Wada, Yusuf
(2022)
Development of multiplex polymerase
chain reaction (PCR) assay for the
simultaneous detection of
vancomycin and linezolid resistant
genes in enterococcus.
PhD thesis, Universiti Sains Malaysia.
Abstract
Enterococci are Gram-positive cocci found in the guts of humans and
animals. The introduction and dissemination of vancomycin-resistant Enterococcus
(VRE) and Linezolid-Resistant Enterococcus (LZRE) in healthcare settings has
increased patient management risks and difficulties. No multiplex PCR has been
developed for the simultaneous detection of both vancomycin and linezolid resistant
genes in Enterococcus. 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 all at the same time. Primers used in this study were specifically designed for
the detection of vancomycin and linezolid resistant genes in Enterococcus. These
genes are; 16S rRNA of Enterococcus genus, vanA – vanB – vanC - vanD for
vancomycin, cfr methyltransferase, optrA and poxtA; an adenosine triphosphatebinding
cassette (ABC) transporter for linezolid. A Vibrio cholerae ctxA (Internal
amplification control) was included. Optimization of primer concentrations, MgCl2,
dNTPs, Taq DNA polymerase and primers annealing temperature was also done.
This was followed by evaluating the sensitivity and specificity of the optimized
multiplex PCR and their analytical sensitivity both at the genomic and bacteria
level. Final Primer concentrations was optimized as follows; 16S rRNA is 1.0
pmol/μl, vanA 1.0 pmol/μl, optrA 1.0 pmol/μl, cfr 1.0 pmol/μl, poxtA 0.1 pmol/μl,
vanB 0.08 pmol/μl, ctxA 0.07 pmol/μl, vanC 0.8 pmol/μl and vanD 0.1 pmol/μl.
Further, a MgCl2, dNTPs and Taq DNA polymerase optimized concentration was
2.5 mM, 0.16 mM and 0.75 units respectively. An annealing temperature of 64.5℃,
a LOD of 100 pg at the genomic level and as low as 105 CFU/ml at the bacteria level
were utilized and evaluated in the developed multiplex PCR assay. In this study, the
sensitivity, specificity, NPV, PPV and accuracy of the developed multiplex PCR
assay in the detection of VRE genes were 76.32% (CI: 59.76% - 88.56%), 100%
(CI: 87.23% - 100.00%), 75% (CI: 62.90% - 84.15%), 100% and 86.15% (CI:
75.34% - 93.47%) respectively. Similarly, the sensitivity, specificity, NPV, PPV
and accuracy of the developed multiplex PCR assay in the detection of LZRE genes
were 88.89% (CI: 51.75% - 99.72%), 100% (CI: 86.77% - 100.00%), 96.30% (CI:
80.38% - 99.40%), 100% and 97.14% (CI: 85.08% - 99.93%) respectively. This
developed multiplex PCR is sensitive, species-specific, rapid and capable of
detecting vancomycin and linezolid resistant Enterococcus genes in clinical and
environmental settings. The development of a multiplex PCR assay that will take
into account all known VRE genes and linezolid mutation so that they would not be
missed during routine laboratory diagnosis is highly recommended.
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