AmoyDx PIK3CA Five Mutations Detection Kit

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1 AmoyDx PIK3CA Five Mutations Detection Kit Detection of five mutations in PIK3CA gene Instruction for Use Instruction Version: B 1.0 Revision Date: May 2013 Store at -20±2 Manufacturer: Amoy Diagnostics Co., LTD Website:

2 Background For: ADx-PI01-RG72 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 the detection of five mutations in PIK3CA gene (Table 1). Our company s patented technology allows detection of 1% mutant DNA in a background of 99% normal DNA (except PI-M1, sensitivity 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), and additional PIK3CA Mixed Standard DNA for positive control reactions. The PIK3CA Taq Mix contains the Taq DNA polymerase for PCR amplification and uracil-n-glycosylase which works at room temperature to prevent PCR amplicon carryover contamination. The Reaction Mix in tube 1-5 contains the reagents for the detection of PIK3CA mutation and internal control, the PIK3CA mutation is indicated by FAM signal, and the internal control is indicated by HEX signal. The Reaction Mix in tube 6 is external control, which is used to assess the DNA quality and indicated by FAM signal. Table 2 Kit Contents Tube No. Reagents Supplied Volume (μl) 1 H1047R Reaction Mix H1047L Reaction Mix E542K Reaction Mix E545K Reaction Mix E545D Reaction Mix PIK3CA External Control Reaction Mix PIK3CA Taq Mix 80 8 PIK3CA Mixed Standard 250 1/5

3 Equipment and Reagents Not Supplied With Kit 1. Compatible PCR instruments are 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. 4. Sterile, nuclease-free H 2 O. Shipping and Storage For: ADx-PI01-RG72 The kit requires cold-chain-transportation. All contents of the kit should be stored immediately upon receipt at -20±2 in the dark in a constant temperature freezer. Avoid unnecessary freezing and thawing of the kit contents more than 5 times. Once opened, this reagent is stable at -20±2 until the expiry. 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 blood, or formalin-fixed paraffin-embedded (FFPE) tissue prior to use and stored at -20±2. High DNA quality 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 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 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 Mix and PIK3CA Mixed Standard at room temperature. 2. Centrifuge PIK3CA Reaction Mix, PIK3CA Mixed Standard and PIK3CA Taq Mix prior to use. 3. According to the ratio of 0.25 μl PIK3CA Taq Mix to 35 μl H1047L Reaction Mix per sample, and 0.35 μl PIK3CA Taq Mix to 35μL PIK3CA Reaction Mix (or PIK3CA External Control Reaction Mix) per sample, transfer the appropriate amount of PIK3CA Taq Mix and PIK3CA Reaction Mix (or PIK3CA External Control Reaction Mix) into a sterile tube. 4. Mix the solution thoroughly by gently pipetting it up and down. Note: avoid vortexing solutions with PIK3CA Taq Mix. 5. Transfer 35 μl of the above mixed solution 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 reaction tubes. 5. 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, frozen pathological sections, non-heparin anticoagulant blood plasma, blood serum and non-heparin anticoagulant blood. 2/5

4 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 time. i. Use 10 ng template DNA for samples less than 3 years. ii. Use 15 ng template DNA for samples more than 3 years. Notes: We recommend use of 1 TE (ph = 8.0) for extracted DNA dilution. Since Taq Mix is viscous, please pay attention to the centrifugation and pipetting process. Minimize the contact interface between the pipette tip and PIK3CA Taq Mix to avoid adding excess enzyme. 7. Seal the PCR tubes. 8. Place the PCR tubes into the real-time PCR instrument. 9. Carry out real-time PCR using the cycling conditions described in Table 3. Note 1: Prior to the operation, please set up the PCR program according to 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. Note 2: Make sure the total volume of solution in each well is 40 µl (35 µl reagent plus 5 µl DNA). Table 3 Cycling Parameters Figure 1 Temperature Time Cycles Stage min 1 Stage s 64 20s 72 20s 15 Stage s 60 35s Data collection of FAM and HEX 72 20s 31 Figure 2 Figure 3 3/5

5 Sample Data Analysis 1. The FAM signal of the mutation detection system indicates the mutation status of the sample. The HEX signal indicates the internal control status. The internal control amplifies and detects a region of genomic DNA adjacent to the PIK3CA gene. 2. Check the FAM signal from the external control assay: (1) The Ct value should be between 10~19. (2) If the requirements of item (1) are satisfied, further analysis should be carried out. However, if the external control Ct < 10, it indicates that the DNA is overloaded, the procedure should be repeated with reduced DNA. If all the results of five tubes (1-5) are negative, the sample is classified as negative. (3) If the external control Ct > 19, it indicates that the DNA template contains PCR inhibitors, thus the DNA need to be re-extracted. If the result in any of the five tubes (1-5) is positive, the sample is classified as positive. 3. The HEX signal in the internal control is also used as control. If the HEX signal fails but the FAM signal works well, continue with the analysis. If both the HEX and FAM signals fail, the obtained data must be discarded and the experiment should be repeated. 4. Analyze one sample at a time. It s necessary to choose the reaction wells for positive control, no-template control and sample simultaneously. Then users can 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 21, but variation may occur due to different threshold settings on different instruments. 6. Analysis of mutation assay results (see Table 4): (1) The calculation of Ct: Ct = mutant FAM Ct value external control FAM Ct value. The mutant FAM Ct value indicates the Ct value of the sample s FAM signal; the internal control HEX Ct value indicates the Ct value of the sample s HEX signal. (2) If the FAM Ct value is in the Positive range, the Ct of the reaction tube shall be calculated to confirm the result. If the Ct value is less than or equal to the corresponding Cut-off value of Ct, the sample is confirmed as positive. If the Ct value is greater than the Cut-off Ct value, the sample is classified as negative or below the detection limit of the kit. (3) If the sample FAM Ct value is greater than or equal to the critical negative value shown in the Negative row in Table 4, the sample is classified as negative or below the detection limit of the kit. Table 4 Results Determination Mutation Name H1047R H1047L E542K E545K E545D Mutant Ct Value Ct<28 Ct<27 Ct<28 Ct<28 Ct<27 Positive Ct Cut-off value Negative Mutant Ct Value Ct 28 Ct 27 Ct 28 Ct 28 Ct 27 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 4/5

6 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 filtered pipette tips to add DNA template 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 could use this kit. Please wear suitable lab coat and disposable gloves. The used kit should be disposed of properly. 9. The product is CE-marked according to the European Union In Vitro Diagnostic Medical Devices Directive 98/79/EC. 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, Nature Rev. Cancer. 10: Yap TA, et al Curr Opin Pharmacol. 8: Sartore-Bianchi A, et al Cancer Res. 69: Serra V, et al Cancer Res. 68: /5