Development of Detection System Using Multiplex PCR and Liquid Beadarray for Stacked Genetically Modified Rice Event (LS28×Cry1Ac)

Abstract

A multiplex system was developed to assess detection of stacked genetically modified (GM) rice (LS28 × Cry1Ac) based on multiplex polymerase chain reaction (PCR) and liquid beadarray, and the accuracy of the system was analyzed. Standard and specific bulging specific (SBS) primers with standard primers were used to simultaneously detect multiple targets in stacked events of rice. Five sets of primers for the stacked events were applied to amplify their targets, and were separated distinctly in agarose gel. A liquid beadarray assay for the stacked GM rice was performed using the multiplex PCR products, followed by target biotinylation and hybridization between biotinylated-tagged target and anti-tagged bead. Fluorescent signals of the hybridized target sequences were detected by the Luminex system. The signaling patterns were analyzed by their mean fluorescent intensity (MFI) value. Results showed that liquid beadarrays with standard and SBS primers were in complete agreement with the PCR data, and detection of the different target elements was found to be very specific with no cross reaction among samples. Therefore, our detection system developed for stacked GM crop using multiplex PCR and liquid beadarray can be a useful and efficient system for screening and analyzing multiple transgenes in a single tube for qualitative analysis.