Freeze-dried human body ethanol metabolism gene detection reagent and preparation and detection methods thereof

Abstract

The invention discloses a freeze-drying reagent for human body ethanol metabolism gene detection and preparation and detection methods, the reagent is obtained by freeze-drying a reaction system, and the reaction system is a q-PCR detection reaction tube containing a reagent A and a reagent B. Compared with the existing liquid reagent, the freeze-drying reagent provided by the invention has the characteristics of supporting normal-temperature storage, being convenient to transport, being capable of getting rid of the problem of liquid cold chain transportation, being stored for more than 2 years, and having the positive effects of being not limited by a detection environment, getting rid of the problem of dependence on laboratory conditions, and being free from operation and interpretation of professionals; the method is convenient for non-professionals to operate, the detection result is not influenced, and the use requirement of the freeze-drying reagent is met, so that the use scene of the freeze-drying reagent can be expanded, and the method has great market competitiveness.

Description

Technical Field

[0001] The present invention relates to the technical field of gene detection, and in particular to a freeze-dried reagent for detecting human ethanol metabolism genes and a preparation and detection method thereof. Background Art

[0002] Alcohol, chemically known as ethanol, is absorbed into the bloodstream through the stomach and intestines after consumption. 90% to 95% of the alcohol is metabolized in the liver, with the remainder excreted through urine, sweat, and respiration. Ethanol undergoes two primary metabolic reactions in the liver: oxidation to acetaldehyde by alcohol dehydrogenase ADH1B; and then oxidation of acetaldehyde to acetic acid by acetaldehyde dehydrogenase. Alcohol dehydrogenase is primarily encoded by the ADH1B gene, while acetaldehyde dehydrogenase is primarily encoded by the ALDH2 gene. During alcohol metabolism, individuals with high acetaldehyde dehydrogenase activity have a strong ability to metabolize alcohol, while those with low acetaldehyde dehydrogenase activity have a weak ability to metabolize alcohol. This weakened ability to metabolize alcohol leads to elevated blood levels of ethanol or acetaldehyde after drinking, which can lead to conditions such as alcohol poisoning, alcoholic liver disease, and gastrointestinal cancers.

[0003] Alcohol consumption, a long-standing Chinese tradition, is an unavoidable part of daily life, work, and interpersonal interactions. After drinking, the metabolism of ethanol in the human body relies primarily on the catalysis of two enzymes: alcohol dehydrogenase and acetaldehyde dehydrogenase. Most of the ethanol is converted to acetaldehyde by alcohol dehydrogenase, which then converts it to acetic acid. This acetaldehyde enters the oxidation cycle and is ultimately metabolized into carbon dioxide and water for excretion. Aldehyde dehydrogenase 2 (ALDH2), a key enzyme in alcohol metabolism, is located at the Glu504Lys polymorphism (also known as rs671) in exon 12. This site undergoes a base substitution, replacing guanine (G, wild type) with adenine (A, mutant), resulting in a change in amino acid position 504 of the enzyme: a glutamic acid (Glu) to lysine (Lys). There are three types of ALDH2 genotypes in the human population: the wild homozygous type (GG type, also known as ALDH2*1 / *1), which produces an enzyme with normal catalytic activity; the mutant heterozygous type (GA type, also known as ALDH2*1 / *2), which produces an enzyme with decreased catalytic activity, only 10%-45% of the wild-type enzyme activity; and the mutant homozygous type (AA type, also known as ALDH2*2 / *2), which produces an enzyme activity of only 1%-5% of the normal level.

[0004] The principle of alcohol tolerance gene testing is to assess an individual's alcohol metabolism ability by detecting single nucleotide polymorphism (SNP) sites (such as ALDH2rs671 and ADH1B rs1229984). Current testing technology mainly relies on conventional liquid reagents, which have the following problems:

[0005] 1. The whole process needs to be stored at low temperature (2-8℃), which has high transportation costs and is prone to failure;

[0006] 2. The enzyme activity in liquid reagents is easily affected by temperature fluctuations, leading to the risk of false negatives / positives;

[0007] 3. Professionals are required to use centrifuges, PCR instruments and other equipment, making it difficult to achieve on-site point-of-care testing (POCT).

[0008] It can be seen from this that using the existing detection method, since most of the detection reagents are liquid reagents, not only do they need to be frozen during transportation, resulting in extremely high transportation costs, but they also need to be prepared and used immediately at the detection site, which places certain requirements on the experimental personnel's operational capabilities.

[0009] Therefore, if the existing detection reagents are made into freeze-dried reagents that are stable at room temperature, such as microspheres or freeze-dried powders, through the freeze-drying process, it will not only solve the problem of dependence on cold chain transportation, but also allow them to be stored at room temperature after freeze-drying, and the shelf life will be extended to more than 2 years. This can greatly reduce transportation and storage costs. At the same time, it can also break away from the limitations of the detection environment and the laboratory, and realize portable on-site detection, which has obvious advantages over liquid reagents, thus expanding its application scenarios. Summary of the Invention

[0010] The technical problem to be solved by the present invention is to address the problems existing in the background technology. In order to solve the problem that the detection reagent needs to be stored at low temperature and the enzyme activity is unstable, a freeze-dried reagent is provided, and the detection reagent is made into a form that is stable at room temperature - freeze-dried powder, so that the detection reagent can be stored at room temperature and the shelf life is extended. At the same time, a preparation method of the freeze-dried reagent and a method for detecting human ethanol metabolism genes using the freeze-dried reagent are also provided, so that portable on-site detection can be realized. Specifically, a freeze-dried reagent for detecting human ethanol metabolism genes and a preparation and detection method are provided.

[0011] To solve the above technical problems, the technical solution adopted by the present invention is: a freeze-dried reagent for human ethanol metabolism gene detection, which is obtained by freeze-drying the following reaction system, wherein the reaction system is a q-PCR detection reaction tube containing reagent A and reagent B.

[0012] Furthermore, the reagents described herein are used, wherein the reagent A is a mixture containing 0.5-1.25 U / μL Taq polymerase, 0.2-0.4 mM dNTPs deoxynucleotides, 1.5-3.5 mM magnesium chloride solution, 10-20 mM Tris-HCl buffer, 0.1-1 μM primer I, 0.1-0.5 μM probe I, 50-65 mM HCl solution, 0.5 μg / μL BSA bovine serum albumin, and 3-5% volume percent trehalose solution; and the reagent B is a mixture containing 0.5-1.25 U / μL Taq polymerase, 0.2-0.4 mM dNTPs deoxynucleotides, 1.5-3.5 mM magnesium chloride solution, 10-20 mM Tris-HCl buffer, 0.1-1 μM primer II, 0.1-0.5 μM probe II, 50-65 mM A mixture of HCl solution, 0.5 μg / μL BSA bovine serum albumin, and trehalose solution with a volume percentage concentration of 3-5%.

[0013] Furthermore, using the reagent described herein, the primer I includes a primer set for amplifying the rs1229984 polymorphic site of the ADH1 B gene, the primer II includes a primer set for amplifying the rs671 polymorphic site of the ALDH2 gene, and the probe I includes a probe set for detecting the rs1229984 polymorphic site of the ADH1 B gene, and the probe II includes a probe set for detecting the rs671 polymorphic site of the ALDH2 gene; wherein the primer set in the primer I includes a forward primer I and a reverse primer I, and the primer set in the primer II includes a forward primer II and a reverse primer II, the probe set in the probe I includes a probe-C and a probe-T, and the probe set in the probe II includes a probe-G and a probe-A; the sequences of the forward primer I, reverse primer I, forward primer II, reverse primer II, and probe-C, probe-T, probe-G, and probe-A are respectively:

[0014] Forward primer I sequence: 5′-ACACAATTTCAGGAATTTGGGTATG-3′;

[0015] Reverse primer I sequence: 5′-TCACCAGGTTGCCACTAACCA-3′;

[0016] Forward primer II sequence: 5′-GGGAGTTGGGCGAGTACG-3′;

[0017] Reverse primer II sequence: 5′-AGACCCTCAAGCCCCAACAG-3′;

[0018] Probe-C sequence: 5′-FAM-ACGTACGTACGTACGT-BHQ1-3′;

[0019] Probe-T sequence: 5′-VIC-GTACGTACGTACGTACG-BHQ1-3′;

[0020] Probe-G sequence: 5′-FAM-ACGTACGTACGTACGT-BHQ1-3′;

[0021] Probe-A sequence: 5′-VIC-GTACGTACGTACGTACG-BHQ1-3′;

[0022] Wherein, the FAM and VIC are fluorescent groups, and BHQ1 is a quenching group.

[0023] The present invention also provides a method for preparing the above-mentioned freeze-dried reagent for human ethanol metabolism gene detection, wherein the preparation method comprises adding reagent A and reagent B into q-PCR reaction tubes respectively, and using a processing system with a cold trap function, wherein the processing system includes a freeze dryer with a compressor and a vacuum pump, and first pre-cooling the q-PCR reaction tubes, and then immediately placing them into the freeze dryer for freeze-drying after the pre-cooling treatment, and after freeze-drying, obtaining a freeze-dried powder reagent containing reagent A and reagent B.

[0024] Furthermore, the preparation method of the present invention comprises the following steps:

[0025] S1. Place the q-PCR reaction tubes containing Reagent A and Reagent B into a drying tray, which is then placed on a pre-freeze rack in a lyophilizer.

[0026] S2. Install the insulation cover with a pressure-type cover, and ensure that there is space for the insulation cover.

[0027] S3. Close the plexiglass cover on the freeze dryer.

[0028] S4. Turn on the freeze dryer and the compressor to rapidly lower the temperature. Use a cold trap to pre-cool the q-PCR reaction tubes in the pre-freeze rack.

[0029] S5. After pre-freezing is completed, remove the pre-freezing rack from the cold trap device;

[0030] S6. Quickly transfer the drying tray to the freeze-drying rack, install the plexiglass cover, and seal it with an O-ring.

[0031] S7. Turn on the vacuum pump and freeze-dry the sealed sample under vacuum;

[0032] S8. After the sample is freeze-dried, quickly cover the reaction tube with the lid used to seal it. This will give the freeze-dried powder reagent containing Reagent A and Reagent B. Finally, turn off the vacuum pump and compressor.

[0033] Furthermore, in the preparation method of the present invention, the vacuum environment requires maintaining the cavity pressure less than 10Pa to ensure efficient sublimation; in the refrigeration process of starting the compressor to rapidly lower the temperature, it is required to provide a cold trap temperature below -80°C to capture sublimated water vapor; and in the freeze-drying process of turning on the vacuum pump, the heating system used can control the shelf temperature between -50°C and +50°C to achieve stage-by-stage adjustment of the drying temperature for sublimation and desorption.

[0034] Furthermore, using the preparation method of the present invention, the specific control procedure of the freeze-drying process is:

[0035] S1. Precooling: Precool the plate to 2-4°C and hold for 10-12 minutes; then cool to 45--35°C and hold for 180-210 minutes;

[0036] S2 sublimation drying: first freeze-dry at -35 to -25°C for 210 to 240 minutes; then, heat to -15 to -10°C and freeze-dry for 480 to 540 minutes;

[0037] S3. Desorption and drying: Heat to 25-30°C and maintain for 240-300 minutes.

[0038] The present invention also provides a detection method for detecting human ethanol metabolism genes using the above reagent, the detection method comprising the following steps:

[0039] S1. Collect oral saliva;

[0040] S2. Add the collected oral saliva to a sample lysis buffer, vortex, and allow to stand to obtain a sample DNA solution, wherein the sample lysis buffer comprises 10-50 mM Tris-HCl buffer (pH 8.0-8.5), 1-10 mM EDTA, 100-500 mM NaCl, 0.1-2% SDS, 0.1-1% Triton X-100, and 1-4 M urea;

[0041] S3. The DNA solution obtained in step S2 is placed into the reagent containing reagent A and reagent B, and mixed thoroughly to completely dissolve the lyophilized powder to obtain a detection solution;

[0042] S4. The test liquid obtained in step S3 is tested on a machine to obtain and analyze the results.

[0043] Furthermore, using the detection method described herein, in step S2, the amount of the sample lysate used is 200 μL, the vortex time is 5 to 10 s, the standing time is 20 s, and the sample DNA solution is obtained after standing; and in step S3, the amount of the DNA solution added is 25 to 40 μL.

[0044] Furthermore, using the detection method described herein, during the on-machine detection process in step S4, the specific operation method is to place the obtained qPCR reaction tube into the qPCR instrument and perform the reaction according to the PCR program. The PCR program includes: pre-denaturation at a temperature of 95°C for 2 minutes, 1 cycle; detection at a temperature of 95°C for 5 seconds, detection at a temperature of 60°C for 35 seconds, 40 cycles, and collection of FAM and VIC fluorescence signals at a temperature of 60°C, requiring the qPCR heating and cooling rate to be 6°C; finally, based on the data results collected by the qPCR instrument, the sample gene data is obtained, and based on the relationship between the gene data and the alcohol consumption, the alcohol consumption of the sample is judged.

[0045] The freeze-dried reagent for human ethanol metabolism gene detection and its preparation and detection method described in the present invention have the following beneficial effects compared with the existing technology: the freeze-dried reagent has the advantage of reducing the time for adding reagents from the original 1 minute per reagent to only needing to take out the freeze-dried powder tube for use; it is characterized by supporting room temperature storage, easy transportation, and being able to get rid of the problem of liquid cold chain transportation, and the storage time can be up to more than 2 years; its positive effect is that it is not restricted by the detection environment and gets rid of the problem of dependence on laboratory conditions; in addition, the freeze-dried reagent described in the present invention is used for human ethanol metabolism gene detection, and portable instruments can be used directly for experiments, thereby expanding the use scenarios. The sample only needs to be added to the freeze-dried reagent and shaken to mix before it can be put into the machine. No professional operation and interpretation are required, which is convenient for non-professional operation. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] The present invention will be further described in detail below with reference to the accompanying drawings.

[0047] Figure 1 is the fluorescence signal result of sample 1 in Example 4;

[0048] Figure 2 The fluorescence signal result of sample 2 in Example 4;

[0049] Figure 3 This is the fluorescence signal result of sample 3 in Example 4;

[0050] Figure 4 is the fluorescence signal result of sample 4 in Example 4;

[0051] Figure 5 This is the fluorescence signal result of sample 5 in Example 4;

[0052] Figure 6 is the fluorescence signal result of sample 1 in Example 5;

[0053] Figure 7 The fluorescence signal result of sample 2 in Example 5;

[0054] Figure 8 This is the fluorescence signal result of sample 3 in Example 5;

[0055] Figure 9 is the fluorescence signal result of sample 4 in Example 5;

[0056] Figure 10 is the fluorescence signal result of sample 5 in Example 5;

[0057] Figure 11 This is a physical structural diagram of the processing system product used in the present invention. DETAILED DESCRIPTION

[0058] The following describes the implementation of the present invention through specific embodiments. People skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0059] It should be noted that the structures, proportions, sizes, etc. depicted in the drawings of this specification are only used to match the contents disclosed in the specification for understanding and reading by those familiar with this technology, and are not used to limit the conditions under which the present invention can be implemented. Therefore, they have no substantial technical significance. Any modification of the structure, change in the proportional relationship, or adjustment of the size should still fall within the scope of the technical content disclosed by the present invention without affecting the efficacy and purpose that can be achieved by the present invention. At the same time, the terms such as "upper", "lower", "left", and "right" quoted in this specification are only for the convenience of description and are not used to limit the scope of the implementation of the present invention. Changes or adjustments in their relative relationships should also be considered as the scope of the implementation of the present invention without substantially changing the technical content.

[0060] In the description of the present invention, it should be noted that, unless otherwise expressly specified or limited, the terms "connected" and "provided with" should be understood in a broad sense. For example, they may refer to fixed connections, detachable connections, or integral connections; mechanical connections, electrical connections; and direct connections or indirect connections through an intermediary. Those skilled in the art will understand the specific meanings of the above terms in the present invention.

[0061] It should be noted that the term "comprise" or any other variation is intended to cover non-exclusive inclusion, so that a process, method, article or apparatus that includes a series of elements includes not only those elements, but also includes other elements not explicitly listed, or also includes elements that are inherent to such process, method, article or apparatus.

[0062] Example 1

[0063] This embodiment provides a reagent obtained by freeze-drying the following reaction system, wherein the reaction system is a reaction tube for q-PCR detection containing reagent A and reagent B; wherein reagent A is a mixture containing 0.5-1.25 U / μL Taq polymerase, 0.2-0.4 mM dNTPs deoxynucleotides, 1.5-3.5 mM magnesium chloride solution, 10-20 mM Tris-HCl buffer, 0.1-1 μM primer I, 0.1-0.5 μM probe I, 50-65 mM HCl solution, 0.5 μg / μL BSA bovine serum albumin, and 3-5% by volume trehalose solution;

[0064] The reagent B is a mixture containing 0.5-1.25 U / μL Taq polymerase, 0.2-0.4 mM dNTPs deoxynucleotides, 1.5-3.5 mM magnesium chloride solution, 10-20 mM Tris-HCl buffer, 0.1-1 μM primer II, 0.1-0.5 μM probe II, 50-65 mM HCl solution, 0.5 μg / μL BSA bovine serum albumin, and 3-5% by volume trehalose solution.

[0065] Furthermore, using the reagents provided in this embodiment, the primer I includes a primer set for amplifying the rs1229984 polymorphic site of the ADH1 B gene, the primer II includes a primer set for amplifying the rs671 polymorphic site of the ALDH2 gene, and the probe I includes a probe set for detecting the rs1229984 polymorphic site of the ADH1 B gene, and the probe II includes a probe set for detecting the rs671 polymorphic site of the ALDH2 gene; wherein the primer set in the primer I includes a forward primer I and a reverse primer I, and the primer set in the primer II includes a forward primer II and a reverse primer II, the probe set in the probe I includes a probe-C and a probe-T, and the probe set in the probe II includes a probe-G and a probe-A; the sequences of the forward primer I, reverse primer I, forward primer II, reverse primer II, and probe-C, probe-T, probe-G, and probe-A are respectively:

[0066] Forward primer I sequence: 5′-ACACAATTTCAGGAATTTGGGTATG-3′;

[0067] Reverse primer I sequence: 5′-TCACCAGGTTGCCACTAACCA-3′;

[0068] Forward primer II sequence: 5′-GGGAGTTGGGCGAGTACG-3′;

[0069] Reverse primer II sequence: 5′-AGACCCTCAAGCCCCAACAG-3′;

[0070] Probe-C sequence: 5′-FAM-ACGTACGTACGTACGT-BHQ1-3′;

[0071] Probe-T sequence: 5′-VIC-GTACGTACGTACGTACG-BHQ1-3′;

[0072] Probe-G sequence: 5′-FAM-ACGTACGTACGTACGT-BHQ1-3′;

[0073] Probe-A sequence: 5′-VIC-GTACGTACGTACGTACG-BHQ1-3′;

[0074] Wherein, the FAM and VIC are fluorescent groups, and BHQ1 is a quenching group.

[0075] For the convenience of description, the primer set used to amplify the rs1229984 polymorphic site of the ADH1 B gene and the primer set used to amplify the rs671 polymorphic site of the ALDH2 gene, as well as the primer and probe sequences involved, are listed in Table 1.

[0076] Table 1 Primer and probe sequence list

[0077] serial number name sequence Forward primer I ADH1Brs1229984-F 5'-ACACAATTTCAGGAATTTGGGTATG-3' Reverse primer I ADH1Brs1229894-R 5'-TCACCAGGTTGCCACTAACCA-3' Probe I ADH1Brs1229894 probe-C 5'-FAM-ACGTACGTACGTACGT-BHQ1-3' Probe I ADH1Brs1229894 probe-T 5'-VIC-GTACGTACGTACGTACG-BHQ1-3' Forward primer II ALDH2rs671-F 5'-GGGAGTTGGGCGAGTACG-3' Reverse primer II ALDH2rs671-R 5'-AGACCCTCAAGCCCCAACAG-3' Probe II ALDH2rs671 Probe-G 5'-FAM-ACGTACGTACGTACGT-BHQ1-3' Probe II ALDH2rs671 Probe-A 5'-VIC-GTACGTACGTACGTACG-BHQ1-3'

[0078] Note: FAM and VIC described in Table 1 are fluorescent groups, while BHQ1 is a quenching group.

[0079] Example 2

[0080] This embodiment provides a preparation method for preparing the above-mentioned freeze-dried human ethanol metabolism gene detection reagent, wherein the preparation method is to add reagent A and reagent B into q-PCR reaction tubes respectively, and adopt a processing system with a cold trap function, wherein the processing system includes a freeze dryer with a compressor and a vacuum pump 1, the freeze dryer includes an exhaust hole 2, a material tray 3, a plexiglass cover 4, a shelf 5, an O-ring 6, a touch screen 7 and a heat dissipation port 8, and the vacuum pump 1 is connected to the freeze dryer through the exhaust hole 2. The actual product of the processing system is shown in FIG. Figure 11 As shown, the q-PCR reaction tube is first pre-cooled and then immediately placed in a freeze dryer for freeze drying. After freeze drying, a freeze-dried powder reagent containing reagent A and reagent B is obtained. The preparation method specifically includes the following steps:

[0081] S1. Place the q-PCR reaction tubes containing Reagent A and Reagent B into a drying tray, which is then placed on a pre-freeze rack in a lyophilizer.

[0082] S2. Install the insulation cover with a pressure-type cover, and ensure that there is space for the insulation cover.

[0083] S3. Close the plexiglass cover on the freeze dryer.

[0084] S4. Turn on the freeze dryer and the compressor to rapidly lower the temperature. Use a cold trap to pre-cool the q-PCR reaction tubes in the pre-freeze rack.

[0085] S5. After pre-freezing is completed, remove the pre-freezing rack from the cold trap device;

[0086] S6. Quickly transfer the drying tray to the freeze-drying rack, install the plexiglass cover, and seal it with an O-ring.

[0087] S7. Turn on the vacuum pump and freeze-dry the sealed sample under vacuum;

[0088] S8. After the sample is freeze-dried, quickly cover the reaction tube with the lid used to seal it. This will give the freeze-dried powder reagent containing Reagent A and Reagent B. Finally, turn off the vacuum pump and compressor.

[0089] Using the preparation method provided in this embodiment, in the specific preparation process, the vacuum environment requires maintaining the cavity pressure less than 10Pa to ensure the efficient sublimation process; in the refrigeration process of starting the compressor to rapidly lower the temperature, it is required to provide a cold trap temperature below -80°C to capture the sublimated water vapor; and in the freeze-drying process when the vacuum pump is turned on, the heating system used can control the shelf temperature between -50°C and +50°C to achieve stage-by-stage adjustment of the drying temperature for sublimation and desorption.

[0090] Furthermore, using the preparation method provided in this embodiment, the specific control procedure of the freeze-drying process is:

[0091] S1. Precooling: Precool the plate to 2-4°C and hold for 10-12 minutes; then cool to 45--35°C and hold for 180-210 minutes;

[0092] S2 sublimation drying: first freeze-dry at -35 to -25°C for 210 to 240 minutes; then, heat to -15 to -10°C and freeze-dry for 480 to 540 minutes;

[0093] S3. Desorption and drying: Heat to 25-30°C and maintain for 240-300 minutes.

[0094] Example 3

[0095] This example provides a method for detecting human ethanol metabolism genes using a reagent prepared by the method described in Example 2. The detection method comprises the following steps:

[0096] S1. Collect oral saliva;

[0097] S2. Add 200 μL of the collected oral saliva to a sample lysis buffer, vortex for 5 to 10 seconds, and let stand for 20 seconds to obtain a sample DNA solution. The sample lysis buffer is a mixture consisting of 10 to 50 mM Tris-HCl buffer (pH 8.0 to 8.5), 1 to 10 mM EDTA, 100 to 500 mM NaCl, 0.1 to 2% SDS, 0.1 to 1% Triton X-100, and 1 to 4 M urea.

[0098] S3. Take 25 to 40 μL of the DNA solution obtained in step S2 and add it to the reagent containing reagent A and reagent B, mix thoroughly to completely dissolve the lyophilized powder to obtain a detection solution;

[0099] S4. The test liquid obtained in step S3 is tested on a machine to obtain and analyze the results. During the on-machine testing process, the specific operation method is: the obtained qPCR reaction tube is placed in the qPCR instrument, and the reaction is carried out according to the PCR program. The PCR program includes: pre-denaturation at a temperature of 95°C for 2 minutes, 1 cycle; detection at a temperature of 95°C for 5 seconds, detection at a temperature of 60°C for 35 seconds, 40 cycles, and collection of FAM and VIC fluorescence signals at a temperature of 60°C. The qPCR heating and cooling rate is required to be 6°C; finally, the sample gene data is obtained based on the data results collected by the qPCR instrument, and the alcohol consumption of the sample is judged based on the relationship between the gene data and the alcohol consumption.

[0100] Example 4

[0101] According to the detection method described in Example 3, in order to better illustrate the advantages of the present invention, a single nucleotide polymorphism (SNP) site (ADH1 Brs1229984) was actually tested to evaluate the alcohol metabolism ability of an individual.

[0102] 1000 samples with known alcohol tolerance genotypes were tested. Only 5 samples are listed in this invention. The detailed experimental results are shown in Table 2. The fluorescence signal results are shown in Table 2. Figures 1 to 5 shown.

[0103] Table 2 ADH1 B rs1229984 genotype interpretation

[0104]

[0105] The experiment collected fluorescence signals from two channels, FAM and VIC. FAM and VIC correspond to the T / C genotype of ADH1Brs1229984, respectively. When the Ct value of the FAM channel is read but the Ct value of the VIC channel is not (N / A), the genotype is determined to be TT. When the Ct values ​​of both the FAM and VIC channels are read, the genotype is determined to be T / C. When the Ct value of the FAM channel is not (N / A) but the Ct value of the VIC channel is read, the genotype is determined to be C / C. The Ct values ​​obtained from the FAM / VIC channel assays can be used to determine the ADH1Brs1229984 genotype in the sample. The experimental results are consistent with the sequencing results of the sample, demonstrating that this method can accurately detect the ADH1B rs1229984 genotype in the sample. Each assay takes approximately 20 minutes.

[0106] Example 5

[0107] According to the detection method described in Example 3, in order to better illustrate the advantages of the present invention, a single nucleotide polymorphism (SNP) site (ALDH2rs671) was actually tested to evaluate the alcohol metabolism ability of an individual.

[0108] 1000 samples with known alcohol tolerance genotypes were tested. Only 5 samples are listed in this invention. The detailed experimental results are shown in Table 3. The fluorescence signal results are shown in Table 3. Figures 6 to 10 shown.

[0109] Table 3 ALDH2rs671 genotype interpretation

[0110]

[0111] The experiment collected fluorescence signals from two channels, FAM and VIC. FAM and VIC correspond to the T / C genotype of ALDH2 rs671, respectively. When the Ct value of the FAM channel is read but the Ct value of the VIC channel is not (N / A), the genotype is determined to be G / G; when the Ct values ​​of both the FAM and VIC channels are read, the genotype is determined to be G / A; when the Ct value of the FAM channel is not (N / A) but the Ct value of the VIC channel is read, the genotype is determined to be A / A. Based on the Ct values ​​obtained by the FAM / VIC channel detection, the ALDH2 rs671 genotype in the sample can be determined. The experimental results are consistent with the sequencing results of the sample, demonstrating that this method can accurately detect the ALDH2 rs671 genotype of the sample. Each test takes approximately 20 minutes.

[0112] Based on the detailed experimental results obtained in Example 4 and Example 5, a comprehensive judgment of the sample genes was performed, and the results are shown in Table 4.

[0113] Table 4 Comprehensive judgment of sample genes

[0114]

[0115] Result analysis:

[0116] Since samples 1-5 in Tables 2 and 3 are from the same group of samples, the alcohol tolerance genotype of the samples can be obtained according to the experimental results in Tables 2 and 3, as well as the rules for interpreting the alcohol tolerance genotype test results. The test results are the same as the known sample gene results, indicating that the detection accuracy of this experiment is 100%.

[0117] It can be seen that the freeze-dried reagent provided by the present invention, compared with the existing liquid reagent, is characterized by supporting room temperature storage, easy transportation, and being able to get rid of the problem of liquid cold chain transportation, and the storage time can be up to more than 2 years. Its positive effect is that it is not restricted by the detection environment, gets rid of the problem of dependence on laboratory conditions, does not require professional operation and interpretation, is convenient for non-professional operation, and the test results are not affected, meeting the use requirements of the freeze-dried reagent, thereby being able to expand its use scenarios and having great market competitiveness.

[0118] Other aspects of the present invention that are not described in detail are all conventional techniques known to those skilled in the art.

[0119] The protection scope of the present invention is not limited to the technical solutions disclosed in the specific implementation methods. The above description is only a preferred implementation method of the present invention and does not limit the present invention. Any minor modifications, equivalent replacements and improvements made based on the technical solutions of the present invention should be included in the protection scope of the technical solutions of the present invention.

Claims

1. A freeze-dried reagent for detecting human ethanol metabolism genes, characterized in that: The reagent is obtained by freeze-drying the following reaction system, which is a reaction tube for q-PCR detection containing reagent A and reagent B.

2. The reagent according to claim 1, characterized in that: The reagent A is a mixture containing 0.5-1.25 U / μL Taq polymerase, 0.2-0.4 mM dNTPs deoxynucleotides, 1.5-3.5 mM magnesium chloride solution, 10-20 mM Tris-HCl buffer, 0.1-1 μM primer I, 0.1-0.5 μM probe I, 50-65 mM HCl solution, 0.5 μg / μL BSA bovine serum albumin, and 3-5% by volume trehalose solution; while the reagent B is a mixture containing 0.5-1.25 U / μL Taq polymerase, 0.2-0.4 mM dNTPs deoxynucleotides, 1.5-3.5 mM magnesium chloride solution, 10-20 mM Tris-HCl buffer, 0.1-1 μM primer II, 0.1-0.5 μM probe II, 50-65 mM HCl solution, 0.5 μg / μL The mixed solution consists of BSA bovine serum albumin and trehalose solution with a volume percentage concentration of 3-5%.

3. The reagent according to claim 2, characterized in that: The primer I includes a primer set for amplifying the rs1229984 polymorphic site of the ADH1 B gene, the primer II includes a primer set for amplifying the rs671 polymorphic site of the ALDH2 gene, and the probe I includes a probe set for detecting the rs1229984 polymorphic site of the ADH1 B gene, and the probe II includes a probe set for detecting the rs671 polymorphic site of the ALDH2 gene; wherein the primer set in the primer I includes a forward primer I and a reverse primer I, and the primer set in the primer II includes a forward primer II and a reverse primer II, the probe set in the probe I includes a probe-C and a probe-T, and the probe set in the probe II includes a probe-G and a probe-A; the sequences of the forward primer I, reverse primer I, forward primer II, reverse primer II, and probe-C, probe-T, probe-G, and probe-A are respectively: Forward primer I sequence: 5′-ACACAATTTCAGGAATTTGGGTATG-3′; Reverse primer I sequence: 5′-TCACCAGGTTGCCACTAACCA-3′; Forward primer II sequence: 5′-GGGAGTTGGGCGAGTACG-3′; Reverse primer II sequence: 5′-AGACCCTCAAGCCCCAACAG-3′; Probe-C sequence: 5′-FAM-ACGTACGTACGTACGT-BHQ1-3′; Probe-T sequence: 5′-VIC-GTACGTACGTACGTACG-BHQ1-3′; Probe-G sequence: 5′-FAM-ACGTACGTACGTACGT-BHQ1-3′; Probe-A sequence: 5′-VIC-GTACGTACGTACGTACG-BHQ1-3′; Wherein, the FAM and VIC are fluorescent groups, and BHQ1 is a quenching group.

4. A method for preparing the reagent according to any one of claims 1 to 3, characterized in that: The preparation method comprises the following steps: adding reagent A and reagent B to a q-PCR reaction tube, respectively; and using a processing system with a cold trap function, wherein the processing system includes a freeze dryer with a compressor and a vacuum pump. The q-PCR reaction tube is first pre-cooled and then immediately placed in the freeze dryer for freeze-drying. After freeze-drying, a freeze-dried powder reagent containing reagent A and reagent B is obtained.

5. The preparation method according to claim 4, characterized in that The preparation method comprises the following steps: S1. Place the q-PCR reaction tubes containing Reagent A and Reagent B into a drying tray, which is then placed on a pre-freeze rack in a lyophilizer. S2. Install the insulation cover with a pressure-type cover, and ensure that there is space for the insulation cover. S3. Close the plexiglass cover on the freeze dryer. S4. Turn on the freeze dryer and the compressor to rapidly lower the temperature. Use a cold trap to pre-cool the q-PCR reaction tubes in the pre-freeze rack. S5. After pre-freezing is completed, remove the pre-freezing rack from the cold trap device; S6. Quickly transfer the drying tray to the freeze-drying rack, install the plexiglass cover, and seal it with an O-ring. S7. Turn on the vacuum pump and freeze-dry the sealed sample under vacuum; S8. After the sample is freeze-dried, quickly cover the reaction tube with the lid used to seal it. This will give the freeze-dried powder reagent containing Reagent A and Reagent B. Finally, turn off the vacuum pump and compressor.

6. The preparation method according to claim 5, characterized in that: The vacuum environment requires maintaining the cavity pressure below 10 Pa to ensure efficient sublimation. During the refrigeration process when the compressor is started to rapidly lower the temperature, a cold trap temperature below -80°C is required to be provided in order to capture sublimated water vapor. During the freeze-drying process when the vacuum pump is turned on, the heating system used can control the shelf temperature between -50°C and +50°C, so as to achieve stage-by-stage adjustment of the drying temperature for sublimation and desorption.

7. The preparation method according to claim 6, characterized in that: The specific control procedures of the freeze-drying process are: S1. Precooling: Precool the plate to 2-4°C and hold for 10-12 minutes; then cool to 45--35°C and hold for 180-210 minutes; S2 sublimation drying: first freeze-dry at -35 to -25°C for 210 to 240 minutes; then, heat to -15 to -10°C and freeze-dry for 480 to 540 minutes; S3. Desorption and drying: Heat to 25-30°C and maintain for 240-300 minutes.

8. A method for detecting human ethanol metabolism genes, characterized in that: The detection method uses the reagent according to any one of claims 1 to 4, and the detection method comprises the following steps: S1. Collect oral saliva; S2. Add the collected oral saliva to a sample lysis buffer, vortex, and allow to stand to obtain a sample DNA solution, wherein the sample lysis buffer comprises 10-50 mM Tris-HCl buffer (pH 8.0-8.5), 1-10 mM EDTA, 100-500 mM NaCl, 0.1-2% SDS, 0.1-1% Triton X-100, and 1-4 M urea; S3. The DNA solution obtained in step S2 is placed into the reagent containing reagent A and reagent B, and mixed thoroughly to completely dissolve the lyophilized powder to obtain a detection solution; S4. The test liquid obtained in step S3 is tested on a machine to obtain and analyze the results.

9. The detection method according to claim 8, wherein: In step S2, the amount of the sample lysate used is 200 μL, the vortex time is 5-10 s, and the standing time is 20 s. After standing, a sample DNA solution is obtained; and in step S3, the amount of the DNA solution added is 25-40 μL.

10. The detection method according to claim 8, wherein: During the on-machine detection process in step S4, the specific operation method is to place the obtained qPCR reaction tube into the qPCR instrument and react according to the PCR program. The PCR program includes: pre-denaturation at a temperature of 95°C for 2 minutes, 1 cycle; detection at a temperature of 95°C for 5 seconds, detection at a temperature of 60°C for 35 seconds, 40 cycles, and collection of FAM and VIC fluorescence signals at a temperature of 60°C. The qPCR heating and cooling rate is required to be 6°C; finally, the sample gene data is obtained based on the data results collected by the qPCR instrument, and the alcohol consumption of the sample is judged based on the relationship between the gene data and the alcohol consumption.

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