User Manual (201409)
Contents
1. 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 Babesiosis 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 Canine Babesiosis Detection Kit DATA INTERPRETATION One example of results shown on the monitor 520 nm Interpretation Babesia Positive Babesia Negative Repeat reaction with freshly prepared nucleic acid ANYLYTICAL SENSITIVITY The detection limit of PetNADTM Canine Babesiosis Detection Kit is about 10 copies reaction 10 PetNAD Canine Babesiosis Detection Kit TROUBLESHOOTING Problems i Possible causes i Solutions False Posit2. Materials Provided 24 tests kit Component Contents or Purpose Amount Premix Pack M Canine Babesiosis Premix 24 bags 1 Canine lyophilized pellet containing Babesiosis Premix dNTPs primers probe and vial and 1 desiccating enzyme 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 Canine Babesiosis 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 Canine Babesiosis 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 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
3. PetNAD Canine Babesiosis Detection Kit For Babesia canis Babesia gibsoni 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 Babesiosis 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 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 EEE ER EE PEE UER AERA PER Un 5 Ld 0 89 VO M Jo gen et dre 5 LIMITATIONS i gesiecsvao coe oa bp cin sp rpev o eee R Eee Sea ae PE PEE eden seb cV agregue n n 6 PROCEDURE cerei qve est n uocari eade ai peor EAE DNE PR EU Era uiae 7 A PetNAD M Canine Babesiosis Detection Kit Quick Guide 7 Bi Procedure nsns iUa i IPs 8 DATA INTERPRETATION eeoseeveesvsenvennenneneesnnenn
4. ed procedure may lead to suboptimal results Quality of the extracts should be validated by the users D For PetNAD Canine Babesiosis Detection Kit it is strongly recommended to use freshly prepared nucleic acid within 1 hour after extraction to achieve optimal results PetNAD Canine Babesiosis Detection Kit PROCEDURE A PetNAD Canine Babesiosis 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 on Put R tube into Nucleic Acid Analyzer and press RUN 6 PetNAD Canine Babesiosis 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
5. enneseneenevsnnnnnensennee 10 PetNAD Canine Babesiosis Detection Kit ANALYTICAL SENSITIVITY ssicssvsossessssstassssinonsscvvssasesvensesssvseness 10 TROUBLESHOOTING eter er SE ei Urs en SES en UE rov x puer Kt Ion E REV Un ERU V 11 REFERENCE passe 13 ii PetNAD Canine Babesiosis Detection Kit INTENDED USE PetNAD Canine Babesiosis Detection Kit is intended for in vitro detection of Canine Babesia 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 Babesiosis Detection Kit SUMMARY AND EXPLANATION Canine Babesiosis is a disease ca
6. extracted from whole blood PetNAD Canine Babesiosis Detection Kit RECOMMENDED NUCLEIC 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 Canine Babesiosis Detection Kit E To get optimal fluorescence detection 1 Wear powder free gloves to EJ 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 recommend
7. ive 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 11 PetNAD Canine Babesiosis Detection Kit Problems Possible causes Solutions False i 1 Nucleic acid B Consult manual of nucleic acid Negative extraction failed extraction kit 2 a 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 MT Leakage or spill of TW Consult with a GeneReach ET contamination reaction from R technical support representative or of amplico
8. ns tube into reaction local distributor in reaction chamber of chamber of POCKIT POCKIT PetNAD Canine Babesiosis Detection Kit REFERENCE 1 Abdullahi S U Mohammed A A and Trimnell A R 1990 Clinical and haematological findings in 70 naturally occurring cases of canine babesiosis J Small Anim Pract 31 145 147 2 Botros B A Moch R W and Barsoum I S 1975 Some observations on experimentally induced infection of dogs with Babesia gibsoni Am J Vet Res 36 293 296 3 Breitschwerdt E B 1990 Infectious diseases of the dog and cat ed 1 WB Saunders Philadelphia PA 4 Casapulla R Baldi L and Avallone V 1998 Canine piroplasmosis due to Babesia gibsoni clinical and morphological aspects Vet Rec 142 168 169 5 Ceron J J Eckersall P D and Martynez Subiela S 2005 Acute phase proteins in dogs and cats current knowledge and future perspectives Vet Clin Pathol 34 8599 6 Chang H F G Tsai Y L Tsai C E 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 d 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 TG e45278 doi 10 1371 journal
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10. used by the intraerythrocytic protozoan parasites Babesia species throughout the world There are varieties of symptoms of Canine Babesiosis including pyrexia weakness depression lymphadenopathy splenomegaly and general malaise It is known that Babesia canis B canis and Babesia gibsoni B gibsoni are two organisms commonly known to infect dogs Moreover it is suggested that B canis might be subdivided into 3 subspecies and B gibsoni might subdivided into 2 isolates PCR is one of the most commonly accepted methods that provide high sensitivity and specificity for Canine Babesia 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 Canine Babesiosis 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 Canine Babesiosis Detection Kit PRINCIPLES OF 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 18S rRNA gene and do not cross react with nucleic acid from host and other feline pathogens PRODUCT DESCRIPTION A
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