Instructions for use - KIT FOR EXTRACTION OF RNA AND SUBSEQUENT REAL TIME PCR DETECTION AND QUANTIFICATION OF HEPATITIS C VIRUS RNA Attention! Please read the information about quantification process carefully! Research Use Only (RUO) RealLine HCV quantitative Uni Format incl. extraction VBD0799 48 Tests valid from: April 2017 Rev20042017_EN Page 1 of 16
Explanation of symbols used in labeling IVD LOT REF For in vitro diagnostic use Batch code Catalogue number Amount of tests Expiry Date Temperature limitation Consult instructions for use Keep out of sunlight Manufacturer BIORON Diagnostics GmbH Rheinhorststr. 18 67071 Ludwigshafen (Germany) Phone +49 621 545 900 70 Fax: +49 621 545 900 68 info@bioron.de Legals: Limited Product Warranty: This warranty limits our liability for the replacement of this product. No warranties of any kind, express or implied, including, without limitation, implied warranties of merchantability or fitness for a particular purpose, are provided. BIORON Diagnostics GmbH shall have no liability for any direct, indirect, consequential, or incidental damages arising out of the use, the results of use, or the inability to use this product. Trademarks: FAM, HEX, JOE and ROX are trademarks of Applera Corporation or its subsidiaries in the US and certain other countries. iq and CFX are trademarks of Bio-Rad Laboratories, Inc. Rotor-Gene is a registered trademark of Qiagen Group, Germany. Rev20042017_EN Page 2 of 16
TABLE OF CONTENT: 1. STORAGE AND TRANSPORTATION 4 2. KIT CONTENTS 4 3. INTRODUCTION 5 4. PRINCIPLES OF THE PROCEDURE 6 5. PRECAUTIONS 6 6. ADDITIONAL MATERIALS AND DEVICES REQUIRED 7 7. SPECIMEN TRANSPORT AND STORAGE 7 8. REAGENT PREPARATION 8 9. PROCEDURE PROTOCOL 9 10. DATA ANALYSIS 13 11. ATTACHMENT 1: CALCULATION OF VIRAL NA CONCENTRATION. 15 12. ATTACHMENT 2: ANALYTICAL SYSTEM VALIDATION. 15 VBD0799 Page 3 of 16
KIT FOR EXTRACTION OF RNA AND SUBSEQUENT REAL TIME PCR DETECTION AND QUANTITATION OF HEPATITIS C VIRUS RNA Research Use Only 1. STORAGE AND TRANSPORTATION Store assay kit at (2-8) С in the manufacturer s packing. Transportation at 25 С for 10 days is allowed. Do not freeze reagents. Shelf life of the kit as printed on the label of the outside box. Do not pool reagents from different lots or from different vials of the same lot. Strictly follow the Instruction manual for reliable results. 2. KIT CONTENTS Specimen Preparation Reagents Concentrating Solution Lysis Reagent 1 Lysis Reagent 2 Sorbent (suspension of magnetic particles) Solution for DNA/RNA Precipitation Wash Solution 1 Wash Solution 2 Specimen Diluent Control Samples Recovery Solution for Controls (RSC) Positive Control (PC) sample Negative Control (NC) sample Internal control (IC) sample, lyophilized Samples for calibration, freeze-dried (CS1 and CS2) - are used when the adequacy of analytical system has to be checked, see the Attachment 2 Passport for the Concentration of PC Amplification reagents Ready Master Mix for reverse transcription and PCR, freeze-dried (RMM) 4 vials -14 ml each 4 vials - 4 ml each 4 vials - 7 ml each 1 vial 4 vials -12 ml each 4 vials - 8 ml each 4 vials - 5 ml each 4 vials - 3 ml each 2 vials - 4 ml each 2 vial 2 vials - 12 ml each 2 vials 1 vial each 48 test tubes Rev20042017_EN Page 4 of 16
3. INTRODUCTION The assay kit RealLine HCV quantitative is intended for the quantitative detection of hepatitis C virus (HCV) RNA in patient plasma and serum. The method is based on the reverse transcription of viral RNA to generate complementary DNA (cdna), with subsequent amplification of target cdna by Polymerase Chain Reaction (PCR) with fluorescent detection of amplified DNA in the real-time mode. The assay kit RealLine HCV quantitative is intended for use in conjunction with clinical practice for diagnosis of hepatitis C disease and for clinical management of HCV infected patients disease progress. The assay kit is adapted for real-time PCR detection systems like iq icycler, iq5 icycler, CFX96 (Bio-Rad, USA), DT-96 (DNA-technology, Russia); Rotor-Gene 3000, Rotor-Gene 6000 (Corbett Research, Australia) or their analogues. The assay kit contains reagents sufficient for 4 12-test runs, which may be performed separately or simultaneously. It is strongly recommended to use three replicas of Positive Control sample and one Negative Control sample in each test run. The assay kit can be used with either of two specimen preparation procedures, the Standard Procedure or the UltraSensitive procedure. In the Standard specimen preparation procedure, HCV RNA is isolated from 100 μl of serum (plasma). In the UltraSensitive specimen preparation procedure, HCV viral particles in serum (plasma) are concentrated by Concentrating Solution of 1 ml of serum (plasma). Specificity: The assay kit is designed for in vitro determination of HCV genotypes: 1a, 1b, 2a, 2b, 2c, 2i, 3, 4, 5a, 6 regardless of subtype. The samples containing HIV RNA with concentration above the detection limit will be determined as positive. If specimen does not contain HIV RNA, analysis will give negative result (in 100 % of cases). Dynamic range of estimated concentration (linearity area): from 15 IU / ml to 10 8 IE / ml HCV RNA for the UltraSensitive procedure (or from 150 IU/ml to 10 8 IU/ml for the Standard procedure). 1 IU = 2.5 copies of HCV RNA (National Institute of Biological Standards and Control for WHO international standard for Hepatitis С virus NIBSC Code: 96/798). Sensitivity: The assay kit securely determines HCV RNA in concentration not less than 15 IU / ml for UltraSensitive specimen preparation procedure (or not less than 150 IU / ml for the Standard procedure). VBD0799 Page 5 of 16
4. PRINCIPLES OF THE PROCEDURE The principle of analysis is based on isolation of nucleic acids directly from serum (plasma) jointly with preliminarily added Internal Control, the reverse transcription of viral RNA with subsequent PCR amplification of target cdna by PCR with fluorescent detection of amplified DNA in the real-time mode. Quantification of Hepatitis C virus RNA is provided by application of the Positive Control (PC) in each test run. Positive Control is characterized in international units (IU/ml) by World Health Organization (WHO international standard for Hepatitis С virus NIBSC Code: 96/798), and serves as a Quantitation Standard for calculation of viral quantity. Threshold cycle value Ct is the cycle number at which fluorescence generated within a reaction crosses fluorescence threshold, a fluorescent signal significantly above the background fluorescence. Quantity of viral RNA in initial sample is calculated by comparison of the threshold cycle value of the analysed sample and Positive Control. Also efficiency of sample preparation and reverse transcription should be considered. For convenience of the user it is recommended to use a magnetic rack through workout. Specimen preparation for 1 ml or 100 μl of serum (plasma) is allowed 5. WARNING AND PRECAUTIONS For in vitro use only. The kits must be used by skilled personnel only. When handling the kit, follow the national safety requirements for working with pathogens. To prevent contamination, the stages of DNA isolation and PCR test run must be spatially separated. Avoid microbial and ribonuclease contamination of reagents when removing aliquots from reagent vials. Wear protective disposable gloves, laboratory coats and eye protection when handling specimens and kit reagents. Every workplace must be provided with its own set of variable-volume pipettes, necessary auxiliary materials and equipment. It is prohibited to relocate them to other workplaces. The use of sterile disposable pipette tips is recommended. Never use the same tips for different samples. Do not pool reagents from different lots or from different vials of the same lot. Dispose unused reagents and waste in accordance with country, federal, state and local regulations. Do not use the kit after the expiration date. Rev20042017_EN Page 6 of 16
6. ADDITIONAL MATERIALS AND DEVICES REQUIRED RealLine Pathogen Diagnostic Kits Real time PCR device iq/iq5 icycler, CFX96 (Bio-Rad, USA), DT-96 (DNA-technology, Russia); Rotor-Gene 3000/6000 (Corbet Research, Australia) or equivalent; Plates or tubes for PCR, if the uni-format tube are not suitable for the device Microcentrifuge (min RCF 13000 rpm), or equivalent; Vortex mixer; Thermo Shaker Disposable gloves, powder-free; Pipettes (capacity 10-100 μl, 100-1000 μl) with filters (aerosol barriers); Disposable DNAse/RNase-free tips with filters; Displacement tips; 2.0 ml polypropylene tubes, sterile, non-siliconised Vacuum aspirator; 2.0 ml and 0.2 ml microtube racks; Magnetic Rack for nucleic acids isolation 7. SPECIMEN TRANSPORT AND STORAGE Attention! Specimens anticoagulated with heparin are unsuitable for this test. Blood should be collected in sterile tubes (or tubes, using EDTA or ACD as the anticoagulant). Separate serum (plasma) from whole blood within 6 hours of collection. Transfer serum (plasma) to a sterile polypropylene tube. Specimens can be transported and stored: At room temperature up to 2 hours; At (2-8) С up to 24 hours; Frozen at 20 С up to 2 weeks. Do not freeze - thaw samples repeatedly! Before use centrifuge specimens of serum (plasma) at 13000 rpm for 5 minutes at room temperature. VBD0799 Page 7 of 16
8. REAGENT PREPARATION 8.1. Prior to use, bring up reagents to room temperature (18-25) С for 30 minutes. Add 1 ml of Recovery Solution of Controls (RSC) into a vial with Internal Control (IC) sample, mix gently, keep for 15 minutes, then carefully mix once again. IC should be stored at (2-8) С and used within 1 month after preparation. 8.2. Add 1 ml of Recovery Solution for Control (RSC) into a vial with Positive Control (PC) sample, mix gently, keep for 15 minutes, then carefully mix once again. PC should be stored at (2-8) С and used within 1 month after preparation. 8.3. Negative Control (NC) sample is ready to use. Once opened, NC should be stored at (2-8) С and used within 1 month. 8.4. Prior to use, warm Lysis Reagents 1 and 2 at (50-60) С and mix thoroughly to dissolve the precipitated material. Vortex Sorbent to a condition of homogeneous suspension. Add 80 μl of Sorbent suspension into a vial with Lysis Reagent 2. Mix carefully. Attention! Once opened, any unused portion of Lysis Reagent 2 should be discarded. For the quantitative determination, the RNA extraction is conducted from 100 µl of serum (plasma) or whole blood using this kit. The Internal Control must be used with the Extraction, if another RNA Extraction kit I used, please add RealLine Internal Control (REF VBC8881) to the extraction procedure Each set of samples must contain 3 PC and 1 NC sample. The elution volume have to be 200 µl. Rev20042017_EN Page 8 of 16
9. PROCEDURE PROTOCOL Ultra Sensitive procedure: RNA isolation from 1 ml of serum (plasma) 9.1. Determine the appropriate number of reaction tubes needed for patient specimen and control testing. It is recommended to use three replicas of Positive Control sample and one Negative Control sample in each test run. 9.2. Label each 2.0 ml tube for each patient specimen and control sample. 9.3. Add 30 μl of IC to each tube. 9.4. For NC, add to the tube, marked NC, 1000 μl of Negative Control sample. 9.5. For PC, add to the tube, marked PC, 970 μl of Negative Control sample, and 30 μl of Positive Control sample. 9.6. Add 1000 μl of each patient specimen to the appropriately labeled tube. 9.7. For each specimen or control tube, add 1 ml of Concentrating Solution. 9.8. Close the tubes and vortex for 3-5 seconds. Leave the tubes for 10 minutes at room temperature, then centrifuge for 5 minutes, 3000 rpm. 9.9. Using a new tip for each sample, carefully remove the supernatant without disturbing the pellet. The pellet should be clearly visible at this step. 9.10. Add 200 μl of Lysis Reagent 1 to each tube. Vortex vigorously for 10-15 seconds. Some insoluble material may remain. Leave tubes for 5 minutes at room temperature. 9.11. Add 500 μl or Lysis Reagent 2 with Sorbent to each tube. Vortex for 5-10 seconds. Place the tubes into Thermo Shaker, and incubate for 10 minutes at 56 C and 1300 rpm. 9.12. Add 750 μl of Solution for DNA/RNA Precipitation in each tube. Vortex for 10-15 seconds. Incubate 5 minutes at room temperature. 9.13. Centrifuge at 13000 rpm for 5 minutes at room temperature. 9.14. Trying not to shake up a pellet, place the tubes to Magnetic Rack. Using a new tip for each sample, carefully remove the supernatant without disturbing the pellet. 9.15. Add 500 μl or Wash Solution 1 in each tube. Vortex vigorously for 10-15 seconds. Centrifuge at 13000 rpm for 5 minutes. VBD0799 Page 9 of 16
9.16. Trying not to shake up a pellet, place the tubes to Magnetic Rack. Using a new tip for each sample, carefully remove the supernatant without disturbing the pellet. 9.17. Add 300 μl or Wash Solution 2 to each tube. Vortex vigorously for 10-15 seconds. Centrifuge at 13000 rpm for 5 minutes. 9.18. Trying not to shake up a pellet, place the tubes to Magnetic Rack. Using a new tip for each sample, carefully remove the supernatant without disturbing the pellet. 9.19. Dry the pellet with open caps for 2-3 minutes at room temperature (18-25) С. 9.20. Add 200 μl of Specimen Diluent to each tube. Vortex vigorously for 10-15 seconds. Place the tubes into Thermo Shaker, and incubate for 10 minutes at 56 C and 1300 rpm. Then centrifuge for 1 minute at 13000 rpm. 9.21. Samples are ready for reverse transcription and PCR. Attention! In the case of RNA isolation start reverse transcription and PCR within 2 hours of specimen and control preparation. Isolated DNA can be stored at (2-8) С for 24 hours. Standard protocol: RNA isolation from 100 μl of serum (plasma) 9.22. Determine the appropriate number of reaction tubes needed for patient specimen and control testing It is recommended to use three replicas of Positive Control sample and one Negative Control sample in each test run. 9.23. Label each 2.0 ml tube for each patient specimen and control sample. 9.24. Add 30 μl of IC to each tube. 9.25. For NC, add to the tube, marked NC, 100 μl of Negative Control sample. 9.26. For PC, add to the tube, marked PC, 70 μl of Negative Control sample, and 30 μl of Positive Control sample. 9.27. Add 100 μl of each patient specimen to the appropriately labeled tube. 9.28. Add 500 μl or Lysis Reagent 2 with Sorbent to each tube. Vortex for 5-10 seconds. Place the tubes into Thermo Shaker, and incubate for 10 minutes at 56 C and 1300 rpm. 9.29. Add 600 μl of Solution for DNA/RNA Precipitation in each tube. Vortex for 10-15 seconds. Incubate 5 minutes at room temperature. 9.30. Centrifuge at 13000 rpm for 5 minutes at room temperature. Rev20042017_EN Page 10 of 16
9.31. Trying not to shake up a pellet, place the tubes to Magnetic Rack. Using a new tip for each sample, carefully remove the supernatant without disturbing the pellet. 9.32. Add 500 μl or Wash Solution 1 in each tube. Vortex vigorously for 10-15 seconds. Centrifuge at 13000 rpm for 3 minutes. 9.33. Trying not to shake up a pellet, place the tubes to Magnetic Rack. Using a new tip for each sample, carefully remove the supernatant without disturbing the pellet. 9.34. Add 300 μl or Wash Solution 2 to each tube. Vortex vigorously for 10-15 seconds. Centrifuge at 13000 rpm for 3 minutes. 9.35. Trying not to shake up a pellet, place the tubes to Magnetic Rack. Using a new tip for each sample, carefully remove the supernatant without disturbing the pellet. 9.36. Dry the pellet with open caps for 2-3 minutes at room temperature (18-25) С. 9.37. Add 200 μl of Specimen Diluent to each tube. Vortex vigorously for 10-15 seconds. Place the tubes into Thermo Shaker, and incubate for 10 minutes at 56 o C and 1300 rpm. Then centrifuge for 1 minute at 13000 rpm. 9.38. Samples are ready for reverse transcription and PCR. Attention! In the case of RNA isolation start reverse transcription and PCR within 2 hours of specimen and control preparation. Isolated DNA can be stored at (2-8) С for 24 hours. Reverse transcription and PCR. 9.39. Place the tubes with processed specimens and controls to Magnetic Rack. 9.40. Prepare an appropriate number of reaction tubes with Ready Master Mix (RMM). Label each reaction tube for each patient specimen and control. Attention! For iq/iq5 icycler and CFX96 put marks on the lateral part of a reaction tube. For Rotor-Gene 3000/6000 put marks on the cap on the reaction tube. 9.41. Add 50 μl of each processed specimen and control to the appropriately labeled reaction tube using a new RNase-free tip with aerosol barrier for each sample. Do not grasp a sorbent particles! 9.42. Place reaction tubes into the thermal block of real time PCR device. VBD0799 Page 11 of 16
9.43. Program real time PCR device as follows: For iq/iq5 icycler, CFX96, DT-96 Stage 1: 45 С, 30 min; Stage 2: 94 С, 1 min; Stage 3: 94 C, 10 sec 50 cycles 60 C, 20 sec Fluorescence measurements should be done at 60 С. For Rotor-Gene 3000 (6000) Stage 1: 45 С, 30 min; Stage 2: 94 С, 1 min; Stage 3: 94 C, 10 sec 50 cycles 60 C, 40 sec Fluorescence measurements should be done at 60 С. 9.44. Collect real-time PCR data through the FAM channel for detection of amplification of IC cdna. 9.45. Collect real-time PCR data through the ROX channel for detection of amplification of HCV cdna. For Rotor-Gene 3000 (6000) In the New Run Wizard window click Calibrate. The window Auto Gain Calibration Setup opens. In the line Channel Settings choose ROX and FAM channels. Make sure that of Tube position is 1. Tick off Perform Calibration Before 1st Acquisition. Click Close button. 9.46. Record the positions of the controls and specimens According to the instruction to the used device. 9.47. Start the reverse transcription and PCR program. Rev20042017_EN Page 12 of 16
10. DATA ANALYSIS For Rotor-Gene 3000 (6000) Results for Internal Control DNA amplification 10.1. Click Analysis button, choose Quantitation from the list, choose Cycling А. FAM, click Show button. 10.2. Click ОK button, and cancel automatic Threshold determination. 10.3. Click Linear scale button. 10.4. In the Quantitation analysis menu buttons Dynamic tube and Slope Correct should be pressed. 10.5. Click More Settings (Outlier removal) button, determine NTC threshold value as 5%. 10.6. In the column CT Calculation (right part of the window) determine Threshold value as 0,04. 10.7. In the result table (Quant. Results window) Ct will be displayed. Results for HCV cdna amplification 10.8. Click Analysis button, choose Quantitation from the list, choose Cycling А. ROX click, Show button. 10.9. Click ОK button, and cancel automatic Threshold determination. 10.10. Click Linear scale button. 10.11. In the Quantitation analysis menu buttons Dynamic tube and Slope Correct should be pressed. 10.12. Click More Settings (Outlier removal) button, determine NTC threshold value as 5%. 10.13. In the column CT Calculation (right part of the window) determine Threshold value as 0,04. 10.14. In the result table (Quant. Results window) Ct will be displayed. 10.15. In Positive Control sample the program should detect: ROX fluorescent signal increase and Сt value (HCV cdna amplification); FAM fluorescent signal increase and Сt value (IC cdna amplification). 10.16. In Negative Control sample the program should detect: FAM fluorescent signal increase and Ct value, and no significant ROX fluorescent increase should appear. If Ct value for NC along ROX channel is less than 40, this indicates the presence of contamination. 10.17. The program should detect amplification signal increase for IC cdna (channel FAM) in each sample and define Ct for IC. Probe analysis is valid if Ct of IC for this sample is equal to or less than 40. 10.18. In the case of contamination (when NC is determined as positive) all positive results in this test should be repeated from the RNA extraction stage. Negative samples of such test run are considered reliable. VBD0799 Page 13 of 16
10.19. Calculate (IC Ct) m as the average Ct value of IC for all samples (including PC and NC). Samples with Ct of IC, that differ from (IC Ct) m by more than 2 fold, should be ignored. After screening, recalculate (IC Ct) m for remaining samples. 10.20. The sample is considered negative if Ct value along the ROX channel exceeds 40 or is not determined. If Ct of IC for this sample differs from (IC Ct) m by more than 2, then result for this sample should be considered as equivocal. The test should be repeated from the sample RNA extraction stage. 10.21. The sample is considered positive if Ct value along the ROX channel does not exceed 40. If Ct of IC for this sample differs from (IC Ct) m more then 2, the sample is considered as positive without quantitative analysis. For quantitative analysis, repeat the test beginning from the RNA isolation stage. 10.22. The test results are considered reliable only when Positive and Negative controls perform as expected. HCV quantitation analysis. 10.23. For quantitative analysis calculate the HCV RNA concentration in analyzed samples in accordance with Attachment 1. Attention! For RNA isolation from 100 μl result should be multiplied by 10. 10.24. If calculated HCV RNA concentration is in dynamic range from 100 IU / ml to 10 8 IU / ml result should be reported as positive with indication of calculated HCV RNA concentration in the sample (in IU / ml). 10.25. Test results greater than 10 8 IU / ml are above the upper limit of quantitation of the Standard test and should be reported as Greater than 10 8 IU / ml. 10.26. Test results less than 100 IU / ml are below the lower limit of quantitation of the Standard test and should be reported as HCV RNA detected, less than 100 IU / ml. 10.27. Analyzed sample is considered as negative if obtains no Ct value along the ROX channel or Ct value exceed 40. For further information and help, ask us at techsupport@bioron.de. We can provide you a calculation sheet for an easy evaluation of your data. Note: For precise calculation of viral NA concentration in analyzed samples, the validation of the analytical system should be done by comparison of Positive Control PC concentration, specified in the passport of the assay kit with PC concentration, calculated using calibration graph. For this purpose three independent Positive Control samples are the needed. Rev20042017_EN Page 14 of 16
11. ATTACHMENT 1: CALCULATION OF VIRAL NA CONCENTRATION. Calculate the viral NA concentration using the following equation: С sampk = С PC 2(Сt PC Сt SAMPk) 2(Сt ICk Сt ICPC), Where: k sample number; С PC PC concentration, specified in the passport of Assay kit; Сt PC and Сt IC PC Сt value of the PC sample ROX and FAM channels, accordingly; Сt SAMP k and Сt IC k Сt value of the sample numbered k along ROX and FAM channels, accordingly, if amplification efficiency is (Еа) =100 %. 12. ATTACHMENT 2: ANALYTICAL SYSTEM VALIDATION. Calculation of viral NA concentration for PC using calibration graph. For precise calculation of viral NA concentration in analyzed samples, the validation of the analytical system should be done by comparison of Positive Control PC concentration, specified in the passport of the assay kit with PC concentration, calculated using calibration graph. Prior to use, warm reagents at room temperature (18-25) С for 30 minutes. Prepare NC, PC and IC as recommended in the instruction. Add 1 ml of Solution for Restoration of Control samples (SRC) into a vial with Calibration sample 1 (CS1) and Calibration sample 2 (CS2). Mix gently, keep for 15 minutes, then carefully mix once again. For RealLine Extraction 1000 (1 ml plasma) use following amounts: Prepare 10 2.0ml tubes one for NC, 9 for 3 repeats of PC, CS1 and CS2. Label the tubes. Add 30 μl of IC to each tube. For NC, add to the tube, marked NC, 1 ml of Negative Control. For PC, add to each of three tubes, marked PC, 970 μl of Negative Control, and 30 μl of Positive Control. For CS1 add to each of three tubes marked CS1 970 μl of Negative Control, and 30 μl of Calibration sample 1. For CS2 add to each of three tubes marked CS2 970 μl of Negative Control, and 30 μl of Calibration sample 2. For each specimen or control tube, add 1 ml of Concentrating Solution. Run the isolation of NA as recommended in the instruction. VBD0799 Page 15 of 16
For RealLine Extraction 100 (100 µl plasma) use following amounts: Prepare 10 tubes one for NC, 9 for 3 repeats of PC, CS1 and CS2. Label the tubes. Add 30 μl of IC to each tube. For NC, add to the tube, marked NC, 100µl of Negative Control. For PC, add to each of three tubes, marked PC, 70 μl of Negative Control, and 30 μl of Positive Control. For CS1 add to each of three tubes marked CS1 70 μl of Negative Control, and 30 μl of Calibration sample 1. For CS2 add to each of three tubes marked CS2 70 μl of Negative Control, and 30 μl of Calibration sample 2. Run the isolation of NA as recommended in the instruction. Run the PCR (or reversed PCR). Insert concentration of CS1 and CS2, specified in the passport of Assay kit, as it recommended in the instruction of PCR instrument. For each sample calculate correct Ct value, using the following equation: Z = Сt SAMP (Сt ICm Сt IC). Calculate average Z value for PC, CS1 and CS2 (Z PCm, Z CS1m, Z CS2m). Samples with Z that differ from Zm more than 2, should be ignored. After screening, recalculate Z m for samples remained. Calculate B coefficient for calculating of specific viral NA, using following equation: B = [Lg(C CS1) Lg(C CS2)] / (Z CS2m Z CS1m), Where: C CS1 and C CS2 specific viral NA concentration in Calibration samples (specified in the passport of the assay kit); For amplification efficiency 100 % B = Lg2 = 0.3. Using received results, calculate concentration of specific viral NA for Positive Control sample, using the following analytical equation: С PC = 10 XPC (IU/ml) Where: X PC = Lg (C CS1) + B (Z CS1m Z PCm); Z CS1m average Z value for Calibration Sample 1 repeats; Z PCm average Z value for Positive Control sample repeats; If the calculated value of specific virus NA concentration in PC differs no more than in 2 from the value specified in the passport of a set, PC can be used as the sample of comparison for the further calculations of specific virus NA concentration in investigated samples. At performance of all specified conditions it is possible to spend calculation of concentration specific virus NA (in IU/ml) in investigated samples under the formula (item 11) using factor B value, calculated on calibration graph. For further information and help, ask us at techsupport@bioron.de. We can provide you a calculation sheet for an easy evaluation of your data. Rev20042017_EN Page 16 of 16