Detection and identification of transgenic elements by fluorescent-PCR-based capillary gel electrophoresis in genetically modified cotton and soybean.

摘要

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 four targets such as Sad1, Cry1Ac, 35S, and Nos in Bt-cotton. Four novel PCR primers were designed to customize amplicon size for multiplexing for better visualization of 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 grain powder (0.1%, w/w). This study demonstrates a PCR-CGE-based method for the qualitative detection of 35S, Nos and Cry1Ac targets associated with genetically modified products.