Produção e purificação da proteína Cas13a de Leptotrichia wadeii e análise de sua aplicabilidade para detecção de Plasmodium vivax e vírus Zika

Abstract

Infectious diseases remain a major health problem in the world, making a significant part of the morbidity and mortality data. The early recognition of these diseases, through the use of rapid and accurate diagnostic tests, speeds up the treatment of the patient, thus allowing for better clinical care. Malaria and Zika virus fever (ZIKV) are infectious diseases typical of tropical regions of the world, such as the Brazilian Amazon. Traditional tools for identifying these diseases have disadvantages that limit detection, such as low specificity and sensitivity. Therefore, the search for new methods capable of overcoming these difficulties has been pursued by many public and private research groups. In this field, the CRISPR-Cas system has been gaining prominence in the last 5 years, contributing to important advances in the development of techniques for the accurate detection of molecular targets. The general objective of this work was to perform the expression and purification of the protein of the CRISPR-Cas13a system, called LwCas13a, as well as to validate its enzymatic activity using tools for the diagnosis of malaria caused by Plasmodium vivax and for Zika virus. As a result, we successfully obtained the expression and purification of the recombinant protein LwCas13a, production of CRISPR RNA (crRNA) and synthetic target RNA (ssRNA) specific for Plasmodium vivax and ZIKV. Which we observed reactivity of the enzyme LwCas31a with an increase in fluorescence intensity as the ssRNA concentrations in the system increased. An inversely proportional relationship was observed when dsDNA was used. Clinical samples from patients infected with ZIKV showed positive reactivity using the method described. No satisfactory results were obtained for the diagnosis of P. vivax malaria using the tools produced. Future work will apply the tools produced to a larger sample of patients infected by ZIKV and new molecular targets that allow efficient detection of Plasmodium vivax using the CRISPRCas13a system.