Abstract
Chronic hepatitis C virus (HCV) infection is a leading cause of cirrhosis, hepatocellular carcinoma, and liver
transplantation. With the successful introduction in 2013 of a direct-acting antiviral (DAA) drug with few side
effects, an affordable price and a >90% cure rate for the treatment of HCV infection, WHO adopted the first-ever
global strategy for eliminating viral hepatitis as a public health problem by 2030. This ambitious goal will require
major efforts, not only to prevent new infections but also to diagnose those who are not yet aware of being
infected. It is estimated that of the 71 million people who are living with HCV infection, half are unaware of their
infection and are thus untreated. A key to providing medical care to this untreated population, and thus to
stopping spread of the infection, is to initiate immediate treatment on site once the infection is diagnosed during
a patient's regular visit to a doctor's office. This will require a rapid, cost-effective diagnostic platform that can be
used at the point of care (POC) and provide diagnostic results in <30 minutes. The current diagnostics, an initial
HCV antibody (Ab) test to document exposure followed by the more complex and expensive HCV RNA test to
confirm viremia, cannot meet the POC need. Since serum HCV RNA can be detected as early as 1-2 weeks
after infection, polymerase chain reaction (PCR) based RNA analysis is the gold standard method for HCV
diagnosis. However, implementation of PCR-based analysis at the POC is limited by its slow turnaround time
and high cost/efficacy ratio, especially in resource-poor settings. This proposal is aimed at filling the gap by
developing a novel diagnostic platform capable of identifying HCV infection in <20 minutes by integrating
recombinase polymerase amplification (RPA), CRISPR-Cas12a and a positively charged gold nanoparticle
(+GNP) based lateral flow assay (LFA) into a unified single-step assay for nucleic acid amplification (NAA) test.
To achieve the objective and address the technology challenges facing an LFA-based POC NAA test, this project
will implement several principal innovations: 1) use of RNA primers for RPA nucleic acid amplification to address
the incompatibility issue in unifying RPA and CRISPR-12a reactions, 2) selective transporting out of the charged
+GNPs released by the CRISPR amplification process to address the reagent/enzyme washout issue, 3) design
of novel target test lines with graded target-binding and an intrinsic reaction “timer” to address the quantification
issue associated with LFA-based nucleic acid diagnostic technology, and 4) miniaturized smartphone-based
detection, which will make HCV diagnosis more POC and mobile health compatible. We expect that successful
completion of this project will lead to a novel, robust, cost-effective POC technology for the rapid NAA diagnosis
of HCV infection, which will have broad positive impacts on the early diagnosis and treatment of HCV and other
infectious diseases.
