User Manual (201409)
Contents
1. PetNAD FHV Detection Kit TROUBLESHOOTING Problems i Possible causes i Solutions False Positive 1 Reuse of micro E Micro centrifuge tubes tips R centrifuge tubes tubes and Premix are for single use tips R tubes and only Reusing these accessories Premix would cause cross contamination and therefore false positive results M Used micro centrifuge tubes tips R tubes and Premix should be collected and discarded according to local regulation Do not place the waste close to the working area to l i prevent cross contamination 2 Contaminated M Use aerosol free tps micropipette 3 Contaminated W Consult with a GeneReach reagent technical support representative or local distributor Feet dem EPSP sere O 1 4 Contaminated i M Consult with a GeneReach working area i technical support representative on how to clean up working area 12 PetNAD FHV Detection Kit Problems Possible causes Solutions False i 1 Nucleic acid B Consult manual of nucleic acid Negative extraction failed extraction kit 2 PCR inhibition W Do not overload PCR with too much nucleic acid M Spike nucleic acid sample 5 ul into a P Control reaction for a parallel PCR reaction Negative results indicate the presence of inhibitors in the nucleic acid In that case prepare another nucleic acid extract Heavy MT Leakage or spill of TW Consult with a GeneReach ET2. Premix vial and 1 primers probe and enzyme desiccating for amplification agent bag E Desiccating agent pack Premix Buffer M Reaction buffer to re dissolve 2 vials 1 3 ml vial B the lyophilized pellet P Control E Dried plasmid containing vial FHV partial sequence as positive control P Control a Reaction buffer to re dissolve I vial 110 ul vial Buffer P Control PetNAD FHV Detection Kit 1 bag 24 pieces bag 1 bag 24 pieces bag User Manual 1 copy B Materials and Equipment Required but Not Provided 1 PetNAD Nucleic Acid Co prep Kit or taco Automatic Nucleic Acid Extraction System 2 POCKIT M Nucleic Acid Analyzer POCKIT PetNADTM compatible instrument 3 cubee M Mini Centrifuge cubeeTM 4 Micropipette and filter tips C Storage and Stability 1 The kit should be stored at 4 C and is stable until the expiration date stated on the label 2 Store Premix vials in sealed Premix Pack to avoid hydration of lyophilized components 3 Reconstituted P Control is stable for 6 months at 4 C Aliquot reconstituted P Control to avoid degradation of nucleic acid D Sample Type DNA extracted from whole blood or swab sample e g oropharyngeal conjunctival and nasal swab PetNAD FHV Detection Kit RECOMMENDED NUCLEIC ACID EXTRACTION METHODS A PetNAD Nucleic Acid Co prep Kit taco DNA RNA Extraction Kit compatible inst
3. NAD FHV Detection Kit ANALYTICAL SENSITIVITY i sissovsossessssctessvsvnossssvsvescsesvensesesteeness 11 TROUBLESHOOTING eter ente ei argen Rs en s eser Du eC E co SE REV unen E iU 12 REFERENCE passe 14 PetNAD FHV Detection Kit INTENDED USE PetNAD FHV Detection Kit is intended for in vitro detection of the Feline Herpesvirus 1 FHV 1 DNA based on insulated isothermal polymerase chain reaction iiPCR technology This kit is designed specially to be used with an insulated isothermal iiPCR compatible instrument POCKIT Nucleic Acid Analyzer The assay is intended for use by veterinarians or technicians with basic laboratory skills This kit is intended for research use only SCIENTIFIC MEANINGS Antibody induced by vaccine or obtained from maternal immunity could lead to false positive interpretation in antibody based diagnostic procedures Detecting pathogen s nucleic acids not antibody PCR based methods can avoid the false positive results described above Furthermore with higher analytical sensitivity PCR can detect lower levels of viral signals than most if not all diagnostic methods It can reduce the chance of false negative results at early infection stage and shorten the window period between time of infection and detection PetNAD FHV Detection Kit SUMMARY AND EXPLANATION FHV 1 an alphaherpesvirus causes mainly keratoconjunctivitis and rhinitis Povey 1979 that lead to mortality exce
4. PetNAD FHV Detection Kit For Feline Herpesvirus 1 User Manual For Research Use Only Manufacturer GeneReach Biotechnology Corporation TEL 886 4 24639869 FAX 886 4 24638255 No 19 Keyuan 2 Road Central Taiwan Science Park Taichung City Taiwan 407 Web Site www petnad com 2014 09 PetNAD FHV Detection Kit Content INTENDED USE eeeee esee esee entes etate snae ta sesto seta stes essen sees tnu 1 SCIENTIFIC MEANINGS e eese esee esee e eese essen tn seta ssa toas ta an 1 SUMMARY AND EXPLANATION seseveeveevevsvseneevevsensnnensessenenensennee 2 PRINCIPLES OF THE PROCEDURE cese reset tn nennt 3 PRODUCT DESCRIPTION sesseevessvveneeveesnnnnvennensenenennennenseneneeneenennne 3 A Materials Provided ap ea n eite cheeses 3 B Materials and Equipments Required but Not Provided 4 C Storage and Stability seisein niina oroi n E 4 D Sample Type eint EU Eri eS 4 RECOMMENDED NUCLEIC ACID EXTRACTION METHODS E E ER EE Ne 5 Ld 0 89 VO M Jo gen et dre 5 LIMITATIONS sscsesscssssensisiscesssatevcsectesetatcessunssisvoceesesbeocsnebesetatcedeveneusie 6 PROCEDURE ie qe ces tton uoi pk eadera eene a PEEL PUNDR DR ti oss Vra RR E 8 A PetNAD FHV Detection Kit Quick Guide sssssss 8 Bi Procedure ue nsus ee pd eue p ees 9 DATA INTERPRETATION eeoseeveesvsenvennennenevsnnennevseneneeneesennnvensennee 11 Pet
5. contamination reaction from R technical support representative or of amplicons tube into reaction local distributor in reaction chamber of chamber of POCKIT POCKIT PetNAD FHV Detection Kit REFERENCE 1 Chang H F G Tsai Y L Tsai C F Lin C K Lee P Y Teng P H Su C and Jeng C C 2012 A thermally baffled device for highly stabilized convective PCR Biotechnology Journal 7 5 662 666 doi 10 1002 biot 201100453 2 Gaskell R M P E Dennis L E Goddard F M Cocker and J M Wills 1985 Journal of General Virology Vol 66 No 2 3 Povey R C 1979 A review of feline rhinotracheitis feline herpesvirus 1 infection Comp Immun Microbiol Infect Dis 2 373 387 4 Mickman M A Reubel G H Hoffman D E et al 1994 An epizootic of feline herpesvirus type 1 in a large specific pathogen free cat colony and attempts to eradicate the infection by identification and culling of carriers Lab Anim 28 320 329 5 Tsai Y L Wang H T T Chang H F G Tsai C E Lin C K Teng P H Su C and Jeng C C 2012 Development of TaqMan probe based insulated isothermal PCR PCR for sensitive and specific on site pathogen detection PLoS ONE 7 9 e45278 doi 10 1371 journal pone 0045278
6. eding 6046 in kitten Mickman et al 1994 In the acute phase of infection large amounts of infectious virus are excreted in nasal and ocular discharges Gaskell et al 1984 Recovered animals remain latently infected with virus in sensory nerve ganalia During this stage virus shedding may be induced by stress or occur spontaneously PCR is one of the most commonly accepted methods that provide high sensitivity and specificity for FHV detection However conventional PCR assays could take three to four hours and require sophisticated thermocyclers and well trained technicians to perform GeneReach has developed PetNAD FHV Detection Kit based on iiPCR technology which significantly reduces reaction time and offers sensitivity and specificity comparable to those of conventional nested PCR Tsai 2012 Chang 2012 Furthermore this simple and easy assay is completed rapidly in a portable POCKIT Nucleic Acid Analyzer PetNAD FHV Detection Kit PRINCIPLES OF THE PROCEDURE In iiPCR hydrolysis probe based chemistry is used to generate fluorescent signal during amplification of target DNA The primers and probe target thymidine kinase TK gene and do not cross react with nucleic acid from host and other major feline upper respiratory pathogens PRODUCT DESCRIPTION A Materials Provided 24 tests kit Component Contents or Purpose Premix Pack B FHV Premix lyophilized 24 bags 1 FHV pellet containing dNTPs
7. mix tube Note When the pellet is not found at the bottom of the tube spin tube briefly to bring it down 3 Add 50 ul Premix Buffer B to each Premix tube 4 Add 5 ul nucleic acid extract or P Control to each Premix tube Spin Premix tube for 10 seconds in a mini centrifuge such as cubeeTM 5 Transfer 50 ul Premix sample mixture into R tube 6 Seal top of each R tube with a cap Make sure R tube is capped tightly 7 Place R tube into the holder of POCKITTM 8 Spin tube briefly in cubee to make sure all solution is PetNAD FHV Detection Kit collected at the bottom of R tube Note Make sure there are no bubbles in the solution Note Start reaction within 1 hour to prevent nucleic acid degradation and non specific reaction 9 POCKIT reaction a Select 520 nm b When System READY is displayed place the holder with R tube s into the reaction chamber c Tap cap of each R tube to make sure the tube is positioned properly 10 Close lid and press Run to start reaction program 11 Test results are shown on the monitor after reaction is completed PetNAD FHV Detection Kit DATA INTERPRETATION One example of results shown on the monitor 520nm Interpretation FHV Positive FHV Negative Repeat reaction with freshly prepared nucleic acid ANYLYTICAL SENSITIVITY The detection limit of PetNADTM FHV Detection Kit is about 10 copies reaction
8. n to achieve optimal results E Vaccination with a modified live FHV vaccine may result in positive PCR results for a few weeks after vaccination Killed or vectored recombinant vaccines will not interfere with PCR testing PetNAD FHV Detection Kit PetNAD is recommended in sick animals with clinical signs and or laboratory abnormalities consistent with infection or in an animal with a suspected subclinical infection as based upon history physical examination and clinical laboratory findings PetNAD FHV Detection Kit PROCEDURE A PetNAD FHV Detection Kit Quick Guide Take one Premix Add 50 pil Premix Add 5 pl nucleic acid from Premix Pack Buffer extract then spin w Transfer 50 ul mixture into Spin R tube for 10 seconds R tube 4 5 H Results are shown on monitor in 1 hour cii Put R tube into Nucleic Acid Analyzer and press RUN 6 PetNAD FHV Detection Kit Procedure Note Before preparing the reactions for iiPCR testing turn on POCKIT to initiate the calibration for the instrument The device will complete self test within 5 minutes Please refer to the user manual of POCKIT for further details Note Before using for the first time add 100 ul P Control Buffer to P Control Store reconstituted P Control at 4 C 1 Label R tube s in the label area 2 Prepare one Premix for each sample Premix tube is in Premix Pack Each Premix Pack contains one Pre
9. rument taco M Automatic Nucleic Acid Extraction System Note Please follow the instruction manual of above extraction methods to obtain optimal results It is the user s responsibility to validate the combination of this reagent set with nucleic acids extracted by other methods for any particular application PRECAUTIONS A Do not open R tube s after reaction to prevent any carryover contamination B Perform extraction and amplification in two independent spaces to minimize contamination C Do not reuse R tube and Premix D Include the P Control to 1 Ensure POCKIT is working normally 2 Ensure detection kit performance after storage PetNAD FHV Detection Kit E To get optimal fluorescence detection 1 Wear powder free gloves to handle R tubes ji Labeling area 2 Do not label in the detection area Detection area of R tube LIMITATIONS A The test should be used only for testing nucleic acid extracted from animal specimens Do not add specimens e g whole blood directly into Premix B PetNAD Nucleic Acid Co prep Kit and taco mini Automatic Nucleic Acid Extraction System are recommended for nucleic acid extraction C Any deviations from the recommended procedure may lead to suboptimal results Quality of the extracts should be validated by the users D For PetNAD M FHV Detection Kit it is strongly recommended to use freshly prepared nucleic acid within 1 hour after extractio
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