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Standardization of Sherlock technique (Specific High-Sensitivity Enzymatic Reporter Unlocking) using the Cas13a protein for the diagnosis of respiratory syncytial virus in pediatric patients

Grant number: 18/23680-0
Support type:Scholarships in Brazil - Master
Effective date (Start): April 01, 2019
Effective date (End): November 30, 2020
Field of knowledge:Biological Sciences - Microbiology - Applied Microbiology
Principal researcher:Edison Luiz Durigon
Grantee:Camila Pereira Soares
Home Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

RSV is a virus widely distributed in the world and is the main virus involved in lower respiratory tract infections. The population at risk for these infections are children under 5 years of age, immunocompromised patients and the elderly. The RSV belongs to the family Pneumoviridae, its genome is composed of a single stranded RNA molecule and encodes 11 proteins. Divided into 2 antigenic groups based on G (A and B) genotyping, it has several well characterized genotypes within each group. The similarity between the symptoms caused by RSV infection with other respiratory viruses impairs the accurate clinical diagnosis. In this context, laboratory diagnostic methods are extremely important. Firstly, because they prevent the common practice of antibiotic prescribing and because patient isolation measures can be applied by decreasing the degree of viral spread. Some novelties have arisen in recent years regarding molecular diagnosis, however, one has been presented in a very promising way: the CRISPR-Cas technique. The technique that uses the Cas9 bacterial endonuclease to edit DNA sequences has already been widely used, and now new proteins have started competing in the spotlight. Two articles published in early 2018 showed the success of two techniques that used proteins like Cas13 and Cas12 in the diagnosis of viruses such as Zika and HPV. The SHERLOCK technique (Specific High-Sensitivity Enzymatic Reporter Unlocking) was able to detect high sensitivity and specificity of viral RNA samples in a few minutes using the Cas13a protein. A characteristic of this technique that makes it simple and fast is the use of the RPA (Reaction Polymerase Amplification) technique as a method of amplification of the genetic material that, unlike PCR, is isothermal, and does not require the use of thermocycling devices. Our objective in this work is to standardize the SHERLOCK technique, using the Leptrotricia wadeii Cas13a protein to detect Human Respiratory Syncytial Virus in samples of children with acute respiratory disease and compare with the standard technique of Real Time PCR

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Scientific publications (7)
(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)
WENDEL, SILVANO; FACHINI, ROBERTA; FONTAO-WENDEL, RITA C. L.; MELLO, RALYRIA; VELASQUEZ, CARLOS VINICIUS; GUARAGNA MACHADO, RAFAEL RAHAL; BRITO, MAYRA ALTOBELLI; AMARAL, MARCELO; SOARES, CAMILA PEREIRA; ACHKAR, RUTH; SCURACCHIO, PATRICIA; MIYAJI, SANDRA CRISTINA; ERDENS, MELINA SANTOS; DURIGON, EDISON LUIZ. urrogate test performance for SARS-CoV-2 neutralizing antibodies (nAbs) for convalescent plasma (CCP): How useful could they be. Transfusion, v. 61, n. 12 OCT 2021. Web of Science Citations: 0.
DE FREITAS DUTRA, VALERIA; BONET-BUB, CAROLINA; YOKOYAMA, ANA PAULA H.; ACHKAR, RUTH; MACHADO, RAFAEL RAHAL GUARAGNA; ASSUNCAO, MURILO; CANDELARIA, GABRIELA; SOARES, CAMILA PEREIRA; FACHINI, ROBERTA MARIA; FONTAO-WENDEL, RITA; HAMERSCHLAK, NELSON; REIS, LUIZ FERNANDO LIMA; ARAUJO, DANIELLE BASTOS; NUDELMAN, VICTOR; PINHO, JOAO R. R.; RIZZO, LUIZ V.; SAKASHITA, ARACI M.; SCURACCHIO, PATRICIA; DURIGON, EDISON LUIZ; WENDEL, SILVANO; KUTNER, JOSE M. Anti-A and SARS-CoV-2: an intriguing association. Vox Sanguinis, v. 116, n. 5 MAR 2021. Web of Science Citations: 1.
BATISTA DOS SANTOS, JULIANA DE MELO; SOARES, CAMILA PEREIRA; MONTEIRO, FERNANDA RODRIGUES; MELLO, RALYRIA; DO AMARAL, JONATAS BUSSADOR; AGUIAR, ANDRESSA SIMOES; SOLEDADE, MARIANA PEREIRA; SUCUPIRA, CAROLINA; DE PAULIS, MILENA; ANDRADE, JULIANA BANNWART; ALMEIDA, FLAVIA JAQUELINE; PALAZZI SAFADI, MARCO AURELIO; MAU, LUCIANA BECKER; BRASIL, JAMILE MENEZES; RAMALHO, THERESA; LOURES, V, FLAVIO; VIEIRA, RODOLFO PAULA; DURIGON, EDISON LUIZ; LEAL DE OLIVEIRA, DANIELLE BRUNA; LACERDA BACHI, ANDRE LUIS. In Nasal Mucosal Secretions, Distinct IFN and IgA Responses Are Found in Severe and Mild SARS-CoV-2 Infection. FRONTIERS IN IMMUNOLOGY, v. 12, FEB 25 2021. Web of Science Citations: 0.
WENDEL, SILVANO; FONTAO-WENDEL, RITA; FACHINI, ROBERTA; CANDELARIA, GABRIELA; SCURACCHIO, PATRICIA; ACHKAR, RUTH; BRITO, MAYRA; REIS, LUIZ FERNANDO; CAMARGO, ANAMARIA; AMANO, MARIANE; MACHADO, RAFAEL; ARAUJO, DANIELLE; SOARES, CAMILA; DURIGON, EDISON. A longitudinal study of convalescent plasma (CCP) donors and correlation of ABO group, initial neutralizing antibodies (nAb), and body mass index (BMI) with nAb and anti-nucleocapsid (NP) SARS-CoV-2 antibody kinetics: Proposals for better quality of CCP collections. Transfusion, v. 61, n. 5 FEB 2021. Web of Science Citations: 4.
WENDEL, SILVANO; KUTNER, JOSE MAURO; MACHADO, RAFAEL; FONTAO-WENDEL, RITA; BUB, CAROLINA; FACHINI, ROBERTA; YOKOYAMA, ANA; CANDELARIA, GABRIELA; SAKASHITA, ARACI; ACHKAR, RUTH; HAMERSCHLAK, NELSON; SCURACCHIO, PATRICIA; AMARAL, MARCELO; DAL BEN, MIRIAN; ARAUJO, DANIELLE; SOARES, CAMILA; CAMARGO, ANAMARIA; KALLAS, ESPER; DURIGON, EDISON; REIS, LUIZ FERNANDO; RIZZO, LUIZ VICENTE. Screening forSARS-CoV-2 antibodies in convalescent plasma in Brazil: Preliminary lessons from a voluntary convalescent donor program. Transfusion, v. 60, n. 12, p. 2938-2951, DEC 2020. Web of Science Citations: 1.
DORLASS, ERICK GUSTAVO; MONTEIRO, CAIRO OLIVEIRA; VIANA, AMANDA OLIVEIRA; SOARES, CAMILA PEREIRA; GUARAGNA MACHADO, RAFAEL RAHAL; THOMAZELLI, LUCIANO MATSUMIYA; ARAUJO, DANIELLE BASTOS; LEAL, FABYANO BRUNO; CANDIDO, ERIKA DONIZETTE; TELEZYNSKI, BRUNA LAROTONDA; VALERIO, CAMILA ARAUJO; CHALUP, VANESSA NASCIMENTO; MELLO, RALYRIA; ALMEIDA, FLAVIA JAQUELINE; AGUIAR, ANDRESSA SIMOES; MOTT BARRIENTOS, ANNA CARLOTTA; SUCUPIRA, CAROLINA; DE PAULIS, MILENA; PALAZZI SAFADI, MARCO AURELIO; BONFIM PRADO SILVA, DANIELLA GREGORIO; MARTINS SODRE, JANAINA JOICE; SOLEDADE, MARIANA PEREIRA; MATOS, SAMANTHA FARIA; FERREIRA, SABRINA RODRIGUES; NUNEZ PINEZ, CELIA MIRANDA; BUONAFINE, CAROLINA PALAMIN; FERREIRA PIERONI, LETICIA NERY; MALTA, FERNANDA MELLO; FERRAZ SANTANA, RUBIA ANITA; SOUZA, ELOISA CORREA; FOCK, RICARDO AMBROSIO; REBELO PINHO, JOAO RENATO; SOUZA FERREIRA, LUIS CARLOS; BOTOSSO, VIVIANE FONGARO; DURIGON, EDISON LUIZ; LEAL OLIVEIRA, DANIELLE BRUNA. Lower cost alternatives for molecular diagnosis of COVID-19: conventional RT-PCR and SYBR Green-based RT-qPCR. Brazilian Journal of Microbiology, v. 51, n. 3 AUG 2020. Web of Science Citations: 0.
DANIELLE BASTOS ARAUJO; RAFAEL RAHAL GUARAGNA MACHADO; DEYVID EMANUEL AMGARTEN; FERNANDA DE MELLO MALTA; GABRIEL GUARANY DE ARAUJO; CAIRO OLIVEIRA MONTEIRO; ERIKA DONIZETTI CANDIDO; CAMILA PEREIRA SOARES; FERNANDO GATTI DE MENEZES; ANA CAROLINA CORNACHIONI PIRES; RÚBIA ANITA FERRAZ SANTANA; AMANDA DE OLIVEIRA VIANA; ERICK DORLASS; LUCIANO THOMAZELLI; LUIS CARLOS DE SOUSA FERREIRA; VIVIANE FONGARO BOTOSSO; CRISTIANE RODRIGUES GUZZO CARVALHO; DANIELLE BRUNA LEAL OLIVEIRA; JOÃO RENATO REBELLO PINHO; EDISON LUIZ DURIGON. SARS-CoV-2 isolation from the first reported patients in Brazil and establishment of a coordinated task network. Memórias do Instituto Oswaldo Cruz, v. 115, p. -, 2020.

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