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Protocol
Quantitation of maize 59122
Keywords: GMO, maize, 59122, RT-PCR, TaqMan, DNA
Event-specific method for the quantitation of maize 59122 using real-time PCR
This protocol is also found on the official website of the "Community Reference Laboratory for GM Food and Feed" (CRL-GMFF), established in the context of Regulation (EC) No 1829/2003 on GM Food and Feed.
The core task of the CRL-GMFF is the scientific assessment and validation of detection methods for GM Food and Feed as part of the EU authorization procedure. The Joint Research Centre (JRC) of the European Commission and, more precisely, the Molecular Biology and Genomics Unit of the Institute for Health and Consumer Protection (IHCP), has been given the mandate for the operation of the CRL-GMFF. Activities are carried out in close collaboration with European Network of GMO Laboratories (ENGL).
The CRL-GMFF operates according to a quality management system certified and accredited under ISO 9001 and ISO 17025.
Method development: Pioneer Hi-Bred International GeneScan Analytics GmbH
Method validation: Joint Research Centre – European Commission Biotechnology & GMOs Unit, Community Reference Laboratory for GM Food and Feed
Instructions
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General information and summary of the methodology
This protocol describes an event-specific real-time quantitative TaqMan® PCR procedure for the determination of the relative content of event DAS-59122-7 DNA to total maize DNA in a sample.
The PCR assay has been optimised for use in real-time PCR instruments for plastic reaction vessels. Glass capillaries are not recommended for the buffer composition described in this method.
Template DNA extracted by means of suitable methods should be tested for quality and quantity prior to the use in PCR assay. Tests for the presence of PCR inhibitors (e.g. monitor run, use of DNA spikes) are recommended.
For specific detection of event DAS-59122-7 genomic DNA, an 86-bp fragment of the recombination region of parts of the construct inserted into the plant genome is amplified using two specific primers. PCR products are measured during each cycle (real-time) by means of a target-specific oligonucleotide probe labelled with two fluorescent dyes: FAM as a reporter dye at its 5′ end and TAMRA as a quencher dye at its 3′ end.
For relative quantitation of event DAS-59122-7 DNA, a maize-specific reference system amplifies a 79-bp fragment of the High Mobility Group (Hmg) gene, a maize endogenous gene, using a pair of Hmg gene-specific primers and an Hmg gene-specific probe labelled with FAM and TAMRA as described above.
The measured fluorescence signal passes a threshold value after a certain number of cycles. This threshold cycle is called the “Ct” value. For quantitation of the amount of event DAS-59122-7 DNA in a test sample, event DAS-59122-7 and Hmg Ct values are determined for the sample. Standard curves are then used to calculate the relative content of event DAS-59122-7 DNA to total maize DNA.
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Validation status and performance characteristics
General The method has been optimised for DNA extracted from seeds, containing mixtures of genetically modified and conventional maize.
The reproducibility and trueness of the method was tested through collaborative trial using samples at different GMO contents.
Collaborative trial The method was validated in a collaborative trial by the Joint Research Centre (JRC) of the European Commission. The study was undertaken with 14 laboratories.
Each participant received twenty unknown samples containing DAS-59122-7 maize genomic DNA at five concentration levels, between 0.10 % and 4.5 %.
Each test sample was analyzed by PCR in three repetitions. The study was designed as a blind quadruplicate collaborative trial; each laboratory received each level of GM DAS-59122-7 in four unknown samples. Two replicates of each GM level were analyzed on the same PCR plate.
A detailed validation report can be found under http://gmo-crl.jrc.it/statusofdoss.htm
Limit of detection According to the method developer, the relative LOD of the method is 0.045%. The relative LOD was not assessed in a collaborative trial. The lowest relative concentration of the target sequence included in collaborative trial was 0.10%.
Limit of quantitation According to the method developer, the relative LOQ of the method is 0.09%. The lowest relative concentration of the target sequence included in collaborative trial was 0.10%.
Molecular specificity The method utilizes a unique DNA sequence of the recombination region of parts of the construct inserted into the plant genome. The sequence is specific to DAS-59122-7 and thus imparts event-specificity to the detection method.
The specificity was assessed by Blastsearch at the National Center for Biotechnology Information (NCBI) with the “Standard nucleotide-nucleotide BLAST [blastn]” (www.ncbi.nlm.nih.gov/blast/Blast.cgi) on the amplicon resulting from the event-specific amplification of the transition region of the sugar beet genomic DNA into the specific event.
No 100% match with other plant GMO sequences was found.
The specificity was experimentally tested against DNA extracted from plant materials containing the specific targets (at least 1000 genomic copies/reaction) of the T25 maize, 1507 maize, Bt176 maize, Bt11 maize, NK603 maize, GA21 maize, MON810 maize, RR soy, RR rape, potato “new leaf”. In addition DNA extracted from non GM-wheat and -rice was also tested.None of the materials yielded detectable amplification, apart from the event DAS-59122-7.
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Preocedures (1T9TPLP)
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General instructions and precautions
All handling of reagents and controls should occur in an ISO 9001:2000 or ISO 17025 environment or equivalent.
• The procedures require experience of working under sterile conditions.• Laboratory organization, e.g. “flow direction” during PCR-setup, should follow the guidelines given by relevant authorities like e.g. ISO, CEN, Codex alimentarius commission.
• PCR-reagents shall be stored and handled in a separate room and freezer and in equipment where no nucleic acids (with exception of PCR primers or probes) or DNA degrading or modifying enzymes have been handled previously. All handling of PCR reagents and controls requires dedicated equipment – especially pipettes.
• All the equipment used must be sterilized prior to use and any residue of DNA has to be removed. All material used (e.g. vials, containers, pipette tips, etc.) must be suitable for PCR and molecular biology applications. They must be DNase-free, DNA-free, sterile and shall not adsorb protein or DNA.
• In order to avoid contamination, filter pipette tips protected against aerosol should be used.
• Use only powder-free gloves and change them frequently.
• Clean lab-benches and equipment periodically with 10% sodium hypochloride solution (bleach).
• Pipettes should be checked regularly for precision and calibrated, if necessary.
• All handling steps - unless specified otherwise - shall be carried out at 0 - 4°C.
• In order to avoid repeated freeze/thaw cycles, aliquots should be prepared.
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General instructions and precautions
All handling of reagents and controls should occur in an ISO 9001:2000 or ISO 17025 environment or equivalent.
• The procedures require experience of working under sterile conditions.• Laboratory organization, e.g. “flow direction” during PCR-setup, should follow the guidelines given by relevant authorities like e.g. ISO, CEN, Codex alimentarius commission.
• PCR-reagents shall be stored and handled in a separate room and freezer and in equipment where no nucleic acids (with exception of PCR primers or probes) or DNA degrading or modifying enzymes have been handled previously. All handling of PCR reagents and controls requires dedicated equipment – especially pipettes.
• All the equipment used must be sterilized prior to use and any residue of DNA has to be removed. All material used (e.g. vials, containers, pipette tips, etc.) must be suitable for PCR and molecular biology applications. They must be DNase-free, DNA-free, sterile and shall not adsorb protein or DNA.
• In order to avoid contamination, filter pipette tips protected against aerosol should be used.
• Use only powder-free gloves and change them frequently.
• Clean lab-benches and equipment periodically with 10% sodium hypochloride solution (bleach).
• Pipettes should be checked regularly for precision and calibrated, if necessary.
• All handling steps - unless specified otherwise - shall be carried out at 0 - 4°C.
• In order to avoid repeated freeze/thaw cycles, aliquots should be prepared.
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Data analysis
Subsequent to the real-time PCR, analyse the run following the procedure below:
a) Set the threshold: display the amplification curves of one system (e.g. DAS-59122-7) in logarithmic mode. Locate the threshold line in the area where the amplification profiles are parallel (exponential phase of PCR) and where there is no “fork effect” between repetitions of the same sample. Press the update button to ensure changes affect Ct values. Switch to the linear view mode by clicking on the Y axis of the amplification plot, and check that the threshold previously set falls within the geometric phase of the curves.
b) Set the baseline: determine the cycle number at which the threshold line crosses the first amplification curve and set the baseline three cycles before that value (e.g. earliest Ct = 25, set the baseline crossing at Ct = 25 – 3 = 22).
c) Save the settings
d) Repeat the procedure described in a) and b) on the amplification plots of the other system (e.g. Hmg system).
e) Save the settings and export all the data into an Excel file for further calculations.
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Calculation of results
After having defined a threshold value within the logarithmic phase of amplification as described above, the instruments software calculated the Ct-values for each reaction.
The standard curves are generated both for the Hmg and DAS-59122-7 specific system by plotting the Ct-values measured for the calibration points against the logarithm of the DNA copy numbers, and by fitting a linear regression line into these data.
Thereafter, the standard curves are used to estimate the copy numbers in the unknown sample DNA by interpolation from the standard curves.
For the determination of the amount of DAS-59122-7 DNA in the unknown sample, the DAS-59122-7 copy number is divided by the copy number of the maize reference gene (Hmg) and multiplied by 100 to obtain the percentage value (GM% = DAS-59122-7/Hmg * 100).
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General information and summary of the methodology
This protocol describes an event-specific real-time quantitative TaqMan® PCR procedure for the determination of the relative content of event DAS-59122-7 DNA to total maize DNA in a sample.
The PCR assay has been optimised for use in real-time PCR instruments for plastic reaction vessels. Glass capillaries are not recommended for the buffer composition described in this method.
Template DNA extracted by means of suitable methods should be tested for quality and quantity prior to the use in PCR assay. Tests for the presence of PCR inhibitors (e.g. monitor run, use of DNA spikes) are recommended.
For specific detection of event DAS-59122-7 genomic DNA, an 86-bp fragment of the recombination region of parts of the construct inserted into the plant genome is amplified using two specific primers. PCR products are measured during each cycle (real-time) by means of a target-specific oligonucleotide probe labelled with two fluorescent dyes: FAM as a reporter dye at its 5′ end and TAMRA as a quencher dye at its 3′ end.
For relative quantitation of event DAS-59122-7 DNA, a maize-specific reference system amplifies a 79-bp fragment of the High Mobility Group (Hmg) gene, a maize endogenous gene, using a pair of Hmg gene-specific primers and an Hmg gene-specific probe labelled with FAM and TAMRA as described above.
The measured fluorescence signal passes a threshold value after a certain number of cycles. This threshold cycle is called the “Ct” value. For quantitation of the amount of event DAS-59122-7 DNA in a test sample, event DAS-59122-7 and Hmg Ct values are determined for the sample. Standard curves are then used to calculate the relative content of event DAS-59122-7 DNA to total maize DNA.
