User Manual PetNAD
Contents
1. PetNAD FCV Detection Kit For Feline calicivirus 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 10 PetNAD FCV 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 cosa OEIS 7 A PetNAD FCV Detection Kit Quick Guid ocoicinnonininnininnnnicinncnss 7 Be Procedi o dacrsanciestuera Raiden cacacainds 8 DATA INTERPRETATION ococoncoconoononosnonosnonosnonasconasnoncsnoncsnocanoness 10 ANALYTICAL SENSITIVITY tii 10 TROUBLESHOOTING esesesessosososcsssesesessosososossesseseosososososessessese 11 REFERENCE unid 13 PetNAD FCV Detection Kit INTENDED USE PetNAD FCV Detection Kit is intended for in vitro detection of FCV RNA based on insulated isothermal polymerase chain reaction PCR technology This kit is designed specially to be used with an iiPCR compat2. URE In uPCR hydrolysis probe based chemistry is used to generate fluorescent signal during amplification of target RNA The primers and probe target ORFI gene and do not cross react with nucleic acid from host and other feline upper respiratory pathogens PetNAD FCV Detection Kit PRODUCT DESCRIPTION A Materials Provided 24 tests kit Premix Pack W FFCV Premix lyophilized 24 bags 1 FCV 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 oe FCV 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 FCV 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 s
3. ed 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 FCV 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 FCV 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 Oo FCV Positive a FCV Negative e Repeat reaction with freshly prepared nucleic acid ANYLYTICAL SENSITIVITY The detection limit of PetNAD FCV Detection Kit is about 10 copies reaction 10 PetNAD FCV 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 tips R tubes and single use only Reus
4. ible 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 Feline calicivirus FCV is highly contagious and widespread in the general cat population The virus is shed predominantly in oral and nasal secretions in acute phase when cats show symptoms including fever conjunctivitis nasal discharge sneezing and oral ulceration During recovery many cats continue shedding until at least 30 days post infection only a few for several years Wardley 1976 Disease prevalence is proportional to the number of cats in the household with the highest prevalence usually seen where large groups are PetNAD FCV Detection Kit accommodated together Wardley et al 1974 PCR is one of the most commonly accepted methods that provide high sensitivity and specificity for FCV detection However conventional PCR assays take three to four hours and require sophisticated thermocyclers and well trained technicians to perform GeneReach has developed PetNAD FCV 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 could be completed rapidly in a portable POCKIT Nucleic Acid Analyzer PRINCIPLES OF THE PROCED
5. ing 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 FCV 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 E Please refer to Troubleshooting acid concentration section of PetNAD Nucleic Acid too high 3 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 AA AAA 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 A ee ee eee eee eo 3 PCR inhibition Do not overload nucleic acid 3 NW Spike nucleic acid sample into P Standard reaction for a parallel PCR reaction Negative res
6. tory abnormalities consistent with infection or in an animal with a suspected subclinical infection as based upon history physical examination and clinical laboratory findings PetNAD FCV Detection Kit W Take Premix from Add 50 ul Premix Add 5 pl nucleic acid Premix Pack Buffer extract PROCEDURE A PetNAD FCV Detection Kit Quick Guide Mix by pipetting Spin R tube for 10 seconds Transfer 50 ul mixture into R tube 4 5 Results are shownon monitor in 1 hour Poca 10 162057 1908 7011 Completo o NY Se aA 00 ivane ue Om A A o d gt p i r LA A Put R tube into POCKIT O Positive Nucleic Acid Analyzer and press 0 Negative F Repeat assay using RUN 6 freshly prepared i 5 E PetNAD FCV 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 capp
7. ults 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 FCV 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 De 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 journal pone 0045278 3 Wardley RC 1976 Feline calicivirus carrier state A study of host virus relationship Arch Virol 52 243 249 4 Wardley RC Gaskell RM Povey RC 1974 Feline respiratory viruses their prevalence in clinically healthy cats J Small Anim Pract 15 579 586 13
8. wab sample i e oropharyngeal conjunctival and nasal swab PetNAD FCV Detection Kit PRECAUTIONS A Do not open R tube s after reaction to prevent any carryover contamination Perform extraction and amplification in two independent spaces to minimize contamination Do not reuse R tube and Premix Include the P Standard to 1 Ensure POCKIT Nucleic Acid Analyzer is working normally 2 Ensure detection kit performance after storage To get optimal fluorescence detection 1 Wear powder free gloves to handle Al abetos R tubes Sl 2 Do not label in the detection area of R Aibe Detection area PetNAD FCV 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 1 hour after extraction to achieve optimal results with PetNAD FCV Detection Kit E Vaccination with a modified live FCV 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 labora
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