CN117230214A - Long-fragment universal primer composition for identifying animal-derived components in food, detection kit and identification method - Google Patents
Long-fragment universal primer composition for identifying animal-derived components in food, detection kit and identification method Download PDFInfo
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Abstract
The invention relates to a long-fragment universal primer composition for identifying animal-derived components in food, a detection kit and an identification method, and belongs to the field of biological detection. The invention provides a primer composition for identifying animal-derived components in food. The invention also provides a detection kit for identifying animal-derived components in food and a method for identifying animal-derived components in food. The primer combination can realize stable amplification of the same target sequence in the DNA of different animal species, and can realize screening and identification of different animal components through a pair of primers, thereby improving the detection efficiency of animal species components.
Description
Technical Field
The invention relates to a long-fragment universal primer composition for identifying animal-derived components in food, a detection kit and an identification method, and belongs to the field of biological detection.
Background
There are many food safety hazards in meat (product) adulteration, so there is a need for an accurate and rapid detection method for identifying adulterated meat (product), tracing animal-derived components in meat (product), especially unknown meat products, determining the authenticity and accuracy of meat components in food, and providing clear and reliable information of meat and meat products to consumers.
Seed source identification occupies a very important position in the food safety field, and current animal seed source identification methods are mainly based on 3 major classes of morphological methods, protein methods (enzyme-linked immunosorbent methods, high performance liquid chromatography) and molecular biotechnology. The morphological seed source identification method mainly judges sensory indexes such as texture structures of tissues such as fur, tooth bones and the like and smell and the like; the methods such as enzyme-linked immunosorbent assay, high performance liquid chromatography and the like are mostly based on the affinity of soluble proteins for analysis and identification; the method can not effectively and accurately identify the species sources of samples with similar varieties or appearance morphological characteristic damage or characteristic protein denaturation caused by processing treatment, and has great limitation in practical application.
The DNA information is large, the difference of species can be directly reflected on the DNA sequence, and the molecular biological method applied to the seed source identification at present mainly comprises a specific PCR method and a DNA bar code method. The specific PCR method is the most commonly used method at present, different species are identified by the existence or length of a specific amplification product, one pair of primers can only identify one specific species, and the unknown and blind detection samples can be effectively identified by multiple pairs of specific primers through multiple detection, so that the detection efficiency is low. The DNA bar code method carries out species identification through a section of short representative DNA sequence, and because applicable universal primers are very limited, the screening and identification of multiple species can not be carried out by using unified fragments, and the method is realized by combining a plurality of DNA bar codes or carrying out multiple experiments; meanwhile, the DNA bar code target sequence is short, and the resolution ratio of the species is lower than that of the long fragment. Therefore, a pair of long fragment sequence universal primers which can be used for screening and identifying animal species components in food is needed to be screened, so that rapid, efficient and accurate detection of different animal species components in food is realized.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a pair of long-fragment universal primer combinations suitable for detecting animal-derived components in food and an application method thereof. Compared with the traditional detection method, the target sequence of the universal primer in the invention is 12SrRNA+tRNA Val+16SrRNA gene region in animal mitochondrial genome, is a multicopy gene sequence, has higher thermal stability, is not limited by morphological environment, and meanwhile, the abundant interspecies variation can realize effective identification of different species.
The invention provides a long fragment universal primer composition for identifying animal-derived components in food, wherein the primer comprises a forward primer and a reverse primer, the nucleotide sequence of the forward primer is shown as SEQ ID No.1, the nucleotide sequence of the reverse primer is shown as SEQ ID No.2, wherein a base R in the sequence SEQ ID No.1 represents a degenerate base A/G, a base Y represents a degenerate base C/T, a base R in the sequence SEQ ID No.2 represents a degenerate base A/G, and a base D represents A/G/T;
SEQ ID No.1：GAGRYAAGTCGTAACAAGGT；
SEQ ID No.2：ATCACGTARGACTDTAATCGTTG。
wherein, the target gene sequence of the primer pair is positioned in the 12SrRNA+tRNA Val+16SrRNA region of mitochondria, and the primer combination is obtained by amplifying SEQ ID No.1 and SEQ ID No.2, and the length of the target gene sequence in different animal species is 1400-1700bp.
The invention also provides application of the primer composition in identifying animal-derived components in food.
The invention also provides a detection kit for identifying animal-derived components in food, which comprises the primer composition and 2 x high-fidelity enzyme premix (TaKaRa Taq TM HS Perfect Mix）、ddH 2 O。
The invention provides a method for identifying animal-derived components in food, which adopts the detection kit to detect the animal-derived components in the food, and comprises the following steps:
a. extracting genomic DNA of a meat product sample;
b. PCR amplification of the extracted DNA with primer set SEQ ID No.1 and SEQ ID No.2, and ddH with blank control 2 O replaces the DNA template;
c. b, taking a small amount of the PCR products in the step b, performing agarose gel electrophoresis detection, and judging the results;
d. purifying the PCR product remained in the step b;
e. constructing a sequencing library from the purified amplified product, and carrying out third generation single molecule nanopore sequencing:
f. and (3) performing quality control, filtering, shearing and classification annotation analysis on the sequencing result to determine animal-derived components in the sample.
Wherein, the PCR reaction system in the step b adopts a 50 mu L system comprising TaKaRa Taq TM HS Perfect Mix 25 [ mu ] L, 10mmol forward primer 1 [ mu ] L (SEQ ID No. 1), 10mmol reverse primer 1 [ mu ] L (SEQ ID No. 1), DNA template 1 [ mu ] L, 1ddH 2 O 22µL。
Wherein, the PCR reaction procedure in the step b is as follows: denaturation at 94℃for 1min, denaturation at 94℃for 5sec, annealing at 60℃for 2sec, extension at 68℃for 40 sec, cycling 35 times, and final extension for 1min.
Wherein, the PCR amplification result in the step c is judged according to the following method: for the electrophoresis result of PCR, the sample is amplified into about 1400-1700bp bands, and a blank control has no amplified bands, which indicates that the PCR reaction system works normally and can carry out the subsequent steps, otherwise, the PCR system needs to be reconfigured for detection.
In the step f, sequencing data processing is carried out by using sequencing platform self-contained sequencing software QPreasy to complete base identification, sequencing data quality evaluation and data filtering; removing sequence joints by using a directop or data analysis tool with the same function, and filtering sequencing data which are too short and too long; then removing the chimeric sequences in the data using yacrd or equivalent data analysis tools; and finally, comparing the sequencing data with NCBI database by using Minmap2 or a data analysis tool with the same function, and carrying out species classification annotation on the representative DNA sequences in the result file, wherein the species with the highest similarity is the species source component corresponding to the DNA sequences.
The beneficial effects of the invention are as follows:
compared with a specific PCR method and an existing DNA bar code method, the universal primer combination can realize stable amplification of the same target sequence in DNAs of different animal species, and screening and identification of different animal components are realized through a pair of primers, so that the detection efficiency of animal species components is improved. The target sequence of the primer pair has the length of 1400-1700bp in different animal species, contains partial variable regions of 12S rRNA and 16S rRNA genes, contains more information sites and has higher species resolution compared with DNA bar codes of short fragments.
Drawings
FIG. 1 is a diagram of reference sequence alignment of target sequences of interest in 12 representative animal species of example 1;
FIG. 2 is a graph showing the results of PCR amplification of the primer pairs SEQ ID No.1 and SEQ ID No.2 in the meat product collected in example 2;
FIG. 3 is a graph showing the results of PCR amplification of the standard DNA barcode primer pair LCO1490 and HCO2198 of example 2 on meat products collected;
FIG. 4 is a graph showing the alignment of the primer pairs SEQ ID No.1 and SEQ ID No.2 of example 2 to target sequences of interest in meat products collected.
Detailed Description
The following examples are provided to further illustrate the invention but are not to be construed as limiting the invention, and modifications or alterations to the invention are within the scope of the invention without departing from the spirit and nature of the invention.
Example 1 design of primers for detection and screening of animal Components in food and screening of target Gene sequences
The animal species are rich and various, some representative animals are selected from animal species which are common in foods, such as livestock, poultry and aquatic animals, the reference target sequences of the animals are downloaded, the reference target sequences of the animals are compared and analyzed, the comparison result is shown in figure 1, the target sequences show the characteristics of conservation at two ends and changeability in middle areas among different animal species, and a pair of universal primers are designed in the conservation areas at two ends of the sequences by taking the target sequences as templates. The forward primer and the reverse primer respectively contain 2 mutation sites in reference sequences of different animal species, wherein the mutation sites adopt degenerate bases, base R represents degenerate base A/G, base Y represents degenerate base C/T, and base D represents A/G/T. The amplified region of the universal primer has high interspecific diversity, different species have different mutation sites, and each species can be distinguished according to the sequence. Wherein the sequences of the forward and reverse universal primers are shown as SEQ ID No.1 and SEQ ID No.2 in the sequence table.
SEQ ID No.1：GAGRYAAGTCGTAACAAGGT
SEQ ID No.2：ATCACGTARGACTDTAATCGTTG
The mitochondrial sequence has a rapid evolution rate, has wide polymorphism in species and among species, can be applied to the identification of similar species, has good stability because mitochondrial DNA is a circular DNA molecule with multiple copy numbers, and is suitable for the detection of processed foods. The prior art often performs species identification by amplifying a short DNA sequence on mitochondria, which has limited species resolution. As shown in FIG. 1, the target gene sequence of the universal primer pair provided by the invention is positioned in the 12SrRNA+tRNA Val+16SrRNA region, the length of the target gene sequence in different animal species is 1400-1700bp, more information sites are covered, and the target species can be better distinguished.
Example 2: general primer pair universality and accuracy verification
1. Sample collection and DNA extraction
12 parts of single-ingredient meat products with different source ingredients are collected from the market, and the source ingredients of the 12 parts of meat products are respectively: cattle, buffalo, yaks, pigs, goats, sheep, horses, deer, chickens, ducks, geese, atlantic salmon. Genomic DNA of these 12 meat samples was extracted using the blood/cell/tissue genomic DNA extraction kit from the company, phylogenetic sciences, ltd, with reference to the extraction kit instructions, or other well-known DNA extraction methods or commercial DNA extraction kits with the same efficacy. After the extraction of the DNA template is completed, the concentration of the DNA template is measured, and the genomic DNA of each sample is diluted to 20-30 ng/. Mu.L for subsequent procedures.
2. PCR amplification
The target sequence of PCR amplification is mitochondrial 12SrRNA+tRNA Val+16SrRNA gene fragment, and the forward and reverse general primer sequences are shown as SEQ ID No.1 and SEQ ID No. 2. The reaction system was 50. Mu.L, comprising 2 XPCR high-fidelity enzyme premix (TaKaRa Taq, takara Shuzo) TM HS Perfect Mix) 25 [ mu ] L, 10mmol forward primer 1 [ mu ] L (SEQ ID No. 1), 10mmol reverse primer 1 [ mu ] L (SEQ ID No. 1), sample DNA template 1 [ mu ] L (ddH is used for blank control) 2 O is used as a template) ddH 2 O 22µL。
PCR reaction conditions: denaturation at 94℃for 1min, denaturation at 94℃for 5sec, annealing at 60℃for 2sec, extension at 68℃for 40 sec, cycling 35 times, and final extension for 1min.
Meanwhile, the general primer pair LCO1490 (SEQ ID No.3: GGTCAACAAATCATAAAGATATTGG) and HCO2198 (SEQ ID No.4: TAAACTTCAGGGTGACCAAAAAATCA) which are widely applied at present are adopted for PCR amplification, the reaction system is referred to the reaction system, the PCR reaction conditions are 94 ℃ denaturation of 1min,94 ℃ denaturation of 5sec,55 ℃ annealing of 2sec,68 ℃ extension of 30 sec, 35 times of circulation and final extension of 1min.
3. Agarose gel electrophoresis analysis
And 5 mu L of PCR products are sucked for 1.5% agarose gel electrophoresis detection, and the amplification products are observed under an ultraviolet gel imaging system, wherein the amplification results of the universal primer pair SEQ ID No.1 and SEQ ID No.2 are shown in figure 2, and the amplification results of the universal primer pair SEQ ID No.3 and SEQ ID No.4 are shown in figure 3. Lanes M are DNA markers, and lanes 1-12 are amplified bands of 12 parts of meat products of different animal species source components, respectively, using a universal primer pair.
As can be seen from FIGS. 2 and 3, the universal primer pair SEQ ID No.1 and SEQ ID No.2 can amplify DNA fragments of 1400-1700bp in different animal species, and the DNA bands are bright and have similar brightness, and the blank control has no amplified band. The general primer pair SEQ ID No.1 and SEQ ID No.2 has good amplification efficiency and similar amplification efficiency for different animal genome DNA.
4. General primer accuracy verification
The amplified PCR products of the general primer pairs SEQ ID No.1 and SEQ ID No.2 of example 2.2 were subjected to sanger sequencing verification, the sequencing result of the target sequence in 12 meat products is shown in SEQ ID No.5-SEQ ID No.16, the sequencing sequence comparison result is shown in FIG. 4, and the obtained DNA sequence sequencing result is subjected to comparison annotation with the sequence of NCBI database, and the comparison annotation result is shown in the following table:
sequencing results of amplified products
| Sequence number | Sample name | Sequence numbering | Authentication result | Similarity degree |
| 1 | (Cattle) | SEQ ID No.5 | Bos taurus | 99.34% |
| 2 | Buffalo (Buffalo) | SEQ ID No.6 | Bubalus bubalis | 99.54% |
| 3 | Yak | SEQ ID No.7 | Bos grunniens | 99.61% |
| 4 | Pig | SEQ ID No.8 | Sus scrofa | 99.60% |
| 5 | Chicken (chicken) | SEQ ID No.9 | Gallus gallus | 99.49% |
| 6 | Duck | SEQ ID No.10 | Anas platyrhynchos | 99.35% |
| 7 | Goose-like body | SEQ ID No.11 | Anser cygnoides | 99.36% |
| 8 | Sheep | SEQ ID No.12 | Ovis aries | 99.41% |
| 9 | Goat | SEQ ID No.13 | Capra hircus | 99.08% |
| 10 | Horse | SEQ ID No.14 | Equus caballus | 99.67% |
| 11 | (Red Deer) | SEQ ID No.15 | Cervus canadensis | 98.56% |
| 12 | Atlantic salmon | SEQ ID No.16 | Salmo salar | 99.69% |
Example 3A test kit for the identification of animal derived components in food products
The detection kit consists of the following 4 parts:
| kit composition | Storage temperature | 50 times |
| Universal forward primer (SEQ ID No. 1) | -20℃ | 50µL |
| Universal reverse primer (SEQ ID No. 2) | -20℃ | 50µL |
| 2 XPCR high-fidelity enzyme premix | -20℃ | 1250µL |
| ddH 2 O | Normal temperature | 1200µL |
The application method of the kit comprises the following steps:
1 DNA extraction
The extraction method of the blood/cell/tissue genome DNA of Tiangen Biochemical technology Co., ltd was used with reference to the extraction kit instruction.
2 PCR amplification
The PCR amplification system is 50 mu L, and comprises PCR high-fidelity enzyme premix (TaKaRa Taq of Baozhen TM HS Perfect Mix) 25 [ mu ] L, 10mmol forward primer (SEQ ID No. 1) 1 [ mu ] L, 10mmol reverse primer (SEQ ID No. 2) 1 [ mu ] L, sample DNA template 1 [ mu ] L (ddH is used for blank control 2 O is used as a template) ddH 2 O 22µL。
PCR reaction conditions: denaturation at 94℃for 1min, denaturation at 94℃for 5sec, annealing at 60℃for 2sec, extension at 68℃for 40 sec, cycling 35 times, and final extension for 1min.
3. Electrophoretic analysis
And (3) taking a small amount of PCR amplification products to carry out electrophoresis detection by using a electrophoresis agarose gel, and observing the conditions of the amplification products under an ultraviolet gel imaging system. The blank control has no band, and the sample has a bright band near 1400-1700bp, so that the sample can be used for subsequent experiments; blank control has no strip, and a sample has no strip, so that PCR amplification is required to be carried out again; the blank has bands, and the sample has bright bands at 1400-1700bp, so that the detection test needs to be carried out again.
4. Third generation sequencing: the PCR amplification products were purified using the Agencourt AMPure XP magnetic bead method or other methods recognized to have equivalent efficacy. And then constructing a PCR product sequencing library by sequentially applying a library-building kit and a sequencing kit, and performing third-generation sequencing by using a nanopore sequencing platform.
5. Data analysis
The sequencing platform is used for completing base identification, sequencing data quality evaluation and data filtering by using tools such as sequencing software QPreasy; then, the pore is used for removing the reserved sequence linker, and sequencing data which is too short and too long is filtered; then removing the chimeric sequences in the data using yacrd; and finally, comparing the sequencing data with an NCBI database by using Minmap2, and carrying out species classification annotation on the representative DNA sequences in the result file, wherein the species with the highest similarity is the species source component corresponding to the sequences.
Example 4 screening and identification of animal species derived Components in commercial samples Using detection kit
1. Sample collection and DNA extraction
5 parts of common meat products are purchased from the market and comprise 2 parts of single-component meat source samples, namely 1 part of mutton soup can (goat component), 1 part of air-dried yak meat (yak component), 3 parts of multi-component mixed meat source samples, 1 part of pork sausage (chicken and beef component), 1 part of fine meat egg roll (pig and chicken components) and flavor meat strings (ducks and sheep). 5 samples are broken into evenly mixed meat paste, and the genomic DNA of the samples is extracted by adopting a blood/cell/tissue genomic DNA extraction kit of Tiangen biochemical technology Co.
2 PCR amplification
The PCR amplification system and the PCR reaction procedure were as in example 3.2.
3. Electrophoretic analysis
The PCR products from step 2 were analyzed by electrophoresis, and the specific procedure was described in section 3 of example 3.
4. Three-generation sequencing
Product purification, library construction and sequencing were as described in example 3.4.
5. Data analysis
Sequencing data analysis was the same as in section 5 of example 3, and the final analysis and identification results are shown in the following table. Through kit detection and data analysis, animal components marked in 5 sample labels are successfully detected, and the invention proves that the method can be used for efficiently and accurately screening and identifying animal species source components in foods.
Screening and identifying result of animal seed source components in commercial sample
| Sample name | Labeling of seed source components | Authentication result | |
| 1 | Mutton soup can | Goat | Capra hircus |
| 2 | Air-dried yak meat | Yak | Capra hircus |
| 3 | Sausage with meat flower | Chicken and cow | Gallus gallus、Bos taurus |
| 4 | Refined meat egg roll | Pig and chicken | Sus scrofa 、Gallus gallus |
| 5 | Flavor big meat shashlik | Duck, sheep | Anas platyrhynchos、Ovis aries |
Compared with the existing method for identifying animal sources of foods, the technology of the invention carries out seed source identification detection based on mitochondrial genome DNA sequences, has better thermal stability and enough interspecific variation, can accurately identify the animal sources without depending on the appearance and shape of the samples, can stably detect processed foods, and widens the sample range of the existing seed source identification. Meanwhile, compared with other methods, a pair of detection primer pairs can only identify and screen a specific animal component, the universal primer provided by the invention has better species universality and accuracy, and detection of the same target sequence in different animal species can be completed through a pair of universal primer pairs, so that simultaneous screening and identification of multiple animal species source components in a single reaction can be realized, and higher detection efficiency is realized. In addition, the length of the target sequence of the universal primer pair in different animals reaches about 1400-1700bp, more site information is covered, and higher species resolution is achieved. In addition, the invention combines the long-fragment universal primer with the third-generation single-molecule nanopore sequencing technology, realizes splice-free rapid analysis of detection data and real-time output of results, solves the problems of high difficulty and the like of the blind detection technology in the prior detection technology, and improves the detection efficiency and screening and identification accuracy of animal-derived components in food.
Claims (9)
1. A long fragment universal primer composition for identifying animal-derived components in food products, characterized in that: the primer comprises a forward primer and a reverse primer, wherein the nucleotide sequence of the forward primer is shown as SEQ ID No.1, the nucleotide sequence of the reverse primer is shown as SEQ ID No.2, wherein a base R in the sequence SEQ ID No.1 represents a degenerate base A/G, a base Y represents a degenerate base C/T, a base R in the sequence SEQ ID No.2 represents a degenerate base A/G, and a base D represents A/G/T;
SEQ ID No.1：GAGRYAAGTCGTAACAAGGT；
SEQ ID No.2：ATCACGTARGACTDTAATCGTTG。
2. the primer composition according to claim 1, wherein: the target gene sequence of the primer pair is located in the 12SrRNA+tRNA Val+16SrRNA region of mitochondria, and is obtained by amplifying SEQ ID No.1 and SEQ ID No.2 by the primer combination, and the length of the target gene sequence in different animal species is 1400-1700bp.
3. Use of the primer composition according to claim 1 or 2 for identifying animal-derived components in food.
4. A test kit for identifying animal-derived components in a food product, characterized by: comprising the primer composition according to claim 1 or 2, 2 XHi-Fi enzyme premix TaKaRa Taq TM HS Perfect Mix、ddH 2 O。
5. A method of identifying animal derived components in a food product, comprising: the method for detecting animal-derived components in food by using the detection kit of claim 4 comprises the following steps:
a. extracting genomic DNA of a meat product sample;
b. PCR amplification of the extracted DNA with primer set SEQ ID No.1 and SEQ ID No.2, and ddH with blank control 2 O replaces the DNA template;
c. b, taking a small amount of the PCR products in the step b, performing agarose gel electrophoresis detection, and judging the results;
d. purifying the PCR product remained in the step b;
e. constructing a sequencing library from the purified amplified product, and carrying out third generation single molecule nanopore sequencing:
f. and (3) performing quality control, filtering, shearing and classification annotation analysis on the sequencing result to determine animal-derived components in the sample.
6. The method of identifying animal derived ingredients in food products according to claim 5, wherein: in the step b, a PCR reaction system adopts a 50 mu L system, and comprises TaKaRa Taq TM HS Perfect Mix 25 [ mu ] L, 10mmol forward primer 1 [ mu ] L (SEQ ID No. 1), 10mmol reverse primer 1 [ mu ] L (SEQ ID No. 2), DNA template 1 [ mu ] L, 1ddH 2 O 22µL。
7. The method of identifying animal derived ingredients in food products according to claim 5, wherein: the PCR reaction procedure in step b is: denaturation at 94℃for 1min, denaturation at 94℃for 5sec, annealing at 60℃for 2sec, extension at 68℃for 40 sec, cycling 35 times, and final extension for 1min.
8. The method of identifying animal derived ingredients in food products according to claim 5, wherein: the PCR amplification result in step c is judged as follows: for the electrophoresis result of PCR, the sample is amplified into about 1400-1700bp bands, and a blank control has no amplified bands, which indicates that the PCR reaction system works normally and can carry out the subsequent steps, otherwise, the PCR system needs to be reconfigured for detection.
9. The method of identifying animal derived ingredients in food products according to claim 5, wherein: in the step f, sequencing data processing is carried out by using sequencing platform self-contained sequencing software QPreasy to complete base identification, sequencing data quality evaluation and data filtering; removing sequence joints by using a directop or data analysis tool with the same function, and filtering sequencing data which are too short and too long; then removing the chimeric sequences in the data using yacrd or equivalent data analysis tools; and finally, comparing the sequencing data with NCBI database by using Minmap2 or a data analysis tool with equivalent function, and carrying out species classification annotation on the representative DNA sequences in the result file, wherein the species with the highest similarity is the species source component corresponding to the sequences in the sample.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102876805A (en) * | 2012-10-25 | 2013-01-16 | 苏州市红冠庄国药饮片有限公司 | Primer system for PCR (polymerase chain reaction) identification for deer, pig, cow, sheep, horse, donkey, rabbit and chicken |
| CN104178570A (en) * | 2014-08-12 | 2014-12-03 | 苏州红冠庄国药股份有限公司 | Primer system for PCR (Polymerase Chain Reaction) identification of bone tissue of animal, such as deer, pig, horse, donkey, cattle and sheep |
| CN110029177A (en) * | 2019-05-14 | 2019-07-19 | 中国计量大学 | It is a kind of to identify cephalopodous DNA bar code primer and its application method |
| CN110283885A (en) * | 2019-07-10 | 2019-09-27 | 福建省水产研究所（福建水产病害防治中心） | Clown fish mitochondria 16S rRNA gene complete sequence amplimer and its amplification method |
| CN113430279A (en) * | 2021-08-04 | 2021-09-24 | 华中农业大学 | DNA (deoxyribonucleic acid) macro-barcode detection target sequence, detection kit and detection method for screening animal provenance components in meat products |
-
2023
- 2023-11-16 CN CN202311523425.6A patent/CN117230214B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102876805A (en) * | 2012-10-25 | 2013-01-16 | 苏州市红冠庄国药饮片有限公司 | Primer system for PCR (polymerase chain reaction) identification for deer, pig, cow, sheep, horse, donkey, rabbit and chicken |
| CN104178570A (en) * | 2014-08-12 | 2014-12-03 | 苏州红冠庄国药股份有限公司 | Primer system for PCR (Polymerase Chain Reaction) identification of bone tissue of animal, such as deer, pig, horse, donkey, cattle and sheep |
| CN110029177A (en) * | 2019-05-14 | 2019-07-19 | 中国计量大学 | It is a kind of to identify cephalopodous DNA bar code primer and its application method |
| CN110283885A (en) * | 2019-07-10 | 2019-09-27 | 福建省水产研究所（福建水产病害防治中心） | Clown fish mitochondria 16S rRNA gene complete sequence amplimer and its amplification method |
| CN113430279A (en) * | 2021-08-04 | 2021-09-24 | 华中农业大学 | DNA (deoxyribonucleic acid) macro-barcode detection target sequence, detection kit and detection method for screening animal provenance components in meat products |
Non-Patent Citations (9)
| Title |
|---|
| LI YANG等: "Species identification through mitochondrial rRNA genetic analysis", SCI REP, vol. 4, pages 4089 * |
| MICHAEL L. ROMAGNOLI等: "Evolutionary Relationships Among Old World Fruitbats (Megachiroptera: Pteropodidae) Based on 12S rRNA, tRNA Valine, and 16S rRNA Gene Sequences", JOURNAL OF MAMMALIAN EVOLUTION, vol. 7, no. 4, pages 259 - 284 * |
| SAPTO ANDRIYONO等: "The Jawa and Bali Island Marine Fish Molecular Identification to Improve 12S rRNA-tRNA Valin-16S rRNA Partial Region Sequences on the GenBank Database", THALASSAS: AN INTERNATIONAL JOURNAL OF MARINE SCIENCES, vol. 36, no. 2, pages 343 * |
| THOMAS ZIEGLER等: "Molecular phylogeny and evolutionary history of Southeast Asian macaques forming the M-silenus group", MOL PHYLOGENET EVOL, vol. 42, no. 3, pages 807 - 816, XP005910866, DOI: 10.1016/j.ympev.2006.11.015 * |
| 刘元峰: "宏条形码分子标记12S rRNA和16S rRNA在哺乳动物混合检材种源鉴定中的应用", 中国优秀硕士学位论文全文数据库基础科学辑, no. 2, pages 006 - 3086 * |
| 唐善虎等: "用LAMP法快速鉴别熟制牦牛肉饼中的猪肉和鸡肉成分", 西南民族大学学报(自然科学版), vol. 44, no. 3, pages 237 - 242 * |
| 戴凡炜等: "三株真菌拮抗菌的鉴定及其对桑椹菌核病的生防效果", 广东蚕业, vol. 51, no. 07, pages 1 - 4 * |
| 王楠: "基于DNA条码技术的食品中鲑科鱼物种成分鉴别研究", 中国优秀硕士学位论文全文数据库工程科技Ⅰ辑, no. 1, pages 024 - 529 * |
| 马媛媛: "蓝蛸Octopus cyanea和栗色蛸Octopus conispadiceus的线粒体基因组研究", 中国优秀硕士学位论文全文数据库农业科技辑, no. 7, pages 052 - 38 * |
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