Diagnostic method for SARS-CoV-2 based on Ribozymes and DNA beacon

The present proposal aims to develop a fast, low cost and easy detection method for the diagnosis of SARS-CoV-2. The method consists of sampling fluids from the upper airways, RNA extraction, reaction and fluorescence detection. The reaction and detection strategy based on three main steps. The first consists of the recognition of the viral RNA molecule by two hammerhead ribozymes. The target regions of activity for each of the two ribozymes will be conserved and specific SARS-CoV-2 sequences obtained after genomic alignment of the different viral strains deposited in a specific database and comparisons with genetic material from other SARS and human viruses. This recognition will lead to the breakdown of the viral RNA molecule and obtaining a fragment called a signal primer. The second step consists of the connection of the initiator molecule to a reporter DNA beacon, which has a clamp-like structure with a fluorophore at one end and a quencher molecule at the other. When the DNA beacon is in the form of a clamp, the fluorophore does not emit fluorescence, however when this molecule interacts with the initiator fragment, the clamp opens, distancing the quencher from the fluorophore and in this molecular configuration will allow the loss of the quenching effect resulting in fluorescence of that DNA reporter. The third stage provides for an increase in stoichiometric fluorescence 1: 1 by inducing a cascade of DNA beacons, so that the signal will increase as a result of the fluorescence of several multiplexed reporter molecules. which has a clamp-like structure with a fluorophore at one end and a quencher molecule at the other. When the DNA beacon is in the form of a clamp, the fluorophore does not emit fluorescence, however when this molecule interacts with the initiator fragment, the clamp opens, distancing the quencher from the fluorophore and in this molecular configuration will allow the loss of the quenching effect resulting in fluorescence of that DNA reporter. The third stage provides for an increase in stoichiometric fluorescence 1: 1 by inducing a cascade of DNA beacons, so that the signal will increase as a result of the fluorescence of several multiplexed reporter molecules. which has a clamp-like structure with a fluorophore at one end and a quencher molecule at the other. When the DNA beacon is in the form of a clamp, the fluorophore does not emit fluorescence, however when this molecule interacts with the initiator fragment, the clamp opens, distancing the quencher from the fluorophore and in this molecular configuration will allow the loss of the quenching effect resulting in fluorescence of that DNA reporter. The third stage provides for an increase in stoichiometric fluorescence 1: 1 by inducing a cascade of DNA beacons, so that the signal will increase as a result of the fluorescence of several multiplexed reporter molecules. When the DNA beacon is in the form of a clamp, the fluorophore does not emit fluorescence, however when this molecule interacts with the initiator fragment, the clamp opens, distancing the quencher from the fluorophore and in this molecular configuration will allow the loss of the quenching effect resulting in fluorescence of that DNA reporter. The third stage provides for an increase in stoichiometric fluorescence 1: 1 by inducing a cascade of DNA beacons, so that the signal will increase as a result of the fluorescence of several multiplexed reporter molecules. When the DNA beacon is in the form of a clamp, the fluorophore does not emit fluorescence, however when this molecule interacts with the initiator fragment, the clamp opens, distancing the quencher from the fluorophore and in this molecular configuration will allow the loss of the quenching effect resulting in fluorescence of that DNA reporter. The third stage provides for an increase in stoichiometric fluorescence 1: 1 by inducing a cascade of DNA beacons, so that the signal will increase as a result of the fluorescence of several multiplexed reporter molecules.