阅读说明：本技术 一种一管式aldh2基因分型试剂盒及其检测方法 (One-tube type A L DH2 genotyping kit and detection method thereof ) 是由 王继国 陈普 于 2019-01-07 设计创作，主要内容包括：本发明公开了一种简单经济的一管式ALDH2基因分型试剂盒,包括如下组分：含三条引物组的直扩染料法qPCR预混液,阳性质粒对照及阴性对照,其特征在于所述三条引物组：ALDH2-F上游引物如SEQ ID NO：1所示；ALDH2-R下游引物如SEQ ID NO：2所示；ALDH2-MR突变型下游引物如SEQ ID NO：3所示。本发明所示试剂盒采取了一管式qPCR反应,省去了样品的抽提步骤,这不仅极大的节省了测试成本,而且避免了抽提过程中的污染风险。本试剂盒由于准确、快速、成本低廉,特别适合用于饮酒行为、酒精型肝硬化、肝癌等遗传因素的危险性分析,也是临床硝酸甘油指导用药的参考方法。(The invention discloses a simple and economical one-tube type A L DH2 gene typing kit, which comprises a direct dye amplification method qPCR premixed solution containing three primer groups, a positive plasmid control and a negative control, and is characterized in that the three primer groups are A L DH2-F upstream primer shown as SEQ ID NO: 1, A L DH2-R downstream primer shown as SEQ ID NO: 2 and A L DH2-MR mutant downstream primer shown as SEQ ID NO: 3.)

1. A tubular A L DH2 genotyping kit, which is characterized by comprising the following components:

three primer groups are used for preparing the primer set,

qPCR buffer solution by a direct dye amplification method,

a positive plasmid control and a negative plasmid control,

wherein, the three primer groups are A L DH2-F upstream primer shown as SEQ ID NO. 1;

the downstream primer of A L DH2-R is shown as SEQ ID NO. 2;

the A L DH2-MR mutant downstream primer is shown as SEQ ID NO. 3.

2. The one-tube A L DH2 genotyping kit of claim 1, wherein the three primer sets and the direct dye method qPCR reaction buffer are mixed to prepare a direct dye method qPCR premix.

3. The one-tube type A L DH2 genotyping kit of claim 2, wherein the direct dye method qPCR premix is prepared from 10 volumes of SYBR Green qPCR Master Mix or EvaGreen qPCR Master Mix, 3 volumes of primer set mixture, wherein the final concentration of each primer reaction system is controlled to be 0.2uM, and finally the 15 volumes are filled with sterile water.

4. The one-tube type A L DH2 genotyping kit of claim 2, wherein the direct dye method qPCR premix comprises 10 parts by volume of KOD SYBR qPCR Mix, 3 parts by volume of primer set mixture, 0.2uM of final concentration of each primer reaction system, 0.04-0.4 part by volume of 50 × ROX, and sterile water for 15 parts by volume.

5. The one-tube type A L DH2 genotyping kit of claim 2, wherein the direct dye method qPCR premix comprises 0.5 volume parts of 5U/ul hot start TthDNApolymerase or 5U/ul TTX DNApolymerase, 10 volume parts of 2 × Reaction Buffer, 3 volume parts of primer set mixture, wherein the final concentration of each primer Reaction system is controlled to be 0.2uM, 0.04-0.4 volume part of 50 × ROX, 0.4 volume part of 1U/ul UDG enzyme, 0.4 volume part of 50 × er SuperGreen, and sterile water to make up to 15 volume parts.

6. The one-tube A L DH2 genotyping kit of claim 1, wherein the positive plasmid control is a positive control plasmid constructed by whole gene synthesis, the gene sequence of the positive plasmid of A L DH 2X 1 is shown in SEQ ID NO. 4, and the gene sequence of the positive plasmid of A L DH 2X 2 is shown in SEQ ID NO. 5.

7. The one-tube A L DH2 genotyping kit of claim 1, wherein the negative control is sterile water.

8. The method for detecting A L DH2 genotyping according to any one of claims 1 to 7, comprising the steps of:

a) sampling human oral epithelial cells or peripheral blood cells;

b) diluting collected human oral epithelial cells or peripheral blood cells into a sample, and performing amplification on quantitative PCR by using the one-tube type A L DH2 genotyping kit of claim 1, and simultaneously performing amplification on a positive control and a negative control of two genotypes;

c) and (3) melting curve analysis: negative control amplification is used for eliminating external environment and reagent pollution, two genotypes are determined by annealing temperatures from two positive controls, and one positive control and one negative control can be adopted in the same batch of reaction.

9. The method of claim 8, wherein the sampling of human oral epithelial cells is performed by sampling oral mucosa with a cotton swab or a medical swab and suspending the samples in 200ul of sterile water.

10. The use according to claim 8, wherein the sampling of human peripheral blood cell samples is performed by taking blood from a blood taking needle from a fingertip or an earlobe at 1 to 3ul, diluting and mixing with 48ul of sterile water.

Technical Field

The invention belongs to the field of medical detection, and particularly relates to a simple and economic one-tube A L DH2 genotyping kit and a detection method thereof.

Background

A L DH2 is named aldehyde dehydrogenase 2family (mitochondrial) and the gene A L DH2 is located at position 12q24.2 of chromosome 12, and its major polymorphism is rs671, i.e. G1510A located in exon 12, where a single base mutation from G to A occurs, so that Glu504 is mutated to L ys504, which is associated with the spatial structure of the A L DH2 protein and thus affects the activity of the enzyme, the normal allele is named A L DH2 1, the single base mutant allele is named A L DH 2. 2. Glu504 homozygote (A L DH2 1/1) shows normal enzymatic activity, the Glu 504/L ys heterozygote (A L DH2 1/2) only has about 6% of its normal activity, while the A3548 ys 585 (A35L) has no substantial enzymatic activity for degrading aldehyde dehydrogenase.

A L DH2 gene is a gene for coding mitochondrial acetaldehyde dehydrogenase, has dehydrogenase activity, and plays a main role in the oxidation of acetaldehyde in human body (namely, is directly related to the capability of metabolizing alcohol in vivo). The A L DH2 genotyping test is particularly suitable for risk analysis of genetic factors such as drinking behavior, alcoholic cirrhosis, liver cancer and the like, meanwhile, A L DH2 also has esterase activity and can catalyze nitroglycerin hydrolysis to generate NO. nitroglycerin for treating angina pectoris and heart failure for 130 years, sublingual nitroglycerin tablets are the conventional preferred prescription for resisting acute attack of coronary heart disease and angina pectoris, but the clinical effectiveness of the sublingual nitroglycerin tablets is influenced by A L DH2 genotype, and the A L DH2 genotyping kit is used as a main method for clinical nitroglycerin medication guidance.

The A L DH2 gene typing kit which is clinically used and invented at present generally comprises methods such as a DNA microarray chip method, a PCR capillary electrophoresis method, a PCR fluorescent probe method, a PCR-high resolution melting curve method and the like.

Disclosure of Invention

The invention aims to provide a simple and economic one-tube A L DH2 genotyping kit, which is a main method for clinical nitroglycerin medication guidance in the future.

The second objective of the invention is to provide a method for detecting A L DH2 genotyping.

The invention relates to a set of primers specially used for A L DH2 gene typing designed and screened according to an allele specific gene PCR method, and A L DH2 gene typing can be realized by fluorescence quantitative PCR melting curve by using the primer set.

The invention constructs a qPCR reaction solution system by a direct dye amplification method, and QPCR quantification can be carried out by the system without genome DNA extraction.

The invention constructs a method for distinguishing three genotypes by different annealing temperatures in melting curves of a dye-based quantitative PCR method.

Therefore, the invention discloses a simple and economic one-tube type A L DH2 genotyping kit, which comprises qPCR premixed solution by a direct-amplification dye method containing three primer groups, positive plasmid control and negative control, and is characterized in that the kit comprises the following three primer groups:

a L DH2-F upstream primer GTACGGGCTGCAGGCATAC;

a L DH2-R downstream primer GCCGCCCTGCCCGCCACACTCACAGTTTTC

ACTGCA；

A L DH2-MR mutant downstream primer CCCACACTCACAGTTTTCACTATA.

Further, the formula of the direct dye amplification qPCR premix solution is as follows: 10 volumes of SYBR Green qPCRMaster Mix or EvaGreen qPCR Master Mix; 3 parts of primer group mixture by volume, wherein the final concentration of each primer reaction system is controlled to be about 0.2 uM; finally make up to 15 volumes with sterile water.

Further, the formula of the direct dye amplification qPCR premix solution comprises 10 parts by volume of KOD SYBR qPCR Mix, 3 parts by volume of a primer group mixture, 0.2uM or so of the final concentration of each primer reaction system, 0.04-0.4 part by volume of 50 × ROX, and finally sterile water is used for supplementing to 15 parts by volume.

Further, the formula of the direct dye method qPCR premix solution comprises 0.5 part by volume of 5U/ul hot start TthDNApolymerase or 5U/ul TTX DNA Polymerase, 10 parts by volume of 2 × Reaction Buffer (containing dUTP), 3 parts by volume of primer group mixture, 0.04-0.4 part by volume of 50 × ROX, 0.4 part by volume of 1U/ul UDG enzyme, 0.4 part by volume of 50 × Super EvaGreen, and finally sterile water is used for supplementing to 15 parts by volume.

Further, the positive plasmid control is a positive control plasmid constructed by means of whole gene synthesis, and the gene sequence in the positive plasmid of A L DH2 x 1

CGAGTACGGGCTGCAGGCATACACTGAAGTGAAAACTGTGAGTGTGGGACC, gene sequence in a L DH2 x 2 positive plasmid:

CGAGTACGGGCTGCAGGCATACACTAAAGTGAAAACTGTGAGTGTGGGACC。

further, the negative control was sterile water.

On the other hand, the invention also discloses a detection method for A L DH2 genotyping, which comprises the following steps:

a) sampling human oral epithelial cells or peripheral blood cells;

b) adding 5ul of the collected human oral epithelial cells or peripheral blood cells and diluted samples into 15ul of 'direct dye amplification method qPCR premixed solution', performing amplification on quantitative PCR, and simultaneously performing amplification on positive control and negative control of two genotypes;

c) and (3) melting curve analysis: negative control amplification is used for eliminating pollution of 'direct dye method qPCR premix', two genotypes are determined by annealing temperatures from two positive controls, and one positive control and one negative control can be adopted in the same batch of reaction.

The main innovation points of the invention are as follows:

the invention utilizes human oral epithelial cells or peripheral blood cells to be directly added into a fluorescent quantitative PCR amplification system, utilizes optimized allele specific PCR primers to enable two different genotypes to generate different annealing temperatures, and can sensitively distinguish the two A L DH2 genotypes through a quantitative PCR dissolution curve.

Drawings

FIG. 1 melting curve of positive plasmid A L DH2 x 1.

FIG. 2 melting curve of positive plasmid A L DH2 x 2.

FIG. 3 shows a melting curve of Glu504 homozygote (A L DH 2. multidot. 1/1).

FIG. 4 shows melting curves of Glu 504/L ys504 heterozygote (A L DH2 × 1/2).

FIG. 5 melting curves of 5L ys504 homozygote (A L DH2 × 2/2).

Detailed Description

Acetaldehyde dehydrogenase 2(A L DH2) is one of NAD < + > dependent acetaldehyde dehydrogenase superfamily members, 19 acetaldehyde dehydrogenase family members have been found at present, acetaldehyde dehydrogenase 2 is an important aldehyde oxidase positioned in mitochondria and can oxidize acetaldehyde which is an ethanol metabolism intermediate product into acetic acid, meanwhile, acetaldehyde dehydrogenase 2 can decompose 4-hydroxynonenal which is an acetaldehyde metabolite and relieve the oxidative damage of cells by acetaldehyde and the acetaldehyde metabolite, the acetaldehyde dehydrogenase is a tetrameric enzyme and is abundantly expressed in tissues such as liver, kidney and lung, and is also expressed in heart and brain tissues with active mitochondrial function, recent studies show that the activation of the acetaldehyde dehydrogenase 2 has obvious protective effect on myocardial cell death caused by myocardial ischemia reperfusion, the acetaldehyde dehydrogenase 2 gene has polymorphism, the 487 amino acid residue of the expression product can be glutamic acid (Glu487) or lysine (L ys487), therefore, the gene can have Glu487 homozygote (acetaldehyde dehydrogenase 2 1/1), Glu/L ys (acetaldehyde dehydrogenase 2 ys487 2) or lysine (L ys487), and the acetaldehyde dehydrogenase gene has obvious mutation rate of acetaldehyde dehydrogenase carried by a mutant gene 125 percent of acetaldehyde dehydrogenase 2, the acetaldehyde dehydrogenase gene which is shown in the statistics of more than that the acetaldehyde dehydrogenase carried by A5 percent of acetaldehyde dehydrogenase carried by a plurality of acetaldehyde dehydrogenase mutant strains of acetaldehyde dehydrogenase 2, the acetaldehyde dehydrogenase gene of the normal cancer patients with more than 7, the acetaldehyde dehydrogenase 1-387 dehydrogenase gene, the acetaldehyde dehydrogenase 2, the acetaldehyde dehydrogenase gene of the normal cancer patients with more than 10-387 homozygote mutation rate of normal cancer, the normal cancer patients with more than 10-387 mutation rate of the normal cancer patients with more than the acetaldehyde dehydrogenase 2.

The main detection methods of acetaldehyde dehydrogenase 2 gene Polymorphism include restriction Fragment length Polymorphism (Restriction Fragment L ength Polymorphism), single-strand conformation Polymorphism, PCR-ASO probe method, PCR-SSO method, PCR-SSP method, PCR-fluorescence method, PCR fingerprinting method, gene chip method, AF L P (amplification Fragment L ength Photomorphism) method, DGGE (differentiation coding amplification) method, RAPD (random amplified polymorphic DNA) method, including but not limited to methods and detection kits shown in CN105177159A, CN102758008A, CN103184268A and CN 104120180A.

The invention is directed to a method for performing dye-based quantitative PCR directly without extracting human oral epithelial cell or peripheral blood cell samples, which are not limited to the above sources.

The preparation components of the direct dye amplification qPCR reaction solution adopt a plurality of one-tube type premixed solutions with strong inhibition resistance and prepared by PCR, and the form of the reagent appears in any form of liquid or freeze-dried powder.

The PCR enzyme having a high inhibitory activity used in the present invention is not limited to KOD series and rTth series, and includes PCR Mix having a high inhibitory activity based on Taq enzyme.

The dye method qPCR method used in the present invention is not limited to SYBR Green and Super EvaGreen, but includes other dye methods for quantitative PCR.

The following examples further illustrate specific embodiments of the present invention.

1. Three primer sets designed according to the principle of allele-specific PCR, A L DH2 two genotypes are even if the length of amplified fragments is the same, the Tm value of a specific amplification product can be improved by more than 5 ℃ by adding a tail sequence (GC tail) with high GC content at the 5' end of a specific primer, so the two genotypes can be distinguished by melting curve analysis of quantitative PCR by a dye method, therefore, the following four primer sets are designed according to the principle of ASP-PCR and the sequence of A L DH2 gene:

by comparing four groups of primers designed by two bases near the site of the rs671 mutation primer as the SNP genotyping site, the other Set I, II and III primer groups can not generate obvious difference of the temperature of the melting curve, so the following Set IV primer group is screened as the optimal primer sequence:

a L DH2-F upstream primer GTACGGGCTGCAGGCATAC shown as SEQ ID NO: 1;

a L DH2-R downstream primer GCCGCCCTGCCCGCCACACTCACAGTTTTCACTGCA shown as SEQ ID NO: 2;

a L DH2-MR mutant downstream primer CCCACACTCACAGTTTTCACTATA shown as SEQ ID NO 3;

2. positive plasmid synthesis:

the gene sequence of the positive control plasmid is a reference SNP sequence of https:// www.ncbi.nlm.nih.gov/SNP/671 in NCBI database, the gene sequence CGAGTACGGGCTGCAGGCATACACTGAAGTGAAAACTGTGAGTGTGGGACC in the positive plasmid of A L DH2 x 1 is shown as SEQ ID NO. 4, the SNP genotyping site is rs671, the gene sequence in the positive plasmid of A L DH2 x 2 is CGAGTACGGGCTGCAGGCATACACTAAAGTGAAAACTGTGAGTGTGGGACC, and is shown as SEQ ID NO. 5;

the plasmid is used for constructing a positive control plasmid in a whole gene synthesis mode.

While sterile water was used as a negative control to exclude contamination of the reaction system.

3. Collecting samples: the method for sampling the human oral epithelial cells or the peripheral blood cells comprises the following steps: the oral mucosa was collected with a cotton swab or a medical buccal swab and suspended in 200ul of sterile water.

Human peripheral blood cell sampling: blood was collected from a blood collection needle from a fingertip or an earlobe in an amount of about 2ul, diluted with 48ul of sterile water, and mixed.

4. The formula of the qPCR premix liquid by the direct dye amplification method comprises the following steps:

scheme one of the direct dye amplification qPCR premix:

10 volumes of SYBR Green qPCR Master Mix (QST-100, TOROIVD formula: 100-500mM Tris-HCl, pH8.0-9.0, 0.1% Tween20, 100-200mM (NH4)2SO4,50-80mM MgCl2, 5Mbetaine,1.5M trehalose, 0.66umSYBR Green I, hot start Taq enzyme 100U/ml, 2 MdNTPs)

) Or EvaGreen qPCR Master Mix (QET-100, TOROIVD, formulation composition: 100-500mM Tris-HCl, pH8.0-9.0, 0.1% Tween20, 100-200mM (NH4)2SO4,50-80mM MgCl2, 5M beta, 1.5M trehalo, 2.66uM Eva Green, 100U/ml hot start Taq enzyme, 2mM dNTPs), 3 volumes of primer set mixture (final concentration of each primer reaction system is controlled at about 0.2 uM), and finally 15 volumes of sterile water are filled.

A second scheme of direct dye amplification qPCR premix solution:

10 volumes of KOD SYBR qPCR Mix (QKD-201, TOYOBO, 100-500mM Tris-HCl, pH8.0-9.0, 0.1% Tween20, 100-200mM (NH4)2SO4,50-80mM MgCl2, 5M betaine,1.5M trehalose, 0.66. mu. SYBR Green I, 100U/ml hot start KOD enzyme, 2mM dNTPs), 3 volumes of primer set Mix (final concentration per primer reaction system controlled around 0.2 uM), 0.4 volumes of 50 × ROX (some quantitative PCR instruments require 0.04 volumes), and finally 15 volumes with sterile water.

A third formula of the qPCR premix liquid by the direct dye amplification method:

featuring contamination-resistant and saturation-type nucleic acid dyes, 0.5 volume of hot-start TthDNA Polymerase (5U/ul) or TTX DNA Polymerase (5U/ul) (TOYOBO), 10 volumes of 2 × Reaction Buffer (containing dUTP) (formula composition 100- & lt 500mM Tris-HCl, pH8.0-9.0, 0.1% Tween20, 100- & lt 200mM (NH4)2SO4,50-80mM MgCl2, 5M beta, 1.5M trehalose, 2mM dNTPs and dUTP), 3 volumes of primer set mix (final concentration of each primer set controlled around 0.2 uM), 0.4 volume of 50 × ROX (some quantitative PCR instruments require 0.04 volume), 0.4 volume of UDG enzyme (concentration 1U/ul) (TOYO), 0.4 volume of Super 50 USa5915 volumes of water, and finally EvAtlanter 15 volumes of Green Satured.

And 5, PCR reaction system configuration:

and (3) performing quantitative PCR reaction by a dye method, namely adding 5ul of the collected human oral epithelial cells or peripheral blood cells and diluted samples into 15ul of 'qPCR reaction solution by a direct dye amplification method', performing amplification on the quantitative PCR, and simultaneously performing amplification on positive control and negative control of two genotypes.

Setting PCR reaction conditions:

for formulation one, the PCR reaction conditions were as follows:

PCR cycling conditions: 95 deg.C, 2 minutes → (95 deg.C, 15 seconds; 60 deg.C, 45 seconds) 50 cycles → melting curve analysis

For formulation two, the PCR reaction conditions were as follows:

PCR cycling conditions: 95 ℃ 2 min → (98 ℃,10 sec; 60 ℃,10 sec; 68 ℃,30 sec) 50 cycles → melting curve analysis

For formulation three, the PCR reaction conditions were as follows:

PCR cycling conditions: 95 deg.C, 2 minutes → (95 deg.C, 15 seconds; 60 deg.C, 45 seconds) 50 cycles → melting curve analysis

7. And (3) melting curve analysis:

negative control amplification is used for eliminating pollution of 'direct dye method qPCR reaction liquid', two genotypes are determined by annealing temperatures from two positive controls, One positive control and One negative control can be adopted for the same batch of reaction, the two genotypes of A L DH2 are distinguished by the difference of the annealing temperatures in a melting curve, and an instrument used in the method is step One plus of ABI company.

FIG. 1 shows the melting curve of positive plasmid A L DH 2X 1 with annealing temperature around 81.5 deg.C, FIG. 2 shows the melting curve of positive plasmid A L DH 2X 2 with annealing temperature around 74.7 deg.C, FIG. 3 shows Glu504 homozygote (A L DH 2X 1/1) with annealing temperature around 81.5 deg.C, FIG. 4 shows Glu 504/L ys504 heterozygote (A L DH 2X 1/2) with annealing temperature around 81.5 deg.C and one peak around 74.7 deg.C, and FIG. 5 shows L ys504 homozygote (A L DH2 DH 2/2) with annealing temperature around 74.7 deg.C.

Results analysis that the annealing temperatures of the same sample in the same machine and the same reaction system are similar for PCR amplification, the Glu504 homozygote (A L DH2 x 1/1) with the main peak appearing only around 81.5 ℃ or other temperatures according to different enzyme reaction systems relative to two positive control plasmids, and the Glu 504/L ys504 heterozygote (A L DH2 x 1/2) with one peak appearing around 81.5 ℃ or other temperatures and 74.7 ℃ or other temperatures according to different enzyme reaction systems, wherein the 74.7 ℃ peak is obviously lower than the 81.5 ℃ peak.

The foregoing description of specific embodiments of the present invention has been presented. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes or modifications may be made by one skilled in the art within the scope of the appended claims without departing from the spirit of the invention.

Sequence listing

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