User Manual PetNAD
Contents
1. PetNAD Nucleic Acid Co prep Kit and taco mini Automatic Nucleic Acid Extraction System are recommended for nucleic acid extraction Any deviations from the recommended procedure may lead to 6 PetNAD Bordetella bronchiceptica Detection Kit suboptimal results Quality of the extracts should be validated by the users D For PetNAD Bordetella bronchiceptica Detection Kit it is strongly recommended to use freshly prepared nucleic acid within 1 hour after extraction to achieve optimal results PetNAD Bordetella bronchiceptica Detection Kit PROCEDURE A PetNAD Bordetella bronchiceptica Detection Kit Quick Guide Take one Premix Add 50 pil Premix Add 5 pl nucleic acid from Premix Pack Buffer extract then spin down for 10 seconds w Transfer 50 ul mixture into Spin R tube for 10 seconds R tube 4 5 H Results are shown on A monitor in 1 hour Put R tube into Nucleic Acid Analyzer and press RUN 6 PetNAD Bordetella bronchiceptica 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 pl P Control Buffer to P Control Store reconstituted P Control at 4 C 1 Label R tube s in the label area 2 Prepare2. 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 DNA extracted from upper respiratory tract swabs RECOMMENDED NUCLEIC ACID EXTRACTION METHODS A PetNAD Nucleic Acid Co prep Kit B 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 PetNADT Bordetella bronchiceptica Detection 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 Control to 1 Ensure POCKIT is working normally 2 Ensure detection kit performance after storage E To get optimal fluorescence detection E 1 Wear powder free gloves to EX Labeling area handle R tubes E 2 Do not label in the detection area Ja of R tube LIMITATIONS A The test should be used only for testing nucleic acid extracted from cats and dogs specimens Do not add specimens e g whole blood directly into Premix
3. and probe target fimA gene and do not cross react with nucleic acid from host and other canine amp feline upper respiratory pathogens PetNADT Bordetella bronchiceptica Detection Kit PRODUCT DESCRIPTION A Materials Provided 24 tests kit Component Premix Pack Contents or Purpose B bronchiceptica Premix lyophilized pellet containing dNTPs primers probe and enzyme for amplification Desiccating agent pack 24 bags 1 B bronchiceptica Premix vial and 1 desiccating agent bag Premix Buffer B Reaction buffer to re dissolve the lyophilized pellet 2 vials 1 3 ml vial P Control Dried plasmid containing Parvovirus partial sequence as positive control 1 vial P Control Buffer Reaction buffer to re dissolve P Control 1 vial 110 ul vial R tube User Manual I bag 24 pieces bag I bag 24 pieces bag 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 Pet NADTM compatible instrument 3 cubee M Mini Centrifuge cubee 4 Micropipette and filter tips 4 PetNAD Bordetella bronchiceptica Detection Kit 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
4. PetNAD Bordetella bronchiceptica Detection Kit 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 11 PetNAD Bordetella bronchiceptica Detection Kit Content INTENDED USE siscscssesuocscstsictnestexeanitstiovasesuoauis be evo FER ctus EUM er Miele eld 1 SCIENTIFIC MEANINGS iic sonkece transiens ror unb ed vh sinere ETE Une 1 SUMMARY AND EXPLANATION eene en eren etate neenon tan 2 PRINCIPLES OF THE PROCEDURE c eeeeee eene tn nttnnnnn 3 PRODUCT DESCRIP TION scsossscescssessssvastiuocscssstcssostsuasuetessccsatesuenses 4 A Materials Provided eset eter nete iege 4 B Materials and Equipments Required but Not Provided 4 C Storage and Stability sees 5 D Sample Type enmt OU RR EEEo 5 RECOMMENDED NUCLEIC ACID EXTRACTION METHODS E E ER EE PER EUER AERA REUS 5 Ld 0 89 V0 M Jo gen eM ERU 6 LIMITATIONS 3 gesieesvao coit oa up cin sp eset o Peu Eee Sea oe PE PEP eden seb cv agregue ndn 6 PROCEDURE eicee c qusc eest enpuocu piede xi eun up EPA PUER PETENTE Ee aee SE 8 A PetNAD M Bordetella bronchiceptica Detection Kit Quick Guide 8 Bi Procedure soupe eue nV 9 DATA INTERPRETATION eeeeeee eene etn sensns enata ttn atn sta
5. acterium that replicates in the respiratory tract B bronchiceptica is a contagious respiratory disease through cats and dogs causing infectious bronchitis transmission through direct or indirect with infected animals The disease is commonly seen in boarding kennels shelters pet exhibitions and vet clinics Datz 2003 The clinical sign for dogs is mainly coughing whereas cats may display nonspecific signs of upper respiratory infection Datz 2003 Schulz et al 2014 The bacterium can be found in oral and nasal secretion of infected animals and presence of contaminated formites in the environments PCR is one of the most commonly accepted methods that provide high sensitivity and specificity for B bronchiceptica 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 Bordetella bronchiceptica 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 PetNADT Bordetella bronchiceptica Detection Kit POCKIT Nucleic Acid Analyzer PRINCIPLES OF THE PROCEDURE In iiPCR hydrolysis probe based chemistry is used to generate fluorescent signal during amplification of target DNA The primers
6. ction 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 M contamination reaction from R technical support representative or of amplicons tube into reaction local distributor in reaction chamber of chamber of POCKIT POCKIT PetNADT Bordetella bronchiceptica 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 Athermally baffled device for highly stabilized convective PCR Biotechnology Journal 7 5 662 666 doi 10 1002 biot 201100453 2 Datz C Bordetella Infections in Dogs and Cats Pathogenesis Clinical Signs and Diagnosis Compendium 2003 Vol 25 No 12 3 Schulz BS Kurz S Weber K Balzer HJ Hartmann K 2014 Detection of respiratory viruses and Bordetella bronchiseptica in dogs with acute respiratory tract infections Vet J Sep 201 3 365 9 doi 10 1016 4 Tsai Y L Wang H T T Chang H EG 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 0045278
7. eaction with freshly prepared nucleic acid ANYLYTICAL SENSITIVITY The detection limit of PetNAD Bordetella bronchiceptica Detection Kit is about 10 copies reaction PetNADT Bordetella bronchiceptica 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 and therefore false positive results M 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 micropipette 3 Contaminated W Consult with a GeneReach reagent technical support representative or local distributor SEE 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 12 PetNAD Bordetella bronchiceptica Detection Kit Problems Possible causes Solutions False i 1 Nucleic acid i B Consult manual of nucleic acid Negative extraction failed extraction kit 2 PCR inhibition E Do not overload PCR with too much nucleic acid M Spike nucleic acid sample 5 ul into a P Control rea
8. n 11 PetNAD Bordetella bronchiceptica Detection Kit ANALYTICAL SENSITIVITY scicsovssssesssssvasssssnossssvvesesessousesesteenese 11 TROUBLESHOOTING sivsnsesssesticcsssecscsastesnsenseassossoestucosucosscuveesansons 12 REFERENCE passe 14 PetNAD Parvovirus Detection Kit INTENDED USE PetNAD Bordetella bronchiceptica Detection Kit is intended for in vitro detection of the DNA based on insulated isothermal polymerase chain reaction iiPCR technology This kit is designed specially to be used with 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 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 PetNADT Bordetella bronchiceptica Detection Kit SUMMARY AND EXPLANATION Bordetella bronchiceptica B bronchiceptica is a gram negative rod shaped b
9. 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 mixture 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 POCKITTM 8 Spin tube briefly in cubee to make sure all solution is collected at the bottom of R tube PetNAD Bordetella bronchiceptica Detection Kit 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 Bordetella bronchiceptica Detection Kit DATA INTERPRETATION One example of results shown on the monitor 520 nm Interpretation o B bronchiceptica Positive e B bronchiceptica Negative E Repeat r
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