Method and kit for guiding atorvastatin individual medication gene

文档序号：796648 发布日期：2021-04-13 浏览：19次中文

阅读说明：本技术 阿托伐他汀个体化用药基因的指导方法及试剂盒 (Method and kit for guiding atorvastatin individual medication gene ) 是由陈涛张静王珊霞林金飞于 2020-12-28 设计创作，主要内容包括：本发明提供了阿托伐他汀个体化用药基因的指导方法及试剂盒,具体的本发明筛选出了与阿托伐他汀个体化用药相关基因的SNP位点的组合,利用核酸质谱仪对阿托伐他汀相关的遗传学标识进行广泛(高通量检测位点、高通量检测样本)筛查和检验。通过本发明的方法检出成功率高、技术重现性好、性价比高,可实现单个小样本多基因的检测,满足小样本最大化使用；本发明的方法具有高准确性、高灵敏性的技术优势,检测结果稳定,提高了检测阳性率。(The invention provides a method and a kit for guiding an atorvastatin individualized medication gene, in particular to a method and a kit for screening out a combination of SNP sites of the atorvastatin individualized medication related gene, and utilizing a nucleic acid mass spectrometer to carry out wide (high-throughput detection sites and high-throughput detection samples) screening and inspection on atorvastatin related genetic markers. The method has high detection success rate, good technical reproducibility and high cost performance, can realize the detection of multiple genes of a single small sample, and meets the maximum use of the small sample; the method has the technical advantages of high accuracy and high sensitivity, the detection result is stable, and the detection positive rate is improved.)

1. An atorvastatin personalized medicine gene guiding kit, which is characterized by comprising a PCR amplification primer pair group, wherein the PCR amplification primer pair group comprises a primer pair for specifically amplifying SNP sites selected from the following groups: rs2032582, rs662799, rs2266788, rs320, rs708272, rs8192870 and rs 4148222.

2. The kit of claim 1, further comprising a single base extension primer set.

3. The kit of claim 1, wherein the PCR amplification primer pair set comprises:

the primer pair for specific amplification of rs2032582 is shown as SEQ ID NO.1 to SEQ ID NO. 2;

the primer pair for specific amplification of rs662799 is shown in SEQ ID NO. 3-SEQ ID NO. 4;

the primer pair for specifically amplifying rs2266788 is shown as SEQ ID NO.5 to SEQ ID NO. 6;

the primer pair for specific amplification of rs320 is shown as SEQ ID NO.7 to SEQ ID NO. 8;

the primer pair for specifically amplifying rs708272 is shown as SEQ ID NO.9 to SEQ ID NO. 10;

the primer pair for specifically amplifying rs8192870 is shown as SEQ ID NO.11 to SEQ ID NO. 12; and/or

The primer pair for specifically amplifying rs4148222 is shown as SEQ ID NO.13 to SEQ ID NO. 14.

4. The kit of claim 2, wherein in the set of single base extension primers:

the extension primer aiming at rs2032582 is shown as SEQ ID NO. 15;

the extension primer aiming at rs662799 is shown as SEQ ID NO. 16;

the extension primer aiming at rs2266788 is shown as SEQ ID NO. 17;

the extension primer aiming at rs320 is shown as SEQ ID NO. 18;

the extension primer aiming at rs708272 is shown as SEQ ID NO. 19;

the extension primer aiming at rs8192870 is shown as SEQ ID NO. 20; and/or

The extension primer aiming at rs4148222 is shown as SEQ ID NO. 21.

5. The kit of claim 2, wherein the kit comprises a first container containing the PCR amplification primer pair set; and/or

The kit comprises a second container, and the single-base extension primer group is contained in the second container.

6. A method for detecting an SNP gene mutation site of an atorvastatin personalized medicine gene based on multiple PCR flight time mass spectra is characterized by comprising the following steps:

(1) carrying out PCR amplification by taking the peripheral blood genome DNA of a sample to be detected as a template to obtain an amplification product;

(2) SAP treatment of the amplification product of step (1) with shrimp alkaline phosphatase;

(3) carrying out single base extension reaction on the purified product in the step (2) by using an extension primer to obtain an extension product;

(4) purifying the extension product with desalting resin;

(5) and (5) detecting and analyzing by a mass spectrum platform, and judging whether genetic variation exists.

7. The method according to claim 6, wherein in the step (1), the SNP sites selected from the group consisting of: rs2032582, rs662799, rs2266788, rs320, rs708272, rs8192870 and rs 4148222.

8. The method according to claim 6, wherein in the step (1), the amplification primer pair group is used for PCR amplification during PCR amplification; and/or

In the step (3), a single-base extension reaction is performed using the single-base extension primer set.

The application of the PCR amplification primer pair group is characterized in that the PCR amplification primer pair group is used for preparing a detection kit, and the detection kit is used for detecting the SNP gene mutation site of the atorvastatin individualized medication gene;

the primer pair group comprises primers with sequences shown as SEQ ID NO.1 to SEQ ID NO. 14.

10. The application of the single base extension primer group is characterized in that the single base extension primer group is used for preparing a detection kit, and the detection kit is used for detecting the SNP gene mutation site of the atorvastatin individualized medication gene;

the single-base extension primer group comprises extension primers with sequences shown as SEQ ID NO.15 to SEQ ID NO. 21.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a guiding method and a kit for atorvastatin individual drug gene administration.

Background

The statins are first-line medicines for treating clinical cardiovascular diseases, and belong to a class of oral lipid-lowering medicines widely used in clinic. At present, with the continuous update of clinical application guidelines, the application of the composition is applied to the treatment of metabolic diseases, cerebrovascular diseases and cardiovascular diseases. However, the response to these drugs shows higher individual variation, and some patients reject or discontinue statins due to concerns about adverse reactions. Therefore, it is of great significance to research the medication guidance method of statins in clinical medication and confirm the use safety of statins.

Single Nucleotide Polymorphisms (SNPs) are genetic markers, which refer to polymorphisms in DNA sequences at the genomic level due to single nucleotide variations. The occurrence frequency in the population is more than 1 percent, and the expression comprises the conversion and inversion of single bases, the insertion or deletion of the single bases and the like, is a new genetic marker, and can provide reliable and effective scientific basis for the prediction, diagnosis and treatment of diseases and the development of novel medicaments.

SNP occurs in a coding region, can affect protein functions, thereby affecting human health or drug metabolism, and is mainly reflected in individual difference in disease susceptibility, individual difference in therapeutic effect of the same drug and individual difference in adverse reaction of the same drug. Pharmacogenetics or pharmacogenomics approaches are very important to address individual differences in drug efficacy, especially in the precise medical era.

Statins are first line therapeutic drugs that lower plasma cholesterol levels. Despite being safe and showing several beneficial non-cholesterol dependent pleiotropic effects, significant differences in therapeutic goals of statins have been reported in the literature. Lipid lowering therapy shows a high degree of variability in clinical response and there is evidence that variability in drug response among individuals is due to genetic factors. Referring to the results of gene detection, doctors can provide individualized medication schemes suitable for each patient in aspects of medicine selection, dosage control, combined medication and the like when prescribing, so that the aims of improving the curative effect of the medicine, reducing the toxicity of the medicine, enabling the patient to go out of a medication blind area, taking the medicine accurately, taking the medicine well and holding the optimal treatment period are finally achieved. At present, no very effective atorvastatin medication guiding method exists in clinic, so a high-sensitivity, economic and simple molecular technology screening method needs to be established urgently, and the domestic blank is filled.

Disclosure of Invention

The invention aims to provide a method and a kit for guiding atorvastatin personalized medicine genes.

In a first aspect of the present invention, there is provided a guiding kit for atorvastatin personalized medicine genes, the kit comprising a PCR amplification primer pair group, the PCR amplification primer pair group comprising a primer pair for specifically amplifying SNP sites selected from the following groups: rs2032582, rs662799, rs2266788, rs320, rs708272, rs8192870 and rs 4148222.

In another preferred embodiment, the kit is used for detecting the SNP gene mutation site of the atorvastatin personalized medicine gene based on multiple PCR flight time mass spectrometry.

In another preferred embodiment, the kit further comprises a single base extension primer set.

In another preferred example, in the PCR amplification primer pair group, the primer pair for specifically amplifying rs2032582 is shown as SEQ ID No.1 to SEQ ID No. 2.

In another preferred example, in the PCR amplification primer pair group, the primer pair for specifically amplifying rs662799 is shown in SEQ ID No.3 to SEQ ID No. 4.

In another preferred example, in the PCR amplification primer pair group, the primer pair for specifically amplifying rs2266788 is shown as SEQ ID No.5 to SEQ ID No. 6.

In another preferred example, in the PCR amplification primer pair group, the primer pair for specifically amplifying rs320 is shown as SEQ ID No.7 to SEQ ID No. 8.

In another preferred example, in the PCR amplification primer pair group, the primer pair specifically amplifying rs708272 is shown as SEQ ID No.9 to SEQ ID No. 10.

In another preferred example, in the PCR amplification primer pair group, the primer pair for specifically amplifying rs8192870 is shown as SEQ ID NO.11 to SEQ ID NO. 12.

In another preferred example, in the PCR amplification primer pair group, the primer pair for specifically amplifying rs4148222 is shown as SEQ ID No.13 to SEQ ID No. 14.

In another preferred example, in the single-base extension primer set, the extension primer for rs2032582 is shown as SEQ ID NO. 15.

In another preferred embodiment, in the single-base extension primer set, the extension primer for rs662799 is shown as SEQ ID No. 16.

In another preferred embodiment, in the single-base extension primer set, the extension primer for rs2266788 is shown as SEQ ID NO. 17.

In another preferred embodiment, in the single-base extension primer set, the extension primer for rs320 is shown as SEQ ID NO. 18.

In another preferred embodiment, in the single-base extension primer set, the extension primer for rs708272 is shown as SEQ ID NO. 19.

In another preferred embodiment, in the single base extension primer set, the extension primer for rs8192870 is shown as SEQ ID NO. 20.

In another preferred embodiment, in the single base extension primer set, the extension primer for rs4148222 is shown as SEQ ID NO. 21.

In another preferred embodiment, the kit comprises a first container, and the PCR amplification primer pair group is contained in the first container.

In another preferred embodiment, the kit comprises a second container, and the single-base extension primer set is contained in the second container.

In another preferred embodiment, the kit comprises a third container, wherein a PCR premix is contained in the third container, and the PCR premix mainly comprises hot-start Taq enzyme, dNTPs and MgCl₂And PCR buffer solution.

In another preferred embodiment, the kit comprises a fourth container containing shrimp alkaline phosphatase (SAPEnzyme).

In another preferred embodiment, the kit comprises a fifth container, and the fifth container contains an SAP buffer solution.

In another preferred embodiment, the kit comprises a sixth container containing an elongase (iPLEX Enzyme) therein.

In another preferred embodiment, the kit comprises a seventh container comprising ddNTPs.

In another preferred embodiment, the kit comprises an eighth container containing an extension reaction buffer.

In another preferred example, the kit further comprises pure water.

In a second aspect of the invention, a method for detecting SNP gene mutation sites of atorvastatin personalized medicine genes based on multiple PCR time-of-flight mass spectrometry is provided, which comprises the following steps:

(1) carrying out PCR amplification by taking the peripheral blood genome DNA of a sample to be detected as a template to obtain an amplification product;

(2) SAP treatment of the amplification product of step (1) with shrimp alkaline phosphatase;

(3) carrying out single base extension reaction on the purified product in the step (2) by using an extension primer to obtain an extension product;

(4) purifying the extension product with desalting resin;

(5) and (5) detecting and analyzing by a mass spectrum platform, and judging whether genetic variation exists.

In another preferred example, in the step (1), during the PCR amplification, SNP sites selected from the following group are specifically amplified: rs2032582, rs662799, rs2266788, rs320, rs708272, rs8192870 and rs 4148222.

In another preferred example, in the step (1), during the PCR amplification, the amplification primer pair group is used for PCR amplification.

In another preferred example, in the step (3), the single-base extension reaction is performed using the single-base extension primer set.

In the third aspect of the invention, the application of the PCR amplification primer pair group is provided, and the PCR amplification primer pair group is used for preparing a detection kit, wherein the detection kit is used for detecting the SNP gene mutation site of the atorvastatin personalized medicine gene;

the primer pair group comprises primers with sequences shown as SEQ ID NO.1 to SEQ ID NO. 14.

In the fourth aspect of the invention, the application of the single base extension primer group is provided, and the single base extension primer group is used for preparing a detection kit, wherein the detection kit is used for detecting the SNP gene mutation site of the atorvastatin personalized medicine gene;

the single-base extension primer group comprises extension primers with sequences shown as SEQ ID NO.15 to SEQ ID NO. 21.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Detailed Description

The invention provides a method and a kit for guiding atorvastatin individualized drug gene, which screen out 7 SNP sites of atorvastatin individualized drug related genes, and further can utilize a nucleic acid mass spectrometer to carry out wide (high-throughput detection sites and high-throughput detection samples) screening and inspection on atorvastatin related genetic markers. Through multi-round screening, a multiplex PCR amplification primer pair which can carry out high-efficiency multiplex amplification on the 7 SNP loci and is suitable for mass spectrometric detection of MassARRAY nucleic acid is obtained, and a suitable extension primer is obtained through screening, so that high-accuracy and high-sensitivity detection on the 7 SNP loci is realized, the detection result is stable, and the detection positive rate is improved.

The determination method of the application is used for detecting the SNP gene mutation site of the atorvastatin individualized medication gene based on a multiplex PCR technology and a MassARRAY nucleic acid mass spectrum technology, and can be used for simultaneously detecting 7 sites.

Multiplex PCR (multiplex PCR), also called multiplex PCR or multiplex PCR, is a PCR reaction in which two or more pairs of primers are added to the same PCR reaction system to simultaneously amplify multiple nucleic acid fragments, and the reaction principle, reaction reagents and operation process are the same as those of ordinary PCR.

There are many factors that affect multiplex PCR reactions, such as:

(1) the imbalance of the reaction system causes some dominant primers and templates thereof to be rapidly amplified in the previous rounds of reactions, and a large amount of amplification products are obtained, and the amplification products are good inhibitors of DNA polymerase. Therefore, the polymerization ability of polymerase is more and more strongly inhibited with the occurrence of a large amount of amplification products, and thus, primers and templates thereof which are at a disadvantage in the early stage are more difficult to react, and finally, the amount of amplification products is so small that they cannot be detected.

(2) The primer specificity, if the primer has stronger binding force with other non-target gene fragments in the system, the ability of the target gene to bind the primer is contended, thereby leading to the reduction of the amplification efficiency.

(3) The optimal annealing temperatures are different, a plurality of pairs of primers are placed in a system for amplification, and the optimal annealing temperatures of each pair of primers are required to be close to each other because the annealing temperatures for PCR reaction are the same.

(4) Primer dimers, including dimers between primers and hairpin structures formed by the primers themselves, are third-party DNA-mediated dimers, and these dimers, like non-specific primers, interfere with the competition between primers and target binding sites, affecting amplification efficiency.

Although several factors affecting amplification efficiency are mentioned above, more are not clear. To date, there is no effective method for clearly predicting amplification efficiency.

Although the multiplex PCR-time-of-flight mass spectrometry detection technology can carry out ultrahigh-flux detection, the requirement on the quality of a PCR amplification product is high. The inventor finds in research that the existing amplification primer and extension primer capable of carrying out detection by a multiplex fluorescence PCR method are directly applied to multiplex PCR-time-of-flight mass spectrometry, and have many defects, such as false negative of mass spectrometry caused by incapability of carrying out single base extension reaction, low sensitivity and poor repeatability, which are difficult to meet clinical application. Therefore, the inventor redesigns a plurality of pairs of amplification primers and extension primers aiming at each detection site, performs multiple combined detection verification under the condition that single-site detection can meet the requirement, and finally obtains a multiple PCR detection system and extension primers which have high sensitivity, good specificity and stable detection result and are suitable for flight time mass spectrometry detection through a large amount of test screening.

The invention adopts a multiplex PCR method to amplify a target sequence, artificially designs a plurality of pairs of primers, optimally selects and verifies the primers, and finally determines a nucleic acid detection kit which contains the following amplification primers and is used for detecting 7 sites in total of SNP gene mutation sites of the atorvastatin personalized medicine gene.

TABLE 1 amplification primers

Wherein, F is an upstream primer, and R is a downstream primer.

The extension primers are shown in table 2:

TABLE 2 extension primers

The primer sequences listed in tables 1 and 2 can be synthesized by conventional polynucleotide synthesis methods.

In addition to the amplification primer and the extension primer, the invention also provides a kit for detecting the SNP gene mutation site of the atorvastatin personalized medicine gene, wherein the specific contents of the components in the kit are as follows:

TABLE 3 kit Components

The invention also provides a method for detecting the SNP gene mutation site of the atorvastatin personalized medicine gene based on the multiple PCR flight time mass spectrum, which comprises the following steps:

(1) carrying out PCR amplification by taking the peripheral blood genome DNA of a sample to be detected as a template to obtain an amplification product;

(2) SAP treatment of the amplification product of step (1) with shrimp alkaline phosphatase;

(3) carrying out single base extension reaction on the purified product in the step (2) by using an extension primer to obtain an extension product;

(4) purifying the extension product with desalting resin;

(5) and (5) detecting and analyzing by a mass spectrum platform, and judging whether genetic variation exists.

Further, in the step (1), during the PCR amplification process, SNP sites selected from the following group are specifically amplified: rs2032582, rs662799, rs2266788, rs320, rs708272, rs8192870 and rs 4148222.

Further, in the step (1), in the PCR amplification process, the amplification primer pair group is used for PCR amplification.

Further, in the step (3), a single base extension reaction is performed using the extension primer set.

Further, the amplification conditions of step (1) are as follows: 95 deg.C for 3 min; 95 deg.C, 15s, 52 deg.C, 15s, 72 deg.C, 1min, 45 cycles; keeping at 72 deg.C for 5 min.

Further, the SAP treatment conditions of the step (2) are as follows: 57 ℃ for 40min and 65 ℃ for 5 min.

Further, the conditions of the extension reaction of step (3) are as follows: 95 ℃ for 30 s; 95 ℃, 5s, (51 ℃, 5s, 72 ℃, 5s, 5 cycles), 35 cycles; keeping at 72 deg.C for 5 min.

The main advantages of the invention are:

the nucleic acid mass spectrum guidance method of the atorvastatin individualized medication gene considers the differences of atorvastatin medication of different sick people, the detected individualized medication gene is more advanced, and a plurality of SNP sites for the atorvastatin individualized medication are included, and the sites have high detection success rate, good technical reproducibility and high cost performance;

the detection technology provided by the invention has obvious price advantage, and overcomes the disadvantages of high price, long time consumption, complicated operation and the like of the traditional single-base detection. The sensitivity in the aspect of the atorvastatin individual medication gene guide detection is higher, the flux is larger, the detection of multiple genes in a single small sample can be realized, and the maximum use of the small sample is met.

The nucleic acid mass spectrometry method for detecting the atorvastatin individual medication gene guidance based on the MassARRAY nucleic acid mass spectrometry technology has the technical advantages of high accuracy and high sensitivity, is stable in detection result, has obvious advantages compared with Sanger sequencing, and improves the detection positive rate.

The present invention will be described in further detail with reference to the following examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures for conditions not specified in detail in the following examples are generally carried out under conventional conditions such as those described in molecular cloning, A laboratory Manual (Huang Petang et al, Beijing: scientific Press, 2002) by Sambrook. J, USA, or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are by weight. The test materials and reagents used in the following examples are commercially available without specific reference.

Example 1

Feasibility analysis of SNP site screening of human atorvastatin personalized medication related gene

Through searching for the atorvastatin-wide association study (GWAS) related sites verified in the large-scale pathology control group clinical research by NCBI (national and international research institute of medicine-wide association), the inventor screens and evaluates the sites, selects 7 single nucleotide polymorphic sites which are obviously related to the atorvastatin personalized medicine, and the sites are independent of each other and have no linkage disequilibrium, so that the site selection provided by the invention has representativeness, independence and risk value accumulation, and can be used for guiding the personalized medicine of atorvastatin.

The selected SNP sites are as follows:

rs2032582、rs662799、rs2266788、rs320、rs708272、rs8192870、rs4148222。

example 2 system verification

The system verification comprises accuracy, specificity, sensitivity, precision, inter-personnel comparison and the like.

An accuracy verification scheme: 20 sites were tested and compared to Sanger sequencing, the expected target was 95%.

Specificity verification scheme: included in the accuracy is the expected target of 95%.

Sensitive verification protocol: the positive sample of human genome DNA is taken as a template, and the content of the labeled sample DNA is 1 ng/muL, 5 ng/muL, 10 ng/muL, 50 ng/muL and 100 ng/muL respectively for sensitivity investigation.

Precision validation protocol (including intra-batch, inter-batch, personnel comparisons, not involving inter-instrument comparisons) expected target 95%.

Internal precision: the same batch was repeated 3 times for each sample and the intra-batch precision was compared.

Batch precision: the same operator examines the same samples in multiple batches and compares the batch-to-batch precision.

The personnel alignment: 2 operators tested the same samples and compared the differences in results between the individuals.

The specific test steps are as follows:

1. DNA extraction: human peripheral blood genomic DNA (50 mu LddH) was prepared according to the procedures provided in the instruction manual of a blood DNA extraction kit (commercially available kit for rapid and efficient extraction of genomic DNA) independently developed by southern medicine₂Eluting with oxygen;

2. PCR procedure

(1) Samples were diluted to 20 ng/. mu.L;

(2) the following table was used to prepare a PCR reaction system (hereinafter, a single sample amount, 40ng of sample DNA in total)

TABLE 4 PCR reaction System

Reagent	W1(μL)	W2(μL)
			Water, ddH₂O	0.8	0.8
10PCR Buffer with 20mM	0.5	0.5
			25mM MgCl₂	0.4	0.4
25mM dNTP mixture	0.1	0.1
			25 mu M amplification primer mixture	1	1
5U/. mu.L PCR Taq enzyme	0.2	0.2
			20ng/μL DNA	2	2
Total volume	5.00	5.00

(3) Sealing the membrane, mixing with vortex for 30 seconds, and centrifuging at 500g for 1 minute;

(4) place the plate on a PCR instrument for the following thermal cycling:

95 ℃ for 3 minutes

45 cycles:

(95 ℃ for 15 seconds)

52 ℃ for 15 seconds

72 ℃ for 1 minute

5 minutes at 72 DEG C

Keeping the temperature at 4 DEG C

2. SAP flow scheme

(1) Taking out the PCR plate, and centrifuging for 3 minutes at 500 g;

(2) SAP reaction systems (individual sample amounts below) were formulated as follows;

TABLE 5 SAP reaction System

Reagent	Per well sample (μ L)	×2
			ddH₂O	1.53	3.06
SAP buffer	0.17	0.34
			SAP enzyme (1.7U/. mu.L)	0.3	0.6
Total volume	2.00	4.00

(3) Adding 2 mu LSAP mixed solution into each hole;

(4) sealing the membrane, mixing with vortex for 30 seconds, and centrifuging at 500g for 1 minute;

(5) place the plate on a PCR instrument for the following thermal cycling:

40 minutes at 57 DEG C

5 minutes at 65 DEG C

Keeping the temperature at 4 DEG C

3. EXT (Single base extension) protocol

(1) Taking out the PCR plate, and centrifuging for 3 minutes at 500 g;

(2) the following table was followed to formulate the EXT reaction system (individual sample amounts below);

TABLE 6 EXT reaction System

Reagent	W1(μL)	W2(μL)
			ddH₂O	0.62	0.62
iPLEX buffer solution	0.2	0.2
			ddNTP mixed liquor	0.2	0.2
Extension primer mixture	0.94	0.94
			iPLEX enzyme	0.04	0.04
Total volume	2.00	2.00

(3) Adding 2 mu L of iPLEX extension mixed solution;

(4) sealing the membrane, mixing with vortex for 30 seconds, and centrifuging at 500g for 1 minute;

(5) place the plate on a PCR instrument for the following thermal cycling:

95℃30s

35 cycles:

(95℃、5s

5 cycles:

(51℃5s

72℃5s))

72℃5min

keeping the temperature at 4 DEG C

4. Resin desalination

Taking out the PCR plate, and centrifuging for 3 minutes at 500 g; spreading clean Resin (Resin) on the sample plate hole, and air-drying for at least 10 min; adding 10uL of water into each hole with the sample in the sample plate; plate closed, vortex10 sec, 500g centrifuge for 1 min; slightly inverting the sample plate in a volley manner, placing the sample plate on the sample plate with the resin, and then inverting the sample plate together with the sample plate (the two quick plates cannot move horizontally in the process) to allow the resin to fall into the holes; taking down the sample plate, sealing the sample plate, and shaking up for 3 minutes with the rotator upside down; centrifuge at 2000g for 5 minutes.

5. Dispensing spotting

MALDI-TOF (matrix assisted laser Desorption ionization-time of flight) mass spectrometer was used to obtain a clustering plot (clear homopolymeric) of each site of the data.

And (3) test results: the accuracy verification results of 1 sample are shown in table 7.

TABLE 7 accuracy verification (comparison of first generation sequencing with MassARRAY results)

SNP_ID	First generation sequencing results	MassARRAY results
			rs2032582	AC	AC
rs662799	GG	GG
			rs2266788	GG	GG
rs320	TG	TG
			rs708272	GG	GG
rs8192870	TT	TT
			rs4148222	CC	CC

The rs4148222 site of the sample is used as an example, and the precision verification results are shown in table 5.

TABLE 8 verification of precision of rs4148222 site

	Repetition of 1	Repetition 2	Repetition of 3
				Batch 1	CC	CC	CC
Batch 2	CC	CC	CC
				Batch 3	CC	CC	CC
Batch 4	CC	CC	CC
				Batch 5	CC	CC	CC

On the whole, all the sites of the method are clustered clearly, basically have no gray areas, and the false detection is possibly small. The accuracy (including sensitivity and specificity) and precision of each site detection of the present application were verified and are shown in table 9.

TABLE 9 verification results of accuracy, sensitivity, specificity

SNP_ID

Accuracy of

Sensitivity of the probe

Specificity of

Precision in batch

Inter-batch precision

Comparison of persons

rs2032582

100％

1ng/μL

100％

rs662799

100％

1ng/μL

100％

rs2266788

100％

1ng/μL

100％

rs320

100％

1ng/μL

100％

rs708272

100％

1ng/μL

100％

rs8192870

100％

1ng/μL

100％

rs4148222

100％

1ng/μL

100％

In the above table, the accuracy of 100% indicates that all positive samples were correctly detected and consistent with Sanger sequencing results; the specificity of 100 percent indicates that no false positive result appears in the detected sample; the batch precision is 100%, which indicates that the repeated detection results of the same batch of samples can be kept consistent; the batch precision is 100 percent, which indicates that the detection results of the same operator for detecting the same sample in multiple batches can be kept consistent; the personnel comparison of 100 percent shows that the detection results of 2 operators detecting the same sample can be kept consistent.

In conclusion, the nucleic acid mass spectrum guidance method for the atorvastatin individualized medication gene considers the differences of atorvastatin medication of different sick people, detects the individualized medication gene more forward, and incorporates a plurality of SNP sites which are used for the atorvastatin individualized medication, and the sites have high detection success rate, good technical reproducibility and high cost performance.

Comparative example 1 screening of PCR amplification primer set and extension primer

Aiming at each site, the inventor designs several to ten pairs of amplification primers and extension primers, and then verifies and optimizes the amplification primers and the extension primers to finally establish a multiplex PCR amplification primer and extension primer combination which can be used for MassARRAY nucleic acid mass spectrometry technology detection.

In this comparative example, the rs662799 site is taken as an example, and an amplification primer and an extension primer having partially unsatisfactory effects are exemplified.

Control primer pair 1:

F-1：ACGTTGGATGGAACAAGCAAGGGAAGC(SEQ ID NO.:22)

R-1：ACGTTGGATGTGGTAGTGGAAATGGAGG(SEQ ID NO.:23)

control primer pair 2:

F-2：ACGTTGGATGTGGAACAAGCAAGGGAA(SEQ ID NO.:3)

R-2：ACGTTGGATGTGGTAGTGGAAATGGAGG(SEQ ID NO.:23)

control primer pair 3:

F-3：ACGTTGGATGGAACAAGCAAGGGAAGC(SEQ ID NO.:22)

R-3：ACGTTGGATGGGACCAAGAATCGGGAG(SEQ ID NO.:24)

control extension primer 1:

Y-1：GGAACTGGAGCGAAAGT(SEQ ID NO.:25)

control extension primer 2:

Y-2：CAGGAACTGGAGCGAAAGT(SEQ ID NO.:26)

the primer pair of the invention comprises: SEQ ID NO.3 and 4

The invention extends primer: SEQ ID NO.16

In the single-fold screening experiment, different extension primers are used for single base extension after single-fold PCR amplification, and then mass spectrometry detection is carried out on extension products, and the result of the single-fold detection shows that the control primer pairs 2 and 3 can normally work in the single-fold system, but positive results cannot be obtained in the multiple system.

In a multiplex system, the detection sensitivity of the combination of the control primer pair 1 and the control extension primers 1 and 2 is respectively 50 ng/muL and 50 ng/muL; the detection sensitivity of the control primer pair 1 and the extension primer combination shown in SEQ ID NO.16 is 5 ng/mu L; the combination of the primer pairs shown in SEQ ID NO.3 and 4 and the control extension probe 1 resulted in a detection sensitivity of only 50 ng/. mu.L. And the combination of the primer pairs shown in SEQ ID NO.3 and 4 and the extension primer shown in SEQ ID NO.16 can reach the detection sensitivity of 1 ng/muL.

The results indicate that the control primer pair 2, 3 cannot effectively amplify the target nucleic acid sequence in the detection system and therefore cannot work in the detection system; control primer pair 1 can work in multiple detection lines, but the sensitivity is poor; the control extension probes 1, 2, while also working in extension, are also less sensitive. The combination of the primer pair (SEQ ID NO.3 and 4) and the extension probe (SEQ ID NO.16) can normally work in a multiple detection system, and the sensitivity is high and reaches 1 ng/muL.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

<110> Guangdong Min Chi medical science and technology Co., Ltd

<120> atorvastatin personalized medicine gene guiding method and kit

<130> 200511

<160> 26

<170> PatentIn version 3.5

<210> 1

<211> 28

<212> DNA