METADATA last updated: 2026-03-05 by BA file_name: 2021-03-22_Instructions for Use - FloodLAMP EasyPCR COVID-19 Test v1.0.md file_date: 2021-03-22 title: Instructions for Use - FloodLAMP EasyPCR COVID-19 Test v1.0 category: regulatory subcategory: fl-fda-submissions tags: source_file_type: gdoc xfile_type: docx gfile_url: https://docs.google.com/document/d/19kCNgAQKcM_fpSBXlOyPBOYG1BPIp1Zpv_WpF5WwebY xfile_github_download_url: https://raw.githubusercontent.com/FocusOnFoundationsNonprofit/floodlamp-archive/main/regulatory/fl-fda-submissions/2021-03-22_Instructions%20for%20Use%20-%20FloodLAMP%20EasyPCR%20COVID-19%20Test%20v1.0.docx pdf_gdrive_url: https://drive.google.com/file/d/1C2VtxuYhMxZ7SyTDB-6te8LNi9ed2J5i pdf_github_url: https://github.com/FocusOnFoundationsNonprofit/floodlamp-archive/blob/main/regulatory/fl-fda-submissions/2021-03-22_Instructions%20for%20Use%20-%20FloodLAMP%20EasyPCR%20COVID-19%20Test%20v1.0.pdf conversion_input_file_type: gdoc conversion: gdoc markdown license: CC BY 4.0 - https://creativecommons.org/licenses/by/4.0/ tokens: 13972 words: 8769 notes: summary_short: The Instructions for Use for the FloodLAMP EasyPCR COVID-19 Test v1.0 provides CLIA high-complexity labs with the full workflow for an extraction-free, duplex RT-qPCR assay using CDC N1 and RNaseP targets after TCEP-based chemical plus heat inactivation. It specifies required reagents and equipment, contamination controls, instrument setup/templates, step-by-step plate and run procedures, and Ct-based result interpretation with control acceptance criteria. It also summarizes stated analytical/clinical performance (including a 3,100 copies/mL LoD and 97.5%/100% agreement in an 80-specimen evaluation) and required reporting/recordkeeping under EUA conditions. CONTENT ***INTERNAL TITLE:*** FloodLAMP EasyPCR(TM) COVID-19 Test Instructions for Use v1.0 IVD COVID-19 Emergency Use Authorization Only For *in vitro* diagnostic (IVD) Use [www.floodlamp.bio](http://www.floodlamp.bio) FloodLAMP Biotechnologies, PBC | 930 Brittan Ave. San Carlos, CA 94070 USA ## Table of Contents | | | |---------|------| | Intended Use | 3 | | Principles of Procedure | 3 | | Materials Provided and Storage | 4 | | Materials Required but Not Provided | 4 | | • Standard Lab Equipment and Consumables | 5 | | Warnings and Precautions | 6 | | • General Precautions | 6 | | • Contamination Precautions | 7 | | Limitations | 7 | | Conditions of Authorization for the Laboratory | 8 | | Specimen Collection and Storage | 9 | | Running Tests | 10 | | • Reagent Preparation | 10 | | • Controls Preparation | 11 | | • PCR Primer Stock Preparation | 12 | | • Sample Preparation | 14 | | • Sample Inactivation | 14 | | • Preparing to Run Assay for the First Time | 14 | | • Create the Plate Layout Map | 16 | | • PCR Amplification Reaction Preparation | 19 | | • Sample Addition | 19 | | • Run the Assay | 20 | | • Analyzing Data | 22 | | • Results Interpretation | 23 | | • • Test Controls | 23 | | • • Patient Specimen Results Interpretation | 23 | | Performance Evaluation | 24 | | • Analytical Sensitivity: Limit of Detection (LoD) | 24 | | • Analytical Sensitivity: Inclusivity/Cross-Reactivity (in silico) | 24 | | • Analytical Specificity: Cross-Reactivity | 25 | | • Analytical Specificity: Interfering Substances | 25 | | Clinical Evaluation | 26 | | Support | 26 | || FloodLAMP EasyPCR(TM) COVID-19 Test For COVID-19 Emergency Use Authorization Only Instructions for Use ## Intended Use FloodLAMP EasyPCR(TM) COVID-19 Test is a reverse transcriptase polymerase chain reaction (RT-PCR) assay intended for the qualitative detection of RNA from SARS-CoV-2 in upper respiratory specimens including nasopharyngeal swabs, anterior nasal and mid-turbinate nasal swabs from individuals suspected of COVID-19 by their healthcare provider and from individuals without symptoms or other epidemiological reasons to suspect COVID-19 infection, when tested at a weekly interval with no more than 9 days between tests. Testing is limited to laboratories certified under the Clinical Laboratory Improvement Amendments of 1988 (CLIA), 42 U.S.C. §263a, to perform high complexity tests, or by similarly qualified non-U.S. laboratories. Results are for the identification of SARS-CoV-2 RNA. The SARS-CoV-2 RNA is generally detectable in upper respiratory specimens including nasopharyngeal swabs, anterior nasal and mid-turbinate nasal swabs during the acute phase of infection. Positive results are indicative of the presence of SARS-CoV-2 RNA; clinical correlation with patient history and other diagnostic information is necessary to determine patient infection status. Positive results do not rule out bacterial infection or co-infection with other viruses. The agent detected may not be the definite cause of disease. Laboratories within the United States and its territories are required to report all test results to the appropriate public health authorities. Negative results do not preclude SARS-CoV-2 infection and should not be used as the sole basis for patient management decisions. Negative results must be combined with clinical observations, patient history, and epidemiological information. The FloodLAMP EasyPCR(TM) COVID-19 Test is intended for use by qualified and trained clinical laboratory personnel specifically instructed and trained in the techniques of *in vitro* diagnostic procedures. The FloodLAMP EasyPCR(TM) COVID-19 Test is only for use under the Food and Drug Administration’s Emergency Use Authorization. ## Principles of Procedure The FloodLAMP EasyPCR(TM) COVID-19 Test is an RNA-extraction-free, dualplexed RT-qPCR assay which indicates whether SARS-CoV-2 RNA is present. It can widely and rapidly be scaled because 1) it does not require nucleic acid extraction equipment, 2) it utilizes reagents and supplies readily available in large quantities, and 3) is a very straightforward protocol with minimal steps that can be executed quickly and reliably. It also utilizes the same streamlined sample preparation as the FloodLAMP QuickColor(TM) test which is isothermal, does not require any instrumentation, and has a visual readout. Together the two tests can be used in an integrated program for screening and rapid confirmation in large populations by a broad range of laboratories. In the FloodLAMP EasyPCR(TM) COVID-19 Test samples are first treated with a TCEP-based Inactivation Solution followed by a heat inactivation step, and the resulting inactivated sample is directly used as input in the dualplexed RT-qPCR test. The test does not use new primers and probes for RT-qPCR testing, but rather uses previously validated primer and probe sets (2019-nCoV\_N1 and RP) developed by the US CDC, which are readily available from multiple commercial suppliers. 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. ## Materials Provided and Storage The FloodLAMP EasyPCR(TM) COVID-19 Test utilizes standard chemicals available from multiple vendors, with the exception of the PCR Master Mix. Designated CLIA labs may order components directly from vendors. ## Materials Required but Not Provided The FloodLAMP EasyPCR(TM) COVID-19 Test is to be used with the reagents or equivalents listed in Table 1. The FloodLAMP EasyPCR(TM) COVID-19 Test is to be used with RT-PCR Instruments such as Applied Biosystems QuantStudio(TM) Systems and Bio-Rad CFX(TM) Systems. #### Table 1: Validated reagents used with Test | **Item** | **Concentration** | **Chemical Composition** | **Vendor** | **Catalog Number** | |---|---|---|---|---| | TCEP | 0.5 M | tris(2-carboxyethyl)phosphine hydrochloride | Sigma-Aldrich / Millipore Sigma | 646547-10X1ML | | EDTA | 0.5 M | Ethylenediaminetetraacetic acid | Thermo Fisher | 15575020 | | NaOH | 10 N | Sodium Hydroxide | Sigma-Aldrich | SX0607N-6 | | Nuclease-free Water | | Ultrapure Water, nuclease free | Thermo Fisher | 10977015 | | NaCl | 5 M | Sodium Chloride | Thermo Fisher | 24740011 | | PCR Kit\* | | LunaⓇ Universal Probe One-Step RT-qPCR Kit | New England Biolabs | E3006 | || \* Item may not be substituted for equivalent. The FloodLAMP EasyPCR(TM) COVID-19 Test uses the validated primer and probe sets (2019-nCoV\_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, detecting the 2 targets in a single well. This configuration is described in the SalivaDirect(TM) EUA Authorized test ([www.fda.gov/media/141192/download](http://www.fda.gov/media/141192/download)). All four primers for both CoV-19 and RNase P detection may be purchased from the vendor Eurofins Genomics using the catalog number 12YS-010YST. This product contains primers and probes suspended at 100μM and is enough for 12,500 reactions. This kit can be mixed along with nuclease-free water to create the primer stock used in the FloodLAMP EasyPCR(TM) COVID-19 Test. See Table 6 below for details. A larger volume of primer-probe stock can be prepared in advance and stored at -20°C for up to 1 year. #### Table 2: Primers and probes | **Target** | **Primer/Probe** | **Sequence** | |---|---|---| | CDC-N1 | 2019-nCoV\_N1-F | GACCCCAAAATCAGCGAAAT | | CDC-N1 | 2019-nCoV\_N1-R | TCTGGTTACTGCCAGTTGAATCTG | | CDC-N1 | 2019-nCoV\_N1-P | **FAM**-ACCCCGCATTACGTTTGGTGGACC-**IBFQ** | | Human RNAseP | RP-F | AGATTTGGACCTGCGAGCG | | Human RNAseP | RP-R | GAGCGGCTGTCTCCACAAGT | | Human RNAseP | RP-P | **Cy5**-TTCTGACCTGAAGGCTCTGCGCG-**IBRQ** | || ### Standard Lab Equipment and Consumables - 70% ethanol - 10% bleach, prepared daily - 96-well PCR reaction plates (Applied Biosystems # 4346906, 4366932, 4346907, Eppendorf # 951020303 or equivalent) - Optical strip caps (Applied Biosystems # 4323032 or equivalent) - Optical plate seal (Applied Biosystems # 4311971 or equivalent) - PCR strip tubes and caps (USA Scientific catalog # 1402-2500 or equivalent) - 5 mL transport tubes or equivalent (sterile) - 1.5 mL microcentrifuge tubes or equivalent (nuclease-free) - Aerosol resistant micropipette tips (nuclease-free) - Micropipettes (calibrated) - Bottle top dispenser for 1 mL volume (optional, calibrated) - 96-well cold block - Cold blocks for 5 mL and 1.5 mL - 2.0 mL tubes, or ice - Vortex mixer - 96-well plate centrifuge or equivalent - Mini centrifuge for 1.5 mL tubes or equivalent - Thermal cycler, water bath, dry heat bath or equivalent (calibrated) - Class II Biological Safety Cabinet (BSC) - PCR Instrument (choose one) - QuantStudio(TM) 6 Flex - QuantStudio(TM) 7 Pro - Bio-Rad CFX96 Touch(TM) ## Warnings and Precautions Materials or chemicals required for the use of the FloodLAMP EasyPCR(TM) COVID-19 Test should be closely examined by the user. The user should carefully read all warnings, instructions or Safety Data Sheets provided by the supplier and follow the general safety precautions when handling biohazards, chemicals and other materials. ### General Precautions - The FloodLAMP EasyPCR(TM) COVID-19 Test is for *in vitro* diagnostic use (IVD) only. Rx Only. - For use under COVID-19 Emergency Use Authorization Only. - Standard precautions and procedures should be taken when handling and disposing of human samples. - This test has not been FDA cleared or approved; the test has been authorized by FDA under an Emergency Use Authorization (EUA) for use by laboratories certified under the Clinical Laboratory Improvement Amendments (CLIA) of 1988, 42 U.S.C. §263a, to perform high complexity tests. - This test has been authorized only for the detection of nucleic acid from SARS-CoV-2, not for any other viruses or pathogens. - This test is only authorized for the duration of the declaration that circumstances exist justifying the authorization of emergency use of *in vitro* diagnostic tests for detection and/or diagnosis of COVID19 under Section 564(b)(1) of the Act, 21 U.S.C. § 360bbb-3(b)(1), unless the authorization is terminated or revoked sooner. - Standard precautions and procedures should be taken when handling and extracting human samples. - Standard precautions and procedures should be taken when using laboratory equipment. - Standard precautions and procedures should be taken when disposing of waste. - Dispose of reagents according to local regulations. - Do not use reagents after their recommended stability time frame. - Ensure reagents are stored at the recommended temperatures as described below and in the vendor product information and manuals. ### Contamination Precautions - Avoid contamination by following good laboratory practices, wearing proper personal protective equipment, segregating workflow, and decontaminating workspace appropriately. - Ensure that surfaces and equipment used for all test steps have been properly cleaned with 10% bleach and 70% ethanol. - Ensure all consumables are DNase and RNase free except for sample collection tubes which may be sterile. - Use only calibrated pipettes and filter tips that are sterile and PCR clean. - After completion of the test, dispose of the amplification reaction plates or tubes. Do not open tubes or remove the seals on plates after heating amplification reactions. ## Limitations - The use of this assay as an *in vitro* diagnostic under the FDA COVID-19 Emergency Use Authorization (EUA) is limited to laboratories that are certified under the Clinical Laboratory Improvement Amendments of 1988 (CLIA), 42 U.S.C. § 263a, to perform high complexity tests by Rx only. - Use of this assay is limited to personnel who are trained in the procedure. Failure to follow these instructions may lead to erroneous results. - The performance of the FloodLAMP EasyPCR(TM) COVID-19 Test was established using Nasopharyngeal Swab specimen type collected in saline. Nasal swabs, oropharyngeal swabs, mid-turbinate nasal swabs specimens are also considered acceptable specimen types for use with the test but performance has not been established. - Samples must be collected according to recommended protocols and transported and stored as described herein. - Samples should not be collected in U(TM) or V(TM) or Liquid Amies transport media. - The effect of vaccines, antiviral therapeutics, antibiotics, chemotherapeutic or immunosuppressant drugs have not been evaluated. - Detection of SARS-CoV-2 RNA may be affected by sample collection methods, patient factors (e.g., presence of symptoms), and/or stage of infection. - False-positive results may arise from various reasons, including, but not limited to the following: - Contamination during specimen collection, handling, or preparation - Contamination during assay preparation - Incorrect sample labeling - False-negative results may arise from various reasons, including, but not limited to the following: - Improper sample collection or storage - Degradation of SARS-CoV-2 RNA - Presence of inhibitory substances - Use of extraction reagents or instrumentation not approved with this assay - Incorrect sampling window - Failure to follow instructions for use - Mutations in SARS-CoV-2 target sequences - Nucleic acid may persist even after the virus is no longer viable. - This test cannot rule out diseases caused by other bacterial or viral pathogens. - Performance has not yet been established in asymptomatic individuals and will be established during a post-authorization study. - Use of the test in a general, asymptomatic population for serial screening is intended to be used as part of an infection control plan, that may include additional preventative measures, such as a predefined serial testing plan or directed testing of high-risk individuals. Negative results should not be treated as definitive and do not preclude current or future infection obtained through community transmission or other exposures. Negative results must be considered in the context of an individual’s recent exposures, history, and presence of clinical signs and symptoms consistent with COVID-19. - This test should not be used within 30 minutes of administering nasal or throat sprays. - Positive results must be reported to appropriate public health authorities, following state and national guidelines. - The clinical performance of the test has not been established in all circulating variants, and test performance may vary depending on the prevalence of variants circulating at the time of patient testing. - Negative test results do not exclude possibility of exposure to or infection with SARS-CoV-2 virus. Patient handling will be directed by healthcare professionals. ## Conditions of Authorization for the Laboratory The FloodLAMP EasyPCR(TM) COVID-19 Test Letter of Authorization, along with the authorized Fact Sheet for Healthcare Providers, the authorized Fact Sheet for Patients, and authorized labeling are available on the FDA website: [https://www.fda.gov/medical-devices/coronavirus-disease-2019-covid-19-emergency-use-authorizations-medical-devices/vitro-diagnostics-euas](https://www.fda.gov/medical-devices/coronavirus-disease-2019-covid-19-emergency-use-authorizations-medical-devices/vitro-diagnostics-euas) However, to assist clinical laboratories running the FloodLAMP EasyPCR(TM) COVID-19 Test, the relevant Conditions of Authorization are listed below: - Authorized laboratories1 using the FloodLAMP EasyPCR(TM) COVID-19 Test will include all authorized Fact Sheets with test result reports. Under exigent circumstances, other appropriate methods for disseminating these Fact Sheets may be used, which may include mass media. - Authorized laboratories1 using the FloodLAMP EasyPCR(TM) COVID-19 Test will use the FloodLAMP EasyPCR(TM) COVID-19 Test as outlined in the FloodLAMP EasyPCR(TM) COVID-19 Test Instructions for Use. Deviations from the authorized procedures, including the authorized clinical specimen types, authorized control materials, authorized other ancillary reagents and authorized materials required to perform the test are not permitted. - Authorized laboratories must notify the relevant public health authorities of their intent to run the test prior to initiating testing. - Authorized laboratories using the FloodLAMP EasyPCR(TM) COVID-19 Test will have a process in place for reporting test results to healthcare providers and relevant public health authorities, as appropriate. - Authorized laboratories will collect information on the performance of the test and report to DMD/OHT7-OIR/OPEQ/CDRH (via email: CDRH-EUA-Reporting@fda.hhs.gov) and FloodLAMP Biotechnologies, PBC support center (via email: eua.support@floodlamp.bio) any suspected occurrence of false positive or false negative results and significant deviations from the established performance characteristics of the test of which they become aware. - All laboratory personnel using the test must be appropriately trained in molecular assay techniques and use appropriate laboratory and personal protective equipment when handling these test components, and use the test in accordance with the authorized labeling. - FloodLAMP Biotechnologies, PBC authorized distributors, and authorized laboratories using the FloodLAMP EasyPCR(TM) COVID-19 Test will ensure that any records associated with this EUA are maintained until otherwise notified by FDA. Such records will be made available to FDA for inspection upon request. 1 For ease of reference, this will refer to, “Clinical Laboratory Improvement Amendments of 1988 (CLIA), 42 U.S.C. §263a certified laboratories with FDA Emergency Use Authorization FDA for performing SARS-CoV-2 testing” ## Specimen Collection and Storage Upper respiratory specimens including nasopharyngeal swabs, anterior nasal and mid-turbinate nasal swabs should be collected using standard procedures and recommendations. Swab specimens should be collected in 0.9% saline, PBS, or dry tubes. Specimens should not be collected in UTM, VTM, or Liquid Amies. Please refer to Interim Guidelines for Collecting, Handling, and Testing Clinical Specimens for COVID-19: [https://www.cdc.gov/coronavirus/2019-ncov/lab/guidelines-clinical-specimens.html](https://www.cdc.gov/coronavirus/2019-ncov/lab/guidelines-clinical-specimens.html) The stability study of the nasal swab sample transported in saline has been conducted by Quantigen Biosciences, with support from The Gates Foundation and UnitedHealth Group. Quantigen Biosciences has granted a right of reference to any sponsor wishing to pursue an EUA to leverage their COVID-19 swab stability data as part of that sponsor’s EUA request. - Samples can be stored at room temperature for 56 hours after collection prior to inactivation. - For longer term storage, samples can be stored at ≤-70°C. Note: Specimens must be packaged, shipped, and transported according to the current edition of the International Air Transport Association Dangerous Goods Regulation. Follow shipping regulations for UN 3373 Biological Substance, Category B when sending potential 2019-nCoV specimens. ## Running Tests ### Reagent Preparation The FloodLAMP EasyPCR(TM) COVID-19 Test is to be used with the reagents or equivalents listed in Table 1. #### Table 1: Validated reagents used with Test | **Item** | **Concentration** | **Chemical Composition** | **Vendor** | **Catalog Number** | |---|---|---|---|---| | TCEP | 0.5 M | tris(2-carboxyethyl)phosphine hydrochloride | Sigma-Aldrich / Millipore Sigma | 646547-10X1ML | | EDTA | 0.5 M | Ethylenediaminetetraacetic acid | Thermo Fisher | 15575020 | | NaOH | 10 N | Sodium Hydroxide | Sigma-Aldrich | SX0607N-6 | | Nuclease-free Water | | Ultrapure Water, nuclease-free | Thermo Fisher | 10977015 | | NaCl | 5M | Sodium Chloride | Thermo Fisher | 24740011 | | PCR Kit\* | | LunaⓇ Universal Probe One-Step RT-qPCR Kit | New England Biolabs | E3006 | || \* Item may not be substituted for equivalent. Stocks of TCEP, EDTA, NaOH, and NaCl may be prepared from powder form at the specified concentration using nuclease-free, MilliQ or equivalent molecular biology grade water. 0.9% Saline (154 mM) may be prepared by diluting 15.4 mL of 5 M NaCl in MilliQ or equivalent molecular biology grade water to a final volume of 500 mL. Equivalent preparations or commercial saline products may be utilized, with appropriate validation. A 100X Inactivation Solution is prepared by mixing the components in Table 3 and vortexing for 30 seconds. Equivalent preparations utilizing components with different source concentrations may be used such that the final 100X Concentration is achieved. Aliquots of 100X Inactivation Solution should be stored in the dark at -20° C for up to 3 months. Upon thaw, working aliquots of 100X Inactivation Solution should be stored in the dark at room temperature for up to 1 month. #### Table 3: 100X Inactivation Solution | **Component** | **Source Concentration** | **Volume** | **100X Concentration** | |---|---|---|---| | TCEP | 0.5 M | 10 mL | 250 mM | | EDTA | 0.5 M | 4 mL | 100 mM | | NaOH | 10 N | 2.3 mL | 1.15 N | | Nuclease-free Water | | 3.7 mL | | | **TOTAL VOLUME** | | **20 mL** | | || Swabs that are collected or eluted in 0.9% saline solution or equivalent, the 100X Inactivation Solution should be added at 1/100th the sample solution volume. For dry swabs, a preparation of 1X Inactivation Saline Solution should be prepared per Table 4. 1X Inactivation Saline Solution should be kept at room temperature and used within 24 hours of preparation from components or 100X Inactivation Solution. #### Table 4: 1X Inactivation Saline Solution | **Component** | **Volume** | |---|---| | 0.9% Saline (154 mM NaCl) in MilliQ Water | 1000 mL | | 100X Inactivation Solution | 10 mL | | **TOTAL VOLUME** | **1010 mL** | || ### Controls Preparation One **Positive Template Control** and one **Negative (No Template) Control** will be included on every 96-well plate with up to 94 samples, or with every batch of strip tubes. An **Internal Process Control** is included in every PCR reaction. 1. A **“No Template” (Negative) Control (NTC)** is needed to assure the absence of cross-contamination from positive samples, positive controls, or amplicons and is used to determine if sample results are valid. It consists of nuclease-free water. 2. A **Positive Template Control** is needed to assure proper functioning of reagents and the absence of significant RNAse contamination. It consists of synthetic viral RNA at a concentration of approximately 100,000 cp/mL diluted in total human RNA and nuclease-free water. Stock and working aliquots of the positive control are produced from the sources listed in Table 5 or equivalents. Working aliquots should be diluted prior to use to 100,000 cp/mL. Positive control aliquots should be stored for at most 3 months at -80°C, or at most 1 month at -20°C. 3. An **Internal Process Control** is needed to assure sufficient specimen quantity and quality. It consists of a primer/probe set targeting the human RNaseP gene that is included in a single PCR amplification reaction. The RNAseP Internal Process Control uses a fluorescent reporter in a separate channel from the SARS-CoV-2 channel (i.e. in duplex). #### Table 5. Components for Positive Template Control | **Material** | **Vendor** | **Catalog \#** | **Volume** | |---|---|---|---| | SARS-CoV2 Positive Control RNA | Twist | 102019 | 5 µl | | Total Human RNA | Thermo Fisher | 4307281 | 100 µl | | Nuclease-free Water | Thermo Fisher | 10977015 | 4,895 µl | || ### PCR Primer Stock Preparation The FloodLAMP EasyPCR(TM) COVID-19 Test uses the validated primer and probe sets (2019-nCoV\_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, detecting the 2 targets in a single well. This configuration is described in the SalivaDirect(TM) EUA Authorized test (www.fda.gov/media/141192/download). All four primers for both CoV-19 and RNase P detection may be purchased from the vendor Eurofins Genomics using the catalog number 12YS-010YST. This kit contains primers and probes suspended at 100μM and is enough for 12,500 reactions. This kit can be mixed along with nuclease-free water to create the primer stock used in the FloodLAMP EasyPCR(TM) COVID-19 Test. See Table 6 below for details. #### Table 6: 5X PCR Primer Stock Preparation from Eurofins Genomics Primer and Probe Set | **Component** | **5X PCR Primer Stock Concentration** | **Volume (1 reaction)** | **Volume (3,125 reactions)** | |---|---|---|---| | 2019-nCov_N1-F | 2 µM | 0.4 µl | 1250 µl | | 2019-nCov_N1-R | 2 µM | 0.4 µl | 1250 µl | | 2019-nCov_N1-P | 1 µM | 0.2 µl | 625 µl | | RP-F | 0.75 µM | 0.15 µl | 469 µl | | RP-R | 0.75 µM | 0.15 µl | 469 µl | | RP-P | 1 µM | 0.2 µl | 625 µl | | Nuclease-free water | - | 2.5 µl | 7813 µl | | **Total Volume** | - | **4 µl** | **12500 µl** | || Alternatively to the Eurofins Genomics Kit, primers may be purchased as individual custom oligos. Table 7 below lists the primer products to be ordered. Appropriate validation of primer mixes from custom oligos is required. Primers may be stored at 4°C for up to one month, or at -20°C for up to 1 year. #### Table 7: PCR Primer Stock Components | Vendor | Item | Catalog number | Quantity | # Reactions | |---------|------|----------------|-----------|-------------| | Order one of the following primer and probe sets ||||| | Eurofins Genomics | SalivaDirect™ complete set of 6 primers and probes | 12YS-010YST | 50-100 nmol | 12,500 | | Integrated DNA Technologies | nCoV_N1 Forward Primer Aliquot | 10006821 | 50 nmol | 6,2500 | | Integrated DNA Technologies | nCoV_N1 Forward Primer Aliquot | 10006830 | 100 nmol | 12,500 | | Integrated DNA Technologies | nCoV_N1 Reverse Primer Aliquot | 10006822 | 50 nmol | 6,250 | | Integrated DNA Technologies | nCoV_N1 Reverse Primer Aliquot | 10006831 | 100 nmol | 12,500 | | Integrated DNA Technologies | NCoV_N1 Probe Aliquot | 10006831 | 25 nmol | 6,250 | | Integrated DNA Technologies | NCoV_N1 Probe Aliquot | 10006832 | 50 nmol | 12,500 | | Integrated DNA Technologies | RNaseP Forward Primer Aliquot | 10006827 | 50 nmol | 16,600 | | Integrated DNA Technologies | RNaseP Forward Primer Aliquot | 10006836 | 100 nmol | 33,300 | | Integrated DNA Technologies | RNaseP Reverse Primer Aliquot | 10006828 | 50 nmol | 16,600 | | Integrated DNA Technologies | RNaseP Reverse Primer Aliquot | 10006837 | 100 nmol | 33,300 | | Integrated DNA Technologies | RNase P Probe | Custom order (cy5) | 25 nmol | 6,250 | | Integrated DNA Technologies | RNase P Probe | Custom order (cy5) | 50 nmol | 12,500 | | Integrated DNA Technologies | RNase P Probe | 10007061 (ATTO647) | 25 nmol | 6,250 | | Integrated DNA Technologies | RNase P Probe | 10007062 (ATTO647) | 50 nmol | 12,500 | | LGC Biosearch Technologies | nCoV_N1 Forward Primer | nCoV-N1-F-100 | 100 nmol | 12,500 | | LGC Biosearch Technologies | nCoV_N1 Forward Primer | nCoV-N1-F-1000 | 1000 nmol | 125,000 | | LGC Biosearch Technologies | nCoV_N1 Reverse Primer | nCoV-N1-R-100 | 100 nmol | 12,500 | | LGC Biosearch Technologies | nCoV_N1 Reverse Primer | nCoV-N1-R-1000 | 1000 nmol | 125,000 | | LGC Biosearch Technologies | NCoV_N1 Probe | nCoV-N1-P-25 | 25 nmol | 6,250 | | LGC Biosearch Technologies | NCoV_N1 Probe | nCoV-N1-P-250 | 250 nmol | 62,500 | | LGC Biosearch Technologies | RNaseP Forward Primer | RNP-F-20 | 20 nmol | 6,660 | | LGC Biosearch Technologies | RNaseP Forward Primer | RNP-F-100 | 100 nmol | 33,300 | | LGC Biosearch Technologies | RNaseP Forward Primer | RNP-F-1000 | 1000 nmol | 333,300 | | LGC Biosearch Technologies | RNaseP Reverse Primer | RNP-R-20 | 20 nmol | 6,660 | | LGC Biosearch Technologies | RNaseP Reverse Primer | RNP-R-100 | 100 nmol | 33,300 | | LGC Biosearch Technologies | RNaseP Reverse Primer | RNP-R-1000 | 1000 nmol | 333,300 | | LGC Biosearch Technologies | RNase P Probe | RNP-PQ670-25 | 25 nmol | 6,250 | | LGC Biosearch Technologies | RNase P Probe | RNP-PQ670-250 | 50 nmol | 12,500 | || ### Sample Preparation \* For wet swab specimens (swabs in saline or unprocessed swab elution): 1. If samples are frozen, thaw unless no ice crystals are present and then keep on ice, cold block or at 4°C. 2. Pulse vortex each sample and briefly spin down in a centrifuge to collect the liquid at the bottom of the tube. 3. Wipe the outside of the sample tube with 70% ethanol. For dry swab specimens: 1. Wipe the outside of the sample tube with 70% ethanol. ### Sample Inactivation 1. Place the inactivation heater (a thermal cycler, water bath, dry heat bath or equivalent) in the BSC, turn on, and set the temperature to hold at 100°C. 2. \* For wet swab specimens: transfer 1 mL or available volume of each sample to an appropriately labeled 1.5 mL or 5 mL tube and securely cap. 3. \* For wet swab specimens: add 10µL per 1 mL sample volume of 100X Inactivation Solution to each sample tube. 4. For dry swab specimens (DO NOT DO FOR WET SWAB SPECIMENS): add 1 mL of 1X Inactivation Solution to each sample tube. 5. Vortex for 30 seconds. 6. Place sample tubes into the inactivation heater for 8 minutes. 7. Remove sample tubes from the inactivation heater and let cool at room temperature for 10 minutes. 8. Place sample tubes on ice, in the refrigerator, or on a cold block at 4°C until ready to perform amplification reaction. Note: Testing of inactivated specimens must be conducted the same day inactivation is performed. For long term storage, keep the original specimen at ≤-70°C. ### Preparing to Run Assay for the First Time *Note: Any instrument running the FloodLAMP EasyPCR(TM) COVID-19 Test must be calibrated for the following dyes: FAM and Cy5.* #### Download the Template Run File The Template Run File contains all the parameters preconfigured to run the FloodLAMP EasyPCR(TM) COVID-19 Test. These parameters can be seen in more detail under “Create the Run File ...” headings below. To download the Template Run File: 1. Go to [www.floodlamp.bio/ifus](http://www.floodlamp.bio/ifus) 2. Download the Template Run file(s) for the instrument type and assay to be run. #### Table 7: RT-PCR Instrument Template Run Files | **Instrument** | **Setup Template Filename** | |---|-----------------------------------------------------------| | ABI QuantStudio(TM) 7 Pro | FloodLAMP_QS7Pro_PCR_template_run.edt | | ABI QuantStudio(TM) 6 Flex | FloodLAMP_QS6Flx_PCR_template_run.edt | | Bio-Rad CFX96 Touch(TM) | FloodLAMP_BRCFX_PCR_protocol.prcl | | Bio-Rad CFX96 Touch(TM) | FloodLAMP_BRCFX_PCR_template_run.pltd | || *Note: Template Run Files only need to be downloaded once upon first use.* #### Alternatively Create the Template Run File on QuantStudio(TM) 6 Flex or 7 Pro 1. Open the Design and Analysis Software. 2. Select the “SET UP PLATE” option. 3. From the sidebar on the screen, select the following properties to filter: - Instrument – choose the appropriate instrument - Block – choose the appropriate block - RunMode – Standard - Analysis options are left blank 4. From the plate sections present on the screen, select the correct System Template and the system will automatically navigate to the "Run Method" tab. - "Presence/Absence" for QuantStudio(TM) 7 Pro - “Presence-Absence Standard Pre PCR Post” for QuantStudio(TM) 6 Flex 5. Change Run Parameters as shown below: - **Run Method:** - 20μL Rxn Vol. - 105° C Heated Cover Temp - Ramp Rate: 1.6° C/s #### Table 8 : Thermal cycling and plate read steps for QuantStudio(TM) Systems for PCR | **Stage** | **Temperature** | **Time** | **Reps** | |---|---|---|---| | 1 | 52° C | 10 min | 1 | | 2 | 95° C | 2 min | 1 | | 3 | 95° C | 10 sec | 44 | | 3 | 55° C\* | 30 sec | 44 | | 4 | 55° C\*\* | 30 sec | 1 | || \*This step should be the optical read step \*\*This step is only required for QuantStudio(TM) 6 Flex - **Plate Setup** - Targets: FAM (N1) & Cy5 (RP) - Quencher: None - Passive Reference: ROX 1. Once done setting up the template, go to “Actions” in the top right corner and hit “Save As”: - On Connect: Save to template folder. - Offline: Save to preferred location. #### Create the Template Run File on Bio-Rad CFX96 Touch(TM) 1. Launch the CFX96 Touch(TM) software package. 2. In the Startup Wizard pop-up window select the instrument “CFX96” from the drop down menu. 3. Under “Select Run Type” press the “User-defined” button. 4. Create a new thermocycler protocol by selecting “Create New” from the Run Setup window. 5. Under Tools in the top left toolbar select “Run Time Calculator” and check “96 Wells-All Channels”. 6. Make the following changes to the cycling conditions in the Protocol Editor: - Sample Volume to 20 µL - Lid Setting to 105°C - Change cycles to be as shown below: #### Table 9 : Thermal cycling and plate read steps for the Bio-Rad CFX96 Touch(TM) | **Stage** | **Temperature** | **Time** | **Reps** | |---|---|---|---| | 1 | 52° C | 10 min | 1 | | 2 | 95° C | 2 min | 1 | | 3 | 95° C | 10 sec | 44 | | 3 | 55° C\* | 30 sec | 44 | || \*This step should be the optical read step 7. Click “OK” to save the protocol as type Protocol File (\*.prcl) and return to the Protocol tab in Run Setup. ### Create the Plate Layout Map #### QuantStudio(TM) 6 Flex or 7 Pro Option 2: Sample Name Input For this option, sample names (i.e. specimen IDs) are directly input into the instrument software prior to starting the run. 1. Open template in Design and Analysis app and go to the “Plate Setup” tab. 2. On the right side of the screen ensure the “Samples” tab is highlighted and press the addition icon to add the number of samples being tested. 3. Click on the “Sample 1” box to rename the sample. - Repeat this step for all subsequent samples being entered. 4. Click the well located in the plate map then check the box next to the sample name from the right side bar to associate the name to the well. - There is also the option to highlight the well location in the plate map and click on the “Enter sample” box. Enter the sample ID and press tab to continue to the next well. 5. Once the sample names have been entered, the wells may be highlighted by left clicking the mouse over the starting well and dragging the mouse across all wells. The targets are then chosen by clicking the check boxes next to each target in the sidebar. - FAM & Cy5 targets should be chosen and named “N1” and “RP” respectively. 6. Once done setting up the template, go to “Actions” in the top right corner and hit “Save As,” a pop-up window will appear directing the user to title the file according to information pertaining to the sample run. - Connect: Save to the desired folder (only applicable for 7 Pro). - Offline: Save to a USB that is inserted into the computer. 7. Use your plate layout to load your samples and controls after preparing the amplification reaction mix. #### QuantStudio(TM) 6 Flex or 7 Pro Option 2: Lookup Based on Well Position For this option, a single generic sample name is applied to all wells, and subsequently, outside of the instrument software, the results are linked to the actual sample name via a lookup table to the well position. 1. Open the template in the Design and Analysis app and go to the “Plate Setup” tab. 2. Highlight the entire plate and add 1 sample to all wells, with the same sample name in every well. 3. Once the sample name has been entered, the targets are chosen by clicking the check boxes next to each target in the sidebar. - FAM & Cy5 targets should be chosen and named “N1” and “RP” respectively. 4. Go to “Actions” in the top right corner and hit “Save As” and name the Template Run File as desired. The software will automatically save as a .edt file. - Connect: Save to desired location (only applicable for QuantStudio 7 Pro). - Offline: Save to a USB that is inserted into the computer. This process only needs to be done once – all subsequent runs can use the same Template Run File. #### Bio-Rad CFX96 Touch(TM): 1. Launch the CFX96 Touch(TM) software package and open the correct protocol template. 2. Review the details of the protocol. If correct, click “Next” to proceed to the Plate tab. 3. On the Plate tab, click “Create New” and the Plate Editor appears. 4. Use the Plate Editor to create a new plate. 5. To ensure the correct plate size is selected, go to “Settings > Plate Size” and check that 96-well or 384-well is selected. 6. To set the plate type, select “Settings > Plate Type” and select “BR Clear” or “BR White” from the drop-down menu. 7. To set the scan mode, select the “All Channels” scan mode from the Scan Mode drop-down list. 8. Select the “Select Fluorophores” button on the upper right of the Plate Editor window. - De-select all default fluorophores and select “FAM” and “Cy5” and click OK. 9. In the Plate Editor window highlight the whole plate and click the checkbox in front of FAM and Cy5. 10. Select the “Experiment Settings” button in order to define the Targets. - In the lower left of the Experiment Settings window in the New box, type in “N1” and select “Add”. - Repeat and type in “RP”. - Select “OK”. 11. In the Plate Editor window next to FAM in the drop-down menu under Target Name select “N1” and for Cy5 select “RP”. 12. Click OK to save changes and close the “Select Fluorophores” dialog box. #### Bio-Rad CFX96 Touch(TM) Option 1: Sample Name Input 1. Load the appropriate sample type to each well by selecting the well and selecting the appropriate Sample Type (Unknown, NTC, or Positive Control) from the drop-down menu. 2. Multiple wells can be selected at once to load the sample type. - Note: The EC can be listed as an Unknown sample. 3. In the “Target Names” section confirm that the necessary fluorophores are assigned to each well. 4. Name each well by typing in the sample name and pressing “Enter” in the Sample Names dropdown list. 5. Click OK to save the Plate File (\*.pltd) and return to the Plate tab in Run Setup. When prompted, specify a name for the plate and a save location. #### Bio-Rad CFX96 Touch(TM) Option 2: Lookup Based on Well Position 1. Name the file as desired and save as type “Plate File (\*.pltd)”. 2. Select “Save”, click “OK” in the Plate Editor window and exit the software. This process only needs to be done once – all subsequent runs can use the same Template Plate File. ### PCR Amplification Reaction Preparation 1. Place a 96-well PCR plate or PCR strip tubes onto a cold block or ice. 2. Thaw frozen reagents until ice crystals are not present. 3. Pulse vortex thawed reagents for 3 seconds and briefly spin down in a centrifuge to collect the liquid at the bottom of the tube. 4. Store on ice or in the refrigerator or on a cold block at 4°C until ready to use. 5. Prepare the PCR Amplification Reaction Mix by combining the components listed below in Table 10. NOTE: Component volumes should be scaled proportionally for the number of reactions. 6. Vortex the PCR Amplification Reaction Solution for 10 seconds and briefly spin down in a centrifuge. 7. Add 18 µL of the PCR Amplification Reaction Solution into the wells of the PCR plate or PCR strip tubes. #### Table 10: PCR Amplification Reaction Mix | **Component** | **Volume (1 reaction)** | **Volume (1 reaction x 100)
1 x 96-plate w/ 4% overage** | |---|---|---| | 5X PCR Primer Stock | 4 µL | 400 µL | | Nuclease-free Water | 3 µL | 300 µL | | PCR Master Mix\* | 10 µL | 1000 µL | | PCR RT\* | 1 µL | 100 µL | | **SUBTOTAL VOLUME** | **18 µL** | **1800 µL** | | Sample | 2 µL | - | | **REACTION VOLUME** | **20 µL** | - | || \* in LunaⓇ Universal Probe One-Step RT-qPCR Kit ### Sample Addition NOTE: Ensure that positive and negative controls are included in each batch run (i.e. in each PCR plate or group of strip tubes that are heated together). 1. Add 2 μL of each sample into a separate tube in the amplification reaction PCR plate or strip tubes. 2. Mix by pipetting. 3. If using PCR plate, seal with optical film (optionally using heat sealer). If using PCR strip tubes, cap strips. 4. Pulse vortex and briefly spin down in a centrifuge to collect the liquid at the bottom. 5. Continue to section “Run the Assay”. ### Run the Assay Refer to Specific Instrument User Manuals for full system usage and maintenance details. #### On QuantStudio(TM) 6 Flex 1. To transfer templates from a USB drive, plug a USB drive into the USB port below the touchscreen. 2. If the instrument is in standby, touch the touchscreen to activate it and then press the green power icon. 3. In the Main Menu, press “View Templates”. 4. In the Browse Experiments screen, select the template: - Press the Folder icon, then choose “USB”. - Press the desired template, then press “Save”. 5. In the Save Experiment As screen, set the name for the file. - Press the “New Template Name” field, then enter a name for the copied file. - Press the “Save to Folder” field, then select the folder to receive the file. - Hit “Save”. 6. Press the Home icon to return to the Main Menu. 7. Navigate to the Main Menu screen, then press the red eject icon. 8. When the side door opens, load the appropriate plate or PCR strips. Ensure that the consumable is properly aligned. 9. In the Main Menu, press “Browse Experiments”. 10. In the Experiments screen, choose the desired experiment and then click the Folder icon to choose where to save the experiment. 11. Then press “Start Run” to start the run immediately. #### On QuantStudio(TM) 7 Pro 1. Log into user on instrument. 2. USB: Plug in USB with saved template on it. 3. From the options on the instrument’s screen press “Load plate file”. - The QuantStudio(TM) 7 is a touchscreen device. 4. From the “Run Queue” screen, - USB: press “USB drive” on the left side. - Connect: press Cloud icon on the left side. 5. This will bring up any plate files saved. 6. Press the plate file associated with the run to be performed. 7. A new window will appear requesting location of results once the run is complete. - Connect: Press the “Cloud Connect” icon, press again to verify location and then press “Done”. - USB: Press the “USB drive Connected” if not already highlighted and press “Done”. 8. Press the double-arrow icon at the top right corner of the screen. - The instrument drawer will open from the front. 9. Place the plate/strips into the plate holder ensuring proper orientation of the plate (A1 well top-left). 10. Press “Start Run” on the screen of the instrument. - A pop-up window will appear asking the user to confirm the plate has been loaded. - If the plate has been loaded, press “Start Run” again or press “Open Drawer” to place the plate into the block and then press “Start Run”. #### On Bio-Rad CFX96 Touch(TM) 1. Open the correct .pcrl file and review the protocol details. If correct, click “Next” to proceed to the Plate tab. 2. When prompted, open the correct .pltd file and review the plate details in the Run Information section. 3. Select the checkbox for the appropriate block (CFX96 or CFX384) on which to perform the run. 4. To insert the plate or 8-tube strips into the block, click Open Lid. 5. Insert the plate or 8-tube strips into the block, ensuring proper orientation. 6. Click Close Lid. 7. Click Start Run at the bottom right of the screen. 8. When prompted, save the data file (.pcrd) to the desired location. ### Analyzing Data #### Exporting Data from QuantStudio(TM) 6 Flex or 7 Pro ##### Using USB 1. Confirm Quant says “File Transferred - USB”. 2. Take USB from Quant and plug it into computer. 3. Export data off of USB onto computer. ##### Using Cloud Connect with QuantStudio(TM) 7 Pro 1. Go to [Cloud Connect](https://apps.thermofisher.com/apps/spa/#/dashboard) and log in. 2. Go to files and find the data that was just uploaded by the Quant, in the folder chosen previously. 3. Download .xlsx file. #### Exporting Data from Bio-Rad CFX96 Touch(TM) 1. After the run has completed, open the data file (.pcrd) by going to Select File > Open > Data File in the Home window and locating the desired data file. Adjust the following settings as described below. 2. Select Settings > Cq Determination mode and select Single Threshold. 3. Select Settings > Baseline Setting and select Baseline Subtracted. 4. Select Settings > Analysis Mode and select analysis by fluorophore. 5. Select Settings > Cycles to Analyze and confirm that all cycles are analyzed, then click “OK”. 6. Cq values of each well are displayed in the Quantification Data tab. 7. Export .xlsx files and select Export > Export all Data Sheets to Excel (Cq values are in "Quantification Plate View Results"). #### Compiling Results Option 1: Lookup Based on Well Position For this option, outside of the instrument software the results are linked to the actual sample name via a lookup table to the well position. An example spreadsheet to perform this lookup and results compilation is available with instructions at [www.floodlamp.bio/ifus](http://www.floodlamp.bio/ifus). #### Compiling Results Option 2: Sample Name Input For this option, sample names (i.e. specimen IDs) are directly input into the instrument software prior to starting the run. Open the results file and continue to “Analyzing Data” section to score results. ### Results Interpretation #### Test Controls All test controls should be examined prior to interpretation of patient specimen results. If the controls are not valid and the expected result, the specimen results cannot be interpreted. Target results for the controls will be interpreted according to Table 5 below. 1. The **No Template (Negative) Control (NTC)** should yield a negative “not detected” result for both the N1 and RNaseP targets. 2. The **Positive Template Control** should yield a positive “detected” result for the N1 target and a negative “not detected” for the RNaseP control. 3. The **Internal Process Control** should yield a positive "detected" result for RNaseP. Detection of RNaseP is required to report a negative SARS-CoV-2 result. If the negative and positive controls do not appear as expected, the specimen results of the corresponding plate or batch should be considered invalid. In the event of a failure of either the positive or negative control, the lab should discard some or all of the consumables utilized for associated run, including the filter tips, tubes, plates, seals, and aliquots of reagents. Additionally, all pipettes, BSC, and appropriate lab surfaces should be thoroughly cleaned with freshly made 10% bleach solution, 70% ethanol, and (optionally) RNAseZAP™ product. In the event of the failure of the positive control, the working aliquot of positive control material should be discarded. Additionally, the lab should review the expiration of the batch of positive control aliquots and verify their integrity by performing qualification reactions of one or more positive control aliquots. If controls continue to fail, labs should not perform additional tests on clinical specimens or report results. Invalid test results should be repeated by performing another amplification reaction. #### Patient Specimen Results Interpretation NOTE: Results can only be interpreted if the positive and negative controls in the plate or group of strip tubes have the expected results. Use Table 11 below to assign a result to each sample. #### Table 11: Interpretation of Assay Results | **ABI QuantStudio(TM) 7 Pro** | | | |---|---|---| | **Result** | **Ct Value: N1** | **Ct Value RP** | | Positive | <38.0 | Any Value | | Negative | ≥38.0 | <35.0 | | *Invalid | ≥38.0 | ≥35.0 | | **Bio-Rad CFX96 Touch(TM)** / **ABI QuantStudio(TM) 6 Flex** | | | | **Result** | **Ct Value: N1** | **Ct Value RP** | | Positive | <40.0 | Any Value | | Negative | ≥40.0 | <35.0 | | *Invalid | ≥40.0 | ≥35.0 | || \*Invalid test results should be repeated by rerunning the primary sample if available, otherwise the inactivated sample. Results from retested samples will follow the same interpretation. If the final interpretation of the test result is invalid, then "Invalid/Inconclusive" should be reported and retesting of the individual is recommended. ## Performance Evaluation ### Analytical Sensitivity: Limit of Detection (LoD) The Limit of Detection (LoD) for the FloodLAMP EasyPCR(TM) COVID-19 Test was established using gamma-irradiated SARS-CoV-2 virus cell lysate (BEI NR-52287) spiked into negative real specimens. The gamma-irradiated virus was spiked into the specimen prior to the heat inactivation step, and carried through the entire assay. The concentration of spike was such that the contrived positive sample was at 100,000 copies/mL after the inactivation step. The stock contrived positive was diluted into inactivated negative sample matrix to produce the concentrations for the LoD study. A preliminary LoD run was performed using the concentrations ranging from 100,000 copies/mL to 3,100 copies/mL. Concentrations of 3,100 and 1,600 were selected for confirmatory LoD runs. LoD run details are provided in Supporting Data, with the results summarized below in Table 12. The LoD, defined as the concentration at which at least 95% of the samples are positive, was determined at 3,100 copies/mL. #### Table 12: Confirmatory LoD Data Results | **Instrument** | **LoD** | **Positive Replicates** | |---|---|---| | ABI QuantStudio(TM) 7 Pro | 3,100 copies/mL | 95% (20/21) | | ABI QuantStudio(TM) 6 Flex | 3,100 copies/mL | 100% (21/21) | | Bio-Rad CFX96 Touch(TM) | 3,100 copies/mL | 95% (20/21) | || ### Analytical Sensitivity: Inclusivity FloodLAMP EasyPCR(TM) COVID-19 Test includes a modified RT-qPCR assay by dualplexing the previously authorized CDC N1 and human RNase P primer-probe sets. Inclusivity was tested in the original US CDC EUA with all publicly available SARS-CoV-2 genomes as of 1 February 2020. The initial analysis showed 100% homology between the N1 primer-probe set and available genomes, except for one low frequency mismatch with the N1 forward primer. However, this was not expected to affect performance of the primer-probe set due to annealing temperatures of 55°C which tolerate 1-2 mismatches. Indeed, performance of the N1 primer-probe set was shown to be high in the previous comparison of primer-probes sets ([https://www.nature.com/articles/s41564-020-0761-6](https://www.nature.com/articles/s41564-020-0761-6)). GISAID continuously evaluates mismatches between newly available SARS-CoV-2 genomes and primer-probe sets and confirms a low frequency of nucleotide mismatches (<5%) with the N1 primer-probe set. ### Analytical Specificity: Cross-Reactivity The primer and probe sets used in FloodLAMP’s dualplex assay were developed by the US CDC and have been EUA certified. The CDC reported no cross-reactivity with other human coronaviruses (229E, OC43, NL63, and HKU1), MERS-coronavirus, SARS-coronavirus, and 14 additional human respiratory viruses (see [https://www.fda.gov/media/134922/download](https://www.fda.gov/media/134922/download)). ### Analytical Specificity: Interfering Substances Exogenous and endogenous substances were tested for potential interference with the FloodLAMP EasyPCR(TM) COVID-19 Test. Gamma-irradiated SARS-CoV-2 virus cell lysate (BEI NR-52287) was spiked onto dried AN swab specimens to produce contrived Positive Controls. Negative Control dried swabs obtained simultaneously were confirmed to be SARS-CoV-2 negative by PCR using the CDC primers. The gamma-irradiated SARS-CoV-2 virus and interfering substances were spiked into the dried swabs prior to the heat inactivation step, and carried through the full test protocol. All interfering substance testing showed no disagreement with expected positive and negative results, as shown in Table 13. #### Table 13: Interfering Substances Results | **Interfering Substance** | **Active Ingredient** | **Concentration** | **% Agreement with Expected Results** (Positive Control) | **% Agreement with Expected Results** (Negative Control) | |---|---|---|---|---| | Blood | N/A | 1% v/v | 100% (3/3) | 100% (3/3) | | Nasal Congestion Spray | Acetaminophen, Guaifenesin, Phenylephrine HCI | 20% v/v | 100% (3/3) | 100% (3/3) | | Nasal Allergy Spray | Oxymetazoline HCl | 15% v/v | 100% (3/3) | 100% (3/3) | | Lozenges | Menthol | 10% w/v | 100% (3/3) | 100% (3/3) | | Mucin | N/A | 0.5% w/v | 100% (3/3) | 100% (3/3) | || ## Clinical Evaluation The clinical evaluation of the FloodLAMP EasyPCR(TM) COVID-19 Test utilized confirmed clinical anterior nares swab specimens. 40 positive and 40 negative clinical specimens were evaluated and compared to a high sensitivity EUA authorized test run on the original fresh samples. The FloodLAMP EasyPCR(TM) COVID-19 Test showed a positive agreement of 97.5% and a negative agreement of 100%. The single false negative result was a specimen with a high Ct value as previously measured by the comparator test, indicating low viral load. A summary of the clinical performance is shown in Table 14. #### Table 14: Clinical Evaluation Results | **FloodLAMP EasyPCR(TM) COVID-19 Test Results** | **Comparator - High Sensitivity EUA Authorized Test** | | | |---|---|---|---| | | **Positive** | **Negative** | **Total** | | Positive | 39 | 0 | 39 | | Negative | 1 | 40 | 41 | | Total | 40 | 40 | 80 | | Positive Agreement | 97.5% (39/40) 95% CI: 86.8% to 99.9% | | | | Negative Agreement | 100% (40/40) 95% CI: 91.2% to 100% | | | || ## Support FloodLAMP Biotechnologies, PBC support center [eua.support@floodlamp.bio](mailto:eua.support@floodlamp.bio) 650-394-5233 QuantStudio is a trademark of Thermo Fisher Scientific (NYSE: TMO) Bio-Rad and Bio-Rad CFX96 Touch is a trademark of Bio-Rad Laboratories, Inc. (NYSE: BIO)