RealLine HPV HCR Genotype Quantitative Str-Format

Transcription

1 Instructions for use Quantitative Str-Format QUANTITATIVE ASSAY KIT FOR THE DIFFERENTIAL DETERMINATION AND QUANTITATION OF DNA OF HUMAN PAPILLOMAVIRUS TYPES 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 AND 59 BY REAL TIME PCR In vitro Diagnostics Quantitative (Str Format) VBD Tests valid from April 2017 Rev _EN Page 1 of 15

2 Explanation of symbols used in labeling IVD LOT REF Σ For in vitro diagnostic use Batch code Catalogue number Content of number of tests Expiry date Temperature limitation Consult instructions for use Manufacturer Keep out of sunlight 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. Cy5 is a registered trademarks of Amersham Biosciences Corp., Part of GE, USA. iq and CFX are trademarks of Bio-Rad Laboratories, Inc.. Rotor-Gene is a registered trademark of Qiagen Group, Germany. Rev _EN Page 2 of 15

3 Table of content: 1. INTRODUCTION 4 2. KIT CONTENTS 5 3. PRINCIPLE OF THE METHOD 6 4. SPECIFICATION 7 5. WARNING AND PRECAUTIONS 8 6. ADDITIONAL MATERIALS AND DEVICES REQUIRED BUT NOT SUPPLIED 8 7. PREPARATION OF THE ANALYSED SAMPLES AND REAGENTS 9 8. PROCEDURE DATA ANALYSIS AND INTERPRETATION STORAGE AND TRANSPORTATION 15 VBD8478 Page 3 of 15

4 ASSAY KIT FOR THE DIFFERENTIAL DETERMINATION AND QUANTITATION OF HUMAN PAPILLOMAVIRUS TYPES 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 AND 59 DNA BY REAL TIME PCR In vitro diagnostics 1. INTRODUCTION Clinical information: Human Papilloma Viruses HPV are DNA-Viruses and more than 100 different types are known. While the majority of HPVs cause no symptoms, some can cause low severe symptoms like warts and a few are known to cause cancer. HPV types that are more likely to lead to the development of cancer are referred as high-risk types HPV. High-risk HPV-types are known to cause the vast majority of cervical cancers which cause death in women with an annual incidence of around half a million and a mortality of almost 50 %. The Quantitative assay kit is designed for differential determination and the quantitation of DNA of high carcinogenic cancer human papillomavirus (HPV) types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 and 59 isolated from clinical specimens using the extraction kits: RealLine DNA-Express (REF VBC8899) RealLine DNA-Extraction 3 (REF VBC8889) RealLine Extraction 100 (REF VBC8896) Quantitative kit is designed for the analysis of clinical materials: scrapings of the epithelial cells, semen, prostatic secretions, and urine. The assay is based on the real-time polymerase chain reaction (PCR) method with fluorescent detection of the amplified product. The Str-Format Kit contains 5 x 96 tubes (0.2 ml each) in strips with lyophilized Mastermix. For the test 50 µl of extracted DNA have to be pipetted into the tube and the ready mastermix is diluted. The kit contains reagents required for 96 tests, including control samples and the positive control sample. For each clinical specimen there is a differential detection of the described high carcinogenic risk viruses (paragraph.3). The kit is validated for use with: iq 5 icycler (Bio-Rad, USA. The kit is compatible with real-time PCR systems such as iq icycler, CFX 96 (Bio-Rad, USA) and DT-96 (DNA-Technology, Russia). Rev _EN Page 4 of 15

5 The use of:! Extraction Kits for nucleic acids from clinical specimen from other supplier! other real-time PCR devices! appropriate reaction volumes, other than 50 µl has to be validated in the lab by the user. The special notes regarding the internal control IC have to be strongly followed. 2. KIT CONTENTS Positive Control sample (PC) based on plasmid DNA with integrated DNA fragments of human papilloma viruses, including HPV DNA types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 and 59 of high cancer risk, lyophilized Ready Master Mix (RMM 1, 2, 3, 4), lyophilized Ready Master Mix (RMM 5 Sample validation), lyophilized Recovery Solution for Controls (RSC) 2 vials 4 microplates, 96 test-tubes each (12 strips x 8 tubes) 96 test-tubes (12 strips x 8 tubes); 1 vial, 4 ml The kit is additionally supplied with PCR optical-quality film. VBD8478 Page 5 of 15

6 3. PRINCIPLE OF THE METHOD Real Time PCR is based on the detection of the fluorescence, produced by a reporter molecule, which increases as the reaction proceeds. Reporter molecule is dual-labeled DNA-probe, which specifically binds to the target region of pathogen DNA. Fluorescent signal increases due to the fluorescent dye and quencher separating by Taq DNA-polymerase exonuclease activity during amplification. PCR process consists of repeated cycles: temperature denaturation of DNA, primer annealing and complementary chain synthesis. Threshold cycle value Ct is the cycle number at which the fluorescence generated within a reaction crosses the fluorescence threshold, a fluorescent signal rises significantly above the background fluorescence. Ct depends on initial quantity of pathogen DNA template. The use of Internal Control (IC) prevents generation of false negative results associated with possible loss of DNA template during specimen preparation. IC indicates if PCR inhibitors occur in the reaction mixture. IC template should be added in each single sample (including control samples) prior to DNA extraction procedure. The amplification and detection of IC does not influence the sensitivity or specificity of the target DNA PCR. To analyze each sample for the content of HPV DNA one half of the strip has to be used - the amplification is carried out in four tubes (RMM1, RMM2, RMM3, RMM4) for detection of DNA; three of the 12 HPV types can be detected in each tube (see Table 1). The efficacy of DNA extraction from the samples is controlled by isolating an Internal Control IC from clinical samples together with DNA of infectious agent. The subsequent detection occurs in all Mastermixes MM by the Cy5 (Red) channel. Analyzed samples are additionally assessed for the human HMBS gene DNA content in order to validate the quality of the material sampling and improve the reliability of the results of the quantitative determination of human papillomaviruses of high cancer risk (HPV HCR). To analyze each sample for the content of HPV DNA half of the strip is used - the amplification is carried out in four tubes (RMM1, RMM2, RMM3, RMM4) with RMM 1, 2, 3, 4 for detection of DNA of three of the 12 HPV types in each tube (see Table 1). Additionally one tube with RMM 5 is used for the assessment of human HMBS gene DNA content in the sample. Table 1 Test-tube HPV types RMM1 16, 18, 39 RMM2 33, 45, 56 RMM3 31, 35, 58 RMM4 52, 51, 59 Rev _EN Page 6 of 15

7 4. SPECIFICATION I. Sensitivity: Sensitivity control was performed four samples containing 100 copies of DNA of each type of HPV per sample prepared from the corresponding Standard Reference Samples (SRS) (Standard Reference Samples containing DNA of HPV HCR types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 and 59), The sensitivity equals 100 %. II. Sensitivity of HMBS gene detection: Sensitivity control of HMBS gene detection was performed four samples containing 250 copies of HMBS gene DNA per sample prepared from HMBS Standard Reference Samples (HMBS SRS). The sensitivity equals 100 %. III. Specificity: Specificity of DNA detection for each type of HPV was determined using the samples negative for DNA for this type of HPV and positive for DNA of other eight types of HPV. Samples were prepared in four repeats from the standard reference panel of negative DNA-extracts (SRP HPV) and corresponding HPV DNA SRS. Specificity equals 100 %. IV. Specificity of HMBS gene detection: Specificity of HMBS gene detection was determined using negative DNA-extracts of the Standard Reference Panel, consisting of samples containing IC DNA and not containing DNA of Papillomavirus infections agents. Specificity of HMBS gene detection equals 100 %. V. Linearity Linearity correlation of the measured value and prescribed value (calculated taking into account the dilution factor) of the logarithm of the concentration of HPV DNA - in the range of %. VI. Coefficient Coefficient of variation for the values of logarithm of the concentration of HPV DNA not above 10 %. VII. Diagnostic evaluation: Diagnostic evaluation was performed on the following clinical samples: 10 clinical samples obtained from healthy donors; 5 clinical samples obtained from patients with STD symptoms but without laboratory signs of the papillomavirus infection; 3 clinical samples (No ) containing human papillomavirus types 6 and 11; 30 clinical samples (No ) obtained from patients with diagnosed type of HPV HCR. All evaluation tests were carried out with a CE-marked reference kit. The data obtained show a 100 % correlation of specificity and sensitivity results presented by quantitative (Str-Format) kit and the CE-marked reference kit. VBD8478 Page 7 of 15

8 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 nuclease 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, like described in p.1 DNA Extraction Kit: RealLine DNA-Express, RealLine DNA-Extraction 3 or RealLine Extraction 100; Internal Control reagent (VBC8881) and Negative Control Sample or H2O (molecular biology grade) if the kit is used with the extraction kits of other supplier; 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; razor or scalpel. Rev _EN Page 8 of 15

9 7. PREPARATION OF THE ANALYSED SAMPLES AND REAGENTS Each group of samples undergoing the procedure of DNA isolation must include a Positive Control sample (PC) from this kit and a Negative Control sample (NC) which is a component of the DNA extraction kit. We strongly recommend the implementation of the Internal Control IC, the Negative Control NC and Positive Control PC samples to the extraction procedure. When using a kit of another supplier for the extraction of nucleic acids as recommended in paragraph 1, add 20 μl of IC (VBC8881) to each tube: For the NC use 100 µl of Negative Control Sample or H 2O (molecular biology grade). For the PC use 70 μl of Negative Control Sample or H 2O (molecular biology grade) and 30 μl of Positive Control to the tube marked PC Sample preparation Prepare the samples for the assay using RealLine DNA-Express, RealLine DNA-Extraction 3 or RealLine Extraction 100 extraction kits according their instruction manuals. If samples of isolated DNA were stored frozen prior the assay, thaw them and keep at least 30 minutes at a temperature of (18 25) C. The isolated DNA can be stored at (2 8) C for 2 days. After initial opening shelf life of Positive Control sample at (2 8) C is 1 month Preparation of the reagents. Prior to the test take the kit out of the refrigerator and keep the Ready Master Mix (RMM 1, 2, 3, 4 and RMM 5) closed in the package at (18 25) C for at least 30 minutes. One RMM 1, 2, 3, 4 plate is sufficient for analysis of 22 specimens. Then open the package and cut the necessary number of tubes in strips with RMM1, 2, 3, 4 and RMM 5 (including prepared samples and controls: 1 NC and 1 PC) with the scalpel. For analysis of each sample (including controls) 4 tubes with RMM 1, 2, 3, 4 (half of the strip containing RMM 1, RMM 2, RMM 3, RMM 4) and 1 tube with RMM 5 is necessary. The tube with RMM 1 is indicated with a line. Cut the tubes together with the covering film. Put the remaining strips immediately back into the foil pouch, squeeze the air out and tightly close with the clip. After initial opening the shelf life of RMM at (2 8) C is 3 months. Open the vial with PC. Add 1 ml of RSC to the vial with PC and carefully mix. Keep at room temperature for 15 min then re-mix. PC is ready for PCR. Store diluted PC at (2 8) C for 1 month. VBD8478 Page 9 of 15

10 Recommended order of tubes in the cycler (Sample validation) RMM5 The first tube for each patient is indicated with the line Sample 17 Sample 15 Sample 13 Sample 11 Sample 9 PC NC Sample 16 Sample 14 Sample 12 Sample 10 Sample 7 Sample 8 Sample 5 Sample 6 Sample 3 Sample 4 Sample 1 Sample 2 PC PC PC PC PC NC NC NC NC NC Analysis of each sample is performed using one strip of four tubes RMM 1, 2, 3, 4 and one tube of RMM 5 Rev _EN Page 10 of 15

11 8. PROCEDURE 8.1. Label the tubes with RMM for each specimen and control. Attention! Labels should be placed on the lateral side of the tubes Add 50 µl of corresponding isolated DNA solution to each tube using a separate pipette tip with filter. Do not touch the pellet! Tightly close the tubes with caps or seal with the PCR transparent film Place the tubes into the Real Time PCR system. Program Real Time PCR system as follows: Stage 1: 50 C 2min Stage 2: 95 C 2min Stage 3: 94 C 10 sec 60 C* 20 sec 50 cycles * Measure the fluorescence at 60 C in FAM, HEX, ROX and Cy Select the amplification detection channels. For RMM 1, 2, 3, 4: Cy5 registration of IC DNA signal; FAM, HEX, ROX registration of DNA signal of HPV of high cancer risk. For RMM 5: Cy5 registration of IC DNA signal; ROX registration of human HMBS gene DNA signal Program the positions of test tubes with samples, positive and negative controls according to the instruction manual for the Real Time PCR system in use Run the program. VBD8478 Page 11 of 15

12 9. DATA ANALYSIS AND INTERPRETATION When using iq5 icycler and CFX96 (Bio-Rad, USA) or similar Real Time PCR systems analysis and culling is carried out in accordance with the enclosed instruction manual For Positive Control PC in each of RMM 1, 2, 3, 4 the program should detect: increase of the IC DNA amplification signal (channel Cy5) and determine the threshold cycle, IC Ct; increase of the HPV DNA amplification signal (channels FAM, HEX, ROX) and determine the PC Ct value for each channel. For PC in RMM 5 the program should detect: increase of the IC DNA amplification signal (channel Cy5) and determine the threshold cycle, IC Ct; increase of the human HMBS gene DNA amplification signal (channel ROX) and determine the PC Ct value for each channel For NC in each of RMM 1, 2, 3, 4 the program should detect: the increase of the amplification signal of IC DNA (channel Cy5) and determine the threshold cycle, IC Ct. No FAM, HEX and ROX fluorescent increase should appear (no HPV DNA amplification) up to Ct 35 for HPV types 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and up to Ct 40 for HPV type 16 and 18. When Ct value for NC in FAM, ROX or HEX channels (for HPV types 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 is less than or equal to Ct 35 or for HPV Types 16 and 18 is less than or equal to Ct 40, this indicates the presence of contamination (see paragraph 9.8) For NC in RMM 5 the program should detect: increase of the amplification signal of IC DNA (channel FAM) and determine the threshold cycle, IC Ct. No ROX fluorescent increase should appear (no HMBS DNA amplification) up to Ct 32. When Ct value for NC through ROX channel is less than or equal to Ct 32 this indicates the presence of contamination (see paragraph 9.8.). Rev _EN Page 12 of 15

13 9.4. Analysis of the sample validation results For each sample in RMM 5 an increase in IC DNA amplification signal (channel FAM) should be recorded Calculate (IC Ct)av as an average IC Ct of all analyzed samples (including PC and NC). IC Ct values that differ by more than 2 from the (IC Ct)av should be ignored. Recalculate the (IC Ct)av for the remaining values after the screening. In case IC Ct values for analyzed sample differ by more than 2 from (IC Ct)av an additional analysis of this sample is necessary starting with the DNA isolation step The sample is considered valid if the Ct value for this sample via ROX in RMM 5 is less than or equal to Ct For samples found to be invalid (Ct through the ROX channel is above Ct 32), a repeated collection of specimens is required Analysis of results of HPV HCR detection For each sample in RMM 1, 2, 3, 4 an increase of the amplification signal of IC DNA (channel Cy5) should be detected and IC Ct determined Calculate (IC Ct)av as an average IC Ct of all analyzed samples (including PC and NC) independently for each of RMM 1, 2, 3, 4. IC Ct values that differ by more than 2 cycles from the (IC Ct)av should be ignored. Recalculate the (IC Ct)av for the remaining values after the screening The sample is considered negative (not containing HPV DNA of the corresponding type), if Ct value via FAM, HEX and ROX channels for this sample is above for HPV types 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and above 40 for HPV type 16 and 18 or is not determined. When IC Ct value for such sample differs from the (IC Ct)av value by more than 2, the result is regarded as equivocal. A repeated analysis of the sample, starting with the DNA isolation step is necessary. VBD8478 Page 13 of 15

14 9.7. The sample is considered positive, i.e. contains HPV DNA, when Ct value via FAM, HEX and ROX channels for this sample (in any of RMM 1,2,3 4 tubes) is less than or equals to Ct 35 for HPV types 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and less than or equals to Ct 40 for HPV type 16 and 18. Correspondence of results with HPV types is presented in the table. Positive detection signal in the channel Tube FAM HEX ROX MM1 type 16 type 39 type 18 MM2 type 33 type 56 type 45 MM3 type 31 type 58 type 35 MM4 type 52 type 59 type 51 Yellow: cut-off is Ct 40 for types 16 and 18 Green: cut-off is Ct 35 for types 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 Note: Analyzed samples may contain HPV DNA of one or several types in any combinations. Attention! Analysis of the sample is considered valid only when the validation analysis proves the sample valid (see p. 9.3). Sample validation enables evaluation of the accuracy of the sample collection and its storage. In case the epithelial scraping sample was obtained wrong (lack of epithelial cells) or stored in conditions that do not ensure the safety of the DNA, the amplification signal of human HMBS gene will be too low, and the sample would be found invalid. A negative result in detection of HPV HCR DNA in this case may be false. Rev _EN Page 14 of 15

15 9.7. Quantitative analysis Quantitative analysis of the HPV HCR DNA with sample validation (quantity of DNA copies per human cells). For a quantitative analysis calculate the concentration of HPV HCR DNA according to the formula: lg C = [(Ct HMBS Ct HPV) + (Ct ICav HMBS Ct IC HMBS) + (Ct IC HPV Ct ICav HPV)] where: Ct HMBS = Ct of the sample through ROX channel in RMM 5; Ct HPV = Ct through of the FAM, HEX, ROX channels in RMM 1-4; Ct ICav HMBS = average Ct in RMM 5; Ct IC HPV = IC Ct of the sample in the RMM 1-4 where the sample was found positive; Ct ICav HPV = average IC Ct in the RMM 1-4 where the sample was found positive. The obtained value corresponds to the decimal logarithm of the concentration of HPV HCR DNA, normalized to human cells In the case of contamination all positive results of this individual PCR run are considered equivocal. Actions are required to identify and eliminate the source of contamination, and repeat the analysis of all samples of this run that were identified as positive. Samples that showed negative results in this run should be considered as negative. 10. STORAGE AND TRANSPORTATION Store the assay kit at (2-8) С in the manufacturer s packing. Transportation at 25 С for up to 10 days is allowed. Do not freeze the kit! Do not pool reagents from different lots or from different vials of the same lot. Strictly follow the instruction manual for reliable results. Do not use kits with damaged inner packages and get in contact with BIORON Diagnostics GmbH. Storage and shelf life of solutions and components of the kit after initial opening: Positive Control sample diluted: 1 month at (2 8) C. Ready Master Mix (RMM): 3 months at (2 8) C Technical Support: techsupport@bioron.de. VBD8478 Page 15 of 15