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Specific real-time fluorescence detection method and kit for trionychinae biotic components

A real-time fluorescence, kit technology, applied in the field of PCR detection

Active Publication Date: 2017-06-09
SHANGHAI ENTRY EXIT INSPECTION & QUARANTINE BUREAU OF P R C
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are no public reports at home and abroad on the identification method of the biological components of the soft-shelled turtle subfamily.

Method used

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  • Specific real-time fluorescence detection method and kit for trionychinae biotic components
  • Specific real-time fluorescence detection method and kit for trionychinae biotic components
  • Specific real-time fluorescence detection method and kit for trionychinae biotic components

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0099] Embodiment 1, nucleic acid extraction result

[0100] All extracted animal and plant genomic DNA was detected by micro-spectrophotometer and 0.8% gel electrophoresis. The DNA concentration was between 500-600ng / μL, and the OD260 / OD280 value was between 1.8-2.0, indicating that the integrity of the genomic DNA was good.

[0101] The results showed that the extracted genomic DNA met the requirements of further quantitative experiments.

Embodiment 2

[0102] Embodiment 2, specificity experiment

[0103] The present invention uses common animal and plant genomic DNA as a template to test the specificity of the established softshell subfamily biological quantitative PCR detection method. When the PCR amplification reaction is carried out with eukaryotic 18SrRNA primers / probes, all animal and plant samples Significant S-type amplification curves appeared in all of them, but not in negative control and blank control. When using primers / probes of Trichoinae for PCR amplification reaction, only Trichoinae biological samples had obvious S-type amplification curves, while other common animals and plants, negative controls and blank controls did not, such as figure 1 .

[0104] Therefore, the real-time fluorescent quantitative PCR detection method established by the present invention is highly specific to the turtle subfamily organisms.

Embodiment 3

[0105] Embodiment 3, establishment of standard curve

[0106] In this example, 5 concentration gradient dilutions of the soft-shelled turtle subfamily DNA were used as templates (10, 1, 0.1, 0.01, 0.001 ng / μL), such as Chinese soft-shelled turtle (Jiangsu), respectively, to amplify and establish a standard curve . Such as figure 2 , the linear equation of the standard curve of the soft-shelled turtle subfamily is y=-3.763x+23.97, and the regression coefficient R 2 The value was 0.999, and the reaction efficiency E was 84.5%.

[0107] The results show that the primer probes designed by the present invention have high reaction efficiency to the biological components of Trichoinae, and the established standard curve has good linearity, which can be used for the detection of the biological components of Trichoinae.

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Abstract

The invention relates to a specific real-time fluorescence detection method and kit for trionychinae biotic components. The invention discloses a primer capable of specifically identifying the trionychinae biotic components. The primer can generate specific amplification aiming at DNA containing the trionychinae biotic components and does not generate specific amplification aiming at DNA which does not contain the trionychinae biotic components. The invention further provides a simplified PCR detection method which can be well applied to identification of the trionychinae biotic components and is good in reproducibility and sensitivity.

Description

technical field [0001] The invention belongs to the field of PCR detection, and more specifically, the invention relates to a specific real-time fluorescence detection method and a kit thereof for the biological components of the softshell subfamily. Background technique [0002] Soft-shelled turtles are commonly known as soft-shelled turtles, belonging to the order Reptilia (Testudines) and Trionychidae (Trionychidae), and they are collectively referred to as soft-shelled aquatic turtles. Soft-shelled soft-shelled turtle (Palea steindachneri), etc., among which the most common one is soft-shelled soft-shelled turtle, which is the most popular, has the largest demand, and has the highest economic value. [0003] Turtle subfamily is not only delicious, high in protein and low in fat, but also a nourishing treasure containing a variety of vitamins and trace elements, which can enhance the body's ability to resist diseases and regulate the endocrine function of the human body. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/6888C12Q2561/101C12Q2561/113C12Q2545/101
Inventor 李想苗丽王梦怡高琴
Owner SHANGHAI ENTRY EXIT INSPECTION & QUARANTINE BUREAU OF P R C
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