No. Reagent Name Specification & Qty. Main Ingredients. 1 HCV Genotype Extraction Solution ml/vial 1 vial GuScN (1 mol/l), magnetic beads

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1 Reference Number S3034E Product Name Hepatitis C Virus Diagnostic Kit (PCR-Fluorescence Probing) Package Specification 12 tests/kit Intended Use This Hepatitis C Virus Diagnostic Kit is designed for qualitative test of hepatitis C virus genotypes (including genotypes 1b, 1, 2, 3, 4, 5 and 6) from RNA positive samples, by applying real-time fluorescence quantitative PCR technology, and the test results can be used as an aid in the diagnosis of genotypes. Hepatitis C is mainly caused by infection and transmitted through blood. Chronic infection of Hepatitis C Virus can lead to chronic inflammation of liver and fibrosis, and some patients may develop into liver cirrhosis, even Hepatocellular Carcinoma (HCC), which greatly injures patients' health and life quality. Researches have shown that patients clinical manifestations and hepatitis disease severity and chronicity are associated with genotypes, especially that the genotype 1 has much stronger pathogenicity compared to other genotypes,. Test result of this diagnostic kit can be used only for clinical reference. The result alone can not be used as an evidence in confirming or excluding the disease. Test result of the kit can be used to identify genotypes, but not for negative or positive determination of. Test Principle The diagnostic kit uses magnetic bead technology to extract -RNA from serum.by applying one-step technology, the kit uses several specific pairs of primers to target conserved regions of different genotypes, including genotypes 1b, 1, 2, 3, 4, 5 and 6, as well as Taqman fluorescence probes to achieve genotyping detection of RNA through fluorescent signal changes. The PCR detection system uses an internal control to monitor the presence of PCR inhibitors by detecting whether the internal control is normal or not, in order to avoid a false negative result. The PCR detection system uses ROX reference dye to essentially eliminate the variations arising from sample adding and differences among tubes. It facilitates the automatic analysis of the ratio between reported fluorescence and ROX (internal reference fluorescence), to achieve more accurate detection. Components of the Diagnostic Kit Extraction Reagents Amplification Reaction Reagents No. Reagent Name Specification & Qty. Main Ingredients SDS (0.5%), Triton X-100 (2%), 1 Extraction Solution ml/vial 1 vial GuScN (1 mol/l), magnetic beads (200 µg/ml) 2 Extraction Solution ml/tube 1 tube HEPES (200 mmol/l), NaCl (200 mmol/l) 3 Extraction Solution ml/vial 1 vial Triton X-100 (0.2%), NaCl (200 mmol/l) 4 Extraction Solution ml/tube 1 tube Mineral Oil 1 Elution Buffer 0.65 ml/tube 1 tube Tris (1mol/L), EDTA (0.5 mol/l) Primer (8-10 pmol), Probe(5 pmol), 2 1/1b PCR Mix 516 μl/tube 1 tube dntps (3.3 mm), 5 x PCR buffer (10 μl), Tth (10 U), H-Taq (2.5 U) Primer (8-10 pmol), Probe ( /6 PCR Mix 516 μl/tube 1 tube pmol), dntps (3.3 mm), 5 x PCR buffer (10 μl), Tth (10 U), H-Taq Doc. Version: V01 Revision Date: Page 1 / 6

2 (2.5 U) Primer (8-16 pmol), Probe (8pmol), 4 3/5 PCR Mix 516 μl/tube 1 tube dntps (3.3 mm), 5 x PCR buffer (10 μl), Tth (10 U), H-Taq (2.5 U) Primer (8-10 pmol), Probe(5-6 4/Internal control pmol), dntps (3.3 mm), 5 x PCR μl/tube 1 tube PCR Mix buffer (10μL), Tth (10 U), H-Taq (2.5 U) 6 Enhancer 60 μl/tube 1 tube Mn(Oac) 2 (150 mm) negative serum (inactivated ) 7 Negative Control 0.5 ml/tube 1 tube (0 IU/mL) positive serum (inactivated) 8 Positive Control 0.5 ml/tube 1 tube (4.00E+05 IU/mL) Diluted Plasmid solution (2.0E Internal Control 20 μl/tube 1 tube copies/ml) Note: 1. Do not mix components from different kit lots. 2. All biological samples in the detection kit should be handled as if infectious even though they have been inactivated. 3. Additional materials: sterile 1.5 ml centrifuge tubes (RNase Free), 0.2 ml PCR reaction tubes, desktop centrifuge; desktop vortex mixer; separator and various models of pipettes and sterile pipette tips (10 μl, 200 μl, 1000 μl). Storage The extraction reagents (in a bigger box) should be stored in a sealed pouch at 2-8 C and amplification reaction reagents (in a smaller box) at -20±5 C, and protected from light. The shelf life of the kit is 12 months. 1. These reagents can be stored for a period within the manufacturing date specified on the outside of the package and keep stable when not used. The freeze-thaw cycles of amplification reaction reagent should not exceed These reagents keep valid within 5 days of transportation. Compatible Instrument This diagnostic kit is applicable to Stratagene Mx3000P and Slan 96P. Specimen Requirements 1. Applicable specimen type: serum. 2. Collection of serum: disinfect partial skin, use a sterile syringe (or a vacuum blood collection tube) to draw 5~10 ml of venous blood from patients and place it at room temperature for 1-2 hours. When the blood coagulates and clots shrinks, centrifuge it at rpm for 15 minutes to separate serum from the rest of blood, and then transfer the serum to a 1.5 ml sterile tube for future use. 3. Storage and transport of specimens Specimens collected via the above-mentioned method can be used for immediate detection, or stored at 4 C for 3 days, or stored below -20 C for 3 months or stored below -70 C for a long term. Caution should be taken to avoid re-freezing and re-thawing. Specimens should be transported in a sealed frozen pitcher with ice or in a sealed foam box with ice. Test Method 1. Preparation of reagents (performed at reagent preparation region ) 1.1 Take out all components from the two kit boxes and place them at room temperature. When the components reach room temperature, vortex them thoroughly for future use. 1.2 Refer to the quantities of negative control, positive control and specimens, and pipette appropriate quantities of Extraction solution 1 and internal control (Extraction solution 1: 600 μl/test + Internal control: 1 μl/test). Fully mix them to make an Extraction solution 1-mix, and then centrifuge it instantaneously for future use. Components Volume Doc. Version: V01 Revision Date: Page 2 / 6

3 genotype extraction solution 1 N x 600 genotype internal control N x 1 Note: N = Number of Samples+2. Samples may include unknown specimens, negative control, positive control. The above configuration is for your reference and to ensure enough volume of the Extraction solution 1-mix, more volume of the actual pipetting may be required. 1.3 Refer to the quantity of under-test specimen, negative control and positive control, and pipette appropriate quantity of these four PCR mixes and in four centrifuge tubes, according to the ratio of PCR mix 43 μl/test μl/test. Fully mix them to make PCR-Mastermixes, and centrifuge it instantaneously for future use. PCR-Mastermix 1 PCR-Mastermix 2 PCR-Mastermix 3 PCR-Mastermix 4 Compone nts 1/1b PCR 2/6 PCR 3/5 PCR 4/Internal control PCR Volume N x 43 N x 1.2 N x 43 N x 1.2 N x 43 N x 1.2 N x 43 N x 1.2 Note: N = Number of Samples+2. Samples may include unknown specimens, negative control, positive control. The above configuration is just for your reference and to ensure enough volume of the PCR reaction mix, more volume of the theoretical pipetting may be required. 1.4 Transfer the reagents prepared as above to the specimen processing region for future use. 2. Processing of specimens (performed at specimen processing region ) (negative control, positive control and unknown specimens are processed together) 2.1 Specimens processing Prepare appropriate number of 1.5 ml sterile centrifuge tubes. Mark negative control, positive control and under-test specimen. Add 600 μl of Extraction solution 1-mix into each tube Add respectively 300 μl of under-test specimen, negative control and positive control to the above marked tubes Add 100 μl of Extraction solution 2 into each tube (mix them thoroughly before the pipetting). Vortex them for 10 seconds to mix them thoroughly and then hold them at room temperature for 30 minutes Centrifuge them instantaneously and then place the centrifuge tubes on the magnetic bead separator and keep them there for 3 minutes, then aspirate the solution out gently (Be careful not to touch the brown matters bound to the tube wall) Add 600 μl of Extraction solution 3 and 200 μl of Extraction solution 4 into each tube. Vortex them for 5 seconds, centrifuge them instantaneously, and place them on the magnetic bead separator again After 3 minutes, aspirate the solution out by putting the tip into the bottom of the tube. Hold it for 1 minute, and then aspirate all the residual liquid out and discard it Add 50 μl of Elution buffer into each tube and wash down the magnetic beads bound to the tube wall by aspirating and injecting the elution buffer 3~4 times. Hold them for 10 minutes at room temperature and then put the tubes on the magnetic bead separator again for 3 minutes, then transfer the eluted RNA to a new 1.5 ml sterile centrifuge tube. 2.2 Specimens loading Refer to the quantities of under-test specimens, negative control and positive control. Prepare four PCR reaction tubes for each under-test specimen, and add 44.2 μl of the four kinds of PCR-Mastermixes prepared according to step 1.3 of this section into the four PCR reaction tubes Add 10 μl of eluted RNA, negative control and positive control into the four PCR reaction tubes, cover the tubes lid and transfer them to the amplification region. 3. PCR Amplification (performed at amplification and analysis region ) (refer to the user manual of each instrument to Doc. Version: V01 Revision Date: Page 3 / 6

4 adjust the settings) 3.1 Put the PCR reaction tubes into sample wells of amplification instrument. Set up the negative control, positive control and unknown samples in the corresponding sequence and input sample information. 3.2 Select PCR fluorescent channels: For Stratagene and Slan series: Select FAM, HEX or VIC channel to test the RNA genotypes of and internal control; Set passive reference: ROX. 3.3 Set cycle parameters: Step Temperature Time Cycle No. 1 Pre-denaturation and enzyme activation 95 C 1 min. 1 2 Reverse transcription 60 C 30 min. 1 3 cdna-predenaturation 95 C 1 min. 1 4 Denaturation 95 C 15 sec. Annealing, extension, fluorescence collection 60 C 30 sec Device cooling 25 C 10 sec. 1 When settings are completed, save settings and carry out the reaction procedure. 4. Result Analysis (refer to the user manual of each instrument to adjust the settings) When the reactions are completed, results will be saved automatically. Analyze the curve. After analysis, adjust Start, End and Threshold values of Baseline of the graph (Users can adjust according to actual situations. Start value can be set between 3-15, and End value between Adjust the amplification curve of negative control to be flat or below threshold). Click Analysis to implement the analysis so that the parameters comply with the requirements of 5. Quality Control, then go to the Results window to record the detected results. 5. Quality Control The test result is treated as valid if all the conditions in the table bellow are met for the same test. Otherwise the test result is treated as invalid and needs to be re-tested. Positive Control Negative Control Internal Control Ct value 36 N/A 36 Positive Reference Value After research on reference values, the Ct reference value of target gene is determined to be 36. Explanation of Detection Result Serial # & PCR mix If the Ct value in FAM 36, it is If the Ct value in HEX 36, it is 1 1/1b PCR mix 1 1b 2 2/6 PCR mix /5 PCR mix /Internal control PCR mix 4 / For all tests, if there is no Ct value or the Ct value is > 36, report that the genotype 1b, 1, 2, 3, 4, 5 and 6 are negative. Limitations of Detection Method 1. Test results of the diagnostic kit can be used only for clinical reference. The patients symptoms/manifestations, disease histories, other laboratory tests and treatment reactions should all be considered when providing clinical treatment for the patients. 2. Analysis on possibilities of false negative results: 2.1 Incorrect specimen collection, delivery and processing, and low concentration of in the specimen all may cause a false negative result. 2.2 Gene mutation of target sequence or sequence change caused by other reasons may cause a false negative result. Doc. Version: V01 Revision Date: Page 4 / 6

5 2.3 Inappropriate storage of reagents may cause a false negative result. 2.4 Other invalidated interference or PCR inhibitors may cause a false negative result. 2.5 This diagnostic kit is specifically designed for genotype 1 (1b, 1a), 2 (2a), 3 (3a, 3b), 4 (4a), 5 (5a), 6 (6a). If it is used to test other subtypes which are not included in this kit, a false negative result may occur. 3. Crossing contamination during sample processing may cause a false positive result. 4. Clinical laboratory should be equipped with the appropriate devices and staff according to the requirements of Clinical Gene Amplification Laboratory Work Practice. The operations should strictly follow the requirements of the manual. 5. This diagnostic kit can only be used for serum samples on the fluorescence PCR machine including Stratagene Mx3000P, and Slan 96P. Product Performance Index 1. Accuracy: while testing enterprise s positive reference of genotypes, the result of the corresponding genotype is positive. 2. Specificity: while testing enterprise s positive reference of genotypes, the detection results are all negative and the internal control is positive. 3. Repeatability: repeated detection of precision reference samples for 10 times, the detection results are all positive and Ct variation coefficient (CV) is below 10%. 4. Detection limit: the precision reference with detection limit 1000 IU/mL is positive. The lower limit of detection is 1000 IU/mL by testing the lower detection limit. Precautions 1. The product can only be used for in vitro diagnosis. Please read the product manual carefully before operation. 2. Please learn and be familiar with the operation procedures and precautions for each instrument before test. Please make sure quality control for each test. 3. Laboratory management shall strictly follow management practices of PCR gene amplification laboratory; laboratory personnel must receive professional training; test processes must be performed in separated regions; all consumables should be for single use only after sterilization; special instruments and devices should be used for every process; all lab devices used in different processes and regions should not be cross-used. 4. All specimens for detection should be handled as if infectious. Wear laboratory coats, protective disposable gloves and change the gloves often to avoid cross-contamination between samples. Handling of specimens and waste must meet relevant requirements outlined in Biosafety Common Criteria in Microbiological and Biomedical Laboratories and Medical Waste Management Regulations by Ministry of Health. 5. Before use, all reagents must be thawed at room temperature and mixed well, to make sure the temperature of the extraction solution 1, 2, 3 and 4 have reached room temperature or above before experiments (it is recommended to place them for at least 1 hour at room temperature or incubate them for more than 30 minutes in 30 C water bath). The extraction solution 1 may form brown precipitation and must be mixed thoroughly before use. The PCR mix,, elution buffer and internal control can be easily bound to the tube wall, which must be centrifuged instantaneously for several seconds before use. 6. If a real time PCR instrument only has two fluorescent channels, i.e. FAM and HEX, the monitoring of passive reference ROX can be omitted. 7. For the specimen detected as negative, make sure the amplification signal of genotype internal control is normal. If the internal control is not normal, the result is a false negative one, which may be caused by laboratory operation, test reagents, inhibitor samples, etc. For a false negative sample, a retest is recommended or retest after dilution. For positive detection samples, the amplification signal of the internal control is not considered. 8. Since is a RNA virus, cautions should be taken to avoid the RNA degradation by RNase during operation. Choose the RNase-free laboratory supplies. Bibliography Doc. Version: V01 Revision Date: Page 5 / 6

6 1. Lindh M, Hannoun C, et al. Genotyping of hepatitis C virus by Taqman real-time PCR. J Clin Virol Oct;34(2): Nakatani SM, Santos CA, et al.development of Hepatitis C Virus Genotyping by Real-Time PCR Based on the NS5B Region PLoS One.2010 Apr 13;5(4):e Rolfe KJ, Wreghitt TG, et al. A real-time Taqman method for hepatitis C virus genotyping and methods for further subtyping of isolates. Methods Mol Biol. 2009:510: Manufacturer s Information Manufacturer s Name Registered Production Address Inc. No. 680, Lusong Road, Yuelu District, Changsha, Hunan Province, PEOPLE S REPUBLIC OF CHINA Phone Number Fax Number Registration Information Medical Device Manufacturer Production License Number Symbols No of Production License by Medical Device Division of Hunan Food and Drug Administration Symbols Meanings Symbols Meanings In Vitro Diagnostic Medical Device Date of Manufacture Use By Caution Temperature Limitation Manufacturer Lot Number Reference Number Number of tests Doc. Version: V01 Revision Date: Page 6 / 6