CN111778328A - Fluorescent PCR method for detecting nucleotide polymorphism of 1165 site of human ADRB1 gene - Google Patents

Fluorescent PCR method for detecting nucleotide polymorphism of 1165 site of human ADRB1 gene Download PDF

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CN111778328A
CN111778328A CN202010805972.3A CN202010805972A CN111778328A CN 111778328 A CN111778328 A CN 111778328A CN 202010805972 A CN202010805972 A CN 202010805972A CN 111778328 A CN111778328 A CN 111778328A
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裴小锐
顾梅
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Suzhou Municipal Hospital
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Abstract

The invention provides a primer, a probe and a fluorescent PCR detection method for detecting ADRB1 gene c.1165 site (G/C) nucleotide polymorphism. According to the theory of allele specific PCR, the nucleotide type of the c.1165 site of the beta 1adrenergic receptor ADRB1 gene is detected on a real-time fluorescent quantitative PCR technical platform, and the method has the advantages of simple and convenient operation, rapidness, low cost and the like.

Description

Fluorescent PCR method for detecting nucleotide polymorphism of 1165 site of human ADRB1 gene
Technical Field
The invention belongs to the field of biomedical clinical molecular detection, and particularly relates to a fluorescence PCR method for detecting nucleotide polymorphism of c.1165 locus of human ADRB1 gene.
Background
With the development of social economy and the change of life style of residents, chronic non-infectious diseases become a great public health problem affecting the health of China and even residents all over the world, and hypertension is one of chronic diseases with high morbidity and is also the most important risk factor of cardiovascular and cerebrovascular diseases. According to the statistics of the world health organization, the death rate of cardiovascular diseases in the whole world is 1700 thousands of death rate, which accounts for 46% of the death rate of chronic diseases, and in which 940 thousands of death rate are hypertension complications, hypertension is a primary risk factor affecting the global disease burden. Data published by the national institutes of health in 2016 show: the prevalence rate of hypertension of adults 18 years old and older in China is 25.2%. Although the awareness rate, treatment rate and control rate of hypertension of people in China have been improved in recent years, the awareness rate, treatment rate and control rate of hypertension of people in China are still at a lower level. Although the state actively advocates that healthy lifestyle, low salt diet, intensive physical exercise, etc. may reduce the prevalence of risk factors for hypertension, controlling blood pressure levels and preventing their complications through drugs remains a major intervention. The common medicines for treating hypertension can be calcium channel antagonist, vasodilator, diuretic, renin-angiotensin system inhibitor and sympathetic nerve inhibitor. Years of clinical research shows that the clinical curative effect of the medicines is greatly different among different patients and is possibly closely related to the difference of specific physiological states and genetic backgrounds of the patients. The pharmacogenetic studies indicate that the main cause of the variation in the efficacy of drugs among individuals results from genetic polymorphisms among individuals, which lead to relevant factors in drug action: drug transporters, drug metabolizing enzymes and drug action targets (receptors) objectively differ among different individuals, so that the blood concentration and drug sensitivity among individuals are remarkably different.
Adrenoreceptors are a class of G-protein coupled tissue receptors that mediate the action of catecholamines and are classified into two classes, alpha and beta. Beta-adrenoceptor (beta-adrenoceptor beta-AR), like the alpha-adrenoceptor, uses epinephrine and norepinephrine as endogenous ligands and mediates the regulation of the function of most organs and tissues by regulating intracellular cAMP levels, and many drugs also exert their effects directly or indirectly through this receptor.
The beta 1-adrenergic receptor (ADRB 1) is involved in the regulation of human blood pressure, and the polymorphism of the gene is closely related to hypertension. G1165C is the polymorphic site on ADRB1 gene related to cardiovascular disease. G1165C causes the polymorphism of a receptor protein Gly389Arg, which is the coupling site of the receptor and the G protein and is the key structure of post-receptor information transmission. The frequency of the 389Arg allele is 0.58 for black people and 0.72-0.76 for white and Asian people. There was a significant difference in C allele frequency among black people and other ethnic groups. Competitive binding experiments by Liggett et al in chinese hamster fibroblasts (CHW) expressing Gly389 and Arg389 type receptors, respectively, showed that there was no difference in the affinity of the two receptors for agonist binding, but this genetic polymorphism significantly altered the coupling of the receptor to G protein: the Arg 389-type receptor is significantly elevated, both in basal adenylate cyclase activity and in agonist-stimulated maximal adenylate cyclase activity. Overall level studies indicate that significant differences exist in cardiovascular responses between Gly389 homozygote and Arg389 homozygote healthy subjects after different doses of metoprolol, as evidenced by significantly higher reductions in resting heart rate, exercise heart rate, and systolic blood pressure for Arg389 homozygote than for Gly389 subjects. Further clinical tests show that the treatment effect of the beta 1 receptor blocking drug on the hypertension is related to the genotype of beta 1adrenergic receptor Gly389Arg polymorphism mutation, and the curative effect of metoprolol on hypertension patients carrying different genotypes has gene dose effect, so that the beta 1 receptor haplotype can be used as a prediction factor of the curative effect of the drug on the hypertension, and the administration dose can be adjusted according to the genotype of the patients clinically to improve the curative effect and reduce adverse reactions. In 2017, related contents of pharmacogenomics are increased in reasonable medication guidelines for hypertension (2 nd edition) jointly issued by the national institutes of health council of medicine experts and the national association of physicians and hypertension technical committee, and it is clearly indicated that ADRB1 gene polymorphism can influence the curative effect of beta-blockers (such as metoprolol).
For many years, the ADRB1 polymorphism genotype has been analyzed at home and abroad, and a direct sequencing method after DNA amplification is mostly adopted. The method is complex to operate, long in detection period and low in flux, and is not suitable for large-scale rapid crowd screening. In addition, it is difficult to realize large-scale popularization because it requires expensive equipment.
Disclosure of Invention
Aiming at the defects of high price, long period, low efficiency, complicated data analysis and the like when the ADRB1 gene polymorphism is detected in the prior art, the invention provides a primer, a fluorescent probe and a detection method for detecting the ADRB1 gene polymorphism based on a real-time fluorescent quantitative PCR technical platform, which are used for detecting the polymorphism of the c.1165 site (G/C) of the human ADRB1 gene.
In order to achieve the purpose, the invention adopts the technical scheme that:
a primer and a corresponding probe for detecting the nucleotide polymorphism of the 1165 site of the human ADRB1 gene are designed according to the polymorphism of the c.1165 site (G/C) of the ADRB1 gene, and the sequences are as follows:
reverse primer:
ADRB1-1165G:5’-CGCGCAGCAGAGCAGTCC-3’(SEQ ID No.1)；
ADRB1-1165C:5’-CGCGCAGCAGAGCAGTCG-3’(SEQ ID No.2)；
a forward primer:
ADRB1-1165F:5’-CCGACCGCCTCTTCGTCTTCTT-3’(SEQ ID No.3)；
a fluorescent probe:
ADRB1-1165P 5 'fluorophore-CCTTGCGGAAGTCGGGGCTGCGGCA-3' quencher (SEQ ID No. 4).
The invention also provides an internal standard primer pair and a corresponding fluorescent probe for detecting the polymorphism of the human ADRB1 gene, wherein the sequences are as follows:
an internal standard primer pair:
a forward primer: ICF 5'-TGCAACAGAAGCCCAGCTAT-3' (SEQ ID No. 5);
reverse primer: ICR 5'-AACCTGGAAGCCAACGAAAGA-3' (SEQ ID No. 6);
internal standard fluorescent probe:
ICP 5 'fluorophore-GACACAAATCAAACCATGCCACCACCT-3' quencher (SEQ ID No. 7).
The probe for detecting polymorphism of c.1165 site (G/C) of human ADRB1 gene and the fluorescent group at the 5 'end of the fluorescent probe corresponding to the internal standard primer are conventional fluorescent reporter groups suitable for fluorescent PCR quantitative analysis, preferably FAM, TET, VIC, HEX or ROX, the quenching group at the 3' end is a conventional fluorescent quenching group suitable for fluorescent PCR quantitative analysis, preferably BHQ-1, BHQ-2, Dabcyl, Eclipse or TAMRA, and the more preferable scheme is that the fluorescent group connected with the 5 'end of the fluorescent probe is FAM and the quenching group connected with the 3' end is Dabcyl; the fluorescent group connected with the 5 'end of the internal standard fluorescent probe is ROX, and the quenching group connected with the 3' end of the internal standard fluorescent probe is BHQ-2.
The invention also provides a fluorescent PCR detection system for detecting the polymorphism of the human ADRB1 gene, which comprises the detection primer for detecting the polymorphism of the c.1165 site (G/C) of the human ADRB1 gene, a corresponding fluorescent probe and an instruction book. Specifically, the method comprises two specific reaction liquids for detecting the polymorphism of the c.1165 site (G/C) of the human ADRB1 gene: ADRB1-1165G PCR reaction liquid and ADRB1-1165C PCR reaction liquid, wherein the PCR reaction liquid contains buffer liquid, magnesium ions, dNTP and other substances which are necessary for PCR reaction, and correspondingly contains the detection primer and the probe. The sequences of the primers and the probes contained in the PCR reaction solution for each specific detection are as follows:
(1) ADRB1-1165G PCR reaction solution
ADRB1-1165G:5’-CGCGCAGCAGAGCAGTCC-3’(SEQ ID No.1)；
ADRB1-1165F:5’-CCGACCGCCTCTTCGTCTTCTT-3’(SEQ ID No.3)；
ADRB1-1165P 5 'fluorophore-CCTTGCGGAAGTCGGGGCTGCGGCA-3' quencher (SEQ ID No. 4).
(2) ADRB1-1165C PCR reaction solution
ADRB1-1165C:5’-CGCGCAGCAGAGCAGTCG-3’(SEQ ID No.2)；
ADRB1-1165F:5’-CCGACCGCCTCTTCGTCTTCTT-3’(SEQ ID No.3)；
ADRB1-1165P 5 'fluorophore-CCTTGCGGAAGTCGGGGCTGCGGCA-3' quencher (SEQ ID No. 4).
In the preferred embodiment of the PCR reaction solution, the PCR reaction solution comprises a pair of internal standard primers and corresponding fluorescent probes in addition to a detection primer and corresponding fluorescent probes for detecting the polymorphism of the c.1165 site (G/C) of the human ADRB1 gene, wherein the sequences of the internal standard primers and the probes are as follows:
an internal standard primer:
a forward primer: IC-F: 5'-TGCAACAGAAGCCCAGCTAT-3' (SEQ ID No. 5);
reverse primer: IC-R: 5'-AACCTGGAAGCCAACGAAAGA-3' (SEQ ID No. 6);
internal standard fluorescent probe:
IC-P5 'fluorophore-GACACAAATCAAACCATGCCACCACCT-3' quencher (SEQ ID No. 7).
The probe for detecting polymorphism of c.1165 site (G/C) of human ADRB1 gene, the probe for detecting polymorphism of c.1286 site (A/C) of human ADRB1 gene and the fluorescent group at the 5 'end of the fluorescent probe corresponding to the internal standard primer in the kit can be a conventionally used fluorescent reporter group suitable for fluorescent PCR quantitative analysis, preferably a FAM, TET, VIC, HEX or ROX, the quencher at the 3' end is a conventionally used fluorescent quencher suitable for fluorescent PCR quantitative analysis, preferably BHQ-1, BHQ-2, Dabcyl, Eclipse or TAMRA, and the more preferable scheme is that the fluorescent group connected with the 5 'end of the fluorescent probe is FAM and the quencher connected with the 3' end is Dabcyl; the fluorescent group connected with the 5 'end of the internal standard fluorescent probe is ROX, and the quenching group connected with the 3' end of the internal standard fluorescent probe is BHQ-2.
The instructions for using the kit comprise descriptions of PCR amplification conditions, preferably:
the PCR reaction consists of a first stage and a second stage:
the conditions for the first stage of pre-denaturation are: the temperature is 95 ℃ and the time is 3 minutes;
the second phase consists of 40 amplification cycles with the conditions:
denaturation: the temperature is 95 ℃ and the time is 10 seconds;
annealing/extending: the temperature is 60 ℃, and the time is 40 seconds; (setting fluorescent Signal Collection)
The invention also provides a method for detecting ADRB1 gene polymorphism by using the detection primer and the fluorescent probe or the contained fluorescent PCR kit for detecting ADRB1 gene polymorphism, which comprises the following steps:
(1) sample processing and template extraction
a. Obtaining a saliva sample from a subject to be tested;
b. obtaining DNA from saliva samples, and recommending the use of commercial kits for extracting saliva DNA;
c. determining the concentration and purity of DNA, wherein the concentration is required to be more than 10 ng/muL; OD260nm/OD280nm＝1.6～2.0。
(2) Fluorescent PCR amplification:
adding a template DNA to be detected and a certain amount of Taq DNA polymerase into a detection primer containing the polymorphism of the c.1165 site (G/C) of the ADRB1 gene of the human being and a corresponding fluorescent probe PCR reaction solution, uniformly mixing and centrifuging in a fluorescent PCR tube, then putting the fluorescent PCR tube into a fluorescent quantitative PCR instrument to perform PCR reaction according to a specific temperature cycle and signal acquisition program, preferably adding the template DNA to be detected and a certain amount of Taq DNA polymerase into the PCR reaction solution of the preferred scheme of the kit containing an internal standard primer and a corresponding fluorescent probe, uniformly mixing and centrifuging in the fluorescent PCR tube, then putting the fluorescent quantitative PCR instrument into the fluorescent quantitative PCR instrument to perform PCR reaction according to the specific temperature cycle and signal acquisition program, and monitoring the reaction effectiveness.
The above-described fluorescent PCR amplification protocol preferably employs the following temperature cycling and signal acquisition procedures for PCR reactions:
the PCR reaction consists of a first stage and a second stage:
the conditions for the first stage of pre-denaturation are: the temperature is 95 ℃ and the time is 3 minutes;
the second phase consists of 40 amplification cycles with the conditions:
denaturation: the temperature is 95 ℃ and the time is 10 seconds;
annealing/extending: the temperature is 60 ℃, and the time is 40 seconds; (setting fluorescent Signal Collection)
(3) Analysis results
And under the conditions of the PCR reaction system and the temperature cycle program, observing whether the target detection fluorescent signal in the specific PCR reaction system forms a logarithmic amplification S-shaped curve or not, and if the logarithmic amplification S-shaped curve is formed, determining that the DNA sample to be detected is positive to the polymorphism type represented by the specific reaction system.
The invention also provides a detection primer for detecting the polymorphism of the c.1165 site (G/C) of the human ADRB1 gene, a corresponding fluorescent probe, an internal standard primer and application of the corresponding probe in detecting the polymorphism of the human ADRB1 gene.
The invention also provides a detection primer for detecting the polymorphism of the c.1165 site (G/C) of the human ADRB1 gene, a corresponding fluorescent probe, and application of an internal standard primer and a detection system of the corresponding probe in detecting the polymorphism of the human ADRB1 gene.
The technical scheme of the invention is explained as follows.
1. The working principle of the invention is as follows: the basic principle of Allele Specific PCR (ASPCR), also known as Allele Specific Amplification (ASA) or Amplification hindered Mutation System (ARMS-PCR), is that when PCR is performed, if the 3 ' end of the primer is mismatched, the chain extension reaction is hindered due to the barrier formed by the 3 ', 5 ' -phosphodiester bond, resulting in a rapid decrease in the amount of PCR product, and no Specific Amplification product can be detected within a certain Amplification cycle; on the contrary, the 3' end is matched to detect the amplification product. Then, for a known polymorphic site, the polymorphic base is designed at the 3' end of the detection primer, and after the amplification reaction, the presence or absence of the product is observed by gel electrophoresis, whereby the base type of the polymorphic site can be determined.
Fluorescent quantitative PCR is a new quantitative experimental technique introduced by Applied Biosystems in 1996, wherein a pair of primers is added during PCR amplification, and a specific fluorescent probe, called as TaqMan probe, is added at the same time, and is an oligonucleotide, and two ends of the oligonucleotide are respectively marked with a reporter fluorophore and a quenching fluorophore. When the probe is complete, the fluorescent signal emitted by the reporter group is absorbed by the quenching group; initially, the probe is bound to any single strand of DNA; during PCR amplification, the 5 '→ 3' exonuclease activity of Taq enzyme cuts and degrades the probe, so that the reporter fluorescent group and the quenching fluorescent group are separated, a fluorescence monitoring system can receive a fluorescence signal, namely, one fluorescent molecule is formed when one DNA chain is amplified, and the fluorescent signal accumulation and the PCR product formation are completely synchronous.
The products of the allele specific PCR reaction system are detected by a real-time fluorescent PCR technology based on a fluorescent probe, and the base type of the corresponding target site of the template in the corresponding detection system can be judged according to the cycle number (Ct value) of a fluorescent signal forming an amplification curve at a specific threshold value.
The design of the primers and the probes in the technical scheme of the invention is as follows: according to the information of the reference sequence (NG-012187.1, NM-000684.3) of ADRB1 gene disclosed by GeneBank of nucleic acid sequence database of NCBI of the national center for Biotechnology information, and the c.1165 site (G/C) polymorphism (SNP ID: rs1801253) disclosed by dbSNP database, primers and probes for detecting the c.1165 site (G/C) polymorphism of ADRB1 gene were designed by using Primer Express 3.0 software of ABI. In order to monitor the effectiveness of a reaction system, an internal standard Primer and a probe are added into a detection system, a segment of sequence of human conserved gene beta-globin is selected, and the detection Primer and the probe are designed by adopting Primer Express 3.0 software of ABI company.
In the technical scheme of the invention, the primer (primer) refers to an oligonucleotide sequence consisting of a certain amount of dNTP, and is generally synthesized by a DNA synthesizer and purified by polyacrylamide gel electrophoresis or other suitable methods after synthesis. In the polymerase chain reaction, a primer can bind to a region complementary to a nucleic acid strand of interest to be amplified, and functions as a starting point for nucleotide polymerization, and at the 3 '-OH of the primer, nucleotides are synthesized in the form of a diester bond, so that the 3' -OH of the primer must be free. The DNA polymerase can begin to extend from its 3' end and synthesize a new nucleic acid strand.
In the technical scheme of the invention, the fluorescent probe is an oligonucleotide probe, a fluorescent group is connected to the 5 'end of the probe, a quenching group is arranged at the 3' end, the fluorescent probe is generally artificially synthesized by a DNA synthesizer, and the fluorescent probe is purified by polyacrylamide gel electrophoresis or other suitable methods after synthesis. Currently, the commonly used fluorescent groups include FAM, TET, VIC, HEX, ROX, etc. As the quencher, there may be mentioned BHQ-1, BHQ-2, Dabcyl, Eclipse, TAMRA and the like.
In the technical scheme of the invention, the internal standard primer and the corresponding probe thereof can be used for monitoring the index of reaction effectiveness and judging whether the test result is influenced by factors such as template quality, machine faults, reagent stability and the like. Normally, the exponential amplification curve formed by the normal amplification of the PCR is vividly described as an 'S' -shaped amplification curve, which indicates that the system is normally amplified. When the target detection fluorescent signal in the specific PCR reaction system forms a logarithmic amplification S-shaped curve, the curve is a positive result of polymorphism type, the amplification of the PCR system is also indicated to be normal, and an internal standard primer and a corresponding probe thereof are not required to be adopted for verifying the result. However, when the objective detection fluorescent signal in the specific PCR reaction system does not form a logarithmic amplification "S" curve, and the detection result is a negative result of the polymorphism type represented by the specific reaction system, but may also be a result of the test influenced by factors such as template quality, machine failure, reagent stability, etc., in this case, the internal standard primer and the corresponding probe can be used for elimination. When the objective detection fluorescent signal in the specific PCR reaction system does not form a logarithmic amplification S-shaped curve, and the internal standard signal forms a normal logarithmic amplification S-shaped curve, the accuracy of the negative result of the polymorphism type can be verified. When the objective detection fluorescent signal in the specific PCR reaction system does not form a logarithmic amplification 'S' -shaped curve and the internal standard signal does not form a normal logarithmic amplification 'S' -shaped curve, the problem occurring on the experimental condition or the instrument is proved to influence the test result, but not the negative result of the polymorphism type.
Due to the application of the technical scheme, compared with the prior art, the invention has the following advantages and effects:
1. the detection primer for detecting the polymorphism of the c.1165 site (G/C) of the ADRB1 gene, the corresponding fluorescent probe, the internal standard primer and the corresponding probe in the technical scheme have very high PCR detection specificity, and the detection result is easy to interpret by adopting a real-time fluorescent PCR technology.
2. The detection primers and the probes in the technical scheme of the invention have low price, and the sequencing is not needed in the experimental process, so that the detection cost is saved, the detection period is greatly shortened, and the detection efficiency is improved.
3. The detection primers and the probes in the technical scheme of the invention adopt a real-time fluorescence PCR technical platform, and can realize high-throughput detection.
Drawings
FIG. 1 is a C.1165 locus (CC homozygous) kit detection map of ADRB1 gene of a certain sample
FIG. 2 is a sequence diagram of the c.1165 site (CC homozygote type) of ADRB1 gene of a sample
Detailed Description
Terms used in the present invention generally have meanings commonly understood by those of ordinary skill in the art, unless otherwise specified.
The present invention is described in further detail below with reference to specific examples and with reference to the data. It will be understood that these examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way.
In the following examples, various procedures and methods not described in detail are conventional methods well known in the art, generally employing conventional conditions such as Sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: Cold Spring harbor laboratory Press, 1989), or according to the manufacturer's recommended procedures. The model of the fluorescent quantitative PCR instrument used in the invention is ABI 7500.
Example 1 primer and probe design:
according to the information of the reference sequences (NG-012187.1, NM-000684.3) of ADRB1 gene disclosed by GeneBank of nucleic acid sequence database of NCBI of the national center for Biotechnology information and the c.1165 site (G/C) polymorphism (SNP ID: rs1801253) disclosed by dbSNP database, primers and probes for detecting the C.1165 site (G/C) polymorphism of ADRB1 gene are designed by using Primer Express 3.0 software of ABI company, which are as follows:
the sequences of primers and probes for detecting C.1165 site (G/C) polymorphism of ADRB1 gene are shown as follows:
reverse primer:
ADRB1-1165G:5’-CGCGCAGCAGAGCAGTCC-3’(SEQ ID No.1)；
ADRB1-1165C:5’-CGCGCAGCAGAGCAGTCG-3’(SEQ ID No.2)；
a forward primer:
ADRB1-1165F:5’-CCGACCGCCTCTTCGTCTTCTT-3’(SEQ ID No.3)；
a fluorescent probe:
ADRB1-1165P:5’FAM-CCTTGCGGAAGTCGGGGCTGCGGCA-3’Dabcyl(SEQ ID No.4)。
in order to monitor the effectiveness of a reaction system, an internal standard Primer and a probe are added into a detection system, a section of sequence of human conserved gene beta-globin is selected, and Primer Express 3.0 software of ABI company is adopted to design the detection Primer and the probe, wherein the specific sequence is as follows:
an internal standard primer pair:
a forward primer: IC-F: 5'-TGCAACAGAAGCCCAGCTAT-3' (SEQ ID No. 5);
reverse primer: IC-R: 5'-AACCTGGAAGCCAACGAAAGA-3' (SEQ ID No. 6);
internal standard fluorescent probe:
IC-P:5’ROX-GACACAAATCAAACCATGCCACCACCT-3’BHQ2(SEQ ID No.7)。
example 2 preparation of primers
The designed primer and probe sequence are synthesized by a synthesis company, generally, instruments are adopted for automatic chemical synthesis, and a synthesis inspection qualified report needs to be provided.
Example 3: preparation of reaction System for detecting polymorphism of human ADRB1 Gene by fluorescent PCR two specific reaction solutions for detecting polymorphism of c.1165 site (G/C) of human ADRB1 Gene were prepared: ADRB1-1165G PCR reaction liquid and ADRB1-1165CPCR reaction liquid, wherein the PCR reaction liquid contains buffer liquid, magnesium ions, dNTP and other substances which are necessary for PCR reaction, and also contains a detection primer and a probe and an internal standard primer and a probe. The concentration of each PCR reaction solution component specifically detected and the sequence information of the included primers and probes are as follows:
TABLE 1 PCR reaction solution Components
Figure BDA0002629131350000081
Figure BDA0002629131350000091
(1) ADRB1-1165G PCR reaction solution
ADRB1-1165G:5’-CGCGCAGCAGAGCAGTCC-3’；
ADRB1-1165F:5’-CCGACCGCCTCTTCGTCTTCTT-3’；
ADRB1-1165P:5’FAM-CCTTGCGGAAGTCGGGGCTGCGGCA-3’Dabcyl
IC-F:5’-TGCAACAGAAGCCCAGCTAT-3’；
IC-R:5’-AACCTGGAAGCCAACGAAAGA-3’；
IC-P:5’ROX-GACACAAATCAAACCATGCCACCACCT-3’BHQ2
(2) ADRB1-1165C PCR reaction solution
ADRB1-1165C:5’-CGCGCAGCAGAGCAGTCG-3’；
ADRB1-1165F:5’-CCGACCGCCTCTTCGTCTTCTT-3’；
ADRB1-1165P:5’FAM-CCTTGCGGAAGTCGGGGCTGCGGCA-3’Dabcyl
IC-F:5’-TGCAACAGAAGCCCAGCTAT-3’；
IC-R:5’-AACCTGGAAGCCAACGAAAGA-3’；
IC-P:5’ROX-GACACAAATCAAACCATGCCACCACCT-3’BHQ2
Example 4: method for detecting c.1165 site (G/C) polymorphism of human ADRB1 gene by fluorescence PCR (polymerase chain reaction)
The first step is as follows: preparation of DNA
a. Obtaining a saliva sample from a subject to be tested;
b. obtaining DNA from saliva samples, and recommending the use of commercial kits for extracting saliva DNA;
c. determining the concentration and purity of DNA, wherein the concentration is required to be more than 10 ng/muL; OD260nm/OD280nm＝1.6～2.0。
The second step is that: preparation of PCR reaction system
PCR reaction system is prepared in a tube special for fluorescent quantitative PCR according to the following table
TABLE 2 PCR reaction System composition
ddH2O Adding to 25 μ L
PCR reaction solution 2.5μL
DNA polymerase (2.5U/. mu.L) 0.2μL
Genomic DNA About 80ng
Paraffin oil 20μL
The PCR reaction solution can be prepared by the preparation method provided in the table 1 of the example 3, and contains a detection primer and a probe for detecting the polymorphism of the c.1165 site (G/C) of the ADRB1 gene, and a PCR reaction solution containing an internal standard primer and a probe;
the third step: detection on machine
Temperature cycling and signal acquisition procedures were set according to the following table.
TABLE 3 PCR reaction procedure
Figure BDA0002629131350000101
Note: FAM and ROX dual channels are arranged at the position of "+" to collect fluorescence signals.
The fourth step: analysis results
And under the conditions of the PCR reaction system and the temperature cycle program, observing whether the target detection fluorescent signal in the specific PCR reaction system forms a logarithmic amplification S-shaped curve or not under the precondition that the internal standard signal forms a normal logarithmic amplification S-shaped curve, and if the logarithmic amplification S-shaped curve is formed, determining that the DNA sample to be detected is positive to the polymorphism type represented by the specific reaction system. For example: a certain sample to be detected forms a logarithmic amplification S-shaped curve in fluorescence signal detection (FAM channel) of an ADRB1-1165G reaction system and an ADRB1-1165C reaction system, and the sample is an ADRB1-1165G/C heterozygous type.
FIG. 1 is a detection diagram of a c.1165 locus kit of ADRB1 gene of a certain sample, wherein the detection diagram shows that internal standard fluorescent signals in ADRB1-1165G reaction system and ADRB1-1165C reaction system are both qualified, no amplification curve is formed when the fluorescent signal (FAM channel) is detected in the ADRB1-1165G reaction system, and a logarithmic amplification "S" type curve is formed when the fluorescent signal (FAM channel) is detected in the ADRB1-1165C reaction system, so that the sample is judged to be ADRB11165C/C homozygous.
FIG. 2 is a sequence chart of the sample ADRB1 gene c.1165 site in FIG. 1, and the sequence result shows that the sample is homozygous for ADRB 11165C/C. The detection result of the kit is consistent with the sequencing result.
Note that:
the above embodiments are merely illustrative of the technical ideas and features of the present invention, and the purpose thereof is to enable those skilled in the art to understand the contents of the present invention and implement the present invention, and not to limit the protection scope of the present invention. All equivalent changes and modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.

Claims (2)

1. A fluorescence PCR method for detecting the nucleotide polymorphism of the 1165 site of the ADRB1 gene is characterized in that a detection system of the fluorescence PCR method comprises the following primers and fluorescence probes, and the sequences are as follows:
reverse primer 1:
ADRB1 1165G:5’-CGCGCAGCAGAGCAGTCC-3’(SEQ ID No.1)；
reverse primer 2:
ADRB1 1165C:5’-CGCGCAGCAGAGCAGTCG-3’(SEQ ID No.2)；
a forward primer:
ADRB1 1165F:5’-CCGACCGCCTCTTCGTCTTCTT-3’(SEQ ID No.3)；
a fluorescent probe:
ADRB 11165P 5 'fluorophore-CCTTGCGGAAGTCGGGGCTGCGGCA-3' quencher (SEQ ID No. 4).
2. The fluorescent probe of claim 1, wherein the fluorescent group at the 5 'end of the fluorescent probe is FAM, TET, VIC, HEX or ROX, and the quencher at the 3' end is BHQ-1, BHQ-2, Dabcyl, Eclipse, or TAMRA.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106520979A (en) * 2016-11-30 2017-03-22 武汉海吉力生物科技有限公司 Nucleic acid, kit and method for detecting G1165C polymorphism of human ADRB1 gene
CN106834466A (en) * 2017-02-08 2017-06-13 重庆迪威纳生物技术有限公司 The method and kit in a kind of detection ADRB1 gene single nucleotide polymorphisms site
CN109943629A (en) * 2019-03-22 2019-06-28 踏石生物科技（苏州）有限公司 A kind of ADRB1 G1165C genotype detection kit and its detection method

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106520979A (en) * 2016-11-30 2017-03-22 武汉海吉力生物科技有限公司 Nucleic acid, kit and method for detecting G1165C polymorphism of human ADRB1 gene
CN106834466A (en) * 2017-02-08 2017-06-13 重庆迪威纳生物技术有限公司 The method and kit in a kind of detection ADRB1 gene single nucleotide polymorphisms site
CN109943629A (en) * 2019-03-22 2019-06-28 踏石生物科技（苏州）有限公司 A kind of ADRB1 G1165C genotype detection kit and its detection method

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