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Turtle and bovine derived fluorescence PCR detection primer in tortoise shell glue, probe, kit, detection method and application

A technology for detection kits and detection primers, applied in biochemical equipment and methods, measurement/testing of microorganisms, DNA/RNA fragments, etc., can solve the problem of high sensitivity and false positive rate, and the identification method cannot meet the authenticity of tortoise shell gum Requirements, inaccurate determination, etc., to achieve good specificity, overcome incomplete extraction difficulties, and high application value

Active Publication Date: 2017-06-09
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 identifying the differences of polypeptides in tortoise shell glue, and due to technical limitations, the sensitivity and false positive rate are relatively high, and the judgment is not accurate enough
Therefore, the traditional identification methods cannot meet the current requirements for authenticity identification of tortoise shell gum.
However, the use of real-time fluorescent quantitative PCR to detect the origin of turtles and cattle in tortoise shell glue has not yet been reported in China.

Method used

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  • Turtle and bovine derived fluorescence PCR detection primer in tortoise shell glue, probe, kit, detection method and application
  • Turtle and bovine derived fluorescence PCR detection primer in tortoise shell glue, probe, kit, detection method and application
  • Turtle and bovine derived fluorescence PCR detection primer in tortoise shell glue, probe, kit, detection method and application

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Experimental program
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Effect test

Embodiment 1

[0062] The DNA of the tortoise shell glue sample was extracted using the method disclosed in the patent 201410317118.7 (a kit for rapidly extracting DNA from donkey-hide gelatin and its extraction method). and concentration. The measured OD260 / OD280 values ​​were all about 1.8-1.9, and the concentration was above 10ng / μl, which indicated that the DNA had a high purity and a moderate concentration, which met the requirements of PCR amplification.

[0063] 1. Selection of target genes and design of primers: Compared with the genome, the copy number of mitochondria in tissues is higher, and the damage degree of tortoise shell glue is relatively small after deep processing, so the mitochondrial 16SrDNA gene is preferred. The outer and inner primers are designed for turtles and cattle, and the amplified fragments are small, making it easier for the primers to bind to the target. The sequences of primers and probes are listed in Table 1.

[0064] 2. Quantitative detection of torto...

Embodiment 2

[0067] Example 2 Specificity Verification

[0068] Using the primers and probes designed by the present invention, the skins or fresh tissues of deer, turtle, fish, cow, donkey, horse, fox, mink, raccoon dog, dog, rabbit, chicken, duck, goose, camel, corn, etc. The total genomic DNA was used as a template for 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 study have strong specificity.

[0069] Table 3. Specificity verification tests

[0070]

Embodiment 3

[0071] Embodiment 3 Sensitivity experiment

[0072] Quantify tortoise and bovine genomic DNA to 50 ng respectively, and dilute by 10× gradient (10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 ), each gradient took 2.0 μL as the template amount, (ie: 10 ng, 1 ng, 0.1 ng, 0.01 ng, 0.001 ng) for real-time fluorescence quantitative PCR detection, and evaluated the detection limit of the present invention. See Figure 4 and Figure 5 , the result shows that the quantitative detection limit of this method is 0.1ng, illustrates that the method provided by the present invention has very high sensitivity.

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Abstract

The invention discloses a turtle and bovine derived fluorescence PCR detection primer in tortoise shell glue, a probe, a kit, a detection method and application. The kit disclosed by the invention is high in detection sensitivity and high in specificity, and rapid detection of turtle and bovine derived components in the tortoise shell glue can be realized. According to the detection method disclosed by the invention, the tortoise shell glue and turtle and bovine derivation in products thereof can be rapidly detected so as to distinguish the true from the false, and the detection method has the characteristics of high repeatability, high specificity, high accuracy degree, large flux, convenient use and short consumed time.

Description

technical field [0001] The invention relates to a detection method, in particular to a fluorescent PCR detection primer, probe, test kit, detection method and application of tortoise and bovine-derived fluorescence in tortoise shell glue. The invention belongs to the technical field of animal-derived detection of glue-type traditional Chinese medicines. Background technique [0002] Tortoise shell glue is a solid glue made from the carapace and plastron of turtles in the family Turtleidae, boiled in water and concentrated. Taste salty, sweet, slightly cold, it belongs to the liver, kidney and heart meridian, has the effects of nourishing yin and suppressing yang, nourishing kidney and strengthening bones, nourishing blood and nourishing the heart, mainly used for yin deficiency and dampness, bone steaming night sweats, dizziness, and internal wind Movement, flaccidity of muscles and bones, guilty conscience and forgetfulness, etc. Long-term consumption of tortoise shell gl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/6888C12Q2600/16C12Q2600/166C12Q2537/143C12Q2563/107C12Q2545/101
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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