scholarly journals Detection of Genetically Modified Maize MON810 and NK603 by Multiplex and Real-Time Polymerase Chain Reaction Methods

2004 ◽  
Vol 52 (11) ◽  
pp. 3264-3268 ◽  
Author(s):  
Hsin-Ying Huang ◽  
Tzu-Ming Pan
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Genetically Modified ◽  
Genetically Modified Maize ◽  
Chain Reaction ◽  
Maize Mon810 ◽  
Polymerase Chain ◽  
Genetically Modified Maize Mon810

Quantitative real-time polymerase chain reaction for the presence of genetically-modified maize in breaded “ready-to-cook” food products

Food Control ◽  
2008 ◽  
Vol 19 (10) ◽  
pp. 1002-1005 ◽  
Author(s):  
Angela Di Pinto ◽  
Flora Alfano ◽  
Angela Giordano ◽  
Federico Capuano ◽  
Terio Valentina ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Genetically Modified ◽  
Food Products ◽  
Genetically Modified Maize ◽  
Chain Reaction ◽  
Polymerase Chain

scholarly journals Real-Time Quantitative Polymerase Chain Reaction Methods for Four Genetically Modified Maize Varieties and Maize DNA Content in Food

2002 ◽  
Vol 85 (3) ◽  
pp. 646-653 ◽  
Author(s):  
Peter D Brodmann ◽  
Evelyn C Ilg ◽  
Hélène Berthoud ◽  
André Herrmann
Keyword(s):  
European Union ◽  
Polymerase Chain Reaction ◽  
Real Time ◽  
Genetically Modified ◽  
Detection Methods ◽  
Genetically Modified Maize ◽  
The European Union ◽  
Chain Reaction ◽  
Maize Varieties ◽  
Polymerase Chain

Abstract Quantitative detection methods are needed for enforcement of the recently introduced labeling threshold for genetically modified organisms (GMOs) in food ingredients. This labeling threshold, which is set to 1% in the European Union and Switzerland, must be applied to all approved GMOs. Four different varieties of maize are approved in the European Union: the insect-resistant Bt176 maize (Maximizer), Bt11 maize, Mon810 (YieldGard) maize, and the herbicide-tolerant T25 (Liberty Link™) maize. Because the labeling must be considered individually for each ingredient, a quantitation system for the endogenous maize content is needed in addition to the GMO-specific detection systems. Quantitative real-time polymerase chain reaction detection methods were developed for the 4 approved genetically modified maize varieties and for an endogenous maize (invertase) gene system.

scholarly journals Quantitation of 35S Promoter in Maize DNA Extracts from Genetically Modified Organisms Using Real-Time Polymerase Chain Reaction, Part 2: Interlaboratory Study

2005 ◽  
Vol 88 (2) ◽  
pp. 558-573 ◽  
Author(s):  
Max Feinberg ◽  
Sophie Fernandez ◽  
Sylvanie Cassard ◽  
Chrystèle Charles-Delobel ◽  
Yves Bertheau ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Genetically Modified Organisms ◽  
Genetically Modified ◽  
Interlaboratory Study ◽  
Performance Criteria ◽  
Tolerance Interval ◽  
Chain Reaction ◽  
Qrt Pcr ◽  
Polymerase Chain

Abstract The European Committee for Standardization (CEN) and the European Network of GMO Working Laboratories have proposed development of a modular strategy for stepwise validation of complex analytical techniques. When applied to the quantitation of genetically modified organisms (GMOs) in food products, the instrumental quantitation step of the technique is separately validated from the DNA extraction step to better control the sources of uncertainty and facilitate the validation of GMO-specific polymerase chain reaction (PCR) tests. This paper presents the results of an interlaboratory study on the quantitation step of the method standardized by CEN for the detection of a regulatory element commonly inserted in GMO maize-based foods. This is focused on the quantitation of P35S promoter through using the quantitative real-time PCR (QRT-PCR). Fifteen French laboratories participated in the interlaboratory study of the P35S quantitation operating procedure on DNA extract samples using either the thermal cycler ABI Prism® 7700 (Applied Biosystems, Foster City, CA) or Light Cycler® (Roche Diagnostics, Indianapolis, IN). Attention was focused on DNA extract samples used to calibrate the method and unknown extract samples. Data were processed according to the recommendations of ISO 5725 standard. Performance criteria, obtained using the robust algorithm, were compared to the classic data processing after rejection of outliers by the Cochran and Grubbs tests. Two laboratories were detected as outliers by the Grubbs test. The robust precision criteria gave values between the classical values estimated before and after rejection of the outliers. Using the robust method, the relative expanded uncertainty by the quantitation method is about 20% for a 1% Bt176 content, whereas it can reach 40% for a 0.1% Bt176. The performances of the quantitation assay are relevant to the application of the European regulation, which has an accepted tolerance interval of about ±50%. These data were fitted to a power model (r2 = 0.96). Thanks to this model, it is possible to propose an estimation of uncertainty of the QRT-PCR quantitation step and an uncertainty budget depending on the analytical conditions.

scholarly journals Real-Time Polymerase Chain Reaction Detection of Cauliflower mosaic virus to Complement the 35S Screening Assay for Genetically Modified Organisms

2005 ◽  
Vol 88 (3) ◽  
pp. 814-822 ◽  
Author(s):  
Katarina Cankar ◽  
Maja Ravnikar ◽  
Jana Žel ◽  
Kristina Gruden ◽  
Nataša Toplak
Keyword(s):  
Polymerase Chain Reaction ◽  
Mosaic Virus ◽  
Real Time ◽  
Genetically Modified Organisms ◽  
Genetically Modified ◽  
Pcr Amplification ◽  
Cauliflower Mosaic Virus ◽  
Chain Reaction ◽  
Screening Assay ◽  
Polymerase Chain

Abstract Labeling of genetically modified organisms (GMOs) is now in place in many countries, including the European Union, in order to guarantee the consumer's choice between GM and non-GM products. Screening of samples is performed by polymerase chain reaction (PCR) amplification of regulatory sequences frequently introduced into genetically modified plants. Primers for the 35S promoter from Cauliflower mosaic virus (CaMV) are those most frequently used. In virus-infected plants or in samples contaminated with plant material carrying the virus, false-positive results can consequently occur. A system for real-time PCR using a TaqMan minor groove binder probe was designed that allows recognition of virus coat protein in the sample, thus allowing differentiation between transgenic and virus-infected samples. We measured the efficiency of PCR amplification, limits of detection and quantification, range of linearity, and repeatability of the assay in order to assess the applicability of the assay for routine analysis. The specificity of the detection system was tested on various virus isolates and plant species. All 8 CaMV isolates were successfully amplified using the designed system. No cross-reactivity was detected with DNA from 3 isolates of the closely related Carnation etched ring virus. Primers do not amplify plant DNA from available genetically modified maize and soybean lines or from different species of Brassicaceae or Solanaceae that are natural hosts for CaMV. We evaluated the assay for different food matrixes by spiking CaMV DNA into DNA from food samples and have successfully amplified CaMV from all samples. The assay was tested on rapeseed samples from routine GMO testing that were positive in the 35S screening assay, and the presence of the virus was confirmed.

scholarly journals Real-Time Polymerase Chain Reaction-Based Approach for Quantification of the pat Gene in the T25 Zea mays Event

2004 ◽  
Vol 87 (6) ◽  
pp. 1342-1355 ◽  
Author(s):  
Florian Weighardt ◽  
Cristina Barbati ◽  
Claudia Paoletti ◽  
Maddalena Querci ◽  
Simon Kay ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Zea Mays ◽  
Real Time ◽  
Real Time Pcr ◽  
Reference Genes ◽  
Genetically Modified ◽  
Mean Value ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Gmo Quantification

Abstract In Europe, a growing interest for reliable techniques for the quantification of genetically modified component(s) of food matrixes is arising from the need to comply with the European legislative framework on novel food products. Real-time polymerase chain reaction (PCR) is currently the most powerful technique for the quantification of specific nucleic acid sequences. Several real-time PCR methodologies based on different molecular principles have been developed for this purpose. The most frequently used approach in the field of genetically modified organism (GMO) quantification in food or feed samples is based on the 5′-3′-exonuclease activity of Taq DNA polymerase on specific degradation probes (TaqMan® principle). A novel approach was developed for the establishment of a TaqMan quantification system assessing GMO contents around the 1% threshold stipulated under European Union (EU) legislation for the labeling of food products. The Zea mays T25eliteeventwas chosen as a model for the development of the novel GMO quantification approach. The most innovative aspect of the system is represented by the use of sequences cloned in plasmids as reference standards. In the field of GMO quantification, plasmids are an easy to use, cheap, and reliable alternative to Certified Reference Materials (CRMs), which are only available for a few of the GMOs authorized in Europe, have a relatively high production cost, and require further processing to be suitable for analysis. Strengths and weaknesses of the use of novel plasmid-based standards are addressed in detail. In addition, the quantification system was designed to avoid the use of a reference gene (e.g., a single copy, species-specific gene) as normalizer, i.e., to perform a GMO quantification based on an absolute instead of a relative measurement. In fact, experimental evidences show that the use of reference genes adds variability to the measurement system because a second independent real-time PCR-based measurement must be performed. Moreover, for some reference genes no sufficient information on copy number in and among genomes of different lines is available, making adequate quantification difficult. Once developed, the method was subsequently validated according to IUPAC and ISO 5725 guidelines. Thirteen laboratories from 8 EU countries participated in the trial. Eleven laboratories provided results complying with the predefined study requirements. Repeatability (RSDr)values ranged from 8.7 to 15.9%, with a mean value of 12%. Reproducibility (RSDR) values ranged from 16.3 to 25.5%, with a mean value of 21%. Following Codex Alimentarius Committee guidelines, both the limits of detection and quantitation were determined to be <0.1%.

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