Development of a Fast and Affordable Diagnostic System for RNA Viruses Using Loop-Mediated Isothermal Amplification and DNA-Nanoprobe Detection

Cea-Callejo, Pablo^1,2†; Arca-Lafuente, Sonia¹†; Gomez-Lucia, Esperanza^2,3; Doménech, Ana^2,3, Biarnés, Mar⁴; Blanco, Angela⁴; Benítez, Laura^2,5ǂ and Madrid, Ricardo^1,2,5ǂ.

¹BioAssays SL. Parque Científico de Madrid, Campus de Cantoblanco, Madrid, Spain

²Research Group of “Animal Viruses” of Complutense University of Madrid

³Deparment of Animal Health, Veterinary Faculty, Complutense University of Madrid (UCM), Spain

⁴Centro de Sanidad Avícola de Cataluña y Aragón (CESAC)

⁵Department of Genetics, Physiology, and Microbiology, School of Biology, Complutense University of Madrid (UCM), Spain

†These authors contributed equally to this work and share first authorship

ǂ These authors contributed equally to this work and share last authorship

* Correspondence: Ricardo Madrid González (rimadrid@ucm.com), Laura Benítez Rico (lbenitez@ucm.es)

Keywords: molecular detection, RT-LAMP, nanoprobes, SARS-CoV-2, aMPV, POC-test.

Abstract

Airbone viral pathogens can rapidly spread and become a global menace, including both human and animal viruses which can trigger important socioeconomical and health effects. Therefore, to prevent and contain potential epidemic outbreaks, accurate, fast, and affordable diagnostic point-of-care tests (POCT) are required. As a proof of concept, we have developed a molecular detection system based in Loop-mediated isothermal AMPlification technique (LAMP) for two different RNA airborne viruses: the well-known human SARS-CoV-2, and the avian metapneumovirus (aMPV), the aetiologic agent of a communicable disease infecting mainly turkeys and chickens. To obtain a POC diagnostic system, we coupled the LAMP technique to DNA-functionalized gold nanoparticles detection. Validation of this system was carried out in 140 pharyngeal swabs from COVID-19 symptomatic and asymptomatic patients, and in 50 different samples (pharyngeal swabs and tracheal tissue samples) collected from aMPV infected chickens and turkeys. The system allows viral detection in about 60 minutes by the naked eye with 100% specificity, and 97.22% and 87.88% sensitivity for SARS-CoV-2 and aMPV, respectively. In summary, this novel detection system based on the coupling of RT-LAMP to DNA-nanoprobes allows suitable virus testing in the field, with accurate levels very close to conventional qRT-PCR based diagnosis.