COVID-19 RT-PCR Kit

For Emergency Use Authorization (EUA) Only

User Manual

Catalog #
M02-06-72
M02-06-73

OMC Healthcare (Pvt.) Ltd.

Rupnagar Industrial Area
Dhaka, Bangladesh

Tel: +8801729286624 Fax: +88 02 58156739

Web: www.omchcare.com, Email: [email protected]

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 INTRODUCTION

RealDetectTM COVID-19 RT-PCR Detection Kit is a multiplex Taqman fluorescent probe-based rRT-PCR assay
system for qualitative detection of 2019-Novel Coronavirus (2019-nCoV). The Taqman fluorescent probe is based
on a reporter-quencher mechanism where the 5’-end is labeled with a fluorophore and the 3’-end is labeled with
a quencher. When the probe is intact, the fluorescence emitted by the fluorophore is absorbed by the quencher
and no fluorescent signal is detected. As the fluorescent reporter and the quencher are cleaved by the 5'->3’
exonuclease activity of Taq DNA polymerase during amplification of the template and thus separated, a
fluorescent signal can be detected. Real-time monitoring of the entire PCR process can be assessed by
monitoring the accumulation of fluorescent signals.

This product provides a single tube multiplex-detection of N1 and N2 gene specific for 2019-nCoV and an
endogenous control which targets the human RNase P gene to assess specimen quality. Specific primers and
probes were designed for the detection of conserved region of 2019-nCoV’s N1 and N2 gene avoiding nonspecific
interference of SARS2003 and BatSARS-like virus strains. Thus, the approach does not give any false positives
from other coronaviruses or human microflora. Internal control human housekeeping gene, RNase P (RP) serves
as an endogenous reference control to monitor sample collection, RNA extraction, and amplification. Positive
control (2019-nCoV nucleocapsid gene) provides a nucleic acid extraction and a reverse transcription control to
validate the entire procedure and reagent integrity.

PACKAGING SPECIFICATIONS
Cat# Pack Size
M02-06-72 50 tests / box
M02-06-73 100 tests / box

PURPOSE

The kit is for emergency authorization use only and will be used for the qualitative detection in vitro of novel
coronavirus (SARS-CoV-2) RNA in respiratory specimens (including nasopharyngeal swabs and alveolar
lavage fluid samples) of COVID-19 patients.

Kit Contents

Cat # Cat #
Color
Name Components M02-06-72 M02-06-73
Code
(50 Tests) (100 Tests)
PCR Buffer, dNTP’s, MgCl2, Reverse transcriptase
Master mix 1 x 500 µL 1 x 1000 µL
(RT) enzyme, DNA polymerase

P&P Mix Primers & probe for N1, N2 & RNase P 1 x 75 µL 1 x 150 µL

Positive 2019-nCoV nucleocapsid gene and Hs_RPP30

1 x 50 µL 1 x 100 µL
Control gene
1 x 250 µL 1 x 500 µL
Water Nuclease-free water

Note: Do not mix reagents from different lots. All PCR component/reagents are ready to use. No need to
reconstitute or preparation

STORAGE AND EXPIRATION

1. All reagents should be stored at -15°C to -30°C with protection from light in a constant temperature
(non-frost-free) freezer through the expiration date.
2. The expiration date will not change if the kit is transported with icepacks and stored at the
recommended condition.
3. Upon opening, the reagents are stable for 6 months when stored at the recommended condition.
4. More than 3 freeze-thaw cycles are not permitted.
5. Minimize the number of freeze-thaw cycles (specially for the master mix and P&P solutions) by
storing in small single use aliquots. For day-to-day use, we recommend keeping an aliquot at
4°C.

REQUIRED MATERIALS (NOT INCLUDED)

● Fluorescence qPCR instrument capable of reading FAM, VIC and Cy5 or equivalent channels
● Vortex Mixer & Centrifuge machine

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 ● Pipettes and Sterile nuclease-free pipette tips and microcentrifuge tubes
● RNA extraction kit

SAMPLE REQUIREMENTS

1. Sample types: Total RNA extracts from nasopharyngeal swab using kit based solid phase extraction or
lysis based liquid phase extraction method.
Note: Sample collection should be conducted according to the relevant guidelines of the government.
2. Samples should be considered as highly infectious and handled in a BSL-2 microbiological and biomedical
laboratory to protect the operator from possible exposure during handling.
3. The collected specimen should be used for detection within the same day. Otherwise, please store the
specimen as follows:
● Store at 2°C - 8°C for no more than 24 hours
● Store at < -20°C for no more than 10 days
● Store at < -70°C for long-term, avoiding repeated freeze-thaw
● The specimen should be transported maintaining cold chain

GENERAL CONSIDERATIONS

1. To prevent contamination of PCR reactions, it is highly advisable to clean and decontaminate all working
surfaces such as biosafety cabinet, master mix preparation room, working bench tops, centrifuges, pipettes, and
other equipment with 10% bleach followed by 70% ethanol and RNase Away®/RNA ZAP.
2. Sample processing and RNA extraction area must be separated from the PCR assay setup area.
3. Positive control should be added after all other additions are done except sealing and centrifugation of the
plate.

TESTING METHOD

1. Sample Processing (Sample Processing Area)

1.1. Sample preservation solution must inactivate the virus and preserve RNA.
1.2. Commercially available viral nucleic acid extraction/purification kits or Lysis Buffer based or Trizol-
based extraction kit may be used to extract viral RNA.

2. Assay Assembly (PCR Assay Setup Area)

2.1. Thaw reagents: Remove each component from the kit, and completely thaw on ice. Collect contents
at the bottom of vial by brief centrifugation. Mix each component gently by pipetting or tapping slightly.
P&P mix must not be subjected to vortexing, tapping or light exposure at any steps of the assay to
prevent the degradation of probes.
2.2. Calculate number of reactions needed: The number of reactions (#) prepared per PCR run maybe
calculated by number of samples +2. Here “2” indicates that 1 positive and 1 no-template control tests
should be added to the samples.
NOTE: 1 or 2 excess reactions might be needed for compensation of pipetting error.
2.3. PCR Reaction Mix preparation:
2.3.1. Each reaction should contain 10µL Master mix 1.5µL P&P solution mix and 3.5µL water. Calculate
the total reaction volume and reaction number according to the 2.2 & table 1.

Table 1: PCR Reaction Mix Calculation

Required volume
Component Volume per reaction
for # no of reactions
Master Mix 10 µL 10x# µL
P&P Solution Mix 1.5µL 1.5x# µL
Water 3.5µL 3.5x# µL
Total Volume 15 µL µL

● Prepare PCR reaction mix(es) on ice by adding the appropriate volume of each component into a
tube, mix gently by pipetting.
● Distribute 15 µL PCR reaction mix to each wells/tube.

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3. Sample Loading (PCR Assay Setup Area)

3.1. Add 5 µL of sample to each well/tube containing 15 µL PCR reaction mix and pipette up and down at
least five (5) times to mix.
3.2. Add 5 µL of positive control in one tube/well and 5 µL nuclease-free water in one tube or well as no-
template control (NTC) instead of sample.

Table 2: Reaction assembly for Samples or Positive Control or no-template control (NTC)

Component PCR Reaction Mix

PCR Reaction Mix 15 µL
Sample or Positive control or
Nuclease free water 5 µL

Total Reaction Volume 20 µL

3.3. Seal the plate or tubes tightly.

3.4. Briefly centrifuge the plate or tube at low speed.

4. PCR Amplification (PCR Assay Setup Area)

4.1 Sample setup: Set the sample number, targets, negative control, and positive control accordingly to
your plate setup.

4.2 Fluorescence Channel Selection:

● FAM (For N1 gene), VIC (For N2 gene) and Cy5 (For RNase P gene) or equivalent channel.
● The reference fluorescence (passive reference) should be set to “None”.

4.3 Run method

4.3.1 Protocol for MIC (Magnetic Induction Cycler) Real-Time PCR System and Software
Operation

● Run ‘mic-PCR software’ on the computer connected to the MIC Real-Time PCR instrument > Click
on ‘New’ at the upper left corner > Select ‘Assay’.
● Click on ‘Information’ at the Assay Setup > go to ‘Chemistry Type’ and select ‘Hydrolysis Probes’ >
Add targets and Select ‘FAM’ for N1, ‘VIC’ for N2 and ‘CY5’ for RNase P > Set ‘Total Volume (µl)’ at
20µl and keep the ‘Temperature Control’ at Fast TAQ (v3).
● Click on ‘Profile’ > Set the run profile according to Table 3.1> click ‘Save’ or ‘Save As’ and save the
assay with a unique name.
● Again click on New > Select ‘Run’ > Click on ‘Assays +’ > Select the saved assay from ‘My Assay’
section > Go to ‘Run setup’> Samples > Provide sample names on the respective wells.
● Click on instrument serial number on the upper right section> start Run.

Table 3.1: MIC RT-PCR Program (Run time: ~39 minutes)

Number of
Steps Temp. (°C) Time
Cycles
55 2 min
Hold Stages 1
95 30 sec
95 02 sec
Cycling Stages 40
60 08 sec

4.3.2 Protocol for QuantStudioTM 5 Real-Time PCR System and Software Operation

● Run QuantStudioTM Software on the computer connected to the QuantStudioTM 5. Click on “Create
New Experiment”. Provide a unique name of experiment each time. Select sequentially
QuantStudioTM 5 system > 96 Well 0.2ml Block > Standard curve > TaqMan@ Reagents >
Fast/Standard.
● Go to Method > Provide 20μL reaction volume > Input the PCR conditions as shown in Table 3.2>
Set the cycle 40 > Set the RAMP rate at 2.5 for every step. Data collection should be on at the last
step.

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 ● Go to Plate > Select “None” from “Passive Reference” in quick setup>go to ‘Advanced Setup’. Add
New Target> Set reporters as shown in 4.2 (Fluorescence Channel Selection).
● Assign Targets and Samples to the desired well positions accordingly.
● Either save the protocol from ‘Save’ option for template; or Go to “START RUN’’ to start amplification
directly as well as saving run file.

Table 3.2: QuantStudioTM 5 RT-PCR Program (Run time: ~49 minutes)

Temperature Number of
Steps Temp. (°C) Time
Ramp Rate Cycles
55 5 min 2.5°C/sec
RT Steps 1
95 1 min 2.5°C/sec
95 7 sec 2.5°C/sec
PCR Steps 40
60 30 sec 2.5°C/sec

4.3.3 Protocol for ABI 7500 Fast / Fast Dx Real-Time PCR System and Software Operation

● Run 7500 Fast DX Software on the computer connected to the ABI 7500 Fast DX. Click on Advance
Setup. Provide a unique name of experiment each time. Select sequentially 7500 Fast (96 well) >
Quantitation-Standard curve > TaqMan@ Reagents > Fast (40 minutes to complete a run).
● Go to Plate Setup. Define and Assign targets according to 4.2 (Fluorescence Channel Selection).
● Go to Run Method > Input the PCR conditions as shown in Table 3.3. Setting with Tabular View is easier
than with Graphical View. Set “Reaction volume Per Well” to 20μL (The RAMP rate must remain 100%
and data collection should be at the last step).
● Save the protocol from File > Save As, then Go to Run and click “START RUN’’ to start amplification.

Table 3.3: ABI 7500 Fast / Fast Dx RT-PCR Program (Run time: ~48 minutes)

Number of
Steps Temp. (°C) Time Temperature Ramp Rate
Cycles
55 8 min Fast mode (100%)
RT Steps 1
95 1 min Fast mode (100%)
95 5 sec Fast mode (100%)
PCR Steps 40
60 30 sec Fast mode (100%)

4.3.4 Protocol for CFX96 Touch Real-Time PCR Detection System and Software Operation

• Open the Bio-Rad CFX Manager software on the computer connected to the BIO-RAD CFX 96 Real-Time
PCR Instrument.
• Go to User Defined >Protocol>Create New>Input the run information as shown in Table 3.4 >Set the
sample volume to 20 μL.
• Go to Plate > Create New > Select wells > Set Fluorophores > Select fluorescence channel FAM, VIC
and CY5. Wells with clinical specimens should be specified as Unknown. Load the sample names and
fluorophores on the selected wells.
• Go to Settings > Plate type > Select BR Clear (if the plate wells are transparent)/ BR White (if the plate
wells are not transparent or white in color)
• Then select OK to save the designed Plate.
• Click Open Lid (Bottom left of the software)> Set the plate on the Block> click Close Lid > Go to Start Run
> Save the program. Then the program will be started automatically.

Table 3.4: CFX96 Touch RT-PCR Program (Run time: ~61 minutes)

Number of
Steps Temp. (°C) Time Temperature Ramp Rate
Cycles
55 8 min 5°C/sec
RT Steps 1
95 1 min 5°C/sec
95 5 sec 5°C/sec
PCR Steps 40
60 30 sec 5°C/sec

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4.3.5 Universal protocol

For other Real Time PCR instruments including Exicycler 96 (Bioneer); LightCycler 96 (Roche); Rotor-Gene
Q (Qiagen); DTprime (DNA-Technology), qTower3 (Analytik Jena) having FAM (For N1), VIC (For N2) and
Cy5 (For RP) or equivalent channel.

Table 3.5: Universal RT-PCR Program

Temperature Number of
Steps Temp. (°C) Time
Ramp Rate Cycles
55 10 min 1.6°C/sec
RT Steps 1
95 1 min 1.6°C/sec
95 10 sec 1.6°C/sec
PCR Steps 40
60 1 min 1.6°C/sec

5. Quality Controls

PCR reactions for the positive and negative controls are considered valid if the negative and positive
controls meet the criteria listed in Table 4.

Table 4: Validation of PCR reactions with quality controls

Target Positive Control No Template Control (NTC)

N1 Ct ≤ 32 Ct > 35 or no Amplification
N2 Ct ≤ 32 Ct > 35 or no Amplification
RP Ct ≤ 32 Ct > 35 or no Amplification

6. Result and Analysis

If the criteria of QUALITY CONTROLs are met, the PCR reaction results of the test samples are explained
according to Tables 5 and 6.

Table 5: Interpretation of PCR Reactions

PCR reaction results Ct

+ Ct ≤35 in N1, N2 and RP
No amplification, or
-
Ct >35 in N1, N2 and RP

Suspected Ct values (Ct value within 33 to 35 showing no typical S-shape curve) may indicate poor PCR
performance due to low RNA amount or contaminants present in the reaction. Retesting is recommended
to confirm results.

Table 6: Interpretation of Test Samples

N1 N2 RP Result Interpretation Report

- - + 2019-nCoV not detected Negative for COVID-19
+ + +/- 2019-nCoV detected Positive for COVID-19
If only one of the two targets is Inconclusive result, repeat
+/- Presumptive positive
positive test
- - - Invalid Result Invalid, repeat test

Positive Result: The sample contains the target genes (1) N1, N2 and RP (2) N1 and N2.
Negative Result: The sample does not contain the target genes N1, N2 but contains RP.
Inconclusive / Invalid Result: Indicate poor PCR performance due to contaminants, presence of certain
inhibitors, low RNA amount, degradation of extracted RNA or error in sample collection. Retesting, re-
extraction of RNA and re-collection of samples (if necessary) is recommended to confirm results.

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LIMITATIONS

1. The detection result of this product is only for clinical reference, and it should not be used as the only
evidence for clinical diagnosis and treatment. The clinical management of patients should be considered in
combination with their symptoms/signs, history, other laboratory tests and treatment responses.

2. A “negative result” may be a false negative (i.e., a sample contains genetic material of SARS-CoV-2).
Possible causes of a false negative result:
● Faulty sample collection, processing, transportation or low sample concentration
● Variations in the target sequence of the 2019-nCoV novel coronavirus or sequence changes
caused by other reasons
● Improper reagent storage
● Other unverified interferences or PCR inhibitors
● Cross-contamination during sample processing

PRODUCT PERFORMANCE INDEX

1. Limit of Detection: 5 copies /reaction.

2. Repeatability: Precision testing showed that the coefficients of variation of the precision Ct values of this kit
lot are ≤ 5%.
3. Accuracy: The kit was tested with a third-party sourced positive reference product and negative reference
product. The compliance rate was 100% for both cases.

REFERENCES

1. Petrillo, S., Carrà, G., Bottino, P., Zanotto, E., De Santis, M. C., Margaria, J. P., Giorgio, A., Mandili, G., Martini,
M., Cavallo, R., Barberio, D., & Altruda, F. (2020). A Novel Multiplex qRT-PCR Assay to Detect SARS-CoV-2
Infection: High Sensitivity and Increased Testing Capacity. Microorganisms, 8(7), 1064.
https://doi.org/10.3390/microorganisms8071064
2. Elien, of Mo Ë S, Leen Vijgen, E LS Keyaerts, et Al. A Novel pancoronavirus RT-the PCR Assay: Frequent
Detection of Human coronavirus NL63 in Children Hospitalized with Respiratory tract infections in Belgium.
The BMC Infectious Diseases, 2005, 5.
3. Ishige T, Murata S, Taniguchi T, Miyabe A, Kitamura K, Kawasaki K, Nishimura M, Igari H, Matsushita K.
Highly sensitive detection of SARS-CoV-2 RNA by multiplex rRT-PCR for molecular diagnosis of COVID-19
by clinical laboratories. Clin Chim Acta. 2020 Aug; 507:139-142. doi: 10.1016/j.cca.2020.04.023. Epub 2020
Apr 23. PMID: 32335089; PMCID: PMC7179514.

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INDEX OF SYMBOLS
Do not reuse Batch code

Use by
In vitro diagnostic medical device

Temperature limitation Contains sufficient for < n > tests

Caution Catalog number

Manufacturer Consult instructions for use

MANUFACTURER CONTACT INFORMATION

OMC Healthcare (Pvt.) Ltd.

Dhaka-1216, Bangladesh
For Inquire: Dial 0172-928-OMCH (6624)
Fax: +880-2-8156739
www.omchcare.com M02-06-72, M02-06-73

N1FN2SRPA_LU2.1
[email protected]