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At the same time, since there is no detection method for this strain in the current national standards and industry standards, the establishment of a detection method for the COT102 strain will also provide technical support for the improvement of relevant standards for the detection of genetically modified components in cotton and its source products.
Method used
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[0114] Genomic DNA samples of all extracted plant materials (including all plant materials listed in the "Test Materials" of the aforementioned "Materials and Methods") were determined by a micro-spectrophotometer, and the concentration was between 30 and 200 ng / μL, OD 260 / OD 280 When the ratio is between 1.7 and 1.9, the concentration and purity of DNA are good. Subsequently, in order to determine whether the extracted DNA samples were suitable for PCR amplification, the plant endogenous gene 18S rDNA was amplified. The reaction system is shown in Table 3, and the amplification curve is shown in Table 3. figure 1 shown.
[0115] Table 3. PCR reaction system for amplification of plant endogenous gene 18S rDNA
[0116]
[0117] The results showed that when primers and probe 18S rDNA-F / R / P were used to perform single-plex real-time fluorescent PCR amplification using the extracted genomic DNA of each plant material as a template, all ...
Embodiment 2
[0118] Embodiment 2, screening and optimization of detection reagent and detection system
[0119] 1. Primer and probe optimization screening
[0120] The present inventors analyzed the genome of the transgenic cotton COT102 line (Genbank accession number CQ814630.1), its transgenic region and its flanking sequences at both ends, conducted research on a variety of site regions, and compared them with other transgenic and non-transgenic species for sequence comparison. After in-depth analysis, some interesting locations were selected, mainly as follows:
[0121] Primer COT102-3-FP / RP and probe COT102-3-P were designed and synthesized for the 3' specific flanking sequence region.
[0122] Aiming at the partial region of the 5' end-specific flanking sequence, primers COT102-5-FP1 / RP1, COT102-5-FP12 / RP12, COT102-5-FP13 / RP13 and COT102-5-FP2 / RP2 were designed and synthesized, and probes Pins COT102-5-P1 and COT102-5-P2.
[0123] In addition, the inventors further synthesized sp...
Embodiment 3
[0150] Embodiment 3, specificity and sensitivity determination
[0151] The present invention uses the genomic DNA of 19 kinds of plant materials (see the "test material" part of the aforementioned "materials and methods") as a template to test the specificity of the established detection method, and the amplification curve is as follows: image 3 shown.
[0153] Genomic DNA of 19 kinds of plant materials were used as templates for PCR amplification.
[0154] The amplification results of the cotton endogenous gene ACP1 showed that only the genomic DNA samples of cotton lines had obvious amplification curves, while the genomic DNA samples of other plant lines had no amplification curves ( image 3 A). The amplification results using the specific sequence of the transgenic cotton COT102 line showed that only the genomic DNA samples of the transgenic cotton COT102 line had a significant amplification curve, while the genomic DNA...
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Abstract
The invention provides dual fluorescent quantitative PCR detection of a transgenic cotton line COT102. The method comprises the steps that amplification is conducted on two target genes at the same time, wherein the two target genes include a gene COT102 and a gene ACP1; and the existence condition or the existence amount of the transgenic cotton line COT102 or the existence proportion of the transgenic line is determined according to the amplification condition. According to the method, on one hand, the problem that a dual PCR quantitative detection method for the cotton line COT102 does notexist in the prior art is solved; and on the other hand, aiming at the characteristics of the transgenic line, a detection reagent with excellent effect is determined, the detection method is optimized, and the detection efficiency is very high. The method has important practical value and demonstration effect on construction and improvement of a quantitative detection technical system of Chinesetransgenic products, and can be used as a technical reserve for threshold management.
Description
technical field [0001] The invention belongs to the field of PCR fluorescence detection, and more specifically, the invention relates to a double fluorescence quantitative PCR detection method of transgenic cotton COT102 strain. Background technique [0002] Genetically modified crops have been planted on a commercial scale since 1996, and by the end of 2018, the cumulative global planting area reached 2.5 billion hectares 2 . By the end of 2018, a total of 26 countries and regions around the world planted genetically modified crops, with an area of 191.7 million ha 2 , about 113 times that of 1996; another 44 countries and regions imported genetically modified agricultural products. Among them, the planting area of transgenic cotton is 24.9 million hectares 2 , accounting for 76% of the total global cotton acreage. With the large-scale planting and consumption of genetically modified crops around the world, more and more genetically modified crops are used in the pr...
Claims
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