For Professional Use Only

Allplex™
SARS-CoV-2 fast PCR Assay
(Cat. No. RV10344X, RV10345Z)

Multiplex real-time one-step RT-PCR system for detection of SARS-CoV-2 from

nasopharyngeal swab, saliva, and combo-swab (nasal swab + oral swab).

For use with

1. CFX96™ Real-time PCR Detection System (CFX Manager™ Software-IVD v1.6)
2. CFX96™ Dx System (CFX Manager™ Dx Software v3.1)
3. Applied Biosystems™ 7500 (7500 Software v2.0.5 / v2.3)
4. SGRT (Seegene Real-time Thermocycler) (SGRT Manager Lite v1.00)

For in vitro diagnostic use only

RV10344X RV10345Z

Seegene Inc.
Taewon Bldg., 91 Ogeum-ro, Songpa-gu, Seoul, Republic of Korea 05548

Medical Technology Promedt Consulting GmbH

Ernst-Heckel-Straß e 7, 66386 St. Ingbert, Germany

Not available in the U.S.

 Allplex™ SARS-CoV-2 fast PCR Assay

TABLE OF CONTENTS

NOTICES ----------------------------------------------------------------------------------------- 3

INTENDED USE -------------------------------------------------------------------------------- 5

PRINCIPLES AND PROCEDURE OVERVIEW ------------------------------------------- 6

BACKGROUND INFORMATION ----------------------------------------------------------- 7

REAGENTS -------------------------------------------------------------------------------------- 8

STORAGE AND HANDLING ---------------------------------------------------------------- 10

MATERIALS REQUIRED BUT NOT PROVIDED -------------------------------------- 10

PROTOCOL -------------------------------------------------------------------------------------- 12

REAL-TIME PCR INSTRUMENT SET UP AND RESULTS ANALYSIS ---------- 25

RESULTS ------------------------------------------------------------------------------------------ 62

TROUBLESHOOTINGS --------------------------------------------------------------------------- 66

PERFORMANCE ---------------------------------------------------------------------------------- 68

REFERENCES ------------------------------------------------------------------------------------- 78

KEY TO SYMBOLS -------------------------------------------------------------------------------- 80

ORDERING INFORMATION ------------------------------------------------------------------- 81

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NOTICES

⚫ For in vitro diagnostic use only.

⚫ The Allplex™ SARS-CoV-2 fast PCR Assay should be performed by qualified, trained
personnel.
⚫ Reliability of the results depends on adequate specimen collection, storage, transport, and
processing procedure.
⚫ If this product is used with Microlab NIMBUS IVD, Microlab STARlet IVD, Seegene
NIMBUS, and Seegene STARlet, it provides maximum 5 separate runs.
⚫ This test has been validated for the following specimen type: nasopharyngeal swab,
saliva, and combo-swab (nasal swab + oral swab). This test has not been validated for
any other types of specimens.
⚫ Store RNA samples at ≤ -20°C until use and keep on ice during use.
⚫ Sensitivity of the assay may decrease if samples are repeatedly frozen/thawed or stored for
a longer period of time.
⚫ Workflow in a laboratory should proceed in a unidirectional manner.
⚫ Wear disposable gloves and change them before entering different areas. Change gloves
immediately if contaminated or treat them with DNA decontaminating reagent.
⚫ Supplies and equipment must be dedicated to working areas and should not be moved from
one area to another.
⚫ Do not pipette by mouth.
⚫ Do not eat, drink, or smoke in laboratory work areas. Wear disposable powder-free gloves,
laboratory coats and eye protections when handling specimens and reagents. Wash hands
thoroughly after handling specimens and test reagents.
⚫ Avoid contamination of reagents when removing aliquots from reagent tubes. Use of
sterilized aerosol resistant disposable pipette tips is recommended.
⚫ Do not pool reagents from different lots or from different tubes of the same lot.
⚫ Do not use the product after its expiry date.
⚫ Do not reuse all disposable items.
⚫ Use screw-capped tubes and prevent any potential splashing or cross-contamination of
specimens during preparation.
⚫ Be careful not to contaminate reagents with extracted nucleic acids, PCR products, and
positive controls. To prevent contamination of reagents, the use of filter-tips is
recommended.

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⚫ Use separated and segregated working areas for each experiment.

⚫ To avoid contamination of working areas with amplified products, open PCR reaction tubes
or strips only at designated working areas after amplification.
⚫ Store positive materials separately from the kit’s reagents.
⚫ Laboratory safety procedures (refer to Biosafety in Microbiological and Biomedical
Laboratories & CLSI Documents) must be taken when handling specimens. Thoroughly
clean and disinfect all work surfaces with 0.5% sodium hypochlorite (in de-ionized or distilled
water). Product components including product residuals and packaging can be considered
as laboratory waste. Dispose of unused reagents and waste in accordance with applicable
federal, state, and local regulations.
⚫ Expiry date is 13 months from the date of manufacture at ≤ -20°C. Please refer to label for
the expiry date.
⚫ Clinical correlation with patient history and other diagnostic information is necessary to
determine patient infection status.
⚫ Seegene NIMBUS and Seegene STARlet are the same equipment as the Microlab NIMBUS
IVD and Microlab STARlet IVD, respectively, although the manufacturers are different from
each other. Since there are no hardware changes on the devices, the test results are the
same for those.
⚫ The brand name of “CFX96™ Real-time PCR Detection System-IVD” has been changed to
“CFX96™ Dx system”. Since there are no hardware changes on the systems, it will be
expected to obtain the same results from both systems.
⚫ “CFX Manager™ Dx Software v3.1” is an upgrade version of “CFX Manager™ Software-IVD
v1.6”. The upgraded software includes enhancements to the “Run” menu. These
enhancements do not impact the results of data analysis; therefore, the results will be the
same.
⚫ This kit is a qualitative in vitro test for the single or multiple detection of 3 types of gene (E
gene, RdRP gene, and N gene).
⚫ Negative results do not preclude COVID-19 and should not be used as the sole basis for
patient management decisions. Negative results must be combined with clinical
observations, patient history, exposures and epidemiological information.
⚫ AIOS combines Seegene STARlet sold by Seegene with real-time PCR equipment (CFX96
Dx, Manufacturer: Bio-Rad) and plate sealer (Manufacturer: SAMICK THK) to form an
automated linkage structure of nucleic acid extraction to PCR.

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 Allplex™ SARS-CoV-2 fast PCR Assay

INTENDED USE

Allplex™ SARS-CoV-2 fast PCR Assay is a qualitative real-time one-step RT-PCR in vitro
diagnostic test for detection of SARS-CoV-2 target genes (E gene, RdRP gene and N gene) in
nasopharyngeal swab, saliva, and combo-swab (nasal swab + oral swab) obtained from
individuals suspected of COVID-19 by their healthcare provider, and those without symptoms or
other reasons to suspect COVID-19. The test is intended for use as an aid in the diagnosis of
respiratory infection if used in conjunction with other clinical and epidemiological information.
Allplex™ SARS-CoV-2 fast PCR Assay is intended for professional use only.

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 Allplex™ SARS-CoV-2 fast PCR Assay

PRINCIPLES AND PROCEDURE OVERVIEW

1. Principles

Allplex™ SARS-CoV-2 fast PCR Assay is a multiplex real-time RT-PCR assay that enables
simultaneous amplification and detection of 3 types of SARS-CoV-2 gene (E gene, RdRP gene,
and N gene) with Internal Control (IC). The presence of specific gene sequences in the reaction is
reported as a Ct value through Seegene Viewer analysis software.
An endogenous gene is used as Internal Control (IC) to monitor the whole process of sample
collection, nucleic acid extraction and to check for any possible PCR inhibition.
To prevent amplification product from acting as potential contaminants, Uracil-DNA glycosylase
(UDG)-dUTP system is employed in Allplex™ SARS-CoV-2 fast PCR Assay. The UDG-dUTP
system is commonly used when performing PCR to eliminate amplicon carry-over using UDG to
excise uracil residues from DNA by cleaving the N-glycosylic bond.

2. Procedure Overview

Samples
(Nasopharyngeal swab, Saliva,
and Combo-swab (nasal swab + oral swab))

Nucleic acid extraction

Nucleic acid

Real-time RT-PCR using

Allplex™ system

Analysis of Results

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 Allplex™ SARS-CoV-2 fast PCR Assay

BACKGROUND INFORMATION

1. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)

Coronavirus is an RNA virus which contains approximately 27~32 kb of positive single-

stranded RNA genome. In human, it causes respiratory tract infections that can range from mild
to lethal. In December 2019, a number of patients with pneumonia of unknown aetiology
emerged in Wuhan, China and Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-
2), a novel coronavirus has been identified as the causative agent. Coronavirus disease 19
(COVID-19) has been spreading around the world and the World Health Organization (WHO)
has declared COVID-19 pandemic in March 2020. By March 2021, more than 100,000,000
confirmed cases and more than 2,500,000 deaths of COVID-19 have been reported to the WHO
worldwide.
SARS-CoV-2 is transmitted through droplets from infected patients. The COVID-19 infected
patients have clinical manifestation which includes the fever and cough as primary clinical
presentations and others are shortness of breath and myalgia etc. However, asymptomatic
infection cases are also emerging, so it is very important to quickly diagnose and take
quarantine measure.

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REAGENTS

The reagents contained in one kit are sufficient for 100 reactions.
Order information ( Ca RV10344X)

Allplex™ SARS-CoV-2 fast PCR Assay

Symbol Contents Volume Description

Oligo Mix:
SC2F MOM 500 L
- Amplification and detection reagent

- RTase
- DNA polymerase
SEMXR2 500 L - Uracil-DNA glycosylase (UDG)
- Buffer containing BSA and Glycerol

Buffer for Real-time PCR

SEMXR2 Buffer 500 L - Buffer containing dNTPs

Positive Control (PC):

SC2F PC 50 L
- Mixture of pathogen and IC clones

RNase-free Water 1,000 L Ultrapure quality, PCR-grade

User manual

Accessory product – analysis software

Seegene Viewer*

* The analysis software is provided by Seegene Inc. or regional manager. Please use Seegene
Viewer beyond V3.

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 Allplex™ SARS-CoV-2 fast PCR Assay

The reagents contained in one kit are sufficient for 25 reactions.

Order information ( Ca RV10345Z)

Allplex™ SARS-CoV-2 fast PCR Assay

Symbol Contents Volume Description

Oligo Mix:
SC2F MOM 125 L
- Amplification and detection reagent

- RTase
- DNA polymerase
SEMXR2 125 L - Uracil-DNA glycosylase (UDG)
- Buffer containing BSA and Glycerol

Buffer for Real-time PCR

SEMXR2 Buffer 125 L - Buffer containing dNTPs

Positive Control (PC):

SC2F PC 50 L
- Mixture of pathogen and IC clones

RNase-free Water 1,000 L Ultrapure quality, PCR-grade

User manual

Accessory product – analysis software

Seegene Viewer*

* The analysis software is provided by Seegene Inc. or regional manager. Please use Seegene
Viewer beyond V3.

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 Allplex™ SARS-CoV-2 fast PCR Assay

STORAGE AND HANDLING

All components of Allplex™ SARS-CoV-2 fast PCR Assay should be stored at ≤ -20°C. All
components are stable under recommended storage conditions until the expiry date stated on
the label. The performance of kit components is not affected for up to 5 freezing and thawing. If
the reagents are to be used only intermittently, they should be stored in aliquots.

MATERIALS REQUIRED BUT NOT PROVIDED

⚫ Disposable powder free gloves (latex or nitrile)

⚫ Pipettes (adjustable) and Sterile pipette tips
⚫ 1.5 mL microcentrifuge tubes
⚫ Nucleic acid extraction kit (see Nucleic Acid Extraction)
⚫ Clean bench
⚫ Ice Maker
⚫ Desktop centrifuge
⚫ Vortex mixer
⚫ CFX96™ Real-time PCR Detection system (Bio-Rad)
⚫ CFX96™ Dx System (Bio-Rad)
⚫ Applied Biosystems™ 7500 (Thermo Fisher Scientific)
⚫ SGRT (Seegene Real-time Thermocycler) (Seegene)
⚫ AIOS (Cat. No. SG72100, Seegene)
⚫ Pierceable cap (Cat. No. 922119, SPL) (for AIOS use only)
⚫ PX1 PCR plate sealer (auto-sealer, Cat. No. 181-4000, Bio-Rad)*
⚫ PCR consumables**

Manufacturer PCR tube Applicable Cover

Low-Profile 0.2 mL 8-Tube Strips without Optical Flat 8-Cap Strips
Caps (white color, Cat. No. TLS0851) (Cat. No. TCS0803)
Optical Flat 8-Cap Strips
Hard-Shell® 96-Well PCR Plates, low
(Cat. No. TCS0803)
profile, thin wall, skirted, white/white
Permanent Clear Heat Seal
Bio-Rad (Cat. No. HSP9655)
(Cat. No. 1814035)*
®
Hard-Shell 96-Well PCR Plates, low Optical Flat 8-Cap Strips
profile, thin wall, skirted, white/white, (Cat. No. TCS0803)
barcoded Permanent Clear Heat Seal
(Cat. No. HSP9955) (Cat. No. 1814035)*
EU 0.1mL 8-tube strip, LP, W, Extra EU Optical Wide area 8-Cap Strip
Robust (Cat. No. B72719) (Cat. No. B57801)
BIOplastics
96 x 0.1mL Plate, LP, W, FULL, 96 well Opti-Seal Optical Sealing Sheet
plate (Cat. No. B70679) (Cat. No. 157300)

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MicroAmp™ Optical 8-Tube Strip, 0.2 MicroAmp™ Optical 8-Cap Strips

mL (Cat No. 4316567) (Cat No. 4323032)
MicroAmp™ Optical 8-Cap Strips
MicroAmp® Optical 96-Well Reaction (Cat No. 4323032)
Thermo
Plate (Cat. No. N8010560) Optical Adhesive Covers
Fisher
(Cat No. 4360954)
Scientific §
MicroAmp™ Optical 8-Cap Strips
MicroAmp™ Optical 96-Well Reaction (Cat No. 4323032)
Plate with Barcode (Cat. No. 4306737) Optical Adhesive Covers
(Cat No. 4360954)
* Make sure to use the heat seal and the plate sealer listed above together.
** Make sure to use the plate/8-tube strip with compatible cover shown in the table.
§ Products for Applied Biosystems™ 7500

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 Allplex™ SARS-CoV-2 fast PCR Assay

PROTOCOL

1. Specimen Collection, Storage, and Transport

Note: All samples should be treated as potentially infectious materials. Only permitted are those
sample materials, which are collected, transported and stored by attending strictly to the following
rules and instructions.
Note: To ensure high quality of samples, samples should be transported as fast as possible at
indicated temperature.

A. Specimen Collection

Nasopharyngeal swab
⚫ Nasopharyngeal swab is routinely examined for common respiratory pathogens.
⚫ Obtaining respiratory specimens may be difficult in some patients. In such cases,
nasopharyngeal swabs may be collected simply and efficiently using new nylon flocked
swabs (COPAN, Italy) and Universal Transport Medium (UTM).

Manufacturer Specimen collection device Cat. No.

COPAN ESwab 482CE

COPAN ENAT PM 2ML PERNASAL APPLICATOR** 606CS01P*

ENAT KIT PNR CAP 2ML+ REGULAR
COPAN FLOQSWAB BP80** 6U063S01***
ENAT KIT PNR CAP 2ML+
COPAN PROGR.FLEXIBLE FLOQSWAB BP80** 6U082S01***

COPAN UTM with Flocked Swabs 360C / 305C

SG Medical GeneTM Set (GTS2)** T5001

SG Medical GeneTM Set (GTS1)** T5002

SG Medical ALLTM Set (ATS 1) T5003

SG Medical ALLTM Set (ATS 2) T5004

SG Medical ALLTM Medium T5103

*Please use catalog numbers shown above to purchase products from Seegene Inc.
**ENAT PM 2ML PERNASAL APPLICATOR, ENAT KIT PNR CAP 2ML+REGULAR FLOQSWAB
BP80, ENAT KIT PNR CAP 2ML+PROGR.FLEXIBLE FLOQSWAB BP80, GeneTM Set (GTS1) and
GeneTM Set (GTS2) are not applicable to extraction-free method.
***Reseal with a replacement cap (Cat. No. 2E003S500, COPAN) after use for further storage.

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Saliva
⚫ Do not eat, drink, smoke, brush teeth, or chew gum for 30 min before sample collection.
⚫ Wash your hands thoroughly for 30 sec.
⚫ Remove the cap of the sterilized empty tube.
Note: Do not touch the inside of the cap and the inside of the sterilized tube.
⚫ Gently expel saliva into the sterilized tube until 1~2 mL has been collected. Be very careful
not to splash or aerosols.
Note: If saliva gets on the outside of the tube, wipe it with an alcohol cotton pad.
⚫ Screw the cap of the specimen tube tightly.

Combo-swab (nasal swab + oral swab)

⚫ Give instruction to patients not to eat, drink, smoke, brush teeth, or chew gum within 1
hour before collecting combo-swab specimens.

a. Oral swab
⚫ Before collecting the specimen, take a deep breath (while wearing a mask) and cough 3
to 5 times.
⚫ Rub the collection tip of the swab over the inside of both cheeks, above and below the
tongue, on both outer (gums), and on the hard palate for total of 20 seconds.
⚫ Place swab, tip first, into the transport tube provided. Break the swab shaft at the
scoreline and then close the lid.

b. Nasal swab
⚫ Insert the entire collection tip of the swab approximately 3 to 4 cm inside the nostril.
⚫ Firmly sample the nasal wall by rotating the swab in a circular path against the nasal
wall for 5~10 seconds.
⚫ Repeat in the other nostril using the same swab.
⚫ Place swab, tip first, into the oral swab-containing transport tube provided. Break the
swab shaft at the scoreline and then close the lid.

Manufacturer Specimen collection device Cat. No.

SG Medical SELTM Set (STS2)* T5021
* SELTM Set (STS2) consists of nasal swab, oral swab, and ALLTM Medium. Also, it is not
applicable to extraction-free method.

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B. Specimen Storage & Transport

Storage & Transport

Specimen Note
Temp. Duration*

Nasopharyngeal swab
- Performance may be affected

2~8°C 3 days by prolonged storage of

specimens.
Combo-swab
- Specimens should also adhere
(nasal swab + oral swab)
to local and national instructions
for transport of pathogenic
2~8°C 4 days
materials.
Saliva
15~25°C 2 days

* Duration: The time period from the specimen collection to the final test (includes transport and
storage of specimens prior to tests).

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2. Nucleic Acid Extraction

[Extraction methods in different specimens]

Note: Please use the automated extraction system according to the specimen shown in the
following table.

Specimen

Automated Extraction System Combo-swab

Nasopharyngeal
Saliva (nasal swab +
swab*
oral swab)

Universal Cartridge Kit O O O

Microlab
Viral DNA/RNA 200 C Kit O O O
NIMBUS IVD / STARlet IVD

STARMag™ N/S Kit O O O

Universal Cartridge Kit O O O

Seegene
Viral DNA/RNA 200 C Kit O O O
NIMBUS / STARlet
STARMag™ N/S Kit O O O

Seegene STARlet 96MPH STARMag™ S96H Kit O O O

MagNA Pure 96 O O O

TANBead® Nucleic Acid

O O O
Extraction Kit
Maelstrom™ 9600
STARMag™ M96 Kit O O O

STARMag 96 ProPrep O O O

SEEPREP32 STARMag 96 ProPrep C O O O

STARMag™ SP32 Kit O O O

Extraction-free O O X

* Nasopharyngeal swab specimen is validated with extraction-free method. However, swab

specimens using ENAT PM 2ML PERNASAL APPLICATOR, ENAT KIT PNR CAP
2ML+REGULAR FLOQSWAB BP80, ENAT KIT PNR CAP 2ML+PROGR.FLEXIBLE FLOQSWAB
BP80, GeneTM Set (GTS1) and GeneTM Set (GTS2) are not applicable to extraction-free
method.

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2-1. Standard Extraction

A. Pre-treatment of specimen

Saliva

⚫ Add 1 volume of 1X PBS to the 1 volume specimen in the sterilized tube and vortex
thoroughly to disperse the sample.
⚫ Transfer recommended volume (See Recommended Vol. of 2-B) of sample to a new
tube.
⚫ Follow the extraction kit protocol.

B. Automated Nucleic Acid Extraction System

Note: Please use the recommended volumes of specimen and elution as indicated below. For
other matters, refer to the manufacturer’s manual.

B-1. Microlab NIMBUS IVD

Note: See Microlab NIMBUS IVD operation manual.

Automated Extraction System Manufacturer Cat. No. Recommended Vol.

Microlab NIMBUS IVD Hamilton 65415-02* -

744300.4. Specimen: 300 L

STARMag 96 X 4 Universal Cartridge Kit Seegene
UC384 Elution: 100 L
STARMag 96 X 4 Viral DNA/RNA Specimen: 300 L
Seegene EX00013C
200 C Kit Elution: 100 L
Specimen: 300 L
STARMag™ N/S Kit Seegene EX00031C
Elution: 100 L
* Please use catalog numbers shown above to purchase products from Seegene Inc.

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B-2. Microlab STARlet IVD

Note: See Microlab STARlet IVD operation manual.

Automated Extraction System Manufacturer Cat. No. Recommended Vol.

Microlab STARlet IVD Hamilton 173000-075* -

744300.4. Specimen: 300 L

STARMag 96 X 4 Universal Cartridge Kit Seegene
UC384 Elution: 100 L
STARMag 96 X 4 Viral DNA/RNA Specimen: 300 L
Seegene EX00013C
200 C Kit Elution: 100 L
Specimen: 300 L
STARMag™ N/S Kit Seegene EX00031C
Elution: 100 L
* Please use catalog numbers shown above to purchase products from Seegene Inc.

B-3. Seegene NIMBUS

Note: See Seegene NIMBUS operation manual.

Automated Extraction System Manufacturer Cat. No. Recommended Vol.

Seegene NIMBUS Seegene 65415-03 -

744300.4. Specimen: 300 L

STARMag 96 X 4 Universal Cartridge Kit Seegene
UC384 Elution: 100 L
STARMag 96 X 4 Viral DNA/RNA Specimen: 300 L
Seegene EX00013C
200 C Kit Elution: 100 L
Specimen: 300 L
STARMag™ N/S Kit Seegene EX00031C
Elution: 100 L

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B-4. Seegene STARlet

Option: Automated Linkage Structure (See AIOS operation manual)

Automated Linkage Structure Manufacturer Cat. No.

AIOS Seegene SG72100

Note: Replace the cap of the Positive Control (PC) with a pierceable cap. After finishing the
operation, replace the cap of the Positive Control (PC) with the original cap.
Note: The pierceable cap is a single-use product and must be disposed of after one use.
Note: If this product is used with AIOS maximum 3 separate runs.

Note: See Seegene STARlet operation manual.

Automated Extraction System Manufacturer Cat. No. Recommended Vol.

Seegene STARlet Seegene 67930-03 -

744300.4. Specimen: 300 L

STARMag 96 X 4 Universal Cartridge Kit Seegene
UC384 Elution: 100 L
STARMag 96 X 4 Viral DNA/RNA Specimen: 300 L
Seegene EX00013C
200 C Kit Elution: 100 L
Specimen: 300 L
STARMag™ N/S Kit Seegene EX00031C
Elution: 100 L

B-5. Seegene STARlet 96MPH

Note: See Seegene STARlet 96MPH operation manual.

Automated Extraction System Manufacturer Cat. No. Recommended Vol.

Seegene STARlet 96MPH Seegene SG71101 -

EX00032P,
EX00033P, Specimen: 300 L
STARMag™ S96H Kit Seegene
EX00034P, Elution: 100 L
EX00035P

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B-6. MagNA Pure 96

Note: See MagNA Pure 96 operation manual.

Automated Extraction System Manufacturer Cat. No. Recommended Vol.

Roche
MagNA Pure 96 06541089001 -
Diagnostics
MagNA Pure 96 DNA and Viral NA Roche Specimen: 200 L
06543588001
Small Volume Kit Diagnostics Elution: 100 L

B-7. Maelstrom™ 9600

⚫ Proceed the extraction process using ‘665-Rapid’ protocol.

Automated Extraction System Manufacturer Cat. No. Recommended Vol.

Taiwan
Maelstrom™ 9600 Advanced M9600* -
Nanotech Inc.
Taiwan
TANBead® Nucleic Acid Extraction Kit Specimen: 300 L
Advanced W665S66
OptiPure Viral Auto Tube Elution: 80 L
Nanotech Inc.
Taiwan
TANBead® Nucleic Acid Extraction Kit Specimen: 300 L
Advanced W665A46
OptiPure Viral Auto Plate Elution: 80 L
Nanotech Inc.
Taiwan W665A10,
TANBead® Nucleic Acid Extraction Kit Specimen: 300 L
Advanced W665A10-
OptiPure Viral Bulk Plate Elution: 80 L
Nanotech Inc. SG15
* Please use catalog numbers shown above to purchase products from Seegene Inc.

⚫ Proceed the extraction process using ‘STARMAGM96’ protocol.

Automated Extraction System Manufacturer Cat. No. Recommended Vol.

Taiwan
Maelstrom™ 9600 Advanced M9600* -
Nanotech Inc.
EX00029P, Specimen: 200 L
STARMag™ M96 Kit Seegene
EX00030P Elution: 100 L
* Please use catalog numbers shown above to purchase products from Seegene Inc.

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B-8. SEEPREP32

⚫ Proceed the extraction process using ‘Pro-Protocol A’.

Automated Extraction System Manufacturer Cat. No. Recommended Vol.

SEEPREP32 Seegene SG71100 -

Specimen: 200 μL
STARMag 96 ProPrep (Plate Type) Seegene EX00009P
Elution: 100 μL
Specimen: 200 μL
STARMag 96 ProPrep (Tube Type) Seegene EX00009T
Elution: 100 μL
Specimen: 200 μL
STARMag 96 ProPrep C (Plate Type) Seegene EX00017P
Elution: 100 μL
Specimen: 200 μL
STARMag 96 ProPrep C (Tube Type) Seegene EX00017T
Elution: 100 μL

⚫ Proceed the extraction process using ‘STARMag_SP32’ protocol.

Automated Extraction System Manufacturer Cat. No. Recommended Vol.

SEEPREP32 Seegene SG71100 -

Specimen: 200 L
STARMag™ SP32 Kit (Plate Type) Seegene EX00028P
Elution: 100 L
Specimen: 200 L
STARMag™ SP32 Kit (Tube Type) Seegene EX00028T
Elution: 100 L

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2-2. Extraction-free

A. Extraction-free method for Swab

1) Prepare mixture* for Extraction-free method.

40 L Nuclease-free water
5 L Proteinase K(PK) (20 mg/mL)**

45 L Total volume of reaction

* Note: Calculate the total amount of each reagent needed based on the number of samples.
** Note: Proteinase K-Solution of Blirt (Cat No. RP107B) (20 mg/mL) is recommended,
though any kinds of Proteinase K can be used.
If PK stock concentration is not 20 mg/mL, adjust the volume of PK stock solution and
Nuclease-free water to meet the final concentration (2.22 mg/mL in total 45 μL).
2) Mix by quick vortexing, and briefly spin down.
3) Aliquot 45 L of mixture into PCR tubes.
4) Add 15 L of each sample into the tube containing aliquot of the mixture.
5) Close the cap, quick vortex and briefly spin down.
6) Incubate at 98℃ for 5 min***.
7) Chill at 4℃ for 3 min***.
*** Note: It is recommended to perform steps 6)~7) on PCR instrument using PCR tube
attached with individual caps.

Note: Immediately initiate the PCR after Extraction-free step.

When it is inevitable, please keep the lysate tube on ice.
Note: If you need retesting, start with the original sample not the lysate.

B. Extraction-free method for Saliva

1) Prepare mixture* for Extraction-free method.

40 L TE buffer (pH 8.0)
5 L Proteinase K(PK) (20 mg/mL)**

45 L Total volume of reaction

* Note: Calculate the total amount of each reagent needed based on the number of samples.
** Note: Proteinase K-Solution of Blirt (Cat No. RP107B) (20 mg/mL) is recommended,
though any kinds of Proteinase K can be used.

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If PK stock concentration is not 20 mg/mL, adjust the volume of PK stock solution and
Nuclease-free water to meet the final concentration (2.22 mg/mL in total 45 μL).
2) Mix by quick vortexing, and briefly centrifuge. spin down.
3) Aliquot 45 L of mixture into PCR tubes.
4) Add 15 L of each sample into the tube containing aliquot of the mixture.
5) Close the cap, vortex for 15 sec and briefly centrifuge. spin down.
6) Incubate at 55℃ for 5 min***.
7) Incubate at 98℃ for 15 min***.
8) Chill at 4℃ for 3 min***.
*** Note: It is recommended to perform steps 6)~8) on PCR instrument using PCR tube
attached with individual caps.

Note: Immediately initiate the PCR after Extraction-free step.

When it is inevitable, please keep the lysate tube on ice.
Note: If you need retesting, start with the original sample not the lysate.

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3. Preparation for Real-time One-step RT-PCR

Note: Correct tubes and caps must be used (see MATERIALS REQUIRED BUT NOT
PROVIDED).
Note: Aerosol resistant filter tips and tight gloves must be used when preparing One-step RT-
PCR reactions. Use extreme care to prevent cross-contamination.
Note: Completely thaw all reagents on ice.
Note: Briefly spin down reagent tubes to collect residual drops inside of the cap.
Note: The steps A~D are automatically processed on Microlab NIMBUS IVD, Microlab
STARlet IVD, Seegene NIMBUS and Seegene STARlet. Refer to each operation manual.

A. Prepare the Reaction Mastermix

5 L SC2F MOM
5 L SEMXR2
5 L SEMXR2 Buffer
15 L Total volume of Mastermix
Note: Calculate the total amount of each reagent needed based on the number of reactions
including samples and controls.

B. Mix by quick vortexing, and briefly spin down.

C. Aliquot 15 L of Reaction Mastermix into PCR tubes.
D. Add 5 L of each sample’s nucleic acids into the tube containing Reaction Mastermix.

15 L Reaction Mastermix
5 L Sample’s nucleic acid

20 L Total volume of reaction

E. Close and briefly spin down the PCR tubes.

F. Verify that the liquid containing all PCR components is at the bottom of each PCR tube. If not,
centrifuge again at a higher rpm for a longer time.

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Note: It is recommended to centrifuge PCR tubes before PCR to eliminate air bubbles and
collect all residual liquids at the bottom of tubes.
Correct Incorrect

Note: Use a new sterile pipette tip for each sample.

Note: For Negative Control (NC), use 5 L of “RNase-free Water” instead of sample’s nucleic
acid.
Note: For Positive Control (PC), use 5 L of “SC2F PC” instead of sample’s nucleic acid.
Note: Be careful not to cross-contaminate the Reaction Mastermix and samples with the Positive
Control.
Note: Do not label the reaction tube on its cap. Fluorescence is detected from the top of each
reaction tube.

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REAL-TIME PCR INSTRUMENT SET UP AND RESULT ANALYSIS

1. CFX96™ Real-time PCR Detection System (CFX Manager™ Software-IVD v1.6)

1.1. Real-time PCR Instrument Setup

Note: CFX96™ Real-time PCR Detection System (Bio-Rad) experiment setup can be divided
into three steps: Protocol Setup, Plate Setup, and Start run.

A. Protocol Setup

1) In the main menu, select “File” → “New” → “Protocol” to open “Protocol Editor”.

Fig. 1. Protocol Setup

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2) In “Protocol Editor”, define the thermal profile as follows:

No. of cycles Temperature Duration
50°C 10 min
1
95°C 3 min
95°C 5 sec
5
60°C 20 sec
95°C 2 sec
40
60°C* 5 sec
Note*: Plate Read Step. Fluorescence is detected at 60°C (Step 7).

Fig. 2. Protocol Editor

Note: Click the “Insert GOTO” and type in “GOTO 3, 4 more times” at Step 5.
Note: Click the “Insert GOTO” and type in “GOTO 6, 39 more times” at Step 8.

3) Click the box next to “Sample Volume” to directly input 20 L.

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4) Click “OK” and save the protocol to open the “Experiment Setup” window.

Fig. 3. Experiment Setup: Protocol

B. Plate Setup

1) From “Plate” tab in “Experiment Setup”, click “Create New” to open “Plate Editor” window.

Fig. 4. Plate Editor

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2) Click “Select Fluorophores” to indicate the fluorophores (FAM, HEX, Cal Red 610, and
Quasar 670) that will be used and click “OK”.

Fig. 5. Select Fluorophores (FAM, HEX, Cal Red 610, and Quasar 670)

3) Select the wells where the PCR tube will be placed and select their sample types from the
“Sample Type” drop-down menu.
- Unknown: Clinical samples
- Negative Control
- Positive Control
4) Click on the appropriate checkboxes (FAM, HEX, Cal Red 610, and Quasar 670) to specify
the fluorophores to be detected in the selected wells.
5) Type “Sample Name” and press enter key.

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6) In “Settings” of the “Plate Editor” main menu, choose the “Plate Size” (96 wells) and
“Plate Type” (BR White).

Fig. 6. Plate Setup

7) Click “OK” to save the new plate.

8) You will be returned to the “Experiment Setup” window.

Fig. 7. Experiment Setup: Plate

9) Click “Next” to start run.

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C. Start Run

1) From “Start Run” tab in “Experiment Setup”, click “Close Lid” to close the instrument lid.

Fig. 8. “Close Lid”

2) Click “Start Run”.

3) Store the run file either in My Documents or in a designated folder. Input the file name, click
“SAVE”, and the run will start.

1.2. Data Analysis

A. Create folders for data export

1) To save data of all detection steps of amplification curves from the result file, create one
folder.
2) Folder name may be as desired by user (For ‘Seegene Export’ function, folder “QuantStep7”
is automatically created to save amplification curve data under the folder created by user).

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B. Pre-settings for Data Analysis in CFX Manager™

1) After the test, click the “Quantitation” tab to see the amplification curve results.

Fig. 9. Amplification curve results

2) Select “No Baseline Subtraction” from Analysis Mode of Settings menu.

Fig. 10. No Baseline Subtraction

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3) Select “Seegene Export” from Tools menu.

Fig. 11. Seegene Export

4) Choose a location to save data and click “OK”.

Fig. 12. Seegene Export to designated folder

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C. Settings for Data Analysis in Seegene Viewer

1) Open Seegene Viewer program, and click “Option” to select CFX96 in the “Instrument”.

Fig. 13. Seegene Viewer

2) Click “Open” to find the saved file in folder “QuantStep7”, open the results file, and select the
test kit from the “PRODUCT” menu.

Fig. 14. Settings for Data Analysis in Seegene Viewer

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3) Check the result for each well.

Fig. 15. Test result on Seegene Viewer

4) Validity Criteria of Control Results

a. Valid Assay Run

To check the validity of experiments, the PCR runs should be accompanied with PC (Positive
Control) and NC (Negative Control). Assay run is determined as valid when all of the following
criteria are met:
Seegene Viewer Result

Control FAM (Ct) Cal Red 610 (Ct) Quasar 670 (Ct) HEX (Ct)
Auto Interpretation
E gene RdRP gene N gene IC

Positive Control 10 ≤ Ct ≤ 23 10 ≤ Ct ≤ 23 10 ≤ Ct ≤ 23 10 ≤ Ct ≤ 23 Positive Control(+)

Negative Control N/A N/A N/A N/A Negative Control(-)

b. Invalid Assay Run

In case of a validity failure, the results should not be interpreted or reported. And the PCR
reaction must be repeated.

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2. CFX96™ Dx System (CFX Manager™ Dx Software v3.1)

2.1 Real-time PCR Instrument Setup

Note: CFX96™ Dx System (Bio-Rad) experiment setup can be divided into three steps:
Protocol Setup, Plate Setup, and Start Run.

A. Protocol Setup

1) In the main menu, select “File” → “New” → “Protocol” to open “Protocol Editor”.

Fig. 1. Protocol Setup

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2) In “Protocol Editor”, define the thermal profile as follows:

No. of cycles Temperature Duration
50°C 10 min
1
95°C 3 min
95°C 5 sec
5
60°C 20 sec
95°C 2 sec
40
60°C* 5 sec
Note*: Plate Read Step. Fluorescence is detected at 60°C (Step 7).

Fig. 2. Protocol Editor

Note: Click the “Insert GOTO” and type in “GOTO 3, 4 more times” at Step 5.
Note: Click the “Insert GOTO” and type in “GOTO 6, 39 more times” at Step 8.

3) Click the box next to “Sample Volume” to directly input 20 L.

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4) Click “OK” and save the protocol to open the “Run Setup” window.

Fig. 3. Run Setup: Protocol

B. Plate Setup

1) From “Plate” tab in “Run Setup”, click “Create New” to open “Plate Editor” window.

Fig. 4. Plate Editor

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2) Click “Select Fluorophores” to indicate the fluorophores (FAM, HEX, Cal Red 610, and
Quasar 670) that will be used and click “OK”.

Fig. 5. “Select Fluorophores” (FAM, HEX, Cal Red 610, and Quasar 670)

3) Select the wells where the PCR tube will be placed and select their sample types from the
“Sample Type” drop-down menu.
- Unknown: Clinical samples
- Negative Control
- Positive Control
4) Click on the appropriate checkboxes (FAM, HEX, Cal Red 610, and Quasar 670) to specify
the fluorophores to be detected in the selected wells.
5) Type “Sample Name” and press enter key.

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6) In “Settings” of the “Plate Editor” main menu, choose the “Plate Size” (96 wells) and
“Plate Type” (BR White).

Fig. 6. Plate Setup

7) Click “OK” to save the new plate.

8) You will be returned to the “Run Setup” window.

Fig. 7. Run Setup: Plate

9) Click “Next” to start run.

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C. Start Run

1) From “Start Run” tab in “Run Setup”, click “Close Lid” to close the instrument lid.

Fig. 8. Close Lid

2) Click “Start Run”.

3) Store the run file either in My Documents or in a designated folder. Input the file name, click
“SAVE”, and the run will start.

2.2. Data Analysis

A. Create folders for data export

1) To save data of all detection steps of amplification curves from the result file, create one
folder.
2) Folder name may be as desired by user (For ‘Seegene Export’ function, folder “QuantStep7”
is automatically created to save amplification curve data under the folder created by user).

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B. Pre-settings for Data Analysis in CFX96™

1) After the test, click the “Quantification” tab to see the amplification curve results.

Fig. 9. Amplification curve results

2) Select “No Baseline Subtraction” from Baseline Setting of Settings menu.

Fig. 10. No Baseline Subtraction

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3) Select “Seegene Export” from Export menu.

Fig. 11. “Seegene Export”

4) Choose a location to save data and click “OK”.

Fig. 12. Seegene Export to designated folder

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C. Settings for Data Analysis in Seegene Viewer

1) Open Seegene Viewer program, and click “Option” to select CFX96 Dx in the “Instrument”.

Fig. 13. Seegene Viewer

2) Click “Open” to find the saved file in folder “QuantStep7”, open the results file, and select the
test kit from the “PRODUCT” menu.

Fig. 14. Settings for Data Analysis in Seegene Viewer

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3) Check the result for each well.

Fig. 15. Test result on Seegene Viewer

4) Validity Criteria of Control Results

a. Valid Assay Run

To check the validity of experiments, the PCR runs should be accompanied with PC (Positive
Control) and NC (Negative Control). Assay run is determined as valid when all of the following
criteria are met:
Seegene Viewer Result

Control FAM (Ct) Cal Red 610 (Ct) Quasar 670 (Ct) HEX (Ct)
Auto Interpretation
E gene RdRP gene N gene IC

Positive Control 10 ≤ Ct ≤ 23 10 ≤ Ct ≤ 23 10 ≤ Ct ≤ 23 10 ≤ Ct ≤ 23 Positive Control(+)

Negative Control N/A N/A N/A N/A Negative Control(-)

b. Invalid Assay Run

In case of a validity failure, the results should not be interpreted or reported. And the PCR
reaction must be repeated.

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3. Applied Biosystems™ 7500 (7500 Software v2.0.5 / v2.3)

3.1. Real-time PCR Instrument set up

Note: The instrument must be calibrated before use.

Note: Applied Biosystems™ 7500 (Thermo Fisher Scientific) experiment setup can be divided into
two steps: Setup and Run

A. Setup

1) In the main menu, select “Set Up” → “Advanced Setup”

Fig 1. Set up

2) In the “Experiment properties” tab, enter “Experiment Name” and select Instrument,
“Experiment type”, “Reagents”, and “Ramp speed” as follows.

Instrument 7500 (96 Wells)

Experiment type Quantitation – Standard Curve

Reagents Taqman® Reagents

Ramp speed Standard

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Fig. 2. Experiment properties tab

3) Click on “Plate setup” tab. In the “Define Targets and Samples” tab, enter “Target
Name” and select “Reporter” and “Quencher” as follows.

Target Name Reporter Quencher

E gene FAM None
IC VIC None
RdRP gene ROX None
N gene CY5 None

Fig. 3. Define Targets and Samples tab

4) Click on “Assign Targets and Samples” tab, select wells where the PCR tube will be

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placed and assign targets. Select None for Passive reference.

NOTE: If a well without sample or mastermix is selected, signal noise may be observed.
Ensure that only wells containing samples or mastermix are selected.

Fig. 4. Assign Targets and Samples tab

5) Click on “Run Method”. In the “Graphical View” or “Tabular View tab”, enter 20 µL as the
“Reaction Volume per Well” field. Define the thermal profile as table below.
No. of cycles Temperature Duration
50°C 10 min
1
95°C 3 min
95°C 5 sec
5
60°C 20 sec
95°C 2 sec
40
60°C* 30 sec
Note*: Plate Read Step. Fluorescence is detected at 60°C.

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Fig. 5. Graphical View tab

6) Click on “File” → “Save as Template” to save the new “template” file in “.edt” format.
Enter the file name, select a location for the template, then click “Save”. The saved “template”
can be used for future testing.

Fig. 6. Save as Template (.edt)

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B. Run

1) Turn on the laptop and Applied Biosystems™ 7500 real-time PCR system. Ensure that
the laptop is connected to the instrument.
2) Push the tray door to open the instrument. Load the PCR plate onto the plate holder of
the instrument.
3) Push the tray door to close the instrument.
4) Click on “File” → “Save as Template” to save the new template file in “.eds” format.
Enter the file name, select a location for the template, then click “Save”.

Fig. 7. Save as (.eds)

5) Click START RUN.

Fig. 8. START RUN

3.2. Data export and analysis

A. Pre-settings for Data export and analysis

1) Create a folder to save data for all of amplification curve detection steps from the result file.
2) Enter folder name as necessary.

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3) Click on “File” → “Export”

Fig. 9. File export

4) Click on the “Export Properties” tab (default) and select “Sample Setup”, “Raw data”,
“Amplification Data”, “Results”, and “Multicomponent Data” under “1. Select data to
export”.

Fig. 10. 1. Select data to export

5) Select “One File” under “2. Select one or separate files:”.

Fig. 11. 2. Select one or separate files

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6) Enter “Export File Name”, then select “Export File Location”. Select “.xls” in the “File
Type” drop-down list.

Fig. 12. 3. Enter export file properties

7) Click “Start Export”.

Fig. 13. Start Export

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B. Set up for Data analysis in Seegene Viewer

1) Open the Seegene Viewer software installed on the laptop connected to the Applied
Biosystems™ 7500. Click on “Option” to select AB7500 v2.0.5 from the “Instrument”.

Fig. 14. Seegene Viewer

2) Click on the “Open” icon and locate the Applied Biosystems™ 7500 export data where the
Applied Biosystems™ 7500 data was saved. After opening the results file, select ‘Allplex™ SARS-
CoV-2 fast PCR Assay’ from the PRODUCT menu.

Fig. 15. Settings for Data Analysis in Seegene Viewer

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3) Assign Positive and Negative control accordingly by selecting PC, NC under the Type drop-down
menu.

Fig. 16. Settings for Sample Type in Seegene Viewer

4) View test results. The auto-interpreted results for each sample can be viewed by clicking on
each well.
5) Validity Criteria of Control Results

a. Valid Assay Run

To check the validity of experiments, the PCR runs should be accompanied with PC (Positive
Control) and NC (Negative Control). Assay run is determined as valid when all of the following
criteria are met:
Seegene Viewer Result

Control FAM (Ct) ROX (Ct) CY5 (Ct) VIC (Ct)

Auto Interpretation
E gene RdRP gene N gene IC

Positive Control 10 ≤ Ct ≤ 23 10 ≤ Ct ≤ 23 10 ≤ Ct ≤ 23 10 ≤ Ct ≤ 23 Positive Control(+)

Negative Control N/A N/A N/A N/A Negative Control(-)

b. Invalid Assay Run

In case of a validity failure, the results should not be interpreted or reported. And the PCR reaction
must be repeated.

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4. SGRT (Seegene Real-time Thermocycler) (SGRT Manager Lite v1.00)

4.1 Real-time PCR Instrument Setup

Note: SGRT (Seegene Real-time Thermocycler) (Seegene) experiment setup can be divided
into three steps: Protocol Setup, Plate Setup, and Start Run.

A. Protocol Setup

1) In the main menu, select “File” → “PCR Setup” to open PCR Setup window.

Fig. 1. PCR Setup

2) From PCR Setup Window, click “Edit Protocol” button to edit protocol.

Fig. 2. PCR Setup Window

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3) In “Protocol Editor”, define the thermal profile as follows:

No. of cycles Temperature Duration
50°C 10 min
1
95°C 3 min
95°C 5 sec
5
60°C 20 sec
95°C 2 sec
40
60°C* 5 sec
Note*: Plate Read Step. Fluorescence is detected at 60°C (Step 6).

Fig. 3. Edit Protocol

Note: From Step 4 “Properties” option, select Step 3 for “Goto” and type 5 Time(s) for the box
next to “Repeat”.
Note: From Step 6 “Properties” option, select Step 5 for “Goto” and type 40 Time(s) for the
box next to “Repeat”.

4) Click the box next to “Sample Volume” to directly input 20 L.

5) Click “OK” and save the protocol to return to the “PCR Setup” window.

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B. Plate Setup
1) Click “Edit Plate” to begin Plate Setup.

Fig. 4. PCR Setup: Edit Plate

2) From plate editing window, select “Edit” → “Select Channels”.

Fig. 5. Edit Plate

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3) Select the fluorophores (FAM, HEX, Cal Red 610, and Quasar 670) that will be used and
click “OK”.

Fig. 6. Select Fluorophores (FAM, HEX, Cal Red 610, and Quasar 670)

4) Select the wells where the PCR tube will be placed and select their sample types from the
“Type” drop-down menu.
- Sample: Clinical samples
- NC: Negative Control
- PC: Positive Control
- None: Empty wells
5) Click on the appropriate checkboxes (FAM, HEX, Cal Red 610, and Quasar 670) to specify
the fluorophores to be detected in the selected wells.
6) Type “Name” and press enter key.

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7) Click “OK” to save the new plate.

Fig. 7. Plate Setup

8) You will be returned to the “PCR Setup” window.

Fig. 8. PCR Setup window

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C. Start Run
1) Click “Setting” → “Configuration” to open the “Configuration” window.
2) Click “Browse” to set a default result file save location after the run. Then, click “Save”.

Fig. 9. Configuration window

3) Click “Open Drawer” to open the drawer of the instrument. After loading the PCR tubes onto the
instrument, click “Close Drawer” to close the drawer. Enter the experiment name.
4) Click “Run” button on the top ribbon menu of the “Devices” window to run the experiment.

Fig. 10. Devices

5) Result will be saved in “.sgdz” format at the default result file save location which has been set
from “Configuration” window.

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4.2. Data Analysis

Note: “.sgdz” file can be opened directly from Seegene Viewer without additional data export step
from SGRT Manager Lite.

A. Settings for Data Analysis in Seegene Viewer

1) Open Seegene Viewer program, and click “Option” to select SGRT in the “Instrument”.

Fig. 11. Seegene Viewer

2) Click “Open” to find the “.sgdz” result file, and select the test kit from the “PRODUCT” menu.

Fig. 12. Settings for Data Analysis in Seegene Viewer

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3) Check the result for each well.

Fig. 13. Test result on Seegene Viewer

4) Validity Criteria of Control Results

a. Valid Assay Run

To check the validity of experiments, the PCR runs should be accompanied with PC (Positive
Control) and NC (Negative Control). Assay run is determined as valid when all of the following
criteria are met:
Seegene Viewer Result

Control FAM (Ct) Cal Red 610 (Ct) Quasar 670 (Ct) HEX (Ct)
Auto Interpretation
E gene RdRP gene N gene IC

Positive Control 10 ≤ Ct ≤ 23 10 ≤ Ct ≤ 23 10 ≤ Ct ≤ 23 10 ≤ Ct ≤ 23 Positive Control(+)

Negative Control N/A N/A N/A N/A Negative Control(-)

b. Invalid Assay Run

In case of a validity failure, the results should not be interpreted or reported. And the PCR reaction
must be repeated.

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RESULTS

1. Analyte Information

CFX96 / SGRT AB7500

Analytes
Fluorophores Fluorophores

FAM FAM E gene

HEX VIC IC

Cal Red 610 ROX RdRP gene

Quasar 670 CY5 N gene

2. Interpretation of Results

Target
Analytes
E gene RdRP gene N gene IC

Detected (+) ≤ 38 ≤ 38 ≤ 38 ≤ 35

Not detected (-) > 38 or N/A > 38 or N/A > 38 or N/A > 35 or N/A

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Target Result
IC Auto-
E RdRP N Description
Result* interpretation
gene gene gene

All target results were valid. Result

+ + + +/-
for SARS-CoV-2 RNA is detected.

- All target results were valid. Result

+ - + +/-
for SARS-CoV-2 RNA is detected.
- Negative target results are
- + + +/- suggestive of
SARS-CoV-2 1) A sample at concentrations near
or below the limit of detection of the
+ + - +/-
test,
2) A mutation in the corresponding
- + - +/-
target region,
3) Other factors.
- - + +/-

- All target results were valid. Result

for Sarbecovirus RNA is detected.
Result for SARS-CoV-2 RNA is
Presumptive Positive.
- Negative target results are
suggestive of
1) A sample at concentrations near
or below the limit of detection of the
SARS-CoV-2
test,
+ - - +/- Presumptive
2) A mutation in the corresponding
positive
target region,
3) Other factors.
- Repeat test with more nucleic acid
- For samples with the same result
on the repeated test, additional
confirmatory testing may be
conducted, if it is necessary to
differentiate between SARS-CoV-2

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and other Sarbecovirus currently

unknown to infect humans, for
epidemiological purposes or clinical
management.

All target results were valid. Result

- - - + Not detected (-) for SARS-CoV-2 RNA is not
detected.

- Results suggest inadequate

specimen collection or processes or
the presence of PCR inhibitors.
- Repeat the test from the nucleic
- - - - Invalid** acid extraction using another aliquot
of the original specimen.
- If the same result is shown in the
diluted nucleic acid, please collect
samples again.

* High level of target nucleic acids may cause interference in Internal Control detection and
readout. Invalid IC signal does not indicate that the positive results for targets are invalid.
** See TROUBLESHOOTINGS section for the detailed instruction.

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3. Application to Clinical Samples

Clinical Sample 1

Clinical Sample 2

CFX96 FAM Cal Red 610 Quasar 670 HEX

AB7500 FAM ROX CY5 VIC Auto
RdRP Interpretation
Sample E gene C(t) C(t) N gene C(t) IC C(t)
gene

1 + 14.39 + 14.95 + 13.36 + 22.56 SARS-CoV-2

2 + 30.51 - N/A + 30.09 + 18.35 SARS-CoV-2

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TROUBLESHOOTINGS

Allplex™ SARS-CoV-2 fast PCR Assay

OBSERVATION PROBABLE CAUSES SOLUTION

The fluorophores for data

Select the correct fluorophores for data
analysis do not comply
analysis.
with the protocol

Incorrect setting of real- Please check the thermal cycling conditions

time thermal cycler and repeat the test under the correct settings.

No signal Please check the storage conditions (See

Incorrect storage or
page 10) and the expiry date (refer to label) of
expiration of the test kit
the test kit and use a new kit if necessary.

Please check the storage condition and expiry

Nucleic acid extraction
date of the extraction kit and use a new kit if
failure
necessary.

If target pathogen signal is observed but not

IC, then IC amplification may have been
High load of pathogen's inhibited by high titer of target pathogen. If you
nucleic acid want to observe IC signal, dilute the specimen
(1/3~1/10) in saline buffer and repeat the test
from extraction step.

No Internal Control Please dilute the extracted nucleic acid

signal (1/2~1/5) in RNase-free water and repeat the
test from RT-PCR step.
Presence of PCR Inhibitor
Please dilute the specimen (1/3~1/10) in
saline buffer and repeat the test from
extraction step.

Please collect the sample again and repeat

Incorrect sample collection
the test from extraction step.

Decontaminate all surfaces and instruments

Putative false positive with sodium hypochlorite and ethanol. Only
or target signal(s) use filter tips throughout the procedure and
Contamination
observed in Negative change tips between tubes. Repeat the entire
Control procedure from nucleic acid extraction with the
new set of reagents.

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 Allplex™ SARS-CoV-2 fast PCR Assay

Allplex™ SARS-CoV-2 fast PCR Assay

OBSERVATION PROBABLE CAUSES SOLUTION

Error in specimen Please check the specimen collection method,

collection and re-collect the specimen.

Please re-collect the specimen and repeat the

Incorrect storage of the
entire procedure. Ensure that the specimen is
specimen
stored as recommended.

Please check the nucleic acid extraction

Error in nucleic acid
procedure as well as nucleic acid
extraction
concentration, and re-extract the nucleic acid.
Putative False negative or
Check the sample numbers of tubes
no signal observed in
Error in adding nucleic containing nucleic acid and make sure to add
Positive Control acid to correct PCR tubes nucleic acid into the correct PCR tubes and
carefully repeat the test if necessary.

Please dilute the specimen (1/3~1/10) in

Presence of inhibitor saline buffer and repeat the test from
extraction step.

Confirm that all components are added to the

reaction mixture (Sensitivity is compromised
Incorrect PCR mixture
with pre-composed premix). All reagents must
be homogenized and spun down before use.

Spikes in any cycles of

Bubble in the PCR tube Centrifuge the PCR tube before run.
amplification curve

Please check the specimen collection device.

ENAT PM 2ML PERNASAL APPLICATOR,

ENAT KIT PNR CAP 2ML+REGULAR

No signal from
Presence of PCR Inhibitor FLOQSWAB BP80, ENAT KIT PNR CAP
extraction-free method
2ML+PROGR.FLEXIBLE FLOQSWAB BP80,

GeneTM Set (GTS1) and GeneTM Set (GTS2)

are not applicable to extraction-free method.

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PERFORMANCE

1. Analytical Specificity

The high specificity of Allplex™ SARS-CoV-2 fast PCR Assay is ensured by the oligos
designed specifically for the targets of interest. Allplex™ SARS-CoV-2 fast PCR Assay was
tested for cross-reactivity to 108 different pathogens, and PCR amplification and detection
were only identified for the specified targets.

No. Organism Source Isolate No. Result†

First WHO International Standard for SARS- E, RdRP, N gene
1 NIBSC 20/146
CoV-2 RNA (Isolate: England/02/2020) Detected
Twist Synthetic SARS-CoV-2 RNA Control 2 TWIST E, RdRP, N gene
2 102024
(MN908947.3) BIOSCIENCE Detected
E, RdRP, N gene
3 SARS-CoV-2 (Isolate USA-WA1/2020) ZMC 0810587CFHI
Detected
SARS-CoV-2 (Isolate: Hong E, RdRP, N gene
4 ZMC 0810590CFHI
Kong/VM20001061/2020) Detected
E, RdRP, N gene
5 SARS-CoV-2 (Isolate: Italy-INMI1) ZMC 0810589CFHI
Detected
E, RdRP, N gene
6 SARS-CoV-2 strain Germany/BavPat1/2020 ATCC VR-1994D
Detected
Twist Synthetic SARS-CoV-2 RNA Control 14 TWIST E, RdRP, N gene
7 103907
(B.1.1.7_710528) BIOSCIENCE Detected
Twist Synthetic SARS-CoV-2 RNA Control 16 TWIST E, RdRP, N gene
8 104043
(B.1.351_678597) BIOSCIENCE Detected
Twist Synthetic SARS-CoV-2 RNA Control 17 TWIST E, RdRP, N gene
9 104044
(P.1_792683) BIOSCIENCE Detected
AMPLIRUN® SARS-CoV-2 B.1.617.2 RNA E, RdRP, N gene
10 VIRCELL MBC141-R
CONTROL Detected
E, RdRP, N gene
11 SARS-CoV-2 (GH/452R.V1, B.1.427 lineage) NCCP 43384
Detected
SARS-CoV-2 (GH/452R.V1; GH clade E, RdRP, N gene
12 NCCP 43385
(B.1.429 lineage)) Detected
E, RdRP, N gene
13 SARS-CoV-2 (G/483K.V3, B.1.525 lineage) NCCP 43386
Detected
E, RdRP, N gene
14 SARS-CoV-2 (GH clade (B.1.526 lineage)) NCCP 43387
Detected
E, RdRP, N gene
15 SARS-CoV-2 (GR clade (P.2 lineage)) NCCP 43383
Detected
SARS-Related Coronavirus 2, Isolate USA- BEI E, RdRP, N gene
16 NR-52287
WA1/2020, Gamma-Irradiated Resources Detected
SARS-CoV-2 (GK clade, B.1.617.2 lineage E, RdRP, N gene
17 NCCP 43405
with K417N) Detected
E, RdRP, N gene
18 SARS-CoV-2 (GK clade, AY.1 lineage) NCCP 43406
Detected

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 Allplex™ SARS-CoV-2 fast PCR Assay

No. Organism Source Isolate No. Result†

E, RdRP, N gene
19 SARS-CoV-2 (GH clade, B.1.621 lineage) NCCP 43407
Detected
E, RdRP, N gene
20 SARS-CoV-2 (GRA clade, B.1.1.529 lineage) NCCP 43408
Detected
21 Human adenovirus 1 ATCC VR-1 Not Detected

22 Human adenovirus 2 KBPV VR-58 Not Detected

23 Human adenovirus 3 ATCC VR-3 Not Detected

24 Human adenovirus 4 ATCC VR-1572 Not Detected

25 Human adenovirus 5 KBPV VR-61 Not Detected

26 Human adenovirus 7 ATCC VR-7 Not Detected

27 Human adenovirus 8 ATCC VR-1368 Not Detected

28 Human adenovirus 14 ATCC VR-15 Not Detected

29 Human adenovirus 18 ATCC VR-1095 Not Detected

30 Human adenovirus 22 ATCC VR-1100 Not Detected

31 Human adenovirus 23 ATCC VR-1101 Not Detected

32 Human adenovirus 31 ATCC VR-1109 Not Detected

33 Influenza A virus (H1N1) ATCC VR-95 Not Detected

34 Influenza A virus (H1N1) ATCC VR-97 Not Detected

35 Influenza A virus (H1N1) ATCC VR-219 Not Detected

36 Influenza A virus (H1N1) ATCC VR-825 Not Detected

37 Influenza A virus (H1N1) ATCC VR-897 Not Detected

38 Influenza A virus (H1N1) ATCC VR-1683 Not Detected

39 Influenza A virus (H1N1) ATCC VR-1736 Not Detected

40 Influenza A virus (H3N2) ATCC VR-544 Not Detected

41 Influenza A virus (H3N2) ATCC VR-547 Not Detected

42 Influenza A virus (H3N2) ATCC VR-810 Not Detected

43 Influenza A virus (H3N2) ATCC VR-1680 Not Detected

44 Influenza A virus (H3N2) ATCC VR-822 Not Detected

45 Influenza B virus ATCC VR-101 Not Detected

46 Influenza B virus ATCC VR-102 Not Detected

47 Influenza B virus ATCC VR-103 Not Detected

48 Influenza B virus ATCC VR-295 Not Detected

49 Influenza B virus ATCC VR-523 Not Detected

50 Influenza B virus ATCC VR-786 Not Detected

51 Influenza B virus ATCC VR-823 Not Detected

52 Influenza B virus ATCC VR-1804 Not Detected

53 Human respiratory syncytial virus A ATCC VR-26 Not Detected

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 Allplex™ SARS-CoV-2 fast PCR Assay

No. Organism Source Isolate No. Result†

54 Human respiratory syncytial virus B ATCC VR-955 Not Detected

55 Human parainfluenza virus 1 ATCC VR-1380 Not Detected

56 Human parainfluenza virus 2 ATCC VR-92 Not Detected

57 Human parainfluenza virus 3 ATCC VR-93 Not Detected

58 Human parainfluenza virus 4A ATCC VR-1378 Not Detected

59 Human parainfluenza virus 4B ATCC VR-1377 Not Detected

60 Human rhinovirus 14 ATCC VR-284 Not Detected

61 Human rhinovirus 16 ATCC VR-283 Not Detected

62 Human rhinovirus 21 ATCC VR-40 Not Detected

63 Human rhinovirus 42 ATCC VR-338 Not Detected

64 Human rhinovirus 90 ATCC VR-1291 Not Detected

65 Human coronavirus NL63 ZMC 0810228CF Not Detected

66 Human coronavirus 229E ATCC VR-740 Not Detected

67 Human coronavirus OC43 KBPV VR-8 Not Detected

68 Human coxsackievirus A9 KBPV VR-11 Not Detected

69 Human coxsackievirus A24 ATCC VR-583 Not Detected

70 Human coxsackievirus B1 KBPV VR-13 Not Detected

71 Human coxsackievirus B2 KBPV VR-14 Not Detected

72 Human coxsackievirus B3 KBPV VR-15 Not Detected

73 Human coxsackievirus B4 KBPV VR-16 Not Detected

74 Human coxsackievirus B5 KBPV VR-17 Not Detected

75 Human coxsackievirus B6 KBPV VR-18 Not Detected

76 Human echovirus 6 KBPV VR-19 Not Detected

77 Human echovirus 7 KBPV VR-20 Not Detected

78 Human echovirus 9 ATCC VR-39 Not Detected

79 Human echovirus 11 KBPV VR-22 Not Detected

80 Human echovirus 22 KBPV VR-23 Not Detected

81 Human echovirus 25 KBPV VR-24 Not Detected

82 Human enterovirus 71 ATCC VR-784 Not Detected

83 Human herpesvirus 1 KBPV VR-52 Not Detected

84 Human cytomegalovirus KBPV VR-7 Not Detected

85 Epstein-Barr virus ATCC VR-1491 Not Detected

86 Mumps virus ATCC VR-106 Not Detected

87 Bordetella pertussis ATCC 9797 Not Detected

88 Candida albicans KCCM 50651 Not Detected

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 Allplex™ SARS-CoV-2 fast PCR Assay

No. Organism Source Isolate No. Result†

89 Chlamydia trachomatis type A ATCC VR-571B Not Detected

90 Lactobacillus acidophilus KCTC 3140 Not Detected

91 Legionella pneumophila ATCC 12009 Not Detected

92 Moraxella catarrhalis ATCC 25238 Not Detected

93 Mycoplasma hominis ATCC 23114 Not Detected

94 Mycoplasma genitalium ATCC 49895 Not Detected

95 Streptococcus salivarius KCTC 5512 Not Detected

96 Pseudomonas aeruginosa KCTC 2004 Not Detected

97 SARS-coronavirus ZMC NATSARS-ST E gene Detected

98 MERS-coronavirus ZMC NATMERS-ST Not Detected

99 Human coronavirus HKU1 Korean isolate Not Detected

100 Human Metapneumovirus (hMPV) KBPV VR-87 Not Detected

101 Chlamydia pneumoniae ATCC 53592 Not Detected

102 Haemophilus influenzae ATCC 51907 Not Detected

103 Streptococcus pneumoniae KCCM 40410 Not Detected

104 Streptococcus pyogenes ATCC 19615 Not Detected

105 Mycoplasma pneumoniae ATCC 15293 Not Detected

106 Pneumocystis jirovecii (PJP) Korean isolate Not Detected

107 Pooled human nasal wash Korean isolate Not Detected

108 Staphylococcus epidermidis KCCM 40416 Not Detected

† Specificity tests were repeated 3 times.

※ ATCC: American Type Culture Collection,
KBPV: Korea Bank for Pathogenic Viruses
ZMC: ZeptoMetrix Corporation
KCTC : Korean Collection for Type Cultures
NIBSC : National Institute for Biological Standards and Control
NCCP : National Culture Collection for Pathogens
KCCM: Korean Culture Center of Microorganisms
BEI Resources: Biodefense and Emerging Infections Research Resources Repository

2. Analytical Sensitivity

In order to determine the sensitivity of Allplex™ SARS-CoV-2 fast PCR Assay, inactivated
SARS-CoV-2 virus of the NIBSC (First WHO International Standard for SARS-CoV-2 RNA,
Code No. 20/146) and inactivated SARS-CoV-2 virus of the BEI (SARS-Related Coronavirus 2,
Isolate USA-WA1/2020, Gamma-Irradiated, Cat. No. NR-52287) were spiked into negative
sample matrix and serially diluted.

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2-1. Standard extraction

Nucleic acids were extracted from each dilution using automated extraction system (Microlab
NIMBUS IVD, Universal Cartridge kit) and analyzed with Allplex™ SARS-CoV-2 fast PCR
Assay.

NIBSC BEI
(Code No. 20/146) (Cat. NR-52287)
Analyte
Limit of Detection (IU/mL) Limit of Detection (GE/mL)*

E gene 449 168

RdRP gene 645 226

N gene 655 264

SARS-CoV-2** 195 61
* GE/mL= copies/mL
** SARS-CoV-2 is considered as “detected” if the auto-interpretation in Seegene Viewer
appears as “SARS-CoV-2” or “SARS-CoV-2 Presumptive positive”

2-2. Extraction-free
After processing specimens as described in extraction-free method for swab and saliva, nucleic
acids were analyzed with Allplex™ SARS-CoV-2 fast PCR Assay.

NIBSC
(Code No. 20/146)
Analyte Limit of Detection (IU/mL) Limit of Detection (IU/mL)

Swab Saliva

E gene 3785 7363

RdRP gene 6621 13050

N gene 5649 8853

SARS-CoV-2* 1107 4264

* SARS-CoV-2 is considered as “detected” if the auto-interpretation in Seegene Viewer appears as
“SARS-CoV-2” or “SARS-CoV-2 Presumptive positive”

3. Reproducibility

The reproducibility test was prepared including High negative (0.1X LoD), Low positive (1X
LoD) and Moderate positive (3X LoD) samples. At each testing site, the kit was tested for five
days, two runs per day by two different experimenters and triplicate of each target. The
positive rates were observed for each target for reproducibility study: 100.0% for Moderate

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 Allplex™ SARS-CoV-2 fast PCR Assay

positive samples, ≥95% for Low positive samples. The reproducibility of the Allplex™ SARS-
CoV-2 fast PCR Assay was evaluated between runs, sites and product lots. Positive rates for
all concentrations and CV values met criteria of less than or equal to 5%(≤5%).
The results were satisfied with the criteria set above, thus confirming the reproducible
performances of Allplex™ SARS-CoV-2 fast PCR Assay.

4. Interfering substances

There were no effects on the results by adding the substance: non-specific detections or
inhibitions on target amplification. Based on the results, 16 interfering substances had no effect
on Allplex™ SARS-CoV-2 fast PCR Assay results.

No. Interfering Substances Source Test Concentration

Sigma-Aldrich
1 Mucin (bovine submaxillary gland, type I-S) (Cat.No.M3895) 60 g/mL
Sigma-Aldrich
2 Mupirocin (Antibiotic, nasal ointment) (Cat.No.1448901) 6.6 mg/mL
Sigma-Aldrich
3 Oxymetazoline (Afrin Nasal Spray) (Cat.No.O2378) 15% (v/v)

4 Blood Human 2% (v/v)

Sigma-Aldrich
5 Tobramycin (Antibacterial, systemic) (Cat.No.T4014) 4.0 g/mL
Sigma-Aldrich
6 Zanamivir (Anti-viral drug-Relenza) 3.3 mg/mL
(Cat.No.SML0492)
Sigma-Aldrich
7 Oseltamivir (Anti-viral drug-Tamiflu) 25 mg/mL
(Cat.No.1479304)
AstraZeneca
8 Pulmicort® (Nasal corticosteroid) - Budesonide 125 µg/mL
(Cat.No.199403233)
Sigma-Aldrich
9 Benzocaine (Throat lozenges) 25 mg/mL
(Cat.No.E1501)
Sigma-Aldrich
10 Menthol (Throat lozenges) 25 mg/mL
(Cat.No.M2772)
Sigma-Aldrich
11 Zinc sulfate 0.55 mg/mL
(Cat.No.83265)
Sigma-Aldrich
12 Sodium chloride 0.8% (w/v)
(Cat.No.S3014)
Sigma-Aldrich
13 Phenylephrine hydrochloride (0.5%) 15% (v/v)
(Cat.No. P6126)
Nicotine Sigma-Aldrich
14* 0.12 mg/mL
(Cat. No. N3876)
15* Toothpaste Colgate® 0.5% (v/v)

16* Mouthwash Listerine® 20% (v/v)

* These interfering substances, which could be found in saliva sample, were tested using saliva
specimen only.

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 Allplex™ SARS-CoV-2 fast PCR Assay

5. Clinical performance

5-1. Standard extraction

[Comparison study with CE-IVD approved comparator]
A total of 640 specimens were included in this clinical performance. The specimens consist of
640 nasopharyngeal swab (NPS) samples. Clinical performance of the Allplex™ SARS-CoV-2
fast PCR Assay was evaluated through the comparison with other SARS-CoV-2 Real-Time
RT-PCR Diagnostic Panel which is CE-IVD approved before. This comparison test shows
more than 95% rate of agreement in clinical sample. Therefore, it is confirmed that the quality
of Allplex™ SARS-CoV-2 fast PCR Assay is valid. The performance is summarized in the
table.

CE-IVD Approved Comparator Result

Positive Negative Total
Allplex™ Positive 120 1* 121
SARS-CoV-2 fast Negative 1* 518 519
PCR Assay Total 121 519 640
- PPA (Positive Percent Agreement): 99.17% (95% CI: 95.48% to 99.98%)
- NPA (Negative Percent Agreement): 99.81% (95% CI: 98.93% to 100.00%)
- OPA (Overall Percent Agreement): 99.69% (95% CI: 98.88% to 99.96%)
- Kappa value: 0.990 (95% CI: 0.976 to 1.000)
* Samples were confirmed true positive by sequencing.

Additional clinical performance using specimens collected from symptomatic/asymptomatic

patients suspected of COVID-19 was conducted. A total of 125 specimens which are
nasopharyngeal swab (NPS) were included in this clinical performance comparison test.
Clinical performance of the Allplex™ SARS-CoV-2 fast PCR Assay was evaluated through the
comparison with other SARS-CoV-2 Real-Time RT-PCR Diagnostic Panel which is CE-IVD
approved before. This comparison test shows more than 95% rate of agreement in clinical
sample. Therefore, it is confirmed that the quality of Allplex™ SARS-CoV-2 fast PCR Assay is
valid for NPS samples collected from symptomatic/asymptomatic patients. The performance is
summarized in the table.

CE-IVD Approved Comparator Result

Positive Negative Total
Allplex™ Positive 50 2* 52
SARS-CoV-2 fast Negative 2* 68 70
PCR Assay Total 52 70 122
- PPA (Positive Percent Agreement): 96.15% (95% CI: 86.79% to 99.53%)
- NPA (Negative Percent Agreement): 97.14% (95% CI: 90.06% to 99.65%)

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 Allplex™ SARS-CoV-2 fast PCR Assay

- OPA (Overall Percent Agreement): 96.72% (95% CI: 91.82% to 99.10%)

- Kappa value: 0.933 (95% CI: 0.868 to 0.998)
* Samples confirmed true positive by sequencing

[Comparison between nasopharyngeal swab (NPS), saliva and combo-swab (nasal

swab + oral swab)]
A total of 351 clinical specimens (117 NPS, 117 saliva, 117 combo-swab) were included in the
clinical performance of standard extraction for saliva and combo-swab. The specimens were
collected from symptomatic/asymptomatic patients. Three types of specimens
(nasopharyngeal swab, saliva and combo-swab) were taken per patient. Each sample was
tested with Allplex™ SARS-CoV-2 fast PCR Assay by standard extraction. In order to prove
the clinical validity of standard extraction for saliva and combo-swab, it was evaluated by
comparing with standard extraction for nasopharyngeal swab. This comparison test showed
higher than 95% rate of agreement in clinical sample. Therefore, it is confirmed that the quality
of standard extraction for saliva and combo-swab for Allplex™ SARS-CoV-2 fast PCR Assay
is valid. The performance is summarized in the tables below.

<Saliva>
Standard extraction for NPS
Positive Negative Total
Positive 47 2* 49
Standard extraction
Negative 2* 66 68
for Saliva
Total 49 68 117
- PPA (Positive Percent Agreement): 95.92% (95% CI: 86.02% to 99.50%)
- NPA (Negative Percent Agreement): 97.06% (95% CI: 89.78% to 99.64%)
- OPA (Overall Percent Agreement): 96.58% (95% CI: 91.48% to 99.06%)
- Kappa value: 0.930 (95% CI: 0.862 to 0.997)
* Samples confirmed true positive by sequencing

<Combo-swab>
Standard extraction for NPS
Positive Negative Total
Standard extraction
Positive 48 2* 50
for combo-swab (nasal Negative 1* 66 67
swab + oral swab)
Total 49 68 117
- PPA (Positive Percent Agreement): 97.96% (95% CI: 89.15% to 99.95%)
- NPA (Negative Percent Agreement): 97.06% (95% CI: 89.78% to 99.64%)
- OPA (Overall Percent Agreement): 97.44% (95% CI: 92.69% to 99.47%)
- Kappa value: 0.947 (95% CI: 0.889 to 1.000)

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 Allplex™ SARS-CoV-2 fast PCR Assay

* Samples confirmed true positive by sequencing

5-2. Extraction-free for swab

[Comparison study with CE-IVD approved comparator]

A total of 110 specimens were included in this clinical performance. The specimens consist of
110 nasopharyngeal swab (NPS) specimens. Clinical performance equivalence of Seegene’s
AllplexTM SARS-CoV-2 fast PCR Assay in extraction-free method for the NPS specimens was
assessed. The results were compared with results obtained from the samples prepared by
extraction method which has already been CE marked. This comparison test shows more than
90% rate of agreement in clinical samples. Therefore, it is confirmed that the quality of
AllplexTM SARS-CoV-2 fast PCR Assay in extraction-free method for NPS specimens is valid.
The performance is summarized in the table.

Extraction
Positive Negative Total
Positive 70 0 70
Extraction-free Negative 6 34 40
Total 76 34 110
- PPA (Positive Percent Agreement): 92.11% (83.60% to 97.05%)
- NPA (Negative Percent Agreement): 100.00% (89.72% to 100.00%)
- OPA (Overall Percent Agreement): 94.55% (95% CI: 88.51% to 97.97%)
- Kappa value: 0.878 (95% CI: 0.784 to 0.972)

Additional clinical performance using specimens collected from symptomatic/asymptomatic

patients suspected of COVID-19 was conducted. A total of 125 specimens which are
nasopharyngeal swab (NPS) were included in this clinical performance comparison test.
Clinical performance equivalence of Seegene’s Allplex TM SARS-CoV-2 fast PCR Assay in
extraction-free method for the NPS specimens was assessed. The results were compared with
results obtained from the samples prepared by extraction method which has already been CE
marked. This comparison test shows more than 95% rate of agreement in clinical samples.
Therefore, it is confirmed that the quality of Allplex TM SARS-CoV-2 fast PCR Assay in
extraction-free method is valid for NPS specimens collected from symptomatic/asymptomatic
patients. The performance is summarized in the table.

Extraction
Positive Negative Total
Positive 35 3* 38
Extraction-free Negative 1* 86 87
Total 36 89 125

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 Allplex™ SARS-CoV-2 fast PCR Assay

- PPA (Positive Percent Agreement): 97.22% (95% CI: 85.47% to 99.93%)

- NPA (Negative Percent Agreement): 96.63% (95% CI: 90.46% to 99.30%)
- OPA (Overall Percent Agreement): 96.80% (95% CI: 92.01% to 99.12%)
- Kappa value: 0.923 (95% CI: 0.849 to 0.997)
* Samples confirmed true positive by sequencing

5-3. Extraction-free for saliva

[Comparison study with CE-IVD approved comparator]

A total of 89 specimens collected from symptomatic/asymptomatic patient were included in this

clinical performance comparison test. Clinical performance equivalence of Seegene’s Allplex TM
SARS-CoV-2 fast PCR Assay in extraction-free method for the saliva specimens was
assessed. The results were compared with the results obtained from the samples prepared by
extraction method which has already been CE marked. This comparison test shows more than
95% rate of agreement in clinical samples. Therefore, it is confirmed that the quality of
AllplexTM SARS-CoV-2 fast PCR Assay in extraction-free method for saliva specimens is valid.
The performance is summarized in the table.
Extraction
Positive Negative Total
Positive 31 2* 33
Extraction-free Negative 1* 55 56
Total 32 57 89
- PPA (Positive Percent Agreement): 96.88% (95% CI: 83.78% to 99.92%)
- NPA (Negative Percent Agreement): 96.49% (95% CI: 87.89% to 99.57%)
- OPA (Overall Percent Agreement): 96.63% (95% CI: 90.46% to 99.30%)
- Kappa value: 0.927 (95% CI: 0.846 to 1.000)
* Samples confirmed true positive by sequencing

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2. D. H. Lee. [TOCE: Innovative Technology for High Multiplex Real-time PCR] Seegene Bulletin.

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3. Y. J. Lee, et al. [Single-channel multiplexing without melting curve analysis in real-time PCR]

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4. J. Y. Chun, et al. [Dual priming oligonucleotide system for the multiplex detection of respiratory

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5. A. E. Gobalenya, et al. [The species Severe acute respiratory syndrome-related coronavirus:

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6. [Coronavirus disease named Covid-19] BBC News Online. (2020)

7. [Surveillance case definitions for human infection with novel coronavirus (nCoV): interim guidance

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9. [About Novel Coronavirus (2019-nCoV)] United States Centers for Disease Control and Prevention

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10. [CoV2020] GISAID EpifluDB. (2020)

11. [WHO Director-General's opening remarks at the media briefing on COVID-19 - 11 March 2020]

World Health Organization (WHO). (2020)

12. [W.H.O. Declares Global Emergency as Wuhan Coronavirus Spreads] The New York Times. (2020)

13. J. F. Chan, et al. [A familial cluster of pneumonia associated with the 2019 novel coronavirus

indicating person-to-person transmission: a study of a family cluster] Lancet. (2020) 395(10223):

514–523

14. P. Zhou, et al. [A pneumonia outbreak associated with a new coronavirus of probable bat origin]

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15. S. Perlman. [Another Decade, Another Coronavirus] The New England Journal of Medicine. (2020)

382(8): 760–762

16. D. Benvenuto, et al. [The 2019-new coronavirus epidemic: Evidence for virus evolution] Journal of

Medical Virology. (2020) 92(4): 455–459

17. [Novel Coronavirus (2019-nCoV): situation report, 22 (Report)] World Health Organization. (2020)

18. [Coronavirus: From bats to pangolins, how do viruses reach us?] Deutsche Welle. (2020)

19. D. S. Hui, et al. [The continuing 2019-nCoV epidemic threat of novel coronaviruses to global health –

The latest 2019 novel coronavirus outbreak in Wuhan, China] The International Journal of Infectious

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Diseases. (2020) 91: 264–266

20. M. J. Loeffelholz, et al. [Laboratory diagnosis of emerging human coronavirus infections – the state of

the art] Emerging Microbes & Infections. (2020) 9(1): 747-756

21. H. J. Huh, et al. [Surveillance of Coronavirus Disease 2019 (COVID-19) Testing in Clinical

Laboratories in Korea] Ann Lab Med. (2021) 41(2): 225-229

22. A. Piccioni, et al. [Patient safety recommendations and management in patients with COVID-19

pneumonia suspicion: a retrospective study] Clin Ter. (2021) 172(3): 225-230

23. [Overview of Testing for SARS-CoV-2 (COVID-19)] United States Centers for Disease Control and

Prevention (CDC). (2021)

24. [Laboratory testing for coronavirus disease (COVID-19) in suspected human cases: Interim guidance

19 March 2020 (Report)] World Health Organization. (2020)

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SYMBOLS

Key to symbols used in the manual and labels.

Symbol Explanation

In vitro diagnostic medical device

Batch code

Catalogue number

Use-by date

Upper limit of temperature

Oligonucleotide mix for amplification and detection

Enzyme mix

Buffer

RNase-free Water

Positive Control (PC)

Consult instructions for use

Manufacturer

Date of manufacture

Authorized representative in the European Community

Caution

Contains sufficient for <n> tests

Unique Device Identifier

Reaction barcode for automated extraction system

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ORDERING INFORMATION

Cat. No. Product Size

Allplex™ series
RP10244Y Allplex™ 2019-nCoV Assay 50 rxns
RP10243X Allplex™ 2019-nCoV Assay 100 rxns
RV10351Z Allplex™ SARS-CoV-2 Assay 25 rxns
RV10248X Allplex™ SARS-CoV-2 Assay 100 rxns
RV10349Z Allplex™ SARS-CoV-2/FluA/FluB/RSV Assay 25 rxns
RV10259X Allplex™ SARS-CoV-2/FluA/FluB/RSV Assay 100 rxns
RV10382Z Allplex™ SARS-CoV-2 Master Assay 25 rxns
RV10284X Allplex™ SARS-CoV-2 Master Assay 100 rxns
RV10381Z Allplex™ SARS-CoV-2 Variants I Assay 25 rxns
RV10286X Allplex™ SARS-CoV-2 Variants I Assay 100 rxns
RV10388Z Allplex™ SARS-CoV-2 Variants II Assay 25 rxns
RV10305X Allplex™ SARS-CoV-2 Variants II Assay 100 rxns
RV10363Z Allplex™ RV Master Assay 25 rxns
RV10307X Allplex™ RV Master Assay 100 rxns
RV10345Z Allplex™ SARS-CoV-2 fast PCR Assay 25 rxns
RV10344X Allplex™ SARS-CoV-2 fast PCR Assay 100 rxns
RV10354X Allplex™ SARS-CoV-2 fast MDx Assay 100 rxns

Automated extraction systems

65415-02 Microlab NIMBUS IVD EA
173000-075 Microlab STARlet IVD EA
65415-03 Seegene NIMBUS EA
67930-03 Seegene STARlet EA
SG72100 AIOS EA
744300.4.UC384 STARMag 96 X 4 Universal Cartridge Kit 384 T / 1 box
EX00013C STARMag 96 X 4 Viral DNA/RNA 200 C Kit 384 T / 1 box
EX00031C STARMag™ N/S Kit 384 T / 1 box
SG71100 SEEPREP32 EA
EX00009P STARMag 96 ProPrep (Plate Type) 96 T / 1 box
EX00009T STARMag 96 ProPrep (Tube Type) 96 T / 1 box

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EX00017P STARMag 96 ProPrep C (Plate Type) 96 T / 1 box

EX00017T STARMag 96 ProPrep C (Tube Type) 96 T / 1 box
EX00028P STARMag™ SP32 Kit (Plate Type) 96 T / 1 box
EX00028T STARMag™ SP32 Kit (Tube Type) 96 T / 1 box
SG71101 Seegene STARlet 96MPH EA

EX00032P STARMag™ S96H Kit 48 T / 1 box

EX00033P STARMag™ S96H Kit 480 T / 1 crt

EX00034P STARMag™ S96H Kit 96 T / 1 box

EX00035P STARMag™ S96H Kit 960 T / 1 crt

M9600 Maelstrom™ 9600 EA

TANBead® Nucleic Acid Extraction Kit

W665S66 72 T / 1 box
OptiPure Viral Auto Tube
W665A10 TANBead® Nucleic Acid Extraction Kit
960 T / 1 crt
W665A10-SG15 OptiPure Viral Bulk Plate
EX00029P STARMag™ M96 Kit 96 T / 1 box

EX00030P STARMag™ M96 Kit 960 T / 1 crt

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