MBPCR242 Hi-PCR® Coronavirus (SARS-CoV-2) Probe PCR Kit

Description Coronaviruses are enveloped non-segmented positive-sense RNA viruses belonging to the family Coronaviridae, order Nidovirales which are broadly distributed in humans and other mammals. Although most human coronavirus infections are mild, the epidemics of the two betacoronaviruses, severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS- CoV), have severe mortality in humans. In December, 2019, a series of pneumonia cases of unknown cause emerged in Wuhan, Hubei, China, with clinical presentations greatly resembling viral pneumonia. Deep sequencing analysis from lower respiratory tract samples indicated a novel coronavirus, which was named as 2019 novel coronavirus (2019-nCoV) which was later official named as COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). COVID-19 caused clusters of fatal pneumonia with clinical presentation greatly resembling SARS-CoV. Patients infected with COVID-19 are thought to develop acute respiratory distress syndrome, have a high likelihood of admission to intensive care and might die. There is currently no vaccine to prevent COVID-19 infection. Hi-PCR® Coronavirus (SARS-CoV-2) Probe PCR Kit is developed for the qualitative detection of nucleic acid from SARS-CoV-2 in various respiratory samples from individuals suspected of COVID-19 infection by healthcare providers.

NOTE: Hi-PCR® Coronavirus (SARS-CoV-2) Probe PCR Kit is for in-vitro use only.

Intended Use Hi-PCR® Coronavirus (SARS-CoV-2) Probe PCR Kit is intended for use by qualified clinical laboratory personnel trained in the techniques of real-time PCR and in-vitro diagnostic procedures. The kit is recommended for sensitive and specific detection of SARS-CoV-2 in clinical samples.

Product Description Hi-PCR® Coronavirus (SARS-CoV-2) Probe PCR Kit includes primer/probe sets specific to detect SARS- CoV-2 genomic region and internal process control. Kit also provides synthetic positive controls for validity of the test.

Positive control This is a control reaction using a known template (target pathogen). A positive control is usually used to check that the primers have been designed properly and the PCR conditions have been set up correctly.

Internal Control This is a control sequence that should amplify in all clinical samples which indicates the presence of sufficient RNA from human RNase P gene indicating the specimen is of acceptable quality. An internal control is often used to detect the failure of amplification in cases where the target sequence is not amplified.

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Unz i p p i n g G en e s P r o d u c t I n f o r m a t i o n

The information contained herein is believed to be accurate and complete. However no warranty or guarantee whatsoever is madeor is to be implied with respect to such information or with respect to any product, method or apparatus referred to herein

Tel: 00-91-22-6147 1919Fax: 6147 1920, 2500 5764

Email : info@himedialabs.comWeb : www.himedialabs.com

A-516, Swastik Disha Business Park, Via Vadhani Indl. Est., LBS Marg, Mumbai - 400 086, India

23, Vadhani Industrial Estate,LBS Marg, Mumbai - 400 086, India. Tel. : (022) 4017 9797 / 2500 1607 Fax : (022) 2500 2286

Commercial Office Registered Office :

WHO GMP

CERTIFIED

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Principle Real-time polymerase chain reaction, also called quantitative Polymerase Chain Reaction (qPCR) or kinetic Polymerase Chain Reaction, is a laboratory technique based on the principle of PCR. This technique is used to amplify a targeted DNA sequence by use of hydrolysis probes that are short oligonucleotides that have a fluorescent reporter dye attached to the 5' end and a quencher dye to the 3' end. Hi-PCR® Coronavirus (SARS-CoV-2) Probe PCR Kit is designed to detect RNA-dependent RNA polymerase gene (RdRp) specific for SARS-CoV-

2 in FAM channel and human RNase P (RNP) gene that serves as an internal control in FAM channel. The kit allows sensitive and specific detection of SARS-CoV-2 in a single tube reaction.

Diagrammatic representation of preferential binding of probe specific to DNA fragments in Real-time PCR

While the probe is intact, the proximity of the quencher dye greatly reduces the fluorescence emitted by the reporter dye by fluorescence resonance energy transfer (FRET). The probes are designed such that they anneal within a DNA region amplified by a specific set of primers. During PCR amplification, these probes will hybridize to the target sequences located in the amplicon i.e. the DNA. As the Taq DNA polymerase replicates the template with the bound probe, the 5'- nuclease activity of the polymerase enzyme cleaves the fluorescent probe. The end result in cleavage of the probe is separation of the reporter dye from the quencher dye and increasing the reporter dye signal. As the probe is removed from the target strand, primer extension continues to the end of the template strand. Hence, fluorescence detected in the quantitative PCR thermal cycler is directly proportional to the fluorophore released and the amount of DNA template present in the PCR. Thus, inclusion of the probe does not inhibit the overall PCR process.

Features Fast and Simple – samples to results within 2 hours Highly sensitive and specific for SARS-CoV-2 detection Includes all reagents and controls. Synthetic positive controls provided for validity of the test Guaranteed reproducible results

Types of Specimen: Bronchoalveolar lavage, tracheal aspirate, sputum, nasopharyngeal swab, oropharyngeal swab, nasopharyngeal wash/aspirate or nasal aspirate, serum. Before extraction, specimens can be stored at 4˚C up to 48 hours after collection. If any delay is expected in extraction, it is recommended to store specimens at -20 ˚C or lower. After extraction, store the extracted RNA samples at -80 ˚C.

Specimen collection and Handling Follow appropriate techniques for handling specimens; after use, contaminated materials must be sterilized by autoclaving before discarding. Standard precautions as per established guidelines should be followed while handling clinical specimens and items contaminated with other body fluids. Safety guidelines may be referred in individual safety data sheets.

Polymerization: A fluorescent reporter (R) dye and a quencher (Q) are attached to the 5’ and 3’ end of the probe respectively

Strand displacement: When the probe is intact, the report dye emission is quenched.

Cleavage: During each extension cycle,the DNA polymerae cleaves the reporter dye from the probe

Polymerization completed: Once separated from the quencher, the reporter dye emits its characteristic fluorescence

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Storage and Shelf life

The provided kit has a shelf-life of 12 months when stored between -10C to -20C. Repeated thawing and freezing of PCR reagents should be avoided, as this may reduce the sensitivity. If the reagents are to be used multiple times, we recommend storing reagents as aliquots to avoid repeated freeze and thaw. Degradation of sample RNA specimens can also reduce the sensitivity of the assay. HiMedia Laboratories does not recommend using the kit after the expiry date stated on pack.

Kit Contents: The provided PCR kit contains:

Components Product Code Reagents provided for (reactions)* (μL) 10R 50R

RT Buffer DS0221 0.12 mL 0.6 mL 10X Solution H DS0222 0.06 mL 0.3 mL

M-MuLV Reverse Transcriptase DS0220 0.024 mL 0.12 mL nCoV_RdRp Primer-Probe Mix DS1000 0.012 mL 0.06 mL

RNP Primer Probe Mix DS0670 0.012 mL 0.06 mL nCoV_RdRp Positive Control DS1001 0.06 mL 0.3 mL

RNP Positive Control (Internal Control) DS0678 0.06 mL 0.3 mL Water DS0440 0.5 mL 2.5 mL

* For a 25 μL PCR reaction

Materials needed but not provided

All materials are available through www.himedialabs.com

Item Product Code

Real-time PCR Instrument and equipment Insta Q48® M2: Real time PCR System, 48 well block, 2 Channels LA1024

Insta Q48® M4: Real time PCR System, 48 well block, 4 Channels LA1023

Insta Q96® Real time PCR System, 96 well block, 5 channels LA1012

Insta Q96® Plus Real time PCR System, 96 well block, 5 channels LA1073

Insta Q96® - 6.0 Real time PCR System, 96 well block, 6 channels LA1074

TabSpin™ Microcentrifuge Rotor capacity: 12 x 0.2/0.5/1.5 or 2.0 mL tubes LA1001

Automated nucleic acid extraction system and materials

Insta NX® Instrument - fully automated nucleic acid purification system utilizing the Innovative Super -S membrane column method

LA1056

Insta NX® Viral RNA Purification Kit MBIN013

Kits and Reagents

HiPurA® Viral RNA Purification Kit MB615

Tubes, plates and other consumables

Varivol II Micropipette-10 (Capacity: 0.5 to 10 µl) LA611

Varivol II Micropipette-100 (Capacity: 10 to 100 µl) LA615

Varivol II Micropipette-1000 (Capacity: 200 to 1000 µl) LA614

Barrier Tips, Maximum capacity 10 µl LA749

Barrier Tips, Maximum capacity 200 µl LA751

Barrier Tips, Maximum capacity 1000 µl LA859

8-strip tubes & optically clear flat caps for PCR PR17

PCR Tubes, 0.2 ml PW1255

PCR Plates PR2/PR3/PR19

Optical Sealing film PR18

RNase KilTM ML162

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Warning and Precautions Read the procedure carefully before beginning the protocol. Wear protective gloves/protective clothing/eye protection/face protection. Follow good clinical laboratory practices while handling clinical samples. Standard precautions should be followed as per established guidelines. Safety guidelines may be referred in safety data sheets of the product.

Limitations Although rare, mutations within the highly conserved regions of the targets genes covered by the kit’s primers and/or probe may result in under quantitation or failure to detect the presence of the target regions in these cases. Validity and performance of the assay design are revised at regular intervals.

General Preparation Instructions Before use all PCR components should be completely thawed on ice (4°C). Perform the amplification reactions in a clean area, preferably in a biosafety cabinet. Use of aerosol barrier pipette tips is recommended to reduce contamination risks from extraneous

DNA templates. Extract and store positive control sample (if used) separately from all other reagents to avoid

contamination and add it to the reaction mix in a separate area.

A. Protocol for PCR Master Mix Preparation

1. Label 2 ml microcentrifuge tube for each primer/probe set (as mentioned in the table below).

Components

Volume (µL) to be added for 1R (for a 25 µL reaction)

Tube 1 Tube 2

RT Buffer 5 5

10X Solution H 2.5 2.5

M-MuLV Reverse Transcriptase 1 1

nCoV_RdRp Primer-Probe Mix 1 -

RNP Primer-Probe Mix - 1

Test RNA / Positive Control / Negative Control 5 5

Water 10.5 10.5

Total volume 25 25

2. Determine the number of reactions to set up per assay. Include sufficient reactions for the negativetemplate control (NTC), positive template control (PTC) and pipetting error. 3. Calculate the amount of each reagent to be added for each primer/probe set reaction master mix.4. Prepare a master mix by serially dispensing components to each tube and centrifuge for 5 secs to collectcontents at bottom of the tube. 5. Dispense 20μl of each master mix into corresponding wells in PCR tubes, strips or plates going across thecolumns as shown below.

Example Test Setup

1 2 3 4 5 6 7 8 A nCoV_RdRp RNP nCoV_RdRp RNP nCoV_RdRp RNP nCoV_RdRp RNP B nCoV_RdRp RNP nCoV_RdRp RNP nCoV_RdRp RNP nCoV_RdRp RNP C nCoV_RdRp RNP nCoV_RdRp RNP nCoV_RdRp RNP nCoV_RdRp RNP D nCoV_RdRp RNP nCoV_RdRp RNP nCoV_RdRp RNP nCoV_RdRp RNP E F G H

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Example Sample Setup

1 2 3 4 5 6 7 8

A NTC NTC S4 S4 S8 S8 S12 S12

B S1 S1 S5 S5 S9 S9 S13 S13

C S2 S2 S6 S6 S10 S10 S14 S14

D S3 S3 S7 S7 S11 S11 PTC PTC

E F G H

6. Pipette 5μl of Water into the NTC wells. Cap NTC well. 7. Cover the reaction plate and move the reaction plate to the nucleic acid handling area. 8. As shown in sample setup table, samples can be added by rows. Pipette 5μl of each sample into all the wells labelled for that sample (for example, Sample “S1” as shown above). Change tips after each addition. 9. Cap the rows to which the sample has been added. This will help to prevent sample cross contamination and enable you to keep track of where you are on the plate. 10. Finally, pipette 5μl of positive template control. 11. Centrifuge the tube briefly at 6000 rpm for about 10 seconds. Place the tubes in Real-time PCR machine and set the recommended PCR program (mentioned below). Interpret the data from the amplification plot (observe the Ct values).

B. Recommended PCR program

1. cDNA Synthesis : 50°C for 15 minutes

2. Initial denaturation : 95°C for 03 minutes 3. Denaturation : 95°C for 15 seconds 4. Annealing : 60°C for 20 seconds (Plate Read) No. of cycles: 40 5. Channel : FAM 6. Hold : 4°C for ∞

Data Analysis

The following conditions should be met for a valid diagnostic test:

Control Detection cha nnel

FAM (nCoV_RdRp) FAM (RNP/Internal Control) Positive Control + +

Negative Control - -

Data Interpretation

Detection Channel Result Interpretation FAM

(nCoV_RdRp) FAM

(RNP/Internal Control)

≤ 35 cycles ≤ 35 cycles Positive for SARS-CoV-2

> 36 cycles ≤ 35 cycles Negative for SARS-CoV-2

No Ct No Ct Invalid Result.

Repeat the PCR or extraction

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Note

Positive results are indicative of the presence of SARS-CoV-2 RNA. However, clinical correlation along

with patient history is necessary to determine patient infection status. Positive results also do not

rule out bacterial infection or co-infection with other viruses.

Negative results must be combined with clinical observations and patient history. Negative results do

not exclude SARS-CoV-2 infection and should not be used as the sole basis for patient management.

Amplification Data

Image representing probe based real-time amplification of nCoV_RdRp and RNP gene with Ct values (provided in table). The results completely depend upon sample types.

Performance characteristics

Limit of Detection (LoD)

The preliminary LoD that can be detected by Hi-PCR® Coronavirus (SARS-CoV-2) Probe PCR Kit in healthy individuals’ nasal specimen type was established to be approximately 10 copies of SARS-CoV-2.

Cross-reactivity

The analytical specificity of Hi-PCR® Coronavirus (SARS-CoV-2) Probe PCR Kit was evaluated by testing

following mentioned organisms. No cross-reaction was observed with any strains when using Hi-PCR®

Coronavirus (SARS-CoV-2) Probe PCR Kit.

Staphylococcus aureus (ATCC 43300) Bacillus subtilis (ATCC 6051)

Staphylococcus epidermidis (ATCC 12228) Candida albicans (ATCC 10231)

Pseudomonas aeruginosa (ATCC 27853) Influenza A (H1N1) pdm 09 (virus isolate)

Mycobacterium tuberculosis (clinical isolate) Seasonal influenza A (H1N1) (virus isolate)

Legionella pneumophila (ATCC 33152) Seasonal influenza A (H3N2) (virus isolate)

Streptococcus pneumoniae (ATCC 49619) Seasonal influenza B (Brisbane) (virus isolate)

Hemophilus influenzae (ATCC 49247) Seasonal influenza B (Wisconsin) (virus isolate)

Neisseria meningitides (ATCC 13090)

Sr. No.

Sample Ct value

1. nCoV_RdRp positive control

19.2

2. RNP positive control 12.67

Evaluation

Each lot of Hi-PCR® Coronavirus (SARS-CoV-2) Probe PCR Kit is tested against predetermined specifications

to ensure consistent product quality.

Quality Control Each lot of Hi-PCR® Coronavirus (SARS-CoV-2) Probe PCR Kit is assayed for contaminating endonuclease, exonuclease and non-specific DNase activities. Functionally tested in DNA amplification.

Troubleshooting Guide

Sr. No.

Problem Cause Solution

1. No amplification

Degraded samples Use freshly prepared RNA to ensure the availability of intact template sequence for efficient amplification.

Error in protocol setup

Verify that the correct reagent volumes, dilutions and storage conditions have been used.

2. Variability between replicates

Error in reaction set-up Prepare a large volume master mix, vortex thoroughly and aliquot into reaction tubes.

Air bubbles in reaction mix

Briefly centrifuge reaction samples/plate prior to running on a real-time PCR instrument.

Pipetting error Ct values of replicates can show increased variation due to poor laboratory technique or imprecise pipettes.

3. Amplification in negative control

Reagents contaminated 1. Replace all critical solutions.2. Repeat the analysis of all tests with fresh

aliquots of critical reagents.

4. No signal with

positive controls

Incorrect programming of the temperature

profile of the thermal cycler

Compare the temperature profile to the manual.

Safety Information Hi-PCR® Coronavirus (SARS-CoV-2) Probe PCR Kit is for laboratory use only, not for drug, household or other uses. Take appropriate laboratory safety measures and wear gloves when handling.

Disposal

User must ensure safe disposal by autoclaving and/or incineration of used or unusable preparations of this

product. Follow established laboratory procedures while disposing the infectious materials. Material that

comes into contact with clinical sample must be decontaminated and disposed of in accordance with current

laboratory techniques.

Technical Assistance At HiMedia, we pride ourselves on the quality and availability of our technical support. For any kind of technical assistance, mail at mb@himedialabs.com

7 Please refer disclaimer Overleaf.

-20°C

-10°C Storage temperature

Do not use if package is damaged

HiMedia Laboratories Pvt. Limited,

23 Vadhani Industrial Estate,

LBS Marg,Mumbai-86,MS,India

PIMBPCR242_O/0320 MBPCR242-00

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Disclaimer :

User must ensure suitability of the product(s) in their application prior to use. Products conform solely to the information contained inthis and other related HiMedia™ publications. The information contained in this publication is based on our research and developmentwork and is to the best of our knowledge true and accurate. HiMedia™ Laboratories Pvt Ltd reserves the right to make changes tospecifications and information related to the products at any time. Products are not intended for human or animal or therapeutic use butfor laboratory,diagnostic, research or further manufacturing use only, unless otherwise specified. Statements contained herein should notbe considered as a warranty of any kind, expressed or implied, and no liability is accepted for infringement of any patents.

HiMedia Laboratories Pvt. Ltd. Reg.office : 23, Vadhani Ind.Est., LBS Marg, Mumbai-400086, India. Customer care No.: 022-6116 9797 Corporate office : A-516,Swastik Disha Business Park,Via Vadhani Ind. Est., LBS Marg, Mumbai-400086, India. Customer care No.: 022-6147 1919 Email: techhelp@himedialabs.com Website: www.himedialabs.com