ARMS-PCR primer group for breeding disease-resistant cattle based on gene polymorphism and application of ARMS-PCR primer group
By designing the ARMS-PCR primer group combined with ordinary multiplex PCR and electrophoretic detection technology, the problem of long and high cost of insertion/deletion polymorphism detection in PRNP intronic region is solved, and efficient and low-cost polymorphism site identification is achieved, supporting cattle gene breeding and animal product risk assessment.
Patent Information
- Application Number
- CN202510704730.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-29
- Publication Date
- 2025-08-15
AI Technical Summary
The existing PRNP intronic region 12bp insertion/deletion polymorphism detection methods are time-consuming, costly or insufficient resolution, making it difficult to meet the immediate detection needs of grassroots laboratories.
A ARMS-PCR primer set is designed, including upstream external primer 12-PF3, upstream internal primer 12-IF2, downstream internal primer 12-Rin and downstream external primer 12-LR1, combined with ordinary multiplex PCR and electrophoretic detection technology, to specifically detect 12bp insertion/deletion polymorphic sites in the intron region of bovine prion protein gene.
It simplifies the detection process, reduces costs, and improves detection efficiency. It is suitable for most molecular testing laboratory equipment, facilitates testing, and provides technical support for assisted breeding of cattle genes and early warning of livestock products.
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Figure CN120485390A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of gene-assisted breeding, and in particular to an ARMS-PCR primer set for breeding disease-resistant cattle based on gene polymorphism and an application thereof. Background Art
[0002] Bovine transmissible spongiform encephalopathy (BSE), commonly known as "mad cow disease," is a fatal neurodegenerative disease caused by structural mutations in the prion protein (PrP). First identified in the United Kingdom in 1986, the disease rapidly spread to numerous countries worldwide, devastating the livestock industry and triggering a global public health crisis. With the implementation of global prevention and control measures and the ban on the use of cattle and sheep-derived ingredients in animal feed, the global incidence of BSE has declined significantly. However, the scientific mechanisms and control of the disease, including its onset and transmission, remain challenging. Pathogenic prions are highly resistant to conventional disinfection methods and high temperatures. They can also be found in a variety of infectious sources, including processed dairy products and meat from infected cattle, and even prions excreted in soil, water, and other environmental sources of BSE, making their elimination extremely challenging. The bovine prion protein gene (PRNP), a core element regulating prion protein expression, exhibits polymorphisms that influence susceptibility and inheritance of BSE. Since the full sequencing of bovine PRNP in 2001, researchers have systematically explored its genetic polymorphisms. Through testing and genetic analysis of multiple cattle breeds, a 12bp insertion / deletion polymorphism within the intron region was found to be significantly associated with BSE resistance. Research is currently underway to improve resistance through PRNP gene editing. The 12bp insertion / deletion polymorphism site is a binding site for the transcriptional repressor SP1. The 12bp deletion eliminates the SP1 binding site from the genome, leading to upregulated prion protein expression in individuals carrying the deletion, shortening the incubation period after infection and increasing susceptibility. Individuals homozygous for the deletion are even more susceptible. Therefore, PRNP genotype testing can be used to assess individual susceptibility to BSE, provide molecular markers for disease-resistant breeding, guide the screening of high-risk groups and formulate prevention and control strategies, which is of great significance to ensuring the safety of animal husbandry and public health.
[0003] Currently, detection of the 12bp insertion / deletion polymorphism in the PRNP intron region primarily relies on Sanger sequencing, as well as PCR-RFLP, real-time fluorescence quantitative PCR (qPCR), high-throughput sequencing (NGS), and gene chip methods. However, these methods all have limitations, such as time-consuming, high-cost, or insufficient resolution. For example, Sanger sequencing can accurately interpret sequence information, but the detection is costly and time-consuming. PCR-RFLP relies on the site-specificity of restriction enzymes, but incomplete enzyme digestion can affect experimental accuracy. NGS enables multi-target parallel analysis, but the equipment investment and data analysis complexity make it difficult to meet the needs of point-of-care testing in primary care laboratories. Therefore, there is an urgent need to develop a low-cost and universally applicable DNA detection method for the detection of the 12bp insertion / deletion polymorphism in the bovine PRNP intron region. Summary of the Invention
[0004] The purpose of the present invention is to provide an ARMS-PCR primer set for breeding disease-resistant cattle based on gene polymorphism. The primer set of the present invention can specifically detect the 12bp insertion / deletion polymorphic site in the intron region of the bovine prion protein gene, providing a scientific basis for the genotype identification of cattle and their products and the assessment of susceptibility to mad cow disease.
[0005] The present invention provides an ARMS-PCR primer set for breeding disease-resistant cattle based on gene polymorphism, wherein the primer set comprises an upstream outer primer 12-PF3, an upstream inner primer 12-IF2, a downstream inner primer 12-Rin and a downstream outer primer 12-LR1;
[0006] The nucleotide sequence of the upstream external primer 12-PF3 is shown in SEQ ID NO: 1;
[0007] The nucleotide sequence of the upstream inner primer 12-IF2 is shown in SEQ ID NO: 2;
[0008] The nucleotide sequence of the downstream inner primer 12-Rin is shown in SEQ ID NO: 3;
[0009] The nucleotide sequence of the downstream outer primer 12-LR1 is shown in SEQ ID NO: 4;
[0010] The disease-resistant cattle are resistant to mad cow disease.
[0011] Preferably, the target gene of the primer set is a 12 bp insertion / deletion polymorphism site in the intron region of the bovine prion protein gene PRNP.
[0012] The present invention also provides a kit containing the ARMS-PCR primer set, wherein the kit comprises the primer set and a detection reagent.
[0013] Preferably, the detection reagent includes Taq enzyme, PCR buffer, dNTP mixture, MgCl2 and ddH2O.
[0014] Preferably, the concentrations of the primers in the kit are: 0.2-0.8 μmol / L for the upstream outer primer 12-PF3, 0.4-1.0 μmol / L for the upstream inner primer 12-IF2, 0.3-0.6 μmol / L for the downstream inner primer 12-Rin, and 0.6-1.2 μmol / L for the downstream outer primer 12-LR1.
[0015] The present invention also provides the use of the ARMS-PCR primer set or the kit in preparing a product for detecting bovine-derived ingredients, comprising the following steps:
[0016] 1) Extracting genomic DNA from the cattle to be tested or the product to be tested;
[0017] 2) using the genomic DNA extracted in step 1) as a template, performing PCR amplification using the ARMS-PCR primer set to obtain an amplified product;
[0018] 3) performing electrophoresis detection on the amplified product;
[0019] 4) When the amplified product has only two electrophoretic bands of 598 bp and 153 bp, it is determined to be a homozygous deletion type (- / -); when the amplified product has only two electrophoretic bands of 610 bp and 472 bp, it is determined to be a homozygous insertion type (+ / +); when the amplified product has only three electrophoretic bands of 610 bp, 472 bp and 153 bp, it is determined to be an insertion-deletion heterozygous type (+ / -).
[0020] Preferably, the products to be tested in step 1) include blood, hair, embryonic cells, beef, milk, milk powder and cheese.
[0021] Preferably, the standard for judging mad cow disease resistance is: the insertion homozygous type (+ / +) has higher disease resistance than the insertion-deletion heterozygous type (+ / -), and the insertion-deletion heterozygous type (+ / -) has higher disease resistance than the deletion homozygous type (- / -).
[0022] The ARMS-PCR primer set and application for breeding disease-resistant cattle based on gene polymorphism provided by the present invention simplify the detection process of the 12bp insertion / deletion polymorphic site in the intron region of cattle PRNP, reduce detection costs, and solve the current technical problem of the cumbersome identification process of this polymorphic site. It is an important supplement to the current detection technology of the 12bp insertion / deletion polymorphic site in the intron region of cattle PRNP. The technology used in the present invention is based on a common multiplex PCR and electrophoresis detection technology platform, and almost all molecular detection laboratories meet the required equipment requirements, facilitating the development of detection applications. The present invention can effectively identify all samples that may come from cattle and cattle products, with a simple method, high efficiency, and low cost. It provides strong technical support for cattle gene-assisted breeding and livestock product risk warning, which is conducive to the healthy development of animal husbandry and the safety assessment and risk warning of imported cattle products. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 The genotype sequencing results of different insertion / deletion polymorphism sites at the 12bp site of the PRNP gene of cattle in Example 1;
[0024] Figure 2 Schematic diagram of the primer design principle in Example 2;
[0025] Figure 3 The results of genotyping at different insertion / deletion polymorphisms at the 12 bp site of the PRNP gene in cattle using the primer set of the present invention in Example 3 are shown, wherein lane M is DNA Marker C (100-1200 bp), lane 1 is a negative control, lanes 2-3 are homozygous insertions, lanes 4-5 are homozygous deletions, and lanes 6-7 are heterozygous insertion-deletion types.
[0026] Figure 4 This is the result of identifying commercially available cattle products using the method of the present invention in Example 4, wherein lane M is DNA Marker C (100-1200 bp), lanes 1, 4, 9, 15, 17, and 18 are insertion-deletion heterozygous types, lanes 2, 5, 6, 8, 10, 12, 14, 16, 19, 21, 23, and 24 are deletion homozygous types, and lanes 3, 7, 11, 13, 20, and 22 are insertion homozygous types. DETAILED DESCRIPTION
[0027] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.
[0028] Example 1 Sequencing confirmation of 12 bp insertion / deletion polymorphism in the intron region of the bovine PRNP gene
[0029] This example provides specific sites and genotypes in the intron region of the bovine PRNP gene, and the specific steps are as follows:
[0030] (1) Six blood samples of cattle were collected (with complete farm pedigree records and confirmed by Sanger sequencing) and genomic DNA was extracted according to the instructions of the animal tissue kit (purchased from Hangzhou Xinjing Biotechnology Co., Ltd.).
[0031] (2) Using the genomic DNA extracted in step (1) as a template, PCR amplification was performed using PRNP gene identification primers to obtain an amplified product. PRNP gene identification was performed according to the literature (Imran M, Mahmood S, Babar ME, et al. PRNP gene variation in Pakistani cattle and buffaloes [J]. Gene, 2012, 505(1): 180-185.). The primers are as follows:
[0032] Upstream primer 12indelF (SEQ ID NO. 5): 5′-GTGCTCGTTGGTTTTTGC-3′;
[0033] Downstream primer 12indelR (SEQ ID NO. 6): 5′-TCCTACACACCACCCACATA-3′.
[0034] (3) The amplified product obtained in step (2) is subjected to agarose gel electrophoresis detection, wherein the initial concentration of agarose during the agarose gel electrophoresis detection is 1.5 wt %, and the agarose gel also contains 0.1% by weight of the nucleic acid dye Gelred.
[0035] (4) When the amplified product has only one electrophoretic band of 119bp, it is determined to be a homozygous individual with a 12bp deletion in the PRNP intron region; when the amplified product has only two electrophoretic bands of 119bp and 131bp, it is determined to be a heterozygous individual with a 12bp insertion-deletion in the PRNP intron region. When the amplified product has only one electrophoretic band of 131bp, it is determined to be a homozygous individual with a 12bp insertion in the PRNP intron region. Based on the above identification, the amplified cattle PRNP intron 12bp insertion / deletion polymorphism sequence product was subjected to Sanger sequencing, and the PRNP sequence obtained by cattle sequencing was aligned using MegAlign software. It was found that there was a 12bp insertion / deletion polymorphism in the intron region. This site appeared heterozygous in the sample, with deletion homozygous type (- / -), insertion-deletion heterozygous type (+ / -) and insertion homozygous type (+ / +). The results are as follows: Figure 1 shown.
[0036] Example 2 Design of ARMS-PCR Primers for the 12 bp Insertion / Deletion Polymorphism in the Intronic Region of Bovine PRNP
[0037] An ARMS-PCR detection method is established based on the 12bp insertion / deletion polymorphism in the intron region of the PRNP sequence. In order to introduce a positive control in the ARMS-PCR reaction, the present invention uses a pair of outer primer amplification products as positive control primers, and the two upstream and downstream inner primers respectively form a detection specific primer set with the downstream and upstream outer primer sequences. When designing the inner primers, the 3' ends of the upstream and downstream inner primers fall on the 12bp insertion / deletion polymorphism site in the intron region, and are paired with two different genotypes respectively. After amplification in combination with the outer primers, fragments of different sizes are generated for identifying the insertion / deletion polymorphism. In order to improve the specificity of the primers, the bases of the 12bp insertion-type detection inner primers are designed to be complementary to the insertion sequence, and the primer sequences are shown in Table 1. The schematic diagram of the primer design principle is shown in FIG. Figure 2 As shown:
[0038] Table 1 Primer sequences
[0039]
[0040] Note: Lowercase letters represent bases that are completely complementary to the inserted sequence.
[0041] Example 3 Optimization of ARMS-PCR detection system
[0042] (1) The genomic DNA of Example 1 was used as a PCR template.
[0043] (2) Using the primer set in Table 1, perform PCR amplification on the DNA from step (1) to obtain an amplified product.
[0044] (3) Amplification was performed using a basic PCR system, and the PCR annealing temperature and number of cycles were optimized. The basic procedure was: pre-denaturation at 95°C for 5 min; denaturation at 94°C for 35 s; annealing (at 60°C, 63°C, 66°C, 69°C, and 72°C) for 40 s; extension at 72°C for 1 min, for 30, 32, 35, and 38 cycles, and a final extension at 72°C for 10 min. Preferably, the annealing temperature was 69°C and the number of cycles was 35.
[0045] (4) Under the preferred conditions of step (3), Mg 2+ The concentration (1.5, 2.0, 2.5 mmol / L) and Taq enzyme (5 U / μL, ThermoFisher) dosage (0.2, 0.3, 0.4, 0.5 and 0.6 μL) were optimized. 2+ The concentration is 2.0mmol / L, and the amount of Taq enzyme is 0.4μL
[0046] (5) Under the conditions of (3) and (4), the concentrations of the inner and outer primers (outer primer to inner primer ratio of 3:1, 2:1, 1:1, 1:2, 1:3) were optimized and fine-tuned. All optimization results were based on the specificity of the 1.5 w% agarose gel electrophoresis test results and the amount of PCR product amplification.
[0047] The preferred PCR amplification reaction system, based on 20 μL, includes the following components: 2.0 μL of 10× buffer, 1.6 μL of 25 mmol / L dNTP, 1.6 μL of 25 mmol / L MgCl2, 1.0 μL of 10 μmol / L upstream outer primer 12-PF3, 1.6 μL of 10 μmol / L upstream inner primer 12-IF2, 0.8 μL of 10 μmol / L downstream inner primer 12-Rin, 11.6 μL of 10 μmol / L downstream outer primer 12-LR, 0.4 μL of 5 U / μL Taq enzyme, 3.0 μL of template DNA, and 6.4 μL of ddH2O.
[0048] The PCR amplification program was as follows: pre-denaturation at 95°C for 5 min; denaturation at 94°C for 35 s, annealing at 69°C for 40 s, extension at 72°C for 1 min, 35 cycles of denaturation, annealing, and extension; and final extension at 72°C for 10 min.
[0049] (6) The amplified product obtained in step (5) is subjected to agarose gel electrophoresis detection, wherein the initial concentration of agarose during the agarose gel electrophoresis detection is 1.5 wt %, and the agarose gel also contains 0.1% by weight of the nucleic acid dye Gelred.
[0050] (7) When the amplified product has only two electrophoretic bands of 598 bp and 153 bp, it is determined to be a deletion homozygous type (- / -); when the amplified product has only two electrophoretic bands of 610 bp and 472 bp, it is determined to be an insertion homozygous type (+ / +); when the amplified product has only three electrophoretic bands of 610 bp, 472 bp and 153 bp, it is determined to be an insertion-deletion heterozygous type (+ / -). The results of genotyping of the 12 bp insertion / deletion polymorphism site in the intron region of the PRNP gene of cattle using the above primer set are shown in Figure 3 .
[0051] Example 4 Detection of commercially available samples using ARMS-PCR
[0052] The ARMS-PCR primer set and kit for breeding disease-resistant cattle based on gene polymorphism of the present invention and their application method were used to detect and identify 24 commercially available cattle products. The accuracy of the results was verified by combining Sanger sequencing identification. Sanger sequencing identification was performed with reference to the literature (Imran M, Mahmood S, Babar ME, et al. PRNP gene variation in Pakistani attle and buffaloes [J]. Gene, 2012, 505(1): 180-185.).
[0053] The main steps of the ARMS-PCR method established using the present invention are as follows:
[0054] (1) Use an animal tissue DNA extraction kit (purchased from Hangzhou Xinjing Biotechnology Co., Ltd.) to extract 24 samples of genomic DNA from the cattle products to be tested.
[0055] (2) Using the genomic DNA extracted in step (1) as a template, PCR amplification was performed using the primer set in Table 1 to obtain an amplified product.
[0056] (3) The reaction system and PCR procedure for PCR amplification in step (2) use the PCR system and procedure preferred in Example 3.
[0057] (4) The amplified product obtained in step (2) was subjected to agarose gel electrophoresis. The initial concentration of agarose during the agarose gel electrophoresis was 1.5 wt %. The agarose gel also contained 1 / 10,000 of the nucleic acid dye Gelred. The ARMS-PCR electrophoresis results are shown in FIG. Figure 4 shown.
[0058] (5) According to the present invention, when the amplified product has only two electrophoretic bands, 598 bp and 153 bp, it is determined to be homozygous for deletion (- / -); when the amplified product has only two electrophoretic bands, 610 bp and 472 bp, it is determined to be homozygous for insertion (+ / +); when the amplified product has only three electrophoretic bands, 610 bp, 472 bp, and 153 bp, it is determined to be heterozygous for insertion-deletion (+ / -). The identification results of the 24 samples are shown in Table 2.
[0059] The results show that the present invention can conveniently detect the 12bp polymorphism of PRNP in cattle products and identify genotypes. The detection process has the advantages of simple operation and compatibility with most testing laboratory instrument platforms. It is an important supplement to the screening and prevention and control detection technology for high-risk populations for BSE, providing certain technical support for the identification of the 12bp polymorphism of PRNP in cattle, which is of great significance. It also provides more options and technical support for the identification of the 12bp polymorphic site of PRNP in cattle and cattle products.
[0060] Table 2 Identification results
[0061]
[0062]
[0063] Note: (- / -) indicates a homozygous deletion genome, (+ / -) indicates an insertion-deletion heterozygous genome, and (+ / +) indicates an insertion homozygous genome.
[0064] As can be seen from the above embodiments and experimental examples, the present invention provides an ARMS-PCR primer set and kit and its application method for breeding disease-resistant cattle and risk assessment of cattle products based on genetic polymorphism. The primer set and identification method of the present invention can specifically detect the 12bp insertion / deletion polymorphism in the PRNP intron region of cattle products, providing a scientific basis for genotype identification of cattle products and susceptibility assessment of mad cow disease.
[0065] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.
Claims
1. An ARMS-PCR primer set for breeding disease-resistant cattle based on gene polymorphism, characterized in that: The primer set includes an upstream outer primer 12-PF3, an upstream inner primer 12-IF2, a downstream inner primer 12-Rin and a downstream outer primer 12-LR1; The nucleotide sequence of the upstream external primer 12-PF3 is shown in SEQ ID NO.1; The nucleotide sequence of the upstream inner primer 12-IF2 is shown in SEQ ID NO.2; The nucleotide sequence of the downstream inner primer 12-Rin is shown in SEQ ID NO.3; The nucleotide sequence of the downstream outer primer 12-LR1 is shown in SEQ ID NO.4; The disease-resistant cattle are resistant to mad cow disease.
2. The ARMS-PCR primer set according to claim 1, wherein The target gene of the primer set is a 12 bp insertion / deletion polymorphism site in the intron region of the bovine prion protein gene PRNP.
3. A kit containing the ARMS-PCR primer set according to claim 1 or 2, characterized in that: The kit includes the primer set and a detection reagent.
4. The kit according to claim 3, wherein The detection reagent includes Taq enzyme, PCR buffer, dNTP mixture, MgCl2 and ddH2O.
5. The kit according to claim 3, wherein The concentrations of the primers in the kit are as follows: upstream outer primer 12-PF3 is 0.2-0.8 μmol / L, upstream inner primer 12-IF2 is 0.4-1.0 μmol / L, downstream inner primer 12-Rin is 0.3-0.6 μmol / L, and downstream outer primer 12-LR1 is 0.6-1.2 μmol / L.
6. Use of the ARMS-PCR primer set according to claim 1 or 2 or the kit according to any one of claims 3 to 5 in preparing a product for detecting bovine-derived ingredients, characterized in that: The following steps are involved: 1) Extracting genomic DNA from the cattle to be tested or the product to be tested; 2) using the genomic DNA extracted in step 1) as a template, performing PCR amplification using the ARMS-PCR primer set to obtain an amplified product; 3) performing electrophoresis detection on the amplified product; 4) When the amplified product has only two electrophoretic bands of 598 bp and 153 bp, it is determined to be a homozygous deletion type (- / -); when the amplified product has only two electrophoretic bands of 610 bp and 472 bp, it is determined to be a homozygous insertion type (+ / +); when the amplified product has only three electrophoretic bands of 610 bp, 472 bp and 153 bp, it is determined to be an insertion-deletion heterozygous type (+ / -).
7. The use according to claim 6, characterized in that The products to be tested in step 1) include blood, hair, embryonic cells, beef, milk, milk powder and cheese.
8. The use according to claim 6, characterized in that The criteria for judging mad cow disease resistance are: the insertion homozygous type (+ / +) has higher resistance than the insertion-deletion heterozygous type (+ / -), and the insertion-deletion heterozygous type (+ / -) has higher resistance than the deletion homozygous type (- / -).