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RT-RPA Assisted CRISPR/Cas12a Based One-Pot Rapid and Visual Detection of the Pan-Dengue Virus
Pooja Bhardwaj1, Preeti Dhangur1, Alagarasu Kalichamy2
1JE-AES Apex Laboratory, ICMR-Regional Medical Research Centre, BRD Medical College Campus, Gorakhpur, India.
Journal of Medical Virology
|February 14, 2025
Summary
A new CRISPR/Cas12a assay rapidly detects all four dengue virus (DENV) serotypes, offering a vital diagnostic tool for resource-constrained settings. This point-of-care test aids early treatment and reduces the global dengue burden.
Area of Science:
- Molecular Biology
- Virology
- Biotechnology
Background:
- Dengue virus (DENV) poses a significant global health risk, with billions at potential risk and frequent seasonal outbreaks.
- Current molecular diagnostic methods for DENV are often impractical in resource-constrained settings (RCSs).
- Accurate and timely diagnosis is crucial for managing severe morbidity and mortality associated with undiagnosed DENV infections.
Purpose of the Study:
- To develop a simple, rapid, and sensitive fluorescence detection platform for all four DENV serotypes (Pan-DENV) suitable for RCSs.
- To enable early diagnosis and differential diagnosis of DENV infections, supporting prompt patient treatment.
Main Methods:
- Development of a fluorescence detection platform utilizing Reverse Transcriptase Recombinase Polymerase Amplification (RT-RPA) and CRISPR/Cas12a technology.
- Targeting of specific DENV genes (NS1 for DENV-1, 2, 3; E for DENV-2) and design of serotype-specific crRNAs for CRISPR/Cas12a detection.
- Analytical sensitivity determination using synthetic RNA and DENV genomes, followed by clinical validation with AES/AFI patient samples.
Main Results:
- The CRISPR/Cas12a assay successfully detected all four DENV serotypes (1-4) in a single reaction with high analytical sensitivity (ranging from fg to pg levels).
- Clinical validation using 76 samples showed 93.7% sensitivity, 100% specificity, and 98.7% overall accuracy, comparable to RT-PCR.
- The assay demonstrated no cross-reactivity with other etiological agents causing similar clinical presentations (AFI/AES).
Conclusions:
- The developed CRISPR/Cas12a-based fluorescence detection platform offers a rapid, accurate, and user-friendly method for Pan-DENV diagnosis.
- Its suitability as a point-of-care test in resource-constrained settings can significantly reduce healthcare burden and improve patient outcomes.
- Field deployment of this assay can facilitate differential diagnosis and prompt initiation of treatment for dengue patients.
