CN112746070A - Primer pair and kit for early sex identification of chicken and application of primer pair and kit - Google Patents
Primer pair and kit for early sex identification of chicken and application of primer pair and kit Download PDFInfo
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- CN112746070A CN112746070A CN202110118532.5A CN202110118532A CN112746070A CN 112746070 A CN112746070 A CN 112746070A CN 202110118532 A CN202110118532 A CN 202110118532A CN 112746070 A CN112746070 A CN 112746070A
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- 241000287828 Gallus gallus Species 0.000 title claims abstract description 43
- 108020004414 DNA Proteins 0.000 claims abstract description 23
- 230000003321 amplification Effects 0.000 claims abstract description 17
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 17
- 238000000034 method Methods 0.000 claims abstract description 16
- 238000012408 PCR amplification Methods 0.000 claims abstract description 12
- 102000053602 DNA Human genes 0.000 claims abstract description 10
- 108020004682 Single-Stranded DNA Proteins 0.000 claims abstract description 10
- 235000013330 chicken meat Nutrition 0.000 claims description 35
- 238000002360 preparation method Methods 0.000 claims description 2
- 238000001962 electrophoresis Methods 0.000 abstract description 7
- 230000008901 benefit Effects 0.000 abstract description 5
- 229920000936 Agarose Polymers 0.000 abstract description 3
- 210000000436 anus Anatomy 0.000 description 4
- 210000003746 feather Anatomy 0.000 description 4
- 230000020509 sex determination Effects 0.000 description 4
- 238000012360 testing method Methods 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 244000144977 poultry Species 0.000 description 2
- 235000013594 poultry meat Nutrition 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 229910021642 ultra pure water Inorganic materials 0.000 description 2
- 239000012498 ultrapure water Substances 0.000 description 2
- 241000209507 Camellia Species 0.000 description 1
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 101150069823 chd gene Proteins 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
- 235000018597 common camellia Nutrition 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 210000004392 genitalia Anatomy 0.000 description 1
- 231100000086 high toxicity Toxicity 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000001850 reproductive effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000001568 sexual effect Effects 0.000 description 1
- 229910052709 silver Inorganic materials 0.000 description 1
- 239000004332 silver Substances 0.000 description 1
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- 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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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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]
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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
- C12Q1/6879—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for sex determination
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Abstract
The invention discloses a primer pair and a kit for identifying early sex of chicken and application thereof. The invention provides a specific primer pair, which 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. The invention also provides a method for identifying the sex of the chicken, which comprises the following steps: and (3) performing PCR amplification by using the specific primer pair by using the genome DNA of the chicken to be detected as a template, wherein if one amplification product is obtained, the chicken to be detected is a cock or a candidate, and if two amplification products are obtained, the chicken to be detected is a hen or a candidate, the chicken to be detected is a hen. Compared with the prior art, the invention has the following advantages: the cock and the hen have the difference of the number of the strips, the difference of the characteristic strips is about 400bp, the identification can be carried out by agarose electrophoresis, the operation is simple and convenient, the misjudgment is not easy, the identification result is quick and accurate, the basic popularization and the use are convenient, and the wide market application prospect is realized.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a primer pair and a kit for early sex determination of chickens and application thereof.
Background
Most commercial poultry varieties are produced in a matched line mode, the father line generally grows faster, the mother line generally has higher breeding efficiency, so that the sex identification is required to be carried out early, the father line needs to eliminate the mother chicks in advance, and the mother line needs to eliminate the male chicks in advance. The commercial broiler cock has the advantages that the growth speed is high, the cock and the mother are bred in groups, and the breeding benefit is better. Only the female chicks are kept when the commercial laying hens are hatched, and the male chicks are directly eliminated. Therefore, early chicken sex determination has important significance in production.
At present, the sex of the chicken in the early stage is identified by observing the reproductive protrusion through turning the anus besides methods such as sex-linked inheritance (golden and silver feather, transverse spot feather, fast and slow feather) and the like. However, the anus turning identification needs to be carried out within 24 hours of the birth of the chick, and beyond the time period, the chick anus is difficult to turn, the genital protrusion is atrophied and even falls into the deep part of the cloaca, so that the observation is inconvenient. The labor intensity of anus overturning identification is high, the working environment is poor, the specialty is high, and the false identification rate is high. The method has important significance for rapidly and accurately identifying the early sex of the chicken by a molecular biological means.
Current molecular sex determination typically uses a chromosomal helix protein gene (CHD) with two homologous copies in female individuals and only one copy in male individuals. However, two homologous copy sequences of the female individual CHD gene are relatively conservative, generally only differ by 100-200bp, agarose electrophoresis is utilized, the resolution is low, misjudgment is easy, and the polyacrylamide gel electrode with high resolution is utilized, so that the experimental process is complicated and has high toxicity.
Therefore, it is necessary to develop a simple and convenient method capable of rapidly and accurately identifying the sex of the chicken.
Disclosure of Invention
The invention aims to provide a primer pair and a kit for early sex determination of chicken and application thereof.
The invention aims to provide a PCR primer pair for quickly identifying early chicken sex, and provides a kit and a method for identifying chicken sex by PCR by using the primer pair.
The invention provides a specific primer pair, which 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. The specific primer pair is used for identifying or assisting in identifying the sex of the chicken.
The invention also provides a kit for identifying or assisting in identifying the sex of the chicken, which comprises the specific primer pair.
The invention also protects the application of the kit in identification or auxiliary identification of chicken sex.
The invention also protects the application of the specific primer pair in identification or auxiliary identification of chicken sex.
The invention also protects the application of the specific primer pair in the preparation of a kit for identifying or assisting in identifying the sex of the chicken.
The invention also provides a method for identifying the sex of the chicken, which comprises the following steps:
taking the genome DNA of the chicken to be detected as a template, and carrying out PCR amplification by adopting a specific primer pair, wherein if one amplification product is obtained, the chicken to be detected is a cock or a candidate, and if two amplification products are obtained, the chicken to be detected is a hen or a candidate is a hen; 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.
The one amplification product is a characteristic amplification product.
The size of the characteristic amplification product is 538 bp;
the two amplification products are two characteristic amplification products.
The sizes of the two characteristic amplification products are 960bp and 538bp respectively.
The genomic DNA may be extracted from chicken feathers or blood.
The genomic DNA may be extracted from chicken tissue.
The genomic DNA may be extracted from an organ of chicken.
The reaction system for PCR amplification specifically may be: 2 XPCR Mix (Nanjing Bolddy Bio Inc.) 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, 21. mu.L ultrapure water.
The reaction procedure of the PCR amplification can be specifically as follows: 5min at 95 ℃; 30s at 95 ℃, 30s at 60 ℃ and 30s at 72 ℃ for 35 cycles; 10min at 72 ℃.
Any one of the chicken may be Camellia chicken or Tibetan chicken.
Compared with the prior art, the invention has the following advantages: the cock and the hen have the difference of the number of the strips, the difference of the characteristic strips is about 400bp, the identification can be carried out by agarose electrophoresis, the operation is simple and convenient, the misjudgment is not easy, the identification result is quick and accurate, the basic popularization and the use are convenient, and the wide market application prospect is realized. 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 in example 1.
FIG. 2 is an electropherogram of the PCR amplification product in example 2.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 establishment of the method
Supplying a sample book: 10 adult tea-colored chickens of known sex, 5 of which the numbers are 1-5 are cocks, and 5 of which the numbers are 6-10 are hens.
1. The genomic DNA is extracted from blood of a test sample.
2. And (3) performing PCR amplification by using the genomic DNA obtained in the step (1) as a template DNA.
The primer pairs used for PCR amplification were as follows:
forward primer (SEQ ID No. 1): 5'-TGTATTTTGGTTCTACAGGC-3', respectively;
reverse primer (SEQ ID NO. 2): 5'-ATCATCATAACCATAAATCT-3' are provided.
Reaction system for PCR amplification (50 μ L): 2 XPCR Mix (Nanjing Bolddy Bio Inc.) 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, 21. mu.L ultrapure water.
Reaction procedure for PCR amplification: 5min at 95 ℃; 30s at 95 ℃, 30s at 60 ℃ and 30s at 72 ℃ for 35 cycles; 10min at 72 ℃.
3. After completion of step 2, the PCR amplification product was subjected to 1.5% agarose gel electrophoresis.
The electrophoretogram is shown in FIG. 1. In fig. 1, lanes 1 to 10 correspond to test samples numbered 1 to 10 in sequence. All cocks showed a single characteristic band (538 bp by sequencing). All hens showed two characteristic bands (one was 960bp and the other was 538bp by sequencing).
The result shows that the primer pair and the corresponding PCR detection method can quickly and accurately identify the sex of the chicken, and are simple and easy to operate.
Example 2 application of the method
Supplying a sample book: 17 Tibetan chicks of unknown sex.
The procedure is as in example 1.
The electrophoretogram is shown in FIG. 2. In FIG. 2, lanes 1 to 17 represent 17 test samples in sequence. Among them, the sample was judged to be a cock when it partially showed a single characteristic band of 538bp ( lanes 1, 3, 6, 7, 8, 10, 11, 15, 17), and was judged to be a hen when it partially showed two characteristic bands of 960bp and 538bp ( lanes 2, 4, 5, 9, 12, 13, 14, 16).
The sample is dissected and observed in sexual organs, and the identification result is completely correct.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the 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 reference to specific embodiments, it will be appreciated that the invention can 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 use of some of the essential features is possible within the scope of the claims attached below.
Sequence listing
<110> scientific research institute for poultry in Jiangsu province
<120> primer pair and kit for early sex identification of chicken and application thereof
<130> GNCYX210396
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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tgtattttgg ttctacaggc 20
<210> 2
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
atcatcataa ccataaatct 20
Claims (7)
1. 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.
2. A kit for identifying or assisting in identifying the sex of a chicken, which comprises the specific primer pair of claim 1.
3. The use of the specific primer pair of claim 1 for identification or assisted identification of chicken sex.
4. Use of the specific primer pair of claim 1 in the preparation of a kit for identification or assisted identification of chicken gender.
5. Use of the kit according to claim 2 for the identification or assisted identification of the sex of chickens.
6. A method for identifying the sex of chicken comprises the following steps:
taking the genome DNA of the chicken to be detected as a template, and carrying out PCR amplification by adopting a specific primer pair, wherein if one amplification product is obtained, the chicken to be detected is a cock or a candidate, and if two amplification products are obtained, the chicken to be detected is a hen or a candidate is a hen;
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.
7. The method of claim 6, wherein:
the amplification product is a characteristic amplification product, and the size is 538 bp;
the two amplification products are two characteristic amplification products, and the sizes of the two characteristic amplification products are 960bp and 538bp respectively.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110118532.5A CN112746070A (en) | 2021-01-28 | 2021-01-28 | Primer pair and kit for early sex identification of chicken and application of primer pair and kit |
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| Application Number | Priority Date | Filing Date | Title |
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| CN202110118532.5A CN112746070A (en) | 2021-01-28 | 2021-01-28 | Primer pair and kit for early sex identification of chicken and application of primer pair and kit |
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| CN112746070A true CN112746070A (en) | 2021-05-04 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113981106A (en) * | 2021-11-05 | 2022-01-28 | 江苏省家禽科学研究所 | Method for performing early sex identification on phasianidae animals, matched kit and special primer pair |
| CN114045347A (en) * | 2021-11-09 | 2022-02-15 | 江苏省家禽科学研究所 | PCR primer, kit and method for sex identification of special poultry |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101130816A (en) * | 2007-07-27 | 2008-02-27 | 华中农业大学 | Method for appraising gender of chicken blastoderm by using multiple PCR |
| CN102181521A (en) * | 2011-03-04 | 2011-09-14 | 中国农业大学 | Polymerase chain reaction (PCR) amplification primer and method used for identifying sex of early embryo of chicken |
| CN203200273U (en) * | 2013-02-19 | 2013-09-18 | 江苏省家禽科学研究所 | Quick chicken gender identification kit |
| CN106834460A (en) * | 2017-01-23 | 2017-06-13 | 扬州大学 | A kind of embryo in egg sex rapid identification method |
| CN110257495A (en) * | 2019-07-31 | 2019-09-20 | 上海交通大学 | A kind of method that based on PCR technology carries out the identification of Chinese ring-necked pheasant early sex |
-
2021
- 2021-01-28 CN CN202110118532.5A patent/CN112746070A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101130816A (en) * | 2007-07-27 | 2008-02-27 | 华中农业大学 | Method for appraising gender of chicken blastoderm by using multiple PCR |
| CN102181521A (en) * | 2011-03-04 | 2011-09-14 | 中国农业大学 | Polymerase chain reaction (PCR) amplification primer and method used for identifying sex of early embryo of chicken |
| CN203200273U (en) * | 2013-02-19 | 2013-09-18 | 江苏省家禽科学研究所 | Quick chicken gender identification kit |
| CN106834460A (en) * | 2017-01-23 | 2017-06-13 | 扬州大学 | A kind of embryo in egg sex rapid identification method |
| CN110257495A (en) * | 2019-07-31 | 2019-09-20 | 上海交通大学 | A kind of method that based on PCR technology carries out the identification of Chinese ring-necked pheasant early sex |
Non-Patent Citations (3)
| Title |
|---|
| KIWOONG NAM ET AL.: "The Chicken (Gallus gallus) Z Chromosome Contains at Least Three The Chicken (Gallus gallus) Z Chromosome Contains at Least Three", 《GENETICS 》 * |
| MICHAEL N. ROMANOV ET AL.: "Widely Applicable PCR Markers for Sex Identification in Birds", 《RUSSIAN JOURNAL OF GENETICS》 * |
| 吴丽丽等: "基于CHD基因利用PCR技术进行鸡早期性别鉴定", 《热带农业工程》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113981106A (en) * | 2021-11-05 | 2022-01-28 | 江苏省家禽科学研究所 | Method for performing early sex identification on phasianidae animals, matched kit and special primer pair |
| 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 |
| CN114045347A (en) * | 2021-11-09 | 2022-02-15 | 江苏省家禽科学研究所 | PCR primer, kit and method for sex identification of special poultry |
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Application publication date: 20210504 |