A kind of multiple fluorescent PCR detection primers, probes, kits, detection method and application of deer and bovine origin in deer horn glue

A detection kit and a technology for detection primers, which are applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., can solve the problem that the identification method cannot fully meet the requirements for authenticity identification of deer horn glue, the determination is not accurate enough, Problems such as high sensitivity and false positive rate, to overcome incomplete extraction difficulties, high application value, and good specificity

Active Publication Date: 2020-07-31
VEGETABLE RES INST OF SHANDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods are aimed at the identification of polypeptide differences in deer horn glue, and due to technical limitations, the sensitivity and false positive rate are relatively high, and the determination is not accurate enough
Therefore, the traditional identification method can not fully meet the current identification requirements of the authenticity of antler glue
However, the use of real-time fluorescent quantitative PCR to detect deer and bovine origin in antler glue has not yet been reported in China.

Method used

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  • A kind of multiple fluorescent PCR detection primers, probes, kits, detection method and application of deer and bovine origin in deer horn glue
  • A kind of multiple fluorescent PCR detection primers, probes, kits, detection method and application of deer and bovine origin in deer horn glue
  • A kind of multiple fluorescent PCR detection primers, probes, kits, detection method and application of deer and bovine origin in deer horn glue

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Experimental program
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Effect test

Embodiment 1

[0059] The DNA extraction of antler gum samples was performed using the method disclosed in the patent 201410317118.7 (a kit for rapid DNA extraction from donkey-hide gelatin and its extraction method). The steps will not be repeated. The purity of the extracted genomic DNA was determined by UV spectrophotometer. And concentration. The measured OD260 / OD280 values ​​are about 1.8 to 1.9, and the concentration is above 10ng / μl, indicating that the DNA purity is high and the concentration is moderate, which meets the requirements of PCR amplification.

[0060] 1. Target gene selection and primer design: Compared with genome, mitochondria have a higher copy number in tissues, and the degree of damage of antagonal gum is relatively small after deep processing, so mitochondrial 16S DNA gene is preferred. Design deer and cattle universal outer primer and inner primer, the amplified fragment is small, so that the primer and the target are easier to bind. The primer probe sequence is sho...

Embodiment 2

[0064] Example 2 Specificity verification

[0065] Using the primers and probes designed in the present invention, the skins or fresh tissues of animals such as deer, turtle, fish, cow, donkey, horse, fox, mink, raccoon, dog, rabbit, chicken, duck, goose, camel, corn, etc. The total genomic DNA is used as a template to perform real-time fluorescent PCR detection to verify the specificity of its primers and probes. The results are shown in Table 3. The results show that the probes and primers designed in this research have strong specificity.

[0066] Table 3. Specificity verification test

[0067]

Embodiment 3

[0068] Example 3 Sensitivity Experiment

[0069] Quantify the genomic DNA of deer and cattle to 50 ng respectively, and dilute them in a 10 × gradient (10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 ), each gradient takes 2.0 μL as the template amount, (ie: 10 ng, 1 ng, 0.1 ng, 0.01 ng, 0.001 ng) for real-time fluorescent quantitative PCR detection to evaluate the detection limit of the present invention. see Figure 4 with Figure 5 The results show that the quantitative detection limit of this method is 0.1 ng, indicating that the method provided by the present invention has high sensitivity.

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Abstract

The invention discloses a fluorescent PCR detection primer, a probe, a kit and a detection method for simultaneously detecting deer and bovine derived ingredients in colla cornus cervi and an application. The kit provided by the invention is high in detection sensitivity and good in specificity; and rapid detection of the deer and bovine derived ingredients in the colla cornus cervi can be achieved. According to the invention, it can rapidly detect the deer and bovine derived ingredients in the colla cornus cervi and a finished product thereof, so as to distinguish true from false; and the invention has the characteristics of being good in repeatability, strong in specificity, high in accuracy, high in flux, convenient to use and short in consumed time.

Description

Technical field [0001] The invention relates to a detection method, in particular to a deer and bovine-derived multiple fluorescent PCR detection primer, probe, kit, detection method and application in antler gum. It belongs to the technical field of animal-derived detection of glue Chinese medicine. Background technique [0002] Antler gum is a deer family animal sika deer ( Cevusnippon Temminck ) Or red deer ( Cevusnippon elaphusl The solid glue made by decoction and concentration of the angle of) was originally published in "Shen Nong's Materia Medica". It is called white glue and is listed as the top grade alongside donkey-hide glue. It is called Lujiao in "Materia Medica Fengyuan". It is sweet, salty, warm in nature, enters the liver and kidneys, and has the effects of warming the liver and kidney, nourishing essence and blood. Long-term use can nourish and strengthen, replenish blood and stop bleeding, resist aging and prevent aging, and is suitable for a wide range of p...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6888C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/6888C12Q2600/16C12Q2561/113C12Q2563/107C12Q2545/114C12Q2537/143
Inventor 步迅刘艳艳范阳阳胡悦张全芳
Owner VEGETABLE RES INST OF SHANDONG ACADEMY OF AGRI SCI
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