DiaPlexQ™ Novel Coronavirus (2019-nCoV) Detection Kit

INSTRUCTIONS FOR USE (IFU)

Revision 2.12 │01.2022

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 Table of Contents
Symbols ................................................................................................................................................... 3

Intended Use .......................................................................................................................................... 4

Product description ............................................................................................................................... 5

Precautions and handing requirements ............................................................................................ 7

Contents .................................................................................................................................................. 9

Storage and Handling........................................................................................................................... 9

Material to be supplied by User....................................................................................................... 10

Compatible Real-Time PCR thermocycler....................................................................................... 10

Process ................................................................................................................................................... 11

Analysis and results ............................................................................................................................ 14

Quality Control ..................................................................................................................................... 18

Trouble Shooting ................................................................................................................................. 19

Performance Characteristics .............................................................................................................. 20

Appendix ............................................................................................................................................... 26

References ............................................................................................................................................. 34

Ordering Information.......................................................................................................................... 35

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 Symbols

Please note that component labels do not contain CE mark, IVD symbol, and manufacturer's complete address due to label
size limitations.

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Intended Use

The DiaPlexQ™ Novel Coronavirus (2019-nCoV) Detection Kit is a real-time reverse transcriptase (RT)-PCR
test intended for the qualitative detection of nucleic acid from SARS-CoV-2 in nasopharyngeal swabs,
oropharyngeal (throat) swabs, and sputum from individuals suspected of COVID-19 by their healthcare
provider.

Results are for the identification of SARS-CoV-2 RNA. The SARS-CoV-2 RNA is generally detectable in
respiratory specimens during the acute phase of infection. Positive results are indicative of the presence of
SARS-CoV-2 RNA; clinical correlation with patient history and other diagnostic information is necessary to
determine patient infection status. The agent detected may not be the definite cause of disease. Positive
results do not rule out bacterial infection or co-infection with other viruses.

Negative results do not preclude SARS-CoV-2 infection and should not be used as the sole basis for patient
management decisions. Negative results must be combined with clinical observations, patient history, and
epidemiological information.

The DiaPlexQ™ Novel Coronavirus (2019-nCoV) Detection Kit is intended for use by qualified clinical
laboratory personnel specifically instructed and trained in the techniques of real-time PCR and in vitro
diagnostic procedures.

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Product description

1. Summary and Explanation

Respiratory infections caused by viruses appears mainly in children, the elderly and immunocompromised
patients. The major respiratory infection viruses are known as Influenza virus, Parainfluenza virus (PIV),
Respiratory syncytial virus (RSVs), Enterovirus, Adenovirus, etc. In recent years, respiratory infections of
Rhinovirus, Coronavirus and Metapneumovirus (MPV) have been increasing, and Bocavirus has been
included in the major respiratory virus tests.
Coronavirus is a virus that can infect animals and humans. There are six known coronaviruses that can infect
humans. Four of them are known as viruses that cause diseases such as the common cold, and the other
two are MERS-CoV (Middle East Respiratory Syndrome Coronavirus) and SARS-CoV (Severe Acute
Respiratory Syndrome Coronavirus), which have been fatal to humans.

SARS-COV-2 is transmissible between humans, with up to a 14-day incubation period, and has been
reported to have a lower mortality rate and a higher incidence than SARS-CoV or MERS-CoV. Sequencing
of the virus revealed that SARS-CoV-2 was 89.1% homologous to bat-derived SARS (severe acute respiratory
syndrome)-like coronaviruses, bat-SL-CoVZC45, bat- SL-CoVZXC21), and 79% homologous to SARS-CoV. It
is also very important to accurately diagnose COVID-19, since it has sequences similar to viruses of the
same genus.
Respiratory virus testing requires the selection of appropriate test methods depending on the characteristics
of the hospital's patient population and laboratory conditions. Antigen testing, virus culture, and molecular
biological methods have been used to detect viruses. Among them, the molecular biological method using
Reverse Transcription Polymerase Chain Reaction (RT-PCR) is analytically highly sensitive and is recognized
as a standard method for detecting viruses that cannot be cultured or that exist in low concentrations.
Recently, one-step RT-PCR, in which reverse transcription and polymerase chain reaction (PCR) amplification
can be performed in one tube, allows for the accurate identification of many viruses. The DiaPlexQ™ Novel
Coronavirus (2019-nCoV) Detection Kit is a real-time RT-PCR test intended for the qualitative detection of
SARS-CoV-2 nucleic acid extracted from nasopharyngeal swabs, oropharyngeal (throat) swabs, and sputum.

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2. Principles of the Procedure

DiaPlexQ™ Novel Coronavirus (2019-nCoV) Detection Kit is an in vitro diagnostic reagent for qualitative
detection of ORF1a and the N gene of SARS-CoV-2 by processing through Multiplex OneStep qRT-PCR.

< Detection Target Information >

Target virus Target genes

N gene

SARS-CoV-2 ORF1a

PCRC

The kit includes 2X OneStep qRT-PCR Buffer, OneStep qRT-PCR Enzyme mix (including reverse transcriptase,
DNA polymerase and RNase inhibitor) and Primer & Probe Mixture. We also provide a DNA-based Control
Template (2019-nCoV), to monitor PCR process and reagent integrity.

< Fluorescence Information >

Target genes 5’ Fluorophore 3’ Quencher

N gene FAM BHQ1

ORF1a JOE BHQ1

PCRC Cal Fluor Red 610 BHQ2

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Precautions and handing requirements

– This product is used for In Vitro Diagnosis (IVD) in European Union.

– The results should be interpreted in accordance with the result analysis section of the IFU after
processing on a Compatible Real-Time PCR thermocycler.
– Only use PCR tubes that are compatible with the applicable PCR machine.
– Wear protective disposable powder-free gloves, a laboratory coat, and eye protection when handling
specimens.
– Always wear protective disposable powder-free gloves when handling kit components in order to avoid
any contamination that can affect the test result.
– Do not reuse disposable tips, gloves, test tubes etc.
– Be careful not to let the reagents in this test come into contact with skin, eyes or mucous membranes.
If contact occurs, wash off immediately with plenty of water.
– The work area should be disinfected prior to and after use.
– All reagents should be stored by following the specified storage conditions before and after use.
– Do not leave the reagent cap open.
– Only use sterile pipette tips.
– Dispose of unused kit reagents and human specimens according to your institution’s safety guidelines
for hazardous material. Do not smoke, drink, eat, handle contact lenses or apply make-up in areas
where kit reagents and/or human specimens are being used. Follow universal precautions and treat all
specimens, samples and used kit components as potentially infectious.
– Store Control Template separately in order to prevent contamination.
– Please thaw the product on ice.
– When using the product, do not mix components from different kit lots.
– If an item arrives broken or damaged during transport, contact SolGent Co., Ltd.

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Product warranty and liability
– The product expiry date is 18 months after the manufacturing date.
– Only use the protocol described in this package insert. Deviations from the protocol may
give erroneous results.
– Exchange is not possible in case of a problem due to the user’s carelessness or fault.
– Do not repeat freeze-thaw over 5 times.

Safety warnings and first aid measures

– Avoid contact with eyes, skin and respiratory system.
– Eye contact: Wash eyes with lots of flowing water.
– Skin contact: Wash affected skin area thoroughly with soap and water.
– Consult with physician in case of irritation.

Precautions
– Do not use product after expiration date.
– Immediately use this kit after opening.
– Specimen quality and the integrity of the extracted nucleic acid may affect test results.
– False results may occur due to contamination.
– Dispose of used devices, pipette tips and specimen tubes according to your institution’s
safety guidelines for hazardous material.

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Contents

Components SQD52-K100

OneStep qRT-PCR Enzyme mix (2019-nCoV) 200 µL x 1 ea

2X OneStep qRT-PCR Buffer (2019-nCoV) 1 mL x 1 ea

Primer & Probe Mixture (2019-nCoV) 300 µL x 1 ea

Control Template (2019-nCoV) 100 µL x 1 ea

RNase free Water 1 mL x 1 ea

Storage and Handling

• DiaPlexQ™ Novel Coronavirus (2019-nCoV) Detection Kit should be stored at -20℃ ± 5℃ and kept
away from sunlight. All components should be stored under recommended storage conditions.

Model Name Storage Period of use

DiaPlexQ™ Novel Coronavirus (2019-nCoV) Detection Kit -20℃ ± 5℃ 18 months

– The expiry date of each component of the product is 18 months from the date of manufacture.
– Do not use product beyond the expiration date.
– Please thaw the product on the ice.
– Do not freeze-thaw more than 5 times.
– If there are or have been transportation problems, or the protective packaging is damaged, do not use the
kit and contact SolGent Co., Ltd. for guidance.

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Material to be supplied by User

– Micro-centrifuge tube
– Micro-centrifuge
– Vortexer
– Pipettes/ pipette filter tips
– Laboratory freezers
– Disposable latex
– Cooling device or ice
– Tubes, plates, and other consumables
– QIAGEN QIAamp DSP Viral RNA Mini Kit (Cat. # 61904) or the MagNA Pure 96 nucleic acid extraction
system with software V3.1 and the MagNA Pure 96 DNA and Viral NA Small Volume Kit (Cat. #06 543 588
001) with External Lysis Buffer (Cat. #06 374 913 001)
– Real-Time PCR Instrument System and data analysis software
– AccuPlex™ SARS-CoV-2 Reference Material Kit (Cat. No. 0505-0126)

Compatible Real-Time PCR thermocycler

– Applied Biosystems™ 7500 Real-Time PCR Instrument System with software V2.0.6
– Applied Biosystems™ 7500 Fast Real-Time PCR Instrument System with software V2.0.6
– Bio-Rad CFX96™ Real-time PCR Detection System with software V3.1

Note:
1. Use film for the plate and cap for strip.
2. Use the dedicated PCR tube for the PCR machine.

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Process

This test process is optimized for DiaPlexQ™ Novel Coronavirus (2019-nCoV) Detection Kit. Use of this
kit is limited to qualified clinical laboratory personnel specifically instructed and trained in the techniques
of real-time PCR and in vitro diagnostic procedures.

Overview

1. Sample collection

The kit can be used for nasopharyngeal swabs, oropharyngeal (throat) swabs, and sputum. Specimens should
be collected, transported and stored according to standard procedures.

2. RNA Isolation

RNA should be extracted from nasopharyngeal swabs, oropharyngeal (throat) swabs, and sputum using the
QIAamp DSP Viral RNA Mini Kit (QIAGEN, catalog # 61904) or MAgNa Pure 96 (Roche, 576 Extraction (06
543 588 001), External Lysis Buffer (06 374 913 001)). Other RNA extraction kits have not been qualified or
validated.
An External Positive Control should be processed in parallel with each batch of patient samples to monitor
for RNA recovery and as a control for reverse transcription. Refer to the Quality Control Section below for
information on how to prepare an appropriate External Positive Control.

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3. Multiplex OneStep qRT-PCR

1) Please thaw all reagents on the ice. After vortex, spin down.
2) Prepare PCR Master Mix by adding the following reagents.
3) The amount of Master mix should be prepared by calculating overage corresponding to at least 1~2
reactions more than the number of samples and controls (including the PCR Positive Control (control
template (2019-nCoV)), NTC (No Template Control) and External Positive Control).
4) Mix master mix using vortex and spin down.

Component 1 rxn 4 rxns 5 rxns 6 rxns 10 rxns

2x OneStep qRT-PCR Buffer (2019-nCoV) 10 µL 40 µL 50 µL 60 µL 100 µL

OneStep qRT-PCR Enzyme mix (2019-nCoV) 2 µL 8 µL 10 µL 12 µL 20 µL

Primer & Probe Mixture (2019-nCoV) 3 µL 12 µL 15 µL 18 µL 30 µL

Total master mix volume 15 µL 60 µL 75 µL 90 µL 150 µL

Note:
Protect the Probe from the light. When the Probe is exposed to the light for a long time, fluorescence may be
reduced and may affect the result.

5) Dispense 15 µL into a plate or strip tube suitable for the equipment using the manufactured master mix.
6) Add Template 5 µL.

Component Volume

PCR master mix 15 µL

Template 5 µL

Total volume 20 µL

Note:
- A PCR Positive Control (Control Template (2019-nCoV)) and NTC (No Template Control) should be included in
each PCR run to check the normal function of the product and contamination of the laboratory environment.
- The PCR Positive Control uses Control Template (2019-nCoV) as template.
- NTC (No Template Control) uses RNase free water as template.
- An External Positive Control comprised of package viral RNA must also be tested in parallel with each batch of
samples to monitor for RNA recovery and reverse transcription (refer to Quality Control section).

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 7) After sealing with cap or film, spin down.
8) Place the prepared PCR mixture on the instrument and proceed with PCR under the following
conditions.
* Refer to Appendix for device setup and Run

No. Step Temperature Acquisition Time Cycles

1 Reverse transcription 50°C - 15 min 1

2 Initial PCR activation 95°C - 15 min 1

3 Denaturation 95°C - 20 sec

45
4 Annealing/Extension 60°C √ 40 sec

< Select the Fluorophore on PCR instruments >

Target genes ABI 7500 / 7500 Fast CFX96™

N gene FAM FAM

ORF1a gene JOE VIC

PCRC Texas Red Cal Fluor Red 610

The kit does not include a “Reference dye”

(Set up the reference dye to “None” in the ABI 7500 / 7500 Fast program)

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Analysis and results

1. Amplicon information
As shown in the following figure, you can check the detection of SARS-CoV-2 by comparing with the result
of amplification of Control Template (2019-nCoV).

Figure 1. DiaPlexQ™ Novel Coronavirus (2019-nCoV) Detection Kit Diagram.

Green is N gene (FAM), Blue is Orf1a gene (JOE/VIC), Red is PCRC (Texas Red/Cal Fluor Red
610)

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2. Cut-off value
① If you are using ABI 7500 or ABI7500 FAST, you can check result of Ct value as follows:
A. Plate and Film: Set the Threshold 20,000
B. Tube and Cap: Set the Threshold 20,000
② If you are using CFX96™, you can check result of Ct value as follows:
A. Plate and Film: Set the Threshold 300
B. Tube and Cap: Set the Threshold 300
③ The amplification plots for the assay controls must satisfy the following conditions.

Control N Orf1a PCRC Expected Ct values of target

No Template Control - - + PCRC ≤26

PCR Positive Control or External N, Orf1a ≤40

+ + +
Positive Control PCRC ≤26

(*If the results show 40 < Ct ≤ 45, perform the experiment again.)

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3. Result Interpretation for Patient Samples
Ct Value
Interpretation
N Gene ORF1a PCRC

≤ 40 Any Any Positive

Any ≤ 40 Any Positive

≤ 40 ≤ 40 Any Positive

> 40 None Any Inconclusive1

None > 40 Any Inconclusive1

> 40 > 40 Any Inconclusive1

None None ≤ 26 Negative

None None > 26 or None Invalid2

1 Repeat RT-PCR
2 Repeat extraction and RT-PCR

Note:

1. Even if the target is detected (Ct ≤40) and the PCRC is not detected, the result is still valid because:
– If the sample is high concentration, PCRC may not amplify.
– If PCR inhibitors are present, the PCRC may not amplify.
2. When the No Template Control test result is positive, all samples must be retested.

※ PCRC (PCR Control)

Erroneous results may occur due to a variety of factors - for example, PCR mixture mix error, PCR condition
error, PCR equipment use error etc. The PCR control is intended to monitor for the success of the PCR
process. If the PCRC fails unexpectedly all experimental procedures and steps should be checked.

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4. Required re-experiment

Figure 2. The failed result of curve pattern

Note :

If PCRC is not amplified, the purity of the RNA sample is not good. Thus, check it by dilute the sample (10 ~ 100 times)
or extract RNA again.

This kit is for the detection of SARS-CoV-2 RNA. Clinical correlation with patient history and other
diagnostic information is necessary to determine patient infection status.

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Quality Control

In accordance with ISO-certified Quality Management System of SolGent, each lot of DiaPlexQ™ Novel
Coronavirus (2019-nCoV) Detection Kit is tested against predetermined specifications to ensure consistent
product quality. External controls are not provided with the DiaPlexQ™ Novel Coronavirus (2019-nCoV)
Detection Kit.

The following external control materials are available:

AccuPlex™ SARS-CoV-2 Reference Material Kit (Cat. No. 0505-0126)

Positive External Controls should be prepared by diluting the stock of virus particles in PBS to a final
concentration of 1,000 copies/200 μL. External Positive Controls should be processed like patient samples
to monitor RNA extraction, reverse transcription, PCR amplification and detection.
At least one External Positive Control should be processed with every batch of patient samples. The expected
result must be obtained with the External Positive Control, as well as the Positive (Template) and Negative
(No Template) PCR Controls in order to interpret the results obtained with patient samples.

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Trouble Shooting
Problem Possible Causes Solution

Check storage temperature for the reagents and obtain new

Wrong Storage condition
reagents if needed
Too short time for Enzyme
Check that the “initial PCR activation” is set for 15min at 95℃
activation in PCR reaction
Expired shelf life Check the expiration date and obtain new reagents if needed
Check primers & probes by swapping with a new primer and
Primer and probe degraded
probe mixture from the same kit lot.
Low template quality Check template quality using spectrophotometer
No or weak PCR Check the processing conditions for the sample. Repeat extraction as
Inhibitors in Template
product appropriate.
fluorescent Inappropriate Nucleic acid Check the concentration of nucleic acid. Re-extract the nucleic acid if
preparation needed.
Template degradation Re-extract template
Reagents stored at room Do not leave reagents at room temperature for extended periods of
temperature time. Obtain new reagents as needed.
Re-test after activating plate read in the steps provided in the IFU when
Deactivation of Plate read
setting PCR conditions in the PCR machine
Unassigned Fluorophore in
Assign the correct fluorophore following the IFU and reanalyze the data.
sample well

Check to confirm that the laboratory environment and

Contamination of PCR mixture
equipment have not been contaminated. Clean as needed.
Non-specific PCR
Amplification Contamination If environment and equipment have not been contaminated,
from the extraction process replace RNA extraction PCR reagents
Contamination of Water Obtain new nuclease free water
False positive /
PCR product with Use filter tips, screw-cap tubes and latex gloves. Perform assay set-up in a
Cross-contamination
No Template hood in a clean environment.
Control(NTC)
Conflicting or Pipette volume error Check the Pipettes and calibrate as needed
unexpected results Cross contamination Be careful when you add samples to the PCR tubes
for different If there are foreign objects on
Remove any debris with a soft cloth before performing the PCR.
optical channels the PCR tubes or caps
Template degradation Do no repeatedly freeze-thaw the positive control (plasmid DNA)
Incorrect storage Check storage condition for kit and use a new kit if needed
No PCR product Inappropriate Nucleic acid Check the concentration of nucleic acid. Re-extract a fresh aliquot of
with positive preparation the sample if needed
control or false Incorrect PCR mixture (primer Check the volumes used for the mixture in case of pipetting
negative & premix) volume error
Storage of the reagents at Do not store the reagents at room temperature and obtain new
room temperature reagents if needed

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Performance Characteristics

1. Limit of Detection (LoD) – Analytical Sensitivity

The preliminary LoD was established by testing serial dilutions of SARS-CoV-2 packaged viral RNA using
the ABI 7500 Fast system. The samples of serial dilutions (4,000 copies/mL, 400 copies/mL, 200 copies/mL,
40 copies/mL) were prepared by spiking the quantified SARS-CoV-2 packaged viral RNA into negative
respiratory clinical matrices (nasopharyngeal swab and sputum). Each replicate was extracted using the
Qiagen QIAamp DSP Viral RNA Mini Kit. For both matrices, the lowest target level at which all five replicates
produced positive results was 200 copies/mL. The LoD was confirmed by testing 20 replicates at the
estimated LoD concentration. All 20/20 test results with both nasopharyngeal swabs and sputum were
positive. The LoD of the DiaPlexQ™ Novel Coronavirus (2019-nCoV) Detection Kit with nasopharyngeal
swabs and sputum was confirmed to be 200 copies/mL.

Final LoD confirmation with SARS-CoV-2 packaged viral RNA [Nasopharyngeal swab]
Instrument Extraction method Target Results Mean Ct (n=20) LoD (copies/mL)
N gene 20/20 33.3 200
Applied Biosystems Qiagen kit
ORF1a gene 20/20 34.5 200
7500 Fast Real-Time
N gene 20/20 33.2 200
PCR Instrument System MAgNa Pure 96
ORF1a gene 20/20 34.0 200
N gene 20/20 34.5 200
Applied Biosystems Qiagen kit
ORF1a gene 20/20 34.9 200
7500 Real-Time PCR
N gene 20/20 33.6 200
Instrument System MAgNa Pure 96
ORF1a gene 20/20 32.9 200
N gene 20/20 35.3 200
Qiagen kit
CFX96_IVD real-time ORF1a gene 20/20 35.1 200
PCR detection system N gene 20/20 34.1 200
MAgNa Pure 96
ORF1a gene 20/20 33.2 200

Final LoD confirmation with SARS-CoV-2 packaged viral RNA [Sputum]

Instrument Extraction method Target Results Mean Ct (n=20) LoD (copies/mL)
N gene 20/20 33.2 200
Applied Biosystems Qiagen kit
ORF1a gene 20/20 34.2 200
7500 Fast Real-Time
N gene 20/20 33.6 200
PCR Instrument System MAgNa Pure 96
ORF1a gene 20/20 34.1 200
N gene 20/20 34.7 200
Applied Biosystems Qiagen kit
ORF1a gene 20/20 34.7 200
7500 Real-Time PCR
N gene 20/20 34.2 200
Instrument System MAgNa Pure 96
ORF1a gene 20/20 33.7 200
N gene 20/20 35.0 200
Qiagen kit
CFX96_IVD real-time ORF1a gene 20/20 35.4 200
PCR detection system N gene 20/20 34.0 200
MAgNa Pure 96
ORF1a gene 20/20 33.2 200

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2. Cross-reactivity
To demonstrate the analytical specificity of the DiaPlexQ™ Novel Coronavirus (2019-nCoV) Detection Kit
testing was performed using high concentrations (>105 genomic equivalents/mL) of purified RNA or DNA
from organisms and viruses that may be found in the respiratory tract. (Table). No cross reaction was
observed.

No. Viruses / Bacteria Ct Value No. Viruses / Bacteria Ct Value

1 Parainfluenza I N/A 21 Klebsiella pneumoniae N/A

2 Parainfluenza II N/A 22 Legionella pneumophila N/A
3 Parainfluenza III N/A 23 Moraxella catarrhalis N/A
4 Parainfluenza IV N/A 24 Mycoplasma pnemoniae N/A
5 Influenza A N/A 25 Pseudomonas aeruginosa N/A
6 Influenza B N/A 26 Serratia marcescens N/A
7 Adenovirus N/A 27 Staphylococcus aureus N/A
8 Respiratory syncytial virus A N/A 28 Stenotrophomonas maltophilia N/A
9 Respiratory syncytial virus B N/A 29 Streptococcus pneumoniae N/A
10 Rhino 8, A N/A 30 Mycobacterium abscessus N/A
11 Bocavirus N/A 31 Mycobacterium avium N/A
12 Metapneumovirus N/A 32 Mycobacterium bovis N/A
13 Beta Coronavirus OC43 N/A 33 Mycobacterium chelonae N/A
14 Alpha Coronavirus 229E N/A 34 Mycobacterium intracellulare N/A
15 Enterovirus N/A 35 Mycobacterium kansasii N/A
16 Acinetobacter baumannii N/A 36 Mycobacterium scrofulaceum N/A
17 Bordetella parapertussis N/A 37 Mycobacterium tuberculosis N/A
18 Bordetella pertussis N/A 38 Human total RNA (10ng/μl) N/A
19 Chlamydophila pneumoniae N/A 39 SARS-CoV-2 packaged viral RNA Detection
20 Haemophilus influenza N/A

N/A: Not applicable

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3. Precision Test

1) Repeatability
SARS-CoV-2 packaged viral RNA was tested at 3xLoD, 1xLoD, negative concentration, once a day with 3
lots of reagents, 2 replicates per run, 3 instruments and repeated for 10 days, and CV was less than 5%.
CV (%)
Precision Target gene
3xLoD 1xLoD Negative
N gene 1.631 2.621 N/D
Between run ORF1a gene 1.342 2.071 N/D
PCRC 1.862 1.514 1.405
N gene 1.937 2.350 N/D
Between lot ORF1a gene 1.685 2.241 N/D
PCRC 1.300 1.218 1.124
N gene 1.422 2.247 N/D
Between instrument ORF1a gene 2.167 2.637 N/D
PCRC 3.238 3.301 2.546

2) Reproducibility (between testers)

3 testers tested SARS-CoV-2 packaged viral RNA at 3xLoD, 1xLoD, negative concentration, once a day with
1 lot of reagents, 2 replicates per run and repeated for 5 days, and CV was less than 5%.
CV (%)
Precision Target
3xLoD 1xLoD Negative
N gene 0.881 1.480 N/D
Between tester ORF1a gene 0.745 1.553 N/D
PCRC 0.556 0.703 0.559

3) Reproducibility (between sites)

At 3 sites, SARS-CoV-2 packaged viral RNA at 3xLoD, 1xLoD, negative concentration, once a day with 1 lot
of reagents, 2 replicates per run and repeated for 5 days, and CV was less than 5%.
CV (%)
Precision Target
3xLoD 1xLoD Negative
N gene 1.658 2.016 N/D
Between site ORF1a gene 1.438 2.215 N/D
PCRC 3.375 3.521 3.761

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4. Clinical Evaluation

1) Clinical evaluation test 1

The results for clinical sensitivity and specificity with 77 positive upper respiratory samples and 121 negative
upper respiratory samples were shown 98.7% (95% confidence interval: 93.0%-99.8%) and 100% (95%
confidence interval: 96.9%-100%). The lower limit values of the confidence interval for sensitivity and
specificity have over lower limit values (Sensitivity: 90% & Specificity: 95%) of the confidence interval for
clinical objectives, so it has confirmed that it has clinical effectiveness.

The results of the diagnosis device

Total
Positive Negative

DiaPlexQ™ Novel Coronavirus Positive 76 0 76

(2019-nCoV) Detection Kit Negative 1 121 122

Total 77 121 198

Clinical sensitivity 98.7% (95% confidence interval: 93.0%-99.8%)

Clinical specificity 100% (95% confidence interval: 96.9%-100%)

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2) Clinical evaluation test 2
The results for clinical sensitivity and specificity with 165 positive and 94 negative upper respiratory samples
(nasopharynx, oropharynx) were shown 100% clinical sensitivity (95% confidence interval: 97.7%-100%) and
100% clinical specificity (95% confidence interval: 96.1%-100%). This result is effective because the target
clinical sensitivity is over 95% (95% CI, lower limit ≥ 90%) and the target clinical specificity is over 98% (95%
CI, lower limit ≥ 95%).

The results of the diagnosis device

Total
Positive Negative

DiaPlexQ™ Novel Coronavirus Positive 165 0 165

(2019-nCoV) Detection Kit Negative 0 94 94

Total 165 94 259

Clinical sensitivity 100% (95% confidence interval: 97.7%-100%)

Clinical specificity 100% (95% confidence interval: 96.1%-100%)

The results for clinical sensitivity and specificity with 165 positive and 94 negative lower respiratory samples
(sputum) were shown 98% clinical sensitivity (95% confidence interval: 94.8%-99.4%) and 100% clinical
specificity (95% confidence interval: 96.1%-100%). This result is effective because the target clinical sensitivity
is over 95% (95% CI, lower limit ≥ 90%) and the target clinical specificity is over 98% (95% CI, lower limit ≥
95%).

The results of the diagnosis device

Total
Positive Negative

DiaPlexQ™ Novel Coronavirus Positive 162 0 162

(2019-nCoV) Detection Kit Negative 3 94 97

Total 165 94 259

Clinical sensitivity 98% (95% confidence interval: 94.8%-99.4%)

Clinical specificity 100% (95% confidence interval: 96.1%-100%)

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3) Clinical evaluation test 3
The results for clinical sensitivity and specificity with 77 positive lower respiratory samples (sputum) and 121
negative lower respiratory samples (sputum) were shown 100% (95% confidence interval: 95.2%-100%) and
100% (95% confidence interval: 96.9%-100%). The lower limit values of the confidence interval for sensitivity
and specificity have over lower limit values (Sensitivity: 90% & Specificity: 95%) of the confidence interval
for clinical objectives, so it has confirmed that it has clinical effectiveness.

The results of the diagnosis device

Total
Positive Negative

DiaPlexQ™ Novel Coronavirus Positive 77 0 77

(2019-nCoV) Detection Kit Negative 0 121 121

Total 77 77 121

Clinical sensitivity 100% (95% confidence interval: 95.2%-100%)

Clinical specificity 100% (95% confidence interval: 96.9%-100%)

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Appendix

 Applied Biosystems™ 7500 / 7500Fast Real-Time PCR Instrument System Set up and Run

1. Click ‘Advanced Setup’ on the main screen.

Figure 3. Main

2. Enter the file name (or Experiment Properties screen).

Figure 4. Experiment Properties

2-1. Fill in “Experiment Name”
2-2. “Which instrument are you using to run the experiment?”
→ Check 7500 (96 Wells) or 7500 Fast (96 Wells)
2-3. “What type of experiment do you want to set up?”
→ Check Quantitation – Standard Curve
2-4. “Which reagents do you want to use to detect the target sequence?”
→ Check TaqMan® Reagents
2-5. “Which ramp speed do you want to use in the instrument run?”
→ Check Standard (~ 2 hours to complete a run) or Fast (~40 minutes to complete a run)

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 3. At the ‘Define Targets and Samples’ Tap in Plate Setup screen, please set up as follows.

Figure 5. Plate Setup - Define Targets and Samples

3-1. Click ‘Add New Target’ at Define Targets. Setup ‘Reporter’ and ‘Quencher’ as follows:
(Target Name and Color can be setup randomly.)
Reporter Quencher

FAM none

JOE none

Texas Red none

The Kit does not include a “Reference dye”
(Set up the reference dye to “None” in the ABI 7500 / 7500 Fast program)

3-2. If you want to fill out sample name, you can assign randomly at ‘Define Samples’.

4. At ‘Assign Targets and Samples’ Tap in ‘Plate Setup’ screen, please set up as follows.

Figure 6. Plate Setup - Assign Targets and Samples

4-1. “View Plate Layout” Select well according to the position of the PCR mixture reaction solution.
4-2. “Assign target(s) to the selected wells” Select Target (3-1).
4-3. “Assign samples(s) to the selected wells” Select Sample (3-2).
4-4. “Select the dye to use as the passive reference” Select None.

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 5. Set the PCR temperature condition as follows, enter the reaction volume as 20 ㎕ and click ‘Start Run’.
No. Step Temperature Acquisition Time Cycles

1 Reverse transcription 50°C - 15 min 1

2 Initial PCR activation 95°C - 15 min 1

3 Denaturation 95°C - 20 sec

45
4 Annealing/Extension 60°C √ 40 sec

Figure 7. Run Method

Note: In Step 6, check Collect Data on Hold to collect data.

6. Select ‘START RUN’ and set the location where the data will be saved.

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  CFX96™ Dx System Setup and Run

1. Turn on the instrument.

2. Run Bio-Rad CFX Manager.
3. Click ‘File’ → ‘New’ → ‘Protocol’.

Figure 8. Main
4. In the Protocol Editor screen, enter Sample Volume 20 ㎕, set the PCR Condition, and click ‘OK’
No. Step Temperature Acquisition Time Cycles

1 Reverse transcription 50°C - 15 min 1

2 Initial PCR activation 95°C - 15 min 1

3 Denaturation 95°C - 20 sec

45
4 Annealing/Extension 60°C √ 40 sec

Figure 9. Protocol Editor

Note: In Step 4, check ‘Collect Data on Hold’ to collect data.

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 5. Click ‘Create New’ in the plate tap. In ‘Plate Editor’ screen, click ‘Select Fluorophores’ and setup
fluorophore.

Figure 10. Plate Editor - 1

Fluorophore

FAM

VIC

Cal Fluor Red 610

6. After selecting well according to the position of PCR mixture reaction solution, designate ‘Sample
Type’ and ‘Fluorophore'.

Figure 11. Plate Editor - 2

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 7. Settings → Plate Type → click ‘BR White’ or ‘BR Clear’ according to the type you are using.

Figure 12. Plate Editor - 3

8. On the ‘Start Run’ tab in ‘Run Setup’, click ‘Close Lid’ to close the lid of the instrument, select the
active ‘Start Run’ and set the location where the data will be saved.

Figure 13. Run Setup

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  Applied Biosystems™ 7500 / 7500Fast Real-Time PCR Instrument System setup for result
analysis

1. After Real-Time PCR is finished, set ‘Plot Settings’ on the ‘Amplification Plot screen’ as below and
select ‘Analysis Settings’.

Figure 14. Amplification

Plot 1-1. “Plot Type” ΔRn vs Cycle / “Graph Type” Linear / “Color” Target

2. In ‘Analysis Settings’, specify the Threshold value for each Fluorophore, and then click ‘Apply Analysis
Settings’. * Threshold: 20,000 (Plate / Strip tube)

Figure 15. Analysis Settings

3. Interpret the results by referring to the result analysis.

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  CFX96™ Dx System Setup for result analysis

1. After Real-Time PCR is finished, check ‘Fluorophore’ in Data Analysis screen and click Settings →
Baseline Threshold.

Figure 16. Data Analysis

2. In the Baseline Threshold Screen, specify the Threshold value for each fluorophore and click OK.
* Threshold: 300 (Plate / Strip tube)

Figure 17. Baseline Threshold

3. Interpret the results by referring to the result analysis.

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References

1. Roujian Lu, Xiang Zhao, Wenjie Tan, et al. Genomic characterisation and epidemiology of 2019 novel
coronavirus: implications for virus origins and receptor binding (2020).
2. Victor Corman, Tobias Bleicker, et al. Diagnostic detection of 2019-nCoV by real-time RT-PCR (2020).
3. LKS Faculty of medicine of the University of Hong Kong. Detection of 2019 novel coronavirus (2019-nCoV) in
suspected human cases by RT-PCR (2020).
4. WHO. Laboratory Testing for Middle East Respiratory Syndrome Coronavirus (2018)
5. CDC. Real-Time RT-PCR Panel for Detection 2019-Novel Coronavirus (2020)
6. KCDC. 2019-nCoV detection real-time RT-PCR protocol (KCDC v1.5)
7. KCDC. COVID-19 Response Procedure
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human pneumovirus isolated from young children with respiratory tract disease. Nat Med 7:719-24 (2001).
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Med Virol 11: 351-4 (2001).
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assay with the R-mix viral culture and immunofluorescence techniques for detection of eight respiratory viruses.
Ann Clin Lab Sci 38:41-6 (2008).10. Forbes BA, et al. Bailey & Scott's Diagnostic Microbiology 12th Edition. St.
Lois Mosby 2007
16. Mahony JB, Petrich A, Smieja M. Molecular diagnosis of respiratory virus infections. Crit Rev Clin Lab Sci.
2011 Sep-Dec;48(5-6):217-49.
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Viruses. Semin Respir Crit Care Med 2011; 32(4): 512-526
18. Sigvard Olofsson, Robin Brittain-Long, Lars Magnus Andersson, Johan Westin1 and Magnus Lindh. PCR for
detection of respiratory viruses: seasonal variations of virus infections. Expert Review of Anti-infective Therapy.
2011, Vol. 9(8), P 615-626.
19. Shu Zhang, Wenhong Zhang and Yi-Wei Tang. Molecular Diagnosis of Viral Respiratory Infections. Current
Infectious Disease Reports. Volume 13, Number 2 (2011), 149-158,
20. Mahony JB. Detection of respiratory viruses by molecular methods. Clin Microbiol Rev. 2008;21:716–747.
21. Arden KE, McErlean P, Nissen MD, Sloots TP, Mackay IM. Frequent detection of human rhinoviruses,
paramyxoviruses, coronaviruses, and bocavirus during acute respiratory tract infections. J Med Virol.
2006;78:1232–1240.
22. Larson HE, Reed SE, Tyrrell DA. Isolation of rhinoviruses and coronaviruses from 38 colds in adults. J Med
Virol 5:221-9 (1980).

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Ordering Information

Cat. No. Name Size

SQD52-K100 DiaPlexQ™ Novel Coronavirus (2019-CoV) Detection Kit 100 reactions/Kit

35 | Revision. 2.12│IFU-61-EN
 SolGent Co., Ltd. ║43-10, Techno 5-ro, Yuseong-gu, Daejeon, 34014, Korea
솔젠트㈜ ║34014, 대전광역시 유성구 테크노5로 43-10
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