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Specific primers, kit and identification method for identifying donkey-derived components in donkey-hide gelatin

A donkey-derived, donkey-hide gelatin technology, applied in the field of identification of traditional Chinese medicinal materials, can solve problems such as difficult amplification, low DNA content, and DNA damage, and achieve accurate identification results, small molecular weight, and high specificity

Active Publication Date: 2022-05-17
TONG REN TANG TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Both of the above two patent documents identify the direct products of donkeys (donkey hide or donkey meat), which is relatively difficult and high-quality DNA can be obtained by direct extraction. However, donkey-hide gelatin is a deep-processed product. During the production of donkey-hide gelatin, donkey The skin is decocted at high temperature for a long time, and the DNA is severely damaged. The DNA content in the finished product is small and the fragments are extremely short. Moreover, the main components of the finished gelatin are various collagen peptides. The existence of a large amount of protein will seriously affect the efficiency of DNA extraction.
In addition, in the process of preparing the finished donkey-hide gelatin, it not only needs to be boiled for a long time, but also auxiliary materials such as rice wine and soybean oil are added in the later stage. The addition of these auxiliary materials will increase the difficulty of extracting the DNA of the finished donkey-hide gelatin and introduce various components that inhibit the PCR reaction. The above reasons make the identification of donkey-derived components in donkey-hide gelatin very difficult. When using the method of the above prior art to identify donkey-derived components in donkey-hide gelatin, it is easy to have problems such as amplification difficulties and miscellaneous bands, and the accuracy and sensitivity are low. Poor, are not suitable for the identification of donkey-hide gelatin

Method used

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  • Specific primers, kit and identification method for identifying donkey-derived components in donkey-hide gelatin
  • Specific primers, kit and identification method for identifying donkey-derived components in donkey-hide gelatin
  • Specific primers, kit and identification method for identifying donkey-derived components in donkey-hide gelatin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0068] Example 2 Kit

[0069] This embodiment provides a kit for identifying donkey-derived components in donkey-hide gelatin, each of which includes a primer solution part and a PCR reaction solution part that are individually packaged; the primer solution contains the The specific primers in 1; the PCR reaction solution part contains amplification buffer, dNTPs, Taq DNA polymerase and sterile ultrapure water for PCR amplification, and the formula of each kit is :

[0070] Upstream primer, 10 μM, 1 μL;

[0071] Downstream primer, 10 μM, 1 μL;

[0072] Taq DNA polymerase, 5U / μL, 0.25μL;

[0073] dNTPs, 10mM, 0.5μL;

[0074] 10×PCR amplification buffer, 2.5 μL;

[0075] Make up to 25 μL with sterile ultrapure water.

Embodiment 3

[0076] Embodiment 3 identification method

[0077] (1) Dialysis pretreatment of samples to be tested

[0078] Take donkey-hide gelatin finished product 1, donkey-hide gelatin finished product 2 and donkey-hide gelatin finished product 3, and perform pretreatment according to the following steps respectively, to obtain pretreated sample 1, pretreated sample 2 and pretreated sample 3.

[0079] Take 4g of donkey-hide gelatin finished product, add 30ml of deionized water, and melt in a warm bath to obtain a sample solution. The sample solution is placed in a dialysis bag with a molecular weight of 1.5kd, and the sample solution is dialyzed with deionized water as the dialysis medium. Dialyze 3 times at room temperature, using 800ml of dialysis medium each time. The first time was 3 hours, the second time was 3 hours, the third time was 12 hours, dialyzed overnight, the combined dialyzate was collected, concentrated at 80°C and evaporated to dryness to obtain the pretreated sample...

Embodiment 4

[0107] Embodiment 4 identification method

[0108] This embodiment provides a method for identifying donkey-derived components in donkey-hide gelatin, including the following steps:

[0109] (1) Ejiao sample dialysis pretreatment

[0110] Take 3.5g of donkey-hide gelatin finished product 1, add 40ml of deionized water, and melt in a warm bath to obtain a sample solution. The sample solution is placed in a dialysis bag with a molecular weight of 1.8kd, and the sample solution is dialyzed with deionized water as the dialysis medium. Dialyze twice at room temperature, using 850ml of dialysis medium each time. The first time was 3 hours, the second time was 12 hours, dialyzed overnight, the combined dialyzate was collected, concentrated at 80°C and evaporated to dryness to obtain the pretreated sample.

[0111] (2) DNA extraction

[0112] Take the pretreated sample and use the GMO Food DNA kit to extract the total DNA, operate according to the instructions, and the extraction ste...

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Abstract

The invention provides a specific primer, a kit and an identification method for identifying donkey-derived components in donkey-hide gelatin. A large number of primers are designed based on the CYT B and COX-1 genes of donkey, and through a large number of primer screening studies, the selected Species-specific primers can be used to identify donkey-derived components in donkey-hide gelatin, and can accurately separate donkeys from other confounding products, and the PCR amplification product is only 70-120bp, with a small molecular weight, ensuring It can identify a large number of donkey-derived components in donkey-hide gelatin in a short period of time, and the identification results are accurate, stable, reliable and highly sensitive.

Description

technical field [0001] The invention belongs to the technical field of identification of traditional Chinese medicinal materials, and in particular relates to a specific primer, a kit and an identification method for identifying donkey-derived components in donkey-hide gelatin. Background technique [0002] Donkey-hide gelatin (ASINI CORII COLLA) is a solid glue made from the dry skin or fresh skin of an equine animal donkey (Equus asinm L.), which is boiled and concentrated, and can be added with an appropriate amount of rice wine, rock sugar and soybean oil. It has the functions of nourishing blood and nourishing yin. Moisturizes dryness and stops bleeding, and is used to treat symptoms such as blood deficiency and chlorosis, dizziness, palpitations, muscle flaccidity and weakness, restlessness and insomnia. Ejiao is a variety included in the 2015 edition of the "Chinese Pharmacopoeia". Its medicinal use has a long history. It is an important source species, especially ef...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6888C12N15/11C12N15/10
CPCC12N15/1003C12Q1/6888
Inventor 姚璐魏弘宜赵晨彭鹏
Owner TONG REN TANG TECH CO LTD
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