Home | LGC Biosearch Technologies
Advancing the World of Health | BD
COPAN Diagnostics Inc. | Collection and Transport Systems LeaderInstructions for LGC models including: Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCRTest
RNaseP reference material, 6 × 1.76 mL tubes per box ... the oKtopure user manual after every run to avoid risk of cross-contamination.
Instructions for use Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCRTest For Emergency Use Authorization. | For Rx Only. | For In Vitro Diagnostic Use. | GEN/861/SW/1120/v3/03242021 LGC 2905 Parmenter Street Middleton, WI 53562 USA Instructions for use Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Test Contents Intended use 3 Summary and explanation of test 3 Special instrument requirements 3 Principles of the procedure 4 Reagents, Equipment and Consumables used in the workflow 5 Material and Reagent consumables 5 Equipment, software and materials 7 Components manufactured by Biosearch Technologies and other suppliers and specified in the Test 7 Control material(s) to be used with Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Test 10 Warnings and precautions 11 Sample collection, handling and storage 12 Instructions for use 12 Minimize the risk of contamination 12 Biosearch Technologies SARS-CoV-2 detection workflow 13 IntelliQube real-time RT-PCR detection of SARS-CoV-2 22 Interpretation of results using the real-time RT-PCR workflow 31 IntelliQube end-point detection of SARS-CoV-2 40 Interpretation of results using the end-point RT-PCR workflow 51 Limitations 59 Conditions of Authorization for the Laboratory 60 Performance evaluation 61 Analytical sensitivity 61 Inclusivity (analytical sensitivity) 62 Cross-reactivity (analytical specificity) 62 Clinical evaluation 62 Summary of changes 63 2 For Emergency Use Authorization. | Rx Only | For In Vitro Diagnostic Use. Instructions for use Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Test Intended use The Biosearch Technologies SAR-CoV-2 Real-Time and End-Point RT-PCR Testis a reverse transcriptase (RT)-polymerase chain reaction (PCR) test intended for the qualitative detection of nucleic acid from SARSCoV-2 in anterior or mid-turbinate nasal swabs, nasopharyngeal swabs, oropharyngeal swabs, and nasopharyngeal washes/aspirates or nasal aspirates collected from individuals suspected of COVID-19 by their healthcare provider.Testing is limited to laboratories certified under the Clinical Laboratory Improvement Amendments of 1988 (CLIA), 42 U.S.C. §263a that meet requirements to perform high complexity tests. Results are for the identification of SARS-CoV-2 RNA. The SARS-CoV-2 RNA is generally detectable in respiratory specimens 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 the patient management decisions. Negative results must be combined with clinical observations, patient history and epidemiological information. The Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Testis intended for use by qualified clinical laboratory personnel specifically instructed and trained in the techniques of real-time and/or end-point PCR and in vitro diagnostic procedures. The Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Testis only for use under the Food and Drug Administration's Emergency Use Authorization (EUA). Summary and explanation of test Special instrument requirements The Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR test is to be used with the Biosearch Technologies oKtopureTM , IntelliQubeTM and Hydrocycler2TM instruments. These instruments will be subject to an on-site qualification process performed by LGC technicians after installation to verify critical instrument parameters, prior to reporting of patient results. The Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Testis a molecular, reverse transcription polymerase chain reaction (RT-PCR), in vitro diagnostic test that is based on the widely used nucleic acid amplification technology. The Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Testcontains primers and probes and internal controls used in RT-PCR for the in vitro qualitative detection of SARS-CoV-2 RNA in anterior or mid-turbinate nasal swabs, nasopharyngeal swabs, oropharyngeal swabs and nasopharyngeal washes/aspirates or nasal aspirates. 3 For Emergency Use Authorization. | Rx Only | For In Vitro Diagnostic Use. Instructions for use Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Test Principles of the procedure Nucleic acids are isolated and purified from upper respiratory specimens using the Biosearch Technologies oKtopure in combination with sbeadexTM viral RNA purification kits. The sbeadex viral RNA purification chemistry uses a magnetic bead-based approach to purify the viral RNA from 200 µL of swab specimen with a final elution volume of 50 µL. Using a one-step RT-PCR approach, the viral RNA template is converted to cDNA and subsequently amplified in either the IntelliQube PCR System (real-time workflow) or in the Hydrocycler2 (end-point workflow) following dispense of 0.8 µL of assay mix containing RapiDxFireTM qPCR Master Mix, EpiScriptTM RNase H- Reverse Transcriptase, SuperROXTM, and BHQTM Probes and primers. In the process, the probe anneals to a specific target sequence located between the f orward and reverse primers. During the extension phase of the PCR cycle, the 5' nuclease activity of Taq polymerase degrades the probe, causing the reporter dye to separate from the quencher dye,generating a f luorescent signal. With each cycle, additional reporter dye molecules are cleaved from their respective probes, increasing the fluorescence intensity. Fluorescence intensity is monitored either at each PCR cycle or at end-point by the IntelliQube PCR System. 4 For Emergency Use Authorization. | Rx Only | For In Vitro Diagnostic Use. Instructions for use Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Test Components manufactured by Biosearch Technologies and specified in the Test Material and reagent consumables: Component oKtopure tips oKtopure filters Waste collection plate Lysis and MagMix plate Elution and PCR sample p l ate Lysis plate seal Elution and assay plate seal RNA purification kit Protease solution Positive control IntelliQube sample pipette ti p s Array TapeTM Cover seal for Array Tape Source Storage condition Description KBS-0010-003 KBS-09-MS027 KBS-7001-044 KBS-7001-031 KBS-7001-130 KBS-7001-139 KBS-7001-132 KBS-7001-133 KBS-0606-002 KBS-0600-002 NAP40-028-04 NAP30-002-02 NAP30-002-03 0505-0211 AX843799 AX840999 AXIT38413WP050CC AXIT76813WP050CC AX8591CVRTCC Ambi en t 96-tips per tray, 200 µL, box of 10 trays Ambi en t oKtopure mandrel filters, 100 filters/pack Ambi en t Ambi en t Ambi en t Ambi en t Ambi en t Ambi en t Ambi en t Ambi en t s bead ex Suspension: 2-8 °C All Others: Ambient 2-8 °C 96-well deep-well plate, 2 mL well volume, 50 plates per box 96-well deep-well plate, 2 mL well volume, 50 plates per box 96-well deep-well plate, 1.2 mL well volume, 50 plates per box 384-well plate, 120 µl well volume, 100 plates per box 384-well plate, 100 µl well volume, 50 plates per box 384-well plate, 200 µl well volume, 50 plates per box Adhesive PCR film, 135 mm × 80 mm, 100 sheets per box Adhesive PCR foil seal, 135 mm × 80 mm, 100 sheets per box sbeadex viral RNA kit, 200 µl sample input, 10,000 purifications Protease solution, 20 mg/mL, 10 mL 2-8 °C Protease solution, 20 mg/mL, 100 mL 2-8 °C Ambi en t AccuPlexTM SARS-CoV2 full genome with RNaseP reference material, 6 × 1.76 mL tubes per box 384-tips per tray, 10 µL Ambi en t 384-tips per tray, 40 µL Ambi en t Ambi en t Ambi en t IntelliQube Array Tape, Clean Consumable (DNAse-, RNAse-, Pyrogen-free), 384-well, 2 reels, 50 arrays per reel IntelliQube Array Tape, Clean Consumable (DNAse-, RNAse-, Pyrogen-free), 768-well, 2 reels, 50 arrays per reel IntelliQube Cover Seal, Clean Consumable (DNAse-, RNAse-, Pyrogen-free) Pressure Sensitive, 30 meter roll with 350 seals 5 For Emergency Use Authorization. | Rx Only | For In Vitro Diagnostic Use. Instructions for use Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Test PCR assay oligo blends N1RNP-1010 N2RNP-1010 30050-1 5X Master Mix 30050-2 30050-100ML ERT12925K-ENZ Reverse Transcriptase ERT12925K-1.25ML Passive reference dye ERT12925K-12ML SR-1000-1 SR-1000-10 -20 °C Storage 2-8 °C Daily Use -20 °C Storage 2-8 °C Daily Use -20 °C Storage 2-8 °C Daily Use -20 °C Storage 2-8 °C Daily Use -20 °C Storage 2-8 °C Daily Use -20 °C Storage 2-8 °C Daily Use -20 °C Storage 2-8 °C Daily Use -20 °C Storage 2-8 °C Daily Use 2 - 8 °C 2 - 8 °C 2019 n Co V N1/Rn P Blen d -100X Sto ck, 1,010 µL 2019 n Co V N2/Rn P Blen d -100X Sto ck, 1,010 µL RapiDxFire qPCR 5X Master Mix GF, 1 mL RapiDxFire qPCR 5X Master Mix GF, 10 mL RapiDxFire qPCR 5X Master Mix GF, 100 mL Episcript RNase H- Reverse Transcriptase, 200 U/µL,0.125 mL Episcript RNase H- Reverse Transcriptase, 200 U/µL, 1.25 mL Episcript RNase H- Reverse Transcriptase, 200 U/µL, 12 mL SuperROX, 15 µM, 1 mL SuperROX, 15 µM, 10 mL Table 1. Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Test Consumables included with the Test. 6 For Emergency Use Authorization. | Rx Only | For In Vitro Diagnostic Use. Instructions for use Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Test Equipment, software and materials: Component Source Description Automation for RNA purification KBS-0009-001 oKtopure high-throughput nucleic acid purification robot RNA purification reagent reservoir oKtopure tip blocks KBS-09-050 KBS-0009-006 KBS-09-127 Maxi buffer reservoir Standard buffer reservoir 192-hole tip blocks for the oKtopure Handheld barcode scanner KBS-0025-003 Various vendors Handheld, USB linear barcode scanner PCR automation Bulk thermal cycling (end-point only) AXDS-0002-100 KBS-0028-001 IntelliQube integrated inline liquid handling, assay processing and analytical system Hy d ro c y c ler2 KBS-0099-101 75 L oven Heat source for sample lysis KBS-0099-102 40 L oven Real-Time Data Analysis So ftware End-Point Data Analysis So ftware Sanosil Super 25 Various vendors Forced convection laboratory oven capable of reaching 95 ºC UgenTec FastFinder Analysis V4.0.1 and Assay Plugin "LG C-EUA-SARS-Co V-2-RT v1.1" UgenTec FastFinder Analysis V4.0.1 and Assay Plugin "LGC- EUA-SARS-CoV-2-EP v1.1" UgenTec Software FastFinder Analysis 4.0.1 with Real Time Assay Plugin 1.1 UgenTec Software FastFinder Analysis 4.0.1 with End-Point Assay Plugin 1.1 San o s i l Highly concentrated water disinfectant Hal o Mi s t Halosil International Water disinfectant Sanostrips 200 San o s i l Measuring Strips MS200 for determination of H2O2 c o n c en trati on Table 2. Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Test equipment, software and materials specified in the Test. Components manufactured by Biosearch Technologies and other suppliers and specified in the Test Component Source Description PCR reagent diluent and NTC Various vendors Molecular or PCR grade water Assay tubes ThermoFisher 4170 0.75 mL blank matrix tubes Assay tube rack ThermoFisher 4896 Empty latch rack for 0.75 mL matrix tubes 7 For Emergency Use Authorization. | Rx Only | For In Vitro Diagnostic Use. Instructions for use Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Test Assay tube seal Assay plate (alternative to ThermoFisher's 4170, 4896, 4463) Adhesive barcode labels ThermoFisher 4463 KBS-7001-131 KBS-7001-231 Greiner Bio-one 786201 KBS-0750-100 Various vendors SepraSeal caps for assay tubes recommend purchase in two colors 96-well V-bottom plate, 0.8 mL well volume 96-well storage plates, 0.8 mL (case 50 plates) Masterblock®, 96-well V-bottom plate, 0.7 mL well volume Adhesive plate barcodes (roll of 5000) Adhesive barcodes Sterilizing basin 5% Sodium hypochlorite Sodium hypochlorite concentration test strips (both required) Pi p etto rs Pipette tips PCR reagent preparation tubes Tube racks Cold storage Ice or cold block Flammable cabinet Co mp res s o r RO water supply Di s p o s abl e, powder free gloves Cen tri fug e Vo rtex Eth an o l Is o p ro p anol Mi c ro c en tri fug e Various vendors Various vendors LaMotte 3002 Deardoff Fitzsimmons 77085 Various vendors Various vendors ~20 L volume, polypropylene sterilizing basins for oKtopure tip blocks Sodium hypochlorite for tip and tip block decontamination Insta-Test Analytic High Range Chlorine Dioxide Test Strips Active High-Level Chlorine Strips Single and multichannel adjustable pipettors (2 µL to 1,000 µL) Filtered, disposable pipette tips Various vendors 1.5 mL centrifuge tubes, 15 mL and 50 mL conical tubes Various vendors Various vendors Various vendors Various vendors Various vendors Various vendors Racks for 1.5 mL, 15 mL and 50 mL tubes Laboratory fridges and freezers (4 ºC, -20 ºC) Various vendors Various vendors Various vendors Various vendors Various vendors Various vendors Centrifuge with rotors compatible with standard and deep-well plates Vortex including plate adapter 70-80% ethanol, molecular biology grade or equivalent 70% isopropanol, molecular biology grade or equivalent Table 3. Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Test other equipment and materials required. 8 For Emergency Use Authorization. | Rx Only | For In Vitro Diagnostic Use. Instructions for use Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Test Control material(s) to be used with Biosearch Technologies SARS-CoV-2 Real-Time and EndPoint RT-PCR Test Component Source: ref # Positive Control 05050211 Description AccuPlex SARS-CoV-2 RNaseP and viral template at 15,000 copies/mL of N1/N2 Target and 30,000 copies/mL of RP target, with p o lyA carrier RNA. Purpose Full Process Positive Control (PC) material Negative Control Major laboratory s up p l i ers Internal Control N/A Molecular or PCR grade water. Negative Template Control (NTC) The human RNase P gene should be Extraction control for present in a properly collected and each individual extracted sample. patient sample Frequency One per 96-well source plate One per 96well source p l ate Analyzed in every patient sample well Table 4. Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Test control materials. Controls that are provided with the test include: a. A "no template" (negative) control is needed to ensure there are no unexpected amplification events that may indicate a f ailure in the assay, a contamination event in the lab, or other mishandling of samples as part of the extraction, purification and amplification process. This control is included as an input into onewell of each 96-wellsource platethatcontainsthe raw clinical samples prior to lysis or purificationand shouldbe identified in theplatemap fileas "NTC." b. The positive template control material is a SARS-CoV-2 Full Genome and human RNase P with spiked carrier RNA. The control material contains the whole genome of the 2019-Coronavirus strain (SARS-CoV-2) corresponding to GenBank sequence NC_045512.2., using LGC SeraCare Life Sciences proprietary AccuPlex Technology.The concentration of this control has been set to a viral load of 15,000 ± 2,000 cp/mL SARS-CoV-2; and 30,000 ± 4,000 cp/mL RNase P verified through digital PCR with an additional inclusion of carrier RNA at a concentration of ~5 g/mL. This concentration represents a target concentration for N1, N2, and RP of between 2X and 3X the LoD of this method. This control is needed to provide assurance that the extraction and purification process was executed as expected and generated an acceptable purified sample concentration as well as a verification that the RT-PCR process is functioning as expected. This control is included as an input into one well of each 96-well source plate that contains the raw clinical samples prior to lysis or purification and should be identified in the plate map f ile as "PC." Human RNase P serves as an internal/extraction control and is detected using the 2019-nCoV N1/RnP and 2019-nCoV N2/RnP assay blends. Detection of this target indicates that human nucleic acid is present and implies that human biological material was collected and successfully extracted and amplified. It does not necessarily indicate that the specimen is of appropriate 10 For Emergency UseAuthorization.| Rx Only | For In Vitro Diagnostic Use. Instructions for use Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Test quality to enable detection of SARS-CoV-2. This RNase P control can be used to flag samples that have a low concentration of purified nucleic acid for re-extraction and retesting as needed. Warnings and precautions As with any test procedure, good laboratory practice is essential to the proper performance of this assay. Due to the high sensitivity of this test, care should be taken to keep reagents and amplification mixtures free of contamination. The Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Test workflow should be performed by qualified and trained staff to avoid the risk of erroneous results. · For in vitro diagnosticuse only. · For prescription useonly. · For Emergency Use Authorization (EUA) Only. · The Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR 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, that meet requirements 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 diagnostics for detection and/or diagnosis of COVID-19 under Section 564(b)(1) of the Federal Food, Drug, and Cosmetic Act, 21 U.S.C. § 360bbb-3(b)(1), unless the authorization is terminated or revoked sooner. · Specimens should always be treated as if infectious and/or biohazardous in accordance with safe laboratory procedures. Refer to Interim Laboratory Biosafety Guidelines for Handling and Processing Specimens Associated withSARS-CoV-2. · Follow necessary precautions when handling specimens. Use personal protective equipment (PPE) consistent with current guidelines for the handling of potentially infectious samples. Refer to Biosafety in Microbiological and Biomedical Laboratories (BMBL)5thEdition CDC. · Do not eat, drink,smoke, apply cosmeticsor handlecontactlenses in areas where reagents and human specimens arehandled. · Modifications to assay reagents,assay protocol or instrumentationare in violationof the product Emergency Use Authorization. 11 For Emergency UseAuthorization.| Rx Only | For In Vitro Diagnostic Use. Instructions for use Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Test · Do not use thekit aftertheindicatedexpirydate. Please see Table 1, material and reagent consumables specified in the Test, for storage conditions. · Avoid freeze/thaw of thePCRreagents. · Disposeof waste in compliancewithlocal,state, and federal regulations. · Safety DataSheets (SDS)are availableuponrequest. · Positive results are indicative of thepresenceof SARS-CoV-2 RNA. · Handle all samples and controlsas if they are capableof transmittinginfectiousagents. Sample collection, handling and storage Proper specimen collection, storage and transport are critical to the performance of this test. Inadequate specimen collection, improper specimen handling and/or transport may yield a f alse result. Sample handling and storage should be consistent with CDC guidelines. The Biosearch Technologies SARS-CoV-2 RealTime and End-Point RT-PCR Testhas been validated for use with anterior and mid-turbinate nasal swabs, nasopharyngeal swabs, oropharyngeal swabs and nasopharyngeal washes/aspirates or nasal aspirates. The collected samples should be handled and stored according to the CDC's recommendations (2-8°C for 72 hours, -70°C for >72 hours). SAFETY WARNING Handle all samples and controls as if they are capable of transmitting infectious agents. Refer to the CDC Interim Guidelines for Collecting, Handling, and Testing Clinical Specimens from Persons Under Investigation (PUIs) f or Coronavirus Disease 2019 (COVID-19). Instructions for use Minimize the risk of contamination · Use appropriatebiosafetyenvironmental containment forsampleand reagent handling. · Always use cautionwhentransferringspecimens fromprimarycontainersto secondarytube(s). · Precautions must be takento preventcross contaminationof samples.This entailsonly re-using consumables were appropriate and using aseptic pipetting techniques. 12 For Emergency UseAuthorization.| Rx Only | For In Vitro Diagnostic Use. Instructions for use Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Test Biosearch Technologies SARS-CoV-2 detection workflow RNA purification oKtopure preparation: 1) Load new mandrel filters(KBS-09-MS027)intothe oKtopurehead.If new filtersare already present, skip this step. IMPORTANT: Mandrel filters should be replaced prior to every run to prevent cross contamination and ensure proper dispense volumes if previous filters encountered fluids. 2) Load oKtopuretip blocks (KBS-09-127)onto theinstrument. 3) Load new oKtopuretips (KBS-0010-003)into thetip blocks. Thismay be performedthroughthe oKtopure software user interface. a. For the first run of the day, load tips into tip positions1-12. Forsubsequent runs, onlythe buffertips should be reused,new tips should beloaded intotip positions 5-12. IMPORTANT: Buffer only tips should be reused for no more than 3 runs. b. When performingtiptransferusingtheoKtopuresoftwareuserinterface: i. Prior to loadingtipsto positions1-12verify"Skip washbuffer tips"box is not checkedFigure1. ii. If only loadingtips to positions 5-12,verify"Skip washbuffertips"is checkedFigure1. 13 For Emergency UseAuthorization.| Rx Only | For In Vitro Diagnostic Use. Instructions for use Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Test Figure 1. Load oKtopure Tip Racks. Extraction reagents preparation: Lysis Buffer Mixture Prepare the required amount of Lysis Buf fer Mix within 4 hours of intended use. 1) Prepare the Lysis BufferMix. a. For the number of required 96-well plate extractions, prepare the Lysis Buffer Mix containing Lysis Buf fer SB f ound in the sbeadex viral RNA purification kit (NAP40-028-04) and Protease solution (NAP30-002-02 or NAP30-002-03) according to Table5. 14 For Emergency UseAuthorization.| Rx Only | For In Vitro Diagnostic Use. Instructions for use Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Test Component Lysis Buffer SB Protease Solution (20 mg/mL) Total volume per plate Table 5. Lysis Buffer SB and Protease Solution volumes for 1 plate. 1 Include percent overage required for liquid handling. Volume per 96-well plate1 19.2 mL 0.48 mL 19.68 mL b. Using the oKtopure to prefill 8 sample plates requires 180 mL of Lysis Buffer Mix using a standard buffer reservoir (KBS-0009-006). Table 6 below references how to prepare Lysis Buffer Mixfor 8 sample plates. Component Volume Lysis Buffer SB 175.61 mL Protease Solution (20 mg/mL) Total volume 4.39 mL 180 mL Table 6. Lysis Buffer SB and Protease Solution volumes for 8 plates. 2) Mix well by inversion. Thesolutionwill remain stable at roomtemperatureforup to 4 hours. Binding Bead Mix 1) Thoroughlymix sbeadex particlesuspension by vortexinguntilsolution is homogenous. 2) Combine and mix the Binding Buffer SB and sbeadex particle suspension following the table below. a. For the number of required 96-well plate extractions, prepare the Binding Bead Mix according to Table 7. Component Binding Buffer SB sbeadex particle suspension Total volume per plate Table 7. Binding Buffer SB and sbeadex particle suspension volumes for 1 plate. 1 Include percent overage required for liquid handling. Volume per 96-well plate1 30.72 mL 1.92 mL 32.64 mL 15 For Emergency UseAuthorization.| Rx Only | For In Vitro Diagnostic Use. Instructions for use Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Test b. Using the oKtopureto prefill 8 sample platesrequires 306 mL of BindingBead Mixusing astandard buf f er reservoir (KBS-0009-006). Prepare the Binding Bead Mix according to Table 8. IMPORTANT: The standard buffer reservoir (KBS-0009-006) is required for this action to keep Binding Bead Mix homogenous.Failure to do so may cause bead settling and inconsistent bead transfer. Component Binding Buffer SB sbeadex particle suspension Total volume Table 8. Binding Buffer SB and sbeadex particle suspension for 8 plates. 3) Mix well by inversion, thenstoreat roomtemperatureuntil use. Volume 288 mL 18 mL 306 mL Prepare sample plate: 1) Combinethefollowing intoeach reactionwell of a96-wellMagMix plate(KBS-7001-130). a. Add 205 µL of LysisBufferMixprepared previously. i. Optional: Pre-fill plate function on the oKtopure may be used to add Lysis Buffer Mix. Be sure to designatethe dispensevolume to 205 µL and select the appropriate numberof platesto be filled. Figure2 may be used as a reference. 16 For Emergency UseAuthorization.| Rx Only | For In Vitro Diagnostic Use. Instructions for use Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Test Figure 2. oKtopure pre-fill plates layout for dispensing Lysis Buffer Mix. ii. Replaceused tips aftercompletionof Pre-fill routine. b. Add 200 µL of sample to each well. i. Record samplepositionwithin theplate. c. Add extractioncontrols to each96-well MagMixplate(KBS-7001-130): i. Add 200 µL of the positivecontrol material (0505-0211).This positivecontrol must be named "PC" in the plate map files usedto reference sample locations. ii. Add 200 µL of nucleasefree water.This negativecontrol must be named"NTC"in the plate-map files used to referencesample locations. iii. The usermay determinethelocationof thecontrols,but they mustbe labeledas "PC" and "NTC" in the sample plate file. 2) Seal the 96-well MagMix plateswith an adhesive PCR film (KBS-0606-002).Firmlypressseal to the 96-well MagMix plates using a roller or equivalent method. IMPORTANT: The adhesive PCR film (KBS-0606-002) must be used to seal the 96-well MagMix plate (KBS-7001-130) at this step. Alternative seals may not tolerate the 95 °C incubation, resulting in cross-contamination risk. 17 For Emergency UseAuthorization.| Rx Only | For In Vitro Diagnostic Use. Instructions for use Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Test 3) Vortexthe 96-well MagMixplatesfor 30 seconds. Avoid splashing liquidonto theseal to prevent potential contamination whenunsealingthe plateafter incubation. 4) Centrifugethe96-well MagMix plates at 2,500× g for 1 minute. 5) Incubatethe 96-well MagMix plates at 95 °C for 30 minutesin an oven. Use cautionwhenremovingthe plates as they may be very warm. 6) Allow 96-well MagMix platesto cool at ambient temperature(20 ± 5 °C) foraminimumof 10 minutes. 7) Centrifugethe96-well MagMix plates at 2,500× g for 1 minute. 8) Leave the plates sealedand set the96-well MagMix platesasideuntil readyto add BindingBeadMix. 9) Carefully unseal plate. 10) Add 340 µL of Binding Bead Mix. a. Optional: Pre-fill plate functionon the oKtopure may be used to add Binding Bead Mix. Designatethe dispense volume to 340 µL and select the appropriate number of plates to be filled. Figure 3 may be used as a reference. IMPORTANT: Buffer reservoir (KBS-0009-006) is required for this action to keep Binding Bead mix homogenous. Failure to do so may cause bead settling and inconsistent bead transfer. Figure 3. oKtopure pre-fill plates layout for dispensing Binding Bead Mix. b. Replaceused tips aftercompletionof Pre-fill routine. 18 For Emergency UseAuthorization.| Rx Only | For In Vitro Diagnostic Use. Instructions for use Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Test Performing the oKtopure run: 1) Place the 96-well MagMix plates (KBS-7001-130) now containing sample, Lysis Buffer Mix, and Binding Bead Mix onto magnet locations 9-12 and 21-24. If less than 8 plates are being extracted, refer to oKtopuresoftwarefor designatedplatelocations. 2) Apply an adhesivefoil seal (KBS-0600-002)to two 384-well destinationplates (KBS-7001-139, KBS7001-132, or KBS-7001-133) and add plates to locations 13 and 25. 3) Add and fill the wash buffer reservoirs (KBS-09-050) with the appropriate volumes of each wash buffer based on the equations below.The standardbufferreservoir (KBS-0009-006)is requiredfor the Elution AMP. Table 9 can be used to referencevolumes requiredforan 8 plate extraction. NOTE: Graduated cylinders should be used to measure the appropriate buffer volumes and carefully pour into the reservoirs. Alternatively, a pre-determined minimum fill line can be marked on the side of the reservoir instead of measuring with agraduated cylinder. IMPORTANT: Buffer reservoir (KBS-0009-006) is required for the Elution AMP. Heating the maxi buffer troughs (KBS-09-050) will cause troughs to warp and no longer seat properly into the oKtopure deck positions. a. BN1 volume (mL)= # of plates × 96 wells/plate× 0.3 mL/well + 45 mL overage b. TN1 volume (mL) = # of plates × 96 wells/plate × 0.24mL/well + 45 mL overage c. TN2 volume (mL) = # of plates × 96 wells/plate × 0.34mL/well + 45 mL overage d. ElutionAMP volume(mL)= # of plates × 96 wells/plate× 0.05 mL/well+ 45 mL overage Component oKtopure deck position Wash Buffer BN1 1 Wash Buffer TN1 2 Wash Buffer TN2 3 Elution AMP 4 Table 9. Wash and Elution Buffer volumes for 8 plates. Volume for 8 plate extraction 275 mL 230 mL 306 mL 83 mL 19 For Emergency UseAuthorization.| Rx Only | For In Vitro Diagnostic Use. Instructions for use Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Test 4) Seal the buffer trough containing Elution AMP with an adhesive foil seal (KBS-0600-002) and place in a 65 °C oven. The instrument will prompt the user to add the elution reservoir trough at the elution step. 5) Add the waste collectionplates (KBS-7001-031or KBS-7001-044 or KBS-7001-244)onto thedeck locationsdirectlyleft of the MAG/MIX plate locations containing a 96-well MagMix plate (KBS7001-130) for extraction. 6) Using a handheldbarcodescanner,scan the platebarcodes in the MAG/MIX locationsand Destination Plate locations as shown in Figure 4 below. If previous barcodes are present, the "Clear purification plates" button can be selected to clear the barcodes. IMPORTANT: Always scan barcodes in the order they are loaded onto the instrument deck and verify proper plate orientation. Failure to do so will result in incorrect plate mapping. Figure 4. oKtopure plate barcode entry. 7) Fromthe workingpage,select the "Sbx ViralRNA 200ul 384 8.8" protocol.Confirmthe selected number of plates is correct and start the run. 8) Confirmtheappropriatebarcodeshave been scanned and continue. 9) When prompted, transfer theheatedelutionbufferreservoir fromthe 65 °C ovento oKtopuredeck position 4 and remove seal. 10) Remove the seals from the 384-well destination plates. 11) Select "Resume." 20 For Emergency UseAuthorization.| Rx Only | For In Vitro Diagnostic Use. Instructions for use Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Test 12) Upon completionof the run,seal the plateswith theadhesive foil seal (KBS-0600-002)and keep on ice or at 4 °C until use. IMPORTANT: Use caution when applying adhesive seals to ensure pressure is applied evenly across the plate for proper sealing across all wells. Failure to do so can lead to potential well-to-well contamination during plate handling. 13) Remove the usedtip blocks fromthe deckand disposeof the used sample tips.The bufferonlytipscan be reusedin subsequentrunsthroughout the day. IMPORTANT: Buffer only tips should be reused for no more than 3 runs. IMPORTANT: oKtopure tip blocks used to hold the samples tips must be decontaminated according to the oKtopure user manual after every run to avoid risk of cross-contamination. 14) Remove themandrel filters fromthedispense head. 15) If this is the first run of theday, then proceedto the instrumentdaily startuproutinesection.If thedaily startup routines were already completed, then proceed to the respective real-time and end-point RT-PCR sections depending on the desired workflow. 21 For Emergency UseAuthorization.| Rx Only | For In Vitro Diagnostic Use. Instructions for use Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Test IntelliQube real-time RT-PCR detection of SARS-CoV-2 NOTE: This section describes the real-time RT-PCR modality of operation. For end-point operation, see p. 40. Instrument daily startup routine: IntelliQube 1) Check Carboys. a. Empty Waste Carboy. b. Fill Source WaterCarboy. c. Check Bleach Carboy;refill if low. 2) Purge DispenseJet. a. Manual Control > Maintenance >Jet Purge 3) Wash Dispense Jet andre-pressurize. a. Manual Control> Jet > "EUA Jet Configuration"> Cycles:200> Wash Tips b. Manual Control> Jet > Pressurizeto atarget pressureof 1.6 psi 4) Clean Dispense Jet withEthanol. a. Prepare96-well deep-well plateor matrixtube rackwith70-80%ethanol. i. Fill wells A1, B1, C1,and D1 with 700µLof 70-80% ethanol. ii. Place on deck position1. b. Manual Control> Jet > SelectAll Tips > Select Plate Deck 1 > SelectAppropriate Plateware > Select Full Dispense Pattern > Aspirate. Refer to Figure 5 c. Wait 5-10 minutes. 22 For Emergency UseAuthorization.| Rx Only | For In Vitro Diagnostic Use. Instructions for use Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Test Figure 5. IntelliQube Dispense Jet manual control. 5) Flush the DispenseJet. a. Manual Control> Jet > Cycles: 200 > Wash Tips 6) Check the sodium hypochlorite concentration in the pipette wash protocol. a. Under the Pipette tab in manual control, select the EUA PipetteWash configurationand EUA 384 Tip 10 µL. b. Select Wash Tips. c. Once the basinhas filledand the DispensePipettebegins to move downto start theaspiration process, fault the instrument by opening the guard door. d. Test the bleach concentrationby movingthetest stripacross the wash fluidin the basin. e. Verify the bleach strip measures2500ppm. f. Close the guarddoorand recover the instrument. g. Select Water Only Pipette Wash configuration and perform 2 washes. h. Repeat steps 6a and 6b. i. Allow the wash to continue to thelast flush cycle. j. Once the basinhas filledand the DispensePipettebegins to move downto start theaspiration process, fault the instrument by opening the guard door. k. Test the bleach concentrationby movingthetest stripacross the wash fluid in the basin. l. Verify the bleach strip measures0 ppm. m. Close the guarddoorand recover the instrument. n. Make any adjustmentsneededto the wash patternand repeatto verify adjustedsettings are correct. 23 For Emergency UseAuthorization.| Rx Only | For In Vitro Diagnostic Use. Instructions for use Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Test 2X PCR reagent preparation for a two 384-well sample platerun: *Note: If preparing less than two full 384-well plates, consult Table 10 below to determine the appropriate volumes of reagents to be used based on sample number. 1) Thaw all reagents and keepon ice or at 4 °C until use. 2) Gently vortexreagentsand briefly centrifuge beforeuse. 3) Calculate amount of assay to prepare. a. For less than 384 samples, take the (# of samples x 0.8 µL + 98 µL) x 1.05 = total volume of assay required. · Example: (96 samples x 0.8 µL + 98 µL) x 1.05 = 183.5 µL b. For a single 384-well sample plate, 405 µL × 1.05 = total volume of assay required. c. For multiple 384-well sample plates, 405 µL × # of plates × 1.05 = total volume of assay required. 4) Preparethe2X mixtureof PCR reagentsusing a1.5-2mL tubereferencingTable10. Table 11 and Table 12 give examples assuming 2 tubes required for each assay. 5) Example calculation for the preparation of enough reagent to test a single 384-well plate, the total volume of reagent needed would be: 405 µL x 1 X 1.05 = 425.25 µL of each N1 or N2 reagent total. Using Table 10 below the individual component volumes would be: 425.25 / 2.5 = 170.1 µL - RapiDxFire qPCR 5X Master Mix GF 425.25 / 33.3 = 12.8 µL - EpiScript RNase H- Reverse Transcriptase 425.25 / 50 = 8.5 µL - 2019-nCoV N1/RnP or N2/RnP blend 425.25 / 100 = 4.25 µL - SuperROX Reference Dye 425.25 170.1 12.8 8.5 4.25 = 229.6 µL - Molecular grade water 24 For Emergency UseAuthorization.| Rx Only | For In Vitro Diagnostic Use. Instructions for use Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Test RapiDxFire qPCR 5X Master Mix GF1 EpiScript RNase H- Reverse Transcriptase2 2019-nCoV N13 or N24/RnP blend SuperROX Reference Dye5 Molecular grade water Stock concentration 5X 200 U/µL 100X 15 µM - Table 10. Variable reagent volume calculations. 1 Part numbers: 30050-1, 30050-2, or 30050-100ML 2 Part numbers: ERT12925K-ENZ, ERT12925K-1.25ML or ERT12925K-12ML 3 Part number: N1RNP-1010 2019-nCoV N1/RnP Blend 4 Part number: N2RNP-1010 2019-nCoV N2/RnP Blend 5 Part numbers: SR-1000-1 or SR-1000-10 RapiDxFire qPCR 5X Master Mix GF EpiScript RNase H- Reverse Transcriptase 2019-nCoV N1/RnP blend SuperROX Reference Dye Molecular grade water Stock concentration 5X 200 U/µL 100X 15 µM - Table 11. N1/RnP reagent volumes for 2 x 384-well sample plate run. RapiDxFire qPCR 5X Master Mix GF EpiScript RNase H- Reverse Transcriptase 2019-nCoV N2/RnP blend SuperROX Reference Dye Molecular grade water Stock concentration 5X 200 U/µL 100X 15 µM - Table 12. N2/RnP reagent volumes for 2 x 384-well sample plate run. Working 2X concentration 2X 6 U/µL 2X 150 nM - To tal Volume (µL) =To tal /2.5 =To tal /33.3 =To tal /50 =To tal /100 =Total sum of all c o mp o n en ts xxxx Working 2X concentration 2X 6 U/µL 2X 150 nM To tal Volume (µL) 340.2 25.5 17.0 8.5 459.3 850.5 Working 2X concentration 2X 6 U/µL 2X 150 nM To tal Volume (µL) 340.2 25.5 17.0 8.5 459.3 850.5 25 For Emergency UseAuthorization.| Rx Only | For In Vitro Diagnostic Use. Instructions for use Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Test 6) Vortex to mix, briefly centrifuge, transfer 405 µL of the assay mixture into 0.75 mL matrix tubes (ThermoFisher 4170)and seal with aSepraSeal cap (ThermoFisher 4463). Ifrunninglessthan384 samples, the assay mixturevolume shouldbeadjustedaccordinglyas describedabove. If assay mixturewill not be immediately used,storeon ice or at 4 °C until ready foruse. The assay mixture is stable at 2-8°C for 24 hours. 7) When readyto beginthe IntelliQube run,place thematrix tubes containingthe 2X PCR reagent mixture into the matrix rack (ThermoFisher 4896) into the positions indicated by the IntelliQube protocol. The assay plate layout can be viewed in the Intellics software by clicking on the protocol in the protocols list and selecting "Quick Review" "PlateSummary." a. For example, when running two 384-well plates, place the matrix tubes containing the 2019-nCoV N1/RnP blend mixtureinto positionsA1 and C1 of thematrixrack (ThermoFisher 4896).Place the matrix tubes containingthe2019-nCoV N2/RnP blend into positions B1 and D1 of the same matrix rack. IMPORTANT: Always confirm proper placement of assay tubes based on the protocol prior to initiating a run. Improperly positioned reagents can lead to misclassification of sample and control results. NOTE: As an alternative to matrix tubes and matrix rack, a 96-well plate (KBS-7001-131, KBS7001-231 or Greiner Bio-one 786201) may be used. The assay mixtures must be pipetted into the same well locations designated above A1 and C1 for 2019-nCoV N1/RnP; B1 and D1 for 2019nCoV N2/RnP. Plates should be sealed with an adhesive foil seal (KBS-0600-002) and stored on ice or at 4 °C until readyfor use. Creating an IntelliQube protocol from template: 1) Start by selectingprotocol "EUA COVID TEMPLATE INLINE"and thenselect "Useas Template." 2) Check all the boxesshownin Figure6 below and select "Create." Figure 6. Create protocol from template. 26 For Emergency UseAuthorization.| Rx Only | For In Vitro Diagnostic Use. Instructions for use Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Test 3) Edit the protocol name to reflect therun being performedand verify all settings match Figure7 below. The Dye Calibration Setselected willbe specific to each instrumentand preset as a default. Figure 7. IntelliQube real-time protocol settings summary. 4) ReferencingFigure 8, importthesample plateinformationfor theprotocol.This can be done by selecting "Browse" and directing the software to the correct plateware .csv f iles. Figure 8. IntelliQube real-time sample plate import. 27 For Emergency UseAuthorization.| Rx Only | For In Vitro Diagnostic Use. Instructions for use Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Test 5) Verify assays2019-nCoV N1/RnP and 2019-nCoV N2/RnP are includedas shown in Figures9aand 9b. Figure 9a. IntelliQube sssay selection screen. Figure 9b. IntelliQube assay information details. 28 For Emergency UseAuthorization.| Rx Only | For In Vitro Diagnostic Use. Instructions for use Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Test 6) Using Figure 10as a reference,select all sample plates by bothassays and click therightarrow to create thecombinations. Figure 10. IntelliQube real-time sample/assay liquid handling. 7) Assign the assay plate barcodeby typingor scanningthebarcodeinto theprovidedspace and selecting "Apply Barcode" Figure11. Figure 11. IntelliQube assay plate barcode definition. 29 For Emergency UseAuthorization.| Rx Only | For In Vitro Diagnostic Use. Instructions for use Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Test 8) Review protocol plate layoutsin the "Array Setup"page and select "Next"or "Skipto Review"to progress to the "Review" page. 9) Review the "Protocol Summary"to ensureprotocolwas createdcorrectly.Figure12 can be used as a reference. Figure 12. IntelliQube real-time protocol summary. 10) A layout of thesample and assay platesincluding required volumescan be viewedand printedby selecting the "Print PlateLayouts." 11) Select "Finish" to complete the protocol setup. Loading the IntelliQube to begin a run: 1) If this is the firstrun of theday,performall daily startuproutine procedures. 2) Before each run, confirm there is an adequate supply of Array Tape(AXIT768-13WP050CC), cover seal (AX8591CVRTCC),sodiumhypochlorite,and RO sourcewater.If thewaste line is not directly plumbedto a drain, confirm the waste carboyis empty. 3) It is recommendedto replacetheDispensePipette tips (AX843799 or AX840999)on a daily basis at a minimum. 4) The two 384-well extractedRNA sampleplates should be centrifugedat 2,500 × g for 1 min. 5) Each sample plate shouldbe barcoded to match thebarcodes specifiedin the IntelliQubeprotocol.The required barcodes for a given protocol are visible on the instrument HMI. 6) After carefully removingthe plateseals,the sampleplates can be placed in any location within the sample plate stacker. IMPORTANT: Use caution when removing adhesive seals. Removal at a 45° angle will minimize the risk of sample transfer between wells. 30 For Emergency UseAuthorization.| Rx Only | For In Vitro Diagnostic Use. Instructions for use Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Test 7) The matrix tuberack containingthe prepared2X PCR reagentmixturesmust also be barcodedto match the barcode specified in the IntelliQube protocol. 8) Place the matrix tuberack in the Plate Deck1 positionand removethematrix tube caps usinga decapper tool. 9) Fromthe IntelliQubeHMI, selectthe appropriateprotocolfromthe listand hit "Run." 10) Upon completionof therun, thedye cycle detailsdatawill be automaticallyexportedanduploaded to FastFinderAnalysissoftware. 11) Proceedto the nextsection for the interpretation of results using the real-time RT-PCR workflow. Interpretation of results using the real-time RT-PCR workflow This test utilizes the UgenTecFastFinder Analysis software to implement the threshold and decision logic described below. Figure 13. FastFinder login. Figure 14. FastFinder Analysesmodule. 31 For Emergency UseAuthorization.| Rx Only | For In Vitro Diagnostic Use. Instructions for use Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Test Figure 15. FastFinder import and view results. Figure 16. FastFinder review and authorize results. Figure 17. FastFinder export results. 32 For Emergency UseAuthorization.| Rx Only | For In Vitro Diagnostic Use. Instructions for use Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Test Figure 18. FastFinder example csv results export file. 33 For Emergency UseAuthorization.| Rx Only | For In Vitro Diagnostic Use. Instructions for use Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Test The test utilizes a control scheme that requires the addition of one positive and one negative control into each 96-well sample source plate. The plates are processed on the oKtopure as four separate 96-well sample plates that are combined into a single 384-well elution plate.The IntelliQube real-time RT-PCR process generates fluorescence data f rom the amplification of the targets of interest. The fluorescence values are exported to the FastFinder Analysis software. Validation of the results is performed automatically by the FastFinder Analysis software based on performance of the Positive and Negative Controls, the passive reference dye, presence of the RNase P target, and concordance of the N1 and N2 targets. The outcomes generated can take the values of "Detected," "Not Detected" or "Void" in the case where one or more of the targetresults wereinvalidor inconclusive. Real-time: Control well individual target interpretation Target + Present Not present Passive Reference Dye RFU >= 0.2 RFU <0.2 N1 Cq <= 36 None or Cq >36 N2 Cq <= 36 None or Cq >36 N1-RP and N2RP Cq <= 37 None or Cq >37 Table 13. Interpretation of individual target results for the PC and NTC controls. 34 For Emergency UseAuthorization.| Rx Only | For In Vitro Diagnostic Use. Instructions for use Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Test Positive and Negative controls are examined by the FastFinder Analysis software and are evaluated against the following control scenarios shown in Table 14 prior to interpretation of clinical results. Failed control wells are automatically flagged in the FastFinder analysis software. If any of the included controls are not marked as Valid, the corresponding patient results from the same source plate cannot be interpreted and will be marked as "Void" by default with a warning message related to the type of failure. Positive and Negative Control wells must be run for each 96-well sample extraction plate that is represented in analysis. Each control well corresponding to a 96-well source plate must pass for the samples analyzed with that plate to be considered valid. If there are failed control wells, the corresponding source plates can be removed as described below in order to complete the analysis for samples with valid controls. Cq value interpretation is definedbythe UgenTec FastFinder software scoring algorithms. Control Outcome for real- Scenario N1 N1RP N2 N2- Pass Control time or RP Ref [1] results end-point clinical samples All N gene and RNase P targets + detected + + + As scored by clinical + Valid sample decision logic Positive Control (PC) Any N gene All wells marked or RNase P target not ? ? ? ? ? Invalid as "Invalid detected Assay Controls" All N gene and RNase P targets not - detected - - - As scored by clinical + Valid sample decision logic No Template Control (NTC) Any N gene target or RNase P ? target detected ? ? ? All wells marked ? Invalid as "Invalid Assay Controls" Table 14. Interpretation of PC and NTC control results. [1] If the passive reference fails, the test can be repeated from the RT-PCR stage without re-extracting the samples. [2] A single control failure may be resolved by repeating RT-PCR, otherwise re-extract the original samples and repeat RT-PCR Suggested user action Report result. Option to mark any sample for re-test as needed. Repeat test by repeating RTPCR or reextracting the original samples associated with the f ailed source plates and repeating RTPCR[2]. Report result. Option to mark any sample for re-test as needed. Repeat test by repeating RTPCR or reextracting the original samples associated with the f ailed source plates and repeating RTPCR[2]. 35 For Emergency UseAuthorization.| Rx Only | For In Vitro Diagnostic Use. Instructions for use Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Test For any 96-well source plate that contains a control failure, it is possible in the FastFinder application to manually omit this source plate from analysis through the PCR setup tab inside the Analysis module such that only valid source plates are used for analysis and generation of results. This allows for valid results to still be analyzed while invalid results are omitted from analysis. The procedure is as follows: 1) Identify the source plate where the control failure is present. (Figure 19,20) 2) Remove the selected assignment of wells from the PCR setup tab. (Figure 21) 3) Reanalyze the results. (Figure 22) 4) Review and authorize the final result containing only the source plates with valid controls (Figure 23) Figure 19. Identify control failures and select assay details in the analysis summary tab. 36 For Emergency UseAuthorization.| Rx Only | For In Vitro Diagnostic Use. Instructions for use Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Test Figure 20. Identify the control failure well location in the details tab. Figure 21. Select and remove assignment for all N1 and N2 wells for the associated 96-well source plate in PCR setup tab. 37 For Emergency UseAuthorization.| Rx Only | For In Vitro Diagnostic Use. Instructions for use Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Test Figure 22. Reanalyze after removal of all wells associated with the specific source plate. After all controls included in analysis have been identified as Valid, the analysis pipeline evaluates the results of the individual patient samples and assigns an overall call of "Detected," "Not Detected" or "Void" to each sample set per the Positive/Negative calling threshold and decision logic. Figure 23. Review and authorize sample results. 38 For Emergency UseAuthorization.| Rx Only | For In Vitro Diagnostic Use. Instructions for use Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Test Real-time: Clinical sample individual target interpretation Target + "Positive" "Negative" Passive Reference RFU >= 0.2 RFU <0.2 N1 Cq Value <= 37.9 None or Cq Value >37.9 N2 Cq Value <= 37.5 None or Cq Value >37.5 N1-RP and N2-RP Cq Value <= 31.7 None or Cq Value >31.7 Table 15. Interpretation of clinical sample individual target results Clinical samples are examined by the FastFinder analysis software and results from Passive Ref erence Dyes and combinations of N1,N1_RP, N2, and N2_RP individual target results are automatically combined and evaluated against the following decision logic shown in Table 16 to define the clinical result. If any result other than "Detected" or "Not Detected" is determined for a given sample, the overall call for the clinical sample will be "Void" and it is recommended to repeat thetestby re-extractingthe original sampleand repeatingthe RT-PCR. 39 For Emergency UseAuthorization.| Rx Only | For In Vitro Diagnostic Use. Instructions for use Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Test Scenario Passive Ref N1 N1 N1_RP Passive Ref N2 Assay Result N2 N2_RP Outcome (User Message Interface) User action Neg ati v e s amp l e or + c o n c en trati on below LOD - + + - + No t d etec ted SARS-Co V-2 not detected N/A Rep o rt results Po s i ti v e sample above + LOD + + + + + Detected SARS-Co V-2 d etec ted N/A Rep o rt results RNase P amp l i fi c ati o n absent, but + + - both N1 and N2 are p res en t. + + - Detected SARS-Co V-2 d etec ted Ex trac ti o n control not d etec ted Rep o rt results N1 or N2 are present, but ? not both, or, any other scenario not rep res en ted abo v e ? ? ? ? ? Vo i d In c o n c l us iv e Vari o us Rep eat test by reex trac ti n g the original s amp l es an d rep eati n g the RT- PCR[1] Table 16. Clinical sample Real-Time decision logic. [1] A single inconclusive result may be evaluated by re-extracting the original sample and repeating RT-PCR. If a second inconclusive result is obtained, the result should be reported as "Inconclusive" and a request to recollect sample should be made. IntelliQube end-point RT-PCR detection of SARS-CoV-2 NOTE: This section describes the end-point RT-PCR modality of operation. For real-time operation, see p. 22. Instrument daily startup routine: IntelliQube 1) Check Carboys. a. Empty Waste Carboy. b. Fill Source WaterCarboy. c. Check Bleach Carboy;refill if low. 2) Purge DispenseJet. a. Manual Control > Maintenance >Jet Purge 3) Wash Dispense Jet andre-pressurize. a. Manual Control> Jet > "EUA Jet Configuration"> Cycles:200> Wash Tips b. Manual Control> Jet > Pressurizeto atarget pressureof 1.6 psi 40 For Emergency UseAuthorization.| Rx Only | For In Vitro Diagnostic Use. Instructions for use Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Test 4) Clean Dispense Jet withEthanol. a. Prepare96-well deep-well plateor matrixtube rackwith70-80%ethanol. i. Fill wells A1, B1, C1,and D1 with 700µLof 70-80% ethanol. ii. Place on deck position1. b. Manual Control> Jet > SelectAll Tips > Select Plate Deck 1 > SelectAppropriate Plateware > Select Full Dispense Pattern > Aspirate. Refer to Figure 24. c. Wait 5-10 minutes. Figure 24. IntelliQube daily startup up routine operations control screen. 5) Flush the DispenseJet. a. Manual Control> Jet > Cycles: 200 > Wash Tips 41 For Emergency UseAuthorization.| Rx Only | For In Vitro Diagnostic Use. Instructions for use Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Test 6) Check the sodium hypochlorite concentration in the pipette wash protocol. a. Under the Pipette tab in manual control, select the EUA PipetteWash configurationand EUA 384 Tip 10 µL. b. Select Wash Tips. c. Once the basinhas filledand the DispensePipettebegins to move downto start theaspiration process, fault the instrument by opening the guard door. d. Test the bleach concentrationby movingthetest stripacross the wash fluidin the basin. e. Verify the bleach strip measures2500 ppm. f. Close the guarddoorand recover the instrument. g. Select Water Only Pipette Wash configuration and perform 2 washes. h. Repeat steps 6a and 6b. i. Allow the wash to continue to thelast flush cycle. j. Once the basinhas filledand the DispensePipettebegins to move downto start theaspiration process, fault the instrument by opening the guard door. k. Test the bleach concentrationby movingthetest stripacross the wash fluid in the basin. l. Verify the bleach strip measures0 ppm. m. Close the guarddoorand recover the instrument. n. Make any adjustmentsneededto the wash patternand repeatto verify adjustedsettings are correct. Hydrocycler2 preparation 1) Check water level. a. Add water to Ballast tank if required. 2) Pre-heattanks. a. Locate theEUA Thermal Profile and verifysetting matchFigure25. b. Select "Pre-heatTanks." Figure 25. Hydrocycler2 thermal cycling settings. 42 For Emergency UseAuthorization.| Rx Only | For In Vitro Diagnostic Use. Instructions for use Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Test 2X PCR reagent preparation 1x to 10x 384-well sample plate run: 1) Thaw all reagentsand keep on ice orat 4 °C until use. 2) Gently vortexreagentsand briefly centrifuge beforeuse. 3) Calculate amount of 2X assay to prepare. a. For less than 384 samples, take the (# of samples x 0.8 µL + 98 µL) x 1.05 = total volume of assay required. · Example: (96 samples x 0.8 µL + 98 µL) x 1.05 = 183.5 µL b. For a single 384-well sample plate, 405 µL × 1.05 = total volume of assay required. c. For multiple 384-well sample plates, 405 µL × # of plates × 1.05 = total volume of assay required. 4) Preparethe 2X mixtureof PCR reagentsusinga5-15 mL tube referencing Table17. Table 18 and Table 19 give examples assuming 10 tubes required for each assay. 5) Example calculation for the preparation of enough reagent to test a single 384-well plate, the total volume of reagent needed would be: 405 µL x 1 X 1.05 = 425.25 µL of each N1 or N2 reagent total. Using Table 10 below the individual component volumes would be: 425.25 / 2.5 = 170.1 µL - RapiDxFire qPCR 5X Master Mix GF 425.25 / 33.3 = 12.8 µL - EpiScript RNase H- Reverse Transcriptase 425.25 / 50 = 8.5 µL - 2019-nCoV N1/RnP or N2/RnP blend 425.25 / 100 = 4.25 µL - SuperROX Reference Dye 425.25 170.1 12.8 8.5 4.25 = 229.6 µL - Molecular grade water RapiDxFire qPCR 5X Master Mix GF1 EpiScript RNase H- Reverse Transcriptase2 2019-nCoV N13 or N24/RnP blend SuperROX Reference Dye5 Molecular grade water Stock concentration 5X 200 U/µL 100X 15 µM - Table 17. Variable reagent volume calculations. 1 Part numbers: 30050-1, 30050-2, or 30050-100ML 2 Part numbers: ERT12925K-ENZ, ERT12925K-1.25ML or ERT12925K-12ML 3 Part number: N1RNP-1010 2019-nCoV N1/RnP Blend 4 Part number: N2RNP-1010 2019-nCoV N2/RnP Blend 5 Part numbers: SR-1000-1 or SR-1000-10 Working 2X concentration 2X 6 U/µL 2X 150 nM - To tal Volume (µL) =To tal /2.5 =To tal /33.3 =To tal /50 =To tal /100 =Total sum of all components xxxx 43 For Emergency UseAuthorization.| Rx Only | For In Vitro Diagnostic Use. Instructions for use Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Test RapiDxFire qPCR 5X Master Mix GF EpiScript RNase H- Reverse Transcriptase 2019-nCoV N1/RnP blend SuperROX Reference Dye Molecular grade water Stock concentration 5X 200 U/µL 100X 15 µM - Table 18. N1/RnP reagent volumes for 10 x 384-well plate sample run. Working 2X concentration 2X 6 U/µL 2X 150 nM To tal Volume (µL) 1701 127.7 85.1 42.5 2296.2 4252.5 RapiDxFire qPCR 5X Master Mix GF EpiScript RNase H- Reverse Transcriptase 2019-nCoV N2/RnP blend SuperROX Reference Dye Molecular grade water Stock concentration 5X 200 U/µL 100X 15 µM - Table 19. N2/RnP reagent volumes for 10 x 384-well plate sample run. Working 2X concentration 2X 6 U/µL 2X 150 nM To tal Volume (µL) 1701 127.7 85.1 42.5 2296.2 4252.5 6) Vortex to mix, briefly centrifuge, transfer 405 µL of the assay mixture into 0.75 mL matrix tubes (ThermoFisher 4170)and seal with aSepraSeal cap (ThermoFisher 4463). Ifrunninglessthan384 samples, the assay mixturevolume shouldbeadjustedaccordinglyas describedabove. If assay mixturewill not be immediately used,storeon ice or at 4 °C until ready foruse. 7) When readyto beginthe IntelliQube run,place thematrix tubes containingthe 2X PCR reagent mixture into the matrix rack (ThermoFisher 4896) into the positions indicated by the IntelliQube protocol. The assay plate layout can be viewed in the Intellics software by clicking on the protocol in the protocols list and selecting "Quick Review" "PlateSummary." IMPORTANT: Always confirm proper placement of assay tubes based on the protocol prior to initiating a run. Improperly positioned reagents can lead to misclassification of sample and control results. NOTE: As an alternative to matrix tubes and matrix rack, a 96-well plate (KBS-7001-131 or Greiner Bio-one 786201) may be used. The assay mixtures must be pipetted into the wells designated in the IntelliQube protocol. Plates should be sealed with an adhesive foil seal (KBS-0600-002) and stored on ice or at 4 °C until ready for use. 44 For Emergency UseAuthorization.| Rx Only | For In Vitro Diagnostic Use. Instructions for use Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Test Creating an IntelliQube protocol from template: 1) Start by selectingprotocol "EUA COVID TEMPLATE OFFLINE"and selecting "Use as Template." 2) Check all the boxesshownin Figure26 belowand select "Create." Figure 26. IntelliQube create protocol from template. 3) Edit the protocol name to reflect therun being performedand verifyall settings match Figure27 below. The Dye Calibration Setselectedwill be specific to each instrumentand preset as a default. Figure 27. IntelliQube end-point protocol setup screen. 45 For Emergency UseAuthorization.| Rx Only | For In Vitro Diagnostic Use. Instructions for use Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Test 4) ReferencingFigure 28, importthesample plateinformationfor theprotocol. Thiscan be done by selecting "Browse" and directing the software to the correct plateware .csv f iles. Figure 28. IntelliQube end-point sample plate import. 5) Verify assays2019-nCoV N1/RnP and 2019-nCoV N2/RnP are includedas shown in Figure 29. Figure 29. IntelliQube assay selection screen. 46 For Emergency UseAuthorization.| Rx Only | For In Vitro Diagnostic Use. Instructions for use Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Test 6) Using Figure 30 as a reference, select all sample plates by both assaysand click therightarrow to create thecombinations. Figure 30. IntelliQube end-point sample/assay liquid handling. 7) Assign the assay plate barcodeby typingor scanningthebarcodeinto theprovidedspace and selecting "Apply Barcode" as shown in Figure 31. Figure 31. IntelliQube assay plate barcode definition. 47 For Emergency UseAuthorization.| Rx Only | For In Vitro Diagnostic Use. Instructions for use Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Test 8) Review the "Protocol Summary"to ensureprotocolwas createdcorrectly.Figure32 can be used as a reference. Figure 32. End-point protocol summary. 9) A layout of thesample and assay platesincluding required volumescan be viewedand printedby selecting the "Print PlateLayouts." 10) Select "Finish" to complete theprotocol setup. Loading the IntelliQube to begin the run: 1) If this is the firstrun of theday,performall daily startuproutine procedures. 2) Before each run, confirm there is an adequate supply of Array Tape(AXIT384-13WP050CC), cover seal (AX8591CVRTCC),sodiumhypochlorite,and RO sourcewater.If thewaste line is not directly plumbedto a drain, confirm the waste carboyis empty. 3) It is recommendedto replacetheDispensePipette tips (AX843799 or AX840999)on a daily basis at a minimum. 4) The extracted384-wellRNA sampleplatesshould becentrifuged at 2,500 × g for 1 min. 5) Each sample plate shouldbe barcoded to match thebarcodes specifiedin the IntelliQubeprotocol.The required barcodes for a given protocol are visible on the instrument HMI. 6) After carefully removingthe plateseals,the sampleplates can be placed in any location within the sample plate stacker. IMPORTANT: Use caution when removing adhesive seals. Removal at a 45° angle will minimize the risk of sample transfer between wells. 48 For Emergency UseAuthorization.| Rx Only | For In Vitro Diagnostic Use. Instructions for use Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Test 7) The matrix tuberack containingthe prepared2X PCR reagentmixturesmust also be barcodedto match the barcode specified in the IntelliQube protocol. 8) Place the matrix tuberack in the Plate Deck1 positionand removethematrix tube caps usinga decapper tool. 9) Fromthe IntelliQube HMI, select theappropriateprotocolfromthe listand hit "Run."A window will pop up informingthat this is the dispensingportionof the twopartprotocol. 10) As the tape is being dispensed and beginning to exit the IntelliQube, attach the Array Tape to the rewind spool using a piece of tape. 11) After theIntelliQubehas completed thedispensing portionof the protocol,detachthe rewindspool from the IntelliQube. 12) Using a spare piece of emptyArray Tapecontaining four arrays, wrap theoutsideof the dispensed arrays containingsampleswiththesparepieceof tape. 13) Use two clips to secure the spare piece of tape holding the sample containing arrays in place on the spool. 14) Attach the spool containingthesamplesto theHydrocycler2basket. 15) Insert the basket intothe Hydrocycler2. 16) Verify the EUA Thermal Profileis selected(Figure33)in the Hydrocycler2 softwareand select "Next." Figure 33. Thermal cycling parameters for the Hydrocycler2. 17) Select "Run." 18) Aftercompletionof thethermal cycleprotocol, remove the basketand detach the spool. 19) Unwind thearraysfromthespooland drywith apaper towel. 20) Re-spool thedriedArray Tapeontoan empty Array Tapespool. 21) Insert the spool back intothe IntelliQube. 22) Fromthe IntelliQube HMI, select theappropriateprotocolfromthe listand hit "Run."A window will pop up informingthat this is the detectionportion of thetwo part protocol. 23) Upon completionof therun, thedye cycle details datawill be automatically exported and uploadedto FastFinder Analysissoftware. 24) Proceedto the nextsection for theinterpretationof results using the end-point RT-PCR workflow. 49 For Emergency UseAuthorization.| Rx Only | For In Vitro Diagnostic Use. Instructions for use Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Test Interpretation of results using the end-point RT-PCR workflow This test utilizes the UgenTecFastFinder Analysis software to implement the threshold and decision logic described below. Figure 34. FastFinder login. Figure 35. Analysismodule. Figure 36. Import and view results. 50 For Emergency UseAuthorization.| Rx Only | For In Vitro Diagnostic Use. Instructions for use Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Test Figure 37. Review and authorize results. Figure 38. Export results. 51 For Emergency UseAuthorization.| Rx Only | For In Vitro Diagnostic Use. Instructions for use Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Test Figure 39. Example csv results export file. 52 For Emergency UseAuthorization.| Rx Only | For In Vitro Diagnostic Use. Instructions for use Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Test The test utilizes a control scheme that requires the addition of one positive and one negative control into each 96-well sample source plate. The plates are processed on the oKtopure as four separate 96-well sample plates that are combined into a single 384-well elution plate.The IntelliQube end-point RT-PCR process generates fluorescence data f rom the amplification of the targets of interest. The fluorescence values are exported to the FastFinder Analysis software. Validation of the results is performed automatically by the FastFinder Analysis software based on performance of the Positive and Negative Controls, the passive reference dye, presence of the RNase P target and concordance of the N1 and N2 targets. The outcomes generated can take the values of "Detected," "Not Detected" or "Void" in the case where one or more of the targetresults wereinvalidor inconclusive. End-point: Control well individual target interpretation Target + Present Not present ? Inconclusive Passive Reference Dye RFU >= 0.2 RFU <0.2 N/A N1 Part of positive cluster Part of negative cluster N2 Part of positive cluster Part of negative cluster Not part of either cluster N1-RP and N2- RP Part of positive cluster Part of negative cluster Table 20. Interpretation of individual target results for the PC and NTC controls. Positive and Negative controls are examined by the FastFinder analysis software and are evaluated against the following control scenarios shown in Table 21 prior to interpretation of unknown clinical results. Failed control wells are automatically flagged in the FastFinder analysis software. If any of the included controls are not marked as Valid, the corresponding patient results from the same source plate cannot be interpreted and will be marked as "Void" by default with a warning message related to the type of failure. Positive and Negative Control wells must be run for each 96-well sample extraction plate that is represented in analysis. Each control well corresponding to a 96-well source plate must pass for the samples analyzed with that plate to be considered valid. If there are failed control wells, the corresponding source plates can be removed as described below in order to complete the analysis for samples with valid controls. End-point cluster calling is definedbythe UgenTec FastFinder software scoring algorithms. 53 For Emergency UseAuthorization.| Rx Only | For In Vitro Diagnostic Use. Instructions for use Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Test Control Po s i ti v e Co n tro l (PC) No Temp l ate Co n tro l (NTC) Scenario All N gene and RNase P targ ets d etec ted Any N gene or RNase P target not d etec ted All N gene and RNase P targets not d etec ted Any N gene or RNase P targ et d etec ted , N1 N1RP N2 N2RP Pass Ref [1] Control results Outcome for real-time or endpoint clinical Suggested user action sample + + + + + Valid As scored by clinical sample decision logic Report result. Option to mark any sample for re-test as needed. Repeat test by re- All wells marked as extracting the ? ? ? ? ? In v al i d "Invalid Assay original samples Co n tro l s " and repeating the RT-PCR - - - - + Valid As scored by clinical sample decision logic Report result. Option to mark any sample for re-test as needed. Repeat test by re- All wells marked as extracting the ? ? ? ? ? In v al i d "Invalid Assay original samples Co n tro l s " and repeating the RT-PCR Table 21. Interpretation of PC and NTC control results. [1] If the passive reference fails, the test can be repeated from the RT-PCR stage without re-extracting the samples. For any 96-well source plate that contains a control failure, it is possible in the FastFinder application to manually omit this source plate from analysis through the PCR setup tab inside the Analysis module such that only valid source plates are used for analysis and generation of results. This allows for valid results to still be analyzed while invalid results are omitted from analysis. The procedure is as follows: 1) Identify the source plate where the control failure is present. (Figure 40,41) 2) Remove the selected assignment of wells from the PCR setup tab. (Figure 42,43,44) 3) Reanalyze the results. (Figure 45,46) 4) Review and authorize the final result containing only the source plates with valid controls. (Figure 47) 54 For Emergency UseAuthorization.| Rx Only | For In Vitro Diagnostic Use. Instructions for use Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Test Figure 40. Identify control failures and select assay details in the analysis summary tab. Figure 41. Identify the control failure well location in the details tab. 55 For Emergency UseAuthorization.| Rx Only | For In Vitro Diagnostic Use. Instructions for use Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Test Figure 42. Select and remove assignment for N1 wells in PCR setup tab. Figure 43. Select and remove assignment for N2 wells in PCR setup tab. 56 For Emergency UseAuthorization.| Rx Only | For In Vitro Diagnostic Use. Instructions for use Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Test Figure 44. Reanalyze after removal of all wells associated with the specific source plate. After all controls included in analysis have been identified as Valid, the analysis pipeline evaluates the results of the individual patient samples and assigns an overall call of "Detected," "Not Detected" or "Void" to each sample set per the Positive/Negative calling threshold and decision logic. Figure 45. Review and authorize sample results. 57 For Emergency UseAuthorization.| Rx Only | For In Vitro Diagnostic Use. Instructions for use Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Test End-point: Clinical sample individual target interpretation Target + "Positive" "Negative" ? Inconclusive Passive Referen c e RFU >= 0.2 RFU <0.2 N/A N1 N2 N1-RP and N2-RP Part of positive cluster Part of negative cluster Not in positive or negative cluster Table 22. Interpretation of clinical sample individual target results. Clinical samples are examined by the FastFinder Analysis software and results from Passive Reference Dyes and combinations of N1,N1_RP, N2, and N2_RP individual target results are automatically combined and evaluated against the following decision logic shown in Table 23 to define the clinical result. If any result other than "Detected" or "Not Detected" is determined for a given sample, the overall call for the clinical sample will be "Void" and it is recommended to repeat thetestby re-extractingthe original sampleand repeatingthe RT-PCR. End-point cluster calling is defined by the UgenTec FastFinder software scoring algorithms. 58 For Emergency UseAuthorization.| Rx Only | For In Vitro Diagnostic Use. Instructions for use Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Test Scenario Passive Ref N1 N1 N1_RP Passive Ref N2 Assay result N2 N2_RP Outcome (User Message Interface) User action Neg ati v e s amp l e or + c o n c en trati on below LoD - + + - + No t d etec ted SARS-Co V-2 not detected N/A Rep o rt results Po s i ti v e sample above + Lo D + + + + + Detected SARS-Co V-2 d etec ted N/A Rep o rt results RNase P amp l i fi c ati o n absent, but + + - both N1 and N2 are p res en t. + + - Detected SARS-Co V-2 d etec ted Ex trac ti o n control not d etec ted Rep o rt results N1 or N2 are present, but not both, or, any other scenario ? not represented abo v e ? ? ? ? ? Vo i d In c o n c l us iv e Vari o us Rep eat test by reex trac ti n g the original s amp l es an d rep eati n g the RT- PCR[1] Table 23. Clinical sample end-point decisionlogic. [1] A single inconclusive result may be evaluated by re-extracting the original sample and repeating RT-PCR. If a second inconclusive result is obtained, the result should be reported as "Inconclusive" and a request to recollect sample should be made. Limitations · The use of this assay as an in vitro diagnostic under the FDA 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. · The SARS-CoV-2 Real-Time and End-Point RT-PCR Test may only be performed using the oKtopure automated extraction instrument, as well as the IntelliQube and/or Hydrocycler2 qPCR instruments using clinical specimens that have been collected as per testing lab procedures or f ollowing vendor instructions. · The performance of the SARS-CoV-2 Real-Time and End-Point RT-PCR Test was established using archived nasopharyngeal swab specimens. Anterior or mid-turbinate nasal swabs, nasopharyngeal swabs, oropharyngeal swabs and nasopharyngeal wash/aspirates or nasal aspirates are also considered acceptable specimen types for use with the SARS-CoV-2 Real-Time and End-Point RT-PCR Test. 59 For Emergency UseAuthorization.| Rx Only | For In Vitro Diagnostic Use. Instructions for use Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Test · The clinical perf ormance has not been established in all circulating variants but is anticipated to be ref lective of the prevalent variants in circulation at the time and location of the clinical evaluation. Perf ormance at the time of testing may vary depending on the variants circulating, including newly emerging strains of SARS-CoV-2 and their prevalence, which change over time. · Validation studies f or non-saliva respiratory specimens were performed using BD Universal Viral Transport media (www.bd.com, phone number 201-847-6800) and UTM (Copan Universal Transport Medium, www.copanusa.com, phone number: (800)-216-4016). Compatibility with other specimen collection media and/or transport media has not been evaluated. Use of this assay is limited to personnel who have been trained in the procedure. Failure to follow the instructions provided in this package insert may cause erroneous results. · Reliable results are dependent on adequate specimen collection. Because the collection and transport system does not allow f or microscopic assessment of specimen adequacy, training of clinicians in proper specimen collection techniques is necessary. · Caref ul compliance with the instructions in this package insert is necessary to avoid erroneous results. Conditions of Authorization for the Laboratory To assist clinical laboratories running the SARS-CoV-2 Real-Time and End-Point RT-PCR Test, the relevant Conditions of Authorization are listed below: A. Authorized laboratories1 using the SARS-CoV-2 Real-Time and End-Point RT-PCR Test must include with test result reports all authorized Fact Sheets. Under exigent circumstances, other appropriate methods for disseminating these Fact Sheets may be used, which may include mass media. B. Authorized laboratories using the SARS-CoV-2 Real-Time and End-Point RT-PCR Test must use the SARS-CoV-2 Real-Time and End-Point RT-PCR Test as outlined in the authorized labeling. Deviations from the authorized procedures, including the authorized instruments, authorized extraction methods, authorized clinical specimen types, authorized control materials, authorized other ancillary reagents and authorized materials required to perform the SARS-CoV-2 Real-Time and End-Point RT-PCR Test are not permitted. C. Authorized laboratories that receive the SARS-CoV-2 Real-Time and End-Point RT-PCR Test must notify the relevant public health authorities of their intent to run the test prior to initiating testing. D. Authorized laboratories using the SARS-CoV-2 Real-Time and End-Point RT-PCR Test must have a process in place f or reporting test results to healthcare providers and relevant public health authorities, as appropriate. E. Authorized laboratories must collect information on the performance of the SARS-CoV-2 Real-Time and End-Point RT-PCR Test and report to DMD/OHT7-OIR/OPEQ/CDRH (via email: CDRH-EUAReporting@fda.hhs.gov) and LGC, Biosearch Technologies (techsupport@lgcgroup.com) any suspected occurrence of false positive or false negative results and significant deviations from the established perf ormance characteristics of the test of which they become aware. 60 For Emergency UseAuthorization.| Rx Only | For In Vitro Diagnostic Use. Instructions for use Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Test F. All laboratory personnel using the test must be appropriately trained in PCR techniques and use appropriate laboratory and personal protective equipment when handling this kit and use the test in accordance with the authorized labeling. G. LGC, Biosearch Technologies, its authorized distributor(s) and authorized laboratories using the SARSCoV-2 Real-Time and End-Point RT-PCR Test must 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. 1For ease of reference, this letter will refer to, "Laboratories certified under the Clinical Laboratory Improvement Amendments of 1988 (CLIA), 42 U.S.C. §263a, that meet requirements to perform high complexity tests" as "authorized laboratories." Performance evaluation Analytical sensitivity Limit of Detection (LoD) studies determine the lowest detectable concentration of SARS-CoV-2 at which greater than or equal to 95% of all (true positive) replicates test positive. To determine the LoD, recombinant virus containing a f ull length SARS-CoV-2 RNA (LGC SeraCare, AccuPlex SARS-CoV-2, Cat: 0505-0168, 100,000 copies/mL stock as determined by reverse transcription digital PCR) was diluted in pooled nasopharyngeal swab specimen matrix previously confirmed to be negative by another FDA-authorized assay. The final LoD was confirmed by testing 5 panel members with target concentrations at 10,000, 7,500, 5,000, 2,500 and 1,250 copies/mL, determined prior to extraction, tested in replicates of 40. The results are summarized in Table 24 and Table 25. The lowest concentration level with observed positive rates 95% for both the N1 and N2 targets was 5,000 viral genome copies/mL for the realtime workflow and 5,000 viral genome copies/mL for the end-point workflow. Sample Concentration Total (copies/mL) replicates 10,000 40 7,500 40 AccuPlex 5,000 40 SARS- Co V-2 2,500 40 1,250 40 0 (blank) 40 Table 24. LoD determination with the real-time workflow. N1 100% 100% 100% 95% 72.5% 0% Positive rate (%) N2 N1 and N2 100% 100% 100% 100% 100% 100% 97.5% 92.5% 75% 55% 0% 0% Mean Cq N1 N2 29.36 29.06 29.60 29.33 29.99 29.71 31.13 30.92 32.11 31.62 - - 61 For Emergency UseAuthorization.| Rx Only | For In Vitro Diagnostic Use. Instructions for use Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Test Sample Concentration (copies/mL) Total replicates 10,000 40 7,500 40 AccuPlex 5,000 40 SARS-Co V-2 2,500 40 1,250 40 0 (blank) 40 Table 25: LoD determination with the end-pointworkflow. N1 100% 100% 100% 82.5% 77.5% 0% Positive rate (%) N2 N1 and N2 100% 100% 100% 100% 100% 100% 100% 82.5% 90% 72.5% 0% 0% Inclusivity (analytical sensitivity) and Cross-reactivity (analytical specificity) The Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Testuses the same primers and probes for the N1 and N2 regions of the nucleocapsid gene as the CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel, manufactured by Biosearch Technologies. In silico analysis of primer and probe inclusivity and specificity was performed by CDC. The CDC has granted a right of ref erence to the performance data contained in the CDC's EUA request to any entity seeking an FDA EUA for a COVID-19 diagnostic device. No additional laboratory testing to evaluate inclusivity and analytical specificity was performed with the Biosearch Technologies SARSCoV-2 Real-Time and End-Point RT-PCR Test. Clinical evaluation A clinical evaluation study was perf ormed to evaluate perf ormance of the Biosearch Technologies SARSCoV-2 Real-Time and End-Point RT-PCR Test using upper respiratory specimens including nasopharyngeal, mid-turbinate, and anterior nares swabs (banked and acquired f rom a clinical laboratory). A total of 128 individual clinical specimens (91 were nasopharyngeal and 47 were mid-turbinate and anterior nares samples) were analyzed using the Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Test and an FDA-authorized comparator assay.Positive Percent Agreement(PPA) was 100.0% for both specimen types for both real-time and end-point workflows,and the Negative Percent Agreement (NPA) was 100.0% for both real-time and end-point workflows.Real-time results are summarized in Tables 26 and 27, and end-pointresults are summarized in Tables 28 and 29. 62 For Emergency UseAuthorization.| Rx Only | For In Vitro Diagnostic Use. Instructions for use Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Test Biosearch Technologies SARS-CoV-2 End-Point RT-PCR Test FDA-authorized Comparator Assay Positive Negative Total Po s i ti v e 39 0 39 In c o n c l us iv e 11 0 1 Neg ati v e 0 51 51 To tal 40 51 91 PPA 100.0% (91.0% - 100.0%) NPA 100.0% (93.0% - 100.0%) Inconclusive Rate 1.1% (1/91) Table 26. Real-Time Clinical Performance Nasopharyngeal (NP) Swab Samples. 1 Samples with repeat inconclusive results were excluded from the calculation of PPA and NPA. Biosearch Technologies SARS-CoV-2 Real-Time RT-PCR Test Positive FDA-authorized Comparator Assay Negative Total Po s i ti v e 24 0 24 In c o n c l us iv e 0 0 0 Neg ati v e 0 13 13 To tal 24 13 37 PPA 100.0% (86.2% - 100.0%) NPA 100.0% (77.2% - 100.0%) Table 27. Real-Time Clinical Performance Mid-turbinate and Anterior Nasal Swab Specimens Biosearch Technologies SARS-CoV-2 End-Point RTPCR Test Po s i ti v e In c o n c l us iv e Positive 40 0 FDA-authorized Comparator Assay Negative 0 0 Total 40 0 Neg ati v e To tal PPA 0 51 51 40 51 91 100.0% (91.2% - 100.0%) NPA 100.0% (93.0% - 100.0%) Table 28. End-point Clinical Performance - Nasopharyngeal (NP) Swab Samples 63 For Emergency UseAuthorization.| Rx Only | For In Vitro Diagnostic Use. Instructions for use Biosearch Technologies SARS-CoV-2 Real-Time and End-Point RT-PCR Test Biosearch Technologies FDA-authorized Comparator Assay SARS-CoV-2 End-Point RT- Positive Negative Total PCR Test Po s i ti v e 24 0 24 In c o n c l us iv e 0 0 0 Neg ati v e 0 13 13 To tal 24 13 37 PPA 100.0% (86.2% - 100.0%) NPA 100.0% (77.2% - 100.0%) Table 29. End-point Clinical Performance Mid-turbinate and Anterior Nasal Swab Specimens Summary of changes Revision Doc ID number GEN/861/SW/1120 GEN/861/SW/1120/v2/02082021 GEN/861/SW/1120/v2/03242021 Description of change In itial release. No tificatio n to FDA o f co mp letio n o f clinical validation. Version 2 release. FDA review. Version 3 release. FDA authorization. Date November 2020 Feb 8, 2021 May 17th, 2021 64 For Emergency UseAuthorization.| Rx Only | For In Vitro Diagnostic Use. @LGCBiosearch biosearchtech.com All trademarks and registered trademarks mentioned herein are the property of their respective owners. All other trademarks and registered trademarks are the property of LGC and its subsidiaries. Specifications, terms and pricing are subject to change. Not all products are available in all countries. Please consultyour local sales representative for details. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording or any retrieval system, withoutthe written permission of the copyright holder. © LGC Limited, 2021. All rights reserved. GEN/861/SW/1120/v2/03242021