Kit for evaluating human coordination agility using SNP rs117047321 as a detection target

Abstract

The present invention discloses a kit for evaluating human coordination agility using SNP rs117047321 as a detection target. The present invention provides the use of a substance for detecting a subject's genotype based on SNP rs117047321 in the preparation of a product; the function of the product is to evaluate the coordination agility of the subject. The agility coordination of CC genotype subjects is higher than that of CG genotype subjects. The present invention can be used to further understand the genetic mechanism of coordination agility, which is helpful for training and selecting excellent athletes. It can also be combined with the achievements of precision sports medicine in clinical practice to integrate genetics and genomics into actual applications such as athlete training and rehabilitation.

Description

Technical Field

[0001] The invention belongs to the field of gene detection, and relates to a kit for evaluating human coordination agility, which utilizes SNP rs117047321 as a detection target. Background Art

[0002] Personal agility and coordination are of great significance in sports competitions and talent selection. Almost every game requires a certain level of coordination and agility, which is affected by many factors such as genetics, age, training and environment.

[0003] In recent years, a series of studies on human sports performance have been conducted at home and abroad, indicating that human coordination and agility have obvious genetic susceptibility tendencies and are closely related to individual susceptibility genes. Therefore, exploring the corresponding genes may serve as an important molecular marker to further understand the genetic mechanism of this sports performance, which will help to cultivate and screen excellent athletes. It can also be combined with the results of precision sports medicine in clinical practice to integrate genetics and genomics into actual applications such as athlete training and rehabilitation. Summary of the invention

[0004] The present invention provides a kit for evaluating human coordination agility using SNP rs117047321 as a detection target.

[0005] The present invention provides the use of a substance for detecting a subject's genotype based on SNP rs117047321 in preparing a product; the function of the product is to evaluate the coordination agility of the subject.

[0006] The subjects with CC genotype had higher agility and coordination than those with CG genotype.

[0007] Exemplarily, the substance for detecting the genotype of the subject based on SNP rs117047321 is a primer set. Specifically, the primer set consists of three primers, which are the single-stranded DNA molecule shown in sequence 1 of the sequence list, the single-stranded DNA molecule shown in sequence 2 of the sequence list, and the single-stranded DNA molecule shown in sequence 3 of the sequence list.

[0008] The present invention also protects a product, comprising a substance for detecting a subject's genotype based on SNP rs117047321; the function of the product is to evaluate the coordination agility of the subject.

[0009] The subjects with CC genotype had higher agility and coordination than those with CG genotype.

[0010] Exemplarily, the substance for detecting the genotype of the subject based on SNP rs117047321 is a primer set. Specifically, the primer set consists of three primers, which are the single-stranded DNA molecule shown in sequence 1 of the sequence list, the single-stranded DNA molecule shown in sequence 2 of the sequence list, and the single-stranded DNA molecule shown in sequence 3 of the sequence list.

[0011] The present invention also protects the use of SNP rs117047321 as a detection target in developing products; the function of the product is to evaluate the coordination agility of a subject.

[0012] SNP rs117047321 is located in human genomic DNA, and its position corresponds to the 301st nucleotide in sequence 4 of the sequence listing, and is represented by S.

[0013] Any of the above products can be a kit.

[0014] Any of the above subjects may be Chinese.

[0015] Any of the above-mentioned subjects may be Chinese Han nationality.

[0016] Any of the above-mentioned subjects may be Chinese Han males.

[0017] Any of the above-mentioned subjects can be Chinese Han males over 18 years old.

[0018] Any of the above subjects may be Chinese Han males aged 18 to 35 years old.

[0019] Any of the above-mentioned substances for detecting the genotype of a subject based on SNP rs117047321 may be a substance for detecting the genotype of a subject based on SNP rs117047321 based on any of the following technologies: DNA sequencing, restriction fragment length polymorphism, single-strand conformation polymorphism, denaturing high performance liquid chromatography, SNP chip, etc. The SNP chip may include a chip based on nucleic acid hybridization reaction, a chip based on a single base extension reaction, a chip based on an allele-specific primer extension reaction, a chip based on a "one-step" reaction, a chip based on a primer ligation reaction, a chip based on a restriction endonuclease reaction, a chip based on a protein DNA binding reaction, a chip based on a fluorescent molecule DNA binding reaction, etc.

[0020] Any of the above substances for detecting the genotype of a subject based on SNP rs117047321 includes a primer set. The primer set consists of three primers, one of which is a single base extension primer, and the other two are used to amplify a DNA fragment including SNPrs117047321. There are no special requirements for the sequences of the two amplification primers, as long as they can amplify a genomic DNA fragment including SNPrs117047321. The single base extension primer can be designed according to the upstream or downstream of SNPrs117047321 in the human genome (excluding the SNP site), and the nucleotide extended by the single base extension primer corresponds to SNP rs117047321 in the human genome, that is, the 3' terminal nucleotide of the single base extension primer corresponds to the adjacent nucleotide of SNP rs117047321 in the human genome (that is, the previous nucleotide of SNP rs117047321 or the latter nucleotide of rs117047321).

[0021] Any of the above-mentioned materials for detecting the genotype of a subject based on SNP rs117047321 may also include PCR reagents, DNA sequencing reagents, and DNA sequencers.

[0022] Coordination agility is reflected by the completion time of the hexagonal agility test and the completion time of the T-run test.

[0023] High agility and coordination are reflected in the short completion time of the hexagonal agility test and the short completion time of the T-run test.

[0024] The present invention found that the genotype based on SNP rs117047321 is related to coordination agility. The agility coordination of CC genotype subjects is higher than that of CG genotype subjects. In Example 4 and Example 5, the present invention uses samples from different populations as examples to exemplify the correlation between the genotype of SNP rs117047321 and coordination agility. The present invention can be used to further understand the genetic mechanism of coordination agility, help train and screen excellent athletes, and can also be combined with the achievements of precision sports medicine in clinical practice to integrate genetics and genomics into actual applications such as athlete training and rehabilitation. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of the hexagonal sensitivity test.

[0026] Figure 2 This is a schematic diagram of the T-type running test. DETAILED DESCRIPTION

[0027] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0028] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial channels. p-value: Personx 2 test. OR: odds ratio.

[0029] Ethics statement: Each subject signed an informed consent, and this study was approved by the Medical Ethics Committee of the General Hospital of the Chinese People's Liberation Army.

[0030] Embodiment 1, hexagonal sensitivity test and T-type run test method

[0031] The test method is derived from the standard specification process written by the Beijing Institute of Sports Science.

[0032] 1. Hexagon Test

[0033] See the schematic diagram Figure 1 .

[0034] 1. Test preparation

[0035] Use breathable tape or chalk to draw a hexagon on a flat surface. The side length of the hexagon is 30cm and the side length is marked with letters. Each angle is 120°.

[0036] 2. Start testing

[0037] (1) Stand in the middle of the hexagon, facing line A;

[0038] (2) Keep your body facing the A line throughout the test;

[0039] (3) When the tester says start, jump out of line A and quickly jump back to the center, then jump out of line B and quickly jump back to the center, then jump out of line C and quickly jump back to the center, then jump out of line D and quickly jump back to the center, then jump out of line E and quickly jump back to the center, then jump out of line F and quickly jump back to the center; then jump out and jump back in the above order again to complete 3 circles of jumping.

[0040] (4) If you jump the wrong line, you need to restart the test.

[0041] 3. Test standards

[0042] Each subject had to complete three tests with 2-3 minutes rest between each test.

[0043] Each time you step on the line, 0.5 seconds will be added to your score.

[0044] If you jump incorrectly, your score will be invalid.

[0045] 4. Score calculation

[0046] The test result is the average of 3 tests, in seconds.

[0047] 2. Agility T-Test

[0048] See the schematic diagram Figure 2 .

[0049] Venue and equipment: 100 square meters of flat venue, 4 marking barrels, photoelectric door and measuring tape.

[0050] 1. The subject stands at the starting point (i.e., barrel A), facing the running direction, and generally adopts a high start method.

[0051] 2. Athletes can be reminded to start by a countdown. If electronic devices are used, a countdown can be used, or the subject can start and the electronic device can be activated.

[0052] 3. After the subject starts running, he / she first runs to point B and touches the B marker barrel with his / her right hand. Then, he / she slides sideways to point C and touches the C marker barrel with his / her left hand. Then, he / she slides sideways to point D and touches the D marker barrel with his / her right hand. Finally, he / she slides sideways from point D to point B and touches the B marker barrel with his / her left hand, and runs backward to point A (starting point). During the test, the subject always faces forward and cannot use a crossover step when sliding sideways; in addition, when moving from point C to point D, the body cannot touch the marker barrel at point B. The test time is measured in seconds, accurate to two decimal places.

[0053] Each subject underwent two tests with an interval of 3-5 minutes between them to ensure that the subject could fully recover.

[0054] The test result is the average of 2 tests, in seconds.

[0055] Example 2: Screening for SNPs associated with human coordination agility

[0056] The subject population consisted of healthy adult Han Chinese males.

[0057] A large number of whole genome sequencing and sequence comparison analysis were performed on the subject group to obtain a large number of differential SNPs. The allele frequency and genotype frequency of each differential SNP in the subject group were verified in turn to perform association analysis with coordination agility. Coordination sensitivity is reflected by the results of the hexagonal sensitivity test and the results of the T-type run test. The test method is shown in Example 1.

[0058] SNP rs117047321, i.e., the SNP numbered rs117047321 in NCBI, is located in an intron of the MYO5B gene, specifically at position 47429380 (GRCh37.p13) of human chromosome 18, and the polymorphic form is C / G. In human genomic DNA, SNP rs117047321 and its surrounding nucleotides are shown in Sequence 4 of the sequence listing (SNP rs117047321 is the 301st nucleotide in Sequence 4 of the sequence listing, represented by S).

[0059] No diagnostic use of SNP rs117047321 has been found in the prior art.

[0060] The inventors found through the aforementioned extensive work that SNP rs117047321 is correlated with human coordination agility, and CC genotype subjects have higher coordination agility than CG genotype subjects.

[0061] High coordination agility was reflected by a shorter time to complete the hexagonal agility test and a shorter time to complete the T-run test.

[0062] Example 3: Establishment of a method for detecting the genotype of a subject based on SNP rs117047321

[0063] 1. Take peripheral blood from the subjects and extract genomic DNA.

[0064] 2. Using the genomic DNA extracted in step 1 as a template, perform PCR amplification using specific primer pairs.

[0065] The specific primer pair consists of primer F1 and primer R1.

[0066] Primer F1 (sequence 1 of the sequence listing): 5′-acgttggatgTTCCCTGTGGATATGTAGGC-3′;

[0067] Primer R1 (sequence 2 in the sequence listing): 5'-acgttggatgTTACACAGACAATGTGCCCC-3'.

[0068] PCR amplification was performed in a 384-well plate, with one reaction system in each well.

[0069] The composition of the PCR amplification reaction system (5 μL): 0.5 μL 10× PCR buffer, 0.4 μL 25 mM MgCl 2 Solution, 0.1 μL 25 mM dNTP mix, genomic DNA, HotStart Taq DNA polymerase, primer F1, primer R1, and make up the volume with ultrapure water. In the 5 μL system, the content of genomic DNA is 20-50 ng, the content of HotStart Taq DNA polymerase is 0.5 U, the content of primer F1 is 0.5 pmol, and the content of primer R1 is 0.5 pmol.

[0070] The reaction program of PCR amplification was as follows: 94°C for 4 minutes; 94°C for 20 seconds, 56°C for 30 seconds, 72°C for 1 minute, 45 cycles; 72°C for 3 minutes; and 4°C hold.

[0071] 3. Perform alkaline phosphatase treatment (to remove free dNTPs in the system).

[0072] Take the 384-well plate that has completed step 2, prepare the reaction system and then carry out the reaction.

[0073] The composition of the reaction system (7 μL): 5 μL of the product solution obtained in step 2, 0.3 μL of SAP, 0.17 μL of 10×SAP buffer, and 1.53 μL of ultrapure water.

[0074] SAP (shrimp alkaline phosphatase): product specification is 1.7U / μl; Agena company, product number is 10002.1. 10×SAP buffer is the matching buffer for SAP.

[0075] Reaction conditions: 37°C for 40 minutes; 85°C for 5 minutes; maintain at 4°C.

[0076] 4. Perform single base extension.

[0077] Take the 384-well plate that has completed step 3, prepare the reaction system, and then perform a single base extension reaction.

[0078] The composition of the single base extension reaction system (10 μL): 7 μL of the product solution obtained in step 3, 0.3 μL of the single base extension reaction enzyme, 0.17 μL of 10× single base extension reaction buffer, 1 μL of the single base extension primer, and 1.53 μL of ultrapure water. In 1 μL of the single base extension primer, the concentration of the single base extension primer is 5 μM.

[0079] Single base extension reaction enzyme: product specification is 1.7U / μl; Agena Company, item number is 1432. 10× single base extension reaction buffer is the matching buffer for single base extension reaction enzyme.

[0080] Single base extension primer (sequence 3 in the sequence listing): 5'-AGGCGCAGAACCTCG-3'.

[0081] The reaction procedure for single base extension is as follows:

[0082] ①94℃ 30 seconds;

[0083] ②94℃ 5 seconds;

[0084] ③52℃ 5 seconds, 80℃ 5 seconds, 5 cycles;

[0085] ④94℃ 5 seconds, 52℃ 5 seconds, 80℃ 5 seconds, 40 cycles;

[0086] ⑤72℃ 3 minutes;

[0087] ⑥Maintain at 4℃.

[0088] 5. Perform resin purification.

[0089] Take the 384-well plate that has completed step 4, add 16 μl of water to each well, then add Clean Resin resin (Agena, USA) to each well, seal the film, rotate vertically at a low speed for 30 minutes to allow the resin to fully contact with the reactants, and then centrifuge to allow the resin to sink to the bottom of the well. The supernatant is the extension product after resin purification.

[0090] 6. Chip spotting.

[0091] Start the MassARRAY Nanodispenser RS1000 (Agena) and transfer the resin-purified extension product to a 384-well SpectroCHIP chip (Agena).

[0092] 7. Mass spectrometry detection.

[0093] The spotted SpectroCHIP chip was analyzed using MALDI-TOF, and the detection results were typed and output using TYPER 4.0 software (Agena).

[0094] Example 4: Analysis of SNP rs117047321 and coordination agility

[0095] Peripheral blood samples were collected from September 2018 to September 2022. 143 peripheral blood samples were obtained from 143 subjects, all of whom were unrelated Chinese male Han people. All selected testers did not exercise vigorously before the experiment, were healthy people, had no congenital disabilities, had not undergone external trauma or other surgeries, had no history of smoking, drinking, and had no lung-related diseases, etc., and all participated voluntarily. The age range of the 143 subjects was 18-31 years old (average 22.2±3.0 years old).

[0096] The operation was performed according to the method of Example 3. The genotype of the subject based on SNP rs117047321 was detected.

[0097] The hexagonal sensitivity test and T-type run test were performed according to the method of Example 1.

[0098] There were 127 individuals with CC genotype (genotype frequency was 88.8%), and the average time to complete the hexagonal sensitivity test was 15.6 s, and the average time to complete the T-run test was 12.8 s.

[0099] There were 16 individuals with CG genotype (genotype frequency was 11.2%), and the average time to complete the hexagonal sensitivity test was 17.6 s, and the average time to complete the T-run test was 13.3 s.

[0100] There are no individuals with genotype GG.

[0101] The experimental data were processed and analyzed using R 4.0.5 statistical software. After linear regression analysis and meta-analysis, the significant P values ​​of the SNP variation and the hexagonal sensitivity test results and T-type run test results were 0.009 and 4.11E-5, respectively, and the effect values ​​were 1.44 and 0.98, respectively; the meta-analysis results showed that the significant P value was 2.28E-6 and the effect value was 1.01. The results showed that SNP rs117047321 was a SNP related to coordination agility, and the subjects with the CC genotype had higher agility coordination than those with the CG genotype.

[0102] Example 5. Analysis of SNP rs117047321 and coordination agility

[0103] Peripheral blood samples were collected from September 2018 to September 2022. 318 peripheral blood samples were obtained from 318 subjects, all of whom were unrelated Chinese male Han people. All selected testers did not exercise vigorously before the experiment, were healthy people, had no congenital disabilities, had not undergone external trauma or other surgeries, had no history of smoking, drinking, and had no lung-related diseases, etc., and all participated voluntarily. The age range of the 318 subjects was 18-25 years old (average 20.1±2.2 years old).

[0104] The operation was performed according to the method of Example 3. The genotype of the subject based on SNP rs117047321 was detected.

[0105] The hexagonal sensitivity test and T-type run test were performed according to the method of Example 1.

[0106] There were 281 individuals with CC genotype (genotype frequency was 88.4%), and the average time to complete the hexagonal sensitivity test was 16.5 s, and the average time to complete the T-run test was 10.4 s.

[0107] There were 37 individuals with CG genotype (genotype frequency was 11.6%), and the average time to complete the hexagonal sensitivity test was 17.9 s, and the average time to complete the T-run test was 10.7 s.

[0108] There are no individuals with genotype GG.

[0109] The experimental data were processed and analyzed using R 4.0.5 statistical software. After linear regression analysis and meta-analysis, the significant P values ​​of the SNP variation and the hexagonal sensitivity test results and T-type run test results were 0.03 and 0.15, respectively, and the effect values ​​were 0.95 and 0.19, respectively; the meta-analysis results showed that the significant P value was 0.042 and the effect value was 0.27. The results showed that SNPrs117047321 was a SNP related to coordination agility, and the subjects with the CC genotype had higher agility coordination than those with the CG genotype.

[0110] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. Use of a substance for detecting a subject's genotype based on SNPrs117047321 in the preparation of a product, wherein the function of the product is to evaluate the coordination agility of the subject.

2. The use according to claim 1, characterized in that: The substance used to detect the subject's genotype based on SNPrs117047321 is a primer set, which consists of three primers, namely the single-stranded DNA molecule shown in SEQ ID NO: 1 in the sequence listing, the single-stranded DNA molecule shown in SEQ ID NO: 2 in the sequence listing, and the single-stranded DNA molecule shown in SEQ ID NO: 3 in the sequence listing.

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