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

Tigst Demeke,Sung-Jong Lee,Monika Eng

doi:10.3390/biology11020201

Abstract

Simple SummaryDigital 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.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.

Highlights

The number of genetically modified (GM) events has been increasing since the start of commercialization of GM crops in the mid-1990s [1]
The concentrations of primers and probes and the probe labels used for duplex, triplex, and tetraplex droplet digital PCR (ddPCR) are shown in Supplementary Tables S1–S3
Mixtures of equal amounts of FAM- and HEX-labeled probes were used for MON88302 and DP305423 GM events in order to obtain the third position for triplex ddPCR

Summary

Introduction

The number of genetically modified (GM) events has been increasing since the start of commercialization of GM crops in the mid-1990s [1]. Many countries have a regulatory approval process for new GM events and effective detection methods are required for monitoring [2,3,4]. The main reason for enforcement is because of consumers’ rights to know what is in their food New breeding technologies, such as CRISPR-Cas, are being used for crop improvement and information about genetic modification is required if there is a need for monitoring [5]. Real-time qualitative and quantitative PCR assays have been widely used for the detection and quantitative analysis of GM events [6,7,8]. The real-time PCR assay relies on the availability of certified reference materials and use of appropriate standard curves. The minimum required performance limit (MRPL), which is the lowest level of GM material required for the validation of quantitative methods, has been set by European Union Reference Laboratory at 0.1% [10]

Objectives

Methods

Results

Conclusion