CN112695101B - Quail early sex identification method, kit and special primer pair - Google Patents
Quail early sex identification method, kit and special primer pair Download PDFInfo
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- CN112695101B CN112695101B CN202110117751.1A CN202110117751A CN112695101B CN 112695101 B CN112695101 B CN 112695101B CN 202110117751 A CN202110117751 A CN 202110117751A CN 112695101 B CN112695101 B CN 112695101B
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- 241000286209 Phasianidae Species 0.000 title claims abstract description 73
- 238000000034 method Methods 0.000 title claims abstract description 24
- 108020004414 DNA Proteins 0.000 claims abstract description 31
- 238000012408 PCR amplification Methods 0.000 claims abstract description 16
- 230000003321 amplification Effects 0.000 claims abstract description 16
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 16
- 102000053602 DNA Human genes 0.000 claims abstract description 14
- 108020004682 Single-Stranded DNA Proteins 0.000 claims abstract description 14
- 238000001962 electrophoresis Methods 0.000 abstract description 7
- 230000008901 benefit Effects 0.000 abstract description 5
- 238000001514 detection method Methods 0.000 abstract description 5
- 229920000936 Agarose Polymers 0.000 abstract description 3
- 241000288030 Coturnix coturnix Species 0.000 abstract description 2
- 238000006243 chemical reaction Methods 0.000 description 7
- 235000013601 eggs Nutrition 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 230000001351 cycling effect Effects 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 244000144977 poultry Species 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 229910021642 ultra pure water Inorganic materials 0.000 description 3
- 239000012498 ultrapure water Substances 0.000 description 3
- 241000334119 Coturnix japonica Species 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 210000000436 anus Anatomy 0.000 description 2
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 235000013372 meat Nutrition 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 230000001568 sexual effect Effects 0.000 description 2
- 230000004568 DNA-binding Effects 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 108060004795 Methyltransferase Proteins 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000002759 chromosomal effect Effects 0.000 description 1
- 210000003555 cloaca Anatomy 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000002224 dissection Methods 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 210000003746 feather Anatomy 0.000 description 1
- 230000035558 fertility Effects 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 210000004392 genitalia Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000013011 mating Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 235000021049 nutrient content Nutrition 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 238000003307 slaughter Methods 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
Classifications
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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
- C12Q1/6876—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
-
- 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
- C12Q1/686—Polymerase chain reaction [PCR]
-
- 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
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6879—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for sex determination
-
- 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
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
Abstract
The invention discloses a quail early sex identification method, a kit matched with the method and a special primer pair. The invention provides a method for identifying sex of quails, which comprises the following steps: taking genome DNA of quail to be detected as a template, adopting a specific primer pair to carry out PCR amplification, if one amplification product is obtained, the quail to be detected is a male quail or a candidate is a female quail, and if two amplification products are obtained, the quail to be detected is a female quail or a candidate is a female quail; the specific primer pair consists of a single-stranded DNA molecule shown as SEQ ID NO.1 and a single-stranded DNA molecule shown as SEQ ID NO. 2. Compared with the prior art, the invention has the following advantages: the common quail and the female quail have the difference of the number of the strips, the difference of the characteristic strips is about 400bp, the detection can be realized through agarose electrophoresis, the operation is simple and convenient, the misjudgment is not easy, the identification result is quick and accurate, the popularization and the use of the base layer are convenient, and the method has wide market application prospect.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for early sex identification of quails, a kit matched with the method and a special primer pair.
Background
Quail belongs to the category of special economic poultry, and the nutrient contents of the quail eggs and the quail meat are basically higher than those of eggs and chicken, so that the quail has extremely high edible value. The quail has early sexual maturity, high growth speed, strong fertility and high feed conversion rate, and has better breeding prospect.
At present, commercial poultry are mostly produced in a mating line mode, the male parent generally grows faster, and the female parent generally breeds more efficiently, so that sex identification needs to be carried out early, the female parent needs to be eliminated in advance, and the female parent needs to be eliminated in advance. The growth speed of the quails of the meat quails is high, the quails are bred in groups, and the breeding benefit is better. When the eggs are hatched with quails, only the mother chicks are reserved, and the mother chicks are directly eliminated. Therefore, the early sex identification of quails has important significance in production.
At present, early sex identification of quails is mainly carried out by observing reproduction protrusion through anal turning. However, the identification of the anus turning is carried out within 24 hours of the birth of young quails, and beyond the period of time, the anus of the young quails is difficult to turn open, and the genital prominence is atrophic and even falls into the deep part of the cloaca, so that the observation is inconvenient. The anal turning identification has the advantages of high labor intensity, poor working environment, high specificity and higher false identification rate. The rapid and accurate early sex identification of quails by means of molecular biology has important significance.
Molecular sexing is currently generally performed using chromosomal helical protein genes (chromobox helicase DNA binding gene, CHD) with two homologous copies in female individuals and only one copy in male individuals. However, two homologous copy sequences of CHD genes of female individuals are relatively conserved and generally only differ by 100-200bp, agarose electrophoresis is utilized, resolution is low, misjudgment is easy, polyacrylamide gel electricity with high resolution is utilized, the test process is too complicated, and toxicity is very high.
Along with the rapid development of quail industry in China, a method for rapidly and accurately identifying the sex of quails is needed to be developed simply and conveniently.
Disclosure of Invention
The invention aims to provide a method for identifying early sex of quails, a kit matched with the method and a special primer pair.
The invention provides a method for identifying sex of quails, which comprises the following steps:
taking genome DNA of quail to be detected as a template, adopting a specific primer pair to carry out PCR amplification, if one amplification product is obtained, the quail to be detected is a male quail or a candidate is a female quail, and if two amplification products are obtained, the quail to be detected is a female quail or a candidate is a female quail;
the specific primer pair consists of a single-stranded DNA molecule shown as SEQ ID NO.1 and a single-stranded DNA molecule shown as SEQ ID NO. 2.
The amplification product is a characteristic amplification product, and the size of the amplification product is 537bp;
the two amplification products are two characteristic amplification products, and the sizes of the two amplification products are 941bp and 537bp respectively.
The genomic DNA may be extracted from feathers or blood of quails.
The genomic DNA may be extracted from quail tissue.
The genomic DNA may be extracted from an organ of quail.
The reaction system for PCR amplification specifically comprises: 2 XPCR Mix (Nanjing Boltd.) 25. Mu.L, 10. Mu. Mol/L forward primer 1. Mu.L, 10. Mu. Mol/L reverse primer 1. Mu.L, 50-100. Mu.g/ml template DNA 2. Mu.L, and ultra pure water 21. Mu.L.
The reaction procedure of PCR amplification can be specifically as follows: 95 ℃ for 5min; cycling at 95 ℃ for 30s, 60 ℃ for 30s, 72 ℃ for 30s and 35; and at 72℃for 10min.
The invention also protects a specific primer pair, which consists of a single-stranded DNA molecule shown as SEQ ID NO.1 and a single-stranded DNA molecule shown as SEQ ID NO. 2. The specific primer pair is used for identifying or assisting in identifying the sex of the quail.
The invention also protects the application of the specific primer pair in identification or auxiliary identification of quail gender.
The invention also protects application of the specific primer pair in preparing a kit for identifying or assisting in identifying the sex of quail.
The invention also provides a kit for identifying or assisting in identifying the sex of quails, which comprises the specific primer pair.
The invention also protects the application of the kit in identification or auxiliary identification of quail gender.
Any of the above quails may be specifically Korean quail or Japanese quail.
The invention aims to solve the technical problem of providing a PCR primer pair for quick identification of early sex of quail and a kit and a method for identifying sex of quail by PCR by using the primer pair.
Compared with the prior art, the invention has the following advantages: the common quail and the female quail have the difference of the number of the strips, the difference of the characteristic strips is about 400bp, the detection can be realized through agarose electrophoresis, the operation is simple and convenient, the misjudgment is not easy, the identification result is quick and accurate, the popularization and the use of the base layer are convenient, and the method has wide market application prospect. The detection kit developed based on the method can generate considerable economic benefit and good social value.
Drawings
FIG. 1 is an electropherogram of the PCR amplification product of example 1.
FIG. 2 is an electropherogram of the PCR amplified product of example 2.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The literature describing quails is as follows: living detection and prediction of the slaughter performance of chestnut-feather egg quails, mu Chunyu, shang Qingping, zhang Rui, yongsheng, chang Lingling, bo Zhu, anhui agricultural science, J.Anhui Agric.Sci.2020, 48 (1): 92-95.
Example 1, establishment of the method
Sample supply: 10 adult Korean quails of known gender, 5 male quails numbered 1-5 and 5 female quails numbered 6-10.
1. Taking blood of a test sample, and extracting genome DNA.
2. And (3) performing PCR amplification by taking the genomic DNA obtained in the step (1) as a template DNA.
Primer pairs used for PCR amplification were as follows:
forward primer (SEQ ID No. 1): 5'-TGTATTTTGGTTCTACAGGC-3';
reverse primer (SEQ ID NO. 2): 5'-ATCATCATAACCATAAATCT-3'.
PCR amplification reaction System (50. Mu.L): 2 XPCR Mix (Nanjing Boltd.) 25. Mu.L, 10. Mu. Mol/L forward primer 1. Mu.L, 10. Mu. Mol/L reverse primer 1. Mu.L, 50-100. Mu.g/ml template DNA 2. Mu.L, and ultra pure water 21. Mu.L.
Reaction procedure for PCR amplification: 95 ℃ for 5min; cycling at 95 ℃ for 30s, 60 ℃ for 30s, 72 ℃ for 30s and 35; and at 72℃for 10min.
3. After completion of step 2, the PCR amplification product was subjected to 1.5% agarose gel electrophoresis.
The electrophoresis diagram is shown in fig. 1. In FIG. 1, lanes 1 to 10 correspond to test samples numbered 1 to 10 in sequence. All quails showed a single characteristic band (537 bp by sequencing). All parent quails showed two characteristic bands (one 941bp and the other 537bp by sequencing).
The result shows that the primer pair and the corresponding PCR detection method can rapidly and accurately carry out sex identification on quails, and are simple and easy to operate.
Example 2 application of the method
Sample supply: 17 Japanese quails of unknown gender were used.
1. Taking blood of a test sample, and extracting genome DNA.
2. And (3) performing PCR amplification by taking the genomic DNA obtained in the step (1) as a template DNA.
Primer pairs used for PCR amplification were as follows:
forward primer (SEQ ID No. 1): 5'-TGTATTTTGGTTCTACAGGC-3';
reverse primer (SEQ ID NO. 2): 5'-ATCATCATAACCATAAATCT-3'.
PCR amplification reaction System (50. Mu.L): 2 XPCR Mix (Nanjing Boltd.) 25. Mu.L, 10. Mu. Mol/L forward primer 1. Mu.L, 10. Mu. Mol/L reverse primer 1. Mu.L, 50-100. Mu.g/ml template DNA 2. Mu.L, and ultra pure water 21. Mu.L.
Reaction procedure for PCR amplification: 95 ℃ for 5min; cycling at 95 ℃ for 30s, 60 ℃ for 30s, 72 ℃ for 30s and 35; and at 72℃for 10min.
3. After completion of step 2, the PCR amplification product was subjected to 1.5% agarose gel electrophoresis.
The electrophoresis diagram is shown in fig. 2. In FIG. 2, lanes 1 through 17 represent 17 samples tested in sequence. Wherein, a single characteristic band of 537bp was partially displayed for the sample (lanes 3, 4, 6, 7, 8, 10, 11) and judged as a quail, and two characteristic bands of 941bp and 537bp were partially displayed for the sample (lanes 1, 2, 5, 9, 12, 13, 14, 15, 16, 17) and judged as a quail. The feature band size was verified by sequencing.
The sample is subjected to dissection and is subjected to observation of a sexual organ, and the identification result is completely correct.
The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.
Sequence listing
<110> Jiangsu province poultry science institute
<120> method for early sex identification of quail, kit and primer pair therefor
<130> GNCYX210395
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
tgtattttgg ttctacaggc 20
<210> 2
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
atcatcataa ccataaatct 20
Claims (4)
1. A method for identifying the sex of quails, comprising the steps of:
taking genome DNA of quail to be detected as a template, adopting a specific primer pair to carry out PCR amplification, if one amplification product is obtained, the quail to be detected is a male quail or a candidate is a female quail, and if two amplification products are obtained, the quail to be detected is a female quail or a candidate is a female quail;
the specific primer pair consists of a single-stranded DNA molecule shown as SEQ ID NO.1 and a single-stranded DNA molecule shown as SEQ ID NO. 2;
the one amplification product is a characteristic amplification product with a size of 537bp;
the two amplification products were two characteristic amplification products, 941bp and 537bp in size, respectively.
2. An application of a specific primer pair in identification or auxiliary identification of quail gender;
the specific primer pair consists of a single-stranded DNA molecule shown as SEQ ID NO.1 and a single-stranded DNA molecule shown as SEQ ID NO. 2.
3. Application of specific primer pair in preparing kit for identifying or assisting in identifying quail sex;
the specific primer pair consists of a single-stranded DNA molecule shown as SEQ ID NO.1 and a single-stranded DNA molecule shown as SEQ ID NO. 2.
4. The kit is applied to identification or auxiliary identification of the sex of quails;
the kit comprises a specific primer pair, wherein the specific primer pair consists of a single-stranded DNA molecule shown in SEQ ID NO.1 and a single-stranded DNA molecule shown in SEQ ID NO. 2.
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| CN202110117751.1A CN112695101B (en) | 2021-01-28 | 2021-01-28 | Quail early sex identification method, kit and special primer pair |
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| CN202110117751.1A CN112695101B (en) | 2021-01-28 | 2021-01-28 | Quail early sex identification method, kit and special primer pair |
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| CN112695101A CN112695101A (en) | 2021-04-23 |
| CN112695101B true CN112695101B (en) | 2024-03-15 |
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Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113981106B (en) * | 2021-11-05 | 2024-04-16 | 江苏省家禽科学研究所 | Method for early sex identification of Phasianidae animals, kit matched with method and special primer pair |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101962677A (en) * | 2010-09-15 | 2011-02-02 | 华南农业大学 | Method for identifying poultry gender |
| TW201317357A (en) * | 2011-10-27 | 2013-05-01 | Univ Kaohsiung Medical | Method for Phasianidae gender identification, nucleotide sequence for Phasianidae gender and nucleotide primer pair for Phasianidae gender |
| CN106244689A (en) * | 2016-08-02 | 2016-12-21 | 湖北省农业科学院畜牧兽医研究所 | A kind of method that PCR-based technology carries out Carnis Coturnicis japonicae sex identification |
-
2021
- 2021-01-28 CN CN202110117751.1A patent/CN112695101B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101962677A (en) * | 2010-09-15 | 2011-02-02 | 华南农业大学 | Method for identifying poultry gender |
| TW201317357A (en) * | 2011-10-27 | 2013-05-01 | Univ Kaohsiung Medical | Method for Phasianidae gender identification, nucleotide sequence for Phasianidae gender and nucleotide primer pair for Phasianidae gender |
| CN106244689A (en) * | 2016-08-02 | 2016-12-21 | 湖北省农业科学院畜牧兽医研究所 | A kind of method that PCR-based technology carries out Carnis Coturnicis japonicae sex identification |
Non-Patent Citations (3)
| Title |
|---|
| Comprehensive identification of sexual dimorphism-associated differentially expressed genes in two-way factorial designed RNA-Seq data on Japanese quail (Coturnix coturnix japonica);Kelsey Caetano-Anolles等;PLOS ONE;第10卷(第9期);标题、摘要、第8页表2、第11页、第13页conclusion * |
| Identification of female specific genes in the W chromosome that are expressed during gonadal differentiation in the chicken;Harikrishna Reddy Rallabandi等;Korean J. Poult. Sci.;第46卷(第4期);Supplemental Table S1 * |
| 鹌鹑性别的分子鉴定方法研究;张小辉等;中国家禽;第42卷(第5期);20-23页 * |
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