Instruction Manual for appliedbiosystems models including: TaqPath COVID-19 PoolingKit
TaqPath COVID-19 Pooling Kit Instructions for Use
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Thermo Fisher Scientific (25 May 2021)
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TaqPath COVID-19 Pooling Kit Instructions for Use
25 mag 2021 — CHAPTER 1 TaqPath™ COVID-19 Pooling Kit product information ..................... 6 ... Extract RNA—Manual method (400-μL sample input volume) .
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EUA-ThermoF-Pooling-ifu TaqPathTM COVID-19 Pooling Kit
INSTRUCTIONS FOR USE Multiplex real-time RT-PCR test intended for the qualitative detection of nucleic acid from SARSCoV2 in pooled samples
Catalog Number A49918 Publication
Number MAN0019598Revision A.0
For In Vitro Diagnostic Use. For Emergency Use Authorization Only | Rx Only
Life Technologies Corporation | 6055 Sunol Blvd | Pleasanton, California 94566 USA For descriptions of symbols on product labels or product documents, go to thermofisher.com/symbols-definition.
The customer is responsible for compliance with regulatory requirements that pertain to their procedures and uses of the instrument.The
information in this guide is subject to change without notice.
DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BELIABLE FOR SPECIAL, INCIDENTAL, INDIRECT,PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH ORARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.
Revision history: Pub. No. MAN0019598
Revision A.0
Date 25 May 2021
Description New Instructions for Use for the TaqPathTM COVID-19 Pooling Kit.
TRADEMARKS: All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. TaqManis a registered trademark of Roche Molecular Systems, Inc., used under permission and license.Everlywell is a trademark of Everly Well, Inc.Nasacort is a trademark of AVENTISUB LLC. Dymista is a trademark of Meda Pharmaceuticals Inc. NeilMed and Nasogel are trademarks of NeilMed Products, Inc. Chloraseptic is a trademark of Medtech Products Inc. Bactroban is a trademark of GLAXOSMITHKLINELLC. Similasan is a trademark of Similasan AG Corporation Switzerland.
©2021 Thermo Fisher Scientific Inc. All rights reserved.
Contents
CHAPTER 1 TaqPathTM COVID-19 Pooling Kit product information ..................... 6
Intended Use...........................................................................................................6 Product description ..................................................................................................7 Contents and storage ...............................................................................................7 Required materials not supplied .................................................................................8 Instrument and software compatibility....................................................................... 10 Warnings and precautions....................................................................................... 11 Assay limitations.................................................................................................... 12 General laboratory recommendations........................................................................ 14 Samples and controls ............................................................................................. 14 Workflow .............................................................................................................. 15
CHAPTER 2 Pool samples .........................................................................................17
Sample collection, transport, and storage ................................................................. 17 Defining a sample pooling strategy ........................................................................... 17 Monitor the pooling strategy..................................................................................... 18 Pool patient samples .............................................................................................. 19
CHAPTER 3 Extract RNA (automated method)......................................................20
Contents
Before you begin.................................................................................................... 20
Extract RNA--Automated method (400L sample input volume).................................. 21 Set up the instrument (400L sample input volume) ............................................ 21 Prepare the processing plates (400L sample input volume)................................. 22 Prepare Binding Bead Mix (400L sample input volume)...................................... 22 Prepare sample plate (400L sample input volume) ............................................ 23 Process the samples (400L sample input volume)............................................. 23
TaqPathTM COVID-19 Pooling Kit Instructions for Use
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CHAPTER 4 Extract RNA (manual method)............................................................24
Before you begin ....................................................................................................24 Extract RNA--Manual method (400L sample input volume)........................................24
Prepare Binding Bead Mix (400L sample input volume).......................................25 Digest with Proteinase K (400L sample input volume).........................................25 Wash the beads (400L sample input volume) ....................................................26 Elute the nucleic acid (400L sample input volume) .............................................26
CHAPTER 5 Prepare RT-PCR reactions..................................................................27
Guidelines for RT-PCR............................................................................................27 Prepare RTPCR reactions (384well reaction plate) ....................................................27
CHAPTER 6 Perform RT-PCR using the Applied BiosystemsTM
QuantStudioTM 7 Flex Real-Time PCR Instrument (384-well block) ....................30
Dye calibration for the QuantStudioTM 7 Flex Real-Time PCR Instrument..........................30 Transf er the template (EDT) file for the QuantStudioTM 7 Flex Real-Time PCR
Instrument (384well block) ..................................................................................30 Set up and run the QuantStudioTM 7 Flex Real-Time PCR Instrument (384well block).......31
CHAPTER 7 Analysis and results..............................................................................33
Obtain the Applied BiosystemsTM COVID19 Interpretive Software ..................................33 Analyze the data.....................................................................................................33 Interpretation of the results.......................................................................................35
CHAPTER 8 Conditions of authorization for labs....................................................38 CHAPTER 9 Performance characteristics ................................................................40
Limit of detection (LoD) for RT-PCR with 14.0 µL of purified sample RNA........................40 Reactivity (Inclusivity)..............................................................................................41 Interf ering substances .............................................................................................41 Cross-reactivity ......................................................................................................44 Clinical evaluation...................................................................................................45
Contents
APPENDIX A Ct cutoff values for assay targets .....................................................48
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TaqPathTM COVID-19 Pooling Kit Instructions for Use
APPENDIX B EUO label for RUO instrument.........................................................49 APPENDIX C Safety ....................................................................................................50
Chemical safety..................................................................................................... 50 Biological hazard safety .......................................................................................... 51
APPENDIX D Documentation and support..............................................................52
Related documentation........................................................................................... 52 Customer and technical support ............................................................................... 52 Limited product warranty......................................................................................... 53
TaqPathTM COVID-19 Pooling Kit Instructions for Use
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TaqPathTM COVID-19 Pooling Kit
product information
Intended Use ....................................................................................................................6 Product description............................................................................................................7 Contents and storage.........................................................................................................7
Required materials not supplied...........................................................................................8 Instrument and software compatibility ................................................................................ 10 Warnings and precautions ................................................................................................ 11
Assay limitations ............................................................................................................. 12
General laboratory recommendations ................................................................................. 14 Samples and controls....................................................................................................... 14 Workf low........................................................................................................................ 15
Intended Use
The TaqPathTM COVID-19 Pooling Kit contains the assays and controls for a real-time reverse transcription polymerase chain reaction (RTPCR) test that is intended for the qualitative detection of nucleic acid from SARS-CoV-2 in pooled samples containing up to 5 individual nasopharyngeal, oropharyngeal, anterior nasal, or mid-turbinate nasal swabs where each specimen is collected by a healthcare provider using individual vials containing transport media from individuals suspected of COVID-19. 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.
Negative results from pooled testing should not be treated as definitive. If a patient's clinical signs and symptoms are inconsistent with a negative result or results are necessary for patient management, then the patient should be considered for individual testing. Specimens included in pools with positive, inconclusive, or invalid results must be tested individually using the TaqPathTM COVID-19 Pooling Kit prior to reporting a result. Specimens with low viral loads may not be detected in sample pools due to the decreased sensitivity of pooled testing.
Results are f or the identification of SARS-CoV-2 RNA. The SARS-CoV-2 RNA is generally detectable in upper 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 for individual samples to the appropriate public health authorities.
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TaqPathTM COVID-19 Pooling Kit Instructions for Use
1 Chapter 1 TaqPathTM COVID-19 Pooling Kit product information Product description
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 TaqPathTM COVID-19 Pooling Kit is intended to be used with 14.0 µL of nucleic acid template extracted from an individual sample or sample pool and run on the Applied BiosystemsTM QuantStudioTM7 Flex Real-Time PCR Instrument (384well block) by qualified clinical laboratory personnel specifically instructed and trained in the techniques of real-time PCR and in vitro diagnostic procedures.
The TaqPathTM COVID-19 Pooling Kit is only for use under the Food and Drug Administration's Emergency Use Authorization.
Product description
The TaqPathTM COVID-19 Pooling Kit includes the assays and controls for a multiplex real-time RT-PCR test f or the qualitative detection of RNA from SARS-CoV-2 in pooled upper respiratory specimens (such as nasopharyngeal, oropharyngeal, anterior nasal, and mid-turbinate swabs) specimens from individuals suspected of COVID-19 by their healthcare provider.
The kit includes the following components:
· TaqPathTM COVID-19 Pooling Assay Kit TaqPathTM COVID-19 Pooling Multiplex Assay--Multiplexed assays that contain three primer/probe sets specific to different SARS-CoV-2 genomic regions and primers/probes for bacteriophage MS2 MS2 Phage Control--Internal process control for nucleic acid extraction
· TaqPathTM COVID19 Control--RNA control that contains targets specific to the SARS-CoV-2 genomic regions targeted by the assays
· TaqPathTM COVID19 Control Dilution Buffer--Buffer formulation for diluting the TaqPathTM COVID19 Control
Contents and storage
Table 1 TaqPathTM COVID-19 Pooling Kit, 200 reactions (Cat. No. A49918)
Box
Com ponents
TaqPathTM COVID-19 Pooling Assay Kit
TaqPathTM COVID-19 Pooling Multiplex Assay
(ORF1ab, N gene, S gene, MS2)
Am ount 300 µL
MS2 Phage Control TaqPathTM COVID19 Control (1 x 104 copies/µL) TaqPathTM COVID19 Control Dilution Buffer
2 × 1,000 µL 2 × 10 µL 2 × 250 µL
Storage
30°C to 10°C
30°C to 10°C 70°C
30°C to 10°C
TaqPathTM COVID-19 Pooling Kit Instructions for Use
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Chapter 1 TaqPathTM COVID-19 Pooling Kit product information Required materials not supplied
Required materials not supplied
Unless otherwise indicated, all materials are available through thermofisher.com. "MLS" indicates that the material is available from fisherscientific.com or another major laboratory supplier.
Catalog numbers that appear as links open the web pages for those products.
Item
Source
Real-time PCR instrument
Applied BiosystemsTM QuantStudioTM7 Flex Real-Time PCR Instrument, 384well block (used with QuantStudioTM RealTime PCR Software v1.3)
4485695 (with laptop computer) 4485701 (with desktop computer)
Equipm ent
Laboratory freezers · 30°C to 10°C · 70°C
MLS
Centrifuge, with a rotor that accommodates standard and deepwell microplates
Microcentrifuge
Laboratory mixer, vortex or equivalent
Single and multichannel adjustable pipettors (1.00 µL to 1,000.0 µL)
Cold block (96well or 384well) or ice Automated nucleic acid extraction system and materials KingFisherTM Flex Magnetic Particle Processor with 96 DeepWell Head KingFisherTM Flex 96 Deep-Well Heating Block KingFisherTM 96 Deep-Well Plate
MLS MLS MLS MLS MLS
5400630 24075430 95040450, A48305, A48424, 95040455
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TaqPathTM COVID-19 Pooling Kit Instructions for Use
1 Chapter 1 TaqPathTM COVID-19 Pooling Kit product information Required materials not supplied
(continued)
Item
96well plate for the tip comb, one of the following: · KingFisherTM 96 KF microplate · Tip Comb Presenting Plate for KF 96 · NuncTM MicroWellTM 96Well Microplate, Flat Bottom · NuncTM MicroWellTM 96Well Microplate, barcoded · ABgeneTM 96Well Polypropylene Storage Microplate · ABgeneTM 96Well 1.2mL Polypropylene DeepwellStorage Plate · NuncTM F96 MicroWellTM Black Polystyrene Plate · NuncTM F96 MicroWellTM White Polystyrene Plate · KingFisherTM 96 Deep-Well Plate
Source
· 97002540 · 267600 · 167008 · 269787 · AB0796 · AB1127
· 137101 · 136101 · 95040450, A48305, A48424, 95040455
KingFisherTM 96 tip comb for DW magnets
Manual nucleic acid extraction system and materials
Magnetic Stand-96
Compact Digital Microplate Shaker
Incubator capable of reaching 65°C with slatted shelves
KingFisherTM 96 Deep-Well Plate
Standard 96well plate for the eluate, one of the following: · KingFisherTM 96 KF microplate · MicroAmpTM Fast Optical 96Well Reaction Plate with Barcode, 0.1 mL · MicroAmpTM Fast Optical 96-Well Reaction Plate, 0.1 mL · MicroAm pTM Optical 96Well Reaction Plate with Barcode, 0.2 mL · MicroAmpTM Optical 96-Well Reaction Plate, 0.2 mL
97002534, A48438, A48414
AM10027 AM10050 88882005
MLS 95040450, A48305, A48424, 95040455
· 97002540 · 4346906, 4366932 · 4346907 · 4306737, 4326659 · N8010560, 4316813
MicroAm pTM Clear Adhesive Film Kits and reagents MagMAXTM Viral/Pathogen Nucleic Acid Isolation Kit
MagMAXTM Viral/Pathogen II Nucleic Acid Isolation Kit TaqPathTM 1Step Multiplex Master Mix (No ROXTM)
4306311
A42352 A48310 A48383 A28521, A28522, A28523
TaqPathTM COVID-19 Pooling Kit Instructions for Use
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Chapter 1 TaqPathTM COVID-19 Pooling Kit product information Instrument and software compatibility
(continued) Item
Fisher BioReagentsTM Ethanol, Absolute, Molecular Biology Grade[1], or equivalent
Source BP2818100, BP2818500, BP28184
Nuclease-free Water (not DEPC-Treated) Calibration plates(QuantStudioTM 7 Flex Real-Time PCR Instrument)
MLS
ABYTM Dye Spectral Calibration Plate for Multiplex qPCR, 384well
A24736
JUNTM Dye Spectral Calibration Plate for Multiplex qPCR, 384well
A24733
Tubes, plates, and other consumables MicroAm pTM Optical 384-Well Reaction Plate with Barcode MicroAm pTM Optical 384-Well Reaction Plate MicroAm pTM Clear Adhesive Film
4309849, 4326270, 4343814 4343370 4306311
MicroAm pTM Optical Adhesive Film MicroAm pTM Adhesive Film Applicator
4311971, 4360954 4333183
Nonstick, RNase-free microcentrifuge tubes (1.5 mL and 2.0 mL)
thermofisher.com/plastics
Sterile aerosol barrier (filtered) pipette tips
[1] Available at fisherscientific.com.
thermofisher.com/pipettetips
Instrument and software compatibility
The f ollowing table lists the version of the COVID19 Interpretive Software that is compatible with your instrument and pooling of samples.
Note: The TaqPathTM COVID-19 Pooling Kit is not compatible with samples collected using the EverlywellTM COVID-19 Test Home Collection Kit, and therefore does not use the RNase P test as part of the workflow. Select the COVID-19 Test button when you sign in to the COVID19 Interpretive Software.
For inf ormation on how to obtain the COVID19 Interpretive Software, see "Obtain the Applied BiosystemsTM COVID19 Interpretive Software" on page 33.
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TaqPathTM COVID-19 Pooling Kit Instructions for Use
1 Chapter 1 TaqPathTM COVID-19 Pooling Kit product information Warnings and precautions
To obtain the analysis software or firmware for your realtime PCR instrument, go to thermofisher.com/ qpcrsoftware, then select your instrument in the Real-Time PCR section.
Instrum ent
Analysis software used with the instrum ent
Compatible COVID19 Interpretive Software version
QuantStudioTM 7 Flex Real-Tim e PCR
Instrument with instrument firmware v1.0.4 · 384-well block
QuantStudioTM RealTim e PCR Software v1.3
v2.5
Warnings and precautions
The TaqPathTM COVID-19 Pooling Kit workflow should be performed by qualified and trained staff to avoid the risk of erroneous results. Use separate areas for the preparation of patient samples and controls to prevent false positive results. Samples and reagents must be handled in a biological safety cabinet.
· For in vitro diagnostic use under the FDA Emergency Use Authorization Only.
· This product has not been FDA cleared or approved but has been authorized for emergency use by FDA under an Emergency Use Authorization (EUA) for use by laboratories certified underthe Clinical Laboratory Improvement Amendments of 1988 (CLIA), 42 U.S.C. §263a, that meet requirements to perform high complexity tests.
· This product has been authorized only for the detection of nucleic acid from SARS-CoV-2, not for any other viruses or pathogens.
· This emergency use of this product is only authorized for the duration of the declaration that circumstances exist justifying the authorization of emergency use of in vitro diagnostics under Section 564(b)(1) of the Act, 21 U.S.C. § 360bbb-3(b)(1), unless the declaration is terminated or the authorization is revoked sooner.
· Samples and controls should always be treated as if infectious and/or biohazardous in accordance with saf e laboratory procedures.
· Follow necessary precautions when handling specimens. Use personal protective equipment (PPE) consistent with current guidelines for the handling of potentially infectious samples.
· Always use pipette tips with aerosol barriers. Tips that are used must be sterile and free from DNases and RNases.
· Do not eat, drink, smoke, or apply cosmetic products in the work areas.
· Modifications to assay reagents, assay protocol, or instrumentation are not permitted, and are in violation of the product Emergency Use Authorization.
· Reagents must be stored and handled as specified in Table 1 on page 7. · Do not use the kit after the indicated expiry date. · Dispose of waste in compliance with local, state, and federal regulations.
· Saf ety Data Sheets are available upon request.
· Laboratories within the United States and its territories are required to report all results to the appropriate public health authorities.
TaqPathTM COVID-19 Pooling Kit Instructions for Use
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Chapter 1 TaqPathTM COVID-19 Pooling Kit product information Assay limitations
· A positive result for a sample pool indicates the presence of SARS-CoV-2 RNA in at least one of the samples that were pooled. Deconvolution by testing the individual samples is required.
· An invalid or inconclusive result for a sample pool requires deconvolution by testing the individual samples.
Assay 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, that meet requirements to perform high complexity tests.
· Analytical performance of the TaqPathTM COVID-19 Pooling Kit was determined by leveraging limit of detection (LoD), interfering substances, and cross-reactivity data for individual samples using the TaqPathTM COVID-19 Combo Kit and TaqPathTM COVID-19 Combo Kit Advanced.
· TaqPathTM COVID-19 Pooling Kit performance was established using nasopharyngeal swab samples only. Anterior nasal swabs, oropharyngeal swabs, and mid-turbinate nasal swabs are considered acceptable specimen types for use with the TaqPathTM COVID-19 Pooling Kit, but performance with these specimen types has not been established. Refer to FDA's FAQs on Diagnostic Testing for SARS-CoV-2 f or additional information. Specimen types other than nasopharyngeal, anterior nasal, oropharyngeal, and mid-turbinate nasal swabs should not be tested with this assay.
· Samples should only be pooled when testing exceeds laboratory capacity and/or when testing reagents are in short supply.
· TaqPathTM COVID-19 Pooling Kit performance was established using only the TaqPathTM COVID-19 Combo Kit Advanced workflow performed on the Applied BiosystemsTM QuantStudioTM 7 Flex RealTime PCR Instrument, 384-well block (with QuantStudioTM RealTime PCR Software v1.3). The TaqPathTM COVID-19 Combo Kit and the TaqPathTM COVID-19 Combo Kit Advanced workflows on the f ollowing instruments have not been validated for pooling: Applied BiosystemsTM 7500 Fast Dx RealTime PCR Instrument Applied BiosystemsTM 7500 Fast RealTime PCR Instrument Applied BiosystemsTM 7500 RealTime PCR Instrument Applied BiosystemsTM QuantStudioTM 5 RealTime PCR Instrument (96-well plates, 0.1-mL block)
Applied BiosystemsTM QuantStudioTM 5 RealTime PCR Instrument (96-well plates, 0.2-mL block)
Applied BiosystemsTM QuantStudioTM 5 RealTime PCR Instrument (384-well plates)
· Samples must be collected, transported, and stored using appropriate procedures and conditions. Improper collection, transport, or storage of specimens may hinder the ability of the assay to detect the target sequences.
· Extraction and amplification of nucleic acid from clinical samples or sample pools must be perf ormed according the specified methods listed in this procedure. Other extraction approaches and processing systems have not been evaluated.
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TaqPathTM COVID-19 Pooling Kit Instructions for Use
1 Chapter 1 TaqPathTM COVID-19 Pooling Kit product information Assay limitations
· False-negative results may arise from: Improper sample collection Degradation of the SARS-CoV-2 RNA during shipping/storage Specimen collection after SARS-CoV-2 RNA can no longer be found in the specimen matrix
Using unauthorized extraction or assay reagents The presence of RT-PCR inhibitors Mutation in the SARS-CoV-2 virus Failure to f ollow instructions for use · False-positive results may arise from: Cross contamination during specimen handling or preparation Cross contamination between patient samples Specimen mix-up
RNA contamination during product handling Improper vortexing and centrifuging when preparing RT-PCR reactions.
· The impacts of vaccines, antiviral therapeutics, antibiotics, chemotherapeutic or immunosuppressant drugs have not been evaluated. The TaqPathTM COVID-19 Pooling Kit cannot rule out diseases caused by other bacterial or viral pathogens.
· Negative results do not preclude infection with SARS-CoV-2 virus, and should not be the sole basis of a patient management decision.
· Positive, inconclusive, and invalid results for pooled samples require deconvolution by testing the individual samples in the pool.
· Specimens with low SARS-CoV-2 RNA concentrations may not be detected in sample pools due to the decreased sensitivity of pooled testing.
· Laboratories are required to report all SARS-CoV-2 results for individual samples to the appropriate public health authorities.
· The clinical performance 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.
· Certain mutations may affect detection of individual targets with the TaqPathTM COVID-19 Pooling Kit, including the B.1.1.7 variant of SARS-CoV-2, which may exhibit positive results for the N and ORF1ab targets and negative results for the S-gene target. If such a pattern of detection is observed, further characterization of the specimen by sequence analysis should be considered. If such services are not readily available, local or state clinical laboratories should consider contacting the Centers for Disease Control and Prevention at EOCenter177@cdc.gov for additional information.
TaqPathTM COVID-19 Pooling Kit Instructions for Use
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Chapter 1 TaqPathTM COVID-19 Pooling Kit product information General laboratory recommendations
General laboratory recommendations
· Implement standard operating procedures in your laboratory to prevent contamination, such as the f ollowing: Frequent glove changes
Frequent decontamination of surfaces, equipment, and pipettes with 10% bleach or decontamination solution, followed by 70% ethanol
Use of ultraviolet light during biosafety cabinet decontamination (when available)
· To prevent degradation, keep eluted sample RNA, master mixes, assays, and controls on ice or in cold blocks while in use.
· Limit f reeze-thaw cycles. · Aliquot reagents to prevent stock contamination and reduce the number of freeze-thaw cycles. · Af ter each run, review the amplification curves in the interpretive software for signs of inadequate
vortexing or centrifugation. Contact your Applications Support team for additional information or training on data QC in your instrument software.
Samples and controls
Patient samples must be collected according to appropriate laboratory guidelines. Positive and negative test controls must be included to accurately interpret patient test results.
Store patient samples according to CDC guidelines. See the CDC website: https://www.cdc.gov/ coronavirus/2019-ncov/lab/guidelines-clinical-specimens.html.
Include the f ollowing controls:
Control
Positive Control (TaqPathTM COVID19Control)
Used to monitor
Assays
RT-PCR reaction setup and reagent integrity
All three SARS-CoV-2 assays
MS2 Phage Control
RNA extraction
MS2 assay
Negative Control
Cross-contamination during RNA extraction and reaction setup
All three SARS-CoV-2 assays MS2 assay
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TaqPathTM COVID-19 Pooling Kit Instructions for Use
1 Chapter 1 TaqPathTM COVID-19 Pooling Kit product information Workflow
Workflow
Pool patient samples
Extract RNA from the pooled samples
Perform RT-PCR
Analyze the data using the Applied Biosystem sTM COVID19 Interpretive Software
Review the run control results
Review the results interpretation for the pool
For pools that test positive or are invalid or inconclusive, extract and test the individual patient samples in each pool
The workf low begins by pooling up to 5 patient samples into a single tube at a total volume of 400 µL, using an equal volume from each sample to create the pool. Samples may include upper respiratory specimens (nasopharyngeal, oropharyngeal, anterior nasal, and mid-turbinate swabs) that arrive in the testing site in transport media. Nucleic acids are isolated and purified from the pooled samples using the MagMAXTM Viral/Pathogen Nucleic Acid Isolation Kit or the MagMAXTM Viral/Pathogen II Nucleic Acid Isolation Kit. Nucleic acid isolation can be performed manually or via an automated process using the KingFisherTM Flex Purification System (KingFisher). For more information about using the kit, see Appendix D, "Documentation and support". The nucleic acid is reverse transcribed into cDNA and amplified using the TaqPathTM COVID-19 Pooling Kit and the Applied BiosystemsTM QuantStudioTM 7 Flex Real-Time PCR Instrument, 384well block. In the process, the probes anneal to three (3) specific SARS-CoV-2 target sequences located between three (3) unique f orward and reverse primers for the following genes:
· ORF1ab · N Gene · S Gene
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 fluorescent signal. With each cycle, additional reporter dye molecules are cleaved from their respective probes, increasi ng the f luorescence intensity. Fluorescence intensity is monitored at each PCR cycle by the real-time P CR instrument. The data are analyzed, then interpreted by the Applied BiosystemsTM COVID19 Interpretive Software.
TaqPathTM COVID-19 Pooling Kit Instructions for Use
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Chapter 1 TaqPathTM COVID-19 Pooling Kit product information Workflow
For pools that test negative, then each sample is reported as negative with the following statement added to the report:
"Negative results are not definitive and, if inconsistent with clinical signs and symptoms or necessary for patient management, pooled samples should be tested individually. Negative results do not preclude SARS-CoV-2 infection and must not be used as the sole basis for patient management decisions. Negative results must be considered in the context of a patient's recent exposures, history, and presence of clinical signs and symptoms consistent with COVID-19."
For pools that test positive, invalid, or inconclusive, the patient samples in each pool must be extracted and tested individually using only the TaqPathTM COVID-19 Pooling Kit and the individual result reported.
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TaqPathTM COVID-19 Pooling Kit Instructions for Use
2
Pool samples
Sample collection, transport, and storage
Note: Handle all patient samples and controls as if they are capable of transmitting infectious agents.
Defining a sample pooling strategy
Sample pooling is generally recommended only if laboratories have backlogs in testing patient samples, resulting in delayed test result reporting. Pooling can lead to a reduction in the sensitivity for detecting SARS-CoV-2 RNA in a patient sample, because patient samples are combined, leading to overall sample dilution. In general, the larger the sample pool, the greater the risk of generating a false-negative result f rom testing pooled samples.[1,2] The TaqPathTM COVID-19 Pooling Kit has been validated for pool sizes ranging from 2 to 5 samples per pool.
Before Implementation of Pooling: Determine Appropriate Pool Size
Bef ore a pooling strategy is implemented, a laboratory should determine the appropriate pool size based on percent positivity rate and desired testing efficiency. If historical laboratory data for individual specimens are available:
· If historical data for individual specimens from the previous 7-10 days are available, estimate the percent positivity rate (Pindividual) based on individual results. (Pindividual) = (Number of positive specimens over chosen date range ÷ Total number of specimens tested over chosen date range) × 100
· Using the calculated Pindividual and Table 2, identify the appropriate n number of samples to pool. · If Pindividual is less than 7%, the maximum pool size validated, (n=5), should be selected to
maximize the efficiency of specimen pooling. Pooling with greater than 5 samples has not been validated and should not be performed. · If Pindividual is greater than 25%, pooling of patient specimens is not efficient and should not be implemented.
If historical laboratory data for individual specimens are unavailable: · If historical data from the previous 7-10 days are unavailable, 5-, 4-, or 3-specimen pooling may still be implemented. · Note: without calculating Pindividual, the pooling size implemented may not maximize pooling ef f iciency.
[1] https://www.who.int/bulletin/volumes/98/9/20-257188/en/ [2] https://www.cap.org/covid-19/pooled-testing-guidance-from-cap-microbiology-committee
TaqPathTM COVID-19 Pooling Kit Instructions for Use
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Chapter 2 Pool samples Monitor the pooling strategy
Table 2 Number of samples to pool based on the percent of positive individuals in the population inorder to achieve the maximum efficiency
(Pindividual) Percent of positive individuals in the tested population
(nmaxefficency) Number of samplesto pool for m axim um efficiency
(F) Efficiency of pooling n sam ples
0.1%6%
5
4.992.15
7%12%
4
1.991.54
13%25%
3
1.481.10
Monitor the pooling strategy
If historical laboratory data for individual specimens are available:
· Af ter implementing a pooling strategy, evaluate the performance of pooled testing by comparing the percent positivity rate of pooled testing to that of individual testing.
· Calculate the percent positivity rate among patient specimens during specimen pooling (Ppools) on a daily basis using a moving average of the data from the previous 710 days of testing. (Ppools) = (Number of patient specimens with a positive result as determined by individual specimen ref lex testing of positive pools over chosen date range ÷ Total number of patient specimens testedin pools over chosen date range) × 100
· Compare Ppools to Pindividual. If Ppools is less than 85% of Pindividual, (Ppools < 0.85 × Pindividual ), it is recommended that the pool size be reassessed and adjusted to maximize pooling efficiency (if necessary), according to the criteria in Table 2 on page 18.
· To ensure maximum pooling efficiency, it is recommended that nmaxefficiency be reassessed periodically while sample pooling is implemented by the laboratory.
If historical laboratory data for individual specimens are unavailable:
· Af ter initiating a pooling strategy, evaluate the performance of pooled testing by calculating the initial percent positivity rate f or pooled specimens (Ppools-initial). (Ppools-initial) is the percent positivity rate f or pooled specimens for the f irst 710 days of pooled testing.
· Calculate the initial percent positivity rate f or individual specimens f rom pool testing (Ppools-initial) f rom the first 710 days of testing. Ppools-initial = (Number of patient specimens with a positive result as determined by individual specimen reflex testing of positive pools in first 710[3] days ÷ Total number of patient specimens tested in pools in the f irst 710 days) × 100
· If Ppools-initial is greater than 25%, pooling of patient specimens is not efficient and should be discontinued until the percent positivity rate decreases.
· If Ppools-initial is less than or equal to 25%, pooling of patient specimens can be continued.
[3] Authorized laboratories must keep records of their sample pooling strategies, including type of strategy, date implemented, quantities tested, and test result data generated as part of monitoring the pooling strategy.
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TaqPathTM COVID-19 Pooling Kit Instructions for Use
2 Chapter 2 Pool samples Pool patient samples
· Continue to monitor pooling strategy by calculating the percent positivity rate among patient specimens during specimen pooling (Ppools-x) f or subsequent 710 day periods. (Ppools-x) should be updated daily using a moving average.
· Compare Ppools-x to Ppools-initial. If Ppools-x is less than 90% of Ppools-initial (Ppools-x < 0.90 × Ppoolsinitial), it is recommended that the pool size be reassessed and potentially adjusted to maximize pooling efficiency.
· To ensure maximum pooling efficiency, it is recommended that nmaxefficiency be reassessed periodically while sample pooling is implemented by the laboratory.
Note: When resource availability is sufficient to meet testing demand, the FDA recommends that laboratories consider whether the risks of reduced test sensitivity with pooling outweigh the benefits of resource conservation.
Pool patient samples
Up to 5 patient samples can be combined into a single pool before RNA extraction. Each RT-PCR reaction plate can contain pools of different numbers of samples (25) as well as individual samples. The volume of each individual patient sample or sample pool used in RNA extraction is 400 µL.
Note: The pooling of samples has only been validated using the automated and manual extraction methods with 400 µL of pooled clinical samples.
Use your laboratory protocols and systems to name and track the samples in pools throughout the workf low.
IMPORTANT! Pools with test results that are positive, invalid, or inconclusive require extraction and testing of the individual patient samples.
1. Combine equal volumes of up to 5 patient samples into a single tube at a total volume of 400 µL. Example volumes are shown in the following table.
Table 3 Example volumes of pooled samples
Sam ple
N = 5
Volum e N = 4
N = 3
1
100 µL
120 µL
150 µL
2
100 µL
120 µL
150 µL
3
100 µL
120 µL
150 µL
4
100 µL
120 µL
--
5
100 µL
--
--
Total volume
500 µL
480 µL
450 µL
2. Store the remaining unpooled volumes of patient samples according to CDC guidelines.
TaqPathTM COVID-19 Pooling Kit Instructions for Use
19
3
Extract RNA (automated method)
Before you begin.............................................................................................................. 20 Extract RNA--Automated method (400L sample input volume) ............................................ 21
Automated RNA extraction is performed using the KingFisherTM Flex Magnetic Particle Processor with 96 Deep-Well Head and the MagMAXTM Viral/Pathogen Nucleic Acid Isolation Kit or MagMAXTM Viral/Pathogen II Nucleic Acid Isolation Kit with an individual or pooled sample input volume of 400 µL.
Before you begin
IMPORTANT! The Binding Bead Mix is not compatible with bleach. For more information, see the SDS.
Note: During the wash steps, the Wash Solution may develop inert white or brown particulates that f loat in solution. This is not a cause for concern and does not negatively affect performance.
· Determine the number of required reactions based on the number of individual patient samples or sample pools to be processed, plus one Negative Control per plate.
· Prepare 1 mL of fresh 80% Ethanol per reaction using Ethanol, Absolute, Molecular Biology Grade and Nuclease-free Water (not DEPC-Treated) for the required number of reactions, plus 10% overage.
· Label the short side of each KingFisherTM 96 Deep-Well Plate (4):
Label
Number of plates
Sample plate
1
Wash 1
1
Wash 2
1
Elution plate
1
· Label the short side of the KingFisherTM 96 KF microplate (1):
Label
Number of plates
Tip comb
1
20
TaqPathTM COVID-19 Pooling Kit Instructions for Use
Chapter 3 Extract RNA (automated method) Extract RNA--Automated method (400L sample input volume)
3
Note: The f ollowing items can be used to hold the tip comb instead of the KingFisherTM 96 KF microplate:
· Tip Comb Presenting Plate for KF 96 · NuncTM MicroWellTM 96Well Microplate, Flat Bottom · NuncTM MicroWellTM 96Well Microplate, barcoded · ABgeneTM 96Well Polypropylene Storage Microplate · ABgeneTM 96Well 1.2mL Polypropylene Deepwell Storage Plate · NuncTM F96 MicroWellTM Black Polystyrene Plate · NuncTM F96 MicroWellTM White Polystyrene Plate · KingFisherTM 96 Deep-Well Plate
· Mark the Negative Control well on the plate.
Extract RNA--Automated method (400L sample input volume)
The f ollowing procedure uses components from the MagMAXTM Viral/Pathogen Nucleic Acid Isolation Kit or the MagMAXTM Viral/Pathogen II Nucleic Acid Isolation Kit.
Set up the instrument (400L sample input volume)
1. Ensure that the KingFisherTM Flex Magnetic Particle Processor with 96 Deep-Well Head is set up with the KingFisherTM Flex 96 Deep-Well Heating Block.
IMPORTANT! Failure to use the proper magnetic head and heat block results in lower yields and potential harm to the instrument.
2. Ensure that the MVP_2Wash_400_Flex program has been downloaded from the MagMAXTM Viral/Pathogen II Nucleic Acid Isolation Kit product page at www.thermofisher.com and loaded onto the instrument.
TaqPathTM COVID-19 Pooling Kit Instructions for Use
21
3
Chapter 3 Extract RNA (automated method) Extract RNA--Automated method (400L sample input volume)
Prepare the processing plates (400L sample input volume)
Prepare the processing plates according to the following table. Cover the plates with a temporary seal (such as MicroAmpTM Clear Adhesive Film), then store at room temperature for up to 1 hour while you setup
the sample plate.
Plate ID
Plate position
Plate type
Reagent
Volume per well
Wash 1 Plate Wash 2 Plate
2
Wash Solution
1,000 µL
3
KingFisherTM 96 Deep-Well Plate
80% Ethanol
1,000 µL
Elution Plate
4
Elution Solution
50 µL
Tip Comb Plate
Place a KingFisherTM 96 tip comb for DW magnets in a KingFisherTM 96
5
KF microplate
Note: The f ollowing items can be used to hold the tip comb instead of the KingFisherTM 96 KF microplate:
· Tip Comb Presenting Plate for KF 96 · NuncTM MicroWellTM 96Well Microplate, Flat Bottom · NuncTM MicroWellTM 96Well Microplate, barcoded · ABgeneTM 96Well Polypropylene Storage Microplate · ABgeneTM 96Well 1.2mL Polypropylene Deepwell Storage Plate · NuncTM F96 MicroWellTM Black Polystyrene Plate · NuncTM F96 MicroWellTM White Polystyrene Plate · KingFisherTM 96 Deep-Well Plate
Prepare Binding Bead Mix (400L sample input volume)
Prepare the required amount of Binding Bead Mix on each day of use. 1. Vortex the Total Nucleic Acid Magnetic Beads to ensure that the bead mixture is homogeneous.
2. For the number of required reactions, prepare the Binding Bead Mix according to the following table:
Com ponent
Volume per well[1]
Binding Solution
530 µL
Total Nucleic Acid Magnetic Beads
20 µL
Total volume per well
550 µL
[1] Include 10% overage when making the Binding Bead Mix for use with multiple reactions.
3. Mix well by inversion, then store at room temperature.
22
TaqPathTM COVID-19 Pooling Kit Instructions for Use
Chapter 3 Extract RNA (automated method) Extract RNA--Automated method (400L sample input volume)
3
Prepare sample plate (400L sample input volume)
This section provides volumes for the sample plate. Your plate layout will depend on the number of samples you run.
1. Add 10 µL of Proteinase K to each well in the KingFisherTM 96 Deep-Well Plate labeled "Sample Plate".
2. Add 400 µL of individual patient sample or sample pool to each sample well.
3. Add 400 µL of Nuclease-free Water (not DEPC-Treated) to the Negative Control well.
4. Invert the Binding Bead Mix 5 times gently to mix, then add 550 µL to each sample well and the Negative Control well in the Sample Plate.
Note: Remix the Binding Bead Mix by inversion frequently during pipetting to ensure even distribution of beads to all samples or wells. The Binding Bead Mix is viscous, so pipet slowly to ensure that the correct amount is added. DO NOT reuse pipette tips to add Binding Bead Mix to the samples, as the high viscosity will cause variations in the volumes added.
5. Add 10 µL of MS2 Phage Control to each sample well and to the Negative Control well.
Process the samples (400L sample input volume)
1. Select the MVP_2Wash_400_Flex on the KingFisherTM Flex Magnetic Particle Processor with 96 Deep-Well Head.
2. Start the run, then load the prepared plates into position when prompted by the instrument.
3. Af ter the run is complete (~24 minutes after start), immediately remove the Elution Plate from the instrument, then cover the plate with MicroAmpTM Clear Adhesive Film. IMPORTANT! To prevent evaporation, seal the plate containing the eluate immediately.
The samples are eluted in 50 µL of Elution Solution (see "Prepare the processing plates (400L sample input volume)" on page 22).
Note:
· Significant bead carry over may adversely impact RT-PCR performance. If bead carry over is
observed, repeat the test by re-extracting a new aliquot of the sample.
· To ensure reliable performance of the KingFisherTM Flex Magnetic Particle Processor, perform
preventive maintenance as instructed by the manufacturer.
Place the Elution Plate on ice for immediate use in real-time RTPCR.
TaqPathTM COVID-19 Pooling Kit Instructions for Use
23
4
Extract RNA (manual method)
Before you begin.............................................................................................................. 24 Extract RNA--Manual method (400L sample input volume) ................................................. 24
Manual RNA extraction can be performed from an individual or pooled sample input volume of 400 µL using either the MagMAXTM Viral/Pathogen Nucleic Acid Isolation Kit or the MagMAXTM Viral/Pathogen II Nucleic Acid Isolation Kit.
Before you begin
IMPORTANT! The Binding Bead Mix is not compatible with bleach. For more information, see the SDS.
Note: During the wash steps, the Wash Solution may develop inert white or brown particulates that f loat in solution. This is not a cause for concern and does not negatively affect performance.
· Determine the number of required reactions based on the number of individual patient samples or sample pools to be processed, plus one Negative Control per plate.
· Prepare 1.5 mL of fresh 80% Ethanol per reaction using Ethanol, Absolute, Molecular Biology Grade and Nuclease-free Water (not DEPC-Treated) for the required number of reactions, plus 10% overage.
· Mark the Negative Control well on the plate.
Extract RNA--Manual method (400L sample input volume)
The f ollowing procedure uses components from the MagMAXTM Viral/Pathogen Nucleic Acid Isolation Kit or the MagMAXTM Viral/Pathogen II Nucleic Acid Isolation Kit.
24
TaqPathTM COVID-19 Pooling Kit Instructions for Use
4 Chapter 4 Extract RNA (manual method)
Extract RNA--Manual method (400L sample input volume)
Prepare Binding Bead Mix (400L sample input volume)
Prepare the required amount of Binding Bead Mix on each day of use. 1. Vortex the Total Nucleic Acid Magnetic Beads to ensure that the bead mixture is homogeneous.
2. For the number of required reactions, prepare the Binding Bead Mix according to the following table:
Com ponent
Volume per well[1]
Binding Solution
530 µL
Total Nucleic Acid Magnetic Beads
20 µL
Total volume per well
550 µL
[1] Include 10% overage when making the Binding Bead Mix for use with multiple reactions.
3. Mix well by inversion, then store at room temperature.
Digest with Proteinase K (400L sample input volume)
This section provides volumes for the sample plate. Your plate layout will depend on the number of samples you run.
1. Add 10 µL of Proteinase K to each well of a KingFisherTM 96 Deep-Well Plate.
2. Add 400 µL of individual patient sample or sample pool to each sample well.
3. Add 400 µL of Nuclease-free Water (not DEPC-Treated) to the Negative Control well.
4. Invert the Binding Bead Mix 5 times gently to mix, then add 550 µL to each sample well and Negative Control well.
Note: Remix the Binding Bead Mix by inversion frequently during pipetting to ensure even distribution of beads to all samples or wells. The Binding Bead Mix is viscous, so pipet slowly to ensure that the correct amount is added. DO NOT reuse pipette tips to add Binding Bead Mix to the samples, as the high viscosity will cause variations in the volumes added.
5. Add 10 µL of MS2 Phage Control to each sample well and to the Negative Control well.
6. Seal the plate with MicroAmpTM Clear Adhesive Film, then shake the sealed plate at 1,050 rpm for 2 minutes.
7. Incubate the sealed plate at 65°C for 5 minutes (ensure the bottom of the plate is uncovered), then shake the plate at 1,050 rpm for 5 minutes.
8. Place the sealed plate on the magnetic stand for 10 minutes or until all of the beads have collected.
TaqPathTM COVID-19 Pooling Kit Instructions for Use
25
4
Chapter 4 Extract RNA (manual method) Extract RNA--Manual method (400L sample input volume)
Wash the beads (400L sample input volume)
1. Keeping the plate on the magnet, carefully remove the cover, then discard the supernatant from each well. IMPORTANT! Avoid disturbing the beads.
2. Remove the plate from the magnetic stand, then add 1 mL of Wash Solution to each sample.
3. Reseal the plate, then shake at 1,050 rpm for 1 minute.
4. Place the plate back on the magnetic stand for 2 minutes, or until all the beads have collected.
5. Keeping the plate on the magnet, carefully remove the cover, then discard the supernatant from each well. IMPORTANT! Avoid disturbing the beads.
6. Repeat step 2 to step 5 using 1 mL of 80% Ethanol.
7. Repeat step 2 to step 5 using 500 µL of 80% Ethanol.
8. Dry the beads by shaking the plate (uncovered) at 1,050 rpm for 2 minutes.
Elute the nucleic acid (400L sample input volume)
1. Add 50 µL of Elution Solution to each sample, then seal the plate with MicroAmpTM Clear Adhesive Film.
2. Shake the sealed plate at 1,050 rpm for 5 minutes.
3. Place the plate in an incubator at 65°C for 10 minutes.
4. Remove the plate from the incubator, then shake the plate at 1,050 rpm for 5 minutes.
5. Place the sealed plate on the magnetic stand for 3 minutes or until clear to collect the beads against the magnets.
6. Keeping the plate on the magnet, carefully remove the seal, transfer the eluates to a fresh standard (not deep-well) 96well plate, then seal the plate with MicroAmpTM Clear Adhesive Film. IMPORTANT! To prevent evaporation, seal the plate containing the eluate immediately after the transf ers are complete.
Note: Significant bead carry over may adversely impact RT-PCR performance. If bead carry over is observed, repeat the test by re-extracting a new aliquot of the sample. Place the plate on ice for immediate use in realtime RTPCR.
26
TaqPathTM COVID-19 Pooling Kit Instructions for Use
5
Prepare RT-PCR reactions
Guidelines for RT-PCR
IMPORTANT!
· For each RT-PCR reaction plate, include the following controls: · One Positive Control · One Negative Control from each extraction run.
For example, if RNA samples from 4 extraction runs are combined on one 384-well RT-PCR reaction plate, then 4 Negative Control wells must be run on that 384-well reaction plate.
· Prepare the RT-PCR reaction plate on ice and keep it on ice until it is loaded into the real-time PCR
instrument.
· Run the plate immediately after preparation. Failure to do so could result in degraded RNA samples. · To prevent contamination, prepare reagents in a PCR workstation or equivalent amplicon-free area.
Do not use the same pipette for controls and RNA samples, and always use aerosol barrier pipet te tips.
· Maintain an RNase-f ree environment. · Protect assays from light. · Keep RNA samples and components on ice during use.
Prepare RTPCR reactions (384well reaction plate)
Use this procedure to prepare RT-PCR reactions for the Applied BiosystemsTM QuantStudioTM 7 Flex Real-Time PCR Instrument (384-well block).
1. If f rozen, thaw the reagents on ice.
2. Gently vortex the reagents, then centrifuge briefly to collect liquid at the bottom of the tube.
3. Dilute TaqPathTM COVID19 Control (1 × 104 copies/µL) to a working stock of 25 copies/µL: a. Pipet 98 µL of TaqPathTM COVID19 Control Dilution Buffer into a microcentrifuge tube, then add 2 µL of TaqPathTM COVID19 Control. Mix well, then centrifuge briefly.
b. Pipet 87.5 µL of TaqPathTM COVID19 Control Dilution Buffer into a second microcentrifuge tube, then add 12.5 µL of the previous dilution. Mix well, then centrifuge briefly.
Note: The TaqPathTM COVID19 Control does not contain the MS2 template.
TaqPathTM COVID-19 Pooling Kit Instructions for Use
27
5
Chapter 5 Prepare RT-PCR reactions Prepare RTPCR reactions (384well reaction plate)
4. Prepare the Reaction Mix.
a. For each run, combine the following components sufficient for the number of RNA samples, plus one Positive Control per 384-well real-time RT-PCR plate, and one Negative Control from each extraction run.
For example, if RNA samples from 4 extraction runs are being combined on one 384-well realtime RT-PCR plate, then 4 Negative Control wells need to be run on that 384-well real-time RT-PCR plate.
All volumes include 10% overage for pipette error.
Com ponent
Volum e per RNA Sam ple or control
Volume for n RNA Sam ples plus y Negative Controls plus 1 Positive
Control
Volume for 379 RNA Sam ples plus 4 Negative Controls plus 1 Positive
Control
TaqPathTM 1Step Multiplex Master Mix (No ROXTM) (4X) TaqPathTM COVID-19 Pooling Multiplex Assay
Total Reaction Mix volume
5.00 µL
1.00 µL 6.0 µL
5.50 x (n + y + 1) µL
1.10 x (n + y + 1) µL --
2112.0 µL
422.4 µL 2534.4 µL
5. Set up the reaction plate: a. Pipet 6.0 µL of the Reaction Mix prepared in step 4 into each well of a MicroAmpTM Optical 384-Well Reaction Plate with Barcode. Plates without a barcode can be used (see "Required materials not supplied" on page 8).
b. Gently vortex the sealed plate containing the purified sample RNA and Negative Control from the RNA extraction procedure, then centrifuge briefly to collect liquid at the bottom of the plate.
c. Unseal the plate containing the purified sample RNA and Negative Control from the RNA extraction procedure. Add either sample RNA, Negative Control, or Positive Control to each well of the reaction plate according to Table 4 on page 29.
IMPORTANT! To prevent sample contamination, unseal one extraction plate at a time, then reseal it af ter adding the samples to the RT-PCR reaction plate.
d. Seal the plate thoroughly with MicroAmpTM Optical Adhesive Film.
IMPORTANT! When applying the MicroAmpTM Optical Adhesive Film, ensure that pressure is applied across the entire plate and that there is a tight seal across every individual well. Failure to do so runs the risk of an improperly sealed well, leading to potential well-to-well contamination during vortexing and evaporation during PCR.
e. Vortex the plate at the highest setting speed for 1030 seconds with medium pressure. Move the plate around to ensure equal contact on the vortex mixer platform.
IMPORTANT! Vortex for 1030 seconds to ensure proper mixing. Failure to do so might result in f alse classification of samples.
28
TaqPathTM COVID-19 Pooling Kit Instructions for Use
5 Chapter 5 Prepare RT-PCR reactions
Prepare RTPCR reactions (384well reaction plate)
f. Centrif uge the reaction plate for 12 minutes at 650 × g (650 RCF) to remove bubbles and to collect the liquid at the bottom of the reaction plate.
Table 4 Reaction plate volumes
Com ponent
Volume per reaction RNA Sample reaction Positive Control reaction
Negative Control reaction
Reaction Mix
Purified sample RNA (from RNA extraction)
6.0 µL 14.0 µL
6.0 µL --
6.0 µL --
Positive Control (diluted
TaqPathTM COVID19 Control
--
2.0 µL
--
from step 3)
Nuclease-free water
--
Purified Negative Control (from RNA extraction)
--
12.0 µL --
-- 14.0 µL
Total volume
20.0 µL
20.0 µL
20.0 µL
TaqPathTM COVID-19 Pooling Kit Instructions for Use
29
6
Perform RT-PCR using the Applied BiosystemsTM QuantStudioTM 7 Flex
Real-Time PCR Instrument (384-
well
block)
Dye calibration for the QuantStudioTM 7 Flex Real-Time PCR Instrument................................... 30 Transf er the template (EDT) f ile for the QuantStudioTM 7 Flex Real-Time PCR Instrument
(384well block)............................................................................................................... 30
Set up and run the QuantStudioTM 7 Flex Real-Time PCR Instrument (384well block)................ 31
Dye calibration for the QuantStudioTM 7 Flex Real-Time PCR Instrument
A maintained instrument will be calibrated for many dyes. In addition to those dyes, the instrument operator must calibrate the instrument for ABYTM dye and JUNTM dye that are used with this kit. For all other assays, refer to the standard calibration process.
Transfer the template (EDT) file for the QuantStudioTM 7 Flex Real-Time PCR Instrument (384well block)
The template (EDT) f ile contains the settings for the instrument run. It is installed on the computer with Applied BiosystemsTM COVID19 Interpretive Software, and must be transferred via a USB drive or other method to the computer on which the QuantStudioTM RealTime PCR Software v1.3 is installed.
IMPORTANT! Be caref ul to select the correct template file. Failure to do so can cause errors in the analysis.
1. On the computer with Applied BiosystemsTM COVID19 Interpretive Software, navigate to the f ollowing directory (where <...> is the installation directory): <...>\Applied Biosystems\COVID-19 Interpretive Software\Client\docs\User Documents\COVID-19
2. Select the EDT f ile: TaqPath COVID-19 Kit Template QS7 384 1_3 v2-2.edt
3. Transf er the EDT file to the computer with QuantStudioTM RealTime PCR Software v1.3, using a USB drive or other method.
30
TaqPathTM COVID-19 Pooling Kit Instructions for Use
6 Chapter 6 Perform RT-PCR using the Applied BiosystemsTM QuantStudioTM 7 Flex Real-Time PCR Instrument (384-well block) Set up and run the QuantStudioTM 7 Flex Real-Time PCR Instrument (384well block)
Set up and run the QuantStudioTM 7 Flex Real-Time PCR Instrument (384well block)
For more information about the instrument, see the documents listed in Appendix D, "Documentation and support".
1. In the QuantStudioTM RealTime PCR Software v1.3 home screen, click Template.
2. Browse to, then open the EDT file that you transferred in "Transfer the template (EDT) file for the QuantStudioTM 7 Flex Real-Time PCR Instrument (384well block)" on page 30.
IMPORTANT! Be caref ul to select the correct template file. Failure to do so can cause errors in the analysis.
3. In the Experiment Properties tab, enter or confirm the following.
· Experiment Name: Enter a unique name · Instrument type: QuantStudioTM 7 Flex System · Block: 384well · Type of Experiment: Standard Curve · Reagents: TaqManTM · Properties: Standard
4. In the Define tab, in the Targets pane, confirm that the targets, reporter dyes, and quenchers are listed correctly.
Target
Reporter dye
Quencher
ORF1ab
FAM
None
N gene
VIC
None
S gene
ABY
None
MS2
JUN
None
5. In the Define tab, in the Samples pane, define a unique sample name for each well in the physical plate.
Note: Wells that do not have a sample name will not be analyzed by the software.
IMPORTANT! Do not use a patient name or associated identifying information in the sample name.
6. In the Define tab, confirm that the Passive Reference is set to None.
7. In the Assign tab, confirm that four targets are assigned to each well in the plate layout. To assign a target to a well, select the well, then check the Assign checkbox.
TaqPathTM COVID-19 Pooling Kit Instructions for Use
31
6
Chapter 6 Perform RT-PCR using the Applied BiosystemsTM QuantStudioTM 7 Flex Real-Time PCR Instrument (384-well block)
Set up and run the QuantStudioTM 7 Flex Real-Time PCR Instrument (384well block)
8. In the Assign tab, in the Samples pane, confirm the labeling of the control wells.
· The template has one positive control (PC) and one negative control (NC) assigned to wells for ref erence.
· Move the control well assignments by copying the existing control wells and pasting them according to their location on the physical plate.
Note: If the 384-well plate includes additional negative controls (from combined extraction runs), def ine each additional negative control and assign it to a well to match the physical plate. Give each negative control a unique name (for example, NC1, NC2, NC3, and NC4).
9. In the Assign tab, confirm the Task assignments.
· For wells with a Positive Control (PC), confirm that the Task is set to S for Standard for all of the targets.
· For wells with a Negative Control (NC), confirm that the Task is set to N for Negative for all of the targets.
· For the wells with a patient sample, confirm that the Task is set to U f or Unknown for all of the targets.
10. In the Assign tab, assign a sample name to each well to match the physical plate. To assign a sample to a well, select the well, then check the Assign checkbox.
11. In the Run Method tab, confirm that the Reaction Volume Per Well is 20 µL, then confirm the thermal protocol.
Step
Tem perature
Tim e
Num ber of cycles
UNG incubation
25°C
2 minutes
1
Reverse transcription
53°C
10 minutes
1
Activation
95°C
2 minutes
1
Denaturation
95°C
3 seconds
40
Anneal / extension
60°C
30 seconds
12. Load the prepared and sealed RT-PCR plate into the real-time PCR instrument. 13. In the Run tab, click Start Run, then select your instrument from the drop-down list. 14. Enter a f ile name in the dialog box that prompts you to save the run file, then save the file.
32
TaqPathTM COVID-19 Pooling Kit Instructions for Use
7
Analysis and results
Obtain the Applied BiosystemsTM COVID19 Interpretive Software
To perf orm data analysis and results interpretation, you must use the Applied BiosystemsTM COVID19 Interpretive Software. To obtain the software, contact your local instrument service team. Go to https:// www.thermofisher.com/contactus.
Analyze the data
For detailed instructions about using the software, click the Help menu in the COVID19 Interpretive Sof tware.
1. Using a USB drive or other method, transfer the SDS or EDS files from the computer with the data collection software to the computer with the COVID19 Interpretive Software.
2. Sign in to the COVID19 Interpretive Software, then click COVID-19 Test.
Note: The TaqPathTM COVID-19 Pooling Kit is not compatible with samples collected using the EverlywellTM COVID-19 Test Home Collection Kit, and therefore does not use the COVID19 + RNase P Test option in the software.
3. In the Home screen, click the Import Samples button.
4. Select the SDS files or the EDS files to import, then click Open. Af ter import, the software analyzes the run data, performs Quality Check (QC) analysis, and calculates the interpretive results for each sample and control.
5. In the Home screen, under Batches, click the <Batch ID> link f or the batch of interest to display the Batch Details screen. · In the Batch Details screen, view the status and result for each sample in the Sample Information tab. · Optional: To identify signs of inadequate vortexing or centrifugation, select the Amplification Plot tab to view the amplification curve for each sample. · Optional: To enter a comment for the sample, select the Amplification Plot tab, select the Comment tab, enter a comment, then click Add. The comment will appear in all exports (CSV files) and reports (PDF files). It will include the user name and date.
TaqPathTM COVID-19 Pooling Kit Instructions for Use
33
7
Chapter 7 Analysis and results Analyze the data
6. To generate a Batch Export (CSV file), return to the Home screen, select the checkbox for the batch, then click Export Batch at the top of the Home screen. Click Open folder location in the dialog box to locate and open the CSV file.
7. To generate a Batch Report (PDF file), select the checkbox for the batch, then click Report Batch at the top of the Home screen. Click Open folder location in the dialog box to locate and open the PDF f ile.
34
TaqPathTM COVID-19 Pooling Kit Instructions for Use
7 Chapter 7 Analysis and results Interpretation of the results
Interpretation of the results
Interpretation of the results is performed by the COVID19 Interpretive Software. For information about the Ct values that are used by the software to interpret results, see Appendix A, "Ct cutoff values for
assay targets".
Quality control and validity of results
A minimum of one Negative Control and one Positive Control must be present for each run.
If multiple extraction plates are combined on an RT-PCR plate, one Negative Control well must be included for each extraction plate. All control wells must pass for the RT-PCR plate to be considered valid.
Validation of results is performed automatically by the COVID19 Interpretive Software based on perf ormance of the Positive and Negative Controls. If the controls do not pass, the run is considered invalid and must be repeated.
Table 5 Result interpretation for pooled patient samples and individual samples if applicable
ORF1ab N gene S gene MS2 Status
Result
Action
NEG NEG NEG NEG INVALID
Pooled samples
Individual sample
NA
Deconvolute by
Repeat the test
extracting and testing by re-extracting the
the individual samples original sample and
in each pool.[1]
repeating the RT-
(Proceed to the
PCR. If the repeat
"Individual sample" result remains invalid,
Action column.)
consider collecting a
new specimen.
TaqPathTM COVID-19 Pooling Kit Instructions for Use
35
7
Chapter 7 Analysis and results Interpretation of the results
Table 5 Result interpretation for pooled patient samples and individual samples if applicable (continued)
ORF1ab N gene S gene MS2
Status
Result
Action
NEG NEG NEG POS
VALID
Pooled samples
Individual sample
SARS-CoV-2 Not Detected
Report the results to the healthcare provider and appropriate public health authorities. Consider testing for other viruses.
N/A[2]
Testing of individual specimens that make up the negative pool is not necessary.
Negative results are not definitive and, if inconsistent with clinical signs and sym ptom s or necessary for patient management, pooled sam ples should be
tested individually.
Negative results do not preclude SARSCoV-2 infection and must not be used as the sole basis for patient management decisions. Negative results must be considered in the context of a patient's recent exposures, history, presence of clinical signs, and
sym ptom s consistent with COVID-19.
36
TaqPathTM COVID-19 Pooling Kit Instructions for Use
7 Chapter 7 Analysis and results Interpretation of the results
Table 5 Result interpretation for pooled patient samples and individual samples if applicable (continued)
ORF1ab N gene S gene MS2 Status
Result
Action
Pooled samples
Individual sample
Only one SARSCoV2 target = POS
POS or VALID NEG
SARS-CoV-2 Inconclusive
Deconvolute by extracting and testing the individual samples in each pool.[1] (Proceed to the "Individual sample" Action column.)
1. Repeat the test by re-extracting the original sample and repeating the RTPCR.
2. After retesting one time, report the results to the healthcare provider and appropriate public health authorities.
If the repeat result rem ains inconclusive, the healthcare provider should conduct additional confirmation testing with a new specimen, if clinically indicated.
Two or more SARS-CoV-2 POS or VALID
targets = POS
NEG
Positive SARS-CoV-2
Deconvolute by
Repeat the test by re-
extracting and testing extracting the original
the individual samples samples that make
in each pool.[1]
up the pool and
(Proceed to the
repeating the RT-
"Individual sample" PCR. If the repeat
Action column.)
result is invalid for
any individual
specimen, consider
collecting a new
specim en.
[1] Individual samples must only be tested using the workflow validated for the TaqPathTM COVID-19 Pooling Kit (400 µL of clinical sample for extraction, 50 µL eluate volume, 14 µL of purified sample RNA, 20µL final PCR volume, run on the QuantStudioTM 7 Flex Real-Time PCR Instrument, 384well block)
[2] N/A: Testing scenario is not applicable.
TaqPathTM COVID-19 Pooling Kit Instructions for Use
37
8
Conditions of authorization for labs
The TaqPathTM COVID-19 Pooling Kit 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-19emergency-use-authorizations-medical-devices/vitro-diagnostics-euas.
However, to assist clinical laboratories using the TaqPathTM COVID-19 Pooling Kit, the relevant Conditions of Authorization are listed below.
· Authorized laboratories[4] using the TaqPathTM COVID-19 Pooling Kit must include with 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.
· Authorized laboratories using specimen pooling strategies when testing patient specimens with the TaqPathTM COVID-19 Pooling Kit must include with negative test result reports for specific patients whose specimen(s) were the subject of pooling, a notice that pooling was used during testing and that "Patient specimens with low viral loads may not be detected in sample pools due to the decreased sensitivity of pooled testing."
· Authorized laboratories must keep records of specimen pooling strategies implemented, including type of strategy, date implemented, quantities tested, and test result data generated as part of "Monitor the pooling strategy" on page 18. For the first 12 months from the date of their creation, such records must be made available to FDA within 48 business hours for inspection upon request. Af ter 12 months from the date of their creation, upon FDA's request, such records must be made available for inspection within a reasonable time.
· Authorized laboratories using the TaqPathTM COVID-19 Pooling Kit must perform the procedures as outlined in the TaqPathTM COVID-19 Pooling Kit Instructions for Use. 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 TaqPathTM COVID-19 Pooling Kit are not permitted.
· Authorized laboratories implementing pooling strategies for testing patient specimens must use the inf ormation provided in Chapter 2, "Pool samples" in the TaqPathTM COVID-19 Pooling Kit Instructions for Use to evaluate the appropriateness of continuing to use such strategies based on the recommendations in the protocol.
· Authorized laboratories that receive the TaqPathTM COVID-19 Pooling Kit must notify the relevant public health authorities of their intent to run the test prior to initiating testing.
· Authorized laboratories using the TaqPathTM COVID-19 Pooling Kit must have a process in place for reporting test results to healthcare providers and relevant public health authorities, as appropriate.
[4] For 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 l abo rato ri es ."
38
TaqPathTM COVID-19 Pooling Kit Instructions for Use
8 Chapter 8 Conditions of authorization for labs Interpretation of the results
· Authorized laboratories must 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 Thermo Fisher Scientific (techservices@thermofisher.com; 1 800 955 6288) any suspected occurrence of f alse 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 TaqPathTM COVID-19 Pooling Kit must be appropriately trained in RT-PCR techniques and use appropriate laboratory and personal protective equipment when handling this kit, and use the test in accordance with the authorized labeling.
· Thermo Fisher Scientific, its authorized distributor(s), and authorized laboratories using the TaqPathTM COVID-19 Pooling Kit must ensure that any records associated with this EUA are maintained until otherwise notified by FDA. Such records will be made available to FDA f or inspection upon request.
TaqPathTM COVID-19 Pooling Kit Instructions for Use
39
9
Performance characteristics
Analytical performance of the TaqPathTM COVID-19 Pooling Kit was determined by leveraging limit of detection (LoD), interfering substances, and cross-reactivity data for individual samples using the TaqPathTM COVID-19 Combo Kit and TaqPathTM COVID-19 Combo Kit Advanced, as described in the
f ollowing sections.
Limit of detection (LoD) for RT-PCR with 14.0 µL of purified sample RNA
The study results in this section were generated using the TaqPathTM COVID-19 Combo Kit Advanced workf low described in the TaqPathTM COVID-19 Combo Kit and TaqPathTM COVID-19 Combo Kit Advanced Instructions for Use. Note that the LoD results in Table 6 are f or the unpooled TaqPathTM COVID-19 Combo Kit Advanced workflow. The TaqPathTM COVID-19 Pooling Kit utilizes the same RNA sample input and PCR workflow.
The LoD study with 14.0 µL of purified sample RNA established the lowest SARS-CoV-2 viral concentration (Genomic Copy Equivalents or GCE) that can be detected by the TaqPathTM COVID-19 Combo Kit Advanced at least 95% of the time. Negative nasopharyngeal swab (NP) matrix was spiked with gamma-irradiated SARS-CoV-2 virus at several concentrations and processed through the TaqPathTM COVID-19 Combo Kit Advanced workflow. A three-phase approach was used to determine the LoD for each specimen type. In phases I and II, the preliminary LoD was established and confirmed in phase III by testing 20 replicates.
Table 6 QuantStudioTM 7 Flex Real-Time PCR Instrument (384-well), 14.0 µL of purified sample RNA
Concentration Replicate
1
2
3
50 GCE/mL
4
(5.6 GCE/
5
reaction)
6
7
8
9
ORF1ab 33.064 34.727 33.364 32.872 33.359 33.379 33.738 33.430 34.121
N gene 33.380 33.488 33.584 33.518 34.923 Not detected 33.190 34.375 35.457
S gene 34.410 33.894 33.596 34.151 33.267 35.909 36.431 33.968 34.250
MS2 Interpretation
22.847
Positive
22.465
Positive
22.569
Positive
22.291
Positive
22.099
Positive
22.514
Positive
21.908
Positive
21.933
Positive
21.829
Positive
% Positive 100%
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TaqPathTM COVID-19 Pooling Kit Instructions for Use
9 Chapter 9 Performance characteristics Reactivity (Inclusivity)
Table 6 QuantStudio 7 Flex Real-Time PCR Instrument (384-well), 14.0 µL of purified sample RNA (continued)
Concentration Replicate ORF1ab
N gene
S gene MS2 Interpretation
10
32.608
34.032
34.510 21.825
Positive
11
32.958
Not detected 33.301 22.152
Positive
12
33.760
33.723
33.910 22.473
Positive
13
33.657
33.837
35.406 22.137
Positive
50 GCE/mL
(5.6 GCE/ reaction)
14
Not detected
34.200
15
33.560
35.388
16
34.338
34.091
33.961 33.713 34.007
21.745 22.517 21.838
Positive Positive Positive
17
33.350
34.315
34.410 21.657
Positive
18
34.025
33.251
34.292 21.584
Positive
19
33.277
34.955
34.468 21.541
Positive
20
33.562
33.137
34.488 21.551
Positive
% Positive 100%
Reactivity (Inclusivity)
In silico analysis was updated using 1,116,456 sequenced genomes in the GISAID database as of 10 May 2021, which include instances of emerging SARS-CoV-2 variants that are under global surveillance. Mismatch melting temperature analysis was performed, in which a positive is called when at least two of the three target assays (ORF1ab, S gene, and N gene) show a melting temperature higher than the annealing temperature. Based on the melting temperature analysis, the TaqPathTM COVID-19 Pooling Kit correctly identified 1,098,871 (98.4%) of known SARS-CoV-2 strains/isolates in GISAID. Evaluation of assay components that did not match 100% to target sequences indicated that most of the primer or probe mismatches are unlikely to impact assay function, thus the COVID-19 testis expected to be highly inclusive for SARS-CoV-2 strain detection.
Interfering substances
The study results in this section were generated using the TaqPathTM COVID-19 Combo Kit workflow described in the TaqPathTM COVID-19 Combo Kit and TaqPathTM COVID-19 Combo Kit Advanced Instructions for Use. The TaqPathTM COVID-19 Pooling Kit also utilizes the ORF1ab, S gene, and N gene, theref ore the results of the study are applicable.
Pooled SARS-CoV-2-negative nasopharyngeal swab and bronchoalveolar lavage specimens were spiked with purified SARS-CoV-2 viral RNA at 3X the limit of detection (30 GCE/reaction) and potential interf ering substances at the concentrations listed in Table 7 on page 43. Each substance was tested with triplicate extractions. The results are presented in Table 7 on page 43.
TaqPathTM COVID-19 Pooling Kit Instructions for Use
41
9 Chapter 9 Performance characteristics Interfering substances
Pooled SARS-CoV-2-negative nasopharyngeal swab and bronchoalveolar lavage specimens were spiked with potential interfering substances at the concentrations above. Each substance was tested with triplicate extractions. No false positive results were observed for any of the substances at the concentrations tested.
42
TaqPathTM COVID-19 Pooling Kit Instructions for Use
43
TaqPathTM COVID-19 Pooling Kit Instructions for Use
Table 7 Interf ering substances
Interfering substance
None Mucin: bovine submaxillary gland, type I-S Blood (human) Nasal sprays or drops-- NasacortTM
Final concentration in sample
N/A 0.1 mg/mL
1% v/v 10% v/v
Positive BAL sam ples 100%[1] 100%[2]
100%[3] 100%[4]
Agreement with expected results
Positive NP sam ples
Negative BAL sam ples
100%
100%
100%
100%
100% 100%[4]
100% 100%
Nasal corticosteroids--DymistaTM
5 µg/mL
NeilMedTM NasogelTM
1% w/v
Influenza A H1N1 Brisbane/59/07 1 × 105 TCID50/m L
Throat lozenges, oral anesthetic and analgesic--ChlorasepticTM
1% w/v
100%[2] 100%[2] 100%[2] 100%[3]
100% 100% 100% 100%
100% 100% 100% 100%
Oseltamivir phosphate
Antibiotic, nasal ointment-- BactrobanTM
Antibacterial, systemic-- Tobram ycin
33 µg/mL 5 µg/mL
0.6 mg/mL
100%[2] 100%[2]
100%[2]
100% 100%
100%
100% 100%
100%
Hom eopathic allergy relief m edicine--SimilasanTM Nasal
10% v/v
100%
100%
100%
[1] Two of six replicates produced a Ct >37 or Undetermined for S Gene, but all replicates were called Positive based on the interpretation algorithm. [2] Two of three replicates produced a Ct >37 or Undetermined for S Gene, but all replicates were called Positive based on the interpretation algorithm. [3] All three replicates produced a Ct >37 or Undetermined for S Gene but were called Positive based on the interpretation algorithm. [4] One of three replicates produced a Ct >37 or Undetermined for S Gene, but all replicates were called Positive based on the interpretation algorithm
Negative NP sam ples 100% 100%
100% 100%
100% 100% 100% 100%
100% 100%
100%
100%
Chapter 9 Performance characteristics
9 Interfering substances
9 Chapter 9 Performance characteristics Cross-reactivity
Cross-reactivity
In silico analysis of the following forty-three (43) organisms was performed.
Table 8 Organisms used for in silico cross-reactivity analysis
Human coronavirus 229E
Rhinovirus/Enterovirus
Human coronavirus OC43
Parechovirus
Human coronavirus HKU1
Candida albicans
Human coronavirus NL63
Corynebacterium diphtheriae
SARS-coronavirus
Legionella (non-pneumophila)
MERS-coronavirus
Bacillus anthracis (Anthrax)
Adenovirus
Moraxella catarrhalis
Hum an Metapneumovirus (hMPV)
Neisseria elongata and Neisseria meningitidis
Parainfluenza 1
Pseudomonas aeruginosa
Parainfluenza 2
Staphylococcus epidermidis
Parainfluenza 3
Streptococcus salivarius
Parainfluenza 4
Leptospira sp.
Influenza A
Chlamydophila pneumoniae
Influenza B
Chlamydophila psittaci
Influenza C
Coxiella burnetii (Q-Fever)
Enterovirus
Staphylococcus aureus
Respiratory Syncytial Virus A
Haemophilus influenzae
Respiratory Syncytial Virus B
Legionella pneumophila
Bordetella pertussis
Mycobacterium tuberculosis
Mycoplasma pneumoniae
Streptococcus pneumoniae
Pneumocystis jirovecii (PJP)
Streptococcus pyogenes
Among the tested organisms, Neisseria elongata showed homology for the forward and reverse primers and probe for the N gene. The f orward primer showed 80% homology while the reverse primer and probe showed 36% homology. The N gene reverse primer and probe show low homology, therefore the likelihood of non-specific amplification is low.
Blast analysis showed 80% homology for one assay component (forward primer, reverse primer, or probe) for select isolates. Despite 80% homology of one assay component for select isolates, t here is no anticipated amplification because hybridization of all three assay components are necessary t o generate a signal. We also found multiple instances where different assay components had 80%
44
TaqPathTM COVID-19 Pooling Kit Instructions for Use
9 Chapter 9 Performance characteristics Clinical evaluation
homology to different isolates of the same species. For example, Bacillus anthracis strain AFS029987 had 80% homology to the ORF1ab forward primer while strain MCCC 1A01412 had 80% homology to the ORF1ab reverse primer. Since these are two different organisms, amplification is not likely to occur. The in silico analysis indicates that significant amplification of non-target sequences that result incrossreactivity or potentially interfere with detection of SARS-CoV-2 is not likely to occur.
Clinical evaluation
A study evaluated the performance of the TaqPathTM COVID-19 Pooling Kit by testing pools of 3 samples and 5 samples containing a mix of positive and negative nasopharyngeal swab specimens, and comparing the results to tests of the individual positive specimens in those pools.
The f ollowing samples and sample pools were used: · 42 individual SARS-CoV-2-positive clinical specimens
· 42 3-sample pools, formulated using material from two negative specimens and one positive specimen
· 42 5-sample pools, formulated using material from four negative specimens and one positive specimen
The tests were performed using the TaqPathTM COVID-19 Pooling Kit on the QuantStudioTM 7 Flex Real-Time PCR Instrument (384-well block). The positive percent agreement (PPA) was calculated by comparing the results for each sample pool to the corresponding results for the individual positive specimens used to formulate each pool. The negative percent agreement (NPA) was calculated by comparing the results for each sample pool to the corresponding results for the individual negative specimens used to formulate each pool.
The concordance between all positive pool results and individual positive specimen results was 100%.
Table 9 Clinical evaluation study
Pool type
Number of pools
Mean Ct (individual)
Mean Ct (pooled)
PPA
NPA
3-sam ple positive pool
ORF1ab = 19.9 ORF1ab = 21.8
100% (95% CI
42
N gene = 20.7 N gene = 22.3
[91.6%
N/A[2]
100%])[1]
S gene = 20.6 S gene = 22.3
5-sam ple positive pool
5-sam ple negative pool[4]
41[3] 29
ORF1ab = 19.9 ORF1ab = 22.3
100% (95% CI N gene = 20.7 N gene = 22.9 [91.4%100%])
N/A
S gene = 20.6 S gene = 23.0
ORF1ab = ND[5] ORF1ab = ND
N/A
100% (95% CI [88.8%100%])
TaqPathTM COVID-19 Pooling Kit Instructions for Use
45
9 Chapter 9 Performance characteristics Clinical evaluation
Table 9 Clinical evaluation study (continued)
Pool type
Number of pools
Mean Ct (individual)
Mean Ct (pooled)
PPA
NPA
5-sam ple negative pool[4]
29
N gene = ND S gene = ND
N gene = ND S gene = ND
N/A
100% (95% CI
[88.8%100%])
[1] 95% CI = Two-sided 95% score confidence interval. [2] N/A: Not applicable. [3] One pool was called invalid on the QuantStudioTM 7 Flex Real-Time PCR Instrument and removed from performance calculations. [4] Only 5-sample negative pools were evaluated (worst case), since validation for 5sample pooling is considered validated for 2-, 3-, or
4sample pools. [5] ND: Not Detected (Negative).
A linear relationship was observed between individual and pooled specimens with an expected Ct shift f or each target. By using a Passing-Bablok linear regression model, the slope and y-intercept were calculated for each target (Table 10 on page 46).
Table 10 Ct shif ts for 3- and 5-sample pools
Pool type
Target
Slope
Y-intercept
ORF1ab
1.01
1.54
3-sample pool
N gene
0.99
1.73
S gene
1.02
1.37
ORF1ab
1.02
2.09
5-sample pool
N gene
1.02
2.11
S gene
1.06
1.54
In silico analysis was conducted to evaluate the effect of 3-sample and 5-sample pooling on the clinical sensitivity of the TaqPathTM COVID-19 Pooling Kit using historical data consisting of 251 individually tested consecutive positive nasopharyngeal and anterior nasal swab specimens collected from four geographic locations. This analysis used the Passing-Bablok regression coefficients (slope and intercept) generated from a pooling validation study to estimate the Ct shift for each gene target for individual samples due to N-sample pooling.
For each gene target, the Ct values at the sample-level limit of detection (95% detection) and at the calling threshold (50% detection) were used to estimate detection in dilution-adjusted samples by generating a piecewise linear approximation to a sigmoid curve that estimates the percent detection for f our different Ct Zones, defined as follows:
· Zone 1: Ct 40 to Calling Threshold minus Shift
· Zone 2: Calling Threshold minus Shift to LoD
· Zone 3: LoD to LoD minus Shift
· Zone 4: LoD minus Shift to Ct 0
The estimated percent detection for each Ct Zone due to pooling was then applied to individual sample testing data collected across four sites (384-well formats), where the observed positivity rate was between 9% and 25%.
46
TaqPathTM COVID-19 Pooling Kit Instructions for Use
9 Chapter 9 Performance characteristics Clinical evaluation
Table 11 In silico analysis results using samples collected from various testing sites (United States)
Site
N samples
Total num ber of Positive samples
PPA for Positive N=3[1]
PPA for Positive N=5[1]
Site 1
368
76
99.9%
99.3%
Site 2
368
93
100.0%
99.5%
Site 3
370
48
100.0%
100.0%
Site 4
376
34
99.7%
99.0%
Total
1,482
251
99.9%
99.4%
[1] PPA calculations exclude inconclusive samples. Inconclusive samples should be retested according to Table 5 on page 35.
TaqPathTM COVID-19 Pooling Kit Instructions for Use
47
A
Ct cutoff values for assay targets
The Applied BiosystemsTM COVID19 Interpretive Software uses the following Ct cutoff values for assay targets during interpretation of the results.
Table 12 Ct cutoff values
Sample or Control
Target
Ct cutoff
Positive Control
MS2 Viral targets
Valid Ct values are >37[1] Valid Ct values are 37
Negative Control
MS2 Viral targets
Valid Ct values are 32 Valid Ct values are >37[2]
Clinical samples
MS2 Viral targets
Valid Ct values are 32[3] Positive Ct values are 37
[1] The software will report Negative for the MS2 target. [2] The software will report Negative for each viral target, and a result of "SARS-CoV-2 Not Detected".
[3] If any of the viral targets is positive, the Ct for MS2 can be >32 (reported as Negative in the software)
48
TaqPathTM COVID-19 Pooling Kit Instructions for Use
B
EUO label for RUO instrument
For the Applied BiosystemsTM QuantStudioTM 7 Flex Real-Time PCR Instrument, affix the Emergency Use Only (EUO) label on each instrument and retain this labeling throughout the Emergency Use Authorization (EUA) use of the QuantStudioTM 7 Flex Real-Time PCR Instrument.
1. Download or print the following EUO label:
Emergency Use Only
This instrument is authorized for use with Thermo Fisher Scientific assays that have
received Emergency Use Authorization
2. Visibly affix the EUO instrument verification label on your instrument. If the instrument includes labeling indicating "For Research Use Only", cover with the EUO instrument verification label.
TaqPathTM COVID-19 Pooling Kit Instructions for Use
49
C
Safety
WARNING! GENERAL SAFETY. Using this product in a manner not specified in the user documentation may result in personal injury or damage to the instrument or device. Ensure that anyone using this product has received instructions in general safety practices for laboratories and the saf ety information provided in this document.
· Bef ore using an instrument or device, read and understand the safety information provided in the
user documentation provided by the manufacturer of the instrument or device.
· Bef ore handling chemicals, read and understand all applicable Safety Data Sheets (SDSs) and use
appropriate personal protective equipment (gloves, gowns, eye protection, and so on). To obtain SDSs, see the "Documentation and Support" section in this document.
Chemical safety
WARNING! GENERAL CHEMICAL HANDLING. To minimize hazards, ensure laboratory personnel read and practice the general safety guidelines for chemical usage, storage, and waste provided below. Consult the relevant SDS for specific precautions and instructions:
· Read and understand the Safety Data Sheets (SDSs) provided by the chemical manufacturer
bef ore you store, handle, or work with any chemicals or hazardous materials. To obtain SDSs, see the "Documentation and Support" section in this document.
· Minimize contact with chemicals. Wear appropriate personal protective equipment when handling
chemicals (for example, safety glasses, gloves, or protective clothing).
· Minimize the inhalation of chemicals. Do not leave chemical containers open. Use only with
suf ficient ventilation (for example, fume hood).
· Check regularly for chemical leaks or spills. If a leak or spill occurs, follow the manufacturer
cleanup procedures as recommended in the SDS.
· Handle chemical wastes in a fume hood. · Ensure use of primary and secondary waste containers. (A primary waste container holds the
immediate waste. A secondary container contains spills or leaks from the primary container. Both containers must be compatible with the waste material and meet federal, state, and local requirements for container storage.)
· Af ter emptying a waste container, seal it with the cap provided. · Characterize (by analysis if needed) the waste generated by the particular applications, reagents,
and substrates used in your laboratory.
· Ensure that the waste is stored, transferred, transported, and disposed of according to all local,
state/provincial, and/or national regulations.
· IMPORTANT! Radioactive or biohazardous materials may require special handling, and disposal
limitations may apply.
50
TaqPathTM COVID-19 Pooling Kit Instructions for Use
C Appendix C Safety
Biological hazard safety
Biological hazard safety
WARNING! Potential Biohazard. Depending on the samples used on this instrument, the surface may be considered a biohazard. Use appropriate decontamination methods when working with biohazards.
WARNING! BIOHAZARD. Biological samples such as tissues, body fluids, infectious agents,and blood of humans and other animals have the potential to transmit infectious diseases. Conduct all work in properly equipped facilities with the appropriate safety equipment (for example, physical containment devices). Safety equipment can also include items for personal protection, such as gloves, coats, gowns, shoe covers, boots, respirators, face shields, safety glasses, or goggles. Individuals should be trained according to applicable regulatory and company/ institution requirements before working with potentially biohazardous materials. Follow all applicable local, state/provincial, and/or national regulations. The f ollowing references provide general guidelines when handling biological samples in laboratory environment.
· U.S. Department of Health and Human Services, Biosafety in Microbiological and Biomedical
Laboratories (BMBL), 6th Edition, HHS Publication No. (CDC) 300859, Revised June 2020; found at: https://www.cdc.gov/labs/pdf/CDC-BiosafetyMicrobiologicalBiomedicalLaboratories-2020P.pdf
· Laboratory biosafety manual, fourth edition. Geneva: World Health Organization; 2020 (Laboratory
biosafety manual, fourth edition and associated monographs); found at: www.who.int/publications/i/item/9789240011311
TaqPathTM COVID-19 Pooling Kit Instructions for Use
51
D
Documentation and support
Related documentation
Docum ent
QuantStudioTM 6 and 7 Flex Real-Time PCR Systems Maintenance and Administration Guide QuantStudioTM 6 and 7 Flex Real-Time PCR Systems Quick Reference QuantStudioTM RealTime PCR Software Getting Started Guide MagMAXTM Viral/Pathogen II Nucleic Acid Isolation Kit Instructions For Use MagMAXTM Viral/Pathogen Nucleic Acid Isolation Kit (automated extraction) User Guide MagMAXTM Viral/Pathogen Nucleic Acid Isolation Kit (manual extraction) User Guide Thermo ScientificTM KingFisherTM Flex User Manual COVID19 Interpretive Software Installation Quick Reference
Publication Number
4489821
4489826 4489822 MAN0019746 MAN0018073 MAN0018072 MAN0019870 MAN0019257
Customer and technical support
For additional documentation and information about this kit, visit: https://www.thermofisher.com/covid19eua
For download instructions for the COVID19 Interpretive Software, see "Obtain the Applied BiosystemsTM COVID19 Interpretive Software" on page 33.
Ref er to the Software Release Notes provided with the COVID19 Interpretive Software before contacting support for the software.
Visit: https://www.thermofisher.com/contactus for service and support information for this kit, including the following:
· Worldwide contact telephone numbers · Product support information · Order and web support
52
TaqPathTM COVID-19 Pooling Kit Instructions for Use
D Appendix D Documentation and support Limited product warranty
· Product documentation such as: Certif icates of Analysis Saf ety Data Sheets (SDSs; also known as MSDSs)
Note: For SDSs for reagents and chemicals from other manufacturers, contact the manuf acturer.
Limited product warranty
Lif e Technologies Corporation and/or its affiliate(s) warrant their products as set forth in the Lif e Technologies' General Terms and Conditions of Sale at www.thermofisher.com/us/en/hom e/ global/terms-and-conditions.html. If you have any questions, please contact Life Technologies at www.thermofisher.com/support.
TaqPathTM COVID-19 Pooling Kit Instructions for Use
53
thermofisher.com/support| thermofisher.com/askaquestion thermofisher.com
25 May 2021
References
Adobe PDF Library 17.11.238
Lab Equipment and Lab Supplies | Fisher Scientific
Thermo Fisher Scientific - AU
Thermo Fisher Scientific - US
Laboratory biosafety manual, 4th edition
Pooled Testing: Guidance from the… | College of American Pathologists Group CAP_logo_rgb[1] User Down Arrow Shopping Cart CAP Logo Down Arrow Down Arrow Down Arrow Down Arrow Down Arrow Down Arrow CAP Logo User Login Down Arrow Down Arrow Down Arrow Down A
Centers for Disease Control and Prevention