PROTOCOL. ViralSEQ Mouse Minute Virus (MMV) Real-Time PCR Detection Kit

Transcription

1 PROTOCOL ViralSEQ Mouse Minute Virus (MMV) Real-Time PCR Detection Kit Publication Part Number Rev. D Revision Date March 2011

2 For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. Information in this document is subject to change without notice. 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. TO THE FULLEST EXTENT ALLOWED BY LAW, IN NO EVENT SHALL APPLIED BIOSYSTEMS BE LIABLE, WHETHER IN CONTRACT, TORT, WARRANTY, OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING BUT NOT LIMITED TO THE USE THEREOF, WHETHER OR NOT FORESEEABLE AND WHETHER OR NOT APPLIED BIOSYSTEMS IS ADVISED OF THE POSSIBILITY OF SUCH DAMAGES. Limited Use Label License The purchase of this product conveys the right to use only this amount of product solely for environmental testing, quality control/quality assurance testing, food and agricultural testing, including 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 and also for the purchaser s own internal research and development activities. No additional rights for any other purpose 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. For information on obtaining additional rights, please contact outlicensing@lifetech.com or Out Licensing, Life Technologies, 5791 Van Allen Way, Carlsbad, California Human diagnostic uses require a separate license from Roche. For information on obtaining additional rights, please contact outlicensing@lifetech.com or Out Licensing, Life Technologies, 5791 Van Allen Way, Carlsbad, California Trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners. TaqMan and Amplitaq Gold are a registered trademarks of Roche Molecular Systems, Inc Life Technologies Corporation. All rights reserved Rev. D

3 Contents About This Guide Purpose Safety information Safety alert words SDSs PROTOCOL ViralSEQ Mouse Minute Virus (MMV) Real-Time PCR Detection Kit Product information Purpose of the product Materials and equipment required Workflow Procedure Prepare the sample Prepare for PCR Prepare the PCR reactions Seal the plates Perform PCR Analyze the plate document View and verify the results Examples of Results Controls Test samples Troubleshooting APPENDIX A Ordering Information How to order Kit contents Materials and equipment not included APPENDIX B Chemistry Overview PCR reaction components Polymerase chain reaction (PCR) TaqMan Probes ViralSEQ Mouse Minute Virus (MMV) Real-Time PCR Detection Kit Protocol 3

4 Contents APPENDIX C Kit Specificity Inclusivity detectable species Exclusivity undetectable organisms APPENDIX D Good Laboratory Practices Prevent PCR contamination Avoiding false positives due to cross-contamination Plate layout suggestions APPENDIX E Safety Chemical safety General chemical safety SDSs Chemical waste safety Biological hazard safety General safety alerts for all chemicals Documentation and Support Related documentation Obtaining support Glossary Index ViralSEQ Mouse Minute Virus (MMV) Real-Time PCR Detection Kit Protocol

5 About This Guide Purpose This protocol provides: A list of materials and equipment that can be used with the ViralSEQ Mouse Minute Virus (MMV) Real-Time PCR Detection Kit Guidelines for sample preparation Instructions for preparing reaction plates and performing PCR using the ViralSEQ Mouse Minute Virus (MMV) Real-Time PCR Detection Kit on Applied Biosystems Real-Time PCR Systems General troubleshooting guidelines Background information about the detection of MMV strains Safety information Note: For general safety information, see this section and Appendix E, Safety on page 37. When a hazard symbol and hazard type appear by a chemical name or instrument hazard, see the Safety Appendix for the complete alert on the chemical or instrument. Safety alert words Four safety alert words appear in Applied Biosystems user documentation at points in the document where you need to be aware of relevant hazards. Each alert word IMPORTANT, CAUTION, WARNING, DANGER implies a particular level of observation or action, as defined below: IMPORTANT! Indicates information that is necessary for proper instrument operation, accurate chemistry kit use, or safe use of a chemical. CAUTION! Indicates a potentially hazardous situation that, if not avoided, may result in minor or moderate injury. It may also be used to alert against unsafe practices. WARNING! Indicates a potentially hazardous situation that, if not avoided, could result in death or serious injury. ViralSEQ Mouse Minute Virus (MMV) Real-Time PCR Detection Kit Protocol 5

6 About This Guide Safety information DANGER! Indicates an imminently hazardous situation that, if not avoided, will result in death or serious injury. This signal word is to be limited to the most extreme situations. SDSs The Safety Data Sheets (SDSs) for any chemicals supplied by Applied Biosystems or Ambion are available to you free 24 hours a day. For instructions on obtaining SDSs, see SDSs on page 39. IMPORTANT! For the SDSs of chemicals not distributed by Applied Biosystems or Ambion contact the chemical manufacturer. 6 ViralSEQ Mouse Minute Virus (MMV) Real-Time PCR Detection Kit Protocol

7 PROTOCOL ViralSEQ Mouse Minute Virus (MMV) Real- Time PCR Detection Kit Product information Purpose of the product The ViralSEQ Mouse Minute Virus (MMV) Real-Time PCR Detection Kit is a TaqMan -based Real-Time PCR kit for highly sensitive detection of MMV in cell culture samples. The kit contains TaqMan probe and primer mix, TaqMan Environmental Master Mix 2.0, negative control, and a dual-purpose positive control. Assays for MMV, Vesivirus, and Mycoplasma can be set up and run in the same plate. Sensitivity Kit specificity We recommend using the PrepSEQ sample preparation procedure in the PrepSEQ Sample Preparation Kits Protocol (PN ), which allows you to detect approximately TCID[50] per ml of sample. (TCID[50] is the median tissue culture infective dose, that is, the amount of a pathogenic agent that will produce pathological change in 50% of cell cultures inoculated.) Note: TCID[50] may vary depending on accuracy of cell culture assay. The ViralSEQ Mouse Minute Virus (MMV) Real-Time PCR Detection Kit can detect all known MMV strains as (see Appendix C, Kit Specificity on page 33). The kit does not detect other genera or cell-line DNA. ViralSEQ Mouse Minute Virus (MMV) Real-Time PCR Detection Kit Protocol 7

8 ViralSEQ Mouse Minute Virus (MMV) Real-Time PCR Detection Kit Materials and equipment required Materials and equipment required The table below includes materials required for using (but not included in) the ViralSEQ Mouse Minute Virus (MMV) Real-Time PCR Detection Kit. Unless otherwise indicated, many of the listed items are available from major laboratory suppliers (MLS). Instruments, equipment, consumables, and reagents Item Source Instruments 7500 Fast Real-Time PCR System Contact your local Applied Biosystems sales office 7500 Real-Time PCR System Contact your local Applied Biosystems sales office 7900HT Fast Real-Time PCR System Equipment Heat block MLS Consumables Disposable gloves Aerosol-resistant pipette tips Pipettors: Positive-displacement Air-displacement Multichannel MicroAmp Optical 96-Well Reaction Plate with Barcode, 20 plates, 0.2-mL well; for use with Applied Biosystems 7300, 7500, and 7900HT Real-Time PCR Systems MicroAmp Fast Optical 96-Well Reaction Plate with Barcode, 20 plates, 0.1-mL well; for use with Applied Biosystems 7500 Fast Real-Time PCR System MicroAmp Optical 96-Well Reaction Plate with Barcode and Optical Adhesive Films, 100 plates with covers; for use with 7300 and 7500 Real-Time PCR Systems Contact your local Applied Biosystems sales office MLS MLS MLS Applied Biosystems (PN ) Not recommended for use with the 7500 Fast system. For 7500 Fast system reactions, use PN Applied Biosystems (PN ) Applied Biosystems (PN ) MicroAmp Optical 8-Cap Strip, 300 strips Applied Biosystems (PN ) 8 ViralSEQ Mouse Minute Virus (MMV) Real-Time PCR Detection Kit Protocol

9 ViralSEQ Mouse Minute Virus (MMV) Real-Time PCR Detection Kit Materials and equipment required Instruments, equipment, consumables, and reagents (continued) Item MicroAmp Optical Adhesive Film Kit, 20 covers, 1 applicator, 1 optical cover compression pad Source Applied Biosystems (PN ) MicroAmp Optical Adhesive Film, 25 covers Applied Biosystems (PN ) Reagents DNase-free, sterile-filtered water MLS For the SDS of any chemical not distributed by Applied Biosystems, contact the chemical manufacturer. Before handling any chemicals, refer to the SDS provided by the manufacturer, and observe all relevant precautions. ViralSEQ Mouse Minute Virus (MMV) Real-Time PCR Detection Kit Protocol 9

10 ViralSEQ Mouse Minute Virus (MMV) Real-Time PCR Detection Kit Workflow Workflow A simplified workflow for using the ViralSEQ Mouse Minute Virus (MMV) Real- Time PCR Detection Kit is shown below. IMPORTANT! For information on how to avoid PCR contamination, see Appendix D, Good Laboratory Practices on page 35. Prepare the sample (page 11) Prepare for PCR Prepare the plate document (page 11) Prepare the kit reagents and premix solution (page 12) Prepare the PCR reactions (page 13) Perform PCR (page 15) Analyze the plate document (page 15) View and verify results (page 16) 10 ViralSEQ Mouse Minute Virus (MMV) Real-Time PCR Detection Kit Protocol

11 ViralSEQ Mouse Minute Virus (MMV) Real-Time PCR Detection Kit Procedure Procedure Prepare the sample Refer to the PrepSEQ Sample Preparation Kits Protocol (PN ) for details on sample preparation. Prepare for PCR Prepare the plate document 1. During setup of the plate document, in the Assay drop-down list, select Absolute Quantification. 2. Select FAM, VIC, and NED detectors with: Quencher Dye set to (none) or (Non Fluorescent) Passive Reference set to ROX 3. Set thermal-cycling conditions as shown in the table and figure below. Step AmpliTaq Gold enzyme activation PCR HOLD Cycle (40 cycles) Denature Anneal/extend Temp 95 C 95 C 60 C Time 10 min 15 sec 1 min Set Sample Volume to 30 µl For the 7500 Fast system, set Run Mode to Standard 7500 Set Data Collection to Stage 2, Step 2 (60.0 C 1:00) For details, refer to the: 7300/7500/7500 Fast Real-Time PCR System Absolute Quantitation Using Standard Curve Getting Started Guide (PN ) 7900HT Fast Real-Time PCR System Absolute Quantitation Using Standard Curve Getting Started Guide (PN ) ViralSEQ Mouse Minute Virus (MMV) Real-Time PCR Detection Kit Protocol 11

12 ViralSEQ Mouse Minute Virus (MMV) Real-Time PCR Detection Kit Procedure Plate document settings Prepare the kit reagents and premix solution 1. Thaw all kit reagents completely. Applied Biosystems recommends thawing the positive control at 37 C for 5 minutes to ensure consistent results. 2. Vortex, then spin down the reagents. 3. Label a microcentrifuge tube for the premix solution, and for each sample and control reaction. 4. Prepare the Premix Solution according to the following table. IMPORTANT! Use a separate pipette tip for each component. Component for Premix Solution Volume for one 30-µL reaction Volume for four 30-µL reactions Environmental Master Mix 2.0 (2 ) 15.0 µl 66.0 µl MMV Real-Time PCR Assay Mix (10 ) 3.0 µl 13.2 µl Negative Control (water) 2.0 µl 8.8 µl Total Premix Solution Volume 20 µl 88 µl Includes 10% excess to compensate for pipetting errors. 12 ViralSEQ Mouse Minute Virus (MMV) Real-Time PCR Detection Kit Protocol

13 ViralSEQ Mouse Minute Virus (MMV) Real-Time PCR Detection Kit Procedure 5. Mix the Premix Solution by gently pipetting up and down, then cap the tube. Prepare the PCR reactions Into labeled appropriate-sized PCR tubes or the wells of a reaction plate, pipet the reagent volumes specified in the following table. To prepare... Negative-control reaction Your unknown sample reaction Positive-control reaction Combine in each tube or well µl of Premix Solution 10 µl of Negative Control (water) 20 µl of Premix Solution 10 µl of unknown sample 20 µl of Premix Solution 2 µl of MMV Real-Time PCR DNA Control (positive control) 8 µl of Negative Control (water) 1. Dispense 20 µl of Premix Solution into each well to be used, gently pipetting at the bottom of the well. For the: 7500 Fast system Dispense into a Fast optical 96-well plate (PN ) 7500 and 7900HT Fast (standard block) systems Dispense into a standard optical 96-well plate (PN ) 7900HT Fast system (Fast block) Dispense into a Fast optical 96-well plate (PN ) 7300 system Dispense into a standard optical 96-well plate (PN ) 2. For each row of wells that you use, place in sequence from left to right the negative control, unknown sample, then positive control. See Plate layout suggestions on page 36 for more information. IMPORTANT! Use at least one negative and one positive control per run. IMPORTANT! Mix each sample very gently by placing the pipette tip at the bottom of the tube and pipetting up and down to minimize aerosol formation and crosscontamination. IMPORTANT! Use a new tip for each well, even when aliquoting the same solution. 3. If you are using tubes, cap the tubes and proceed to Perform PCR on page 15. If you are using plates, continue to the next section. ViralSEQ Mouse Minute Virus (MMV) Real-Time PCR Detection Kit Protocol 13

14 ViralSEQ Mouse Minute Virus (MMV) Real-Time PCR Detection Kit Procedure Seal the 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 to completely seal 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. Long edge of plate Applicator 3. Rub the edge of the applicator horizontally and vertically between all wells. Short edge of plate 4. Rub the edge of the applicator around all outside edges of the plate using small back and forth motions to completely seal around the outside wells. 5. Vortex the plate on the low setting for 5 seconds. If you see liquid on the well sidewalls, spin down the plate at 2000 g for 20 seconds using a centrifuge with a plate adapter. IMPORTANT! Make sure that reagents are in the bottom of the wells. 14 ViralSEQ Mouse Minute Virus (MMV) Real-Time PCR Detection Kit Protocol

15 ViralSEQ Mouse Minute Virus (MMV) Real-Time PCR Detection Kit Procedure Perform PCR On an Applied Biosystems Real-Time PCR System: 1. Open the plate document that corresponds to the reaction plate you created on page Load the reaction plate into the real-time PCR system. 3. Start the run. Analyze the plate document For specific instructions on analyzing your results, refer to the user guide for your realtime PCR instrument. 1. Open the plate document. 2. Select the wells to analyze. 3. In the Results tab, click Analysis. Select Analyze in the drop-down list. 4. In the Analysis Settings dialog box, make sure the settings for all reactions are: C T Start (cycle) End (cycle) Threshold Manual ViralSEQ Mouse Minute Virus (MMV) Real-Time PCR Detection Kit Protocol 15

16 ViralSEQ Mouse Minute Virus (MMV) Real-Time PCR Detection Kit Procedure View and verify the results View amplification plot 1. In the Results tab, select the Amplification Plot tab. 2. In the drop-down list in the Detector field at the top right corner of the plot screen, select the appropriate detectors. 3. Evaluate the results for wells according to Table 1 on page 17. Interpret results Examine the wells for these characteristics: IPC signal (NED dye) should be present in all wells. For most instruments, the IPC in all wells should have a C T between 28 and 36 at the manual C T threshold of 0.2. The IPC signal can be inhibited or absent in test samples if there is a large amount of viral DNA due to competition. In a test sample, samples with viral DNA competition show: Low C T for the target-specific signal (FAM dye) C T > 36 or Undetermined for the IPC signal (NED dye) No positive control signal (VIC dye) If there is viral inhibition and the target-specific signal (FAM dye) is present, the IPC signal (NED dye) can be ignored and the sample considered positive for the presence of MMV. Negative controls: PCR negative control (NTC) and extraction negative control (ENC) Only IPC signal (NED dye), should be present in the negative controls. If either VIC dye or FAM dye is present, the control shows contamination and it is necessary to repeat the experiment with freshly prepared samples and reagents. Positive controls: PCR positive control (PPC) and extraction positive control (EPC) Target-specific signal (FAM dye), positive control signal (VIC dye), and IPC signal (NED dye) should be present in the positive controls. If they are not, repeat the experiment with freshly prepared samples and reagents. Test samples (Unknowns): If the only signal detected is IPC (NED dye), the test sample is negative for the presence of MMV. If both IPC (NED dye) and targetspecific (FAM dye) signals are detected, the test sample is positive for the presence of MMV. If positive control signal (VIC dye) is present, the test sample shows contamination with the positive control and it is necessary to repeat the experiment with freshly prepared samples and reagents. The information in the following table applies to results generated on any Applied Biosystems Real-Time PCR System. C T values may vary among instrument platforms. 16 ViralSEQ Mouse Minute Virus (MMV) Real-Time PCR Detection Kit Protocol

17 ViralSEQ Mouse Minute Virus (MMV) Real-Time PCR Detection Kit Procedure Table 1 Acceptance criteria Sample Type FAM C T value VIC C T value NED signal Call PCR Positive Control (PPC) (PCR, 2,000 copies/reaction) Extraction Positive Control (EPC) (Sample DNA extraction) PCR Negative Control (NTC) (PCR) Extraction Negative Control (ENC) (Sample DNA extraction) C T 32 C T 32 Present Positive for PPC (Example: Positive controls: FAM and VIC present ) C T C T Present Positive for EPC (Example: Positive controls: FAM and VIC present ) Undetermined Undetermined Present Negative (Example: Negative controls: FAM and VIC undetermined ) Undetermined Undetermined Present Negative (Example: Negative controls: FAM and VIC undetermined ) Test sample C T Undetermined Present Positive for MMV (Example: Positive result ) Test sample Undetermined Undetermined Present Negative for MMV (Example: Negative result ) The average FAM C T for 20 copies of MMV genomic DNA is 36.7 ± 0.7 The average VIC C T for 10 copies of MMV Positive Control is 37.1 ± 0.3. ViralSEQ Mouse Minute Virus (MMV) Real-Time PCR Detection Kit Protocol 17

18 ViralSEQ Mouse Minute Virus (MMV) Real-Time PCR Detection Kit Examples of Results Examples of Results The figures in this section show examples of results that meet or do not meet criteria for interpretation that are set in our laboratories. Users should interpret results according to their laboratory protocols. See Troubleshooting on page 24 if signals do not meet the appropriate criteria. Amplification plots were generated on the 7500-Fast Real-Time PCR System with SDS Software v1.4. Controls Positive controls: FAM and VIC present The criteria determined in Applied Biosystems laboratories for positive controls are: FAM dye (red, MMV) C T VIC dye (green, PC) C T NED dye (blue, IPC) C T The amplitude curves and reports below meet these criteria. PCR Positive Control (2,000 copies of positive control) Extraction Positive Control (EPC) Note: Users should determine criteria for their laboratory according to their laboratory protocols. 18 ViralSEQ Mouse Minute Virus (MMV) Real-Time PCR Detection Kit Protocol

19 ViralSEQ Mouse Minute Virus (MMV) Real-Time PCR Detection Kit Examples of Results Negative controls: FAM and VIC undetermined The criteria determined in Applied Biosystems laboratories for negative controls are: FAM dye (red, MMV) C T undetermined VIC dye (green, PC) C T undetermined NED dye (blue, IPC) C T The amplitude curves and reports below meet these criteria. Non-template Control (NTC) Extraction Negative Control (ENC) Note: Users should determine criteria for their laboratory according to their laboratory protocols. ViralSEQ Mouse Minute Virus (MMV) Real-Time PCR Detection Kit Protocol 19

20 ViralSEQ Mouse Minute Virus (MMV) Real-Time PCR Detection Kit Examples of Results Controls indicating contamination FAM and VIC dye signal in the unknown sample or negative control reaction indicates contamination by the Positive Control. If FAM and VIC dye signal appears in these wells, the reactions should be repeated. The amplitude curves and reports below show results that do not meet our criteria. Positive control in negative control wells: FAM dye (red, MMV) Present VIC dye (green, PC) Present Positive control in test sample: FAM dye (red, MMV) Present VIC dye (green, PC) Present In these cases, we recommend repeating the experiment with freshly prepared sample and reagents. Positive control in ENC Positive control in test sample 20 ViralSEQ Mouse Minute Virus (MMV) Real-Time PCR Detection Kit Protocol

21 ViralSEQ Mouse Minute Virus (MMV) Real-Time PCR Detection Kit Examples of Results Test samples Positive result The criteria determined in Applied Biosystems laboratories for positive results are: FAM dye (red, MMV) C T VIC dye (green, PC) C T undetermined NED dye (blue, IPC) C T Present The IPC signal can be inhibited or absent if there is a large amount of viral DNA due to competition. The amplitude curve and report below meet our criteria for positive results. MMV-positive result MMV-positive result Note: Users should determine criteria for their laboratory according to their laboratory protocols. ViralSEQ Mouse Minute Virus (MMV) Real-Time PCR Detection Kit Protocol 21

22 ViralSEQ Mouse Minute Virus (MMV) Real-Time PCR Detection Kit Examples of Results Negative result The criteria determined in Applied Biosystems laboratories for negative results are: FAM dye (red, MMV) C T undetermined VIC dye (green, PC) C T undetermined NED dye (blue, IPC) Present The amplitude curve and report below meet our criteria for negative results. MMV-negative result Note: Users should determine criteria for their laboratory according to their laboratory protocols. 22 ViralSEQ Mouse Minute Virus (MMV) Real-Time PCR Detection Kit Protocol

23 ViralSEQ Mouse Minute Virus (MMV) Real-Time PCR Detection Kit Examples of Results Inconclusive result The amplitude curve and report below show undetermined signal for all dyes. This result is inconclusive. We recommend repeating the experiment with freshly prepared reagents and freshly prepared re-purified sample. Inconclusive result ViralSEQ Mouse Minute Virus (MMV) Real-Time PCR Detection Kit Protocol 23

24 ViralSEQ Mouse Minute Virus (MMV) Real-Time PCR Detection Kit Troubleshooting Troubleshooting Observation Possible cause Action No MMV target-specific signal (FAM dye) is detected in PCR positive control and/or extraction positive control wells. No positive control signal (VIC dye) is detected in PCR positive control and extraction positive control wells. No IPC (NED ) or targetspecific (FAM ) dye signal is detected in unknown wells IPC signal C T > 36 in unknown or positive control wells IPC signal C T is inconsistent between unknown wells No IPC signal is detected, but target-specific signal is detected No IPC signal is detected Target-specific signal or positive-control signal is detected in negative-control wells No target-specific signal detected: background noise crosses the threshold FAM detector not selected VIC detector not selected Pipetting error (no positive control added) Inhibition of PCR Inhibition of PCR Your instrument may have intrinsic well-to-well variability High copy number of target DNA resulting in preferential amplification of the targetspecific DNA NED dye detector not selected Carryover contamination High background noise Make sure FAM detector is selected in the plate document (see Prepare the plate document on page 11). Repeat the analysis with the FAM detector selected. Make sure VIC detector is selected in the plate document (see Prepare the plate document on page 11). Repeat the analysis with the VIC detector selected. Repeat the assay. Make sure to add positive control into all positive-control wells. Repeat the sample preparation, then repeat the assay. If PCR remains inhibited, dilute the sample (for example, 1:5 or 1:10) to dilute inhibitors. Repeat the sample preparation, then repeat the assay. If PCR inhibition continues, dilute the sample (for example, 1:5 or 1:10) to dilute inhibitors. Repeat assay at different position in the heating block. Dilute the sample (for example, 1:5 or 1:10), then repeat assay reactions. Make sure NED detector is selected in the plate document (see Prepare the plate document on page 11). Repeat the analysis with the NED detector selected. 1. Repeat the assay using fresh aliquots of all reagents and clean pipetting equipment. 2. If the negative control continues to show contamination, repeat the assay using a new kit. 3. If the negative control still continues to show contamination, contact Applied Biosystems Technical Support. Manually set the threshold slightly above any negativecontrol signal. However, setting the threshold too high above background noise may result in decreased detection sensitivity of positive samples. If the new threshold does not cross the exponential phase in the positive wells, decrease the threshold. Reanalyze. Note: Setting the threshold above the negative control signal changes the CT of the negative controls to Undetermined. 24 ViralSEQ Mouse Minute Virus (MMV) Real-Time PCR Detection Kit Protocol

25 ViralSEQ Mouse Minute Virus (MMV) Real-Time PCR Detection Kit Troubleshooting Observation (continued) Possible cause Action Replicate results for sample are inconsistent No dye signal in test sample well Your instrument may have intrinsic well-to-well variability Error in preparation If more than two replicates yield the same result (for example, you ran three replicates and two replicates are negative, but one replicate is positive), the result associated with the larger number of replicates is probably accurate. However, your laboratory protocol may dictate that you repeat the assay using fresh samples and reagents. If you ran only two replicates and results are not consistent, repeat the assay using fresh samples and reagents. If instrument variability is suspected, try repeating the assay at a different position in the heating block. In addition, check the lamp status on the instrument. Repeat the sample preparation and perform a new PCR reaction, using freshly prepared sample and reagents. ViralSEQ Mouse Minute Virus (MMV) Real-Time PCR Detection Kit Protocol 25

26 ViralSEQ Mouse Minute Virus (MMV) Real-Time PCR Detection Kit Troubleshooting 26 ViralSEQ Mouse Minute Virus (MMV) Real-Time PCR Detection Kit Protocol

27 APPENDIX A Ordering Information How to order To order kits and supplies, go to Kit contents The table shows components of the ViralSEQ Mouse Minute Virus (MMV) Real- Time PCR Detection Kit (PN ). Part Number Part Cap Color Component Quantity Storage Conditions Box 1, SEQ Real-Time PCR Core Kit, containing: 1 box Protect from light Environmental Master Mix tubes Store ml/tube components in light orange the box at 15 to 25 C until Negative Control (water) 1 tube first use. After 1.0 ml first use, store at 2 to 8 C. white Box 2, MMV Real-Time PCR Assay Mix, containing: 1 box Protect from light MMV Assay Mix, 1 tube 1 tube Store ml components in the box at 15 yellow to 25 C Box 3, MMV Discriminatory Positive/Extraction control, containing: red MMV Positive Control, 1 tube 1 box Store components in the box at 15 1 tube to 25 C ml ViralSEQ Mouse Minute Virus (MMV) Real-Time PCR Detection Kit Protocol 27

28 Appendix A Ordering Information Materials and equipment not included Materials and equipment not included The table below includes materials required for using (but not included in) the ViralSEQ Mouse Minute Virus (MMV) Real-Time PCR Detection Kit. Unless otherwise indicated, many of the listed items are available from major laboratory suppliers (MLS). Instruments, equipment, consumables, and reagents Item Source Instruments 7500 Fast Real-Time PCR System Contact your local Applied Biosystems sales office 7500 Real-Time PCR System Contact your local Applied Biosystems sales office 7900HT Fast Real-Time PCR System Equipment Block heater MLS Consumables Disposable gloves Aerosol-resistant pipette tips Pipettors: Positive-displacement Air-displacement Multichannel MicroAmp Optical 96-Well Reaction Plate with Barcode, 20 plates, 0.2-mL well; for use with Applied Biosystems 7300, 7500, and 7900HT Real-Time PCR Systems MicroAmp Fast Optical 96-Well Reaction Plate with Barcode, 20 plates, 0.1-mL well; for use with Applied Biosystems 7500 Fast Real-Time PCR System MicroAmp Optical 96-Well Reaction Plate with Barcode and Optical Adhesive Films, 100 plates with covers; for use with 7300 and 7500 Real-Time PCR Systems Contact your local Applied Biosystems sales office MLS MLS MLS Applied Biosystems (PN ) Not recommended for use with the 7500 Fast system. For 7500 Fast system reactions, use PN Applied Biosystems (PN ) Applied Biosystems (PN ) MicroAmp Optical 8-Cap Strip, 300 strips Applied Biosystems (PN ) 28 ViralSEQ Mouse Minute Virus (MMV) Real-Time PCR Detection Kit Protocol

29 Appendix A Ordering Information Materials and equipment not included Instruments, equipment, consumables, and reagents (continued) Item MicroAmp Optical Adhesive Film Kit, 20 covers, 1 applicator, 1 optical cover compression pad Source Applied Biosystems (PN ) MicroAmp Optical Adhesive Film, 25 covers Applied Biosystems (PN ) Reagents DNase-free, sterile-filtered water MLS For the SDS of any chemical not distributed by Applied Biosystems, contact the chemical manufacturer. Before handling any chemicals, refer to the SDS provided by the manufacturer, and observe all relevant precautions. ViralSEQ Mouse Minute Virus (MMV) Real-Time PCR Detection Kit Protocol 29

30 Appendix A Ordering Information Materials and equipment not included 30 ViralSEQ Mouse Minute Virus (MMV) Real-Time PCR Detection Kit Protocol

31 APPENDIX B Chemistry Overview PCR reaction components Environmental Master Mix 2.0 (2 ) MMV Real-Time PCR Primer Mix (10 ) DNA isolated from media, cell culture, or other sources Polymerase chain reaction (PCR) 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 solution containing DNA polymerase, nucleotides, and primers that are complementary to the target DNA sequence. When this solution is heated, the dsdna denatures, separating into two separate strands. As the solution cools, the primers anneal to the target sequences in the separated DNA strand. The DNA polymerase then forms a new strand by extending the primers with nucleotides, creating a complimentary copy of the target 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 Probes The TaqMan probe contains a fluorescent reporter dye at the 5 end of the probe and a quencher dye at the 3 end of the probe. When the probe is intact, the proximity of the reporter dye to the quencher dye suppresses the reporter fluorescence. Probe cleavage during the PCR reaction spatially separates the reporter dye from the quencher moiety and allows detection of the reporter dye fluorescence. ViralSEQ Mouse Minute Virus (MMV) Real-Time PCR Detection Kit Protocol 31

32 Appendix B Chemistry Overview TaqMan Probes 32 ViralSEQ Mouse Minute Virus (MMV) Real-Time PCR Detection Kit Protocol

33 APPENDIX C Kit Specificity Inclusivity detectable species Bioinformatic analysis indicates that the ViralSEQ Mouse Minute Virus (MMV) Real- Time PCR Detection Kit can detect four MMV strains: MMVi MMVp MMVm MMVc Exclusivity undetectable organisms The table below shows species that the kit is not designed to detect. Organism Strain/source Human Mouse Rat Chinese Hamster Ovary (CHO)-K1 Streptococcus pneumoniae Saccharomyces cerevisiae Staphylococcus enterica Staphylococcus aureus Escherichia coli Promega Novagen Novagen Lofstrand ATCC ATCC ATCC ATCC ATCC ViralSEQ Mouse Minute Virus (MMV) Real-Time PCR Detection Kit Protocol 33

34 Appendix C Kit Specificity Exclusivity undetectable organisms 34 ViralSEQ Mouse Minute Virus (MMV) Real-Time PCR Detection Kit Protocol

35 APPENDIX D Good Laboratory Practices Prevent PCR contamination PCR assays require special laboratory practices to avoid false positive amplifications. The efficiency and high sensitivity of these assays can lead to amplification of a single DNA molecule. When preparing samples for PCR amplification: Wear clean gloves and a clean lab coat (not previously worn while handling amplified PCR products or used during sample preparation) when preparing samples for PCR amplification. Change gloves whenever you suspect that they are contaminated. Maintain separate work areas and dedicated equipment and supplies for: Sample preparation PCR setup PCR amplification Analysis of PCR products Never bring amplified PCR products into the PCR setup area. 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 a positive-displacement pipette or aerosol-resistant pipette tips. Clean lab benches and equipment after use with freshly diluted 10% bleach solution. Avoiding false positives due to cross-contamination To avoid false positives due to cross-contamination: Prepare and close all negative control and unknown sample tubes before pipetting the positive control. Do not open tubes after amplification. Use different sets of pipettors when pipetting negative control, unknown, and positive control samples. ViralSEQ Mouse Minute Virus (MMV) Real-Time PCR Detection Kit Protocol 35

36 Appendix D Good Laboratory Practices Plate layout suggestions Plate layout suggestions For each plate row, dispense in sequence from left to right the: negative controls, unknown samples, and positive controls (at the end of the row or column). Place positive controls in one of the outer columns. If possible, separate all samples from each other by at least one well; if space is limiting, place at least one well between unknown samples and controls. Be aware that caps come in strips of 8 or ViralSEQ Mouse Minute Virus (MMV) Real-Time PCR Detection Kit Protocol

37 APPENDIX E Safety This appendix covers: Chemical safety General chemical safety SDSs Chemical waste safety Biological hazard safety General safety alerts for all chemicals ViralSEQ Mouse Minute Virus (MMV) Real-Time PCR Detection Kit Protocol 37

38 Appendix E Safety Chemical safety Chemical safety General chemical safety Chemical hazard warning WARNING! CHEMICAL HAZARD. Before handling any chemicals, refer to the Safety Data Sheet (SDS) provided by the manufacturer, and observe all relevant precautions. WARNING! CHEMICAL HAZARD. All chemicals in the instrument, including liquid in the lines, are potentially hazardous. Always determine what chemicals have been used in the instrument before changing reagents or instrument components. Wear appropriate eyewear, protective clothing, and gloves when working on the instrument. WARNING! CHEMICAL HAZARD. Four-liter reagent and waste bottles can crack and leak. Each 4-liter bottle should be secured in a low-density polyethylene safety container with the cover fastened and the handles locked in the upright position. Wear appropriate eyewear, clothing, and gloves when handling reagent and waste bottles. WARNING! CHEMICAL STORAGE HAZARD. Never collect or store waste in a glass container because of the risk of breaking or shattering. Reagent and waste bottles can crack and leak. Each waste bottle should be secured in a lowdensity polyethylene safety container with the cover fastened and the handles locked in the upright position. Wear appropriate eyewear, clothing, and gloves when handling reagent and waste bottles. Chemical safety guidelines To minimize the hazards of chemicals: 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. (See About SDSs on page 39.) 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 SDS. 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 SDS. Check regularly for chemical leaks or spills. If a leak or spill occurs, follow the manufacturer s cleanup procedures as recommended in the SDS. Comply with all local, state/provincial, or national laws and regulations related to chemical storage, handling, and disposal. 38 ViralSEQ Mouse Minute Virus (MMV) Real-Time PCR Detection Kit Protocol

39 Appendix E Safety Chemical safety SDSs About SDSs Chemical manufacturers supply current Safety Data Sheets (SDSs) with shipments of hazardous chemicals to new customers. They also provide SDSs with the first shipment of a hazardous chemical to a customer after an SDS has been updated. SDSs provide the safety information you need to store, handle, transport, and dispose of the chemicals safely. Each time you receive a new SDS packaged with a hazardous chemical, be sure to replace the appropriate SDS in your files. Obtaining SDSs The SDS for any chemical supplied by Applied Biosystems is available to you free 24 hours a day. To obtain SDSs: 1. Go to click Support, then select SDS. 2. In the Keyword Search field, enter the chemical name, product name, SDS part number, or other information that appears in the SDS 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 Note: For the SDSs of chemicals not distributed by Applied Biosystems, contact the chemical manufacturer. Chemical waste safety Chemical waste hazards CAUTION! HAZARDOUS WASTE. Refer to Safety Data Sheets and local regulations for handling and disposal. WARNING! CHEMICAL WASTE HAZARD. Wastes produced by Applied Biosystems instruments are potentially hazardous and can cause injury, illness, or death. WARNING! CHEMICAL STORAGE HAZARD. Never collect or store waste in a glass container because of the risk of breaking or shattering. Reagent and waste bottles can crack and leak. Each waste bottle should be secured in a lowdensity polyethylene safety container with the cover fastened and the handles locked in the upright position. Wear appropriate eyewear, clothing, and gloves when handling reagent and waste bottles. ViralSEQ Mouse Minute Virus (MMV) Real-Time PCR Detection Kit Protocol 39

40 Appendix E Safety Chemical safety Chemical waste safety guidelines Waste disposal To minimize the hazards of chemical waste: Read and understand the Safety Data Sheets (SDSs) 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 SDS. 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 SDS. Handle chemical wastes in a fume hood. After emptying a waste container, seal it with the cap provided. Dispose of the contents of the waste tray and waste bottle in accordance with good laboratory practices and local, state/provincial, or national environmental and health regulations. 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. 40 ViralSEQ Mouse Minute Virus (MMV) Real-Time PCR Detection Kit Protocol

41 Appendix E Safety Chemical safety Biological hazard safety General biohazard WARNING! BIOHAZARD. Biological samples such as tissues, body fluids, infectious agents, and blood of humans and other animals have the potential to transmit infectious diseases. Follow all applicable local, state/provincial, and/or national regulations. Wear appropriate protective equipment, which includes but is not limited to: protective eyewear, face shield, clothing/lab coat, and gloves. All work should be conducted in properly equipped facilities using the appropriate safety equipment (for example, physical containment devices). Individuals should be trained according to applicable regulatory and company/ institution requirements before working with potentially infectious materials. Read and follow the applicable guidelines and/or regulatory requirements in the following: U.S. Department of Health and Human Services guidelines published in Biosafety in Microbiological and Biomedical Laboratories found at: Your company s/institution s Biosafety Program protocols for working with/ handling potentially infectious materials. Additional information about biohazard guidelines is available at: ViralSEQ Mouse Minute Virus (MMV) Real-Time PCR Detection Kit Protocol 41

42 Appendix E Safety General safety alerts for all chemicals General safety alerts for all chemicals Avoid contact with skin, eyes, and/or clothing. Read the SDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. 42 ViralSEQ Mouse Minute Virus (MMV) Real-Time PCR Detection Kit Protocol

43 Documentation and Support Related documentation Document ViralSEQ Mouse Minute Virus (MMV) Real-Time PCR Detection Kit Quick Reference Card Part number PrepSEQ Sample Preparation Kits Protocol /7500/7500 Fast Real-Time PCR System Absolute Quantitation Using Standard Curve Getting Started Guide 7900HT Fast Real-Time PCR System Absolute Quantitation Using Standard Curve Getting Started Guide Obtaining support For the latest services and support information for all locations, go to: At the Applied Biosystems web site, you can: Access worldwide telephone and fax numbers to contact Applied Biosystems Technical Support and Sales facilities. Search through frequently asked questions (FAQs). Submit a question directly to Technical Support. Order Applied Biosystems user documents, SDSs, certificates of analysis, and other related documents. Download PDF documents. Obtain information about customer training. Download software updates and patches. ViralSEQ Mouse Minute Virus (MMV) Real-Time PCR Detection Kit Protocol 43

44 Documentation and Support Obtaining support 44 ViralSEQ Mouse Minute Virus (MMV) Real-Time PCR Detection Kit Protocol

45 Glossary Amplification Polymerase Chain Reaction (PCR) Negative Control Internal Positive Control (IPC) MMV Discriminatory Positive Control (PCR/Extraction Positive Control) The process of making copies of and thereby increasing the amount of a specific DNA sequence. Technology used to increase the amount of a DNA sequence. A reaction solution that lacks a target sequence. Monitors for contamination (unexpected amplification in the absence of a target) and reagent integrity. At least one negative control is required per run. A control used to monitor PCR amplification, allowing accurate interpretation of negative sample results. The IPC assay consists of: IPC template DNA (a synthetic sequence not found in nature) Two primers for amplifying the 130 base IPC template DNA One TaqMan MGB probe labeled with NED dye for detecting the amplified IPC DNA A specially designed plasmid DNA used as the positive control and as an extraction control. When used as: A positive control, amplification of the plasmid mimics the expected amplification of a target. Target signal that is not detected in a positive-control well indicates a pipetting error or a problem with amplification. At least one positive control is required per run. An extraction control, recovery of the plasmid from samples is used to monitor DNA extraction efficiency. The control plasmid is designed to give dual signals in a PCR reaction. This allows for detection of accidental contamination of samples with the positive control. Primer Target Unknown Sample A segment of DNA that is complementary to the target DNA sequence and is needed to start amplification. The pathogen being tested. A DNA sample from media, cell culture, or other source that you are testing for the presence of MMV. ViralSEQ Mouse Minute Virus (MMV) Real-Time PCR Detection Kit Protocol 45