scholarly journals Evaluation of Modified PCR Quantitation of Genetically Modified Maize and Soybean Using Reference Molecules: Interlaboratory Study

2009 ◽  
Vol 92 (1) ◽  
pp. 223-233 ◽  
Author(s):  
Takashi Kodama ◽  
Hideo Kuribara ◽  
Yasutaka Minegishi ◽  
Satoshi Futo ◽  
Masatoshi Watai ◽  
...  
Keyword(s):  
Quantitative Methods ◽  
Conversion Factor ◽  
Recombinant Dna ◽  
Genetically Modified ◽  
Interlaboratory Study ◽  
Chain Reaction ◽  
Specific Sequence ◽  
Reference Plasmid ◽  
Repeatability And Reproducibility ◽  
Polymerase Chain

Abstract Real-time polymerase chain reaction (PCR)-based quantitative methods were previously developed and validated for genetically modified (GM) maize or soy. In this study, the quantification step of the validated methods was modified, and an interlaboratory study was conducted. The modification included the introduction of the PCR system SSIIb 3 instead of SSIIb 1 for the detection of the taxon-specific sequence of maize, as well as the adoption of colE1 as a carrier included in a reference plasmid solution as a replacement for salmon testis. The interlaboratory study was conducted with the ABI PRISM<sup/> 7700 and consisted of 2 separate stages: (1) the measurement of conversion factor (Cf) value, which is the ratio of recombinant DNA (r-DNA) sequence to taxon-specific sequence in each genuine GM seed, and (2) the quantification of blind samples. Additionally, Cf values of other instruments, such as the ABI PRISM 7900 and the ABI PRISM 7000, were measured in a multilaboratory trial. After outlier laboratories were eliminated, the repeatability and reproducibility for 5.0 samples were <15.8 and 20.6, respectively. The quantitation limits of these methods were 0.5 for Bt11, T25, and MON810, and 0.1 for GA21, Event176, and RR soy. The quantitation limits, trueness, and precision of the current modified methods were equivalent to those of the previous methods. Therefore, it was concluded that the modified methods would be a suitable replacement for the validated methods.

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

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.

scholarly journals Detection for suspected Genetically Modified Maize and Soybean crops in the selcted places of Ethiopia using PCR machine

2019 ◽  
Author(s):  
Adugnaw Admas ◽  
Smegnew Melese ◽  
Amare Genetu
Keyword(s):  
Genetically Modified Organisms ◽  
Seed Quality ◽  
Recombinant Dna ◽  
Genetically Modified ◽  
Positive Control ◽  
Chain Reaction ◽  
Development Activity ◽  
The Past ◽  
Polymerase Chain ◽  
Thermal Cycler

AbstractDetection of genetically modified organisms (GMOs) in crops is an important issue for all the subjects involved in seed quality control and customer’s right. Due to the increasing number of GMOs research and development activity in the globe during the past few years, it has become necessary to screen and regulate highely practiced GMO crops. In this study, following the extraction of genomic DNA from agriculture farming land and commercial areas collected samples that suspected for GMOs such as Soybean and maize crops, the recombinant DNA target sequences were targeted to detect for transgene such as CaMV35s gene using thermal cycler Polymerase Chain Reaction (PCR). In addition, for positive control taxone specfic Invertase and Lectine gene of maize and soybean have done. Based on the gel electrophoresis results, Invertase and Lectine gene events were detected in all maize and soybean samples respectively. How ever, fortunately in all collected samples of maize and soybean in different places of the countery trans-genes were not detected. Finally, this reserch result concludes that in the studied area soybean and maize crops are GMO free.

Evaluation of polymerase chain reaction and DNA isolation protocols for detection of genetically modified soybean

2007 ◽  
Vol 42 (10) ◽  
pp. 1249-1255 ◽  
Author(s):  
Cibele dos Santos Ferrari ◽  
Luciana Lehmkuhl Valente ◽  
Fábio Cristiano Angonesi Brod ◽  
Caroline Tagliari ◽  
Ernani Sebastião Sant'Anna ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Isolation ◽  
Genetically Modified ◽  
Chain Reaction ◽  
Genetically Modified Soybean ◽  
Polymerase Chain

The fate of a genetically modifiedPseudomonasstrain and its transgene during the composting of poultry manure

2004 ◽  
Vol 50 (6) ◽  
pp. 415-421 ◽  
Author(s):  
J Guan ◽  
J L Spencer ◽  
M Sampath ◽  
J Devenish
Keyword(s):  
Polymerase Chain Reaction ◽  
Incubation Temperature ◽  
Genetically Modified ◽  
Chicken Manure ◽  
Detection Sensitivity ◽  
Plate Count ◽  
Chain Reaction ◽  
Nested Polymerase Chain Reaction ◽  
Soil Microcosms ◽  
Polymerase Chain

The fate of the genetically modified (GM) Pseudomonas chlororaphis strain 3732 RN-L11 and its transgene (lacZ insert) during composting of chicken manure was studied using plate count and nested polymerase chain reaction (PCR) methods. The detection sensitivity of the nested PCR method was 165 copies of the modified gene per gram of moist compost or soil. Compost microcosms consisted of a 100-g mixture of chicken manure and peat, whereas soil microcosms were 100-g samples of sandy clay loam. Each microcosm was inoculated with 4 × 1010CFU of P. chlororaphis RN-L11. In controlled temperature studies, neither P. chlororaphis RN-L11 nor its transgene could be detected in compost microcosms after incubation temperature was elevated to 45 °C or above for one or more days. In contrast, in the compost microcosms incubated at 23 °C, the target organism was not detected by the plate count method after 6 days, but its transgene was detectable for at least 45 days. In compost bins, the target organism was not recovered from compost microcosms or soil microcosms at different levels in the bins for 29 days. However, the transgene was detected in 8 of the 9 soil microcosms and in only 1 of the 9 compost microcosms. The compost microcosm in which transgene was detected was at the lower level of the bin where temperatures remained below 45 °C. The findings indicated that composting of organic wastes could be used to reduce or degrade heat sensitive GM microorganisms and their transgenes.Key words: composting, genetically modified Pseudomonas strain, transgene, polymerase chain reaction.

scholarly journals Extração de DNA a partir de sangue humano coagulado para aplicação nas técnicas de genotipagem de antígenos leucocitários humanos e de receptores semelhantes à imunoglobulina

2009 ◽  
Vol 42 (6) ◽  
pp. 651-656 ◽  
Author(s):  
Daniela Maira Cardozo ◽  
Gláucia Andréia Guelsin ◽  
Samaia Laface Clementino ◽  
Fabiano Cavalcante de Melo ◽  
Marco Antônio Braga ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Killer Cell ◽  
Human Leukocyte ◽  
Reaction Sequence ◽  
Chain Reaction ◽  
Specific Sequence ◽  
Salting Out ◽  
Leukocyte Antigens ◽  
Polymerase Chain ◽  
Made In

O objetivo deste estudo foi padronizar uma metodologia de extração de DNA de alta qualidade a partir de amostras de sangue coagulado. Quarenta e oito amostras de sangue humano coagulado foram utilizadas para a extração de DNA pelo kit comercial EZ-DNA® (Biological Industries, Beit Haemek, Israel), pelo kit de coluna Neoscience® (One Lambda Inc., San Diego, CA) e pelo método modificado de salting out. Apenas o método de salting out foi capaz de extrair altas concentrações de DNA (média, 180ng/µL), as quais foram medidas pelo detector de fluorescência Qubit® (Invitrogen, USA). Este método permitiu a amplificação dos genes HLA (human leukocyte antigens) pela tecnologia PCR-SSO (polymerase chain reaction - specific sequence of oligonucleotides) Luminex, a qual exige DNA de boa qualidade, e de genes KIR (killer cell immunoglobulin-like receptors) pela técnica made in house PCR-SSP (polymerase chain reaction-sequence specific of primers), a qual demanda uma concentração específica de DNA (10ng/µL). Concluímos que a técnica de salting out modificada foi muito eficiente, simples e rápida para a extração de DNA de amostras de sangue humano coagulado, com o objetivo de realizar a genotipagem de genes HLA e KIR.

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