Canine Tick-Borne Diseases Panel
Contents
1. PetNAD Canine Tick Borne Disease Panel DATA INTERPRETATION One example of results shown on the monitor Pockit 1 0 16 28 57 17 08 2011 Reaction Completed 0000 60 Buzzer Off 520nm Interpretation Positive infection o Negative infection o Repeat reaction with freshly prepared nucleic acid ANYLYTICAL SENSITIVITY The detection limit of PetNAD Canine Tick Borne Disease Panel is about 10 copies reaction TROUBLESHOOTING PetNAD Canine Tick Borne Disease Panel Problems Possible causes i Solutions False Positive 1 Reuse of micro E Micro centrifuge tubes tips centrifuge tubes R tubes and Premix are for tips R tubes and single use only Reusing these Premix accessories would cause cross contamination E 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 prevent cross contamination b M EM M i 2 Contaminated i M Disassemble and clean up micropipette micropipette E Use aerosol free tips 3 Contaminated LE m Consult with a GeneReach j reagent technical support representative or local distributor 4 Contaminated W Consult withaGeneReach j working area technical support representative on how to clean up working area Problems i Possible causes i S2. ANALYTICAL SENSITIVITY eeeeee eene eese notant nn nan 9 TROUBLESHOOTING e eere sees seen enean osoro seta sens ense ta so 10 PetNAD Canine Tick Borne Disease Panel ii PetNAD Canine Tick Borne Disease 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 panel is designed specially to be used with an iiPCR compatible instrument POCKIT Nucleic Acid Analyzer The assay is intended for use by people with basic laboratory skills This kit is intended for research use only SUMMARY AND EXPLANATION Tick borne diseases caused by Babesia canis Babesia gibsoni Ehrlichia canis and Anaplasma platys formerly Ehrlichia platys often occur in dog whose main vector is the 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 PetNAD Canine Tick Borne Disease Panel PCR is one of the most commonly accepted methods that provide high sensitivity and specificity for canine tick borne disease detection However conventional PCR assays take three to four hours and require sophisticated thermocycler
3. Equipments Required but Not Provided 1 PetNAD Nucleic Acid Co prep Kit 2 POCKIT Nucleic Acid Analyzer PetNAD compatible instrument 3 cubee Mini Centrifuge cubee 4 Micropipette and tips C PetNAD Canine Tick Borne Disease Panel Storage and Stability 1 The kit should be stored at 4 C and is stable until the expiration date which is stated on the label 2 Store Premix vials in sealed Premix Pack to avoid hydration of lyophilized components 3 Reconstituted P Standard is stable for 6 months at 4 C Aliquot reconstituted P Standard to avoid degradation and contamination of nucleic acid D Sample Type Nucleic acid extracted from whole blood 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 Standard to 1 Ensure POCKIT Nucleic Acid Analyzer is working normally 2 Ensure detection kit performance after storage PetNAD Canine Tick Borne Disease Panel E To get optimal fluorescence detection E DA 1 Label area 1 Wear powder free gloves to handle V R tubes 2 Do not label in the detection area of Detection area R tube LIMITATIONS A The test should be used only for testing nucleic acid extracted from animal specimen Do not add specimen i e whole blood
4. PetNAD gt 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 Rd Central Taiwan Science Park Taichung City Taiwan 407 Web Site www petnad com 2013 07 PetNAD Canine Tick Borne Disease Panel Content INTENDED USE e eeeeee esee enses eese senatu seta sts toss tastes osion 1 SUMMARY AND EXPLANATION crecen eren eene neenon enata an 1 PRINCIPLES OF THE PROCEDURE eee eeee eene tn attnanun 2 PRODUCT DESCRIPTION eese ee eene ne tastes senses sns tn seen tnu 3 A Materials Provided mossen neaei getto p Recon 3 B Materials and Equipments Required but Not Provided 3 C Storage and Stability ecese a e eas eein 4 D Sample Type oue eda REIS ee 4 PRECAUTIONS eeeeeee cesse enses enose terro Is seta stes sse to sets tnu 4 LIMITATIONS e eeeeee esee sees eene enata stato seta sens sone tastes sensns seoses 5 Id ULL UT O 6 A PetNAD Canine Tick Borne Disease Panel Quick Guide 6 B P Standard Preparation eese 7 C Procedute ette Er E ge oto n estemicetbe uis 7 DATA INTERPRETATION crees eee ne testen neta sensns etus ets tnus 9
5. directly into Premix B PetNAD Nucleic Acid Co prep Kit is recommended for nucleic acid extraction C Any deviation from recommended procedure may not achieve the optimal results and should be validated by the users D It is strongly recommended to use freshly prepared nucleic acid within 1 hour after extraction to achieve optimal results with PetNAD Canine Tick Borne Disease Panel PetNAD Canine Tick Borne Disease Panel PROCEDURE A PetNAD Canine Tick Borne Disease Panel Quick Guide v For each target Add 50 ul Premix Add 5 pl nucleic acid Take one Premix Buffer extract from Premix Pack TAE Mix by pipetting Spin R tube for 10 seconds Transfer 50 ul mixture into R tube 4 5 Results are shown on monitor in 1 hour mae mem Put R tube into POCKIT Nucleic Acid Analyzer and press l RUN 6 PetNAD Canine Tick Borne Disease Panel P Standard Preparation Note Before using for the first time add 100 pl Standard Buffer to Panel P Standard Store reconstituted P Standard at 4 C 1 2 Label R tube s in the label area Prepare one P Standard Premix for each run Premix tube is in the Panel P Standard 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 Standard to the Premix tube Mix by p
6. ipetting up and down 5 Follow Procedure C Step 5 to proceed P Standard preparation Procedure 1 Label R tube s in the label area 2 For each target prepare one Premix tube Premix tube is in the Premix Pack Each Premix Pack contains eight Premix tubes Note If the pellet is not found at the bottom of the tube spin tube briefly to bring it down 3 4 5 6 7 8 9 PetNAD Canine Tick Borne Disease Panel Add 50 ul Premix Buffer B to each Premix tube Add 5 ul nucleic acid extract to each Premix tube Mix by pipetting up and down Transfer 50 ul Premix sample mixture into R tube Seal top of each R tube with a cap Make sure R tube is capped tightly Place R tube into the holder of POCKIT M Spin tube briefly in cubee to make sure all solution is collected at the bottom of R tube Note Start reaction within 1 hour to prevent nucleic acid degradation Note Make sure there are no bubbles in the tube POCKIT reaction Note Please see the user manual of POCKIT for details a Turn on POCKIT which should complete self testing within 5 minutes b Select 520 nm c When System READY is displayed place the holder with R tube s into the reaction chamber d 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
7. olutions False 1 Nucleic acid W Consult manual of nucleic acid PetNAD Canine Tick Borne Disease Panel Negative i extraction failed extraction kit i 2 Bad nucleic acid M Check sample storage condition quality or nucleic E Please refer to Troubleshooting acid concentration section of Pet NAD Nucleic Acid too high Co prep Kit E Ifaspectrophotometer is available check OD 260 280 ratio This ratio should be between 1 4 and 2 0 W Do not overload nucleic acid i 3 PCR inhibition E Spike nucleic acid sample into P Standard 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 il Consult witha GeneReach contamination reaction from technical support representative or of amplicons R tube into local distributor in reaction j reaction chamber chamberof of POCKIT POCKIT M REFERENCE 1 Bourdoiseau G 2006 Canine babesiosis in France Vet 11 PetNAD Canine Tick Borne Disease Panel Parasitol 138 118 125 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 Harrus S Bark H and Waner T 1997 Canine monocytic ehrlichiosis An
8. s and well trained technicians to perform GeneReach has developed PetNAD M Canine Tick Borne Disease Panel based on iiPCR technology which significantly reduces reaction time and offers sensitivity and specificity comparables to those of conventional nested PCR Tsai 2012 Chang 2012 Furthermore this simple and easy assay could be completed rapidly in a portable 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 and probe target specific genes and do not cross react with nucleic acid from host and other tick borne pathogens PetNAD Canine Tick Borne Disease Panel PRODUCT DESCRIPTION A Materials Provided 4 combo tests for 8 dogs Component Premix Pack Contents or Purpose Canine Babesia Premix Babesia gibsoni Premix Ehrlichia canis Premix and Anaplasma platys Premix lyophilized pellet containing dNTPs primers probe and enzyme for amplification Panel P Standard Premix Desiccating agent pack 5 bags 8 tubes and 1 desiccating bag Premix Buffer B Reaction buffer to re dissolve the lyophilized pellet 2 vials 1 3 ml vial P Standard Dried P control template 1 vial Standard Buffer Reaction buffer to re dissolve P Standard vial 110 pl vial User Manual 1 copy B Materials and
9. update Compendium of Continuing Education for the Veterinary Practitioner 19 4 431 444 Lobetti R G 1998 Canine babesiosis Compend Cont Educ Pract Vet 20 418 431 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 Tsai Y L Wang H T T Chang H E G Tsai C F Lin C K Teng P H Su C and Jeng C C 2012 Development of TagMan 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 12
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