PLEASE READ THIS PACKAGE INSERT CAREFULLY BEFORE USE

For in vitro diagnostic use only

Prepared: June 2021 (1st version)

SARS Coronavirus nucleic acid detection kit

KANEKA Direct RT-PCR kit SARS-CoV-2[Important basic precautions][Intended use]

1. Negative results from this test do not preclude SARS-CoV2 infection.

For detection of SARS-CoV-2 RNA in biological samples(To help diagnose SARS-CoV-2 infection)

2. Results from this test should not be used as the sole basis
to diagnose SARS-CoV-2 infection; infection status shouldbe determined comprehensively by including the patient'sclinical symptoms. Refer to the updated information for
medical institutions and inspection agencies released by
the Ministry of Health, Labour and Welfare.
3. Use the necessary biohazard measures when collecting
and handling specimens.
4. For specimens to be used for testing, please refer to the"Guidelines for Novel Coronavirus Infection (COVID-19)
Pathogen Testing" published by the Ministry of Health,Labour and Welfare.
[General precautions]
1. KANEKA Direct RT-PCR kit SARS-CoV-2 (the Product) is an invitro diagnostic Product. The Product should not be used for any
intended uses other than that described in this Package Insert.
2. The Product should be used in accordance with the intended use andoperation method described in this Package Insert. Performance is notguaranteed if the Product is used for any purpose or by any operating
method not described in this Package Insert.
3. Always wear protective clothing, masks, safety glasses, gloves, etc.
when using the Product, and take care to prevent the reagents fromcontacting your eyes, skin, and mucosa. If such contact occurs, rinse
thoroughly with plenty of water and consult a physician as necessary.
4. Regarding instruments and equipment to be used, please read thisPackage Insert or the Instructions for Use. They must have been
properly serviced and calibrated.
5. When using the Product, this test should be performed under theguidance of personnel who are proficient in performing virus andbacteria testing.
[Configuration and structure (Kit content)]
1. RNA Extraction Reagent:

400 μL, 1 vial (for 100 tests)

2. RT-PCR Enzyme Mix:
600 μL, 1 vial (for 100 tests)
Reverse transcriptase
DNA polymeraseDeoxyribonucleoside triphosphate
3. Primers & Probes Mix:
2019-nCoV-N1 F-Primer
2019-nCoV-N1 R-Primer
2019-nCoV-N1 Probe
2019-nCoV-N2 F-Primer
2019-nCoV-N2 R-Primer
2019-nCoV-N2 Probe
IC F-Primer
IC R-Primer
IC Probe
IC Template DNA

500 μL, 1 vial (for 100 tests)

4. RNase Free Water:

900 μL, 1 vial (for 100 tests)(Precautions for use related to the intended use)
Read the sections [Precautions for operation] and (Clinical performance testresults) to familiarize yourself with the contents, and be sure to understandthe benefit of the Product before selecting the specimen samples.
[Test principle]
The Product uses reagents to detect SARS-CoV-2 genes contained inbiological samples by 1-step RT-PCR (the Reverse Transcription Polymerase
Chain Reaction method) with a fluorogenic probe. The Product's Primers &Probes Mix includes primers and fluorogenic probes consisting of thesequence specific to SARS-CoV-2 RNA, and also primers and fluorogenic
probes consisting of internal control (IC) DNA and their specific sequences
to confirm that the PCR amplification step is working properly. Regarding
the primers and probes for detecting SARS-CoV-2, the Product utilizes thesequences described in "2019-Novel Coronavirus (2019-nCoV) Real-timerRT-PCR Panel Primers and Probes (Effective: 24 Jan 2020)," issued by theUS Centers for Disease Control (CDC).
The Product can extract RNA contained in biological samples by mixing
biological samples and the Product's RNA Extraction Reagents, and heating
the mixture. After using reverse transcriptase to synthesize cDNA fromRNA, it amplifies the target sequence in the cDNA with DNA polymerase.
The fluorogenic probe contained in the reaction solution is labelled withquencher at the 3'-terminal side and with fluorescent material (for detectingSARS, FAM; for detecting IC, ROX) at the 5'-terminal side. During theprocess of DNA elongation process that incorporates four types of
deoxyribonucleoside triphosphate, the fluorogenic probe decomposes, andthe fluorescent material previously quenched by the quencher is released,producing fluorescence. The fluorescence intensity is measured at each PCR
cycle by the real-time PCR system. The presence or absence of SARS-CoV2 RNA in each sample is determined based on the Ct value of the FAMfluorescence of the probe for detecting SARS-CoV-2 and the Ct value of theROX fluorescence of the probe for detecting IC.
[Precautions for operation]
1. Specimen collection
To collect and transport each patient's specimen, please refer to the"Guidelines for Novel Coronavirus Infection (COVID-19) Pathogen
Testing" published by the Ministry of Health, Labour and Welfare andthe "Manual for Collection/Transportation of Specimens Obtained fromPatients Suspected of Having 2019-nCoV (Novel Coronavirus)
Infection” by the National Institute of Infectious Diseases (NIID).
Improper storage or freezing and thawing may affect the test result.
2. Interfering substances/drugs
The Product does not purify nucleic acids during the pretreatment step.
Therefore, when a specimen is suspended in a solution containing aprotein-denaturing agent (such as guanidine) or ethanol, the suspension
may affect the PCR reaction. If necessary, confirm in advance thesuitability with the Product (such as the presence or absence of PCRinhibition) before use.
* If the solution contains a protein-denaturing agent (such as guanidine),ethanol, PBS, or high concentration of DTT, it is recommended toperform the test after pretreatment for RNA purification.

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3. Cross-reactivity
RNA or DNA from a total of 11 types of viruses and bacteria(commercial product) were examined. All test results were negative, andno cross-reactivity was observed.
Types of viruses and bacteria
Evaluation result
Influenza A H1
Negative
Influenza A H5
Negative
Influenza B
Negative
Human coronavirus OC43
Negative
Respiratory Syncytial Virus A
Negative
Respiratory Syncytial Virus B
Negative
Mycoplasma pneumoniae
Negative
Streptococcus pneumoniae
Negative
Legionella pneumophilaNegative
Bordetella parapertussis
Negative
Haemophilus influenzae
Negative
In in silico analysis using BLAST to predict the cross-reactivity of N1and N2 primers/probes, no cross-reactivity with pathogens other than
SARS-CoV-2 was observed.

[Dosage and administration (Operating method)]
1. Necessary instruments and equipment(1) Real-time PCR system (LightCycler 96, manufactured by Roche
Diagnostics, or an equivalent device should be used)(2) Container for real-time PCR (8 consecutive tubes, 96 well plates,etc.)(3) Heating block (limited to blocks that can be set to 95°C)(4) Micropipette(5) Tip with filter for micropipette(6) Microtube (RNase- and DNase-free)(7) Desktop centrifuge (For spin-down)(8) Vortex mixer(9) Positive control [recommended product separately sold by: Nihon
Gene Research Laboratories, Inc., SARS-CoV-2 positive control
RNA (US CDC Real-time RT-PCR N1/N2 PC mix)]
2. Pretreatment (RNA extraction)(1) Mix 16 μL of specimen with 4 μL of RNA Extraction Reagent in amicrotube.

Types of viruses and bacteria used for in silico analysis (Excerpts)
Types of viruses and bacteria
Human coronavirus
SARS-coronavirus
MERS-coronavirus
Adenovirus
Human Metapneumovirus (hMPV)
Human parainfluenza virus
Influenza A virus
Influenza B
Influenza CEnterovirus
Respiratory Syncytial Virus A
Respiratory Syncytial Virus B
Rhinovirus
Parechovirus
Bordetella pertussis
Candida albicans
Corynebacterium diphtheriae
Coxiella burnetiid (Q-Fever)Chlamydophila pneumoniae
Chlamydophila psittaci
Haemophilus influenzae
Legionella (non-pneumophila)
Legionella pneumophila
Leptospira sp.
Mycobacterium tuberculosis
Mycoplasma pneumoniae
Neisseria elongate
Neisseria meningitidis
Pseudomonas aeruginosa
Staphylococcus aureus
Staphylococcus epidermidis
Streptococcus salivarius
Streptococcus pneumoniae
Streptococcus pyogenes(2) Perform heat treatment with a device such as a heating block at 95°C
for 5 minutes.
* Immediately after heat treatment, add the mixtures to the real-time
RT-PCR reaction solution, and start the reaction. If the pretreatedsolution is to be stored temporarily, place it on ice or at 4°C.
3. Settings for the real-time PCR system(1) Set up the following conditions according to the documented
procedures for the real-time PCR system to be used.
i) Detection channel: SARS-CoV-2 FAM, internal control (IC) ROXii) Amount of RT-PCR reaction solution: 25 μLiii) Conditions for RT-PCR reaction
Steps

Temperature

1252°C
95°C
95°C
60°C

3Reaction
time300 sec
20 sec5 sec
30 secNumberof cycles
1145

Fluorescence
detection
OFFOFFOFFON

4. Preparation of Master Mix for real-time RT-PCR
Prepare the Master Mix according to the composition shown in the tablebelow. The amount prepared should be increased 10% above what is
actually needed (Number of specimens + control reactions).
Reagent name
RT-PCR Enzyme Mix
Primers & Probes Mix
RNase Free Water(Total amount)

Additive amount
6 μL
5 μL
9 μL
20 μL

5. Preparation of the RT-PCR reaction solution<Specimens>
After adding 5 µL of the RNA extract obtained in Step 2 to a tube, add 20
μL of the Master Mix prepared in Step 4 to the tube, and mix bypipetting.

4. Coexisting substances
It was confirmed that the coexisting substances shown in the table below
had no effect even if contained in test specimens.
Coexisting substances
Acceptable concentration*
Clarithromycin
2 mg/mL
Ceftazidime
2 mg/mL
Levofloxacin
2 mg/mL
Acetaminophen
2 mg/mL
Loxoprofensodium
2 mg/mL* The maximum concentration that would not cause any problems evenwhen the above substances are present in RT-PCR reaction solution.
This has been confirmed experimentally.

<Control reaction solution>
For proper evaluation of results, prepare a reaction solution that contains
a negative control and a positive control (Prepare solution for at least one
reaction in each PCR run).
Use the RNase Free Water supplied with the Product as the negativecontrol, and solution prepared at 103 copies/μL from commerciallyavailable artificially synthesized RNA as the positive control.
After adding 5 μL of negative control or positive control to a tube, add 20
μL of the Master Mix, and mix thoroughly by pipetting.

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6. RT-PCR reaction
Start running the reaction under the conditions set in Step 3.
7. Data analysis
Analyze data according to the documented procedures for the analysis
software of the real-time PCR system used, and calculate the Ct value.
[How to evaluate the test result]

2. Salivary specimens<Test result of the control reactions>
After data analysis, confirm that the test result of the control reactions
met the following requirements. If it did not meet the requirements,
retesting is recommended.

Negative control
Positive control

SARS-CoV-2(FAM)
Ct > 40or Not detectableCt ≤ 30

IC(ROX)
Ct ≤ 35
Ct ≤ 35<Test results of specimens>
Determine a positive or negative test result based on the calculated Ct
value according to the following decision table.

IC(ROX)≤ 35>35 or Notdetectable

NIID method
Nasopharyngeal swab
Total(Actual specimens)
Positive
Negative
Positive
10010TheProduct
Negative
01515
Total
101525
Positive agreement rate: 100% (10/10); Negative agreement rate: 100%(15/15)

SARS-CoV-2 (FAM)>40 or Not≤ 40
detectable
Positive
Negative
Retest is
Positiverecommend

Positive specimens from nasopharyngeal swabs were spiked into
negative salivary specimens to prepare simulated specimens. Using thesesimulated specimens, the Product was compared with the RNA
purification method described in the "Manual for the Detection ofPathogen 2019-nCoV Ver. 2.9.1"2), issued by the National Institute of
Infectious Diseases. The comparison test showed a positive agreement
rate of 100% and a negative agreement rate of 100%, with a total
agreement rate of 100%.
NIID method
SalivaTotal(Simulated specimens)
Positive
Negative
Positive
10010TheProduct
Negative
01515
Total
101525
Positive agreement rate: 100% (10/10); Negative agreement rate: 100%(15/15)[Performance]
1. Performance(1) Sensitivity<Precautions for evaluation>
A tested specimen may be negative for any of the following reasons.
Therefore, a negative result from this test does not preclude the presenceof SARS-CoV-2. The patient's attending physician should make acomprehensive clinical diagnosis based on the patient's clinicalsymptoms as well as the test result.

When the positive control specimens (50 copies/reaction) are tested,
SARS-CoV-2 genes are detected within 38 cycles, and internal
control genes within 35 cycles.
(2) Accuracy
When the positive control specimens (50 copies/reaction) are tested,
they yield a positive result. When the negative control specimen istested, they yield a negative result.

1. If viral the load of the specimen is below the minimum detection
sensitivity.
2. If a mutation, deletion or insertion of SARS-CoV-2 genes occurred in theregion corresponding to the Product's primers/probes.

(3) Within-run reproducibility
When control specimens are tested three times within a run, all of thepositive control specimens yield a positive result and all of thenegative control specimens show a negative result.

3. If the shape of an amplification curve is abnormal.
[Clinical significance](4) Minimum detection sensitivity (Example)

Novel coronavirus infection (COVID-19) is a viral respiratory infection
caused by SARS-CoV-2. The virus has been spreading rapidly across theworld and many persons infected with the virus have been confirmed since
its emergence was identified in Wuhan City, Hubei Province, China inDecember 2019. Early diagnosis is essential to prevent further spread of theinfection, and RT-PCR testing to amplify SARS-CoV-2 genes is being
conducted for this purpose. This Product is an in vitro diagnostic product for
detecting SARS-CoV-2 genes by the real-time RT-PCR method. The featureof the Product is that it does not require RNA purification because a simple
specimen pretreatment method is adopted. Thereby, a test will be completed
within about an hour. Furthermore, the Product can detect internal controls
simultaneously, and therefore prevent false-negative results that may arise
due to PCR inhibition. For the above reasons, the Product will be useful forassisting in the diagnosis of COVID-19.

50 copies/reaction
2. Reference material for calibration
Positive control specimens: Two types of artificially synthesized RNA,each containing about 100 bases of SARS-CoV-2 N genes, are mixed for
use.
Negative control specimens: Sterile purified water (DNA-, RNA-, andNuclease-free)[Precautions for use and handling]
1. Precautions for handling (hazard prevention)(1) Use the Product after taking measures against biohazards under
the guidance of personnel who are proficient at handling viruses
and bacteria.

(Clinical performance test results)(2) Always wear protective clothing, masks, safety glasses, gloves,etc. when handling the Product.

1. Nasopharyngeal swab specimens
By using positive specimens from 10 patients and negative specimensfrom 15 patients, the Product was compared with the RNA purification
method described in the "Manual for the Detection of Pathogen 2019nCoV Ver. 2.9.1"2), issued by the National Institute of Infectious
Diseases. The comparison test showed a positive agreement rate of 100%and a negative agreement rate of 100%, with a total agreement rate of100%.

2. Precautions for use

3/4 (D805-0)(1) Do not use the Product in combination with other reagents.
(2) Store the Product in accordance with the storage condition.
Sediment may be found when thawed, but this will not affect thequality. Confirm that reagents are completely thawed before use.
After use, they should be stored immediately in accordance withthe storage condition.

(3) Do not use expired Products.
(4) Do not replenish reagents of different lots. The lot number isprinted on the labels of the outer package and the contents.
(5) The reagent may be repeatedly frozen and thawed no more than
10 times.
(6) Spin down before placing the RT-PCR reaction solution in thereal-time PCR system.
(7) Ensure that there is no adhesion of RT-PCR reaction solution or
air bubbles on the lid and wall surface of the real-time PCRcontainer.
(8) Perform the test by real-time PCR immediately after preparing
the RT-PCR reaction solution.
3. Precautions for disposal(1) All specimens must be handled as if they contain infectious
agents, and discarded in accordance with the safety regulations ofyour institution.
(2) Dispose of the instruments used for tests in accordance with theinternal rules of your institution after heat sterilization or otherappropriate procedures.
(3) Discard specimens without opening the lid of the reaction
container after the PCR reaction. To prevent possible
contamination by PCR amplification product, do not autoclavethem.
(4) Disposal of containers and other materials used should be inaccordance with the Waste Management and Public Cleansing
Act and the internal rules of your institution.
[Storage condition and shelf life]
1. Storage condition: −10°C to −30°C
2. Shelf life: 12 months (The expiry date is indicated on the outer package.)[Packaging unit]
KANEKA Direct RT-PCR Kit SARS-CoV-2 for 100 tests(For more details on the constituent reagents, please refer to the section[Configuration and structure (Kit content)].
[References]
1. 2019-Novel Coronavirus (2019-nCoV) Real-time rRT-PCR Panel
Primers and Probes, issued by the US Centers for Disease Control (CDC)(Effective date: 24 Jan 2020)
2. Manual for the Detection of Pathogen 2019-nCoV Ver. 2.9.1, by theNational Institute of Infectious Diseases[Conditions for approval]
1. As extremely limited data was available at the time of approval, an
appropriate study to evaluate the clinical performance of the Product is tobe performed after marketing.
2. A real-condition stability study is to be performed after marketing.
[Contact information]
For inquiries, contact
KANEKA CORPORATION (Medical Solutions Vehicle)
Mail: KANEKA_IVD_Team@kaneka.co.jp[Name and address of manufacturer]
KANEKA CORPORATION
3-18, 2-Chome, Nakanoshima, Kita-ku, Osaka-city, Osaka, 530-8288
JAPAN4/4 (D805-0)