Multiplex real-time PCR for detection of <i>qac A/B</i> and <i>smr</i> genes in Gram-positive bacteria

Background. Disinfectants are effective means of non-specific prevention of infections associated with the provision of medical care. Violation of disinfectant use regimes leads to the formation of microorganism resistance to them. To monitor the spread of clinically significant microorganisms resistant to disinfectants, the development of methods for their detection, including molecular genetic methods, remains relevant. The aim of the study was to develop a multiplex real-time PCR for the identification of qacA/B and smr genes, the determinants of resistance to cationic biocides, in Gram-positive bacteria. Materials and methods. Conserved regions of qacA, qacB and smr genes were searched, and primers and probes were designed using BLASTN, GeneRunner, and Multiple Primer Analyzer programs. To evaluate the analytical sensitivity of the multiplex PCR, plasmids pTZ57-qacA/B, pTZ57-smr, and pTZ57-16S containing qacA/B, smr and 16S rRNA gene fragments of 197 bp, 127 bp, and 287 bp, respectively, were constructed. The method was tested on clinical isolates of Gram-positive bacteria (n = 30). Results. A multiplex real-time PCR using TaqMan probes was developed for the detection of qacA/B and smr genes in Gram-positive bacteria. The 16S rRNA gene was used as an internal amplification control. The sensitivity of the multiplex PCR was 103 copies for all genes. Multiplex PCR validation showed that qacA/B genes were present in 30%, smr genes were present in 10% of the isolates tested. The reproducibility of the results was 100%. Conclusion. The developed multiplex PCR differs from existing assays by high specificity and short turnaround time, as well as by the presence of an internal amplification control. It can be used for the detection of Gram-positive bacteria potentially resistant to cationic biocides.