CN113604548B - PCR primer, kit and method for sex identification of geese - Google Patents
PCR primer, kit and method for sex identification of geese Download PDFInfo
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Abstract
The invention provides a PCR primer, a kit and a method for sex identification of geese, wherein the sequence of the PCR primer for sex identification of poultry is shown as SEQ ID NO.1 and/or SEQ ID NO. 2. The PCR primer is used for PCR amplification and agarose gel electrophoresis, the sex of the duck, the chicken, the pigeon, the muscovy duck, the goose or the quail is determined by utilizing the number of bands of the electrophoresis result, the method has the advantages of simple and convenient operation, quick and accurate identification result, convenience for popularization and use of a base layer, and wide market application prospect, and in addition, the detection kit developed based on the method can generate considerable economic benefit and good social value.
Description
The application is divided application of China patent invention, wherein the application date is 2021, 01, 28, 202110120742.8 and the invention name is PCR primer, kit and method for identifying the sex of poultry.
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a PCR primer, a kit and a method for sex identification of geese.
Background
Commercial poultry varieties are 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 male and female poultry of the meat poultry generally grow faster, and the male and female poultry are bred in groups, so that the breeding benefit is better. When the egg-used poultry is hatched, only the mother chick is reserved, and the mother chick is directly eliminated. Early sexing of poultry is therefore of great importance in production.
At present, sex identification in early stage of poultry is mainly carried out by observing reproduction protrusion through anal turning except methods such as concomitant inheritance (gold and silver feather, transverse spot feather, rapid and slow feather) and the like. However, the identification of the anus of the young birds needs to be carried out within 24 hours after the birth of the young birds, and beyond the period of time, the anus of the young birds is difficult to open, the reproduction protrusion is atrophic and even falls into the deep part of the cloaca, so that the labor intensity of the identification of the anus of the young birds is high, the working environment is poor, the specialization is high, and the false identification rate is high.
Therefore, there is a need to develop a simple, convenient method for rapidly and accurately identifying the sex of poultry.
Disclosure of Invention
The invention aims to solve the technical problem of providing a PCR primer which is reasonable in design and can be used for rapid identification of early sex of poultry aiming at the defects of the prior art.
The invention achieves the aim of the invention through the following technical scheme:
the invention aims at providing a PCR primer for sex identification, and the primer sequence is shown as SEQ ID NO.1/SEQ ID NO. 2. Namely, the nucleotide sequence of the primer is as follows:
forward primer (SEQ ID No. 1): 5'-ATGAAAGAGTTTCAGCACTTGA-3';
reverse primer (SEQ ID NO. 2): 5'-TTCATAATAGGAGCTCGAGCCA-3'.
The second object of the present invention is to provide a kit comprising the PCR primer for sex determination of the present invention.
In one embodiment of the present invention, the kit of the present invention comprises the PCR primer of the present invention, a PCR reaction solution, and in some embodiments, a DNA extraction solution.
The third purpose of the invention is to provide the application of the PCR primer or the kit in the breeding industry.
The fourth purpose of the invention is to provide an application of the PCR primer or the kit in duck sex identification.
The application of the primer in sex identification of ducks in the invention carries out PCR amplification, and the electrophoresis result shows that one strip of 1147bp is a male duck, and the two strips of 1147bp and 851bp are female ducks.
The invention also provides the application of the PCR primer or the kit in chicken sex identification.
The primer of the invention is used for PCR amplification, and the electrophoresis result shows that a 983bp band is a cock, and the electrophoresis result shows that a 983bp band and a 798bp band are hens.
The invention aims at providing the application of the PCR primer or the kit in pigeon sex identification.
The primer of the invention is used for PCR amplification, and the electrophoresis result shows that one band of 937bp is male pigeon, and two bands of 937bp and 1153bp are female pigeon.
The invention aims at providing the application of the PCR primer or the kit in sex identification of the muscovy ducks.
The primer of the invention is used for PCR amplification, and the electrophoresis result shows that one band of 1153bp is male muscovy ducks, and two bands of 1153bp and 832bp are female muscovy ducks.
The invention aims at providing the application of the PCR primer or the kit in sex identification of geese.
The application of the invention in sex identification of geese shows that a 1145bp band is male geese and that 1145bp and 833bp bands are female geese.
The invention aims at providing the application of the PCR primer or the kit in quail sex identification.
The electrophoresis result shows that one band of 1068bp is the male quail, and two bands of 1068bp and 791bp are the female quail.
The invention also provides a method for rapidly identifying the sex of the ducks, chickens, pigeons, muscovy ducks, geese or quails, which comprises the following steps:
1) Extracting genome DNA of poultry to be detected;
2) Taking the DNA extracted in the step 1) as a template, and carrying out PCR amplification reaction by using the primer pair;
3) The PCR product is subjected to agarose gel electrophoresis detection,
when the poultry to be detected is a duck, the electrophoresis result shows that one band of 1147bp is a cock of a male duck, and the electrophoresis result shows that two bands of 1147bp and 851bp are female ducks;
when the poultry to be detected is a chicken, the electrophoresis result shows that one band of 983bp is a cock, and the poultry to be detected shows that two bands of 983bp and 798bp are hens;
when the poultry to be detected is a pigeon, the electrophoresis result shows that a 937bp band is a male pigeon, and shows that a 937bp band and a 1153bp band are female pigeons;
when the poultry to be detected is a muscovy duck, performing agarose gel electrophoresis detection on the electrophoresis result PCR product, wherein the electrophoresis result shows that one band of 1153bp is a male muscovy duck, and shows that two bands of 1153bp and 832bp are female muscovy duck;
when the poultry to be detected is a goose, the electrophoresis result shows that a 1145bp band is a male goose, and shows that two 1145bp and 833bp bands are female geese;
when the poultry to be detected is quail, the electrophoresis result shows that one band of 1068bp is male quail, and two bands of 1068bp and 791bp are female quail.
The method of the present invention, step 1) of extracting the genomic DNA of the poultry to be tested may be performed according to a conventional method in the art, for example, the extraction of the genomic DNA of the poultry to be tested may be performed by selecting the materials including, but not limited to, feathers, blood, tissues, organs, etc.
The PCR reaction system in step 2) of the method of the present invention may be a conventional system in the art, and in a specific embodiment of the present invention, it is: 2 XPCR Mix (Nanjinozan biological Co., ltd.) 25. Mu.L, 10. Mu. Mol/L forward and reverse primers each 1. Mu.L, 50-100. Mu.g/ml template DNA 2. Mu.L, and ultra-pure water 21. Mu.L.
The PCR reaction procedure in step 2) of the method of the present invention may be a conventional system in the art, and in a specific embodiment of the present invention, it is: the reaction was circulated at 95℃for 5min, (95℃for 30s,60℃for 30s, and 72℃for 30 s) 35 and at 72℃for 10min.
Compared with the prior art, the invention has the following advantages: the invention carries out PCR amplification and agarose gel electrophoresis through the designed PCR primer, utilizes the number of strips of the electrophoresis result to determine the sex of poultry, has simple and convenient operation, quick and accurate identification result, is convenient for popularization and use in a basic layer, has wide market application prospect, and can generate considerable economic benefit and good social value based on the detection kit developed by the method.
The primer of the invention has small restriction on the variety of poultry, so the specific variety of the ducks, chickens, pigeons, muscovy ducks, geese or quail is not limited, wherein the ducks in the invention refer to green-head ducks of Duck genus in the Duck family, the muscovy ducks are also called foreign ducks, and the ducks in the Duck family and the genus Duck are verruca nasus ducks.
Drawings
FIG. 1 is an electropherogram of the PCR amplified product of duck of example 1 of the present invention.
FIG. 2 is an electropherogram of the PCR amplified product of duck of example 2 of the present invention.
FIG. 3 is an electropherogram of the PCR amplified product of chicken of example 3 of the present invention.
FIG. 4 is an electropherogram of the PCR amplified product of chicken of example 4 of the present invention.
FIG. 5 shows an electropherogram of the PCR amplified product of pigeon according to example 6 of the present invention.
FIG. 6 is an electropherogram of the PCR amplified product of pigeon according to example 7 of the present invention.
FIG. 7 is an electropherogram of PCR amplified products of Muscovy Duck of example 8 of the present invention.
FIG. 8 is an electropherogram of PCR amplified products of Muscovy Duck of example 9 of the present invention.
FIG. 9 is an electropherogram of the PCR amplified product of goose of example 10 of the present invention.
FIG. 10 is an electropherogram of PCR amplified products of goose of example 11 of the present invention.
FIG. 11 is an electropherogram of PCR amplified product of quail of example 12 of the present invention.
FIG. 12 is an electropherogram of PCR amplified product of quail of example 13 of the present invention.
Detailed Description
The following examples facilitate a better understanding of the present invention, but are not intended to limit the same. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores.
Example 1
1 identification of Duck of known sex
1.1 sample collection
10 adult Beijing ducks with known sexes are collected, the numbers 1-5 are male ducks, and the numbers 6-10 are female ducks, and the designed primers are used for amplifying duck genome.
Forward primer (SEQ ID No. 1): 5'-ATGAAAGAGTTTCAGCACTTGA-3';
reverse primer (SEQ ID NO. 2): 5'-TTCATAATAGGAGCTCGAGCCA-3'.
1.2 PCR amplification
The PCR reaction system is as follows:
2 XPCR Mix (Nanjinozan biological Co., ltd.) 25. Mu.L, 10. Mu. Mol/L forward primer 1. Mu.L each, 50-100. Mu.g/ml template DNA 2. Mu.L, and ultra pure water 21. Mu.L.
The PCR reaction procedure was: the reaction was circulated at 95℃for 5min, (95℃for 30s,60℃for 30s, and 72℃for 30 s) 35 and at 72℃for 10min.
1.3 electrophoresis detection
After the completion of the reaction, the reaction mixture was subjected to 1.5% agarose gel electrophoresis. The electrophoresis detection of the duckling only has one 1147bp band, and the electrophoresis detection of the female duck has two 1147bp bands and 851bp bands, and the result is shown in figure 1 (1-5 are duckling and 6-10 are female duckling).
The results show that the sex identification method can quickly and accurately carry out sex identification on ducks, and is simple and easy to operate.
Example 2
2 identification of unknown sex ducks
2.1 sample collection
16 Shaoxing ducklings of unknown gender were collected and the duck genome was amplified using the primers designed according to the invention (SEQ ID NO. 1) and (SEQ ID NO. 2).
2.2 PCR amplification
The PCR reaction system is as follows:
2 XPCR Mix (Nanjinozan biological Co., ltd.) 25. Mu.L, 10. Mu. Mol/L forward primer 1. Mu.L each, 50-100. Mu.g/ml template DNA 2. Mu.L, and ultra pure water 21. Mu.L.
The PCR reaction procedure was: the reaction was circulated at 95℃for 5min, (95℃for 30s,60℃for 30s, and 72℃for 30 s) 35 and at 72℃for 10min.
2.3 electrophoresis detection
After the completion of the reaction, the reaction mixture was subjected to 1.5% agarose gel electrophoresis. The electrophoresis detection of the duckling only has one 1147bp band, and the electrophoresis detection of the female duck has two 1147bp bands and 851bp bands, and the results are shown in figure 2 (wherein 3, 4, 6, 7, 11, 12, 15 and 16 are duckling, and 1, 2, 5, 8, 9, 10, 13 and 14 are female duckling).
The sample is subjected to dissection and is subjected to observation of a sexual organ, and the identification result is completely correct.
Example 3
1 identification of known sexes
1.1 sample collection
8 adult Chongren Ma chickens of known gender are collected, the number 1-4 is cock, and the number 5-8 is hen, and the primers designed by the invention are used for amplifying the genome of the chickens.
Forward primer (SEQ ID No. 1): 5'-ATGAAAGAGTTTCAGCACTTGA-3';
reverse primer (SEQ ID NO. 2): 5'-TTCATAATAGGAGCTCGAGCCA-3'.
1.2 PCR amplification
The PCR reaction system is as follows:
2 XPCR Mix (Nanjinozan biological Co., ltd.) 25. Mu.L, 10. Mu. Mol/L forward primer 1. Mu.L each, 50-100. Mu.g/ml template DNA 2. Mu.L, and ultra pure water 21. Mu.L.
The PCR reaction procedure was: the reaction was circulated at 95℃for 5min, (95℃for 30s,60℃for 30s, and 72℃for 30 s) 35 and at 72℃for 10min.
1.3 electrophoresis detection
After the completion of the reaction, the reaction mixture was subjected to 1.5% agarose gel electrophoresis. The hen electrophoresis detection only has one 983bp band, and the hen electrophoresis detection has two 983bp bands and 798bp bands, and the result is shown in figure 3 (wherein 1-4 are hens and 5-8 are hens).
The results show that the sex identification method can rapidly and accurately carry out sex identification on chickens, and is simple and easy to operate.
Example 4
2 identification of unknown sex chickens
2.1 sample collection
Chicks of 24 recessive white chickens of unknown sex were collected and the chicken genome was amplified using the primers designed according to the invention (SEQ ID NO. 1) and (SEQ ID NO. 2).
2.2 PCR amplification
The PCR reaction system is as follows:
2 XPCR Mix (Nanjinozan biological Co., ltd.) 25. Mu.L, 10. Mu. Mol/L forward primer 1. Mu.L each, 50-100. Mu.g/ml template DNA 2. Mu.L, and ultra pure water 21. Mu.L.
The PCR reaction procedure was: the reaction was circulated at 95℃for 5min, (95℃for 30s,60℃for 30s, and 72℃for 30 s) 35 and at 72℃for 10min.
2.3 electrophoresis detection
After the completion of the reaction, the reaction mixture was subjected to 1.5% agarose gel electrophoresis. The rooster electrophoresis detection only has one 983bp band, and the hen electrophoresis detection has two 983bp bands and 798bp bands, and the results are shown in figure 4 (wherein 1, 4, 5, 7, 8, 9, 12, 14, 17, 20, 21, 22 are roosters, 2, 3, 6, 10, 11, 13, 15, 16, 18, 19, 23, 24 are hens).
The sample is subjected to dissection and is subjected to observation of a sexual organ, and the identification result is completely correct.
Example 5
3 resource protection application
3.1 the primers (SEQ ID NO. 1) and (SEQ ID NO. 2) were designed to amplify the chicken genome using 19 varieties of Dagu chicken, white-ear yellow chicken, henan nugget chicken, beijing oil chicken, xianju chicken, theat chicken, silk feather black-bone chicken, langshan chicken, tibetan chicken, qingyuan Ma chicken, wai gray chicken, jinhu black-bone chicken, theat chicken, shou chicken, deer garden chicken, dongxiang black chicken, edge chicken, wenchang chicken, gushi chicken and the like in the blood sample gene library of the local unit local chicken.
3.2 PCR amplification
The PCR reaction system is as follows:
2 XPCR Mix (Nanjinozan biological Co., ltd.) 25. Mu.L, 10. Mu. Mol/L forward primer 1. Mu.L each, 50-100. Mu.g/ml template DNA 2. Mu.L, and ultra pure water 21. Mu.L.
The PCR reaction procedure was: the reaction was circulated at 95℃for 5min, (95℃for 30s,60℃for 30s, and 72℃for 30 s) 35 and at 72℃for 10min.
3.3 electrophoresis detection
After the completion of the reaction, the reaction mixture was subjected to 1.5% agarose gel electrophoresis. The rooster electrophoresis detection only has a 983bp band, and the hen electrophoresis detection has two 983bp bands and 798bp bands, and the identification result is consistent with the result of individual gender record, as shown in Table 1.
TABLE 1 detection results of 19 different local breeds of chickens
Example 6
1 identification of pigeon of known sex Bai Yuwang
1.1 sample collection
8 adult Bai Yuwang pigeons with known sexes are collected, the number 1-4 is male pigeon, the number 5-8 is female pigeon, and the genome of the pigeon is amplified by using the primers designed by the invention.
Forward primer (SEQ ID No. 1): 5'-ATGAAAGAGTTTCAGCACTTGA-3';
reverse primer (SEQ ID NO. 2): 5'-TTCATAATAGGAGCTCGAGCCA-3'.
1.2 PCR amplification
The PCR reaction system is as follows:
2 XPCR Mix (Nanjinozan biological Co., ltd.) 25. Mu.L, 10. Mu. Mol/L forward primer 1. Mu.L each, 50-100. Mu.g/ml template DNA 2. Mu.L, and ultra pure water 21. Mu.L.
The PCR reaction procedure was: the reaction was circulated at 95℃for 5min, (95℃for 30s,60℃for 30s, and 72℃for 30 s) 35 and at 72℃for 10min.
1.3 electrophoresis detection
After the completion of the reaction, the reaction mixture was subjected to 1.5% agarose gel electrophoresis. The electrophoresis detection of the male pigeon only has one 937bp band, and the electrophoresis detection of the female pigeon has two 937bp bands and 1153bp bands, and the result is shown in figure 5 (wherein 1-4 are male pigeons and 5-8 are female pigeons).
The results show that the sex identification method can rapidly and accurately identify the sex of the pigeons, and is simple and easy to operate.
Example 7
2 identification of European meat pigeons of unknown sex
2.1 sample collection
17 young pigeons of European meat pigeons of unknown sex were collected and the pigeon genome was amplified using the primers designed according to the invention (SEQ ID NO. 1) and (SEQ ID NO. 2).
2.2 PCR amplification
The PCR reaction system is as follows:
2 XPCR Mix (Nanjinozan biological Co., ltd.) 25. Mu.L, 10. Mu. Mol/L forward primer 1. Mu.L each, 50-100. Mu.g/ml template DNA 2. Mu.L, and ultra pure water 21. Mu.L.
The PCR reaction procedure was: the reaction was circulated at 95℃for 5min, (95℃for 30s,60℃for 30s, and 72℃for 30 s) 35 and at 72℃for 10min.
2.3 electrophoresis detection
After the completion of the reaction, the reaction mixture was subjected to 1.5% agarose gel electrophoresis. The male pigeon electrophoresis detection only has one 937bp band, and the female pigeon electrophoresis detection has two 937bp bands and 1153bp bands, and the results are shown in figure 6 (wherein 1, 2, 4, 7, 8, 12, 14 and 15 are male pigeons, and 3, 5, 6, 9, 10, 11, 13, 16 and 17 are female pigeons).
The sample is subjected to dissection and is subjected to observation of a sexual organ, and the identification result is completely correct.
Example 8
1 identification of white-feather Muscovy Duck of known sex
1.1 sample collection
10 adult white-feather muscovy ducks with known sexes are collected, the numbers 1-5 are male muscovy ducks, and the numbers 6-10 are female muscovy ducks, and the genome of the muscovy ducks is amplified by using the designed primers.
Forward primer (SEQ ID No. 1): 5'-ATGAAAGAGTTTCAGCACTTGA-3';
reverse primer (SEQ ID NO. 2): 5'-TTCATAATAGGAGCTCGAGCCA-3'.
1.2 PCR amplification
The PCR reaction system is as follows:
2 XPCR Mix (Nanjinozan biological Co., ltd.) 25. Mu.L, 10. Mu. Mol/L forward primer 1. Mu.L each, 50-100. Mu.g/ml template DNA 2. Mu.L, and ultra pure water 21. Mu.L.
The PCR reaction procedure was: the reaction was circulated at 95℃for 5min, (95℃for 30s,60℃for 30s, and 72℃for 30 s) 35 and at 72℃for 10min.
1.3 electrophoresis detection
After the completion of the reaction, the reaction mixture was subjected to 1.5% agarose gel electrophoresis. The electrophoresis detection of the male Muscovy duck only has one 1153bp band, and the electrophoresis detection of the female Muscovy duck has two 1153bp and 832bp bands, and the result is shown in figure 7 (wherein 1-5 are male Muscovy ducks and 6-10 are female Muscovy ducks).
The results show that the sex identification method can rapidly and accurately identify the sex of the muscovy ducks, and is simple and easy to operate.
Example 9
2 identification of unknown sex black-feather muscovy ducks
2.1 sample collection
17 black-feather muscovy ducks of unknown sex were collected and the muscovy duck genome was amplified using the primers designed according to the invention (SEQ ID NO. 1) and (SEQ ID NO. 2).
2.2 PCR amplification
The PCR reaction system is as follows:
2 XPCR Mix (Nanjinozan biological Co., ltd.) 25. Mu.L, 10. Mu. Mol/L forward primer 1. Mu.L each, 50-100. Mu.g/ml template DNA 2. Mu.L, and ultra pure water 21. Mu.L.
The PCR reaction procedure was: the reaction was circulated at 95℃for 5min, (95℃for 30s,60℃for 30s, and 72℃for 30 s) 35 and at 72℃for 10min.
2.3 electrophoresis detection
After the completion of the reaction, the reaction mixture was subjected to 1.5% agarose gel electrophoresis. The electrophoresis detection of the male Muscovy duck only has one 1153bp band, and the electrophoresis detection of the female Muscovy duck has two 1153bp and 832bp bands, and the result is shown in figure 8 (wherein 2, 3, 5, 7, 10, 11, 13, 14 and 17 are male Muscovy ducks, and 1, 4, 6, 8, 9, 12, 15 and 16 are female Muscovy ducks).
The sample is subjected to dissection and is subjected to observation of a sexual organ, and the identification result is completely correct.
Example 10
1 identification of known sex of Yangzhou geese
1.1 sample collection
10 adult Yangzhou geese of known sex are collected, the number 1-5 is male geese, and the number 6-10 is female geese, and the genome of the geese is amplified by using the primers designed by the invention.
Forward primer (SEQ ID No. 1): 5'-ATGAAAGAGTTTCAGCACTTGA-3';
reverse primer (SEQ ID NO. 2): 5'-TTCATAATAGGAGCTCGAGCCA-3'.
1.2 PCR amplification
The PCR reaction system is as follows:
2 XPCR Mix (Nanjinozan biological Co., ltd.) 25. Mu.L, 10. Mu. Mol/L forward primer 1. Mu.L each, 50-100. Mu.g/ml template DNA 2. Mu.L, and ultra pure water 21. Mu.L.
The PCR reaction procedure was: the reaction was circulated at 95℃for 5min, (95℃for 30s,60℃for 30s, and 72℃for 30 s) 35 and at 72℃for 10min.
1.3 electrophoresis detection
After the completion of the reaction, the reaction mixture was subjected to 1.5% agarose gel electrophoresis. The electrophoresis detection of the male goose only has a 1145bp band, and the electrophoresis detection of the female goose has two 1145bp bands and 833bp bands, and the result is shown in figure 9 (wherein 1-5 are male geese and 6-10 are female geese).
The results show that the sex identification method can rapidly and accurately identify the sex of the geese, and is simple and easy to operate.
Example 11
2 identification of unknown sex of Rhine geese
2.1 sample collection
17 non-sex-known Rhine geese were harvested and the goose genome was amplified using the primers designed according to the invention (SEQ ID NO. 1) and (SEQ ID NO. 2).
2.2 PCR amplification
The PCR reaction system is as follows:
2 XPCR Mix (Nanjinozan biological Co., ltd.) 25. Mu.L, 10. Mu. Mol/L forward primer 1. Mu.L each, 50-100. Mu.g/ml template DNA 2. Mu.L, and ultra pure water 21. Mu.L.
The PCR reaction procedure was: the reaction was circulated at 95℃for 5min, (95℃for 30s,60℃for 30s, and 72℃for 30 s) 35 and at 72℃for 10min.
2.3 electrophoresis detection
After the completion of the reaction, the reaction mixture was subjected to 1.5% agarose gel electrophoresis. The electrophoresis detection of the male geese only has one 1145bp band, and the electrophoresis detection of the female geese has two 1145bp bands and 833bp bands, and the results are shown in figure 10 (wherein 1, 4, 7, 8, 11, 13, 15 and 16 are male geese, and 2, 3, 5, 6, 9, 10, 12, 14 and 17 are female geese).
The sample is subjected to dissection and is subjected to observation of a sexual organ, and the identification result is completely correct.
Example 12
1 identification of Japanese quail of known sex
1.1 sample collection
8 adult Japanese quails of known sex are collected, the numbers 1-4 are male quails, and the numbers 5-8 are female quails, and the genome of the quails is amplified by using the primers designed by the invention.
Forward primer (SEQ ID No. 1): 5'-ATGAAAGAGTTTCAGCACTTGA-3';
reverse primer (SEQ ID NO. 2): 5'-TTCATAATAGGAGCTCGAGCCA-3'.
1.2 PCR amplification
The PCR reaction system is as follows:
2 XPCR Mix (Nanjinozan biological Co., ltd.) 25. Mu.L, 10. Mu. Mol/L forward primer 1. Mu.L each, 50-100. Mu.g/ml template DNA 2. Mu.L, and ultra pure water 21. Mu.L.
The PCR reaction procedure was: the reaction was circulated at 95℃for 5min, (95℃for 30s,60℃for 30s, and 72℃for 30 s) 35 and at 72℃for 10min.
1.3 electrophoresis detection
After the completion of the reaction, the reaction mixture was subjected to 1.5% agarose gel electrophoresis. The electrophoresis detection of the male quail only has one 1068bp band, and the electrophoresis detection of the female quail has two 1068bp bands and 791bp bands, and the results are shown in FIG. 11 (wherein 1-4 are the male quails and 5-8 are the female quails).
The results show that the sex identification method can quickly and accurately identify the sex of the quails, and is simple and easy to operate.
Example 13
2 identification of unknown sex of Chinese white feather quails
2.1 sample collection
17 Chinese white feather quails of unknown gender were collected and the primers (SEQ ID NO. 1) and (SEQ ID NO. 2) designed by the invention were used to amplify the quail genome.
2.2 PCR amplification
The PCR reaction system is as follows:
2 XPCR Mix (Nanjinozan biological Co., ltd.) 25. Mu.L, 10. Mu. Mol/L forward primer 1. Mu.L each, 50-100. Mu.g/ml template DNA 2. Mu.L, and ultra pure water 21. Mu.L.
The PCR reaction procedure was: the reaction was circulated at 95℃for 5min, (95℃for 30s,60℃for 30s, and 72℃for 30 s) 35 and at 72℃for 10min.
2.3 electrophoresis detection
After the completion of the reaction, the reaction mixture was subjected to 1.5% agarose gel electrophoresis. The electrophoresis detection of the male quail only has one 1068bp band, and the electrophoresis detection of the female quail has two 1068bp bands and 791bp bands, and the results are shown in FIG. 12 (wherein 3, 5, 6, 9, 10, 11, 13 and 16 are male quails, and 1, 2, 4, 7, 8, 12, 14, 15 and 17 are female quails).
The sample is subjected to dissection and is subjected to observation of a sexual organ, and the identification result is completely correct.
Sequence listing
<110> Jiangsu province poultry science institute
<120> PCR primer, kit and method for sex identification of geese
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
atgaaagagt ttcagcactt ga 22
<210> 2
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
ttcataatag gagctcgagc ca 22
Claims (6)
1. The application of a PCR primer or a kit containing the PCR primer in sex identification of geese is specifically that PCR amplification is carried out by the PCR primer or the kit, the electrophoresis result shows that one 1145bp band is a male geese, and the two 1145bp and 833bp bands are female geese; the nucleotide sequence of the primer is as follows: the forward primer sequence is shown as SEQ ID NO.1, and the reverse primer sequence is shown as SEQ ID NO. 2.
2. The use according to claim 1, wherein the kit further comprises a PCR reaction solution and/or a DNA extraction solution.
3. The rapid sex identification method for geese is characterized by comprising the following steps of:
1) Extracting genome DNA of the goose to be detected;
2) Using the DNA extracted in the step 1) as a template, and carrying out PCR amplification reaction by using a primer; the nucleotide sequence of the primer is as follows: the forward primer sequence is shown as SEQ ID NO.1, and the reverse primer sequence is shown as SEQ ID NO. 2;
3) The PCR product is subjected to agarose gel electrophoresis detection,
the electrophoresis result shows that one band of 1145bp is a male goose, and the two bands of 1145bp and 833bp are a female goose.
4. The method according to claim 3, wherein step 1) extracting the genomic DNA of the goose to be tested uses feathers, blood, tissues or organs for extracting the genomic DNA.
5. The method according to claim 3, wherein the PCR reaction system in step 2) is: 2 XPCRMIX 25. Mu.L, 10. Mu. Mol/L forward and reverse primers each 1. Mu.L, 50-100. Mu.g/ml template DNA 2. Mu.L, and ultra pure water 21. Mu.L.
6. The method of claim 3, wherein the PCR reaction procedure in step 2) is as follows: the cycle was 5min at 95℃for 30s,60℃for 30s, and 72℃for 30s 35, and 10min at 72 ℃.
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| CN114045347B (en) * | 2021-11-09 | 2022-08-16 | 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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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102618659A (en) * | 2012-04-18 | 2012-08-01 | 江苏省家禽科学研究所 | Polymerase chain reaction (PCR) primers, method and kit for identifying duck gender |
| CN103468825A (en) * | 2013-10-14 | 2013-12-25 | 扬州大学 | Primer, kit and method used for goose sex identification |
| CN109913556A (en) * | 2019-01-08 | 2019-06-21 | 北京市农林科学院 | A kind of primer, kit and its method for Rapid identification dove gender |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101962677A (en) * | 2010-09-15 | 2011-02-02 | 华南农业大学 | Method for identifying poultry gender |
| CN203200273U (en) * | 2013-02-19 | 2013-09-18 | 江苏省家禽科学研究所 | Quick chicken gender identification kit |
| CN204079950U (en) * | 2014-06-16 | 2015-01-07 | 江苏省家禽科学研究所 | Fowl Sex Rapid identification test kit |
| EP3371322B1 (en) * | 2015-11-03 | 2019-11-27 | Planton GmbH | Method for gender identification in domestic chicken |
| CN105925690B (en) * | 2016-05-16 | 2019-07-19 | 华南农业大学 | It is a kind of for identifying the primer, kit and its discrimination method of pigeon gender |
| CN106244689B (en) * | 2016-08-02 | 2019-06-18 | 湖北省农业科学院畜牧兽医研究所 | A kind of method that based on PCR technology carries out quail sex identification |
| CN106435008A (en) * | 2016-12-26 | 2017-02-22 | 河南科技大学 | Primers, kit and detection method for detecting genders of cotuenix coturnix |
| CN108588204A (en) * | 2018-06-07 | 2018-09-28 | 浙江大学 | Dove early sex PCR identification kits, application and method |
| CN111979308B (en) * | 2020-10-10 | 2023-05-05 | 广东科贸职业学院 | Primer composition, kit and method for identifying early sex of pigeons |
-
2021
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102618659A (en) * | 2012-04-18 | 2012-08-01 | 江苏省家禽科学研究所 | Polymerase chain reaction (PCR) primers, method and kit for identifying duck gender |
| CN103468825A (en) * | 2013-10-14 | 2013-12-25 | 扬州大学 | Primer, kit and method used for goose sex identification |
| CN109913556A (en) * | 2019-01-08 | 2019-06-21 | 北京市农林科学院 | A kind of primer, kit and its method for Rapid identification dove gender |
Non-Patent Citations (2)
| Title |
|---|
| Molecular techniques for sex identification of captive birds;Medania Purwaningrum等;《Veterinary World》;第12卷(第9期);第1506-1513页 * |
| 快速鉴定主要禽类性别的一对新的通用引物;刘宏祥等;《农业生物技术学报》;第22卷(第12期);第1567-1574页 * |
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