Plant GMO Screen (35S, NOS) Real-TM

Real Time PCR kit for the detection of GENETICALLY MODIFIED ORGANISMS (GMO)

for use with RotorGene 3000/6000 (Corbett Research), SmartCycler (Cepheid), iQ iCycler and iQ5 (Biorad), MX3000P and MX3005P (Stratagene), Applied Biosystems 7300/7500 Real Time PCR Systems (Applera)

REF GM411-50FRT

VER 05.08.2010

50

NAME Plant GMO Screen (35S, Nos) Real-TM INTRODUCTION A genetically modified organism (GMO) or genetically engineered organism (GEO) is an organism whose genetic material has been altered using genetic engineering techniques. GMO are used in biological and medical research, production of pharmaceutical drugs, experimental medicine (e.g. gene therapy), and agriculture (e.g. golden rice). For instance, corn MON810 contains Cry1Ab gene which expresses a protein toxic for Lepidoptera insects (e.g. corn borer) and Soya GTS 40-3-2 contains CP4 Epsps gene whose proteic product confers tolerance to the non selective herbicide glyphosate. INTENDED USE Plant GMO Screen (35S, Nos) Real-TM kit is a Real-Time PCR test for the detection of GENETICALLY MODIFIED ORGANISMS (GMO) in the material of plant origin. Plant GMO Screen (35S, Nos) Real-TM kit allow to determine, through the nucleic acid amplification in Real Time PCR, the presence of: Plant presence on the Joe(Yellow)/Cy3/HEX 35S promoter (P-35S) on the Rox(Orange)/TexasRed Terminator Nos on the Fam(Green) channel Positive inhibition Internal Control (Pos IC) on the Cy5(Red) channel PRINCIPLE OF ASSAY Plant GMO Screen (35S, Nos) Real-TM kit is a Real-Time PCR test for the detection of GENETICALLY MODIFIED ORGANISMS (GMO) in the plant origin material. DNA is extracted from samples, amplified using Real Time Amplification and detected using fluorescent reporter dye probes specific for Plant origin material, 35S promoter, Terminator Nos and Pos IC. In Plant GMO Screen (35S, Nos) Real-TM kits there are four independent reactions running in parallel in each tube: the first detects Plant presence on the Joe(Yellow)/Cy3/HEX, the second 35S promoter (P-35S) on the Rox(Orange)/TexasRed, the third Terminator Nos on the Fam(Green) channel and the forth (Pos IC) on the Cy5(Red) channel which allows excluding unreliable results. In real-time PCR, the fluorescent signal is generated from the presence of an oligonucleotide probe specific for target DNA sequence. The probe contains a fluorescent dye molecule on its 5 end and a quencher molecule on its 3 end. The probe hybridizes with one of the chains of the amplified fragment. During synthesis of a complementary chain, Taq DNA polymerase cleaves the probe due to its 5-3 nuclease activity. As a result, the fluorescent dye molecule becomes separated from the quencher, and the total fluorescence of reaction volume increases in direct proportion to the number of amplicon copies synthesized during PCR. The fluorescent signal is measured in each cycle of reaction, and the threshold cycle value is determined by the obtained curve. The threshold cycle is proportional to the initial number of DNA copies in a sample, and its value allows quantitative comparisons of analyzed and control samples. MATERIALS PROVIDED Part N 1 Plant GMO Screen (35S, Nos) Real-TM: PCR-mix-1 (Plant,35S,Nos), 2 x 0,55 ml; Taq Polymerase, 0,05 ml; Pos C+ (Soya GMO 1%), 0,1 ml; Negative Control C-, 0,1 ml; Contains reagents for 50 samples. MATERIALS REQUIRED BUT NOT PROVIDED DNA purification kit (see DNA isolation) Real Time Thermalcycler Tubes or PCR plate Workstation Pipettors (capacity 0,5-10 l; 5-40 l) with aerosol barrier Tube racks

WARNINGS AND PRECAUTIONS 1. Wear disposable gloves, laboratory coats and eye protection when handling specimens and reagents. Thoroughly wash hands afterward. 2. Use routine laboratory precautions. Do not eat, drink, smoke, apply cosmetics, or handle contact lenses in laboratory work areas. Do not pipette by mouth. 3. Do not use a kit after its expiration date. 4. Do not mix reagents from different kits. 5. Dispose all specimens and unused reagents in accordance with local regulations. 6. Heparin has been shown to inhibit reaction. The use of heparinized specimens is not recommended. 7. Avoid repeated thawing and freezing of the reagents, this may reduce the sensitivity of the test. 8. Once the reagents have been thawed, vortex and centrifuge briefly the tubes. 9. Prepare quickly the Reaction mix. 10. Specimens may be infectious. Use Universal Precautions when performing the assay. 11. Specimens and controls should be prepared in a laminar flow hood. 12. Handle all materials containing specimens or controls according to Good Laboratory Practices in order to prevent cross-contamination of specimens or controls. 13. Clean and disinfect all spills of specimens or reagents using a disinfectant such as 0,5% sodium hypochlorite, or other suitable disinfectant. Follow by wiping down the surface with 70% ethanol. 14. Avoid contact of specimens and reagents with the skin, eyes and mucous membranes. If these solutions come into contact, rinse immediately with water and seek medical advice immediately. 15. Material Safety Data Sheets (MSDS) are available on request. 16. Use of this product should be limited to personnel trained in the techniques of amplification. 17. Workflow in the laboratory must proceed in a uni-directional manner, beginning in the Extraction Area and moving to the Amplification Area. Do not return samples, equipment and reagents in the area where you performed previous step. Personnel should be using proper anti-contamination safeguards when moving between areas. STORAGE INSTRUCTIONS Store kit at -20C. The kit can be shipped at 2-8C but should be stored -20C immediately on receipt. STABILITY Plant GMO Screen (35S, Nos) Real-TM is stable up to the expiration date indicated on the kit label. The product will maintain performance through the control date printed on the label. Exposure to light, heat or humidity may affect the shelf life of some of the kit components and should be avoided. Repeated thawing and freezing of these reagents should be avoided, as this may reduce the sensitivity. Components stored under conditions other than those stated on the labels may not perform properly and may adversely affect the assay results. QUALITY CONTROL The complete kit has been tested on an RotorGene 6000 (Corbett Research). Certificates of Analyses are available on request at info@sacace.com. SAMPLE COLLECTION, STORAGE AND TRANSPORT Plant GMO Screen (35S, Nos) Real-TM can analyze DNA extracted with DNA-Sorb-C (REF K-1-1/B) from: 1. Soya and corn flour, soya protein concentrate, animal and poultry feed, pet food: Take 10 portions of approx. 100 gm each of the sample being tested and put in a clean bag, using disposable gloves; Agitate the contents of the bag and take a 20 gm sample; this must be divided into 2 parts: the first is sent to the laboratory for detection of GMO (Genetically Modified Organism) and the second is stored for 2-3 months at 2-8 C for possible discrepancies in the results interpretation. Take 5-10 gm of the sample and homogenise/crush in a clean mortar 2. Meat processed with soya, soya products (mince, cooked salami, frankfurter, sausage, soya salami, soya pie, soya ragout sauce, soya ball, soya buttermilk curd, soya cheese, soya chocolate): Take 5-10 gm of the sample, chop up with scissors and homogenise in a clean mortar. 3. Soya milk, soya Mothers milk substitute do not require any pre-extraction treatment. Specimens can be stored at +2-8C for no longer than 48 hours, or freeze at -20C to -80C. DNA ISOLATION The following kit is recommended: DNA-Sorb-C (Sacace, REF K-1-1/C) Please carry out DNA extraction according to the manufactures instruction.

PROTOCOL: 1. Prepare required quantity of reaction tubes (or PCR plate) for samples and controls. 2. Prepare in the new sterile tube for each sample Reaction Mix: 20*N l of PCR-mix-1 and 0,5*N of Taq DNA Polymerase. Vortex and centrifuge briefly. 3. Add to each tube 20 l of Reaction Mix. 4. Add 5,0 l of extracted DNA sample to appropriate tube with Reaction Mix. 5. Prepare for each run 1 Pos Control and 1 Neg Control: add 5,0 l of Pos C+ to the tube labeled PCR Pos Control; add 5,0 l of Negative Control to the tube labeled PCR Negative Control; 6. Insert the tubes in the thermalcycler. Create a temperature profile on your Real-time instrument as follows:
Stage Hold emp, C 94 94 Cycling 59 50 secs Time 3 mins 15 secs Fam (Green) , ROX(Orange)/Texas Red, JOE (Yellow)/HEX/Cy3, Cy5 (red) 45 Fluorescence detection Cycle repeats 1

The results are interpreted through the presence of crossing of fluorescence curve with the threshold line. RESULTS INTERPRETATION Plant presence is detected on the Joe(Yellow)/Cy3/HEX, 35S promoter (P-35S) on the Rox(Orange)/TexasRed, Terminator Nos on the Fam(Green) channel and Positive inhibition Internal Control (Pos IC) on the Cy5(Red) channel.

Sacace Biotechnologies Srl 44 Scalabrini, str 22100 Como, Italy

*PCR: The Polymerase Chain Reaction (PCR) process is covered by patents owned by Hoffmann-La Roche and applicable in certain countries. Sacace does not encourage or support the unauthorized or unlicensed use of the PCR process. Use of this kit is recommended for persons that either have a license to perform PCR or are not required to obtain a license