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Specific primer and probe as well as real-time fluorescence quantification PCR kit used for detecting DNA of sunflower

A real-time fluorescent quantitative and specific technology, applied in 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 primer and probe as well as real-time fluorescence quantification PCR kit used for detecting DNA of sunflower
  • Specific primer and probe as well as real-time fluorescence quantification PCR kit used for detecting DNA of sunflower
  • Specific primer and probe as well as real-time fluorescence quantification PCR kit used for detecting DNA of sunflower

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Preparation of sunflower 11S rDNA gene standard

[0038]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 sunflower 11S rDNA as the target sequence. This example mainly uses PCR technology to amplify the 11S rDNA gene of sunflower seeds, and uses gene recombination technology to connect it to the plasmid vector pMD18-T to construct the recombinant plasmid pMD18-T-11S, 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.

[0039] 1. Preparation of template DNA

[0040] Genomic DNA was extracted from sunflower seeds and used as a template for PCR amplification of 11S rDNA gene. ...

Embodiment 2

[0087] Example 2 Real-time fluorescent quantitative PCR kit

[0088] 1. Design and synthesis of specific primers and probes

[0089]Taking the conserved fragment of the 11S rDNA gene of sunflower selected above as the target, a set of real-time fluorescent quantitative PCR primers and probes were designed using Primer Premier 5 software.

[0090] In order to distinguish sunflower from common vegetable oils such as peanut, corn, soybean, potato, sesame, rapeseed, flax, walnut and olive, the sunflower oil detection region selected in the present invention is the 11S globule gene on the RBCL gene, which is a synthetic sunflower seed oil. The key gene of the protein is highly conserved. The sunflower oil 11S gene is blasted in NCBI, and the results are as follows figure 1 .

[0091] The comparison results showed that the sunflower 11S globular egg gene was highly conserved, and it existed in chromosome 1 of Burkholderia and in the genome of Schizophrenia, with partial homology....

Embodiment 3

[0139] Example 3 Quantitative detection method of the sample to be tested by the real-time fluorescent quantitative PCR kit

[0140] The 11S rDNA 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.

[0141] The PCR reaction system is as follows:

[0142]

[0143] 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 a specific primer and a probe for detecting DNA of sunflower. The sequence of the probe is as follows: 5'-ACGCTCTCCTCGCCAACAACAA-3'; the specific primer has the following sequences or the complementary chain sequences of the following sequences: the upstream primer sequence: 5'-CAAATAGTCCGCCCACCACAA-3', and the downstream primer sequence: 5'-5'-TGGGAAGGGTTGTCAATGTTC-3'. Theinvention further provides a corresponding real-time fluorescence quantification PCR kit. The specific primer and the probe as well as the corresponding kit can be used for the real-time fluorescencequantification PCR detection, and the established method has high sensitivity and high specificity, and can be used for specifically identifying sunflower oil from various oil crops including peanut,flax, maize, soybean, potato, sesame, oilseed rape, walnut, sunflower, olive and the like.

Description

technical field [0001] The invention relates to the technical field of molecular biology and in vitro diagnostic reagents, in particular to a real-time fluorescent PCR kit for sunflower DNA detection, and in particular to specific primers and probes for sunflower DNA detection. 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 ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6851C12Q1/6895C12Q1/686C12N15/11
CPCC12Q1/6851C12Q1/686C12Q1/6895C12Q2531/113C12Q2563/107C12Q2545/113
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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