Construction of an ultrasensitive biosensor for CRISPR-Cas12-based diagnosis for SARS-CoV-2 and variants detection without nucleic acid amplification

Abstract

The real-time quantitative PCR (qPCR) methodology has become, in recent years, the reference diagnostic technology for various infections, including the current COVID-19 pandemic. However, dependence on specialized equipment and highly trained personnel restrict this method to centralized laboratories. Alternative methods of amplifying and detecting nucleic acids, based on isothermal amplification technologies (such as LAMP, NASBA, and RPA), have emerged in current years as alternatives for decentralizing diagnostic methods based on amplification and detection of nucleic acids. However, the detection specificity of amplified products still represents a challenge for these methodologies. Diagnostic techniques based on CRISPR (short grouped and regularly interspaced palindromic repetitions) have emerged as an alternative to traditional nucleic acid identification methods, as they attempt to deliver the minimum qualifications for point-of-care settings. Nevertheless, those tests either still depend on DNA amplification methods or have low sensitivity, which prevents their utilization in real-world applications. This project aims to develop a biosensor technology based on CRISPR-Cas12, combining different strategies to increase test sensitivity without DNA amplification. This strategy seeks to enhance the test signal in three ways: 1) the use of gold nanoparticles to amplify the fluorescent signal emitted as a result of the collateral cleavage of the fluorescent substrates; 2) apply magnetic microspheres with a high capacity for binding and distributing those targets; 3) the use of multiple sgRNA that will be directed to different fragments in the same target, to increase the sensitivity of target recognition. Thus, it is expected that such a test will be more sensitive and have an improved detection limit than those already developed so far. It is also expected that the test will have high accuracy when compared to real-time PCR. (AU)