Rapid detection of Pseudomonas aeruginosa by recombinase polymerase amplification combined with CRISPR-Cas12a

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Shuang Liu1, Siyuan Huang1, Fang Li1

  • 1Department of Critical Medicine, Children's Hospital Affiliated Capital Institute of Pediatrics, Beijing, China.

Frontiers in Cellular and Infection Microbiology
|August 28, 2023
PubMed

Insights

A new assay combines recombinase polymerase amplification (RPA) and CRISPR-Cas12a for rapid Pseudomonas aeruginosa detection. This sensitive and specific method offers a promising tool for early diagnosis and control of P. aeruginosa infections.

Area of Science:

  • Microbiology
  • Molecular Diagnostics
  • Biotechnology

Background:

  • Pseudomonas aeruginosa is a significant bacterial pathogen causing diverse infections and contributing to antimicrobial resistance.
  • Rapid and accurate diagnostic methods are crucial for effective management and control of P. aeruginosa infections.
  • Existing diagnostic techniques may lack the speed, sensitivity, or specificity required for timely intervention.

Purpose of the Study:

  • To develop a simple, rapid, sensitive, and specific diagnostic platform for P. aeruginosa infection.
  • To integrate recombinase polymerase amplification (RPA) with a CRISPR-Cas12a biosensing system for P. aeruginosa detection.
  • To evaluate the performance and clinical feasibility of the developed assay.

Main Methods:

  • Development of a novel assay termed P. aeruginosa-CRISPR-RPA, integrating RPA and CRISPR-Cas12a technology.
  • Optimization of reaction conditions, including temperature and time for both RPA and CRISPR-Cas12a steps.
  • Assessment of sensitivity using serial dilutions of P. aeruginosa genomic DNA and specificity against non-P. aeruginosa strains.
  • Validation of clinical feasibility using clinical samples and comparison with a microfluidic chip method.

Main Results:

  • The P. aeruginosa-CRISPR-RPA assay achieved P. aeruginosa detection within 30 minutes (20 min RPA at 42°C, 10 min CRISPR-Cas12a at 37°C).
  • Demonstrated high sensitivity with a limit of detection of 60 fg per reaction (~8 copies).
  • Exhibited excellent specificity (100%) and consistent results with clinical samples compared to a microfluidic chip method.

Conclusions:

  • The P. aeruginosa-CRISPR-RPA assay is a reliable and rapid tool for P. aeruginosa detection.
  • This assay shows significant promise for early and swift diagnosis of P. aeruginosa infections.
  • The developed platform can aid in controlling the spread of P. aeruginosa, particularly in hospital settings.
Keywords:
CRISPR-Cas12aPseudomonas aeruginosaRPAoprLrecombinase polymerase amplification

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