A novel One-pot rapid diagnostic technology for COVID-19

Area of Science:

• Virology
• Molecular Diagnostics
• Biotechnology

Background:

• The COVID-19 pandemic, caused by SARS-CoV-2, necessitates accurate and accessible diagnostic tools.
• Current RT-qPCR diagnostics for SARS-CoV-2 are effective but costly, labor-intensive, and require specialized equipment.
• There is a need for rapid, low-cost, and user-friendly diagnostic methods for widespread COVID-19 testing.

Purpose of the Study:

• To develop and validate a novel, one-tube, rapid diagnostic approach for SARS-CoV-2 detection.
• To establish a colorimetric RT-LAMP assay that bypasses the need for viral RNA extraction.
• To evaluate the sensitivity, specificity, and clinical applicability of the developed diagnostic method.

Main Methods:

• A one-tube, formamide-based, colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was established.
• The assay was optimized for visual detection of SARS-CoV-2 within 45 minutes.
• Performance was assessed using extracted RNA, viral transport medium, and clinical nasopharyngeal swab samples, with comparisons to RT-qPCR.

Main Results:

• The One-Pot RT-LAMP assay detected SARS-CoV-2 visually within 45 minutes.
• The limit of detection was 5 copies/reaction for extracted RNA and approximately 7.66 virus copies/μL in viral transport medium.
• The assay demonstrated high specificity, with no cross-reactivity against 12 other viruses, and yielded results consistent with RT-qPCR in 45 clinical samples.

Conclusions:

• The One-Pot RT-LAMP assay is a sensitive, specific, and rapid method for SARS-CoV-2 identification.
• This technology offers a low-cost alternative to RT-qPCR, eliminating the need for viral RNA extraction.
• The developed assay holds potential as a valuable tool for high-throughput, accessible COVID-19 diagnostics.