Primer group for detecting polymorphism of ADH1B gene and ALDH2 gene as well as kit and method for detecting alcohol metabolism capability

Abstract

The invention provides a primer group for detecting ADH1B gene and ALDH2 gene polymorphism, and a kit and a method for detecting alcohol metabolism capability, and relates to the technical field of gene detection. According to the method for detecting the alcohol metabolism capability, genes are extracted through nucleic acid release liquid, target genes are amplified through an isothermal amplification reagent, and then gene amplification products are detected through a dual nucleic acid chromatography test strip. Compared with an existing PCR technology, the one-step method nucleic acid release liquid is higher in release speed, and amplification can be carried out after incubation is carried out for 0-2 minutes; an amplification reagent is a freeze-dried reagent, so that more samples can be added, and the detection stability is improved; the result of an amplification product is displayed through a chromatographic test strip, so that the equipment cost is reduced; genotype results displayed by the chromatography test strip are clear and concise, and operation and interpretation by professionals are not needed.

Description

Technical Field

[0001] The present application relates to the field of gene detection technology, and in particular to a primer set for detecting polymorphisms of ADH1B gene and ALDH2 gene, and a kit and method for detecting alcohol metabolism ability. Background Art

[0002] As a long-standing tradition of the Chinese nation, drinking culture is something that everyone cannot avoid in daily life, work, and interpersonal communication. However, some people can drink a lot without getting drunk, while some people get red-faced or even poisoned by alcohol when they drink. The reason is that everyone's alcohol metabolism-related genes have different alcohol degradation abilities, which in turn affects the amount of alcohol they can drink.

[0003] When developing a genetic test kit for alcohol metabolism, gene loci related to alcohol metabolism are usually tested, mainly including the gene polymorphisms of alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH). The following are some common technical solutions:

[0004] Fluorescence PCR-melting curve method: This method evaluates alcohol metabolism ability by detecting gene polymorphism. It has the characteristics of no extraction, small sample volume, high sensitivity, and automatic interpretation, but it requires specific instruments and equipment, and the equipment investment cost is high;

[0005] High-throughput sequencing (NGS): This method can detect multiple gene loci at the same time and is suitable for large panel detection, but it is costly, time-consuming, and requires high technical skills of equipment and experimenters;

[0006] Nucleic acid mass spectrometry detection method: This method designs specific primers, uses PCR and single base extension technology, and combines nucleic acid mass spectrometer for detection and analysis. It has the characteristics of high accuracy and high throughput, but requires specific mass spectrometer equipment and has certain requirements on the technical level of the operator.

[0007] In summary, the prior art lacks a simple, efficient and low-cost method for detecting alcohol metabolism ability. Summary of the invention

[0008] The purpose of the present application is to provide a primer set for detecting polymorphisms of the ADH1B gene and the ALDH2 gene, a kit and a method for detecting alcohol metabolism ability, aiming to solve the problems existing in the existing methods for detecting alcohol metabolism ability.

[0009] To achieve the above objectives, the present application provides a primer set for detecting polymorphisms of the ADH1B gene and the ALDH2 gene, including a primer set for amplifying the rs1229984 polymorphic site of the ADH1B gene and a primer set for amplifying the rs671 polymorphic site of the ALDH2 gene;

[0010] The primer set for amplifying the rs1229984 polymorphic site of the ADH1B gene includes a T-type upstream primer, a C-type upstream primer, and a first common downstream primer; the T-type upstream primer and the C-type upstream primer are single-stranded DNAs with a length of 25-30 nucleotides, the 5' end of the T-type upstream primer is labeled with FAM, and the 5' end of the C-type upstream primer is labeled with digoxigenin;

[0011] The primer set for amplifying the rs671 polymorphic site of the ALDH2 gene includes a G-type upstream primer, an A-type upstream primer, and a second common downstream primer; the G-type upstream primer and the A-type upstream primer are single-stranded DNAs with a length of 25-30 nucleotides, the 5' end of the G-type upstream primer is labeled with FAM, and the 5' end of the A-type upstream primer is labeled with digoxigenin;

[0012] The first common downstream primer and the second common downstream primer are single-stranded DNA with a length of 40-50 nucleotides, with biotin labeled at the 5' end, the 16th nucleotide from the 3' end replaced with tetrahydrofuran, and C3 Spacer labeled at the 3' end.

[0013] In some embodiments, the C-type upstream primer and the A-type upstream primer introduce a mismatched base next to the SNP site.

[0014] In some embodiments, the T-type upstream primer is shown as SEQ ID No.1, and the C-type upstream primer is shown as SEQ ID No.2.

[0015] In some embodiments, the G-type upstream primer is shown as SEQ ID No.10, and the A-type upstream primer is shown as SEQ ID No.11.

[0016] In some embodiments, the first common downstream primer is shown as SEQ ID No.3, and the second common downstream primer is shown as SEQ ID No.12.

[0017] The present application also provides a kit for detecting alcohol metabolism ability, comprising a nucleic acid release solution, a constant temperature amplification freeze-dried reagent, and a double nucleic acid chromatography test strip, wherein the constant temperature amplification freeze-dried reagent comprises the primer set for detecting ADH1B gene and ALDH2 gene polymorphism.

[0018] In some embodiments, the isothermal amplification lyophilized reagent includes a first isothermal amplification lyophilized reagent and a second isothermal amplification lyophilized reagent;

[0019] The first isothermal amplification freeze-dried reagent includes a first freeze-dried ball A and a freeze-dried ball B, wherein the first freeze-dried ball A includes a 2× isothermal amplification reagent and a primer set for amplifying the rs1229984 polymorphic site of the ADH1B gene;

[0020] The second isothermal amplification freeze-dried reagent includes a second freeze-dried ball A and a freeze-dried ball B, and the second freeze-dried ball A includes a 2× isothermal amplification reagent and the primer set for amplifying the rs671 polymorphic site of the ALDH2 gene;

[0021] The lyophilized ball B includes 5% by mass of PEG 35K and 60 mM of magnesium acetate.

[0022] In some embodiments, the final molar concentration ratio of the T-type upstream primer, the C-type upstream primer, and the first common downstream primer is 0.6:2:0.6;

[0023] The final molar concentration ratio of the G-type upstream primer, the A-type upstream primer and the second common downstream primer is 1:1.5:0.3.

[0024] In some embodiments, the nucleic acid release solution includes 50 mM NaOH, 2 mM EDTA, 0.5% by mass of SDS, and 1% by volume of Triton X100;

[0025] The double nucleic acid chromatography test strip comprises a bottom plate, a sample pad, a binding pad, a nitrocellulose membrane, and a water absorbent pad; a quality control line, a detection line 1, and a detection line 2 are fixed on the nitrocellulose membrane, the detection line 1 is coated with anti-FITC / FAM antibodies, and the detection line 2 is coated with anti-digoxin antibodies.

[0026] During the test, the treated sample to be tested is dripped into the reagent injection area. The two types of nucleic acid amplification products with markers contained in the sample first bind to the biotin ligand labeled with the latex complex, and then the conjugate is chromatographed upward (at the absorbent paper end) under the capillary effect. One will be bound by the anti-digoxigenin antibody fixed on the membrane, and a red band will appear on the test line 2. The other will be bound by the anti-FITC / FAM antibody fixed on the membrane, and a red band will appear on the test line 1. When the sample does not contain the nucleic acid amplification product with markers or the nucleic acid amplification product with markers contained is less than the minimum detection limit, there will be no red bands on the test line 1 and the test line 2. Regardless of whether the nucleic acid amplification product with markers exists in the sample, a blue band will appear on the quality control line. The blue band presented by the quality control line is the standard for judging whether there is enough sample and whether the chromatography process is normal. It also serves as the internal control standard of the test strip.

[0027] The present application also provides a method for detecting alcohol metabolism ability, using the above-mentioned kit for detecting alcohol metabolism ability, comprising:

[0028] Collect oral mucosal cells;

[0029] inserting a buccal swab into the nucleic acid releasing fluid;

[0030] Taking the nucleic acid release solution and adding it into the isothermal amplification freeze-dried reagent to obtain an amplification product;

[0031] The amplified product is added to the double nucleic acid chromatography test strip for typing detection to determine the genotype.

[0032] Compared with the prior art, the beneficial effects of this application include:

[0033] The method for detecting alcohol metabolism ability provided by the present application is to extract genes by nucleic acid release liquid, amplify target genes by constant temperature amplification reagent, and then detect gene amplification products by double nucleic acid chromatography test strips. Compared with the existing PCR technology, the one-step nucleic acid release liquid of the present application has a faster release speed, and amplification can be performed after incubation for 0-2 minutes; the amplification reagent is a freeze-dried reagent, and more samples can be added to improve the detection stability; the amplification product is displayed by the chromatography test strip, which reduces the equipment cost; the chromatography test strip displays the genotype results clearly and concisely, and does not require professional operation and interpretation.

[0034] The method for detecting alcohol metabolism ability provided by the present application can reduce the cost of experimental equipment. Compared with the currently available PCR technology, it is not limited to qPCR instruments, and PCR, metal baths, water baths and other instruments that can provide a constant temperature environment can also be used for experiments, thereby reducing the cost of experiments; it is easy to carry, and portable instruments can be directly used for experiments, and the usage scenarios are not limited to laboratories; the experimental operation is simpler, and only the sample needs to be added to the constant temperature amplification freeze-dried reagent, and the experiment can be carried out on the machine after oscillation and mixing, which is convenient for non-professionals to operate; the genotype interpretation is simple, and the genotype interpretation can be performed through the bands displayed on the test strips, which is convenient for non-professionals to interpret by themselves. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present application and therefore should not be regarded as limiting the scope of the present application.

[0036] Figure 1 A comparison chart of different reaction times for nucleic acid release solution of ADH1B gene;

[0037] Figure 2 A comparison chart of different reaction times for nucleic acid release solution of ALDH2 gene;

[0038] Figure 3 This is a schematic diagram of the interpretation results of the ADH1B gene;

[0039] Figure 4 This is a schematic diagram of the interpretation results of the ALDH2 gene;

[0040] Figure 5This is the experimental result diagram of sample solutions of ADH1B gene with different dilution multiples;

[0041] Figure 6 This is the experimental result diagram of sample solutions with different dilution multiples of ALDH2 gene;

[0042] Figure 7 ADH1B gene kit repeatability test results

[0043] Figure 8 This is a graph showing the repeatability test results of the ALDH2 gene kit;

[0044] Fig. 9 This is a diagram showing the stability test results of the ADH1B gene kit;

[0045] Fig.10 This is a graph showing the stability test results of the ALDH2 gene kit. DETAILED DESCRIPTION

[0046] As used herein:

[0047] "Prepared from" is synonymous with "comprising." As used herein, the terms "comprising," "including," "having," "containing," or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises the listed elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

[0048] The conjunction "consisting of excludes any unspecified element, step, or component. If used in a claim, this phrase renders the claim closed-ended so that it does not include materials other than those described, except for conventional impurities associated therewith. When the phrase "consisting of" appears in a clause of the body of a claim rather than immediately following the subject matter, it limits only the elements described in that clause; other elements are not excluded from the claim as a whole.

[0049] When an amount, concentration, or other value or parameter is expressed as a range, a preferred range, or a range defined by a series of upper preferred values ​​and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pairing of any range upper limit or preferred value with any range lower limit or preferred value, regardless of whether the range is disclosed separately. For example, when a range of "1 to 5" is disclosed, the described range should be interpreted as including ranges "1 to 4", "1 to 3", "1 to 2", "1 to 2 and 4 to 5", "1 to 3 and 5", etc. When a numerical range is described in this article, unless otherwise stated, the range is intended to include its end values ​​and all integers and fractions within the range.

[0050] In these examples, parts and percentages are by mass unless otherwise indicated.

[0051] "Parts by mass" refers to the basic unit of measurement for expressing the mass ratio of multiple components. 1 part can represent any unit mass, such as 1g or 2.689g. If we say that the mass of component A is a parts and the mass of component B is b parts, it means the ratio of the mass of component A to the mass of component B is a:b. Alternatively, it means that the mass of component A is aK and the mass of component B is bK (K is an arbitrary number, indicating a multiple factor). It should not be misunderstood that, unlike the mass parts, the sum of the mass of all components is not limited to 100 parts.

[0052] “And / or” is used to indicate that one or both of the stated situations may occur, for example, A and / or B includes (A and B) and (A or B).

[0053] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. In the description of the present invention, the meaning of "plurality" is two or more, unless otherwise clearly and specifically defined.

[0054] The embodiments of the present application will be described in detail below in conjunction with specific examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present application and should not be considered as limiting the scope of the present application. If specific conditions are not specified in the examples, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0055] The NaOH (item number: A620617), EDTA (item number: A600437), and SDS (item number: A600485) used to prepare the one-step nucleic acid release solution in the examples of the present application were all purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0056] The primers and probes used in the examples of this application were all synthesized by Sangon Biotechnology (Shanghai) Co., Ltd.

[0057] In the examples of the present application, the ADH1B gene T-type recombinant plasmid (number: PUC57-ETOH1-T), the ADH1B gene C-type recombinant plasmid (number: PUC57-ETOH1-C), the ALDH2 gene G-type recombinant plasmid (number: PUC57-ETOH2-G), and the ALDH2 gene A-type recombinant plasmid (number: PUC57-ETOH2-A) were synthesized and constructed by Sangon Biotech (Shanghai) Co., Ltd.

[0058] The isothermal amplification reagent (article number: B631002) and the double nucleic acid chromatography test strip (article number: B110201) in the examples of the present application were purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0059] The metal bath (article number: G100518), oscillator (article number: G002032), and centrifuge (article number: G508010) used in the examples of the present application were all purchased from Sangon Biotechnology (Shanghai) Co., Ltd.

[0060] Example 1 Screening of primer sets for detecting polymorphisms of ADH1B gene and ALDH2 gene

[0061] The primer set of the present application is a primer set for the ADH1B gene and a primer set for the ALDH2 gene. Primers are designed according to the SNP sites obtained by screening in order to identify the genotypes of the polymorphic sites of the ADH1B gene (rs 1229984, c.143T>C) and the ALDH2 gene (rs671, c.1510G>A). All primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and the primer set sequences designed in this application are shown in Tables 1 and 2 below.

[0062] All T-type upstream primers were labeled with FAM (carboxyfluorescein) at the 5' end, C-type upstream primers were labeled with digoxigenin at the 5' end, T / C shared downstream primers were labeled with biotin at the 5' end and C3 Spacer at the 3' end, and the 16th nucleotide from the 3' end was replaced with a tetrahydrofuran site.

[0063] All G-type upstream primers were labeled with FAM (carboxyfluorescein) at the 5' end, and A-type upstream primers were labeled with digoxigenin at the 5' end. The G / A shared downstream primers were labeled with biotin at the 5' end and C3 Spacer at the 3' end. The 16th nucleotide from the 3' end was replaced with a tetrahydrofuran site.

[0064] Table 1 Primer set for ADH1B gene

[0065]

[0066]

[0067] Table 2 ALDH2 gene primer set

[0068]

[0069]

[0070] The test was performed using the detection scheme of this application.

[0071] 1. Different primer combinations were configured into isothermal amplification reaction reagents. The reaction system was: 1) 2× isothermal amplification reagent 12.5 μL;

[0072] 2) 1.25 μL of magnesium acetate solution;

[0073] 3) 10 μM G-type upstream primer 1.0 μL;

[0074] 4) 10 μM type A upstream primer 1.0 μL;

[0075] 5) 0.3 μL of 10 μM G / A common downstream primer;

[0076] 6) 1 μL of plasmid template or nuclease-free water;

[0077] 7) Add nuclease-free water to make up to 25 μL.

[0078] 2. For the ADH1B gene (rs 1229984, c.143T>C), 3000 copies / μL T-type recombinant plasmid, 3000 copies / μL C-type recombinant plasmid, 3000 copies / μL TC-type recombinant plasmid (3000 copies / μL T-type recombinant plasmid and 3000 copies / μL C-type recombinant plasmid 1:1 mixture) were used as positive templates. For the ALDH2 gene (rs671, c.1510G>A), 3000 copies / μL G-type recombinant plasmid, 3000 copies / μL A-type recombinant plasmid, 3000 copies / μL GA-type recombinant plasmid (3000 copies / μL G-type recombinant plasmid and 3000 copies / μL A-type recombinant plasmid 1:1 mixture) were used as positive templates. The above 6 genotypes were used as positive templates in this experimental example, and nuclease-free water was used as a negative control in this experimental example.

[0079] 3. Place the constant temperature amplification reagent in a metal bath at 40°C for 15 minutes.

[0080] 4. Dilute the amplification solution 16 times with pure water, take 80ul and add it to the sample pad of the nucleic acid test strip, chromatograph for 10 minutes and read the results.

[0081] The detection results of different primer combinations are shown in Table 3.

[0082] Table 3 Detection results of different primer combinations

[0083]

[0084]

[0085] Note: “-” means no stripe, “+” means stripe is displayed.

[0086] As can be seen from the above table, for the ADH1B gene, the primer set of combination 1 meets the requirements and will not produce false positives or false negatives. For the ALDH2 gene, combination 4 meets the requirements.

[0087] Therefore, the present application provides detection primers for the polymorphic site of the ADH1B gene (rs 1229984, c.143T>C): a T-type upstream primer as shown in SEQ ID No.1, a C-type upstream primer as shown in SEQ ID No.2, and a T / C common downstream primer as shown in SEQ ID No.3, and the G base near the SNP site of the C-type upstream primer is mismatched to a T base; detection primers for the polymorphic site of the ALDH2 gene (rs671, c.1510G>A): a G-type upstream primer as shown in SEQ ID No.10, an A-type upstream primer as shown in SEQ ID No.11, and a G / A common downstream primer as shown in SEQ ID No.12, and the A base near the SNP site of the A-type upstream primer is mismatched to a G base.

[0088] Example 2 Concentration of primer sets for detecting polymorphisms of ADH1B gene and ALDH2 gene

[0089] The detection primers for the polymorphic site of the ADH1B gene (rs 1229984, c.143T>C) include a T-type upstream primer as shown in SEQ ID No.1, a C-type upstream primer as shown in SEQ ID No.2, and a T / C common downstream primer as shown in SEQ ID No.3. The input ratios are adjusted as shown in Table 4.

[0090] Table 4 ADH1B different primer input ratio

[0091] Primer name Group 1 Group 2 Group 3 984-F1-w(10μM) 1μL 0.6μL 1μL 984-F1-m(10μM) 1μL 2μL 3μL 984-P1(10μM) 0.3μL 0.6μL 0.6μL

[0092] The detection primers for the polymorphic site of the ALDH2 gene (rs671, c.1510G>A) include a G-type upstream primer as shown in SEQ ID No.10, an A-type upstream primer as shown in SEQ ID No.11, and a G / A common downstream primer as shown in SEQ ID No.12. The input ratios are adjusted as shown in Table 5.

[0093] Table 5 ALDH2 different primer input ratio

[0094] Primer name Group 4 Group 5 Group 6 671-F1-w(10μM) 1μL 1μL 1.4μL 671-F1-m(10μM) 1μL 1.5μL 2μL 671-P1(10μM) 0.3μL 0.3μL 0.3μL

[0095] The test results of different primer input amounts are shown in Table 6.

[0096] Table 6 Detection results of different primer input amounts

[0097]

[0098]

[0099] Note: “-” indicates no band, “+” indicates band is present, and “++” indicates obvious band.

[0100] According to Table 6, for the ADH1B gene, the best effect was achieved when the final molar concentration ratio of the T-type upstream primer, the C-type upstream primer and the T / C shared downstream primer was 0.6:2:0.6; for the ALDH2 gene, the best effect was achieved when the final molar concentration ratio of the G-type upstream primer, the A-type upstream primer and the G / A shared downstream primer was 1:1.5:0.3.

[0101] Example 3 Nucleic acid release solution for detecting alcohol metabolism ability

[0102] The components of the nucleic acid release solution for detecting alcohol metabolism genes of the present application are shown in Table 7 below.

[0103] Table 7 Nucleic acid release solution composition

[0104]

[0105]

[0106] Oral mucosal cells were collected using oral swabs, inserted into the one-step nucleic acid release solution in Table 7, mixed, and then immediately sampled and reacted for 1 minute, 2 minutes, 5 minutes, and 20 minutes. 25 μL of each sample was sampled for constant temperature amplification and release performance was tested. The results are as follows: Figure 1 and Figure 2 As shown, Figure 1 The test result is the homozygous CC genotype of ADH1B gene. Figure 2 This is the detection result of the homozygous GG genotype of the ALDH2 gene. It can be seen that there is no obvious difference between the results of immediate sampling and the sampling results after a few minutes of reaction. The nucleic acid release solution of the present application can achieve the effect of quickly releasing the required detection nucleic acid.

[0107] Example 4 Kit for detecting alcohol metabolism ability

[0108] The kit for detecting alcohol metabolism ability of the present application includes the nucleic acid release solution of Example 3, the isothermal amplification freeze-dried reagent, and the double nucleic acid chromatography test strip. The isothermal amplification freeze-dried reagent includes the primer set for detecting ADH1B gene and ALDH2 gene polymorphism of Example 2.

[0109] The isothermal amplification freeze-dried reagent is divided into freeze-dried ball A and freeze-dried ball B, and the formula is shown in Table 8.

[0110] Table 8 Components of isothermal amplification lyophilized reagents

[0111]

[0112] Further, the freeze-dried pellet A reagent prepared according to the above formula was frozen into a spherical shape in liquid nitrogen at a volume of 16 μL / drop. The freeze-dried pellet B reagent prepared according to the above formula was frozen into a spherical shape in liquid nitrogen at a volume of 6 μL / drop.

[0113] Furthermore, the frozen lyophilized balls A and lyophilized balls B are poured into lyophilization trays, respectively, placed in a lyophilizer, and the lyophilization program is set according to Table 9.

[0114] Table 9 Freeze-drying procedure

[0115]

[0116] After freeze-drying is completed, freeze-dried balls A and B are sequentially loaded into reaction tubes, the reaction tubes are covered with lids, and vacuum-packed in aluminum foil bags to keep them in a sealed and dry environment.

[0117] Example 5 Method for detecting alcohol metabolism ability

[0118] The detection method provided in this embodiment uses the kit of Example 4, comprising the following steps:

[0119] 1. Collect oral mucosal cells.

[0120] 2. Insert the oral swab into the one-step nucleic acid release solution, mix well, and quickly release the genomic DNA.

[0121] 3. Take 25 μL of the release solution and add the isothermal amplification freeze-dried reagent, amplify at 40°C for 15 minutes to obtain the amplified product.

[0122] 4. Add the amplified product to the double nucleic acid chromatography test strip for chromatography and interpret the results within 10 minutes.

[0123] 5. Determine the genotype.

[0124] like Figure 3 and Figure 4 As shown, when test line 1 shows no band and test line 2 shows no band, it is judged as negative and needs to be re-sampled for testing; when test line 1 shows a band and test line 2 shows no band, it is judged as TT type homozygous genotype or GG type homozygous genotype; when test line 1 shows a band and test line 2 also shows a band, it is judged as TC type heterozygous genotype or GA type heterozygous genotype; when test line 1 shows no band and test line 2 shows a band, it is judged as CC type homozygous genotype or AA type homozygous genotype.

[0125] Among them, the liver enzyme activity of TT type people is 40 times that of CC type. They have a good alcohol tolerance, are not easily drunk, and their body metabolizes alcohol quickly; the alcohol metabolism rate of TC type is lower than that of TT type, but the liver can still oxidize ethanol into acetaldehyde faster, and can quickly adapt to the physical discomfort after drinking. After drinking, they have a sense of "tips" and can rationally control their words and deeds; CC type is the least efficient in converting ethanol into acetaldehyde. After drinking, ethanol accumulates in the body for a long time and cannot be eliminated. It only relies on the liver's enzymes to slowly break down ethanol. The liver load increases accordingly, increasing the probability of inflammatory damage to liver cells and fatty degeneration. Such people are not recommended to drink.

[0126] The acetaldehyde dehydrogenase activity of GG type people is 100%, and they have good alcohol metabolism ability, so they can drink in moderation. The acetaldehyde dehydrogenase activity of GA type people decreases by about 45%, and their alcohol metabolism ability is poor, so it is best not to drink. The gene of AA type people is completely mutated, and the enzyme activity decreases by 90%, basically losing the ability to metabolize the toxic substance acetaldehyde. For people with complete ALDH2 gene mutation, the risk of developing alcohol-related colon cancer is about 2 times higher than other people; the risk of developing alcohol-related esophageal cancer is 4.13 times higher than other people; and they are prone to cirrhosis, so drinking is not recommended.

[0127] Example 6 Detection Limit of the Kit for Detecting Alcohol Metabolism

[0128] 1. The first generation sequencing was performed on 10 individuals to determine their genotypes. The results are shown in Table 10 below.

[0129] Table 10 First-generation sequencing genotypes

[0130]

[0131]

[0132] 2. Collect oral samples from 3 individuals (LBT, WXQ, NCY) covering all genotypes; then use one-step nucleic acid release solution to extract the genome of the collected samples, divide the obtained sample solution into 3 parts, keep one original sample solution, and use lysis solution to dilute the other two parts by 5 times and 10 times respectively, load the samples of the above three concentration gradients with gene amplification, and perform test strip typing detection. The experimental results are shown in Figure 5 , Figure 6 , Figure 5 The test results of ADH1B gene. Figure 6 This is the detection result of ALDH2 gene, and it can be seen that its detection limit is 5 times diluted sample solution.

[0133] Example 7 Repeatability of the kit for detecting alcohol metabolism ability

[0134] The double heterozygous samples (NCY) in Table 10 were tested for repeatability. The genome of the collected samples was extracted using a one-step nucleic acid release solution. The real samples were used as repeatability references to test the reagents. Each reference was repeated 10 times, and the results all met the requirements. The experimental results are shown in Figure 7 , Figure 8 , Figure 7 The test results of ADH1B gene. Figure 8 This is the test result of ALDH2 gene.

[0135] Example 8 Stability of the kit for detecting alcohol metabolism ability

[0136] The test kit within the validity period was placed at 4°C and 37°C for 7 days. Oral samples were collected from 3 individuals (LBT, WXQ, NCY) with all genotypes covered in Table 10; after the samples were loaded for gene amplification, the test strip typing test was performed. All tests were in line with expectations. The experimental results are shown in Fig. 9 , Fig.10 , Fig. 9 The test results of ADH1B gene. Fig.10 This is the test result of ALDH2 gene.

[0137] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit it. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present application.

[0138] In addition, those skilled in the art will appreciate that, although some embodiments herein include certain features included in other embodiments but not other features, the combination of features of different embodiments is meant to be within the scope of the present application and form different embodiments. For example, in the above claims, any one of the claimed embodiments may be used in any combination. The information disclosed in this background technology section is intended only to deepen the understanding of the overall background technology of the present application and should not be regarded as an admission or in any form of implication that the information constitutes prior art known to those skilled in the art.

Claims

1. A primer set for detecting polymorphisms of ADH1B gene and ALDH2 gene, characterized in that: It includes a primer set for amplifying the rs1229984 polymorphic site of the ADH1B gene and a primer set for amplifying the rs671 polymorphic site of the ALDH2 gene; The primer set for amplifying the rs1229984 polymorphic site of the ADH1B gene includes a T-type upstream primer, a C-type upstream primer, and a first common downstream primer; the T-type upstream primer and the C-type upstream primer are single-stranded DNAs with a length of 25-30 nucleotides, the 5' end of the T-type upstream primer is labeled with FAM, and the 5' end of the C-type upstream primer is labeled with digoxigenin; The primer set for amplifying the rs671 polymorphic site of the ALDH2 gene includes a G-type upstream primer, an A-type upstream primer, and a second common downstream primer; the G-type upstream primer and the A-type upstream primer are single-stranded DNAs with a length of 25-30 nucleotides, the 5' end of the G-type upstream primer is labeled with FAM, and the 5' end of the A-type upstream primer is labeled with digoxigenin; The first common downstream primer and the second common downstream primer are single-stranded DNA with a length of 40-50 nucleotides, with biotin labeled at the 5' end, the 16th nucleotide from the 3' end replaced with tetrahydrofuran, and C3 Spacer labeled at the 3' end.

2. The primer set for detecting polymorphisms of ADH1B gene and ALDH2 gene according to claim 1, characterized in that: The C-type upstream primer and the A-type upstream primer introduce a mismatched base next to the SNP site.

3. The primer set for detecting polymorphisms of ADH1B gene and ALDH2 gene according to claim 2, characterized in that: The T-type upstream primer is shown as SEQ ID No.1, and the C-type upstream primer is shown as SEQ ID No.

2.

4. The primer set for detecting polymorphisms of ADH1B gene and ALDH2 gene according to claim 2, characterized in that: The G-type upstream primer is shown as SEQ ID No.10, and the A-type upstream primer is shown as SEQ ID No.

11.

5. The primer set for detecting polymorphisms of ADH1B gene and ALDH2 gene according to claim 1, characterized in that: The first common downstream primer is shown as SEQ ID No.3, and the second common downstream primer is shown as SEQ ID No.

12.

6. A kit for detecting alcohol metabolism ability, characterized in that: The invention comprises a nucleic acid releasing solution, a constant temperature amplification freeze-dried reagent, and a double nucleic acid chromatography test strip, wherein the constant temperature amplification freeze-dried reagent comprises a primer set for detecting polymorphisms of the ADH1B gene and the ALDH2 gene according to any one of claims 1 to 5.

7. The kit for detecting alcohol metabolism ability according to claim 6, characterized in that: The isothermal amplification freeze-dried reagent comprises a first isothermal amplification freeze-dried reagent and a second isothermal amplification freeze-dried reagent; The first isothermal amplification freeze-dried reagent includes a first freeze-dried ball A and a freeze-dried ball B, wherein the first freeze-dried ball A includes a 2× isothermal amplification reagent and a primer set for amplifying the rs1229984 polymorphic site of the ADH1B gene; The second isothermal amplification freeze-dried reagent includes a second freeze-dried ball A and a freeze-dried ball B, and the second freeze-dried ball A includes a 2× isothermal amplification reagent and the primer set for amplifying the rs671 polymorphic site of the ALDH2 gene; The lyophilized ball B includes 5% by mass of PEG 35K and 60 mM of magnesium acetate.

8. The kit for detecting alcohol metabolism ability according to claim 7, characterized in that: The final molar concentration ratio of the T-type upstream primer, the C-type upstream primer and the first common downstream primer is 0.6:2:0.6; The final molar concentration ratio of the G-type upstream primer, the A-type upstream primer and the second common downstream primer is 1:1.5:0.

3.

9. The kit for detecting alcohol metabolism ability according to claim 6, characterized in that: The nucleic acid releasing solution comprises 50 mM NaOH, 2 mM EDTA, 0.5% by mass of SDS, and 1% by volume of Triton X100; The double nucleic acid chromatography test strip comprises a bottom plate, a sample pad, a conjugation pad, a nitrocellulose membrane, and a water-absorbing pad; a detection line 1 and a detection line 2 are fixed on the nitrocellulose membrane, the detection line 1 is coated with an anti-FITC / FAM antibody, the detection line 2 is coated with an anti-digoxigenin antibody, and the conjugation pad is fixed with a biotin ligand latex complex.

10. A method for detecting alcohol metabolism ability, characterized in that: The kit for detecting alcohol metabolism ability according to any one of claims 6 to 9 comprises: Collect oral mucosal cells; inserting a buccal swab into the nucleic acid releasing fluid; Taking the nucleic acid release solution and adding it into the isothermal amplification freeze-dried reagent to obtain an amplification product; The amplified product is added to the double nucleic acid chromatography test strip for typing detection to determine the genotype.

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