CN112831557A

CN112831557A - Primer group and method for detecting drug metabolism related SNP (Single nucleotide polymorphism) sites

Info

Publication number: CN112831557A
Application number: CN202110260045.2A
Authority: CN
Inventors: 梅艳巧; 谢桂贞; 陈佳颖; 杨帆; 王博
Original assignee: Shanghai Genomepilot Institutes Of Genomics And Human Health
Current assignee: Shanghai Dashan Biotechnology Co ltd
Priority date: 2021-03-10
Filing date: 2021-03-10
Publication date: 2021-05-25
Anticipated expiration: 2041-03-10
Also published as: CN112831557B

Abstract

The invention discloses a primer group for detecting drug metabolism related SNP sites and a method thereof, belonging to the technical field of biology, wherein the method is used for detecting whether 14 SNP sites related to specific drug metabolism of any crowd are mutated, and the primer sequence is shown as SEQIDNo.1-42. The primer group and the method for detecting the SNP sites related to drug metabolism provided by the invention can not only provide accurate medication for sick people, but also provide personalized medication reminding from various aspects such as metering, drug effect and adverse reaction, improve the curative effect of patients and reduce or avoid the side effect of drugs; risk assessment can be provided for healthy people, and potential improper risks of medication can be effectively avoided. The method can achieve the effects of measuring 14 sites through one reaction, high accuracy, high flux, low cost, quickness and suitability for all people.

Description

Primer group and method for detecting drug metabolism related SNP (Single nucleotide polymorphism) sites

Technical Field

The invention relates to the technical field of biology, in particular to a primer group for detecting SNP sites related to drug metabolism and a method thereof.

Background

The world health organization makes statistics that 1/3 death cause of death cases is irrational medication. If all people can safely take the medicine, tens of millions of people can be prevented from dying every year.

The personalized medicine gene detection is to detect the genotype of the medicine sensitive site of different individuals by using a gene sequencing technology, and provide reference information related to the curative effect and metabolism of the medicine for diagnosis and medicine application, so that personalized 'medicine application to the symptom' is realized. Through genetic testing, the physician can: (1) according to the gene polymorphism condition of the metabolic enzyme or the drug action receptor or target point, guiding proper dosage; (2) identifying patients with certain genotypes as likely to develop adverse drug reactions; (3) patients who have confirmed certain genetic characteristics may benefit more readily with certain treatment regimens and may be guided in drug selection compliance and dose adjustments to achieve desired therapeutic effects; (4) virus resistance is detected and appropriate drugs are selected. By revealing the genetic difference of individuals and guiding the formulation of individual medical treatment schemes, the safety and effectiveness of medication are improved, serious adverse reactions are avoided, and the risk and cost of medication are reduced.

The basic principle of the Sequenom SNP detection system is a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) technology. The method is mainly characterized in that SNP sequence specific extension primers are added into a product obtained after PCR amplification, and 1 base is extended on an SNP locus. The prepared sample analyte was then co-crystallized with the chip matrix, the crystal was placed into the vacuum tube of a mass spectrometer, and transient nanoseconds (10) were used^-9s) strong laser excitation, because the energy absorbed by the matrix molecule by radiation leads to energy accumulation and rapid heat generation, thus leading the matrix crystal to sublimate, the nucleic acid molecule will be desorbed and converted into metastable state ion, the generated ion is mostly single charge ion, the single charge ion obtains the same kinetic energy in the accelerating electric field, and then is separated according to the mass-to-charge ratio in a non-electric field drift region, and flies in a vacuum tubule to reach the detector. MALDI-generated ions are often detected using a Time-of-Flight (TOF) detector, with the smaller the mass of the ion, the smaller the mass of the ionThe faster the arrival. Theoretically, there is no upper limit on the number of mass molecules that can be detected by the TOF detector, as long as the flight tube length is sufficient. MASSARRAY SNP mass spectra were measured in the range of 5000 to 8500 Dalton. Because the Mass spectrum technology is combined with the multi-primer extension technology and the Mass ARRAY technology for use, a plurality of mutation sites can be simultaneously detected in one reaction system, the workload is greatly reduced, the detection flux is improved, and the detection cost is reduced.

Disclosure of Invention

In view of the defects of unreasonable medication of human beings at present, the invention provides a primer group for detecting SNP sites related to drug metabolism and a method thereof

The SNP genotype analysis is carried out by extending the primer by a single base, and the effects of detecting 14 sites by one reaction, high accuracy, high flux, low cost, quickness and suitability for all people can be achieved.

In order to achieve the purpose, the invention adopts the following technical scheme:

a primer set for detecting a drug metabolism-associated SNP site, comprising: 12s rRNA, rs267606617, rs1876828, rs4244285, rs4986893, rs1799853, rs1057910, rs1065852, rs776746, rs37973, rs3909184, rs2844682, rs730012 and rs 3812718; the sequence of the primer group is shown as SEQ ID NO. 1-28.

According to one aspect of the invention, the primer set further comprises 14 single-base extension primers shown in SEQ ID NO. 29-42.

A method for detecting SNP sites related to drug metabolism comprises the following steps: synthesizing a primer sequence: synthesizing the primer set for detecting SNP sites related to drug metabolism according to any one of claims 1 to 2; amplifying SNP locus gene fragments by multiplex PCR: amplifying the synthesized primer group once through a multiplex PCR amplification system to cover the personalized medicine gene fragment for detecting the SNP locus related to the medicine metabolism; single base extension SNP site: extending SNP sites of personalized medicine genes at one time through a multiple single base extension system; detecting the genotype on a computer: and detecting data and analyzing the genotype of each mutation site by a matrix-assisted laser desorption ionization time-of-flight mass spectrometry method.

According to one aspect of the present invention, the method further comprises a shrimp alkaline phosphatase digestion reaction step of performing a shrimp alkaline phosphatase digestion reaction after amplifying the SNP site gene fragment by multiplex PCR, and performing a single base extension of the SNP site after performing the shrimp alkaline phosphatase digestion reaction.

According to one aspect of the present invention, the multiplex PCR amplification system is an amplification reaction system in which a plurality of components are mixed in a single well.

According to one aspect of the invention, the reagents required for the multiplex PCR amplification system include nucleic-Free Water, 10 XPCR Buffer, MgCl₂dNTP Mix, PCR amplification primer Mix, genomic DNA and PCR Enzyme.

According to one aspect of the invention, the reagents required for the multiplex single base extension system include nucleic-Free Water, iPLX Buffer Plus, iPLX Termination Mix, single base extension primer Mix and iPLX Enzyme.

In accordance with one aspect of the invention, the reagents required for the Shrimp alkaline Phosphatase digestion reaction include Nuclear-Free Water, SAP Buffer, and Shrimp Alkal ine Phosphomutase Enzyme.

Compared with the prior art, the invention has the following beneficial effects:

1) the primer group provided by the invention is an SNP locus of a specific drug metabolism related gene of all people.

2) The invention can detect 14 SNP sites by one reaction.

3) The SNP sites selected by the method are genetic materials existing in human cells, polymorphism exists in people, the extended product fragments are between 15 and 30bp, and the genotype of 14 SNP sites is analyzed according to the flight time of the extended product in a vacuum tubule.

4) The invention not only can provide accurate medication for the sick people, give personalized medication reminding from various aspects such as metering, drug effect and adverse reaction, improve the curative effect of patients and reduce or avoid the side effect of the drug, but also can provide risk assessment for healthy people and effectively avoid the improper risk of potential medication.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to these drawings without creative efforts.

FIG. 1 is a diagram of mass spectrometry of a normal sample of the SNP site of rs 1057910;

FIG. 2 is a diagram of mass spectrometry of a sample of a heterozygous mutation at the SNP site of rs1057910 in example 2;

FIG. 3 is a diagram of mass spectrometry of a normal sample of the SNP site of rs 1065852;

FIG. 4 is a diagram of mass spectrometry of a sample of a heterozygous mutation at the SNP site of rs1065852 in example 2;

FIG. 5 is a diagram of mass spectrometry of a normal sample of the SNP site of rs 2844682;

FIG. 6 is a diagram of mass spectrometry of a sample of a heterozygous mutation at the SNP site of rs2844682 in example 2;

FIG. 7 is a diagram of mass spectrometry of a normal sample of the SNP site of rs 37973;

FIG. 8 is a diagram of mass spectrometry of a sample of a heterozygous mutation at the SNP site of rs37973 in example 2;

FIG. 9 is a mass spectrometry plot of a normal sample of the SNP site of rs 3812718;

FIG. 10 is a diagram of mass spectrometry of a sample of a heterozygous mutation at the SNP site of rs3812718 in example 2;

fig. 11 is a mass spectrometry plot of a normal sample of the SNP site of rs 730012;

fig. 12 is a diagram of mass spectrometry of a heterozygous mutation sample at the SNP point of rs730012 in example 2.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

A primer set for detecting a drug metabolism-associated SNP site, comprising: 12s rRNA, rs267606617, rs1876828, rs4244285, rs4986893, rs1799853, rs1057910, rs1065852, rs776746, rs37973, rs3909184, rs2844682, rs730012 and rs3812718, wherein the sequence of the primer group is shown as SEQ ID NO. 1-28; the primer group also comprises 14 single-base extension primers shown in SEQ ID NO. 29-42.

The PCR amplification primer and the single base extension primer can amplify a plurality of SNPs sites and extend a plurality of SNPs sites with equal efficiency in one hole, the detection genes related to the personalized medicine are shown in table 1, 14 SNP sites are related, and the PCR amplification primer and the single base extension primer used in 14 sites are shown in table 2. Wherein, the PCR amplification primers are shown in Table 3; single base extension primers are shown in Table 4.

TABLE 1 personalized medicine correspondence detection genes

PCR amplification primers and Single base extension primers for the 214 SNP sites in Table

TABLE 3 PCR amplification primer sequences

The multiplex PCR amplification system of the present application is an amplification reaction system in which a plurality of components are mixed in a single well.

TABLE 4 Single-base extension primers

Example 1

A kit for detecting whether 14 SNP sites related to personalized medicine metabolism are mutated or not by one person comprises the following components:

(1) PCR amplification primers for 14 SNP sites, 1OD for each primer, and the primer sequences are shown in Table 3.

(2) Primers were extended for a single base at 14 SNP sites, 1OD per primer, and the primer sequences are shown in Table 4.

(3) Reagent: PCR Reagent Set, Large (agena bioscience),

Gold Reagent Set, large (agena bioscience), absolute ethanol.

The kit is stored at-20 ℃ and repeated freeze thawing is reduced as much as possible.

Example 2

The use step of the kit for detecting whether 14 SNP sites related to personalized medicine metabolism are mutated or not by one person comprises the following steps:

(1) extracting genomic DNA of a sample

Genomic DNA samples (blood, buccal swabs, dried blood slides) from 1 adult male (45 years old) were extracted and assayed for concentration using Nanodrop. Diluted with Nuclean-Free Water to a final concentration of 10-20 ng/ul. The diluted samples were stored at-20 ℃. Before use, the mixture can be thawed by centrifugation at 2000rpm for 2-5 min.

(2) Preparation of PCR amplification primer Mix and single base extension primer Mix

The primer dry powder in Table 2 was centrifuged at 12000rpm for 2min, and nucleic-Free Water was added as a primer synthesis solution. Diluting all PCR amplification primers to 100uM, diluting single base extension primers to 400uM, fully shaking and mixing uniformly, standing at room temperature for 15-30min, shaking, mixing uniformly again, centrifuging, and storing at-20 ℃.

Preparing a PCR amplification primer Mix: the final concentration of PCR primers Mix was 0.5uM, i.e., each 100uM primer was diluted 200-fold. Shaking, mixing, centrifuging, and storing at-20 deg.C.

Preparation of single-base extension primer Mix: each single base extension (UEP) primer was stored at a concentration of 400 uM. Then, the calculation was carried out according to the "single-base extension primer dilution Table". Shaking, mixing, centrifuging, and storing at-20 deg.C.

(3) Multiplex PCR amplification of SNP locus gene fragment

The multiplex PCR amplification system is shown in table 5:

TABLE 5 multiplex PCR amplification System

Reagent	Reaction volume (uL)
		Nuclease-FreeWater	1.8
10×PCRBuffer	0.5
		MgCl2	0.4
dNTPMix	0.1
		PCR amplification primer Mix	1
Genomic DNA (10-20ng/ul)	1
		PCREnzyme	0.2
Total amount of	5

The multiplex PCR amplification reaction conditions are shown in table 6:

TABLE 6 multiplex PCR amplification reaction conditions

(4) Shrimp alkaline phosphatase digestion (SAP) reaction

The shrimp alkaline phosphatase digestion reaction system is shown in table 7:

TABLE 7 shrimp alkaline phosphatase digestion reaction System

Reagent	Reaction volume (uL)
		Nuclease-FreeWater	1.53
SAPBuffer	0.17
		ShrimpAlkalinePhosphataseEnzyme	0.3
Total amount of	2

The shrimp alkaline phosphatase digestion reaction conditions are shown in table 8:

TABLE 8 shrimp alkaline phosphatase digestion reaction conditions

Reaction temperature	Reaction time	Number of cycles
			37℃	40min	1
85℃	5min	1
			4℃	hold	1

(5) Single base extension reaction

The single-base extension reaction system is shown in Table 9:

TABLE 9 Single-base extension reaction System

The conditions for the single-base extension reaction are shown in Table 10:

TABLE 10 Single-base extension reaction conditions

(6) Detecting the genotype by using a mass spectrometer: and detecting data and analyzing the genotype of each mutation site by a matrix-assisted laser desorption ionization time-of-flight mass spectrometry method.

The single base extension product was subjected to resin purification desalting reaction and centrifugation to deposit the resin on the bottom of 384 well plates. The 384 well plate is then placed on the automatic spotting machine and the chip is placed in the corresponding position. 60uL of sample is taken from each well and added to the corresponding position of the chip for detecting the quality of the chip and the sample application used in the experiment.

And adding the sample in the 384-well to the corresponding position of the chip, then placing the chip after adding the sample in a mass spectrometer, editing the hole to be detected, and clicking START until the whole process is finished.

(7) Analysis of results

And opening the software Mass ARRAY Typers 4.0, checking experimental detection data and performing data analysis. Analyzing a mass spectrum analysis chart of the SNP locus genotype according to a signal value detected by a mass spectrometer, wherein a map result shows that 6 SNP locus mutations exist in the experimenter, and the 6 mutated SNP loci are respectively as follows: rs1057910, rs1065852, rs2844682, rs37973, rs3812718 and rs 730012. Among them, 6 mutated SNP sites are shown in FIG. 2, FIG. 4, FIG. 6, FIG. 8, FIG. 10 and FIG. 12, respectively.

The invention can determine 14 SNP loci of the specific drug metabolism related genes through a reaction, provide accurate medication for a detector by detecting whether the 14 SNP loci of the specific drug metabolism related genes are mutated or not, give personalized medication reminding from multiple aspects such as metering, drug effect and adverse reaction, improve the curative effect of patients and reduce or avoid the side effect of drugs; risk assessment can be provided for healthy people, and potential improper risks of medication can be effectively avoided.

The above description is only an embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention disclosed herein are intended to be covered by the scope of the present invention.

Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.

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Claims

1. A primer set for detecting a drug metabolism-associated SNP site, comprising: 12s rRNA, rs267606617, rs1876828, rs4244285, rs4986893, rs1799853, rs1057910, rs1065852, rs776746, rs37973, rs3909184, rs2844682, rs730012 and rs 3812718; the sequence of the primer group is shown as SEQ ID NO. 1-28.

2. The primer set for detecting SNP sites related to drug metabolism according to claim 1, wherein the primer set further comprises 14 single-base extension primers shown in SEQ ID No.29 to 42.

3. A method for detecting SNP sites associated with drug metabolism, characterized in that the method for detecting SNP sites associated with drug metabolism comprises the following steps:

synthesizing a primer sequence: synthesizing the primer set for detecting SNP sites related to drug metabolism according to any one of claims 1 to 2;

amplifying SNP locus gene fragments by multiplex PCR: amplifying the synthesized primer group once through a multiplex PCR amplification system to cover the personalized medicine gene fragment for detecting the SNP locus related to the medicine metabolism;

single base extension SNP site: extending SNP sites of personalized medicine genes at one time through a multiple single base extension system;

detecting the genotype on a computer: and detecting data and analyzing the genotype of each mutation site by a matrix-assisted laser desorption ionization time-of-flight mass spectrometry method.

4. The method for detecting SNP sites associated with drug metabolism according to claim 3, wherein: the method also comprises a shrimp alkaline phosphatase digestion reaction step, wherein the shrimp alkaline phosphatase digestion reaction is carried out after the SNP locus gene fragment is amplified by multiple PCR, and the SNP locus is extended by single base after the shrimp alkaline phosphatase digestion reaction is carried out.

5. The method for detecting SNP sites associated with drug metabolism according to claim 3, wherein: the multiplex PCR amplification system is an amplification reaction system for mixing a plurality of components in a single hole.

6. The method for detecting SNP sites associated with drug metabolism according to claim 3, wherein: the reagents required by the multiplex PCR amplification system comprise nucleic-Free Water, 10 XPCR Buffer and MgCl₂dNTP Mix, PCR amplification primer Mix, genomic DNA and PCR Enzyme.

7. The method for detecting SNP sites associated with drug metabolism according to claim 3, wherein: reagents required for the multiplex single base extension system include nucleic-Free Water, iPLEX Buffer Plus, iPLEX Termination Mix, single base extension primer Mix and iPLEX Enzyme.

8. The method for detecting SNP sites associated with drug metabolism according to claim 4, wherein: reagents required for the Shrimp alkaline Phosphatase digestion reaction include nucleic-Free Water, SAP Buffer, and Shrimp Alkal ine Phosphomutase Enzyme.