CN110951858A

CN110951858A - Primer-probe combination for guiding detection of genes related to glibenclamide drug personalized administration, kit and application

Info

Publication number: CN110951858A
Application number: CN201911267923.2A
Authority: CN
Inventors: 刘颖; 宣涛; 杜晴晴; 孙子奎
Original assignee: Shanghai Personal Biotechnology Co ltd
Current assignee: Shanghai Personal Biotechnology Co ltd
Priority date: 2019-12-11
Filing date: 2019-12-11
Publication date: 2020-04-03

Abstract

The invention discloses a specific primer probe combination for guiding a glibenclamide drug individualized medication related gene SNP, which is characterized by comprising specific primer probe combinations aiming at rs5219 and rs889299 gene SNPs. The invention also provides a kit containing the primer combination and application thereof. The invention has the beneficial effects that: the rapid genotyping kit for guiding the SNP of the gene related to the individualized medication of the glyburide medicament and the application thereof are provided, and the detection result obtained by applying the kit is accurate and reliable, has lower cost and better practicability.

Description

Primer-probe combination for guiding detection of genes related to glibenclamide drug personalized administration, kit and application

Technical Field

The invention relates to the technical field of in-vitro diagnosis, in particular to a primer probe combination for guiding detection of genes related to personalized medication of glibenclamide, a kit and application.

Background

According to the statistics of the international diabetes alliance, the number of global Diabetes Mellitus (DM) patients in 2006 is up to 2.46 hundred million, and the number of global Diabetes Mellitus (DM) patients in 2025 is expected to reach 3.8 hundred million. DM can reduce the life quality of patients, shorten the life span and increase the fatality rate. Therefore, active control is very important.

Glibenclamide (INN), also known as glibenclamide (USAN), is a sulfonylurea hypoglycemic agent. Glibenclamide inhibits hepatic glycogenolysis and gluconeogenesis, and decreases hepatic production and output glucose by increasing portal insulin levels or acting directly on the liver; the oral administration has quick absorption, the protein combination rate is high and is 95 percent, the blood concentration reaches the peak value 2 to 5 hours after the oral administration, and the continuous action lasts for 24 hours. T1/2 was 10 hours. Metabolized in the liver, being excreted by the liver and kidneys in about 50% each. Glibenclamide (excellent hypoglycemic and dalan therapy) is one of the second generation oral sulfonylureas hypoglycemic drugs with the longest action time and the strongest hypoglycemic action, which is 100 times of D-860. The oral administration can be carried out for about 16-24 h, but the product is easy to have hypoglycemia reaction and local and general allergic reaction.

Technical guidelines for the detection of drug-metabolizing enzymes and drug-action target genes (trial) indicate that: the detection of drug metabolizing enzyme and drug target gene can guide the patient to select proper drug and administration dosage to realize 'individual' administration, and improve the effectiveness and safety of drug treatment. According to the relation between related genes and adverse drug reactions and dosage, the KCNJ11 and SCNN1B genotypes are recommended to be detected so as to guide the accurate treatment of the glibenclamide.

Adverse reactions resulting from overdose of glyburide include hypoglycemia, gastrointestinal reactions (e.g., abdominal bloating, diarrhea, nausea, etc.), skin reactions, blood system reactions (hemolytic anemia), increased risk of cardiovascular death, and macrovascular events. The therapeutic effect of the glibenclamide has obvious individual difference, and the subsequent toxic and side effect seriously harms the health of patients. By detecting the metabolism, transportation or action target gene of the glyburide, the difference of curative effect and toxic and side effect of the glyburide on different individuals can be discovered as soon as possible, so that the 'individual' administration of the glyburide is realized, the treatment effect is improved, and the risk caused by improper administration dosage is reduced.

The current gene polymorphism detection technology adopted in the market is PCR-RFLP, the main principle is that a PCR amplification product is digested by using specific restriction endonuclease, and whether variation exists or not is judged according to whether a digestion site disappears, but the method is complex to operate, cross contamination of the PCR product is easily caused when the sample amount is large, false negative or false positive results are easily caused due to insufficient enzyme digestion or excessive enzyme digestion, and the reliability is low. Although the specificity of the multiplex PCR method is improved, the principle of the method is still based on the principle of common PCR, and the results can be influenced by the primer specificity, low-fidelity Taq enzyme and other factors.

Although the DNA sequencing method is the gold standard for performing gene diagnosis at present, the steps are complicated, the process is complex, the reagent price is high, and the sequencing failure is easily caused by cross contamination among samples; in addition, the equipment of the sequencer is beyond the tolerance of a general clinical laboratory. The high-resolution melting curve method is a rapid, simple, economical and practical genotyping method, but genotyping depends on the precision of instrument temperature control, and false positive is high. The method of the Taqman probe adopts a specific fluorescence-labeled probe, and has the advantages of strong specificity, high sensitivity, convenient operation and high speed.

Disclosure of Invention

The kit aims to solve the technical problems that clinical adverse reactions are easy to occur when the traditional clinical experience medical treatment mode is used for diagnosis and the glibenclamide medicament is used, and a proper kit product is not available.

One of the purposes of the invention is to provide a primer probe combination for guiding the SNP of genes related to the personalized medication of the glyburide medicine.

Another object of the present invention is to provide a kit comprising a primer probe set for SNP of the related gene

The invention also aims to provide application of the kit.

In order to realize one of the purposes of the invention, the adopted technical scheme is as follows:

a specific primer probe combination for guiding genes SNP related to the personalized medication of glibenclamide medicines comprises specific primer probe combinations aiming at rs5219 and rs889299 gene SNP, and specifically comprises the following steps:

the rs5219 primer probe sequence is:

rs5219-F：5'-CTGCTCCCGGATGTTCTTGT-3'；

rs5219-R：5'-CCGAGGAATACGTGCTGACA-3'；

rs5219-P1：5'FAM-TACCTGGGCTTGGC-MGB 3'；

rs5219-P2：5'VIC-TACCTGGGCTCGGCA-MGB 3'；

the probe sequence of the rs889299 primer is as follows:

rs889299-F：5'-CGAGGTGTCAGGGTCTTGGT-3'；

rs889299-R：5'-GTCCAGTCCCTTCCCTTGAGA-3'；

rs889299-P1：5'FAM-TGCCTCATTTGTGTGATTGCTCTTTTATTG-TAMRA 3'；

rs889299-P2：5'VIC-CTGCCTCATTTGTGTGATTGTTCTTTTATTG-TAMRA 3'。

in order to realize the second purpose of the invention, the adopted technical scheme is as follows:

a fluorescent quantitative PCR kit for guiding genes SNP related to the personalized medication of glyburide medicines comprises a primer probe combination, wherein the sequence of the primer probe combination is shown as SEQ ID NO. 1-8.

In a preferred embodiment of the present invention, the fluorescent quantitative PCR kit further comprises a PCR reaction solution including the specific primer probe combination, the PCR reaction solution is rs5219 and rs889299 PCR reaction solutions respectively, the PCR reaction solution further comprises 2 × NuHi SNP Mix and ultrapure water, the NuHi SNP Mix comprises Taq enzyme, dNTPs, MgCl, and the like₂PCR buffer or ROX reference fluorescence.

In a preferred embodiment of the present invention, the final concentrations of the components of the PCR reaction solution are: 1 XNuHi SNPMix, 0.5. mu.M of each specific primer and 0.2. mu.M of probe.

In a preferred embodiment of the invention, the kit further comprises a positive control substance I, a positive control substance II or a positive control substance III;

the positive control substance I comprises TT type or GG type plasmids of rs5219 or rs889299 gene loci;

the positive control II comprises rs5219 gene locus TT type and CC type or rs889299 gene locus GG type and AA type according to the weight ratio of 1: 1 number ratio of heterozygous plasmids;

the positive control product III is CC type or AA type plasmid of rs5219 or rs889299 gene locus respectively.

In order to realize the third purpose of the invention, the adopted technical scheme is as follows:

the application of the kit is to perform fluorescent quantitative PCR reaction on human genome DNA by using the fluorescent quantitative PCR kit, distinguish polymorphism of corresponding SNP sites according to a fluorescent signal generated in the reaction process, and guide the personalized medication of the glyburide medicament.

The invention has the beneficial effects that:

the rapid genotyping kit for guiding the SNP of the gene related to the individualized medication of the glyburide medicament and the application thereof are provided, and the detection result obtained by applying the kit is accurate and reliable, has lower cost and better practicability.

Drawings

FIG. 1: the amplification curve diagram (FAM above) of the TT genotype of rs5219 in the embodiment of the invention;

FIG. 2: the amplification curve diagram (FAM below) of the TC genotype of rs5219 in the embodiment of the invention;

FIG. 3: the amplification curve diagram (FAM below) of the CC genotype of rs5219 in the embodiment of the invention;

FIG. 4: a PCR fluorescence analysis chart of the rs5219 genotyping result in the embodiment of the invention;

FIG. 5: the amplification curve diagram (FAM above) of GG genotype of rs889299 in the embodiment of the invention;

FIG. 6: the amplification curve diagram (FAM above) of GA genotype of rs889299 in the embodiment of the invention;

FIG. 7: the amplification curve diagram of the AA genotype of rs889299 in the embodiment of the invention (FAM below);

FIG. 8: a PCR fluorescence analysis chart of the rs889299 genotyping result in the embodiment of the invention;

FIG. 9: amplification curve diagram (FAM above) of TT genotype of primer probe combination rs5219-2 in comparative example;

FIG. 10: amplification profile of TC genotype of primer probe combination rs5219-2 in comparative example (FAM below);

FIG. 11: the amplification curve (FAM above) of CC genotype of primer probe combination rs5219-2 in the comparative example;

FIG. 12: a PCR fluorescence analysis chart of the primer probe combination rs5219-2 genotyping result in the comparative example;

FIG. 13: the amplification curve diagram of GG genotype of primer probe combination rs889299-2 in the comparative example (FAM above);

FIG. 14: amplification profile of GA genotype of primer probe combination rs889299-2 in comparative example (FAM under);

FIG. 15: amplification profile of AA genotype of primer probe combination rs889299-2 in comparative example (FAM below);

FIG. 16: PCR fluorescence analysis chart of primer probe combination rs889299-2 genotyping result in comparative example.

Detailed Description

The principle of the invention is mainly as follows: the kit is a detection kit combining a Taqman probe and a fluorescent quantitative PCR technology, and is suitable for clinical popularization and industrialization.

The PCR reaction system of the TaqMan probe method comprises a pair of PCR primers and a pair of probes. The probe binds specifically only to the template, with the binding site between the two primers. The 5 'end of the probe is marked with a reporter gene, the 3' end of the probe is marked with a fluorescence quenching group, when the probe is complete, the fluorescence energy emitted by the reporter gene is absorbed by the quenching group, the signal can not be detected by an instrument, along with the PCR, when Taq enzyme meets the probe combined with a template in the chain extension process, the 3 '-5' exonuclease activity cuts off the probe, the reporter group is far away from the quenching group, and the energy can not be absorbed, namely, a fluorescence signal is generated. The pair of probes is respectively marked by two different fluorescent dyes (such as FAM, HEX and VIC), and the genotype judgment of a single SNP locus can be completed in one PCR reaction aiming at different genotypes of the biallelic SNP.

The present invention is further illustrated by the following examples, which should not be construed as limiting the invention thereto.

Example 1

100 samples of the anticoagulated blood of the population were taken as samples of the PCR template, and extracted using a commercial DNA extraction kit. The specific extraction steps are as follows: 1. taking 400ul of whole blood, adding 800ul of cell lysate CL, mixing uniformly, centrifuging at 10000rpm/11500 Xg for 1min, and discarding the supernatant, wherein the process can be repeated once if the lysis is not complete.

2. Adding 200ul buffer solution GS into the precipitate, mixing uniformly, adding 20ul protease K, and mixing uniformly.

3. 200ul of buffer GB was added and mixed well at 56 ℃ for 10min, with the phases reversed several times until the solution became clear (if not clear, the time could be extended to clear).

4. Adding 200ul of absolute ethyl alcohol, mixing uniformly, and centrifuging for a short time.

5. The sample mixture was transferred to adsorption column CB3, 13400 Xg/12000 rpm, and centrifuged for 30 seconds, and the filtrate was discarded.

6. 500ul of buffer GD was added to adsorption column CB3, 13400 Xg/12000 rpm, and centrifuged for 30s, and the filtrate was discarded.

7. 600ul of buffer PW was added to adsorption column CB3, 13400 Xg/12000 rpm, and the mixture was centrifuged for 30 seconds, and the filtrate was discarded and repeated once.

8. Placing the adsorption column CB3 into a clean centrifuge tube, centrifuging at 13400 Xg/12000 rpm for 2min, removing the filtrate, standing for 3 min at room temperature, and drying in the air.

9. Adding 100ul ddH₂O, incubating at room temperature for 2-5min,13400 Xg/12000 rpm, centrifuging for 2min, collecting eluted products, and storing at-20 ℃.

Design and synthesis of primers and probes: 1. specific primers and probes were designed for the rs5219 and rs889299 gene loci (see the published human whole genome sequence in NCBI database) in human genome using Primer Premier 5.0 and Primer express3.0 software, respectively.

Specific primer and probe sequences were prepared as shown in table 1 below:

TABLE 1

Note: the primer names are named by rs numbers corresponding to the genes; f represents an upstream primer, R represents a downstream primer, P1 represents a FAM probe, and P2 represents a VIC probe.

The PCR reaction solution was prepared as follows:

the first group of primer probes with gene locus rs5219 are added, the primer sequences are SEQ ID NO.1 and SEQ ID NO.2, and the probe sequences are SEQ ID NO.3 and SEQ ID NO. 4;

the second group adds the primer probe with gene locus rs889299, the primer sequence is SEQ ID NO.5 and SEQ ID NO.6, the probe sequence is SEQ ID NO.7 and SEQ ID NO. 8;

each PCR reaction solution also includes 2 XNuHi SNP Mix (Taq enzyme, dNTPs, MgCl)₂PCR buffer, ROX reference fluorescence) and ultrapure water;

the final concentration of the PCR reaction solution components is as follows: 1 XNuHi SNP Mix, 0.5. mu.M of each specific primer and 0.2. mu.M of probe.

The PCR reaction system is shown in the following table 2:

TABLE 2

PCR amplification procedure, as shown in table 3 below:

TABLE 3

An ABI SteponePlus fluorescence quantitative PCR instrument (Applied Biosystems, Applied Biotechnology, USA) was used.

Fourthly, experimental results:

after the PCR reaction was completed, analysis was performed using Stepone software V2.3(Applied Biosystems, Applied Biotechnology Co., Ltd., USA), and 100 cases of the results were classified, and 6 genotypes of 2 SNP sites were collectively distinguished according to the probe of the present invention.

The amplification curve graphs of the three genotypes of rs5219 are shown in figures 1-3, and the PCR fluorescence analysis graph is shown in figure 4; the amplification curve diagrams of the three genotypes of rs889299 are shown in figures 5-7, and the PCR fluorescence analysis diagram is shown in figure 8.

The application of the detection result of the kit in guiding the medication of the glibenclamide medicament is as follows:

and determining the genotypes of the rs5219 locus and the rs889299 locus according to the result in the fluorescent quantitative PCR. The relationship between genotype and glibenclamide drug individualized administration is shown in the following table 4:

TABLE 4

Comparative example 1:

and (3) changing the sequence information of the primer probe combination to detect the genotyping of the human anti-coagulation tissue sample.

100 samples of the anticoagulated blood of the population were taken as samples of the PCR template, and extracted using a commercial DNA extraction kit. The specific extraction procedure was the same as that of human DNA in example 1.

Design and synthesis of primers and probes:

1. two sets of Primer probes were designed for the rs5219 and rs889299 gene sites in the human genome (see the sequence of the human whole genome published in NCBI database), respectively, using Primer Premier 5.0 and Primer Express3.0 software.

The specific primer probe sequences and the comparative primer probe sequences of the present invention are shown in table 5 below:

TABLE 5

The PCR reaction solution was prepared as follows:

the first group is primer probe combination rs 5219-1: adding primer sequences of SEQ ID NO.1 and SEQ ID NO.2, and probe sequences of SEQ ID NO.3 and SEQ ID NO. 4;

the second group is a primer probe combination rs 5219-2: adding primer sequences of SEQ ID NO.9 and SEQ ID NO.10, and probe sequences of SEQ ID NO.11 and SEQ ID NO. 12;

the third group is primer probe combination rs 889299-1: adding primer sequences of SEQ ID NO.5 and SEQ ID NO.6, and probe sequences of SEQ ID NO.7 and SEQ ID NO. 8;

the fourth group is primer probe combination rs 889299-2: adding primer sequences of SEQ ID NO.13 and SEQ ID NO.14, and probe sequences of SEQ ID NO.15 and SEQ ID NO. 16;

The procedure of the PCR reaction system is shown in Table 2 in example 1.

The procedure of the PCR amplification procedure is shown in Table 3 in example 1. An ABI SteponePlus fluorescence quantitative PCR instrument (Applied Biosystems, Applied Biotechnology, USA) was used.

The experimental results are as follows:

after the PCR reaction was completed, analysis was performed using Stepone software V2.3(Applied Biosystems, Applied Biotechnology Co., Ltd., USA) and the analysis results were as follows:

the amplification curve diagrams of the three genotypes of the primer probe combination rs5219-1 are shown in figures 1-3, the PCR fluorescence analysis diagram is shown in figure 4, and the amplification curve diagrams and the PCR fluorescence analysis diagram are well distinguished;

the amplification curve graphs of the three genotypes of the primer probe combination rs5219-2 are shown in figures 9-11, the PCR fluorescence analysis graph is shown in figure 12, and the amplification curve graphs and the PCR fluorescence analysis graph have poor distinguishing effect;

the amplification curve graphs of the three genotypes of the primer probe combination rs889299-1 are shown in figures 5-7, the PCR fluorescence analysis graph is shown in figure 8, and the amplification curve graph and the PCR fluorescence analysis graph are well distinguished;

the amplification curve diagrams of the three genotypes of the primer probe combination rs889299-2 are shown in figures 13-15, the PCR fluorescence analysis diagram is shown in figure 16, and the amplification curve diagrams and the PCR fluorescence analysis diagram have poor distinguishing effects.

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Claims

1. A specific primer probe combination for guiding genes SNP related to the personalized medication of glibenclamide medicines is characterized by comprising specific primer probe combinations aiming at rs5219 and rs889299 gene SNP, and the specific primer probe combinations are as follows:

the rs5219 primer probe sequence is:

rs5219-F：5'-CTGCTCCCGGATGTTCTTGT-3'；

rs5219-R：5'-CCGAGGAATACGTGCTGACA-3'；

rs5219-P1：5'FAM-TACCTGGGCTTGGC-MGB 3'；

rs5219-P2：5'VIC-TACCTGGGCTCGGCA-MGB 3'；

the probe sequence of the rs889299 primer is as follows:

rs889299-F：5'-CGAGGTGTCAGGGTCTTGGT-3'；

rs889299-R：5'-GTCCAGTCCCTTCCCTTGAGA-3'；

rs889299-P1：5'FAM-TGCCTCATTTGTGTGATTGCTCTTTTATTG-TAMRA 3'；

rs889299-P2：5'VIC-CTGCCTCATTTGTGTGATTGTTCTTTTATTG-TAMRA 3'。

2. the fluorescent quantitative PCR kit for guiding the SNP of the genes related to the personalized medicine application of the glyburide according to claim 1, which comprises the primer probe combination, wherein the sequence of the primer probe combination is shown as SEQ ID No. 1-8.

3. The fluorescent quantitative PCR kit for guiding the SNP of genes related to the drug individualization of glyburide according to claim 1, further comprising a PCR reaction solution including the specific primer probe combination, wherein the PCR reaction solution is rs5219 or rs889299, the PCR reaction solution further comprises 2 XNuHi SNP Mix and ultrapure water, and the NuHi SNP Mix comprises Taq enzyme, dNTPs, MgCl and MgCl₂PCRbuffer or ROX reference fluorescence.

4. The fluorescent quantitative PCR kit for guiding the SNP (single nucleotide polymorphism) of the genes related to the drug personalized medication of the glyburide according to claim 1, wherein the final concentrations of the components of the PCR reaction solution are as follows: 1 XNuHi SNP Mix, 0.5. mu.M of each specific primer and 0.2. mu.M of probe.

5. The fluorescent quantitative PCR kit for guiding the SNP of the genes related to the personalized medicine application of the glyburide according to claim 1, wherein the kit further comprises a positive control I, a positive control II or a positive control III;

6. The use of the kit according to any one of claims 2 to 5, wherein the fluorescent quantitative PCR kit is used for carrying out fluorescent quantitative PCR reaction on human genome DNA, and distinguishing polymorphism of corresponding SNP sites according to a fluorescent signal generated in the reaction process for guiding the personalized medication of the glyburide medicament.