CN112980942A

CN112980942A - Sildenafil signal pathway gene detection kit and detection method and application thereof

Info

Publication number: CN112980942A
Application number: CN202110275827.3A
Authority: CN
Inventors: 李铁鹰
Original assignee: Hunan Puang Biotechnology Co ltd
Current assignee: Hunan Puang Biotechnology Co ltd
Priority date: 2021-03-15
Filing date: 2021-03-15
Publication date: 2021-06-18

Abstract

The invention discloses a sildenafil signal path gene detection kit, a detection method and application thereof, wherein the detection kit designs specific amplification primers for polymorphisms of three genes, namely GNB3, NOS3 and NOS1, and is used for fluorescent quantitative PCR detection, and the kit comprises the following components: GNB3 reaction solution, NOS3 reaction solution, NOS1 reaction solution, positive control, blank control and enzyme. The invention adopts a fluorescent quantitative PCR method to detect sildenafil signal path genes, the kit can simultaneously detect 3 core sites, and can more directly determine whether the level of the sample source for the NO signal path enhancing capability is normal or high by detecting the genotypes of the core sites GNB3(C825T), NOS3(T-786C) and NOS1(C276T), thereby providing a gene angle suggestion for clinical personalized medication.

Description

Sildenafil signal pathway gene detection kit and detection method and application thereof

Technical Field

The invention relates to a sildenafil signal pathway gene detection kit and a detection method and application thereof, belonging to the field of gene detection.

Background

Erectile Dysfunction (ED) is clinically defined as the inability to achieve and maintain sufficient erections to allow satisfactory intercourse, with organic, psychological and mixed etiologies. While ED is a non-threatening obstacle, it can affect physical and psychosocial health, having a significant impact on the quality of life of patients and their partners. The risk of ED has been shown to be associated with co-morbidities such as diabetes, obesity, cardiovascular disease, hypertension and/or dyslipidemia, and may also be due to nerve damage following radical prostatectomy.

Nitric Oxide (NO) is a key mediator involved in penile erection, and this molecule is produced by l-arginine through various NO Synthases (NOs), including neurons (nNOS), endothelium (eNOS), and inducible NOs, which predominate over other mediators in the erectile-inducing action. Peripherally acting oral drugs such as sildenafil, which is a PDE5 inhibitor, a specific phosphodiesterase inhibitor that increases intracellular cyclic guanosine monophosphate levels, enhances NO signaling pathways, and has been used to treat ED, are currently the most widely used treatment. Gene polymorphisms encoding eNOS and nNOS affect the response to PDE-5 inhibitors.

Studies have shown that multiple SNP sites are associated with differences in sildenafil response. Polymorphism of neural nitric oxide synthase (NOS1/nNOS) and rs2682826(C276T) locus of NOS1 gene are related to the curative effect of sildenafil. In ED Patients (PED) after prostatectomy, CT-type and TT-type are associated with better response; in clinical ED patients (CED), type TT is associated with a better response. Endothelial nitric oxide synthase (NOS3/eNOS), whose primary function is to participate in arginine and proline metabolism and to catalyze the production of Nitric Oxide (NO). The eNOS gene-786C > T site polymorphism affects the transcriptional activity of endothelial nitric oxide synthase, thereby allowing individuals to respond differently to sildenafil. A study on 118 ED patients found that the C allele was associated with a better response of postoperative ED patients to sildenafil. GNB3 is a G protein β 3 subunit that mediates communication between cell membranes and effector proteins. The 825C > T polymorphism results in deletion of nucleotide 498-620 in exon 9, representing a truncated but functional splice variant GNB3, which allows for enhanced signal transduction and easier drug action when sildenafil is used. A study of 113 patients with erectile dysfunction treated with sildenafil (25-100mg) showed that the erectile response rate was significantly correlated with the GNB3 gene C825T genotype. A response rate of 90.9% was observed in patients with TT, while response rates were 50.9% and 48.9% in patients with CC and CT, respectively.

With the progress of research, the relationship between the SNP sites and the response difference of drug metabolism is more and more clear and definite, and the relationship can be effectively applied to the aspects of gene screening and personalized medicine. At present, the polymorphism of three genes, namely GNB3(C825T), NOS3(T-786C) and NOS1(C276T), is not detected to judge the metabolic capability of clinical ED patients and ED patients after prostatectomy on sildenafil and other drugs, so that certain guidance reference is provided for personalized medication.

Disclosure of Invention

Aiming at the problems in the prior art, the invention aims to obtain a sildenafil signal pathway gene detection kit based on fluorescent quantitative PCR, and a detection method and application thereof.

In order to realize one of the above objects, the technical scheme of the sildenafil signal pathway gene detection kit adopted by the invention is as follows:

the kit provided by the invention is used for designing specific amplification primers aiming at the polymorphism of three genes, namely GNB3(C825T), NOS3(T-786C) and NOS1(C276T), and is used for fluorescent quantitative PCR detection, and the kit comprises the following components: GNB3 reaction solution, NOS3 reaction solution, NOS1 reaction solution, positive control, blank control and enzyme.

Preferably, a large number of specific primers are designed, as shown in the following table:

preferably, the sequence of the specific primer group of GNB3 is shown in sequence tables SEQ ID NO. 1-SEQ ID NO. 4. The sequence is the optimal selection, and the sequence of the specific primer of GNB3 is also shown in sequence tables SEQ ID NO. 5-SEQ ID NO. 7.

Preferably, the specific primer group sequence of the NOS3 is shown in a sequence table SEQ ID NO. 8-SEQ ID NO. 11. The sequence is the optimal selection, and the specific primer sequence of the NOS1 is also shown in a sequence table SEQ ID NO. 125-SEQ ID NO. 15.

Preferably, the specific primer group sequence of the NOS3 is shown in a sequence table SEQ ID NO. 16-SEQ ID NO. 19. The sequence is the optimal selection, and the specific primer sequence of the NOS3 is also shown in a sequence table SEQ ID NO. 20-SEQ ID NO. 22.

Preferably, the positive control is an XDNF positive control comprising three sites 10, GNB3, NOS1 and NOS3⁴The copies wild type plasmids are mixed in equal proportion and three sites are 10⁵The ratio of the wild type and the mutant plasmid of the copies is mixed and the three sites are 10⁴The copies mutant plasmids were mixed in equal proportions.

Preferably, the enzyme is HS Taq enzyme.

The volume of the reaction solution in the kit is preferably within 500 mu L, the volume of the positive control in the kit is preferably within 100 mu L, and the reagent is taken out in advance according to the reaction number and melted at room temperature to avoid repeated freezing and thawing. Before the test, the components are recommended to be vortexed for 15 seconds, and the components of the kit are centrifuged at low speed for 15 seconds for standby.

Preferably, the reaction number N, N is the number of samples to be tested (N) + the number of quality control products (3) + the blank. And positive control and blank control analysis are simultaneously carried out in each PCR experiment.

The invention also discloses a sildenafil signal channel gene detection method adopting the kit, the detection method detects three SNP sites of GNB3, NOS1 and NOS3 in a human whole blood sample through fluorescent quantitative PCR, and sample DNA, positive control and blank control are respectively added into a PCR reaction tube according to the sample loading of 1.5 mu L to be detected during detection.

The preferred components of the reaction solution are as follows:

GNB3(C825T) reaction solution: GNB3 pre-primer (0.25uM), GNB3 post-primer (0.25uM), GNB3(C825T) C allele probe GNB3-CP (0.15uM), GNB3(C825T) T allele probe GNB3-TP (0.3uM), PCR Buffer (1.5X), dNTPS (0.3mM), HS-Taq enzyme (1U), BSA (0.05mg/ml), trehalose (0.2%), nuclease-free water (supplement system to 18.5. mu.L).

NOS3(T-786C) reaction solution: NOS3 pre-primer (0.2uM), NOS3 post-primer (0.2uM), NOS3(T-786C) C allele probe NOS3-CP (0.2uM), NOS3(T-786C) T allele probe NOS3-TP (0.3uM), PCR Buffer (1.5X), dNTPS (0.3mM), HS-Taq enzyme (1U), BSA (0.05mg/ml), trehalose (0.2%), nuclease-free water (system was supplemented to 18.5. mu.L).

NOS1(C276T) reaction solution: NOS1 pre-primer (0.2uM), NOS1 post-primer (0.2uM), NOS1(C276T) C allele probe NOS1-CP (0.15uM), NOS1(C276T) T allele probe NOS1-TP (0.25uM), PCR Buffer (1.25X), dNTPS (0.3mM), HS-Taq enzyme (1U), BSA (0.05mg/ml), trehalose (0.2%), nuclease-free water (system was supplemented to 18.5. mu.L).

The amplification conditions were 95 ℃ for 5min, 95 ℃ for 15sec, 60 ℃ for 30sec, and 40cycles were amplified. The fluorescence intensity was thresholded at the inflection point where the FAM amplification curve rises, and the VIC threshold was the same as the FAM.

Preferably, the invention uses human whole blood sample for detection, the whole blood sample is EDTA anticoagulated whole blood, and uses DNA extraction kit to extract whole blood genome DNA. The concentration of the extracted genome DNA should be more than 10 ng/mu L, the purity is detected by an ultraviolet spectrophotometer, the ratio of A260/A280 should be between 1.7 and 2.0, and the ratio of A260/A230 should be between 1.9 and 2.5.

The present invention summarizes the level of enhanced competence of the sildenafil signaling pathway by analyzing genotypic combinations of genetic tests.

The invention also discloses application of the sildenafil signal path gene detection kit, and the detection kit performs fluorescent quantitative PCR detection on three core genes of the sildenafil signal path, namely GNB3(C825T), NOS3(T-786C) and NOS1(C276T), so as to obtain the enhanced capability level of the sildenafil NO signal path.

Compared with the prior art, the invention adopts a fluorescent quantitative PCR method to detect sildenafil signal pathway genes, the kit can simultaneously detect 3 core sites, and can relatively directly determine that the level of the sample source for the NO signal pathway enhancement capability is normal or high by detecting the genotypes of the core sites GNB3(C825T), NOS3(T-786C) and NOS1(C276T), thereby providing a gene angle suggestion for clinical personalized medication.

Drawings

FIG. 1 is a graph showing an example of the result of the quantitative fluorescence detection of GNB3(C825T) provided by the present invention;

FIG. 2 is a diagram showing an example of the fluorescent quantitative detection result of NOS1(C276T) provided by the present invention;

FIG. 3 is a diagram showing an example of the fluorescent quantitative detection result of NOS3(T-786C) according to the present invention;

FIG. 4 is an exemplary graph of the fluorescence quantitative detection result of the primer combination 1 of GNB3 provided by the present invention;

FIG. 5 is an exemplary graph of the fluorescent quantitative detection result of primer combination 2 of GNB3 provided by the present invention;

FIG. 6 is an exemplary graph of the fluorescent quantitative detection result of the primer combination 3 of GNB3 provided by the present invention;

FIG. 7 is an exemplary graph of the fluorescent quantitative detection result of the primer combination 4 of GNB3 provided by the present invention;

FIG. 8 is an exemplary view of the fluorescent quantitative detection result of primer combination 1 NOS1 according to the present invention;

FIG. 9 is an exemplary illustration of the fluorescent quantitative detection result of primer combination 2 NOS1 according to the present invention;

FIG. 10 is an exemplary illustration of the fluorescent quantitative detection result of primer combination 3 NOS1 according to the present invention;

FIG. 11 is an exemplary illustration of fluorescent quantitative determination results of primer combination 4 NOS1 according to the present invention;

FIG. 12 is an exemplary view of the fluorescent quantitative determination result of primer combination 1 NOS3 according to the present invention;

FIG. 13 is an exemplary view of the fluorescent quantitative determination result of primer combination 2 NOS3 according to the present invention;

FIG. 14 is an exemplary illustration of the fluorescent quantitative detection result of primer combination 3 NOS3 according to the present invention;

FIG. 15 is an exemplary view of fluorescent quantitative detection results of primer combination 4 NOS3 provided by the present invention.

Detailed Description

The sildenafil signaling pathway gene detection kit provided by the present invention, the detection method thereof and the use thereof will be described in detail and in full below with reference to examples. The following examples are illustrative only and are not to be construed as limiting the invention.

The experimental procedures in the following examples are conventional unless otherwise specified. The experimental materials used in the following examples were all commercially available unless otherwise specified.

The kit of the invention designs a large number of specific amplification primers aiming at the polymorphism of three genes, namely GNB3(C825T), NOS3(T-786C) and NOS1(C276T), and is used for fluorescent quantitative PCR detection. Gene polymorphism sequences are subject to published sequences in Genebank.

The kit of the invention optimizes the combination of the primer probes and selects the optimal primer probe combination

The optimized primer combination is as follows:

the optimal primer probe combination for GNB3(C825T) gene detection is as follows: GNB3(C825T) forward primer GNB3-QF1 shown in SEQ ID NO.2, GNB3(C825T) reverse primer GNB3-QR1 shown in SEQ ID NO.5, GNB3(C825T) C allele probe GNB3-CP shown in SEQ ID NO.6 and GNB3(C825T) T allele probe GNB3-TP shown in SEQ ID NO. 7;

the optimal primer probe combination for detecting the NOS1(C276T) gene is as follows: NOS1(C276T) forward primer NOS1-QF1 shown in SEQ ID NO.9, NOS1(C276T) reverse primer NOS1-QR shown in SEQ ID NO.11, NOS1(C276T) C allele probe NOS1-CP shown in SEQ ID NO.14 and NOS1(C276T) T allele probe NOS1-TP shown in SEQ ID NO. 15;

the optimal primer probe combination for detecting the NOS3(T-786C) gene is as follows: NOS3(T-786C) forward primer NOS3-QF2 as shown in SEQ ID NO.9, NOS3(T-786C) reverse primer NOS3-QR1 as shown in SEQ ID NO.11, NOS3(T-786C) C allele probe NOS3-CP as shown in SEQ ID NO.14 and NOS3(T-786C) T allele probe NOS3-TP as shown in SEQ ID NO. 15;

the detection kit of the embodiment comprises the following components:

the invention has no special restriction on the plasmid, and the conventional plasmid is selected to carry out the construction method of the gene recombinant plasmid. For example, the plasmid is preferably a T-vector which is ligated with the wild-type and mutant gene fragments of the above five genes, respectively, and then transformed into the complete Escherichia coli JM 109. The three positive controls correspond to three types of detected gene loci, provide reference for type determination of unknown samples, and simultaneously carry out quality control on the effectiveness of the reaction solution.

The single-person preparation system of 3 reaction solutions of the detection kit of the embodiment is as follows:

the above primer probes were purchased from Biotech, dNTP (2.5mM) from Novozam, 10 XPCR buffer, HS Taq (5U/. mu.L) from TAKARA.

The detection kit of the embodiment comprises the following detection methods:

first, sample preparation

The kit is used for detecting a human whole blood sample, EDTA (ethylene diamine tetraacetic acid) anticoagulated whole blood is adopted as the sample, a DNA extraction kit is used for extracting whole blood genome DNA, the DNA extraction kit is extracted by adopting a conventional human peripheral blood genome DNA extraction kit on the market, the specific operation is carried out according to the kit specification, the concentration of the extracted genome DNA is more than 10 ng/mu L, an ultraviolet spectrophotometer is used for detecting the purity, the ratio of A260/A280 is between 1.7 and 2.0, and the ratio of A260/A230 is between 1.9 and 2.5. If the quality or concentration of the DNA does not meet the requirements, the sample is drawn again or the sample size is enlarged and the extraction is carried out again.

Second, reagent preparation

The reagents were removed 30 minutes in advance, thawed at room temperature, and the components vortexed for 15 seconds, and the kit components were centrifuged at low speed for 15 seconds until use.

And determining the reaction number N, wherein N is the number of samples to be detected (N), the number of quality control products (3) and a blank control. It is recommended that positive control and blank control analyses be performed simultaneously for each PCR experiment. The amounts of each reagent added to the reaction mixture were calculated, as shown in the table below,

composition (I)	Reaction volume (μ L)	The using amount of each component
			PCR reaction solution (1 to 3)	18.3μL	18.3×N
Enzyme mixture	0.2μL	0.2×N

3 sterilized centrifuge tubes are taken to be configured with the 3 reaction systems, after all the reagents are added, vortex oscillation is carried out for 15 seconds, and low-speed centrifugation is carried out for 15 seconds. Then, the prepared reaction solution was dispensed into PCR reaction tubes at 18.5. mu.L/tube.

Third, sample adding detection

Adding the sample DNA, the positive control and the blank control into a PCR reaction tube according to the sample adding amount of 1.5 mu L, covering the tube cover tightly, centrifuging at low speed for 15 seconds to completely throw liquid on the tube wall to the tube bottom, and immediately carrying out PCR amplification reaction.

The kit adopts an ABI 7500 type fluorescent quantitative PCR instrument to carry out fluorescent quantitative detection, the PCR reaction system is 20 mu L, and the amplification conditions are as follows:

fourthly, interpretation of results

4.1 validity judgment:

setting a threshold value: different models are divided differently according to the integral fluorescence intensity threshold, the threshold can be defined according to the rising inflection point of the FAM amplification curve, and the VIC threshold is the same as the FAM.

The detection rate of the blank reference substance of the kit is 0(Ct value is more than or equal to 38 or no Ct value), and the detection results of the positive reference substances 1, 2 and 3 respectively accord with homozygous wild, heterozygous mutation and homozygous mutation.

4.2 criteria for determination of results

Fifth, the performance of the kit is detected

5.1. Specificity of

The result of the detection of 16 specific samples (including non-human DNA templates and dilutions of amplification products of different sites or homologous sites of the same human gene) is negative.

5.2. Accuracy of

The detection of 12 reference samples (including GNB3(C825T) × 3, NOS3(T-786C) and NOS1 (C276T)) of different genotypes in the kit range should detect the corresponding genotypes.

5.3. Minimum detection limit

The minimum detection limit should not be higher than 10 ng/ul.

5.4. Repeatability of

In the detection kit, each reference substance is subjected to 10 times of detection, the results are corresponding mutation types, and the Coefficient of Variation (CV) of the Ct value of a corresponding detection channel is less than or equal to 5.0%.

5.5. Correlation of gene detection results with sildenafil metabolism

In the table, "+" indicates that the patient and patient had taken sildenafil normally; "+ + +" indicates that the patient is taking sildenafil with enhanced response. However, the response results in the table are only for reference, and the actual drug selection should be combined with the clinic.

Finally, it must be said here that: the above embodiments are only used for further detailed description of the technical solutions of the present invention, and should not be understood as limiting the scope of the present invention, and the insubstantial modifications and adaptations made by those skilled in the art according to the above descriptions of the present invention are within the scope of the present invention.

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Claims

1. The sildenafil signal path gene detection kit is characterized in that the detection kit designs specific amplification primers for polymorphism of three genes, namely GNB3, NOS3 and NOS1, and is used for fluorescent quantitative PCR detection, and the kit comprises the following components: GNB3 reaction solution, NOS3 reaction solution, NOS1 reaction solution, positive control, blank control and enzyme.

2. The sildenafil signal pathway gene detection kit as claimed in claim 1, wherein the positive control is an XDNF positive control comprising three sites 10 of GNB3, NOS1 and NOS3⁴The copies wild type plasmids are mixed in equal proportion and three sites are 10⁵The ratio of the wild type and the mutant plasmid of the copies is mixed and the three sites are 10⁴The copies mutant plasmids were mixed in equal proportions.

3. The sildenafil signal pathway gene detection kit according to claim 1, wherein the sequence of the specific primer group of GNB3 is shown in sequence tables SEQ ID NO. 1-SEQ ID NO. 4.

4. The sildenafil signal pathway gene detection kit as claimed in claim 1, wherein the sequence of the specific primer set of NOS1 is shown in SEQ ID NO 8-SEQ ID NO 11 of the sequence Listing.

5. The sildenafil signal pathway gene detection kit as claimed in claim 1, wherein the sequence of the specific primer set of NOS3 is shown in SEQ ID NO. 16-SEQ ID NO. 19 of the sequence Listing.

6. A gene detection method of the sildenafil signal path gene detection kit according to any one of claims 1 to 5, characterized in that the gene detection method adopts fluorescent quantitative PCR to detect three SNP sites of GNB3, NOS1 and NOS3 in a human whole blood sample, and the amplification system is a 20 μ L system.

7. The sildenafil signal pathway gene detection method according to claim 6, wherein the amplification system comprises the following components:

GNB3(C825T) reaction solution: GNB3 pre-primer (0.25uM), GNB3 post-primer (0.25uM), GNB3(C825T) C allele probe GNB3-CP (0.15uM), GNB3(C825T) T allele probe GNB3-TP (0.3uM), PCR Buffer (1.5X), dNTPS (0.3mM), HS-Taq enzyme (1U), BSA (0.05mg/ml), trehalose (0.2%), nuclease-free water (system was supplemented to 18.5. mu.L);

NOS3(T-786C) reaction solution: NOS3 pre-primer (0.2uM), NOS3 post-primer (0.2uM), NOS3(T-786C) C allele probe NOS3-CP (0.2uM), NOS3(T-786C) T allele probe NOS3-TP (0.3uM), PCR Buffer (1.5X), dNTPS (0.3mM), HS-Taq enzyme (1U), BSA (0.05mg/ml), trehalose (0.2%), nuclease-free water (system was supplemented to 18.5. mu.L);

8. The sildenafil signaling pathway gene detection method as set forth in claim 6, wherein the amplification conditions are 95 ℃ for 5min, 95 ℃ for 15sec, 60 ℃ for 30sec, and 40cycles of amplification.

9. The sildenafil signal pathway gene detection method as recited in claim 6, wherein the human whole blood sample is subjected to whole blood genomic DNA extraction by using a DNA extraction kit, the concentration of the extracted genomic DNA is greater than 10ng/μ L, the A260/A280 ratio is between 1.7 and 2.0, and the A260/A230 ratio is between 1.9 and 2.5.

10. The use of the sildenafil signal pathway gene detection kit as claimed in any one of claims 1 to 9, wherein the detection kit performs fluorescence quantitative PCR detection on three core genes of the sildenafil signal pathway, namely GNB3(C825T), NOS3(T-786C) and NOS1(C276T), so as to obtain the enhanced capability level of the sildenafil NO signal pathway.