Rapid detection and differentiation of mobile colistin resistance (mcr-1 to mcr-10) genes by real-time PCR and melt-curve analysis

粘菌素 MCR-1型 熔化曲线分析 基因 生物信息学 底漆(化妆品) 多路复用 质粒 多重聚合酶链反应 聚合酶链反应 生物 医学 微生物学 遗传学 抗生素 大肠杆菌 肠杆菌科 化学 有机化学
作者
M. Mentasti,Sophia David,Kirsty Sands,S. Khan,Leanne Davies,Luke Turner,Mandy Wootton
出处
期刊:Journal of Hospital Infection [Elsevier BV]
卷期号:110: 148-155 被引量:15
标识
DOI:10.1016/j.jhin.2021.01.010
摘要

Summary

Background

The emergence of multi-drug-resistant (MDR) micro-organisms prompted new interest in older antibiotics, such as colistin, that had been abandoned previously due to limited efficacy or high toxicity. Over the years, several chromosomal-encoded colistin resistance mechanisms have been described; more recently, 10 plasmid-mediated mobile colistin resistance (mcr) genes have been identified. Spread of these genes among MDR Gram-negative bacteria is a matter of serious concern; therefore, reliable and timely mcr detection is paramount.

Aim

To design and validate a multiplex real-time polymerase chain reaction (PCR) assay for detection and differentiation of mcr genes.

Methods

All available mcr alleles were downloaded from the National Center for Biotechnology Information Reference Gene Catalogue, aligned with Clustal Omega and primers designed using Primer-BLAST. Real-time PCR monoplexes were optimized and validated using a panel of 120 characterized Gram-negative strains carrying a wide range of resistance genes, often in combination. Melt-curve analysis was used to confirm positive results.

Findings

In-silico analysis enabled the design of a 'screening' assay for detection of mcr-1/2/6, mcr-3, mcr-4, mcr-5, mcr-7, mcr-8 and mcr-9/10, paired with an internal control assay to discount inhibition. A 'supplementary' assay was subsequently designed to differentiate mcr-1, mcr-2, mcr-6, mcr-9 and mcr-10. Expected results were obtained for all strains (100% sensitivity and specificity). Melt-curve analysis showed consistent melting temperature results. Inhibition was not observed.

Conclusions

The assay is rapid and easy to perform, enabling unequivocal mcr detection and differentiation even when more than one variant is present. Adoption by clinical and veterinary microbiology laboratories would aid the surveillance of mcr genes amongst Gram-negative bacteria.
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