Argonaute-integrated isothermal amplification for rapid, portable, multiplex detection of SARS-CoV-2 and influenza viruses

Ye, Xingyu; Zhou, Haiwei; Guo, Xiang; Liu, Donglai; Li, Zhonglei; Jian, Sun; Huang, Jun; Liu, Tao; Zhao, Pengshu; Xu, Huiqin; Li, Kai; Wang, Hanming; Wang, Jihua; Wang, Li; Zhao, Weili; Liu, Qian; Xu, Sihong; Feng, Yue

doi:10.1016/j.bios.2022.114169

Cited by 68 publications

(40 citation statements)

References 34 publications

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“…Inspired by the stepwise Pf Ago activity, we then developed the renewed gDNA-assisted DNA cleavage with Argonaute (RADAR) method that demonstrated rapid and sensitive multiplex virus detection, enabling efficient genotype diagnosis at a single-nucleotide resolution (Xun et al, 2021a ). The multiplex Ago-based nucleic acid detection system (MULAN) method integrated with LAMP and RADAR allowed simultaneous detection of SARS-CoV-2 and influenza viruses (Ye et al, 2022 ). Zhao et al have developed Pf Ago-mediated nucleic acid detection (PAND) for SARS-CoV-2 detection based on a similar principle, which integrated Pf Ago cleavage and LAMP with prefabricated capillaries to enable a single-tube reaction (Xun et al, 2021b ).…”

Section: Discussionmentioning

confidence: 99%

“…In our previous study, Pf Ago was able to cleave the target sequence with a gDNA carrying a single-nucleotide mismatch (Liu et al, 2021a ; Xun et al, 2021a ; Ye et al, 2022 ). We then introduced an extra mismatch in the gDNA that yielded two contiguous mismatches to the target, which significantly decreased cleavage activity.…”

Section: Discussionmentioning

confidence: 99%

“…We then introduced an extra mismatch in the gDNA that yielded two contiguous mismatches to the target, which significantly decreased cleavage activity. Therefore, the MULAN method was developed to discriminate targets at single-base resolution, such as SARS-CoV-2 and its D614G mutant (Ye et al, 2022 ). Considering that both Km Ago and Pb Ago can distinguish targets with mismatches at certain positions (Dong et al, 2021 ; Liu et al, 2021c ), we could introduce a mismatch in the reporter that resulted in a single-nucleotide mismatch with the target DNA, but two consecutive mismatches with the mutant DNA.…”

Section: Discussionmentioning

confidence: 99%

“…Benefiting from its cleavage activity does not require specific recognition sequences, Ago can target arbitrary sequences with gDNAs that are easy to design and synthesize at a low cost (Swarts et al, 2015 ; Enghiad and Zhao, 2017 ). In recent years, thermophilic Agos have stimulated the development of novel detection techniques (Hegge et al, 2019 ; Song et al, 2020 ; Brandolini et al, 2021 ; Liu et al, 2021a ; Wang et al, 2021a ; Xun et al, 2021a , b ; Qin et al, 2022 ; Ye et al, 2022 ). Compared with CRISPR-Cas enzymes, thermophilic Agos present distinct advantages in accurate recognition of single-nucleotide differential sequences and the orthogonal capability of a single enzyme to achieve multiplex detection for multiple targets (Wang et al, 2021a ; Xun et al, 2021b ; Ye et al, 2022 ).…”

Section: Introductionmentioning

confidence: 99%

“…In recent years, thermophilic Agos have stimulated the development of novel detection techniques (Hegge et al, 2019 ; Song et al, 2020 ; Brandolini et al, 2021 ; Liu et al, 2021a ; Wang et al, 2021a ; Xun et al, 2021a , b ; Qin et al, 2022 ; Ye et al, 2022 ). Compared with CRISPR-Cas enzymes, thermophilic Agos present distinct advantages in accurate recognition of single-nucleotide differential sequences and the orthogonal capability of a single enzyme to achieve multiplex detection for multiple targets (Wang et al, 2021a ; Xun et al, 2021b ; Ye et al, 2022 ). Nevertheless, the existing detection methods based on thermophilic Agos still require heating equipment to maintain Ago activity, which restricts POCT application (Qin et al, 2022 ).…”

Section: Introductionmentioning

confidence: 99%

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

Dong

Guo

et al. 2022

Front. Microbiol.

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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.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Dong

Guo

et al. 2022

Front. Microbiol.

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Mn²⁺‐Mediated Modulation of PfAgo Activity for Biosensing

Fu,

Hou,

Wang

et al. 2024

Adv Healthcare Materials

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Argonaute (Ago) as a powerful enzyme has provided new insights into biosensing due to its programmability, high sensitivity, and user‐friendly operation. However, current strategies mainly rely on phosphorylated guide DNA to modulate the cleavage activity of Ago, which are limited in versatility and simplicity. Herein, we reported Mn2+‐enhanced cleavage activity of Ago and employed Mn ions with variable valence to regulate the activity of Pyrococcus furiosus Ago (PfAgo) for biosensing applications. The conversion of Mn ions with different valence states through MnO2 nanoflowers enables the sensitive detection of ascorbic acid, alkaline phosphatase, and arsenic with limits of detection of 2.5 nmol L−1, 0.009 U L−1, and 0.4 ng mL−1, respectively. We further developed a PfAgo‐based immunoassay that allows for the detection of diverse targets, thus providing a promising toolbox to broaden PfAgo‐based sensors into versatile bioanalytical and biomedical applications.This article is protected by copyright. All rights reserved

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Ultra‐Fast Single‐Nucleotide‐Variation Detection Enabled by Argonaute‐Mediated Transistor Platform

Kong,

Zhang,

Guo

et al. 2023

Advanced Materials

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Abstract“Test‐and‐go” single‐nucleotide variation (SNV) detection within several minutes remains challenging, especially in low‐abundance samples, since existing methods face a trade‐off between sensitivity and testing speed. Sensitive detection usually relies on complex and time‐consuming nucleic acid amplification or sequencing. Here, we develop a graphene field‐effect transistor (GFET) platform mediated by Argonaute protein that enables rapid, sensitive and specific SNV detection. The Argonaute protein provides a nanoscale binding channel to preorganize the DNA probe, accelerating target binding and rapidly recognizing SNVs with single‐nucleotide resolution in unamplified tumor‐associated microRNA, circulating tumor DNA, virus RNA and reverse transcribed cDNA when a mismatch occurs in the seed region. An integrated microchip simultaneously detects multiple SNVs in agreement with sequencing results within 5 min, achieving the fastest SNV detection in a “test‐and‐go” manner without the requirement of nucleic acid extraction, reverse transcription and amplification.This article is protected by copyright. All rights reserved

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Argonaute-integrated isothermal amplification for rapid, portable, multiplex detection of SARS-CoV-2 and influenza viruses

Cited by 68 publications

References 34 publications

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

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

Mn²⁺‐Mediated Modulation of PfAgo Activity for Biosensing

Ultra‐Fast Single‐Nucleotide‐Variation Detection Enabled by Argonaute‐Mediated Transistor Platform

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Argonaute-integrated isothermal amplification for rapid, portable, multiplex detection of SARS-CoV-2 and influenza viruses

Cited by 68 publications

References 34 publications

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

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

Mn2+‐Mediated Modulation of PfAgo Activity for Biosensing

Ultra‐Fast Single‐Nucleotide‐Variation Detection Enabled by Argonaute‐Mediated Transistor Platform

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Product

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Mn²⁺‐Mediated Modulation of PfAgo Activity for Biosensing