No. Reagent Name Specification & Qty Main Ingredients

Transcription

1 Product Name Human Immunodeficiency Virus Type 1 RNA Quantitative Fluorescence Diagnostic Kit (PCR-Fluorescence Probing) Packaging Specification 24 tests/kit Intended Use Human Immunodeficiency Virus Type 1 (HIV-1) RNA Quantitative Fluorescence Diagnostic Kit is based on real-time PCR method and designed for HIV quantification by amplifying viral RNA fragments extracted from clinical human serum or plasma. The product is intended for assisting HIV-1 infection diagnosis, drug response, monitoring and prognostic evaluation. This kit has not been validated for blood screening or HIV-1 infection confirmation. Clinical background: Human immunodeficiency virus (HIV) is a retrovirus that infects human immune system. So far, two kinds of HIV (HIV-1 and HIV-2) have been identified. HIV-1 is widely spread in the world and causes prevalence of acquired immune deficiency syndrome worldwide. The virus destroys human immune system and results in the loss of abilities of the immune system, thus, many kinds of diseases and cancers are able to exist in the body, eventually leading to AIDS. (Acquired Immunodeficiency Syndrome) HIV detection methods in laboratory mainly include: immunological method to test HIV antibody, detection of HIV P24 antigen, detection of CD4 lymphocyte through cell culture and testing, real-time PCR testing of nucleic acid, RT-PCR, side chain DNA, the nucleic acid sequence-dependent amplification, transcription-mediated amplification and gene chip technology. Test Principle The diagnostic kit uses magnetic bead technology to extract HIV-1 RNA from clinical serum or plasma. By applying real-time fluorescence quantitative PCR technology, this test uses two pairs of specific primers which are designed to target a conserved sequence of HIV-1 RNA and a specific fluorescence probe, accompanied with other ingredients in PCR-Mastermix,, to achieve quantitative detection of HIV-1 RNA through fluorescent signal changes. The PCR detection system uses an internal positive control to monitor the presence of PCR inhibitors in test specimens by detecting whether the internal control is normal or not in order to avoid a false PCR negative result. The PCR detection system uses ROX reference dye to eliminate variations occurring in specimen adding and existing among different tubes, as well as to facilitate the instrument s automatic analysis of the ratio between reported fluorescence and the internal reference fluorescence (ROX), to achieve more accurate quantification. Components of the Diagnostic Kit No. Reagent Name Specification & Qty Main Ingredients Extraction Solution 1 Lysis buffer 15 ml/vial 1 tube SDS, triton X-100, Guanidine isothiocyanate, magnetic beads 2 Nucleic acid washing buffer 1 12 ml/vial 1 tube Triton X-100,Sodium chloride 3 Nucleic acid washing buffer 2 10 ml/vial 1 tube mineral oil 4 Elution buffer 0.6 ml x 1 tube Tris, EDTA 1 HIV Internal Control 50 μl /tube 1 tube Cloning Plasmid 2 HIV PCR Mix 1.2 ml/tube 1 tube primer, probe, dntps, buffer system 3 HIV RT-PCR enhancer 25 μl /tube 1 tube Manganese acetate Amplification Reaction Solution 4 HIV Quantitative Reference A 0.5 ml/tube 1 tube 5 HIV Quantitative Reference B 0.5 ml/tube 1 tube 6 HIV Quantitative Reference C 0.5 ml/tube 1 tube 7 HIV Quantitative Reference D 0.5 ml/tube 1 tube 8 HIV Negative Control 0.5 ml/tube 1 tube HIV negative plasma (inactivated) 9 HIV Strong Positive Control 0.5 ml/tube 1 tube Doc. #: HIV Doc. Version: V01 Revision Date: Page 1 / 5

2 10 HIV Weak Positive Control 0.5 ml/tube 1 tube Note: 1. Do not mix components from different kit lots. 2. All biological samples in the kit have been inactivated. For the specific concentration of HIV quantitative references, please refer to the internal surface of the package box of the diagnostic kit. 3. Self-prepared equipment and consumables: 1.5 ml centrifuge tubes (DNase or RNase free), 0.2 ml PCR reaction tube, tips of 10 μl, 200 μl, and 1000 μl filtered tips, centrifuge, vortex mixer, magnetic bead separator, pipettes. Storage The diagnostic kit should be stored in a sealed pouch, protected from light. The extraction solution should be stored at 2-8, and the amplification reaction solution at -20±5. The shelf life of the kit is 12 months. Warning: 1. The kit stays stable within its shelf life. Once it is opened, do not carry out multiple freeze-thaw cycles for up to 3 times. 2. The kit stays stable within 5 days during the shipment. Compatible Instrument The kit is compatible to fluorescence PCR instruments such as ABI 7500 and Stratagene Mx3000P. Specimen Requirements 1. Applicable specimen type: human serum or plasma 2. Collection of specimen: Collection of serum: sterilize part of skin and use disposable syringe (or Monoject TM Blood Collection Tubes) to draw 5-10 ml venous blood from patients, and place it at room temperature for 1-2 hours until the blood is solidified and clotted. Centrifuge it at rpm for 15 minutes, and then transfer the serum to a 1.5 ml centrifuge tube for later use. Collection of plasma: sterilize part of skin and use Monoject TM Blood Collection Tubes with anticoagulant (EDTA sodium or potassium salt, sodium citrate) to draw proper volume of venous blood, or use disposable syringe to draw venous blood, transfer it to a tube with anticoagulant. Mix them by 6-8 times of gentle inversion. Centrifuge it at rpm for 15 minutes. Transfer the plasma in the upper to a 1.5 ml centrifuge tube for future use. 3. Storage, delivery and acceptance of specimen: Specimen collected via the above mentioned method can be stored at 4 for 4 days, or stored below -20 for 3 months or below -70 for a long term. Caution should be taken to avoid re-freezing and re-thawing. Samples for viral load testing should be transported below -20. The transportation and acceptance of specimens should comply with the requirements of related laws and regulations. The transportation should be escorted by qualified staff approved by relevant department. Use three-layer container to pack the specimen. A check list corresponding to the unique coding of the specimen should be accompanied with the specimen. The check list should indicate the tester, specimen type and other information and should be placed between the second the third layer of the container. Specimens should be packed in a laboratory which is able to treat infectious materials and should be opened in a biological safety cabinet by trained staff with protective clothing, mask and protective goggles. Used package should be sterilized immediately. Test Method 1. Preparation of reagent (performed at reagent preparation region ) 1.1 Take out each component from the detection kit. When each component has fully thawed and there is no precipitate or crystal, mix them for future use. 1.2 Refer to quantities of test specimens, negative controls, strong positive controls, weak positive controls, quantitative references A-D, pipette appropriate volume of Lysis Buffer and HIV internal control (Lysis buffer 600 μl/test+ HIV internal control 0.4 μl/test). Fully mix them into Lysis buffer-mix. Centrifuge it instantaneously for later use. 1.3 Refer to quantities of test specimens, negative controls, strong positive controls, weak positive controls and quantitative references A-D, pipette appropriate volume of HIV PCR mix and RT-PCR enhancer (PCR mix 39 μl/test + RT-PCR enhancer 1 μl/test). Fully mix them and centrifuge it instantaneously for future use. 1.4 Transfer the above-prepared reagents to the specimen processing region for later use. 2. Processing of specimens (performed at specimen processing region ) (negative control, positive control, quantitative reference materials and specimens are processed together) 2.1 Prepare an appropriate number of 1.5 ml sterile centrifuge tubes and mark them respectively with negative control, positive control, quantitative references A to D and test specimen. Add 600 μl of Lysis buffer-mix into each tube. 2.2 Centrifuge the specimens at 6000 rpm for 5 minutes. Add respectively 500 μl of specimen, negative control, positive Doc. #: HIV Doc. Version: V01 Revision Date: Page 2 / 5

3 control, quantitative references A to D into each tube. Cover the tube lid. 2.3 Vortex them for 10 seconds and hold it at room temperature for 30 minutes. 2.4 Place the tubes on the magnetic bead separator for 3 minutes, pipette the solution out gently (Be aware not to touch the brown material bound to the tube wall). 2.5 Add respectively 500 μl of Nucleic acid washing buffer 1 and 400 μl Nucleic acid washing buffer 2 into each tube. Vortex them for 5 seconds. Centrifuge them instantaneously and place them on the magnetic bead separator again. 2.6 After 3 minutes, aspirate the solution out completely by putting the pipette tip into the bottom of the tube and discard it, wait for 1 minute, and then draw all the residual liquid out completely and discard it. 2.7 Add 25μl elution buffer to washed down the magnetic beads to the tube bottom. Pipette them up and down for 3-4 times.place the tubes under room temperature for 10 minutes, then put them on the magnetic-bead separator and keep them there for 3 minutes. Trasnsfer the eluent to a new 1.5 ml nuclease-free centrifuge tube. Mix 20uL the eluted Nucleic acid with 40uL of PCR-Mastermix for amplification. 3. PCR Amplification (performed at amplification and analysis region ) (refer to user s manual for each instrument to do the settings) 3.1 Place PCR reaction tube into the specimen wells of the amplification device. Set up the negative control, positive control, quantitative references A-D and unknown samples in the corresponding sequence and input sample information and concentration of quantitative references A-D. 3.2 Select PCR test channel: For ABI, Stratagene series A. Select FAM channel (Reporter: FAM, Quencher: None) to test HIV-1 RNA; B. Select HEX or VIC channel (Reporter: HEX/VIC, Quencher: None) to test HIV-1 internal control; C. Set reference dye: ROX; D. Set sample volume: Setting of cycle parameters (take ABI, Stratagene series for example) Step Temperature Time Cycle No. 1 Predenaturation and enzyme activation 95 1 min. 1 2 Reverse transcription min. 1 3 cdna predenaturation 95 1 min. 1 Denaturation sec Annealing, extension, fluorescence collection sec. 5 Device cooling sec. 1 Note: Because of the technical specifications of ABI7500, it can not be set at 30 seconds but 31 seconds. 4. Result Analysis (refer to the user manual of each instrument to adjust the settings) After the reactions are completed, results will be saved automatically. Analyze the HIV curve and the HIV internal control curve respectively. After analysis, adjust Start, End and Threshold values of Baseline of the graph (user can adjust according to actual situation. 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 Analyze to implement the analysis and make sure each parameter conform to 5. Quality Control. Then go to Plate window to record quantitative result. 5. Quality Control 5.1 Four HIV-1 Quantitative References: all are detected positive, coefficient of correlation of the standard curve R ; 5.2 HIV-1 Negative Control: no display of Ct value; HIV-1 internal control is detected as positive and Ct value 40; 5.3 HIV-1 Strong Positive Control: detection concentration ranges from 1.58E to 1.58E+006 IU/mL. 5.4 HIV-1 Weak Positive Control: detection concentration ranges from 1.58E to 1.58E+003 IU/mL. 5.5 The above requirements must be satisfied at the same time in the same test, otherwise, the test is treated as invalid, and a retest is needed. Reference Range Through the research on reference values, the lower detection limit of this detection kit is determined to be 50 IU/mL. The Ct reference value of internal control is determined to be 40. Explanation of Detection Result 1. For specimens whose detected values are between 5.0E + 01 IU/mL - 1.0E + 08 IU/mL, the test results can be applied and reported. 2. For specimens whose detected values > 1.0E IU/mL, make a footnote >1.0E IU/mL in the report. If precise Doc. #: HIV Doc. Version: V01 Revision Date: Page 3 / 5

4 quantification is required, dilute the specimen below 1.0E IU /ml and retest it. 3. For specimens whose detected values < 5.0E + 01 IU/mL and the detected internal control is positive (Ct value 40), report that HIV-1 RNA is below the detection limit. If internal control is abnormal (Ct>40 or no display), the detection result is invalid. Measures should be taken to find out reasons and retest it (if repeated tests still show invalid result, please contact Sansure.) Limitations of Detection Method 1. The detection result is only for clinical reference. Clinical diagnosis should take symptoms/physical sign, medical history, lab test and therapeutic response into account. 2. Possible reasons for a false negative result 2.1 Improper specimen collection, processing, delivery and/or low virus titer may cause a false negative result. 2.2 Variation in HIV-1 target sequence and sequence change caused by other reasons may lead to a false negative result. 2.3 Improper reagent storage may lead to a false negative result. 2.4 Other unverified interference or PCR inhibitors may lead to a false negative result. 3. Cross-contamination during specimen processing may also result in a false positive result. 4. Clinical laboratory should provide equipment and staff according to the Guideline for Clinical Gene Amplification Laboratory and National Guideline for Detection of HIV/AIDS. Operations should strictly follow the user s manual. 5. The test is only applicable to serum or plasma sample using ABI7500, Stratagene Mx3000P and other PCR instruments. Product Performance Index 1. Accuracy When the kit is used to test the linear reference of HIV nucleic acid national reference by China Drug and Biological Product Institute or standardized enterprise s reference, the quantitative result conforms to quality standard. Linear tests indicate that the linear range of the kit is 5.0E+001 IU/mL - 1.0E+008IU/mL. 2. Analytical specificity When the kit is used to test the the specificity reference of HIV nucleic acid national reference by China s Drug and Biological Product Institute or negative reference of enterprise s reference, the result is negative and the coincidence rate is 100%. The result is negative when testing other common pathogens(hepatitis A Virus, Hepatitis B Virus, Hepatitis C Virus, Cytomegalovirus, Eptein-Barr Virus and Treponema pallidum) 3. Subtype detection capability It is able to conduct quantification of HIV-1 subtype M and O. When it is used to test positive reference of HIV nucleic acid national reference by China s Drug and Biological Product Institute or enterprise s reference, the result is positive and coincidence rate is 100%. 4. Precision The coefficient of variation for logarithmic value of Intra-batch precision (CV%) 5%. 5. Detection limit When the kit is used to test HIV nucleic acid national reference by China s Drug and Biological Product Institute or enterprise s reference for detection limit, the test result conforms to quality standard. Tests indicate that the detection limit of this kit is 50 IU/mL. 6. Interference material The presence of bilirubin, triglycerides, hemoglobin and total IgG in serum or plasma shows no obvious influence on detection result. When drugs are added to serum or plasma in proportion such as 1.5 μg/ml lamivudine, 3 μg/ml lamivudine, 3 μg/ml zidovudine, 300 U/mL α-interferon or 6 μg/ml didanosine, no influence on detection result is found. 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 shall be handled as if infectious. Wear protective disposable gloves, laboratory coats and eye protection when handling specimens and kit reagents. Wash hands thoroughly after handling specimens and test reagents. Doc. #: HIV Doc. Version: V01 Revision Date: Page 4 / 5

5 Change gloves when necessary to avoid cross-contamination. Handling of waste must meet relevant requirements outlined in Common Criteria of Bio-safety for Microbiology Biomedical Laboratory and Medical Wastes Management Regulations by Health Department. 5. Before use, all reagents must be fully thawed at room temperature and mixed thoroughly; before the specimen is extracted, please make sure the temperature of Lysis buffer, Nucleic acid washing buffer 1 and Nucleic acid washing buffer 2 has reached room temperature or above (suggestion: place them for at least 1 hour at room temperature or incubate them more than 30 min. in 30 water bath). The lysis buffer may produce a brown precipitate, do mix it thoroughly before use. The HIV enhancer easily sticks to the tube wall. Centrifuge it instantaneously for a couple of seconds before use. 6. If there are only two test channels for fluorescence PCR LightCycler (such as FAM and ROX), monitoring of internal control can be omitted. Do not add internal control in step 2.2 of Section 2. Processing of specimens to avoid multicolor fluorescence interference. 7. For the specimen detected as negative, make sure the amplification signal of HIV-1 internal control is normal so as to ensure that the test is operated properly and test reagents are used correctly and that no PCR inhibitors are generated, in order to avoid a false negative result. For the specimen detected as positive, the amplification signal of the internal control need not be considered. 8. As HIV is a RNA virus, use specific vessels and pipettes, disposable centrifuge tubes, PCR tubes and tips which should be DNase and RNase free to avoid degradation. Bibliography 1. Palmer S, Wiegand AP, Maldarelli F, et al. New real-time reverse transcriptase-initiated PCR assay with single-copy sensitivity for human immunodeficiency virus type 1 RNA in plasma. J Clin Microbiol. 2003;41(10): Yun Z, Fredriksson E, Sönnerborg A. Quantification of human immunodeficiency virus type 1 proviral DNA by the TaqMan real-time PCR assay. J Clin Microbiol. 2002;40(10): Sierra S, Kupfer B, Kaiser R. Basics of the virology of HIV-1 and its replication. J Clin Virol. 2005;34(4): Pasternak AO, Adema KW, Bakker M, et al. Highly sensitive methods based on seminested real-time reverse transcription-pcr for quantitation of human immunodeficiency virus type 1 unspliced and multiply spliced RNA and proviral DNA. J Clin Microbiol. 2008;46(7): Piwowar-Manning EM, Henderson TA, Brisbin L, et al. A modified ultrasensitive assay to detect quantified HIV-1 RNA of fewer than 50 copies per milliliter. Am J Clin Pathol. 2003;120(2): 李金明,RNA 病毒扩增检测的质控品和标准品研究进展. 中华检验医学杂志 2004 年第 27 卷第 12 期. Manufacturer s Information Manufacturer s Name Registered Production Address Sansure Biotech Inc. REPUBLIC OF CHINA Phone Number Fax Number Registration Information No. 680, Lusong Road, Yuelu District, Changsha, Hunan Province, PEOPLE S 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 Number of tests Doc. #: HIV Doc. Version: V01 Revision Date: Page 5 / 5