TaqMan Pseudomonas aeruginosa Detection Kit

Transcription

1 TaqMan Pseudomonas aeruginosa Detection Kit Protocol November 26, :08 pm, TMB Pseud_Title.fm

2 Copyright 2005, 2007, 2010 Applied Biosystems. All rights reserved. NOTICE TO PURCHASER: LIMITED LICENSE Use of the TaqMan Pseudomonas aeruginosa Detection Kit 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, quality control/quality assurance testing, and food testing] applications 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. 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. TRADEMARKS: Applied Biosystems, ABI PRISM, PrepMan, and VIC are registered trademarks and AB (Design), Applera, and FAM are trademarks of Applied Biosystems or its subsidiaries in the U.S. and/or certain other countries. AmpliTaq Gold and TaqMan are registered trademarks of Roche Molecular Systems, Inc. All other trademarks are the sole property of their respective owners. Part Number Rev. C 06/2010 November 26, :08 pm, TMB Pseud_Title.fm

3 Contents Preface v Safety v How to Obtain More Information ix How to Obtain Support ix 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 Preventing Contamination Overview Preventing PCR Contamination Plate Layout Suggestions Pathogen Detection Procedure Overview Enriching Samples Overview Validating Your Own Enrichment Procedure Sample Handling Preparing DNA Samples Overview Validating Your Own Sample Preparation Procedure Sample Preparation Process TaqMan Pseudomonas aeruginosa Detection Kit Protocol November 26, :08 pm, TMB PseudTOC.fm iii

4 Preparing PCR Overview Creating the Plate Document Preparing the Reaction Mix Sealing Plates and Tubes Running a Plate (Performing PCR) Overview Before You Begin Running a Plate Viewing Results Overview Viewing Results Appendix A: Troubleshooting References iv TaqMan Pseudomonas aeruginosa Detection Kit Protocol November 26, :08 pm, TMB PseudTOC.fm

5 Preface This preface contains: Safety v How to Obtain More Information ix How to Obtain Support ix 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 Pseudomonas aeruginosa Detection Kit Protocol November 26, :07 pm, TMB Pseud_Pref_Protocol.fm v

6 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 below.) 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 vi TaqMan Pseudomonas aeruginosa Detection Kit Protocol November 26, :07 pm, TMB Pseud_Pref_Protocol.fm

7 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 Pseudomonas aeruginosa Detection Kit Protocol November 26, :07 pm, TMB Pseud_Pref_Protocol.fm vii

8 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: viii TaqMan Pseudomonas aeruginosa Detection Kit Protocol November 26, :07 pm, TMB Pseud_Pref_Protocol.fm

9 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 PrepMan Ultra Sample Preparation Protocol PrepMan Ultra Sample Preparation Quick Reference Card 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 TaqMan Pseudomonas aeruginosa Detection Kit Protocol November 26, :07 pm, TMB Pseud_Pref_Protocol.fm ix

10 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 x TaqMan Pseudomonas aeruginosa Detection Kit Protocol November 26, :07 pm, TMB Pseud_Pref_Protocol.fm

11 Product Overview Product Description 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. The pathogen detection assay utilizes: The polymerase chain reaction (PCR) to amplify a 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. New kits are currently in development. Visit the Applied Biosystems website at for the latest information on kits. Each kit is designed using Applied Biosystems sophisticated bioinformatic pipeline. Each assay has been verified according to our Bronze 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 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 Pseudomonas aeruginosa Detection Kit Protocol 1

12 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 Environmental Master Mix and Target Assay Mix to your template DNA, then run on an Applied Biosystems Real-Time PCR platform of your choice. Assays can be performed on mixtures of samples containing different bacteria, and can use enriched, unprocessed, or uncultured samples. All kits use universal cycling conditions, allowing you to run any combination of kits together in the same run. No post-pcr processing is required. Definitions of Terms Amplification The process of making copies of, and thereby increasing the amount of, a specific DNA sequence. Environmental Master Mix (EMM) A common reagent for all pathogen detection assays. The EMM 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 Monitors 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. Polymerase Chain Reaction (PCR) Technology used to amplify, or increase the amount of a DNA sequence. Positive control Monitors 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 you prepare that contains Environmental Master Mix (EMM) and Target Assay Mix (TAM). Primer A segment of DNA that is complementary to the target DNA sequence or Internal Positive Control DNA sequence. Needed to initiate amplification. 2 TaqMan Pseudomonas aeruginosa Detection Kit Protocol

13 Chemistry Overview 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. Unknown sample A DNA sample from a substance that you are testing for the presence of one or more pathogens in food testing, animal testing, environmental studies, or epidemiological studies. Chemistry Overview Reaction Components Polymerase Chain Reaction (PCR) Reaction components include: Target Assay Mix Environmental Master Mix DNA isolated from enrichment culture of raw materials or food, animal, environmental, or epidemiological samples, which you supply PCR is a method used to amplify, or increase the amount of, a 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 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. TaqMan Pseudomonas aeruginosa Detection Kit Protocol 3

14 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. The inclusion of the IPC in each reaction avoids false negatives due to the presence of substances inhibitory to 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 Pseudomonas aeruginosa Detection Kit (PN ) contains reagents for 100 reactions (30 µl reaction volume). Cap Color Reagent Volume Blue 10X Pseudomonas aeruginosa Target Assay Mix, 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 Yellow/Gold 2X Environmental Master Mix, 2tubes 750 µl 4 TaqMan Pseudomonas aeruginosa Detection Kit Protocol

15 Materials and Equipment Storage Upon receipt, store the 10 Pseudomonas aeruginosa Target Assay Mix at 20 C and protect from light. Excessive exposure to light may affect the fluorescent probes. Store the Negative Control and 2 Environmental Master Mix at 2 C to 8 C. Minimize freeze-thaw cycles. Equipment and Materials Not Included Instruments from Applied Biosystems Instruments Applied Biosystems 7300 Real-Time PCR System Applied Biosystems 7500 Real-Time PCR System Source Contact your local Applied Biosystems sales office. User-supplied materials Materials Source ABI PRISM 96-Well Optical Reaction Plate with Barcode (code 128), 20 plates ABI PRISM Optical Adhesive Covers and ABI PRISM 96-Well Optical reaction Plate with Barcode (code 128), 100 plates with covers ABI PRISM Optical Tubes ABI PRISM Optical Caps, (8 caps/strip), 300 strips ABI PRISM Optical Adhesive Cover Starter Kit Applied Biosystems Optical Adhesive Covers, 25 covers MicroAmp Splash free support base Enrichment reagent and protocol appropriate for the pathogen you are testing for Applied Biosystems (PN ) Applied Biosystems (PN ) Applied Biosystems (PN ) Applied Biosystems (PN ) Applied Biosystems (PN ) Applied Biosystems (PN ) Applied Biosystems (PN ) Major Laboratory Suppliers (MLS) TaqMan Pseudomonas aeruginosa Detection Kit Protocol 5

16 Sample preparation reagent and protocol, for example, PrepMan Ultra Sample Preparation Reagent Benchtop microcentrifuge Disposable gloves Heat block Heat sealer Incubator shaker Pipet tips, aerosol resistant Pipettors: Positive-displacement Air-displacement Multichannel Materials Sterile microcentrifuge tubes with attached screw-cap lid RNase-free, sterile-filtered water Pseudomonas aeruginosa (Source strain 633) Applied Biosystems (PN ) MLS MLS MLS MLS MLS MLS MLS MLS MLS Source ATCC (PN 17933D) For use as a positive control. Applied Biosystems does not recommend the use of positive controls with TaqMan Pathogen Detection Kits. Please refer to Internal Positive Control (IPC) on page 4 for more information. 6 TaqMan Pseudomonas aeruginosa Detection Kit Protocol

17 Preventing Contamination Preventing Contamination 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 Pseudomonas aeruginosa Detection Kit Protocol 7

18 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 Plate/Tube Layout A TARG1 UNKN 1 TARG1 UNKN 1 TARG1 UNKN 1 TARG1 UNKN 2 TARG1 UNKN 2 TARG1 UNKN 2 NEG CTRL NEG CTRL POS CTRL B C TAR21 UNKN 1 TAR21 UNKN 1 TAR21 UNKN 1 TARG2 UNKN 2 TARG2 UNKN 2 TARG2 UNKN 2 NEG CTRL NEG CTRL 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 Pseudomonas aeruginosa Detection Kit Protocol

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

20 Enriching Samples Overview Validating Your Own Enrichment Procedure Sample Handling Depending on your sample type, enrichment may be the first step in using the TaqMan Pseudomonas aeruginosa Detection Kit. Enrichment consists of growing the specific pathogen from a selected sample. Applied Biosystems has not validated an enrichment procedure for use with the TaqMan Pseudomonas aeruginosa Detection Kit. Most standard enrichment procedures can be used with the TaqMan Pseudomonas aeruginosa Detection Kit, for example, the USDA FSIS or FDA BAM methods. However, you must optimize the time needed to enrich your specific sample. Note: Enrichment time for this PCR-based pathogen detection kit can be reduced significantly from standard microbiology enrichment protocols. 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: 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 ; 01.html). Your company/institution s Biosafety Program protocols for working/handling potentially infections materials. Additional information about biohazard guidelines is available at: 10 TaqMan Pseudomonas aeruginosa Detection Kit Protocol

21 Preparing DNA Samples Preparing DNA Samples Overview Validating Your Own Sample Preparation Procedure Sample Preparation Process Sample preparation is the second step (if you enriched your samples) in using the TaqMan Pseudomonas aeruginosa Detection Kit. Sample preparation consists of preparing the sample for PCR. Applied Biosystems has not validated a sample preparation procedure for use with the TaqMan Pseudomonas aeruginosa Detection Kit. However, the PrepMan Ultra Sample Preparation Reagent has been shown to work on a broad range of Gram positive and Gram negative organisms. The following procedure for preparing enriched samples is recommended as a starting point to validate your own sample preparation procedure. For more information on preparing bacteria from a liquid or a solid-phase culture, see the PrepMan Ultra Sample Preparation Reagent Protocol. CHEMICAL HAZARD. PrepMan Ultra contains a material that may cause eye, skin, and respiratory tract irritation, and adverse effects on the kidneys and blood and central nervous system. Read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. To prepare the enriched sample: 1. Transfer 1 ml of your enriched sample from the sample stomacher bag to a sterile microcentrifuge tube with attached screw-cap lid, then cap the tube. Use caution for biohazard waste. Note: Sterile microcentrifuge tubes with attached screw-cap lids are recommended in order to avoid: Lids opening during sample heating Sample cross-contamination (resulting from lids being placed on the wrong sample tubes) 2. Centrifuge the tube at maximum speed in a microcentrifuge for 3 minutes to spin down the contents. TaqMan Pseudomonas aeruginosa Detection Kit Protocol 11

22 To prepare the enriched sample: (continued) 3. Remove the supernatant from the tube, leaving the pellet behind. Dispose of the supernatant according to biohazardous material disposal procedures. Note: Do not disturb the pellet during supernatant extraction. 4. Resuspend the pellet in 100 µl of PrepMan Ultra Sample Preparation Reagent, then briefly vortex the tube to mix the contents. 5. Heat the tube in a heat block at C for 10 minutes, then briefly vortex the tube to mix the contents. 6. Centrifuge the tube at maximum speed in a microcentrifuge for 3 minutes to spin down the contents. 7. Transfer 50 µl of the supernatant into a new microcentrifuge tube. The sample can be stored at 4 C for up to a month. Note: Do not disturb the pellet during supernatant extraction. 8. Transfer 10 µl of the sample DNA to a new tube containing 90 µl of RNase-free water. 9. Briefly vortex the new tube to mix the contents. The sample DNA is now ready for PCR. 12 TaqMan Pseudomonas aeruginosa Detection Kit Protocol

23 Preparing PCR Preparing PCR Overview Creating the Plate Document Preparing PCR is the third step in using the TaqMan Pseudomonas aeruginosa Detection Kit. Preparing PCR consists of creating the plate document and the reaction mix. Note: All TaqMan Pathogen Detection Kits use universal cycling conditions, allowing you to run any combination of kits together in the same run. To create the plate document: 1. Create a plate document (Assay type = Relative Quantification or Absolute Quantification without standard curve). 2. Specify FAM and VIC detectors for each reaction well you will use in the assay. 3. Set thermal cycling conditions specified below. Refer to the appropriate instrument user guide for details. Step AmpliTaq Gold Enzyme Activation HOLD PCR Cycle (45 Cycles) Denature Anneal/ Extend Time 10 min 15 sec 1 min Temp 95 C 95 C 60 C Preparing the Reaction Mix To prepare the reaction mix: 1. Thaw all reagents completely. 2. Label a tube with the target name. TaqMan Pseudomonas aeruginosa Detection Kit Protocol 13

24 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. 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 Environmental Master Mix (EMM) 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 EMM 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. 14 TaqMan Pseudomonas aeruginosa Detection Kit Protocol

25 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: a. Applied Biosystems recommends diluting the positive control with RNase-free water to 1 pg per µl, resulting in approximately 2,000 copies per reaction. 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. 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 16). 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 Pseudomonas aeruginosa Detection Kit Protocol 15

26 Sealing Plates and Tubes 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. 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. 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. 16 TaqMan Pseudomonas aeruginosa Detection Kit Protocol

27 Preparing PCR To seal tubes: 1. Place strip caps on the tubes. 2. IMPORTANT! Apply significant downward pressure on the sealing tool in all steps 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 Pseudomonas aeruginosa Detection Kit Protocol 17

28 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 Pseudomonas aeruginosa Detection Kit. Running a plate consists of using an Applied Biosystems 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 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. 18 TaqMan Pseudomonas aeruginosa Detection Kit Protocol

29 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. To view 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 (target) Present VIC signal (IPC) Present or Absent Result Positive Absent Present Negative Absent Absent See Appendix A: Troubleshooting on page 20 TaqMan Pseudomonas aeruginosa Detection Kit Protocol 19

30 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 in unknown wells Target-specific signal detected in negative control wells PCR inhibited Environmental 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. 20 TaqMan Pseudomonas aeruginosa Detection Kit Protocol

31 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 Environmental 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 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 Pseudomonas aeruginosa Detection Kit Protocol 21

32 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 Pseudomonas aeruginosa Detection Kit Protocol

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36 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. C