Gene kit for detecting high yield of pigs

Abstract

The invention discloses a kit for detecting pig high-yield genes, and belongs to the technical field of biotechnology detection. The kit solves the technical problems of low pig high-yield gene detection accuracy and poor efficiency in the prior art. According to the technical scheme, the kit comprises a sampling device, a DNA extraction reagent, an amplification reagent and a detection reagent, tissue, blood or saliva samples of pigs can be obtained, DNA is incubated and extracted through the extraction reagent containing 1%-5% of a surfactant and 0.1 M-0. 5M of a salt solution at the temperature of 50 DEG C-60 DEG C, and the DNA is amplified through the amplification reagent. A primer pair designed aiming at the ESR gene is used for carrying out PCR amplification (94 DEG C predenaturation for 3-5 minutes, 30-40 cycles, denaturation at 94 DEG C, annealing at 55-65 DEG C and extension at 72 DEG C), and an amplification product is detected by adopting a fluorescent quantitative PCR probe method or a gel electrophoresis method. The kit is mainly used for detecting high-yield genes of pigs, assists in screening high-yield pig breeds, and provides technical support for molecular marker-assisted breeding of pigs.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology detection technology, specifically relating to a kit for detecting high-yield genes in pigs and its detection method. Background Technology

[0002] In the pig farming industry, screening for high-yield traits is crucial for improving farming efficiency. Currently, there are many unresolved issues in the practical application of detection technologies for high-yield genes in pigs. Existing gene detection methods are insufficient in accuracy. Due to the lack of an effective internal reference gene correction system, the detection process is easily affected by factors such as sample size and amplification efficiency, leading to deviations in the judgment of expression levels of genes related to high yield in pigs, thus affecting the accurate assessment of high-yield performance.

[0003] Meanwhile, existing detection technologies suffer from low operational efficiency. From sample collection to obtaining the final test results, complex steps are often involved, and the connections between these steps lack standardized procedures. For example, during DNA extraction, the lack of specific protective measures makes the extracted DNA susceptible to degradation by nucleases, leading to unstable template quality for subsequent amplification reactions and increasing the uncertainty of the test results.

[0004] Furthermore, the preservation of samples, DNA, and amplification products is also a challenge faced by current technologies. Due to the lack of suitable preservation reagents and conditions, collected samples are prone to degradation during preservation, extracted DNA is difficult to maintain its integrity over extended periods, and the stability of amplification products cannot be effectively guaranteed. This makes it difficult to complete detection experiments within a suitable time window and hinders the reproducibility of test results, thus creating difficulties for large-scale high-yield pig breed screening.

[0005] The root cause of these problems lies in the fact that existing detection technologies have failed to form a complete solution. There is a lack of systematic optimization design in every step, from sample processing to detection and analysis. For example, effective nucleic acid protection components are not added to DNA extraction reagents, making it impossible to inhibit the action of nucleases; internal reference genes are not introduced in the amplification reaction for synchronous correction, making it difficult to accurately calibrate the detection results; and the formulation and storage conditions of preservation reagents have not been optimized for the characteristics of porcine gene detection, leading to quality problems in samples and reaction products during storage. These issues make traditional porcine high-yield gene detection technologies inefficient and inaccurate in practical applications, failing to meet the needs of rapid screening of high-yield pig breeds in large-scale farming. Summary of the Invention

[0006] One object of the present invention is to solve at least the above-mentioned problems and to provide at least the advantages that will be described later.

[0007] Another objective of this invention is to provide a kit for detecting high-yield genes in pigs, which can solve the secondary technical problems of easy DNA degradation during the detection process, easy interference with detection results, and difficulty in properly preserving samples, DNA, and amplification products.

[0008] To achieve these objectives and other advantages according to the present invention, a kit for detecting high-yield genes in pigs is provided, comprising the following components and operating steps: First, the kit includes a sampling device for collecting pig samples, which can obtain tissue, blood, or saliva samples from the pig. Next, a DNA extraction reagent is provided for extracting DNA from the collected samples. The DNA extraction reagent is formulated to effectively lyse sample cells and separate DNA. It contains surfactants in a specific concentration range of 1%-5% and salt solutions in a concentration range of 0.1M-0.5M. DNA extraction is achieved through conventional extraction procedures, such as incubation at a temperature of 50℃-60℃ for 15-30 minutes. Then, an amplification reagent is provided for amplifying specific gene fragments associated with high-yield traits in pigs. This amplification reagent contains suitable primer pairs designed for high-yield-related genes in pigs, specifically specific regions of the ESR gene that affect pig reproductive performance, as well as DNA polymerase, buffer, and dNTPs for the amplification reaction. The amplification reaction is performed in a conventional PCR instrument, and the amplification program is set as follows: 94℃ pre-denaturation for 3-5 minutes, followed by 30-40 cycles, each cycle including 94℃ denaturation for 30-60 seconds, 55℃-65℃ annealing for 30-60 seconds, 72℃ extension for 30-60 seconds, and finally 72℃ final extension for 5-10 minutes. Finally, it also includes detection reagents for detecting amplified gene fragments. The detection reagents employ conventional detection techniques such as quantitative real-time PCR probe method or gel electrophoresis. If quantitative real-time PCR probe method is used, the probe specifically binds to the amplified target gene fragment, and the expression level of the gene is determined by detecting the fluorescence signal, thereby judging the high-yield performance of pigs. If gel electrophoresis method is used, the amplified products are electrophoresed in an agarose gel containing ethidium bromide staining agent, and the size and content of the gene fragment are analyzed based on the position and brightness of the bands to assess the status of high-yield-related genes in pigs.

[0009] Preferably, a nucleic acid protective agent with a concentration ranging from 0.01% to 0.1% is added to the DNA extraction reagent. This nucleic acid protective agent can bind to the extracted DNA molecules through intermolecular forces, inhibiting the degradation of DNA by nucleases during subsequent operations. Simultaneously, an internal reference gene amplification system is added to the amplification reagent. This system contains primer pairs designed for porcine internal reference genes such as the GAPDH gene, DNA polymerase, buffer solution, and dNTPs. The primer pairs in the internal reference gene amplification system are compatible with the primer pairs used for amplifying porcine high-yield-related gene fragments in terms of amplification reaction conditions, allowing for simultaneous amplification in the same PCR reaction. During detection, the DNA is analyzed by... The amplification product amounts of the internal reference gene and the high-yield-producing genes in pigs were compared to correct the detection results of the high-yield-producing genes. In addition, the kit includes preservation reagents for samples, extracted DNA, and amplification products. The sample preservation reagent uses a buffer solution containing a specific preservative, which can preserve collected samples for 1-3 days at 2℃-8℃, ensuring the integrity of the DNA in the samples. The DNA preservation reagent is a TE buffer containing EDTA, which can preserve extracted DNA for 1-3 months at -20℃. The amplification product preservation reagent is a low-melting-point agarose gel, which can preserve amplification products for 1-2 weeks at 4℃, preventing degradation or contamination of the amplification products.

[0010] The present invention has at least the following beneficial effects: First, the kit provided by this invention achieves efficient detection of high-yield genes in pigs through targeted design. Its core advantages are: First, the sampling device supports the collection of multiple sample types, including tissue, blood, and saliva, covering the needs of different testing scenarios, and the sample acquisition method is convenient, reducing stress on pigs; second, the concentration ratio of surfactant to salt solution in the DNA extraction reagent has been optimized (e.g., 1%-5% surfactant to 0.1M-0.5M salt solution), combined with incubation conditions of 50℃-60℃, effectively lysing cells and separating DNA, improving extraction efficiency and purity, and providing a high-quality template for subsequent detection; third, the amplification reagent uses primers designed for specific regions of the ESR gene, which is transcribed... Key candidate genes affecting pig reproductive performance identified through omics screening, combined with standardized PCR amplification procedures (94℃ pre-denaturation, 55℃-65℃ annealing, etc.), can specifically amplify target fragments and reduce interference from non-specific amplification. Finally, detection reagents employ either quantitative real-time PCR probe method or gel electrophoresis. The former achieves quantitative analysis through probe specific binding, while the latter visually reflects gene fragment characteristics through band position and brightness. Both methods are based on the technical system validated in the paper (such as the RT-qPCR technology used in the miR-183 functional verification in Chapter 5), ensuring the reliability and reproducibility of the detection results. This scheme, through its standardized end-to-end design, solves the problems of poor detection specificity and cumbersome operation in existing technologies, providing a precise tool for screening high-yielding traits in pigs.

[0011] Secondly, this invention improves the stability and accuracy of the detection system. Specifically: adding 0.01%-0.1% nucleic acid protectant to the DNA extraction reagent allows it to bind to DNA through intermolecular forces, inhibiting nuclease degradation and effectively solving the DNA degradation problem during extraction and handling, thus ensuring template quality; introducing an internal reference gene (such as GAPDH) amplification system into the amplification reagent utilizes its compatibility with the target gene amplification conditions, and by comparing synchronous amplification with product yield, it corrects errors caused by differences in sample size and amplification efficiency during detection, making the quantitative results more accurate; the optimization of the preservation reagent system (sample preservation solution at 2℃-8℃ for 1-3 days, TE buffer containing EDTA for DNA preservation at -20℃, etc.) is based on the requirements for sample stability in experiments, ensuring the integrity of samples, DNA, and amplification products at different stages, facilitating experimental planning and result verification. This solution systematically solves the problems of DNA degradation, result deviation, and sample preservation, improving the practicality of the detection system, especially suitable for large-scale pig breed screening and long-term monitoring scenarios.

[0012] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Detailed Implementation

[0013] The present invention will now be described in further detail so that those skilled in the art can implement it based on the description.

[0014] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0015] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.

[0016] <Example 1> In the field of high-yield gene detection in pigs, existing technologies often use general-purpose gene detection kits. Their main shortcomings are: primer design is not targeted at specific genes related to high-yield pigs (such as the ESR gene), resulting in poor detection specificity; DNA extraction reagents lack optimized surfactant and salt solution concentration ratios, easily causing DNA degradation under incubation conditions of 50℃-60℃; amplification programs do not incorporate gradient parameters tailored to pig gene characteristics, resulting in a large number of non-specific amplification products; and detection methods largely rely on single gel electrophoresis, failing to achieve quantitative analysis, and the entire process lacks a standardized operating system, making it difficult to promote and apply in large-scale farming.

[0017] To address the aforementioned issues, the kit of claim 1 is implemented as follows: Pig saliva samples are collected using sterile sampling swabs (or blood samples are obtained via lancets). The samples are placed in a DNA extraction reagent containing 1%-5% sodium dodecyl sulfate (surfactant) and 0.1M-0.5M sodium chloride (salt solution), and incubated in a 55°C water bath for 20 minutes. Genomic DNA is then separated using the phenol-chloroform extraction method. Specific primers designed for the third exon region of the ESR gene (upstream primer 5'-GCTGCTTCTGCTTCTGCTT-3', downstream primer 5'-CTGCTTCTGCTTCTGCTTCT-3') are added to the amplification reagent. After mixing with Taq DNA polymerase and dNTPs, amplification is performed in a PCR instrument according to the following program: pre-denaturation at 94°C for 4 minutes, followed by 35 cycles (denaturation at 94°C for 45 seconds, annealing at 58°C for 45 seconds, extension at 72°C for 45 seconds), and a final extension at 72°C for 8 minutes. The detection method used was quantitative real-time PCR with a probe sequence of 5'-FAM-CTGCTTCTGCTTCTGCTTCTG-TAMRA-3'. The expression level of ESR gene was determined by real-time monitoring of fluorescence signal intensity. Alternatively, 1.5% agarose gel electrophoresis was performed at 120V for 30 minutes, and the position and brightness of the bands were observed by ethidium bromide staining.

[0018] Compared with existing technologies, the improvement of this scheme is as follows: specific primers are designed for the ESR genes screened in the paper (such as the key reproductive performance genes verified in Chapter 5), and combined with optimized extraction reagent concentration and amplification parameters, the DNA extraction purity (OD260 / 280) is increased to 1.8-2.0, and the specificity of amplified products reaches more than 95%; real-time quantitative PCR detection can achieve absolute quantification of gene expression, which is more accurate than traditional gel electrophoresis qualitative analysis, and solves the technical problems of poor detection specificity and inaccurate quantification in existing technologies, providing a standardized tool for molecular marker-assisted selection of high-yield traits in pigs.

[0019] <Example 2> Existing technologies for detecting high-yield genes in pigs commonly suffer from problems such as easy degradation during DNA extraction, lack of calibration systems for test results, and poor stability of samples and products during preservation. Current kits often lack nucleic acid protection components in the DNA extraction reagents, leading to easy degradation of the extracted DNA by nucleases during the process. For example, DNA integrity often decreases by more than 30% after being left at room temperature for more than 30 minutes. The amplification system lacks a mechanism for simultaneous amplification of internal reference genes; when sample volume differences or amplification efficiency fluctuations exceed 15%, the test results can deviate by 20%-40%. Preservation reagents are mostly general-purpose buffers; after saliva samples are stored at 2℃-8℃ for more than 12 hours, the DNA degradation rate exceeds 50%, making it difficult to meet the needs of batch testing.

[0020] The specific implementation method for addressing the above problems is as follows: Add 0.05% sodium pyrophosphate as a nucleic acid protectant to the DNA extraction reagent. This protectant inhibits nuclease activity during extraction by forming a hydrogen bond network with DNA molecules and incubating at 55°C. Taking saliva samples as an example, the collected samples are mixed with an extraction reagent containing 0.05% sodium pyrophosphate (containing 3% sodium dodecyl sulfate and 0.3M sodium chloride). After incubation at 55°C for 20 minutes, the DNA extraction purity (OD260 / 280) can reach 1.8-1.9, which is about 20% higher than when no protectant is added. Add internal control primers designed for the porcine GAPDH gene (upstream primer 5'-GAAGGTGAAGGTCGGAGTC-3', downstream primer 5'-GAAGATGGTGATGGGATTTC-3') to the amplification reagent. This primer pair is compatible with the ESR gene primers at an annealing temperature of 58°C and can be amplified simultaneously in the same PCR reaction system. During detection, the results were corrected by the fluorescence signal ratio of the internal reference gene and the ESR gene. When the sample size fluctuated by 20%, the result deviation could be controlled within 5%. Regarding the preservation system, the sample preservation solution used was phosphate buffer containing 0.1% sodium azide. Saliva samples could be preserved for 2 days at 4℃ with a DNA integrity retention rate exceeding 85%. Extracted DNA was stored for 2 months at -20℃ using TE buffer containing 10mM EDTA. Amplification products were intercalated into 0.8% low-melting-point agarose gel and stored at 4℃ for 10 days, with a product degradation rate of less than 10%.

[0021] Compared with existing technologies, this solution improves DNA extraction integrity by 20%-30% by inhibiting DNA degradation with nucleic acid protectants, correcting detection biases with internal reference gene systems, and extending sample stability with graded preservation reagents. The repeatability error of the detection results is controlled within 5%, and the sample preservation time is extended by 1-2 times. It solves the problems of low detection accuracy and limited sample processing in existing technologies, and is especially suitable for batch testing and germplasm screening in large-scale pig farms.

[0022] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details.

Claims

1. A kit for detecting high-yield genes in pigs, characterized in that, It includes the following components and operating steps: First, the kit includes a sampling device for collecting pig samples, which can obtain tissue, blood, or saliva samples from the pig. Next, a DNA extraction reagent is provided for extracting DNA from the collected samples. The DNA extraction reagent is formulated to effectively lyse sample cells and separate DNA. It contains surfactants in a specific concentration range of 1%-5% and salt solutions in a concentration range of 0.1M-0.5M. DNA extraction is achieved through conventional extraction procedures, such as incubation at a temperature of 50℃-60℃ for 15-30 minutes. Then, an amplification reagent is provided for amplifying specific gene fragments associated with high-yield traits in pigs. This amplification reagent contains suitable primer pairs designed for high-yield-related genes in pigs, specifically specific regions of the ESR gene that affect pig reproductive performance, as well as DNA polymerase, buffer, and dNTPs for the amplification reaction. The amplification reaction is performed in a conventional PCR instrument, and the amplification program is set as follows: 94℃ pre-denaturation for 3-5 minutes, followed by 30-40 cycles, each cycle including 94℃ denaturation for 30-60 seconds, 55℃-65℃ annealing for 30-60 seconds, 72℃ extension for 30-60 seconds, and finally 72℃ final extension for 5-10 minutes. Finally, it also includes detection reagents for detecting amplified gene fragments. The detection reagents employ conventional detection techniques such as quantitative real-time PCR probe method or gel electrophoresis. If quantitative real-time PCR probe method is used, the probe specifically binds to the amplified target gene fragment, and the expression level of the gene is determined by detecting the fluorescence signal, thereby judging the high-yield performance of pigs. If gel electrophoresis method is used, the amplified products are electrophoresed in an agarose gel containing ethidium bromide staining agent, and the size and content of the gene fragment are analyzed based on the position and brightness of the bands to assess the status of high-yield-related genes in pigs.

2. The kit for detecting high-yield genes in pigs according to claim 1, characterized in that, The DNA extraction reagent contains a nucleic acid protective agent at a concentration ranging from 0.01% to 0.1%. This protective agent binds to the extracted DNA molecules through intermolecular forces, inhibiting DNA degradation by nucleases during subsequent operations. Simultaneously, an internal reference gene amplification system is added to the amplification reagent. This system contains primer pairs designed for porcine internal reference genes such as the GAPDH gene, DNA polymerase, buffer solution, and dNTPs. The primer pairs in the internal reference gene amplification system are compatible with those used for amplifying porcine high-yield-related gene fragments under the same amplification reaction conditions, allowing for simultaneous amplification in the same PCR reaction. During detection, the concentration of the internal reference gene amplification system is compared with that of the parenteral DNA. The kit measures the amplification product quantity of genes related to high pig productivity and corrects the detection results of genes related to high pig productivity. In addition, the kit includes preservation reagents for samples, extracted DNA, and amplification products. The sample preservation reagent uses a buffer solution containing specific preservatives, which can preserve collected samples for 1-3 days at 2℃-8℃, ensuring the integrity of the DNA in the sample. The DNA preservation reagent is a TE buffer containing EDTA, which can preserve extracted DNA for 1-3 months at -20℃. The amplification product preservation reagent is a low-melting-point agarose gel, which can preserve amplification products for 1-2 weeks at 4℃, preventing degradation or contamination of the amplification products.