AmpliSens HCV-EPh PCR kit

Transcription

1 For Professional Use Only AmpliSens HCV-EPh PCR kit Instruction Manual AmpliSens 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 CONTENTS 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...11 REF V1-100-R0,5-CE; REF V1-100-R0,2-CE / VER / page 2 of 12

3 1. INTENDED USE AmpliSens HCV-EPh PCR kit is an in vitro nucleic acid amplification test for qualitative detection of hepatitis C virus (HCV) RNA in the clinical material (blood plasma) 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 Hepatitis C virus detection by the polymerase chain reaction (PCR) is based on the amplification of pathogen cdna specific region using specific hepatitis C virus primers. AmpliSens HCV-EPh 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 a wax layer. Wax melts and reaction components mix only at 95 C. 3. CONTENTS AmpliSens HCV-EPh PCR kit is produced in 2 forms: AmpliSens HCV-EPh variant 100 R (0.5-ml tubes) REF V1-100-R0,5-CE; AmpliSens HCV-EPh variant 100 R (0.2-ml tubes) REF V1-100-R0,2-CE; AmpliSens HCV-EPh variant 100 R includes: Reagent Description Volume, ml Quantity PCR-mix-1-R HCV ready-to-use single-dose test tubes (under wax) colorless clear liquid tubes of 0.5 or 0.2 ml PCR-mix-2 blue blue clear liquid tube Mineral oil for PCR colorless viscous liquid vial Positive Control cdna HCV (C+ HCV ) colorless clear liquid tube DNA-buffer colorless clear liquid tube Negative Control (C )* colorless clear liquid tube Positive Control HCV-rec** colorless clear liquid tubes Internal Control (HCV+HGV+HDV)- rec (IC)*** colorless clear liquid tubes * must be used in the extraction procedure as Negative Control of Extraction. ** serves as Positive Control of Extraction (PCE). *** add 5 µl of Internal Control during the RNA extraction procedure directly to the REF V1-100-R0,5-CE; REF V1-100-R0,2-CE / VER / page 3 of 12

4 sample/lysis mixture (see RIBO-sorb, REF K CE or RIBO-prep, REF K CE protocols). AmpliSens HCV-EPh PCR kit variant 100 R is intended for 110 reactions (including controls). 4. ADDITIONAL REQUIREMENTS RNA extraction kit Reverse transcription kit Agarose gel detection kit Disposable powder-free gloves and laboratory coat. Pipettes (adjustable) Sterile RNase-free pipette tips with aerosol barriers (up to 200 µl) Vortex mixer Thermostatic bath or dry block for tubes with controlled temperature and capability to incubate at C Tube racks. PCR box Personal thermocycler (for example, Terzik (DNA-Technology, Russia), Gradient Palm Cycler (Corbett Research, Australia), MaxyGene (Axygen Scientific, USA), GeneAmp PCR System 2700 (Applied Biosystems, USA), Biometra, MiniCycler, PCR-100 (MJ Research) or equivalent) Disposable polypropylene microtubes for PCR (0.5- or 0.2-ml; for example, Axygen, USA). Refrigerator for 2 8 C. Deep-freezer for 16 C. Waste bin for used tips. 5. GENERAL PRECAUTIONS The user should always pay attention to the following: Use sterile pipette tips with aerosol barriers and use new tip for every procedure. Store and handle amplicons away from all other reagents. Thaw all components thoroughly at room temperature before starting detection. When thawed, mix the components and centrifuge briefly. Use disposable gloves, laboratory coats, 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 REF V1-100-R0,5-CE; REF V1-100-R0,2-CE / VER / page 4 of 12

5 areas. Do not use a kit after its expiration date. Dispose of all samples and unused reagents in compliance with local authorities requirements. 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 contact with the skin, eyes, and mucosa. If skin, eyes, or mucosa contact, immediately flush with water and seek medical attention. Material Safety Data Sheets (MSDS) are available on request. Use of this product should be limited to personnel trained in DNA amplification techniques. The laboratory process must be one-directional, it should begin in the Extraction Area and then move to the Amplification and Detection Areas. Do not return samples, equipment, and reagents to 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. 6. SAMPLING AND HANDLING AmpliSens Obtaining samples of biological materials for PCR-analysis, transportation and storage are described in manufacturer s handbook [1]. It is recommended to read this handbook before starting work. extraction kits from: Peripheral blood plasma HCV-EPh PCR kit is intended for analysis of RNA extracted with RNA Peripheral blood plasma. Blood should be collected in a tube (for instance, Vacuette) that contains 6 % EDTA solution (50 µl of EDTA per 1.0 ml of blood) after overnight fasting. After the tube is filled, invert it several times to ensure adequate mixing. Spin the tube at 3,000 rpm for 10 min. Remove and transfer plasma to a 1.5-ml tube using a tips with aerosol barrier. Plasma should be collected within 6 h from the time of blood taking. Storage of samples: at 2-8 С for 1 week; at or below minus 68 С for 1 year. 7. WORKING CONDITIONS AmpliSens HCV-EPh PCR kit should be used at C. REF V1-100-R0,5-CE; REF V1-100-R0,2-CE / VER / page 5 of 12

6 8. PROTOCOL 8.1 RNA extraction It is recommended to use the following nucleic acid extraction kit: RIBO-sorb, REF K2-1-Et-100-CE; RIBO-prep, REF K2-9-Et-100-CE. Extract RNA in compliance with the manufacturer s protocol. The volume of clinical sample is 100 µl. The volume of Negative Control (C ) is 100 µl. During RNA extraction, add 5 µl of IC to every tube. Prepare the tube with positive control of extraction: add 10 µl of Positive Control HCV-rec and 90 µl of Negative Control (C ) Reverse transcription It is recommended to use the following kit for complementary DNA (cdna) synthesis from RNA: REVERTA-L, REF K CE. Carry out the reverse transcription in compliance with the manufacturer s protocol. The volume of RNA sample is 10 µl Preparing PCR Total reaction volume is 25 µl, volume of cdna sample is 10 µl Preparing tubes for PCR 1. Prepare the required number of tubes with PCR-mix-1-R HCV with wax for amplification of cdna from clinical and control samples. 2. Add 10 µl of PCR-mix-2 blue to the surface of the wax layer of each tube ensuring that it does not fall under the wax and mix with PCR-mix-1-R HCV. 3. Add above 1 drop of mineral oil for PCR (about 25 µl). 4. Add 10 µl of cdna obtained from clinical or control samples using tips with aerosol barrier. 5. Carry out 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 cdna HCV to the tube labeled C+ HCV (Positive REF V1-100-R0,5-CE; REF V1-100-R0,2-CE / VER / page 6 of 12

7 Control of Amplification) Amplification Run the following program on the thermocycler (see Tables 1, 2, 3). When the temperature reaches 95 C (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 centrifuging (1 3 s) before placing them in the thermocycler. Programming thermocyclers for HCV cdna amplification Thermocyclers with active temperature adjustment: Terzik (DNA-Technology) Gradient Palm Cycler (Corbett Research) Step Temperature Time Cycles Temperature Time Cycles 0 95 С pause 95 С pause 1 95 С 5 min 1 95 С 5 min 1 95 С 10 s 95 С 20 s 2 67 С 10 s С 20 s С 10 s 72 С 20 s 3 72 С 1 min 1 72 С 1 min С storage 4 С storage Programming thermocyclers HCV cdna amplification Thermocyclers with active temperature adjustment: MaxyGene (Axygen) GeneAmp PCR System 2700 (Applied Biosystems) Step Temperature Time Cycles Temperature Time Cycles 0 95 С pause 95 С 1 95 С 5 min 1 95 С 5 min 1 95 С 30 s 95 С 30 s 2 67 С 45 s С 30 s С 45 s 72 С 30 s 3 72 С 1 min 1 72 С 1 min С storage 4 С storage Table 1 Table 2 REF V1-100-R0,5-CE; REF V1-100-R0,2-CE / VER / page 7 of 12

8 Programming thermocyclers HCV cdna amplification Thermocyclers with block temperature adjustment: Biometra, MiniCycler, PTC-100 (MJ Research) Step Temperature time cycles 0 95 С pause 1 95 С 5 min 1 95 С 1 min 2 67 С 1 min С 1 min 3 72 С 1 min С storage Table 3 Amplification in thermocyclers with block temperature adjustment lasts 2 h; in thermocyclers with active temperature adjustment, for 1 h 30 min. After the reaction is finished, PCR tubes must be collected and transferred to the room for PCR product analysis. The amplified samples can be stored at room temperature for 16 h or at 2 8 С for 1 week (make sure that samples are warmed the to room temperature before running electrophoresis). 9. DATA ANALYSIS It is recommended that the following detection agarose kit is used: EPh variant 200, REF K5-200-CE. Each gel row should obligatory include C+ control and advisory include a DNA molecular weight marker. Analysis of results is based on the presence or absence of specific bands of amplified cdna in 1.7% agarose gel (agarose for high-resolution DNA electrophoresis is used). The lengths of specific amplified cdna fragments are as follows: HCV 240 bp Internal Control (HCV+HGV+HDV)-rec 550 bp 9.1. Interpretation of results Results for controls Control Step for control Specific bands in the agarose gel 240 bp 550 bp PCE RNA extraction Yes Yes C RNA extraction No Yes NCA PCR No No C+ PCR Yes No Table 4 1. The lane corresponding to Positive Control of Extraction (PCE) must contain 2 bright orange bands: the 240-bp specific band and the 550-bp internal control band. REF V1-100-R0,5-CE; REF V1-100-R0,2-CE / VER / page 8 of 12

9 2. The lane corresponding to Negative Control of Extraction (C ) must contain only 550- bp internal control band. 3. The lain corresponding to Positive Control of Amplification (C+ HCV ) must contain the 240-bp bright orange specific band. 4. The lain corresponding to Negative Control of Amplification (NCA) must not contain any bands. 5. The sample is considered as positive if the 240-bp specific band of high of low intensity is present in the agarose gel, irrespective of presence of the internal control band. The internal control band can be absent in samples with a high concentration of HCV RNA. 6. The sample is considered as negative if it contains only the 550-bp band of high or low intensity. Besides the 240-bp and 550-bp bands, the fuzzy bands of primer dimers may appear in the lanes below the 100-bp level. 10. TROUBLESHOOTING Analysis results are not taken into account in the following cases: 1. If results of the controls do not match with those listed above (Table 4), the appropriate step of the test should be repeated. 2. If none of the 240-bp and 550-bp bands is observed in the lane of the clinical sample, the result of analysis for this sample is invalid, the test should be repeated from the first step (RNA extraction). It can be caused by errors in sample processing, which led to DNA/RNA loss or inhibition of reverse transcription and/or PCR. 3. If both 240-bp and 550-bp bands are absent in lanes corresponding to any sample whereas the nonspecific bands are present, the result of analysis for this sample is considered invalid and PCR should be repeated. It can be caused by the lack of hot start or by incorrect temperature profile of the thermocycler. 4. If the 240-bp specific band appears in lanes corresponding to a Negative Control (NCA, C ), this indicates contamination of reagents or samples. In this case, the results of the analysis of all samples are considered invalid. The test should be repeated from the first step (RNA extraction) and measures to detect and eliminate the source of contamination should be taken. 11. TRANSPORTATION AmpliSens HCV-EPh PCR kit should be transported at 2 8 C for no longer than 5 days. 12. STABILITY AND STORAGE All components of AmpliSens HCV-EPh PCR kit should be stored at 2 8 C when not in REF V1-100-R0,5-CE; REF V1-100-R0,2-CE / VER / page 9 of 12

10 use. All components of the 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. 13. SPECIFICATIONS Sensitivity Analytical Sensitivity of AmpliSens HCV-EPh PCR kit is 10 3 IU/ml (International Units per 1 ml of a peripheral blood plasma) Specificity The claimed analytical features of AmpliSens HCV-genotype-EPh PCR kit are guaranteed only when additional kits of reagents, RIBO-sorb or RIBO-prep, REVERTA-L, and EPh (manufactured by Federal Budget Institute of Science Central Research Institute for Epidemiology ) are used. Specificity of AmpliSens HCV-EPh PCR kit is ensured by selection of specific primers and strict reaction conditions as well as laboratory and clinical trials. 14. REFERENCES Manual Sampling, Transportation and Storage of Clinical Material for PCR Diagnostics, developed by Federal Budget Institute of Science Central Research Institute for Epidemiology, Moscow, QUALITY CONTROL In compliance with Federal Budget Institute of Science Central Research Institute for Epidemiology ISO Certified Quality Management System, each lot of AmpliSens HCV-EPh PCR kit is tested against predetermined specifications to ensure consistent product quality. REF V1-100-R0,5-CE; REF V1-100-R0,2-CE / VER / page 10 of 12

11 16. KEY TO SYMBOLS USED Catalogue number Caution Batch code Sufficient for Research use only Expiration Date Version Temperature limitation Manufacturer PCE NCA Consult instructions for use Positive Control of Extraction Negative control of amplification Date of manufacture C IC Internal control C+ Negative control of extraction Positive control of amplification REF V1-100-R0,5-CE; REF V1-100-R0,2-CE / VER / page 11 of 12

12 VER VV BM IvI List of Changes Made in the Instruction Manual Location of changes Cover page, text Text, Table 4 Title page, Key to symbols used Essence of changes The name of Institute was changed to Federal Budget Institute of Science Central Research Institute for Epidemiology The length of specific amplified fragment of internal control cdna was changed from 440 to 550 bp Symbol IVD in vitro diagnostic medical device was changed to RUO research use only REF V1-100-R0,5-CE; REF V1-100-R0,2-CE / VER / page 12 of 12