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Public health strategies to reduce viral transmission are based on widespread diagnostic testing to detect and isolate contagious patients. Several reverse transcription (RT)-PCR tests, along with other SARS-CoV-2 diagnostic assays, are available to attempt to cover the global demand. Loop-mediated isothermal amplification (LAMP) based methods have been established as rapid, accurate, point of care diagnostic tests for viral infections; hence, they represent an excellent alternative for SARS-CoV-2 detection. The aim of this study was to develop and describe molecular detection systems for SARS-CoV-2 based on RT-LAMP. Recombinant DNA polymerase from<jats:italic>Bacillus stearothermophilus<\/jats:italic>and thermostable engineered reverse transcriptase from Moloney Murine Leukemia Virus were expressed using a prokaryotic system and purified by fast protein liquid chromatography. These enzymes were used to set up fluorometric real time and colorimetric end-point RT-LAMP assays. Several reaction conditions were optimized such as reaction temperature, Tris-HCl concentration, and pH of the diagnostic tests. The key enzymes for RT-LAMP were purified and their enzymatic activity was determined. Standardized reaction conditions for both RT-LAMP assays were 65\u00b0C and a Tris-HCl-free buffer at pH 8.8. Colorimetric end-point RT-LAMP assay was successfully used for viral detection from clinical saliva samples with 100% sensitivity and 100% specificity compared to the results obtained by RT-qPCR based diagnostic protocols with Ct values until 30. The developed RT-LAMP diagnostic tests based on purified recombinant enzymes allowed a sensitive and specific detection of the nucleocapsid gene of SARS-CoV-2.<\/jats:p>","DOI":"10.1371\/journal.pone.0279681","type":"journal-article","created":{"date-parts":[[2023,1,26]],"date-time":"2023-01-26T18:41:17Z","timestamp":1674758477000},"page":"e0279681","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":13,"title":["Starting from scratch: Step-by-step development of diagnostic tests for SARS-CoV-2 detection by RT-LAMP"],"prefix":"10.1371","volume":"18","author":[{"given":"Diana Ang\u00e9lica","family":"Tapia-Sidas","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Brenda Yazm\u00edn","family":"Vargas-Hern\u00e1ndez","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jos\u00e9 Abrah\u00e1n","family":"Ram\u00edrez-Pool","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Leandro Alberto","family":"N\u00fa\u00f1ez-Mu\u00f1oz","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6144-1411","authenticated-orcid":true,"given":"Berenice","family":"Calder\u00f3n-P\u00e9rez","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5761-0633","authenticated-orcid":true,"given":"Rogelio","family":"Gonz\u00e1lez-Gonz\u00e1lez","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Luis Gabriel","family":"Brieba","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6812-9471","authenticated-orcid":true,"given":"Rosal\u00eda","family":"Lira-Carmona","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Eduardo","family":"Ferat-Osorio","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0530-492X","authenticated-orcid":true,"given":"Constantino","family":"L\u00f3pez-Mac\u00edas","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Roberto","family":"Ruiz-Medrano","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7366-7725","authenticated-orcid":true,"given":"Beatriz","family":"Xoconostle-C\u00e1zares","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"340","published-online":{"date-parts":[[2023,1,26]]},"reference":[{"key":"pone.0279681.ref001","unstructured":"WHO. Rolling updates on coronavirus disease (COVID-19). World Health Organ 2020. https:\/\/www.who.int\/emergencies\/diseases\/novel-coronavirus-2019\/events-as-they-happen."},{"key":"pone.0279681.ref002","doi-asserted-by":"crossref","first-page":"727","DOI":"10.1056\/NEJMoa2001017","article-title":"A novel coronavirus from patients with pneumonia in China, 2019","volume":"382","author":"N Zhu","year":"2020","journal-title":"N Engl J Med"},{"key":"pone.0279681.ref003","unstructured":"CDC. Coronavirus Disease 2019 (COVID-19): Interim Clinical Guidance for Management of Patients with Confirmed Coronavirus Disease (COVID-19). Cent Dis Control Prev 2020. https:\/\/www.cdc.gov\/coronavirus\/2019-ncov\/hcp\/clinical-guidance-management-patients.html."},{"key":"pone.0279681.ref004","doi-asserted-by":"crossref","first-page":"497","DOI":"10.1016\/S0140-6736(20)30183-5","article-title":"Clinical features of patients infected with 2019 novel coronavirus in Wuhan","volume":"395","author":"C Huang","year":"2020","journal-title":"China. The Lancet"},{"key":"pone.0279681.ref005","first-page":"1","article-title":"Prevalence of Asymptomatic SARS-CoV-2 Infection","author":"DP Oran","year":"2020","journal-title":"Ann Intern Med"},{"key":"pone.0279681.ref006","first-page":"1","article-title":"Unclear but present danger: An asymptomatic SARS-CoV-2 carrier","author":"X Yu","year":"2020","journal-title":"Genes Dis"},{"key":"pone.0279681.ref007","doi-asserted-by":"crossref","first-page":"1248","DOI":"10.1016\/j.cmi.2020.06.009","article-title":"Large-scale implementation of pooled RNA extraction and RT-PCR for SARS-CoV-2 detection","volume":"26","author":"R Ben-Ami","year":"2020","journal-title":"Clin Microbiol Infect"},{"key":"pone.0279681.ref008","article-title":"SalivaDirect: A simplified and flexible platform to enhance SARS-CoV-2 testing capacity","volume":"2","author":"CBF Vogels","year":"2021","journal-title":"Med"},{"key":"pone.0279681.ref009","doi-asserted-by":"crossref","first-page":"901","DOI":"10.1016\/j.jiph.2020.06.005","article-title":"Laboratory diagnosis of SARS-CoV-2\u2014A review of current methods","volume":"13","author":"JP Mathuria","year":"2020","journal-title":"J Infect Public Health"},{"key":"pone.0279681.ref010","doi-asserted-by":"crossref","first-page":"771","DOI":"10.1261\/rna.076232.120","article-title":"Overcoming the bottleneck to widespread testing: A rapid review of nucleic acid testing approaches for COVID-19 detection","volume":"26","author":"MN Esbin","year":"2020","journal-title":"RNA"},{"key":"pone.0279681.ref011","doi-asserted-by":"crossref","first-page":"516","DOI":"10.1038\/d41587-020-00010-2","article-title":"Fast, portable tests come online to curb coronavirus pandemic","volume":"38","author":"C Sheridan","year":"2020","journal-title":"Nat Biotechnol"},{"key":"pone.0279681.ref012","doi-asserted-by":"crossref","first-page":"281","DOI":"10.1080\/20477724.2021.1933335","article-title":"Reverse Transcriptase Loop Mediated Isothermal Amplification (RT-LAMP) for COVID-19 diagnosis: a systematic review and meta-analysis","volume":"115","author":"AD Subali","year":"2021","journal-title":"Pathog Glob Health"},{"key":"pone.0279681.ref013","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.mehy.2020.109786","article-title":"Loop mediated isothermal ampli fi cation (LAMP) assays as a rapid diagnostic for COVID-19","volume":"141","author":"J Kashir","year":"2020","journal-title":"Med Hypotheses"},{"key":"pone.0279681.ref014","doi-asserted-by":"crossref","first-page":"121","DOI":"10.21315\/mjms2018.25.1.15","article-title":"LAMP method as one of the best candidates for replacing with PCR method","volume":"25","author":"M. 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Nkengasong","year":"2020","journal-title":"Nature"},{"key":"pone.0279681.ref027","doi-asserted-by":"crossref","first-page":"91","DOI":"10.1016\/j.jviromet.2018.02.012","article-title":"A multiplexed RT-LAMP assay for detection of group M HIV-1 in plasma or whole blood","volume":"255","author":"KA Curtis","year":"2018","journal-title":"J Virol Methods"},{"key":"pone.0279681.ref028","doi-asserted-by":"crossref","first-page":"773","DOI":"10.1016\/j.cmi.2020.04.001","article-title":"Rapid and visual detection of 2019 novel coronavirus (SARS-CoV-2) by a reverse transcription loop-mediated isothermal amplification assay","volume":"26","author":"C Yan","year":"2020","journal-title":"Clin Microbiol Infect"},{"key":"pone.0279681.ref029","doi-asserted-by":"crossref","first-page":"269","DOI":"10.1016\/j.bbrc.2014.10.044","article-title":"Amino acid substitutions away from the RNase H catalytic site increase the thermal stability of Moloney murine leukemia virus reverse transcriptase through RNase H inactivation","volume":"454","author":"A Konishi","year":"2014","journal-title":"Biochem Biophys Res Commun"},{"key":"pone.0279681.ref030","doi-asserted-by":"crossref","first-page":"299","DOI":"10.1016\/j.jbiotec.2010.09.961","article-title":"Increase in thermal stability of Moloney murine leukaemia virus reverse transcriptase by site-directed mutagenesis","volume":"150","author":"K Yasukawa","year":"2010","journal-title":"J Biotechnol"},{"key":"pone.0279681.ref031","first-page":"7","article-title":"A Quick and Easy Method for Making Competent Escherichia coli Cells for Transformation Using Rubidium Chloride","author":"N Sharma","year":"2017","journal-title":"BIO-Protoc"},{"key":"pone.0279681.ref032","unstructured":"Centers for Disease Control and Prevention (CDC). Research Use Only 2019-Novel Coronavirus (2019-nCoV) Real-time RT-PCR Primers and Probes. Res Use 2019-Nov Coronavirus 2019-NCoV Real-Time RT-PCR Primer Probes 2020. https:\/\/www.cdc.gov\/coronavirus\/2019-ncov\/lab\/rt-pcr-panel-primer-probes.html."},{"key":"pone.0279681.ref033","first-page":"25","article-title":"Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR","author":"VM Corman","year":"2020","journal-title":"Eurosurveillance"},{"key":"pone.0279681.ref034","first-page":"5","article-title":"Sensitivity, Specificity, and Predictive Values: Foundations, Pliabilities, and Pitfalls in Research and Practice","author":"R Trevethan","year":"2017","journal-title":"Front Public Health"},{"key":"pone.0279681.ref035","doi-asserted-by":"crossref","first-page":"593","DOI":"10.1038\/s41563-020-00906-z","article-title":"Diagnostics for SARS-CoV-2 infections","volume":"20","author":"BD Kevadiya","year":"2021","journal-title":"Nat 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Sarbecovirus genomes","author":"I Jungreis","year":"2021","journal-title":"Nat Commun"},{"key":"pone.0279681.ref044","doi-asserted-by":"crossref","first-page":"3487","DOI":"10.1038\/s41598-021-83105-3","article-title":"Genomic mutations and changes in protein secondary structure and solvent accessibility of SARS-CoV-2 (COVID-19 virus)","volume":"11","author":"TT Nguyen","year":"2021","journal-title":"Sci Rep"},{"key":"pone.0279681.ref045","doi-asserted-by":"crossref","first-page":"100033","DOI":"10.1016\/j.snr.2021.100033","article-title":"A review of reaction enhancement strategies for isothermal nucleic acid amplification reactions","volume":"3","author":"B \u00d6zay","year":"2021","journal-title":"Sens Actuators Rep"},{"key":"pone.0279681.ref046","doi-asserted-by":"crossref","first-page":"973","DOI":"10.1002\/slct.201702542","article-title":"PCR under Low Ionic Concentration Buffer Conditions","volume":"3","author":"F Takei","year":"2018","journal-title":"ChemistrySelect"},{"key":"pone.0279681.ref047","first-page":"1","article-title":"Enhancing colorimetric LAMP amplification speed and sensitivity with guanidine chloride","volume":"69","author":"Y Zhang","year":"2020","journal-title":"bioRxiv"},{"key":"pone.0279681.ref048","doi-asserted-by":"crossref","first-page":"6026","DOI":"10.1039\/D1AN01043C","article-title":"All-in-one in situ colorimetric RT-LAMP assay for point-of-care testing of SARS-CoV-2","volume":"146","author":"Y He","year":"2021","journal-title":"The Analyst"},{"key":"pone.0279681.ref049","first-page":"1","article-title":"Loop-mediated isothermal amplification (LAMP) reaction as viable PCR substitute for diagnostic applications: A comparative analysis study of LAMP, conventional PCR, nested PCR (nPCR) and real-time PCR (qPCR) based on Entamoeba histolytica DNA derived from","volume":"20","author":"PC Foo","year":"2020","journal-title":"BMC 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Yang","year":"2020","journal-title":"medRxiv"},{"key":"pone.0279681.ref053","doi-asserted-by":"crossref","first-page":"924","DOI":"10.1002\/ejp.1722","article-title":"Nasopharyngeal swab\u2010induced pain for SARS\u2010CoV\u20102 screening: A randomised controlled trial of conventional and self\u2010swabbing","volume":"25","author":"X Moisset","year":"2021","journal-title":"Eur J Pain"},{"key":"pone.0279681.ref054","doi-asserted-by":"crossref","first-page":"1619","DOI":"10.1007\/s00784-020-03248-x","article-title":"Coronavirus COVID-19 impacts to dentistry and potential salivary diagnosis","volume":"24","author":"R Sabino-Silva","year":"2020","journal-title":"Clin Oral Investig"},{"key":"pone.0279681.ref055","unstructured":"ECDC EC for DP and C. Considerations for the use of saliva as sample material for COVID-19 testing, May 2021. 2021."},{"key":"pone.0279681.ref056","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1038\/s41598-020-67825-6","article-title":"Overcoming challenges in human saliva gene expression measurements","volume":"10","author":"P Ostheim","year":"2020","journal-title":"Sci Rep"},{"key":"pone.0279681.ref057","doi-asserted-by":"crossref","first-page":"74","DOI":"10.1016\/j.jviromet.2007.10.020","article-title":"Comparison between serum and saliva for the detection of hepatitis A virus RNA","volume":"148","author":"LA Amado","year":"2008","journal-title":"J Virol Methods"},{"key":"pone.0279681.ref058","doi-asserted-by":"crossref","first-page":"365","DOI":"10.1101\/gr.3.6.365","article-title":"Inhibitory effect of salivary fluids on PCR: Potency and removal","volume":"3","author":"AS Ochert","year":"1994","journal-title":"PCR Methods Appl"},{"key":"pone.0279681.ref059","first-page":"9","article-title":"Oral Microbiome Alterations and SARS-CoV-2 Saliva Viral Load in Patients with COVID-19","author":"EH Miller","year":"2021","journal-title":"Microbiol Spectr"},{"key":"pone.0279681.ref060","doi-asserted-by":"crossref","first-page":"102","DOI":"10.1016\/j.jviromet.2017.08.013","article-title":"Defining the relative performance of isothermal assays that can be used for rapid and sensitive detection of foot-and-mouth disease virus","volume":"249","author":"ELA Howson","year":"2017","journal-title":"J Virol Methods"},{"key":"pone.0279681.ref061","first-page":"11","article-title":"Saliva is more sensitive than nasopharyngeal or nasal swabs for diagnosis of asymptomatic and mild COVID-19 infection","author":"AKJ Teo","year":"2021","journal-title":"Sci Rep"},{"key":"pone.0279681.ref062","article-title":"Saliva swabs are the preferred sample for Omicron detection","author":"G Marais","year":"2021","journal-title":"medRXiv"},{"key":"pone.0279681.ref063","first-page":"12","article-title":"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","author":"PA Alves","year":"2021","journal-title":"Front Microbiol"},{"key":"pone.0279681.ref064","doi-asserted-by":"crossref","first-page":"114392","DOI":"10.1016\/j.jviromet.2021.114392","article-title":"The screening value of RT-LAMP and RT-PCR in the diagnosis of COVID-19: systematic review and meta-analysis","volume":"300","author":"R Pu","year":"2022","journal-title":"J Virol Methods"},{"key":"pone.0279681.ref065","doi-asserted-by":"crossref","first-page":"e0266703","DOI":"10.1371\/journal.pone.0266703","article-title":"A rapid, specific, extraction-less, and cost-effective RT-LAMP test for the detection of SARS-CoV-2 in 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