CN110643689A - TaqMan probe real-time fluorescent PCR method for detecting rs6313 site of HTR2A gene and primer probe combination thereof - Google Patents
TaqMan probe real-time fluorescent PCR method for detecting rs6313 site of HTR2A gene and primer probe combination thereof Download PDFInfo
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Abstract
The invention belongs to the field of gene diagnosis, and discloses a TaqMan probe real-time fluorescence PCR method for detecting an rs6313 locus (HTR2A, 102C > T) of an HTR2A gene and a primer probe combination thereof. Wherein: an upstream primer FpC-T: 5'-GCTCTACAGTAATGACTTTAACTTC-3', an upstream primer FpT-T: 5'-GCTCTACAGTAATGACTTTAACTTT-3', a downstream primer Rp: 5'-GATGAAGTAAGGAGAGACACGAC-3', Probe Probe: 5 '-FAM-TAACACTTCTGATGCATTTAACTGGAC-BHQ 2-3'. After the PCR reaction is completed, the result can be analyzed by the presence or absence of an amplification curve. The invention has the advantages of simple operation, high resolution, no pollution, high flux and the like.
Description
Technical Field
The invention belongs to the field of pharmacogenomics and gene detection, and particularly relates to a specific primer and probe design for an rs6313 locus allele of an HTR2A gene and a detection method.
Background
Recent statistical data published by the World Health Organization (WHO) show that the total population suffering from global depression is about 3.22 hundred million, and the population proportion of global depression patients is estimated to be 4.4%. The prevalence rate of Chinese depression is 4.2%, and the conservative estimation shows that the prevalence rate of Chinese depression currently exceeds 5800 ten thousand. Depression is now the fourth disease in the world, and antidepressant drugs such as fluoxetine (fluooxetine), paroxetine (parooxetine), fluvoxamine (fluvoxamine) and the like are the main therapies for depression treatment. Despite the wide variety of drugs, approximately 30-50% of patients do not respond to treatment. A random control study finds that after 6-8 weeks, only 35-45% of major depressive patients receive the clinical standard dose treatment of antidepressant drugs, and the patients can return to the functional level without any significant depressive symptom before onset.
Among the many factors that affect differences in the effects of action, genetic polymorphisms are recognized as one of the key factors. Anti-depression pharmacogenomics research discovers some genes related to depression drug response, wherein HTR2A (5-hydroxytryptamine receptor 2A, 5-hydroxytryptamine 2A receptor) is one of the hot spots of research. The serotonin 2A receptor encoded by the HTR2A gene is a member of the serotonin receptor family, is expressed in all neocortical areas of the brain, and is widely distributed in and around the central nervous system. 5-HTR2A may mediate the 5-hydroxytryptamine signaling system and play an important role in regulating mood, mood and stress in the central nervous system.
The HTR2A gene has significant genetic polymorphisms, the most common of which is the variation at c.102t > C (rs 6313). Clinical studies show that the HTR2A T102C gene polymorphism is closely related to the curative effect of the current most clinically used antidepressant drug serotonin reuptake inhibitor (SSRIs). Kato M et al (2006) have found that after 4 weeks of treatment with fluoxetine in 100 depressed patients, patients with the C allele had better improvement in depression symptoms than patients with the T allele (P0.022) and patients with the CC genotype responded better to fluoxetine. The correlation of the T102C gene polymorphism with SSRIs treatment response was also confirmed in a study of 265 major depressive patients by Kishi T et al (2010). In addition, a meta-analysis of 1775 subjects from 11 studies also showed that the HTR2A T102C gene polymorphism was significantly associated with high response rates to antidepressants, and that patients carrying the C allele responded better to SSRIs. Therefore, developing a simple, rapid and sensitive genotyping technology of HTR2A T102C locus has important clinical value for determining the genetic characteristics of patients, predicting the response of the patients to antidepressant drugs and establishing individualized treatment schemes.
The current real-time fluorescent quantitative PCR method is widely applied to gene polymorphism typing detection, and is a method which adds fluorescent groups or fluorescent dyes into a common PCR reaction system, monitors the progress of the whole PCR reaction in real time by utilizing fluorescent signal accumulation monitoring, and finally carries out quantification or qualitative detection by a standard curve or a fluorescence value measured in an exponential amplification period. The combination of the TaqMan probe and the real-time fluorescent PCR is the most common genotyping method at present, and has the advantages of high sensitivity, strong specificity, good stability, realization of multiple reactions, no need of uncovering after the reactions, avoidance of secondary pollution and the like. At present, a gene detection technology aiming at HTR2Ars6313 locus typing and commercial kit products are rare, so that the development of a rapid, simple and reliable gene typing technology of the HTR2Ars6313 locus based on a real-time fluorescence PCR method of a TaqMan probe has very important clinical significance and value.
Disclosure of Invention
The invention aims to provide a more simple, convenient, rapid, high-throughput and high-specificity real-time fluorescence PCR method for detecting human HTR2A gene rs6313 locus allele on the basis of the existing PCR technology so as to overcome the defects existing in the prior art; and correspondingly obtaining specific primer and probe combination.
Specifically, the invention mainly designs the following specific primers and probe combinations:
upstream primer FpC: 5'-GCTCTACAGTAATGACTTTAACTTC-3'
Upstream primer FpT: 5'-GCTCTACAGTAATGACTTTAACTTT-3'
A downstream primer Rp: 5'-GATGAAGTAAGGAGAGACACGAC-3'
Probe Probe: 5 '-FAM-TAACACTTCTGATGCATTTAACTGGAC-BHQ 2-3'
Wherein, FAM is 6-carboxyfluoroscein; BHQ2 is Black Hole Quencher-2;
based on the specific primer and the probe combination, a corresponding detection kit can be prepared; a detection module (which can be combined with computer information processing) and even a genotyping detection instrument aiming at the rs6313 locus allele of the HTR2A gene can also be constructed.
The TaqMan probe real-time fluorescence PCR method for detecting rs6313 locus allele provided by the invention comprises the following steps:
(1) designing three specific primers of a target gene, wherein the three specific primers comprise two upstream primers, a downstream primer and a specific probe;
(2) extracting the genome DNA of a sample to be detected;
(3) configuring a reaction system
Respectively adding corresponding specific upstream primer, general downstream primer and probe, primer and probe of internal reference gene, genomic DNA of tested sample, 5 XHS HiTaq Buffer (Mg)2+Plus), Solution I (10X), dNTP (2.5mM), hotspot HiTaq DNA polymerase, mixed according to a certain proportion (Mix);
(4) PCR reaction
The prepared reaction system is put into a real-time fluorescent PCR instrument ViiATM7, amplifying, and judging the genotype of the detected sample by the existence of an amplification curve long after the PCR is finished.
Preparing a quality control sample and a detection sample:
(1) quality control sample type
And selecting a sample of rs6313 locus detected by first-generation sequencing, wherein the samples with the genotypes of CC, CT and TT are respectively used as quality control samples.
(2) Preparation of test samples
Extracting a blood sample or a tissue sample by using a DNA extraction kit to obtain genome DNA, and detecting the concentration and the quality of the DNA by using an ultraviolet spectrophotometer NanoDrop; the quality-qualified DNA was diluted to 50 ng/. mu.l or 10 ng/. mu.l for use.
In order to achieve the best effect, the invention is also optimized and limited as follows.
Adding 200nM of specific upstream primer, 200nM of general downstream primer, 100nM of fluorescent probe, 150-200 nM of upstream and downstream primers of internal reference gene, 100nM of probe, 10-50 ng of detected sample genome DNA, 0.1375 μ l of hotspot HiTaq DNA polymerase and 5 XHS HiTaq Buffer (Mg) into a reaction tube by 10 μ l of a single-tube reaction system2+Plus)2μl、Solution I(10×)1μl、dNTP(2.5mM)1μl、ddH2O was replenished to a final volume of 10. mu.L.
The PCR parameters were set as follows: pre-denaturation at 95 ℃ for 10 min; denaturation at 95 ℃ for 15sec, and annealing at 60 ℃ for 40 sec; for a total of 40 cycles.
Compared with the prior art, the method has the obvious advantages that:
1. the specificity is strong. In the invention, the specific ARMS primer and probe combination designed aiming at the rs6313 locus has high specificity, and the specific amplification of a template is ensured.
2. The sensitivity is high. In the present invention, the ARMS primers used, in addition to their basic functions, and in combination with the probes, serve to block the amplification of other genotype templates. Meanwhile, only 10ng-20ng of genome DNA is needed to carry out accurate rs6313 locus allele detection.
3. Time and material saving
The invention saves time and consumables to a great extent, is simple to operate, combines with designed reaction programs, only needs 50min to 1h in the detection process, and can completely complete the whole experiment within 2 h.
4. High flux
By using the method, 96 or 384 samples can be simultaneously detected at one time by combining the configuration condition of the current instrument, and the rs6313 locus allele detection is carried out at high flux, so that the detection efficiency is improved.
5. Simple analysis of results
Compared with the traditional PCR detection method, the method disclosed by the invention is easier to analyze the experimental result: the genotype of each sample can be judged by observing the presence or absence of the amplification curve.
Drawings
FIG. 1 shows the result of amplification of a sample with CC genotype at locus rs 6313. In the sample, the reference gene ACTB is normally amplified, the FAM channel in the C reaction system is normally amplified, and the FAM channel in the T reaction system is not amplified (or the FAM channel of the sample in the C reaction system and the FAM channel of the T reaction system are amplified, but Ct (A reaction) -Ct (C reaction) >3), so that the genotype of the sample is CC (complementary strand GG) homozygous.
FIG. 2 shows the result of amplification of a sample with CT as the genotype at the rs6313 locus. The reference gene ACTB in the sample is normally amplified, FAM channels in a C reaction system and a T reaction system show normal amplification, and the absolute value of the difference between the Ct values of the two reactions is less than 3, so that the sample is a genotype CT (complementary strand GA) heterozygote.
FIG. 3 shows the result of amplification of a sample with the genotype of the rs6313 locus TT. In the sample, the reference gene ACTB is normally amplified, the FAM channel in the T reaction system is normally amplified, and the FAM channel in the C reaction system is not amplified (or the FAM channel of the sample in the C reaction system and the T reaction system is amplified, but Ct (A reaction) -Ct (C reaction) >3), so that the genotype of the sample is TT (complementary strand AA) homozygous.
Detailed Description
The invention is described in further detail below with reference to the figures and specific examples.
The specific embodiment is as follows: and detecting the rs6313 locus allele by using a TaqMan probe real-time fluorescent PCR method.
1. Extraction and dilution of DNA samples
Collecting venous Blood with vacuum Blood collection tube anticoagulated with Ethylene Diamine Tetraacetic Acid (EDTA) according to conventional method, and extracting DNA with QIAamp DNA Mini Blood Kit (Qiagen, Germany); the concentration of the extracted DNA was measured using NanoDrop 2000 (A260/280: 1.95-2.15). Using the above method, 104 samples of the Boolean DNA were each measured for concentration, and then PCR-grade H was used2O diluted the sample to 10 ng/. mu.l.
2. Design of primers and probes
Specific upstream primers: c allele: 5'-GCTCTACAGTAATGACTTTAACTTC-3', respectively; t allele: 5'-GCTCTACAGTAATGACTTTAACTTT-3', respectively;
a universal downstream primer: 5'-GATGAAGTAAGGAGAGACACGAC-3', respectively;
and (3) probe: 5 '-FAM-TAACACTTCTGATGCATTTAACTGGAC-BHQ 2-3';
wherein, FAM is 6-carboxyfluoroscein; BHQ2 is Black Hole Quencher-2.
The composition is entrusted to Shanghai company.
3. Sample detection
In a real-time fluorescent PCR instrument ViiATM7, for the same sample, respectively carrying out two-channel fluorescence collection on two specific upstream primers, a universal downstream primer and a probe, and a primer and a probe of an internal reference gene by respectively utilizing a FAM channel and a VIC channel, wherein the reaction system comprises: 200nM of upstream primer (allele C or T), 200nM of downstream primer, 100nM of fluorescent probe, 150-200 nM of upstream primer and downstream primer of internal reference gene respectively, 100nM of probe, 10-50 ng of detected sample genome DNA, 0.1375 μ l of Hotstart HiTaq DNA polymerase (Fipeng Biotech Co., Ltd.), and 5 XHSHiTaq Buffer (Mg)2+Plus)2μl、Solution I(10×)1μl、dNTP(2.5mM)1μl、ddH2O was replenished to a final volume of 10. mu.L.
4. Analysis of Experimental results
The internal reference gene is used as quality control, and an amplification curve must appear to represent that the quality of the DNA sample is qualified.
If the FAM channel in the C reaction system presents normal amplification and the FAM channel in the T reaction system does not have amplification (or the FAM channels of the sample in the C reaction system and the T reaction system are amplified, but Ct (A reaction) -Ct (C reaction) >3), the genotype of the sample is CC (complementary strand GG) homozygote;
if FAM channels in the C reaction system and the T reaction system show normal amplification and the absolute value of the difference of the two reaction Ct values is less than 3, the sample is a genotype CT (complementary strand GA) heterozygote;
if the FAM channel in the T reaction system shows normal amplification and the FAM channel in the C reaction system does not amplify (or the FAM channels of the sample in the C reaction system and the T reaction system are amplified, but Ct (A reaction) -Ct (C reaction) >3), the genotype of the sample is TT (complementary strand AA) homozygous.
And (3) verification experiment: mass sequencing of 100 samples and comparison of rs6313 locus allele detection method
100 samples were randomly drawn for mass sequencing, and the sequencing results were displayed using Excel tables, so as to recheck the experimental results of the present invention. Comparing the Massary sequencing result with the detection method result of the invention (see Table 1), the negative and positive coincidence rate between the two is 100%.
TABLE 1
In the embodiment, the standard substances known as CC and TT allele homozygous type and CT heterozygous type are prepared in the early stage and used as the positive control of the detection system, so that the accuracy of judging the homozygous type or the heterozygous type of the rs6313 locus allele of the sample to be detected is further improved.
The embodiment can be suitable for detecting the rs6313 allele of the human peripheral blood whole genome DNA sample, has important guiding significance for improving the curative effect of the antidepressant and evaluating the occurrence of depression, and is beneficial to effectively utilizing the antidepressant guided under the rs6313 allele.
<110> Shaanxi Baimei Gene GmbH
<120> TaqMan probe real-time fluorescence PCR method for detecting rs6313 locus of HTR2A gene and primer probe combination thereof
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Claims (7)
1. A specific primer probe combination applied to a TaqMan probe real-time fluorescence PCR method for detecting rs6313 locus allele of HTR2A gene is characterized by comprising the following specific upstream primer (ARMS), universal downstream primer and probe sequence:
an upstream primer FpC: 5'-GCTCTACAGTAATGACTTTAACTTC-3';
an upstream primer FpT: 5'-GCTCTACAGTAATGACTTTAACTTT-3';
a downstream primer Rp: 5'-GATGAAGTAAGGAGAGACACGAC-3', respectively;
probe Probe: 5 '-FAM-TAACACTTCTGATGCATTTAACTGGAC-BHQ 2-3';
wherein, FAM is 6-carboxyfluoroscein; BHQ2 is Black Hole Quencher-2.
2. Use of the specific primer probe combination of claim 1 for preparing a detection kit for rs6313 locus allele of HTR2A gene.
3. Use of the specific primer probe combination of claim 1 in constructing a detection module or a genotyping detection instrument for the rs6313 locus allele of the HTR2A gene.
4. A TaqMan probe real-time fluorescence PCR method for detecting rs6313 locus allele of HTR2A gene, which is used for non-disease diagnosis purposes, comprises the following steps:
(1) taking a specific primer probe combination of claim 1;
(2) extracting the genome DNA of a sample to be detected;
(3) configuring a reaction system
Respectively constructing the same reaction systems in the two reaction tubes, and carrying out double-channel fluorescence acquisition on the FAM channel and the VIC channel; the reaction system is that the specific primer probe combination, the primer and the probe of the internal reference gene, the genome DNA of the tested sample and 5 XHS HiTaq Buffer (Mg) are added into a reaction tube2+Plus), Solution I (10X), dNTP, hotspot HiTaq DNA polymerase, mixing;
(4) PCR reaction
The prepared reaction system is put into a real-time fluorescent PCR instrument ViiATM7, amplifying, and judging the genotype of the detected sample according to the existence of an amplification curve after PCR is finished.
5. The method of claim 4, wherein:
the selected reference gene is ACTB, and the designed reference gene primers and probes are as follows:
an upstream primer ACTB-F: 5'-CAGCAGATGTGGATCAGCAAG-3';
the downstream primer ACTB-R: 5'-GCATTTGCGGTGGACGAT-3', respectively;
probe ACTB-P: 5 '-HEX-AGGAGTATGACGAGTCCGGCCCC-BHQ 2-3';
wherein HEX is 6-carboxy-hexachlorofluoroescein; BHQ2 is Black Hole Quencher-2.
6. The method of claim 4, wherein:
adding a specific upstream primer of 200nM, a general downstream primer of 200nM and a fluorescent probe of 100nM into a reaction tube by taking 10 mul of a single-tube reaction system; 150-200 nM of upstream and downstream primers of internal reference gene respectively, 100nM of probe, 10-50 ng of detected sample genome DNA, 0.1375 ul of Hotstart HiTaq DNA polymerase and 5 XHS HiTaq Buffer (Mg)2+Plus)2μl、Solution I(10×)1μl、2.5mM dNTP1μl、ddH2O was replenished to a final volume of 10. mu.L.
7. The method of claim 6, wherein:
the PCR reaction parameters were set as follows: pre-denaturation at 95 ℃ for 10 min; denaturation at 95 ℃ for 15sec, and annealing at 60 ℃ for 40 sec; for a total of 40 cycles.
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|---|---|---|---|
| CN201911037273.2A CN110643689A (en) | 2019-10-29 | 2019-10-29 | TaqMan probe real-time fluorescent PCR method for detecting rs6313 site of HTR2A gene and primer probe combination thereof |
| PCT/CN2020/084692 WO2021082358A1 (en) | 2019-10-29 | 2020-04-14 | Taqman probe real-time fluorescent pcr method for detecting rs6313 site of htr2a gene and primer and probe combination thereof |
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| CN201911037273.2A CN110643689A (en) | 2019-10-29 | 2019-10-29 | TaqMan probe real-time fluorescent PCR method for detecting rs6313 site of HTR2A gene and primer probe combination thereof |
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| WO2021082358A1 (en) * | 2019-10-29 | 2021-05-06 | 陕西佰美基因股份有限公司 | Taqman probe real-time fluorescent pcr method for detecting rs6313 site of htr2a gene and primer and probe combination thereof |
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| US8401801B2 (en) * | 2003-02-20 | 2013-03-19 | Mayo Foundation For Medical Education And Research | Methods for selecting medications |
| US7387887B2 (en) * | 2004-04-20 | 2008-06-17 | University Of Utah Research Foundation | Nucleic acid melting analysis with saturation dyes |
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| WO2021082358A1 (en) * | 2019-10-29 | 2021-05-06 | 陕西佰美基因股份有限公司 | Taqman probe real-time fluorescent pcr method for detecting rs6313 site of htr2a gene and primer and probe combination thereof |
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