Sansure Biotech. Wellkang Ltd Suite B, 29 Harley Street London W1G 9QR UNITED KINGDOM. For Professional Use Only

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1 Wellkang Ltd Suite B, 29 Harley Street London W1G 9QR UNITED KINGDOM For Professional Use Only Reference Number S3002E Product Name Herpes Simplex Virus Type 2 DNA Fluorescence Diagnostic Kit (PCR-Fluorescence Probing) Package Specification 48 tests/kit Intended Use This diagnostic kit is used to detect the presence of Herpes Simplex Virus-2 (HSV-2) DNA in samples such as male urethral swab and female cervical swab. The detection result can be used as an aid in the diagnosis of an HSV-2 infection. The test provides a molecular diagnostics-based solution for early diagnosis of venereal disease and for preliminary screening of high-risk venereal disease groups. Test Principle The diagnostic kit adopts a nucleic acid lysis buffer to allow rapid lysis and release of HSV-2 DNA from a reproductive tract secretion sample. By applying real-time fluorescence quantitative PCR technology, this test uses a pair of specific primers which are designed to target a conserved region of HSV-2 DNA, and a specific fluorescence probe, accompanied with other ingredients in PCR mix, to achieve quick detection of HSV-2 DNA through the change in fluorescent signals. The PCR detection system uses UNG enzyme + dutp contamination-proof system which is able to fully degrade possible unwanted side-products to avoid a false positive result. The PCR detection system uses an internal control, which monitors the presence of PCR inhibitors in test samples by detecting whether the internal control signal is normal, to avoid a false negative result. Components of the Diagnostic Kit No. Reagent Name Specification & Qty. 1 HSV-2-Lysis Buffer 2.5 ml/tube 1 tube 2 HSV-2-Internal Control 50 μl/tube 1 tube 3 HSV-2-Enzyme Mix 96 μl/tube 1 tube 4 HSV-2-PCR Mix 912 μl/tube 2 tubes 5 HSV-2-Positive Reference A (2.00E+07 copies/ml) 50 μl/tube 1 tube 6 HSV-2-Positive Reference B (2.00E+06 copies/ml) 50 μl/tube 1 tube 7 HSV-2-Positive Reference C (2.00E+05 copies/ml) 50 μl/tube 1 tube 8 HSV-2-Positive Reference D (2.00E+04 copies/ml) 50 μl/tube 1 tube 9 HSV-2-Negative Control 50 μl/tube 1 tube 10 HSV-2-Positive Control 50 μl/tube 1 tube Note: 1. Do not mix components from different kit lots. 2. All biological samples inside the detection kit have been inactivated. 3. Self-prepared reagent: sterile saline. Storage The detection kit should be stored at -20±5 C and protected from light. The shelf life of the kit is 12 months. Multiple freeze/thaw cycles should be avoided. Doc. #: HSV-2 Manual Doc. Version: V01 Revision Date: Page 1 / 5

2 Compatible Instrument The product applies to fluorescence PCR instruments such as ABI 7500, Stratagene Mx3000P and Roche Light Cycler 480. Specimen Requirements 1. Applicable specimen type: reproductive tract secretion or semen. 2. Collection of specimen: (Patient is required not to urinate in 2 hours before specimen collection. Refer to Bibliography 1 for collection details.) For male reproductive tract secretion: Clean urethra with sterile saline, then insert sterile calcium alginate swab into urethra for 2-3 cm, rotate the swab 1-2 circles to get the specimen. For female reproductive tract secretion: Clean excessive secretion at cervical mouth, then insert sterile calcium alginate swab 2-3 cm deep in the cervix, rotate the swab for 1-2 circles to get the specimen. The specimen should be collected containing some mucosa. Place the collection swab in a sterile collection tube, seal it and send it for detection. 3. Storage and delivery of specimens: Specimens collected via the above-mentioned method can be used for immediate detection, or stored at 2-8 C for less than 24 hours. If a long term storage is required, the specimen should be stored at -20 C or below. Specimens should be transported in a sealed frozen pitcher with ice or in a sealed foam box with ice. Test Method 1. Pretreatment of specimens (performed at specimen processing region ) 1.1 For reproductive tract secretions Add 1mL of sterile saline to the specimen collection tube, vortex it to wash the cotton swab, and then press to dry the cotton swab against the tube wall and discard it Transfer 500 μl of specimen (if there are excessive secretions or the specimen is turbid, centrifuge it at low speed before transferring.) from the collection tube to a 1.5 ml sterile centrifuge tube. Then centrifuge it at 13,000 rpm for 5 minutes, pipette the supernatant up and discard it. Add 50 μl of lysis buffer to the centrifuge tube. Hold it for 10 minutes to allow lysis. Centrifuge it instantaneously for later. 1.2 For semen: Add 1 ml of sterile saline to the specimen collection tube, vortex it thoroughly, then centrifuge it at 3500 rpm for 1 minute. Transfer 100 μl of specimen to a 1.5 ml sterile centrifuge tube; centrifuge it at 13,000 rpm for 5 minutes; pipette the supernatant up and discard it Add 50 μl of lysis buffer to the centrifuge tube, and hold it for 10 minutes then centrifuge it instantaneously for later. Remarks: the specimens processed via the above steps can be used to test the CT-DNA, UU-DNA, NG-DNA and HSV-DNA. 2. Preparation of reagent (performed at reagent preparation region ) 2.1 Take out each component from the detection kit and place them at room temperature. When the components have reached room temperature, mix them for later. 2.2 Refer to quantities of pretreated specimens, negative controls and positive controls and positive references A-D, pipette appropriate quantities of PCR mix, enzyme mix and internal control (PCR mix 38 μl/test+ enzyme mix 2 μl/test+ internal control 1 μl/test), fully mix them to make a PCR- Mastermix and centrifuge it instantaneously for later. 1 sample 10 samples 24 samples 48 samples PCR mix (μl) Enzyme mix (μl) Internal control (μl) Note: The above configuration is just for your reference and to ensure enough volume of the PCR-Mastermix, more volume of the actual pipetting may be required. 3. Loading of pretreated specimen/ controls/ references (performed at sample processing region ) 3.1 Add 5 μl of pretreated specimen to each PCR reaction tube. Processing of negative control, positive control and quantitative references: add respectively 5 μl of negative control, positive control and quantitative references(containing lysis buffer) to each PCR reaction tube. Doc. #: HSV-2 Manual Doc. Version: V01 Revision Date: Page 2 / 5

3 3.2 Add 40 μl of PCR-Mastermix to each tube, cap it (flick the tube to remove bubbles if exist), centrifuge it at 2000 rpm for 30 seconds, or waggle the tube until it has no obvious liquid beads. 4. PCR Amplification (performed at amplification and analysis region ) (refer to user manual of each instrument to adjust the settings) 4.1 Place PCR reaction tubes into the sample well of the amplification device.set up the negative control, positive control, positive references A-D and unknown samples in the corresponding sequence and input sample information and concentration of positive references A-D. 4.2 Select PCR test channel For ABI and Stratagene series: a. Select FAM channel (Reporter: FAM, Quencher: None) to test HSV-2 DNA. b. Select HEX or VIC channel (Reporter: HEX/VIC, Quencher: None) to test the internal control. c. Set passive reference: None. d. Set sample volume: 50. For Roche LightCycler 480: Choose "New Experiment", click "Dual Color Hydrolysis Probe/UPL Probe" in the drop-down menu of Detection format under Setup panel, then go to the drop-down menu of "Customize": a. Select FAM channel to test HSV-2 DNA. b. Select VIC/HEX/Yellow 555 channel to test the internal control. c. Set reaction volume: Set cycle parameters (the time parameter varies according to different instruments): For ABI, Stratagene series: Step Temperature Time No. of cycle 1 UNG enzyme reaction 50 C 2 min. 1 2 Taq enzyme activation 94 C 5 min. 1 3 Denaturation 94 C 15 sec. 45 Annealing, extension, fluorescence collection 57 C 30 sec.* 4 Device cooling (optional) 25 C 10 sec. 1 Note: Due to the device ABI 7500 s technical specification, it can not be set at 30 sec. but 31 sec. For LightCycler 480 (default value should be chosen for the un-listed parameters) Program Target( C) Acquisition Mode Hold(hh:mm:ss) Cycles Analysis Mode 1 50 None 00:02:00 1 None 2 94 None 00:02:00 1 None 3 94 None 00:00:05 45 Quantification 57 Single 00:00:30 4 (optional) 25 None 00:00:10 1 None When the setting is completed, save settings and carry out the reaction procedure. 5. Result Analysis (refer to user manual of each instrument to adjust the settings) When the reactions are completed, results will be saved automatically. After analysis, adjust Start, End and Threshold values of Baseline (Users can adjust them according to the 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 Analyze to implement the analysis, make sure each parameter satisfy the requirement of the below mentioned 6. Quality Control, then go to Plate window to record the Ct value. 6. 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 Positive References Doc. #: HSV-2 Manual Doc. Version: V01 Revision Date: Page 3 / 5

4 (A, B, C, D) Detection concentration 4.59E+04 ~ 3.65E+05 N/A 40 39, R or Ct value copies/ml Reference Range Through the research on reference values, the Ct reference value for detecting target gene is determined to be 39. The Ct reference value for detecting internal control is 40. Explanation of Detection Result 1. Determination of negative and positive results Conclusion Ct value of Ct value of internal Amplification curve of sample control sample Positive 39 - like "S" Negative N/A 40 like "--" Out of limit (concentration < 4.00E+02 >39 40 like "S" copies/ml) Invalid* - > 40 or N/A - Note: *This suggests an investigation should be carried out to find out the reasons when Ct value of the internal control is > 40 or N/A and retest it. (If repeated tests still produce invalid results, please contact Sansure Biotech at info@sansure.com.cn) 2. Quantification result: 2.1 For samples detected with a value between 4.00E+02 copies/ml E+10 copies/ml, the test results can be applied and reported. 2.2 For samples detected with a value > 4.00E+10 copies/ml, report it with a note "> 4.00E+10 copies/ml". If precise quantification is required, dilute the sample below 4.00E+10 copies/ml and retest it. 2.3 For samples which are detected with a value <4.00E+2 copies/ml, and the internal control is detected positive (Ct value 40), it means that the HSV-2 DNA is below the lower detection limit of the kit. If the internal control's Ct value > 40 or there is no display of Ct value, the detection result is invalid. An investigation should be performed to find out the reasons and then retest it.(if repeated tests still show invalid results, please contact Sansure Biotech.) This kit is only used for qualitative detection of Herpse Simplex Virus-2 (HSV-2) DNA. The quantitative results can only be used for reference and cannot serve as a basis for clinical report. Limitation of Detection Method Detection result is related to sample collection, processing, delivery and storage quality. Any deviation from the stated procedure will lead to an inaccurate detection result. Cross-contamination during specimen processing may result in a false positive result. Product Performance Index When the kit is used to detect enterprise work references, the conformity rate for both negative and positive reaches 100%. Precision test shows excellent reproducibility in both intra-batch and inter-batches with its coefficient of variation of Ct value <10%, and its coefficient of variation of concentration < 50%. The detection limit of this kit is 4.00E+02 copies/ml. It shows no cross-contamination with common sexually transmitted pathogens (CT, UU, NG, HPV, etc.). 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 Doc. #: HSV-2 Manual Doc. Version: V01 Revision Date: Page 4 / 5

5 devices used in different processes and regions should not be cross-used. Sansure Biotech 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 fully thawed at room temperature and mixed thoroughly. 6. If there is no HEX or VIC test channel for fluorescence PCR Light-Cycler, monitoring of internal control can be omitted. Do not add internal control at the above step 2.2 in item 2. Preparation of reagent to avoid interference from extra multicolor fluorescence. 7. Because of the nonuniformity in secretion collection, the quantitative result can only be used for reference. Bibliography 1. Li Jinming. Real-time fluorescence PCR technology [M]. Beijing: People s Military Medical Press, 2007: Gao YIng, Zhao Youyun, Wang Chunxiang. Application of FQ-PCR technology for pregnant woman with Herpes Simplex Virus Type II infections [J] 2008,22(2): StocherM, LebV, BozicM, et al. Parallel detection of five human herpes virus DNAs by a set of Real-time polymerase chain reactions in a single run[j]. J Clin Virol, 2003, 26(1): Weidmann M, Meyer-Koning U, Hufert FT. Rapid detection of herpes simplex virus and varicella-zoster virus infections by Real-time PCR[J]. J Clin Microbiol, 2003, 41(4): 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 No of Production License by Medical Device Division of Hunan Food and Drug Administration Symbols Symbols Meanings Symbols Meanings In Vitro Diagnostic Medical Device Date of Manufacture Use By Caution Temperature Limitation Manufacturer Lot Number Authorized representative in the European Community Number of tests Reference Number Doc. #: HSV-2 Manual Doc. Version: V01 Revision Date: Page 5 / 5