CN111187821A

CN111187821A - Method for distinguishing Nicol lol personalized medicine by using primer composition to perform mass spectrometry

Info

Publication number: CN111187821A
Application number: CN201811356540.8A
Authority: CN
Inventors: 马庆伟; 钟逾; 张海燕; 刘昕超
Original assignee: Beijing Clin Bochuang Biotechnology Co Ltd
Current assignee: Beijing Clin Bochuang Biotechnology Co Ltd; Beijing Yixin Bochuang Biological Technology Co Ltd
Priority date: 2018-11-15
Filing date: 2018-11-15
Publication date: 2020-05-22

Abstract

The invention provides a method for distinguishing Nichol personalized medicine type by performing mass spectrometry on a primer composition, which comprises the following steps: respectively designing a multiplex amplification primer and an extension primer according to 10 target SNP sites to be detected; preparing a multiple amplification primer reaction system and an extension primer reaction system; in a reaction system, a plurality of sets of primers are used for simultaneously and respectively carrying out amplification and single base extension reaction on 10 target SNP sites; and (3) performing flight time mass spectrometry on the product obtained after the single base extension reaction, identifying the genotype of SNP (single nucleotide polymorphism) related to different drug metabolism according to the products of the extension primers with different molecular weights represented by mass spectrum peaks, and guiding the administration of the antihypertensive drug Nichol. The kit can simultaneously detect 10 SNP sites related to the metabolism of the antihypertensive drug Nicholol, and has the advantages of low cost, no need of synthesizing probes, short time consumption, simple and convenient result analysis and extremely wide application field.

Description

Method for distinguishing Nicol lol personalized medicine by using primer composition to perform mass spectrometry

Technical Field

The invention belongs to the field of molecular biology detection, and relates to a method for detecting multiple PCR single-base extension products by using a mass spectrum characteristic peak diagram and a product thereof. More specifically, the method utilizes different time-of-flight mass spectrum characteristic peak maps generated by different target oligonucleotide fragments in the mass spectrum typing process to simultaneously detect a plurality of target SNP sites so as to guide the medication of the antihypertensive drug Nichol.

Background

The genetic information of human beings is stored in the genome, and the international cooperation project human genome project is finally completed in 2002, so that a fine map of the structure of the human genome is drawn, and a reference sequence is provided for related research. Human genomic sequences consist of 30 hundred million bases in total, and a number of studies have shown that > 1% of genetic differences in the population account for approximately 0.1% of genomic sequences, i.e., differences of about 300 kilobases, with variation frequencies in the population of more than 1%. These differences are most likely genetic factors contributing to individual differences between two individuals. The sites can be widely applied to the research fields of genetic marker research, individual genetic relationship evaluation, species evolution evaluation and the like.

In the human genome, about 90% of the differences are expressed in the form of Single nucleotides, and such difference sites are called Single Nucleotide Polymorphisms (SNPs). SNP has two distinct characteristics: firstly, the number of the SNPs is large, the number of the SNPs in the human genome is estimated to be about 1100 ten thousand, and at present, the number of the SNPs which are collected from the human genome in the NCBI dbSNP database reaches 1083 ten thousand, wherein 644 ten thousand are confirmed (8 months and 22 days in 2007), so that a large number of the SNPs are convenient for selecting proper sites to study diseases, and most of the genome regions are within the SNP coverage range; another characteristic is that most SNP sites are bimodal, i.e., only two manifestations (genotypes) are present at one site, so that a high-throughput automated detection method can be easily designed. Because of these two characteristics, the SNP site is more and more favored by researchers and is known as the third generation molecular marker following Restriction Fragment Length Polymorphism (RFLP) and Short Tandem Repeat (STR). Before and after the completion of human genome project, the focus of genomics research is gradually turning to the field of SNP, and therefore, SNP has important applications in the fields of genetic marker science, biological population genetics, biological taxonomy, genetic breeding science, species or human chemistry, law science, pharmacogenomics, and the like.

Cardiovascular disease is the primary disease burden of human health worldwide. The prevalence rate of cardiovascular diseases in China is in a continuously rising stage. Hypertension is one of the most common cardiovascular diseases and also a major risk factor for cardiovascular diseases. The number of cardiovascular diseases in China is calculated to be 2.9 hundred million, wherein the high blood pressure accounts for 2.7 hundred million. About 70% of strokes and 50% of myocardial infarction occur in association with elevated blood pressure, and at least half of cardiovascular deaths are associated with hypertension. According to the national large-scale hypertension prevalence rate survey of the past, the hypertension prevalence rate of residents of more than or equal to 18 years old is increased from 5.1% to 25.2% from 1959 to 2012, the increasing trend is obvious, and the disease prevalence rate is an important disease burden in China. Therefore, prevention and treatment of hypertension have become important requirements for prevention of cardiovascular diseases and protection of national health.

Two methods are mainly used for predicting the curative effect of the antihypertensive drug clinically, one method is doctor empirical prediction, a doctor selects the antihypertensive drug empirically according to general clinical examination of a patient, and after a period of time, the addition, the change of the drugs or the combined administration of the drugs is considered if the curative effect is poor or the side effect is large; the other prediction method is blood pressure monitoring, and the static state is developed into dynamic blood pressure monitoring, so that the blood pressure can be effectively monitored in real time, but the current dynamic blood pressure monitoring technology has limitations, the dynamic blood pressure value has no unified standard, the examination cost is high, the blood pressure reduction effect of a certain medicine cannot be truly predicted, and accurate individualized information cannot be provided for a doctor to select the medicine. In conclusion, the two existing methods have certain hysteresis and blindness, which means that doctors cannot select, combine and determine the dosage according to individual differences during medication, so that the efficiency and safety of clinical medication and treatment are relatively reduced, and the risk of toxic and side effects and economic burden is increased. Pharmacogenomics has become an important tool for guiding clinical individualized medication and evaluating the risk of serious adverse drug reactions. Through detecting drug metabolizing enzyme and drug target gene, the clinician can be guided to select proper antihypertensive drug and administration dosage for specific patients, and the effectiveness and safety of treatment of the antihypertensive drug are improved. Each segment of the drug's action may exhibit significant differential responses due to genetic variation.

The calcium antagonists have some disputes in clinical application Furberg et al ("nifedipine. dose-related involved in the cell transmembrane signaling process, mediate the coupling of excitation-contraction and excitation-secretion, maintain the normal morphology and functional integrity of cells, regulate the contractile activity of vascular smooth muscles", et al ". Circulati et al" with short-acting calcium antagonists increase the risk of coronary heart disease onset and even death, as well as increase the risk of cancer and digestive tract hemorrhage. Pahort et al ("interstitial tissue associated with coronary heart disease), et al" with the significant risk of cardiovascular failure, β. the major background of cardiovascular events and the use of these antagonists is one of the major pharmacological agents responsible for the differences in cardiovascular events, β.

The pharmacogenomic knowledge base (PharmGKB, pharmacogenetics & pharmacogenetics knowledge base) created by the National Institute of Health (NIH), which collects and systematically classifies the most complete genotypic and phenotypic information related to the pharmacogenomic, is currently the most important pharmacogenomic knowledge base worldwide, and the Clinical pharmacogenomic implementation collaboration Consortium (CPIC) established based thereon is the main criterion for pharmacogenomic Clinical deployment compliance.

Research shows that the antihypertensive drug Nigrol has a plurality of metabolism-related gene loci. For example, the guidelines for rational administration for hypertension (2 nd edition), which are commonly written by the national committee for health council and the national institute of physicians, hypertension professional committee, screened 10 polymorphic site information on 8 loci (AGTR1, KCNH2, PROX1, GALNT2, ABCB1, 9p23, CACNA1C, LDLR) associated with absorption, transport, metabolism, and effects of nilla, which were included in PharmGKB and CPIC. And (5) carrying out gene detection guided by blood pressure lowering drug selection. According to the medication suggestion pointed by the detected genotype result, the selection of the type and the dosage of the antihypertensive drug more suitable for the individual genetic background is carried out, so that the effectiveness and the safety of the treatment of the antihypertensive drug are improved. In order to guide the medication more comprehensively, the SNP sites need to be analyzed simultaneously, and more reliable and effective medication guidance can be given by comprehensive consideration. Therefore, SNP detection methods characterized by high sensitivity and high throughput are the most preferred candidates for achieving gene-based personalized medicine.

Different from the traditional SNP detection method (fluorescent quantitative PCR, gene chip, Sanger method), the novel soft ionization biological Mass spectrum-Matrix assisted laser Desorption ionization Time-Of-Flight Mass spectrum (MALDI-TOF MS) [1, 2] developed in recent years has the advantages Of high sensitivity, high flux, good specificity, low cost, simple operation, environmental protection and the like. The MALDI-TOFMS instrument mainly comprises two parts: matrix-assisted laser desorption ionization ion source (MALDI) and time-of-flight mass analyzer (TOF). The principle of MALDI is the process of irradiating a co-crystallized thin film formed by a sample and a matrix with laser light, the matrix absorbing energy from the laser light to be transferred to biomolecules, and the ionization process transferring protons to or from the biomolecules to ionize them. Therefore, the method is a soft ionization technology and is suitable for measuring mixtures and biomacromolecules. The principle of TOF is that ions are accelerated to fly through a flight tube under the action of an electric field, and are detected according to different flight times of arriving at a detector, namely, the mass-to-charge ratio (M/Z) of the ions is measured to be in direct proportion to the flight time of the ions, and the ions are detected. MALDI-TOF, a soft ionization technique, produces no or fewer fragment ions. Since molecular weight is the most basic physicochemical property parameter of organic compounds, the correctness of molecular weight usually represents the correctness of the structure of the organic compound and biomacromolecule to be measured.

MALDI-TOF MS genotyping technology is based on "primer extension method", i.e. extending primer to extend one or several bases on SNP site to be detected, and then determining its genotype according to the difference of fluorescence carried by extension product or the difference of its molecular mass (FIG. 1).

The hME and iPLEX methods of Sequenom, USA, GOOD assay method of Bruker, Germany, and the RFMP method of GeneMatrix, Korea are specific SNP detection methods developed based on MALDI-TOF MS. The principle of the methods is to design an oligonucleotide probe matched with the genome sequence at the upstream of the SNP locus to be detected, and according to the genotype difference of the SNP locus, the probe is connected with different deoxynucleotides at the SNP locus to be detected. Wherein the molecular weights of the four deoxynucleotides constituting the DNA fragment are respectively: dAMP 313.21Da, dCMP 289.19Da, dGMP 329.21Da, dTMPP 304.20Da. There is a molecular weight difference between the different deoxynucleotides, with the smallest difference being 9.01Da between dAMP and dTMP and the largest difference being 40.02Da between dCMP and dGMP, which can be detected by a mass spectrometer to distinguish between probes of different molecular weights. The detection effect of the mass spectrometer is related to the molecular weight of the probe, and the shorter the probe fragment is, the smaller the molecular weight is, the more obvious the distinguishing effect is: for example, in FIG. 2, the two peaks indicated in FIG. 2-A correspond to two oligonucleotide fragments of 5'-ACGT-3' and 5'-ACGA-3', molecular weights of 1174.699 and 1183.770Da, respectively, differing by 9.071Da, the two peaks indicated in the B diagram, the two oligonucleotide fragments of 5 '-ACGTACGT-3' and 5'-ACGTACGA-3', molecular weights of 2410.628 and 2419.821Da, respectively, differing by 9.193Da, the two peaks indicated in FIG. 2-C correspond to two oligonucleotide fragments of 5'-ACGTACGTACGT-3' and 5'-ACGTACGTACGA-3', molecular weights of 3645.917 and 3654.736Da, differing by 8.819Da, also differing by about 9Da, and thus, MALDI-TOF MS can sensitively and accurately measure single SNP differences. However, as shown in the figure, the difference between the two peaks is most significant in fig. 2-a, and relatively insignificant in fig. 2-B, that is, the smaller the molecular weight, the more significant the mass spectrometer discrimination, i.e., the higher the resolution. In order to improve the resolution of the mass spectrometer, the detection of SNP sites tends to detect oligonucleotide fragments with smaller molecular weight, for example, RFMP method detects oligonucleotide fragments with about 2000-4000 Da by restriction enzyme digestion of PCR products containing SNP sites, while GOOD assay method detects oligonucleotide fragments with SNP sites by Phosphodiesterase (PDE) into small fragments with about 1000-2000 Da.

Chinese patent application CN101748196A, "SNP rs762551 of CYP1A2 gene and application thereof in relevant drug metabolic activity detection" discloses a method for judging drug metabolic activity in advance by detecting SNP rs762551 of CYP1A2 gene, which comprises detecting whether variation exists in cytochrome oxidase P4501A2 gene (CYP1A2) and transcript of human individual compared with normal, and the existence of variation indicates that the drug metabolic activity of the individual is different from that of ordinary people or normal people. The invention also discloses a corresponding detection kit for pre-judging the drug metabolic activity of an individual. However, the invention of the patent is specific to the antihypertensive drug Nipporo, the Nipporo part is metabolized by CYP1A2, and the clinical significance of CYP1A2 on the drug effect of Nipporo is unknown or has weak influence. In addition, the invention can only predict the SNP detection and the drug effect aiming at the polymorphic site (rs762551) of a single gene (CYP1A2), the detection cost in clinical application is higher, and the detection effect has certain limitation.

As the closest prior art, Chinese patent application CN1810986A, "a method for predicting the drug effect of calcium antagonist of antihypertensive drugs and application thereof", discloses a method for predicting the drug effect of calcium antagonist antihypertensive drugs by determining the genotype of polymorphic sites of adrenergic receptor genes of subjects, wherein genomic DNA is extracted from whole blood or saliva, and then three polymorphic sites of Arg347Cys, Arg389Gly and Gln27Glu are detected respectively by PCR or fluorescent quantitative PCR, and the corresponding medication opinions are given according to the results. However, the detection method and the kit provided by the patent of the invention only aim at the polymorphic sites of the adrenergic receptor gene, and the detection object has limitation. In addition, the method mainly adopts a fluorescent quantitative PCR detection method, and because the fluorescent quantitative PCR needs to design a specific probe aiming at SNP site variation, the method has low flux, only one polymorphic site can be detected in one experiment, and the method is not suitable for detecting multiple SNPs. In addition, if information on all related SNPs is to be obtained, a plurality of detection tests are required, which leads to an increase in cost.

Therefore, a kit and a detection method matched with the kit are needed, which can detect polymorphic sites of all genes related to target drug metabolism, improve the effective rate of drug use, reduce the incidence rate of adverse reactions, and facilitate the clinical formulation of a more reasonable and effective individual administration scheme.

Disclosure of Invention

Individual differences in drug response are a complex phenotypic trait that results from the interaction of multiple genetic factors and multiple environmental factors. In the future, an individual administration scheme designed according to the genetic environment characteristics of individual patients can comprehensively expect the response of the patients to specific medicines according to various genetic characteristics of the patients, select the individual optimal treatment scheme for the corresponding patients and guide doctors to provide individual medical services for the patients. Therefore, the SNP genotyping detection based on MALDI-TOF MS has the characteristics of sensitivity, accuracy and high flux, is suitable for the Nichol dose selection of individual genetic background, and improves the effectiveness and safety of Nichol treatment.

The principle of the invention is as follows: according to the research results of modern human genetics and pharmacogenomics, the genetic characteristics of Chinese population can be represented in a specific gene region to reflect the change of drug absorption and metabolism efficiency, and SNP sites with high frequency variation in the Chinese population are selected to form a set of SNP combination with 10 sites, and the SNP combination can effectively perform the drug type discrimination work of the antihypertensive drug Nichol in the Chinese population. Based on the basis, the invention creatively discovers and selects a detection system consisting of 10 SNP sites through a large amount of experimental groves to be used for distinguishing the individual drug type of the antihypertensive drug Nichol.

Therefore, the first object of the present invention is to provide a primer composition for detecting human SNP markers for discriminating the drug type of Nichol, a antihypertensive drug, characterized in that the primer composition comprises a PCR primer pair and an extension primer for 10 SNP markers capable of discriminating the drug type of Nichol, wherein the PCR primer sequence pair is selected from the sequences shown in SEQ ID NO. 1a-10a, and the extension primer sequence is selected from the sequences shown in SEQ ID NO. 1b-10 b.

Wherein the SNP is selected from rs5186, rs1137617, rs340874, rs2144297, rs2144300, rs3213619, rs12346562, rs1104514, rs1051375 and rs 688.

In one embodiment, the primer composition is divided into 3 independently performed multiplex PCR reaction primer compositions and the sequences are shown in table 1.

TABLE 1

Wherein, the site represented by the sequence of SEQ1-3 constitutes the first complete reaction system to be carried out independently, and the reaction system comprises two steps: multiplex PCR reaction and single base extension reaction, wherein the primer pair SEQ1a-SEQ3a participates in the multiplex PCR reaction, and the primer pair SEQ1b-SEQ3b participates in the single base extension reaction. The sites represented by the sequence of SEQ4-6 constitute a second, independently performed complete reaction system, which also comprises two steps, a multiplex PCR reaction and a single base extension reaction: the primer pair of SEQ4a-SEQ6a was involved in a multiplex PCR reaction, and the primer of SEQ4b-SEQ6b was involved in a single base extension reaction. The sites represented by the sequences of SEQ7-10 constitute a third, independently performed complete reaction system, which also comprises two steps, a multiplex PCR reaction and a single base extension reaction: the primer pair of SEQ7a-SEQ10a was involved in a multiplex PCR reaction, and the primer of SEQ7b-SEQ10b was involved in a single base extension reaction.

The sequence of the SNP loci rs5186, rs1137617, rs340874, rs2144297, rs2144300, rs3213619, rs12346562, rs1104514, rs1051375 and rs688 can be obtained from a known public human genome sequence library.

In another embodiment, the molecular weights of the extension primers and the extension products corresponding to each site are shown in Table 2.

TABLE 2

In one embodiment, the PCR primer sequence is a core sequence, which may include a protective base sequence, preferably 5-15 bases, at the 5' end. In a specific embodiment, the protective base sequence is selected from tag (ACGTTGGATG) added 10bp from the 5' stretch. In another embodiment, the 5' end of the extension primer may also be added with a base sequence as an adaptor.

The second purpose of the invention is to provide a product prepared by the primer composition and used for distinguishing the medication type related polymorphic sites of the antihypertensive drug Nigrol.

In one embodiment, the product is a test kit comprising:

(1) a reaction reagent for PCR comprising: the amplification primer pair, high-temperature resistant DNA polymerase, dNTPs and PCR reaction buffer solution;

(2) reagents for PCR product purification;

(3) a reagent for single base extension reactions comprising: the extension primer, high temperature resistant single base extension enzyme (SAP enzyme), ddNTPs, extension reaction buffer.

In one embodiment, the product further comprises a vector for mass spectrometric detection and protection of the SNP combination, including but not limited to: chip and target plate.

In one embodiment, the kit may further comprise: negative quality control material, positive quality control material, resin for purification, target sheet for sample application and mass spectrum detection, exonuclease, human genome DNA extraction reagent and other reagents. In another embodiment, the reagents for PCR product purification: alkaline phosphatase, or alkaline phosphatase and exonuclease ExoI, or an electrophoresis gel recovery reagent, or a PCR product purification column. Wherein the PCR primers used do not need to include a protective base when alkaline phosphatase and exoI purification reagents are included.

The third purpose of the invention is to provide a method for distinguishing the drug type of Nigroll by using the primer composition or the product and through different mass spectrum peak patterns of different SNP sites, which comprises the following steps:

(1) multiplex PCR: using the PCR primer pair to simultaneously amplify the DNA regions where the 10 SNP sites are located in two reaction systems to obtain a PCR product containing the DNA regions where the 10 polymorphic sites are located;

(2) and (3) PCR product purification: purifying the PCR product obtained in the step (1) to reduce the interference to the subsequent reaction;

(3) single base extension: performing multiple single-base extension on the purified PCR product obtained in the step (2) by using the 10 specific extension primers, wherein the extension primers extend one nucleotide at the corresponding SNP site, and the nucleotide is complementarily paired with the genotype at the SNP site;

(4) and (3) purification of an extension product: purifying the extension product obtained in the step (3) to obtain a high-purity extension product, and avoiding the influence of impurities such as salt ions on subsequent detection;

(5) mass spectrometer detection: and (4) spotting the purified product obtained in the step (4) on a target plate containing a matrix, and putting the target plate into a mass spectrometer for detection.

In one embodiment, the purification process of step 2 may be selected from alkaline phosphatase digestion, alkaline phosphatase and exonuclease ExoI digestion, gel cutting purification, PCR purification column chromatography, and the like. In one embodiment, the purification is performed by high temperature enzyme inactivation after alkaline phosphatase digestion, or alkaline phosphatase and exonuclease ExoI digestion.

The fourth purpose of the invention is to provide an application of the primer composition or the product for distinguishing the drug type of the antihypertensive drug Nipagol.

In one embodiment, the use comprises: guiding the individualized medication of Nichol and the genetic susceptibility research of human pharmacogenomics, and the like.

Technical effects

Through the detection of the genes related to the antihypertensive drugs, a more reasonable and effective personalized drug delivery scheme can be made according to the genotype of a patient. The problem that the treatment effect cannot be achieved due to insufficient dosage of the medicine, or adverse reaction easily occurs due to excessive dosage of the medicine, and the economic burden of a patient is aggravated due to improper dosage types is avoided. Under the trend that the medical mode is changed from public medical treatment to individual medical treatment, the detection of the drug related genes provides better medical service for patients, and simultaneously greatly promotes the process of reasonable medication. The invention has the following technical effects:

1. the mass spectrum technology and the multiple PCR technology are combined, the high-flux automatic detection of the SNP can be realized, the output result is directly interpreted by a computer, the detection speed is increased, and the artificial subjective error is avoided; 2. in the experimental period, the method for detecting the SNP mainly comprises multiple PCR reactions, SAP enzyme digestion reactions, single base extension reactions, purification, sample application and mass spectrometer typing. Wherein the multiple PCR reaction, SAP enzyme digestion reaction, single base extension reaction and purification respectively take 68min, 25min, 53.5min and 30min at least in theory;

3. compared with the traditional method, the method has the advantages of low cost (no need of synthesizing a probe), short time consumption (36 target SNP sites can be detected in one reaction, at most 380 samples can be detected at one time), simple and convenient result analysis (each SNP site has only two alleles, and three genotypes are combined), and extremely wide application field;

4. and (3) sensitivity: the invention integrates the technologies of multiplex PCR, single base extension, mass spectrum detection and the like, can amplify a detection template through the PCR technology, can detect a trace sample through the mass spectrum technology, integrates the advantages of the two technologies, and is far superior to the method for detecting polymorphic SNP by using PCR alone, so the detection sensitivity is very high;

5. specifically: the single base extension is also called as micro sequencing, and uses a specific probe to identify DNA molecules, so that the method has the characteristics of high accuracy, good specificity, low false positive and the like of a sequencing technology, and the technology only extends a single base and has lower error probability;

6. the operation is simple and safe, the automation degree is high, and the pollution is prevented;

7. high flux, can complete the detection of hundreds of samples within 5-6 hours;

8. the invention can detect a plurality of individuals to be detected to respectively obtain detection results with different SNP sites, wherein the examinee can have mutation variation of a single SNP site or a plurality of SNP sites, which indicates that the examinee can carry one or a plurality of SNP mutation variations.

Principles and definitions

The invention provides a detection scheme for detecting and distinguishing drug type related polymorphic Sites (SNP) by combining technologies such as multiplex PCR, single base extension, mass spectrum detection and the like. The principle is as follows:

in the multiplex PCR step, by designing and using appropriate primers, DNA fragments containing 10 SNP sites can be amplified simultaneously.

In the single base extension step, the products of the previous multiplex PCR are sequentially purified and subjected to multiplex single base extension. Wherein, the extension primers have 10 pieces, which correspond to 10 SNP sites respectively, and extend one nucleotide at the corresponding SNP site, and the nucleotide is complementary to the genotype at the SNP site (for example, if the SNP site is an A genotype, a T nucleotide is extended on the corresponding extension primer). In the single base extension step, ddNTPs are used instead of dNTPs, so that extension of the primer will terminate after one base has been extended.

In the mass spectrometric detection process, the single base extension product, after purification, is spotted onto a target plate containing a matrix, and is excited by a laser in a vacuum environment, passing through a flight tube to a detector. The time for different substances to pass through the flight tube is inversely related to their molecular weight, i.e. the higher the molecular weight, the slower the flight speed and the later the time to reach the detector.

The terms "rs 5186, rs1137617, rs340874, rs2144297, rs2144300, rs3213619, rs12346562, rs1104514, rs1051375, rs 688" are all the uniform numbering of human SNP sites in the NCBI db _ SNP database.

The term "primer composition" refers to a system or composition of the present invention consisting of 10 pairs of PCR amplification primer pairs and 10 extension primers. The primer is designed according to 200bp sequences around each site.

The term "alkaline phosphatase digestion" is used to degrade residual dNTPs in the system after PCR reaction, and the principle is that the 5'-P end of dNTP is converted into 5' -OH end, so that the ability of primer extension caused by primer binding is lost, and the influence on the next single base extension is avoided.

The term "exonuclease ExoI digestion" has the function of catalyzing the hydrolysis of 3, 5-phosphodiester bonds between dNTPs constituting a single-stranded DNA in order from one end of the DNA, to finally hydrolyze the single-stranded DNA to dNTPs. The method is used for degrading the residual PCR primers after the PCR reaction. Since the exonuclease cleaves single-stranded PCR primers and does not appear in the detection window, the PCR primers used do not need to include a protective base when the exonuclease is used.

The term "single base extension", also called micro sequencing (mini sequence), refers to adding extension primer and ddNTP into the system, the ddNTP is connected with 3' end of the extension primer to form extension product (i.e. primer extends by one base), according to base complementary pairing principle, the genotype at SNP site determines which kind of ddNTP is connected specifically, the process is similar to that dNTP is added to PCR primer one by one according to base composition of complementary strand in PCR process. Since the "ddNTP" is different from ordinary dNTP in that a hydroxyl group is lacked at the 3' position of deoxyribose, and a phosphodiester bond cannot be formed with the subsequent ddNTP, the extension primer is only connected with one ddNTP at the SNP site and cannot be continuously extended downwards like PCR, so that the extension primer is called single-base extension. The single base extension is very similar to the sequencing process, the sequencing system is added with a mixture of dNTP and ddNTP, the sequencing primer is continuously extended after being connected with the dNTP, and the extension is terminated only after being connected with the ddNTP, so that the mixture of nucleotide fragments with different lengths is generated by sequencing; the single base extension system adds only ddNTP, the extension primer can only be connected with one ddNTP, and the extension is stopped, so that the single base extension generates a nucleotide fragment for extending the extension primer by only one base.

The term "detection product" refers to any conventional product for detecting the genotype of a SNP site, including: detection reagents, detection chips (such as gene chips, liquid chips and the like), detection carriers, detection kits and the like.

The term "ddNTP" is a specific nucleotide which has a molecular weight difference, such as the molecular weights of ddATP, ddCTP, ddGTP, and ddTTP are 271.2Da, 247.2Da, 287.2Da, 327.1Da, respectively (wherein ddTTP is the modified molecular weight). When the extension primer extends different nucleotides depending on the genotype of the SNP site, a difference in molecular weight will be formed. This difference can be resolved by mass spectrometric detection. For example, if a SNP site is G/A polymorphism, the length of the corresponding extension primer is 20 bases (molecular weight 6153Da), when the SNP site is G genotype, the extension primer will extend one C nucleotide and terminate the extension, forming an extension product with 23 bases and molecular weight of 6400.2Da, when the SNP site is A genotype, the extension primer will extend one T nucleotide and terminate the extension, forming an extension product with 23 bases and molecular weight of 6480.1Da, and there is a molecular weight difference of 80.1Da between the two products. That is, for this SNP site, if the extension primer of 6153Da is used, the G genotype will correspond to the mass spectrum peak of 6400.2Da, and the A genotype will correspond to the mass spectrum peak of 6480.1 Da. In the actual detection process, a user can observe three places of 6153Da, 6400.2Da and 6480.1Da through software: if mass spectrum peak appears at 6153Da, it is that part or all of the extended primer is not combined with ddNTP; whether a mass spectrum peak appears at 6153Da or not, if only one mass spectrum peak appears at 6400.2Da and 6480.1Da, the genotype of the SNP site is homozygous and corresponds to the position of the mass spectrum peak, as mentioned above, the mass spectrum peak of 6400.2Da corresponds to the G genotype, and the mass spectrum peak of 6480.1Da corresponds to the A genotype; if the mass spectrum peaks of 6400.2Da and 6480.1Da appear, the genotype of the SNP site is heterozygous; if neither mass spectrum peak of 6400.2Da nor 6480.1Da appears, the experiment fails.

The term "purification" refers to a treatment step that serves to reduce the effect of other substances in the system being tested on subsequent reactions. The PCR product of the invention can be purified in two ways: firstly, the impurities are separated and discarded, and secondly, the impurities are inactivated. Wherein, gel cutting purification, purification column chromatography and the like are used for separating impurities through electrophoresis, purification column chromatography and the like, and relatively pure PCR products are recovered, which can be regarded as a first purification mode, and the mode generally consumes time and is complex to operate, particularly when the sample amount is large; alkaline phosphatase acts to degrade (also called "digest") dNTPs so that they do not continue to participate in the PCR or single base extension reaction as substrates for DNA polymerase or single base extension enzyme, thereby not interfering with the subsequent reaction, and can be considered a second mode of purification. It should be noted that ExoI alone does not play a role in purification, and when it is used in combination with alkaline phosphatase, it plays a role in degrading single-stranded DNA (mainly the remaining PCR primers in the PCR product system after completion of the reaction) into dNTPs in advance, and then the dNTPs are further degraded by the alkaline phosphatase. Since the PCR primers are degraded, the final mass spectrometric detection step is not entered, and therefore, if the ExoI exonuclease treatment is added to the planned purification step, PCR primers with protected bases do not need to be used. In addition, since both exonuclease and alkaline phosphatase are inactivated by high temperature before the single base extension step, it does not degrade the single-stranded extension primer, ddNTP, etc. added in the single base extension step, thus avoiding influence on the subsequent experiments.

The term "detection window" refers to the range of nucleotide molecular weights that can be used for mass spectrometric detection, and generally refers to the design reference range of primers. When designing the extension primer, for different SNP sites, the extension primer and the extension product with different molecular weights can be designed according to the sequence characteristics of the DNA region where the sites are located and the genotype of the SNP sites, so that the interference between the different extension primers and the products due to the proximity of the molecular weights can be avoided, and the detection of a plurality of SNP sites can be realized in a relatively wide detection window, such as 4000-.

The term "SNP" genotype refers to a type of single nucleotide polymorphism in the human genome. In practical examination, the genotype used for detection as a control can be from a control human genome or from a vector tool cloned into a plasmid, and the latter has the advantages of convenient replication and preservation and stable source, and is popular with practical users.

The term "SNP mutation variation frequency" refers to the probability of a mutation variation occurring at a SNP site. Theoretically, the invention can simultaneously detect 10 SNP mutation variations in a single individual. However, in practice, researchers have found that different SNP mutation variations have a certain mutation variation frequency in individuals.

The variation frequency in the Chinese population of 10 SNP sites (HCB, Han Chinese Beijing) is seen in:

https://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi？rs＝5186

https://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi？rs＝1137617

https://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi？rs＝340874

https://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi？rs＝2144297

https://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi？rs＝2144300

https://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi？rs＝3213619

https://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi？rs＝12346562

https://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi？rs＝1104514

https://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi？rs＝1051375

https://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi？rs＝688

the term "SEQ ID NO: 1-10" refers to 200bp sequences around 10 sites, the sequences of which are shown in the sequence listing.

Drawings

FIG. 1: schematic representation of the SNP principle of MALDI-TOF-MS typing;

FIG. 2: respectively, are a graphical representation of the relationship of sequence molecular weights of different lengths with the same SNP (T/A) to mass spectrometer resolution. Wherein, the legend A is the difference of molecular weight mass spectrometry detection of 5bp length (5 '-ACGT-3' and 5 '-ACGA-3'), the legend B is the difference of molecular weight mass spectrometry detection of 8bp length (5 '-ACGTACGT-3' and 5 '-ACGTACGA-3'), and the legend C is the difference of molecular weight mass spectrometry detection of 12bp length (5'-ACGTACGTACGT-3' and 5'-ACGTACGTACGA-3');

FIG. 3 (A-D): the mass spectrum peak chart result of simultaneous detection of a plurality of sites of Nigrol, wherein the abscissa unit is molecular weight, the extended primer of each site has possibility of generating mass spectrum peaks at two positions, and the mass spectrum peaks of each site are not overlapped;

FIG. 4: mass spectrum peak diagrams of wild type plasmids (FIG. 4-A, FIG. 4-B, FIG. 4-C and FIG. 4-D) and mutant plasmids (FIG. 4-E, FIG. 4-F, FIG. 4-G and FIG. 4-H).

Reference to the literature

1.Eleutherobin:Defining a structure-activity profile and patterns ofcross-resistance in taxol-resistant cell lines.Proceedings of the AmericanAssociation for Cancer Research Annual Meeting,1999.40:p.623.

2.THE MULTIDRUG TRANSPORTER,A DOUBLE-EDGED SWORD.Journal ofBiological Chemistry,1988.263(25):p.12163-12166.

Detailed Description

The first embodiment is as follows: primer design and Synthesis

The SNP peripheral sequences of interest of the kit are queried in db _ SNP (built 132) and Hapmap (Rel 28, Phase II + III, Aug 10) databases, and these sequences are used to design multiplex PCR primers and single base extension primers.

Aiming at 10 polymorphic sites related to the drug type for discrimination, such as rs5186, rs1137617, rs340874, rs2144297 and the like, a corresponding specific PCR primer core sequence (SEQ 1a-SEQ 10a) and a specific extension primer core sequence (SEQ 1b-SEQ 10b) are designed. 10 pairs of PCR primers and 10 extension primers (SEQ1a/b to SEQ10a/b) make up 3 independently performed reaction systems. SEQ1a/b to SEQ3a/b constitute a first reaction system, SEQ4a/b to SEQ7a/b constitute a second reaction system, and SEQ8a/b to SEQ10a/b constitute a third reaction system. In these 3 independently performed reaction systems, SEQ1a to SEQ3a, SEQ4a to SEQ6a, SEQ7a to SEQ10a are involved in 3 independent multiplex PCR reactions, SEQ1b to SEQ3b, SEQ4b to SEQ6b, and SEQ7b to SEQ10b are respectively involved in 3 subsequent independent single base extension reactions.

The relevant primers were synthesized by Biotechnology engineering (Shanghai) Co., Ltd.

Example two: sample DNA extraction

DNA samples of the general Chinese were collected in 10 cases and labeled A1-A10. Wherein, the human venous blood is collected according to the specification requirements, such as sample collection, DNA extraction and the like, and is collected by an EDTA anticoagulation tube. According to the requirements of the specification, collected blood should be stored at 2-8 ℃ for no more than one week, stored at-20 ℃ for no more than one month, and can be transported by using ice in an ice kettle or a foam box and ice sealing, and the extraction of genome DNA by using fresh blood is recommended to be carried out as much as possible. Since the kit does not provide a human genomic DNA extraction reagent, human genomic DNA was extracted from 200. mu.l of whole Blood of each patient using a commercial nucleic acid extraction kit (e.g., DNeasy Blood and tissue kit from QIAGEN), quantified by NanoDrop 2000(Thermo Co.), and normalized to 30 ng/. mu.l (A1-A10, respectively). The kit recommends the detection of human genome DNA with the concentration of 30 ng/. mu.l, but a comparative experiment shows that the kit can detect a positive result on human genome DNA with the concentration as low as 10 ng/. mu.l. According to the requirements of the specification, the extracted human genome DNA is stored at the temperature of 2-8 ℃ for not more than one week, stored at the temperature of-20 ℃ for not more than two years, stored at the temperature of-80 ℃ for a long time, prevented from being repeatedly frozen and thawed, and placed in an ice box for transportation.

Example three: biological experiments

The polymorphic sites of 10 and the type of the drug to be distinguished were examined using an ABI9700 type PCR instrument according to the instructions.

The components used for PCR, PCR product purification and single base extension in the kit are:

serial number	Component name	Principal Components	Specification of
				1	PCR primer mixture	PCR primer		24 μ l/tube x1 tube
2	PCR reaction solution	Taq enzyme, dNTP	72 μ l/tube x1 tube
				3	Enzyme digestion reaction solution	SAP enzymes	48 μ l/tube x1 tube
4	Extension primer mixture	Extension primer						24 μ l/tube x1 tube
				5	Extension reaction solution	Single base elongases, ddNTPs	24 μ l/tube x1 tube
6	Positive quality control product	Human genome DNA (30 ng/. mu.l)	10 μ l/tube x1 tube

Wherein the concentration of each primer pair is 500 nmol/L.

According to the specification, the specific operation method is as follows:

PCR amplification

1.1 preparing 200ul PCR reaction tubes in a PCR solution preparation area according to the number of samples to be detected (containing positive quality control substances, negative control and blank control), and marking sample numbers on the tubes;

1.2 taking out the PCR primer mixed solution and the PCR reaction solution from the kit, naturally thawing the PCR primer mixed solution and the PCR reaction solution, performing vortex oscillation to fully mix the PCR primer mixed solution and the PCR reaction solution, and performing instantaneous centrifugation to the bottom of a tube;

1.3 according to the number of samples, taking out the PCR primer mixed solution and the PCR reaction solution according to the proportion in the following table, placing the mixture in a centrifuge tube, uniformly mixing, adding 4ul of mixture into each PCR reaction tube, and subpackaging. Because of the residual pipette tip during dispensing, which may be insufficient to dispense the desired number of portions, it is recommended to properly scale up the volume of the mixture to be dispensed. For example, when 10 parts of the sample to be tested is present, the mixture can be prepared from 10.5 to 11 parts of the sample.

Component name	Single reaction volume
		PCR primer mixture	1μl
PCR reaction solution	3μl
		Total up to	4μl

The total reaction system of the PCR reaction is as follows:

1.4 Add 1. mu.l of the sample to be tested to each tube of the mixture in the PCR amplification zone, making the total volume of each PCR reaction system 5. mu.l. Wherein, the negative control is purified water, and the blank control is without template.

1.5 the PCR reaction tube was placed in a PCR amplification apparatus and the PCR amplification reaction was carried out according to the procedure of the following table.

The reaction steps are as follows: after 120s activation of DNA polymerase at 94 ℃ the cycles were 45 times with denaturation at 94 ℃ for 10s, annealing at 56 ℃ for 10s and extension at 72 ℃ for 60 s.

Enzymatic digestion of SAP

The SAP enzyme reaction system is:

mu.l of the digestion reaction solution was added to the PCR reaction tube, and then the PCR reaction tube was placed in a PCR amplification apparatus, and the following procedure was performed.

Temperature of	Time (minutes)	Number of cycles
			85	5	1
37	20	1

The reaction steps are as follows: adding 2 μ l of 0.3U SAP enzyme into the reaction system, standing at 85 deg.C for 5min for activation, and standing at 37 deg.C for 20min for excess base reduction reaction.

3. Extension of

3.1 in the PCR solution preparation area, taking out the extension primer mixed solution and the extension reaction solution according to the proportion of the following table according to the number of samples, and placing the extension primer mixed solution and the extension reaction solution in a centrifuge tube for uniformly mixing. Because of the residual pipette tip during dispensing, which may be insufficient to dispense the desired number of portions, it is recommended to properly scale up the volume of the mixture to be dispensed. For example, when 10 parts of the enzyme-cleaved product is present, a mixture can be prepared in 10.5 to 11 parts of the sample.

Component name	Single reaction volume
		Extension primer mixture	1μl
Extension reaction solution	1μl
		Total up to	2μl

The overall reaction system for single base extension reaction is:

3.2 in the PCR amplification area, 2. mu.l of the mixture is added to each tube of the enzyme digestion product for split charging.

3.3 the PCR reaction tube was placed in a PCR amplification apparatus and the extension reaction was performed according to the procedure of the following table.

The reaction steps are as follows: after 30s activation of DNA polymerase at 94 ℃, denaturation at 94 ℃ for 5s, annealing at 52 ℃ for 5s and extension at 80 ℃ for 5s, 200 cycles. The final step was an extension of 180s at 72 ℃.

4. Purification of

Add 16. mu.l purified water, 6mg resin to each tube extension in PCR amplification zone and mix by inversion for 30 min.

5. Spotting is carried out

Using a micropipette, 1. mu.l of the purified product was pipetted and spotted onto the target plate.

Example four: on-machine detection and result interpretation

And (3) detecting the spotted target plate and judging the result by using a Clin-TOF type flight time mass spectrometer produced by Yixinxing (Beijing) science and technology Limited company.

The mass spectrum peak diagram is a diagram obtained by spotting a reaction product on a mass spectrum chip and analyzing the reaction product by a mass spectrometer after the single base extension reaction is finished. The extension primers at each site have different molecular weights, and after single base extension is carried out on two alleles at each site, two extension products with different molecular weights can be generated, so that different samples and different sites generate different mass spectrum peaks on different molecular weight horizontal coordinates. . Further, wild-type controls B1-B10 and mutant-type controls C1-C10 were provided at the above positions, respectively. Wherein, the wild type control B1-B10 and the mutant variant control C1-C10 are respectively from artificial plasmids which are sold in the market or deposited in the laboratory. The wild-type control plasmid B1-B10 and the mutant control plasmid C1-C10 used in the present invention are constructed by inserting PCR products into pMD18-T Vector after PCR is performed with primers and normal human DNA according to the conventional method described in molecular cloning on the basis of the commercial plasmid pMD18-T Vector (Takara Co., Ltd.), thereby constructing the wild-type plasmid B1-B10, and then performing site-specific mutagenesis respectively to construct 10 mutant plasmids C1-C10. The plasmids B1-B10 and C1-C10 can be stored in glycerol at-20 deg.C for a long period, activated and extracted plasmid DNA when used.

As shown in Table 2, the 10 extension primers and the extension products thereof generated at the 10 polymorphic sites according to the respective genotypes have different molecular weights corresponding to the respective mass spectrum peaks, and when a mass spectrum peak appears at a certain molecular weight, it is judged that a substance (extension primer or product) corresponding to the molecular weight exists:

and (4) judging the standard:

(1) if the mass spectrum peaks corresponding to the wild type and the mutant variant do not appear, judging that the experiment fails regardless of the existence of the mass spectrum peak corresponding to the extended primer;

(2) if only one mass spectrum peak corresponding to the wild type or the mutant variant appears, the genotype corresponding to the appeared mass spectrum peak is judged to be homozygous;

(3) and if the mass spectrum peaks corresponding to the wild type or the mutant variant appear, judging the hybrid type.

The mass spectrum results are shown in FIG. 4, in which FIGS. 4-A-4-D show the mass spectra of the wild-type plasmid for 10 SNP sites, and FIGS. 4-E-4-H show the mass spectra of the variant plasmid for 10 SNP sites.

The results of mass spectrometry of samples A1-A10 were examined for the molecular weight of the extension primers and extension products at each site shown in Table 2, and the genotypes of the SNP sites were determined, as shown in Table 3:

TABLE 3

Comparative example A

First, the SNP sites disclosed in example 1 were sequenced by a conventional PCR detection method using the following primers shown in Table 4:

TABLE 4

Second, sample DNA source

To make the data generated between the different experiments comparable, the human genomic DNA extracted from the venous blood collected from 10 subjects to be examined in example two (A1-A10) was used for sequencing verification.

Thirdly, sequencing identification

1. The PCR reaction system was 25. mu.l

Filled with ddHO.

2. Reaction conditions are as follows: the reaction was carried out on a 9700 thermal cycler from ABI under conditions of 94 ℃ pre-denaturation for 5 minutes, 94 ℃ denaturation for 30 seconds, 55 ℃ annealing for 30 seconds, 72 ℃ extension for 40 seconds, 35 cycles, 72 ℃ extension for 7 minutes after the reaction was completed, and storage at 4 ℃.

3. PCR product purification and sequencing

(1) Add 50. mu.l of wash buffer to the 96-well plate containing the PCR product and mix well.

(2) This was transferred to a Millipore purification plate and placed on a vacuum pump to suction filter for about 3 minutes, seeing that there was no water in the purification plate.

(3) Add 50. mu.l of wash buffer to the purification plate again and continue the suction filtration until there is no water in the purification plate.

(4) The purification plate was removed from the vacuum pump, and 20. mu.l of deionized water was added to the plate and allowed to stand for 15 minutes.

(5) Shake for another 15 minutes and then pipette into a new 96-well plate.

The reagents required for the sequencing reaction should be freshly prepared, and the reagents that need to be autoclaved must be sterilized before use. The equipment required for the sequencing reaction (e.g., 384-well plates, tip heads, etc.) should also be clean and sterile.

(6) In order to ensure the freshness of the sequencing sample and the reaction reagent, the sample should be loaded on ice.

(7) The sequencing reaction system is 5 mul, and the addition amount of various reagents is as follows: PCR products 3-10ng, BigDyev 3.10.25. mu.l, 5. mu.g BigDye buffer 0.875. mu.l, primer 1.6 pmol;

(8) the samples were placed on a PCR instrument for the following reactions:

the method comprises the following steps: at 95 ℃ for 5 min;

95 ℃ for 10 seconds; at 60 ℃, 4 minutes; repeat 30 cycles;

held at 4 ℃ until ready for purification.

(9) Add 20. mu.l 80% ethanol to each well, centrifuge at 4,000rpm for 30 min;

(10) putting the sample plate on a folded paper towel, and reversely throwing the sample plate in a centrifugal machine at the speed of 1000 rpm;

(11) adding 30 mul of 70% ethanol into each hole, centrifuging at 4000rpm for 10min, and reversely throwing;

(12) repeating the operation of the step 11 for 2 times;

(13) placing the sample plate in a clean drawer, and drying for 30min in a dark place;

(14) adding 5 mul formamide, sealing the membrane, centrifuging and placing in a refrigerator at the temperature of minus 20 ℃;

(15) denaturation 5min at 95 ℃ before sequencer, ice-free for 2 min, centrifugation and loading.

(16) Sequencing Using ABI3730xl type genetic Analyzer

Fourthly, the result

For A1-A10 samples, the primers described in the table above were used to sequence the 10 SNP sites rs5186, rs1137617, rs340874, rs2144297, rs2144300, rs3213619, rs12346562, rs1104514, rs1051375 and rs688 (respectively numbered NO:1-10), and the final sequencing results are shown in Table 5:

TABLE 5

By comparison, the results in Table 3 are completely consistent with those in Table 5, indicating the accuracy of the detection method of the present invention.

Sequence listing

<110> Beijing resolute Xinbo Chuang Biotech Co., Ltd

<120> method for discriminating Nicol lol personalized medicine by using primer composition to perform mass spectrometry

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acgttggatg tcagagcttt agaaaagtcg 30

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acgttggatg agaacattcc tctgcagcac 30

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<213> Artificial Sequence (Artificial Sequence)

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tcaattctga aaagtagcta a 21

<210>4

<211>30

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acgttggatg gagactgaga cactgacctg 30

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acgttggatg atgatggccg acacgttgc 29

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ccccagccct catgta 16

<210>7

<211>30

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

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acgttggatg gctctccgcc cttttaaatg 30

<210>8

<211>30

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<213> Artificial Sequence (Artificial Sequence)

<400>8

acgttggatg catataagtt agcgccagcc 30

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<400>9

cgttgtaagg tgtggaaagg tata 24

<210>10

<211>30

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<213> Artificial Sequence (Artificial Sequence)

<400>10

acgttggatg ggcctcatgg gtattggtaa 30

<210>11

<211>30

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<400>11

acgttggatg ggatgatttg tcccaaatgc 30

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<400>12

tgggtattgg taaggattc 19

<210>13

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acgttggatg tttgagttta cccagccgtg 30

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<400>14

acgttggatg ccaaaaccct gtcaattccc 30

<210>15

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<400>15

cgttggaaag gacgctgtac c 21

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acgttggatg gctctctttg ccacaggaag 30

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<400>17

acgttggatg ccgactttag tggaaagacc 30

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<400>18

gcctgagctc attcgag 17

<210>19

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<213> Artificial Sequence (Artificial Sequence)

<400>19

acgttggatg attgtgtgtg ggtcatctgg 30

<210>20

<211>29

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<213> Artificial Sequence (Artificial Sequence)

<400>20

acgttggatg ctcaatagta aagtgttgg 29

<210>21

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<213> Artificial Sequence (Artificial Sequence)

<400>21

catctcttca catgtttttt t 21

<210>22

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<400>22

acgttggatg atagcaatgc cccaccaatc 30

<210>23

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<400>23

acgttggatg ggacgatgag cccattaaac 30

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<400>24

ccccgcatta ggctattctt gc 22

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<400>25

acgttggatg ttggagctga gcttccacg 29

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acgttggatg tcaacaacgc caacaacacc 30

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<400>27

ctccacagtg ctgac 15

<210>28

<211>30

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<400>28

acgttggatg tcactccatc tcaagcatcg 30

<210>29

<211>30

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<213> Artificial Sequence (Artificial Sequence)

<400>29

acgttggatg atctcgtacg taagccacac 30

<210>30

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tctcaagcat cgatgtcaa 19

Claims

1. A method for distinguishing Nichol personalized medicine for reducing hypertension by using a mass spectrometry method through a primer composition, wherein the primer composition comprises a PCR primer pair aiming at 10 SNP markers capable of distinguishing the Nichol personalized medicine type and an extension primer, wherein the PCR primer sequence pair is selected from sequences shown as SEQ ID NO. 1a-10a, and the extension primer sequence is selected from sequences shown as SEQ ID NO. 1b-10 b.

2. The method of claim 1, wherein the primer composition is divided into 3 independently performed multiplex PCR reaction primer compositions and the sequences are as follows:

。

3. the method of claim 2, wherein the molecular weights of the extension primers and the extension products corresponding to each locus are as follows:

。

4. the method of claim 3, wherein the PCR primer sequence is a core sequence, which may comprise 5-15 protected nucleotide sequences at the 5' end, preferably 10bp tag: ACGTTGGATG are provided.

5. The method according to claim 3, wherein the base sequence as a linker can be added to the 5' end of the extended primer, preferably the base sequence of the linker is 1 to 15 bases.

6. The method of claim 5, wherein the linker base sequence is 1-3 bases.

7. The method of any of claims 1-6, comprising the steps of:

(3) single base extension: performing multiple single-base extension on the purified PCR product obtained in the step (2) in two reaction systems by using the 10 specific extension primers, wherein the extension primers extend one nucleotide at the corresponding SNP site, and the nucleotide is complementarily paired with the genotype at the SNP site;

(5) mass spectrometer detection: and (5) spotting the purified product obtained in the step (4) on a target sheet containing a matrix, and putting the target sheet into a mass spectrometer for detection.

8. The method of claim 7, wherein the purification process of step 2 is selected from the group consisting of alkaline phosphatase digestion, alkaline phosphatase and exonuclease ExoI digestion, gel cutting purification, PCR purification column chromatography.