A paper-based assay for the colorimetric detection of SARS-CoV-2 variants at single-nucleotide resolution

Zhang, Ting; Deng, Ruijie; Wang, Yuxi; Wu, Chunlin; Zhang, Kaixiang; Wang, Chunyan; Gong, Ningqiang; Ledesma-Amaro, Rodrigo; Teng, Xianliang; Yang, Chunrong; Xue, Ting; Zhang, Yong; Hu, Yun; He, Qiang; Li, Wei Min; Li, Shanpeng

doi:10.1038/s41551-022-00907-0

Cited by 130 publications

(90 citation statements)

References 47 publications

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“… 34 − 36 According to the reports, the nucleic acid amplification and probe-based assays had sensitivities in the range of tens to hundreds of copies per reaction. 37 − 39 The CRISPR-based assays had sensitivities of around 10 copies per reaction. 21 , 40 By fully utilizing Pf Ago’s specific recognition and cleavage property, the RPA- Pf Ago method exhibited a solid advantage in terms of sensitivity with which a single copy could be sufficient for the mutation identification ( Figure 3 ).…”

Section: Resultsmentioning

confidence: 99%

“…According to the fluorescence signals, with as low as 10 0 copies of the SARS-CoV-2 mutant (MT) RNA, the L452R mutation could be identified (Figure ). The single-copy sensitivity is the highest sensitivity that can be achieved theoretically, and is also the highest sensitivity as compared to the available SARS-CoV-2 genotyping assays. − According to the reports, the nucleic acid amplification and probe-based assays had sensitivities in the range of tens to hundreds of copies per reaction. − The CRISPR-based assays had sensitivities of around 10 copies per reaction. , By fully utilizing Pf Ago’s specific recognition and cleavage property, the RPA- Pf Ago method exhibited a solid advantage in terms of sensitivity with which a single copy could be sufficient for the mutation identification (Figure ). The single-copy sensitivity is the highest sensitivity that can be achieved theoretically and is also the highest sensitivity as compared to the available SARS-CoV-2.…”

Section: Resultsmentioning

confidence: 99%

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Rapid and Sensitive Genotyping of SARS-CoV-2 Key Mutation L452R with an RPA-PfAgo Method

Zhao¹,

Yang²,

Zhang³

et al. 2022

Anal. Chem.

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In the two years of COVID-19 pandemic, the SARS-CoV-2 variants have caused waves of infections one after another, and the pandemic is not ending. The key mutations on the S protein enable the variants with enhanced viral infectivity, immune evasion, and/or antibody neutralization resistance, bringing difficulties to epidemic prevention and control. In support of precise epidemic control and precision medicine of the virus, a fast and simple genotyping method for the key mutations of SARS-CoV-2 variants needs to be developed. By utilizing the specific recognition and cleavage property of the nuclease Argonaute from Pyrococcus f uriosus (PfAgo), we developed a recombinase polymerase amplification (RPA) and PfAgo combined method for a rapid and sensitive genotyping of SARS-CoV-2 key mutation L452R. With a delicate design of the strategy, careful screening of the RPA primers and PfAgo gDNA, and optimization of the reaction, the method achieves a high sensitivity of a single copy per reaction, which is validated with the pseudovirus. This is the highest sensitivity that can be achieved theoretically and the highest sensitivity as compared to the available SARS-CoV-2 genotyping assays. Using RPA, the procedure of the method is finished within 1.5 h and only needs a minimum laboratorial support, suggesting that the method can be easily applied locally or on-site. The RPA-PfAgo method established in this study provides a strong support to the precise epidemic control and precision medicine of SARS-CoV-2 variants and can be readily developed for the simultaneous genotyping of multiple SARS-CoV-2 mutations.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Rapid and Sensitive Genotyping of SARS-CoV-2 Key Mutation L452R with an RPA-PfAgo Method

Zhao¹,

Yang²,

Zhang³

et al. 2022

Anal. Chem.

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“…Especially, the wireless data transfer via the strategies such as Bluetooth, near‐field communication, Wi‐Fi, 4G cellular data service, and even 4G cellular data service can transmit the acquired smartphone data to cloud or professionals, which greatly benefit the development of biosensors for POC test (X. Huang et al., 2018 ; X. Zhang, Wu, Wu, & Li, 2021 ). Benefit from its superior performances, smartphone has been successfully integrated with CRISPR/Cas system‐based biosensors for the POC assay of SARS‐CoV‐2 (Abbasi, 2021 ; Arizti‐Sanz et al., 2020 ; Fozouni et al., 2021 ; Samacoits, Nimsamer, Mayuramart, Chantaravisoot, Sitthi‐Amorn, Nakhakes, Luangkamchorn, Tongcham, Zahm, & Suphanpayak, 2021 ; Silva et al., 2021 ; Tian, Qiu, Jiang, Zhu, & Zhou, 2022 ; T. Zhang et al., 2022 ). For instance, Fozouni et al.…”

Section: Crispr/cas Systems‐based Sars‐cov‐2 Detectionmentioning

confidence: 99%

Point‐of‐care CRISPR/Cas biosensing technology: A promising tool for preventing the possible COVID‐19 resurgence caused by contaminated cold‐chain food and packaging

et al. 2022

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The ongoing coronavirus disease 2019 (COVID‐19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) has caused great public health concern and has been a global threat due to its high transmissibility and morbidity. Although the SARS‐CoV‐2 transmission mainly relies on the person‐to‐person route through the respiratory droplets, the possible transmission through the contaminated cold‐chain food and packaging to humans has raised widespread concerns. This review discussed the possibility of SARS‐CoV‐2 transmission via the contaminated cold‐chain food and packaging by tracing the occurrence, the survival of SARS‐CoV‐2 in the contaminated cold‐chain food and packaging, as well as the transmission and outbreaks related to the contaminated cold‐chain food and packaging. Rapid, accurate, and reliable diagnostics of SARS‐CoV‐2 is of great importance for preventing and controlling the COVID‐19 resurgence. Therefore, we summarized the recent advances on the emerging clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system‐based biosensing technology that is promising and powerful for preventing the possible COVID‐19 resurgence caused by the contaminated cold‐chain food and packaging during the COVID‐19 pandemic, including CRISPR/Cas system‐based biosensors and their integration with portable devices (e.g., smartphone, lateral flow assays, microfluidic chips, and nanopores). Impressively, this review not only provided an insight on the possibility of SARS‐CoV‐2 transmission through the food supply chain, but also proposed the future opportunities and challenges on the development of CRISPR/Cas system‐based detection methods for the diagnosis of SARS‐CoV‐2.

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“…Pandemic outbreaks, particularly coronavirus disease 2019 (COVID-19) epidemic, highlight the demand to enhance surveillance systems and diagnostic capacities for pathogenic biosafety [1]. The global COVID-19 pandemic has infected 627 million cumulative cases and caused 6.56 million cumulative deaths, and it still results in nearly 80 thousand new cases per day, as of November 1, 2022 (WHO Coronavirus Dashboard, https://covid19.who.int/).…”

Section: Introductionmentioning

confidence: 99%

CRISPR-based nucleic acid diagnostics for pathogens

Yang

Zhang

Teng

et al. 2022

Preprint

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Pathogenic infections remain the primary threat for human health, especially the global COVID-19 pandemic. It is important to develop rapid, sensitive and multiplexed tools for detecting pathogens and their mutations, particularly the tailor-made strategies for point-of-care diagnosis allowing for use in resource-constrained settings. The rapidly evolving CRISPR/Cas systems have provided a powerful toolbox for pathogenic diagnostics via nucleic acid tests. In this review, we first describe the resultant promising class 2 (single, multidomain effector) and recently explored class 1 (multisubunit effector complexes) CRISPR tools. We present the diverse engineering nucleic acid diagnostics based on CRISPR/Cas systems for pathogenic viruses, bacteria and fungi, and highlight the application for detecting viral variants and drug-resistant bacteria enabled by CRISPR-based mutation profiling. Finally, we discuss the challenges such as the development of preamplification-free diagnostic assays and present the emerging CRISPR systems and CRISPR cascade that potentially enable multiplexed and preamplification-free pathogenic diagnostics.

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A paper-based assay for the colorimetric detection of SARS-CoV-2 variants at single-nucleotide resolution

Abstract: March 2021Ethics oversight 2020(100)).Note that full information on the approval of the study protocol must also be provided in the manuscript.

Cited by 130 publications

References 47 publications

Rapid and Sensitive Genotyping of SARS-CoV-2 Key Mutation L452R with an RPA-PfAgo Method

Rapid and Sensitive Genotyping of SARS-CoV-2 Key Mutation L452R with an RPA-PfAgo Method

Point‐of‐care CRISPR/Cas biosensing technology: A promising tool for preventing the possible COVID‐19 resurgence caused by contaminated cold‐chain food and packaging

CRISPR-based nucleic acid diagnostics for pathogens

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