A rapid CRISPR/Cas12a-based assay for the detection of HIV-1 Indian Clade-C infections

Early detection of HIV-1 infection is crucial to initiate anti-retroviral therapy (ART) to suppress viremia and disease progression. Herein, we developed a CRISPR/Cas12a-based HIV-1 detection assay by optimizing components for a coupled isothermal preamplification by recombinase polymerase amplification (RPA). The HIV-1 Indian Clade-C-specific conserved pol region was targeted by crRNA designed for Clade-specific detection. The CRISPR/Cas12a cleavage of the viral cDNA input is displayed as a single visually detectable outcome due to the collateral cleavage of the ssDNA-FAM-BQ reporter, enabling the rapid detection of HIV-1. The performance of the assay was evaluated by testing sera of 41 Indian Clade C HIV-1 seropositive individuals, which included 28 HIV-1 infected infant samples, HIV-1 Indian clade C genome plasmid, viral disease control DNA/RNA samples (Influenza, RSV, Parvovirus, HPIV, CMV, and HBV), and 31 healthy donor sera samples. With 96% sensitivity and 92.65% specificity for HIV-1C detection, with fluorescence and visual readout, and a capability of detection using lateral flow dipsticks, our CRISPR/Cas12a-based HIV-1 C detection assay demonstrates the potential to be developed into a robust point-of-care molecular diagnostic test for HIV-1C. Moreover, it may serve as a potential rapid NAT alternative in detecting mother-to-child transmission (MCT) of HIV-1C in infants (<2 years of age), where rapid antibody-based serology tests are rendered ineffective due to the presence of maternal antibodies.