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Composition, kit and method for identifying authenticity of hide glue and application thereof

A composition and kit technology, which is applied in biochemical equipment and methods, recombinant DNA technology, and microbial determination/inspection, etc., can solve the problem that the detection object cannot directly target Ejiao products, and it is difficult to amplify long-fragment PCR products. Unfavorable market supervision of Ejiao products, etc., to achieve the effects of reliable results, accurate and sensitive results, and simple operation

Inactive Publication Date: 2012-02-29
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In recent years, domestic scientific research institutions have carried out research on the PCR-RFLP method, but the detection objects cannot directly target donkey-hide gelatin products
Because donkey-hide gelatin is subjected to high-intensity treatment such as high-temperature boiling during the production process, the DNA is severely degraded, and it is difficult to amplify long-segment PCR products.
This situation is very unfavorable for the supervision of Ejiao products in the market

Method used

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  • Composition, kit and method for identifying authenticity of hide glue and application thereof
  • Composition, kit and method for identifying authenticity of hide glue and application thereof
  • Composition, kit and method for identifying authenticity of hide glue and application thereof

Examples

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Embodiment 1

[0031] In this example, the specificity and sensitivity of the primers and probes of donkey-hide gelatin were verified through the following tests.

[0032] By detecting the mitochondrial DNA (mtDNA) cytochrome b region sequence, the specificity and detection sensitivity of the combination of specific oligonucleotide primers and probes used to identify the authenticity of donkey-hide gelatin can be determined. The reaction system is: Mastermix 12.5 μL; probe (10 μM) 2 μL; upstream and downstream primers (10 μM) 1 μL each; template DNA 5 μL; add ddH 2 O to a total volume of 25 μL. The reaction program was 95°C for 10 min; 95°C for 15 s; 60°C for 1 min, 50 cycles.

[0033] The used donkey-specific oligonucleotide primer pair is composed of an upstream primer and a downstream primer, the base sequence of the upstream primer is SEQ ID No.1, and the base sequence of the downstream primer is SEQ ID No.2; The base sequence of the probe used is SEQ ID No.3, a fluorescent quenching g...

Embodiment 2

[0068] The inventors of the present invention verified the commercially available donkey-hide gelatin samples through the following tests.

[0069] Seven commercially available donkey-hide gelatin samples were selected for real-time fluorescent PCR reaction to determine the feasibility and specificity of the established real-time fluorescent PCR method.

[0070] Among the 7 commercially available samples, samples 1-5 were known to be true donkey-hide gelatin, while samples 6 and 7 were false donkey-hide gelatin.

[0071] In this embodiment, the donkey-specific oligonucleotide primer pair used is SEQ ID No.1 and SEQ ID No.2 and the probe is SEQ ID No.3, and a The fluorescent quenching group TAMRA is connected with a fluorescent reporting group FAM at the 5' end. In addition, in order to further confirm the accuracy of the detection and identify the source of the sample, in this embodiment, the horse-specific oligonucleotide primer pair is SEQ ID No.4 and SEQ ID No.5 and the pr...

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Abstract

The invention relates to a composition for identifying the authenticity of hide glue. The composition comprises an oligonucleotide primer and a probe. The invention also relates to a real-time fluorescence polymerase chain reaction (PCR) detection method for identifying the authenticity of the hide glue, and the method comprises using the specific oligonucleotide primer and the probe of the invention. The invention also relates to a PCR detection kit for identifying the authenticity of the hide glue, and the kit comprises the specific oligonucleotide primer and the probe of the invention. Theinvention also relates to application of the specific oligonucleotide primer and the probe of the invention in the identification of the authenticity of the hide glue. The identification of the authenticity of the hide glue by PCR detection method is simple, quick, specific and flexible.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular, the present invention relates to a composition comprising oligonucleotide primers and probes for identifying the authenticity of donkey-hide gelatin, a real-time fluorescent PCR detection method for identifying the authenticity of donkey-hide gelatin, and the identification of donkey-hide gelatin. Application of specific oligonucleotide primers and probes in identifying the authenticity of donkey-hide gelatin. Background technique [0002] Donkey-hide gelatin has been eaten for thousands of years in China. It has the effects of nourishing blood and stopping bleeding, nourishing yin and nourishing deficiency. It is a special Chinese medicinal material formed by boiling donkey skin, and its main component is collagen. Pure donkey-hide gelatin must come from 100% donkey skin in order to ensure the nutritional efficacy of the product. In terms of product characteristics, donkey-hide gela...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68G01N21/64C12N15/11
Inventor 陈颖吴亚君王斌韩建勋张海亮
Owner CHINESE ACAD OF INSPECTION & QUARANTINE