Primer probe composition and kit for direct spread detection of hypertension drug gene polymorphism and application method

Abstract

The application provides a primer probe composition and kit for direct spread detection of hypertension drug gene polymorphism and an application method, and belongs to the field of molecular biology and in-vitro diagnosis technology. The application is directed to five hypertension clinical core drug gene sites of ACE-rs1799752, AGTR1-rs5186, ADRB1-rs1801253, CYP2D6*10-rs1065852 and CYP2C9*3-rs1057910, and a specific primer and a fluorescent probe are combined; combined with whole blood direct spread PCR and fluorescent probe melting curve technology, five-site gene polymorphism detection is realized in a single reaction tube. The scheme does not need to extract and purify the nucleic acid of the blood sample, is closed tube operation throughout, effectively avoids aerosol cross contamination, has the advantages of simple operation, rapid detection, low cost, accurate typing, and can be adapted to a conventional fluorescent quantitative PCR instrument, and provides a reliable molecular diagnosis basis for clinical hypertension precise medication.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and in particular to a primer-probe composition, kit, and application method for direct amplification detection of gene polymorphisms in hypertension medications. Background Technology

[0002] Hypertension is one of the most prevalent chronic diseases worldwide and a core high-risk factor for serious cardiovascular and cerebrovascular complications such as stroke, myocardial infarction, and renal failure. Clinical treatment primarily involves controlling blood pressure with antihypertensive drugs, such as beta-blockers, angiotensin-converting enzyme inhibitors, angiotensin II receptor antagonists, and drug-metabolizing enzymes. However, significant individual differences exist among patients regarding the antihypertensive efficacy, metabolic rate, and adverse reactions of the same drug. Extensive clinical pharmacogenomics studies have revealed that differences in the efficacy and safety of antihypertensive drugs are mainly influenced by pharmacogenomic polymorphisms in patients, such as the polymorphic distribution of key gene loci like ACE, AGTR1, ADRB1, and CYP2D6*10. These polymorphisms directly affect the target response sensitivity and metabolic clearance efficiency of relevant antihypertensive drugs in the patient's body.

[0003] Therefore, conducting relevant pharmacogenomic polymorphism (pMP) testing before initiating hypertension drug treatment helps to provide precise medication for different patients, thereby improving drug efficacy and reducing drug toxicity and side effects. Currently, the detection of pMPs for hypertension mainly relies on gene sequencing technology and traditional quantitative PCR (qPCR) technology. The testing process usually requires collecting peripheral anticoagulated whole blood samples from patients, and then using nucleic acid extraction kits to extract and purify genomic DNA from the blood samples through cumbersome steps such as cell lysis, nucleic acid binding, rinsing, and elution to obtain nucleic acid templates that meet the requirements of subsequent amplification reactions. During gene sequencing, fragments containing core target sites need to be amplified by PCR and then sequenced and compared. Traditional qPCR technology usually requires designing specific primers and double-labeled hydrolysis probes for each polymorphic site, and real-time amplification monitoring is performed in multiple reaction tubes or using a single tube with multicolor fluorescence channels. The patient's genotype is determined based on the Ct value or the characteristics of the amplification curve.

[0004] However, the aforementioned methods all heavily rely on cumbersome sample pretreatment processes. Nucleic acid extraction and purification are not only time-consuming and highly manual, but also significantly limit their immediacy in clinical use. Furthermore, there is a significant trade-off between the throughput and reaction cost of these technologies. Commonly used qPCR kits typically employ single-tube single-site or combined detection of a few sites. To maximize coverage of hypertension drug-related genes, multiple reaction tubes need to be used independently, which not only reduces detection efficiency but also increases reagent consumption and testing costs. In addition, traditional detection procedures often involve numerous capping operations during amplification product analysis or multi-tube sample loading. Frequent capping can easily generate high concentrations of amplification product aerosols in the laboratory, leading to severe cross-contamination between samples and directly jeopardizing the accuracy of the test results. Therefore, a solution is urgently needed to address these issues. Summary of the Invention

[0005] The purpose of this invention is to provide a primer and probe composition, kit, and application method for direct amplification detection of polymorphisms in hypertension drug genes. This invention uses five sets of primer and probe compositions for simultaneous amplification in the same reaction tube, which not only has high binding specificity for each but also avoids mutual interference. It can complete the simultaneous detection of polymorphisms at five target sites in a single reaction tube, effectively reducing detection costs and improving detection convenience.

[0006] In a first aspect, the present invention provides a primer and probe composition for direct amplification detection of gene polymorphisms related to hypertension medications, comprising primers and probes for detecting the ACE-rs1799752 gene locus, AGTR1-rs5186 gene locus, ADRB1-rs1801253 gene locus, CYP2D6*10-rs1065852 gene locus, and CYP2C9*3-rs1057910 gene locus, respectively; the difference between each pair of melting temperatures (Tm) of the detection probes corresponding to each gene locus is ≥6℃.

[0007] Optionally, the forward primer sequence for the primer probe used to detect the ACE-rs1799752 gene locus is shown in SEQ ID NO.1, the reverse primer sequence is shown in SEQ ID NO.2, and the detection probe sequence is shown in SEQ ID NO.3.

[0008] Optionally, the forward primer sequence for the primer probe used to detect the AGTR1-rs5186 gene locus is shown in SEQ ID NO.4, the reverse primer sequence is shown in SEQ ID NO.5, and the detection probe sequence is shown in SEQ ID NO.6.

[0009] Optionally, the forward primer sequence for the primer probe used to detect the ADRB1-rs1801253 gene locus is shown in SEQ ID NO.7, the reverse primer sequence is shown in SEQ ID NO.8, and the detection probe sequence is shown in SEQ ID NO.9.

[0010] Optionally, the forward primer sequence for the primer probe used to detect the CYP2D6*10-rs1065852 gene locus is shown in SEQ ID NO.10, the reverse primer sequence is shown in SEQ ID NO.11, and the detection probe sequence is shown in SEQ ID NO.12.

[0011] Optionally, the forward primer sequence for the primer probe used to detect the CYP2C9*3-rs1057910 gene locus is shown in SEQ ID NO.13, the reverse primer sequence is shown in SEQ ID NO.14, and the detection probe sequence is shown in SEQ ID NO.15.

[0012] Secondly, the present invention also provides a kit for direct amplification detection of gene polymorphisms in hypertension medications, comprising a primer-probe mixture having the primer-probe composition as described in claim 1 or 2 and a reaction buffer, wherein the reaction buffer comprises a basal buffer and a dissolved whole blood endogenous inhibitory component thereof, wherein the whole blood endogenous inhibitory component comprises a heme inhibitor, spermidine, trehalose and an active agent.

[0013] Optionally, in a 20 μL reaction system, the final concentration of trehalose is 0.2 mol / L-0.6 mol / L, the final concentration of the activator is 0.5 wt%-1.5 wt%, the final concentration of the heme inhibitor is 10 μg / mL-40 μg / mL, and the final concentration of spermidine is 0.5 mmol / L-2.0 mmol / L.

[0014] Optionally, the reaction buffer comprises Taq DNA polymerase, dNTPs, Tris-HCl buffer, KCl, MgCl2, and a stabilizer; wherein: in a 20 μL reaction system: the amount of Taq DNA polymerase added is 0.8 U-1.2 U; the amount of dNTPs added is 0.2 mmol / L-0.4 mmol / L; the final concentration of the Tris-HCl buffer is 15 mmol / L-25 mmol / L; the final concentration of KCl is 40 mmol / L-60 mmol / L; the final concentration of MgCl2 is 1.5 mmol / L-2.5 mmol / L; and the final concentration of the stabilizer is 0.05 wt%-0.20 wt%.

[0015] Optionally, the primer-probe mixture contains primers with a final concentration of 100 nmol / L-250 nmol / L and probes with a final concentration of 80 nmol / L-120 nmol / L; it also includes PCR reaction solution, positive control, and blank control. The positive control includes recombinant plasmid standards with wild-type and mutant sequences of five detection sites, and the blank control includes purified water without nucleic acid.

[0016] Thirdly, the present invention also provides a method for applying any of the above-mentioned optional reagent kits in the detection of gene polymorphism in hypertension medication, comprising: directly adding EDTA anticoagulated fresh whole blood samples to a PCR tube containing primer probe mixture and reaction buffer, without nucleic acid extraction and purification steps, mixing well and directly performing PCR thermal cycling amplification, and after the amplification is completed, starting the closed tube melting program to collect fluorescence signals in real time, and analyzing and determining the genotype of the target detection site based on the difference in Tm values ​​of the melting curves of each channel in the reaction system.

[0017] Optionally, after mixing the sample, incubate it at 37℃ for 5 min for UNG enzyme degradation, then pre-denature it at 94℃ for 5 min, and then perform 40 cycles of PCR amplification. The program for each cycle is: 94℃ for 15 seconds, 58℃ for 40 seconds, and 72℃ for 30 seconds. After amplification, incubate it at 94℃ for 2 min, then at 38℃ for 2 min, and then increase the temperature from 38℃ to 75℃. During the temperature increase, continuously collect fluorescence signals from the FAM, HEX, ROX, and CY5 fluorescence channels and generate corresponding melting curves.

[0018] Optionally, when analyzing and determining the genotype of the target detection site based on the differences in Tm values ​​of the melting curves of each channel in the reaction system, the genotyping criteria include: (1) For the CYP2D6*10-rs1065852 site, in the FAM channel: a Tm value of 52.31℃±1.5℃ indicates wild homozygous type, a Tm value of 60.60℃±1.5℃ indicates mutant homozygous type, and the presence of two melting peaks at 52.31℃±1.5℃ and 60.60℃±1.5℃ indicates heterozygous type; (2) For the CYP2C9*3-rs1057910 site, in the HEX channel: a Tm value of 51.62℃±1.5℃ indicates wild homozygous type, a Tm value of 60.07℃±1.5℃ indicates mutant homozygous type, and the presence of two melting peaks at 51.62℃±1.5℃ and 60.07℃±1.5℃ indicates heterozygous type; (3) For the ACE-rs1799752 site, in the ROX channel: a Tm value of 47.89℃±1.5℃ indicates DD homozygous type, a Tm value of 63.68℃±1.5℃ indicates II homozygous type, and the simultaneous appearance of two melting peaks at 47.89℃±1.5℃ and 63.68℃±1.5℃ indicates ID heterozygous type; (4) For the AGTR1-rs5186 site and the ADRB1-rs1801253 site, the two sites share the CY5 channel for independent differentiation and interpretation of dual targets: a Tm value of 41.36℃±1.5℃ indicates AGTR1 wild-type homozygous, a Tm value of 54.58℃±1.5℃ indicates AGTR1 mutant homozygous, and the presence of two melting peaks at 41.36℃±1.5℃ and 54.58℃±1.5℃ indicates AGTR1 heterozygous; a Tm value of 60.93℃±1.5℃ indicates ADRB1 wild-type homozygous, a Tm value of 70.09℃±1.5℃ indicates ADRB1 mutant homozygous, and the presence of two melting peaks at 60.93℃±1.5℃ and 70.09℃±1.5℃ indicates ADRB1 heterozygous.

[0019] The present invention provides a primer and probe composition, kit, and application method for direct amplification detection of gene polymorphisms related to hypertension medications, which has at least one of the following advantages compared to the prior art: 1. By adding heme inhibitors, spermidine, trehalose, and activators to the basic buffer of the reaction buffer, the heme inhibitors can chelate and inactivate heme and endogenous nucleases in whole blood samples during the amplification process, inhibiting the influence of endogenous substances in whole blood on Taq DNA polymerase activity. At the same time, the combination of spermidine and trehalose can improve the stability of polymerase conformation during high-temperature denaturation, thus enabling direct single-tube in situ amplification of EDTA-anticoagulated whole blood, effectively shortening the overall detection cycle and improving the immediacy and convenience of detection. 2. By adjusting the base sequence of each detection probe to ensure that the difference between each pair of melting temperatures (Tm) is ≥6℃, it helps to form independent genotyping spaces that do not overlap and have clear boundaries in the melting curve. In addition, by simultaneously accommodating the polymorphic genotyping interpretation of two independent gene loci, AGTR1 and ADRB1, in a single CY5 fluorescence channel, and avoiding interference from poor signals by utilizing a higher Tm value span, it effectively improves the problem of limited channels in traditional multiplex PCR, helps to reduce reagent consumption and labor costs during detection, and facilitates large-scale drug screening in clinical practice. Attached Figure Description

[0020] Figure 1 This is a typical fluorescent probe melting curve typing diagram of the CYP2D6*10-rs1065852 gene locus in the FAM channel in an embodiment of the present invention; Figure 2 This is a typical fluorescent probe melting curve typing diagram of the CYP2C9*3-rs1057910 gene locus in the HEX channel in an embodiment of the present invention; Figure 3 This is a typical fluorescent probe melting curve typing diagram of the ACE-rs1799752 gene locus in the ROX channel in an embodiment of the present invention; Figure 4 This is a typical fluorescent probe melting curve typing diagram in the embodiment of the present invention, showing the independent differentiation of the AGTR1-rs5186 and ADRB1-rs1801253 gene loci in a single channel under the CY5 channel. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise defined, the technical or scientific terms used herein should have the ordinary meaning understood by those skilled in the art to which this invention pertains.

[0022] This invention provides a primer-probe composition for direct amplification detection of gene polymorphisms related to hypertension medications, comprising primers and probes for detecting the ACE-rs1799752, AGTR1-rs5186, ADRB1-rs1801253, CYP2D6*10-rs1065852, and CYP2C9*3-rs1057910 gene loci, respectively. Furthermore, the difference between each pair of melting temperatures (Tm) of the detection probes corresponding to each gene locus is ≥6℃. In fact, by adjusting the base sequence of each detection probe to ensure that the difference between each pair of melting temperatures (Tm) is ≥6℃, it helps to form independent genotyping spaces that are non-overlapping and have clear boundaries in the melting curve.

[0023] Furthermore, the forward primer sequence for the primer probe used to detect the ACE-rs1799752 gene locus is shown in SEQ ID NO.1, the reverse primer sequence is shown in SEQ ID NO.2, and the detection probe sequence is shown in SEQ ID NO.3.

[0024] Furthermore, the forward primer sequence for the primer probe used to detect the AGTR1-rs5186 gene locus is shown in SEQ ID NO.4, the reverse primer sequence is shown in SEQ ID NO.5, and the detection probe sequence is shown in SEQ ID NO.6.

[0025] Furthermore, the forward primer sequence for the primer probe used to detect the ADRB1-rs1801253 gene locus is shown in SEQ ID NO.7, the reverse primer sequence is shown in SEQ ID NO.8, and the detection probe sequence is shown in SEQ ID NO.9.

[0026] Furthermore, the forward primer sequence for the primer probe used to detect the CYP2D6*10-rs1065852 gene locus is shown in SEQ ID NO.10, the reverse primer sequence is shown in SEQ ID NO.11, and the detection probe sequence is shown in SEQ ID NO.12.

[0027] Furthermore, the forward primer sequence for the primer probe used to detect the CYP2C9*3-rs1057910 gene locus is shown in SEQ ID NO.13, the reverse primer sequence is shown in SEQ ID NO.14, and the detection probe sequence is shown in SEQ ID NO.15.

[0028] Specifically, the sequences of SEQ ID NO.1 to SEQ ID NO.15 are shown in Table 1 below: Table 1 Sequences of SEQ ID NO.1 to SEQ ID NO.15

[0029] In fact, this invention also provides a kit for direct amplification detection of hypertension drug gene polymorphisms, comprising a primer-probe mixture having the primer-probe composition of any of the above embodiments and a reaction buffer. The reaction buffer includes a basal buffer and its dissolved whole blood endogenous inhibitory component. Furthermore, the whole blood endogenous inhibitory component includes a heme inhibitor, spermidine, trehalose, and an active agent. Specifically, the active agent used is polyvinylpyrrolidone (PVP), and commercially available conventional products such as K30 and K60 can be selected.

[0030] Specifically, the preparation method of the primer-probe mixture includes: diluting the five sets of primers and their corresponding fluorescent probes with purified water, and then mixing them according to a final primer concentration of 100 nmol / L-250 nmol / L and a final probe concentration of 80 nmol / L-120 nmol / L to obtain the primer-probe mixture. Specifically, the ACE-rs1799752 probe is ROX fluorescently labeled, the CYP2D6*10-rs1065852 probe is FAM fluorescently labeled, the CYP2C9*3-rs1057910 probe is HEX fluorescently labeled, and the AGTR1-rs5186 and ADRB1-rs1801253 probes share the same CY5 fluorescent label.

[0031] Specifically, the preparation method of the reaction buffer includes dissolving the endogenous inhibitory component of whole blood in a basic buffer solution by stirring. Further, in a 20 μL reaction system, the amounts and final concentrations of each component in the reaction buffer can be as follows: trehalose final concentration of 0.2 mol / L-0.6 mol / L, activator final concentration of 0.5 wt%-1.5 wt%, heme inhibitor final concentration of 10 μg / mL-40 μg / mL, and spermidine final concentration of 0.5 mmol / L-2.0 mmol / L.

[0032] Furthermore, in a 20 μL reaction system, the amounts and final concentrations of each component of the basal buffer can be as follows: Taq DNA polymerase added at 0.8 U-1.2 U; dNTPs added at 0.2 mmol / L-0.4 mmol / L; Tris-HCl buffer at a final concentration of 15 mmol / L-25 mmol / L; KCl at a final concentration of 40 mmol / L-60 mmol / L; MgCl2 at a final concentration of 1.5 mmol / L-2.5 mmol / L; and stabilizer at a final concentration of 0.05 wt%-0.20 wt%. In some embodiments, 0.2 U of UNG enzyme can also be added to the basal buffer. Specifically, the stabilizer used can be gelatin.

[0033] In fact, heme inhibitors and Taq DNA polymerase can be premixed and commercially available products can be used, such as TP-04123 from Chengdu Fanjing Biotechnology Co., Ltd., IM-01011, IM-01012, and IM-01013 from Chengdu Fuji Biotechnology Co., Ltd., and CW2634M and CW2563M from Jiangsu Kangwei Century Biotechnology Co., Ltd. All of these products contain premixed heme inhibitors and Taq DNA polymerase at the time of manufacture.

[0034] In fact, the present invention also provides a method for applying the kit in any of the above embodiments to detect gene polymorphisms in hypertension medication, including: directly adding EDTA-anticoagulated fresh whole blood samples to a PCR tube containing primer-probe mixture and reaction buffer, without nucleic acid extraction and purification steps, mixing well and directly performing PCR thermal cycling amplification, and starting a closed-tube melting program to collect fluorescence signals in real time after amplification, and analyzing and determining the genotype of the target detection site based on the Tm value difference of the melting curves of each channel in the reaction system.

[0035] Furthermore, after mixing the sample and incubating it at 37℃ for 5 min for UNG enzyme degradation, the sample was pre-denatured at 94℃ for 5 min and then subjected to 40 cycles of PCR amplification. The program for each cycle was: 94℃ for 15 seconds, 58℃ for 40 seconds, and 72℃ for 30 seconds. After amplification, the sample was incubated at 94℃ for 2 min and then at 38℃ for 2 min. The temperature was then increased from 38℃ to 75℃. During the temperature increase, the fluorescence signals of the FAM, HEX, ROX, and CY5 fluorescence channels were continuously collected and the corresponding melting curves were generated.

[0036] In fact, when analyzing and determining the genotype of the target detection site based on the differences in Tm values ​​of the melting curves of each channel in the reaction system, the genotyping criteria include: (1) For the CYP2D6*10-rs1065852 site, in the FAM channel: a Tm value of 52.31℃±1.5℃ indicates wild homozygous type, a Tm value of 60.60℃±1.5℃ indicates mutant homozygous type, and the presence of two melting peaks at 52.31℃±1.5℃ and 60.60℃±1.5℃ indicates heterozygous type; (2) For the CYP2C9*3-rs1057910 site, in the HEX channel: a Tm value of 51.62℃±1.5℃ indicates wild homozygous type, a Tm value of 60.07℃±1.5℃ indicates mutant homozygous type, and the presence of two melting peaks at 51.62℃±1.5℃ and 60.07℃±1.5℃ indicates heterozygous type; (3) For the ACE-rs1799752 site, in the ROX channel: a Tm value of 47.89℃±1.5℃ indicates DD homozygous type, a Tm value of 63.68℃±1.5℃ indicates II homozygous type, and the simultaneous appearance of two melting peaks at 47.89℃±1.5℃ and 63.68℃±1.5℃ indicates ID heterozygous type; (4) For the AGTR1-rs5186 site and the ADRB1-rs1801253 site, the two sites share the CY5 channel for independent differentiation and interpretation of dual targets: a Tm value of 41.36℃±1.5℃ indicates AGTR1 wild-type homozygous, a Tm value of 54.58℃±1.5℃ indicates AGTR1 mutant homozygous, and the presence of two melting peaks at 41.36℃±1.5℃ and 54.58℃±1.5℃ indicates AGTR1 heterozygous; a Tm value of 60.93℃±1.5℃ indicates ADRB1 wild-type homozygous, a Tm value of 70.09℃±1.5℃ indicates ADRB1 mutant homozygous, and the presence of two melting peaks at 60.93℃±1.5℃ and 70.09℃±1.5℃ indicates ADRB1 heterozygous.

[0037] Example 1: A reaction buffer, wherein the amounts and final concentrations of each component in a 20 μL reaction system are as follows: 1.0 U hot-start Taq DNA polymerase, 0.3 mmol / L dNTPs (containing dUTP), 20 mmol / L Tris-HCl buffer, 50 mmol / L KCl, 2.0 mmol / L MgCl2, 0.10 wt% stabilizer (gelatin), 0.4 mol / L trehalose, 1.0 wt% activator (PVP, K30), 25 μg / mL heme inhibitor, and 1.2 mmol / L spermidine; wherein the Taq DNA polymerase and heme inhibitor are premixed and purchased from Chengdu Fanjing Biotechnology Co., Ltd., TP-04123.

[0038] Comparative Example 1: A reaction buffer in a 20 μL reaction system, the amounts and final concentrations of each component are as follows: 1.0 U hot-start Taq DNA polymerase, 0.3 mmol / L dNTPs (containing dUTP), 20 mmol / L Tris-HCl buffer, 50 mmol / L KCl, 2.0 mmol / L MgCl2, 0.10 wt% stabilizer (gelatin), 25 μg / mL heme inhibitor, and 1.2 mmol / L spermidine.

[0039] Comparative Example 2: A reaction buffer in a 20 μL reaction system, the amounts and final concentrations of each component were as follows: 1.0 U hot-start Taq DNA polymerase, 0.3 mmol / L dNTPs (containing dUTP), 20 mmol / L Tris-HCl buffer, 50 mmol / L KCl, 2.0 mmol / L MgCl2, 0.10 wt% stabilizer (gelatin), 0.4 mol / L trehalose, and 1.0 wt% activator (PVP).

[0040] The reaction buffers prepared in Example 1 and Comparative Examples 1 to 2 were combined with primer-probe mixtures, PCR reaction solutions, positive controls, and blank controls to form kits. The following tests were performed on each kit: the primer-probe mixture had a final primer concentration of 150 nmol / L and a final probe concentration of 100 nmol / L in a 20 μL reaction system; the positive controls included recombinant plasmid standards with wild-type and mutant sequences of five detection sites; and the blank controls included purified water without nucleic acids.

[0041] 1. Accuracy verification of the reagent kit: EDTA-anticoagulated peripheral whole blood samples were collected from 50 hypertensive patients. Genomic DNA was extracted from the whole blood samples using a nucleic acid extraction kit (QIAamp DNA Blood Mini Kit). The target detection sites (ACE-rs1799752, AGTR1-rs5186, ADRB1-rs1801253, CYP2D6*10-rs1065852, CYP2C9*3-rs1057910) were sequenced using Sanger sequencing. The sequencing results were used as the gold standard for detection. The peripheral whole blood samples described above were subjected to extraction-free direct amplification typing detection using the kits corresponding to Example 1 and Comparative Examples 1 to 2, respectively. The process included: adding 18 μL of detection mixture (containing reaction buffer, primer-probe mixture, and PCR reaction solution) to each well of a 96-well PCR plate; directly adding 2 μL of anticoagulated peripheral whole blood sample to the corresponding well; gently mixing with a pipette; sealing with an optical seal film; transferring to a centrifuge for instantaneous centrifugation; and then transferring to a quantitative real-time PCR device for PCR amplification. Cycling was performed (after incubating at 37℃ for 5 min, the temperature was increased to 94℃ for 5 min of pre-denaturation; 40 PCR thermal cycles were performed, each cycle including denaturation at 94℃ for 15 s, annealing and extension at 58℃ for 40 s, and extension at 72℃ for 30 s); after amplification, continuous heating was started directly: first incubating at 94℃ for 2 min, then incubating at 38℃ for 2 min, and finally increasing the temperature from 38℃ to 75℃ at a rate of 0.04℃ / s, and collecting the fluorescence intensity changes of the four fluorescence channels FAM, HEX, ROX, and CY5 in real time during the heating process; After converting the fluorescence intensity change curve into a negative derivative melting curve, the genotype interpretation results corresponding to the kit are output based on the deviation of the Tm value corresponding to the characteristic melting peak of each fluorescence channel and the following genotyping criteria. The results are shown in Table 2 below.

[0042] Criteria for classification: (1) For the CYP2D6*10-rs1065852 site, in the FAM channel: a Tm value of 52.31℃±1.5℃ indicates wild homozygous type, a Tm value of 60.60℃±1.5℃ indicates mutant homozygous type, and the presence of two melting peaks at 52.31℃±1.5℃ and 60.60℃±1.5℃ indicates heterozygous type; (2) For the CYP2C9*3-rs1057910 site, in the HEX channel: a Tm value of 51.62℃±1.5℃ indicates wild homozygous type, a Tm value of 60.07℃±1.5℃ indicates mutant homozygous type, and the presence of two melting peaks at 51.62℃±1.5℃ and 60.07℃±1.5℃ indicates heterozygous type; (3) For the ACE-rs1799752 site, in the ROX channel: a Tm value of 47.89℃±1.5℃ indicates DD homozygous type, a Tm value of 63.68℃±1.5℃ indicates II homozygous type, and the simultaneous appearance of two melting peaks at 47.89℃±1.5℃ and 63.68℃±1.5℃ indicates ID heterozygous type; (4) For the AGTR1-rs5186 site and the ADRB1-rs1801253 site, the two sites share the CY5 channel for independent differentiation and interpretation of dual targets: a Tm value of 41.36℃±1.5℃ indicates AGTR1 wild-type homozygous, a Tm value of 54.58℃±1.5℃ indicates AGTR1 mutant homozygous, and the presence of two melting peaks at 41.36℃±1.5℃ and 54.58℃±1.5℃ indicates AGTR1 heterozygous; a Tm value of 60.93℃±1.5℃ indicates ADRB1 wild-type homozygous, a Tm value of 70.09℃±1.5℃ indicates ADRB1 mutant homozygous, and the presence of two melting peaks at 60.93℃±1.5℃ and 70.09℃±1.5℃ indicates ADRB1 heterozygous.

[0043] Table 2. Accuracy verification of the reagent kits corresponding to Example 1, Comparative Examples 1 to 2.

[0044] 2. Sensitivity verification of the reagent kit: Fresh, healthy human negative whole blood (pre-confirmed by Sanger sequencing to be free of target mutant sequences) was used as the dilution background. Recombinant plasmid standards containing wild-type and mutant target sequences of five detection sites (ACE, AGTR1, ADRB1, CYP2D6*10, and CYP2C9*3) were serially diluted using the dilution background to obtain gradient dilutions with concentrations of 5000 copies / μL, 1000 copies / μL, 500 copies / μL, 100 copies / μL, and 50 copies / μL, respectively. The kits corresponding to Example 1 and Comparative Example 1 were used to detect the gradient dilutions according to the direct amplification typing detection method described above. Each sample was tested in parallel 20 times, and the detection rate of samples after different gradient dilutions was calculated. The results are shown in Table 3 below.

[0045] Table 3. Sensitivity verification of the reagent kits corresponding to Example 1 and Comparative Example 1

[0046] 3. Reproducibility verification of the reagent kit: Three representative clinical human whole blood samples were obtained through pre-screening using Sanger sequencing: Sample A, with all target loci being wild-type homozygous; Sample B, with all target loci being mutant homozygous; and Sample C, with all target loci being heterozygous. The corresponding kits from Example 1 and Comparative Example 1 (each using three batches, namely batch A, batch B, and batch C) were used to detect the samples according to the direct amplification typing method described above. Each sample was tested in parallel 20 times, and the coefficient of variation of the melting peak Tm value was calculated. The results are shown in Table 4 below.

[0047] Table 4. Repeatability verification of the kits corresponding to Example 1 and Comparative Example 1

[0048] While embodiments of the present invention have been described in detail above, it will be apparent to those skilled in the art that various modifications and variations can be made to these embodiments. However, it should be understood that such modifications and variations fall within the scope and spirit of the invention as set forth in the claims. Furthermore, the invention described herein may have other embodiments and can be implemented or carried out in various ways.

Claims

1. A primer-probe composition for direct amplification detection of gene polymorphisms related to hypertension drugs, characterized in that, This includes primers and probes for detecting the ACE-rs1799752 gene locus, AGTR1-rs5186 gene locus, ADRB1-rs1801253 gene locus, CYP2D6*10-rs1065852 gene locus, and CYP2C9*3-rs1057910 gene locus, respectively; the difference between any two pairs of melting temperatures (Tm) of the detection probes corresponding to each gene locus is ≥6℃.

2. The primer-probe composition according to claim 1, characterized in that: The forward primer sequence for the primer probe used to detect the ACE-rs1799752 gene locus is shown in SEQ ID NO.1, the reverse primer sequence is shown in SEQ ID NO.2, and the detection probe sequence is shown in SEQ ID NO.3; and / or, the forward primer sequence for the primer probe used to detect the AGTR1-rs5186 gene locus is shown in SEQ ID NO.4, the reverse primer sequence is shown in SEQ ID NO.5, and the detection probe sequence is shown in SEQ ID NO.6; and / or, the forward primer sequence for the primer probe used to detect the ADRB1-rs1801253 gene locus is shown in SEQ ID NO.7, the reverse primer sequence is shown in SEQ ID NO.8, and the detection probe sequence is shown in SEQ ID NO.9; and / or, the forward primer sequence for the primer probe used to detect the CYP2D6*10-rs1065852 gene locus is shown in SEQ ID NO.10, the reverse primer sequence is shown in SEQ ID NO.11, and the detection probe sequence is shown in SEQ ID NO. As shown in NO.12; and / or, the forward primer sequence for the primer probe used to detect the CYP2C9*3-rs1057910 gene locus is shown in SEQ ID NO.13, the reverse primer sequence is shown in SEQ ID NO.14, and the detection probe sequence is shown in SEQ ID NO.

15.

3. A kit for direct amplification detection of gene polymorphisms related to hypertension medications, characterized in that, The mixture includes a primer-probe mixture having the primer-probe composition as described in claim 1 or 2 and a reaction buffer, wherein the reaction buffer comprises a base buffer and a dissolved whole blood endogenous inhibitory component, wherein the whole blood endogenous inhibitory component comprises a heme inhibitor, spermidine, trehalose and an active agent.

4. The reagent kit according to claim 3, characterized in that, In a 20 μL reaction system, the final concentration of trehalose is 0.2 mol / L-0.6 mol / L, the final concentration of the activator is 0.5 wt%-1.5 wt%, the final concentration of the heme inhibitor is 10 μg / mL-40 μg / mL, and the final concentration of spermidine is 0.5 mmol / L-2.0 mmol / L.

5. The reagent kit according to claim 3, characterized in that, The reaction buffer comprises Taq DNA polymerase, dNTPs, Tris-HCl buffer, KCl, MgCl2, and a stabilizer; wherein: in a 20 μL reaction system: the amount of Taq DNA polymerase added is 0.8 U-1.2 U; the amount of dNTPs added is 0.2 mmol / L-0.4 mmol / L; the final concentration of the Tris-HCl buffer is 15 mmol / L-25 mmol / L; the final concentration of KCl is 40 mmol / L-60 mmol / L; the final concentration of MgCl2 is 1.5 mmol / L-2.5 mmol / L; and the final concentration of the stabilizer is 0.05 wt%-0.20 wt%.

6. The reagent kit according to claim 3, characterized in that, The primer-probe mixture contains primers with a final concentration of 100 nmol / L-250 nmol / L and probes with a final concentration of 80 nmol / L-120 nmol / L. It also includes PCR reaction solution, positive control, and blank control. The positive control includes recombinant plasmid standards with wild-type and mutant sequences for five detection sites. The blank control includes purified water free of nucleic acids.

7. A method for using the kit as described in any one of claims 3 to 6 in the detection of gene polymorphisms in hypertension medications, comprising: Fresh whole blood samples anticoagulated with EDTA were added directly to PCR tubes containing primer-probe mixture and reaction buffer. No nucleic acid extraction and purification steps were required. After mixing, the samples were directly loaded into the PCR instrument for thermal cycling amplification. After amplification, a closed-tube melting program was started to collect fluorescence signals in real time. The genotype of the target detection site was analyzed and determined based on the difference in Tm values ​​of the melting curves of each channel in the reaction system.

8. The application method according to claim 7, characterized in that: After mixing the sample, it was incubated at 37℃ for 5 min for UNG enzyme degradation, followed by pre-denaturation at 94℃ for 5 min, and then 40 cycles of PCR amplification were performed. The program for each cycle was: 94℃ for 15 seconds, 58℃ for 40 seconds, and 72℃ for 30 seconds. After amplification, the sample was incubated at 94℃ for 2 min, then at 38℃ for 2 min, and then the temperature was increased from 38℃ to 75℃. During the temperature increase, the fluorescence signals of the FAM, HEX, ROX, and CY5 fluorescence channels were continuously collected and the corresponding melting curves were generated.

9. The application method according to claim 7, characterized in that: When analyzing and determining the genotype of the target detection site based on the differences in Tm values ​​of the melting curves of each channel in the reaction system, the genotyping criteria include: (1) For the CYP2D6*10-rs1065852 site, in the FAM channel: a Tm value of 52.31℃±1.5℃ indicates wild homozygous type, a Tm value of 60.60℃±1.5℃ indicates mutant homozygous type, and the presence of two melting peaks at 52.31℃±1.5℃ and 60.60℃±1.5℃ indicates heterozygous type; (2) For the CYP2C9*3-rs1057910 site, in the HEX channel: a Tm value of 51.62℃±1.5℃ indicates wild homozygous type, a Tm value of 60.07℃±1.5℃ indicates mutant homozygous type, and the presence of two melting peaks at 51.62℃±1.5℃ and 60.07℃±1.5℃ indicates heterozygous type; (3) For the ACE-rs1799752 site, in the ROX channel: a Tm value of 47.89℃±1.5℃ indicates DD homozygous type, a Tm value of 63.68℃±1.5℃ indicates II homozygous type, and the simultaneous appearance of two melting peaks at 47.89℃±1.5℃ and 63.68℃±1.5℃ indicates ID heterozygous type; (4) For the AGTR1-rs5186 site and the ADRB1-rs1801253 site, the two sites share the CY5 channel for independent differentiation and interpretation of dual targets: a Tm value of 41.36℃±1.5℃ indicates AGTR1 wild-type homozygous, a Tm value of 54.58℃±1.5℃ indicates AGTR1 mutant homozygous, and the presence of two melting peaks at 41.36℃±1.5℃ and 54.58℃±1.5℃ indicates AGTR1 heterozygous; a Tm value of 60.93℃±1.5℃ indicates ADRB1 wild-type homozygous, a Tm value of 70.09℃±1.5℃ indicates ADRB1 mutant homozygous, and the presence of two melting peaks at 60.93℃±1.5℃ and 70.09℃±1.5℃ indicates ADRB1 heterozygous.

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