Duplex Real-time Quantitative PCR Method for Detection of Genetically Modified Cotton (Gossypium hirsutum) Event COT102
PAN Zhi-Lin1,2, ZUO Cui-Hua1, WU Qi-Ming1, QIAN Chang-Yuan1, YIN Lu1, LIU Yue-Ming1, LI Xiang1,*
1 Technical Center for Animal, Plant and Food Inspection and Quarantine of Shanghai Customs, Shanghai 200135, China; 2 College of Food Science and Technology, Shanghai Ocean University, Shanghai 201306, China
Abstract Cotton (Gossypium hirsutum) is an important cash crop, by the end of 2019, 76% of the total cotton acreage was cultivated on genetically modified (GM) cotton globally. GM cotton event COT102 is a transgenic cotton event with insect resistance developed by Syngenta, Switzerland. This event has been approved by the Ministry of Agriculture of China, but has no detection method in relevant standards in China. In order to ensure the development of cotton industry in China and the orderly conduct of import and export trade, it is very important to establish an effective detection method for GM cotton event COT102. In this study, multiple sets of primers and probes were designed according to the exogenous inserts and adjacent sequences of genomic DNA of GM cotton event COT102 based on duplex real-time PCR technology. After screening and optimizing the reaction conditions, the proportion of primers and probes and other indicators, a duplex real-time quantitative PCR method was developed for the detection of GM cotton event COT102. The specificity, sensitivity and reproducibility of the method were measured. The results showed that the method was specific for the detection of GM cotton event COT102, without any nonspecific amplification. The sensitivity of the method was high, and the lower limit of detection and quantification was as low as 5 copies of cotton genomic DNA. The deviation between repeats of multiple PCR amplification was small and reproducibility was good. The results showed that the bias between the measured value and the expected value was between -0.42% and 4.63%. The standard deviation (SD) was less than 0.5, and the relative standard deviation (RSD) was less than 25%, both were within the acceptable range. Therefore, the duplex real-time quantitative PCR method established in this study was suitable for qualitative and quantitative detection of the components of GM cotton event COT102 in cotton and its products. The high-throughput detection method could effectively improve the detection efficiency, reduce the detection cost, and improve the customs clearance efficiency. This method provides effective technical support for the effective supervision of imported GM cotton and its products, and promotes the implementation of GM product labeling policies in China, meanwhile provides necessary technical support for the smooth and orderly conduct of import and export trade.
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