RUNCOV: a one-pot triplex real-time RT-LAMP as a point-of-care diagnostic tool for detecting SARS-CoV-2

Area of Science:

• Molecular Biology
• Infectious Diseases
• Diagnostics

Background:

• The emergence of zoonotic diseases, including SARS-CoV-2 variants, necessitates rapid diagnostic solutions.
• Current diagnostic methods may not be sufficiently fast or accessible for widespread point-of-care use.

Purpose of the Study:

• To develop and validate a rapid, sensitive, and specific molecular diagnostic assay for SARS-CoV-2 detection.
• To create a portable, point-of-care tool for infectious disease surveillance and control.

Main Methods:

• Development of a one-pot triplex real-time RT-LAMP assay (RUNCOV) targeting SARS-CoV-2 Orf1ab and N genes, with a human RNase P internal control.
• Comparison of RUNCOV with gold-standard RT-qPCR using clinical samples.
• Extensive *in silico* analysis of over 14 million SARS-CoV-2 genomes to assess variant detection capabilities.

Main Results:

• RUNCOV demonstrated high diagnostic sensitivity (97.44%) and specificity (100%) compared to RT-qPCR.
• The assay has a low limit of detection (5-20 copies/reaction) and is rapid (under 40 minutes).
• *In silico* analysis confirmed RUNCOV's ability to detect all known SARS-CoV-2 variants, including KP.3.1.1 and BA2.86.1.

Conclusions:

• The RUNCOV assay is a rapid, sensitive, and specific molecular diagnostic tool suitable for point-of-care applications.
• Its portability and broad variant detection capability make it valuable for controlling the spread of infectious diseases, including COVID-19.
• This assay offers a promising solution for on-site diagnostics in diverse settings.