CN104774957B - Diagnose the method and kit of people SHOX2 genes and people's RASSF1A gene methylations - Google Patents
Diagnose the method and kit of people SHOX2 genes and people's RASSF1A gene methylations Download PDFInfo
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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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- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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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Abstract
The present invention relates to the method and kit of diagnosis people SHOX2 genes and people's RASSF1A gene methylations.A kind of methylate DNA by two kinds of lung cancer markers of cooperation detection is disclosed first come diagnosing, the method for improving lung cancer recall rate, and described lung cancer marker is people SHOX2 genes and people's RASSF1A genes.Present invention also offers the detection SHOX2 genes of optimization and people's RASSF1A gene methylations DNA reagent, detection method.
Description
Technical field
The invention belongs to gene diagnosis field, more particularly it relates to a kind of diagnosis people SHOX2 genes and people
The method and kit of RASSF1A gene methylations.
Background technology
Lung cancer has become one of human cancer main causes of death.It is incidence of disease highest cancer in lung cancer in China,
And morbidity and mortality are increased rapidly.The incidence and the death rate of lung cancer are highests in all tumours, but lung cancer is but not
It is most tumours of diagnosis, in the U.S., tumor of breast and tumor of prostate have higher diagnosis, and reason is to examine in early days
It is disconnected to substantially increase 5 years survival rates (being respectively 89 and 99%) so as to early treatment, and 5 years survival rates of lung cancer are
15%.
In clinical practice, lung cancer early diagnosis is always difficult point, early detection effectively the controlling to cancer patient of cancer
Treatment is very important.Diagnosis Main Basiss clinical symptoms, iconography detection and histopathological examination at present to cancer etc.,
But there have many cancer clinical symptoms to occur to be later, and living body sampling detection is also difficult, has had a strong impact on the early diagnosis of cancer
With the prognosis of patient.Relative to tissue biopsy, the sample such as bronchoalveolar lavage fluid, blood plasma, sputum is readily available, and is not also had to person under inspection
Wound.At present, in the circulating of periphery, the DNA of same gene abnormal methylation only accounts for the small part of all DNA, about
0.1%~1%, these non-methylate DNAs and methylate DNA only exist small difference, it is necessary to from highly complex " background "
In detect abnormal methylate DNA.It (is usually tens to one hundred polybase that DNA in other circulating, which has generally degraded,
Base to) fragment, so needing special extracting and detection technique to obtain higher sensitivity.
With technical fast development, the tumour that tumor markers develops into after diagnostic imaging, pathological diagnosis
The new field of diagnoses and treatment, diagnosis, monitoring and treatment to tumour generate significant impact.Tumor markers can in body fluid or
Detected in tissue, can reflect that therapeutic effect etc. is estimated and judged in presence, differentiation degree, the prognosis of tumour.The early stage of lung cancer by
Hardly discovered in often without characteristic symptom by doctor and patient, be also difficult to early detection with conventional diagnostic method and early stage is fixed
Property diagnosis, in addition some tumor markers be only capable of pointing out as the primary dcreening operation or auxiliary diagnosis of lung cancer and can not make a definite diagnosis, therefore lung cancer
Early diagnosis it is more difficult.At present, as gene diagnosis technology is continued to develop, the early diagnosis to lung cancer brings hope,
One of field is exactly methylate DNA detection.DNA methylation almost has generation in all tumours, becomes tumour
One reliable target of diagnosis.DNA methylation is a tumorigenic earliest events, with regard to that can be detected before disease is made a definite diagnosis
Come, be the potential index of early diagnosis of tumor, risk prediction, clinical disease course monitoring and curative effect evaluation.
DNA methylation is as a kind of novel molecular mark, and the importance in diagnosing tumor is increasingly subject to pay attention to, its advantage
Predominantly：First, in neoplastic process, the occurrence frequency of promoter supermethylation is very high, even above gene mutation, its
In be no lack of and form relevant important gene with tumour；Second, it is the critical event of tumour generation early stage to methylate；Third, DNA first
Base is stabilized, and can be detected by PCR enlarge-effects.Therefore, DNA methylation assay has potentially to early diagnosis of tumor
Application value.Recently research both at home and abroad finds that the contents level of the methylate DNA of some genes in blood plasma is used as early diagnosis, quick
Perception is better than existing protein sera mark, wherein having the cyclin- related to the early stage of lung cancer than more prominent
The genes such as dependent kinase inhibitor 2A gene p16 (p16), H-cadherin (CDH13).Smoking is lung
The main reason of cancer, is found that several genes often occurred in Human Lung Cancer are different in the respiratory tract of normal smoking population
Often.Wherein, what research was more is p16 Methylation of Gene.Cast-off cells in normal smoking population sputum specimen are examined
During survey, it is found that p16 Methylation of Gene frequencies are higher, and the change do not found then in normal non-smokers' sputum specimen,
Illustrate that this gene alteration may be closely related with early stage respiratory tumor generating process.Related gene promoter methyl in sputum
Change degree is as tumour danger increases and increases, and 3 years that CDKN2A/p16 and/or mgmt gene are diagnosed in clear and definite lung squamous cancer
It is preceding to be detected in phlegm.At present, there are many research detection blood, sputums, cell or DNA methylation in bronchoalveolar lavage fluid
State, to find the mark early diagnosed for lung cancer.
Although having discovered that the DNA methylation of some genes and the correlation of lung cancer in the prior art, at present originally
In field there is still a need for further research can effectively be applied to pulmonary cancer diagnosis related gene, and develop with compared with
The detection reagent of high detection accuracy.
The content of the invention
It is an object of the invention to provide a kind of method for diagnosing people SHOX2 genes and people's RASSF1A gene methylations and
Kit.
In the first aspect of the present invention there is provided a kind of kit for being used to detect lung cancer, described kit includes：It is special
The detection reagent of the DNA methylation of opposite sex detection people's SHOX2 genes；With the DNA methylation of specific detection people's RASSF1A genes
Detection reagent.
In a preference, described DNA methylation sequence contains CpG dinucleotides structures, and CpG dinucleotides
The C bases of structure carry methyl group.
In another preference, in kit, the detection of the DNA methylation of described specific detection people's SHOX2 genes
Reagent is the primer and probe for people's SHOX2 genes；It is preferred that described primer and probe is directed to SEQ in people's SHOX2 genes
ID NO:3 sequence (the SEQ ID NO after bisulfites or weight bisulfites or hydrazonium salt modification:4)；Or it is described special
Property detection people's RASSF1A genes the detection reagent of DNA methylation be primer and probe for people's RASSF1A genes；Preferably
Ground, described primer and probe is directed to SEQ ID NO in people's RASSF1A genes:1 through bisulfites or weight bisulfites or
Sequence (SEQ ID NO after hydrazonium salt modification:2).
In another preference, in kit, the detection of the DNA methylation of described specific detection people's SHOX2 genes
Reagent is：SEQ ID NO:14 and SEQ ID NO:Primer and SEQ ID NO shown in 18:Probe shown in 25；Or it is described
The detection reagents of DNA methylation of specific detection people's RASSF1A genes be：SEQ ID NO:7 and SEQ ID NO:10 institutes
The primer and SEQ ID NO shown:Probe shown in 22.
In another preference, in kit, also include in described kit：The detection of specific detection reference gene
Reagent；It is preferred that the detection reagent of described specific detection reference gene is primer and the spy for people's β-actin genes
Pin；More preferably, described primer and probe is directed to SEQ ID NO in people's β-actin genes:5 through bisulfites or weight sulfurous
Sequence (SEQ ID NO after sour hydrogen salt or hydrazonium salt modification:6)；More preferably, the DNA first of described specific detection reference gene
The detection reagent of base is：SEQ ID NO:19 and SEQ ID NO:Primer and SEQ ID NO shown in 21:Spy shown in 26
Pin.
In another preference, described reference gene is used for the quality for indicating that DNA is extracted and modified.
In another preference, in kit, specific fluorophor is connected with for the probe of each gene；Preferably
Ground, marks VIC fluoresceins, for the probe flag F AM fluoresceins of people's RASSF1A genes, pin for the probe of people's SHOX2 genes
CY5 fluoresceins are marked to the probe of people's β-actin genes.
In another preference, in the kit, for the ratio (primer of the primer and probe of people's SHOX2 genes:Visit
Pin) be：0.2：0.06 in kit (for example, design PCR amplification system primer final concentration:Probe final concentration=0.2uM：
0.06uM)；Or in the kit, for the ratio (primer of the primer and probe of people's RASSF1A genes:Probe) be：0.2：
0.06 in kit (for example, design PCR amplification system primer final concentration:Probe final concentration=0.2uM：0.06uM)；Or should
In kit, for the ratio (primer of the primer and probe of people's β-actin genes:Probe) be：0.67：0.02 (for example, in examination
PCR amplification system primer final concentration is designed in agent box:Probe final concentration=0.67uM：0.02uM).
In another preference, in the kit, also include but is not limited to：Archaeal dna polymerase (such as Taq enzyme), dNTPs,
Mg2+Ion includes archaeal dna polymerase (such as Taq enzyme), dNTPs, Mg2+The PCR system of ion；It is preferred that described includes DNA
Polymerase, dNTPs, Mg2+In the PCR system of ion, described Mg2+Ion concentration be 3.5 ± 0.5mM (preferably 3.5 ±
0.3mM)；Or described dNTPs concentration is 0.25 ± 0.3mM (preferably 0.25 ± 0.1mM)；Or described archaeal dna polymerase is
1.5 ± 0.2U (preferably 1.5 ± 0.1U).It is preferred that also including in described kit：Bisulfites or weight bisulfite
Salt or hydrazonium salt.
In another preference, also include but is not limited in described kit：Quality-control product, negative control and/or say
Bright book.
There is provided specific detection people SHOX2 genes and the DNA methyl of people's RASSF1A genes in another aspect of this invention
The purposes of the reagent of change, the kit for preparing detection lung cancer.
In a preference, the detection reagent of the DNA methylation of described specific detection people's SHOX2 genes is to be directed to
The primer and probe of people's SHOX2 genes；It is preferred that described primer and probe is directed to SEQ ID NO in people's SHOX2 genes:3 warps
Sequence (SEQ ID NO after bisulfites or weight bisulfites or hydrazonium salt modification:4)；More preferably, described specificity inspection
Surveying the detection reagent of the DNA methylation of people's SHOX2 genes is：SEQ ID NO:14 and SEQ ID NO:Primer shown in 18 and
SEQ ID NO:Probe shown in 25；Or the detection reagent of the DNA methylation of described specific detection people's RASSF1A genes is
For the primer and probe of people's RASSF1A genes；It is preferred that described primer and probe is directed to SEQ in people's RASSF1A genes
ID NO:1 sequence (the SEQ ID NO after bisulfites or weight bisulfites or hydrazonium salt modification:2)；More preferably, it is described
The detection reagents of DNA methylation of specific detection people's RASSF1A genes be：SEQ ID NO:7 and SEQ ID NO:10 institutes
The primer and SEQ ID NO shown:Probe shown in 22.
People SHOX2 genes and people's RASSF1A bases are detected there is provided a kind of external nondiagnostic in another aspect of this invention
The method of the DNA methylation of cause, methods described includes：
(1) testing sample is subjected to bisulfites or weight bisulfites or hydrazonium salt is handled, obtained through the to be measured of modification
Sample；
(2) testing sample through modification of step (1) is carried out using the kit for being described previously for detecting lung cancer
Detection, obtains the DNA methylation situation of people's SHOX2 genes and the DNA methylation situation of people's RASSF1A genes.
In a preference, if the DNA methyl of people's SHOX2 gene DNAs methylation positive and/or people's RASSF1A genes
Change the positive, then the supplier for showing testing sample is High Risk of Lung Cancer or patients with lung cancer.
In another preference, multiple nucleic acid fragment detections are carried out in a reaction tube；And simultaneously in a reaction tube
It is interior to carry out multiple wave band fluorescence signal detections, different DNA fragmentations are distinguished by different fluorescence signals.Or, can be not
Different nucleic acid fragment detections are carried out in same reaction tube；And single wave band fluorescence signal inspection is carried out in a reaction tube simultaneously
Survey, different DNA fragmentations are distinguished by fluorescence signal.
In another preference, described sample of nucleic acid is derived from：The nucleic acid of sample extraction containing cell, such as alveolar are filled
Washing lotion, is derived from the tissue of diseased region, hydrothorax, sputum etc.；Or the sample containing the nucleic acid from cell, such as blood plasma, serum
Deng.
The other side of the present invention, due to this disclosure, is apparent to those skilled in the art
's.
Brief description of the drawings
Fig. 1, people SHOX2 genes and people's RASSF1A gene methylations DNA detection specificity.
Digital table in Fig. 2, people SHOX2 genes and people's RASSF1A gene methylation DNA detection sensitivities (VIC signals), figure
Show cell number.
Digital table in Fig. 3, people SHOX2 genes and people's RASSF1A gene methylation DNA detection sensitivities (FAM signals), figure
Show cell number.
Embodiment
The present inventor passes through in-depth study, and a kind of methylating by two kinds of lung cancer markers of cooperation detection is disclosed first
DNA is come the method for improving lung cancer recall rate, and described lung cancer marker is people SHOX2 genes and people's RASSF1A genes.The present invention
People also optimizes pcr amplification reaction program, further increases amplification efficiency.The present inventor also optimizes detection people's SHOX2 bases
Cause and people's RASSF1A gene methylations DNA reagent, detection method.The present invention is completed on this basis.
As used herein, " sample to be tested ", " testing sample " or " determined nucleic acid (such as DNA) sample " refers to core to be detected
Acid sample, wherein containing a kind of nucleic acid or multiple nucleic acids, it is to be understood that wherein with the presence or absence of target nucleic acid.It is described in the present invention
Sample to be tested can be：Bronchoalveolar lavage fluid, is derived from the tissue of diseased region, hydrothorax, sputum etc.；Or containing from cell
The sample of nucleic acid, such as blood plasma, serum etc..
As used herein, " target nucleic acid " refers to nucleic acid fragment interested, and it is people SHOX2 genes and people RASSF1A
Gene methylation DNA specific fragments.
As used herein, " probe " refers to a kind of single-chain nucleic acid with known nucleotide sequence, and it has and target core
The nucleotide sequence structure that acid is substantially complementary, can be with " target nucleic acid " formation double-strand.Described " probe " can carry mark
Remember thing.For example, label can be connected to the 5 ' ends or 3 ' ends of probe.
People SHOX2 (short stature homeobox 2) gene is a member of short and small hox genes family, its core
Nucleotide sequence can be with GenBank accession number：NC_000003 sequence is essentially identical.Its gene expression regulation and allelotaxis
It is closely related.Research finds that SHOX2 genes are expressed in mesoderm and ectoderm in embryonic stage, to bone, heart and nerveous system
The development of system plays an important role.SHOX2 genes abnormal expression in a variety of entity tumors, such as lung cancer, breast cancer, kidney.
People RASSF1A (Ras-association domain family 1A, RASSFlA) gene is Ras relevant ranges
Family's lA genes, its nucleotide sequence can be with GenBank accession number：DQ444319 sequence is essentially identical.RASSFlA regulates and controls
Target gene be related to the aspects of contents such as genetic transcription, signal transduction, cytoskeleton, cell cycle, cell adhesion and apoptosis, have
There is extensive biological action.And RASSFlA is as a kind of novel tumour suppressor gene, its tumour occur and evolution in
Effect it is particularly important.It is now recognized that RASSFlA gene expressions inactivate hyper-methylation mainly abnormal with promoter region, heterozygosis
Property missing and chromosome deficiency it is relevant.
Present inventors have surprisingly found that, in clinical patients with lung cancer, tumor specimen negative SHOX2 gene methylations DNA
In, most samples occur that RASSF1A gene methylations DNA is positive, joint-detection people SHOX2 genes and people's RASSF1A genes
Methylate DNA more individually detects two kinds of genes, and the accuracy rate (recall rate) of lung cancer detection can be significantly improved.
There is provided specific detection people SHOX2 genes and people's RASSF1A genes for above-mentioned new discovery based on the present inventor
The purposes of the reagent of DNA methylation, the kit for preparing detection lung cancer.
Further, it is particularly suitable for people's SHOX2 genes there is provided a class present inventor has performed screening and repetition test
The primer and probe detected with people RASSF1A gene methylation DNA specific fragments.The selection in gene methylation site directly with
Clinical detection sensitivity is related, typically all in the promoter region of gene, but many genes methylation sites related to tumour
It is also possible to be located at First Intron and First Exon region.By it is substantial amounts of experiment and compare, the present inventor have chosen people
SHOX2 genes and people's RASSF1A genes specifically methylate region, such as correspond to the SEQ ID NO of people's SHOX2 genes:3 sequences
Row, or corresponding to the SEQ ID NO of RASSF1A genes:1 sequence.On this basis, the present inventor have also been devised high special
Primer is recognized to amplify different purpose fragments with the probe of high special.
The successful design of primed probe is an extremely important ring for real-time PCR detection, is promoted after sulphite modification
" C " in DNA is converted into " U ", cause G/C content to reduce, PCR is occurred continuous " T " of length after reacting in the sequence, hold
Easily cause the fracture of DNA.In addition, also resulting in DNA loss in purge process.The inventors discovered that, the length of PCR primer
During more than 300bp, it is difficult to the DNA after being modified using sulphite and is expanded as template, 200bp length is proper；With
Unlike standard PCR, sulphite modifies the G/C content for reducing template DNA, makes continuous " T " for occurring length in sequence, leads
Cause to be difficult selection with suitable Tm values and stable primer；On the other hand, modified and without sulfurous to distinguish sulphite
Hydrochlorate moditied processing and the DNA of not complete sulphite modification, it is necessary to primer has sufficient amount of " C ", each of which increases
The difficulty of the stable primer of selection.Difficult for these, the present inventor is on the basis of sufficient sequence research, and selection optimizes suitable
Section, devise the amplimer and probe of high special, establish a kind of high people SHOX2 genes of sensitivity and people
RASSF1A gene methylation DNA method for detecting specificity and kit.
As the preferred embodiment of the present invention, in method of the invention or kit, also indicated using internal reference DNA extract and
The quality of modification.Described internal reference is, for example, preferably β-actin.
, it is necessary to be chemically modified to the DNA of extraction in the method for the present invention, bisulfites or weight bisulfites or
Hydrazonium salt modification can be applied to this kind of chemical modification.In research process, the present inventor is compared by sulphite sequence measurement
The sulphite modification kit of QIAGEN companies and ZYMO companies, on conversion ratio and the rate of recovery, the two does not have significant difference,
From cost consideration, the sulphite modification kit of selection ZYMO companies.
The need for according to clinical detection, pedestrian SHOX2 genes and people's RASSF1A genes are entered using real-time fluorescence PCR technology
The detection of methylate DNA specific fragment is more preferred.When being detected using real-time fluorescence PCR, described probe connects
It is connected to the fluorophor for being suitable for screening different genes methylated DNA fragments.The front end of probe is marked with fluorophor, the other end
It is marked with quenching group, wherein quenching group can the fluorescence that sends of quenching fluorescence group.When pcr amplification reaction is carried out, utilize
Polymerase it is positive it is exo-acting the base with fluorophor is cut off, dissociate after fluorophor no longer by quenching group
Influence, can launch the fluorescence signal of certain wavelength in the presence of exciting light.With the continuous accumulation of PCR primer, fluorescence signal
Constantly strengthen, so as to detect the presence of special methylate DNA.
As the preferred embodiment of the present invention, for convenience of Clinical practice, when being detected, using in same reaction tube
Add three kinds of three kinds of different fluorophors marks specific probes (corresponding to people's SHOX2 genes, people RASSF1A genes and β-
Actin genes), indicate the presence situation of 3 kinds of purpose methylated DNA fragments simultaneously in a reaction tube.
As the preferred embodiment of the present invention, for convenience of Clinical practice, the fluorophor of detection probe mark can be VIC,
ROX, FAM, Cy5, HEX, TET, JOE, NED etc.；Quenching group can be TAMRA, BHQ, MGB, Dabcyl.With suitable for current
The conventional multichannel PCR detection system of clinical detection, realizes and multicolor fluorescence detection is carried out in a reaction tube.
In some preferred embodiments of the present invention, the present inventor also optimizes pcr amplification reaction, further increases amplification
Efficiency.Consumption, dNTPs consumptions including optimizing enzyme, concentration of the primed probe in PCR system so that after PCR reactions, Ct values
Relatively centralized, reaches ideal effect.It should be understood that technique effect can be realized in the case where not optimizing, and enter performing PCR amplification journey
After the optimization of sequence and amplification system, then effect is even more ideal.
The detection reagent of the present invention is placed in kit, consequently facilitating people apply.Except primer and probe reagent, examination
It can also be included in agent box：Bisulfites or weight bisulfites or hydrazonium salt, PCR reaction solutions, quality-control product, negative control and/or
Operation instructions.
The method and kit of the present invention is particularly suitable for lung cancer auxiliary diagnosis and early diagnosis, with high sensitivity, height
It is specific, easy to operate, the features such as detection cycle is short.Detection accuracy can be effectively improved, and reduces the false negative of result.
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention
Rather than limitation the scope of the present invention.The experimental method of unreceipted actual conditions in the following example, generally according to conventional strip
Part such as J. Pehanorm Brookers etc. are write, Molecular Cloning:A Laboratory guide, the third edition, Science Press, the condition described in 2002, or
According to the condition proposed by manufacturer.
Embodiment 1, the selection for detecting target gene
Methylate DNA has clear advantage as detection target, and compared to protein-based mark, DNA is to expand
, and be readily possible to detect；Compared with being mutated class mark, methylate DNA is all located at the privileged site of gene, is typically opening
Mover area, makes detection become easier to and conveniently.In order to complete the present invention, inventor has consulted lot of documents, report with
In the related gene of lung cancer, representational p16, H-cadherin (CDH13), SHOX2, HOXA9, RAR β, RASSF1A are selected
As the detection gene of candidate, each gene methylation site distribution situation is studied, the primed probe of design detection is respectively used to
Detection.Each genetic test primed probe is as follows：
P16 detection primer and probe be：
P16 primers Fs：ACGTCGTGAGCGAGTGTTC(SEQ ID NO:27)；
P16 primers R：TACCAACGCTAACTCTAACGAA(SEQ ID NO:28)；
P16 probes：ROX-CTTCCGACTAATACCCCCGAAA-BHQ(SEQ ID NO:29)；
H-cadherin (CDH13) detection primer and probe be：
Primers F：GAAAATATGTTTAGTGTAGTCGCGT(SEQ ID NO:30)；
Primer R：ACGCACAAAACGAACGAAAT(SEQ ID NO:31)；
Probe：CY5-TTTTATCCGACTAGAAACGCCCG-BHQ(SEQ ID NO:32)；
SHOX2 detection primer and probe be：
Primers F：TTGTTTTTGGGTTCGGGTT(SEQ ID NO:12)
Primer R：CATAACGTAAACGCCTATACTCG(SEQ ID NO:16)
Probe：VIC-ATCGAACAAACGAAACGAAAATTACC-BHQ(SEQ ID NO:24)
HOXA9 detection primer and probe be：
Primers F：GGTATATCGTAGCGGGTATAGC(SEQ ID NO:33)；
Primer R：AACTTCCAATCCAAAACGACG(SEQ ID NO:34)；
Probe：FAM-TTCGTCGCGTGTATTGGGTTTTAC-BHQ(SEQ ID NO:35)；
RAR β detection primer and probe be：
Primers F：CGAGAACGCGAGCGATTC(SEQ ID NO:36)；
Primer R：AACCTTCCGAATACGTTCCG(SEQ ID NO:37)；
Probe：VIC-TCCTACCCCGACGATACCCAAAC-BHQ(SEQ ID NO:38)；
RASSF1A detection primer and probe be：
Primers F：CGGGGTTCGTTTTGTGGTTTC(SEQ ID NO:7)
Primer R：CCGATTAAATCCGTACTTCGC(SEQ ID NO:10)
Probe：FAM-TCGCGTTTGTTAGCGTTTAAAGT-BHQ(SEQ ID NO:22)
Experimentation：
1st, DNA is extracted
The sample and non-tumor patient sample for making a definite diagnosis patients with lung cancer are collected, cell is separated, with Tiangeng biochemical technology (Beijing)
Exemplified by Co., Ltd's genome DNA extracting reagent kit (DP304), DNA is extracted.
2nd, DNA modification
Said with ZYMO RESEARCH biotech firms kit EZ DNA Methylation-DirectTM KIT (D5020)
Bright sulphite modification.
3rd, amplification and detection
It is following to prepare 10 × primed probe mix.
It is following to prepare reaction system (20 μ L)：
PCR amplification programs are as follows：
First stage：95 DEG C, 10min, 1 circulation；
Second stage：95 DEG C, 15sec, 60 DEG C of 30sec, 5 circulations；
Phase III：95 DEG C, 15sec, 57 DEG C of 30sec, 40 circulations；
Signal collection：FAM, VIC, ROX, CY5 signal are collected during 60 DEG C of phase III.
4th, testing result
According to each amplification situation, testing result is shown in Table 1.
Table 1
| p16 | CDH13 | SHOX2 | HOXA9 | RARβ | RASSF1A | |
| Tumor specimen 1 | - | + | + | - | + | + |
| Tumor specimen 2 | + | - | + | + | + | + |
| Tumor specimen 3 | - | - | - | - | - | - |
| Tumor specimen 4 | + | + | + | + | - | + |
| Tumor specimen 5 | + | + | + | + | + | + |
| Tumor specimen 6 | - | - | + | + | + | - |
| Tumor specimen 7 | - | + | + | - | + | + |
| Tumor specimen 8 | - | - | - | - | - | - |
| Tumor specimen 9 | + | + | + | - | + | - |
| Tumor specimen 10 | + | + | + | + | + | + |
| Tumor specimen 11 | - | - | - | - | + | + |
| Tumor specimen 12 | + | + | + | - | + | + |
| Tumor specimen 13 | - | - | + | + | + | + |
| Tumor specimen 14 | + | - | + | + | - | |
| Tumor specimen 15 | + | - | + | + | + | + |
| Tumor specimen 16 | + | - | - | - | - | + |
| Tumor specimen 17 | + | + | + | + | - | - |
| Tumor specimen 18 | - | - | + | + | + | + |
| Tumor specimen 19 | + | + | + | - | - | + |
| Tumor specimen 20 | + | + | + | - | - | + |
| Non- tumor specimen 1 | - | - | - | - | - | - |
| Non- tumor specimen 2 | - | - | - | - | - | - |
| Non- tumor specimen 3 | - | - | - | - | + | - |
| Non- tumor specimen 4 | - | - | - | - | - | - |
| Non- tumor specimen 5 | - | - | - | - | - | - |
Note："+" is that methylate DNA detects positive, and "-" is that methylate DNA detects negative
As can be seen from the above results, at the same detection when, SHOX2 methylation positive rate highests, CDH13, HOXA9, RAR β
Gene pairs improves system Positive rate and not acted synergistically, and wherein RAR β have a detection positive in non-tumor specimen, with
Its joint-detection influences testing result on the contrary, reduces Detection accuracy；RASSF1A test positive in sample 11,16, and mark
This 11 and sample 16 be that SHOX2 DNA methylation assays are negative, therefore, the positive rate that RASSF1A gene pairs improves system detection has association
Same-action.P16 is made there is also similar collaboration with (p16 detects the positive to sample 16, and SHOX2 detections are negative), but its collaboration
With not as RASSF1A it is notable.Therefore selection RASSF1A carries out cooperation detection with SHOX2 gene methylations.
Therefore, inventor expands detection range, 50 tumor samples and 5 non-tumor sample (part Experiment numbers are have collected
According to seeing embodiment 7), further whether checking RASSF1A and SHOX2 cooperation detections contribute to the raising of Positive rate, help
In the accuracy for improving diagnosis.As a result find, in the tumor sample that feminine gender is presented that methylated in 4 SHOX2,3 RASSF1A are in
The existing positive.This more determines RASSF1A and SHOX2 cooperation detections and is favorably improved Positive rate.
According to result above, inventor determines that selection people SHOX2 and people RASSF1A is used as couple candidate detection gene.
Embodiment 2, primer and probe design
It is applied to detect people SHOX2 genes and people's RASSF1A gene methylations DNA detection reagent simultaneously to optimize,
The present inventor has made intensive studies for each gene order, after studying comparison repeatedly, selectes the amplification region of each gene
Sequence, as shown in table 2.
Table 2
The region according to determined by table 2, the present inventor further optimization design specific primer and detection probe.It is set
The primer and detection probe of meter are as shown in table 3.All primer and probes are by the raw limited public affairs of work bioengineering (Shanghai) share
Department's synthesis.
Table 3
Embodiment 3, the experiment of different primers probe combinations
DNA is extracted, sulphite is modified, detection architecture is prepared, PCR amplification programs be the same as Example 1, and amplification object is modification
Sample DNA afterwards and comparison DNA (SHOX2, RASS1FA DNA fragmentation).Test after end of run, analytical procedure is as follows：
(1) determine whether experiment is credible：
(a) CY5 has signal, and CY5 signal Ct value >=12, then realizes credible；
If Ct values ＜ 12, point out the DNA added excessive, answer and done again after dilution DNA；
If (b) without CY5 signals, pointing out the DNA added to contain PCR inhibitor or DNA processing failures, it is necessary to extract again
DNA and sulphite processing；
Wherein, positive quality control product reaction tube FAM, VIC and CY5 should have signal, and negative quality-control product should expand without FAM and VIC
Increase signal, no CY5 signals.
(2) confirm non-selected correction fluorescence reference, set and analyze FAM fluorescence signals, it is necessary to simultaneous selection positive quality control
Product reaction tube and example reaction pipe, the flex point of amplification curve is determined according to actual amplification curve situation, is adjusted baseline position, is obtained
The Ct values of FAM fluorescence signals.
(a) amplification curve of FAM fluorescence signals is smooth ' S ' shape, and Ct values ＜ 35, prompter's RASSF1A gene first
Baseization is positive；
(b) Ct value >=35, prompter RASSF1A gene methylations are negative or methylation is less than minimum detectability.
(3) confirm non-selected correction fluorescence reference, set and analyze VIC fluorescence signals, it is necessary to simultaneous selection positive quality control
Product reaction tube and example reaction pipe, the flex point of amplification curve is determined according to actual amplification curve situation, is adjusted baseline position, is obtained
The Ct values of VIC fluorescence signals.
(a) amplification curve of VIC fluorescence signals is smooth ' S ' shape, and Ct values ＜ 32, prompter's SHOX2 gene methyl
Change positive；
(b) Ct value >=32, prompter SHOX2 gene methylations are negative or methylation is less than minimum detectability.
Result judgement method such as table 4.
Table 4
Each primer combination of probe and testing result (CT values) such as table 5.
Table 5
| Group | Primer combination of probe | The cell CT values of 500 cell CT values/5000 |
| Group 1 | RASS F1, RASS R1, RASS P1 | 23/19.8 |
| Group 2 | RASS F1, RASS R1, RASS P2 | 23.8/20.1 |
| Group 3 | RASS F1, RASS R2, RASS P1 | 24.2/21.6 |
| Group 4 | RASS F1, RASS R2, RASS P2 | 23.5/20.8 |
| Group 5 | RASS F2, RASS R1, RASS P1 | 24.1/21.5 |
| Group 6 | RASS F2, RASS R1, RASS P2 | 23.8/22.5 |
| Group 7 | RASS F2, RASS R2, RASS P1 | 24.6/20.9 |
| Group 8 | RASS F2, RASS R2, RASS P2 | 24.3/21.5 |
| Group 9 | RASS F3, RASS R1, RASS P1 | 23.5/20.2 |
| Group 10 | RASS F3, RASS R1, RASS P2 | 24/20.5 |
| Group 11 | RASS F3, RASS R2, RASS P1 | 23.6/20 |
| Group 12 | RASS F3, RASS R2, RASS P2 | 24.8/21 |
| Group 13 | SH F-n1, SH R-n1, SH P-n3.1 | 23.6/19.5 |
| Group 14 | SH F-n1, SH R-n1, SH P-n3.2 | 24.5/20.5 |
| Group 15 | SH F-n1, SH R-n2, SH P-n3.1 | 24.5/21 |
| Group 16 | SH F-n1, SH R-n2, SH P-n3.2 | 24.1/20.5 |
| Group 17 | SH F-n1, SH R-n3, SH P-n3.1 | 23.7/20.6 |
| Group 18 | SH F-n1, SH R-n3, SH P-n3.2 | 24.1/21.5 |
| Group 19 | SH F-n2, SH R-n1, SH P-n3.1 | 23.3/21.4 |
| Group 20 | SH F-n2, SH R-n1, SH P-n3.2 | 23.3/22.5 |
| Group 21 | SH F-n2, SH R-n2, SH P-n3.1 | 22.8/21.4 |
| Group 22 | SH F-n2, SH R-n2, SH P-n3.2 | 23.5/21.5 |
| Group 23 | SH F-n2, SH R-n3, SH P-n3.1 | 23.5/20.8 |
| Group 24 | SH F-n2, SH R-n3, SH P-n3.2 | 24.1/21.2 |
| Group 25 | SH F-n3, SH R-n1, SH P-n3.1 | 23.8/22.1 |
| Group 26 | SH F-n3, SH R-n1, SH P-n3.2 | 23.5/22.5 |
| Group 27 | SH F-n3, SH R-n2, SH P-n3.1 | 23.5/21.8 |
| Group 28 | SH F-n3, SH R-n2, SH P-n3.2 | 22.8/22.2 |
| Group 29 | SH F-n3, SH R-n3, SH P-n3.1 | 23.1/21.5 |
| Group 30 | SH F-n3, SH R-n3, SH P-n3.2 | 22.1/19.5 |
| Group 31 | SH F-n4, SH R-n1, SH P-n3.1 | 24.5/22.8 |
| Group 32 | SH F-n4, SH R-n1, SH P-n3.2 | 23.6/22.2 |
| Group 33 | SH F-n4, SH R-n2, SH P-n3.1 | 24.8/23.1 |
| Group 34 | SH F-n4, SH R-n2, SH P-n3.2 | 23.8/22.5 |
| Group 35 | SH F-n4, SH R-n3, SH P-n3.1 | 24.2/22.8 |
| Group 36 | SH F-n4, SH R-n3, SH P-n3.2 | 24.6/23.5 |
| Group 37 | AC-F, AC-R1, AC-P | 21.7/19.8 |
| Group 38 | AC-F, AC-R2, AC-P | 21.3/19.0 |
According to each group amplification curve and CT values, primer probe sequence such as table 6 preferably.
Table 6
Embodiment 4, detection architecture optimizing research
Purpose：Primed probe concentration, hot start Taq polymerase addition in optimizing reaction system, dNTPs, Mg2+Ion is dense
Degree, and then SSR-PCR optimization, improve detection sensitivity and specificity.
1st, SHOX primed probes ratio is screened
Purpose：Ratio and amount to SHOX primers and probe are screened.
Primed probe ratio screening concentration is：SHOX (primers:Probe) final concentration 0.1uM：0.03uM、0.2uM：
0.06uM、0.3uM：0.1uM, 3 concentration are screened.
Wild type human after the SHOX2 reference products of 50 copies/μ L after being modified with sulphite, 500 copies/μ L processing
Genome is used as DNA profiling to be checked.
Conclusion：SHOX primed probe concentration ratio screening experiment results show, SHOX2 primers:Concentration and probe concentration is 0.2uM：
Sensitivity and specificity are best during 0.06uM.
2nd, RASSF1A primed probes ratio is screened
Purpose：Ratio and amount to RASS primers and probe carry out screening
Primed probe ratio screening concentration is：RASS (primers：Probe) final concentration 0.1uM：0.03uM、0.2uM：
0.06uM、0.3uM：0.1uM, 3 concentration are screened.
Wild type after the RASSF1A reference products of 50 copies/μ L after being modified with sulphite, 500 copies/μ L processing
Human genome is used as DNA profiling to be checked.
Conclusion：RASSF1A primed probe concentration ratio screening experiment results show, RASSF1A primers：Concentration and probe concentration is
0.2uM：Sensitivity and specificity are best during 0.06uM.
3rd, β-actin primed probes ratio is screened
Purpose：Screen AC primed probe ratios.
β-actin, as internal reference, can indicate the effect of DNA extraction efficiencies and sulphite modification in 3 weight PCR,
In reaction system, resource had better not be striven with index S HOX, RASS, the primed probe amplification efficiency of AC designs itself is compared with two
High 1 Ct of index or so, so AC concentration in screening is relatively low, and will be combined screening to primer with RASS, SHOX 2.
So by AC primers：Concentration and probe concentration first prepares 0.2uM：0.06uM.Screening concentration is respectively 1/3 original content, 1/2 original content and matched somebody with somebody
The original content of system.
Conclusion：From experimental result as can be seen that the amplification efficiency of reduction AC concentration declines 1 Ct or so, result is not influenceed
Judge；RASSF1A and SHOX2 amplification efficiencies increased after reduction AC primed probe concentration, so the reduction of AC concentration can be more preferably
Raising kit amplification efficiency.Determine that AC primed probe ratios are：0.67uM：0.02uM.
4、Mg2+Ion concentration optimizes
Purpose：To Mg in system2+Ion concentration is optimized, and screens Mg2+Ion concentration is 2.5mM (in 10 × buffer
Carry without addition), 3.5mM, 4.5mM, 5.5mM, 25mM Mg2+Ion addition is respectively 0 μ L, 0.8 μ L, 1.6 μ L, 2.4 μ
L。
After RASSF1A the and SHOX2 reference products of 50 copies/μ L after being modified with sulphite, 500 copies/μ L processing
Wild type human genome be used as DNA profiling to be checked.
Conclusion：Mg2+Ion concentration increase can improve amplification efficiency also can be increasingly severe but non-specific, considers
3.5mM Mg2+Ion concentration amplification best suits requirement.
5th, dNTP concentration optimizations
Purpose：DNTP concentration in system is optimized, screening dNTP concentration 0.15mM, 0.2mM, 0.25mM, 0.3mM are used
The dNTP solution of 2.5mM concentration is separately added into 1.2 μ L, 1.6 μ L, 2 μ L, 2.4 μ L.
After RASSF1A the and SHOX2 reference products of 50 copies/μ L after being modified with sulphite, 500 copies/μ L processing
Wild type human genome be used as DNA profiling to be checked.
Conclusion：The amplification efficiency of detection architecture is improved as dNTPs concentration increases, when dNTPs concentration reaches 0.25mM
When reach saturation state.It is 0.25mM to determine dNTPs concentration.
6th, Taq polymerase concentration optimization
Experiment purpose：The enzyme concentration in PCR reaction systems, Jin Eryou are adjusted by adjusting the amount of addition Taq polymerase
Change PCR reaction systems.
The enzyme amount of screening is that the enzyme amount that 0.5U, 1U, 1.5U, 2U are added in system is respectively：0.1μL、0.2μL、0.3μ
L、0.4μL。
After RASSF1A the and SHOX2 reference products of 50 copies/μ L after being modified with sulphite, 500 copies/μ L processing
Wild type human genome be used as DNA profiling to be checked.
Conclusion：From experimental result as can be seen that enzyme concentration increase amplification efficiency is also with increased, when the usage amount of enzyme
Amplification efficiency is no longer improved when reaching 1.5U units, it is determined that enzyme addition be 1.5U.
Embodiment 5, specificity experiments
Clinical sample is collected, lung cancer is excluded and is diagnosed as pneumonia, pulmonary abscess, pulmonary tuberculosis, bronchiectasis, lung Aspergillus
The congenital dysplastic bronchoalveolar lavage fluid sample 8 of the infectious diseases such as disease, hydatid cyst of lung and lung, is centrifuged thin
Born of the same parents.
DNA is extracted, sulphite is modified, detection selects the (table of embodiment 3 with analyzing process be the same as Example 3, primer, probe
6) preferred scheme.
Testing result is as shown in Figure 1.Positive quality control PC and 8 non-tumor patient samples, can detect the (instruction of CY5 signals
β-actin genes), in addition to people SHOX2 genes in positive control reaction tube and people's RASSF1A gene methylations DNA detections are positive,
People VIC signals (assignor SHOX2 genes) and FAM signals (assignor RASSF1A genes) first in remaining reaction tube of sample 1~8
Base DNA detections are feminine gender, illustrate good, the of the invention detection architecture non-false positive phenomenon of kit specificity.
Embodiment 6, sensitivity for analysis experiment
The lung cancer cell line HCC827 cell counts of culture, take 50,100,1000,10000 cells, are separately added into 1ml
It is derived from the peripheral blood of Healthy People, is used as the simulated samples of sensitivity experiment.DNA is extracted, sulphite is modified, detection is with analyzing
Process be the same as Example 3, from preferred primer, the probe sequence of embodiment 3 (table 6).
Shown in testing result such as Fig. 2 (VIC signals), Fig. 3 (FAM signals), 50 HCC827 cell specimens, people's SHOX2 bases
Cause and people's RASSF1A gene methylations DNA detections are positive, show that reaction system sensitivity for analysis of the invention can reach 50 thin
Born of the same parents/reaction.
Embodiment 7, clinical samples detection
Bronchoalveolar lavage fluid sample totally 25 of certain Cancer Hospital clinical definite for lung cancer and non-lung cancer patient is collected, from
The heart separates cell.DNA is extracted, sulphite is modified, detected with analyzing process be the same as Example 3, from the excellent of embodiment 3 (table 6)
Select primer, probe sequence.
Testing result is as shown in table 7.
Table 7
FAM channel Cs t >=35, or VIC channel Cs t >=32, are indicated with "/".
The result of table 7 shows, in 25 samples, and 20 are lung cancer specimen, and detection methylate DNA is positive 19, missing inspection 1.
In positive test symbol, people's SHOX2 gene methylations DNA detections are positive 16, people's RASSF1A gene methylations DNA detection sun
Property 12, the detection negative RA SSF1A gene methylations DNA detections of 3 SHOX2 methylate DNAs are positive, single inspection SHOX2 gene first
Base DNA Detection accuracy is 80%, after people SHOX2 and people's RASSF1A gene methylation DNA joint-detections, positive
Accuracy rate improves 15%, substantially increases Detection accuracy, demonstrates again that RASSF1A methylates to SHOX2 genes in lung cancer
Synergy in DNA detections.In 5 non-lung cancer patient's samples, the positive is not detected, illustrates that specificity is good.
Embodiment 8, the kit for detecting lung cancer
Kit is provided, including：
PCR reaction solutions：The amplimer of 0.2uM RASSF1A genes is to (SEQ ID NO:14,SEQ ID NO:18)、
0.06uM RASSF1A detection probe (SEQ ID NO:25), the amplimer of 0.2uM SHOX2 genes is to (SEQ ID NO:
7, SEQ ID NO:10), 0.06uM SHOX2 detection probe (SEQ ID NO:, and 0.67uM β-actin genes 22)
Amplimer is to (SEQ ID NO:19, SEQ ID NO:21) with detection probe (the SEQ ID NO of 0.02uM-actin genes:
26)；3.5mM MgCl2、0.25mM dNTP；
Polymerase：200U Taqman polymerases；
Positive quality control product (500 cell/μ L are the DNA extracted after non-small cell lung cancer HCC827 cell lines are counted)；
Negative control：Aqua sterilisa.
DNA modification liquid：3M sodium hydrogensulfites (pH5.0).
All documents referred in the present invention are all incorporated as reference in this application, independent just as each document
It is incorporated as with reference to such.In addition, it is to be understood that after the above-mentioned instruction content of the present invention has been read, those skilled in the art can
To be made various changes or modifications to the present invention, these equivalent form of values equally fall within the model that the application appended claims are limited
Enclose.
Claims (12)
1. a kind of kit for being used to detect lung cancer, it is characterised in that described kit includes：
The detection reagent of the DNA methylation of specific detection people's SHOX2 genes, it is：SEQ ID NO:14 and SEQ ID NO:
Primer and SEQ ID NO shown in 18:Probe shown in 25；With
The detection reagent of the DNA methylation of specific detection people's RASSF1A genes, it is：SEQ ID NO:7 and SEQ ID NO:
Primer and SEQ ID NO shown in 10:Probe shown in 22.
2. kit as claimed in claim 1, it is characterised in that also include in described kit：Specific detection internal reference
The detection reagent of gene, it is the primer and probe for people's β-actin genes.
3. kit as claimed in claim 2, it is characterised in that described primer and probe is directed in people's β-actin genes
SEQ ID NO:5 sequence after bisulfites or weight bisulfites or hydrazonium salt modification.
4. kit as claimed in claim 3, it is characterised in that the DNA methylation of described specific detection reference gene
Detection reagent be：SEQ ID NO:19 and SEQ ID NO:Primer and SEQ ID NO shown in 21:Probe shown in 26.
5. the kit as described in claim 1-4 is any, it is characterised in that be connected with specifically for the probe of each gene
Fluorophor.
6. kit as claimed in claim 5, it is characterised in that mark VIC fluoresceins for the probe of people's SHOX2 genes,
For the probe flag F AM fluoresceins of people's RASSF1A genes, CY5 fluoresceins are marked for the probe of people's β-actin genes.
7. the kit as described in claim 1-4 is any, it is characterised in that in the kit, for drawing for people's SHOX2 genes
The ratio of thing and probe is：0.2：0.06；Or
In the kit, the ratio for the primer and probe of people's RASSF1A genes is：0.2：0.06；Or
In the kit, the ratio for the primer and probe of people's β-actin genes is：0.67：0.02.
8. any described kits of claim 1-4, it is characterised in that in the kit, in addition to：Archaeal dna polymerase,
dNTPs、Mg2+Ion includes archaeal dna polymerase, dNTPs, Mg2+The PCR system of ion.
9. the kit described in claim 8, it is characterised in that described includes archaeal dna polymerase, dNTPs, Mg2+The PCR of ion
In system, described Mg2+Ion concentration is 3.5 ± 0.5mM；Or described dNTPs concentration is 0.25 ± 0.3mM；Or it is described
Archaeal dna polymerase is 1.5 ± 0.2U.
10. the kit described in claim 8, it is characterised in that also include in described kit：Bisulfites or weight are sub-
Disulfate or hydrazonium salt.
11. the purposes of the reagent of the DNA methylation of specific detection people SHOX2 genes and people's RASSF1A genes, for preparing inspection
Survey the kit of lung cancer；The detection reagent of the DNA methylation of described specific detection people's SHOX2 genes is：SEQ ID NO:
14 and SEQ ID NO:Primer and SEQ ID NO shown in 18:Probe shown in 25；
The detection reagent of the DNA methylation of described specific detection people's RASSF1A genes is：SEQ IDNO:7 and SEQ ID
NO:Primer and SEQ ID NO shown in 10:Probe shown in 22.
12. detecting to a kind of external nondiagnostic the method for the DNA methylation of people SHOX2 genes and people's RASSF1A genes, it is special
Levy and be, methods described includes：
(1) testing sample is subjected to bisulfites or weight bisulfites or hydrazonium salt is handled, obtain the testing sample through modification；
(2) using any described kits for being used to detect lung cancer of claim 1-10 to step (1) through the to be measured of modification
Sample is detected, obtains the DNA methylation situation of people's SHOX2 genes and the DNA methylation situation of people's RASSF1A genes.
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Application publication date: 20150715 Assignee: SHANGHAI TELLGEN LIFE SCIENCE CO.,LTD. Assignor: Shanghai Jiayu Life Technology Co.,Ltd. Contract record no.: X2023990000987 Denomination of invention: Method and kit for diagnosing methylation of human SHOX2 gene and human RASSF1A gene Granted publication date: 20170721 License type: Exclusive License Record date: 20231218 |