User Manual
Contents
1. Pork DNA detected Invalid result See Chapter 3 Troubleshooting This result is expected in reactions that have a strong FAM signal See Chapter 3 Troubleshooting Quantitative analysis For samples with positive results the percentage of pork DNA with respect to total animal DNA can be quantified using the RapidFinder Pork ID Kit in combination with the RapidFinder Quant Multi Meat Set Cat no A24399 TM The RapidFinder Quant Multi Meat Set includes the Multi Meat Standard a plasmid DNA quantitation standard containing individual species specific DNA targets and a highly conserved animal specific mitochondrial genomic region total animal DNA target The kit also includes a TaqMan assay for the total animal DNA target Both pork and total animal DNA present in each sample can be quantified relative to the Multi Meat Standard using primers and probe from both kits For detailed instructions refer to the RapidFinder Quant Multi Meat Set User Guide Pub no MAN0009930 Note The Positive Control included in the RapidFinder Pork ID Kit is not intended for use as a quantitation standard RapidFinder Pork ID Kit User Guide 9 Troubleshooting Observation Possible cause Recommended action In the Positive Control wells no target specific and no IPC signals are detected PCR amplification failure Check that the thermal cycler settings and amplification2. and a clean lab coat not previously worn while handling amplified 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 and reaction setup Amplification and analysis of products Do not bring amplified products into the reaction setup area Open and close all sample tubes carefully Avoid splashing or spraying samples Keep reactions and components capped as much as possible Use a positive displacement pipettor or aerosol resistant barrier pipette tips Clean lab benches and equipment periodically with 10 bleach solution or DNAZap Solutions Cat no AM9890 For additional information refer to ISO 22174 2005 Plate layout suggestions 12 Separate different targets by a row if enough space is available Put at least one well between unknown samples and controls if possible Separate negative and positive controls by one well if possible Place replicates of one sample for the same target next to each other Start with the unknown samples and put controls at the end of the row or column Put positive controls in one of the outer rows or columns if possible Consider that caps for PCR tubes come in strips of 8 or 12 RapidFinder Pork ID Kit User Guide Safety WARNING GENERAL SAFETY Using this product in a manner not specified in the user documentation may result in
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4. of data analysis depend on the real time PCR instrument that you use refer to the appropriate user guide for instructions on how to analyze your data 1 View the amplification plots for all reactions to make sure that they appear normal 2 Use the Auto instrument setting for e Baseline e FAM threshold e VIC threshold 3 Check that the results obtained in all control wells are as expected Reaction type FAM channel VIC channel Pork IPc Et Positive Control i R Negative extraction control F No template control F RapidFinder Pork ID Kit User Guide 11 In all reactions the Cy of the IPC should be similar to the Cy of the Positive Control Chapter 2 Methods Se Ja Interpretation of results For unexpected control results refer to the troubleshooting section of this guide 4 Establish the positive cut off value for the test samples and assign results Cr cut off 7 Cr Positive Control 3 32 Sample Cy value Sample result Cr gt Cr cut off Negative Cr lt CH cut off Positive Ml For fresh or minimally processed meat samples the cut off values corresponds to approximately 0 01 pork DNA when the DNA sample concentration is 10 ng pL Interpretation of results Interpret unknown sample results according to the following table FAM channel VIC channel Interpretation Pork IPC Pork DNA not detected
5. personal injury or damage to the instrument or device Ensure that anyone using this product has received instructions in general safety practices for laboratories and the safety information provided in this document Before using an instrument or device read and understand the safety information provided in the user documentation provided by the manufacturer of the instrument or device Before handling chemicals read and understand all applicable Safety Data Sheets SDSs and use appropriate personal protective equipment gloves gowns eye protection etc To obtain SDSs see the Documentation and Support section in this document RapidFinder Pork ID Kit User Guide 13 Chemical safety 14 Appendix C Safety WARNING GENERAL CHEMICAL HANDLING To minimize hazards ensure laboratory personnel read and practice the general safety guidelines for chemical usage storage and waste provided below and consult the relevant SDS for specific precautions and instructions Read and understand the Safety Data Sheets SDSs provided by the chemical manufacturer before you store handle or work with any chemicals or hazardous materials To obtain SDSs see the Documentation and Support section in this document Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing Minimize the inhalation of chemic
6. 17 RapidFinder Pork ID Kit User Guide 3 Product information Product description Identification of meat species present in food samples is an essential step for verification of origin and traceability of raw materials as well as for quality control of handling and cleaning processes in production lines The RapidFinder Pork ID Kit enables real time PCR detection of DNA from pork Sus scrofa that is present in food and feed samples The kit includes e All reagents necessary for the real time PCR reaction specific FAM labeled probe and primers for pork mitochondrial DNA enzyme and other buffer components e An internal positive control IPC VIC labeled probe primers and template to monitor for PCR inhibition included in the reagents e A Positive Control to confirm pork DNA detection Unknown samples and control samples are provided by the investigator Kit contents and storage Reagents sufficient for 48 reactions are provided Component Amount Storage Pork Master Mix green pad 360 pL 20 C General Master Mix white pad 600 uL 4 C Positive Control green cap 100 pL 20 C Ml Refer to the expiration date on the box Materials required but not provided Unless otherwise specified all materials are available from Life Technologies MLS major laboratory supplier Item Source Instrument and equipment 4 RapidFinder Pork ID Kit User Gui
7. The result is considered positive In unknown wells no IPC or target specific signal is Excess sample DNA in the PCR the recommended maximum is Repeat the PCR with the correct amount of DNA detected 250 ng PCR inhibitors in the sample Repeat the DNA extraction If the problem DNA persists contact Technical Support 10 RapidFinder Pork ID Kit User Guide The detection limit was calculated with standard samples consisting of mixtures of Kit sensitivity and specificity raw pork meat and other species RapidFinder Pork ID Kit can detect blends with as little as 0 01 w w of pork meat The limit of detection in processed samples varies depending upon the composition and food processing The kit specificity was tested by comparison of the probe and primer sequences with the NCBI database and it was also experimentally tested on a collection of reference DNAs with the following results Meat species Result Sheep Not detected Goat Not detected Horse Not detected Beef Not detected Pork Detected Buffalo Not detected Deer Not detected Chicken Not detected Turkey Not detected Duck Not detected Ostrich Not detected Goose Not detected Wild boar Detected Human Not detected RapidFinder Pork ID Kit User Guide 11 Good laboratory practices for PCR and RT PCR When preparing samples for PCR or RT PCR amplification Wear clean gloves
8. USER GUIDE P technologies RapidFinder Pork ID Kit Real time PCR detection of pork DNA in food and feed samples Catalog Number A24392 Publication Number MANOQ009924 Revision A 0 ImMegen For testing of Food and Environmental samples only technologies For testing of Food and Environmental samples only The information in this guide is subject to change without notice DISCLAIMER LIFE TECHNOLOGIES CORPORATION AND OR ITS AFFILIATE S DISCLAIM ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT EXPRESSED OR IMPLIED INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY FITNESS FOR A PARTICULAR PURPOSE OR NON INFRINGEMENT TO THE EXTENT ALLOWED BY LAW IN NO EVENT SHALL LIFE TECHNOLOGIES AND OR ITS AFFILIATE S 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 LIMITED USE LABEL LICENSE No 492 Environmental Testing Quality Control Quality Assurance Testing Food and Agricultural Testing Notice to Purchaser The purchase of this product conveys to the purchaser the limited non transferable right to use the purchased amount of the product a to perform internal research for the sole benefit of the purchaser and b for environmental testing quality control quality assurance testing food and agricultural testing includ
9. actions 1 Combine the following components for the number of reactions required plus 10 overage Component Volume per reaction Pork Master Mix green pad 7 5 uL Master Mix General white pad 12 5 uL 2 Mix thoroughly by vortexing and distribute 20 uL to each reaction well or tube 3 Add 5 uL of DNA sample 10 ng uL mock purified sample negative extraction control Nuclease free Water no template control or Positive Control to the appropriate wells 4 Seal each plate or tube mix then centrifuge briefly to bring the contents to the bottom RapidFinder Pork ID Kit User Guide 7 Chapter 2 Methods Set up and run the real time PCR instrument Set up and run the real time PCR instrument 1 Following the manufacturer s instructions set up the run using the following parameters e Reaction volume 25 uL e ROX passive reference dye included e TaqMan probe reporter dyes and quenchers Target Reporter Quencher Pork FAM dye MGB IPC VIC dye MGB e Thermal cycler settings F Stage 1 Stage 2 Sett Da Enzyme activation PCR Number of cycles 1 Hold 36 Denature Anneal extend Temperature 95 C 95 C 60 C Time 10 minutes 15 seconds 1 minute 2 Load the reactions run the thermal cycler program and collect real time amplification data Analyze results The general process for analyzing results is described in this section The details
10. als Do not leave chemical containers open Use only with adequate ventilation for example fume hood Check regularly for chemical leaks or spills If a leak or spill occurs follow the manufacturer s cleanup procedures as recommended in the SDS e Handle chemical wastes in a fume hood Ensure use of 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 After emptying a waste container seal it with the cap provided Characterize by analysis if necessary the waste generated by the particular applications reagents and substrates used in your laboratory Ensure that the waste is 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 RapidFinder Pork ID Kit User Guide Appendix C Safety Biological hazard safety Biological hazard safety AX 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 All work should be conducted in properly equipped facilities using th
11. de Chapter 1 Product information a Materials required but not provided SAB Item Source Applied Biosystems real time PCR thermal cycler and Contact your local sales required accessories office e StepOne Real Time PCR System e StepOnePlus Real Time PCR System e 7500 Fast Real Time PCR System e 7500 Real Time PCR System Adjustable micropipettors 10 uL 20 uL 200 uL MLS Benchtop microcentrifuge with adaptors for PCR plates MLS and or tubes Laboratory mixer Vortex mixer or equivalent MLS Optical reaction plates and covers or optical PCR tubes and caps as appropriate for your instrument For use with the 7500 Real Time PCR System MicroAmp Optical Reaction Plate Cat no 4306737 MicroAmp Optical Adhesive Film Cat no 4311971 MicroAmp Fast 8 Tube Strip 0 1 mL Cat no 4358293 See below for caps For use with the StepOne Real Time PCR System MicroAmp Fast Optical 48 Well Reaction Plate Cat no 4375816 MicroAmp 48 Well Optical Adhesive Film Cat no 4375323 MicroAmp Optical 8 Tube Strip Cat no 4316567 See below for caps For use with the StepOnePlus Real Time PCR System or 7500 Fast Real Time PCR System MicroAmp Fast Optical 96 Well Reaction Plate 0 1 mL Cat no 4346907 MicroAmp Optical Adhesive Film Cat no 4311971 MicroAmp Fast 8 Tube Strip 0 1 mL Cat no 4358293 See below for caps For use with all
12. e appropriate safety equipment for example physical containment devices Safety equipment also may include items for personal protection such as gloves coats gowns shoe covers boots respirators face shields safety glasses or goggles Individuals should be trained according to applicable regulatory and company institution requirements before working with potentially biohazardous materials Follow all applicable local state provincial and or national regulations The following references provide general guidelines when handling biological samples in laboratory environment U S Department of Health and Human Services Biosafety in Microbiological and Biomedical Laboratories BMBL 5th Edition HHS Publication No CDC 21 1112 Revised December 2009 found at www cdc gov biosafety publications bmb15 BMBL pdf World Health Organization Laboratory Biosafety Manual 3rd Edition WHO CDS CSR LYO 2004 11 found at www who int csr resources publications biosafety Biosafety7 pdf RapidFinder Pork ID Kit User Guide 15 Documentation and support Obtaining SDSs Safety Data Sheets SDSs are available from www lifetechnologies com support Note For the SDSs of chemicals not distributed by Thermo Fisher Scientific contact the chemical manufacturer Obtaining Certificates of Analysis The Certificate of Analysis provides detailed quality control and product qualification information for each product Certificates of Analy
13. ing reporting results of purchaser s activities in environmental testing quality control quality assurance testing food and agricultural testing for a fee or other commercial consideration No other right is hereby granted expressly by implication or by estoppel This product is for environmental testing quality control quality assurance testing food and agricultural testing and research purposes only The purchase of this product does not grant the purchaser any additional rights including without limitation the right to transfer or resell the product in any form or the right to use the product as a therapeutic agent or diagnostics test component For information on obtaining additional rights please contact outlicensing alifetech com or Out Licensing Life Technologies 5791 Van Allen Way Carlsbad California 92008 Trademarks All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified TaqMan is a registered trademark of Roche Molecular Systems Inc used under permission and license 2014 Thermo Fisher Scientific Inc All rights reserved Contents m CHAPTER1 Product information ccceeeeee ee eee eee eens 4 Product deSCriptiOMN sc s2e esc seek eon bie weed nap Le koe a a EE bee ee 4 Kit contents and storage 2 02 e ee eee eee eee 4 Materials required but not provided 0 cece eee eee ee eee eens 4 M CHAPTER2 Methods c cece eee e
14. ontact Life Technologies at www lifetechnologies com support RapidFinder Pork ID Kit User Guide 17 For support visit lifetechnologies com support or email techsupportfdlifetech com lifetechnologies com 28 March 2014 technologies
15. program are correct In the negative extraction control wells target specific and IPC signals are detected Contamination during the DNA extraction procedure Contamination may be due to errors in sample handling reagent contamination or environmental contamination e Check that the DNA extraction protocol was performed correctly e Take care to avoid contamination during sample homogenization decontaminate grinding equipment or homogenizer with 10 bleach or DNAZap Solutions Cat no AM9890 e Decontaminate benchtop surfaces and other equipment where the DNA extraction process is performed with 10 bleach or DNAZap Solutions e If necessary use fresh reagents and repeat the DNA extraction In the no template control wells target specific and IPC signals are detected Contamination of the PCR Contamination may be due to errors in sample handling reagent contamination or environmental contamination e Decontaminate benchtop surfaces and other equipment where PCR is performed with 10 bleach or DNAZap Solutions Cat no AM9890 e Use fresh reagents and repeat the PCR e Setup the Positive Control PCR reactions last to avoid cross contamination In unknown wells no IPC signal is detected but target specific signal is detected A high copy number of target DNA exists in samples resulting in preferential amplification of the target specific DNA No action is required
16. sis are available on our website Go to www lifetechnologies com support and search for the Certificate of Analysis by product lot number which is printed on the box Obtaining support For the latest services and support information for all locations go to www lifetechnologies com support At the website you can e Access worldwide telephone and fax numbers to contact Technical Support and Sales facilities e Search through frequently asked questions FAQs e Search for user documents SDSs vector maps and sequences application notes formulations handbooks certificates of analysis citations and other product support documents e Obtain information about customer training e Download software updates and patches Food Safety support Website www lifetechnologies com foodsafety Support email foodsafety lifetech com Phone number in North America 1 800 500 6855 16 RapidFinder Pork ID Kit User Guide Documentation and support Limited product warranty Phone number outside of North America Visit www lifetechnologies com support select the link for phone support and select the appropriate country from the dropdown menu Limited product warranty Life Technologies Corporation and or its affiliate s warrant their products as set forth in the Life Technologies General Terms and Conditions of Sale found on Life Technologies website at www lifetechnologies com termsandconditions If you have any questions please c
17. specified real time PCR systems MicroAmp Optical 8 Cap Strips Cat no 4323032 Other plastics and consumables Aerosol resistant pipette tips MLS 1 5 mL nuclease free microcentrifuge tubes MLS RapidFinder Pork ID Kit User Guide 5 Chapter 1 Product information eu Materials required but not provided gt Item Source Powder free disposable gloves MLS Reagents GMO Extraction Kit recommended for DNA isolation Cat no 4466336 Nuclease free Water Cat no AM9938 6 RapidFinder Pork ID Kit User Guide LIN D j oy we Methods 7 NG E z E x Input DNA requirements e Prepare the DNA sample with a method that allows processing of 10 20 g of food sample The GMO Extraction Kit is recommended e Prepare at least one mock purified sample as an extraction negative control processed with the same DNA isolation method that is used for test samples e Dilute the final DNA sample to 10 ng uL for the PCR Determine the number of reactions and thaw the reagents 1 Plan to include the following reactions e Duplicate reactions for each test sample e Duplicate control reactions Positive Control included in the kit Negative extraction control mock purified samples No template control reactions use Nuclease free Water in place of sample DNA 2 Thaw all reagents vortex to mix thoroughly and place on ice Set up the PCR re
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