{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,13]],"date-time":"2026-05-13T19:40:32Z","timestamp":1778701232346,"version":"3.51.4"},"reference-count":39,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2021,4,23]],"date-time":"2021-04-23T00:00:00Z","timestamp":1619136000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Just One Giant Lab (JOGL)","award":["N\/A"],"award-info":[{"award-number":["N\/A"]}]},{"name":"UC Berkeley Center for Emerging and Neglected Diseases","award":["COVID-CATALYST Grant"],"award-info":[{"award-number":["COVID-CATALYST Grant"]}]},{"name":"ANID \u2013 Millennium Science Initiative Program","award":["ICN17_022"],"award-info":[{"award-number":["ICN17_022"]}]},{"name":"ANID CONCYTEC","award":["covbio0012"],"award-info":[{"award-number":["covbio0012"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>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\/\u03bcL) and specificity (&gt;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.<\/jats:p>","DOI":"10.3390\/v13050742","type":"journal-article","created":{"date-parts":[[2021,4,25]],"date-time":"2021-04-25T02:12:57Z","timestamp":1619316777000},"page":"742","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":17,"title":["Accessible LAMP-Enabled Rapid Test (ALERT) for Detecting SARS-CoV-2"],"prefix":"10.3390","volume":"13","author":[{"given":"Ali","family":"Bekta\u015f","sequence":"first","affiliation":[{"name":"Oakland Genomics Center, 355 30th Street, Oakland, CA 94609, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Michael F.","family":"Covington","sequence":"additional","affiliation":[{"name":"Oakland Genomics Center, 355 30th Street, Oakland, CA 94609, USA"},{"name":"Amaryllis Nucleics, 355 30th Street, Oakland, CA 94609, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Guy","family":"Aidelberg","sequence":"additional","affiliation":[{"name":"Center for Research and Interdisciplinarity (CRI), Universit\u00e9 de Paris, INSERM U1284, F-75006 Paris, France"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Anibal","family":"Arce","sequence":"additional","affiliation":[{"name":"Institute for Biological and Medical Engineering, Schools of Engineering, Biology and Medicine, Pontificia Universidad Cat\u00f3lica de Chile, Santiago 7820244, Chile"},{"name":"FONDAP Center for Genome Regulation, ANID\u2013Millennium Science Initiative Program\u2013Millennium Institute for Integrative Biology (iBIO), Santiago 8331150, Chile"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0486-985X","authenticated-orcid":false,"given":"Tamara","family":"Matute","sequence":"additional","affiliation":[{"name":"Institute for Biological and Medical Engineering, Schools of Engineering, Biology and Medicine, Pontificia Universidad Cat\u00f3lica de Chile, Santiago 7820244, Chile"},{"name":"FONDAP Center for Genome Regulation, ANID\u2013Millennium Science Initiative Program\u2013Millennium Institute for Integrative Biology (iBIO), Santiago 8331150, Chile"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4439-3745","authenticated-orcid":false,"given":"Isaac","family":"N\u00fa\u00f1ez","sequence":"additional","affiliation":[{"name":"Institute for Biological and Medical Engineering, Schools of Engineering, Biology and Medicine, Pontificia Universidad Cat\u00f3lica de Chile, Santiago 7820244, Chile"},{"name":"FONDAP Center for Genome Regulation, ANID\u2013Millennium Science Initiative Program\u2013Millennium Institute for Integrative Biology (iBIO), Santiago 8331150, Chile"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Julia","family":"Walsh","sequence":"additional","affiliation":[{"name":"School of Public Health, University of California Berkeley, Berkeley, CA 94720, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5006-8279","authenticated-orcid":false,"given":"David","family":"Boutboul","sequence":"additional","affiliation":[{"name":"Clinical Immunology Department, U976 HIPI, H\u00f4pital Saint Louis, Universit\u00e9 de Paris, F-75006 Paris, France"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Constance","family":"Delaugerre","sequence":"additional","affiliation":[{"name":"Clinical Immunology Department, U976 HIPI, H\u00f4pital Saint Louis, Universit\u00e9 de Paris, F-75006 Paris, France"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ariel B.","family":"Lindner","sequence":"additional","affiliation":[{"name":"Center for Research and Interdisciplinarity (CRI), Universit\u00e9 de Paris, INSERM U1284, F-75006 Paris, France"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Fern\u00e1n","family":"Federici","sequence":"additional","affiliation":[{"name":"Institute for Biological and Medical Engineering, Schools of Engineering, Biology and Medicine, Pontificia Universidad Cat\u00f3lica de Chile, Santiago 7820244, Chile"},{"name":"FONDAP Center for Genome Regulation, ANID\u2013Millennium Science Initiative Program\u2013Millennium Institute for Integrative Biology (iBIO), Santiago 8331150, Chile"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Anitha D.","family":"Jayaprakash","sequence":"additional","affiliation":[{"name":"Oakland Genomics Center, 355 30th Street, Oakland, CA 94609, USA"},{"name":"Girihlet Inc., 355 30th Street, Oakland, CA 94609, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2021,4,23]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Etievant, S., Bal, A., Escuret, V., Brengel-Pesce, K., Bouscambert, M., Cheynet, V., Generenaz, L., Oriol, G., Destras, G., and Billaud, G. (2020). Performance Assessment of SARS-CoV-2 PCR Assays Developed by WHO Referral Laboratories. J. Clin. Med., 9.","DOI":"10.1101\/2020.05.03.20072207"},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Graham, T.G.W., Dugast-Darzacq, C., Dailey, G.M., Nguyenla, X.H., Dis, E.V., Esbin, M.N., Abidi, A., Stanley, S.A., Darzacq, X., and Tjian, R. (2020). Inexpensive, versatile and open-source methods for SARS-CoV-2 detection. MedRxiv.","DOI":"10.1101\/2020.09.16.20193466"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"4638","DOI":"10.1021\/acs.biochem.0c00778","article-title":"One-Enzyme Reverse Transcription qPCR Using Taq DNA Polymerase","volume":"59","author":"Bhadra","year":"2020","journal-title":"Biochemistry"},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Bhadra, S., Maranhao, A., Paik, I., and Ellington, A. (2021). A one-enzyme RT-qPCR Assay for SARS-CoV-2, and Procedures for Reagent Production. Bioprotocol, 11.","DOI":"10.21769\/BioProtoc.3898"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"15438","DOI":"10.1074\/jbc.RA120.015434","article-title":"A blueprint for academic laboratories to produce SARS-CoV-2 quantitative RT-PCR test kits","volume":"295","author":"Mascuch","year":"2020","journal-title":"J. Biol. Chem."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"e63","DOI":"10.1093\/nar\/28.12.e63","article-title":"Loop-mediated isothermal amplification of DNA","volume":"28","author":"Notomi","year":"2000","journal-title":"Nucleic Acids Res."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"717","DOI":"10.1039\/C9AY02246E","article-title":"Loop-mediated isothermal amplification (LAMP)\u2014review and classification of methods for sequence-specific detection","volume":"12","author":"Becherer","year":"2020","journal-title":"Anal. Methods"},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Zhang, Y., Odiwuor, N., Xiong, J., Sun, L., Nyaruaba, R.O., Wei, H., and Tanner, N.A. (2020). Rapid Molecular Detection of SARS-CoV-2 (COVID-19) Virus RNA Using Colorimetric LAMP. MedRxiv.","DOI":"10.1101\/2020.02.26.20028373"},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"El-Tholoth, M., Bau, H.H., and Song, J. (2020). A Singel and Two-Stafge, Closed-Tube, Molecular Test for the 2019 Novel Coronavirus (COVID-19) at Home, Clinic, and Points of Entry. ChemRxiv.","DOI":"10.26434\/chemrxiv.11860137.v1"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"870","DOI":"10.1038\/s41587-020-0513-4","article-title":"CRISPR\u2013Cas12-based detection of SARS-CoV-2","volume":"38","author":"Broughton","year":"2020","journal-title":"Nat. Biotechnol."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Spackman, E., and Suarez, D.L. (2008). Avian Influenza Virus, Humana Press.","DOI":"10.1007\/978-1-59745-279-3"},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Kellner, M.J., Ross, J.J., Schnabl, J., Dekens, M.P.S., Heinen, R., Grishkovskaya, I., Bauer, B., Stadlmann, J., Men\u00e9ndez-Arias, L., and Fritsche-Polanz, R. (2020). A rapid, highly sensitive and open-access SARS-CoV-2 detection assay for laboratory and home testing. BioRxiv.","DOI":"10.1101\/2020.06.23.166397"},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Saferali, A., Tang, A.C., Strug, L.J., Quon, B.S., Zlosnik, J., Sandford, A.J., and Turvey, S.E. (2020). Immunomodulatory function of the cystic fibrosis modifier gene BPIFA1. PLoS ONE, 15.","DOI":"10.1371\/journal.pone.0227067"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"2645","DOI":"10.2147\/IJN.S132670","article-title":"Detection of influenza viruses by coupling multiplex reverse-transcription loop-mediated isothermal amplification with cascade invasive reaction using nanoparticles as a sensor","volume":"12","author":"Ge","year":"2017","journal-title":"Int. J. Nanomed."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"3562","DOI":"10.1021\/acs.analchem.5b04054","article-title":"Quenching of Unincorporated Amplification Signal Reporters in Reverse-Transcription Loop-Mediated Isothermal Amplification Enabling Bright, Single-Step, Closed-Tube, and Multiplexed Detection of RNA Viruses","volume":"88","author":"Ball","year":"2016","journal-title":"Anal. Chem."},{"key":"ref_16","unstructured":"Aidelberg, G. (2021). Towards Democratization of Nucleic Acid Detection. [Submitted Thesis, Universite de Paris]."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"1021","DOI":"10.1038\/s41587-020-0655-4","article-title":"The COVID-19 XPRIZE and the need for scalable, fast, and widespread testing","volume":"38","author":"MacKay","year":"2020","journal-title":"Nat. Biotechnol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"24450","DOI":"10.1073\/pnas.2011221117","article-title":"SARS-CoV-2 detection using isothermal amplification and a rapid, inexpensive protocol for sample inactivation and purification","volume":"117","author":"Rabe","year":"2020","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_19","unstructured":"Agrawal, S., Fanton, A., Chandrasekaran, S.S., Charrez, B., Escajeda, A.M., Son, S., Mcintosh, R., Bhuiya, A., Derby, M.D.D., and Switz, N.A. (2020). Rapid detection of SARS-CoV-2 with Cas13. MedRxiv Prepr. Serv. Health Sci."},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Fowler, V.L., Armson, B., Gonzales, J.L., Wise, E.L., Howson, E.L., and Vincent-Mistiaen, Z. (2020). A highly effective reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay for the rapid detection of SARS-CoV-2 infection. MedRxiv.","DOI":"10.1016\/j.jinf.2020.10.039"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"113972","DOI":"10.1016\/j.jviromet.2020.113972","article-title":"Optimization and clinical validation of dual-target RT-LAMP for SARS-CoV-2","volume":"286","author":"Mohon","year":"2020","journal-title":"J. Virol. Methods."},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Yaren, O., McCarter, J., Phadke, N., Bradley, K.M., Overton, B., Yang, Z., Ranade, S., Patil, K., Bangale, R., and Benner, S.A. (2021). Ultra-rapid detection of SARS-CoV-2 in public workspace environments. PLoS ONE, 16.","DOI":"10.1371\/journal.pone.0240524"},{"key":"ref_23","unstructured":"Aidelberg, G., Aronoff, R., Eliseeva, T., Quero, F.J., Vielfaure, H., Codyre, M., Hadasch, K., and Lindner, A.B. (2021). Corona Detective: An RT-LAMP -based, simple, scalable and robust SARS-CoV-2 detection method. J. Biomol. Tech."},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Diego, J.G.-B., Fern\u00e1ndez-Soto, P., Dom\u00ednghez-Gil, M., Belhassen-Garc\u00eda, M., Bellido, J.L.M., and Muro, A. (2020). A Simple, Affordable, Rapid, Stabilized, Colorimetric, Versatile RT-LAMP assay to detect SARS-CoV-2. Diagnostics.","DOI":"10.21203\/rs.3.rs-103047\/v1"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"495","DOI":"10.1007\/s00253-006-0675-0","article-title":"Magnetic particles for the separation and purification of nucleic acids","volume":"73","author":"Berensmeier","year":"2006","journal-title":"Appl. Microbiol. Biot."},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Oberacker, P., Stepper, P., Bond, D.M., H\u00f6hn, S., Focken, J., Meyer, V., Schelle, L., Sugrue, V.J., Jeunen, G., and Moser, T. (2019). Bio-On-Magnetic-Beads (BOMB): Open platform for high-throughput nucleic acid extraction and manipulation. PLoS Biol., 17.","DOI":"10.1371\/journal.pbio.3000107"},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Dahdouh, E., L\u00e1zaro-Perona, F., Romero-G\u00f3mez, M.P., Mingorance, J., Garc\u00eda-Rodriguez, J., and Group S-C-2, W. (2020). Ct values from SARS-CoV-2 diagnostic PCR assays should not be used as direct estimate of viral load. J. Infection.","DOI":"10.1016\/j.jinf.2020.10.017"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"114030","DOI":"10.1016\/j.jviromet.2020.114030","article-title":"Comparison of three TaqMan real-time reverse transcription-PCR assays in detecting SARS-CoV-2","volume":"288","author":"Xiao","year":"2021","journal-title":"J. Virol. Methods."},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Sherrill-Mix, S., Hwang, Y., Roche, A.M., Weiss, S.R., Li, Y., Graham-Wooten, J., Taylor, L.J., Collman, R.G., van Duyne, G.D., and Bushman, F.D. (2020). LAMP-BEAC: Detection of SARS-CoV-2 RNA Using RT-LAMP and Molecular Beacons. MedRxiv.","DOI":"10.1101\/2020.08.13.20173757"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"150","DOI":"10.1006\/bbrc.2001.5921","article-title":"Detection of Loop-Mediated Isothermal Amplification Reaction by Turbidity Derived from Magnesium Pyrophosphate Formation","volume":"289","author":"Mori","year":"2001","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"eabc7075","DOI":"10.1126\/scitranslmed.abc7075","article-title":"A colorimetric RT-LAMP assay and LAMP-sequencing for detecting SARS-CoV-2 RNA in clinical samples","volume":"12","author":"Thi","year":"2020","journal-title":"Sci. Transl. Med."},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Zhang, Y., Ren, G., Buss, J., Barry, A.J., Patton, G.C., and Tanner, N.A. (2020). Enhancing Colorimetric LAMP Amplification Speed and Sensitivity with Guanidine Chloride. BioRxiv.","DOI":"10.1101\/2020.06.03.132894"},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Zhang, Y., and Tanner, N.A. (2020). Development of Multiplexed RT-LAMP for Detection of SARS-CoV-2 and Influenza Viral RNA. MedRxiv.","DOI":"10.1101\/2020.10.26.20219972"},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Bhadra, S., Riedel, T.E., Lakhotia, S., Tran, N.D., and Ellington, A.D. (2020). High-surety isothermal amplification and detection of SARS-CoV-2, including with crude enzymes. BioRxiv.","DOI":"10.1101\/2020.04.13.039941"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"22","DOI":"10.1056\/NEJMp2025631","article-title":"Rethinking Covid-19 Test Sensitivity\u2014A strategy for containment","volume":"383","author":"Mina","year":"2020","journal-title":"N. Engl. J. Med."},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Maranhao, A., Bhadra, S., Paik, I., Walker, D., and Ellington, A.D. (2020). An improved and readily available version of Bst DNA Polymerase for LAMP; and applications to COVID-19 diagnostics. MedRxiv.","DOI":"10.1101\/2020.10.02.20203356"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"114041","DOI":"10.1016\/j.jviromet.2020.114041","article-title":"Rapid and simple colorimetric loop-mediated isothermal amplification (LAMP) assay for the detection of Bovine alphaherpesvirus 1","volume":"289","author":"Peltzer","year":"2021","journal-title":"J. Virol. Methods."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"1928","DOI":"10.4014\/jmb.1806.06016","article-title":"Simple, Rapid and Sensitive Portable Molecular Diagnosis of SFTS Virus Using Reverse Transcriptional Loop-Mediated Isothermal Amplification (RT-LAMP)","volume":"28","author":"Baek","year":"2018","journal-title":"J. Microbiol. Biotechn."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"686","DOI":"10.1007\/s12161-017-1041-9","article-title":"A Multiplex, Fluorescent, and Isothermal Method for Detecting Genetically Modified Maize","volume":"11","year":"2018","journal-title":"Food Anal. Method."}],"container-title":["Viruses"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4915\/13\/5\/742\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T05:52:03Z","timestamp":1760161923000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4915\/13\/5\/742"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,4,23]]},"references-count":39,"journal-issue":{"issue":"5","published-online":{"date-parts":[[2021,5]]}},"alternative-id":["v13050742"],"URL":"https:\/\/doi.org\/10.3390\/v13050742","relation":{"has-preprint":[{"id-type":"doi","id":"10.1101\/2021.02.18.21251793","asserted-by":"object"}]},"ISSN":["1999-4915"],"issn-type":[{"value":"1999-4915","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,4,23]]}}}