Development of simple and rapid detection of Hepatitis C virus using RT-Lamp-Coupled Crispr-Cas12 assay

Abstract

Hepatitis C virus (HCV) infection can be recently cured with pan-genotypic direct-acting antiviral agents. However, identifying individuals with current hepatitis C remains a major challenge in resource-limited settings where access to or availability of molecular test is still limited. We developed a molecular assay that allows rapid detection of HCV RNA without the requirements for sophisticated and expensive equipment. It is based on a combination of reverse transcription loop-mediated isothermal amplification (RTLAMP) method with the clustered regularly interspaced short palindromic repeats CRISPR associated protein 12a (CRISPR-Cas12a) cleavage assay that allows a recognition of specific HCV nucleic acid sequence. Products obtained from cleavage reactions can be visualized on lateral flow strips or can be measured with a fluorescence detector. When tested on clinical samples from individuals infected with HCV, HIV or HBV or from healthy donors, the RT-LAMP-CRISPR-Cas12 assay yielded 96% sensitivity, 100% specificity and 97% agreement as compared to the reference method (Roche COBAS AmpliPrep/COBAS TaqMan HCV Test). This assay could detect HCV RNA concentration as low as 10 ng/µL. Therefore, this sensitive and specific assay may represent a promising, affordable and reliable point-of-care test for the identification of individuals with active hepatitis C in low-resource settings.