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Master's Dissertation
DOI
https://doi.org/10.11606/D.42.2023.tde-07062024-145303
Document
Author
Full name
Camila Pereira Soares
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2023
Supervisor
Committee
Durigon, Edison Luiz (President)
Almeida, Flavia Jacqueline
Araujo, Jansen de
Pinho, João Renato Rebello
Title in Portuguese
Padronização da técnica SHERLOCK (Specific High-Sensitivity Enzymatic Reporter Unlocking) utilizando a proteína Cas13a para diagnóstico do Vírus Respiratório Sincicial em pacientes pediátricos
Keywords in Portuguese
CRISPR-Cas
Diagnóstico
ELISA
RSV
SARS-CoV-2
Abstract in Portuguese
O RSV é um vírus amplamente distribuído no mundo e é o principal vírus envolvido em infecções respiratórias do trato inferior. A principal população de risco dessas infecções são crianças menores de 5 anos. Nesse contexto, os métodos de diagnósticos laboratoriais se apresentam extremamente importantes devido a similaridade entre os sintomas causados pela infecção do RSV com outros vírus respiratórios. Esse trabalho propôs utilizar a enzima LwCas13a como detectora do RSV em amostras de pacientes pediátricos tendo como base a técnica SHERLOCK. A proteína recombinante foi obtida utilizando cromatografia de afinidade e gel filtração para purificação. O RNA alvo foi desenhado com base em uma região conservada no gene M do RSV. Em amostras clínicas previamente amplificadas por PCR convencional, 100% foram detectadas pela enzima e em amostras sem amplificação prévia 45,3% foram detectadas. Não houve reação cruzada em nenhum dos testes com outros vírus de importância respiratória. Durante o enfrentamento à Covid-19 em 2020 e 2021, produzimos um teste de ELISA para detectar anticorpos IgG/IgA/IgM contra duas nucleoproteínas recombinantes, uma comercial e uma produzida in house. Os teste foi padronizado em comparação ao ensaio padrão ouro de neutralização viral (VNT100 ). Para todas as três imunoglobulinas obtivemos valores acima de 88% de sensibilidade e 94% de especificidade. O ensaio foi empregado na triagem de doadores e receptores de plasma convalescente, no diagnóstico fornecido para 7 hospitais público da cidade de São Paulo. Além disso, padronizamos um ensaio ELISA para IgG utilizando sangue capilar seco coletado em papel de filtro. Na tentativa de facilitar a coleta de amostras em um cenário de pandemia.
Title in English
Standardization of SHERLOCK technique (Specific High Sensitivity Enzymatic Reporter Unlocking) using the Cas13a protein for the diagnosis of Respiratory Syncytial Virus in pediatric patients
Keywords in English
CRISPR-Cas
Diagnosis
ELISA
RSV
SARS-CoV-2
Abstract in English
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.
 
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Release Date
2026-06-07
Publishing Date
2024-06-11
 
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