User Manual PetNAD
Contents
1. PetNAD FeLV 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 pap Dunham S P and Graham E 2008 Retroviral infections of small animals Veterinary Clinics of North America Small Animal Practice 38 4 879 901 3 Gomes Keller M A G nczi E and Tandon R et al 2006 Detection of feline leukemia virus RNA in saliva from naturally infected cats and correlation of PCR results with those of current diagnostic methods J Clin Microbiol 44 916 922 4 Kapil S and Lamm C G 2008 Emergency and Reemerging Viruses of Dogs and Cats Veterinary Clinics of North America Small Animal Practice 38 4 x111 X1V 5 Levy J Crawford C Hartmann K Hofman Lehmann R Little S Sundahl E and Thayer V 2008 American Association of Feline Practitioners feline retrovirus management guidelines Journal of Feline Medicine and Surgery 10 300 316 6 Hartmann K 2009 Feline leukemia virus and feline immunodeficiency virus Current Veterinary Therapy XIV W B Saunders Co Philadelphia PA 1278 1283 7 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 iPCR for Sensitive andSpecific On Site Path2. 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 I working area I I I I I how to clean up working E 11 PetNAD FeLV 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 nucleic 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
3. 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 FeLV 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 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 POC
4. KIT which should complete PetNAD FeLV 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 FeLV 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 Oo FeLV Positive a FeLV Negative e Repeat reaction with freshly prepared nucleic acid ANYLYTICAL SENSITIVITY The detection limit of PetNAD FeLV Detection Kit is about 10 copies reaction 10 PetNAD FeLV 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 cross 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
5. PetNAD FeLV Detection Kit For Feline Leukemia Virus 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 03 PetNAD FeLV 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 cosas T OES 7 A PetNAD FeLV Detection Kit Quick Guide coicininnininnininnnnicinncnss 7 Bi Procedi eddie 8 DATA INTERPRETATION ococoncoconoononosnonosnonosnonasconasnoncsnoncsnocanoness 10 ANALYTICAL SENSITIVITY tii 10 TROUBLESHOOTING esesesessosososcsssesesessosososossesseseosososososessessese 11 REFERENCE unid 13 PetNAD FeLV Detection Kit INTENDED USE PetNAD FeLV Detection Kit is intended for in vitro detection of Feline Leukemia Virus FeLV RNA based on insulated isothermal polymerase chain reaction PCR technology This kit is designed specially to be used with an wuPCR com
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7. orm extraction and amplification in two independent spaces to minimize contamination Do not reuse R tube and Premix Include the P Standard to 1 Ensure POCKIT Nucleic Acid Analyzer is working normally 2 Ensure detection kit performance after storage A Label area Detection area To get optimal fluorescence detection 1 Wear powder free gloves to handle R tubes q 2 Do not label in the detection area of R tube A __ i PetNAD FeLV 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 hour after extraction to achieve optimal results with PetNAD FeLV Detection Kit E Vaccination with a modified live FeLV 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 FeLV Detection Kit W Take Premix from Add 50 ul Premix Add 5 pl nucleic acid Premix Pack Buffer extract PROCEDURE A PetNAD FeLV Detection Kit Quick Guide Mix by pipetting Spin R
8. patible 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 SUMMARY AND EXPLANATION FeLV is a retrovirus that causes the most common fatal disease in cats Large amounts of virus are excreted in FeLV infected cat saliva Transmission of virus is mainly through cat fights biting and or mutual grooming FeLV could also be transmitted vertically during pregnancy delivery or postpartum Clinical signs of the disease vary widely including loss of appetite fever weight loss pale mucous membranes and enlarged lymph nodes PCR is one of the most commonly accepted methods that provide high sensitivity and specificity for FeLV detection However conventional PetNAD FeLV Detection Kit PCR assays take three to four hours and require sophisticated thermocyclers and well trained technicians to perform GeneReach has developed PetNAD FeLV 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 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 RNA The primers and
9. probe target LTR gene and do not cross react with nucleic acid from host and other feline pathogens PetNAD FeLV Detection Kit PRODUCT DESCRIPTION A Materials Provided 24 tests kit Premix Pack E FeLV Premix lyophilized 24 bags 1 FeLV 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 FeLV 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 FeLV 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 of nucleic acid D Sample Type Nucleic acid extracted from whole blood PetNAD FeLV Detection Kit PRECAUTIONS A Do not open R tube s after reaction to prevent any carryover contamination Perf
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