Thermo Fisher Scientific Real-Time PCR User Manual
Real-Time PCR Detection of SARS-CoV-2 on
Food Packaging and Environmental Surfaces
USER GUIDE
for use with:
PrepSEQ™ Nucleic Acid Extraction Kit for Food and Environmental Testing
KingFisher™ Flex Purification System with 96 Deep-Well Head
MagMAX™ Express‑96 Deep Well Magnetic Particle Processor
TaqMan™ 2019-nCoV Assay Kit v1
TaqMan™ 2019-nCoV Control Kit v1
RNA UltraSense™ One-Step qPCR Master Mix
Applied Biosystems™ QuantStudio™ Design and Analysis Software v1.5.1 or later
Applied Biosystems™ 7500 Software SDS v.1.4.2 or later
Publication Number MAN0019499
Revision E.0
For testing of Food and Environmental samples only.
Life Technologies Corporation | 6055 Sunol Blvd | Pleasanton, CA 94566
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. MAN0019499
Revision Date Description
E.0 22 January 2021 Minor edits from the technical board.
D.0 05 December 2020 Updated the review results topic, and added Appendix B software
C.0 12 October 2020 Addition of RNA UltraSense™ One-Step qPCR Master Mix
B.0 02 October 2020 Correction of sample volume on page 15
A.0 11 September 2020 New document.
examples.
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. Thermomixer is a trademark of Eppendorf.
©2021 Thermo Fisher Scientific Inc. All rights reserved.
Contents
■
CHAPTER 1 Product information .................................................. 5
Product description ............................................................. 5
Required materials .............................................................. 6
Materials for sample preparation and nucleic acid extraction ..................... 6
Materials for PCR detection and analysis ...................................... 7
Procedural guidelines ............................................................ 9
Guidelines for sample collection and storage ................................... 9
Guidelines for the 2019-nCoV assay .......................................... 9
Workflow overview ............................................................. 10
■
CHAPTER 2 Methods ............................................................. 11
Prepare the samples and extract RNA using PrepSEQ™ Nucleic Acid Extraction
Kit for Food and Environmental Testing ......................................... 11
Perform 1-Step RT-PCR ........................................................ 13
Analyze data .................................................................. 14
Review results ................................................................. 15
Adjust the QuantStudio software to simplify the interpretation ................... 16
Adjust the 7500 software to simplify the interpretation ......................... 17
■
Troubleshooting ..................................................................... 18
■
APPENDIX A Supplemental information ......................................... 20
Good laboratory practices for PCR ............................................... 20
Symbol definitions ............................................................. 21
■
APPENDIX B Software examples ................................................ 22
QuantStudio Design and Analysis Software v1.5.1 examples ........................ 22
7500 Software SDS v1.4.2 examples ............................................. 28
■
APPENDIX C Safety ............................................................... 32
Chemical safety ................................................................ 33
Biological hazard safety ......................................................... 34
RT-PCR Detection of SARS-CoV-2 on Environmental Surfaces User Guide
3
Contents
■
APPENDIX D Documentation and support ...................................... 35
Food Safety support ............................................................ 35
Related documentation ......................................................... 35
Reference ..................................................................... 35
4
RT-PCR Detection of SARS-CoV-2 on Environmental Surfaces User Guide
1
IMPORTANT! Before using this product, read and understand the information in the “Safety” appendix
in this document.
Product description
This guide describes the following Performance Tested workflow for detection of SARS-CoV-2 in food
packaging and environmental surfaces with the Applied Biosystems™ TaqMan™ 2019-nCoV Assay Kit
v1 (Cat. No. A47532). This kit includes three TaqMan™ RT‑PCR assays that target SARS-CoV-2 genes,
and one positive control assay that targets the Human RNase P RPPH1 gene:
• Assays target three dierent viral genomic regions, reducing the risk of false negatives.
• Assays have undergone bioinformatic selection and analysis to specifically target sequences that
are unique to SARS-CoV-2.
• The RNase P assay is run in duplex with the combined 2019-nCoV assays as an internal positive
control.
Product information
The kit is used with the TaqMan™ 2019-nCoV Control Kit v1 (Cat. No. A47533) to monitor assay-specific
amplification.
For more information about genetic analysis solutions available for SARS-CoV-2, go to
thermofisher.com/coronavirus.
RT-PCR Detection of SARS-CoV-2 on Environmental Surfaces User Guide
5
Chapter 1 Product information
1
Required materials
Required materials
Unless otherwise indicated, all materials are available through the Thermo Fisher Microbiology ordering
process or thermofisher.com. MLS: Fisher Scientific (fisherscientific.com) or other major laboratory
supplier.
Note: Parts may ship separately depending on configuration and storage conditions.
Materials for sample preparation and nucleic acid extraction
Table 1 PrepSEQ™ Nucleic Acid Extraction Kit
Contents
Lysis Buer 2 × 50 mL 6 × 50 mL
Magnetic Particles 2 × 1.5 mL 6 × 1.5 mL
Binding Solution (Isopropanol)
Wash Buer Concentrate
Elution Buer 25 mL 3 × 25 mL
Proteinase K (PK) Buer 50 mL 3 × 50 mL
Proteinase K, 20 mg/mL 1.25 mL 3 × 1.25 mL –25°C to –15°C
[1]
Refer to the product label for the expiration date.
[2]
Add ~35 mL of 100% isopropanol to the empty bottle before use.
[3]
Add 74 mL of 95% ethanol before use.
[2]
[3]
Cat. No. 4480466
(100 reactions)
1 empty bottle 3 empty bottles
2 × 26 mL 6 × 26 mL
Cat. No. 4428176
(300 reactions)
Storage
15°C to 30°C
[1]
Table 2 Magnetic particle processor
Item
KingFisher™ Flex instrument and accessories
KingFisher™ Flex Purification System with 96 Deep-Well
Head with BindIt™ Software
Source
A32681
KingFisher™ Flex 96 Deep-Well Heating Block 24075430 (included in A32681)
KingFisher™ Deep-Well 96 Plate, V-bottom 95040450
KingFisher™ 96 tip comb for DW magnets 97002534
MagMAX™ Express-96 instrument and accessories
MagMAX™ Express‑96 Deep Well Magnetic Particle
Processor
MagMAX™ Express-96 Deep Well Plates
MagMAX™ Express-96 Deep Well Tip Combs
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RT-PCR Detection of SARS-CoV-2 on Environmental Surfaces User Guide
Contact your local sales representative.
Materials for PCR detection and analysis
Table 3 TaqMan™ 2019-nCoV Assay Kit v1 (Cat. No. A47532)
Chapter 1 Product information
Required materials
1
Contents Dye Amount
[1]
Concentration Storage
2019-nCoV (ORF1ab) (Tube 1) FAM™ dye 75 µL 20X
2019-nCoV (S Protein) (Tube 2) FAM™ dye 75 µL 20X
2019-nCoV (N Protein) (Tube 3) FAM™ dye 75 µL 20X
RNase P Assay (Tube 4) VIC™ dye 250 µL 20X
[1]
Sufficient for 50 × 25-µL reactions.
Table 4 TaqMan™ 2019-nCoV Control Kit v1 (Cat. No. A47533)
Contents Target Sequence Amount
[1]
Concentration Storage
ORF1ab
S protein
2019-nCoV Control v1
50 µL 1 × 104 copies/µL –30°C to –10°C
N protein
Human RNase P RPPH1
[1]
Sufficient for 50 positive-control reactions.
Table 5 Other required materials
–30°C to –10°C
Item
Source
Master Mix
Thermo Scientific™ RNA UltraSense™ One-Step qPCR Master
Mix:
•
250 μL RNA UltraSense™ Enzyme Mix
•
1 mL RNA UltraSense™ 5X Reaction Mix
•
300 μL 20X Bovine Serum Albumin (BSA)
•
1 mL 50-mM Magnesium Sulfate (MgSO4)
•
100 μL ROX™ Reference Dye
11732927
Total RNA Control (Human) 4307281, or equivalent
Real-Time PCR Instrument (one of the following)
Applied Biosystems™ QuantStudio™ 5 Food Safety Real-Time
PCR System
Applied Biosystems™ 7500 Fast Food Safety Real-Time PCR
System
A36328 or A36320
A30299 or A30304
RT-PCR Detection of SARS-CoV-2 on Environmental Surfaces User Guide
7
Chapter 1
1
Required materials
Product information
Table 5 Other required materials (continued)
Item Source
Real-Time PCR Instrument Software (one of the following)
Applied Biosystems™ QuantStudio™ Design and Analysis
Software v1.5.1 or later
[1]
Included with QuantStudio™ 5 Real‑Time PCR
System
Applied Biosystems™ 7500 Software SDS v.1.4.2 or later Included with 7500 Real‑Time PCR Instrument
Equipment
Laboratory freezers -30℃ to -10℃ and ≤-70℃
Centrifuge, with a rotor that accommodates deepwell
microplates
Microcentrifuge
MLS
Laboratory mixer, vortex or equivalent
Single and multichannel adjustable pipettors (1 μL to 1,000 μL)
Cold block (96‑well or 384‑well) or ice
Tubes, plates, and other consumables
MicroAmp™ Fast Optical 96-Well Reaction Plate, 0.1 mL 4346907
MicroAmp™ Optical Adhesive Film Kit 4313663
MicroAmp™ Optical 8-Cap Strips 4323032
MicroAmp™ Fast 8-Tube Strip, 0.1 mL 4358293
Disposable gloves
Micropipette tips, aerosol resistant
MLS
Swabs with synthetic tip and aluminium or plastic shaft,
preferably large macrofoam tipped applicator
Reagents
Thermo Scientific™ Oxoid™ Phosphate Buered Saline BR0014G
Ethanol, 95%
Isopropanol, 100%
MLS
Nuclease-free water
[1]
Real-Time PCR Instrument Software is used to control the instrument and to collect instrument run data.
8
RT-PCR Detection of SARS-CoV-2 on Environmental Surfaces User Guide
Procedural guidelines
Guidelines for sample collection and storage
Sample collection:
•
Pre-moisten a non-cellulose swab with synthetic tip with Phosphate Buered Saline (PBS).
•
When sampling, apply pressure with the wet swab onto the surface, move in at least two dierent
directions while rotating the swab stick. Avoid letting the swab dry completely. Swab an area up to
2 x 2 inches, ensuring that the full area is covered in the sampling.
•
Place the swab back into the transport tube containing 2 mL of PBS.
Note: Viral Transport Media (VTM) can be used instead of PBS according to World Health Organization
protocol on Surface sampling of coronavirus disease (COVID-19): A practical "how to" protocol for
health care and public health professionals, Version: 1.1, February 2020 (www.who.int).
Storage:
•
Swabs should be placed at refrigeration temperature (2-8°C) no more than 15 minutes postsampling and maintain refrigeration until analysis.
•
Analysis of swabs should occur within 24 hours from sample collection (48 hours maximum). If the
swabs are not analyzed with the 48 hour maximum, store the swab between -70°C and -80°C and
ship on dry ice.
Chapter 1 Product information
Procedural guidelines
1
Note: Analyze the swabs preferably in the coming 24 hours. If the swabs are not analyzed within
48 hours maximum, store the swabs preferably between -70°C and -80°C, and ship on dry ice.
Guidelines for the 2019-nCoV assay
•
Use purified, non-degraded total nucleic acid that is free of RNase activity and RT-PCR inhibitors.
•
Protect the assays and master mix from light.
•
For each research sample, include the primers and probes for all three 2019-nCoV targets (FAM
assay) in multiplex with the RNase P target (VIC™ assay).
•
Before you begin, determine the number of required reactions. In addition to the nucleic acid
research samples, include the following reactions for the combined 2019-nCoV assay:
–
One negative extraction control (NEC) per plate
–
One 2019-nCoV Control v1 reaction per plate
–
One no-template control (NTC) per plate
™
RT-PCR Detection of SARS-CoV-2 on Environmental Surfaces User Guide
9
Chapter 1 Product information
1
Workflow overview
Workflow overview
SARS-CoV-2 Detection on Food Packaging and Environmental Surfaces
Sampling and Transport
Simple sample collection with noncellulose swabs and Viral Transport
Medium or Phosphate Buered Saline
Sample Preparation
Options for automated
medium to high-throughput
and manual low-throughput
PCR and Data Analysis
Master mix and Real-Time PCR
instrumentation with software
designed to run environmental
PCR range of products
10
RT-PCR Detection of SARS-CoV-2 on Environmental Surfaces User Guide
2
Methods
Prepare the samples and extract RNA using PrepSEQ
™
Nucleic Acid Extraction Kit for Food and Environmental
Testing
The KingFisher™ Flex Purification System with 96 Deep-Well Head or the MagMAX™ Express‑96
Deep Well Magnetic Particle Processor can be used to extract RNA with the PrepSEQ™ Nucleic Acid
Extraction Kit for Food and Environmental Testing.
•
Ensure that the KingFisher™ Flex Purification System with 96 Deep-Well Head is set up with the
KingFisher™ Flex 96 Deep-Well Heating Block.
•
Ensure that the PSNA_Flex_300ul has been downloaded from the product page and loaded onto
the instrument using the BindIt™ software.
Note: Use PSNA_MagMAX_300ul if you are using MagMAX™ Express‑96 Deep Well Magnetic
Particle Processor.
•
Add 74 mL 95% ethanol the Wash Buer Concentrate bottle.
•
Add 35 mL 100% isopropanol to an empty Binding Solution bottle.
1.
Prepare and label the side of the processing plates according to the following table.
Plate ID
Plate type Reagent Volume per well
Sample
Wash Plate 1 Wash Buer 300 μL
Wash Plate 2 Wash Buer 300 μL
Elution Plate Elution Buer 100 μL
Tip Comb Plate
2.
Cover the plates with an adhesive film, then store at room temperature while setting up the sample
plate (up to 1 hour).
3.
Vortex the Magnetic Beads to ensure the bead mixture is homogenous.
If a white precipitate has formed, first incubate the tube of Magnetic Particles at 37±1°C for
approximately 10 minutes and re-vortex.
RT-PCR Detection of SARS-CoV-2 on Environmental Surfaces User Guide
KingFisher™ Deep-Well
96 Plates
Place a KingFisher™ 96 tip comb for Deep Well magnets in a KingFisher™ 96
microplate
Binding Bead Mix 700 μL
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