CN107034296A - A kind of composition and its application for early stage of lung cancer non-invasive screening - Google Patents
A kind of composition and its application for early stage of lung cancer non-invasive screening Download PDFInfo
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Abstract
The invention discloses a kind of composition for early stage of lung cancer non-invasive screening and its application, composition includes 3 pairs of Methylation-specific primer probes, 3 pairs of Methylation-specific primers of PTGER4 genes and the 1 pair of internal reference gene primer probe of SHOX2 genes, in addition to the corresponding MGB probes of 3 of the corresponding MGB probes of 3 of the SHOX2 genes non-target gene that the methylate and PTGER4 genes non-target gene that methylate.Methylation detection kit for early stage of lung cancer non-invasive screening includes said composition, by detecting 3 target site methylation status in lung cancer High risk group blood in the SHOX2 genes of dissociative DNA and PTGER4 gene promoters area, gene code and non-coding region come examination early stage of lung cancer patient, and aid in the early diagnosis of lung cancer.It is easy to operate, time-consuming shorter for PCR (Methylation specific PCR, MSP) detection method of methylation-specific, as a result it is easy to analysis.
Description
Technical field
It is particularly a kind of based on plasma DNA the present invention relates to lung cancer extreme early examination detection technique field
For the non-invasive screening technology of the phase of lung cancer 0 and I phases.
Background technology
According to the newest data display of national tumour Register, national lung cancer new cases about 73.3 ten thousand occupy institute
Have first (19.9%) of Cancer Mortality；Death about 59.1 ten thousand, accounts for the of whole Malignant Tumor Deads
One (26.5%).Under current medical system, the medium and advanced lung cancer patient survival rate of 5 years is only 10-20%, but for
The treatment of early stage of lung cancer patient, there are about more than 80-90% by the method for surgery excision can cure.Therefore early screening of lung cancer meaning
Great, effective early screening and therapeutic intervention can make the incidence of lung cancer decline 60%, and case fatality rate declines 80%.But it is big absolutely
The most early stage of lung cancer (especially 0 phase and I phases) patients are without any obvious clinical symptoms.Patient goes out lung cancer in initial diagnosis
When, 85% is middle and advanced stage, the ratio only about 14% of early diagnosis.The early stage of lung cancer does not have obvious clinical symptoms, correlation to appear
It has been the middle and advanced stage of lung cancer during clinical symptoms, only about 15.8% patient survival can reach more than 5 years, now patient
Clinical symptoms are more obvious, and related external auxiliary diagnosis is without too big meaning.
" preventing trouble before it happens " is the core objective under current precisely medical treatment and medical big data historical background.The early stage of lung cancer
It was found that, early diagnosis, early treatment be reduce lung cancer death number important measures.But the extreme early diagnosis skill of current lung cancer
The great challenge of art, has many technological difficulties not yet to overcome.The method for lung cancer early screening has a variety of at present, such as low dosage
CT diagnosis, the pulmonary cancer diagnosis of histocyte aspect, the pulmonary cancer diagnosis of gene aspect etc..But there is many disadvantages in these technological means
End：There is more false positive in low-dose CT detection, and testing cost is high；The meeting in terms of sampling of the pulmonary cancer diagnosis of tissue aspect
Larger pain is brought to patient, and is likely to result in cancer cell diffusion or shifts；The tumor-specific molecule mark of gene aspect
A kind of means that analyte detection can be early diagnosed as lung cancer, but technology is not yet ripe.
Liquid biopsy is a kind of Non-invasive detection technology of great potential applicability in clinical practice, can be detected by taking a blood sample in blood
Free ctDNA, and the tumour strong with early stage of lung cancer correlation is above all found in the clinical practice of tumour early screening
Mark, the purpose of early detection tumour is realized by the technological means of genetic test.In recent years, multiple clinical trial results tables
It is bright, in tumour generating process the methylation of some specific genes can as diagnosing tumor a kind of molecular marker, and this
It is specific to tumour to plant change.Early stage in tumour occurs for DNA methylation, it is thus possible to utilized initial stage in tumour formation
Genetic test means find that the prevention, treatment, diseases monitoring and prognosis for after provide an intuitively potential finger in advance
Mark.In the present invention, we are using the first in the Circulating tumor DNA (ctDNA) in detection blood with early stage of lung cancer related gene
Whether base condition adjudgement occurs tumour.DNA methylation is the expression of epigenetics level gene, cell development and cell differentiation
Deng the important way of process adjustment.Abnormal DNA methylation plays an important role in development of cancer.DNA methylation is main
Occur in-C- phosphoric acid-G- (CpG) site.The research that methylates by object of cancerous lung tissue or cell finds, SHOX2 and
The methylation level of PTGER4 CpG islands is more significantly raised than normal structure or cell, i.e. supermethylation phenomenon.In addition, there is report
Road shows that in the cancer of different times and the tumour of different grade malignancies the DNA of supermethylation occurs for cancer related gene
Target sequence is variant, illustrates the generation of the early stage of lung cancer and has certain sequence-specific.
This patent is related to the related mark of two lung cancer：
1st, the short and small hox genes SHOX2 of people (short stature homebox2) is the one of hox genes family
Member, it is expressed in mesoderm and ectoderm, and the development to bone, heart and nervous system is played an important role.There is research to send out
Existing, high methylation occurs for 96% patients with lung cancer SHOX2, and substantially related to gene copy number.And newest research
Show, methylating for SHOX2 genes is closely related with lung cancer, and this has also pointed out methylating for SHOX2 genes to be likely to become lung cancer
Early screening index.And there is correlative study to show to arrive in -800bp to -900bp, -1550bp to -1650bp and+2700bp
The probability that several site SHOX2 genes such as+2800bp methylate is higher.
2nd, prostaglandin E receptor gene PTGER4 (prostaglandin E receptor 4gene) methylations with
The generation of lung cancer is closely related, and is detected with SHOX2 gene associations, it is possible to increase lung cancer recall rate and accuracy.For
With lung cancer the partial sequence that the closely related region that methylates is located at the stub area of Second Exon 3 ' occurs for PTGER4 genes.
DNA abnormal methylations are the common changes of human tumor.Generation is different on the CpG islands of promoter region and gene internal
It is the most common mechanism of gene inactivation often to methylate.Detection DNA methylation detection method has at present：The PCR of methylation-specific
(Methylation-specific PCR, MSP), bisulfite sequencing (Bisulfite sequencing PCR,
BSP), high-resolution melting curve method (High Resolution Melting, HRM) and high-flux sequence method etc..Relative to
For other detection methods, bisulfite sequencing (Bisulfite sequencing PCR, BSP) is DNA methylation
" goldstandard " in detection, detects more accurate, but its detection sensitivity is relatively low, and operates cumbersome, cost high.High score
Resolution melting curve method (High Resolution Melting, HRM) is in PCR amplifications by observing the change of melting curve
To judge whether DNA methylates, such methods and resultses analysis is relative complex.High-flux sequence detection method is directed primarily to
The cost arrived is too high, is unfavorable for popularization clinically.
With tumor research is goed deep into, gradually find to organize Biopsy to have one in the diagnosis of cancer and therapeutic process
Fixed limitation.It is mainly shown as：Tumour has heterogeneity, for cancer cell has occurred and that the patient of transfer, Jin Jinqu
The tumor tissues at some position, can not reflect the overall condition of patient, but all tumor tissues are not all sampled with detection again not
Correspond to reality；Some patients situation of itself determines that he is not suitable for doing tissue biopsy；After the disturbance performed the operation, some tumours
There is the risk for accelerating to shift；It is also unfavorable to organize treatment of the hysteresis quality of biopsy to patient.Therefore for cancer diagnosis and
Detection technique has higher requirement.
The appearance of liquid Biopsy, the problem of solving above-mentioned is also advanced by the Diagnostic Time of cancer.Liquid biopsy is
A kind of technology, even more a kind of clinical solution.There can be many applications under the background of liquid biopsy, including it is clinical and face
Consumer.Mainly mutation, gene magnification, Gene Fusion, the missing/insertion including DNA etc. of the target spot of current genetic test, it is this
It is a kind of irreversible change.And DNA change runs far deeper than these, some apparent modifications can produce influence to gene function,
And then influence cell function and individual health, the generation that methylates of such as gene promoter area can suppress as a rule transcription because
The function of the combination of son and promoter, activation or suppressor.By a common example explanation, many people like body-building to mould
Body, in the strengthening process of muscle, just contains the dynamic changing process that methylates of specific gene.This dynamic monitoring process,
Tissue can not possibly be repeatedly obtained, multiple, real-time monitoring can be realized by noninvasive liquid biopsy.We can gradually have found this
Kind of technology can many aspects application.
The advantage of liquid biopsy, which is that to sample by Noninvasive, reduces the harm of biopsy, and effectively extends patient
Life cycle, cost performance is high.Relative organization is easier to obtain, and noninvasive to patient.But cycling tumor DNA amount is less in blood plasma, and
Easily degrade.Therefore, the difficulty that methylates of gene is relatively large in detection blood plasma, not only needs to obtain high-quality tumour to follow
DNA after circular DNA and the conversion of high-quality bisulfite, and need the high sensitivity that methylates, the high accuracy of gene
Ground is detected.Therefore, the exploitation for such detection kit needs to have the requirement of two aspects：High specific and height
Sensitivity.
The content of the invention
It is an object of the invention to provide one kind for detection of early lung cancer have high sensitivity and specific composition and
Its kit, by detecting the SHOX2 genes of dissociative DNA and PTGER4 gene promoters area, base in lung cancer High risk group blood
Because 3 target site methylation status in coding and non-coding region carry out examination early stage of lung cancer patient, and aid in the morning of lung cancer
Phase diagnoses.For PCR (Methylation-specific PCR, MSP) detection method of methylation-specific, easy to operate,
It is time-consuming shorter, as a result it is easy to analysis.
To achieve these goals, a kind of composition for early stage of lung cancer non-invasive screening that the present invention is provided, described group
Compound includes the nucleic acid for being respectively used to detect 3 SHOX2 genes in the region that methylates related to the early stage of lung cancer in measuring samples
The nucleotide sequence of sequence, the nucleotide sequence of 3 PTGER4 genes and 1 reference gene, wherein, for 3 pairs of first of SHOX2 genes
The sequence of base specific primer, 3 pairs of Methylation-specific primers for PTGER4 genes and 1 pair of internal reference gene primer is such as
Under：
The forward primer F of SHOX2 genes 1：TGATGTTTTTTTGTCGGAG,
The reverse primer R of SHOX2 genes 1：TCAAAACAAAAAACCGC；
The forward primer F of SHOX2 genes 2：GTTTGTATTTTTTTCGAT,
The reverse primer R of SHOX2 genes 2：CACGAATAATAAAACGAAT；
The forward primer F of SHOX2 genes 3：TTTTTTGGGTAGTTAATATGG,
The reverse primer R of SHOX2 genes 3：CCAAATAATCTCCGTCC；
The forward primer F of PTGER4 genes 1：TAGTTTTGTCGCGTTTTAG
The reverse primer R of PTGER4 genes 1：TAACCATCTAAATCTCGACG
The forward primer F of PTGER4 genes 2：GAGGTAGCGGGGTGGTTT
The reverse primer R of PTGER4 genes 2：ACCTCGCTAAACACCGAACAA
The forward primer F of PTGER4 genes 3：GTTTTCGGGTTATTGGTTT
The reverse primer R of PTGER4 genes 3：ACCGACCTATTAAACACTTT
Reference gene (β-actin) forward primer F：GTGATGGAGGAGGTTTAG,
Reference gene (β-actin) reverse primer R：AAATTACAAAAACCACAA.
Preferably, the composition also includes the corresponding MGB probes of 3 methylation sites, the PTGER4 bases of SHOX2 genes
The corresponding MGB probes of 3 methylation sites and the corresponding MGB probes of reference gene of cause, specific nucleotide sequence are as follows：
The probe of SHOX2 genes 1：FAM-AGCGACGTTGCGT-MGB-BHQ1；
The probe of SHOX2 genes 2：FAM-TTCGGTTCGTAGGT-MGB-BHQ1；
The probe of SHOX2 genes 3：FAM-TGCGATTTCGGTCG-MGB-BHQ1；
The probe of PTGER4 genes 1：CY5-TTCGGCGTCGTCG-MGB-BHQ2
The probe of PTGER4 genes 2：CY5-GCGGTCGCGGTCG-MGB-BHQ2
The probe of PTGER4 genes 3：CY5-CGTGGCGATGGTTATCMGB-BHQ2
Reference gene (β-actin) probe：VIC-CACCACCCAACACACAAT-MGB-BHQ1；
Wherein mark fluorescent reporter groups, 3 ' end mark quenching groups are held in the 5 ' of probe sequence.
Preferably, the composition also including the corresponding MGB probes of 3 of the SHOX2 genes non-target gene that methylate and
The corresponding MGB probes of 3 of the PTGER4 genes non-target gene that methylate, specific nucleotide sequence is as follows：
The degeneracy of SHOX2 genes 1 closes primer：AGYGAYGTTGYGT-CY3-spacer；
The degeneracy of SHOX2 genes 2 closes primer：TTYGGTTYGTAGGT-CY3-spacer；
The degeneracy of SHOX2 genes 3 closes primer：TGYGATTTYGGTYG-CY3-spacer；
The degeneracy of PTGER4 genes 1 closes primer：TTYGGYGTYGTYGGAGT-CY3-spacer；
The degeneracy of PTGER4 genes 2 closes primer：TYGAGGYGGTYGYGGTYG-CY3-spacer；
The degeneracy of PTGER4 genes 3 closes primer：AYGTGGYGATGGTTATYGGG-CY3-spacer；
Wherein mark fluorescent reporter groups, 3 ' end mark quenching groups are held in the 5 ' of probe sequence.
3 pairs of methylation-specifics for the methylation detection kit of early stage of lung cancer non-invasive screening, including SHOX2 genes
The 3 pairs of Methylation-specific primers and 1 pair of internal reference gene primer in addition to 3 first of SHOX2 genes of primer and PTGER4 genes
The corresponding MGB probes in base site are corresponding with the corresponding MGB probes of 3 methylation sites and reference gene of PTGER4 genes
MGB probes, include 3 of the corresponding MGB probes of 3 of the SHOX2 genes non-target gene that methylate and PTGER4 genes
The corresponding MGB probes of the non-target gene that methylates.
Preferably, the kit includes PCR reaction solutions, UDG enzymes.
Preferably, the PCR reaction solutions include DNA Taq polymerases, dNTPs and Mg2+, 10 × archaeal dna polymerase buffer.
Preferably, its interpretation method be detect the site of SHOX2 and PTGER4 genes at least two methylate or
At least one site of SHOX2 and PTGER4 methylates simultaneously, the generation for assessing the early stage of lung cancer.
For the sample processing method of the methylation detection kit of early stage of lung cancer non-invasive screening, including：
(1) sample is provided, the samples sources are in the peripheral blood of Healthy People, High Risk of Lung Cancer crowd and early stage of lung cancer patient
Or isolated blood plasma；
(2) plasma sample carries out the extraction of dissociative DNA in step (1), carries out DNA quality monitorings, chooses OD260/280
DNA between 1.8~2.0；
(3) dissociative DNA obtained in step (2), carries out bisulfite conversion, makes what is do not methylated in DNA
5 ' Cytosines are uracil, and the 5 ' cytimidines methylated do not change, and finally obtain Bis-DNA；
(4) Bis-DNA obtained in step (3) is template, uses being used for early any one of claim 1-3
The composition of phase lung cancer non-invasive screening enters performing PCR amplification.
Preferably, in step (4) PCR expand, it is preferable that PCR mixtures include 75-150ng Bis-DNA templates and
200-400nM primers, 100-300nM probes, 1UTaq polymerases, 1-10mM MgCl2Deng the PCR courses of reaction are：37
UDG enzyme reactions 2min at DEG C, 95 DEG C of pre-degeneration 3min；94 DEG C of denaturation 15s, 60 DEG C of annealing extension 35s, 45 circulations.
A kind of composition and its application for early stage of lung cancer non-invasive screening that the present invention is provided, with following beneficial effect
Really.
The site of kit detection SHOX2 and PTGER4 gene methylations of the present invention not only includes the promoter region of gene
Domain, includes the coding region and noncoding region of gene, so carries out multizone detection and is conducive to finding and early stage of lung cancer height
Related SHOX2 genes and the target sequence of PTGER4 genes, while can also improve the accuracy and specificity of detection, are improved
The recall rate of SHOX2 and PTGER4 gene methylations.The kit has very strong for lung cancer early screening on a molecular scale
Specific aim, has the advantages that the specific good, sensitivity of detection is high and of good reliability, so for early stage possible patients with lung cancer and
Early progress prevents and takes corresponding remedy measures.
Kit of the present invention mainly uses the means of Taqman-MGB probe in detecting, special with methylated DNA fragments by probe
Property combine, and optimization reaction system, SHOX2 and PTGER4 gene methylations site is accurately detected.In contrast to special with PCR
Methylation sites are identified by the method that specific primer is detected using probe, with higher sensitivity, accuracy.
And on the basis of MGB probes, introduce degeneracy closing primer technique.The present invention carries out the first of related gene using this technology
Baseization is detected, by noninvasive liquid biopsy, easy to operate, be easy to interpretation, to the less demanding of instrument, and whole PCR mistakes
Cheng Caiyong totally-enclosed forms, it is to avoid the possibility of cross pollution, make result more accurate.The high sensitivity of kit detection is applicable
In the early screening of lung cancer.
Brief description of the drawings
Fig. 1 is the testing result of embodiment 1.
Embodiment
In order that those skilled in the art more fully understand the present invention program, with reference to embodiment to this hair
It is bright to be described in further detail.
Embodiment 1：
SHOX2 and PTGER4 genes methylates in detection I phase Plasma of The Patients With Lung Cancer dissociative DNAs.
Reagent constituents such as following table：
Choose the plasma sample of 5 Healthy Peoples, and 5 tumour plasma samples that known pathology information is the lung cancer I phases.
1st, extraction dissociative DNA is carried out to above-mentioned 10 plasma samples.
2nd, dissociative DNA, which is extracted, is completed, and bisulfite conversion, unmethylated cytimidine (C) are carried out to the DNA extracted
It is changed into uracil (U), and the cytimidine (C) methylated is constant.Obtain purified Bis-DNA.
3rd, PCR reaction solutions are prepared：Archaeal dna polymerase, dNTPs, Mg2+, 10 × archaeal dna polymerase buffer, SHOX2 and PTGER4
The primed probe of gene 1, the primed probe of SHOX2 and PTGER4 genes 2, the primed probe of SHOX2 and PTGER4 genes 3, reference gene
Primed probe, the degeneracy of SHOX2 and PTGER4 genes 1 closing primer, the degeneracy of SHOX2 and PTGER4 genes 2 closing primer, SHOX2
Primer is closed with the degeneracy of PTGER4 genes 3.
4th, PCR reaction conditions are：37 DEG C of UDG enzyme reactions 2min；95 DEG C of pre-degeneration 3min；94 DEG C of denaturation 15s, 60 DEG C of annealing
Extend 35s, 45 circulations；
5th, the configuration of PCR reaction systems
DNA PCR expand MIX:
| Component | One person-portion addition (μ L) |
| Archaeal dna polymerase | 0.25 |
| dNTPs | 2 |
| Mg2+ | 3-3.5 |
| 10 × archaeal dna polymerase buffer | 2.5 |
| UDG enzymes | 0.1 |
| dUTP | 0.01 |
Pcr amplification reaction system:
| Component | One person-portion addition (μ l) |
| DNA PCR expand MIX | 12.5 |
| SHOX2 genes 1/2/3-F (10 μM) | 0.5 |
| SHOX2 genes 1/2/3-R (10 μM) | 0.5 |
| SHOX2 genes 1/2/3-FP (10 μM) | 0.2 |
| PTGER4 genes 1/2/3-F (10 μM) | 0.5 |
| PTGER4 genes 1/2/3-R (10 μM) | 0.5 |
| PTGER4 genes 1/2/3-FP (10 μM) | 0.2 |
| SHOX2 genes Blocker1/2/3 (20 μM) | 0.2 |
| PTGER4 genes Blocker1/2/3 (20 μM) | 0.2 |
| Reference gene-F (10 μM) | 0.3 |
| Reference gene-R (10 μM) | 0.3 |
| Reference gene-FP (10 μM) | 0.2 |
| Purified water | 6.9 |
| Template | 2 |
PCR amplification programs are as follows：
First amplification stage：37 DEG C of UDG enzyme reactions 2min；
Second amplification stage：95 DEG C of pre-degeneration 3min；
3rd amplification stage：94 DEG C of denaturation 15s, 60 DEG C of annealing extension 35s, 45 circulations；
Signal collection, 60 DEG C of collection FAM, VIC and CY5 signals of phase III.
6th, Analysis of test results
Testing result is as shown in Figure 1 to be detected to 10 samples using above-mentioned reaction system.Display kit of the present invention
5 lung cancer I phase positive samples can accurately be detected, the difference of target gene 1/2/3Ct values and reference gene Ct values is respectively less than 8, says
Bright SHOX2 and PTGER4 genes methylate.There is no amplification curve, target gene 1/2/3Ct to remaining 5 negative samples
The difference of value and reference gene Ct values is all higher than 8, illustrates that SHOX2 and PTGER4 do not methylate.
7th, result interpretation analysis (such as table 1)
1) meeting internal standard passage has S type amplification curves, and Ct values≤32, SHOX2 and PTGER4 Gene channels are expanded without S types
Curve or Ct values ＞ 40, △ Ct values ＞ 8, and then result of determination is feminine gender；
2) meeting internal standard passage has S type amplification curves, and Ct values≤32, SHOX2 or PTGER4 Gene channels at least two
Methylate region detection Ct value≤40, △ Ct value≤8, then result of determination is the positive；
3) meeting internal standard passage has S type amplification curves, and Ct values≤32, SHOX2 and PTGER4 Gene channels at least one
The individual region that methylates is while meet detection Ct value≤40, △ Ct value≤8, then result of determination is the positive；
4) if internal standard passage is without S types amplification curve or Ct values ＞ 32, result of determination is invalid, it is proposed that extracts sample again and enters
Row detection.
The result interpretation of table 1 is referred to
Embodiment 2：
Collect the plasma sample of 93 early stage of lung cancer patients (0 phase 27, I phases 66) from Shanghai Fei Ke hospitals and come
From 100 human normal plasma's samples of Shandong Shandong hospital, SHOX2 and PTGER4 gene first is carried out to this 193 plasma samples
The detection of base, concrete operation step is as described in case study on implementation 1.In 100 normal plasma pattern detection results, 95 samples
The difference of target gene 1/2/3Ct values and reference gene Ct values is all higher than 8, and specificity reaches 95%；And for 93 early stages of lung cancer
Plasma sample, detects that the difference of 88 sample results target gene 1/2/3Ct values and reference gene Ct values is respectively less than 8, and it is sensitive
Property reaches 95%, wherein it is 97% that 0 phase lung cancer, which is 89%, I phases lung cancer,.
Specific case used herein is elaborated to inventive concept, and the explanation of above example is only intended to
Help to understand core concept of the invention.It should be pointed out that for those skilled in the art, not departing from this
On the premise of inventive concept, any obvious modification, equivalent substitution or other improvement made should be included in the present invention
Protection domain within.
SEQUENCE LISTING
<110>Beijing Xinnuo Meidi Gene Inspection Technology Co., Ltd.
<120>A kind of composition and its application for early stage of lung cancer non-invasive screening
<130> P20170065
<160> 27
<170> PatentIn version 3.3
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<400> 16
ttcggttcgt aggt 14
<210> 17
<211> 14
<212> DNA
<213>Artificial sequence
<400> 17
tgcgatttcg gtcg 14
<210> 18
<211> 13
<212> DNA
<213>Artificial sequence
<400> 18
ttcggcgtcg tcg 13
<210> 19
<211> 13
<212> DNA
<213>Artificial sequence
<400> 19
gcggtcgcgg tcg 13
<210> 20
<211> 16
<212> DNA
<213>Artificial sequence
<400> 20
cgtggcgatg gttatc 16
<210> 21
<211> 18
<212> DNA
<213>Artificial sequence
<400> 21
caccacccaa cacacaat 18
<210> 22
<211> 13
<212> DNA
<213>Artificial sequence
<400> 22
agygaygttg ygt 13
<210> 23
<211> 14
<212> DNA
<213>Artificial sequence
<400> 23
ttyggttygt aggt 14
<210> 24
<211> 14
<212> DNA
<213>Artificial sequence
<400> 24
tgygatttyg gtyg 14
<210> 25
<211> 17
<212> DNA
<213>Artificial sequence
<400> 25
ttyggygtyg tyggagt 17
<210> 26
<211> 18
<212> DNA
<213>Artificial sequence
<400> 26
tygaggyggt ygyggtyg 18
<210> 27
<211> 20
<212> DNA
<213>Artificial sequence
<400> 27
aygtggygat ggttatyggg 20
Claims (10)
1. a kind of composition for early stage of lung cancer non-invasive screening, it is characterised in that the composition includes being respectively used to detection
The nucleotide sequence of 3 SHOX2 genes in the methylate region related to the early stage of lung cancer in measuring samples, 3 PTGER4 genes
The nucleotide sequence of nucleotide sequence and 1 reference gene, wherein, 3 pairs of Methylation-specific primers for SHOX2 genes, it is directed to
3 pairs of Methylation-specific primers of PTGER4 genes and the sequence of 1 pair of internal reference gene primer are as follows：
The forward primer F of SHOX2 genes 1：SEQ ID NO.1,
The reverse primer R of SHOX2 genes 1：SEQ ID NO.2；
The forward primer F of SHOX2 genes 2：SEQ ID NO.3,
The reverse primer R of SHOX2 genes 2：SEQ ID NO.4；
The forward primer F of SHOX2 genes 3：SEQ ID NO.5,
The reverse primer R of SHOX2 genes 3：SEQ ID NO.6；
The forward primer F of PTGER4 genes 1：SEQ ID NO.7,
The reverse primer R of PTGER4 genes 1：SEQ ID NO.8；
The forward primer F of PTGER4 genes 2：SEQ ID NO.9,
The reverse primer R of PTGER4 genes 2：SEQ ID NO.10；
The forward primer F of PTGER4 genes 3：SEQ ID NO.11,
The reverse primer R of PTGER4 genes 3：SEQ ID NO.12；
Reference gene (β-actin) forward primer F：SEQ ID NO.13,
Reference gene (β-actin) reverse primer R：SEQ ID NO.14.
2. the composition according to claim 1 for early stage of lung cancer non-invasive screening, it is characterised in that the composition is also
The corresponding MGB of 3 methylation sites of the corresponding MGB probes of 3 methylation sites, PTGER4 genes including SHOX2 genes
Probe and the corresponding MGB probes of reference gene, specific nucleotide sequence are as follows：
The probe of SHOX2 genes 1：SEQ ID NO.15；
The probe of SHOX2 genes 2：SEQ ID NO.16；
The probe of SHOX2 genes 3：SEQ ID NO.17；
The probe of PTGER4 genes 1：SEQ ID NO.18；
The probe of PTGER4 genes 2：SEQ ID NO.19；
The probe of PTGER4 genes 3：SEQ ID NO.20；
Reference gene (β-actin) probe：SEQ ID NO.21；
Wherein mark fluorescent reporter groups, 3 ' end mark quenching groups are held in the 5 ' of probe sequence.
3. the composition according to claim 2 for early stage of lung cancer non-invasive screening, it is characterised in that the composition is also
3 non-purposes that methylate of the corresponding MGB probes of 3 including the SHOX2 genes non-target gene that methylate and PTGER4 genes
The corresponding MGB probes of gene, specific nucleotide sequence is as follows：
The degeneracy of SHOX2 genes 1 closes primer：SEQ ID NO.22；
The degeneracy of SHOX2 genes 2 closes primer：SEQ ID NO.23；
The degeneracy of SHOX2 genes 3 closes primer：SEQ ID NO.24；
The degeneracy of PTGER4 genes 1 closes primer：SEQ ID NO.25；
The degeneracy of PTGER4 genes 2 closes primer：SEQ ID NO.26；
The degeneracy of PTGER4 genes 3 closes primer：SEQ ID NO.27；
Wherein mark fluorescent reporter groups, 3 ' end mark quenching groups are held in the 5 ' of probe sequence.
4. the composition for early stage of lung cancer non-invasive screening according to any one of claim 1-3 was prepared for early stage
Application in the reagent of lung cancer non-invasive screening.
5. a kind of methylation detection kit for early stage of lung cancer non-invasive screening, it is characterised in that the kit includes power
Profit requires the composition for early stage of lung cancer non-invasive screening any one of 1-3.
6. the methylation detection kit according to claim 5 for early stage of lung cancer non-invasive screening, it is characterised in that institute
Stating kit includes PCR reaction solutions and UDG enzymes.
7. the methylation detection kit according to claim 6 for early stage of lung cancer non-invasive screening, it is characterised in that institute
Stating PCR reaction solutions includes DNATaq polymerases, dNTPs, Mg2+With 10 × archaeal dna polymerase buffer.
8. the methylation detection kit according to claim 7 for early stage of lung cancer non-invasive screening, it is characterised in that its
Interpretation method is, the site of SHOX2 and PTGER4 genes at least two methylate or SHOX2 and PTGER4 at least one
Site methylates simultaneously, the generation for assessing the early stage of lung cancer.
9. a kind of sample process of the methylation detection kit according to claim 8 for early stage of lung cancer non-invasive screening
Method, it is characterised in that the sample processing method includes：
(1) provide sample, the samples sources in Healthy People, the peripheral blood of High Risk of Lung Cancer crowd and early stage of lung cancer patient or
Isolated blood plasma；
(2) extraction of dissociative DNA is carried out to plasma sample in step (1), DNA quality monitorings are carried out, OD260/280 is chosen and exists
DNA between 1.8~2.0；
(3) bisulfite conversion is carried out to the dissociative DNA obtained in step (2), makes the 5 ' born of the same parents not methylated in DNA phonetic
Pyridine is converted into uracil, and the 5 ' cytimidines methylated do not change, and finally obtains Bis-DNA；
(4) Bis-DNA obtained using in step (3) is used for early stage lung as template using any one of claim 1-3
The composition of cancer non-invasive screening enters performing PCR amplification.
10. the sample process side of the methylation detection kit according to claim 9 for early stage of lung cancer non-invasive screening
Method, it is characterised in that in the step (4), the Bis-DNA templates of PCR mixtures including 75-150ng, 200-400nM primers,
The MgCl of 100-300nM probes, 1UTaq polymerases and 1-10mM2；PCR courses of reaction are：UDG enzyme reactions 2min, 95 at 37 DEG C
DEG C pre-degeneration 3min, 94 DEG C of denaturation 15s, 60 DEG C of annealing extension 35s, 45 circulations.
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