Hexaplex PCR assay and liquid bead array for detection of stacked genetically modified cotton event...

A hexaplex system based on multiplex polymerase chain reaction (PCR) coupled with liquid bead array was developed to assist detection of stacked genetically modified (GM) cotton event 281-24-236 × 3006-210-23 (Widestrike) expressing two kinds of endotoxin from Bacillus thuringiensis (Bt). The efficiency of this multiplex detection system was assessed. Specific primer sets for simultaneous detection of six targets in the stacked GM cotton event were constructed and used for the PCR assay. Each of the six targets was amplified, and the amplicons could be separated as discrete bands by agarose gel electrophoresis. A liquid bead array assay for the stacked GM cotton was performed using the hexaplex PCR products followed by hybridization between the biotinylated targets and anti-tagged microsphere beads. The hybridization products produced fluorescent signals that were detected by the Luminex system. Signal strengths were analyzed by their median fluorescent intensity values. Comparison of the assays showed that results from the liquid bead array using specific probes agreed with those from the PCR, and detection of the different target elements was found to be very specific with no cross-reaction. Therefore, the combination of hexaplex PCR and liquid bead array for detection of stacked GM events can be a useful and efficient system for screening and analyzing multiple transgenes for simultaneous qualitative analysis.