User Manual PetNAD
Contents
1. ANYLYTICAL SENSITIVITY The detection limit of PetNAD CDV Detection Kit is up to 10 copies reaction 10 TROUBLESHOOTING PetNAD CDV Detection Kit Observation or Problems False Positive Possible Causes 1 2 3 4 The reuse of micro centrifuge tubes tips R tubes and Premix Micropipette contaminated Reagent contaminated Working area contaminated 11 Comment and Suggestions The micro centrifuge tubes tips R tubes and Premix are for one time use only Reuse of these accessories will cause contamination Once used the micro centrifuge tubes tips R tubes and Premix should be collected and discarded according to the local regulation Do not place the waste close to the working area to prevent contamination Disassemble pipette and do clean up We recommend using aerosol free tips Consult with GeneReach or local distributor Consult with GeneReach for working area clean up Observation or Problems False Negative Possible Causes 1 Nucleic acid extraction failed PetNAD CDV Detection Kit Comment and Suggestions 2 Bad nucleic acid quality or nucleic acid concentration too high 3 PCR inhibition 12 E Check nucleic acid extraction procedure Please check the sample storage condition Please refer to the Troubleshooting section of PetNAD Nucleic Acid Co prep Kit If a spectrophotometer is available check2. PetNAD Canine Distemper Virus CDV Detection Kit is intended for in vitro detection of CDV RNA based on the insulated isothermal polymerase chain reaction PCR technology This kit is specially designed to be used with a compatible iiPCR instrument POCKIT Nucleic Acid Analyzer The intended user of this kit is veterinarians or lab technicians who have basic laboratory skills This kit is intended for research use 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 PetNAD CDV Detection Kit may be useful but poses a problem in young puppies due to uncertainty caused by maternal antibody interference CDV detection by PCR is the most sensitive and specific method However conventional PCR takes three to four hours and requires delicate machines as well as well trained technicians to perform the test GeneReach has developed PetNAD CDV Detection Kit based on PCR technology which significantly reduces the reaction time and is as sensitive and specific as the convent
3. Kit LIMITATIONS A The test should only be used for testing nucleic acid extracted from animal specimen Do not add specimen i e whole blood directly into the 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 Freshly prepared nucleic acid samples within 1 hour after extraction are strongly recommended to be used for PetNAD CDV Detection Kit to achieve optimal results PetNAD CDV Detection Kit OPERATION PROCEDURE A PetNAD CDV Detection Kit Quick Guide V Open the Add 50 ul Add5 pl Premix Pack Premix nucleic and take out Buffer B acid thePremix 1 2 extract Mix by pipetting Transfer 50 ul mixture into the R tube A Result will show within 1 hour Spin the R tubes for 10 secs 5 Put the R tube Disease Positive into the POCKIT Disease Negative and press RUN 6 Use fresh sample and repeat the test PetNAD CDV Detection Kit Procedure Note Please dissolve the P Standard by 100 ul Standard Buffer at first time use The dissolved P Standard should be stored at 4 C 1 Open the Premix Pack according to the sample number and take out the Premix Note If the pellet is not at the bottom please spin it down 2 Open the cap add 50 ul Premix Buffer B into each Premix tube 3 Add 5 ul nucleic aci
4. OD 260 280 ratio Normally this ratio should be 1 4 to 2 0 Do not add too much nucleic acid Please follow the operation procedure Spike P Standard for a parallel PCR reaction If the one with P Standard showed positive then the inhibition was ruled out If P Standard was negative then there was inhibition User need to prepare another nucleic acid extraction PetNAD CDV Detection Kit Observation or Possible Causes Comment and Suggestions Problems Solution or other R tube broken or E Consult with GeneReach or interferences fall solution spilled in the your local distributor into the reaction reaction chamber of chamber of POCKIT POCKIT 13 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 Bennett D 1991 Canine distemper viral antigens and antibodies in dogs with rheumatoid arthritis Res Vet Sci 50 64 68 3 Iwatsuki K Miyashita N Yoshida E Gemma T Shin Y S Mori T Hirayama N Kai C Mikami T 1997 Molecular and phylogenetic analyses of the haemagglutinin proteins of field isolates of canine distemper virus from naturally infected dogs Journal of General Virology 78 373 380 4 Saito T B Alfieri A A Wosiacki S R Negrao FJ Morais H S Alfieri A F 2006 Detection of canine distemper virus by reverse transcriptase polymerase chain reaction in the
5. 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 one Rd Central Taiwan Science Park Taichung City Taiwan 407 Web Site www petnad com 2011 11 PetNAD CDV Detection Kit Content INTENDED USE sesseneneeenenennenennesenenenesenenesses 1 SUMMARY AND EXPLANATION ssssemennsesenense 1 PRINCIPLES OF THE PROCEDURE csssssssessessseesessenseeeee 2 PRODUCT DESCRIPTION ssccscsssssscscsssssssssesscsessensesssesseneeseee 3 Ay Mat rials Provided 2 5525 fie saab dna Minime 3 B Materials and Equipments Required but Not Provided s ssessseseseee 3 C Storage and Stability ss 4 D Sample TYPE dant me Ar en 4 PRECAUTIONS senneenennnenenneneneneneeneneses 5 LIMITATIONS ssssennnenennenenneseeeneseeneneses 6 OPERATION PROCEDURE cscsssssssssessssssessesssssssscsssesscrscsseees 7 A PetNAD CDV Detection Kit Quick Guide c cc cccceesesseeeeeeeseeeeees 7 Be Proc dure 252 frs a Aisi nr ne de E tent oes 8 DATA INTERPRETATION ssesenneennesenenense 10 ANALYTICAL SENSITIVITY sssssessennsennersenenesse 10 TROUBLESHOOTING sseenssessenenneseneneneenenesenenesse 11 PetNAD CDV Detection Kit REFERENCE sis ccccaisessiuicacuscsusanbecsseasesebetsevestisisnausbesasebcsnebasetateevondiess 14 ii PetNAD CDV Detection Kit INTENDED USE
6. d extract or dissolved P Standard into each Premix tube Mix by pipetting up and down 4 Transfer 50 ul of the Premix mixture into the R tube 5 Cap the R tube put into the holder of POCKIT and use cubee to spin down the solution Note Please make sure all solution has been spun down to the bottom of the R tube Perform the following amplification reaction within 1 hour to prevent nucleic acid degradation Note Please make sure there is no bubble in the tube Please see the user manual of POCKIT for details 6 Turn on POCKIT The analyzer will complete self testing within 5 minutes Select 520 nm for use System READY will be displayed Note Please see the user manual of POCKIT for details 7 8 9 PetNAD CDV Detection Kit Place the holder containing the R tube s into the reaction chamber of POCKIT and tap the cap of each R tube to make sure the tube is properly positioned in the reaction chamber Close the lid and press Run to start the reaction program The test result will be shown on the screen after the reaction PetNAD CDV Detection Kit DATA INTERPRETATION Please check the results on the screen after the reaction For example from the screen Pockit 1 0 16 28 57 17 08 2011 Reaction Completed 1 2 3 4 5 6 7 8 2170000000 Buzzer off 520nm Interpretation CDV Positive CDV Negative O Recheck with fresh sample
7. ional PCR for CDV detection The assay has been simplified for easier and faster operation using compact equipments for CDV detection in the clinic PRINCIPLES OF THE PROCEDURE The assay is based on iiPCR In addition to specific primers fluorogenic probe hydrolysis chemistry is used to generate a fluorescent signal when specific CDV RNA is presented in samples The primers and probe target the nucleocapsid protein gene N gene specific to CDV and will not react with canine genomic DNA and nucleic acid of other pathogens PetNAD CDV Detection Kit PRODUCT DESCRIPTION A Materials Provided 24 tests kit Component Contents or Purpose Amount Premix Pack Each pack contains 1 pack of 1 zip lock bag desiccating agent and Premix containing 24 vial with a lyophilized pellet individually sealed containing dNTPs CDV specific packs primers fluorescent probes and enzyme Premix Buffer B Reaction buffer to re dissolve 700 l vial 2 vials the lyophilized pellet P Standard Dry plasmid pellet containing CDV partial sequence Standard Buffer Reaction buffer to re dissolve 110 pl vial 1 vial the CDV P Standard R tube 24 Pieces bag 1 bag Cap 24 Pieces bag 1 bag User Manual 1 B Materials and Equipments Required but Not Provided 1 PetNAD Nucleic Acid Co prep Kit 2 POCKIT the compatible instrument for PetNAD 3 cubee Mini centrifuge cubee 4 Microp
8. ipette 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 Premix vials should be kept in the sealed Premix Pack to avoid the rehydration of lyophilized pellet 3 Dissolved P Standard can be stored at 4 C for up to 6 months To avoid the degradation of P Standard it is recommended to aliquot the dissolved P Standard into several vials D Sample Type This kit is suitable for detecting nucleic acid extracted from whole blood urine and swab sample PetNAD CDV Detection Kit PRECAUTIONS A Do not open the R tube after the amplification reaction to prevent any carryover contamination We strongly recommend that the working area for extraction procedure and amplification procedure should be separated into two independent spaces to avoid any possible contamination Do not reuse the R tube and Premix The P Standard is used to 1 Confirm the operation procedure after installation or when any uncertain result has occurred 2 Ensure the kit performance after storage In order to get optimal fluorescence TE IN detection please wear powder free lape area gloves to handle the R tube and do not 7 mark and or label the detection area of Detection area the R tube The label area and detection area of the R tube are indicated as shown PetNAD CDV Detection
9. urine of dogs with clinical signs of distemper encephalitis Res Vet Sci 80 116 119 5 Vandevelde M Zurbriggen A 1995 The neuro biology of canine distemper virus infection Vet Microbiol 44 271 280 14
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