WO2024037252A1 - Pcr primer for sex identification of young pigeons, use thereof, and method for sex identification of young pigeons - Google Patents

Abstract

Provided are a PCR primer for sex identification of young pigeons, use thereof, and a method for sex identification of young pigeons. The primer is designed for a specific EE0.6 sequence fragment on the W chromosome of female pigeons. During detection, Biotin and FAM are used for modifying the primer. Firstly, an efficient nucleic acid lysis solution is used for rapidly extracting DNA in feather pulp, thereby simplifying the DNA-obtaining process; then specific PCR amplification is carried out by using the modified primer, and an amplified PCR product is used for rapidly identifying female individuals by means of the presence or absence of a band on a colloidal gold test strip. The method has the advantages of high specificity, high accuracy rate, simple and convenient operation, rapid and accurate identification result, small damage to animals, and the like, thereby reducing the time and economic cost of pigeon sex identification, and having wide market application prospects. The detection result of the method is consistent with the result of the colloidal gold test strip, and the accuracy rate is 100%.

Description

A PCR primer for gender identification of young pigeons and its application and a method for gender identification of young pigeons

This application requires the priority of the Chinese patent application submitted to the China Patent Office on August 16, 2022, with the application number CN202210980622.X and the invention title "A method for gender identification of young pigeons and PCR primers for identification", all of which The contents are incorporated into this application by reference.

Technical field

The invention belongs to the field of biotechnology, and specifically relates to a PCR primer for gender identification of young pigeons and its application and a method for gender identification of young pigeons.

Background technique

China's pigeon breeding industry is developing rapidly. However, due to insufficient investment in scientific research and the unique biological characteristics of pigeons, meat pigeon breeding technology lags behind other poultry. Among them, the problem of gender identification in the production of breeding pigeons still needs to be solved. Gendering is particularly important in pigeon breeding and controlling breeding costs. Individuals of different genders have different nutritional needs. Early identification of gender will help save feeding costs and improve breeding efficiency. For breeding pigeons, a reasonable sex ratio is required. If the sex ratio is inappropriate, it will not be conducive to breeding. If the sex of pigeons can be accurately separated and paired in a timely manner at an early stage, it will not only improve the production efficiency of the pigeon farm and increase production efficiency, but also eliminate unnecessary male pigeons early and reduce production costs, especially in the young pigeon period. Males and females are more prominent in the laying pigeon industry. Therefore, early gender identification has important practical significance and economic value.

However, pigeons are sexually monomorphic birds, and it is difficult to distinguish whether they are young pigeons or adult pigeons in terms of appearance or other behaviors. In addition, pigeons are late adult birds, and the sex of the born chicks cannot be determined by turning the anus like ordinary poultry. . At present, in terms of gender identification of young pigeons, pigeon gender identification is mainly carried out through methods such as appearance and behavioral characteristics, laparoscopy, fecal steroid detection and karyotype. However, these methods are not accurate and the identification process is cumbersome and difficult to use in production. Mass promotion. When applying the anal turning method on pigeons, the accuracy rate is only about 70%. With the development of molecular biology, some molecular biology methods can be selected for pigeon sex identification, which have the advantages of high accuracy and less harm to animals. However, most of the reported identification methods require tedious genome extraction steps and agarose gel electrophoresis gel analysis results, which are difficult to promote and apply in production.

Contents of the invention

The technical problem to be solved by the present invention is to provide a PCR primer for gender identification of young pigeons and its application and a method for gender identification of young pigeons, which can identify the gender of young pigeons simply, quickly and accurately.

In order to solve the above technical problems, embodiments of the present invention provide a method for gender identification of young pigeons, including Including the following steps:

S1. Plug out 1 to 2 new feathers with hair follicles on the squab;

S2. Use genomic DNA extraction reagent to extract the genomic DNA of the squab to be tested. The operation steps are as follows: Mix lysis solution A and lysis solution B according to 24:1, and then add the feather sample with feather pith into the mixed lysis solution. , heat at 55°C for 15 minutes, heat at 95°C for 5 minutes, then add stop solution, and take the supernatant to obtain the genomic DNA of pigeon feather marrow;

S3. Use the extracted DNA as a template and use Biotin and FAM-modified specific upstream and downstream primers to perform PCR amplification; among them, the PCR reaction system is: 2×Taq Master Mix 10μL, with a concentration of 10μmol/L forward and reverse. 0.8 μL of each primer, 1 μL of template DNA, and 7.4 μL of ultrapure water; the PCR reaction program is: pre-denaturation at 95°C for 3 minutes; extension for 30 cycles of 95°C for 15 seconds, 60°C for 15 seconds, and 72°C for 60 seconds; incubation at 72°C for 5 minutes; 4 ℃ ends.

S4. Take 1 to 2 μL of the amplified PCR primer and dilute it 50 to 100 times with the special sample diluent of the colloidal gold test strip. Add 50 μL to the sampling hole of the colloidal gold test strip and react for 2 to 5 minutes. Male squabs correspond to After reacting with the reagent, only one band appeared on the quality control line of the colloidal gold test strip, and there was no band at the detection line; after reacting with the reagent corresponding to the female squab, there was a band on the quality control line and the detection line of the colloidal gold test strip. One band appears on each side to identify the sex of the squab.

The invention also provides a PCR primer for gender identification of young pigeons, including:

Upstream primer sequence: 5’-TCCTATGCCTACCACCTTCCT-3’ (SEQ ID NO.1);

Downstream primer sequence: 5’-TTGAGAAAGAATCTGACCTGAACC-3’ (SEQ ID NO. 2).

The invention also provides an application of the PCR primer for gender identification of young pigeons in gender identification of young pigeons.

The invention also provides the application of a PCR primer for gender identification of young pigeons in preparing a kit for gender identification of young pigeons.

The invention also provides a kit for gender identification of young pigeons, which includes the PCR primers for gender identification of young pigeons as described in the above technical solution, and also includes: genomic DNA extraction reagent, 2×Taq Master Mix, ultrapure water, Special sample diluent for colloidal gold test strips.

The beneficial effects of the above technical solutions of the present invention are as follows:

The invention provides a method for gender identification of young pigeons and a pair of PCR primers for identification. The differences between the sex chromosomes ZW (female) and ZZ (male) of male and female pigeons are used to design primers for the unique EE0.6 sequence fragment on the W chromosome of female pigeons. , and uses Biotin and FAM to modify the upstream and downstream primers. The primers have the advantages of good specificity, high resolution, and intuitive amplification results.

The present invention uses the exploratory lysis solution to obtain the genomic DNA of young pigeon feather marrow simply and quickly, eliminating the extraction steps of commercial DNA extraction kits and saving time and economic costs.

In the present invention, the PCR product is directly judged as male or female by the number of bands on the colloidal gold rapid detection test strip, and the results are intuitive and clear. It does not require the time-consuming (20-30 min) agarose gel electrophoresis method to determine the results. It meets the requirements of fast, accurate and economical detection, and is suitable for promotion and application in large-scale pigeon production and breeding.

The present invention uses feathers to extract DNA, which causes little harm to pigeons. It uses PCR amplification technology and colloidal gold rapid detection test strips to identify the sex of pigeons, thereby shortening the detection time, improving the detection rate, lowering the technical threshold, and playing a key role in the breeding and development of pigeons. It is highly innovative in reproduction.

Description of drawings

In order to explain the embodiments of the present invention or the technical solutions in the prior art more clearly, the drawings needed to be used in the embodiments will be briefly introduced below.

Figure 1 is a schematic diagram of a 21-day-old squab in Example 1;

Figure 2 shows the rapid detection results of PCR products in 21-day-old squabs using colloidal gold test paper in Example 1;

Figure 3 is an anatomical verification diagram when the 21-day-old squab is raised to 28 days old in Example 1;

Figure 4 is a graph showing the nucleic acid electrophoresis gel results of gender identification of 21-day-old squabs of unknown male and female in Example 1;

Figure 5 is a schematic diagram of a 6-day-old squab in Example 2;

Figure 6 is a graph showing the results of rapid detection of PCR products in 6-day-old squabs using colloidal gold test paper in Example 2;

Figure 7 is a graph showing the nucleic acid electrophoresis gel results of gender identification of 6-day-old squabs of unknown male and female in Example 2;

Figure 8 is a graph showing the rapid detection results of known male and female breeding pigeons using colloidal gold test paper in Example 3.

Detailed ways

In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

The invention provides a method for gender identification of young pigeons, which includes the following steps:

S1. Plug out 1 to 2 new feathers with hair follicles on the squab;

S2. Use genomic DNA extraction reagent to extract the genomic DNA of the squab to be tested; the operation steps are as follows: Mix lysis solution A and lysis solution B according to 24:1, and then add the feather sample with feather pith into the mixed lysis solution. , heat at 55°C for 15 minutes, heat at 95°C for 5 minutes, then add stop solution, and take the supernatant to obtain the genomic DNA of pigeon feather marrow. Take 1 μL of the supernatant for PCR amplification, and store the rest at -20°C for later use.

S3. Use the extracted DNA as a template and use specific upstream and downstream primers modified by Biotin and FAM. Perform PCR amplification; the PCR reaction system is: 2×Taq Master Mix 10 μL, forward and reverse primers with a concentration of 10 μmol/L 0.8 μL each, and template DNA with a concentration of 40 to 400 μg/mL extracted in the previous step 1 μL , 7.4 μL of ultrapure water; the PCR reaction program is: pre-denaturation at 95°C for 3 minutes; extension for 30 cycles of 95°C for 15s, 60°C for 15s, and 72°C for 60s; incubation at 72°C for 5 minutes; end at 4°C. The specific primers in this step are designed for the unique EE0.6 sequence fragment on the pigeon W chromosome.

S4. Take 1 to 2 μL of the amplified PCR primer, dilute it 50 to 100 times with the colloidal gold test strip special sample diluent, add 50 μL to the sample hole of the colloidal gold test strip, and react for 2 to 5 minutes. After the reaction of the reagent corresponding to the pigeon, only one band appeared on the quality control line of the colloidal gold test strip, and there was no band on the detection line; after the reaction of the reagent corresponding to the female squab, there was a band on the detection line and quality control line of the colloidal gold test strip. A strip appears on each line to identify the sex of the squab.

Detailed steps are as follows:

(1) Plug out 1 to 2 new feathers with hair follicles on the wings or tail of each squab;

(2) Use genomic DNA rapid extraction reagent to extract the genomic DNA of the pigeon to be tested (20 minutes);

(3) Using the extracted DNA as a template, use Biotin and FAM-modified specific primers upstream and downstream primers to perform PCR amplification. The PCR reaction system is: 2×Taq Master Mix (Nanjing Novezan Biotechnology Co., Ltd.) 10 μL, 0.8 μL each forward and reverse primer with a concentration of 10 μmol/L, 1 μL template DNA with a concentration of 40-400 μg/mL, ultra 7.4μL of pure water. The PCR reaction program is: pre-denaturation at 95°C for 3 minutes; extension for 30 cycles at 95°C for 15 seconds, 60°C for 15 seconds, and 72°C for 60 seconds; incubation at 72°C for 5 minutes; end at 4°C.

(4) After diluting the amplified PCR primer 50 to 100 times, add 50uL to the sampling hole of the colloidal gold detection test strip, and react for 2 to 5 minutes. Only female pigeons will appear visible to the naked eye on the detection line. Colloidal gold strip, and the quality control line also has a strip. The PCR product of male pigeons has no band on the detection line and only one band on the quality control line. This method can achieve the purpose of quickly identifying female individuals.

Note: Please use the test paper as soon as possible after opening it; the result should be judged within the specified time, preferably within 5 minutes. Reading after more than 10 minutes may cause false positives.

The technical solution of the present invention will be further described below with reference to several embodiments.

Example 1

Test time: 20220420~20220430, test location: A pigeon industry Co., Ltd.

1. Randomly select 30 21-day-old young pigeons (healthy pigeons of unknown gender), wear numbered anklets (as shown in Figure 1), and then sample. The sampler needs to wear disposable gloves to pick out each pigeon. There are 1 to 2 new feathers with hair follicles on the wings or tail of squabs, which are used to identify their gender.

2. Use genomic DNA rapid extraction reagent to extract DNA, use DNA as a template, and pass through the above The primers were used for PCR amplification, and then the PCR product was diluted 1:100. 50 μL of the diluted product was added to the sampling part of the colloidal gold test strip. The results were read after 2 minutes. Only the PCR product of the female squab was in the Only on the detection line will the colloidal gold strips visible to the naked eye appear. As shown in Figure 2, the colloidal gold test paper test results showed that there were 16 female pigeons and 14 male pigeons.

3. When the young pigeons are released to the market at the age of 28 days, they will be euthanized and dissected, and the male gonads and one pair of white testicles (Figure 3) will be used as the criterion. A total of 14 had testicles and 16 had no testicles. Comparing the coincidence rate between the gonad testicle observation results (Figure 3) and the identification results of the colloidal gold test paper (Figure 2), the detection results of the colloidal gold test paper are completely consistent with the slaughter verification results, showing that the accuracy of the colloidal gold test paper method is 100%. Simple and efficient.

4. Detect the PCR product by 1.5% agarose gel electrophoresis. If the electrophoresis result (Figure 4) shows a 360bp band, it is a female pigeon, and if there is no band, it is a male pigeon. The results are consistent with the results of colloidal gold test strips and slaughter verification.

Example 2

Test time: 20220505～20220602, test location: A pigeon industry Co., Ltd.

Randomly select 20 6-day-old young pigeons (healthy pigeons of unknown gender), wear the numbered anklet tags, and then conduct sampling. The sampler needs to wear disposable gloves to pick out the hair follicles on the wings or tail of each squab. There are 1 to 2 new feathers (as shown in Figure 5), which are used to identify its gender. Feather marrow DNA was extracted, and after specific PCR amplification, the PCR product was diluted 1:100 and added to the colloidal gold test strip. The results were read after 2 minutes and showed 9 females and 11 males (Figure 6). When they were released to the market at 28 days of age, they were euthanized and dissected. It was found that 11 animals had one pair of testicles and 9 had no testicles. The coincidence rate of the observation results of gonads and testicles and the identification results of colloidal gold test paper was compared, and the accuracy rate was 100%. The PCR product was tested by 1.5% agarose gel electrophoresis (the results are shown in Figure 7), and the results were also consistent with the colloidal gold test paper results.

Example 3

Test time: 20220606~20220609, test location: A pigeon industry Co., Ltd.

Select 5 pairs of male and female breeding pigeons (350 to 360 days old) that have been paired successfully and have known sex. Plug out 1 feather with a hair follicle under each wing. DNA is extracted. After specific PCR amplification, the PCR primers are 1:100. Dilute it, add it to the colloidal gold test strip, and read the result after 2 minutes (the result is shown in Figure 8). The test strip results showed 5 male pigeons and 5 female pigeons, which was completely in line with expectations.

The above is the preferred embodiment of the present invention. It should be pointed out that for those of ordinary skill in the art, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications can also be made. should be regarded as the protection scope of the present invention.

Claims (7)

1. A PCR primer for gender identification of young pigeons, characterized in that the PCR primer includes an upstream primer and a downstream primer;

   The nucleotide sequence of the upstream primer is shown in SEQ ID NO.1;

   The nucleotide sequence of the downstream primer is shown in SEQ ID NO.2.
2. Application of the PCR primer for gender identification of young pigeons according to claim 1 in the preparation of a kit for gender identification of young pigeons.
3. A kit for gender identification of young pigeons, characterized in that the kit includes genomic DNA extraction reagent, 2×Taq Master Mix, ultrapure water, colloidal gold test strip special sample diluent and claim 1 PCR primers for gender identification of young pigeons.
4. The kit according to claim 3, characterized in that the kit further includes a colloidal gold detection test strip.
5. Application of the PCR primer for gender identification of young pigeons according to claim 1 or the kit for gender identification of young pigeons according to claim 3 or 4 in the gender identification of young pigeons.
6. A method for gender identification of young pigeons, which is characterized by including the following steps:

   S1. Plug out 1 to 2 new feathers with hair follicles on the squab;

   S2. Use genomic DNA extraction reagent to extract the genomic DNA of the squab to be tested. The operation steps are as follows: Mix lysis solution A and lysis solution B according to 24:1, and then add the feather sample with feather pith into the mixed lysis solution. , heat at 55°C for 15 minutes, heat at 95°C for 5 minutes, then add stop solution, and take the supernatant to obtain the genomic DNA of pigeon feather marrow;

   S3. Use the extracted genomic DNA as a template and use Biotin and FAM-modified specific upstream and downstream primers to perform PCR amplification; the PCR reaction system is: 2×Taq Master Mix 10μL, with a concentration of 10μmol/L upstream primers and downstream primers. 0.8 μL of each primer, 1 μL of template DNA, and 7.4 μL of ultrapure water; the PCR reaction program is: pre-denaturation at 95°C for 3 minutes; extension for 30 cycles of 95°C for 15s, 60°C for 15s, and 72°C for 60s; incubation at 72°C for 5 minutes; 4 ℃ end;

   The specific upstream and downstream primers are the upstream and downstream primers in the PCR primers of claim 1.

   S4. Take 1 to 2 μL of the amplified PCR product and dilute it 50 to 100 times with the colloidal gold test strip’s special sample diluent. Add 50 μL to the sampling hole of the colloidal gold test strip and react for 2 to 5 minutes. After the reagent reaction, only A band appears on the quality control line of the colloidal gold test strip, but there is no band at the detection line. It is a male squab. After the reagent reaction, a band appears on the quality control line and the detection line of the colloidal gold test strip. , a female squab.
7. [Correction 15.09.2023 under Rule 91]
   The method for gender identification of young pigeons according to claim 6, characterized in that the concentration of template DNA in the S3 is 40-400 μg/mL.