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CN-121975951-A - Bezoar-based antigen identification primer pair and application thereof

CN121975951ACN 121975951 ACN121975951 ACN 121975951ACN-121975951-A

Abstract

The invention relates to a primer pair for identifying bezoar-based antigen, which is selected from any one of a primer pair 1, a forward primer, a reverse primer and a primer pair 2, wherein the nucleotide sequence of the forward primer is shown as SEQ ID NO. 1, the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 2, the nucleotide sequence of the forward primer is shown as SEQ ID NO. 3, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 4. The primer pair can effectively identify bezoar-based antigen and has good specificity, sensitivity and anti-interference performance.

Inventors

  • LIU JINXIN
  • ZENG LINGCHAO
  • SHI LINCHUN
  • Zang Erhuan
  • MA DENGXIU
  • ZHOU LISI
  • ZHANG YANG
  • MA TINGYU
  • LUO LILIAN
  • WANG BIN

Assignees

  • 中央民族大学
  • 中国医学科学院药用植物研究所
  • 远大岐黄生物科技(北京)有限公司

Dates

Publication Date
20260505
Application Date
20260324

Claims (13)

  1. 1. A primer pair for identifying bezoar-based antigen, wherein the primer pair is selected from any one of the following primer pairs: the nucleotide sequence of the forward primer of the primer pair 1 is shown as SEQ ID NO. 1, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 2; The nucleotide sequence of the primer pair 2 is shown as SEQ ID NO. 3, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 4.
  2. 2. A kit for identifying bezoar-based antigen, comprising the primer pair of claim 1.
  3. 3. The kit according to claim 2, further comprising a DNA extraction reagent and a PCR amplification reagent.
  4. 4. Use of the primer pair of claim 1, the kit of any one of claims 2-3 for identifying bezoar primordia.
  5. 5. A method for identifying bezoar-based antigen, comprising the steps of: (1) Amplifying a DNA sample of a sample to be tested using the primer set of claim 1; (2) And judging whether the sample to be detected is bezoar or not according to the amplification result.
  6. 6. The method of claim 5, wherein step (2) is: And (3) carrying out electrophoresis analysis on the amplified product, wherein the sample with the specific band is bezoar.
  7. 7. The method of claim 5, wherein step (2) is: sequencing the amplified product to obtain the sample with the nucleotide sequence shown as SEQ ID No. 5 or SEQ ID No. 6 as bezoar.
  8. 8. An extraction method of calculus bovis DNA, characterized in that the extraction method comprises the step of cooperatively lysing a sample by using a buffer 1 containing CDAB and a buffer 2 containing SDS.
  9. 9. The method of extraction according to claim 8, characterized in that said co-lysing sample comprises in particular the following steps: (1) Taking a bezoar sample, and grinding; (2) Adding a buffer solution 1, and incubating; (3) Buffer 2 was added and incubated.
  10. 10. The method according to claim 8, wherein the ratio of the amount of the bezoar sample to the amount of the buffer 1 to the amount of the buffer 2 is 30-150 mg:600-1000. Mu.L and 100-300. Mu.L.
  11. 11. The method according to claim 8, wherein the CTBA content in buffer 1 is 0.5-3% and the SDS content in buffer 2 is 8-12%.
  12. 12. The method according to claim 8, wherein the method further comprises a step of separating DNA magnetic beads having a particle diameter of 300 to 600nm after the sample is lysed.
  13. 13. A method for extracting calculus bovis DNA and identifying primordia, which is characterized by comprising the following steps: (A) Extracting a DNA sample from a sample to be tested: Extracting a DNA sample using the extraction method of any one of claims 8-12; (B) Identifying bezoar base: Bezoar-based antigen identification using the method according to any one of claims 5-7.

Description

Bezoar-based antigen identification primer pair and application thereof Technical Field The invention belongs to the technical field of traditional Chinese medicine identification, and particularly relates to a primer pair for identifying bezoar base and application thereof. Background Natural bezoar is a pathological product in cattle, and is a substitute for natural bezoar prepared by artificial means because the natural yield is very rare and the market demand is difficult to meet, and the natural bezoar is mainly divided into two types, namely, in-vitro cultured bezoar prepared by taking fresh bile of cattle as mother liquor and adding deoxycholic acid, cholic acid, compound bilirubin calcium and other components, and the other type is artificial bezoar prepared by taking ox gall powder, cholic acid, hyodeoxycholic acid, bilirubin and the like as raw materials. At present, bezoar products in the market are relatively confusing in sources, and a plurality of cases exist that substances such as pig gall yellow are used as natural bezoar. The basic source identification is a method for identifying the source of the traditional Chinese medicine by applying morphological and taxonomic knowledge of plants, animals or minerals and determining the correct animal and plant academic names and mineral names so as to ensure the accuracy of the applied varieties. Currently, traditional Chinese medicine identification methods include traditional methods, namely source identification, character identification, microscopic identification, physicochemical identification, and modern DNA molecular identification methods. However, bezoar belongs to a dry calcified calculus sample, the cell structure is seriously damaged, the DNA is highly degraded and the content is extremely low, a sufficient and complete template cannot be provided for molecular identification, and in addition, the sample is rich in bilirubin, cholic acid, calcium salt and other impurities, which can strongly interfere enzymatic reactions such as PCR and the like, so that an effective DNA molecular identification technical means is not established for the primordial identification of bezoar until now. For example, patent CN106480206a discloses a primer pair for identifying beef, but because more impurities and other interfering substances exist in a cow-bezoar DNA sample, the primer pair cannot be used for identifying cow-bezoar, CN107881243a discloses a primer pair for identifying bovine-derived components, but the primer pair also has the problems of insufficient anti-interference capability and poor sensitivity in the process of identifying cow-bezoar by PCR. Disclosure of Invention In order to solve the technical problems, the application provides a primer pair for identifying bezoar base, which can effectively identify bezoar base and has better specificity, sensitivity and anti-interference performance. According to one aspect of the present application there is provided a primer pair for identifying bezoar base, said primer pair being selected from any one of the following primer pairs: the nucleotide sequence of the forward primer of the primer pair 1 is shown as SEQ ID NO. 1, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 2; The nucleotide sequence of the primer pair 2 is shown as SEQ ID NO. 3, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 4. According to another aspect of the present application there is provided a kit for identifying bezoar based antigen, the kit comprising a primer pair as described above. Optionally, the kit further comprises a DNA extraction reagent and a PCR amplification reagent. According to another aspect of the application, there is provided the use of the above primer pair, the above kit for identifying bezoar primordia. According to another aspect of the present application, there is provided a method for identifying bezoar based antigen, the method comprising the steps of: (1) Amplifying a DNA sample of a sample to be tested using the primer set of claim 1; (2) And judging whether the sample to be detected is bezoar or not according to the amplification result. Optionally, the step (2) is: And (3) carrying out electrophoresis analysis on the amplified product, wherein the sample with the specific band is bezoar. Optionally, the step (2) is: sequencing the amplified product to obtain the sample with the nucleotide sequence shown as SEQ ID No. 5 or SEQ ID No. 6 as bezoar. According to another aspect of the present application, there is provided a method for extracting bovine bezoar DNA, comprising the step of co-lysing a sample using a buffer 1 containing CDAB and a buffer 2 containing SDS. Optionally, the co-lysis sample specifically comprises the following steps: (1) Taking a bezoar sample, and grinding; (2) Adding a buffer solution 1, and incubating; (3) Buffer 2 was added and incubated. Optionally, the ratio of the bezoar sample to the buffer 1to the buffer 2 is