User Manual (201409)
Contents
1. 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 E Dried plasmid containing M vial felis partial sequence as positive control P Control a Reaction buffer to re dissolve 1 vial 110 ul vial Buffer P Control R tube I bag 24 pieces bag PetNAD Mycoplasma felis Detection Kit Cap 1 bag 24 pieces bag User Manual 1 copy B Materials and Equipment Required but Not Provided 1 PetNADTM 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 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 Nucleic acid extracted from ocular swab or oropharyngeal swab PetNAD Mycoplasma felis Detection Kit RECOMMENDED NUCLEIC ACID EXTRACTION METHODS A PetNAD Nucleic Acid Co prep Kit taco DNA RNA Extraction Kit compatible instrument ta2. of them are regarded as harmless to animals However M felis can be pathogenic to both cats and horses In case of shelter cats isolation of M felis from oropharyngeal swabs has been associated with upper respiratory tract infection and conjunctivitis Bannasch et al 2005 PCR is one of the most commonly accepted methods that provide high sensitivity and specificity for M felis 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 Mycoplasma felis 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 Mycoplasma felis Detection Kit PRINCIPLES OF THE PROCEDURE In iiPCR hydrolysis probe based chemistry is used to generate fluorescent signal during amplification of target gene The primers and probe target the gag 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 Purpose Amount Premix Pack m Mz felis Premix lyophilized 24 bags 1 M felis pellet containing dNTPs Premix vial and 1 primers probe
3. 1 hour after extraction to achieve optimal results PetNAD Mycoplasma felis Detection Kit PROCEDURE A PetNAD Mycoplasma felis 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 5 4 H Results are shown on A monitor in 1 hour Put R tube into Nucleic Acid Analyzer and press RUN 6 PetNAD Mycoplasma felis 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 mixture into R tube 6 Seal top of each R tube with a cap Ma
4. PetNAD Mycoplasma felis 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 09 PetNAD Mycoplasma felis Detection Kit Content INTENDED USE esooovevvennensenevennennennennneenessennnnennensenenennennensennneeneenennne 1 SCIENTIFIC MEANINGS eessoevesevensesnenevsnnennenseneneenersensnnensennnnenensennee 1 SUMMARY AND EXPLANATION seseveeveevevsvseneevevsensnnensessenenensennee 2 PRINCIPLES OF THE PROCEDURE cese reset tn nennt 3 PRODUCT DESCRIPTION sesseevessvveneeveesnnnnvennensenenennennenseneneeneenennne 3 A Materials Provided ap ea n FWYD thee FFAN 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 EEE RR RR a PER Un 5 Ld 0 89 VO M Jo gen et dre 5 LIMITATIONS i ieueinc Fo cin ddo o eee R Eee Sea ae PE PEE eden seb cV agregue n n 6 PROCEDURE ceres quis eset puotu bk SDN YAWN neb 7 A _ PetNAD M Mycoplasma felis Detection Kit Quick Guide 7 Bi Procedure o nous UH REPRE 8 DATA INTERPRETATION eeoseeveesvsenvennennenevennennenseseneeneesnnnnnensennee 10 PetNAD Mycoplasma felis Detection Kit ANALYTICAL SENSI
5. TIVITY i cicsovcossessssstassoscnossosvsvssesessencesssnseness 10 TROUBLESHOOTING eter rr SE ei rsen RS en UE rov px puer Kt Io SE HEY Un ERU V 11 REFERENCE passe 13 PetNAD Mycoplasma felis Detection Kit INTENDED USE PetNAD Mycoplasma felis Detection Kit is intended for in vitro detection of Mycoplasma felis M felis based on insulated isothermal polymerase chain reaction iiPCR technology This kit is designed specially to be used with an insulated isothermal 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 PetNAD Mycoplasma felis Detection Kit SUMMARY AND EXPLANATION Mycoplasma spp are gram negative bacteria Several Mycoplasma species are part of the normal bacterial flora of animals many
6. co 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 Mycoplasma felis Detection Kit E _ To get optimal fluorescence detection 1 Wear powder free gloves to SI 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 users D For PetNAD Mycoplasma felis Detection Kit it is strongly recommended to use freshly prepared nucleic acid within
7. 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 SS micropipette 3 Contaminated i W Consult with a GeneReach reagent technical support representative or local distributor SEE Pu E P A 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 Mycoplasma felis Detection Kit Problems Possible causes 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 M 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 1 Leakage or spill of TW Consult with a GeneReach HE contamination reaction from R technical support representative or of amplicons tube into reaction local distributor in reaction chamber of chamber of POCKIT POCKIT PetNAD Mycoplasma felis Detection Kit REFERENCE 1 Bannasch M J Foley J E 2005 Epidemiologic evaluation of multiple res
8. ke 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 PetNAD Mycoplasma felis 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 Mycoplasma felis Detection Kit DATA INTERPRETATION One example of results shown on the monitor 520 nm Interpretation ao M felis Positive e M felis Negative 2 Repeat reaction with freshly prepared nucleic acid ANYLYTICAL SENSITIVITY The detection limit of PetNADTM Mycoplasma felis Detection Kit is about 10 copies reaction PetNAD Mycoplasma felis 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
9. piratory pathogens in cats in animal shelters J Feline Med Surg 7 109 119 2 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 3 _ S derlund R B lske G Holst B S and Asp n A 2011 Development and evaluation of a real time polymerase chain reaction method for the detection of Mycoplasma felis J VET Diagn Invest 23 890 doi 10 1177 1040638711407479 4 Tsai Y L Wang H T T Chang H F G Tsai C E 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
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