Post on 02-Oct-2021
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For prescription use only. For in vitro diagnostic use only.
This manual is proprietary to PerkinElmer, Inc., and intended only for customer use in connection with the product(s) described herein and for no other purpose. This document and its contents shall not be used or distributed for any other purpose without the prior written consent of PerkinElmer. Follow the protocol included with the kit.
Instructions for PKamp™ Respiratory SARS-CoV-2 RT-PCR Panel
V 3.1
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Key to symbols used
European conformity
In vitro diagnostic medical device
Store at -25℃ to -15℃
Consult instructions for use
This way up
Recyclable
Contains sufficient for (n) test
Catalogue number
Lot number
Manufacturer
Use by date
Fragile
Authorized Representative in the European
Community
Date of manufacture
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Table of Contents Key to symbols used ....................................................................................................................... 2
Product Name .................................................................................................................................. 4
Kit Contents ..................................................................................................................................... 4
Intended Use .................................................................................................................................... 4
Principles of the Assay.................................................................................................................... 5
Kit Components and Packaging Specifications ............................................................................. 5
Materials Required but Not Provided .............................................................................................. 6
Storage & Handing Requirements .................................................................................................. 7
Warnings and Precautions .............................................................................................................. 7
Collection, Storage & Shipment of Specimens .............................................................................. 8
Assay Procedure ............................................................................................................................. 9
Nucleic Acid Extraction and PCR Setup .................................................................................... 9
Extraction on chemagic 360 .......................................................................................................................... 9
Setup PCR Manually for 30μL PCR Reactions on 96-Well Instrument with FAM™, HEX™/VIC™, ROX™,
Cy®5 and Cy5.5® Channels .........................................................................................................................11
Setup PCR Manually for 30μL PCR Reactions on 96-Well Instrument with FAM™, HEX™/VIC™, ROX™
and Cy®5 Channels......................................................................................................................................12
Setup PCR Manually for 15μL PCR Reactions in 384-well Plate on Instrument with FAM™,
HEX™/VIC™,ROX™, Cy®5 and Cy5.5® Channels .....................................................................................13
Setup PCR Manually for 15μL PCR Reactions in 384-well Plate on Instrument with FAM™,
HEX™/VIC™,ROX™ and Cy®5 Channels .................................................................................................14
Amplification............................................................................................................................. 14
Interpretation of Results ................................................................................................................ 15
Kit Limitations ................................................................................................................................ 21
Assay Performance ....................................................................................................................... 22
Limit of Detection (LoD) ........................................................................................................... 22
Co-infection Sensitivity ............................................................................................................ 24
Analytical Reactivity (Inclusivity) ............................................................................................. 25
Analytical Specificity (Cross-reactivity) .................................................................................. 27
Interfering Substances Study .................................................................................................. 32
Equivalency between negative clinical matrix and viral transport medium plus human cells ...................................................................................................................................................34
Precision (Repeatability) .......................................................................................................... 34
Appendix ........................................................................................................................................ 38
Applied Biosystems QuantStudio Dx Calibration ................................................................... 38
References ..................................................................................................................................... 41
Basic Information .......................................................................................................................... 42
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Product Name
PKamp™ Respiratory SARS-CoV-2 RT-PCR Panel
Kit Contents
96 Tests or 384 Tests
Intended Use
The PKamp Respiratory SARS-CoV-2 RT-PCR Panel assay is a real-time RT-PCR
multiplexed test intended for the simultaneous qualitative detection and
differentiation of RNA from SARS-CoV-2, influenza A viruses, influenza B viruses
and respiratory syncytial virus (RSV) or simultaneous qualitative detection and
differentiation of RNA from SARS-CoV-2, influenza A viruses and influenza B
viruses or detection of SARS-CoV-2 RNA only in upper respiratory specimens
(nasopharyngeal, oropharyngeal and nasal swabs) collected from individuals
suspected of respiratory viral infection consistent with COVID-19 by a healthcare
provider. The PKamp Respiratory SARS-CoV-2 RT-PCR Panel assay is not
intended to detect RNA from influenza C viruses.
The qualitative detection is based on the following fluorescence detections: SARS-
CoV-2 (FAM), Influenza A viruses (ROX), Influenza B viruses (Cy5), respiratory
syncytial virus (Cy5.5), human endogenous internal control gene RNase P
(HEX/VIC). The use of the product will be defined by the available fluorescence
detectors of the specific instrument. For the instruments without Cy5.5 fluorescence
detectors, the product is not intended to detect respiratory syncytial virus RNA and
the results of respiratory syncytial virus will not be reported.
Positive results are indicative of the presence of the corresponding viral RNA but
do not rule out infection or co-infection with other microorganisms. Clinical
correlation with patient history and other diagnostic information is necessary to
determine patient infection status. The viral RNA detected may not be the definite
cause of disease.
Negative Respiratory SARS-CoV-2 RT-PCR Panel assay results do not preclude
SARS-CoV-2, influenza A viruses, influenza B viruses and respiratory syncytial
virus infection and should not be used as the sole basis for diagnosis, treatment or
other patient management decisions. Negative results interpretation must be
combined with clinical observations, patient history and epidemiological
information.
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Negative results obtained from individuals who are not exhibiting clinical signs and
symptoms associated with respiratory viral infection at the time of specimen
collections should be interpreted with caution. Negative results in asymptomatic
individuals cannot be used as definitive evidence that an individual has not been
exposed to or infected with SARS-CoV-2, influenza viruses or respiratory syncytial
virus.
Testing with the PKamp Respiratory SARS-CoV-2 RT-PCR Panel assay is intended
for use by trained laboratory personnel who are proficient in performing real-time
RT-PCR assays.
Principles of the Assay
The PKamp Respiratory SARS-CoV-2 RT-PCR Panel assay is a real-time reverse
transcription polymerase chain reaction (rRT-PCR) test. The SARS-CoV-2,
Influenza A viruses, Influenza B viruses and RSV primer and probe sets are
designed to detect RNA from these viruses in upper respiratory tract
(nasopharyngeal or oropharyngeal swabs) from patients suspected of COVID-19
by their healthcare provider.
The oligonucleotide primers and probes for detection of SARS-CoV-2 were
selected from the regions of the virus nucleocapsid (N) gene and ORF1ab gene.
The primers and probes for detection of influenza A viruses and RSV were selected
from the regions of matrix protein. The primers and probes for detection of influenza
B viruses were selected from the regions of nuclear export protein (NEP) and
nonstructural protein 1 (NS1) genes. An additional primer/probe set to detect the
endogenous control gene RNase P is also included in the test. It is used for
monitoring sample collection and extraction efficiency.
The TaqMan probes for the amplicons of SARS-CoV-2, Influenza A, Influenza B,
RSV and RNase P are labeled with FAM, ROX, Cy5, Cy5.5 and HEX/VIC
fluorescent dyes respectively to generate target-specific signal.
The assay also uses a dUTP/UNG carryover prevention system to avoid
contamination of PCR products and subsequent false positive results.
Kit Components and Packaging Specifications
Catalog Number for 30 μL PCR reaction: SDX-56392 (96 tests per kit)
Component Name Specifications & Loading
Main Ingredients Storage Conditions
nCoV Reagent A 950 μL × 1 tube Buffers, dNTPs, Mg2+ -25 to -15°C
nCoV Resp Reagent B1 200 μL × 1 tube TE buffer, primers, probes -25 to -15°C
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nCoV Resp Reagent B2 40 μL × 1 tube TE buffer, primers, probes -25 to -15°C
nCoV Enzyme Mix 170 μL × 1 tube Taq DNA polymerase, MMLV,
RNasin, UNG -25 to -15°C
nCoV Resp Positive Control
1.4 mL
× 1 tube
SARS-CoV-2, Influenza A,
Influenza B, RSV and RNaseP
RNA fragments capsulated in
bacteriophage envelop
-25 to -15°C
nCoV Negative Control 1.4 mL × 2 tubes TE buffer -25 to -15°C
Note: nCoV Enzyme Mix contains 0.027% (v/v) Nonidet® P 40 (NP-40).
Catalog Number for 15 μL PCR reaction: SDX-56393 (384 tests per kit)
Component Name Specifications & Loading
Main Ingredients Storage Conditions
nCoV Reagent A 950 μL × 2 tubes Buffers, dNTPs, Mg2+ -25 to -15°C
nCoV Resp Reagent B1 200 μL × 2 tubes TE buffer, primers, probes -25 to -15°C
nCoV Resp Reagent B2 40 μL × 2 tubes TE buffer, primers, probes -25 to -15°C
nCoV Enzyme Mix 170 μL × 2 tubes Taq DNA polymerase, MMLV,
RNasin, UNG -25 to -15°C
nCoV Resp Positive Control
1.4 mL
× 4 tubes
SARS-CoV-2, Influenza A,
Influenza B, RSV and RNaseP
RNA fragments capsulated in
bacteriophage envelop
-25 to -15°C
nCoV Negative Control 1.4 mL × 2 tubes TE buffer -25 to -15°C
Note: nCoV Enzyme Mix contains 0.027% (v/v) Nonidet® P 40 (NP-40).
Materials Required but Not Provided
1. RNA extraction reagents and instrument
RNA extraction reagents, instrument and related software that have been
validated with the PKamp™ Respiratory SARS-CoV-2 RT-PCR Panel:
chemagic™ Viral DNA/RNA 300 Kit special H96 (CMG-1033 or CMG-1033-
S) and chemagic™ 360 (2024-0020) with chemagic™ Rod Head Set 96
(CMG-371) (chemagic™ MSM I software version 6.1.0.5)
2. Real-time RT-PCR:
Instruments with FAM™, HEX™/VIC™, ROX™, Cy®5 and Cy5.5® channels
(e.g. Applied BiosystemsTM 7500 Real-Time PCR System, Applied
BiosystemsTM 7500 Fast Dx Real-Time PCR System, Applied BiosystemsTM
QuantStudioTM Dx Real-Time Instruments, BioRad® CFX384TM, BioRad®
CFX96TM Touch Real-Time PCR Detection System, qTower3G Real-Time PCR
Detection System, qTower3G 84 Real-Time PCR Detection System)
3. Additional tools and consumables
a. Consumables for automated nucleic acid extraction using chemagic™ 360.
2 mL deep-well-plate (riplate SW) (chemagen, CMG-555)
Low-well-plate (chemagen, CMG-555-1)
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Magnetic rods disposable tips (chemagen, CMG-550)
Hanging Tip Racks (PerkinElmer, CMG-425)
b. Tools and consumables
Centrifuge (Eppendorf, Epp 5810/ 5810 R)
Vortex Mixer (VWR, 97043-562)
c. Micropipettors (range between 1-20 μL, 20-200 μL and 100-1000 μL)
d. Non-aerosol pipette tips
Storage & Handing Requirements
1. Store all reagents at -25 to -15°C.
2. Completely thaw reagents before use. Reagent A may precipitate upon
thawing. Mix reagent at room temperature until fully dissolved.
3. The reagents are stable for at least 18 days with 6 cycles of freeze-thaw after
opening.
4. The reagents are estimated to be stable at -25 to -15°C for at least 12 months.
Note: The shelf-life, in-use and freeze-thaw stability of the PKamp Respiratory SARS-CoV-
2 RT-PCR Panel kit is estimated based on data from accelerated stability study, in-use and
freeze-thaw stability study of a similar product PerkinElmer® SARS-CoV-2 Real-time RT-
PCR Assay.
Warnings and Precautions
General Precautions 1. Corresponding positive results are indicative of the presence of SARS-CoV-2
RNA, Influenza A virus RNA, Influenza B virus RNA and/or RSV RNA.
2. Laboratories within the European Union and its territories are required to report
all positive results to the appropriate public health authorities.
3. Specimens should be collected with appropriate infection control precautions.
4. Do not use reagents after their expiration dates.
5. Do not use the kit if the outer box sealing label is broken upon arrival.
6. Do not use reagents if the tube caps are open or broken upon arrival.
7. Follow the assay procedures indicated in this document to ensure optimal test
performance.
8. Dispose of waste according to local, state, and federal regulations.
9. The PCR instruments used for this assay should be calibrated regularly
according to instruments’ instructions to eliminate crosstalk between channels.
Safety Precautions 1. Wear appropriate Personal Protective Equipment (PPE), including (but not
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limited to) disposable powder-free gloves, hats, protective lab coats and goggles.
Change gloves often when handling reagents or samples.
2. Wash hands thoroughly after handling specimens and reagents.
3. Specimen for SARS-CoV-2 testing should be inactivated before use, by
incubating the specimen at 56℃ for 30 minutes.
4. Handle the Positive Control, all specimens and waste materials as if they could
transmit infectious agents in accordance with Universal Precautions.
5. Follow national biological safety recommendations for handling biological
samples.
6. Refer to the Clinical and Laboratory Standards Institute (CLSI) Protection of
Laboratory Workers from Occupationally Acquired Infections; Approved
Guideline (M29), for safety precautions.
7. Safety Data Sheets are available upon request.
Laboratory Precautions for Contamination Prevention 1. Do not handle samples in a biosafety cabinet which is used for other culturing or
testing purpose with SARS-CoV-2, influenza A viruses, influenza B viruses and
RSV.
2. Prior to processing samples, thoroughly clean the work area with freshly
prepared 10% bleach or 75% ethanol. Then wipe the work area with water.
3. Avoid excessive handling of the Positive Control to avoid contamination.
4. Change gloves after handling the Positive Control.
5. If spillage of the specimen and the Positive Control occurs, immediately disinfect
the area with freshly prepared 0.5% sodium hypochlorite (bleach) or follow
appropriate laboratory biosafety procedures.
6. After amplification is complete, immediately place the PCR tubes/plates in a
sealable bag, ensure the bag is sealed, then discard the tubes/plates in a
biohazard container.
7. Change gloves after handling after-run PCR tubes/plates.
8. All materials used in one area should remain in that area and should not be
moved or used in other areas. Never bring after-run PCR tubes/plates to other
areas (i.e. PCR set up area and sample preparation area).
Collection, Storage & Shipment of Specimens
1. Specimen Collection
Use only synthetic fiber swabs with plastic shafts. Do not use calcium alginate
swabs or swabs with wooden shafts, as they may contain substances that
inactivate some viruses and inhibit PCR testing. Place swabs immediately into
sterile tubes containing 3 ml of viral transport media. For initial testing,
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nasopharyngeal swab specimens are recommended. Collection of
oropharyngeal swabs or anterior nasal swabs is an alternative if
nasopharyngeal swab specimens are not available.
• Nasopharyngeal swab (NP): Insert a swab into nostril parallel to the
palate. Swab should reach depth equal to distance from nostrils to outer
opening of the ear. Leave swab in place for several seconds to absorb
secretions. Slowly remove swab while rotating it.
• Oropharyngeal swab (OP): Swab the posterior pharynx, avoiding the
tongue.
• Anterior Nasal Swab (NS): Using a flocked or spun polyester swab, insert
the swab at least 1 cm (0.5 inch) inside the nostril (naris) and firmly
sample the nasal membrane by rotating the swab and leaving in place
for 10 to 15 seconds. Sample both nostrils with same swab.
2. Storage
Store specimens at 2-8°C up to 72 hours after collection or for 48 hours if
shipped overnight on ice packs. If a delay in testing or shipping is expected,
store specimens at -70°C or below. Avoid repeated freeze-thaw of the
specimens and do not freeze-thaw the specimens for more than 2 cycles.
3. Shipping
Specimens PUI’s must be packaged, shipped, and transported according to the
current edition of the International Air Transport Association (IATA) Dangerous
Goods Regulation External Icon. Store specimens at 2-8°C and ship overnight
to the lab on ice pack. If a specimen is frozen at -70°C ship overnight to the lab
on dry ice.
Note: Sample storage and shipping conditions are recommended based on SARS-CoV-
2 sample stability study using the PerkinElmer® SARS-CoV-2 Real-time RT-PCR Assay.
It is also based on the guideline from US CDC (4,5).
Assay Procedure
Nucleic Acid Extraction and PCR Setup
Extraction on chemagic 360
Please follow chemagic 360 User Manual for extraction setup. A quick-start
instruction is described below.
1) 300 µL nCoV Resp Positive Control is required to be included in each extraction
run.
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2) Place specimens and nCoV Resp Positive Control in a biological safety cabinet.
If the specimen is frozen, completely thaw it at room temperature before use.
3) In a 2 mL deep-well-plate (riplate SW), add 300 µL Lysis buffer, 300 µL
specimen, 4 µL Poly (A) RNA and 10 µL Proteinase K to each well in a sequential
order.
Please note:
i. Dissolve lyophilized Poly(A) RNA by adding 440 μL of the Poly(A) RNA
Buffer to the Poly(A) RNA tube and mix thoroughly before use.
ii. Dissolve lyophilized Proteinase K in H2O before use (volume is given on
the label).
iii. An extraction master mix of Poly(A) and Proteinase K can be made for ease
of use in sample addition (14 µL of premix is added to each well).
4) In a low-well-plate, add 150 µL magnetic beads into each well.
5) In a new deep-well-plate (riplate SW), add 60 µL Elution Buffer 6 into each well;
6) Turn on the chemagic 360, double click the software icon “chemagic_360”, select
username and enter password to start. Follow the chemagic 360 User Manual to
select the appropriate protocol.
7) Load the magnetic rods disposable tips box onto the tracking system (table)
according to the instructions given by the chemagic software, the tip rack should
be in the position indicated in the table below.
8) Load the plates manually onto the tracking system (table) according to the
instructions given by the chemagic software. The plates should be in the
positions indicated in the table below.
Please note:
i. Specimens and Magnetic Beads should be thoroughly vortex mixed before
use.
ii. Never move the tracking system (table) manually. All movements must be
performed with the [Turn Table] function in the instrument software.
chemagic 360 layout:
Position 1 Magnetic rods disposable tips
Position 2 Low-well-plate (MICROTITER SYSTEM) prefilled
with 150 μL Magnetic Beads
Position 3 Deep-well-plate (riplate SW) containing:
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300 μL Lysis Buffer 1
300 μL specimen
4 μL Poly(A) RNA
10 μL Proteinase K
• Binding Buffer 2 (added automatically)
Position 4 Empty deep-well-plate (riplate SW) [Wash Buffer 3
added automatically]
Position 5 Empty deep-well-plate (riplate SW) [Wash Buffer 4
added automatically]
Position 6 Empty deep-well-plate (riplate SW) [Wash Buffer 5
added automatically]
Position 7 Deep-well-plate (riplate SW) prefilled with 60 μL
Elution Buffer 6
9) Double check the positions and directions of all consumables according to the
tracking system.
10) Click “Start” to start the extraction process.
11) Proceed to downstream assay with the extracted nucleic acids or store the
nucleic acids at -25°C to -15°C.
Setup PCR Manually for 30μL PCR Reactions on 96-Well Instrument with FAM™, HEX™/VIC™, ROX™, Cy®5 and Cy5.5® Channels
Note: In order to ensure the performance of multiplex real-time PCR, it requires:
• Prepare PCR mix always when the sample extraction close to finish or
finished
• After setting up PCR, start PCR run immediately on the instrument.
Setup PCR manually according the procedures described below after nucleic acid
extraction using chemagic 360.
1) Prepare PCR mix in Reagent Preparation Area according to the following table.
It is recommended to prepare 110% of the calculated amount of PCR mix to
account for pipetting carryovers.
Note: Reagent A may precipitate. Keep it at room temperature and mix well to
ensure complete resuspension.
Component Volume/
test
Volume for N Samples and
Positive Control 110% of volume
nCoV Reagent A 7.5 μL 7.5 x (n + 1) μL 8.25 x (n + 1) μL
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nCoV Resp Reagent B1 1.25 μL 1.25 x (n + 1) μL 1.375 x (n + 1) μL
NCoV Resp Reagent B2 0.25 μL 0.25 x (n + 1) μL 0.275 x (n + 1) μL
nCoV Enzyme mix 1 μL 1 x (n + 1) μL 1.1 x (n + 1) μL
2) Vortex the prepared PCR mix to ensure it is fully mixed, then centrifuge briefly
to collect in the bottom of the tube.
3) Pipette 10 μL into each well of a 96-well PCR plate.
4) Add 20 μL of extracted nucleic acid (including Positive Control) into each well
containing PCR mix.
5) Add 20 μL of nCoV Negative Control into the remaining well containing PCR
mix.
6) Seal the PCR plate with an appropriate film.
7) Vortex plate for 10-20 seconds, and centrifuge for 5 minutes at 350x g.
Setup PCR Manually for 30μL PCR Reactions Reactions on 96-Well Instrument with FAM™, HEX™/VIC™, ROX™ and Cy®5 Channels
Note: In order to ensure the performance of multiplex real-time PCR, it requires:
• Prepare PCR mix always when the sample extraction close to finish or
finished
• After setting up PCR, start PCR run immediately on the instrument.
Setup PCR manually according the procedures described below after nucleic acid
extraction using chemagic 360.
1) Prepare PCR mix in Reagent Preparation Area according to the following table.
It is recommended to prepare 110% of the calculated amount of PCR mix to
account for pipetting carryovers.
Note: Reagent A may precipitate. Keep it at room temperature and mix well to
ensure complete resuspension.
Component Volume/
test
Volume for N Samples and
Positive Control 110% of volume
nCoV Reagent A 7.5 μL 7.5 x (n + 1) μL 8.25 x (n + 1) μL
nCoV Resp Reagent B1 1.25 μL 1.25 x (n + 1) μL 1.375 x (n + 1) μL
TE Buffer 0.25 μL 0.25 x (n + 1) μL 0.275 x (n + 1) μL
nCoV Enzyme mix 1 μL 1 x (n + 1) μL 1.1 x (n + 1) μL
Note: nCoV negative control is TE buffer, can be used for PCR mix preparation.
2) Vortex the prepared PCR mix to ensure it is fully mixed, then centrifuge briefly
to collect in the bottom of the tube.
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3) Pipette 10 μL into each well of a 96-well PCR plate.
4) Add 20 μL of extracted nucleic acid (including Positive Control) into each well
containing PCR mix.
5) Add 20 μL of nCoV Negative Control into the remaining well containing PCR
mix.
6) Seal the PCR plate with an appropriate film.
7) Vortex plate for 10-20 seconds, and centrifuge for 5 minutes at 350x g.
Setup PCR Manually for 15μL PCR Reactions in 384-well Plate on Instrument with FAM™, HEX™/VIC™, ROX™, Cy®5 and Cy5.5® Channels
Note: In order to ensure the performance of multiplex real-time PCR, it requires:
• Prepare PCR mix always when the sample extraction close to finish or
finished
• After setting up PCR, start PCR run immediately on the instrument.
Setup PCR manually according the procedures described below after nucleic acid
extraction using chemagic 360.
1) Prepare PCR mix in Reagent Preparation Area according to the following table.
It is recommended to prepare 110% of the calculated amount of PCR mix to
account for pipetting carryovers.
Note: Reagent A may precipitate. Keep it at room temperature and mix well to
ensure complete resuspension.
Component Volume/ test Volume for N Samples
and Positive Control 110% of volume
nCoV Reagent A 3.75 μL 3.75 x (n + 1) μL 4.125 x (n + 1) μL
nCoV Resp Reagent B1 0.625 μL 0.625 x (n + 1) μL 0.6875 x (n + 1) μL
NCoV Resp Reagent B2 0.125 μL 0.125 x (n + 1) μL 0.1375 x (n + 1) μL
nCoV Enzyme mix 0.5 μL 0.5 x (n + 1) μL 0.55 x (n + 1) μL
2) Vortex the prepared PCR mix to ensure it is fully mixed, then centrifuge briefly
to collect in the bottom of the tube.
3) Pipette 5 μL into each well of a 384-well PCR plate.
4) Add 10 μL of extracted nucleic acid (including Positive Control) into each well
containing PCR mix.
5) Add 10 μL of nCoV Negative Control into the remaining well containing PCR
mix.
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6) Seal PCR the plate with an appropriate film.
7) Vortex plate for 10-20 seconds, and centrifuge for 5 minutes at 350x g.
Setup PCR Manually for 15μL PCR Reactions on in 384-well Plate on Instrument with FAM™, HEX™/VIC™, ROX™, and Cy®5 Channels
Note: In order to ensure the performance of multiplex real-time PCR, it requires:
• Prepare PCR mix always when the sample extraction close to finish or
finished
• After setting up PCR, start PCR run immediately on the instrument.
Setup PCR manually according the procedures described below after nucleic acid
extraction using chemagic 360.
1) Prepare PCR mix in Reagent Preparation Area according to the following table.
It is recommended to prepare 110% of the calculated amount of PCR mix to
account for pipetting carryovers.
Note: Reagent A may precipitate. Keep it at room temperature and mix well to
ensure complete resuspension.
Component Volume/ test Volume for N Samples
and Positive Control 110% of volume
nCoV Reagent A 3.75 μL 3.75 x (n + 1) μL 4.125 x (n + 1) μL
nCoV Resp Reagent B1 0.625 μL 0.625 x (n + 1) μL 0.6875 x (n + 1) μL
TE Buffer 0.125 μL 0.125 x (n + 1) μL 0.1375 x (n + 1) μL
nCoV Enzyme mix 0.5 μL 0.5 x (n + 1) μL 0.55 x (n + 1) μL
Note: nCoV negative control is TE buffer, can be used for PCR mix preparation.
2) Vortex the prepared PCR mix to ensure it is fully mixed, then centrifuge briefly
to collect in the bottom of the tube.
3) Pipette 5 μL into each well of a 384-well PCR plate.
4) Add 10 μL of extracted nucleic acid (including Positive Control) into each well
containing PCR mix.
5) Add 10 μL of nCoV Negative Control into the remaining well containing PCR
mix.
6) Seal the PCR plate with an appropriate film.
7) Vortex plate for 10-20 seconds, and centrifuge for 5 minutes at 350x g.
Amplification
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1) Set up and run the PCR instrument according to the instrument reference guide.
2) Set the thermal cycling condition as below.
Step Temperature Time Number of Cycles
1 37°C 2 minutes 1
2 50°C 15 minutes 1
3 94°C 10 minutes 1
4
94°C 10 seconds 45a
55°C 15 seconds
65°C* 45 seconds
* Collect fluorescence signal during the final 65°C step. a. 45 cycles are used for validation study. Based on Ct cutoff, 40 or 42 cycles can be
applied based on instrument Ct cutoff
3) Make sure the reaction volume setting is correct (30μL or 15uL).
4) Fluorophore settings
For instruments with FAM™, HEX™/VIC™, ROX™, Cy®5 and Cy5.5®
Channels, select the fluorophore setting as below.
Target Name or Detector Channel
SARS CoV-2 FAM
Influenza A ROX
Influenza B Cy5
RSV Cy5.5/Quasar 705
RNase P VIC
For instruments with FAM™, HEX™/VIC™, ROX™ and Cy®5 Channels,
select the fluorophore setting as below.
Target Name or Detector Channel
SARS CoV-2 FAM
Influenza A ROX
Influenza B Cy5
RNase P VIC
5) Double check all settings and start the run.
Note: If suppressing the results for a particular target is desired, do not select to
read the reporter of that target. If use ABI 7500 Fast instrument, fast mode is
applied.
Interpretation of Results
1. Baseline and threshold setting
After the run completion, save and analyze the data according to PCR
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instrument instructions.
1) Set baseline for each target
The horizontal part of the baseline is used for the baseline range, which
normally starts from 3-5 cycles and ends at 15-20 cycles. Baseline setting
is normally automatically done by instrument. Manual baseline 3-15 is
recommended as general.
2) Set threshold for each target
Thresholds should be adjusted to fall within exponential phase of the
fluorescence curves and above any background signal (refer to the
background signal of true negative samples). The threshold value for
different instruments varies due to different signal intensities. Two examples
from different instruments are given below.
3) Interpret the results based on the tables listed in “Quality Control” and
“Examination and Interpretation of Specimen Results”.
2. Quality Control
The provided Negative Control (TE buffer) and Positive Control monitor the
reliability of the results for the entire batch of specimens. All test controls should
be examined prior to interpretation of patient results. Positive Control and
Negative Control should meet the requirements listed in the below table to
ensure valid results. If the controls are not valid, the patient results cannot be
interpreted.
1) Negative Control: A “no template” (negative) control is needed to monitor
reagent and/or environment contamination. One PCR plate should include
one Negative Control. It is not included in the sample extraction process.
The Ct requirements are listed in the following table. If one of the targets
fails the Ct requirements, the negative control is invalid.
2) Positive Control: A positive control is needed to monitor the overall process
of extraction, reverse transcriptase and PCR amplification signals for each
target at different detection channel/color module. It contains RNA
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fragemnts of SARS-CoV-2, Influenza A viruses, Influenza B viruses, RSV
and RNase P capusulated with bacteriophage envelope.
It can be used to assist the threshold setup to differentiate the amplification
signal vs. instrument background noises or signal drift. The Ct requirements
are listed in the following table. If one of the targets fails the Ct
requirements, the positive control is invalid.
3) Endogenous control (internal control): The RNase P serves as an
endogenous internal control for the assay that is used together with the data
from other targets for interpretation of an individual specimen. RNase P
should be positive (<=35 Ct) for all clinical specimens in the absence of
signals for all the viral targets.
If RNase P is negative in the presence of a positive result for one or multiple
viral target(s), the viral target result should be considered valid.
However, if all viral targets generate negative results and RNase P is also
negative (> 35 Ct), the test is considered as invalid. Failure to detect RNase
P in clinical specimens could indicate:
• Insufficient nucleic acid extraction from clinical samples
• Poor specimen quality or loss of specimen integrity
• Improper assay execution
• Reagent or equipment malfunction
For specimens with invalid results, repeat testing of specimen nucleic acid
or re-extract and repeat RT-PCR. If all targets are negative after retest,
report the specimen as invalid. Collection of a new specimen and
subsequent testing should be considered.
Instrument with active FAM, HEX/VIC, ROX, Cy5 and Cy5.5/Quasar705
detection capability
Control
name
Ct
SARS-CoV-2
(FAM)
Influenza A
(ROX)
Influenza B
(Cy5)
RSV
(Cy5.5)
RNase P (endogenous control)
(HEX/VIC)
Negative
control
Undet/blank
or > 40
Undet/blank
or > 40
Undet/blank
or > 40
Undet/blank
or > 40
Undet/blank or > 35
Positive
control
≤ 35 ≤ 35 ≤ 35 ≤ 35 ≤ 35
Undet: Undetermined
Page 18 of 42
CFX 96 specifically
Control
name
Ct
SARS-CoV-2
(FAM)
Influenza A
(ROX)
Influenza B
(Cy5)
RSV
(Cy5.5)
RNase P (endogenous control)
(HEX/VIC)
Negative
control
Undet/blank
or > 40
Undet/blank
or > 40
Undet/blank
or > 42
Undet/blank
or > 42
Undet/blank or > 35
Positive
control
≤ 35 ≤ 35 ≤ 35 ≤ 35 ≤ 35
Undet: Undetermined
Note: CFX96 Cy5 threshold > 300 RFU
Instrument with active FAM, HEX/VIC, ROX and Cy5 detection capability
Control name Ct
SARS-CoV-2
(FAM)
Influenza A
(ROX)
Influenza B
(Cy5)
RNase P (endogenous control)
(HEX/VIC)
Negative control Undet/blank
or > 40
Undet/blank
or > 40
Undet/blank
or > 40
Undet/blank or > 35
Positive control ≤ 35 ≤ 35 ≤ 35 ≤ 35
Undet: Undetermined
The final report will be based on the fluorescence detectors selected in the
specific instrument.
3. Examination and Interpretation of Patient Specimen Results
Assessment of clinical specimen test results should be performed after the
positive and negative controls have been examined and confirmed to be valid
and acceptable. If the controls are not valid, the patient results cannot be
interpreted. However, if the whole PCR plate is with valid negative control and
valid internal control (RNAse P) for more than 95% clinical samples, only the
positive control is out of specification, the whole PCR plate is valid and patient
results can still be interpreted. The issue could be with positive control or
extraction, further investigation on positive control should be performed.
The table below lists the expected results for the assay with valid positive
control and negative control.
Ct cutoff for the instrument with FAM, HEX/VIC, ROX, Cy5 and
Cy5.5/Quasar705 detection capability
Sample
Ct Cutoff
SARS-CoV-2
(FAM)
Influenza A
(ROX)
Influenza B
(Cy5)
RSV
(Cy5.5/Quasa
r705)
RNase P (endogenous
control)
(HEX/VIC)
Clinical
sample
Positive ≤ 40
Positive ≤ 40
Positive ≤ 40
Positive ≤ 40
Positive ≤ 35
If anyone of the viral targets
is positive, there is no
Page 19 of 42
requirements on the Ct
value
Ct cutoff for CFX 96 specifically
Sample
Ct Cutoff
SARS-CoV-2
(FAM)
Influenza A
(ROX)
Influenza B
(Cy5)
RSV
(Cy5.5/Quasa
r705)
RNase P (endogenous
control)
(HEX/VIC)
Clinical
sample
Positive ≤ 40
Positive ≤ 40
Positive ≤ 42
Positive ≤ 42
Positive ≤ 35
If anyone of the viral targets
is positive, there is no
requirements on the Ct
value
Ct cutoff for the instrument with FAM, HEX/VIC, ROX and Cy5 detection
capability
Sample
Ct Cutoff
SARS-CoV-2
(FAM)
Influenza A
(ROX) Influenza B (Cy5)
RNase P (endogenous control)
(HEX/VIC)
Clinical
sample
Positive ≤ 40
Positive ≤ 40
Positive ≤ 40
Positive ≤ 35
If anyone of the viral targets is
positive, there is no requirements
on the Ct value
Result interpretation for the instrument with FAM, HEX/VIC, ROX, Cy5 and
Cy5.5 detection capability
Ct Result interpretation
IC
(VIC/HEX)
RNase P
SARS-
CoV-2
(FAM)
Influenza A
(ROX)
Influenza B
(Cy5)
RSV
(Cy 5.5/
Quasar705)
Detected
Not Detected
≤35
_
_
_
_
None
SARS-CoV-2,
Influenza A, Influenza
B and RSV
/ + _ _ _
SARS-CoV-2
Influenza A, Influenza
B and RSV
/ _ + _ _
Influenza A
SARS-CoV-2,
Influenza B and RSV
/ _ _ + _ Influenza B
SARS-CoV-2,
Influenza A and RSV
/
_
_
_
+
RSV
SARS-CoV-2,
Influenza A and
Influenza B
/ + + _ _ SARS-CoV-2,
Influenza A
Influenza B, RSV
/ + _ + _ SARS-CoV-2,
Influenza B
Influenza A, RSV
/ + _ _ + SARS-CoV-2,
RSV
Influenza A,
Influenza B
/ _ + + _ Influenza A,
Page 20 of 42
Influenza B SARS-CoV-2, RSV
/ _ + _ + Influenza A, RSV SARS-CoV-2,
Influenza B
/ _ _ + + Influenza B, RSV SARS-CoV-2,
Influenza A
/
+
+
+
_ SARS-CoV-2,
Influenza A, Influenza B
RSV
/ + + _ + SARS-CoV-2,
Influenza A, RSV
Influenza B
/ + _ + + SARS-CoV-2,
Influenza B, RSV
Influenza A
/ _ + + + Influenza A,
Influenza B, RSV
SARS-CoV-2
/
+
+
+
+
SARS-CoV-2,
Influenza A,
Influenza B, RSV
None
>35 or
Undet/blank
_
_
_
_
Invalid result, specimen needs to be re-
tested from re-extraction or re-collected
from patient for test.
-: Ct > target cutoff or Undet/blank; +: Ct <= target cutoff
/: No requirements on the Ct value; Undet: Undetermined
Result interpretation for the instrument with FAM, HEX/VIC, ROX and Cy5 detection capability
Ct Result interpretation
IC (VIC/HEX)
RNase P
SARS-
CoV-2
(FAM)
Influenza A
(ROX)
Influenza B
(Cy5)
Detected
Not Detected
Unknown
≤35
_
_
_
None
SARS-CoV-2,
Influenza A,
Influenza B
RSV
/ + _ _ SARS-CoV-2 Influenza A,
Influenza B
RSV
/ _ + _ Influenza A SARS-CoV-2,
Influenza B
RSV
/ _ _ + Influenza B SARS-CoV-2,
Influenza A
RSV
/ + + _ SARS-CoV-2,
Influenza A Influenza B
RSV
/ + _ + SARS-CoV-2,
Influenza B Influenza A
RSV
/ _ + + Influenza A,
Influenza B SARS-CoV-2
RSV
/
+
+
+
SARS-CoV-2,
Influenza A,
Influenza B
None
RSV
Page 21 of 42
>35 or
Undet _ _ _
Invalid result, specimen needs to be re-tested from
re-extraction or re-collected from patient for test.
-: Ct > target cutoff or Undet/blank; +: Ct <= target cutoff
/: No requirements on the Ct value.
Undet: Undetermined
The final report will be based on the fluorescence detectors selected in the specific
instrument.
Confirmation of low copy number virus detection
If the Ct of target virus is more than 37, it is recommended to do retest either by
repeating extraction (if there is enough sample left) or repeating PCR. If both tests
are positive, it will be reported as positive for the corresponding virus. Otherwise, it
will be reported as inconclusive. Collection of a new specimen and subsequent
testing should be considered for patient with cinclusive results.
Kit Limitations
1. The PKamp™ Respiratory SARS-CoV-2 RT-PCR Panel assay is for in vitro
diagnostic use.
2. This kit is used for qualitative detection of RNA from SARS-CoV-2, influenza A
viruses, influenza B viruses (and RSV depending on instruments) from human
oropharyngeal swabs, nasopharyngeal swabs and anterior nasal swabs. The
results cannot directly reflect the viral load in the original specimens.
3. The PKamp™ Respiratory SARS-CoV-2 RT-PCR Panel performance has only
been established with the specimen types described in the Intended Use
section. Testing other types of specimen may cause inaccurate results. Note
that the clinical study was performed using nasopharyngeal swabs as this
specimen type is known to be the most challenging upper respiratory matrix (6).
4. The specimens to be tested shall be collected, processed, stored and
transported in accordance with the conditions specified in the instructions.
Inappropriate specimen preparation and operation may lead to inaccurate
results.
5. RNA extraction methods other than that listed in the Assay Procedure have not
been evaluated. Users should verify any other extraction methods before using
them with the PKamp Respiratory SARS-CoV-2 RT-PCR Panel.
6. The limit of detection (LoD) is determined based on a 95% confidence of
detection. When viral target presents at or above the LoD concentration in the
test specimen, there will be a low probability that viral target is not detected.
When viral target presents below the LoD concentration in the test specimen,
Page 22 of 42
there will also be certain probability that viral target can be detected.
7. Primers and probes for this kit target highly conserved regions within the
genome of SARS-CoV-2, Influenza A, Influenza B and RSV. Mutations
occurred in these conserved regions (although rare) may result in RNA being
undetectable.
8. This kit uses an UNG/dUTP PCR products carryover prevention system which
can prevent contamination caused by PCR products. However, in the actual
operation process, the amplicon contamination can be avoided only by strictly
following the instructions of PCR laboratories.
9. Negative results do not preclude SARS-CoV-2, Influenza A viruses, Influenza
B viruses and RSV infections and should not be used as the sole basis for
treatment or other management decisions.
10. This kit was not tested for all known/unknown cross-reactants i.e. some fungi,
bacteria and virus.
11. The impacts of vaccines, antiviral therapeutics, antibiotics, chemotherapeutics
or immunosuppressant drugs have not been evaluated.
12. Laboratories are required to report all positive results to the appropriate public
health authorities.
Assay Performance
Limit of Detection (LoD)
Analytical sensitivity of the PKamp Respiratory SARS-CoV-2 RT-PCR Panel kit
was determined in limit of detection studies using viral stocks of an influenza A
virus, an influenza B virus, a RSV virus and a SARS-CoV-2 virus (provided from
BEI Resources) to determine the lowest concentration of each virus at which ≥95%
of all replicates are positive. Each virus was prepared with a diluent consisting of a
suspension of human A549 cells and viral transport medium (VTM).
Table: Virus used in LoD study
Viral target Strain name Stock titer (TCID /mL)
50
BEI catalog number
Influenza A A/Georgia/T51700/2012 (H1N1)pdm09
8
2.8 x 10 NR-42940
Influenza B B/Brisbane/60/2008 (Victoria Lineage)
7
1.8 x 10 NR-42005
Human respiratory syncytial virus
A2001/3-12 8
1.6 × 10 NR-28526
Page 23 of 42
SARS-coronavirus-2 Isolate USA-WA1/2020, Heat Inactivated
5
1.6 × 10 NR-52286
Range-finding (triplicates of 10-fold dilution, five replicates of 3-fold dilution) was
done to determine an estimated LoD. Each serial dilution was tested by extracting
300 µL of each virus dilution, eluting 60 µL elution buffer, and 20 µL was used as
input for the PCR reactions on Bio-Rad CFX96, Applied Biosystems 7500 standard,
Applied Biosystems 7500 Fast Dx, QuantStudioTM Dx Real-Time Instrument (96-
well fast block), and qTower3G Real-Time PCR Detection System, 10 µL was used
as input for the PCR reactions on Bio-Rad CFX384, Applied Biosystems
QuantStudio Dx 384 and qTower3G 84 Real-Time PCR Detection System.
LoD was subsequently confirmed by testing 20 replicates at estimated LoD, listed
in the following table (D: Detected, ND: Not Detected). The LoD was further
confirmed by testing 20 replicates at 3-fold dilution till a <95% detection rate
concentration to show that LoDs were correctly deteremined.
The summary is listed in the following two tables. In summary, the validated
instruments have comparable LoD.
Table. LoD study summary (TCID50/mL) SARS-CoV-2 Influenza A Influenza B RSV
QuantStudio 384 0.11 3.33 0.33 3.33
CFX 96 0.11 3.33 0.33 3.33
CFX 384 0.11 9.99 0.33 NA
ABI 7500 Standard 0.11 3.33 0.33 NA
ABI 7500 Fast Dx 0.11 3.33 0.33 NA
QuantStudio 96 0.11 3.33 0.11 3.33
qTower 96 0.11 3.33 0.11 3.33
qTower 384 0.11 6.66 0.33 9.99
NA: Not applicable
0.11 TCID50/mL of SARS-CoV-2 is around 258 copies/mL per supplier’s CoA.
Table. Statistical summary of Ct values of LoD study
Instrument SARS-CoV-2 Influenza A Influenza B RSV
Mean SD Rate Mean SD Rate Mean SD Rate Mean SD Rate
QuantStudio 384 36.87 0.93 20/20 35.73 0.87 19/20 36.37 0.95 19/20 36.37 0.95 19/20
CFX 96 37.76 0.47 20/20 37.01 1.52 20/20 39.27 0.61 20/20 37.87 1.47 20/20
CFX 384 36.19 0.66 20/20 34.76 0.88 20/20 36.71 0.95 20/20 NA NA NA
ABI 7500 Fast Dx 35.39 0.40 20/20 37.20 1.07 20/20 37.66 0.70 20/20 NA NA NA
ABI 7500 Standard 36.36 0.44 20/20 37.48 0.81 20/20 37.92 0.70 20/20 NA NA NA
QuantStudio 96 36.47 0.57 20/20 36.65 0.67 20/20 36.29 0.64 19/20 37.62 1.14 19/20
qTower 96 36.67 0.49 20/20 37.94 0.63 19/20 37.45 0.69 20/20 38.22 1.12 20/20
qTower 384 36.52 1.05 20/20 36.74 0.69 20/20 36.64 0.89 20/20 36.50 1.52 20/20
Page 24 of 42
Co-infection Sensitivity
Analytical sensitivity of the PKamp Respiratory SARS-CoV-2 RT-PCR Panel kit in
the context of a co-infection scenario was evaluated by the following co-infection
panel. One representative strain of each targeted organism (SARS-CoV-2,
Influenza A viruses, Influenza B viruses and RSV) was tested. Co-infection Panels
were made by spiking one target organism at high concentration (3000X LoD) and
another target organism at low concentration (3X LoD) in a simulated clinical
matrix. Additional panel members were made with one target at high concentration
(3000X LoD) and another target at low concentration (10X LoD), if the competitive
interference was observed. It was evaluated on 5 instruments, with the same virus
strains used for LoD study. Each condition was tested as triplicates from sample
extraction to PCR.
Table. Study for the instruments with FAM, HEX/VIC, ROX, Cy5 and Cy5.5
detection capability.
Condition SARS-CoV-2 Influenza A Influenza B RSV
1 3000XLoD 3XLoD 3000XLoD 3000XLoD
2 3000XLoD 3000XLoD 3XLoD 3000XLoD
3 3000XLoD 3000XLoD 3000XLoD 3XLoD
4 3XLoD 3000XLoD 3000XLoD 3000XLoD
Table. Study for the instruments with FAM, HEX/VIC, ROX and Cy5 detection capability. Condition SARS-CoV-2 Influenza A Influenza B RSV
1 3000XLoD 3XLoD 3000XLoD 3000XLoD
2 3000XLoD 3000XLoD 3XLoD 3000XLoD
3 3XLoD 3000XLoD 3000XLoD 3000XLoD
Interference was observed for Influenza A and RSV with Applied Biosystems
QuantStudio Dx 384: 2/3 were detected as positive when 3XLoD influenza A co-
infected with 3000XLoD of SARS-CoV-2, influenza B and RSV, 2/3 were detected
as positive when with 3000XLoD of SARS-CoV-2, influenza B and RSV. There was
no interference observed on CFX384 and CFX96 instruments for evaluated
conditions.
Applied Biosystems QuantStudio Dx 384 was further evaluated with two new
panels: 1) 10XLoD influenza A co-infected with 3000XLoD of SARS-CoV-2,
influenza B and RSV. 2) 10XLoD RSV co-infected with 3000XLoD of SARS-CoV-
2, influenza A and influenza B. There was no interference observed. This is
acceptable as 10X LoD is a titer that is much lower than what is commonly found
in clinical samples. The statistical summary is listed in the following table.
Table. Co-infection study statistical summary
Page 25 of 42
Instrument
SARS-CoV-2 Influenza A Influenza B RSV RNase P
Panel name Mean SD N Mean SD N Mean SD N Mean SD N Mean SD N
Applied Biosystems QuantStudio Dx 384
InfA 10XLOD 25.27 0.30 3 34.69 0.41 3 26.13 0.31 3 23.95 0.93 3 30.33 0.05 3
InfA 3XLOD 25.30 0.32 3 38.73 0.66 2 25.50 0.84 3 24.94 1.42 3 30.35 1.77 3
InfB 3XLOD 25.60 0.27 3 26.80 0.32 3 36.12 0.61 3 25.14 0.37 3 29.76 0.26 3
RSV 10XLOD 25.36 0.07 3 25.57 0.04 3 26.21 0.10 3 38.21 0.87 3 30.21 0.15 3
RSV 3XLOD 25.47 0.22 3 26.80 0.22 3 25.26 0.18 3 40.06 1.38 2 29.88 0.18 3
SC2 3XLOD 36.08 0.76 3 26.66 0.09 3 25.16 0.18 3 24.22 1.08 3 29.16 0.11 3
Bio-Rad CFX96
InfA 3XLOD 27.28 0.2 3 35.86 1.50 3 28.53 0.47 3 24.61 1.25 3 31.10 1.54 3
InfB 3XLOD 26.97 0.24 3 24.62 0.38 3 39.62 0.32 3 24.74 0.50 3 29.88 0.37 3
RSV 3XLOD 26.96 0.24 3 24.58 0.12 3 28.37 0.12 3 38.50 0.49 3 29.94 0.14 3
SC2 3XLOD 37.00 0.4 3 24.43 0.04 3 27.78 0.12 3 23.81 0.76 3 28.92 0.04 3
Bio-Rad CFX384
InfA 3XLOD 26.72 0.21 3 35.06 1.61 3 25.81 0.21 3 N/A N/A N/A 30.03 0.03 3 InfB 3XLOD 26.78 0.52 3 25.37 0.41 3 35.64 1.46 3 N/A N/A N/A 29.59 0.47 3 SC2 3XLOD 35.81 0.64 3 24.85 0.06 3 25.25 0.10 3 N/A N/A N/A 29.11 0.04 3
Applied Bioystems7500 Standard 96
InfA 3XLOD 25.45 0.23 3 36.15 0.99 3 26.61 0.23 3 N/A N/A N/A 29.98 0.19 3
InfB 3XLOD 25.52 0.44 3 26.06 0.42 3 37.68 0.24 3 N/A N/A N/A 30.28 0.45 3
SC2 3XLOD 35.14 0.42 3 26 0.03 3 27.01 0.14 3 N/A N/A N/A 29.42 0.08 3
Applied BIosystes 7500 Fast Dx 96
InfA 3XLOD 24.66 0.11 3 36.03 0.92 3 25.27 0.38 3 N/A N/A N/A 30.82 0.29 3
InfB 3XLOD 25.06 0.5 3 25.96 0.47 3 36.49 0.22 3 N/A N/A N/A 30.84 0.37 3
SC2 3XLOD 34.97 0.49 3 25.99 0.2 3 26.01 0.17 3 N/A N/A N/A 29.36 0.24 3
N/A: Not applicable
Analytical Reactivity (Inclusivity)
In-silico analysis of inclusivity of SARS-CoV-2:
The inclusivity of the primers/probes have been routinely evaluated based on NCBI
and GISAID database. The most recent evaluation was in August 2020.
There are many sequences being added to the NCBI and GISAID database daily,
especially for SARS-CoV-2. Some of the sequences are not full-length genomic
sequences and some are with lower quality builds and contain many N base pairs.
Using incomplete and low-quality sequences during alignment can lead to lower
alignment rates, and make the homology seem lower than it is. Therefore, three
filters were applied on the database browser before downloading in order to avoid
this kind of misleading analysis. The filter definitions are listed as following:
• Complete: Genomes with >29000bp
• High: Entries with <1% Ns and <0.05% unique amino acid mutations (not seen
in other sequences in the database) and no indels unless verified by the
submitter
• Low coverage exclusion: Excludes entries with >5% Ns
SARS-Cov-2 summary: More than 52,000 sequences in GISAID and 10,000
sequences in NCBI are defined as qualified with filters as "complete", "high
coverage", "low coverage exclusion". The primers/probe for ORF1ab, reverse
primer and probe for N gene have showed perfect match for more than 98% of the
sequences. 5' variants have been identified in some of forward primer sequences
of N gene (around 30% of the qualified sequences). However, it has less impacts
Page 26 of 42
on Tm which will impact primer binding efficiency. Moreover, the detection on
SARS-CoV-2 is based on N or ORF1ab gene: as long as one of the genes (N, ORFI
ab) is amplified/detected, the target virus is detected. Therefore, the identified
sequence variances have less impacts on inclusivity for SARS-CoV-2.
Two wet testing studies were performed on QuantStudio Dx 384.
Inclusivity study 1: 18-panel evaluation
The inclusivity of the kit was evaluated using 11 influenza A, 5 influenza B and 2
RSV viruses representing temporal, geographic, and genetic diversity within the
subtype and lineage, and one SARS-CoV-2 virus. Each panel member was diluted
and extracted using the chemagic 360 instrument and tested in triplicate. Results
are presented in the following table.
Table: 18-panel for inclusivity study.
Number Lineage Strain Evaluation
Concentration Unit Vendor
Part No.
1 Influenza A (H1N1) A/Brisbane/59/2007 (H1N1) 8.90E+03
CEID50/mL BEI NR-12282
2 Influenza A (H1N1) A/Denver/1/1957 (H1N1) 1.60E+02
CEID50/mL BEI NR-4229
3 Influenza A (H1N2) A/swine/Ohio/09SW1484E/2
009 (H1N2) 1.60E+03 TCID50/mL BEI NR-36703
4 Influenza A (H2N2) A/Japan/305/1957 (H2N2) 3.26E-06 ng/μL BEI NR-2775
5 Influenza A/H3N2 A/Wisconsin/67/2005 (H3N2) 2.80E+02
CEID50/mL BEI NR-41800
6 Influenza A (H3N8) A/equine/Pennsylvania/1/200
7 (H3N8) 1.60E-01
CEID50/mL BEI NR-13426
7 Influenza A (H5N2) A/duck/Pennsylvania/10218/
1984 (H5N2) 2.55E-03 ng/μL BEI NR-2763
8 Influenza A (H6N2) A/turkey/Massachusetts/374
0/1965 (H6N2) 1.10E+03
CEID50/mL BEI NR-21654
9
Influenza A (H7N2)
A/equine/Prague/1/1956 (HA) x A/Aichi/2/1968 (NA) x A/Puerto Rico/8/1934 (H7N2), Reassortant X-32
3.65E-04
ng/μL
BEI
NR-9680
10
Influenza A (H7N7)
Genomic RNA from Influenza A Virus, A/equine/Prague/1956 (H7N7)
1.50E-03
ng/μL
BEI
NR-35976
11 Influenza A (H9N2) A/turkey/Wisconsin/1/1966 (H9N2) 1.60E+03
CEID50/mL BEI NR-21659
12 Influenza B B/Florida/4/2006 (Yamagata
Lineage) 1.60E+02
CEID50/mL BEI NR-41795
13
B-YAM Influenza B Virus, B/Texas/06/2011 (Yamagata Lineage)
8.90E+02
CEID50/mL
BEI
NR-44024
14
Influenza B Influenza B Virus, B/Hong Kong/330/2001 (Victoria Lineage)
1.80E+01
CEID50/mL
BEI
NR-41802
15 Influenza B B/Malaysia/2506/2004 1.58E+01
CEID50/mL BEI NR-9723
16 Influenza B B/Ohio/01/2005 (Victoria Lineage)
1.60E+03
CEID50/mL BEI NR-41801
17 Human respiratory syncytial virus
B1 8.90E+00 TCID50/mL BEI NR-4052
18 Human respiratory syncytial virus
A2 Concentration not
available
Concentrat ion not
available
BEI
NR-12149
Table: 18-panel inclusivity study result summary Number InfA InfA InfA InfB InfB InfB RSV RSV RSV CoV2 CoV2 CoV2
1 31.18 31.71 31.39 ND ND ND ND ND ND ND ND ND
2 34.03 34.29 34.41 ND ND ND ND ND ND ND ND ND
3 33.77 33.72 33.32 ND ND ND ND ND ND ND ND ND
4 38.10 39.47 38.99 ND ND ND ND ND ND ND ND ND
5 31.50 32.15 32.37 ND ND ND ND ND ND ND ND ND
Page 27 of 42
6 37.05 37.12 36.79 ND ND ND ND ND ND ND ND ND
7 36.58 36.63 36.69 ND ND ND ND ND ND ND ND ND
8 36.05 33.72 36.46 ND ND ND ND ND ND ND ND ND
9 37.43 36.71 36.81 ND ND ND ND ND ND ND ND ND
10 36.97 36.41 36.44 ND ND ND ND ND ND ND ND ND
11 30.89 31.13 31.07 ND ND ND ND ND ND ND ND ND
12 ND ND ND 32.21 32.54 33.06 ND ND ND ND ND ND
13 ND ND ND 33.71 33.54 32.88 ND ND ND ND ND ND
14 ND ND ND 31.42 30.21 30.18 ND ND ND ND ND ND
15 ND ND ND 30.26 30.20 30.30 ND ND ND ND ND ND
16 ND ND ND 31.89 32.79 32.21 ND ND ND ND ND ND
17 ND ND ND ND ND ND 35.32 32.33 33.16 ND ND ND
18 ND ND ND ND ND ND 35.29 35.28 35.02 ND ND ND
ND: Not detected.
Note a. It is purified RNA. Due to the sample instability with the extracting process, it
was tested directly by PCR.
Inclusivity study 2: CDC 2020 human influenza virus panel
The panel for 2020-2021 Influenza annual reactivity panel was used for this study following the CDC protocol. For each virus stock, 5-fold serial dilution were prepared. For each concentration, 5 replicates were tested on QuantStudio Dx 384. The results are summarized in the following table.
Table: CDC panel LoD summary
Virus
Strain Designation
HA titer
EID50 titer/ml
Stock
concentrati on (ID50/mL)
LoD
Concentrat ion (ID50/mL)
LoD Ct Mean
LoD
Ct SD
Influenza A (H3N2) A/Perth/16/2009 256 1E9.3 2.00E+09 5.11E+02 32.46 0.16
Influenza A (H3N2) A/Hong
Kong/2671/2019 64 1E7.5 3.16E+07 8.10E+00 34.99 1.73
Influenza A (H1N1)pdm09
A/Christ Church/16/2010
2048 1E10.2 1.58E+10 8.11E+02 35.53 0.42
Influenza A (H1N1)pdm09
A/Guandong- Maonan/1536/2019
2048 1E9.1 1.26E+09 6.45E+01 36.67 0.42
Influenza B (Victoria lineage)
B/Michigan/09/2011 32 1E6.9 7.94E+06 8.13E-02 36.23 0.95
Influenza B (Victoria lineage)
B/Washington/02/2019 256 1E9.2 7.94E+09 3.25E+00 36.71 1.14
Influenza B (Yamagata lineage) B/Texas/81/2016 128 1E8.3 2.00E+08 2.04E+00 34.18 3.12
Influenza B (Yamagata lineage)
B/Phuket/3073/2013 128 1E9.9 1.58E+09 1.63E+01 37.15 0.74
In summary, the results from wet lab study 1 and 2 demonstrated that the kit can
detect multiple clinically relevant strains of influenza A viruses, influenza B viruses,
and RSV, including the strains from 2020 CDC influenza panel.
Analytical Specificity (Cross-reactivity)
Cross-reactivity of the PKamp Respiratory SARS-CoV-2 RT-PCR Panel Kit was
evaluated using both in silico analysis and wet testing against normal and
pathogenic organisms found in the respiratory tract.
Page 28 of 42
The primer/probe design in this kit for SARS-CoV-2 is identical to the CE approved
kit (PerkinElmer SARS-CoV-2 Real-time RT-PCR assay, product No.: COVID-19-
PCR-AUS-C).
BLASTn analysis queries of the SARS-CoV-2 primers and probes targeting N and
ORF1ab gene were performed against public domain nucleotide sequences with
default settings. The database search parameters were as follows:
• The match and mismatch scores were 1 and -3, respectively.
• The penalty to create and extend a gap in an alignment was 5 and 2, respectively.
• The search parameters automatically adjusted for short input sequences and the expected threshold was 1000.
In summary no organisms, including other related SARS-coronaviruses, exhibited
>80% homology to the forward primer, reverse primer, and probe for either the
ORF1ab or N target. The results of the in silico analysis suggest the kit is designed
for the specific detection of SARS-CoV-2, with no expected cross reactivity to the
human genome, other coronaviruses, or human microflora that would predict
potential false positive RT-PCR results.
The sequence alignment for cross-reactivity for primers and probes for influenza A
viruses, influenza B viruses and RSV is listed in the following table. The Influenza
B reverse primer shows 85% homologous to Candida albicans. However, there is
poor alignment on forward primer and probe. Therefore, the risk of cross-reactivity
to Candida albicans is very low. It was further confirmed in the following wet testing.
Table: In silico analysis of primers and probes of influenza A, influenza B and RSV
Pathogen
Strain GenBank
Acc#
% Homology Test
FluA- F
FluA- R
FluA- P
FluB- F
FluB- R
FluB- P
RSV- F
RSV- R
RSV- P
Human coronavirus 229E
229E NC_002645.1 43.2 47.9 37.5 54.6 52.4 40.7 41.7 41.1 37.0
Human coronavirus
OC43 ATCC VR-759 NC_006213.1 40.9 40.6 33.3 59.1 52.4 48.2 41.7 33.9 33.3
Human coronavirus HKU1 HCoV-HKU1 NC_006577.2 38.6 37.5 37.5 36.4 52.4 44.4 45.8 35.7 40.7
Human coronavirus NL63 NL63 NC_005831.2 38.6 40.6 33.3 36.4 47.6 37.0 37.5 37.5 29.6
SARS-coronavirus None listed NC_004718.3 40.9 52.1 50.0 45.5 57.1 48.2 41.7 35.7 37.0
MERS-coronavirus NL140455 MG987421.1 54.6 43.8 37.5 40.9 47.6 44.4 41.7 35.7 33.3
Adenovirus (e.g. C1 Ad. 71)
type 2 J01917.1 40.9 44.8 37.5 40.9 42.9 37.0 50.0 32.1 37.0
Human
Metapneumovirus (hMPV)
CAN97-83
NC_039199.1
36.4
35.4
33.3
40.9
47.6
40.7
41.7
30.4
44.4
Parainfluenza virus 1 (Human respirovirus 1)
HPIV1/Los_Angeles/USA/CHLA 36/2016
MK167043.1
40.9
37.5
33.3
40.9
42.9
33.3
41.7
35.7
37.0
Parainfluenza virus
2 (Human rubulavirus 2)
HPIV2/Seattle/USA/SC9949/20 18
MN369034.1
36.4
38.5
37.5
36.4
52.4
33.3
50.0
30.4
33.3
Parainfluenza virus 3 (Human respirovirus 3)
NIV1721711
MH330335.1
43.2
39.6
33.3
36.4
42.9
29.6
45.8
46.4
44.4
Parainfluenza virus
4a (Human
rubulavirus 4a)
4a M-25
NC_021928.1
45.5
44.8
29.2
45.5
38.1
48.2
54.2
33.9
33.3
Influenza A
New York/392/2004(H3N2)
NC_007373.1
, NC_007372.1 ,
NC_007371.1 , NC_007366.1
, NC_007369.1 ,
93.2
93.8
100.
0
36.4
52.4
29.6
37.5
53.6
33.3
Page 29 of 42
NC_007368.1
,
NC_007367.1 , NC_007370.1
Influenza B
B/Lee/1940
NC_002205.1
, NC_002206.1
, NC_002207.1
, NC_002208.1 ,
NC_002209.1 , NC_002210.1
, NC_002211.1 , NC_002204.1
40.9
37.5
33.3
100.
0
100.
0
100.
0
50.0
37.5
37.0
Enterovirus (e.g. EV68)
coxsackievirus B1 NC_001472.1 38.6 44.8 41.7 40.9 42.9 37.0 33.3 32.1 29.6
Respiratory syncytial virus
RSS-2 NC_001803.1 36.4 39.6 33.3 45.5 42.9 33.3 100.
0 92.9 92.6
Rhinovirus 1 strain ATCC VR-1559 NC_038311.1 38.6 41.7 37.5 54.6 52.4 33.3 37.5 42.9 33.3
Chlamydia pneumonia CWL029 NC_000922.1 50.0 50.0 54.2 68.2 57.1 48.2 58.3 57.1 44.4
Haemophilus influenzae
Rd KW20 NC_000907.1 56.8 54.2 45.8 54.6 61.9 48.2 54.2 44.6 44.4
Legionella pneumophila
Philadelphia 1 NC_002942.5 63.6 61.5 58.3 54.6 61.9 44.4 58.3 50.0 44.4
Mycobacterium tuberculosis
H37Rv NC_000962.3 56.8 60.4 66.7 59.1 61.9 59.3 50.0 50.0 48.2
Streptococcus
pneumoniae R6 NC_003098.1 59.1 59.4 50.0 50.0 61.9 48.2 54.2 50.0 48.2
Streptococcus pyogenes M1 GAS NC_002737.2 56.8 54.2 45.8 68.2 61.9 51.9 54.2 53.6 70.4
Bordetella pertussis Tohama I NC_002929.2 54.5 62.5 50.0 54.6 76.2 48.2 45.8 41.1 48.2
Mycoplasma pneumoniae
M129 NC_000912.1 50.0 53.1 45.8 54.6 57.1 40.7 50.0 46.4 59.3
Pneumocystis
jirovecii RU7
NW_0172647
75.1 54.6 56.3 45.8 63.6 57.1 48.2 62.5 51.8 48.2
Candida albicans SC5314 NC_032089.1 56.8 62.5 50.0 63.6 85.7 48.2 66.7 53.6 48.2
Pseudomonas
aeruginosa PAO1 NC_002516.2 63.6 50.0 58.3 59.1 66.7 51.9 54.2 44.6 59.3
Wet testing against normal and pathogenic organisms of the respiratory tract was
performed to confirm the results of the in silico analysis. Each organism identified
in the table below was tested in triplicates with the kit at the concentrations
indicated on Applied Biosystems QuantStudio Dx 384. All results were negative
(The evaluation unit refers to the sample concentration before sample extraction).
Table: Organisms tested for cross-reactivity with the PKamp Respiratory SARS-
CoV-2 RT-PCR Panel
Name/Description Evaluation
concentration Evaluation
Unit
Vendor
Part No.
Human coronavirus 229E 1.60E+05 TCID50/mL BEI NR-52726
Human coronavirus OC43 (gRNA) 6.34E+08 copies/mL ATCC VR-1558D
Human coronavirus HKU1 (gRNA) 5.40E+07 copies/mL ATCC VR-3262SD
Human coronavirus NL63 1.60E+04 TCID50/mL BEI NR-470
SARS-coronavirus
N/A (10-fold dilution of Stock)
N/A ZeptoMetrix
NATSARS- ST
MERS-coronavirus 8.90E+04 TCID50/mL BEI NR-50549
Adenovirus (e.g. C1 Ad. 71) 1.25E+06 TCID50/mL BEI NR-51436
Human Metapneumovirus (hMPV) (gRNA)
1.82E+08 copies/mL BEI NR-49122
Human parainfluenza virus 1 RNA 2.48E+08 copies/mL ATCC VR-94D
Human parainfluenza virus 2 5.00E+06 TCID50/mL BEI NR-3229
Human parainfluenza virus 3 (gRNA) 2.43E+07 copies/mL ATCC VR-93D
Huamn parainfluenza virus 4b 2.50E+05 TCID50/mL BEI NR-3238
Enterovirus (EV71) RNA 1.46E+09 copies/mL BEI NR-4960
Rhinovirus 1.00E+05 TCID50/mL BEI NR-51453
Page 30 of 42
Haemophilus influenzae gDNA 4.20E+06 copies/mL ATCC 51907DQ
Legionella pneumophila gDNA 3.27E+07 copies/mL ATCC 33152D-5
Mycobacterium tuberculosis gDNA 6.78E+08 copies/mL BEI NR-14867
Streptococcus pneumoniae gDNA 8.70E+07 copies/mL ATCC 6308D-5
Streptococcus pyogenes gDNA 6.18E+07 copies/mL ATCC BAA-572D-5
Bordetella pertussis gDNA 2.70E+07 copies/mL ATCC 9797D-5
Mycoplasma pneumoniae gDNA 1.24E+07 copies/mL ATCC 15531D
Pneumocystis jirovecii (PJP)
N/A (5-fold dilution of Stock)
N/A
ZeptoMetrix NATPJI-
ERC
Candida albicans gDNA 1.62E+07 copies/mL BEI NR-50361
Pseudomonas aeruginosa gDNA 2.33E+07 copies/mL ATCC 9027D-5
Staphylococcus epidermis gDNA 4.50E+07 copies/mL ATCC 12228D-5
Streptococcus salivarius gDNA 2.20E+07 copies/mL BEI HM-121D
Herpes Simplex virus
1.25E+05 copies/mL Thermo/AcroMet
rix 954501
Varicella-zoster virus
2.5E+04 copies/mL Thermo/AcroMet
rix 954512
Epstein Barr virus gDNA 1.01E+09 copies/mL ATCC CRL-5957D
Measles Virus gDNA 6.60E+08 copies/mL BEI NR-44104
Mumps virus 1.60E+06 TCID50/mL BEI NR-3846
Cytomegalovirus 5.00E+04 IU/mL WHO 09/162
Corynebacterium diphtheriae gDNA 9.34E+07 copies/mL ATCC 700971D-5
Escherichia coli 9.06E+07 copies/mL BEI NR-9281
Lactobacillus plantarum 3.16E+06 cfu/mL ATCC BAA-793
Moraxella catarrhalis gDNA 2.21E+08 copies/mL ATCC 25240D-5
Staphylococcus aureus gDNA 1.13E+08 copies/mL BEI NR-10320
Neisseria elongata
N/A (20-fold dilution of Stock)
N/A ATCC 25295
Neisseria meningitidis gDNA 2.53E+08 copies/mL BEI NR-48806
The copy number was calculated based on concentration (ng) and genome size.
Cross-reactivity (non-targeted influenza types, coronavirus types and RSV)
Cross-reactivity of each primer probe set to viruses targeted by another component
of the kit was also evaluated at high titer concentration of each virus as part of the
cross-reactivity study and during testing of the panel of viruses provided by US
CDC. The performance was evaluated on 5 instruments (Applied Biosystems
QuantStudio Dx 384, Bio-Rad CFX96, Bio-Rad CFX384, Applied Biosystems 7500
Standard, Applied Biosystems 7500 Fast Dx) by either triplicates or five replicates
(for the CDC 2020 human influenza virus panel).
For each evaluated primer/probe set, it only generated positive results on the
corresponding detection target, with negative results on the other targets. For
Page 31 of 42
example, the SARS-CoV-2 primer and probe set generated negative results for
influenza A and influenza B and RSV viruses. Result summary is shown in the
following tables.
Table. Applied Biosystems QuantStudio Dx 384 summary
Virus
Strain
Designation
Evaluation concentratio
n (ID50/mL)
SARS-CoV-2
Influenza A
Influenza B
RSV
Mean SD N Mean SD N Mean SD N Mean SD N
Influenza A (H3N2)
A/Perth/16/20 09
3.99E+07 0 0 0 16.64 0.11 5 0 0 0 0 0 0
Influenza A (H3N2)
A/Hong Kong/2671/2 019
6.32E+05
0
0
0
19.05
0.42
5
0
0
0
0
0
0
Influenza A (H1N1)pdm0 9
A/Christ Church/16/20 10
3.17E+08
0
0
0
15.63
0.16
5
0
0
0
0
0
0
Influenza A (H1N1)pdm0 9
A/Guandong- Maonan/1536 /2019
2.52E+07
0
0
0
16.93
0.78
5
0
0
0
0
0
0
Influenza B
(Victoria lineage)
B/Michigan/0
9/2011
1.59E+05
0
0
0
0
15.87
0.1
5
0
0
0
Influenza B
(Victoria
lineage)
B/Washingto n/02/2019
3.17E+07
0
0
0
0
14.94
0.16
5
0
0
0
Influenza B (Yamagata lineage)
B/Texas/81/2 016
3.99E+06
0
0
0
0
15.32
0.09
5
0
0
0
Influenza B (Yamagata lineage)
B/Phuket/307 3/2013
1.59E+08
0
0
0
43.81
0
15.27
0.16
5
0
0
0
Human respiratory
syncytial virus
A2001/3-12
3.20E+06
0
0
0
0
0
0
0
0
3
15.81
0.04
3
SARS- coronavirus-2
Isolate USA-
WA1/2020, Heat
Inactivated
3.20E+03
20.87
0.73
3
0
0
0
Table. Bio-Rad CFX96 summary
Virus
Strain
Designation
Evaluation concentrati on (ID50/mL)
SARS-CoV-2 Influenza A Influenza B RSV
Mean SD N Mean SD N Mean SD N Mean SD N
Influenza A (H3N2)
A/Perth/16/20 09
3.99E+07 0 0 0 18.86 0.26 5 0 0 0 0 0 0
Influenza A (H3N2)
A/Hong
Kong/2671/2 019
6.32E+05
0
0
0
20.89
0.31
5
0
0
0
0
0
0
Influenza A (H1N1)pdm0 9
A/Christ Church/16/20 10
3.17E+08
0
0
0
16.87
0.20
5
0
0
0
0
0
0
Influenza A
(H1N1)pdm0 9
A/Guandong-
Maonan/1536 /2019
2.52E+07
0
0
0
18.03
0.64
5
0
0
0
0
0
0
Influenza B
(Victoria
lineage)
B/Michigan/0 9/2011
1.59E+05
0
0
0
0
0
0
17.02
0.09
5
0
0
0
Influenza B
(Victoria
lineage)
B/Washingto n/02/2019
3.17E+07
0
0
0
0
0
0
15.09
0.41
5
0
0
0
Influenza B
(Yamagata lineage)
B/Texas/81/2 016
3.99E+06
0
0
0
0
0
0
15.93
0.17
5
0
0
0
Influenza B (Yamagata lineage)
B/Phuket/307 3/2013
1.59E+08
0
0
0
0
0
0
15.59
0.35
5
0
0
0
Human respiratory syncytial virus
A2001/3-12
3.20E+06
17.11
1.1
3
SARS-
coronavirus-2
Isolate USA- WA1/2020,
Heat
Inactivated
3.20E+03
22.19
0.96
3
0
0
0
Table. Bio-Rad CFX384 summary
Virus
Strain
Designation
Evaluation concentratio n (ID50/mL)
SARS-CoV-2 Influenza A Influenza B RSV
Mean SD N Mean SD N Mean SD N Mean SD N
Influenza A (H3N2)
A/Perth/16/2 009 3.99E+07 0 0 0 18.32 0.33 5 0 0 0 NA NA NA
Influenza A (H3N2)
A/Hong Kong/2671/ 2019
6.32E+05
0
0
0
20.26
0.27
5
0
0
0
NA
NA
NA
Influenza A (H1N1)pdm 09
A/Christ Church/16/2 010
3.17E+08
0
0
0
16.45
0.18
5
0
0
0
NA
NA
NA
Page 32 of 42
Influenza A (H1N1)pdm 09
A/Guandon g- Maonan/153 6/2019
2.52E+07
0
0
0
17.73
0.75
5
0
0
0
NA
NA
NA
Influenza B (Victoria lineage)
B/Michigan/ 09/2011
1.59E+05
0
0
0
0
0
0
18.91
0.18
5
NA
NA
NA
Influenza B (Victoria lineage)
B/Washingt on/02/2019
3.17E+07
0
0
0
0
0
0
16.82
0.29
5
NA
NA
NA
Influenza B (Yamagata lineage)
B/Texas/81/ 2016
3.99E+06
0
0
0
0
0
0
17.54
0.17
5
NA
NA
NA
Influenza B (Yamagata lineage)
B/Phuket/30 73/2013
1.59E+08
0
0
0
0
0
0
17.22
0.21
5
NA
NA
NA
Human respiratory syncytial virus
A2001/3-12
3.20E+06
0
0
0
0
0
0
NA
NA
NA
SARS- coronavirus -2
Isolate USA- WA1/2020, Heat Inactivated
3.20E+03
22.37
0.66
3
0
0
NA
NA
NA
Table. Applied Biosystems 7500 Standard summary
Virus
Strain
Designation
Evaluation concentratio n (ID50/mL)
SARS-CoV-2 Influenza A Influenza B RSV
Mean SD N Mean SD N Mean SD N Me an
S D
N
Influenza A (H1N1)pdm09
A/Christ Church/16/2 010
3.17E+08
0.00
0.00
0
15.96
0.22
3
0
0
0
NA N A
N A
Influenza B (Victoria lineage)
B/Washingt on/02/2019
3.17E+07
0
0
0
0
0
0
14.78
0.31
3
NA N A
N A
Human respiratory syncytial virus
A2001/3-12
3.20E+06
0
0
0
0
0
0
0
0
0
NA N A
N A
SARS- coronavirus-2
Isolate USA- WA1/2020, Heat Inactivated
3.20E+03
21.23
0.67
3
0
0
0
0
0
0
NA
N A
N A
Table. Applied Biosystems 7500 Fast Dx summary
Virus
Strain Designation
Evaluation concentrati on (ID50/mL)
SARS-CoV-2 Influenza A Influenza B RSV
Mean SD N Mean SD N Mean SD N Mean SD N
Influenza A (H1N1)pdm09
A/Christ Church/16/201 0
3.17E+08
0
0
0
14.38
0.42
3
0
0
0
NA
NA
NA
Influenza B (Victoria lineage)
B/Washington/ 02/2019
3.17E+07
0
0
0
0
0
0
14.68
0.24
3
NA
NA
NA
Human respiratory syncytial virus
A2001/3-12
3.20E+06
0
0
0
0
0
0
0
0
0
NA
NA
NA
SARS- coronavirus-2
Isolate USA- WA1/2020, Heat Inactivated
3.20E+03
21.42
0.49
3
0
0
0
0
0
0
NA
NA
NA
Interfering Substances Study
The performance of the kit was evaluated with potentially interfering substances
that may be present in the upper respiratory tract. The following potential interfering
substances listed in the table were studied in the absence (negative samples) or
presence (positive samples) of targets on Applied Biosystems QuantStudio Dx 384.
Four-plex mixture of inactivated cultured virus in 3XLoD was used as positive
samples, which is considered as more challenging study than single-plex
evaluation. Each condition was tested in triplicates.
Table: Substances tested for interference with the PKamp Respiratory SARS-
Page 33 of 42
CoV-2 RT-PCR Panel
Substances Evaluation Concentration
Oxymetazoline 15% v/v
Blood 0.5% v/v
Benzocaine, Menthol 3.5 mg/mL
Fluticasonea 5% v/v
Menthol 8 mg/mL
Triamcinolone 5% v/v
Phenylephrine hydrochloride 5% v/v
Saline (sodium chloride) 15% v/v
Galphimia glauca, Luffa operculata, Sabadilla 5% v/v
Mucin protein 60 ug/mL
Menthol, thymol, methyl salicylate, and eucalyptol 5% v/v
Budesonide (Glucocorticoid)b 1% v/v
Oseltamivira 2.5 mg/mL
Tobramycin 4.0 ug/mL
Zanamivir 3.3 mg/mL
Mupirocina 10 mg/mL
Peramivir 45 ng/mL
Note: Blood and peramivir were evaluated with single-plex template, triplicates for each
condition.
For all listed above substances, all negative samples were negative at the
concentration tested. 3/3 of positive samples were detected for all targets for the
evaluated substances with the following exceptions:
• Group a: substances of fluticasone, Oseltamivir, Mupirocin, only RSV was 2/3
detected. All other targets (Influenza A, influenza B, SARS-CoV-2) were 3/3
detected.
• Group b: substances of Budesonide, 2/3 of influenza A, 2/3 influenza B, 2/3
SRAS-CoV-2 and 2/3 RSV was detected.
For the substances of fluticasone, Oseltamivir, Mupirocin and Budesonide, further
evaluation was performed on single target with observed interference at 3XLOD
and/or 10XLOD as triplicates. The results are summarized in the following table.
This is acceptable as 10X LoD is a titer that is much lower than what is commonly
found in clinical samples.
Substances
Evaluation Concentration
SARS-CoV-2 Influenza A Influenza B RSV
Fluticasonea 5% v/v NA NA NA 3/3 detected at 10XLOD
Budesonide (Glucocorticoid)b 1% v/v
3/3 detected at 10XLOD
3/3 detected at 3XLOD
3/3 detected at 3XLOD
3/3 detected at 10XLOD
Oseltamivira 2.5 mg/mL NA NA NA 3/3 detected at 3XLOD
Mupirocina 10 mg/mL NA NA NA 3/3 detected at 3XLOD
Page 34 of 42
NA: not tested as single target because of 3/3 detected in co-infection conditions
Equivalency between negative clinical matrix and viral transport medium plus human cells
The purpose of this study was to show that the simulated clinical matrix (viral
transport medium plus human cells) was equivalent to the negative pooled clinical
nasopharyngeal swab (NP) when testing SARS-CoV2, Influenza A viruses,
Influenza B viruses and RSV with this kit.
The virus used in the LoD study were diluted in simulated clinical matrix or negative
pooled clinical matrix to around 3XLOD, go through the same sample extraction
and PCR process (to minimize the run-to-run variations). Each condition was tested
with 6 replicates. The study was performed on 3 instruments. The results are
summarized in the following table. It has demonstrated that the equivalence of the
two dilution matrices.
Instrument Virus Target Name
Pooled NP swab (Clinica l) VTM + human cells
Mean Std Dev N Mean Std Dev N
QS384
InfA-1 3XLOD FluA 35.14 0.36 6 36.14 1.49 6
InfB-2 3XLOD FluB 33.81 0.14 6 34.72 0.71 6
RSV-1 3XLOD RSV 37.36 0.57 6 37.37 1.49 6
SC2 3XLOD CoV 33.25 0.21 6 35.30 1.08 6
CFX384
InfA-1 3XLOD FluA 33.44 0.34 6 33.98 0.58 6
InfB-2 3XLOD FluB 33.53 0.32 6 35.23 0.94 6
SC2 3XLOD CoV 33.55 0.23 6 35.46 0.71 6
CFX96
InfA-1 3XLOD FluA 36.73 0.45 6 37.44 0.54 6
InfB-2 3XLOD FluB 35.60 0.14 6 36.60 0.81 6
RSV-1 3XLOD RSV 37.65 0.43 6 38.16 1.87 6
SC2 3XLOD CoV 34.16 0.12 6 35.78 0.59 6
Precision (Repeatability and Reproducibility)
Within-laboratory precision was examined using a panel composed of co-spiked
influenza A (A/Georgia/T51700/2012 (H1N1)pdm09), influenza B
(B/Brisbane/60/2008 (Victoria Lineage), SARS-CoV-2 (USA-WA1/2020, heat-
inactivated) and/or RSV (A2001/3-12) cultures diluted in simulated clinical matrix
(viral transport medium plus human A549 cells). Two studies on eight instruments
(ABI 7500 Standard, ABI 7500 Fast Dx, CFX 384, CFX 96, QuantStudio Dx 384,
QuantStudio Dx 96, qTower 96, qTower 384) were performed. Sources of variability
were examined with a panel consisting of three concentration levels: Negative
samples, 2-3 LoD samples of co-infection mixture, 5-7 LoD samples of co-infection
mixture (4-plex for CFX 96, QuantStudio Dx 384, QuantStudio Dx 96, qTower 96
and qTower 384, 3-plex for ABI 7500 Standard, ABI 7500 Fast Dx, CFX 384).
Study 1 (reproducibility) used two lots of reagents over a time course of 3 days by
Page 35 of 42
at least 2 operators. Each evaluation sample was tested as duplicated for each lot
and each instrument of each run. The total is: 3X2X2X8X3 = 288, as listed in the
following table.
Study 2 (repeatability) was designed to evaluate 20 replicates of each panel for
each instrument by one lot of reagent.
Study 1: All negative panel members tested negative, and more than 98% positive
samples tested positive throughout the study. The statistical summary for 8
instruments is listed in the following table.
Panel Target Mean(Ct) Std Dev(Ct) CV %(Ct) Agreement
Percent Agreement, 95% CI (LL - UL)
3x LOD
SARS-CoV-2 35.47 0.91 2.57% 96/96 100.0% (96.2 -100.0%)
Influenza A 36.10 1.32 3.64% 96/96 100.0% (96.2 -100.0%)
Influenza B 34.74 1.35 3.89% 96/96 100.0% (96.2 -100.0%)
RNase P 29.54 1.20 4.07% 96/96 100.0% (96.2 -100.0%)
RSV 35.74 2.15 6.02% 59/60* 98.3 (91.1-99.7%)
6x LOD
SARS-CoV-2 34.47 1.02 2.96% 96/96 100.0% (96.2 -100.0%)
Influenza A 34.94 1.28 3.67% 96/96 100.0% (96.2 -100.0%)
Influenza B 33.53 1.45 4.32% 96/96 100.0% (96.2 -100.0%)
RNase P 29.63 1.21 4.10% 96/96 100.0% (96.2 -100.0%)
RSV 34.80 2.13 6.13% 60/60 100.0% (93.9 -100.0%)
Negative RNase P 29.16 1.00 3.43% 96/96 100.0% (96.2 -100.0%)
*Further investigation on the RSV results suggested that additional freeze-thaw cycles of the
samples may attribute to the negative result of one of the replicates.
Study 2: All negative panel members tested negative, and more than 95% positive
samples tested positive throughout the study. The statistical summary is listed in
the following table.
Instrument
Panel
Target Name Mean
(CT)
Std Dev
(CT)
CV %
(CT)
Agree
ment
Percent Agreement,
95% CI (LL - UL)
Applied Biosystems 7500 Fast Dx
3x LOD SARS-CoV-2 35.27 0.33 0.93 20/20 100% (83.9%-100.0%)
3x LOD Influenza A 36.06 0.30 0.83 20/20 100% (83.9%-100.0%)
3x LOD Influenza B 33.16 0.88 2.66 20/20 100% (83.9%-100.0%)
3x LOD RNase P 29.40 0.31 1.07 20/20 100% (83.9%-100.0%)
6x LOD SARS-CoV-2 34.87 0.33 0.96 20/20 100% (83.9%-100.0%)
6x LOD Influenza A 35.26 0.48 1.37 20/20 100% (83.9%-100.0%)
6x LOD Influenza B 32.01 1.05 3.28 20/20 100% (83.9%-100.0%)
6x LOD RNase P 29.22 0.23 0.78 20/20 100% (83.9%-100.0%)
Negative RNase P 28.88 0.23 0.78 20/20 100% (83.9%-100.0%)
Applied Biosystems 7500 Standard
3x LOD SARS-CoV-2 35.29 0.41 1.15 20/20 100% (83.9%-100.0%)
3x LOD Influenza A 35.50 0.37 1.03 20/20 100% (83.9%-100.0%)
3x LOD Influenza B 33.31 0.90 2.71 20/20 100% (83.9%-100.0%)
3x LOD RNase P 29.36 0.29 0.98 20/20 100% (83.9%-100.0%)
6x LOD SARS-CoV-2 34.76 0.32 0.91 20/20 100% (83.9%-100.0%)
6x LOD Influenza A 34.70 0.48 1.38 20/20 100% (83.9%-100.0%)
6x LOD Influenza B 32.14 1.20 3.74 20/20 100% (83.9%-100.0%)
6x LOD RNase P 29.19 0.25 0.87 20/20 100% (83.9%-100.0%)
Negative RNase P 29.73 0.71 2.39 20/20 100% (83.9%-100.0%)
Bio-Rad CFX384 3x LOD SARS-CoV-2 35.60 0.45 1.26 20/20 100% (83.9%-100.0%)
3x LOD Influenza A 34.93 0.37 1.07 20/20 100% (83.9%-100.0%)
3x LOD Influenza B 33.77 0.51 1.52 20/20 100% (83.9%-100.0%)
3x LOD RNase P 29.11 0.17 0.58 20/20 100% (83.9%-100.0%)
Page 36 of 42
6x LOD SARS-CoV-2 34.72 0.37 1.07 20/20 100% (83.9%-100.0%)
6x LOD Influenza A 33.73 0.28 0.83 20/20 100% (83.9%-100.0%)
6x LOD Influenza B 31.83 0.69 2.16 20/20 100% (83.9%-100.0%)
6x LOD RNase P 29.17 0.34 1.18 20/20 100% (83.9%-100.0%)
Negative RNase P 29.26 0.19 0.64 20/20 100% (83.9%-100.0%)
Bio-Rad CFX96 3x LOD SARS-CoV-2 35.37 0.39 1.09 20/20 100% (83.9%-100.0%)
3x LOD Influenza A 36.63 0.34 0.92 20/20 100% (83.9%-100.0%)
3x LOD Influenza B 35.02 0.80 2.29 20/20 100% (83.9%-100.0%)
3x LOD RNase P 29.46 0.27 0.90 20/20 100% (83.9%-100.0%)
3x LOD RSV 36.21 2.14 5.91 20/20 100% (83.9%-100.0%)
6x LOD SARS-CoV-2 34.53 0.39 1.13 20/20 100% (83.9%-100.0%)
6x LOD Influenza A 35.74 0.47 1.33 20/20 100% (83.9%-100.0%)
6x LOD Influenza B 34.16 0.80 2.33 20/20 100% (83.9%-100.0%)
6x LOD RNase P 29.49 0.87 2.93 20/20 100% (83.9%-100.0%)
6x LOD RSV 35.75 0.42 1.19 20/20 100% (83.9%-100.0%)
Negative RNase P 29.63 0.29 0.99 20/20 100% (83.9%-100.0%)
Applied Biosystem Quantstudio Dx 384
3x LOD SARS-CoV-2 34.59 0.32 0.92 20/20 100% (83.9%-100.0%)
3x LOD Influenza A 35.67 0.55 1.55 20/20 100% (83.9%-100.0%)
3x LOD Influenza B 32.34 0.92 2.84 20/20 100% (83.9%-100.0%)
3x LOD RNase P 28.95 0.19 0.64 20/20 100% (83.9%-100.0%)
3x LOD RSV 32.72 1.90 5.82 20/20 100% (83.9%-100.0%)
6x LOD SARS-CoV-2 33.61 0.34 1.00 20/20 100% (83.9%-100.0%)
6x LOD Influenza A 34.63 0.48 1.38 20/20 100% (83.9%-100.0%)
6x LOD Influenza B 31.30 0.86 2.75 20/20 100% (83.9%-100.0%)
6x LOD RNase P 29.09 0.76 2.61 20/20 100% (83.9%-100.0%)
6x LOD RSV 32.34 0.49 1.50 20/20 100% (83.9%-100.0%)
Negative RNase P 28.96 0.18 0.64 20/20 100% (83.9%-100.0%)
QuantStudio Dx 96
3x LOD SARS-CoV-2 35.21 0.51 1.46 20/20 100% (83.9%-100.0%)
3x LOD Influenza A 35.87 0.95 2.66 20/20 100% (83.9%-100.0%)
3x LOD Influenza B 36.54 0.85 2.32 20/20 100% (83.9%-100.0%)
3x LOD RNase P 27.22 0.36 1.32 20/20 100% (83.9%-100.0%)
3x LOD RSV 34.19 1.18 3.45 20/20 100% (83.9%-100.0%)
6x LOD SARS-CoV-2 34.50 0.33 0.95 19/19* 100% (83.2%-100.0%)
6x LOD Influenza A 35.23 0.53 1.51 19/19* 100% (83.2%-100.0%)
6x LOD Influenza B 35.50 0.62 1.74 19/19* 100% (83.2%-100.0%)
6x LOD RNase P 27.62 0.34 1.25 19/19* 100% (83.2%-100.0%)
6x LOD RSV 34.31 0.68 1.97 19/19* 100% (83.2%-100.0%)
Negative RNase P 27.49 0.33 1.19 20/20 100% (83.9%-100.0%)
qTower 96 3x LOD SARS-CoV-2 35.46 1.07 3.01 20/20 100% (83.9%-100.0%)
3x LOD Influenza A 36.84 1.07 2.92 20/20 100% (83.9%-100.0%)
3x LOD Influenza B 37.18 0.80 2.15 19/20 95.0% (76.4%-99.1%)
3x LOD RNase P 28.31 0.95 3.37 20/20 100% (83.9%-100.0%)
3x LOD RSV 36.52 1.36 3.73 20/20 100% (83.9%-100.0%)
6x LOD SARS-CoV-2 34.68 0.44 1.27 20/20 100% (83.9%-100.0%)
6x LOD Influenza A 36.16 0.58 1.60 20/20 100% (83.9%-100.0%)
6x LOD Influenza B 36.29 0.60 1.67 20/20 100% (83.9%-100.0%)
6x LOD RNase P 28.75 0.50 1.73 20/20 100% (83.9%-100.0%)
6x LOD RSV 36.06 0.93 2.59 20/20 100% (83.9%-100.0%)
Negative RNase P 28.93 0.39 1.35 20/20 100% (83.9%-100.0%)
qTower 384 3x LOD SARS-CoV-2 36.13 0.69 1.91 20/20 100% (83.9%-100.0%)
3x LOD Influenza A 35.82 0.87 2.44 20/20 100% (83.9%-100.0%)
3x LOD Influenza B 35.84 0.95 2.65 20/20 100% (83.9%-100.0%)
3x LOD RNase P 28.43 0.35 1.23 20/20 100% (83.9%-100.0%)
3x LOD RSV 36.06 0.83 2.31 20/20 100% (83.9%-100.0%)
6x LOD SARS-CoV-2 34.98 0.44 1.25 20/20 100% (83.9%-100.0%)
6x LOD Influenza A 34.65 0.66 1.90 20/20 100% (83.9%-100.0%)
6x LOD Influenza B 35.28 1.19 3.38 20/20 100% (83.9%-100.0%)
6x LOD RNase P 28.53 0.26 0.93 20/20 100% (83.9%-100.0%)
6x LOD RSV 34.90 0.73 2.10 20/20 100% (83.9%-100.0%)
Negative RNase P 27.87 0.48 1.72 20/20 100% (83.9%-100.0%)
* One of the replicates was invalid due to multichannel pipette operational error.
Clinical study
Clinical performance of the PKamp™ Respiratory SARS-CoV-2 RT-PCR Panel
Page 37 of 42
was evaluated using residual nasopharyngeal swab (NPS) collected from patients
who were suspected to have a respiratory viral infection by a healthcare provider.
All positive and negative archived NPS samples were previously tested with FDA
EUA authorized or cleared method for Influenza A, Influenza B, SARS-CoV-2, and
respiratory syncytial virus (RSV). The PKamp™ Respiratory SARS-CoV-2 RT-PCR
Panel test results were compared with the archived test results or parallel test result
using a FDA cleared method.
The clinical screening performance summary of results for the 373 NPS clinical
samples analyzed with the PKamp™ Respiratory SARS-CoV-2 RT-PCR Panel are
shown in the following table. Overall, the assay demonstrated good sensitivity,
specificity and agreement with the comparator tests for the detection of SARS-CoV-
2, Influenza A viruses, Influenza B viruses and RSV. Influenza B and RSV showed
100% for both PPA and NPA; Influenza A established PPA>90% and NPA=100%;
SARS-CoV-2 demonstrated >95% for both PPA and NPA.
The PKamp™ Respiratory SARS-CoV-2 RT-PCR Panel assay was derived from the
same reagent platform as the FDA EUA authorized PerkinElmer New Coronavirus
Nucleic Acid Detection Kit (EUA2000055), which has demonstrated the best
detection sensitivity in the approved FDA EUA kits for SARS-CoV-2 detection (7). It
was thought that the 5 discordant positive sample for Influenza A and the 4
discordant positive samples for SARS-CoV-2 are due to other cause (e.g. sample
stability) than lack of sensitivity for the PKamp™ Respiratory SARS-CoV-2 RT-PCR
Panel assay, however this was not verified as the residual samples were insufficient
for further testing.
Virus
Number
of samples
Test results Agreement Statistics
Concordant Positive (N)
Discordant
Positive (N)
Concordant Negative(N)
Discordant
Negative (N)
Agreement Parameter
Percent
Agreement (%)
95% CI LCL, UCL
Influenza A
125
53
5
67
0
PPA 91.4% 81.4%,96.3%
NPA 100% 94.6%, 100%
Influenza B
103
36
0
67
0
PPA 100% 90.4%, 100%
NPA 100% 94.6%, 100%
RSV
114
55
0
59
0
PPA 100% 93.5%, 100%
NPA 100% 94.0%, 100%
SARS- CoV-2
156
79
4
70
3
PPA 95.2% 88.3%, 98.1%
NPA 95.9% 88.6%, 98.6%
Total 223 9 263 3
Page 38 of 42
Appendix
Applied Biosystems QuantStudio Dx Calibration
This PCR assay utilizes all the detector channels available in the instrument
including Cy5.5. This plate is only available through PerkinElmer. PerkinElmer
also recommends using our Cy5 calibration plate order to ensure Cy5 detection
quality.
Part Number Description
4178-0010 384-well Spectral Calibration Plate with Cy5 Dye
4179-0010 384-well Spectral Calibration Plate with Cy5.5 Dye
4172-0010 96-well Spectral Calibration Plate with Cy5 Dye
4173-0010 96-well Spectral Calibration Plate with Cy5.5 Dye
Additionally, special attention must be given to the calibration curve results for the
background calibration. Even if the background calibration passes, there must be
no outlier well curves in the Background Calibration. Outlier wells should be
identified, and the block should be removed and cleaned thoroughly before
proceeding with additional calibrations. A new ROI and background calibration are
recommended at that point.
Calibration procedure:
NOTE: Calibration plates should be thawed 30 minutes before use and should not be used after 2 hours of being thawed so do not thaw more than 2-3 plates at a time. Calibration plates cannot be frozen/re-used again.
NOTE: Before any calibration, vortex the calibration plate for 5 seconds and centrifuge the plate for 2 minutes at < 1500 rpm.
NOTE: Do not allow the bottoms of the plates to become dirty. Fluids and other contaminants that adhere to the bottoms of the consumables can contaminate the sample block and cause an abnormally high background signal.
Calibrate the instrument in IVD or RUO mode following the specific plate order and
instructions below (if it is calibrated in IVD mode, it can cover both IVD and RUO
mode):
1. ROI calibration
2. Background calibration – regardless of the pass/fail outcome of the calibration,
all the traces should be in a tight bundle. Check for outlier wells in the
calibration results (I.e. any well whose calibration trace is significantly different
from the rest of the wells, see Figure below). Even if the calibration “Passes”,
Page 39 of 42
wells with curves that are obviously higher than most curves may produce a
shift in the Ct calls for specific analytes. If outlier wells are apparent, the block
and/or cover must be cleaned, and the ROI and Background calibration
performed again using the same plates but inverting them for the repeat
calibration.
NOTE: Recommended cleaning procedure:
• Identify outlier well(s) in the block (rotated 180° if compared with the plate
calibration results). Visually check if there’s any debris in the well(s) and clean
that well(s) with appropriate QTIP.
• Pipette 20µL of 10% bleach solution into the well. Let it sit for 1 minute and
dry/clean with appropriate QTIP.
• Repeat the previous step with water.
• Repeat the previous step with 70% Ethanol.
Figure: example of outlier in Background Calibration:
If after cleaning the block the background calibration results in the same outlier(s), the optical cover/path may need to be cleaned/checked.
3. Uniformity calibration
Note: Change the instrument to RUO mode to check if Uniformity transferred. If not, repeat Uniformity calibration in RUO mode. The switch back to IVD mode.
4. CY5 calibration (PerkinElmer Brand calibration plate)*
5. OTHER calibration – CY5.5 (PerkinElmer Brand calibration plate)*
Page 40 of 42
6. Calibrate FAM, VIC and ROX.
7. Normalization calibrations (FAM/ROX and VIC/ROX)
* If calibration fails, check the QC tab and check whether there is any outlier in the
calibration plot. Make sure that the bottom of the plate is clean and re-run the
calibration inverting the plate up to two additional times. If the calibration still fails
after two repeated attempts, a new CY5 or CY5.5 plate must be used. If failure still
occurs with the new plate(s), clean the instrument block and start over from Step 1.
Page 41 of 42
References
1. Laboratory testing for 2019 novel coronavirus (2019-nCoV) in suspected human cases: World Health Organization; 2020.
2. Innis, MA, et Al, and the PCR Protocols Applications: A Laboratory Manual, Academic, New York, 1989.
3. Mahony JB. Detection of Respiratory Viruses by Molecular Methods. Clinical Microbiology Reviews. 2008; 21 (4): 716-747.
4. CDC specimen collection guidelines: https://www.cdc.gov/urdo/downloads/speccollectionguidelines.pdf
5. Interim Guidelines for Collecting, Handling, and Testing Clinical Specimens for COVID-19: https://www.cdc.gov/coronavirus/2019-ncov/lab/guidelines-clinical- specimens.html
6. FDA regulation on SARS-CoV-2: https://www.fda.gov/media/135900/download (July 28, 2020 version).
7. https://www.fda.gov/medical-devices/coronavirus-covid-19-and-medical- devices/sars-cov-2-reference-panel-comparative-data.
Page 42 of 42
Basic Information
Emergo Europe, Prinsessegracht 20 2514 AP The Hague, The Netherlands
Suzhou Sym-Bio Lifescience Co., Ltd.
No. 115, North Taiping Road, Taicang, Jiangsu Province, 215400, China
Contact Information: +86 0512 53378788
Company Website: www.perkinelmer.com
After-Sales Unit
PerkinElmer Medical Diagnostic Product (Shanghai) Co., Ltd.
Contact information:
+86 800 820 5046 +86 400 820 5046
Fax: +86 021 60645858
E-mail: service.symbio@perkinelmer.com
Modification date and version: 2020-11-10 V3.1
Approval date : 2020-11-12