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

Anal Chem. 2019 Feb 5;91(3):2013-2020. doi: 10.1021/acs.analchem.8b04412. Epub 2019 Jan 24.

Abstract

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: blaOXA-48, blaNDM, blaVIM, and blaKPC, which account for 97% of the UK's reported carbapenemase-producing Enterobacteriaceae.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Anti-Bacterial Agents / pharmacology*
  • Carbapenems / pharmacology*
  • DNA, Bacterial / drug effects
  • DNA, Bacterial / genetics
  • Drug Resistance, Bacterial / drug effects*
  • Enterobacteriaceae / drug effects*
  • Enterobacteriaceae / genetics
  • Escherichia coli / drug effects*
  • Klebsiella pneumoniae / drug effects*
  • Microbial Sensitivity Tests
  • Nucleic Acids / genetics*
  • Real-Time Polymerase Chain Reaction / standards

Substances

  • Anti-Bacterial Agents
  • Carbapenems
  • DNA, Bacterial
  • Nucleic Acids