Multiplex High-Resolution Melting Analysis as a Diagnostic Tool

This study was aimed to develop a multiplex RT-PCR with high resolution melting (HRM) analysis for the rapid and accurate detection of five carbapenemasehousekeeping gene 16S rRNA. Six pairs of primers and RT-PCR conditions were designed according to Beacon designer software. The identification of the genes was conducted by melting curves and temperatures with Multiplex RT-PCR using a dedicated HRM reagent. This is the first assay detection the main carbapenemase genes into a two-tube assay, enabling the simultaneous detection of 5 of the most important markers of resistance to 3rd generation cephalosporins and carbapenems.In the first panel tube, the reaction included bla-IMP, bla-VIM and bla-NDM. The results revealed that the melting temperature (TM) values were (81.1 oC, 84.5 oC and 86.1 oC), respectively, while the second panel tube contain the reaction of 16S rRNA, bla-OXA 48, and bla-KPC . The the melting temperature (TM) values were (84.5 oC,81.7 oC and 89.35 oC), respectively at in one run with 2 hours. Result most prevalent genes were bla-IMP 38.75%, bla-OXA48 36.25%, and bla-VIM 36.25%, followed by bla-KPC 21.25%, and bla-NDM 13.75%.