Detection of nonauthorized genetically modified organisms using differential quantitative polymerase chain reaction: application to 35S in maize

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.

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Detection of Genetic Modification in Cured Tobacco Leaf: Proficiency Testing Using Polymerase Chain Reaction-Based Methods

Journal of AOAC International ◽

10.1093/jaoac/89.3.693 ◽

2006 ◽

Vol 89 (3) ◽

pp. 693-707

Author(s):

Ferruccio Gadani ◽

Martin Ward ◽

Sue Black ◽

Neil Harris ◽

David McDowell ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Proficiency Testing ◽

Quantitative Methods ◽

Genetically Modified Organisms ◽

Genetically Modified ◽

Cauliflower Mosaic Virus ◽

Chain Reaction ◽

Tobacco Leaf ◽

Qualitative And Quantitative ◽

Polymerase Chain

Abstract The Cooperation Centre for Scientific Research Relative to Tobacco (CORESTA; Paris, France) Task Force Genetically Modified TobaccoDetection Methods investigated the performance of qualitative and quantitative methods based on the polymerase chain reaction (PCR) for the detection and quantitation of genetically modified (GM) tobacco. In the 4 successful rounds of proficiency testing, the cauliflower mosaic virus 35S RNA promoter (CaMV 35S) and the Agrobacterium tumefaciens nopaline synthase terminator (NOS) were selected as target sequences. Blind-coded reference materials containing from 0.1 to 5.0% and from 0.15 to 4% GM tobacco were used in 2 rounds of qualitative and quantitative PCR, respectively. Eighteen laboratories from 10 countries participated in this study. Considering all methods and 2 rounds, the different laboratories were able to detect GM tobacco at the 0.1% level in 46 out of 58 tests in qualitative assays. The results of the proficiency test indicate that both end point screening and real-time quantitative methods are suitable for the detection of genetically modified organisms in tobacco leaf samples having a GM content of 0.1% or higher. The CORESTA proficiency study represents a first step towards the interlaboratory evaluation of accuracy and precision of PCR-based GM tobacco detection, which may lead to the harmonization of analytical procedures and to the enhancement of comparability of testing results produced by different laboratories.

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Development of AFLP-derived, functionally specific markers for environmental persistence studies of fungal strains

Canadian Journal of Microbiology ◽

10.1139/w05-140 ◽

2006 ◽

Vol 52 (5) ◽

pp. 451-461 ◽

Cited By ~ 8

Author(s):

S S Hynes ◽

O Chaudhry ◽

M A Providenti ◽

M L Smith

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Genetically Modified Organisms ◽

Quantitative Polymerase Chain Reaction ◽

Pcr Primers ◽

Chain Reaction ◽

Environmental Persistence ◽

Fungal Strains ◽

Target Dna ◽

Polymerase Chain

The ability to rapidly identify and quantify a microbial strain in a complex environmental sample has widespread applications in ecology, epidemiology, and industry. In this study, we describe a rapid method to obtain functionally specific genetic markers that can be used in conjunction with standard or real-time polymerase chain reaction (PCR) to determine the abundance of target fungal strains in selected environmental samples. The method involves sequencing of randomly cloned AFLP (amplified fragment length polymorphism) products from the target organism and the design of PCR primers internal to the AFLP fragments. The strain-specific markers were used to determine the fate of three industrially relevant fungi, Aspergillus niger, Aspergillus oryzae, and Chaetomium globosum, during a 4 month soil microcosm experiment. The persistence of each of the three fungal strains inoculated separately into intact soil microcosms was determined by PCR analyses of DNA directly extracted from soil. Presence and absence data based on standard PCR and quantification of the target DNA by real-time PCR showed that all three strains declined after inoculation (~14-, 32-, and 4-fold for A. niger, A. oryzae, and C. globosum, respectively) but remained detectable at the end of the experiment, suggesting that these strains would survive for extended periods if released into nature.Key words: Canada domestic substances list (DSL), Canadian Environmental Protection Act (CEPA), genetically modified organisms (GMO), quantitative polymerase chain reaction (qPCR).

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Electrochemiluminescence polymerase chain reaction detection of genetically modified organisms

Analytica Chimica Acta ◽

10.1016/j.aca.2005.01.011 ◽

2005 ◽

Vol 537 (1-2) ◽

pp. 119-123 ◽

Cited By ~ 15

Author(s):

Jinfeng Liu ◽

Da Xing ◽

Xingyan Shen ◽

Debin Zhu

Keyword(s):

Polymerase Chain Reaction ◽

Genetically Modified Organisms ◽

Genetically Modified ◽

Polymerase Chain Reaction Detection ◽

Chain Reaction ◽

Polymerase Chain

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The validation of methods based on polymerase chain reaction for the detection of genetically modified organisms in food

Analytica Chimica Acta ◽

10.1016/s0003-2670(98)00824-1 ◽

1999 ◽

Vol 393 (1-3) ◽

pp. 177-179 ◽

Cited By ~ 9

Author(s):

Elke Anklam

Keyword(s):

Polymerase Chain Reaction ◽

Genetically Modified Organisms ◽

Genetically Modified ◽

Chain Reaction ◽

Validation Of Methods ◽

Polymerase Chain

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

Journal of AOAC International ◽

10.1093/jaoac/88.3.814 ◽

2005 ◽

Vol 88 (3) ◽

pp. 814-822 ◽

Cited By ~ 20

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.

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