Increasing the Efficiency of Canola and Soybean GMO Detection and Quantification Using Multiplex Droplet Digital PCR

SIMPLE SUMMARY: Digital PCR (dPCR) technology has been used for absolute quantification of genetically modified (GM) events. Duplex dPCR consisting of a target gene and a reference gene is mostly used for absolute quantification of GM events. We investigated the feasibility of absolute quantification of two, three, and four GM canola and soybean events at the same time using the QX200 Droplet Digital PCR (ddPCR) system. Adjustments of the probe concentrations and labels for some of the assays were needed for successful multiplex ddPCR. Absolute quantification of GM canola and soybean events was achieved for duplex, triplex, and tetraplex ddPCR at 0.1%, 1%, and 5% concentrations. ABSTRACT: The number of genetically modified (GM) events for canola, maize, and soybean has been steadily increasing. Real-time PCR is widely used for the detection and quantification of individual GM events. Digital PCR (dPCR) has also been used for absolute quantification of GM events. A duplex dPCR assay consisting of one reference gene and one GM event has been carried out in most cases. The detection of more than one GM event in a single assay will increase the efficiency of dPCR. The feasibility of detection and quantification of two, three, and four GM canola and soybean events at the same time was investigated at 0.1%, 1%, and 5% levels using the QX200 Droplet Digital PCR (ddPCR) system. The reference gene assay was carried out on the same plate but in different wells. For some of the assays, optimization of the probe concentrations and labels was needed for successful ddPCR. Results close to the expected result were achieved for duplex, triplex, and tetraplex ddPCR assays for GM canola events. Similar ddPCR results were also achieved for some GM soybean events with some exceptions. Overall, absolute quantification of up to four GM events at the same time improves the efficiency of GM detection.