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

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 ( group, 12; group, 6; group, 5; group, 3; group, 3; -like group, 1 strain; and group + , 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 group and group co-coding strain) and all 20 of 20 isolates, respectively. Melting temperature (Tm) values of the various genes were as follows: group, 81.2±0.5°C; group, 91.8±0.4°C; group, 85.4±0.3°C; group, 90.5±0.3°C; group, 94.1±0.5°C; and -like group, 82.1°C. Identification of the various genotypes was possible from the Tm values. However, only a peak derived from the group could be detected in the strains producing both group and 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.