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Specific primer and probe for detecting sesame DNA and real-time fluorescent quantitative PCR kit

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

Inactive Publication Date: 2018-06-15
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 for detecting sesame DNA and real-time fluorescent quantitative PCR kit
  • Specific primer and probe for detecting sesame DNA and real-time fluorescent quantitative PCR kit
  • Specific primer and probe for detecting sesame DNA and real-time fluorescent quantitative PCR kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Preparation of sesame 2s gene standard

[0039]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 sesame 2s rDNA as the target sequence. This example mainly uses PCR technology to amplify the 2s rDNA gene of sesame seeds, and uses gene recombination technology to connect it to the plasmid vector pMD18-T to construct the recombinant plasmid pMD18-T-2s, 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.

[0040] 1. Preparation of template DNA

[0041] Genomic DNA was extracted from sesame seeds and used as a template for PCR amplification of 2s rDNA gene. The Ezup column type p...

Embodiment 2

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

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

[0089]A set of real-time fluorescent quantitative PCR primers and probes were designed by using the conserved fragment of the 2s rDNA gene of sesame selected above as the target, using PrimerPremier5 software.

[0090] In order to distinguish sesame from the detection of common vegetable oils such as peanut, corn, soybean, potato, flax, rape, walnut, sunflower and olive, the detection region of sesame oil selected in the present invention is 2s rDNA gene, and sesame oil 2s rDNA is the endogenous gene in sesame seeds. genes, which are more stable and highly conserved in processed sesame oil. The sesame 2s rDNA gene was blasted in NCBI, and the results were as follows figure 1 .

[0091] figure 1 It is the result of blast comparison between sesame oil 2s gene and other vegetable oil genes.

[0092] The comparison results show that the 2S CDS sequence...

Embodiment 3

[0140] Example 3 Quantitative detection method of the sample to be tested by the real-time fluorescent quantitative PCR kit

[0141] The 2S 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.

[0142] The PCR reaction system is as follows:

[0143]

[0144] 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 probe for detecting sesame DNA. The sequence of the probe is 5'-CTCACAGCGGCACCTCTCGTCCAC-3'; the specific primer adopts the following sequence or a complementary chain sequence of the following sequence: the sequence of an upstream primer is 5'-ACGATGAAGCCAACCAGCAGA-3'; the sequence of a downstream primer is 5'-GCTGCCTCACTGCTTGCCTAAT-3'. The invention further provides a corresponding real-time fluorescent quantitative PCR kit. The specific primer and probe and the corresponding kit can be used for real-time fluorescent quantitative PCR detection, and ensures that an established method has high sensitivity and high specificity, and can specifically identify sesame oil from various oil plant, such as peanut, flax, corn, soy, potato, sesame, rape,walnut, sunflower and olive.

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 sesame DNA, in particular to specific primers and probes for detecting sesame 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. By establishing a species-specific PCR method to detect this species in processed food products, it has gradually become an important means of food quality detection. Amplified fragment size and amplified gene composition are important for PCR amplification of refined edible oil DNA. The smaller the amplified fragment, the higher the success rate of PCR amplification, and DNA with high GC content is more stable in processing engineering, and endogenous genes are more stable in processing than exogenous genes. Therefore, endogenous genome fragments with sm...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12N15/11
CPCC12Q1/6895
Inventor 石文徐红刘宇琴车团结沈颂东陈游张镭李亚鹏
Owner SUZHOU BAIYUAN GENT CO LTD
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