RT-LAMPとCRISPR-Cas12bベースの戦略を組み合わせたHCV RNA検出のための迅速アッセイ

Hepatitis C virus (HCV) diagnosis usually requires detection of antibody followed by HCV RNA. The requirement for two tests leads to major drop-offs in the cascade of care. Existing near-care HCV RNA tests have slow turnaround time and are expensive with limited availability. We aim to develop a cost-effective, rapid and sensitive test for detection of HCV RNA to enhance screening, particularly in marginalized and remote populations.

After RNA extraction from plasma, HCV RNA is reverse transcribed and amplified using loop-mediated isothermal amplification (RT-LAMP) with HCV-specific primers. The amplified HCV DNA is then detected via CRISPR-Cas12b with a fluorescence readout.

HCV RNA from patient samples with genotypes 1a, 1b, 2, 3a and 4 was detected with high sensitivity and specificity. The lower limit of detection (LLOD) with HCV JFH1 plasmid (genotype 2) is 250 plasmid copies/ml (∼100 IU/ml). For clinical samples, we determined the LLOD for genotypes 1 and 3, the most common in North America. Using 500 μl plasma, genotype 1 RNA ≥100 IU/ml was detected within 40-45 min, while genotype 3 had an LLOD of 5,000 IU/ml. Clinical sensitivity was 100% in 72 HCV patient samples, including acute HCV and HCV/HBV co-infection. Specificity was 100%, with no false positives in 33 HCV-negative samples, including those with HBV or HIV/HBV co-infection.

Our assay shows high specificity and sensitivity to detect HCV RNA directly from plasma within 45 min, and hence could be utilized for efficient screening and diagnosis of HCV infection globally.