peaks were observed in the 20 AmpC and 20 ESBL-producing Gram-negative bacteria, and even when Tm values were detected, the dF/dT values were low and easily differentiated. This method appears to be very useful as a rapid and inexpensive test that can provide detection in about 2 hours.