Parvovirus Detection Kit User Manual
Contents
1. 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 Micropipette and tips PetNAD Parvovirus 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 or rectal swab PetNAD Parvovirus Detection Kit PRECAUTIONS A Do not open the R tube after the amplification reaction to prevent any carryover contamination B 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 C 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 E In order to get optimal fluorescence detection please wear powder free glove2. extract 3 Mix by pipetting Transfer 50 ul mixture into the R tube A Result will show within 1 hour i ao 00000 so Put the R tube Disease Positive into the POCKIT Disease Negative and press RUN 5 Spin the R tubes for 10 secs 5 Use fresh sample b and repeat the test PetNAD Parvovirus 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 acid 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
3. 11 PetNAD Parvovirus Detection Kit REFERENCE eeter e steenen eneen aaea odd et ee eta 14 ii PetNAD Parvovirus Detection Kit INTENDED USE PetNAD Parvovirus Detection Kit is intended for in vitro detection of parvovirus DNA based on the insulated isothermal polymerase chain reaction iiPCR 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 Parvovirus is a highly contagious single strand DNA virus The virus is very stable in the environment able to withstand wide pH ranges and high temperature It is resistant to a number of common disinfectants and may survive for several months in contaminated areas It can be especially severe in puppies that are not protected by maternal antibodies or vaccination In the sever stage of infection dogs can die within 48 to 72 hours if not treated with fluid or antibiotics PetNAD Parvovirus Detection Kit Parvovirus 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 Parvovirus Detection Kit based on iiPCR technology which significantly reduces the reaction t
4. GeneReach or your local distributor PetNAD Parvovirus 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 Biotech J 7 662 666 2 Parrish C R 1995 Pathogenesis of feline panleukopenia virus and canine parvovirus Baillieres Clin Haematol 8 57 71 3 Pollock R V and Coyne M J 1993 Canine parvovirus Vet Clin North Am Small Anim Pract 23 555 568 4 Tijssen P 1999 Molecular and structural basis of the evolution of parvovirus tropism Acta Vet Hung 47 379 394 5 Truyen U 1994 Canine parvovirus recent knowledge of the origin and development of a viral pathogen Tierarztl Prax 22 579 584 14
5. PetNAD Parvovirus Detection Kit 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 2012 10 PetNAD Parvovirus Detection Kit Content INTENDED USE siscisdecicestatcnsssceccnscasnistouteccsedsusostuoescecsascesosssnuiscesensteen 1 SUMMARY AND EXPLANATION asen eeansoncenseorsonvenseereeovensensee 1 PRINCIPLES OF THE PROCEDURE aaan oor soncenseor vene 2 PRODUCT DESCRIPTION ssivisssssavessssesitutssicsevesssvostaebiestvesevnbetseave 3 Ay Materials Provided r a aa dna Aaa 3 B Materials and Equipments Required but Not Provided 3 C Storage and Stability ennen enveenvennerneerseernvennvennvenn 4 De Sample Type pe r r neee re ar a a Sa EE Sa e S SRS 4 PRECAUTIONS sssssesessescsssssososssssecversrosscecocsestserovo sssesereseocsesssesre isos 5 LIMITATIONS cacsustsctasesssncstessestuctacsvassustnssstonscassossesssustasensicunsuessbaiees 6 OPERATION PROCEDURE ease onsoovenoosvensonoononsescensesveneene 7 A PetNAD Parvovirus Detection Kit Quick Guide sees 7 B Procedure neend nne A A ee ae 8 DATA INTERPRETATION an sanvenveonoooveonceneooreencenseerensvennensee 10 ANALYTICAL SENSITIVITY ssasoosonseorensosvensoseononsencenseoreneene 10 TROUBLESHOOTING eneen ernversnersn eenn eeenvenn
6. ime and is as sensitive and specific as conventional PCR Chang H F G Tsai Y L Tsai C F Lin C K Lee P Y et al 2012 for parvovirus detection The assay has been simplified for easier and faster operation using compact equipments for parvovirus 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 parvovirus DNA is presented in samples The primers and probe target the gene specific to parvovirus sequences and will not react with canine and feline genomic DNA and nucleic acid of other pathogens PetNAD Parvovirus Detection Kit PRODUCT DESCRIPTION A Materials Provided 24 tests kit Component Premix Pack Contents or Purpose Each pack contains pack of desiccating agent and Premix vial with a lyophilized pellet containing dNTPs parvovirus specific primers fluorescent probes and enzyme Amount 1 zip lock bag containing 24 individually sealed packs Premix Buffer B Reaction buffer to re dissolve the lyophilized pellet 700 ul vial 2 vials P Standard Dry plasmid pellet containing parvovirus partial sequence Standard Buffer Reaction buffer to re dissolve the parvovirus P Standard 110 ul vial 1 vial R tube Cap User Manual 24 Pieces bag bag 24 Pieces bag bag
7. manual of POCKIT for details 7 8 9 PetNAD Parvovirus 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 Parvovirus Detection Kit DATA INTERPRETATION Please check the results on the screen after the reaction For example from the screen Pockit1 1 0202 16 28 57 17 08 2011 Reaction Completed 0000o o Buzzer Off 520nm Interpretation Parvovirus Positive 6 Parvovirus Negative Recheck with fresh sample ANALYTICAL SENSITIVITY The detection limit of PetNAD Parvovirus Detection Kit is up to 10 copies reaction 10 TROUBLESHOOTING PetNAD Parvovirus Detection Kit Observations 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 contamin ation Once used the micro centrifuge tubes tips R tubes and Premix should be c
8. ollected 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 Observations or Problems False Negative Possible Causes 1 Nucleic acid extraction failed 2 Bad nucleic acid quality or concentration too high 3 PCR inhibition 12 PetNAD Parvovirus Detection Kit Comment and Suggestions 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 check 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 Observations or Problems Solution or other interferences fall into the reaction chamber of POCKIT Possible Causes R tube broken or solution spilled in the reaction chamber of POCKIT 13 PetNAD Parvovirus Detection Kit Comment and Suggestions B Consult with
9. s and or label the detection area of the to handle the R tube and do not mark R tube The label area and detection area TI of the R tube are indicated as shown Label area Detection area PetNAD Parvovirus Detection 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 Parvovirus Detection Kit to achieve optimal results E Vaccination with a modified live parvovirus 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 Parvovirus Detection Kit OPERATION PROCEDURE A PetNAD Parvovirus Detection Kit Quick Guide Kad Y Open the Add 50 pl Add 5 pl Premix Pack Premix nucleic and take out Buffer B acid the Premix
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