scholarly journals Validation Studies and Proficiency Testing

2002 ◽  
Vol 85 (3) ◽  
pp. 809-816 ◽  
Author(s):  
Elke Anklam ◽  
Petra Heinze ◽  
Simon Kay ◽  
Guy van den Eede ◽  
Bert Popping
Keyword(s):  
Validation Studies ◽  
Genetically Modified Organisms ◽  
Quantitative Polymerase Chain Reaction ◽  
Extraction Methods ◽  
International Level ◽  
Testing Methods ◽  
Chain Reaction ◽  
Formal Validation ◽  
Food Market ◽  
Polymerase Chain

Abstract Genetically modified organisms (GMOs) entered the European food market in 1996. Current legislation demands the labeling of food products if they contain <1% GMO, as assessed for each ingredient of the product. To create confidence in the testing methods and to complement enforcement requirements, there is an urgent need for internationally validated methods, which could serve as reference methods. To date, several methods have been submitted to validation trials at an international level; approaches now exist that can be used in different circumstances and for different food matrixes. Moreover, the requirement for the formal validation of methods is clearly accepted; several national and international bodies are active in organizing studies. Further validation studies, especially on the quantitative polymerase chain reaction methods, need to be performed to cover the rising demand for new extraction methods and other background matrixes, as well as for novel GMO constructs.

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

2006 ◽  
Vol 52 (5) ◽  
pp. 451-461 ◽  
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).

scholarly journals Development of a Systematic qPCR Array for Screening GM Soybeans

Foods ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 610
Author(s):  
Saet-Byul Park ◽  
Ji-Yeong Kim ◽  
Do-Geun Lee ◽  
Jae-Hwan Kim ◽  
Min-Ki Shin ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Genetically Modified Organisms ◽  
Quantitative Polymerase Chain Reaction ◽  
Screening Method ◽  
35S Promoter ◽  
Prediction System ◽  
Chain Reaction ◽  
Qpcr Array ◽  
Polymerase Chain ◽  
Gm Soybean

A screening method using the 35S promoter and nos terminator for genetically modified organisms (GMOs) is not sufficient to cover all GM soybean events. In this study, a real-time polymerase chain reaction (also known as quantitative polymerase chain reaction, qPCR) array targeting eight screening assays combined with a prediction system was developed for the rapid tracking of GM soybeans. Each assay’s specificity was tested and confirmed using 17 GM soybean events that have been approved in Korea. The sensitivity of each assay was determined to range from 0.01% to 0.05% using DNA mixtures with different GM ratios, and it was validated by the results of three experimenters. The applicability of this study was tested by monitoring 23 processed foods containing soybeans. It was figured out that 13 of the 23 samples included GM soybeans. The prediction system combined with screening results will be helpful to trace the absence/presence of GM soybean events. This new qPCR array and prediction system for GM soybean detection provides rapid, convenient and reliable results to users.

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

2008 ◽  
Vol 376 (2) ◽  
pp. 189-199 ◽  
Author(s):  
Katarina Cankar ◽  
Valérie Chauvensy-Ancel ◽  
Marie-Noelle Fortabat ◽  
Kristina Gruden ◽  
André Kobilinsky ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Genetically Modified Organisms ◽  
Genetically Modified ◽  
Quantitative Polymerase Chain Reaction ◽  
Chain Reaction ◽  
Polymerase Chain

Determination of low bacterial concentrations in hyperarid Atacama soils: comparison of biochemical and microscopy methods with real-time quantitative PCR

2011 ◽  
Vol 57 (11) ◽  
pp. 953-963 ◽  
Author(s):  
Lauren E. Fletcher ◽  
Catharine A. Conley ◽  
Julio E. Valdivia-Silva ◽  
Saul Perez-Montaño ◽  
Renee Condori-Apaza ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Fatty Acid ◽  
Real Time ◽  
Phospholipid Fatty Acid ◽  
Quantitative Polymerase Chain Reaction ◽  
Extraction Methods ◽  
The Other ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Number Of Bacteria

Hyperarid Atacama soils are reported to contain significantly reduced numbers of microbes per gram of soil relative to soils from other environments. Molecular methods have been used to evaluate microbial populations in hyperarid Atacama soils; however, conflicting results across the various studies, possibly caused by this low number of microorganisms and consequent biomass, suggest that knowledge of expected DNA concentrations in these soils becomes important to interpreting data from any method regarding microbial concentrations and diversity. In this paper we compare the number of bacteria per gram of Atacama Desert soils determined by real-time quantitative polymerase chain reaction with the number of bacteria estimated by the standard methods of phospholipids fatty acid analysis, adenine composition (determined by liquid chromatography – time-of-flight mass spectrometry), and SYBR-green microscopy. The number determined by real-time quantitative polymerase chain reaction as implemented in this study was several orders of magnitude lower than that determined by the other three methods and probably underestimates the concentrations of soil bacteria, most likely because of soil binding during the DNA extraction methods. However, the other methods very possibly overestimate the bacteria concentrations owing to desiccated, intact organisms, which would stain positive in microscopy and preserve both adenine and phospholipid fatty acid for the other methods.

AN IMPROVED METHOD FOR INHIBITOR FREE NUCLEIC ACIDS FROM POLYPHENOL RICH LEAVES OF MUSA SPP

2017 ◽  
Vol 23 (1) ◽  
Author(s):  
N.NANDHA KUMAR ◽  
K. SOURIANATHA SUNDARAM ◽  
D. SUDHAKAR ◽  
K.K. KUMAR
Keyword(s):  
Polymerase Chain Reaction ◽  
Quantitative Polymerase Chain Reaction ◽  
Proteinase K ◽  
Chain Reaction ◽  
Banana Plant ◽  
High Molecular Weight Dna ◽  
Musa Spp ◽  
Leaf Tissues ◽  
Polymerase Chain

Excessive presence of polysaccharides, polyphenol and secondary metabolites in banana plant affects the quality of DNA and it leads to difficult in isolating good quality of DNA. An optimized modified CTAB protocol for the isolation of high quality and quantity of DNA obtained from banana leaf tissues has been developed. In this protocol a slight increased salt (NaCl) concentration (2.0M) was used in the extraction buffer. Polyvinylpyrrolidone (PVP) and Octanol were used for the removal of polyphenols and polymerase chain reaction (PCR) inhibitors. Proteins like various enzymes were degraded by Proteinase K and removed by centrifugation from plant extract during the isolation process resulting in pure genomic DNA, ready to use in downstream applications including PCR, quantitative polymerase chain reaction (qPCR), ligation, restriction and sequencing. This protocol yielded a high molecular weight DNA isolated from polyphenols rich leaves of Musa spp which was free from contamination and colour. The average yields of total DNA from leaf ranged from 917.4 to 1860.9 ng/ìL. This modified CTAB protocol reported here is less time consuming 4-5h, reproducible and can be used for a broad spectrum of plant species which have polyphenol and polysaccharide compounds.

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