Amplificação isotérmica associada a CRISPR-Cas12 para diagnóstico rápido e específico da COVID-19
Data
2023-04-01
Autores
Sousa, Ana Caroline de Castro Nascimento 
Orientadores
Moretti, Nilmar Silvio
Tipo
Dissertação de mestrado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
A pandemia causada pelo SARS-CoV-2 (COVID-19) criou um novo desafio de saúde pública para as autoridades em todo o mundo. O alto custo de testes diagnósticos confiáveis e a dependência de insumos importados para a execução destes testes, impossibilitando assim a testagem massiva da população, torna fundamental o desenvolvimento de novos métodos de diagnóstico rápidos e precisos para o SARS-CoV-2. Entre as estratégias recentemente testadas, e aprovadas pelo U.S Food and Drug Administration (FDA), está o uso da metodologia de LAMP (Loop-Mediated Isothermal Amplification), isolada ou combinada à tecnologia de CRISPR/Cas12. Neste trabalho, através da validação da metodologia de LAMP, verificamos a eficiência de enzimas nacionais parceiras na detecção do gene N, E e RdRp do SARS-CoV-2 nas amostras, utilizando tanto RNA como DNA como molde, demonstrando assim uma performance compatível à dos kits comerciais importados já utilizados. Além disso, visando diminuir os custos para realização destes ensaios, realizamos em nosso laboratório a padronização de todas as etapas de expressão e purificação de enzimas Cas12a provenientes de diferentes espécies de bactérias, assim como a síntese de seus sgRNAs, comparando sua eficiência na detecção do SARS-CoV-2 em ensaios in vitro. Em seguida, validamos os ensaios de detecção do SARS-CoV-2 que utilizam a atividade inespecífica da Cas12a após detecção do alvo de interesse, acoplada a uma molécula fluorescente para confirmação do diagnóstico e determinamos assim a dependência do mecanismo à correta formação do complexo RNP-Cas12a, além de sua especificidade e sensibilidade, na presença de plasmídeos carregando a sequência dos genes de diferentes vírus e utilizando diferentes concentrações de um plasmídeo contendo a sequência do gene N de SARS-CoV-2. Verificamos, adicionalmente, a termoestabilidade das enzimas expressas em nosso laboratório, que mostraram funcionalidade em variadas temperaturas (25-75°C), revelando assim seu potencial para aplicação no campo biotecnológico. Finalmente, realizamos uma prova de conceito desta metodologia, utilizando amostras de pacientes com COVID-19 previamente confirmadas por diagnóstico molecular por qRT-PCR, e demonstramos que o sistema é capaz de detectar com especificidade diferentes tipos de alvo, promovendo um grande aumento no sinal fluorescente nas amostras contendo produtos de LAMP em relação ao controle. Nossos resultados indicam que é possível implementar a metodologia de LAMP-Cas12a utilizando tecnologia 100% nacional, como alternativa as metodologias já descritas, aumentando a sensibilidade e diminuindo o tempo e os custos para o diagnóstico molecular laboratorial do SARS-CoV-2. Esses achados apontam ainda para o seu potencial em abordagens que utilizam o conceito point of care (P.O.C) e que, futuramente, podem se estender ao diagnóstico de outras doenças infecciosas de importância econômica e sanitária.
The pandemic caused by SARSCoV2 (COVID19) has created a new public health challenge for authorities around the world. The high cost of reliable diagnostic tests and the dependence on imported reagents to perform these tests, thus making it impossible to mass test the population, makes it essential to develop new rapid and accurate diagnostic methods for SARSCoV2. Among the strategies recently tested and approved by the U.S Food and Drug Administration (FDA), is the use of the LAMP (LoopMediated Isothermal Amplification) methodology, alone or combined with CRISPR/Cas12 technology. In this work, through the validation of the LAMP methodology, we verified the efficiency of national partner enzymes in the detection of the N, E and RdRp gene of SARSCoV2 in the samples, using both RNA and DNA as a template, thus demonstrating a performance compatible with the of already used imported commercial kits. In addition, in order to further reduce the costs of carrying out these tests, we established in our laboratory all steps of expression and purification of Cas12a enzymes from different species of bacteria, as well as the synthesis of their sgRNAs, comparing their efficiency in detection of SARSCoV2 in vitro assays. Then, we validate the SARSCoV2 detection assays that use the unspecific activity of Cas12a after detection of the target of interest, coupled to a fluorescent molecule to confirm the diagnosis, and thus determine the dependence of the mechanism on the correct formation of the RNP complexCas12a, in addition to its specificity and sensitivity, in the presence of plasmids carrying the sequence of genes from different viruses and using different concentrations of a plasmid containing the N gene sequence of SARSCoV2. We additionally verified the thermostability of the enzymes expressed in our laboratory, which showed functionality in diferente temperatures (2575ºC), thus revealing their potential for application in the biotechnological field. Finally, we performed a proof of concept of this methodology, using samples from patients with COVID19 previously confirmed by molecular diagnosis by qRTPCR, and demonstrated that the system is capable of detecting different types of target with specificity, promoting a large increase in signal fluorescent in the samples containing LAMP products relative to the control. Our results indicate that it is possible to implement the LAMPCas12a methodology using 100% national technology, as an alternative to the methodologies already described, increasing its sensitivity and reducing the time and cost for molecular laboratory diagnosis of SARSCoV2. These findings also demosntrates the potential to use this methods for point of care (P.O.C) diagnosis in the future and to expand the use for the diagnosis of other infectious diseases of economic and health importance.
The pandemic caused by SARSCoV2 (COVID19) has created a new public health challenge for authorities around the world. The high cost of reliable diagnostic tests and the dependence on imported reagents to perform these tests, thus making it impossible to mass test the population, makes it essential to develop new rapid and accurate diagnostic methods for SARSCoV2. Among the strategies recently tested and approved by the U.S Food and Drug Administration (FDA), is the use of the LAMP (LoopMediated Isothermal Amplification) methodology, alone or combined with CRISPR/Cas12 technology. In this work, through the validation of the LAMP methodology, we verified the efficiency of national partner enzymes in the detection of the N, E and RdRp gene of SARSCoV2 in the samples, using both RNA and DNA as a template, thus demonstrating a performance compatible with the of already used imported commercial kits. In addition, in order to further reduce the costs of carrying out these tests, we established in our laboratory all steps of expression and purification of Cas12a enzymes from different species of bacteria, as well as the synthesis of their sgRNAs, comparing their efficiency in detection of SARSCoV2 in vitro assays. Then, we validate the SARSCoV2 detection assays that use the unspecific activity of Cas12a after detection of the target of interest, coupled to a fluorescent molecule to confirm the diagnosis, and thus determine the dependence of the mechanism on the correct formation of the RNP complexCas12a, in addition to its specificity and sensitivity, in the presence of plasmids carrying the sequence of genes from different viruses and using different concentrations of a plasmid containing the N gene sequence of SARSCoV2. We additionally verified the thermostability of the enzymes expressed in our laboratory, which showed functionality in diferente temperatures (2575ºC), thus revealing their potential for application in the biotechnological field. Finally, we performed a proof of concept of this methodology, using samples from patients with COVID19 previously confirmed by molecular diagnosis by qRTPCR, and demonstrated that the system is capable of detecting different types of target with specificity, promoting a large increase in signal fluorescent in the samples containing LAMP products relative to the control. Our results indicate that it is possible to implement the LAMPCas12a methodology using 100% national technology, as an alternative to the methodologies already described, increasing its sensitivity and reducing the time and cost for molecular laboratory diagnosis of SARSCoV2. These findings also demosntrates the potential to use this methods for point of care (P.O.C) diagnosis in the future and to expand the use for the diagnosis of other infectious diseases of economic and health importance.