AmpliSens HPV 6/11-FRT PCR kit

Transcription

1 For Professional Use Only AmpliSens HPV 6/11-FRT PCR kit Instruction Manual AmpliSens Ecoli s.r.o., Studenohorska Bratislava 47 Slovak Republic Tel.: Fax: Federal Budget Institute of Science Central Research Institute for Epidemiology 3A Novogireevskaya Street Moscow Russia

2 TABLE OF CONTENTS 1. INTENDED USE PRINCIPLE OF PCR DETECTION CONTENT ADDITIONAL REQUIREMENTS GENERAL PRECAUTIONS SAMPLING AND HANDLING WORKING CONDITIONS PROTOCOL DATA ANALYSIS TROUBLESHOOTING TRANSPORTATION STABILITY AND STORAGE SPECIFICATIONS REFERENCES QUALITY CONTROL KEY TO SYMBOLS USED REF R-V11(RG,iQ,Mx)-CE / VER / Page 2 of 16

3 1. INTENDED USE AmpliSens HPV 6/11-FRT PCR kit is an in vitro nucleic acid amplification test for qualitative detection and differentiation of Human Papillomavirus (HPV) types 6 and 11 DNA in the clinical material (cervical and urethral scrapes) using real-time hybridizationfluorescence detection of amplified products. The results of PCR analysis are taken into account in complex diagnostics of disease. 2. PRINCIPLE OF PCR DETECTION HPV types 6 and 11 detection by the polymerase chain reaction (PCR) is based on the amplification of a pathogen genome specific region using special HPV 6/11 primers. In the real-time PCR, the amplified product is detected with the use of 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 the realtime PCR allows the detection of accumulating product without re-opening the reaction tubes after the PCR run. The test is based on simultaneous amplification (multiplex-pcr) of DNA fragments of HPV and a fragment of β-globin gene which is used as an internal endogenous control. PCR analysis for HPV types 6 and 11 DNA detection is carried out in one tube. The DNA-target selected as endogenous internal control is a human genome fragment. It must be present in a sample (cervical scrape) in sufficient amount equivalent to the number of cells in the sample ( genomes). Thus, the use of an endogenous internal control makes it possible not only to monitor test stages (DNA extraction and amplification) but also to assess the adequacy of sampling and storage of clinical material. If epithelial swab was taken incorrectly (the number of epithelial cells is insufficient), the amplification signal of β- globin gene will be weak. AmpliSens HPV 6/11-FRT PCR kit uses hot-start, which greatly reduces frequency of nonspecifically primed reactions. Hot-start is guaranteed by using chemically modified polymerase (TaqF). The chemically modified polymerase (TaqF) is activated by heating at 95 ºC for 15 min. HPV genotypes 6 and 11 are low carcinogenic risk viruses that are not associated with cervical carcinoma. Low risk HPV types have a productive effect on cells. These viruses are responsible for onset of genital warts (genital pointed condyloma), recurrent respiratory throat papillomatosis; and more than 95% of these pathologies are associated with REF R-V11(RG,iQ,Mx)-CE / VER / Page 3 of 16

4 genotypes 6 and 11. Necessary to mention, that genotypes 6 and 11 are the part of quadrivalent vaccine which protects from HPV 6/11/16/18 infections. Therefore, detection of HPV types 6 and 11 can help in evaluating of the efficacy of vaccinal prevention from cervical cancer and benign genital tumors. 3. CONTENT AmpliSens HPV 6/11-FRT PCR kit is produced in 1 form: AmpliSens HPV 6/11-FRT PCR kit variant FRT REF R-V11(RG,iQ,Mx)-CE. AmpliSens HPV 6/11-FRT PCR kit, variant FRT includes: Reagent Description Volume, ml Quantity PCR-mix-1-FEP/FRT HPV 6/11 colorless clear liquid tubes PCR-mix-2-FRT colorless clear liquid tubes Polymerase (TaqF) colorless clear liquid tubes Positive Control DNA HPV types 6,11 and human DNA (C+ HPV 6,11 ) colorless clear liquid tube DNA-buffer colorless clear liquid tube Negative Control (C )* colorless clear liquid tube * must be used in the extraction procedure as Negative Control of Extraction (see DNAsorb-AM REF K CE protocol or DNA-sorb-B REF K CE protocol or DNA-sorb-C REF K CE protocol or AmpliSens DNA-sorb-D REF К CE). AmpliSens HPV 6/11-FRT PCR kit is intended for 120 reactions, including controls. 4. ADDITIONAL REQUIREMENTS DNA extraction kit Disposable powder-free gloves and a laboratory coat Pipettes (adjustable) Sterile pipette tips with aerosol filters (up to 200 µl) Tube racks Vortex mixer Desktop centrifuge with rotor for 2 ml reaction tubes REF R-V11(RG,iQ,Mx)-CE / VER / Page 4 of 16

5 PCR box Real-time instruments (for example, Rotor-Gene 3000/6000 (Corbett Research, Australia); icycler iq or icycler iq5 (Bio-Rad, USA); Mx3000P, Mx3005P (Stratagene, USA). Disposable polypropylene PCR tubes (0.1- or 0.2-ml): a) 0.2-ml striped or nonstriped PCR tubes with domed caps or PCR plates with optically transparent thermolabile adhesive films if a plate-type instrument is used; b) 0.2-ml nonstriped PCR tubes with flat caps if a rotor-type instrument is used. Refrigerator for 2 8 C Deep-freezer for the temperature from minus 24 to minus 16 C. Reservoir for used tips. 5. GENERAL PRECAUTIONS The user should always pay attention to the following: Use sterile pipette tips with aerosol filters and use a new tip for every procedure. Store all extracted positive material (specimens, controls and amplicons) away from all other reagents and add it to the reaction mix in a distantly separated facility. Thaw all components thoroughly at room temperature before starting an assay. When thawed, mix the components and centrifuge briefly. Use disposable protective gloves and laboratory cloths, and protect eyes while samples and reagents handling. Thoroughly wash hands afterwards. Do not eat, drink, smoke, apply cosmetics, or handle contact lenses in laboratory work areas. Do not use the PCR kit if the internal packaging was damaged or its appearance was changed. Do not use the PCR kit if the transportation and storage conditions according to the Instruction Manual were not observed. Do not use a kit after its expiration date. Dispose of all specimens and unused reagents in accordance with local regulations. Samples should be considered potentially infectious and handled in biological cabinet in compliance with appropriate biosafety practices. Clean and disinfect all samples or reagents spills using a disinfectant, such as 0.5 % sodium hypochlorite or another suitable disinfectant. Avoid samples and reagents contact with the skin, eyes, and mucous membranes. If these solutions come into contact, rinse the injured area immediately with water and REF R-V11(RG,iQ,Mx)-CE / VER / Page 5 of 16

6 seek medical advice immediately. Safety Data Sheets (SDS) are available on request. The PCR kit is intended for single use for PCR analysis of specified number of samples (see the section Content ). The PCR kit is ready for use in accordance with the Instruction Manual. Use the PCR kit strictly for intended purpose. Use of this product should be limited to personnel trained in DNA amplification techniques. Workflow in the laboratory must be one-directional, beginning in the Extraction Area and moving to the Amplification and Detection Area. Do not return samples, equipment and reagents in the area where 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 Obtaining samples of biological materials for the PCR-analysis, transportation and storage are described in the manufacturer s handbook [1]. It is recommended that this handbook is read before starting work. AmpliSens HPV 6/11-FRT PCR kit is intended for analysis of the DNA extracted with the DNA extraction kits from the clinical material (cervical or urethral scrapes). Female: samples of epithelial cells should be obtained as for cytological examination: Method 1 - The sampling kit with one/two cervical cytobrushes and 2-ml tube with 0.5 ml of Transport Medium with Mucolytic Agent REF 953-CE are used. Place the cervical epithelial scrape (endocervix) taken with the first cervical cytobrush and/or the superficial cervical scrape (ectocervix) taken with the second cervical cytobrush to the tube with transport medium. Break the lower part of the cytobrush and leave it in the tube with transport medium. Method 2 - The Digene cervical sampler (USA), which contains cervical cytobrush and a tube with 1.0 ml of Digene transport medium, is used. Place the cervical epithelial scrape (endocervix) obtained with cytobrush into the tube with Digene transport medium. Method 3 - The sampling kit, which contains the combined gynecological probe for simultaneously taking epithelium from endocervix and ectocervix and 5-ml tube with 2.0 ml of Transport Medium with Mucolytic Agent REF 952-CE, is used. REF R-V11(RG,iQ,Mx)-CE / VER / Page 6 of 16

7 Place the cervical epithelial scrape (endocervix) and superficial cervical scrape (ectocervix) into the tube with transport medium. Break the lower part of the probe and leave it in the tube with transport medium. Method 4 The sampling kit, which contains a combined gynecological probe for simultaneously taking epithelial samples from endocervix and ectocervix and a liquidbased cytology vial with CytoScreen (Italy) or PreservCyt (USA) transport medium, is used. Place the cervical epithelial scrape (endocervix) and superficial cervical scrape (ectocervix) into the tube with transport medium. Break the lower part of the probe and leave it in the vial with transport medium. Male: Obtain urethral epithelial scrape by universal probe, place it into the 2-ml tube with 0.5 ml of Transport Medium with Mucolytic Agent REF 953-CE. Storage conditions: at the temperature from 18 to 25 С no more than 5 days; at the temperature from 2 to 8 С no more than 20 days; at the temperature from minus 24 to minus 16 С for 1 year. Only one freeze-thawing cycle is allowed; in the transport medium for liquid-based cytology at room temperature for 1 year. 7. WORKING CONDITIONS AmpliSens HPV 6/11-FRT PCR kit should be used at C. 8. PROTOCOL 8.1. DNA extraction It is recommended to use the following nucleic acid extraction kits: DNA-sorb-AM REF K CE (for clinical material obtained by the 1 st, 2 nd and 3 rd methods); DNA-sorb-B REF K CE (for clinical material obtained by the 1 st, 2 nd and 3 rd methods); DNA-sorb-C REF K CE (for biopsy of mucous); AmpliSens DNA-sorb-D REF К CE CE (for liquid-based cytology samples). Extract the DNA according to the manufacturer s protocol. REF R-V11(RG,iQ,Mx)-CE / VER / Page 7 of 16

8 8.2. Preparing the PCR Total reaction volume is 25 µl, the volume of DNA sample is 10 µl Preparing tubes for PCR 1. Prepare the mixture of PCR-mix-2-FRT and Polymerase (TaqF). Transfer the content of the tube with Polymerase (TaqF) (30 µl) into the tube with PCR-mix-2-FRT (300 µl). Vortex carefully to avoid foaming. Indicate the mix preparation time on the tube. Prepared mixture is intended for 60 samples. Store at 2-8 C for 3 months and use as required. 2. Prepare the reaction mixture (see Table 1). Add two control reactions (negative and positive controls) and one extra reaction when calculating the reaction mixture volume. Each PCR reaction requires: - 10 µl of PCR-mix-1-FEP/FRT HPV 6/11; - 5 µ of mixture of PCR-mix-2-FRT and Polymerase (TaqF). Number of samples PCR-mix-1- FEP/FRT HPV 6/11, μl Mixture of PCR-mix-2-FRT and polymerase (TaqF), μl Number of samples PCR-mix-1- FEP/FRT HPV 6/11, μl Mixture of PCR-mix-2-FRT and polymerase (TaqF), μl Scheme of reaction mixture preparation REF R-V11(RG,iQ,Mx)-CE / VER / Page 8 of 16 Table Note Given calculated volumes include 2 controls and 1 extra reaction. 3. Take the required number of tubes for amplification for the clinical and control samples. 4. Add 15 µl of prepared reaction mixture into the tubes. 5. Add 10 µl of DNA samples obtained at the DNA extraction stage into prepared tubes. 6. Carry the control amplification reactions: NCA Add 10 µl of DNA-buffer to the tube labeled NCA (Negative Control of Amplification). C+ Add 10 µl of Positive Control DNA HPV types 6,11 and human DNA (C+ HPV 6,11 ) to the tube labeled C+ (Positive Control of Amplification).

9 C Add 10 µl of the sample extracted from the Negative Control (C ) reagent to the tube labeled C (Negative control of Extraction) Amplification. 1. Create a temperature profile on your instrument as follows: Table 2 Amplification program for HPV types 6 and 11 DNA Step Rotor-type instruments 1 Plate-type instruments 2 Temperature, С Time Cycles Temperature, С Time Cycles min min s s 2 35 s 45 1 min 60 Fluorescence 60 Fluorescence 45 detection detection Fluorescent signal is detected in the channels for the FAM, JOE and ROX fluorophores. This program is unified program for both PCR kits AmpliSens HPV 6/11-FRT and AmpliSens HPV 16/18-FRT. AmpliSens-1 universal program and HPV HCR 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 DNA amplification program for the AmpliSens HPV HCR screen-titre-fl PCR kit can also be used (see tables 3, 4, 5, 6). Any combination of the tests (for example, with the tests for detection of STI pathogens DNA) can be performed in one instrument simultaneously with the use of the universal amplification program. The tests for detection of HPV HCR DNA and HPV 6, 11 DNA can be performed simultaneously with the use of HPV HCR 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 DNA amplification program. The analytical features of the PCR kit does not change with the use of AmpliSens-1 universal program and HPV HCR 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 DNA amplification program. REF R-V11(RG,iQ,Mx)-CE / VER / Page 9 of 16 Table 3 AmpliSens-1 amplification program Step Rotor-type instruments 1 Plate-type instruments 2 Temperature, С Time Cycles Temperature, С Time Cycles min min s 95 5 s s s s s 95 5 s 95 5 s s Fluorescence detection s Fluorescence detection s s Fluorescent signal is detected in the channels for the FAM, JOE and ROX fluorophores. The channel for the Cy5 fluorophore is enabled if tests in multiprime format are carried out. 1 For example, Rotor-Gene 3000/Rotor-Gene 6000 (Corbett Research, Australia), Rotor-Gene (QIAGEN, Germany). 2 For example, icycler iq, icycler iq5 (Bio-Rad, USA), Mx3000P, Mx3005P (Stratagene, USA).

10 Table 4 HPV HCR 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 DNA amplification program for rotor-type instruments Step Temperature, С Time Fluorescence detection Cycle repeats Hold min 1 Hold min s Cycling 64 Touchdown: 25 s 5 1 deg. per cycle s s s Cycling 2 40 FAM, JOE, s ROX, Cy5 Table 5 HPV HCR 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 DNA amplification program for icycler iq5 (Bio-Rad, USA) instrument Step Temperature, С Time Fluorescence detection Cycle repeats Cycle min s Cycle 3 65 Touchdown: 55 s 6 1 deg. per cycle s s Cycle s FAM, JOE, ROX, Cy s Table 6 HPV HCR 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 DNA amplification program for Mx3000P (Stratagene, USA) instrument Step Temperature, С Time Fluorescence detection Cycle repeats Segment min 1 Segment min s 64 Segment 3 Touchdown: (Cycling) 1 deg. per cycle 25 s s Segment 4 (Cycling) s s s FAM, JOE, ROX, Cy5 2. Adjust the fluorescence channel sensitivity according to the Important Product 40 REF R-V11(RG,iQ,Mx)-CE / VER / Page 10 of 16

11 Information Bulletin and Guidelines [2]. 3. Insert tubes into the reaction module of the device. 4. Run the amplification program with fluorescence detection. 5. Analyze results after the amplification program is completed. 9. DATA ANALYSIS Analysis of results is performed by the software of the real-time PCR instrument used by measuring fluorescence signal accumulation in three channels: The signal of the HPV type 6 DNA amplification product is detected in the channel for the FAM fluorophore. The signal of the HPV type 11 DNA amplification product is detected in the channel for the JOE fluorophore. The signal of the IC DNA amplification product is detected in the channel for the ROX fluorophore. Results are interpreted by the crossing (or not-crossing) the fluorescence curve with the threshold line set at the specific level that corresponds to the presence (or absence) of a Ct value of the DNA sample in the corresponding column of the results grid. Principle of interpretation is the following: HPV type 6 DNA is detected if the Ct value determined in the results grid in the channel for the FAM fluorophore is less than the boundary Ct value specified in the Important Product Information Bulletin. HPV type 11 DNA is detected if the Ct value determined in the results grid in the channel for the JOE fluorophore is less than the boundary Ct value specified in the Important Product Information Bulletin. The Ct value determined in the channel for the ROX fluorophore can exceed the boundary value or be absent only for the samples for which the positive signals (not equivocal) was determined in the channels for the FAM and JOE fluorophores. HPV type 6 and 11 are not detected in a sample if the Ct value is not determined (absent) in the channels for FAM and JOE fluorophores, whereas the Ct value determined in the channel for the ROX fluorophore is less than the boundary Ct value specified in the Important Product Information Bulletin. The result is equivocal if the Ct value determined in the channel for the FAM or JOE fluorophore is greater than the boundary Ct value specified in the Important Product Information Bulletin, whereas the Ct value determined in the channel for the ROX fluorophore is less than the boundary Ct value specified in the Important Product REF R-V11(RG,iQ,Mx)-CE / VER / Page 11 of 16

12 Information Bulletin. In such cases, the amplification and detection should be repeated. If the positive or equivocal result is obtained in the second run then the result is considered positive. In other case, the result is negative. The result is invalid if the Ct value is not determined (absent) or exceeds the boundary value in the channels for FAM, ROX fluorophores, whereas the Ct value in the channel for the ROX fluorophore is not determined (absent) or greater than the specified boundary Ct value. In such cases, the PCR analysis should be repeated starting from the DNA extraction stage or even re-sampling of material is recommended. Boundary Ct values are specified in the Important Product Information Bulletin enclosed to the PCR kit. See also Guidelines [2] The result of the analysis is considered reliable only if the results obtained for Positive and Negative Controls of amplification as well as for the Negative Control of extraction are correct (see Table 7). Control Stage for control Results for controls Сt value in the channel for fluorophore FAM JOE ROX C DNA/RNA extraction Absent Absent Absent NCA PCR Absent Absent Absent Table 7 С+ PCR <boundary value <boundary value <boundary value 10. TROUBLESHOOTING Results of analysis are not being registered in the following cases: 1. The Ct value determined in the channel for the ROX fluorophore (human DNA (IC) detection) is greater than the boundary value or absent. Insufficient amount of clinical material was collected or an error had place during the sample treatment. The PCR analysis should be repeated starting from the DNA extraction stage or re-sampling of material is recommended. The Ct value determined in the channel for the ROX fluorophore can exceed the boundary value or be absent only for the samples for which the positive signals (not equivocal) was determined in the channels for the FAM and JOE fluorophores. 2. The Ct value is determined for the Negative Control of Amplification (NCA) and/or Negative Control of Extraction (C ) in the channels for the FAM, JOE or ROX fluorophores. It indicates the contamination of samples or reagents. In this case the analysis results are considered to be invalid. The analysis of all the samples should be REF R-V11(RG,iQ,Mx)-CE / VER / Page 12 of 16

13 repeated. The measures for detection and elimination of contamination source must be taken. If you have any further questions or if you encounter problems, please contact our Authorized representative in the European Community. 11. TRANSPORTATION AmpliSens HPV 6/11-FRT PCR kit should be transported at 2 8 ºC for no longer than 5 days. 12. STABILITY AND STORAGE All components of the AmpliSens HPV 6/11-FRT PCR kit are to be stored at 2 8 ºC when not in use(except for Polymerase (TaqF), PCR-mix-1-FEP/FRT HPV 6/11 and PCR-mix-2- FRT). All components of the AmpliSens HPV 6/11-FRT PCR kit are stable until the expiry date stated on the label. The shelf life of reagents before and after the first use is the same, unless otherwise stated. Polymerase (TaqF), PCR-mix-1-FEP/FRT HPV 6/11 and PCR-mix-2-FRT are to be stored at temperature from minus 24 to minus 16 ºC. PCR-mix-1-FEP/FRT HPV 6/11 is to be kept away from light 13. SPECIFICATIONS Sensitivity Analytical sensitivity of AmpliSens HPV 6/11-FRT PCR kit is no less than 1х10 3 genome equivalents per 1 ml of sample (GE/ml) Specificity The claimed analytical features of AmpliSens HPV 6/11-FRT PCR kit are guaranteed only when additional reagents kits DNA-sorb-AM, DNA-sorb-B, or DNA-sorb-C, AmpliSens DNA-sorb-D (manufactured by Federal Budget Institute of Science Central Research Institute for Epidemiology ) are used. The analytical specificity of AmpliSens HPV 6/11-FRT PCR kit is ensured by selection of specific primers and probes, as well as the selection of strict reaction conditions. The primers and probes were checked for possible homologies to all in gene banks published sequences by sequence comparison analysis. The clinical specificity of AmpliSens HPV 6/11-FRT PCR kit was confirmed in laboratory clinical trials. REF R-V11(RG,iQ,Mx)-CE / VER / Page 13 of 16

14 14. REFERENCES 1. Handbook Sampling, transportation, storage of clinical material for PCR diagnostics, developed by Federal Budget Institute of Science Central Research Institute for Epidemiology of Federal Service for Surveillance on Consumers Rights Protection and Human Well-Being, Moscow, Guidelines to the AmpliSens HPV 6/11-FRT PCR kit for qualitative detection and differentiation of Human Papillomavirus (HPV) types 6 and 11 DNA in the clinical material by polymerase chain reaction (PCR) with real-time hybridization-fluorescence detection, developed by Federal Budget Institute of Science Central Research Institute for Epidemiology. 15. QUALITY CONTROL In compliance with Federal Budget Institute of Science Central Research Institute for Epidemiology ISO Certified Quality Management System, each lot of the AmpliSens HPV 6/11-FRT PCR kit has been tested against predetermined specifications to ensure consistent product quality. REF R-V11(RG,iQ,Mx)-CE / VER / Page 14 of 16

15 16. KEY TO SYMBOLS USED Catalogue number Caution Batch code Sufficient for In vitro diagnostic medical device Expiration Date Version Temperature limitation Manufacturer NCA Consult instructions for use Keep away from sunlight Negative control of amplification Date of manufacture C Negative control of extraction Authorised representative in the European Community C+ Positive control of amplification IC Internal control REF R-V11(RG,iQ,Mx)-CE / VER / Page 15 of 16

16 VER KM List of Changes Made in the Instruction Manual Location of changes Cover page Intended use Content Stability and Storage Key to Symbols Used RT Cover page, text LA ME 3. Content Text Essence of changes The phrase For Professional Use Only was added The phrase The results of PCR analysis are taken into account in complex diagnostics of disease was added New sections Working Conditions and Transportation were added The Explanation of Symbols section was renamed to Key to Symbols Used The information about the shelf life of reagents before and after the first use was added Information that PCR-mix-1-FEP/FRT HPV 6/11 is to be kept away from light was added The explanation of symbols was corrected The name of Institute was changed to Federal Budget Institute of Science Central Research Institute for Epidemiology Short designation of the positive control of amplification was corrected: C+ HPV 6,11 instead of C+ 6,11 Corrections according to the template. Grammar corrections AmpliSens DNA-sorb-D nucleic acid extraction kit was 8.1. RNA extraction added Preparing The table Scheme of reaction mixture preparation was tubes for PCR added Amplification 9. Data analysis 10. The sections were rewritten Troubleshooting 14. References The reference to Guidelines was added REF R-V11(RG,iQ,Mx)-CE / VER / Page 16 of 16