Simultaneous Single-Channel Multiplexing and Quantification of Carbapenem-Resistant Genes Using Multidimensional Standard Curves

Multiplexing and quantification of nucleic acids, both have, in their own right, significant and extensive use in biomedical related fields. Currently, the ability to detect several nucleic acid targets in a single-reaction scales linearly with the number of targets; an expensive and time-consuming feat. Here, we propose a new methodology based on multidimensional standard curves that extends the use of real-time PCR data obtained by common qPCR instruments. By applying this novel methodology, we achieve simultaneous single-channel multiplexing and enhanced quantification of multiple targets using only real-time amplification data. This is obtained without the need of fluorescent probes, agarose gels, melting curves or sequencing analysis. Given the importance and demand for tackling challenges in antimicrobial resistance, the proposed method is applied to four of the most prominent carbapenem-resistant genes: bla_OXA‑48, bla_NDM, bla_VIM, and bla_KPC, which account for 97% of the UK’s reported carbapenemase-producing Enterobacteriaceae.