CN109097475A - Adenocarcinoma of lung detection kit and the method for detecting CCND1 gene methylation level - Google Patents
Adenocarcinoma of lung detection kit and the method for detecting CCND1 gene methylation level Download PDFInfo
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Abstract
The invention discloses a kind of adenocarcinoma of lung detection kit and the methods for detecting CCND1 gene methylation level.Adenocarcinoma of lung detection kit of the invention includes the CCND1 gene forward primer as shown in SEQ ID NO:1 sequence, the CCND1 gene reverse primer as shown in SEQ ID NO:2 sequence, methylate probe, the non-methylation probe as shown in SEQ ID NO:4 sequence as shown in SEQ ID NO:3 sequence；With real-time fluorescence quantitative PCR detection reagent.The difference that the present invention passes through CCND1 gene methylation level and cancer beside organism CCND1 gene methylation level in detection cancerous tissue, it can be determined that go out the risk that person to be detected suffers from adenocarcinoma of lung.
Description
Technical field
The invention belongs to field of biomedicine technology, and in particular to a kind of adenocarcinoma of lung detection kit and detection CCND1 base
Because of the method for methylation level.
Background technique
Lung cancer is that morbidity and mortality growth is most fast, to one of population health and the maximum malignant tumour of life threat.
World Health Organization's International Cancer Research Center (IARC/WHO) report, global lung cancer male's disease incidence is 35.5/10 within 2002
Ten thousand, fall ill 970,000 people, and the death rate is 31.2/10 ten thousand, dead 850,000 people；Women disease incidence is 12.1/10 ten thousand, and fall ill 390,000 people,
The death rate is 10.3/10 ten thousand, dead 330,000 people." the Third National death original announced the Ministry of Public Health of China in April, 2008
Because of investigation " display, in past 30 years, China's lung cancer morbidity rate increment is larger, and being substituted liver cancer becomes the first pernicious swollen
The tumor cause of death.According to the pathological analysis of WHO, lung cancer is divided into four kinds of major tissue types: Small Cell Lung Cancer and non-small cell
Lung cancer, the latter can also be further divided into adenocarcinoma of lung, phosphorus columnar epithelium cancer and large cell carcinoma.
In order to be diagnosed to lung cancer, general all a variety of methods of integrated use.Up to the present, by primary swollen to lung
Tumor and metastatic tumo(u)r (including lymph node and other histoorgans) carry out pathological biopsy and are carried out by ImmunohistochemistryMethods Methods
Analysis is the current internationally recognized goldstandard to diagnose the illness.Further, it is also possible to disconnected in conjunction with radiography of chest, chest computer
Layer scanning, branchofiberoscope etc. carry out auxiliary diagnosis.Compared with pathological diagnosis, other aided diagnosis methods have different lack
It falling into and insufficient, such as uses chest computed tomography, the size of lung cancer, which must reach 0.1cm or more, to be measured,
In this period, cancer is likely to be transferred into other tissues.Therefore, a kind of early stage, efficient diagnosis lung cancer method ten
Divide necessity.
DNA methylation (DNA methylation) is a kind of form of DNA chemical modification, can not change DNA sequence dna
Under the premise of, change Genetic Performance, is a part of epigenetics.In mammals, DNA methylation process can be such that methyl adds
It is added on the 5' carbon of the cytimidine ring of DNA molecular, DNA methylation typically occurs in the CpG for accounting for human genome about 1%~2%
On site.It is 5-methylcytosine through dnmt rna catalysis Cytosines.The CpG of about 80%-90% in human gene
Site has been methylated, but in certain specific regions, is not methylated if the island CpG rich in cytimidine and guanine.This
It is related with comprising the promoter in 56% mammalian genes including all wide expression genes.It is found that epigenetic
It also plays an important role during the occurrence and development of tumour, it is also more important that wherein the change of DNA methylation level, which is relatively early,
Epigenetic change one of.Effect of the DNA methylation in tumour is mainly manifested in the following aspects: first is that methylation
Cytimidine in the dinucleotides of the island CpG becomes thymidine with higher frequency deamination, causes gene mutation；Second is that suppression cancer base
Silencing due to DNA-repair gene due to supermethylation；Third is that oncogene methylation level is reduced and is activated；Fourth is that genome is total
The reduction of body methylation level makes transposons, repetitive sequence activation that chromosome stability be caused to decline.These factors are to lead to tumour
Development, deteriorates the major reason for eventually leading to death at transfer.The overall methylated level of DNA is (i.e. methylome) and specific
The change of gene methylation degree can be used as diagnosing tumor index.The occurrence and development of DNA methylation and cancer are closely related, therefore grind
Study carefully what DNA methylation relevant to cancer occurrence and development methylated with important clinical meaning, specific gene promoter region
Methylation state can be used as the complementary mark of the clinical diagnosis of cancer to predict the occurrence and development and prognosis of cancer.
Methylation chip detection is carried out by 189 adenocarcinomas of lung and 149 normal human bloods, is found in adenocarcinoma of lung blood plasma
The hypomethylation of Cyclin D1 encoding gene (i.e. cyclin D-1 gene, abbreviation CCND1 gene) may be with lung gland
Generation, the development of cancer are related.It can be used as a specific molecular marker of adenocarcinoma of lung early diagnosis.Detect CCND1 gene
Methylation state is of great significance to the diagnosis, treatment, Index for diagnosis etc. of adenocarcinoma of lung.
The methylation state of traditional accurate detection exact site is to be handled by bisulfite DNA sample, right
Do not methylate modification cytimidine carry out desamination reaction, so that it is converted into sulfonate group, finally in the effect of sodium hydroxide
Under the cytimidine that is changed into uracil, and methylates can not be remained unchanged by bisulfite processing.Pass through the methods of PCR
The cytimidine of methylation with non-methylation can be distinguished.This method is cumbersome, processing the time be up to 16~18h, be not suitable with
The needs quickly detected.Therefore, it is necessary to develop new CCND1 gene methylation detection kit, so as to more efficiently accurately
Detect the methylation state of CCND1 gene.
Summary of the invention
The technical problem that the present invention is cumbersome for adenocarcinoma of lung detection method in the prior art, the processing time is long, it is therefore intended that
A kind of new adenocarcinoma of lung detection kit is provided, which passes through CCND1 gene first in detection biological sample
Base level judges adenocarcinoma of lung.
Applicant is had found by carrying out methylation chip detection to 20 adenocarcinoma of lung crowd cancerous tissues and cancer beside organism in lung
In gland cancer cancerous tissue the hypomethylation of Cyclin D1 encoding gene (CCND1) may with the generation of adenocarcinoma of lung, develop phase
It closes.Cyclin D1 encoding gene (CCND1) can be used as a specific molecular marker of adenocarcinoma of lung early diagnosis, detection
CCND1 gene methylation state is of great significance to the diagnosis, treatment, Index for diagnosis etc. of adenocarcinoma of lung.
Adenocarcinoma of lung detection kit of the invention, including the CCND1 gene forward direction as shown in SEQ ID NO:1 sequence are drawn
Object, the CCND1 gene reverse primer as shown in SEQ ID NO:2 sequence methylate as shown in SEQ ID NO:3 sequence and visit
Needle, the non-methylation probe as shown in SEQ ID NO:4 sequence；With real-time fluorescence quantitative PCR detection reagent.
The primer and probe of 1 adenocarcinoma of lung detection kit of table
Y=C/T；R=A/G
The real-time fluorescence quantitative PCR detection reagent be archaeal dna polymerase premixed liquid ( MethyLight PCR
Kit).The specific ingredient of archaeal dna polymerase premixed liquid are as follows: archaeal dna polymerase (Plus), PCR buffer,
DNTP mixed liquor and fluorescent material such as ROX (carboxyrhodamine) dyestuff.
The volume of the CCND1 gene forward primer is 1~3 μ L preferably 2 μ L, and concentration is 0.3~0.5 μm of preferably 0.4 μ
m；The volume of CCND1 gene reverse primer is 1~3 μ L preferably 2 μ L, and concentration is 0.3~0.5 μm preferably 0.4 μm；Methylation is visited
The volume of needle is 0.4~0.6 μ L preferably 0.5 μ L, and concentration is 0.1~0.3 μm preferably 0.2 μm；With the volume of non-methylation probe
For 0.4~0.6 μ L preferably 0.5 μ L, concentration is 0.05~0.2 μm preferably 0.1 μm.
The 25 μ L of volume of the archaeal dna polymerase premixed liquid.
Another object of the present invention is to provide a kind of method for detecting CCND1 gene methylation level, this method includes
Following steps:
S1 extracts the DNA in biological sample；
S2, bisulfite convert the DNA extracted；
S3, the DNA after being converted using adenocarcinoma of lung detection kit real-time fluorescence quantitative PCR amplification described in claim 1,
It is horizontal to detect CCND1 gene methylation.
The DNA in blood sample is extracted using paramagnetic particle method in step S1.
Real-time fluorescence quantitative PCR amplification condition is that 95 DEG C of 30min of initial denaturation are recycled 1 time；95 DEG C of 10s are denaturalized, annealing extends
56 DEG C of 30s are recycled 50 times.
The present invention is horizontal by detecting CCND1 gene methylation in suspicious cancerous tissue sample, according to the suspicious cancer of patient itself
Horizontal, the more between the two difference of side tissue CCND1 gene methylation, it can be determined that go out the wind that person to be detected suffers from adenocarcinoma of lung
Danger；If the suspicious relatively suspicious cancer beside organism of cancerous tissue CCND1 gene methylation level is low, the risk for suffering from adenocarcinoma of lung is high, if
Suspicious cancerous tissue CCND1 gene methylation level is comparatively high, then the risk for suffering from adenocarcinoma of lung is low.CCND1 gene methylation water
The flat marker that can be used as adenocarcinoma of lung early diagnosis, potential applicability in clinical practice is good, sentences to the diagnosis, treatment, prognosis of adenocarcinoma of lung
It is disconnected etc. to be of great significance.
It is described further below with reference to technical effect of the attached drawing to design of the invention, specific structure and generation, with
It is fully understood from the purpose of the present invention, feature and effect.
Detailed description of the invention
Fig. 1 is to make a definite diagnosis adenocarcinoma of lung cancerous tissue and cancer beside organism using real-time fluorescence quantitative PCR augmentation detection CCND1 gene
Methylation level compares figure；
Fig. 2 is that suspicious patients with lung cancer is compared using real-time fluorescence quantitative PCR augmentation detection CCND1 gene methylation level
Figure；
Fig. 3 A is suspicious cancerous lung tissue pathological examination result (HE dyes × 200 times)；
Fig. 3 B is suspicious cancer beside organism's pathological examination result (HE dyes × 200 times).
Specific embodiment
Embodiment 1
20 cancerous tissues for being diagnosed as patients with lung cancer and cancer beside organism's (being provided by Hunan Provincial Tumour Hospital) are provided
Step S1 paramagnetic particle method extracts the DNA in tissue samples
In conjunction with liquid: 4.62M guanidine thiocyanate, 10mM Tris-Cl (pH 8.0), 10mM EDTA (pH8.0) and 20%
Triton x-100；Cleaning solution: 20mM Tris-Cl (pH8.0), 1mM EDTA and 70% ethyl alcohol；Eluent: 10mM Tris-
Cl(pH 8.5)。
Specific step is as follows: 5 μ L protease, 56 DEG C of processing 10min are added into 2g tissue.100 μ L magnetic bead solution are added
And 15mL combination liquid, room temperature concussion are incubated for 60min.4min is placed on magnet stand, discards supernatant liquid.One is cleaned with 3mL cleaning solution
It is secondary.4min is placed on magnet stand, discards supernatant liquid.The step of centrifugation can be added, completely remove cleaning solution.In 56 DEG C of dry 5min.
100 μ L eluents are added, 65 DEG C of concussions are incubated for 15min.It is transferred in a clean EP pipe and (takes appropriate detectable concentration).
Step S2 bisulfite converts (rapid conversion method)
Conversion fluid: by 2.08g NaHSO3、0.67g(NH4)2SO3·H2O is dissolved in 50% (NH of 5.0mL4)HSO3In, add
Heat is cooled to room temperature to 90 degree to being completely dissolved, and is detected and is adjusted pH value of solution to 5.2~5.3.
Specific step is as follows: 50 μ L conversion fluids are added in 25 μ L 500ng DNA samples.90 DEG C of processing 10min, it is cooling
To room temperature.500 μ L combination liquid are added, are transferred in QIA quick centrifugal column, 12000rpm is centrifuged 1min.With 500 μ L cleaning solutions
Cleaning 2 times.50 μ L eluents are added to be eluted.DNA after collecting conversion.
It is horizontal that step S3 real-time fluorescence quantitative PCR detects CCND1 gene methylation
The each component of QIAGEN EpiTect MethyLight PCR (59930) kit shown in table 2 is added to
It is expanded on the qPCR instrument of Bio-Rad CFX Connect, setting reaction condition is as follows: real-time fluorescence quantitative PCR amplification
Condition is that 95 DEG C of 30min of initial denaturation are recycled 1 time；95 DEG C of 10s are denaturalized, annealing extends 56 DEG C of 30s, recycles 50 times.It collects at circulation end
Fluorescence signal.
Table 2QIAGEN EpiTect MethyLight PCR (59930) kit qPCR system composition.
| 2x EpiTect MethyLight Master Mix | 25μL |
| CCND1 gene forward primer SEQ ID NO:1 | 0.4μM |
| CCND1 gene reverse primer SEQ ID NO:2 | 0.4μM |
| Methylate probe SEQ ID NO:3 | 0.1μM |
| Non- methylation probe SEQ ID NO:4 | 0.1μM |
| DNA after conversion | ≤100ng |
| H2O | It is supplemented to 50 μ L |
Experimental result: as seen from Figure 1, compared with cancer beside organism, CCND1 in the cancerous tissue sample for suffering from patients with lung adenocarcinoma is made a definite diagnosis
Gene methylation level is very low, significant difference.Illustrate that adenocarcinoma of lung is horizontal related to CCND1 gene methylation.
Embodiment 2
It acquires 5 suspicious patients with lung adenocarcinoma cancerous tissues and suspicious cancer beside organism detection CCND1 gene methylation is horizontal, experiment
Step and experiment condition are the same as embodiment 1.
The detection of CCND1 gene methylation level is carried out to the suspicious cancerous tissue of suspicious patients with lung adenocarcinoma, acquired results are such as
Shown in Fig. 2, the suspicious relatively suspicious cancer beside organism of cancerous tissue CCND1 gene methylation level is very low, significant difference, suspects patient
With adenocarcinoma of lung.
Further detected using pathological examination.By suspicious cancerous tissue and suspicious cancer beside organism, (thickness is no more than 0.5
Centimetre) the protein denaturation solidification for making tissue, cell in the fixer prepared in advance (10% formalin) is put into, it is thin to prevent
Born of the same parents' self-dissolving after death or the decomposition of bacterium, thus the morphosis for keeping cell original；Tissue after fixation is put into embedded box,
Flowing water rinses (fixer in removal tissue) 30 minutes, and makees dehydrating agent with 100% and 90% concentration alcohol respectively, gradually
Slough the moisture content in tissue block；Transparent tissue block is placed in the paraffin dissolved, 65 DEG C of wax-dissolving box heat preservations are put into；To stone
Wax is embedded after being completely immersed in tissue block, be cooled and solidified into blocks；Embedded wax stone is fixed on slicer, is cut into
5~8 microns of thickness pieces, are affixed on glass slide, put in 45 DEG C of insulating boxs and dry.Pathological section HE dyes (leica automatic staining
Instrument) specific steps are as follows: xylene soak 5min repeats to impregnate three times, and slice dewaxing is more thorough；100% ethyl alcohol impregnates 2min；
5) 90% ethyl alcohol impregnates 2min；80% ethyl alcohol impregnates 2min；70% ethyl alcohol impregnates 2min；Flowing water rinses 5min；Bush sperm dye
Color 5min；Flowing water slightly rinses 1~3s of bush sperm；1% acidic alcohol impregnates 1~3s；Flowing water, which is crossed, is washed till anti-indigo plant；0.5% Yihong
Liquid dyes 1~3s；Distilled water slightly washes 1~2s；80% ethyl alcohol slightly washes 1~2s；95% ethyl alcohol slightly washes 2~3s；100% ethyl alcohol is slightly
Wash 3~5s；Xylene soak 5min repeats to impregnate three times, and slice dewaxing is more thorough；Neutral gum sealing.It is seen under microscope
It examines, as a result as shown in Fig. 3 A and Fig. 3 B, the suspicious cancerous lung tissue of Fig. 3 A passes through microscopy, and oncocyte heteromorphism is obvious, and structure is different,
Part cell is in reality agglomerate or small streak arrangement, it is seen that lumen of gland is formed, and part cell arrangement is at tubulose or adenoid structure, symbol
Close the pathological change of gland cancer；The suspicious cancer beside organism of Fig. 3 B shows as normal cell.
To sum up, the present invention is horizontal by detecting CCND1 gene methylation in suspicious cancerous tissue sample, suspicious with patient itself
Cancer beside organism CCND1 gene methylation level carries out comparison in difference, it can be determined that goes out the risk that person to be detected suffers from adenocarcinoma of lung；If can
Doubtful cancerous tissue CCND1 gene methylation level is low, then the risk for suffering from adenocarcinoma of lung is high, if suspicious cancerous tissue CCND1 gene methylation
Horizontal high, then the risk for suffering from adenocarcinoma of lung is low.CCND1 gene methylation level can be used as the marker of adenocarcinoma of lung early diagnosis,
Potential applicability in clinical practice is good, is of great significance to the diagnosis, treatment, Index for diagnosis etc. of adenocarcinoma of lung.
Sequence table
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Claims (9)
1. a kind of adenocarcinoma of lung detection kit, it is characterised in that the adenocarcinoma of lung detection kit includes such as SEQ ID NO:1 sequence
CCND1 gene forward primer shown in column, CCND1 gene reverse primer, such as SEQ ID as shown in SEQ ID NO:2 sequence
Methylate probe, the non-methylation probe as shown in SEQ ID NO:4 sequence shown in NO:3 sequence；And real time fluorescent quantitative
PCR detection reagent.
2. adenocarcinoma of lung detection kit as described in claim 1, it is characterised in that the real-time fluorescence quantitative PCR detection reagent
For archaeal dna polymerase premixed liquid.
3. adenocarcinoma of lung detection kit as described in claim 1, it is characterised in that the archaeal dna polymerase premixed liquid specifically:
Archaeal dna polymerase, PCR buffer, dNTP mixed liquor and fluorescent material dyestuff.
4. adenocarcinoma of lung detection kit as described in claim 1, it is characterised in that the volume of the CCND1 gene forward primer
For 1~3 μ L preferably 2 μ L, concentration is 0.3~0.5 μm preferably 0.4 μm；The volume of CCND1 gene reverse primer is that 1~3 μ L is preferred
2 μ L, concentration are 0.3~0.5 μm preferably 0.4 μm；The volume of methylation probe is 0.4~0.6 μ L preferably 0.5 μ L, concentration 0.1
~0.3 μm preferably 0.2 μm；Volume with non-methylation probe is 0.4~0.6 μ L preferably 0.5 μ L, and concentration is 0.05~0.2 μm
It is preferred that 0.1 μm.
5. adenocarcinoma of lung detection kit as claimed in claim 2, it is characterised in that the 25 μ L of archaeal dna polymerase premixed liquid.
6. a kind of method for detecting CCND1 gene methylation level, it is characterised in that include the following steps:
S1 extracts the DNA in biological sample；
S2, bisulfite convert the DNA extracted；
S3, using the DNA after adenocarcinoma of lung detection kit real-time fluorescence quantitative PCR amplification conversion described in claim 1, with inspection
It is horizontal to survey CCND1 gene methylation.
7. method as claimed in claim 6, it is characterised in that extract the DNA in biological sample using paramagnetic particle method in step S1.
8. method as claimed in claim 6, it is characterised in that real-time fluorescence quantitative PCR amplification condition is 95 DEG C of initial denaturation
30min is recycled 1 time；95 DEG C of 10s are denaturalized, annealing extends 56 DEG C of 30s, recycles 50 times.
9. method as claimed in claim 6, it is characterised in that the biological sample is group by suspicious cancerous tissue and suspicious cancer
It knits.
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Application publication date: 20181228 |