One copy in one-pot for rapid and accurate SFTSV testing by LAC12b-2M.

Area of Science:

• Molecular Biology
• Virology
• Biotechnology

Background:

• Severe Fever with Thrombocytopenia Syndrome virus (SFTSV) is a dangerous tick-borne pathogen with high fatality rates.
• SFTSV prevalence in resource-limited, mountainous regions necessitates improved diagnostic tools.

Purpose of the Study:

• To develop a rapid, sensitive, and convenient SFTSV detection method for low-resource environments.
• To evaluate the performance of the novel LAC12b-2M assay.

Main Methods:

• A one-pot, visualized loop-mediated isothermal amplification assay (LAC12b-2M) utilizing CRISPR/Cas12b was developed.
• The method's sensitivity, specificity, accuracy, and limit of detection (LOD) were assessed.
• Performance was compared against clinical RT-qPCR using SFTSV RNA samples and clinical serum samples.

Main Results:

• The LAC12b-2M assay achieved a LOD of 1 copy/μL at 61°C within 30 minutes.
• High diagnostic performance was observed: 98.8% sensitivity, 100% specificity, and 99.4% accuracy in cohort 1.
• 100% accuracy was achieved in cohort 2, indicating robust performance across different sample groups.

Conclusions:

• The LAC12b-2M method offers a promising solution for point-of-care SFTSV testing.
• This assay is particularly suitable for healthcare settings in low-resource regions endemic for SFTSV.
• The developed method addresses the urgent need for accessible SFTSV diagnostics.