CN111118132A

CN111118132A - Primer composition for distinguishing individual drug type of enalapril

Info

Publication number: CN111118132A
Application number: CN201811281277.0A
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: 2018-10-31
Filing date: 2018-10-31
Publication date: 2020-05-08

Abstract

The invention provides an extension primer with different molecular weights at different Single Nucleotide Polymorphism (SNP) sites, so that a plurality of sites are simultaneously detected by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), and finally a primer composition for distinguishing the individual drug type of enalapril is obtained. The preparation method of the primer composition comprises the following steps: respectively designing a multiple amplification primer and an extension primer according to a plurality of 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 a plurality of target SNP sites; and (3) carrying out 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 enalapril. The invention can simultaneously detect 6 SNP sites related to enalapril drug metabolism 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

Primer composition for distinguishing individual drug type of enalapril

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 administration of antihypertensive drug enalapril.

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. Hypertension patients have about 6.9 hundred million people all over the world, and the incidence rate is up to 31.3 percent. Epidemiological studies have shown that blood pressure levels are linearly related to cardiovascular disease incidence; elevated blood pressure is an independent risk factor for stroke and coronary heart disease. Hypertension is a major cause of life-threatening cardiovascular and cerebrovascular diseases such as myocardial infarction, stroke, renal insufficiency, etc. Therefore, the effective control of blood pressure has great clinical significance for preventing the occurrence of cardiovascular and cerebrovascular complications of hypertensive patients.

The antihypertensive drug enalapril is an Angiotensin Converting Enzyme Inhibitor (ACEI), and is hydrolyzed into enalaprilat in vivo after oral administration, has strong inhibiting effect on angiotensin converting enzyme, reduces the content of angiotensin II, causes systemic vasodilatation, and reduces blood pressure, and is a common drug for treating hypertension.

Pharmacogenomics has become an important tool for guiding clinical 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. Differences in drug action are manifested as differences in pharmacokinetics and pharmacodynamics.

The pharmacogenomic knowledge base (phase GKB, the pharmaceutical and Pharmacogenomics knowledge base) created by the National Institute of Health (NIH), which collects the most complete genotypic and phenotypic information related to the Pharmacogenomics in history and systematically classifies these information, is currently the most important pharmacogenomic knowledge base worldwide, and the Clinical Pharmacogenomics implementation collaboration group (CPIC) established based on it is the main criterion for Pharmacogenomics Clinical deployment to follow.

The studies show that enalapril has a plurality of gene loci related to metabolism. For example, according to the guideline for rational drug administration for hypertension (2 nd edition) written by the committee for national health council of physicians and the council of the society of physicians for hypertension, 6 polymorphic site information of 6 loci (PTGER3, AGT, MME, NR3C2, 9q34.2, BDKRB2) related to absorption, transport, metabolism and effect of enalapril in PharmGKB and CPIC were screened. 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.

Different from the traditional SNP detection 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 friendliness and the like. The MALDI-TOF MS 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). Some specific SNP detection methods developed on the market based on MALDI-TOF MS are as follows: the method of hME and iPLEX by Sequenom, usa, the method of GOOD assay by Bruker, germany, and the method of RFMP by GeneMatrix, korea. 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, dCMP289.19Da, dGMP 329.21Da and dTMP 304.20 Da. 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 "kit for predicting action effect of angiotensin converting enzyme inhibitor class drugs" (patent No: CN101063166) discloses a method for predicting action effect of angiotensin converting enzyme inhibitor class drugs by using relationship between mononucleotide polymorphism site E112D genotype of important enzyme proline carboxypeptidase (PRCP) gene on blood vessel and endothelial function regulating pathway and action effect of angiotensin converting enzyme inhibitor class drugs. The kit contains the polymorphism typing oligonucleotide of E112D polymorphism site genotype for detecting PRCP gene in a biological sample and related reagents, can guide doctors to implement prediction medication, selects according to individual difference, improves the effectiveness and safety of clinical medication, and provides a basis for developing new drugs for treating hypertension acting on PRCP. However, the invention needs to judge the reasonability of medication according to the effect of lowering blood pressure and other toxic and side effects generated after patients with different genotypes take the medicine for a period of time, the time consumption is long in clinical application, and the prediction result is greatly influenced by various physiological parameters (such as sex, age, BMI, smoking, drinking, baseline homocysteine and other factors) of the patients.

As the closest prior art, Chinese patent application "application of polymorphic site genotype to the application, method and kit of the effect of angiotensin converting enzyme inhibitor drugs" (patent No. 200510130528.1) relates to the application of polymorphic site genotype of key enzyme genes on the homocysteine metabolic pathway to the prediction of the effect of Angiotensin Converting Enzyme Inhibitor (ACEI) drugs, the polymorphic typing oligonucleotides for determining the polymorphic site genotype of key enzyme genes on the homocysteine metabolic pathway, and the method and kit of the effect of ACEI drugs by determining the polymorphic site genotype of key enzyme genes on the homocysteine metabolic pathway. However, the detection method and the kit provided by the invention only aim at the polymorphic sites of the key enzyme genes on the homocysteine metabolic pathway, the detection objects have limitations, the operation is complicated, and the clinical rapid and accurate requirements are difficult to meet.

Therefore, a kit and a detection method matched with the kit are needed, and the kit can be used for quickly and effectively detecting polymorphic sites of all genes related to target drug metabolism, improving the effective rate of drug use, reducing the incidence rate of adverse reactions and facilitating the clinical formulation of a more reasonable and effective drug administration scheme.

Disclosure of Invention

At present, a large body of literature and representative studies in modern genetics indicate that SNPs are the most abundant genetic markers in the human genome, covering all 23 pairs of chromosomes in humans in high density and all directions. The different pharmacogenomic information of patients is an important reason for the difference in dosage of enalapril. In the future, the medicine scheme designed according to the genetic environment characteristics of individual patients can comprehensively expect the reaction of the patients to specific medicines according to various genetic characteristics of the patients, select the optimal treatment scheme for the corresponding individual patients and guide doctors to provide 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 enalapril dosage selection of individual genetic background, and improves the effectiveness and safety of enalapril treatment.

The principle of the invention is as follows: according to the research results of modern human genetics and pharmacogenomics, SNP sites which can reflect the change of drug absorption and metabolism efficiency and have high frequency variation in Chinese population are selected in a specific gene region to form a set of SNP combination with 6 sites, and the SNP combination can be used for effectively carrying out the drug type discrimination work of enalapril in Chinese population. The use of this rapid and accurate genotyping method will provide individualized treatment regimens for patients in need of enalapril treatment.

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 enalapril, a antihypertensive drug, characterized in that the primer composition comprises PCR primer pairs for 6 SNP markers capable of discriminating the drug type of enalapril and extension primers, wherein the PCR primer sequence pairs are selected from the sequences shown in SEQ ID NO. 1a-6a, and the extension primer sequences are selected from the sequences shown in SEQ ID NO. 1b-6 b.

Wherein the SNP is selected from rs699, rs2016848, rs5522, rs11209716, rs8012552 and rs 495828.

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 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 participates in the multiplex PCR reaction, and the primer of SEQ4b participates in the single base extension reaction; the locus sequence represented by the sequence of SEQ5-6 constitutes a third, independently performed complete reaction system, which again comprises two steps of multiplex PCR and single base extension: the primer pair of SEQ5a-SEQ6a was involved in a multiplex PCR reaction, and the primer of SEQ5b-SEQ6b was involved in a single base extension reaction.

The human SNP loci rs699, rs2016848, rs5522, rs11209716, rs8012552 and rs495828 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 of the drug type-related polymorphic site prepared by the primer composition for distinguishing the antihypertensive drug.

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 object of the present invention is to provide a method for discriminating a drug type by using the above primer composition or product through different mass spectrum peak patterns of different SNP sites, comprising:

(1) multiplex PCR: using the PCR primer pair to simultaneously amplify the DNA regions where the 6 SNP sites are located in two reaction systems to obtain a PCR product containing the DNA regions where the 6 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) in two reaction systems by using the 6 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 (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.

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, 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 medication type of the antihypertensive drug.

In one embodiment, the use comprises: guiding the individualized medication of enalapril 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 detected person can have variation of a single SNP site or a plurality of SNP sites, which means that the detected person can carry one or a plurality of SNP 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 with up to 6 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 6 pieces, which correspond to 6 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 2016848, rs 11209716", etc., 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 6 pairs of PCR amplification primer pairs and 6 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 variation frequency" refers to the probability of variation at a SNP site. Theoretically, the invention can simultaneously detect 6 SNP variations in a single individual. However, in practice, researchers have found that different SNP variations have a certain variation frequency among individuals.

The variation frequency in the Chinese population of 6 SNP sites (HCB, Han Chinese Beijing) is seen in:

http://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi？rs＝11209716

http://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi？rs＝2016848

http://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi？rs＝495828

http://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi？rs＝5522

http://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi？rs＝699

http://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi？rs＝8012552

the term "SEQ ID NO: 1-6" refers to 200bp sequences around 6 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: mass spectrum peak chart results when a plurality of sites of a certain sample are detected simultaneously;

wherein the horizontal coordinate unit is molecular weight, the extended primer of each site has possibility of mass spectrum peaks at two positions, and the mass spectrum peaks of each site have no overlap;

FIG. 4: wild type plasmid B1-B6 and mutant plasmid C1-C6 mass spectrum peak diagrams.

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

3.STR typing of buccal swabs for paternity testing with reference toJapanese popμLation data on the D20S85,D14S118,and D14S543 loci.Nihon HoigakuZasshi.1996 Dec；50(6):404-11.

4.Production of monoclonal anti-thymine-dimer antibody and its usefor detection of STR alleles.Nihon Hoigaku Zasshi.1996 Oct；50(5):343-8.

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 6 polymorphic sites related to the discrimination drug type, such as rs699, rs2016848, rs5522, rs11209716, rs8012552, rs495828 and the like, a corresponding specific PCR primer core sequence (SEQ1a to SEQ6a) and a specific extension primer core sequence (SEQ1b to SEQ6b) are designed. 6 pairs of PCR primers and 6 extension primers constitute 3 independently performed reaction systems: SEQ1a/b to SEQ3a/b constitute a first reaction system, SEQ4a/b constitutes a second reaction system, and SEQ5a/b to SEQ6a/b constitute a third reaction system. In the 3 independently performed reaction systems, SEQ1a to SEQ3a, SEQ4a, SEQ5a to SEQ6a are respectively involved in 3 independent multiplex PCR reactions, and SEQ1b to SEQ3b, SEQ4b, SEQ5b to SEQ6b are respectively involved in the subsequent 3 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 6 and the discrimination medication types were examined using ABI 9700 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 mu 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 in a PCR liquid preparation area, preparing a 200 mu L PCR reaction tube 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 tube;

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, mixing the mixture uniformly, adding 4 mu L 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 adding 1 μ L of sample to be tested into each tube of mixture in the PCR amplification area, so that the total volume of each PCR reaction system is 5 μ 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 enzyme-cleaved reaction solution was added to the PCR reaction tube, and then the PCR reaction tube was placed in a PCR amplification apparatus to carry out the following procedures.

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 μ 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 of purified water, 6mg of resin to each tube of extension product 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 primer molecular weight of each site is different, and after the extension of single base of two alleles of 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-B6 and mutant controls C1-C6 were provided at the above positions, respectively. Wherein, the wild control B1-B6 and the variant control C1-C6 are respectively from artificial plasmids which are sold in the market or deposited in the laboratory. The wild-type control plasmid B1-B6 and the variant control plasmid C1-C6 used in the present invention are constructed by inserting PCR products into pMD18-T Vector after PCR is carried out 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-B6, and then carrying out site-specific variation respectively, thereby constructing 6 variant plasmids C1-C6. The plasmids B1-B6 and C1-C6 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 6 extension primers and the extension products thereof generated at the 6 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 variant do not appear, judging that the experiment fails no matter whether the mass spectrum peak corresponding to the extended primer exists or not;

(2) if only one mass spectrum peak corresponding to the wild type or the 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 variant appear, judging the hybrid type.

The mass spectrum results are shown in FIG. 4, wherein FIGS. 4-A and 4-B are mass spectra of plasmid B1-B5 in which 6 SNP sites are all wild-type, and FIGS. 4-C and 4-D are mass spectra of plasmid C1-C6 in which 6 SNP sites are all variant.

The results of mass spectrometry of samples A1-A6 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 is 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 mu L, and the addition amount of various reagents is as follows: PCR products 3-10ng, BigDyev 3.10.25. mu.L, 5. mu.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 μ L70% ethanol into each well, centrifuging at 4000rpm for 10min, and back-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 mu L of 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 the A1-A10 samples, the primers described in the above table were used to sequence the above 6 SNP sites rs699, rs2016848, rs5522, rs11209716, rs8012552, rs495828 (numbering NO:1-6 respectively), 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. However, the conventional sequencing detection method needs to detect 6 sites and is difficult to be optimized by combining software, so that the process is too cumbersome and inefficient.

Sequence listing

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Claims

1. A primer combination for distinguishing the individualized drug type of enalapril which is a antihypertensive drug is characterized by comprising a PCR primer pair and an extension primer, wherein the PCR primer pair aims at 6 SNP markers capable of distinguishing the drug type, the sequence of the PCR primer pair is selected from the sequences shown in SEQ ID NO. 1a-6a, and the sequence of the extension primer is selected from the sequences shown in SEQ ID NO. 1b-6 b.

2. The primer composition of claim 1, wherein the SNPs are each selected from the group consisting of:

rs699、rs2016848、rs5522、rs11209716、rs8012552、rs495828。

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

4. the primer composition of claim 3, wherein the molecular weights of the extension primer and the extension product corresponding to each site are as follows:

5. the primer composition of claim 4, wherein the PCR primer sequence is a core sequence, which may include 5 to 15 protecting base sequences at the 5' end.

6. The primer composition of claim 5, wherein the protective base sequence is a10 bp tag: ACGTTGGATG are provided.

7. The primer composition as claimed in claim 6, wherein the base sequence as an adaptor can be added to the 5' end of the extended primer.

8. The primer composition of claim 7, wherein the linker base sequence is 1-15 bases.

9. The primer composition of claim 8, wherein the linker base sequence is 1-3 bases.