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Specific primers and probe for detecting flax DNA and real-time fluorescence quantitative PCR kit

A real-time fluorescence quantitative and specific technology, applied in the direction of DNA / RNA fragments, recombinant DNA technology, biochemical equipment and methods, etc., can solve the problems of low content and difficult DNA extraction, and achieve high sensitivity and high specificity Effect

Inactive Publication Date: 2018-06-01
SUZHOU BAIYUAN GENT CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since the production of refined edible oil requires high temperature and high pressure, the DNA in it is degraded into fragments of different sizes, and the content is extremely low, which makes DNA extraction very difficult.

Method used

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  • Specific primers and probe for detecting flax DNA and real-time fluorescence quantitative PCR kit
  • Specific primers and probe for detecting flax DNA and real-time fluorescence quantitative PCR kit
  • Specific primers and probe for detecting flax DNA and real-time fluorescence quantitative PCR kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] The preparation of embodiment 1 FAD3A gene standard product

[0037] To establish a real-time fluorescent quantitative PCR method, the external standard required by the method must first be prepared. The standard should contain highly conserved and specific sequences, and high specificity of the reaction must be ensured. The present invention uses flax FAD3A rDNA as the target sequence. This example mainly uses PCR technology to amplify the FAD3A rDNA gene of flax seeds, and uses gene recombination technology to connect it to the plasmid vector pMD18-T to construct the recombinant plasmid pMD18-T-FAD3A, and carry out corresponding PCR identification and sequencing identification, Finally, it is quantified as a standard for the method to be established, laying the foundation for the next method and evaluation.

[0038] 1. Preparation of template DNA

[0039] Genomic DNA was extracted from flax seeds and used as a template for PCR amplification of FAD3A rDNA gene. The ...

Embodiment 2

[0085]Embodiment 2 real-time fluorescent quantitative PCR kit

[0086] 1. Design and synthesis of specific primers and probes

[0087] Taking the conserved fragment of the FAD3A rDNA gene of flax selected above as the target, a set of real-time fluorescent quantitative PCR primers and probes were designed using Primer Premier5 software.

[0088] In order to distinguish flax from common vegetable oils such as peanut, corn, soybean, potato, sesame, rapeseed, walnut, sunflower and olive, the detection region of flax oil selected in the present invention is the fatty acid desaturase 3a gene (FAD3a) on the RBCL gene. The transcripts can be detected in the leaves and fruit balls of flax, which is the main gene of fatty acid synthesis in flax, and has a highly conserved type in flax. The flax oil FAD3a gene was blasted in NCBI, the results are as follows figure 1 .

[0089] The comparison results showed that the flax FAD3a gene was highly conserved, but the genome of Corynebacteri...

Embodiment 3

[0136] Example 3 The quantitative detection method of the sample to be tested by the real-time fluorescent quantitative PCR kit

[0137] Respectively with the FAD3A gene series concentration standard substance (1.00 * 10 8 copies / ml, 1.00×10 7 copies / ml, 1.00×10 6 copies / ml, 1.00×10 5 copies / ml,, 1.00×10 4 copies / ml) as a template, use the primers and probes in the kit to perform fluorescent quantitative PCR amplification, and set up positive and negative controls at the same time.

[0138] The PCR reaction system is as follows:

[0139]

[0140] Reaction conditions: 94°C pre-denaturation for 2 minutes, 94°C for 15 seconds, 60°C for 40s and collecting fluorescence signals, 40 cycles. Rapid quantitative detection through the standard curve and the Ct value of the sample to be tested.

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Abstract

The invention provides specific primers and a probe for detecting flax DNA. A sequence of the probe is shown as 5'-ATGACAATTTTGGACCCGACCTGGT-3'; and the specific primers adopt the following sequencesor complementary chain sequences of the sequences: the sequence of an upstream primer is 5'-CTCCCAATGTTTGCGTATCCTA-3', and the sequence of a downstream primer is 5'-ATATCGACCCGAGTCAACCAAA-3'. The invention also provides a corresponding a real-time fluorescence quantitative PCR kit. The specific primers and the probe as well as the corresponding kit provided by the invention are applicable to real-time fluorescence quantitative PCR detection; an established method is high in sensitivity and specificity; and the specific identification of flax seed oil from various oil crops as peanuts, flax, corns, soybeans, potatoes, sesames, rapes, walnuts, sunflowers, olives, flax and the like can be achieved.

Description

technical field [0001] The invention relates to the technical fields of molecular biology and in vitro diagnostic reagents, in particular to a real-time fluorescent PCR kit for detecting flax DNA, and in particular to specific primers and probes for detecting flax DNA. Background technique [0002] The genes used to detect the DNA quality of edible oil mainly include chloroplast ATP, RBCL genes and species-specific genes. The designed primers were all derived from the rbcL gene sequence on the chloroplast. Chloroplast is an important organelle in plant cells, and chloroplast DNA is widely used in the study of phylogenetic evolution. Chloroplast genes are highly conserved, and the rbcL gene is widely used in the study of plant phylogeny because of its evolutionary characteristics. Some of its coding regions are relatively slow and conservative in the evolution process, while some non-coding regions are relatively fast in evolution among different species, and have certain i...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/6895C12Q2561/113C12Q2563/107C12Q2545/114
Inventor 刘宇琴徐红车团结沈颂东陈游石文张镭李亚鹏
Owner SUZHOU BAIYUAN GENT CO LTD
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