CN107974503A - Multiple lung cancer related genes methylate combined detection kit, associated detecting method and application - Google Patents

Multiple lung cancer related genes methylate combined detection kit, associated detecting method and application Download PDF

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CN107974503A
CN107974503A CN201610913371.8A CN201610913371A CN107974503A CN 107974503 A CN107974503 A CN 107974503A CN 201610913371 A CN201610913371 A CN 201610913371A CN 107974503 A CN107974503 A CN 107974503A
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王方金
陈静文
郭安亮
姚见儿
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Shanghai Tellgen Diagnosis Science Co Ltd
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Abstract

The present invention provides a kind of multiple lung cancer related genes and methylates combined detection kit, including：The detection reagent of the DNA methylation of specific detection people's SHOX2 genes；The detection reagent of the DNA methylation of specific detection people's RASSF1A genes；The detection reagent of the DNA methylation of specific detection people's ANKRD18B genes；With the detection reagent of the DNA methylation of specific detection people's MPDZ genes.It is preferred that detection reagent is primer and probe.Additionally provide multiple lung cancer related genes and methylate combined detection kit in the application for carrying out multiple lung cancer related genes and methylating in joint-detection, and multiple lung cancer related genes methylate associated detecting method.Multiple lung cancer related genes of the present invention methylate combined detection kit, its can the multiple lung cancer related genes of joint-detection methylate, can significantly improve the recall rate of tumour, it is ingenious in design, it is simple for structure, suitable for large-scale promotion application.

Description

Multiple lung cancer related genes methylate combined detection kit, associated detecting method and Using
Technical field
The present invention relates to technical field of gene detection, more particularly to gene methylation detection technique field, in particular to one Kind multiple lung cancer related genes methylate combined detection kit, associated detecting method and application.
Background technology
Lung cancer has become one of human cancer main causes of death.It is the highest cancer of incidence in lung cancer in China, And morbidity and mortality increase rapidly.The incidence and the death rate of lung cancer are highest 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 the diagnosis of higher, the reason is that can examine in early days Break so as to early treatment, substantially increase 5 years survival rates (being respectively 89 and 99%), and 5 years survival rates of lung cancer are 15%.
In clinical practice, lung cancer early diagnosis is always difficult point, and the effective of cancer patient is controlled in the early detection of cancer Treatment is very important.Diagnosis Main Basiss clinical symptoms to cancer, iconography detection and histopathological examination etc. at present, But there have many cancer clinical symptoms to occur to be later, and living body sampling detection is also difficult, has seriously affected the early diagnosis of cancer With the prognosis of patient.It is readily available relative to the tissue sample such as biopsy, bronchoalveolar lavage fluid, blood plasma, sputum, does not also have 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.In addition it (is usually tens to one hundred polybase that the DNA in circulating, which has usually degraded, Base to) fragment, so needing special extracting and detection technique to obtain higher sensitivity.
With technical fast development, tumor markers develops into the tumour 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 primary dcreening operation or the auxiliary diagnosis prompting as lung cancer and cannot make a definite diagnosis, therefore lung cancer Early diagnosis it is more difficult.At present, as gene diagnosis technology continues 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.As soon as DNA methylation is tumorigenic earliest events, 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.
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 marker, wherein more relevant cyclin- with the early stage of lung cancer than more prominent having The gene 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, it is p16 Methylation of Gene to study more.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, sputum, cell or DNA methylation in bronchoalveolar lavage fluid State, to find the marker for lung cancer early diagnosis.
DNA methylation is as a kind of novel molecular mark, and the importance in diagnosing tumor is paid more and more attention, 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 related 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 early diagnosis of tumor potentially Application value.
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, different genes methylation procedure take part in the different phase of tumor development, therefore The different phase of tumor development can be directed to, joint carries out polygenes DNA methylation assay, can significantly improve tumour Recall rate.
The combined detection kit accordingly, it is desirable to provide a kind of multiple lung cancer related genes methylate, it being capable of joint-detection Multiple lung cancer related genes methylate, and can significantly improve the recall rate of tumour.
The content of the invention
In order to overcome it is above-mentioned in the prior art the shortcomings that, it is related it is an object of the present invention to provide a kind of multiple lung cancer Gene methylation combined detection kit, its can the multiple lung cancer related genes of joint-detection methylate, can significantly improve The recall rate of tumour, suitable for large-scale promotion application.
Another object of the present invention is to provide a kind of multiple lung cancer related genes to methylate combined detection kit, it sets Count it is ingenious, it is simple for structure, suitable for large-scale promotion application.
Another object of the present invention is to provide a kind of multiple lung cancer related genes to methylate associated detecting method, it can The multiple lung cancer related genes of joint-detection methylate, and the recall rate of tumour can be significantly improved, suitable for large-scale promotion application.
Another object of the present invention is to provide a kind of multiple lung cancer related genes to methylate associated detecting method, it is designed It is ingenious, it is easy to operate, suitable for large-scale promotion application.
Another object of the present invention is to provide a kind of multiple lung cancer related genes methylate combined detection kit should With it is used for the multiple lung cancer related genes of joint-detection and methylates, and the recall rate of tumour can be significantly improved, suitable for extensive Promote and apply.
Another object of the present invention is to provide a kind of multiple lung cancer related genes methylate combined detection kit should With its is ingenious in design, easy to operate, suitable for large-scale promotion application.
To achieve the above objectives, in the first aspect of the present invention, there is provided a kind of multiple lung cancer related genes methylate joint Detection kit, its main feature is that, including：The detection reagent of the DNA methylation of specific detection people's SHOX2 genes；Specificity inspection Survey the detection reagent of the DNA methylation of people's RASSF1A genes；The inspection of the DNA methylation of specific detection people's ANKRD18B genes Test agent；With the detection reagent of the DNA methylation of specific detection people's MPDZ genes.
It is preferred that the sequence of the DNA methylation contains CpG dinucleotides structures, and CpG dinucleotides structures C bases carry methyl group.
It is preferred that the detection reagent of the DNA methylation of specific detection people's SHOX2 genes is to be directed to people SHOX2 The primer and probe of gene；Or the detection reagent of the DNA methylation of specific detection people's RASSF1A genes is to be directed to people The primer and probe of RASSF1A genes；Or the detection reagent of the DNA methylation of specific detection people's ANKRD18B genes It is the primer and probe for people's ANKRD18B genes；Or the inspection of the DNA methylation of specific detection people's MPDZ genes Test agent is the primer and probe for people's MPDZ genes.
More preferably, the primer and probe for people's SHOX2 genes is to be directed to SEQID NO in people's SHOX2 genes:3 The primer and probe of sequence after bisulfites or weight bisulfites or hydrazonium salt modification；Or described it is directed to people The primer and probe of RASSF1A genes is to be directed to SEQ ID NO in people's RASSF1A genes:1 through bisulfites or weight sulfurous acid The primer and probe of sequence after hydrogen salt or hydrazonium salt modification；Or the primer and probe for people's ANKRD18B genes is pin To SEQ ID NO in people's ANKRD18B genes:5 sequence after bisulfites or weight bisulfites or hydrazonium salt modification is drawn Thing and probe；Or the primer and probe for people's MPDZ genes is to be directed to SEQ ID NO in people's MPDZ genes:7 through Asia The primer and probe of sequence after disulfate or weight bisulfites or hydrazonium salt modification.
More preferably, the detection reagent of the DNA methylation of specific detection people's SHOX2 genes is：SEQ ID NO: 18 and SEQ ID NO:Primer and SEQ ID NO shown in 22:Probe shown in 38；Or the specific detection people The detection reagent of the DNA methylation of RASSF1A genes is：SEQ ID NO:11 and SEQ ID NO:Primer shown in 14 and SEQ ID NO:Probe shown in 35；Or the detection reagent of the DNA methylation of specific detection people's ANKRD18B genes It is；SEQ ID NO:24 and SEQ ID NO:Primer and SEQ ID NO shown in 25:Probe shown in 39；Or the spy The detection reagent of DNA methylation of opposite sex detection people's MPDZ genes is：SEQ ID NO:29 and SEQ ID NO:Drawing shown in 31 Thing and SEQ ID NO:Probe shown in 40.
It is preferred that the multiple lung cancer related gene methylates, combined detection kit further includes：Specific detection internal reference The detection reagent of gene.
More preferably, the detection reagent of the specific detection reference gene be for people's β-actin genes primer and Probe.
Further, the primer and probe for people's β-actin genes is directed in people's β-actin genes SEQ ID NO:The primer and probe of 9 sequence after bisulfites or weight bisulfites or hydrazonium salt modification.
More preferably, the detection reagent of the specific detection reference gene is：SEQ ID NO:32 and SEQ ID NO: Primer and SEQ ID NO shown in 34:Probe shown in 41.
More preferably, the reference gene is used for the quality for indicating DNA extractions and modification.
More preferably, it is respectively connected with fluorophor for the probe of each gene.If four genes and reference gene are all The detection in a pipe, then the probe for each gene is respectively connected with different fluorophors；If it is divided at two Detection, then be respectively connected with different fluorophors for 2 genes in each pipe and the probe of reference gene in pipe, for example, VIC fluoresceins are marked for the probe of people SHOX2 genes, MPDZ genes, for people RASSF1A genes, ANKRD18B genes Probe flag F AM fluoresceins, CY5 fluoresceins are marked for the probe of people's β-actin genes.
More preferably, for people's SHOX2 genes primer and probe molar concentration ratio (primer:Probe) be：0.2： 0.06 (for example, design PCR amplification system primer final concentration:Probe final concentration=0.2uM：0.06uM)；Or for people RASSF1A Molar concentration ratio (the primer of the primer and probe of gene:Probe) be：0.2：0.06 (for example, design PCR amplification system primer Final concentration:Probe final concentration=0.2uM：0.06uM)；Or the molar concentration rate of the primer and probe for people's ANKRD18B genes Example (primer:Probe) be：0.2：0.06 (for example, design PCR amplification system primer final concentration:Probe final concentration=0.2uM： 0.06uM)；Or molar concentration ratio (the primer of the primer and probe for people's MPDZ genes:Probe) be：0.2：0.06 (example Such as, PCR amplification system primer final concentration is designed:Probe final concentration=0.2uM：0.06uM)；Or for people's β-actin genes Molar concentration ratio (the primer of primer and probe:Probe) be：0.67：0.02 (for example, design PCR amplification system primer is dense eventually Degree:Probe final concentration=0.67uM：0.02uM).
It is preferred that further include (but not limited to)：Archaeal dna polymerase (such as Taq enzyme), dNTPs, Mg2+Ion polymerize comprising DNA Enzyme (such as Taq enzyme), dNTPs, Mg2+The PCR system of ion；It is preferred that described includes archaeal dna polymerase, dNTPs, Mg2+Ion PCR system in, the Mg2+Ion concentration is 3.5 ± 0.5mM (preferably 3.5 ± 0.3mM)；Or the dNTPs concentration For 0.25 ± 0.3mM (preferably 0.25 ± 0.1mM)；Or the archaeal dna polymerase for 1.5 ± 0.2U (preferably 1.5 ± 0.1U)。
It is preferred that further included in the kit：Bisulfites or weight bisulfites or hydrazonium salt.
It is preferred that further include (but not limited to) in the kit：Quality-control product, negative control and/or specification.
In the second aspect of the present invention, there is provided above-mentioned multiple lung cancer related genes combined detection kit that methylates exists Carry out the application that multiple lung cancer related genes methylate in joint-detection.
In the third aspect of the present invention, there is provided a kind of multiple lung cancer related genes methylate associated detecting method, it is special Point is to comprise the following steps：
(1) sample to be tested is subjected to bisulfites or weight bisulfites or hydrazonium salt is handled, obtained modified to be measured Sample；
(2) combined detection kit is methylated to described modified to be measured using above-mentioned multiple lung cancer related genes Sample is detected, and obtains the DNA methylation situation of people's SHOX2 genes, the DNA methylation situation of people's RASSF1A genes, people The DNA methylation situation of ANKRD18B genes and the DNA methylation situation of people's MPDZ genes.
It is preferred that if one or more methylation positives occur in four gene DNAs to be checked, can tentatively judge to be checked Object may be High Risk of Lung Cancer or patients with lung cancer.
It is preferred that multiple nucleic acid fragment detections are carried out in a reaction tube, and progress is more in a reaction tube at the same time A wave band fluorescence signal detection, different DNA fragmentations is distinguished by different fluorescence signals；Alternatively, can be in different reactions Different nucleic acid fragment detections are carried out in pipe, and carry out single wave band fluorescence signal detection in a reaction tube at the same time, by glimmering Optical signal distinguishes different DNA fragmentations；Alternatively, multiple nucleic acid fragment detections can be carried out in different reaction tubes, and at the same time Multiple wave band fluorescence signal detections are carried out in a reaction tube, different DNA fragmentations is distinguished by fluorescence signal.
It is preferred that the sample to be tested derives from：The nucleic acid of sample extraction containing cell, such as bronchoalveolar lavage fluid, are derived from The tissue of diseased region, hydrothorax, sputum etc.；Or the sample containing the nucleic acid from cell, such as blood plasma, serum etc..
Advantageous effect of the invention essentially consists in：
1st, multiple lung cancer related genes of the invention combined detection kit that methylates includes：Specific detection people SHOX2 The detection reagent of the DNA methylation of gene；The detection reagent of the DNA methylation of specific detection people's RASSF1A genes；Specificity Detect the detection reagent of the DNA methylation of people's ANKRD18B genes；With the inspection of the DNA methylation of specific detection people's MPDZ genes Test agent, so as to 4 lung cancer related gene methylation status of joint inspection, therefore, it is possible to the multiple lung cancer dependency basis of joint-detection Because methylating, the recall rate of tumour can be significantly improved, suitable for large-scale promotion application.
2nd, multiple lung cancer related genes of the invention combined detection kit that methylates includes：Specific detection people SHOX2 The detection reagent of the DNA methylation of gene；The detection reagent of the DNA methylation of specific detection people's RASSF1A genes；Specificity Detect the detection reagent of the DNA methylation of people's ANKRD18B genes；With the inspection of the DNA methylation of specific detection people's MPDZ genes Test agent, it is ingenious in design so as to 4 lung cancer related gene methylation status of joint inspection, it is simple for structure, suitable for large-scale promotion Using.
3rd, multiple lung cancer related genes of the invention associated detecting method that methylates comprises the following steps：(1) obtain to be checked The DNA of object, carries out bisulfites or weight bisulfites by the DNA or hydrazonium salt is handled, acquisition is modified to treat test sample Product；(2) combined detection kit is methylated to the modified sample to be tested using above-mentioned multiple lung cancer related genes It is detected, obtains the DNA methylation situation of people's SHOX2 genes, the DNA methylation situation of people's RASSF1A genes, people The DNA methylation situation of ANKRD18B genes and the DNA methylation situation of people's MPDZ genes, it is multiple therefore, it is possible to joint-detection Lung cancer related gene methylates, and the recall rate of tumour can be significantly improved, suitable for large-scale promotion application.
4th, multiple lung cancer related genes of the invention associated detecting method that methylates comprises the following steps：(1) obtain to be checked The DNA of object, carries out bisulfites or weight bisulfites by the DNA or hydrazonium salt is handled, acquisition is modified to treat test sample Product；(2) combined detection kit is methylated to the modified sample to be tested using above-mentioned multiple lung cancer related genes It is detected, obtains the DNA methylation situation of people's SHOX2 genes, the DNA methylation situation of people's RASSF1A genes, people The DNA methylation situation of ANKRD18B genes and the DNA methylation situation of people's MPDZ genes, it is ingenious in design, it is easy to operate, it is suitable for Large-scale promotion application.
5th, multiple lung cancer related genes of the invention methylate combined detection kit in the multiple lung cancer related genes of progress Application in the joint-detection that methylates, methylates for the multiple lung cancer related genes of joint-detection, can significantly improve tumour Recall rate, suitable for large-scale promotion application.
6th, multiple lung cancer related genes of the invention methylate combined detection kit in the multiple lung cancer related genes of progress Application in the joint-detection that methylates, methylates for the multiple lung cancer related genes of joint-detection, ingenious in design, easy to operate, Suitable for large-scale promotion application.
These and other objects, feature and the advantage of the present invention, is filled by following detailed descriptions and claim Split shows, and can be achieved by means, device and the combinations thereof specially pointed out in appended claims.
Embodiment
The present inventor passes through in-depth study, discloses a kind of methyl by four kinds of lung cancer related genes of cooperation detection first Change DNA to improve the method for lung cancer recall rate, the lung cancer related gene is people SHOX2 genes, people RASSF1A genes, people ANKRD18B genes and people's MPDZ genes.The present inventor also optimizes pcr amplification reaction program, further increases amplification effect Rate.The present inventor also optimizes detection people SHOX2 genes, people RASSF1A genes, people ANKRD18B genes and people's MPDZ gene first The reagent of base DNA, detection method.The present invention is completed on this basis.
As used herein, " sample to be tested ", " sample to be tested " 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, it is people SHOX2 genes, people's RASSF1A bases The methylate DNA specific fragment of cause, people ANKRD18B genes and people's MPDZ genes.
As used herein, " probe " refers to a kind of single-chain nucleic acid with known nucleotide sequence, it has and target core The nucleotide sequence structure that acid is substantially complementary, can form double-strand with " target nucleic acid "." 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：The sequence of NC_000003 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：The sequence of DQ444319 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 that.It is now recognized that RASSFlA gene expressions inactivate the mainly hyper-methylation with promoter region exception, heterozygosis Property missing and chromosome deficiency it is related.
Ankyrin repeat is a kind of protein sequence die body being prevalent in eucaryon, protokaryon and virus.Typical case ANK repetitive sequences typically contain 33 residues, different number of ANK units are together in series, formed permitted great ANK domains (ANK repeat domain), ankyrin repeat function are mainly to participate in the interaction of protein-protein.Due to ANK has differences on number, primary sequence and space structure in protein families member so that ANK die bodys can Combined with multiple ligands, including DNA binding protein, sequence associated proteins, cell cycle regulating protein, transcription factor etc., extensively The vital movements such as cell growth, cell cycle regulating, signal transduction are take part in, it is also closely related with the generation of a variety of diseases.People ANKRDl8B (Ankyrin repeat domain18B) gene contains typical ANK structural regions (ANK domains), has 13 A CpG islands site, research find ANKRD18B genes obvious hyper-methylation in lung cancer generating process, prompt the gene unconventionality first Baseization may be related with lung cancer generation.
People MPDZ (multiple PDZ domain protein) gene contains 47 extrons, encodes 2070 amino The protein of acid, protein contain 13 typical PDZ domains, which is by memebrane protein and cytoskeleton Grappling, form protein complex, participates in the conduction of signal.MPDZ as close connection GAP-associated protein GAP, take part in epithelial cell, The close-connected formation such as endothelial cell, and it is related to cell polarity and Premeabilisation of cells pressure adjusting etc..With tumour machine occurs for MPDZ In terms of system, MPDZ albumen can be with the protein binding such as coxsackie adenovirus receptor and tumor suppressor gene FAT4, thus it is speculated that its The suppression of the carcinogenic effect of virus and the execution of FAT4 cancer suppressing actions may be take part in.There is research to confirm MPDZ genes in lung cancer group There is upper frequency to methylate in knitting, and do not detect to methylate in the cancer beside organism of distal end, prompt MPDZ genes may The generation of lung cancer is take part in, and is likely to be the tumor suppressor gene of a candidate.
Present inventors have surprisingly found that in clinical patients with lung cancer, the tumor specimen of SHOX2 gene methylations DNA feminine genders In, the RASSF1A gene methylations DNA positives, joint-detection people SHOX2 genes and people's RASSF1A genes occurs in most samples Methylate DNA more individually detects two kinds of genes, and the accuracy rate (recall rate) of lung cancer detection can significantly improve.Subsequent In clinical trial, it is found that partial clinical is made a definite diagnosis in the patient of lung cancer, people SHOX2 genes and people's RASSF1A gene methylations DNA inspections Survey is feminine gender.To these ' negative ' specimens, inventor have detected the methylation state of other genes, finder's ANKRD18B genes With people MPDZ genes in the sample of part test positive.
Above-mentioned new discovery based on the present inventor, there is provided specific detection people SHOX2 genes, people RASSF1A genes, people The associated detecting method of the DNA methylation of ANKRD18B genes and people's MPDZ genes, for significantly improving the accurate of lung cancer detection Rate (recall rate).
Further, present inventor has performed screening and repetition test, there is provided one kind is particularly suitable for people's SHOX2 bases Cause, the primer of people RASSF1A genes, people ANKRD18B genes and the detection of people MPDZ gene methylation DNA specific fragments and spy Pin.The selection in gene methylation site is directly related to clinical detection sensitivity, generally all in the promoter region of gene, but very Polygenes is also possible to positioned at First Intron and First Exon region with the relevant methylation sites of tumour.By substantial amounts of Test and compare, the present inventor have chosen people SHOX2 genes, people RASSF1A genes, people ANKRD18B genes and people's MPDZ genes The specific region that methylates, such as corresponds to the SEQ ID NO of people's SHOX2 genes:3 sequences, or corresponding to RASSF1A genes SEQ IDNO:1 sequence, or the SEQ ID NO corresponding to ANKRD18B genes:5 sequences, or the SEQ ID corresponding to MPDZ genes NO:7 sequences.On this basis, the present inventor have also been devised the primer of high special to amplify different purpose fragments, be used in combination The probe of high special identifies.
The successful design of primed probe is an extremely important ring for real time fluorescent PCR detection method, is promoted after sulphite modification " C " in DNA chain 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 chain.In addition, the loss of DNA is also resulted in purge process.The inventors discovered that the length of PCR product During more than 300bp, it is difficult to the DNA after being modified using sulphite and is expanded as template, the length of 200bp 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 the primer stablized；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 primer is stablized in selection.Difficult for these, on the basis of sufficient sequence research, selection optimizes suitable the present inventor Section, devise the amplimer and probe of high special, establish people SHOX2 genes, the people of a kind of high sensitivity RASSF1A genes, people ANKRD18B genes and people MPDZ 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 using internal reference come indicate DNA extractions and The quality of modification.The 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.Research process, the present inventor are compared by sulphite sequencing approach The sulphite of QIAGEN companies and ZYMO companies modifies kit, and on conversion ratio and the rate of recovery, the two does not have significant difference, From cost consideration, the sulphite of ZYMO companies is selected to modify kit.
According to the needs of clinical detection, using real-time fluorescence PCR technology carry out the detection of methylate DNA specific fragment be compared with To be preferable.When using real-time fluorescence PCR be detected when, the probe be connected be suitable for screen different genes methylate The fluorophor of DNA fragmentation.The front end of probe is marked with fluorophor, and the other end is marked with quenching group, wherein quenching group Can the fluorescence that sends of quenching fluorescence group.When pcr amplification reaction carries out, using polymerase it is positive it is exo-acting will carry it is glimmering The base of light group is cut off, and the fluorophor after dissociating no longer is influenced by quenching group, can be launched under the action of exciting light The fluorescence signal of certain wavelength.With the continuous accumulation of PCR product, fluorescence signal constantly strengthens, so as to detect spy The presence of different methylate DNA.
As the preferred embodiment of the present invention, for convenience of Clinical practice, when being detected, using in same reaction tube Three kinds of specific probes for adding three kinds of different fluorophor marks (correspond respectively to people SHOX2 genes, people's RASSF1A genes With β-actin genes；And people MPDZ genes, people ANKRD18B genes and β-actin genes), in a reaction tube at the same time Indicate 3 kinds of purpose methylated DNA fragments there are situation, in this way, using two reaction tubes, it is possible to detect 4 lung cancer correlations Detected in methylation-specific gene and reference gene, i.e. a reaction tube people SHOX2 genes, people RASSF1A genes and people β- Actin genes；People ANKRD18B genes, people MPDZ genes and people's β-actin genes are detected in another reaction tube.
As the present invention preferred embodiment, for convenience of Clinical practice, detection probe mark fluorophor can be VIC, ROX, FAM, Cy5, HEX, TET, JOE, NED etc.；Quenching group can be TAMRA, BHQ, MGB, Dabcyl.With suitable for current The common multichannel PCR detection system of clinical detection, realizes the progress multicolor fluorescence detection in a reaction tube.
In some preferred embodiments of the present invention, the present inventor also optimizes pcr amplification reaction, further increases amplification Efficiency.Dosage, dNTPs dosages including optimizing enzyme, concentration of the primed probe in PCR system so that after PCR reactions, Ct values Relatively centralized, reaches ideal effect.It is to be understood that can realize technique effect in the case where not optimizing, and carry out 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 solution, quality-control product, negative control and/or Operation instructions.
The method and kit of the present invention has high sensitivity, height particularly suitable for lung cancer auxiliary diagnosis and early diagnosis The features such as specific, easy to operate, 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 further explained.It is to be understood that these embodiments are merely to illustrate the present invention Rather than limit the scope of the invention.The experimental method of actual conditions is not specified in the following example, usually 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 marker, DNA is to expand , and be readily possible to detect；Compared with being mutated class marker, methylate DNA is all located at the privileged site of gene, is generally opening Mover area, makes detection become easier to and conveniently.Different genes methylate DNA testing result has certain collaboration in early-stage study Effect, in order to further improve positive clinical recall rate, on the original basis (p16, H-cadherin (CDH13), SHOX2, HOXA9, RAR β, RASSF1A) detection gene of new ANKRD18B, MPDZ gene as candidate has been added, further research is each A gene methylation site distribution situation, the primed probe for designing detection are respectively used to detect.Each genetic test primed probe is such as Under：
The detection primer and probe of p16 be：
P16 primers Fs：ACGTCGTGAGCGAGTGTTC(SEQ ID NO:42)；
P16 primers R：TACCAACGCTAACTCTAACGAA(SEQ ID NO:43)；
P16 probes：ROX-CTTCCGACTAATACCCCCGAAA-BHQ(SEQ ID NO:44)；
The detection primer and probe of H-cadherin (CDH13) be：
Primers F：GAAAATATGTTTAGTGTAGTCGCGT(SEQ ID NO:45)；
Primer R：ACGCACAAAACGAACGAAAT(SEQ ID NO:46)；
Probe：CY5-TTTTATCCGACTAGAAACGCCCG-BHQ(SEQ ID NO:47)；
The detection primer and probe of SHOX2 be：
Primers F：TTGTTTTTGGGTTCGGGTT(SEQ ID NO:16)
Primer R：CATAACGTAAACGCCTATACTCG(SEQ ID NO:20)
Probe：VIC-ATCGAACAAACGAAACGAAAATTACC-BHQ(SEQ ID NO:37)
The detection primer and probe of HOXA9 be：
Primers F：GGTATATCGTAGCGGGTATAGC(SEQ ID NO:48)；
Primer R：AACTTCCAATCCAAAACGACG(SEQ ID NO:49)；
Probe：FAM-TTCGTCGCGTGTATTGGGTTTTAC-BHQ(SEQ ID NO:50)；
The detection primer and probe of RAR β be：
Primers F：CGAGAACGCGAGCGATTC(SEQ ID NO:51)；
Primer R：AACCTTCCGAATACGTTCCG(SEQ ID NO:52)；
Probe：VIC-TCCTACCCCGACGATACCCAAAC-BHQ(SEQ ID NO:53)；
The detection primer and probe of RASSF1A be：
Primers F：CGGGGTTCGTTTTGTGGTTTC(SEQ ID NO:11)
Primer R：CCGATTAAATCCGTACTTCGC(SEQ ID NO:14)
Probe：FAM-TCGCGTTTGTTAGCGTTTAAAGT-BHQ(SEQ ID NO:35)
The detection primer and probe of ANKRD18B be：
Primers F:TTGAGATTTCGGAACGTTTC(SEQ ID NO:23)
Primer R:AAAACGCCCTCGAACTCG(SEQ ID NO:25)
Probe：FAM-TAACTACAACTCGAACTCAAACGCC-BHQ(SEQ ID NO:39)
The detection primer and probe of MPDZ be：
Primers F:GTTTTTGGATGTTTCGTTTCG(SEQ ID NO:27)
Primer R:CGACGAAACGCTAAACTAACG(SEQ ID NO:30)
Probe：FAM-CACCGCGAAATTAAATTTCTCAAT-BHQ(SEQ ID NO:40)
Experimentation：
1st, DNA is extracted
The sample of patients with lung cancer and non-tumor patient sample are made a definite diagnosis in collection, cell are 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 book carries out 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 program is as follows：
First stage：95 DEG C, 10min, 1 circulations；
Second stage：95 DEG C, 15sec, 60 DEG C 30sec, 5 circulations；
Phase III：95 DEG C, 15sec, 57 DEG C 30sec, 40 circulations；
Signal collection：Fluorescence signal is collected during 57 DEG C of phase III.
4th, testing result
According to each amplification situation, testing result is shown in Table 1.
Table 1
Note："+" is positive for methylate DNA detection, and "-" is negative for methylate DNA detection
As can be seen from the above results, when while detecting, SHOX2 methylation positive rate highests, CDH13, HOXA9, RAR β Gene pairs improves system Positive rate and does not act synergistically, wherein RAR β false positives in non-tumor specimen, inspection in conjunction Survey influences testing result on the contrary, reduces Detection accuracy；RASSF1A test positive in sample 11,16, and sample 11 and mark Originally 16 is negative for SHOX2 DNA methylation assays, and therefore, the positive rate that RASSF1A gene pairs improves system detection has synergistic effect. There is also similar collaboration with (p16 detects sample 16 positive, and SHOX2 detections are negative) by p16, but its synergistic effect is not so good as RASSF1A is notable, therefore primarily determines that RASSF1A and SHOX2 has and compare assistance synergistic effect；Sample 3,8 and 22, Qi Tajian Index (including SHOX2, RASSF1A) is surveyed as feminine gender, and ANKRD18B (to the sample 3 and 8) detections that methylate are positive, MPDZ methyl It is positive to change (sample 8 and 22) detection；In addition, the DNA methylation assay result of this 2 gene pairs sample 23 and 27 is also the positive, it is SHOX2 and RASSF1A detections feminine gender is supplemented.As it can be seen that SHOX2, RASSF1A, ANKRD18B and MPDZ carry out cooperation detection Help to improve tumour recall rate.
For this reason, inventor expands detection range, 50 tumor samples and 5 non-tumor sample (part Experiment numbers are have collected According to seeing embodiment 7), further verify whether RASSF1A, SHOX2, ANKRD18B and MPDZ cooperation detection contribute to detection positive The raising of rate, helps to improve the accuracy of diagnosis.
According to result above, inventor determine selection people SHOX2 genes, people RASSF1A genes, people ANKRD18B genes and People MPDZ genes are as couple candidate detection gene.
Embodiment 2, primer and probe design
In order to optimize suitable for while detect people SHOX2 genes, people RASSF1A genes, people ANKRD18B genes and people The detection reagent of MPDZ gene methylations DNA, the present inventor has made intensive studies for each gene order, by studying repeatedly After comparison, the amplification region sequence of each gene is selected, as shown in table 2.
Table 2
According to region determined by table 2, the present inventor's 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
With embodiment 1, amplification object is modification for DNA extractions, sulphite modification, detection architecture preparation, PCR amplification program Sample DNA afterwards and comparison DNA (DNA fragmentation of SHOX2, RASS1FA, ANKRD18B and MPDZ).After testing 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 tests credible；
If Ct values ＜ 12, prompt the DNA added excessive, done again after answering dilution DNA；
If (b) DNA added is prompted to contain PCR inhibitor or DNA processing failures, it is necessary to extract again without CY5 signals 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 RASSF1A gene, People ANKRD18B gene methylations are positive；
(b) Ct value >=35, prompter RASSF1A, people ANKRD18B gene methylations are negative or methylation is less than most Low detection limits.
(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 ＜ 35, prompter SHOX2 gene, people MPDZ gene methylations are positive；
(b) Ct value >=35, prompter SHOX2 genes, people MPDZ gene methylations are negative or methylation is less than minimum Detection limit.
When clinical samples detect, selected 4 genes at least one for detection it is positive when, which is judged as methyl It is positive to change DNA；When 4 genes all detect negative, which is judged as methylate DNA feminine gender.
Each primer combination of probe and testing result (CT values) such as table 5.
Table 5
According to each group amplification curve and CT values, preferable primer probe sequence such as table 6.
Table 6
Embodiment 4, detection architecture optimizing research
Purpose：Primed probe (see the table 6 of embodiment 3) concentration, hot start Taq polymerase addition in optimizing reaction system, dNTPs、Mg2+Ion concentration, and then SSR-PCR optimization, improve detection sensitivity and specificity.
1st, SHOX primed probes ratio is screened
Purpose：The ratio and amount of 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, the processing of 500 copies/μ L Genome is as DNA profiling to be checked.
Conclusion：SHOX primed probe concentration ratio screening experiments are the results show that 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：Screening is carried out to the ratio and amount of RASS primers and probe
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, the processing of 500 copies/μ L Human genome is as DNA profiling to be checked.
Conclusion：RASSF1A primed probe concentration ratio screening experiments are the results show that RASSF1A primers：Concentration and probe concentration is 0.2uM：Sensitivity and specificity are best during 0.06uM.
3rd, ANKRD18B primed probes ratio is screened
Purpose：The ratio and amount of ANKRD18B primers and probe are screened.
Primed probe ratio screening concentration is：ANKRD18B (primers:Probe) final concentration 0.1uM：0.03uM、0.2uM： 0.06uM、0.3uM：0.1uM, 3 concentration are screened.
It is wild after the ANKRD18B reference products of 50 copies/μ L after being modified with sulphite, the processing of 500 copies/μ L Type human genome is as DNA profiling to be checked.
Conclusion：ANKRD18B primed probe concentration ratio screening experiments are the results show that ANKRD18B primers:Concentration and probe concentration is 0.2uM：Sensitivity and specificity are best during 0.06uM.
4th, MPDZ primed probes ratio is screened
Purpose：The ratio and amount of MPDZ primers and probe are screened.
Primed probe ratio screening concentration is：MPDZ (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 MPDZ reference products of 50 copies/μ L after being modified with sulphite, the processing of 500 copies/μ L Genome is as DNA profiling to be checked.
Conclusion：MPDZ primed probe concentration ratio screening experiments are the results show that MPDZ primers:Concentration and probe concentration is 0.2uM： Sensitivity and specificity are best during 0.06uM.
5th, β-actin primed probes ratio is screened
Purpose：Screen AC primed probe ratios.
β-actin are used as internal reference in 5 weight PCR, can indicate DNA extraction efficiencies and the effect of sulphite modification, In the reaction system, resource had better not be striven with index S HOX, RASS, ANKRD18B and MPDZ, the primer of AC designs itself is visited Pin amplification efficiency 1 Ct high compared with two indices or so, so AC concentration in screening is relatively low, and will with RASS, SHOX 2, ANKRD18B and people's MPDZ primers are combined screening.So by AC primers：Concentration and probe concentration first prepares 0.2uM：0.06uM.Screening Concentration is respectively the original content of 1/3 original content, 1/2 original content and preparation.
Conclusion：According to experimental result, the amplification efficiency for reducing AC concentration declines 1 Ct or so, does not influence result judgement；Drop Low AC primed probes concentration does not influence RASS, SHOX 2, ANKRD18B and people's MPDZ amplification efficiencies, so the reduction of AC concentration can be more The amplification efficiency of good raising kit.Determine that AC primed probe ratios are：0.67uM：0.02uM.
6、Mg2+Ion concentration optimizes
Purpose：To Mg in system2+Ion concentration optimizes, and screens Mg2+Ion concentration is 2.5mM (in 10 × buffer Carry without addition), 3.5mM, 4.5mM, 5.5mM, the Mg of 25mM2+Ion addition is respectively 0 μ L, 0.8 μ L, 1.6 μ L, 2.4 μ L。
RASS, SHOX 2 of 50 copies/μ L after being modified with sulphite, ANKRD18B and MPDZ reference products, 500 are copied Wild type human genome after the processing of shellfish/μ L is 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.
7th, 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.
RASS, SHOX 2 of 50 copies/μ L after being modified with sulphite, ANKRD18B and MPDZ reference products, 500 are copied Wild type human genome after the processing of shellfish/μ L is 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.
8th, Taq polymerase concentration optimization
Experiment purpose：The enzyme concentration in PCR reaction systems, Jin Eryou are adjusted by adjusting the amount for adding 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。
RASS, SHOX 2 of 50 copies/μ L after being modified with sulphite, ANKRD18B and MPDZ reference products, 500 are copied Wild type human genome after the processing of shellfish/μ L is as DNA profiling to be checked.
Conclusion：Enzyme concentration increase amplification efficiency is also with increased, is expanded when the usage amount of enzyme reaches 1.5U units Efficiency no longer improves, and definite enzyme addition is 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, centrifuges thin Born of the same parents.
DNA extractions, sulphite modification, detection select 3 (table of embodiment with analytic process with embodiment 3, primer, probe 6) preferred solution.
Testing result：Positive quality control PC and 8 non-tumor patient samples, can detect CY5 signals (instruction β-actin bases Cause), in addition to the corresponding gene methylate DNA detection positive in positive control reaction tube, the detection of sample corresponding gene methylate DNA is equal For feminine gender, illustrate good, the of the invention detection architecture non-false positive phenomenon of kit specificity.
Embodiment 6, sensitivity for analysis experiment
Lung cancer cell line HCC827 cell (Chinese Academy of Sciences's cell banks of culture：HCC827 Non-small cell lung carcinomas cell) Count, take 50,100,1000,10000 cells, be separately added into 1ml and be derived from the peripheral blood of Healthy People, as sensitivity experiment Simulated samples.DNA extractions, sulphite modification, detection, with embodiment 3, select the excellent of embodiment 3 (table 6) with analytic process Select primer, probe sequence.
Testing result：50 HCC827 cell specimens, people SHOX2 genes, people RASSF1A genes, people ANKRD18B and people MPDZ gene methylations DNA detections are positive, show that the reaction system sensitivity for analysis of the present invention can reach 50 cells/reaction.
Embodiment 7, clinical samples detection
Certain Cancer Hospital clinical definite is collected as lung cancer and the bronchoalveolar lavage fluid sample totally 25 of non-lung cancer patient, from The heart separates cell.DNA extractions, sulphite modification, detection, with embodiment 3, select the excellent of embodiment 3 (table 6) with analytic process Select primer, probe sequence.
Testing result is as shown in table 7.
Table 7
In table, Ct >=35 are indicated with "-".
Table 7 the result shows that, in 25 samples, 20 are lung cancer specimen, positive 19 of detection methylate DNA.Positive detection As a result in, people's SHOX2 gene methylations DNA detections are 13 positive, and people's RASSF1A gene methylations DNA detections are 9 positive, people Positive 10 of ANKRD18B gene methylations DNA detections, people's MPDZ gene methylations DNA detections are 8 positive, 3 SHOX2 and RASSF1A methylate DNAs detect ' negative ' specimens, and ANKRD18B DNA methylation assays are the positive, and 1 SHOX2 and RASSF1A methylates DNA is detected as feminine gender, and the test positive of MPDZ methylate DNAs, illustrates that there is ANKRD18B and MPDZ certain supplement to assist Same-action, can further improve lung cancer recall rate.It is positive without detection in 5 non-lung cancer patient's samples, illustrate specificity It is good.
Embodiment 8, the kit for detecting lung cancer
Kit is provided, including：
PCR reaction solution：The amplimer of 0.2uM RASSF1A genes is to (SEQ ID NO:11,SEQ ID NO:14)、 Detection probe (the SEQ ID NO of 0.06uM RASSF1A:35), the amplimer of 0.2uM SHOX2 genes is to (SEQ ID NO: 18, SEQ ID NO:22), detection probe (the SEQ ID NO of 0.06uM SHOX2:38), the expansion of 0.2uM ANKRD18B genes Increase primer pair (SEQ ID NO:24, SEQ ID NO:25), detection probe (the SEQ ID NO of 0.06uMANKRD18B:39), The amplimer of 0.2uM MPDZ genes is to (SEQ ID NO:29, SEQ ID NO:31), the detection probe of 0.06uM MPDZ (SEQ ID NO:40), and 0.67uM β-actin genes amplimer to (SEQ ID NO:32, SEQ ID NO:34) and Detection probe (the SEQ ID NO of 0.02uM-actin genes:41)；3.5mM MgCl2、0.25mM dNTP；
Polymerase：200U Taqman polymerases；
Positive quality control product (500 cells/μ L, the DNA extracted after being counted for non-small cell lung cancer HCC827 cell lines)；
Negative control：Aqua sterilisa.
DNA modification liquid：3M sodium hydrogensulfites (pH5.0).
Therefore, the present invention relates to one kind detection people SHOX2 genes, people RASSF1A genes, people ANKRD18B genes and people The detection method of MPDZ gene methylations, coherent detection reagent, and the related kit prepared by the detection reagent, pass through Joint carries out polygenes DNA methylation assay, can significantly improve the recall rate of tumour.
To sum up, multiple lung cancer related genes of the invention methylate combined detection kit, its can joint-detection it is multiple Lung cancer related gene methylates, and can significantly improve the recall rate of tumour, ingenious in design, simple for structure, suitable for pushing away on a large scale Wide application.
It can be seen from the above that the purpose of the present invention is achieved completely and effectively.The function and structural principle of the present invention Shown and illustrated in embodiment, under without departing substantially from the principle, embodiment can make any modification.So this hair The bright all variant embodiments included based on claim spirit and right.

Claims (11)

  1. The combined detection kit 1. a kind of multiple lung cancer related genes methylate, it is characterised in that including：
    The detection reagent of the DNA methylation of specific detection people's SHOX2 genes；
    The detection reagent of the DNA methylation of specific detection people's RASSF1A genes；
    The detection reagent of the DNA methylation of specific detection people's ANKRD18B genes；With
    The detection reagent of the DNA methylation of specific detection people's MPDZ genes.
  2. The combined detection kit 2. multiple lung cancer related genes as claimed in claim 1 methylate, it is characterised in that described The detection reagent of the DNA methylation of specific detection people's SHOX2 genes is the primer and probe for people's SHOX2 genes；Or
    The detection reagent of the DNA methylation of specific detection people's RASSF1A genes is drawing for people's RASSF1A genes Thing and probe；Or
    The detection reagent of the DNA methylation of specific detection people's ANKRD18B genes is for people's ANKRD18B genes Primer and probe；Or
    The detection reagent of the DNA methylation of specific detection people's MPDZ genes is the primer and spy for people's MPDZ genes Pin.
  3. The combined detection kit 3. multiple lung cancer related genes as claimed in claim 2 methylate, it is characterised in that described Primer and probe for people's SHOX2 genes is to be directed to SEQ ID NO in people's SHOX2 genes:3 through bisulfites or weight sulfurous The primer and probe of sequence after sour hydrogen salt or hydrazonium salt modification；Or
    The primer and probe for people's RASSF1A genes is to be directed to SEQ ID NO in people's RASSF1A genes:1 through sulfurous The primer and probe of sequence after sour hydrogen salt or weight bisulfites or hydrazonium salt modification；Or
    The primer and probe for people's ANKRD18B genes is to be directed to SEQ ID NO in people's ANKRD18B genes:5 through Asia The primer and probe of sequence after disulfate or weight bisulfites or hydrazonium salt modification；Or
    The primer and probe for people's MPDZ genes is to be directed to SEQ ID NO in people's MPDZ genes:7 through bisulfites Or the primer and probe of the sequence after weight bisulfites or hydrazonium salt modification.
  4. The combined detection kit 4. multiple lung cancer related genes as claimed in claim 2 methylate, it is characterised in that described The detection reagent of the DNA methylation of specific detection people's SHOX2 genes is：SEQ ID NO:18 and SEQ ID NO:Shown in 22 Primer and SEQ ID NO:Probe shown in 38；Or
    The detection reagent of the DNA methylation of specific detection people's RASSF1A genes is：SEQ ID NO:11 and SEQ ID NO:Primer and SEQ ID NO shown in 14:Probe shown in 35；Or
    The detection reagent of the DNA methylation of specific detection people's ANKRD18B genes is；SEQ ID NO:24 and SEQ ID NO:Primer and SEQ ID NO shown in 25:Probe shown in 39；Or
    The detection reagent of the DNA methylation of specific detection people's MPDZ genes is：SEQ ID NO:29 and SEQ ID NO:Primer and SEQ ID NO shown in 31:Probe shown in 40.
  5. The combined detection kit 5. multiple lung cancer related genes as claimed in claim 1 methylate, it is characterised in that described more A lung cancer related gene combined detection kit that methylates further includes：The detection reagent of specific detection reference gene.
  6. The combined detection kit 6. multiple lung cancer related genes as claimed in claim 5 methylate, it is characterised in that described The detection reagent of specific detection reference gene is the primer and probe for people's β-actin genes.
  7. The combined detection kit 7. multiple lung cancer related genes as claimed in claim 6 methylate, it is characterised in that described Primer and probe for people's β-actin genes is to be directed to SEQ ID NO in people's β-actin genes:9 through bisulfites or again The primer and probe of sequence after bisulfites or hydrazonium salt modification.
  8. The combined detection kit 8. multiple lung cancer related genes as claimed in claim 6 methylate, it is characterised in that described The detection reagent of specific detection reference gene is：SEQ ID NO:32 and SEQ ID NO:Primer and SEQ ID shown in 34 NO:Probe shown in 41.
  9. The combined detection kit 9. multiple lung cancer related genes as claimed in claim 2 methylate, it is characterised in that for every The probe of a gene is respectively connected with fluorophor.
  10. 10. such as multiple lung cancer related genes of claim 1-9 any one of them methylate, combined detection kit is more in progress A lung cancer related gene methylates the application in joint-detection.
  11. The associated detecting method 11. a kind of multiple lung cancer related genes methylate, it is characterised in that comprise the following steps：
    (1) sample to be tested is subjected to bisulfites or weight bisulfites or hydrazonium salt is handled, obtain modified sample to be tested；
    (2) combined detection kit is methylated to institute using such as the multiple lung cancer related genes of claim 1-9 any one of them The modified sample to be tested stated is detected, and obtains the DNA methylation situations of people's SHOX2 genes, people's RASSF1A genes The DNA methylation situation of DNA methylation situation, the DNA methylation situation of people's ANKRD18B genes and people's MPDZ genes.
CN201610913371.8A 2016-10-20 2016-10-20 Multiple lung cancer related genes methylate combined detection kit, associated detecting method and application Pending CN107974503A (en)

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Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108118092A (en) * 2018-01-17 2018-06-05 王景杰 The processing method of polygene combined faeces DNA DNA methylation assay data
CN109097474A (en) * 2018-08-29 2018-12-28 天津智鱼生物科技有限公司 A kind of primer combination of probe and its application of RASSF1A gene and the detection of P16 gene methylation
CN109825581A (en) * 2019-02-28 2019-05-31 昆明理工大学 A kind of primer, detection method, kit and application detecting RASSF1A gene methylation
CN110283913A (en) * 2019-07-21 2019-09-27 广州奥百阕谱生物科技有限公司 RASSF1A gene methylation state detection kit and its application
WO2019233449A1 (en) * 2018-06-07 2019-12-12 深圳市圣必智科技开发有限公司 Dna methylation marker for predicting early lung cancer and application thereof
WO2019233448A1 (en) * 2018-06-07 2019-12-12 深圳市圣必智科技开发有限公司 Test kit for detecting early stage dna methylation markers in lung cancer and detection method
CN110923320A (en) * 2019-12-26 2020-03-27 益善生物技术股份有限公司 Nucleic acid composition, kit and detection method for detecting methylation of lung cancer related genes
CN110964812A (en) * 2018-09-29 2020-04-07 广州市康立明生物科技有限责任公司 Application of HOXA9 methylation detection reagent in preparation of lung cancer diagnosis reagent
CN111363817A (en) * 2018-12-26 2020-07-03 广州市康立明生物科技有限责任公司 Lung cancer diagnostic agent and kit based on HOXD12 gene
CN111363814A (en) * 2018-12-25 2020-07-03 广州市康立明生物科技有限责任公司 Lung cancer diagnostic reagent and kit based on DMRTA2 and FOXD3 genes
CN111363816A (en) * 2018-12-26 2020-07-03 广州市康立明生物科技有限责任公司 Lung cancer diagnostic reagent and kit based on PAX3 and ZIC4 genes
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CN111676287A (en) * 2020-06-03 2020-09-18 广州市康立明生物科技有限责任公司 Gene marker combination and application thereof
CN111676286A (en) * 2020-05-29 2020-09-18 武汉爱基百客生物科技有限公司 Multiplex PCR primer system for detecting free DNA methylation of lung cancer plasma, detection method and application
CN112094912A (en) * 2020-10-16 2020-12-18 中国药科大学 Plasma free DNA methylation gene combination for identifying benign and malignant pulmonary nodules and application thereof
CN112501288A (en) * 2020-08-19 2021-03-16 深圳思凝一云科技有限公司 Method for detecting methylation of SHOX2 gene in lung tumor tissue DNA
CN113106151A (en) * 2021-03-25 2021-07-13 杭州瑞普基因科技有限公司 Nucleic acid composition and kit for detecting methylation of pulmonary nodule based on qPCR (quantitative polymerase chain reaction)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1854313A (en) * 2002-09-30 2006-11-01 肿瘤疗法科学股份有限公司 Method for diagnosing non-small cell lung cancers
CN104774957A (en) * 2015-04-24 2015-07-15 上海透景生命科技股份有限公司 Method for diagnosing methylation of human SHOX2 gene and human RASSF1A gene and kit thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1854313A (en) * 2002-09-30 2006-11-01 肿瘤疗法科学股份有限公司 Method for diagnosing non-small cell lung cancers
CN104774957A (en) * 2015-04-24 2015-07-15 上海透景生命科技股份有限公司 Method for diagnosing methylation of human SHOX2 gene and human RASSF1A gene and kit thereof

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
WEN-BIN LIU等: "Epigenetic regulation of ANKRD18B in lung cancer", 《MOLECULAR CARCINOGENESIS》 *
封杰等: "肺癌患者MPDZ基因异常甲基化的研究", 《癌变畸变突变》 *
肖雨等: "表达谱基因芯片筛选结直肠癌转移中Tiam1相关基因", 《临床与实验病理学杂志》 *

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WO2019233449A1 (en) * 2018-06-07 2019-12-12 深圳市圣必智科技开发有限公司 Dna methylation marker for predicting early lung cancer and application thereof
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