Method for identifying allergens of crustacean aquatic products based on 16S rRNA (ribosomal ribonucleic acid) micro bar codes

Abstract

The invention relates to a DNA micro-bar code for accurately identifying allergen species of crustacean aquatic products. The DNA micro-bar code is an animal mitochondrial genome sequence fragment 16SrRNA. Compared with a traditional COI animal general bar code, the 16S rRNA micro bar code used in the method can be better applied to identification of the allergens of the crustacean aquatic products in commercially available food, the detection requirements of market supervision departments and food production enterprises can be met, and more comprehensive food allergen information can be provided for consumers.

Description

Technical Field

[0001] The invention relates to the field of food detection, and in particular to a method for identifying allergens in crustacean aquatic products. Background Art

[0002] Allergy to crustacean products is mainly a type I hypersensitivity reaction mediated by IgE. Even trace amounts of crustacean food ingredients can trigger allergic reactions. Crustacean products mainly include shrimp, prawns, lobsters and crabs. They are one of the most consumed foods in the world. Their sensitization rate is higher than other aquatic products. About 2.5% of the world's population is allergic to crustacean products. Allergic reactions caused by crustacean products usually occur in adults. When patients come into contact with crustacean allergens, they may experience skin redness, swelling, itching, nausea and vomiting, etc. This reaction may accompany them for a lifetime.

[0003] DNA barcoding technology can use a pair of short, homologous DNA fragments to quickly and accurately identify species. Traditional animal universal DNA barcodes generally use the 658bp mitochondrial cytochrome c oxidase I (COI) gene to identify species. Crustacean aquatic products are often used in deep food processing. High pressure, high temperature, shearing and other processing technologies can easily cause DNA breakage and degradation, and the amplification effect of longer DNA sequences will be affected. Therefore, some components of processed food species may be difficult to identify using traditional COI barcodes (658bp) due to high DNA degradation. Although short fragments of the 16S ribosomal RNA (rRNA) gene are gradually being used to identify species components in food, there are no reports on the use of DNA microbarcoding to identify crustacean food allergens. Summary of the invention

[0004] To solve the above technical problems, the present invention includes the following aspects:

[0005] The first aspect of the present invention provides a DNA microbarcode for identifying crustacean aquatic product allergen species, wherein the DNA microbarcode is an animal mitochondrial genome sequence fragment 16S rRNA.

[0006] Preferably, the crustacean aquatic product is shrimp or crab.

[0007] More preferably, the crustacean aquatic products are Japanese crab, three-tuberculate swimming crab, olive green crab, Chinese mitten crab, deep-sea swimming crab, northern long-browed shrimp, Procambarus clarkii, American lobster, corrugated lobster, Japanese shrimp, whiteleg shrimp, Macrobrachium rosenbergii, giant tiger prawn, white shrimp and hair shrimp, etc.

[0008] The second aspect of the present invention provides the use of the above-mentioned DNA microbarcode in identifying the allergen species of crustacean aquatic products.

[0009] Preferably, the crustacean aquatic product is shrimp or crab.

[0010] More preferably, the crustacean aquatic products are Japanese crab, three-tuberculate swimming crab, olive green crab, Chinese mitten crab, deep-sea swimming crab, northern long-browed shrimp, Procambarus clarkii, American lobster, corrugated lobster, Japanese shrimp, whiteleg shrimp, Macrobrachium rosenbergii, giant tiger prawn, white shrimp and hair shrimp, etc.

[0011] The third aspect of the present invention provides a primer for identifying DNA micro-barcode 16SrRNA of crustacean aquatic product allergen species, the primer comprising:

[0012] Forward primer 16S rRNA-F: 5′-GGGACGATAAGACCCTATAAA-3′ (SEQ ID NO. 1); and

[0013] Reverse primer 16S rRNA-R: 5′-TAATTCAACATCGAGGTCGC-3′ (SEQ ID NO. 2).

[0014] Preferably, the crustacean aquatic product is shrimp or crab.

[0015] More preferably, the crustacean aquatic products are Japanese crab, three-tuberculate swimming crab, olive green crab, Chinese mitten crab, deep-sea swimming crab, northern long-browed shrimp, Procambarus clarkii, American lobster, corrugated lobster, Japanese shrimp, whiteleg shrimp, Macrobrachium rosenbergii, giant tiger prawn, white shrimp and hair shrimp, etc.

[0016] A fourth aspect of the present invention provides a kit for identifying allergen species of crustacean aquatic products, the kit comprising the above-mentioned DNA microbarcode 16S rRNA primers.

[0017] Preferably, the crustacean aquatic product is shrimp or crab.

[0018] More preferably, the crustacean aquatic products are Japanese crab, three-tuberculate swimming crab, olive green crab, Chinese mitten crab, deep-sea swimming crab, northern long-browed shrimp, Procambarus clarkii, American lobster, corrugated lobster, Japanese shrimp, whiteleg shrimp, Macrobrachium rosenbergii, giant tiger prawn, white shrimp and hair shrimp, etc.

[0019] The fifth aspect of the present invention provides the use of the above primers in the preparation of a kit for identifying allergen species of crustacean aquatic products.

[0020] Preferably, the crustacean aquatic product is shrimp or crab.

[0021] More preferably, the crustacean aquatic products are Japanese crab, three-tuberculate swimming crab, olive green crab, Chinese mitten crab, deep-sea swimming crab, northern long-browed shrimp, Procambarus clarkii, American lobster, corrugated lobster, Japanese shrimp, whiteleg shrimp, Macrobrachium rosenbergii, giant tiger prawn, white shrimp and hair shrimp, etc.

[0022] A sixth aspect of the present invention provides a method for identifying species of crustacean aquatic product allergens, the method comprising the following steps:

[0023] (1) Extracting DNA from the species to be tested;

[0024] (2) performing PCR amplification on the extracted DNA using the above primers to obtain an amplified product;

[0025] (3) The amplified products were sequenced and spliced ​​to obtain complete COI and 16S rRNA sequences. The processed sequences were searched for similarity in the Blast database (http: / / blast.ncbi.nlm.nih.gov) of the National Center for Biotechnology Information (NCBI) of the United States to confirm the species of crustacean aquatic product allergens.

[0026] Preferably, the crustacean aquatic product is shrimp or crab.

[0027] More preferably, the crustacean aquatic products are Japanese crab, three-tuberculate swimming crab, olive green crab, Chinese mitten crab, deep-sea swimming crab, northern long-browed shrimp, Procambarus clarkii, American lobster, corrugated lobster, Japanese shrimp, whiteleg shrimp, Macrobrachium rosenbergii, giant tiger prawn, white shrimp and hair shrimp, etc.

[0028] The technical effects produced by the present invention are:

[0029] Compared with the traditional COI animal universal barcode, the 16S rRNA microbarcode used in the present invention can be better used for the identification of crustacean aquatic product allergens in commercially available foods. It can not only meet the detection needs of market regulatory departments and food production companies, but also provide consumers with more comprehensive food allergen information. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the electrophoresis of the PCR amplification product of the COI fragment of crustacean aquatic product allergen (M: Marker; CK: blank control; 1: Swimming crab trituberculatus; 2: Green crab; 3: Eriocheir sinensis; 4: Swimming crab; 5: Northern long-necked shrimp; 6: Procambarus clarkii; 7: Macrobrachium rosenbergii; 8: American lobster; 9: Homarus americanus; 10: Japanese crab; 11: Japanese shrimp; 12: Penaeus vannamei; 13: Penaeus monodon; 14: White shrimp; 15: Hair shrimp).

[0031] Figure 2This is the electrophoresis of the PCR amplification product of the 16S rRNA fragment of the crustacean aquatic product allergen (M: Marker; CK: blank control; 1: Swimming crab trituberculatus; 2: Green crab; 3: Eriocheir sinensis; 4: Swimming crab; 5: Northern long-necked shrimp; 6: Procambarus clarkii; 7: Macrobrachium rosenbergii; 8: American lobster; 9: Homarus americanus; 10: Japanese crab; 11: Japanese shrimp; 12: Litopenaeus vannamei; 13: Penaeus monodon; 14: White shrimp; 15: Hair shrimp).

[0032] Figure 3 It is the electrophoresis of the PCR amplification product of COI fragment of commercially available food of crustacean aquatic product allergen (M: Marker; CK: blank control; 1: gold label crab paste; 2: crab claws with sauce; 3: drunken crab with Huadiao wine; 4: small green dragon with garlic sauce; 5: steamed Boston lobster; 6: garlic prawns; 7: spicy crab chunks; 8: spicy crayfish; 9: garlic butter lobster; 10: nine-section dried shrimp; 11: grilled shrimp; 12: deep-fried prawns; 13: spicy crab chunks; 14: frozen Arctic sweet shrimp);

[0033] Figure 4 It is the electrophoresis of the PCR amplification products of the 16S rRNA fragment of the crustacean aquatic product allergen commercially available food (M: Marker; CK: blank control; 1: Jinbiao crab paste; 2: crab claws with sauce; 3: Huadiao drunken crab; 4: small green dragon with garlic sauce; 5: steamed Boston lobster; 6: garlic prawns; 7: spicy crab chunks; 8: spicy crayfish; 9: garlic butter lobster; 10: nine-section dried shrimp; 11: grilled shrimp; 12: deep-fried prawns; 13: spicy crab chunks; 14: frozen Arctic sweet shrimp);

[0034] Figure 5 The COI gene sequence of the crustacean aquatic product allergen species;

[0035] Figure 6 It is the 16S rRNA gene sequence of crustacean aquatic product allergen species. DETAILED DESCRIPTION

[0036] The present invention is further described below in conjunction with examples, but the embodiments of the present invention are not limited thereto. The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0037] Experimental Example 1: Detection method of crustacean aquatic product allergens based on 16S rRNA microbarcode

[0038] 1. Test materials

[0039] Common crustacean allergens were collected, and commercially available foods containing a single target allergen were purchased. Before grinding each sample in a ball mill, the sample surface was rinsed 2-3 times with double distilled water and anhydrous ethanol to remove impurities and avoid fungal contamination during the amplification process. Finally, the ground samples were transferred to centrifuge tubes, labeled, and stored in a -20°C refrigerator for later use.

[0040] 2. Test methods

[0041] 2.1 DNA extraction

[0042] 2.1.1 DNA extraction from raw materials

[0043] The genomic DNA of the raw material samples was extracted using the Animal Genomic DNA Kit (Tiangen Biotechnology Co., Ltd., China). The excess salt ions in the DNA extraction process were eluted using the rinse buffer PW in the kit. The operation was repeated twice, and finally the DNA was eluted twice with 100 μL elution buffer TE. Finally, the absorbance values ​​at A230, A260, and A280 were measured using a NanoDrop ONE spectrophotometer to determine the DNA concentration of the extracted samples. All genomic DNA samples were stored at -20°C for future use.

[0044] 2.1.2 DNA extraction from commercially available foods

[0045] use The genomic DNA of processed foods containing a single crustacean allergen was extracted using the Food kit (Machereye-Nagel, Germany). The DNA lysis time was 60 min, and the DNA was eluted twice using 100 μL elution buffer CE. Finally, the absorbance values ​​at A230, A260, and A280 were measured using a NanoDrop ONE spectrophotometer to determine the DNA concentration of the extracted samples. All genomic DNA samples were stored at -20°C for future use.

[0046] 2.2 PCR amplification

[0047] 2.2.1. PCR amplification primers and screening:

[0048] The universal primers for 16S rRNA are shown in Table 1.

[0049] Table 1 Universal primers for 16S rRNA and COI

[0050]

[0051]

[0052] 2.2.2 PCR reaction system

[0053] The PCR reaction system was 25 μL, including 12.5 μL 2×Taq PCR StarMix, 0.5 μL each of upstream and downstream primers (10 μmol / mL), 1.5 μL DNA template (10 ng / μL), and the rest was made up to 25 μL with ddH2O. A negative control without DNA template was set up.

[0054] 2.2.3 PCR amplification procedure

[0055] Pre-denaturation at 94°C for 5 minutes; then 35 cycles were performed, each cycle including denaturation at 94°C for 30 seconds, annealing temperature at 58°C for 30 seconds, and then extension at 72°C for 45 seconds; finally, extension at 72°C for another 10 minutes.

[0056] PCR amplification was performed using a PCR thermal cycler, and the amplified products were subjected to gel electrophoresis using 1.5% agarose gel. The amplified products were analyzed using an ultraviolet gel imaging system to observe whether the amplified COI target band was 658 bp and whether the length of the 16S rRNA target band was 260 bp.

[0057] 2.3. Splicing 16S rRNA sequences

[0058] 2.3.1 Sequencing

[0059] The qualified amplified products were sequenced bidirectionally, and then the sequences were spliced ​​using SeqMan.

[0060] 2.3.2 BLAST analysis:

[0061] Blast comparison using complete COI and 16S rRNA sequences showed that all samples were target allergens.

[0062] 2.4. Verification of commercially available food

[0063] Commercially available foods containing a single crustacean allergen were purchased and identified using the above method. The results showed that 16S rRNA microbarcoding was more suitable for detecting crustacean allergens in commercially available foods.

[0064] 3. Test results

[0065] 3.1 PCR amplification results

[0066] Single raw material of crustacean allergens: The PCR amplification rate of COI sequence of crustacean allergens was 80%, and the PCR amplification rate of 16S rRNA sequence was 100%. The PCR products were detected by 1.5% agarose gel electrophoresis. The results showed that COI barcode could amplify the target band, but some of them had non-specific amplification, while the target bands amplified by 16S rRNA microbarcode were single and bright (see gel electrophoresis results for details). Figure 1 and 2 ).

[0067] Commercially available foods containing crustacean allergens: The PCR amplification rate of COI sequence in commercially available foods containing crustacean allergens was 64.29%, and the PCR amplification rate of 16S rRNA sequence was 100%. The PCR products were detected by 1.5% agarose gel electrophoresis. The results showed that the COI barcode was able to amplify 9 samples with target bands, but some had non-specific amplification; 5 samples failed to amplify successfully. The 16S rRNA microbarcode was able to amplify all commercially available foods containing crustacean allergens, and the target bands were all single and bright (see the gel electrophoresis results for details). Figure 3 and 4 ).

[0068] 3.2 Sequence Analysis

[0069] The products obtained by COI barcode amplification were purified by gel excision and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The products of 16S rRNA microbarcode amplification were directly sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results were spliced, and the length of the obtained complete COI sequence was about 658bp, and the length of the 16S rRNA sequence was about 260bp. The complete 16S rRNA sequence was used for Blast comparison in the GenBank database of NCBI. The results showed that for the single raw material of crustacean aquatic product allergens, all samples were the target allergens (the identification results are shown in Tables 2 and 3), the sequencing success rate of COI sequence was 80%, and the sequencing success rate of 16S rRNA sequence was 100%.

[0070] Table 2 Identification of crustacean allergens using COI barcodes

[0071]

[0072] Note: “——” indicates sequencing failure

[0073] Table 3 Identification of crustacean allergens using 16S rRNA microbarcoding

[0074]

[0075] Commercially available foods of crustacean aquatic product allergens: The sequencing success rate of COI sequence was 50%, and the sequencing success rate of 16S rRNA sequence was 100% (the identification results are shown in Table 4). Among them, the COI barcode can only detect spicy crab pieces, steamed Boston lobster, Huadiao drunken crab, crab claws with sauce, golden crab paste, frozen Arctic sweet shrimp and nine-section dried shrimp, a total of 7 commercially available foods. This may be because the processing methods of these 7 samples are relatively simple, and the degree of DNA degradation is low, so the longer COI fragments can also be sequenced successfully; the remaining 7 samples may be due to the more complex processing technology, high temperature baking, frying, or high pressure, etc., which makes the DNA breakage or degradation more serious, making it difficult to amplify the longer COI fragments. The identification success rate of 16S rRNA microbarcode reached 100%, which may be because the DNA fragments used are relatively short, although the DNA of processed foods is degraded, it can still be effectively amplified. The purchased spicy crab chunks, garlic-fried crab and crab claws were identified as crustaceans that were not target allergens, but the corresponding species could still be identified, indicating that the 16SrRNA microbarcode in the present invention has universal applicability in identifying crustaceans.

[0076] Table 4 Identification of crustacean allergens in commercial foods using COI and 16S rRNA barcoding Note: “——” indicates sequencing failure

[0077] In summary, the 16S rRNA microbarcode of the present invention can identify crustacean allergens composed of a single sample, and the result is accurate and efficient, and can be applied to commercially available foods containing a single crustacean allergen.

[0078] Experimental Example 2: 16S rRNA microbarcode and primer screening test

[0079] COI gene and 16S rRNA gene are widely used in the classification and identification analysis of animal species in DNA barcoding technology because of their strong interspecies identification ability. COI gene is a protein coding gene of mitochondrial genome, and its full name is cytochrome c oxidase subunit I. 16S rRNA gene is the DNA sequence encoding 16S rRNA on ribosome. In this test case, the COI sequence and 16S rRNA sequence of all crustacean aquatic product allergen species involved were downloaded from the National Center for Biotechnology Information (https: / / www.ncbi.nlm.nih.gov / ) of the United States, and manually compared by MEGA 11.0 software to observe whether it can be used to design microbarcode universal primers.

[0080] Through comparison, it was found that there is no conserved region in the COI region of crustacean allergen species for designing universal primers (such as Figure 5 By comparing the 16S rRNA regions of crustacean allergen species, there are conserved regions for designing universal primers (such as Figure 6 As shown), we can try to design relevant micro-barcode universal primers.

[0081] Principles of 16S rRNA barcode design: All DNA sequences of crustacean aquatic product allergen species were downloaded from the National Center for Biotechnology Information (NCBI) and manually aligned using MEGA 11.0 software. According to the principles of primer design, a total of 6 pairs of universal primers that met the requirements were designed, as shown in Table 5.

[0082] Table 5 Primer information for PCR amplification of crustacean allergens

[0083]

[0084] As shown in Table 5 above, the species product amplified by the 16S rRNA primer No. 1 has an appropriate length (the product length is 200-400 bp, which is the optimal length for the microbarcode), but its downstream primer has a lower GC content than other downstream primers. The low GC content is not conducive to initiating the reaction, so the primer No. 1 is not the best choice for the 16S rRNA primer.

[0085] The species product amplified by the 16S rRNA primer No. 2 has an appropriate length, and its downstream primer has a moderate GC content compared with other downstream primers. Therefore, the primer No. 2 is the best choice for 16S rRNA primers.

[0086] The species product amplified by the 16S rRNA primer No. 3 has an appropriate length, but its downstream primer has the lowest GC content compared with other downstream primers. Low GC content is not conducive to initiating the reaction. Therefore, the primer No. 3 is not the best choice for 16S rRNA primers.

[0087] The species product amplified by the 16S rRNA primer No. 4 is too short (60 bp), which will result in low species resolution and may not be able to distinguish closely related species, so it is excluded.

[0088] The species product amplified by the 16S rRNA primer No. 5 is too short (130 bp), which will result in low species resolution and may not be able to distinguish closely related species, so it is excluded.

[0089] The species product amplified by the 16S rRNA primer with sequence number 6 is too short (72bp), which will result in low species resolution and may not be able to distinguish closely related species, so it is excluded.

[0090] In summary, the 16S rRNA primer No. 2 was finally selected for subsequent identification of crustacean aquatic product allergen species.

[0091] Although specific embodiments of the present invention have been described, it will be appreciated by those skilled in the art that various changes and modifications may be made to the present invention without departing from the scope or spirit of the present invention. Therefore, the present invention is intended to cover all such changes and modifications that fall within the scope of the appended claims and their equivalents.

Claims

1. A DNA microbarcode for identifying allergen species in crustacean aquatic products, characterized in that: The DNA microbarcode is an animal mitochondrial genome sequence fragment 16SrRNA.

2. Application of DNA microbarcodes in identifying allergen species of crustacean aquatic products, characterized in that: The DNA microbarcode is an animal mitochondrial genome sequence fragment 16SrRNA.

3. A DNA microbarcode 16SrRNA primer for identifying crustacean aquatic product allergen species, characterized in that: The primers include: Forward primer 16SrRNA-F: 5′-GGGACGATAAGACCCTATAAA-3′ (SEQ ID NO. 1); and Reverse primer 16SrRNA-R: 5′-TAATTCAACATCGAGGTCGC-3′ (SEQ ID NO. 2).

4. A kit for identifying allergen species of crustacean aquatic products, characterized in that: The kit comprises the primers for the DNA microbarcode 16SrRNA according to claim 3.

5. Use of the DNA microbarcode 16SrRNA primers according to claim 3 in the preparation of a kit for identifying allergen species of crustacean aquatic products.

6. A method for identifying allergen species of crustacean aquatic products, characterized in that: The method comprises the following steps: (1) Extracting DNA from the species to be tested; (2) performing PCR amplification on the extracted DNA using the primers described in claim 3 to obtain an amplified product; (3) The amplified products were sequenced and spliced ​​to obtain complete COI and 16S rRNA sequences. The processed sequences were searched for similarity in the Blast database of the National Center for Biotechnology Information (NCBI) to confirm the species of crustacean aquatic product allergens.

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