COVID 19 Rapid Testing

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COVID 19 Rapid Testing

StemnovateÔ Quantitative Polymerase Chain Reaction

kit and protocol for

in vitro diagnostics (IVD) and in vitro 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 Q- PCR Kit

Catalogue Number STEM 17002

Product Kit Contains

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

3. Ready to use Internal Control - RPL13A

4. PCR Grade Water

Product Insert

SARS Cov-2 RT-Q-PCR-Quant version 1.0-2020

Product Features

• RT-Q-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 faster and better diagnostics and drug discovery. The company is among one to watch companies of 2020 in the biotech industry in the UK.

COVID 19 was declared a pandemic by the World Health Organisation in March 2020, and since then the global deaths count has passed over one million, and the recurrence of infection remains a matter of concern for all countries. The containment strategy requires the implementation of accurate testing for 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 quantitative 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 quantitative RT-QPCR kit-Quant in a user-friendly format to ensure accuracy for COVID diagnosis.

We aim to alleviate the COVID testing problems and support vital programs run by central government and private testing providers by manufacturing the high quality rapid diagnostic kits in the UK. These are accurate as well as economical for mass testing.

The highlights are,

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

The diagnostic accuracy includes validation with SARS CoV-2 and cross reactivity study 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 validation 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, common cold; therefore, our emphasise on specificity and accuracy.

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

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2 Stemnovate kits are manufactured under strict quality control in Cambridge UK. The test 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’s 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 simple and with a ready to use reaction preparation user friendly over multitube reagents.

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

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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.

Usage Limitations

1. The kit is for in vitro diagnostics for clinical reference and in vitro 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)

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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 qPCR 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

5. Tissue samples

RNA extraction

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

KIT COMPONENTS for Stemnovate’s SARS CoV-2 RT-qPCR

Content Description Volume

Amplification master mix

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

500 microlitre

Amplification master mix For detection of Nucleocapsid

High fidelity Taq polymerase, nucleocapsid forward and reverse primer, buffer, MgCl2 and dNTPS mix (all four bases) and cofactors.

500 microlitre

Amplification master mix For detection of endogenous housekeeping; RPL13A

High fidelity Taq polymerase, RPL13A forward and reverse primer, buffer, MgCl2 and dNTPS mix (all four bases) and cofactors

500 microlitre

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Protocol Steps

Before you begin:

1. Gently thaw the ready to use qPCR 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.

3. Place your 96-well plate on a cooler rack.

4. Customise and design your plate setup and parameters using the associated qPCR software e.g. for relative RNA expression assay use comparative ΔΔCt method. Select your desired targets, reporters and quenchers (if used). Designate your reference and endogenous controls using the software’s drop column. Plate sample number, replicates and negative controls (NTC) should be entered into the cells.

5. Save your experimental method.

Method:

1. Add 18 µl of SARS-CoV-2 RDRP, nucleocapsid and endogenous control ready to use mix into the wells of your sample plate.

2. Add 2 µl of your target cDNA and NTC sample into the respective wells. 3. Gently apply optical adhesive film to the seal the plate wells.

4. Centrifuge 96- well plates for 2mins at 500 x g at 4o_{C to collect content to the bottom of the}

wells.

5. Transfer sample plates to the stage of the real time quantitative instrument for amplification. 6. Locate your saved experimental qPCR method file.

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6 8. After amplification has elapsed, save the run and analyse the collected data using the analysis tools available on the software. Below is a typical result obtained using the SARS CoV-2 ready to use qPCR mix on a human patient’s saliva and Nasal specimen RNA compared to a Known SARS- CoV-2 positive specimen.

Data interpretation and results

Based on WHO’s SARS-CoV-2 qPCR assessment criteria, low cycle threshold (Ct) values < 20 are considered to have a high viral load and is considered a definite positive.

Sample data

Typical data generated using the SARS Cov-2 Ready to use qPCR mix on a human patient

Figure A: Amplification plot graph showing expression of housekeeping gene (RPL13A) and SARS’s CoV-2’s Nucleocapsid in Saliva, Nasal, RT-Negative and known SARS- CoV-2 positive sample.

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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.

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 genome sample, received in kind from MRC-Centre for Virus research, UK).

Limitations

• The detection sensitivity of amplified nucleic acids by the fluorescent 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

Safety warning (Pl refer to MSDS)

Disclaimer: Stemnovate labs do not culture SARS CoV-2 being a notifiable disease and the genomic samples have only been obtained in kind from MRC Centre for virus research for sole purpose of assay validation.

The user must assess risk in handling human samples for upstream processes.

The kit design, validation and data are property of Stemnovate Limited and should not be replicated in any form.

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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

Coming soon SARS CoV-2 IVD on a chip

This novel technology is for decentralised, point of care rapid testing, for more information contact [email protected]

COVID 19 Rapid Testing