Kit and detection method for detecting human gene mutation related to alcohol metabolism capability

Abstract

The invention relates to the technical field of gene mutation detection, and discloses a kit for detecting human gene mutation related to alcohol metabolism capability, which comprises a nucleic acid releasing agent and a PCR (Polymerase Chain Reaction) reaction solution, the nucleic acid releasing agent is prepared from the following components: 10 to 20 mM of Tris, 20 to 100 mM of NaCl, 2 to 10 mM of EDTA (Ethylene Diamine Tetraacetic Acid), 0.1 to 1 percent of SDS (w / v), 5 to 20 mg / mL of protease K and 0.005 to 0.02 percent of TritonX-100 (v / v). The human throat swab serves as a detection sample, sampling is easy and rapid, meanwhile, compared with complex components of a whole blood sample, components in the throat swab sample are simple, subsequent amplification detection can be conducted after nucleic acid is released through the nucleic acid releasing agent, and tedious extraction and purification processes are not needed.

Description

Technical Field

[0001] The present invention relates to the technical field of gene mutation detection, and in particular to a kit and a detection method for detecting human gene mutations related to alcohol metabolism ability. Background Art

[0002] Alcohol (ethanol) is mainly metabolized by the liver in the human body. After ethanol enters the human body, it is decomposed into acetaldehyde by the action of alcohol dehydrogenase, and then converted into acetic acid by acetaldehyde dehydrogenase, and then decomposed into water and carbon dioxide and excreted from the body. Among them, acetaldehyde is harmful to the human body and will stimulate the nerves. It is listed as a Class I carcinogen by the World Health Organization; if it cannot be decomposed in time, it will accumulate in the body, causing drunkenness phenomena such as blushing, dizziness, vomiting, and even threatening life. Alcohol dehydrogenase and acetaldehyde dehydrogenase are one of the important metabolic enzymes in the human body, encoded by ADH1B and ALDH2 genes, respectively. If the ADH1B and ALDH2 gene sequences mutate, such as base deletions, base insertions, and single base mutations, the activity of the alcohol dehydrogenase and acetaldehyde dehydrogenase encoded by them may be reduced or inactive, thereby affecting the ability to metabolize and decompose ethanol in the body. For East Asian and Han Chinese populations, the most common mutation site of ADH1B is rs1229984 (T / C), and its frequencies in East Asian populations are T (70.45%) and C (29.58%) respectively; the most common mutation site of ALDH2 is rs671 (G / A), and its frequencies in East Asian populations are G (77.53%) and A (22.47%) respectively. The ADH1B rs1229984 site can be divided into three genotypes according to the type of bases: homozygous TT type, homozygous CC type, and heterozygous TC type. Studies have shown that the TT type is a normal metabolic type, that is, the activity of alcohol dehydrogenase is normal; the activity of alcohol dehydrogenase of the TC type is slightly reduced, which is an intermediate metabolic type; the activity of alcohol dehydrogenase of the CC type is weak, which is a slow metabolic type, and its ability to metabolize ethanol is weak. The ALDH2 rs671 locus can be divided into three genotypes according to the type of base: homozygous GG type, homozygous AA type, and heterozygous GA type. Studies have shown that the GG type is a normal metabolic type, that is, the activity of acetaldehyde dehydrogenase is 100%; the activity of acetaldehyde dehydrogenase of the GA type is reduced, but it still has an activity of 13% to 14%, which is an intermediate metabolic type; the activity of acetaldehyde dehydrogenase of the AA type is the weakest, only about 2%, which is a slow metabolic type, and the ability to metabolize acetaldehyde is weak.

[0003] At present, the commonly used methods for detecting base mutations at the ADH1B rs1229984 or ALDH2 rs671 sites on the market include sequencing, PCR melting curve method, PCR capillary electrophoresis method and fluorescence PCR method.

[0004] Fluorescence PCR technology is currently the most widely used detection technology in the field of molecular diagnosis. Due to its high detection sensitivity and strong specificity, it is widely used in many fields such as pathogen detection, early tumor screening, and food testing. Its core principle is PCR amplification and fluorescence signal detection. The PCR amplification process simulates the semi-conservative replication process of DNA in the body. In the presence of biological enzymes, dNTPs, metal ions and other substances, the primers that are complementary to the target gene nucleic acid sequence are used as guides to synthesize new nucleic acid fragments. The reaction conditions include denaturation, annealing and extension. The fluorescent signal is detected by a probe that is complementary to the target sequence and labeled with a reporter group and a quencher group at the 5' and 3' ends respectively. Common probes include the most commonly used Taqman probes, molecular beacon probes, scorpion probes, MGB probes, etc. Different types of probes are used in different specific detection fields.

[0005] Among them, molecular beacon probes are currently the most commonly used detection probes for the detection of single-base mutations in alcohol metabolism-related genes or other genes because of their stronger specificity than Taqman probes. Molecular beacon is a nucleotide sequence that can form a stem-loop structure under normal conditions. Some bases at its 5' end (labeled with a luminescent group) and 3' end (labeled with a quenching group) can complement each other to form a stem structure, and the middle section forms a ring structure, so that it forms a relatively stable stem-loop structure under natural conditions. Its advantages are: a) strong specificity. Compared with ordinary Taqman probes, it can detect mismatches, deletions or insertion mutations of a single base; b) low background signal. Due to its stem structure, the reflective group of the 5' segment and the quenching group of the 3' segment are shorter in space, and the fluorescence resonance energy transfer effect is stronger, so that the background signal of its detection is lower and the signal-to-noise ratio is stronger.

[0006] 1) The existing fluorescent PCR products have a low extension speed of about 1 kb / minute due to the core raw material Taq enzyme. For PCR amplification of a product length of about 200 bp to 300 bp, at least 15 seconds of extension time is required. Due to the complexity of the PCR reaction and the fluorescence reading time, it generally takes longer than 30 seconds or more.

[0007] 2) Existing fluorescent PCR products are basically based on common PCR instrument platforms, with a heating and cooling speed of only about 2.5°C / second, which means that each cycle of denaturation-annealing / extension (taking 95°C denaturation and 60°C annealing / extension as an example) takes nearly 30 seconds, and the total heating and cooling time for 40 cycles is nearly 20 minutes;

[0008] 3) Most of the existing fluorescent PCR products use molecular beacon probes, which have complex probe design and high requirements. If the requirements are not met, it is easy to cause the generation of non-specific signals;.

[0009] 4) Existing products mainly use whole blood samples, and the sampling process is relatively complicated and requires highly professional technicians to operate. Due to the complexity of whole blood samples, they need to go through a complicated nucleic acid extraction process when used for subsequent PCR testing, which is time-consuming and labor-intensive. Summary of the invention

[0010] The purpose of the present invention is to solve the problems existing in the prior art and to propose a kit and a detection method for detecting human gene mutations related to alcohol metabolism ability.

[0011] In order to achieve the above object, the present invention adopts the following technical solutions:

[0012] A kit for detecting human gene mutations related to alcohol metabolism ability, comprising: a nucleic acid release agent and a PCR reaction solution;

[0013] The nucleic acid releaser comprises: 10-20 mM Tris, 20-100 mM NaCl, 2-10 mM EDTA, 0.1-1% SDS (w / v), 5-20 mg / mL proteinase K, and 0.005-0.02% TritonX-100 (v / v);

[0014] The PCR reaction solution comprises: rs1229984 site-specific primers, rs671 site-specific probes, 0.05-0.4 U / μl fast Taq enzyme, PCR Buffer matched with the Taq enzyme, and 0.01-0.02 U / μl UDG enzyme.

[0015] Preferably, the PCR reaction solution comprises 0.2-0.8 μM rs1229984 site-specific primer and 0.1-0.4 μM rs671 site-specific probe.

[0016] Preferably, the rs1229984 site-specific primer comprises:

[0017] Upstream primer 5′-TCACCAGGTTGCCACTAACCA-3′;

[0018] Downstream primer 5′-ACACAATTTCAGGAATTTGGGTATG-3′;

[0019] Probe 1 5′-TCTGTGCGACAGAT-3′;

[0020] Probe 2 5′-TCTGTGTGACAGAT-3′.

[0021] Preferably, the rs671 site-specific primer comprises:

[0022] Upstream primer 5′-GGGAGTTGGGCGAGTACG-3′;

[0023] Downstream primer 5′-AGACCCTCAAGCCCCAACAG-3′;

[0024] Probe 1 5′-TACACTGAAGTGAA-3′;

[0025] Probe 2 5′-TACACTAAAGTGAA-3′.

[0026] Preferably, the 5' end of the probe is labeled with one of the FAM / VI C / ROX / CY5 luminescent groups, the 3' end is connected with the MGB group, and the 7th base from the 5'-3' end (ie, the base at the mutation site to be detected) is LNA-modified.

[0027] A method for detecting a human gene mutation related to alcohol metabolism ability, using a kit for detecting a human gene mutation related to alcohol metabolism ability, comprises the following steps:

[0028] a) Use a sterile swab to collect pharyngeal secretions and immerse the swab head in the nucleic acid release agent;

[0029] b) Take PCR reaction solution and dispense it into PCR reaction tubes at 15 μl / tube;

[0030] c) taking 5 μl of the nucleic acid releasing agent in step a) and adding it to the reaction tube containing the PCR reaction solution, mixing and centrifuging;

[0031] d) Perform on-machine detection on a fluorescent PCR instrument, and the reaction program is set as follows: ① 95°C-98°C for 30 seconds-2 minutes; ② 98°C for 0-2 seconds, 60°C for 2-5 seconds (read fluorescence: FAM / VIC / ROX / CY5), 40-45 cycles;

[0032] e) After PCR amplification, the analysis software of the fluorescent PCR instrument is used to analyze the results of the tested samples, and the experimental validity and the genotype of the samples are determined by the fluorescence signal value of the FAM / VIC / ROX / CY5 channel.

[0033] Preferably, in step d), when the on-machine detection is performed on a fluorescent PCR instrument, the reaction program is set to: ① 95°C for 30 seconds; ② 98°C for 1 second, 60°C for 2 seconds (reading fluorescence: FAM / VIC / ROX / CY5), 40 cycles

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] 1) The present invention uses human throat swabs as test samples, which is simple and fast to sample. Compared with the complex components of whole blood samples, the components of throat swab samples are simple, so that subsequent amplification detection can be performed after the nucleic acid is released by the nucleic acid releaser, without the need for cumbersome extraction and purification processes;

[0036] 2) The present invention uses the improved rapid Taq enzyme as the core raw material, and can achieve full-process detection within 10 minutes in combination with a rapid fluorescent PCR instrument, which shortens the detection time and increases the application efficiency;

[0037] 3) The present invention is based on the principle of fluorescent PCR method of LNA+MGB probe, has a simpler design and higher specificity for single base mutation (SNP) detection. DETAILED DESCRIPTION

[0038] The technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0039] Example 1

[0040] A kit for detecting human gene mutations related to alcohol metabolism ability,

[0041] Including: nucleic acid release agent and PCR reaction solution;

[0042] The nucleic acid release agent is a reagent used to quickly lyse cells in human throat swab samples and release nucleic acids for subsequent testing. The components include: 10mM Tris, 20mM NaCl, 2mM EDTA, 1% SDS (w / v), 5mg / mL proteinase K, 0.005% TritonX-100 (v / v);

[0043] The PCR reaction solution is an important component of PCR amplification. After being mixed with the nucleic acid in the nucleic acid releaser in a certain proportion, amplification detection is directly performed and the results are analyzed and displayed; the PCR reaction solution includes: 0.2μM rs1229984 site-specific primer, 0.1rs671 site-specific probe, 0.05U / μl commercial fast Taq enzyme, commercial PCRBuffer matched with Taq enzyme, and 0.01U / μl commercial UDG enzyme.

[0044] In this embodiment, the rs1229984 site-specific primer includes:

[0045] Upstream primer 5′-TCACCAGGTTGCCACTAACCA-3′;

[0046] Downstream primer 5′-ACACAATTTCAGGAATTTGGGTATG-3′;

[0047] Probe 1 5′-TCTGTGCGACAGAT-3′;

[0048] Probe 2 5′-TCTGTGTGACAGAT-3′.

[0049] In this embodiment, the rs671 site-specific primer includes:

[0050] Upstream primer 5′-GGGAGTTGGGCGAGTACG-3′;

[0051] Downstream primer 5′-AGACCCTCAAGCCCCAACAG-3′;

[0052] Probe 1 5′-TACACTGAAGTGAA-3′;

[0053] Probe 2 5′-TACACTAAAGTGAA-3′.

[0054] In this embodiment, the 5' end of the probe is labeled with one of the FAM / VIC / ROX / CY5 luminescent groups, the 3' end is connected with the MGB group, and the 7th base from the 5'-3' end (ie, the mutation site base to be detected) is LNA modified.

[0055] A method for detecting a human gene mutation related to alcohol metabolism ability, using a kit for detecting a human gene mutation related to alcohol metabolism ability, comprises the following steps:

[0056] a) Use a sterile swab to collect pharyngeal secretions and immerse the swab head in the nucleic acid release agent;

[0057] b) Take PCR reaction solution and dispense it into PCR reaction tubes at 15 μl / tube;

[0058] c) taking 5 μl of the nucleic acid releasing agent in step a) and adding it to the reaction tube containing the PCR reaction solution, mixing and centrifuging;

[0059] d) Perform the test on a fluorescent PCR instrument, and set the reaction program as follows: ① 95°C for 30 seconds; ② 98°C for 2 seconds, 60°C for 2 seconds (read fluorescence: FAM / VIC / ROX / CY5), 40 cycles;

[0060] e) After PCR amplification, the analysis software of the fluorescent PCR instrument is used to analyze the results of the tested samples, and the experimental validity and the genotype of the samples are determined by the fluorescence signal value of the FAM / VIC / ROX / CY5 channel.

[0061] Compared with the prior art, the present invention:

[0062] Human throat swabs are used as test samples. Sampling is simple and fast. Compared with the complex components of whole blood samples, the components of throat swab samples are simple, so that subsequent amplification detection can be performed after the nucleic acid is released by the nucleic acid release agent, without the need for cumbersome extraction and purification processes.

[0063] Using the improved fast Taq enzyme as the core raw material, combined with a fast fluorescent PCR instrument, the whole process detection can be achieved within 10 minutes, with short detection time and higher application efficiency;

[0064] Based on the principle of fluorescent PCR method with LNA+MGB probe, the design is simpler and the specificity for single base mutation (SNP) detection is higher.

[0065] Among them, the LNA+MGB probe is a new type of specific fluorescent PCR probe: its 5' end is labeled with a conventional fluorescent reporter group, such as FAM / VIC / HEX / ROX / CY5, etc.; in addition to being labeled with a non-luminescent quencher group (NFQ), the 3' end is also connected to a minor groove binder (MGB). The modification of MGB can significantly increase the Tm value of the probe (10-20°C), thereby increasing the specificity of binding to the nucleic acid template; locked nucleic acid (LNA) is a class of modified high-affinity RNA analogs. The addition of LNA nucleotides can improve the thermal stability of the probe binding to the nucleic acid template (2-8°C), and has higher recognition and affinity for the target, thereby improving the specificity of detection.

[0066] Ultrafast PCR fluorescence detection technology is a detection method that relies on fast Taq polymerase and ultrafast PCR instrument platform. It can complete 40 cycles of PCR amplification in about 5 minutes at the shortest. Fast Taq enzyme is a genetically modified Taq DNA polymerase. Compared with conventional Taq DNA polymerase, its extension speed is increased by 10 times to 1kb / second, which shortens the PCR extension time from the conventional 15 to 45 seconds to about 2 seconds, thereby shortening the entire PCR cycle time. Currently, there are many commercial fast Taq enzymes on the market. Compared with conventional fluorescent PCR instruments, the advantage of ultrafast PCR instruments is that the heating and cooling speed is faster, which can reach 15 to 20℃ / second, while the heating and cooling speed of conventional fluorescent PCR instruments is only 1.5 to 5℃ / second. Therefore, ultrafast PCR instruments can significantly reduce the heating and cooling time in the PCR cycle, thereby shortening the entire PCR amplification time.

[0067] The present invention is described below in conjunction with the defects in the prior art:

[0068] 1) The existing fluorescent PCR products have a low extension rate of about 1 kb / minute due to the core raw material Taq enzyme. For PCR amplification of a product length of about 200 bp to 300 bp, at least 15 seconds of extension time is required. Due to the complexity of the PCR reaction and the fluorescence reading time, a longer extension time of 30 seconds or more is generally required. The fast Taq enzyme in the present invention can achieve an extension rate of 1 kb / second, and the extension time can be shortened to 2 seconds, so that the total extension time of the 40-cycle PCR process is shortened by nearly 20 minutes; in addition, the kit and the detection method of the present invention also shorten the denaturation time from the conventional 3 to 10 seconds to 0 seconds, but the detection effect is not affected, further shortening the total amplification time.

[0069] 2) Existing fluorescent PCR products basically use ordinary PCR instrument platforms, and their heating and cooling speed is only about 2.5°C / second, so that the time consumption of each cycle of denaturation-annealing / extension (taking 95°C denaturation and 60°C annealing / extension as an example) is close to 30 seconds, and the total heating and cooling time of 40 cycles is close to 20 minutes; while the kit and detection method of the present invention use an ultra-fast PCR instrument platform, and its heating and cooling speed reaches 15°C to 20°C or above, and the total heating and cooling time of 40 cycles is only 2 to 3 minutes, which is 15 to 20 minutes shorter than that of conventional PCR instrument platforms.

[0070] 3) Most existing fluorescent PCR products use molecular beacon probes, which have complex probe design and high requirements. If the requirements are not met, non-specific signals are easily generated. The kit of the present invention uses LNA+MGB probe technology, which has a simple design, better stability, and stronger detection specificity for single-base mutations. The 5' end is labeled with a conventional fluorescent reporter group, such as FAM / VIC / HEX / ROX / CY5, etc.; the 3' end is labeled with a non-luminescent quenching group (NFQ) and is also connected to a minor groove binder (MGB). The modification of MGB can significantly increase the Tm value of the probe (10-20°C), thereby increasing the specificity of binding to the nucleic acid template; locked nucleic acid (LNA) is a class of modified high-affinity RNA analogs. The addition of LNA nucleotides can improve the thermal stability of the probe binding to the nucleic acid template (2-8°C), and has higher recognition and affinity for the target, thereby improving the specificity of detection.

[0071] 4) Existing products mainly use whole blood samples, and the sampling process is relatively complicated and requires highly professional technicians to operate. Due to the complexity of whole blood samples, they need to go through a complex nucleic acid extraction process when used for subsequent PCR testing, which is time-consuming and laborious. The present invention provides a kit and a detection method that can use human throat swab samples for testing. The samples do not need to be subjected to nucleic acid extraction, and can be directly used for testing after a nucleic acid release step.

[0072] In general, the purpose of the present invention is to solve the problems of existing products: 1) the detection time is too long and the efficiency is low; 2) the molecular beacon probe design has high requirements and is prone to non-specific signals; 3) the sampling and processing process of the sample type used is cumbersome and requires high professional skills.

[0073] Example 2

[0074] A kit for detecting human gene mutations related to alcohol metabolism ability,

[0075] Including: nucleic acid release agent and PCR reaction solution;

[0076] The nucleic acid release agent is a reagent used to quickly lyse cells in human throat swab samples and release nucleic acids for subsequent testing. The components include: 10mM Tris, 20mM NaCl, 2mM EDTA, 1% SDS (w / v), 5mg / mL proteinase K, 0.005% TritonX-100 (v / v);

[0077] The PCR reaction solution is an important component of PCR amplification. After being mixed with the nucleic acid in the nucleic acid releaser in a certain proportion, amplification detection is directly performed and the results are analyzed and displayed; the PCR reaction solution includes: 0.8 μM rs1229984 site-specific primer, 0.4 μM rs671 site-specific probe, 0.4 U / μl commercial fast Taq enzyme, commercial PCR Buffer matched with Taq enzyme, and 0.02 U / μl commercial UDG enzyme.

[0078] In this embodiment, the rs1229984 site-specific primer includes:

[0079] Upstream primer 5′-TCACCAGGTTGCCACTAACCA-3′;

[0080] Downstream primer 5′-ACACAATTTCAGGAATTTGGGTATG-3′;

[0081] Probe 1 5′-TCTGTGCGACAGAT-3′;

[0082] Probe 2 5′-TCTGTGTGACAGAT-3′.

[0083] In this embodiment, the rs671 site-specific primer includes:

[0084] Upstream primer 5′-GGGAGTTGGGCGAGTACG-3′;

[0085] Downstream primer 5′-AGACCCTCAAGCCCCAACAG-3′;

[0086] Probe 1 5′-TACACTGAAGTGAA-3′;

[0087] Probe 2 5′-TACACTAAAGTGAA-3′.

[0088] In this embodiment, the probes are labeled with one of the FAM / VIC / ROX / CY5 luminescent groups at the 5' end, connected with the MGB group at the 3' end, and the 7th base from the 5'-3' end (ie, the base at the mutation site to be detected) is modified with LNA.

[0089] A method for detecting a human gene mutation related to alcohol metabolism ability, using a kit for detecting a human gene mutation related to alcohol metabolism ability, comprises the following steps:

[0090] a) Use a sterile swab to collect pharyngeal secretions and immerse the swab head in the nucleic acid release agent;

[0091] b) Take PCR reaction solution and dispense it into PCR reaction tubes at 15 μl / tube;

[0092] c) taking 5 μl of the nucleic acid releasing agent in step a) and adding it to the reaction tube containing the PCR reaction solution, mixing and centrifuging;

[0093] d) Perform the test on a fluorescence PCR instrument, and the reaction program is set as follows: ① 98°C for 2 minutes; ② 98°C for 2 seconds, 60°C for 5 seconds (read fluorescence: FAM / VI C / ROX / CY5), 45 cycles;

[0094] e) After PCR amplification, the analysis software of the fluorescent PCR instrument is used to analyze the results of the tested samples, and the experimental validity and the genotype of the samples are determined by the fluorescence signal value of the FAM / VIC / ROX / CY5 channel.

[0095] Example 3

[0096] A kit for detecting human gene mutations related to alcohol metabolism ability,

[0097] Including: nucleic acid release agent and PCR reaction solution;

[0098] The nucleic acid release agent is a reagent used to quickly lyse cells in human throat swab samples and release nucleic acids for subsequent testing. The components include: 10mM Tris, 20mM NaCl, 2mM EDTA, 1% SDS (w / v), 5mg / mL proteinase K, 0.005% TritonX-100 (v / v);

[0099] The PCR reaction solution is an important component of PCR amplification. After being mixed with the nucleic acid in the nucleic acid releaser in a certain proportion, amplification detection is directly performed and the results are analyzed and displayed; the PCR reaction solution includes: the PCR reaction solution includes: 0.2rs671 site-specific primer, 0.1μM rs1229984 site-specific probe, 0.05U / μl commercial fast Taq enzyme, commercial PCR Buffer matched with Taq enzyme, and 0.01U / μl commercial UDG enzyme.

[0100] In this embodiment, the rs1229984 site-specific primer includes:

[0101] Upstream primer 5′-TCACCAGGTTGCCACTAACCA-3′;

[0102] Downstream primer 5′-ACACAATTTCAGGAATTTGGGTATG-3′;

[0103] Probe 1 5′-TCTGTGCGACAGAT-3′;

[0104] Probe 2 5′-TCTGTGTGACAGAT-3′.

[0105] In this embodiment, the rs671 site-specific primer includes:

[0106] Upstream primer 5′-GGGAGTTGGGCGAGTACG-3′;

[0107] Downstream primer 5′-AGACCCTCAAGCCCCAACAG-3′;

[0108] Probe 1 5′-TACACTGAAGTGAA-3′;

[0109] Probe 2 5′-TACACTAAAGTGAA-3′.

[0110] In this embodiment, the 5' end of the probe is labeled with one of the FAM / VIC / ROX / CY5 luminescent groups, the 3' end is connected with the MGB group, and the 7th base from the 5'-3' end (ie, the mutation site base to be detected) is LNA modified.

[0111] A method for detecting a human gene mutation related to alcohol metabolism ability, using a kit for detecting a human gene mutation related to alcohol metabolism ability, comprises the following steps:

[0112] a) Use a sterile swab to collect pharyngeal secretions and immerse the swab head in the nucleic acid release agent;

[0113] b) Take PCR reaction solution and dispense it into PCR reaction tubes at 15 μl / tube;

[0114] c) taking 5 μl of the nucleic acid releasing agent in step a) and adding it to the reaction tube containing the PCR reaction solution, mixing and centrifuging;

[0115] d) Perform on-machine detection on a fluorescent PCR instrument, and the reaction program is set as follows: ① 98°C for 2 minutes; ② 98°C for 2 seconds, 60°C for 5 seconds (read fluorescence: FAM / VIC / ROX / CY5), 45 cycles;

[0116] e) After PCR amplification, the analysis software of the fluorescent PCR instrument is used to analyze the results of the tested samples, and the experimental validity and the genotype of the samples are determined by the fluorescence signal value of the FAM / VIC / ROX / CY5 channel.

[0117] Example 4

[0118] A kit for detecting human gene mutations related to alcohol metabolism ability,

[0119] Including: nucleic acid release agent and PCR reaction solution;

[0120] The nucleic acid release agent is a reagent used to quickly lyse cells in human throat swab samples and release nucleic acids for subsequent testing. The components include: 10mM Tris, 20mM NaCl, 2mM EDTA, 1% SDS (w / v), 5mg / mL proteinase K, 0.005% TritonX-100 (v / v);

[0121] The PCR reaction solution is an important component of PCR amplification. After being mixed with the nucleic acid in the nucleic acid releaser in a certain proportion, amplification detection is directly performed and the results are analyzed and displayed; the PCR reaction solution includes: the PCR reaction solution includes: 0.8μMrs671 site-specific primer, 0.4μM rs1229984 site-specific probe, 0.4U / μl commercial fast Taq enzyme, commercial PCR Buffer matched with Taq enzyme, and 0.02U / μl commercial UDG enzyme.

[0122] In this embodiment, the rs1229984 site-specific primer includes:

[0123] Upstream primer 5′-TCACCAGGTTGCCACTAACCA-3′;

[0124] Downstream primer 5′-ACACAATTTCAGGAATTTGGGTATG-3′;

[0125] Probe 1 5′-TCTGTGCGACAGAT-3′;

[0126] Probe 2 5′-TCTGTGTGACAGAT-3′.

[0127] In this embodiment, the rs671 site-specific primer includes:

[0128] Upstream primer 5′-GGGAGTTGGGCGAGTACG-3′;

[0129] Downstream primer 5′-AGACCCTCAAGCCCCAACAG-3′;

[0130] Probe 1 5′-TACACTGAAGTGAA-3′;

[0131] Probe 2 5′-TACACTAAAGTGAA-3′.

[0132] In this embodiment, the probes are labeled with one of the FAM / VIC / ROX / CY5 luminescent groups at the 5' end, connected with the MGB group at the 3' end, and the 7th base from the 5'-3' end (ie, the base at the mutation site to be detected) is modified with LNA.

[0133] A method for detecting a human gene mutation related to alcohol metabolism ability, using a kit for detecting a human gene mutation related to alcohol metabolism ability, comprises the following steps:

[0134] a) Use a sterile swab to collect pharyngeal secretions and immerse the swab head in the nucleic acid release agent;

[0135] b) Take PCR reaction solution and dispense it into PCR reaction tubes at 15 μl / tube;

[0136] c) taking 5 μl of the nucleic acid releasing agent in step a) and adding it to the reaction tube containing the PCR reaction solution, mixing and centrifuging;

[0137] d) Perform on-machine detection on a fluorescent PCR instrument, and the reaction program is set as follows: ① 98°C for 2 minutes; ② 98°C for 2 seconds, 60°C for 5 seconds (read fluorescence: FAM / VIC / ROX / CY5), 45 cycles;

[0138] e) After PCR amplification, the analysis software of the fluorescent PCR instrument is used to analyze the results of the tested samples, and the experimental validity and the genotype of the samples are determined by the fluorescence signal value of the FAM / VIC / ROX / CY5 channel.

[0139] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A kit for detecting human gene mutations related to alcohol metabolism ability, characterized in that: include: Nucleic acid release agent and PCR reaction solution; The nucleic acid release agent comprises: 10-20 mM Tris, 20-100 mM NaCl, 2-10 mM EDTA, 0.1-1% SDS (w / v), 5-20 mg / mL proteinase K, and 0.005-0.02% TritonX-100 (v / v); The PCR reaction solution comprises: rs1229984 site-specific primers, rs671 site-specific probes, 0.05-0.4 U / μl fast Taq enzyme, PCR Buffer matched with the Taq enzyme, and 0.01-0.02 U / μl UDG enzyme.

2. The kit for detecting human gene mutations related to alcohol metabolism ability according to claim 1, characterized in that: The PCR reaction solution includes 0.2-0.8 μM rs1229984 site-specific primer and 0.1-0.4 μM rs671 site-specific probe.

3. The kit for detecting human gene mutations related to alcohol metabolism ability according to claim 1, characterized in that: The PCR reaction solution includes 0.2-0.8 μM rs671 site-specific primer and 0.1-0.4 μM rs1229984 site-specific probe.

4. The kit and method for detecting human gene mutations related to alcohol metabolism ability according to claim 1, characterized in that: The rs1229984 site-specific primers include: Upstream primer 5′-TCACCAGGTTGCCACTAACCA-3′; Downstream primer 5′-ACACAATTTCAGGAATTTGGGTATG-3′; Probe 1 5′-TCTGTGCGACAGAT-3′; Probe 2 5′-TCTGTGTGACAGAT-3′.

5. The kit for detecting human gene mutations related to alcohol metabolism ability according to claim 4, characterized in that: The rs671 site-specific primers include: Upstream primer 5′-GGGAGTTGGGCGAGTACG-3′; Downstream primer 5′-AGACCCTCAAGCCCCAACAG-3′; Probe 1 5′-TACACTGAAGTGAA-3′; Probe 2 5′-TACACTAAAGTGAA-3′.

6. The kit for detecting human gene mutations related to alcohol metabolism ability according to claim 5, characterized in that: The 5' end of the probe is labeled with one of the FAM / VIC / ROX / CY5 luminescent groups, the 3' end is connected with the MGB group, and the 7th base from the 5'-3' end (ie the base of the mutation site to be detected) is LNA-modified.

7. A method for detecting a human gene mutation related to alcohol metabolism ability, using the kit for detecting a human gene mutation related to alcohol metabolism ability according to any one of claims 1 to 6, characterized in that: The following steps are involved: a) Use a sterile swab to collect pharyngeal secretions and immerse the swab head in the nucleic acid release agent; b) Take PCR reaction solution and dispense it into PCR reaction tubes at 15 μl / tube; c) taking 5 μl of the nucleic acid releasing agent in step a) and adding it to the reaction tube containing the PCR reaction solution, mixing and centrifuging; d) Perform on-machine detection on a fluorescent PCR instrument, and the reaction program is set as follows: ① 95°C-98°C for 30 seconds-2 minutes; ② 98°C for 0-2 seconds, 60°C for 2-5 seconds (read fluorescence: FAM / VIC / ROX / CY5), 40-45 cycles; e) After PCR amplification, the analysis software of the fluorescent PCR instrument is used to analyze the results of the tested samples, and the experimental validity and the genotype of the samples are determined by the fluorescence signal value of the FAM / VIC / ROX / CY5 channel.

8. The method for detecting human gene mutations related to alcohol metabolism ability according to claim 7, characterized in that: In the step d), when the on-machine detection is performed on the fluorescence PCR instrument, the reaction program is set as follows: ① 95°C for 30 seconds; ② 98°C for 1 second, 60°C for 2 seconds (reading fluorescence: FAM / VIC / ROX / CY5), 40 cycles.

9. The method for detecting human gene mutations related to alcohol metabolism ability according to claim 7, characterized in that: In the step d), when the on-machine detection is performed on the fluorescent PCR instrument, the reaction program is set as: ① 98° C. for 2 minutes; ② 98° C. for 2 seconds, 60° C. for 5 seconds (reading fluorescence: FAM / VIC / ROX / CY5), 45 cycles.