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CN-122012789-A - Primer, method and application for identifying peach kernels and mountain peach kernels

CN122012789ACN 122012789 ACN122012789 ACN 122012789ACN-122012789-A

Abstract

The invention belongs to the technical field of traditional Chinese medicines, and discloses a primer, a method and application for identifying peach kernels and mountain peach kernels, wherein the primer comprises a primer petA-F and a primer psbJ-R, the gene sequence of the primer petA-F is SEQ ID NO.1, and the gene sequence of the primer psbJ-R is SEQ ID NO.2. The invention establishes a PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) intraspecific identification method of peach kernels, applies a site-specific PCR technology to select primers, designs restriction enzymes by utilizing different sites, and adopts an enzyme cutting method to identify peach kernels and mountain peach kernels. Can rapidly identify the peach kernels and the mountain peach kernels, and provides a new technical means for quality control of the peach kernel related products.

Inventors

  • REN GUIQI
  • HU CHUNLAN
  • CHEN YUANYUAN
  • LI JUN
  • WANG MEI
  • WANG JINHUAN
  • LIU YAJING

Assignees

  • 颈复康药业集团有限公司

Dates

Publication Date
20260512
Application Date
20260313

Claims (7)

  1. 1. A primer for identifying peach kernels and mountain peach kernels is characterized by comprising a primer petA-F and a primer psbJ-R, wherein the gene sequence of the primer petA-F is SEQ ID NO.1, and the gene sequence of the primer psbJ-R is SEQ ID NO.2.
  2. 2. The use of the primer according to claim 1 for identifying peach kernel and mountain peach kernel.
  3. 3. The method for identifying peach kernels and mountain peach kernels by using the primers as claimed in claim 1 is characterized in that the method uses a site-specific PCR technology to select the primers, uses different sites to design restriction enzymes, and adopts an enzyme digestion method to identify peach kernels and mountain peach kernels.
  4. 4. The method according to claim 3, comprising the steps of: Randomly sampling in a peach kernel sample, extracting genome as a template, carrying out PCR amplification, purifying, connecting to a pMD20-T vector, converting into DH5 alpha competent cells, culturing in a 37 ℃ incubator in LB Amp + plate culture medium for 16h, picking up positive clones, putting into 400 mu L of LB Amp + liquid culture medium for 2h under shaking culture at 37 ℃, sequencing after PCR detection of recombinants, carrying out enzyme digestion by EcoRV, carrying out enzyme digestion on each 10 mu L of enzyme digestion system, containing 2 mu L of PCR products, 0.3 mu L of EcoRV enzyme, 1 mu L of 10 XBuffer, 6.7 mu L of sterilized deionized water, mixing uniformly, putting into a 37 ℃ incubator for 1h, carrying out electrophoresis detection on the enzyme digestion products by using 3.0% agarose gel, and carrying out enzyme digestion on peach kernels without enzyme digestion sites, wherein the peach kernels are digested by EcoRV into two fragments.
  5. 5. The method according to claim 4, wherein the amount of 5. Mu.L of Genstar polymerase per 10. Mu.L of amplification system of 1U. Mu.L -1 , 0.2. Mu.L of each of the upstream and downstream primers petA-F and psbJ-R, and 0.3. Mu.L of template DNA are added with sterile deionized water to a total volume of 10. Mu.L.
  6. 6. The method according to claim 4, wherein the amplification conditions in PCR amplification are 94℃for 30s, 57℃for 30s, 72℃for 1 min,34 cycles, 72℃for 5min and 4℃for preservation after a pre-denaturation at 94 ℃.
  7. 7. The method according to any one of claims 4 to 6, wherein each 1L of LB Amp + plate medium is prepared from 10g of tryptone, 5g of yeast extract, 10g of NaCl, 1.5g of agar and 1L of deionized water; each 1L of LB Amp + liquid culture medium comprises 10g of tryptone, 5g of yeast extract and 10g of NaCl, and is prepared into 1L by deionized water.

Description

Primer, method and application for identifying peach kernels and mountain peach kernels Technical Field The invention belongs to the technical field of traditional Chinese medicines, and in particular relates to a primer, a method and application for identifying peach kernels and mountain peach kernels. Background Semen Persicae is dry mature seed of Prunus persica (L.) Batsch or Prunus persica Prunus davidiana (Carr.) Franch of Rosaceae, and has effects of promoting blood circulation, removing blood stasis, loosening bowel to relieve constipation, relieving cough and asthma. Peach and mountain peach belong to the same family and genus plant seeds, the appearance and the size are very similar, and the peach and mountain peach are more difficult to distinguish after peeling. The literature describes that there are significant differences in the chemical composition of peach kernel and mountain peach kernel. The content of amino acid is used as an evaluation index, the peach kernel is superior to peach kernel, and the fat-soluble component is used as an evaluation index, and the two are obviously different. At present, the quality control standard of traditional Chinese medicinal materials is limited to main components and microscopic identification, the medicinal materials with similar appearance characteristics cannot be accurately identified, peach kernels are seed medicinal materials, the peach kernels are rich in more grease and easy to go away and deteriorate, the result is affected by sensory differences and detection environments, and the operability in mass industrial production is poor. The conventional common identification methods such as character identification, microscopic identification, physicochemical identification, chemical component analysis identification and the like have the defects of strong subjectivity, long period, complex operation and high cost. The molecular biology technology has the advantages of rapidness, trace quantity, strong specificity, accuracy and reliability, is not influenced by factors such as the growth and development stage of the sample, the tested part, the environmental condition, the storage and the like, and is very suitable for researching the Chinese medicinal material sample. At present, the analysis and detection of traditional Chinese medicinal materials by utilizing a molecular biology technology have been studied in a large number of tens of varieties of traditional Chinese medicinal materials, and a lot of results are obtained. Therefore, the establishment of the peach kernel basic source identification method is an urgent requirement for ensuring the clinical safety and effective application of peach kernel medicinal materials. Disclosure of Invention The invention aims to overcome the defects in the prior art and provides a primer, a method and application for identifying peach kernels and mountain peach kernels. The technical scheme adopted for solving the technical problems is as follows: A primer for identifying peach kernels and mountain peach kernels comprises a primer petA-F and a primer psbJ-R, wherein the gene sequence of the primer petA-F is SEQ ID NO.1, and the gene sequence of the primer psbJ-R is SEQ ID NO.2. The primer is applied to the identification of peach kernels and mountain peach kernels. The method for identifying peach kernels and mountain peach kernels by using the primers comprises the steps of selecting the primers by using a site-specific PCR technology, designing restriction enzymes by using different sites, and identifying the peach kernels and the mountain peach kernels by using an enzyme digestion method. Further, the method comprises the following steps: Randomly sampling in a peach kernel sample, extracting genome as a template, carrying out PCR amplification, purifying, connecting to a pMD20-T vector, converting into DH5 alpha competent cells, culturing in a 37 ℃ incubator in LB Amp + plate culture medium for 16h, picking up positive clones, putting into 400 mu L of LB Amp + liquid culture medium for 2h under shaking culture at 37 ℃, sequencing after PCR detection of recombinants, carrying out enzyme digestion by EcoRV, carrying out enzyme digestion on each 10 mu L of enzyme digestion system, containing 2 mu L of PCR products, 0.3 mu L of EcoRV enzyme, 1 mu L of 10 XBuffer, 6.7 mu L of sterilized deionized water, mixing uniformly, putting into a 37 ℃ incubator for 1h, carrying out electrophoresis detection on the enzyme digestion products by using 3.0% agarose gel, and carrying out enzyme digestion on peach kernels without enzyme digestion sites, wherein the peach kernels are digested by EcoRV into two fragments. Further, in the PCR amplification, 5. Mu.L of Genstar polymerase of 1U. Mu.L -1, 0.2. Mu.L of each of the upstream and downstream primers, namely, the primers petA-F and psbJ-R, and 0.3. Mu.L of template DNA were added to a total volume of 10. Mu.L by adding sterile deionized water. Further, the amplificatio