A Nanoplex PCR Assay for the Simultaneous Detection of Vancomycin- and Linezolid-Resistant Genes in <i>Enterococcus</i>

Background: Enterococci are Gram-positive cocci found in the guts of humans and animals. The goal of this research is to develop a multiplex PCR assay that can detect the Enterococcus genus, four VRE genes, and three LZRE genes simultaneously. Methods: Primers used in this study were specifically designed for the detection of 16S rRNA of Enterococcus genus, vanA—vanB—vanC—vanD for vancomycin, cfr methyltransferase, and optrA, and poxtA, as well as an adenosine triphosphate-binding cassette (ABC) transporter for linezolid. A Vibrio cholerae ctxA (internal amplification control) was included. Optimization of primer concentrations and PCR components was also done. This was followed by evaluating the sensitivity and specificity of the optimized multiplex PCR. Results: Final Primer concentrations were optimized as follows: 16S rRNA is 1.0 pmol/μL, vanA is 1.0 pmol/μL, optrA is 1.0 pmol/μL, cfr is 1.0 pmol/μL, poxtA is 0.1 pmol/μL, vanB is 0.08 pmol/μL, ctxA is 0.07 pmol/μL, vanC is 0.8 pmol/μL, and vanD is 0.1 pmol/μL. Further, the optimized concentrations for MgCl2, dNTPs and Taq DNA polymerase were 2.5 mM, 0.16 mM, and 0.75 units respectively, and an annealing temperature of 64.5 °C. Conclusions: The developed multiplex PCR is sensitive and species-specific. The development of a multiplex PCR assay that will take into account all known VRE genes and linezolid mutation is highly recommended