Kit for rapidly detecting human ALDH2 genotype and use method thereof

Abstract

The invention discloses a kit for rapidly detecting human ALDH2 genotypes and a use method thereof. The kit comprises a group of primers and probes for real-time fluorescent quantitative PCR detection of ALDH2, a high-stress-resistance rapidly-amplified DNA polymerase obtained by site-directed mutagenesis screening based on wild Taq enzyme, and a sample detection extraction-free reagent. By utilizing the combination, the rapid detection of the human ALDH2 gene can be realized, and a single reaction can be completed within 20 minutes, so that the detection efficiency is greatly improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of nucleic acid detection, and relates to a kit for rapidly detecting the genotype of human ALDH2 and a method for using the same. Background Art

[0002] The ALDH2 gene is located on chromosome 12 (12q24.2), and its main polymorphism is located at the G1510A site of exon 12. This leads to the replacement of glutamic acid at position 504 of the amino acid sequence by lysine (Glu504Lys). Among them, the wild type with catalytic activity is called the G allele (ALDH2*1), and the mutant type with inactivated catalytic ability is called the A allele (ALDH2*2). In the yellow race population, ALDH2*2 is the most frequent and important mutant type in ALDH2 polymorphisms. Nitroglycerin is a classic drug for treating angina pectoris. The vasodilatory effect of nitroglycerin is mediated by the release of nitric oxide (NO). Studies have found that the aldehyde dehydrogenase 2 gene (ALDH2) is closely related to the conversion of nitroglycerin to NO. The curative effect of nitroglycerin in treating angina pectoris in patients with the ALDH2*1 genotype is significantly better than that in patients with ALDH2*2, and the rapid effective rate of the former is also significantly higher than that of the latter. However, the inefficiency rate of patients with the ALDH2*2 mutant type reaches 42.4%, which is about 3 times that of the ALDH2 wild type. This results in the fact that some patients clinically cannot quickly and effectively relieve angina pectoris by sublingual administration of nitroglycerin, aggravating severe myocardial ischemia. Therefore, the dosage and frequency of nitroglycerin in patients with ALDH2*2 should be increased accordingly based on the wild type, and the medication cycle should be appropriately extended. In addition, ALDH2 is highly expressed in the liver and stomach and is one of the key enzymes in the human ethanol metabolism pathway. In the drinking population, especially the heavy drinking population, the ALDH2 gene polymorphism is closely related to diseases such as alcoholism, alcoholic liver disease, and digestive tract cancer.

[0003] Deoxyribonucleic acid (DNA) is a biological genetic code with different sequences for different species. DNA polymerase (DNA Polymerase), which participates in DNA synthesis in cells, plays a crucial role in realizing the functions of DNA molecule replication and genetic information transmission. DNA polymerase enables the DNA replication process to maintain a high degree of accuracy through the complementarity between sequence bases and the recognition of base structural characteristics. DNA polymerase can be divided into three types: I, II, and III, and can be further divided into three families of polymerases: A, B, and C according to the similarity of amino acid sequences. Among them, family A (such as Taq) has two functional domains, a polymerase domain from the 5' end to the 3' end and an exonuclease domain from the 5' end to the 3' end, while family B (such as Pfu) also has two functional domains, a polymerase domain from the 5' end to the 3' end and an exonuclease domain from the 3' end to the 5' end. In terms of structure and actual use, the binding degree of family A enzymes to the DNA template, the length of the amplified product, and the accuracy of the amplified product are weaker than those of family B enzymes. However, family A enzymes have better adaptability to different primers than family B enzymes. Therefore, the amplification reaction conditions are lower, and the experimental reaction is more likely to succeed. It is currently the most widely used DNA polymerase.

[0004] Molecular diagnostic technology uses genes as detection targets. qPCR technology is a relatively mature one in molecular diagnostic technology. Based on two primers, a TaqMan probe is added. After binding to specific sites, the Taq enzyme's cleavage activity is used to release fluorescent groups. The machine collects fluorescent signals in real time and presents image results. The detection time is usually within 2 hours, with the characteristics of rapid detection, high sensitivity, and strong specificity. Summary of the Invention

[0005] Object of the Invention: The object of the present invention is to provide a detection method for rapidly detecting the human ALDH2 genotype. By performing site-directed mutagenesis on wild-type Taq enzyme, a Taq enzyme with high stress resistance and rapid amplification (mutant Taq enzyme) is obtained, which can cooperate with a reagent for nucleic acid extraction-free to rapidly detect the human ALDH2 genotype.

[0006] Technical Solution: To achieve the above object of the invention, the present invention provides a detection reagent and a detection method for rapidly detecting the human ALDH2 genotype.

[0007] On the one hand, the present invention relates to a kit for rapidly detecting the human ALDH2 genotype, comprising an ALDH2 genotype detection reagent and a nucleic acid extraction-free reagent;

[0008] The ALDH2 genotype detection reagent includes: a primer-probe group for detecting the ALDH2 genotype, a PCR buffer, dNTPs, mutant Taq enzyme, UNG enzyme, spermidine, betaine, and allyl isothiocyanate;

[0009] The nucleic acid extraction-free reagent includes: cetostearyl polyoxyethylene ether, Tris-HCl, EDTA, ε-polylysine citrate.

[0010] Furthermore, the primer-probe set for detecting the ALDH2 genotype includes:

[0011] The primer-probe set for detecting the wild-type ALDH2 gene sequence:

[0012] The ALDH2-F2 primer, having the nucleotide sequence shown in SEQ ID NO:1,

[0013] The ALDH2-R2 primer, having the nucleotide sequence shown in SEQ ID NO:2,

[0014] The ALDH2-P2 probe, having the nucleotide sequence shown in SEQ ID NO:3,

[0015] And / or, the primer-probe set for detecting the mutant ALDH2 gene sequence:

[0016] The ALDH2-F5 primer, having the nucleotide sequence shown in SEQ ID NO:4,

[0017] The ALDH2-R5 primer, having the nucleotide sequence shown in SEQ ID NO:5,

[0018] The ALDH2-P5 probe, having the nucleotide sequence shown in SEQ ID NO:6,

[0019] Wherein, the ALDH2-P2 probe and the ALDH2-P5 probe have 5'-end fluorescent group modification and / or 3'-end quenching group modification, and the fluorescent group includes any one of FAM, HEX, VIC, CY5, ROX, TAMRA, JOE, ABY, CM-DYE, JUN, NED, MUSTANG PURPLE, SYBR and Alexa Fluor dyes; the quenching group includes any one of BHQ1, BHQ2, BHQ3, DABCYL, MGBNFQ, MGB and TAMRA.

[0020] Furthermore, the ALDH2-P2 probe is labeled with FAM, and the ALDH2-P5 probe is labeled with ROX.

[0021] On the other hand, the ALDH2 genotype detection reagent in the kit of the present invention comprises: a reaction solution composed of PCR buffer, dNTPs, mutant Taq enzyme, UNG enzyme, spermidine, betaine, and allyl isothiocyanate. The concentration of spermidine in the reaction solution is 25 mM, the concentration of betaine in the reaction solution is 0.5 M, and the concentration of allyl isothiocyanate in the reaction solution is 0.12% (by volume).

[0022] Further, the amino acid sequence of the mutant Taq enzyme is as shown in SEQ ID NO: 20;

[0023] The mutant Taq enzyme is obtained by site-directed mutation of the wild-type Taq enzyme. Specifically, valine at the 257th position is mutated to glutamate, threonine at the 386th position is mutated to alanine, proline at the 580th position is mutated to threonine, alanine at the 690th position is mutated to arginine, and glutamate at the 821st position is mutated to glycine.

[0024] In the nucleic acid extraction-free reagent, the concentration of cetostearyl polyoxyethylene ether is 1% (by volume), the concentration of Tris-HCl is 10 mM, the concentration of EDTA is 1 mM, the concentration of ε-polylysine citrate is 2% (by mass), and the pH is 7.0 - 8.0.

[0025] The nucleic acid extraction methods include but are not limited to blood extraction, oropharyngeal swabs, nasopharyngeal swabs, and anal swabs, etc.

[0026] The present invention also provides a method for using the above-mentioned kit for rapid detection of human ALDH2 genotype, comprising the following steps:

[0027] (1) Extract DNA: After rinsing the mouth with clean water, use a sterile sampling swab to scrape the oral epithelial cells on the upper palate of the mouth back and forth 10 - 20 times. Break the swab head and put it into the nucleic acid extraction-free system. Shake it up and down to make the epithelial cells fall off the swab. After standing for 2 minutes, take the supernatant as the PCR template. Or directly take 20 μl of whole blood and add it to 180 μl of the nucleic acid extraction-free system. Shake it up and down and mix well, then stand for 2 minutes. Take the supernatant as the PCR template;

[0028] (2) Use the ALDH2 genotype detection reagent to mix with the PCR template for PCR amplification reaction to obtain the detection result.

[0029] Compared with the prior art, the present invention has the following beneficial effects or advantages:

[0030] The present invention provides a primer-probe set for detecting human ALDH2 genotype, which is designed based on the human ALDH2 gene sequence and can effectively distinguish the wild-type, mutant, and heterozygous types of ALDH2.

[0031] In view of the characteristics of the need for rapid identification of ALDH2 genotypes in clinical emergencies, a Taq enzyme with a fast amplification speed and strong anti-interference ability was obtained by directed mutation screening of Taq enzymes. This enzyme, in combination with an optimized nucleic acid extraction-free reagent and an ALDH2 genotype detection reagent, can rapidly detect the ALDH2 genotype, greatly improving the detection efficiency. At the same time, it is applicable to two sample types, oral swabs and blood samples, meeting different clinical diagnostic needs. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 It is the amplification curve of the primers and probes in Example 1;

[0033] Figure 2 It is the electrophoresis pattern of the amplification products of the point mutant Taq enzyme, wild-type Taq enzyme and Taq DNA polymerase (recombinant) of ThermoFisher Company in Example 2;

[0034] Figure 3 It is the amplification curve of the experimental group 9, control groups 10 and 11 for the diluted 10-fold oral upper wall swabs in Example 4;

[0035] Figure 4 It is the amplification curve of the experimental group 10, control groups 12 and 13 for the diluted 10-fold blood in Example 4;

[0036] Figure 5 It is the detection flow chart in the actual application in Example 5. DETAILED DESCRIPTION OF THE INVENTION

[0037] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be described below with reference to the accompanying drawings. However, the following embodiments are only used to explain the present invention and are not used to limit the present invention.

[0038] Example 1

[0039] This example provides the design and screening of primers and probes for detecting the human ALDH2 genotype.

[0040] In order to ensure the detection of the human ALDH2 genotype and avoid false positives or false negatives, the conserved region of the human ALDH2 gene sequence was selected as the target for primer design.

[0041] Multiple groups of primers were designed at different positions of the human ALDH2 gene in the present invention, and each group of primer probes was tested and screened. Based on a large amount of primer design and screening work, a group of primer probe groups with excellent performance was finally selected.

[0042] Primer-probe sets for detecting the ALDH2 wild-type (G allele) gene sequence: ALDH2-F2 primer, having the nucleotide sequence shown in SEQ ID NO:1; ALDH2-R2 primer, having the nucleotide sequence shown in SEQ ID NO:2; ALDH2-P2 probe, having the nucleotide sequence shown in SEQ ID NO:3.

[0043] Primer-probe sets for detecting the ALDH2 mutant (A allele) gene sequence: ALDH2-F5 primer, having the nucleotide sequence shown in SEQ ID NO:4; ALDH2-R5 primer, having the nucleotide sequence shown in SEQ ID NO:5; ALDH2-P5 probe, having the nucleotide sequence shown in SEQ ID NO:6.

[0044] Due to excessive content, only some results are presented as comparative examples:

[0045] Comparative Example 1

[0046] Primer-probe sets for detecting the ALDH2 wild-type (G allele) gene sequence: ALDH2-F7 primer, having the nucleotide sequence shown in SEQ ID NO:7; ALDH2-R7 primer, having the nucleotide sequence shown in SEQ ID NO:8; ALDH2-P7 probe, having the nucleotide sequence shown in SEQ ID NO:9.

[0047] Primer-probe sets for detecting the ALDH2 mutant (A allele) gene sequence: ALDH2-F9 primer, having the nucleotide sequence shown in SEQ ID NO:10; ALDH2-R9 primer, having the nucleotide sequence shown in SEQ ID NO:11; ALDH2-P9 probe, having the nucleotide sequence shown in SEQ ID NO:12.

[0048] Comparative Example 2

[0049] Primer-probe sets for detecting the ALDH2 wild-type (G allele) gene sequence: ALDH2-F11 primer, having the nucleotide sequence shown in SEQ ID NO:13; ALDH2-R11 primer, having the nucleotide sequence shown in SEQ ID NO:14; ALDH2-P11 probe, having the nucleotide sequence shown in SEQ ID NO:15.

[0050] Primer-probe set for detecting ALDH2 mutant (A allele) gene sequence: ALDH2-F15 primer, having the nucleotide sequence shown in SEQ ID NO:16, ALDH2-R15 primer, having the nucleotide sequence shown in SEQ ID NO:17, and ALDH2-P15 probe, having the nucleotide sequence shown in SEQ ID NO:18.

[0051] Table 1 Primers and probes described in Example 1 and Comparative Examples 1 and 2

[0052]

[0053] 1. Sample acquisition and processing

[0054] Obtain oral upper wall swabs, extract them with the Novoprotein VAMNE Magnetic Pathogen DNA Kit, and store for subsequent steps.

[0055] Construct plasmids containing ALDH2 wild-type and mutant gene fragments and dilute them.

[0056] 2. Reaction system and reaction program

[0057] Table 2 50 μL reaction system

[0058]

[0059] The above reaction solution can be adapted to a variety of fluorescence quantitative PCR instruments. In this example, taking the common rapid fluorescence quantitative PCR on the market as an example, the reaction program is 25 °C: 2 minutes to activate UNG enzyme; 95 °C: 30 s pre-denaturation; 95 °C: 5 s denaturation, 65 °C: 10 s annealing and collecting fluorescence signals, 45 cycles.

[0060] Set a positive control and a negative control for each group of reactions.

[0061] 3. Interpretation of reaction results

[0062] The instrument collects fluorescence-labeled probe signals of different wavelengths, and after being processed by software, amplification curves of two fluorescence channels, FAM and ROX, are presented. The ALDH2 genotype is judged according to different fluorescence signals and CT values. Among them, the positive control is positive and the negative control is negative, indicating that the results are valid.

[0063] The detection results are as Figure 1 and shown in Table 3. In Example 1, typical amplification curves were presented for both the detection of ALDH2 wild-type and mutant, and the CT values were below 40. There was no non-specific amplification in the negative control, indicating that the present invention has good ability to detect ALDH2 genotype.

[0064] Table 3 Detection results of primers and probes described in Example 1 and Comparative Examples 1 and 2

[0065] Primer-probe combination Wild-type template Mutant template Negative control ALDH2-F2+R2+P2 Detected Not detected Not detected ALDH2-F5+R5+P5 Not detected Detected Not detected ALDH2-F7+R7+P7 Not detected Not detected Not detected ALDH2-F9+R9+P9 Detected Detected Not detected ALDH2-F11+R11+P11 Detected Detected Not detected ALDH2-F15+R15+P15 Detected Detected Not detected

[0066] Example 2

[0067] This example provides a process for constructing a point-mutated Taq enzyme for detecting the human ALDH2 genotype and a performance comparison experiment.

[0068] Process for constructing the point-mutated Taq enzyme:

[0069] Using gene synthesis method, synthesize the pET-30a recombinant plasmid containing the sequence of SEQ ID NO:19, transform the recombinant plasmid into Escherichia coli BL21(DE3) competent cells, select positive clone strains on the resistant plate, inoculate the positive clone strains into LB medium and culture at 37 °C and 250 rpm until the OD600 of the medium is about 0.5, add IPTG with a final concentration of 1 mM for induction, collect the bacterial cells after overnight induction and perform ultrasonic disruption, centrifuge to collect the supernatant after 45 minutes of water bath at 70 °C, filter with a 0.22 μm filter membrane to obtain the crude enzyme solution, pass the crude enzyme solution through a nickel ion affinity chromatography column, wash the miscellaneous proteins and elute the target protein with buffer solutions of different imidazole concentrations. Place the enzyme solution of the target protein in a 30KD dialysis bag and dialyze overnight. Define the amount of enzyme required to incorporate 10 nmol of deoxynucleotides into DNA within 30 minutes at 74 °C as 1 activity unit (U). Obtain the point-mutated Taq enzyme, which has the amino acid sequence shown in SEQ ID NO:20 and the enzyme concentration in the preservation solution is 200 U / μl.

[0070] Table 4: Nucleotide and amino acid sequences of the point-mutated Taq enzyme

[0071]

[0072]

[0073] Evaluate the tolerance ability of the point-mutated Taq enzyme to common interferents:

[0074] Add different concentration gradients of interferents to the reaction system of Example 1 for amplification. Taq DNA polymerase (wild type) is used as Control Group 1, Taq DNA polymerase (recombinant) from ThermoFisher (product number: 10342053) is used as Control Group 2, and the point-mutated Taq enzyme is used as Experimental Group 1. Amplify the mutant plasmid with a fixed copy number (1000 copies / test) using the method described in Example 1, record its CT value, calculate the half-inhibitory concentration (IC50) of each DNA polymerase to the interferent through the CT value, and further obtain the tolerance ability of each Taq polymerase to the interferent.

[0075] Table 5: IC50 Concentrations of Taq Enzyme against Different Interferents

[0076]

[0077] As can be seen from the results in Table 5, the ability (IC50) of the Taq enzyme optimized by point mutation to tolerate different interferents is better than that of the wild-type Taq enzyme and the commercially available general Taq enzyme.

[0078] Evaluate the amplification performance of the point-mutated Taq enzyme:

[0079] Using the ALDH2 gene mutant as a template, amplification was carried out using the point-mutated Taq enzyme as experimental group 2, the Taq DNA polymerase (recombinant) of Thermo Fisher as control group 3, and the wild-type Taq enzyme as control group 4. The reaction system and procedure refer to Example 1. After the reaction, the results were detected by electrophoresis on a 1% agarose gel, and the results are as Figure 2 shown.

[0080] The results show that under the same reaction system, a large amount of target fragments can be amplified by the point-mutated Taq enzyme, and the band brightness is higher than that of the wild-type Taq enzyme and the Taq DNA polymerase (recombinant) of Thermo Fisher. This indicates that the amplification performance of the point-mutated Taq enzyme is significantly better than that of the wild-type Taq enzyme or the Taq DNA polymerase (recombinant) of Thermo Fisher.

[0081] Example 3

[0082] This example provides a sensitivity test for detecting the human ALDH2 genotype and a partial optimization process of the reaction system.

[0083] Dilute the mutant plasmid to 10 6 copies / mL, 10 5 copies / mL, 10 4 copies / mL, 10 3 copies / mL, 10 2 copies / mL, and store for use in subsequent steps.

[0084] Referring to the multiplex reaction system in Example 1, set the following experimental groups.

[0085] Experimental group 3: Add spermidine to the reaction system so that the final concentration of spermidine in the reaction system is 25 mM.

[0086] Experimental group 4: Add betaine to the reaction system so that the final concentration of betaine in the reaction system is 0.5 M.

[0087] Experimental group 5: Allyl isothiocyanate was added to the reaction system so that the final concentration of allyl isothiocyanate in the reaction system was 0.12%.

[0088] Experimental group 6: Spermidine, betaine, and allyl isothiocyanate were added to the reaction system so that the final concentration of spermidine in the reaction system was 25 mM, the final concentration of betaine in the reaction system was 0.5 M, and the final concentration of allyl isothiocyanate in the reaction system was 0.12%.

[0089] Experimental group 7: Hemin was added to the reaction system so that the final concentration of hemin in the reaction system was 7 mmol / L.

[0090] Experimental group 8: Hemin was added to the reaction system so that the final concentration of hemin in the reaction system was 7 mmol / L. At the same time, spermidine, betaine, and allyl isothiocyanate were added to the reaction system so that the final concentration of spermidine in the reaction system was 25 mM, the final concentration of betaine in the reaction system was 0.5 M, and the final concentration of allyl isothiocyanate in the reaction system was 0.12%.

[0091] Control group 5: No additional addition.

[0092] The above-diluted samples were detected using the method described in Example 1. Each group of samples was repeated 20 times. The results with a CT value below 40 were statistically analyzed, and the detection rate was calculated. The results are shown in Table 6.

[0093] Table 6: Sensitivity test results of different reaction systems

[0094]

[0095]

[0096] From the results of experimental groups 3 - 6 and control group 5, it can be seen that adding spermidine, betaine, and allyl isothiocyanate to the reaction system has an enhancing effect on the detection of human ALDH2 genotype, and the enhancing effect of adding multiple components is higher than that of adding them alone. From the results of experimental groups 7 - 8 and control group 5, it can be seen that the optimized human ALDH2 genotype detection system has good resistance to the inhibitory effect of hemin.

[0097] Example 4

[0098] This example provides the optimization of the extraction-free system for detecting human ALDH2 genotype in the present invention and a comparative experiment on nucleic acid extraction with the column extraction method system and the magnetic bead extraction method system.

[0099] Obtain oral upper wall swabs with a known mutant ALDH2 genotype, and elute and dilute them 10-fold, 100-fold, and 1000-fold using TE solution.

[0100] Obtain blood with a known mutant ALDH2 genotype and dilute it 10-fold, 100-fold, and 1000-fold using TE solution.

[0101] Experimental group 9: Prepare a nucleic acid extraction-free reagent, where the concentration of cetostearyl polyoxyethylene ether is 1%, the concentration of Tris-HCl is 10 mM, the concentration of EDTA is 1 mM, the concentration of ε-polylysine citrate is 2%, and the pH is 7.0 - 8.0. Take 100 μL of the nucleic acid extraction-free system for tongue swabs, add 100 μL of the eluate from oral upper wall swabs to it, shake and mix well for 20 s, let it stand for 2 minutes, and take 15 μL of the supernatant and add it to the multiplex reaction system in Example 1 for detection.

[0102] Experimental group 10: The experimental method is the same as that in Experiment 9, except that 20 μL of the diluted blood is added to 180 μL of the nucleic acid extraction-free system for tongue swabs, shake and mix well for 20 s, let it stand for 2 minutes, and take 15 μL of the supernatant and add it to the multiplex reaction system in Example 1 for detection.

[0103] Control group 6: The experimental method is the same as that in Experimental group 9, except that cetostearyl polyoxyethylene ether is not added.

[0104] Control group 7: The experimental method is the same as that in Experimental group 9, except that ε-polylysine citrate is not added.

[0105] Control group 8: The experimental method is the same as that in Experimental group 10, except that cetostearyl polyoxyethylene ether is not added.

[0106] Control group 9: The experimental method is the same as that in Experimental group 10, except that ε-polylysine citrate is not added.

[0107] Control group 10: The column extraction method refers to the extraction using the Novizan FastPure Cell / Tissue DNA Isolation mini Kit. Add 100 μL of the eluate from oral upper wall swabs to it, elute with 200 μL of TE, and then take 15 μL and add it to the multiplex reaction system in Example 1 for detection.

[0108] Control group 11: The magnetic bead extraction method refers to the extraction using the Novizan VAMNE Magnetic Pathogen DNA Kit. Add 100 μL of the eluate from oral upper wall swabs to it, elute with 200 μL of TE, and then take 15 μL and add it to the multiplex reaction system in Example 1 for detection.

[0109] Control group 12: The column extraction method was referred to the extraction method of Novoprotein FastPure Cell / Tissue DNA Isolation mini Kit. 20 μL of diluted blood was added to it, and after elution with 200 μL of TE, 15 μL was taken and added to the multiplex reaction system of Example 1 for detection.

[0110] Control group 13: The magnetic bead extraction method was referred to the extraction method of Novoprotein VAMNE Magnetic Pathogen DNA Kit. 20 μL of diluted blood was added to it, and after elution with 200 μL of TE, 15 μL was taken and added to the multiplex reaction system of Example 1 for detection.

[0111] The above detections were repeated 20 times for each group of samples. The results with a CT value below 40 were statistically analyzed to calculate the detection rate. The statistical results are shown in Table 7. The amplification results of experimental group 9 and control groups 10 and 11 are as Figure 3 shown. The amplification results of experimental group 10 and control groups 12 and 13 are as Figure 4 shown.

[0112] As can be seen from Table 7, adding cetostearyl polyoxyethylene ether and ε-polylysine citrate to the reagent-free extraction can promote the lysis of oral epithelial cells and improve the detection rate of low-concentration samples. Among them, the addition of ε-polylysine citrate can effectively remove the influence of impurities in blood samples on PCR.

[0113] As can be seen from Table 7 and Figure 3-4 it can be known that the reagent-free extraction system for detecting human ALDH2 genotype in the present invention has an extraction efficiency of nucleic acid equivalent to that of the magnetic bead method and is superior to the column extraction method. Compared with the column extraction method and the magnetic bead method, the tongue swab nucleic acid reagent-free extraction system provided by the present invention has fewer steps in the lysis process, reduces the chance of contamination, and reduces the loss of nucleic acid.

[0114] Table 7: Statistical results of detection rates for different sample treatment methods

[0115] Experimental grouping Diluted 10-fold Diluted 100-fold Diluted 1000-fold NTC Experimental group 9 100％ 100％ 95％ 0％ Experimental group 10 100％ 100％ 85％ 0％ Control group 6 100％ 95％ 65％ 0％ Control group 7 100％ 100％ 75％ 0％ Control group 8 85％ 75％ 45％ 0％ Control group 9 65％ 75％ Not detected 0％ Control group 10 100％ 75％ 25％ 10％ Control group 11 100％ 95％ 80％ 5％ Control group 12 100％ 65％ 25％ 5％ Control group 13 100％ 95％ 75％ 15％

[0116] Example 5

[0117] This example provides an application test for rapid detection of human ALDH2 genotype.

[0118] Experimental samples: Clinical samples (80 cases) provided by the hospital, oral upper wall swab samples (scraped back and forth on the oral upper wall about 10 - 20 times) and blood samples were taken from the same patients. Taking the Sanger sequencing results of sample nucleic acid as the gold standard, the accuracy of the ALDH2 genotype detection method provided by the present invention was judged.

[0119] Experimental group 11: Detection was carried out with reference to experimental groups 9 and 10 in Example 4.

[0120] Control group 14: Detection was carried out with reference to experimental groups 11 and 13 in Example 4.

[0121] Detection was carried out with reference to the reaction procedure in Example 1, and the compliance rates of wild-type results, mutant results, and heterozygous results were counted.

[0122] Table 8: Statistics of application test results

[0123]

[0124] As can be seen from Table 8, the reagent for rapid detection of human ALDH2 without extraction provided by the present invention is applicable to the detection of oral upper wall swab samples and blood samples.

[0125] In summary, a detection method for rapid detection of human ALDH2 genotype provided by the present invention uses a highly stress-resistant and rapidly amplified Taq enzyme obtained by site-directed mutagenesis optimization of wild-type Taq enzyme. Using this mutant Taq enzyme and adding 25 mM spermidine, 0.5 M betaine, and 0.12% allyl isothiocyanate to the reaction system can effectively eliminate the inhibitory effect of blood impurities on PCR. At the same time, by adding 1% cetostearyl polyoxyethylene ether and 2% ε-polylysine citrate to the reagent without extraction, the lysis of sample cells is promoted, the inhibitory effect of sample impurities on amplification is reduced, and the detection rate of low-concentration samples is increased. Using this rapid detection method can rapidly detect the human ALDH2 genotype, and is applicable to various sample types such as blood and oral upper wall swabs, meeting different clinical detection needs.

[0126] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A kit for rapid detection of human ALDH2 genotype, characterized in that, The kit includes an ALDH2 genotype detection reagent and a nucleic acid extraction-free reagent; The ALDH2 genotype detection reagent includes: a primer-probe set for detecting the ALDH2 genotype, a PCR buffer, dNTPs, mutant Taq enzyme, UNG enzyme, spermidine, betaine, allyl isothiocyanate; The nucleic acid extraction-free reagent includes: cetostearyl polyoxyethylene ether, Tris-HCl, EDTA, ε-polylysine citrate.

2. The kit for rapid detection of human ALDH2 genotype according to claim 1, wherein The primer-probe set for detecting the ALDH2 genotype includes: A primer-probe set for detecting the wild-type ALDH2 gene sequence: The ALDH2-F2 primer has the nucleotide sequence shown in SEQ ID NO:1, The ALDH2-R2 primer has the nucleotide sequence shown in SEQ ID NO:2, The ALDH2-P2 probe has the nucleotide sequence shown in SEQ ID NO:3, And / or, the primer-probe set for detecting the mutant ALDH2 gene sequence: The ALDH2-F5 primer has the nucleotide sequence shown in SEQ ID NO:4, The ALDH2-R5 primer has the nucleotide sequence shown in SEQ ID NO:5, The ALDH2-P5 probe has the nucleotide sequence shown in SEQ ID NO:6, Wherein, the ALDH2-P2 probe and the ALDH2-P5 probe have 5'-end fluorescent group modification and / or 3'-end quenching group modification.

3. A kit for rapidly detecting the human ALDH2 genotype according to claim 1, characterized in that, The fluorescent group includes any one of FAM, HEX, VIC, CY5, ROX, TAMRA, JOE, ABY, CM-DYE, JUN, NED, MUSTANG PURPLE, SYBR and Alexa Fluor dyes; The quenching group includes any one of BHQ1, BHQ2, BHQ3, DABCYL, MGBNFQ, MGB and TAMRA.

4. A kit for rapid detection of human ALDH2 genotype according to claim 1, characterized in that, The ALDH2-P2 probe is labeled with FAM, and the ALDH2-P5 probe is labeled with ROX.

5. A kit for rapidly detecting the genotype of human ALDH2 as described in claim 1, characterized in that, The ALDH2 genotype detection reagent includes: using a PCR buffer, dNTPs, mutant Taq enzyme, UNG enzyme, spermidine, betaine, and allyl isothiocyanate to form a reaction solution, wherein the concentration of spermidine in the reaction solution is 25 mM, the concentration of betaine in the reaction solution is 0.5 M, and the concentration of allyl isothiocyanate in the reaction solution is 0.12% (by volume).

6. The kit for rapid detection of human ALDH2 genotype according to claim 1, characterized in that, The amino acid sequence of the mutant Taq enzyme is shown in SEQ ID NO:20; The wild-type Taq enzyme is subjected to site-directed mutagenesis, with valine at the 257th position mutated to glutamate, threonine at the 386th position mutated to alanine, proline at the 580th position mutated to threonine, alanine at the 690th position mutated to arginine, and glutamate at the 821st position mutated to glycine to obtain the mutant Taq enzyme.

7. A kit for rapidly detecting the genotype of human ALDH2 as described in claim 1, characterized in that, In the nucleic acid extraction-free reagent, the concentration of cetostearyl polyoxyethylene ether is 1% (by volume), the concentration of Tris-HCl is 10 mM, the concentration of EDTA is 1 mM, the concentration of ε-polylysine citrate is 2% (by mass), and the pH is 7.0 - 8.

0.

8. A method for using a kit for rapid detection of human ALDH2 genotype according to claim 1, characterized in that, It includes the following steps: After rinsing the mouth with clear water, use a sterile sampling swab to scrape the oral epithelial cells on the upper palate, scrape back and forth 10 - 20 times on the upper wall of the oral cavity, break the swab head and put it into the nucleic acid extraction-free system, shake it up and down to make the epithelial cells fall off the swab, and take the supernatant after standing for 2 minutes as the PCR template. Or directly add 20 µl of whole blood into 180 µl of the nucleic acid extraction-free system, shake it up and down to mix evenly, stand for 2 minutes, take the supernatant as the PCR template, and then use the ALDH2 genotype detection reagent to perform the PCR amplification reaction.

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