CN115747308A - Method for rapidly detecting polymorphism of drug gene locus for hypertension disease - Google Patents
Method for rapidly detecting polymorphism of drug gene locus for hypertension disease Download PDFInfo
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Abstract
The invention relates to the technical field of gene detection, in particular to a method for rapidly detecting the polymorphism of a drug-taking gene locus of a hypertension disease, which takes genome DNA of a sample to be detected as a template, utilizes a designed and synthesized specific PCR primer pair to carry out PCR amplification reaction, and carries out ASPE reaction and hybridization reaction on a purified PCR product; and finally detecting the fluorescence signal. Compared with the prior art, the invention can simultaneously, quickly and accurately detect the types of the target gene polymorphic sites related to various antihypertensive drugs by using the liquid-phase chip technology, has high detection flux, short time and stable and reliable result, and provides laboratory basis for clinically selecting proper antihypertensive drugs, reducing adverse reactions and improving drug effects, so the invention has great clinical application value.
Description
Technical Field
The invention relates to the technical field of gene detection, in particular to a method for rapidly detecting polymorphism of a drug gene locus for hypertension disease.
Background
Cardiovascular disease is a common disease that seriously threatens the health of humans, especially the middle-aged and elderly people over 50 years old, and includes coronary heart disease, cerebrovascular disease, hypertension, peripheral vascular disease, rheumatic heart disease, congenital heart disease, heart failure and cardiomyopathy, wherein hypertension is the most major risk factor for cardiovascular disease. The control of blood pressure by drug therapy to reduce complications of hypertension is currently the primary treatment for hypertension. However, the individual reaction difference of the medicine for treating hypertension is quite common in clinic, and the blood pressure of 20-50% of patients receiving the medicine treatment is not well controlled. The main reason for this is also due to genetic variation in drug-associated drug-metabolizing enzymes and receptors. The current research on the genetic variation of hypertension drug metabolizing enzyme and receptor mainly focuses on CYP2C9 x 3, ADRBI (1165G > C), AGTRI (166A > C), CYP2D6 x 10, ACE (I/D) and other genes, and the mutation of these genes is the root cause of individual difference of hypertension patient drug response.
The five types of antihypertensive drugs, namely beta receptor blocking drugs, angiotensin Converting Enzyme Inhibitors (ACEI), angiotensin II receptor Antagonists (ARB), diuretics and calcium antagonists (CCB), which are commonly used at present cause different and even opposite drug effect differences among different individuals due to activity differences of drug metabolic enzymes. Therefore, before the medicine is taken, the gene type of the patient can be detected by a clinician to know which specific blood pressure lowering medicine the patient is effective on, so that the optimal treatment scheme is worked out to ensure that the patient is treated most effectively.
In the prior art, methods commonly used for detecting Single Nucleotide Polymorphism (SNP) mainly comprise a PCR-direct sequencing method, a PCR-pyrophosphoric acid sequencing method, a fluorescent quantitative PCR method, a PCR-high resolution melting curve method, an allele specific PCR method, a PCR-restriction fragment length polymorphism method, a solid chip method and the like, but the methods are mostly low in flexibility, complex to operate, long in detection period and incapable of carrying out high-throughput multi-site simultaneous detection, so that the polymorphism detection technology is still not ideal in clinical application at present, and the clinical detection requirement cannot be met all the time.
Disclosure of Invention
The invention aims to provide a method for rapidly detecting the polymorphism of the drug gene sites of the hypertension disease aiming at the defects of the prior art, the method can rapidly and accurately detect the types of the polymorphism sites of the target genes related to various antihypertensive drugs, has high detection flux, short detection time and stable and reliable results, and provides a laboratory basis for clinical reasonable drug administration.
The purpose of the invention is realized by the following technical scheme:
provides a method for rapidly detecting the polymorphism of the drug gene locus for hypertension diseases, which comprises the following steps:
step A, extracting genome DNA of a sample to be detected;
b, designing and synthesizing a plurality of groups of primer groups, wherein each group of primer group comprises a pair of specific PCR primers for detecting polymorphism of a target gene and ASPE primers respectively aiming at the target gene; the detection sites for detecting the target gene polymorphism comprise CYP2D6 x 10, AGTR1-1166A/C, ACE (I/D), NPPA-2238T/C and CYP3A5;
step C, taking the genome DNA of the step A as a template, carrying out PCR amplification reaction by adopting the specific PCR primer pair of the step B, and purifying a PCR product after the reaction;
step D, carrying out ASPE reaction on the purified PCR product, carrying out extension reaction when the first base at the 3' end of the primer is complementary with the amplified target sequence detection site under the action of DNA polymerase, and terminating the extension reaction if the first base is not complementary;
step E, mixing the 1.5 XTMAC hybridization buffer solution with the microspheres, and then carrying out hybridization reaction with the ASPE reaction product in the step D;
step F, suspending the microspheres subjected to the hybridization reaction in 100 mu l of 1 XTMAC hybridization buffer solution containing 7.5 mu g/ml streptavidin-phycoerythrin, and incubating for 20min at 37 ℃;
and G, detecting a fluorescence signal by adopting a Luminex 200 instrument.
In the technical scheme, the PCR primer for detecting CYP2D6 x 10 locus has sequences shown in SEQ ID NO.1 and SEQ ID NO.2, and the ASPE primer has sequences shown in SEQ ID NO.3 and SEQ ID NO. 4;
the PCR primer for detecting the AGTR1-1166A/C locus has the sequences shown by SEQ ID NO.5 and SEQ ID NO.6, and the ASPE primer has the sequences shown by SEQ ID NO.7 and SEQ ID NO. 8;
PCR primers for detecting ACE (I/D) sites have sequences shown in SEQ ID NO.9 and SEQ ID NO.10, and ASPE primers thereof have sequences shown in SEQ ID NO.11 and SEQ ID NO. 12;
the PCR primer for detecting NPPA-2238T/C locus has the sequences shown in SEQ ID NO.13 and SEQ ID NO.14, and the ASPE primer has the sequences shown in SEQ ID NO.15 and SEQ ID NO. 16;
the PCR primer for detecting the CYP3A5 locus has sequences shown in SEQ ID NO.17 and SEQ ID NO.18, and the ASPE primer thereof has sequences shown in SEQ ID NO.19 and SEQ ID NO. 20.
In the above technical scheme, in the step C, the total volume of the PCR reaction system is 50 μ l, wherein the total volume of the PCR reaction system contains 2 μ l of DNA sample, 10 XTaq TM Hot Star DNA polymerase buffer 5. Mu.l, 5U/. Mu.l Taq TM 0.25. Mu.l of Hot Start polymerase, 4. Mu.l of 10mmol/L dNTP, 8. Mu.l of upstream and downstream mixed primers, and 30.75. Mu.l of deionized water.
In the above technical scheme, the specific conditions of the PCR amplification reaction are as follows: pre-denaturation at 94 ℃ for 30s, circulation at 94 ℃ for 30s, annealing at 57 ℃ for 30s, extension at 72 ℃ for 30s, and 5 times of circulation; then carrying out 30 cycles of 94 ℃ for 30s, annealing at 55 ℃ for 30s and extension at 72 ℃ for 30 s; finally, the extension was terminated at 72 ℃ for 10 min.
In the above technical scheme, in the step D, the total volume of the ASPE reaction system is 20 μ l, wherein the total volume contains 2 μ l of purified PCR product, 10 XTaq TM 5. Mu.l of Hot Star DNA polymerase buffer, 0.3. Mu.l of 400. Mu.M Biotin-dCTP, and 100. Mu.M each of 300. Mu.M dATP, dGTP and dTTP, in total 1. Mu.l; 0.4. Mu.l of 100mM dCTP, 0.1. Mu.l of TaqTMHotStart enzyme, 500nM each of the TAG-ASPE primers, 1. Mu.l in total, and 13.55. Mu.l of deionized water.
In the above technical scheme, the specific conditions of the ASPE reaction are as follows: 30s at 94 ℃, 30s at 57 ℃, 1min at 74 ℃ and 40 cycles.
In the above technical scheme, in step E, the specific conditions of the hybridization reaction are: 96 ℃ deformation 90s,37 ℃ incubation 45min.
The invention has the advantages of
The method for rapidly detecting the polymorphism of the drug gene locus for the hypertension disease provided by the invention can simultaneously, rapidly and accurately detect the types of the polymorphism loci of the target genes related to various antihypertensive drugs by using a liquid chip technology, has high detection flux, short time and stable and reliable results, and provides a laboratory basis for clinically selecting appropriate antihypertensive drugs, reducing adverse reactions and improving drug effects, so the method has great clinical application value.
Detailed Description
The invention is further described with reference to the following examples.
1. Reagents and instrumentation:
multiplex PCR kit Taq TM HotStart Version, available from TaKaRa, japan;
DNA extraction Kit TIAnamp Genomic DNA Kit, purchased from Beijing Tiangen Biotechnology, inc.);
exonucleases I-shrimp alkaline phosphatase (ExoSAP-IT), platinum TM GenoType Tsp DNA polymerase, streptavidin-bathochromic protein ((SAPE), biotin-dCTP, dNTPs, etc. are all available from Seimer Feishel scientific, USA;
microspheres and Luminex 200 available from Luminex corporation, usa;
a LifeECO gene amplification instrument purchased from China perspective Life technologies, inc.;
BioDrop protein nucleic acid analyzers were purchased from haworth biotechnology ltd;
model DYY-6C electrophoresis apparatus, available from six biological Limited companies in Beijing.
2. A sample to be tested:
the biological sample adopted in the embodiment is the residual human anticoagulation of the hypertensive patient detected in medical inspection of Dongguan Ming Zhi during 2021 years and 5-12 months. All hypertension patients meet the hypertension diagnosis standard issued by WHO.
3. The experimental method comprises the following steps:
the method for rapidly detecting the polymorphism of the gene locus for the hypertension disease comprises the following steps:
step A, extracting genome DNA of a sample to be detected:
three human anticoagulation samples are taken, numbered 1, 2 and 3, the genomic DNA of a sample to be detected is extracted by using a self-prepared or commercial nucleic acid extraction kit, and A260/A280 detection is carried out by using a NanoDrop 2000 ultramicro ultraviolet spectrophotometer, and the concentration C (ng/mu l) of a sample is quantified by using a Qubit. A260/a280=1.7 to 1.9, and the sample concentrations of nos. 1, 2, and 3 were 18.6 ng/. Mu.l, 23.5 ng/. Mu.l, and 26.1 ng/. Mu.l, respectively.
And step B, designing and synthesizing a plurality of groups of primer groups for detecting the polymorphism of the locus, wherein each group of primer groups comprises a pair of specific PCR primers for detecting the polymorphism of a target gene and ASPE primers respectively aiming at the target gene:
primers were designed using Primer3.0 based on the human whole genome sequence disclosed by NCBI (national center for Biotechnology information). Because a plurality of primers and templates in the multiplex PCR system exist in the same reaction tube, mutual interference is easy to cause in the experimental process, and dimers are easy to form among the primers, thereby influencing the extension of the ASPE primers, therefore, the design of the primers is the key of the invention and is also the basis for Luminex liquid phase chip detection.
The detection sites for detecting the target gene polymorphism in the embodiment comprise CYP2D6 x 10, AGTR1-1166A/C, ACE (I/D), NPPA-2238T/C and CYP3A5. Specific PCR primers for detecting the site polymorphisms are designed according to the nucleotide sequences of the site polymorphisms of the target genes, are used for amplifying DNA fragments of the target gene sites, and are designed into ASPE primer sequences, which are detailed in Table 1.
TABLE 1 specific PCR primers and ASPE primers
Step C, taking the genome DNA of the sample to be detected as a template, and adopting the specific PCR primer pair in the step B to carry out PCR amplification reaction, wherein the total volume is 50 mul, the DNA sample contained in the PCR amplification reaction is 2 mul, and the volume is 10 XTaq TM Hot Star DNA polymerase buffer5μl，5U/μl Taq TM 0.25 mul of Hot Start polymerase, 4 mul of 10mmol/L dNTP, 8 mul of upstream and downstream mixed primer and 30.75 mul of deionized water;
the conditions of the PCR amplification reaction are as follows: pre-denaturation at 94 ℃ for 30s, circulation at 94 ℃ for 30s, annealing at 57 ℃ for 30s, extension at 72 ℃ for 30s, and 5 times of circulation; then carrying out 30 cycles of 94 ℃ for 30s, annealing at 55 ℃ for 30s and extension at 72 ℃ for 30 s; finally, the extension is stopped at 72 ℃ for 10 min;
after PCR amplification reaction, purification is carried out, and redundant dNTP, primers and single-stranded products in the PCR product are digested by exonuclease and alkaline phosphatase.
Step D, carrying out ASPE reaction on the purified PCR product, carrying out extension reaction when the first base at the 3' end of the primer is complementary with the detection site of the amplified target sequence under the action of DNA polymerase, and terminating the extension reaction if the first base is not complementary;
in an ASPE reaction system: the total volume was 20. Mu.l containing 2. Mu.l of the purified PCR product, 10 XTaq TM 5. Mu.l of Hot Star DNA polymerase buffer, 0.3. Mu.l of 400. Mu.M Biotin-dCTP, and 100. Mu.M each of 300. Mu.M dATP, dGTP and dTTP, in total 1. Mu.l; 0.4 mul of 100mM dCTP, 0.1 mul of TaqTMHotStart enzyme, 500nM of each TAG-ASPE primer, 1 mul in total, and 13.55 mul of deionized water;
the conditions for the ASPE reaction were: 30s at 94 ℃, 30s at 57 ℃, 1min at 74 ℃ and 40 cycles.
Step E, mixing microspheres with 1.5 XTMAC hybridization buffer solution, and then taking 20 mul of the mixed solution to perform hybridization reaction with 2 mul of the ASPE reaction product obtained in the step D;
the hybridization reaction conditions were 96 ℃ modified 90s, and 37 ℃ incubation for 45min.
Step F, suspending the microspheres subjected to the hybridization reaction in 100 mu l of 1 XTMAC hybridization buffer solution containing 7.5 mu g/ml streptavidin-phycoerythrin, and incubating for 20min at 37 ℃;
and G, detecting a fluorescence signal on a Luminex 200 instrument, wherein the detection result is shown in Table 2.
TABLE 2 detection results of polymorphism of target genomic loci
As can be seen from the results in Table 2, the detection method of the invention can simultaneously, rapidly and accurately detect the types of the target gene polymorphic sites related to various antihypertensive drugs, has high detection flux, short detection time and stable and reliable results, and provides laboratory basis for clinically selecting appropriate antihypertensive drugs, reducing adverse reactions and improving drug effects, so the invention has great clinical application value.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention, and not for limiting the protection scope of the present invention, although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (7)
1. A method for rapidly detecting the polymorphism of a drug gene locus for hypertension diseases is characterized in that: the method comprises the following steps:
a, extracting genome DNA of a sample to be detected;
b, designing and synthesizing a plurality of groups of primer groups, wherein each group of primer groups comprises a pair of specific PCR primers for detecting polymorphism of a target gene and ASPE primers respectively aiming at the target gene; the detection sites for detecting the target gene polymorphism comprise CYP2D6 x 10, AGTR1-1166A/C, ACE (I/D), NPPA-2238T/C and CYP3A5;
step C, taking the genome DNA of the step A as a template, carrying out PCR amplification reaction by adopting the specific PCR primer pair of the step B, and purifying a PCR product after the reaction;
step D, carrying out ASPE reaction on the purified PCR product, carrying out extension reaction when the first base at the 3' end of the primer is complementary with the detection site of the amplified target sequence under the action of DNA polymerase, and terminating the extension reaction if the first base is not complementary;
step E, mixing 1.5 times TMAC hybridization buffer solution with the microspheres, and then carrying out hybridization reaction with the ASPE reaction product in the step D;
step F, suspending the microspheres subjected to the hybridization reaction in 100 mu l of 1 XTMAC hybridization buffer solution containing 7.5 mu g/ml streptavidin-phycoerythrin, and incubating for 20min at 37 ℃;
and G, detecting a fluorescence signal by adopting a Luminex 200 instrument.
2. The method for rapidly detecting the polymorphism of the drug gene site for hypertension according to claim 1, which is characterized in that: the PCR primer for detecting CYP2D6 x 10 locus has sequences shown in SEQ ID NO.1 and SEQ ID NO.2, and the ASPE primer thereof has sequences shown in SEQ ID NO.3 and SEQ ID NO. 4;
the PCR primer for detecting the AGTR1-1166A/C locus has the sequences shown by SEQ ID NO.5 and SEQ ID NO.6, and the ASPE primer has the sequences shown by SEQ ID NO.7 and SEQ ID NO. 8;
the PCR primer for detecting the ACE (I/D) locus has sequences shown in SEQ ID NO.9 and SEQ ID NO.10, and the ASPE primer has sequences shown in SEQ ID NO.11 and SEQ ID NO. 12;
the PCR primer for detecting NPPA-2238T/C locus has the sequences shown in SEQ ID NO.13 and SEQ ID NO.14, and the ASPE primer has the sequences shown in SEQ ID NO.15 and SEQ ID NO. 16;
the PCR primer for detecting the CYP3A5 site has sequences shown in SEQ ID NO.17 and SEQ ID NO.18, and the ASPE primer has sequences shown in SEQ ID NO.19 and SEQ ID NO. 20.
3. The method for rapidly detecting the polymorphism of the gene locus for hypertension treatment according to claim 1, wherein the method comprises the following steps: in step C, the total volume of the PCR reaction system is 50. Mu.l, containing 2. Mu.l of DNA sample, 10 XTaq TM Hot Star DNA polymerase buffer 5. Mu.l, 5U/. Mu.l Taq TM 0.25. Mu.l of Hot Start polymerase, 4. Mu.l of 10mmol/L dNTP, 8. Mu.l of upstream and downstream mixed primers, and 30.75. Mu.l of deionized water.
4. The method for rapidly detecting the polymorphism of the gene locus for hypertension treatment according to claim 3, wherein the method comprises the following steps: the specific conditions of the PCR amplification reaction are as follows: pre-denaturation at 94 ℃ for 30s, circulation at 94 ℃ for 30s, annealing at 57 ℃ for 30s, and extension at 72 ℃ for 30s for 5 cycles; then carrying out 30 cycles of 94 ℃ for 30s, annealing at 55 ℃ for 30s and extension at 72 ℃ for 30 s; finally, the extension was terminated at 72 ℃ for 10 min.
5. The method for rapidly detecting the polymorphism of the gene locus for hypertension treatment according to claim 1, wherein the method comprises the following steps: in step D, the total volume of the ASPE reaction system is 20. Mu.l, which contains 2. Mu.l of the purified PCR product, 10 XTaq TM 5. Mu.l of Hot Star DNA polymerase buffer, 0.3. Mu.l of 400. Mu.M Biotin-dCTP, and 100. Mu.M of each of 300. Mu.M dATP, dGTP and dTTP, in total 1. Mu.l; 0.4. Mu.l of 100mM dCTP, 0.1. Mu.l of TaqTMHotStart enzyme, 500nM each of the TAG-ASPE primers, 1. Mu.l in total, and 13.55. Mu.l of deionized water.
6. The method for rapidly detecting the polymorphism of the drug gene site for hypertensive disease according to claim 5, wherein the method comprises the following steps: the specific conditions of the ASPE reaction are as follows: 30s at 94 ℃, 30s at 57 ℃, 1min at 74 ℃ and 40 cycles.
7. The method for rapidly detecting the polymorphism of the drug gene site for hypertension according to claim 1, which is characterized in that: in step E, the specific conditions of the hybridization reaction are as follows: 96 ℃ deformation 90s,37 ℃ incubation 45min.
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