PCR (Polymerase Chain Reaction) primer, kit and method for identifying sex of pigeons
By designing specific PCR primers and electrophoresis methods, the problem of rapid and accurate pigeon sex identification was solved, and the simple and easy identification of pigeon sex was achieved, thereby improving the production efficiency and economic benefits of the pigeon breeding industry.
Patent Information
- Application Number
- CN202510880287.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-27
- Publication Date
- 2025-10-17
Smart Images
Figure HDA0005472217410000011 
Figure HDA0005472217410000012
Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the field of biotechnology, and particularly relates to a PCR primer, a kit and a method for pigeon gender identification. BACKGROUND
[0002] Pigeon belongs to the category of special economic poultry, and pigeon meat and pigeon eggs are rich in nutrients and are known as "animal ginseng". Since ancient times, there has been the saying "one pigeon is better than nine chickens". At present, the number of breeding pigeons in China reaches more than 60 million pairs, and the total number of annual production of squab reaches more than 800 million, and the total production has accounted for more than 80% of the world total, and the meat pigeon industry has become a characteristic industry and sunrise industry with the most development prospects in China.
[0003] Pigeon is a monomorphic bird, and it is difficult to distinguish between male and female from appearance and other behaviors at both the young bird and adult bird stages. Meanwhile, pigeon is a "one-man-one-wife" system, and only pairing can breed the next generation or produce by-products (squab or pigeon eggs), so the identification of male and female has important significance in the pigeon breeding industry.
[0004] At present, the gender identification of pigeon is mainly in pairing (5-6 months), and experienced technicians roughly judge the gender of pigeon for pairing by observing and body appearance, and the misidentification rate is high. Therefore, a simple and convenient method for quickly and accurately identifying the gender of pigeon needs to be developed. SUMMARY
[0005] The technical problem to be solved by the present application is to provide a PCR primer for early gender identification of pigeon, which is reasonable in design.
[0006] To solve the above technical problems, the technical scheme adopted by the present application is that a PCR primer for pigeon gender identification, the primer sequence 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 application also provides a kit comprising the PCR primer for pigeon gender identification.
[0010] In one embodiment of the present application, the kit of the present application comprises the PCR primer of the present application, the PCR reaction solution, and in some embodiments, the DNA extraction solution.
[0011] The present application also provides the use of the PCR primer of the present application or the kit of the present application in pigeon breeding industry.
[0012] The present application also provides the use of the PCR primer of the present application or the kit of the present application in pigeon gender identification.
[0013] The pigeon of the present application includes all breeds of pigeons.
[0014] The use of the present application is that the PCR amplification is performed by the primer of the present application, and the electrophoresis result presents one band of 978bp for male pigeon, and presents two bands of 978bp and 568bp for female pigeon.
[0015] The present application also provides a rapid pigeon gender identification method, comprising the following steps:
[0016] 1) extracting the genomic DNA of the pigeon to be tested;
[0017] 2) using the DNA extracted in step 1) as a template, and performing PCR amplification reaction by using the primer pair of the present application;
[0018] 3) performing agarose gel electrophoresis detection on the PCR product, and the electrophoresis result presents one band of 978bp for male pigeon, and presents two bands of 978bp and 568bp for female pigeon.
[0019] The method of the present application, step 1) of extracting the genomic DNA of the pigeon to be tested can be extracted according to the conventional method in the art, for example, the genomic DNA of the pigeon to be tested can be extracted from feather, blood, tissue, organ, etc.
[0020] The method of the present application, the PCR reaction system in step 2) can be the conventional system in the art, and in one specific embodiment of the present application, it is: 2x PCR Mix (Nanjing Novozyme Biochem Co., Ltd.) 25 μL, 10 μmol / L forward and reverse primer 1 μL each, 50-100 μg / ml template DNA 2 μL, ultrapure water 21 μL.
[0021] The method of the present application, the PCR reaction program in step 2) can be the conventional system in the art, and in one specific embodiment of the present application, it is: 95℃ 5min, (95℃ 30s, 60℃ 30s, 72℃ 30s) 35 cycles, 72℃ 10min.
[0022] Compared with the prior art, the present application has the following advantages: the present application determines the gender of pigeons by designing PCR primers, performing PCR amplification and agarose gel electrophoresis, and using the band number of the electrophoresis result, has the advantages of simple and convenient operation, rapid and accurate identification result, easy popularization and use at the basic level, and wide market application prospect, in addition, the detection kit developed based on the method of the present application can produce considerable economic benefits and good social value. BRIEF DESCRIPTION OF DRAWINGS
[0023] Figure 1 Electrophoretogram of the PCR amplification product of Example 1 of the present application.
[0024] Figure 2 Electrophoretogram of the PCR amplification product of Example 2 of the present application. DETAILED DESCRIPTION
[0025] The following examples facilitate better understanding of the present application, but do not limit the present application. In the following examples, the experimental methods are conventional methods unless otherwise specified. The test materials used in the following examples are commercially available from conventional biochemical reagent stores unless otherwise specified.
[0026] Example 1
[0027] 1 Known gender pigeon identification
[0028] 1.1 Sample collection
[0029] Eight known gender Taishen pigeons were collected, numbered 1-4 as male pigeons, and 5-8 as female pigeons, and the pigeon genome was amplified using the primer designed in the present application.
[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:
[0034] 2x PCR Mix (Nanjing Novozyme Biochem Co., Ltd.) 25 μL, 10 μmol / L forward primer 1 μL each, 50-100 μg / ml template DNA 2 μL, ultrapure water 21 μL.
[0035] The PCR reaction program is: 95℃ for 5min, (95℃ for 30s, 60℃ for 30s, 72℃ for 30s) for 35 cycles, 72℃ for 10min.
[0036] 1.3 Electrophoresis detection
[0037] After the reaction, 1.5% agarose gel electrophoresis was used for detection. The male pigeon had only one band of 978bp, and the female pigeon had two bands of 978bp and 568bp, as shown in the results below Figure 1 (1-4 are male pigeons, and 5-8 are female pigeons).
[0038] The above results show that the application can quickly and accurately identify the gender of pigeons, and is simple and easy to operate.
[0039] Example 2
[0040] 2. Unknown gender pigeon identification
[0041] 2.1 Sample collection
[0042] Forty-six white-feathered Wang pigeon chicks of unknown gender were collected, and the pigeon genome was amplified using the primers (SEQ ID NO. 1) and (SEQ ID NO. 2) designed in the application.
[0043] 2.2 PCR amplification
[0044] The PCR reaction system is as follows:
[0045] 2x PCR Mix (Nanjing Novozyme Biochem Co., Ltd.) 25 μL, 10 μmol / L forward primer 1 μL each, 50-100 μg / ml template DNA 2 μL, ultrapure water 21 μL.
[0046] The PCR reaction program is as follows: 95℃ for 5min, (95℃ for 30s, 60℃ for 30s, 72℃ for 30s) for 35 cycles, and 72℃ for 10min.
[0047] 2.3 Electrophoresis detection
[0048] After the reaction, 1.5% agarose gel electrophoresis was used for detection. The male pigeon had only one band of 978bp, and the female pigeon had two bands of 978bp and 568bp, as shown in the results below Figure 2 (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, 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 sex organs were observed, and the above identification results were completely correct.
Claims
1. A PCR primer for pigeon sex identification, characterized in that: The primer sequences are shown in SEQ ID NO.1 and SEQ ID NO.
2.
2. a test kit comprising the pigeon sex identification PCR primers according to claim 1.
3. The kit according to claim 2, wherein The kit further comprises a PCR reaction solution and / or a DNA extraction solution.
4. Use of the PCR primer according to claim 1 or the test kit according to claim 2 or 3 in pigeon breeding.
5. Application of the PCR primer according to claim 1 or the test kit according to claim 2 or 3 in pigeon sex identification.
6. The use according to claim 5, characterized in that PCR amplification is performed using the PCR primers described in claim 1 or the kit described in claim 2 or 3. If the electrophoresis result shows a band of 978 bp, it is a male pigeon, and if two bands of 978 bp and 568 bp are shown, it is a female pigeon.
7. A method for rapid sex identification of pigeons, characterized in that: The following steps are involved: 1) Extracting genomic DNA from the pigeon to be tested; 2) using the DNA extracted in step 1) as a template and the primer pair according to claim 1, performing a PCR amplification reaction; 3) The PCR products were detected by agarose gel electrophoresis. If the electrophoresis results showed a single band of 978 bp, it was a male pigeon; if two bands of 978 bp and 568 bp were present, it was a female pigeon.
8. The identification method according to claim 7, characterized in that Step 1) Extracting genomic DNA from the pigeon to be tested: Extracting genomic DNA from feathers, blood, tissues or organs.
9. The identification method according to claim 7, characterized in that In step 2), the PCR reaction system 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.
10. The identification method according to claim 7, characterized in that The PCR reaction program in step 2) was: 95°C for 5 min, 35 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s, and 72°C for 10 min.