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Multiplex real-time fluorescence quantitative PCR detection kit for transgenic rapeseed RF1, Oxy235 and RF3 and application

A real-time fluorescent quantitative, oxy235-f technology, applied in recombinant DNA technology, DNA/RNA fragments, etc., can solve problems such as low efficiency, achieve high throughput, low cost, and save experimental samples and drugs.

Inactive Publication Date: 2017-11-24
AGRO ENVIRONMENTAL PROTECTION INST OF MIN OF AGRI
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  • Abstract
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  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the detection of genetically modified ingredients in genetically modified crops and their derivative products mainly relies on single-plex PCR detection, which is inefficient

Method used

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  • Multiplex real-time fluorescence quantitative PCR detection kit for transgenic rapeseed RF1, Oxy235 and RF3 and application
  • Multiplex real-time fluorescence quantitative PCR detection kit for transgenic rapeseed RF1, Oxy235 and RF3 and application
  • Multiplex real-time fluorescence quantitative PCR detection kit for transgenic rapeseed RF1, Oxy235 and RF3 and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Establishment of multiple real-time fluorescent quantitative PCR detection methods for transgenic rapeseed RF1, Oxy235, and RF3

[0042] (1) Synthesize the following primers and probes:

[0043] Rapeseed cruciferinA (CruA) gene in National Standard Announcement No. 2031 of the Ministry of Agriculture-9-2013 "Detection of Rapeseed Internal Standard Gene Qualitative PCR Method for Components of Transgenic Plants and Their Products" was used as the internal standard gene. According to the flanking sequence information of RF1, Oxy235, and RF3 published in the GenBank nucleic acid database, the primers were designed and analyzed using Oligo V7.0 software, and synthesized by Sangon Biopower (Shanghai) Co., Ltd.

[0044] Table 1 Screening results of multiplex real-time fluorescent quantitative PCR primers

[0045]

[0046]

[0047] RF1-F: 5'-AATTTGGCCTGTAGACCT-3', its nucleotide sequence is shown in SEQ ID NO.1;

[0048] RF1-R: 5'-TACACGCGACTCATCATCC-3', its ...

Embodiment 2

[0070] Embodiment 2 specificity test

[0071] In order to evaluate and verify the specificity of multiple real-time fluorescent quantitative PCR primers and probes, genomic DNA was extracted from transgenic rapeseed, transgenic maize, transgenic soybean, transgenic rice, transgenic cotton and other transgenic crops with different transformation vectors for verification. The results showed that the expected amplification curve was obtained only in the transgenic rapeseed containing the objective transformation vector.

[0072] Conclusion: by figure 2 The results show that according to the established multiple real-time fluorescent PCR method for specificity testing, CruA, Oxy235, RF3 and RF1 have expected amplification curves within 36 Ct values, indicating that this method can detect CruA, Oxy235, RF3 and RF1 target components.

Embodiment 3

[0073] Embodiment 3 sensitivity test

[0074] Genomic DNA standard substances of transgenic rapeseed with three different transformation vectors were diluted with non-transgenic rapeseed genomic DNA, mixed thoroughly, and prepared into mass fraction ratios of 10%, 5%, 1%, 0.5%, 0.1%, 0.05%, and 0.025% and 0.01% etc. a series of genomic DNA templates. 100 ng each was taken as a template (20 μL system) for real-time fluorescent PCR test, and each diluted sample was set for 3 replicates. Find the Ct value of 3 repetitions SD and RSD. The standard curve and regression equation between Ct value and template amount were fitted to determine the linearity and sensitivity of the established multiple real-time fluorescent PCR method.

[0075] According to the provisions of the European Network of Transgenic Laboratories on the detection method of genetically modified components (2011), it is required that the slope of the standard curve between the Ct value of real-time fluorescent ...

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Abstract

The invention discloses a multiplex real-time fluorescence quantitative PCR detection kit for transgenic rapeseed RF1, Oxy235 and RF3 and an application thereof. The detection kit comprises the following primers and probes: CruA-F / R, CruA-Probe, RF1-F / R, RF1-Probe, Oxy235-F / R, Oxy235-Probe, RF3-F / R and RF3-Probe. By extracting the DNA of the sample to be tested as a template, the primers and probes are added, the real-time fluorescence quantitative PCR detection is conducted, and whether the test samples contain the RF1, Oxy235 or RF3 transformant components is judged according to the markers on the probes, the Ct value and an expected amplification curve. According to the multiplex real-time fluorescence quantitative PCR detection kit, the qualitative and quantitative detection of the rapeseed internal standard gene and 3 kinds of transgenic rapeseed strains can be rapidly and accurately achieved through only one PCR reaction; the PCR detection kit has the advantages of low cost and high flux.

Description

technical field [0001] The invention belongs to the technical field of transgene detection, in particular to a multiple real-time fluorescent quantitative PCR detection kit for transgenic rapeseed RF1, Oxy235 and RF3 and its application. Background technique [0002] With the rapid growth of transgenic rapeseed planting area and increasing market share, public concern, worry and controversy about the safety of genetically modified rapeseed and its products have been aroused. At present, there is an urgent need to establish simple, fast, efficient and accurate identification and detection technology for genetically modified rapeseed and its products, to strengthen the safety supervision of genetically modified rapeseed and its products, to effectively implement the labeling system, to protect the property rights of genetically modified varieties, and to protect consumers right to know and to choose. [0003] At present, the commonly used transgenic detection techniques are d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 修伟明鲁军李刚赵建宁杨殿林
Owner AGRO ENVIRONMENTAL PROTECTION INST OF MIN OF AGRI
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