CN109609658A - Detect horse, donkey, mule derived component fluorescence quantifying PCR method and kit - Google Patents
Detect horse, donkey, mule derived component fluorescence quantifying PCR method and kit Download PDFInfo
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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
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Abstract
The present invention provide it is a kind of detection horse, donkey, mule derived component fluorescence quantifying PCR method and kit.Contain detection horse, donkey, the primer of mule derived component and probe (SEQ ID NO:1-4) in the kit.Kit specificity of the present invention is good, and detection method is quick and easy, and accuracy is high, and sensitivity is good, and the detection for horse, donkey, mule derived component in poultry meat and its product provides new method.Fluorescence quantitative PCR detection technique provided by the invention can quickly and accurately realize the identification to mule derived component in poultry meat and its product; detection can be completed in 1-2h; have many advantages, such as that quick, special, sensitive, high-throughput, stability is good; it is not only to detect horse, donkey, mule derived food to provide new method; and can effectively resist that meat products is adulterated, increase the protection to consumer's interests.
Description
Technical field
The present invention relates to fluorescence quantitative PCR detection techniques, specifically, being related to a kind of detection horse, donkey, mule derived component
Fluorescence quantifying PCR method and kit.
Background technique
Donkey meat and its product are favored by people due to high nutritive value and unique taste.Donkey is raw simultaneously
Long period is long, therefore donkey meat resource shortage, and price is constantly soaring.Since horse, donkey, mule belong to equid, it is difficult from flesh
Meat tissue configuration aspects carry out accurate recognition, and the adulterated problem of donkey meat is following, with the horseflesh and mule meat of price inexpensively
It is the most universal instead of the adulteration of donkey meat.Therefore, foundation is quickly, accurate, reliably identification horse, donkey, mule meat method seem outstanding
It is important and urgent.
Currently, increasingly mature based on the animal derived detection technique based on molecular biology both at home and abroad.These technology bases
It originally is using animal mitochondria as target.Because there is variable region in the existing conserved region of mitochondria again, detection sensitivity is can be improved in multicopy,
It is suitable for the analysis of fabricated product.These detection techniques based on DNA breach the limitation according to animal origin structural form,
Taxonomic identification with more objectivity and accuracy, suitable for species.The country is for donkey meat and its product derived component detection mark
Quasi- method mainly has: " the 5th part of identification method of common domestic animals kind in SN/T 3730.5-2013 food and feed: Ma Cheng
Sorting surveys real-time fluorescence PCR method "；" the identification method of common domestic animals kind the 4th in SN/T 3730.4-2013 food and feed
Point: donkey composition detection real-time fluorescence PCR method ".Relevant Research Literature also has to be delivered successively.These detection methods can be fast respectively
Speed, accurate identification donkey source property and horse derived component.
Be directed to some specialized species, as hybrid animal mule identification when, based on mitochondria be target object detection method
In the presence of limitation to a certain degree.Mitochondria is in stringent matrilinear inheritance.Mule is that horse and donkey reciprocal cross are given birth to, the matter base of fertilized eggs
Because of difference, mule is mainly provided by the mitochondria of mare egg cell, and hinny is mainly provided by the mitochondria of jenny ass egg cell.Cause
This can only distinguish between donkey and horse source property using chondriogen, can not identify mule source property.Therefore, using mitochondria as the inspection of target
Survey method cannot achieve the detection identification of mule source property.
Currently, there is no both at home and abroad using karyogene as target detection horse, donkey, mule meat source property fluorescence quantitative PCR method correlation
Report.
Summary of the invention
The object of the present invention is to provide it is a kind of detection horse, donkey, mule derived component fluorescence quantifying PCR method and kit.
In order to achieve the object of the present invention, inventor starts with from the Analysis of Genetic Background of donkey, horse and mule, using karyogene as target
Mark carries out horse, donkey and the property detection method research of mule source.No matter hinny or mule are all from the product of horse Yu donkey reciprocal cross, fertilization
The karyogene of ovum is identical, is provided by 32 chromosomes of horse and 31 chromosomes of donkey；Karyogene is especially some house keeper's bases
Cause such as encodes flesh type creatine kinase gene (Creatine Kinase Muscle type, CKM), has well-conserved, list
Copy, horse and this house-keeping gene homologous sequence heredity of donkey have notable difference.Based on this, using horse and donkey CKM gene as target,
Horse, donkey and mule derived component multiple fluorescence quantitative PCR detection method are established, provides technical support and guarantor for food safety Regulation
Barrier.
In a first aspect, the application provides the universal primer of detection horse, donkey derived component, including upstream primer and downstream are drawn
Object, nucleotide sequence is respectively as shown in SEQ ID NO:1-2.
Second aspect, the application provide the fluorescence probe being used cooperatively with above-mentioned universal primer, for detect horse source property at
The fluorescence probe divided is 5 '-F1-AAACAACCGGATCTTTCAAGT-Q1-3 ', for detecting the fluorescence probe of donkey derived component
For 5 '-F2-AAATAACTGGATCTTTTAAGT-Q2-3 '.
Wherein, F1, F2 are different fluorophors, and Q1, Q2 are fluorescent quenching group.
In the specific embodiment of the present invention, F1 FAM, F2 VIC, Q1 and Q2 are MGB.
The third aspect, the application provide the kit of detection horse, donkey derived component, contain in the kit above-mentioned general
Primer and/or above-mentioned fluorescence probe.
Fourth aspect, the application provide following any application of above-mentioned universal primer or above-mentioned fluorescence probe:
1) horse, the application in donkey derived component in detection poultry meat and its product；
2) application in preparation horse, the detection reagent of donkey derived component or kit.
5th aspect, the application provide a kind of detection horse, donkey, mule derived component fluorescence quantifying PCR method, utilization is above-mentioned
Universal primer and above-mentioned fluorescence probe carry out fluorescence quantitative PCR detection to single source components Sample.
It the described method comprises the following steps:
1) DNA in sample to be tested is extracted；
2) using the DNA extracted in step 1) as template, quantitative fluorescent PCR reaction is carried out；
3) species according to corresponding to single derived component in amplification judgement sample.
Preferably, the reaction system of quantitative fluorescent PCR reaction are as follows:
The response procedures of quantitative fluorescent PCR reaction are as follows: 95 DEG C of 3min；95 DEG C of 3s, 62 DEG C of 32s, 40 circulations.
This method must set up negative control and positive control when detecting sample every time.Negative control is believed without FAM and VIC fluorescence
Number detection, no Ct value, no amplification curve；In positive control, only there is FAM, VIC fluorescence signal in horse, donkey positive control correspondence, respectively
Fluorescence signal has obvious amplification curve, and value < 35.0 Ct；There is FAM, VIC fluorescence signal, each fluorescence letter simultaneously in mule positive control
Number there are obvious amplification curve, and value < 35.0 Ct；If negative control and positive control conditions are unsatisfactory for above-mentioned condition, this time test
It is invalid to be considered as.
The judgment criteria of step 3) amplification is as follows: if the corresponding amplification curve of detection horse source property probe, no donkey source property
The corresponding amplification curve of probe, and Ct value < 35.0 show to contain horse derived component in sample to be tested；If detecting donkey source property probe
Corresponding amplification curve, the corresponding amplification curve of no horse source property probe, and Ct value < 35.0, show to contain donkey source in sample to be tested
Property ingredient；If detecting horse source property probe and the corresponding amplification curve of donkey source property probe simultaneously, and Ct value < 35.0, show to test sample
Contain mule derived component in product；
If value >=35.0 Ct are determined as feminine gender, or without horse source property, the corresponding amplification curve of donkey source property probe, and without Ct
Value, is determined as feminine gender, shows in sample to be tested without containing target animal derived component.
6th aspect, the application provide detection horse, donkey, mule derived component kit, at least contain in the kit
Above-mentioned universal primer, above-mentioned fluorescence probe and positive template, the positive template be horse genomic DNA, donkey genomic DNA and
Mule genomic DNA.
Further, the application provides kit horse, donkey, mule in fluorescence quantitative PCR detection poultry meat and its product
Application in derived component.
Kit of the invention can be to be formed by multiple partitions, fixed one or more such as pipe or bottle to accommodate
Container.One of these containers or it is multiple primer and fluorescence probe of the invention can be housed, as needed primer and glimmering
The state that light probe can be lyophilized form or be dissolved in buffer.In addition, can also include being used in kit of the invention
One or more enzyme/reagents of quantitative fluorescent PCR reaction, and implement other ingredients and apparatus required for the present invention, such as
DNA lysate, fluorescent quantitation reaction solution, negative template, positive template, the feminine gender template is distilled water, the positive template
For horse genomic DNA, donkey genomic DNA, mule genomic DNA.
The present invention provides it is a kind of detection horse, donkey, mule derived component double fluorescent PCR method, this method is with meat products
DNA is template, carries out double fluorescent quantitative PCR amplification using universal primer and fluorescence probe, determines result according to Ct value.
By above-mentioned technical proposal, the present invention at least have following advantages and the utility model has the advantages that
Kit specificity of the present invention is good, and detection method is quick and easy, and accuracy is high, and sensitivity is good, is poultry meat and its system
The detection of horse, donkey, mule derived component provides new method in product.Fluorescence quantitative PCR detection technique provided by the invention can be with
It quickly and accurately realizes the identification to mule derived component in poultry meat and its product, detection can be completed in 1-2h, have quick, special
Different, sensitive, high-throughput, the advantages that stability is good is not only that detection horse, donkey, mule derived food provide new method, and can have
Effect resists that meat products is adulterated, increases the protection to consumer's interests.
Detailed description of the invention
Fig. 1 is the double quantitative pcr amplification curve of horse CKM gene in the embodiment of the present invention 2.
Fig. 2 is the double quantitative pcr amplification curve of donkey CKM gene in the embodiment of the present invention 2.
Fig. 3 is the double quantitative pcr amplification curve of mule CKM gene in the embodiment of the present invention 2.
Fig. 4 is primer and probe specific amplification curve in the embodiment of the present invention 3.
Fig. 5 A- Fig. 5 C is respectively double fluorescence quantitative PCR method detection horse, donkey, mule derived component in the embodiment of the present invention 4
Sensitive amplification curve.
Fig. 6 is horse, donkey CKM Gene Partial sequence alignment result in the embodiment of the present invention 1.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment
According to conventional laboratory conditions, such as Sambrook molecular cloning experiment handbook (Sambrook J&Russell DW,
Molecular Cloning:a Laboratory Manual, 2001), or according to the condition of manufacturer's specification suggestion.
The design of 1 primer and probe of embodiment
Horse and donkey belong to equus, there is many genes relevant to movement, flesh type creatine kinase in its skeletal muscle
Gene (creatine kinase muscle CKM) belongs in scheming and the highly expressed gene of skeletal muscle, and the creatine of coding swashs
ADP phosphorylation is generated ATP by enzymatic phosphocreatine, is energized for contraction of muscle, is that transcript profile expresses gene the most abundant, with
This is drone design detection primer and probe.The many animals CKM genes such as horse, donkey, ox, sheep are searched from the website NCBI GenBank
Sequence is simultaneously downloaded, and is carried out sequence alignment using BioEdit software, is determined target region.Meanwhile considering that horse and donkey belong to Equus, parent
Edge relationship is close, chooses common conserved sequence design primer universal primer, reduces to the greatest extent dry between primer in multiplex PCR system
It disturbs.Design MGB probe enhancing horse and donkey probe specificity.3 primers and 3 probes are devised altogether, by screening and optimizing, finally
Determine that primer and probe detection effect shown in SEQ ID NO:1-4 is best.Expanding fragment length is 217bp, is located at CKM gene
(GenBank:HQ336263.1) between the 281st~497 bit base.This section of sequence both (could containing the shared conserved sequence of horse, donkey
Universal primer is designed accordingly), and contain variable base sequence (for designing respective probe).Horse, this section of sequence of donkey comparison knot
Fruit sees Fig. 6.
Horse, donkey CKM specific primer to and Taqman MGB probe, be respectively designated as horse donkey CKM:F (P1), horse donkey CKM:
R (P2), horse karyogene CKM probe (P3), donkey karyogene CKM probe (P4).Expand for dual Taqman MGB real-time fluorescence PCR
The primer and probe sequence of increasing is following (SEQ ID NO:1-4):
Horse, donkey karyogene CKM universal primer:
F:5’-TCATCGTAGCATCGAGGGGA-3’
R:5’-AGCAGAGCAGGAAGGGAGT-3’
Horse probe P1:5 '-FAM-AAACAACCGGATCTTTCAAGT-MGB-3 '
Donkey probe P2:5 '-VIC-AAATAACTGGATCTTTTAAGT-MGB-3 '
The foundation of 2 fluorescent quantitative PCR detection method of embodiment
(1) sample DNA is extracted
1. taking mule this 0.2g of meat sample, shred as far as possible.It is placed in 1.5ml centrifuge tube and the cell lysis buffer solution of 1ml is added, 20
μ l Proteinase K (500ug/ml) mixes.The water-bath 30min in 65 DEG C of thermostat water baths, intermittent oscillation centrifuge tube is for several times.In platform
Formula centrifuge is centrifuged 5min with 12000rpm, and supernatant is taken to enter in another centrifuge tube.
2. adding the phenol of equivalent, the oscillation of chloroform mixed liquor is mixed, and 12000rpm is centrifuged 10min.
3. taking upper solution to another pipe, isometric chloroform is added, oscillation mixes, and 12000rpm is centrifuged 5min.
4. taking upper solution to another pipe, the 3M sodium acetate of 1/10 volume and the dehydrated alcohol of 2 times of volumes is added, after mixing
Precipitation at room temperature 10min, 12000rpm are centrifuged 10min.
5. carefully being exhausted supernatant with liquid-transfering gun.
6. using 75% ethanol washing sediment of 1ml, 12000rpm is centrifuged 5min.
7. repeating step 5 and 6.
8. drying at room temperature 5min will be precipitated with liquid-transfering gun device exhaustion supernatant.
9. adding 50ul TE or ddH2O re-dissolves sediment, is subsequently placed in 4 DEG C or -20 DEG C save backup.
(2) universal primer and 2 probes for using embodiment 1, using above-mentioned DNA as template, with horse genomic DNA and donkey
Genomic DNA is positive control, using free nucleic acid distilled water as negative control, establishes fluorescence quantitative PCR detection system.
20 μ L reaction systems are as follows:
Response procedures are as follows: 95 DEG C of 3min；95 DEG C of 3s, 62 DEG C of 32s, 40 circulations.After amplification, background fluorescence letter is deducted
Same Threshold Analysis data are taken after number, determine the Ct value of each sample.
This method must set up negative control and positive control when detecting sample every time.Negative control is believed without FAM and VIC fluorescence
Number detection, no Ct value, no amplification curve；In positive control, only there is FAM, VIC fluorescence signal in horse, donkey positive control correspondence, respectively
Fluorescence signal has obvious amplification curve, and value < 35.0 Ct；There is FAM, VIC fluorescence signal, each fluorescence letter simultaneously in mule positive control
Number there are obvious amplification curve, and value < 35.0 Ct；If negative control and positive control conditions are unsatisfactory for above-mentioned condition, this time test
It is invalid to be considered as.
The judgment criteria of amplification is as follows: if the corresponding amplification curve of detection horse source property probe, no donkey source property probe pair
The amplification curve answered, and Ct value < 35.0 show to contain horse derived component (Fig. 1) in sample to be tested；If detecting donkey source property probe
Corresponding amplification curve, the corresponding amplification curve of no horse source property probe, and Ct value < 35.0, show to contain donkey source in sample to be tested
Property ingredient (Fig. 2)；If detecting horse source property probe and the corresponding amplification curve of donkey source property probe simultaneously, and Ct value < 35.0, show
Contain mule derived component (Fig. 3) in sample to be tested.
If value >=35.0 Ct are determined as feminine gender, or without horse source property, the corresponding amplification curve of donkey source property probe, and without Ct
Value, is determined as feminine gender, shows in sample to be tested without containing target animal derived component.
3 specific test of embodiment
Using the primer and probe of embodiment 1, using horse, donkey, mule genomic DNA as positive control, with pig, ox, sheep, chicken,
Duck, goose, deer, fox DNA be negative control, the specificity of detection probe and primer.
Specific experiment method is referring to embodiment 2.
Experimental result removes horse, donkey, mule as shown in Figure 4 amplification curve and Ct value < 30, other equal nothings of animal origin template
Amplification.It can be seen that primer and probe specificity provided by the invention is good, with other species no cross reactions.
4 sensitivity test of embodiment
Double quantitative fluorescent PCR system sensitivity and repeatability measurement:
Horse, donkey and mule genome are extracted respectively, it is quantitative to 100ng with Nanodrop, do 10 times of gradient dilutions, each gradient
Taking 2 μ L is template quantity, according to the test reaction system and condition optimized, detects horse, donkey and mule derived component respectively to examine
Examine the detection sensitivity of this method.3 Duplicate Samples are set up into every group of detection, each sample is repeated 3 times test, the knot tested every time
Fruit unanimously just can determine that detection limit sensitivity；The DNA of mule is extracted as template, according to the reaction system in embodiment 2 and instead
Condition is answered to carry out real-time fluorescence PCR reaction, same template carries out 3 secondary responses simultaneously, and it every other day does once, is repeated 15 times altogether,
The variance for analyzing the same sample carrys out the reproducibility and repeatability of appraisement system.
Experimental result is as shown in figure 5, as the minimum 0.01ng of template DNA dosage, horseflesh, donkey meat and mule meat karyogene
CKM has apparent amplification curve, when template DNA dosage is 0.001ng without amplification, thereby determines that the detection of this method is sensitive
Degree is 0.01ng.
5 repetitive test of embodiment
Same template carries out 3 quantitative fluorescent PCR reactions simultaneously, every other day does once, is repeated 15 times altogether, analyzes same
The variance of a sample carrys out the reproducibility and repeatability of appraisement system.
Experimental result is shown in Table 1, horse, donkey derived component Ct value average value be respectively 24.4 and 26.3, standard deviation difference
For 0.2 and 0.3, the coefficient of variation 0.8%, 1.1%.The above result shows that this method detection limit amplification is precisely stablized, it can
For horse, donkey, the detection of mule derived component.
Table 1
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be modified or is improved, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.
Sequence table
<110>Beijing's food security supervision and risk assessment center (Beijing's Food Inspection institute)
<120>detect horse, donkey, mule derived component fluorescence quantifying PCR method and kit
<130> KHP181116436.1
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
tcatcgtagc atcgagggga 20
<210> 2
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
agcagagcag gaagggagt 19
<210> 3
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
aaacaaccgg atctttcaag t 21
<210> 4
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
aaataactgg atcttttaag t 21
Claims (10)
1. detecting the universal primer of horse, donkey derived component, which is characterized in that including upstream primer and downstream primer, nucleotides sequence
Column are respectively as shown in SEQ ID NO:1-2.
2. the fluorescence probe being used cooperatively with universal primer described in claim 1, which is characterized in that for detecting horse derived component
Fluorescence probe be 5 '-F1-AAACAACCGGATCTTTCAAGT-Q1-3 ', the fluorescence probe for detecting donkey derived component is
5'-F2-AAATAACTGGATCTTTTAAGT-Q2-3'；
Wherein, F1, F2 are different fluorophors, and Q1, Q2 are fluorescent quenching group.
3. detecting the kit of horse, donkey derived component, which is characterized in that containing general described in claim 1 in the kit
Fluorescence probe described in primer and/or claim 2.
4. following any application of fluorescence probe described in universal primer or claim 2 described in claim 1:
1) horse, the application in donkey derived component in detection poultry meat and its product；
2) application in preparation horse, the detection reagent of donkey derived component or kit.
5. detect horse, donkey, mule derived component fluorescence quantifying PCR method, which is characterized in that using general described in claim 1
Fluorescence probe described in primer and claim 2 carries out fluorescence quantitative PCR detection to single source components Sample.
6. according to the method described in claim 5, characterized by comprising the following steps:
1) DNA in sample to be tested is extracted；
2) using the DNA extracted in step 1) as template, quantitative fluorescent PCR reaction is carried out；
3) species according to corresponding to single derived component in amplification judgement sample.
7. according to the method described in claim 6, it is characterized in that, the reaction system of quantitative fluorescent PCR reaction are as follows:
The response procedures of quantitative fluorescent PCR reaction are as follows: 95 DEG C of 3min；95 DEG C of 3s, 62 DEG C of 32s, 40 circulations.
8. method according to claim 6 or 7, which is characterized in that step 3) specifically: if detection horse source property probe is corresponding
Amplification curve, the corresponding amplification curve of no donkey source property probe, and Ct value < 35.0, show in sample to be tested containing horse source property at
Point；If the corresponding amplification curve of detection donkey source property probe, the corresponding amplification curve of no horse source property probe, and Ct value < 35.0, table
Contain donkey derived component in bright sample to be tested；If horse source property probe and the corresponding amplification curve of donkey source property probe are detected simultaneously, and
Ct value < 35.0 shows to contain mule derived component in sample to be tested；
If value >=35.0 Ct are determined as feminine gender, or without horse source property, the corresponding amplification curve of donkey source property probe, and without Ct value, sentence
It is set to feminine gender, shows in sample to be tested without containing target animal derived component.
9. detect horse, donkey, mule derived component kit, which is characterized in that in the kit at least contain claim 1 institute
Fluorescence probe described in universal primer, claim 2, negative template and positive template are stated, the positive template is horse genome
DNA, donkey genomic DNA and mule genomic DNA, negative template are pig genomic DNA.
10. kit described in claim 9 in fluorescence quantitative PCR detection poultry meat and its product horse, donkey, in mule derived component
Using.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110964837A (en) * | 2019-12-19 | 2020-04-07 | 内蒙古农业大学 | Primer group and detection kit for detecting horse, donkey, horse mule and donkey mule-derived components |
| CN111763714A (en) * | 2020-07-21 | 2020-10-13 | 山东省农业科学院畜牧兽医研究所 | Kit for rapidly identifying donkey and horse-derived components and donkey and horse-derived components in product and application of kit |
| CN113755607A (en) * | 2021-09-15 | 2021-12-07 | 华中农业大学 | Universal primer for specifically identifying donkey-hide gelatin and donkey-horse and mule-derived components in raw materials of donkey-hide gelatin, detection method and application |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2702701A1 (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 |
| CN101659996A (en) * | 2009-07-27 | 2010-03-03 | 花群义 | Fluorescence PCR detection reagent capable of discriminating source components of ruminant animal, preparation method and application thereof |
| US20110030072A1 (en) * | 2008-12-04 | 2011-02-03 | Sigma-Aldrich Co. | Genome editing of immunodeficiency genes in animals |
| CN103451304A (en) * | 2013-09-16 | 2013-12-18 | 中华人民共和国吉林出入境检验检疫局 | Primer probe and method for performing real-time fluorescent polymerase chain reaction (PCR) detection on origin ingredients of raccoon |
| CN104046700A (en) * | 2014-07-08 | 2014-09-17 | 山东省农业科学院生物技术研究中心 | Detection kit for quickly identifying donkey skin, horse skin and mule skin |
| CN104561327A (en) * | 2015-01-16 | 2015-04-29 | 北京市食品安全监控和风险评估中心 | Dual-fluorescence PCR method for simultaneously detecting original components of horse and donkey |
| CN106119390A (en) * | 2016-08-24 | 2016-11-16 | 济南诺贝莱生物科技有限公司 | A kind of based on MGB probe identification donkey skin, Corium Equi and the test kit of mule skin and detection method thereof |
| CN108342493A (en) * | 2018-05-08 | 2018-07-31 | 四川华汉三创生物科技有限公司 | One breeding class composition detection kit and method |
-
2018
- 2018-12-20 CN CN201811563519.5A patent/CN109609658A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2702701A1 (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 |
| US20110030072A1 (en) * | 2008-12-04 | 2011-02-03 | Sigma-Aldrich Co. | Genome editing of immunodeficiency genes in animals |
| CN101659996A (en) * | 2009-07-27 | 2010-03-03 | 花群义 | Fluorescence PCR detection reagent capable of discriminating source components of ruminant animal, preparation method and application thereof |
| CN103451304A (en) * | 2013-09-16 | 2013-12-18 | 中华人民共和国吉林出入境检验检疫局 | Primer probe and method for performing real-time fluorescent polymerase chain reaction (PCR) detection on origin ingredients of raccoon |
| CN104046700A (en) * | 2014-07-08 | 2014-09-17 | 山东省农业科学院生物技术研究中心 | Detection kit for quickly identifying donkey skin, horse skin and mule skin |
| CN104561327A (en) * | 2015-01-16 | 2015-04-29 | 北京市食品安全监控和风险评估中心 | Dual-fluorescence PCR method for simultaneously detecting original components of horse and donkey |
| CN106119390A (en) * | 2016-08-24 | 2016-11-16 | 济南诺贝莱生物科技有限公司 | A kind of based on MGB probe identification donkey skin, Corium Equi and the test kit of mule skin and detection method thereof |
| CN108342493A (en) * | 2018-05-08 | 2018-07-31 | 四川华汉三创生物科技有限公司 | One breeding class composition detection kit and method |
Non-Patent Citations (1)
| Title |
|---|
| 刘艳艳: "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", 《药学研究》 * |
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| CN110964837B (en) * | 2019-12-19 | 2022-06-21 | 内蒙古农业大学 | Primer group and detection kit for detecting horse, donkey, horse mule and donkey mule-derived components |
| CN111763714A (en) * | 2020-07-21 | 2020-10-13 | 山东省农业科学院畜牧兽医研究所 | Kit for rapidly identifying donkey and horse-derived components and donkey and horse-derived components in product and application of kit |
| CN111763714B (en) * | 2020-07-21 | 2023-06-13 | 山东省农业科学院畜牧兽医研究所 | Kit for rapidly identifying donkey and horse-derived components and donkey and horse-derived components in product and application of kit |
| CN113755607A (en) * | 2021-09-15 | 2021-12-07 | 华中农业大学 | Universal primer for specifically identifying donkey-hide gelatin and donkey-horse and mule-derived components in raw materials of donkey-hide gelatin, detection method and application |
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