Quantitative Detection of Soil Persistence of Exogenous and Reference Gene from Genetically Modified Maize (Zea mays) 'CC-2' by Droplet Digital PCR
DONG Shan-Shan1,*, XIAO Ze-Hua1,2,*, ZHANG Di-Ni1, LIU Yan1,**
1 Key Laboratory for Biosafety of Environmental Protection, Nanjing Institute of Environmental Science, Ministry of Ecology and Environment, Nanjing 210042, China; 2 Institute of Biodiversity Science, Fudan University, Shanghai 200438, China
Abstract：Monitoring the persistence and dynamic changes of transgenes in the soil from genetically modified (GM) crop is an important issue in assessing the potential risks of GM crops. The present study applied a novel droplet digital polymerase chain reaction (ddPCR) method to quantitatively detect the copy number concentrations of exogenous gene CP4-EPSPS (Agrobacterium tumefaciens strain CP4 5-enolpyruvyl shikimate-3-phosphate synthase) and reference gene zSSIIb (Zea mays starch synthase isoform zSTSII-2) fragments in the rhizosphere soil of herbicide-resistant transgenic maize (Zea mays) 'CC-2' and its non-transgenic control line 'Zheng-58', at different growth stages of maize (seedling stage, jointing stage, silking stage, milk-ripe stage and full-ripe stage). The limit of quantitation (LOQ) of ddPCR for the CP4-EPSPS and zSSIIb system was (0.48±0.07) and (0.22±0.04) copies/µL, respectively. The copy number concentrations of CP4-EPSPS were lower than the limit of quantitation in the rhizosphere soil of GM maize 'CC-2' at all growth stages, and the positive detection rate of CP4-EPSPS obviously declined with increasing growth stage. There was no significant difference between CP4-EPSPS and zSSIIb concentrations in rhizosphere soil of 'CC-2'. At different growth stage, there was a similar change tendency in the positive detection rate and concentration of zSSIIb gene fragments in rhizosphere soil of 'CC-2' and 'Zheng-58', but the copy number concentration of zSSIIb in rhizosphere soil of 'Zheng-58' was significantly higher than that of 'CC-2' at the jointing stage (P<0.05). Test results showed that the transgene fragments of GM maize could enter into the soil environment through root exudates, but the concentration was very low and showed a decreasing trend with the growth stage, which indicates that the ddPCR method is suitable for sensitive and precise quantitative analysis of the persistent plant DNA fragments in soil environment. This study provided a novel method and reference for quantitative detection of transgene from GM crops in soil.
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