GUIDELINES AmpliSens Florocenosis / Aerobes-FRT AmpliSens

Transcription

1 For Professional Use Only GUIDELINES to AmpliSens Florocenosis / Aerobes-FRT PCR kit for quantitative detection of the DNA of Enterobacteriaceae (E.coli, Klebsiella spp., Proteus spp. etc.), Staphylococcus spp. and Streptococcus spp. in the biological material by the polymerase chain reaction (PCR) with real-time hybridization-fluorescence detection AmpliSens Ecoli s.r.o., Studenohorska Bratislava 47 Slovak Republic Tel.: Fax: Federal Budget Institute of Science Central Research Institute for Epidemiology 3A Novogireevskaya Street Moscow Russia

2 TABLE OF CONTENTS INTENDED USE... 3 AMPLIFICATION AND DATA ANALYSIS USING Rotor-Gene 3000/6000 (Corbett Research, Australia) AND Rotor-Gene Q (QIAGEN, Germany) INSTRUMENTS... 3 AMPLIFICATION AND DATA ANALYSIS USING CFX96 (Bio-Rad, USA) AMPLIFICATION AND DATA ANALYSIS USING icycler iq and iq5 (Bio-Rad, USA) INSTRUMENTS REF R-B FT-CE / VER / Page 2 of 22

3 INTENDED USE The guidelines describe the procedure of using the AmpliSens Florocenosis / Aerobes- FRT PCR kit for quantitative detection of the DNA of Enterobacteriaceae (E.coli, Klebsiella spp., Proteus spp. etc.), Staphylococcus spp. and Streptococcus spp. in the biological material by the polymerase chain reaction (PCR) with real-time hybridization-fluorescence detection using the following instruments: Rotor-Gene 3000, Rotor-Gene 6000 (Corbett Research, Australia); Rotor-Gene Q (QIAGEN, Germany); icycler iq, iq5 (Bio-Rad, USA); CFX96 (Bio-Rad, USA). AMPLIFICATION AND DATA ANALYSIS USING Rotor-Gene 3000/6000 (Corbett Research, Australia) AND Rotor-Gene Q (QIAGEN, Germany) INSTRUMENTS When working with Rotor-Gene 3000 one should use the Rotor-Gene Version 6 versions 6.1 (or higher) software and the Rotor-Gene 6000 versions 1.7 (build 67) software or higher for Rotor-Gene 6000 and Rotor-Gene Q instruments. Hereinafter, all the terms corresponding to different instruments and software are indicated in the following order: for Rotor-Gene 3000 / for Rotor-Gene 6000/Q. Carry out the sample pretreatment and reaction mixture preparation stages according to the PCR kit Instruction Manual. Use the DNA-sorb-AM REF K CE kit for the extraction of DNA from the biological material. Extraction of DNA by express methods (for example, EDEM REF K CE) is unallowable. When carrying out the amplification it is recommended to use thin-walled PCR tubes (0.2 ml) with flat caps (e.g. Axygen, USA), or Rotor-Gene PCR tubes (0.1 ml) with caps from the four-pieces-strips (e.g. Corbett Research, Australia; QIAGEN, Germany) (detection through the bottom of the tube). Programming the thermocycler 1. Turn the instrument on, run the Rotor-Gene software. 2. Insert the tubes or strips into the rotor of the Rotor-Gene 3000/6000/Q instrument beginning from the first well; insert the rotor into the instrument, close the lid (the rotor wells are numbered, the numbers are used for the further programming of the samples order in the thermocycler). REF R-B FT-CE / VER / Page 3 of 22

4 Balance the rotor of the instrument if it is not loaded entirely. Fill the spare wells with empty tubes (don t use the tubes left after previous experiments). Well 1 must be filled with any studied tube except for an empty one. 3. Program the instrument according to the Instruction Manual given by the manufacturer of the instrument. 4. Click the New button in the main program menu. To create the template select the Advanced tab in the opened window New run. 5. Select the TwoStep/Hidrolysis Probes template in the tab for edition and click The New button. 6. In the opened window select the 36-Well Rotor (or 72-Well Rotor) and tick the No Domed Tubes / Locking Ring Attached option. Click the Next button. 7. In the opened window enter the operator name, select the reaction volume 25 µl. Tick the 15 µl oil layer volume option. Click the Next button. 8. In the New Run Wizard window set the temperature profile of the experiment. To do this click the Edit profile button and set the amplification program: AmpliSens unified amplification program Step Temperature, С Time Fluorescent Cycle signal detection repeats min min s FAM/Green, 3 JOE/Yellow, s ROX/Orange, Cy5/Red Any combination of the tests can be performed in one instrument simultaneously with the use of the unified amplification program. If only the tests for pathogen agent DNA detection are performed in one instrument then the first step (50 C 15 minutes) can be deleted from the program for time saving. AmpliSens-1 amplification program for rotor-type instruments Step Temperature, С Time Fluorescent signal detection Cycles min s s s 95 5 s s FAM/Green, JOE/Yellow, ROX/Orange, Cy5/Red s REF R-B FT-CE / VER / Page 4 of 22

5 AmpliSens-1 program is a universal amplification program for carrying out tests with the help of AmpliSens kits to identify the DNA of STI pathogens and other reproductive organs infections. All the tests and any tests combinations can be carried out in one instrument simultaneously. Note Channel for the Cy5.5 fluorophore is enabled when required if the multiprime format tests are performed. Fluorescence detection is assigned in the second step (60 ºC) of the cycling block (Step 3) (Acquiring to Cycling A) in the FAM/Green, JOE/Yellow, ROX/Orange, Cy5/Red channels. 9. Click OK after stating the amplification and detection programs. 10. Set automatic calibration for choosing Gain parameter. In New Run Wizard window, Channel Setup window click Calibrate/Gain Optimisation. In the opened window Auto Gain Calibration Setup click Calibrate Acquiring/Optimize Acquiring. Set the channels calibration, for FAM/Green channel enter 5 into the Min Reading box and 10 into Max Reading. For JOE/Yellow, ROX/Orange, Cy5/Red channels enter 4 into the Min Reading box and 8 in Max Reading. Perform the calibration in FAM/Green, JOE/Yellow, ROX/Orange and Cy5/Red channels before the first detection (tick the Perform Calibration Before 1 st Acquisition/ Perform Optimisation Before 1 st Close button. REF R-B FT-CE / VER / Page 5 of 22 Acquisition button). Click the Note Go to the next window. Click Save Template to save the template. Name the template according to the amplification program. Save the file to the folder Templates (the inside folder Quick Start Templates), close New Run Wizard window. After that the template will appear in the list of templates in New Run window. To run the test with the use of the ready template, choose New Run from the menu, select Advanced tab on top of the New Run Wizard window. Select the needed template with the amplification program in the list of templates in this window. 11. Click the Next button. For saving the programmed template it is necessary to click the Save Template button and enter the template file name, corresponding to the amplification program AmpliSens. Save the file into a proposed folder: Templates\Quick Start Templates; close the New Run Wizard window. After that the programmed template will appear in the template list in the New Run window. 12. Name the experiment and save it to the disk (results of the run will be automatically saved in this file).

6 13. In the samples table window set the order of the samples in the rotor by specifying name for each sample. Set the type Unknown opposite all the test samples. Set the type Standard for DNA calibrators and indicate their concentration. Set the type None for the cells matching with the corresponding empty tubes. Click OK. Samples indicated as None won t be analyzed. The table of samples can be edited before the start. For this, choose Edit Samples Before Run Started in File menu in submenu User preferences. Data analysis The analysis of the results is performed separately (sequentially) for each channel according to the Instruction Manual and given description. Analysis of the results is performed by the Rotor-Gene software with subsequent calculation of concentrations using the software in Microsoft Excel format. The fluorescence signal accumulation curves are analyzed in four channels: The signal of the Enterobacteriaceae DNA amplification product is detected in the FAM/Green channel. The signal of the Staphylococcus spp. DNA amplification product is detected in the JOE/Yellow channel. The signal of the Streptococcus spp. DNA amplification product is detected in the ROX/Orange channel. The signal of the IC DNA amplification product is detected in the Cy5/Red channel. Results are interpreted by the crossing (or not crossing) of the fluorescence curve with the threshold line that set at the level of exponential growth of fluorescence. It corresponds to the presence (or absence) of Ct (cycle threshold) value for the DNA-target in the appropriate cell of the result grid. The calibration curve is plotted and DNA concentrations of each group of microorganisms are calculated automatically by the used software based on the obtained Ct values in the respective channels and specified values of DNA calibrators. 1. Make sure that DNA calibrators K1 and K2 are set in the sample table. 2. Activate the button Analysis in the menu, select the mode of the analysis Quantitation, activate the buttons Cycling A. JOE/Cycling A. Yellow, Show, Cycling A. FAM/Cycling A. Green, Show, Cycling A. ROX/Cycling A. Orange, Show and Cycling A. Cy5/Cycling A. Red, Show. 3. Cancel the automatic choice of the threshold line level for each of the main open windows (FAM/Green, JOE/Yellow, ROX/Orange and Cy5/Red) by activating the REF R-B FT-CE / VER / Page 6 of 22

7 Threshold button. 4. Choose Linear scale. 5. Activate the Dynamic tube and Slope Correct buttons in the menu of each main window (Quantitation analysis). 6. In CT Calculation menu (at the right side of the window), set Threshold = Select the More settings/outlier Removal option and set NTC Threshold (see Table 1). Fluorescent channel FAM/Green JOE/Yellow ROX/Orange Table 1 Parameters for analysing results Detecting DNA target Threshold NTC Threshold Slope Correct Enterobacteriaceae DNA % On Staphylococcus spp. DNA % On Streptococcus spp. DNA % On Cy5/Red IC DNA % On 8. Ct values and calculated concentration values (Calc Conc.) will appear in the results grid (Quantitation Results window). 9. Calculate the results by the software in Microsoft Excel format. To do this, consistently for each channel copy the Ct values column to the appropriate column of the software in Microsoft Excel format. Mark the control samples and DNA calibrators according to the instruction for the software and click Calculate. Interpretation of results The results of PCR are reliable if the results of control samples are correct. Otherwise see Troubleshooting. The following results for controls are correct: 1. The Ct values determined for DNA calibrator K2 in four channels should not exceed the boundary values specified in the table 4 or 5 (depending on the amplification program). 2. Efficiency coefficient Е in the standard plot window in the FAM/Green, JOE/Yellow and ROX/Orange channels should be in the range (1.0 ± 0.2), specified in the Important Product Information Bulletin for the PCR kit. 3. The results for negative control samples should correspond to the results specified in the tables 2, 3. The concentrations values for the Negative Control of Amplification (NCA) should be absent or be not greater than the boundary value. The concentrations values for the Negative Control of Extraction (C ) should be absent or be not greater than the boundary value in the FAM/Green, JOE/Yellow and ROX/Orange channels. The Ct value determined for Negative Control of Extraction (C ) in Cy5/Red channel REF R-B FT-CE / VER / Page 7 of 22

8 should be not greater than the boundary value. Results for controls with the use of AmpliSens unified amplification program Control Stage for control Calculated concentration (GE/ml) or Ct value in FAM/Green, JOE/Yellow, in Cy5/Red channel ROX/Orange channels K2 PCR Ct < 38 Ct < 40 C DNA extraction NCA PCR absent or < 2х10 3 Ct < 40 absent or < 1х10 3 Ct value is absent Results for controls with the use of AmpliSens-1 amplification program Control Stage for control Calculated concentration (GE/ml) or Ct value in FAM/Green, JOE/Yellow, in Cy5/Red channel ROX/Orange channels K2 PCR Ct < 33 Ct < 35 C DNA Extraction NCA PCR absent or < 2х10 3 Ct < 35 absent or < 1х10 3 Ct value is absent Table 2 Table 3 Obtained values of number of copies of Enterobacteriaceae, Staphylococcus spp. and Streptococcus spp. DNA in the reaction tube are calculated automatically by the software of the instrument and given in the corresponding column of the result grid. Those values are used for calculation of number of genomic equivalents of the microorganisms DNA contained in 1 ml of the initial sample of biological material according to the formula: Number of microorganisms DNA copies x K = Number of genomic equivalents per 1 ml (GE/ml) The coefficient K for calculation of the result in GE/ml is specified in the Important Product Information Bulletin for the PCR kit. Results of the test samples are interpreted according to the following: 1. The result is considered to be reliable for the test sample, in which the calculated concentration of DNA of Enterobacteriaceae, Staphylococcus spp. and Streptococcus spp. is less than 1x10 4 GE/ml or is absent, only if the Ct value detected in the Cy5/Red channel (the channel for detection of the Internal Control) is not greater than 40 if the AmpliSens amplification program is used and 35 if the AmpliSens-1 amplification program is used. Otherwise, result is invalid (see Troubleshooting). REF R-B FT-CE / VER / Page 8 of 22

9 2. If the obtained concentration value is from 2х10 3 to 1x10 4 GE/ml then the result is interpreted as less than 1x10 4 GE/ml. If the obtained value is greater than 1х10 8 GE/ml then the result is interpreted as greater than 1х10 8 GE/ml (according to the linear range of the kit). 3. If the obtained concentration value for the test sample is less than 2x10 3 GE/ml, then the result is interpreted as [microorganisms group] DNA is not detected The clinical interpretation of the test results should be carried out by the doctor only on the basis of complex examination of the patient according to the anamnesis data, clinical and epidemiological status in accordance with the existing clinical and methodological recommendations. REF R-B FT-CE / VER / Page 9 of 22

10 AMPLIFICATION AND DATA ANALYSIS USING CFX96 (Bio-Rad, USA) Carry out the sample pretreatment and reaction mixture preparation stages according to the PCR kit Instruction Manual. When carrying out the amplification it is recommended to use thin-walled PCR tubes (0.2 ml) with domed or flat optically transparent caps or tubes (0.2 ml) with transparent caps from the eight-pieces-strips (detection through the cap of the tube). Monitor the tubes. There must not be drops left on the walls of the tubes as falling drops during the amplification process may lead to the signal failure and complicate the results analysis. Programming the thermocycler 1. Turn on the instrument and start the program Bio-Rad CFX Manager. 2. Program the instrument according to the Instruction Manual given by the manufacturer of the instrument. 3. Select Create a new Run in the start window. Select the universal amplification program AmpliSens or AmpliSens-1. Select or create this program in Experiment Setup module in Protocol Editor window. Put Sample Volume 25 μl. AmpliSens unified amplification program Step Temperature, С Time Fluorescent signal Cycle detection repeats min min s 3 FAM, HEX, s ROX, Cy5 Any combination of the tests can be performed in one instrument simultaneously with the use of the unified amplification program. If only the tests for pathogen agent DNA detection are performed in one instrument then the first step (50 C 15 minutes) can be deleted from the program for time saving. AmpliSens-1 amplification program for plate-type instruments Step Temperature, С Time Fluorescent signal detection Cycles min s s s 95 5 s s FAM, HEX, ROX, Cy s REF R-B FT-CE / VER / Page 10 of 22

11 Set Ramp Rate 2,5 С/s by clicking the Step Options button for each step of cycling (see the figure below). Click OK. (fig. 1) Figure 1 AmpliSens-1 program is a universal amplification program for carrying out tests with the help of AmpliSens kits to identify the DNA of STI pathogens and other reproductive organs infections. All the tests and any tests combinations can be carried out in one instrument simultaneously. Note Channel for the Cy5.5 fluorophore is enabled when required if the multiprime format tests are performed. 4. Save the protocol: in the Protocol Editor New window select File, then Save As, name the file and click Save. 5. In the next window (Plate module) set the plate setup: set the samples order in the reaction block and select fluorescent detection in four channels FAM, HEX, ROX and Cy5 for all samples. Click Select Fluorophores, tick the needed fluorophores in the list. For all DNA samples obtained from clinical samples and negative controls select Unknown in Sample type field. Specify the identifiers in Sample name field. Mark the Sample type Standard for DNA calibrators K1 and K2 in all the channels and enter its concentration in Concentration field according to the Important Product Information Bulletin for the PCR kit. Save file with the plate setup, click OK. Set the calibrators K1 and K2 for all the channels as Sample type Standard. Enter a concentration in the Concentration box according to the Important Product Information Bulletin enclosed to the PCR kit, moreover the Load function is to be ticked. 6. Save the plate setup: select File and then Save as in the Plate Editor New window. Enter the file name, click Save. 7. Select the Start Run tab. Open the cap of the instrument using Open Lid button. Insert the reaction tubes into the instrument wells according to the plate setup. Close the cap of the instrument using Close Lid button. REF R-B FT-CE / VER / Page 11 of 22

12 8. Click the Start Run button and start the program with the selected plate setup. Select the directory for the file saving, name the file, click Save. 9. After the program has finished, proceed to the results analysis. Using the ready template for the run The test parameters and the plate setup set earlier can be used for the further runs. To do this: click the Select Existing button in the Run Setup window of the Protocol tab. Select the needed file with the amplification program in the Select Protocol window. Click Open. go to the Plate tab in the Run Setup window. Click the Select Existing button. Select the needed file with the plate setup in the Select Plate window. Click Open. Click the Edit selected button to edit the plate setup. Data analysis Analysis of the results is performed by the CFX96 software with subsequent calculation of concentrations using the software in Microsoft Excel format. The fluorescence signal accumulation curves are analyzed in four channels: The signal of the Enterobacteriaceae DNA amplification product is detected in the FAM channel. The signal of the Staphylococcus spp. DNA amplification product is detected in the HEX channel. The signal of the Streptococcus spp. DNA amplification product is detected in the ROX channel. The signal of the IC DNA amplification product is detected in the Cy5 channel. Results are interpreted by the crossing (or not crossing) of the fluorescence curve with the threshold line that set at the level of exponential growth of fluorescence. It corresponds to the presence (or absence) of Ct (cycle threshold) value for the DNA-target in the appropriate cell of the result grid. The calibration curve is plotted and DNA concentrations of each group of microorganisms are calculated automatically by the used software based on the obtained Ct values in the respective channels and specified values of DNA calibrators. 1. Start the software and open the saved file. To do this, select File in the menu, then Open and Data file and select the needed file. 2. Analyze data separately for each channel turning off other channels (clear the tick box with channel disighnation under the main window with the Amplification curves). 3. Set the threshold line for each channel at the correct level (or check the automatic threshold line selection). The threshold line is to cross the curves of signal accumulation REF R-B FT-CE / VER / Page 12 of 22

13 of positive samples and controls only in the zone of the exponential growth and is not to cross curves without the characteristic exponential growth (base line fluctuations). Switch on the logarithmic scale, set the threshold line level at the level, where the curves are linear, and higher than base line fluctuations. Otherwise, the level can be determined in the range of % of maximum fluorescence obtained for the DNA-calibrator K1 in the last amplification cycle (turning off the logarithmic scale). Zone of linearity of fluorescence curves Threshold line 4. In the result grid Cq values and calculated concentrations SQ for the analysed channel will appear. 5. Calculate the results by the software in Microsoft Excel format. To do this, consistently for each channel copy the Ct values column to the appropriate column of the software in Microsoft Excel format. Mark the control samples and DNA calibrators according to the instruction for the software and click Calculate. Interpretation of results The results of PCR are reliable if the results of control samples are correct. Otherwise see Troubleshooting. The following results for controls are correct: 1. The Cq values determined for DNA calibrator K2 in four channels should not exceed the boundary values specified in the table 13 or 14 (depending on the amplification program). 2. Efficiency coefficient Е in the standard plot window in the FAM, HEX and ROX channels should be in the range (1.0 ± 0.2), specified in the Important Product Information Bulletin for the PCR kit. 3. The results for negative control samples should correspond to the results specified in the tables 4, 5. The concentrations values for the Negative Control of Amplification (NCA) should be absent or be not greater than the boundary value. The concentrations values for the Negative Control of Extraction (C ) should be absent or be not greater REF R-B FT-CE / VER / Page 13 of 22

14 than the boundary value in the FAM, HEX and ROX channels. The Cq value determined for Negative Control of Extraction (C ) in Cy5 channel should be not greater than the boundary value. Results for controls with the use of AmpliSens unified amplification program Control Stage for control Calculated concentration (GE/ml) or Cq (Ct) value in FAM, HEX, ROX in Cy5 channel channels K2 PCR Ct < 41 Ct < 43 C DNA extraction NCA PCR absent or < 2х10 3 Ct < 43 absent or < 1х10 3 Ct value is absent Table 4 Results for controls with the use of AmpliSens-1 amplification program Control Stage for control Calculated concentration (GE/ml) or Cq (Ct) value in FAM/Green, JOE/Yellow, in Cy5/Red channel ROX/Orange channels K2 PCR Ct < 36 Ct < 38 C DNA Extraction NCA PCR absent or < 2х10 3 Ct < 38 absent or < 1х10 3 Ct value is absent Table 5 Obtained values of number of copies of Enterobacteriaceae, Staphylococcus spp. and Streptococcus spp. DNA in the reaction tube are calculated automatically by the software of the instrument and given in the corresponding column of the result grid. Those values are used for calculation of number of genomic equivalents of the microorganisms DNA contained in 1 ml of the initial sample of biological material according to the formula: Number of microorganisms DNA copies x K = Number of genomic equivalents per 1 ml (GE/ml) The coefficient K for calculation of the result in GE/ml is specified in the Important Product Information Bulletin for the PCR kit. Results of the test samples are interpreted according to the following: 1. The result is considered to be reliable for the test sample, in which the calculated concentration of DNA of Enterobacteriaceae, Staphylococcus spp. and Streptococcus spp. is less than 1x10 4 GE/ml or is absent, only if the Ct value detected in the Cy5 channel (the channel for detection of the Internal Control) is not greater than 43 if the REF R-B FT-CE / VER / Page 14 of 22

15 AmpliSens amplification program is used and 38 if the AmpliSens-1 amplification program is used. Otherwise, result is invalid (see Troubleshooting). 2. If the obtained concentration value is from 2х10 3 to 1x10 4 GE/ml then the result is interpreted as less than 1x10 4 GE/ml. If the obtained value is greater than 1х10 8 GE/ml then the result is interpreted as greater than 1х10 8 GE/ml (according to the linear range of the kit). 3. If the obtained concentration value for the test sample is less than 2x10 3 GE/ml, then the result is interpreted as [microorganisms group] DNA is not detected The clinical interpretation of the test results should be carried out by the doctor only on the basis of complex examination of the patient according to the anamnesis data, clinical and epidemiological status in accordance with the existing clinical and methodological recommendations. AMPLIFICATION AND DATA ANALYSIS USING icycler iq and iq5 (Bio-Rad, USA) INSTRUMENTS Carry out the sample pretreatment and reaction mixture preparation stages according to the PCR kit Instruction Manual. When carrying out the amplification it is recommended to use thin-walled PCR tubes (0.2 ml) with domed or flat optically transparent caps, or tubes (0.2 ml) with transparent caps from the eight-pieces-strips (e.g. Axygen, USA) (detection through the cap of the tube). Programming the thermocycler 1. Switch on the instrument and the power supply unit of the optical block of the instrument. The lamp is to be warmed up during 15 min before starting the experiment. 2. Start the program icycler iq/iq5. 3. Insert the tubes or strips into the reaction module of the thermocycler and program the instrument. Monitor the tubes. There must not be drops left on the walls of the tubes as falling drops during the amplification process may lead to the signal failure and complicate the results analysis. Don t turn the strips upside down while inserting them into the instrument. Program the thermocycler only according to the Instruction Manual given by the manufacturer of the instrument: 1. Set the plate setup (set the order of the tubes in the reaction chamber and the detection of fluorescent signal). For icycler iq5 click the Create New or Edit buttons in the Selected Plate Setup REF R-B FT-CE / VER / Page 15 of 22

16 window of the Workshop module. One can edit the plate setup in the Whole Plate loading mode. Set the reaction volume (Sample Volume) as 25 µl, the caps type (Seal Type) as Domed Cap, and the tubes type (Vessel Type) as Tubes. Select the fluorescent signal detection in the FAM, JOE/HEX, ROX and Cy5 channels. Save the set plate setup by clicking the Save&Exit Plate Editing button. For icycler iq select the order of the samples in the reaction module in the Samples: Whole Plate Loading option in the Edit Plate Setup window of the Workshop module. Name each sample in the Sample Identifier window. Set the fluorescence signal detection in all the tubes in the FAM, JOE/HEX, ROX and Cy5 channels. Save the plate setup by naming the file in the Plate Setup Filename window (with.pts filename suffix) and clicking the Save this plate setup button (in the upper part of the screen). One can edit the plate setup which was used before. To do this, choose View Plate Setup in the Library window, select the needed setup in Plate Setup (the file with.pts filename suffix) and click the Edit button to the right. It is necessary to save the edited file before using. Set the using of the given plate setup by clicking the Run with selected protocol button. 2. Set all the test samples as Unknown. Set the type Standard for DNA calibrators and indicate their concentration in accordance to the Important Product Information Bulletin for the PCR kit. Save the plate setup. 3. Select the universal amplification program AmpliSens or AmpliSens-1. Select or create this program in Protocol module (View Protocols for icycler iq) click Run with selected Plate Setup. AmpliSens unified amplification program Step Temperature, С Time Fluorescence detection Number of cycles Hold min 1 Hold min 1 Cycling s s FAM, JOE/HEX, ROX, Cy5 Any combination of the tests can be performed in one instrument simultaneously with the use of the unified amplification program. If only the tests for pathogen agent DNA detection are performed in one instrument then the first step (50 C 15 minutes) can be deleted from the program for time saving. 45 AmpliSens-1 amplification program for plate-type instruments Step Temperature, С Time Fluorescent signal detection Cycles min 1 REF R-B FT-CE / VER / Page 16 of 22

17 s s s 95 5 s s FAM, JOE/HEX, ROX, Cy s 5 40 AmpliSens-1 program is a universal amplification program for carrying out tests with the help of AmpliSens kits to identify the DNA of STI pathogens and other reproductive organs infections. All the tests and any tests combinations can be carried out in one instrument simultaneously. It is not recommended running the tests with different combinations of detection channels simultaneously in the icycler iq Instrument. If these tests are to be conducted within the same run, then the External Well Factors Plate option should be selected for detection of a well factor and the tube kit with a special External Well Factor Solution (Bio-Rad) should be used for start up. Before programming the icycler iq instrument, make sure that the dinamicwf.tmo protocol is set as follows (standard). If this protocol was changed and did not correspond to the specified one, it must be corrected: dinamicwf.tmo protocol for icycler iq: 4. Start the selected program with the created plate setup. icycler iq5 instrument. To start the program, click the Run button. Select the Collect Well Factors from Experimental Plate option (set by default) for detection of a well factor. icycler iq instrument. In the Run Prep window select the Experimental Plate option (set by default) below the Select well factor source line to determine the well factor (see item 2). Set the reaction mix volume as 25 µl. Press Run to start. 5. Proceed to data analysis when the program is completed. 6. At the end of the work, close the program and switch off the instrument (thermocycler and an optical system). REF R-B FT-CE / VER / Page 17 of 22

18 Using the ready template for the run The test parameters and the plate setup set earlier can be used for the further runs. To do this: select the needed file with the run in the upper left window of the Workshop module; click the Edit button in the Selected Plate Setup area of the Workshop module and edit the plate setup (the files of protocols are saved in the SampleFiles folder by default); click the Edit button in the Selected Protocol area of the Workshop module and check the correctness of the selected protocol (the files of protocols are saved in the SampleFiles folder by default). Data analysis Analysis of the results is performed by the icycler iq5 / iq software with subsequent calculation of concentrations using the software in Microsoft Excel format. The fluorescence signal accumulation curves are analyzed in four channels: The signal of the Enterobacteriaceae DNA amplification product is detected in the FAM channel. The signal of the Staphylococcus spp. DNA amplification product is detected in the JOE/HEX channel. The signal of the Streptococcus spp. DNA amplification product is detected in the ROX channel. The signal of the IC DNA amplification product is detected in the Cy5 channel. The results are interpreted according to the crossing (or not-crossing) of the S-shaped fluorescence curve with the threshold line (set in the middle of the linear fragment of fluorescence growth of the positive control in the log scale) that corresponds to the presence (or absence) of the Ct (threshold cycle) value in the corresponding column of the results table. The calibration curve is plotted and DNA concentrations of each group of microorganisms are calculated automatically by the used software based on the obtained Ct values in the respective channels and specified values of DNA calibrators. 1. Start the program and open the saved file. To do this: for icycler iq5: select the needed file for the analysis in the Data File window of the Workshop module, and click the Analyze button; for icycler iq: activate the View Post-Run Data in the Library module. Select the needed file with the analysis data and click the Analyse Data button. 2. Browse the data. To do this: REF R-B FT-CE / VER / Page 18 of 22

19 for icycler iq5: select Analysis Mode: PCR Base Line Subtracted Curve Fit (selected by default) for icycler iq: in the PCR Quantification tab in the Select a Reporter menu select symbol of channel (must be selected PCR Base Line Subtracted Curve Fit mode). 3. The data for each channel are to be browsed separately. 4. Set the threshold line for each channel at the correct level (or check the automatic threshold line selection). The threshold line is to cross only the S-shaped curves of signal accumulation of positive samples and controls and is not to cross the base line. In another case the threshold level should be raised by clicking Log View button and setting the threshold lines level (with the left mouse button) so that the fluorescence curves are linear and do not cross with the curves of the negative samples. Otherwise, the level can be determined in the range of % of maximum fluorescence obtained for the DNA-calibrator K1 in the last amplification cycle (turning off the logarithmic scale). Zone of linearity of fluorescence curves Threshold line 5. In order to analyze the results click the PCR Quant button (for icycler iq) or activate the Results button which is situated under the buttons with the fluorophores names (for icycler iq5). 6. In the result grid Ct values and calculated concentrations for the analysed channel will appear. 7. Calculate the results by the software in Microsoft Excel format. To do this, consistently for each channel copy the Ct values column to the appropriate column of the software in Microsoft Excel format. Mark the control samples and DNA calibrators according to the instruction for the software and click Calculate. Interpretation of results The results of PCR are reliable if the results of control samples are correct. Otherwise see Troubleshooting. The following results for controls are correct: REF R-B FT-CE / VER / Page 19 of 22

20 1. The Ct values determined for DNA calibrator K2 in four channels should not exceed the boundary values specified in the table 6 or 7 (depending on the amplification program). 2. Efficiency coefficient Е in the FAM, JOE/HEX and ROX channels should be in the range ( %), specified in the Important Product Information Bulletin for the PCR kit. 3. The results for negative control samples should correspond to the results specified in the tables 6, 7. The concentrations values for the Negative Control of Amplification (NCA) should be absent or be not greater than the boundary value. The concentrations values for the Negative Control of Extraction (C ) should be absent or be not greater than the boundary value in the FAM, JOE/HEX and ROX channels. The Ct value determined for Negative Control of Extraction (C ) in Cy5 channel should be not greater than the boundary value. Results for controls with the use of AmpliSens unified amplification program Control Stage for control Calculated concentration (GE/ml) or Ct value in FAM, HEX, ROX in Cy5 channel channels K2 PCR Ct < 41 Ct < 43 C DNA extraction NCA PCR absent or < 2х10 3 Ct < 43 absent or < 1х10 3 Ct value is absent Table 6 Results for controls with the use of AmpliSens-1 amplification program Control Stage for control Calculated concentration (GE/ml) or Ct value in FAM/Green, JOE/Yellow, in Cy5/Red channel ROX/Orange channels K2 PCR Ct < 36 Ct < 38 C DNA Extraction NCA PCR absent or < 2х10 3 Ct < 38 absent or < 1х10 3 Ct value is absent Table 7 Obtained values of number of copies of Enterobacteriaceae, Staphylococcus spp. and Streptococcus spp. DNA in the reaction tube are calculated automatically by the software of the instrument and given in the corresponding column of the result grid. Those values are used for calculation of number of genomic equivalents of the microorganisms DNA REF R-B FT-CE / VER / Page 20 of 22

21 contained in 1 ml of the initial sample of biological material according to the formula: Number of microorganisms DNA copies x K = Number of genomic equivalents per 1 ml (GE/ml) The coefficient K for calculation of the result in GE/ml is specified in the Important Product Information Bulletin for the PCR kit. Results of the test samples are interpreted according to the following: 1. The result is considered to be reliable for the test sample, in which the calculated concentration of DNA of Enterobacteriaceae, Staphylococcus spp. and Streptococcus spp. is less than 1x10 4 GE/ml or is absent, only if the Ct value detected in the Cy5 channel (the channel for detection of the Internal Control) is not greater than 43 if the AmpliSens amplification program is used and 38 if the AmpliSens-1 amplification program is used. Otherwise, result is invalid (see Troubleshooting). 2. If the obtained concentration value is from 2х10 3 to 1x10 4 GE/ml then the result is interpreted as less than 1x10 4 GE/ml. If the obtained value is greater than 1х10 8 GE/ml then the result is interpreted as greater than 1х10 8 GE/ml (according to the linear range of the kit). 3. If the obtained concentration value for the test sample is less than 2x10 3 GE/ml, then the result is interpreted as [microorganisms group] DNA is not detected The clinical interpretation of the test results should be carried out by the doctor only on the basis of complex examination of the patient according to the anamnesis data, clinical and epidemiological status in accordance with the existing clinical and methodological recommendations. REF R-B FT-CE / VER / Page 21 of 22

22 VER ME ME EM Location of changes Text Amplification and data analysis using LineGene 9660 (BIOER TECHNOLOGY CO., LTD, China) instrument Amplification and data analysis with the use of icycler iq and iq5 (Bio- Rad, USA) instruments Amplification and data analysis using LineGene 9660 (BIOER TECHNOLOGY CO., LTD, China) instrument Amplification and data analysis using Rotor-Gene 3000/6000 (Corbett Research, Australia) and Rotor-Gene Q (QIAGEN, Germany) instruments Amplification and data analysis using CFX96 (Bio-Rad, USA) Amplification and data analysis using icycler iq and iq5 (Bio-Rad, USA) instruments Text List of Changes Made in the Guidelines Grammar corrections The chapter was added Essence of changes The note Channel for the Cy5.5 fluorophore is activated when it necessary, if tests are conducted in the multiprime format that use the channel was deleted The chapter was deleted In the tables with results for the controls with the use of AmpliSens and AmpliSens-1 amplification programs the result for the NCA sample in the Cy5 channel was changed from ignored to Ct value is absent All the chapters were corrected according to the Russian Guidelines REF R-B FT-CE / VER / Page 22 of 22