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Updated: May 22, 2026
Point-of-care CRISPR-based Diagnostics with Premixed and Freeze-dried Reagents
Published on: August 16, 2024
Self-amplifying CRISPR-based one-pot ultrasensitive testing for rapid SARS-CoV-2 and its variant detection
Jayeon Song1, Mikyung Kang2, Baekdong Cha3
1Bionanotechnology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Republic of Korea; Center for Systems Biology, Massachusetts General Hospital Research Institute, Boston, MA, USA; Department of Radiology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA; Department of Forensic Sciences, Sungkyunkwan University (SKKU), Suwon, Republic of Korea; School of Pharmacy, SKKU, Suwon, Republic of Korea.
A new CRISPR-Cas13a assay rapidly detects SARS-CoV-2 RNA and its variants in under 40 minutes. This one-tube test offers attomolar sensitivity and field-deployable operation for point-of-care diagnostics.
Area of Science:
- Molecular Biology
- Biotechnology
- Infectious Disease Diagnostics
Background:
- Rapid and accessible molecular testing for viral variants is crucial.
- Existing methods often require complex pre-amplification steps.
- Distinguishing between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants is a significant challenge.
Purpose of the Study:
- To develop a rapid, one-tube CRISPR-Cas13a assay for SARS-CoV-2 detection and variant discrimination.
- To achieve high sensitivity and specificity without nucleic acid extraction.
- To enable field-deployable point-of-care (POC) testing.
Main Methods:
- A one-tube clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 13a (Cas13a) assay was designed.
- The assay couples target recognition with a T7-promoter-driven self-amplifying loop for fluorescence amplification.
- Detection of SARS-CoV-2 RNAs (ORF1a, N, S, E) and specific spike (S) mutations was performed.
- A compact reader and smartphone application facilitated real-time quantification.
Main Results:
- The assay achieved exponential fluorescence amplification at 37°C within 40 minutes.
- Limits of detection (LoDs) ranged from 0.32-0.96 copies/μL, demonstrating attomolar sensitivity.
- The system successfully discriminated key SARS-CoV-2 S gene mutations (e.g., D614G, L452R).
- Clinical evaluation (n=105) showed concordance with reverse transcription quantitative polymerase chain reaction (RT-qPCR).
Conclusions:
- A minimal-handling, extraction-free workflow for SARS-CoV-2 detection and mutation resolution was established.
- The developed CRISPR-Cas13a assay is suitable for rapid, field-deployable POC diagnostics.
- This platform offers a generalizable architecture for nucleic acid testing.
