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

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

Journal of AOAC International ◽

10.1093/jaoac/92.3.765 ◽

2009 ◽

Vol 92 (3) ◽

pp. 765-772 ◽

Cited By ~ 27

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.

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Determination of eight genetically modified maize events by quantitative, multiplex PCR and fluorescence capillary gel electrophoresis

European Food Research and Technology ◽

10.1007/s00217-008-0828-8 ◽

2008 ◽

Vol 227 (4) ◽

pp. 1125-1137 ◽

Cited By ~ 30

Author(s):

Bjarte R. Heide ◽

Signe M. Drømtorp ◽

Knut Rudi ◽

Even Heir ◽

Askild L. Holck

Keyword(s):

Multiplex Pcr ◽

Gel Electrophoresis ◽

Genetically Modified ◽

Genetically Modified Maize ◽

Capillary Gel Electrophoresis

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

Journal of AOAC International ◽

10.5740/jaoacint.13-027 ◽

2014 ◽

Vol 97 (1) ◽

pp. 159-165 ◽

Cited By ~ 14

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.

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