title: A Novel Pretreatment-Free Duplex Chamber Digital PCR Detection System for the Absolute Quantitation of GMO Samples.
abstract: Digital polymerase chain reaction (PCR) has developed rapidly since it was first reported in the 1990s. However, pretreatments are often required during preparation for digital PCR, which can increase operation error. The single-plex amplification of both the target and reference genes may cause uncertainties due to the different reaction volumes and the matrix effect. In the current study, a quantitative detection system based on the pretreatment-free duplex chamber digital PCR was developed. The dynamic range, limit of quantitation (LOQ), sensitivity and specificity were evaluated taking the GA21 event as the experimental object. Moreover, to determine the factors that may influence the stability of the duplex system, we evaluated whether the pretreatments, the primary and secondary structures of the probes and the SNP effect influence the detection. The results showed that the LOQ was 0.5% and the sensitivity was 0.1%. We also found that genome digestion and single nucleotide polymorphism (SNP) sites affect the detection results, whereas the unspecific hybridization within different probes had little side effect. This indicated that the detection system was suited for both chamber-based and droplet-based digital PCR. In conclusion, we have provided a simple and flexible way of achieving absolute quantitation for genetically modified organism (GMO) genome samples using commercial digital PCR detection systems.
authors.affiliation: Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Food Science &amp; Nutritional Engineering, China Agricultural University, Beijing 100083, China. zhupengyu3638@163.com.
authors.forename: Pengyu
authors.initials: P
authors.lastname: Zhu
authors.affiliation: Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Food Science &amp; Nutritional Engineering, China Agricultural University, Beijing 100083, China. italy10.wang@gmail.com.
authors.forename: Chenguang
authors.initials: C
authors.lastname: Wang
authors.affiliation: Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Food Science &amp; Nutritional Engineering, China Agricultural University, Beijing 100083, China. huangkl009@sina.com.|Beijing Laboratory for Food Quality and Safety, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China. huangkl009@sina.com.
authors.forename: Kunlun
authors.initials: K
authors.lastname: Huang
authors.affiliation: Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Food Science &amp; Nutritional Engineering, China Agricultural University, Beijing 100083, China. lyb@cau.edu.cn.|Beijing Laboratory for Food Quality and Safety, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China. lyb@cau.edu.cn.
authors.forename: Yunbo
authors.initials: Y
authors.lastname: Luo
authors.affiliation: Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Food Science &amp; Nutritional Engineering, China Agricultural University, Beijing 100083, China. xuwentao@cau.edu.cn.|Beijing Laboratory for Food Quality and Safety, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China. xuwentao@cau.edu.cn.
authors.forename: Wentao
authors.initials: W
authors.lastname: Xu
mesh_terms: D018744:DNA, Plant / Q000737:chemistry / Q000235:genetics*; D009693:Nucleic Acid Hybridization; D030821:Plants, Genetically Modified / Q000145:classification* / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide
chemical_list: D018744:DNA, Plant
keywords: GMO; absolute quantitative detection method; digital PCR; pretreatment-free