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Rapid Detection of blaKPC in Carbapenem-Resistant Enterobacterales Based on CRISPR/Cas13a

Mingjun Liang1, Bin Xiao2, Lidan Chen1

  • 1Department of Laboratory Medicine, General Hospital of Southern Theater Command, No. 111, Liuhua Road, Yuexiu District, Guangzhou City, 510010, Guangdong Province, China.

Current Microbiology
|September 22, 2023
PubMed

Insights

A new RPA-Cas13a assay rapidly detects the blaKPC gene, crucial for carbapenem resistance in Enterobacterales. This simple, one-hour test aids in controlling challenging superbug infections.

Area of Science:

  • Molecular Biology
  • Antimicrobial Resistance
  • Diagnostic Development

Background:

  • Klebsiella pneumoniae carbapenemase (KPC) is a key enzyme driving carbapenem resistance in Enterobacterales.
  • Infections caused by carbapenem-resistant Enterobacterales (CREs) pose significant treatment challenges.
  • Rapid and accurate KPC detection is essential for controlling CRE prevalence.

Purpose of the Study:

  • To develop a novel, rapid, and accurate method for detecting the blaKPC gene.
  • To establish a recombinase polymerase amplification (RPA) and CRISPR/Cas13a-based assay for KPC detection.

Main Methods:

  • Developed a blaKPC detection assay using recombinase polymerase amplification (RPA) and CRISPR/Cas13a with fluorophore activation (RPA-Cas13a).
  • Optimized amplification primers for blaKPC and determined the lower limit of detection using recombinant plasmids.
  • Validated the assay on 57 blaKPC-positive CRE strains and 311 sputum samples.

Main Results:

  • Achieved a lower limit of detection of approximately 2.5 copies/μL for the blaKPC gene.
  • The RPA-Cas13a assay demonstrated 96.5% sensitivity and 100% specificity against DNA sequencing.
  • Results from 311 sputum samples showed high consistency (Kappa = 0.978) between the assay and antibiotic susceptibility testing.

Conclusions:

  • The RPA-Cas13a system offers a simple, efficient, one-hour method for detecting the blaKPC gene.
  • This technology can aid in the rapid identification of potentially fatal antibiotic resistance genes.
  • The assay shows promise for controlling the spread of carbapenem-resistant superbugs.

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