Prepared in September 2021 (1st edition)

Instructions for Use
KANEKA RT-PCR Kit "SARS-CoV-2 (L452R/E484Q/E484K/N501Y)"
Precautions for use⚫

Wear personal protective equipment (such as rubber gloves, safety glasses, and masks) before using
KANEKA RT-PCR Kit.

⚫

The results obtained from KANEKA RT-PCR Kit should be evaluated and used at your own discretion.
Kaneka Corporation is not responsible, whether directly or indirectly, for any damages or loss which
may be caused by the evaluation or use of the obtained results.

⚫

You are responsible for verifying the validity of any detection results obtained using an operatingprocedure that is not indicated in these instructions for use.

⚫

KANEKA RT-PCR Kit is a set of reagents intended for research applications and is not to be used todiagnose, or help to diagnose, a disease.

1.

Product description
KANEKA RT-PCR Kit is a set of reagents to detect L452R mutation, E484Q mutation, E484K mutation, andN501Y mutation, among mutations found in the SARS-CoV-2 spike protein, using 1-step RT-PCR (the reversetranscription polymerase chain reaction method) with fluorogenic probes.
The fluorogenic probes contained in the reaction solution are labelled with quencher on the 3' end and withfluorescent material (FAM for detecting L452R, Yakima Yellow ™* for E484Q, ROX for E484K, and Cyanine 5 for
N501Y) on the 5' end. During the process of DNA elongation that incorporates four types of deoxynucleotide
triphosphate, the fluorogenic probes decompose and the fluorescent material previously quenched by thequencher is released, producing fluorescence. The fluorescence in each cycle of PCR is measured using a realtime PCR system, and the presence/absence of any SARS-CoV-2 spike protein gene variants is evaluatedbased on the Ct value of each fluorescence.
* "Yakima Yellow" is a trademark of Elitech Group.

2.

Product structure/storage conditions

Component

RT-PCR Enzyme Mix*1
L452R/E484Q/E484K/N501Y
Primer & Probe Mix
RNase Free Water*2

Volume (100 tests)

Storage temperature

Expiration date

−10˚C to −30˚C

One year after
manufacturing

600 µL x 1 vial
500 µL x 1 vial
1 mL x 1 vial

*1. This mix contains enzymes, substrates, etc.
*2. Store this mix away from light because it contains fluorescent labeled probes.

Prepared in September 2021 (1st edition)
3.

How to use
3.1. Instruments and equipment that are necessary but not included in KANEKA RT-PCR Kit• Micropipette and micropipette tips with filters• Real-time PCR system (a system that can detect FAM, Yakima Yellow™, ROX and Cyanine 5)• PCR tubes (0.2 mL)• Desktop centrifuge• Positive control (sold separately)
3.2. Preparation of samples (purification of RNA from the specimen)
The RNA extracted from the specimen is used as a sample in KANEKA RT-PCR Kit. Samples should beprepared according to "Manual for the Detection of Pathogen 2019-nCoV), published by the National
Institute of Infectious Diseases.
3.3. Preparation of the master mix(1) Thaw the RT-PCR enzyme mix completely by leaving it on ice. Thaw other reagents completely at room
temperature (0˚C or higher). After thawing, mix each reagent thoroughly by inverting or pipetting, thenspin down before opening the lid.
(2) Prepare the master mix according to the composition shown in the table below.
Master mix for one reactionReagent name
RT-PCR Enzyme Mix
L452R/E484Q/E484K/N501Y
Primer & Probe Mix
RNase Free Water

Additive amount
6 µL
5 µL

(Total amount)

9 µL
20 µL

3.4. Preparation of the RT-PCR reaction solution<Specimens>
Add 20 μL of the master mix to a tube containing the sample prepared in 3.2 and mix thoroughly bypipetting. *3*3. Perform RT-PCR reaction immediately after preparing the reaction solution.
<Control reaction solution>
For proper evaluation of the result, prepare reaction solutions to which the negative control, positive control
1 (N501Y/E484K), *4 or positive control 2 (L452R/E484Q) *5 has been added. (Prepare at least one of eachsolution for each PCR run.)(1) Use the RNase Free Water supplied with the kit as the negative control, and use a solution prepared
to 103 copies/μL from commercially available artificially synthesized RNA as positive controls 1 and 2.
(2) After adding 5 μL of the negative control or a positive control to a tube, add 20 μL of the master mix,
and mix thoroughly by pipetting.

Prepared in September 2021 (1st edition)*4. We have confirmed that the following products can be used as positive control 1 (N501Y/E484K):• SARS-CoV-2 Gamma Strain Positive Control RNA (NIHON GENE RESEARCH LABORATORIES,INC.)• Twist Synthetic SARS-CoV-2 RNA Control 16 (B.1.351) (Twist Bioscience)*5. We have confirmed that the following product can be used as positive control 2 (L452R/E484Q):• Twist Synthetic SARS-CoV-2 RNA Control 18 (B.1.617.1) (Twist Bioscience)
3.5. RT-PCR reaction
Place the PCR tubes containing the reaction solutions prepared in 3.4 on the real-time PCR system andperform RT-PCR reaction with the settings listed in the table below.
To detect fluorescence, read the instructions for use of the real-time PCR system and select a channel thatallows the measurement of the following fluorescence.
FAM:

wavelength of maximum absorbance = 495 nm, wavelength of maximum emissions =520 nm

Yakima Yellow™:

wavelength of maximum absorbance = 525 nm, wavelength of maximum emissions =549 nm

ROX:wavelength of maximum absorbance = 576 nm, wavelength of maximum emissions =602 nm

Cyanine 5:

wavelength of maximum absorbance = 649 nm, wavelength of maximum emissions =666 nm

StepTemperature

1252˚C
95˚C
95˚C
60˚C

34.

Reactiontime300 sec
20 sec5 sec
30 secNumber ofcycles
1145

Fluorescence
detection
OFFOFFOFFON

How to evaluate
After performing RT-PCR reaction, check the amplification curve and confirm that the analysis parameters
have been properly programmed according to the instructions for use of the real-time PCR system, thencalculate the Ct values. Evaluate the results according to the criteria listed in the table below.

Test result of the control reactions

Negative control*6
Positive control 1(N501Y/E484K)
Positive control 2(L452R/E484Q)

FAM(L452R detectionsystem)
Not detected

Yakima Yellow™
(E484Q detectionsystem)
Not detected

ROX(E484K detectionsystem)
Not detected

Cyanine5(N501Y detectionsystem)
Not detected

Not detected

Not detectedCt ≤ 35*7

Ct ≤ 35*7

Ct ≤ 35*7

Ct ≤ 35*7

Not detected

Not detectedPrepared in September 2021 (1st edition)*6. Confirm that the negative control was not detected. If a Ct value is calculated, it means that there is apossibility of contamination, so decontaminate the instruments and the work environment and run thetest again.
*7. Confirm that the Ct value of the positive control is 35 or less. If the Ct value is higher than 35 or is notdetected, check the settings of the equipment and run the test again.
Test results of specimens

Ct value*8

Evaluation resultNot detected

Negative or below detection limit*9< 42

Positive≥ 42

Retest is recommended*10*8. Some real-time PCR system models may not calculate the Ct value for some amplification curves
correctly. Always check the shape of the amplification curves and, if necessary, manually set thesettings according to the instructions for use of the real-time PCR system.
*9. Even when the result is determined to be below the detection limit, a low copy number of the novel
coronavirus gene may be present.
*10. If the Ct value ≥ 42, a retest is recommended. If the same result is obtained after a retest, it isrecommended to determine the result as positive.

Examples of test results and evaluation result
L452R

E484Q

E484KN501Y(FAM)(Yakima Yellow™)(ROX)(Cyanine5)−−−−−+++++−+−−−−

Evaluation result
Alpha strain
Beta strain, Gammastrain
Delta strain
Kappa strain(Positive: +, Negative: −)5.

Precautions for use⚫

Always follow the storage conditions and expiration date indicated in these instructions for use.

⚫

The specifications of KANEKA RT-PCR Kit are subject to change without prior notice.

⚫

Follow the instructions specified by the manufacturers of the instruments and equipment being used.

⚫

Replace the micropipette tip after each use in order to prevent contamination. It is recommended to use
micropipette tips with filters.

Prepared in September 2021 (1st edition)⚫

Clean the experiment table, etc., before and after using KANEKA RT-PCR Kit with 0.55% sodium hypochlorite,
DNA removal agent, ultraviolet (UV) light, or other methods.

⚫

In order to prevent false results caused by contamination, it is recommended that the RNA extraction, preparationof the RT-PCR reaction solution, and amplification and detection using a real-time PCR system be done inseparate areas. Do not open and close the reaction tubes after amplification.

⚫

Before opening tubes containing reagents, RNA extract, etc., spin down the tubes in a desktop centrifuge orother equipment (to prevent the content from dispersing).

⚫

If contamination is confirmed, clean the instruments and equipment used by following the methods specified bytheir manufacturers.

⚫

Reagents should not be frozen and thawed more than 15 times.

⚫

Dispose of KANEKA RT-PCR Kit, RT-PCR reaction solution, RNA extract, and other materials in accordancewith the rules concerning waste in your region and institution while considering hygiene and environmentalfactors.

⚫

We have confirmed the performance of KANEKA RT-PCR Kit with the following real-time PCR system. If youare using other models, you are responsible for verifying the validity of the detected results yourself.
CronoSTAR® 96 Real-Time PCR System (4ch) (Takara Bio USA)
LightCycler® 96 System (Roche Diagnostics K.K.)
LightCycler® 480 System II (Roche Diagnostics K.K.)* "CronoSTAR" is a registered trademark of Takara Bio Inc.
* "LightCycler" is a registered trademark of Roche Diagnostics K.K.

<Contact information>
Kaneka Corporation: Medical Solutions Vehicle
2-3-18, Nakanoshima, Kita-ku, Osaka-shi, Osaka, 530-8288 JAPANURL: https://www.kaneka-labtest.com/contact-us/