Highly Specific and Rapid Detection of Hepatitis C Virus Using RT-LAMP-Coupled CRISPR–Cas12 Assay

Abstract

Hepatitis C virus (HCV) infection can be cured with pan-genotypic direct-acting antiviral agents. However, identifying individuals with current hepatitis C remains a major challenge, especially in resource-limited settings where access to or availability of molecular tests is still limited. The goal of this study was to develop and validate a molecular assay for the rapid detection of HCV RNA in resource-limited settings. It is based on a combination of reverse transcription loop-mediated isothermal amplification (RT-LAMP) with the clustered regularly interspaced short palindromic repeats–CRISPR-associated protein 12a (CRISPR–Cas12a) cleavage assay that allows the recognition of specific HCV nucleic acid sequences. Amplified products after the cleavage reactions can be visualized on lateral flow strips or 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-coupled 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 concentrations as low as 10 ng/µL (an estimated 2.38 Log10 IU/mL). Therefore, this sensitive and specific assay may represent an affordable and reliable point-of-care test for the identification of individuals with active hepatitis C in low-resource settings.

PrimedSherlock: a tool for rapid design of highly specific CRISPR-Cas12 crRNAs

“…In terms of crRNA specificity for target sequences, as few as two mismatches can cause a significant reduction in detection efficiency [ 24 – 26 ]. Furthermore, mismatches in the seed region, or bases 1–6 proximal to the PAM site, can negatively impact on-target recognition [ 3 , 26 – 28 ]. Our design logic accounts for these mismatches to increase crRNA efficiency.…”

PrimedSherlock: A tool for rapid design of highly specific CRISPR-Cas12 crRNAs

“…In terms of crRNA specificity for target sequences, as few as two mismatches can cause a significant reduction in detection efficiency [24, 25, 26]. Furthermore, mismatches in the seed region, or bases 1-6 proximal to the PAM site, can negatively impact on-target recognition [3, 26, 27, 28]. Our design logic accounts for these mismatches to increase crRNA efficiency.…”

CRISPR-Cas-Integrated LAMP

“…To detect emerging variants of SARS-CoV-2, a variant-specific amplification and detection platform was developed [ 50 ]. In addition to SARS-CoV-2 diagnostic tests, PoC platforms targeting other infectious viral pathogens, including Influenza A and B [ 53 ], Human Papillomavirus 16 and 18 [ 54 ], and Hepatitis C Virus [ 55 ] were also created using Cas12 or Cas13 integrated LAMP or RT-LAMP technologies. Similarly, pathogenic bacteria detection, including Shigella flexneri [ 40 ], Neisseria meningitidis [ 56 ], and Klebsiella pneumonia carbapenemase [ 57 ], was also achieved using Cas-integrated LAMP.…”