User Manual (201409)
Contents
1. PLANATION Feline immunodeficiency virus FIV is a lentivirus associated with AIDS like illness in cats Sodora et al 1994 Five subtypes A to F have been classified based on sequence diversity Madhu et al 2010 In the initial or acute phases FIV infection is often accompanied by mild symptoms such as fever lethargy anorexia pyrexia and Iymphadenopathy Janet et al 2010 FIV infections are not necessarily fatal Cats could live a relatively healthy life and serve as FIV carriers for years The primary routes of FIV transmission are through deep bite wounds and scratches where the infected cat s saliva enters the other cat s bloodstream American Association of Feline Practitioners 2002 FIV could also be transmitted vertically in uterus PCR is one of the most commonly accepted methods that provide high sensitivity and specificity for FIV 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 FIV 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 FIV Detection Kit PRINCIPLES OF THE PROCEDURE In iiPCR hydrolysis probe based ch2. PetNAD FIV Detection Kit For Feline Immunodeficiency Virus Types A B C E and F 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 FIV 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 sscsessosssseistsiscesssatevcsectasetatcessunssisvocensesbeocsnebesetatcedenenessie 6 PROCEDURE eceecs quic ette poti p eiue xix een oa osov o seseo ERN EE Era uid 7 A PetNAD FIV Detection Kit Quick Guide ess
3. SHOOTING 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 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 tps micropipette 3 Contaminated W Consult with a GeneReach reagent technical support representative or local distributor Feet dem EPSP E O 1 4 Contaminated i M Consult with a GeneReach working area i technical support representative on how to clean up working area 11 PetNAD FIV 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 W Do not overload PCR with too much 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 suppor
4. emistry is used to generate fluorescent signal during amplification of target RNA The primers and probe target the gag 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 FIV Premix lyophilized 24 bags 1 FIV pellet containing dNTPs Premix vial and 1 primers probe and enzyme desiccating for amplification agent bag E Desiccatng agent pack Premix Buffer M Reaction buffer to re dissolve 2 vials 1 3 ml vial AO the lyophilized pellet P Control E Dried plasmid containing FIV 1 vial partial sequence as positive control P Control a Reaction buffer to re dissolve 1 vial 110 ul vial Buffer P Control R tube 1 bag 24 pieces bag PetNAD FIV Detection Kit Cap 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 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 stated on the label 2 Store Premix vials in sealed Premix Pack to avoid hydration of lyo
5. es 7 Bi Procedure sns oue genu oa In ue Rees 8 DATA INTERPRETATION eeeeeee teen tn stessa tn setas tn atn a eta an 10 PetNAD FIV Detection Kit ANALYTICAL SENSITIVITY occisis raso sooo reae atro ao srt uod 10 TROUBLESHOOTING eteren esseri argen been sees xu eC E co SE REV Un ERU UE 11 30 0d DL Dior 13 PetNAD FIV Detection Kit INTENDED USE PetNAD FIV Detection Kit is intended for in vitro detection of the Feline Immunodeficiency Virus RNA 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 FIV Detection Kit SUMMARY AND EX
6. om of the tube spin tube briefly to bring it down 3 Add 50 ul Premix Buffer AO 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 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 FIV 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 FIV Detection Kit DATA INTERPRETATION One example of results shown on the monitor 520 nm Interpretation FIV Positive FIV Negative Repeat reaction with freshly prepared nucleic acid ANYLYTICAL SENSITIVITY The detection limit of PetNADTM FIV Detection Kit is about 10 copies reaction 10 PetNAD FIV Detection Kit TROUBLE
7. philized 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 saliva or mucosa swab PetNAD FIV Detection Kit RECOMMENDED NUCLEIC ACID EXTRACTION METHODS A PetNAD Nucleic Acid Co prep Kit taco DNA RNA 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 FIV Detection Kit E To get optimal fluorescence detection M 1 Wear powder free gloves to SI handle R tubes T 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 Premi
8. t representative or of amplicons tube into reaction local distributor in reaction chamber of chamber of POCKIT POCKIT PetNAD FIV Detection Kit REFERENCE 1 American Association of Feline Practitioners 2002 Feline Immunodeficiency Virus Cornell Feline Health Center Cornell University College of Veterinary Medicine Retrieved 2008 11 12 2 Chang H E 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 3 Janet Y Sanou M Abbott J and Coleman J 2010 Feline immunodeficiency virus model for designing HIV AIDS vaccines Current HIV Research 8 14 25 4 Sodora D L Shpaer E G Kitchell B E Dow S W Hoover E A and Mullins J I 1994 Identification of three feline immunodeficiency virus FIV env gene subtypes and comparison of the FIV and human immunodeficiency virus type 1 evolutionary patterns J Virol April 1994 vol 68 no 4 2230 2238 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 iiPCR for sensitive and specific on site pathogen detection PLoS ONE 7 9 e45278 doi 10 1371 journal pone 0045278
9. x 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 FIV Detection Kit it is strongly recommended to use freshly prepared nucleic acid within 1 hour after extraction to achieve optimal results PetNAD FIV Detection Kit PROCEDURE A PetNAD FIV 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 E c Put R tube into Nucleic Acid Analyzer and press RUN 6 PetNAD FIV 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 bott
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