For

For use onlyPerformance

under Emergency Cat. No. RP10250X
Use Authorization
Evaluation Only
AllplexTM 2019-nCoV Assay For U.S. ONLY

Intended Use
The Allplex 2019-nCoV Assay is an in vitro diagnostic (IVD) real-time reverse transcriptase polymerase chain reaction (RT-PCR) test intended for the qualitative detection of
nucleic acid from severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) in human nasopharyngeal swab, oropharyngeal swab, anterior nasal swab, mid-
turbinate and sputum specimens from individuals with signs and symptoms of infection who are suspected of COVID-19 by their health care provider. Testing is limited to U.S.
laboratories certified under the Clinical Laboratory Improvement Amendments of 1988 (CLIA), 42 U.S.C. §263a, to perform high complexity tests.

Kit stability Nucleic Acid Extraction

• Expiry date is 8 months from the date of manufacture at ≤ -20℃. NOTE: Vortex specimen before use. If the specimen is still viscous, let it cool down or
Please refer to product label for final expiry date. add saline solution.
• This product can be used for 30 days after opening the vials. NOTE: Refer to volume of internal control (IC) in Seegene Launcher program.
• This product can be used for maximum 7 repeats of freezing and thawing. Seegene NIMBUS/STARlet
Extraction reagent : STARMag™ 96 X 4 Universal Cartridge kit*
Specimen Handling and Storage
Required (or Minimum) specimen volume : 300 μL
• Specimens can be stored at 4 ℃ for up to 72 hours after collection. If Elution volume : 100 μL
any delay in extraction is expected, store specimens at -70℃ or lower.
Proceed the extraction step following ‘Tutorial’ of Seegene Launcher program.
• Extracted nucleic acids should be stored at -20 ℃ or lower.
RP-V IC tube must be loaded on extraction equipment before nucleic acid extraction.

Amplification and Detection (CFX96 Touch™, Bio-Rad)

NOTE: If the RP-V IC is not added during extraction, the negative sample is interpreted as ‘Invalid’.

1. Preparation for Real-time PCR 2. Real-time PCR Instrument set up

NOTE: After completely thawing all reagents stored at ≤ -20℃, centrifugation ① Protocol Setup
must be performed.
NOTE: Positive control and clinical samples require special caution in order to - In the main menu, select File  New  Protocol to open
avoid carry-over contamination. protocol Editor.
NOTE: PCR setup can be performed automatically via Seegene NIMBUS or - In Protocol Editor, define the thermal profile as table below.
STARlet. The following is a guide for manual users.
Step No. of cycles Temperature Duration
① Prepare following reagents in a labeled sterile 1.5 mL tube. 1 1 50℃ 20 min
Set up all reagents on ice.
2 1 95℃ 15 min
No. of Reactions 1 2 3 4 5
3 94℃ 15 sec
2019-nCoV MOM 5 10 15 20 25 45
4* 58℃ 30 sec
RNase-free Water 5 10 15 20 25 5 GOTO Step 3, 44 more times
5X Real-time One-step Buffer 5 10 15 20 25 * Plate Read at Step 4. Fluorescence is detected at 58°C.
Real-time One-step Enzyme 2 4 6 8 10 - Click the box next to Sample Volume to directly input 25 μL.
② Mix by inverting the tube 5 times or quick vortex, and briefly - Click OK and save the protocol to open the Experiment Setup window.
centrifuge. ② Plate Setup
③ Aliquot 17 μL of the One-step RT-PCR Mastermix into PCR tubes*. - From Plate tab in Experiment Setup, click Create New to open Plate
④ Add 8 μL of each sample’s nucleic acids, 2019-nCoV PC and NC Editor window.
(RNase-free Water) into the tube containing an aliquot of the One-
- Click Select Fluorophores to indicate the fluorophores (FAM, HEX,
step RT-PCR Mastermix.
⑤ Close the cap, and briefly centrifuge the PCR tubes. Cal Red 610 and Quasar 670) that will be used and click OK.
⑥ Verify that the liquid containing all PCR components is at the - Select the desired well(s) and then its sample type from the Sample
bottom of each PCR tube. If not, centrifuge again at a higher rpm Type drop-down menu.
and for a longer time. - Unknown : Clinical samples
⑦ Immediately initiate PCR. - Negative Control
NOTE: Be sure to centrifuge the PCR tube before running PCR reaction in - Positive Control
order to set the liquid to the bottom and to eliminate air bubbles. - Click on the appropriate checkboxes (FAM, HEX, Cal Red 610 and
* Available PCR Tube Quasar 670) to specify the fluorophores to be detected in the selected
Low-Profile 0.2 mL 8-Tube Strips without Caps (white color, Cat. No. TLS0851, Bio-Rad) wells.
Optical Flat 8-Cap Strips (Cat No. TCS0803, Bio-Rad) - Type in Sample Name and press enter key.
Hard-Shell® PCR plates 96-well WHT/WHT (Cat. No. HSP9655, Bio-Rad)
Permanent Clear Heat Seal (Cat. No. 1814035, Bio-Rad)* - In Settings of the Plate Editor main menu, choose Plate Size (96
PX1 PCR plate sealer (auto-sealer, Cat. No. 181-4000, Bio-Rad)* wells) and Plate Type (BR White).
* The above mentioned heat seal and plate sealer must be used in combination.
- Click OK to save the new plate.
[ Analytes ] - You will be returned to the Experiment Setup window.
Fluorophore Analyte ③ Start Run
- From Start Run tab in Experiment Setup, click Close Lid to close the
FAM E gene
instrument lid.
HEX Internal Control (IC) - Click Start Run.
Cal Red 610 RdRP gene - Store the run file either in My Documents or in a designated folder.
Input the file name, click SAVE, and the run will start.
Quasar 670 N gene
* Required, but not provided (Cat. No. 744300.4.UC384)

04/2020 V1.0
 For
For use onlyPerformance
under Emergency Cat. No. RP10250X
Use Authorization
Evaluation Only
AllplexTM 2019-nCoV Assay For U.S. ONLY

Data Analysis (CFX96™ Touch, Bio-Rad)

1. Pre-setting for Data Analysis

A. Create folders for data export
① Create a folder to save amplification curve detection results.
② The location and name of the folder is specified by user, but in case of using ‘Seegene Export’ function, folder named “QuantStep4” is
created automatically in selected location.

B-1. Pre-settings for Data Analysis in CFX Manager™ Software V3.1 of CFX96™ Touch

① After the PCR reaction, select No Baseline ② Select Excel 2007 from export All ③ Choose a location to save data and
Subtraction from Baseline Setting of Data Sheets from Export menu. click OK.
Settings menu.

B-2. Pre-settings for Data Analysis in CFX Maestro™ Software of CFX96™ Touch

① After the PCR reaction, select No Baseline ② Select Excel 2007 from export All ③ Choose a location to save data and
Subtraction from Baseline Setting of Data Sheets from Export menu. click OK.
Settings menu.

2. Settings for Data Analysis in Seegene Viewer

① Open Seegene Viewer program and click ② After opening the results file, select the ③ Check the result for each well.
Open, select the exported data. ‘Allplex™ 2019-nCoV Assay’ from the
PRODUCT menu.

04/2020 V1.0
 For
For use onlyPerformance
under Emergency Cat. No. RP10250X
Use Authorization
Evaluation Only
AllplexTM 2019-nCoV Assay For U.S. ONLY

Interpretation

RdRP
Target IC E gene N gene
gene Auto-
Results
CalRed Quasar interpretation
Fluorophore HEX FAM
610 670
2019-nCoV
Case 1 +/- + + + All Target Results are valid. Result for SARS-CoV-2 RNA is Detected.
Detected
Case 2 +/- + - +
All Target Results are valid. Result for SARS-CoV-2 RNA is Detected.
Case 3 +/- + + -
Negative target results are suggestive of
2019-nCoV
Case 4 +/- - + + 1) a sample at concentrations near or below the limit of detection of the test,
Detected
2) a mutation in the corresponding target region, or
Case 5 +/- - - +
3) other factors.
Case 6 +/- - + -
All Target Results are valid. Result for Sarbecovirus RNA is detected. Result for
SARS-CoV RNA is Presumptive Positive.
Negative target results are suggestive of
1) a sample at concentrations near or below the limit of detection of the test,
Presumptive 2) a mutation in the corresponding target region, or
Case 7 +/- + - - 3) other factors.
positive
Repeat test with more nucleic acids (up to 13 μL) instead of RNase-free Water.
For sample with the same result on a repeated test, additional confirmatory
testing may be conducted, if it is necessary to differentiate between SARS-CoV
-2 and SARS-CoV-1 or other Sarbecovirus currently unknown to infect humans,
for epidemiological purposes or clinical management.

Case 8 + - - - Negative All Target Results are valid. Result for SARS-CoV-2 RNA is Not Detected.

Results are invalid. Repeat test.

Case 9 - - - - Invalid
If the result is still invalid, a new specimen should be obtained.

[Cut-off]

For all targets,

Ct value Result

≤ 40 Detected

> 40 or N/A Not detected NOTE: If Ct value of IC is ‘> 40’, please conduct a re-test.

04/2020 V1.0