Detection and Identification of Transgenic Elements by Fluorescent-PCR-Based Capillary Gel Electrophoresis in Genetically Modified Cotton and Soybean

2014 ◽  
Vol 97 (1) ◽  
pp. 159-165 ◽  
Author(s):  
Sanjay Basak ◽  
Nasreen Z Ehtesham ◽  
Boindala Sesikeran ◽  
Sudip Ghosh
Keyword(s):  
Multiplex Pcr ◽  
Dna Sequences ◽  
Gel Electrophoresis ◽  
Genetically Modified Organisms ◽  
Genetically Modified ◽  
Bt Cotton ◽  
Regulatory Requirement ◽  
Sequencing Platform ◽  
Capillary Gel Electrophoresis ◽  
Multiplex Pcr Assay

Abstract A detection method for genetically modified foods is an essential regulatory requirement for many countries. The present study is aimed at developing a qualitative method for detection of genetically modified organisms by combining PCR methodology with capillary gel electrophoresis (PCR-CGE) in a sequencing platform to detect Bacillus thuringiensis (Bt)-cotton (MON 531)and Roundup Ready (RR) soybean (GTS 40-3-2). A sensitive duplex PCR-CGE method was developed in which target DNA sequences (35S and Nos) were separated both by size and color to detect 0.01% Cry1Ac DNA (w/w) in Bt-cotton. A multiplex PCR-CGE method was developed to simultaneously detect fourtargets such as Sad1, Cry1Ac, 35S, and Nos in Bt-cotton. Four novel PCR primers were designed to customize amplicon size for multiplexing for better visualizationof multiple peaks. The LOD for Cry1Ac DNA specific PCR was 0.01% for Bt-cotton. The LOD for multiplex PCR assay was 0.05% for Bt-cotton. A singleplex PCR-CGE method was developed to detect Lec, 35S and Nos in a trace sample of RR soybean grainpowder (0.1%, w/w). This study demonstrates aPCR-CGE-based method for the qualitative detection of35S, Nos and Cry1Ac targets associated with genetically modifiedproducts.

scholarly journals Multiplex Polymerase Chain Reaction-Capillary Gel Electrophoresis: A Promising Tool for GMO ScreeningAssay for Simultaneous Detection of Five Genetically Modified Cotton Events and Species

2009 ◽  
Vol 92 (3) ◽  
pp. 765-772 ◽  
Author(s):  
Anna Nadal ◽  
Teresa Esteve ◽  
Maria Pla
Keyword(s):  
Polymerase Chain Reaction ◽  
Multiplex Pcr ◽  
Gel Electrophoresis ◽  
Multiplex Polymerase Chain Reaction ◽  
Genetically Modified ◽  
Simultaneous Detection ◽  
Chain Reaction ◽  
Capillary Gel Electrophoresis ◽  
Cotton Species ◽  
Polymerase Chain

Abstract A multiplex polymerase chain reaction assay coupled to capillary gel electrophoresis for amplicon identification by size and color (multiplex PCR-CGE-SC) was developed for simultaneous detection of cotton species and 5 events of genetically modified (GM) cotton. Validated real-time-PCR reactions targeting Bollgard, Bollgard II, Roundup Ready, 3006-210-23, and 281-24-236 junction sequences, and the cotton reference gene acp1 were adapted to detect more than half of the European Union-approved individual or stacked GM cotton events in one reaction. The assay was fully specific (<1.7 of false classification rate), with limit of detection values of 0.1 for each event, which were also achieved with simulated mixtures at different relative percentages of targets. The assay was further combined with a second multiplex PCR-CGE-SC assay to allow simultaneous detection of 6 cotton and 5 maize targets (two endogenous genes and 9 GM events) in two multiplex PCRs and a single CGE, making the approach more economic. Besides allowing simultaneous detection of many targets with adequate specificity and sensitivity, the multiplex PCR-CGE-SC approach has high throughput and automation capabilities, while keeping a very simple protocol, e.g., amplification and labeling in one step. Thus, it is an easy and inexpensive tool for initial screening, to be complemented with quantitative assays if necessary.

Establishment of a Decaplex PCR-Capillary Gel Electrophoresis Method for the Simultaneous Detection of Six Kinds of Genetically Modified Animals

2018 ◽  
Vol 101 (2) ◽  
pp. 601-606 ◽  
Author(s):  
Xiaofei Liu ◽  
Songyin Qiu ◽  
Xiaolin Li ◽  
Dandan Liu ◽  
Hongli Jing ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Gel Electrophoresis ◽  
Detection System ◽  
Genetically Modified ◽  
Simultaneous Detection ◽  
Fatty Acid Desaturase ◽  
Capillary Gel Electrophoresis ◽  
Inspection And Quarantine ◽  
Electrophoresis Method ◽  
Gm Animals

Abstract This study aimed to establish an event-specific multiplex PCR system using microsatellite markers and fluorescently labeled primers to detect six different genetically modified (GM) animal lines, including human lactoferrin GM cattle, human lysozyme GM cattle, human α-lactalbumin GM cattle, myostatin knockout pigs, phytase GM pigs, and ω-3 fatty acid desaturase gene GM pigs. Four different microsatellite loci for species identification, along with six GM animal-specific fragments, were selected as targets for primer design. The capillary gel electrophoresis results of multiplex PCR showed that the target fragments were amplified successfully. This high-throughput multiplex PCR detection system can be applied for the inspection and quarantine of GM animals.

A general multiplex-PCR assay for the general detection of genetically modified soya and maize

Food Control ◽  
2005 ◽  
Vol 16 (6) ◽  
pp. 535-539 ◽  
Author(s):  
V.T. Forte ◽  
A. Di Pinto ◽  
C. Martino ◽  
G.M. Tantillo ◽  
G. Grasso ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Genetically Modified ◽  
Pcr Assay ◽  
Multiplex Pcr Assay

scholarly journals Current PCR Methods for the Detection, Identification and Quantification of Genetically Modified Organisms(GMOs): a Brief Review

2000 ◽  
Vol 19 (2) ◽  
pp. 85-96 ◽  
Author(s):  
F Gadani ◽  
G Bindler ◽  
H Pijenburg ◽  
L Rossi ◽  
J Zuber
Keyword(s):  
Dna Sequences ◽  
Method Development ◽  
Genetically Modified Organisms ◽  
Genetically Modified ◽  
Value Added ◽  
Detection Methods ◽  
Agricultural Crops ◽  
The European Union ◽  
Gmo Detection ◽  
Food Ingredients

AbstractAnalytical methods based on the polymerase chain reaction (PCR) technology are increasingly used for the detection of deoxyribonucleic acid (DNA) sequences associated with genetically modified organisms (GMOs). In the European Union and Switzerland, mandatory labeling of novel foods and food ingredients consisting of, or containing GMOs is required according to food regulations and is triggered by the presence of newly introduced foreign DNA sequences, or newly expressed proteins. In order to meet regulatory and consumer demand, numerous PCR-based methods have been developed which can detect, identify and quantify GMOs in agricultural crops, food and feed. Moreover, the determination of genetic identity allows for segregation and traceability (identity preservation) throughout the supply chain of GM crops that have been enhanced with value-added quality traits. Prerequisites for GMO detection include a minimum amount of the target gene and prior knowledge of the type of genetic modification, such as virus or insect resistance traits, including controlling elements (promoters and terminators). Moreover, DNA extraction and purification is a critical step for the preparation of PCR-quality samples, particularly for processed agricultural crops such as tobacco. This paper reviews the state-of-the-art of PCR-based method development for the qualitative and quantitative determination and identification of GMOs, and includes a short summary of official and validated GMO detection methods.

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