Development of RPA and nested-RPA based CRISPR/Cas13a diagnostic platform for the identification of HBV DNA and HCV RNA in Indian patient cohort.

BACKGROUND

Among the Indian population, hepatitis B virus (HBV) is one of the major burdens and the hepatitis C virus (HCV) chronically infects around 1% Indian population. CRISPR-based detection platforms have shown to be a novel low-cost technology with high sensitivity and specificity. In the presence of target nucleic acids, Cas13a molecule is activated to trans-cleave the fluorophore quencher (FQ)-labeled ssRNA reporter, and illuminate detectable fluorescent signals.

METHODS

Leptotrichia wadei (Lwa) cas13a was expressed and purified. Recombinase Polymerase Amplification (RPA) was implemented to produce T7 RNA polymerase appended amplicons of conserved regions of HBV and HCV at 37°C and 42°C respectively. Corresponding crRNAs have been designed against amplified regions and produced using In-Vitro Transcription (IVT). With T7 RNA polymerase, the RPA-amplified HBV and HCV templates are transcribed into ssRNAs, which are further used in detection assay containing expressed Cas protein, crRNA, and fluorescent probes. This detection was performed in the microplate reader in kinetic format.

RESULTS

LwaCas13a was expressed and was purified using the strep tag. Conserved regions among Indian HBV and HCV genotypes are selected as targets of detection. RPA and Nested-RPA was performed using primers against conjunct region of HBV polymerase and surface antigen and RNA-dependent RNA polymerase (RdRp) region of HCV. The detection assay was performed from 45 HBV and 30 HCV human samples, out of which it could differentiate positive and healthy samples.

CONCLUSION

This approach can be a better alternative to be used in rural India and at the same time with high sensitivity, for a rapid detection of HBV and HCV, which could be used as a novel a low-cost diagnostics platform for identification of HBV DNA and HCV RNA.