SureFood GMO. LibertyLink Canola. real-time PCR. Kit for quantitative analysis of LibertyLink canola DNA 2004/01 E

Transcription

1 SureFood GMO LibertyLink Canola real-time PCR 2004/01 E Kit for quantitative analysis of LibertyLink canola DNA Art.-No. S 2018

2 Distribution of SureFood products: R-Biopharm AG Landwehrstrasse 54, D Darmstadt / Germany Tel.: +49 (0) , Fax: +49 (0) info@r-biopharm.de orders@r-biopharm.de Production of SureFood kits: CONGEN Biotechnologie GmbH Robert Rössle Str Berlin / Germany Tel.: +49 (0) Fax: +49 (0) congen@congen.de Web: 2 SureFood GMO real-time PCR LibertyLink Canola

3 SureFood GMO LibertyLink Canola Contents Kit Components 4 Introduction 5 Preparations 6 Protocol 6 Interpretation of results 11 Ordering information 12 SureFood Kits are developed and manufactured by CONGEN Biotechnology GmbH within ISO 9001:2000 quality management system. The Polymerase Chain Reaction (PCR) is protected by a patent. For the commercial use of this method in the area of in vitro diagnostics a license from Hoffmann- LaRoche, Basel is required. The purchase of a SureFood GMO kit does not give the right for the buyer or user to carry out PCR without a license. SureFood GMO real-time PCR LibertyLink Canola 3

4 Kit Components The reagents of the kit are sufficient for 42 quantitative single determinations (including standards and positive controls) of the relative LibertyLink canola DNA content in food, feed and seeds. With this kit 2 x 50 PCRs can be performed. Each kit contains: 1x Standard DNA (25 µl) (Code 1) 1x Positive Control DNA, 2% GMO (25 µl) (Code 2) 1x Cru Reaction Mix (1,0 ml) (Code 3) 1x Glu Reaction Mix (1,0 ml) (Code 4) 1x FDE (120 µl) (Code 5) 1x Taq Polymerase (15 µl) (Code 6) 1x TE Buffer (1,3 ml) (Code 7) The code numbers are indicated on the caps and the tube labels. On receipt of the kit all reagents shall be stored at 20 C. Tubes 3 and 4 contain light sensitive probes and should be stored in the dark. 4 SureFood GMO real-time PCR LibertyLink Canola

5 Introduction This kit is used for the quantitative determination of LibertyLink rape seeds (canola) DNA. It contains two PCR systems, one specific for the genetic modification (Glu detection gene) and one specific for the plant rape seed (Cru reference gene). Sample Preparation The quality of DNA extraction from the samples and from the GMOstandards effects the quantitation. DNA extraction kits from different suppliers are sometimes leading to different results in the quantitation. We recommend to use the identical sample preparation for the GMO standards and the samples. CONGEN offers a sample preparation kit for the use in combination with the SureFood GMO kits SureFood PREP Plant (see Ordering Information on page 12). Detection and quantitation limit The detection limit of the real-time PCR systems specific for Glu and Cru is 2 copies of the target sequence, respectively. The practical limit of quantitation depends on the concentration of the DNA used in the analysis. For example, when copies of the Cru gene are measured, the quantitation limit for the genetic modification is approx. at 0.1 % relative GMO content. SureFood GMO real-time PCR LibertyLink Canola 5

6 Preparations 1. Thaw the reagents slowly at room temperature. 2. Before opening the reagent tubes and pipetting the solutions shortly mix (Vortex) and centrifuge in a mini-centrifuge. Protocol 1. Calculate the number of the reactions needed for Cru and Glu detection. 5 reactions are needed for the standard curve for Cru. 5 reactions are needed for the standard curve for Glu. One no-template control and two positive controls (Code 2) for Cru has to be performed. One no-template control and two positive controls (Code 2) for Glu has to be performed. For each sample DNA 4 reactions (double measurements of Cru and Glu) are needed. for Cru: n x 2 for Glu: n x 2 (n = number of samples) 2. Preparation of the Cru PCR-mix: For each reaction for detection of the reference gene mix 17,0 µl of the Cru reaction mix (Code 3) with 1,0 µl FDE (Code 5) and 0,1 µl of Taq Polymerase (Code 6). Make sure that the reagents are well mixed. * Due to pipetting loss of reagents, it is advised to prepare approx. 10 % Cru mix more than actually needed. 6 SureFood GMO real-time PCR LibertyLink Canola

7 Example for 10 reactions: Components 1 reaction 10 reactions (+10 %) Cru reaction mix 17,0 µl 187 µl FDE 1,0 µl 11 µl Taq Polymerase 0,1 µl 1,1 µl 3. Preparation of the Glu mix For each reaction of the Glu detection (GMO detection gene) mix 17,0 µl of Glu reaction mix (Code 4) with 1,0 µl FDE (Code 5) and 0,1 µl of Taq Polymerase (Code 6). Make sure that the reagents are well mixed. * Due to pipetting loss of reagents, it is advised to prepare approx. 10 % Glu mix more than actually needed. Example for 10 reactions: Components 1 reaction 10 reactions (+10 %) Glu reaction mix 17,0 µl 187 µl FDE 1,0 µl 11 µl Taq Polymerase 0,1 µl 1,1 µl 4. Dilute the standard DNA (Code 1) in 1: 10 steps in TE Buffer (Code 7) in order to prepare different DNA concentrations for the standard curves of the reference gene (Cru) and the detection gene (Glu). Five different dilutions will be needed. For the standard curves the dilution levels 1 to 5 will be used (see Table 1). It is recommended to prepare the dilutions directly before the PCR is performed. SureFood GMO real-time PCR LibertyLink Canola 7

8 * The standard DNA (Code 1) provided with the kit contains 10 6 copies of both targets (Cru and Glu). The copy numbers of the dilutions have to be entered in the analysis software of the realtime PCR detection system. Table 1: dilution step* Cru (copies/µl) Glu (copies/µl) 1x10 5 1x10 4 1x10 3 1x10 2 1x10 1 1x10 5 1x10 4 1x10 3 1x10 2 1x10 1 *Step 1 is the first dilution of the standard DNA taken from stock solution contained in tube 1 (Code 1). For PCR 2 µl are used per reaction. The copy numbers per reaction are entered in calculation software of the instrument. 5. Prepare the right amount of tubes for the reference gene. Pipette 18 µl of the Cru mix into the tube. Close the first tube (notemplate control) immediately. Pipette 2 µl of positive control DNA (Code 2) into the next tube. Pipette 2 µl of standard DNAs (different dilutions from step 4) into the following 5 tubes. Then pipette 2 µl of the sample DNA into the next tubes. Close the tube. Repeat the same procedure for the detection gene (Glu). Centrifuge the tubes briefly (lowest rpm) according to the recommendation of the real-time PCR instrument manual. 8 SureFood GMO real-time PCR LibertyLink Canola

9 6. Cycler conditions SureFood GMO real-time PCR kits can be used on all usual realtime PCR instruments. When using a LightCycler instrument specific heating and ramping times have to be selected because of the extremely high heating speed. For block-type real-time PCR instruments use the conditions described in section LightCycler Setup (Roche): a) PCR protocol (amplification) Experiment File: Initiatil Denaturation : 1 min 95 C Acquisition mode: none Denaturation : 5 s 95 C Acquisition mode: none Annealing : 10 s 62 C Acquisition mode: none Elongation : 15 s 65 C Acquisition mode: single 45 cycles Cooling : 10 s 40 C Acquisition mode: none (Temp. Transition: 20 C per second) For older LightCycler Software versions: Fluorimeter gains: Channel 1: 20 (has to be adjusted after measuring the fluorescence level in the real-time fluorimeter option). Channel conditions: F1/F2 SureFood GMO real-time PCR LibertyLink Canola 9

10 6.2 ABI PRISM 7000/7700/7900 SDS (Applied Biosystems) Mx3000P, Mx4000 (Stratagene) Rotor-Gene (Corbett Research) Opticon (MJ Research / Oxoid) icycler (Bio-Rad) Smart Cycler (Cepheid) a) PCR protocol (amplification) initial denaturation: 5min, 95 C (HOLD) denaturation: 10s, 95 C annealing: 15s, 62 C elongation: 30s, 65 C 45 cycles b) Detection Ramp rate: Fluorescence detection: Reporter dye: Quencher dye: Reference dye: maximal end of the elongation phase FAM TAMRA none For fluorescence detection and data analysis use a typical realtime PCR detection program (single reporter) with standard conditions as prescribed by the instrument manufacturer. 10 SureFood GMO real-time PCR LibertyLink Canola

11 Interpretation of results The calculation for both reactions (Glu and Cru) has to be made separately. Mark the standards, the controls and the samples for the specific system. The CT-values of the standards should have constant distances, the distance should be at approx. 3,2 to 4 cycles (±0,2) between two dilution steps. If the analysis software of the instrument provides a standard curve calculation method this should be used to calculate the copy numbers for the samples and the positive control and taken from the data report. By using the calculated copy numbers for Glu and Cru the relative GMO content can be determined in the following way: Positive control DNA Glu Positive control DNA Cru 400 copies copies Divide the Glu copy number by the Cru copy number and multiply by 100 to obtain the percentage. For the given example the numbers lead to a LibertyLink canola GMO content of 1,4 %. For a control of the quantitation results the percentage calculated for the the positive control DNA has to be considered. In case of a clear deviation of the calculated GMO % for the 2% LibertyLink canola positive control in the present run caused by a run-to-run fluctuation, it is possible to correct the values for the samples by introducing a correction factor K. K is calculated in the following way: K = GMO content reference DNA / calculated number K (example) = 2,0 % / 1,4 % = 1,4 The calculated GMO content of the samples can be corrected with this factor. The calculated value for the sample is then multiplied with K to obtain a corrected value. GMO content sample = calculated content of sample x K. Example: 3,0% x 1,4 = 4,2%. SureFood GMO real-time PCR LibertyLink Canola 11

12 Ordering information Further SureFood GMO kits for DNA preparation and qualitative and quantitative analysis: Product Description Amount Art-No. PREP-Plant Extraction of plant DNA from food and feed 2 x 50 Prep. S 1002 PREP-Plant X Extraction of plant DNA from highly processed food and feed 2 x 50 Prep. S 1006 RuR Soya Bt176 Corn Bt11 Corn T25 Corn MON810 Corn LibertyLink Canola 35S Promoter NOS Terminator 35S + NOS RuR Soya Bt176 Corn Bt11 Corn T25 Corn LibertyLink Canola MON810 Corn 35S Corn Screening PCR-ELISA for qualitative analysis of Roundup Ready soya DNA PCR-ELISA for qualitative analysis of Bt176 corn DNA PCR-ELISA for qualitative analysis of Bt11 corn DNA PCR-ELISA for qualitative analysis of T25 corn DNA PCR-ELISA for qualitative analysis of MON810 corn DNA PCR-ELISA for qualitative analysis of LibertyLink rapeseed DNA PCR-ELISA for qualitative analysis of 35S promoter DNA (Screening) PCR-ELISA for qualitative analysis of NOS terminator DNA (Screening) PCR-ELISA for qualitative analysis of 35S + NOS DNA (Screening) Roundup Ready Soya DNA Bt176 corn DNA Bt11 corn DNA T25 corn DNA LibertyLink rapeseed DNA MON810 corn DNA 35S promoter in corn DNA (Screening) 24 x 8 Rct. S x 8 Rct. S x 8 Rct. S x 8 Rct. S x 8 Rct. S x 8 Rct. S x 8 Rct. S x 8 Rct. S x 8 Rct. S x 50 Rct. S x 50 Rct. S x 50 Rct. S x 50 Rct. S x 50 Rct. S x 50 Rct. S x 50 Rct. S 2020 For ordering contact: R-Biopharm AG, Darmstadt, Germany Tel.: +49 (0) / Telefax: +49 (0) SureFood GMO real-time PCR LibertyLink Canola