Thermo Fisher Scientific SARS-CoV-2 User Manual
TaqMan™ SARS-CoV-2 Mutation Panel
USER GUIDE
Publication Number MAN0024768
Revision A.0
For Research Use Only. Not for use in diagnostic procedures.
Life Technologies Corporation | 6055 Sunol Blvd | Pleasanton, California 94566 USA
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The information in this guide is subject to change without notice.
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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. MAN0024768
Revision Date Description
A.0 5 March 2021 New document for the TaqMan™ SARS-CoV-2 Mutation Panel.
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.
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registered trademark of Roche Molecular Systems, Inc., used under permission and license.
©2021 Thermo Fisher Scientific Inc. All rights reserved.
Contents
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CHAPTER 1 Product information .................................................. 5
About this guide ................................................................ 5
Product description ............................................................. 6
Context sequences ......................................................... 6
Storage and stability ............................................................. 7
Required materials not supplied ................................................... 7
Reagents .................................................................. 8
Workflow ....................................................................... 9
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CHAPTER 2 Ordering information ................................................ 10
Order the assays ............................................................... 10
Shipment contents ............................................................. 10
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CHAPTER 3 Methods ............................................................. 11
Guidelines for Positive Controls .................................................. 11
Prepare RT-PCR reactions ...................................................... 12
Set up and run the real-time PCR instrument ...................................... 13
Analyze the data ............................................................... 15
KK417T and K417N mutation assay analysis ................................. 17
Export the data ................................................................ 19
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APPENDIX A Supplemental information ......................................... 20
Assay description .............................................................. 20
Signals and sample genotype ................................................... 22
Overview of TaqMan™ MGB probes .............................................. 23
Good laboratory practices for PCR and RT-PCR ................................... 23
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APPENDIX B Safety ............................................................... 24
Chemical safety ................................................................ 25
Biological hazard safety ......................................................... 26
TaqMan
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SARS-CoV-2 Mutation Panel User Guide
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Contents
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APPENDIX C Documentation and support ...................................... 27
Related documentation ......................................................... 27
Customer and technical support ................................................. 27
Limited product warranty ........................................................ 27
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TaqMan™ SARS-CoV-2 Mutation Panel User Guide
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About this guide ....................................................................... 5
■
Product description .................................................................... 6
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Storage and stability ................................................................... 7
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Required materials not supplied ......................................................... 7
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Workflow ............................................................................. 9
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IMPORTANT! Before using this product, read and understand the information in the “Safety” appendix
in this document.
About this guide
Product information
This document provides guidance for the detection of emerging SARS-CoV-2 mutants in respiratory
tract samples that have previously had SARS-CoV-2 nucleic acid detected. The Applied Biosystems
TaqMan™ SARS-CoV-2 Mutation Panel assays contain probes to detect reference and mutation
sequences. The assays in the TaqMan™ SARS-CoV-2 Mutation Panel are combined with TaqPath
1-Step RT-qPCR Master Mix, CG and samples that contain SARS-CoV-2 nucleic acid.
This procedure is optimal for a minimum of 6–8 samples.
Real-time RT-PCR is performed with a QuantStudio™ 5 Real‑Time PCR Instrument or an equivalent
Applied Biosystems™ real-time PCR instrument.
Data are analyzed with the QuantStudio™ Design and Analysis Software v2.5 or later and the
Genotyping Analysis Module.
The TaqMan™ SARS-CoV-2 Mutation Panel is a research use only tool for surveillance.
IMPORTANT! The procedures in this document have not been optimized for detection of SARS-CoV-2
mutations. It is the responsibility of laboratories to design and validate their own experimental design
and analysis parameters.
™
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TaqMan™ SARS-CoV-2 Mutation Panel User Guide
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Chapter 1 Product information
1
Product description
Product description
The Applied Biosystems™ TaqMan™ SARS-CoV-2 Mutation Panel use TaqMan™ 5' nuclease chemistry
for amplifying and detecting specific variants in purified genomic RNA samples.
Each TaqMan™ SARS-CoV-2 Mutation Panel assay contains the following items:
•
Sequence-specific forward and reverse primers to amplify the target sequence region.
The reverse primer also primes reverse transcription of the SARS-CoV-2 genomic RNA sequences.
•
Two TaqMan™ probes with a minor groove binder (MGB), a non-fluorescent quencher (NFQ), and
the following 5' reporter dyes:
–
One VIC™ dye‑labeled probe to detect the reference sequence
–
One FAM™ dye‑labeled probe to detect the mutation sequence
Go to https://www.thermofisher.com/mutationpanel for a list of available assays. The following
information is provided for each assay:
•
Mutation
•
Gene
•
Assay ID
Table 1 Configurations of the TaqMan™ SARS-CoV-2 Mutation Panel assays
Scale
Small 40X 4332077
Medium 40X 4332075
Context sequences
Reporter dye information for the TaqMan™ SARS-CoV-2 Mutation Panel is represented in the assay
context sequence, which is the nucleotide sequence surrounding the mutation site in the SARS-CoV-2
reference genome (hCoV-19/Wuhan/WIV04/2019; GISAID EPI_ISL_402124). The reference and variant
nucleotide alleles are included in brackets, where the order of the alleles corresponds to the association
with probe reporter dyes, where [VIC™ dye / FAM™ dye].
An example is shown in the following table. The context sequence for the N501Y mutation is:
CCAACCCACT[A/T]ATGGTGTTG.
It is recommended to use the mutation target name as the SNP Assay name in the software.
Mutation
N501Y ANPRYZA A T (U in the RNA genome)
Assay ID
Formulation Cat. No.
VIC™ dye probe (reference
allele)
FAM™ dye probe (mutant
allele)
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TaqMan™ SARS-CoV-2 Mutation Panel User Guide
Storage and stability
Store the TaqMan™ SARS-CoV-2 Mutation Panel assays at –25°C to –15°C in the dark.
IMPORTANT! Protect the assays from direct exposure to light. Excessive exposure to light can aect
the fluorescent probes.
Limit freeze-thaw cycles. If you expect to freeze-thaw multiple times, consider creating aliquots of the
assay to minimize freeze-thaw cycles.
The assays are stable for up to 5 years after the manufacturing date. The manufacturing date is printed
on each assay tube.
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.
Chapter 1 Product information
Storage and stability
1
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 VIC™ dye and FAM™ dye.
The assay has been tested with the Applied Biosystems
QuantStudio™ 5 Real‑Time PCR Instrument.
Software
QuantStudio™ Design and Analysis Software v2.5 or later with
the Genotyping Analysis Module
Equipment
Centrifuge with plate adapter MLS
Microcentrifuge MLS
™
Contact your local sales oce
thermofisher.com/qpcrsoftware
Laboratory mixer, vortex or equivalent MLS
Single and multichannel adjustable pipettors (1.00 µL to
1,000.0 µL)
Tubes, plates, and other consumables
Real-time PCR plate thermofisher.com/plastics
TaqMan™ SARS-CoV-2 Mutation Panel User Guide
MLS
7
Chapter 1 Product information
1
Required materials not supplied
(continued)
Item Source
MicroAmp™ Optical Adhesive Film 4311971, 4360954
MicroAmp™ Adhesive Film Applicator 4333183
Nonstick, RNase-free microcentrifuge tubes thermofisher.com/plastics
Sterile aerosol barrier (filtered) pipette tips thermofisher.com/pipettetips
Nuclease-free Water (not DEPC-Treated) MLS
Reagents
Reagents
TaqPath™ 1-Step RT-qPCR Master Mix, CG
Optional controls
AcroMetrix™ Coronavirus 2019 (COVID-19) RNA Control
(RUO)
GeneArt™ DNA string or plasmid controls https://www.thermofisher.com/geneart
MEGAscript™ T7 Transcription Kit (used with GeneArt
strings to generate RNA)
Synthetic viral controls Vendor of choice
™
Cat. No.
A15299 (5 × 1 mL)
A15300 (1 x 10 mL)
954519
AMB13345
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TaqMan™ SARS-CoV-2 Mutation Panel User Guide
Workflow
Chapter 1 Product information
Workflow
TaqMan™ SARS-CoV-2 Mutation Panel workflow
Order the assay for the variants of interest
See Chapter 2, “Ordering information”.
Start with isolated RNA from respiratory tract samples with SARS-CoV-2 nucleic acid detected
Prepare RT-PCR reactions (page 12)
Set up and run the real-time PCR instrument (page 13)
Analyze the data (page 15)
1
(Optional) Export the data (page 19)
TaqMan™ SARS-CoV-2 Mutation Panel User Guide
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