Plant-derived food allergen identification method based on trnL (UAA) micro bar code

Abstract

The invention relates to a DNA (Deoxyribose Nucleic Acid) micro-bar code for identifying common plant-derived food allergen species, the DNA micro-bar code is a chloroplast genome sequence fragment trnL (UAA), and primers of the DNA micro-bar code trnL (UAA) are shown as SEQ ID NO.1 and SEQ ID NO.2. The invention further relates to a method for identifying the plant-derived food allergen species. By adopting the trnL (UAA) bar code identification method provided by the invention, most target allergens can be identified, meanwhile, the detection requirements of market supervision departments and food production enterprises can be met, more comprehensive food allergen information can be provided for consumers, and allergic events are reduced.

Description

Technical Field

[0001] The present invention relates to the field of food analysis, and in particular to a method for identifying plant-derived food allergens. Background Art

[0002] Plant-derived food allergens are considered to be the most important source of food allergy reactions and the most common cause of systemic allergic reactions and fatal allergic reactions in humans. Plant-derived food allergens mainly include sesame, nuts, cereals, beans, etc. When food allergy patients come into contact with allergenic ingredients, they are generally accompanied by acute systemic allergic reactions, and severe cases may even cause shock and death. Currently, the prevalence of food allergies is on the rise worldwide. The Food and Agriculture Organization of the United Nations has identified it as a global food safety and public health issue, which seriously affects people's daily lives.

[0003] Usually, food allergens mainly come from food ingredients, food sources, and cross-contamination during production and transportation. In actual processing and production, the initial form of the raw materials is usually destroyed, which brings difficulties to morphological identification. In addition, polyphenols, polysaccharides, lipids and other substances exist in certain organs of the target allergens, and complex food processing technology can also cause DNA degradation, which brings certain difficulties to DNA extraction and PCR amplification. Therefore, it is of great significance to establish an accurate, highly sensitive, highly stable and easy-to-operate allergen detection method.

[0004] DNA barcoding technology has been a hot topic and research direction in biological classification and identification in recent years. DNA barcoding technology is based on traditional DNA sequence labeling technology. It can accurately identify samples by PCR amplification and sequencing of a pair of short homologous DNA sequences from different individuals. The trnL (UAA) gene is located in the chloroplast genome of plants and has a highly conserved region. It can amplify highly degraded DNA samples and has successfully identified a variety of Chinese herbal medicines. Although the chloroplast genomes of common plant-derived food allergen species have been revealed, the use of DNA microbarcoding to identify common plant-derived food allergens has not been reported. Summary of the invention

[0005] To solve the above technical problems, the present invention includes the following aspects:

[0006] A first aspect of the present invention provides a DNA microbarcode for identifying common plant-derived food allergen species. The DNA microbarcode is a chloroplast genome sequence fragment trnL (UAA) of common plant-derived food allergen species.

[0007] Preferably, the common plant-derived food allergen species include white kidney beans, chickpeas, lupines, white beans, lentils, sesame seeds, flax seeds, mustard seeds, soybeans, peanuts, mung beans, peas, sweet buckwheat, bitter buckwheat, barley, wheat, oats, macadamia nuts, cashews, almonds, almonds, pecans, walnuts, pistachios, hazelnuts and chestnuts, etc.

[0008] The second aspect of the present invention provides the use of the above-mentioned DNA microbarcode in identifying common plant-derived food allergen species.

[0009] Preferably, the common plant-derived food allergen species include white kidney beans, chickpeas, lupines, white beans, lentils, sesame seeds, flax seeds, mustard seeds, soybeans, peanuts, mung beans, peas, sweet buckwheat, bitter buckwheat, barley, wheat, oats, macadamia nuts, cashews, almonds, almonds, pecans, walnuts, pistachios, hazelnuts and chestnuts, etc.

[0010] The third aspect of the present invention provides a primer for identifying the DNA microbarcode trnL (UAA) of common plant-derived food allergen species:

[0011] Forward primer trnL(UAA)-F: 5'-GAGACTCAATGGAAGCTGTTCT-3' (SEQ ID NO. 1),

[0012] Reverse primer trnL(UAA)-R: 5′-TTGACACGTAGAATGGGACTCT-3′ (SEQ ID NO. 2).

[0013] Preferably, the common plant-derived food allergen species include white kidney beans, chickpeas, lupines, white beans, lentils, sesame seeds, flax seeds, mustard seeds, soybeans, peanuts, mung beans, peas, sweet buckwheat, bitter buckwheat, barley, wheat, oats, macadamia nuts, cashews, almonds, almonds, pecans, walnuts, pistachios, hazelnuts and chestnuts, etc.

[0014] A fourth aspect of the present invention provides a kit for identifying common plant-derived food allergen species, the kit comprising primers of the above-mentioned DNA microbarcode trnL(UAA).

[0015] Preferably, the common plant-derived food allergen species include white kidney beans, chickpeas, lupines, white beans, lentils, sesame seeds, flax seeds, mustard seeds, soybeans, peanuts, mung beans, peas, sweet buckwheat, bitter buckwheat, barley, wheat, oats, macadamia nuts, cashews, almonds, almonds, pecans, walnuts, pistachios, hazelnuts and chestnuts, etc.

[0016] The fifth aspect of the present invention provides the use of the above primers in preparing a kit for identifying common plant-derived food allergen species.

[0017] Preferably, the common plant-derived food allergen species include white kidney beans, chickpeas, lupines, white beans, lentils, sesame seeds, flax seeds, mustard seeds, soybeans, peanuts, mung beans, peas, sweet buckwheat, bitter buckwheat, barley, wheat, oats, macadamia nuts, cashews, almonds, almonds, pecans, walnuts, pistachios, hazelnuts and chestnuts, etc.

[0018] The technical effects produced by the present invention are:

[0019] Plant-derived food allergen raw materials in commercially available foods do not have complete morphological characteristics (such as wheat used to make biscuits), making them difficult to identify by morphology. Complex food processing techniques lead to DNA degradation and polyphenols and polysaccharides in the raw materials also make DNA extraction and PCR amplification difficult. In view of the above problems, the trnL (UAA) microbarcode identification method of the present invention can identify most of the target allergens, meet the testing needs of market regulatory authorities and food production companies, and provide consumers with more comprehensive food allergen information to reduce the occurrence of allergic events. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The electrophoresis diagram of PCR amplification products of common plant-derived food allergens (M: Marker; CK: blank control; 1: white kidney bean; 2: chickpea; 3: lupine; 4: white bean; 5: lentil; 6: sesame; 7: flax seed; 8: mustard seed; 9: soybean; 10: peanut; 11: mung bean; 12: pea; 13: sweet buckwheat; 14: bitter buckwheat; 15: barley; 16: wheat; 17: oat; 18: macadamia; 19: cashew; 20: almond; 21: almond; 22: pecan; 23: walnut; 24: pistachio; 25: hazelnut; 26: chestnut);

[0021] Figure 2 The NJ tree was constructed based on the trnL (UAA) sequences of common plant-derived food allergens;

[0022] Figure 3 is the sequence map of rbcL gene of barley and wheat;

[0023] Figure 4 The rbcL gene sequence diagram of sweet buckwheat and bitter buckwheat;

[0024] Figure 5 The matK gene sequence map of barley and wheat;

[0025] Figure 6 The matK gene sequence map of sweet buckwheat and bitter buckwheat;

[0026] Figure 7The terminal sequence map of the matK gene of barley and wheat;

[0027] Figure 8 The psbA-trnH gene sequence map of barley and wheat;

[0028] Fig. 9 The psbA-trnH gene sequence diagram of sweet buckwheat and bitter buckwheat;

[0029] Fig.10 The conserved sequence of the trnL (UAA) gene of barley and wheat;

[0030] Fig.11 The conserved sequence of trnL (UAA) gene of sweet buckwheat and bitter buckwheat;

[0031] Fig.12 The trnL (UAA) gene sequence map of barley and wheat;

[0032] Fig.13 This is the trnL (UAA) gene sequence map of sweet buckwheat and bitter buckwheat. DETAILED DESCRIPTION

[0033] 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.

[0034] Experimental Example 1: Application of trnL (UAA) gene as DNA microbarcode in identifying common plant-derived food allergen species

[0035] 1. Test materials

[0036] Common plant-derived food allergens were collected, and commercially available foods containing a single target allergen were purchased. Each sample was crushed using a grinder (to reduce cross contamination, the grinder was cleaned with distilled water and wiped dry with kitchen paper after processing each sample). The crushed sample powder was transferred to a centrifuge tube, labeled, and stored in a -20°C refrigerator for later use.

[0037] 2. Test methods

[0038] 2.1 DNA extraction

[0039] 2.1.1 DNA extraction from raw materials

[0040] Genomic DNA of raw material samples was extracted using a plant genomic DNA kit (Tiangen Biotechnology Co., Ltd.). Nuts, peanuts, and sesame samples were lysed for 60 min, and 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.

[0041] 2.1.2 DNA extraction from commercially available foods

[0042] use The genomic DNA of processed foods containing a single 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.

[0043] 2.2 PCR amplification

[0044] 2.2.1. PCR amplification primers and screening

[0045] The universal primers for trnL(UAA) are shown in Table 1.

[0046] Table 1 trnL (UAA) universal primer

[0047]

[0048] 2.2.2 PCR reaction system

[0049] 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), 10 ng DNA template, and the rest was made up to 25 μL with ddH2O. A negative control without DNA template was set up.

[0050] 2.2.3 PCR amplification procedure

[0051] 94℃, 5min; 94℃, 30s; 58℃, 30s; 72℃, 45s, 30 cycles; 72℃, 10min.

[0052] The PCR products were detected by 1.5% agarose gel electrophoresis and the amplification results were observed under an ultraviolet gel imaging system. The amplification results showed that the length of the trnL (UAA) sequence was between 100-400 bp.

[0053] 2.3. Splicing trnL (UAA) sequence

[0054] 2.3.1 Sequencing

[0055] The qualified amplified products were sequenced bidirectionally, and then the sequences were spliced ​​using SeqMan.

[0056] 2.3.2 BLAST analysis

[0057] Blast comparison using the complete trnL(UAA) sequence showed that all samples were target allergens.

[0058] 2.4. Verification of commercially available food

[0059] Commercially available foods containing a single target allergen were purchased and identified using the above method. The results showed that the target allergen could be detected in commercially available foods.

[0060] 3. Test results

[0061] 3.1 Sequence analysis

[0062] The PCR amplification rate of trnL (UAA) sequences of various common plant-derived food allergens was 100%. The PCR products were detected by 1.5% agarose gel electrophoresis, and the results showed that all samples were able to amplify a single, bright band (see the gel electrophoresis results for details). Figure 1 ). The sequencing results were spliced, and the length of the obtained complete trnL (UAA) sequence was between 100-400 bp. The complete trnL (UAA) sequence was used for Blast comparison. The results showed that all samples were target allergens (identification results are shown in Table 2), and the sequencing success rate was 96.15%. The specific results are shown in Table 2.

[0063] Table 2 Identification results of common plant-derived food allergens

[0064]

[0065]

[0066] Note: “—” indicates sequencing failure

[0067] 3.2 Analysis of genetic distances between and within species

[0068] MEGA11.0 software was used to calculate the genetic distances within and between species for the trnL (UAA) sequences of all common plant-derived food allergens based on the K2P model, and the results are shown in Table 3. The average genetic distance within the species was 0.0000, and the average genetic distance between species was 0.0122-1.0641, of which the maximum genetic distance within the species was 0.0000, and the minimum genetic distance between species was 0.0122. The maximum genetic distance within the species was smaller than the minimum genetic distance between species, and there was a barcoding gap. Therefore, the trnL (UAA) sequence was able to distinguish the target allergens in this study.

[0069] Table 3 Analysis of genetic distances between and within common plant-derived food allergens

[0070]

[0071]

[0072] The K2P model was used to construct the NJ developmental tree (e.g. Figure 2 The phylogenetic tree constructed based on trnL(UAA) sequences can separate common plant-derived food allergen species. Figure 2 It can be seen that barley, wheat, and oats all belong to the Poaceae family and are clustered into a small branch, almonds and almonds all belong to the Rosaceae family and are clustered into a small branch, sweet buckwheat and bitter buckwheat all belong to the Polygonaceae family and are clustered into a small branch, and peas and lentils all belong to the Leguminosae family and are clustered into a small branch, indicating that their trnL (UAA) gene sequences are highly similar and closely related. Mung beans and white kidney beans all belong to the Leguminosae family and are clustered into one branch, and white kidney beans, peas, and lentils all belong to the Leguminosae family and are clustered into one branch, indicating that their trnL (UAA) gene sequences are highly similar and closely related.

[0073] 3.3 Analysis of processed food

[0074] For the common plant-derived food allergens selected in this test example, corresponding processed foods containing single allergens were selected for verification, and the sequencing success rate reached 92% (specific results are shown in Table 4).

[0075] Table 4 Identification results of processed foods containing single plant-derived food allergens

[0076]

[0077] Note: “—” indicates sequencing failure

[0078] According to the sequencing results, the trnL (UAA) sequences of the purchased common plant-derived food allergens have a similarity of more than 99% with their control sequences. Therefore, the trnL (UAA) barcode sequence obtained by the present invention can be used as a standard detection sequence for a variety of common plant-derived food allergen identification methods, and can accurately, stably and efficiently identify a variety of common plant-derived food allergens.

[0079] Test Example 2: DNA barcode screening test

[0080] 1. Screening of commonly used DNA barcode types

[0081] Commonly used plant DNA barcodes include rbcL, trnL (UAA), matK, psbA-trnH, etc. Therefore, all relevant DNA sequences of common plant-derived food allergen species were downloaded from the National Center for Biotechnology Information (NCBI) and manually aligned using Mega 11.0 software.

[0082] rbcL region: It exists in the circular chromosome of the chloroplast of plant cells and is part of the genome. It is 1300bp in length. The primers are highly universal and easy to amplify, but the evolution rate is slow. It is mainly used for identification research at the family and genus level. In this test example, the rbcL sequences of some species of commonly used plant-derived food allergens are highly conserved among species, and there are conserved regions for designing universal primers. However, the sequences of some species, such as sweet buckwheat and bitter buckwheat, barley and wheat, are extremely similar (e.g. Figure 3 and 4 Therefore, it is not possible to design rbcL barcodes to identify common plant-derived food allergen species.

[0083] matK region: Located in the intron between two highly conserved exons of the chloroplast lysine tRNA gene (trnK), the sequence length is about 1500bp, it is a single-copy coding gene, the primers have poor universality, and different primers are often required in different groups, and the amplification efficiency is low. In this test example, the matK sequences of some commonly used plant-derived food allergen species are highly conserved among species, and there are conserved regions for designing universal primers, but the sequences of some species such as sweet buckwheat and bitter buckwheat, barley and wheat species are extremely similar (such as Figure 5 and 6 The difference between barley and wheat sequences is high at the terminal of matK region (as shown in Figure 7 However, there is no conserved region and primers cannot be designed. Therefore, it is not possible to design matK barcodes for identifying common plant-derived food allergen species.

[0084] psbA-trnH region: a non-coding region between the chloroplast genes psbA and trnH. The length of the sequences obtained after amplification of most plants is between 340-660bp, and there are a large number of insertion / deletion regions, which makes it difficult to compare species of different genera. In this test example, the psbA-trnH sequences of some common plant-derived food allergen species are highly conserved among species, and there are conserved regions for designing universal primers, but the sequences of some species such as sweet buckwheat and bitter buckwheat, barley and wheat are extremely similar (e.g. Figure 8 and 9 Therefore, it is not possible to design a psbA-trnH barcode to identify common plant-derived food allergen species.

[0085] trnL (UAA) region: Located in the intron region of the chloroplast gene trnL, it belongs to the non-coding region, with a sequence length of 254-767bp, and has a conserved region and an alternate variable region. In this test example, the trnL (UAA) sequences of commonly used plant-derived food allergen species have highly conserved regions (such as Fig.10 and 11 As shown in ), universal primers can be designed. At the same time, the sequence differences between species are large, such as barley and wheat, sweet buckwheat and bitter buckwheat (as shown in Fig.12 and 13 ), predicted to be distinguishable by trnL(UAA) sequences. Therefore, trnL(UAA) barcodes can be designed to identify common plant-derived food allergen species.

[0086] 2. Barcode design principles for trnL(UAA)

[0087] All DNA sequences of common plant-derived food 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.

[0088] Table 5 Primer information for PCR amplification of common plant-derived food allergens

[0089]

[0090] The trnL (UAA) primer with sequence number 1 is highly conserved among some species. For example, the sequences of sweet buckwheat and bitter buckwheat, barley and wheat are extremely similar and cannot be distinguished by primers, so it is excluded.

[0091] The trnL (UAA) primer with serial number 2 has large sequence differences among species, but the product is long (500-600bp) and is not suitable for the detection of commercially available foods containing common plant-derived food allergens, so it is excluded.

[0092] The trnL (UAA) primer with sequence number 3 has large sequence differences among species, but the product is long (700 bp) and is not suitable for the detection of commercially available foods containing common plant-derived food allergens, so it is excluded.

[0093] The trnL (UAA) primer with sequence number 4 has a product length of 100-400bp and a moderate sequence length. It is suitable for the detection of commercially available foods containing common plant-derived food allergens, and the sequences vary greatly among species, so it can be selected.

[0094] The trnL (UAA) primer with sequence number 5 has large sequence differences among species, but the product is long (700 bp) and is not suitable for the detection of commercially available foods containing common plant-derived food allergens, so it is excluded.

[0095] The trnL (UAA) primer with sequence number 6 is highly conserved among some species. For example, the sequences of sweet buckwheat and bitter buckwheat, barley and wheat are extremely similar and cannot be distinguished by primers, so it is excluded.

[0096] In summary, the trnL (UAA) primer with sequence number 4 was selected for the identification of common plant-derived food allergens.

[0097] 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 common plant-derived food allergen species, characterized in that: The DNA microbarcode is a chloroplast genome sequence fragment trnL (UAA) of a common plant-derived food allergen species.

2. Application of DNA barcoding in identifying common plant-derived food allergen species, characterized in that: The DNA microbarcode is a chloroplast genome sequence fragment trnL (UAA) of a common plant-derived food allergen species.

3. A primer for identifying DNA microbarcode trnL (UAA) of common plant-derived food allergen species, characterized in that: The primers include: Forward primer trnL(UAA)-F: 5'-GAGACTCAATGGAAGCTGTTCT-3' (SEQ ID NO. 1), and reverse primer trnL(UAA)-R: 5'-TTGACACGTAGAATGGGACTCT-3' (SEQ ID NO. 2).

4. A kit for identifying common plant-derived food allergen species, characterized in that: The kit comprises the primers of the DNA microbarcode trnL (UAA) according to claim 3.

5. Use of the primers of the DNA microbarcode trnL (UAA) according to claim 3 in preparing a kit for identifying common plant-derived food allergen species.

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