ViroBOAR Spike 1.0 RT-PCR Kit (SARS-CoV-2)User ManualFor research use onlyFor use with Roche LightCycler 480 II InstrumentEurofins Genomics Europe Synthesis GmbHwww.eurofinsgenomics.comAnzinger Str. 7a85560 EbersbergGermany6000-ViroBoSPIKE1

ContentsContents . 21.Introduction . 32.lntended Use . 43.Principle of the RT-PCR Assay . 44.Material Provided . 55.Stability and Storage. 56.Additionally Required Materials and Devices but not provided . 57.Sample Collection and Preparation . 68.Assay Procedure . 68.1Reaction Setup . 68.2Programming the Roche LightCycler 480 II Instrument . 89.Data Analysis and Interpretation . 910.Specific Performance Characteristics . 1011.Quality Control . 1112.Trademarks and Disclaimers . 1113.Precautions and Warnings. 1214.Disposal Considerations . 1315.Ordering Information . 1316.Bibliography. 142

1. IntroductionEnd of 2019, a novel respiratory disease emerged in the city of Wuhan, Hubei Province of thePeople’s Republic of China, and soon spread rapidly within the country and worldwide. The causativeagent was identified as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2(2019-nCoV), like the closely related SARS coronavirus (SARS-CoV), belongs to the genusBetacoronavirus within the family of coronaviruses. The zoonotic reservoir of the virus appears to bebats.Coronaviruses are enveloped, positive single‐stranded large RNA viruses that infect humans, but alsoa wide range of animals. The common human coronaviruses NL63, 229E, OC43 and HKU1 arewidespread especially throughout the winter months. They are responsible for up to one third of allacute respiratory diseases, typically with mild symptoms (common cold). More than 80 % of the adultpopulation have antibodies against human coronaviruses. The immunity from previous infectionslasts only for a short period of time. Therefore, reinfections with the same pathogen are possible justafter one year.SARS-CoV-2 is predominantly transmitted by droplet infection via coughing or sneezing and throughclose contact with infected patients. In theory, smear infection and infection through the conjunctivaof the eyes are also possible. The incubation period is in the median 5–6 days (and up to 14 daysmaximum).The clinical manifestations of SARS‐CoV‐2‐related COVID-19 disease include fever, cough, respiratoryproblems and fatigue. In most patients the infection manifests with symptoms of a mild febrile illnesswith irregular lung infiltrates.The initial clinical sign of COVID‐19 which allowed case detection was pneumonia. But it turned outthat the course of the disease is non-specific and varies widely, from asymptomatic courses to severepneumonia with lung failure and death. However, based on current knowledge, around 80 % of theillnesses are mild to moderate.Although severe courses of the disease also occur in younger patients and people without previousillness, the following groups of people have an increased risk of serious forms of the disease: elderlypeople (with a steadily increasing risk from around 50-60 years of age), smokers and people withcertain diseases of the cardiovascular system or the lungs, patients with chronic liver diseases,diabetes mellitus, cancer, or patients with a weakened immune system (e.g. due to immunedeficiencies or by taking drugs that suppress the immune system).The presence of pathogen or infection may be identified by Nucleic acid testing (NAT): e.g. RT-PCR Serology: detection of antibodies by e.g. ELISAEnd of 2020 two SARS-CoV-2 strains were identified that show mutations in different positions of theS gene, coding for the spike protein. Some of these mutations lead to a higher infectivity. Especiallymutation N501Y is identified as one root cause. This mutation is found in virus isolates in UK (known3

as virus variant B.1.1.7) and in South Africa (virus variant B.1.351). These two strains can bedistinguished by the mutation A570D which is present only in the strain from UK.2. lntended UseThe ViroBOAR SPIKE 1.0 RT-PCR Kit is used for simultaneous qualitative detection of SARS-CoV-2 Sgene variants in codon 501 and 570 for discrimination of SARS-CoV-2 wildtype virus and strainsB1.1.7 and B1.351 (genomic RNA) extracted from human respiratory specimen (e.g. pharynx garglelavage, nasal wash/swab, nasopharyngeal wash/swab and oropharyngeal swab as described in WHOinterim guidance “Laboratory testing for 2019 novel coronavirus (2019-nCoV) in suspected humancases”) and already pretested positive by RT-PCR method. The ViroBOAR SPIKE 1.0 RT-PCR Kit isintended for use by trained laboratory personnel only.3. Principle of the RT-PCR AssayThe kit contains a specific ready-to-use system for the detection of Coronavirus SARS-CoV-2 byReverse Transcription Polymerase Chain Reaction (RT-PCR) in the real-time PCR system. The reactionis done in one tube two step real-time RT-PCR. The first step is a reverse transcription (RT), duringwhich the virus RNA is transcribed into cDNA. Afterwards, a thermostable DNA polymerase is used toamplify the specific gene fragments by means of polymerase chain reaction (PCR). Fluorescence isemitted during PCR and measured by the real-time systems optical unit. The detection of amplifiedvirus DNA fragment is performed in fluorimeter channel FAM (for the detection of S gene mutationN501Y and A570D) and HEX (for the detection of S gene wildtype version of N501Y and A570D) withMGBEQ Quencher.Depending on user preference, the assays for codon 501 and 570 can be detected together in aduplex reaction or separately in single reactions.Since this kit is used for confirmatory analysis of extracts that have already tested positive for thepresence of corona virus RNA, it does not include a positive control that would detect inhibition.The principle of the real-time detection is based on the fluorogenic 5’ nuclease assay. During the PCRreaction, the DNA polymerase cleaves the probe at the 5’ end and separates the reporter dye fromthe quencher dye only when the probe hybridizes to the target DNA. This cleavage results in thefluorescent signal generated by the cleaved reporter dye, which is monitored real-time by the PCRdetection system. The increasing fluorescence signal generated during the PCR reaction isproportional to the amount of the specific PCR product. Monitoring the fluorescence intensities inreal-time allows the detection of the accumulating product without having to re-open the reactiontube after the amplification.4

4. Material ProvidedPresentation (1000 rxns) Presentation (5000 rxns)Component Nr.Kit ComponentsPresentation (100 rxns)12x qPCR Mix1 vial; 700 µl1 vial; 7,0 ml1 vial; 35 ml2Oligo Mix 5011 vial; 20 µl1 vial; 200 µl1 vial; 1,0 ml3Oligo Mix 5701 vial; 20 µl1 vial; 200 µl1 vial; 1,0 ml420x Rtase1 vial; 100 µl1 vial; 1000 µl1 vial; 5,0 ml5ddH201 vial; 110 µl1 vial; 1100 µl1 vial; 5,5 ml5. Stability and StorageThe ViroBOAR SPIKE 1.0 RT-PCR Kit is shipped on dry ice and all components should arrive frozen. All components have to be stored at -20 C upon arrival.Storage at 4 C is not recommended for longer than 3 hoursMore than one repeated freeze thaw cycles of reagents should be avoided, since this mightaffect the performance of the kit. Reagents should be frozen in aliquots if they are usedintermittently.Keep unfrozen storage (e.g. storage on ice) as short as possible.Keep the kit components in the freezer, until you are ready to use it.Protect the Oligo Mixes (Comp. 2 and Comp. 3) from light.6. Additionally Required Materials and Devices but not provided Biological cabinet/Laminar AirflowVortex mixerCryo-containerSterile filter tips for micro pipetsDisposable gloves, powderlessRefrigerator and freezerRoche LightCycler 480 II InstrumentPipets (0.5μl – 1000μl)Sterile microtubesBiohazard waste containerTube racksDesktop microcentrifuge for “Eppendorf” type tubes (RCF max. 16,000 x g)Extraction deviceViral RNA extraction kit (e.g. PurePrep Pathogen from Molgen)5

7. Sample Collection and Preparation Regarding sample collection and shipment please refer to the WHO interim guidance“Laboratory testing for 2019 novel coronavirus (2019-nCoV) in suspected human cases”. Extracted RNA from human respiratory specimen types is the starting material for theViroBOAR SPIKE1.0 RT-PCR Kit. The quality of the extracted RNA has a crucial effect on theperformance of the entire RT-PCR test system. Make sure that the nucleic acid extractionmethod is compatible with real-time PCR technology. For nucleic acid extraction a method suitable for extracting virus RNA from humanrespiratory specimen should be used. During kit development and validation, the PurePrepPathogens Kit from Molgen was used. Since ethanol is a strong real-time PCR inhibitor, it is necessary to completely eliminate itprior to the elution of the nucleic acid during extraction. If using spin columns with washingbuffers containing ethanol, it is highly recommended to perform an additional centrifugationstep of 10 min at approximately 17,000 x g ( 13,000 rpm) before eluting the RNA. For thisadditional centrifugation step, use a new collection tube.8. Assay Procedure8.1Reaction Setup Please read the instructions for use carefully before performing the assay. Reliability ofresults depends on following strictly the instructions for use. Before use make sure that all samples and reagents are thawed completely, mixed by up anddown pipetting or vortexing and centrifuged briefly. Prepare quickly the Reaction Mix on ice or in the cooling block. If possible, run samples and controls in duplicates or triplicates. Pipette kit componentsslowly and carefully and use pipette tips suitable for pipetting viscous liquids. The use of ddH2O (Comp. 5) as no template control (NTC) is highly recommended. Define the positions of the wells on the plate for samples and controls (positive control orNTC). Multiply the volumes of 2xqPCR Mix (Comp. 1), Oligo Mix 501 (Comp. 2), Oligo Mix 570(Comp. 3), 20xRTase (Comp. 4), ddH2O (Comp. 5) per reaction with the number of plannedPCR reactions, which includes the number of NTCs and RNA extracts from patient samplesprepared. ddH2O (Comp. 5) is set into the RT-PCR as no template control. For reasons ofunprecise pipetting, always add an extra virtual sample. Mix completely and then spin downbriefly with a centrifuge.6

Pipette 8μl Master Mix with a micropipette and sterile filter tips to each of the real-time PCRreaction plates/tubes. Separately add 4μl template (nucleic acid extracted from negativecontrol (NTC) or RNA extracts from patient samples, positive control RNA with no extraction)to different reaction plates/tubes. Immediately close the plates/tubes to avoid crosscontamination. Spin down briefly in order to collect the Master Mix in the bottom of the reaction tubes. Perform the following protocol in the instrument of Roche LightCycler 480 II instrument.Singleplex-reaction:Duplex-reaction:7

8.2Programming the Roche LightCycler 480 II InstrumentRegarding setup and programming of Roche LightCycler 480 II instrument, please use thecorresponding manual provided by the manufacturer.RT-PCR Run Settings:Before starting the test run, please check the settings for cycles, temperature and time.Fluorescent Detectors/Dyes:DetectionGenDyeQuencherDetection channel (Excitation/Emission)SARS-CoV-2S gene: N501Y mutationFAMMGBEQ465nm/510nmSARS-CoV-2S gene: N501Y wildtypeHEXMGBEQ533nm/580nmSARS-CoV-2S gene: A570D mutationFAMMGBEQ465nm/510nmSARS-CoV-2S gene: A570D wildtypeHEXMGBEQ533nm/580nm8

9. Data Analysis and InterpretationData analysis should be performed with the software of the Roche LightCycler 480 II instrumentaccording to manufacturer’s instructions.Diagnosis of an infectious disease should not be established only on the basis of a single test result. Aprecise diagnosis should take into consideration clinical history, symptomatology as well as otherlaboratory diagnostics.The probes for the “wildtype” sequences are HEX-labelled, the probes for the mutated sequences areFAM labelled.If assay for discrimination of mutation on codon 501 is used in single confirmation assay withoutother controls signals must be detected in the following channels:N501YTarget:Dye:WildtypeB 1.1.7B.1.351S MutantFAMno signalsignalsignalS WildtypeHEXsignalno signalno signalIf assay for discrimination of mutation on codon 570 is used in combination with the existing qPCRfor detection of N gene and E gene signals must be detected in the following channels:A570DTarget:Dye:WildtypeB 1.1.7B.1.351S MutantFAMno signalsignalno signalS WildtypeHEXsignalno signalsignalIf both assay for discrimination of mutation on codon 501 and 570 are used in combination with theexisting qPCR for detection of N gene and E gene signals must be detected in the following channels:N501Y / A570DS MutantTarget:Dye:FAMWildtypeno signalB 1.1.7signalB.1.351signalS WildtypeHEXsignalno signalsignal9

These duplex assays can be used on all qPCR instruments that can measure into a FAM channel aswell as a VIC/HEX channel. Besides Roche and Agilent, these are for example the instruments frome.g. Bio-RAD and Thermo Scientific. These duplex assays have also been used at Eurofins Genomics sofar only on the Roche LC 480 II. For other instruments there are currently no analysis data availableat Eurofins Genomics.Discrimination of virus strains with respect to S gene codons 501 and 570 is only valid for sampleswhich were already pretested positive for the presence of SARS-CoV-2 RNA and if the Cp value in oneor both of the S gene channels is below 34,5.10.Specific Performance CharacteristicsSpecific performance characteristics were determined with the Roche LightCycler 480 II inaccordance with the manufacturer’s recommendations.Other commercially available SARS-CoV-2 kits were used as reference method for the evaluation ofperformance characteristics.To establish performance characteristics, RNA extraction was performed using the PurePrepPathogens Kit from Molgen (Utrecht, Netherlands; Art.No.OE00290096) on KingFisher Flexinstruments (ThermoFisher Scientific).The use of other extraction kits may only be released after internal validation and after consultationof the kit manufacturer.The use of other RealTime Instruments and resulting cut offs may only be released after internalvalidation and after consultation of the kit manufacturer.ValidationParameterResultsAcceptance criteriaStatus100 % of all samples pretested orSpecificity /Sensitivityconfirmed by NGS as wildtype or100 % fulfilledmutant must be determined asPassedwildtype or mutant by qPCR100 % of all samples which show aLimit of DetectionCp value of 34,5 or lower with E gene100% fulfilledassay must show a signal with S genePassedassay2Linearity2The correlation coefficient R is The correlation coefficient R must0, 0,9810Passed

The accuracy must be 100%: for theAccuracy100 % ednofalse“wildtype” results must be producedon these samples. For the NGSPassedconfirmed “wildtype” samples, nofalse “mutant” results must beproduced on these samples.The same RNA extracts must bePrecision100% fulfilleddetermined in 100 % of the qPCRPassedanalyses as “wildtype” or “mutant”.All positive samples must bedetermined as “wildtype” or“mutant” in both assays.Matrix Effect100% fulfilledAll samples must be positive ornegative for Corona virus,Passedrespectively in both assays. Cp valuesshould differ by not more than 3 Cpvalues.The validation results are based on a sample size of 291 patient samples, among which 190 patientsample were tested positive for SARS-CoV-2 N gene and E gene.11.Quality ControlIn accordance with Eurofins Genomics Europe Synthesis GmbH Quality Management System (ISO13485:2016), each lot of the ViroBoSPIKE 1.0 RT-PCR Kit has been tested against predeterminedspecifications to ensure consistent product quality. A certificate of Analysis is provided with the kiton demand.12.Trademarks and DisclaimersLightCycler 480 Instrument II (Roche)KingFisher Flex Purification System (ThermoFisher Scientific)PurePrep Pathogens Kit from Molgen (Utrecht, Netherlands; Art.No.OE00290096)11

Registered names, trademarks, etc. used in this document are to be considered protected by laweven if not specifically marked as such.13.Precautions and Warnings The test procedure, the information, the precautions and warnings in the instructions for usehave to be strictly followed. The use of the test kit with other equipment than listed in “10. Specific PerformanceCharacteristics” needs to be validated prior to routine diagnostic application. Any deviation from the test procedure as well as any use in combination with other productsnot approved by the manufacturer is not authorized; the user himself is responsible for suchchanges. The manufacturer is not liable for false results and incidents for these reasons. Only for in-vitro diagnostic use. Do not interchange reagents of different production lots. No reagents of other manufacturers should be used along with reagents of this test kit. Do not use reagents beyond expiry date stated on the label. Wear disposable powder-free gloves, a laboratory coat and eye protection when handlingspecimens. Always use DNase/RNase-free disposable reaction tubes and sterile pipette tips with aerosolbarriers. Avoid microbial and nuclease (DNase/RNase) contamination of the specimen and thecomponents of the kit. In order to avoid contamination of working space with nucleic acids, reaction tubes/platesshould not be re-opened after amplification. RT-PCR is highly sensitive to nucleic acid contamination. Therefore, positive/potentiallypositive material needs to be stored separate from all other components of the kit. Dedicate kit components and equipment to the separate working areas and do not movethem from one area to another. This assay must not be used on the specimen directly; prior to using this assay, the nucleicacid has to be extracted with suitable extraction methods from the original specimen. The result of this RT-PCR kit may be influenced by potential mutations in the genome of thepathogen if they are located in the primer / probe binding region. Underestimation and/orfailure to detect the pathogen may occur.12

PCR inhibitors may also elicit underestimation, false negative results or invalid runs.Therefore, only use nucleic acids extraction kits (PurePrep Pathogens Kit from Molgen),which remove PCR inhibitors and which are dedicated for downstream PCR processes. The RT-PCR is only designed for qualified personnel who are familiar with good laboratorypractice and trained in Real Time-PCR technology. Avoid repeated thawing and freezing of reagents as this may reduce the sensitivity of thetest. Avoid unnecessary light exposure from Oligo Mixes (Comp. 2 and Comp. 3)14.Disposal ConsiderationsResidues of chemicals and preparations are generally considered as hazardous waste. The disposal ofthis kind of waste is regulated through national and regional laws and regulations. Contact your localauthorities or waste management companies which will give advice on how to dispose hazardouswaste.15.Prod. No.:Ordering Information6100-ViroBoSPIKECorona Conformation Assay 1.0 RT-PCR Kit, 100 rxns6200-ViroBoSPIKECorona Conformation Assay 1.0 RT-PCR Kit, 1000 rxns6300-ViroBoSPIKECorona Conformation Assay 1.0 RT-PCR Kit, 5000 rxns13

16.BibliographyBennett S, Davidson RS, Gunson RN. Comparison of gargle samples and throat swab samples for thedetection of respiratory pathogens. J Virol Methods 2017; 248:83–6.Gargle Lavage as a Safe and Sensitive Alternative to Swab Samples to Diagnose COVID-19: A CaseReport in Japan Clin Infect Dis . 2020 Jul 28;71(15):893-894. doi: 10.1093/cid/ciaa377.Gorbalenya, Alexander E.; Baker, Susan C.; Baric, Ralph S.; Groot, Raoul J. de; Drosten, Christian;Gulyaeva, Anastasia A. et al. (2020): Severe acute respiratory syndrome-related coronavirus: Thespecies and its viruses – a statement of the Coronavirus Study Group (14). bioRxiv2020.02.07.937862; doi: , Lisa E.; Menachery, Vineet D. (2020): Return of the Coronavirus: 2019-nCoV. In Viruses 12(2). DOI: 10.3390/v12020135.RKI (Ed.): SARS-CoV-2 Steckbrief zur Coronavirus-Krankheit-2019 (COVID-19). Available online at Coronavirus/Steckbrief.html. (accessed:20.01.2021)RKI (Ed.): Hinweise zur Testung von Patienten auf Infektion mit dem neuartigen Coronavirus SARSCoV-2. Available online at Coronavirus/Vorl Testung nCoV.html.(accessed: 20.01.2020)Wang, Guan; Jin, Xian (2020): The progress of 2019 novel coronavirus event in China. In Journal ofmedical virology 92 (5), pp. 468–472. DOI: 10.1002/jmv.25705.WHO: Clinical management of severe acute respiratory infection (SARI) when COVID-19 disease issuspected. Interim guidance. 27 May 2020. Available online at n-novel-coronavirus-(ncov)infection-is-suspected, accessed: 20.01.2020.WHO: Laboratory testing for coronavirus disease 2019 (COVID-19) in suspected human cases: interimguidance, 19 March 2020. Available online at man-cases-20200117, accessed: 20.01.2021.Wu,F., Zhao,S., Yu,B., Chen,Y.M., Wang,W., Song,Z.G., Hu,Y.,Tao,Z.W., Tian,J.H., Pei,Y.Y., Yuan,M.L.,Zhang,Y.L., Dai,F.H.,Liu,Y., Wang,Q.M., Zheng,J.J., Xu,L., Holmes,E.C. and Zhang,Y.Z: A newcoronavirus associated with human respiratory disease in China. Nature 579 (7798), 265-269 (2020).Zhou, Peng; Yang, Xing-Lou; Wang, Xian-Guang; Hu, Ben; Zhang, Lei; Zhang, Wei et al. (2020): Apneumonia outbreak associated with a new coronavirus of probable bat origin. In Nature 579 (7798),pp. 270–273. DOI: 10.1038/s41586-020-2012-7.14

SYMBOLS KEYEurofins Genomics Europe Synthesis GmbHAnzinger Str. 7a85560 EbersbergGermanyInternet: eurofinsgenomics.comEmail: [email protected] No.: EGE210122 / Issue Date: Jan 22th, 202115

8.2 Programming the Roche LightCycler 480 II Instrument . Regarding setup and programming of Roche LightCycler 480 II instrument, please use the corresponding manual provided by the manufacturer. RT-PCR Run Settings: Before starting the test run, please check the settings for cycles, temper