Accessible LAMP-Enabled Rapid Test (ALERT) for Detecting SARS-CoV-2.
Ali Bektaş,Michael F. Covington,Guy Aidelberg,Anibal Arce,Tamara Matute,Tamara Matute,Isaac Núñez,Isaac Núñez,Julia Walsh,David Boutboul,Constance Delaugerre,Ariel B. Lindner,Fernán Federici,Fernán Federici,Anitha D. Jayaprakash +14 more
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TLDR
In this paper, the authors proposed an accessible LAMP-enabled rapid test (ALERT) for detecting viral RNA from nasal swabs or saliva with high sensitivity (0.1 to 2 viral particles/μL) and specificity (97% true negative rate) utilizing reverse transcription loop-mediated isothermal amplification (RT-LAMP).Abstract:
The coronavirus disease 2019 (COVID-19) pandemic has highlighted bottlenecks in large-scale, frequent testing of populations for infections. Polymerase chain reaction (PCR)-based diagnostic tests are expensive, reliant on centralized labs, can take days to deliver results, and are prone to backlogs and supply shortages. Antigen tests that bind and detect the surface proteins of a virus are rapid and scalable but suffer from high false negative rates. To address this problem, an inexpensive, simple, and robust 60-minute do-it-yourself (DIY) workflow to detect viral RNA from nasal swabs or saliva with high sensitivity (0.1 to 2 viral particles/μL) and specificity (>97% true negative rate) utilizing reverse transcription loop-mediated isothermal amplification (RT-LAMP) was developed. ALERT (Accessible LAMP-Enabled Rapid Test) incorporates the following features: (1) increased shelf-life and ambient temperature storage, compared to liquid reaction mixes, by using wax layers to isolate enzymes from other reagents; (2) improved specificity compared to other LAMP end-point reporting methods, by using sequence-specific QUASR (quenching of unincorporated amplification signal reporters); (3) increased sensitivity, compared to methods without purification through use of a magnetic wand to enable pipette-free concentration of sample RNA and cell debris removal; (4) quality control with a nasopharyngeal-specific mRNA target; and (5) co-detection of other respiratory viruses, such as influenza B, by multiplexing QUASR-modified RT-LAMP primer sets. The flexible nature of the ALERT workflow allows easy, at-home and point-of-care testing for individuals and higher-throughput processing for labs and hospitals. With minimal effort, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific primer sets can be swapped out for other targets to repurpose ALERT to detect other viruses, microorganisms, or nucleic acid-based markers.read more
Citations
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Journal ArticleDOI
Detection of SARS-CoV-2 RNA using RT-LAMP and molecular beacons.
Scott Sherrill-Mix,Young Hwang,Aoife M. Roche,Abigail L. Glascock,Susan R. Weiss,Yize Li,Leila Haddad,Peter V. Deraska,Caitlin Monahan,Andrew Kromer,Jevon Graham-Wooten,Louis J. Taylor,Benjamin S. Abella,Arupa Ganguly,Ronald G. Collman,Gregory D. Van Duyne,Frederic D. Bushman +16 more
TL;DR: In this paper, the authors describe the design and testing of molecular beacons, which allow sequence-specific detection of SARS-CoV-2 genomes with improved discrimination in simple reaction mixtures.
Posted ContentDOI
LAMP-BEAC: Detection of SARS-CoV-2 RNA Using RT-LAMP and Molecular Beacons
Scott Sherrill-Mix,Young Hwang,Aoife M. Roche,Abigail L. Glascock,Susan R. Weiss,Yize Li,Leila Haddad,Peter V. Deraska,Caitlin Monahan,Andrew Kromer,Jevon Graham-Wooten,Louis J. Taylor,Benjamin S. Abella,Arupa Ganguly,Ronald G. Collman,Gregory D. Van Duyne,Frederic D. Bushman +16 more
TL;DR: The design and testing of molecular beacons are described, which allow sequence-specific detection of SARS-CoV-2 genomes with improved discrimination in simple reaction mixtures and how beacons with different fluorescent labels can allow convenient multiplex detection of several amplicons in "single pot" reactions.
Journal ArticleDOI
Optimization and Clinical Validation of Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification, a Fast, Highly Sensitive and Specific COVID-19 Molecular Diagnostic Tool That Is Robust to Detect SARS-CoV-2 Variants of Concern
Pedro Augusto Alves,Ellen G. de Oliveira,Ana Paula Moreira Franco-Luiz,Leticia T. Almeida,Amanda B. Goncalves,Iara A. Borges,Flávia de S. Rocha,Raissa Prado Rocha,Matheus Filgueira Bezerra,Pâmella Miranda,Flavio D. Capanema,Henrique R. Martins,Gerald Weber,Santuza M. R. Teixeira,Gabriel Luz Wallau,Rubens L. Monte-Neto +15 more
Journal ArticleDOI
Sample-to-answer, extraction-free, real-time RT-LAMP test for SARS-CoV-2 in nasopharyngeal, nasal, and saliva samples: Implications and use for surveillance testing
Kathryn Kundrod,Mary E. Natoli,Megan Chang,Chelsey A. Smith,Sai Paul,Dereq Ogoe,Christopher Goh,Akshaya Santhanaraj,Anthony Price,Karen Eldin,Keyur Patel,Ellen Baker,Kathleen M. Schmeler,Rebecca Richards-Kortum +13 more
TL;DR: Successful development, validation, and scaling of this sample-to-answer, extraction-free real-time RT-LAMP test for SARS-CoV-2 adds a highly adaptable tool to efforts to control the COVID-19 pandemic, and can inform test development strategies for future infectious disease threats.
Journal ArticleDOI
The Complexity of SARS-CoV-2 Infection and the COVID-19 Pandemic
Maria Karoliny da Silva Torres,Carlos David Araújo Bichara,Maria de Nazaré do Socorro de Almeida,Mariana Cayres Vallinoto,Maria Alice Freitas Queiroz,Izaura Maria Vieira Cayres Vallinoto,Eduardo José Melo dos Santos,Carlos Alberto Marques de Carvalho,Antonio Carlos Rosário Vallinoto +8 more
TL;DR: An extensive literature review is carried out in order to bring together, in a single article, the biological, social, genetic, diagnostic, therapeutic, immunization, and even socioeconomic aspects that impact the SAR-CoV-2 pandemic.
References
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Journal ArticleDOI
Loop-mediated isothermal amplification of DNA
Tsugunori Notomi,Hiroto Okayama,Harumi Otawara-shi Masubuchi,Toshihiro Yonekawa,Keiko Watanabe,Nobuyuki Amino,Tetsu Hase +6 more
TL;DR: A novel method that amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions that employs a DNA polymerase and a set of four specially designed primers that recognize a total of six distinct sequences on the target DNA.
Journal ArticleDOI
CRISPR-Cas12-based detection of SARS-CoV-2.
James Paul Broughton,Xianding Deng,Guixia Yu,Clare L Fasching,Venice Servellita,Jasmeet Singh,Xin Miao,Jessica Streithorst,Andrea Granados,Alicia Sotomayor-Gonzalez,Kelsey C. Zorn,Allan Gopez,Elaine Hsu,Wei Gu,Steve Miller,Chao Yang Pan,Hugo Guevara,Debra A. Wadford,Janice S. Chen,Charles Y. Chiu +19 more
TL;DR: The CRISPR-based DETECTR assay provides a visual and faster alternative to the US Centers for Disease Control and Prevention SARS-CoV-2 real-time RT–PCR assay, with 95% positive predictive agreement and 100% negative predictive agreement.
Journal ArticleDOI
Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation.
TL;DR: Real-time monitoring of the LAMP reaction was achieved by real-time measurement of turbidity, which indicated an increase in the turbidity of the reaction mixture according to the production of precipitate correlates with the amount of DNA synthesized.
Journal ArticleDOI
Rethinking Covid-19 Test Sensitivity - A Strategy for Containment.
TL;DR: A simple point-of-care test that is inexpensive enough to use frequently, even if it lacks sensitivity, is proposed for Covid-19 cases.
Journal ArticleDOI
A colorimetric RT-LAMP assay and LAMP-sequencing for detecting SARS-CoV-2 RNA in clinical samples.
Viet Loan Dao Thi,Konrad Herbst,Kathleen Boerner,Matthias Meurer,Lukas P.M. Kremer,Lukas P.M. Kremer,Daniel Kirrmaier,Daniel Kirrmaier,Andrew Freistaedter,Dimitrios Papagiannidis,Carla V. Galmozzi,Megan L. Stanifer,Steeve Boulant,Steeve Boulant,Steffen Klein,Petr Chlanda,Dina Khalid,Isabel Barreto Miranda,Paul Schnitzler,Hans-Georg Kräusslich,Michael Knop,Michael Knop,Simon Anders +22 more
TL;DR: The sensitivity and specificity of the RT-LAMP assay for detecting SARS-CoV-2 viral RNA was determined and a multiplexed sequencing protocol (LAMP-sequencing) was developed as a diagnostic validation procedure to detect and record the outcome of RT- LAMP reactions.
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