Method, primer and probe for quantitatively detecting transgenic maize IE034 in animal tissue based on ddPCR and applications of method

Abstract

The invention discloses a method, a primer and a probe for quantitatively detecting transgenic maize IE034 in animal tissue based on ddPCR and applications of the method, relating to the technical field of molecular biology and transgenic detection. The method comprises the following steps: (1) extracting genome DNA of an animal tissue sample; (2) carrying out microdroplet type digital PCR amplification by utilizing specific primers and probes of an exogenous gene Cry1Ie and a reference gene rubisco; and (3) detecting and analyzing the PCR product. The method is independent of a standard curve, is free of the influences of a PCR inhibitor and the PCR amplification efficiency, and has relatively high sensitivity and stability compared with the conventional PCR, the lower limit of detectioncan be greatly reduced, the false negative probability can be reduced, the transgenic ingredient taken into the animal tissue can be accurately and quantitatively detected, and the method belongs to an effective method for quantitatively detecting horizontal transfer of the exogenous gene.

Description

technical field [0001] The invention belongs to the technical field of molecular biology and transgene detection, and in particular relates to a droplet digital PCR quantitative detection method for transgenic corn IE034 ingested in animal tissues. Background technique [0002] In 2016, the global planting area of ​​genetically modified crops increased from 1.7 million hectares in 1996 to 185 million hectares, of which the planting area of ​​genetically modified corn was 60.6 million hectares, accounting for 64% of the total global corn planting area, and the application rate was 26%. As of 2016, regulators in 40 countries have approved the use of GM crops as food and / or feed and their release into the environment, with 1238 of the 3768 regulatory approvals granted covering feed use, GM for feed The crop share is about 33%. Corn is an important feed raw material. With the growth of feed processing and industrial alcohol demand for corn, the proportion of genetically modifie...

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Problems solved by technology

At present, most of the above studies use the conventional real-time fluorescent quantitative PCR (qPCR) method, and increase the sensitivity and accuracy of detection by increasing samples and technical repetitions. However, the real-time fluorescent quantitative PCR method has certain defects: 1) In the detection complex 2) Real-time fluorescent quantitative PCR must rely on the standard curve and genes with known copy numbers when analyzing the copy number of exogenous genes, which is only a relative quantitative method, and the standard curve The quality of DNA is easily affected by many factors such as DNA purity, concentration of primers and probes, and reaction inhibitors, so that the detection sensitivity and accuracy cannot meet the requirements of accurate quantification, which may lead to underestimation or overestimation of the content of transgenes

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