Published July 19, 2023 | Version v1
Journal article Open

CRISPR-Cas13a-Based Assay for Accurate Detection of OXA-48 and GES Carbapenemases

Authors/Creators

  • 1. Multidisciplinary and Translational Microbiology Group (MicroTM), Biomedical Research Institute of A Coruña (INIBIC), Microbiology Service, University Hospital of A Coruña (CHUAC), University of A Coruña (UDC), A Coruña, Spain, Study Group on Mechanisms of Action and Resistance to Antimicrobials (GEMARA) on behalf of the Spanish Society of Infectious Diseases and Clinical Microbiology (SEIMC), Madrid, Spain
  • 2. Microbiology Service, University Hospital Marqués de Valdecilla – IDIVAL, Santander, Spain
  • 3. Microbiology Service, University Hospital Central de Asturias. Translational Microbiology Group, ISPA, Oviedo, Spain, CIBER de Enfermedades Respiratorias (CIBERES), Instituto de Salud Carlos III, Madrid, Spain
  • 4. Reference and Research Laboratory for Antibiotic Resistance and Health Care Infections, National Centre for Microbiology, Institute of Health Carlos III, Majadahonda, Madrid, Spain, CIBER de Enfermedades Infecciosas (CIBERINFEC), Instituto de Salud Carlos III, Madrid, Spain
  • 5. Microbiology Service, University Hospital Son Espases and Health Research Institute Illes Balears (IdISBa), Palma de Mallorca, Spain, CIBER de Enfermedades Infecciosas (CIBERINFEC), Instituto de Salud Carlos III, Madrid, Spain
  • 6. Clinical Unit of Infectious Diseases and Microbiology, University Hospital Virgen Macarena, Institute of Biomedicine of Sevilla (University Hospital Virgen Macarena/CSIC/University of Sevilla), Sevilla, Spain
  • 7. Microbiology Service, University Hospital Ramón y Cajal and Ramón y Cajal Health Research Institute (IRYCIS), Madrid, Spain, CIBER de Enfermedades Infecciosas (CIBERINFEC), Instituto de Salud Carlos III, Madrid, Spain
  • 8. Microbiology Service, University Hospital Marqués de Valdecilla – IDIVAL, Santander, Spain, CIBER de Enfermedades Infecciosas (CIBERINFEC), Instituto de Salud Carlos III, Madrid, Spain

Description

Carbapenem-resistant pathogens have been recognized as a health concern
as they are both difficult to treat and detect in clinical microbiology laboratories.
Researchers are making great efforts to develop highly specific, sensitive, accurate, and
rapid diagnostic techniques, required to prevent the spread of these microorganisms and
improve the prognosis of patients. In this context, CRISPR-Cas systems are proposed as
promising tools for the development of diagnostic methods due to their high specificity;
the Cas13a endonuclease can discriminate single nucleotide changes and displays collateral
cleavage activity against single-stranded RNA molecules when activated. This technology
is usually combined with isothermal pre-amplification reactions in order to increase its
sensitivity. We have developed a new LAMP-CRISPR-Cas13a-based assay for the detection
of OXA-48 and GES carbapenemases in clinical samples without the need for nucleic acid
purification and concentration. To evaluate the assay, we used 68 OXA-48-like-producing
Klebsiella pneumoniae clinical isolates as well as 64 Enterobacter cloacae complex GES-6,
14 Pseudomonas aeruginosa GES-5, 9 Serratia marcescens GES-6, 5 P. aeruginosa GES-6,
and 3 P. aeruginosa (GES-15, GES-27, and GES-40) and 1 K. pneumoniae GES-2 isolates. The
assay, which takes less than 2 h and costs approximately 10 e per reaction, exhibited
100% specificity and sensitivity (99% confidence interval [CI]) for both OXA-48 and all GES
carbapenemases.

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Journal article: 37466441/PMID (Handle)