CN112662787A - PCR primer, kit and method for poultry sex identification - Google Patents
PCR primer, kit and method for poultry sex identification Download PDFInfo
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Abstract
The invention provides a PCR primer, a kit and a method for identifying poultry sex, wherein the sequence of the PCR primer for identifying poultry sex 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, and the sex of the duck, chicken, pigeon, Muscovy duck, goose or quail is determined by the number of bands of the electrophoresis result, so that the method has the advantages of simple and convenient operation, quick and accurate identification result, convenience for basic popularization and use, wide market application prospect, and considerable economic benefit and good social value based on the detection kit developed by the method.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a PCR primer, a kit and a method for poultry gender identification.
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 male poultry and the female poultry of the meat poultry generally grow faster, and the male poultry and the female poultry are bred in groups, so that the breeding benefit is better. When the laying poultry is hatched, only the female poultry is kept and the male poultry is directly eliminated. Early sexing of poultry is therefore of great importance in production.
At present, sex identification of early poultry is carried out by observing reproductive protrusion through turning anus except methods such as sex-linked inheritance (golden and silver feather, transverse spot feather, fast and slow feather) and the like. However, the flip anus identification needs to be carried out within 24 hours of the birth of the young poultry, and beyond the time period, the anus of the young poultry is difficult to flip, the genital protuberance is atrophied and even falls into the deep part of the cloaca, the flip anus identification is inconvenient to observe, the labor intensity is high, the working environment is poor, the specialty is high, and the misidentification rate is high.
Therefore, it is necessary to develop a simple and convenient method capable of 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 quickly identifying early sex of poultry aiming at the defects of the prior art.
The invention realizes the purpose of the invention by the following technical scheme:
one purpose of the invention is to provide a PCR primer for identifying the sex, and the sequence of the primer 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', respectively;
reverse primer (SEQ ID NO. 2): 5'-TTCATAATAGGAGCTCGAGCCA-3' are provided.
Another 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 invention also aims to provide the application of the PCR primer or the kit in the breeding industry.
The fourth purpose of the invention is to provide the application of the PCR primer or the kit in duck sex identification.
The application in duck sex identification provided by the invention is that PCR amplification is carried out by the primer provided by the invention, and the duck presents a male duck with one band of 1147bp and a female duck with two bands of 1147bp and 851bp as electrophoresis results.
The fifth purpose of the invention is to provide the application of the PCR primer or the kit in chicken sex identification.
The application in chicken sex identification provided by the invention is that PCR amplification is carried out by the primer provided by the invention, and the cock is represented by one band of 983bp and the hen is represented by two bands of 983bp and 798bp in electrophoresis results.
The sixth purpose of the invention is to provide the application of the PCR primer or the kit in pigeon sex identification.
The primer is used for PCR amplification, and the male pigeon with one 937bp band and the female pigeon with two 937bp and 1153bp bands are obtained by electrophoresis.
The seventh purpose of the invention is to provide the application of the PCR primer or the kit in identifying the sex of Muscovy ducks.
The application in identifying the sex of Muscovy ducks is to perform PCR amplification through the primer, and the Muscovy ducks are male Muscovy ducks with one band of 1153bp and female Muscovy ducks with two bands of 1153bp and 832 bp.
The eighth purpose of the invention is to provide the application of the PCR primer or the kit in goose sex identification.
The invention relates to an application in goose sex identification, wherein an electrophoresis result shows that a male goose is represented by one band of 1145bp, and a female goose is represented by two bands of 1145bp and 833 bp.
The ninth purpose of the invention is to provide the application of the PCR primer or the kit in the quail sex identification.
The application of the invention in the sex determination of quails has the electrophoresis result that male quails are presented with 1068bp one band, and female quails are presented with 1068bp and 791bp two bands.
The invention also provides a method for rapidly identifying the sex of the duck, the chicken, the pigeon, the muscovy duck, the goose or the quail, which comprises the following steps:
1) extracting the genomic DNA of the poultry to be detected;
2) carrying out PCR amplification reaction by using the DNA extracted in the step 1) as a template and using the primer pair disclosed by the invention;
3) the PCR product is detected by agarose gel electrophoresis,
when the poultry to be detected is a duck, the cock of the duck is presented with one band of 1147bp by the electrophoresis result, and the female duck is presented with two bands of 1147bp and 851 bp;
when the poultry to be detected is a chicken, the cock is represented by one band of 983bp in the electrophoresis result, and the hen is represented by two bands of 983bp and 798bp in the electrophoresis result;
when the poultry to be detected is a pigeon, the pigeon is a male pigeon with one 937bp band presented by the electrophoresis result, and the pigeon is a female pigeon with two 937bp and 1153bp bands presented by the electrophoresis result;
when the poultry to be detected is Muscovy duck, carrying out agarose gel electrophoresis detection on the PCR product of the electrophoresis result, wherein the male Muscovy duck is shown as an 1153bp band in the electrophoresis result, and the female Muscovy duck is shown as two 1153bp and 832bp bands in the electrophoresis result;
when the poultry to be detected is a goose, the goose is a male goose if an 1145bp strip is presented as an electrophoresis result, and the goose is a female goose if two strips of 1145bp and 833bp are presented as electrophoresis results;
when the poultry to be detected is a quail, the male quail appears in a 1068bp strip as an electrophoresis result, and the female quail appears in two strips of 1068bp and 791 bp.
The method of the present invention, step 1) of extracting the genomic DNA of the poultry to be tested, can be extracting according to conventional methods in the art, for example, the extraction of the genomic DNA of the poultry to be tested can be performed by using, but not limited to, feather, blood, tissue, organ, etc.
The method of the present invention, the PCR reaction system in step 2) may be a conventional system in the art, and in a specific embodiment of the present invention, the PCR reaction system is: 2 XPCR Mix (Nanjing Novozam Bio Inc.) 25. mu.L, 10. mu. mol/L forward and reverse primers 1. mu.L each, 50-100. mu.g/ml template DNA 2. mu.L, 21. mu.L ultrapure water.
The method of the present invention, the PCR reaction procedure in step 2) may be a routine system in the art, and in a specific embodiment of the present invention, is: at 95 ℃ for 5min, (30 s at 95 ℃, 30s at 60 ℃ and 30s at 72 ℃) for 35 cycles, and at 72 ℃ for 10 min.
Compared with the prior art, the invention has the following advantages: the invention carries out PCR amplification and agarose gel electrophoresis by the designed PCR primer, determines the sex of the poultry by utilizing the number of bands of an electrophoresis result, has simple and convenient operation, quick and accurate identification result, is convenient for basic popularization and use, 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 little restriction on the poultry variety, so that the duck, chicken, pigeon, Muscovy duck, goose or quail of the invention has no specific variety restriction, wherein the duck is a green duck species in the duck genus of the duck family, and the Muscovy duck is also called foreign duck and is a wart and rhinomyza duck in the duck family and the duck genus.
Drawings
FIG. 1 is an electropherogram of the PCR amplification product of duck of example 1 of the present invention.
FIG. 2 is an electropherogram of the PCR amplification product of duck of example 2 of the present invention.
FIG. 3 is an electropherogram of PCR amplification products of chickens of example 3 of the present invention.
FIG. 4 is an electropherogram of PCR amplification products of chickens of example 4 of the present invention.
FIG. 5 is an electropherogram of PCR amplification products of pigeons according to example 6 of the present invention.
FIG. 6 is an electropherogram of PCR amplification product of pigeon in example 7 of the present invention.
FIG. 7 is an electropherogram of PCR amplification products of Muscovy duck of example 8 of the present invention.
FIG. 8 is an electropherogram of PCR amplification products of Muscovy duck of example 9 of the present invention.
FIG. 9 is an electropherogram of the PCR amplification product of goose in example 10 of the present invention.
FIG. 10 is an electropherogram of the PCR amplification product of goose of example 11 of the present invention.
FIG. 11 is an electrophoretogram of PCR amplification products of quails according to example 12 of the present invention.
FIG. 12 is an electrophoretogram of PCR amplification products of quails according to example 13 of the present invention.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.
Example 1
1 identification of known sexed Duck
1.1 sample Collection
10 adult Beijing ducks with known sexes are collected, the male ducks are numbered 1-5, the female ducks are numbered 6-10, and the designed primers are used for amplifying duck genomes.
Forward primer (SEQ ID No. 1): 5'-ATGAAAGAGTTTCAGCACTTGA-3', respectively;
reverse primer (SEQ ID NO. 2): 5'-TTCATAATAGGAGCTCGAGCCA-3' are provided.
1.2PCR amplification
The PCR reaction system is as follows:
2 XPCR Mix (Nanjing NuoZan Bio Inc.) 25. mu.L, 10. mu. mol/L forward primers 1. mu.L each, 50-100. mu.g/ml template DNA 2. mu.L, ultrapure water 21. mu.L.
The PCR reaction program is: at 95 ℃ for 5min, (30 s at 95 ℃, 30s at 60 ℃ and 30s at 72 ℃) for 35 cycles, and at 72 ℃ for 10 min.
1.3 electrophoretic detection
After the reaction was completed, the detection was carried out by 1.5% agarose gel electrophoresis. The electrophoresis detection of the drake has only one 1147bp band, and the electrophoresis detection of the duck has two 1147bp and 851bp bands, and the results are shown in figure 1 (wherein 1-5 are drakes, and 6-10 are ducks).
The results show that the sex identification method can be used for quickly and accurately identifying the sex of the duck, and is simple and easy to operate.
Example 2
Identification of duck of unknown sex
2.1 sample Collection
16 Shaoxing duck ducklings of unknown sex are collected, and the duck genome is amplified by using the designed primers (SEQ ID NO.1) and (SEQ ID NO.2) of the invention.
2.2PCR amplification
The PCR reaction system is as follows:
2 XPCR Mix (Nanjing NuoZan Bio Inc.) 25. mu.L, 10. mu. mol/L forward primers 1. mu.L each, 50-100. mu.g/ml template DNA 2. mu.L, ultrapure water 21. mu.L.
The PCR reaction program is: at 95 ℃ for 5min, (30 s at 95 ℃, 30s at 60 ℃ and 30s at 72 ℃) for 35 cycles, and at 72 ℃ for 10 min.
2.3 electrophoretic detection
After the reaction was completed, the detection was carried out by 1.5% agarose gel electrophoresis. The electrophoresis detection of the male duck only has one 1147bp band, the electrophoresis detection of the female duck has two 1147bp and 851bp bands, and the results are shown in figure 2 (wherein 3, 4, 6, 7, 11, 12, 15 and 16 are male ducks, and 1, 2, 5, 8, 9, 10, 13 and 14 are female ducks).
The sample is dissected and observed in sexual organs, and the identification result is completely correct.
Example 3
1 identification of known sexed chickens
1.1 sample Collection
8 adult Chongren Ma chickens with known sex are collected, the numbers 1-4 are cock chickens, the numbers 5-8 are hen chickens, and the primer designed by the invention is used for amplifying the chicken genome.
Forward primer (SEQ ID No. 1): 5'-ATGAAAGAGTTTCAGCACTTGA-3', respectively;
reverse primer (SEQ ID NO. 2): 5'-TTCATAATAGGAGCTCGAGCCA-3' are provided.
1.2PCR amplification
The PCR reaction system is as follows:
2 XPCR Mix (Nanjing NuoZan Bio Inc.) 25. mu.L, 10. mu. mol/L forward primers 1. mu.L each, 50-100. mu.g/ml template DNA 2. mu.L, ultrapure water 21. mu.L.
The PCR reaction program is: at 95 ℃ for 5min, (30 s at 95 ℃, 30s at 60 ℃ and 30s at 72 ℃) for 35 cycles, and at 72 ℃ for 10 min.
1.3 electrophoretic detection
After the reaction was completed, the detection was carried out by 1.5% agarose gel electrophoresis. The electrophoresis detection of the cock only has one band of 983bp, and the electrophoresis detection of the hen has two bands of 983bp and 798bp, and the results are shown in figure 3 (wherein 1-4 are cocks, and 5-8 are hens).
The results show that the sex identification method can be used for quickly and accurately identifying the sex of the chicken, and is simple and easy to operate.
Example 4
2 identification of chickens of unknown sex
2.1 sample Collection
The chick of 24 recessive white chickens with unknown sex is collected, and the chick genome is amplified by using the designed primers (SEQ ID NO.1) and (SEQ ID NO.2) of the invention.
2.2PCR amplification
The PCR reaction system is as follows:
2 XPCR Mix (Nanjing NuoZan Bio Inc.) 25. mu.L, 10. mu. mol/L forward primers 1. mu.L each, 50-100. mu.g/ml template DNA 2. mu.L, ultrapure water 21. mu.L.
The PCR reaction program is: at 95 ℃ for 5min, (30 s at 95 ℃, 30s at 60 ℃ and 30s at 72 ℃) for 35 cycles, and at 72 ℃ for 10 min.
2.3 electrophoretic detection
After the reaction was completed, the detection was carried out by 1.5% agarose gel electrophoresis. The electrophoresis of the cock only has one band of 983bp, the electrophoresis of the hen has two bands of 983bp and 798bp, and the results are shown in figure 4 (wherein 1, 4, 5, 7, 8, 9, 12, 14, 17, 20, 21 and 22 are cocks, and 2, 3, 6, 10, 11, 13, 15, 16, 18, 19, 23 and 24 are hens).
The sample is dissected and observed in sexual organs, and the identification result is completely correct.
Example 5
3 resource protection applications
3.1 the chicken genome is amplified by using 19 varieties such as big bone chicken, white-eared yellow chicken, Henan Daji, Beijing fatty chicken, Xiju chicken, tea chicken, silky-feather black-bone chicken, Langshan chicken, Tibetan chicken, Qingyuan chicken, tile ash chicken, Jinhu black-bone chicken, tea chicken, Shouguang chicken, Luyuan chicken, Dongxiang black chicken, Bian chicken, Wenchang chicken, Gushi chicken and the like in the local chicken blood sample gene bank of the unit by using the designed primers (SEQ ID NO.1) and (SEQ ID NO.2) of the invention.
3.2PCR amplification
The PCR reaction system is as follows:
2 XPCR Mix (Nanjing NuoZan Bio Inc.) 25. mu.L, 10. mu. mol/L forward primers 1. mu.L each, 50-100. mu.g/ml template DNA 2. mu.L, ultrapure water 21. mu.L.
The PCR reaction program is: at 95 ℃ for 5min, (30 s at 95 ℃, 30s at 60 ℃ and 30s at 72 ℃) for 35 cycles, and at 72 ℃ for 10 min.
3.3 electrophoretic detection
After the reaction was completed, the detection was carried out by 1.5% agarose gel electrophoresis. The cock electrophoresis detection only has one band of 983bp, the hen electrophoresis detection has two bands of 983bp and 798bp, and the identification result is consistent with the result recorded by individual sex, which is shown in table 1.
Table 119 local variety detection results of different chickens
Example 6
1 identification of white-feather pigeon of known sex
1.1 sample Collection
8 adult white-feather king pigeons with known sex are collected, the number of the adult white-feather king pigeons is 1-4, the number of the adult white-feather king pigeons is male pigeons, the number of the adult white-feather king pigeons is 5-8, the female pigeons is female pigeons, and the designed primer is used for amplifying the pigeon.
Forward primer (SEQ ID No. 1): 5'-ATGAAAGAGTTTCAGCACTTGA-3', respectively;
reverse primer (SEQ ID NO. 2): 5'-TTCATAATAGGAGCTCGAGCCA-3' are provided.
1.2PCR amplification
The PCR reaction system is as follows:
2 XPCR Mix (Nanjing NuoZan Bio Inc.) 25. mu.L, 10. mu. mol/L forward primers 1. mu.L each, 50-100. mu.g/ml template DNA 2. mu.L, ultrapure water 21. mu.L.
The PCR reaction program is: at 95 ℃ for 5min, (30 s at 95 ℃, 30s at 60 ℃ and 30s at 72 ℃) for 35 cycles, and at 72 ℃ for 10 min.
1.3 electrophoretic detection
After the reaction was completed, the detection was carried out by 1.5% agarose gel electrophoresis. The electrophoresis detection of the male pigeons only has one 937bp strip, and the electrophoresis detection of the female pigeons has two 937bp and 1153bp strips, and the results are 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 be used for quickly and accurately identifying the sex of the pigeons, and is simple and easy to operate.
Example 7
2 identification of European meat pigeon of unknown sex
2.1 sample Collection
The squab of 17 European meat pigeons of unknown sex was collected and the pigeon genome was amplified using the primers designed according to the invention (SEQ ID NO.1) and (SEQ ID NO. 2).
2.2PCR amplification
The PCR reaction system is as follows:
2 XPCR Mix (Nanjing NuoZan Bio Inc.) 25. mu.L, 10. mu. mol/L forward primers 1. mu.L each, 50-100. mu.g/ml template DNA 2. mu.L, ultrapure water 21. mu.L.
The PCR reaction program is: at 95 ℃ for 5min, (30 s at 95 ℃, 30s at 60 ℃ and 30s at 72 ℃) for 35 cycles, and at 72 ℃ for 10 min.
2.3 electrophoretic detection
After the reaction was completed, the detection was carried out by 1.5% agarose gel electrophoresis. The electrophoresis detection of male pigeons only has one 937bp band, the electrophoresis detection of female pigeons has two 937bp 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 dissected and observed in sexual organs, and the identification result is completely correct.
Example 8
1 identification of Muscovy ducks with known sex
1.1 sample Collection
10 adult white muscovy ducks with known sexes are collected, the male muscovy ducks are numbered 1-5, the female muscovy ducks are numbered 6-10, and the primers designed by the invention are used for amplifying muscovy duck genomes.
Forward primer (SEQ ID No. 1): 5'-ATGAAAGAGTTTCAGCACTTGA-3', respectively;
reverse primer (SEQ ID NO. 2): 5'-TTCATAATAGGAGCTCGAGCCA-3' are provided.
1.2PCR amplification
The PCR reaction system is as follows:
2 XPCR Mix (Nanjing NuoZan Bio Inc.) 25. mu.L, 10. mu. mol/L forward primers 1. mu.L each, 50-100. mu.g/ml template DNA 2. mu.L, ultrapure water 21. mu.L.
The PCR reaction program is: at 95 ℃ for 5min, (30 s at 95 ℃, 30s at 60 ℃ and 30s at 72 ℃) for 35 cycles, and at 72 ℃ for 10 min.
1.3 electrophoretic detection
After the reaction was completed, the detection was carried out by 1.5% agarose gel electrophoresis. The male Muscovy duck has only one 1153bp band in electrophoresis detection, and the female Muscovy duck has two 1153bp and 832bp bands in electrophoresis detection, and the results are shown in figure 7 (wherein 1-5 are male Muscovy ducks and 6-10 are female Muscovy ducks).
The results show that the method can be used for quickly and accurately identifying the sex of the Muscovy ducks, and is simple and easy to operate.
Example 9
2 identification of Black Muscovy Duck of unknown sex
2.1 sample Collection
17 black feather Muscovy ducks with unknown sexes are collected, and Muscovy duck genome is amplified by using the designed primers (SEQ ID NO.1) and (SEQ ID NO.2) of the invention.
2.2PCR amplification
The PCR reaction system is as follows:
2 XPCR Mix (Nanjing NuoZan Bio Inc.) 25. mu.L, 10. mu. mol/L forward primers 1. mu.L each, 50-100. mu.g/ml template DNA 2. mu.L, ultrapure water 21. mu.L.
The PCR reaction program is: at 95 ℃ for 5min, (30 s at 95 ℃, 30s at 60 ℃ and 30s at 72 ℃) for 35 cycles, and at 72 ℃ for 10 min.
2.3 electrophoretic detection
After the reaction was completed, the detection was carried out by 1.5% agarose gel electrophoresis. The electrophoresis detection of male Muscovy ducks only has one 1153bp band, and the electrophoresis detection of female Muscovy ducks has two 1153bp and 832bp bands, and the results are 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 dissected and observed in sexual organs, and the identification result is completely correct.
Example 10
1 identification of Yangzhou goose with known sex
1.1 sample Collection
10 adult Yangzhou geese with known sex are collected, the number of the adult Yangzhou geese is 1-5, the number of the adult Yangzhou geese is male goose, the number of the adult Yangzhou geese is 6-10, and the designed primer is used for amplifying goose genome.
Forward primer (SEQ ID No. 1): 5'-ATGAAAGAGTTTCAGCACTTGA-3', respectively;
reverse primer (SEQ ID NO. 2): 5'-TTCATAATAGGAGCTCGAGCCA-3' are provided.
1.2PCR amplification
The PCR reaction system is as follows:
2 XPCR Mix (Nanjing NuoZan Bio Inc.) 25. mu.L, 10. mu. mol/L forward primers 1. mu.L each, 50-100. mu.g/ml template DNA 2. mu.L, ultrapure water 21. mu.L.
The PCR reaction program is: at 95 ℃ for 5min, (30 s at 95 ℃, 30s at 60 ℃ and 30s at 72 ℃) for 35 cycles, and at 72 ℃ for 10 min.
1.3 electrophoretic detection
After the reaction was completed, the detection was carried out by 1.5% agarose gel electrophoresis. The electrophoresis detection of the male goose has only one 1145bp band, and the electrophoresis detection of the female goose has two 1145bp and 833bp bands, and the results are shown in FIG. 9 (wherein 1-5 are male geese and 6-10 are female geese).
The results show that the method can be used for quickly and accurately identifying the sex of the goose, and is simple and easy to operate.
Example 11
2 identification of Rhein goose of unknown sex
2.1 sample Collection
17 gosling of the Rhine of unknown sex were collected, and the genome of the goose was amplified using the primers (SEQ ID NO.1) and (SEQ ID NO.2) designed according to the present invention.
2.2PCR amplification
The PCR reaction system is as follows:
2 XPCR Mix (Nanjing NuoZan Bio Inc.) 25. mu.L, 10. mu. mol/L forward primers 1. mu.L each, 50-100. mu.g/ml template DNA 2. mu.L, ultrapure water 21. mu.L.
The PCR reaction program is: at 95 ℃ for 5min, (30 s at 95 ℃, 30s at 60 ℃ and 30s at 72 ℃) for 35 cycles, and at 72 ℃ for 10 min.
2.3 electrophoretic detection
After the reaction was completed, the detection was carried out by 1.5% agarose gel electrophoresis. The electrophoresis detection of the male goose has only one 1145bp band, the electrophoresis detection of the female goose has two 1145bp and 833bp bands, and the results are shown in FIG. 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 dissected and observed in sexual organs, 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, male quails numbered 1-4 and female quails numbered 5-8 were collected, and the quail genome was amplified using the primers designed according to the present invention.
Forward primer (SEQ ID No. 1): 5'-ATGAAAGAGTTTCAGCACTTGA-3', respectively;
reverse primer (SEQ ID NO. 2): 5'-TTCATAATAGGAGCTCGAGCCA-3' are provided.
1.2PCR amplification
The PCR reaction system is as follows:
2 XPCR Mix (Nanjing NuoZan Bio Inc.) 25. mu.L, 10. mu. mol/L forward primers 1. mu.L each, 50-100. mu.g/ml template DNA 2. mu.L, ultrapure water 21. mu.L.
The PCR reaction program is: at 95 ℃ for 5min, (30 s at 95 ℃, 30s at 60 ℃ and 30s at 72 ℃) for 35 cycles, and at 72 ℃ for 10 min.
1.3 electrophoretic detection
After the reaction was completed, the detection was carried out by 1.5% agarose gel electrophoresis. The male quail electrophoresis detection has only one 1068bp strip, and the female quail electrophoresis detection has two 1068bp and 791bp strips, and the results are shown in FIG. 11 (wherein 1-4 are male quails, and 5-8 are female quails).
The results show that the sex determination method can be used for rapidly and accurately performing sex determination on the quails, and is simple and easy to operate.
Example 13
Identification of Chinese white feather quails of unknown sex
2.1 sample Collection
17 Chinese white feather quail young quails of unknown sex are collected, and the quail genome is amplified by using the designed primers (SEQ ID NO.1) and (SEQ ID NO.2) of the invention.
2.2PCR amplification
The PCR reaction system is as follows:
2 XPCR Mix (Nanjing NuoZan Bio Inc.) 25. mu.L, 10. mu. mol/L forward primers 1. mu.L each, 50-100. mu.g/ml template DNA 2. mu.L, ultrapure water 21. mu.L.
The PCR reaction program is: at 95 ℃ for 5min, (30 s at 95 ℃, 30s at 60 ℃ and 30s at 72 ℃) for 35 cycles, and at 72 ℃ for 10 min.
2.3 electrophoretic detection
After the reaction was completed, the detection was carried out by 1.5% agarose gel electrophoresis. The electrophoresis detection of male quail only has one 1068bp band, and the electrophoresis detection of female quail has two 1068bp and 791bp bands, and the results are shown in FIG. 12 (wherein 3, 5, 6, 9, 10, 11, 13, 16 are male quails, and 1, 2, 4, 7, 8, 12, 14, 15, 17 are female quails).
The sample is dissected and observed in sexual organs, and the identification result is completely correct.
Sequence listing
<110> scientific research institute for poultry in Jiangsu province
<120> PCR primer, kit and method for poultry sex identification
<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
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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ttcataatag gagctcgagc ca 22
Claims (10)
1. A PCR primer for identifying the sex is characterized in that the sequence of the primer is shown as SEQ ID NO.1/SEQ ID NO. 2.
2. A kit comprising the PCR primer for sex determination according to claim 1; preferably, the kit further comprises a PCR reaction solution and/or a DNA extraction solution.
3. Use of the PCR primer of claim 1 or the kit of claim 2 for duck gender identification; preferably, the application is specifically to perform PCR amplification through the PCR primer of claim 1 or the kit of claim 2 or 3, and the duck is a male duck when an electrophoresis result shows that one band of 1147bp is displayed, and the duck is a female duck when two bands of 1147bp and 851bp are displayed.
4. Use of the PCR primer of claim 1 or the kit of claim 2 for the identification of chicken gender; preferably, the application is specifically to perform PCR amplification through the PCR primer of claim 1 or the kit of claim 2 or 3, wherein the cock is represented by one band of 983bp and the hen is represented by two bands of 983bp and 798 bp.
5. Use of the PCR primer of claim 1 or the kit of claim 2 for pigeon sex determination; preferably, the application is specifically to perform PCR amplification through the PCR primer of claim 1 or the kit of claim 2 or 3, and the male pigeon is shown as one 937bp band and the female pigeon is shown as two 937bp and 1153bp bands as electrophoresis results.
6. The use of the PCR primer of claim 1 or the kit of claim 2 for identifying the sex of muscovy ducks; preferably, the application is specifically that PCR amplification is carried out by the PCR primer of claim 1 or the kit of claim 2 or 3, and the male Muscovy duck is obtained when one band of 1153bp is presented as an electrophoresis result, and the female Muscovy duck is obtained when two bands of 1153bp and 832bp are presented.
7. The use of the PCR primer of claim 1 or the kit of claim 2 for goose gender identification; preferably, the application specifically comprises the step of performing PCR amplification through the PCR primer of claim 1 or the kit of claim 2 or 3, wherein a male goose is represented by one 1145bp band and a female goose is represented by two 1145bp and 833bp bands as electrophoresis results.
8. Use of the PCR primer of claim 1 or the kit of claim 2 for pigeon sex determination; preferably, the application is PCR amplification carried out by the PCR primer of claim 1 or the kit of claim 2 or 3, and male quails appear when 1068bp bands appear as electrophoresis results, and female quails appear when 1068bp and 791bp bands appear as electrophoresis results.
9. A method for rapidly identifying the sex of a duck, a chicken, a pigeon, a Muscovy duck, a goose or a quail is characterized by comprising the following steps:
1) extracting the genomic DNA of the poultry to be detected;
2) carrying out PCR amplification reaction by using the DNA extracted in the step 1) as a template and the primer pair of claim 1;
3) the PCR product is detected by agarose gel electrophoresis,
when the poultry to be detected is a duck, the cock of the duck is presented with one band of 1147bp by the electrophoresis result, and the female duck is presented with two bands of 1147bp and 851 bp;
when the poultry to be detected is a chicken, the cock is represented by one band of 983bp in the electrophoresis result, and the hen is represented by two bands of 983bp and 798bp in the electrophoresis result;
when the poultry to be detected is a pigeon, the pigeon is a male pigeon with one 937bp band presented by the electrophoresis result, and the pigeon is a female pigeon with two 937bp and 1153bp bands presented by the electrophoresis result;
when the poultry to be detected is Muscovy duck, the poultry to be detected is male Muscovy duck when the poultry to be detected presents 1153bp one strip, and is female Muscovy duck when the poultry to be detected presents 1153bp and 832bp two strips;
when the poultry to be detected is a goose, the goose is a male goose if an 1145bp strip is presented as an electrophoresis result, and the goose is a female goose if two strips of 1145bp and 833bp are presented as electrophoresis results;
when the poultry to be detected is a quail, the male quail appears in a 1068bp strip as an electrophoresis result, and the female quail appears in two strips of 1068bp and 791 bp.
10. The identification method of claim 9, wherein the step 1) of extracting the genomic DNA of the poultry to be tested is carried out by extracting the genomic DNA from feathers, blood, tissues or organs; preferably, the PCR reaction system in step 2) is: 2 XPCR Mix 25 uL, 10 umol/L forward and reverse primers each 1 uL, 50-100 ug/ml template DNA 2 uL, ultrapure water 21 uL; preferably, the PCR reaction procedure in step 2) is as follows: 5min at 95 ℃, namely 30s at 95 ℃, 30s at 60 ℃, 30s at 72 ℃ for 35 cycles, and 10min at 72 ℃.
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| CN202110900994.2A CN113604548B (en) | 2021-01-28 | 2021-01-28 | PCR primer, kit and method for sex identification of geese |
| CN202110900670.9A CN113621712B (en) | 2021-01-28 | 2021-01-28 | PCR primer, kit and method for sex identification of muscovy ducks |
| CN202110900950.XA CN113481304B (en) | 2021-01-28 | 2021-01-28 | PCR primer, kit and method for chicken sex identification |
| CN202110120742.8A CN112662787B (en) | 2021-01-28 | 2021-01-28 | PCR primer, kit and method for poultry sex identification |
| CN202110901014.0A CN113621694B (en) | 2021-01-28 | 2021-01-28 | PCR primer, kit and method for quail sex identification |
| CN202110900668.1A CN113403407B (en) | 2021-01-28 | 2021-01-28 | PCR primer, kit and method for pigeon sex identification |
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| CN202110900668.1A Division CN113403407B (en) | 2021-01-28 | 2021-01-28 | PCR primer, kit and method for pigeon sex identification |
| CN202110900670.9A Division CN113621712B (en) | 2021-01-28 | 2021-01-28 | PCR primer, kit and method for sex identification of muscovy ducks |
| CN202110900994.2A Division CN113604548B (en) | 2021-01-28 | 2021-01-28 | PCR primer, kit and method for sex identification of geese |
| CN202110901014.0A Division CN113621694B (en) | 2021-01-28 | 2021-01-28 | PCR primer, kit and method for quail sex identification |
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| CN202110900950.XA Active CN113481304B (en) | 2021-01-28 | 2021-01-28 | PCR primer, kit and method for chicken sex identification |
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| 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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| CN111621574B (en) * | 2020-05-29 | 2022-11-11 | 广州动物园 | Primer for identifying sex of ostrich animals, sex identification method and kit |
| CN116622859B (en) * | 2023-05-30 | 2023-11-14 | 江苏省家禽科学研究所 | Molecular biological identification method and application of pure-bred tea-flower chicken |
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| CN113403407B (en) | 2023-06-09 |
| CN113604548B (en) | 2023-05-12 |
| CN113403407A (en) | 2021-09-17 |
| CN113621694B (en) | 2023-05-23 |
| CN113481304A (en) | 2021-10-08 |
| CN113604548A (en) | 2021-11-05 |
| CN112662787B (en) | 2021-09-07 |
| CN113621694A (en) | 2021-11-09 |
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| CN113621712A (en) | 2021-11-09 |
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