User Manual PetNAD
Contents
1. D FHV Detection Kit 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 Standard to 1 Ensure POCKIT Nucleic Acid Analyzer is working normally 2 Ensure detection kit performance after storage E To get optimal fluorescence detection 1 Wear powder free gloves to handle a R tubes Label area 2 Do not label in the detection area of R tube i Detection area PetNAD FHV Detection Kit LIMITATIONS A The test should be used only for testing nucleic acid extracted from animal specimen Do not add specimen i e whole blood directly into Premix B PetNAD Nucleic Acid Co prep Kit is recommended for nucleic acid extraction C Any deviation from recommended procedure may not achieve the optimal results and should be validated by the users D It is strongly recommended to use freshly prepared nucleic acid within 1 hour after extraction to achieve optimal results with PetNAD FHV Detection Kit 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 is recommended in sick animals with clinical signs and or laboratory abnormalities consistent with infection or2. 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 one Rd Central Taiwan Science Park Taichung City Taiwan 407 Web Site www petnad com 2013 06 PetNAD FHV Detection Kit Content INTENDED USE neoan n a 1 SUMMARY AND EXPLANATION sesesesesescororoseseseseseeeoecsosesesesess 1 PRINCIPLES OF THE PROCEDURE csccccsssssssssssssssessssesessers 2 PRODUCT DESCRIPTION iia aii 3 A Materials Provided isla ii ii 3 B Materials and Equipments Required but Not Provided oiciccco 3 C Storage and Stability ccccccsccssscscssssscscsssescsssssescsssssssssssssseesssssesesseees 4 De Sampe Pei iia 4 PRECAUTIONS Sua E EN AA 5 LIMITATIONS ensena ana ES 6 PROCEDURE sra OEIS 7 A PetNAD FHV Detection Kit Quick Guide ocinicinnonininnininnonicinncnos 7 Be Procedi o dacrsanciestuera Raiden cacacainds 8 DATA INTERPRETATION ococoncoconoononosnonosnonosnonasconasnoncsnoncsnocanoness 10 ANALYTICAL SENSITIVITY tii 10 TROUBLESHOOTING esesesessosososcsssesesessosososossesseseosososososessessese 11 REFERENCE unid 13 PetNAD FHV Detection Kit INTENDED USE PetNAD FHV Detection Kit is intended for in vitro detection of feline herpes virus 1 FHV 1 DNA based on insulated isothermal polymerase chain reaction PCR technology This kit is designed specially to
3. be used with an wuPCR compatible instrument POCKIT Nucleic Acid Analyzer The assay is intended for use by people with basic laboratory skills This kit is intended for research use only SUMMARY AND EXPLANATION FHV 1 an alphaherpesvirus causes mainly keratoconjunctivitis and rhinitis Povey 1979 that lead to mortality exceeding 60 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 PetNAD FHV Detection Kit sensitivity and specificity for FHV detection However conventional PCR assays take three to four hours and require sophisticated thermocyclers and well trained technicians to perform GeneReach has developed PetNAD FHV Detection Kit based on PCR technology which significantly reduces reaction time and offers sensitivity and specificity comparables to those of conventional nested PCR Tsai 2012 Chang 2012 Furthermore this simple and easy assay could be completed rapidly in a portable POCKIT Nucleic Acid Analyzer PRINCIPLES OF THE PROCEDURE In uPCR hydrolysis probe based chemistry is used to generate fluorescent signal during amplification of target DNA The
4. eic acid In that case prepare another nucleic acid extract Heavy 3 1 Leakage or spill of Ml Consult with a GeneReach contamination 3 reaction from 3 technical support representative or of amplicons R tube into local distributor in reaction 3 reaction chamber chamber of 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 De 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 F 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 13
5. in an animal with a suspected subclinical infection as based upon history physical examination and clinical laboratory findings PetNAD FHV Detection Kit W Take Premix from Add 50 ul Premix Add 5 pl nucleic acid Premix Pack Buffer extract PROCEDURE A PetNAD FHV Detection Kit Quick Guide Mix by pipetting Spin R tube for 10 seconds Transfer 50 ul mixture into R tube 4 5 Results are shownon monitor in 1 hour Poca 10 162057 1908 7011 Completo o NY Se aA 00 ivane ue Om A A o d gt p i r LA A Put R tube into POCKIT O Positive Nucleic Acid Analyzer and press 0 Negative F Repeat assay using RUN 6 freshly prepared i 5 E PetNAD FHV Detection Kit Procedure Note Before using for the first time add 100 ul Standard Buffer to P Standard Store reconstituted P Standard 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 Premix Note If 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 Standard to each Premix tube Mix by pipetting up and down 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
6. of POCKIT 8 Spin tube briefly in cubee to make sure all solution is collected at the bottom of R tube Note Start reaction within 1 hour to prevent nucleic acid degradation Note Make sure there are no bubbles in the tube 9 POCKIT reaction Note Please see the user manual of POCKIT for details a Turn on POCKIT which should complete PetNAD FHV Detection Kit self testing within 5 minutes b Select 520 nm c When System READY is displayed place the holder with R tube s into the reaction chamber d 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 Pockit 1 0 16 28 57 17 08 2011 Reaction Completed 1 2 3 a 6 7 8 90009 90 Buzzer Off Q FHV Positive a FHV Negative e Repeat reaction with freshly prepared nucleic acid ANYLYTICAL SENSITIVITY The detection limit of PetNAD FHV Detection Kit is about 10 copies reaction 10 PetNAD FHV Detection Kit TROUBLESHOOTING Problems Possible causes Solutions False Positive 1 Reuse of micro NW Micro centrifuge tubes tips centrifuge tubes R tubes and Premix are for tips R tubes and single use only Reusing these Premix accessories would cause cros
7. 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 PetNAD FHV Detection Kit PRODUCT DESCRIPTION A Materials Provided 24 tests kit Premix Pack W FHV Premix lyophilized 24 bags 1 FHV Premix pellet containing dNTPs vial and desiccating primers probe and enzyme agent bag for amplification Desiccating agent pack Premix Buffer B Reaction buffer to re dissolve 2 vials 1 3 ml vial the lyophilized pellet P Standard Dried plasmid containing oe FHV partial sequence Standard Buffer Reaction buffer to re dissolve 1 vial 110 ul vial P Standard B Materials and Equipments Required but Not Provided 1 PetNAD Nucleic Acid Co prep Kit 2 POCKIT Nucleic Acid Analyzer PetNAD compatible instrument 3 cubee Mini Centrifuge cubee 4 Micropipette and tips PetNAD FHV Detection Kit C Storage and Stability 1 The kit should be stored at 4 C and is stable until the expiration date which is stated on the label 2 Store Premix vials in sealed Premix Pack to avoid hydration of lyophilized components 3 Reconstituted P Standard is stable for 6 months at 4 C Aliquot reconstituted P Standard to avoid degradation and contamination of nucleic acid D Sample Type Nucleic acid extracted from whole blood or swab sample i e oropharyngeal conjunctival and nasal swab PetNA
8. s contamination E 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 prevent cross contamination E Disassemble and clean up 2 Contaminated micropipette micropipette E Use aerosol free tips 3 Contaminated Consult withaGeneReach reagent technical support representative or local distributor NW Consult with a GeneReach 1 Mf ym mt tytn ttt e 4 Contaminated technical support representative on I I I I I working area i I I I how to clean up working area S S SS 11 PetNAD FHV Detection Kit Problems Possible causes Solutions False 1 Nucleic acid Consult manual of nucleic acid Negative extraction failed 3 extraction kit 2 Bad nucleic acid ll Check sample storage condition quality or nucleic E Please refer to Troubleshooting acid concentration section of PetNAD Nucleic Acid too high 3 Co prep Kit E If a spectrophotometer is available check OD 260 280 ratio This ratio should be between 1 4 and 2 0 AAA AAA AA A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A ee eee 3 PCR inhibition Do not overload nucleic acid 3 E Spike nucleic acid sample into P Standard reaction for a parallel PCR reaction Negative results indicate the presence of inhibitors in the nucl
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