User Manual (201409)
Contents
1. 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 Hartmann K 2005 Feline infectious peritonitis Vet Clin North Am Small Anim Pract 2005 Jan 35 1 39 79 3 Lai MMC Perlman S Anderson L J 2007 Coronaviridae In Knipe DM Howley PM Griffin DE Lamb RA Martin MA Roizman B Straus SE eds Fields virology Lippincott Williams amp Wilkins Philadelphia PA 1305 1335 4 Pedersen N C Boyle J F Floyd K Fudge A and Barker J 1981 An enteric coronavirus infection of cats and its relationship to feline infectious peritonitis Am J Vet Res 42 368 377 5 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 00452782. Purpose Amount Premix Pack B FCoV Premix lyophilized 24 bags 1 FCoV pellet containing dNTPs Premix vial and 1 primers probe and enzyme desiccating for amplification agent bag E Desiccatng agent pack Premix Buffer B Reaction buffer to re dissolve 2 vials 1 3 ml vial B the lyophilized pellet P Control B Dried plasmid containing vial FCoV partial sequence as positive control P Control a Reaction buffer to re dissolve 1 vial 110 pl vial Buffer P Control R tube 1 bag 24 pieces bag PetNAD FCoV Detection Kit Cap 1 bag 24 pieces bag User Manual 1 copy B Materials and Equipment Required but Not Provided 1 PetNAD Nucleic Acid Co prep Kit or taco Automatic Nucleic Acid Extraction System 2 POCKIT M Nucleic Acid Analyzer POCKIT PetNADTM compatible instrument 3 cubee M Mini Centrifuge cubee 4 Micropipette and filter tips C Storage and Stability 1 The kit should be stored at 4 C and is stable until the expiration date stated on the label 2 Store Premix vials in sealed Premix Pack to avoid hydration of lyophilized components 3 Reconstituted P Control is stable for 6 months at 4 C Aliquot reconstituted P Control to avoid degradation of nucleic acid D Sample Type RNA extracted from whole blood rectal swab and pleural and abdominal effusion PetNAD FCoV Detection Kit RECOMMENDED NUCL
3. DATA INTERPRETATION eeeeeee eene testen ena tn setas tn atn s etas 10 PetNAD FCoV Detection Kit ANALYTICAL SENSITIVITY ssissovsossesssssvsssvssnossssvevesesessousesesdveness 10 TROUBLESHOOTING eteren esseri argen been sees xu eC E co SE REV Un ERU UE 11 30 0d DL Dior 13 PetNAD FCoV Detection Kit INTENDED USE PetNAD FCoV Detection Kit is intended for in vitro detection of the Feline Coronavirus RNA based on insulated isothermal polymerase chain reaction iiPCR technology This kit is designed specially to be used with an insulated isothermal PCR 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 SCIENTIFIC MEANINGS Antibody induced by vaccine or obtained from maternal immunity could lead to false positive interpretation in antibody based diagnostic procedures Detecting pathogen s nucleic acids not antibody PCR based methods can avoid the false positive results described above Furthermore with higher analytical sensitivity PCR can detect lower levels of viral signals than most if not all diagnostic methods It can reduce the chance of false negative results at early infection stage and shorten the window period between time of infection and detection PetNAD FCoV Detection Kit SUMMARY AND EXPLANATION FCoV is an important feline patho
4. EIC ACID EXTRACTION METHODS A PetNAD Nucleic Acid Co prep Kit taco DNA RNA Extraction Kit compatible instrument taco Automatic Nucleic Acid Extraction System Note Please follow the instruction manual of above extraction methods to obtain optimal results It is the user s responsibility to validate the combination of this reagent set with nucleic acids extracted by other methods for any particular application 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 Control to 1 Ensure POCKIT is working normally 2 Ensure detection kit performance after storage PetNAD FCoV Detection Kit E To get optimal fluorescence detection M 1 Wear powder free gloves to EA handle R tubes T Labeling area 2 Do not label in the detection area Detection area of R tube LIMITATIONS A The test should be used only for testing nucleic acid extracted from animal specimens Do not add specimens e g whole blood directly into Premix B PetNAD Nucleic Acid Co prep Kit and taco mini Automatic Nucleic Acid Extraction System are recommended for nucleic acid extraction C Any deviations from the recommended procedure may lead to suboptimal results Quality of the extracts should be validated by the u
5. PetNAD FCoV Detection Kit For Feline Coronavirus User Manual For Research Use Only Manufacturer GeneReach Biotechnology Corporation TEL 886 4 24639869 FAX 886 4 24638255 No 19 Keyuan 2 Road Central Taiwan Science Park Taichung City Taiwan 407 Web Site www petnad com 2014 09 PetNAD FCoV Detection Kit Content INTENDED USE 0 cscsssessssccsccnsecescsscncesecsscsssssscsscssssencesesssseseeeoes 1 SCIENTIFIC MEANINGS e eeeeee esee enses enata sesto seta statuae tans 1 SUMMARY AND EXPLANATION eene ener en een en stanno aeta 2 PRINCIPLES OF THE PROCEDURE cese reset tn nennt 3 PRODUCT DESCRIPTION ceres eene tne n enne tn stesse natuss tnus 3 A Materials Provided ap ea n eite cheeses 3 B Materials and Equipments Required but Not Provided 4 C Storage and Stability seisein niina oroi n E 4 D Sample Type eint EU tire OUR 4 RECOMMENDED NUCLEIC ACID EXTRACTION METHODS sabssedudeusasedtsnescesteussvesssansusnsasesuassassssdecsssesussasedesdausavecessasesseses PEE UER AERA PER Un 5 Ld 0 89 VO M Jo gen et rors eseis reet rinasa 5 LIMITATIONS i gesiecsvao coe ea bp cin spes ev o Pee R ES e Sea a Ce PE PP eden seb cV agregue nn 6 PROCEDURE ce dvi renati pk eadera iu een a pEPE EA PUNDN DR ERE Era uS 7 A PetNAD FCoV Detection Kit Quick Guide sues 7 Bi Procedure oues ug e ue Pee 8
6. gen world wide The enteric form of infection is limited to the gastrointestinal tract leading to asymptomatic infections mild enteritis or fatal infectious peritonitis in cats Pedersen et al 1981 FCoV an enveloped positive sense single stranded RNA virus Lai et al 2007 is highly contagious among cats by the fecal oral route The virus is typically shed in feces by healthy recovered cats and can survive in the environment for up to 7 weeks Hartmann 2005 PCR is one of the most commonly accepted methods that provide high sensitivity and specificity for FCoV detection However conventional PCR assays could take three to four hours and require sophisticated thermocyclers and well trained technicians to perform GeneReach has developed PetNAD M FCoV 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 is completed rapidly in a portable POCKIT Nucleic Acid Analyzer PetNAD FCoV Detection Kit PRINCIPLES OF THE PROCEDURE In iPCR hydrolysis probe based chemistry is used to generate fluorescent signal during amplification of target RNA The primers and probe target the M gene and do not cross react with nucleic acid from host and other feline pathogens PRODUCT DESCRIPTION A Materials Provided 24 tests kit Component Contents or
7. ixture 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 PetNAD FCoV Detection Kit collected at the bottom of R tube Note Make sure there are no bubbles in the solution Note Start reaction within 1 hour to prevent nucleic acid degradation and non specific reaction 9 POCKIT reaction a Select 520 nm b When System READY is displayed place the holder with R tube s into the reaction chamber c 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 FCoV Detection Kit DATA INTERPRETATION One example of results shown on the monitor 520 nm Interpretation FCoV Positive FCoV Negative Repeat reaction with freshly prepared nucleic acid ANYLYTICAL SENSITIVITY The detection limit of PeCNAD FCoV Detection Kit is about 10 copies reaction 10 PetNAD FCoV Detection Kit TROUBLESHOOTING Problems i Possible causes i Solutions False Positive 1 Reuse of micro E Micro centrifuge tubes tips R centrifuge tubes tubes and Premix are for single use tips R tubes and only Reusing these accessories Premix would cause cross contamination a
8. nd therefore false positive results 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 l i prevent cross contamination 2 Contaminated M Use aerosol free tips SSS micropipette 3 Contaminated W Consult with a GeneReach reagent technical support representative or local distributor Feet dem EPSP E O 1 4 Contaminated i M Consult with a GeneReach working area i technical support representative on how to clean up working area 11 PetNAD FCoV Detection Kit Problems Possible causes t Solutions False i 1 Nucleic acid B Consult manual of nucleic acid Negative extraction failed extraction kit 2 PCR inhibition W Do not overload PCR with too much nucleic acid E Spike nucleic acid sample 5 ul into a P Control 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 I Leakage or spill of TW Consult with a GeneReach a contamination reaction from R technical support representative or of amplicons tube into reaction local distributor in reaction chamber of chamber of POCKIT POCKIT PetNAD FCoV Detection Kit REFERENCE 1 Chang H F G Tsai Y L Tsai C E Lin C K Lee P Y Teng P H Su C and
9. sers D For PetNAD FCoV Detection Kit it is strongly recommended to use freshly prepared nucleic acid within 1 hour after extraction to achieve optimal results PetNAD FCoV Detection Kit PROCEDURE A PetNAD FCoV Detection Kit Quick Guide Take one Premix Add 50 pil Premix Add 5 pl nucleic acid from Premix Pack Buffer extract then spin w Transfer 50 pil mixture into Spin R tube for 10 seconds R tube 4 5 H Results are shown on monitor in 1 hour cii Put R tube into Nucleic Acid Analyzer and press RUN 6 PetNAD FCoV Detection Kit Procedure Note Before preparing the reactions for iiPCR testing turn on POCKIT to initiate the calibration for the instrument The device will complete self test within 5 minutes Please refer to the user manual of POCKIT for further details Note Before using for the first time add 100 ul P Control Buffer to P Control Store reconstituted P Control 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 tube Note When 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 Control to each Premix tube Spin Premix tube for 10 seconds in a mini centrifuge such as cubee 5 Transfer 50 ul Premix sample m
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