IQ Plus™ WSSV Kit with POCKIT System
Contents
1. E Please repeat the test E Check the expiration date and storage condition E Check to make sure the Premix pellet is dry and has not been rehydrated before use E Do not add too much nucleic acid Please follow the recommendation in this user manual E Spike 5 ul nucleic acid sample into a positive 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 E We recommend using aerosol free tips E Replace reagent s E Consult with GeneReach for lab clean up IQ Plus WSSV Kit with POCKIT System Problems Possible causes Comments or solution Reaction solution or 1 R tube broken or solution W Please contact local other objects found spilled in the reaction distributor for further in the reaction chamber of the assistance chamber of the POCKIT Nucleic Acid POCKIT Nucleic Analyzer Acid Analyzer IQ Plus WSSV Kit with POCKIT System REFERENCE 1 Chang PS Lo CF Wang YC Kou GH 1996 Identification of white spot syndrome associated baculovirus WSSV target organs in shrimp Penaeus monodon by in situ hybridization Dis Aquat Org 27 131 139 2 Chang H G Tsai Y Tsai C Lin C Lee P Teng P et al 2012 A thermally baffled device for highly stabilized convective PCR Biotechnology Journal 7 5 662 666 doi 10 1002 biot 201100453 3 Chou HY Hu2. Validated and certified by the OIE as fit for the y purposes defined in this kit insert provided with this kit Registration number 20130108 IQ Plus WSSV Kit with POCKIT System For White Spot Syndrome Virus Detection Manufacturer Address TEL FAX Web Site User Manual in vitro use only 2014 11 GeneReach Biotechnology Corporation No 19 Keyuan 2 Road Central Taiwan Science Park Taichung City 407 Taiwan 886 4 24639869 886 4 24638255 gt www iq2000kit com IQ Plus WSSV Kit with POCKIT System INTENDED USE IQ Plus WSSV Kit with POCKIT System uses insulated isothermal polymerase chain reaction PCR technology to detect the DNA of white spot syndrome virus WSSV Chang et al 2012 Tsai et al 2012 This detection kit is specially designed to be used on an uPCR compatible instrument POCKIT Nucleic Acid Analyzer The intended users of this detection kit are aquaculture technicians who have basic laboratory skills This detection kit is intended for in vitro use only SUMMARY AND EXPLANATION White spot syndrome virus WSSV is a major shrimp disease which has caused high mortality rate and economic losses to major shrimp farming countries It is a pathogen found in different penaeid shrimp species including P monodon P japonicus and L vannamei as well as other crustaceans such as crab and crayfish WSSV is highly lethal and contagious Within only few days of WSSV
3. 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 PCR and fluorescent probe detection principles is designed to work in an 1PCR compatible instrument POCKIT The cut off for POCKIT device were determined on the basis of fluorescent signal of numerous NTC and positive reactions of 11PCR 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 repeated 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 IQ Plus WSSV Kit with POCKIT System samples were sampled and tested in this study Negative reference animals selected by IQ2000 WSSV DPS were also subjected to analysis by IQ Plus WSSV Kit with POCKIT System 1Q2000 WSSV DPS Positive Negative IQ Plus WSSV Positive 374 9 Kit with POCKIT System Negative 26 291 In summa
4. e45278 doi 10 1371 journal pone 0045278 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 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 18 IQ Plus WSSV Kit with POCKIT System 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 Alan Agresti Categorical Data Analysis 2nd edition Wiley Press 2002
5. sub optimal results Performance of the modified protocol should be validated by the users 4 Itis strongly recommended to use freshly prepared nucleic acids within 1 hour after extraction to achieve optimal results with 1Q Plus WSSV Kit with POCKIT System IQ Plus WSSV Kit with POCKIT System SAMPLE TYPE This detection kit is intended for analyzing nucleic acids extracted from shrimp tissue as below Pleopod of broodstocks or juvenile shrimps Post larvae PL OPERATION PROCEDURE 1 2 3 4 NOTE Before preparing the reactions for iiPCR testing turn on POCKIT Nucleic Acid Analyzer to initiate the calibration for the instrument The device will complete self test within 5 minutes Please refer to the user manual of POCKIT Nucleic Acid Analyzer 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 Label R tube s in the labeling area Prepare one Premix for each sample Premix tubes are in Premix Pack Each Premix Pack contains eight Premix tubes E NOTE When the pellet is not found at the bottom of the tube spin tube briefly to bring it down Add 50 ul Premix Buffer B to each Premix tube Use the inoculating loop take nucleic acid extracts or dissolved P Control into each Premix tube Spin Premix tubes briefly in a mini centrifuge such as cubee Mini Centrifuge E NOTE Please
6. 12 13 14 15 16 IQ Plus WSSV Kit with POCKIT System Rouge Lightner D V 2011 Virus disease of farmed shrimp in the Western Hemishpere the Americas A review J Invertebr Pathol 106 110 130 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 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 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 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 Peng S N et al 1998 Detection of white spot syndrome baculovirus WSSV in giant freshwater prawn Macrobrachium rosenbergii using polymerase chain reaction Aquaculture 164 253 262 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 Tsai Y L et al 2012b Development of TaqMan Probe Based Insulated Isothermal PCR iiPCR for Sensitive and Specific On Site Pathogen Detection PLoS ONE 7 9
7. 550m OOO 072060 Buzzer OFF 520 nm 550 nm Interpretation WSSV positive WSSV positive WSSV negative E For shrimp tissue please recheck see Troubleshooting E For non shrimp tissue such as pond dirt the sample shows WSSV negative result WSSV positive Repeat reaction with freshly prepared nucleic acid Repeat reaction with freshly prepared nucleic acid Repeat reaction with freshly prepared nucleic acid OOOO 8 099 00O9O 0 909 Repeat reaction with freshly prepared nucleic acid IQ Plus WSSV Kit with POCKIT System ANALYTICAL SENSITIVITY 1 The detection limit of IQ Plus WSSV Kit with POCKIT System is up to 10 copies reaction 2 The sensitivity of IC of IQ Plus WSSV Kit with POCKIT System is 20 ng genomic nucleic acid reaction IQ Plus WSSV Kit with POCKIT System TROUBLESHOOTING Problems Possible causes Comments or solution Negative in 550 nm and 520 nm internal control is negative No template control showed positive result 1 Poor nucleic acid quality 2 Premix pellet was not reconstituted to correct volume 3 No nucleic acid added 4 Deterioration of the reagents 5 PCR inhibition 1 Micropipette contaminated 2 Reagent contaminated 3 Lab contaminated E Please see the Troubleshooting section in the IQ Plus Extraction Kit manual E Check the preparation procedure of the Premix
8. repeatedly dip the inoculating loop into solution three times to collect the correct solution volume IQ Plus WSSV Kit with POCKIT System 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 Nucleic Acid Analyzer 8 Spin tube holder set briefly in cubee Mini Centrifuge to make sure all solution is collected at the bottom of R tube E NOTE Make sure there are no bubbles in the solution E NOTE Start reaction within 1 hour to prevent nucleic acid degradation to prevent nucleic acid degradation and non specific reaction 9 POCKIT Nucleic Acid Analyzer reaction a Select 520 nm 550 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 the reaction is complete IQ Plus WSSV Kit with POCKIT System DATA INTERPRETATION 520 nm fluorescent signal is used to detect nucleic acid sequence of virus 550 nm fluorescent signal as the internal control IC is used to target a house keeping gene of Penaeid shrimps The following example is 1PCR reaction results shown on the monitor POCKIT 1 0 1020 20 33 15 01 09 2012 a Completed 2 3 456 7 8 z2m0O0000000
9. ang CY Wang CH Chiang HC Lo CF 1995 Pathogenicity of a baculovirus infection causing white spot syndrome in cultured penaeid shrimp in Taiwan Dis Aquat Org 23 165 173 4 Lightner DV ed 1996 A handbook of pathology and diagnostic procedures for diseases of penaeid shrimp World Aquaculture Soc Baton Rouge Section 3 11 5 Lo CF Ho CH Chen CH Liu KF Chiu YL Yeh PY Peng SE Hsu HC Liu HC Chang CF Su MS Wang CH Kou GH 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 6 Lo CF Ho CH Peng SE Chen CH Hsu HC Chiu YL Chang CF Liu KF Su MS Wang CH Kou GH 1996 b White spot syndrome baculovirus WSSV detected in cultured and captured shrimps crabs and other arthropods Dis Aquat Org 27 215 225 7 Lo CF Leu JH Ho CH Chen CH Peng SE Chen YT Chou CM Yeh PY Huang CJ Chou HY Wang CH and Kou GH 1996 a Detection of baculovirus associated with white spot syndrome WSSV in penaeid shrimps using polymerase chain reaction Dis Aquat Org 25 133 141 8 Peng SN Lo CF Ho CH Chang CF Kou GH 1996 Detection of white spot syndrome baculovirus WSSV in giant freshwater prawn Macrobrachium rosenbergii using polymerase chain reaction Aquaculture 164 253 262 9 Tsai Y Wang H T Chang H G Tsai C Lin C Teng P et al 2012 Development o
10. ble instrument for IQ Plus Detection Kit 2 cubee Mini Centrifuge 3 Micropipette and filter tips IQ Plus WSSV Kit with POCKIT System 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 PRECAUTIONS 1 Do not open R tube s after reaction to prevent any carryover contamination 2 Perform extraction and amplification in two independent spaces to minimize contamination 3 Bring Premix Pack to room temperature before use 4 Do not reuse R tube and Premix 5 Include the P Control to Ensure POCKIT Nucleic Acid Analyzer is working Ensure detection kit performance after storage 6 To get optimal fluorescence detection Labelinsana Wear powder free gloves to handle R tubes Detection area Do not label in the detection area of R tube eS IQ Plus WSSV Kit with POCKIT System LIMITATION 1 IQ Plus Extraction Kit or taco mini Automatic Nucleic Acid Extraction System is recommended for nucleic acid extraction 2 The test should only be used for testing nucleic acid extracts Do not add specimens directly into the Premix 3 Any deviation from the recommended procedures may lead to
11. eping gene of Penaeid shrimps The assay has been simplified for easy and fast operation in POCKIT for pond site WSSV DNA detection 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 IQ2000 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 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 pWSSVI 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
12. f TaqMan Probe Based Insulated Isothermal PCR iiPCR for Sensitive and Specific On Site Pathogen Detection PLoS ONE 7 9 e45278 doi 10 137 1 journal pone 0045278 IQ Plus WSSV Kit with POCKIT System 10 Wang CH Lo CF Leu JH Chou CM Yeh PY Chou HY Tung MC Chang CF Su 11 MS Kou GH 1995 Purification and genomic analysis of baculovirus associated with white spot syndrome WSSV of Penaeus monodon Dis Aquat Org 23 239 242 Wongteerasupaya C Vickers JE Sriurairatana S Nash GL Akarajamorn A Boonsaeng V Panyim S Tassanakajon A Withyachumnarnkul B Flegel TW 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 IQ Plus WSSV Kit with POCKIT System APPENDIX Oie 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
13. 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 A Information on the kit Information can be found by emailing sales genereach com or by visiting http www iq2000kit com products_2 php bgid 3 amp gid 6 amp sgid 34 IQ Plus WSSV Kit with POCKIT System In summary IQ Plus WSSV Kit with POCKIT System was designed for qualitative detection of WSSV DNA based on multiplex insulated isothermal PCR technology 1iPCR 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 1PCRcompatible 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 ke
14. infection mass mortalities occur and can affect the entire populations of many shrimp farms resulting in large economic losses to the shrimp farming industry Since there is no effective therapeutic method available the only way to prevent WSSV infection is through screening Various molecular methods such as microtomy immuo assay hybridization and PCR for WSSV detection have been developed At present PCR method is recognized to be the most effective diagnostic tool for WSSV GeneReach has developed IQ Plus WSSV Kit with POCKIT 1 IQ Plus WSSV Kit with POCKIT System System based on 11PCR technology which is highly sensitive and specific for WSSV detection IQ Plus WSSV Kit with POCKIT System is specially designed for on site viral detection in the farm The assay has been simplified for easy and fast operation with the use of compact and portable equipment for WSSV detection at pond side The validation data for this kit have been certified by the OIE based on expert review as fit for 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 PRINCIPLE OF THE PROCEDURE The assay is based on multiplex 1 PCR for qua
15. litative detection of white spot syndrome virus WSSV Fluorogenic probe hydrolysis chemistry is used to generate fluorescent signal when a specific DNA sequence of WSSV is amplified The primers and probe target specific sequences of WSSV and do not react with nucleic acids of other pathogens In addition internal control IC primers and probe are used to target a house keeping gene of Penaeid shrimps IQ Plus WSSV Kit with POCKIT System PRODUCT DESCRIPTION A Materials Provided 1 IQ Plus WSSV Kit with POCKIT System 48 tests kit Component Contents Amount WSSV Premix E Vials with lyophilized pellet 6 individually sealed zip Pack containing dNTPs WSSV lock packs 8 vials pack specific primers fluorescent probes and enzyme E Desiccating agent pack Premix Buffer B E Reaction buffer to re dissolve 2 vials 1 3 ml vial the lyophilized pellet WSSV P E Dried plasmid pellet containing 1 vial Control WSSV partial sequence as positive control P Control E Reaction buffer to re dissolve 1 vial 110 pl vial Buffer the WSSV P Control Inoculating Loops 3 packs 20 pieces pack User Manual 1 copy Operation Guide 1 copy 2 IQ Plus Extraction Kit 50 tests kit Please refer to the IQ Plus Extraction Kit user manual for details 3 R tube 48 tubes pack B Materials and Equipment Required but Not Provided 1 POCKIT Nucleic Acid Analyzer the 11PCR compati
16. ll three laboratories Each sample was analysed with 3 batches of IQ Plus 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 References 1 Armbruster D A and Pry T 2008 Limit of Blank Limit of Detection and Limit of Quantitation Clin Biochem Rev 29 S49 S52 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 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 10 11
17. ry 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 E Comparative performance See Diagnostic sensitivity DSn and specificity DSp estimates E Agreement and discrepancies The results revealed that when compared to IQ2000 WSSV DPS with defined reference animals the diagnostic sensitivity of IQ Plus 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 POCKIT 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 IQ Plus WSSV Kit with POCKIT System 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 a
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