Standardization of SHERLOCK technique (Specific High Sensitivity Enzymatic Reporter Unlocking) using the Cas13a protein for the diagnosis of Respiratory Syncytial Virus in pediatric patients

Author(s)

Camila Pereira Soares

• Document type: Master's Dissertation
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Instituto de Ciências Biomédicas (ICB/SDI)
• Defense date: 2023-06-06

Examining board members

Edison Luiz Durigon; Flavia Jacqueline Almeida; Jansen de Araujo; João Renato Rebello Pinho

• Advisor: Edison Luiz Durigon

Abstract

RSV is a widely distributed virus worldwide and is the main virus involved in lower respiratory tract infections. The main population at risk for these infections are children under 5 years of age. In this context, laboratory diagnostic methods are extremely important due to the similarity between the symptoms caused by RSV infection and other respiratory viruses. This work proposed to use the enzyme LwCas13a as a detector of RSV in samples from pediatric patients based on the SHERLOCK technique. Recombinant protein was obtained using affinity chromatography and gel filtration for purification. The target RNA was designed based on a conserved region in the RSV M gene. In clinical samples previously amplified by conventional PCR, 100% were detected by the enzyme and in samples without prior amplification 45.3% were detected. There was no cross-reaction in any of the tests with other viruses of respiratory importance. During the Covid-19 pandemic in 2020 and 2021 we produced an ELISA test to detect IgG/IgA/IgM antibodies against two recombinant nucleoproteins, one commercial and one produced in house. The test was standardized against the gold standard viral neutralization assay (VNT100 ). For all three immunoglobulins we obtained values above 88% sensitivity and 94% specificity. The assay was used in the screening of convalescent plasma donors and recipients, in the diagnosis provided to 7 public hospitals in the city of São Paulo. In addition, we have standardized an ELISA assay for IgG using dried capillary blood collected on filter paper. To facilitate sample collection in a pandemic scenario. (AU)

• FAPESP's process: 18/23680-0 - Standardization of Sherlock technique (Specific High-Sensitivity Enzymatic Reporter Unlocking) using the Cas13a protein for the diagnosis of respiratory syncytial virus in pediatric patients
• Grantee: Camila Pereira Soares
• Support Opportunities: Scholarships in Brazil - Master