AmpliSens HPV 6/11-EPh PCR kit

Transcription

1 For Professional Use Only AmpliSens HPV 6/11-EPh 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 Page 2 of 13

3 1. INTENDED USE AmpliSens HPV 6/11-EPh PCR kit is an in vitro nucleic acid amplification test for qualitative detection and differentiation of Human Papillomavirus types 6 and 11 DNA in the clinical material using electrophoretic detection of the amplified products in agarose gel. The results of PCR analysis are taken into account in complex diagnostics of disease. 2. PRINCIPLE OF PCR DETECTION Detection of Human papillomavirus types 6 and 11 DNA by the polymerase chain reaction (PCR) is based on the amplification of pathogen DNA specific region using specific HPV 6/11 primers. After PCR, the amplified product is detected in agarose gel. AmpliSens HPV 6/11-EPh PCR kit is based on simultaneous amplification (in multiplex PCR) of DNA fragments of human papillomavirus and β-globin gene, which is used as an endogenous internal control. PCR test for HPV types 6 and 11 DNA is performed in a single tube. The DNA-target selected as an endogenous internal control is a human genome fragment. It must be present in the sample (cervical scrape) in sufficient quantities (not less than genome equivalents). Therefore, the endogenous internal control not only allows monitoring of the test stages (DNA extraction and PCR analysis) but also evaluating the clinical material collection and storage adequacy. If the quantity of epithelial cells in the sample is insufficient, the band corresponding to the internal control will be absent in agarose gel. AmpliSens HPV 6/11-EPh PCR kit uses hot-start, which greatly reduces frequency of nonspecifically primed reactions. Hot-start is guaranteed by separation of nucleotides and Taq-polymerase by using wax layer or chemically modified polymerase (TaqF). Wax melts and reaction components mix only at 95 ºC. Chemically modified polymerase (TaqF) activates by heating at 95 ºC for 15 min. 3. CONTENT AmpliSens HPV 6/11-EPh PCR kit is produced in 3 forms: AmpliSens HPV 6/11-EPh PCR kit variant 100 R (0.5 ml tubes), REF V R0,5-CE. AmpliSens HPV 6/11-EPh PCR kit variant 100 R (0.2 ml tubes), REF V R0,2-CE. AmpliSens HPV 6/11-EPh PCR kit variant 50 F, REF V11-50F-CE. AmpliSens HPV 6/11-EPh PCR kit variant 100 R includes: Page 3 of 13

4 Reagent Description Volume, ml Quantity PCR-mix-1-R HPV 6/11 ready-touse single-dose test tubes (under wax) colorless clear liquid tubes of 0.5 or 0.2 ml PCR-mix-2 blue clear blue liquid tube Mineral oil for PCR colorless viscous liquid 4.0 Positive Control DNA HPV types 6,11 and human DNA (C+ HPV 6,11 ) 1 dropper bottle colorless clear liquid tube DNA-buffer colorless clear liquid tube AmpliSens HPV 6/11-EPh PCR kit variant 100 R is intended for 110 reactions, including controls AmpliSens HPV 6/11-EPh PCR kit variant 50 F includes: Reagent Description Volume, ml Quantity PCR-mix-1 HPV 6/11 colorless clear liquid tube 2.5x PCR-buffer blue clear blue liquid tube Polymerase (TaqF) colorless clear liquid tube Mineral oil for PCR Positive Control DNA HPV types 6, 11 and human DNA (C+ HPV 6,11 ) colorless viscous liquid tube colorless clear liquid tube DNA-buffer colorless clear liquid tube AmpliSens HPV 6/11-EPh PCR kit variant 50 F is intended for 55 reactions, including controls. 4. ADDITIONAL REQUIREMENTS DNA extraction kit. Disposable powder-free gloves and laboratory coat. Detection agarose kit. Pipettes (adjustable). Sterile pipette tips with aerosol filters (up to 200 µl). Vortex mixer. Desktop centrifuge with a rotor for 2-ml reaction tubes. Tube racks. PCR box. Page 4 of 13

5 Personal thermocyclers (for example, Gradient Palm Cycler (Corbett Research, Australia), GeneAmp PCR System 2400, GeneAmp PCR System 2700, (Applied Biosystems), Biometra, MiniCycler, PTC-100 (MJ Research), MaxyGene (Axygen). Disposable polypropylene microtubes for PCR with 0.5 ml (0.2) capacity (for example, Axygen, USA). 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 and handle amplicons away from all other reagents. Thaw all components thoroughly at room temperature before starting an assay. When thawed, mix the components and centrifuge briefly. Use disposable protective gloves, laboratory coats, and protect eyes while samples and reagents handling. Thoroughly wash hands afterward. 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 samples and unused reagents in compliance with local regulations. Samples should be considered potentially infectious and handled in a biological cabinet in accordance with appropriate biosafety practices. Clean and disinfect all sample or reagent 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, 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 to the area where the previous step was performed. Page 5 of 13

6 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 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-EPh PCR kit is intended for analysis of DNA extracted with DNA extraction kits from: Cervical or urethral swabs For women: epithelial samples are taken in the same way as for cytological analysis: Method 1. The sampling kit consists of one or two cervical cytobrushes and a 2-ml tube with 0.5 ml of Transport Medium with Mucolytic REF 953-CE. Place the cervical epithelial swab (endocervix) taken with the first cervical cytobrush and/or the superficial cervical swab (ectocervix) taken with the second cervical cytobrush to the tube with transport medium. The working part of the cytobrush should to be broken off and left in the tube with transport medium. Method 2. The sampling kit (Digene, USA) consists of a cervical cytobrush and a tube with 1.0 ml of Digene transport medium. Place the cervical epithelial swab (endocervix) taken with the cervical cytobrush to the tube with Digene transportation medium. Method 3. The sampling kit consists of a combined gynecological probe for simultaneously taking epithelium from endocervix and ectocervix and a 5-ml tube with 2.0 ml of Transport Medium with Mucolytic REF 953-CE. Place the cervical epithelial swab (endocervix) and the superficial cervical swab (ectocervix) into the tube with the transport medium. The working part of the probe is to be broken off and left in the tube with the transport medium. Method 4. The sampling kit consists of a combined gynecological probe for simultaneously taking epithelial samples from endocervix and ectocervix and a jar with transport fixation medium for fluid cytology purchased from CytoScreen (Italy) or PreservCyt (USA). Place the cervical epithelial swab (endocervix) and the superficial cervical swab (ectocervix) into the tube with transport fixation medium. The working part of the probe is to be broken off and left in the tube with the medium. Page 6 of 13

7 For men: Place the urethral epithelial swab taken with a universal probe to a 2.0-ml tube with 0.5 ml of Transport Medium with Mucolytic REF 953-CE. Sample storage conditions: at temperature from 18 to 25 С no more than 5 days; at temperature from 2 to 8 С no more than 20 days; at temperature from minus 24 to minus 16 С no more than 1 year; Only one freeze thaw cycle of biological material is allowed. Material in the transport and fixing environment for liquid cytology stored at room temperature throughout the year. 7. WORKING CONDITIONS AmpliSens HPV 6/11-EPh PCR kit should be used at C. 8. PROTOCOL 8.1. DNA Extraction It s recommended to use following nucleic acid extraction kit: DNA-sorb-AM, REF K CE. Extract the DNA according to the manufacturer s protocol Preparing the PCR. Total reaction volume - 25 µl, volume of DNA sample - 10 µl Preparing tubes for PCR. AmpliSens HPV 6/11-EPh PCR kit variant 100 R: 1. Collect the required number of tubes prepared as described above or tubes with PCRmix-1-R HPV 6/11 and wax for amplification of DNA from clinical and control samples. 2. Add 10 µl of PCR-mix-2 blue to the surface of wax layer, ensuring that it does not fall under the wax and mix with reagents in the tube. 3. Add above 1 drop of mineral oil for PCR (about 25 µl). When using thermocycler with heating cover this step could be omitted. AmpliSens HPV 6/11-EPh PCR kit variant 50 F: 1. Prepare the reaction mix for N reactions: 5(N+1) µl of PCR-mix-1 HPV 6/11 Page 7 of 13

8 9.5(N+1) µl of 2.5x PCR-buffer blue 0.5(N+1) µl of polymerase (TaqF) When calculating reaction mix volume additional reactions should be included: two controls and one extra reaction. It is permitted to mix 2.5x PCR-buffer blue and polymerase (TaqF) in advance. Transfer polymerase (TaqF) (0.03 ml) into the tube with 2.5x PCR-buffer blue (0.6 ml). Spin the tube carefully. Mark the tube indicating the date of preparation. Prepared mix is sufficient for 60 reactions. Store at 2-8 С for up to 3 months. 2. Spin the tube with the reaction mix. Pipette 15 µl of the reaction mix into PCR tubes. 3. Add above 1 drop of mineral oil for PCR. When using thermocycler with heating cover this step could be omitted Amplification 1. Use prepared tubes for PCR. Using tips with aerosol filters add 10 µl of DNA samples obtained from clinical or control samples at the DNA extraction stage, under oil or directly on the level of oil. 2. Carry control amplification reactions: NCA -Add 10 µl of DNA-buffer to the tube for Negative Control of Amplification (NCA). C+ -Add 10 µl of Positive Control DNA HPV types 6, 11 and human DNA the tube for Positive Control of Amplification. 3. Run the following program on the thermocycler (see Tables 1 and 2). When the temperature reaches 95ºC (pause mode), insert tubes into the cells of the thermocycler and press the button to continue. It is recommended to sediment drops from walls of tubes by short centrifugation (1 3 s) before placing them into thermocycler. Page 8 of 13 Table 1 Programming thermocyclers for amplification of HPV types 6 and 11 DNA (variant 100 R) Thermocyclers with active temperature adjustment: GeneAmp PCR System 2400 (Applied Biosystems) GeneAmp PCR System 2700 (Applied Biosystems), Gradient Palm Cycler (Corbett Research) Thermocyclers with block temperature adjustment: Biometra, MiniCycler, PTC-100 (MJ Research) Step Temperature Time Cycles Temperature Time Cycles Temperature Time Cycles 0 95 С pause 95 С pause 95 С pause 1 95 С 5 min 1 95 С 5 min 1 95 С 5 min 1 95 С 10 s 95 С 15 s 95 С 1 min 2 65 С 10 s С 25 s С 1 min С 10 s 72 С 25 s 72 С 1 min 3 72 С 1 min 1 72 С 1 min 1 72 С 1 min С storage 4 С storage 10 С storage

9 Table 2 Programming thermocyclers for amplification of HPV types 6 and 11 DNA (variant 50 F) Thermocyclers with active temperature adjustment: GeneAmp PCR System 2400 (Applied Biosystems) GeneAmp PCR System 2700 (Applied Biosystems), Gradient Palm Cycler (Corbett Research), MaxyGene (Axygen) Thermocyclers with block temperature adjustment: Biometra, MiniCycler, PTC-100 (MJ Research) Step Temperature Time Cycles Temperature Time Cycles Temperature Time Cycles 0 95 С pause 95 С pause 95 С pause 1 95 С 15 min 1 95 С 15 min 1 95 С 15min 1 95 С 10 s 95 С 15 s 95 С 1 min 2 65 С 10 s С 25 s С 1 min С 10 s 72 С 25 s 72 С 1 min 3 72 С 1 min 1 72 С 1 min 1 72 С 1 min С storage 4 С storage 10 С storage 4. Amplification in thermocycler with block temperature adjustment lasts 2 h 30 min, in thermocycler with active temperature adjustment 1 h 50 min. 5. After the reaction is finished, PCR tubes must be collected and transferred to the room for PCR products analysis. The amplification products are analyzed by separation of DNA fragments in agarose gel. The amplified samples can be stored at room temperature for 16 h and at 2-8 С for 1 week (be sure to warm the samples to room temperature before running electrophoresis). 9. DATA ANALYSIS It s recommended to use the following detection agarose kit: EPh variant 200, REF K5-200-CE. Use EPh kit according to the manufacturer s protocol. Analysis of results is based on the presence or absence of specific bands of amplified DNA in agarose gel (1.7%). The length of specific amplified DNA fragments is: HPV type bp HPV type bp Internal Control (fragment of β-globine gene) 723 bp Put on a protective mask or use a glass filter while watching and photographing the gel. Page 9 of 13

10 9.1 Interpretation of results Table 3 Results for controls Control Specific bands in the Controlled agarose gel stage 260 bp 425 bp 723 bp Interpretation NCA Amplification No No No OK C+ Amplification Yes Yes Yes OK The sample is considered to be positive for HPV types 6 and 11 DNA if 260-bp and 425-bp bands are present in agarose gel. Their intensity does not depend on the presence or absence of the Internal Control band (723 bp). The sample is considered to be negative for HPV types 6 and 11 DNA if 260-bp and 425-bp bands are absent whereas the 723-bp band is present. In addition to the specific bands, fuzzy bands corresponding to primer dimers may appear in lanes below the 100-bp level. 10. TROUBLESHOOTING Results of analysis are not taken into account in the following cases: If results of analysis of control points do not correspond to those listed above (Table 3), the tests should be repeated. Discard any reagents that may be suspect. The absence of the band of Internal Control (723 bp) in lanes corresponding to biological samples suggests insufficient quantity of biological material or errors in biological material processing, DNA extraction, or PCR run. The appearance of nonspecific bands of different molecular weight in lanes may be caused by the lack of hot start or an inappropriate temperature regime in the thermocycler. In this case, the results of analysis are invalid. The appearance of specific 325-bp or 425-bp bands in the lanes corresponding to negative controls (NCA, C ) suggests contamination of reagents or samples. In such cases, the results of analysis are considered to be invalid. Analysis of all samples must be repeated and measures to detect and eliminate the source of contamination must be taken. If you have any further questions or if you encounter problems, please contact our Authorized representative in the European Community. Page 10 of 13

11 11. TRANSPORTATION AmpliSens HPV 6/11-EPh 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-EPh PCR kit (except polymerase Taq (F)) are to be stored at 2 8 ºC when not in use. All components of the AmpliSens HPV 6/11- EPh PCR kit are to be stable until labeled expiration date. The shelf life of reagents before and after the first use is the same, unless otherwise stated. Polymerase (Taq F) is to be stored at temperature from minus 24 to minus 16 ºC when not in use. 13. SPECIFICATIONS Sensitivity The analytical Sensitivity of AmpliSens HPV 6/11-EPh PCR kit is 5000 copies per 1 ml for HPV type 6 DNA detection and 1000 copies per 1 ml for HPV type 11 DNA detection Specificity The claimed analytical features of AmpliSens HPV 6/11-EPh PCR kit are guaranteed only when additional kits of reagents, DNA-sorb-AM and EPh (manufactured by Federal Budget Institute of Science Central Research Institute for Epidemiology ) are used. The analytical specificity of AmpliSens HPV 6/11-EPh PCR kit is ensured by selection of specific primers and stringent reaction conditions. The clinical specificity of this 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, QUALITY CONTROL In compliance with the Federal Budget Institute of Science Central Research Institute for Epidemiology ISO Certified Quality Management System, each lot of the AmpliSens HPV 6/11-EPh PCR kit has been tested against predetermined specifications to ensure consistent product quality. Page 11 of 13

12 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 Page 12 of 13

13 List of Changes Made in the Instruction Manual VER Location of changes Essence of changes Through the text Records about PCR kit variant 200 are deleted Cover page The phrase For Professional Use Only was added Intended use 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 Content The Explanation of Symbols section was renamed to Key KM to Symbols Used VV KM Stability and Storage Key to Symbols Used Cover page, text Specifications References Text The information about the shelf life of open reagents 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 Analytical Sensitivity was changed to 5000 copies per 1 ml for HPV type 6 DNA detection and 1000 copies per 1 ml for HPV type 11 DNA detection Section was added Corrections according to the template Page 13 of 13