GUIDELINES. to AmpliSens HPV 6/11-FRT PCR kit

Transcription

1 For Professional Use Only GUIDELINES to AmpliSens HPV 6/11-FRT PCR kit for qualitative detection and differentiation of types 6 and 11 of human papillomavirus (HPV) DNA in clinical material by the polymerase chain reaction (PCR) with real-time hybridizationfluorescence detection 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 WITH Rotor-Gene 3000/6000 (Corbett Research, Australia) INSTRUMENTS... 3 AMPLIFICATION AND DATA ANALYSIS WITH icycler iq5 (Bio-Rad, USA) INSTRUMENTS AMPLIFICATION AND DATA ANALYSIS WITH Mx3000P (Stratagene, USA) INSTRUMENT REF R-V11(RG,iQ,Mx)-CE / VER / Page 2 of 22

3 INTENDED USE Guidelines describe the procedure of the use of AmpliSens HPV 6/11-FRT PCR kit for qualitative detection and differentiation of types 6 and 11 of human papillomavirus (HPV) DNA by means of real-time hybridization-fluorescence detection with the following realtime PCR instruments: Rotor-Gene 3000, Rotor-Gene 6000 (Corbett Research, Australia), icycler iq5 (Bio-Rad, USA), Mx3000P (Stratagene, USA). AMPLIFICATION AND DATA ANALYSIS WITH Rotor-Gene 3000/6000 (Corbett Research, Australia) INSTRUMENTS Carry out sample pretreatment and reaction mixture preparation stages according to the instruction manual. Transparent no domed 0.2-ml PCR tubes (detection through the bottom of a tube) or 0.1-ml tubes are recommended for use. Hereinafter, all terms corresponding to different instruments and software are indicated in the following order: for Rotor-Gene 3000/for Rotor-Gene When working with Rotor-Gene 3000 instrument use the program Rotor-Gene version 6. When working with Rotor-Gene 6000 instrument use the program Rotor-Gene 6000 versions 1.7 (build 67) or higher. Programming the instrument 1. Turn the instrument on and start Rotor-Gene program. 2. Place the tubes in the rotor so that the first tube is in No. 1 well, insert the rotor in the reaction module, and secure the cover (the rotor cells are numbered and these numbers are used for sample order programming). Balance the rotor if it is not loaded entirely. Fill unused wells with empty tubes. Do not use tubes from previous runs. No.1 well should be loaded with a test tube from the current experiment (not empty). 3. Program the instrument to run amplification and detection: In the opened window select Advanced mode. Activate the New button. Select 36-Well Rotor (or 72-Well Rotor) and No Domed Tubes/Locking ring attached. Click Next. In the opened window define the operator and set the reaction volume: Reaction volume is 25 µl. Tick off the 15 µl oil layer volume option. set amplification program. REF R-V11(RG,iQ,Mx)-CE / VER / Page 3 of 22

4 DNA HPV types 6 and 11 amplification program for Rotor-Gene 3000/6000 (Corbett Research, Australia) Stage Тemperature, С Time Fluorescence Cycle detection repeats Hold min s Cycling ROX/Orange FAM/Green, s JOE/Yellow, 45 Table 1 This program can be applied for both AmpliSens HPV 6/11-FRT and AmpliSens HPV 16/18-FRT PCR kits. Following amplification programs can be used as well: AmpliSens-1 RG (see Table 2) and amplification program for HPV HCR DNA types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 (see Table 3) (this program is applied for AmpliSens HPV HCR screen-titre-frt PCR kit). AmpliSens-1 RG amplification program is a universal program and it can be used for running different tests within one run (for example tests for detection of STIs). For detection of DNA of HCR HPV and HPV DNA types 6, 11 within one run AmpliSens FRT HR HPV ScreenRG4x Program can be applied. Using amplification program for HPV HCR DNA types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 (from AmpliSens HPV HCR screen-titre-frt PCR kit) does not affect analytical performances of this PCR kit. AmpliSens-1 RG amplification program Table 2 Step Temperature, С Time Fluorescence Cycle detection repeats Hold min s Cycling s s 95 5 s Cycling s FAM/Green, JOE/Yellow, ROX/Orange, Cy5/Red s Cy5/Red detection channel should be enabled for running multiplex tests (if required). REF R-V11(RG,iQ,Mx)-CE / VER / Page 4 of 22

5 Table 3 Amplification program for HPV HCR DNA types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 Step Temperature, С Time Fluorescence detection Cycle repeats Hold min 1 Hold min s Cycling 64 Touchdown: 25 s 5 1 deg. per cycle s s s FAM/Green, Cycling 2 40 JOE/Yellow, s ROX/Orange, Cy5/Red The file AmpliSens FRT HR HPV Screen RG4x Program.pro enclosed to AmpliSens HPV HCR screen-titre-frt PCR kit can be used for programming. Click the Calibrate/Gain Optimisation button in the New Run Wizard window. a) for detection in FAM/Green, JOE/Yellow, ROX/Orange, and Cy5/Red channels select Calibrate Acquiring/Optimise Acquiring; b) click Perform Calibration Before 1 st Acquisition/Perform Optimisation Before 1 st Acquisition. c) click the Edit button in the Auto gain calibration channel settings window and set Min Reading as 4 and Max Reading as 8 for all channels. 4. Select the Start run button for amplification to run. Name the experiment and save it to the disk (results of the run will be automatically saved in this file). 5. Enter data in the table of samples (opens by default when the run begins). Define names/numbers of clinical samples in the Name column. Define the Negative Control of amplification as NCA and the Positive Control of amplification as C+. Enter the Unknown type for all clinical and control samples. Indicate the None type for empty wells. The sample will not be analyzed if its type is defined as None. Due to the fact that the PCR kit applies a multiplex control including several DNA matrixes (HPV type 6, HPV type 11 and human DNA) several pages should be created in protocol. To do this, indicate HPV 6 (FAM/Green channel) in the Page area of the Name field. Click New. A new page for information about samples will appear. It contains the same names of samples as in previous page. Indicate HPV 11 (JOE/Yellow channel) in the Page area REF R-V11(RG,iQ,Mx)-CE / VER / Page 5 of 22

6 of the Name field. Click New. Third page for information about samples will appear. Indicate Human (ROX/Orange channel) in the Page area of the Name field. Data analysis Review row data in all three channels (FAM/Green, JOE/Yellow and ROX/Orange) by selecting Cycling A buttons on the Channels bar. Pay attention to the followings: among all signals detected in the channel at least one should be positive (a positive signal has typical S-shape, see Fig.1). If the experiment is correct, the signal of positive control C+ must be detected. If positive signals were not detected in a channel the result of data analysis can be incorrect. If background signal of some samples greatly differs from all other samples it indicates incorrect dispensing reaction mixture or DNA sample during tube preparation (see Fig. 2a). 1. Make sure that all analyzed samples are activated on the legend at the right. 2. Activate the Analysis button then select the Quantitation mode. Activate the Cycling A. FAM/Cycling A. Green button; click Show. 3. Set Threshold = Select Linear scale. Activate the Dynamic tube and Slope Correct buttons in the main window menu (Quantitation analysis). 5. Select the More Settings/Outlier Removal parameter and set the NTC threshold value as 10 %. 6. Carry out data analysis for JOE/Yellow and ROX/Orange channels similarly to that for FAM/Green channel. In some rare cases due to descending character of the curves at the beginning of the experiment, fluorescent curves can cross the threshold line at first cycles and Rotor-Gene program can accept these results as positive (Fig. 7). Use Eliminate cycles before option and enter For data analysis select Analysis then Quantitation. REF R-V11(RG,iQ,Mx)-CE / VER / Page 6 of 22

7 TROUBLESHOOTING Review this section before running the experiment Problem Cause How to identify Ways to eliminate Negative samples are analyzed as positive samples by Rotor- Gene program Use the Ignore First option: select 5 cycles. If it does not help, increase this value by 1-5 Drop of sensitivity due to contamination of instrument lenses Drop of sensitivity due to deterioration of probes Incorrect mathematical processing of negative samples in the presence of drop of fluorescence at the initial cycles (Fig. 2a) The threshold line crosses descending fluorescence curves at the initial cycles (Fig. 6) Lenses contamination leads to reduction of effectiveness of fluorescence excitation and detection. Primarily this has an impact on the samples containing small quantity of specific DNA which show low fluorescence growth Incorrect storage or utilizing kit components (high temperature, multiple opening reagent tubes, foul working conditions) can cause oligonucleotides destruction A normal positive sample has a S- shaped curve of fluorescence accumulation (Fig. 1, 3-5). The negative samples processed incorrectly appear as quite straight bottomup lines (Fig. 7) The red threshold line crosses (or touches) fluorescence curves at the left (initial cycles) on the chart of processed fluorescence curves (Fig. 6) Low background signal in all four detection channels (<1) along with maximum value of the gain parameter (10) Probe destruction can be find out as a result of comparison of experimental data obtained at the first use of the reagents and after a period of time or in comparison with the reagents of the same lot stored properly. It can be revealed as a reduction of value of the gain multiplying coefficient (selected automatically) by more than 2 units in different experiments (in case the same instrument is used). Note! Effect of the gain parameter Use the Eliminate cycles before option: select 5 (crossing the threshold with fluorescence curve is ignored for the first 5 cycles) Clean horizontal and vertical lenses of the instrument with a dry cotton pad at least once a month Use mixtures stored under stated conditions until labeled expire date (see part Stability and Storage in the Instruction Manual) REF R-V11(RG,iQ,Mx)-CE / VER / Page 7 of 22

8 Desensitization due to decrease of polymerase (TaqF) activity Incorrect storage or contamination cause enzyme deterioration increase can appear after cleansing the instrument lenses from sever contamination It appears as a fail of the positive control or if a boundary Ct value of the positive control is greater than the threshold of weak samples Use the reagent stored under required condition (see part Stability and Storage in the Instruction Manual) or use a new reagent Note! A report on all parameters specified as well as a report on auto-calibration is available if the Settings button is activated. In particular, the Messages tab, the Autocalibration Log Messages option. Problem Feature Ways to eliminate Contamination of a specific DNA Detection of a signal of the negative control in any channel Repeat the experiment. Take measures to detect and eliminate the source of The volume of a DNA sample added to a reaction tube is less than required/dna samples is not added The volume of the reaction mixture added to a reaction tube is less than required/reaction mixture is not added/the volume of a DNA sample added to a reaction tube is greater than required The autocalibration parameter from 4Fl to 8Fl is not set or it is a failure of the first tube in the rotor (the No.1 well is empty; a volume of DNA sample or reaction mixture added to the tube is incorrect) Background signal of a sample is much stronger than the others (see raw curves, Fig. 2b). The sample is negative Background signal of a sample is much lower than the others (see raw curves, Fig. 2b). Most of the fluorescence background signals are less than 1 or greater than 20 contamination Repeat PCR for this sample If the sample is negative, repeat the test Set the parameter in the next run. In case of off-scale reading or if the signals are too weak (no positive signals after processing; a background is less than 0.5) the experiment should be repeated beginning with PCR Polymerase (TaqF) was not added to a reaction mixture Positive signals are absent for all samples including a positive control Repeat the experiment beginning with PCR. Ensure appropriate preparation of reaction mixtures REF R-V11(RG,iQ,Mx)-CE / VER / Page 8 of 22

9 Unprocessed data (raw channel) Fig. 1. Raw curves are normal Fig. 2а. Raw curves have a bend (drop of fluorescence at the initial cycles) 1 2 Fig. 2b. Level of background signal points on the possible error: DNA sample was not added to the tube 1; double volume of DNA sample was added to the tube 2 Processed data (Quantitation analysis) а) processed curves are normal (S-shaped, threshold line crosses curves at the area of growth of fluorescence) Fig. 3. FAM channel Fig. 4. JOE channel REF R-V11(RG,iQ,Mx)-CE / VER / Page 9 of 22

10 Fig. 5. ROX channel internal control (β-globin gene) b) curves are processed incorrectly Fig. 6. Threshold line crosses fluorescence curves twice Fig. 7. Incorrect processing of the curves with bend (from Fig. 2) REF R-V11(RG,iQ,Mx)-CE / VER / Page 10 of 22

11 AMPLIFICATION AND DATA ANALYSIS WITH icycler iq5 (Bio-Rad, USA) INSTRUMENTS Carry out sample pretreatment and reaction mixture preparation stages according to the instruction manual. Transparent domed 0.2-ml PCR tubes (detection through the cap of the tube) are recommended for use. 1. Switch on the instrument and the power supply of the optical module. The lamp should be warmed up for at least 15 min before the experiment starts. 2. Open the iq5 program. DNA HPV types 6 and 11 amplification program for icycler iq, iq5 (Bio-Rad, USA) Fluorescence Cycle Step Temperature, С Time detection repeats min s min FAM, HEX, ROX Table 4 This program can be applied for both AmpliSens HPV 6/11-FRT and AmpliSens HPV 16/18-FRT PCR kits. Following amplification programs can be used as well: AmpliSens-1 iq (see Table 5) and amplification program for HPV HCR DNA types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 (see Table 6) (this program is applied for AmpliSens HPV HCR screen-titre-frt PCR kit). AmpliSens-1 iq amplification program is universal program and it can be used for running different tests within one run (for example tests for detection of STIs). For detection of DNA of HCR HPV and HPV DNA types 6, 11 within one run AmpliSens FRT HR HPV ScreenRG4x Program can be applied. Using amplification program for HPV HCR DNA types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 (from AmpliSens HPV HCR screen-titre-frt PCR kit) does not affect analytical performances of this PCR kit AmpliSens-1 iq amplification program Step Temperature, С Time Fluorescence Cycle detection repeats min s s s 95 5 s s FAM, HEX, ROX, Cy s Table 5 REF R-V11(RG,iQ,Mx)-CE / VER / Page 11 of 22

12 Cy5/Red detection channel should be enabled for running multiplex tests (if required). Amplification program for HPV HCR DNA types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 Step Temperature, С Time Fluorescence Cycle detection repeats Cycle min s Cycle 3 65 Touchdown: 55 s 6 1 deg. per cycle s s Cycle s Real-time s Table 6 The AmpliSens FRT HR HPV Screen iq5.tmo file enclosed to AmpliSens HPV HCR screen-titre-frt PCR kit can be used for programming. Create a new plate setup in the Edit Plate Setup tab. Indicate samples as Unknown. Proceed to the Select fluorofores tab and set fluorescence detection for all tubes in the FAM, HEX and ROX channels. Go back to the Samples: Whole plate loading and click the Per Dye Layer button. Click Run. Select Well factor (both well factor by experiment tubes and fixed well factor are acceptable for use but the latter is recommended). Run the experiment. Data analysis 1. Proceed to the PCR Quantification tab in the Data Analysis mode. 2. Review data in one channel at a time. 3. Ensure that the threshold line was set correctly for each channel. If it is required, activate User Defined, 2 through 10 cycles parameter. Normally threshold line should cross s-shaped curves of signal accumulation of positive samples and controls and not cross the base line. Otherwise, raise the threshold line up to the level where it crosses positive samples only. 4. Activate the Results button (below the fluorofore buttons). 5. Proceed to the PCR Standard Curve tab, review parameters of the reaction, copy the results. 6. Click on the results grid using the mouse right button. Select Export to Excel from the drop-down menu. Agree to save the file. If Microsoft Excel program is loaded on the computer it will open automatically (Microsoft Excel program is required for further REF R-V11(RG,iQ,Mx)-CE / VER / Page 12 of 22

13 data analysis). Results in the FAM, HEX, ROX, and then Cy5 channels follow consistently. TROUBLESHOOTING Problem Cause How to identify Ways to eliminate Incorrect storage or Use mixtures stored utilizing kit under stated components (high conditions until temperature, labeled expire date multiple opening (see part Stability reagent tubes, foul and Storage in the working conditions) Instruction Manual) can cause destroy of oligonucleotides Drop of sensitivity due to deterioration of probes Drop of sensitivity due to decrease of activity of polymerase (TaqF) Incorrect storage or contamination cause enzyme deterioration Probe destruction can be find out as a result of comparison of experimental data obtained at the first use of the reagents and after a period of time or in comparison with the reagents of the same lot stored properly. It is appeared as an increase of background fluorescence (fluorescence is assessed in the Background subtracted mode at the beginning of the experiment) twice as much in different experiments (in case the same instrument is used). Note! Effect of fluorescence increase can appear in case the lamp was changed, optical system was cleaned, or calibration was done) It appears as a fail of the positive control or if a boundary Ct value of the positive control is much greater than the normal one Use the reagent stored under required condition (see part Stability and Storage in the Instruction Manual) or use a new reagent Problem Feature Ways to eliminate Contamination of a specific DNA Detection of a signal for a negative control in any channel Repeat the experiment. Take measures to detect and eliminate the source of The volume of a DNA sample added to a reaction tube is less than required/dna samples is not added Background signal of the sample is much stronger than the others (see raw curves Background subtracted mode). The sample is negative contamination Repeat PCR for this sample REF R-V11(RG,iQ,Mx)-CE / VER / Page 13 of 22

14 The volume of a reaction mixture added to a reaction tube is less than required/reaction mixture is not added or the volume of a DNA sample added to a reaction tube is greater than required Incorrect adjustment of the threshold level Drops were not removed from the tube walls before experiment started Polymerase (TaqF) was not added to a reaction mixture Background signal of the sample is much lower than the others (see raw curves - Background subtracted mode) Threshold line is located along with negative samples or above some or all positive curves (S-shaped) Curves of fluorescence accumulation have negative or positive steps (Fig. 2) Positive signals are absent for all samples including a positive control If the sample is negative, repeat the test Set the threshold line so that it crosses only sigmoid curves of fluorescence accumulation or at the level of ¼ between final values of negative and positive samples Select the BaseLine Threshold window (the right mouse button on the graph of fluorescence) and indicate that base line is to be calculated beginning with the first cycle after the step Repeat the experiment beginning with PCR. Ensure appropriate preparation of reaction mixtures Unprocessed (raw) data (Background subtracted mode). Err Fig. 1 Raw curves are normal (FAM) Fig. 2. Baseline has a step : drops were not removed from the tube walls before the experiment in the sample indicated as Err REF R-V11(RG,iQ,Mx)-CE / VER / Page 14 of 22

15 Processed data Processed curves are normal; S-shaped; thresholds are located correctly (PCR Base Line Subtracted Curve Fit mode) Fig. 3. FAM channel Fig.4. HEX channel Fig 5. ROX channel internal control (β-globin gene) REF R-V11(RG,iQ,Mx)-CE / VER / Page 15 of 22

16 AMPLIFICATION AND DATA ANALYSIS WITH Mx3000P (Stratagene, USA) INSTRUMENT Carry out sample pretreatment and reaction mixture preparation stages according to the instruction manual. Transparent domed 0.2-ml PCR tubes (detection through the cap of the tube) are recommended for use. 1. Switch on the instrument and open the Mx3000P program. 2. In the New Experiment Options window select Quantitative PCR (Multiple Standards) and select Turn lamp on for warm-up. The lamp should be warmed up for at least 15 min before the experiment starts. 3. Place the tubes in the instrument, secure the locking tool and close the lid. 4. Select Optics Configuration in the Options menu. In the Dye Assignment tab indicate JOE next to the HEX/JOE filter set option; indicate FAM next to the FAM filter set option. 5. In the Instrument menu of the Mx3000P program select the Filter Set Gain Settings option. In the opened window set the following parameters of multiplication factors: Cy5 ROX HEX/JOE FAM x4 x1 x4 x4 Prior to analysis, remove drops from tube walls, because drops falling during amplification cause signal errors and complicate data analysis. Do not turn strip/plate over when loading into the PCR instrument. 6. Define parameters of fluorescence acquiring in the Plate Setup menu. To do this: a) hold Ctrl and use the mouse to select all cells in which test tubes are inserted. b) define all selected cells as Unknown in the Well type window. In the Collect fluorescence data option select FAM, JOE, ROX, and Cy5. Double click each cell and enter a sample name (Well Information window). Indicate the positive control as +, the negative control as. Sample names can be entered during or after the amplification run in the Plate Setup menu. 7. Proceed to the Thermal Profile Setup tab and set the amplification program (see Table 7): REF R-V11(RG,iQ,Mx)-CE / VER / Page 16 of 22

17 DNA HPV types 6 and 11 amplification program for Mx3000P, Mx3005P (Stratagene, USA) Fluorescence Cycle Step Temperature, С Time detection repeats min s min FAM, HEX/JOE, ROX Table 7 This program can be applied for both AmpliSens HPV 6/11-FRT and AmpliSens HPV 16/18-FRT PCR kits. Following amplification programs can be used as well: AmpliSens-1 Mx (see Table 2) and amplification program for HPV HCR DNA types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 (see Table 9) (this program is applied for AmpliSens HPV HCR screen-titre-frt PCR kit). AmpliSens-1 Mx amplification program is universal program and it can be used for running different tests within one run (for example tests for detection of STIs). For detection of DNA of HPV HCR and HPV DNA types 6, 11 within one run AmpliSens FRT HR HPV ScreenRG4x Program can be applied. Using amplification program for HPV HCR DNA types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 (from AmpliSens HPV HCR screen-titre-frt PCR kit) does not affect analytical performances of this PCR kit AmpliSens-1 Mx amplification program Fluorescence Cycle Step Temperature, С Time detection repeats Segment min s Segment s 5 (Cycling) s 95 5 s Segment 3 FAM, HEX/JOE, ROX, s 40 (Cycling) Cy s Table 8 Cy5/Red detection channel should be enabled for running multiplex tests (if required). REF R-V11(RG,iQ,Mx)-CE / VER / Page 17 of 22

18 Table 9 Amplification program for HPV HCR DNA types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 Step Temperature, С Time Fluorescence Cycle detection repeats Segment min 1 Segment min s 64 Segment 3 Touchdown: (Cycling) 1 deg. per cycle 25 s s Segment 4 (Cycling) s s s Cy5, FAM, HEX/JOE, ROX The AmpliSens FRT HR HPV Screen Mx.mxp file enclosed to AmpliSens HPV HCR screen-titre-frt PCR kit can be used for programming. 8. In the Plate Setup tab of the Setup mode indicate names of the samples. Define all samples as Unknown. Select FAM, JOE/HEX, ROX dyes for all samples. 9. Run the program. 10. Proceed to the analysis of results after the end of the run. Data analysis 1. Proceed to the Analysis item by clicking the button on the toolbar in the Mx300P program. 2. Ensure that all test samples are enabled (the cells are tinted) in the opened Analysis Selection/Setup tab. Otherwise, hold Ctrl and select all test samples with the mouse. 3. Proceed to the Results tab. 4. On the Dyes shown toolbar activate displaying FAM channel and deactivate displaying all others channels. Set 150 in the Threshold fluorescence field for FAM channel. One at a time activate next channel and deactivate the previous one and enter threshold values according to the table: Cy5 150 ROX 150 HEX/JOE 150 FAM Activate displaying all channels (FAM, HEX and ROX buttons should be selected in the Dyes Shown field at the bottom of the program window). 6. Ensure that the JOE, FAM and ROX fluorescence channels are ticked off in the Threshold fluorescence field. REF R-V11(RG,iQ,Mx)-CE / VER / Page 18 of 22 40

19 7. Ensure that the threshold line is set correctly. Normally, a threshold line should cross S-shaped curves of signal accumulation of positive samples and controls and should not cross the base line. Otherwise, the threshold should be raised. 8. Select Text Report option in the Area to analyze field. Review the results and export data for further analysis (for example export to Excel: File>Export Instrument Data>Export Instrument Data to Excel). TROUBLESHOOTING Problem Cause How to identify Ways to eliminate Incorrect storage or Use mixtures stored utilizing kit under stated components (high conditions until temperature, labeled expire date multiple opening (see part Stability reagent tubes, foul and Storage in the working conditions) Instruction Manual) can cause destroy of oligonucleotides Drop of sensitivity due to deterioration of probes Drop of sensitivity due to decrease of polymerase (TaqF) activity Incorrect storage or contamination cause enzyme deterioration Probe destruction can be find out as a result of comparison of experimental data obtained at the first use of the reagents and after a period of time or in comparison with the reagents of the same lot stored properly. It is appeared as an increase of background fluorescence (fluorescence is assessed in the R mode at the beginning of the experiment) twice as much in different experiments (in case the same instrument is used). Note! Effect of fluorescence increase can appear after cleansing the optical system It appears as a fail of the positive control or if a boundary Ct value of the positive control is greater than the threshold of weak samples Use the reagent stored under required condition (see part Stability and Storage in the Instruction Manual) or use a new reagent REF R-V11(RG,iQ,Mx)-CE / VER / Page 19 of 22

20 Problem Feature Ways to eliminate Contamination of a specific DNA Detection of a signal for a negative control in any channel Repeat the experiment. Take measures to detect and eliminate the source of The volume of a DNA sample added to a reaction tube is less than required/dna samples is not added The volume of a reaction mixture added to a reaction tube is less than required/reaction mixture is not added or the volume of a DNA sample added to a reaction tube is greater than required Incorrect adjustment of the threshold level Polymerase (TaqF) was not added to a reaction mixture Background signal of a sample is much stronger than the others (see raw curves R-multicomponent view mode) The sample is negative Background signal of a sample is much lower than the others (see raw curves - R- multicomponent view mode) Threshold line is located along with negative samples or above some or all positive curves (S-shaped) Positive signals are absent for all samples including a positive control contamination Repeat PCR for this sample If the sample is negative, repeat the test Set the threshold line so that it crosses only sigmoid curves of fluorescence accumulation or at the level of ¼ between final values of negative and positive samples Repeat the experiment beginning with PCR. Ensure appropriate preparation of reaction mixtures REF R-V11(RG,iQ,Mx)-CE / VER / Page 20 of 22

21 Unprocessed data (R mode) Err Fig. 1. Raw curves are normal Processed data Normal curves that were processed, dr mode (S-shaped) Fig. 2. Level of background signal point on a possible problem: DNA sample was not added to the Err tube Fig. 3. FAM channel Fig. 4. HEX channel Fig. 5. ROX channel internal control (β-globin gene) REF R-V11(RG,iQ,Mx)-CE / VER / Page 21 of 22

22 List of Changes Made in the Instruction Manual VER SA Location of changes Cover page Footer Essence of changes Address of European representative was added REF R-V11(RG,iQ,Mx)-СЕ-B was deleted REF R-V11(RG,iQ,Mx)-CE / VER / Page 22 of 22