Method, primer and probe for quantitative detection of transgenic maize IE034 in fodder based on ddPCR and application

Abstract

The invention discloses a method, a primer and a probe for quantitative detection of transgenic maize IE034 in a fodder based on ddPCR and application and relates to the technical fields of molecularbiology and transgenic test. The primer comprises exogenous gene Cry1Ie primers, and the probe comprises reference gene rubisco probes. The method comprises the following steps: (1) extracting genomicDNA of a fodder sample; (2) utilizing the specific primers and probes of exogenous genes Cry1Ie and reference genes to perform ddPCR amplification on the diluted genomic DNA; and (3) detecting and analyzing a PCR product. The method does not depend on a standard curve, is not influenced by a PCR inhibitor or the PCR amplification efficiency, has higher sensitivity and stability compared with conventional PCR, can accurately and quantitatively detect the copy number concentration of a transgene in the fodder, and can quantitatively analyze the transgenic maize IE034 in the fodder.

Description

technical field [0001] The invention belongs to the technical field of molecular biology and transgene detection and detection, and in particular relates to a droplet digital PCR quantitative detection method for transgenic corn IE034 in feed. Background technique [0002] In 2016, the global planting area of ​​genetically modified crops increased from 1.7 million hectares in 1996 to 185.1 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 had approved GM crops for use as food and / or feed and for release into the environment, with 1238 of the 3768 regulatory approvals granted covering feed uses, including direct use or For processing purposes, the proportion of genetically modified crops used for feed is about 33%. Corn is an important feed raw material. With the growth of feed processing and ...

AI Technical Summary

Problems solved by technology

After retrieval, the related technical solutions have been published in the prior art. Chinese patent number 201410457457.5, the application with the publication date of 2014.12.10 discloses the transformant-specific quantitative PCR detection primers and probes and methods of transgenic maize IE034. Its design The upstream primer sequence, downstream primer sequence and fluorescent probe sequence were used for PCR amplification. Although this real-time fluorescent quantitative PCR method can perform specific quantitative detection of transgenic corn IE034 and its derivatives, there are still certain defects: in the analysis For exogenous gene copy number, real-time fluorescent quantitative PCR must rely on the standard curve and genes with known copy number, which is only a relative quantitative method, and the quality of the standard curve is easily affected by DNA purity, concentration of primers and probes, and reaction inhibition Influenced by many factors such as factors, the sensitivity and accuracy of the detection cannot meet the requirements of accurate quantification, which may lead to underestimation or overestimation of the content of the transgene

Moreover, since exogenous genes are usually integrated into the recipient genome at low copy numbers (1–2), qPCR is susceptible to interference from background DNA when detecting transgenic components in complex substances

The droplet digital PCR (ddPCR) that has emerged in recent years is a new absolute quantitative technology, which achieves theoretical single-molecule amplification through extreme dilution, so that rare target nucleic acid sequences can be separated from a large amount of background DNA, and their relative content, thereby improving the sensitivity and repeatability of the detection. At present, this technology has not been applied in the field of detection of transgenic corn IE034

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