Thermo Fisher Scientific CoviPath User Manual
CoviPath™ COVID-19 RT-PCR Kit
USER GUIDE
Multiplex real-time RT-PCR test intended for the qualitative
detection of nucleic acid from SARS‑CoV‑2
Catalog Number A50780
Publication Number MAN0019860
Revision A.0
For In Vitro Diagnostic Use.
Invitrogen Bioservices Private Ltd. | Citius Block, First Technology Place, No. 3 EPIP | Whitefield, Bangalore 560066, India
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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 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. MAN0019860
Revision Date Description
A.0 27 January 2021 New user guide for the CoviPath™ COVID-19 RT-PCR Kit.
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Contents
■
CHAPTER 1 Product information .................................................. 5
Intended Use ................................................................... 5
Product description ............................................................. 5
Contents and storage ............................................................ 6
Required materials not supplied ................................................... 6
General laboratory recommendations .............................................. 7
Assay limitations ................................................................ 8
Samples and controls ............................................................ 9
Workflow ....................................................................... 9
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CHAPTER 2 Before you begin .................................................... 10
Warnings and precautions ....................................................... 10
Sample collection, transport, and storage ........................................ 10
■
CHAPTER 3 Guidelines for RNA extraction ..................................... 11
Sample input and elution volumes for RNA extraction .............................. 11
Negative control for RNA extraction .............................................. 11
■
CHAPTER 4 Prepare RT-PCR reactions ......................................... 12
Guidelines for RT-PCR .......................................................... 12
Optional RT-PCR controls ....................................................... 12
TE buer ...................................................................... 13
Prepare the RT‑PCR reactions (200‑µL sample input volume) ........................ 13
Prepare the RT‑PCR reactions (400‑µL sample input volume) ........................ 15
■
CHAPTER 5 Set up and run the real-time RT-PCR ............................. 18
Dye calibration ................................................................. 18
Example RT-PCR setup and run instructions ...................................... 18
Analyze data .................................................................. 20
Interpretation of the results ...................................................... 21
CoviPath
™
COVID-19 RT-PCR Kit User Guide
3
Contents
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■
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CHAPTER 6 Performance characteristics ....................................... 22
Limit of detection (LoD) ......................................................... 22
Reactivity (Inclusivity) ........................................................... 23
Interfering substances .......................................................... 23
Clinical sensitivity and specificity ................................................. 25
APPENDIX A Safety ............................................................... 26
Chemical safety ................................................................ 26
Biological hazard safety ......................................................... 27
APPENDIX B Documentation and support ...................................... 28
Customer and technical support ................................................. 28
Limited product warranty ........................................................ 28
4
CoviPath™ COVID-19 RT-PCR Kit User Guide
1
Intended Use ......................................................................... 5
■
Product description .................................................................... 5
■
Contents and storage .................................................................. 6
■
Required materials not supplied ......................................................... 6
■
General laboratory recommendations .................................................... 7
■
Assay limitations ...................................................................... 8
■
Samples and controls .................................................................. 9
■
Workflow ............................................................................. 9
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Intended Use
The CoviPath™ COVID-19 RT-PCR Kit contains the reagents and controls for a real-time reverse
transcription polymerase chain reaction (RT-PCR) test intended for the qualitative detection of nucleic
acid from SARS-CoV-2 in nasopharyngeal swab, nasopharyngeal aspirate, and bronchoalveolar lavage
(BAL) specimens from individuals suspected of COVID-19.
Product information
The CoviPath™ COVID-19 RT-PCR Kit is intended to be used as part of a customer defined and
validated workflow that generates a reportable result for detection of SARS-CoV-2. The customer’s
workflow will need to include a sample preparation method, a choice of a real time PCR instrument and
analysis software, as well as the CoviPath™ COVID-19 RT-PCR Kit.
Product description
CoviPath™ COVID-19 RT-PCR Kit (200 reactions) includes the following components:
•
CoviPath™ COVID‑19 Assay Multiplex—Multiplexed assay that contains the following:
–
Two primer/probe sets specific to dierent SARS-CoV-2 genomic regions (ORF1ab and N
gene)
–
One primer/probe set targeting the RNase P gene. The RNase P gene serves as an internal
positive control to monitor the sample source.
•
CoviPath™ COVID‑19 Control—RNA control that contains targets specific to the SARS-CoV-2
genomic regions targeted by the assay.
•
CoviPath™ 1‑Step Multiplex Master Mix (No ROX™)
CoviPath™ COVID-19 RT-PCR Kit User Guide
5
Chapter 1 Product information
1
Contents and storage
Contents and storage
Table 1 CoviPath™ COVID-19 RT-PCR Kit, 200 reactions (Cat. No. A50780)
Component Quantity
CoviPath™ COVID‑19 Assay
Multiplex (ORF1ab, N gene,
RNase P gene)
CoviPath™ COVID‑19 Control 2 tubes
CoviPath™ 1‑Step Multiplex
Master Mix (No ROX™)
[1]
The shelf life of the kit is determined by the component with the shortest shelf life.
2 tubes 150 µL 100099244
2 tubes 625 µL 100099246
Volume per
tube
10 µL
(2 x 103 copies
/ μL)
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
Unit part
number
100099245
Storage Shelf life
–30°C to
–20°C
–30°C to
–20°C
–30°C to
–20°C
Source
6 months
6 months
12 months
[1]
Equipment
Applied Biosystems™ real-time PCR instrument, calibrated for FAM™ dye,
VIC™ dye, and JUN™ dye
Other real-time PCR instrument with the following filter wavelengths:
•
Excitation of 494 nm ± 5 nm and emission of 518 ± 5 nm (for FAM™ dye)
•
Excitation of 538 nm ± 5 nm and emission of 554 ± 5 nm (for VIC™ dye)
•
Excitation of 580 nm ± 5 nm and emission of 605 ± 5 nm (for JUN™ dye)
Laboratory freezer (–30°C to –10°C) MLS
Centrifuge, with a rotor for microplates MLS
Microcentrifuge MLS
Laboratory mixer, vortex or equivalent MLS
Single and multichannel adjustable pipettors (1.00 µL to 1,000.0 µL) MLS
Cold block (96‑well) or ice MLS
Kits and reagents
Nuclease-free Water (not DEPC-Treated) AM9937
Contact your local sales oce for
Applied Biosystems™ real-time PCR
instruments
6
CoviPath™ COVID-19 RT-PCR Kit User Guide
Chapter 1
General laboratory recommendations
Product information
(continued)
Item Source
TE Buer (RNase-free) 12090015
1
1 M Tris, pH 8.0 and 0.5 M EDTA, pH 8.0 (to prepare TE Buer, if it is not
purchased)
Tubes, plates, and other consumables
Optical 96-well reaction plate and adhesive film compatible with your realtime PCR instrument and block. The following plates and film are compatible
with QuantStudio™ 5 Real‑Time PCR Instrument with a 96-well, 0.2-mL
block:
•
MicroAmp™ Optical 96‑Well Reaction Plate with Barcode, 0.2 mL, or
equivalent
•
MicroAmp™ Optical 96-Well Reaction Plate, 0.2 mL, or equivalent
•
MicroAmp™ Optical Adhesive Film, or equivalent
Nonstick, RNase-free microcentrifuge tubes (1.5 mL and 2.0 mL) thermofisher.com/plastics
Sterile aerosol barrier (filtered) pipette tips thermofisher.com/pipettetips
Optional controls
TaqMan™ Control Genomic DNA 4312660
Universal Human Reference RNA QS0639
N8010560, 4316813
MLS
4306737, 4326659
4311971, 4360954
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 fresh 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 for signs of inadequate vortexing or centrifugation.
CoviPath™ COVID-19 RT-PCR Kit User Guide
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1
Chapter 1
Assay limitations
Product information
Assay limitations
•
This assay is intended to be used for in vitro diagnostic purposes only. Follow good laboratory
practices and all precautions and guidelines in these user guides to avoid cross-contamination
between samples.
•
The CoviPath™ COVID-19 RT-PCR Kit performance was established using nasopharyngeal
and oropharyngeal swab, nasopharyngeal aspirate, and bronchoalveolar lavage samples only.
Nasal swabs and mid-turbinate swabs are considered acceptable specimen types according to
the Interim Guidelines for Collecting, Handling, and Testing Clinical Specimens from Persons
for Coronavirus Disease 2019 (COVID-19) published by the Centers for Disease Control and
Prevention, but performance of the CoviPath™ COVID-19 RT-PCR Kit with these specimen types
has not been established. Testing of nasal and mid-turbinate nasal swabs (self-collected under
supervision of or collected by a healthcare provider) is limited to patients with symptoms of
COVID-19. Specimen types other than nasopharyngeal, oropharyngeal, nasal and mid-turbinate
nasal swabs, nasopharyngeal aspirate and bronchoalveolar lavage should not be tested with this
assay.
•
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.
•
Refer to the Interim Guidelines for Collecting, Handling, and Testing Clinical Specimens from
Persons for Coronavirus Disease 2019 (COVID-19), published by the Centers for Disease Control
and Prevention, for specimen collection and storage guidelines.
•
Specimens must be packaged, shipped, and transported according to the current edition of
the International Air Transport Association (IATA) Dangerous Goods Regulations (iata.org/en/
programs/cargo/dgr).
•
This kit uses purified RNA as a sample for the analysis. The quality of the RNA recovered from
biological samples is essential for the quality of the results generated with this kit.
•
False-negative results may arise from:
–
Improper sample collection
–
Degradation of the viral RNA during shipping/storage
–
Specimen collection after nucleic acid can no longer be found in the specimen matrix
–
Using poor extraction method
–
The presence of RT-PCR inhibitors
–
Mutation in the SARS-CoV-2 virus
–
Failure to follow 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
•
The impacts of vaccines, antiviral therapeutics, antibiotics, chemotherapeutic or
immunosuppressant drugs have not been evaluated. The CoviPath™ COVID-19 RT-PCR Kit cannot
rule out diseases caused by other bacterial or viral pathogens.
8
CoviPath™ COVID-19 RT-PCR Kit User Guide
•
Negative results do not preclude infection with SARS-CoV-2 virus, and should not be the sole basis
of a patient management decision.
•
Laboratories may be required to report all positive results to the appropriate Competent Health
Authorities.
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.
Include the following controls:
Control Used to monitor
Positive Control (CoviPath™ COVID‑19 Control) RT-PCR reaction setup and reagent integrity
Chapter 1 Product information
Samples and controls
1
Negative Control
Workflow
The workflow begins with nucleic acid purified from upper respiratory specimens and BAL specimens,
which is reverse transcribed into cDNA and amplified using a real-time PCR instrument with appropriate
filter wavelengths (see “Required materials not supplied” on page 6). In the process, probes anneal to
two target sequences that are specific to SARS-CoV-2 (ORF1ab and N gene), and one target sequence
that is specific to RNase P.
During the extension phase of the PCR cycle, the 5’ nuclease activity of Taq polymerase degrades the
probe. This degradation causes the reporter dye to separate from the quencher dye, which generates a
fluorescent signal. With each cycle, additional reporter dye molecules are cleaved from their respective
probes, which increases the fluorescence intensity. Fluorescence intensity is monitored at each PCR
cycle by the real-time PCR instrument.
The data are analyzed and interpreted using the analytical software associated with your real-time PCR
instrument.
Cross-contamination during RNA extraction and reaction
setup
CoviPath™ COVID-19 RT-PCR Kit User Guide
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