CN111763714B - Kit for rapidly identifying donkey and horse-derived components and donkey and horse-derived components in product and application of kit - Google Patents
Kit for rapidly identifying donkey and horse-derived components and donkey and horse-derived components in product and application of kit Download PDFInfo
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- 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
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- 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
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
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- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention provides a kit for rapidly identifying donkey-derived components and donkey-derived components in a product thereof, and belongs to the technical field of animal-derived component molecular detection. The kit comprises primers shown in SEQ-ID-No. 1-3, a probe, a RAA fluorescent universal reaction reagent, a reaction buffer solution and positive and negative quality control products. In addition, the primer and the probe comprise horse specific primers and probes, and the nucleotide sequence of the primers is shown as SEQ-ID-No. 4-6. The invention adopts RAA fluorescence method to carry out isothermal amplification at 30-42 ℃ and complete detection for 5-20min, and can judge whether the sample contains donkey source components or not and whether the sample is doped with horse source components or not. The kit is simple to operate, and can provide a rapid, sensitive and accurate detection method for molecular identification of donkey source components in meat and skin and detection of adulterated components in meat and skin; the device is matched with a small portable instrument, is suitable for large-scale detection in a laboratory, is also suitable for rapid detection of a base layer and a site, and has good application prospect.
Description
Technical Field
The invention relates to the field of animal molecular biology, in particular to a kit for rapidly identifying donkey and horse-derived components in donkey and horse-derived components and products and application thereof.
Background
Donkey meat enjoys the reputation of 'Tianshang dragon meat and on-ground donkey meat' from ancient times, and is enough to meet the absolute beauty of the taste and mouthfeel of donkey meat. In fact, the donkey meat has a nutritional value not lower than or even higher than that of beef, mutton or pork. It has the characteristics of high protein, high essential amino acid, high essential fatty acid, low fat, low cholesterol and low calorie, and is an unattainable healthy meat product. The donkey meat is rich in nutrition and delicious in taste, and has good effects of preventing obesity and cardiovascular diseases.
With the rapid development of economy and the improvement of the living standard of people, the material and spirit consumption demands begin to be diversified, and the requirements on the quality of animal products are higher and higher. Donkey meat is delicious in meat quality and unique in flavor, and related meat products are deeply favored by consumers, so that the demand is increased. Meanwhile, the quality safety problem of donkey meat and products is also an important point of attention of society and regulatory authorities. Adulteration of donkey meat and products has become a serious social problem, called "economic benefit driven adulteration". In order to pursue benefits, some illegal vendors use low-quality horse meat to impersonate high-quality donkey meat, and the safety of donkey meat product sources has become one of the most concerned problems of consumers. Therefore, it is necessary to establish a rapid, sensitive and accurate detection method for donkey-derived products.
Methods for identifying animal products by means of sensory and empirical means have not achieved the objective of controlling and supervising the adulteration of donkey meat and its processed products. In recent years, DNA having high stability and identity in different tissues has been used as a target for detection of animal-derived components. The method comprises a main method for identifying animal-derived components by using a common Polymerase Chain Reaction (PCR) technology and a fluorescent quantitative PCR technology (RT-PCR) (Cao Chenfu, zong Hui, rainbow and the like; a real-time fluorescent PCR detection method for donkey or donkey-derived components, a primer and a probe for detection; chinese patent CN102311999A, a quick fluorescent quantitative PCR detection method for a Taqman probe for pork or chicken components in foods added with an amplified internal standard, PCR and RT-PCR have a common defect, namely, the time is long, and the result can be obtained usually more than 2 hours, so that a detection method for identifying meat adulteration more quickly, sensitively and accurately is urgent, and the quick, sensitive and accurate DNA amplification technology for amplifying can be completed only by 5-20 minutes under the condition of 35-42 ℃.
At present, a rapid detection method for identifying donkey and Ma Rouyuan sex components at the same time has not been reported. In addition, adulteration of animal-derived components in donkey skin is also very common. Therefore, the establishment of a detection method for rapidly identifying donkey and horse source components is significant for the safety supervision of donkey meat foods and products.
Disclosure of Invention
Aiming at the defect of long time consumption in the prior art, the invention provides a primer, a probe or a kit for rapidly identifying donkey source components by using an RAA fluorescence method, which can realize sample detection within 30min, has the characteristics of rapidness, sensitivity and accuracy, and can meet the requirements of large-scale detection in a laboratory and rapid detection in the field.
The invention also aims to provide a rapid detection method for identifying the donkey-source product adulterated horse meat.
In order to achieve the above purpose, the invention screens donkey genome specific DNA sequences from aspects of intraspecies consistency and stability, interspecific specificity, copy number and the like, and screens donkey specific DNA fragments which are not present in horses or other animal species or have lower homology as detection targets through detection in donkey and horse genome DNA. Through a large number of analyses and experiments, donkey specific DNA sequences for detection are finally obtained.
The invention firstly provides a primer or a probe for rapidly detecting donkey specific DNA fragments by an RAA fluorescence method, wherein the primer or the probe has the following sequence:
EA-A：5’-CAAGATGCTCCCATAATTGGAACACCTGATG-3’，SEQ-ID-NO.1；
EA-B：5’-GTAGAGCCTTGTGAAGCAGTGTCTGAGAAC-3’，SEQ-ID-NO.2。
EA-C：5’- CCACAACTGACGGAGACCTGTGCACTGGC[FAM-dT]G[THF]C[BHG1-dT]GGACCCACAGAAGC[C3-spacer]-3’；SEQ-ID-NO.3；
the probe is modified by adopting a fluorescence reporter group FAM-dT and a fluorescence quenching group BHG1-dT, wherein the fluorescence reporter group is modified at a position 30bp away from the 5' -end base of the probe sequence; the fluorescence quenching group is modified at the position 15bp away from the 3 'end base of the probe sequence, 3 bases GCC are arranged between the fluorescence reporting group and the quenching group, the middle base C is replaced by tetrahydrofuran residue THF, and the 3' end of the probe EA-C is connected with a C3-spacer.
Further, the present invention provides a detection kit containing the above specific primer or probe.
Further, the kit further comprises the following primers or probes:
primer or probe for detecting horse specific DNA fragment by RAA fluorescence method, wherein the primer or probe has the following sequence:
EC-D：5'-CGGCCTCCATTCTCAGTGACAAGGCGGTGAA-3'，SEQ-ID-NO.4；
EC-E：5'-CGCAGGACGCAGTTCTCGAACGTCTTGGGTG-3'，SEQ-ID-NO.5。
EA-F：5’-tctcctcccaccttgtgggtatcccgggc[fam-dt]c[thf]a[bhg1-dt]gcggatgctgccag[c3-spacer]-3’；SEQ-ID-NO.6；
the probe EA-F is modified by adopting a fluorescence reporter group FAM-dT and a fluorescence quenching group BHG1-dT, wherein the fluorescence reporter group is modified at a position 30bp away from the 5' -end base number of the probe sequence; the fluorescence quenching group is modified at the position 15bp away from the 3 'end base of the probe sequence, 3 bases CAA are separated between the fluorescence reporting group and the quenching group, the middle base A is replaced by tetrahydrofuran residue THF, and the 3' end of the probe EA-F is connected with a C3-spacer.
Further, the kit also comprises one or more of the following reagents: general reaction reagent, negative and positive quality control products of RAA fluorescence method; the positive quality control product is donkey genome DNA template, and the negative quality control product is ddH 2 O or purified water.
Further, the positive quality control also comprises a horse genome DNA template.
Furthermore, the invention provides application of the primer or the probe or the detection kit for donkey specificity in the RAA fluorescence method in donkey source component identification or donkey source product adulteration detection.
The invention also provides a method for detecting donkey-source components and horse-source components in the product by using the RAA fluorescence method, which comprises the following steps:
1) Preparing a sample genome DNA template;
2) Isothermal amplification by RAA fluorescence: isothermal amplification is carried out for 5-20min at 30-42 ℃;
a) Primer for detecting donkey-source component by RAA fluorescence method and probe for detecting donkey-source component by RAA fluorescence method are used for carrying out RAA fluorescence method detection on sample, and donkey genome DNA template is used as positive control, ddH is used 2 O or purified water as negative control; obtaining a kit;
b) Detecting the sample by using the kit of A) through RAA fluorescence method, and using one or more of donkey genome DNA template and horse DNA template as positive control, and using ddH 2 O or purified water as negative control;
3) And (5) analyzing and judging the result.
The beneficial effects of the invention are that
1. Rapid detection method for simultaneously identifying donkey and Ma Rouyuan sex components
The method can realize the completion of sample detection within 30min, has the characteristics of rapidness, sensitivity and accuracy, and can meet the requirements of large-scale laboratory detection and on-site rapid detection. The kit is simple to operate, and can provide a rapid, sensitive and accurate detection method for molecular identification of donkey source components in meat and skin and detection of adulterated components in meat and skin; the device is matched with a small portable instrument, is suitable for large-scale detection in a laboratory, is also suitable for rapid detection of a base layer and a site, and has good application prospect.
2. Function in food safety supervision
Adulteration of animal-derived components in donkey skin is also very common. Therefore, the establishment of a detection method for rapidly identifying donkey and horse source components is significant for the safety supervision of donkey meat foods and products.
Detailed Description
The following examples are provided to further illustrate the present invention, but should not be construed as limiting the invention, and modifications or color rendering of the invention are within the scope of the invention without departing from the spirit and nature of the invention.
Example 1 specific detection of donkey-derived Components in samples
1. Preparation and preservation of samples
1.1 sampling
1g of donkey meat sample is collected and stored at the temperature of minus 20 ℃ for standby.
1.2 preparation of DNA templates
The DNA template can be prepared by using a conventional extraction method or a commercially available extraction-free kit, and the prepared template is stored at 4 ℃ for standby or at-20 ℃ for standby.
2. Primer design
The DNA sequence of the primer or probe of the embodiment is shown as SEQ-ID-NO: 1-3.
The donkey specific primer probe designed by the invention
EA-A：5’-CAAGATGCTCCCATAATTGGAACACCTGATG-3’，SEQ-ID-NO.1；
EA-B：5’-GTAGAGCCTTGTGAAGCAGTGTCTGAGAAC-3’，SEQ-ID-NO.2。
EA-C：5’- CCACAACTGACGGAGACCTGTGCACTGGC[FAM-dT]G[THF]C[BHG1-dT]GGACCCACAGAAGC[C3-spacer]-3’；SEQ-ID-NO.3；
3. Selecting a fluorescence reporter group and a fluorescence quencher group
A RAA-F1620 fluorescence gene detector manufactured by Wuxi Tianqi biosciences instruments Co., ltd is adopted, and fluorescence detected is FAM fluorescence, so that a fluorescence reporting group is selected as FAM, and a fluorescence quenching group is selected as BHQ1.
The fluorescence reporter group may also be selected as HEX, TET, JOE, VIC, ROX, cy or Cy5 depending on the performance of the instrument to detect fluorescence. The fluorescence quenching group is TAMRA, eclipse, BHQ, BHQ3 or DABCYL. However, the fluorescent reporter groups are preferably FAM and HEX, and the fluorescent quenching groups are preferably BHQ1 and BHQ2.
The method for modifying the probe preferably comprises: the fluorescent reporter group is modified at a position 30bp away from the 5' end base of the probe sequence; the fluorescence quenching group is modified at the position 15bp away from the 3' -end base of the probe sequence, and 3 bases GCC are separated between the fluorescence reporting group and the quenching group, wherein the base C at the middle position is replaced by tetrahydrofuran residue. The modified probes EA-C were: CCACAACTGACGGAGACCTGTGCACTGGC [ FAM-dT ] G [ THF ] C [ BHG1-dT ] GGACCCACAGAAGC [ C3-spacer ].
4 RAA fluorescence detection
In this example, the RAA fluorescent universal reagent was purchased from Jiangsu Qitide Biotechnology Co., ltd, cat# F00001, and was operated strictly according to the specification. The specific operation steps are as follows:
1) The power supply of the instrument (RAA-F1620) is connected for preheating, and the reaction parameters are set: the temperature is 39 ℃ and the time is 20min.
2) The reaction system was prepared as shown in Table 3 with the re-dissolved RAA reaction buffer, and the negative and positive controls were set.
TABLE 3RAA fluorescence reaction system
| Name of the name | Volume (mul) |
| RAA universal reaction buffer | 25 |
| ddH2O | 15.7 |
| EA-F | 2.1 |
| EA-R | 2.1 |
| EA-p | 0.6 |
| Magnesium sulfate | 2.5 |
| DNA template | 2 |
| Totalizing | 50 |
3) And (3) fully and uniformly mixing the reaction system in the step (2), and then placing the mixture into an instrument RAA-F1620 for detection.
5 analysis and determination of detection results
5.1 analysis of control detection results
According to the positive judging method of the RAA-F1620 instrument, judging that the slope value K is more than or equal to 20 and is positive; the slope value K is less than 20, and the negative is judged; the positive control should have a slope value K more than or equal to 20, and the negative control should have a slope value K less than 20, which indicates that the reaction system is normal, and the result can be judged.
5.2 analysis and determination of the results of the sample detection
If the sample slope value K is more than or equal to 20, judging positive, namely detecting donkey-derived components in the sample; if the slope value K is less than 20, the result is negative, namely 'donkey source component is not detected in the sample'.
Example 2 sensitivity test
The detection was performed under the conditions of example 1 using donkey genomic DNA templates at different concentrations of 0.1 ng/. Mu.L, 1 ng/. Mu.L, 10 ng/. Mu.L, 100 ng/. Mu.L, respectively. The template concentration of 1 ng/. Mu.L is amplified, which shows that the amplified fragment of the specific primer has higher sensitivity.
Example 3 primer set for detecting horse-derived component incorporated in donkey-derived products
The invention detects donkey and horse source components, and judges whether the horse source components are doped or not by detecting the donkey and horse source components by using a RAA fluorescence method by using corresponding primer probes of each species.
1 preparation of sample DNA templates
The preparation and preservation methods of the DNA templates of each species were as described in example 1.
2 design of primers
The DNA sequence of the primer or probe of the embodiment is shown as SEQ-ID-NO: 4-6.
The invention designs a horse specificity primer probe
EC-D：5'-CGGCCTCCATTCTCAGTGACAAGGCGGTGAA-3'，SEQ-ID-NO.4；
EC-E：5'-CGCAGGACGCAGTTCTCGAACGTCTTGGGTG-3'，SEQ-ID-NO.5。
EA-F：5’-tctcctcccaccttgtgggtatcccgggc[fam-dt]c[thf]a[bhg1-dt]gcggatgctgccag[c3-spacer]-3’；SEQ-ID-NO.6；
3. Selecting a fluorescence reporter group and a fluorescence quencher group
Selection of fluorescent reporter groups and fluorescence quencher groups the selection of the modified probes is as described in example 1 above, as follows: EC-F: TCTCCTCCCACCTTGTGGGTATCCCGGGC [ FAM-dT ] C [ THF ] A [ BHG1-dT ] GCGGATGCTGCCAG [ C3-spacer ] -3'.
4 RAA fluorescence detection
The detection method of this embodiment is the same as that described in the previous embodiment 1.
5 analysis and determination of detection results
The results show that: the specific primers can only amplify the DNA templates of corresponding species, and have good specificity.
Example 4A kit for detecting donkey and horse derived Components
The kit comprises the following components:
primers and probes (donkey, horse primers and probes SEQ ID NO. 1-6 and one part each);
general buffer solution for RAA fluorescence method;
positive quality control (one for each donkey, horse genome DNA template);
ddH20。
the method of application of the kit is as described in example 1 or example 3.
Example 5 application of a kit for detecting donkey and horse-derived Components
1. Preparation of sample DNA template, detection by RAA fluorescence and analytical determination of the results were as described in example 1 or example 3.
2. The detection method is applied to detection of donkey meat products on the market, donkey meat products such as donkey meat burning and the like of different merchants are purchased, basic information is recorded, a DNA template is prepared, RAA fluorescence detection is carried out, and real components in the donkey meat products are detected.
3. The kit provided by the invention is used for detecting 13 groups of samples obtained on the market, and the results show that only 6 products are qualified products, 7 groups of PCR detection results are different from food labels, and the detection results show that 7 products have the adulteration phenomenon of horse meat.
The method provided by the invention has a wide prospect in application to donkey-derived product detection. Besides the detection of donkey meat or donkey meat products, the method can also be widely applied to related fields of donkey products such as skin products and traditional Chinese medicine component detection.
Sequence listing
<110> Shandong agricultural sciences laboratory animal husbandry and veterinary institute
<120> kit for rapidly identifying donkey and horse-derived components and donkey and horse-derived components in product and application thereof
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 31
<212> DNA
<213> Artificial sequence (artiartificial sequence)
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caagatgctc ccataattgg aacacctgat g 31
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<211> 30
<212> DNA
<213> Artificial sequence (artiartificial sequence)
<400> 2
gtagagcctt gtgaagcagt gtctgagaac 30
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<211> 45
<212> DNA
<213> Artificial sequence (artiartificial sequence)
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ccacaactga cggagacctg tgcactggcg cggacccaca gaagc 45
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<213> Artificial sequence (artiartificial sequence)
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cggcctccat tctcagtgac aaggcggtga a 31
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<212> DNA
<213> Artificial sequence (artiartificial sequence)
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cgcaggacgc agttctcgaa cgtcttgggt g 31
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<212> DNA
<213> Artificial sequence (artiartificial sequence)
<400> 6
tctcctccca ccttgtgggt atcccgggcc agcggatgct gccag 45
Claims (9)
1. A primer for detecting donkey-source components by using an RAA fluorescence method, which is characterized by comprising the following sequences:
eA-A:5'-CAAGATGCTCCCATAATTGGAACACCTGATG-3', SEQ-ID-NO.1; and EA-B:5'-GTAGAGCCTTGTGAAGCAGTGTCTGAGAAC-3', SEQ-ID-NO.2.
2. A probe for detecting donkey-source components by using an RAA fluorescence method, which is characterized by comprising the following sequences:
EA-C：5’- CCACAACTGACGGAGACCTGTGCACTGGC[FAM-dT]G[THF]C[BHG1-dT]GGACCCACAGAAGC[C3-spacer]-3’；SEQ-ID-NO.3；
the probe EA-C is modified by adopting a fluorescence reporter group FAM-dT and a fluorescence quenching group BHG1-dT, wherein the fluorescence reporter group is modified at a position 30bp away from the 5' -end base of the probe sequence; the fluorescence quenching group is modified at the position 15bp away from the 3 'end base of the probe sequence, 3 bases GCC are arranged between the fluorescence reporting group and the quenching group, the middle base C is replaced by tetrahydrofuran residue THF, and the 3' end of the probe EA-C is connected with a C3-spacer.
3. A kit comprising the primer according to claim 1 and the probe according to claim 2.
4. The test kit of claim 3, further comprising the following primers: primer pairs for amplifying horse genome DNA to generate horse specific amplified fragments:
EC-D：5'-CGGCCTCCATTCTCAGTGACAAGGCGGTGAA-3'，SEQ-ID-NO.4；
EC-E：5'-CGCAGGACGCAGTTCTCGAACGTCTTGGGTG-3'，SEQ-ID-NO.5。
5. the test kit of claim 3, further comprising the following probes: horse source component probe:
EA-F：5’-tctcctcccaccttgtgggtatcccgggc[fam-dt]c[thf]a[bhg1-dt]gcggatgctgccag[c3-spacer]-3’；SEQ-ID-NO.6；
the probe EA-F is modified by adopting a fluorescence reporter group FAM-dT and a fluorescence quenching group BHG1-dT, wherein the fluorescence reporter group is modified at a position 30bp away from the 5' -end base number of the probe sequence; the fluorescence quenching group is modified at the position 15bp away from the 3 'end base of the probe sequence, 3 bases CAA are separated between the fluorescence reporting group and the quenching group, the middle base A is replaced by tetrahydrofuran residue THF, and the 3' end of the probe EA-F is connected with a C3-spacer.
6. The test kit of any one of claims 3-5, further comprising one or more of the following reagents: general reaction reagent, negative and positive quality control products of RAA fluorescence method; the positive quality control product is donkey genome DNA template, and the negative quality control product is ddH 2 O or purified water.
7. The kit of claim 6, wherein the positive quality control further comprises a equine genomic DNA template.
8. Use of a kit according to any one of claims 3 to 7 in the identification of donkey-derived components or in the detection of adulteration of donkey-derived products.
9. Use of the kit of claim 7 for simultaneous detection of donkey, equine derived ingredients or donkey derived product adulterated horse meat.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009035792A1 (en) * | 2007-08-03 | 2009-03-19 | Melba Ketchum | Compositions, methods and systems for the simultaneous determination of parentage, identity, sex, genotype and/or phenotype and breed determination in animals |
| CA2806307A1 (en) * | 2010-07-23 | 2012-01-26 | Yeditepe Universitesi | A kit for detection of donkey meat in meat products |
| CN104774958A (en) * | 2015-04-27 | 2015-07-15 | 山东省农业科学院生物技术研究中心 | Primer probe composition for identifying sources of animals including donkeys, horses and foxes, kit and multiplex real-time fluorescence quantitative PCR detecting method |
| CN105132543A (en) * | 2015-08-26 | 2015-12-09 | 山东省农业科学院生物技术研究中心 | Primers, probe composition, kit and multiplex fluorescence PCR detection method for detecting ass-derived component, horse-derived component and bovine-derived component in cosmetics |
| CN105907852A (en) * | 2016-04-28 | 2016-08-31 | 山东省农业科学院生物技术研究中心 | Nested fluorescent PCR detection primer, probe, kit and detection method of donkey-origin, horse-origin, swine-origin and bovine-origin in donkey-hide gelatin, and applications thereof |
| CN109609658A (en) * | 2018-12-20 | 2019-04-12 | 北京市食品安全监控和风险评估中心（北京市食品检验所） | Detect horse, donkey, mule derived component fluorescence quantifying PCR method and kit |
| RU2726555C1 (en) * | 2019-10-16 | 2020-07-14 | Федеральное государственное бюджетное образовательное учреждение высшего образования "Кубанский государственный аграрный университет имени И.Т. Трубилина" | Test system for detecting dna of donkey (equus asinus) tissue in dry fodder and semi-finished meat products |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104250667B (en) * | 2014-10-09 | 2017-01-18 | 北京农学院 | Detecting method for horse source elements in meat and meat products and detecting method for donkey source element in meat and meat products |
| CN104561327B (en) * | 2015-01-16 | 2017-07-04 | 北京市食品安全监控和风险评估中心 | It is a kind of while detecting the double fluorescent PCR method of horse and donkey derived component |
| CN108330168B (en) * | 2017-09-22 | 2021-08-17 | 中国肉类食品综合研究中心 | Primer combination for synchronously detecting 14 animal-derived components in meat or meat product and application thereof |
-
2020
- 2020-07-21 CN CN202010703884.2A patent/CN111763714B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009035792A1 (en) * | 2007-08-03 | 2009-03-19 | Melba Ketchum | Compositions, methods and systems for the simultaneous determination of parentage, identity, sex, genotype and/or phenotype and breed determination in animals |
| CA2806307A1 (en) * | 2010-07-23 | 2012-01-26 | Yeditepe Universitesi | A kit for detection of donkey meat in meat products |
| CN104774958A (en) * | 2015-04-27 | 2015-07-15 | 山东省农业科学院生物技术研究中心 | Primer probe composition for identifying sources of animals including donkeys, horses and foxes, kit and multiplex real-time fluorescence quantitative PCR detecting method |
| CN105132543A (en) * | 2015-08-26 | 2015-12-09 | 山东省农业科学院生物技术研究中心 | Primers, probe composition, kit and multiplex fluorescence PCR detection method for detecting ass-derived component, horse-derived component and bovine-derived component in cosmetics |
| CN105907852A (en) * | 2016-04-28 | 2016-08-31 | 山东省农业科学院生物技术研究中心 | Nested fluorescent PCR detection primer, probe, kit and detection method of donkey-origin, horse-origin, swine-origin and bovine-origin in donkey-hide gelatin, and applications thereof |
| CN109609658A (en) * | 2018-12-20 | 2019-04-12 | 北京市食品安全监控和风险评估中心（北京市食品检验所） | Detect horse, donkey, mule derived component fluorescence quantifying PCR method and kit |
| RU2726555C1 (en) * | 2019-10-16 | 2020-07-14 | Федеральное государственное бюджетное образовательное учреждение высшего образования "Кубанский государственный аграрный университет имени И.Т. Трубилина" | Test system for detecting dna of donkey (equus asinus) tissue in dry fodder and semi-finished meat products |
Non-Patent Citations (3)
| Title |
|---|
| Development of an isothermal recombinase polymerase amplification assay for rapid detection of pseudorabies virus;Yang Yang等;《Molecular and Cellular Probes》;第33卷;第32-35页 * |
| 一种驴和马及狐狸源性成分快速检测方法的研究;卞如如;范阳阳;刘艳艳;霍胜楠;盛清凯;谭晴晴;张全芳;张卉;步迅;;中国畜牧杂志(第01期);第107-111页 * |
| 利用多重荧光定量PCR检测阿胶原料驴、马、驴骡和马骡皮张的源性;刘艳艳;张全芳;卞如如;陈杰;步迅;;药学研究(第10期);第15-20页 * |
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