CRISPR-Cas12 Application for the Detection of Pneumocystis jirovecii in Immunodepression Patients Through Fluorescent and Lateral Flow Colorimetric Assay

Pneumonia caused by <i>Pneumocystis jirovecii</i> poses a serious threat, particularly to immunocompromised patients such as those with HIV/AIDS, transplant recipients, or individuals undergoing chemotherapy. Its diagnosis is challenging because current methods, such as microscopy and certain molecular tests, have limitations in sensitivity and specificity, and require specialized equipment, which delays treatment initiation. In this context, CRISPR-Cas12-based methods offer a promising alternative: they are rapid, highly specific, sensitive, and low-cost, enabling more timely and accessible detection, even in resource-limited settings. We developed a simple and rapid detection platform based on the CRISPR-Cas12 coupled with lateral flow strips. A guide RNA was designed against <i>DHPS</i>, <i>β-tubulin</i>, and <i>mtLSU rRNA</i> genes. The guide corresponding to <i>β-tubulin</i> showed high sensitivity in the detection of <i>P. jirovecii</i> to produce a detectable fluorescence signal within the first 20-30 min. In addition, it demonstrated high specificity for <i>P. jirovecii</i> when DNA from other microorganisms was used. When coupled with lateral flow strips, high sensitivity and specificity were also observed for detecting positive samples, without the need for genetic amplification. CRISPR-Cas12 successfully detected <i>P. jirovecii</i> infection in an initial diagnostic application, demonstrating the potential of this method for integration into public health diagnostic systems, particularly in field, due to its adaptability, speed, and ease of use.