药物和营养代谢能力检测snp位点引物组合物及应用

SNP (Single nucleotide polymorphism) site primer composition for detecting drug and nutrient metabolic capability and application

文档序号：3379 发布日期：2021-09-17 浏览：41次中文

1. A SNP site primer composition for detecting drug and nutrient metabolizability, characterized by: the SNP loci comprise: rs4693075, rs7412, rs1057910, rs11676382, rs1799853, rs2108622, rs2359612, rs4917639, rs56165452, rs61742245, rs7196161, rs7294, rs8050894, rs9923231, rs9934438, rs10509681, rs17244841, rs20455, rs16960228, rs4149601, rs7297610, rs1346268, rs1719247, rs17244841, rs4149056, rs1801133, rs70991108, rs 12299999984, rs1799899, rs1800562, rs197273, rs3811647, rs 17671 8, rs1049296, rs10849915, rs 15341418966, rs 1789676702, rs2168784, rs35951, rs 784457858, rs 445776720, rs 75767656300, rs 18276549, and 1828859.

2. The primer composition of claim 1, wherein: the primer composition comprises:

3. the primer composition of claim 2, wherein: the medicine comprises warfarin, hydrochlorothiazide, atorvastatin, pravastatin, simvastatin and rosiglitazone; the nutrient substances include alcohol, lactose, folic acid and caffeine.

4. Use of the primer composition of claim 1 for the preparation of products for testing the metabolic capacity of drugs and nutrients.

5. A product for testing the metabolic capacity of drugs and nutrients, characterized in that: the product comprises the SNP site primer composition according to claim 1.

6. The product of claim 5, wherein: the product is an independent reagent or a kit.

7. Use of the primer composition of claim 1 or the product of claim 5 for detecting SNP sites associated with metabolic capacity for drugs and nutrients.

8. A method for detecting SNP sites associated with drug and nutrient metabolic capabilities, said method being a non-disease diagnostic and therapeutic method characterized by: the method comprises detecting SNP sites in the genome of a sample to be detected by using the primer composition of claim 1 or the product of claim 5.

9. The method of claim 8, wherein: the method comprises the following steps:

(1) extracting genome DNA;

(2) 1, performing multiplex PCR reaction;

(3) magnetic bead purification of round 1;

(4) performing multiplex PCR reaction of 2 nd round;

(5) magnetic bead purification for round 2;

(6) quantifying the library;

(7) detecting the quality of the library;

(8) and performing computer sequencing on the library to obtain a fastaq sequence file, and performing quality evaluation, comparison and SNP containment to obtain the genotype information of the SNP of the capture region of the sample to be detected.

10. The method of claim 9, wherein the quality assessment software in step (8) is FASTQC software, the alignment software is BWA software, and the SNP calling software is GATK software.

Background

Drug metabolism refers to the process of changing the chemical structure of a drug under the action of various drug-metabolizing enzymes (especially liver drug enzymes) in vivo, and is also called biotransformation or drug metabolism, and the biotransformation and excretion of the drug are called elimination. There are two outcomes of the drug after biotransformation in vivo: firstly, the medicine is inactivated and becomes a medicine without pharmacological activity; secondly, the activation, the non-pharmacological activity becomes a metabolite with pharmacological activity or generates a toxic metabolite, or the original pharmacological action is still kept after the metabolism. Nutrient metabolism also relies on the breakdown of various metabolic enzymes in the body to convert nutrients into compounds that are functional to the body and provide the body with the necessary nutrients.

The metabolism of drugs and nutrients is dependent on the activation of metabolic enzymes in vivo, genes related to the metabolism play a very important role in the metabolic process of the substances, and genetic mutations occurring in the genes, such as single nucleotide polymorphism, gene deletion or repeated molecular structure variation, may cause genetic variation at the levels of action targets, transport proteins, metabolic enzymes and the like, thereby affecting the metabolic efficiency of the organisms on the substances, and some mutations may cause higher side effects or toxicity. Therefore, the health of human body can be better maintained by guiding the use of medicine and the intake of nutrient substances based on genetic factors.

A Single Nucleotide Polymorphism (SNP) is a polymorphism in a DNA sequence caused by a variation of a single nucleotide (including four forms of transition, transversion, deletion and insertion), and generally, only two forms of transition and transversion occur, and the ratio of the two forms is about 1: 2. SNPs are widely present in the human genome, and occur in about every five hundred to one thousand bases, accounting for more than 90% of all known genetic polymorphisms, and are the most common heritable variations in humans. Single nucleotide polymorphisms occur most often in CG sequences, with C-allelic switching to T-allelic being most common, and this may be related to the ease with which cytosine is methylated to form thymine. The single nucleotide polymorphism is used as a third generation DNA genetic marker which is explored, has the advantages of large quantity, large density, wide distribution, strong stability, easy detection and the like compared with the first two generations of markers, and is beneficial to the application and popularization of the single nucleotide polymorphism in the medical field.

Patent CN110205393B discloses 51 SNP sites and primer compositions thereof for detecting nutrition metabolic capacity, which can be used for detecting metabolic capacity of vitamin A, vitamin B6, vitamin B12, vitamin C, microorganism D, vitamin E, calcium, iron, zinc, selenium, iodine, sodium, alcohol, folic acid, lactose, unsaturated fatty acid, saturated fatty acid, cholesterol, carbohydrate and caffeine, and the detection result can predict the demand of a certain nutrient substance, comprehensively evaluate the absorption capacity of various nutrient substances and provide scientific reference for healthy diet of individuals. Patent CN110423803A discloses a clopidogrel, statin and aspirin related drug metabolism genotyping detection multiplex amplification system, which comprises detection primers of 8 SNP sites and can jointly carry out auxiliary diagnosis on individual medication of clopidogrel, statin and aspirin. However, the prior art can only detect a single metabolism of nutrient metabolism or drug metabolism, and cannot comprehensively detect the metabolism of nutrient and drug at one time.

Therefore, it is highly desirable to construct a system and a method for simultaneously detecting nutrients and drug metabolism, which is convenient for better maintaining the health of human body.

Disclosure of Invention

Aiming at the defects, the invention provides the SNP locus and the primer composition thereof for simultaneously detecting the metabolism of nutrient substances and medicines. The invention designs and develops a group of SNP locus amplification primers by an NGS method, and detects the gene structure change related to metabolism in an individual body by a bioinformatics analysis method, thereby guiding people to select and use medicines and intake and select nutrient substances. The primer composition and the detection method have the advantages of low detection cost, simplicity and convenience in operation, good sensitivity, high accuracy and good repeatability, and have great clinical application value.

In order to achieve the above object, the technical solution of the present invention is as follows:

in one aspect, the present invention provides a primer composition for detecting SNP sites for drug and nutritional metabolizing ability, the SNP sites comprising: rs4693075, rs7412, rs1057910, rs11676382, rs1799853, rs2108622, rs2359612, rs4917639, rs56165452, rs61742245, rs7196161, rs7294, rs8050894, rs9923231, rs9934438, rs10509681, rs17244841, rs20455, rs16960228, rs4149601, rs7297610, rs1346268, rs1719247, rs17244841, rs4149056, rs1801133, rs70991108, rs 12299999984, rs1799899, rs1800562, rs197273, rs3811647, rs 17671 8, rs1049296, rs10849915, rs 15341418966, rs 1789676702, rs2168784, rs35951, rs 784457858, rs 445776720, rs 75767656300, rs 18276549, and 18276549.

Specifically, the primer compositions are shown in table 1 below.

TABLE 1 primer compositions

Specifically, the medicine comprises warfarin, hydrochlorothiazide, atorvastatin, pravastatin, simvastatin, rosiglitazone and the like; the nutrient substances comprise alcohol, lactose, folic acid, caffeine and the like.

On the other hand, the invention provides the application of the SNP locus primer composition in preparing products for detecting the drug and nutrition metabolic capability.

In still another aspect, the present invention provides a product for measuring the metabolic capability of drugs and nutrients, which comprises the above SNP site primer composition.

Specifically, the product is an independent reagent or a kit.

In still another aspect, the invention provides an application of the SNP site primer composition or the product in detecting SNP sites related to drug and nutrition metabolic capability.

In still another aspect, the present invention provides a method for detecting SNP sites associated with drug and nutritional metabolic abilities, which is a non-disease diagnosis and treatment method, comprising detecting SNP sites in the genome of a sample to be tested using the above primer composition or product.

Specifically, the method comprises the following steps:

(1) extracting genome DNA;

(2) 1, performing multiplex PCR reaction;

(3) magnetic bead purification of round 1;

(4) performing multiplex PCR reaction of 2 nd round;

(5) magnetic bead purification for round 2;

(6) quantifying the library;

(7) detecting the quality of the library;

Further specifically, the quality evaluation software in the step (8) is FASTQC software, the alignment software is BWA software, and the SNP calling software is GATK software.

Compared with the prior art, the invention has the advantages that:

1. the invention provides an SNP locus primer composition for detecting metabolic capacities of medicines and nutrients, which can simultaneously detect the metabolic capacities of the medicines and the nutrients, wherein the medicines comprise warfarin, hydrochlorothiazide, atorvastatin, pravastatin, simvastatin, rosiglitazone and the like, the nutrients comprise alcohol, lactose, folic acid, caffeine and the like, the metabolism of a certain medicine can be predicted, the metabolism of a certain nutrient can also be predicted, the metabolic capacities of various medicines or nutrients can be comprehensively evaluated, and scientific references are provided for individual medicine use or healthy diet.

2. The SNP locus primer composition and the detection method provided by the invention can obtain enough genetic information from trace peripheral blood for genetic risk assessment, have good sensitivity, simultaneously, the efficient and specific amplification primer can accurately obtain the SNP locus information, avoid non-specific amplification, greatly improve the accuracy, reduce the experiment cost and have simple and convenient operation. And calculating and predicting the future possible disease risk of the individual from the obtained SNP combination through a specific analysis method. Has great clinical application value.

3. The SNP locus information contained in the invention covers a plurality of drug and nutrient metabolic loci, and for each drug/nutrient type, including one or a plurality of SNP loci, each SNP locus has the possibility of three genotypes, and according to the genotyping result of individual SNP, the SNP locus information is compared with the established drug and nutrient database to obtain the drug and nutrient metabolic condition corresponding to the genotype of the locus of the individual.

Drawings

FIG. 1 is a graph of in-flight sequencing data.

Detailed Description

The present invention will be further illustrated in detail with reference to the following specific examples, which are not intended to limit the present invention but are merely illustrative thereof. The experimental methods used in the following examples are not specifically described, and the materials, reagents and the like used in the following examples are generally commercially available under the usual conditions without specific descriptions.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

In certain embodiments, details of the reagents or instruments used are as follows:

anhydrous ethanol: the Beijing chemical industry is analytically pure;

AgencourtAMPure XP Kit：Beckman(A63881)；

QubitdsDNA HS Assay Kit：Life Technologies(Q32851)；

Nuclease-free water：Ambion(AM9930)；

High Sensitivity DNA Kit：Agilent(p/n 5067-4626)；

custom Panel: agutaikang biotechnology (beijing) ltd (IGMU062), including Box 1(-20 ℃ storage): IGT-I7 (5. mu.M), IGT-EM101 polymerase mix, Enhancer NB (1N) and Box 2(-20 ℃ storage): YF buffer B (4 ℃ storage), IGT-I5 (5. mu.M);

PCR instrument: ABI 9700 by Life corporation;

DynaMag-96 Side：Life Technologies(cat.no.12331D)；

3.0 Fluorometer：Thermo(Q33216)；

Agilent 2100 Bioanalyzer system：Agilent(G2939AA)。

example 1 primer composition

The primer compositions described herein are specifically shown in table 2 below.

TABLE 2 primer compositions

Example 2 library construction and detection

1. Genomic DNA extraction

Saliva or blood genomic DNA (gDNA) extraction was performed according to commercial genomic DNA extraction kit procedures and quantified using the Qubit (Life technologies).

2. 1 st round of multiplex PCR reaction

1.1. The reaction system is shown in table 3 below.

TABLE 3 reaction System

Reagent	Volume(μL)
		ddH₂O	4-x
Enhancer NB(1N)	5
		Primer (Table 2)	2.4
gDNA	X(30ng)
		IGT-EM101 polymerase mixture	3.6

Wherein the final concentration of each Primer in the Primer mixture is 0.5 pmol.

1.2. The reaction procedure is as follows: 30s at 95 deg.C for 3 min; 18 cycles at 98 ℃ for 20s and 60 ℃ for 8 min; 5min at 72 ℃.

3. Round 1 magnetic bead purification

3.1. Adding 15 mu L of AMPure XP magnetic beads which are balanced at room temperature into 15 mu L of PCR products, and sucking and uniformly mixing the products for a plurality of times by using a pipettor;

3.2. after incubation for 5min at room temperature, the PCR tube was placed on a DynaMag-96 Side magnetic frame for 3 min;

3.3. thoroughly removing the supernatant, taking the PCR tube off the magnetic frame, adding 40 mu LYF buffer B into the tube, and sucking and mixing the mixture for several times by using a pipettor;

3.4. after incubation for 5min at room temperature, the PCR tube was placed on a DynaMag-96 Side magnetic frame for 3 min;

3.5. removing the supernatant, continuously placing the PCR tube on a magnetic frame, adding 180 mu L of 80% ethanol solution into the PCR tube, and standing for 30 s;

3.6. removing the supernatant, continuously placing the PCR tube on a magnetic frame, adding 180 mu L of 80% ethanol solution into the PCR tube, standing for 30s, and completely removing the supernatant;

3.7. standing at room temperature for 10min to completely volatilize residual ethanol;

3.8. taking down the PCR tube from the magnetic frame, adding 22 mu L of nucleic-free water, gently sucking a pipette to beat the heavy suspension magnetic beads to avoid generating bubbles, and standing for 2min at room temperature;

3.9. placing the PCR tube on the magnetic frame again, and standing for 3 min;

3.10. pipette 9.4. mu.L of the supernatant, and transfer to a new 200. mu.L PCR tube, where the supernatant is the multiplex PCR product.

4. Multiplex PCR reaction round 2

4.1. The reaction system is shown in table 4 below.

TABLE 4 reaction System

Reagent	Volume(μL)
		Step 3.10 multiplex PCR reaction product mixture	9.4
IGT-I5(5μM)	1
		IGT-I7(5μM)	1
IGT-EM101 polymerase mixture	3.6

4.2. The reaction procedure is as follows: 30s at 95 deg.C for 3 min; 20s at 98 ℃, 1min at 58 ℃, 30s at 72 ℃ and 8 cycles; 5min at 72 ℃.

5. Round 2 magnetic bead purification

5.1. Adding 13.5 mu L of AMPure XP magnetic beads which are balanced at room temperature into 15 mu L of PCR products, and sucking and uniformly mixing the mixture for a plurality of times by using a pipettor;

5.2. after incubation for 5min at room temperature, the PCR tube was placed on a DynaMag-96 Side magnetic frame for 3 min;

5.3. thoroughly removing the supernatant, taking down the PCR tube from the magnetic frame, adding 40 mu L YF buffer B into the PCR tube, and sucking and mixing the mixture by a pipettor for several times;

5.4. after incubation for 5min at room temperature, the PCR tube was placed on a DynaMag-96 Side magnetic frame for 3 min;

5.5. removing the supernatant, continuously placing the PCR tube on a magnetic frame, adding 180 mu L of 80% ethanol solution into the PCR tube, and standing for 30 s;

5.6. removing the supernatant, continuously placing the PCR tube on a magnetic frame, adding 180 mu L of 80% ethanol solution into the PCR tube, standing for 30s, and completely removing the supernatant;

5.7. standing at room temperature for 10min to completely volatilize residual ethanol;

5.8. taking down the PCR tube from the magnetic frame, adding 24 μ L of nucleic-free water or 1 × TE buffer (pH 8.0), gently pipetting the resuspended magnetic beads by a pipette to avoid generating bubbles, and standing at room temperature for 2 min;

5.9. placing the PCR tube on the magnetic frame again, and standing for 3 min;

5.10. pipette 20. mu.L of the supernatant, which is the prepared multiplex PCR library, into a new PCR tube.

6. Library quantification

2 μ L of library samples were used3.0fluorometer (QubitdsDNA HS Assay kit) to determine the library concentration, record the library concentration.

7. Library quality detection

Using Agilent 2100Bioanalyzer system (High Sensitivity DNA Kit), 1 μ L of library sample was taken for library fragment length and purity measurement, the target fragment distribution interval of the normal library was between 300 and 400bp, and the main peak was around 360 bp.

8. And performing computer sequencing on the library to obtain a fastaq sequence file. And obtaining the genotype information of the SNP of the capture area of the sample to be detected through the steps of quality evaluation, comparison, SNP calling and the like, wherein FASTQC software is used for quality evaluation, BWA software is used for comparison, and GATK software is used for SNP calling.

Example 3

The correspondence between the drug or nutrient metabolism ability and the gene and the test site is shown in Table 5 below.

TABLE 5

Experimental example 1 accuracy test

The 48 sites were tested using the assay method described herein and simultaneously verified by Sanger sequencing, the results of which are shown in Table 6 below.

TABLE 6 accuracy test results

As can be seen from Table 6, the accuracy of the primer composition and the detection method described in the present application was 100%.

Experimental example 2 repeatability test

1 of the samples A and 1 of the samples B were selected, and each sample was tested in a batch by repeating 3 times, and the test results are shown in Table 7 below.

TABLE 7 results of repeated measurements

Remarking: sample A-1 is sample A at the 1 st test.

As shown in the test results in Table 7, the repeatability of the primer composition and the test method disclosed in the present application is 100%.

Experimental example 3 detection of precision

1 of the samples A and 1 of the samples B were selected, and each sample was tested with three batches of the primer composition, and the test results are shown in Table 8 below.

TABLE 8 results of precision measurements

As is clear from the results in Table 8, the precision of the primer composition and the detection method described in the present application was 100%.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Sequence listing

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