Instruction Manual

Product # STLC22-50

Batch #047036

50 Reactions

Room Temperature

Feb, 2023
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Contents
1. INTENDED USE ........................................................................................................................................ 2
2. PRINCIPLE OF DIAGNOSIS ...................................................................................................................... 2
3. REAGENTS PROVIDED ............................................................................................................................ 2
4. PREREQUISITES ...................................................................................................................................... 2
5. COMPATIBLE INSTRUMENTS .................................................................................................................. 3
6. SHIPMENT AND STORAGE CONDITIONS ................................................................................................. 3
7. PRECAUTIONS FOR USERS ..................................................................................................................... 3
8. TEST PROCEDURE .................................................................................................................................. 4
9. RESULT INTERPRETATION ...................................................................................................................... 6
10. LIMITATIONS OF THE TEST ..................................................................................................................... 7
11. QUALITY CONTROL ................................................................................................................................. 8
12. PLATE TEMPLATE: (EXAMPLE ONLY). .................................................................................................... 8
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1. INTENDED USE
Stellence Covid-19 RT-qPCR Kit is used for the qualitative detection of a novel coronavirus in suspected Test specimens from
upper respiratory tract specimens (nasopharyngeal extracts, deep cough sputum, etc.) and lower respiratory tract specimens
(alveoli irrigation fluid, etc.) by real time PCR systems. This test is intended for use as an aid in the diagnosis of COVID 19 in
combination with clinical and epidemiological risk factors. RNA is extracted from respiratory specimens, amplified using RT-
PCR and detected using fluorescent reporter dye probes specific for COVID 19.

2. PRINCIPLE OF DIAGNOSIS
Stellence Covid-19 RT-qPCR Kit is designed for the diagnosis of COVID 19 using the RNA extracted from respiratory samples.
The detection is done in two well multiplex real time RT format where the reverse transcription and the subsequent
amplification of specific target sequence occur in the same reaction well. The isolated RNA target is transcribed generating
complementary DNA by reverse transcriptase which is followed by the amplification of a conserved region of ORF1ab, and N
genes for COVID 19 using specific primers , primers for RNAse P housekeeping gene along with labelled probe.
Stellence Covid-19 RT-qPCR Kit is based on the 5´ exonuclease activity of DNA polymerase. During DNA amplification, this
enzyme cleaves and degrades the probe bounded to the complementary DNA sequence, which results in separating the
quencher dye from the reporter. This reaction generates an increase in the fluorescent signal of the reporter dye which is
proportional to the quantity of target template. This fluorescence can be measured on Real Time PCR platforms.
Stellence Covid-19 RT-qPCR Kit contains the components necessary for real time PCR assay (specific primers/probes, One
step concoction and a test positive control). N gene is amplified and detected in HEX/VIC channel, ORF1ab gene is amplified
and detected in FAM channel and the internal control (RNase P) in ROX channel.

3. REAGENTS PROVIDED

Reagent Name Description Storage

Master Mix RT One step concoction for RT QPCR Room Temperature

PCR Mix - 1 Primer and probes for N and Orf 1ab Room Temperature

PCR Mix - 2 Primer and probe mix for RNase P Room Temperature

Positive control (PC) Positive Control template Room Temperature

Negative control (NC) Negative control Room Temperature

4. PREREQUISITES
 Vortex mixer
 Micro centrifuge
 Micropipettes (2, 10, 100, 200 and 1000 µL).
 Racks for micro centrifuge tubes
 2X 96 well 20 deg C cold block
 7500 real time PCR systems/Applied Biosystems Step one RT QPCR / Applied Biosystems QS3 &5 or equivalent.
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 Nuclease free water

 Surface sanitizer
 QIA AMP Viral RNA mini kit or equivalent
 Aerosol Barrier pipette tips
 1.5mL centrifuge tube DNase and RNase free PCR vials
 Laminar air flow hood
 96- 100 % Ethanol
 PPEs

5. COMPATIBLE INSTRUMENTS
Stellence Covid-19 RT-qPCR Kit can be used with the following equipment’s: Applied Biosystems 7500 Fast Real-Time PCR
System, Applied Biosystems StepOne™ Real-Time PCR System, and QS 3/5 or equivalent.

6. SHIPMENT AND STORAGE CONDITIONS

 The kits can be shipped and stored at Room temperature until the expiry date which is stated on the label.
 However It is recommended to store all components at +2ºC to +8ºC once opened.
 Keep components away from sunlight.
Note: Storage temperature has been assigned based on similar product commercially available and upon literature
information.

7. PRECAUTIONS FOR USERS

 The product is indented for use by professional users only, such as laboratory or health professionals and technicians,
trained in molecular biological techniques.
 Do not use past expiration date.
 Do not mix reagents from different envelopes and / or kits and / or lots and / or another supplier.
 Protect reagents against from humidity. Prolonged exposure to humidity may affect product performance.
 Design a unidirectional workflow. It should begin in the Extraction Area and then move to the Amplification and Detection
Area. Do not return samples, equipment and reagents to the area in which the previous step was performed.
 Follow Good Laboratory Practices. Wear protective clothing, use disposable gloves, goggles and mask. Do not eat, drink
or smoke in the working area. Once you finish the test wash your hands.
 Specimens must be treated as potentially infectious, as well as all the reagents and materials that have been exposed
to the samples and they must be handled according to the national safety regulations. Take necessary precautions
during the collection, storage, treatment and disposal of samples.
 Decontamination of commonly used equipment is recommended, especially micropipettes and work surfaces.
 Consult safety data sheets, upon request.
 Consult each Real Time PCR instrument's reference manual for additional warnings, precautions and procedures.
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8. TEST PROCEDURE
RNA extraction
Perform the sample preparation according to the recommendations appearing in the instructions for use of the extraction kit
used.
For RNA extraction from respiratory samples manual or automatic routine optimized system can be used. Also, any
commercially available RNA extraction kit can be used by following the manufacturer´s instructions.
Reaction and plate set up:
Note: Keep all reagents on cold rack during experiment set up.
1) Take and thaw all the components thoroughly and before using it. Mix gently, spin down the content for 5 seconds and
then test it immediately.
2) Assay controls should be run concurrently with all test samples.
 PC- Positive template control with an expected Ct value range
 NTC- no template control added during RT- PCR reaction set-up
 NC- Negative control
3) Determine the number of reactions (n) to perform for each experiment. It is necessary to make excess reaction cocktail
to allow for control reactions and pipetting error.
4) Prepare the below reaction mix for each sample using the components listed below
5) For each primer/probe set, calculate the amount of each reagent to be added for each reaction mixture (n+1).
Reagent Volume per reaction
Master mix RT 8.75 µl
PCR Mix -1 1.25 µl
Total reaction Mix volume 10 µl

and
Reagent Volume per reaction
Master mix RT 8.75 µl
PCR Mix -2 1.25 µl
Total reaction Mix volume 10 µl

a) Mix reaction mixtures by pipetting up and down. Do not vortex.

b) Centrifuge for few sec to collect contents at the tube, and then place the tube in cold rack.
c) Set up reaction strip tubes or plates in 96-well cooler rack.
d) Dispense 10 µL of each reaction mix into the appropriate wells of plates/strips.
e) Pipette 10 µL of nuclease- free water into the NTC sample wells. Securely cap NTC wells before proceeding.
f) Prior to moving to the nucleic acid handling area, prepare the Negative control (NC) reactions in to Negative control
wells in the assay preparation area.
g) Cover the entire reaction plate and move the reaction plate to the specimen nucleic acid handling area.
Addition of Template
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a. Gently vortex nucleic acid sample tubes for approximately 5 seconds.

b. After centrifugation, place extracted nucleic acid sample tubes in the cold rack.
c. Samples should be added to designated sample wells. Carefully pipette 10 µL of the sample into all the wells labelled
for that sample. Keep other sample wells covered during addition. Change tips after each addition.
d. Securely cap the column to which the sample has been added to prevent cross contamination and to ensure sample
tracking.
e. Change gloves often and when necessary to avoid contamination.
f. Repeat steps c and d for the remaining samples.
g. When necessary, add 10 µL of human specimen Control (NC) extracted sample to the NC wells securely cap wells after
addition.
h. Cover the entire reaction plate and move the reaction plate to the positive template control handling area.
Addition of Assay control
A. Pipette 10 µL of positive control to the positive control wells. Securely cap wells after addition.
B. NOTE: If using 8-tube strips, label the TAB of each strip to indicate sample position.
C. DO NOT LABEL THE TOPS OF THE REACTION TUBES!
D. Briefly centrifuge reaction tube strips for 10-15 seconds. After Centrifugation return to cold rack.
E. NOTE: If using 96- well plates for 30 seconds at 500 x g, 4℃.
Setting up the Equipment
NOTE: Please ensure that the instruments have been installed, calibrated, checked and maintained according to the
manufacturers recommendations
a. Clean and decontaminate all work surfaces, pipettes, centrifuges and other equipment prior to use using RNase Away
or 10% freshly prepared bleach.
b. Turn on real time instrument and allow block to reach optimal temperature.
c. Perform plate set up and select cycling protocol on the instrument.
d. Instrument Settings:
Run Mode: Standard Sample Volume: 20 µL
Passive Reference: None; Channel section: As following:

Wells or Sample Targets Reporter Quencher

N HEX / VIC
1 Select None
Orf 1ab FAM

Wells or Sample Targets Reporter Quencher

2 RNase P ROX Select None

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RT PCR cycling parameters:

Stages Cycles Temperature Time

cDNA Synthesis 1 48℃ 15 minutes
RT Inactivation 1 95℃ 3 minutes
95℃ 15 seconds
DNA Amplification 40 30 seconds*
60℃
(*Data collection)
 Fluorescence data should be collected during the 60℃ for 30 seconds.
 After completion of the run, save the run and analyze the collected data.

9. RESULT INTERPRETATION
The use of positive and negative controls in each run validates the reaction by checking the absence of signal in the negative
control well and the presence of signal for in the positive control well. Check Internal Control signal to verify the correct
functioning of the amplification mix. The analysis of the samples is done by the software of the used real time PCR equipment
itself according to manufacturer´s instructions. Using the following table read and analyse the results:
Note: Before interpretation of positive control and sample results, adjust threshold of all gene targets of NTC to undetermined
when necessary.

Assay interpretation:

N Orf1 ab RNase P
Target Ct
(HEX) (FAM) (ROX)
Positive Ct value ≤35 for N and Orf 1ab genes
control + + ± & Ct value ≤35 for RNase P
No template
control - - - -
Sample interpretation:

Detection of N Detection of Detection of

Results
(HEX/VIC) Orf 1ab (FAM) RNase P ( ROX)
+ +
± Covid-19 RNA is detected
Ct value ≤35 Ct value ≤35
+
- Ct value ≤35
± Covid-19 RNA is detected
+ +
± Covid-19 RNA is detected
Ct value ≤35 Ct value ≤35
+ + Covid-19 RNA is presumptive
Ct value ≤35 - Ct value ≤35 positive

- - ± COVID-19 NOT detected

+
Ct value ≤35 - ± COVID-19 NOT detected

+
- - Ct value ≤35
COVID-19 NOT detected

- - - Invalid result
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Ignore non-specific Ct in NTC in rare cases, then adjust Threshold as appropriate.

+: Amplification curve and Ct ≤32
- : No amplification curve.
* The single-gene amplification or even random positive results is suggestive of:
a) Low amount of RNA template of the sample near or below the detection limit of the reactions
b) Mutations in the nucleic acid sequences targeted.
c) Slightly different amplification yield of the targets regions,
d) Due to differential sensitivity of the target genes and due to multiplex format of assay, in rare cases, weak positive
samples may show signal in either of targets.
Or
e) Other factors.
A sample is considered positive if the Ct value obtained is less than or equal to 32 and 35 for the internal control (RNase
P) shows or not shows an amplification signal. Sometimes, the detection of internal control is not necessary because a
high copy number of target can cause preferential amplification of target-specific nucleic acids.
A sample is considered negative, if the sample shows no amplification signal in the detection system but the internal
control is positive. An inhibition of the PCR reaction can be excluded by the amplification of internal control. In case of a
continued ambiguous result, it is recommended to verify the correct performance of each of the steps and review the
parameters and the sigmoid shape of the curve. It is also recommended to repeat the assay, preferably in duplicate. The
result of the test should be evaluated by a health care professional in the context of medical history, clinical symptoms
and other diagnostic tests.

10. LIMITATIONS OF THE TEST

The results of the test should be evaluated by a health care professional in the context of medical history, clinical
symptoms and other diagnostic tests. Although this assay can be used with other types of samples it has been validated
only with RNA extracted from respiratory samples (nasopharyngeal swab and oropharyngeal swab).

 The quality of the test depends on the quality of the sample; proper extracted nucleic acid from clinical samples
must be extracted.
 Extremely low levels of target below the limit of detection might be detected, but results may not be reproducible.
 There is a possibility of false positive results due to cross-contamination by SARS-CoV-2, either samples containing
high concentrations of target RNA or contamination due to PCR products from previous reactions.
 The specific primer and probe combinations for detection of the ORF1ab and N genes used in in the Kit designed
for the detection of SARS-CoV-2, do not show significant combined homologies with the human genome, human
microflora, SARS-CoV or other coronaviruses (with the exception of some N gene sequences from coronaviruses
identified in bats and pangolin), which might result in predictable false positive.
 False Negative results may arise from several factors and their combinations, including: Improper specimens’
collection, transport, storage, and/or handling methods.
o Improper processing procedures (including RNA extraction).
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o Degradation of the viral RNA during sample shipping/storage and/or processing.

o Mutations or polymorphisms in primer or probe binding regions may affect detection of new or unknown SARS-CoV-
2 variants.
o A viral load in the specimen below the limit of detection for the assay.
o The presence of RT-qPCR inhibitors or other types of interfering substances. The impacts of vaccines, antiviral
therapeutics, antibiotics, and chemotherapeutics or immunosuppressant drugs used to prevent COVID-19 or used
during the treatment of the infection have not been evaluated.
o Failure to follow instructions for use and the assay procedure.
In case of a doubt, it is recommended referring to a reference laboratory for further testing.
 A positive test result does not necessarily indicate the presence of viable viruses and does not imply that these
viruses are infectious or are the causative agents for clinical symptoms. However, a positive result is indicative of
the presence of targets viral sequences (ORF1ab and/or N genes).
 Negative results do not preclude SARS-CoV-2 infection and should not be used as the sole basis for treatment or
other patient management decisions. Optimum specimen types and timing for peak viral levels during infections
caused by SARS-CoV-2 have not been determined. The collection of multiple specimens (types and time points)
from the same patient may be necessary to detect the virus.
 If diagnostic tests for other respiratory illnesses are negative and the patient’s clinical presentation and
epidemiological information suggest that SARS-CoV-2 infection is possible, then a false negative result should be
considered, and a re-testing of the patient should be discussed

11. QUALITY CONTROL

Stellence Covid-19 RT-qPCR Kit should always be used along with positive and a negative control in each run to correctly
interpret the results. Also, the internal control (RNase P) in each well confirms the correct performance of the PCR and
presence of RNA template in sample. In accordance with Stellence quality systems, each lot of kit is tested against
identified specifications to ensure consistent product quality.

12. PLATE TEMPLATE: (EXAMPLE ONLY).

1 2 3 4 5 6 7 8 9 10 11 12
Positive
A control
8 16 24 32 40 48 56 64 72 80 88
B 1 9 17 25 33 41 49 57 65 73 81 89
C 2 10 18 26 34 42 50 58 66 74 82 90
D 3 11 19 27 35 43 51 59 67 75 83 91
E 4 12 20 28 36 44 52 60 68 76 84 92
F 5 13 21 29 37 45 53 61 69 77 85 93
Negative
G 6 14 22 30 38 46 54 62 70 78 86 Control
H 7 15 23 31 39 47 55 63 71 79 87 NTC