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Authors: John-Sebastian Eden, Eby Sim
Group: Coronavirus Method Development Community
Summary: This protocol describes methods to sequence SARS-CoV-2 with pooled amplicons (14 x 2.5kb) using Illumina Platforms.

Proper citation: John-Sebastian Eden, Eby Sim 2020. SARS-CoV-2 Genome Sequencing Using Long Pooled Amplicons on Illumina Platforms. protocols.io dx.doi.org/10.17504/protocols.io.befyjbpw Copy   

•   Source: Protocols.io

Authors: peijun he
Group: Coronavirus Method Development Community, XPRIZE Rapid Covid Testing, Highfield Diagnostics
Summary: Coronavirus Lateral Flow Assay (LFA) operation protocol

Proper citation: peijun he 2020. Coronavirus Lateral Flow Assay (LFA) operation protocol. protocols.io dx.doi.org/10.17504/protocols.io.bksjkwcn Copy   

•   Source: Protocols.io

Authors: Diego Lai
Group: Coronavirus Method Development Community, XPRIZE Rapid Covid Testing
Summary: LooK SPOT COVID-19 antigen rapid test is a lateral flow immunoassay intended for the qualitative detection of nucleocapsid protein antigen from SARS-CoV-2 in nasal swabs from patients suspected of COVID-19 within the first seven (7) days of symptom onset. Testing is limited to laboratories certified under the Clinical Laboratory Improvement Amendments of 1988 (CLIA), 42 U.S.C. §263a, that meet the requirements to perform moderate complexity tests. This test is authorized for use at the Point of Care (POC), i.e., in a patient care settings operating under a CLIA Certificate of Waiver, Certificate of Compliance, or Certificate of Accreditation.].Results are for the identification of SARS-CoV-2 nucleocapsid protein antigen. The antigen is generally detectable in nasal samples during the acute phase of infection. Positive results indicate the presence of viral antigens, but the clinical correlation with patient history and other diagnostic information is necessary to determine infection status. Positive results do not rule out a bacterial infection or co-infection with other viruses. The agent detected may not be the definite cause of thedisease. Laboratories within the United States and its territories are required to report all positive results to the appropriate public health authorities.Negative results should be treated as presumptive, and do not rule out SARS-CoV-2 infection, and should not be used as the sole basis for treatment or patient management decisions, including infection control decisions. Negative results should be considered in the context of a patient's recent exposures, history, and the presence of clinical signs and symptoms consistent with COVID-19, and confirmed with a molecular assay, if necessary, for patient management. LooK SPOT COVID-19 Antigen Rapid Test can deliver a diagnosis of the SARS-CoV-2 virus detection between 5 to 8 minutes by using machine learning AI. LooK SPOT’s AI algorithm has high accuracy and can identify the fluorescence response when human eyes cannot identify the low positive cases. Healthcare responders in the COVID-19 test sites often need to make time-sensitive decisions to determine the test results during the time many patients are within their vicinity. But the tempo, volume, stress, fatigue, lighting, fear, and various other factors can overwhelm healthcare responders when making the visual interpretation of antigen test results. It is of paramount importance to reduce healthcare responders' cognitive load by providing accurate test results in an easy-to-read format. LooK SPOT COVID-19 Antigen Rapid Test is a COVID-19 rapid test solution designed with a tactical edge to fight COVID-19. The LooK SPOT COVID-19 Antigen Rapid Test is intended for use at the Point of Care (POC) settings by trained personnel specifically instructed and trained in vitro diagnostic procedures. It is only for use under the Food and Drug Administration's Emergency Use Authorization.

Proper citation: Diego Lai 2020. LooK SPOT Antigen Rapid Test System. protocols.io dx.doi.org/10.17504/protocols.io.bp32mqqe Copy   

•   Source: Protocols.io

Authors: Stephanie Loeb, Katy Graham, Marlene Wolfe, Krista Wigginton, Alexandria Boehm
Group: Coronavirus Method Development Community, Wastewater-based epidemiology working group
Summary: OverviewThis SOP describes pre-analytical procedures to be followed for the isolation and identification of SARS-CoV-2 RNA in primary settled solids samples from wastewater treatment plants. This protocal should be paired with our digital PCR protocal. The protocol follows the approximate workflow:Separate and aliquot sample (~4 hrs)Spike with BCoV (~1 hr)Extract RNA (~ 12 hours total, may be split into 2-3 steps/days)Inhibitor removal (~1 hr)Biosafety ConcernsConcentration and extraction procedures that utilize raw samples must adhere to strict Biosafety Level 2+ procedures. These procedures should be performed in a dedicated room. Downstream products may be handled using standard laboratory safety guidelines. This protocol was developed by Stephanie Loeb, Katy Graham, Marlene Wolfe, Krista Wigginton, and Alexandria Boehm at Stanford University and University of Michigan.

Proper citation: Stephanie Loeb, Katy Graham, Marlene Wolfe, Krista Wigginton, Alexandria Boehm 2020. Extraction of RNA from Wastewater Primary Solids Using a Direct Extraction Method for Downstream SARS-CoV-2 RNA Quantification. protocols.io dx.doi.org/10.17504/protocols.io.bi6skhee Copy   

•   Source: Protocols.io

Authors: Chantal Vogels, Joseph Fauver, Isabel Ott, Nathan Grubaugh
Group: Coronavirus Method Development Community
Summary: The protocol describes how to generate high-quality single-stranded RNA transcript standards (starting from a virus RNA stock) targeting the nsp10, RdRp, nsp14, envelope (E), and nucleocapsid (N) coding regions for use with China CDC, Hong Kong University (HKU), Corman et al. (Berlin), and US CDC SARS-CoV-2 primer and probe sets for qRT-PCR. (Sequences for transcripts generated, along with their corresponding assays, are provided under 'Guidelines.')

Proper citation: Chantal Vogels, Joseph Fauver, Isabel Ott, Nathan Grubaugh 2020. Generation of SARS-COV-2 RNA transcript standards for qRT-PCR detection assays. protocols.io dx.doi.org/10.17504/protocols.io.bdv6i69e Copy   

•   Source: Protocols.io

Authors: Oscar Whitney, Basem Al-Shayeb, Alex Crits-Cristoph, Mira Chaplin, Vinson Fan, Hannah Greenwald, Adrian Hinkle, Rose Kantor, Lauren Kennedy, Anna Maurer, Robert Tjian, Kara L. Nelson, UC Berkeley Wastewater-based epidemiology consortium
Group: Coronavirus Method Development Community
Summary: This protocol describes the procedure of the "4S" (Sewage, Salt, Silica and SARS-CoV-2) method for SARS-CoV-2 RNA extraction from wastewater. Offering a highly efficient, modular and economical alternative to existing wastewater RNA purification methods, this procedure lowers the barrier to entry for SARS-CoV-2 wastewater-based epidemiology. This procedure is intended to be carried out in a BSL2+ laboratory space, with precautions when handling raw wastewater samples.

Proper citation: Oscar Whitney, Basem Al-Shayeb, Alex Crits-Cristoph, Mira Chaplin, Vinson Fan, Hannah Greenwald, Adrian Hinkle, Rose Kantor, Lauren Kennedy, Anna Maurer, Robert Tjian, Kara L. Nelson, UC Berkeley Wastewater-based epidemiology consortium 2020. V.4 - Direct wastewater RNA capture and purification via the "Sewage, Salt, Silica and SARS-CoV-2 (4S)" method. protocols.io dx.doi.org/10.17504/protocols.io.bpdfmi3n Copy   

•   Source: Protocols.io

Authors: Monica Simion, Alexandru Salceanu
Group: Coronavirus Method Development Community, covid19-test, XPRIZE Rapid Covid Testing

Proper citation: Monica Simion, Alexandru Salceanu 2020. microfluidic sars cov 2 lamp protocol. protocols.io dx.doi.org/10.17504/protocols.io.bkjjkukn Copy   

•   Source: Protocols.io

Authors: Lyle J. Arnold Ph.D., Stella M. Sung Ph.D.
Group: Coronavirus Method Development Community, XPRIZE Rapid Covid Testing
Summary: This protocol is for the Aegea Biotechnologies rapid PCR-based SARS-CoV-2 test. This assay uses patented "Switch-Blocker" technology as well as taqman probes to test for presence of SARS-CoV-2 and simultaneously orthogonally validate. A single amplification reaction is performed, and "Switch Blocker" is used on the forward strand and taqman is used on the reverse strand. The assay design has high sensitivity & specificity--single nucleotide level. Moreover, it is able to detect the SARS-CoV-2 L strain vs the SARS-CoV-2 S strain. The test can be adapted to point of care (Roche LIAT) as well as for different SARS-CoV-2 strains as the virus mutates. A next generation version of the assay could identify the presence of the SARS-CoV-2 L/S strains vs. influenza A/B. Finally, because of the sensitivity and specificity, the Aegea PCR-based SARS-CoV-2 test should be able to use saliva samples, and it is suitable for pooled testing. This protocol is designed for high throughput PCR (96 or 384 well plate formats).Keywords: PCR, COVID-19, coronavirus, SARS-CoV-2, high throughput, multiplex, Switch-Blocker, taqman, high sensitivity, high specificity, accurate, pooling, saliva, strain types, L-strain, S-strain, combination SARS-CoV-2 and influenza

Proper citation: Lyle J. Arnold Ph.D., Stella M. Sung Ph.D. 2020. Aegea Biotechnologies rapid PCR SARS-CoV-2 test (high sensitivity & specificity; able to detect different strain types). protocols.io dx.doi.org/10.17504/protocols.io.bk2dkya6 Copy   

•   Source: Protocols.io

Authors: Emma Betteridge, Naomi Park, Keith James, Jillian Durham, Josh Quick
Group: Coronavirus Method Development Community
Summary: This SOP describes the procedure for generating cDNA from SARS-CoV-2 viral nucleic acid extracts and subsequently producing 275nt amplicons. This is followed by library construction, equivolume pooling of samples and quantitation, prior to sequencing on the Illumina NovaSeq. A key benefit of the short amplicon method is that it can be utilised on any Illumina platform with a 300 cycle (or greater) kit.It is an adaptation of the 400nt COVID-19 ARTIC v3 amplicon protocol which can be found here:https://www.protocols.io/view/covid-19-artic-v3-illumina-library-construction-an-bgxjjxknBoth the above protocols were adapted from the nCov-2019 sequencing protocol: https://www.protocols.io/view/ncov-2019-sequencing-protocol-bbmuik6w

Proper citation: Emma Betteridge, Naomi Park, Keith James, Jillian Durham, Josh Quick 2020. COVID-19 ARTIC v3 Illumina library construction and sequencing protocol - short amplicons (275bp). protocols.io dx.doi.org/10.17504/protocols.io.bh4zj8x6 Copy   

•   Source: Protocols.io

Authors: Nabil-Fareed Alikhan, Emma Griffiths, Ruth Timme, Duncan MacCannell
Group: Coronavirus Method Development Community, PHA4GE
Summary: Guidance on how to populate the extended PHA4GE metadata package for SARS-CoV-2 submissions, maximizing interoperability for covid-19 surveillance.

Proper citation: Nabil-Fareed Alikhan, Emma Griffiths, Ruth Timme, Duncan MacCannell 2020. SOP for populating EBI submission templates (ENA). protocols.io dx.doi.org/10.17504/protocols.io.bh5dj826 Copy   

•   Source: Protocols.io

Authors: Max Marrone
Group: Coronavirus Method Development Community, Opentrons COVID-19 Testing

Proper citation: Max Marrone 2020. Operating an OT-2 for COVID-19 testing. protocols.io dx.doi.org/10.17504/protocols.io.bejejcje Copy   

•   Source: Protocols.io

Authors: Mario Thomas, Jasmine Sollen, Natalia Ivanova, Michelle Feng, Reda Fayek, Heidi Abdilla, Stephanie Lim, Amanda Naaum
Group: Coronavirus Method Development Community, XPRIZE Rapid Covid Testing, SANATA
Summary: This procedure outlines the protocol for testing for SARS-CoV-2 using a saliva sample collected from an individual. The purpose of this test is to detect low levels of SARS-CoV-2 antigen at a higher sensitivity. Precision Biomonitoring Inc. developed an ultra-rapid digital, disposable, highly-sensitive and inexpensive testing device used for screening purposes. The mobile app complementary to this medical device is connected through Bluetooth. Using this innovation, the user can be tested at point-of-care (POC) by a health care professional, and obtain qualitative results.

Proper citation: Mario Thomas, Jasmine Sollen, Natalia Ivanova, Michelle Feng, Reda Fayek, Heidi Abdilla, Stephanie Lim, Amanda Naaum 2020. XPRIZE SANATA Protocol for Saliva LFIA Test. protocols.io dx.doi.org/10.17504/protocols.io.bqfrmtm6 Copy   

•   Source: Protocols.io

Authors: Nicola O'Reilly, Svend Kjaer, Maria Greco
Group: Coronavirus Method Development Community, Crick COVID-19 Consortium
Summary: Purpose of examination / Clinical relevanceAt the end of 2019, several pneumonia cases were reported in Wuhan, China and the pathogen was confirmed as a new viral strain. World Health organization has named the newly identified coronavirus as 2019-nCoV, also known as COVID19. The disease developed into a dangerous pandemic, posing major challenges to the NHS. Although more research is necessary to better understand the virus, in response to the emergency, simple and rapid testing is essential to identify the virus in infected individuals. This will aid the implementation of efficient interventions to contain the spread, and distinguish healthcare workers who have been infected, and are required to self-isolate, from those showing similar symptoms but which are not 2019-nCoV associated. The latter category may continue to work, alleviating stress on hard-pressed healthcare resources. 2019-nCoV is an RNA virus, and the diagnostic tests detect viral RNA in swabs from patient airways using a reverse transcriptase PCR assay. Samples are submitted to HSL, an accredited reporting laboratory, and transferred to FCI for testing.Principles of ExaminationThis procedure involves the preparation of binding buffer for the RNA extraction protocol.

Proper citation: Nicola O'Reilly, Svend Kjaer, Maria Greco 2020. Preparation of Binding Buffer (BB). protocols.io dx.doi.org/10.17504/protocols.io.bfd2ji8e Copy   

•   Source: Protocols.io

Authors: John Daniels
Group: Coronavirus Method Development Community, Front Line Technologies, XPRIZE Rapid Covid Testing, Team Exhaled Breath Condensate
Summary: The Kinaptic mask-based COVID-19 testing system uses a unique Exhaled Breath Condensate (EBC) collector that converts breath vapor to a liquid biosample for rapid testing and wireless automatic contact tracing. Exhaled Breath Condensate (EBC) is collected from a mask-based EBC collector as a low cost disposable component. Our electronic biosensor detects even low concentrations of virus biomarkers if they are present in the EBC sample. Corona viruses have slowly evolving N-protein and quickly evolving S-protein. N-Protein testing screens for any corona virus infection / S-Protein screens for only COVID-19 infection. The Kinaptic mask-based COVID-19 testing system is a “two-stage filter”: 1) catch only of N-protein biomarker = trigger warning to get more stringent diagnostic and contact trace; 2) catch both N- and S-, or just S- biomarkers = more urgent need for immediate quarantine and medical attention. As the target biomarkers bind to the capture molecules, the electrical conductivity of the electronic biosensor changes. This change in conductive is amplified by our bluetooth electronics, analyzed to determine the presence and quantity of the target biomarker, and the test result wireless transmitted to a smartphone or directly to the cloud.

Proper citation: John Daniels 2020. Mask-Based Covid-10 testing system using Exhaled Breath Condensate. protocols.io dx.doi.org/10.17504/protocols.io.bkzzkx76 Copy   

•   Source: Protocols.io

Authors: Chris Ockenhouse, Chris Gast, Renee Holt, Jorge Flores
Group: Coronavirus Method Development Community, PATH
Summary: This is Part 9 of "Phase 3 randomized, double-blinded, placebo-controlled trial to evaluate the safety, immunogenicity, and efficacy of Vaccine Candidate against COVID-19 in adults > 18 years of age"This generic Phase 3 protocol was developed by the PATH team with support of the Bill and Melinda Gates Foundation. The aim of the collection is to share recommended best practices in designing and implementing a Phase 3 study of a COVID-19 vaccine candidate. As Phase 3 trials of different Vaccine Candidates proceed around the world, following the same protocols will ensure consistency and comparability of the Phase 3 trial results.Please note that this is an evolving document, to be versioned and updated, based on community feedback and new data.

Proper citation: Chris Ockenhouse, Chris Gast, Renee Holt, Jorge Flores 2020. Statistical Considerations (Part 9 of Phase 3 study of Vaccine Candidate for COVID-19). protocols.io dx.doi.org/10.17504/protocols.io.bj55kq86 Copy   

•   Source: Protocols.io

Authors: Josh Quick
Group: Diaz-Munoz Lab, Coronavirus Method Development Community, Szentagothai Research Centre Virology
Summary: This is a subprotocol for performing adapter ligation with AMII

Proper citation: Josh Quick 2020. Adapter ligation with AMII. protocols.io dx.doi.org/10.17504/protocols.io.bdp9i5r6 Copy   

•   Source: Protocols.io

Authors: Shripal Gandhi, Santo Purnama, Keyur Patel, Praveen Sukumara, Dr Rinu R Ravi
Group: Coronavirus Method Development Community, SensingSelfMission, XPRIZE Rapid Covid Testing
Summary: Summary:The S1 COVID-19 Rapid Antigen test kit developed by Sensing Self Pte. Ltd. can quickly and qualitatively detect the spike glycoprotein of novel coronavirus (SARS-COV-2) in human saliva/sputum/stool samples. It can be used as an aid for COVID-19 diagnosis.Objective:According to the clinical trial plan, the S1 COVID-19 Rapid Antigen test kit or “test reagent”, is to test saliva/sputum/stool samples from healthy subjects and confirmed COVID-19 patients. Test results are compared with another commercial SARS-COV-2 nucleic acid detection kit with CFDA approval, which is defined as the “gold standard”. The sensitivity, specificity, and total agreement rate are used to evaluate the feasibility of the test reagent in clinical applications.Method:A collection of clinical samples were examined by the S1 COVID-19 Rapid Antigen test kit and the gold standard SARS-COV-2 nucleic acid detection kit in parallel, to calculate the clinical sensitivity, clinical specificity and total agreement rate of the test reagent.Results:Compared to the gold standard, the clinical sensitivity of test reagent reached 90.0%, the clinical specificity reached 100.0%, and the total coincidence rate reached 95.0%.Conclusions:The performance of test reagent has a high agreement rate with the gold standard, proving its good feasibility in diagnosing suspected COVID-19 cases.

Proper citation: Shripal Gandhi, Santo Purnama, Keyur Patel, Praveen Sukumara, Dr Rinu R Ravi 2020. SensingSelf S1 Rapid Antigen Test (Saliva/Sputum/Stool). protocols.io dx.doi.org/10.17504/protocols.io.bj6ykrfw Copy   

•   Source: Protocols.io

Authors: New England Biolabs
Group: Coronavirus Method Development Community
Summary: This protocol details methods for the NEBNext® ARTIC SARS-CoV-2 Companion Kit (Oxford Nanopore Technologies®), NEB #E7660S/L 24/96 reactions.

Proper citation: New England Biolabs 2021. NEBNext® ARTIC SARS-CoV-2 Companion Kit (Oxford Nanopore Technologies®) E7660. protocols.io dx.doi.org/10.17504/protocols.io.btcenite Copy   

•   Source: Protocols.io

Authors: Josh Quick
Group: Diaz-Munoz Lab, Coronavirus Method Development Community, Szentagothai Research Centre Virology

Proper citation: Josh Quick 2019. Starting a MinION sequencing run using MinKNOW. protocols.io dx.doi.org/10.17504/protocols.io.7q6hmze Copy   

•   Source: Protocols.io

Authors: steve.puts
Group: Coronavirus Method Development Community, Mmolecular, XPRIZE Rapid Covid Testing
Summary: The Direct One-Step RT-qPCR COVID-19 Test Kit is designed for quantitativereal-time analysis of target RNA directly from, swabs without the requirement of any priorRNA purification steps.Shipping: shipped on blue ice Storage Conditions: store at -20 °C Additional Storage Conditions: avoid freeze/thaw cycles stable at 4 °C for up to 4 weeks Shelf Life: 12 monthsForm: liquid / lyophilized

Proper citation: steve.puts 2020. Mmolecular COVID-19 Extraction-Free Direct-One-Step Fast Cycling Protocol. protocols.io dx.doi.org/10.17504/protocols.io.bkjpkumn Copy   

•   Source: Protocols.io