TaqMan Bacillus anthracis Detection Kit

Transcription

1 TaqMan Bacillus anthracis Detection Kit Protocol This product is for environmental testing, quality assurance/quality control testing and for research use

2 Copyright 2007, 2010 Applied Biosystems. All rights reserved. only. Not for use in diagnostic procedures. 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 using only this amount of product solely in environmental testing and quality control/quality assurance testing, 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. TaqMan Pathogen Detection Kits provide a simple, reliable, and rapid procedure for detecting the presence of a specific bacterial pathogen. These kits have been validated by Applied Biosystems internal design and development standards, however they have not been validated for any specific application or use. It is the user s responsibility to validate these kits for the user s particular application or use. This Product has not been cleared or otherwise approved by the United States Food and Drug Administration or by any other regulatory body in any country, or under the European IVD Directive, for human diagnostic or any other clinical purposes. The user of this product agrees not to use this product for human diagnostic or other clinical purposes. For Research Use Only. Not for use in diagnostic purposes. Information in this document is subject to change without notice. Applied Biosystems assumes no responsibility for any errors that may appear in this document. This document is believed to be complete and accurate at the time of publication. In no event shall Applied Biosystems be liable for incidental, special, multiple, or consequential damages in connection with or arising from the use of this document. 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, INCLUDING 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, INCID- ENTAL, INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING BUT NOT LIMITED TO THE USE THEREOF. TRADEMARKS: AB (Design), Applera, Applied Biosystems, ABI PRISM, PrepMan, and VIC are registered trademarks and FAM and StepOne are trademarks of Applied Biosystems or its subsidiaries in the U.S. and/or certain other countries. AmpliTaq and TaqMan are registered trademarks of Roche Molecular Systems, Inc. All other trademarks are the sole property of their respective owners. Part Number Rev B 6/2010

3 TaqMan Bacillus anthracis Detection Kit IMPORTANT NOTICE PLEASE READ BEFORE USING THIS PRODUCT PLEASE READ THE IMPORTANT INFORMATION BELOW, WHICH INCLUDES FIELD OF USE LIMITATIONS, THE PRODUCT S LIMITED WARRANTY, AND LIMITATIONS OF LIABILITY AND REMEDIES. 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 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 pathogen 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 Bacillus anthracis as named on the product label. 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 (i) that this product will always detect the presence of the designated pathogen or the nucleic acid sequence it is designed to detect, even if the sample tested contains such sequence, (ii) that the nucleic acid sequence that this product is designed to detect is not present in other organisms, or (iii) that it will not indicate a positive finding when tested against other organisms. This product should not be used as the sole basis for determining the presence or absence of the designated pathogen. The user should always confirm and validate the presence or absence of Bacillus anthracis. Applied Biosystems will not be responsible or liable for failures to detect the designated pathogen or for false positive indications. LIMITED WARRANTY AND LIMITATION OF LIABILITY AND REMEDIES. Applied Biosystems warrants that this 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 ing 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

4 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 maintenance, shipping or handling, inappropriate storage or maintenance, or storage or maintenance not in accordance with Applied Biosystems' instructions, improper or abnormal use, adulteration, degradation, any unauthorized change or modification to the product, or any 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 FROM A 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 OF (i) REFUND OF THE PURCHASE PRICE OR REPLACEMENT OF THE PRODUCT OR (ii) US $2, 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 General 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. This product has not been cleared or otherwise approved by the United States Food and Drug Administration or by any other regulatory body in any country, or under the European IVD Directives, for human diagnostic or any other clinical purposes. The user of this product agrees not to use this product for human diagnostic or other clinical purposes. For research use only. Not for use in diagnostic procedures. Information in this document is subject to change without notice. Applied Biosystems assumes no responsibility for any errors that may appear in this document. This document is believed to be complete and accurate at the time of publication. In no event shall Applied Biosystems be liable for incidental, special, multiple, or consequential damages in connection with or arising from the use of this document.

5 Contents Preface vii Safety vii How to Obtain More Information xi How to Obtain Support xi Product Overview Product Description Benefits Definitions of Terms Chemistry Overview Reaction Components Polymerase Chain Reaction (PCR) Fluorescent Detection Internal Positive Control (IPC) Materials and Equipment Kit Contents Storage Equipment and Materials Not Included Good Laboratory Practices Overview Preventing PCR Contamination Plate Layout Suggestions Pathogen Detection Procedure Overview Preparing DNA Samples Overview Validating Your Own Sample Preparation Procedure Preparing PCR Overview Creating the Plate Document Preparing the Reaction Mix Sealing Plates and Tubes Running a Plate (Performing PCR) Overview Before You Begin TaqMan Bacillus anthracis Detection Kit v

6 Running a Plate Viewing Results Overview Viewing Results Appendix A: Troubleshooting References vi TaqMan Bacillus anthracis Detection Kit

7 Preface This preface contains: Safety vii How to Obtain More Information xi How to Obtain Support xi Safety 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. Indicates a potentially hazardous situation that, if not avoided, may result in minor or moderate injury. It may also be used to alert against unsafe practices. Indicates a potentially hazardous situation that, if not avoided, could result in death or serious injury. 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. Chemical Hazard Warning CHEMICAL HAZARD. Some of the chemicals used with Applied Biosystems instruments and protocols are potentially hazardous and can cause injury, illness, or death. TaqMan Bacillus anthracis Detection Kit vii

8 Chemical Safety Guidelines To minimize the hazards of chemicals: Read and understand the Material Safety Data Sheets (MSDS) provided by the chemical manufacturer before you store, handle, or work with any chemicals or hazardous materials. (See About MSDSs on page viii.) 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 on the MSDS. Comply with all local, state/provincial, or national laws and regulations related to chemical storage, handling, and disposal. 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 time you receive a new MSDS packaged with a hazardous chemical, be sure to replace the appropriate MSDS in your files. Obtaining MSDSs You can obtain from Applied Biosystems the MSDS for any chemical supplied by Applied Biosystems. This service is free and available 24 hours a day. To obtain MSDSs: 1. Go to viii TaqMan Bacillus anthracis Detection Kit

9 2. In the Search field, type in the chemical name, 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 choose 4. To have a copy of a document sent by fax or , select Fax or to the left of the document title in the Search Results page, then click RETRIEVE DOCUMENTS at the end of the document list. 5. After you enter the required information, click View/Deliver Selected Documents Now. Chemical Waste Hazard CHEMICAL WASTE HAZARD. Some wastes produced by the operation of the instrument or system are potentially hazardous and can cause injury, illness, or death. Chemical Waste Safety Guidelines 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. TaqMan Bacillus anthracis Detection Kit ix

10 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 the waste container, seal it with the cap provided. Dispose of the contents of the waste tray and waste bottle in accordance with good laboratory practices and local, state/provincial, or national environmental and health regulations. Waste Disposal If potentially hazardous waste is generated when you operate the instrument, you must: Characterize (by analysis if 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 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. Biological Hazard Safety 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 utilizing the appropriate safety equipment (for example, physical containment devices). Individuals should be trained in accordance with applicable regulatory and company/institution requirements before working with potentially infectious materials. Read and adhere to the following guidelines and/or regulatory requirements as applicable: x TaqMan Bacillus anthracis Detection Kit

11 U.S. Department of Health and Human Services guidelines published in Biosafety in Microbiological and Biomedical Laboratories (stock no ; Occupational Safety and Health Standards, Bloodborne Pathogens (29 CFR ; waisidx_01/29cfr1910a_01.html). Your company/institution s Biosafety Program protocols for working/handling potentially infections materials. Additional information about biohazard guidelines is available at: How to Obtain More Information Related Documentation See the following related documents for more information on the topics in this guide: User Guide for your Applied Biosystems or Real-Time PCR System Send Us Your Comments Applied Biosystems welcomes your comments and suggestions for improving its user documentation. You can your comments to: techpubs@appliedbiosystems.com How to Obtain Support For the latest services and support information for all locations, go to then click the link for Support. At the Support page, you can: Obtain 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 TaqMan Bacillus anthracis Detection Kit xi

12 Product Overview Product Description TaqMan Pathogen Detection Kits provide a simple, reliable, rapid procedure for detecting the presence of a specific bacterial pathogen. These kits have been validated using Applied Biosystems' internal design and development standards; however, they have not been validated for any specific application or use. It is your responsibility to validate these kits for your particular application or use. The pathogen detection assay utilizes: Polymerase chain reaction (PCR) to amplify a gene target unique to that microorganism. TaqMan probes to detect the presence of the specific organism. An Internal Positive Control (IPC) to check for the presence of PCR-inhibitors and to ensure the reliability of a negative result. Applied Biosystems Real-Time PCR System to perform the PCR and detect the probes. Each kit is designed using Applied Biosystems bioinformatic pipeline. Each assay has been verified by a process to demonstrate that: DNA of the target organism(s) yields a positive signal DNA of two to five closely related organisms yields a negative signal Negative controls yield a negative signal This product is for environmental testing, quality assurance/quality control testing and for research use only. Not for use in diagnostic procedures. Benefits Gold-standard TaqMan probe-based chemistry provides unmatched sensitivity, specificity, and reproducibility, allowing for data comparisons across experiments and labs. Pre-designed kits save the time, money, and effort involved in primer design, synthesis, and formulation of bacteria-specific assays. TaqMan Bacillus anthracis Detection Kit 1

13 Single, closed tube analysis simplifies the assay and minimizes contamination. Assays are delivered ready-to-use: The kit contains all reagents needed for PCR. You add TaqMan Fast PCR Master Mix and Target Assay Mix to your template DNA, then run the analysis on an Applied Biosystems Fast Real-Time PCR platform of your choice. You can perform assays on mixtures of samples containing different bacteria, using enriched, unprocessed, or uncultured samples. The kit uses Fast PCR cycling conditions, allowing you to obtain results within an hour. No post-pcr processing is required. The TaqMan Bacillus anthracis Detection Kit contains two specific Taqman primer/probe sets for more accurate detection. Definitions of Terms Amplification he process of making copies of a specific DNA sequence, thereby increasing the amount of DNA. Fast PCR Master Mix (FMM) A common reagent for pathogen detection assays. The Master Mix is used to prepare the premix solution. It contains the polymerase enzyme that initiates PCR in the presence of the necessary primers and DNA sample. Internal Positive Control (IPC) A control present in all reaction wells (contained in the Target Assay Mix). The IPC should always yield a positive result. If it does not, there may be a problem with amplification. For more information, see Internal Positive Control (IPC) on page 4. Negative control Monitor for contamination (unexpected amplification in the absence of a target) and reagent integrity (IPC signal should be present). One negative control is required for each target assay. Positive control Monitor for the expected amplification of a target. Target signal not detected in a positive control well indicates a pipetting error or a problem with amplification. A positive control is optional for each target assay and is not recommended because it can cause cross-contamination. Premix solution A solution that you prepare containing Fast PCR Master Mix (FMM) and Target Assay Mix (TAM). 2 TaqMan Bacillus anthracis Detection Kit

14 Chemistry Overview Primer A segment of DNA that is complementary to the target DNA sequence or Internal Positive Control DNA sequence. Primer is needed to initiate amplification. Probe A segment of DNA that is complementary to the target DNA sequence or Internal Positive Control DNA sequence. The probe is labeled with a reporter dye. When the probe binds to its target or Internal Positive Control, a reaction detected by the Real-Time PCR System indicates the presence of the target or Internal Positive Control. Target The pathogen being tested. Target Assay Mix (TAM) Target-specific reaction for the pathogen detection assay. TAM is used to prepare the premix solution. TAM contains specific primers and probes for the target and the IPC. The TaqMan Bacillus anthracis Detection Kit contains two specific TaqMan primer/probe sets, TAM1 (pxo1) and TAM2 (pxo2), labeled with the same dye (FAM) for specific detection of the target pathogen, Unknown sample A DNA sample from a substance that you are testing for the presence of one or more pathogens environmental testing or quality control/quality assurance testing. Chemistry Overview Reaction Components Polymerase Chain Reaction (PCR) Reaction components include: Two Target Assay Mixes designed to detect two Bacillus anthracis specific targets Fast PCR Master Mix DNA isolated from environmental or epidemiological samples, which you supply PCR is a method used to amplify, or increase the number of copies of specific DNA sequence. Typically, the target DNA sequence is amplified using a reaction containing DNA polymerase, nucleotides, and primers complementary to that DNA sequence. When this solution is heated, the DNA sequence denatures, separating it into separate strands. As the solution cools, the primers anneal, or bind, to the target sequences in the separated DNA strand. The DNA polymerase then creates a new strand by extending the primers with TaqMan Bacillus anthracis Detection Kit 3

15 nucleotides, creating a copy of the DNA sequence. When repeated, this cycle of denaturing, annealing, and extending exponentially increases the number of target DNA sequences. Ideally, no amplification occurs if the target DNA sequence is not present. Fluorescent Detection Internal Positive Control (IPC) The TaqMan probe contains a fluorescent dye on one end and a quencher that suppresses fluorescence on the other. If the target sequence is present during the PCR, then the probe is degraded, resulting in an increase in fluorescence. The instrument detects accumulation of PCR products by monitoring the increase in fluorescence. The increase in fluorescence signal is detected only if the target sequence is complementary to the probe and is amplified during PCR. Because of these requirements, separate nonspecific amplification is not detected. Applied Biosystems includes an IPC in the Target Assay Mix in the TaqMan Pathogen Detection Kits. The inclusion of the IPC in each reaction avoids false negatives due to the presence of substances that inhibit PCR. The IPC also demonstrates whether PCR reagents are working and amplifying properly. This IPC eliminates the need for a positive control, reducing the risk of cross contamination in unknown samples. Materials and Equipment Kit Contents The TaqMan Bacillus anthracis Detection Kit (PN ) contains reagents for 100 reactions (30-µl reaction volume). Cap Color Reagent Volume Purple 10X Bacillus anthracis Target Assay pxo1, 1 tube Contains primers and probes for amplification and detection of target (FAM -dye labeled probe) and IPC (VIC -dye labeled probe) 300 µl 4 TaqMan Bacillus anthracis Detection Kit

16 Materials and Equipment Cap Color Reagent Volume Green 10X Bacillus anthracis Target Assay pxo2, 1 tube Contains primers and probes for amplification and detection of target (FAM -dye labeled probe) and IPC (VIC -dye labeled probe) 300 µl White Negative Control, 1 tube 1000 µl Red 2X Fast PCR Master Mix, 2 tubes 1500 µl Storage Upon receipt, store the 10 Bacillus anthracis Target Assay pxo1 and pxo2 at 20 C and protect it from light. Excessive exposure to light may affect the fluorescent probes. Store the Negative Control and 2 Fast PCR Master Mix at 2 C to 8 C. Minimize freeze-thaw cycles. Equipment and Materials Not Included Instruments from Applied Biosystems Instruments Applied Biosystems 7500 Fast Real-Time PCR System Applied Biosystems StepOne Real-Time PCR System Applied Biosystems 7500 Real-Time PCR System* Applied Biosystems 7300 Real-Time PCR System* Source Contact your local Applied Biosystems sales office. * These instruments must be used with their respective consumables. TaqMan Bacillus anthracis Detection Kit 5

17 User-supplied materials Materials MicroAmp Fast Optical 96-Well Reaction Plate with Barcode, 20 plates Source Applied Biosystems (PN ) ABI PRISM Optical Adhesive Cover Starter Kit Applied Biosystems Optical Adhesive Covers, 25 covers MicroAmp Fast 96-Well Tray Benchtop microcentrifuge Disposable gloves Heat block Heat sealer Incubator shaker Pipet tips, aerosol resistant Pipettors: Positive-displacement Air-displacement Multichannel Sterile microcentrifuge tubes with attached screw-cap lid RNase-free, sterile-filtered water Applied Biosystems (PN ) Applied Biosystems (PN ) Applied Biosystems (PN ) Major Laboratory Suppliers (MLS) MLS MLS MLS MLS MLS MLS MLS MLS 6 TaqMan Bacillus anthracis Detection Kit

18 Good Laboratory Practices Good Laboratory Practices Overview Preventing PCR Contamination PCR assays require special laboratory practices to avoid false positive amplifications (Kwok and Higuchi, 1989). The high sensitivity of these assays can lead to amplification of a single DNA molecule (Saiki et al., 1985; Mullis and Faloona, 1987). If possible, maintain separate work areas, dedicated equipment, and supplies for: Sample preparation PCR setup PCR amplification Analysis of PCR products Note: Rooms can be simulated using a clean bench or PCR bench available from major laboratory suppliers. Do not bring amplified PCR products into the PCR setup area. Wear a clean lab coat (not previously worn while handling amplified PCR products or used during sample preparation) and clean gloves when preparing samples for PCR amplification. Change gloves whenever you suspect that they are contaminated and before leaving the work area. To avoid false positives due to cross contamination, do not include a positive control unless required. If a positive control is necessary, close all unknown sample tubes before pipetting the positive control. Open and close all sample tubes and reaction plates carefully. Try not to splash or spray PCR samples. Keep reactions and components capped as much as possible. Use positive-displacement pipets or aerosol-resistant pipet tips. Clean lab benches and equipment after use with freshly diluted 10% bleach solution. IMPORTANT! To avoid false positives due to amplified material in your work area, do not open tubes after amplification. TaqMan Bacillus anthracis Detection Kit 7

19 Plate Layout Suggestions Place unknown samples at the beginning of a row or column and place controls at the end of the row or column. Place replicates of one sample for the same target next to each other. If enough space is available: Separate different targets by one row. Separate unknown samples and controls by one well. Separate negative and positive controls by one well. Place positive controls in an outer row or column. Example Portion of a Plate/Tube Layout A Sample 1 TAM1 Sample 1 TAM1 Sample 1 TAM1 Sample 1 TAM2 Sample 1 TAM2 Sample 1 TAM2 NEG CTRL TAM1 NEG CTRL TAM2 POS CTRL B C Sample 2 TAM1 Sample 2 TAM1 Sample 2 TAM1 Sample 2 TAM2 Sample 2 TAM2 Sample 2 TAM2 NEG CTRL TAM1 NEG CTRL TAM2 POS CTRL D etc... Positive control not recommended, but if required, leave a blank well preceding it and locate it at the end of a row. 8 TaqMan Bacillus anthracis Detection Kit

20 Pathogen Detection Procedure Overview Pathogen Detection Procedure Overview Sample Enrich samples (if needed) Prepare DNA Samples A Prepare PCR B C D E F G H Premix solution 96-well plate Run the plate (perform PCR) Real-Time PCR instrument View Results TaqMan Bacillus anthracis Detection Kit 9

21 Preparing DNA Samples Overview Validating Your Own Sample Preparation Procedure Sample preparation is the first step in using the TaqMan Bacillus anthracis Detection Kit. Preparing DNA samples consists of preparing the samples for PCR. Applied Biosystems has not validated a sample preparation procedure for use with the TaqMan Bacillus anthracis Detection Kit. It is recommended that you validate your own sample preparation procedure as a starting point. In general, sample preparation procedures specific for spore-forming organisms should deliver the appropriate amount of DNA to run this test. Preparing PCR Overview Creating the Plate Document Preparing PCR is the second step in using the TaqMan Bacillus anthracis Detection Kit. Preparing PCR consists of creating the plate document and the reaction mix. Note: The TaqMan Bacillus anthracis Detection Kit uses Fast cycling conditions and consists of two TAMs (pxo1 and pxo2), designed for different target sequences. Both TAMs contain a FAMlabeled TaqMan probe. Applied Biosystems recommends that you run both TAMs for specific identification and confirmation of the presence of Bacillus anthracis. To create the plate document: 1. In the software, create a plate document and specify an assay type of Relative Quantification or Absolute Quantification without standard curve. 2. Specify FAM and VIC detectors for each reaction well you will use in the assay. 10 TaqMan Bacillus anthracis Detection Kit

22 Preparing PCR To create the plate document: (continued) 3. Set thermal cycling conditions specified below. Refer to the appropriate instrument user guide for details. Step Enzyme Activation PCR HOLD Cycle (45 Cycles) Denature Anneal/ Extend Time 20 sec. 3 sec. 30 sec. Temp 95 C 95 C 60 C Preparing the Reaction Mix CHEMICAL HAZARD. 2 Fast PCR Master Mix may cause eye and skin irritation. Exposure may cause discomfort if swallowed or inhaled. Read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. To prepare the reaction mix: 1. Thaw all reagents completely. 2. Label a tube with the target name. TaqMan Bacillus anthracis Detection Kit 11

23 To prepare the reaction mix: (continued) 3. Calculate the volume of Premix Solution components needed for the number of samples, replicates, and controls for your run. Note: Prepare separate Premix solutions for each TAM and label the tube appropriately. Applied Biosystems recommends performing three replicates for each sample. At least one negative control is required per target organism tested. Add one 30 µl reaction per positive control, negative control, or replicate. Component Vol (µl) for One 30-µL Reaction Vol (µl) for Four 30-µL Reactions 2 Fast PCR Master Mix (FMM) 10 Target Assay Mix (TAM) Total Volume Includes 10% overage to compensate for pipetting errors. 4. Pipette the Premix Solution components into the labeled tube. Use a new tip when pipetting FMM and TAM. 5. Mix the solution by gently pipetting up and down, then cap the tube. 6. Repeat steps 2 through 5 for each target assay. 7. Refer to Plate Layout Suggestions on page 8, then gently pipette 18 µl of Premix Solution into the bottom of each well. Note: Use a new tip for each Target Premix Solution. 12 TaqMan Bacillus anthracis Detection Kit

24 Preparing PCR To prepare the reaction mix: (continued) 8. Transfer 12 µl of unknown sample into each sample well, then gently pipette up and down to mix the solution. IMPORTANT! Mix very gently with the pipette tip at the bottom of the tube to minimize aerosol formation and cross-contamination. Note: Use a new tip for each well, even when pipetting the same sample. 9. Transfer 12 µl of negative control into each negative control well, then gently pipette up and down to mix the solution. IMPORTANT! Mix very gently with the pipette tip at the bottom of the tube to minimize aerosol formation and cross-contamination. Note: Use a new tip for each negative control. 10. (Optional) If you are using a positive control, dilute the positive control: a. Add RNase-free water to 1 pg per µl. b. Close all unknown sample and negative control tubes. c. Transfer 12 µl of positive control into each positive control well, then gently pipette up and down to mix the solution. The result of the dilution is approximately 2,000 copies per reaction. IMPORTANT! Mix very gently with the pipette tip at the bottom of the tube to minimize aerosol formation and cross-contamination. Note: Use a new tip for each well, even when pipetting the same positive control. 11. Close tubes or apply an optical cover to the plate (see Sealing Plates and Tubes on page 14). 12. Make sure reagents are in the bottom of the wells. If available, use a centrifuge with a plate adapter to briefly centrifuge the plate. TaqMan Bacillus anthracis Detection Kit 13

25 Sealing Plates and Tubes IMPORTANT! Apply significant downward pressure on the applicator in all steps to form a complete seal on top of the wells. Pressure is required to activate the adhesive on the optical cover To seal plates: 1. Place an optical adhesive cover on the plate, then rub the flat edge of the applicator back and forth along the long edge of the plate. 2. Rub the flat edge of the applicator back and forth along the short edge (width) of the plate. 3. Rub the end of the applicator horizontally and vertically between all wells. 4. Rub the end of the applicator around all outside edges of the plate using small back and forth motions to form a complete seal around the outside wells. 14 TaqMan Bacillus anthracis Detection Kit

26 Preparing PCR To seal tubes: 1. Place strip caps on the tubes. 2. IMPORTANT! Apply significant downward pressure on the sealing tool to form a complete seal on top of the tubes. If you are using the rolling capping tool: Roll the capping tool across all strips of caps on the short edge, then the long edge, of the tray. Roll the capping tool around all outer rows of strips of caps. If you are using the rocking capping tool: Slip your fingers through the handle with the holes in the tool facing down. Place the holes in the tool over the first eight caps in a row. Rock the tool back and forth a few times to seal the caps. Repeat for remaining caps in the row, then for all remaining rows. TaqMan Bacillus anthracis Detection Kit 15

27 Running a Plate (Performing PCR) Overview Before You Begin Running a Plate Running the plate to perform PCR is the fourth step in using the TaqMan Bacillus anthracis Detection Kit. Running a plate consists of using an Applied Biosystems Fast Real-Time PCR System to analyze your sample. Ensure that your instrument is properly installed and calibrated. For calibration information, see the documentation provided with your instrument. To run the plate: 1. Open the plate document you created on page Load the reaction plate into the Fast Real-Time PCR System. 3. Start the run. IMPORTANT! To avoid false positives due to amplified material in your work area, do not open tubes after amplification. 16 TaqMan Bacillus anthracis Detection Kit

28 Viewing Results Viewing Results Overview Viewing Results The steps you perform to view results depend on the instrument you use. Refer to the appropriate instrument user guide for instructions on how to analyze data and view your results. 1. View the amplification plots for the entire plate. 2. Set the baseline and threshold values. 3. Check each sample for FAM dye signal (target specific signal) and VIC dye signal (IPC), then interpret results: FAM signal (TAM1) FAM signal (TAM2) VIC signal (IPC) Result Present Present Present or Absent Presumptive Positive Absent Absent Present Presumptive Negative Absent Absent Absent See Appendix A: Troubleshooting on page 18. Absent Present Present or Absent Present Absent Present or Absent Nondeterminant Repeat experiment Nondeterminant Repeat experiment This product should not be used as the sole basis for assessing the presence or absence of any particular pathogen, and should be used only by or under the supervision of technically qualified persons. TaqMan Bacillus anthracis Detection Kit 17

29 Appendix A: Troubleshooting Observation Possible Cause Action No IPC or target-specific signal detected in unknown wells No IPC detected, but target-specific signal detected unknown wells Target-specific signal detected in negative control wells PCR inhibited Fast PCR Master Mix not stored properly Target specific 10 Assay Mix not stored properly Pipetting Error (no premix solution added) High copy number of target DNA resulting in preferential amplification of the target-specific DNA Carryover contamination Repeat sample preparation, then repeat assay. If PCR is still inhibited, dilute the sample (for example, 1:5 or 1:10) to dilute inhibitors. Alternatively, use a Bacterial Genomic DNA Purification Kit (Major Laboratory Supplier) to remove inhibitors. Repeat the assay using properly stored assay components. Avoid freezing and thawing assay components. Protect the Assay Mix from light. Repeat the assay. Make sure to pipette premix solution into all wells. No action required. Repeat the assay using fresh aliquots of all reagents and clean pipetting equipment. If the negative control still shows contamination, repeat the assay using a new kit. If the negative control still shows contamination, contact Applied Biosystems Technical Support. 18 TaqMan Bacillus anthracis Detection Kit

30 Appendix A: Troubleshooting Observation Possible Cause Action Target-specific signal and no IPC signal detected in negative control wells No IPC or target-specific signal in positive control wells No target-specific signal detected in positive control wells No IPC signal but targetspecific signal detected in positive control wells Replicate results for this sample are inconsistent Carryover contamination and one of the following: High copy number of target DNA resulting in preferential amplification of the targetspecific DNA Problem with IPC amplification Fast PCR Master Mix not stored properly Target specific 10 Assay Mix not stored properly Pipetting error (no positive control added) High copy number of target DNA resulting in preferential amplification of the targetspecific DNA All replicate wells for a sample do not have the same result. 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 using fresh aliquots of all reagents and clean pipetting equipment. Repeat the assay using properly stored assay components. Avoid freezing and thawing assay components. Protect the Assay Mix from light. Repeat the assay. Make sure to pipette positive control into all positive control wells. No action required. If more than two replicates yield the same result (for example, you ran 3 replicates and 2 replicates are negative, but 1 replicate is positive), it is probable that the result associated with the larger number of replicates is 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. TaqMan Bacillus anthracis Detection Kit 19

31 References Kwok, S. and Higuchi, R Avoiding false positives with PCR. Nature 339: Mullis, K.B. and Faloona, F.A Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol. 155: Saiki, R.K., Scharf, S., Faloona, F., et al Enzymatic amplification of β-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230: TaqMan Bacillus anthracis Detection Kit

32 Worldwide Sales and Support Applied Biosystems vast distribution and service network, composed of highly trained support and applications personnel, reaches 150 countries on six continents. For sales office locations and technical support, please call our local office or refer to our Web site at Applied Biosystems is committed to providing the world s leading technology and information for life scientists Headquarters 850 Lincoln Centre Drive Foster City, CA USA Phone: Toll Free (In North America): Fax: / Part Number Rev. B