CN112695101A - Method for identifying early sex of quail, matched kit and special primer pair - Google Patents
Method for identifying early sex of quail, matched kit and special primer pair Download PDFInfo
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- 241000286209 Phasianidae Species 0.000 title claims abstract description 73
- 238000000034 method Methods 0.000 title claims abstract description 21
- 108020004414 DNA Proteins 0.000 claims abstract description 27
- 238000012408 PCR amplification Methods 0.000 claims abstract description 17
- 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 10
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- 229920000936 Agarose Polymers 0.000 abstract description 3
- 238000006243 chemical reaction Methods 0.000 description 7
- 238000012360 testing method Methods 0.000 description 5
- 210000000436 anus Anatomy 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 3
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- 238000009395 breeding Methods 0.000 description 3
- 230000001488 breeding effect Effects 0.000 description 3
- 235000013601 eggs Nutrition 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
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- 235000013372 meat Nutrition 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 238000011160 research Methods 0.000 description 2
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- 241001070941 Castanea Species 0.000 description 1
- 235000014036 Castanea Nutrition 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 244000207740 Lemna minor Species 0.000 description 1
- 235000006439 Lemna minor Nutrition 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 101150069823 chd gene Proteins 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
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- 210000003555 cloaca Anatomy 0.000 description 1
- 238000011161 development Methods 0.000 description 1
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- 230000005611 electricity Effects 0.000 description 1
- 238000002474 experimental method Methods 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
- 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
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- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000001850 reproductive effect Effects 0.000 description 1
- 230000020509 sex determination Effects 0.000 description 1
- 238000003307 slaughter Methods 0.000 description 1
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Abstract
The invention discloses a method for identifying early sex of quails, a matched kit and a special primer pair. The invention provides a method for identifying the sex of quails, which comprises the following steps: taking the genome DNA of a quail 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 quail to be detected is or is a candidate of a male quail, and if two amplification products are obtained, the quail to be detected is or is a candidate of a female quail; 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. Compared with the prior art, the invention has the following advantages: the male 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 by 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 a basic level are convenient, and the wide market application prospect is realized.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for identifying early sex of quails, a matched kit and a special primer pair.
Background
Quail belongs to the category of special economic poultry, the nutrient content of quail eggs and quail meat is basically higher than that of eggs and chicken, and the quail has extremely high edible value. The quail has the advantages of early sexual maturity, high growth speed, strong fecundity, high feed conversion rate and better breeding prospect.
At present, most of commercial poultry are produced in a complete set mode, the father line generally grows faster, the mother line generally has higher breeding efficiency, so that the early sex identification is needed, the father line needs to eliminate the mother chicks in advance, and the mother line needs to eliminate the male chicks in advance. The male quails and the female quails for meat quails grow fast, and the male quails and the female quails are bred in groups, so that the breeding benefit is better. When the laying quails are hatched, only the female chicks are kept, and the male chicks are directly eliminated. Therefore, the early sex identification of the quails has important significance in production.
At present, the early sex identification of quails is mainly carried out by observing reproductive protuberances through anus turning. However, the anus turning identification needs to be carried out within 24 hours of the young quails, and beyond the time period, the anus of the young quails is difficult to turn, and the genital protuberance is atrophied and even falls into the deep 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 quail 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 electricity with high resolution is utilized, so that the test process is complicated and has high toxicity.
With the rapid development of the quail industry in China, a method which is simple and convenient and can rapidly and accurately identify the gender of the quail is urgently needed to be developed.
Disclosure of Invention
The invention aims to provide a method for identifying early sex of quail, a matched kit and a special primer pair.
The invention provides a method for identifying the sex of quails, which comprises the following steps:
taking the genome DNA of a quail 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 quail to be detected is or is a candidate of a male quail, and if two amplification products are obtained, the quail to be detected is or is a candidate of a female quail;
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 amplification product is a characteristic amplification product and has the size of 537 bp;
the two amplification products are two characteristic amplification products, and the sizes of the two characteristic amplification products are 941bp and 537bp respectively.
The genomic DNA may be extracted from quail feathers or blood.
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 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 ℃.
The invention also protects a specific primer pair, which consists of a single-stranded DNA molecule shown by SEQ ID NO.1 and a single-stranded DNA molecule shown by 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 identifying or assisting in identifying the sex of the quail.
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 quail.
The invention also provides a kit for identifying or assisting in identifying the sex of the quail, which comprises the specific primer pair.
The invention also protects the application of the kit in identifying or assisting in identifying the sex of the quail.
Any of the above quails may be Korean quail or Japanese quail.
The invention aims to provide a PCR primer pair for quickly identifying the early sex of quails, and provides a kit and a method for identifying the sex of the quails by PCR by using the primer pair.
Compared with the prior art, the invention has the following advantages: the male 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 by 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 a basic level 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.
The following documents are described for quails: living body detection research for predicting slaughtering performance of chestnut feather egg quails, Mochuyu, Lemna minor, Zhan stamen, Paoyongyong, Changling, Bozhu, Anhui agricultural science, J.Anhui agric.Sci.2020, 48(1): 92-95).
Example 1 establishment of the method
Supplying a sample book: 10 adult Korean quails of known gender, 5 male quails numbered 1-5 and 5 female quails numbered 6-10.
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 sample numbers 1 to 10 in sequence. All male quails showed a single characteristic band (537 bp by sequencing). All female quails showed two characteristic bands (one is 941bp and the other is 537bp by sequencing).
The result shows that the primer pair and the corresponding PCR detection method can quickly and accurately identify the sex of the quail, and are simple and easy to operate.
Example 2 application of the method
Supplying a sample book: 17 Japanese quail young quails of unknown sex.
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. 2. In FIG. 2, lanes 1 to 17 represent 17 test samples in sequence. Among them, the sample was judged to be male quails by showing 537bp single characteristic bands ( lanes 3, 4, 6, 7, 8, 10, and 11), and was judged to be female quails by showing 941bp and 537bp two characteristic bands ( lanes 1, 2, 5, 9, 12, 13, 14, 15, 16, and 17). The size of the characteristic band was verified by sequencing.
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> method for identifying early sex of quail, matched kit and special primer pair
<130> GNCYX210395
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Claims (7)
1. A method for identifying the sex of quails comprises the following steps:
taking the genome DNA of a quail 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 quail to be detected is or is a candidate of a male quail, and if two amplification products are obtained, the quail to be detected is or is a candidate of a female quail;
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. The method of claim 1, wherein:
the amplification product is a characteristic amplification product and has the size of 537 bp;
the two amplification products are two characteristic amplification products, and the sizes of the two characteristic amplification products are 941bp and 537bp respectively.
3. 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.
4. The use of the specific primer pair of claim 3 for identifying or assisting in identifying the sex of quail.
5. Use of the specific primer pair of claim 3 in the preparation of a kit for identifying or assisting in identifying quail gender.
6. A kit for identifying or assisting in identifying quail gender, comprising the specific primer pair of claim 3.
7. Use of the kit according to claim 6 for identification or assisted identification of quail gender.
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Cited By (1)
| 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 |
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|---|---|---|---|---|
| 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 |
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2021
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Patent Citations (3)
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|---|---|---|---|---|
| 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 |
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| HARIKRISHNA REDDY RALLABANDI等: "Identification of female specific genes in the W chromosome that are expressed during gonadal differentiation in the chicken", KOREAN J. POULT. SCI., vol. 46, no. 4, pages 1 * |
| KELSEY CAETANO-ANOLLES等: "Comprehensive identification of sexual dimorphism-associated differentially expressed genes in two-way factorial designed RNA-Seq data on Japanese quail (Coturnix coturnix japonica)", PLOS ONE, vol. 10, no. 9, pages 8 * |
| 张小辉等: "鹌鹑性别的分子鉴定方法研究", 中国家禽, vol. 42, no. 5, pages 20 - 23 * |
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 |
| 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 |
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