biostudies-literatureUnknown15Gong X<h4>Objective</h4>Recently, 10 plasmid-mediated mobile colistin resistance genes, <i>mcr-1</i> to <i>mcr-10</i>, and their variants have been identified, posing a new threat to the treatment of clinical infections caused by Gram-negative bacteria. Our objective was to develop a rapid, sensitive, and accurate molecular assay for detecting <i>mcr</i> genes in clinical isolates.<h4>Methods</h4>The primers and corresponding TaqMan-MGB probes were designed based on the sequence characteristics of all reported MCR family genes, multiplex Taqman-MGB probe-based qPCR assays were developed and optimized, and the sensitivity, specificity and reproducibility of the method were evaluated. The assay contained 8 sets of primers and probes in 4 reaction tubes, each containing 2 sets of primers and probes.<h4>Results</h4>The standard curves for both the single and multiplex systems showed good linearity (R² > 0.99) between the starting template amount and the Ct value, with a lower limit of detection of 10² copies/μL. The specificity test showed positive amplification results only for strains containing the <i>mcr</i> genes, whereas the other strains were negative. The results of intra-and inter-group repeatability experiments demonstrated the stability and reliability of the newly developed method. It was used to detect <i>mcr</i> genes in 467 clinically-obtained Gram-negative isolates, which were multidrug-resistant. Twelve strains containing the <i>mcr</i> genes were detected (seven isolates carrying <i>mcr-1</i>, four isolates carrying <i>mcr-10</i>, and one isolate carrying <i>mcr-9</i>). The products amplified by the full-length PCR primer were identified by sequencing, and the results were consistent with those of the multiplex qPCR method.<h4>Conclusion</h4>The assay developed in this study has the advantages of high specificity, sensitivity, and reproducibility. It can be used to specifically detect drug-resistant clinical isolates carrying the <i>mcr</i> genes (<i>mcr-1 to mcr-10</i>), thus providing a better basis for clinical drug treatment and drug resistance research.Frontiers in microbiology1279186https://www.ebi.ac.uk/biostudies/studies/S-EPMC10967403biostudies-literatureA multiplex TaqMan real-time PCR assays for the rapid detection of mobile colistin resistance (<i>mcr-1</i> to <i>mcr-10</i>) genes.PMC10967403Zou XYang GLi ZYu RDuan CJia XWu DZou DWang YGong XLiu WfalseA multiplex TaqMan real-time PCR assays for the rapid detection of mobile colistin resistance (<i>mcr-1</i> to <i>mcr-10</i>) genes.<h4>Objective</h4>Recently, 10 plasmid-mediated mobile colistin resistance genes, <i>mcr-1</i> to <i>mcr-10</i>, and their variants have been identified, posing a new threat to the treatment of clinical infections caused by Gram-negative bacteria. Our objective was to develop a rapid, sensitive, and accurate molecular assay for detecting <i>mcr</i> genes in clinical isolates.<h4>Methods</h4>The primers and corresponding TaqMan-MGB probes were designed based on the sequence characteristics of all reported MCR family genes, multiplex Taqman-MGB probe-based qPCR assays were developed and optimized, and the sensitivity, specificity and reproducibility of the method were evaluated. The assay contained 8 sets of primers and probes in 4 reaction tubes, each containing 2 sets of primers and probes.<h4>Results</h4>The standard curves for both the single and multiplex systems showed good linearity (R² > 0.99) between the starting template amount and the Ct value, with a lower limit of detection of 10² copies/μL. The specificity test showed positive amplification results only for strains containing the <i>mcr</i> genes, whereas the other strains were negative. The results of intra-and inter-group repeatability experiments demonstrated the stability and reliability of the newly developed method. It was used to detect <i>mcr</i> genes in 467 clinically-obtained Gram-negative isolates, which were multidrug-resistant. Twelve strains containing the <i>mcr</i> genes were detected (seven isolates carrying <i>mcr-1</i>, four isolates carrying <i>mcr-10</i>, and one isolate carrying <i>mcr-9</i>). The products amplified by the full-length PCR primer were identified by sequencing, and the results were consistent with those of the multiplex qPCR method.<h4>Conclusion</h4>The assay developed in this study has the advantages of high specificity, sensitivity, and reproducibility. It can be used to specifically detect drug-resistant clinical isolates carrying the <i>mcr</i> genes (<i>mcr-1 to mcr-10</i>), thus providing a better basis for clinical drug treatment and drug resistance research.2024-01-01T00:00:00Z20242025-04-04T23:53:30.15Z2025-04-04T23:53:30.15ZS-EPMC109674033854486210.3389/fmicb.2024.1279186