Method for detecting gelatin in sea cucumber based on fluorescent PCR (polymerase chain reaction) technology

Abstract

The invention discloses a method for detecting gelatin in sea cucumber based on a fluorescent PCR (Polymerase Chain Reaction) technology. The method comprises the following steps: determining a target and designing a primer and a probe; determining a target gene: selecting a mitochondrial gene aiming at the DNA which is easy to degrade after processing, and designing a primer and a probe for amplifying a short fragment; obtaining a specific sequence: obtaining target gene sequences of the target sea cucumber and common adulterated species from an NCBI gene database, and finding a unique specific sequence region of the sea cucumber through comparison; synthesizing a primer and a probe: designing and synthesizing the primer and the TaqMan probe on the basis of the specific region; preparing a reference substance; sample pretreatment and DNA extraction; preparing a fluorescent PCR reaction system and adding a sample; running a fluorescent PCR amplification program: placing the reaction tube in a real-time fluorescent quantitative PCR instrument; result analysis and interpretation: quality control inspection and sample result interpretation. According to the invention, a primer and a probe which are only complementarily combined with sea cucumber DNA are designed, and a fluorescence signal is generated in PCR amplification, so that specific detection can be realized.

Description

Technical Field

[0001] This invention relates to the field of gelatin detection technology, specifically to a method for detecting gelatin in sea cucumbers based on fluorescence PCR technology. Background Technology

[0002] Sea cucumbers, belonging to the class Holothuroidea, are marine echinoderms that live from the seashore to depths of 8,000 meters. They have existed for over 600 million years, feeding on seabed algae and plankton. Covered in fleshy spines, sea cucumbers are widely distributed in oceans worldwide. The South my country Sea coast has a relatively large variety, with over twenty species available for consumption. Sea cucumbers are considered one of the world's eight most precious delicacies, alongside ginseng, bird's nest, and shark fin. Beyond being a valuable food, sea cucumbers are also a prized medicinal ingredient. According to the *Compendium of Materia Medica*, sea cucumbers are sweet and salty, nourishing the kidneys, replenishing essence, controlling urination, and strengthening yang to treat impotence. Their warming and nourishing properties are comparable to ginseng, hence the name "sea ginseng." Sea cucumbers are also believed to improve memory, delay gonadal aging, prevent arteriosclerosis, and have anti-tumor effects. With increasing awareness of the value of sea cucumbers, they are gradually becoming a staple on people's tables.

[0003] When sea cucumbers are removed from seawater, they produce an autolytic enzyme that causes them to dissolve into a liquid, watery state, within 6-7 hours, leaving no trace. Some data indicates that sea cucumbers can also spontaneously dissolve in the ocean after growing for 10-15 years. Furthermore, autolysis can occur not only when removed from seawater but also when their environment is polluted. Because sea cucumbers contain this autolytic enzyme, they require prompt processing after capture.

[0004] Currently, sea cucumbers are mainly processed in two ways: drying and ready-to-eat. However, gelatin is added during the existing sea cucumber processing. Gelatin, which is usually used to make jelly and other desserts, is made from boiled animal bones, skin and tendons. Therefore, a method for detecting gelatin in sea cucumbers based on fluorescent PCR technology was designed to detect it in existing sea cucumber products. Summary of the Invention

[0005] The purpose of this invention is to provide a method for detecting gelatin in sea cucumbers based on fluorescence PCR technology, so as to solve the problems mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a method for detecting gelatin in sea cucumbers based on fluorescence PCR technology, comprising the following steps: Step S1: Define the target and design primers and probes; Step S11: Identify target genes: For DNA that is easily degraded after processing, select mitochondrial genes and design primers and probes to amplify short fragments; Step S12: Obtain specific sequences: Obtain the target gene sequences of the target sea cucumber and common adulterant species from the NCBI gene database, and find the unique specific sequence regions of sea cucumber by comparison; Step S13, Synthesis of primers and probes: Based on the specific region, primers and TaqMan probes are designed and synthesized; Step S2: Prepare the control sample; Step S3: Sample pretreatment and DNA extraction; Step S4: Preparation and addition of the fluorescent PCR reaction system: Prepare the reaction system; Sample addition: Dispense the mixture of the above-prepared reaction system into PCR tubes, and then add the DNA sample to be tested, positive control, negative control and blank control respectively; Step S5: Running the fluorescent PCR amplification program: Place the reaction tube in the real-time quantitative PCR instrument; Step S6, Result Analysis and Interpretation: Quality control inspection and sample result interpretation.

[0007] Preferably, step S2, preparing the reference standard, includes: Positive control: Known, purified sea cucumber DNA samples; Negative control: DNA from non-sea cucumber species; Blank control: Use sterile water instead of DNA template.

[0008] Preferably, step S3, sample pretreatment and DNA extraction, includes: Step S31, Sample processing: Grind dried sea cucumber, ready-to-eat products, or samples containing gelatin into fine powder to increase the lysis surface area; Step S32, DNA extraction: For gelatin deep-processing samples, DNA is extracted using a column-based genomic DNA extraction kit suitable for animal tissue blood. Step S33, DNA quality detection: Use a micro spectrophotometer to measure DNA concentration and purity, and observe DNA fragment size using 1.5% agarose gel electrophoresis.

[0009] Preferably, step S4 is performed in a clean bench or an environment equipped with an aerosol-proof suction head to prevent contamination.

[0010] Preferably, the components of the prepared reaction system are: 10 μL of 2×TaqMan premix, 0.8 μL of forward primer, 0.8 μL of reverse primer, 0.4 μL of TaqMan probe, 2-5 μL of DNA template, and 20 μL of sterile nuclease-free water.

[0011] Preferably, the quality control inspection includes: Positive control: A typical S-type amplification curve must be observed, and the Ct value must be within a reasonable range; Negative control and blank control: There must be no amplification curve. Any abnormality indicates contamination, and the results of this experiment are invalid.

[0012] Preferably, the sample result interpretation is as follows: Positive result: The sample showed a clear S-shaped amplification curve and the Ct value was ≤ 35, indicating that sea cucumber-specific DNA fragments were detected; Negative result: The sample has no amplification curve or the Ct value is greater than the set threshold, indicating that sea cucumber DNA was not detected.

[0013] Preferably, the short segment is 100-150 bp.

[0014] Compared with the prior art, the beneficial effects of the present invention are: 1. By detecting species-specific DNA fragments of sea cucumbers, it is possible to indirectly identify whether the product contains gelatin or collagen derived from sea cucumbers. 2. By designing primers and probes that bind only to sea cucumber DNA, fluorescent signals can be generated during PCR amplification, thus enabling specific detection. Attached Figure Description

[0015] Figure 1 This is a schematic diagram of the process of the present invention; Figure 2 This invention clarifies the objectives and designs a schematic diagram of the primer and probe process; Figure 3 This is a schematic diagram of the sample pretreatment and DNA extraction process of the present invention. Detailed Implementation

[0016] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0017] Please see Figure 1-3 This invention provides a technical solution: a method for detecting gelatin in sea cucumbers based on fluorescence PCR technology, comprising the following steps: Step S1: Define the target and design primers and probes; Step S11: Identify target genes: For DNA that is easily degraded after processing, select mitochondrial genes and design primers and probes to amplify short fragments; Step S12: Obtain specific sequences: Obtain the target gene sequences of the target sea cucumber and common adulterant species from the NCBI gene database, and find the unique specific sequence regions of sea cucumber by comparison; Step S13, Synthesis of primers and probes: Based on the specific region, primers and TaqMan probes are designed and synthesized; Step S2: Prepare the control sample; Positive control: Known, purified sea cucumber DNA samples; Negative control: DNA from non-sea cucumber species; Blank control: Use sterile water instead of DNA template.

[0018] Step S3: Sample pretreatment and DNA extraction; Step S31, Sample processing: Grind dried sea cucumber, ready-to-eat products, or samples containing gelatin into fine powder to increase the lysis surface area; Step S32, DNA extraction: For gelatin deep-processing samples, DNA is extracted using a column-based genomic DNA extraction kit suitable for animal tissue blood. Step S33, DNA quality detection: Use a micro spectrophotometer to measure DNA concentration and purity, and observe DNA fragment size using 1.5% agarose gel electrophoresis.

[0019] Step S4: Preparation and addition of the fluorescent PCR reaction system: Prepare the reaction system; Sample addition: Dispense the mixture of the above-prepared reaction system into PCR tubes, and then add the DNA sample to be tested, positive control, negative control and blank control respectively; Step S5: Running the fluorescent PCR amplification program: Place the reaction tube in the real-time quantitative PCR instrument; Step S6, Result Analysis and Interpretation: Quality control inspection and sample result interpretation; The quality control inspection: Positive control: A typical S-type amplification curve must be observed, and the Ct value must be within a reasonable range; Negative control and blank control: There must be no amplification curve. Any abnormality indicates contamination, and the results of this experiment are invalid.

[0020] Interpretation of the sample results: Positive result: The sample showed a clear S-shaped amplification curve and the Ct value was ≤ 35, indicating that sea cucumber-specific DNA fragments were detected; Negative result: The sample has no amplification curve or the Ct value is greater than the set threshold, indicating that sea cucumber DNA was not detected.

[0021] In this invention, step S2, preparing the reference standard, includes: In this invention, step S4 is performed in a clean bench or an environment equipped with an aerosol-proof suction head to prevent contamination.

[0022] In this invention, the components of the prepared reaction system are: 10 μL of 2×TaqMan premix, 0.8 μL of forward primer, 0.8 μL of reverse primer, 0.4 μL of TaqMan probe, 2-5 μL of DNA template, and 20 μL of sterile nuclease-free water.

[0023] In this invention, the short segment is 100-150 bp.

[0024] This invention: Clearly defines the target and designs primers and probes; targeting easily degradable DNA after processing, mitochondrial genes are selected, and primers and probes for amplifying short fragments are designed; target gene sequences of the target sea cucumber and common adulterant species are obtained from the NCBI gene database, and specific sequence regions unique to sea cucumbers are identified through comparison; based on these specific regions, primers and TaqMan probes are designed and synthesized; controls are prepared; positive control: known, pure sea cucumber DNA samples; negative control: DNA from non-sea cucumber species; blank control: sterile water is used instead of DNA template; dried sea cucumber products, ready-to-eat products, or samples containing gelatin are thoroughly ground into a fine powder to increase the lysis surface area; DNA extraction is performed on samples containing gelatin using a column-based genomic DNA extraction kit suitable for animal tissues and blood; micro-... DNA concentration and purity were measured using a spectrophotometer, and DNA fragment size was observed using 1.5% agarose gel electrophoresis. Fluorescent PCR reaction system preparation and sample loading: The reaction system was prepared; Sample loading: The mixture of the prepared reaction system was dispensed into PCR tubes, and then the sample DNA, positive control, negative control, and blank control were added respectively; Fluorescent PCR amplification program execution: The reaction tubes were placed in a real-time quantitative PCR instrument; Quality control checks and sample result interpretation; Quality control checks: Positive control: A typical S-shaped amplification curve must appear, and the Ct value must be within a reasonable range; Negative control and blank control: No amplification curve must appear; any abnormality indicates contamination, and the experimental results are invalid; Sample result interpretation: Positive result: The sample shows a clear S-shaped amplification curve, and the Ct value ≤ 35, indicating that sea cucumber-specific DNA fragments were detected; Negative result: The sample has no amplification curve or the Ct value is greater than the set threshold, indicating that sea cucumber DNA was not detected.

[0025] The contents not described in detail in this specification are prior art known to those skilled in the art. Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for detecting gelatin in sea cucumbers based on fluorescent PCR technology, characterized in that: Includes the following steps: Step S1: Define the target and design primers and probes; Step S11: Identify target genes: For DNA that is easily degraded after processing, select mitochondrial genes and design primers and probes to amplify short fragments; Step S12: Obtain specific sequences: Obtain the target gene sequences of the target sea cucumber and common adulterant species from the NCBI gene database, and find the unique specific sequence regions of sea cucumber by comparison; Step S13, Synthesis of primers and probes: Based on the specific region, primers and TaqMan probes are designed and synthesized; Step S2: Prepare the control sample; Step S3: Sample pretreatment and DNA extraction; Step S4: Preparation and addition of the fluorescent PCR reaction system: Prepare the reaction system; Sample addition: Dispense the mixture of the above-prepared reaction system into PCR tubes, and then add the DNA sample to be tested, positive control, negative control and blank control respectively; Step S5: Running the fluorescent PCR amplification program: Place the reaction tube in the real-time quantitative PCR instrument; Step S6, Result Analysis and Interpretation: Quality control inspection and sample result interpretation.

2. The method for detecting gelatin in sea cucumbers based on fluorescent PCR technology according to claim 1, characterized in that: Step S2, preparing the reference standard, includes: Positive control: Known, purified sea cucumber DNA samples; Negative control: DNA from non-sea cucumber species; Blank control: Use sterile water instead of DNA template.

3. The method for detecting gelatin in sea cucumbers based on fluorescence PCR technology according to claim 1, characterized in that: Step S3, sample pretreatment and DNA extraction, includes: Step S31, Sample processing: Grind dried sea cucumber, ready-to-eat products, or samples containing gelatin into fine powder to increase the lysis surface area; Step S32, DNA extraction: For gelatin deep-processing samples, DNA is extracted using a column-based genomic DNA extraction kit suitable for animal tissue blood. Step S33, DNA quality detection: Use a micro spectrophotometer to measure DNA concentration and purity, and observe DNA fragment size using 1.5% agarose gel electrophoresis.

4. The method for detecting gelatin in sea cucumbers based on fluorescent PCR technology according to claim 1, characterized in that: Step S4 is performed in a clean bench or an environment equipped with an aerosol-proof suction head to prevent contamination.

5. The method for detecting gelatin in sea cucumbers based on fluorescent PCR technology according to claim 1, characterized in that: The components of the prepared reaction system are: 10 μL of 2×TaqMan premix, 0.8 μL of forward primer, 0.8 μL of reverse primer, 0.4 μL of TaqMan probe, 2-5 μL of DNA template, and 20 μL of sterile nuclease-free water.

6. The method for detecting gelatin in sea cucumbers based on fluorescent PCR technology according to claim 1, characterized in that: The quality control inspection: Positive control: A typical S-type amplification curve must be observed, and the Ct value must be within a reasonable range; Negative control and blank control: There must be no amplification curve. Any abnormality indicates contamination, and the results of this experiment are invalid.

7. The method for detecting gelatin in sea cucumbers based on fluorescent PCR technology according to claim 1, characterized in that: Interpretation of the sample results: Positive result: The sample showed a clear S-shaped amplification curve and the Ct value was ≤ 35, indicating that sea cucumber-specific DNA fragments were detected; Negative result: The sample has no amplification curve or the Ct value is greater than the set threshold, indicating that sea cucumber DNA was not detected.

8. The method for detecting gelatin in sea cucumbers based on fluorescent PCR technology according to claim 1, characterized in that: The short fragment is 100-150 bp.

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