QIAGEN SARS-CoV-2 User Manual
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45114
QIAGEN GmbH
QIAGEN Strasse 1,
R1
April 2021
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SARS-CoV-2 Prep&Amp
UM Kit Instructions for Use
(Handbook)
®
768
3072
Version 1
For In Vitro Diagnostic Use Only. Validation of this test has not been reviewed
by the FDA. Review under the EUA program is pending. The test is distributed in
accordance with the guidance on Policy for Coronavirus Disease-2019 Tests
During the Public Health Emergency, Section IV.C.2.
℞
For Prescription Use Only
For use on Rotor-Gene
®
Q MDx and ABI® 7500 Fast Dx instruments
40, 4511449
40724 Hilden, GERMANY
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SARS-CoV-2 Prep&Amp UM Kit Instructions for Use (Handbook) 04/2021
Contents
Intended Use .............................................................................................................. 4
Description and Principle ............................................................................................. 5
Pathogen information ........................................................................................ 5
Summary and explanation ................................................................................. 6
Materials Provided ...................................................................................................... 9
Kit contents ...................................................................................................... 9
Kit components .............................................................................................. 10
Platforms and software .................................................................................... 11
Materials Required but Not Provided .......................................................................... 12
Consumables ................................................................................................. 12
Equipment ..................................................................................................... 12
Warnings and Precautions ......................................................................................... 13
Safety information .......................................................................................... 13
Precautions .................................................................................................... 14
Reagent Storage and Handling .................................................................................. 15
Specimen Transport, Storage and Handling ................................................................. 15
Specimen collection, transport and storage ........................................................ 15
Protocol: Sample preparation and SARS-CoV-2 detection on the RGQ MDx .................... 16
Protocol: Sample Preparation and SARS-CoV-2 Detection on ABI 7500 Fast Dx ............... 21
Results ..................................................................................................................... 25
Interpretation of Results .............................................................................................. 27
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SARS-CoV-2 Prep&Amp UM Kit Instructions for Use (Handbook) 04/2021
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Limitations ................................................................................................................ 29
Performance ............................................................................................................. 30
Analytical sensitivity (Limit of detection) ............................................................. 30
Analytical specificity studies (Inclusivity and exclusivity/cross-reactivity) ................ 30
Interfering substances ...................................................................................... 36
Precision ....................................................................................................... 37
Clinical performance ...................................................................................... 38
References ............................................................................................................... 40
Troubleshooting Guide .............................................................................................. 41
Symbols ................................................................................................................... 43
Contact Information .................................................................................................. 44
Ordering Information ................................................................................................ 45
Document Revision History ......................................................................................... 46
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SARS-CoV-2 Prep&Amp UM Kit Instructions for Use (Handbook) 04/2021
Intended Use
The
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SARS-CoV-2 Prep&Amp UM Kit is a real-time RT-PCR test intended for the qualitative
detection of nucleic acid from the SARS-CoV-2 in nasopharyngeal swabs (NPS), nasal swabs,
and oropharyngeal swabs from individuals with signs and symptoms of infection who are
suspected of COVID-19. Testing is limited to laboratories certified under the Clinical
Laboratory Improvement Amendments of 1988 (CLIA), 42 U.S.C. §263a, to perform high
complexity tests or by similarly qualified non-U.S. laboratories.
Results are for the identification of SARS-CoV-2 RNA. The SARS-CoV-2 RNA is generally
detectable in NPS, nasal swabs, and oropharyngeal swabs during the acute phase of
infection. Positive results are indicative of active infection. Laboratories within the United States
and its territories are required to report all positive results to the appropriate public health
authorities.
Negative results do not preclude SARS-CoV-2 infection and should not be used as the sole
basis for patient management decisions. Negative results must be combined with clinical
observations, patient history, and epidemiological information.
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The
personnel specifically instructed and trained in the techniques of real-time PCR and
diagnostic procedures. Validation of this test has not been reviewed by the FDA. Review under
the EUA program is pending. The test is distributed in accordance with the guidance on Policy
for Coronavirus Disease-2019 Tests During the Public Health Emergency, Section IV.C.2.
The
MDx System or the ABI 7500 Fast Dx as RT-PCR instruments.
SARS-CoV-2 Prep&Amp UM Kit is intended for use by trained clinical laboratory
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SARS-CoV-2 PCR assay test is intended to be used with the Rotor-Gene Q (RGQ)
in vitro
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SARS-CoV-2 Prep&Amp UM Kit Instructions for Use (Handbook) 04/2021
5
Description and Principle
Pathogen information
Coronaviruses, a genus in the family
RNA viruses that cause highly virulent disease in humans and domestic animals (1).
Coronaviruses are known to infect humans account for one-third of common cold infections
and are also a well-known cause of nosocomial upper respiratory infections in premature
infants (2).
A novel member of the coronavirus family caused an outbreak of respiratory disease in
Wuhan City in China (1, 3). First named novel coronavirus (2019-nCoV), SARS-CoV-2 differs
from the SARS-CoV (1, 3), which was responsible for the 2003 outbreak, and the MERS-CoV,
which has been circulating in the Middle East since 2012. SARS-CoV-2 is the causative agent
of COVID-19. The SARS-CoV-2 RNA is detectable during the early and acute phases of the
infection from various upper respiratory tract specimens (nasal, oropharyngeal, and
nasopharyngeal swabs) (3).
The SARS-CoV-2 Prep&Amp UM assay targets 2 viral genes (N1 and N2 genes) detected with
the same fluorescence channel. The two gene targets are not differentiated, and amplification
of either or both gene targets leads to a fluorescence signal. Positive results are indicative of
the presence of the SARS-CoV-2 virus, but do not rule out co-infection with other pathogens.
On the other hand, negative RT-PCR results do not exclude a possible infection.
Coronaviridae
, are large enveloped, positive-stranded
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SARS-CoV-2 Prep&Amp UM Kit Instructions for Use (Handbook) 04/2021
Summary and explanation
The
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SARS-CoV-2 Prep&Amp UM Kit constitutes a ready-to-use system with a simple sample
preparation step followed by detection of the SARS-CoV-2 RNA using RT-PCR on either the
RGQ MDx system or on ABI 7500 Fast Dx platform (Figure 1). The SARS-CoV-2 UM Amp Buffer
contains reagents and enzymes for the specific amplification of a 72 base pair (bp) and a 67 bp
regions of the SARS-CoV-2 RNA genome and for their direct detection in the “Green” fluorescence
channel of the RGQ MDx instruments and with the fluorescent filter A/1 of the ABI 7500 Fast Dx.
The Primers and Probes mix of the
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SARS-CoV-2 Prep&Amp UM Kit also contains the
oligonucleotides required for the RNAse P amplifications. When detected in the “Yellow”
fluorescence channel of the RGQ MDx instrument or with the fluorescent filter B/2 of the ABI 7500
Fast Dx, those amplicons assure that enough biological sample has been collected on the swab.
This control is critical to ensure the presence of biological samples in SARS-CoV-2 negative samples.
An amplification should always be detectable; otherwise, it questions the sample quality.
The
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SARS-CoV-2 Prep&Amp UM Kit also contains a third heterologous amplification system
to reveal possible RT-PCR inhibition. This is detected as an internal RNA control (IC) in the “Red”
fluorescence channel of the RGQ MDx instruments and with the fluorescence filter E/5 of the
ABI 7500 Fast Dx. Because the IC is included in the SARS--CoV--2 Amp Primers Mix, its
amplification should be constant, unless an RT-PCR inhibitor is present in the sample or in the RT-PCR
reaction, which delays or prevents amplification.
External positive and negative controls (SARS-CoV-2 Positive Control and nuclease-free water used
as NTC, respectively) are supplied in the
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SARS-CoV-2 Prep&Amp UM Kit to attest of the
performance of the PCR step. A no extraction control (SARS-CoV-2 UM Prep Buffer used as NEC)
is strongly recommended to verify the absence of RT-PCR inhibitors in the preparation buffer.
Taken together, the efficiency of the reverse transcription and the PCR steps are monitored by these
controls.
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SARS-CoV-2 Prep&Amp UM Kit Instructions for Use (Handbook) 04/2021
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SARS-CoV-2 Prep&Amp UM Kit Workflow
(Continued on next page)
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SARS-CoV-2 Prep&Amp UM Kit Instructions for Use (Handbook) 04/2021
RGQ MDx
(Continued from previous page)
Figure 1.
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SARS-CoV-2 Prep&Amp UM Kit workflow.
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SARS-CoV-2 Prep&Amp UM Kit Instructions for Use (Handbook) 04/2021
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SARS-CoV-2 Prep&Amp UM Kit
Materials Provided
Kit contents
Catalog no.
Number of reactions
Tube color Lid Color Identity Tube ID Volume (µl) Volume (µl)
Clear
Clear
Clear
Clear
Clear Red SARS-CoV-2 Positive Control Positive Control 1 x 220
Clear
Clear
Yellow
Blue
Purple
Green
Clear
Clear
SARS-CoV-2 UM Prep Buffer Preparation Buffer 2 x 930
SARS-CoV-2 UM Amp Buffer
SARS-CoV-2 Amp Primers
SARS-CoV-2 Internal Control
Water for NTC Water (NTC) 1 x 1900
ROX Reference Dye ROX Dye 1 x 210
Master Mix
Primers and Probes
Internal Control (IC)
4511440
768
4 x 1440
4 x 1680
1 x 1390
4511449
3072
8 x 930
16 x 1440
16 x 1680
4 x 1390
4 x 220
4 x 1900
4 x 210
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SARS-CoV-2 Prep&Amp UM Kit Instructions for Use (Handbook) 04/2021
Kit components
Reagents
In each tube, the reagent volumes have been optimized for 8 batches of 96 samples (for the
768 reactions kit) or 32 batches of 96 reactions (for the 3072 reactions kit), including a
positive control (PC), a no template control (NTC), and a no extraction control (NEC).
Fewer or a greater number of samples may be run, but there will be sub-optimal reagent usage.
It is recommended to avoid multiple freeze−thaw cycles. Reagents may be aliquoted to avoid
multiple freeze−thaw cycles.
Primers and probes
Primers and probes targeting the SARS-CoV-2 sequences are based on the primers and probes
designed by the Centers for Disease Control and Prevention (CDC).
Controls and calibrators
The assay contains 5 controls to monitor the RT-PCR efficiency.
Internal control (IC): The internal control is a single-strand IVT RNA that verifies the presence of
contaminants that could inhibit the reverse transcription. The internal control also monitors the
reverse transcription efficiency in the no template control (NTC) and no extraction control (NEC).
No template control (NTC): The no template control is composed of nuclease-free water. It is
added to the PCR plate to verify introduction of contaminants during the PCR plate preparation
that could lead to misinterpretation of the SARS-CoV-2 targets.
Positive control (PC): The positive control is a double-strand DNA amplified with the
SARS-CoV-2 Primers and Probes (P&P mix). Its detection verifies the efficiency of the reagent
involved in the PCR amplification step.
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SARS-CoV-2 Prep&Amp UM Kit Instructions for Use (Handbook) 04/2021
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No extraction step (NEC): The no extraction control is composed of the SARS-CoV-2 UM Prep
Buffer. It is processed in parallel with the clinical samples to verify introduction of contaminants
during the sample preparation that could lead to misinterpretation of the SARS-CoV-2 targets.
Sampling Control: The Sampling Control detects the RNAse P gene and is critical to ensure
the presence of biological samples in SARS-CoV-2 negative samples. Amplification of the
sampling control should always be detectable; otherwise, it questions the sample quality.
Platforms and software
Prior to use, ensure that instruments have been maintained and calibrated according to the
manufacturer’s recommendations. This kit can be used in two workflows that require the use of the
Rotor-Gene Q MDx or of the ABI 7500 Fast Dx instruments and their appropriate software:
Rotor-Gene Q MDx: Rotor-Gene Q software version 2.3.1 or higher
ABI 7500 Fast Dx: SDS software version 1.4.1 or higher
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SARS-CoV-2 Prep&Amp UM Kit Instructions for Use (Handbook) 04/2021
Materials Required but Not Provided
Consumables
Disposable powder-free gloves
Sterile and nuclease-free pipette tips with filters
1.5 ml or 2 ml PCR-free tubes
0.1 ml PCR tubes for use with the Rotor-Gene Q MDx (Strip Tubes and Caps, 0.1 ml,
cat. no. 981103)
96-Well MicroAmp™ for use with the ABI 7500 Fast Dx qPCR platform
(Applied Biosystems 96-well plate, cat. no. N8010560)
MicroAmp Optical Adhesive film for use with the ABI 7500 Fast Dx qPCR platform
(Applied Biosystems, cat. no. 4360954)
Equipment*
Desktop centrifuge with rotor for 2 ml reaction tubes
Pipettes (adjustable)
Vortex mixer
Block heater
Rotor-Gene Q MDx (cat. no. 9002035 or 9002036) with Rotor-Gene Q software
version 2.3.1 or higher
Rotor-Disc 72 Rotor (cat. no. 9018899)
Rotor-Disc 72 Locking Ring (cat.no. 9018900)
72-well Loading Block (loading block 72 x 0.1 ml tubes, cat. no 9018901)
Alternatively: ABI 7500 Fast Dx qPCR platform (Thermo Fisher Scientific,
cat. no 4406985) with software version 1.4.1 or higher and a 96-well plate centrifuge
* Prior to use and when applicable, ensure that instruments have been checked and calibrated according to the
manufacturer’s recommendations.
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SARS-CoV-2 Prep&Amp UM Kit Instructions for Use (Handbook) 04/2021
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Warnings and Precautions
Please be aware that you may be required to consult your local regulations for reporting
serious incidents that have occurred to the device to the manufacturer and the regulatory
authority in which the user and/or the patient is established.
Safety information
When working with chemicals, always wear a suitable lab coat, disposable gloves, and
protective goggles. For more information, please consult the appropriate safety data sheets
(SDSs). These are available online in convenient and compact PDF format at
www.qiagen.com/safety, where you can find, view, and print the SDS for each QIAGEN kit
and kit component.
Always wear appropriate personal protective equipment, including but not limited to
disposable powder-free gloves, a lab coat, and protective eyewear. Protect skin, eyes, and
mucus membranes. Change gloves often when handling samples.
All samples should be treated as potentially hazardous. Always observe safety precautions as
outlined in relevant guidelines, such as the Clinical and Laboratory Standards Institute
Protection of Laboratory Workers from Occupationally Acquired Infections; Approved
Guideline
(M29), or other appropriate documents.
Specimens and samples are potentially infectious. Discard sample and assay waste
according to your local safety procedures.
®
(CLSI)
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SARS-CoV-2 Prep&Amp UM Kit Instructions for Use (Handbook) 04/2021
Precautions
Observe standard laboratory procedures for keeping the working area clean and
contamination-free. Dedicate an area with specific equipment to manipulate RNA.
Follow good laboratory practices to minimize cross-contamination.
Pay attention to avoid contamination with RNAse during the experiment and use
RNAse-free plasticware.
Make sure to have a good traceability with records, especially for sample identification.
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SARS-CoV-2 Prep&Amp UM Kit Instructions for Use (Handbook) 04/2021
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Reagent Storage and Handling
Attention should be paid to expiration dates and storage conditions printed on the box and
all components’ labels. Do not use expired or incorrectly stored components.
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The
until expiry date.
SARS-CoV-2 Prep&Amp UM Kit can be kept at −30°C to −15°C for 6 months, or
Specimen Transport, Storage and Handling
The
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SARS-CoV-2 Prep&Amp UM Kit is for use with nasopharyngeal, nasal, and
oropharyngeal swabs. All samples should be treated as potentially hazardous.
The Centers for Disease Control and Prevention (CDC) and Public Health England have
provided guidelines for sample collection, handling, and testing clinical specimens. Refer to
these guidelines or to other relevant national reference laboratory protocols for additional
information.
Specimen collection, transport and storage
For swab specimen collection, storage, and transport, please refer to the supplier’s
recommendations. Swabs must be fully immersed in transport media to maintain specimen
integrity.
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