Innovations and Molecular Diagnosis
GMO DETECTION IN 24 AND 48 HOUR!!
In Florianopolis, Brazil, GENOLIFE do Brazil is a part of GENOLIFE France company founded in 1996 dedicated to the application of DNA analysis technology improving the safety and quality of foodstuffs.
GENOLIFE's goals are, first, to dramatically increase the speed of diagnosis and analysis by combining the benefits of genetic and recent advances in instrument technology (automatic DNA extractions, real-time PCR, micro-arrays, DNA chips). GENOLIFE is assembling a portfolio of proprietary technology platforms to enable complete, cost-effective automation of complex DNA detection, from sample preparation, real-time PCR detection, labchip and DNA chip to High Throughput Screening format.
Broad market applications range from toxicological diagnosis, food quality testing and environmental testing to research and development activities in molecular biology:
ü Plants, species and GMO's detection (Genetically Modified Organisms)
ü Microbial contamination diagnosis in food, pharmaceutical and cosmetic products
ü Genotoxicologic assays for HTS drug-discovery programs.
One focus of our activities is to detect genetically modified organisms and derived product. The field of plant biotechnology has changed dramatically over the last 5 –10 years and continues to evolve at a rapid pace, especially in USA and Canada. Therefore, following EC policy, 138 countries signed the bio-safety protocol dated January 2000 in Montreal for controlling the import and export of genetically modified organisms. According to the agreement, deliveries of raw materials containing genetically modified ingredients, must be labelled as GMO products. It was also fixed, that the threshold limit of 1% is viewed to represent the upper limit for presence of EC authorised GMO in foodstuffs.
Efforts were made to establish quantitative methods for detecting genetically modified in various foodstuffs.
We are working on various transgenic crops (maize, soya, canola, sugar beet, bacteria...) but GMO testing is currently done in our laboratories on corn and soya.
Each of our PCR reaction can identify at numerous targets which could be present in the transgenic product, including targets proposed by the screening method (S35 and Nos). Only "S35" and "Nos" from screening method are not enough to give a good and safe results. "Nos" is not present in BT 176 and "S35" in Ga21, and "S35" could give some false positives especially on derivative products like starch, lecithin, beer and non transgenic wheat...
1) GENOLIFE organisation
The layout and organisation of the GENOLIFE laboratories is based on the "forward flow" principle for samples to ensure that samples to be analysed and PCR products remain segregated.
The analytical laboratory contain four separate rooms:
- one for sample grinding, blending and fractionating,
- one for DNA extraction,
- one for the preparation of the master mixes and PCR machines (classical),
- one for post-PCR operations and PCR semi-quantification (Agilent BioAnalyser).
Precise quantification is still done in France with real time PCR Light Cycler equipment.
Genolife Staff wear different types of lab coats for each type of operation working area. Talc-coated Powdered gloves and starch-talc-coated Powdered gloves should not be used for pre-PCR operations since they can inhibit PCR reactions or can give false positive (starch from OGM corn).
Waste glass are 0,5N soaked in chlorine bleach containing 1.7% of active chlorine, 1N hydrochloric acid, 0,5N sodium hydroxide. All disposable equipment (pipette tips, pipettes, etc.) and equipment that can be recycled before cleaning (including the PCR tube holders), are stored temporarily by immersion in chlorine bleach containing 1.7% of active chlorine or a product (DNA clean up) at least as effective before being removed from the processing rooms.
2) Sampling and test portion
The size (weight) of laboratory samples depends on the type of product, and more specifically on the size of the particles and their level of uniformity.
Products Minimum laboratory sample size
q Grain 1) (corn, soybean, oilseed rape (canola), rice, etc.) 10,000 grains or 3 kg
q Powder (semolina, flour, grits, oilcake, etc.) 1 kg
q Liquids 500 ml
q Paste or viscous products 500 g or 500 ml
q Finished products (packaged) 2 individual packages or 100 g
An aliquot of the laboratory test sample is retained for six months for subsequent analysis under the same conditions as for the test sample. When the sample is received, weight is check and also the condition of the product, and the packaging.
The size of the test portion required for extraction is 1 g.
3) DNA Extraction - purification protocol and systems
Now it happens that quality of PCR is tightly related to quality of DNA extraction. This step determines quality of DNA used in PCR, and by the way, sensitivity and reliability of PCR. If DNA is damaged it is impossible to realise an efficient PCR. However DNA contaminants (carbohydrates, proteins, lipids...) could decrease PCR sensitivity. For example, these contaminants could impair specific annealing of primers on DNA, resulting in artefactual PCR products and false results (false positives). In the opposite, PCR inhibition with these contaminants could lead to false negatives.
The basic principle of nucleic acid extraction consists in releasing the DNA present, possibly by lysing the cells present and concurrently or subsequently purifying the DNA by removing all other components, particularly proteins and the PCR inhibitors.
In order to extract a quality DNA, it is advised, where relevant, to remove all polysaccharides (pectin, cellulose, hemi-cellulose, starch, etc.) with depolymerases (pectinase, cellulase, hemi-cellulase, a -amylase, b -amylase, etc.) and proteins (Proteinase K) during or after extraction. Further purification may be achieved. Using DNA extraction methods based on affinity resin column for these kinds of applications witht specific modification (filtration, change of buffer volume, new lysis buffer, agent for protein precipitation...) appear really efficient.
Our Brazil laboratory is able to manage 48 DNA extraction samples a day at this time.
A precise dosage of the extracted DNA solution is essential for reproducibility of subsequent quantification of PCR products, therefore this is not always possible due to the low DNA content of some samples (such as products that have been extensively processed) .
4) DNA amplification (Polymerase Chain Reaction)
In addition of constitutive gene primers (positive control of DNA extraction and PCR, 100% of corn or soy), Genolife is using numerous different pairs of primers for transgenic construction detection.
With these genetic targets, we can detect EC authorised corn and soya and also non EC authorised corn and soya produce in north and south America (products having pending regulatory issues with EPA or USDA/APHIS.
If there is a doubt about the qualitative result, we even go back and prepare new DNA using additional steps to remove materials that might be interfering with the PCR reaction.
For negative PCR (and therefore for universal sequences, plants and GMO) an inhibition control internal PCR control is used to determine the inhibiting effect of the solution of DNA extracted.
In order to account for different food processing methods, the size of DNA fragments that can be amplified is between 80 and 200 base pairs.
The sensitivity of the test is less than 0,01% on seeds, powder, flour
Limit of quantification is 0.1%.
Our primers are capable of detecting all corn and soya varieties (cultivars) GMO, EC authorised and non authorised.
Status of GMO (corn and soya)
Event approved by Brazil
Events approved by the European Union and Argentina
Events not approved by European Union, Brasil or Argentina
5) Quantitative PCR for T25, Bt 176, Bt 11, Mon 810 and RR soya (EC authorised)
The first step of our analysis is a complete qualitative PCR of the DNA coming from the customer sample with all the possible transgenic targets listed above.
If qualitative PCR give us a positive result , a quantitative PCR is done using a specific target like Cry, EPSPS, bar. If the detected GMO is a non authorised construction in EC, quantification is not done. Using a target like S35for quantitative PCR will be possible only on pure soya (one copy). On a complex product(soya + corn) or corn product, quantitative PCR with S35 target will give wrong results, because it could be more than one copy of S35 promoter in corn sample.
Qualitative result are read with the Agilent Labchip microcapillary system.
For our quantitative result, we are working with the Roche Light cycler system and the Perkin Elmer 7700 system using Taqman technology and hybridization probes (others systems will arrive on the market like Ecycler and Smartcycler) .
We are doing quantitative PCR using standard (GMO DNA) extracted from soy or corn from FLUKA (Bt11 + 176 + Soya RR) and from other sources (T25 + Mon 810).
Real time PCR
Real time PCR works with flourescence probes between the two primers. Genolife is using the Light Cycler and and hybridisation probes.
The system has a high specificity for the accumulation of the GMO targets.
Amplicon size is around 100 bp .
Using real time PCR are done to quantify only one variety by exemple Bt11 . Using DNA targets like S35 promotor is only possible on product contamined only by one GMO because the amount of these S35 sequence is different between each GMO.
RR soya -
1copy of S35
GENOLIFE DO BRASIL
RUA SANTO INÁCIO DE
LOYOLA, 149 - CENTRO
CLERMONT-LIMAGNE - 63 360 SAINT BEAUZIRE - FRANCE
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