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Kit for extracting high-purity DNA of colla corii asini and derivative products of colla corii asini, as well as extraction method

A technology of derivative products and kits, which is applied in the extraction kits of donkey-hide gelatin and its derivative products of high-purity DNA and its extraction field, which can solve the problems of pollution, DNA detection failure, high price, etc., and achieve improved DNA purity and good safety , the effect of a wide range of sources

Inactive Publication Date: 2016-02-03
INST OF PLANT PROTECTION SHANDONG ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] DNA extracted from donkey-hide gelatin and its derivatives with complex components contains inhibitors that can interfere with PCR, which can easily lead to DNA detection failure (false negative)
Most PCR inhibitors (proteins, polysaccharides, phenols, urea, humic acid, and heme, etc.) have similar solubility to DNA, and classical DNA extraction methods (CTAB / SDS, protease, and phenol-chloroform-isoamyl alcohol Processing) is not easy to completely remove, these substances become pollutants in the final extraction solution of DNA
In addition, organic reagents such as phenol and chloroform used in classical DNA extraction methods are highly toxic and corrosive, and pose a risk of harming the physical and mental health of test personnel.
In addition to the classic DNA extraction method, the invention patent (application number: CN201410317118.7) also discloses a kit for quickly extracting DNA from donkey-hide gelatin and its extraction method, but the kit uses many types of reagents and is expensive. Moreover, individual reagents are scarce and not easy to purchase, which increases detection and time costs

Method used

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  • Kit for extracting high-purity DNA of colla corii asini and derivative products of colla corii asini, as well as extraction method
  • Kit for extracting high-purity DNA of colla corii asini and derivative products of colla corii asini, as well as extraction method
  • Kit for extracting high-purity DNA of colla corii asini and derivative products of colla corii asini, as well as extraction method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Using the extraction kit of the present invention to extract DNA from donkey-hide gelatin and its derivatives

[0036] 1. Extraction kit:

[0037] Composed of component I, component II, proteinase K solution and RNase solution;

[0038] The composition of component I is: 0.1MSDS, 1.0MTris-HCl, 0.5MNa 2 EDTA, PH=8.0;

[0039] The composition of component II is: 8.3MCsCl, 10mMTris-HCl, PH=8.0;

[0040] The concentration of proteinase K solution is 20 mgmL -1 ; The concentration of RNase solution is 100mgmL -1 .

[0041] 2. Samples to be tested:

[0042] Ejiao, donkey-hide gelatin cake and donkey-hide gelatin liquid; (all are commercially available products).

[0043] 3. Extraction method:

[0044] (1) Take 1.0g of donkey-hide gelatin or donkey-hide gelatin cake or 1.0mL of donkey-hide gelatin solution to be tested into a 2mL centrifuge tube. Set 3 replicates for each sample. Add 1mL component I and 5μL proteinase K (final concentration is 0.1mgmL) -1 ), 5μL RNase (final conc...

Embodiment 2

[0049] Example 2: The DNA of donkey-hide gelatin and its accessory products extracted by the extraction kit of the present invention is used for detection of donkey components by real-time fluorescent PCR

[0050] Real-time fluorescent PCR was used to detect donkey components in the DNA of donkey-hide gelatin and its accessory products mentioned in Example 1.

[0051] The primers and probes were synthesized by Shenggong Bioengineering (Shanghai) Co., Ltd. and diluted to 10μM for use.

[0052] Donkey component-specific primers and probes adopt national standard primers and probes (SN / T3730.4-2013), and their sequence is as follows:

[0053] Upstream primer F: 5'-AATAGCTCTAGCCGTACGGCTAACT-3'; (SEQIDNO.1)

[0054] Downstream primer R: 5'-CAGGATAATGAATGTAATAAGGGCTG-3'; (SEQIDNO.2)

[0055] Probe P: FAM-5'-TGCCGGACATCTTCTAATCCACCTT-3'-TAMRA. (SEQIDNO.3)

[0056] The amplification reaction volume is 20μL, containing 2×PremixExTaq(TaKaRa)10μL, the upstream primer F0.4μL with a concentration of ...

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Abstract

The invention discloses a kit for extracting high-purity DNA of colla corii asini and derivative products of colla corii asini. The kit consists of a component I, a component II, a protease K solution and an RNA enzyme solution, wherein the component I consists of 0.08-0.12 M SDS, 0.8-1.2 M Tris-HCl and 0.4-0.6 M Na2EDTA, and the PH of the component I is 7.5-8.5; the component II consists of 8.0-8.5 M CsCl and 8-12 mM Tris-HCl, and the PH of the component II is 7.5-8.5. The invention further discloses a method for extracting the high-purity DNA of colla corii asini and the derivative products of colla corii asini by adopting the kit. The kit is simple in component and low in reagent cost, and can effectively reduce the detection cost. The extracted DNA is high in purity, and is suitable for next molecular biological detection; poisonous reagents of phenol / chloroform and the like are not used in the extraction process, so that damage to the human body by the dangerous chemical reagent can be reduced.

Description

Technical field [0001] The invention belongs to the technical field of food detection, and specifically relates to an extraction kit for high-purity DNA of donkey-hide gelatin and its derivative products and an extraction method thereof. Background technique [0002] The modern pharmacopoeia defines donkey-hide gelatin as a solid glue made from the skin of equine donkeys by decoction and concentration. Its taste is sweet and flat, enters the lung, liver, and kidney meridians, has the effects of nourishing blood to stop bleeding, nourishing yin and moisturizing dryness, and is used as medicine and food. At present, the market price of donkey-hide gelatin has risen sharply, and substitutes made from other animal skins have appeared everywhere. Fake glues made from bone glue and miscellaneous skins not only have poor clinical effects, but may also be poisoned. From the appearance of donkey skin glue, horse skin glue or cowhide glue, they are very similar and difficult to distinguis...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 张广远路兴波孙红炜李凡杨淑珂徐晓辉高瑞刘玲雪
Owner INST OF PLANT PROTECTION SHANDONG ACAD OF AGRI SCI
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