Novel Quadruplex PCR for detecting and genotyping mobile colistin resistance genes in human samples

Area of Science:

• Clinical microbiology
• Molecular diagnostics
• Antimicrobial resistance

Background:

• Mobile colistin resistance (mcr) genes pose a significant threat to public health.
• Specific mcr genes (mcr-1, mcr-3, mcr-8, mcr-10) directly from clinical samples represent a heightened risk.
• Accurate and rapid detection of these genes in clinical settings is crucial.

Purpose of the Study:

• To develop and validate a novel Quadruplex polymerase chain reaction (Quad-PCR) protocol.
• To enable simultaneous detection and genotyping of clinically relevant mcr genes (mcr-1, mcr-3, mcr-8, mcr-10) in Enterobacteria.
• To provide a tool for clinical laboratory use.

Main Methods:

• Development of a Quadruplex PCR (Quad-PCR) assay.
• Validation using clinical isolates of Escherichia coli and Klebsiella.
• Comparison of Quad-PCR results with whole-genome sequencing data.

Main Results:

• The Quad-PCR protocol successfully detected and genotyped target mcr genes.
• The assay demonstrated full concordance with whole-genome sequencing.
• High sensitivity and 100% specificity were achieved.
• The method could identify single and mixed mcr gene types.

Conclusions:

• The Quad-PCR assay is a sensitive, specific, and reliable method for detecting and genotyping clinically significant mcr genes.
• This protocol offers a valuable tool for clinical laboratories to manage the threat of colistin resistance.
• The assay facilitates rapid identification of high-risk mcr alleles in Enterobacteria from human samples.