CN112301120A - Probe, primer and kit for detecting ADRB1 gene polymorphism - Google Patents
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Abstract
The invention provides a probe for detecting ADRB1 gene polymorphism, wherein the sequence of the probe is shown in SEQ ID No. 1, or both ends of the sequence of the SEQ ID No. 1 are modified by groups. The invention also provides a primer for detecting ADRB1 gene polymorphism, and the sequence of the primer is shown in SEQ ID NO. 2 and SEQ ID NO. 3. The probe and the primer for detecting the ADRB1 gene polymorphism have the advantages of high sensitivity, good specificity, rapidness, high-throughput detection and the like when the ADRB1 gene polymorphism is detected.
Description
Technical Field
The invention relates to the field of molecular biology, in particular to a probe, a primer and a kit for detecting ADRB1 gene polymorphism.
Background
The beta-adrenoceptor (beta-adrenoceptor) is a subfamily of adrenoceptors, belonging to the G-protein coupled receptor superfamily, comprising beta1、β2And beta3Three different subtypes. The receptor regulates cAMP and L type Ca in cells by coupling with Gs protein2+The opening frequency of the channel is the target of the action of beta receptor agonist and beta receptor blocker. Beta is a1Polymorphism of the receptor-encoding gene ADRB1 affecting beta receptor blockers such as metoprololHas therapeutic effect. The ADRB1Gly389Arg (rs1801253) polymorphism results in two types of receptors at positions Arg389 and Gly389, wherein the coupling efficiency of the Arg389 type receptor to the G protein is higher than that of the Gly389 type receptor. The degree of blood pressure reduction of an Arg389 homozygote hypertensive patient after metoprolol application is 3 times that of an individual with a Gly389Arg heterozygote genotype; the improvement of left ventricular ejection fraction is better after the Arg389 homozygote genotype heart failure patient is treated by carvedilol and metoprolol. The clinician is advised to apply beta1ADRB1 polymorphism detection is carried out before receptor blocking drugs, and the dosage is adjusted according to the genotype of the receptor blocking drugs, so that the curative effect is improved, and the occurrence of adverse reactions is reduced.
The mutation detection method commonly applied at present is a direct DNA sequencing method, and the PCR product is directly subjected to DNA sequence analysis, so that mutation sites can be defined, but the defects of time and labor waste, high cost, inapplicability to detection of a large number of samples and the like exist. Therefore, it is necessary to develop a method suitable for the detection of large-scale samples.
Disclosure of Invention
The invention aims to provide a probe for detecting ADRB1 gene polymorphism, wherein the sequence of the probe is shown in SEQ ID NO. 1, or groups are modified at two ends of the sequence of the SEQ ID NO. 1;
wherein the modifying group is: the 5' modifying group is a fluorophore, such as FAM; 3' modifying groups are quenching groups, such as Dabcyl;
specifically, the probe for detecting ADRB1 gene polymorphism provided by the invention is as follows: 5' -FAM-CCGCAAGGCCTTCCAGNGACTGCTCTGCGG-Dabcyl-3′；
Wherein N is a variant base, specifically G > C;
the probe comprises a loop sequence and a stem sequence, wherein the loop sequence is 6-25bp (5' -AGGCCTTCCAG)NGACTGCTC-3 ') flanked by two reverse complementary stem sequences (5' -CCGCA … … TGCGG-3).
The design principle of the probe is as follows: according to the ADRB1 gene polymorphism rs1801253, probes and primers are designed, and the probes are required to be in a stem-loop state when the DNA template does not exist at the annealing temperature. The quenching group adopted by the probe is Dabcyl, the quenching space range is very small, and the fluorescence can be well quenched only when the molecular beacon is in a stem-loop structure by matching with a fluorescent group with short emission wavelength, such as FAM. When the loop sequence is complementary with the target DNA sequence, the optimal PCR primer is determined to be 40-45bp in size, and the length of the PCR product is 100-150 bp.
The invention also provides a primer for detecting ADRB1 gene polymorphism, wherein the sequence of the primer is shown as SEQ ID NO. 2 (primer 1) and SEQ ID NO. 3 (primer 2):
primer 15 '-AGGTGACACTATAGAATAGCCTTCAACCCCATCATCTA-3'
Primer 25 '-GCAAGCCCTCACGTAGCGAACAGGCTCGAGTCGCTGTC-3'.
The present invention also provides a kit for detecting ADRB1 gene polymorphism, which comprises the probe and/or primer as described above;
the kit further comprises Taq enzyme, dNTP, and/or Mg2+And instructions for use.
The present invention also provides a method for detecting ADRB1 gene polymorphism, which comprises the steps of:
(1) collecting a sample and extracting DNA;
(2) carrying out fluorescent quantitative PCR reaction by using the probe and the primer;
(3) analyzing the result by using the matched software of the PCR instrument, defining proper base lines and threshold values according to an amplification curve, and displaying different genotypes based on the difference of Tm values displayed by formed hybridization peaks;
wherein the Tm value is 67 ℃ for the wild type, and the Tm value is 74 ℃ for the mutant type.
Wherein the total amount of PCR reaction is 15ul (including PCR Mix 7.5ul, forward primer solution 0.5ul and reverse primer solution 0.5ul, probe 0.1ul, sample DNA 2ul, sterilized double distilled water 4.4 ul); carrying out reaction on a fluorescent quantitative PCR instrument, wherein the PCR reaction condition is pre-denaturation at 92-97 ℃ for 5-15 minutes; denaturation at 92-97 deg.C for 10-30 s, annealing at 57-65 deg.C for 10-30 s, extension at 70-75 deg.C for 10-30 s, and 40-50 cycles; extension at 72 ℃ for 10 min; denaturation at 92-97 deg.C for 1 min, renaturation at 40 deg.C for 1 min, and real-time monitoring of fluorescence signal at 45-80 deg.C, and recording 5 times at 1 deg.C per time.
The invention detects ADRB1 gene polymorphism, can realize rapid screening of ADRB1 gene polymorphism, and assists clinical adjustment of drug dosage for patients according to genotypes of the patients, so as to improve curative effect and reduce adverse reactions.
The invention utilizes a fluorescent probe capable of specifically identifying a nucleic acid sequence, releases fluorescent dye through conformational change after hybridization with a target sequence, and judges a typing result according to peak patterns at different temperatures generated after hybridization. Under the condition that no target DNA exists, the fluorescent group and the quenching group can be stably combined together, and no fluorescent signal can be detected; when the target DNA exists, the structure of the fluorescence labeling probe is damaged, and the fluorescent group and the quenching group are separated from each other, so that the fluorescence signal can be detected. Compared with other genetic typing techniques, the method is simple to operate, can finish 96 cases of detection within 2-3 hours, has the advantages of high sensitivity, good specificity, rapidness, high-flux detection and the like, obtains a detection result by directly detecting a fluorescent signal in a PCR process, has clear genotyping, does not need PCR post-treatment or electrophoresis detection, and can realize real closed-tube operation.
Drawings
Three peak types (GG type, CG type, CC type) of rs1801253 locus in figure 1
FIG. 2 shows the results of precision experiments (CG type for two peaks and GG type for one peak)
FIG. 3, results of detection Limit experiments
FIG. 4 is a graph showing melting curves of multiple samples
Detailed Description
Example 1: probe and primer design
The invention designs a probe and a primer sequence aiming at ADRB1 SNP locus. The specific principle is that the fluorescent probe and a target sequence are hybridized to release fluorescent dye through conformational change, and a genotyping result is judged according to peak graphs and Tm values of different temperatures generated after hybridization. Under the condition that no target DNA exists, the fluorescent group and the quenching group can be stably combined together, and no fluorescent signal can be detected; when the target DNA exists, the structure of the fluorescence labeling probe is damaged, and the fluorescent group and the quenching group are separated from each other, so that the fluorescence signal can be detected.
Designing a probe and a primer to realize that the probe is in a stem-loop state when the template does not exist at the annealing temperature, wherein the probe comprises a loop sequence and stem sequences with two sides in reverse complementarity, the total length is 30bp, and the loop sequence is 6-25bp (5' -AGGCCTTCCAG)NGACTGCTC-3'), wherein 5 bases at two ends form a stem sequence; and the stem sequences at the two ends are just complementary; the quenching group adopted is Dabcyl, and is matched with a fluorescent group FAM with short emission wavelength. The loop sequence is complementary with the target DNA sequence, the size of the PCR primer is determined to be 38-40bp, and the length of the PCR product is 179 bp.
The primer and probe sequences were as follows:
sequence of primer 1: 5'-AGGTGACACTATAGAATAGCCTTCAACCCCATCATCTA-3', respectively;
sequence of primer 2: 5'-GCAAGCCCTCACGTAGCGAACAGGCTCGAGTCGCTGTC-3', respectively;
sequence of the probe: 5' -FAM-CCGCAAGGCCTTCCAGNGACTGCTCTGCGG-Dabcyl-3′。
Wherein N is a variant base, specifically G > C;
the above-mentioned probe and primer were synthesized by Biotechnology engineering (Shanghai) Ltd.
Example 2: standard substance for detecting different genotypes
1. The plasmid ADRB1 is used for constructing and preparing a wild type standard plasmid containing a target gene rs1801253 locus and a mutant type standard plasmid (a plasmid source and the synthesis of the plasmid containing the target gene are synthesized by the company of Biotechnology engineering (Shanghai); the accuracy of the sequence is determined by sanger sequencing, the wild type standard plasmid rs1801253 genotype is GG, the mutant type standard plasmid rs1801253 genotype is CC, and the DNA concentration of the standard plasmid is normalized to 10 ng/ul.
2. The probe and primer in example 1 were used.
3. And (3) PCR reaction system:
1) sequentially adding 7.5ul of PCR Mix, 0.5uM of primer 1 solution, 0.5uM of primer 2 solution and 0.1uM of probe into each PCR reaction hole, then respectively adding 2ul of wild type standard plasmid DNA, mutant type standard plasmid DNA and mixed type DNA (the wild type standard plasmid and the mutant type standard plasmid are mixed according to a ratio of 1: 1) into 3 different PCR reaction holes, and supplementing 15ul of sterilized redistilled water;
2) carrying out reaction on a fluorescent quantitative PCR instrument, wherein the PCR reaction condition is pre-denaturation at 95 ℃ for 5 minutes; denaturation at 95 ℃ for 30 seconds, annealing at 60 ℃ for 30 seconds, extension at 72 ℃ for 30 seconds, and 45 cycles; extension at 72 ℃ for 10 min; denaturation at 95 ℃ for 1 min, renaturation at 40 ℃ for 1 min, and real-time monitoring of fluorescence signals at a melting temperature of 45-80 ℃ with 5 recordings at 1 ℃ per temperature rise.
4. The results of analysis by SLAN software equipped with a PCR instrument revealed different genotypes based on the difference in Tm values exhibited by the formed hybridization peaks. The peak of the rs1801253 site appears at 67 ℃ and is GG genotype, the peak appears at 74 ℃ and is CC genotype, and the peaks at both positions are CG genotype (figure 1).
Example 3: performance analysis experiment of detection method
1. Precision experiment
Wild type standard plasmid DNA and mixed type DNA (rs1801253 plasmid source, and synthesis of plasmid containing target gene synthesized by Biotechnology engineering (Shanghai) GmbH. wild type standard plasmid and mutant type standard plasmid are mixed at a ratio of 1: 1) are taken, each part is detected for 3 times every day for 5 days continuously, the amplification reaction procedure adopts the method in example 2, and the result is shown in figure 2. The fluorescent PCR amplification reaction has good repeatability (the coincidence rate is more than 95 percent, and the Ct value variation coefficient CV of the detection result is less than 5 percent).
2. Coincidence rate experiment
DNA samples of 20 healthy volunteers in Shanghai regions are selected, the rs1801253 locus detection is carried out by applying the method of the embodiment 2, meanwhile, the sanger sequencing method is applied for verification, the consistency of the detection results of the two methods is compared, and the result shows that the consistency degree of the typing result of the embodiment 2 and the conformance rate experiment of the sanger sequencing method is 100%, and the consistency is excellent.
3. Limit of detection experiment
A portion of mixed DNA (wild type standard plasmid and mutant type standard plasmid mixed at a ratio of 1: 1) with known concentration was diluted to four concentrations of 10ng/ul, 5ng/ul, 2ng/ul and 1ng/ul, and each concentration was assayed in 3 tubes in parallel. The results show that the corresponding genotypes can be detected when the sample concentration is 10ng/ul, 5ng/ul, 2ng/ul and 1ng/ul, namely the lowest detectable concentration is 1ng/ul, and the results are shown in figure 3.
Example 4: detection of DNA in oral epithelial cell samples
1. And extracting the oral epithelial cell genome DNA of 92 healthy volunteers in Shanghai region by a silica gel adsorption method, detecting the concentration and purity of the DNA by an electrophoresis gel imaging method, and marking the DNA concentration of a sample to be detected to 10 ng/ul.
2. The detection method comprises the following steps: sequentially adding 7.5ul of PCR Mix, 0.5uM of forward primer solution, 0.5uM of reverse primer solution and 0.1uM of probe into each PCR reaction hole, simultaneously detecting a weak positive control (the genotype is CG), a negative control (the genotype is GG) and a sample to be detected, adding 2ul of DNA into each reaction hole, and supplementing 15ul of sterilized redistilled water; carrying out reaction on a fluorescent quantitative PCR detector, wherein the PCR reaction condition is pre-denaturation at 95 ℃ for 5 minutes; denaturation at 95 ℃ for 30 seconds, annealing at 60 ℃ for 30 seconds, extension at 72 ℃ for 30 seconds, and 45 cycles; extension at 72 ℃ for 10 min; denaturation at 95 ℃ for 1 min, renaturation at 40 ℃ for 1 min, and real-time monitoring of fluorescence signals at a melting temperature of 45-80 ℃ with 5 recordings at 1 ℃ per temperature rise.
3. The analysis results of SLAN by using the matched software show different genotypes based on the Tm value difference displayed by the formed hybridization peaks. The peak of the rs1801253 locus appears at 67 ℃ and is GG genotype, the peak appears at 74 ℃ and is CC genotype, and the peaks at the two positions are CG genotype. As shown in fig. 4, the melting curve of the multi-sample is shown, and the success rate of typing reaches 100%.
4. The 92 cases of oral epithelial cell genome DNA were subjected to sanger sequencing at the same time, and the detection results completely coincided with those of the present invention, thereby demonstrating that the accuracy of the result of the method of the present invention for detecting ADRB1 polymorphism is high.
Sequence listing
<110> Shanghai Zhongyou precise medical science and technology GmbH
<120> a probe, primer and kit for detecting ADRB1 gene polymorphism
<160> 3
<170> SIPOSequenceListing 1.0
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<213> Artificial Sequence (Artificial Sequence)
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<223> n = g or c
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ccgcaaggcc ttccagngac tgctctgcgg 30
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aggtgacact atagaatagc cttcggcccc atcatcta 38
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<213> Artificial Sequence (Artificial Sequence)
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gcaagccctc acgtagcgaa caggctcgag tcgctgtc 38
Claims (7)
1. A probe for detecting ADRB1 gene polymorphism, the sequence of the probe is shown as SEQ ID No. 1; or modified by groups at both ends of the sequence of SEQ ID No. 1.
2. The probe of claim 1, wherein the modifying group is: the modifying group of 5' is a fluorescent group FAM; the modifying group at 3' is a quencher Dabcyl.
3. The probe of claim 1, wherein the probe comprises a loop sequence and a stem sequence, wherein the loop sequence is 6-25bp, and is flanked by two reverse complementary stem sequences.
4. A primer for detecting ADRB1 gene polymorphism is characterized in that the sequence of the primer is shown as SEQ ID NO. 2 and SEQ ID NO. 3.
5. A kit for detecting ADRB1 gene polymorphism, characterized in that the kit comprises the probe of any one of claims 1 to 3 and/or the primer of claim 4.
6. The kit of claim 5, wherein the kit further comprises Taq enzyme, dNTP, and/or Mg2+And instructions for use.
7. A method for detecting ADRB1 gene polymorphism, characterized in that the method comprises the steps of:
(1) collecting a sample and extracting DNA;
(2) performing a fluorescent quantitative PCR reaction using the probe of any one of claims 1 to 3 and the primer of claim 4;
(3) analyzing the result by using the matched software of the PCR instrument, defining proper base lines and threshold values according to an amplification curve, and displaying different genotypes based on the difference of Tm values displayed by formed hybridization peaks; wherein the Tm value is 67 ℃ for the wild type, and the Tm value is 74 ℃ for the mutant type.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113981069A (en) * | 2021-11-10 | 2022-01-28 | 郑州华沃生物科技有限公司 | Primer and kit for detecting ADRB1 gene G1165C polymorphism, detection method and application thereof |
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