PCR primer, kit and method for pigeon gender identification

Abstract

The application provides a PCR primer, a kit and a method for pigeon gender identification, wherein the sequence of the PCR primer for pigeon gender identification is shown as SEQ ID NO. 1 and SEQ ID NO. 2. The method comprises the following steps: performing PCR amplification and agarose gel electrophoresis by using the PCR primer of the application; and determining the gender of the pigeon by using the band number of the electrophoresis result. The method has the advantages of simple operation, rapid and accurate identification result, easy popularization and use, and wide market application prospect. In addition, the detection kit developed based on the method of the application can generate considerable economic benefits and good social value.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a PCR primer, kit, and method for sex identification of pigeons. Background Technology

[0002] Pigeons belong to the category of special economic poultry. Pigeon meat and eggs are rich in nutrients and are known as "animal ginseng." There is an old saying that "one pigeon is better than nine chickens." Currently, the number of breeding pigeons in China has reached more than 60 million pairs, with an annual production of more than 800 million squabs. The total production accounts for more than 80% of the world's total, and it is growing rapidly at a "high speed" of 20% per year. The pigeon meat industry has become one of the most promising characteristic industries and sunrise industries in my country.

[0003] Pigeons are monomorphic birds, making it difficult to distinguish between young and adult birds based on appearance and behavior alone. They are also monogamous, meaning they can only reproduce or produce byproducts (squabs or eggs) by pairing. Therefore, sexing pigeons is of great importance in pigeon farming.

[0004] Currently, pigeon sex determination mainly occurs during pairing (5-6 months of age). Experienced technicians roughly determine the sex of pigeons by observing their vent and physical appearance before pairing them, but this method has a high misidentification rate. Therefore, there is a need to develop a simple, convenient, fast, and accurate method for identifying the sex of pigeons. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a reasonably designed PCR primer that can be used for rapid early sex identification of pigeons, addressing the shortcomings of the prior art.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a PCR primer for pigeon sex identification, the primer sequence of which is shown in SEQ ID NO.1 and SEQ ID NO.2. That is, the nucleotide sequence of the primer is:

[0007] Forward primer (SEQ ID NO.1): 5'-AGTGTTCCACCCACAAGCAA-3';

[0008] Reverse primer (SEQ ID NO.2): 5'-GCAGTCACTAATTTGATGGCACA-3'.

[0009] The present invention also provides a kit containing the PCR primers for pigeon sex identification described in this invention.

[0010] In one embodiment of the present invention, the kit of the present invention comprises the PCR primers and PCR reaction solution of the present invention. In some embodiments, it may also comprise DNA extraction solution.

[0011] This invention also provides the application of the PCR primers or kits described herein in pigeon farming.

[0012] This invention also provides the application of the PCR primers or kits described herein in pigeon sex identification.

[0013] The pigeons described in this invention include all breeds of pigeons.

[0014] In the application described in this invention, PCR amplification is performed using the primers described in this invention. The electrophoresis result showing a single band of 978bp indicates a male pigeon, while the result showing two bands of 978bp and 568bp indicates a female pigeon.

[0015] This invention also provides a rapid method for determining the sex of pigeons, comprising the following steps:

[0016] 1) Extract genomic DNA from the pigeons to be tested;

[0017] 2) Using the DNA extracted in step 1) as a template, PCR amplification reaction is performed using the primer pair described in this invention;

[0018] 3) The PCR products were detected by agarose gel electrophoresis. The pigeons with a single band of 978bp were male, and the pigeons with two bands of 978bp and 568bp were female.

[0019] The method described in this invention, step 1) extracting the genomic DNA of the pigeon to be tested can be performed using conventional methods in the art, such as using materials including but not limited to feathers, blood, tissues, and organs to extract the genomic DNA of the pigeon to be tested.

[0020] The PCR reaction system in step 2) of the method described in this invention can be a conventional system in the art. In a specific embodiment of this invention, it is: 25 μL of 2×PCR Mix (Nanjing Novizan Biotechnology Co., Ltd.), 1 μL each of 10 μmol / L forward and reverse primers, 2 μL of 50-100 μg / ml template DNA, and 21 μL of ultrapure water.

[0021] The PCR reaction program in step 2) of the method described in this invention can be a conventional system in the art. In a specific embodiment of this invention, it is: 95℃ for 5 min, (95℃ for 30 s, 60℃ for 30 s, 72℃ for 30 s) for 35 cycles, and 72℃ for 10 min.

[0022] Compared with the prior art, the present invention has the following advantages: The present invention uses designed PCR primers to perform PCR amplification and agarose gel electrophoresis, and uses the number of bands in the electrophoresis results to determine the sex of pigeons. It is simple and convenient to operate, and the identification results are fast and accurate. It is easy to promote and use at the grassroots level and has broad market application prospects. In addition, the detection kit developed based on the method of the present invention can generate considerable economic benefits and good social value. Attached Figure Description

[0023] Figure 1 This is the electrophoretic pattern of the PCR amplification product of Example 1 of the present invention.

[0024] Figure 2 This is the electrophoretic pattern of the PCR amplification product of Example 2 of the present invention. Detailed Implementation

[0025] The following examples are provided to better understand the present invention, but are not intended to limit the invention. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent stores.

[0026] Example 1

[0027] 1. Identification of pigeons with known sex

[0028] 1.1 Sample Collection

[0029] Eight Thai pigeons of known sex were collected, numbered 1-4 as males and 5-8 as females, and the pigeon genome was amplified using primers designed in this invention.

[0030] Forward primer (SEQ ID NO.1): 5'-AGTGTTCCACCCACAAGCAA-3';

[0031] Reverse primer (SEQ ID NO.2): 5'-GCAGTCACTAATTTGATGGCACA-3'.

[0032] 1.2 PCR amplification

[0033] The PCR reaction system is as follows:

[0034] 25 μL of 2×PCR Mix (Nanjing Novizan Biotechnology Co., Ltd.), 1 μL each of 10 μmol / L forward primers, 2 μL of 50-100 μg / ml template DNA, and 21 μL of ultrapure water.

[0035] The PCR reaction program was as follows: 95℃ for 5 min, (95℃ for 30 s, 60℃ for 30 s, 72℃ for 30 s) for 35 cycles, and 72℃ for 10 min.

[0036] 1.3 Electrophoretic detection

[0037] After the reaction, the samples were analyzed by 1.5% agarose gel electrophoresis. The male pigeon showed only one 978bp band in the electrophoresis results, while the female pigeon showed two bands at 978bp and 568bp, as shown in the results below. Figure 1 (Where 1-4 are male pigeons, and 5-8 are female pigeons).

[0038] The above results indicate that the present invention can quickly and accurately identify the sex of pigeons, and is simple and easy to operate.

[0039] Example 2

[0040] 2. Identification of pigeons of unknown sex

[0041] 2.1 Sample Collection

[0042] Forty-six white-feathered king pigeon chicks of unknown sex were collected, and the pigeon genome was amplified using primers (SEQ ID NO.1) and (SEQ ID NO.2) designed in this invention.

[0043] 2.2 PCR amplification

[0044] The PCR reaction system is as follows:

[0045] 25 μL of 2×PCR Mix (Nanjing Novizan Biotechnology Co., Ltd.), 1 μL each of 10 μmol / L forward primers, 2 μL of 50-100 μg / ml template DNA, and 21 μL of ultrapure water.

[0046] The PCR reaction program was as follows: 95℃ for 5 min, (95℃ for 30 s, 60℃ for 30 s, 72℃ for 30 s) for 35 cycles, and 72℃ for 10 min.

[0047] 2.3 Electrophoresis detection

[0048] After the reaction, the samples were analyzed by 1.5% agarose gel electrophoresis. The male pigeon showed only one 978bp band in the electrophoresis results, while the female pigeon showed two bands at 978bp and 568bp, as shown in the results below. Figure 2 (Where 1, 2, 4, 5, 10, 11, 16, 17, 18, 19, 21, 24, 26, 28, 30, 32, 37, 38, 39, 42, 43, 46 are male pigeons, and 3, 6, 7, 8, 9, 12, 13, 14, 15, 20, 22, 23, 25, 27, 29, 31, 33, 34, 35, 36, 40, 41, 44, 45 are female pigeons).

[0049] The test samples were dissected and the sexual organs were observed. The above identification results are completely correct.

Claims

1. The application of the primers shown in SEQ ID NO.1 and SEQ ID NO.2, or a kit containing said primers, in pigeon sex determination, characterized in that, The pigeons are either Thai Deep Pigeons or White Feather King Pigeons. PCR amplification is performed using the PCR primers or the kit described above. Pigeons showing a single band of 978 bp in the electrophoresis result are male, while pigeons showing two bands of 978 bp and 568 bp are female.

2. A rapid method for determining the sex of pigeons, characterized in that, Includes the following steps: 1) Extract genomic DNA from the pigeon to be tested; the pigeon is either a Tai-Shen pigeon or a White King pigeon; 2) Using the DNA extracted in step 1) as a template, PCR amplification was performed using the primer pair shown in SEQ ID NO.1 and SEQ ID NO.2; 3) The PCR products were detected by agarose gel electrophoresis. The pigeons with a single band of 978 bp were male, and the pigeons with two bands of 978 bp and 568 bp were female.

3. The identification method according to claim 2, characterized in that, Step 1) Extract genomic DNA from the pigeon to be tested. Select feathers, blood, tissues or organs to extract genomic DNA.

4. The identification method according to claim 2, characterized in that, The PCR reaction system in step 2) is as follows: 25 μL of 2×PCR Mix, 1 μL each of 10 μmol / L forward and reverse primers, 2 μL of 50-100 μg / ml template DNA, and 21 μL of ultrapure water.

5. The identification method according to claim 2, characterized in that, The PCR reaction program in step 2) is as follows: 95℃ for 5 min, 95℃ for 30 s, 60℃ for 30 s, 72℃ for 30 s for 35 cycles, and 72℃ for 10 min.

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