AmoyDx JAK2 Mutation Detection Kit

Transcription

1 AmoyDx JAK2 Mutation Detection Kit Detection of V617F mutation in the JAK2 oncogene Instructions For Use For Research Use Only and For Reference Only Instructions Version: B1.2 Date of Revision: June 2016 Store at -20±5

2 Background For: ADx-JA01- RG72-R The JAK family of non-receptor tyrosine kinases, includes JAK1, JAK2, JAK3, and TYK2. Growth factors and cytokines can regulate gene transcription through JAK-dependent activation of signal transducer and activator of transcription (STAT). The JAK-STAT signal transduction pathway regulates cell proliferation, differentiation, apoptosis and immune regulation. Activation of JAK2 by mutation of the amino acid at position 617 (V617F ) is associated with myeloproliferative disorders, including polycythemia vera (mutations found in 90% of cases), essential thrombocythemia (50%) and idiopathic myelofibrosis (50%). JAK2 mutation is the main diagnostic indicator of MPD. The AmoyDx JAK2 Mutation Detection Kit is a highly selective and sensitive assay for detecting the V617F mutation in the JAK2 oncogene. Our company s patented technology allows detection of 1% mutant DNA in a background of 99% normal DNA, while ensuring that false negatives are minimized. The procedure is easily adapted for use in high-throughput sample processing. Intended Use AmoyDx JAK2 Mutation Detection Kit is intended for research use only. Kit Contents This kit contains sufficient reagents to carry out 24 tests (Table 1), and additional mixed standard DNA (genomic DNA plus V617F mutated plasmid DNA) for positive control reactions. Table1 Kit Contents Reagents Supplied Volume (μl) V617F Reaction Mix 700 JAK2 Taq DNA Polymerase 15 JAK2 Positive Control 150 Equipment and Reagents Not Supplied With Kit 1. Compatible PCR instrument is Rotor-Gene 6000 (72 Wells) and Rotor-Gene Q (72 Wells). 2. Sterile, nuclease-free tubes. 3. Dedicated pipette and filter pipette tips for handling DNA template. 4. Sterile, nuclease-free H 2 O. Shipping and Storage The kit requires cold-chain-transportation. All contents of the kit should be stored immediately upon receipt at -20±5 in the dark in a constant temperature freezer. Avoid unnecessary freezing and thawing of the contents of the kit. Stability The shelf-life of the kit is twelve months when stored under the recommended conditions and in the original packaging. Do not use the kit after the stated expiry date. Specimen Material Human genomic DNA must be extracted from blood and stored at -20±5 prior to use. High DNA quality is essential and we recommend use of AmoyDx DNA extraction kit (AmoyDx Blood DNA Kit (Spin Column), Cat No. ADx-BL01, for blood specimens). The OD value of DNA samples should be measured using a spectrophotometer after extraction. The Thermo Fisher NanoDrop 1000/2000 spectrophotometer is recommended. Make sure A 260 /A 280 value is between 1.8 and 2.0. Technological Principles 1/5

3 The kit uses novel, proprietary primers and probes in a real-time PCR assay to detect the JAK2 V617F mutation in human genomic DNA. The mutant JAK2 V617F gene DNA is amplified by the specific primers, and detected by the novel probes. Protocol 1. Each reaction tube includes a mutation detection system and internal control system. The mutation detection system is used to detect the mutation status of the JAK2 gene. The internal control system is designed for detecting the presence of inhibitors, which may lead to false negative results. 2. The threshold at which the signal is detected above background fluorescence is called the Cycle threshold (Ct). The Ct values used to determine if a sample is positive or negative are based on extensive validation. If the Ct value falls within the appointed range (see below), the sample is classed as mutation positive. If the Ct value is outside the appointed range, the sample is classed as negative or below the detection limit of the kit. 3. The JAK2 Positive Control contains a recombinant JAK2 gene with the V617F mutation, and normal human genomic DNA. 4. The mutation assay for each sample and control assay must be analyzed within the same PCR run to avoid run-to-run variations in threshold settings. It is recommended that the JAK2 Positive Control should be analyzed during each PCR run, along with no-template controls (NTC). Experimental Procedure 1. Thaw V617F Reaction Mix and JAK2 Positive Control at room temperature. When the reagents completely thawed, mix the reagents by inverting the tube 10 times and centrifuge briefly to collect the contents at the bottom of the tube. 2. Briefly centrifuge JAK2 Taq DNA polymerase prior to use. 3. According to the ratio of 0.25 μl JAK2 Taq DNA Polymerase to 22 μl V617F Reaction Mix per sample, transfer the appropriate amount of JAK2 Taq DNA Polymerase and V617F Reaction Mix into a sterile tube. Note: The volumes given for each reaction mix have been optimized and validated. Changing volumes of any reagent may result in a loss of performance. Do not store user-prepared mixes, use immediately. Since Taq DNA Polymerase is viscous, please pay attention to the centrifugation and pipetting process. Minimize the contact interface between the pipette tip and Taq DNA Polymerase to avoid adding excess enzyme. 4. Mix the solution thoroughly by gently pipeting it up and down more than 10 times, and then centrifuge briefly. Note: avoid vortexing solutions with Taq DNA polymerase. 5. Transfer 22 μl of the above master mix into the appropriate PCR tubes. 6. Add 3 μl sample DNA (2~3 ng/µl), 3 μl JAK2 Positive Control (PC), or 3 μl ddh 2 O (NTC) to the appropriate PCR tubes. 7. Seal the PCR tubes. 8. Spin down the PCR tubes gently to collect the reagents at the bottom of tubes. Note: this spin step is essential for proper mixing of the reagents. 9. Place the PCR tubes into the real-time PCR instrument. Note: place the PCR tubes into the real-time PCR instrument and start to run immediately. If not, please store the PCR tubes at 4 for no more than 12 hours. 2/5

4 10. Carry out real-time PCR using the cycling conditions described in Table2. Note: For: ADx-JA01- RG72-R Prior to the operation, please set up the PCR program by the following steps: 1select Gain Optimisation, the Auto Gain Optimisation Setup window will open; 2 Click Perform Calibration Before 1st Acquisition and Optimise Acquiring. 3Click OK, then click Close to continue. Please see the following Figure 1, 2 and 3 for more details. Make sure the total volume of solution in each well is 25 µl (22 µl reagents plus 3 µl DNA). Table 2 Cycling Parameters Figure 1 Temperature Time Cycles Stage min 1 Stage s 64 10s s Stage s 60 15s Data collection of FAM and HEX 72 8s 31 Figure 2 Figure 3 Sample Data Analysis 1. The FAM signal indicates the mutation status of sample and the HEX signal indicates the internal control status. 2. Make sure that each well gives a HEX signal except the non-template control. i. The Ct value should be between 13 ~ 20 for paraffin embedded specimens; and between 11~17 ii. iii. for non-paraffin embedded specimens. If the requirements of i) are satisfied, further analysis should be carried out. However, if Ct value is below the indicated range, the DNA is overloaded. The procedure should be repeated with reduced DNA. If the internal control assay has failed or above the indicated range, the DNA template contains PCR inhibitors or input DNA less than requirement. In this case, the DNA should be re-extracted 3/5

5 or input more DNA, and the whole experiment should be carried out again. iv. If the HEX signal assay has failed but the FAM test has worked well, continue with the analysis. 1. Ensure the calibration fluorescence is unselected. Select the sample and control positions as a group. Then adjust the Threshold for FAM amplification curves to obtain the Ct values of the samples and controls. 2. The Positive Control FAM Ct value should be less than 21, but variation may occur due to different threshold settings on different instruments. 5. Analysis of mutation assay results. a) Check the FAM Ct value for each sample. Based on different mutant Ct values, the detection results are divided into strong positive, weak positive or negative. b) Negative: If the sample FAM Ct value is greater than or equal to 26 (the critical negative value), the sample is classified as negative or below the limits of the kit. c) If the FAM Ct value is less than 26 (the critical negative value), please analyze the results by the following approaches: i) Strong Positive: If the sample FAM Ct value is less than 23 (critical positive value), the sample is classified as strong positive. ii) If the sample FAM Ct value is greater than or equal to 23 (critical positive value), the Ct of the reaction tube shall be calculated to confirm the result. If the Ct value is less than 5, the sample is confirmed as weak positive. If the Ct value is greater than or equal to 5, the sample is classified as negative or below the limits of the kit. d) The calculation of Ct: Ct = mutant FAM Ct value internal control HEX Ct value. The mutant Ct value indicates the Ct value of the sample mutant FAM signal; the internal control Ct value indicates the Ct value of internal control HEX signal of the sample. Warnings and Precautions 1. Please read the instruction carefully and become familiar with all components of the kit prior to use. 2. The product specified above does not contain any virus, reagent by-product of the same, or metabolic by-product of Hepatitis A, B, C, D or HIV. 3. Do not exchange and mix up the kit contents with different batches. 4. The kit and its contents cannot be resold or modified for resale without the written approval of manufacturer. 5. Using other sources of reagents is not recommended. Strictly distinguish the reagents from mixed standard to avoid contamination. Otherwise, false positive may be produced. 6. Do the experiments with attention to prevent exogenous DNA contamination to reagents. It is recommended that users have separate, dedicated pipettes and filter pipette tips to add DNA template and during the preparation of reagents. 7. To optimize the activity and performance, mixtures should always be protected from light to avoid photo bleaching. 8. Only trained professionals can use this kit. Please wear suitable lab coat and disposable gloves. The used kit should be disposed of properly. 4/5

6 Notes 9. Symbol for "KEEP DRY" 10. Symbol for "THIS WAY UP" 11. Symbol for "FRAGILE,HANDLE WITH CARE" References 1. Goldin LR, et al. Genome Med : Tefferi A, Leuk Lymphoma : Kilpivaara O, Nat Genet : /5