An experimental approach for diagnosing cercosporosis using RPA+CRISPR/Cas12a

Abstract

The article demonstrates the prospects for using a new experimental approach for the detection of the fungus Cercospora Sojina Hara. The main principle underlying the presented method is a combination of two technologies: RPA (Recombinase polymerase amplification) and CRISPR/Cas12a. RPA - is an alternative to classical PCR, with features in the form of a faster reaction rate and its passage under isothermal conditions. Using RPA technology will reduce amplification to 15-30 minutes. Amplified genomic DNA can be detected fluorescently labeled with CRISPR/Cas12a. The difficulties of this method lie in the selection of specific primers and the selection of spacers for the guide RNA within the amplicon. As a result of the work carried out, using the primer3 plugin on the Unipro Ugene platform, it was possible to select a specific pair of primers that would make it possible to identify this particular phytopathogen. The genome spacer was identified in the ChopChop web toolkit. The resulting primer pair and spacer having complementarity exclusively to the CP036215 locus in Cercospora Sojina H.