User Manual (201409)
Contents
1. PetNAD Canine Leishmaniasis 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 I 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 Canine Leishmaniasis Detection Kit REFERENCE 1 Alvar J Yactayo S Bern C 2006 Leishmaniasis and poverty Trends Parasitol 2006 22 552 557 2 Chai J J Zuo X P Zhang S Zhao J P Zhang J M 1999 The Desert type kala azar in Xinjiang China In Epidemiology and Control of Leishmaniasis in Central Eurasia Edited by Matsumoto Y Tokyo International Press Editing Centre Incorporation 1999 15 19 3 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 4 Companion Animal Parasite C2. 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 A 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 PetNAD Canine Leishmaniasis 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 sh
3. PetNAD Canine Leishmaniasis Detection Kit For Leishmania donovani complex 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 Canine Leishmaniasis 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 DESCRIPTION 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 einem OU OR VERE RERO 5 RECOMMENDED NUCLEIC ACID EXTRACTION METHODS E E ER EE Ne 5 Ld 0 89 V8 M Jo gen e srooot ssrt isse 6 LIMITATIONS gestiens coe oa bp cin sp epev o Pee R EG ena o Ce PE PP eS een sp eb cv tios riist 7 PROCEDURE ceres quic est n uota bk enudces xi een aV CER PIER PR EE roroot 8 A PetNAD M Canine Leishmaniasis Detection Kit Quick Guide 8 Bi Procedure ease eene EU RE ees 9 DATA INTERPRETATION eeoseeveesvsenvennennenevsnne
4. THE PROCEDURE In iiPCR hydrolysis probe based chemistry is used to generate fluorescent signal during amplification of target DNA The primers and probe target the internal transcribed spacer 1 ITS1 and do not cross react with nucleic acid from host and other canine pathogens PetNAD Canine Leishmaniasis Detection Kit PRODUCT DESCRIPTION A Materials Provided 24 tests kit Component Premix Pack Contents or Purpose Canine leishmaniasis Premix lyophilized pellet containing dNTPs primers probe and enzyme for amplification Desiccating agent pack 24 bags 1 canine leishmaniasis Premix vial and 1 desiccating agent bag Premix Buffer A Reaction buffer to re dissolve the lyophilized pellet 2 vials 1 3 ml vial P Control Dried plasmid containing canine leishmaniasis partial sequence as positive control 1 vial P Control Buffer Reaction buffer to re dissolve P Control I vial 110 ul vial R tube User Manual 1 bag 24 pieces bag 1 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 Nucleic Acid Analyzer POCKIT PetNAD compatible instrument 3 cubee M Mini Centrifuge cubee PetNAD Canine Leishmaniasis Detection Kit 4 Micropipette and filter tips C Storage and Stab
5. eishmaniasis Detection Kit LIMITATIONS A PetNAD Canine Leishmaniasis Detection Kit targets Leishmania donovani complex sequence and may also detect Leishmania aethiopica Leishmania major and Leishmania tropica B 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 C PetNAD Nucleic Acid Co prep Kit and taco mini Automatic Nucleic Acid Extraction System are recommended for nucleic acid extraction D Any deviations from the recommended procedure may lead to suboptimal results Quality of the extracts should be validated by the users E For PetNAD Canine Leishmaniasis Detection Kit it is strongly recommended to use freshly prepared nucleic acid within 1 hour after extraction to achieve optimal results PetNAD Canine Leishmaniasis Detection Kit PROCEDURE A PetNAD Canine Leishmaniasis 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 on Put R tube into Nucleic Acid Analyzer and press RUN 6 PetNAD Canine Leishmaniasis Detection Kit Procedure Note Before preparing the reactions for iiPCR testing turn on POCKIT to initiate the calibration for the instrument The
6. ility 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 DNA extracted from whole blood or conjunctival swab 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 PetNAD Canine Leishmaniasis 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 1 Wear powder free gloves to handle R tubes T Labeling area 2 Do not label in the detection area i Detection area of R tube PetNAD Canine L
7. ishmaniasis CanL is a vector borne disease caused by Leishmania parasites Dereure et al 1999 CanL could be categorized into three forms cutaneous visceral and mucocutaneous leishmaniasis based on clinical symptoms Visceral leishmaniasis VL endemic in 61 countries is caused by Leishmania donovani complex VL is a severe zoonotic disease and an important public health issue worldwide Alvar et al 2006 Chia et al 1999 CanL is transmitted among dogs through the bites of infected female Phlebotomine sandflies Clinical diagnosis of CanL is difficult at the asymptomatic stage and the acute stage due to its wide range of symptoms Therefore a sensitive and specific identification tool is especially important for canine leishmaniaisis detection PCR is one of the most commonly accepted methods that provide high sensitivity and specificity for canine leishmaniasis 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 Canine Leishmaniasis 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 PetNAD Canine Leishmaniasis Detection Kit simple and easy assay is completed rapidly in a portable POCKIT Nucleic Acid Analyzer PRINCIPLES OF
8. nnenneneneeneesennnnensennee 11 PetNAD Canine Leishmaniasis Detection Kit ANALYTICAL SENSITIVITY ssissossossessessvasssscnossscvevescsessousesesteaneds 11 TROUBLESHOOTING eter ente ei argen Rs en s eser Du eC E co SE REV unen E iU 12 REFERENCE passe 14 PetNAD Canine Leishmaniasis Detection Kit INTENDED USE PetNAD Canine Leishmaniasis Detection Kit is intended for in vitro detection of the Leishmania donovani complex DNA 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 Canine Leishmaniasis Detection Kit SUMMARY AND EXPLANATION Canine le
9. ouncil Capcvet org Retrieved 2012 10 04 5 Dereure J Pratlong F Dedet J P 1999 Geographical distribution and the identification of parasites causing canine leishmaniasis in the Mediterranean Basin Canine leishmaniasis an update Proceedings of the International Canine Leishmaniasis Forum Barcelona Spain 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 iiPCR for sensitive and specific on site pathogen detection PLoS ONE 7 9 e45278 doi 10 137 1 journal pone 0045278
10. own on the monitor after reaction is completed PetNAD Canine Leishmaniasis Detection Kit DATA INTERPRETATION One example of results shown on the monitor am GGG eee Buzzer OFF 520 nm Interpretation ao Canine Leishmaniasis Positive eS Canine Leishmaniasis Negative 2 Repeat reaction with freshly prepared nucleic acid ANYLYTICAL SENSITIVITY The detection limit of Pet NADTM Canine Leishmaniasis Detection Kit is about 10 copies reaction PetNAD Canine Leishmaniasis 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 SSS micropipette 3 Contaminated W Consult with a GeneReach reagent technical support representative or local distributor SEE aan 1 4 Contaminated i M Consult with a GeneReach working area i technical support representative on how to clean up working area 12
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