CN113774115A

CN113774115A - Method, kit, primer pair, probe, gene chip and application for detecting ALDH2 gene

Info

Publication number: CN113774115A
Application number: CN202110891033.XA
Authority: CN
Inventors: 张晓柯; 李洪波; 邢军芬; 朱滨
Original assignee: Shanghai Baio Technology Co ltd
Current assignee: Shanghai Baio Technology Co ltd
Priority date: 2021-08-04
Filing date: 2021-08-04
Publication date: 2021-12-10

Abstract

The invention discloses a method, a kit, a primer pair, a probe, a gene chip and application for detecting ALDH2 gene. The kit comprises a PCR reaction system and a gene chip, wherein the PCR reaction system comprises a primer pair consisting of an upstream primer and a downstream primer, and the nucleotide sequences of the primer pair are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2; and/or the nucleotide sequence of the probe is shown as SEQ ID NO.3 and/or SEQ ID NO. 4. When the method, the kit, the primer pair and the probe are used for detecting the ALDH2 gene, the method is simple to operate, low in cost, high in sensitivity and easy to read and accurate in result.

Description

Method, kit, primer pair, probe, gene chip and application for detecting ALDH2 gene

Technical Field

The invention relates to the field of in-vitro diagnostic reagents, in particular to a method, a kit, a primer pair, a probe, a gene chip and application for detecting ALDH2 gene.

Background

Blood is an important biological sample, and contains a large amount of DNA polymerase inhibitory substances such as heme, hetero-protein, fat, and the like. In order to complete the PCR amplification of nucleic acid from blood samples, the nucleic acid material must be purified from the blood. Generally, the method comprises three processes, namely, rupturing cell membranes and nuclear membranes; secondly, nucleic acid is purified, namely impurities such as protein and the like which influence the subsequent PCR reaction are removed; thirdly, eluting nucleic acid. Although nucleic acid with higher purity can be obtained through extraction treatment, the nucleic acid extraction process often results in a great amount of nucleic acid loss, and the complicated extraction and purification steps increase the risk of cross contamination among samples, thereby increasing the possibility of failure of subsequent PCR amplification. In addition, the extraction process is long in time consumption and high in cost, and some extraction methods also need phenol and other harmful reagents, so that the risk of infection of operators is increased, and high-throughput detection is difficult to realize. Therefore, the blood sample can be directly used as a template or simply treated to be used as a template for PCR amplification detection, which is a problem to be solved for blood sample amplification.

Human acetaldehyde dehydrogenase (ALDH), a quadruplex protein, catalyzes the oxidation of acetaldehyde and other aliphatic aldehydes. It has been found that there are 19 ALDH genes, mainly 4: ALDH1, ALDH2, ALDH3, and ALDH4, are encoded by separate gene loci located in four different chromosomes. Among them, ALDH2 is the most active member among 19 members of the acetaldehyde dehydrogenase family, and ALDH2 has the strongest ability to oxidize acetaldehyde, an intermediate metabolite of alcohol, to acetic acid in vivo, and is involved in acetaldehyde metabolism of 60% or more. ALDH2 is located in mitochondria, and its gene is located at 12q24.2 position of chromosome 12, with 43,438bp total length, and encodes polypeptide consisting of 517 amino acid residues.

The ALDH2 gene has different genotypes distributed in the population, and the most common single nucleotide polymorphism (rs671) of the gene is caused by mutation of a glutamic acid residue at exon 12 of the coding ALDH2 gene into lysine (Glu504 Lys). This polymorphism allows ALDH2 to develop three different genotypes: ALDH2 × 1/' 1 (wild homozygous), ALDH2 × 1/' 2 (heterozygous), ALDH2 × 2/' 2 (mutant homozygous). Carrying different genotypes will present different ALDH2 enzyme activities: the activity of the wild homozygote enzyme is normal, the activity of the heterozygote enzyme is reduced by 30 to 50 percent, and the activity of the mutant homozygote enzyme basically disappears.

At present, many technical means such as fluorescent quantitative PCR, high-resolution melting curve, gene sequencing and the like are adopted for ALDH2 genotyping detection. The gene sequencing method is suitable for high-throughput multi-site detection, but is long in operation time and low in sensitivity, and is not suitable for rapid clinical detection; and the amount of information obtained by sequencing is large, much information is useless for patients, some professional knowledge is needed for judging the sequencing result, and the cost is relatively high. The high-resolution dissolution curve method has special requirements on equipment, can be used only by a machine which is provided with high-resolution software and sensitive to temperature, and has certain difficulty in clinical popularization.

Regarding a fluorescent quantitative PCR method, CN201811034949 discloses a human ALDH2 gene polymorphism detection kit, which comprises a PCR premixed reaction solution for amplifying ALDH2 gene G1510A locus, a positive control and a negative control; ARMs primers with tag sequences carrying different fluorescence can specifically recognize and amplify the wild and mutant genotype templates, release more fluorescence along with the consumption of the primers, and collect the fluorescence of each channel at the end of the cycle to generate a fluorescence curve. The ALDH2 genotyping detection performed by the methods has the problems of high instrument purchase cost, need of extracting genome DNA, complex operation procedures and the like in different degrees, and is not beneficial to improving the detection efficiency and realizing clinical popularization. The detection requirement is huge at present, so that an efficient detection means with a price easily accepted by patients is found, and the detection method has important practical significance for ALDH2 genotyping detection.

Furthermore, it is well known in the art that primer design takes into account the sequence composition (GC content) of the amplification product containing the SNP site, the size of the fragment, the position of the SNP site in the product, and the like, which are important factors affecting the hybridization kinetics of the PCR product with the probe, in genotyping. The size of a PCR product fragment with good hybridization kinetics is generally 200-300 bp, the steric hindrance of the PCR product with a larger fragment (for example, more than 500bp) is not favorable for hybridization with the probe, the hybridization efficiency is reduced, the hybridization efficiency of the PCR product with a smaller fragment (for example, less than 100bp) is higher, but the specific hybridization difficulty of wild and mutant probes is increased. The PCR product with higher GC content has higher hybridization efficiency due to stronger hydrogen bonding capability among bases, while the PCR product with lower GC content has lower efficiency under the same hybridization conditions. The position of the SNP site in the PCR product is also an important factor influencing the steric hindrance of hybridization. These factors make it not particularly easy to design primer pairs that can be used to genotype a particular SNP site. Similarly, the design of the probe needs to overcome many difficulties, such as ensuring that the hybridization conditions of the wild-type probe and the mutant-type probe are as consistent as possible. A great deal of experimental verification is needed to design better primer pairs and probes.

Disclosure of Invention

The invention aims to solve the technical problems of low amplification efficiency, high cost, complex and time-consuming operation procedures, difficulty in popularization and increase in cross contamination and toxic reagent use in genotyping in the prior art, and provides a method, a kit, a primer pair, a probe and application thereof for detecting ALDH2 gene, which have the advantages of high amplification efficiency, low cost, simplicity in operation and easiness in popularization. The method, the kit, the primer pair and the probe can be used for detecting the ALDH2 genotype, have high accuracy and can accurately distinguish mutation sites. The gene chip in the kit has good signal-to-noise ratio, is not easily interfered by other interferents in samples to be detected, such as whole blood, oral exfoliative cells and the like, and solves the problem that amplification products of the samples to be detected, such as the whole blood, the oral exfoliative cells and the like, containing various interferents influence the hybridization of the chip. Wherein the probe and the primer pair have good specificity. The method and the kit have the advantages of simple operation and low cost, have high sensitivity (in a preferred embodiment of the invention, the detection limit can be as low as 130 white blood cells), greatly shorten the detection time, have easily-readable and accurate results, are very visual in judgment, and are more suitable for clinical detection and popularization. The method of the invention can preferably use the sample to be detected of whole blood as PCR template directly, and can avoid extracting genome DNA, so that the method does not need to purchase extra genome DNA extraction kit, saves cost, reduces pollution chance, and is suitable for large-scale sample detection. And the required samples are few, and only 1 microliter of blood is needed at least (while 200 microliter of blood is generally needed in the prior art for DNA extraction), and the samples to be detected such as whole blood and the like stored in a refrigerator at 4 ℃ within 7 days have stable detection results, are beneficial to reexamination, or other samples to be detected such as whole blood and the like detected by a patient can be utilized, and the patient does not need to sample specially, thereby relieving the pain of the patient. The invention can realize the detection of ALDH2 gene by directly hybridizing the sample to be detected after PCR. The hybridization step in the method can be realized through a full-automatic hybridization process, so that the method is more convenient and quicker and avoids a plurality of uncertain factors existing in the manual operation process. The method, the kit, the primer pair, the probe and the application thereof provided by the invention are used for detecting the ALDH2 genotype of a patient, and a simple and easy solution is provided.

The existing method for genotyping generally adopts fluorescent quantitative PCR, even if strong-tolerance DNase is used, a sample to be detected can be used in the PCR only by certain pretreatment, and the requirement on the sample to be detected is high and the dosage is large. In addition, as known to those skilled in the art, in the environment of a sample to be tested (such as whole blood, exfoliated cells in the mouth, etc.) with an interfering substance, the design and the proportion of a primer pair and a probe are relatively difficult. In the hybridization reaction, the sequence composition, the lengths of the target DNA molecule and probe, the hybridization temperature, salts, etc. all affect the hybridization efficiency and strength. The amplification product used for genotyping cannot be too long, which would affect the hybridization speed, and when the amplification product is short, there is very little room for primer selection, while it is necessary to ensure sufficient amplification efficiency, and the design difficulty is very large in consideration of the cooperation with the primer. In addition, because the probe is fixed on the glass slide, and the glass slide has a certain repulsive force, the spatial structure of the probe needs to be considered when the probe is designed, so that the repulsive force is reduced; meanwhile, the designed probe is ensured to be capable of hybridizing with the amplification product; and the specificity of probe hybridization needs to be considered, and the homozygous mutant sample needs to ensure that no signal is generated when the wild-type probe is hybridized, and the mutant probe can generate a signal. The wild type and mutant type probes in the invention only have difference of a few bases, and the design difficulty is very large. The present inventors have made extensive attempts to design primer pairs and probes of the present invention, and have prepared gene chips based thereon, and by using them in combination with a strongly tolerated DNA polymerase, detection results with high accuracy, sensitivity and specificity can be obtained even with ordinary non-fluorescent quantitative PCR. The detection method is simple, rapid and low in cost, and samples such as whole blood, oral exfoliative cells and the like to be detected can be directly added for detection without any pretreatment.

In order to solve the technical problems, the invention aims to provide a kit for detecting ALDH2 gene, which comprises a PCR reaction system and a gene chip, wherein the PCR reaction system comprises a primer pair consisting of an upstream primer and a downstream primer, and the gene chip comprises a probe; wherein the content of the first and second substances,

the nucleotide sequences of the primer pairs are shown as SEQ ID NO.1 (5'-gggagttgggcgagtacgg-3') and SEQ ID NO.2 (5'-ccctctcttgtcacttctcaggc-3');

and/or the nucleotide sequence of the probe is shown as SEQ ID NO.3 (5'-GGCATACACTgAAGTGAAAACTG-3') and/or SEQ ID NO.4 (5'-GGCATACACTAAAGTGAAAACTGTG-3').

In the present invention, the gene chip may be conventional in the art, and generally comprises a solid support and specific oligonucleotide probes immobilized on the solid support.

Preferably, the concentration of the primers in the PCR reaction system is 0.06-0.76. mu.M, preferably 0.18-0.76. mu.M, and more preferably 0.24-0.76. mu.M.

More preferably, the concentration of the upstream primer in the PCR reaction system is 0.06-0.24. mu.M, preferably 0.24. mu.M; and/or the concentration of the downstream primer in the PCR reaction system is 0.18-0.76 mu M, preferably 0.76 mu M.

Preferably, the concentration of the probe in the gene chip is 1.67-20 μ M, more preferably 5 μ M.

In the present invention, unless otherwise specified, the concentration of the primer or probe is generally referred to as the working concentration.

Preferably, the primer pair is a primer pair modified by a biological detection marker at the 5' end, and the biological detection marker is preferably biotin, digoxin, fluorescein, a fluorescein derivative, a fluorescent molecule, alkaline phosphatase, horseradish peroxidase or the like, such as biotin. By adopting the biological detection marker modification of different primer pairs, the corresponding detection method can be changed during the subsequent hybridization detection. These detection methods may be any known in the art.

In a preferred embodiment of the present invention, the primer pair is a primer pair with biotin modification at the 5' end, and the subsequent hybridization detection is also performed by using an anti-biotin antibody solution and a developing solution. The method specifically comprises the following steps: biotin (Biotin) on the amplification product first hybridized with the detection probe first reacts with Streptavidin-alkaline phosphatase complex (Streptavidin-AP) to form a new complex: Biotin-Streptavidin-AP, which undergoes a color reaction as follows:

Biotin-Streptavidin-AP+BCI-P→BCI-OH+Pi(pH 7.5)

BCI-OH + NBT → bluish purple precipitate

Wherein BCIP is 5 bromo-4 chloro-3 indolyl phosphate; NBT is nitro blue tetrazolium. The ALDH2 genotype was detected by hybridizing the probe to the amplified region.

Preferably, the probe is a probe modified at the 5' end with a group which is bound to the solid support of the gene chip, preferably a probe modified with an amino group, more preferably a probe modified with an amino group and a plurality of bases T, and still more preferably a probe modified with an amino group and 16 bases T. After amino modification, the probe can be better connected with a solid phase support (such as a glass slide or a silicon chip) of the gene chip modified by aldehyde group.

Preferably, the PCR reaction system further comprises PCR reaction buffer and/or deoxynucleoside triphosphate (dNTP).

In the present invention, the PCR reactionThe reaction system may also include a strongly tolerant DNA polymerase. The highly tolerant DNA polymerase, PCR reaction buffer, deoxynucleotide triphosphate 3 may be obtained commercially, for example in the form of a commercially available 2 XPCR Mix. For example, the 2 × PCR Mix is selected from one or more of the following: 2 XDirect PCR Mix, 2 XTransDirect^TMPCR Supermix, Phusion Blood Direct PCR Master Mix (2X). Therefore, when the detected sample is whole blood and the like, the whole blood sample does not need any pretreatment, the PCR reaction system in the kit can be directly used for carrying out PCR amplification on the whole blood, and the extraction of genome DNA is not needed, so that the additional purchase of the genome DNA extraction kit is not needed, the cost is saved, the pollution opportunity is reduced, and the kit is suitable for detection of large-scale samples.

Reagent	Purchasable factory
		2×Direct PCR Mix	SANGON BIOTECH (SHANGHAI) Co.,Ltd.
2×TransDirect^TM PCR SuperMix	Beijing Quan Shijin Biotechnology Co., Ltd.
		Phusion Blood Direct PCR Master Mix(2×)	Thermo Scientific

In a preferred embodiment of the present invention, the amount of the PCR reaction system is as follows:

preferably, the sample to be detected for the kit is a biological sample containing genomic DNA, preferably whole blood or oral mucosa exfoliative cells.

When the sample to be tested is oral mucosa exfoliated cells, the oral mucosa exfoliated cells are generally suspended in a solvent to be applied to PCR reaction.

Preferably, the kit also comprises negative probes, and the nucleotide sequences of the negative probes preferably have the nucleotide sequences shown in SEQ ID No.5 (5'-GGCATACACTAcgGTGAAAACTG-3') with the final concentration of 2.5 mu M.

Preferably, the kit further comprises a hybridization solution, wherein the hybridization solution preferably comprises a hybridization buffer solution, a pre-hybridization solution, a hybridization reaction solution and a washing solution, the hybridization solution in the hybridization color development kit is preferably selected, and the hybridization color development kit (with the product number of BST03021) produced by Baiao science and technology, Inc. of Shanghai is more preferably selected.

Preferably, the kit further comprises a solution capable of reacting with the detection marker of the primer pair and a color-developing solution such as tetrazolium blue (NBT), 5-bromo-4-chloro-3-indoxyl-phosphate-4-toluidine salt (BCIP), or Tetramethylbenzidine (TMB). After the PCR product is hybridized with the gene chip and is mixed with a solution which can react with the biological detection markers of the primer pair, the color development solution can react with the solution to develop color so as to detect a hybridization signal.

In the present invention, the PCR may be a PCR technique which is conventional in the art, and may be, for example, a non-fluorescent quantitative PCR.

In order to solve the technical problems, the invention aims to provide a primer pair and/or a probe for detecting ALDH2 gene, wherein the nucleotide sequence of the primer pair is shown as SEQ ID NO.1 and SEQ ID NO.2, and the nucleotide sequence of the probe is shown as SEQ ID NO.3 and/or SEQ ID NO. 4.

Preferably, the probe is immobilized on a solid support of the gene chip. The solid support may be conventional in the art, such as a glass slide or a silicon wafer. In a preferred embodiment, when multiple probes are included, the probes can be immobilized on the solid support of the same gene chip.

Preferably, the assay is a PCR assay.

Preferably, the sample to be detected is a biological sample containing genomic DNA, preferably whole blood or oral mucosa exfoliative cells.

In order to solve the above technical problems, the present invention aims to provide a gene chip, which comprises a solid support and the probes as described above immobilized on the solid support, wherein the gene chip preferably comprises one or more than one block.

In order to solve the technical problems, the invention aims to provide application of the primer pair and/or the probe in detecting ALDH2 gene and preparing a reagent or a kit for detecting ALDH2 gene.

In order to solve the above technical problems, the present invention aims to provide a method for detecting ALDH2 gene, which comprises the following steps: and carrying out PCR reaction on a sample to be detected by using the PCR reaction system, hybridizing the obtained product with the gene chip, and detecting a hybridization signal. The method for detecting the ALDH2 gene in the invention can be a detection method for non-diagnostic purposes, such as detecting ALDH2 gene in an ex vivo sample for research and development.

In the present invention, the oligonucleotide probes can specifically hybridize to different genotypes at the 504 th site of the ALDH2 gene by a chip hybridization method. The specific primer pair can amplify a target region containing a detection target site. The probe is constructed by the principle that the target DNA and the complementary base of the probe are restored to the original double helix structure by hydrogen bonding.

Preferably, the resulting product is mixed with a hybridization buffer prior to hybridization with the gene chip.

Preferably, the gene chip is prehybridized with a prehybridization solution before hybridizing with the gene chip. This step is advantageous for optimizing the background of chip hybridization.

The method for detecting ALDH2 gene for non-diagnosis purpose of the invention has the detection sensitivity of 130 leucocytes for whole blood sample.

In the present invention, the detection of the hybridization signal may be to obtain the information to be detected based on the position, intensity, etc. of the labeled signal (the signal of the biological detection marker on the fragment obtained by PCR amplification) on the gene chip, and the method for detecting the hybridization signal may be based on the basic phosphatase-catalyzed tetrazolium blue (NBT) color reaction, horseradish peroxidase-catalyzed 5-bromo-4-chloro-3-indoxyl-phosphate-4-toluidine salt (BCIP) color reaction, horseradish peroxidase-catalyzed Tetramethylbenzidine (TMB) color reaction and/or fluorescence detection, which is compounded with an avidin antibody, avidin, streptavidin, an anti-digoxigenin antibody, or an anti-fluorescein antibody. The specific method can be found in the manual of genetic diagnosis technology-nonradioactive operation, compiled by Wangshen five kingdoms. If the amplification product is labeled with a fluorophore, the information to be detected can also be obtained by using a fluorescence detection device (such as a confocal laser scanner Scan array 3000). Preferably, the hybridization signal is detected by alkaline phosphatase-catalyzed tetrazolium blue color reaction, horseradish peroxidase-catalyzed 5-bromo-4-chloro-3-indoxyl-phosphate-4-toluidine color reaction, horseradish peroxidase-catalyzed tetramethylbenzidine reaction or fluorescence detection.

In the present invention, the gene chip can be prepared according to a conventional method for manufacturing a biochip. For example, if a modified glass slide or silicon wafer is used as the solid support and the 5' -end of the probe contains a poly-dT chain modified with an amino group, the oligonucleotide probe can be prepared as a solution, and then spotted on the modified glass slide or silicon wafer using a spotting device to arrange it into a predetermined sequence or array, followed by standing overnight for immobilization, to obtain a gene chip. If the oligonucleotide probe does not contain amino group modifications, the method of preparation can also be referred to: wangshen five-main edition "Gene diagnosis technology: non-radioactive operation manual; derisi, JL, equals 1997 in science 278(5338):680 ℃ 686 (Dersi, JL, IyerVR, Brown PO. expanding the metabolic and genetic control of gene expression in the genome of human beings) and Malikan et al (biochips, chemical Industrial Press) eds.

In the present invention, the solid phase hybridization between the amplification product and the gene chip is carried out according to a classical method in the art, or according to the genetic diagnosis technique, which is compiled by Wangshen five: non-radioactive operation manual; J. the scientific Press was compiled in molecular cloning guidelines, by Summebruke et al.

The term "strong tolerance" in the case of a strongly tolerant DNA polymerase means that the polymerase is resistant to DNA polymerase inhibitory substances contained in a sample to be tested and is not inhibited by the DNA polymerase inhibitory substances.

On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.

The reagents and starting materials used in the present invention are commercially available.

The positive progress effects of the invention are as follows:

(1) the method, the kit, the primer pair and the probe can be used for detecting the ALDH2 genotype, have high accuracy and can accurately distinguish 504 mutation sites.

(2) The gene chip in the kit has good signal-to-noise ratio, is not easily interfered by other interferents in samples to be detected, such as whole blood, oral exfoliated cells and the like, solves the problem that amplification products of the samples to be detected, such as the whole blood, the oral exfoliated cells and the like, containing various interferents influence the hybridization of the chip, and the probe and the primer pair have good specificity.

(3) The method and the kit have the advantages of simple operation and low cost, have higher sensitivity (in a preferred embodiment of the invention, the detection limit can be as low as 130 white blood cells), are suitable for directly amplifying a whole blood sample, avoid the step of nucleic acid extraction, greatly shorten the detection time, have easily-readable and accurate results, are very intuitive in judgment, and are more suitable for clinical detection and popularization. The method of the invention preferably can also take the sample to be detected as the PCR template directly, and can avoid extracting the genome DNA, so that the method does not need to purchase a genome DNA extraction kit additionally, saves the cost, reduces the pollution chance and is suitable for the detection of large-scale samples. And the required samples are few, only 1 mu L is needed at least, and the samples to be detected such as whole blood and the like are stored in a refrigerator at 4 ℃ for 7 days, so that the detection result is stable, and the reexamination is facilitated, or the samples to be detected such as other detected whole blood and the like of a patient can be utilized, the special sampling of the patient is not needed, and the pain of the patient is relieved. The invention can realize the detection of ALDH2 gene by directly hybridizing the sample to be detected after PCR.

(4) The hybridization step in the method can be realized through a full-automatic hybridization process, so that the method is more convenient and quicker and avoids a plurality of uncertain factors existing in the manual operation process.

The method, the kit, the primer pair, the probe and the application thereof can be used for detecting the ALDH2 genotype of a patient, and the ALDH2 gene of the patient can be detected, so that a scientific basis is provided for individualized medication, and a simple, convenient and feasible solution is provided.

Drawings

FIG. 1 is a schematic diagram of the ALDH2 gene chip in example 1.

Fig. 2 is a graph showing the scanning results in example 4.

FIG. 3 is a graph showing the results of sequencing verification in example 4, and the sequencing graph at the position 504 is shown by an arrow.

FIG. 4 is a graph showing the results of sensitivity detection in example 5.

FIG. 5 is a graph showing the results of detection in example 6.

FIG. 6 is a graph showing the results of detection in example 7.

FIG. 7 is a graph showing the results of detection in example 8.

Fig. 8 is a graph showing the results of the measurement in comparative example 1.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.

The gene sequence used was derived from NCBI (national center for biotechnology information):

ALDH2 amino acid Glu/Lys at position 504 (rs671 site)

EXAMPLE 1 preparation of Gene chip

Aldehyde-modified glass slides (product number: BSM03011, Baiao science Co., Ltd., Shanghai). A probe (Biotechnology, Shanghai, Ltd.) was artificially synthesized by dissolving the probe in water to a concentration of 100 pmol/. mu.l, and mixing the solution at an equal ratio (volume ratio) of 2 Xsample application buffer (product No.: BST02010, Shanghai Baiao science and technology, Ltd.). Next, an array as shown in the left side of FIG. 1 (right side of FIG. 1 shows each number of probes) was spotted using BD-1 spotter from Baiao science, Shanghai, as described in the specification. The mixture was left at room temperature overnight.

The sequence of a specific oligonucleotide probe detecting site 504Glu is shown in SEQ ID No.3 (underlined):

NH2-TTTTTTTTTTTTTTTTGGCATACACTgAAGTGAAAACTG(ii) a The 5 'end of the poly-16-poly-dT also contains a segment of 5' amino (-NH2) modified poly-deoxythymidylate.

The sequence of the specific oligonucleotide probe for the detection site 504Lys is shown in SEQ ID No.4 (underlined):

NH2-TTTTTTTTTTTTTTTTGGCATACACTAAAGTGAAAACTGTG(ii) a The 5 'end of the poly-16-poly-dT also contains a segment of 5' amino (-NH2) modified poly-deoxythymidylate.

The sequence of the 504 th negative control probe of the ALDH2 gene is shown in SEQ ID No.5 (underlined part):

NH2-TTTTTTTTTTTTTTTTGGCATACACTAcgGTGAAAACTG(ii) a The 5 'end of the poly-16-poly-dT also contains a segment of 5' amino (-NH2) modified poly-deoxythymidylate.

As shown in FIG. 1, a positive quality control probe (purchased from Baiao science and technology Co., Ltd., Shanghai), a specific detection probe, a negative quality control probe and a blank control were spotted on a slide glass to obtain a gene chip. Wherein

A positive quality control probe; the method comprises the following steps: 504Glu probe; secondly, the step of: 504Lys probe;

504 negative control probes;

blank control.

Example 2 amplification of ALDH2 Gene fragment by PCR Using primers provided by the invention

Primers were synthesized by Compton bioengineering (Shanghai) Inc., and the primer information is shown below.

The sequence of the upstream primer is SEQ ID No. 1:

5’-gggagttgggcgagtacgg-3’，

the sequence of the downstream primer is SEQ ID No. 2:

5’-ccctctcttgtcacttctcaggc-3’，

and, the 5' end of the primer is modified with biotin.

Then dissolved in water and diluted to a concentration of 10 pmol/. mu.l.

Preparing a PCR amplification system according to the following formula:

amplification was performed using a PCR apparatus (TC-96/G/H (b) PCR amplification apparatus, available from Bori science, Inc., Hangzhou) according to the following procedure:

50℃5min；94℃10min；94℃25sec，60℃25sec，72℃30sec，40cycles；72℃5min。

SEQ ID NO.6 in the sequence list is the SNP sequence of ALDH2 gene 504 site, the sequence is from NCBI SNP database information, can find ALDH2 gene 504 site sequence through rs671, it is the partial sequence of ALDH2, the amplified target product of the invention is included in this sequence. Wherein R represents a degenerate base, i.e., A/G.

Example 3 hybridization of the Gene chip obtained in example 1 and the PCR product obtained in example 2

Hybridization was performed in a fully automatic hybridization apparatus (BSE 03011) using the hybridization color development kit (BST03021) available from shanghai baiao science and technology gmbh, as follows: the hybridization color development kit is alkaline phosphatase color development reaction.

1) Preparing a hybridization reaction solution: mu.L of hybridization buffer solution in the hybridization color development kit was aspirated, 10. mu.L of the amplification product of the reaction system in example 2 was added, and mixed well.

2) The gene chip prepared in example 1 was placed on a hybridization apparatus and covered tightly.

3) Setting a hybridization program: prehybridization solution, 44 ℃, 5 min; hybridizing the reaction solution at 44 ℃ for 30 min; washing at 1, 44 deg.C for 6 min; washing at 1, 44 deg.C for 6 min; washing at 2, 28 deg.C for 5 min; washing at 2, 28 deg.C for 5 min; antibody solution, 28 deg.C, 20 min; washing at 2, 28 deg.C for 5 min; washing at 2, 28 deg.C for 5 min; washing at 28 deg.C for 3 min; developing solution at 44 ℃ for 30 min; pre-hybridization solution at 28 ℃ for 2 min; pre-hybridization solution at 28 ℃ for 2 min; the specific operation is shown in the instruction book of the instrument. The program is operated, and the hybridization color reaction is automatically carried out.

Example 4 detection results of hybridization signals of Gene chip

The hybridized and washed gene chip was placed on a BaiO BE-3.0 biochip reader (Shanghai Baiao science and technology Co., Ltd.) and scanned to obtain the detection result shown in FIG. 2, which was very visual. The result shows that the ALDH2 genotype of the subject is Lys504Lys, the detection result is verified by sequencing, the sequencing result is shown in figure 3 (the sequencing result of 504 sites at the arrowheads), and the result is completely consistent.

Example 5 results of sensitivity detection

1) Sample preparation

Blood of known ALDH2 genotype (supplied by clinical cooperative, known as Glu540Glu) was taken, diluted with negative control to obtain samples with different leukocyte content as shown in the following table:

name (R)	Leukocyte concentration (10)⁸/L)
		G-1	1.44
G-2	1.3
		G-3	0.96

2) Detection of

The samples were tested as described in examples 1-4.

3) Results

The hybridization results are shown in FIG. 4: where the graph A, B, C corresponds to G-1, G-2, and G-3, respectively.

The detection result is integrated to show that the detection method has high detection sensitivity and can detect 1.3 multiplied by 10⁸Blood at leukocyte concentration, i.e. sensitivity of 130 leukocytes.

Example 6 interference test results

1) Sample preparation

Selecting EDTA anticoagulated whole blood sample with known ALDH2 genotype (provided by clinical cooperative unit, all blood samples with known genotype, and known genotype as Glu504Lys), preparing the sample containing the interferent according to the following method: according to the solubility of the interferent in the blood matrix, the easily soluble substance is prepared by directly adding the interferent solid into the blood, dissolving and uniformly mixing; for insoluble matters, dissolving the insoluble matters by adopting a proper solvent, adding the dissolved matters into a matrix, and uniformly mixing the dissolved matters; samples with different levels of interferents are shown in the following table:

name of interfering substance	Concentration of
		Total Cholesterol	250mg/dL
Triglycerides	3000mg/dL
		Bilirubin	20mg/dL

2) Detection of

The above samples were tested by the gene chip according to the methods of examples 1 to 4.

3) Results

The hybridization results are shown in FIG. 5: wherein FIG. D, E, F corresponds to total cholesterol, triglyceride, and bilirubin, respectively.

250mg/dL total cholesterol in blood; 3000mg/dL triglyceride; 20mg/dL bilirubin does not interfere with the assay of the present invention.

Example 7 detection results under different primer concentration conditions

The known ALDH2 genotype blood (known genotype as Glu504Glu, amplification solutions with different primer concentration ratios) was used for detection according to the methods of examples 1-4, and the detection results are shown in FIG. 6.

The amplification solution primer ratio experiments are shown in the following table:

	primer mix 1	Primer ratio 2	Primer ratio 3
				Upstream primer (mu M)	0.06	0.12	0.24
Downstream primer (μ M)	0.18	0.40	0.76

From the above results, the concentrations of the upstream primer and the downstream primer in the amplification solution are known, and the concentrations of the primers may be in the range of 0.06 to 0.76. mu.M, preferably 0.24. mu.M and 0.76. mu.M.

Example 8 test results at different Probe concentrations

The detection was carried out by the methods of examples 1 to 4 using gene chips prepared by mixing blood of known ALDH2 genotype (known as Glu504Glu) with different probe concentrations. The results of the detection are shown in FIG. 7.

The gene chip 1 probe ratio experiment is shown in the following table:

probe needle	Concentration ratio 1(μ M)	Concentration ratio 2 (mu M)	Concentration ratio 3 (mu M)
				SEQ ID NO:3	10	5	5
SEQ ID NO:4	5	5	10
				SEQ ID NO:5	2.5	2.5	2.5

SEQ ID NO.3 is a 504Glu probe, SEQ ID NO.4 is a 504Lys probe, and SEQ ID NO.5 is a 504 negative probe.

The concentrations of the 504Glu probe, 504Lys probe and the negative probe are preferably 5. mu.M, 5. mu.M and 2.5. mu.M in combination of the above results.

Comparative example

1) Non-preferred probes:

design failure probe for detection of the 504Glu site SEQ ID NO:7 (underlined):

NH2-TTTTTTTTTTTTTTTTAGGCATACACTAAAGTGAAAACTGTG

a chip was prepared using the above 504Glu site non-preferred probe and 504Lys site preferred probe, and whole blood (supplied by clinical Cooperation Unit) of known genotype ALDH2 (known genotype Lys504Lys) was used as a sample to carry out the test according to examples 1 to 4, and the test images are shown in FIG. 8. As can be seen from the figure, the site signal non-specific signal corresponding to the non-preferred probe of the gene chip under the condition of the non-preferred probe is high, and the genotype cannot be accurately judged, thereby affecting the accuracy of the result.

Sequence listing

<110> Shanghai Baiao science and technology, Inc

<120> method, kit, primer pair, probe, gene chip and application for detecting ALDH2 gene

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ccctctcttg tcacttctca ggc 23

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ggcatacact aaagtgaaaa ctgtg 25

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aggcatagtg gcacatactt gttatcttaa ctacttggga ggctgaggca ggaggatcac 60

tgaagaccag gagttggaga ccagcctggg taacataatc agaccctgtc tcttaaaaaa 120

aaatttattg ccaggcgtgg ttgcacgtgc tggtagtcca gctactcagg aagctgaggc 180

aggagaatct cttgaacccc agatgtggag gttgcaacga gccaagatca tgccatggca 240

actccagcct gggcaacaga gaaagattct atctcaaaaa aaaaaatttt tttttaagtt 300

aaaaataaaa taaagacttt ggggcaatac agggggtcct gggagtgtaa cccataaccc 360

ccaagagtga tttctgcaat ctcgtttcaa attacagggt caactgctat gatgtgtttg 420

gagcccagtc accctttggt ggctacaaga tgtcggggag tggccgggag ttgggcgagt 480

acgggctgca ggcatacact raagtgaaaa ctgtgagtgt gggacctgct gggggctcag 540

ggcctgttgg ggcttgaggg tctgctggtg gctcggagcc tgctggggga ttggggtctg 600

ttgggggctc ggggcctgcc agaggttcag gacctgccgg ggactcaggg cctgctggaa 660

gttcaggacc tgctggggat cagggcctgc cagggattta gggtctgctg ggcgggccac 720

cttttggcct ctccctcatg cttgaggcca tcagtgtttc ctactaattt cccattttaa 780

gcctgagaag tgacaagaga gggtaaagac ccagcctctg ctctgtccca tgagaaatac 840

tgagggacgt gcccccatca ggcctatgcg gtcatttgct gggcttcgtt atacgccaag 900

gcctgtaggc ctgagaagag ggagagactt cagggggcgg agcggagagg aaaagcttct 960

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Claims

1. A kit for detecting ALDH2 gene, characterized in that, it comprises PCR reaction system and gene chip, the PCR reaction system includes a primer pair composed of an upstream primer and a downstream primer, the gene chip includes probe; wherein the content of the first and second substances,

the nucleotide sequences of the primer pair are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2;

and/or the nucleotide sequence of the probe is shown as SEQ ID NO.3 and/or SEQ ID NO. 4.

2. The kit of claim 1, wherein the concentration of the primers in the PCR reaction system is 0.06-0.76 μ M, preferably 0.18-0.76 μ M, and more preferably 0.24-0.76 μ M;

and/or the concentration of the probe in the gene chip is 1.67-20 mu M, preferably 5 mu M.

3. The kit of claim 1 or 2, wherein the primer pair is a primer pair modified at the 5' end by a biological detection marker, preferably biotin, digoxigenin, fluorescein, a fluorescein derivative, a fluorescent molecule, alkaline phosphatase, or horseradish peroxidase;

and/or, the probe is a probe whose 5' end is modified by a group, the group is combined with a solid phase support of the gene chip, preferably a probe modified by an amino group, more preferably a probe modified by an amino group and a plurality of bases T, and still more preferably a probe modified by an amino group and 16 bases T;

and/or the PCR reaction system also comprises a strong tolerance DNA polymerase, and the strong tolerance DNA polymerase is preferably Direct PCR Mix and TransDirect^TMOne or more of PCR SuperMix and Phusion Blood Direct PCR Master Mix;

and/or the sample to be detected for the kit is a biological sample containing genomic DNA, preferably whole blood or oral mucosa exfoliative cells.

4. The kit of any one of claims 1 to 3, wherein the PCR reaction system further comprises a PCR reaction buffer and/or deoxynucleoside triphosphates;

and/or, the kit also comprises a negative probe, and the nucleotide sequence of the negative probe is preferably shown as SEQ ID No. 5.

5. The kit according to any one of claims 1 to 4, wherein the kit further comprises a hybridization solution, preferably comprising a hybridization buffer, a prehybridization solution and/or a wash solution; and/or, the kit further comprises a solution capable of reacting with the detection marker of the primer pair and a color developing solution such as tetrazolium blue, 5-bromo-4-chloro-3-indoxyl-phosphate-4-toluidine salt, or tetramethylbenzidine.

6. A primer pair for detecting ALDH2 gene is characterized in that the nucleotide sequence of the primer pair is shown as SEQ ID NO.1 and SEQ ID NO. 2;

preferably, the detection is PCR detection, and/or the detected sample is a biological sample containing genomic DNA, preferably whole blood or oral mucosa exfoliative cells.

7. A probe for detecting ALDH2 gene is characterized in that the nucleotide sequence of the probe is shown as SEQ ID NO.3 and/or SEQ ID NO. 4;

preferably:

the probe is fixed on a solid phase support of the gene chip; and/or, the detection is PCR detection; and/or the detected sample is a biological sample containing genome DNA, preferably whole blood or oral mucosa cast-off cells.

8. A gene chip comprising a solid support and the probe of claim 7 immobilized on the solid support.

9. Use of the primer pair according to claim 6 and/or the probe according to claim 7 for detecting ALDH2 gene, preparing a reagent or a kit for detecting ALDH2 gene.

10. A method for detecting ALDH2 gene for non-diagnostic purposes, comprising the steps of: carrying out PCR reaction on a sample to be detected by using a PCR reaction system of the kit according to any one of claims 1 to 4, hybridizing the obtained product with a gene chip of the kit according to any one of claims 1 to 4, and detecting a hybridization signal; preferably:

mixing the obtained product with a hybridization buffer solution before hybridization with the gene chip;

and/or, the gene chip is pre-hybridized with a pre-hybridization solution before being hybridized with the gene chip;

and/or the method for detecting the hybridization signal is tetrazolium blue color development reaction catalyzed by alkaline phosphatase, 5-bromo-4-chloro-3-indoxyl-phosphate-4-toluidine color development reaction catalyzed by horseradish peroxidase, tetramethylbenzidine reaction catalyzed by horseradish peroxidase and/or fluorescence detection;

and/or the detected sample is a biological sample containing genome DNA, preferably whole blood or oral mucosa exfoliative cells.

11. The method of claim 10, wherein the detection sensitivity for a whole blood sample is 130 leukocytes.