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
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. 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.
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
About this guide ................................................................ 5
Product description ............................................................. 6
Context sequences ......................................................... 6
Storage and stability ............................................................. 7
Required materials not supplied ................................................... 7
Reagents .................................................................. 8
Workflow ....................................................................... 9
■
CHAPTER 2 Ordering information ................................................ 10
Order the assays ............................................................... 10
Shipment contents ............................................................. 10
■
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
■
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
■
APPENDIX B Safety ............................................................... 24
Chemical safety ................................................................ 25
Biological hazard safety ......................................................... 26
TaqMan
™
SARS-CoV-2 Mutation Panel User Guide
3
Contents
■
APPENDIX C Documentation and support ...................................... 27
Related documentation ......................................................... 27
Customer and technical support ................................................. 27
Limited product warranty ........................................................ 27
4
TaqMan™ SARS-CoV-2 Mutation Panel User Guide
1
About this guide ....................................................................... 5
■
Product description .................................................................... 6
■
Storage and stability ................................................................... 7
■
Required materials not supplied ......................................................... 7
■
Workflow ............................................................................. 9
■
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.
™
™
TaqMan™ SARS-CoV-2 Mutation Panel User Guide
5
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)
6
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
8
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
9
2
Order the assays
Order assays at https://www.thermofisher.com/mutationpanel.
The assays that are available are listed. The following items are provided for each assay:
•
Mutation
•
Gene
•
Assay ID
Click on the Assay ID for a specific assay, then select the size.
Shipment contents
The shipment includes the following components:
•
Dispatch Note (Packing List)—Contains your order information, a list of the assays that were
ordered, and the quantity of the assays shipped.
•
Assay tubes—Each assay tube is identified with a label and a 2‑D barcode.
•
Data Sheet—Provides a summary of the assays in your shipment, the assay details, and the
storage conditions.
Ordering information
10
Go to thermofisher.com/taqmanfiles to download the following supporting documents:
•
Assay Information File (AIF)
•
Certificates of Analysis
•
Safety Data Sheets
For detailed information about the shipment and the AIF, see Understanding Your Shipment (Pub.
No. MAN0017153).
TaqMan™ SARS-CoV-2 Mutation Panel User Guide
3
Guidelines for Positive Controls ........................................................ 11
■
Prepare RT-PCR reactions ............................................................. 12
■
Set up and run the real-time PCR instrument ............................................ 13
■
Analyze the data ..................................................................... 15
■
Export the data ...................................................................... 19
■
Guidelines for Positive Controls
Possible sources of positive controls that contain the SARS-CoV-2 assay target mutation include the
following items:
•
A sample in your lab that has been characterized by sequence analysis
•
A commercial RNA control
•
A commercial DNA control, for example GeneArt™ strings or plasmids.
Supply the mutation of interest and 100 nucleotides of flanking sequence to each side of the
mutation.
Methods
Use approximately 1,000 copies of a control per 20 µL reaction. Adjust this amount as needed.
TaqMan™ SARS-CoV-2 Mutation Panel User Guide
11
Chapter 3
3
Prepare RT-PCR reactions
Methods
Prepare RT-PCR reactions
The recommended sample input is based on results from the sample run with an assay to detect
nucleic acid from SARS-CoV-2. The average Cq (Ct) value of the ORF1ab, S gene, and N gene assay
results should have a Cq cuto of ≤30 Cq. This is approximate and can be adjusted as needed to
conserve sample and/or achieve sucient sample amplification.
1.
Calculate the number of reactions to be performed for each assay, including controls.
It is recommended to include at least 6 test samples for each assay, one Positive Control for each
assay (optional), and at least one No Template Control (NTC) for each assay.
2.
Prepare a Reaction Mix in an appropriately-size tube, according to one of the following tables.
Table 2 96-well 0.2-mL plates
Component
TaqPath™ 1-Step RT-qPCR Master
Mix, CG (4X)
TaqMan™ SARS-CoV-2 Mutation
Panel Assay (40X)
Volume per assay
5 µL 528 µL
0.5 µL 52.8 µL
[1]
Volume per 96-well plate
Nuclease-free water 9.5 µL 1,003.2 µL
Total Reaction Mix volume 15 µL 1,584 µL
[1]
Add 10% overage.
[2]
Includes 10% overage.
Table 3 96-well 0.1-mL plates and 384-well plates
Component Volume per assay
Volume per 96-well
[1]
plate
[2]
Volume per 384-well
plate
TaqPath™ 1-Step RTqPCR Master Mix, CG
2.5 µL 264 µL 1,056 µL
(4X)
TaqMan™ SARS-CoV-2
Mutation Panel Assay
0.25 µL 26.4 µL 105.6 µL
(40X)
[2]
[2]
12
Nuclease-free water 4.75 µL 501.6 µL 2,006.4 µL
Total Reaction Mix
volume
[1]
Add 10% overage.
[2]
Includes 10% overage.
3.
Pipet the Reaction Mix into each well of a reaction plate.
•
15 µL of Reaction Mix into each well of a 96-well 0.2-mL plate
•
7.5 µL of Reaction Mix into each well of a 96-well 0.1-mL plate or a 384-well plate
7.5 µL 792 µL 3,168 µL
TaqMan™ SARS-CoV-2 Mutation Panel User Guide
Set up and run the real-time PCR instrument
4.
Add RNA sample or nuclease-free water (No Template Control) in the appropriate well of the
reaction plate.
•
5 µL of RNA or nuclease-free water into each well of a 96-well 0.2-mL plate
•
2.5 µL of RNA or nuclease-free water into each well of a 96-well 0.1-mL plate or a 384-well
plate
Note: The RNA sample input volume can be adjusted as needed. Adjust the volume of nucleasefree water accordingly.
5.
Seal the plate thoroughly with MicroAmp™ Optical Adhesive Film.
6.
Vortex the plate for 10–30 seconds to ensure proper mixing.
7.
Centrifuge the plate for 1–2 minutes at ≥650 × g (≥650 RCF) to remove bubbles and collect the
liquid at the bottom of the wells.
Set up and run the real-time PCR instrument
Chapter 3
Methods
3
For detailed instructions, see the user documentation for your real-time PCR instrument.
Your instrument software can be used to set up and run the experiment using the settings outlined
in this section. Alternatively, QuantStudio™ Design and Analysis Software v2.5 or later can be used to
set-up and run experiments for all QuantStudio™ instruments, except for the QuantStudio™ 12K Flex
Real-Time PCR Instrument.
This section provides an example of instrument set-up and run instructions for the QuantStudio™ 5
Real‑Time PCR Instrument with a 96-well 0.2-mL block and the QuantStudio™ Design and Analysis
Software v2.5. Use the appropriate settings for your own instrument, block, and data collection
software.
For instructions to obtain the Assay Information File (AIF), see “Shipment contents” on page 10.
1.
In the home screen, click Set Up Plate.
The Plate Gallery screen and the System Templates tab is displayed.
2.
In the left pane, apply the appropriate filters for the Instrument, Block, and Run Mode.
3.
In the left pane, select the following filters:
•
Run Mode: Standard
•
Analysis: Genotyping
Note: The run mode depends on the chemistry of the master mix. It does not depend on the
reaction plate.
4.
Select Genotyping_Pre_PCR_Post.
The Run Method tab is displayed.
TaqMan™ SARS-CoV-2 Mutation Panel User Guide
13
Chapter 3
3
Set up and run the real-time PCR instrument
5.
6.
Methods
In the Reaction Volume field, enter the appropriate reaction volume.
•
20 µL for a 96-well 0.2-mL plate
•
10 µL for a 96-well 0.1-mL plate or a 384-well plate
Modify the thermal protocol.
Step Temperature Time
Pre-read 60°C 30 seconds 1
Reverse transcription
Fast DNA polymerase activation
Denaturation 95°C 3 seconds
Anneal / extend 60°C 30 seconds
Post-read 60°C 30 seconds 1
[1]
The optimal temperature for reverse transcription is 48°C to 55°C.
[2]
Required for reverse transcriptase inactivation, initial denaturation, and activation of DNA polymerase.
7.
In the Plate Setup tab, set the Passive Reference to ROX.
8.
(Optional) Click 4Import AIF to import an assay information file (AIF).
[1]
[2]
50°C 10 minutes 1
95°C 2 minutes 1
Number of
cycles
Note: Use the menu above the grid view to import an AIF.
The assays, reporter and quenchers, and the nucleotide alleles from the assay context sequences
will be imported.
9.
In the Samples table, click (Add) to add samples.
45
14
10.
In the Samples table, add a name to the Name field, then select an option from the Type
dropdown list.
11.
In the SNP Assays table, click
12.
In the SNP Assay table, add a name to the SNP Assay field, then define the following parameters.
•
Allele 1 Reporter: VIC
•
Allele 1 Quencher: NFQ-MGB
•
Allele 2 Reporter: FAM
•
Allele 2 Quencher: NFQ-MGB
(Add) to add assays.
Note: The variant allele is detected by FAM™ dye. The reference allele is detected by VIC™ dye.
13.
(Optional) To edit the default Allele 1 and Allele 2 names displayed in the data plots and results,
click
(Edit) in the SNP Assays. Edit the Name or Base(s) fields to contain the nucleotide base
detected by the probes, found in the context sequence, or other name of choice.
If the AIF was imported, the base names for the alleles are imported into the Name or Base(s)
fields.
TaqMan™ SARS-CoV-2 Mutation Panel User Guide
14.
To assign samples and SNP assays to a well, select the well, select the assay from the dropdown
list in the well, then select the checkbox for the appropriate sample in the Samples table.
The Task in the SNP Assays table is automatically assigned.
15.
In the Run Summary tab, review the method and the plate setup.
16.
Select and instrument from the list, then start the run.
Analyze the data
A data file is generated is EDS or SDS format, depending on the real-time PCR instrument.
This section provides guidelines for data analysis using the QuantStudio™ Design and Analysis Software
v2.5. This software supports viewing and analyzing data from all QuantStudio™ instruments (except for
TaqMan™ OpenArray™ Plate files) and from the StepOnePlus™ Instrument, the ViiA™ 7 instrument, the
7500/7500 Fast Real-Time PCR System, and the 7900HT Real-Time PCR Instrument.
After the experiment has been run, use the analysis software associated with your real-time PCR
instrument to process and analyze your data.
Chapter 3
Analyze the data
Methods
3
1.
Open a data file.
Note: QuantStudio™ Design and Analysis Software v2 requires data files created on the
compatible instruments to be saved as a new data file. Click Actions4Save As, then save the
data file with a new name.
2.
Click Actions4Analysis Modules, then select Genotyping.
If the Genotyping tab is not available, click Analyze4Analysis Modules.
3.
In the Analysis Modules dialog box, select Genotyping, then click OK.
4.
Navigate to the Genotyping tab.
5.
In the Actions dropdown list, click Genotyping Analysis Setting.
6.
In the Genotyping Analysis Setting dialog box, select Real Time Data, then click Apply.
This setting enables real-time tract data to be viewed in the Allelic Discrimination Plot.
7.
View the Allelic Discrimination Plot.
Note: The genotype calling algorithm is designed for diploid organism genotype calling.
For the TaqMan SARS-CoV-2 variant assays, the reference allele cluster should be
called as HOMOZYGOUS_ALLELE1 and the variant allele cluster should be called as
HOMOZYGOUS_ALLELE2.
If the clustering algorithm calls one of the expected two clusters as HETEROZYGOUS, select the
cluster samples and manually adjust the call using the dropdown list in the upper right hand corner
of the results table.
Samples that are not called due to weak amplification and that lie along the real-time trajectory of
one of the two clusters can be called manually.
TaqMan™ SARS-CoV-2 Mutation Panel User Guide
15
Chapter 3 Methods
3
Analyze the data
8.
Samples may run o the main 'homozygous' clusters if they contain one of the following items:
A mutation that underlies the assay binding sites
·
A mixture of viruses that have both reference and variant alleles
·
For such samples, further characterization of the sample by sequence analysis is recommended.
Select a well from the plate layout view or the table view to display on the plot.
Samples that carry the reference allele will cluster along the X-axis (Allele 1 VIC™ dye). Samples
that carry the mutant alleles will cluster along the Y-axis (Allele 2 FAM™ dye).
The following figures show genotyping data plots for two TaqMan™ SARS-CoV-2 Mutation Panel
assays.
IVT-RNA control samples for reference and mutant alleles were plated at the following
concentrations:
•
100,000 copies/20 µL reaction
•
10,000 copies/20 µL reaction
•
1,000 copies/20 µL reaction
•
250 copies/20 µL reaction
16
TaqMan™ SARS-CoV-2 Mutation Panel User Guide
Chapter 3 Methods
Analyze the data
3
KK417T and K417N mutation assay analysis
K417T (A22812C) and K417N (G22813T) are independent mutations that are located next to one
another in the SARS-CoV-2 virus genome. This can complicate genotype analysis as the probes of
an assay to one mutation will fail to bind to viral sequences that contain the other, adjacent mutation.
The context sequences for the K417T and K417N assays are shown below.
K417T (A22812C) assay context sequence
AGGGCAAACTGGAA[A/C]GATTGCTGATTATAAT
K417N (G22813T) assay context sequence
AGGGCAAACTGGAAA[G/T]ATTGCTGATTATAAT
K417T (A22812C) - K417N (G22813T)
AGGGCAAACTGGAA[A/C][G/T]ATTGCTGATTATAAT
Example data plots for the K417T and K417N assays are shown in the figures below. Samples that
contain K417T or K417N mutations are identified and circled.
TaqMan™ SARS-CoV-2 Mutation Panel User Guide
17
3
Chapter 3
Analyze the data
Methods
18
Samples that are run with the K417T assay and that contain the K417N mutation, or samples run with
the K417N assay and that contain the K417T mutation, can cluster near the NTCs or exhibit weak
amplification due to probe nonspecific activity. If a mutation containing sample is called as wild type
with the adjacent SNP assay, the call can be manually adjusted to "no amp".
We highly recommend that both K417T and K417N mutation assays are run on the same samples, and
that the results of both assays are compared, to facilitate accurate genotype analysis.
If it is not possible to make a clear genotype call, rerun the experiment or further characterize the
sample by sequence analysis.
TaqMan™ SARS-CoV-2 Mutation Panel User Guide
Export the data
1.
Click Actions4Export.
The Export Plate dialog box is displayed.
2.
(Optional) Edit the following fields: .
•
Export Name
•
File Format
3.
Click Export.
•
Destination
•
Export Setting
Chapter 3 Methods
Export the data
3
TaqMan™ SARS-CoV-2 Mutation Panel User Guide
19
RT
V
F
F
NFQ
MGB
3'
RT
5'
5'
3'
5'
C/T
A
Assay description
The assay is a one-step RT-PCR to detect reference and mutation sequences.
Note: The following figures are general representations of RT-PCR with TaqMan™ MGB probes and
the TaqMan™ SARS-CoV-2 Mutation Panel assays. The sequence regions are not necessarily drawn to
scale.
Supplemental information
RNA
cDNA template
Reverse transcriptase
Forward primer
Reverse primer
Probe
VIC™ reporter dye
FAM™ reporter dye
FAM™ reporter dye fluorescing
Quencher dye
MGB
During reverse transcription (RT), the RNA is reversed transcribed to cDNA (Figure 1 and Figure 2).
The reverse transcription product is single-stranded.
Figure 1 Reverse transcription
Figure 2 Reverse transcription product
After RT, the PCR amplification of the cDNA template begins.
The master mix contains a DNA polymerase. The reaction also contains a forward primer, a reverse
primer, a FAM™-labeled TaqMan™ probe, and a VIC™-labeled TaqMan™ (Figure 3).
20
TaqMan™ SARS-CoV-2 Mutation Panel User Guide
3'
5'
5'
3'
C/T
G/A
F
G
V
A
3'
5'
C
5'
3'
G
V
A
F
G
G
3'
5'
C
5'
3'
V
A
F
G
Appendix A
Supplemental information
Assay description
Figure 3 Components
The cDNA templates are denatured, then the forward and reverse primers anneal to complementary
sequences along the denatured cDNA strands (Figure 4).
A
Figure 4 Primer anneal
A probe anneals to a complementary sequence between the forward and reverse primer sites (Figure 5).
When the probe is intact, the proximity of the reporter dye to the quencher results in suppression of the
fluorescent signal.
Figure 5 Probe anneal
During polymerization, the DNA polymerase only cleaves probes that hybridize to the target sequence.
Cleavage separates the reporter dye from the probe. The separation of the reporter dye from the
quencher dye results in increased fluorescence by the reporter (Figure 6).
21
The increase in fluorescence occurs only if the target sequence is complementary to the probe and
amplified during PCR. Because of these requirements, nonspecific amplification is not detected, and
TaqMan™ SARS-CoV-2 Mutation Panel User Guide
the fluorescence signal indicates which alleles are in the sample
F
3'
5'
C
5'
3'
G
3'
5'
C
5'
3'
G
V
A
3'
5'
C
5'
3'
G
F
V
A
Appendix A Supplemental information
A
Signals and sample genotype
Figure 6 Probe cleavage
Figure 7 Completion
Signals and sample genotype
Signal
VIC™ dye Reference sequence
FAM™ dye Mutation sequence
Sample genotype
22
TaqMan™ SARS-CoV-2 Mutation Panel User Guide
Appendix A Supplemental information
Overview of TaqMan™ MGB probes
Overview of TaqMan™ MGB probes
TaqMan™ MGB probes consist of target-specific oligonucleotides with:
•
A reporter dye at the 5´ end of the probe:
–
VIC™ dye for Allele 1 probe
–
FAM™ dye for Allele 2 probe
•
A non‑fluorescent quencher (NFQ) dye at the 3´ end of the probe.
•
A minor groove binder (MGB) at the 3´ end of the probe that:
–
Increases the melting temperature (Tm) without increasing probe length.
–
Allows for the design of shorter probes.
Good laboratory practices for PCR and RT-PCR
•
Wear clean gloves and a clean lab coat.
–
Do not wear the same gloves and lab coat that you have previously used when handling
amplified products or preparing samples.
•
Change gloves if you suspect that they are contaminated.
•
Maintain separate areas and dedicated equipment and supplies for:
–
Sample preparation and reaction setup.
–
Amplification and analysis of products.
•
Do not bring amplified products into the reaction setup area.
•
Open and close all sample tubes carefully. Avoid splashing or spraying samples.
•
Keep reactions and components capped as much as possible.
•
Use a positive-displacement pipettor or aerosol‑resistant barrier pipette tips.
•
Clean lab benches and equipment periodically with 10% bleach solution or DNA decontamination
solution.
A
TaqMan™ SARS-CoV-2 Mutation Panel User Guide
23
B
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 safety information provided in this document.
Before 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.
Before 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.
Safety
24
TaqMan™ SARS-CoV-2 Mutation Panel User Guide
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
·
before 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
·
sucient 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.)
After 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.
Appendix B Safety
Chemical safety
B
AVERTISSEMENT ! PRÉCAUTIONS GÉNÉRALES EN CAS DE MANIPULATION DE PRODUITS
CHIMIQUES. Pour minimiser les risques, veiller à ce que le personnel du laboratoire lise
attentivement et mette en œuvre les consignes de sécurité générales relatives à l’utilisation et au
stockage des produits chimiques et à la gestion des déchets qui en découlent, décrites ci-dessous.
Consulter également la FDS appropriée pour connaître les précautions et instructions particulières à
respecter :
Lire et comprendre les fiches de données de sécurité (FDS) fournies par le fabricant avant de
·
stocker, de manipuler ou d’utiliser les matériaux dangereux ou les produits chimiques. Pour obtenir
les FDS, se reporter à la section « Documentation et support » du présent document.
Limiter les contacts avec les produits chimiques. Porter des équipements de protection appropriés
·
lors de la manipulation des produits chimiques (par exemple : lunettes de sûreté, gants ou
vêtements de protection).
Limiter l’inhalation des produits chimiques. Ne pas laisser les récipients de produits chimiques
·
ouverts. Ils ne doivent être utilisés qu’avec une ventilation adéquate (par exemple, sorbonne).
Vérifier régulièrement l’absence de fuite ou d’écoulement des produits chimiques. En cas de fuite
·
ou d’écoulement d’un produit, respecter les directives de nettoyage du fabricant recommandées
dans la FDS.
Manipuler les déchets chimiques dans une sorbonne.
·
TaqMan™ SARS-CoV-2 Mutation Panel User Guide
25
Appendix B Safety
B
Biological hazard safety
Veiller à utiliser des récipients à déchets primaire et secondaire. (Le récipient primaire contient
·
les déchets immédiats, le récipient secondaire contient les fuites et les écoulements du récipient
primaire. Les deux récipients doivent être compatibles avec les matériaux mis au rebut et
conformes aux exigences locales, nationales et communautaires en matière de confinement des
récipients.)
Une fois le récipient à déchets vidé, il doit être refermé hermétiquement avec le couvercle fourni.
·
Caractériser (par une analyse si nécessaire) les déchets générés par les applications, les réactifs et
·
les substrats particuliers utilisés dans le laboratoire.
Vérifier que les déchets sont convenablement stockés, transférés, transportés et éliminés en
·
respectant toutes les réglementations locales, nationales et/ou communautaires en vigueur.
IMPORTANT ! Les matériaux représentant un danger biologique ou radioactif exigent parfois une
·
manipulation spéciale, et des limitations peuvent s’appliquer à leur élimination.
Biological hazard safety
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 following 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), 5th Edition, HHS Publication No. (CDC) 21-1112, Revised December 2009;
found at:
https://www.cdc.gov/labs/pdf/CDC-BiosafetymicrobiologicalBiomedicalLaboratories-2009P.pdf
World Health Organization, Laboratory Biosafety Manual, 3rd Edition,
·
WHO/CDS/CSR/LYO/2004.11; found at:
www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf
26
TaqMan™ SARS-CoV-2 Mutation Panel User Guide
Documentation and support
C
Related documentation
Document
TaqPath™ 1-Step RT-qPCR Master Mix, CG User Guide MAN0007959
QuantStudio™ Design and Analysis Software v2 User Guide MAN0018200
QuantStudio™ Design and Analysis Software v2 Genotyping Analysis Module
User Guide
Understanding Your Shipment MAN0017153
Customer and technical support
Visit thermofisher.com/support for the latest service and support information.
•
Worldwide contact telephone numbers
•
Product support information
–
Product FAQs
–
Software, patches, and updates
–
Training for many applications and instruments
•
Order and web support
•
Product documentation
–
User guides, manuals, and protocols
–
Certificates of Analysis
–
Safety Data Sheets (SDSs; also known as MSDSs)
Publication number
MAN0018749
Note: For SDSs for reagents and chemicals from other manufacturers, contact the
manufacturer.
Limited product warranty
Life Technologies Corporation and/or its aliate(s) warrant their products as set forth in the
Life Technologies' General Terms and Conditions of Sale at www.thermofisher.com/us/en/home/
global/terms-and-conditions.html. If you have any questions, please contact Life Technologies at
www.thermofisher.com/support.
TaqMan™ SARS-CoV-2 Mutation Panel User Guide
27
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