Optimization of a novel dengue diagnostic method based on CRISPR-Cas assays

VICENTE JOSEFINA; FAY JESSICA; BOAGLIO M. VICTORIA; IBAÑEZ DAIANA; ESPINDOLA SONIA; TABARES EDIT; CACERES GABRIELA; FERRRERAS JULIAN; MIRETTI MARCOS

Seattle

Jornada; American Society of Tropical Medicine and Hygiene 2022 Annual Meeting; 2022

American Society of Tropical Medicine and Hygiene

Dengue is major arthropod-borne viral disease in humans caused by dengue virus (DENV, Flaviridae) inflicting a vast public health burden. Prevention and mitigation of dengue outbreaks rely on vector control and effective epidemiological surveillance aiming early viral circulation detection using timely and affordable diagnostic tools. Reverse transcriptase real time PCR (RT qPCR) is the gold standard method for DENV identification and serotyping, however, it requires equipment and infrastructure mostly unavailable at the point of care in resource limited endemic urban districts. CRISPR-Cas method emerged as a proven low cost and portable diagnostic tool in viral diseases. We have been working in the proof of concept of a CRISPR-Cas method for dengue diagnostics, as reported previously. In this work, we aimed to evaluate its performance in an increased sample size and by reducing protocol complexity and costs. DENV presence in RNA extracted from febrile patients’ blood samples was initially assayed by RT-qPCR. cDNA from these samples was then amplified by PCR and RPA (Recombinase Polymerase Amplification) and mixedwith dengue specific sgRNA-CRISPR-Cas12 assembly. Dengue infected samples were detected by measuring the fluorescent signal released by the enzyme nuclease collateral activity using single strand oligo reporters. In the comparative analyses we assessed sensitivity, specificity and theKappa concordance index. Results from processing 98 samples with both, RT-qPCR and CRISPR-Cas12 protocols, showed 81.6% concordance, 70% CRISPR-Cas12 assay sensitivity and 97.5% specificity. The kappa index for detection (0.64) indicates moderate agreement between tests. Our results indicate that the CRISPR based diagnostic tool evaluated and improved in this work is highly specific and potentially portable. This initial evaluation allowed us to identify sensitive steps in the original protocol incorporating key solutions. Further experimental work is necessary to increase sensitivity, to determine limit of detection and to achieve complete portability required for the implementation of this assay in early diagnostic surveillance.