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

A real-time fluorescence quantitative and specific technology, applied in DNA/RNA fragments, recombinant DNA technology, biochemical equipment and methods, etc., can solve problems such as false positives, increased fluorescence intensity, and poor specificity

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

Fluorescent dye intercalation technology uses SYBR green I to intercalate DNA double-strands to increase the fluorescence intensity. The fluorescent signal is introduced into double-stranded DNA. The specificity of this method is poor, and the results are often interfered by the presence of primer-dimers, resulting in false positives. and other issues, so it is not suitable for food quick inspection

Method used

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

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] The preparation of embodiment 1 Arah1 gene standard product,

[0036]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 peanut Arah1 rDNA as the target sequence. This example mainly uses PCR technology to amplify the Arah1 rDNA gene of peanut seeds, and uses gene recombination technology to connect it to the plasmid vector pMD18-T to construct the recombinant plasmid pMD18-T-Arah1, 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.

[0037] 1. Preparation of template DNA

[0038] 1. Genomic DNA was extracted from peanut seeds and used as a template for PCR amplification of the Arah1 rDN...

Embodiment 2

[0087] Embodiment 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 using the Primer Premier5 software with the conserved fragment of Arah1 rDNA gene selected above as the target.

[0090] Arah1 gene of peanut oil was downloaded from NCBI, and multiple pairs of Arah1 gene primers and probes were designed by Primer Premier 5 and Becon Designer software, and primer specificity was verified by BLAST in NCBI. see the result Figure 1-Figure 3 .

[0091] Figure 1-Figure 3 It is the comparison result of 3 pairs of primer probe specificity BLAST.

[0092] Each pair of primers for peanuts is screened for primer specificity by ordinary PCR. The selected templates are: peanuts, soybeans, sunflowers, flax, rapeseed, corn, sesame, olives, walnuts, potatoes, negative for water, amplified bands Run the gel, and one pair of primers has the h...

Embodiment 3

[0137] Example 3 The quantitative detection method of the sample to be tested by the real-time fluorescent quantitative PCR kit

[0138] Respectively with the Arah1 rDNA gene series concentration standard substance (serial concentration standard substance series concentration standard substance) prepared by the sample DNA to be tested and embodiment 1: 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.

[0139] The PCR reaction system is as follows:

[0140]

[0141] Reaction conditions: 94°C pre-denaturation for 2 minutes, 94°C for 15 seconds, 60°C for 40s and collecting fluorescence signals, 40 cycles. Draw a standard curve, and perform 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 peanut. The sequence of the probe is as follows: 5'-AAGTCATCAGCAGCCACGGAA-3'; the specific primer has the following sequencesor the complementary chain sequences of the following sequences: the upstream primer sequence: 5'-ATCCTCGTTGTGTCTATGA-3', and the downstream primer sequence: 5'-TGTTCCCCACTCTTGTTCT-3. The invention 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 fluorescence quantification PCR detection of peanuts, and the established method has high sensitivity and high specificity, can be used for specifically identifying peanut oil from various oil crops including peanut, flax, maize, soybean, potato, sesame, oilseed rape, walnut, sunflowers, olive and the like.

Description

technical field [0001] The invention belongs to the technical field of molecular biology and in vitro diagnostic reagents, in particular to specific primers and probes for detecting peanut DNA and a real-time fluorescent quantitative PCR kit. Background technique [0002] The genes used to detect the DNA quality of edible oil mainly include chloroplast ATP, RBCL genes and species-specific genes. 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 inter-species differences and intra-species conservation, so they are suitable for species identification . By de...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/6895C12Q2561/113C12Q2563/107C12Q2545/114
Inventor 张镭徐红刘宇琴车团结沈颂东陈游石文李亚鹏
Owner SUZHOU BAIYUAN GENT CO LTD
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