Mesophilic Argonaute-based isothermal detection of SARS-CoV-2

Abstract

Coronavirus disease (COVID-19), caused by SARS-CoV-2 infection and its mutations, has spread rapidly all over the world and still requires sensitive detection to distinguish mutations. CRISPR-based diagnosis has been regarded as a next-generation detection method; however, it has some limitations, such as the need for specific recognition sequences and multiple enzymes for multiplex detection. Therefore, research on the exploration and development of novel nucleases helps to promote specific and sensitive diagnoses. Prokaryotic Argonaute (Ago) proteins exert directed nuclease activity that can target any sequence. Recently, thermophilic Agos have been developed as new detection techniques achieving multiplexity for multiple targets using a single enzyme, as well as accurate recognition of single-base differential sequences. In this study, to overcome the requirement for high reaction temperature of thermophilic Ago-based methods, we expanded the mining of mesophilic Agos to achieve CRISPR-like isothermal detection, named mesophilic Ago-based isothermal detection method (MAIDEN). The principle of MAIDEN uses mesophilic Ago cleavage combined with reverse transcription, which can provide single-strand DNA as a substrate and allow cleavage of fluorescence probes to sense SARS-CoV-2 at moderate temperature. We first mined and optimized the mesophilic Ago and the fluorescence reporter system and then selected a compatible reverse transcription reaction. Furthermore, we optimized MAIDEN into a one-step reaction that can detect SARS-CoV-2 RNA at the nanomolar concentration at a constant temperature of 42°C within 60 min. Therefore, MAIDEN shows advantageous portability and easy-to-implement operation, avoiding the possibility of open-lid contamination. Our study was the first attempt to demonstrate that mesophilic Agos can be harnessed as diagnostic tools, and MAIDEN was easily extended to detect other pathogens in a rapid and efficient manner.

Keywords: RNA viruses; SARS-CoV-2; isothermal detection; mesophilic Ago; one-step method.

Figure 1

Workflow of the MAIDEN system. (A) Schematic illustration of the enzymatic process of mesophilic Ago-based diagnostics for RNA viruses. (B) Schematic diagram of the procedure for the two-step operation and the one-step operation.

Figure 2

Validation of the stepwise cleavage activity of mesophilic Ago. (A) Schematic diagram of Ago stepwise cleavage. (B) Gel image of PbAgo stepwise cleavage. (C) Fluorescent result of KmAgo stepwise cleavage. Data were collected from three independent experiments and are presented as the mean ± SD.

Figure 3

Optimization of an isothermal detection reaction of mesophilic Agos. (A) Schematic and sequence diagram of stepwise clevage in the isothermal detection system. (B–D) Fluorescence values of PbAgo stepwise cleavage under different concentrations of gDNA (B), PbAgo (C), and tDNA (D).

Figure 4

Sensitivity evaluation of the MAIDEN system. (A) Schematic diagram of the two-step operation. (B) Sensitivity of the PbAgo and KmAgo-mediated two-step assay. (C) Schematic diagram of the one-step operation. (D) Sensitivity of the PbAgo and KmAgo-mediated one-step assay. Data were collected from three independent experiments and are presented as the mean ± SD, and significance was considered as ns ≥ 0.05, *p < 0.05, **p < 0.01, and ***p < 0.001.