COVID-19 RAPID TESTING

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COVID-19 RAPID TESTING

StemnovateÔ Reverse Transcriptase (RT) Polymerase Chain Reaction

kit and protocol for

in vitro diagnostics (IVD) and research for SARS CoV-2

(COVID-19)

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Manual| Version 1.0 -2020.

SARS-CoV 2- RT and Q- PCR: COVID 19 (in vitro diagnostics: Coronavirus detection in humans)

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STEMNOVATE™- SARS CoV2 RT- PCR kit

Catalogue Number STEM 17001

Product Kit Contains

1. Ready to use Reaction Mix – Nucleocapsid 2. Ready to use Reaction Mix - RDRP

3. Ready to use Internal Control - B Actin 4. PCR Grade Water

Product Insert

SARS Cov-2 RT-PCR-Gold version 1.0-2020

Product Features

• RT-PCR validated for SARS CoV-2

• SARS CoV-2 Specific Probes for specificity (RDRP and Nucleocapsid) • Biosafety and Handling BSL-1; BSL-2

Product Development and Manufacturing

Stemnovate, Cambridge, United Kingdom StemnovateÔ is the trading name of Stemnovate Limited, a business registered in England & Wales; registered number 101406

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Introduction

Stemnovate is a biotech company based in Cambridge that provides innovative molecular solutions for fast and accurate diagnostics.

Severe Acute Respiratory Syndrome Coronavirus strain 2 (SARS CoV-2) belongs to a family of RNA viruses that spreads to humans through contact, small droplets, coughing, sneezing or talking. Several drug molecules are under evaluation for SARS CoV-2 also commonly known as COVID-19, but treatment has remained elusive.

The polymerase chain reaction is among the most reliable methods to detect the virus in saliva or nasal swabs. Most commercial in vitro diagnostics (IVD) kits rely on in-silico methods for validation, but in the field cross-reactivity and false negatives are becoming a matter of concern for mass testing. Therefore, Stemnovate is introducing a validated RT-PCR kit-Gold in a user-friendly format to ensure accuracy for COVID diagnosis.

• The kits are CE marked for professional use • Validation with actual SARS CoV-2 genome

The diagnostic accuracy includes specificity for SARS CoV-2 and no cross reactivity with the common cold virus genome (COVID 19 causing SARS CoV-2 virus; and the common cold-causing, Rhinovirus, genomes were sourced in kind from the MRC Centre for Virus research UK).

This is important as there are seven coronavirus strains that affect the human population because of previous SARS and MERS episodes. In addition, some of the respiratory symptoms in the patient can be similar to influenza or common cold therefore, an emphasise on specificity and accuracy.

The other advantage of the kit is the user-friendly ready to use format, adaptability to most standard RT- PCR machines or thermocyclers (see protocol).

Product Description

The assay detects SARS CoV-2 RNA dependent RNA Polymerase (RDRP) and Nucleocapsid (N). These are essential for COVID-19's viral replication and structural integrity. The SARS CoV-2 whole genome sequence was retrieved from NCBI (National Centre for Biotechnology Information) NC_045512.2.

Advantages

Stemnovate rapid PCR kits provide complete solution for accurate testing as a gold standard method for viral genome detection and has an advantage for accuracy and specificity over alternatives such as RT-LAMP. In literature, the problems identified in RT-LAMP include the use of multiple primers (forward-backwards, inner and loop) that can result in unwanted primer to primer interaction and the endpoint colour change discrepancy due to reaction pH change insensitivity. Stemnovate protocol is user friendly as it comes in ready to use format over multitube reagents.

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In vitro Diagnostics

Stemnovate SARS-CoV-2 RDK is intended for in vitro diagnostics of human Coronavirus, (SARS CoV-2, COVID 19) from nucleic acid and requires upstream handling and processing of swab, saliva or blood samples.

Handling and Biosafety

The kit is suitable for use under Good Laboratory Practices (GLP) standards and biosafety level 1 (BSL1). The upstream sampling (swab, saliva, blood) collection and handling should be done under health and safety guidelines (HSE, PHE) for COVID 19 and require BSL2 or BSL3 facility.

Recommendations

o The precaution should be taken while handling samples including labelling and pre-test preparations

o The process should be carried out in a dedicated molecular diagnostic area

o The personal protective equipment (protective clothing, goggles, gloves) should be used for handling, sampling and testing.

o The hazard and risk assessment should be carried out for handling clinical samples

Diagnostic Sensitivity and Specificity

The test includes probes specific for SARS COV-2 RDRP and Nucleocapsid

The genome size of the SARS-CoV-2 varies from 29.8 kb to 29.9 kb and its genome structure is similar to known Coronaviruses. At the 5ʹ more than two-thirds of the genome comprises orf1ab encoding orf1ab polyproteins, while the 3ʹ one third consists of genes encoding structural proteins including surface (S), envelope (E), membrane (M), and nucleocapsid N proteins.

RNA-Dependent RNA Polymerase (RDRP) plays important role in viral replication and transcription, an essential feature for viral survival. It is a conserved protein within RNA viruses.

SARS CoV-2 nucleocapsid (N) is a structural protein that forms complexes with genomic RNA and plays a critical role in virus transcription and assembly.

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Usage Limitations

1. The kit is for in vitro diagnostics, clinical reference and research only.

2. The product is to be used as per protocol (see product insert) and under GLP (Good Lab Practices)

3. The sampling, collection, labelling should be done as per industry standards (follow country specific health and safety guidelines)

4. Store the kit under recommended conditions (see product insert)

5. The kit should be used before the expiry date mentioned on the product insert (check lot information).

6. COVID-19 is a notifiable disease so follow the guidelines (PHE, WHO or country specific regulatory body).

Biosafety

It is recommended to implement health and safety guidelines for working with infectious materials

1. The kit should only be used by trained personnel under good lab practices

2. The personal protective equipment should be used at all times (gloves, laboratory gown, eye protection and face masks for handling potentially infectious specimen).

3. Follow the guidelines on use, storage and disposal

Storage conditions and product stability

Reagents supplied in kit should be stored at -20o_{C on arrival. Avoid repeated freezing/thawing for} optimum stability. The reagents supplied in kit should be thawed on ice and mixed properly before use. When stored under the recommended conditions and handled appropriately, optimum enzymatic activity is retained until the expiry date on the outer box label.

Sampling and upstream processes

The RT-PCR requires upstream processing of samples to extract RNA and copy DNA synthesis The samples suitable for nucleic acid extraction

1. Respiratory specimens (bronchioalveolar lavage, tracheal aspirate) 2. Sputum or salivary samples

3. Nasopharyngeal or Oropharyngeal Swabs 4. Blood, Plasma or Serum

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RNA extraction

QIAamp viral RNA extraction kit, certified commercial extraction protocols or reliable in-lab formulated extraction protocol.

KIT COMPONENTS for SARS CoV-2 RT-PCR

Content Description Volume

Amplification master mix

For detection of RDRP High fidelity Taq polymerase, RDRP forward and reverse primer, buffer, and dNTPS mix (all four bases) and cofactors.

1.2ml

For detection of

Nucleocapsid

High fidelity Taq polymerase, nucleocapsid forward and reverse primer, buffer, and dNTPS mix (all four bases) and cofactors

1.2ml

For detection of

endogenous housekeeping; beta Actin

High fidelity Taq polymerase, B-actin forward and reverse primer, buffer, and dNTPS mix (all four bases) and cofactors.

1.2ml

PCR grade water Nuclease free water _2.0ml

Protocol

Before you begin:

1. Gently thaw the ready to use detection reaction mix at room temperature.

2. Resuspend content of the reaction mix with a P200 pipette and place the vials on ice for subsequent use.

Method:

1. Add 48 µl of SARS-CoV-2 RDRP, nucleocapsid and internal control ready to use mix into labelled sterile 0.2ml PCR tubes.

2. Add 2 µl of your target cDNA sample into the respective PCR tubes. 3. Centrifuge briefly to collect content to the bottom of the PCR tubes.

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5. The following thermal profile can be used for amplification.

Parameter Temperature Time

Initial denaturation 95 o_C _2min

Denaturation 95 o_C _30sec

Annealing 55 o_C _30sec

Extension 72 o_C _1min

Final Extension 72 o_C _{7 min}

Hold 4-15 o_C _∞

Number of cycles = 40

6. After amplification has elapsed, samples can be analysed by gel electrophoresis. Expected band size fragment for each target amplicon is stated in the table below and a typical gel data can be seen below.

Gene Expected band size (bp)

SARS-CoV-2 RDRP 107

SARS-CoV-2 Nucleocapsid 360

Endogenous housekeeping Beta- Actin 840

Data interpretation and results

The SARS CoV-2 known specimen shows amplification for RDRP and Nucleocapsid detected at the expected band size. The primers do not amplify Rhinovirus and are specific for SARS CoV-2. No template control (NTC) is a negative control for the reaction.

Sensitivity

The sensitivity of 99.5% is about 100 copies of RNA genome equivalent per reaction (amount is always detected), it can detect much lower amounts <10 copies. But for diagnostics 100 copies is a recommended standard for accuracy.

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Specificity

The test is validated for SARS CoV-2 (viral genome sourced from MRC-Centre for Virus research, UK) and patient specimen known to be positive for SARS CoV-2. The Cross sensitivity tested with a patient specimen known to be positive for Rhinovirus (common cold virus, in kind, MRC-Centre for Virus research, UK).

Limitations

• The detection sensitivity of amplified nucleic acids by the SYBR dye is 100pmols therefore DNA quantities of lower concentration will be difficult to detect.

• False negative reactions could occur due to errors or residual reagent carryovers in RNA extraction step, cDNA synthesis step or amplification.

In silico Analysis

Seeker probes and oligos for SARS-CoV-2’s nucleocapsid and RDRP were designed using the target gene sequence from NCBI and ExPASy gene bank. Probes and oligos were designed for COVID-19’s conserved nucleotide domains. BLAST analysis on FASTA sequence was routinely carried out on mega blast stringency algorithm to determine the alignments of designed probes and oligos against new and existing SARS-CoV-2 submitted strains and possible sequence homology with other SARS-COV-2 nucleotide sequence to eliminate false positive screening results that may arise from non-specific hybridisation and amplification.

Warning and precautions

The samples must be handled with care and due precautions should be taken for safe handling at all times.

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Safety warning (Pl refer to MSDS)

References

Aerosol and surface stability of HCoV-19 (SARS-CoV-2) compared to SARS-CoV-1 N van Doremalen, T Bushmaker, D Morris, M Holbrook… - medRxiv, 2020 - medrxiv.org

A novel human coronavirus, now named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, referred to as HCoV-19 here) that emerged in Wuhan, China in late 2019 is now causing a pandemic. Here, we analyze the aerosol and surface stability of HCoV-19.

Detection of SARS-CoV-2 in different types of clinical specimens

W Wang, Y Xu, R Gao, R Lu, K Han, G Wu, W Tan - Jama, 2020 - jamanetwork.com

Methods| We investigated the biodistribution of SARS-CoV-2 among different tissues of inpatients with coronavirus disease 2019 (COVID-19) diagnosed based on symptoms and radiology and confirmed by SARS-CoV-2 detection. This study was approved by the ethics.

Clinical characteristics of 140 patients infected with SARS-CoV-2 in Wuhan, China. JJ Zhang, X Dong, YY Cao, YD Yuan, YB Yang… - Allergy, 2020 - europepmc.org

Ordering Information

Please visit for online ordering, www.stemnovate.co.uk Email: [email protected]

T: +44 (0)1223830192 Contact

Stemnovate Limited (Diagnostics) Maia Building 270

Babraham research campus CB22 3AT, Cambridge, United Kingdom

COVID-19 RAPID TESTING