Plant-derived allergen psbA-trnH bar code detection method, system and application

Abstract

The invention discloses a plant-derived allergen psbA-trnH bar code detection method and system and application, and the method comprises the following steps: step 1, crushing or homogenizing a food sample to be detected, and extracting corresponding plant-derived total DNA by using a DNA extraction kit; step 2, carrying out PCR (Polymerase Chain Reaction) amplification on the extracted plant-derived total DNA by using a psbA-trnH bar code universal primer, so as to obtain an amplification product of a psbA-trnH bar code specific DNA fragment; step 3, constructing a high-throughput sequencing library by using an amplification product of the psbA-trnH bar code specific DNA fragment, and carrying out double-end sequencing by adopting an Illumina platform to obtain original sequencing data; and step 4, comparing the original sequencing data with the plant allergen database to obtain various allergen components in the food sample to be detected. The invention aims to realize rapid and accurate detection of various plant-derived allergens.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of food detection, in particular to a plant-derived allergen psbA-trnH barcode detection method, system and application. BACKGROUND

[0002] Plant-derived food allergens are an important global public health problem, with increasing incidence rates that can lead to severe allergic reactions and even death. Common allergens include nuts, peanuts, soybeans, etc., which are often hidden or mislabeled in processed foods, posing a risk to consumer health. Existing allergen detection technologies mainly include proteomics technology and immunodetection methods (such as ELISA), which can identify allergenic proteins but are affected by protein conformation and are prone to failure after high-temperature and high-pressure processing, and cannot detect trace amounts or degraded components. PCR technology: detection is achieved by amplifying DNA fragments, but traditional PCR methods are usually targeted at a single species, lacking the ability to simultaneously screen multiple species. DNA barcode technology: uses short DNA sequences for species identification, with high specificity, standardization, and high-throughput advantages, and has been widely used in food ingredient identification such as meat, nuts, and fish. However, existing DNA barcodes (such as rbcL, matK, psbA-trnH) have limitations in plant-derived allergen identification: some barcodes (such as rbcL) evolve slowly and have small inter-species differences, making it difficult to distinguish closely related species (such as almond and apricot kernel, walnut and hickory). Universal primer design is difficult, with low amplification efficiency, especially when dealing with degraded DNA samples, the performance is unstable. Lack of systematic comparative studies makes it difficult to determine the optimal barcode for multi-species allergen identification. Regulatory requirements are becoming increasingly stringent, such as the European Union, the United States, and China all require accurate labeling of allergens on food labels. The existing CN106119379A patent discloses a peanut DNA barcode standard detection gene and a molecular identification method for peanut species, which develops a peanut-specific barcode sequence and primer to avoid cross-reactions and verifies processed foods such as peanut butter and sesame paste, but is only targeted at peanuts, with a narrow range and unable to efficiently handle complex food matrices and multi-component detection. Therefore, there is an urgent need for an optimized method based on DNA barcodes to quickly and accurately detect multiple plant-derived allergens. SUMMARY

[0003] The main purpose of the present application is to provide a plant-derived allergen psbA-trnH barcode detection method, system and application, which aims to solve the technical problem of the inability to simultaneously detect multiple species of allergens in existing foods.

[0004] To achieve the above-mentioned purpose, the present application provides a plant-derived allergen psbA-trnH barcode detection method, which comprises the following steps:

[0005] Step 1, crushing or homogenizing the food sample to be tested, and extracting the corresponding total plant-derived DNA using a DNA extraction kit;

[0006] Step 2, PCR amplification of the extracted total plant-derived DNA using psbA-trnH barcode universal primers to obtain the amplification product of psbA-trnH barcode-specific DNA fragments;

[0007] Step 3, constructing a high-throughput sequencing library for the amplification product of psbA-trnH DNA fragments, and performing double-end sequencing using the Illumina platform to obtain raw sequencing data;

[0008] Step 4, aligning the raw sequencing data with a plant allergen database to obtain multiple allergen components in the food sample to be tested.

[0009] Optionally, in step 2, the sequence pair of the primers is shown in SEQ ID NO: 1 for the forward primer and SEQ ID NO: 2 for the reverse primer. Forward primer: 5'-GTTATGCATGAACGTAATGCTC-3',

[0010] Reverse primer: 5'-CGCGCATGGTGGATTCACAATCC-3'

[0011] Optionally, in step 2, the reaction program for PCR amplification is as follows: 94℃ pre-denaturation for 5 min; 35 cycles of 94℃ denaturation for 30 s, 58-62℃ annealing for 30 s, 72℃ extension for 45 s; and 72℃ final extension for 10 min.

[0012] Optionally, in step 2, the PCR reaction system is Premix Ex Taq TM Premix 12.5 μL, 0.5 μL of each of the upstream and downstream primers, 5 μL of DNA template, and supplemented with sterile double-distilled water to 25 μL.

[0013] Optionally, in step 3, the construction of the sequencing library includes nucleic acid purification, PCR amplification, and purification steps.

[0014] Optionally, in step (4), the plant allergen database is NCBI RefSeq or a custom database, the alignment tool is BLAST algorithm, and the output result includes species confidence.

[0015] Optionally, the allergen component comprises one or more of hazelnut, brazil nut, macadamia nut, walnut, pecan, almond, pistachio, cashew, pistachio, peanut and soybean. In addition, in order to achieve the above-mentioned purpose, the present application also provides a system for realizing the plant-derived allergen psbA-trnH barcode detection method, which comprises a DNA extraction module, a PCR amplification module, a sequencing library construction module and a data analysis module:

[0016] The DNA extraction module comprises a plant DNA extraction reagent for extracting total plant DNA from a food sample;

[0017] The PCR amplification module comprises a psbA-trnH barcode universal primer pair and a PCR reaction solution;

[0018] The sequencing library construction module comprises high-throughput sequencing library construction reagents for nucleic acid purification, PCR amplification and purification of PCR amplification products;

[0019] The data analysis module comprises a plant allergen database and sequence alignment software for identifying multiple allergen components.

[0020] Optionally, the PCR amplification module further comprises a positive control unit containing mixed plasmid DNA of hazelnut, brazil nut, macadamia nut, walnut, pecan, almond, pistachio, cashew, pistachio, peanut and soybean psbA-trnH sequences.

[0021] In addition, in order to achieve the above-mentioned purpose, the present application also provides the use of psbA-trnH barcode in the preparation of a kit for the simultaneous detection of multiple plant-derived allergen components in food.

[0022] Advantages:

[0023] The present application combines psbA-trnH barcode detection with high-throughput sequencing technology for actual sample detection, solving the limitations of traditional identification methods (such as the inability to distinguish between closely related species), and supporting simultaneous detection of multiple species, achieving simultaneous detection of multiple allergen components. Furthermore, the barcode detection method established by the present application provides a powerful tool for allergen control in the global food supply chain, and is expected to promote the widespread application of DNA molecular detection technology in the field of food authenticity and safety, and help regulatory agencies around the world to improve food safety and security. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 The flowchart of an embodiment of the plant-derived allergen psbA-trnH barcode detection method of the present application.

[0025] Figure 2 The electrophoresis results of different allergen components are shown in the schematic diagram.

[0026] Figure 3 psbA-trnH NJ tree for allergen species;

[0027] Figure 4 trnl NJ tree for allergen species;

[0028] Figure 5 rbcl NJ tree for allergen species.

[0029] The implementation, functional features and advantages of the present application will be further described with reference to the embodiments and the accompanying drawings. DETAILED DESCRIPTION

[0030] It should be understood that the specific embodiments described herein are merely illustrative of the present application and are not intended to limit the present application.

[0031] Referring to Figure 1 The present application provides a plant-derived allergen psbA-trnH barcode detection method, which comprises the following steps:

[0032] Step 1, crushing or homogenizing the food sample to be tested, and extracting the corresponding plant-derived total DNA using a DNA extraction kit.

[0033] Specifically, the food sample to be tested includes raw materials or processed food, and the allergen components contained therein include 1: hazelnut; 2: brazil nut; 3: walnut; 4: almond; 5: pistachio; 6: macadamia nut; 7: cashew nut; 8: pistachio; 9: hickory nut; 10: peanut; 11: soybean. Generally, the sample is crushed into powder, which may contain complex matrixes (such as oil, polysaccharide or polyphenol). The DNA extraction kit uses the DNA extraction kit (item number 69514) of QIANGEN company. After extraction, the DNA concentration and purity are measured by NanoDrop spectrophotometer, and the integrity is verified by agarose gel electrophoresis (a clear band should appear, without degradation). The purpose of this step is to extract plant-derived total DNA from the food sample to be tested, eliminate the interference of inhibitors such as polysaccharide, polyphenol and oil, and provide a high-purity template for subsequent PCR amplification.

[0034] Step 2, using psbA-trnH barcode universal primers to perform PCR amplification on the extracted plant-derived total DNA, to obtain the amplification product of psbA-trnH barcode specific DNA fragments.

[0035] In the PCR reaction system, Premix Ex Taq™ premix solution is 12.5 μL, upstream primer and downstream primer are each 0.5 μL, 5 μL DNA template, and sterile double distilled water is added to 25 μL. The specific preparation operation process is as follows:

[0036] Take a sterile PCR tube and add the following components (total volume 25 μL) in order:

[0037] (1) Premix (such as Premix Ex Taq™): 12.5 μL (containing DNA polymerase, dNTPs, Mg²⁺ and buffer), (2) Forward primer (10 μM): 0.5 μL, (3) Reverse primer (10 μM): 0.5 μL, (4) DNA template (total DNA extracted from plant source in step 1): 5 μL (concentration 20-50 ng / μL), (5) Sterile double distilled water: make up to 25 μL.

[0038] Gently vortex to mix and centrifuge briefly to collect droplets on the tube wall. The order of loading should be consistent, add premix first and DNA template last to reduce the risk of non-specific amplification. The working concentration of primers needs to be accurately controlled, with a final concentration of 0.2 μM.

[0039] and the sequence pair of the primer is the forward primer shown in SEQ ID NO: 1 and the reverse primer shown in SEQ ID NO: 2. Forward primer: 5'-GTTATGCATGAACGTAATGCTC-3', Reverse primer: 5'-CGCGCATGGTGGATTCACAATCC-3'.

[0040] Place the PCR tube in a thermal cycler and set the following program:

[0041] Pre-denaturation: 94℃, 5 minutes (activate hot-start enzyme and ensure complete denaturation of DNA);

[0042] Cycle amplification (35 cycles):

[0043] Denaturation: 94℃, 30 seconds;

[0044] Annealing: 60℃, 30 seconds (optimize temperature to ensure specific binding of primers);

[0045] Extension: 72℃, 45 seconds;

[0046] Final extension: 72℃, 10 minutes (ensure product integrity);

[0047] Storage: 4℃, ∞.

[0048] Start the program and the running time is about 2 hours.

[0049] Amplification product verification, specifically including:

[0050] After PCR, take 5 μL of the amplification product for 1.5% agarose gel electrophoresis (voltage 120 V, time 30 minutes).

[0051] The bands were observed using a gel imaging system after staining with a nucleic acid dye (such as GeneGreen).

[0052] Expected results: Single or multiple clear bands at the 300-700 bp position. Preferably, the PCR products are detected by capillary electrophoresis on a fully automated nucleic acid and protein analysis system (QIAxcel Connect), using a QX Alignment Marker 15 bp / 3 kb, a QX Size Marker 100 bp - 2.5 kb, and a DNA scanning analysis card. Specific electrophoresis results are as follows... Figure 2 As shown, the numbers 1-11 represent different allergens, specifically: 1: hazelnut; 2: Brazil nut; 3: walnut; 4: almond; 5: almond kernel; 6: macadamia nut; 7: cashew; 8: pistachio; 9: pecan; 10: peanut; 11: soybean.

[0053] The reaction process involved running both a positive control (using known peanut DNA) and a negative control (using sterile water) simultaneously to ensure reliable results. If bands were blurred or missing, the reaction conditions needed to be optimized.

[0054] Product preservation: Store the remaining amplification products at -20℃ to avoid repeated freeze-thaw cycles, and use them for subsequent sequencing and library construction.

[0055] Step 2 then utilizes a standardized PCR procedure to achieve efficient amplification of barcode fragments from plant-derived allergens. Key parameters (such as primer sequences and annealing temperature) have been rigorously optimized to ensure method stability and reliability. The amplified products can be directly used for sequencing library preparation in step 3, serving as a bridge in the entire detection process.

[0056] Step 3: Construct a high-throughput sequencing library from the amplification products of the psbA-trnH DNA fragment, and perform paired-end sequencing using the Illumina platform to obtain raw sequencing data;

[0057] The sequencing library construction process is as follows:

[0058] Purification of amplification products: PCR amplification products were purified using AMPure XP magnetic beads at a volume ratio of 1:1 (bead to sample).

[0059] Library construction: Perform PCR amplification for 5-10 cycles using Index primers. Program: 95℃ 3 min → [94℃ 20s, 55℃ 20s, 72℃ 30s] × 5 cycles → 72℃ 5 min.

[0060] Purification: Performed using AMPure XP beads, with a bead-to-sample volume ratio of 1:0.8;

[0061] Furthermore, the quality of the acquired library was assessed using an Agilent 2100 Bioanalyzer to detect fragment distribution, requiring a main peak between 300-700 bp, no nonspecific bands, and a concentration ≥10 nM.

[0062] Further sequencing was performed using Illumina Nextseq 2000, specifically in PE300 mode (300 bp paired-end reads), with a data volume of ≥10 M reads per sample and a cluster density of 200-300 K / mm². The raw sequencing data were in FASTQ format.

[0063] Step 4: Compare the raw sequencing data with a plant allergen database to obtain multiple allergen components in the food sample to be tested.

[0064] Specifically, the raw sequencing data from step 3 is compared with a plant allergen database using the BLAST algorithm. Species are identified based on a 97% sequence similarity threshold, and a list of multiple allergen components and their relative concentrations in the tested food sample is output. The plant allergen database is either NCBI RefSeq or a custom database, containing species annotation information for the psbA-trnH sequences.

[0065] Specific operations:

[0066] Data preprocessing: Quality control: Trimmomatic removes low-quality reads (Q<20) and adapter sequences. Primer removal: Cutadapt removes primer regions, retaining pure psbA-trnH variable regions.

[0067] Sequence alignment: The BLAST algorithm is used to align the reads with the database. The similarity threshold is set to ≥97%, with parameters: e-value <1e-5 and coverage >90%.

[0068] Species identification: Based on alignment results (such as sequence consistency and scores), a species annotation list is generated. Multiple identification: The relative content of each allergen is estimated by reading count (e.g., a high percentage of peanut reads indicates the main component).

[0069] Visualization output: Construct a phylogenetic tree (neighbor-join method) to show the clustering relationships of species.

[0070] Identification results report: List the plant-derived allergen species detected (such as peanuts, soybeans, hazelnuts, etc.), their relative content (based on the proportion of reading segments), and their confidence level (such as comparison score).

[0071] Furthermore, the above methods can be used to efficiently detect allergens in food, which can then be used for the supervision of processed foods, providing a reliable tool for food safety.

[0072] The psbA-trnH barcode detection method for plant-derived allergens of this invention enables multiplex and simultaneous detection of plant-derived allergens, improving detection efficiency and accuracy. Specifically, a single test can simultaneously identify multiple allergens (such as peanuts, soybeans, nuts, etc.), reducing the time cost of multiple tests required by traditional methods (detection time is shortened from several hours to approximately 2 hours). The large interspecies genetic distance based on the psbA-trnH barcode (average 0.5273) ensures species identification specificity and avoids false positives / negatives. It is applicable to processed foods (such as peanut butter and biscuits) and overcomes problems of DNA degradation and matrix interference.

[0073] Furthermore, the present invention also provides a detection system for implementing the psbA-trnH barcode detection method for plant-derived allergens, the system comprising a DNA extraction module, a PCR amplification module, a sequencing library preparation module, and a data analysis module.

[0074] The DNA extraction module includes plant DNA extraction reagents for extracting total plant-derived DNA from food samples.

[0075] The PCR amplification module includes a universal primer pair for psbA-trnH barcoding and a PCR reaction solution; the PCR amplification module also includes a positive control, which is a mixed plasmid DNA containing the psbA-trnH sequences of hazelnuts, Brazil nuts, macadamia nuts, walnuts, pecans, almonds, almond kernels, cashews, pistachios, peanuts and soybeans.

[0076] The sequencing library preparation module includes high-throughput sequencing library construction reagents for nucleic acid purification, PCR amplification, and purification of PCR amplification products;

[0077] The data analysis module includes access to a plant allergen database and sequence alignment software for identifying various allergen components.

[0078] Furthermore, to better illustrate the detection method of the present invention, specific embodiments are described below.

[0079] Step 1: Sample pretreatment and DNA extraction

[0080] 1. After crushing or homogenizing the sample, weigh 200mg of the sample.

[0081] 2. Follow the instructions in the kit:

[0082] Add 1 mL of lysis buffer and 2.5 μL of RNase A, incubate in a 60 °C water bath for 30 min (inverting to mix every 3 min). Centrifuge (14,000 rpm, 5 min) and collect the supernatant.

[0083] Add 500 μL of chloroform, mix well, centrifuge (14,000 rpm, 15 min), and collect the supernatant.

[0084] Add an equal volume of AP, purify by column chromatography, and then elute the DNA with 100 μL of Buffer EB.

[0085] 3. Quality Inspection:

[0086] NanoDrop assays showed DNA concentrations of 1.8 (A260 / A280), and agarose gel electrophoresis revealed clear bands with no degradation.

[0087] Step 2: PCR amplification

[0088] 1. Reaction system (25 μL)

[0089]

[0090] PCR program: 94℃ pre-denaturation for 5 min; 35 cycles: 94℃ 30 s → 60℃ 30 s → 72℃ 45 s; 72℃ final extension for 10 min;

[0091] Amplification product validation:

[0092] 1.5% agarose gel electrophoresis shows single or multiple clear bands (approximately 300 bp-700 bp). Positive controls show bands, while negative controls show no amplification.

[0093] Step 3: Sequencing library construction and sequencing

[0094] Library construction: Purification using AMPure XP magnetic beads, library construction using Index primers, and purification using AMPure XP magnetic beads.

[0095] Sequencing parameters:

[0096] Platform: Illumina Nextseq 2000

[0097] Mode: PE300 (300 bp dual-ended)

[0098] Data volume: 15 M reads per sample.

[0099] Step 4: Bioinformatics Analysis

[0100] Data preprocessing:

[0101] FastQC quality control: Q30=89.5%, GC content=45.2%, connector contamination rate=1.3%.

[0102] Trimmomatic filters low-quality reads (quality value < 20).

[0103] Sequence alignment:

[0104] BLAST comparison with a custom database (parameters: -evalue 1e-5 -perc_identity 97).

[0105] Species identification criteria: similarity ≥ 97%, coverage ≥ 90%.

[0106] The specific results are shown in Table 2.

[0107]

[0108] The statistical results in Table 2 show that some of the allergens detected by the above testing methods are consistent with the sample labels. For example, sample YP-1 claimed to contain gluten and nuts, but almonds were actually detected; YP-3 claimed to contain soy and gluten, but soy was actually detected. Some claimed allergens were not detected. For example, sample YP-2 claimed to contain gluten, nuts, peanuts, and soy, but only peanuts and walnuts were detected, and soy was not detected. There were also allergens not listed on the labels. For example, sample YP-5 claimed to contain soy and nuts, but soy and peanuts were actually detected; sample YP-7 claimed to contain soy, but peanuts and soy were actually detected. These results indicate that there are some discrepancies between the allergen labels and the actual test results for commercially available food samples.

[0109] Furthermore, to better illustrate the effect of the psbA-trnH sequence, the following describes the identification of peanuts, soybeans, and nine kinds of nuts using the psbA-trnH, trnl, and rbcl sequences.

[0110] Materials and reagents

[0111] Eleven species, including hazelnuts, Brazil nuts, macadamia nuts, walnuts, pecans, almonds, almond kernels, cashews, pistachios, peanuts, and soybeans, were selected as research subjects. A total of 128 gene sequences were obtained, including 40 psbA-trnH sequences, 42 trnl sequences, and 46 rbcl sequences. Detailed sample information is shown in Tables 3-5.

[0112]

[0113]

[0114]

[0115] Sequence acquisition and alignment

[0116] The psbA-trnH, trnl, and rbcl sequences of 11 species taxa were downloaded from the NCBI GenBank database. Homology comparison was performed using the BLAST function, and sequences of questionable reliability were removed, resulting in 128 valid sequences. MEGA 11 bioinformatics analysis software was used to align all sequences and remove fragments with irregular base alignment at both ends.

[0117] Genetic distance calculation

[0118] The sequences were systematically analyzed using MEGA11 software, and the intraspecific and interspecific genetic distances of each species were accurately calculated based on the Kimura2-Parameter model.

[0119] NJ phylogenetic tree construction

[0120] Phylogenetic trees were constructed using the neighbor-joining (NJ) method in MEGA11 software. Gene sequence clustering analysis was performed, and the reliability of phylogenetic relationships in each branch was evaluated through Bootstrap 1000-times replication test to ensure the scientific validity and robustness of the results.

[0121] Comparison of genetic distance analysis of candidate barcodes

[0122] Genetic distance calculations were performed using the Kimura2-parameter distance model based on MEGA11 software. The results showed that in the psbA-trnH sequences (Table 6), the intraspecific genetic distance for all species was 0, while the interspecific genetic distance fluctuated between 0.0152 and 1.0507, with an average interspecific genetic distance of 0.5273. The minimum interspecific genetic distance was greater than the maximum intraspecific distance, indicating a significant barcoding gap both intraspecificly and interspecifically. In the trnl sequences (Table 7), except for macadamia nuts where the intraspecific genetic distance was 0.0060, all others showed a distance of 0. The interspecific genetic distance ranged from 0.0143 to 1.3366, with an average interspecific genetic distance of 0.5076, also exhibiting a barcoding gap. In contrast, the rbcl sequences (Table 8) all showed a distance of 0 within each species, while the interspecific genetic distance ranged from 0 to 0.1010. Specifically, the interspecific genetic distance between almonds and walnuts, and between walnuts and hickory nuts, was 0, with an average interspecific genetic distance of 0.0703. A comprehensive comparison (Table 9) revealed that the average interspecific genetic distance of the three sequences showed a decreasing trend: psbA-trnH > trnl > rbcl. The difference between interspecific and intraspecific genetic distances was ranked as psbA-trnH > trnl > rbcl. Furthermore, the average interspecific genetic distance of all three barcodes was greater than the average intraspecific genetic distance, indicating that the psbA-trnH sequence was significantly superior to the trnl and rbcl sequences in species differentiation, with the latter showing decreasing differentiation ability in that order.

[0123]

[0124]

[0125]

[0126]

[0127] Phylogenetic analysis of NJ

[0128] A phylogenetic tree was constructed using the neighbor-joining (NJ) method based on 40 psbA-trnH gene sequences from 11 species, as shown below. Figure 3 As shown in the figure, different individuals of the same species are tightly clustered, and the boundaries between species are clearly distinguishable. The self-spreading support rate of each branch is higher than 91%, and the self-spreading support rate of most species (except almonds, apricots, pecans, and pistachios) is as high as 100%, indicating that the tree structure has high reliability.

[0129] Phylogenetic tree of trnl and rbcl gene sequences as follows Figure 4 and 5Analysis showed that in the phylogenetic tree constructed based on rbcl sequences, almonds and amygdalites clustered in the same branch, as did walnuts and hickory nuts, while other species clustered independently, forming clear species-specific branching structures. Taxonomically, almonds and amygdalites belong to the Rosaceae family, while walnuts and hickory nuts belong to the Juglandaceae family, indicating a close phylogenetic relationship. This meant that rbcl barcodes could not effectively distinguish between almonds and amygdalites, or walnuts and hickory nuts. These results suggest that the closer the phylogenetic relationship between species, the fewer barcodes are available, requiring more extensive screening to find suitable barcodes.

[0130] Further analysis of the psbA-trnH, trnl, and rbcl gene sequences revealed that the psbA-trnH sequence exhibited the greatest interspecific genetic distance variation, with an average interspecific genetic distance higher than that of the trnl and rbcl sequences. The rbcl sequence, however, showed limitations in distinguishing between walnuts and pecans, and between almonds and apricot kernels. Cluster analysis further corroborated this, showing that the NJ phylogenetic tree constructed based on the psbA-trnH and trnl sequences clearly clustered the species; conversely, the rbcl sequence also failed to effectively distinguish between walnuts and pecans, and between almonds and apricot kernels. Based on these combined indicators, the psbA-trnH sequence was identified as the optimal DNA barcode for identifying 11 species, including nuts, peanuts, and soybeans.

[0131] Furthermore, this invention combines psbA-trnH barcode detection with high-throughput sequencing technology for actual sample testing, enabling the simultaneous detection of multiple allergen components. The barcode detection method established by this invention provides a powerful tool for allergen control in the global food supply chain, and is expected to promote the widespread application of DNA molecular detection technology in the field of food authenticity and safety, helping regulatory agencies in various countries improve food safety assurance levels.

[0132] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or system that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or system. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or system that includes that element.

[0133] The sequence numbers of the above embodiments of the present invention are for descriptive purposes only and do not represent the superiority or inferiority of the embodiments.

[0134] The above are merely preferred embodiments of the present invention and do not limit the scope of the patent. Any equivalent structural or procedural transformations made based on the description and drawings of the present invention, or direct or indirect applications in other related technical fields, are similarly included within the scope of patent protection of the present invention.

Claims

1. A method for detecting plant-derived allergens psbA-trnH using barcodes, characterized in that, The method includes the following steps: Step 1: Crush or homogenize the food sample to be tested, and use a DNA extraction kit to extract the corresponding plant-derived total DNA; Step 2: Use the psbA-trnH barcode universal primers to perform PCR amplification on the extracted plant-derived total DNA to obtain the amplification product of the psbA-trnH barcode specific DNA fragment; Step 3: Construct a high-throughput sequencing library from the amplification products of the psbA-trnH barcode-specific DNA fragment, and perform paired-end sequencing using the Illumina platform to obtain raw sequencing data; Step 4: Compare the raw sequencing data with a plant allergen database to obtain multiple allergen components in the food sample to be tested.

2. The method according to claim 1, characterized in that, In step 2, the primer sequence includes the forward primer shown in SEQ ID NO:1 and the reverse primer shown in SEQ ID NO:

2.

3. The method according to claim 1, characterized in that, In step 2, the PCR amplification reaction program was as follows: 94℃ pre-denaturation for 5 min; 35 cycles of 94℃ denaturation for 30 s, 58-62℃ annealing for 30 s, 72℃ extension for 45 s; and 72℃ final extension for 10 min.

4. The method according to claim 1, characterized in that, In step 2, the PCR reaction system is Premix ExTaq. TM 12.5 μL of premix, 0.5 μL each of upstream and downstream primers, 5 μL of DNA template, and bring the volume to 25 μL with sterile double-distilled water.

5. The method according to claim 1, characterized in that, In step 3, the construction of the sequencing library includes nucleic acid purification, PCR amplification, and purification steps.

6. The method according to claim 1, characterized in that, In step (4), the plant allergen database is NCBI RefSeq or a custom database, the comparison tool is the BLAST algorithm, and the output results include species confidence.

7. The method according to claim 1, characterized in that, The allergens include one or more of the following: hazelnuts, Brazil nuts, macadamia nuts, walnuts, pecans, almonds, almond kernels, cashews, pistachios, peanuts, and soybeans.

8. A detection system for implementing the method of any one of claims 1-7, characterized in that, The system includes a DNA extraction module, a PCR amplification module, a sequencing library preparation module, and a data analysis module. The DNA extraction module contains plant DNA extraction reagents for extracting total plant-derived DNA from food samples; The PCR amplification module includes the psbA-trnH barcode universal primer pair and PCR reaction solution; The sequencing library preparation module includes high-throughput sequencing library construction reagents for nucleic acid purification, PCR amplification, and purification of PCR amplification products; The data analysis module includes a plant allergen database and sequence alignment software for identifying various allergen components.

9. The system according to claim 8, characterized in that, The PCR amplification module also includes a positive control unit, which is a mixed plasmid DNA containing the psbA-trnH sequences of hazelnuts, Brazil nuts, macadamia nuts, walnuts, pecans, almonds, almond kernels, cashews, pistachios, peanuts, and soybeans.

10. Application of psbA-trnH barcode in the preparation of a kit for the simultaneous detection of multiple plant-derived allergen components in food.

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