RealLine HCV Genotype quantitative Str-Format

Transcription

1 Instructions for use REAL TIME PCR DETECTION AND DIFFERENTIATION KIT FOR HEPATITIS C VIRUS GENOTYPES 1, 2 AND 3 RNA WITH QUANTIFICATION Research Use Only (RUO) Str Format VBD Tests valid from June 2016 Rev _EN Page 1 of 11

2 Explanation of symbols used in labeling RealLine Pathogen Diagnostic Kits RUO LOT REF Research use only Batch code Catalogue number Amount of tests Expiry Date Temperature limitation Consult instructions for use Keep out of sunlight Manufacturer BIORON Diagnostics GmbH Rheinhorststr Ludwigshafen (Germany) Phone Fax: 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. VBD0797 Page 2 of 11

3 Table of content: 1. STORAGE AND TRANSPORTATION 4 2. KIT CONTENTS 4 3. INTRODUCTION 4 4. PRINCIPLES OF THE PROCEDURE 5 5. WARNING AND PRECAUTIONS 6 6. ADDITIONAL MATERIALS AND DEVICES REQUIRED BUT NOT SUPPLIED 6 7. REAGENT, CONTROL AND SPECIMEN PREPARATION 7 8. PROCEDURE PROTOCOL 8 9. DATA ANALYSIS ATTACHMENT 1: CALCULATION OF VIRAL RNA CONCENTRATION. 11 VBD0797 Page 3 of 11

4 HEPATITIS C VIRUS GENOTYPES 1, 2 AND 3 RNA REAL TIME PCR DETECTION AND DIFFERENTIATION KIT 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 Integrated Positive Control sample (PC), lyophilized Ready Master Mix 1 (RMM1) for reverse transcription and PCR, lyophilized, for genotypying HCV Ready Master Mix 2 (RMM2) for reverse transcription and PCR, lyophilized, for detection and quantitation Recovery Solution for Control samples (RSC) PCR adhesive foils Passport for the concentration of PC 1 vial 48 test tubes (6 strips x 8 tubes) 48 test tubes (6 strips x 8 tubes) 1 vials, 4 ml each; 3. INTRODUCTION The assay kit is intended for the quantitative detection of hepatitis C virus (HCV) RNA and differentiation of HCV genotypes 1, 2 and 3 in patient plasma and serum. The method is based on the reverse transcription of viral RNA to generate complementary DNA (cdna), with subsequent PCR amplification of target cdna by Polymerase Chain Reaction (PCR) with fluorescent detection of amplified DNA in the real-time mode. The assay kit is intended for use in conjunction with clinical practice. HCV RNA isolation from serum (plasma) should be done using extraction kit: RealLine Extraction 1000 or RealLine Extraction 100 Kits. Assay kit is adapted for real-time PCR detection systems like iq icycler, iq5 icycler, CFX96 (Bio-Rad, USA). Assay kit contains reagents sufficient for 48 test runs, including control samples. VBD0797 Page 4 of 11

5 Specificity Assay kit is designed for in vitro determination of HCV genotypes 1a, 1b, 2a, 2b, 2c, 2i, 3, 4, 5a, 6 regardless of subtype. Assay kit allows genotypes 1 (1a, 1b), 2 (2a, 2b, 2c, 2i) and 3 (3a, 3b) differentiation. In samples containing HCV RNA above detection limit concentration of HCV RNA could be determined. If specimen does not contain HCV RNA, analysis must 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 RNA isolation from 1 ml (or from 150 IU/ml to 10 8 IU/ml for RNA isolation from 100 μl). 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 HCV RNA for RNA isolation from 1 ml (or not less 150 IU/ml for RNA isolation from 100 μl). Genotyping: The kit enables to differentiate HCV genotype in RNA concentration not less than 400 IU/ml for RNA isolation from 1 ml (or not less 4000 IU/ml for RNA isolation from 100 μl). 4. PRINCIPLES OF THE PROCEDURE Principle of analysis is based on 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. Each sample is analyzed using two Ready-to-use Master Mixes (RMM-1 and RMM-2). HCV RNA genotyping is provided by independent detection of type-specific HCV genome fragments amplification in different channels. 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) which serves as an 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 analyzed sample and Positive Control. Also efficiency of sample preparation and reverse transcription should be considered. Efficiency of RNA isolation is provided by isolation of nucleic acids from serum (plasma) jointly with preliminarily added Internal Control. VBD0797 Page 5 of 11

6 5. WARNING AND PRECAUTIONS RealLine Pathogen Diagnostic Kits 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. 6. ADDITIONAL MATERIALS AND DEVICES REQUIRED BUT NOT SUPPLIED real time PCR system; laminar safety box; refrigerator; half-automatic variable-volume single-channel pipettes; disposable medical non-sterile powder-free gloves; disposable pipette tips with aerosol barrier; biohazard waste container. VBD0797 Page 6 of 11

7 7. REAGENT, CONTROL AND SPECIMEN PREPARATION RealLine Pathogen Diagnostic Kits 7.1. Specimen preparation. Prepare specimens for the assay with Extraction kit RealLine Extraction 100 or RealLine Extraction 1000 according to Extraction Kit manual. Prior to RNA isolation procedure add 1 ml of Recovery Solution for Control samples (RSC) into a vial with Integrated 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 of preparation. It is recommended to use three replicas of Positive Control sample and one Negative Control sample (NC) in each test run Reagent preparation. Prior to use, warm up reagents (do not open!) to room temperature (18-25) С. Open the package, separate an appropriate number of reaction tubes with RMM-1 and the same number of reaction tubes with RMM-2 using razor or scalpel. Keep the tubes which were not used for the test in the original bag. Try to squeeze excess of the air out of the bag before closing the clip. After initial opening of the package, store the kit at (2-8) С and use within 3 months. VBD0797 Page 7 of 11

8 8. PROCEDURE PROTOCOL 8.1. Label each reaction tube with RMM-1 for each specimen and control. Label each reaction tube with RMM-2 for each specimen and control. Attention! Mark the lateral part of the reaction tube Add 50 µl of each processed specimen and control to the appropriately labeled reaction tube of RMM-1 and RMM-2 using a new tip with aerosol barrier for each sample. Do not touch the pellet! Safely close the tubes by PCR adhesive foils Place reaction tubes into the thermal block of real time PCR device Program real time PCR device as follows: Stage 1: 45 С, 30 min; Stage 2: 94 С, 1 min; Stage 2: 94 C, 10 sec 60 C, 20 sec 50 cycles Fluorescence measurements should be done at 60 С. RMM For detection of amplification of HCV genotype 1 cdna: collect real-time PCR data through the HEX channel 8.6. For detection of amplification of HCV genotype 2 cdna: collect real-time PCR data through the FAM channel 8.7. For detection of amplification of HCV genotype 3 cdna: collect real-time PCR data through the ROX channel RMM For detection of amplification of Interal control IC DNA: collect real-time PCR data through the FAM channel 8.9. For detection of amplification of HCV cdna: collect real-time PCR data through the ROX channel Record the positions of the controls and specimens according to the instruction manual of the operating device Start the PCR program. VBD0797 Page 8 of 11

9 9. DATA ANALYSIS 9.1. The program should detect in the Positive Control sample: for RMM-1 a fluorescent signal increase and a Сt value in the following channels: HEX (HCV genotype 1 cdna amplification) FAM (HCV genotype 2 cdna amplification) ROX (HCV genotype 3 cdna amplification). for RMM-2 a fluorescent signal increase and a Сt value in the following channels: ROX (HCV cdna amplification) FAM (IC cdna amplification) The program should detect in Negative Control sample no significant FAM, HEX, ROX fluorescent increase for RMM-1; for RMM-2 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 FAM, HEX, ROX (RMM-1) and ROX (RMM-2) channel is less than 40, this indicates the presence of contamination 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 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 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 cycles, should be ignored. After screening, recalculate (IC Ct)m for remaining samples 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 differ from (IC Ct)m by more than 2 cycles, then result for this sample should be considered as equivocal. The test should be repeated from the sample RNA extraction stage 9.9. The sample is considered positive if Ct value along the ROX channel (RMM-2) 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. VBD0797 Page 9 of 11

10 9.10. The test results are considered reliable only when Positive and Negative controls perform as expected. HCV genotype definition Analysed sample is considered as positive for HCV genotype 1 if Ct value along the HEX channel (RMM-1) and Ct value along the ROX channel (RMM-2) does not exceed Analysed sample is considered as positive for HCV genotype 2 if Ct value along the FAM channel (RMM-1) and Ct value along the ROX channel (RMM-2) does not exceed Analysed sample is considered as positive for HCV genotype 3 if Ct value along the ROX channel (RMM-1) and Ct value along the ROX channel (RMM-2) does not exceed 40. RMM-1 RMM-2 Channel HEX FAM ROX FAM ROX Detected cdna HCV genotype 1 HCV genotype 2 HCV genotype 3 IC HCV (all types) If HCV RNA concentration is less than 400 IU/ml (RMM-2), it can be not enough for genotype differentiation Sample is considered as containing HCV genotype different from 1 (1a, 1b), 2 (2a, 2b, 2c, 2i), 3 (3a, 3b) if HCV RNA concentration in analyzed sample is more than 400 IU/ml (RMM-2), and there is no significant FAM, HEX, ROX fluorescent increase for RMM-1. VBD0797 Page 10 of 11

11 HCV quantitation analysis 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 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) 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 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 Analyzed sample is considered as negative if obtains no Ct value along the ROX channel or Ct value exceed ATTACHMENT 1: CALCULATION OF VIRAL RNA CONCENTRATION. Calculate the viral RNA 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 average Сt value for three replicas of the PC samples ROX and FAM channels, accordingly; Сt SAMPk and Сt IC k Сt value of the sample numbered k along ROX and FAM channels, accordingly, if amplification efficiency is (Еа) =100%. For an easier calculation we can provide you with a small Excel sheet. Ask us at : techsupport@bioron.de 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 independable Positive Control samples would be the needed. VBD0797 Page 11 of 11