Amplicon-depleted crispr/cas-regulated isothermal molecular assay at skin temperature enabling multi-level, equipment-free, home-based self-testing of viral infections

A method of amplicon-depleted CRISPR/Cas-regulated isothermal molecular assay at skin temperature enabling multi-level, equipment-free, home-based self-testing of viral infections, the method comprising:

• a) obtaining a sample comprising a target nucleic acid sequence containing target region for primer binding;
• b) hybridizing a forward Recombinase Polymerase Loop-mediated Amplification (RPLA) primer to the target region, wherein the forward RPLA primer comprises a complementary sequence to the target region and a protospacer adjacent motif (PAM) code sequence downstream of the complementary sequence;
• c) hybridizing a backward RPLA primer to the primer target region of the displaced sequence;
• d) extending the backward RPLA primer to yield a dumbbell sequence containing a loop structure;
• e) extending the forward and backward RPLA primers to yield a displaced sequence in the loop containing amplicon, wherein the displaced sequence contains the sgRNA binding region;
• f) amplifying the dumbbell sequence by CRISPR/Cas activation that specifically recognizes single guide RNA (sgRNA) binding regions using cis cleavage activity;
• and
• g) cleaving the surrounding reporters by collateral cleavage activity right after activation.