instructions for pkamp™ respiratory sars-cov-2 rt-pcr panel

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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 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



finished









Component Volume/

test

Volume for N Samples and

Positive Control 110% of volume



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.



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containing PCR mix.


mix.



Setup PCR Manually for 15μL PCR Reactions in 384-well Plate on Instrument with FAM™, HEX™/VIC™, ROX™, Cy®5 and Cy5.5® Channels



finished









Component Volume/ test Volume for N Samples

and Positive Control 110% of volume



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





containing PCR mix.


mix.

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6) Seal PCR the plate with an appropriate film.


Setup PCR Manually for 15μL PCR Reactions on in 384-well Plate on Instrument with FAM™, HEX™/VIC™, ROX™, and Cy®5 Channels



finished









Component Volume/ test Volume for N Samples

and Positive Control 110% of volume



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.





containing PCR mix.


mix.



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

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CFX 96 specifically

Control

name

Ct

SARS-CoV-2

(FAM)

Influenza A

(ROX)

Influenza B

(Cy5)

RSV

(Cy5.5)


(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)


(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

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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)


(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,

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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

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>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,

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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

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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

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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




Table. Study for the instruments with FAM, HEX/VIC, ROX and Cy5 detection capability. Condition SARS-CoV-2 Influenza A Influenza B RSV




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

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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

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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


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


6 Influenza A (H3N8) A/equine/Pennsylvania/1/200

7 (H3N8) 1.60E-01


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


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


12 Influenza B B/Florida/4/2006 (Yamagata

Lineage) 1.60E+02


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


16 Influenza B B/Ohio/01/2005 (Victoria Lineage)

1.60E+03


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





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12 ND ND ND 32.21 32.54 33.06 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


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


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.

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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

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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

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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

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 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

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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


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


B/Texas/81/2 016

3.99E+06

0

0

0

0

15.32

0.09

5

0

0

0


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


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



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


B/Phuket/307 3/2013

1.59E+08

0

0

0

0

0

0

15.59

0.35

5

0

0

0


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)



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


3.17E+08

0

0

0

16.45

0.18

5

0

0

0

NA

NA

NA

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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


B/Michigan/ 09/2011

1.59E+05

0

0

0

0

0

0

18.91

0.18

5

NA

NA

NA


B/Washingt on/02/2019

3.17E+07

0

0

0

0

0

0

16.82

0.29

5

NA

NA

NA


B/Texas/81/ 2016

3.99E+06

0

0

0

0

0

0

17.54

0.17

5

NA

NA

NA


B/Phuket/30 73/2013

1.59E+08

0

0

0

0

0

0

17.22

0.21

5

NA

NA

NA


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)


Mean SD N Mean SD N Mean SD N Me an

S D

N



3.17E+08

0.00

0.00

0

15.96

0.22

3

0

0

0

NA N A

N A


B/Washingt on/02/2019

3.17E+07

0

0

0

0

0

0

14.78

0.31

3

NA N A

N A


A2001/3-12

3.20E+06

0

0

0

0

0

0

0

0

0

NA N A

N A

SARS- coronavirus-2


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)





3.17E+08

0

0

0

14.38

0.42

3

0

0

0

NA

NA

NA


B/Washington/ 02/2019

3.17E+07

0

0

0

0

0

0

14.68

0.24

3

NA

NA

NA


A2001/3-12

3.20E+06

0

0

0

0

0

0

0

0

0

NA

NA

NA

SARS- coronavirus-2


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-

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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


Fluticasonea 5% v/v NA NA NA 3/3 detected at 10XLOD

Budesonide (Glucocorticoid)b 1% v/v

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

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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

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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%)

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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

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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

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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”,

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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)*

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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.

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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.

https://www.cdc.gov/urdo/downloads/speccollectionguidelines.pdf

https://www.cdc.gov/urdo/downloads/speccollectionguidelines.pdf

https://www.cdc.gov/coronavirus/2019-ncov/lab/guidelines-clinical-specimens.html



http://www.fda.gov/media/135900/download

http://www.fda.gov/media/135900/download

http://www.fda.gov/medical-devices/coronavirus-covid-19-and-medical-

http://www.fda.gov/medical-devices/coronavirus-covid-19-and-medical-

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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: [email protected]

Modification date and version: 2020-11-10 V3.1

Approval date : 2020-11-12

http://www.perkinelmer.com/

mailto:[email protected]

instructions for pkamp™ respiratory sars-cov-2 rt-pcr panel

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