AmoyDx PIK3CA Five Mutations Detection Kit Detection of five mutations in PIK3CA gene Instruction for Use Instruction Version: B2.2 Revision Date: June 2016 Store at -20±5
Background For: ADx-PI01 Phosphatidylinositol 3-kinases (PI3Ks), are a group of enzymes, involved in a diverse range of cellular functions including cell proliferation, differentiation, survival and intracellular trafficking. The product of the phosphoinositide-3-kinase catalytic alpha (PIK3CA) gene is a downstream signal transducer of the epidermal growth factor receptor (EGFR). PIK3CA is somatically mutated in several types of human cancer, such as colorectal cancers (CRC), breast cancer and lung cancer. Activating mutations of the PIK3CA gene are associated with drug resistance to EGFR-TKIs through persistent activation of PI3K/Akt pathway. Intended Use The AmoyDx PIK3CA Five Mutations Detection Kit is highly selective and sensitive in detection of five mutations in the PIK3CA gene (Table 1). Our company s patented technology allows detection of 1% mutant DNA in a background of 99% normal DNA (except for PI-M3 and PI-M4, whose sensitivity is 2%), while ensuring that false negatives are minimized. The AmoyDx PIK3CA Five Mutations Detection Kit is CE marked for IVD use in Europe. Table 1. Details of five somatic mutations in PIK3CA gene Name Mutation Base Change Cosmic ID PI-M1 H1047R CAT > CGT 775 PI-M2 H1047L CAT > CTT 776 PI-M3 E542K GAA >AAA 760 PI-M4 E545K GAG> AAG 763 PI-M5 E545D GAG > GAT 765 Kit Contents This kit contains sufficient reagents to carry out 24 tests (Table 2). The assay uses six wells per sample. Table 2. Kit Contents Tube No. Reagents Supplied Volume (μl) Channel 1 H1047R Reaction Mix 650 FAM, HEX/VIC 2 H1047L Reaction Mix 650 FAM, HEX/VIC 3 E542K Reaction Mix 650 FAM, HEX/VIC 4 E545K Reaction Mix 650 FAM, HEX/VIC 5 E545D Reaction Mix 650 FAM, HEX/VIC 6 PIK3CA External Control Reaction Mix 650 FAM 7 PIK3CA Taq DNA Polymerase 50 / 8 PIK3CA Mixed Standard 250 / Equipment and Reagents Not Supplied With Kit 1. Compatible PCR instruments are: Stratagene Mx3000P /Mx3005P, ABI7900, ABI7300, ABI7500, ABI StepOnePlus, BioRad-CFX96, and LightCycler480. (1) This kit is compatible with LightCycler480 II instrument. Fluorescence calibration is required for LightCycler480 I instrument. If the fluorescence crossover occurs in the LightCycler480 II instrument, the fluorescence calibration is also needed prior to the operation. (2) For ABI instruments please set up as follows: Reporter Dye: FAM, VIC; Quencher Dye: TAMRA; 1/6
Passive Reference: NONE. (3) To run the assay on ABI7900HT machine, please use the ABI7900HT adaptor, available from BIOplastics, Cat No. B7900RAN. (4) For ABI7900HT, please set up as follows: Instrument: Standard, Ramp speed: Standard, Reaction volume: 25 µl. (5) For Stratagene Mx3000P /Mx3005P, if there s low net fluorescence signal (dr) but high background signal (R), please reduce the signal gain setting of instrument properly. (6) We recommend that all PCR instruments in use should be conducted fluorescence calibration once a year. 2. Sterile, nuclease-free tubes. 3. Dedicated pipette and filtered pipette tips for handling DNA samples. 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. Do not use the reagent after eight freeze-thaw cycles. Stability The shelf-life of the kit is eight 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 tissue or formalin-fixed paraffin-embedded samples (FFPE) and stored at -20±5 prior to use. High quality DNA is essential and we recommend use of DNA extraction kit (AmoyDx FFPE DNA Kit, Cat No. ADx-FF01, for paraffin embedded specimens; AmoyDx Tissue DNA Kit, Cat No. ADx-TI01, for tissue and pleural effusion specimens). The OD value of DNA samples should be measured using the spectrophotometer after extraction. The NanoDrop 1000 /2000 spectrophotometer is recommended. Make sure A260/A280 value between 1.8 and 2.0. Technological Principles The kit uses novel, proprietary primers and probes in a real-time PCR assay to detect PIK3CA mutations in human genomic DNA. The mutant PIK3CA gene is amplified by the specific primers, and detected by the novel probes. Protocol 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 PIK3CA Mixed Standard (STD) should be analyzed during each PCR run, along with no-template controls. 1. Thaw PIK3CA Reaction Mixes and PIK3CA Mixed Standard., Reaction Mixes and Mixed Standard prior to use. 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 PIK3CA Taq DNA polymerase prior to use. 3. According to the ratio of 0.2 μl PIK3CA Taq DNA Polymerase to 20 μl E542K or E545K Reaction Mix per sample, and 0.16 μl PIK3CA Taq DNA Polymerase to 20 μl other Reaction Mix (or External Control Reaction Mix) per sample, transfer the appropriate amount of PIK3CA Taq DNA Polymerase and Reaction Mixes (or External Control Reaction Mix) into a sterile tube. Note: 2/6
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 20 μl of the above master mix into the appropriate PCR tubes. 6. Add 5 μl sample DNA (see following for sample DNA concentrations), 5 μl PIK3CA Mixed Standard or 5 μl ddh 2 O (no-template control, NTC) to the appropriate PCR tubes. 7. According to different sources, samples can be divided into two groups: paraffin embedded and non-paraffin embedded specimens. a) Non-paraffin embedded specimens include fresh tissue, and frozen pathological sections. i. For non-paraffin embedded samples, the recommended DNA amount in each test tube is 2 ~ 5 ng. b) For paraffin embedded samples, we recommend use of 10 or 15 ng template DNA in each PCR tube based on different storage times. i. Use 10 ng of template DNA for samples with less than 3 years storage time. ii. Use 15 ng of template DNA for samples with more than 3 years storage time. Note: we recommend use of TE (ph = 8.0) for extracted DNA dilution. Suggest using at least 5 µl DNA for dilution within ten-fold series, to ensure the accuracy of final concentration. 8. Seal the PCR tubes. 9. Spin down the PCR tubes gently or centrifuge them briefly to collect the reagents at the bottom of tubes. Note: This spin or centrifuge step is essential for proper mixing of the reagents. 10. Place the PCR tubes into the real-time PCR instrument. A recommended plate layout is shown in Table 3. 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. Table 3 Plate Layout (example for 14 tests) Well 1 2 3 4 5 6 7 8 9 10 11 12 A Sample1 Sample1 Sample1 Sample1 Sample1 Sample1 Sample9 Sample9 Sample9 Sample9 Sample9 Sample9 B Sample2 Sample2 Sample2 Sample2 Sample2 Sample2 Sample10 Sample10 Sample10 Sample10 Sample10 Sample10 C Sample3 Sample3 Sample3 Sample3 Sample3 Sample3 Sample11 Sample11 Sample11 Sample11 Sample11 Sample11 D Sample4 Sample4 Sample4 Sample4 Sample4 Sample4 Sample12 Sample12 Sample12 Sample12 Sample12 Sample12 E Sample5 Sample5 Sample5 Sample5 Sample5 Sample5 Sample13 Sample13 Sample13 Sample13 Sample13 Sample13 F Sample6 Sample6 Sample6 Sample6 Sample6 Sample6 Sample14 Sample14 Sample14 Sample14 Sample14 Sample14 G Sample7 Sample7 Sample7 Sample7 Sample7 Sample7 STD STD STD STD STD STD H Sample8 Sample8 Sample8 Sample8 Sample8 Sample8 NTC NTC NTC NTC NTC NTC 11. Carry out real-time PCR using the cycling conditions described in Table 4. Note: The reaction volume is 25 μl per well (20 μl reagents plus 5 μl template). 3/6
Please pack the post-pcr tubes with two disposable gloves and discard properly. Do NOT open the post-pcr tubes to avoid contamination. Table 4 Cycling Parameters Stage Temperature Time Cycles 1 95 5 min 1 95 25 s 2 3 64 20 s 72 20 s 93 25 s 60 35 s Data collection of FAM and HEX/VIC 72 20 s 15 31 Sample Data Analysis 1. The FAM signals of the mutation detection system indicate the mutation status of the sample. The HEX/VIC signals indicate the internal control status. The internal control amplifies and detects a region of genomic DNA adjacent to the PIK3CA gene. 2. Check the FAM signals from the external control assay: i. The Ct value should be between 15 ~ 21 for paraffin embedded specimens; and between 13~19 for non-paraffin embedded specimens. ii. 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. iii. If the external control assay has fails, the DNA template contains PCR inhibitors, indicating that the DNA needs to be re-extracted. 3. The HEX/VIC signals of the internal control are also used as controls. If the HEX/VIC signals assay fails but the FAM test works well, please continue with the analysis. If both the HEX/VIC and FAM signals fails, the data should be discarded to repeat the experiment. 4. Instrument set-up: ensure the calibration fluorescence is unselected, and select single mutation detection for each tube accordingly. It is necessary to choose reaction wells for positive control, no-template control and samples simultaneously. Then users may adjust the Threshold of FAM amplification curve, and obtain the Ct value of mutant group. 5. The PIK3CA Mixed Standard FAM Ct value should be less than 20, but variation may occur due to different threshold settings on different instruments. Analysis of mutation assay results. See Table 5. 1. 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. 2. Strong Positive: If the sample FAM Ct value is less than the Ct value shown in the Strong Positive row in Table5, the sample is classified as strong positive. 3. Weak Positive: If the sample FAM Ct value is in the range shown in the Weak Positive row in Table 5, the sample is provisionally classified as weak positive. a. If the FAM Ct value is in the Weak Positive range, the Ct of the reaction tube shall be calculated to confirm the result. b. If the Ct value is less than the corresponding Cut-off value of Ct, the sample is confirmed as weak 4/6
positive. Otherwise, the sample is classified as negative or below the detection limit of the kit. 4. The calculation of Ct: Formula 1. Ct = mutant FAM Ct value external control FAM Ct value. a. Where: i. The mutant FAM Ct value indicates the Ct value of the mutant FAM signals from a sample. ii. The external control FAM Ct value indicates the Ct value of the FAM signal in external control tube. Table 5 Results Determination Name of Mutation H1047R H1047L E542K E545K E545D Strong Positive Mutant Ct Value Ct<25 Ct<26 Ct<25 Ct<26 Ct<26 Weak Positive Mutant Ct Value 25 Ct <28 26 Ct <29 25 Ct <29 26 Ct <29 26 Ct <29 Ct Cut-off value 11 12 12 12 12 Negative Mutant Ct Value Ct 28 Ct 29 Ct 29 Ct 29 Ct 29 5. Negative: If the sample FAM Ct value is greater than or equal to the critical negative value shown in the Negative row in Table 5, the sample is classified as negative or below the detection limit of the kit. 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 could always be protected from light to avoid photo bleaching. 8. Only trained professionals could use this kit. Please wear suitable lab coat and disposable gloves. The used kit should be disposed of properly. 5/6
Notes 1. Symbol for "AUTHORISED REPRESENTATIVE IN THE EUROPEAN COMMUNITY". 2. Symbol for "IN VITRO DIAGNOSTIC MEDICAL DEVICE". 3. Symbol for "KEEP DRY". 4. Symbol for "THIS WAY UP". 5. Symbol for "FRAGILE,HANDLE WITH CARE". Information of European Authorised Representative Wellkang Ltd t/a Wellkang Tech Consulting Suite B, 29 Harley Street, London W1G 9QR United Kingdom References 1. Bunney TD & Katan M, 2010. Nature Rev. Cancer. 10: 342-352. 2. Yap TA, et al. 2008.Curr Opin Pharmacol. 8: 393-412. 3. Sartore-Bianchi A, et al. 2009.Cancer Res. 69: 1851-57. 4. Serra V, et al. 2008. Cancer Res. 68: 8022-30. 6/6