Chicken embryo sex determination kit directly using blood as template and application thereof

Abstract

The invention discloses a chicken embryo sex determination kit directly using blood as a template and application thereof. Belongs to the technical field of biology. The kit comprises Taq DNA polymerase, an upstream primer, a downstream primer and water. Compared with the prior art, the method has the beneficial effects that a PCR reaction system is optimized by adopting the super-fidelity Taq DNA polymerase, whole blood is diluted and then is directly used as a template for PCR reaction, tedious DNA extraction is not needed, meanwhile, a PCR reaction enhancer and a PCR system stabilizer are not needed to be added into the reaction system, and the operation is simple and convenient. The method has the advantages of simple and convenient operation, less time consumption, rapid identification and the like, and avoids the defects of time and labor consumption and error proneness of a traditional method.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and more particularly to a chicken embryo sex identification kit which directly uses blood as a template and an application thereof. Background Art

[0002] Early sex identification of poultry has important economic and scientific significance for poultry production. In production, sex identification can optimize poultry feeding and management and improve production efficiency. Early sex identification technology can reduce unnecessary waste of resources. For example, during the incubation process, poultry of different sexes can be reasonably allocated and managed according to the sex identification results to improve breeding efficiency. At the same time, this technology can also avoid economic losses caused by incorrect sex identification, especially in the broiler and laying hen industries, where sex identification is crucial for optimizing production strategies. Early sex identification of poultry is of great significance in the field of scientific research. It not only helps to further understand the genetic and molecular mechanisms of sex determination in poultry, but also helps to study poultry reproduction and breeding, genetic disease prevention and control, population structure and population genetic analysis. Through early sex identification technology, researchers can more accurately analyze sex ratios, genetic diversity, and sex-specific trait expressions, thereby providing a scientific basis for the improvement and optimization of poultry breeds.

[0003] Existing methods for identifying the sex of chicken embryos include estrogen determination by allantoic fluid, PCR method, blood vessel thickness, artificial intelligence-driven MRI technology, etc. Due to the disadvantages of high cost and complexity, blood vessel thickness and artificial intelligence-driven MRI technology are not widely promoted and applied at present. Commonly used methods are estrogen determination by allantoic fluid and PCR method. The more mature method of identifying the sex of chicken embryos is to collect allantoic fluid from chicken embryos over 9 days old and determine the estrogen content by ELISA method. The high content is female, and the estrogen-free content is male. This method can only identify chicken embryos over 9 days old.

[0004] The development of molecular biology technology has provided new methods for early sex identification of poultry, such as polymerase chain reaction (PCR) or loop-mediated isothermal amplification (LAMP) technology, which can achieve sex identification by analyzing specific genes on the sex chromosomes of chicken embryos. For example, the sex of chickens can be identified by detecting the difference in the intron gene sequence of the chromosome helical protein gene (CHD1 gene). PCR has been widely used in the field of sex identification due to its rapid, sensitive, simple and high specificity. With the development of technology, PCR sex identification methods have developed from simple PCR, double or multiple PCR, nested PCR to improved two-temperature gradient PCR. In practical applications, blood PCR technology has been used for sex identification of poultry, especially chickens. The PCR method mainly collects chicken embryo tissue to extract DNA and then conducts PCR identification. Studies have shown that the PCR method after extracting DNA from blood, feathers and other materials can accurately and quickly identify the sex of chickens and chicken embryos, which is of great significance for sex control in poultry breeding.

[0005] The above strategies solve the problem of early sex identification of poultry based on DNA extraction from blood or tissue and PCR-agarose gel electrophoresis identification. However, due to the cumbersome DNA extraction process, it is usually applicable to scientific research or the protection of high-value poultry breeds and rare poultry populations and the preservation of genetic resources, which is not conducive to large-scale application in production. There are some problems with direct PCR using blood as a template. Certain components in the blood may inhibit the PCR reaction, such as hemoglobin, lactoferrin and immunoglobulin, which may affect the PCR amplification efficiency and lead to false negative results. In addition, some exogenously added anticoagulants will also have a certain inhibitory effect on the PCR reaction. Therefore, PCR reaction enhancers and PCR system stabilizers need to be introduced into the existing blood PCR system. Commonly used PCR reaction enhancers include betaine, trehalose, dimethyl sulfoxide, gelatin and glycerol, and PCR system stabilizers include Tween20, NP40, Triton X-100, etc. Since chicken embryo blood collection is usually accompanied by other protein contamination, chicken embryo blood PCR needs to be further optimized.

[0006] In summary, how to provide a method for sex identification by direct PCR of chicken embryo blood is an urgent problem to be solved by those skilled in the art. Summary of the invention

[0007] In view of this, the present invention provides a chicken embryo sex identification kit which directly uses blood as a template and its application.

[0008] The method of the present invention can be applied to chicken embryos of all ages, that is, as long as blood can be collected from the chicken embryo, the sex of the chicken embryo can be identified by direct blood PCR.

[0009] In order to achieve the above object, the present invention adopts the following technical solution:

[0010] A chicken embryo sex identification kit directly using blood as a template, comprising Taq DNA polymerase, an upstream primer, a downstream primer and water;

[0011] The primer sequences are as follows:

[0012] Upstream primer: 5'-GTTACTGATTCGTCTACGAGA-3', SEQ ID NO.1;

[0013] Downstream primer: 5'-ATTGAAATGATCCAGTGCTTG-3', SEQ ID NO.2.

[0014] Furthermore, the Taq DNA polymerase is Genestar 2x SurperNova ultra-fidelity PCR premix.

[0015] Furthermore, it includes 10 μL of Genestar 2x SurperNova ultra-fidelity PCR premix, 6 μL of ddH2O, 1 μL of 10 μmol / L upstream primer, and 1 μL of 10 μmol / L downstream primer.

[0016] A method for identifying the sex of chicken embryos directly using blood as a template comprises the above-mentioned kit.

[0017] Further, the following steps are included:

[0018] (1) Draw 10 μL of blood from the chicken embryo and place it in a centrifuge tube containing EDTA;

[0019] (2) Centrifuge and discard the supernatant, add ddH2O to dilute the blood to a red bean paste color, shake vigorously to separate the layers, and take the supernatant as a template;

[0020] (3) Perform PCR amplification using the kit, and then detect the PCR amplification product using agarose gel electrophoresis.

[0021] Furthermore, in step (2), the centrifugation parameter is 1000 × g for 3 min.

[0022] Furthermore, in step (3), the PCR amplification conditions are:

[0023] Step 1: pre-denaturation at 95°C for 5 min;

[0024] Step 2: denaturation at 94°C for 30 s, annealing at 57°C for 45 s, and extension at 72°C for 30 s, for 34 cycles;

[0025] Step 3: Extend at 72°C for 7 min.

[0026] Furthermore, when the amplification product showed only one 550 bp specific amplification band, it was determined to be male, and when the amplification product showed two specific amplification bands of 450 bp and 550 bp at the same time, it was determined to be female.

[0027] It can be seen from the above technical solution that, compared with the prior art, the beneficial effects achieved by the present invention are:

[0028] The present invention adopts ultra-fidelity Taq DNA polymerase to optimize the PCR reaction system. Whole blood is directly used as a template for PCR reaction after dilution, without the need for cumbersome DNA extraction. At the same time, no PCR reaction enhancer and PCR system stabilizer need to be added to the reaction system. It has the advantages of simple and convenient operation, less time consumption, rapid identification, etc., avoiding the disadvantages of traditional methods that are time-consuming, labor-intensive and prone to errors. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0030] Figure 1 The PCR results of three batches of chicken embryo blood at different ages in Example 1 of the present invention are shown in Figure 1, wherein the white numbers are negative and positive controls; the blue numbers are the first batch of samples; the orange numbers are the second batch of samples, and the green numbers are the third batch of samples;

[0031] Figure 2 The PCR results of three batches of chicken embryo blood at different ages in Comparative Example 1 of the present invention are shown in Figure 1, wherein the white numbers are negative and positive controls; the blue numbers are the first batch of samples; the orange numbers are the second batch of samples, and the green numbers are the third batch of samples;

[0032] Figure 3 The PCR results of three batches of chicken embryo blood at different ages in Comparative Example 2 of the present invention are shown in Figure 2, wherein the white numbers are negative and positive controls; the blue numbers are the first batch of samples; the orange numbers are the second batch of samples, and the green numbers are the third batch of samples;

[0033] Figure 4 The PCR results of DNA from chicken embryos of three batches at different ages in Comparative Example 3 of the present invention are shown in Figure 3, wherein the white numbers are negative and positive controls; the blue numbers are the first batch of samples; the orange numbers are the second batch of samples, and the green numbers are the third batch of samples;

[0034] Figure 5These are the PCR results of three batches of chicken embryo tissue lysates of different ages in Comparative Example 4 of the present invention, wherein white numbers are negative and positive controls; blue numbers are the first batch of samples; orange numbers are the second batch of samples, and green numbers are the third batch of samples. DETAILED DESCRIPTION

[0035] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0036] The materials used in the following examples are as follows:

[0037] Sample source

[0038] Chicken embryos were yellow-feathered broilers from the original breeder farm of the Animal Science Institute of Guangdong Academy of Agricultural Sciences. They were incubated in a RcomPRO 50 incubator (Rutoex) at 38.5°C and 60% relative humidity to 6.5, 8.5, and 10.5 days of age.

[0039] Main reagents

[0040] EDTA, Dongsheng 2×PCR Recation Mix enzyme, Acori Bio 2×Pro Taq Master Mix enzyme, Genestar 2x SurperNova ultra-fidelity PCR premix, ddH2O, and agarose gel electrophoresis related reagents.

[0041] Instruments and equipment

[0042] Electrophoresis apparatus, universal gel imager, desktop refrigerated centrifuge, PCR instrument, water bath.

[0043] Primers

[0044] Upstream primer: 5'-GTTACTGATTCGTCTACGAGA-3', SEQ ID NO.1;

[0045] Downstream primer: 5'-ATTGAAATGATCCAGTGCTTG-3', SEQ ID NO.2.

[0046] Example 1

[0047] Blood PCR sex determination

[0048] 10 μL of blood was drawn from chicken embryos at 6.5 d, 8.5 d, and 10.5 d, respectively, and placed in a 1.5 mL centrifuge tube containing 150 μL of EDTA. The tube was centrifuged at 1000 × g for 3 min, the upper plasma was discarded, 150 μL of ddH2O was added to the blood cell pellet to dilute the blood to a bean paste color, and the blood was shaken vigorously to separate the layers. The supernatant was used as a template for PCR amplification using poultry sex identification PCR primers.

[0049] PCR reaction system:

[0050] Genestar 2x SurperNova ultra-fidelity PCR premix 10 μL, ddH2O 6 μL, upstream primer 1 μL (10 μmol / L), downstream primer 1 μL (10 μmol / L), blood template 2 μL, the total reaction system is 20 μL.

[0051] PCR reaction conditions:

[0052] Step 1: pre-denaturation at 95°C for 5 min;

[0053] Step 2: denaturation at 94°C for 30 s, annealing at 57°C for 45 s, and extension at 72°C for 30 s, for 34 cycles;

[0054] Step 3: Extend at 72°C for 7 min.

[0055] The PCR products were stored at 4°C.

[0056] Take an appropriate amount of PCR product for agarose gel electrophoresis at 130 V for 30 min. Identify the gender based on the electrophoresis results of the product. Experimentally identify three batches of different samples.

[0057] The results are as follows Figure 1 As shown. Using Genestar 2x SurperNova ultra-fidelity PCR premix as Taq DNA polymerase for PCR amplification achieved good identification results. The results were consistent with the DNA extraction results (Comparative Example 3), and chicken embryo blood of different ages was suitable for this amplification system.

[0058] Comparative Example 1

[0059] Blood PCR sex determination

[0060] 10 μL of blood was drawn from chicken embryos at 6.5 d, 8.5 d, and 10.5 d, respectively, and placed in a 1.5 mL centrifuge tube containing 150 μL of EDTA. The tube was centrifuged at 1000 × g for 3 min, the upper plasma was discarded, 150 μL of ddH2O was added to the blood cell pellet to dilute the blood to a bean paste color, and the blood was shaken vigorously to separate the layers. The supernatant was used as a template for PCR amplification using poultry sex identification PCR primers.

[0061] PCR reaction system:

[0062] 2×Pro Taq Master Mix enzyme 10 μL, ddH2O 6 μL, upstream primer 1 μL (10 μmol / L), downstream primer 1 μL (10 μmol / L), blood template 2 μL, the total reaction system is 20 μL.

[0063] PCR reaction conditions:

[0064] Step 1: pre-denaturation at 95°C for 5 min;

[0065] Step 2: denaturation at 94°C for 30 s, annealing at 57°C for 45 s, and extension at 72°C for 30 s, for 34 cycles;

[0066] Step 3: Extend at 72°C for 7 min.

[0067] The PCR products were stored at 4°C.

[0068] Take an appropriate amount of PCR product for agarose gel electrophoresis at 130 V for 30 min. Identify the gender based on the electrophoresis results of the product. Experimentally identify three batches of different samples.

[0069] The results are as follows Figure 2 As shown. Figure 2 It can be seen that the target PCR product cannot be obtained when PCR amplification is performed using 2×Pro Taq Master Mix enzyme as Taq DNA polymerase.

[0070] Comparative Example 2

[0071] Blood PCR sex determination

[0072] 10 μL of blood was drawn from chicken embryos at 6.5 d, 8.5 d, and 10.5 d, respectively, and placed in a 1.5 mL centrifuge tube containing 150 μL of EDTA. The tube was centrifuged at 1000 × g for 3 min, the upper plasma was discarded, 150 μL of ddH2O was added to the blood cell pellet to dilute the blood to a bean paste color, and the blood was shaken vigorously to separate the layers. The supernatant was used as a template for PCR amplification using poultry sex identification PCR primers.

[0073] PCR reaction system:

[0074] 2×PCR Recation Mix enzyme 10 μL, ddH2O 6 μL, upstream primer 1 μL (10 μmol / L), downstream primer 1 μL (10 μmol / L), blood template 2 μL, the total reaction system is 20 μL.

[0075] PCR reaction conditions:

[0076] Step 1: pre-denaturation at 95°C for 5 min;

[0077] Step 2: denaturation at 94°C for 30 s, annealing at 57°C for 45 s, and extension at 72°C for 30 s, for 34 cycles;

[0078] Step 3: Extend at 72°C for 7 min.

[0079] The PCR products were stored at 4°C.

[0080] Take an appropriate amount of PCR product for agarose gel electrophoresis at 130 V for 30 min. Identify the gender based on the electrophoresis results of the product. Experimentally identify three batches of different samples.

[0081] The results are as follows Figure 3 As shown. Figure 3 It was found that the target PCR product could not be obtained when PCR amplification was performed using 2×PCR Recation Mix as Taq DNA polymerase.

[0082] Comparative Example 3

[0083] Tissue DNA PCR Sex Identification

[0084] 30 mg of thigh muscle tissue samples were collected from 6.5, 8.5, and 10.5 day old chicken embryos, and DNA was extracted for PCR identification. DNA was extracted using MGBio's HiPure Viral DNA Kit (Cat. No. D3191-03).

[0085] PCR reaction system:

[0086] 2×Pro Taq Master Mix enzyme 10μL, ddH2O 6 μL, upstream primer 1μL (10 μmol / L), downstream primer 1μL (10 μmol / L), DNA template 2 μL, the total reaction system is 20 μL.

[0087] PCR reaction conditions:

[0088] Step 1: pre-denaturation at 95°C for 5 min;

[0089] Step 2: denaturation at 94°C for 30 s, annealing at 57°C for 45 s, and extension at 72°C for 30 s, for 34 cycles;

[0090] Step 3: Extend at 72°C for 7 min.

[0091] The PCR products were stored at 4°C.

[0092] Take an appropriate amount of PCR product for agarose gel electrophoresis at 130 V for 30 min. Identify the gender based on the electrophoresis results of the product. Experimentally identify three batches of different samples.

[0093] The results are as follows Figure 4 As shown. Figure 4 It can be seen that when chicken embryo tissue DNA was used as PCR template, good identification results were obtained when PCR amplification was performed using Acori Bio 2×Pro Taq Master Mix enzyme as Taq DNA polymerase. Bands appeared near Maker 500 bp, of which only the 550 bp band was male ZZ type, and the 450 bp and 550 bp bands were female ZW type.

[0094] Comparative Example 4

[0095] Tissue lysis PCR sex identification

[0096] 30 mg of thigh muscle tissue samples were collected from 6.5 d, 8.5 d, and 10.5 d chicken embryos, and the tissues were lysed. The lysate was used as a template for PCR identification. Tissue lysis was performed using the Bio-Tech Animal Genomic DNA Rapid Extraction Kit (Cat. No. D0065M).

[0097] PCR reaction system:

[0098] 2×Pro Taq Master Mix enzyme 10μL, ddH2O 6 μL, upstream primer 1μL (10 μmol / L), downstream primer 1μL (10 μmol / L), DNA template 2 μL, the total reaction system is 20 μL.

[0099] PCR reaction conditions:

[0100] Step 1: pre-denaturation at 95°C for 5 min;

[0101] Step 2: denaturation at 94°C for 30 s, annealing at 57°C for 45 s, and extension at 72°C for 30 s, for 34 cycles;

[0102] Step 3: Extend at 72°C for 7 min.

[0103] The PCR products were stored at 4°C.

[0104] Take an appropriate amount of PCR product for agarose gel electrophoresis at 130 V for 30 min. Identify the gender based on the electrophoresis results of the product. Experimentally identify three batches of different samples.

[0105] The results are as follows Figure 5 As shown. Figure 5 It can be seen that when chicken embryo tissue lysate is used as a PCR template, the effect of PCR amplification using Acori Bio 2×Pro Taq Master Mix enzyme as Taq DNA polymerase is unstable, and some samples cannot obtain the target PCR product, and the effect is general.

[0106] In summary, PCR gender identification of tissue-extracted DNA is a traditional method, but this method is time-consuming and labor-intensive. Compared with traditional tissue DNA extraction, directly lysing tissue supernatant as a PCR template simplifies the experimental steps to a certain extent, but the PCR results are unstable. The present invention uses Genestar 2x SurperNova ultra-fidelity PCR premix as Taq DNA polymerase to optimize the PCR reaction system, and whole blood is directly used as a template for PCR reaction after dilution, without the need for cumbersome DNA extraction, and at the same time, the reaction system does not need to add PCR reaction enhancers and PCR system stabilizers, which greatly reduces its operational complexity and cost, and has the advantages of simple and convenient operation, less time consumption, and rapid identification.

[0107] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0108] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A chicken embryo sex identification kit directly using blood as a template, characterized in that: Includes Taq DNA polymerase, upstream primer, downstream primer and water; The primer sequences are as follows: Upstream primer: 5'-GTTACTGATTCGTCTACGAGA-3', SEQ ID NO.1; Downstream primer: 5'-ATTGAAATGATCCAGTGCTTG-3', SEQ ID NO.

2.

2. The kit according to claim 1, characterized in that The Taq DNA polymerase is Genestar 2xSurperNova ultra-fidelity PCR premix.

3. The kit according to claim 1, characterized in that Includes 10 μL of Genestar 2x SurperNova ultra-fidelity PCR premix, 6 μL of ddH2O, 1 μL of 10 μmol / L upstream primer, and 1 μL of 10 μmol / L downstream primer.

4. A method for sex identification of chicken embryos directly using blood as a template, characterized in that: Use the kit according to any one of claims 1 to 3.

5. The method according to claim 4, characterized in that The steps include: (1) Draw 10 μL of blood from the chicken embryo and place it in a centrifuge tube containing EDTA; (2) Centrifuge and discard the supernatant, add ddH2O to dilute the blood to a red bean paste color, shake vigorously to separate the layers, and take the supernatant as a template; (3) Perform PCR amplification using the kit, and then detect the PCR amplification product using agarose gel electrophoresis.

6. The method according to claim 5, characterized in that In the step (2), the centrifugation parameter is 1000 × g for 3 min.

7. The method according to claim 5, characterized in that In the step (3), the PCR amplification conditions are: Step 1: pre-denaturation at 95°C for 5 min; Step 2: denaturation at 94°C for 30 s, annealing at 57°C for 45 s, and extension at 72°C for 30 s, for 34 cycles; Step 3: Extend at 72°C for 7 min.

8. The method according to claim 5, characterized in that When only one 550 bp specific amplification band appeared in the amplification product, it was judged as male; when two specific amplification bands of 450 bp and 550 bp appeared in the amplification product at the same time, it was judged as female.

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