CN109136374B - Detection kit for auxiliary diagnosis of intracranial aneurysm and application thereof - Google Patents
Detection kit for auxiliary diagnosis of intracranial aneurysm and application thereof Download PDFInfo
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- CN109136374B CN109136374B CN201811045699.8A CN201811045699A CN109136374B CN 109136374 B CN109136374 B CN 109136374B CN 201811045699 A CN201811045699 A CN 201811045699A CN 109136374 B CN109136374 B CN 109136374B
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/136—Screening for pharmacological compounds
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/154—Methylation markers
Abstract
The invention discloses a detection kit for auxiliary diagnosis of intracranial aneurysm and application thereof, wherein the detection kit comprises a pair ofPNPLA6Methylation specific amplification primers and a methylation specific sequencing primer in a gene promoter region: wherein, the upstream primer has a nucleotide sequence shown as SEQ ID NO.1, the downstream primer has a nucleotide sequence shown as SEQ ID NO.2, and the methylation specificity sequencing primer is shown as SEQ ID NO.3, has the advantages that the diagnosis kit can conveniently and rapidly realize the detection of intracranial aneurysm on the molecular level, has high detection efficiency and strong pertinence, and simultaneously, the diagnosis kit can be used for detecting intracranial aneurysmPNPLA6The medicament taking methylation of the gene promoter region as a target spot is expected to become a new means for auxiliary diagnosis, detection and screening of intracranial aneurysm.
Description
Technical Field
The invention relates to the technical field of auxiliary diagnosis of intracranial aneurysm, in particular to a detection kit for auxiliary diagnosis of intracranial aneurysm and application thereof, and especially relates to a kit for detection of intracranial aneurysm-related antibodyPNPLA6A kit for the degree of gene methylation and application thereof.
Background
Intracranial Aneurysms (IA) refer to the localized abnormal expansion of the inner lumen of an Intracranial artery resulting in a prominent nodular appearance of the vessel wall, which is the leading cause of subarachnoid hemorrhage. Cerebral hemorrhage caused by IA rupture is third in cerebrovascular accident cases, is second to cerebral thrombosis and hypertensive cerebral hemorrhage, and has higher disability rate and mortality rate. After diagnosis of IA in patients, with conservative treatment, approximately 70% of patients die from the aneurysm and then bleed. While the aneurysm ruptures for the first time, the mortality rate is as high as 40%, half of which die within 48 hours after the onset. Even if survived, 1/3 can develop secondary bleeding, with mortality rates as high as 70-80% for those who develop secondary bleeding. Before bleeding due to rupture, 90% of patients with IA have no obvious symptoms and signs, have extremely high latency, and bring huge danger to life of people. Patients may also remain with varying degrees of disability and sequelae after surgery, placing a heavy burden on the patient's family and society. The cause of IA is not well understood. At present, the development and formation of IA are considered to be a complex process of the combined action of polygenic and biological factors. The pathogenesis of the disease comprises a plurality of factors such as genetic genes, cerebral hemodynamics, blood vessel growth conditions, living environment (smoking, alcoholism) and the like. With medical advances, researchers have increasingly tended to study genes and biological factors of IA.
The nerve-targeting lipase (protein-binding protein 6, PNPLA6) is a phospholipase which deacetylates intracellular phosphatidylcholine to produce glycerophosphatidylcholine.PNPLA6Mainly located on the surface of the cytoplasmic endoplasmic reticulum and concentrated in brain neurons, placenta and kidney. The balance of brain PNPLA6 levels plays an important role, and loss of activity results in abnormally elevated levels of phosphatidylcholine in the brain and impairment of secretory pathways in neurons. The current research on gene function finds that the silencingPNPLA6Expression of the gene results in a series of functional pathway changes, which ultimately lead to changes in the nervous and vascular systems of the brain. At present, no intracranial aneurysm and the like are disclosed at home and abroadPNPLA6The research related to gene methylation is reported.
Disclosure of Invention
The invention aims to solve the technical problem of providing a detection kit for auxiliary diagnosis of intracranial aneurysm and application thereof, which has high detection efficiency and strong pertinence, aiming at the current situation of the prior art, whereinPNPLA6GeneMethylation levels are positively correlated with the prevalence of intracranial aneurysms.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a detection kit for auxiliary diagnosis of intracranial aneurysm is provided, wherein the detection kit comprises a pair ofPNPLA6Methylation specific amplification primers and a methylation specific sequencing primer in a gene promoter region:
nucleotide sequence of methylation specific forward primer:
5’- Biotin - GGATTTGGGGGTGGTTAGA -3’；
nucleotide sequence of methylation specific downstream primer:
5’- TACTCCCCCACCAACTCCTTCT -3’；
nucleotide sequence of methylation specific sequencing primer:
5’- ACCAACTCCTTCTTAC -3’。
the application of the detection kit for the auxiliary diagnosis of the intracranial aneurysm can be used for the auxiliary diagnosis, detection or screening of the intracranial aneurysm for medicines.
Compared with the prior art, the invention has the advantages that: the invention discloses for the first time a method for detecting a condition associated with an intracranial aneurysmPNPLA6A detection kit for the methylation degree of genes and application thereof,PNPLA6the high methylation level of the gene results inPNPLA6The low expression of the gene causes the change of related signal paths, thereby causing the disorder of cerebral artery and blood vessel to cause diseases, therefore,PNPLA6the level of intragenic methylation is positively correlated with the prevalence of intracranial aneurysms. To detectPNPLA6The detection kit based on the methylation level in the gene can conveniently and quickly realize the detection of intracranial aneurysm on the molecular level, has high detection efficiency and strong pertinence, and simultaneously, the detection kit based on the methylation level in the gene can realize the detection of intracranial aneurysm on the molecular levelPNPLA6The drug with gene methylation as the target is expected to become a new means for auxiliary diagnosis, detection and screening of intracranial aneurysm.
Drawings
FIG. 1 is a drawing ofPNPLA6The region of the gene where the detected sequence is located (the specific positions are hg19, Chr19:7,615,203-7,615,727), and the detected sequenceThe correlation between 5 CpG sites (for example, the correlation coefficient between CpG1 and CpG2 is 0.958, the correlation coefficient between CpG2 and CpG3 is 0.901, and the correlation coefficient between CpG3 and CpG4 is 0.931);
FIG. 2 isPNPLA6Exemplary results of gene methylation level measurements (percentages shown in the figure are the degree of methylation at corresponding CpG sites, as indicated by CpG1 to CpG5 of 58%, 61%, 50%, 59%, 76%), respectively);
FIG. 3 shows the relationship between intracranial aneurysm and control groupPNPLA6Gene methylation differences (intracranial aneurysm patients)PNPLA6The degree of gene methylation is significantly higher than that of the control group);
FIG. 4 is between case and control groupsPNPLA6Gene methylation difference chart (table 1);
FIG. 5 shows the case control groups after gender groupingPNPLA6Gene methylation difference chart (table 2);
FIG. 6 is a drawing showingPNPLA6Sensitivity and specificity of gene methylation with intracranial aneurysms (AUC = 0.738, sensitivity at the optimal cut-off point 83.3%, specificity 37.5%).
Detailed Description
The invention is described in further detail below with reference to the accompanying examples.
A detection kit for auxiliary diagnosis of intracranial aneurysm is provided, wherein the detection kit comprises a pair ofPNPLA6Methylation specific amplification primers and a methylation specific sequencing primer in a gene promoter region:
nucleotide sequence of methylation specific forward primer:
5’- Biotin - GGATTTGGGGGTGGTTAGA -3’；
nucleotide sequence of methylation specific downstream primer:
5’- TACTCCCCCACCAACTCCTTCT -3’；
nucleotide sequence of methylation specific sequencing primer:
5’- ACCAACTCCTTCTTAC -3’。
the application of the detection kit for the auxiliary diagnosis of the intracranial aneurysm can be used for the auxiliary diagnosis, detection or screening of the intracranial aneurysm for medicines.
1. Collection of study subjects
Collecting volunteers willing to participate in the research, collecting a case group according to the international diagnostic standard of intracranial aneurysm, taking healthy volunteers as a control group, investigating the general condition of the volunteers in the form of questionnaire, and simultaneously taking venous blood to carry out general blood biochemical detection, wherein the specific steps are as follows:
48 patients (24 men; 24 women) with intracranial aneurysms confirmed by magnetic resonance imaging and angiography were collected from a neurosurgical dwelling of a certain hospital, and 48 normal persons of matched gender and similar age were collected as a control group (24 men; 24 women). All subjects involved in the study excluded cardiogenic shock, severe heart failure, severe ventricular arrhythmia, with other severe diseases such as malignant tumors, severe liver and kidney diseases, etc. Recording general biochemical indexes of all research objects such as blood fat and blood sugar in fasting blood drawing detection, simultaneously drawing 3ml of venous blood into an EDTA anticoagulant tube, and storing at the low temperature of-80 ℃ for uniformly extracting genome DNA from a specimen.
2. Extraction of genomic DNA
Whole blood genomic DNA of the study subject was extracted using genomic DNA extraction kit (QIAGEN), and the concentration of the obtained DNA was measured by nucleic acid protein analyzer for usePNPLA6Detection of the methylation level of gene DNA.
3. DNA methylation level determination
The present study adopted pyrosequencing technology pairPNPLA6DNA methylation level analysis was performed at 5 CpG sites (FIG. 1) within the gene (hg19, Chr19:7,615,203-7,615, 727). The basic principle of this technique: after bisulfite treatment of the DNA sample, Polymerase Chain Reaction (PCR) amplification is used to maintain the methylated cytosine (C) base unchanged while the unmethylated C is converted to uracil (U), and PCR sequencing is performed using sequencing primers to determine which site is methylated. In the research, PyroMark Assay Design software is adopted for primer Design, and PCR amplification primers and sequencing primers used for experiments are as follows:
(1) methylation specific Forward primer (Forward primer)
5’- Biotin - GGATTTGGGGGTGGTTAGA -3’(SEQ ID NO.1)；
(2) Methylation specific downstream primer (Reverse primer)
5’- TACTCCCCCACCAACTCCTTCT -3’，(SEQ ID NO.2)；
(3) Methylation specific Sequencing primer (Sequencing primer)
5’- ACCAACTCCTTCTTAC -3’(SEQ ID NO.3)。
The specific experimental steps are as follows:
step A. Bisulfite conversion of the sample DNA was performed using QIAGEN EpiTect Bisulfite treatment Kits (EpiTech Bisulfite Kits; Qiagen; # 59104).
And step B, taking 20ng of the DNA sample converted in the step A, adding the DNA sample into a PCR amplification system, and finally metering to 50 mu L according to the following reagent proportion.
PCR amplification System (50. mu.L)
The PCR amplification procedure was as follows: firstly, denaturation at 95 ℃ for 3 min; then annealing reaction is carried out for 40 cycles of 94 ℃ for 30s, 56 ℃ for 30s and 72 ℃ for 1 min; then the extension reaction was carried out at 72 ℃ for 7 min.
Step c. pyrosequencing:
1. adding 2 mu L of reaction binding beads, 38 mu L of binding buffer solution and 40 mu L of PCR products into a 96-hole PCR reaction plate, and fully and uniformly mixing for 10min at room temperature;
2. starting a vacuum pump to absorb the binding beads and the PCR product suspension, and sequentially immersing the binding beads and the PCR product suspension in 70% ethanol, 0.2M NaOH and washing buffer solution for 5s respectively;
3. turning off the vacuum pump, placing the binding beads on the probe and the PCR product in 40 μ L annealing buffer solution (containing 1.5 μ L of sequencing primer), denaturing at 85 deg.C for 2 min, cooling to room temperature, and allowing the primer and the template to anneal and hybridize;
4. calculating the dosage according to Pyrosequencing software sequence design information, and sequentially adding a substrate mixture, an enzyme mixture and four dNTPs (QIAGEN) into a reagent cabin;
5. the reagent chamber and the 96-well reaction plate were placed in a Pyrosequencing detector (PyroMark Q96 ID, QIAGEN) for reaction, and then the methylation status of each site was automatically analyzed using Pyro Q-CpG software provided in a pyrosequencer (see fig. 2 for the results of methylation level detection).
4. Data analysis
SPSS 16.0 is adopted to carry out sorting analysis on data in the research, and the research finds that the 5 detected CpG sites have obvious correlation (correlation coefficient r)> 0.848，p <0.0001, statistically significant, see fig. 1) (note:p <a value of 0.05 indicates statistical significance, as follows). By case-control group comparison, we found intracranial aneurysm patientsPNPLA6The average methylation degree of the genes is higher than that of the control group (64.40 +/-15.99 vs. 57.52 +/-11.12,p= 0.016, fig. 3), wherein the methylation level at CpG1-4 site is higher than that in the normal human group (see fig. 4 (table 1),p <0.040). Furthermore, we have also found that in male patientsPNPLA6The increased gene methylation was more pronounced (see figure 5 (table 2),p <0.034) in female patientsPNPLA6There were no significant differences in 5 CpG sites within the gene. And isPNPLA6Analysis of sensitivity and specificity of gene methylation with intracranial aneurysms (see fig. 6) showed AUC = 0.738, sensitivity at the optimal cut-off point was 83.3% and specificity was 37.5%.
The detection kit for the auxiliary diagnosis of the intracranial aneurysm has the advantages of accuracy, reliability, flexibility, rapidness, economy and economy. Adopt above-mentioned kit toPNPLA6The gene methylation level is detected, and reference can be rapidly and reliably provided for the auxiliary diagnosis, detection or drug screening of intracranial aneurysm.
While the preferred embodiments of the present invention have been illustrated, various changes and modifications may be made by one skilled in the art without departing from the scope of the present invention.
Sequence listing
<110> Ningbo City first Hospital
<120> detection kit for auxiliary diagnosis of intracranial aneurysm and application thereof
<141> 2018-09-07
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
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<212> DNA
<213> Artificial sequence (Unknown)
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ggatttgggg gtggttaga 19
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<212> DNA
<213> Artificial sequence (Unknown)
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tactccccca ccaactcctt ct 22
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<212> DNA
<213> Artificial sequence (Unknown)
<400> 3
accaactcct tcttac 16
Claims (1)
1. A pair ofPNPLA6The application of the methylation specific amplification primer and the methylation specific sequencing primer in preparing the detection kit for the auxiliary diagnosis of intracranial aneurysm is characterized in that:
nucleotide sequence of methylation specific forward primer:
5’- Biotin - GGATTTGGGGGTGGTTAGA -3’；
nucleotide sequence of methylation specific downstream primer:
5’- TACTCCCCCACCAACTCCTTCT -3’；
nucleotide sequence of methylation specific sequencing primer:
5’- ACCAACTCCTTCTTAC -3’；
PNPLA6the specific positions of the region in which the detected sequence of the gene is located are hg19, Chr19:7,615,203-7,615, 727; pair by pyrosequencing technologyPNPLA6DNA methylation level analysis is carried out at 5 CpG sites in the gene, i.e. the gene A in PNPLA6The level of basal is positively correlated with the prevalence of intracranial aneurysms.
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| CN201811045699.8A CN109136374B (en) | 2018-09-07 | 2018-09-07 | Detection kit for auxiliary diagnosis of intracranial aneurysm and application thereof |
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| CN109136374B true CN109136374B (en) | 2021-07-02 |
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| CN113355417B (en) * | 2021-06-09 | 2022-05-24 | 宁波市第一医院 | Application of MAP3K10 gene fragment and primer in preparation of intracranial aneurysm detection kit |
| CN113249486B (en) * | 2021-06-28 | 2022-04-08 | 宁波市第一医院 | Kit for detecting DNA methylation degree of VGLL3 gene and application thereof |
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Non-Patent Citations (4)
| Title |
|---|
| 《Characterization of the human patatin-like phospholipase family》;Paul A. Wilson等;《Journal of Lipid Research》;20060930;第47卷(第9期);全文 * |
| 《Genomic DNA Methylation Signatures Enable Concurrent Diagnosis and Clinical Genetic Variant Classification in Neurodevelopmental Syndromes》;Erfan Aref-Eshghi等;《The American Journal of Human Genetics》;20180104;第102卷(第1期);参见对比文件1摘要、第162页右栏第4段、"Supplemental Data"第21-25页表S5及其注释、图5及其注释 * |
| 《Hypermethylation reduces the expression of PNPLA7 in hepatocellular carcinoma》;XIAOJIAO ZHANG等;《ONCOLOGY L 670 ETTERS》;20160731;第12卷(第1期);全文 * |
| 《PNPLA3 在实验性非酒精性脂肪肝病中的表达及其 CpG 岛》;欧阳琴;《广州医科大学硕士学位论文 医药卫生科技辑》;20150331;全文 * |
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Effective date of registration: 20220512 Address after: No.396, Mingzhu Road, science and Technology Park, Ningbo City, Zhejiang Province Patentee after: Ningbo Haier Shi Gene Technology Co.,Ltd. Address before: Ningbo first hospital, 59 Liuting street, Haishu District, Ningbo City, Zhejiang Province, 315010 Patentee before: NINGBO FIRST Hospital |
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