User Manual (201409)
Contents
1. D Sample Type Nucleic acid extracted from whole blood PetNAD Canine Tick Borne Diseases Panel 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 DNAs 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 Tick Borne Diseases Panel 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 de2. 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 PetNAD Canine Tick Borne Diseases Panel 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 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 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 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 PetNAD Canine Tick Borne Diseases Panel b When System READY is displayed place the holder with
3. G 1998 Canine babesiosis Compend Cont Educ Pract Vet 20 418 431 6 Murphy G L Ewing S A Whitworth L C Fox J C and Kocan A A 1998 A molecular and serologic survey of Ehrlichia canis E chaffeensis and E ewingii in dogs and ticks from Oklahoma Vet Parasitol 79 325 339 7 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 0045278
4. 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 Tick Borne Diseases Panel DATA INTERPRETATION One example of results shown on the monitor 520 nm Interpretation Positive infection Negative infection Repeat reaction with freshly prepared nucleic acid ANYLYTICAL SENSITIVITY The detection limit of Pet NADTM Canine Tick Borne Disease Panel is about 10 copies reaction PetNADTM Canine Tick Borne Diseases Panel 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 SS micropipette 3 Contaminated W Consult with a GeneReach reagent technical support representative or local distributor SEE aan 1
5. 4 Contaminated i M Consult with a GeneReach working area i technical support representative on how to clean up working area 12 PetNAD Canine Tick Borne Diseases Panel Problems Possible causes Solutions False i 1 Nucleic acid 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 pl 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 iy Leakage or spill of TW Consult with a GeneReach E 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 Tick Borne Diseases Panel REFERENCE 1 Bourdoiseau G 2006 Canine babesiosis in France Vet Parasitol 138 118 125 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 Harrus S Bark H and Waner T 1997 Canine monocytic ehrlichiosis An update Compendium of Continuing Education for the 4 Veterinary Practitioner 19 4 431 444 5 Lobetti R
6. PetNAD Canine Tick Borne Diseases Panel For Canine Babesiosis Babesia gibsoni Ehrlichia canis and Anaplasma platys 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 Tick Borne Diseases Panel 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 geteilt choses 3 B Materials and Equipments Required but Not Provided 4 C Storage and Stability eissii niina eoi 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 Lesser 7 A PetNAD M Canine Tick Borne Diseases Panel Quick Guide 7 B P 4 Control Preparation sooonraronnvnonrnvennnnerrvennnnenrnsennnnerssennenenrssennenee 8 C
7. Procedures uet Guss 8 PetNAD Canine Tick Borne Diseases Panel DATA INTERPRETATION 64 sssssssessessesestveadisvcnavesssvvnscsussousesssasaeads 11 ANALYTICAL SENSITIVITY ssensevseveevensseveneenvenenneensnnensenesnseneene 11 TROUBLESHOOTING spss sessicscssesesnsatonissssncsesestuscscsusnecssniesnuieccsonets 12 REFERENCE Dit trosses rensaseoeaes esoe pisro sossen tooo roe saes oeos0s ss 14 PetNAD Canine Tick Borne Diseases Panel INTENDED USE PetNAD Canine Tick Borne Disease Panel is intended for in vitro detection of canine babesiosis Babesia gibsoni Ehrlichia canis and Anaplasma platys 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 t
8. cleic acid from host and other tick borne pathogens PRODUCT DESCRIPTION A Materials Provided 4 combo tests for 8 dogs Component Contents or Purpose Amount Premix Pack M Canine Babesiosis Premix 5 bags 8 tubes and 1 Babesia gibsoni Premix desiccating Ehrlichia canis Premix and agent bag Anaplasma platys Premix lyophilized pellet containing dNTPs primers probe and enzyme for amplification Desiccating agent pack Premix Buffer M Reaction buffer to re dissolve 2 vials 1 3 ml vial B the lyophilized pellet P Control W Dried P Control template 1 vial P Control M Reaction buffer to re dissolve 1 vial 110 ul vial buffer P Control PetNAD Canine Tick Borne Diseases Panel R tube 48 1 box 48 R tubes and 48 Caps 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 cubeeTM 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
9. he window period between time of infection and detection PetNADTM Canine Tick Borne Diseases Panel SUMMARY AND EXPLANATION Tick borne diseases caused by Babesia canis Babesia gibsoni Ehrlichia canis and Anaplasma platys formerly Ehrlichia platys often occur in dog The disease is mainly transmitted by brown dog tick Rhipicephalus sanguineus Clinical abnormalities associated with tick borne diseases often include lethargy anorexia pale mucosa membranes haemolytic anaemia haemoglobinuria and thrombocytopenia Lobetti 1998 Bourdoiseau 2006 PCR is one of the most commonly accepted methods that provide high sensitivity and specificity for tick borne disease 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 Tick Borne Disease Panel 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 Tick Borne Diseases Panel 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 specific genes and do not cross react with nu
10. viations from the recommended procedure may lead to suboptimal results Quality of the extracts should be validated by the users D For PetNAD M Canine Tick Borne Diseases Panel it is strongly recommended to use freshly prepared nucleic acid within 1 hour after extraction to achieve optimal results PetNAD Canine Tick Borne Diseases Panel PROCEDURE A PetNAD Canine Tick Borne Diseases Panel 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 Canine Tick Borne Diseases Panel P Control Preparation Note Before using for the first time add 100 pl P Control Buffer to Panel P Control Store reconstituted P Control at 4 C 1 Label R tube s in the label area 2 Prepare one P Control Premix for each run Premix tube is in the Panel P Control Premix Pack containing eight Premix tubes Note If 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 the Premix tube 4 Add 5 ul P Control to the Premix tube Spin Premix tube for 10 seconds in a mini centrifuge such as cubeeTM 5 Follow Procedure C Step 5 to proceed P Control preparation
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