User Manual (201411)
Contents
1. ANALYTICAL SENSITIVITY s ssssssesesesesososoesessososeeeeseseso 10 TROUBLESHOOTING eteren esseri argen been sees xu eC E co SE REV Un ERU UE 11 30 0d DL Dior 13 PetNAD Chlamydophila felis Detection Kit INTENDED USE PetNAD Chlamydophila felis Detection Kit is intended for in vitro detection of the Chlamydophila felis C felis DNA 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 the window period between time of infection and detection PetNAD Chlamydophila felis Detection Kit SUMMARY AND EXPLANATION C felis is a gram negative rod shaped bacterium that infects the conjunctiva causing conjunctivitis and upper respirator2. PetNAD Chlamydophila felis Detection Kit 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 11 PetNAD Chlamydophila felis Detection Kit Content INTENDED USE ssccssssssscesccnscccccsscnscsecsscsssssscescsssseneesesssseneeeoes 1 SCIENTIFIC MEANINGS ccsccsssssssscsscessesscssccsscsessscessessesseseeees 1 SUMMARY AND EXPLANATION cccssssssssscsssessescessesserscsseees 2 PRINCIPLES OF THE PROCEDURE ssssesessessesesoeseesesose 3 PRODUCT DESCRIPTION ssccscsscsscssssssssssscsscssssencesssesseseeees 3 A Materials Provided ai kaaa 3 B Materials and Equipments Required but Not Provided 4 C Storage and Stability wihi 4 D Sample Type eint EU tire OUR 4 RECOMMENDED NUCLEIC ACID EXTRACTION METHODS S Eee VENIR ERE Pa HERES LOS o PETER WA BURN AES AA TA NER NUS 5 Ld 0 89 VO M Jo gen et naisikia 5 LIMITATIONS sisimizi aina 6 PROCEDURE ssccsctouisescsatussccusssceceustsseustotdencenssustasuivoceceussaupictoniscccanseoes 7 A PetNAD Chlamydophila felis Detection Kit Quick Guide 7 Bi Procedure uve usi eure Hee 8 DATA INTERPRETATION cccssssssscsssssscssssssssscsssessersnssesseeees 10 PetNAD Chlamydophila felis Detection Kit
3. 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 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 l 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 AA AAA 1 4 Contaminated i M Consult with a GeneReach working area i technical support representative on how to clean up working area 11 PetNAD Chlamydophila felis 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 E 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 a contamination reaction from R technical support repr
4. Type DNA extracted from nasopharyngeal swab PetNAD Chlamydophila felis 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 Chlamydophila felis Detection Kit 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 devia
5. and other feline upper respiratory pathogens PRODUCT DESCRIPTION A Materials Provided 24 tests kit Component Contents or Purpose Premix Pack a C felis Premix lyophilized 24 bags 1 C felis pellet containing dNTPs Premix vial and 1 primers probe and enzyme desiccating for amplification agent bag E Desiccating agent pack Premix Buffer M Reaction buffer to re dissolve 2 vials 1 3 ml vial B the lyophilized pellet P Control E Dried plasmid containing vial Parvovirus partial sequence as positive control P Control a Reaction buffer to re dissolve 1 vial 110 pl vial Buffer P Control PetNAD Chlamydophila felis Detection Kit 1 bag 24 pieces bag 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 PetNADTM 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 D Sample
6. esentative or of amplicons tube into reaction local distributor in reaction chamber of chamber of POCKIT POCKIT PetNAD Chlamydophila felis Detection Kit REFERENCE 1 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 2 Bridger J amp Russell P 2007 Virology Study Guide Royal Veterinary College 3 Ettinger S J amp Feldman E C 2000 Textbook of Veterinary Internal Medicine Diseases of the Dog and Cat Volume 2 Fifth Edition W B Saunders Company Merck amp Co 2008 The Merck Veterinary Manual Eighth Edition Merial 4 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 PCR for sensitive and specific on site pathogen detection PLoS ONE 7 9 e45278 doi 10 1371 journal pone 0045278
7. r 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 POCKIT 8 Spin tube briefly in cubee to make sure all solution is PetNAD Chlamydophila felis 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 Chlamydophila felis Detection Kit DATA INTERPRETATION One example of results shown on the monitor 520 nm Interpretation c C felis Positive Se C felis Negative 2 Repeat reaction with freshly prepared nucleic acid ANYLYTICAL SENSITIVITY The detection limit of PetNAD Chlamydophila felis Detection Kit is about 10 copies reaction 10 PetNAD Chlamydophila felis Detection Kit TROUBLESHOOTING
8. tions from the recommended procedure may lead to suboptimal results Quality of the extracts should be validated by the users D For PetNAD Chlamydophila felis Detection Kit it is strongly recommended to use freshly prepared nucleic acid within 1 hour after extraction to achieve optimal results PetNAD Chlamydophila felis Detection Kit PROCEDURE A PetNAD Chlamydophila felis 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 pil mixture into Spin R tube for 10 seconds R tube 4 5 H Results are shown on WA monitor in 1 hour vici Put R tube into Nucleic Acid Analyzer and press RUN 6 PetNAD Chlamydophila felis 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 Buffe
9. y infections Co infection of C felis with feline herpesvirus and feline calicivirus leads to the feline respiratory complex disease also known as cat flu Transmission is through direct contact with conjunctival or nasal secretion from infected cats Margie et al 2013 Clinical signs include conjunctival congestion ocular discharge blepharospasm sneezing and nasal discharge C felis infection is commonly seen at sites keeping large numbers of cats such as breeding catteries and shelters Wills et al 1987 PCR is one of the most commonly accepted method that provides high sensitivity and specificity for C felis 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 Chlamydophila felis 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 Chlamydophila felis 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 outer membrane protein ompA gene and do not cross react with nucleic acid from host
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