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GUIDELINES Real-Time PCR Detection of STIs and Other

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1. PCR kit Threshold More Settings Slope Correct Outlier Removal Chlamydia trachomatis 0 1 0 off Neisseria gonorrhoeae screen 0 1 0 off Neisseria gonorrhoeae test 0 1 0 off Neisseria gonorrhoeae 0 1 0 off Mycoplasma genitalium 0 1 0 off Ureaplasma species 0 1 0 off Mycoplasma hominis 0 1 0 off HSV Il 0 1 0 off CMV 0 1 0 off Gardnerella vaginalis 0 1 0 off Treponema pallidum 0 1 5 on Trichomonas vaginalis 0 1 5 on Candida albicans 0 1 0 off 4 Principle of interpretation The microorganism DNA is found in a sample if its Ct value is detected in the results grid in the FAM Green channel The fluorescence curve should cross the threshold line at the typical exponential growth phase The microorganism DNA is not found in a sample if its Ct value is not detected in the results grid in the FAM Green channel the fluorescence curve does not cross the threshold line whereas the Ct value detected in the JOE Yellow channel is less than 30 The result is invalid if the Ct value of a sample is not detected in the FAM Green channel whereas the Ct value in the JOE Yellow channel is either absent or greater than 30 Repeat the PCR test for such samples The result of 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 Table 6 7 Table 6 Results for controls Ct in FAM G
2. 2o eshold 5 10 15 20 25 30 35 40 Upon e Lu Quant Results Cycling A Red Page 1 3 lni x 26 11 L qe Ct value for IC DNA 2541 is notidetected IC Quantitation Analysis Cyding Orange P Yellow P lglxi FA Sepe Correct Z ignore Fest dias Settings Save Dents T Bi Spe Corect Z ignore Fst ag More Settings Save Defauts M hom DNA W 7 is Found The result for negative controls C and NCA is negative The Ct value detected for C in the Cy5 Red channel detection of IC is less than 33 The result for positive control C is positive Ct values do not exceed the boundary Ct values in all channels The results for controls correspond with specified values The results for test samples are valid Sample No 2 shows the presence of DNA of the microorganisms that are detected in the FAM Green channel Chlamydia trachomatis in here as well as DNA of the microorganisms detected in the JOE Yellow channel Ureaplasma spp in here Sample No 3 shows the presence of DNA of the microorganisms detected in the JOE Yellow and ROX Orange channels Ureaplasma spp and Mycoplasma hominis respectively in here Sample No 7 shows the presence of DNA of the microorganism detected in the JOE Yellow channel Ureaplasma spp in here Ct values less than 33 are detected for all samples except for sample No 5 in the Cy5 Red channel Sample No 5 shows an invalid result tha
3. DNA of the specific microorganism is not found in samples Nos 2 and 6 Sample No 3 shows an invalid result that is Ct values are absent in both channels Analysis of this sample should be repeated 01 02 11 20 03 11 Page 36 of 48 Example of results obtained using the iCycler iQ instrument I Select Wells Select analysis mode PCR Base Line Subtracted Curve rit gt 3 Reports Threshold Cycle Calculation ei t Baseline Cycles Auto Calculated Recalculate Threshold Cycles C User Defined Threshold Position C Auto Calculated J 0 User dered 8 FAM B B 8 the microorganism DNA is found bos db di dida PCR Base Line Subtracted CF RFU 8 8 L 10 12 14 16 18 20 22 24 26 28 30 X 3 3 Cyde Z t a Select analysis mode PCR Base Line Subtracted Curve Fit 2 pi Sef Reports NOS Threshold Cycle Calculation Baseline Cycles Auto Calculated Recalculate Threshold Cycles C User Defined Threshold Position C Auto Calculated J xo eweme too view 400 400 350 350 i HEX amp 300 ci 5 m Te m IC j g 150 150 10 N A IC C t 100 100 u N A INCA C x Li o 50 0 2 4 6 98 10 12 14 16 18 20 22 24 26 28 30 X3 9 SB Cyde The results for negative controls C and NCA are negative The Ct value detected for C in the HEX channel detection of IC is less than 33 The result for positive control C is positive Ct values in the FAM channel do not exceed the
4. Ureaplasma spp AmpliSens C trachomatis Ureaplasma MULTIPRIME FEP Chlamydia trachomatis Mycoplasma genitalium AmpliSens C trachomatis M genitalium MULTIPRIME FEP Candida albicans Candida glabrata Candida krusei AmpliSens C albicans C glabrata C krusei MULTIPRIME FEP AmpliSens C albicans C glabrata C krusei MULTIPRIME FRT HSV CMV AmpliSens HSV CMV MULTIPRIME FEP AmpliSens HSV CMV MULTIPRIME FRT Mycoplasma hominis Gardnerella vaginalis AmpliSens M hominis G vaginalis MULTIPRIME FEP AmpliSens M hominis G vaginalis MULTIPRIME FRT 01 02 11 20 03 11 Page 4 of 48 1 INTENDED USE Guidelines describe the procedure of detection of STIs and other reproductive tract infections in clinical material by the polymerase chain reaction PCR with real time hybridization fluorescence detection using real time PCR instruments Rotor Gene 3000 and 6000 Corbett Research iCycler iQ and iQ5 Bio Rad Mx3000P and Mx3005 Stratagene 2 PRINCIPLE OF PCR DETECTION Detection of microorganisms by the polymerase chain reaction PCR is based on the amplification of a pathogen genome specific region using specific primers In real time PCR the amplified product is detected using fluorescent dyes These dyes are linked to oligonucleotide probes which bind specifically to the amplified product during thermocycling The real time monitoring of fluorescence intensities during
5. see the DNA sorb AM K1 12 100 CE K1 12 50 CE protocol PCR kit variant FRT 100 F is intended for 110 reactions including controls 01 02 11 20 03 11 Page 6 of 48 4 ADDITIONAL REQUIREMENTS 5 DNA extraction kit Transport medium Disposable powder free gloves and laboratory coat Pipettes adjustable Sterile pipette tips with aerosol barriers up to 200 ul Tube racks Vortex mixer Desktop centrifuge with rotor for 2 ml reaction tubes PCR box Personal thermocyclers for example Rotor Gene 3000 or Rotor Gene 6000 Corbett Research Australia iCycler iQ or iQ5 Bio Rad USA or equivalent Disposable polypropylene tubes for PCR 0 1 or 0 2 ml for example Axygen USA Refrigerator for 2 8 C Deep freezer for lt 16 C Waste bin for used tips GENERAL PRECAUTIONS The user should always pay attention to the following Use sterile pipette tips with aerosol barriers and use a new tip for every procedure Store and handle amplicons away from all other reagents Thaw all components thoroughly at room temperature before starting detection When thawed mix the components and centrifuge briefly Use disposable gloves laboratory coats protect eyes while samples and reagents handling Thoroughly wash hands afterward Do not eat drink smoke apply cosmetics or handle contact lenses in laboratory work areas Do not use a kit after its expiration date Dispose of all samples and u
6. 01 02 11 20 03 11 Page 35 of 48 Table 13 Boundary Ct values for positive control in the FAM channel Ct value in FAM channel PCR kit for C Chlamydia trachomatis HSV I Il CMV Candida albicans Neisseria gonorrhoeae screen Neisseria gonorrhoeae test Neisseria gonorrhoeae 1 and 2 reaction Mycoplasma genitalium Trichomonas vaginalis 36 Treponema pallidum Ureaplasma species 33 Mycoplasma hominis Gardnerella vaginalis Example of results obtained using the iCycler iQ instrument the microorganism DNA s found resholdCyce EU T etter Trestle eu En YES SampleType Carin a LE R A dotze L fi ac 1 9 jJee 0 Os The results for negative controls C and NCA are negative the Ct value detected for C in the HEX channel detection of IC is less than 33 The result for positive control C is positive Ct values in the FAM channel do not exceed the boundary Ct values The results for controls correspond to the boundary Ct values specified The results for test samples are valid DNA of the specific microorganism was found in samples Nos 1 4 and 5 Ct value not exceeding the boundary Ct value 33 is detected in the HEX channel for all samples except for sample No 3
7. Acquisition Perform Optimisation Before 1 Acquisition box Close the Auto Gain Calibration Setup window 5 Proceed to the next window Click the Save Template button Enter the template name corresponding to the name of amplification program Amplisens 1 or 60 45 RG Save the template in the offered Templates folder in the Quick Start Templates subfolder and close the New Run Wizard window The created template will appear in the template list in the New Run window The AmpliSens 1 template can be used for conducting any amplification tests for detection of STIs with use of PCR kits manufactured by CRIE B Use of the created template 1 Place the tubes into the rotor so that the first well is loaded with a tube filled with the reaction mixture prepared for the run see Notes 1 and 2 Fix the locking ring secure the rotor and close the lid To start run with the prepared template select the Advanced tab in the New Run Wizard window of the New Run menu Select the template with AmpliSens 1 amplification program from the drop down list box set as described in section A Creating Template If a PCR kit variant FRT with a wax layer is used the template with the 60 45 RG amplification program can be selected Select 36 Well Rotor or 72 Well Rotor and tick the No Domed 0 2 ml Tubes Locking 01 02 11 20 03 11 Page 23 of 48 ring attached line Proceed to the next window 4 Make sure that the reaction volume
8. DNA extraction N A absent Eee de NCA PCR N A absent N A absent Detected Ct value is less C PCR than the specified E 36 boundary Ct value Table 15 Boundary Ct values for positive control C Ct value in channel POAN FAM HEX ROX C trachomatis Ureaplasma M genitalium 33 36 36 C trachomatis Ureaplasma M hominis 33 36 36 N gonorrhoeae C trachomatis M genitalium 36 33 36 C albicans C glabrata C krusei 36 36 36 U parvum U urealyticum 36 36 36 HS V4yping 36 33 36 T vaginalis N gonorrhoeae 36 36 36 HSV CMV 33 33 36 Example of results obtained using the iCycler iQ instrument for a MULTIPRIME PCR kit Results obtained with AmpliSens C trachomatis Ureaplasma M genitalium MULTIPRIME FRT PCR kit 01 02 11 20 03 11 Page 40 of 48 B 1800 a P mu 2 L amp 1600 7 FAM a Ew T E i ET Z Ser om E 7 7 2 on gt 1 50 b Chl t DNA A C 50 a f 2 E round N NCA 2x0 Z I1 5 5 20 g w H mn E Fa ee pc 100 c ox 7 E A LA dB M lann ann s T L E E E E v Oye E T pdt Cals IPCR Base Ure Are Curve Ft ii mms m Lr d YA d Unis Unde Sr NA asa pem pee ae T Unkn 6 Unis brz un NCA 7421 Ct value for IC DNA eu Em is not detected is not found C C Gm cune B jE aeoe j exem jJ m
9. To obtain the overall results grid activate data in both channels in the Results Amplification Plots window and select the Text Report option in the Area to analyze menu list The results grid can be exported to Excel to do this press the right mouse button and select the Export Text Report to Excel option from the menu displayed 01 02 11 20 03 11 Page 43 of 48 4 Interpretation of results Principle of interpretation The microorganism DNA is found in a sample if its Ct value is detected in the results grid in the FAM channel make sure that the FAM 490 sigh is selected in the Select a Reporter window line in the typical exponential growth phase The microorganism DNA is not found in a sample if N A appears in the results grid in the FAM channel the fluorescence curve does not cross the threshold line The fluorescence curve should cross the threshold whereas the Ct value detected in the JOE channel is less than 33 The result is invalid if the Ct value of the sample is not detected in the FAM channel N A appears whereas the Ct value in the JOE channel is either absent or greater than 33 Repeat the PCR test for such samples The result of analysis is considered reliable only if the results of Positive and Negative Controls of amplification as well as Negative Control of extraction are correct see Tables 17 and 18 Table 17 Result for controls Ct value Control Stage for control F
10. analysis of the other tests carried out with AmpliSens PCR kits for detection of pathogens of sexually transmitted diseases The same threshold level in the HEX channel can be used in further runs conducted on the same Instrument in case a new calibration has not been performed 3 Principle of interpretation The microorganism DNA is found in a sample if its Ct value is detected in the results grid in the FAM channel The fluorescence curve should cross the threshold line in the typical exponential growth phase The microorganism DNA is not found in a sample if N A appears in the results grid in the FAM channel the fluorescence curve does not cross the threshold line whereas the Ct value detected in the HEX channel is less than 33 The result is invalid if the Ct value of the sample is not detected in the FAM channel N A appears whereas the Ct value in the HEX channel is either absent N A or greater than 33 Repeat the PCR test for such 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 see Tables 12 and 13 Table 12 Results for controls Ct Control Stage for control FAM channel HEX channel C DNA extraction N A absent Mosis VANESS NCA PCR N A absent N A absent Detected Ct value is less et PCR than the specified BERRIEN Lapis 33 boundary Ct value
11. fragment of specific microorganisms whereas Ct in the channel assigned for IC DNA is either absent or greater than 36 It is necessary to repeat the PCR test for such a sample For automatic analysis of results the AmpliSens lt PCR kit gt Results Matrix program supplied by the manufacturer can be used The obtained data should be analyzed as 01 02 11 20 03 11 Page 46 of 48 described in items 1 and 2 Ct values should be copied from the results grid in the clipboard and entered in the corresponding column of the program for the automatic result analysis 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 see Table 19 and 20 Table 19 Results for controls Ct value Control Stage for control Channel for detection of v UE specific microorganism DNA amplification amplification Cy5 or ROX C DNA extraction Absent Detected Ct value lt 36 NCA PCR Absent Absent Detected Ct value is less than C PCR the specified boundary Ct Detected Ct value lt 36 value Table 20 Boundary Ct values for Positive Control of Amplification C s Ct value in channel PCR kit test FAM HEX ROX C trachomatis Ureaplasma M genitalium 33 36 36 C trachomatis Ureaplasma M hominis 33 36 36 N gonorrhoeae C trachomatis M genitalium 36 33 36 C albicans C
12. in the FAM channel Moreover the fluorescence curve should cross the threshold line in the area of exponential fluorescence growth e The microorganism DNA is not detected in a sample if its Ct value is not detected in the results grid in the FAM channel the fluorescence curve does not cross the threshold line whereas the Ct value in the JOE channel is less than the boundary Ct value specified e The result is invalid if the Ct value of a sample in the FAM channel is not detected absent whereas the Ct value in the JOE channel is either absent or greater than the boundary Ct value specified Repeat the PCR test for such a sample enclosed in the PCR kit and in the chapter Conducting Real Time PCR with the j Boundary Ct values are specified in the Important Product Information Bulletin Use of Different Instruments of this Guidelines manual 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 Table 2 Table 2 Results for controls Ct value in channel Control Stage for control Interpretation FAM fluorophore JOE fluorophore Pos boundary Ct C DNA extraction Neg value OK NCA Amplification Neg Neg OK nw Pos boundary Ct Pos boundary Ct C Amplification value value OK B If MULTIPRIME PCR kits are used The fluorescent signal intensity is detected in each chan
13. menu and then select the required channel Perform operation for FAM Green channel by selecting Cycling A FAM Cycling A Green perform operation for JOE Yellow channel by selecting Cycling A JOE Cycling A Yellow 2 Data analysis of IC DNA amplification in the JOE Yellow channel 2 1 Select normalized curves in the JOE Yellow channel 2 2 Make sure that the Dynamic tube button is activated set by default Activate the More SettingslOutlier Removal button and enter 5 596 in the text field 2 3 In the CT Calculation menu set Threshold 0 1 2 4 Ct values of each sample in the JOE Yellow channel will appear in the results grid Quant Results Cycling A JOE Quant Results Quant Results Cycling A Yellow 3 Data analysis of the microorganism DNA amplification in the FAM Green channel 3 1 Select normalized curves in the FAM Green channel 3 2 Make sure that the Dynamic tube button is activated set by default The Slope Correct button should be turned off or on as specified in Table 5 Activate the More Settings Outlier Removal button and in the text field enter the value specified in Table 5 3 3 In the CT Calculation menu set Threshold 0 1 3 4 Ct values of each sample in FAM Green channel will appear in the results grid Quant Results Cycling A FAM Quant Results Quant Results Cycling A Green 01 02 11 20 03 11 Page 25 of 48 Table 5 Parameters of analysis of results in the FAM Green channel
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15. reaction chamber in accordance with the created plate setup Secure the instrument 4 Start the AmpliSens 1 program along with the created plate setup 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 a well factor 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 Experimental Plate option set by default below the Select well factor source line see point 1 Set the reaction mix volume as 30 yl Press Run to start 5 Proceed to paragraph 6 if PCR kit variant FRT 100 F is used If PCR kit variant FRT is used press the Pause button when the temperature in the reaction chamber reaches 95 C open the instrument and insert the tubes into the wells in accordance with the created plate setup Close the lid and press the Resume Run button for iCycler iQ5 or Continue Running Protocol button for iCycler iQ Proceed to data analysis when the program is done At the end of the work close the program and shut down the instrument 01 02 11 20 03 11 Page 33 of 48 Data analysis iCycler iQ and iQ5 instruments The obtained data are interpreted with the software of iCycler iQ or iQ5 instruments The results are interpr
16. several MULTIPRIME tests are simultaneously carried out the first well should be loaded with the tube analyzed in the maximum number of channels Note 2 Do not place tubes that already passed amplification run in the rotor iteratively It is acceptable to leave some rotor wells unloaded Note 3 If tests for detection of human papillomavirus HPV DNA and different tests for detection of STI with PCR kits manufactured by CRIE are simultaneously conducted it is necessary to create the second page in the table of samples In this page all samples tested for HPV should be defined while the other samples should have None type This is important for data analysis 01 02 11 20 03 11 Page 24 of 48 Analysis of result obtained with Rotor Gene 3000 Rotor Gene 6000 or Rotor Gen Q instruments Hereinafter the terms specific for different instruments are listed in the following order for the Rotor Gene 3000 instrument for the Rotor Gene 6000 or Rotor Gene Q If terms for different instruments coincide only one term is shown If PCR kits for detection of a single microorganism are used the fluorescence signal is detected in two channels the amplification product of the DNA fragment of the specific microorganism is detected in the FAM Green channel the amplification product of the Internal Control DNA is detected in the JOE Yellow channel 1 Select the Analysis sign in the main menu select the Quantitation tab in the drop down
17. 0 2 tubes TE buffer for DNA elution colorless clear liquid 5 0 1 tube 5 0 2 tubes oae Control complex colorless clear liquid 1 0 1 tube 1 0 1 tube Internal Control FL colorless clear liquid 1 0 1 tube 1 0 1 tube Negative Control colorless clear liquid 1 2 1 tube 1 2 1 tube should be used during DNA extraction procedure if followed by PCR analysis with electrophoretic detection should be used during DNA extraction procedure if followed by PCR analysis with hybridization fluorescent detection Preparation to DNA extraction 1 Turn on the thermostat and set the temperature at 65 C 2 Lysis Solution if stored at 2 8 C should be heated to 65 C until the ice crystals disappear ice crystals can appear at the bottom of the vial 3 Take the required number of 1 5 ml disposable sterile tubes label them and place in a tube rack 4 Centrifuge the tubes with clinical samples at 1500 3000 rpm for 5 s then carefully mix using a vortex mixer and place in a tube rack VER 01 02 11 20 03 11 Page 11 of 48 5 When using the first portion of the morning specimen for analysis pretreat it to obtain the urine pellet in the transport medium as specified in item 8 1 A DNA extraction procedure 1 Add 10 ul of Internal Control complex in case of electrophoretic detection or 10 pl of Internal Control FL in case of hybridization fluorescence detection to each sterile disposable tube If di
18. 11 Page 9 of 48 spinal fluid N Extract DNA according to the manufacturer s instructions A Pretreatment of urine samples with the DNA sorb AM reagent kit for subsequent DNA extraction 1 Shake the vial with the urine 2 Transfer 1 ml of urine to a 1 5 ml sterile disposable tube using a new tip with aerosol barrier for each sample 3 Centrifuge the tube at 10 000 g 12 000 rpm at MiniSpin centrifuge Eppendorf for 5 min If urine contains excess salts resuspend only the upper layer of salt pellet in 1 ml and centrifuge again 4 Discard the supernatant using a vacuum aspirator with a trap flask without disturbing the pellet use a new tip without aerosol barrier for each sample 5 Add the transport medium to the pellet final volume 0 2 ml Mix thoroughly the content of the tubes using a vortex mixer Thus pretreated urine samples urine pellet in the transport medium can be stored at2 8 C for 24 h at lt 20 C for one week ats 68 C for a long time B Pretreatment of urine samples with the EDEM reagent kit for subsequent DNA extraction 1 Shake the vial with urine 2 Transfer 1 ml of urine to a tube with 0 5 ml of TM EDEM transport medium using a new tip with the aerosol barrier for each sample 3 Centrifuge the tubes containing TM EDEM and urine at 12 000 rpm for 5 min in a MiniSpin centrifuge Eppendorf 4 Discard the supernatant using a vacuum aspirator with a trap flask without distu
19. 3 11 Page 41 of 48 CONDUCTING REAL TIME PCR WITH THE USE OF Mx3000P OR Mx3005P INSTRUMENTS 1 Create a plate setup which shows the order of tubes in the module and the settings of fluorescence detection in the tubes in the required channel in the Plate Setup window Indicate all samples as Unknown tick the names of fluorophores to be detected click the Show Well Names button and enter names of samples If a PCR kit for detection of a single microorganism is used enable detection in FAM and JOE channels If a MULTIPRIME PCR kit is use enable detection in FAM JOE ROX and Cy5 channels 2 Assign execution of the Amplisens 1 Mx program To do this select or create the program in the Thermal Profile Setup module Save the file with the specified program and the required plate setup and click the Run button for running Table 16 AmpliSens 1 program Step Temperature C Time Cycles Segment 1 95 15 min 1 95 5s Segment 2 60 20s 5 72 15s 95 5s 30s Segment 3 60 Acquiring 40 72 15s Acquiring fluorescent signal is enabled at 60 C of Segment 3 in the FAM JOE ROX and Cy5 channels with AmpliSens PCR kits Therefore any combination of tests including the tests for identifying human papillomaviruses HPV HCR can be carried out simultaneously in the same PCR instrument AmpliSens 1 is a general program for conducting tests for detection of STIs 3 Transfer the reaction tubes into the we
20. 7 Add 300 pl of Washing Solution 1 to each tube Vortex until the sorbent is completely resuspended 8 Repeat step 6 9 Add 500 yl of Washing Solution 2 to each tube Vortex until sorbent is completely resuspended 10 Centrifuge at 10 000 rpm for 30 s Discard the supernatant using a vacuum aspirator Use a new tip for every tube 11 Repeat steps 9 10 Remove the supernatant entirely 12 Incubate all tubes with open caps at 65 C for 5 10 min 13 Add 50 yl of TE buffer for DNA elution Mix the contents of the tubes on a vortex mixer Incubate the tubes at 65 C for 5 min vortex occasionally while incubating 14 Centrifuge tubes at 12 000 rpm for 1 min The supernatant contains purified DNA and is ready for PCR amplification The purified DNA can be stored at 2 8 C for 1 week at lt 16 C for 1 year C DNA extraction by express method with the EDEM reagent kit Reagent kit for Extraction of DNA by Express Method EDEM is intended for the treatment of different types of clinical materials urogenital oropharyngeal and conjunctival swabs erosive ulcerative lesions of skin and mucous membranes and first portions of human urine samples with subsequent tests for the presence of STIs and other reproductive tract infections by using hybridization fluorescence detection and PCR kits manufactured by CRIE including the MULTIPRIME series kits The reagent kit EDEM is intended for qualitative PCR analysis and primary screeni
21. A fragments of specific microorganisms the fluorescence curve does not cross the threshold line whereas the Ct value for the Internal Control is detected in the appropriate channel and it is less than the boundary Ct value specified The result is invalid if none Ct value is detected in the channels assigned for detection of amplification products of DNA fragments of specific microorganisms whereas the Ct value in the channel for detection of the Internal Control amplification product is either absent or greater than the specified boundary Ct value Repeat the PCR assay for such samples Boundary Ct values are specified in the mportant Product Information Bulletin enclosed in the PCR kit and in the chapter Conducting Real Time PCR with the Use of Different Instruments of this Guidelines manual The results of analysis are considered reliable only if the results of both Positive and Negative Controls of amplification as well as Negative Control of extraction are correct Table 4 Table 4 Results for controls Control Stage for control Ctchannels FAM JOE Ctchannel ROX Interpretation C DNA extraction Neg Pos lt boundary Ct OK value NCA Amplification Ne Ne OK C Amplification Pos lt boundary Ct value Pos Ee Ct OK 10 TROUBLESHOOTING A If PCR kits for detection of a single microorganism are used Results of analysis are not taken into account in the following cases 1 If the Ct val
22. AM JOE on DNA extraction N A absent pla VRBES NCA PCR N A absent N A absent Detected Ct value is less C PCR than the specified Defects Callies 33 boundary Ct value 01 02 11 20 03 11 Page 44 of 48 Table 18 Boundary Ct values for positive control C in FAM channel PCR kit Ct Chlamydia trachomatis HSV I Il CMV Candida albicans Neisseria gonorrhoeae screen Neisseria gonorrhoeae test Neisseria gonorrhoeae 18 and 2 reaction Mycoplasma genitalium Trichomonas vaginalis 36 Treponema pallidum Ureaplasma species 33 Mycoplasma hominis Gardnerella vaginalis If MULTIPRIME PCR kits are used Fluorescence curves are analyzed in all channels used for detection For each specific microorganism the result of its DNA fragment amplification is detected in the specific channel defined in the PCR kit Instruction Manual and in Table 20 FAM or JOE or ROX channels The result of IC DNA amplification is obtained in the Cy5 channel if a PCR kit for detection of three microorganisms is used or in the ROX channel if a PCR kit for detection of two microorganisms duplex is used Interpretation of results is based on the presence or absence of Ct values in the channels in accordance with the channel assignment detection of the specific microorganism DNA or IC DNA specified in Table 3 1 Data analysis of IC DNA amplification 1 1 Select data in the req
23. Curve Fit mode is activated set by default 1 2Set the threshold line at a level of 10 20 of the maximum fluorescence level obtained for the Positive Control C in the last amplification cycle the fluorescence level for the Positive Control is considered as equal to the nearest digital scale mark The fluorescence curve for the Positive Control should contain a segment of a typical exponential growth of fluorescence The threshold line can also be set by default if it fits in this range In order to select Positive Control curve or any other object use the Display Wells Select Wells button or the point tab cursor at a desired curve and double click iCycler iQ instrument To set the threshold line level move it using the left mouse button or select the Baseline Threshold menu in the drop down menu which appears after clicking the right mouse button at window with fluorescence curves then select the User Define option and insert the required value in the Threshold Position text field The results grid will be displayed after clicking the Results button iCycler iQ instrument To set the threshold line level either move it using the left mouse button or select User Defined option enter the required value in the Threshold Position text field and click the Recalculate Threshold Cycles button 01 02 11 20 03 11 Page 38 of 48 Note The selected threshold level can be used for data analysis in further runs performed with the sa
24. For in Vitro Diagnostic Use For Professional Use Only GUIDELINES Real Time PCR Detection of STIs and Other Reproductive Tract Infections Federal State Institution of Science Central Research Institute of Epidemiology 3A Novogireevskaya Street Moscow 111123 Russia TABLE OF CONTENTS TINTENDED US ER 5 2 PRINCIPLE OF POR DE TEC DION tette tote tr ertet e te be edo Serb ee bte Re Patos 5 SS GCGONTEN 22249 edle ese be iPods cat de sid Aces etes a be rhe epe ee add he 5 4 ADDITIONAL REQUIREMENTS eee 7 S GBENEISAL PRECAUTION S cost erieose peer E etx ehe PEE est vines ERE EHE EE etx ep Egbe x e Ey oeeie 7 6 SAMPLING AND HANDLING icto a HH MN ON IU UE 8 7 WORKING CONDITIONS ettet t EUER AUR tdt 9 8 PROTOCOL 2 tela it eae situm Indo Buen adir nuire tetuer d elec 9 A Pretreatment of urine samples with the DNA sorb AM reagent kit for subsequent proxies mec M 10 B Pretreatment of urine samples with the EDEM reagent kit for subsequent DNA Tale el e e REN ROTEN 10 BI DNATEXTISAC DIO o etos heec able cole baeo debe ee a cabled epit eb ntact nte B eB 11 A DNA extraction with DNA sorb AM reagent kit 11 B DNA extraction with DNA sorb B reagent kit 13 C DNA extraction by express method with the EDEM reagent kit 14 9 2 REAL TIME PGR ideis pope rea ea e aee o ipM em QUE pO S 16 A Preparing tubes Tor PCR asses sese cesse eee rara ront natia ra rit rarae 16 Sanlel
25. Kaat EN eoe LZ 8 8 a 8 E jne Subtracted Curve Fit RFU PCR Base L b fi 88 e Ee PEE Cr NA 2642 SemgleType Identifier on The results for negative controls C and NCA are negative the Ct value detected for C in the Cy5 channel detection of IC is less than 36 The result for positive control C is positive Ct values do not exceed the boundary Ct values in all channels The results for controls correspond to the specified Ct values The results for test samples are valid ES Si Ct values less than 36 are detected for all samples except for No 5 in the Cy5 channel Sample No 2 shows the presence of DNA of microorganisms detected in the FAM channel Chlamydia trachomatis in here as well as DNA of microorganisms detected in the HEX channel Ureaplasma spp in here Sample No 3 shows the presence of DNA of microorganisms detected in HEX and ROX channels Ureaplasma spp and Mycoplasma hominis respectively in here Sample No 4 shows the presence of DNA of the microorganism detected in the HEX channel Ureaplasma spp in here Sample No 5 shows an invalid result that is Ct values are absent in all channels None of the specific microorganisms was found in samples Nos 1 5 and 6 01 02 11 20 0
26. ONDUCTING REAL TIME PCR WITH THE USE OF Rotor Gene 3000 Rotor Gene 6000 and Rotor Gene Q INSTRUMENTS When working with the Rotor Gene 3000 instrument use the Rotor Gene 6 program version 6 1 or higher When working with the Rotor Gene 6000 or Rotor Gene Q instruments use the Rotor Gene 6000 program version 1 7 build 67 or higher A Creating a template Hereinafter the terms specific for different instruments are listed in the following order for the Rotor Gene 3000 instrument for the Rotor Gene 6000 or Rotor Gene Q If terms for different instruments coincide only one term is shown 1 In the New Run window select the Advanced mode Select any template for example Dual Labeled Probe Hydrolysis probes for editing and click the New button Select 36 Well Rotor in the next window Tick the No Domed Tubes Locking ring attached line 2 Set the reaction mixture volume Reaction Volume uL 30 for Rotor Gene 3000 25 for Rotor Gene 6000 Tick the 15 uL oil layer volume box to activate this option 3 In the Edit profile window set the AmpliSens 1 amplification program Click OK when finished AmpliSens 1 program Step Temperature C Time Cycles Hold 95 15 min 1 95 5s Cycling 60 20s 5 72 15s 95 5s S 20s Cycling 2 60 Acquiring 40 72 15s Acquiring fluorescent signal is detected at 60 C of stage Cycling 2 Acquiring to Cycling A in the FAM Green JOE Yellow ROX Or
27. RIME FEP AmpliSens C trachomatis Ureaplasma M genitalium MULTIPRIME FRT Chlamydia trachomatis Ureaplasma spp Mycoplasma hominis AmpliSens C trachomatis Ureaplasma M hominis MULTIPRIME FEP AmpliSens C trachomatis Ureaplasma M hominis MULTIPRIME FRT Neisseria gonorrhoeae Chlamydia trachomatis Mycoplasma genitalium Trichomonas vaginalis AmpliSens N gonorrhoeae C trachomatis M genitalium T vaginalis MULTIPRIME FEP AmpliSens N gonorrhoeae C trachomatis M genitalium T vaginalis MULTIPRIME FRT Chlamydia trachomatis Ureaplasma spp Mycoplasma genitalium Mycoplasma hominis AmpliSens C trachomatis Ureaplasma M genitalium M hominis MULTIPRIME FEP AmpliSens C trachomatis Ureaplasma M genitalium M hominis MULTIPRIME FRT Neisseria gonorrhoeae Chlamydia trachomatis Mycoplasma genitalium HSV I HSV II AmpliSens N gonorrhoeae C trachomatis M genitalium MULTIPRIME FEP AmpliSens N gonorrhoeae C trachomatis M genitalium MULTIPRIME FRT AmpliSens HSV typing FEP AmpliSens HSV typing FRT Ureaplasma parvum Ureaplasma urealyticum AmpliSens U parvum U urealyticum FEP AmpliSens U parvum U urealyticum FRT detection and differentiation Trichomonas vaginalis Neisseria gonorrhoeae AmpliSens T vaginalis N gonorrhoeae MULTIPRIME FEP AmpliSens T vaginalis N gonorrhoeae MULTIPRIME FRT Chlamydia trachomatis
28. Steps 3 and 4 are carried out in both variants 3 Add 10 ul of DNA obtained from clinical or control samples at the DNA extraction stage into the prepared tubes using tips with aerosol barrier 4 Carry out the control amplification reactions NCA Add 10 yl of DNA buffer to the tube labeled NCA Negative Control of Amplification 01 02 11 20 03 11 Page 16 of 48 C Add 10 yl of Positive Control complex to the tube labeled C Positive Control of Amplification Add 10 pl of a sample extracted from the Negative Control to the tube labeled C Negative Control of Extraction B Amplification 1 Create a temperature profile in your PCR instrument as follows Table 1 AmpliSens 1 program Rotor type Instruments Plate type Instruments Step Temperature C Time Cycles Temperature C Time Cycles 1 95 15 min 1 95 15 min 1 95 5s 95 5s 2 60 20s 5 60 20s 5 72 15s 72 15s 95 5s 95 5s 20s 30s 3 60 fluorescent 40 60 fluorescent 40 signal detection signal detection 72 15s 72 15s The instrument programming is described in detail below in chapter Conducting Real Time PCR with the Use of Different Instruments of this Guidelines manual 2 Insert tubes into the reaction module of the instrument 3 Run the amplification program with fluorescence detection 4 Analyze results after the amplification program is completed 9 DATA ANALYSIS The analysis of r
29. Tela oW ME p EET 17 S DATACANAEYSIS 2 4 sao icq th cat oto ceat iro stantia von ste A tr toS E I te Ot die tits 17 A If PCR kits for detection of a single microorganism are used ss 18 B If MULTIPRIME PCR kits are Used sss eee 18 10 TROUBLESHOOTING jx cease ZN Za N SR Z EA ee erat ne rene 20 A If PCR kits for detection of a single microorganism are used ss 20 B If MULTIPRIME PCR kits are Used sss eee 21 CONDUCTING REAL TIME PCR WITH THE USE OF DIFFERENT INSTRUMENTS 22 CONDUCTING REAL TIME PCR WITH THE USE OF Rotor Gene 3000 Rotor Gene 6000 and Rotor Gene Q INSTRUMENTS 22 CONDUCTING REAL TIME PCR WITH THE USE OF iCycler iQ or iQ5 INSTRUMENTS32 CONDUCTING REAL TIME PCR WITH THE USE OF Mx3000P OR Mx3005P INSTRUMENTS cu est e aean HR RM IR AS DE RA ue 2 42 01 02 11 20 03 11 Page 2 of 48 The list of reagent kits AmpliSens manufactured by CRIE for detection of STIs and other reproductive tract infections by the polymerase chain reaction PCR with hybridization fluorescence detection Detectable microorganisms infectious agents Reagents kits Chlamydia trachomatis Bacterial infections AmpliSens Chlamydia trachomatis FEP AmpliSens Chlamydia trachomatis FRT MULTIPRIME series kits Neisseria gonorrhoeae Treponema pallidum AmpliSens Neisseria gonorrhoeae screen FEP AmpliSens Neisseria gonorrhoeae screen FRT MULTIPRIME series ki
30. Ureaplasma spp U parvum U urealyticum DNA conjunctival secretion as well as rectal and oropharyngeal swabs are used for detection of Chlamydia trachomatis and Neisseria gonorrhoeae DNA urogenital rectal and oropharyngeal swabs as well as exudate of blisters and erosive ulcerative lesions of skin and mucous membranes are used for detection of HSV I II and Treponema pallidum DNA Whole blood cerebrospinal fluid and conjunctival secretion are used for detection of HSV I II DNA as well urogenital swabs urine saliva and whole blood are used for detection of CMV DNA urogenital and oropharyngeal swabs and urine are used for detection of Candida albicans C glabrata and C kruzei DNA vaginal swabs are used for detection of Gardnerella vaginalis DNA The following transport media manufactured by CRIE are recommended for transportation and storage of clinical material e Transport Medium with Mucolytic Agent REF 952 CE e Transport Medium for Swabs 956 CE 987 CE If DNA is extracted with EDEM Reagent Kit manufactured by CRIE N Transport Medium TM EDEM 1533 CE is recommended for pretreatment of urine samples 7 WORKING CONDITIONS Reagents kits should be used at 18 25 C 8 PROTOCOL It is recommended to use the following nucleic acid extraction kits s DNA sorb AM K1 12 100 CE K1 12 50 CE s DNA sorb B K1 2 100 CE K1 2 50 CE for prostate secretion whole blood and 01 02 11 20 03
31. ange Cy5 Red and Crimson channels A Another program 60 45 RG can be exceptionally applied for PCR kits variant FRT wax AmpliSens 1 is a general program for conducting tests for detection STIs with AmpliSens PCR kits Therefore any combination of tests including tests for identifying human papillomaviruses HPV HCR can be carried out simultaneously in the same instrument layer is used It allows the run time to be to reduced by 10 min To do this create a new template and enter the 60 45 RG program in the Edit Profile window 01 02 11 20 03 11 Page 22 of 48 60 45 RG program Step Temperature C Time Cycles Hold 95 5 min 1 95 5s Cycling 60 20s 5 72 15s 95 5s 20s Cycling 2 60 Acquiring 40 72 15s Acquiring fluorescent signal is enabled as described for AmpliSens 1 program 4 Adjust the fluorescence channel sensitivity In the Channel Setup window select the Calibrate Gain Optimisation button In the opened Auto Gain Calibration Setup window click the Calibrate Acquiring Optimise Acquiring button For the FAM Green channel enter 5 in the Min Reading line and 10 in the Max Reading line For JOE Yellow ROX Orange Cy5 Red and Crimson channels enter 4 in the Min Reading line and 8 in the Max Reading line In the Tube position column specify the number of the test tube as 1 which means automatic selection of the gain parameter Tick the Perform Calibration Before 1
32. boundary Ct values specified The results for controls correspond to the boundary Ct values specified The results for test samples are valid Sample No 10 B3 shows the presence of a specific microorganism DNA of the specific microorganism is not found in samples Nos 9 and 11 15 01 02 11 20 03 11 Page 37 of 48 If MULTIPRIME PCR kits are used Fluorescence signal is detected in all channels enabled for detection The product of amplification of the analyzed microorganism DNA is detected in the channel specified in Table 3 FAM HEX JOE fluorophore or ROX channels The product of amplification of the IC DNA is detected in the Cy5 channel if a PCR kits for detection of three microorganisms is used or in the ROX channel if a PCR kit for detection of two microorganisms duplex is used The interpretation of results is based on data obtained from each channel assigned for detection of analyzed microorganisms as well as for detection of Internal Control in accordance with Table 3 1 Data analysis of the IC DNA amplification 1 1Select data in the channel assigned for detection of IC Cy5 channel if PCR kit for detection of three microorganisms is used for iCycler iQ instrument select the Cy5 635 sign in the Select a Reporter window or ROX channel if two microorganisms duplex are tested for the iCycler iQ instrument select the ROX 575 sign in the Select a Reporter window Make sure that the PCR Base Line Subtracted
33. detected in the results grid in the channel assigned for detection of amplified DNA fragment of this microorganism the fluorescence curve does not cross the threshold line whereas the Ct value detected in the channel assigned for IC DNA is less than 36 Cy5 or ROX channel for tests of the first or second group respectively The result is invalid if the Ct of the sample is not detected in all channels assigned for detection of the amplified DNA fragment of specific microorganisms whereas the Ct value in the channel assigned for the IC DNA is either absent or greater than 36 Repeat the PCR test for such samples 4 For automatic analysis of results the AmpliSens PCR kit gt Results Matrix program supplied by the manufacturer can be used Obtained data should be analyzed as described in items 1 and 2 Ct values should be copied from the results grid in the 01 02 11 20 03 11 Page 39 of 48 clipboard and entered in the corresponding column of the program for automatic analysis of results 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 see Table 14 and 15 Table 14 Results for controls Ct value Channel for detection Channel for Contos stage for control of specific detection of IC DNA microorganism DNA amplification amplification Cy5 or ROX c
34. ect the Quantitation tab in the drop down menu and then select the required channel 3 2 Select window of normalized curves in the required channel 3 3 Make sure that the Dynamic tube button is activated set by default The Slope Correct button should be turned off or on as specified in Table 8 on for Crimson channel off for other channels Activate the More Settings Outlier Removal button and in the text field enter value specified in Table 8 3 4 In the CT Calculation menu set Threshold 0 1 3 5 Ct values of each sample in the required channel will appear in the results grid Quant Results window For convenient interpretation of results we recommend that the Ct value column is copied and entered into the corresponding column in Excel Table 8 Parameters of result analysis for MULTIPRIME PCR kit Detection channel Threshold More Settings Outlier Slope Correct Removal FAM Green 0 1 0 off JOE Yellow 0 1 5 off ROX Orange 0 1 5 off Crimson if used 0 1 5 off Cy5 Red 0 07 5 on 4 Principle of interpretation The microorganism DNA is found in a sample if its Ct value is detected in the results grid in the channel assigned for detection of the given microorganism The fluorescence curve should cross the threshold line in the typical exponential growth phase The microorganism DNA is not found in a sample if its Ct value is not detected in the results grid in the channel assign
35. ed for detection of this microorganism the 01 02 11 20 03 11 Page 28 of 48 fluorescence curve does not cross the threshold line whereas the Ct value in the channel assigned for detection of the internal control amplification product Cy5 Red channel for group 1 PCR kits or ROX Orange channel for group 2 PCR kits is detected and less than 33 The result is invalid if the Ct value of a sample is absent in all channels assigned for detection of specific microorganisms whereas the Ct value detected in the channel assigned for the internal control amplification product is either absent or greater than 33 Repeat the PCR test for such samples 5 For automatic analysis of results the AmpliSens PCR kit gt Results Matrix program supplied by the manufacturer can be used The obtained data should be analyzed as described in items 1 and 2 Ct values should be copied from the results grid to the clipboard and entered in the corresponding column of the program for automatic analysis of results 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 Tables 9 10 Table 9 Results for controls If PCR kit for detection of 3 or 4 microorganisms is used group 1 Ct in channels FAM Green Ct in Cy5 Red ch l Control Stage for control JOE Yellow br E ROX Orange and Crimson if required C DNA e
36. ek at 2 8 C or for one year at lt 16 C it is necessary to vortex and recentrifuge the tube contents according to item 3 if PCR analysis of DNA samples is performed once again EDEM reagent kit repeat DNA extraction procedure To do this 100 ul of clinical material in TM EDEM transport medium should be treated with the DNA sorb AM reagent kit according to its instruction manual In case of invalid or equivocal result of PCR analysis obtained with the use of 8 2 REAL TIME PCR A Preparing tubes for PCR Variant FRT Total reaction volume is 30 pl the volume of DNA sample is 10 pl 1 Prepare the required number of the tubes with PCR mix 1 FL and wax for amplification of DNA from clinical and control samples 2 Add 10 pl of PCR mix 2 FL red to the surface of wax layer of each tube so that it does not fall under the wax and mix with PCR mix 1 FL Variant FRT 100 F Total reaction volume is 25 pl the volume of DNA sample is 10 pl 1 Thaw the PCR mix 2 FRT tube Vortex the tubes with PCR mix 1 FL PCR mix 2 FRT and polymerase TaqF then centrifuge briefly Collect the required number of the tubes strips for amplification of DNA obtained from clinical and control samples 2 ForN reactions including 2 controls mix in a new tube 10 N 1 ul of PCR mix 1 FL 5 0 N 1 ul of PCR mix 2 FRT 0 5 N 1 ul of polymerase TaqF Vortex the tube then centrifuge briefly Transfer 15 ul of the prepared mixture to each tube
37. el for the Positive Control is considered as equal to the nearest digital scale mark The fluorescence curve for the Positive Control should contain a segment of the typical exponential growth of fluorescence The threshold line can also be set by default if it fits in this range To obtain the overall results grid activate data in all channels in the Results Amplification Plots window and select the Text Report option in the Area to analyze menu list The results grid can be exported to Excel to do this press the right mouse button and select the Export Text Report to Excel option from the menu displayed Principle of interpretation The microorganism DNA is found in a sample if its Ct value is detected in the results grid in the channel assigned for detection of amplified DNA fragment of this microorganism Moreover the fluorescence curve should cross the threshold line at the typical exponential growth phase The microorganism DNA is not found in a sample if the Ct value is not detected in the results grid in the channel assigned for detection of an amplified DNA fragment of this microorganism the fluorescence curve does not cross the threshold line whereas the Ct value detected in the channel assigned for the IC DNA is less than 36 Cy5 or ROX channel for tests of the first or second group respectively The result is invalid if the Ct value of the sample is not detected in all channels assigned for detection of amplified DNA
38. ent predrying 9 Add 100 yl of TE buffer for DNA elution using tip with aerosol barrier Vortex until the sorbent is completely resuspended Incubate tubes at 65 C for 5 min The elution volume can be adjusted to 150 ul 10 Centrifuge tubes at 12 000 rpm for 1 min The supernatant contains purified DNA and is ready for PCR amplification The purified DNA can be stored 8t2 8 C for 1 week 8t 16 C for 1 year If samples are analyzed once again mix the content of the tubes on a vortex mixer and repeat centrifugation in accordance with item 10 01 02 11 20 03 11 Page 12 of 48 B DNA extraction with DNA sorb B reagent kit The principle of extraction with the DNA sorb B reagent kit corresponds to the principle specified above for the DNA sorb AM reagent kit DNA sorb B nucleic acid extraction kit variant 50 or 100 includes Variant 50 Variant 100 Reagent Description Volume A i Volume A ml moun mi moun Lysis Solution colorless clear liquid 15 1 vial 30 1 vial Washing Solution 1 colorless clear liquid 15 1 vial 30 1 vial Washing Solution 2 colorless clear liquid 50 1 vial 100 1 vial Universal Sorbent white suspension 1 25 1 tube 1 25 2 tubes TE DUTTGE TOURING colorless clear liquid 50 1tube 50 2tubes elution Preparation to DNA extraction 1 Turn on the thermostat and set temperature at 65 C 2 Lysis Solution and Washing Solution 1 if stored at 2 8 C should be
39. esults was performed by the software of the instrument used The fluorescent signal intensity is detected in the channels assigned for detection of amplification products of DNA fragments of specific microorganisms and in the channel assigned for detection of amplification product of IC DNA The results are interpreted by the crossing or not crossing of the fluorescence curve with the threshold line set at a specific level and are shown as the presence or absence of Ct cycle threshold in the results grid To analyze results in each channel set the threshold line at the required level and activate the required options in accordance with the instrument user manual and the chapter Conducting Real Time PCR with the Use of Different Instruments of this Guidelines manual For example Rotor Gene 3000 Rotor Gene 6000 Rotor Gene Q or equivalent For example iCycler iQ iQ5 Mx3000P Mx3000 DT 96 or equivalent 01 02 11 20 03 11 Page 17 of 48 A If PCR kits for detection of a single microorganism are used The fluorescent signal intensity is detected in two channels e The signal from the amplification product of DNA of the analyzed microorganism is detected in the FAM channel e The signal from the Internal Control amplification product is detected in the JOE channel Interpretation of results Principle of interpretation e The microorganism DNA is detected in a sample if its Ct value is detected in the results grid
40. eted by the crossing or not crossing of the fluorescence curve with the threshold line set at a certain level and it is shown as the presence or absence of a Ct cycle threshold value in the results grid If PCR kits for detection of a single microorganism DNA are used fluorescence signal is detected in two channels amplification product of a DNA fragment of a specific microorganism is detected in the FAM channel and the amplification product of the Internal Control DNA is detected in the HEX channel T Ts Data analysis of the specific microorganism amplification Select data in the FAM channel iCycler iQ or activate the FAM 490 sign in the Select a Reporter window iCycler iQ Make sure that the PCR Base Line Subtracted Curve Fit mode is activated set by default Set the threshold line at the level of 10 20 of maximum level of fluorescence obtained for the Positive Control C in the last amplification cycle the fluorescence level for the Positive Control is considered as equal to the nearest digital scale mark The fluorescence curve for Positive Control should represent typical exponential growth of fluorescence The threshold line can also be set at default if it fits in this range In order to select Positive Control graph or any other object use the Display Wells Select Wells button or point tab cursor on a desired graph and double click iCycler iQ5 instrument To set the threshold line l
41. evel move it using the left mouse button or select the Baseline Threshold menu in the drop down menu which appears after clicking the right mouse button in the fluorescence graph window then select the User Define option and insert required value in the Threshold Position text field Results grid will be displayed after clicking the Results button iCycler iQ instrument To set the threshold line level either move it using the left mouse button or select the User Defined option insert the required value in the Threshold Position text field and press the Recalculate Threshold Cycles button Note Selected threshold level can be used for data analysis in further runs performed with the same PCR kit and conducted on the same Instrument in case the new calibration was not performed 01 02 11 20 03 11 Page 34 of 48 2 Data analysis of the IC amplification Select data in HEX channel iCycler iQ or activate HEX 530 sign in the Select a Reporter window iCycler iQ Select the PCR Base Line Subtracted Curve Fit mode set by default Set the threshold line at the level of 10 20 of the maximum fluorescence intensity recorded for the Positive Control C in the last amplification cycle The fluorescence curve for Positive Control C should contain a section of typical exponential fluorescence growth The threshold line can also be set at default if it fits in this range Note The selected threshold level can be used for IC data
42. fferent detection methods are used within one test run both internal controls can be added 10 ul of each 2 Thoroughly resuspend Universal Sorbent on a vortex mixer Add 20 ul of Universal Sorbent and 300 pl of Lysis Solution to each test tube using tips with aerosol barrier If the number of clinical samples exceeds 50 it is recommended that the whole volume of sorbent and IC are transferred to the tube with Lysis Solution 2 ml of Universal Sorbent and 1 ml of IC per 30 ml of Lysis Solution Thoroughly stir this suspension and transfer 330 ul of it to the tubes The prepared mixture can be stored at room temperature for 2 days Stir well before use 3 Add 100 yl of a sample to the tube using a tip with aerosol barrier Add 100 pl of Negative Control to the tube with Negative Control of Extraction C 4 Tightly close the caps thoroughly mix the tubes on a vortex mixer and incubate them at 65 C for 5 min in a thermostat After incubation mix the contents of the tubes on a vortex once again and incubate at room temperature for another 2 min 5 Centrifuge all tubes at 10 000 rpm for 30 s and carefully remove the supernatant from each tube with a vacuum aspirator without disturbing the pellet Use a new tip without aerosol barrier for every tube 6 Add 1 ml of Washing Buffer into each tube Vortex until the sorbent is completely resuspended 7 Repeat step 5 8 Incubate all tubes with open caps at 65 C for 5 10 min for sorb
43. fore programming the iCycler iQ instrument make sure that the dinamicwf tmo protocol is set as follows standard Viewing Protocol dynamicwf tmo Selected Plate Setup T Selected Plate Setup may be MAX 00 30 95 0 al MAX 00 30 95 0 01 02 11 20 03 11 Page 32 of 48 2 Set the plate setup that is tubes order in the reaction chamber and the detection of fluorescent signal for all tubes in the required channels in the Edit Plate Setup window of the Workshop module If a PCR kit for detection of a single microorganism is used activate the FAM and HEX detection channels If a MULTIPRIME PCR kit is used activate FAM HEX ROX and Cy5 channels Save the plate setup Click the Run with selected protocol button 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 To turn on the second fluorophore use sign Set Sample Volume as 30 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 Press the Run with selected protocol button to save and activate the created plate setup 3 Proceed to item 4 if a PCR kit variant FRT hot start is ensured by using a wax layer is used If a PCR kit variant FRT 100 F TaqF polymerase is applied is used insert the tubes into the
44. glabrata C krusei 36 36 36 U parvum U Urealyticum 36 36 36 HSV typing 36 33 36 T vaginalis N gonorrhoeae 36 36 36 HSV CMV 36 36 36 01 02 11 20 03 11 Page 47 of 48 Example of results obtained with the Mx3000P instrument for a MULTIPRIME PCR kit Results obtained with AmpliSens C trachomatis Ureaplasma M genitalium MULTIPRIME FRT PCR kit ee gt en Z G to Ose e fam je jn Lc r2 2874 S E k L S or ee NCA oe fa Ne cr tian fra Ie The results for negative controls C and NCA are negative the Ct value detected for C in the Cy5 channel detection of IC is less than 36 The result for positive control C is positive Ct values do not exceed the boundary Ct values in all channels The results for controls correspond to the specified Ct values The results for test samples are valid Samples Nos 2 3 and 6 show the presence of DNA of the microorganisms detected in the JOE channel Ureaplasma spp in here Sample No 3 shows the presence of DNA of microorganisms detected in the ROX channel Mycoplasma hominis in here Sample No 5 shows the presence of DNA of the microorganisms detected in the FAM channel Chlamydia trachomatis in here and DNA of the microorganisms detected in the JOE channel Ureaplasma spp in here Samples Nos 1 and 4 shows the absence of DNA of analyzed microorganisms The Ct value in the Cy5 channel IC detection does not exceed 36 01 02 11 20 03 11 Page 48
45. he Quantitation tab in the drop down menu after which select the required channel for example select Cycling A FAM Cycling A Green for the FAM Green channel Cycling A JOE Cycling A Yellow for the JOE Yellow channel etc Data analysis of IC DNA amplification N P Group 1 PCR kits Select normalized curves in the Cy5 Red channel Make sure that Dynamic tube button is activated set by default Activate the Slope Correct button Turn on the More Settings Outlier Removal button and enter 5 5 in the text field In the CT Calculation menu set Threshold 0 07 Ct values of each sample in Cy5 Red channel will appear in the results grid Quant Results Cycling A Cy5 Quant Results Quant Results Cycling A Red 01 02 11 20 03 11 Page 27 of 48 2 2 Group 2 PCR kits duplex Select normalized curves in the ROX Orange channel Make sure that the Dynamic tube button is activated set by default Activate the Slope Correct button Turn on the More Settings Outlier Removal button and enter 5 5 in the text field In the CT Calculation menu set Threshold 0 1 Ct values of each sample in ROX Orange channel will appear in the results grid Quant Results Cycling A ROX Quant Results Quant Results Cycling A Orange 3 Data analysis of the microorganism DNA amplification Results should be consecutively analyzed as described below in each channel used 3 Select the Analysis sign in the main menu sel
46. heated to 65 C until ice crystals disappear 3 Take the required number of 1 5 ml sterile disposable tubes label them and place in a tube rack 4 Centrifuge the tubes with clinical samples at 1500 3000 rpm for 5 s then carefully mix using a vortex mixer and place in a tube rack DNA extraction procedure 1 Add 10 ul of Internal Control complex in case of electrophoretic detection or 10 u of Internal Control FL in case of hybridization fluorescence detection to each sterile disposable tube If different detection methods are used within one test run both internal controls can be added 10 yl of each 2 Add 300 ul of Lysis Solution to each prepared tube 3 Add 100 yl of a sample to the tubes with Internal Control and Lysis Solution Add 100 pl of Negative Control to the tube labeled C 4 Vortex the tubes and then incubate at 65 C for 5 min Centrifuge all tubes at 5 000 rpm for 5 s If a sample hasn t dissolved completely centrifuge the tube at 12000 rpm for 5 min transfer the supernatant to a new tube and use for DNA extraction 5 Thoroughly resuspend Universal Sorbent on vortex mixer into each test tube using a 01 02 11 20 03 11 Page 13 of 48 new tip Vortex the tubes then place them in a tube rack for 2 min Vortex once again and place the tubes for 5 min in a tube rack 6 Centrifuge all tubes at 5000 rpm for 30 s Discard the supernatant using a vacuum aspirator Use a new tip for every tube
47. is Ae ae Neisseria Chlamydia Trichomonas IC 7 BARN gonorrhoeae trachomatis vaginalis T vaginalis ira re Neisseria Chlamydia Mycoplasma IC 7 l As gonorrhoeae trachomatis genitalium M genitalium Coe Candida Candida Candida IC 7 C krusei albicans glabrata krusei PCR kit test group 2 duplex U parvum Ureaplasma Ureaplasma IC 7 7 U urealyticum parvum urealyticum HSV typing HSV II HSV IC T vaginalis Trichomonas Neisseria IC 7 7 N gonorrhoeae vaginalis gonorrhoeae HSV CMV HSV CMV IC The channel for the Cy5 5 fluorophore Crimson channel is available in Rotor Gene 6000 and Rotor Gene Q instruments Principle of interpretation 01 02 11 20 03 11 Page 19 of 48 The microorganism DNA is found in a sample if its Ct value is detected in the results grid in the channel assigned for detection of this microorganism in accordance with instruction manual to PCR kit Moreover the fluorescence curve of this sample should cross the threshold line in the typical exponential growth phase The microorganism DNA is not found in a sample if its Ct value is not detected absent in the results grid in the required channel the fluorescence curve does not cross the threshold line DNA of any analyzed microorganisms is not found in a sample if its Ct values are not detected absent in the results grid in the required channel assigned for detection of amplification products of DN
48. is correct Make sure that 15 uL oil layer volume is selected for Rotor Gene 6000 or Rotor Gene Q Proceed to the next window 5 Check the correctness of the amplification program and automatic optimization gain parameters Note If MULTIPRIME PCR kits are not used for the run unable fluorescence detection in the ROX Orange Cy5 Red and Crimson channels in the Edit Profile window FAM Green and JOE Yellow channels are activated 6 Start the program by clicking the Start button Make sure that rotor is secured and the lid is closed Enter the file name for result data and click Save 7 In the table of samples define the order of samples by entering the name and type Unknown of each sample Click Finish OK Note Rotor Gene 6000 and Rotor Gene Q instruments allow editing the table of samples before the run starts To do this select the Edit Samples Before Run Started button in the User Preferences submenu of the File menu See Note 3 8 Proceed to interpretation of results when the run is completed N When PCR run is completed the tubes should be removed from the rotor and discarded Note 1 The first tube in the rotor is used for automatic optimization of the level of signal therefore the first tube should contain the reaction mixture If several tests for detection of STIs with PCR kits manufactured by CRIE are conducted within the same run any tube containing reaction mixture can be placed in the first well of the rotor If
49. l scale mark The fluorescence curve for the Positive Control should contain a typical segment of the exponential growth of fluorescence The threshold line can also be set by default if it fits in this range To select the curve of the C sample or another sample in the Analysis Selection Setup window select the required well and shift to the Results window Note The selected threshold level can be used for interpretation of results obtained for the same pathogen with the given PCR kit 2 Data analysis of the Internal Control DNA amplification Select data in the JOE channel in the Result Amplification Plots window of the Analysis module Set the threshold line at the level of 10 20 of maximum fluorescence level recorded for the Positive Control C in the last amplification cycle the fluorescence level for the Positive Control is considered as equal to the nearest digital scale mark The fluorescence curve for the Positive Control should contain a segment of the typical exponential growth of fluorescence The threshold line can also be set by default if it fits in this range Note The selected threshold level can be used for interpretation of results of the amplified Internal Control DNA obtained with other STI tests for detection of a single microorganism performed with PCR kits manufactured by CRIE The same threshold level set for the JOE channel can be used for further runs conducted with the same PCR instrument 3
50. lls of the instrument in accordance with the specified plate setup Secure the lid 4 It is recommended that the option of lamp shutdown after the run completion is activated the box is ticked 5 When the run is completed proceed to analysis of results 6 Close the program and switch the instrument off when the work with the instrument is finished Data analysis Mx3000P and Mx3005P instruments The obtained data are interpreted with the software of Mx3000P and Mx3005P PCR instruments The result are interpreted by the crossing or not crossing of the fluorescence 01 02 11 20 03 11 Page 42 of 48 curve with the threshold line and is shown as the presence or absence of the Ct cycle threshold value in the results grid If PCR kits for detection of a single microorganism are used Fluorescence signal is detected in two channels the amplification product of a DNA fragment of a specific microorganism is detected in the FAM channel and the amplification product of the Internal Control DNA is detected in the JOE channel 1 Data analysis of the specific microorganism DNA amplification Select data in the FAM channel in the Result Amplification Plots window of the Analysis module Set the threshold line at the level of 10 20 of the maximum fluorescence level recorded for the Positive Control C in the last amplification cycle the fluorescence level for the Positive Control is considered as equal to the nearest digita
51. me PCR kit and conducted on the same Instrument if a new calibration was not performed 2 Data analysis of the specific microorganism amplification Obtained results should be consistently analyzed as described below for each channel used 2 1 Select the required channel for the iCycler iQ instrument select the sign in the Select a Reporter window in the analysis window Make sure that the PCR Base Line Subtracted Curve Fit mode is activated set by default 2 2Set the threshold line at the level of 10 20 of the maximum fluorescence level recorded for the Positive Control C in the last amplification cycle the fluorescence level for the Positive Control is considered as equal to the nearest digital scale mark The fluorescence curve for the Positive Control should represent typical exponential growth of fluorescence The threshold line can also be set by default if it fits in this range 2 3 For convenient interpretation of results we recommend that the Ct value column is copied and entered into the corresponding column in Excel 3 Interpretation of results Principle of interpretation The microorganism DNA is found in a sample if its Ct value is detected in the results grid in the channel assigned for detection of the amplified DNA fragment of this microorganism The fluorescence curve should cross the threshold line in the typical exponential growth phase The microorganism DNA is not found in a sample if Ct value is not
52. membranes urine sediment use the first portion of the morning specimen and prostate secretion The following clinical material is used 1 Women cervical vaginal and urethral swabs and urine 2 Men urethral swabs urine and prostate secretion 3 Children conjunctival secretion Clinical material should be placed into tubes with a transport medium recommended by CRIE The obtained samples except for urine can be transported and stored under the following conditions e Atthe room temperature for 48 h e At 2 8 C for two weeks e At x 20 C for the month e At s 68 C for a long time Samples placed to the Transport Medium with Mucolytic agent can be transported and stored under the following conditions e Atthe room temperature 18 25 C for 28 days e At 2 8 C for 3 months e At 20 C for a long time 01 02 11 20 03 11 Page 8 of 48 N Only one freeze thaw cycle of clinical material is allowed The obtained urine samples can be transported and stored under the following conditions e Atthe room temperature for 6 h e At 2 8 C for 24 h Transportation of clinical samples is performed in special container with cooling elements The following types of clinical material are used for microorganism DNA detection urogenital swabs urine sediment and prostate secretion are used for detection of Chlamydia trachomatis Neisseria gonorrhoeae Mycoplasma genitalium M hominis Trichomonas vaginalis
53. nels assigned for detection of amplification products of DNA fragments of specific microorganisms and in the channel 01 02 11 20 03 11 Page 18 of 48 assigned for detection of amplification product of the IC DNA Designations of channels are indicated in Table 3 and in the Instruction Manual to the PCR kit used MULTIPRIME PCR kits can be divided in two groups PCR kits for detection of three or four microorganisms group 1 and PCR kits for detection of two microorganisms duplex group 2 The signal of amplification product of IC DNA is detected in the Cy5 channel if PCR kits belonging to group 1 are used The signal of the amplification product of IC DNA is detected in the ROX channel if PCR kits belonging to group 2 duplex are used Table 3 Channels for detection of signal indicating amplification of microorganism DNA and internal control DNA fragments PCR kit test Channel for fluorophore cS group 1 FAM JOE ROX Cy5 Ean N gonorrhoeae C trachomatis Neisseria Chlamydia Mycoplasma IC Trichomonas M genitalium gonorrhoeae trachomatis genitalium vaginalis T vaginalis C trachomatis Ureaplasma Chlamydia Ureaplasma Mycoplasma IC Mycoplasma M genitalium trachomatis spp genitalium hominis M hominis ee Chlamydia Ureaplasma Mycoplasma IC M genitalium trachomatis spp genitalium pce Chlamydia Ureaplasma Mycoplasma IC _ pide trachomatis spp hominis M homin
54. ng of patients This reagents kit is not intended for quantitative PCR analysis or monitoring after treatment for these purposes DNA sorb AM reagents kit is used for DNA extraction EDEM reagent kit contains TM EDEM T Samples must be placed into tubes with TM EDEM transport medium only the Urine samples should be preliminary treated 01 02 11 20 03 11 Page 14 of 48 Clinical material obtained from a patient is transferred into TM EDEM transport medium in which it is stored and transported to a laboratory For DNA extraction an aliquot of a clinical sample is transferred into a tube with IC diluent after which it is thermally processed to destroy cell membranes viral coats and other biopolymer complexes and to ensure DNA release Insoluble components are pelleted on the tube bottom by centrifuging the supernatant containing DNA is used for PCR The internal control sample IC contained in IC diluent is isolated simultaneously with DNA from clinical material and thereby is a quality marker of laboratory analysis of clinical samples EDEM reagent kit includes Reagent Description Volume ml Quantity Transport mediumTM EDEM colorless clear liquid 0 5 100 tubes IC diluent colorless clear liquid 0 3 100 tubes PCR buffer Background colorless clear liquid 0 5 2 tubes Preparation to DNA extraction 1 Switch on the thermostat and set the temperature at 95 C 2 Prepare and place the
55. nused reagents in compliance with local authorities requirements Samples should be considered potentially infectious and handled in a biological cabinet in accordance with appropriate biosafety practices Clean and disinfect all sample or reagent spills using a disinfectant such as 0 5 sodium hypochlorite or another suitable disinfectant Avoid contact with the skin eyes and mucosa If skin eyes or mucosa contact 01 02 11 20 03 11 Page 7 of 48 immediately flush with water and seek medical attention e Material Safety Data Sheets MSDS are available on request e Use of this product should be limited to personnel trained in DNA amplification techniques The laboratory process must be one directional it should begin in the Extraction Area and then move to the Amplification and Detection Areas Do not return samples equipment and reagents to the area in which the previous step was performed Some components of this kit contain sodium azide as a preservative Do not use metal tubing for reagent transfer 6 SAMPLING AND HANDLING storage are described in the manufacturer s handbook 1 It is recommended that this handbook is read before starting work The following material is used for analysis urogenital rectal and oropharyngeal swabs T Obtaining samples of biological materials for PCR analysis transportation and conjunctival secretion exudate of blisters and erosive ulcerative lesions of skin and mucous
56. omologous 1 0 1 tube to known microorganisms and viruses and complementary to the fluorescent probe which is included in the PCR kit must be used in the extraction procedure as Negative Control of Extraction add 10 ul of Internal Control FL during the DNA extraction directly to the sample lysis mixture see DNA sorb AM K1 12 100 CE K1 12 50 CE protocol PCR kit is intended for 110 reactions including controls PCR kit variant FRT 100 F includes Volume Reagent Description ni Amount PCR mix 1 FL Solution containing primers dNTP 12 4 tube and oligonucleotide probes PCR mix 2 FRT Buffer solution containing Taq 0 3 2 tubes polymerase and Mg Polymerase TaqF Solution containing modified Taq 0 03 2 tubes polymerase Positive Control complex C Solution Containing Specie fragments 0 2 1 tube of DNA of analyzed microorganisms DNA buffer Buffer solution 0 5 1 tube Negative Control C Buffer solution 1 2 1 tube Phage Agt67 particle solution containing a cloned genetically engineered construct with an artificial Internal Control FL IC nucleotide sequence nonhomologous 1 0 1 tube to known microorganisms and viruses and complementary to the fluorescent probe which is included in the PCR kit must be used in the extraction procedure as Negative Control of Extraction add 10 ul of Internal Control FL during the DNA extraction directly to the sample lysis mixture
57. rbing the pellet use a new tip without aerosol barrier for each sample 5 Add 0 5 ml of TM EDEM to each tube with urine pellet using a new tip for each tube Close tubes tightly Mix thoroughly the content of the tubes on a vortex mixer to resuspend the pellet Centrifuge at 1500 3000 rpm for 2 3 s to spin down the drops from the walls of the tube and the cap 6 Obtained samples of urine pellet in the TM EDEM should be used for DNA extraction procedure Thus obtained urine pellet in the TM EDEM can be stored 01 02 11 20 03 11 Page 10 of 48 atthe room temperature 18 25 C for 48 h at2 8 C for 14 days at lt 20 C for a long time 8 1 DNA EXTRACTION A DNA extraction with DNA sorb AM reagent kit DNA sorb AM nucleic acid extraction kit is a reagent kit for rapid and efficient manual extraction and purification of DNA from various clinical materials Lysis solution contains a chaotropic agent guanidine chloride that lyses cells and denatures cell proteins The nucleic acids are then adsorbed on silica particles DNA extracted from clinical samples can be used for PCR diagnostic tests DNA sorb AM nucleic acid extraction kit variant 50 or 100 includes Variant 50 Variant 100 i sre Pn ME Quantity EE Quantity Lysis Solution colorless clear liquid 15 1 vial 30 1 vial Washing Buffer colorless clear liquid 50 1 vial 100 1 vial Universal Sorbent white suspension 1 0 1 tube 1
58. reen Ct in JOE Yellow s Control Stage for control channel channel Interpretation C DNA extraction Neg Detected value lt 30 OK NCA Amplification Neg Neg OK C Amplification Pos lt boundary Ct value Detected value lt 30 OK 01 02 11 20 03 11 Page 26 of 48 Boundary Ct value for C in the Green channel Table 7 PCR kit variant FRT Boundary Ct value for C in the Green channel Chlamydia trachomatis HSV I Il CMV Candida albicans 30 Neisseria gonorrhoeae screen Neisseria gonorrhoeae test Neisseria gonorrhoeae 1 and 2 reactions Mycoplasma genitalium Trichomonas vaginalis Treponema pallidum Ureaplasma species Mycoplasma hominis Gardnerella vaginalis 33 If MULTIPRIME PCR kits are used the fluorescent signal is detected in all channels enabled for detection The products of amplification of DNA of the analyzed microorganisms are detected in the channels listed in Table 20 FAM Green JOE Yellow ROX Orange or Crimson channel The product of amplification of IC DNA is detected in the Cy5 Red channel for group 1 PCR kits or in the ROX Orange channel for group 2 PCR kit duplex Interpretation of results is based on the data obtained for each channel assigned for detection of the analyzed microorganisms as well as for detection of Internal Control in accordance with Table 3 1 Select the Analysis sign in the main menu and t
59. required number of tubes with IC diluent into the tube rack and label them Spin down the drops of solution from tube walls and caps by short centrifuging at 1500 3000 rpm for 2 3 s 3 Before starting DNA extraction mix the content of tubes with clinical material in TM EDEM transport medium by vortexing and spin down the drops of material from tube walls and caps by short centrifuging at 1500 000 rpm for 2 3 s Place the prepared tubes into tube rack 4 Urine samples should be preliminary treated in accordance with item 8 1 B to obtain the urine pellet in the TM EDEM transport medium To do this the additional reagent TM EDEM transport medium 50 ml is to be used DNA extraction procedure 1 Transfer 100 ul of clinical material in the TM EDEM transport medium into the prepared tubes with IC diluent using a new tip with aerosol barrier for each sample Add 100 ul of the TM EDEM transport medium into the tube for Negative Control of Extraction C 2 Tightly close all tubes carefully mix the contents by vortexing prevent spraying and incubate in a thermostat at 95 C for 5 min N Close tightly the tubes so that they would not open during heating 3 After the end of incubation place the tubes into a desktop centrifuge and centrifuge at 01 02 11 20 03 11 Page 15 of 48 14 000 rpm for 1 min Thus obtained DNA samples are ready for PCR analysis with hybridization fluorescence detection DNA samples can be stored for one we
60. signed for detection of amplification products of DNA fragments of specific microorganisms PCR should be repeated for all samples for which Ct values in these channels were not detected 2 f a Ct value is present for the Negative Control of Extraction C and or for the Negative Control of Amplification NCA in the channels assigned for detection of amplification products of DNA fragments of specific microorganisms PCR should be repeated for all samples for which a Ct value in these channels was detected If a Ct value is detected for C and or for NCA in the channels assigned for detection of amplification product of microorganism DNA in the second run this N indicates contamination of reagents or samples In such cases the results of analysis must be considered as invalid Test analysis must be repeated and measures to detect and eliminate the source of contamination must be taken 3 If a Ct value of a sample is detected in the results grid in the FAM channel but the fluorescence curve does not have a typical exponential growth phase the curve is linear the result should not be considered as positive This may suggest incorrect setting of the threshold line or other analysis parameters If threshold level is correct as well as other analysis settings amplification should be repeated of such a sample to get correct result 01 02 11 20 03 11 Page 21 of 48 CONDUCTING REAL TIME PCR WITH THE USE OF DIFFERENT INSTRUMENTS C
61. t is Ct values are absent in all channels None of the microorganisms of interest was found in samples Nos 1 4 and 6 01 02 11 20 03 11 Page 31 of 48 CONDUCTING REAL TIME PCR WITH THE USE OF iCycler iQ or iQ5 INSTRUMENTS 1 Setthe AmpliSens 1 Table 11 general amplification and detection program Table 11 AmpliSens 1 program for iCycler iQ or iQ5 instruments Step Temperature C Time Cycle repeats 1 95 15 min 1 95 5s 2 60 20s 5 72 15s 95 5s 30s 3 60 fluorescent signal 40 detection 72 15s To do this select or create this program in the Protocol module View Protocols for iCycler iQ For iCycler iQ5 click the Run with selected Plate Setup button to start the program AmpliSens 1 general program allows simultaneously conducting any combination of tests for detection of DNA of sexually transmitted infections with PCR kits manufactured by CRIE including the tests for identifying and genotyping Human Papillomaviruses HPV HCR It is not recommended running the MULTIPRIME format test and single pathogen detection tests 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 the well factor determination and the tube kit with a special External Well Factor Solution Bio Rad should be used for start up Be
62. the real time PCR allows the detection of accumulating product without re opening the reaction tubes after the PCR run The Internal Control IC is used in the extraction procedure in order to control the extraction process of each individual sample and to identify possible reaction inhibition It is possible to automate data analysis and reduce the subjectivity in the interpretation of results Simultaneous amplification and detection of several DNA targets in a single reaction is possible Optimization ensures a high sensitivity to each DNA target The use of multiplex PCR allows a 3 4 fold increase in the efficiency of analysis without extending the instrument base Reagent kits of the MULTIPRIME series are intended for multiplex PCR analysis 3 CONTENT PCR kit variant FRT includes Reagent Description poe Amount PCR mix 1 FL ir 110 tubes ready to use single dose test Sate ee SA STE 0 01 of tubes under wax MESI H ELA OSS 0 2 ml PCR mix 2 FL red Buffer solution containing Taq 14 1 tube polymerase and Mg Positive Control complex C Solution containing specific fragments 0 2 1 tube p of DNA of analyzed microorganisms DNA buffer Buffer solution 0 5 1 tube 01 02 11 20 03 11 Page 5 of 48 Negative Control C Buffer solution 1 2 1 tube Phage Agt67 particle solution containing a cloned genetically engineered construct with an artificial Internal Control FL IC nucleotide sequence nonh
63. ts AmpliSens Treponema pallidum FEP AmpliSens Treponema pallidum FRT Mycoplasma genitalium AmpliSens Mycoplasma genitalium FEP AmpliSens Mycoplasma genitalium FRT MULTIPRIME series kits Ureaplasma parvum Ureaplasma urealyticum AmpliSens Ureaplasma spp FEP AmpliSens Ureaplasma spp FRT MULTIPRIME series kits Mycoplasma hominis AmpliSens Mycoplasma hominis FEP AmpliSens Mycoplasma hominis FRT MULTIPRIME series kits Gardnerella vaginalis AmpliSens Gardnerella vaginalis FEP AmpliSens Gardnerella vaginalis FRT MULTIPRIME series kits Virus infections herpesviruses HSV I Il CMV AmpliSens HSV I II FEP AmpliSens HSV I II FRT AmpliSens HSV typing FEP AmpliSens HSV typing FRT MULTIPRIME series kits AmpliSens CMV FEP AmpliSens CMV FRT MULTIPRIME series kits Protozoal infections Trichomonas vaginalis AmpliSens Trichomonas vaginalis FEP AmpliSens Trichomonas vaginalis FRT MULTIPRIME series kits Mycotic infections Candida albicans AmpliSens Candida albicans FEP AmpliSens Candida albicans FRT Candida albicans Candida glabrata Candida krusei MULTIPRIME series kits 01 02 11 20 03 11 Page 3 of 48 Simultaneously detectable microorganisms Reagents kits of MULTIPRIME series Chlamydia trachomatis Ureaplasma spp Mycoplasma genitalium AmpliSens C trachomatis Ureaplasma M genitalium MULTIP
64. ue of Positive Control of amplification C in the FAM channel is absent or greater than the boundary Ct value amplification should be repeated for all samples in 01 02 11 20 03 11 Page 20 of 48 which the microorganism DNA was not detected 2 If the Ct value is detected for C and or for NCA in the FAM channel PCR assay should be repeated starting from the DNA extraction stage for all samples in which the microorganism DNA was detected If a Ct value is repeatedly detected for C and or for NCA in the FAM channel it indicates contamination of reagents or samples In such cases the results of N analysis must be considered as invalid Test analysis must be repeated and measures to detect and eliminate the source of contamination must be taken If a Ct value of a sample is detected in the results grid in the FAM channel but the fluorescence curve does not have a typical exponential growth phase the curve is linear the result should not be considered as positive This may suggest incorrect setting of the threshold line or other analysis parameters If threshold level as well as other analysis settings are correct amplification of such samples should be repeated B If MULTIPRIME PCR kits are used Results of analysis are not taken into account in the following cases 1 If no signal is detected for Positive Control of Amplification C or the signal is greater than the specified boundary Ct value in more than one channel as
65. uired channel in the Analysis module of the Results Amplification Plots window the Cy5 channel if a PCR kit for detection of three microorganisms is used or the ROX channel if a PCR kit for detection of two microorganisms duplex is used 1 2 Set the threshold line at the level of 10 20 of the maximum fluorescence level recorded for the Positive Control C in the last amplification cycle the fluorescence level for the Positive Control is considered as equal to the nearest digital scale mark The fluorescence curve for the Positive Control should contain a segment of the typical exponential growth of fluorescence The threshold line can also be set by default if it fits in this range To select positive control curve C or another sample select this sample in the result 01 02 11 20 03 11 Page 45 of 48 2 2 2 4 5 list left to the Amplification Plots window and shift to the Result window To activate all analyzed samples click the Select all button in the right bottom corner below the result list Data analysis of the specific microorganism amplification The obtained results should be consistently analyzed in each channel used as described below Select the required channel in the Results Amplification Plots of the Analysis module Set the threshold line at the level of 10 20 of the maximum fluorescence level recorded for the Positive Control C in the last amplification cycle the fluorescence lev
66. xtraction Neg Detected value lt 33 NCA PCR Neg Neg C PCR Pos lt boundary Ct value Detected value lt 33 If PCR kit for detection of 2 microorganisms is used group 2 C DNA extraction Neg Detected value lt 33 NCA PCR Ne Ne C PCR Pos lt boundary Ct value Detected value lt 33 01 02 11 20 03 11 Page 29 of 48 Table 10 Boundary Ct value for positive control C PCR kit group 1 Boundary Ct value in channel SONR FAM Green JOE Yellow ROX Orange Cy5 Red Crimson N gonorrhoeae C trachomatis M genitalium 35 35 35 33 33 T vaginalis C trachomatis Ureaplasma Mgenitalium M hominis di m m 34 di C trachomatis Ureaplasma M genitalium 39 2 oe id C trachomatis Ureaplasma M hominis id 99 i A i N gonorrhoeae C trachomatis 33 30 33 33 M genitalium C albicans C glabrata 33 33 33 33 C krusei PCR kit group 2 duplex U parvum U urealyticum 22 ii 9 7 E HSV typing 33 30 33 T vaginalis 33 33 33 7 N gonorrhoeae HSV CMV 30 30 33 01 02 11 20 03 11 Page 30 of 48 Examples of obtained results Results obtained with AmpliSens C trachomatis Ureaplasma M genitalium MULTIPRIME FRT PCR kit Ft Quantitation Analysis C CNg ASGreEN 210 x SE z0x B slope Correct gi More Settings d Save Defauts A ignore First Sd More Settings ed Save Defauks ONDON WH

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