A Digital Polymerase Chain Reaction Method Based on Figwort Mosaic Virus 35S Promoter (FMV 35S) for Quantitative Detection of Genetically Modified Crops

Abstract

Using genetically modified (GM) soybean MON87705 containing the figwort mosaic virus 35S promoter (FMV 35S) as a marker gene, a quantitative method for detecting transgenic crops was established based on real-time fluorescent quantitative polymerase chain reaction (real-time PCR) and digital polymerase chain reaction (dPCR). The amplification system was tested by real-time PCR, revealing that the developed method was highly specific to FMV 35S. By determining quantitative parameters using duplex droplet digital PCR (ddPCR), it was found that within the genomic DNA concentration range of 0.005–20 ng/mL, the measured copy number of the target gene had a good linear correlation with the theoretical copy number; the limit of detection (LOD) and the limit of quantification (LOQ) of dPCR for FMV 35S were as low as 5 copies and 0.1%, respectively. Blind samples with different contents of MON87705 were tested on two dPCR platforms, indicating that accurate, reliable and reproducible results were obtained in the duplex and triple detection using a combination of internal reference genes and foreign specific fragments. Therefore, the quantitative dPCR method, which can meet the parameter requirements of the quantitative detection standard for genetically modified components with improved detection efficiency, represents an accurate and simple technology for the quantitative detection of genetically modified crops.

Keywords

• transgenic; screening marker; digital pcr; fmv 35s; quantitative detection