Animal-derived ingredient detection method based on high-throughput sequencing technology and its application

Abstract

The present invention discloses a method for detecting animal-derived ingredients based on high-throughput sequencing technology and its application, belonging to the field of biotechnology; the method comprises the following steps: (1) sample DNA extraction, (2) one-step PCR amplification, (3) MiSeq sequencing, and (4) SAFESeqr Suite data analysis. The method for detecting animal-derived ingredients based on high-throughput sequencing technology of the present invention directly performs high-throughput sequencing analysis on the extracted sample DNA, thereby ensuring a sufficient amount of data, designing universal primers to simultaneously determine meat products adulterated with multiple different species, and to determine the relative content of each species in the meat products, thereby realizing rapid identification of unknown animal-derived ingredients and accurate determination of adulteration and doping. The method can simultaneously perform component and component analysis of 96 mixed samples, with high sensitivity, simple operation, low cost, and short detection time.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a method for detecting animal-derived components based on high-throughput sequencing technology and an application thereof. Background Art

[0002] In recent years, my country's food industry has entered a golden period of rapid development. Poultry and livestock meat products and their products have enriched the dining tables of the general public. Meat products are the main channel for people to obtain high-quality protein. The quality of meat products is closely related to human health. However, in recent years, food adulteration has occurred frequently. Meat product adulteration is a common type of food adulteration phenomenon. It is mainly due to the fact that some illegal vendors add or even replace meat products with similar-shaped and cheaper non-species meat in order to make profits. Taking the "horse meat storm" incident as an example, horse meat ingredients were detected in beef products in many large supermarkets in the UK and other European countries in 2013. Most of these adulterated meats have not been reviewed by the quarantine department, and some are even non-edible meats. These meats of unknown origin may carry unknown pathogens or contain some harmful chemical components, which have serious effects on the health of consumers. In order to more effectively supervise and control the occurrence of such phenomena, and to prevent such adulterated meat from flowing into the hands of consumers as much as possible, the establishment of an effective animal-derived component detection method can not only effectively crack down on illegal vendors, but also take targeted measures to reduce the occurrence of such phenomena.

[0003] Many scholars at home and abroad have also conducted a lot of research on the detection technology of animal-derived ingredients. At present, the genes that can be used to identify the ingredients of meat food are usually located on mitochondria (mtDNA), including cytochrome b (Cytb) gene, cytochrome C oxidase subunit I (COI) gene, 12SrDNA, 16SrDNA, D-loop gene, etc. At present, the adulteration and doping inspection of meat products at home and abroad are mainly based on PCR-RFLP, multiplex PCR, real-time quantitative PCR and other methods. However, these technologies can generally only detect one or two indicators each time, and cannot comprehensively analyze and detect the various animal-derived ingredients involved in the food. When encountering mixed samples of several different varieties, to determine the relative amount of each variety in the mixture, it is necessary to find the only known target sequence in the variety as a reference sequence, and it is very difficult to obtain accurate measurement results. In addition, the existing detection methods have high requirements on the operator's skills and laboratory instrument equipment configuration, and the detection cost is expensive. It is difficult to meet the detection requirements of high efficiency, large batch, large scale, simple operation, etc., and it cannot meet the urgent needs of market supervision and real-time control of the quality of raw materials and finished products of enterprises. Therefore, developing a convenient, fast, highly sensitive, low-cost, short-detection-time, accurate and high-throughput detection method to achieve rapid identification of unknown animal-derived ingredients and precise determination of adulteration and doping will help law enforcement agencies to quickly and accurately identify animal-derived ingredients and improve the detection capabilities of functional departments. Summary of the invention

[0004] The purpose of the present invention is to provide a method for detecting animal-derived ingredients based on high-throughput sequencing technology and its application in view of the deficiencies of the prior art. The detection method directly performs high-throughput sequencing analysis on the extracted sample DNA, ensuring sufficient data volume, and the design of universal primers can simultaneously determine meat products adulterated with multiple different species, and can determine the relative content of each species in the meat products, thereby realizing the rapid identification of unknown animal-derived ingredients and the accurate determination of adulteration and doping.

[0005] To achieve the above object, the present invention adopts the following technical solution:

[0006] The method for detecting animal-derived ingredients based on high-throughput sequencing technology includes the following steps:

[0007] (1) Sample DNA extraction;

[0008] (2) One-step PCR amplification;

[0009] (3) MiSeq sequencing;

[0010] (4) SAFESeqr Suite data analysis;

[0011] The one-step PCR amplification method in step (2) is as follows:

[0012] a1. Design primers PR2-F and PR2-R to amplify short fragment sequences of each sample; wherein,

[0013] Primer PR2-F: 5'- ATAAGACGAGAAGACCCT-3'

[0014] Primer PR2-R: 5'- ACATCGAGGTCGTAAACC-3'

[0015] b1. Add Illumina sequencing adapter and index sequence to the 5' end of amplification primer PR2 to distinguish different samples;

[0016] c1. The PCR reaction system is 25 μL, including 12.5 μL of FailSafe PCR 2×PreMix F, 0.5 μL of FailSafePCR Enzyme Mix, 2 μL of each forward and reverse primer (5 μM), 2 μL of gDNA (1 ng / μL), and 6 μL of ultrapure water. The PCR product is obtained by one-step PCR reaction program {96°C, 5 min; 25 cycles (96°C, 45 s; 45°C, 45 s; 72°C, 45 s); 72°C, 3 min}; ultrapure water is used to replace gDNA in the sample as a negative control (NTC), and three replicates are prepared in parallel;

[0017] d1. The obtained PCR product was subjected to 1% agarose gel electrophoresis. The target fragment size was 400 bp to 500 bp.

[0018] Furthermore, the DNA extraction method of the sample in step (1) is as follows: the meat product is cut into pieces and ground, 100-300 mg of the sample is weighed, and the total DNA of the sample is extracted using a deep-processed food DNA extraction kit.

[0019] Furthermore, the MiSeq sequencing step in step (3) is as follows: purify the PCR products of each sample using AMPure XP beads, confirm whether the impurity fragments are removed by 1% agarose gel electrophoresis, and use Qubit ® The purified PCR products were quantitatively analyzed by Fluorometer and uniformly diluted to 4 nM. Each sample and PhiX Control were combined to form a sequencing sample library. After denaturation treatment, the MiSeq Reagent Kit v2 (500-cycles) was used for sequencing.

[0020] Furthermore, the step (4) of SAFESeqr Suite data analysis is as follows: the FASTQ files of the sample Read1 and Read2 obtained by sequencing are imported into SAFESeqr Suite, and the sample components and component results are analyzed. The relevant introduction of the SAFESeqr Suite software used can be found on the following website:

[0021] Furthermore, the sequences of the primers PR2-F and PR2-R are shown in SEQ ID NO:1 and SEQ ID NO:2.

[0022] The animal-derived ingredient detection method based on high-throughput sequencing technology, the classification database of the method contains animal 16s rRNA gene sequence information of >1500 genera and >2700 species.

[0023] Another object of the present invention is to provide the application of the animal-derived ingredient detection method based on high-throughput sequencing technology in the inspection of adulteration and doping of meat products.

[0024] Compared with the prior art, the animal-derived component detection method based on high-throughput sequencing technology and its application in the present invention have the following advantages:

[0025] (1) The present invention provides a method for detecting animal-derived ingredients based on high-throughput sequencing technology. The method directly performs high-throughput sequencing analysis on the extracted sample DNA, ensuring a sufficient amount of data. The design of universal primers can simultaneously determine meat products adulterated with multiple different species, and can determine the relative content of each species in the meat products, thereby achieving rapid identification of unknown animal-derived ingredients and accurate determination of adulteration and doping.

[0026] (2) The animal-derived component detection method based on high-throughput sequencing technology provided by the present invention has constructed a database containing more than 1,500 genera and more than 2,700 species of bony fish, cartilaginous fish, mammals, birds, reptiles, etc., which can accurately and effectively identify unknown animal-derived components.

[0027] (3) The animal-derived component detection method based on high-throughput sequencing technology described in the present invention improves the detection efficiency by one-step PCR amplification, saving time and labor costs.

[0028] (4) Compared with the existing animal-derived component detection method, the animal-derived component detection method based on high-throughput sequencing technology described in the present invention can simultaneously analyze the components and fractions of 96 mixed samples, with high sensitivity, simple operation, low cost, short detection time, high accuracy, and a detection limit of 0.1% and 1μg. This detection method can meet the detection requirements of high efficiency, large batch, large scale, simple operation, and short detection time, and has significant progress.

[0029] (5) The animal-derived ingredient detection method based on high-throughput sequencing technology of the present invention can solve the technical problem of the difficulty in simultaneously identifying unpredictable animal-derived ingredients in meat products, eliminate the adulteration of meat products from the source, and fundamentally solve the problem of meat product safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The PCR results of primer PR2 without Illumina adapter are shown in the figure, L-KAPA UniversalLadder, C-chicken, B-Beef, P-Pork, N-NTC;

[0031] Figure 2 This is the PCR result of primer PR2 with Illumina adapter added. In the figure, L-KAPA UniversalLadder, C-chicken, B-Beef, P-Pork, N-NTC. DETAILED DESCRIPTION

[0032] The present invention will be described in detail below with reference to the embodiments and the accompanying drawings.

[0033] Example 1

[0034] 1. A method for detecting animal-derived ingredients based on high-throughput sequencing technology, comprising the following steps:

[0035] (1) Sample DNA extraction: Cut the meat product into pieces and grind it. Weigh 100-300 mg of the sample and use a deep-processed food DNA extraction kit to extract the total DNA of the sample.

[0036] (2) One-step PCR amplification:

[0037] a1. Design primers PR2-F and PR2-R to amplify short fragment sequences of each sample; wherein,

[0038] Primer PR2-F: 5'- ATAAGACGAGAAGACCCT-3' (SEQ ID NO: 1);

[0039] Primer PR2-R: 5'- ACATCGAGGTCGTAAACC-3' (SEQ ID NO: 2);

[0040] b1. Add Illumina sequencing adapter and index sequence to the 5' end of amplification primer PR2 to distinguish different samples;

[0041] c1. The PCR reaction system is 25 μL, including 12.5 μL of FailSafe PCR 2×PreMix F, 0.5 μL of FailSafePCR Enzyme Mix, 2 μL of each forward and reverse primer (5 μM), 2 μL of gDNA (1 ng / μL), and 6 μL of ultrapure water. The PCR product is obtained by one-step PCR reaction program {96°C, 5 min; 25 cycles (96°C, 45 s; 45°C, 45 s; 72°C, 45 s); 72°C, 3 min}; ultrapure water is used to replace gDNA in the sample as a negative control (NTC), and three replicates are prepared in parallel;

[0042] d1. The obtained PCR product was subjected to 1% agarose gel electrophoresis. The target fragment size was 400 bp to 500 bp.

[0043] (3) MiSeq sequencing:

[0044] The PCR products of each sample were purified using AMPure XP beads, and the removal of the impurity fragments was confirmed by 1% agarose gel electrophoresis. ® The purified PCR products were quantitatively analyzed by Fluorometer and uniformly diluted to 4 nM. Each sample and PhiX Control were combined to form a sequencing sample library. After denaturation treatment, the MiSeq Reagent Kit v2 (500-cycles) was used for sequencing.

[0045] (4) SAFESeqr Suite data analysis:

[0046] The FASTQ files of sample Read1 and Read2 obtained by sequencing were imported into SAFESeqr Suite, and the sample composition and component results were analyzed.

[0047] The animal-derived ingredient detection method based on high-throughput sequencing technology has a classification database containing animal 16s rRNA gene sequence information of >1500 genera and >2700 species.

[0048] 2. Sample Testing

[0049] Ham sausage, meatballs, meat jerky, beef skewers, etc. were purchased from the market, and the ingredients were detected by fluorescent quantitative PCR and the high-throughput sequencing detection method of the present invention. The detection results of the two methods were consistent, and unlabeled ingredients were detected in some samples, and the content was relatively high, as shown in Table 1.

[0050]

[0051] Example 2 Method Confirmation

[0052] 1. Specificity

[0053] Weigh 10 mg each of single meat samples of cat, pig, mouse, chicken, goose, sheep, buffalo, duck, quail, partridge, dog, donkey, hairtail, grouper, grass carp, sea bass, salmon, cod, sardine and king cobra, and 10 mg each of homemade mixed samples HE (cattle: sheep = 1:1) and HG (cattle: sheep = 3:1), extract sample DNA; prepare DNA samples PR2-RB-3 (cattle), PR2-RC-3 (chicken), PR2-RP-3 (pig), PR2-M-CBP-2 (cattle: chicken: pig = 1:1:1), PR2-M-2-2 (cattle: chicken = 95:5) 2ng and PR2-M-20-2 (cattle: chicken = 95:5) 20ng. The 16s rRNA gene short fragment sequence of all the above sample DNA was amplified and analyzed by high-throughput sequencing. The components and compositions of each sample were basically consistent with the actual samples, as shown in Table 2.

[0054] 2. Sensitivity

[0055] Beef and mutton were prepared into mixed sample HF at a mass ratio of 99:1, and then diluted 10 times to prepare mixed sample HK (999:1), and 1 mg was weighed respectively to extract sample DNA; DNA samples PR2-S-20 (chicken) 20 ng, PR2-S-2 (chicken) 2 ng, and PR2-S-02 (chicken) 0.2 ng were prepared. The 16s rRNA gene short fragment sequence was amplified from the DNA of all the above samples, and high-throughput sequencing analysis was performed. The results were basically consistent with the actual samples, as shown in Table 2, indicating that the detection limit of this method for samples can reach a mixed ratio of 0.1% and an initial mass of 1 μg.

[0056]

[0057] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention. Sequence Listing <110> Guangxi Zhuang Autonomous Region Food and Drug Inspection Institute <120> Animal-derived ingredient detection method based on high-throughput sequencing technology and its application <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <400> 1 ataagacgag aagaccct 18 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <400> 2 acatcgaggt cgtaaacc 18

Claims

1. Application of a PCR primer in the detection of animal-derived ingredients, adulteration of meat products, and adulteration inspection, characterized in that: The PCR primers consist of an upstream primer PR2-F and a downstream primer PR2-R, the nucleotide sequence of the upstream primer PR 2-F is: 5'-ATAAGACGAGAAGACCCT-3', and the nucleotide sequence of the downstream primer PR2-R is: 5'-ACATCGAGGTCGTAAACC-3'; The method for using the PCR primers in animal-derived component detection, meat product adulteration and doping inspection comprises the following steps: (1) DNA extraction from samples; (2) One-step PCR amplification; (3) MiSeq sequencing; (4) SAFESeqr Suite data analysis; The one-step PCR amplification method in step (2) is as follows: a1. Design the upstream primer PR2-F and the downstream primer PR2-R; b1. Add Illumina sequencing adapter and index sequence to the 5' end of amplification primer PR2-R to distinguish different samples; c1. The PCR reaction system is 25 μL, including 12.5 μL of FailSafe PCR 2×PreMix F, 0.5 μL of FailSafe PCREnzyme Mix, 2 μL of 5 μM upstream and downstream primers, 2 μL of 1 ng / μL gDNA, and 6 μL of ultrapure water. The one-step PCR reaction program is 96°C for 5 min; 25 cycles: 96°C for 45 s, 45°C for 45 s, 72°C for 45 s; 72°C for 3 min to obtain the PCR product; ultrapure water is used to replace gDNA in the sample as a negative control, and three replicates are prepared in parallel; d1. The obtained PCR product was subjected to 1% agarose gel electrophoresis, and the target fragment size was 400 bp to 500 bp.

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