CN102864225B - Micro-satellite primer for egg tracing, kit thereof and application - Google Patents
Micro-satellite primer for egg tracing, kit thereof and application Download PDFInfo
- Publication number
- CN102864225B CN102864225B CN201210350903.3A CN201210350903A CN102864225B CN 102864225 B CN102864225 B CN 102864225B CN 201210350903 A CN201210350903 A CN 201210350903A CN 102864225 B CN102864225 B CN 102864225B
- Authority
- CN
- China
- Prior art keywords
- egg
- seq
- dna
- laying hen
- micro
- 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.)
- Expired - Fee Related
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a micro-satellite primer for egg tracing, a kit thereof and application. The micro-satellite primer for egg tracing is a nucleotide sequence which is shown by Seq No ID 1-20. A detection method provided by the invention is easy to operate, quick, low in cost and high in accuracy, and automatic direct detection can be realized. A detection result of the detection kit developed by the method is reliable, stable and accurate, and provides a judgment basis for related responsibility confirmation of a sudden egg safety accident. Genome DNA (Deoxyribonucleic Acid) is extracted from a vitelline membrane; a molecular biological method is used for identifying true and false eggs; biological tracing from eggs to egg chickens is realized; and the quality safety of commercially available eggs can be effectively monitored.
Description
Technical field
The present invention relates to technical field of molecular biology, the micro-satellite primers that is specifically related to trace to the source for egg, its test kit and application.
Background technology
Egg, because of features such as its low price, protein content are high, quality betters, is one of requisite food in people's daily life.Especially the such populous nation of China, egg consumption is very high, but how to ensure that the safety of egg has just become a stubborn problem, and the event such as trimeric cyanamide egg, false egg brings safely problem to especially egg in recent years, in this case, egg product trace to the source imperative.
At present the flow process of generally tracing to the source of egg can be reviewed link by chicken house, processing and packing factory and 3 of distribution logistics and formed, and can tracing information comprises in laying hen hatching information, breeding layer chicken process information, Egg Production of Laying Hens information, egg roughing in postpartum processing control information and egg logistics marketing information etc. with safety and correlation of attributes.The bar codes technique that is mainly that is applicable to egg sign.In numerous bar codes, the application of EAN-128 code is more extensive, and EAN-128 code is, according to EAN/UCC-128 code standard, information table is shown to bar code symbol, has the characteristics such as uniqueness, versatility, integrity, compactness and high-reliability.Because its character set is large, density is high, and represented information can comprise some variable informations in production process, as date manufactured, lot number etc., realized information fast, Obtaining Accurate and transmission.Human consumer can be known the attributes such as the date of laying eggs, culturist, cultivation base, product hierarchy, detection index, the checking but the reliability of its information is had no way of.Microsatellite DNA, is known as again short series winding and repeats or simple repeated sequence, and every element length, between 1～6bp, is the tandem repetitive sequence that joins end to end and form.Based microsatellite instability analysis is at Application of Animal Genetic diversity evaluation, the classification of variety source, preserves and utilizes aspect to play an important role.In addition, can also utilize microsatellite polymorphism to carry out paternity test, take the allelic frequency of a plurality of micro-satellites seat in colony as basis, calculate probability of exclusion and draw.Development and application along with bioinformatics technique at present makes to become possibility based on reviewing of DNA, and especially the superiority of livestock and poultry individual own " hereditary barcode " is immutable, is the ultimate solution that animal products is traced to the source.
At present, application microsatellite DNA technology carries out to egg the method that molecule traces to the source also report.
Summary of the invention
The object of this invention is to provide a kind of micro-satellite primers of tracing to the source for egg.
Another object of the present invention is to provide a kind of detection kit that contains above-mentioned primer.
Still a further object of the present invention is to provide the detection method that a kind of egg is traced to the source.
Another object of the present invention is to provide above-mentioned micro-satellite primers or the application of test kit in judgement egg or laying hen genetic affinity.
The invention provides the micro-satellite primers of tracing to the source for egg, it is included as a pair of or several right in the nucleotide sequence shown in Seq No ID 1 and 2, Seq No ID 3 and 4, Seq No ID 5 and 6, Seq No ID 7 and 8, Seq No ID 9 and 10, Seq No ID 11 and 12, Seq No ID 13 and 14, Seq No ID 15 and 16, SeqNo ID 17 and 18, Seq No ID 19 and 20.
Further, the present invention also provides the test kit that contains above-mentioned primer.This test kit also comprises one or more in following reagent: PCR damping fluid, Mg
2+, archaeal dna polymerase, dNTPs.
The present invention utilizes the microsatellite polymorphism analysis of egg and laying hen sample, analyzes their genetic affinity.Specifically comprise the following steps:
1) extract respectively laying hen DNA and egg DNA;
2) utilize above-mentioned micro-satellite primers, take respectively laying hen DNA in step 1) and egg DNA carries out pcr amplification as template;
3) PCR product is carried out to denaturing polyacrylamide gel electrophoresis analysis, read after glue figure judges genotype, use Dispan computed in software Nai Shi genetic distance (Ds), judge its genetic affinity.
Wherein, described egg DNA extracts and obtains from egg yolk film, and described laying hen DNA for to extract and to obtain from laying hen blood.
The reaction conditions of described pcr amplification is: 95 ℃ of denaturation 5min; 95 ℃ of sex change 30s, 62-65 ℃ of annealing 30s, 72 ℃ are extended 30s, 30-35 circulation; 72 ℃ are extended 7min.
Beneficial effect of the present invention:
1) detection method of the present invention is simple to operate, quick, expense is cheap, accuracy is high, and can realize the direct-detection of automatization.
2) detected result of the inventive method exploitation detection kit is reliable, stable, accurately, for the related responsibility identification of burst egg security incident provides basis for estimation.
3) the present invention extracts genomic dna from vitelline membrane, Genome DNA content reaches 74.91 ± 24.00ng/ μ l, molecular biology method is used for differentiating true and false egg, and the biology of realizing from egg to laying hen reviews, quality safety that can the commercially available egg of effective monitoring.
Accompanying drawing explanation
Fig. 1 is the factor correlation analysis figure of four groups of samples, and A representative is from the Bai Laihang unfertilized egg of China Agricultural University's experiment chicken house; B represents the Bai Laihang laying hen blood sample of China Agricultural University's experiment chicken house; C represents Beijing Bai Laihang laying hen blood sample of chicken house for generations; D represents Beijing chicken house Luo island red eggs chicken blood sample for generations.
Embodiment
Following examples are used for illustrating the present invention, but are not used for limiting the scope of the invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Test materials used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.% in following embodiment, if no special instructions, is quality percentage composition.
Choose at random following testing sample:
A:40 piece of Bai Laihang unfertilized egg, from China Agricultural University's experiment chicken house;
B:30 part Bai Laihang laying hen blood sample, from China Agricultural University's experiment chicken house;
C:30 part Bai Laihang laying hen blood sample, Beijing is chicken house for generations;
D:30 Fen Luo island red eggs chicken blood sample, Beijing is chicken house for generations.
1, the separation of vitelline membrane
From the blunt end of egg, egg is knocked out, with egg-white egg-yellow separator, isolate yolk, the normal saline flushing yolk with 0.9%, puts into culture dish by yolk, scratch vitelline membrane, use again 0.9% normal saline flushing, isolate vitelline membrane, put into afterwards a new culture dish, with 0.9% physiological saline, clean again, repeat so again, until yolk is cleaned up to only remaining lily vitelline membrane.Vitelline membrane after processing is put into 2ml centrifuge tube and-20 ℃ of preservations.
2, the collection of laying hen blood sample
Venous blood collection 1ml under every chicken wings, is placed in the centrifuge tube that fills 0.3ml left and right ACD antithrombotics (1.72% Trisodium Citrate, 1.47% glucose, 0.48% citric acid), mixes and puts into-20 ℃ of preservations.
3, the extraction of DNA
1) in vitelline membrane or the anticoagulant centrifuge tube of 20 μ l ACD are housed, add 700 μ l anticoagulation lysates (100mM TrisHcl(PH8.0), 100mM EDTA(PH8.0), 1%SDS) and 20 μ l 10% Proteinase Ks, 55 ° of C water-baths digestion 4 hours.
2) add the saturated phenol of isopyknic Tris, slowly shake up 15 minutes, centrifugal 12 minutes of 12000rpm, draws supernatant liquor to clean centrifuge tube with rifle head.
3) step repetition 2)
4) add isopyknic phenol imitative, slowly shake up 15 minutes, centrifugal 12 minutes of 12000rpm, draws supernatant liquor to clean centrifuge tube with rifle head.
5) add isopyknic trichloromethane, slowly shake up 15 minutes, centrifugal 12 minutes of 12000rpm, draws supernatant liquor to clean centrifuge tube with rifle head.
6) add 1000 μ l dehydrated alcohols, jog centrifuge tube, standing for some time.
7) 8000rpm is centrifugal 5 minutes, and visible centrifuge tube bottom adularescent precipitation, abandons supernatant, adds 600 μ l 70% ethanol, continues centrifugal 5 minutes, abandons supernatant.
8) vacuum-drying is 2 minutes, adds 20 μ l deionized waters, after room temperature is dissolved, puts into-20 ℃ of preservations.Detect DNA concentration 60ng/ μ l.
4, PCR reaction amplification
10 micro-satellite seats are distributed on the coloured differently body of chicken, polymorphism high specificity and do not have each other linkage relationship.Micro-satellite primers is synthetic by the raw work in Shanghai.The sequence of primer and reaction conditions are in Table 1,2.
The micro-satellites information of table 1
Table 2PCR reaction conditions
By each primer condition, on pcr amplification instrument, increase, PCR reaction system (15 μ l):
DNA 1μl（60ng/μl）；
Taq archaeal dna polymerase 0.5 μ l(0.5U);
dNTP 0.5μl（200μmol/L）；
Each 0.5 μ l(50ng of primer upstream and downstream);
Mg
2＋ 0.5μl（2.5mmol/L）；
10×buffer 1.5μl；
Distilled water 10 μ l.
5, gel electrophoresis
PCR product, through agarose electrophoresis, is defined as on 6% denaturing polyacrylamide gel, carrying out electrophoresis after the positive.A PCR product adds the sex change sample-loading buffer (0.09% dimethylbenzene is blue, 0.09% bromjophenol blue for 95% methane amide, 10mmol EDTA, pH8.0) of 3 parts, after 95 ℃ of sex change 5min, is placed in rapidly cooled on ice.300V voltage, 1 * TBE electrophoresis, 4～5h.
6, silver dyes (method is referring to the Ph D dissertation < < of China Agricultural University Chinese Native Chicken Breeds molecular genetic diversity research > >)
Fixing, wash-out and silver dye process: 25% ethanol, 1% nitric acid, 0.2% Silver Nitrate; 5-30min;
Wash-out: deionized water; 1-5min;
Colour developing: 0.2% formaldehyde, sodium carbonate 3%; 2-5min;
Stop: acetic acid 10%, 2-10min.
7, data analysis
Read after glue figure judges genotype, utilize the genetic distance between two groups of samples of Dispan computed in software, judge its genetic affinity, result is as shown in table 3.
The Nai Shi standard genetic distance (Ds) of four groups of sample rooms of table 3
| Sample | A | B | C |
| B | 0.096 | ||
| C | 0.412 | 0.603 |
| D | 2.200 | 1.356 | 0.755 |
Interpretation of result: the genetic distance of colony can be used for describing genetic construction and the difference of colony, the genetic distance result of 4 groups of samples shows: the genetic distance minimum (0.096) of the white Leghorn egg He Bailaihang laying hen colony of China Agricultural University, and with same kind another one colony---take second place (0.412) in Beijing for generations genetic distance of the Bai Laihang laying hen colony of chicken house, and Beijing genetic distance maximum (2.200) of chicken house Luo island red eggs chicken colony for generations.According to the factor correlation analysis of Fig. 1, can find out, A and B major part are all got together simultaneously, and C distance is slightly far away, D far apart.This proof egg sample comes from Bai Laihang laying hen colony of China Agricultural University, but not other Bai Laihang colony or Luo Daohong colony.
Test kit of the present invention comprises following reagent:
(1) 2.5U/ μ l Taq archaeal dna polymerase;
(2) 10 * pcr amplification damping fluids;
（3）20mmol/L 10×dNTPs；
（4）25mmol/L MgCl
2；
(5) embodiment 1 primer pair used;
（6）ddH
2O。
The specification of test kit is 25 times/box, in every box, the amount of each component is: 1 bottle of 2.5U/ μ l TaqDNA polysaccharase (500 μ l/ bottle), 1 bottle of 10 * pcr amplification damping fluid (500 μ l/ bottle), 1 bottle of 20mmol/L 10 * dNTPs (200 μ l/ bottle), 25mmol/L MgCl
21 bottle (500 μ l/ bottle), the upstream and downstream primer of every pair of primer each 1 bottle (50 μ l/ bottle) and ddH
2o1 bottle (1000 μ l/ bottle).By above-mentioned dosage, each component in test kit is carried out to packing, after packing, obtain the test kit of tracing to the source for egg.
Claims (7)
1. the micro-satellite primers group of tracing to the source for egg, it comprises the nucleotide sequence shown in Seq No ID1 and 2, Seq No ID3 and 4, Seq No ID5 and 6, Seq No ID7 and 8, Seq No ID9 and 10, Seq No ID11 and 12, Seq No ID13 and 14, Seq No ID15 and 16, Seq No ID17 and 18, Seq No ID19 and 20.
2. the test kit that contains primer sets described in claim 1.
3. test kit according to claim 2, is characterized in that, also comprises one or more in following reagent: PCR damping fluid, Mg
2+, archaeal dna polymerase, dNTPs.
4. the detection method that egg is traced to the source, comprises the following steps:
1) extract respectively laying hen DNA and egg DNA; Described egg DNA extracts and obtains from egg yolk film;
2) utilize micro-satellite primers group described in claim 1, take respectively laying hen DNA in step 1) and egg DNA carries out pcr amplification as template;
3) PCR product is carried out to denaturing polyacrylamide gel electrophoresis, analyzing gene type, the genetic affinity of judgement egg and laying hen.
5. detection method according to claim 4, is characterized in that, described laying hen DNA extracts and obtains from laying hen blood.
6. detection method according to claim 4, is characterized in that, the reaction conditions of described pcr amplification is: 95 ℃ of sex change 30s, and 62-65 ℃ of annealing 30s, 72 ℃ are extended 30s, 30-35 circulation.
7. the application of test kit in judgement egg and laying hen genetic affinity described in micro-satellite primers group, claim 2 or 3 described in claim 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210350903.3A CN102864225B (en) | 2012-09-19 | 2012-09-19 | Micro-satellite primer for egg tracing, kit thereof and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210350903.3A CN102864225B (en) | 2012-09-19 | 2012-09-19 | Micro-satellite primer for egg tracing, kit thereof and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102864225A CN102864225A (en) | 2013-01-09 |
| CN102864225B true CN102864225B (en) | 2014-03-19 |
Family
ID=47443350
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210350903.3A Expired - Fee Related CN102864225B (en) | 2012-09-19 | 2012-09-19 | Micro-satellite primer for egg tracing, kit thereof and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102864225B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104007161A (en) * | 2014-05-12 | 2014-08-27 | 塔里木大学 | Polyacrylamide gel solution for microsatellite marker polymorphism detection and silver staining method |
| CN109456962B (en) * | 2018-12-21 | 2022-03-04 | 河北农业大学 | Method and kit for extracting DNA from eggshell |
| CN110283894A (en) * | 2019-06-26 | 2019-09-27 | 扬州大学 | It is a kind of to identify the identification method that PGC transplants offspring chicken by plumage color binding molecule genetic marker |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20060038880A (en) * | 2004-10-30 | 2006-05-04 | 이학교 | Method discrimination for product traceability and identification of chicken using microsatellite dna |
| JP2009225700A (en) * | 2008-03-21 | 2009-10-08 | Bio Regenerations:Kk | Method for dna identification of chicken-derived sample |
-
2012
- 2012-09-19 CN CN201210350903.3A patent/CN102864225B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20060038880A (en) * | 2004-10-30 | 2006-05-04 | 이학교 | Method discrimination for product traceability and identification of chicken using microsatellite dna |
| JP2009225700A (en) * | 2008-03-21 | 2009-10-08 | Bio Regenerations:Kk | Method for dna identification of chicken-derived sample |
Non-Patent Citations (4)
| Title |
|---|
| 利用微卫星标记分析山东地方鸡品种的遗传多样性;陈红菊;《遗传学报》;20030930;第30卷(第9期);第856页表1 * |
| 张莹.鸡蛋卵黄膜DNA的提取及应用.《家禽遗传育种》.2003,475. |
| 陈红菊.利用微卫星标记分析山东地方鸡品种的遗传多样性.《遗传学报》.2003,第30卷(第9期), |
| 鸡蛋卵黄膜DNA的提取及应用;张莹;《家禽遗传育种》;20031231;475 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102864225A (en) | 2013-01-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102776183B (en) | A kind of amplimer of large yellow croaker plastosome whole genome sequence and application thereof | |
| CN105506103A (en) | Mitochondria genome amplified universal primer mixture as well as design and amplification method thereof | |
| CN105648046B (en) | Method for identifying sheep, goat, mink, nutria and duck meat at one time | |
| CN104946790B (en) | A kind of PCR method for 8 kinds of animal derived materials of identification of tracing to the source | |
| CN104946788A (en) | PCR primer and kit for identifying eight kinds of animal-derived components | |
| Abbadi et al. | Species identification of bivalve molluscs by pyrosequencing | |
| CN102864225B (en) | Micro-satellite primer for egg tracing, kit thereof and application | |
| CN104878099A (en) | Method for detecting single-nucleotide polymorphism of goat ATBF1 gene and application of goat ATBF1 gene | |
| CN102876796A (en) | Method for distinguishing green shell egg laying chicken and chicken green shell egg genotype as well as special fragment and primers | |
| CN105821054A (en) | Sinibrama taeniatus DNA barcode standard check sequence and application thereof | |
| CN102643912B (en) | Amplification primer for detecting mink derived ingredients | |
| CN108611405B (en) | Microsatellite fluorescent multiplex PCR (polymerase chain reaction) method for parent-child identification of culter alburnus | |
| CN104450697B (en) | SNP marker associated with oyster antiviral properties and application thereof | |
| CN104131097B (en) | A kind of method detecting beef cattle UCP3 gene mononucleotide polymorphism and application thereof | |
| CN110499371B (en) | Microsatellite primer, kit and identification method for identifying epinephelus coioides, malaba epinephelus malabaricus and first filial generation thereof | |
| CN104342489A (en) | Method for detecting genotype of chicken beard | |
| CN104694538B (en) | The SNP marker related to the more toe characters of chicken and its application | |
| CN103276090B (en) | Jinghai yellow chicken 16-week weight molecular genetic marker and application thereof | |
| CN104988233A (en) | LAMP detecting method for distinguishing dog meat from beef and mutton by utilizing mitochondrion DNA (deoxyribose nucleic acid) | |
| CN104962660A (en) | Ruditapes philippinarum species real-time fluorescent PCR (polymerase chain reaction) specific detection system and application thereof | |
| CN103397100B (en) | Establishment method of Pinctada martensii MSAP (methylation sensitive amplification polymorphism) technical system | |
| Oguntunji et al. | Blood protein polymorphism and genetic diversity in locally adapted Muscovy duck (Cairina moschata) in Nigeria | |
| CN104962650A (en) | PCR method and kit for synchronously identifying animal-derived ingredients | |
| CN111850135B (en) | PCR high-resolution melting detection kit and detection method for 15 animal-derived components | |
| CN110042169B (en) | Molecular marker primer, kit and identification method for group specificity of Fennel fish in Heilongjiang |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20140319 Termination date: 20140919 |
|
| EXPY | Termination of patent right or utility model |