Desenvolvimento de ensaios moleculares baseados no Sistema CRISPR/Cas13a para detecção de Plasmodium vivax

Abstract

Malaria has been considered a global public health problem in humans for years, due to it being one of the main diseases with the greatest impact on morbidity and mortality in many tropical and subtropical countries. The most used methods for diagnosing malaria are microscopic examination, rapid diagnostic tests and polymerase chain reaction, tools limited by the technical knowledge involved, lower sensitivity and high cost of reagents and equipment. Given this, there is a need to develop tools that facilitate the diagnosis of malaria, specifically the Plasmodium species. vivax, in a faster and simpler way to be carried out at service centers (POC) by health agents. Electrochemical biosensors are inserted in the clinical scenario as promising analytical alternatives for diagnosis in POC. Thus, the central objectives of this study were the development of an electrochemical biosensor based on the CRISPR/Cas13a system, using printed carbon electrodes modified with streptavidin (SPCE/STV), in addition to the detection of clinical samples of P. vivax through the assay fluorescence based on the CRISPR/Cas13a system. The Cas13a protein was expressed and purified, and the crRNA sequence was designed to recognize a fragment of the pvmdr-1 gene. For the development of the electrochemical biosensor, a synthetic ssDNA/ssRNA oligonucleotide with the presence of biotin was designed, which was immobilized on the surface of SPCE/STV for subsequent molecular detection. Electrochemical measurements were conducted by differential pulse voltammetry and cyclic voltammetry. As a result, the CRISPR/Cas13a fluorescence test performed well in a clinical setting. Of the 30 positive samples, 27 tested positive with the method, demonstrating considerable clinical sensitivity. Of the 20 negative samples analyzed, the method did not detect the presence of 19 of them, demonstrating acceptable specificity. Furthermore, the test provided a detection limit of 1,35 pM, presenting good sensitivity. In turn, the biosensor developed using the CRISPR/Cas13a system proved to be specific for the target fragment, with changes in the electrochemical response (increase in current) being recorded after the collateral cleavage of Cas13a on the oligonucleotide immobilized on the electrode surface. On the other hand, in the absence of the target, the voltammetric profile did not change, indicating differences between the positive and negative samples. As promising results presented in this work, CRISPR/Cas13a-based molecular tests may provide an alternative approach to identify P. vivax infections in an easy and timely manner.