Single-tube detection of nine bacterial antibiotic-resistance genes by a 2-dimensional multiplex qPCR assay based on fluorescence and melting temperature.

Simple, multiplex qPCR methods are advantages for rapid molecular diagnosis of multiple antibiotics-resistant genes simultaneously. However, the number of genes can be detected in a single reaction tube is often limited by the fluorescence channels of a real-time PCR instrument. In this study, we developed a simple 2-D multiplex qPCR method by combining the probe colors and amplicon Tm values to overcome the mechanical limit of the machine. The principle of the novel assay was validated by detection of nine bacterial antibiotic-resistance genes (KPC, NDM, VIM, OXA-48, GES, CIT, EBC, ACC and DHA) in a single reaction tube. This assay is highly sensitive within a range of 30-3000 copies per reaction. The simplicity, rapidity, high sensitivity and specificity, and low cost of the novel method make it a promising tool for developing clinical diagnostic kits for monitoring resistance and other genetic determinants of infectious diseases.