CRISPR–Cas12-based detection of SARS-CoV-2

Broughton, James P.; Deng, Xianding; Yu, Guixia; Fasching, Clare L.; Servellita, Venice; Singh, Jasmeet; Miao, Xin; Streithorst, Jessica; Granados, Andrea; Sotomayor-González, Alicia; Zorn, Kelsey C.; Gopez, Allan; Hsu, Elaine; Gu, Wei; Miller, Steve; Pan, Chao-Yang; Guevara, Hugo; Wadford, Debra A.; Chen, Janice S.; Chiu, Charles Y.

doi:10.1038/s41587-020-0513-4

Cited by 2,212 publications

(2,444 citation statements)

References 21 publications

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“…30 The DETECTR assay by Mammoth Biosciences relies on the cleavage of reporter RNA by Cas12a to specifically detect viral RNA sequences of the E and N genes, followed by isothermal amplification of the target, resulting in a visual readout with a fluorophore. 31 These CRISPR-based methods, as depicted in Figure 3, do not require complex instrumentation and can be read using paper strips to detect the presence of the SARS-CoV-2 virus without loss of sensitivity or specificity. These tests are both low-cost and can be performed in as little as 1 h. These tests have great potential for point-of-care diagnosis.…”

Section: Reverse Transcription Loop-mediated Isothermal Amplificationmentioning

confidence: 99%

Assay Techniques and Test Development for COVID-19 Diagnosis

et al. 2020

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An ongoing theme of the COVID-19 pandemic is the need for widespread availability of accurate and efficient diagnostic testing for detection of SARS-CoV-2 and antiviral antibodies in infected individuals. This report describes various assay techniques and tests for COVID-19 diagnosis. Most tests for early detection of SARS-CoV-2 RNA rely on the reverse transcription-polymerase chain reaction, but isothermal nucleic acid amplification assays, including transcription-mediated amplification and CRISPR-based methodologies, are promising alternatives. Identification of individuals who have developed antibodies to the SARS-CoV-2 virus requires serological tests, including enzyme-linked immunosorbent assay (ELISA) and lateral flow immunoassay. This report also provides an overview of current development in COVID-19 diagnostic techniques and products to facilitate future improvement and innovation.

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Section: Reverse Transcription Loop-mediated Isothermal Amplificationmentioning

confidence: 99%

Assay Techniques and Test Development for COVID-19 Diagnosis

et al. 2020

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“…They showed that Cas12b behaves similarly to Cas12a for ssDNA reporter cleavage and can achieve a detection limit of 5 copies/ L in 40-60 minutes . Moreover, Broughton et al used LAMP instead of RPA in DETECTR and further reduced the testing time for SARS-CoV-2 RNA to 30-32 minutes while maintaining a low detection limit (10 copies/ L) (Broughton et al 2020).…”

Section: Crispr-based Detectionmentioning

confidence: 99%

Overcoming the bottleneck to widespread testing: a rapid review of nucleic acid testing approaches for COVID-19 detection

Esbin

Whitney

Chong

et al. 2020

RNA

482

474

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The current COVID-19 pandemic presents a serious public health crisis, and a better understanding of the scope and spread of the virus would be aided by more widespread testing. Nucleicacid based tests currently offer the most sensitive and early detection of COVID-19. However, the "gold standard" test pioneered by the United States Center for Disease Control & Prevention, takes several hours to complete and requires extensive human labor, materials such as RNA extraction kits that could become in short supply and relatively scarce qPCR machines. It is clear that a huge effort needs to be made to scale up current COVID-19 testing by orders of magnitude. There is thus a pressing need to evaluate alternative protocols, reagents, and approaches to allow nucleic-acid testing to continue in the face of these potential shortages. There has been a tremendous explosion in the number of papers written within the first weeks of the pandemic evaluating potential advances, comparable reagents, and alternatives to the "gold-standard" CDC RT-PCR test. Here we present a collection of these recent advances in COVID-19 nucleic acid testing, including both peer-reviewed and preprint articles. Due to the rapid developments during this crisis, we have included as many publications as possible, but many of the cited sources have not yet been peer-reviewed, so we urge researchers to further validate results in their own labs. We hope that this review can urgently consolidate and disseminate information to aid researchers in designing and implementing optimized COVID-19 testing protocols to increase the availability, accuracy, and speed of widespread COVID-19 testing.

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“…LAMP has been used to detect many pathogens including Zika virus [3], Mycobacterium tuberculosis [4], malaria [5], and human leishmaniasis [6]. RT-LAMP has also been performed on extracted RNA for subsequent CRISPR-Cas12-based detection of SARS-CoV-2 [7].…”

Section: Introductionmentioning

confidence: 99%

Clinical assessment and validation of a rapid and sensitive SARS-CoV-2 test using reverse-transcription loop-mediated isothermal amplification

Anahtar

McGrath

Rabe

et al. 2020

Preprint

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Amid the enduring COVID-19 pandemic, there is an urgent need for expanded access to rapid and sensitive SARS-CoV-2 testing worldwide. Here we present a simple clinical workflow that uses a sensitive and highly specific colorimetric reverse-transcription loop-mediated isothermal amplification (RT-LAMP) to detect SARS-CoV-2 and takes forty minutes from sample collection to result. This test requires no specialized equipment and costs a few dollars per sample. Nasopharyngeal samples collected in saline were added either directly (unprocessed) to RT-LAMP reactions or first inactivated by a combined chemical and heat treatment step to . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) : medRxiv preprint SARS-CoV-2-specific primer sets and an internal specimen control; the resulting color change was visually interpreted. While direct addition of unprocessed specimens to RT-LAMP reactions could reliably detect samples with abundant SARS-CoV-2, the assay sensitivity markedly increased after the addition of an inactivation step. In 62 clinical samples with a wide range of SARS-CoV-2 nucleic acid concentrations, the assay had 87.5% sensitivity and 100% specificity with a limit of detection at least 25 copies/µL, making it an ideal test to rule in infection. To increase sensitivity, samples that tested negative for SARS-CoV-2 by direct sample addition could be reflexed to a purification step, to increase the effective per-reaction sample input volume. In 40 purified samples, the assay yielded a 90% sensitivity and 100% specificity, with a limit of detection comparable to commercially available real-time PCR-based diagnostics that have received Emergency Use Authorization (EUA) from the FDA. This test for SARS-CoV-2 can be performed in a range of settings for a fraction of the price of other available tests, with limited equipment, and without relying on over-burdened supply chains to increase overall testing capacity.

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CRISPR–Cas12-based detection of SARS-CoV-2

Cited by 2,212 publications

References 21 publications

Assay Techniques and Test Development for COVID-19 Diagnosis

Assay Techniques and Test Development for COVID-19 Diagnosis

Overcoming the bottleneck to widespread testing: a rapid review of nucleic acid testing approaches for COVID-19 detection

Clinical assessment and validation of a rapid and sensitive SARS-CoV-2 test using reverse-transcription loop-mediated isothermal amplification

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