optimization of a novel dengue diagnostic method based on cripsr-cas assays

VICENTE J.; FAY J.; BOAGLIO V.; IBAÑEZ-ALEGRE, DAIANA; ESPINDOLA S.; TABARES E; CACERES G; FERRERAS J.; MIRETTI M.

Seattle

Jornada; ANNUAL MEETING 2022; 2022

american society of tropical medicin and hygiene

Dengue is major arthropod-borne viral disease in humans caused bydengue virus (DENV, Flaviridae) inflicting a vast public health burden.Prevention and mitigation of dengue outbreaks rely on vector controland effective epidemiological surveillance aiming early viral circulation429astmh.orgdetection using timely and affordable diagnostic tools. Reversetranscriptase real time PCR (RT qPCR) is the gold standard method forDENV identification and serotyping, however, it requires equipment andinfrastructure mostly unavailable at the point of care in resource limitedendemic urban districts. CRISPR-Cas method emerged as a proven lowcost and portable diagnostic tool in viral diseases. We have been workingin the proof of concept of a CRISPR-Cas method for dengue diagnostics,as reported previously. In this work, we aimed to evaluate its performancein an increased sample size and by reducing protocol complexity and costs.DENV presence in RNA extracted from febrile patients’ blood samples wasinitially assayed by RT-qPCR. cDNA from these samples was then amplifiedby PCR and RPA (Recombinase Polymerase Amplification) and mixedwith dengue specific sgRNA-CRISPR-Cas12 assembly. Dengue infectedsamples were detected by measuring the fluorescent signal released bythe 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 fordetection (0.64) indicates moderate agreement between tests. Our resultsindicate that the CRISPR based diagnostic tool evaluated and improved inthis work is highly specific and potentially portable. This initial evaluationallowed us to identify sensitive steps in the original protocol incorporatingkey solutions. Further experimental work is necessary to increasesensitivity, to determine limit of detection and to achieve completeportability required for the implementation of this assay in early diagnosticsurveillance.