CN113462767A - FKBP5 gene methylation detection primer and kit based on pyrosequencing technology - Google Patents
FKBP5 gene methylation detection primer and kit based on pyrosequencing technology Download PDFInfo
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Abstract
The invention belongs to the technical field of methylation detection, and particularly relates to FKBP5 gene methylation detection primers and a kit based on a pyrosequencing technology. The FKBP5 gene methylation detection primer based on pyrosequencing technology provided by the invention can be used for amplifying CpG sites which contain methylation and are obviously related to the expression level of FKBP5 gene; the detection primer is prepared into a detection kit for detecting the methylation of the FKBP5 gene based on a pyrosequencing technology, and the methylation of the FKBP5 gene can be simply, conveniently, quickly and low-cost detected, so that the accurate evaluation of the methylation level of the FKBP5 gene is realized. The kit can effectively detect the cg03546163 site methylation of the FKBP5 gene, so as to obtain the methylation degree of the related gene of a sample to be detected, and is beneficial to judging the death risk of coronary heart disease and malignant pleural mesothelioma and the morbidity risk of systemic vasculitis.
Description
Technical Field
The invention belongs to the technical field of methylation detection, and particularly relates to FKBP5 gene methylation detection primers and a kit based on a pyrosequencing technology.
Background
According to statistics, the prevalence rate of cardiovascular diseases is in a continuous rising stage, and the mortality rate of cardiovascular diseases still accounts for the first cause of death of residents. Coronary heart disease is a complex disease caused by the combined action of environmental and genetic factors, and the change of genetic materials plays an important role in the occurrence and development of coronary heart disease. Therefore, the high-risk population of the coronary heart disease is screened according to the genetic risk factors, and the individual precise treatment is assisted, so that the morbidity and mortality of the coronary heart disease are effectively reduced, and the new trend of the future coronary heart disease prevention and treatment work is formed.
Malignant pleural mesothelioma is an invasive malignant tumor that occurs on the pleural and serosal surfaces, and has the disadvantages of easy spread of tumor cells, poor prognosis, and difficult diagnosis and treatment. At present, the diagnosis of the disease mainly depends on imaging and intrathoracic endoscopy, and due to the reasons of trauma, lack of specificity and the like, the diagnosis rate of malignant pleural mesothelioma is low and the death rate is high. Genetic testing can screen patients at high risk of death from early stage, thereby helping clinicians select appropriate treatment and surgical approaches to reduce mortality.
Systemic vasculitis is a high-incidence systemic immune vascular inflammatory disease in east asia, mainly characterized by recurrent oral, ocular, genital and skin lesions, and although the cause of systemic vasculitis is not clear at present, it has been proved to be a polygenic genetic disease significantly related to heredity. The clinical manifestations of systemic vasculitis are complex, the laboratory diagnosis standard and definite histopathological changes are lacked, misdiagnosis and missed diagnosis are easy to occur, and the genetic evaluation of individual suffering risks and disease diagnosis have important significance for prevention and treatment of systemic vasculitis.
FKBP5(FK506 binding protein 5) is deeply studied in aspects of nerve, endocrine system, psychological diseases and the like. Research shows that FKBP5 is closely related to individual depression, anxiety, psychological trauma, lipid metabolism, stress response and the like, and the expression level of FKBP5 gene is expected to be one of laboratory indexes for evaluating individual psychological states, the degree of nervous system function damage and the like. The epigenetic modification plays an important role in the regulation of the expression level of FKBP5, and the methylation degree of a CpG locus cg03546163 positioned in a 5' UTR region is proved to be in significant negative correlation with the expression level of FKBP5, thereby having great significance in the research of FKBP5 and related diseases.
Epigenetic modification refers to heritable changes that occur without changes in the base sequence of DNA, and mainly includes DNA methylation, histone acetylation, miRNA-based mechanisms, and the like. Epigenetic modifications are bridges connecting environmental and genetic factors, and epigenetic changes can affect the expression of genes to further change the functions of cells and organs, thereby causing changes in the internal environment; meanwhile, the change of internal environment can also cause epigenetic change of gene through a series of physiological and biochemical actions. Among them, DNA methylation is an important modification mode in epigenetic change, and is closely related to the regulation of gene expression, the ontogeny of organisms, and the occurrence and development of diseases. DNA methylation mainly refers to the process of covalent substitution of 5' cytosine by methyl within CpG (cytosine-phosphate-guanine) dinucleotide sequences catalyzed by DNA methyltransferases (DNMTs). DNA methylation is regulated by DNMTs and DNA demethylases, is a reversible regulation, can be inherited to progeny following cell division, and plays an important role in gene expression, chromosome stability and parental imprinting.
Epigenetic modifications can also be used as biomarkers, which can reflect the expression level of genes and environmental exposure conditions, and can also predict the risk of disease onset. The whole-genome relevance research reveals that the relation between the death risk of coronary heart disease and malignant pleural mesothelioma, the morbidity risk of systemic vasculitis and the like and the CpG locus cg03546163 of the coding FKBP5 gene is most obvious. Therefore, FKBP5 can be used as an index to assess the risk of morbidity and mortality in coronary heart disease, malignant pleural mesothelioma and systemic vasculitis. In addition, the methylation of FKBP5 can affect the transcription of genes and change the expression of proteins, thereby regulating the physiological function and structure of cells, and the expression of FKBP5 is also proved to be obviously related to various diseases (nerve, endocrine, cardiovascular system and the like), can be used as an index for predicting and evaluating various diseases, and provides basis and reference for the diagnosis and treatment of the diseases.
Therefore, FKBP5 methylation detection is expected to be used as an index of an effective risk prediction model, can screen high-risk populations which benefit most from early intervention treatment, and has important significance for guiding clinical reasonable medication, realizing individualized treatment and improving the life quality and prognosis of patients. Therefore, the method for detecting the methylation of the FKBP5 gene is simple, convenient, rapid and low in cost, and has important application value.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides an FKBP5 gene methylation detection primer based on a pyrosequencing technology, and an FKBP5 gene methylation detection kit prepared from the detection primer has the advantages of simplicity, rapidness, low cost, strong pertinence, high detection accuracy and the like in the methylation detection process of an FKBP5 gene.
In order to achieve the purpose, the invention adopts the technical scheme that:
the invention provides a FKBP5 gene methylation detection primer based on pyrosequencing technology, which comprises specific amplification primers FKBP5-F and FKBP5-R for amplifying a sequencing fragment required by FKBP5 gene and a sequencing primer FKBP5-S matched with the sequencing fragment required by FKBP5 gene, wherein the nucleotide sequences of the FKBP5-F, FKBP5-R and the FKBP5-S are respectively shown as SEQ ID NO 1, SEQ ID NO 2 and SEQ ID NO 3.
Preferably, the FKBP5 gene is a human FKBP5 gene. Specifically, the FKBP5 gene was a nucleotide fragment on chromosome 6 of the human NCBI release 37 genome sequence (chr6: 35655607-.
Preferably, the CpG site detected by the methylation detection primer is cg03546163 site of FKBP5 gene (chr6: 35654363).
The invention also provides application of the FKBP5 gene methylation detection primer based on the pyrosequencing technology in preparing an FKBP5 gene methylation detection product.
The invention also provides a kit for detecting the methylation of the FKBP5 gene based on the pyrosequencing technology, which comprises the primer for detecting the methylation of the FKBP5 gene based on the pyrosequencing technology.
According to the FKBP5 gene methylation detection kit based on the pyrosequencing technology, firstly, unmethylated cytosine (C) is converted into uracil (U) by using a bisulfite conversion mode, methylated cytosine (C) is kept unchanged, and an amplification product with a C/T single base difference is generated after amplification of an amplification primer, so that the aim of distinguishing different methylation conditions of cytosine (C) at the same site is fulfilled. And then quantitative detection of the target site is realized by utilizing a pyrosequencing technology, and the methylation rate of the original unit site is obtained according to the C/T ratio of the detected result, so that the method has the advantages of simplicity, convenience, rapidness, low cost and the like.
The invention has convenient detection, strong pertinence and high detection accuracy, and can be used for detecting the methylation degree of FKBP5 gene related to diseases such as tumor, cardiovascular system and the like. The CpG locus (cg03546163) methylation level of the FKBP5 gene is negatively related to the death risk of coronary heart disease and malignant pleural tumor and the morbidity risk of systemic vasculitis, and the cg03546163 locus can be used as a biomarker for predicting the morbidity risk and prognosis of an individual. Meanwhile, the methylation degree of cg03546163 is obviously and negatively related to the expression level of FKBP5 gene, FKBP5 is closely related to immunity and stress reaction of human body, and is a hot spot of research on nerve, cardiovascular system and psychological disease, and the invention of the methylation degree detection method of cg03546163 has great significance for research on FKBP5 gene related disease. The kit can conveniently and quickly provide effective means and basis for evaluating the death risk of individual coronary heart disease and malignant pleurisy and the potential possibility of the risk of systemic vasculitis on the molecular level, provides a new way and a new method for deeply researching the FKBP5 gene, and has wide application prospect.
Preferably, the kit also comprises a reagent for amplifying a sequencing fragment of the FKBP5 gene and a pyrosequencing reagent.
Further, reagents for amplifying sequencing fragment of FKBP5 gene include Pro Taq DNA Polymerase (5U/. mu.L), 10 XPro Taq PCR Buffer VER.2(Mg2+free)、MgCl2 Solution(50mM)、dNTP Mix(10mM each)。
Further, the pyrosequencing reagent comprises magnetic beads, pyrosequencing binding buffer, pyrosequencing annealing buffer, pyrosequencing denaturation buffer, pyrosequencing elution buffer, pyrosequencing substrate, pyrosequencing enzyme system and four dNTPs.
Preferably, the PCR reaction system of the kit is 50 μ L, including Pro Taq DNA Polymerase (5U/. mu.L) 0.25 μ L, 10 × Pro Taq PCR Buffer VER.2 (Mg)2+free)5μL，MgCl2 Solution(50mM)2μL，dNTP Mix(10mM each)1μL，FKBP5-F 2μL、FKBP5-R 2μL，DNA 50-150ng。
Specifically, the concentration of FKBP5-F and FKBP5-R was 5. mu.M.
Preferably, the PCR reaction procedure of the kit is: pre-denaturation at 95 ℃ for 10 min; denaturation at 95 ℃ for 30 sec; annealing at 60 ℃ for 50 sec; extension at 72 ℃ for 15 sec; a total of 50 cycles; extension at 72 ℃ for 5 min.
The invention also provides a use method of the detection kit for detecting FKBP5 gene methylation based on pyrosequencing technology in detecting AHRR gene methylation, which comprises the following steps:
1) extracting DNA in a sample;
2) performing bisulfite conversion on the extracted DNA;
3) performing PCR amplification by using the sample DNA treated in the step 2) as a template and using FKBP5 gene specific amplification primers FKBP5-F and FKBP 5-R;
4) pyrosequencing the amplification product obtained in the step 3) by using FKBP5 gene methylation sequencing primer FKBP5-S, thereby obtaining the methylation state of CpG sites in the FKBP5 gene sequencing fragment.
Preferably, the sample is selected from human blood, cell or tissue samples.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a primer for detecting FKBP5 gene methylation based on pyrosequencing technology, which can amplify CpG sites containing methylation and obviously related to the expression level of FKBP5 gene; the detection primer is prepared into a detection kit for detecting the methylation of the FKBP5 gene based on a pyrosequencing technology, and the methylation of the FKBP5 gene can be simply, conveniently, quickly and low-cost detected, so that the accurate evaluation of the methylation level of the FKBP5 gene is realized. The kit can effectively detect the cg03546163 site methylation of the FKBP5 gene, so as to obtain the methylation degree of the related gene of a sample to be detected, and is beneficial to judging the death risk of coronary heart disease and malignant pleural mesothelioma and the morbidity risk of systemic vasculitis. The invention has convenient detection, strong pertinence and high detection accuracy, can conveniently and quickly provide effective means and basis for evaluating the death risk of individual coronary heart disease and malignant pleurisy and the potential possibility of the disease risk of systemic vasculitis on the molecular level, provides a new way and a method for deeply researching the FKBP5 gene, and has wide application prospect.
Drawings
FIG. 1 is an agarose gel electrophoresis of the FKBP5 gene amplified fragment [ Lane 1 is a blank control without DNA, Lane 2, 3 are amplified samples, M is Marker (Thermo Fisher Scientific, SM0332) ];
FIG. 2 shows pyrosequencing results of FKBP5 gene.
In FIG. 2, the peak value at each site is the fluorescence value of the site, the target site is the 4 th site in the sequencing sequence, and since the first two sites of the site are both T, the result of the non-methylation at the site is superimposed with the result of the T at the first two sites, and the methylation at the site is calculated as C/[ (C + T + T)/3 ].
Detailed Description
The following further describes the embodiments of the present invention. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
The experimental procedures in the following examples were carried out by conventional methods unless otherwise specified, and the test materials used in the following examples were commercially available by conventional methods unless otherwise specified.
Example 1 establishment of FKBP5 Gene methylation detection kit based on pyrosequencing technology
(1) The kit comprises the following components: mainly comprises the following components:
1) FKBP5 gene specific amplification primers FKBP5-F and FKBP5-R, wherein a sequencing fragment to be amplified is cg03546163 site of FKBP5 gene; FKBP5 gene methylation sequencing primer, and the primer is matched with a required sequencing fragment of the FKBP5 gene.
The amplification primer and the sequencing primer are designed according to cg03546163 target site of FKBP5 gene by adopting Pyromark Assay Design 2.0 software (QIAGEN), and the sequences are as follows:
an amplification primer:
FKBP 5-F: 5 ' -Biotin-TGAAATGTAATTTTATTGTATGTGGTTTAT-3 ' (Biotin Biotin marker is carried at the 5 ' end);
FKBP5-R：5’-AAATAAATTAAACAATAATTCATCCTCAC-3’。
sequencing primer:
FKBP5-S：5’-TTAATTATTTATATAGAATAAGTTT-3’。
2) reagent (eksori bio corporation, Code n0.ag11104) for amplifying FKBP5 gene target region (cg03546163 site): pro Taq DNA Polymerase (5U/. mu.L))、10×Pro Taq PCR Buffer VER.2(Mg2+free)、MgCl2 Solution(50mM)、dNTP Mix(10mM each)。
3) Reagent for detecting methylation of target site of FKBP5 gene: magnetic beads, pyrosequencing binding buffer, pyrosequencing annealing buffer, pyrosequencing denaturation buffer, pyrosequencing elution buffer, pyrosequencing substrate, pyrosequencing enzyme system and four dNTPs.
(2) Optimization of amplification system and procedure:
1) the PCR reaction system was 50. mu.L, wherein Pro Taq DNA Polymerase (5U/. mu.L) was 0.25. mu.L, 10 XPro Taq PCR Buffer VER.2 (Mg)2+free)5μL，MgCl2Solution (50mM) 2. mu.L, dNTP Mix (10mM each) 1. mu.L; the amount of the amplification primers (5. mu.M) added was set to 1. mu.L, 1.5. mu.L, 2. mu.L, 2.5. mu.L, and 3. mu.L in a gradient manner; the amount of DNA added was 50-150ng, and the whole system was made up to 50. mu.L with enzyme-free water. The optimal input amount of the amplification primers is finally determined to be 2 mu L according to the size of the amplified product strip with none or brightness.
2) Optimization of the amplification procedure: the process is mainly optimized for annealing temperature and extension time. According to the Tim value given by software during the design of the amplification primers, the annealing temperature in the PCR process is optimized in a gradient way, and the annealing temperature is 54 ℃, 56 ℃, 58 ℃, 60 ℃ and 62 ℃. After that, the extension times were designed to be 11sec, 13sec, 15sec, 17sec, 19sec, respectively. After a plurality of experimental optimizations, the optimal annealing temperature is finally determined to be 60 ℃ and the extension time is 15 sec.
Finally, the amplification program was determined as: pre-denaturation at 95 ℃ for 10 min; denaturation at 95 ℃ for 30sec, annealing at 60 ℃ for 50sec, and extension at 72 ℃ for 15sec for 50 cycles; extension at 72 ℃ for 5 min.
Example 2 application of FKBP5 gene methylation detection kit based on pyrosequencing technology
(1) Blood cell DNA extraction (TIANAmp Genomic DNA Kit, DP304)
1) 200 μ L of fresh or frozen human blood samples (provided by volunteers diagnosed with coronary heart disease in Min Hospital, Guangdong province) were taken, and less than 200 μ L of the sample was made up with buffer GA.
2) Add 20. mu.L of protease K solution and mix well.
3) Adding 200 μ L buffer GB, mixing thoroughly, standing at 70 deg.C for 10min to clear the solution, and centrifuging briefly to remove water droplets on the inner wall of the tube.
4) Add 200. mu.L of absolute ethanol, mix well for 15sec with shaking, at which time a flocculent precipitate may appear, and centrifuge briefly to remove water droplets on the inner wall of the tube cover.
5) The solution and flocculent precipitate obtained in the previous step are added into an adsorption column, centrifuged at 12,000rpm for 30sec, the waste liquid is decanted, and the adsorption column is returned to the collection tube.
6) Add 500. mu.L of buffer GD to the adsorption column, centrifuge at 12,000rpm for 30sec, discard the waste, and place the adsorption column back into the collection tube.
7) 600. mu.L of the rinsing solution PW was added to the adsorption column, centrifuged at 12,000rpm for 30sec, the waste liquid was discarded, and the adsorption column was returned to the collection tube.
8) Operation 7 is repeated.
9) The column was returned to the collection tube, centrifuged at 12,000rpm for 2min and the waste liquid was decanted. The adsorption column was left at room temperature for several minutes to completely dry the residual rinse solution in the adsorption material.
10) Transferring the adsorption column into a clean centrifuge tube, suspending and dropwise adding 50-200 μ L of elution buffer TE to the middle part of the adsorption membrane, standing at room temperature for 2-5min, centrifuging at 12,000rpm for 2min, collecting the solution in the centrifuge tube, and extracting to obtain DNA.
And (3) measuring the concentration and purity of the extracted DNA, and taking the following components in mass: the DNA sample with OD260/280 being more than or equal to 1.8 and less than 2.0 and OD260/230 being more than 2.0 is qualified DNA for further use.
(2) DNA bisulfite conversion (EZ DNA Methylation-Lightning Kit, D5030T)
Performing bisulfite conversion on the DNA which is extracted in the step (1) and has qualified quality after detection, wherein the specific method comprises the following steps:
1) a20. mu.L sample was taken (the total amount of DNA contained therein was made to be 1. mu.g, and the remaining volume was made up with water).
2) 130 μ L of Lighting Conversion Reagent was added.
3) The system is placed in a PCR instrument, and the setting conditions are as follows: at 98 deg.C for 8 min; 54 ℃ for 60 min; 4 ℃ for 10 min.
4) In a dedicated reaction column, 600. mu. L M-Binding Buffer was added, and the sample was taken out from the PCR apparatus, transferred to the reaction column, mixed by inverting upside down, centrifuged in a centrifuge (12000r/s, 4 ℃, 30sec) and the liquid in the lower tube was removed.
5) 100 μ L M-Wash Buffer was added to the column and centrifuged (12000r/s, 4 ℃ C., 30 sec).
6) Then, 200. mu. L L-Des. mu.Lphosphorylation Buffer was added thereto, and the mixture was left at room temperature for 20min and centrifuged (12000r/s, 4 ℃ C., 30 sec).
7) 200 μ L M-Wash Buffer was added and centrifuged (12000r/s, 4 ℃ C., 30sec), and this step was repeated once.
8) The lower tube was discarded, and the upper tube was placed in a 1.5mL low-adsorption centrifuge tube, and the transformation product in the lower tube was collected after Elution of DNA by centrifugation (12000r/s, 4 ℃ C., 30sec) with 10. mu. L M-elute Buffer.
(3) PCR amplification
1) The PCR reaction (50. mu.L) was configured as follows:
2) amplification of
After the reaction system is prepared, PCR amplification is carried out according to the following amplification procedure:
95℃、15min；50×(95℃、30sec；60℃、50sec；72℃、15sec)；72℃、5min。
3) agarose gel electrophoresis
2.5g of agarose was weighed and dissolved thoroughly by heating with 0.5X TBE buffer 100 mL. And (3) after the solution is cooled to room temperature, adding 10 mu L of special Gel Green dye, shaking and uniformly mixing, pouring the mixture into a prepared Gel making groove, inserting a comb, after Gel is completely condensed and formed, demolding and placing the Gel in an electrophoresis device. The electrophoresis solution was subjected to 0.5X TBE buffer under 130V for 50 min. After electrophoresis is finished, the gel is placed under an ultraviolet projector to observe an electrophoresis result.
As shown in FIG. 1, after the sample DNA was amplified, a product band was evident between 210bp and 230bp (lanes 2 and 3). The control blank had no corresponding product band (lane 1).
(4) Sequencing methylated pyrophosphate of FKBP5 gene
The reagents matched with the pyrosequencing comprise the magnetic beads, pyrosequencing combined buffer solution, pyrosequencing modified buffer solution, pyrosequencing elution buffer solution, pyrosequencing annealing buffer solution, pyrosequencing substrates, pyrosequencing enzyme systems and four dNTPs (dATP alpha S, dTTP, dGTP and dCTP), and are purchased from Xinkayuan Feisite precision medical science and technology Limited company (Xiangchan instruments 20190409) in Hunan.
1) Single strand preparation
First, the corresponding buffer is diluted:
preparing a binding buffer solution: binding buffer: 38 μ L, beads: 2 mu L of the solution;
dilution of denaturation buffer (8 ×): denatured solution (8 ×): 1V, ultrapure water: 7V, and (3) adding water;
dilution wash buffer (10 ×): wash buffer (10 ×): 1V, ultrapure water: 9V.
Adding 40 mu L of binding buffer solution (containing micro-beads) into a clean EP tube, adding 20 mu L of PCR product into the clean EP tube, placing the mixture on a table-type oscillator, and oscillating the mixture for 15min at 1100 rpm;
③ 7,000 Xg, centrifuging for 1 min;
adding 150 mu L of diluted washing buffer into an EP tube, and centrifuging for 1min at 7,000 Xg (the step is operated for 3 times in total);
fifthly, taking a new clean EP tube, adding 6 mu L of annealing buffer solution into the EP tube, and then adding 1 mu L of sequencing primer FKBP 5-S;
sixthly, taking out the purification column, inserting the purification column into an EP tube, adding 22 mu L of working solution of diluted denaturation buffer solution into the purification column, standing for 5min, centrifuging for 1min at 7,000 Xg, collecting a single-chain product by the EP tube, and removing the purification column.
2) Sequencing on computer [ real-time quantitative pyrophosphate sequence analyzer (QIAGEN GmbH, SFD (I)20113404004) ]
Dissolving sequencing enzyme and sequencing substrate: taking out vials of sequencing enzyme and sequencing substrate dry powder, respectively adding 620 mu L of ultrapure water, standing at room temperature for 5-10min, slightly rotating and shaking the vials during the standing period until the vials are completely dissolved, and subpackaging the vials into PCR tubes for later use, wherein each tube is divided into 53 mu L;
transferring the single-chain product in the EP tube to a sequencing tube, and adding 3 mu L of sequencing enzyme and 3 mu L of sequencing substrate into each sequencing tube;
③ taking a dNTP calandria, and adding 20 uL dATP alpha S, 20 uL dTTP, 20 uL dGTP and 20 uL dCTP from the round end to the flat end in sequence. Lightly knocking the bottom of the calandria against the tabletop to enable the bases to be flatly paved at the bottom of the calandria;
preparing a cleaning water tank: adding pure water into a cleaning water tank until the pure water is submerged by a brush, and placing absorbent paper at a specified position of the cleaning water tank;
starting the computer and waiting for the Windows desktop to appear;
sixthly, turning on a power switch of the pyrosequencing instrument and waiting for one minute;
and seventhly, operating iLight sequencing software by double clicking and clicking a tray in and out button. And (5) after the tray is moved out, putting the cleaning water tank, the dNTP discharging pipe and the sequencing pipe into a specified position. Clicking the 'tray in and out' button again to withdraw the tray;
clicking a 'new' button. Selecting a project to be sequenced from an A-I pull-down menu, and inputting the corresponding sample number;
the ninthly clicks the "start sequencing" button. After checking whether the preparation work is finished or not carefully by contrasting the prompt dialog box, clicking a 'confirm' button to start sequencing;
after sequencing on the r, the software pops up an "end" dialog box and clicks the "ok" button. Clicking the 'save' button can save the sequencing result file. Clicking the "output report" button can output the result (excel format).
As shown in fig. 2, the methylation rate of the blood sample at the cg03546163 site is: 39/[ (39+194+0)/3] ═ 50.2%; methylation was successfully detected in the blood sample.
The embodiments of the present invention have been described in detail, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, and the scope of protection is still within the scope of the invention.
Sequence listing
<110> New open Source Crystal Sharp (Guangzhou) biomedical science and technology Co., Ltd
Guangdong province people hospital
<120> FKBP5 gene methylation detection primer and kit based on pyrosequencing technology
<160> 3
<170> SIPOSequenceListing 1.0
<210> 4
<211> 30
<212> DNA
<213> FKBP5-F(Artificial Sequence)
<220>
<221> misc_feature
<223> the 5' end has Biotin Biotin label
<400> 4
tgaaatgtaa ttttattgta tgtggtttat 30
<210> 2
<211> 29
<212> DNA
<213> FKBP5-R(Artificial Sequence)
<400> 2
aaataaatta aacaataatt catcctcac 29
<210> 3
<211> 25
<212> DNA
<213> FKBP5-S(Artificial Sequence)
<400> 3
ttaattattt atatagaata agttt 25
Claims (10)
1. The FKBP5 gene methylation detection primer based on pyrosequencing technology is characterized by comprising specific amplification primers FKBP5-F and FKBP5-R for amplifying a sequencing fragment required by FKBP5 gene and a sequencing primer FKBP5-S matched with the sequencing fragment required by FKBP5 gene, wherein the nucleotide sequences of the FKBP5-F, FKBP5-R and the FKBP5-S are respectively shown as SEQ ID NO 1, SEQ ID NO 2 and SEQ ID NO 3.
2. The primer for detecting the methylation of the FKBP5 gene based on the pyrosequencing technology of claim 1, wherein the FKBP5 gene is a human FKBP5 gene.
3. The primer for detecting the methylation of the FKBP5 gene based on the pyrosequencing technology according to claim 1, wherein the CpG site detected by the primer for detecting the methylation is cg03546163 site of the FKBP5 gene.
4. Use of the primer for detecting the methylation of FKBP5 gene based on pyrosequencing technology of any one of claims 1-3 in the preparation of a product for detecting the methylation of FKBP5 gene.
5. A kit for detecting the methylation of the FKBP5 gene based on pyrosequencing is characterized by comprising the primer for detecting the methylation of the FKBP5 gene based on pyrosequencing of any one of claims 1-3.
6. The kit for detecting the methylation of the FKBP5 gene based on the pyrosequencing technology, according to claim 5, wherein the kit further comprises a reagent for amplifying a sequencing fragment of the FKBP5 gene and a pyrosequencing reagent.
7. The kit for detecting the methylation of FKBP5 gene based on pyrosequencing technology as claimed in claim 6, wherein the reagents for amplifying FKBP5 gene sequencing fragment comprise Pro Taq DNA Polymerase (5U/. mu.L), 10 XPro Taq PCR Buffer VER.2(Mg Taq PCR buffer.2)2+free)、MgCl2 Solution(50mM)、dNTP Mix(10mM each)。
8. The FKBP5 gene methylation detection kit based on pyrosequencing technology according to claim 6, wherein the pyrosequencing reagent comprises magnetic beads, pyrosequencing binding buffer, pyrosequencing annealing buffer, pyrosequencing denaturation buffer, pyrosequencing elution buffer, pyrosequencing substrate, pyrosequencing enzyme system, and four dNTPs.
9. The kit for detecting the methylation of FKBP5 gene based on pyrosequencing technology as claimed in claim 5, wherein the PCR reaction system of the kit is 50 μ L, and comprises Pro Taq DNA Polymerase (5U/. mu.L) 0.25 μ L, 10 XPro Taq PCR Buffer VER.2 (Mg)2+free)5μL，MgCl2 Solution(50mM)2μL，dNTP Mix(10mM each)1μL，FKBP5-F 2μL、FKBP5-R 2μL，DNA 50-150ng。
10. The kit for detecting the methylation of the FKBP5 gene based on the pyrosequencing technology, according to claim 5, wherein the PCR reaction procedure of the kit is as follows: pre-denaturation at 95 ℃ for 10 min; denaturation at 95 ℃ for 30 sec; annealing at 60 ℃ for 50 sec; extension at 72 ℃ for 15 sec; a total of 50 cycles; extension at 72 ℃ for 5 min.
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