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BioMed Research International
Volume 2013, Article ID 134675, 7 pages
Research Article

Development of a Novel Reference Plasmid for Accurate Quantification of Genetically Modified Kefeng6 Rice DNA in Food and Feed Samples

Biotechnology Research Institute, Chinese Agricultural Academy of Sciences, No. 12 Zhongguancun South Street, Haidian District, Beijing 100081, China

Received 28 June 2013; Revised 20 September 2013; Accepted 4 October 2013

Academic Editor: Guihua H. Bai

Copyright © 2013 Liang Li et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


Reference plasmids are an essential tool for the quantification of genetically modified (GM) events. Quantitative real-time PCR (qPCR) is the most commonly used method to characterize and quantify reference plasmids. However, the precision of this method is often limited by calibration curves, and qPCR data can be affected by matrix differences between the standards and samples. Here, we describe a digital PCR (dPCR) approach that can be used to accurately measure the novel reference plasmid pKefeng6 and quantify the unauthorized variety of GM rice Kefeng6, eliminating the issues associated with matrix effects in calibration curves. The pKefeng6 plasmid was used as a calibrant for the quantification of Kefeng6 rice by determining the copy numbers of event- (77 bp) and taxon-specific (68 bp) fragments, their ratios, and their concentrations. The plasmid was diluted to five different concentrations. The third sample (S3) was optimized for the quantification range of dPCR according to previous reports. The ratio between the two fragments was 1.005, which closely approximated the value certified by sequencing, and the concentration was found to be 792 copies/μL. This method was precise, with an RSD of ~3%. These findings demonstrate the advantages of using the dPCR method to characterize reference materials.