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Authors: tenOever Lab
Group: Coronavirus Method Development Community
Summary: Method to plaque the SARS-CoV-2 virus Image attribution:Y tambe / CC BY-SA (http://creativecommons.org/licenses/by-sa/3.0/)Downloaded from: https://commons.wikimedia.org/wiki/File:Plaque_assay_macro.jpg

Proper citation: tenOever Lab 2020. SARS-CoV-2 virus plaque assays [Biosafety Level 3]. protocols.io https://dx.doi.org/10.17504/protocols.io.bdtni6me Copy   

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Authors: samz
Group: Coronavirus Method Development Community, XPRIZE Rapid Covid Testing

Proper citation: samz 2020. AMDx Lateral Flow Assay WIth a Cover Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.bk54ky8w Copy   

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Authors: Alejandro Caro-Quintero, Roxana Yockteng
Group: Coronavirus Method Development Community, Reclone.org (The Reagent Collaboration Network)
Summary: Here we present the second version of an RNA extraction protocol for SARs-cov2 using magnetic beads. This protocol was optimized in AGROSAVIA laboratories and is presented as an alternative to the limitations of commercial viral RNA extraction kits. The viral RNA extraction protocol was adapted from the protocol entitled “SARS-CoV-2 RNA purification from nasal / throat swabs collected in Viral Transfer Media” at https://bomb.bio/ and which is based on methods previously reported by the same group (Oberacker et al 2019). The optimized version here achieves Ct results like the commercial kits and reduces the cost of RNA extraction per sample by 90%, that is, 2000 Colombian pesos or 50 cents per sample. We share this protocol in the hope that it can ensure the sustainability of the diagnosis in Colombia and other countries where it is difficult and expensive to import commercial kits.REFERENCESOberacker, P., Stepper, P., Bond, D. M., Höhn, S., Focken, J., Meyer, V., ... & Hore, S. R. (2019). Bio-On-Magnetic-Beads (BOMB): Open platform for high-throughput nucleic acid extraction and manipulation. PLoS biology, 17(1), e3000107.Yu, F., Qiu, T., Zeng, Y., Wang, Y., Zheng, S., Chen, X., & Chen, Y. (2018). Comparative evaluation of three Preprocessing Methods for extraction and detection of Influenza A Virus Nucleic Acids from sputum. Frontiers in Medicine, 5, 56.He, H., Li, R., Chen, Y., Pan, P., Tong, W., Dong, X., ... & Yu, D. (2017). Integrated DNA and RNA extraction using magnetic beads from viral pathogens causing acute respiratory infections. Scientific reports, 7, 45199.Corman, V.M., Landt, O., Kaiser, M., Molenkamp, R., Meijer, A., Chu, D.K., Bleicker, T.,Brünink, S., Schneider, J., Schmidt, M.L. and Mulders, D.G., 2020. Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Eurosurveillance, 25(3).Kumar, M., Mazur, S., Ork, B.L., Postnikova, E., Hensley, L.E., Jahrling, P.B., Johnson, R.and Holbrook, M.R., 2015. Inactivation and safety testing of Middle East respiratory syndrome coronavirus. Journal of virological methods, 223, pp.13-18

Proper citation: Alejandro Caro-Quintero, Roxana Yockteng 2020. Viral RNA extraction low-cost protocol optimized for SARS-Cov2 at AGROSAVIA. protocols.io https://dx.doi.org/10.17504/protocols.io.bggvjtw6 Copy   

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Authors: Addgene The Nonprofit Plasmid Repository
Group: Coronavirus Method Development Community
Summary: This protocol describes RNA extraction without a kit. To see the full abstract and additional resources, visit https://www.addgene.org/protocols/kit-free-rna-extraction/

Proper citation: Addgene The Nonprofit Plasmid Repository 2020. RNA Extraction Without a Kit. protocols.io https://dx.doi.org/10.17504/protocols.io.beabjaan Copy   

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Authors: Ladislav Nevery
Group: Coronavirus Method Development Community, XPRIZE Rapid Covid Testing, Ladislav Nevery
Summary: By passing saliva sample thru 150nm filter to remove particles larger than avg 125nm sized covid 19 virus particles and concentrating resulting fluid to very small 1 mm2 area of 110nm filter. Pores in this small area should be clogged up fast raising filter air/fluid resistance. Due to high virus density 5.2 log10 ml saliva of infected person should clogg filter much faster than healthy person. Test is setup in such way that we let gravity pass 2l of fluid thru clogged filter and record rate of drops from its bottom as audio on mobile phone placed bellow glass cup. rate of drops thru clogged filter should be measurably slower than clean filter.

Proper citation: Ladislav Nevery 2020. covid 19 indirect detection thru rise of 100nm filter fluid resistance. protocols.io https://dx.doi.org/10.17504/protocols.io.bmazk2f6 Copy   

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Authors: Mario Thomas, Jasmine Sollen, Natalia Ivanova, Michelle Feng, Reda Fayek, Heidi Abdilla, Stephanie Lim, Amanda Naaum, Holly Rumery, Mario Escobar
Group: Coronavirus Method Development Community, SANATA, InnOvOtion By Holly, InnOvOtion by Holly
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, Holly Rumery, Mario Escobar 2021. XPRIZE SANATA Protocol for Saliva LFIA Test. protocols.io https://dx.doi.org/10.17504/protocols.io.brgsm3we Copy   

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Authors: Nikki Freed, Olin Silander
Group: Coronavirus Method Development Community
Summary: To enable faster, easier sequencing of SARS-COV2 genomes with fewer steps than current methods, we use multiplexed 1200 base pair PCR amplicons with the Oxford Nanopore RAPID barcoding kit (RBK004). This is a modification of the ARTIC amplicon V3 sequencing protocol for MinION for nCoV-2019 developed by Josh Quick, which produces 400 base pair amplicons and uses the Oxford Nanopore Ligation barcoding kit (LSK-109). We have increased the size of the amplicons to 1200bp and use the RAPID barcode kit (RBK004), which enables requires less time and fewer reagents than the LSK-109 protocol. The amplicons produced in this protocol could also be used for Illumina sequencing. Primers were all designed using Primal Scheme: http://primal.zibraproject.org/, described here https://www.nature.com/articles/nprot.2017.066.Primer sequences are here: https://docs.google.com/spreadsheets/d/1M5I_C56ZC8_2Ycgm9EFieVlVNqxsP7dXAnGoBZy3nDo/edit?usp=sharingWe can ship a small amount of pooled primers to interested labs for further testing, email [email protected] or [email protected]

Proper citation: Nikki Freed, Olin Silander 2020. nCoV-2019 sequencing protocol (RAPID barcoding, 1200bp amplicon). protocols.io https://dx.doi.org/10.17504/protocols.io.bgc8jszw Copy   

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Authors: Wei-Ting Lu, Ming Jiang, Robert Goldstone, Karen Ambrose, Chris Ekin, Amy Strange, Nnenna Kanu, Paul Grant, Efthymios Fidanis, Jerome Nicod, David Moore, Michael Howell
Group: BGI, 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 the Crick for testing. The first step of the process is sample receipt at the Crick.This protocol describes the preparation of the RT-PCR master mix plate, automated addition of RNA template and the reverse-transcriptase quantitative PCR of viral RNA to detect the Orf1ab region of the viral genome, which has high specificity to the 2019-nCoV virus. Furthermore, the protocol describes the analysis of the COVID-19 test results.Principles of ExaminationThe kit is a qualitative in vitro nucleic acid amplification assay to detect the new coronavirus 2019-nCoV identified in China in 2019 using Reverse transcription PCR of RNA derived from specimen of throat swabs and Bronchoalveolar Lavage Fluid (BALF) from individuals suspected to have been exposed to the virus. The kit is based on in vitro RT-PCR with fluorescent probes. Primers and sequence-specific fluorescence probes were designed tailored to the high conservative ORF1ab region of the 2019-nCoV genome. The probes are oligonucleotides with attached fluorophores at the 5' end (FAM as reporter) and at the 3’ end (quencher). In addition, a specific primer and probe set designed to human b-actin is included as an internal reference with fluorophores VIC/HEX attached at 5’ end as reporter. During the PCR procedure, the DNA polymerase cleaves the probe at the 5’ end and separates the reporter dye from the quencher dye when the probes hybridize to the target DNA. This cleavage results in a fluorescent signal generated by the cleaved reporter dye, which is monitored in real-time by the PCR detection system. Monitoring the fluorescence intensities during Real Time allows the qualitative detection of 2019-nCoV in specimens.Analysis of the COVID19 test results will allow the qualitative detection of the 2019-nCoV nucleic acid in specimens from patients with suspected 2019n-nCoV. This process will enable the assessment of the infection in clinical and public health practice. The workflow for reporting results from this test is illustrated below:

Proper citation: Wei-Ting Lu, Ming Jiang, Robert Goldstone, Karen Ambrose, Chris Ekin, Amy Strange, Nnenna Kanu, Paul Grant, Efthymios Fidanis, Jerome Nicod, David Moore, Michael Howell 2020. SARS-CoV-2 detection using BGI RT-PCR kit. protocols.io https://dx.doi.org/10.17504/protocols.io.bfc8jizw Copy   

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Authors: Smruthi Karthikeyan, Greg Humphrey
Group: Coronavirus Method Development Community
Summary: Large-scale wastewater surveillance has the ability to greatly augment the tracking of infection dynamics especially in communities where the prevalence rates far exceed the testing capacity. However, current methods for viral detection in wastewater are severely lacking in terms of scaling up for high throughput. In the present study, we employed an automated magnetic-bead based concentration approach for viral detection in sewage that can effectively be scaled up for processing 24 samples in a single 40-minute run. The method compared favorably to conventionally used methods for viral wastewater concentrations with a limit of detection of 8.809 viral gene copies/ml from input sample volumes as low as 10ml and can enable the processing of over 100 wastewater samples in a day.

Proper citation: Smruthi Karthikeyan, Greg Humphrey 2020. Automated high throughput viral concentration from wastewater using the KingFisher Flex platform. protocols.io https://dx.doi.org/10.17504/protocols.io.bptemnje Copy   

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Authors: Efthymios Fidanis, Sophia Ward, Laura Cubitt, Amelia Edwards, Debbie Hughes, Michael Hubank, Jerome Nicod
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 SARS-Cov-2. 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 the Crick for testing. The first step of the process is sample receipt at the Crick. This SOP describes the transfer of inactivated virus into plates for RNA extraction.Principles of ExaminationThe assay uses the Hamilton robot to transfer 150 μl of 93 samples of inactivated viral media samples from Barcoded 2 ml tubes (Cat no. 72.694.005) to Barcoded 96 well Nunc plates (Cat no. 260251) maintaining sample identity.--The authors wish to thank Heather Ringrose for support with the Hamilton liquid handlingworkstation.

Proper citation: Efthymios Fidanis, Sophia Ward, Laura Cubitt, Amelia Edwards, Debbie Hughes, Michael Hubank, Jerome Nicod 2020. Automated transfer of inactivated virus samples from individual 2ml tubes to 96-well plate. protocols.io https://dx.doi.org/10.17504/protocols.io.bfb2jiqe Copy   

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Authors: Mustapha Bittaye, Sarah Gilbert, Katie Ewer, Teresa Lambe
Group: Coronavirus Method Development Community
Summary: This Standard Operating Procedure (SOP) describes the techniques used to measure the titres of total human immunoglobulin G (IgG) responses against a full length recombinant clamped MERS-CoV spike glycoprotein (GP) in human plasma and serum samples. The measured antibody titres will serve to indicate the humoral immune response to a candidate ChAdOx1 MERS vaccine encoding the full length spike GP of the MERS-CoV in human subjects. This assay must be performed in accordance with the principles of the International Conference on Harmonisation Tripartite Guideline on Good Clinical Practice and the Human Tissue Act. Informed consent must be in place for samples to be tested in this assay.

Proper citation: Mustapha Bittaye, Sarah Gilbert, Katie Ewer, Teresa Lambe 2020. MERS-CoV Spike Glycoprotein (GP) – ELISA Jenner Clinical SOP Template. protocols.io https://dx.doi.org/10.17504/protocols.io.bgr6jv9e Copy   

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Authors: Christina Newman, Andrea Weiler, Dawn Dudley, Mitchell Ramuta, Amelia Haj, Shelby O'Connor, David O'Connor
Group: Coronavirus Method Development Community

Proper citation: Christina Newman, Andrea Weiler, Dawn Dudley, Mitchell Ramuta, Amelia Haj, Shelby O'Connor, David O'Connor 2020. RT-LAMP SARS-CoV-2 positive control stock. protocols.io https:// Copy   

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Authors: Chris Ockenhouse, Chris Gast, Renee Holt, Jorge Flores
Group: Coronavirus Method Development Community, PATH
Summary: This is Part 10 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. Quality Assurance and Quality Control (Part 10 of Phase 3 study of Vaccine Candidate for COVID-19). protocols.io https://dx.doi.org/10.17504/protocols.io.bj57kq9n Copy   

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Authors: Chantal Vogels, Doug E. Brackney, Chaney Kalinich, Isabel Ott, Nathan Grubaugh, Anne Wyllie
Group: Coronavirus Method Development Community
Summary: SalivaDirect™ is an RNA-extraction free, dual-plexed RT-qPCR method for SARS-CoV-2 detection. It can be broadly implemented as it (1) does not require saliva collection tubes containing preservatives, (2) does not require specialized equipment for RNA extraction, and (3) is validated for use with products from multiple vendors. Thus, the simplicity and flexibility of SalivaDirect™ means that it is not as affected by supply chain bottlenecks as some other assays. Our method is RNA-extraction free which enables testing of low volume and minimally processed saliva in dual-plexed RT-qPCR for SARS-CoV-2 detection. Saliva will be treated with proteinase K followed by a heat inactivation step, and is then directly used as input in the dual-plexed RT-qPCR test. Our aim was not to design new primers and probes for RT-qPCR testing, but rather to use validated primer and probe sets (N1 and RP) developed by the US CDC. The human Ribonuclease P (RP) probe was modified with a different fluorophore so that the primer/probe set could be combined in a dualplex assay, reducing the number of tests to 1 assay with 2 sets.Version 2 includes: Optimized thermocycler conditionsLocally validated alternative options for Proteinase K, RT-qPCR master mix, and thermocyclersUse of 8-strip tubes for sample processing step, due to contamination issues in 96-well plates.Version 3 has been updated to remove steps for sample self-collection.Version 4 has updated Ct thresholds for the ABI 7500 Fast Dx.Version 5 has an updated description for use, additional RP probe with ATTO647 fluorophore, and a detailed table with catalog numbers.

Proper citation: Chantal Vogels, Doug E. Brackney, Chaney Kalinich, Isabel Ott, Nathan Grubaugh, Anne Wyllie 2020. SalivaDirect™: RNA extraction-free SARS-CoV-2 diagnostics. protocols.io https://dx.doi.org/10.17504/protocols.io.bkjgkujw Copy   

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Authors: Aaron Bivins, Warish Ahmed, Devin North, Kyle Bibby
Group: Coronavirus Method Development Community, 2019-nCoV Wastewater Epidemiology
Summary: The following protocol describes the GERM Lab workflow for processing wastewater for SARS-CoV-2 via pH adjustment, MCE filtration, extraction, and assay by droplet digital PCR (ddPCR). It is synonymous to method A as described in Ahmed et al. 2020 (the referenced manuscript) with modifications to the consumables and PCR format. It is intended for use in a BSL2+ lab with extra precautions for processing environmental specimens for SARS-CoV-2 as described in the CDC Interim Laboratory Biosafety Guidelines.

Proper citation: Aaron Bivins, Warish Ahmed, Devin North, Kyle Bibby 2020. Wastewater Concentration by Adsorption and Direct Extraction for SARS-CoV-2 RNA Detection and Quantification using RT-ddPCR. protocols.io https://dx.doi.org/10.17504/protocols.io.bhiuj4ew Copy   

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Authors: Megan Folkerts, mnguyen , Danielle Vazquez, Amber Jones
Group: Coronavirus Method Development Community

Proper citation: Megan Folkerts, mnguyen , Danielle Vazquez, Amber Jones 2020. High-Throughput Tiled Amplicon Sequencing of Sars-CoV2 using Seqwell's plexWell 384. protocols.io https://dx.doi.org/10.17504/protocols.io.bnkimcue Copy   

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Authors: Bei Wang, Wen-Hsin Sandy Lee, Helen Huang, Patricia Ng, Eve Ngoh, Chia Yin Lee, Hwee Ching Tan, Rabiatul Adawiyah, Mun Kuen Soh, Frannie Teo, Yvonne Yeap, Yuanyu Hu, Cheng-I Wang
Group: Coronavirus Method Development Community

Proper citation: Bei Wang, Wen-Hsin Sandy Lee, Helen Huang, Patricia Ng, Eve Ngoh, Chia Yin Lee, Hwee Ching Tan, Rabiatul Adawiyah, Mun Kuen Soh, Frannie Teo, Yvonne Yeap, Yuanyu Hu, Cheng-I Wang 2020. IgG expression and purification. protocols.io https://dx.doi.org/10.17504/protocols.io.bitvken6 Copy   

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Authors: Ariel Cerda, Catalina Ibarra-Henriquez, Valentina Sebastian, Grace Armijo, Liliana Lamig, Carolina Miranda, Marcela Lagos, Sandra Solari, Ana María Guzmán, Teresa Quiroga, Susan Hitschfeld, Eleodoro Riveras, Marcela Ferres, Rodrigo A. Gutiérrez, Patricia García, Aniela Wozniak
Group: Coronavirus Method Development Community, Reclone.org (The Reagent Collaboration Network)

Proper citation: Ariel Cerda, Catalina Ibarra-Henriquez, Valentina Sebastian, Grace Armijo, Liliana Lamig, Carolina Miranda, Marcela Lagos, Sandra Solari, Ana María Guzmán, Teresa Quiroga, Susan Hitschfeld, Eleodoro Riveras, Marcela Ferres, Rodrigo A. Gutiérrez, Patricia García, Aniela Wozniak 2020. Validation of Selected RNA Extraction Method. protocols.io https:// Copy   

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Authors: Adélaïde Roguet, Shuchen Feng
Group: Coronavirus Method Development Community, mclellan lab

Proper citation: Adélaïde Roguet, Shuchen Feng 2021. Nucleic acid extraction - RNeasy PowerMicrobiome Kit (Qiagen). protocols.io https://dx.doi.org/10.17504/protocols.io.bpg5mjy6 Copy   

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Authors: mrwick
Group: Coronavirus Method Development Community, XPRIZE Rapid Covid Testing, BVS, Inc, mrwick
Summary: IVDS is uniquely suited to large volume sampling.Collection from saliva or sputum are straightforward – collection in disposable pipette and transfer to a small vial, dilute as needed, filter, and test, all at under 5 minutes including results.Sample collection, storage and processing are straightforward, not requiring sensitive or unstable solutions.

Proper citation: mrwick 2020. Integrated Virus Detection System - sample collection - prep - processing. protocols.io https://dx.doi.org/10.17504/protocols.io.bktkkwkw Copy   

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