CN110184330B - Method for synchronously detecting various meat-derived components by using cross primer isothermal amplification reaction system - Google Patents
Method for synchronously detecting various meat-derived components by using cross primer isothermal amplification reaction system Download PDFInfo
- Publication number
- CN110184330B CN110184330B CN201910535636.9A CN201910535636A CN110184330B CN 110184330 B CN110184330 B CN 110184330B CN 201910535636 A CN201910535636 A CN 201910535636A CN 110184330 B CN110184330 B CN 110184330B
- Authority
- CN
- China
- Prior art keywords
- meat
- primer
- cpa
- reaction
- primers
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 235000013372 meat Nutrition 0.000 title claims abstract description 45
- 238000006243 chemical reaction Methods 0.000 title claims abstract description 25
- 238000000034 method Methods 0.000 title claims abstract description 17
- 238000011901 isothermal amplification Methods 0.000 title claims abstract description 13
- 238000001514 detection method Methods 0.000 claims abstract description 24
- 238000011330 nucleic acid test Methods 0.000 claims abstract description 10
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims abstract description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 8
- KWIUHFFTVRNATP-UHFFFAOYSA-N Betaine Natural products C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 claims abstract description 4
- KWIUHFFTVRNATP-UHFFFAOYSA-O N,N,N-trimethylglycinium Chemical compound C[N+](C)(C)CC(O)=O KWIUHFFTVRNATP-UHFFFAOYSA-O 0.000 claims abstract description 4
- 229960003237 betaine Drugs 0.000 claims abstract description 4
- 239000007795 chemical reaction product Substances 0.000 claims abstract description 4
- 239000011535 reaction buffer Substances 0.000 claims abstract description 3
- 241000283074 Equus asinus Species 0.000 claims description 20
- 241001494479 Pecora Species 0.000 claims description 18
- 241000282485 Vulpes vulpes Species 0.000 claims description 15
- 241000282421 Canidae Species 0.000 claims description 4
- 239000000523 sample Substances 0.000 claims description 4
- 239000000243 solution Substances 0.000 claims description 3
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 2
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims description 2
- 235000013305 food Nutrition 0.000 abstract description 6
- 238000011160 research Methods 0.000 abstract description 4
- 230000001360 synchronised effect Effects 0.000 abstract description 4
- 238000003745 diagnosis Methods 0.000 abstract description 2
- 108020004414 DNA Proteins 0.000 description 17
- 230000003321 amplification Effects 0.000 description 16
- 238000003199 nucleic acid amplification method Methods 0.000 description 16
- 108090000623 proteins and genes Proteins 0.000 description 10
- 239000000047 product Substances 0.000 description 9
- 238000013461 design Methods 0.000 description 8
- 241000894007 species Species 0.000 description 8
- 108020004707 nucleic acids Proteins 0.000 description 7
- 102000039446 nucleic acids Human genes 0.000 description 7
- 150000007523 nucleic acids Chemical class 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 238000005516 engineering process Methods 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 3
- 108020005196 Mitochondrial DNA Proteins 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 230000027455 binding Effects 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000002093 peripheral effect Effects 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 108010067770 Endopeptidase K Proteins 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 239000005662 Paraffin oil Substances 0.000 description 1
- 239000007984 Tris EDTA buffer Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 235000015278 beef Nutrition 0.000 description 1
- BKHZIBWEHPHYAI-UHFFFAOYSA-N chloroform;3-methylbutan-1-ol Chemical compound ClC(Cl)Cl.CC(C)CCO BKHZIBWEHPHYAI-UHFFFAOYSA-N 0.000 description 1
- ZYWFEOZQIUMEGL-UHFFFAOYSA-N chloroform;3-methylbutan-1-ol;phenol Chemical compound ClC(Cl)Cl.CC(C)CCO.OC1=CC=CC=C1 ZYWFEOZQIUMEGL-UHFFFAOYSA-N 0.000 description 1
- -1 colloidal gold nucleic acid Chemical class 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 230000010460 detection of virus Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 238000002205 phenol-chloroform extraction Methods 0.000 description 1
- 235000015277 pork Nutrition 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000037452 priming Effects 0.000 description 1
- 235000020995 raw meat Nutrition 0.000 description 1
- 210000003705 ribosome Anatomy 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
A method for synchronously detecting various meat-derived components by a cross primer isothermal amplification reaction system belongs to the technical field of molecular diagnosis. Which comprises the following steps: 1) Adding a basic CPA system into a reaction tube:BstDNA polymerase reaction buffer, betaine and Mg 2+ ，BstDNA polymerase, ddH 2 O; a set of CPA primer systems; different meat source DNA; 2) The reaction tube is taken out after being amplified in a water bath kettle with constant temperature of 63 ℃ for 60min, and after the specific externally-labeled primers of each meat source are added for 6s/a-n, the reaction tube is reacted in a water bath with the temperature of 93 ℃ for 3min, and the reaction product is taken out to be tested on a colloidal gold nucleic acid test strip. According to the invention, the number of research objects is not considered, and only one CPA primer system is matched with externally-added marked primers of different species, so that synchronous detection of various meat-derived components is realized, and the method has important reference application value for meeting the current food safety field rapid detection market and supervision.
Description
Technical Field
The invention belongs to the technical field of molecular diagnosis, and particularly relates to a method for synchronously detecting various meat-derived components by a cross primer isothermal amplification reaction system.
Background
Identification of the source of meat animals affects the fields of food and meat production and processing, trade markets, catering industry, and the like. Meat food safety concerns are growing over alternatives to low-priced meats such as pork, and meats such as beef, donkey, mutton, and the like, which are more expensive. Fraudulent labelling of meat compromises consumer interest. Meat verification is one of the main fields of quality and safety of animal-derived foods. The importance of development research based on methods of identification processing and raw meat species is becoming increasingly prominent.
CPA is a nucleic acid isothermal amplification technology originally developed by Hazhou Yongda Biotechnology Co., ltd (WO 2010/080691 A1). Traditional single-cross primer CPA amplification systems rely on DNA polymerase with strand displacement function and a set of primers designed based on target sequences: a cross primer (2 a1 s) for introducing additional priming site amplification during each round of amplification; two labeled primers (2 a, 3 a) are involved in amplification and are used for detection; a pair of peripheral primers (4 s,5 a) separates the single stranded sequences. Amplification produces "antigen" products (2 a x 1s3s/2s1a3a x) with binding to specific binding sites of the nucleic acid strip detection line under temperature conditions suitable for enzymatic amplification.
The research of the CPA technology is applied to the detection of viruses, prokaryotes, eukaryotes and the like in the fields of food medicine and epidemic prevention quarantine. In the application of detecting meat-derived components, the traditional CPA amplification system can only design a specific CPA detection system aiming at one meat source, and the meat source to be detected is distinguished from other meat sources by using a colloidal gold nucleic acid test strip or other detection modes, if the detection of multiple meat-derived components needs to be carried out, the design of a plurality of sets of specific primer systems and multiple experimental operations are needed, and the traditional CPA amplification system has limitations in the aspects of rapid detection of meat source adulteration or other detection applications.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention aims at providing a technical scheme of a method for synchronously detecting various meat-based components by a cross primer isothermal amplification reaction system.
The method for synchronously detecting various meat-derived components by using the cross primer isothermal amplification reaction system is characterized by comprising the following steps of:
1) Adding a basic CPA system into a reaction tube:BstDNA polymerase reaction buffer, betaine and Mg 2+ ，BstDNA polymerase, ddH 2 O; a set of CPA primer systems; different meat source DNA;
2) The reaction tube was amplified in a 63 ℃ thermostat water bath for 60min, then taken out, and after adding the specific additional labeled primers for each meat source 6s/a-n (n=1, 2, 3.) the reaction tube was reacted in a 93 ℃ water bath for 3min, and the reaction product was tested on a colloidal gold nucleic acid test strip.
The method for synchronously detecting a plurality of meat-derived components by using the cross primer isothermal amplification reaction system is characterized in that the CPA primer system in the step 1) is as follows: the 5 universal primers designed by the same gene fragments of different meat sources comprise a cross primer 2a1s, two inner primers 3a and 2a and a pair of peripheral primers 4s and 5a, wherein any one of the inner primers 3a and 2a is marked with one.
The method for synchronously detecting a plurality of meat-derived components by using the cross primer isothermal amplification reaction system is characterized in that in the step 2), the specific externally-labeled primers 6s/a-n of each meat source are externally-labeled detection primers designed according to specific gene fragments of different meat sources to be detected.
The method for synchronously detecting various meat-derived components by using the cross primer isothermal amplification reaction system is characterized in that the detection area of the colloidal gold nucleic acid test strip in the step 2) can be provided with antigens/antibodies combined with different additional markers of the specific additional marker primers 6s/a-n of each meat source so as to block products of different meat sources, thereby displaying results at different detection line positions.
The invention takes various meat-derived DNA as an object, combines a nucleic acid test strip detection technology to establish a CPA system amplification technology, and skillfully changes one mark in the system into a thought of adding a marked primer after amplification reaction. The synchronous detection of various meat-derived components can be realized by only matching one CPA primer system with externally-added marked primers of different species without considering the number of research objects, and the method has important reference application value for meeting the current food safety field rapid detection market and supervision.
Drawings
FIG. 1 is a schematic diagram of a method for synchronously detecting various meat-based components by a CPA reaction system;
the CPA system optimized in FIG. 2 is a diagram of the detection result of the synchronous detection of donkey, sheep and fox meat DNA on a colloidal gold nucleic acid test strip.
0-ddH in the figure 2 O+donkey, sheep, fox plus markers, 1-donkey DNA+donkey plus markers, 2-donkey DNA+sheep plus markers, 3-donkey DNA+fox plus markers, 4-sheep DNA+donkey plus markers,5-sheep DNA+sheep additional mark, 6-sheep DNA+fox additional mark, 7-fox DNA+donkey additional mark, 8-fox DNA+sheep additional mark, 9-fox DNA+fox additional mark.
Detailed Description
The invention is further illustrated by the following examples.
Example 1
1. Material and apparatus
1.1 Experimental materials
Fresh animal meat (mutton, donkey meat purchased from Hangzhou farmer market; fox meat supplied by a farm); STE buffer, SDS, phenol-chloroform-isoamyl alcohol (25:24:1), chloroform-isoamyl alcohol (24:1) (Noryang Biotechnology, hangzhou Co., ltd.); 10×Bst buffer、BstDNA polymerase (beijing bayer di biotechnology limited); absolute ethyl alcohol, primer, proteinase K (12 Mg/mL), 5 mol/L NaCl solution, TE buffer, dNTPs and Mg 2+ 、ddH 2 O, 5 mol/L betaine, paraffin oil (Shanghai engineering Co., ltd.).
1.2 Instrument and equipment
Nanodrop-2000 nucleic acid protein analyzer (Thermo company), thermostatic water bath, centrifuge (Thermo company), XH-C vortex mixer.
2. Experimental method
2.1 Extraction of meat source DNA
Cutting fresh meat, extracting DNA of each meat source to be detected by using an improved phenol-chloroform extraction method, measuring concentration and purity by using a nucleic acid protein analyzer, and obtaining OD (optical density) by using a nucleic acid protein analyzer 260 /OD 280 The value is the reference value, and the proper DNA sample is frozen and stored at-20 ℃ for standby.
2.2 CPA primer for fox, sheep and donkey and externally-labeled primer design
Mitochondrial genes of the species were selected as target segments for the design primers. The whole mitochondrial gene sequences of sheep, fox and donkey are downloaded from NCBI database, DNAMAN software is utilized for comparison, about 100bp difference gene sequences of three species are selected as a section for designing additional labeled Primer design, and 5 universal primers are designed in combination with the Primer software in the consistency region around the sections as primers participating in CPA amplification. The possibility of interaction between each primer is analyzed in Oligo software, and the designed primers are modified according to GC, tm value, deltaG and other parameters to reduce the possibility of the binding of the externally labeled primer and the primer in the detection probe.
2.3 CPA amplification and colloidal gold nucleic acid test strip test
And (3) carrying out effectiveness test on fox, sheep and donkey template DNA by using a CPA reaction system of 20 mu L, adding the 20 mu LCPA reaction system (table 1) into a reaction tube, carrying out isothermal amplification for 60min in a water bath kettle of 63 ℃, adding 3 mu L of externally-added marked primers of different species into the corresponding tube after amplification, taking out after reacting for 3min at 93 ℃, and testing the detection products of 2a1s3 a/6 s-n on a colloidal gold nucleic acid test strip.
Table 1 20 mu LCPA reaction system
。
3. Results and analysis
3.1 CPA primer of fox, sheep and donkey and design result of externally-added labeled primer
Selecting whole mitochondrial gene alignment of three species of fox, sheep and donkey from NCBI database, wherein the gene position is16S ribosomal RNAThe gene segment position was used as the target segment for the design of CPA primers. Three additional labeled primers (table 2) of fox, sheep and donkey are designed by selecting a section of differential gene sequence of about 100bp of the gene sequence position, and the additional labeled primers of each species can be found out by comparing the nucleic acid database. 5 universal primers were designed in their front and rear positions as primers involved in CPA amplification (Table 3).
TABLE 2 sequence of externally labeled primers for fox, sheep and donkey
TABLE 3 primer list of general CPA System for fox, sheep and donkey
。
3.2 CPA amplification and colloidal gold nucleic acid test strip test results
The method comprises the steps of amplifying DNA of foxes, sheep and donkey by using a CPA system, adding corresponding externally-labeled primers into the obtained amplified products, and combining colloidal gold nucleic acid detection test paper strips to verify the effectiveness of the design method, wherein initial CPA system detection results show that target products of foxes, sheep and donkey can be amplified but are not strong in positivity, and the quantity of products complementary with externally-labeled probes in the products is increased and the quantity of 2a1s3s products competing for the strips is reduced by changing the concentration ratio of 3a and 2 a. The results show that under experimental conditions, the corresponding reaction products of the species to be detected can be amplified by using the CPA primer amplification system in a ratio of 2a:3a=1:3, and the specific externally-added labeled primers can be effectively combined with the amplified products and show better positive results on the test strip (fig. 2). Therefore, the CPA detection method which converts one marked primer into an externally marked primer can realize the synchronous detection of the multisource components, and provides a new solution for synchronously detecting the multisource components by isothermal amplification.
Claims (1)
1. The method for synchronously detecting various meat-derived components by using a cross primer isothermal amplification reaction system is characterized by comprising the following steps of:
1) Adding a basic CPA system into a reaction tube: bst DNA polymerase reaction buffer solution, betaine and Mg 2+ BstDNA polymerase, ddH 2 O; general CPA system primer for fox, sheep and donkey; different meat source DNA;
the primers of the general CPA system for foxes, sheep and donkeys are as follows:
wherein the ratio of the inner primer TY2a to the detection probe inner primer TY3a is 1:3;
2) The reaction tube is taken out after being amplified in a water bath kettle with constant temperature of 63 ℃ for 60min, and after the specific externally-added marked primers of 6s < -1 >, 6s < -2 > and 6s < -3 > of each meat source are added, the water bath reaction is carried out for 3min at 93 ℃, and the reaction products are taken out to be tested on a colloidal gold nucleic acid test strip;
the sequence of the externally-added marking primer for foxes, sheep and donkeys is as follows:
。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910535636.9A CN110184330B (en) | 2019-06-20 | 2019-06-20 | Method for synchronously detecting various meat-derived components by using cross primer isothermal amplification reaction system |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910535636.9A CN110184330B (en) | 2019-06-20 | 2019-06-20 | Method for synchronously detecting various meat-derived components by using cross primer isothermal amplification reaction system |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110184330A CN110184330A (en) | 2019-08-30 |
| CN110184330B true CN110184330B (en) | 2024-01-09 |
Family
ID=67722570
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910535636.9A Active CN110184330B (en) | 2019-06-20 | 2019-06-20 | Method for synchronously detecting various meat-derived components by using cross primer isothermal amplification reaction system |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110184330B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101899501A (en) * | 2010-02-10 | 2010-12-01 | 江苏出入境检验检疫局动植物与食品检测中心 | Constant temperature amplification detection kit and method for detecting food allergen crustacean gene |
| CN102758002A (en) * | 2011-04-29 | 2012-10-31 | 宗卉 | Method of using gene chip technology to identify and detect 16 animal-derived compositions |
| CN104388573A (en) * | 2014-12-09 | 2015-03-04 | 中国计量学院 | Method for detecting ingredients of beef source by virtue of cross primers and dual-probe isothermal amplification |
-
2019
- 2019-06-20 CN CN201910535636.9A patent/CN110184330B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101899501A (en) * | 2010-02-10 | 2010-12-01 | 江苏出入境检验检疫局动植物与食品检测中心 | Constant temperature amplification detection kit and method for detecting food allergen crustacean gene |
| CN102758002A (en) * | 2011-04-29 | 2012-10-31 | 宗卉 | Method of using gene chip technology to identify and detect 16 animal-derived compositions |
| CN104388573A (en) * | 2014-12-09 | 2015-03-04 | 中国计量学院 | Method for detecting ingredients of beef source by virtue of cross primers and dual-probe isothermal amplification |
Non-Patent Citations (1)
| Title |
|---|
| 动物源性成分交叉引物等温扩增快速检测方法的研究和建立;冯涛;《中国优秀硕士学位论文全文数据库》;20170415;摘要，第14页第3段，第15页第2段，第20页第1段，第21页表2.2，第57页第1段、图5.1 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110184330A (en) | 2019-08-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Ahmed et al. | Meat species identification based on the loop mediated isothermal amplification and electrochemical DNA sensor | |
| Kang et al. | Comparison of quantitative methods based on SYBR Green real-time qPCR to estimate pork meat adulteration in processed beef products | |
| Ali et al. | Analysis of pork adulteration in commercial burgers targeting porcine-specific mitochondrial cytochrome B gene by TaqMan probe real-time polymerase chain reaction | |
| Rojas et al. | Application of a real-time PCR assay for the detection of ostrich (Struthio camelus) mislabelling in meat products from the retail market | |
| Bai et al. | A novel common primer multiplex PCR (CP-M-PCR) method for the simultaneous detection of meat species | |
| Li et al. | Quantitative determination of mutton adulteration with single-copy nuclear genes by real-time PCR | |
| Zhang et al. | Species-specific TM-LAMP and Trident-like lateral flow biosensor for on-site authenticity detection of horse and donkey meat | |
| CN106048034B (en) | Animal-derived component forms in based on PCR-RLFP-DHPLC technology scanning analysis food | |
| Liu et al. | A multiplex PCR assay mediated by universal primers for the detection of adulterated meat in mutton | |
| CN104946790A (en) | PCR method for tracking source identification of origin components of eight kinds of animals | |
| CN103154268A (en) | A kit for detection of donkey meat in meat products | |
| Tafvizi et al. | Specific identification of chicken and soybean fraud in premium burgers using multiplex-PCR method | |
| CN106521016B (en) | The real-time fluorescence PCR detection method of calf-derived Cyclospora in food and feed is detected using single-copy nuclear gene | |
| Mounika et al. | Identification of sheep (Ovis aries) meat by alkaline lysis-loop mediated isothermal amplification technique targeting mitochondrial D-loop region | |
| Ali et al. | Development of loop-mediated isothermal amplification (LAMP) assay for rapid and direct screening of yellowfin tuna (Thunnus albacares) in commercial fish products | |
| CN110184330B (en) | Method for synchronously detecting various meat-derived components by using cross primer isothermal amplification reaction system | |
| CN105385766A (en) | Typing liquid chip kit for streptococcus suis serotypes | |
| CN106995852B (en) | Real-time fluorescence PCR detection method for detecting sheep-derived components in food and feed by using single-copy nuclear gene | |
| CN107012247B (en) | Real-time fluorescence PCR detection method for detecting goat-derived ingredients in food and feed by using single-copy nuclear gene | |
| CN112852980B (en) | Detection method for detecting nucleic acid of Atlantic salmon based on CRISPR fluorescence method and detection kit thereof | |
| CN104962650A (en) | PCR method and kit for synchronously identifying animal-derived ingredients | |
| AU2019100071A4 (en) | Pcr detection kit for rapidly identifying salmonella of specific serotypes | |
| CN108251534B (en) | Multiple PCR detection kit for rapidly detecting meat-derived food | |
| KR101764128B1 (en) | Primer set for determining identity of shrimp material in food, method of determining identity of shrimp material in food using the same and kit comprising the same | |
| KR101906217B1 (en) | Porcine gelatin detection kit and porcine gelatin detection process using the kit |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |