TBEV, B.burgdorferi sl, A.phagocytophilum, E.chaffeensis / E.muris
Contents
1. Result interpretation of the control samples The result of the analysis is considered reliable only if the results of both Positive and Negative Controls of amplification as well as Negative Control of extraction are correct R V59 RG iQ Mx Dt CE 09 12 10 03 04 14 Page 13 of 24 Table 5 Results of positive and negative controls should not exceed Ct values specified Table 5 Table 5 Results for controls obtained with iCycler iQ and iQ5 Stage for Ct value Interpret Control control FAM HEX ROX ation PCR mix 1 FRT TBEV A ph E ch E m i Detection of Detection of pin of A phagocyto E chaffeensis philum E muris c ANADA Absent Absent Absent OK extraction NCA Amplification Absent Absent Absent OK C TBEV B b sl A ph E ch E m Amplification lt 30 lt 31 lt 30 OK STI PCR mix 1 FRT B b sl IC Detection of Betecuon af IG B burgdorferi sl 7 cC RNA DNA lt 33 Absent OK extraction NCA Amplification Absent Absent OK C TBEV B b sl Aph E ch E m Amplification 30 30 x OK STI Result interpretation of test samples TBEV cDNA is detected in a sample if its Ct value in the results grid in the FAM channel with the use of PCR mix 1 FRT TBEV A ph E ch E m is less than the Ct value specified in Table 6 A phagocytophilum DNA is detected in a sample if its Ct value in the results grid in the HEX channel with the2. Follow the steps perform calibration in the FAM Green JOE Yellow and ROX Orange channels click the Calibrate Acquiring Optimize Acquiring button click the Perform Calibration Before 1 Acquisition Perform Optimization Before 1 Acquisition button set channel calibration from 5FI to 10FI for all channels the Edit button in the Auto gain calibration channel settings window Click Close Click Next Select the Start run button for amplification to run Name the experiment and save it to the disk the results of the run will be automatically saved in this file Set the data in the table of samples opens by default when run begins Define names numbers of clinical and control samples in the Name column Enter None for empty wells N Samples indicated as None will not be analyzed Data analysis The results are interpreted by the Instrument software by the crossing or not crossing of the fluorescence curve with the threshold line and are shown as the presence or absence of Ct cycle threshold in the results grid R V59 RG iQ Mx Dt CE 09 12 10 03 04 14 Page 4 of 24 Data analysis of amplification in the FAM Green channel 1 Select the Analysis button in the main menu select the Quantitation mode Activate the Cycling A FAM Cycling A Green button Click Show 2 Cancel the automatic selection of the Threshold level 3 The Dynamic tube and Slope Correct buttons should be activated in the
3. 2 Start the iCycler iQ iQ5 program 3 Define the plate setup that is a tube order in the reaction chamber and fluorescence signal detection in all tubes in FAM JOE and ROX channels if PCR mix 1 FRT TBEV A ph E ch E m is used or in FAM and JOE channels if PCR mix 1 FRT B b SI IC is used iCycler iQ 5 instrument In the Selected Plate Setup window of the Workshop module press the Create New or Edit button Edit the plate setup in the Whole Plate loading mode Set Sample Volume as 25 ul Seal Type as Domed Cap and Vessel Type as Tubes Press Save amp Exit Plate Editing iCycler iQ instrument Edit the plate setup in the Edit Plate Setup window of the Workshop module To do this define the sample order in the Samples Whole Plate Loading option and enter names of samples in the Sample Identifier window In the Select and Load Fluorophores option assign the detection of fluorescence signal in all tubes in FAM JOE and ROX channels if PCR mix 1 FRT TBEV A ph E ch E m is used or in FAM and JOE channels if PCR mix 1 FRT B b sl IC is used To save the created plate setup name it in the Plate Setup Filename window use pts file extension and click the Save this plate setup button at the top of the display To edit a previously created plate setup open View Plate Setup in the Library window and select the required plate setup pts file and click the Edit button on the right Save the edited file Press th
4. E m should be placed into the rotor first if both PCR mixes 1 are used in the run When working with the Rotor Gene 3000 instrument use the Rotor Gene version 6 software When working with the Rotor Gene 6000 instrument use the Rotor Gene 6000 versions 1 7 build 67 software or higher Hereinafter all terms corresponding to different instruments and software are indicated in the following order for Rotor Gene 3000 for Rotor Gene 6000 Programming the Rotor Gene 3000 6000 Rotor Gene Q instrument 1 Click the New button in the main program menu 2 In the opened window select Advanced and click Dual Labeled Probe Hydrolysis probes Activate the New button 3 Select 36 Well Rotor or 72 Well Rotor and No Domed 0 2 ml Tubes Locking ring attached for Rotor Gene 6000 instrument Click Next R V59 RG iQ Mx Dt CE 09 12 10 03 04 14 Page 3 of 24 11 Reaction volume is 25 ul Enter an operator Tick the 15 ul oil layer volume option for Rotor Gene 6000 Click Next In the opened window click the Edit profile button and set the temperature profile of the experiment as follows Table 1 Amplification program for Rotor Gene 3000 6000 and Rotor Gene Q Step Temperature C Time Ken Cycles 1 95 15 min 1 95 10s 2 60 30s 5 72 15s 95 10s 30 s FAM Green 3 56 JOE Yellow 40 ROX Orange 72 15s Click OK Click the Calibrate Gain Optimization button in the New Run Wizard window
5. Information window 7 In the Plate Setup menu select all cells with the test tubes and select Unknown in the Well type drop down window Activate FAM JOE and ROX fluorophores in the Collect fluorescence data option 8 Set the amplification program see the table below in Thermal Profile Setup Table 7 Amplification program for Mx3000P instrument m Fluorescent signal Step Temperature C Time PAM Mini Cycles 1 95 15 min 1 95 10s 2 60 35s 5 72 15s 95 10s 3 56 35s FAM JOE ROX 40 72 15s R V59 RG iQ Mx Dt CE 09 12 10 03 04 14 Page 19 of 24 9 Select Run in the menu Ensure that amplification program is correct Click Start Tick the Turn lamp off at end of run box Save the experiment Analysis of results Data processing Open the saved data file and shift to Analysis mode 1 2 Activate the Results window in the menu 3 Select the Amplification plots line in the Area to analyze unit 4 In the Threshold fluorescence unit set the threshold line at a level where fluorescence curves are linear It is recommended to set the threshold level equal to 500 for all channels Normally the threshold line should cross only sigmoid curves of positive samples and controls and should not cross the base line Otherwise raise the threshold 5 Select the Text report line in the Area to analyze unit Result interpretation for control samples The result of the analysis is
6. Quantitation analysis window 4 Inthe CT Calculation menu set Threshold 0 03 5 Activate the More Settings Outlier Removal button and enter NTC threshold level as 5 5 in the text field Ct value of each sample in the FAM Green channel will appear in the results grid Quant results window Data analysis of amplification in the JOE Yellow channel 1 Select the Analysis sign in the main menu select the Quantitation mode Activate the Cycling A JOE Cycling A Yellow button Click Show 2 Cancel the automatic selection of the Threshold level 3 The Dynamic tube and Slope Correct buttons should be activated in the Quantitation analysis window 4 Inthe CT Calculation menu set Threshold 0 03 5 Activate the More Settings Outlier Removal button and enter NTC threshold level as 5 5 in the text field Ct value of each sample in the JOE Yellow channel will appear in the results grid Quant results window Data analysis of amplification in the ROX Orange channel 1 Select the Analysis sign in the main menu select the Quantitation mode Activate the Cycling A ROX Cycling A Orange button Click Show 2 Cancel the automatic selection of the Threshold level 3 The Dynamic tube and Slope Correct buttons should be activated in the Quantitation analysis window 4 In the CT Calculation menu set Threshold 0 03 5 Activate the More Settings Outlier Removal button and enter NTC threshold le
7. RG iQ Mx Dt CE 09 12 10 03 04 14 Page 21 of 24 Troubleshooting Results of analysis are not taken into account in the following cases The samples except for NCA that show negative result in all channels should be analyzed once again PCR with detection If the same result is obtained in the second run repeat the test starting from the extraction stage For NCA the negative result detected in all channels is accepted as normal If the Ct value of the Positive Control of amplification C rgev Bo si Aph Ech Em STI IS absent or greater than the specified boundary Ct value in FAM JOE or ROX channels with the use of PCR mix 1 FRT TBEV A ph E ch E m or in FAM and JOE channels with the use of PCR mix 1 FRT B b sl IC PCR should be repeated for all samples in which specific CDNA DNA was not found in the appropriate channel If the Ct value of the Negative Control of extraction C in FAM JOE ROX channels with PCR mix 1 FRT TBEV A ph E ch E m and in the JOE channel with PCR mix 1 FRT B b sl IC and or Negative Control of amplification NCA in any channel is detected in the results grid PCR should be repeated for all samples in which specific cDNA DNA was found in the appropriate channel Examples of obtained results FAM channel amplification of TBEV cDNA for PCR mix 1 FRT TBEV A ph E ch E m and amplificaton of IC cDNA for PCR mix 1 FRT B b sI IC Positive Control of TBEV Fluorescenc
8. considered reliable only if the results of both Positive and Negative Controls of amplification as well as Negative Control of extraction are correct Table 8 Results of positive and negative controls should not exceed Ct values specified in Table 8 Table 8 Results for controls obtained with Mx3000P Ct value Control Stage for Interpret control FAM JOE ROX ation PCR mix 1 FRT TBEV A ph E ch E m Detection of Detection of E o A phagocyto E chaffeensis philum E muris C RNA DNA Absent Absent Absent OK extraction NCA Amplification Absent Absent Absent OK C TBEV B b sl A ph E ch E m Amplification lt 30 lt 31 lt 30 OK STI PCR mix 1 FRT B b sI IC Detection of Detection of IC B burgdorferi sl C RNA DN A lt 34 Absent OK extraction NCA Amplification Absent Absent OK C TBEV B b sl Aph E ch E m Amplification lt 30 lt 30 e OK STI R V59 RG iQ Mx Dt CE 09 12 10 03 04 14 Page 20 of 24 Result interpretation for test samples TBEV cDNA is detected in a sample if its Ct value in the results grid in the FAM channel with the use of PCR mix 1 FRT TBEV A ph E ch E m is less than the Ct value specified in Table 9 A phagocytophilum DNA is detected in a sample if its Ct value in the results grid in the JOE channel with the use of PCR mix 1 FRT TBEV A ph E ch E m is less than the Ct value spec
9. opd 900 800 e eo 600 No e o R V59 RG iQ Mx Dt CE 09 12 10 03 04 14 Page 18 of 24 AMPLIFICATION AND DATA ANALYSIS WITH THE USE OF Mx3000P Stratagene USA INSTRUMENT Carry out sample pretreatment and reaction mixture preparation stages according to the instruction manual Program the instrument according to the user manual provided by the manufacturer 1 Switch on the instrument Start the Stratagene Mx3000P program 2 Select Quantitative PCR Multiple Standards and check Turn lamp on for warm up in the New Experiment Options window N The lamp should be warmed up for at least 15 min before the experiment starts 3 Place experimental tubes into the module and lock it 4 Select Optics Configuration in the Options menu In the Dye Assignment window set FAM next to the FAM filter set line JOE next to the HEX JOE filter set line and ROX next to the ROX filter set line 5 Set fluorescence detection parameters in the Plate Setup menu To do this select all cells with PCR mix 1 FRT TBEV A ph E ch E m test tubes and define them as Unknown in the Well type drop down window Select FAM JOE and ROX fluorophores in the Collect fluorescence data option Then select all cells with PCR mix 1 FRT B b sl IC test tubes and define them as Unknown in the Well type drop down window Select FAM and JOE fluorophores in the Collect fluorescence data option 6 Enter the names of the test samples in the Well
10. For Professional Use Only GUIDELINES to AmpliSens TBEV B burgdorferi sl A phagocytophilum E chaffeensis E muris FRT PCR kit for qualitative detection and differentiation of tick borne infection pathogens RNA of TBEV tick borne encephalitis virus Borrelia burgdorferi sl Ixodes tick borne borreliosis ITB pathogen Ehrlichia chaffeensis and Ehrlichia muris human monocytic ehrlichiosis HME pathogens and DNA of Anaplasma phagocytophilum human granulocytic anaplasmosis HGA pathogen in biological materials by the polymerase chain reaction PCR with real time hybridization fluorescence detection Ecoli s r o Studenohorska Federal Budget Institute of 12 Science Central Research REP 841 03 Bratislava 47 Institute for Epidemiology Slovak Republic 3A Novogireevskaya Street Tel 421 2 6478 9336 Moscow 111123 Russia Fax 421 2 6478 9040 TABLE OF CONTENTS INTENDED USE AMPLIFICATION AND DATA ANALYSIS WITH THE USE OF Rotor Gene 3000 6000 Corbett Research Australia and Rotor Gene Q Qiagen Germany INSTRUMENTS 3 AMPLIFICATION AND DATA ANALYSIS WITH THE USE OF iCycler iQ and iQ5 Bio Rad USA ING RUMEN S220 caletnnmndeseneiisinehoriealesheduntsnilralstildencnedwenn tsb batenceetetebaeshiens 10 AMPLIFICATION AND DATA ANALYSIS WITH THE USE OF Mx3000P Stratagene USA INSTRUMENT 2 sities celta eae eae i Siac eos i aah ec di 19 R V59 RG iQ Mx Dt CE 09 12 10 03 04 14 Page 2 of 24 INTENDED U
11. RT TBEV A ph E ch E m and in the JOE channel with PCR mix 1 R V59 RG iQ Mx Dt CE 09 12 10 03 04 14 Page 7 of 24 FRT B b sl IC and or Negative Control of amplification NCA in any channel is detected in the results grid PCR should be repeated for all samples in which specific CDNA DNA was found in the appropriate channel Examples of obtained results FAM channel amplification of TBEV cDNA for PCR mix 1 FRT TBEV A ph E ch E m and amplificaton of IC cDNA for PCR mix 1 FRT B b sI IC Positive Control Vz AF ad of TBEV r4 A f y 5 10 15 20 25 30 35 40 Linkn R V59 RG iQ Mx Dt CE 09 12 10 03 04 14 Page 8 of 24 JOE channel amplification of A phagocytophilum DNA for PCR mix 1 FRT TBEV A ph E ch E m and amplification of B burgdorferi sl cDNA for PCR mix 1 FRT B b sl 1C PCR mix 1 FRT B burgdorferi sl 1C Positive Control of B burgdorferi sl Positive Control of A phagocytophillum PCR mix 1 FRT TBEV Ap Em Ech ROX channel amplification of E chaffeensis E muris cDNA for PCR mix 1 FRT TBEV A ph E ch E m 0 8 0 6 Positive Control of E chaffeensis E muris 0 4 0 2 R V59 RG iQ Mx Dt CE 09 12 10 03 04 14 Page 9 of 24 AMPLIFICATION AND DATA ANALYSIS WITH THE USE OF iCycler iQ and iQ5 Bio Rad USA INSTRUMENTS 1 Switch on the instrument The lamp should be warmed up for at least 30 min before the experiment starts
12. SE Guidelines describe the procedure of the use of AmpliSens TBEV B burgdorferi sl A phagocytophilum E chaffeensis E muris FRT PCR kit for qualitative detection of RNA of tick borne encephalitis virus TBEV Borrelia burgdorferi sl Ixodes tick borne borreliosis ITB pathogen Ehrlichia chaffeensis and Ehrlichia muris human monocytic ehrlichiosis HME pathogens and DNA of Anaplasma phagocytophilum human granulocytic anaplasmosis HGA pathogen in biological materials ticks blood cerebrospinal fluid and autopsy material by using real time hybridization fluorescence detection with Rotor Gene 3000 three and more channels Corbett Research Australia Rotor Gene 6000 five and six channels Corbett Research Australia Rotor Gene Q Qiagen Germany iCycler iQ iQ5 Bio Rad USA Mx3000P Stratagene USA AMPLIFICATION AND DATA ANALYSIS WITH THE USE OF Rotor Gene 3000 6000 Corbett Research Australia and Rotor Gene Q Qiagen Germany INSTRUMENTS Carry out sample pretreatment and reaction mixture preparation stages according to the instruction manual Transparent flat cap 0 2 ml PCR tubes and 0 1 ml tubes are recommended for use Place the tubes in the rotor so that the first tube is in well No 1 insert the rotor in the reaction module and secure the lid the rotor cells are numbered and these numbers are used for the programming sample order N The tubes with PCR mix 1 FRT TBEV A ph E ch
13. V A phagocytophilum i m ra E ch E m i lt 39 lt 39 lt 39 l Detection of PARCOL B burgdorferi sl 2 B b sl 1C 38 39 Troubleshooting Results of analysis are not taken into account in the following cases e The samples except for NCA that show negative result in all channels should be analyzed once again PCR with detection If the same result is obtained in the second run repeat the test starting from the extraction stage For NCA the Negative result detected in all channels is accepted as normal e f the Ct value of the Positive Control of amplification C 7Bev Bb si Aph Ech Em STI IS absent or greater than the specified boundary Ct value in FAM JOE or ROX channels with the use of PCR mix 1 FRT TBEV A ph E ch E m or in the FAM and JOE channels with the use of PCR mix 1 FRT B b sl IC PCR should be repeated for all samples in which specific CDNA DNA was not found in the appropriate channel e f the Ct value of the Negative Control of extraction C in the FAM JOE ROX channels with PCR mix 1 FRT TBEV A ph E ch E m and in the JOE channel with PCR mix 1 FRT B b sl IC and or Negative Control of amplification NCA in any channel is detected in the results grid PCR should be repeated for all samples in which specific CDNA DNA was found in the appropriate channel R V59 RG iQ Mx Dt CE 09 12 10 03 04 14 Page 15 of 24 Examples of obtained results FAM c
14. dance with the specified tube order Prior to analysis remove drops from tube walls because drops falling during N amplification cause signal errors and complicate data analysis Do not turn strips plate when placing it into the PCR instrument iCycler iQ5 instrument Ensure that the Selected Protocol and Selected Plate Setup are set correctly before starting the program To start the program click the Run button Select the Use Persistent Well Factor option set by default for detection of the well factor Make sure that amplification and calibration are performed with the same type of plastic consumables Click the Begin Run button name the experiment result data will be automatically saved to this file and click OK Select Seal Type Domed cap and Vessel Type Tubes iCycler iQ instrument Ensure that the selected protocol and plate setup are set correctly in the Run Prep window For determination of the well factor select the Persistant Plate option in the Select well factor source line Set the reaction mix volume as 25 ul in the Sample Volume window Click the Begin Run button name the experiment result data will be automatically saved in this file and click OK Proceed to analysis of results after the end of amplification run Analysis of results The results are interpreted by the Instrument software by the crossing or not crossing of the fluorescence curve with a threshold line and are shown as the presence
15. e dR R V59 RG iQ Mx Dt CE 09 12 10 03 04 14 Page 22 of 24 JOE channel amplification of A phagocytophilum DNA for PCR mix 1 FRT TBEV A ph E ch E m and amplification of B burgdorferi sl cDNA for PCR mix 1 FRT B b sI IC Positive Control of Positive Control of A phagocytophillum Fluorescence dR ROX channel amplification of E chaffeensis E muris cDNA for PCR mix 1 FRT TBEV A ph E ch E m Positive Control of E chaffeensis E muris Fluorescence dR R V59 RG iQ Mx Dt CE 09 12 10 03 04 14 Page 23 of 24 List of Changes Made in the Guidelines VER Location of Essence of changes changes 29 06 11 The name of Institute was changed to Federal Budget L A Cover page Institute of Science Central Research Institute for Epidemiology m T Cover page Address of European representative was added R V59 RG iQ Mx Dt CE 09 12 10 03 04 14 Page 24 of 24
16. e Run with selected protocol button to activate the created plate setup Set the amplification program see Table 4 Table 4 Amplification program for iCycler iQ and iQ5 Step Temperature C Time ordren pe Cycles 1 95 15 min 1 95 10s 2 60 35s 5 72 15s 95 10s 3 56 35s FAM HEX ROX 40 72 15s R V59 RG iQ Mx Dt CE 09 12 10 03 04 14 Page 10 of 24 iCycler iQ5 instrument Click the Create New or Edit button in the Selected Protocol window of the Workshop module Set the amplification parameters and click the Save amp Exit Protocol Editing button to save the protocol For further runs a file with this program can be selected from the Protocol unit all files are saved to the Users folder by default iCycler iQ instrument To set the amplification program select the Edit Protocol option of the Workshop module Enter the number of cycles time and temperature in the bottom window Specify the signal acquiring in the window at the right as follows Cycle 3 Step To save the protocol name the file in the Protocol Filename window use the tmo file extention and click the Save this protocol button at the top of the display For further runs a file with this program can be selected from the View Protocol tab of the Library module Press the Run with selected plate setup button to save and activate the created program 5 Insert the prepared tubes into the reaction chamber in accor
17. ed in a sample if its Ct value in the results grid in the FAM Green channel with the use of PCR mix 1 FRT TBEV A ph E ch E m is less than the Ct value specified in Table 3 e A phagocytophilum DNA is detected in a sample if its Ct value in the results grid in the JOE Yellow HEX channel with the use of PCR mix 1 FRT TBEV A ph E ch E m is less than the Ct value specified in Table 3 e E chaffeensis E muris cDNA is detected in a sample if its Ct value in the results grid in the ROX Orange channel with the use of PCR mix 1 FRT TBEV A ph E ch E m is less than the Ct value specified in Table 3 e Borrelia burgdorferi sl cDNA is detected in a sample if its Ct value in the results grid in the JOE Yellow HEX channel with the use of PCR mix 1 FRT B b sl IC is less than R V59 RG iQ Mx Dt CE 09 12 10 03 04 14 Page 6 of 24 the Ct value specified in Table 3 Moreover the fluorescence curve of every studied sample should cross the threshold line at the exponential growth stage e cDNA DNA of the above mentioned microorganisms are not detected in a sample if the Ct value in FAM channel with the use of PCR mix 1 FRT B b sl IC is less than the boundary Ct value specified in Table 3 while no Ct value is detected in the channel assigned for the specific pathogen The result is invalid if the Ct value of a sample is absent in the channels for specific pathogen detection whereas Ct in the FAM Green channel with the u
18. hannel amplification of TBEV cDNA for PCR mix 1 FRT TBEV A ph E ch E m and amplificaton of IC cDNA for PCR mix 1 FRT B b sI IC Amplification Chart Data 2009 04 15 1425 id ud iaadi i ai i 2008 new opd PCR Base Line Subtracted Curve Fit RFU 2000 L L L L J r 1 lI lo Let Positive Control of TBEV PCR Base Line Subtracted Curve Fit RFU R V59 RG iQ Mx Dt CE 09 12 10 03 04 14 Page 16 of 24 JOE channel amplification of A phagocytophilum DNA for PCR mix 1 FRT TBEV A ph E ch E m and amplification of B burgdorferi sl cDNA for PCR mix 1 FRT B b sI IC Amplification Chart Data 2009 04 15 1425 EN T om 2008 new c e o Positive Control of A phagocytophilum 200 PCR Base Line Subtracted Curve Fit RFU Cycle Amplification Chart Data 2009 04 15 1425 l e beisa i ESi a al 2008 new opd 1800 1600 A e eo No e e e e o Positive Control of B burgdorferi sl 800 600 PCR Base Line Subtracted Curve Fit RFU 400 200 100 00 R V59 RG iQ Mx Dt CE 09 12 10 03 04 14 Page 17 of 24 PCR Base Line Subtracted Curve Fit RFU ROX channel amplification of E chaffeensis E muris cDNA for PCR mix 1 FRT TBEV A ph E ch E m Amplification Chart Data 2009 04 15 1425 l amp eisa i i ai i 2008 new
19. ified in Table 9 E chaffeensis E muris cDNA is detected in a sample if its Ct value in the results grid in the ROX channel with the use of PCR mix 1 FRT TBEV A ph E ch E m is less than the Ct value specified in Table 9 Borrelia burgdorferi sl cDNA is detected in a sample if its Ct value in the result grid in the JOE channel with the use of PCR mix 1 FRT B b sl 1C is less than the Ct value specified in Table 9 Moreover the fluorescence curve of each studied sample should cross the threshold line at the exponential growth stage CDNA DNA of the above mentioned microorganisms are not detected in a sample if the Ct value detected in the FAM channel with the use of PCR mix 1 FRT B b s IC is less than boundary Ct value specified in Table 9 while no Ct value is detected in the channel assigned for the specific pathogen The result is invalid if Ct of a sample is absent in the channels for specific pathogen detection whereas in the FAM channel with the use of PCR mix 1 FRT B b sl IC Ct is also absent or greater than the specified boundary Ct value Repeat the PCR test for such samples Table 9 Results for test samples obtained with Mx3000P Signal in channel Ct PCR mix 1 FRT FAM HEX ROX Detection of Detection of E TBEV A ph TBEV A phagocytophilum i E muris E ch E m 39 39 39 Detection of HJetaetio eme B burgdorferi sl 7 B b sl IC 38 39 R V59
20. nalysis menu 2 Select the Baseline Threshold option using the right mouse button on the plot of the fluorescence curves 3 Set following parameters select User Defined Select all and Edit Range in the Base Line Cycles menu and set Start Cycle 2 Ending Cycle 25 select User Defined in the Crossing Threshold menu and set Threshold Position 100 Click OK 4 Ct values will appear in the results grid Results window Analysis of amplification results obtained with PCR mix 1 FRT B b sI I1C Internal Control 1 Click the FAM button in the Data Analysis menu 2 Select the Baseline Threshold option using the right mouse button on the plot of the fluorescence curves 3 Set following parameters select User Defined Select all and Edit Range in the Base Line Cycles menu and set Start Cycle 2 Ending Cycle 25 select User Defined in the Crossing Threshold menu and set Threshold Position 50 Click OK 4 Ct values will appear in the results grid Results window B burgdorferi sl DNA 1 Click the JOE button in the Data Analysis menu 2 Select the Baseline Threshold option using the right mouse button on the plot of the fluorescence curves 3 Set following parameters select User Defined Select all and Edit Range in the Base Line Cycles menu and set Start Cycle 2 Ending Cyclez25 select User Defined in the Crossing Threshold menu and set Threshold Positionz100 Click OK 4 Ct values will appear in the result grid Results window
21. or absence of Ct threshold cycle in the results grid The fluorescence curve for the sample should contain a segment of a typical exponential growth S shape of fluorescence and cross the R V59 RG iQ Mx Dt CE 09 12 10 03 04 14 Page 11 of 24 threshold line once Data processing iCycler iQ instrument Activate the View Post Run window in the Library module Select the required file in the Data Files window and click the Analyze Data button Select the desired channel in the PCR Quantification option of the Select a Reporter menu Make sure that PCR Base Line Subtracted Curve Fit mode is activated selected by default In the Threshold Cycle Calculation menu indicate that the threshold is to be set manually and the base line is to be calculated automatically To do this select Auto Calculated in the Baseline Cycles submenu and select User Define in the Threshold Position submenu To set the threshold line it should be move with the cursor while the left mouse button is pushed When data obtained with PCR mix 1 FRT TBEV A ph E ch E m are processed disable the wells that were analyzed with PCR mix 1 FRT B b s IC in the Display wells menu When data obtained with PCR mix 1 FRT B b sl IC are processed disable the wells that were analyzed with PCR mix 1 FRT TBEV A ph E ch E m in the Display wells menu Click the Recalculate Threshold Cycles button Ct values will appear in the results grid iQ5 instrument Select
22. se of PCR mix 1 FRT B b sl IC is also absent or greater than the specified boundary Ct value It is necessary to repeat the PCR test for such samples Table 3 Results for test samples obtained with Rotor Gene 3000 6000 and Rotor Gene Q Signal in channel Ct PCR mix 1 FRT FAM Green HEX Yellow ROX Orange Detection of Detection of Eo d TBEV A ph TBEV A phagocytophilum E furs E ch E m 38 38 38 Detection of Detection of IC B burgdorferi sl B b sl IC lt 35 lt 38 Troubleshooting Results of analysis are not taken into account in the following cases e The samples except for NCA that show negative result in all channels should be analyzed once again PCR with detection If the same result is obtained in the second run repeat the test starting from the extraction stage For NCA the Negative result detected in all channels is accepted as normal If the Ct value of the Positive Control of amplification C rgev B b si A ph Ech E m STI IS absent or greater than the specified boundary Ct value in FAM JOE or ROX channels with the use of PCR mix 1 FRT TBEV A ph E ch E m or in the FAM and JOE channels with the use of PCR mix 1 FRT B b sl IC PCR should be repeated for all samples in which specific CDNA DNA was not found in the appropriate channel If the Ct value of the Negative Control of extraction C in FAM JOE ROX channels with PCR mix 1 F
23. the required result data file Data File window of the Workshop module and click the Analyze button Select data obtained in the required channel in the window of the module Make sure that the PCR Base Line Subtracted Curve Fit mode is activated selected by default Analysis of amplification results obtained with PCR mix 1 FRT TBEV A ph E ch E m TBEV cDNA T 2 4 Click the FAM button in the Data Analysis menu Select the Baseline Threshold option using the right mouse button on the plot of the fluorescence curves Set following parameters select User Defined Select all and Edit Range in the Base Line Cycles menu and set Start Cycle 2 Ending Cycle 25 select User Defined in the Crossing Threshold menu and set Threshold Positionz200 Click OK Ct values will appear in the results grid Results window A phagocytophilum DNA 1 2 Click the JOE button in the Data Analysis menu Select the Baseline Threshold option using the right mouse button on the plot of the R V59 RG iQ Mx Dt CE 09 12 10 03 04 14 Page 12 of 24 fluorescence curves 3 Set following parameters select User Defined Select all and Edit Range in the Base Line Cycles menu and set Start Cycle 2 Ending Cycle 25 select User Defined in the Crossing Threshold menu and set Threshold Position 100 Click OK 4 Ct values will appear in the results grid Results window E muris E chaffensis cDNA 1 Click the ROX button in the Data A
24. use of PCR mix 1 FRT TBEV A ph E ch E m is less than the Ct value specified in Table 6 E chaffeensis E muris cDNA is detected in a sample if its Ct value in the results grid in the ROX channel with the use of PCR mix 1 FRT TBEV A ph E ch E m is less than the Ct value specified in Table 6 Borrelia burgdorferi sl cDNA is detected in a sample if its Ct value in the results grid in the JOE Yellow HEX channel with the use of PCR mix 1 FRT B b sl IC is less than the Ct value specified in Table 6 Moreover the fluorescence curve of each studied sample should cross the threshold line at the exponential growth stage R V59 RG iQ Mx Dt CE 09 12 10 03 04 14 Page 14 of 24 e cDNA DNA of the above mentioned microorganisms are not detected in a sample if the Ct value detected in FAM channel with the use of PCR mix 1 FRT B b sl IC is less than boundary Ct value specified in Table 6 while no Ct value is detected in the channel assigned for the specific pathogen e The result is invalid if Ct of a sample is absent in the channels for specific pathogen detection whereas in the FAM channel with the use of PCR mix 1 FRT B b sl IC Ct is also absent or greater than the specified boundary Ct value Repeat the PCR test for such samples Table 6 Results for test samples obtained with iCycler iQ and iQ5 Signal in channel Ct PCR mix 1 FRT FAM HEX ROX Detection of Detection of E TBEV A ph TBE
25. vel as 5 5 in the text field Ct value of each sample in the JOE Yellow channel will appear in the results grid Quant results window 10 T If fluorescence curves do not show exponential growth set the NTC threshold as R V59 RG iQ Mx Dt CE 09 12 10 03 04 14 Page 5 of 24 Result interpretation of the control samples The result of the analysis is considered reliable only if the results of Positive and Negative Controls of amplification as well as Negative Control of extraction are correct Table 2 The results of positive and negative controls should not exceed Ct values specified in Table 2 Table 2 Results for controls obtained with Rotor Gene 3000 6000 and Rotor Gene Q Borie Stage for Ct value Interpret control FAM Green JOE Yellow ROX Orange ation PCR mix 1 FRT TBEV A ph E ch E m Detection of Detection of dcos 9 A phagocyto E chaffeensis philum E muris c RNA DNA Absent Absent Absent OK extraction NCA Amplification Absent Absent Absent OK C TBEV B b sl A ph E ch E m Amplification lt 27 lt 27 lt 27 OK STI PCR mix 1 FRT B b sI IC Detection of Detection of _ IC B burgdorferi sl C RNA DNA lt 30 Absent OK extraction NCA Amplification Absent Absent OK C TBEV B b sl A ph E ch E m Amplification 27 27 OK STI Result interpretation for test samples e TBEV cDNA is detect
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