User Manual (201409)
Contents
1. 10 seconds in a mini centrifuge such as cubee 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 PetNAD FeLV 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 FeLV Detection Kit DATA INTERPRETATION One example of results shown on the monitor 520 nm Interpretation FeLV Positive FeLV Negative Repeat reaction with freshly prepared nucleic acid ANYLYTICAL SENSITIVITY The detection limit of PetNADTM FeLV Detection Kit is about 10 copies reaction PetNAD FeLV 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 and2. lyophilized 24 bags 1 FeLV pellet containing dNTPs Premix vial and 1 primers probe and enzyme desiccating for amplification agent bag E Desiccatng agent pack Premix Buffer B Reaction buffer to re dissolve 2 vials 1 3 ml vial B the lyophilized pellet P Control B Dried plasmid containing vial FeLV partial sequence as positive control P Control a Reaction buffer to re dissolve 1 vial 110 pl vial Buffer P Control R tube 1 bag 24 pieces bag PetNAD FeLV Detection Kit Cap 1 bag 24 pieces bag User Manual 1 copy B Materials and Equipments 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 cubee 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 which is 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 RNA extracted from whole blood PetNAD FeLV Detection Kit RECOMMENDED NUCLEIC ACID EXTRACTION METHODS A PetNAD Nucleic Acid Co prep Kit taco DNA RNA
3. only Reusing these accessories Premix would cause cross contamination and therefore false positive results 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 l i prevent cross contamination 2 Contaminated M Use aerosol free tips SSS micropipette 3 Contaminated W Consult with a GeneReach reagent technical support representative or local distributor Feet dem 1 4 Contaminated i M Consult with a GeneReach working area i technical support representative on how to clean up working area 11 PetNAD FeLV Detection Kit Problems Possible causes t Solutions False i 1 Nucleic acid B Consult manual of nucleic acid Negative extraction failed extraction kit 2 PCR inhibition E Do not overload PCR with too much of nucleic acid E 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 I Leakage or spill of TW Consult with a GeneReach a contamination reaction from R technical support representative or of amplicons tube into reaction local distributor in reaction chamber of chamber of POCKIT POCKIT PetNAD FeLV Detection Kit REFERENCE 1 Chang H F
4. pene 8 DATA INTERPRETATION eeeeeeee enne etes ento setas tn tns tao 10 PetNAD FeLV Detection Kit ANALYTICAL SENSITIVITY scissossossessssstassvscnosssstevescsessensessstvenecs 10 TROUBLESHOOTING eteren esseri argen been sees xu eC E co SE REV Un ERU UE 11 30 0d DL Dior 13 PetNAD FeLV Detection Kit INTENDED USE PetNAD FeLV Detection Kit is intended for in vitro detection of the feline leukemia virus FeLV RNA based on insulated isothermal polymerase chain reaction iiPCR technology This kit is designed specially to be used with an 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 FeLV Detection Kit SUMMARY AND EXPLANATION FeLV is a retrovirus that causes feline leu
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6. Extraction Kit compatible instrument taco 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 FeLV 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 recommended procedure may lead to suboptimal results Quality of the extracts should be validated by the users D For PetNAD FeLV Detection Kit it is strongly recommended to use freshly prepared nucle
7. 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 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 xiii xiv 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 EG Tsai C E Lin C K Teng P H Su C and Jeng C C 2012 Development of TaqMan Probe Based Insulated Isothermal PCR iiPCR for Sensitive andSpecific On Site Pathogen Detection PLoS ONE 7 9 e45278 doi 10 1371 journal pone 004
8. 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 2 Road Central Taiwan Science Park Taichung City Taiwan 407 Web Site www petnad com 2014 09 PetNAD FeLV Detection Kit Content INTENDED USE ssccssssssscesccnscccccsscnscsecsscsssssscescsssseneesesssseneeeoes 1 SCIENTIFIC MEANINGS ccsccsssssssscsscessesscssccsscsessscessessesseseeees 1 SUMMARY AND EXPLANATION cccssssssssscsssessescessesserscsseees 2 PRINCIPLES OF THE PROCEDURE cese reset tn nennt 3 PRODUCT DESCRIPTION ceres eene tne n enne tn stesse natuss tnus 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 tire OUR 4 RECOMMENDED NUCLEIC ACID EXTRACTION METHODS sabssedudeusasedtsnescesteussvesssansusnsasesuassassssdecsssesussasedesdausavecessasesseses PEE UER AERA PER Un 5 Ld 0 89 VO M Jo gen et rors eseis reet rinasa 5 LIMITATIONS i gesiecsvao coe ea bp cin spes ev o Pee R ES e Sea a Ce PE PP eden seb cV agregue nn 6 PROCEDURE iere ep ren o a pk eadera ir osson os suvesse soro URN EE Era RR 7 A PetNAD FeLV Detection Kit Quick Guide sss 7 Bi Procedure eo nsu eeu eue
9. ic acid within 1 hour after extraction to achieve optimal results E Vaccination with a modified live FeLV vaccine may result in positive PCR results for a few weeks after vaccination Killed vaccines will not interfere with PCR testing interpretation PetNAD FeLV Detection Kit PROCEDURE A PetNAD FeLV 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 pil 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 FeLV 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 Premix 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. kemia one of the most common fatal diseases in cats Large amounts of virus are excreted in saliva of FeLV infected cat 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 PCR assays could 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 is completed rapidly in a portable POCKIT Nucleic Acid Analyzer PetNAD FeLV Detection Kit PRINCIPLES OF THE PROCEDURE In iiPCR hydrolysis probe based chemistry is used to generate fluorescent signal during amplification of target RNA The primers and probe target the LTR gene and do not cross react with nucleic acid from host and other feline pathogens PRODUCT DESCRIPTION A Materials Provided 24 tests kit Component Contents or Purpose Amount Premix Pack E FeLV Premix
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