CN112725431A

CN112725431A - Primer, probe and kit for detecting polymorphic sites of APOE and SLCO1B1 genes

Info

Publication number: CN112725431A
Application number: CN202011626922.5A
Authority: CN
Inventors: 王博; 王婉君; 葛毅媛; 谢龙旭; 李烈军
Original assignee: Beijing Kaipu Medical Laboratory Co ltd; Zhengzhou Kaipu Medical Laboratory LP; Guangzhou Kaipu Pharmaceutical Technology Co ltd
Current assignee: Beijing Kaipu Medical Laboratory Co ltd; Zhengzhou Kaipu Medical Laboratory LP; Guangzhou Kaipu Pharmaceutical Technology Co ltd
Priority date: 2020-12-30
Filing date: 2020-12-30
Publication date: 2021-04-30
Anticipated expiration: 2040-12-30
Also published as: CN112725431B

Abstract

The invention discloses a primer, a probe and a kit for detecting polymorphic sites of APOE and SLCO1B1 genes, wherein the nucleotide sequences of the primer and the probe are respectively shown in SEQ ID NO. 1-12. The detection kit can simultaneously detect APOE gene APOE c.526C > T, APOE c.388T > C sites and SLCO1B1 gene SLCO1B1 c.388A > G, SLCO1B1 c.521T > C sites by one tube, has the advantages of simple operation, strong specificity, high flux, easy result interpretation, low detection cost and the like, can be used for predicting disease risk and health management, can also guide the reasonable use of statins, and is worthy of popularization.

Description

Primer, probe and kit for detecting polymorphic sites of APOE and SLCO1B1 genes

Technical Field

The invention relates to the technical field of gene detection, in particular to a primer, a probe and a kit for detecting polymorphic sites of APOE and SLCO1B1 genes.

Background

In recent 30 years, the blood lipid level of Chinese people gradually rises, and the prevalence rate of dyslipidemia is obviously increased. The reports of nutrition and chronic disease symptoms of Chinese residents (2015) show that the overall prevalence of dyslipidemia in Chinese adults is as high as 40.40%. Chinese guidelines for the prevention and treatment of dyslipidemia (revised 2016) indicate that LDL-C is a primary target for intervention in lipid-regulating therapy, and lowering LDL-C levels can significantly reduce the risk of morbidity and mortality due to ASCVD. Statins can reduce the synthesis of cholesterol by inhibiting the activity of HMG-CoA reductase, and research shows that statins can reduce the level of LDL-C by 55 percent and reduce cardiovascular events by 20 to 30 percent, can obviously reduce the risk of cardiovascular events in the primary and secondary prevention of ASCVD, and are the most important drugs for preventing and treating the diseases. The statins are drugs with large individual difference in curative effect and adverse reaction in clinical use, the individual difference is related to drug gene polymorphism, and APOE and SLCO1B1 genes are the most hot genes studied in recent years.

APOE participates in lipid metabolism regulation of the body through various ways, and is an important intrinsic factor influencing the blood lipid level of the body. The APOE polymorphism is considered as a susceptibility candidate gene of hyperlipoproteinemia and atherosclerotic vascular disease, and can form 3 haplotypes of APOE3(388T-526C), APOE2(388T-526T) and APOE4(388C-526C), wherein the risk of coronary heart disease of an APOE4 carrier is higher than 40%, and statins are poor or ineffective in curative effect on the APOE4 carrier and are strongest in lipid-lowering effect on the APOE2 carrier. The OATP1B1 organic anion transport polypeptide coded by SLCO1B1 is a main carrier protein for transporting statins into the liver, and the gene polymorphism of SLCO1B1 can influence the blood concentration of the statins. The SLCO1B1 × 5 genotype can reduce the level of statin transported to the liver, and increase the blood concentration of statin, thereby increasing the adverse reaction of statin, resulting in increased risk of rhabdomyolysis and myopathy. Therefore, CPIC guidelines for pharmacogenetics and guidelines for the detection of genes for drug metabolism and drug targets (trial) issued by the national health committee indicate that: SLCO1B1 and APOE gene polymorphism affect metabolism level of statin drugs, and doctors can instruct patients to take statins according to the gene polymorphism, so that the risk of myopathy is reduced.

The current detection method aiming at gene polymorphism mainly comprises the following steps: high resolution melting curve method, chip method, fluorescence PCR method, SNPshot method, direct sequencing method. The high-resolution melting curve method has high requirements on the temperature resolution of equipment, the difference among sample holes is required to be less than 0.1 ℃, the temperature difference among most conventional quantitative PCR instruments is between 0.3 and 0.5 ℃, the conventional quantitative PCR instruments cannot be used for analyzing a high-resolution melting curve (HRM), and the clinical popularization is difficult; the chip method has complex operation, long time consumption and lower automation degree; the common fluorescence PCR method has limited detection flux due to the limitation of a fluorescence channel, and the existing similar kit only detects one genotype of one site in one tube of mixed liquor, so the operation is complicated; the SNPshot and direct sequencing methods require the use of a sequencer, which is expensive and difficult to popularize. Therefore, the development of a kit for detecting polymorphic sites of APOE and SLCO1B1 genes, which has high accuracy and low cost, is urgently needed.

Disclosure of Invention

The invention aims to overcome the defects of the prior art for detecting gene polymorphic sites and provides a kit for detecting APOE and SLCO1B1 gene polymorphic sites, which has the advantages of low detection cost, short detection time, strong specificity and high discrimination of different genotypes.

The first purpose of the invention is to provide a primer group for detecting the polymorphic site of the APOE gene.

The second purpose of the invention is to provide a primer group for detecting the polymorphic site of the SLCO1B1 gene.

The third purpose of the invention is to provide a probe for detecting the polymorphic site of the APOE gene.

The fourth purpose of the invention is to provide a probe for detecting the polymorphic site of the SLCO1B1 gene.

The fifth purpose of the invention is to provide the application of any one of the primer group and/or the probe in the preparation of a detection kit for the polymorphic site of the APOE and/or SLCO1B1 gene.

The sixth purpose of the invention is to provide a kit for detecting the polymorphic sites of the APOE and/or SLCO1B1 genes.

In order to achieve the purpose, the invention is realized by the following technical scheme:

the invention provides a primer group for detecting APOE gene polymorphic sites, which comprises: the nucleotide sequence of the primer group with the detection site APOE c.388T & gtC is shown as SEQ ID NO. 1-2; the nucleotide sequence of the primer group of the detection site APOE c.526C & gtT is shown in SEQ ID NO. 3-4.

A primer group for detecting SLCO1B1 gene polymorphic sites comprises: the nucleotide sequence of the primer group for detecting the site SLCO1B1 c.521T & gtC is shown in SEQ ID NO. 5-6; the nucleotide sequence of the primer group for detecting the sites SLCO1B1 c.388A & gtG is shown as SEQ ID NO. 7-8.

Preferably, the primer components are an upstream primer and a downstream primer, and the nucleotide sequence of the upstream primer is SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5 and SEQ ID NO. 7; the nucleotide sequence of the downstream primer is SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6 and SEQ ID NO. 8.

More preferably, the length of the primer with the sequence shown in SEQ ID 1-8 is between 16-28 bp.

Even more preferably, the length of the primer with the sequence shown in SEQ ID 1-8 is preferably between 20-24 bp.

A probe for detecting polymorphic sites of an APOE gene, which comprises: the nucleotide sequence of the probe for detecting the site APOE c.388T & gtC is shown as SEQ ID NO. 9; the nucleotide sequence of the probe of the detection site APOE c.526C & gtT is shown in SEQ ID NO. 10.

A probe for detecting SLCO1B1 gene polymorphic sites, which comprises: the nucleotide sequence of the probe for detecting the site SLCO1B1 c.521T & gtC is shown as SEQ ID NO. 11; the nucleotide sequence of the probe for detecting the site SLCO1B1 c.388A & gtG is shown as SEQ ID NO. 12.

Preferably, the length of the probe with the sequence shown in SEQ ID NO. 9-12 is between 16-24 bp.

More preferably, the length of the probe with the sequence shown in SEQ ID NO. 9-12 is preferably between 20-22 bp.

Preferably, the 5 'end of the probe is modified by any one of fluorescent groups of FAM, ROX, VIC or CY5, and the 3' end of the probe is modified by any one of quenching groups of BHQ1 or BHQ 2.

More preferably, the 5 'end of the probe of the detection site APOE c.388T & gtC is modified by FAM fluorescent group, and the 3' end is modified by BHQ1 quenching group; the 5 'end of the probe of the detection site APOE c.526C & gtT is modified by a ROX fluorescent group, and the 3' end is modified by a BHQ2 quenching group; the 5 'end of the probe of the detection site SLCO1B1 c.521T & gtC is modified by a VIC fluorescent group, and the 3' end is modified by a BHQ2 quenching group; the 5 'end of the probe with the detection site SLCO1B1 c.388A & gtG is modified by a CY5 fluorescent group, and the 3' end of the probe is modified by a BHQ2 quenching group.

The application of the primer group and/or the probe in preparing a detection kit for the polymorphic site of the APOE and/or SLCO1B1 gene also belongs to the protection scope of the invention.

The invention also claims a kit for detecting the polymorphic sites of the APOE and/or SLCO1B1 genes, which comprises the primer group and the probe.

Preferably, the detection kit comprises PCR reaction liquid, Taq enzyme and quality control substance control plasmid.

Preferably, in the detection kit, the PCR reaction solution comprises a PCR amplification Buffer solution, more than 0.1-1 μ M of the primer group, more than 0.1 μ M of the probe, 1 × GC Buffer and ddH₂O。

More preferably, in the primer set in the PCR reaction solution, the upstream primer is 0.1. mu.M, and the downstream primer is 1. mu.M.

Preferably, the quality control plasmids comprise a plasmid of a wild-type APOE gene and a plasmid of a mutant APOE gene, a plasmid of a wild-type SLCO1B1 gene and a plasmid of a mutant SLCO1B1 gene.

Preferably, the concentration of the quality control plasmid is 1 × 10⁵copies/μL。

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

(1) compared with the traditional fluorescence PCR method, the gene polymorphism site detection kit of the invention adopts a melting curve method, saves half of probes, is simple to operate, can detect four gene sites by one tube only by using 4 probes, has low detection cost, short detection time, strong specificity and good repeatability, has the lowest detection limit of 1 ng/mu L, and has high discrimination between different genotypes of the same site (the difference of Tm values of melting peaks corresponding to a wild type and a mutant type is more than 5).

(2) The gene polymorphism site detection kit can be satisfied by adopting a common fluorescent PCR instrument, is simple and convenient to operate, and can detect 8 types of two genes and one site to be detected in the same reaction tube, so that a result can be obtained more quickly; the difference of the Tm values corresponding to melting peaks in the same channel and different types is more than 5 ℃, and the difference is easy to distinguish and judge; the detection result is easy to interpret by matching with automatic interpretation software; the operation processes are closed tube reactions, so that the pollution is obviously reduced, and the result is ensured to be real and credible; the detection kit can be used for predicting the risk of diseases and managing health and guiding the reasonable use of statins through detecting the polymorphic sites of APOE and SLCO1B1 genes.

Drawings

FIG. 1 shows APOE gene site APOE c.388T & gtC gene amplification melting curve results.

FIG. 2 shows the result of APOE gene locus APOE c.526C > T gene amplification melting curve.

FIG. 3 shows the result of SLCO1B1 gene locus SLCO1B1 c.521T > C gene amplification melting curve.

FIG. 4 shows the result of SLCO1B1 gene locus SLCO1B1 c.388A > G gene amplification melting curve.

Detailed Description

The invention is described in further detail below with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.

Example 1 kit for detecting polymorphic sites in APOE and SLCO1B1 genes

Preparation of detection kit

1. Primer and probe design

Respective primers and probes are designed aiming at the c.526C > T and c.388T > C sites of the APOE gene and the c.388A > G and c.521T > C sites of the SLCO1B1 gene, and the sequences of the primers and the probes are shown in Table 2:

TABLE 1 primers and probes for detecting polymorphic sites of APOE and SLCO1B1 genes

2. Preparation of quality control plasmid

The quality control plasmid comprises APOE gene wild type plasmid and APOE gene mutant type plasmid, SLCO1B1 gene wild type plasmid and SLCO1B1 gene mutant type plasmid, and the specific components are shown in Table 2:

TABLE 2 quality control Material composition

The preparation method comprises the following steps:

(1) clinical samples of wild type and homozygous mutant types of APOE c.526C > T, APOE c.388T > C and SLCO1B1 c.388A > G, SLCO1B1 c.521T > C4 gene loci were collected, DNA was extracted using a Kaemp blood genomic DNA extraction kit, and the specific procedures were according to the instructions provided in the kit.

(2) Respectively taking more than 2 mu L of DNA as templates, and adopting corresponding primer pairs (SEQ ID NO. 1-8) to amplify.

(3) And (3) performing gel cutting recovery on the PCR product by using an OMEGA gel recovery kit, and connecting the purified PCR product with a pMD Vector at 16 ℃ for 2 hours.

(4) mu.L of the ligation product was used to transform E.coli DH 5. alpha. competent cells, plated on LB (Amp +) plate, and cultured overnight at 37 ℃.

(5) White single colonies were picked from the transformed dish and inoculated into a 5mL tube of LB (Amp +) liquid medium and cultured overnight at 37 ℃.

(6) After the bacteria are cultured, extracting plasmid DNA by using an OMEGA plasmid extraction kit, dissolving the plasmid DNA in TE buffer solution, carrying out gel electrophoresis detection, measuring the concentration by using an ultraviolet spectrophotometry, diluting the solution by using the TE buffer solution, subpackaging and storing at the temperature of below 15 ℃.

(7) Sequencing the extracted plasmid DNA to determine the correct plasmid type.

3. Composition of the kit

A simple, fast, high accuracy APOE and SLCO1B1 gene polymorphism site detection kit can detect APOE c.526C > T, APOE c.388T > C and SLCO1B1 c.388A > G, SLCO1B1 c.521T > C gene sites simultaneously in one tube; the main components of the kit are shown in table 3:

TABLE 3 major Components and amounts of the kit

Wherein, the PCR reaction solution comprises the following components: dNTPs, buffer solution, the primers and the probes designed above, and the final concentration and content of each component of the reaction solution are shown in Table 4:

TABLE 4 final concentration and content of each component of PCR reaction solution

Name of each component	Final concentration
		PCR amplification buffer	1×
dNTPs(dATP、dGTP、dCTP、dTTP)	375μM
		SEQ ID NO.1	0.1μM
SEQ ID NO.2	1μM
		SEQ ID NO.9	0.1μM
SEQ ID NO.3	0.1μM
		SEQ ID NO.4	1μM
SEQ ID NO.10	0.1μM
		SEQ ID NO.5	0.1μM
SEQ ID NO.6	1μM
		SEQ ID NO.11	0.1μM
SEQ ID NO.7	0.1μM
		SEQ ID NO.8	1μM
SEQ ID NO.12	0.1μM
		GC Buffer	1×
ddH₂O	/

Second, use method of APOE and SLCO1B1 gene polymorphism site detection kit

1. Preparation of test sample (specimen preparation zone)

(1) Venous whole blood, buccal swab clinical samples were taken and DNA extraction was performed by reference to the procedures of the commercial genomic DNA extraction kit instructions purchased.

(2) Taking 2 mu L of the extracted DNA sample as a template to carry out PCR amplification; the remaining DNA samples were stored below-15 ℃ for future use, with freeze-thaw times not exceeding 5.

2. Amplification reagent preparation (reagent preparation zone)

(1) Taking out the PCR reaction solution from the prepared detection kit, unfreezing the PCR reaction solution at room temperature in a dark place, reversing the reaction solution from top to bottom and mixing the solution uniformly, and centrifuging the reaction solution at 6000 rpm for 10 seconds; taking out DNA polymerase from the kit, and centrifuging at 6000 rpm for 10 seconds; mu.L of the PCR reaction solution and 1. mu.L of DNA polymerase were mixed to prepare 18. mu.L of an amplification reagent.

(2) And fully and uniformly mixing the prepared amplification reagent, centrifuging for 1 minute at 6000 rpm, adding 18 mu L of the prepared amplification reagent into a PCR reaction tube, and transferring to a sample treatment area.

3. Application of sample (specimen preparation zone)

(1) The treated sample DNA, wild type control and mutant control were added to 2. mu.L each, and the tube was closed and centrifuged slightly (total reaction volume 24. mu.L/person).

(2) Transferring to a detection area, putting into a corresponding fluorescent PCR detector, and recording the arrangement sequence of the samples.

4. PCR amplification and melting curve analysis (amplification zone)

The amplification and melting curve analysis procedure was programmed as shown in Table 5, and the fluorescence signal acquisition channels were FAM, VIC, ROX and CY5 channels. The detection mode of the above macro stone SLAN-96S is a standard melting curve, and the detection mode of the Roche LightCycler 480II is Mono Color Hydrolysis Probe/UPL Probe.

TABLE 5 setting of amplification program

5. Determination of detection result

(1) Reference range

The usual value of each Tm value in Table 6 was used as a reference value (as measured on the Shanghai Macro Stone SLAN-96S instrument, when Roche LightCycler 480II was used, the Tm value may vary slightly, based on the Tm values of the wild type control and the mutant type control in the respective tests).

Selecting an effective Tm value with reference to the peak patterns of the wild type control and the mutant type control when the apparatus gives 1 or more Tm values; when the Tm value cannot be automatically given by the instrument, the Tm value can be obtained by adjusting the baseline or directly manually interpreting.

TABLE 6 Tm values reference

(2) Determination of results

The detection result of the wild type control is the wild type of 4 detection sites; the result of the mutant control is homozygous mutant of 4 detection sites. When the melting peak Tm values of the wild type control and the mutant type control are in accordance with the Tm value range shown in Table 6, the sample to be tested can be analyzed:

comparing the melting point (Tm value) difference between the melting peak of each channel of a sample to be detected in a PCR system and the melting peaks of wild type control and mutant type control at corresponding sites to judge whether the sample is mutated:

a. when the melting peak of the sample is a single peak, and the difference between the melting peak of the sample and the melting peak of the wild type control (sample peak-wild peak) is within +/-1.0 ℃, the sample is the wild type;

b. when the melting peak of the sample is a single peak, and the difference between the melting peak of the sample and the melting peak of the mutant control (sample peak-mutant peak) is within +/-1.0 ℃, the sample is the homozygous mutant;

c. when the melting peak of the sample is double peak, the difference between the melting peak of the sample at the left side and the melting peak of the mutant control (sample peak-mutation peak) is within +/-1.0 ℃, and the difference between the melting peak of the sample at the right side and the melting peak of the wild control (sample peak-wild peak) is within +/-1.0 ℃, the heterozygote mutant is obtained.

And secondly, if the detection result has no melting peak, the sample needs to be detected again, and DNA is extracted again for detection if necessary.

6. The result of the detection

The results of the gene amplification melting curve detection are analyzed, and the detection results of the kit on the sample are shown in figures 1-4 and table 7:

TABLE 7 analysis of sample test results

Example 2 detection accuracy of the kit

The detection accuracy of the kit is verified by detecting the following known type samples P1-P7 by using the detection kit and the detection method in the embodiment 1, and finally, three types (wild type, heterozygous mutant type and homozygous mutant type) of each locus are detected at least once. The type composition of the known samples is shown in table 8:

table 8 verification of sample composition

The results of the tests on the validation samples are shown in table 9:

TABLE 9 accuracy test results

The results show that the genotypes of 7 known samples are all consistent with the genotypes detected by the kit on the samples, and the accuracy is 100%.

Example 3 detection specificity and minimum detection Limit of the kit

First, experiment method

1. And (3) specificity verification: the results of detection of 4 non-human genome samples and samples outside the detection range of the kit were shown in Table 11, using the kit and detection method of example 1 (Table 10).

TABLE 10 samples for specific assays

Note: "-" indicates that the reference does not contain the detection region of the site

TABLE 11 results of specific detection

2. And (3) verification of the lowest detection limit: 7 samples (wild type, homozygous mutation, heterozygous mutation covering the assay site, Table 12) not higher than 1 ng/. mu.L were assayed using the kit and assay of example 1, and the assay results are shown in Table 13.

TABLE 12 lowest detection Limit validation samples

TABLE 13 lowest detection Limit verification results

Second, experimental results

1. Specificity: the detection results of the non-human genome sample and the sample outside the detection range of the kit are all undetected, which shows that the detection kit and the detection method have strong specificity.

2. The lowest detection limit is: the detection result of the reference substance with the lowest detection limit (1 ng/. mu.L) accords with the type of the corresponding site, which shows that the detection kit and the detection method have higher sensitivity and are worthy of popularization.

Example 4 reproducibility test of the kit

The test was repeated 10 times on the sample (encompassing all the site-tested heterozygous mutations, table 14) using the kit and test method of example 1, and the test results are shown in table 15.

Table 14 repeatability verification samples

TABLE 15 repeatability test results

The results show that the detection results are consistent when the sample is repeatedly detected for 10 times, which indicates that the detection kit and the detection method have good stability.

It should be finally noted that the above examples are only intended to illustrate the technical solutions of the present invention, and not to limit the scope of the present invention, and that other variations and modifications based on the above description and thought may be made by those skilled in the art, and that all embodiments need not be exhaustive. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.

Sequence listing

<110> Guangzhou Kaipp medicine science and technology Co., Ltd

Zhengzhou Kaipu medical laboratory (limited partnership)

Beijing Kaipu medical laboratory Co.,Ltd.

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Claims

1. A primer group for detecting polymorphic sites of an APOE gene, which is characterized by comprising: the nucleotide sequence of the primer group with the detection site APOE c.388T & gtC is shown as SEQ ID NO. 1-2; the nucleotide sequence of the primer group of the detection site APOE c.526C & gtT is shown in SEQ ID NO. 3-4.

2. A primer group for detecting the polymorphic site of the SLCO1B1 gene, which is characterized by comprising the following components: the nucleotide sequence of a primer group of a detection site SLCO1B1 c.521T > C is shown in SEQ ID NO. 5-6; the nucleotide sequence of a primer group for detecting the sites SLCO1B1 c.388A > G is shown as SEQ ID NO. 7-8.

3. A probe for detecting polymorphic sites of an APOE gene, which is characterized by comprising: the nucleotide sequence of the probe for detecting the site APOE c.388T & gtC is shown as SEQ ID NO. 9; the nucleotide sequence of the probe of the detection site APOE c.526C & gtT is shown in SEQ ID NO. 10.

4. A probe for detecting the polymorphic site of the SLCO1B1 gene, which is characterized by comprising the following components: a probe for detecting a site SLCO1B1 c.521T > C, wherein the nucleotide sequence of the probe is shown as SEQ ID NO. 11; the nucleotide sequence of the probe for detecting the site SLCO1B1 c.388A > G is shown as SEQ ID NO. 12.

5. The probe according to any one of claims 3 to 4, wherein the 5 'end of the probe is modified by a fluorophore selected from FAM, ROX, VIC and CY5, and the 3' end of the probe is modified by a quencher selected from BHQ1 and BHQ 2.

6. Use of the primer set according to claims 1 to 2 and/or the probe according to claims 3 to 4 for preparing a kit for detecting polymorphic sites in APOE and/or SLCO1B1 genes.

7. A kit for detecting polymorphic sites of APOE and/or SLCO1B1 genes, which comprises the primer set of claim 1-2 and the probe of claim 3-4.

8. The detection kit of claim 7, comprising a PCR reaction solution, Taq enzyme and a quality control plasmid.

9. The detection kit according to claim 8, wherein the PCR reaction solution comprises a Buffer solution for PCR amplification, 0.1 to 1. mu.M of the primer set according to claim 1 and 0.1 to 1. mu.M of the primer set according to claim 2, 0.1. mu.M of the probe according to claim 3 and 0.1. mu.M of the probe according to claim 4, 1 XGC Buffer, ddH₂O。

10. The detection kit of claim 8, wherein the quality control plasmids comprise a wild-type APOE gene plasmid and a mutant APOE gene plasmid, a wild-type SLCO1B1 gene plasmid and a mutant SLCO1B1 gene plasmid.