CN112941191A - Multiple gene kit for detecting movement pattern and use method thereof - Google Patents
Multiple gene kit for detecting movement pattern and use method thereof Download PDFInfo
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Abstract
The invention discloses a multiple gene kit for detecting a movement pattern and a using method thereof. The invention adopts a multiple PCR combined electrophoresis analysis method to synchronously and rapidly qualitatively detect 2 Single Nucleotide Polymorphisms (SNP) sites on ACTN3 and ADRB3 genes related to explosive force and muscle endurance. A detection step: (1) collecting oral exfoliated cells, storing the oral exfoliated cells in a cell collection card, or collecting a blood sample and extracting nucleic acid; (2) adding the cell acquisition card or the nucleic acid in the step 1 as a template into a reaction system for PCR amplification; (3) synchronously separating 2 SNP sites, 3 human genome DNA reference genes and 1 PCR reaction reference PCR product according to the length/quality of the PCR product fragment; (4) and analyzing and interpreting the result. The invention has the advantages of rapidness, high sensitivity, good repeatability, strong specificity, high flux and low cost. The fitness plan can be scientifically formulated according to the detection result of the movement mode, the fitness efficiency is improved, and references are provided for increasing the fitness power and the continuous force of the user. Fig. 2 is an abstract attached drawing.
Description
Technical Field
The invention relates to the field of gene detection, in particular to a primer composition of single nucleotide polymorphism related to a movement pattern, a multiple gene detection kit and a using method thereof.
Background
The level of human motor ability is influenced by various factors such as external environmental factors (e.g., nutrition, training level, etc.), genetic factors, etc., and outstanding motor ability is largely determined genetically. In recent years, with the rapid development of biological techniques, related methods and technical means have been attracting attention in the field of research on gene polymorphisms associated with exercise ability (such as explosive predisposition and endurance predisposition). In the research of polymorphism of genes related to the motor ability, the outstanding motor ability is influenced by multiple gene factors, the change of each gene has small influence on a specific motor ability phenotype, but the contribution size is not necessarily the same, and finally, the effect generated by the change of each gene jointly influences the specific motor ability phenotype.
The relationship between the gene polymorphism of ACTN 3R 577X and the burst strength quality is a research hotspot. ACTN3 (actinin 3) is a structural protein expressed in the Z-line of skeletal muscle type II fast muscle fibers, acts as a anchor point, interconnects the filaments, maintains the ordered arrangement of muscle fibers, and functions as a regulator during contraction of muscle fibers. The ACTN3 gene R577X polymorphism (rs1815739) has 3 genotypes, namely RR type, RX type and XX type. Deletion of ACTN3 occurred when ACTN 3R 577X was of the XX genotype. According to researches, the ACTN3 gene R577X polymorphism is associated with the outstanding explosive power quality, the muscle strength and explosive power quality of athletes of RR genotype are considered to be stronger than those of XX type, and therefore, the ACTN 3R 577X is considered by scholars to be possibly used as a molecular marker for selecting excellent speed or strength type athletes.
The research on the association of the outstanding endurance quality and the gene polymorphism is carried out most extensively and most deeply in the research on the outstanding motor ability related gene polymorphism, and related gene sites are the most. The adrenergic receptor genes are among the candidate genes. Wherein ADRB3 gene encodes an adrenergic receptor (ADR), is mainly expressed in adipose tissue, and is associated with metabolism and cardiovascular capacity. The research of Santiago et al reports the relationship between the Trp64Arg polymorphism (rs4994) of ADRB3 and the excellent endurance quality, and also finds that the ratio of Arg alleles of excellent endurance athletes is significantly higher than that of ordinary people. Therefore, a motion mode more suitable for the constitution of the user can be selected through gene detection, a body-building plan is scientifically formulated on the basis, the body-building efficiency is improved, and the body-building power and the continuous force of the user are increased.
Disclosure of Invention
The invention provides a motion mode related gene detection kit which is rapid, high in sensitivity, good in repeatability, high in accuracy, strong in specificity, high in flux and low in cost and a using method thereof.
Specifically, the invention discloses a kit for detecting genes related to a movement pattern, which comprises the following steps:
(1) collecting oral exfoliated cells, storing in a cell collection card, or collecting a blood sample and extracting nucleic acid. The oral exfoliated cells stored on the cell collection card can be directly used for PCR amplification without nucleic acid extraction, so that the time for extracting nucleic acid is saved;
(2) performing multiple PCR amplification by using a cell acquisition card or extracted nucleic acid as a template.
(3) 2 SNP sites and 4 internal references are synchronously separated according to the length/quality of the PCR product fragment. The invention adopts capillary electrophoresis to separate PCR products: prepare electrophoresis sample in 96-well sample plate, take high-purity formamide 8.7 μ L, standard SIZE-5000.3 μ L, PCR product 1 μ L, mix well and centrifuge. The prepared electrophoresis sample was placed in a 3500 gene analyzer and subjected to capillary electrophoresis according to the instructions.
(4) And (4) carrying out result analysis according to the designed fragment length of each detection site. The kit comprises the following components: primer Mix, PCR reaction solution and DNA/RNA-free water.
Wherein the Primer Mix comprises a Primer group for amplifying 2 gene SNP polymorphic sites and a Primer group for 3 human genome DNA internal references and reaction internal references pcDNA, which are detailed in Table 1.
The kit uses a multiplex PCR combined fragment length analysis method, and synchronously and rapidly qualitatively detects the genotypes of 2 detected genes ADRB3 and ACTN3 related to a motion mode in a reaction tube through the design of SNP primers: heterozygotes or homozygotes; the pcDNA is used for detecting a reaction system, monitoring whether the reaction system is effective or not and whether amplification is normal or not; 3 human genome DNA is internally referred to for detecting human samples, so that the human samples are guaranteed to be effective.
Wherein the PCR reaction solution comprises the following components: 2 XPCR buffer, DNA polymerase, dNTPs, potassium chloride, magnesium chloride, etc.
The PCR amplification reaction conditions of the kit are shown in Table 2.
TABLE 2 PCR amplification reaction conditions of the present invention
The amplification product of the kit is analyzed by electrophoresis; the preferred electrophoresis is capillary electrophoresis.
The genotype of each SNP site can be obtained from the detection peak map, and the motion pattern genetic information of the subject is judged by combining the reference information (table 3) corresponding to each gene.
TABLE 3 detection site reference information of the present invention
Compared with the prior art, the invention has the following advantages: the invention establishes a detection scheme for synchronously detecting 2 SNP loci related to a movement mode based on an ABI 3500 gene analyzer; specificity and accuracy can reach qPCR level; the detection of 2 SNP sites of a plurality of samples can be completed simultaneously in a short time (2.5 hours); the use of the DNA internal reference and the reaction internal reference can monitor the efficiency of nucleic acid extraction and PCR reaction, and avoid false negative and false positive.
The SNP detection method adopted by the invention is based on multiplex PCR and Capillary Electrophoresis (CE) separation technology. Adding multiple pairs of specific gene amplification primers and internal reference primers into the same reaction tube at the same time to obtain gene amplification fragments with different sizes, and separating by using capillary electrophoresis to further analyze the SNP genotype. The detection method and the kit adopted by the invention can quickly and effectively detect a plurality of SNP sites, overcome the defects of the traditional method and have the following advantages:
1. high flux: can realize synchronous detection of a plurality of SNP sites.
2. The accuracy is high: the CE technology is adopted to separate PCR products, so that non-specific amplification products, primer dimers and specific amplification products can be separated, and false positive can be reduced to the greatest extent.
3. The sensitivity is high: the system can detect DNA samples with the content as low as 1 ng/reaction and has ultrahigh sensitivity.
4. The method is simple and convenient, and is economical to use: the invention provides a complete set of experimental schemes such as reagent, multiple PCR primer design, result analysis and the like; the detection cost is low, and the large-scale popularization is facilitated.
Drawings
FIG. 1 is a negative control DNA/RNA-free water amplification map of the kit. Only characteristic peaks of pcDNA appear.
FIG. 2 shows the result of the detection of the exfoliated buccal cell mass collecting card sample of the subject 1. As shown in FIG. 2, the abscissa represents the size of the PCR fragment and the ordinate represents the intensity of the fluorescence signal, and the genotype of each site can be obtained from the position of the characteristic peak. The genotype of the user ACTN3 gene rs1815739 site is CC, and the genotype of the user ADRB3 gene rs4994 site is TT. The pcDNA peak and characteristic peaks of human genome DNA (huDNA) internal reference-1, human genome DNA (huDNA) internal reference-3 and human genome DNA (huDNA) internal-8 appear.
FIG. 3 shows the results of blood sample measurement of subject 2. The genotype of the user ACTN3 gene rs1815739 site is CT, and the genotype of the user ADRB3 gene rs4994 site is TT. The pcDNA peak and characteristic peaks of human genome DNA (huDNA) internal reference-1, human genome DNA (huDNA) internal reference-3 and human genome DNA (huDNA) internal-8 appear.
Detailed Description
For a better understanding of the present invention, reference is made to the following detailed description and accompanying drawings. It is to be understood that these examples are for further illustration of the invention and are not intended to limit the scope of the invention. In addition, it should be understood that the invention is not limited to the above-described embodiments, but is capable of various modifications and changes within the scope of the invention.
The reagent stored in the kit body comprises the following steps:
a) collecting the cells dropped off from the oral cavity of a testee, storing the cells in a collection card or extracting DNA nucleic acid from blood;
b) adding a saliva acquisition card sample or an extracted DNA sample of a testee into a reaction system for PCR amplification;
c) running an amplification program;
d) carrying out electrophoretic analysis on the amplification product, and judging according to a peak pattern;
the kit comprises the following components: primer Mix including Primer set, PCR reaction solution and DNA/RNA-free water.
The PCR reaction system Primer Mix comprises a Primer group for amplifying 2 SNP polymorphic sites, 3 personal genome references and pcDNA reaction references, and the details are shown in Table 1.
Wherein the PCR reaction solution comprises the following components: 2 XPCR buffer, DNA polymerase, dNTPs, potassium chloride, magnesium chloride, etc.
In the embodiment, the sample isDNA/RNA-freeWater, a collection card of the cells exfoliated from the oral cavity wall of the testee 1 and DNA extracted from a blood sample of the testee 2.
In the embodiment, the electrophoresis is to carry out electrophoresis on the amplification product through capillary electrophoresis, and an amplification map is given and analyzed by combining analysis software, so that reference is provided for judging the explosive force and the exercise tolerance of the testee.
Example one
(1) The selected samples were: DNA/RNA-free water.
(2) Architecture configuration
According to the specification, preparing a reaction system by the Primer Mix and the PCR reaction solution on ice according to the proportion of the specification, carrying out vortex mixing, centrifuging by a centrifuge, mixing uniformly by a gun head, and subpackaging.
(3) Adding the sample
According to the instructions, using a pipette to take a corresponding volume of DNA/RNA-free water, and adding into the reaction system which is divided.
(4) Amplification procedure
The amplification procedure on the PCR instrument is as in table 2.
(5) Detection of amplification product on 3500DX genetic analyzer
A sample mixture is composed of deionized formamide and an internal molecular weight standard (Size-500) in the system, wherein the sample mixture is (1L Size-500+12L deionized formamide) × (sample injection number) }. Mix 9. mu.L of the sample mixture with 1. mu.L of the amplification product to avoid the formation of bubbles and to perform electrophoresis as soon as possible. Detection and analysis are carried out by an ABI 3500 genetic analyzer (purchased from ABI company of America), and specific analysis parameters are sample injection voltage: 1.2kv, sample injection time: 15s, electrophoresis time 1210-. The results of the detection are shown in FIG. 1.
(6) Analyzing data
The experiment uses DNA/RNA-free water, and the electrophoresis pattern should not have target peaks of each gene locus, and only has characteristic peaks of the pcDNA gene, as follows: the peak appearance corresponding to the pcDNA site is: singlet, showing pcDNA in the map;
as shown in FIG. 1, the electrophoretically analyzed DNA/RNA-free water-amplified product showed only the peak of the internal reference pcDNA of the reaction.
The experiment can effectively verify the amplification effectiveness of the reaction reagent and has no external pollution source.
Example two
(1) The selected samples were: a sample of cells exfoliated from the oral wall of subject 1.
(2) Sample collection
The collection type is as follows: cells are shed from the oral wall.
The acquisition method comprises the following steps: the saliva collecting rod is adopted to wipe the inner wall of the oral cavity back and forth for 4 times, the reverse side of the saliva collecting rod is used to wipe the inner wall of the oral cavity back and forth for 4 times, the saliva collecting rod is taken out, the saliva collecting rod is repeatedly pressed on a saliva sample collecting card, cells on the inner wall of the saliva are transferred to the saliva sample collecting card, and the collected saliva sample collecting card is dried in a pollution-free area.
Valid samples: the area of the saliva sample acquisition card with the pink area changed into light pink or white is the effective saliva sample area.
The sample selecting method comprises the following steps: manual punch sampling was performed using a dabber plastic punch (1.0 mm).
(3) Architecture configuration
According to the specification, preparing a reaction system by the Primer Mix and the PCR reaction solution on ice according to the proportion of the specification, carrying out vortex mixing, centrifuging by a centrifuge, mixing uniformly by a gun head, and subpackaging.
(4) Adding a sample: and taking 1-2 effective samples in the saliva card by using a puncher, and adding the effective samples into the prepared reaction system.
(5) Amplification procedure
The amplification procedure on the PCR instrument is as in table 2.
(6) Detection of amplification product on 3500DX genetic analyzer
A sample mixture consisting of deionized formamide and an internal molecular weight standard in the system (Size-500) { (1. mu.L Size-500+ 12. mu.L deionized formamide) × (number of samples) }. Mix 9. mu.L of the sample mixture with 1. mu.L of the amplification product to avoid the formation of bubbles and to perform electrophoresis as soon as possible. Detection and analysis are carried out by an ABI 3500 genetic analyzer (purchased from ABI company of America), and specific analysis parameters are sample injection voltage: 1.2kv, sample injection time: 15s, electrophoresis time 1210-. The results are shown in FIG. 2.
(7) Analyzing data
The experiment uses a sample of a tested person, and the genotype of the tested person is determined according to the position of each target peak in the map. The genotype of the site rs1815739 of the ACTN3 gene of the subject is CC, and the explosive power quality is good; the genotype of the site rs4994 of the ADRB3 gene is TT, and the endurance quality is general. Referring to table 3, the user is suggested to select a velocity-force type exercise more than once.
EXAMPLE III
(1) The selected samples were: blood sample of subject 2.
(2) Sample type: a blood sample.
(3) And (3) extracting DNA: and (3) carrying out DNA extraction on the blood sample by using a nucleic acid extractor.
(4) Architecture configuration
According to the specification, preparing a reaction system by the Primer Mix and the PCR reaction solution on ice according to the proportion of the specification, carrying out vortex mixing, centrifuging by a centrifuge, mixing uniformly by a gun head, and subpackaging.
(5) Adding a sample: according to the instruction, a certain amount of the extracted DNA sample is taken by a pipette and added to the reaction system.
(6) Amplification procedure
The amplification procedure on the PCR instrument is as in table 2.
(7) Detection of amplification product on 3500DX genetic analyzer
A sample mixture consisting of deionized formamide and an internal molecular weight standard in the system (Size-500) { (1. mu.L Size-500+ 12. mu.L deionized formamide) × (number of samples) }. Mix 9. mu.L of the sample mixture with 1. mu.L of the amplification product to avoid the formation of bubbles and to perform electrophoresis as soon as possible. Detection and analysis are carried out by an ABI 3500 genetic analyzer (purchased from ABI company of America), and specific analysis parameters are sample injection voltage: 1.2kv, sample injection time: 15s, electrophoresis time 1210-. The results of the measurements are shown in FIG. 3.
(8) Analyzing data
Fig. 3 is a blood-extracted DNA sample analysis map of the subject 2. The genotype of the user ACTN3 gene rs1815739 site is CT, and the explosive power quality is general; the genotype of the rs4994 site of the ADRB3 gene is TT, and the endurance quality is general. The user may adjust the fitness campaign with reference to this result.
Claims (7)
1. A multiple gene kit for detecting a movement pattern, comprising the steps of:
a) collecting the cells dropped off from the oral cavity of a testee, storing the cells in a collection card or extracting DNA nucleic acid from a blood sample;
b) adding a saliva acquisition card sample or an extracted DNA sample of a testee into a reaction system for PCR amplification;
c) running an amplification program;
d) performing electrophoretic fragment analysis on the product, and judging according to a peak pattern;
the PCR reaction system comprises primer groups for amplifying 2 gene SNP polymorphic sites, 3 human genome DNA internal references and one PCR reaction internal reference, and the primer groups are shown in table 1, and the following primer sequence directions are all from 5 'end to 3' end.
TABLE 1 detection sites and primers of motion pattern multiple gene detection kit
2. The multiple gene kit for detecting motor patterns according to claim 1, wherein the exfoliated cells in the mouth are exfoliated cells on the inner wall of the mouth, and are preserved on a cell collecting card and can be directly used for PCR amplification.
3. The multiple gene kit for detecting exercise patterns according to claim 1, wherein the blood sample is DNA extracted from a blood sample of a subject or a blood sample collected from a blood storage card and directly used for PCR amplification.
4. The multiple gene kit for detecting a motor pattern according to claim 1, wherein the reaction system comprises Primer Mix including a Primer set, a PCR reaction solution and DNA/RNA-free water.
5. The multiple gene kit for detecting a motor pattern according to claim 1, wherein the Primer Mix in the reaction system comprises primers for detecting all genotypes of SNPs of ACTN3 and ADRB3, and comprises primers for detecting pcDNA and 3 human genomic DNA internal references.
6. The method of using the multiple gene kit for detecting exercise patterns according to claim 1, wherein the PCR reaction solution in the reaction system comprises 2 XPCR buffer, DNA polymerase, dNTPs, potassium chloride, magnesium chloride, etc.
7. The method of using the multiple gene kit for detecting exercise pattern according to claim 1, wherein the amplification products are analyzed by electrophoresis; preferably the electrophoresis is capillary electrophoresis.
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2019
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| US20100136561A1 (en) * | 2008-05-16 | 2010-06-03 | Interleukin Genetics, Inc. | Genetic Markers for Weight Management and Methods of Use Thereof |
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Application publication date: 20210611 |