Kit, method and primer pair for identifying animal-derived ingredients in crude protein-type biochemical material

A technology of animal-derived components and primer pairs, applied in the field of molecular biology, can solve the problems of high reagent cost, unfavorable promotion and application, and waste, and achieve the effects of ensuring drug safety, facilitating promotion and application, and simple operation

Inactive Publication Date: 2017-07-21
SPH NO 1 BIOCHEM & PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The technical problem to be solved by the present invention is to overcome the detection method for the residual DNA in the crude raw material in the prior art that cannot distinguish the ruminant-derived components and the existing detection method has high requirements for instruments and high reagent costs, which is not conducive to In view of the defects of large-scale popularization and application, a kit, method and primer pair for identifying animal, especially artiodactyly, animal-derived components in crude protein biochemical raw materials are provided.
However, the amount of residual DNA is not very clear. If the DNA is extracted directly, it may be impossible to extract an effective amount of DNA because the content is too low; and if the existing fluorescent quantitative PCR with high sensitivity and specificity is used, there will be The detection cost is high, which is not conducive to popularization and application, and its sensitivity is too high, which is much lower than the DNA content in the crude raw material, resulting in unnecessary waste

Method used

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  • Kit, method and primer pair for identifying animal-derived ingredients in crude protein-type biochemical material
  • Kit, method and primer pair for identifying animal-derived ingredients in crude protein-type biochemical material
  • Kit, method and primer pair for identifying animal-derived ingredients in crude protein-type biochemical material

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Extraction of residual genomic DNA in the raw material crude product of embodiment 1

[0039] (1) Grind crude protein biochemical raw materials into powder, add 180 μL of cell lysate 1 for every 25 mg of sample powder, then add 20 μL of proteinase K solution, shake and mix well, and put in a water bath at 56°C for 2 hours until the cells are completely lysed; (2) Add lysate After 200 μL of the solution was fully inverted and mixed, 70 ° C water bath for 10 minutes; (3) After the water bath, centrifuged at 5000 rpm for 5 minutes, and the supernatant was taken into a new tube; (4) 200 μL of absolute ethanol was added to the supernatant, and thoroughly mixed by inversion (5) Put the adsorption column into the collection tube, add all the solution into the adsorption column with a pipette, let it stand for 2 minutes, then centrifuge at 10000rpm room temperature for 1min, and pour off the waste liquid in the collection tube; (6) If the volume of the solution is less than Lar...

Embodiment 2

[0040] Embodiment 2PCR amplification and agarose gel electrophoresis detection

[0041] (1) Primer design

[0042] Design species-specific primers derived from porcine, bovine, and sheep, respectively:

[0043] a. Design primer pair Sus for porcine cytochrome oxidase subunit I gene EU623453.1:

[0044] Forward primer: 5'-GGCACCCTGTACCTACTATTTGG-3' (see SEQ ID NO.1 for details);

[0045] Reverse primer: 5'-ATTCCGAATCCTGGTAAGATGAG-3') See SEQ ID NO.2 for details);

[0046] b. Design primer pair Bos for bovine cytochrome oxidase subunit I gene DQ487094.1:

[0047] Forward primer: 5'-GAGAGTAGTAGTAGTACGGCGGTAAT-3' (see SEQ ID NO.3 for details);

[0048] Reverse primer: 5'-CATCCTCTATAGTTGAAGCTGGG-3' (see SEQ ID NO.4 for details);

[0049] c. Design primer pair Ovis for sheep cytochrome oxidase subunit I gene EU834864.1:

[0050] Forward primer: 5'-TAGATCTAACTATTTTCTCCCCTACATCTG-3' (see SEQ ID NO.5 for details);

[0051] Reverse primer: 5'-GGTGTTGGTATAGGATAGGGTCTC-3' (see SEQ ...

Embodiment 3

[0062] Example 3 Pigs, cattle, and sheep-derived primers carry out PCR amplification of single-species genomic DNA of pigs, cattle, and sheep respectively

[0063] Genomic DNA of pigs, cattle, and sheep were PG-313, BG-313, and SG-313 products of Zyagen Company, and the concentration was 1000ng / μL. Genomic DNA of pigs, cattle, and sheep was diluted with double distilled water to a concentration of 100 ng / μL as a DNA template; a blank control was set, and 1 μL of double distilled water was used to replace the DNA template.

[0064] (1) PCR amplification

[0065] The primers, PCR system and amplification program in Examples 1 and 2 were used for PCR amplification.

[0066] (2) Agarose gel electrophoresis detection

[0067] Take 10 μL of PCR amplification product and add 2 μL of loading buffer (containing 0.25% bromophenol blue, 0.25% xylene aniline, 60% glycerol), and use agarose gel electrophoresis with a concentration of 1% by mass percentage, voltage 120V, electrophoresis f...

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Abstract

The invention discloses a kit, method and primer pair for identifying animal-derived ingredients in crude protein-type biochemical material; the kit comprises a PCR (polymerase chain reaction) system that at least includes one primer pair; the primer pair is designed for cytochrome oxidase subunit I gene in an animal, especially even-toed ungulates. The kit has the advantages of good operational simplicity, low cost, high accuracy, good repeatability and the like, and is especially capable of distinguishing ingredients from ruminant animals, such as pig, cattle, and sheep.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a kit for identifying animal-derived components in crude protein biochemical raw materials, a method thereof and a pair of primers. Background technique [0002] In recent years, with the improvement of pharmacopoeia standards, the source of animal species needs to be clarified for various crude biochemical raw materials. On October 19, 2016, the State Food and Drug Administration issued the "Appendix to the Good Manufacturing Practice for Biochemical Drugs (Draft for Comment)", in which Article 40 on species identification mentioned that the source of raw materials for biochemical drugs should be relatively stable , The species and organ tissues of the animal should be clarified. When necessary, identify the animal species of raw materials. "Animal" as used herein refers to non-human animals. At present, there are more than 100 kinds of biochemical raw material drugs derived ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6888
Inventor 黄臻辉王欣刘弘忍汪雅雯丁金国
Owner SPH NO 1 BIOCHEM & PHARMA CO LTD
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