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Specific primer, kit and identification method for identifying donkey-derived components

A donkey-derived, kit-based technology, applied in the field of identification of Chinese herbal medicines, can solve the problems of less DNA content, DNA damage, and short fragments, and achieve the effects of accurate identification results, high specificity, and small molecular weight

Active Publication Date: 2019-04-09
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 patents are for the identification of direct products of donkeys (donkey skin or donkey meat), which is relatively low difficulty, and high-quality DNA can be obtained by direct extraction. However, donkey-hide gelatin is a deep-processed product. After long-term decoction at high temperature, 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 donkey-hide 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, not only need to go through a long time of decocting, but also add auxiliary materials such as rice wine and soybean oil 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 reason makes it very difficult to identify donkey-derived components in donkey-hide gelatin. When using the above-mentioned prior art methods to identify donkey-derived components in donkey-hide gelatin, problems such as failure to amplify and miscellaneous bands are prone to occur, and the accuracy, sensitivity, and specificity are poor. , are not suitable for the identification of donkey-hide gelatin

Method used

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  • Specific primer, kit and identification method for identifying donkey-derived components
  • Specific primer, kit and identification method for identifying donkey-derived components
  • Specific primer, kit and identification method for identifying donkey-derived components

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0173] Example 2 Kit

[0174] This embodiment provides a kit for identifying donkey-derived components, each of which includes a primer solution part and a PCR reaction solution part that are individually packaged; The specific primers; 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:

[0175] DNA template, 50ng / μL, 3μL;

[0176] Upstream primer, 10 μM, 1 μL;

[0177] Downstream primer, 10 μM, 1 μL;

[0178] Taq DNA polymerase, 5U / μL, 0.4μL;

[0179] dNTPs, 10mM, 0.5μL;

[0180] 10×PCR amplification buffer, 2.5 μL;

[0181] Make up to 25 μL with sterile ultrapure water.

Embodiment 3

[0182] Embodiment 3 identification method

[0183] This example provides a method for identifying donkey-derived components using the specific primers of Example 1 or the kit of Example 2, including the following steps:

[0184] (1) Ejiao to be tested is pretreated with Ni-NTA

[0185] Take 50mg donkey-hide gelatin sample 1, add 5ml Lysis Buffer (NaH 2 PO 4 50mM, NaCl 300mM, NaOH to adjust the pH to 8.0), warm the bath until the sample melts completely, put it in a Ni-NTA prepacked column (the amount of filler is based on 10mg protein per 1ml Ni-NTA filler), shake on ice Combine for 1 hour, collect the breakthrough solution, and evaporate to dryness at 85°C to obtain the pretreated sample.

[0186] (2) DNA extraction

[0187] Take the pretreated sample and use the TAKARA MiniBest DNA kit to extract the total DNA, operate according to the instructions, and the extraction steps are the same as the aforementioned TAKARA MiniBest DNA kit.

[0188] (3) PCR amplification

[0...

Embodiment 4

[0203] Embodiment 4 identification method

[0204] This example provides a method for identifying donkey-derived components using the specific primers of Example 1 or the kit of Example 2, including the following steps:

[0205] (1) Ejiao to be tested is pretreated with Ni-NTA

[0206] Take 100mg donkey-hide gelatin sample 2, add 1ml Lysis Buffer (NaH 2 PO 4 75mM, NaCl 100mM, NaOH to adjust the pH to 10.0), warm the bath until the sample melts completely, place it in a Ni-NTA prepacked column (the amount of filler is based on 8mg protein per 1ml Ni-NTA filler), shake on ice Combine for 2 hours, collect the breakthrough solution, and evaporate to dryness at 80°C to obtain the pretreated sample.

[0207] (2) DNA extraction

[0208] Take the pretreated sample and use the TAKARA MiniBest DNA kit to extract the total DNA, operate according to the instructions, and the extraction steps are the same as the aforementioned TAKARA MiniBest DNA kit.

[0209] (3) PCR amplification ...

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PUM

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Abstract

The invention provides a specific primer, a kit and an identification method for identifying donkey-derived components. A large number of primers are designed based on CYT B and COX-1 genes of a donkey, and are screened and researched to screen out a species-specific primer; and the primer is used for identifying the donkey-derived components in a mixture to be detected. The specific primer, the kit and the identification method provided by the invention can accurately distinguish the donkey-derived components from other adulterants, have a PCR amplification product of only 50-150bp and smallmolecular weight, ensure identification of the donkey-derived components in a large number of mixtures within a short time, and can obtain accurate, stable, reliable and highly sensitive identification results.

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 components derived from donkeys. Background technique [0002] Donkey-hide gelatin (ASINICORIICOLLA) is a solid gelatin made from the dry skin or fresh skin of an equine animal donkey (Equus asinm L.), which is decocted and concentrated, and can be added with an appropriate amount of rice wine, rock sugar and soybean oil. , the effect of hemostasis, for the treatment of symptoms such as blood deficiency and chlorosis, dizziness, palpitations, muscle flaccidity, 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 effective in preventing diseases. There are more than 3,200 medical prescriptions about donkey-hide g...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12N15/11
CPCC12Q1/6888
Inventor 杜菁赵晨姚璐魏弘宜易剑平
Owner TONG REN TANG TECH CO LTD
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