Parvovirus Detection Kit User Manual
Contents
1. 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 Parvovirus 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 Parvovirus Detection Kit DATA INTERPRETATION One example of results shown on the monitor Pockit 1 0 16 28 57 17 08 2011 Reaction Completed 1 2 3 a 6 7 8 90009 90 Buzzer Off Interpretation B ranma rose O a Parvovirus Negative e Repeat reaction with freshly prepared nucleic acid ANYLYTICAL SENSITIVITY The detection limit of PetNAD Parvovirus Detection Kit is about 10 copies reaction 10 PetNAD Parvovirus Detection Kit TROUBLESHOOTING Problems Possible causes Solutions False Positive 1 Reuse of micro NW Micro centrifuge tubes tips centrifuge tubes R tubes and Premix are for tip2. PetNAD gt 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 2013 03 PetNAD Parvovirus Detection Kit Content INTENDED USE neoan n a 1 SUMMARY AND EXPLANATION sesesesesescororoseseseseseeeoecsosesesesess 1 PRINCIPLES OF THE PROCEDURE csccccsssssssssssssssessssesessers 2 PRODUCT DESCRIPTION iia aii 3 A Materials Provided isla ii ii 3 B Materials and Equipments Required but Not Provided oiciccco 3 C Storage and Stability ccccccsccssscscssssscscsssescsssssescsssssssssssssseesssssesesseees 4 De Sampe Pei iia 4 PRECAUTIONS Sua E EN AA 5 LIMITATIONS ensena ana ES 6 PROCEDURE ss OEE 7 A PetNAD Parvovirus Detection Kit Quick Guide oiiniinininninicno 7 Be Procedi o dacrsanciestuera Raiden cacacainds 8 DATA INTERPRETATION ococoncoconoononosnonosnonosnonasconasnoncsnoncsnocanoness 10 ANALYTICAL SENSITIVITY tii 10 TROUBLESHOOTING esesesessosososcsssesesessosososossesseseosososososessessese 11 REFERENCE unid 13 PetNAD Parvovirus Detection Kit INTENDED USE PetNAD Parvovirus Detection Kit is intended for in vitro detection of parvovirus DNA based on insulated isothermal polymerase chain reaction 1PCR technology This kit is designed specially to be used with
3. 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 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 PCR is one of the most commonly accepted methods that provide high PetNAD Parvovirus Detection Kit sensitivity and specificity for parvovirus detection However conventional PCR assays take three to four hours and require sophisticated thermocyclers and well trained technicians to perform GeneReach has developed PetNAD Parvovirus Detection Kit based on 11PCR 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 uPCR hydrolysis probe based chemistry is used to generate fluore
4. 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 3 1 Leakage or spill of Ml Consult with a GeneReach contamination 3 reaction from 3 technical support representative or of amplicons R tube into local distributor in reaction 3 reaction chamber chamber of of POCKIT POCKIT 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 Biotechnology Journal 7 5 662 666 doi 10 1002 biot 201100453 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 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 PCR for sensitive and specific on site pathogen detection PLoS ONE 7 9 e45278 doi 10 1371 jour
5. ion or in an animal with a suspected subclinical infection as based upon history physical examination and clinical laboratory findings PetNAD Parvovirus Detection Kit PROCEDURE A PetNAD Parvovirus Detection Kit Quick Guide Take Premix from Add 50 ul Premix Add 5 pl nucleic acid Premix Pack Buffer extract Mix by pipetting Spin R tube for 10 seconds Transfer 50 ul mixture into R tube 4 5 Results are shownon monitorin Lhour Poca 10 MIBAT 1908 7011 i y na 4 d a i a LA AT Completo 2 2 MIA O T Cd 90 E pl a Put R tube into POCKIT Positive Nucleic Acid Analyzer and press S Negative F Repeat assay using ads 6 freshly prepared 7 nucie ACIC PetNAD Parvovirus Detection Kit Procedure Note Before using for the first time add 100 ul 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 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
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7. s 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 E Disassemble and clean up 2 Contaminated micropipette micropipette E Use aerosol free tips 3 Contaminated Consult withaGeneReach reagent technical support representative or local distributor NW Consult with a GeneReach 1 Mf ym mt tytn ttt e 4 Contaminated technical support representative on I I I I I I working area I I I I I how to clean up working E 11 PetNAD Parvovirus Detection Kit Problems Possible causes Solutions False 1 Nucleic acid Consult manual of nucleic acid Negative extraction failed 3 extraction kit 2 Bad nucleic acid M Check sample storage condition quality or nucleic M Please refer to Troubleshooting acid concentration section of PetNADTM Nucleic too high 3 Acid Co prep Kit E If a spectrophotometer is available check OD 260 280 ratio This ratio should be between 1 4 and 2 0 AAA AAA AA A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A ee eee 3 PCR inhibition Do not overload nucleic acid 3 E Spike nucleic acid sample into P Standard reaction
8. scent signal during amplification of target DNA The primers and probe target the gene and do not cross react with nucleic acid from host and other canine pathogens PetNAD Parvovirus Detection Kit PRODUCT DESCRIPTION A Materials Provided 24 tests kit Premix Pack W Parvovirus Premix 24 bags 1 Parvovirus lyophilized pellet containing Premix vial and dNTPs primers probe and desiccating agent bag enzyme 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 oe parvovirus partial sequence Standard Buffer Reaction buffer to re dissolve 1 vial 110 ul vial P Standard 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 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 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 and rectal swab PetNAD Parvovirus Detec
9. tion 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 Al Label area E To get optimal fluorescence detection rr 1 Wear powder free gloves to handle R tubes 2 Do not label in the detection area of R tube Detection area PetNAD Parvovirus 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 optimal results and should be validated by the users D It is strongly recommended to use freshly prepared nucleic acid within hour after extraction to achieve optimal results with PetNAD PARVOVIRUS Detection Kit 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 infect
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