Recombinase amplified CRISPR enhanced chain reaction (RACECAR) for viral genome detection

Abstract

We have developed a ‘recombinase amplified CRISPR enhanced chain reaction’ (RACECAR) assay that can detect as little as 40 copies of hepatitis B virus (HBV) genome using a benchtop spectrofluorometer. The limit of detection was determined to be 3 copies of HBV genome. The specificity of RACECAR was confirmed against hepatitis A virus (HAV). This assay can detect the genomic targets directly in serum samples without an extraction step. The 4 h-long fluorometric assay was developed by combining three tiers of isothermal amplification processes and can be repurposed for any target of choice. This highly modular reaction setup is an untapped resource that can be incorporated into the front-runners of molecular diagnostics.