Establishment of an RPA-CRISPR/Cas12a combined diagnostic system for Pneumocystis jirovecii pneumonia

Area of Science:

• Molecular diagnostics
• Microbiology
• Immunocompromised patient care

Background:

• Pneumocystis jirovecii pneumonia (PJP) presents a significant mortality risk and economic burden, particularly in immunocompromised individuals.
• Existing diagnostic methods often lack the speed and accessibility required for early detection, especially in resource-limited environments.
• There is a critical need for innovative, point-of-care diagnostic solutions for PJP.

Purpose of the Study:

• To develop and validate a novel, highly sensitive, and rapid diagnostic method for P. jirovecii.
• To create a point-of-care assay suitable for low-resource settings.
• To target the mitochondrial small subunit rRNA (mtSSU rRNA) gene of P. jirovecii for detection.

Main Methods:

• Development of Recombinase Polymerase Amplification (RPA) primers and CRISPR-derived RNAs (crRNAs) targeting the P. jirovecii mtSSU rRNA gene.
• Integration of RPA with CRISPR/Cas12a technology for enhanced detection sensitivity.
• Fluorescence-based detection and validation using 83 clinical samples (39 confirmed, 44 suspected P. jirovecii cases).

Main Results:

• The developed RPA-CRISPR/Cas12a method achieved a limit of detection (LOD) of 1 copy per reaction.
• Sensitivity was superior to current real-time PCR methods when using the combination of crRNA5 and crRNA6.
• The assay demonstrated high specificity, with no cross-reactivity observed against other common respiratory pathogens.

Conclusions:

• The novel RPA-CRISPR/Cas12a assay provides a highly sensitive and rapid tool for early and on-site detection of P. jirovecii pneumonia.
• This diagnostic approach is well-suited for resource-limited settings, addressing a critical unmet need.
• The platform shows promise for broader applications in detecting other human pathogens, enhancing global diagnostic capabilities.