User Manual PetNAD
Contents
1. 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 POCKIT which should complete PetNAD CDV 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 CDV Detection Kit DATA INTERPRETATION One example of results shown on the monitor Pockit 1 0 16 28 57 17 08 2011 Reaction Completed Su Se 3 A 5 6 7 8 QOOOO OO Buzzer Off 520nm Interpr2. section of PetNAD Nucleic Acid too high Co prep Kit W Ifa spectrophotometer is available check OD 260 280 ratio This ratio should be between 1 4 and 2 0 3 PCR inhibition W Do not overload nucleic acid 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 1 Leakage or spill of il Consult witha GeneReach contamination reaction from technical support representative or of amplicons R tube into local distributor in reaction reaction chamber chamber of of POCKIT POCKIT 12 PetNAD CDV Detection Kit REFERENCE 1 Appel M J G 1970 Distemper pathogenesis in dogs J Am Vet Med Assoc 156 1681 1684 2 Bell S C Carter S D and Bennett D 1991 Canine distemper viral antigens and antibodies in dogs with rheumatoid arthritis Res Vet Sci 50 64 68 3 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 4 Iwatsuki K Miyashita N Yoshida E Gemma T Shin Y S Mori T Hirayama N Kai C and Mikami T 1997 Molecular and phylogenetic analyses of the haemagglutinin proteins of field isolates of canine distemper vir
3. PetNAD CDV Detection Kit For Canine Distemper Virus User Manual For Research Use Only Manufacturer GeneReach Biotechnology Corporation TEL 886 4 24639869 FAX 886 4 24638255 No 19 Keyuan gn Rd Central Taiwan Science Park Taichung City Taiwan 407 Web Site www petnad com 2013 03 PetNAD CDV Detection Kit Content INTENDED USE 0 sscssscsssccssccnsccescsscnccsscescsssssscescsssseneesesssseseesees 1 SUMMARY AND EXPLANATION encres eene nennen nennen aetas 1 PRINCIPLES OF THE PROCEDURE eee eren eene tn nentnun 2 PRODUCT DESCRIPTION ceres eene seen en setas tss n stas ees anus 3 A Materials Provided senses eue de peel ederet 3 B Materials and Equipments Required but Not Provided 3 C Storage and Stability essere 4 D Sample Type sietes eh e A ae ee ie es 4 PRECAUTIONS 0 cssssssssssccsccnsscscessensssscescsssssscsscsssseneesesssseneesees 5 LIMITATIONS ootis 6 PROCEDURE eee eee eene enses enata sets sons tn seta seasons tastes sns tasses tnu 7 A PetNAD CDV Detection Kit Quick Guide 7 B Procedure i oon e aU eq ers 8 DATA INTERPRETATION eeeeeeee teens testen sensn seta sensns eta ao 10 ANALYTICAL SENSITIVITY eeeeeeee eee eene aetate tn natat 10 TROUBLESHOOTING eese sees eren enean sna tn aeta
4. e cubee 4 Micropipette and tips PetNAD CDV 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 urine and swab sample PetNAD CDV 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 2 Wear powder free gloves to handle R tubes EJ Label area Do not label in the detection area of R tube si Detection area PetNAD CDV 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 opti
5. etation CDV Positive CDV Negative Q Repeat reaction with freshly prepared nucleic acid ANYLYTICAL SENSITIVITY The detection limit of PetNAD CDV Detection Kit is about 10 copies reaction 10 PetNAD CDV Detection Kit TROUBLESHOOTING Problems i Possible causes i Solutions False Positive 1 Reuse of micro E 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 2 Contaminated W Disassemble and cleanup 7 micropipette micropipette E Use aerosol free tips 3 Contaminated M Consul with a GeneReach j reagent technical support representative or local distributor 4 Contaminated W Consult with a GeneReach j working area technical support representative on how to clean up working PetNAD CDV Detection Kit Problems i Possible causes i Solutions False i 1 Nucleic acid B Consult manual of nucleic acid Negative extraction failed extraction kit i 2 Bad nucleic acid W Check sample storage condition quality or nucleic E Please refer to Troubleshooting acid concentration
6. mal 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 CDV Detection Kit E Vaccination with a modified live CDV 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 or in an animal with a suspected subclinical infection as based upon history physical examination and clinical laboratory findings PetNAD CDV Detection Kit PROCEDURE A PetNAD CDV Detection Kit Quick Guide Take Premix from Add 50 pl Premix Add 5 pl nucleic acid Premix Pack Buffer extract 1 Mix by pipetting Spin R tube for 10 seconds Transfer 50 ul mixture into R tube 4 5 Results are shown on monitor in 1hour rario aeu n mu Ruaction Comgietw 00 o ame acim On Positive Negative Repeat assay using freshly prepa red 7 Put R tube into POCKIT Nucleic Acid Analyzer and press F RUN 6 PetNAD CDV Detection Kit Procedure Note Before using for the first time add 100 pl 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
7. on iiPCR 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 iiPCR hydrolysis probe based chemistry is used to generate fluorescent signal during amplification of target RNA The primers and probe target nucleocapsid protein gene N gene and do not cross react with nucleic acid from host and other canine pathogens PetNAD CDV Detection Kit PRODUCT DESCRIPTION A Materials Provided 24 tests kit Component Contents or Purpose Premix Pack E CDV Premix lyophilized 24 bags 1 CDV 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 1 vial CDV partial sequence Standard Buffer Reaction buffer to re dissolve 1 vial 110 pl vial P Standard R tube 1 bag 24 pieces bag 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 2 POCKIT Nucleic Acid Analyzer PetNAD compatible instrument 3 cubee Mini Centrifug
8. tes snae ta an 11 REFERENCE O Sebeos 13 PetNAD CDV Detection Kit INTENDED USE PetNAD CDV Detection Kit is intended for in vitro detection of CDV RNA based on insulated isothermal polymerase chain reaction PCR 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 SUMMARY AND EXPLANATION CDV is a single stranded RNA virus of the paramyxovirus family It is commonly seen in puppies of 3 to 6 months old and in young unvaccinated dogs Infection in dogs can result in subclinical infection gastrointestinal signs and or respiratory signs frequently with central nervous system CNS involvement high morbidity and mortality Early clinical diagnosis is difficult since the initial symptoms are indistinguishable from those of the kennel cough Serologic detection may be useful but poses a problem in young puppies due to uncertainty caused by maternal antibody interference PetNAD CDV Detection Kit PCR is one of the most commonly accepted methods that provide high sensitivity and specificity for CDV detection However conventional PCR assays take three to four hours and require sophisticated thermocyclers and well trained technicians to perform GeneReach has developed PetNAD CDV Detection Kit based
9. us from naturally infected dogs Journal of General Virology 18 373 380 5 Saito T B Alfieri A A Wosiacki S R Negrao F J Morais H S and Alfieri A F 2006 Detection of canine distemper virus by reverse transcriptase polymerase chain reaction in the urine of dogs with clinical signs of distemper encephalitis Res Vet Sci 80 116 119 6 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 7 Vandevelde M and Zurbriggen A 1995 The neuro biology of canine distemper virus infection Vet Microbiol 44 271 280 13
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