AUTHOR=Gong Xinran , Yang Guang , Liu Wei , Wu Di , Duan Chunyuan , Jia Xinjing , Li Zhiqiang , Zou Xiaocang , Yu Renfeng , Zou Dayang , Wang Yong 

TITLE=A multiplex TaqMan real-time PCR assays for the rapid detection of mobile colistin resistance (mcr-1 to mcr-10) genes

JOURNAL=Frontiers in Microbiology

VOLUME=15

YEAR=2024

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2024.1279186

DOI=10.3389/fmicb.2024.1279186

ISSN=1664-302X

ABSTRACT=<sec id="sec1"><title>Objective</title><p>Recently, 10 plasmid-mediated mobile colistin resistance genes, <italic>mcr-1</italic> to <italic>mcr-10</italic>, 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 <italic>mcr</italic> genes in clinical isolates.</p></sec><sec id="sec2"><title>Methods</title><p>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.</p></sec><sec id="sec3"><title>Results</title><p>The standard curves for both the single and multiplex systems showed good linearity (R<sup>2</sup> &gt; 0.99) between the starting template amount and the Ct value, with a lower limit of detection of 10<sup>2</sup> copies/μL. The specificity test showed positive amplification results only for strains containing the <italic>mcr</italic> 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 <italic>mcr</italic> genes in 467 clinically-obtained Gram-negative isolates, which were multidrug-resistant. Twelve strains containing the <italic>mcr</italic> genes were detected (seven isolates carrying <italic>mcr-1</italic>, four isolates carrying <italic>mcr-10</italic>, and one isolate carrying <italic>mcr-9</italic>). 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.</p></sec><sec id="sec4"><title>Conclusion</title><p>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 <italic>mcr</italic> genes (<italic>mcr-1 to mcr-10</italic>), thus providing a better basis for clinical drug treatment and drug resistance research.</p></sec>