Development and Validation of a CRISPR-Cas12-Based Diagnostic Platform for Rapid Detection of Carbapenemase Encoding Genes

• Grant number: 24/13835-8
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate
• Start date: November 01, 2024
• End date: October 31, 2025
• Field of knowledge: Biological Sciences - Microbiology - Applied Microbiology
• Principal Investigator: Ana Cristina Gales
• Grantee: André Valêncio Siqueira
• Supervisor: Paulo Jose Martins Bispo
• Host Institution: Escola Paulista de Medicina (EPM). Universidade Federal de São Paulo (UNIFESP). Campus São Paulo. São Paulo , SP, Brazil
• Institution abroad: Harvard University, Boston, United States
• Associated to the scholarship: 23/12960-0 - Carbapenemase Detection in Clinical Isolates: Development of Agglutination Tests as a Diagnostic Alternative, BP.DR

Abstract

Klebsiella pneumoniae carbapenemase (KPC) and New Delhi Metallo-beta-lactamase (NDM) genes are frequently identified across diverse geographical regions. Accurate detection and differentiation of KPC and NDM carbapenemases are crucial for directing appropriate antimicrobial treatment. Lateral flow assays, which are rapid and straightforward to perform, offer a practical solution in clinical settings. Currently, immunochromatographic tests (lateral flow assays) depend on monoclonal or polyclonal antibodies for detection. Although effective, this method increases production costs and restricts accessibility in low-income countries. The CRISPR-Cas detection system, noted for its specificity and high sensitivity, may enable the accurate identification of genes responsible for carbapenemase production. Consequently, our objective is to develop, standardize, and validate a cost-effective lateral flow test that could differentiate between blaNDM and blaKPC genes. This will involve designing specific crRNA sequences for these targets and developing specialized primers for gene amplification through recombinase polymerase amplification (RPA). We will test a variety of carbapenemase-producing isolates to determine the test's sensitivity, specificity, and turnaround time. By leveraging the results of these studies, we intend to standardize a rapid and innovative assay for the detection and differentiation of carbapenemases, focusing on carbapenemases of clinical importance in Brazil and worldwide. Thus, these assays could be further employed for development of a diagnostic kit to be used in low resource settings.