tacoTM Instrument user manual
Contents
1. WSSV Detection and Prevention System DPS Signals were generated from only WSSV infected not from IHHNV and HPV infected samples in IQ Plus WSSV Kit with POCKIT System E Analytical sensitivity Analysis using standard plasmid pWSSV1 and purified WSSV genomic DNA of known copy numbers shows that the gt 95 detection rate of IQ Plus WSSV Kit with POCKIT System was 23 7 and 16 9 copies pWSSV1 and WSSV DNA respectively per reaction Furthermore analysis of DNA extracted from WSSV infected L vannamei serially diluted with ddH2O or DNA extracts of SPF L vannamei shows that the detection endpoint 10 dilution of IQ Plus WSSV Kit with POCKIT System was similar to that of IQ2000 WSSV DPS 2 Diagnostic Characteristics E Test Cut off Determination IQ Plus WSSV Kit with POCKIT System based on iiPCR and fluorescent probe detection principles is designed to work in an iiPCR compatible instrument POCKIT The cut off for POCKIT device were determined on the basis of fluorescent signal of numerous NTC and positive reactions of PCR assays developed for various targets at GeneReach confidential data Readouts of the results are determined as follows When is displayed on POCKIT the sample is classified as WSSV positive When is displayed on POCKIT the sample is classified as WSSV negative When is displayed on POCKIT the test result is indeterminate and should be repeate2. Temperature Error please check the environmental temperature POCKIT 1 0 0101 01 01 01 01 01 2011 Camera Error please call for service POCKIT 1 0 0101 01 01 01 01 01 2011 Time Sequence Error please call for service POCKIT 1 0 0101 01 01 01 01 01 2011 SD Card Error please check SD card and press OK POCKIT 1 0 0101 01 01 01 01 01 2011 system Error please call for service POCKIT 1 0 0101 01 01 01 01 01 2011 Create Data Directory Error please check SD card and press OK 14 4 9 Switch Off After the reaction switch off the main power and unplug the SD card to read the raw data on a PC or Mac Main power AX Caution Ensure the reaction has been completed before shutting down the device 15 Section 5 Cleaning and Maintenance 5 1 General Care Use only clean and wet paper towel or cotton to wipe out stains in the reaction chamber Do not disassemble the POCKIT for cleaning as 1t will void the warranty Do not use solvent detergent or bleach to clean the POCKIT surface Note Always wear gloves and always switch off and unplug the POCKIT before cleaning 5 2 Dealing with Spills 1 Wipe out visible spills with disposable paper towel immediately and contact your local distributor or GeneReach directly for further assistance 2 Please use ultraviolet lights UV to irradiate and sterilize POCKIT for 15 minutes 16 Troubleshooting Observation or Problems Comments and Suggestion
3. E E E le meseeatene 16 32 Mal SW tla ONS occ 16 MOUSSE ai die 17 Appendix OIE Procedure for Registration of Diagnostic Kits ccccccccnononccnnnnnnnnononononnnnnnnnnnnnnnnnos 18 Symbols Date of manufacturing Manufacturer Catalogue number Serial number Alternative current Consult instruction before use i Risk of personal injury to the operator or a safety hazard to the Caution surrounding area This product is required to comply with the European Union s WEEE symbol Waste Electrical amp Electronic Equipment WEEE Directive 2002 96 EC D4 gt eee BEL Section 1 Introduction The POCKIT Nucleic Acid Analyzer POCKIT is designed for performing insulated isothermal polymerase chain reaction PCR It provides 11PCR based analysis and is equipped with two optical channels 520 nm 550 nm for multiplex detection The qualitative test results are readily displayed on the LCD monitor and automatically saved in a SD card It generates results for up to 8 nucleic acid samples within 1 hour 1 1 Intended Use POCKIT is intended to provide qualitative detection of nucleic acid targets using fluorescence based 11PCR reagents It is intended for in vitro use only and for research purposes Section 2 General Information 2 1 Product Components e POCKIT Nucleic Acid Analyzer x 1 unit e Power Cord x l piece e User Manual x 1 copy e Warranty Card x 1 copy e Holder x piece 2 2 Specifications Dimensio
4. baculovirus infection causing white spot syndrome in cultured penaeid shrimp in Taiwan Dis Aquat Org 23 165 173 5 Chou P H et al 2011 Real time target specific detection of loop mediated isothermal amplification for white spot syndrome virus using fluorescence energy transfer based probes J Virol Methods 173 67 74 6 Lightner D V ed 1996 A handbook of pathology and diagnostic procedures for diseases of penaeid shrimp World Aquaculture Soc Baton Rouge 7 Lightner D V 2011 Virus disease of farmed shrimp in the Western Hemishpere the Americas A review J Invertebr Pathol 106 110 130 8 Lo C F et al 1996a Detection of baculovirus associated with white spot syndrome WSSV in penaeid shrimps using polymerase chain reaction Dis Aquat Org 25 133 141 9 Lo C F et al 1996b White spot syndrome baculovirus WSSV detected in cultured and captured shrimps crabs and other arthropods Dis Aquat Org 27 215 225 10 Lo C F et al 1997 Detection and tissue tropism of white spot syndrome baculovirus WSSV in captured brooders of Penaeus monodon with a special emphasis on reproductive organs Dis Aquat Org 30 53 72 11 Nunan L M et al 1998 The detection of white spot syndrome virus WSSV and yellow head virus YHV in imported commodity shrimp Aquaculture 160 19 30 12 Peng S N et al 1998 Detection of white spot syndrome baculovirus WSSV in giant freshwater prawn Mac
5. IT System Statistical analysis using one tailed binomial test suggested that results from these two experiments defined reference animals and un defined animals agreed with each other 3 Reproducibility Different lots of IQ Plus WSSV Kit with POCKIT System were sent to three different labs located in Chinese Taipei and USA including two OIE Reference Laboratories to be tested Trunk muscles of total of 64 L vannamei samples were aliquoted preserved in 95 ethanol and sent to all three laboratories Each sample was analysed with 3 batches of IQ Plus Y WSSV Kit with POCKIT System Chi square test for homogeneity was conducted to analyse the experimental results generated from the three labs The results showed that there was no difference among different laboratories with three batches of IQ Plus WSSV Kit with POCKIT System 4 Applications The kit is being used worldwide by different laboratories Private and Public 20 C References 1 Armbruster D A and Pry T 2008 Limit of Blank Limit of Detection and Limit of Quantitation Clin Biochem Rev 29 549 552 2 Chang H F G et al 2012 A thermally baffled device for highly stabilized convective PCR Biotechnol J 7 662 666 3 Chang P S et al 1996 Identification of white spot syndrome associated baculovirus WSSV target organs in shrimp Penaeus monodon by in situ hybridization Dis Aquat Org 131 139 4 Chou H Y et al 1995 Pathogenicity of a
6. PICART naciona anaryzer USER MANUAL Validated and certified by the OIE as fit for the J purposes defined in this kit insert provided with this kit Registration number 20130108 Manufacturer GeneReach Biotechnology Corporation TEL 886 4 24639869 FAX 886 4 24638255 No 19 Keyuan 2nd Road Central Taiwan Science Park Taichung City 407 Taiwan E mail sales genereach com Website www iipcr com 2014 07 CONTENT A O PN RTS en RTS TO vem E err ne re nee l SOCOM Le Odu O S T 2 eTe Use T E ae aie a cece E S 2 Section 2 General M Orma a a E 3 ZA PLOGUCE C PO I a noantonupiandidd utaneeuebsan hunt ncanianuatendadttentaneiadaations 3 2 De CNG ALI ONG nit iodo cos Dosis ta ue oa 3 23 Tesne Capac a a a a a 3 2 4 Equipments and Materials Required But Not Provided cccccooooocnccnnnnnnnnnnnnnnccnnnnnnnonnnananonnnos 3 ZO NS A A 4 PORER E 5 SECON 09 1081016193210 ced ba 01 Se a A O 6 Dies ALCL A IN onna O 0 COOPER OP o eo sta as naan ten olde ga adda din Sev esa lad tein ved lado SN ses acs 6 Section 4 OPE rI O si AA A A A aa 7 SO O E 7 42 Choose Detection Wav Clem MASSA 7 AS Set Up Date and TIME a 9 ARAU I INC Ai 10 Ae MIS lay Me INC SUIT iaa 12 KORVEN ME RAW Dal Tenir ches hcpeni test o ben 13 A oa fed gt ANA A A ea A 13 4 9 Errors occurred durin th Operation dd dia 14 KI SWE OE seene N A a a a N 14 Section o Cleanins and Mantenne a st Mews a E Maatd dnedeedeeenneaensanaades 16 A E WANING A Create NEO ce
7. RARARRARARARRARARARRARARARRARARA k Contact your local distributor or GeneReach for assistance 17 Appendix A OI OIE Procedure for Registration of Diagnostic Kits Abstract sheet Name of the diagnostic kit IQ Plus WSSV Kit with POCKIT System Manufacturer Genereach Biotechnology Corporation OIE Approval number 20130108 Date of Registration May 2013 Disease White Spot Disease Pathogen Agent White Spot Syndrome Virus WSSV Type of Assay Insulated isothermal Polymerase Chain Reaction Purpose of Assay Certified by the OIE in May 2013 as fit for the detection of white spot disease in target tissues Shrimp tissue of ectodermal and mesodermal origin of Litopenaeus vannamei and for the following purposes 1 To certify freedom from infection lt 10 virions reaction in individual animals or products for trade movement purposes 2 To confirm diagnosis of suspect or clinical cases confirmation of a diagnosis by histopathology or clinical signs 3 To estimate prevalence of infection to facilitate risk analysis surveys herd health schemes disease control Species and Specimen Litopenaeus vannamei Shrimp tissue of ectodermal and mesodermal origin Information on the kit Information can be found by emailing sales O genereach com or by visiting http www ig2000kit com products_2 php bgid 3 amp gid 6 amp sgid 34 In summary IQ Plus WSSV Kit with POCKIT System was designed for qualitat
8. d E Diagnostic sensitivity DSn and specificity DSp estimates Diagnostic sensitivity was evaluated by comparing the test results of IQ Plus WSSV reaction of positive reference animals which were identified by IQ2000 WSSV DPS Pleopods of 400 WSSV positive samples were sampled and tested in this study Negative reference animals 19 selected by IQ2000 M WSSV DPS were also subjected to analysis by IQ Plus WSSV Kit with POCKIT System 102000 WSSV DPS IQ Plus WSSV Kit with Positive 374 on In summary this validation testing was conducted on 700 samples The results are sensitivity 93 5 95 confidence interval CI 90 61 95 56 specificity 97 0 95 CI 94 31 98 50 Comparative performance See Diagnostic sensitivity DSn and specificity DSp estimates EH Agreement and discrepancies The results revealed that when compared to IQ2000 WSSV DPS with defined reference animals the diagnostic sensitivity of IQ Plus Y WSSV Kit with POCKIT System was 93 5 with a 95 CI of 90 61 95 56 and the diagnostic specificity was 97 0 with a 95 CI of 94 31 98 50 In addition with un defined reference animal IQ Plus WSSV Kit with POCKIT System showed 100 agreement 100 100 for both sensitivity and specificity For this experiment pleopods of 100 un defined shrimps obtained randomly from a local farm were sampled and examined by both IQ2000 WSSV DPS and IQ Plus WSSV Kit with POCK
9. ety Information DO NOT OPEN LID DURING REACTION Do not touch any plug and electric switch with wet hands This product should be placed in a dry clean and ventilated indoor place Strictly keep away from moisture Switch off and unplug the device before moving it Hold the plug while pulling the power cord out from the electrical outlet Do not pull it by the cord directly Do not place the power cord on any hot surfaces Pack the device carefully before moving and delivering Vibration or crash may cause damage Users should revalidate the device after it has been moved Do not look directly into the LED light in the reaction wells Only use consumables and accessories provided from GeneReach Make sure the ambient temperature during reaction is kept at 15 35 C SD card should not be removed until the device is switched off Do not disassemble the device if any problem occurs Contact your local distributor or GeneReach directly for troubleshooting In case of emergency or abnormal conditions turn off the power switch or unplug Do not block access to power switch or plug When working around the device always wear a lab coat disposable gloves and protective goggles Waste materials such as gloves and R tube may be biohazardous Please discard them carefully according to your local regulation Section 4 Operation 4 1 Switch On Plug in the POCKIT and switch on the main power located in the back The device will com
10. ive detection of WSSV DNA based on multiplex insulated isothermal PCR technology MPCR Chang et al 2012 Tsai et al 2012a Tsai et al 2012b IQ Plus WSSV Kit with POCKIT System is designed to be used with a compact and portable iiPCRcompatible instrument POCKIT Nucleic Acid Analyzer POCKIT IQ Plus WSSV Kit with POCKIT System is highly sensitive and specific for WSSV DNA detection from aquaculture specimen and suitable for onsite viral DNA detection Specific primers and probe 520 nm fluorescent signal are designed to detect WSSV DNA in samples In addition internal control IC primers and probe 550 nm fluorescent signal are used to target a house keeping gene of Penaeid shrimps The assay has been simplified for easy and fast operation in POCKIT for pond site WSSV DNA detection 18 B Summary of validation studies 1 Analytical characteristics E Repeatability Various types including negative and positive WSSV infected shrimps L vannamei were selected and three production batches were tested Each sample was tested in quadruplicates per run The data showed 100 agreement among the test results E Analytical specificity WSSV Infectious Hypodermal and Hematopoietic Necrosis Virus IHHNV or Hepatopancreatic Parvovirus HPV infected L vannamei were used to test the specificity of IQ Plus WSSV Kit with POCKIT System The IHHNV and HPV infected samples were confirmed to be WSSV negative by IQ 2000
11. lid and press Run to start the reaction POCKIT 1 0 0101 01 01 01 01 01 2011 System Ready CH 520 550 nm Note Ensure the holder with R tubes completely fits into the reaction wells A Caution Do not look directly into LED lights in the reaction wells 3 The screen will display the remaining reaction time and the folder name of the reaction POCKIT 1 0 0101 01 01 01 01 01 2011 Remaining 52 min CH 520 550 nm Folder 201101 20110101 010101 MX Caution Should any of the circumstances below occur the results are unreliable E The lid is opened or the SD card is removed during the POCKIT reaction E The ambient temperature is not in the range of 15 35 C during the reaction E The variation of ambient temperature is greater than 5 C during the reaction 10 4 When the reaction complete a beeping tone is made from POCKIT Press Buzzer OFF to turn off the sound POCKIT 1 0 0101 01 01 01 01 01 2011 Reaction Completed 12 3 4 5 6 520m O 550m O O 8 2 Note If there is no beep sound after the completion of the reaction please see Troubleshooting 5 If the reaction temperature is abnormal during the reaction 1t will show Temperature Warning on the screen POCKIT 1 0 0101 01 01 01 01 01 2011 Reaction Completed 12 3 4 5 6 7 8 zm OOO sm BBB Buzzer OFF Caution Fluctuation of ambient temperature should not be more than 5 C during
12. ns 280 mm W x 250 mm D x 85 mm H Net Weight 2 1 kg Power Supply 100 120 200 240 V AC 50 60 Hz 2A Fuses 2A 250V AC Q 3 x 20 mm fast acting low breaking capacity glass tube fuses Operating Temperature 15 35 C 2 3 Testing Capacity Number of Samples 1 8 reactions per run Reaction Volume 45 55 ul per reaction 2 4 Equipments and Materials Required But Not Provided Nucleic acid extraction kit Nucleic acid amplification reagents and kits 200 ul and 1000 ul micropipette tips and 1 5 ml tubes R tubes and Caps cubee mini centrifuge Disposable gloves Note To avoid power failure connecting POCKIT to an uninterrupted power system UPS and or voltage regulator 1s highly recommended 2 5 Front View reaction chamber A touchscreen where users key in the operation commands 4 Control panel and view the results A slot where users insert the SD card to run POCKIT and 5 SD card slot i save reaction raw data Load R tubes into the reaction wells 2 6 Rear View a Pm m mia la do es r l a SS a dl AAA A T To turn on off the main power To connect to the power cord Section 3 Important Notes Check the device upon receiving If any items are missing or harmed please contact your local distributor or GeneReach immediately for replacement Do not use any damage items as they may lead to poor performance of the test or harm the user 3 1 Saf
13. plete self testing within 5 minutes POCKIT 1 0 0101 01 01 01 01 01 2011 PILILKIT Self Testing 4 2 Choose Detection Wavelength 1 After self testing choose the detection wavelength according to the kits or methods used and press OK POCKIT 1 0 0101 01 01 01 01 01 2011 O B20nm O 550nm e 520nm 550 nm 2 System Ready will be shown after choosing the detection wavelength POCKIT 1 0 0101 01 01 01 01 01 2011 System Ready CH 520 550 nm 3 Before the reaction starts to change the detection wavelength press CH Select and choose again POCKIT 1 0 0101 01 01 01 01 01 2011 System Ready CH 520 550 nm 4 3 Set up Date and Time 1 Press Time Setting POCKIT 1 0 0101 01 01 01 01 01 2011 System Ready CH 520 550 nm 2 Touch the numbers that needs to be changed on the screen Press Up and Down buttons to select the desired number Press OK to confirm and return to the System Ready screen Press Cancel to quit and leave the Time Setting screen POCKIT 1 0 0101 01 01 01 01 01 2011 2011 Year 01 Month 01 Day 01 Hour 01 Min 01 Sec Note The data derived from each test is stored in the SD card and in a folder named after the devices default date and time when the user switches on the analyzer 4 4 Run PCR Reaction 1 Place R tubes into a holder 2 Place the holder with R tubes into the reaction well Close the
14. robrachium rosenbergii using polymerase chain reaction Aquaculture 164 253 262 13 Tsai Y L et al 2012a Detection of white spot syndrome virus by polymerase chain reaction performed under insulated isothermal conditions J Virol Methods 181 134 137 14 Tsai Y L et al 2012b Development of TaqMan Probe Based Insulated Isothermal PCR G1PCR for Sensitive and Specific On Site Pathogen Detection PLoS ONE 7 9 e45278 doi 10 1371 journal pone 0045278 15 Wang C H et al 1995 Purification and genomic analysis of baculovirus associated with white spot syndrome WSSV of Penaeus monodon Dis Aquat Org 23 239 242 16 Thakur P C F et al 2002 Estimation of prevalence of white spot syndrome virus WSSV by polymerase chain reaction in Penaeus monodon postlarvae at time of stocking in shrimp farms of Karnataka India a population based study Dis Aquat Org 49 235 243 17 Wongteerasupaya C et al 1995 A non occluded systemic baculovirus that occurs in cells of ectodermal and mesodermal origin and causes high mortality in the black tiger prawn Penaeus monodon Dis Aquat Org 21 69 77 18 Alan Agresti Categorical Data Analysis 2nd edition Wiley Press 2002 21
15. s a Self test runs more than 5 minutes Re start the device 1 Contact your local distributors or GeneReach for assistance nn rr _ a ee E E ml 1 and repeat the reaction If any further problems occur please contact your local distributor or GeneReach for assistance Da gt iaa 1 Contact your local distributor or GeneReach for assistance 1f the problem persists a Pi Tama a ia system Replace fuse 1 Contact your local distributor or GeneReach for assistance n sia Tae ce Ga ee assistance co cae hn ies cece e RI ET ee reaction Contact your local distributor or GeneReach for assistance oe La Contact your local ee or GeneReach for assistance a tes ae ee es ee da assistance hihih G Create data directory error Check storage space is available in SD card Keep number of files folders below 512 in each directory 1 Contact your local distributor or GeneReach for assistance ETT E mm re gt e n assistance AAARARARAPRARARARRARARNARARAANARARAR US GAP RRD PEPO NARARAAAARARPARARRAPAAARARARAPRARARAARARARAPAARARAPRARARAPRARAAAPARAPAAPARARARAPRARARARAARARAPRARARAPRARARAPAAR ARAPA ARAPAARARAPARAARAPRARARAPAARARARRRRARARRAEAIIIRRIRIAARRIRIAAARARARAARARARRARARARRARARARRARARARRARARARRARARARRARARARAARARARAARARARAARARARRARARARRARARARRARARARAARARARRARARARRARARARRARARARAARARARAARARARAARARARAARARARA AARAA ARAA AARARARA AARAA AA RARARAARARARRARARARRARARARRARARARRARARARRARARARRR
16. the reaction 11 4 5 Display the Result 1 An example of results displayed at the end of a single detection wavelength either 520 nm or 550 nm program POCKIT 1 0 0101 01 01 01 01 01 2011 POCKIT 1 0 0101 01 01 01 01 01 2011 Reaction Completed Reaction Completed 123 4 5 6 7 8 123 4 5 6 7 8 520m O A om SO 00000A Buzzer OFF Buzzer OFF 2 An example of results displayed at the end of a double detection wavelength 520 nm 550 nm program POCKIT 1 0 0101 01 01 01 01 01 2011 Reaction Completed 1 2 345678 somm O 0000A som OOO O0O0O0OOA Buzzer OFF 3 Description of the results O eni CA Vii Note Please interpret the results according to the user manual of the kits used Note When you see a A symbol please contact your local distributors or GeneReach for assistance 12 4 6 Review the Raw Data 1 Insert the SD card on a PC or Mac Find the raw csv file in the folder of the reaction 2 Please refer to the kit manual for data interpretation 4 7 Exit the Experiment 1 Press Abort to pause the reaction POCKIT 1 0 0101 01 01 01 01 01 2011 Remaining 52 min CH 520 550 nm Folder 201101 20110101 010101 2 Press Yes to confirm and return to the self testing screen or press No to continue the reaction POCKIT 1 0 0101 01 01 01 01 01 2011 13 4 8 Errors occurred during the operation POCKIT 1 0 0101 01 01 01 01 01 2011
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