Improved protocol for Bst polymerase and reverse transcriptase production and application to a point-of-care diagnostics system

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de Souza, Lucas Rodrigo ; Poly da Silva, Italo Esposti ; Celis-Silva, Gabriele ; Raddatz, Bruna Winkert ; Imamura, Louise Matie ; Kim, Edson Yu Sin ; Valderrama, Gabriel Vieira ; Riedi, Halanna de Paula ; Rogal Jr, Sergio Renato ; Machado de Almeida, Bernardo Montesanti ; Mazega Figueredo, Marcus Vinicius ; Bengtson, Mario Henrique ; Massirer, Katlin Brauer

• Tipo de documento: Artigo Científico
• Fonte: Experimental Biology and Medicine; v. 248, n. 19, p. 13-pg., 2023-12-13.

Resumo

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has raised awareness in the scientific community about the importance of being prepared for sanitary emergencies. Many measures implemented during the COVID pandemic are now being expanded to other applications. In the field of molecular and immunological diagnostics, the need to massively test the population worldwide resulted in the application of a variety of methods to detect viral infection. Besides gold standard reverse transcription quantitative polymerase chain reaction (RT-qPCR), the use of reverse transcription loop-mediated isothermal amplification (RT-LAMP) arose as an alternative and sensitive method to amplify and detect viral genetic material. We have used openly available protocols and have improved the protein production of RT-LAMP enzymes Bst polymerase and HIV-reverse transcriptase. To optimize enzyme production, we tested different protein tags, and we shortened the protein purification protocol, resulting in reduced processing time and handling of the enzymes and, thus, preserved the protein activity with high purity. The enzymes showed significant stability at 4 degrees C and 25 degrees C, over 60 days, and were highly reliable when used as a one-step RT-LAMP reaction in a portable point-of-care device with clinical samples. The enzymes and the reaction setup can be further expanded to detect other infectious diseases agents. (AU)

• Processo FAPESP: 20/16094-8 - Caracterização estrutural de proteínas de ligação a RNA e varredura de bibliotecas de fragmentos químicos por LC-MS cromatografia líquida integrada com espectrometria de massas
• Beneficiário: Lucas Rodrigo de Souza
• Modalidade de apoio: Bolsas no Brasil - Pós-Doutorado
• Processo FAPESP: 20/02006-0 - Caracterização molecular e estrutural de proteínas de ligação a RNA como alvos terapêuticos em câncer e neurologia
• Beneficiário: Katlin Brauer Massirer
• Modalidade de apoio: Auxílio à Pesquisa - Parceria para Inovação Tecnológica - PITE
• Processo FAPESP: 14/50897-0 - INCT 2014: Centro de Química Medicinal de Acesso Aberto
• Beneficiário: Katlin Brauer Massirer
• Modalidade de apoio: Auxílio à Pesquisa - Temático
• Processo FAPESP: 19/11320-2 - Investigação celular de proteínas quinases envolvidas com splicing, por sistema de minigene
• Beneficiário: Gabriel Vieira Valderrama
• Modalidade de apoio: Bolsas no Brasil - Iniciação Científica