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CN-121992110-A - Primer probe combination for detecting shrimp, crab and fish allergens based on double Proofman-LMTIA technology and application thereof

CN121992110ACN 121992110 ACN121992110 ACN 121992110ACN-121992110-A

Abstract

The invention discloses a primer probe combination for detecting shrimp, crab and fish allergens based on a double Proofman-LMTIA technology and application thereof, and belongs to the technical field of molecular detection. The primer probe combination comprises primers and probes with sequences shown as SEQ ID NO. 1-10. The invention designs a specific primer and a probe based on Proofman-LMTIA technology, and constructs a rapid detection system of shrimp, crab and fish allergens. In the links of production, processing, storage and transportation and circulation, the method can accurately identify shrimp, crab and fish allergens possibly mixed in processed foods, provides a powerful technical tool for efficiently controlling allergen risks for food enterprises, can help regulatory departments to carry out more accurate and more efficient spot check and screening on foods containing allergens, reduces the risk of wrong feeding of allergic people from the source, and provides a technical barrier for the food safety and the health of allergic people.

Inventors

  • TIAN BO
  • GUO LINQING
  • WANG DEGUO
  • ZHOU DAN
  • LI JIANPING
  • CHEN YUE
  • WANG ZHIYUAN

Assignees

  • 东北农业大学

Dates

Publication Date
20260508
Application Date
20260215

Claims (9)

  1. 1. A primer Probe combination for detecting shrimp and crab allergens based on a double Proofman-LMTIA technology is characterized by comprising XiaXie-F1 shown as SEQ ID NO.1, xiaXie-B1 shown as SEQ ID NO.2, xiaXie-LF1 shown as SEQ ID NO.3, xiaXie-LB1 shown as SEQ ID NO.4, yuR-F2 shown as SEQ ID NO.5, yuR-B2 shown as SEQ ID NO.6, yuR-LF2 shown as SEQ ID NO.7, yuR-LB2 shown as SEQ ID NO.8, xiaXie-Probe1 shown as SEQ ID NO.9, yuR-Probe2 shown as SEQ ID NO. 10; The XiaXie-Probe1 and YuR-Probe2 are linked to different fluorophores.
  2. 2. Use of the primer probe combination of claim 1 in the preparation of a reagent or kit for detecting shrimp, crab and fish allergens.
  3. 3. A kit for detecting shrimp, crab and fish allergens based on the dual Proofman-LMTIA technology, wherein the kit comprises the primer probe combination of claim 1.
  4. 4. The method for detecting the shrimp, crab and fish allergens is characterized by comprising the following steps: extracting genome DNA of a sample to be detected, taking the genome DNA as a template, performing amplification reaction by using the kit of claim 3, and observing fluorescent signals and amplification curves; If the fluorescent signal and the amplification curve with the same color as the fluorescent group connected with XiaXie-Probe1 are provided, the sample to be detected contains shrimp or crab allergens, and if the fluorescent signal and the amplification curve with the same color as the fluorescent group connected with YuR-Probe2 are provided, the sample to be detected contains fish allergens.
  5. 5. The method according to claim 4, wherein the amplification reaction is performed using 5. Mu.L of 2 Xmix premix and 0.5. Mu.L of the genomic DNA template of DNA polymerase 、0.16 μLXiaXie-F1、0.16 μL XiaXie-B1、0.04 μL XiaXie-LF1、0.04 μL XiaXie-LB1、0.1 μL XiaXie-Probe1、0.16 μL YuR-F2、0.16 μL YuR-B2、0.04 μL YuR-LF2、0.04 μL YuR-LB2、0.4 μL YuR-Probe2、2 μL, and the DEPC-treated water is added to 10. Mu.L.
  6. 6. The method according to claim 5, wherein the concentrations of XiaXie-F1, xiaXie-B1, xiaXie-LF1, xiaXie-LB1, yuR-F2, yuR-B2, yuR-LF2, yuR-LB2 are each 100. Mu.M, and the concentrations of XiaXie-Probe1, yuR-Probe2 are each 10. Mu.M.
  7. 7. The method according to claim 5, wherein the 2 Xmix premix comprises the following components ：40 mM Tris-HCl,20 mM KCl,20 mM (NH 4 ) 2 SO 4 ,12 mM MgSO 4 ,0.2% Triton X-100,2.4 mM dNTP.
  8. 8. The method according to claim 4, wherein the amplification reaction is carried out at a temperature of 61℃for 30 minutes.
  9. 9. Use of the primer probe combination of claim 1 or the kit of claim 3 for detecting shrimp and crab allergens and/or fish allergens in food products.

Description

Primer probe combination for detecting shrimp, crab and fish allergens based on double Proofman-LMTIA technology and application thereof Technical Field The invention relates to the technical field of molecular detection, in particular to a primer probe combination for detecting shrimp, crab and fish allergens based on a double Proofman-LMTIA technology and application thereof. Background Food allergy problems are a significant challenge to the safety and health of all humans, and it is estimated that food allergy affects 5% of adults and 8% of children worldwide, and the proportion is also rising. In addition, eight major food allergens identified by the united nations grain and agriculture organization (FAO) and World Health Organization (WHO) account for 90% of the total number of food allergies, wherein crustaceans (typified by shrimp and crab) and fish are important members of the eight major food allergens, are an IgE-mediated immune disease, and usually trace amounts of allergens can induce allergic symptoms such as skin erythema and itching, respiratory mucosa edema, digestive tract discomfort and even anaphylactic shock in severe cases, thus directly threatening the life safety of allergic people. The shrimp and crab nutrition value is outstanding, the shrimp and crab nutrition value is rich in high-quality digestible proteins, unsaturated fatty acids, various minerals such as calcium, phosphorus, zinc and the like, and simultaneously contains nutrition components such as vitamin B group, vitamin A and the like, thereby being a high-quality protein source in human diet. The shrimp and crab body contains various muscle structural proteins and soluble proteins, wherein more than 20 proteins are considered as potential sensitization proteins, tropomyosin is a core allergen of shrimp and crab allergy, and more than about 90% of shrimp and crab allergy patients can detect specific IgE aiming at the proteins. By carrying out multiple sequence homology comparison analysis on tropomyosin genes of different shrimp and crab species, highly conserved gene fragments are screened out as specific target sequences for subsequent allergen detection. Fish is one of important dietary protein sources for human beings, and is rich in high-quality protein, polyunsaturated fatty acid, vitamin D, selenium and other nutrient substances. The potential sensitization proteins identified in the fish are more than 15, the parvalbumin (Parvalbumin) is the primary allergen for fish allergy, the parvalbumin specific IgE can be detected in about 85% of serum of fish allergic patients, the protein has high thermal stability, and the sensitization of the parvalbumin is difficult to be completely eliminated by conventional cooking processing. However, the gene of the 12S rRNA in the fish has stronger specificity and stability, so that the specific fragment of the gene is selected as a target sequence for detecting the fish allergen. Current allergen detection methods are largely divided into DNA-based and allergen protein-based detection. In the allergen detection technology at the protein level, an enzyme-linked immunosorbent assay (ELISA) and a Lateral Flow Immunochromatography (LFIA) are the two most widely used methods at present, wherein the ELISA can realize qualitative and quantitative analysis of the allergen by means of a dual mechanism of antigen-antibody specific binding and enzymatic signal amplification, and the LFIA relies on antigen-antibody immune reaction and capillary chromatography to realize on-site rapid screening of the allergen. However, the immunological detection method is highly dependent on the specificity and affinity of the antibody, the preparation difficulty of the antibody of the shrimp and crab tropomyosin and fish parvalbumin is high, the antibody is easy to cross react with homologous proteins from other aquatic organisms, and meanwhile, the structural denaturation of the allergen protein is easy to be caused under conditions of high temperature, high pressure and the like in the food processing process, and the structural denaturation of the allergen protein can be caused to be false positive. The mass spectrometry technology realizes allergen detection by accurately identifying characteristic peptide segments, can effectively avoid the limitations of an immunological method, but is usually combined with separation technologies such as high performance liquid chromatography and the like, depends on large-scale precise instruments and professional data analysis teams, and is difficult to meet the actual requirements of on-site rapid detection. In contrast, DNA molecules have the advantage of being chemically stable, yet maintain nucleotide sequence integrity under complex processing conditions. In food allergen detection, commonly used nucleic acid detection methods include real-time fluorescent quantitative PCR (qPCR), digital PCR (dPCR), loop-mediated isothermal amplification (LAMP), and the like.