AmpliSens HPV 16/18-FRT PCR kit

Transcription

1 For Professional Use Only AmpliSens HPV 16/18-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-V12(RG,iQ,Mx)-CE / VER / Page 2 of 14

3 1. INTENDED USE AmpliSens HPV 16/18-FRT PCR kit is an in vitro nucleic acid amplification test for qualitative and quantitative detection and differentiation of Human Papillomavirus (HPV) types 16 and 18 DNA in the clinical material (cervical and urethral scrapes) using real-time hybridization-fluorescence 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 16 and 18 detection by the polymerase chain reaction (PCR) is based on the amplification of the pathogen genome specific region using specific HPV 16/18 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 real-time 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 16 and 18 DNA detection is carried out in one tube. The DNA target selected as an endogenous internal control is a human genome fragment that is present in sample in a sufficient quantity equivalent to that of cells in the sample genomes). 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. AmpliSens HPV 16/18-FRT PCR kit uses hot-start, which greatly reduces the frequency of nonspecifically primed reactions. Hot-start is guaranteed by separation of nucleotides and Taq-polymerase by using chemically modified polymerase (TaqF). The Chemically modified polymerase (TaqF) is activated by heating at 95 ºC for 15 min. AmpliSens HPV 16/18-FRT PCR kit which allows differentiation of two most carcinogenic virus types is recommended as an auxiliary tool for detection of papillomavirus infection. PCR kits that allow diagnostics of a wide range (11-14 genotypes) of highly carcinogenic HPV genotypes such as AmpliSens (electrophoretic detection in agarose gel), AmpliSens REF R-V12(RG,iQ,Mx)-CE / VER / Page 3 of 14 HPV HCR screen-eph HPV HCR screen-fep, AmpliSens HPV HCR screen titre-frt (hybridization fluorescence detection) should be applied first.

4 3. CONTENT AmpliSens HPV 16/18-FRT PCR kit is produced in 1 form: AmpliSens HPV 16/18-FRT PCR kit variant screen-titre-frt REF R-V12(RG,iQ,Mx)-CE. AmpliSens HPV 16/18-FRT PCR kit variant screen-titre-frt includes: Reagent Description Volume, ml Quantity PCR-mix-1-FEP/FRT HPV 16/18 colorless clear liquid tubes PCR-buffer-FRT colorless clear liquid tubes Polymerase (TaqF) colorless clear liquid tubes DNA calibrator C1 HPV 16, 18 colorless clear liquid tubes DNA calibrator C2 HPV 16, 18 colorless clear liquid tubes DNA calibrator C3 HPV 16, 18 colorless clear liquid tubes 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. AmpliSens HPV 16/18-FRT PCR kit is intended for 108 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. PCR box. Real-time instruments (for example, Rotor-Gene 3000/6000 (Corbett Research, Australia), Rotor-Gene Q (QIAGEN, Germany), icycler iq or icycler iq5 (Bio-Rad, USA), Mx3000P or Mx3005P (Stratagene, USA). Disposable polypropylene PCR tubes (0.1- or 0.2-ml). a) 0.2-ml PCR tubes with optical transparent domed or flat caps if a plate-type instrument is used; b) 0.2-ml PCR tubes with flat caps or strips of four 0.1-ml Rotor-Gene PCR tubes if a REF R-V12(RG,iQ,Mx)-CE / VER / Page 4 of 14

5 rotor-type instrument is used. Refrigerator for 2 8 C. Deep-freezer at 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 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 seek medical advice immediately. Safety Data Sheets (SDS) are available on request. 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 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. REF R-V12(RG,iQ,Mx)-CE / VER / Page 5 of 14

6 6. SAMPLING AND HANDLING Obtaining samples of biological materials for PCR-analysis, transportation and storage are described in manufacturer s handbook [1]. It is recommended that this handbook is read before starting the work. AmpliSens HPV 16/18-FRT PCR kit is intended for analysis of the DNA extracted with DNA extraction kits from the clinical material (cervical and 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 2.0-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. Snap off the lower part of the cytobrush and leave it in the tube with transport media. Method 2 The Digene Cervical Sampler (USA), which contains cervical cytobrush and a tube with 1.0-ml of Digene transport medium. 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 simultaneous taking of epithelium from endo-/exocervix and 5.0-ml tube with 2.0 ml of the Transport Medium with Mucolytic Agent REF 953-CE is used. Place the cervical epithelial scrape (endocervix) and superficial cervical scrape (ectocervix) into the tube with the transport medium. Snap off 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 simultaneous taking of epithelium from endo-/exocervix and a vial with CytoScreen (Italy) or PreservCyt (USA) preservation/transportation medium for fluid cytology is used. Place the cervical epithelial scrape (endocervix) and superficial cervical scrape (ectocervix) into the tube with the transport-fixation medium. Snap off the lower part of the probe and leave it in the vial with transport medium. Male: Obtain urethral epithelial scrape by universal probe and place it into the 2.0 ml tube with 0.5 ml of Transport Medium with Mucolytic Agent REF 953-CE. The biological samples can be stored: at the temperature from 18 to 25 С no more than 5 days; REF R-V12(RG,iQ,Mx)-CE / VER / Page 6 of 14

7 at the temperature from 2 to 8 С no more than 20 days; at the temperature below 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 16/18-FRT PCR kit should be used at C. 8. PROTOCOL 8.1. DNA Extraction It s 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 K CE (for liquid-based cytology samples). In the extraction procedure it is necessary to carry out the control reactions as follows: C Add 100 µl of Negative Control (C ) to the tube labelled C (Negative Control of Extraction). Extract the DNA according to the manufacturer s protocol. In case of extracting with the DNA-sorb-AM reagent kit, don t add Internal Control complex (ICc) or Internal Control-FL (IC) Preparing the PCR. The total reaction volume is 25 µl, the volume of the DNA samples is 10 µl Preparing tubes for PCR. 1. Prepare the mixture of PCR-buffer-FRT and polymerase (TaqF). To do this, transfer the whole content of the tube with polymerase (TaqF) (20 µl) into the tube with PCRbuffer-FRT (300 µl). Vortex carefully to avoid foaming. Indicate the date of mixture preparation on the tube. The prepared mixture is intended for analysis of 40 samples. Store at 2-8 ºC for 3 months and use as required. 2. Prepare the reaction mixture (see Table 1). Add four control reactions (negative control REF R-V12(RG,iQ,Mx)-CE / VER / Page 7 of 14

8 and three calibrators) and one extra reaction when calculating the reaction mixture volume. Each PCR reaction requires: 7 µl of PCR-mix-1-FEP/FRT HPV 16/18 8 µl of the mixture of PCR-buffer-FRT and polymerase (TaqF). Number of samples PCR-mix-1- FEP/FRT HPV 16/18, µl Mix of PCRbuffer-FRT and polymerase (TaqF), µl Number of samples PCR-mix-1- FEP/FRT HPV 16/18, µl Mix of PCRbuffer-FRT and polymerase (TaqF), µl Table 1 Scheme of reaction mix preparation If 40 samples are to be simultaneously analysed, the simplified way of reaction mix preparation can be applied. The whole content of one tube with PCR-mix-1- FEP/FRT HPV 16/18 and one tube with polymerase (TaqF) should be transferred into the tube with PCR-buffer-FRT. 4. Take the required number of the tubes for amplification of clinical and control samples. 5. Add 15 µl of prepared reaction mixture into the tube. 6. Add 10 µl of DNA samples obtained at the DNA extraction stage into prepared tubes. 7. Carry out control and calibration amplification reactions: NCA Add 10 µl of DNA-buffer to the tube labeled NCA (Negative Control of Amplification). C Add 10 µl of the sample extracted from Negative Control of Extraction (C ) reagent to the tube labeled C (Negative control of Extraction). DNA calibrators: C1 C2 C3 Add 10 µl of DNA calibrator C1 HPV 16, 18, to the tube labeled C1 Add 10 µl of DNA calibrator C2 HPV 16, 18, to the tube labeled C2 Add 10 µl of DNA calibrator C3 HPV 16, 18, to the tube labeled C3 REF R-V12(RG,iQ,Mx)-CE / VER / Page 8 of 14

9 Amplification 1. Create a temperature profile on your instrument as follows: Amplification program Table 2 Step Rotor-type instruments 1 Plate-type instruments 2 Temperature, С Time Cycles Temperature, С Time Cycles min min s s s Fluorescence detection min Fluorescence detection Fluorescent signal is detected in the channels for the FAM, JOE and ROX fluorophores. AmpliSens-1 amplification program 45 Table 3 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 s s Fluorescence detection s s Fluorescence detection s s Fluorescent signal is detected in the channels for the FAM, JOE and ROX fluorophores. The channels for the ROX and Cy5 fluorophores are enabled if tests in multiprime format are carried out. AmpliSens-1 is the universal amplification program. 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 analytical features of the PCR kit do not change with the use of AmpliSens-1 universal program. 2. Adjust the fluorescence channel sensitivity according to the Important Product 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 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). REF R-V12(RG,iQ,Mx)-CE / VER / Page 9 of 14

10 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 16 DNA amplification product is detected in the channel for the FAM fluorophore; The signal of the HPV type 18 DNA amplification product is detected in the channel for the ROX fluorophore; The signal of the internal endogenous control (IC) β-globin DNA amplification product is detected in the channel for the JOE 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. Based on the obtained Ct values and specified concentration values of DNA calibrators (C1, C2 and C3) a calibration line is plotted and the number of copies of HPV types 16 and 18 DNA as well as human DNA in the PCR sample is calculated. Obtained values are used for calculation of HPV types 16 and/or 18 DNA quantity per 1х10 5 of human cells. HPV DNA concentration values are calculated according to the formula: number of HPV (16 or 18) DNA copies lg [ x2*10 5 ] number of human DNA copies =lg(hpv per 10 5 cells) Concentration values of DNA-calibrators are specified in the Important Product Information Bulletin enclosed to the PCR kit. The obtained result is interpreted according to the table below. Result Results interpretation for the test samples Interpretation <3 lg (HPV per 10 5 human cells) Clinically insignificant value. Clinically significant value. 3-5 lg (HPV per 10 5 human cells) Dysplasia cannot be excluded. Risk of dysplasia development. >5 lg (HPV per 10 5 Clinically significant, increased value human cells) High probability of dysplasia Table TROUBLESHOOTING Results of analysis are not taken into account in the following cases: 1. If human DNA concentration volume is less than 1,000 copies per tube. 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 REF R-V12(RG,iQ,Mx)-CE / VER / Page 10 of 14

11 stage or re-sampling of material is recommended. 2. The correlation coefficient R is less than 0.9 when plotting the calibration curve. The amplification and detection for all the samples should be repeated. 3. If Ct value is determined for the Negative Control of Extraction (C ) in the channels for the FAM, JOE or ROX fluorophores and for the Negative Control of Amplification (NCA) in any of the channels for the FAM, JOE or ROX fluorophores It indicates the contamination of samples or reagents. The analysis of all the samples should be 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 16/18-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 16/18-FRT PCR kit are to be stored at 2 8 ºC when not in use (except for polymerase (TaqF) and PCR-mix-1-FEP/FRT HPV 16/18). All components of the AmpliSens HPV 16/18-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) and PCR-mix-1-FEP/FRT HPV 16/18 are to be stored at the temperature from minus 24 to minus 16 ºC. PCR-mix-1-FEP/FRT HPV 16/18 is to be kept away from light. 13. SPECIFICATIONS Sensitivity Analytical sensitivity of AmpliSens 1х10 3 genome equivalents per 1 ml of sample (GE/ml). HPV 16/18-FRT PCR kit is no less than The claimed analytical features of AmpliSens HPV 16/18-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. REF R-V12(RG,iQ,Mx)-CE / VER / Page 11 of 14

12 13.2. Specificity The analytical specificity of AmpliSens HPV 16/18-FRT PCR kit is ensured by the selection of specific primers and probes as well as stringent reaction conditions. The primers and probes have been checked for possible homologies to all sequences published in gene banks by sequence comparison analysis. The clinical specificity of AmpliSens HPV 16/18-FRT PCR kit was confirmed in laboratory clinical trials. 14. REFERENCES 1. Handbook Sampling, Transportation, and 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 16/18-FRT PCR kit for qualitative and quantitative detection and differentiation of Human Papillomavirus (HPV) types 16 and 18 DNA in the clinical material by the 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 16/18-FRT PCR kit has been tested against predetermined specifications to ensure consistent product quality. REF R-V12(RG,iQ,Mx)-CE / VER / Page 12 of 14

13 16. KEY TO SYMBOLS USED Catalogue number Sufficient for Batch code Expiration Date In vitro diagnostic medical device Consult instructions for use Version Temperature limitation NCA Keep away from sunlight Negative control of amplification Manufacturer C Date of manufacture C+ Negative control of extraction Positive control of amplification Authorised representative in the European Community IC Internal control Caution REF R-V12(RG,iQ,Mx)-CE / VER / Page 13 of 14

14 VER KM RT LA PM PM ME List of Changes Made in the Instruction Manual Location of changes Cover page Intended use Content Stability and Storage Key to Symbols Used 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 The names of DNA calibrators were given in full (e.g. DNA calibrator C1 HPV 16, 18) 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 16/18 is to be kept away from light was added The explanation of symbols was corrected The name of Institute was changed to Federal Budget Cover page, text Institute of Science Central Research Institute for Epidemiology Due to replacing appendix 1, 2, 3 with guidelines, Text references to appendixes were substituted by reference to guidelines PCR kit intended use was specified ( qualitative 1. INTENDED USE detection was changed to qualitative and quantitative detection ) 8. PROTOCOL Information about extraction methods was added. Through the text Text 8.1. DNA extraction Amplification 9. Data analysis 10. Troubleshooting Text Instruction manual was corrected in accordance with the template Corrections according to the template. Grammar corrections AmpliSens DNA-sorb-D nucleic acid extraction kit was added The sections were rewritten The reference number of AmpliSens DNA-sorb-D nucleic acid extraction kit was changed REF R-V12(RG,iQ,Mx)-CE / VER / Page 14 of 14