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Authors: Dr Georgios Gerardos
Group: XPRIZE Rapid Covid Testing

Proper citation: Dr Georgios Gerardos 2020. GNRI's COVID-19 Antigen Lateral Flow Test Device Use Steps. protocols.io https://dx.doi.org/10.17504/protocols.io.bk34kyqw Copy   

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Authors: Suman Chakraborty, Arindam Mondal, Aditya Bandopadhyay, Sujay Kumar Biswas, Saptarshi Banerjee, Nandita Kedia, Subhamoy Chatterjee, Aratrika de, Indranath Banerjee
Group: XPRIZE Rapid Covid Testing
Summary: We have developed an RT-LAMP based protocol for isothermal amplification and subsequent colorimetric detection of the COVID19 infection. This protocol essentially relies upon the RT-LAMP mediated reverse transcription and subsequent amplification into large copies of cDNA products in the first step of the protocol. Subsequently, we have fused a second step where we label the cDNA with a fluorophore based probe to increase the sensitivity and specificity of the results. This step also makes the overall process to be compatible with the paper strip based detection technique where the product from the second step gets captured by the molecule impregnated in the paper strip. This leads to the development of the control and test line which could be detected with a smart phone based app for the prediction and rapid dissemination of the result. .justify:after { content: ""; display:inline-block; width: 100%; }

Proper citation: Suman Chakraborty, Arindam Mondal, Aditya Bandopadhyay, Sujay Kumar Biswas, Saptarshi Banerjee, Nandita Kedia, Subhamoy Chatterjee, Aratrika de, Indranath Banerjee 2020. COVIRAP testing protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.bkzgkx3w Copy   

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Authors: Anne Berking
Group: Coronavirus Method Development Community, XPRIZE Rapid Covid Testing
Summary: The "Rapid Ag test based on Alpaca Nanobodies" is a test developed with single domain antibodies. This type of antibody is capable of effectively recognize the Spike protein on the surface of SARS-CoV2 leading a signal that is 10 times stronger than conventional antibodies. It takes only 16 minutes from sample to result. The procedures for using this test were designed to ensure an easy use and interpretation, making it a quick and simple test.

Proper citation: Anne Berking 2020. Rapid column Ag test. protocols.io https://dx.doi.org/10.17504/protocols.io.bqdjms4n Copy   

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Authors: yliu
Group: OnsiteGene 1, XPRIZE Rapid Covid Testing
Summary: This OnsiteGene protocol is designed for testing the normal saliva samples without nucleic acid extraction. A convenient saliva swab is used to collect about 50 μL of saliva for the detection. The protocol uses the Star Array® Hi-SenseTM COVID-19 Molecular Testing Kit 1.0 in the one-step real-time RT-qPCR test assay to qualitatively detect RNA from SARS-CoV-2 virus in the human respiratory specimen. It combines the reverse transcription technology and real-time PCR method to provide accurate detection of the SARS-CoV-2 coronavirus. Along with the Star Array® SATM Direct Extract Buffer, the protocol supports direct amplification without the need of specific RNA extraction equipment and kit. It significantly reduces the loss of RNA from the sample extraction and purification process, saves time and workload from sample preparation, and minimized the burdens on supply chain. The triplex fluorescence design of the kit simultaneously detects the N1 and Orf1ab genes of the virus and the human RNase P gene as an internal control to ensure the sample quality. This protocol uses the Star Array® XDiveTM Superfast Real-Time PCR instrument to perform 40-cycle PCR in 8 minutes, and can test up to 16 samples or controls in each run. The total protocol time from sample collection to data interpretation is less than 11 minutes.

Proper citation: yliu 2020. OnsiteGene 2 Protocol Saliva Direct. protocols.io https://dx.doi.org/10.17504/protocols.io.bkuikwue Copy   

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Authors: Stanford Microfluidics Lab, Purigen Biosystems
Group: XPRIZE Rapid Covid Testing, ITP-CRISPR detection of SARS-CoV-2 RNA
Summary: The Stanford University and Purigen Biosystems team's SARS-CoV-2 assay leverages on-chip microfluidics to eliminate laborious and time consuming steps associated with standard molecular diagnostics such as solid phase spin-column extraction and PCR amplification. Purification of nucleic acids from a variety of biological sources is achieved in a one-step, automated fashion using on-chip isotachophoresis (ITP). The purified nucleic acids are then amplified using reverse transcription (RT) loop-mediated isothermal amplification (LAMP) in 30 minutes, less than half of the time associated with standard qPCR. We then use CRISPR-Cas12 fluorescent detection to identify amplicons associated with the SARS-CoV-2 genome for enhanced specificity. 

Proper citation: Stanford Microfluidics Lab, Purigen Biosystems 2020. Stanford and Purigen Biosystems Microfluidics Team protocol XPRIZE. protocols.io https://dx.doi.org/10.17504/protocols.io.bqw5mxg6 Copy   

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Authors: Mario Thomas, Jasmine Sollen, Natalia Ivanova, Michelle Feng, Reda Fayek, Heidi Abdilla, Stephanie Lim, Amanda Naaum
Group: 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 home, at work or point-of-care (POC), and obtain qualitative results. It is recommended that positive results from any home-use test should be confirmed by RT-PCR by a healthcare professional.

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 https://dx.doi.org/10.17504/protocols.io.bkvekw3e Copy   

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Authors: Powell RL
Group: XPRIZE Rapid Covid Testing, Powell Lab/Lactiga Collab
Summary: Currently, the COVID-19 diagnostics sector includes Point-of-Care and ELISA tests for IgG and IgM that can assess saliva or blood as described by the CDC [Centers for Disease Control and Prevention]. Similarly, the FDA webpage for EUA Authorized Serology Test Performance makes no reference to IgA COVID-19 assays [Food and Drug Administration]. Taken together, there are no direct competitors in the mucosal IgA quantitative diagnostics space.To address this gap in diagnostic coverage we have generated a novel proof-of-concept dataset on the use of COVID-specific IgA antibodies as a marker of infection. As described below, our assay dataset demonstrates utility across several parameters:-Highly specific-Highly sensitive-Works with undiluted human milk, simplifying sample collection and processing-Defines a novel correlation between COVID-specific IgA and Secretory Component (SC), where SC is a known surrogate for dimeric IgA, the predominant IgA type in mucosal secretions-Establishes that dimeric IgA antibodies detected by the assay are capable of neutralizing SARS-CoV-2

Proper citation: Powell RL 2020. SARS-CoV-2 Milk ELISA. protocols.io https://dx.doi.org/10.17504/protocols.io.bk2skyee Copy   

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Authors: Randy True
Group: FloodLAMP, XPRIZE Rapid Covid Testing
Summary: The Binding Solution includes NaI, HCl, Triton-X100, and water. We have had lots of trouble with it, largely due to a mistake that has been corrected. In our hands currently, the Binding Solution is a pale yellow—a possibility mentioned in the Rabe Cepko paper. It does seem to become cloudy over time (days to weeks). Our current plan is to prepare weighed-out NaI and the other components premixed (stored at 4C) for easier prep, and then make fresh Binding Solution up at least weekly.We have moved to storing the Binding Solution in 5mL amber screw cap tubes for development runs (2.25mL for a single strip of 8 samples), with these in an opaque container. This makes it convenient to add glass milk and use it as a master mix (as suggested by Rabe Cepko). Care must be taken to keep the silica resuspended prior to pipetting for the addition to the sample, hence the mixing by pipetting on every draw.

Proper citation: Randy True 2020. FloodLAMP Binding Solution Prep v1.1. protocols.io https://dx.doi.org/10.17504/protocols.io.bk5sky6e Copy   

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Authors: Diego Lai
Group: Coronavirus Method Development Community, XPRIZE Rapid Covid Testing
Summary: LooK SPOT COVID-19 antigen rapid test uses a smartphone-based LooK SPOT reader, antigen cassette, and LocationNow AI Cloud to qualitatively detect the SARS-CoV-2 Nucleocapsid protein antigen within 5 minutes. The reagent is used to detect the SARS-CoV-2 nucleocapsid protein antigen, which is usually present in the upper respiratory tract sample in the acute phase of infection. When a positive result is presented, it means that there are viral antigens in the sample. The LooK SPOT system consists of 5 key components: LooK SPOT reader LooK SPOT app LooK COVID-19 antigen cassetteLooK PASS appLocationNow AI CloudTo start the test, the patient has to download and register the LooK PASS app from the app stores. The patient scans the QR code of the LooK COVID-19 antigen cassette with the LooK PASS app, and the information is uploaded to the LocationNow AI Cloud for the registration of the test. Then place the nasopharyngeal swab with the sample of the patient into the Extraction Buffer Tube. Stir the nasopharyngeal swab in the Extraction Buffer Tube and wait for one (1) minute. The virus particles in the sample are disrupted and exposing the internal viral nucleoproteins. Use the liquid dropper to extract the liquid inside the tube and apply three drops in the sample window of the antigen cassette. Insert the antigen cassette into LooK SPOT reader attached to a smartphone by using the camera of the smartphone to take pictures from the sample. LooK SPOT reader detects the ID of the LooK antigen cassette and sends these images of the sample reading to LocationNow AI cloud for analysis. LocationNow AI cloud analyzes the images of the fluorescent signal of the sample by using proprietary AI algorithms and returns results of Positive, Negative, or Invalid to the LooK SPOT reader within 5 minutes. The same result is also sent to the patient's smartphone's LooK PASS app. This test procedure is secured with full privacy. LocationNow AI algorithms have reduced the false-negative cases caused by human visual check errors. LooK COVID-19 antigen rapid test is designed for use at the Point of Entry for the revival of the economy.

Proper citation: Diego Lai 2020. LooK SPOT Antigen Rapid Test System. protocols.io https://dx.doi.org/10.17504/protocols.io.bks6kwhe Copy   

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Authors: Maira Rivera, Severine Cazaux, Ariel Cerda, Anibal Arce Medina, Isaac Núñez, Tamara Matute, Alex Brown, Javier Gasulla, Fernan Federici, Cesar A Ramirez-Sarmiento
Group: Reclone.org (The Reagent Collaboration Network), XPRIZE Rapid Covid Testing
Summary: This protocol has been optimized for the recombinant expression of a codon-optimized Bst-LF polymerase.The goal of this protocol was to eliminate the use of large volumes for dyalisis through the use of concentrators for buffer exchange before storage conditions.The plasmid encoding the codon-optimized Bst-LF enzyme used here can be found on reclone.org

Proper citation: Maira Rivera, Severine Cazaux, Ariel Cerda, Anibal Arce Medina, Isaac Núñez, Tamara Matute, Alex Brown, Javier Gasulla, Fernan Federici, Cesar A Ramirez-Sarmiento 2020. Recombinant protein expression and purification of codon-optimized Bst-LF polymerase. protocols.io https://dx.doi.org/10.17504/protocols.io.bksrkwd6 Copy   

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Authors: Sarah Ware, Ellen Jorgensen, Chris Monaco
Group: XPRIZE Rapid Covid Testing, Open Science
Summary: This is a fast (one hour), very simple LAMP-based Covid-19 diagnostic test using readily-available components that is appropriate for point-of-care use and also can be easilty scaled up on a pipetting robot such as an Opentrons OT2 because the protocol requires no cengtrifugation.It leverages a commercially-avaialble swab kit to collect a sample from the mouth of the patient in a safe manner. Viruses are inactivated by the buffer in the swab kit so risk of exposure is minimal. The sample can be safely handled once the swab is placed back in the collection tube. The collection kit has an EUA from FDA.A commercially-available reagent kit called prepIT.Q2A is used to prepare an aliquot of the sample for LAMP analysis. The two kit reagents are added to 100uL of the sample, a precipitate forms and falls to the tube bottom with no centrifugation necessary, and the RNA is pipetted off the top of the liquid in the tube.The RNA solution is diluted 1:10 with water and analyzed by adding 1uL to a preloaded PCR tube with primers and the NEB WarmStart Colorimetric LAMP reagent. Incubation at 68C for 30min produces a color change visible to the eye for a simple yes/no readout.The whole process takes an hour from start to finish.

Proper citation: Sarah Ware, Ellen Jorgensen, Chris Monaco 2020. One Hour Covid Test Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.bktakwie Copy   

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Authors: Daphna Strauss, Ayelet Rahat, Israa Sharkia, Alon Chappleboim, Miriam Adam, Daniel Kitsberg, Gavriel Fialkoff, Matan Lotem, Omer Gershon, Anna-Kristina Schmidtner, Esther Oiknine-Djian, Agnes Klochendler, Ronen Sadeh, Yuval Dor, Dana Wolf, Naomi Habib, Nir Friedman
Group: Coronavirus Method Development Community, XPRIZE Rapid Covid Testing
Summary: The global SARS-CoV-2 pandemic led to a steep increase in the need for viral detection tests worldwide. Most current tests for SARS-CoV-2 are based on RNA extraction followed by quantitative reverse-transcription PCR assays that involve a separate RNA extraction and qPCR reaction for each sample with a fixed cost and reaction time. While automation and improved logistics can increase the capacity of these tests, they cannot exceed this lower bound dictated by one extraction and reaction per sample. Multiplexed next generation sequencing (NGS) assays provide a dramatic increase in throughput, and hold the promise of richer information on viral strains and host immune response.Here, we establish a significant improvement of existing RNA-seq detection protocols. Our workflow, ApharSeq (Amplicon Pooling by Hybridization And RNA-Seq), includes a fast and cheap RNA capture step, that is coupled to barcoding of individual samples, followed by sample-pooling prior to the reverse transcription, PCR and massively parallel sequencing. Thus, only one step is performed before pooling hundreds of barcoded samples for subsequent steps and further analysis. Considering these improvements, our proposed workflow is estimated to reduce costs by 10-50 fold, labor by 5-100 fold, automated liquid handling by 5-10 fold, and reagent requirements by 100-1000 fold compared to existing methods.

Proper citation: Daphna Strauss, Ayelet Rahat, Israa Sharkia, Alon Chappleboim, Miriam Adam, Daniel Kitsberg, Gavriel Fialkoff, Matan Lotem, Omer Gershon, Anna-Kristina Schmidtner, Esther Oiknine-Djian, Agnes Klochendler, Ronen Sadeh, Yuval Dor, Dana Wolf, Naomi Habib, Nir Friedman 2020. SARS-CoV-2 detection with ApharSeq. protocols.io https://dx.doi.org/10.17504/protocols.io.bkdcks2w Copy   

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Authors: Ashwin Ramachandran, Diego A. Huyke, Eesha Sharma, Malaya K. Sahoo, ChunHong Huang, Niaz Banaei, Benjamin A. Pinsky, Juan G. Santiago
Group: XPRIZE Rapid Covid Testing, ITP-CRISPR detection of SARS-CoV-2 RNA
Summary: The rapid spread of COVID-19 across the world has revealed major gaps in our ability to respond to new virulent pathogens. Rapid, accurate, and easily configurable molecular diagnostic tests are imperative to prevent global spread of new diseases. CRISPR-based diagnostic approaches are proving to be useful as field-deployable solutions. In one basic form of this assay, the CRISPR-Cas12 enzyme complexes with a synthetic guide RNA (gRNA).This complex becomes activated only when it specifically binds to target DNA and cleaves it. The activated complex thereafter non-specifically cleaves single-stranded DNA reporter molecules labeled with a fluorophore-quencher pair. We discovered that electric field gradients can be used to control and accelerate this CRISPR assay by co-focusing Cas12-gRNA, reporters, and target within a microfluidic chip. We achieve an appropriate electric field gradient using a selective ionic focusing technique known as isotachophoresis (ITP) implemented on a microfluidic chip. We also use ITP for automated purification of target RNA from raw nasopharyngeal swab samples. We here combine this ITP purification with loop-mediated isothermal amplification (LAMP) and the ITP-enhanced CRISPR assay to achieve detection of SARS-CoV-2 RNA (from raw sample to result) in about 30 min for both contrived and clinical nasopharyngeal swab samples. Our goal is to use validated LAMP primers and gRNAs and implement them on our ITP-CRISPR microfluidic platform. The on-chip electric field control enables a new modality for a suite of microfluidic CRISPR-based diagnostic assays and makes the technology amenable to automation and point-of-care applications.

Proper citation: Ashwin Ramachandran, Diego A. Huyke, Eesha Sharma, Malaya K. Sahoo, ChunHong Huang, Niaz Banaei, Benjamin A. Pinsky, Juan G. Santiago 2020. ITP-CRISPR detection of SARS-CoV-2 RNA. protocols.io https://dx.doi.org/10.17504/protocols.io.bkxnkxme Copy   

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Authors: Albert Yu, Tim Brown, Jasmine Quynh Le, Kristina Galatsis, Michael Rosbash
Group: XPRIZE Rapid Covid Testing
Summary: A protocol for the detection of SARS-CoV-2 from saliva samples featuring a rapid purification step and a high-contrast colorimetric readout. Saliva is first inactivated using a 100x inactivation reagent consisting of 2.5M TCEP, 100 mM EDTA, 1.2N NaOH solution diluted to approximately 1x final concentration and heated to 95C for 5 minutes. RNA is rapidly purified and concentrated with magnetic beads in a PEG/NaCl-based buffer using a 3D-printed magnetic stick that enables selective separation of beads without carryover of saliva contaminants. Beads are eluted directly into an RT-LAMP reaction mix, which uses a novel high contrast dye that turns from purple to clear when acidified by nucleic acid amplification products that enables unambiguous identification of successful amplification. This protocol is sensitive down to 1 copy/µl of SARS-CoV-2 in 300 µl of saliva. This degree of sensitivity enables faithful detection of SARS-CoV-2 even in pooled samples.

Proper citation: Albert Yu, Tim Brown, Jasmine Quynh Le, Kristina Galatsis, Michael Rosbash 2020. Rosbash/Janelia StickLAMP Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.bk89kzz6 Copy   

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Authors: bkatchman
Group: Coronavirus Method Development Community, XPRIZE Rapid Covid Testing
Summary: The DetectX-Rvtest is a test based on end-point reverse transcription polymerase chain reaction (RT-PCR) coupled to DNA microarray hybridization for the detection of multiple genes within SARS-CoV-1 and SARS-CoV-2 viruses.The DNA microarray contains:DetectX-Rv Kit – -DetectX-Rv SARS-CoV-2 Multiplex Assay—contains 5 (five) SARS-CoV-2 primer sets; 4 (four) SARS-CoV-2probes targeting each N1, N2, and N3 genes-DetectX-Rv RNase P Control—internal process control for nucleic acid extraction 2 (two) RNase P primer and probe sets as an internal positive control. DetectX-Rv SARS-CoV-2 Control – RNA control that contains targets specific to the SARS-CoV-2 genomic regions that are targeted by the assay-DetectX-Rv SARS-CoV-2 Multiplex Assay—contains 5 (five) SARS-CoV-2 primer sets; 4 (four) SARS-CoV-2probes targeting each N1, N2, and N3 genes-DetectX-Rv RNase P Control—internal process control for nucleic acid extraction 2 (two) RNase P primer and probe sets as an internal positive control.

Proper citation: bkatchman 2020. DetectXRv Kit. protocols.io https://dx.doi.org/10.17504/protocols.io.bkg4ktyw Copy   

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

Proper citation: peijun he 2020. Coronavirus Lateral Flow Assay (LFA) sample preparation protocol v2. protocols.io https://dx.doi.org/10.17504/protocols.io.bqvnmw5e Copy   

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Authors: Shadi Emam, nasrollahpourmotla.m , n.sun
Group: Coronavirus Method Development Community, XPRIZE Rapid Covid Testing

Proper citation: Shadi Emam, nasrollahpourmotla.m , n.sun 2020. Saliva Vapor Tests in a Home Environment for Point-of-care COVID-19 Tests. protocols.io https://dx.doi.org/10.17504/protocols.io.bkwrkxd6 Copy   

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Authors: Massood Tabib-Azar
Group: XPRIZE Rapid Covid Testing, University of Utah, U of Utah Azar, COVID-19 Rapid Testing
Summary: This protocol is used to operate an electronic rapid (1 min) point-of-care SARS-CoV-2 sensor

Proper citation: Massood Tabib-Azar 2020. Protocol to apply saliva samples to quantum mechanical tunneling current real-time point-of-care COVID-19 sensor.. protocols.io https://dx.doi.org/10.17504/protocols.io.bk2gkybw Copy   

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Authors: Huimin Zhao, stlane2 , Guanhua Xun
Group: XPRIZE Rapid Covid Testing

Proper citation: Huimin Zhao, stlane2 , Guanhua Xun 2020. SPOT2 protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.bk28kyhw Copy   

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Authors: Jon Arizti-Sanz, Catherine A. Freije, Chloe K. Boehm, Sameed M. Siddiqui, Allen M. Goodman, Tinna-Solveig F. Kosoko-Thoroddsen, A'Doriann Y. Bradley, Jeremy Johnson, Pardis C. Sabeti, Cameron Myhrvold
Group: XPRIZE Rapid Covid Testing
Summary: This protocol describes how to perform a SHINE paper-based assay to detect SARS-CoV-2 RNA from a self-collected nasopharyngeal sample. This protocol is intended for in-home use. All enzymatic components are provided as a single-test freeze-dried pellet for shelf-stable storage, and all steps of the protocol are performed at ambient temperature. The protocol presented here is an improved version of the method presented in Arizti-Sanz J*, Freije CA*, et al. Integrated sample inactivation, amplification, and Cas13-based detection of SARS-CoV-2. bioRxiv (2020).

Proper citation: Jon Arizti-Sanz, Catherine A. Freije, Chloe K. Boehm, Sameed M. Siddiqui, Allen M. Goodman, Tinna-Solveig F. Kosoko-Thoroddsen, A'Doriann Y. Bradley, Jeremy Johnson, Pardis C. Sabeti, Cameron Myhrvold 2020. XPRIZE SHINE - Paper-based SARS-CoV-2 NP Test. protocols.io https://dx.doi.org/10.17504/protocols.io.bk3qkymw Copy   

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