CN113293216A - Identification method for animal-derived components in salmonidae products based on high-throughput sequencing - Google Patents
Identification method for animal-derived components in salmonidae products based on high-throughput sequencing Download PDFInfo
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Abstract
The invention relates to the technical field of food quality safety detection, and particularly discloses a method for identifying animal-derived components in salmonids and products thereof based on a high-throughput sequencing technology, which comprises the steps of extracting genome DNA, amplifying by 255 bp with a basic group label16S rRNAThe general primer pair is used for extracting genome DNA to carry out PCR amplification, constructing an amplicon sequencing library, carrying out high-throughput sequencing, carrying out bioinformatics data analysis, judging results and the like. The method can identify various animal-derived components in salmonids and products thereof by one-time detection, and the detection sensitivity is as low as 1%.
Description
Technical Field
The invention belongs to the technical field of food quality safety detection, and particularly relates to a method for identifying animal-derived components in a salmonidae product based on high-throughput sequencing.
Background
Salmonidae (Salmonidae) belongs to the phylum Chordata (Chordata) Salmoniformes (Salmoniformes) and comprises a total of 3 subfamilies (Salmonidae, Corynebaideae and Anethopalidae), 7 genera (Atlantic, hucho, Lepidiobolus, Salmonella, whitefish and Anise), for a total of about 220 species of fish. The salmonidae fish is a deep-sea fish with nutritive value and health care efficacy, has delicious and delicious taste and is deeply loved by the vast consumers.
At present, the most common animal product adulteration screening related nucleic acid detection technology at home and abroad is to detect by utilizing a Sanger sequencing method, a PCR (fluorescent quantitative PCR) method, electrophoresis (or enzyme digestion) and other technologies. However, the existing method can only detect one or a few species at a time, and cannot realize synchronous identification of multiple source components in unknown samples. When the number of samples is large, a large amount of material and labor are required. In addition, when the sample to be tested is a mixture or an unknown sample, the separation of organisms is difficult, and the existing animal product adulteration identification technology is far from meeting the practical requirements in the aspects of related product range, method applicability and the like. Particularly, with the development of modern industry, the types of the salmonid products are more and more, the processing is more and more delicate, the components are more and more complex, the original characteristics of the species disappear, and the adulteration is hidden and difficult to detect. Thus, there is an increasing need for non-targeted screening of multiple species simultaneously. The high-throughput sequencing technology greatly improves the analysis efficiency of DNA, changes the traditional analysis mode, and can detect all species in a mixed sample at one time by selecting a universal primer with a proper amplification interval and length.
The invention provides a method for identifying animal-derived components in a salmonidae fish mixed product by using a high-throughput sequencing technology. So as to provide theoretical basis and technical support for species identification, component detection and the like of commercial salmonids.
Disclosure of Invention
The invention aims to establish a method for identifying animal-derived components in a salmonid product based on a high-throughput sequencing technology, which can accurately and efficiently detect all animal-derived species components in a sample and has the advantages of wide detection range, high detection flux, high sensitivity and the like.
The purpose of the invention can be realized by the following technical scheme:
(a) extracting genome DNA from a sample to be detected;
(b) by means of a belt with upstream and downstream marks16S rRNACarrying out PCR amplification on the genome DNA in the (a) by using the universal primer to obtain a PCR amplification product;
(c) purifying the PCR amplification product and measuring the concentration;
(d) constructing a sequencing library by using the PCR amplification product reaching the concentration requirement in the previous step and carrying out quality inspection;
(e) performing high-throughput sequencing on the sequencing library of the last step to obtain16S rRNAA gene fragment sequence;
(f) obtained in the last step16S rRNAThe sequence of the gene fragment is compared with a public database and the composition of the animal-derived species in the salmonid preparation is determined based on the homology of the sequenced sequence to known sequences in the database.
The salmonid products of the invention include salmonids, salmonid processed products and food products comprising salmonid ingredients.
In the step (b), in order to distinguish the amplification sequences of different samples in the subsequent bioinformatics analysis, different marker sequences are added in front of the 3' ends of the upstream primer and the downstream primer for marking different DNA templates, so that different samples can be conveniently mixed and sequenced.
In the step (b), the step (c),16S rRNAforward and reverse primers for gene amplification are 16 SF: AYAAGACGAGAAGACCC and 16 SR: GATTGCGCTGTTATTCC, the specific reaction conditions are as follows: pre-denaturation at 94 ℃ for 5 min, denaturation at 94 ℃ for 30 s, annealing at 53 ℃ for 30 s, extension at 72 ℃ for 60 s, 35 cycles, and final extension at 72 ℃ for 10 min.
In the step (c), a general PCR product recovery kit is adopted for purifying and recovering the amplification product of each sample.
The step (d) comprises the following steps: DNA end repair of PCR products, DNA fragment selection, 3' end A tail addition, joint bridge amplification and purification by using a Bioanalyser 2100 capillary electrophoresis apparatus and a Qubit®3.0 carry out the quality check of the sequencing library.
In step (e), the high-throughput sequencing is performed by using an Illumina-based second generation high-throughput sequencer HiSeq2500 PE 250.
In step (f), if the Salmonidae products to be tested are amplified16S rRNAThe consistency of a certain sequence of the gene fragment and a certain sequence of a certain animal in a public database is more than or equal to 97 percent, the salmonidae product to be detected contains the animal species corresponding to the sequence in the database, and if the salmonidae product to be detected is amplified, the obtained animal species is used as the salmonidae product16S rRNAAnd if the consistency of a sequence of the gene fragment and a sequence of an animal in the public database is less than 97%, the salmonid product to be detected does not contain or candidate does not contain the animal species corresponding to the sequence in the database.
The threshold value of taxonomic unit clustering (OTU) in the bioinformatics analysis in step (f) is 97%; the species is considered to be present in the sample only if it is detected in at least 2 replicates; for species not labeled in the tag, species are considered present in the sample when their relative abundance is above 2%.
Compared with the prior art, the method has the following advantages and beneficial effects:
most of the existing molecular biology detection methods can not realize synchronous and rapid identification of multiple source components in unknown samples, and only one reaction hole can detect one source component. Based on the research idea of high-throughput sequencing, the method for sequencing by using the amplicon can overcome the problem that the PCR amplification efficiency is low or the amplification cannot be carried out due to DNA degradation by amplifying the short gene segment with the length of only 255 bp, so that all main animal-derived species and impurity species in the salmonidae products can be detected in a non-specific manner, the process can be simplified, and the cost can be reduced.
Drawings
FIG. 1 is based on16S rRNAA histogram of the relative abundance of species components of a known mixture of sequences. M1-M18 correspond to M1-M18, respectively, in Table 1 below. Species abundance refers to the number of species contained in a sample, i.e., the ratio of barcode sequences representing that species to the total sequences in the sample, expressed as a percentage. The species composition and relative content of each sample can be visually seen from the figure.
Detailed Description
The present invention is further illustrated by way of examples, which are intended to be illustrative only and not to be limiting in scope. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. The various chemicals and biologicals used in the examples were all commercially available products.
Example 1
This example is an evaluation of the accuracy and sensitivity of high throughput sequencing techniques in identifying mixed species by the following assay.
1. Preparation of Mixed Salmonidae samples
Each sample was dehydrated and dried in a freeze dryer and ground into powder, and 9 fish samples of known sources and species were collected in different proportions and in different ways (fish species mixing and fish livestock and poultry mixing) based on the mass of the sample to prepare mixed samples (table 1). Meanwhile, the sample mixing ratio of 1% at the minimum is set so as to evaluate the accuracy of the high-throughput sequencing technology in the aspect of species characterization, and the detection flux and sensitivity.
TABLE 1 Salmonidae fish mixed sample preparation protocol
2. Sample genomic DNA extraction
The modified CTAB method is selected to extract the genome DNA of 18 salmonidae mixed samples. 100 mg of each mixed sample was weighed, and 500 μ L of CTAB extract (1.2% w/v CTAB, 10 mM EDTA-Na, 60 Mm Tris, 0.8M NaCl, pH = 8) and 25 μ L of proteinase K (10 mg/mL) were added. The mixture was digested by lysis at 65 ℃ for 24 h and centrifuged at 12000 rpm for 10 min. And taking the supernatant into a new 1.5 mL centrifuge tube, adding 500 mu L chloroform for extraction, and then centrifuging at 12000 rpm for 5 min. The supernatant was taken, added to two volumes of CTAB precipitation buffer (1% w/v CTAB, 10 mM EDTA-Na, 50 mM Tris, 0.8M NaCl, pH = 8), mixed well and centrifuged at 12000 rpm for 10 min. Discarding the supernatant, adding 400 μ L CTAB buffer to the precipitateThe solution (1 mM EDTA-Na, 10 mM Tris, 1M NaCl, pH = 8) was then solubilized for at least 30 min at 65 ℃. And adding 400 mu L of isopropanol into the cooled mixed solution, standing for 10 min, and centrifuging at 12000 rpm for 10 min. Washing and precipitating twice with 500 mu L70% ethanol, and after precipitation is dried, washing and precipitating with 50 mu L ddH2Dissolving O to obtain a sample DNA stock solution.
The absorbance values of the sample DNA at 230, 260 and 280 nm, and A260/A280 and A230/A260 values were measured using a nucleic acid protein analyzer, respectively, to determine the concentration and purity of the extracted DNA. Wherein the A260/A280 value is preferably 1.8-2.0.
3. PCR amplification and purification
Uniformly diluting the DNA of each sample to 10 ng/mu L, taking the DNA of the sample as a template, and adding different barcode sequences16S rRNAThe primers were subjected to PCR amplification.16S rRNAForward and reverse primers for gene amplification are 16 SF: AYAAGACGAGAAGACCC and 16 SR: GATTGCGCTGTTATTCC are provided. The amplification system was as follows: the total volume was 50. mu.L, including 25. mu.L of 2 XPCR reaction mix, 21. mu.L of ddH2O, 2. mu.L of DNA template, 1. mu.L of each of the upstream and downstream primers (10. mu.M concentration). The amplification conditions were: pre-denaturation at 94 ℃ for 4 min; followed by 35 cycles, each cycle comprising 94 ℃ denaturation 30 s, 53 ℃ annealing 30 s, 72 ℃ extension 1 min; finally, extension is carried out for 10 min at 72 ℃. And (3) carrying out gel electrophoresis on the amplification product by using 2% agarose gel, analyzing the PCR amplification product by using a gel imaging system to observe the length of the amplified target band, and preliminarily evaluating the quality and the quantity of the amplification product.
The PCR product was purified using Wizard SV Gel and PCR Clean-Up System kit (Promega, USA), and the specific procedures were performed according to the kit instructions, so that the final purified product concentration reached 20 ng/. mu.L. And (3) determining the concentration of the purified PCR product by using a Qubit 3.0 fluorescent quantitative analyzer.
4. High throughput sequencing
In the present invention, sequencing can be performed using conventional sequencing techniques and platforms.
In the present invention, library fragments that meet the in-silico sequencing standards are sequenced using the Illumina Miseq platform.
The invention is based on an Illumina Miseq sequencing platform to complete the high-throughput sequencing of the 18 samples. And (3) sequentially carrying out DNA fragmentation, target fragment end repair, fragment screening, A tail addition, linker connection, PCR and other steps on each PCR product to construct 14 sequencing libraries, and completing the construction of the sequencing libraries by using the TruSeq DNA PCR-Free Sample Preparation Kit library construction Kit. The constructed library is quantified by Qubit and Q-PCR, and is subjected to on-machine sequencing by using HiSeq2500 PE250 after the library is qualified. The cluster generation was performed on Flow cells By bridge PCR amplification, and finally the species sequences contained in each sample were obtained By Sequencing By Synthesis (SBS). Sequencing databases were constructed by searching the NCBI for the two keywords "metazoa" and "16S" to obtain the 16S nucleotide sequences of the protozoa phylum for species assignment of the quality controlled sequences.
5. Sequencing data analysis
The sequencing data obtained after the machine was unloaded was split according to the primer pairs to which different barcode sequences were added, to correctly assign to the 18 samples. Then removing the barcode and the sequences of the upstream primer and the downstream primer, and splicing the sequencing fragments of the samples by using FLASH to obtain original Tags data (Raw Tags). For the original sequence obtained by sequencing, primer and linker sequences were removed, reads with a length of less than 100 bp (e.g., primer dimer) and low mass fraction (QV < 20) were removed, and valid Tags data (Effective Tags) were obtained. And then carrying out sequence clustering by using 97% sequence consistency through Uperase software to obtain operable classification units (OTUs), taking a sequence with the highest occurrence frequency in each OTU as a representative sequence, carrying out sequence comparison with a pre-constructed sequencing database by adopting a Mothur method and using 0.8 as a set threshold value, and carrying out statistics by using a sample as a unit at the seed (species) level.
6. Analysis of results
Species relative abundance was plotted from the results obtained by high throughput sequencing (figure 1). Due to the efficiency of DNA extraction of different species and the pair16S rRNAThe amplification efficiency of the primers is different, and the number of sequences obtained by high-throughput sequencing does not have correlation with the mass ratio of the species in the actual sample. Furthermore, in the PCR amplification step for the mixed sample,the PCR product is easy to form aerosol when the air and liquid interface rub, and the aerosol can be formed to cause pollution when the reaction tube is shaken violently, the cover is opened and the sample is sucked. Because the copy quantity of the PCR product is very large, and the second-generation sequencing is very sensitive to the analysis of trace substances, the accuracy of the final experimental result is influenced by trace PCR product pollution. Thus, false positive species of different relative abundance were detected in all samples, but no false positive species were present in 18 pooled samples when the species abundance threshold was increased to 2%. Furthermore, all species present were detected in the sample consisting of 8 mixtures of species, and no foreign species were detected. In the aspect of detection sensitivity, the high-throughput sequencing technology established by the invention can detect the species with the mass ratio as low as 1 percent, and has high sensitivity. In a word, the macro-bar code technology established by the invention can be used for detecting all animal-derived species components in the mixed species at one time, and has high flux; the abundance threshold of the species is increased to 2%, all animal-derived species components in the actual sample can be accurately detected, and no other species is detected, so that the accuracy is good; in addition, this technique is highly sensitive and can detect trace species (as low as 1%) present in the mixed sample. Therefore, the method established by the invention can be used for identifying animal-derived species components of commercial salmonid products by adopting a 2% species abundance threshold.
Example 2
This example is a commercial salmonid preparation analyzed for the composition of animal-derived species by the following assay.
1. Experimental methods
A total of 32 salmonidae food products were collected from retailers, supermarkets and e-commerce platforms, essentially covering the type of salmonidae food currently on the market. DNA was extracted, and the rest was identical to example 1.
2. Analysis of results
Of the 32 collected commercial salmonidae fish foods, 29 parts (90.6%) were designated as "salmon" as the food name. The identification results show that the species mainly related to the identification results comprise Atlantic salmon: (S. salar) And humpback salmon (O. gorbuscha) (ii) a A small partThe samples (7, 8) were identified with species common names in addition to salmon, and were found by reference to the relevant introduction of salmon in books "marine species and distribution in china" and "fish taxonomy": the pink salmon is camel-back salmon (O. gorbuscha) The emperor salmon is commonly known as the salmon (O. tshawytscha) Both belong to the genus salmon in the salmonidae family; the tags for the remaining 3 samples (6, 13, 14) were more canonical, identified with a definite taxonomic name of the species, and the sequencing results were consistent with the food tags (table 3).
Samples No. 15-32 were commercially available salmonid preparations, and 17 of the 18 salmonid further processed preparations were detected as species not identified in the tag, i.e., species substitution and adulteration were present to varying degrees. According to the high throughput sequencing results (Table 3), the components of more than one species of animal origin were detected in all samples except sample No. 31. Sample No. 15-21 is a salmon can product. Wherein 15, 16 and 17 are three salmon fish canned products with different tastes and produced by the same manufacturer, and three canned samples are detected to be composed of pork and chicken, and the detected components are obviously different from the labels. Atlantic salmon were detected in samples Nos. 18-21, with only a small amount of Atlantic salmon components (4% -10% relative abundance of species) detected in samples Nos. 18-20; atlantic salmon with a species relative abundance of 61.9% was detected in sample No. 21, but a minor amount of duck meat component was also detected. Samples 22-24 are products of dried fish floss commercially identified as salmon, but all species other than salmonids were detected in the samples. Wherein 30% -40% of Mugil species are detected in No. 22 and No. 24 samples. Sample No. 25 was a dried salmon product, and it was found that pork was detected in addition to whitefish. 26. The No. 27 sample is salmon rice flour produced by two different manufacturers, wherein except for whitefish, pig-derived components are detected in the No. 26 sample; sample No. 27 had more complex species and had pork and chicken detected in addition to whitefish and rainbow trout. The commercial label of sample No. 28 is salmon noodle, and the high-throughput sequencing result shows that the salmon noodle consists of two salmonidae fishes, namely whitefish and rainbow trout; 29. 30 are intestinal preparations of two salmonids, and the detected species compositions of the two samples are consistent, wherein the ingredient table of product No. 29 is marked as consisting of salmon and surimi, but only whitefish among the fish components, chicken and pork are detected. Only sample No. 31, "salmon silk" species was single in composition, consisting of whitefish only. In the No. 32 sample 'salmon in honeydew', except for the detection of common adulterated species such as chicken and pork, 42 percent of seriola quinqueradiata is detected, and the detection caused by pollution on a production line can be eliminated.
TABLE 3 high throughput sequencing identification of commercially available Salmonidae products
While particular embodiments of the present invention have been described, those skilled in the art will recognize that many changes and modifications may be made thereto without departing from the scope or spirit of the invention. Accordingly, it is intended to cover all such changes and modifications that fall within the scope of the appended claims and equivalents thereof.
Claims (4)
1. A detection method for identifying animal-derived species components in a salmonid product by using a high-throughput sequencing technology is characterized by comprising the following steps of:
(a) extracting genome DNA from a sample to be detected;
(b) by means of a belt with upstream and downstream marks16S rRNACarrying out PCR amplification on the genome DNA in the (a) by using the universal primer to obtain a PCR amplification product;
(c) purifying the PCR amplification product and measuring the concentration;
(d) constructing a sequencing library by using the PCR amplification product reaching the concentration requirement in the previous step and carrying out quality inspection;
(e) performing high-throughput sequencing on the sequencing library of the last step to obtain16S rRNAA gene fragment sequence;
(f) obtained in the last step16S rRNAComparing the gene fragment sequence with a public database, and determining animal-derived species components in the salmonid product according to the homology of the sequencing sequence and known sequences in the database;
wherein, in step (f), the Salmonidae products to be tested are amplified16S rRNAThe consistency of a certain sequence of the gene fragment and a certain sequence of a certain animal in a public database is more than or equal to 97 percent, the salmonidae product to be detected contains the animal species corresponding to the sequence in the database, and if the salmonidae product to be detected is amplified, the obtained animal species is used as the salmonidae product16S rRNAAnd if the consistency of a sequence of the gene fragment and a sequence of an animal in the public database is less than 97%, the salmonid product to be detected does not contain or candidate does not contain the animal species corresponding to the sequence in the database.
2. The method of claim 1, wherein the salmonid products comprise salmonids, salmonid processed products, and food products comprising salmonid ingredients.
3. The method of claim 1, wherein in step (b) the top and bottom marker sequences are different marker sequences for identifying different DNA templates, thereby facilitating mixed sequencing of different samples.
4. The method of claim 1, wherein the threshold for taxonomic unit clustering (OTU) in the bioinformatic analysis in step (f) is 97%; the species is considered to be present in the sample only if it is detected in at least 2 replicates; for species not labeled in the tag, species are considered present in the sample when their relative abundance is above 2%.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113897425A (en) * | 2021-11-11 | 2022-01-07 | 广西壮族自治区食品药品检验所 | Animal-derived component detection method based on high-throughput sequencing technology and application thereof |
| CN115171515A (en) * | 2022-07-08 | 2022-10-11 | 华南理工大学 | Label reflecting food freshness and method for detecting food freshness |
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2021
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Non-Patent Citations (3)
| Title |
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| RAN RAN XING: "Application of next generation sequencing for species identification in meat and poultry products: a DNA metabarcoding approach", 《FOOD CONTROL》 * |
| 王楠: "基于DNA条码技术的食品中鲑科鱼物种成分鉴别研究", 《中国优秀学位论文全文数据库 工程科技I辑》 * |
| 王楠等: "DNA条形码技术在鲑科鱼类真伪鉴别中的应用", 《中国食品学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113897425A (en) * | 2021-11-11 | 2022-01-07 | 广西壮族自治区食品药品检验所 | Animal-derived component detection method based on high-throughput sequencing technology and application thereof |
| CN115171515A (en) * | 2022-07-08 | 2022-10-11 | 华南理工大学 | Label reflecting food freshness and method for detecting food freshness |
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Application publication date: 20210824 |
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