Development and evaluation of a crispr-cas13a-based platform for rapid and ultraprecise detection of rna viruses in clinical respiratory samples

Zahraa Ali Mohammed; Nahid Sabah Aldin Saber; Huda Sabah Jabr; RashadImad Al-Zamili; Rahma Majid Al-Allawi; Noor Sabah Jabr

doi:10.70604/

Authors

Zahraa Ali Mohammed Assistant lecture/ Department of Clinical Laboratory Sciences/ Collage of Pharmacy/ Al-Qadisiyah University.
Nahid Sabah Aldin Saber Department of Clinical Laboratory Sciences, College of Pharmacy, University of Al-Qadisiyah, Diwaniyah, Iraq.
Huda Sabah Jabr College of Biotechnology, University of Al-Qadisiyah, Iraq.
RashadImad Al-Zamili College of Biotechnology, University of Al-Qadisiyah, Iraq.
Rahma Majid Al-Allawi College of Biotechnology, University of Al-Qadisiyah, Iraq.
Noor Sabah Jabr Al-Mustaqbal University, College of Health and Medical Technologies, Department of Radiology Technologies, Babil, Iraq.

Abstract

Background: Respiratory RNA virus outbreaks have highlighted the urgent need for rapid, accurate, and affordable diagnostic tools. Traditional molecular diagnostics, while effective, often require complex infrastructure and have long turnaround times, limiting their use in resource-limited and point-of-care settings.

Objective: This study aimed to design and evaluate a CRISPR-Cas13a-based detection system for rapid identification of respiratory RNA viruses in clinical samples, focusing on sensitivity, specificity, and clinical utility.

Methodology: We developed a CRISPR-Cas13a diagnostic platform integrating reverse transcription isothermal amplification and Cas13a trans-cleavage activity. The assay targeted conserved genomic regions of SARS-CoV-2, influenza A/B, respiratory syncytial virus (RSV), and human metapneumovirus (hMPV). Clinical validation was performed on 847 nasopharyngeal swab specimens from symptomatic patients, using RT-qPCR as the reference standard.

Results: The assay demonstrated high analytical sensitivity with detection limits of 10–20 copies/L for all target viruses. Clinical sensitivity was 96.7% (95% CI: 94.2–98.4) and specificity 99.1% (95% CI: 97.8–99.7). It showed 100% concordance with RT-qPCR in high viral load samples (Ct <25) and 92.3% in low viral load samples (Ct 30–35). Results were available within 45 minutes post-sampling.

Conclusion: This CRISPR-Cas13a platform offers rapid, accurate, and infrastructure-light detection of respiratory RNA viruses. Its performance and practicality support its use in point-of-care testing, outbreak response, and routine diagnostics, underscoring the clinical promise of CRISPR-based technologies.

Keywords:

CRISPR-Cas13a, RNA virus detection, respiratory pathogens, molecular diagnostics, point-of-care testing, isothermal amplification

DOI

https://doi.org/10.70604/

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