VLA TaqMan® Influenza A/H5/H7 Detection Kits Protocol (PN
Contents
1. 28 Chemical waste safety 29 Biological hazard safety 31 Chemical AlenS iuc du aee ea 31 Documentation 39 Related documentation w 33 VLA Influenza A H5 H7 Detection Kits Protocol Contents vi VLA TagMan Influenza A H5 H7 Detection Kits Protocol Preface Safety information Note For general safety information see this Preface and Appendix B Safety on page 27 When a hazard symbol and hazard type appear by a chemical name or instrument hazard see the Safety Appendix for the complete alert on the chemical or instrument Refer to the Instrument User Manual provided with the relevant instrument for user related instrument safety information Safety alert words Four safety alert words appear in Applied Biosystems user documentation at points in the document where you need to be aware of relevant hazards Each alert word IMPORTANT CAUTION WARNING DANGER implies a particular level of observation or action as defined below IMPORTANT Indicates information that is necessary for proper instrument operation accurate chemistry kit use or safe use of a chemical CAUTION Indicates a potentially hazardous situation that if not avoided may result in minor or moderate inj2. Pipetting error no positive control added Repeat the assay Make sure to pipette the positive control into all positive control wells VLA Influenza A H5 H7 Detection Kits Protocol 23 VLA TagMan Influenza A H5 H7 Detection Kits Troubleshooting Observation Possible cause Action Replicate results for sample are Your instrument may have intrinsic If more than two replicates yield the same inconsistent well to well variability result for example you ran three replicates and two replicates are negative but one replicate is positive the result associated with the larger number of replicates is probably accurate However your laboratory protocol may dictate that you repeat the assay using fresh samples and reagents If you ran only two replicates and results are not consistent repeat the assay using fresh samples and reagents If instrument variability is suspected try repeating the assay at a different position in the heating block In addition check the lamp status on the instrument No target specific signal High background noise detected background noise crosses the threshold Manually set the threshold slightly above any negative control signal However setting the threshold too high above background noise may result in decreased detection sensitivity of positive samples If the new threshold does not cross the exponential phase in the positive wells decrease the thres
3. Applied Protocol AS Biosystems Copyright 2008 2010 Applied Biosystems rights reserved This product is for Research Use Only Not for use in diagnostic procedures This product is not cleared or approved by the United States Food and Drug Administration or licensed by the United States Department of Agriculture and is not for sale or use in the United States except under a special research use only certification Contact Applied Biosystems for details Not for sale or use in Japan Information in this document is subject to change without notice Applied Biosystems assumes no responsibility for any errors that may appear in this document APPLIED BIOSYSTEMS DISCLAIMS ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT EXPRESSED OR IMPLIED IN CLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE IN NO EVENT SHALL APPLIED BIOSYSTEMS BE LIABLE WHETHER IN CONTRACT TORT WARRANTY OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL INCIDENTAL INDIRECT PUNITIVE MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT INCLUDING BUT NOT LIMITED TO THE USE THEREOF For The VLA TaqMan Influenza A H5 H7 Detection Kits NOTICE TO PURCHASER LIMITED LICENSE Use of this product is covered by US patent claims and corresponding patent claims outside the US The purchase of this product includes a limited non transferable immunity from suit under the foregoing patent claims for usi
4. IMPORTANT Do not remove the desiccant from the storage pouch 2 Remove the appropriate number of individual tubes or 8 tube strips one tube for each reaction you plan to run IMPORTANT Applied Biosystems recommends performing three replicates for each sample 3 Place tubes on ice or in cold block 4 Seal the storage pouch using the zip lock strip and store at 2 C to 8 C Prepare samples and Nuclease free water 1s provided as a negative control and to dilute samples and controls controls The recommended RNA concentration is between 100 to 10 000 copies per reaction 1 12 Thaw all reaction components completely IMPORTANT Keep all tubes on ice For each type of sample or control to be analyzed label a sterile microcentrifuge tube with the sample name Calculate the volume of components needed for the number of diluted samples sample replicates and controls for one assay The table below lists volumes for three sample types unknown RNA sample negative control and RNA positive control Multiply volumes by an appropriate factor for multiple replicates Note Applied Biosystems recommends performing three replicates for each sample the positive control and the no template control 30 uL total volume per tube VLA TaqMan Influenza A H5 H7 Detection Kits Protocol Combine diluted samples and controls with assay beads Next step VLA TaqMan Influenza A H5 H7 Detection Kits Protocol VLA
5. 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 Cycle Number Figure 7 Negative result FAM absent VIC present Amplification plots of target specific FAM and IPC specific VIC signals were generated on the 7500 Fast System VLA TaqMan Influenza A H5 H7 Detection Kits Protocol Troubleshooting VLA TagMan Influenza A H5 H7 Detection Kits Troubleshooting Observation Possible cause Action No IPC VIC or target specific FAM dye signal is detected in unknown wells RT reverse transcription or PCR inhibited Repeat the sample preparation then repeat the assay If RT or PCR remains inhibited dilute the sample for example 1 5 or 1 10 to dilute inhibitors or use alternate RNA purification procedure TagMan Influenza Assay Beads and reagents not stored properly Repeat the assay using properly stored assay components Protect the assay beads from lights IPC signal is greater than 36 in unknown or positive controls wells or PCR inhibited Repeat the sample preparation then repeat the assay If RT or PCR remains inhibited dilute the sample for example 1 5 or 1 10 to dilute inhibitors or use alternate RNA purification procedure IPC signal is inconsistent between unknown wells Your instrument may have intrinsic well to well variability Repeat assay at different position in the heating block No IPC signal is d
6. Influenza A H5 H7 Detection Kits Prepare the RT PCR reactions RNA positive Component Unknown RNA control Negative control 1 4 1 4 1 4 replicate replicates replicate replicates replicate replicates Sample 10 uL 40 uL 10 uL 40 uL Nuclease 20 uL 80 uL 20 uL 80 uL 30 uL 120 uL free water Total 30 uL 120 uL 30 uL 120 uL 30 uL 120 uL 4 Prepare the reagent mix a Pipette the appropriate volumes of components into the labeled sample tube from step 2 on page 12 b Mix the components by vortexing c Briefly spin down tube contents using a microcentrifuge IMPORTANT Keep all tubes on ice Repeat step 3 and step 4 above to prepare a reagent mix for each remaining sample or control Examine the assay beads in the 8 tube strips Gently tap the tubes as needed to move all assay beads to the bottom of all tubes Carefully remove the colored caps from the 8 tube strips containing the assay beads to avoid disturbing the beads from the bottom of the tubes Discard the colored caps IMPORTANT Avoid disturbing the beads from the bottom of the tubes IMPORTANT Do not use colored caps or tubes for kit reactions Colored caps or tubes are not compatible with real time PCR For each sample or control transfer 30 uL of the reagent mix prepared above in step 4 into a tube containing the appropriate influenza assay bead Beads dissolve in 1 to 5 seconds IMPORTA
7. on page viii Real time PCR system Document PN All real time PCR VLA TaqMan Influenza A H5 H7 Detection Kits Quick Reference Card 4400541 Systems 7900 Fast System Applied Biosystems 7900 Fast Real Time PCR System Absolute Quantitation 4364014 Using Standard Curve Getting Started Guide 7300 7500 and Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Absolute 4347825 7500 Fast Systems Quantitation Using Standard Curve Getting Started Guide StepOne and Applied Biosystems StepOne and StepOnePlus Real Time Systems 4376787 StepOnePlus Systems Presence Absence Experiments Getting Started Guide Portable document format PDF versions of this guide and the documents listed above are available at www appliedbiosystems com Note To open the documentation available from the Applied Biosystems web site use the Adobe Acrobat Reader software available at www adobe com VLA TaqMan Influenza A H5 H7 Detection Kits Protocol 33 Documentation Related documentation 34 VLA TaqMan Influenza A H5 H7 Detection Kits Protocol Part Number 4400271 Rev 06 2010 A p i e d Headquarters International Sales Bi t 850 Lincoln Centre Drive Foster City CA 94404 USA For our office locations please call the division 105 5 ems Phone 650 638 5800 Toll Free 800 345 5224 headquarters or refer to our Web site at www appliedbiosystems com www appliedbiosystems com about offices cfm
8. overview probes The TaqMan probe contains a fluorescent reporter dye at the 5 end of the probe and a quencher dye at the 3 end of the probe When the probe is intact the proximity of the reporter dye to the quencher dye suppresses the reporter fluorescence Probe cleavage during the PCR reaction spatially separates the reporter dye from the quencher moiety and allows detection of the reporter dye fluorescence Reverse Primer extends on RNA d 5 cDNA 1st cDNA strand is synthesized 8 5 CONA Primer extends on cDNA __ OUO 5 M 2nd cDNA strand is synthesized a SE 5 D e PCR Forward and reverse primers anneal and extend probe hybridizes bi p 3 gt 5 bi EOU 3 ea 5 DNA polymerase cleaves probe 5 __ e SSS 5 5 Fluorescence d ji 3 5 5 ___ iiiziiii 2 vuoi Wife 5 Figure 1 Reverse transcription polymerase chain reaction RT PCR VLA TaqMan Influenza A H5 H7 Detection Kits Protocol Materials and equipment Kit contents VLA TagMan Influenza A H5 H7 Detection Kits Materials and equipment The VLA TaqMan Influenza A H5 H7 Detection Kits contain reagents for 96 Influenza A or Influenza A subtype HS or subtype H7 RI PCR reactions Each kit contains This protocol One pouc
9. time you receive a new MSDS packaged with a hazardous chemical be sure to replace the appropriate MSDS in your files Obtaining The MSDS for any chemical supplied by Applied Biosystems is available to you MSDSs free 24 hours a day To obtain MSDSs 1 Go to www appliedbiosystems com click Support then select MSDS 2 In the Keyword Search field enter the chemical name product name MSDS part number or other information that appears in the MSDS of interest Select the language of your choice then click Search 3 Find the document of interest right click the document title then select any of the following Open To view the document Print Target To print the document Save Target As To download a PDF version of the document to a destination that you select Note For the MSDSs of chemicals not distributed by Applied Biosystems contact the chemical manufacturer Chemical waste safety Chemical waste CAUTION HAZARDOUS WASTE Refer to Material Safety Data hazards Sheets MSDSs and local regulations for handling and disposal WARNING CHEMICAL WASTE HAZARD Wastes produced by Applied Biosystems instruments are potentially hazardous and can cause injury illness or death WARNING CHEMICAL STORAGE HAZARD Never collect or store waste in a glass container because of the risk of breaking or shattering Reagent and waste bottles can crack and leak Each waste bottle should be secured in a low density polyethy
10. viruses or other organisms In addition to inherent limitations on nucleic acid sequence selection validation verification and testing many other factors can affect detection efficacy and test results including sample preparation laboratory practices equipment use and maintenance reagent storage or loading and operating and environmental conditions Applied Biosystems cannot and does not represent warrant or otherwise guarantee 1 that this product will always detect the presence of the designated influenza virus type and subtype s or the nucleic acid sequence it is designed to detect even if the sample tested contains such type subtype s or sequence i1 that the nucleic acid sequence that this product is designed to detect is not present in other influenza virus type or subtype s or organisms or 111 that it will not indicate a positive finding when tested against other type or subtype s of the influenza virus or other organisms This product should not be used as the sole basis for determining the presence absence of the designated influenza virus type or subtype s The user should always confirm and validate the presence or absence of the designated type or subtype s by other means Applied Biosystems will not be responsible or liable for failures to detect the designated influenza virus type or subtype s or for false positive indications LIMITED WARRANTY AND LIMITATION OF LIABILITY AND REMEDIES Applied Biosystems warrants that t
11. 5 Influenza A subtype H5 1000 copies uL 1 tube 1 5 mL PN 4400535 VLA TaqMan Influenza A Subtype H7 Detection Kit PN 4398277 VLA Influenza A H5 H7 Detection Kits Protocol VLA TagMan Influenza A H5 H7 Detection Kits Materials and equipment Table 1 VLA TaqMan Influenza A H5 H7 Detection Kits Item Cap Color Reagent VLA TaqMan Influenza A Subtype H7 Detection Kit Blue VLA Influenza A Subtype H7 Assay Part 1 of 2 PN 4398295 Beads 96 tubes in 8 tube strips 96 reactions kit N A MicroAmp Optical 8 Cap Strips 96 caps in strips of 8 VLA TaqMan Influenza A Subtype H7 Detection Kit Grey Negative Control nuclease free water Controls Part 2 of 2 PN 4398321 C D 2 tubes 2 0 mL each PN 4340451 Green VLA RNA Positive Control H7 Influenza A subtype H7 1000 copies LL 1 tube 1 5 mL PN 4400536 Storage e Upon receipt store Assay Beads at 2 C to 8 C Protect from light and moisture e Upon receipt store all other components at 15 C to 25 C Minimize freeze thaw cycles Protect assay components from light Excessive exposure to light may affect the fluorescent probes 6 VLA Influenza A H5 H7 Detection Kits Protocol VLA Influenza A H5 H7 Detection Kits Materials and equipment Materials not included The following table includes materials required for using but not included in the in the kit VLA TagMan Influe
12. C IPC A NA S Start cycle e 0 100 End cycle 15 Figure 5 Indeterminate result FAM gt 36 for Influenza A FAM gt 35 for either VLA H5 or H7 assays repeat assay Amplification plots of target specific FAM and IPC specific VIC signals were generated on the 7500 Fast System Delta Rn vs Cycle 000 002 EE Deke Fin ve Cycle Detector u 1 000e 000 FAM SE al 1 000e 001 Threshold fo 1 000e 004 P 4 gt 1 1 000 002 N Start cycle 6 N Lt VIC IPC End cycle 15 1 000e 003 Fo mv mm N Help 1 000e 005 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 Cycle Number Figure6 Invalid result FAM present VIC absent repeat assay Amplification plots of target specific FAM and IPC specific VIC signals were generated on the 7500 Fast System VLA Influenza A H5 H7 Detection Kits Protocol 21 VLA TagMan Influenza A H5 H7 Detection Kits View and verify results 22 Delta Rn Delta Rn vs Cycle d Data Detta Rn vs Cycle Detector Au Line Color Detector Color e Analysis Settings Auto Ct 1 000 CG Manual Ct Threshold fo2 VIC IPC Auto Baseline lt Manual Baseline Start cycle e End cycle 15 Help 0 100 0 010 1 2 3 4 5 6 7 8 10 11 12
13. CR to amplify the viral target TaqMan probes to detect the presence of a specific influenza gene sequence An Internal Positive Control IPC to check for the presence of PCR inhibitors Oneofthe following Applied Biosystems instruments to perform the PCR and detect the probe cleavage Applied Biosystems 7500 Fast Real Time PCR System with SDS software version 1 3 or 1 4 Applied Biosystems 7300 or 7500 Real Time PCR System ABI PRISM 7000 Sequence Detection System Applied Biosystems 7900HT Fast Real Time PCR System using a standard block Applied Biosystems StepOne or StepOnePlus Real Time PCR System The TaqMan primers and probes in the VLA TaqMan Influenza A H5 H7 Detection Kits are based on the existing VLA assay sequences for Influenza A Influenza A subtype H5 and Influenza A subtype H7 Specifically the kits were validated by the VLA to detect the following Detection Kit A all avian influenza A virus subtypes H1 through H16 from all geographic regions Detection Kit H5 Eurasian H5 avian influenza virus subtypes Detection Kit H7 Eurasian H7 avian influenza virus subtypes Ongoing validation monitors the performance of the test against new isolates VLA TaqMan Influenza A H5 H7 Detection Kits Protocol VLA TagMan Influenza A H5 H7 Detection Kits Chemistry overview Chemistry overview Reaction components About negative and positive controls Reaction compo
14. Chemical alerts A au der ege xn dod manes d A Rand kin 31 VLA TagMan Influenza A H5 H7 Detection Kits Protocol 27 Appendix Safety Chemical safety Chemical safety Chemical hazard warning Chemical safety guidelines 28 WARNING CHEMICAL HAZARD Before handling any chemicals refer to the Material Safety Data Sheet MSDS provided by the manufacturer and observe all relevant precautions A WARNING CHEMICAL HAZARD All chemicals in the instrument including liquid in the lines are potentially hazardous Always determine what chemicals have been used in the instrument before changing reagents or instrument components Wear appropriate eyewear protective clothing and gloves when working on the instrument WARNING CHEMICAL HAZARD Four liter reagent and waste bottles AX can crack and leak Each 4 liter bottle should be secured in a low density polyethylene safety container with the cover fastened and the handles locked in the upright position Wear appropriate eyewear clothing and gloves when handling reagent and waste bottles WARNING CHEMICAL STORAGE HAZARD Never collect or store waste in a glass container because of the risk of breaking or shattering Reagent and waste bottles can crack and leak Each waste bottle should be secured in a low density polyethylene safety container with the cover fastened and the handles locked in the upright position Wear appropriate eyewear clot
15. F 1 REFUND OF THE PURCHASE PRICE OR REPLACEMENT OF THE PRODUCT OR 11 US 2 000 00 BUYER OR ANY SUCH USER OF THIS PRODUCT SHALL NOTIFY APPLIED BIOSYSTEMS OF ANY CLAIM WITHIN 30 DAYS THEREOF AND SHALL COMMENCE ANY ACTION AGAINST APPLIED BIOSYSTEMS WITHIN ONE YEAR OF THE EVENT OR OMISSION GIVING RISE TO THE CAUSE OF ACTION OR OTHERWISE SHALL BE BARRED FROM ANY REMEDY APPLIED BIOSYSTEMS SHALL NOT BE RESPONSIBLE FOR ANY COST LOSS LIABILITY OR DAMAGE INCLUDING WITHOUT LIMITATION LOSS OF REVENUE OR PROFIT LOST OR DAMAGED DATA OR OTHER COMMERCIAL OR ECONOMIC LOSS OR ANY OTHER DIRECT OR ANY INDIRECT INCIDENTAL SPECIAL OR CONSEQUENTIAL DAMAGES WHATSOEVER THAT ARISES FROM BUYER S OR ANY SUCH USER S OPERATION OF ITS BUSINESS OR FROM THE USE OF THIS PRODUCT WHETHER SINGLY OR IN COMBINATION WITH OTHER PRODUCTS EXCEPT FOR THE LIMITED REMEDIES SET FORTH ABOVE BUYER AND ANY USER OF THIS PRODUCT ASSUME ALL RISK AND LIABILITY RESULTING FROM USE OF THIS PRODUCT WHETHER USED SINGLY OR IN COMBINATION WITH OTHER PRODUCTS Some countries or jurisdictions limit the scope of or preclude limitations or exclusion of warranties or of remedies or damages In such countries and jurisdictions the limitation or exclusion of warranties or remedies or damages set forth above shall apply to the fullest extent permitted by law and shall not apply only to the extent prohibited by law The terms and conditions set forth above are supplemental to Applied Biosystems Genera
16. NT Use a new pipette tip for each sample After you have combined the assay beads with the samples Prepare tubes for the 7500 Fast System 7500 Fast System only as described on page 14 13 VLA TagMan Influenza A H5 H7 Detection Kits Prepare the RT PCR reactions Or Select the appropriate optical plate for your system and proceed to Transfer tube contents to a plate and seal the plates on page 15 For 7000 7300 7500 7900HT StepOnePlus Systems transfer the reaction contents from the assay bead tubes to a 96 well standard optical plate PN 4306737 For the StepOne System transfer the reaction contents from the assay bead tubes to a 48 well optical plate PN 4375816 Prepare tubes for the 7500 Fast System 7500 Fast System only To prepare tubes for the 7500 Fast System IMPORTANT 8 tube strips containing assay beads are compatible only with 7500 Fast instruments 1 Cap the tubes Seal each tube completely with the transparent optical strip caps provided in the kit PN 4316567 Follow the following instructions carefully to reduce the risk of tubes collapsing when using the MicroAmp Fast 8 Tube strips 0 1 mL To minimize bending or misaligning the tube strips during the capping process place the tube strips in the MicroAmp 96 Well Base PN N8010531 and use the MicroAmp Cap Installing Tool Handle PN 4330015 to cap the tubes Verify that the strips are straight and that each tube 1s in li
17. S and or certain other countries TagMan is a registered trademark of Roche Molecular Systems Inc other trademarks are the sole property of their respective owners Part Number 4400271 Rev B 06 2010 VLA TaqMan Influenza A H5 H7 Detection Kits IMPORTANT NOTICE PLEASE READ BEFORE USING THIS PRODUCT PLEASE READ THE IMPORTANT INFORMATION BELOW INCLUDING THE LIMITATION OF WARRANTY AND LIMITATION OF LIABILITY TERMS BEFORE USING THIS PRODUCT If the terms and conditions limitations and statements are not acceptable notify Applied Biosystems immediately but in any event no later than 5 days from delivery of this product to you and arrangements will be made for return of this product and for refund of the purchase price less shipping costs You must notify Applied Biosystems within 5 days of delivery in order to obtain a refund USE OF THIS PRODUCT CONSTITUTES ACKNOWLEDGMENT THAT YOU HAVE READ AND AGREED TO AND ACCEPT THE INFORMATION AND TERMS BELOW INCLUDING WITHOUT LIMITATION THE LIMITATIONS OF WARRANTY LIABILITY AND REMEDIES Any additional or different terms in Buyer s purchase form s or other documents are material alterations and are hereby rejected Field of Use Limitations This product and data obtained from use of this product should not be used for human diagnostic human treatment or other clinical purposes or for personal family or household purposes This product has not been cleared or otherwise approved by the
18. USDA United States Food and Drug Administration or by any other regulatory body in any country or under the European IVD Directive for diagnostic treatment or any other clinical purpose This product is for Research Use Only Not for use in diagnostic procedures This product should not be used as the sole basis for assessing the presence or absence of any particular organism or virus type or subtype and should be used only by or under supervision of technically qualified persons Read the Limitation of Warranty Liability and Remedies statements below before using this product See additional important information below This product should be used only by technically qualified persons who have their own independent skill and expertise in connection with the selection and use of products such as this product and in strict compliance with good and safe laboratory practices and all applicable instructions warnings and other information in user manuals and other product documentation This product should be used only with appropriate nucleic acid sequence detection equipment that has been properly maintained and calibrated by technically qualified persons This product is designed to test only for the presence of the influenza virus type and subtype s named on the product label This product has also been tested against certain other respiratory pathogens for selectivity However it has not been tested against all influenza types or subtype s
19. accordance with good laboratory practices and local state provincial or national environmental and health regulations If potentially hazardous waste 15 generated when you operate the instrument you must Characterize by analysis 1f necessary the waste generated by the particular applications reagents and substrates used in your laboratory Ensure the health and safety of all personnel in your laboratory Ensure that the instrument waste 1s stored transferred transported and disposed of according to all local state provincial and or national regulations IMPORTANT Radioactive or biohazardous materials may require special handling and disposal limitations may apply VLA TaaMan Influenza A H5 H7 Detection Kits Protocol Appendix B Safety Biological hazard safety Biological hazard safety General biohazard WARNING BIOHAZARD Biological samples such as tissues body fluids infectious agents and blood of humans and other animals have the potential to transmit infectious diseases Follow all applicable local state provincial and or national regulations Wear appropriate protective equipment which includes but is not limited to protective eyewear face shield clothing lab coat and gloves All work should be conducted in properly equipped facilities using the appropriate safety equipment for example physical containment devices Individuals should be trained according to applicable regulatory and company institutio
20. age of the fluorogenic probe During RT PCR see Figure 1 on page 4 RNA is reverse transcribed to cDNA cycles are performed 1 The primers anneal and extend and the probe hybrizes a The DNA polymerase creates a new DNA strand by extending the primers with nucleotides b Ifthe cDNA target of interest 1s present in the amplification product the TaqMan probe hybridizes to the sequence 2 The DNA polymerase cleaves the probe a The 5 to 3 nucleolytic activity of the DNA polymerase cleaves the hybridized probe between the reporter dye and the quencher dye see TaqMan probes below for more information The reporter dye fragments are displaced from the target resulting in an increase in fluorescence Note This step which occurs in every cycle does not interfere with the exponential accumulation of product b The polymerization of the strand continues The 3 end of the probe 15 blocked to prevent extension of the probe during PCR 3 Fluorescence increases The accumulation of PCR products is detected directly by monitoring the increase in fluorescence of the reporter dye Note The increase in fluorescence signal occurs only if the target sequence is complementary to the probe and is amplified during PCR Nonspecific amplification 15 not detected in the absence of a probe binding site VLA Influenza A H5 H7 Detection Kits Protocol VLA TagMan Influenza A H5 H7 Detection Kits Chemistry
21. ay drop down list For information on creating a plate document refer to the documentation provided with your instrument 2 Create or select FAM and VIC dye detectors with the Quencher Dye set to none or Non Fluorescent VLA Influenza A H5 H7 Detection Kits Protocol VLA TagMan Influenza A H5 H7 Detection Kits Prepare the plate document 3 Associate both FAM and VIC dye detectors with each reaction Note The FAM dye is used for detection of the target for example Influenza A subtype H7 and the VIC dye is used for detection of the internal positive control IPC 4 Setthermal cycling conditions according to the following table Step RT PCR CYCLE each of 40 cycles HOLD HOLD Denature Anneal extend Temp 45 C 95 C 95 C 54 C Time 30 min 2 min 15 sec 1 min 5 Set Sample Volume to 30 uL 6 Select the appropriate Run Mode for your system according to the following table System Run mode 7500 and 7500 Fast Standard 7500 7000 Standard 7300 Standard 7900HT Standard StepOne Standard StepOnePlus Standard VLA TaqMan Influenza A H5 H7 Detection Kits Protocol 11 VLA TagMan Influenza A H5 H7 Detection Kits Prepare the RT PCR reactions Prepare the RT PCR reactions Prepare the assay 1 Openthe storage pouch containing the assay beads by cutting at the notch beads located in the upper corner of the storage pouch above the zip lock strip
22. ction as described below Note Provides information that may be of interest or help but 1s not critical to the use of the product IMPORTANT Provides information that is necessary for proper instrument operation accurate chemistry kit use or safe use of a chemical How to obtain support For the latest services and support information for all locations go to www appliedbiosystems com At the Applied Biosystems web site you can viii Access worldwide telephone and fax numbers to contact Applied Biosystems Technical Support and Sales facilities Search through frequently asked questions FAQs Submit a question directly to Technical Support Order Applied Biosystems user documents MSDSs certificates of analysis and other related documents Download PDF documents Obtain information about customer training Download software updates and patches VLA Influenza A H5 H7 Detection Kits Protocol Protocol VLA TagMan Influenza A H5 H7 Detection Kits Product overview Product description The VLA TaqMan Influenza A H5 H7 Detection Kits in a lyophilized reagent format rapidly assay samples for the presence of Influenza type A The kits were validated by the UK Veterinary Laboratory Agency VLA to detect the same strains as the existing VLA assays The VLA TaqMan Influenza A H5 H7 Detection Kits reactions use Reverse transcription to convert viral RNA to cDNA e Polymerase chain reaction P
23. ent consumables and reagents continued Source Plates tubes caps and covers as needed MLS RNase free microfuge tubes 1 5 mL Applied Biosystems PN AM12400 RNase free tips 200 uL Applied Biosystems PN AM12650 HNase free tips 1000 uL Applied Biosystems PN AM12660 MicroAmp Optical 96 Well Reaction Plate with Barcode 20 plates 0 2 mL Applied Biosystems PN 4306737 Not recommended for use with 7500 Fast System For 7500 Fast System reactions use PN 4346906 MicroAmp Fast Optical 96 Well Reaction Plate with Barcode 20 plates 0 1 mL Applied Biosystems PN 4346906 MicroAmp Optical 8 Tube Strip 1000 tubes in strips of 8 0 2 mL Applied Biosystems PN 4316567 Not recommended for use with 7500 Fast System For 7500 Fast System reactions use PN 4358293 MicroAmp Fast 8 Tube Strip 0 1 mL Applied Biosystems PN 4358293 MicroAmp Optical 8 Cap Strip 300 strips Applied Biosystems PN 4323032 MicroAmp Fast Optical 48 Well Reaction Plate 20 plates 0 1 mL Applied Biosystems PN 4375816 For use with StepOne System reactions MicroAmp Optical 48 Well Adhesive Film Applied Biosystems PN 4375323 For use with StepOne system reactions MicroAmp Optical 96 Well Reaction Plate with Barcode and Optical Adhesive Films 100 plates and 100 covers Applied Biosystems PN 4314320 MicroAmp Optical Adhesive Film 25 covers Applied B
24. etected but target specific signal is detected High copy number of target RNA resulting in preferential amplification of the target specific RNA Dilute the sample for example 1 5 or 1 10 then repeat assay reactions Target specific signal is detected in negative control wells Carryover contamination Repeat the assay reactions using fresh aliquots of all reagents and clean pipetting equipment If the negative control still shows contamination repeat assay reactions using a new kit If the negative control still shows contamination contact Applied Biosystems Technical Support Target specific signal and no IPC signal is detected in negative control wells Carryover contamination and one of the following e High copy number of contaminating target resulting in preferential amplification of the target specific e Problem with IPC amplification Examine unknowns to determine if IPC signal is present If IPC signal is present in unknown wells you can rule out a problem with IPC amplification Repeat the assay reactions using fresh aliquots of all reagents and clean pipetting equipment No IPC or target specific signal is detected in positive control wells TagMan Influenza assay beads and reagents not stored properly Repeat the assay using properly stored assay components Protect the assay beads from light No target specific signal is detected in positive control wells
25. h containing Assay beads in tubes for Influenza A or Influenza A subtype H5 or H7 One bag of 12 MicroAmp Optical 8 Cap Strips One box containing RNA Positive Control Influenza A Influenza A subtype H5 or Influenza A subtype H7 Negative control nuclease free water Table1 VLA TaqMan Influenza A H5 H7 Detection Kits Item Cap Color Reagent VLA Influenza A Detection Kit PN 4398276 VLA TagMan Influenza A Detection Kit Part 1 of 2 Dark Green VLA Influenza A Assay Beads 96 tubes PN 4398293 in 8 tube strips 96 reactions kit twelve 8 tube strips MicroAmp Optical 8 Cap Strips 96 caps in strips of 8 VLA Influenza A Detection Kit Controls Part 2 of 2 PN 4398280 Negative Control nuclease free water 2 tubes 2 0 mL each PN 4340451 VLA RNA Positive Control Influenza A 1000 copies LL 1 tube 1 5 mL PN 4400534 VLA TaqMan Influenza A Subtype H5 Detection Kit PN 4398312 VLA TagMan Influenza A Subtype H5 Detection Kit Part 1 of 2 PN 4398278 Orange VLA Influenza A Subtype H5 Assay Beads 96 tubes in 8 tube strips 96 reactions kit N A MicroAmp Optical 8 Cap Strips 96 caps in strips of 8 VLA TagMan Influenza A Subtype H5 Detection Kit Grey Negative Control nuclease free water Controls Part 2 of 2 PN 4398319 2 tubes 2 0 mL each PN 4340451 Yellow VLA RNA Positive Control H
26. he adhesive on the optical cover of plate Rub the flat edge of the applicator back and forth along the short edge width of the plate Short of plate Rub the edge of the applicator horizontally and vertically between all wells Rub the edge of the applicator around all outside edges of the plate using small back and forth motions to completely seal around the outside wells Vortex the plate on the low setting for 5 seconds Spin down the plate at 2000 x g for 20 seconds using a centrifuge with a plate adapter IMPORTANT Make sure reagents are in the bottom of the wells 9 Proceed to Run the reactions on page 16 VLA TaqMan Influenza A H5 H7 Detection Kits Protocol 15 VLA TagMan Influenza A H5 H7 Detection Kits Run the reactions Run the reactions Overview Before you begin Run the 8 tube reactions 7500 Fast Systems only 16 Running tubes or a plate consists of using an Applied Biosystems Sequence Detection System SDS or Real Time PCR System to analyze your sample Ensure that your instrument 15 properly installed and calibrated For calibration information see the documentation provided with your instrument For 8 tube strips in a 7500 Fast System Use of the 7500 Fast Precision Plate Holder for MicroAmp Tube Strips PN 4403809 1s optional When using the MicroAmp 8 tube strips if columns 1 and 12 are not used insert two fully capped empty 8 tube str
27. hing and gloves when handling reagent and waste bottles To minimize the hazards of chemicals Read and understand the Material Safety Data Sheets MSDSs provided by the chemical manufacturer before you store handle or work with any chemicals or hazardous materials See About MSDSs on page 29 Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing For additional safety guidelines consult the MSDS Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood For additional safety guidelines consult the MSDS Check regularly for chemical leaks or spills If a leak or spill occurs follow the manufacturer s cleanup procedures as recommended in the MSDS Comply with all local state provincial or national laws and regulations related to chemical storage handling and disposal VLA Influenza A H5 H7 Detection Kits Protocol Appendix Safety Chemical waste safety About MSDSs Chemical manufacturers supply current Material Safety Data Sheets MSDSs with shipments of hazardous chemicals to new customers They also provide MSDSs with the first shipment of a hazardous chemical to a customer after an MSDS has been updated MSDSs provide the safety information you need to store handle transport and dispose of the chemicals safely Each
28. his product will be free of defects in materials and workmanship for a period of 90 days from the date delivered by Applied Biosystems to the buyer or its agent Warranty Period subject to the conditions set forth below Applied Biosystems will replace free of charge any defective product or refund the purchase price of the defective product in Applied Biosystems discretion if a warranty claim is made within the Warranty Period or within 30 days after the end of the Warranty Period Any warranty claim must be received by Applied Biosystems during the Warranty Period or within 30 days after the end of the Warranty Period This product may not be returned without a return authorization number from Applied Biosystems and then only in the manner prescribed by Applied Biosystems Return authorization numbers may be obtained from Applied Biosystems by calling or e mailing your Applied Biosystems salesperson or representative Expiration dates or shelf life periods with respect to this product are included for informational purposes only and shall not be deemed a period of warranty Applied Biosystems warranty does not cover and Applied Biosystems will not replace or refund the purchase price of any defective product if a defect is caused by failure to use the product in accordance with Applied Biosystems instructions and good laboratory practices by technically qualified persons or by other misuse or neglect including but not limited to improper main
29. hold Reanalyze Note setting the threshold above the negative control signal changes the of the negative controls to Undetermined IPC signal is less than 28 ROX degradation and or ROX fluorescence signal in multicomponent plot clearly decreases over course of reaction see figure below VIC 1234 5 6 7 8 9 10 11 12 12 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 38 37 22 39 40 Cycle Number 1 2000 095 Sa SESS K 80000094 HHHH Repeat the assay reactions Componest s E F ROX rv 89090999 Hitt 20002 004 1234 5 6 7 8 9 1011 12 13 14 15 16 17 18 19 20 24 22 23 24 25 26 27 28 29 30 DI 3233 MH 36 37 38 39 40 Cycles Figure 8 A Amplification plot of sample where and VIC signals were shifted in response to unusual ROX degradation B Multicomponent plot showing linear loss of ROX signal blue curve that is diagnostic of ROX degradation 24 VLA Influenza A H5 H7 Detection Kits Protocol Appendix A Good PCR Practices Prevent contamination and nonspecific amplification PCR assays require special laboratory practices to avoid false positive amplifications The high throughput and repetition of these assays can lead to amplification of one DNA molecule PCR good laboratory When preparing samples for PCR amplificati
30. iosystems PN 4360954 MicroAmp Optical Adhesive Film Kit Applied Biosystems PN 4313663 Reagents Ambion RT PCR Grade Water Applied Biosystems PN AM9935 i Includes recommended usage procedure VLA TaaMan Influenza A H5 H7 Detection Kits Protocol VLA TagMan Influenza A H5 H7 Detection Kits Detection procedure workflow Detection procedure workflow Isolate RNA from samples Prepare plate document Prepare RT PCR reactions Prepare assay beads Prepare samples and controls Combine diluted samples and controls with assay beads Vortex and centrifuge samples 7500 Fast 7000 7300 7500 7900HT StepOne Prepare StepOnePlus tubes Transfer tube contents to appropriate platform compatible plate Run the reactions perform RT PCR View and verify results VLA TaqMan Influenza A H5 H7 Detection Kits Protocol VLA TagMan Influenza A H5 H7 Detection Kits Isolate RNA Isolate RNA Validating your own RNA sample preparation procedure Sample handling precautions Use high purity viral RNA that 1s free of materials that can inhibit amplification in the performance of these assays Most commercially available viral RNA isolation kits satisfy the requirements of the VLA TagMan Influenza A H5 H7 Detection Kits However Applied Biosystems has not validated an RNA isolation procedure for use with this protocol You must validate your own procedure WARNING BIOHAZARD Biological
31. ips into the far left and far right columns Note The empty capped 8 tube strips evenly distribute the clamping load applied to the sample tube strips during processing thereby minimizing the risk of tubes collapsing VLA Influenza A H5 H7 Detection Kits Protocol VLA TagMan Influenza A H5 H7 Detection Kits Run the reactions 1 Carefully insert in pairs the 8 tube strips containing samples starting from the center of the plate holder and moving out Note A minimum of two and a maximum of 12 tube strips can be run at one time IMPORTANT Always use intact 8 tube strips If fewer samples are needed transfer contents to an intact 8 tube strip It 16 alright to have empty wells 2 Open the plate document that corresponds to the reaction plate created Prepare the plate document on page 10 3 Start the run Run the 48 or 96 well 1 Place the appropriate plate adaptor in the Real Time PCR System plate reactions 2 Load the reaction plate into the Real Time PCR System 3 Open the plate document that corresponds to the reaction plate created in Prepare the plate document on page 10 4 Start the run VLA Influenza A H5 H7 Detection Kits Protocol 17 VLA TagMan Influenza A H5 H7 Detection Kits View and verify results View and verify results Overview The steps you perform to view results depend on the instrument you use Refer to the appropriate instrument user guide for ins
32. l Terms and Conditions of Sale or other agreement between Applied Biosystems and buyer In case of a conflict the terms set forth above shall take precedence and prevail Contents mice Ae deg re hoe aa 265 vii Safety information vil How to use this guide viii How to obtain support viii Protocol VLA TaqMan Influenza A H5 H7 Detection Kits 1 Product OVelVIGW sss dado dd EE 1 Chemistry overview LL 2 Materials and equipment 5 Detection procedure workflow 9 Isolate RNA 10 Prepare the plate document 10 Prepare the RT PCR reactions 12 Run the reactionS 16 VIEW verify TeSUultS 18 JFOCDIGSDOOHEIQ 572 Eine 29 Appendix A Good PCR Practices 25 Prevent contamination and nonspecific amplification 25 Appendix B NSafety 27 Chemical safety
33. le Detector n Ee Line Color Detector Color FAM Target gie Analysis Settings Auto Ct 1 000 f lt Manual Ct Threshold o 2 Auto Baseline S Start cycle R 0 100 RW 15 VIC Help Figure 3 Positive result FAM are present with sigmoidal character and the FAM signal crosses the threshold before the cutoff value C lt 36 for Influenza A and C x35 for Influenza A subtypes H5 and H7 Amplification plots of target specific FAM and IPC specific VIC signals were generated on the 7500 Fast System Delta Rn vs Cycle eese EF Analysis Settings Threshold o2 Cramo Bessie 3 i Start cycle 0 100 FAM Target FA TC DN YA End cycle 15 E a 0 010 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 Cycle Number Figure 4 Negative result FAM present with linear character Amplification plots of target specific FAM and IPC specific VIC signals were generated on the 7500 Fast System 20 VLA Influenza A H5 H7 Detection Kits Protocol VLA Influenza A H5 H7 Detection Kits View and verify results Delta Rn vs Cycle 10 000 Data Delta Rn vs Cycle Detector a 1 Line Color Detector Color be Analysis Settings Auto Ct 1 000 CG Manual Ct Threshold fo 2 Auto Baseline VI
34. lene safety container with the cover fastened and the handles locked in the upright position Wear appropriate eyewear clothing and gloves when handling reagent and waste bottles VLA Influenza A H5 H7 Detection Kits Protocol 29 Appendix B Safety Chemical waste safety Chemical waste safety guidelines Waste disposal 30 To minimize the hazards of chemical waste Read and understand the Material Safety Data Sheets MSDSs provided by the manufacturers of the chemicals in the waste container before you store handle or dispose of chemical waste Provide primary and secondary waste containers A primary waste container holds the immediate waste A secondary container contains spills or leaks from the primary container Both containers must be compatible with the waste material and meet federal state and local requirements for container storage Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing For additional safety guidelines consult the MSDS Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood For additional safety guidelines consult the MSDS Handle chemical wastes in a fume hood After emptying a waste container seal it with the cap provided Dispose of the contents of the waste tray and waste bottle in
35. m that kit reagents do not contain any components that amplify Runa process control to confirm that your process does not contain any components that amplify from a sample with no virus Positive controls Runa PCR control to confirm that the kit reagents and instrument function to amplify the expected target A synthetic RNA is provided as a positive control in the kit Runa process control to test the entire process from sample preparation to detection to ensure that your process can generate a positive result from a sample known to contain virus IMPORTANT When you run positive controls cover the sample and negative control wells before pipetting positive controls to avoid cross contamination of samples and negative controls VLA Influenza A H5 H7 Detection Kits Protocol VLA TagMan Influenza A H5 H7 Detection Kits Chemistry overview Internal positive control Applied Biosystems includes an IPC VIC dye in the assay beads A positive IPC IPC signal Demonstrates that PCR reagents amplify as expected e Allows accurate interpretation of negative sample results RT PCR The VLA TaqMan Influenza A and Influenza A subtype H5 and H7 reactions use a one step reverse transcription polymerase chain reaction RT PCR format in a single tube The assay beads contain a thermoreactive reverse transcriptase and a thermostable DNA polymerase that provide the 5 to 3 nuclease activity necessary for cleav
36. n requirements before working with potentially infectious materials Read and follow the applicable guidelines and or regulatory requirements in the following U S Department of Health and Human Services guidelines published in Biosafety in Microbiological and Biomedical Laboratories stock no 017 040 00547 4 bmbl od nih gov Occupational Safety and Health Standards Bloodborne Pathogens 29 CFR 1910 1030 www access gpo gov nara cfr waisidx 01 29cfr1910a 01 html Your company s institution s Biosafety Program protocols for working with handling potentially infectious materials Additional information about biohazard guidelines 1s available at www cdc gov Chemical alerts General alerts for all Avoid contact with skin eyes and or clothing Read the MSDS and follow the chemicals handling instructions Wear appropriate protective eyewear clothing and gloves WARNING The VLA TaqMan Influenza A H5 H7 Detection Kits cause eye skin and respiratory tract irritation Avoid breathing dust Use with adequate ventilation Avoid contact with eyes and skin Read MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves VLA Influenza A H5 H7 Detection Kits Protocol 31 Appendix Safety Chemical alerts 32 VLA TaaMan Influenza A H5 H7 Detection Kits Protocol Documentation Related documentation For additional documentation see How to obtain support
37. nal FAM dye detector in all wells Use the following tables to interpret the results Table information applies to results generated on any Applied Biosystems Real Time PCR System Table 3 Analysis for Influenza A assay View and verify results FAM signal FAM C VIC signal target specific value IPC Result Example Present with lt 36 Present Positive Figure 3 sigmoidal character Present with linear lt 40 Present Negative Figure 4 character Present with gt 36 Present Indeterminate Figure 5 sigmoidal character repeat assay Present with lt 36 Absent Invalid repeat Figure 6 sigmoidal character assay Absent Present Negative Figure 7 Absent Absent Invalid repeat assay Table 4 Analysis for Influenza subtype 5 and 7 assays FAM signal FAM C VIC signal target specific value IPC Result Example Present with lt 35 Present Positive Figure 3 sigmoidal character Present with linear lt 40 Present Negative Figure 4 character Present with gt 35 Present Indeterminate Figure 5 sigmoidal character repeat assay Present lt 35 Absent Invalid repeat Figure 6 assay Absent Present Negative Figure 7 Absent Absent Invalid repeat assay VLA TaqMan Influenza A H5 H7 Detection Kits Protocol 19 VLA TagMan Influenza A H5 H7 Detection Kits View and verify results 10 000 Data betta Rn vs Cyc
38. ne with the adjacent tube 2 Vortex the tubes on the high setting for 5 seconds IMPORTANT Ensure that the caps are on firmly before vortexing 3 Spin down tube contents at 2000 x g for 20 seconds using a centrifuge with a plate adapter IMPORTANT Make sure samples are thoroughly mixed IMPORTANT Make sure reagents are in the bottom of the wells after centrifugation 4 Proceed to Run the reactions on page 16 14 VLA Influenza A H5 H7 Detection Kits Protocol VLA TagMan Influenza A H5 H7 Detection Kits Prepare the RT PCR reactions Transfer tube contents to a plate and seal the plates 1 Verify that the reaction mix 15 at the bottom of each tube If not cap the tubes securely vortex them and spin them as described in Prepare tubes for the 7500 Fast System 7500 Fast System only on page 14 Remove and discard the strip caps Usea pipette to transfer the entire reaction volume of each sample or control to the appropriate plate IMPORTANT Use a new pipette tip for each reaction even when pipetting the same sample or control IMPORTANT Keep all tubes and plates on ice Place an optical adhesive cover PN 4314320 on the plate then rub the flat edge of the applicator PN 4333183 back and forth along the ong edge of the plate Applicator Ow IMPORTANT Apply significant downward pressure on the applicator to completely seal the wells Pressure 15 required to activate t
39. nents of each of the VLA TaqMan Influenza A H5 H7 Detection Kits include Assay Beads Influenza A Influenza A subtype H5 or Influenza A subtype H7 containing enzymes primers probes and other components for reverse transcription RT and polymerase chain reaction PCR e Sample RNA which you supply isolated from environmental or epidemiological samples RNA positive control Negative controls Each kit provides reagents for 96 reactions It is good practice to include PCR specific and process specific controls in your experiment A negative PCR control is a reaction solution that lacks a target sequence At least one negative control 1s required for every run to monitor for reagent integrity and contamination unexpected amplification in the absence of a target A negative control in the form of nuclease free water PN 4340451 1s provided in the kit A positive PCR control contains a template known to be amplified by the primers in the assay In RT PCR the control template is an RNA Target signal FAM dye that is not detected in a positive control well indicates a pipetting error or a problem with amplification Positive RNA controls for Influenza A PN 4400534 the H5 subtype of Influenza A PN 4400535 or the H7 subtype of Influenza A PN 4400536 are provided in the kit At least one positive control 15 required per run e Negative controls RunaPCR control water only no template to confir
40. ng only this amount of product solely in environmental testing ap plications including epidemiological studies including reporting results of purchaser s activities for a fee or other commercial consideration and also for the purchaser s own internal research No right under any other patent claim such as apparatus or system claims for real time PCR is conveyed expressly by implication or by estoppel Further information on purchasing licenses may be obtained from the Director of Licensing Applied Biosystems 850 Lincoln Centre Drive Foster City California 94404 USA For Research Use Only This product and data obtained from use of this product should not be used for human diagnostic human treatment or other clinical purposes or for personal family or household purposes This product has not been cleared or otherwise approved by the USDA United States Food and Drug Administration or by any other regulatory body in any country or under the European IVD Directive for diagnostic treatment or any other clinical purpose This product should not be used as the sole basis for assessing the presence or absence of any particular organism or virus type or subtype and should be used only by or under supervision of technically qualified persons TRADEMARKS Applied Biosystems AB Design ABI PRISM MicroAmp and VIC are registered trademarks and FAM ROX StepOne and StepOnePlus are trademarks of Applied Biosystems Inc or its subsidiaries in the U
41. nza A H5 H7 Detection Kits Unless otherwise noted many of the listed items are available from major laboratory suppliers MLS Table2 Instruments equipment consumables and reagents Item Source Applied Biosystems Instruments ABI PRISM 7000 Sequence Detection Contact your local Applied Blosystems System sales office Applied Biosystems 7300 Real Time PCR System Applied Biosystems 7500 Real Time PCR System Applied Biosystems 7500 Fast Real Time PCR System Applied Biosystems 7900HT Real Time PCR System using a standard block StepOne Real Time PCR System StepOnePlus Real Time PCR System Equipment 96 well cold block MLS Benchtop microcentrifuge MLS Centrifuge with adapter for 96 well plate MLS Ice bucket MLS MicroAmp Adhesive Film Applicator Applied Biosystems PN 4333183 MicroAmp Optical 48 Well Base Adaptor Applied Biosystems PN 4375284 For use with StepOne system reactions MicroAmp Splash Free 96 Well Base Applied Biosystems PN 4312063 Pipettors MLS e Positive displacement e Air displacement e Multichannel 7500 Fast System Precision Plate Holder for Applied Biosystems PN 4403809 MicroAmp Tube Strips Vortexer MLS Consumables Disposable gloves MLS VLA TaqMan Influenza A H5 H7 Detection Kits Protocol VLA TagMan Influenza A H5 H7 Detection Kits Materials and equipment Table 2 Instruments equipm
42. on practices Wear clean gloves and a clean lab coat not previously worn while handling amplified PCR products or used during sample preparation Change gloves whenever you suspect that they are contaminated Maintain separate areas and dedicated equipment and supplies for Sample preparation PCR setup PCR amplification Analysis of PCR products Never bring amplified PCR products into the PCR setup area e Open and close all sample tubes carefully Try not to splash or spray PCR samples e Keep reactions and components capped as much as possible e Use a positive displacement pipette or aerosol resistant pipette tips e Clean lab benches and equipment periodically with 10 bleach solution IMPORTANT To avoid false positives due to cross contamination Prepare and close all negative control and unknown sample tubes before pipetting the positive control Do not open tubes after amplification Use different sets of pipettors when pipetting negative control unknown and positive control samples VLA TagMan Influenza A H5 H7 Detection Kits Protocol 25 Appendix A Good PCR Practices Prevent contamination and nonspecific amplification 26 VLA Influenza A H5 H7 Detection Kits Protocol Appendix oafety This appendix covers E C snum 28 B Chemical waste safety 29 Biological hazard safety 31
43. samples such as tissues body fluids infectious agents and blood of humans and other animals have the potential to transmit infectious diseases Follow all applicable local state provincial and or national regulations Wear appropriate protective equipment which includes but is not limited to protective eyewear face shield clothing lab coat and gloves All work should be conducted in properly equipped facilities using the appropriate safety equipment for example physical containment devices Individuals should be trained according to applicable regulatory and company institution requirements before working with potentially infectious materials Read and follow the applicable guidelines and or regulatory requirements in the following U S Department of Health and Human Services guidelines published in Biosafety in Microbiological and Biomedical Laboratories stock no 017 040 00547 4 bmbl od nih gov Occupational Safety and Health Standards Bloodborne Pathogens 29 CFR 1910 1030 www access gpo gov nara cfr waisidx 01 29cfr1910a 01 html e Your company S institution s Biosafety Program protocols for working with handling potentially infectious materials Additional information about biohazard guidelines 1s available at www cdc gov WARNING CHEMICAL HAZARD VLA TaqMan Influenza A H5 H7 Detection Kits Prepare the plate document 10 1 Create a plate document select Absolute Quantification Standard Curve from Ass
44. tenance shipping or handling inappropriate storage or maintenance or storage or maintenance not in accordance with Applied Biosystems instructions improper abnormal use adulteration degradation any unauthorized change or modification to the product or defect caused by accident THE FOREGOING WARRANTY IS APPLIED BIOSYSTEMS SOLE AND EXCLUSIVE WARRANTY WITH RESPECT TO THIS PRODUCT AND IS IN LIEU OF ALL OTHER WARRANTIES EXPRESSED OR IMPLIED ALL OF WHICH OTHER WARRANTIES ARE EXPRESSLY DISCLAIMED INCLUDING WITHOUT LIMITATION THOSE OF MERCHANTABILITY FITNESS OR SUITABLITY FOR A PARTICULAR PURPOSE OR NON INFRINGEMENT WHETHER ARISING FROMA STATUTE OR OTHERWISE IN LAW OR FROM A COURSE OF DEALING OR USAGE OF TRADE THE REPLACEMENT OF DEFECTIVE PRODUCT OR REFUND OF THE PURCHASE PRICE AS SET FORTH ABOVE IS BUYER S SOLE AND EXCLUSIVE REMEDY WITH RESPECT TO APPLIED BIOSYSTEMS BREACH OF THE FOREGOING WARRANTY OR ANY CLAIMS OF ANY KIND OR NATURE WHATSOEVER RELATING TO DEFECTIVE PRODUCT IN ADDITION TO THE FOREGOING THE SOLE AND EXCLUSIVE REMEDY OF BUYER AND ANY USER OF THIS PRODUCT AND THE SOLE AND EXCLUSIVE LIABILITY OF APPLIED BIOSYSTEMS ITS AFFILIATES DISTRIBUTORS SUPPLIERS LICENSORS OR REPRESENTATIVES FOR ANY AND ALL CLAIMS IN RESPECT THEREOF OTHER THAN WARRANTY CLAIMS WHICH SHALL BE GOVERNED BY THE PRECEDING PARAGRAPH INCLUDING CLAIMS IN TORT CONTRACT STRICT LIABILITY NEGLIGENCE OR OTHERWISE SHALL BE LIMITED TO THE GREATER O
45. tructions on how to analyze data and View your results View results 1 View the amplification plots for all reactions 2 Forall reactions under Analysis Settings Select Manual Baseline e Set Start cycle to 6 Set End cycle to 15 e Set Manual C threshold to 0 20 Click Analyze Verify internal positive Examine the IPC signal VIC dye detector in all wells For most instruments the control IPC results IPC signal for the internal positive control in all wells should yield Cys in the range of 28 to 36 at the manual threshold of 0 2 Delta vs Cycle Delta Rn n Start cycle e 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 Cycle Number Figure 2 Amplification plot of IPC VIC9 dye detector in negative controls for the Influenza A assay on the 7500 Fast Real Time PCR System 8 replicates The circled area indicates where the signal crosses the threshold horizontal green line If you observe any of the following see Troubleshooting on page 23 PC signal is not present in negative control unknown or positive control wells IPC signal is greater than 36 in any well IPC signal C is inconsistent between replicate wells 18 VLA Influenza A H5 H7 Detection Kits Protocol Interpret results of unknown samples VLA TagMan Influenza A H5 H7 Detection Kits Examine the target specific sig
46. ury It may also be used to alert against unsafe practices WARNING Indicates a potentially hazardous situation that if not avoided could result in death or serious injury A DANGER Indicates an imminently hazardous situation that if not avoided will result in death or serious injury This signal word is to be limited to the most extreme situations MSDSs The MSDSs for any chemicals supplied by Applied Biosystems or Ambion available to you free 24 hours a day For instructions on obtaining MSDSs see Appendix B IMPORTANT For the MSDSs of chemicals not distributed by Applied Biosystems or Ambion contact the chemical manufacturer VLA TaqMan Influenza A H5 H7 Detection Kits Protocol vii Preface How to use this guide How to use this guide Text conventions This guide uses the following conventions Bold text indicates user action For example Type 0 then press Enter for each of the remaining fields Italic text indicates new or important words and is also used for emphasis For example Before analyzing always prepare fresh matrix A right arrow symbol separates successive commands you select from a drop down or shortcut menu For example Select File Open Spot Set Right click the sample row then select View Filter gt View Runs User attention words Two user attention words appear in Applied Biosystems user documentation Each word implies a particular level of observation or a
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