Rapid and Sensitive Detection of SARS-CoV-2 Using Clustered Regularly Interspaced Short Palindromic Repeats

Tsou, Jen-Hui; Liu, Hongjie; Stass, Sanford A.; Jiang, Feng

doi:10.3390/biomedicines9030239

Cited by 23 publications

(31 citation statements)

References 22 publications

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“…The 3.84 µL EnGen ® LbaCas12a (NEB, Ipswich, MA, USA) was preassembled with gRNA (3.84 µL) in 22.32 µL of 1x NEBuffer and then mixed with 4.8 µL custom ssDNA-FQ reporter (IDT) in 25.2 µL of 1x NEBuffer [ 12 , 13 , 14 , 15 ]. The preassembled mixture was added directly to the 5 µL RT-RPA reactions in a volume of 20 µL and incubated at 42 °C on a Biotek fluorescence plate reader (Biotek, Winooski, VT, USA).…”

Section: Methodsmentioning

confidence: 99%

“…The preassembled mixture was added directly to the 5 µL RT-RPA reactions in a volume of 20 µL and incubated at 42 °C on a Biotek fluorescence plate reader (Biotek, Winooski, VT, USA). The fluorescence kinetics was measured on the fluorescence plate reader (λex = 485 nM; λem = 535 nM) [ 12 , 13 , 14 , 15 ].…”

Section: Methodsmentioning

confidence: 99%

“…We used the methods described above to perform CRISPR-Cas12a reactions. As described in our previous studies [ 12 , 13 , 14 , 15 ], the visual detection was based on the reaction solution’s fluorescence signal, in which the tubes’ images were captured in the Bio-Rad ChemiDoc™ MP imaging system with its built-in UV channel (Bio-Rad Laboratories, Hercules, CA, USA).…”

Section: Methodsmentioning

confidence: 99%

“…We used the Centers for Disease Control and Prevention (CDC) standard PCR assay to detect the viruses as described in our previous study [ 15 ]. Briefly, the RNA was amplified with a GoTaq ® Probe 1-Step RT-PCR system (Promega, Madison, WI, USA).…”

Section: Methodsmentioning

confidence: 99%

“…Our research has also demonstrated that the CRISPR-based test can rapidly and sensitively detect endogenous DNA mutations of EGFR and KRAS in plasma and tissue specimens of lung cancer patients [ 13 , 14 ]. Recently, we developed CRISPR-Cas12a as a rapid test for sensitive detection of SARS-CoV-2 [ 15 ]. In this study, we investigated if the CRISPR-Cas12a test could simultaneously detect and precisely distinguish different respiratory viruses.…”

Section: Introductionmentioning

confidence: 99%

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Detection and Differentiation of SARS-CoV-2, Influenza, and Respiratory Syncytial Viruses by CRISPR

et al. 2021

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SARS-CoV-2, influenza, and respiratory syncytial viruses (RSVs) cause acute respiratory infections with similar symptoms. Since the treatments and outcomes of these infections are different, the early detection and accurate differentiation of the viruses are clinically important for the prevention and treatment of the diseases. We previously demonstrated that clustered regularly interspaced short palindromic repeats (CRISPR) could rapidly and precisely detect SARS-CoV-2. The objective of this study was to develop CRISPR as a test for simultaneously detecting and accurately distinguishing the viruses. The CRISPR assay with an RNA guide against each virus was performed in the reference standards of SARS-CoV-2, influenza A and B, and RSV. The CRISPR assay had a limit of detection of 1–100 copies/µL for specifically detecting SARS-CoV-2, influenza A and B, and RSV without cross-reaction with other respiratory viruses. The validation of the test in nasopharyngeal specimens showed that it had a 90–100% sensitivity and 100% specificity for the detection of SARS-CoV-2, influenza A and B, and RSV. The CRISPR assay could potentially be used for sensitive detection and specific differentiation of the respiratory viruses.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Detection and Differentiation of SARS-CoV-2, Influenza, and Respiratory Syncytial Viruses by CRISPR

et al. 2021

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Potential of the CRISPR‐Cas system for improved parasite diagnosis

et al. 2022

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CRISPR-Cas technology accelerates development of fast, accurate, and portable diagnostic tools, typified by recent applications in COVID-19 diagnosis. Parasitic helminths cause devastating diseases afflicting 1.5 billion people globally, representing a significant public health and economic burden, especially in developing countries. Currently available diagnostic tests for worm infection are neither sufficiently sensitive nor fieldfriendly for use in low-endemic or resource-poor settings, leading to underestimation of true prevalence rates. Mass drug administration programs are unsustainable longterm, and diagnostic tools -required to be rapid, specific, sensitive, cost-effective, and user-friendly without specialized equipment and expertise -are urgently needed for rapid mapping of helminthic diseases and monitoring control programs. We describe the key features of the CRISPR-Cas12/13 system and emphasise its potential for the development of effective tools for the diagnosis of parasitic and other neglected tropical diseases (NTDs), a key recommendation of the NTDs 2021-2030 roadmap released by the World Health Organization.

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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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Rapid and Sensitive Detection of SARS-CoV-2 Using Clustered Regularly Interspaced Short Palindromic Repeats

Cited by 23 publications

References 22 publications

Detection and Differentiation of SARS-CoV-2, Influenza, and Respiratory Syncytial Viruses by CRISPR

Detection and Differentiation of SARS-CoV-2, Influenza, and Respiratory Syncytial Viruses by CRISPR

Potential of the CRISPR‐Cas system for improved parasite diagnosis

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

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