User Manual PetNAD
Contents
1. Kit Procedure Note Please dissolve the P Standard by 100 ul Standard Buffer at first time use The dissolved P Standard should be stored at 4 C 1 Open the Premix Pack according to the sample number and take out the Premix Note If the pellet is not at the bottom please spin it down 2 Open the cap add 50 ul Premix Buffer B into each Premix tube 3 Add 5 ul nucleic acid extract or dissolved P Standard into each Premix tube Mix by pipetting up and down 4 Transfer 50 ul of the Premix mixture into the R tube 5 Cap the R tube put into the holder of POCKIT and use cubee to spin down the solution Note Please make sure all solution has been spun down to the bottom of the R tube Perform the following amplification reaction within 1 hour to prevent nucleic acid degradation Note Please make sure there is no bubble in the tube Please see the user manual of POCKIT for details 6 Turn on POCKIT The analyzer will complete self testing within 5 minutes Select 520 nm for use System READY will be displayed Note Please see the user manual of POCKIT for details PetNAD Canine Babesiosis Detection Kit 7 Place the holder containing the R tube s into the reaction chamber of POCKIT and tap the cap of each R tube to make sure the tube is properly positioned in the reaction chamber 8 Close the lid and press Run to start the reaction program 9 The test result will be s2. 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 one Rd Central Taiwan Science Park Taichung City Taiwan 407 Web Site www petnad com 2012 05 PetNAD Canine Babesiosis Detection Kit Content INTENDED USE eese esee eese entes etes sossen sts toss tastes sns isois 1 SUMMARY AND EXPLANATION eee en seen eene neenon enata 1 PRINCIPLES OF THE PROCEDURE ecce eren eene tnnt ntnun 2 PRODUCT DESCRIPTION eee ee eene s etate setas tento n senses tnus 3 A Materials Provided sono aida 3 B Materials and Equipments Required but Not Provided 3 C Storage and Stability eeeeeeeeeseeeeeereessersesssrsesssssssessassessessesssesesessansne 4 De Sample Type pe r r eene A 4 PRECAUTIONS aore eeno eiieou ese enses seses sena asesan stato on esoo ie asane 5 LIMITATIONS ae reeeo orres rosses es rot resore orses stato oS ESE Eos on 6 OPERATION PROCEDURE seseesersesosoesecoesossesesossossescseosoesesossossese 7 A PetNAD Canine Babesiosis Detection Kit Quick Guide 7 B Procedure sein epo DRE agens Aedes 8 DATA INTERPRETATION e eeeeee teens testen natns ta sna tn aetas 10 ANALYTICAL SENSITIVITY eere eee ene
3. ard R tube 24 Pieces bag 1 bag 24 Pieces bag 1 bag User Manual 1 B Materials and Equipments Required but Not Provided 1 PetNAD Nucleic Acid Co prep Kit 2 POCKIT the compatible instrument for PetNAD M 3 cubee Mini centrifuge cubee 4 Micropipette and tips PetNAD Canine Babesiosis Detection Kit 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 Premix vials should be kept in the sealed Premix Pack to avoid the rehydration of lyophilized pellet 3 Dissolved P Standard can be stored at 4 C for up to 6 months To avoid the degradation of P Standard it is recommended to aliquot the dissolved P Standard into several vials Sample Type This kit is suitable for detecting nucleic acid extracted from whole blood PetNAD Canine Babesiosis Detection Kit PRECAUTIONS A Do not open the R tube after the amplification reaction to prevent any carryover contamination We strongly recommend that the working area for extraction procedure and amplification procedure should be separated into two independent spaces to avoid any possible contamination Do not reuse the R tube and Premix The P Standard is used to 1 Confirm the operation procedure after installation or when any uncertain result has occurred 2 Ensure the kit performance after storage In order to get optimal fluoresce
4. bservations 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 85 99 14
5. ell as well trained technicians to perform the test GeneReach has developed PetNAD Canine Babesiosis Detection Kit based on iiPCR technology which is highly sensitive and specific for Canine Babesia detection and is comparable to conventional PCR system The assay has been simplified for easier and faster operation with the use of compact equipments for Canine Babesia detection in the clinic PRINCIPLES OF THE PROCEDURE The assay is based on iiPCR In addition to specific primers fluorogenic probe hydrolysis chemistry is used to generate a fluorescent signal when specific Canine Babesia DNA is presented in samples The primers and probe target the 18S rRNA gene specific to Canine Babesia and will not react with canine genomic DNA and the nucleic acid of other canine protozoans PetNAD Canine Babesiosis Detection Kit PRODUCT DESCRIPTION A Materials Provided 24 tests kit Component Contents or Purpose Amount Premix Pack Each pack contains 1 pack of 1 zip lock bag desiccating agent and 1 Premix vial containing 24 with a lyophilized pellet containing individually sealed dNTPs Canine Babesia specific packs primers fluorescent probes and enzymes Premix Reaction buffer to re dissolve the 700 ul vial 2 vials Buffer B lyophilized pellet P Dry plasmid pellet containing Standard Babesia partial sequence Standard Reaction buffer to re dissolve the 110 pl vial 1 vial Buffer Babesia P Stand
6. hown on the screen after the reaction PetNAD Canine Babesiosis Detection Kit DATA INTERPRETATION Please check the results on the screen after the reaction For example from the screen Pockit 1 1 0202 16 28 57 17 08 2011 Reaction Completed 1 2 3 4 5 6 7 8 00000 OO Buzzer off 520nm Interpretation Babesia Positive a Babesia Negative Recheck with fresh sample ANALYTICAL SENSITIVITY The detection limit of PetNAD M Canine Babesiosis Detection Kit is up to 10 copies reaction 10 PetNAD Canine Babesiosis Detection Kit TROUBLESHOOTING Observations Possible Causes or Problems False Positive 1 The reuse of micro centrifuge tubes tips R tubes and Premix 2 Micropipette contaminated 3 Reagent contaminated 4 Working area contaminated 11 Comment and Suggestions The micro centrifuge tubes tips R tubes and Premix are for one time use only Reuse of these accessories will cause contamin ation Once used the micro centrifuge tubes tips R tubes and Premix should be collected and discarded according to the local regulation Do not place the waste close to the working area to X prevent contamination Disassemble pipette and do clean up We recommend using aerosol free tips Consult with GeneReach or local distributor Consult with GeneReach for working area clean up Observations or Problems Fal
7. nce detection please wear powder free gloves Label area gt lt to handle the R tube and do not mark and or label the detection area of the Detection area R tube The label area and detection area of the R tube are indicated as shown PetNAD Canine Babesiosis Detection Kit LIMITATIONS A The test should only be used for testing nucleic acid extracted from animal specimen Do not add specimen i e whole blood directly into Premix PetNAD Nucleic Acid Co prep Kit is recommended for nucleic acid extraction Any deviation from recommended procedure may not achieve the optimal results and should be validated by the users Freshly prepared nucleic acid samples within 1 hour after extraction are strongly recommended to be used for PetNAD Canine Babesiosis Detection Kit to achieve optimal results PetNAD Canine Babesiosis Detection Kit OPERATION PROCEDURE A PetNAD Canine Babesiosis Detection Kit Quick Guide Open the Add 50 ul Add 5 ul Premix Pack Premix nucleic and take out Buffer B acid thePremix 1 J extract Mix by pipetting Transfer 50 ul mixture into the R tube 4 Result will show within 1 hour pm was Dom 00606 60 in Spin the Putthe R tube Q Disease Positive R tubes for into the POCKIT amp Disease Negative 10 secs 5 and press RUN 6 gt Use fresh sample and repeat the test B PetNAD Canine Babesiosis Detection
8. r ne tanen enata 10 TROUBLESHOOTING esee 11 PetNAD Canine Babesiosis Detection Kit REFERENCE ipie UR ogee et ee ae 14 11 PetNAD Canine Babesiosis Detection Kit INTENDED USE PetNAD Canine Babesiosis Detection Kit is intended for in vitro detection of Canine Babesia DNA based on the insulated isothermal polymerase chain reaction iiPCR technology This kit is specially designed to be used with a compatible iiPCR instrument POCKIT Nucleic Acid Analyzer The intended user of this kit is veterinarians or lab technicians who have basic laboratory skills This kit is intended for research use SUMMARY AND EXPLANATION Canine Babesiosis is a disease caused 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 c and Babesia gibsoni B g 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 PetNAD Canine Babesiosis Detection Kit Canine Babesia detection by PCR is the most rapid sensitive and specific method Also it can eliminate false negative and positive cases However conventional PCR takes three to four hours and requires delicate machines as w
9. se Negative PetNAD Canine Babesiosis Detection Kit Possible Causes 1 Nucleic acid extraction failed 2 Bad nucleic acid quality or concentration too high 3 PCR inhibition 12 Comment and Suggestions Check nucleic acid extraction procedure Please check the sample storage condition Please refer to the Troubleshooting section of PetNAD Nucleic Acid Co prep Kit If a spectrophotometer is available check OD 260 280 ratio Normally this ratio should be 1 4 to 2 0 Do not add too much nucleic acid Please follow the operation procedure Spike P Standard for a parallel PCR reaction If the one with P Standard showed positive then the inhibition was ruled out If P Standard was negative then there was inhibition User need to prepare another nucleic acid extraction Observations or Problems Solution or other interferences fall into the reaction chamber of POCKIT PetNAD Canine Babesiosis Detection Kit Possible Causes Comment and Suggestions R tube broken or B Consult with GeneReach or your solution spilled in the local distributor reaction chamber of POCKIT 13 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 L S 1975 Some o
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