Related Experiment Video
Updated: Jun 16, 2026
DNA Virus Detection System Based on RPA-CRISPR/Cas12a-SPM and Deep Learning
Published on: May 10, 2024
Development of RPA and nested-RPA based CRISPR/Cas13a diagnostic platform for the identification of HBV DNA and HCV
Teja Naveen Sata1, Amrendra Kumar Sah1, Md Ismail1
1Faculty of Life Sciences and Biotechnology, South Asian University, New Delhi, India.
Insights
This study presents a novel CRISPR-based diagnostic platform for rapid, low-cost detection of hepatitis B virus (HBV) and hepatitis C virus (HCV) in India. The method successfully differentiated positive and healthy samples, offering a promising solution for rural healthcare.
Area of Science:
- Molecular Biology
- Biotechnology
- Infectious Disease Diagnostics
Background:
- Hepatitis B virus (HBV) and hepatitis C virus (HCV) pose significant health burdens in India.
- CRISPR-based detection offers a sensitive, specific, and low-cost diagnostic approach.
- Cas13a's trans-cleavage activity on ssRNA reporters enables signal generation in the presence of target nucleic acids.
Purpose of the Study:
- To develop and evaluate a CRISPR-Cas13a based platform for the detection of HBV and HCV.
- To assess the feasibility of this low-cost, high-sensitivity method for use in resource-limited settings like rural India.
Main Methods:
- Leptotrichia wadei (Lwa) Cas13a was expressed and purified.
- Recombinase Polymerase Amplification (RPA) was used to amplify conserved regions of HBV and HCV, followed by T7 RNA polymerase transcription.
- CRISPR RNA (crRNA) targeting amplified regions and fluorescent probes were employed in a microplate reader assay.
Main Results:
- LwaCas13a was successfully expressed and purified.
- RPA and Nested-RPA were performed on conserved regions of HBV and HCV.
- The assay successfully differentiated positive and healthy human samples (45 HBV, 30 HCV).
Conclusions:
- The developed CRISPR-Cas13a platform provides a sensitive and rapid method for HBV and HCV detection.
- This approach is a viable low-cost diagnostic alternative for rural India.
- It enables efficient identification of HBV DNA and HCV RNA.
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.
