A multiplex TaqMan real-time PCR assays for the rapid detection of mobile colistin resistance (mcr-1 to mcr-10) genes.
Front Microbiol
; 15: 1279186, 2024.
Article
em En
| MEDLINE
| ID: mdl-38544862
ABSTRACT
Objective:
Recently, 10 plasmid-mediated mobile colistin resistance genes, mcr-1 to mcr-10, 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 mcr genes in clinical isolates.Methods:
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.Results:
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 mcr 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 mcr genes in 467 clinically-obtained Gram-negative isolates, which were multidrug-resistant. Twelve strains containing the mcr genes were detected (seven isolates carrying mcr-1, four isolates carrying mcr-10, and one isolate carrying mcr-9). 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.Conclusion:
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 mcr genes (mcr-1 to mcr-10), thus providing a better basis for clinical drug treatment and drug resistance research.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Idioma:
En
Revista:
Front Microbiol
Ano de publicação:
2024
Tipo de documento:
Article
País de afiliação:
China