Thermo Fisher Scientific TaqMan User Manual
TaqMan™ SARS-CoV-2 Pooling Assay
USER GUIDE
Publication Number MAN0019859
Revision A.0
For Research Use Only. Not for use in diagnostic procedures.
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 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 BE
LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR
ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.
Revision history: Pub. No. MAN0019859
Revision Date Description
A.0 29 January 2021 New document.
Important Licensing Information: This product may be covered by one or more Limited Use Label Licenses. By use of this product,
you accept the terms and conditions of all applicable Limited Use Label Licenses.
Trademarks: All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. TaqMan is a
registered trademark of Roche Molecular Systems, Inc., used under permission and license.
©2021 Thermo Fisher Scientific Inc. All rights reserved.
Contents
■
CHAPTER 1 Product information .................................................. 5
Product description ............................................................. 5
Contents and storage ............................................................ 6
Required materials not supplied ................................................... 7
General laboratory recommendations .............................................. 9
Workflow ..................................................................... 10
■
CHAPTER 2 Pool samples ........................................................ 11
Overview of pooling ............................................................ 11
Pool samples .................................................................. 11
■
CHAPTER 3 Extract RNA ......................................................... 13
Before you begin ............................................................... 13
Set up the instrument ........................................................... 14
Prepare the processing plates ................................................... 15
Prepare Binding Bead Mix ...................................................... 15
Prepare sample plate .......................................................... 16
Process the samples ........................................................... 16
■
CHAPTER 4 Prepare RT-PCR reactions ......................................... 17
Guidelines for RT-PCR .......................................................... 17
Prepare the RT‑PCR reactions (96-well reaction plate) .............................. 17
Prepare RT‑PCR reactions (384‑well reaction plate) ................................. 19
■
CHAPTER 5 Set up and run the real-time PCR instrument ................... 22
Calibration .................................................................... 22
Dye calibration for the QuantStudio™ 5 Real‑Time PCR Instrument ............... 22
Dye calibration for the QuantStudio™ 7 Flex Real-Time PCR Instrument ........... 22
Perform RT-PCR ............................................................... 22
TaqMan
™
SARS-CoV-2 Pooling Assay User Guide
3
Contents
■
■
■
CHAPTER 6 Analyze data ......................................................... 24
Analyze data .................................................................. 24
APPENDIX A Documentation and support ...................................... 25
Related documentation ......................................................... 25
Customer and technical support ................................................. 25
Limited product warranty ........................................................ 26
APPENDIX B Safety ............................................................... 27
Biological hazard safety ......................................................... 27
Chemical safety ................................................................ 28
4
TaqMan™ SARS-CoV-2 Pooling Assay User Guide
1
Product description .................................................................... 5
■
Contents and storage .................................................................. 6
■
Required materials not supplied ......................................................... 7
■
General laboratory recommendations .................................................... 9
■
Workflow ............................................................................ 10
■
IMPORTANT! Before using this product, read and understand the information in the “Safety” appendix
in this document.
Product description
The TaqMan™ SARS-CoV-2 Pooling Assay contains the assays and the controls for a real-time reverse
transcription polymerase chain reaction (RT-PCR) to detect nucleic acid from SARS-CoV-2 in pooled
upper respiratory samples.
Product information
At least one sample in a pool contains SARS-CoV-2 nucleic acid if the SARS-CoV-2 nucleic acid is
detected in the pooled samples. In order to determine which sample contains SARS-CoV-2 nucleic
acid, RT-PCR should be performed on each sample in the pool individually.
If SARS-CoV-2 nucleic acid is not detected in the pooled samples, this indicates that none of the
samples contain SARS-CoV-2 nucleic acid.
The TaqMan™ SARS-CoV-2 Pooling Assay is performed with the following components:
•
TaqMan™ SARS-CoV-2 Pooling Assay Kit
–
TaqMan™ SARS-CoV-2 Pooling Multiplex Assay—Multiplexed assays that contain three primer
and probe sets specific to dierent SARS-CoV-2 genomic regions and primers and probes for
bacteriophage MS2 (see Table 1 on page 6)
–
TaqMan™ MS2 Phage Control—Internal process control for nucleic acid extraction
•
TaqMan™ SARS-CoV-2 Control—RNA control that contains targets specific to the SARS-CoV-2
genomic regions targeted by the assays
•
TaqMan™ Control Dilution Buer—Dilution buer for the control
•
TaqPath™ 1‑Step Multiplex Master Mix
For catalog numbers and storage conditions, see “Contents and storage” on page 6.
TaqMan™ SARS-CoV-2 Pooling Assay User Guide
5
Chapter 1 Product information
1
Contents and storage
Table 1 Dyes, quenchers, and targets
Dye Quencher Target
FAM™ dye QSY™ quencher ORF1ab
VIC™ dye QSY™ quencher N gene
ABY™ dye QSY™ quencher S gene
JUN™ dye QSY™ quencher MS2
IMPORTANT! It is the responsibility of the laboratories using the TaqMan
™
SARS-CoV-2 Pooling Assay
to design and validate their own experimental design and analysis parameters.
Contents and storage
The items listed in the following table are required for the TaqMan™ SARS-CoV-2 Pooling Assay. The
items listed are sucient for 200 reactions.
Kit or product Cat. No.
TaqMan™ SARS-CoV-2 Pooling
Assay Kit
TaqMan™ SARS-CoV-2 Control 956118 — 2 × 10 µL ≤ –70°C
TaqMan™ Control Dilution Buer A49889 — 10 × 250 µL
TaqPath™ 1‑Step Multiplex Master
Mix
[1]
Catalog numbers that appear as links open the web pages for those products.
A50790
A28523 — 10 mL
[1]
TaqMan™ SARS-CoV-2 Pooling
Multiplex Assay
TaqMan™ MS2 Phage Control 2 × 1 mL
Components Amount Storage
300 µL
–30°C to
–10°C
–30°C to
–10°C
–30°C to
–10°C
–30°C to
–10°C
6
TaqMan™ SARS-CoV-2 Pooling Assay User Guide
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.
IMPORTANT! The customer is responsible for performing all of the necessary validations to run this
assay.
Item Source
Real-time PCR instrument
An Applied Biosystems™ real-time PCR instrument compatible
with the four dyes listed in Table 1 on page 6.
The assay has been tested with the following instruments:
Applied Biosystems™ QuantStudio™ 5 Real‑Time PCR
Instrument, 96-well, 0.2-mL block
Chapter 1 Product information
Required materials not supplied
Contact your local sales oce
1
Applied Biosystems™ QuantStudio™ 7 Flex Real-Time PCR
Instrument, 384-well block
Software
QuantStudio™ Design and Analysis Software v2.5 or later thermofisher.com/qpcrsoftware
Equipment
Laboratory freezers
•
–30°C to –10°C
•
≤ –70°C
Centrifuge, with a rotor that accommodates standard and
deepwell microplates
Microcentrifuge MLS
Laboratory mixer, vortex or equivalent MLS
Single and multichannel adjustable pipettors (1.00 µL to
1,000.0 µL)
Cold block (96‑well or 384‑well) or ice MLS
Automated nucleic acid extraction system and materials
MLS
MLS
MLS
KingFisher™ Flex Magnetic Particle Processor with 96 DeepWell Head
KingFisher™ Flex 96 Deep-Well Heating Block 24075430
KingFisher™ Deep-Well 96 Plate 95040450, A48305, A48424, 95040455
TaqMan™ SARS-CoV-2 Pooling Assay User Guide
5400630
7
Chapter 1 Product information
1
Required materials not supplied
(continued)
Item Source
96‑well plate for the tip comb, one of the following:
•
KingFisher™ 96 KF microplate
•
•
Tip Comb Presenting Plate for KF 96
•
Nunc™ MicroWell™ 96‑Well Microplate, Flat Bottom
•
Nunc™ MicroWell™ 96‑Well Microplate, barcoded
•
ABgene™ 96–Well Polypropylene Storage Microplate
•
ABgene™ 96–Well 1.2–mL Polypropylene Deepwell
Storage Plate
•
Nunc™ F96 MicroWell™ Black Polystyrene Plate
•
Nunc™ F96 MicroWell™ White Polystyrene Plate
•
KingFisher™ Deep-Well 96 Plate
KingFisher™ 96 tip comb for DW magnets 97002534, A48438, A48414
97002540
•
267600
•
167008
•
269787
•
AB0796
•
AB1127
•
137101
•
136101
•
95040450, A48305, A48424, 95040455
Kits and reagents
MagMAX™ Viral/Pathogen II Nucleic Acid Isolation Kit A48383R
Fisher BioReagents™ Ethanol, Absolute, Molecular Biology
[1]
Grade
Nuclease-free Water (not DEPC-Treated) MLS
Calibration plates (QuantStudio™ 7 Flex Real-Time PCR Instrument)
ABY™ Dye Spectral Calibration Plate for Multiplex qPCR, 384well
JUN™ Dye Spectral Calibration Plate for Multiplex qPCR, 384well
Tubes, plates, and other consumables
MicroAmp™ Optical 96‑Well Reaction Plate with Barcode,
0.2 mL
MicroAmp™ Optical 96-Well Reaction Plate, 0.2 mL N8010560, 4316813
MicroAmp™ Optical 384-Well Reaction Plate with Barcode 4309849, 4326270, 4343814
MicroAmp™ Optical 384-Well Reaction Plate 4343370
, or equivalent
BP2818100, BP2818500, BP28184
A24736
A24733
4306737, 4326659
MicroAmp™ Clear Adhesive Film 4306311
MicroAmp™ Optical Adhesive Film 4311971, 4360954
MicroAmp™ Adhesive Film Applicator 4333183
8
TaqMan™ SARS-CoV-2 Pooling Assay User Guide
(continued)
Chapter 1 Product information
General laboratory recommendations
Item Source
1
Nonstick, RNase-free microcentrifuge tubes (1.5 mL and
2.0 mL)
Sterile aerosol barrier (filtered) pipette tips thermofisher.com/pipettetips
[1]
Available at fisherscientific.com.
thermofisher.com/plastics
General laboratory recommendations
•
Implement standard operating procedures in your laboratory to prevent contamination, such as the
following:
–
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 freeze-thaw cycles.
•
Aliquot reagents to prevent stock contamination and reduce the number of freeze-thaw cycles.
•
After each run, review the amplification curves in the instrument software according to data QC
standard operating procedures for your lab.
TaqMan™ SARS-CoV-2 Pooling Assay User Guide
9
Loading...