The use of these guideline is dedicated for Argene kits that mentioned the instrument ABI PRISM 7900 in their package insert.

Transcription

1 ABI PRISM 7900 DNA R-gene kits Programming The use of these guideline is dedicated for Argene kits that mentioned the instrument ABI PRISM 7900 in their package insert. Some products have their own amplification program following the same principle. In this case, please refer to their Outlined procedure. Products to be amplified relate to the extracted nucleic acids obtained with the extraction methods recommended in the corresponding datasheet. Plan the experiment as described in the datasheet. AMPLIFICATION PROGRAM: 60 C R-gene Program Steps Time Temperature Cycles Wavelength for signal reading Taq Polymerase Activation 15 min. 95 C 1 - Amplification Denaturation 10 sec. 95 C Annealing Elongation 40 sec. 60 C Note: Temperature transition rate/slope is preset at 100% 45 - FAM and/or VIC end of the annealing 1. Throughout the patient follow-up, it is imperative to use the same protocol and to use the same extraction and amplification instrument.! 2. This guideline is based on ABI PRISM 7900HT, Version 2.2 Software. 3. Check if None is selected in the field Passive reference (see page 6) because amplification premixes do not contain PASSIVE REFERENCE. Page 1 / 10

2 ABI PRISM Programmiing In the room reserved for amplification STARTING THE APPLICATION SDS Switch on the computer, its screen and the ABI PRISM real-time PCR platform. - Enter user name and password. - Launch SDS plate Utility software by clicking its icon SDS2.2 (Fig.1). - A main window SDS2.2 appears. - From the File menu, select New. ENTRY OF THE PROGRAM DATA - A New document dialog box appears (Fig.2). - In the Assay field, select Absolute Quantification (standard curve). - In the Container field, select 96 wells Clear Plate. - In the template field, select Blank Template. - Leave the barcode field blank. - Click on OK. - A window Untitled 1- Absolute Quantification appears (Fig.3). - Click on the Instrument tab. Fig.2 Fig.1 Fig.3 Fig.4 - In the Thermal profile tab, click in the window stage 1 and click on delete step (fig. 5). Fig.5 Page 2 / 10

3 Hot Start Taq Activation: - In the field Stage 1 (Fig.6): Enter the value 95.0 in the top part of the window. Enter the value 15:00 in the bottom part of the window. Denaturation step: - In the left part of the field Stage 2. Enter the value 95.0 in the top part of the window. Enter the value 0:10 in the bottom part of the window. Annealing/Elongation step: - In the right part of the field Stage 2. Enter the value 60.0 in the top part of the window. Fig.6 Enter the value 0:40 in the bottom part of the window. - stage 2 is repeated 45 times, so type 45* in the blank field Repeat. * Number of cycles for amplification - Remove the selection of 9600 emulation and enter 25 in sample volume (μl). - Click on the Ramp Rate tab to check the parameters are 100% (Fig.7). - Click on the data collection tab to check that acquisition is done at 60.0 (Fig.8). Fig.7 Fig.8 - Click on tools then Detector Manager. A window Detector Manager appears. - Click on New to open the Add Detector window and fill in the fields (Fig.9). - Enter the following data: In the field Name enter, FAM R-gene. In the field Group select, Default. Leave the field Description blank. In the field Reporter enter, FAM. In the field Quencher enter, Non Fluorescent. In the field Color, choose green. - Click on OK to validate the creation of the detector FAM R-gene. Fig.9 Page 3 / 10

4 Only for multiple wavelength detection: - Click on New to open the Add Detector window and fill in the fields (Fig.10) - Enter the following data: In the field Name enter, VIC r-gene. In the field Group select, Default. Leave the field Description blank. In the field Reporter enter, VIC. In the field Quencher enter, Non Fluorescent. In the field Color, choose orange. - Click on OK to validate the creation of the detector VIC r-gene. Fig.10 Runniing the Program - Select the detector FAM R-gene then click on Copy to Plate Document. - Select the detector VIC R-gene then click on Copy to Plate Document. m Only for multiple wavelength detection. - Click on Done. DEFINE THE SAMPLE - Click the Set Up tab. - Select each well on the microplate map on the left hand side of the window. - Name the sample in the sample name field. - Select all used wells in the microplate map and then, select the check box Use in the Set up section (fig.11). FAM Rgene VIC Rgene Fig.11! For multiple wavelength detection, the wells need 2 detectors: FAM R-gene and VIC R-gene Page 4 / 10

5 - Specify the sample in the Task column as described below: Quantitative kits Qualitative kits Channels FAM VIC FAM VIC Patient samples unknown unknown unknown unknown Quantification standard (QS) standard unknown Not applicable Sensitivity control (SC) unknown unknown Not applicable Extraction+inhibition control (IC2W0) unknown unknown unknown unknown Positive control (PC) Not applicable unknown unknown Negative Amplification Control (R0) NTC (no template control) unknown NTC (no template control) unknown! If several parameters are detected in the same experiment, each quantification standard range has to be identified one by one. Not applicable for qualitative detection kits. ONLY FOR QUANTIFICATION: - Specify the concentration of the quantification standard in the Quantity. Conditions R-gene Kit Extraction method Specimen Sample volume to be extracted Standard concentration to be entered Elution volume QS1 QS2 QS3 QS EBV R-gene Whole blood 100 µl QIAampDNA 50 µl QIAcube Whole blood 100 µl µl 50 µl MagNA Pure Compact Whole blood 100 µl MagNA Pure LC System 50 µl NucliSENS easymag 50 µl BioRobot EZ1 Workstation Whole blood 350 µl 200 µl m2000sp Abbott 800 µl 250 µl plasma / CSF / (extract 300 µl) (eluate 150 µl) BAL / biopsies Versant kpcr Molecular System SP Plasma 400 µl (extract 250 µl) 65 µl (eluate 50 µl) CMV R-gene CMV HHV6, 7,8 R-gene QIAcube MagNA Pure Compact MagNA Pure LC System NucliSENS easymag m2000sp Abbott Versant kpcr Molecular System SP Whole blood Amniotic Fluid Serum / Whole blood Amniotic Fluid Serum / Whole blood Amniotic Fluid Serum / Whole blood Serum / plasma / BAL / urine / biopsies / amniotic fluid Plasma 200 µl 800 µl (extract 300 µl) 400 µl (extract 250 µl) 250 µl (eluate 150 µl) 65 µl (eluate 50 µl) Page 5 / 10

6 Conditions R-gene Kit Extraction method Specimen Sample volume to be extracted Standard concentration to be entered Elution volume QS1 QS2 QS3 QS HSV1 HSV2 VZV R-gene HSV1 r-gene HSV2 r-gene VZV r-gene QIAcube QIAamp MinElute Virus Spin Kit QIAamp MinElute Virus Spin Kit CSF / BAL / Ophthalmologic specimens / Gynaecological, smears / ENT cutaneous / plasma CSF CSF / BAL / Ophthalmologic specimens / Gynaecological, smears / ENT cutaneous / plasma CSF 200 µl 50 µl MagNA Pure Compact MagNA Pure LC System CSF NucliSENS easymag Versant kpcr Molecular System SP CSF CSF 400 µl (extract 250 µl) 65 µl (eluate 50 µl) samples* / Stool* / biopsies ADENOVIRUS R-gene QIAcube Stool Mini kit Stool Mini kit MagNA Pure Compact MagNA Pure LC System NucliSENS easymag BioRobot M48 QIAGEN m2000sp Abbott Stool* µl samples* / Stool* / biopsies 200 µl Stool* Plasma / samples* Stool* samples* / Stool* samples* biopsies / samples* 800 µl (extract 300 µl) Versant kpcr Molecular 400 µl System SP (extract 250 µl) * For a quantitative detection in cp/pcr, see datasheet, section µl (eluate 150 µl) 65 µl (eluate 50 µl) Page 6 / 10

7 Conditions R-gene Kit Extraction method Specimen Sample volume to be extracted Standard concentration to be entered Elution volume QS1 QS2 QS3 QS BK Virus R-gene QIAcube MagNA Pure Compact Urine Plasma Urine Plasma Urine 200 µl Plasma MagNA Pure LC System Whole blood Plasma / Urine NucliSENS easymag Plasma Urine Versant kpcr Molecular 400 µl 65 µl Plasma / Urine System SP (extract 250 µl) (eluate 50 µl) ! If several parameters are detected in the same experiment, each quantification standard range has to be identified one by one. Not applicable for qualitative detection kits. - Select none in passive Reference (amplification premix does not contain passive reference) (Fig.12). - Click on File in the front screen menu bar, then select Save As. - Name the program ( 60 C Rgene) and choose the folder where you want to save the program. - Validate your choice by clicking the Save button. - Click Instrument tab, click Connect (if necessary), then click on the Open/close button in the Instrument section and insert the microplate in the instrument. - Launch the run by clicking on Start. Fig.12 Page 7 / 10

8 Data Anallysiis - At the end of the experiment, the SDS software automatically opens (Fig.13). - Click OK. - Click the Analysis tab. - Click the Results tab. MANUAL BASELINE ADJUSTMENT: IDENTIFICATION OF THE SAMPLES - Click on Analysis menu then on Analysis Settings then set the following parameters: (Fig.14) - Choose All Detector in the field Detector. - Select Manual CT radio button. - Select the Manual Baseline radio button. - Choose 5 in the Start field and 15 in Stop field. Note: The value 15 is convenient only if all the curves display a CT after the 15 th cycle. - Click on Apply then Ok. - Right click on the Y axis in «Amplification plot», then Display settings and set the following parameters (Fig.15). Fig.13 Fig.14 - Click the Scale tab. o In the X Axis tab: Remove the selection for Autoscale button. Type 0.0 in the minimum field. Type 45.0 in the maximum field. o In the Y Axis tab: Select Log radio button. Remove the selection for Autoscale. Type in the minimum field. Type 100.0* in the maximum field. * This value has to be higher than the maximum of final fluorescence. - Click apply then OK. Fig.15 Page 8 / 10

9 Adjust the noise band (red horizontal line) to a position where it crosses the fluorescence curves of all the samples in their linear part. This step is performed in order to identify the positive samples which correspond to a calculated CT value. Negative samples are defined as Undetermined which is displayed in the CT column by the SDS software. LINEAR MODE: QUANTIFICATION OF THE SAMPLES - Go back to the linear mode by double clicking on the Y axis of the Amplification plot graph and by setting the following parameters (Fig.16) ONLLY FFOR QUANTTIIFFIICATTIION - Click the Scale tab. o o In the X Axis tab: Type 0.0 in the minimum field. Type 45.0 in the maximum field. In the Y Axis tab: Select Linear radio button. Type -1 in the minimum field. Type 60.0* in the maximum field. * This value has to be higher than the maximum of final fluorescence. - Click apply then OK. Fig.16 The quantification results are edited at the end of each experiment. NOTE : The maximum value for the Y axis may be different from one experiment to the other. Therefore, adjust the value of the Y axis in order to display the top of the amplification curves in the graph. Page 9 / 10

10 ANALYSIS OF CONTROLS (IC2sample, IC2W0) - Click on the Detector field then select the detector VIC R-gene (Fig.17).! (DICO Extra r-gene, Argene) has to be read in the FAM channel. Fig.17 - Perform the manual baseline adjustment as described in the previous section. - Read the calculated CT at VIC for each sample inhibition control (IC2sample) and compare its value to the CT value of the reference extraction+ inhibition control (IC2W0). NOTE : Do not read the CT of the quantification standards (QS), sensitivity control (SC), positive control (PC) or negative amplification control (R0) in the VIC channel. The interpretation results is only based on the comparison between the CT obtained for each sample extraction + inhibition control (IC2sample) and the CT obtained with reference extraction+ inhibition control (IC2W0). Interpretatiion of Resullts - Detailed interpretation is described in each corresponding data sheet. BIOMERIEUX, the blue logo, ARGENE, R-gene, r-gene, easymag and NucliSENS are used, pending and/or registered trademarks belonging to biomérieux, or one of its subsidiaries, or one of its companies. Any other name or trademark is the property of its respective owner. biomérieux S.A. Chemin de l Orme Marcy-l Étoile - France RCS LYON Tel.: 33 (0) Fax: 33 (0)