title: Development and application of absolute quantitative detection by duplex chamber-based digital PCR of genetically modified maize events without pretreatment steps.
abstract: The possibility of the absolute quantitation of GMO events by digital PCR was recently reported. However, most absolute quantitation methods based on the digital PCR required pretreatment steps. Meanwhile, singleplex detection could not meet the demand of the absolute quantitation of GMO events that is based on the ratio of foreign fragments and reference genes. Thus, to promote the absolute quantitative detection of different GMO events by digital PCR, we developed a quantitative detection method based on duplex digital PCR without pretreatment. Moreover, we tested 7 GMO events in our study to evaluate the fitness of our method. The optimized combination of foreign and reference primers, limit of quantitation (LOQ), limit of detection (LOD) and specificity were validated. The results showed that the LOQ of our method for different GMO events was 0.5%, while the LOD is 0.1%. Additionally, we found that duplex digital PCR could achieve the detection results with lower RSD compared with singleplex digital PCR. In summary, the duplex digital PCR detection system is a simple and stable way to achieve the absolute quantitation of different GMO events. Moreover, the LOQ and LOD indicated that this method is suitable for the daily detection and quantitation of GMO events.
authors.affiliation: Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Food Science & Nutritional Engineering, China Agricultural University, Beijing, 100083, China.
authors.forename: Pengyu
authors.initials: P
authors.lastname: Zhu
authors.affiliation: The Institute of Plant Quarantine, Chinese Academy of Inspection and Quarantine, Beijing, 100029, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Fu
authors.affiliation: The Institute of Plant Quarantine, Chinese Academy of Inspection and Quarantine, Beijing, 100029, China.
authors.forename: Chenguang
authors.initials: C
authors.lastname: Wang
authors.affiliation: Guangxi Entry-Exit Inspection and Quarantine Bureau, Guangxi, 530028, China.
authors.forename: Zhixin
authors.initials: Z
authors.lastname: Du
authors.affiliation: Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Food Science & Nutritional Engineering, China Agricultural University, Beijing, 100083, China; Beijing Laboratory for Food Quality and Safety, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, 100083, China.
authors.forename: Kunlun
authors.initials: K
authors.lastname: Huang
authors.affiliation: The Institute of Plant Quarantine, Chinese Academy of Inspection and Quarantine, Beijing, 100029, China. Electronic address: zhushf0421@163.com.
authors.forename: Shuifang
authors.initials: S
authors.lastname: Zhu
authors.affiliation: Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Food Science & Nutritional Engineering, China Agricultural University, Beijing, 100083, China; Beijing Laboratory for Food Quality and Safety, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, 100083, China. Electronic address: xuwentaoboy@sina.com.
authors.forename: Wentao
authors.initials: W
authors.lastname: Xu
mesh_terms: D018744:DNA, Plant / Q000235:genetics; D030821:Plants, Genetically Modified; D016133:Polymerase Chain Reaction / Q000379:methods*; D003313:Zea mays / Q000235:genetics*
chemical_list: D018744:DNA, Plant
keywords: Absolute quantitation; Digital polymerase chain reaction; Duplex detection; Pretreatment-free