Multiplex fluorescent ARMS-PCR detection method for genetic typing of genetic defect marker sites of beef cattle

Abstract

The invention belongs to the field of biological medicine, and particularly relates to a multiplex fluorescent ARMS-PCR detection method for genetic typing of genetic defect marker sites of beef cattle, the method comprises a step of using a primer group for multiplex fluorescent ARMS-PCR detection for detection, and target sites detected by the primer group comprise TP, BMS, BH2, FH4, PP, MA, PHA, HY, MH, CL-16, WCS, FH2, MFD, DW, MH1, SH8, ZDL, AS, SH9, CHS, F11, TH, IE, SH5, D2, AM, DD, HS and OS. Based on a single-tube multiple ARMS-PCR technology, a capillary electrophoresis detection mode is integrated, and a detection system capable of being used for beef cattle multi-site genotyping is established. According to the method, as many as 40 sites can be detected through a single tube, and compared with the prior art, the detection method is simple and rapid, low in cost, high in accuracy, capable of flexibly adjusting core sites, high in feasibility and easy to popularize, and a set of comprehensive, efficient and executable detection system is provided for beef cattle breeding performance improvement.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and in particular relates to a multiplex fluorescence ARMS-PCR detection method for genetic typing of genetic defect marker sites in beef cattle. Background Art

[0002] Genetic defects can lead to early embryonic death, calf malformations, reproductive defects, growth and development disorders or metabolic disorders.

[0003] Commonly used molecular detection methods for genetic defects include TaqMan probe method, fluorescent primer PCR product electrophoresis method, PCR-restriction fragment length polymorphism technology (PCR-restriction fragment length polymorphism, PCR-RFLP), competitive allele-specific PCR (Kompetitive allele-specific PCR, KASP), SNP chip detection and other methods. The classic PCR product electrophoresis method and PCR-RFLP technology can complete the detection using conventional instruments, with low cost, but cumbersome steps. TaqMan probe method and KASP are detection methods based on fluorescent signals. Both of them collect fluorescent signals during the PCR process to complete typing, which takes relatively short time and is mostly used for the detection of single-site SNPs, with low detection throughput.

[0004] Amplification-refractory mutation system PCR (ARMS-PCR) is a SNP detection method that introduces additional mismatched bases at the 3' end of the primer for identification. This technology has the advantages of high accuracy, strong specificity and low cost. Combined with agarose gel electrophoresis, ARMS PCR is used to identify mutations associated with genetic defects or economic traits. However, the multiplex detection capability of ARMS PCR is limited by the low resolution of agarose gel electrophoresis, which makes it unable to distinguish DNA fragments of very similar sizes. By integrating capillary electrophoresis, the resolution can be improved, and even single base differences can be detected. Compared with the first-generation sequencing gold standard, the multiplex fluorescence ARMS-PCR method identified wild-type and carrier genotypes with a detection rate of 100%, with extremely high detection accuracy. In the genotyping of specific genetic defects, mf-ARMS PCR is more accurate than KASP, and is an efficient molecular inspection method.

[0005] The prior art CN108410993A discloses a four-primer amplification hindered mutation system PCR (T-ARMS-PCR) detection method for the A316G mutation of the CAPN1 gene in Wagyu three-way hybrid cattle. After PCR amplification with specially designed primers and then identification by agarose gel electrophoresis, the single nucleotide polymorphism of the site can be simply, quickly, low-cost and accurately detected, thereby facilitating the early screening of high-quality hybrid cattle and helping to improve the accuracy of selecting Wagyu three-way hybrid cattle with excellent meat quality traits.

[0006] Prior art CN118389667A discloses an ARMS-PCR primer combination and a kit for detecting nine genetic defect genes and three hornless genes in cattle. The kit of the invention can rapidly, at low cost and with high sensitivity simultaneously detect the genetic defect sites corresponding to the nine genetic defect genes DW, AS, MFD, TP, ZDL, BMS, BH2, FH2, and FH4 in cattle and the mutation sites corresponding to the three hornless genes P202ID, P219ID, and P80K, and can be widely used in the field of cattle breeding and reproduction.

[0007] Prior art CN117210551A discloses an ARMS-PCR primer combination and a kit for detecting 10 genetic defect genes in cattle. The kit of the invention can rapidly, cheaply and highly sensitively simultaneously detect the genetic defect sites corresponding to the 10 genetic defect genes HH1, HH2, HH3, HH4, HH5, HH6, HCD, BLAD, CVM and BS in cattle, and can be widely used in the field of cattle breeding and reproduction.

[0008] However, the sites detected by the above-mentioned prior art are insufficient, and therefore, multiplex ARMS-PCR capable of amplifying more sites continues to be developed. Summary of the invention

[0009] In view of the shortcomings of the prior art, according to the first aspect of the technical solution of the present invention, the present invention provides a primer set for multiplex fluorescence ARMS-PCR detection of genetic defect marker site genotyping in beef cattle, wherein the target sites detected by the primer set include one or more of TP, BMS, BH2, FH4, PP, MA, PHA, HY, MH, CL-16, WCS, FH2, MFD, DW, MH1, SH8, ZDL, AS, SH9, CHS, F11, TH, IE, SH5, D2, AM, DD, HS, and OS.

[0010] In certain embodiments, the target sites detected by the primer set include TP, BMS, BH2, FH4, PP, MA, PHA, HY, MH, CL-16, WCS, FH2, MFD, DW, MH1, SH8, ZDL, AS, SH9, CHS, F11, TH, IE, SH5, D2, AM, DD, HS and OS.

[0011] In certain embodiments, the primer sequence is one or more of SEQ ID Nos: 1-80.

[0012] In certain embodiments, the primer sequences are shown in SEQ ID No: 1-80.

[0013] The present invention also provides the use of the primer set as described above in preparing a product for multiplex fluorescence ARMS-PCR detection of genetic typing of beef cattle genetic defect marker sites.

[0014] In certain embodiments, the article of manufacture comprises a kit.

[0015] The present invention also provides a kit for multiplex fluorescence ARMS-PCR detection of genetic typing of beef cattle genetic defect marker sites, and the kit comprises the above primer set.

[0016] In certain embodiments, the kit further comprises an amplification reagent and a DNA extraction reagent.

[0017] The present invention also provides the use of the primer set or the kit in multiplex fluorescence ARMS-PCR detection for genetic typing of beef cattle genetic defect marker sites.

[0018] Finally, the present invention provides a multiplex fluorescence ARMS-PCR detection method for genetic typing of genetic defect marker sites in beef cattle, the method comprising the step of using the above primer set or the kit described in any one of claims 7-8 for detection.

[0019] Compared with the prior art, the present invention has at least the following beneficial effects:

[0020] The method disclosed in the present invention is based on single-tube multiplex ARMS-PCR technology, integrating capillary electrophoresis detection, and establishing a detection system that can be used for multi-site genotyping of beef cattle. This method can detect up to 40 sites in a single tube. Compared with the existing technology, the detection method is simple, fast, low-cost, and highly accurate. In addition, the detection sites can be flexibly adjusted according to the core sites of the latest research progress in the pasture or the country, which is highly feasible and easy to promote, and helps to provide a comprehensive, efficient and executable detection system for improving the breeding performance of beef cattle. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the peak graph and typing result graph of the wild-type sample;

[0022] Figure 2 This is the peak diagram of the homozygous mutant sample and the typing result diagram;

[0023] Figure 3 This is the peak graph and typing result graph of heterozygous mutant samples;

[0024] Figure 4 This is the peak diagram of the genotyping results of sample 200506;

[0025] Figure 5 This is the peak graph of the genotyping results for sample 200507. DETAILED DESCRIPTION

[0026] In order to make the technical problems, technical solutions and advantages to be solved by the present invention more clear, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0027] Example 1

[0028] (1) Selection of genetic defect marker loci

[0029] Based on domestic and foreign references, genetic defects that may have a high carrying rate in domestic beef cattle breeds and homozygous mutants can lead to early embryonic death, calf malformation, reproductive defects, growth and development disorders or metabolic disorders are screened out. The present invention screened out a total of 29 genetic defect marker sites for beef cattle, which are used to construct a multiplex fluorescence ARMS-PCR detection method.

[0030] (2) Primer design

[0031] Primer5 software was used to design the primers, and the specificity of the primers was compared using the NCBI website. The primer fragment size was designed to be within the range of 90-450 bp, and the Tm value was controlled at (60±2)℃.

[0032] When designing primers, ARMS primers for wild-type templates, the 3' end of the primers terminates at the site to be tested and matches the wild-type sequence, wherein a mismatch base is set at the 2nd to 4th position of the 3' end of the primers; ARMS primers for mutant templates, the 3' end of the primers terminates at the site to be tested and matches the mutant sequence, wherein a mismatch base is set at the 2nd to 4th position of the 3' end of the primers; share an upstream or downstream fluorescent primer. The product fragments generated by each pair of primers are of different sizes.

[0033] Select 29 pairs of primers for single amplification of bovine genomic DNA, and use capillary electrophoresis to check the amplification efficiency of each pair of primers and whether there are non-specific fragments. If the efficiency is low or there are non-specific fragments, it is necessary to replace the primers or optimize the PCR amplification conditions until the efficiency of the 29 pairs of primers is basically the same and no non-specific fragments are generated.

[0034] (3) Primer verification

[0035] 29 pairs of primers were selected for single amplification of bovine genomic DNA, and capillary electrophoresis was used to check the amplification efficiency of each pair of primers, whether there were non-specific fragments, and whether the genotypes could be effectively distinguished. If the efficiency was low or there were non-specific fragments, the primers needed to be replaced until the efficiency of the 29 pairs of primers was basically the same and no non-specific fragments were produced.

[0036] Primer pair composition: TP, BMS, BH2, FH4, PP, MA, PHA, HY, MH, CL-16, WCS, FH2, MFD, DW, MH1, SH8, ZDL, AS, SH9, CHS, F11, TH, IE, SH5, D2, AM, DD, HS, OS.

[0037] (4) Multiple system testing

[0038] The primer pairs of 29 genetic marker sites are divided into four groups, see Table 1. The fluorescent dye markers used by the amplification primers of each site in each group are different from each other. The fluorescent dye marker used by the amplification primers of the first group of genetic defect marker sites is FAM; the fluorescent dye marker used by the amplification primers of the second group of genetic defect marker sites is HEX; the fluorescent dye marker used by the amplification primers of the third group of genetic defect marker sites is TAMRA; the fluorescent dye marker used by the amplification primers of the fourth group of genetic defect marker sites is ROX. Through different fluorescence and PCR amplification product lengths, genotyping of multiple marker sites is achieved.

[0039] Table 1: Group details

[0040] Group Genetic defect marker loci 1 group BMS, TP, BH2, FH4, PP, MA, PHA, HY, MH, CL-16, WCS, FH2 2 groups MFD, DW, MH1, SH8, ZDL, AS, SH9, CHS, F11, TH 3 groups IE, SH5, D2 4 groups AM, DD, HS, OS

[0041] Each primer is mixed in equal proportions, and the mixed primer is used to amplify bovine genomic DNA. The amplification efficiency of each primer and whether non-specific fragments are generated are analyzed by capillary electrophoresis. If non-specific fragments are generated, the primers that produce non-specific fragments are checked and the primers are redesigned. If there is a large difference in amplification efficiency at different sites, the concentration of the primers is adjusted. The ultimate goal: to make the amplification efficiency of each pair of primers basically consistent, without non-specific fragments.

[0042] After optimization, the final concentrations of the primers in each group of the primers in the primer composition in the PCR composite amplification system are different, the final concentrations of the amplification primers for the 12 sites in the first group are 0.125 μmol / L, the final concentrations of the amplification primers for the 10 sites in the second group are 0.125 μmol / L, the final concentrations of the amplification primers for the 3 sites in the third group are 0.25 μmol / L, and the final concentrations of the amplification primers for the 4 sites in the fourth group are 0.25 μmol / L.

[0043] Example

[0044] The multiplex fluorescence ARMS-PCR method for genetic defect marker loci genotyping in beef cattle was used to detect and type 29 genetic defects in beef cattle.

[0045] DNA extraction: Genomic DNA from beef cattle blood samples was extracted using the DP329 magnetic bead blood genome extraction kit from Tiangen Biochemical Biotechnology Co., Ltd. The DNA concentration and quality were tested using the NanoDrop 2000 nucleic acid quality detector and 1% agarose gel electrophoresis.

[0046] PCR amplification: PCR amplification was performed using the above-screened primer combination and the KT109 multiplex PCR amplification kit of Tiangen Biochemical Biotechnology Co., Ltd., with the extracted bovine genomic DNA as a template.

[0047] Capillary electrophoresis detection: Take 1 μL of amplified product, add 8.8 μL HIDI (highly deionized formamide, ABI) and 0.2 μL LIZ500 molecular weight internal standard (ABI), centrifuge instantly, denature at 95°C for 5 minutes, immediately put in an ice bath for 5 minutes, and then put into the ABI3730 sequencer for detection.

[0048] Data analysis: After the test is completed, Genemapper6.0 analysis software (Applied Biosystems) is used for data analysis. The name of each detection site (panel) and the fragment size range of each site (bin) are set according to the product fragment size, and the typing of each site is determined according to the peak position and fluorescence color.

[0049] Table 2: Primers for amplifying genetic defect marker sites

[0050]

[0051]

[0052]

[0053]

[0054]

[0055]

[0056] Table 3: Final concentration of primers in the composite amplification system

[0057]

[0058]

[0059]

[0060]

[0061]

[0062] Table 4 PCR amplification operation program

[0063]

[0064] Results Figure 1-Figure 5 The present invention can realize multiple identification of multiple sites. The detection method of the present invention is simple, fast, low-cost, and highly accurate. The detection sites can be flexibly adjusted according to the core sites of the latest research progress in the pasture or in China. It is highly feasible and easy to promote, and helps to provide a comprehensive, efficient and executable detection system for improving the breeding performance of beef cattle.

[0065] The above is 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 the scope of protection of the present invention.

Claims

1. A primer set for multiplex fluorescence ARMS-PCR detection of genetic defect marker loci for beef cattle, characterized in that: The target sites detected by the primer set include one or more of TP, BMS, BH2, FH4, PP, MA, PHA, HY, MH, CL-16, WCS, FH2, MFD, DW, MH1, SH8, ZDL, AS, SH9, CHS, F11, TH, IE, SH5, D2, AM, DD, HS, and OS.

2. The primer set according to claim 1, characterized in that The target sites detected by the primer set include TP, BMS, BH2, FH4, PP, MA, PHA, HY, MH, CL-16, WCS, FH2, MFD, DW, MH1, SH8, ZDL, AS, SH9, CHS, F11, TH, IE, SH5, D2, AM, DD, HS and OS.

3. The primer set according to claim 1, characterized in that The primer sequence is one or more of those shown in SEQ ID No: 1-80.

4. The primer set according to claim 3, characterized in that: The primer sequences are shown in SEQ ID No: 1-80.

5. Use of the primer set according to any one of claims 1 to 4 in the preparation of a product for multiplex fluorescence ARMS-PCR detection of genotyping of genetic defect marker sites in beef cattle.

6. The use according to claim 5, characterized in that: The product comprises a kit.

7. A kit for multiplex fluorescence ARMS-PCR detection of genetic defect marker loci for beef cattle, characterized in that: The kit comprises the primer set according to any one of claims 1 to 4.

8. The kit according to claim 7, characterized in that The kit also comprises an amplification reagent and a DNA extraction reagent.

9. Use of the primer set according to any one of claims 1 to 4 or the kit according to any one of claims 7 to 8 in multiplex fluorescence ARMS-PCR detection for genotyping of genetic defect marker sites in beef cattle.

10. A multiplex fluorescence ARMS-PCR detection method for genetic typing of genetic defect marker loci in beef cattle, characterized in that: The method comprises the step of using the primer set according to any one of claims 1 to 4 or the kit according to any one of claims 7 to 8 to perform detection.

Images