Primerdesign Ltd TM Hepatitis E virus (HEV)

1 ORF2 capsid protein geneHepatitis E virus (HEV) Primerdesign LtdTM150 testsgenesig Advanced Kit For general laboratory and research use only1 Quantification of Hepatitis E virus (HEV) genomesgenesig Advanced kit handbook Date: 07/11/2018 TheHepatitisEVirusisamemberoftheHepeviri daefamilyandisasingle-stranded,positive- senseRNAviruswithalineargenomeofjustover 7, , and3 , is responsible for causing Hepatitis E, an inflammatory liver ,appetiteloss, to human infection whereas serotypes three and four infect other animals as to Hepatitis E virus (HEV)2 Quantification of Hepatitis E virus (HEV) genomesgenesig Advanced kit handbook Date: 07/11/2018 SpecificityThePrimerdesigngenesigKitforH epatitisEvirus(HEV)(HEV) , theprimersrepresent100%homologywithover9 5%oftheNCBI databasereference sequences available at the time of and when required releases new will answer your of Hepatitis E virus (HEV) genomesgenesig Advanced kit handbook Date.

2 07/11/2018 Kit contents HEV specific primer/probe mix (150 reactions BROWN)FAM labelled HEV positive control template (for Standard curve RED) Internal extraction control primer/probe mix (150 reactions BROWN)VIC labelled as standard Internal extraction control RNA (150 reactions BLUE) Endogenous control primer/probe mix (150 reactions BROWN)FAM labelled RNase/DNase free water (WHITE)for resuspension of primer/probe mixes Template preparation buffer (YELLOW)for resuspension of internal control template, positive control template and standard curvepreparationReagents and equipment to be supplied by the userReal- time PCR InstrumentExtraction ,itisdesignedtoworkwellwithallprocessest hatyieldhighqualityRNAandDNAwithminimalP CR lyophilised OneStep or Precision PLUS OneStep 2X RT-qPCR Master MixContains complete OneStep RT-qPCR master mixPipettors and TipsVortex and centrifugeThin walled ml PCR reaction tubes4 Quantification of Hepatitis E virus (HEV) genomesgenesig Advanced kit handbook Date: 07/11/2018 Kit storage and stabilityThiskitisstableatroomtemperatur ebutshouldbestoredat-20 Cforlongerthan30 from the positive does not recommend using the kit after the expiry date stated on the sample ,concentration,andRNA/DNAintegrity(Anint ernalPCRcontrolissuppliedtotestfornonspe cificPCRinhibitors).

3 ,replacethetemplateRNAsample with RNase/DNase free range of testUnderoptimalPCRconditionsgenesigHEVd etectionkitshaveveryhighprimingefficienc iesof >95% and can detect less than 100 copies of target and disclaimersThisproductisdeveloped, ,1145 AtlanticAvenue,Alameda,CA94501orAppliedB iosystemsbusinessgroupoftheAppleraCorpor ation,850 LincolnCentreDrive,FosterCity, ,the5' ,210,015and5,487,972,ownedbyRocheMolecul ar Systems, Inc, and by Patent 5,538,848, owned by The Perkin-Elmer is a trademark of Primerdesign is a registered trademark of Primerdesign ,683,195,and4,683, ,ABIPRISM GeneAmp andMicroAmp areregisteredtrademarksoftheAppleraGenom ics(AppliedBiosystemsCorporation).BIOMEK isaregisteredtrademarkofBeckmanInstrumen ts,Inc.;iCycler isaregisteredtrademarkofBio-RadLaborator ies, ,TaqMan andAmpliTaqGold areregisteredtrademarksofRocheMolecularS ystems,Inc.,ThepurchaseofthePrimerdesign of Hepatitis E virus (HEV) genomesgenesig Advanced kit handbook Date: 07/11/2018 Principles of the testReal- time , `-dyeanda3` , controlForcopynumberdeterminationandasap ositivecontrolforthePCRsetup,thekitconta insa positive control , controls before pipetting the positive control into the positive control up the of Hepatitis E virus (HEV) genomesgenesig Advanced kit handbook Date: 07/11/2018 Internal RNA extraction controlWhenperformingRNAextraction, at a high +/-3depending on the level of sample controlToconfirmextractionofavalidbiolog icaltemplate, of Hepatitis E virus (HEV) genomesgenesig Advanced kit handbook Date.

4 07/11/2018 Component - resuspend in waterVolumeHEV primer/probe mix (BROWN)165 lInternal extraction control primer/probe mix (BROWN)Endogenous control primer/probe mix (BROWN)Pre-PCR pack165 l165 lResuspension protocolTominimizetheriskofcontamination withforeignDNA, Filter tips are recommended for all pipetting each tube in a centrifuge before opening the ,according to the table below:To ensure complete resuspension, vortex each tube thoroughly.* , extractionTheinternalextractioncontrolRN AcanbeaddedeithertotheRNAlysis/extractio nbufferorto the RNA sample once it has been resuspended in lysis NOT add the internal extraction control RNA directly to the unprocessed biologicalsample as this will lead to degradation and a loss in loftheInternalextractioncontrolRNA(BLUE) toeachsampleinRNAlysis/extraction buffer per RNA extraction according to the manufacturer s preparation buffer supplied, according to the table below:To ensure complete resuspension, vortex each tube Positive Control Template (RED) *500 lPost-PCR heat-sealed foilComponent - resuspend in template preparation bufferVolumeInternal extraction control RNA (BLUE)Pre-PCR heat-sealed foil600 l8 Quantification of Hepatitis E virus (HEV) genomesgenesig Advanced kit handbook Date.

5 07/11/2018 OneStep RT-qPCR detection protocolFor optimum performance and OneStep or PrecisionPLUSOneStep 2X RT-qPCR Master Mix1 lHEV primer/probe mix (BROWN)Final Volume1 l15 l10 lInternal extraction control primer/probe mix (BROWN)RNase/DNase free water (WHITE)3 l2. ForeachRNAsampleprepareanendogenouscontr olreactionaccordingtothetable below (optional) OneStep or PrecisionPLUS OneStep 2X RT-qPCR Master Mix1 lEndogenous control primer/probe mix (BROWN)Final Volume15 l10 lRNase/DNase free water (WHITE)4 l1. For each RNA sample prepare a reaction mix according to the table below:Include sufficient reactions for positive and negative lofthesemixesintoeachwellaccordingtoyour qPCRexperimentalplate set Pipette5 lofRNAtemplateintoeachwell, is 20 of Hepatitis E virus (HEV) genomesgenesig Advanced kit handbook Date: 07/11/2018 OneStep RT-qPCR Amplification ProtocolAmplificationconditionsusingoasi gOneSteporPrecisionPLUSOneStep2 XRT-qPCRM aster of standard curve dilution ) Pipette 90 l of template preparation buffer into 5 tubes and label 2-62) Pipette 10 l of Positive Control Template (RED) into tube 23) Vortex thoroughly4) Change pipette tip and pipette 10 l from tube 2 into tube 35) Vortex thoroughlyRepeat steps 4 and 5 to complete the dilution lofstandardtemplateintoeachwellforthesta ndardcurveaccordingtoyour plate set-upThe final volume in each well is 20 TranscriptionEnzyme activationDenaturationDATA COLLECTION *TimeTemp10 min2 min10 s60 s55 oC95 oC95 oC60 oCCycling x50* Fluorogenic data should be collected during this step through the FAM and VIC channelsComponentVolumeoasig OneStep or PrecisionPLUSOneStep 2X RT-qPCR Master Mix1 lHEV primer/probe mix (BROWN)

6 Final Volume15 l10 lRNase/DNase free water (WHITE)4 to the table below:No international unitsInternational UnitsCopy Number2 x 105 per l2 x 104 per l2 x 103 per l2 x 102 per l20 per l2 per lStandard CurveTube 1 Positive control (RED)Tube 2 Tube 3 Tube 4 Tube 5 Tube 6 Copy Number *Standard Curve*The quantitative results produced by the genesig HEV kit can be converted toInternational Units by multiplying copy numbers by This conversion factor wasdeveloped using RNA/DNA extracted (where applicable) from the WHO InternationalStandard for of this kit are advised that the conversion factor serves as a guide. For the highestlevel of accuracy, it is best practice to calculate conversion factors independently usingthe WHO International Standard. If unsure, please contact your local sales representativefor details regarding the generation of the conversion factor to ensure it is applied in themost appropriate of Hepatitis E virus (HEV) genomesgenesig Advanced kit handbook Date: 07/11/2018 Interpretation of results 30 PositivecontrolNegativecontrolInternalco ntrol(VIC)Target(FAM)Interpretation> 30+ / -+ / --+ / -+---> 35 POSITIVE QUANTITATIVE RESULT calculate copy number+ / -++ / -++++ 35 EXPERIMENT FAILEDdue to test contamination*POSITIVE QUANTITATIVE RESULT calculate copy number> 30 POSITIVE QUALITATIVE RESULTdo not report copy number as thismay be due to poor sample extraction-++-NEGATIVE RESULT--+-SAMPLE PREPARATION FAILED+ / -+ / --+ / -EXPERIMENT FAILED*Wherethetestsampleispositiveandth enegativecontrolispositivewithaCq>35,the sample must be reinterpreted based on the relative signal strength of the two results.

7 SampleNegative control Cq > 5 SAMPLE POSITIVES ampleNegative control Cq < 5 INCONCLUSIVEI fthesampleamplifies>5 Cqearlierthanthenegativecontrolthenthesa mpleshouldbereinterpreted(viathetableabo ve)withthenegativecontrolverifiedas < (RED) of Hepatitis E virus (HEV) genomesgenesig Advanced kit handbook Date: 07/11/2018 Internal PCR controlTheCqvalueobtainedwiththeinternal controlwillvarysignificantlydependingont heextractionefficiency, , is present in the of Hepatitis E virus (HEV) genomesgenesig Advanced kit handbook Date: 07/11/2018