# Two‐Stage Mixed‐Dye‐Based Isothermal Amplification with Ribonuclease‐Cleavable Enhanced Probes for Dual‐Visualization Detection of SARS‐CoV‐2 Variants of Interest

## TL;DR

A new method called REP-TMAP allows for quick and visual detection of SARS-CoV-2 variants using color and fluorescence changes in a single test.

## Contribution

REP-TMAP introduces a dual-visualization isothermal amplification method for rapid and specific detection of SARS-CoV-2 variants.

## Key findings

- REP-TMAP detects SARS-CoV-2 variants in 40 minutes with high sensitivity and specificity.
- The method accurately identified JN.1, BA.2, and BA.4/5 variants from clinical samples.
- Color and fluorescence changes enable dual-visualization for infection and variant identification.

## Abstract

Rapid and visual detection of SARS‐CoV‐2 variants is vital for timely assessment of variant transmission in resource‐limited settings. Here, a closed‐tube, two‐stage, mixed‐dye‐based isothermal amplification method with ribonuclease‐cleavable enhanced probes (REP), termed REP‐TMAP, for dual‐visualization detection of SARS‐CoV‐2 variants including JN.1, BA.2, BA.4/5, and Delta is introduced. The first stage of REP‐TMAP is reverse transcription recombinase polymerase amplification and the second stage is dual‐visualization detection synergistically mediated by the REP and the mixed dyes of cresol red and hydroxy naphthol blue. In REP‐TMAP reaction, the color change under ambient light indicates SARS‐CoV‐2 infection, while the fluorescence change under blue light excitation specifies variant type. On detecting transcribed RNA of SARS‐CoV‐2 spike gene, this assay is rapid (within 40 min), highly sensitive (10–200 copies per reaction), and highly specific (identification of single‐base mutations). Furthermore, the assay has been clinically validated to accurately detect JN.1, BA.2, and BA.4/5 variants from 102 human oropharyngeal swabs. The proposed assay therefore holds great potentials to provide a rapid, dual‐visualization, sensitive, specific, point‐of‐care detection of SARS‐CoV‐2 variants and beyond.

A closed‐tube, two‐stage, mixed‐dye‐based isothermal amplification method coupled with a ribonuclease‐cleavable enhanced probe (REP), termed REP‐TMAP, is developed for dual‐visualization detection of SARS‐CoV‐2 variants of interest. The color changes of REP‐TMAP reactions under ambient light indicate SARS‐CoV‐2‐positive infection, while the fluorescence changes under blue light excitation specify the variant types such as JN.1, BA.2, and BA.4/5.