CN105368945B - A kind of detection of early lung cancer methylation marker and its detection method - Google Patents
A kind of detection of early lung cancer methylation marker and its detection method Download PDFInfo
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- CN105368945B CN105368945B CN201510831357.9A CN201510831357A CN105368945B CN 105368945 B CN105368945 B CN 105368945B CN 201510831357 A CN201510831357 A CN 201510831357A CN 105368945 B CN105368945 B CN 105368945B
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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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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- 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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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/154—Methylation markers
Abstract
The present invention relates to a kind of detection of early lung cancer methylation marker and its detection method, methylation marker includes PCDHGB6, HOXA9, MGMT and microRNA-126 gene.Carrying out joint-detection using marker of the invention has the characteristics that quick, high-throughput, sensitive and specific good, and detection method is applicable not only to the detection of lung cancer early stage, can be used for the detection of advanced lung cancer, has a good application prospect.
Description
Technical field
The invention belongs to lung cancer detection field, in particular to a kind of detection of early lung cancer methylation marker and its detection
Method.
Background technique
Lung cancer, also known as primary bronchogenic carcinoma are to one of maximum malignant tumour of human health risk, and the death rate occupies
First of all kinds of malignant tumours.World Health Organization's prediction there will be over 1,000,000 new cases every year to China in 2025, thus
Greatly compromise the life security and health of China people.Research thinks, total 5 years survival rates of lung cancer only 10%-
5 years survival rates of 16%, I phase patient are up to 65-75%, and the 5 years survival rates of II~IV phase patient always then drop to from 40%
5%.Since the pathogenic factor and pathogenesis of lung cancer are extremely complex, and clinical manifestation has individual difference, and most of patients is medical
When be developed to advanced stage, therefore early discovery, early diagnosis, early treatment are to extending lung cancer malignant tumor patient life cycle and reduce dead
Rate has great significance.
The occurrence and development of lung cancer are extremely complex multifactor, multistage regulation exception a processes, are science of heredity
With the coefficient result of epigenetics.Science of heredity, which regulates and controls, refers to the change of nucleotide sequence, and mutation including base lacks
Lose, be inserted into, recombinate etc.；And epigenetic regulation mainly influences gene by modes such as DNA methylation and histone modifications
Transcriptional activity.DNA methylation refers to that under the action of DNA methyltransferase the methyl (- CH3) of S-adenosylmethionine is covalent
It is integrated on 5 carbon atoms of cytimidine (C) base of DNA molecular, is formed 5-methylcytosine (5mC), but do not change DNA
Sequence.Recent studies indicate that pressing down cancer caused by the occurrence and development of lung cancer and the methylation of the island tumor suppressor gene promoter region CPG
Gene expression inactivation is related, and its early stage for being likely to occur in lung cancer, is the molecule of a very promising early diagnosis lung cancer
Index.Therefore, the methylation state of the multiple gene promoters of joint-detection, diagnosis, treatment, Index for diagnosis etc. to lung cancer
It is of great significance.
Currently, the detection method of methylation mainly has methylation status of PTEN promoter, bisulfite sequencing and methyl fluorescence
Method.However these methods the disadvantages of there are cumbersome, false positive rate is high, higher cost, it is limited in clinical labororatory
It is widely applied.High-resolution solubility curve technology [Wojdacz TK, the Dobrovic A.Methylation- developed in recent years
sensitive high resolution melting(MS-HRM):a new approach for sensitive and
high-throughput assessment of methylation.Nucleic Acids Res.2007；35(6):e41],
[Migheli F,Stoccoro A,CoppedèF,et al.Comparison study of MS-HRM and
pyrosequencing techniques for quantification of APC and CDKN2A gene
methylation.PLoS One.2013；8 (1): e52501] it is to be combined together PCR amplification and solubility curve analysis, by
Cause the change of PCR solution temperature in the minor sequence difference of target fragment, so as to gene mutation, Genotyping, base
Because methylation etc. is detected.
Summary of the invention
Technical problem to be solved by the invention is to provide a kind of detection of early lung cancer methylation markers and its detection
Method, carrying out joint-detection using the marker has the characteristics that quick, high-throughput, sensitive and specific good, and detection method is not
It is only applicable to the detection of lung cancer early stage, the detection of advanced lung cancer is can be used for, has a good application prospect.
A kind of detection of early lung cancer of the invention methylation marker, the methylation marker include PCDHGB6,
HOXA9, MGMT and microRNA-126.
The PCDHGB6 upstream primer sequence is as shown in SEQ ID NO.1, downstream primer sequence such as SEQ ID NO.2 institute
Show；
The HOXA9 upstream primer sequence is as shown in SEQ ID NO.3, and downstream primer sequence is as shown in SEQ ID NO.4；
The MGMT upstream primer sequence is as shown in SEQ ID NO.5, and downstream primer sequence is as shown in SEQ ID NO.6；
The microRNA-126 upstream primer sequence is as shown in SEQ ID NO.7, downstream primer sequence such as SEQ ID
Shown in NO.8.
SEQ1:5-AATTTGAGGGGGATGTATATTT-3 (SEQ ID NO.1)；
SEQ2:5-AAAATCCCAAACCAAAAACT-3 (SEQ ID NO.2)；
SEQ3:5-GAGTTGTGGTTGTTTTTTTTTG-3 (SEQ ID NO.3)；
SEQ4:5-ACCTTTCAAAACTCCTTCCTC-3 (SEQ ID NO.4)；
SEQ5:5-GCGTTTCGGATATGTTGGGATAGT-3 (SEQ ID NO.5)；
SEQ6:5-AACGACCCAAACACTCACCAA-3 (SEQ ID NO.6)；
SEQ7:5-TGGGTTGGTTTTTGTTAGG-3 (SEQ ID NO.7)；
SEQ8:5-TAACCCTCACCTACTCCACAA-3 (SEQ ID NO.8).
The methylation marker forms kit, wherein kit further include: 1 × master and 25mM MgCl2。
The kit further include: positive control and negative control.
A kind of detection of early lung cancer of the invention detection method of methylation marker, includes the following steps:
(1) DNA is extracted from tissue to be checked, and moditied processing is carried out respectively to sample DNA, standard items DNA, after conversion
Template after DNA (at least 20ng) is dissolved with water, as PCR；
(2) pair of primers, Master, MgCl of gene are added according to PCR system ratio2, so that each pair of primer, MgCl2's
Final concentration is respectively 1uM, 4mM；PCR amplification is then carried out, the melting temperature of the standard items DNA according to different methylation ratios is poor
It is different to do standard curve, then according to the PCR of sample as a result, obtaining the opposite methylation level of sample with standard curve.
PCR amplification condition in the step (2) are as follows: 95 degree of denaturation, 10min；45cycles, 95 DEG C of 10s, from 62 DEG C to
51 DEG C of 10s, 72 DEG C of 20s；95 DEG C of 1min, 40 DEG C of 1min, 65 DEG C are warming up to 94 DEG C.
The technology that the present invention uses: in the primer of the DNA methylation assay of a pair of of specificity of promoter region design of gene, so
Amplification vulcanization sample DNA is removed with the primer afterwards, the methylation of sample to be tested is determined according to the relative fluorescence of PCR amplification result
It is horizontal.
Beneficial effect
Compared with conventional method of lung cancer diagnosis, carrying out joint-detection using marker of the invention has quick, high pass
The features such as measuring, is sensitive and specific good, detection method is applicable not only to the detection of lung cancer early stage, can be used for advanced lung cancer
Detection, this method can make the early discovery of lung cancer patient, early treatment, extend life cycle, so that the quality of life of tumour patient is improved,
It has a good application prospect.
Detailed description of the invention
Fig. 1 is the standard curve that four genes are drawn using standard items DNA as the PCR result of template；Wherein, A HOXA9, B
For PCDHGB6, C MGMT, D miR-126；
Fig. 2 is four genes (PCDHGB6, HOXA9, MGMT and microRNA-126) in cancerous lung tissue and normal group of distal end
The DNA methylation assay distribution of results knitted.
Specific embodiment
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.In addition, it should also be understood that, after reading the content taught by the present invention, those skilled in the art
Member can make various changes or modifications the present invention, and such equivalent forms equally fall within the application the appended claims and limited
Range.
Embodiment 1
One group for the pulmonary cancer diagnosis specific primer design of methylation marker:
According to mankind's whole genome sequence disclosed in NCBI (US National Biotechnology Information center), Primer is used
Premier3.0 and Methyl Primer Express v1.0 design, by Shanghai, Sheng Gong Co., Ltd is synthesized.Described
The upstream primer and downstream primer of PCDHGB6 gene are respectively shown in SEQ ID NO.1 and SEQ ID NO.2；The HOXA9
The upstream primer and downstream primer of gene are respectively shown in SEQ ID NO.3 and SEQ ID NO.4；The mgmt gene it is upper
It swims primer and downstream primer is respectively shown in SEQ ID NO.5 and SEQ ID NO.6；The MicroRNA-126 gene it is upper
It swims primer and downstream primer is respectively shown in SEQ ID NO.7 and SEQ ID NO.8.
Embodiment 2
The application of kit of the invention in the methylation level of detection human lung cancer:
The Master that the present invention uses is purchased from Roche Co., Ltd；DNA extraction kit is QIAmp DNA Mini
Kit(QIAGEN)；Conversion reagent box is EZ DNA Methylation kit (Zymo Research, Orange, CA, USA)；
Other reagents are domestic analytical reagents.
Biomaterial of the present invention is all from Shanghai City Zhong Shan hospital.
Method:
One, biological sample:
The tumor tissues of 54 patients with lung cancer and match normal tissue.
Two, the extraction and conversion of tissue DNA:
It takes about 25mg cancerous lung tissue or normal tissue to be shredded with the operating scissors after autoclaving sterilization and is added to 1.5ml's
In centrifuge tube, the DNA of sample to be tested is extracted using DNA extraction kit, is then turned with conversion reagent box to DNA is extracted
Change.
Three, PCR process
1 PCR instrument used is Roche LightCycler480, and Master and magnesium ion are also the reagent of Roche, reaction
System is 20ul；
Shown in the preparation of 2PCR reaction system and the following Tables 1 and 2 of condition:
The preparation of table 1, PCR reaction system
| Component | Volume |
| Master | 10ul |
| 25mM MgCl2 | 3.2ul |
| Aqua sterilisa | 5.8ul |
| Template | 1ul |
Table 2, PCR response procedures:
Four, interpretation of result
1, according to the Comparative dissolution curve difference after the methylation standard items DNA cloning of different proportion, draws each gene
Standard curve (see attached drawing 1).
2 calculate the ratio to methylate in unknown sample according to the standard curve established, and data analysis uses Fisher, really
Probability analysis is cut, P < 0.05 is that difference is statistically significant.
The methylation distribution situation of 3 cancerous lung tissues and 4 genes of distal end normal tissue (see attached drawing 2).
Embodiment 3
Performance curve analyzes (ROC curve analysis):
Building ROC curve compares 4 methylation markers and distinguishes the tumor tissues of lung cancer patient and the diagnosis of normal tissue
Ability.Line Integral is not as follows under the ROC curve of 4 markers: PCDHGB6, and 0.796；HOXA9,0.694；MGMT, 0.594；
Mir-126,0.658.Under optimal cutoff value, the sensibility and specificity of gene is as follows: PCDHGB6,66.7% He
90.7%；HOXA9,42.6% and 96.3%；MGMT, 25.9% and 94.4%；Mir-126,38.9% and 90.7%.This 4
The AUC that marker joins together to detect reaches 0.891, and sensibility and specificity is respectively 85.2% and 81.5% (being shown in Table 3).This
The result shows that, 4 methylation markers in detecting lung cancer have relatively high sensitivity and specificity a bit.
The ROC curve analysis of the methylation marker of table 3
The above description is only a preferred embodiment of the present invention, is merely illustrative for the purpose of the present invention, and not restrictive.
In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can make the present invention various
Changes, modifications or even equivalent change, but fall in protection scope of the present invention.
Claims (3)
1. a kind of detection of early lung cancer methylation marker, it is characterised in that: the methylation marker include PCDHGB6,
HOXA9, MGMT and microRNA-126；
The PCDHGB6 upstream primer sequence is as shown in SEQ ID NO.1, and downstream primer sequence is as shown in SEQ ID NO.2；
The HOXA9 upstream primer sequence is as shown in SEQ ID NO.3, and downstream primer sequence is as shown in SEQ ID NO.4；
The MGMT upstream primer sequence is as shown in SEQ ID NO.5, and downstream primer sequence is as shown in SEQ ID NO.6；
The microRNA-126 upstream primer sequence is as shown in SEQ ID NO.7, downstream primer sequence such as SEQ ID NO.8 institute
Show.
2. a kind of detection of early lung cancer according to claim 1 methylation marker, it is characterised in that: the methylation
Marker is applied to kit, wherein kit further include: 1 × master and 25mM MgCl2。
3. a kind of detection of early lung cancer according to claim 2 methylation marker, it is characterised in that: the kit
Further include: positive control and negative control.
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Families Citing this family (10)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108342477A (en) * | 2017-01-24 | 2018-07-31 | 北京艾克伦医疗科技有限公司 | Detection kit based on multiple gene diagnosis patients with lung cancer |
| CN107475369B (en) * | 2017-07-07 | 2021-05-04 | 南方医科大学 | Application of HOXA9 gene in preparation of medicine for treating skin squamous cell carcinoma |
| CN111630186A (en) * | 2018-01-23 | 2020-09-04 | 北京艾克伦医疗科技有限公司 | Methods and kits for identifying lung cancer status |
| CN110628900A (en) * | 2018-06-22 | 2019-12-31 | 深圳市圣必智科技开发有限公司 | Method for detecting methylation of multiple genes of non-small cell lung cancer |
| CN110964810B (en) * | 2018-09-29 | 2022-04-29 | 广州康立明生物科技股份有限公司 | Application of HOXA7 and HOXA9 methylation detection reagent in preparation of lung cancer diagnostic reagent |
| CN110964811B (en) * | 2018-09-29 | 2022-04-29 | 广州康立明生物科技股份有限公司 | HOXA9 methylation detection reagent |
| CN110964812B (en) * | 2018-09-29 | 2021-10-22 | 广州康立明生物科技股份有限公司 | Application of HOXA9 methylation detection reagent in preparation of lung cancer diagnosis reagent |
| CN110567906A (en) * | 2019-09-12 | 2019-12-13 | 深圳大学 | Method for representing RNA methylation modification and application |
| CN111676286B (en) * | 2020-05-29 | 2023-04-14 | 武汉爱基百客生物科技有限公司 | Multiplex PCR primer system for detecting free DNA methylation of lung cancer plasma, detection method and application |
| CN117625795A (en) * | 2024-01-25 | 2024-03-01 | 北京迈基诺基因科技股份有限公司 | Probe set, kit and detection system for methylation detection of lung cancer and application |
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