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CN-121975944-A - Method for detecting gelatin in sea cucumber based on fluorescence PCR technology

CN121975944ACN 121975944 ACN121975944 ACN 121975944ACN-121975944-A

Abstract

The invention discloses a method for detecting gelatin in sea cucumbers based on a fluorescence PCR technology, which comprises the following steps of determining a target gene, selecting a mitochondrial gene and designing a primer and a probe for amplifying short fragments aiming at DNA which is easy to degrade after processing, obtaining a specific sequence, obtaining target gene sequences of the target sea cucumbers and common adulterated species from an NCBI gene database, comparing to find a specific sequence area unique to the sea cucumbers, synthesizing the primer and the probe, designing and synthesizing the primer and the TaqMan probe based on the specific area, preparing a reference substance, carrying out sample pretreatment and DNA extraction, preparing and adding samples by a fluorescence PCR reaction system, operating a fluorescence PCR amplification program, namely placing a reaction tube in a real-time fluorescence quantitative PCR instrument, and carrying out result analysis and interpretation, quality control inspection and sample result interpretation. According to the invention, by designing the primer and the probe which are only complementarily combined with the sea cucumber DNA, a fluorescent signal is generated in PCR amplification, and specific detection can be realized.

Inventors

  • ZHOU YUCHEN
  • REN GUOJIE
  • WU HAIXIA
  • LI YUEWEN
  • WANG HAITING
  • ZHOU MEILING
  • QIU YUE
  • Geng Jialing

Assignees

  • 大连产品质量检验检测研究院有限公司

Dates

Publication Date
20260505
Application Date
20251225

Claims (8)

  1. 1.A method for detecting gelatin in sea cucumber based on a fluorescence PCR technology is characterized by comprising the following steps: S1, defining a target, and designing a primer and a probe; Step S11, determining target genes, namely selecting mitochondrial genes aiming at DNA which is easy to degrade after processing, and designing primers and probes for amplifying short fragments; s12, acquiring specific sequences, namely acquiring target gene sequences of target sea cucumbers and common adulterated species from NCBI gene database, and finding out unique specific sequence regions of the sea cucumbers through comparison; s13, synthesizing a primer and a probe, namely designing and synthesizing the primer and the TaqMan probe based on the specific region; Step S2, preparing a reference substance; S3, sample pretreatment and DNA extraction; S4, preparing and sampling a fluorescent PCR reaction system: Preparing a reaction system; Sample adding, namely subpackaging the mixed solution of the preparation reaction system into a PCR tube, and then adding DNA of a sample to be detected, positive control, negative control and blank control respectively; S5, performing fluorescence PCR amplification program operation, namely placing the reaction tube in a real-time fluorescence quantitative PCR instrument; And S6, analyzing and judging results, namely quality control inspection and sample result judgment.
  2. 2. The method for detecting gelatin in sea cucumber according to claim 1, wherein the step S2 of preparing the reference substance comprises the steps of: Positive control, known, pure sea cucumber DNA sample; negative control, DNA of non-sea cucumber species; blank control, sterile water was used instead of DNA template.
  3. 3. The method for detecting gelatin in sea cucumber based on fluorescence PCR technology as claimed in claim 1, wherein the step S3 of sample pretreatment and DNA extraction comprises the following steps: Step S31, sample treatment, namely fully grinding dried sea cucumber, instant product or gelatin-containing sample into fine powder so as to increase the cracking surface area; s32, DNA extraction, namely using a column type genome DNA extraction kit suitable for animal tissue blood to extract DNA from a gelatin deep processing sample; Step S33, DNA quality detection, namely measuring the concentration and purity of the DNA by using a micro-spectrophotometer, and observing the size of the DNA fragment by using 1.5% agarose gel electrophoresis.
  4. 4. The method for detecting gelatin in sea cucumber according to claim 1, wherein the step S4 is performed in an ultra clean bench or an environment equipped with an anti-aerosol tip to prevent contamination.
  5. 5. The method for detecting gelatin in sea cucumber based on fluorescence PCR technology as claimed in claim 1, wherein the components of the preparation reaction system are as follows: 10. Mu.L of 2 XTaqMan premix, 0.8. Mu.L of forward primer, 0.8. Mu. L, taqMan probe of reverse primer, 2-5. Mu.L of L, DNA template, and 20. Mu.L of sterile nuclease-free water.
  6. 6. The method for detecting gelatin in sea cucumber based on fluorescence PCR technology as claimed in claim 1, wherein the quality control inspection is characterized in that: Positive control, wherein a typical S-shaped amplification curve is required to appear, and the Ct value is in a reasonable range; the negative control and the blank control are no amplification curve, and any abnormality indicates pollution, so that the experimental result is invalid.
  7. 7. The method for detecting gelatin in sea cucumber based on fluorescence PCR technology as claimed in claim 1, wherein the sample result is interpreted as follows: Positive results show that a sample has an obvious S-shaped amplification curve, and the Ct value is less than or equal to 35, which indicates that the sea cucumber specific DNA fragment is detected; And negative results, wherein the sample has no amplification curve or Ct value is larger than a set threshold value, which indicates that the sea cucumber DNA is not detected.
  8. 8. The method for detecting gelatin in sea cucumber based on fluorescence PCR technology as claimed in claim 1, wherein the short segment is 100-150 bp.

Description

Method for detecting gelatin in sea cucumber based on fluorescence PCR technology Technical Field The invention relates to the technical field of gelatin detection, in particular to a method for detecting gelatin in sea cucumbers based on a fluorescent PCR technology. Background Sea cucumber belongs to sea cucumber class (Holothuroidea), is a marine echinoderm living at sea to 8000 meters, has been six hundred million years old to date, and is fed by seafloor algae and plankton. Sea cucumber grows full of meat thorns throughout the body and is widely spread in the world's oceans. The variety of coastal south China sea is more, more than twenty kinds of sea cucumbers can be eaten, and the sea cucumbers are named as ginseng, bird's nest and shark fins, so that the sea cucumbers are one of eight precious products in the world. Sea cucumber is not only a precious food, but also a precious medicinal material. According to the description in Ben Cao gang mu Shi, sea cucumber is called sea cucumber because it has sweet and salty taste, and is effective in tonifying kidney, replenishing essence and marrow, relieving urination, strengthening yang, treating flaccidity, warming and tonifying nature, and curing ginseng which is a natural enemy. Sea cucumber has effects of improving memory, delaying gonad aging, preventing arteriosclerosis, and resisting tumor. Along with popularization of the value knowledge of sea cucumbers, the sea cucumbers gradually enter common people's dining tables. When sea cucumber leaves sea water, autolytic enzyme is generated in the body, and the sea cucumber can melt into liquid in 6-7 hours, and is in a water state, so that the dissolution area is stable. The data show that sea cucumber can be automatically dissolved in sea after 10-15 years of growth. Meanwhile, the sea water is separated from the sea water, and autolysis phenomenon can occur under the condition that the sea cucumber growth environment is polluted and the like. Because the sea cucumber contains autolyzed enzyme in vivo, the sea cucumber needs to be treated in time after being captured. At present, two processing methods of drying and instant processing are mainly adopted for processing sea cucumbers, however, gelatin (Gelatin) is added in the existing sea cucumber processing process, and is usually used for preparing jelly and other desserts, and the jelly and other desserts are prepared from boiled animal bones, skins and tendons, so that a detection method of Gelatin in sea cucumbers based on a fluorescent PCR technology is designed and used for detecting the existing sea cucumber products. Disclosure of Invention The invention aims to provide a method for detecting gelatin in sea cucumbers based on a fluorescent PCR technology, so as to solve the problems in the background technology. In order to achieve the purpose, the invention provides the following technical scheme that the method for detecting gelatin in sea cucumbers based on the fluorescent PCR technology comprises the following steps: S1, defining a target, and designing a primer and a probe; Step S11, determining target genes, namely selecting mitochondrial genes aiming at DNA which is easy to degrade after processing, and designing primers and probes for amplifying short fragments; s12, acquiring specific sequences, namely acquiring target gene sequences of target sea cucumbers and common adulterated species from NCBI gene database, and finding out unique specific sequence regions of the sea cucumbers through comparison; s13, synthesizing a primer and a probe, namely designing and synthesizing the primer and the TaqMan probe based on the specific region; Step S2, preparing a reference substance; S3, sample pretreatment and DNA extraction; S4, preparing and sampling a fluorescent PCR reaction system: Preparing a reaction system; Sample adding, namely subpackaging the mixed solution of the preparation reaction system into a PCR tube, and then adding DNA of a sample to be detected, positive control, negative control and blank control respectively; S5, performing fluorescence PCR amplification program operation, namely placing the reaction tube in a real-time fluorescence quantitative PCR instrument; And S6, analyzing and judging results, namely quality control inspection and sample result judgment. Preferably, the step S2 of preparing the reference substance includes: Positive control, known, pure sea cucumber DNA sample; negative control, DNA of non-sea cucumber species; blank control, sterile water was used instead of DNA template. Preferably, the step S3 of sample pretreatment and DNA extraction includes: Step S31, sample treatment, namely fully grinding dried sea cucumber, instant product or gelatin-containing sample into fine powder so as to increase the cracking surface area; s32, DNA extraction, namely using a column type genome DNA extraction kit suitable for animal tissue blood to extract DNA from a gelatin deep processing sample; Step S33, DNA qualit