MNC-N10082 In vitro diagnostic medical device for qualitative detection of Novel coronaviruses (2019-nCoV) from RNA extracted from

tative detection of Novel coronaviruses (2019-nCoV) from RNA extracted from human nasopharyngeal swab and sputum

careGENE TM
N-CoV RT-PCR kit In vitro diagnostic medical devices

Intended Use using specific primers and probe specific to the viral 12. It is recommended to use the commercial RNA extraction
genome (2019-nCoV) with the same mechanism of the kit. [QIAamp DSP Virus RNA Mini Kit (QIAGEN, cat no. Number of test Pan-CoV set
careGENETM N-CoV RT-PCR kit is an in vitro
first screening test. 61704)].
diagnostic medical device for qualitative detection of 4X 1 Step RT-PCR Mix 5 μL
13. The final diagnosis should not be based solely on the
novel coronavirus (2019-nCoV) from RNA extracted Pan-CoV primer/probe Mix 5 μL
Materials Provided (100 tests/kit) results of this product. The final diagnosis should be based
from human Nasopharyngeal swab, Oropharyngeal Total 10 μL
on a combination of different test methods and clinical
swab and sputum using real-time RT-PCR (Reverse Components Volume Storage
results at the discretion of the physician. (2) Dispense 10 μL of the Pan-CoV set Master Mix into
transcription-Polymerase Chain Reaction). 4X 1 step RT-PCR Mix 500 μL x 2 Below -20℃
each well of an appropriate optical 96-well reaction
Introduction Pan-CoV primer/probe Mix 500 μL x 1 Below -20℃ Test Procedure
plate or an appropriate optical reaction tube.
N-CoV primer/probe Mix 500 μL x 1 Below -20℃ Specimen collection and handling
The novel coronavirus (2019-nCoV) is the first (3) Add 10 μL of RNA sample into each well of an
positive single-stranded RNA coronavirus reported in Positive control 500 μL x 1 Below -20℃ It is recommended to use the upper and lower respiratory
appropriate optical 96-well reaction plate or an
2019. The sequence is similar to the beta coronavirus Nuclease free water 600 μL x 1 Below -20℃ tract specimens of people with symptoms of novel
found in bats. It is genetically distinct from common coronavirus (2019-nCoV) infection and store them under appropriate optical reaction tube, and mix 2~3
coronaviruses, such as Severe Acute Respiratory the following conditions. times.
Materials Required but Not Provided
Syndrome coronavirus (SARS-CoV) and Middle East (4) Set the PCR machine with appropriate detection
1. Appropriate (optical) 96-well reaction PCR plate or Specimen from upper respiratory tract
Respiratory Syndrome coronavirus (MERS-CoV). channel.
tube 1. Collect nasopharyngeal swabs and oropharyngeal swabs
The outbreak of pneumonia caused by a novel
simultaneously and place them in one virus transport * Fluorescent Reporter
coronavirus in December 2019 in Wuhan City, Hubei 2. Micropipette
medium (VTM).
Province, China, is believed to have occurred in the 3. Centrifuge, Vortex mixer Detection target Reporter (Pan-CoV set)
Wuhan Huanan seafood wholesale market on - Nasopharyngeal swab : scrape the secretion through
4. Disposable powder-free gloves Pan-CoV (E gene) FAM
December 12, 2019. the nostrils from the lower and lower nasal concha
5. Any of following PCR machine Pan-SARS (RdRPP1) Cy5
Symptoms of infection are fever, dry cough, and (oropharyngeal).
Internal Control ROX
shortness of breath, and worsening symptoms can  CFX96 TM
Dx system (Bio-Rad) - Oropharyngeal swab : Press the tongue and scrape the
lead to pneumonia, kidney failure, or death in the case  Applied Biosystems 7500 Real-Time PCR System secretion from the pharyngeal wall. (5) Perform PCR amplification step as follows.
of serious infections. (Thermo Fisher Scientific) ※ VTM is not provided. (Do not set up the passive reference).
In accordance with the WHO literature published
UDG cDNA Pre-
on January 23, 2020, a quarter of those infected 2. To ensure accurate test results, immediately store the Amplification
Warnings and Precautions incubation synthesis denaturation
experienced severe illness, and many of the deaths bottle containing the specimen in the refrigerator (4℃)
1. For Professional Use Only 25℃ 55°C 94°C 94°C 60°C
showed the immune system damage including high until the test.
2. Be careful when handling specimens as they cannot 2 min 10 min 3 min 15 sec 30 sec
blood pressure, diabetes, cardiovascular disease, etc. Specimen from lower respiratory tract
exclude infections such as unknown microorganisms or
There are no known vaccines or treatments to 1. Sputum : Collect sputum into the sterilization container 1 cycle 1 cycle 1 cycle 45 cycles
other infectious diseases.
date, and the incubation periods is known to be 2 to (sputum cans, etc.) by inducing cough so that the collected
3. Wear lab clothing and disposable rubber gloves or vinyl (6) Samples that are positive after PCR are subjected
14 days which is predicted based on the incubation specimen does not contain saliva, etc.
gloves while handling specimen and using this product.
period of 2019-nCoV virus. 2. To ensure accurate test results, immediately store the to a second confirmation test.
(Disposable items are prohibited to reuse.)
4. Do not chat or eat while using the product. bottle containing the specimen in the refrigerator (4℃)
Principle
5. Be careful not to contaminate the specimen or product until the test. 2. Confirmation Test (2019-nCoV)
careGENETM N-CoV RT-PCR kit is developed to
when you open the tube cap or take out the contents. (1) Mix the components following the table below.
use the real-time RT-PCR method using Taqman RNA Sample preparation and storage
6. When processing specimen and testing with the product, Number of test N-CoV set
probe. In the first screening test, RNA extracted from
filter tip should be used to prevent contamination. The RNA sample used for the test is extracted using
patient specimen is converted into the complementary 4X 1 Step RT-PCR Mix 5 μL
7. When using this product, we recommend testing in a QIAamp DSP Virus RNA Mini Kit (QIAGEN, cat no. 61704)
DNA (cDNA) by reverse-transcription and target
clean bench to prevent contamination. and it is recommended to store the extracted RNA below -20℃. N-CoV primer/probe Mix 5 μL
genes are amplified by polymerase chain reaction
8. Mixing with previous lot product is prohibited. ※The specimen should be stored at 4°C up to 2 days
using primers specific to two site at viral genome in Total 10 μL
9. Dispense the reagents and store the reagents after after collection. For longer period of storage, the
order to detect Pan-CoV (E gene) and Pan-SARS
freezing (below -20 ℃) for long term storage. specimen should be stored below -70°C. (2) Dispense 10 μL of the N-CoV set Master Mix into
(RdRPP1) simultaneously. In this process, the
10. Because PCR is a very sensitive method, take care to avoid each well of an appropriate optical 96-well reaction
fluorescence signal decomposed from the Real-time PCR Master Mix set up
carry-over during the test.
fluorescence probe is detected by real-time RT-PCR. 1. Screening Test (Pan-CoV/Pan-SARS) plate or an appropriate optical reaction tube.
11. Wastes generated during the experimental should be
In the second confirmation test, novel coronavirus (3) Add 10 μL of RNA sample into each well of an
discard in the waste container and managed according to (1) Mix the components following the table below.
is confirmed by detection of the fluorescence signal
the waste management regulations. appropriate optical 96-well reaction plate or an

IFU-MNC-EN / Rev. A WELLS BIO, INC. Effective Date: 2020.02.06.

 MNC-N10082 In vitro diagnostic medical device for qualitative detection of Novel coronaviruses (2019-nCoV) from RNA extracted from human nasopharyngeal swab and sputum

careGENE TM
N-CoV RT-PCR kit In vitro diagnostic medical devices

appropriate optical reaction tube, and mix 2~3 - If the test result is negative and if the internal control is negative, (RdRPP2) 100 34.8 20/20 100%
Sensitivity (Sputum)
times. the test is invalid. In this case, retest. 50 35.9 20/20 100%
Result in Percent
- If the test result is strong positive, test result is regarded as copies/uL Mean Ct 10 38.2 20/20 100%
(4) Set the PCR machine with appropriate detection agreement agreement
positive even if the internal control is invalid. 1000 28.9 20/20 100% 5 40.9 19/20 95%
channel.
100 32.2 20/20 100% 1 N/A 0/20 0%
* Fluorescent Reporter Performance Characteristic 50 33.3 20/20 100%
Pan-CoV
Sensitivity (Sputum)
Detection target Reporter (N-CoV set) (E gene) 10 36.0 20/20 100%
Analytical sensitivity (Limit of Detection) Result in Percent
5 36.7 20/20 100% copies/uL Mean Ct
N-CoV (RdRPP2) FAM To determine the analytical sensitivity of careGENETM N- agreement agreement
1 N/A 1/20 5% 1000 31.0 20/20 100%
Internal Control ROX CoV RT-PCR kit, the upper respiratory tract specimens
Result in Percent 100 34.4 20/20 100%
copies/uL Mean Ct
(Nasopharyngeal swab) and the lower respiratory tract agreement agreement
(5) Perform PCR amplification step under the same Pan-CoV 50 35.8 20/20 100%
specimens (Sputum) were diluted with internal standard 1000 29.9 20/20 100%
condition as number (5) of the screening test. (E gene) 10 37.9 20/20 100%
material, and were tested 20 times. The concentration of 100 33.3 20/20 100%
(Do not set up the passive reference) 5 38.5 20/20 100%
Pan-SARS 50 33.8 20/20 100%
95% or more positive result was determined as the 1 N/A 4/20 20%
(RdRPP1) 10 36.8 20/20 100%
minimum detection limit. copies/uL Mean Ct
Result in Percent
Data Analysis 5 37.9 20/20 100% agreement agreement
The limit of detection is 5 copies/µL for both the specimens
1 N/A 1/20 5% 1000 31.5 20/20 100%
1. Analysis setting
from the upper respiratory tract and lower respiratory tract Result in Percent 100 35.2 20/20 100%
(1) Set the baseline of all PCR results using flat copies/uL Mean Ct
regardless of the PCR systems including CFX96TM Dx system agreement agreement
Pan-SARS 50 36.5 20/20 100%
signal in an initiation phase. 1000 29.3 20/20 100%
(Bio-Rad) and Applied Biosystems 7500 Real-Time PCR System (RdRPP1) 10 38.2 20/20 100%
(2) Set up the threshold by PCR system as follows. 100 32.2 20/20 100%
(Thermo Fisher Scientific). 5 39.1 20/20 100%
CFX96TM Dx system N-CoV 50 33.1 20/20 100%
1 N/A 0/20 0%
1. CFX96TM Dx system (Bio-Rad) (RdRPP2) 10 35.7 20/20 100%
- Threshold is 300 (RFU). Result in Percent
5 37.3 20/20 100% copies/uL Mean Ct
Sensitivity (Nasopharyngeal swab) agreement agreement
Applied Biosystems 7500 Real-Time PCR System
Result in Percent 1 N/A 1/20 5% 1000 30.2 20/20 100%
- Threshold is 30,000 (delta Rn). copies/uL Mean Ct
agreement agreement 100 33.8 20/20 100%
1000 28.9 20/20 100% 2. Applied Biosystems 7500 Real-Time PCR System (Thermo
2. Acceptance Criteria N-CoV 50 34.8 20/20 100%
100 32.3 20/20 100% Fisher Scientific) (RdRPP2)
(1) Positive: Ct value of signal is 45 or less. 10 37.0 20/20 100%
Pan-CoV 50 33.2 20/20 100% 5 38.0 20/20 100%
(2) Negative: Ct value is not detected. Sensitivity (Nasopharyngeal swab)
(E gene) 10 36.1 20/20 100%
Result in Percent 1 N/A 2/20 10%
copies/uL Mean Ct
3. Interpretation of Results 5 36.6 19/20 95% agreement agreement
1 N/A 7/20 35% 1000 31.8 20/20 100% Analytical sensitivity (Cut off Value)
(1) Examples of positive/Negative result
Result in Percent 100 35.2 20/20 100% The cut off value was determined as 45 based on the Ct
Results copies/uL Mean Ct
Example Set FAM Cy5 ROX agreement agreement
interpretation Pan-CoV 50 36.5 20/20 100% value, which was set using the LOD (Limit of detection)
1000 30.0 20/20 100%
Pan-CoV Positive / (E gene) 10 38.5 20/20 100% test result value.
1 Positive Positive Positive
Pan-SARS Positive 100 33.3 20/20 100%
5 41.3 19/20 95%
2 Positive Negative Positive Pan-CoV Positive Pan-SARS 50 33.7 20/20 100% Analytical specificity (Cross Reactivity)
Pan-CoV 1 N/A 2/20 10%
3 (RdRPP1) 10 36.9 20/20 100%
Negative Positive Positive Pan-SARS Positive
Result in Percent To evaluate the cross reactivity of careGENETM N-CoV RT-
copies/uL Mean Ct
4 Negative Negative Positive Negative 5 37.8 20/20 100% agreement agreement
PCR kit, the possible cross reactive pathogens as listed in
5 Positive - Positive N-CoV Positive
1 N/A 1/20 5% 1000 32.5 20/20 100%
the table below were tested 3 repeated times.
N-CoV Result in Percent 100 36.0 20/20 100%
6 Negative - Positive N-CoV Negative copies/uL Mean Ct
agreement agreement As a result, no cross reactivity was observed for the
Pan-SARS 50 37.4 20/20 100%
※ Even if the internal control is negative, it is positive if the target 1000 29.3 20/20 100%
(RdRPP1) 10 38.6 20/20 100% pathogens showing the similar symptoms or alpha
fluorescence is positive. 100 32.3 20/20 100%
5 40.1 20/20 100% coronavirus.
※ The test results of both negative and positive controls should be valid. 50 33.1 20/20 100%
N-CoV
1 N/A 1/20 5% Possible reactive pathogens tested
If either one is not valid, retest. (RdRPP2) 10 35.9 20/20 100%
Result in Percent Middle East respiratory syndrome Alphacoronaviruses
copies/uL Mean Ct
5 37.4 20/20 100% agreement agreement
(2) Invalid result
Dengue virus serotype 1 Human coronavirus NL63
1 N/A 1/20 5% N-CoV 1000 31.2 20/20 100%

IFU-MNC-EN / Rev. A WELLS BIO, INC. Effective Date: 2020.02.06.

 MNC-N10082 In vitro diagnostic medical device for qualitative detection of Novel coronaviruses (2019-nCoV) from RNA extracted from human nasopharyngeal swab and sputum

careGENE TM
N-CoV RT-PCR kit In vitro diagnostic medical devices

Dengue virus serotype 2 Human coronavirus 229E strong positive sample(3×LOD), weak positive sample(1×
Dengue virus serotype 3 Zika/Flavivirus LOD) and negative sample.
Dengue virus serotype 4 West Nile Virus As a result, the precision by day and lot was 100%
Chikungunya virus Yellow Fever virus/Flavivirus consistent for each sample. SD and CV are below 0.33 and
Influenza A Respiratory syncytial virus 0.91.
Influenza B Plasmodium falciparum
Storage condition
careGENETM N-CoV RT-PCR kit components: Store below
Analytical specificity (Interference)
-20℃ (sealed). It is stable and can be used for 6 months
To test the effect of the possible interfering substances
from the
date of manufacture.
(Mucin 1%, Acetyl salicylic Acid 15mg/mL, NaCl
7.3mg/mL, Oxymetazoline 20%, Hemoglobin 0.2%, Reference
Whole blood 5%), careGENETM N-CoV RT-PCR kit was
1. https://www.who.int/emergencies/diseases/novel-
tested 3 repeated times using specimen prepared by
coronavirus-2019
adding the materials listed in the table below. As a result,
2. https://www.cdc.gov/coronavirus/2019-
no interfering effect was observed for the substances
ncov/about/symptsym.html
tested.
3. Manual for the Laboratory Diagnosis and Virological
Interfering substances
Surveillance of Influenza, WHO 2011, page 32
Mucin 1% w/v
Endogenous
interfering substances
Acetyl salicylic
15 mg/mL Description of Symbol Used
Acid
Symbol Description Symbol Description
NaCl 7.3 mg/mL
In vitro diagnostic
Exogenous Oxymetazoline 20% Catalogue number
medical device
interfering substances Hemoglobin 0.2%
Whole blood 5% v/v Batch code Caution

Precision (Reproducibility) Use-by date Manufacturer

To evaluate reproducibility of careGENETM N-CoV RT-PCR kit
for nasopharyngeal swab from the upper respiratory tract Upper limit of Consult instructions
and sputum from the lower respiratory tract, two runs of temperature for use

test were performed each day. Each test was repeated

twice with 1 lot by two experimenters in 3 different places
for 5 days. WELLS BIO, INC.
16, Magokjungang 8-ro 1-gil,  +82-2-3660-6900
As a result, the precision between places and between Gangseo-gu, Seoul, 07795,  +82-2-3660-6990
experimenters showed 100% consistency for each Republic of Korea  info@wellsbio.net
 www.wellsbio.net
sample. SD and CV are below 0.08 and 0.22.

Precision (Repeatability)
To evaluate repeatability of careGENETM N-CoV RT-PCR kit
for nasopharyngeal swab from the upper respiratory tract
and sputum from the lower respiratory tract, two runs of
test were performed each day. Each test was repeated
twice with 3 lots for 13 days. Specimens used includes

IFU-MNC-EN / Rev. A WELLS BIO, INC. Effective Date: 2020.02.06.