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CN109022567A - For identifying kit and its application of Lung neoplasm and/or lung cancer status - Google Patents

For identifying kit and its application of Lung neoplasm and/or lung cancer status Download PDF

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Publication number
CN109022567A
CN109022567A CN201810886500.8A CN201810886500A CN109022567A CN 109022567 A CN109022567 A CN 109022567A CN 201810886500 A CN201810886500 A CN 201810886500A CN 109022567 A CN109022567 A CN 109022567A
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primer
sequence
primer sets
dna
lung cancer
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陈彦利
蒲珏
李明明
李淑钰
徐春叶
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Beijing Ai Kelun Medical Science And Technology Co Ltd
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Beijing Ai Kelun Medical Science And Technology Co Ltd
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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
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes

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Abstract

The kit and its application, kit that the present invention relates to a kind of for identifying Lung neoplasm and/or lung cancer status include the primer pair for detecting the methylation level of biomarker genes or respective segments in the biological sample from subject；Primer pair is used for carry out pcr amplification reaction as template through the biomarker genes of bisulf iotate-treated or its segment；Biomarker genes are selected from one of APC, DAPK, FHIT, FOXL2, HOXA9, MGMT, P16, PTGER4, RASSF1A, SHOX2, SOX17 and ZAR1 or a variety of.The present invention passes through joint-detection biomarker genes or the methylation level of respective segments, so that the sensitivity and specificity of Lung neoplasm and lung cancer detection are improved, to ensure that the correctness and reliability of testing result, a quick, reliable and accurate new way is provided for the prediction, diagnosis and assessment of Lung neoplasm and lung cancer.

Description

For identifying kit and its application of Lung neoplasm and/or lung cancer status
Technical field
The present invention relates to field of biotechnology, and in particular to a kind of for identifying the reagent of Lung neoplasm and/or lung cancer status Box and its application.
Background technique
Lung cancer is whole world disease incidence, the highest malignant tumour of the death rate, is the great disease for threatening human life and health Disease.World's cancer of World Health Organization's International Agency for Research on Cancer (IARC) publication in 2014 reports display: global cancer burden It is constantly aggravating, wherein lung cancer tops the list in new hair common cancer in 2012, about 1,800,000, is accounting for common cancer sum 13%；In common dead cancer, lung cancer also ranks first, and about 1,600,000, accounts for the 19.4% of sum.In top ten disease incidence In higher cancer, disease incidence of the lung cancer in male tops the list, and accounts for 23%, and disease incidence accounts for 14.85% in women, is only second to mammary gland The 16.97% of cancer.In addition the death rate of lung cancer also occupies the umber one in male and female, and 29.50% is accounted in male, and women is about 23.89%.
Lung neoplasm is the precancerous lesion of lung cancer common in clinic, is common phenomenon in a kind of clinic, including benign knot Concealment is compared in section and Malignant Nodules, malign lung nodules early detection, if not early intervention, the course of disease is rapid, grade of malignancy is strong, pre- It is poor afterwards.Such small pulmonary nodules venereal disease becomes the pernicious rate for having 73%, predominantly adenocarcinoma of lung, is secondly bronchioalveolar carcinoma； And benign rate is 27%, predominantly hemangioma, purulence ulcer, granuloma and tuberculoma.So finding Lung neoplasm as early as possible and correctly evaluating Lung neoplasm it is good pernicious, help the correct treatment means of selection, improve prognosis.
The prognosis of lung cancer is poor, and it was found that when be mostly middle and advanced stage.If can have in early detection and using certain Effect treatment method can substantially reduce the death rate of lung cancer.Although lung cancer is in the side such as diagnosis, operative treatment and research in recent years Face achieves a series of progress, but its five year survival rate remains at lower 16%.The reason for this is that patient often exists The advanced stage of disease just finds thus to lose optimal therapic opportunity.Foreign study found that the Patients with Non-small-cell Lung of I phase exists After receiving radical surgery treatment, 5 years survival rates can reach 58%-73%, however only 15% patient's energy at present In early detection.This explanation essentially consists in the early diagnosis efficiency of raising patients with lung cancer to improve the prognosis of patients with lung cancer.
Currently, the diagnosis of lung cancer relies primarily on the technologies such as iconography, Sputum and bronchoscopic biopsies, these are examined There is no the death rates for largely reducing patients with lung cancer therefore how to improve the survival rate of patients with lung cancer for disconnected technology, according to Rely the early diagnosis in lung cancer, the biological markers for screening and excavating the valuable early stage of lung cancer become urgently to be resolved and ask Topic.Using some tumor markers, such as CEA (carcinomebryonic antigen), identifies innocent and malignant tumour accuracy rate of diagnosis and also there was only 48%.By Fail to show good effect in lung cancer early screening in Imaging Technology, people start to shift sight to lung cancer early stage The molecular marker of diagnosis, regrettably so far without one higher molecular marker of sensibility and specificity of discovery. Lung cancer Progress of Epigenetic Study is very fast in recent years, especially DNA methylation, and research finds just to deposit early stage lung cancer occurs There is different degrees of methylation state in many distinctive tumor-related genes to change, is the spy of lung cancer early diagnosis marker Rope provides new opportunity.
One of the hot spot of the abnormal generation with tumour of the DNA methylation of genome always medical field research, the cell cycle, DNA reparation, angiogenesis and apoptosis etc. are directed to the methylation of related gene.The most probable regulating and controlling effect of DNA hyper-methylation It is the expression by inhibiting key gene, thus determine the destiny of the cell, it is such as extremely existing for DNA methylation in tumour cell The research of elephant achieves many great progress in kinds of tumors.In mammal, methylation only influences bird in DNA chain Cytimidine (CpG) before purine.The methylation distribution of CpG dinucleotide and inhomogenous, about 50% gene in normal cell In promoter region with the presence of the island CpG of CpG integrated distribution, 0.5~2kb of length is differed, the transcriptional control in the region and gene Close relation.The island the CpG methylation of the certain gene regulatory regions of human body frequently occurs in associated cancer cell tissue, shows It is related to the morbidity of certain tumours, disease progression and prognosis, with Drug Sensitivity etc. out.So far, swollen in most of mankind Gene methylation exception is had found in tumor.Research finds that epigenetic coding gets muddled in cancer cell, shows DNA first Methylation level disorder, also known as methylation are reset.Since the local height on the island tumor suppressor gene CpG methylates earlier than the evil of cell Property hyperplasia, therefore, the detection of DNA methylation can be used for tumorigenic early diagnosis.Cancer related gene, which methylates, is also The earliest events that lung cancer occurs, therefore related gene methylation state becomes early stage of lung cancer risk profile efficiency index.Although such as This, still lacks at present and is effectively detected to the methylation state of these cancer related genes and handled testing result Means.
Currently, in division of respiratory disease oncology there is an urgent need to be minimally invasively to assess and predict Lung neoplasm and lung cancer Existing clinical trial.
Summary of the invention
For the defects in the prior art, it is an object of that present invention to provide one kind for identifying Lung neoplasm and/or lung cancer shape The kit of state, kit include for detecting biomarker genes or respective segments in the biological sample from subject The primer pair of methylation level；Primer pair be used for using through the biomarker genes of bisulf iotate-treated or its segment as template Carry out PCR amplified reaction；Biomarker genes be selected from APC, DAPK, FHIT, FOXL2, HOXA9, MGMT, P16, One of PTGER4, RASSF1A, SHOX2, SOX17 and ZAR1 or a variety of.It should be noted that from extraction from biological material DNA And with bisulf iotate-treated, so that the non-methylated cytosine residue deamination in DNA, and the cytosine residues to methylate are protected It holds constant.
Biomarker genes be selected from APC, DAPK, FHIT, FOXL2, HOXA9, MGMT, P16, PTGER4, RASSF1A, 2 kinds in SHOX2, SOX17 and ZAR1 or two or more；Preferably, biomarker genes be selected from APC, DAPK, FHIT, 5 kinds or 5 kinds or more in FOXL2, HOXA9, MGMT, P16, PTGER4, RASSF1A, SHOX2, SOX17 and ZAR1；It is preferred that Ground, the biomarker genes of Lung neoplasm are FOXL2 and/or P16；Preferably, lung cancer status is lung cancer I phase or II phase, biology Marker gene is HOXA9 and/or RASSF1A；Preferably, lung cancer status is gland cancer, biomarker genes be DAPK and/or MGMT；Preferably, lung cancer status is squamous carcinoma, and biomarker genes are FHIT and/or ZAR1；Preferably, lung cancer status is big Cell lung cancer, biomarker genes are HOXA9 and/or PTGER4；Preferably, lung cancer status is Small Cell Lung Cancer, biology mark Will object gene is SHOX2 and/or SOX17.
Primer pair includes the first primer group, the second primer sets, third primer sets, the 4th primer sets, the 5th primer sets, the 6th Primer sets, the 7th primer sets, the 8th primer sets, the 9th primer sets, the tenth primer sets, the 11st primer sets, the 12nd primer sets, Tenth three-primer group, the 14th primer sets, the 15th primer sets, the 16th primer sets, the 17th primer sets, the 18th primer Group, the 19th primer sets, the 20th primer sets, the 21st primer sets, the 22nd primer sets, the 24th primer sets, It is one or more groups of in 25 primer sets and the 26th primer sets；Each primer sets include upstream primer, downstream primer, Close primer and probe；Wherein, the first primer group includes the primer that DNA shown in sequence 13-16 is formed in sequence table；Second draws Object group includes the primer that DNA shown in sequence 17-20 is formed in sequence table；Third primer sets include sequence 21-24 in sequence table Shown in DNA composition primer；4th primer sets include the primer that DNA shown in sequence 25-28 is formed in sequence table；5th draws Object group includes the primer that DNA shown in sequence 29-32 is formed in sequence table；6th primer sets include sequence 33-36 in sequence table Shown in DNA composition primer；7th primer sets include the primer that DNA shown in sequence 37-40 is formed in sequence table；8th draws Object group includes the primer that DNA shown in sequence 41-44 is formed in sequence table；9th primer sets include sequence 45-48 in sequence table Shown in DNA composition primer；Tenth primer sets include the primer that DNA shown in sequence 49-52 is formed in sequence table；Tenth One primer sets include the primer that DNA shown in sequence 53-56 is formed in sequence table；12nd primer sets include sequence in sequence table The primer of the composition of DNA shown in 57-60；Tenth three-primer group include in sequence table DNA shown in sequence 61-64 form draw Object；14th primer sets include the primer that DNA shown in sequence 65-68 is formed in sequence table；15th primer sets include sequence The primer that DNA shown in sequence 69-72 is formed in table；16th primer sets include DNA group shown in sequence 73-76 in sequence table At primer；17th primer sets include the primer that DNA shown in sequence 77-80 is formed in sequence table；18th primer sets packet Include the primer that DNA shown in sequence 81-84 is formed in sequence table；19th primer sets include in sequence table shown in sequence 85-88 DNA composition primer；20th primer sets include the primer that DNA shown in sequence 89-92 is formed in sequence table；21st Primer sets include the primer that DNA shown in sequence 93-96 is formed in sequence table；22nd primer sets include sequence in sequence table The primer of the composition of DNA shown in 97-100；20th three-primer group includes the composition of DNA shown in sequence 101-104 in sequence table Primer；24th primer sets include the primer that DNA shown in sequence 105-108 is formed in sequence table；25th primer Group includes the primer of the composition of DNA shown in sequence 109-112 in sequence table；26th primer sets include sequence in sequence table The primer of the composition of DNA shown in 113-116.
It should be noted that the first primer group and the second primer sets are used to detect the methylation of apc gene, third primer sets It is used to detect the methylation of DAPK gene, the 5th primer sets and the 6th primer sets with the 4th primer sets for detecting FHIT gene Methylation, the 7th primer sets, the 8th primer sets and the 9th primer sets are used to detect the methylation of FOXL2 gene, the tenth primer sets It is used to detect the methylation of HOXA9 gene, the 12nd primer sets and the tenth three-primer group with the 11st primer sets for detecting The methylation of mgmt gene, the 14th primer sets, the 15th primer sets and the 16th primer sets are used to detect the methyl of P16 gene Change, the 17th primer sets and the 18th primer sets are for detecting the methylation of PTGER4 gene, the 19th primer sets and the 20th Primer sets are used to detect the methylation of RASSF1A gene, the 21st primer sets and the 22nd primer sets for detecting The methylation of SHOX2 gene, the 20th three-primer group and the 24th primer sets are used to detect the methylation of SOX17 gene, the 25 primer sets and the 26th primer sets are used to detect the methylation of ZAR1 gene.
Different primers group detects the methylation of at least one section nucleic acid sequence in corresponding gene target region nucleic acid sequence respectively；Its In, target region is respectively selected from shown in the sequence 1 of sequence table, the sequence 2 of sequence table is shown, sequence table sequence 3 is shown, sequence table Sequence 4 shown in, shown in the sequence 5 of sequence table, shown in the sequence 6 of sequence table, the sequence 7 of sequence table, the sequence of sequence table 8, Sequence 9, the sequence of sequence table 10 of sequence table are shown, sequence table sequence 11 is shown, DNA sequence shown in sequence table sequence 12 The segment of continuous at least 15 bases longs in column；Nucleic acid sequence is equal respectively, complementary or hybridization is in above-mentioned target region.Examination In agent box, every kind of primer sets can be packed individually.
Kit further includes reference gene ACTB (NCBI GenBank sequences number: NM_001101.3) primer sets, reference gene ACTB primer sets be used for using in biological sample after bisulf iotate-treated ACTB gene or its segment be template progress PCR Amplified reaction；Reference gene ACTB primer sets include the primer that DNA shown in sequence 117-119 is formed in sequence table.
Preferably, kit further includes DNA extraction reagent and bisulfite agent, bisulfite agent include sulfurous Sour hydrogen sodium；
Preferably, kit further includes illustrating kit application method and being handled with logistic regression testing result Specification.
The present invention also protects application of the kit in preparation identification Lung neoplasm and/or lung cancer status product.
Preferably, Lung neoplasm and/or lung cancer include Lung neoplasm and/or lung cancer susceptibility, the presence of lung cancer, progress, hypotype And/or by stages.
The present invention also protect a kind of detection APC, DAPK, FHIT, FOXL2, HOXA9, MGMT, P16, PTGER4, The method of the methylation of one of RASSF1A, SHOX2, SOX17 and ZAR1 or several genes and its segment, comprising steps of S1: the biological sample of subject is acquired；S2: using the methylation water of biomarker genes in kit detection biological sample It is flat；Wherein, biomarker genes include APC, DAPK, FHIT, FOXL2, HOXA9, MGMT, P16, PTGER4, RASSF1A, One of SHOX2, SOX17 and ZAR1 or a variety of；S3: the methylation level that will test and biomarker base corresponding in group The normal methyl group level of cause is compared.
It preferably, further include repeating step S1 and S2, the first that then will be obtained twice after subject receives medical treatment The horizontal testing result of baseization is compared, to determine the situation of change of Lung neoplasm and/or lung cancer in subject.
Preferably, in S1, biological sample be selected from the blood of subject, serum, blood plasma, sputum, lymph, cerebrospinal fluid, hydrothorax, One of BAL fluid, urine and tissue biopsy are a variety of.
Preferably, in S2, include the steps that the methylation level for detecting target region in biomarker genes, target region point Not Wei in biomarker genes at least 15 bases longs nucleotide sequence or its complementary series.
It should be noted that the present invention also protects a kind of method for identifying Lung neoplasm and/or lung cancer status, comprising steps of S1: the biological sample of subject is acquired；S2: using the methylation water of biomarker genes in kit detection biological sample It is flat, wherein biomarker genes be selected from Gene A PC, DAPK, FHIT, FOXL2, HOXA9, MGMT, P16, PTGER4, One of RASSF1A, SHOX2, SOX17 and ZAR1 or a variety of；S3: the methylation level that will test biology corresponding to group The normal methyl group level of marker gene is compared, and is returned according to the methylation level logic-based of biomarker genes To judge the Lung neoplasm and/or lung cancer in subject.
Technical solution provided by the invention, by joint using detection APC, DAPK, FHIT, FOXL2, HOXA9, MGMT, One or more genes or the methylation water of its segment in P16, PTGER4, RASSF1A, SHOX2, SOX17 and ZAR1 gene It is flat, so that the sensitivity and specificity of Lung neoplasm and/or lung cancer detection are improved, to ensure that the correct of testing result Property and reliability.Also, by the method for the biomarker genes methylate DNA in detection sample, needle can be conveniently realized To APC, DAPK, FHIT, FOXL2, HOXA9, MGMT, P16, PTGER4, RASSF1A, SHOX2, SOX17 and ZAR1 gene this The detection of 12 biomarkers, is analyzed using logistic regression equation, and quickly and conveniently whether judgement sample is positive and risk Value provides a kind of kit of quickly detection cancer.Method and kit provided herein is Lung neoplasm and/or lung Prediction, diagnosis and the assessment of cancer provide a quick, reliable and accurate new way.
Additional aspect and advantage of the invention will be set forth in part in the description, and will partially become from the following description Obviously, or practice through the invention is recognized.
Detailed description of the invention
Fig. 1 is the Receiver Operating Characteristics (ROC) of 12 kinds of biomarker genes methylation levels in the embodiment of the present invention 4 Curve graph；
Fig. 2 is 6 kinds of biomarker genes APC, DAPK, FHIT, FOXL2, HOXA9 and MGMT in inventive embodiments 4 The horizontal distribution figure to methylate in Lung neoplasm and different stages of lung cancer；
Fig. 3 be inventive embodiments 4 in 6 kinds of biomarker genes P16, PTGER4, RASSF1A, SHOX2, SOX17 and Horizontal distribution figure of the methylation of ZAR1 in different Lung neoplasms and different stages of lung cancer；
Fig. 4 is the methyl of 6 kinds of marker Gene As PC, DAPK, FHIT, FOXL2, HOXA9 and MGMT in inventive embodiments 4 Change the horizontal distribution figure in different lung cancer hypotypes；
Fig. 5 is 6 kinds of marker Gene PTENs, PTGER4, RASSF1A, SHOX2, SOX17 and ZAR1 in inventive embodiments 4 Horizontal distribution figure of the methylation in different lung cancer hypotypes；
Fig. 6 is special using subject's work of the gene constructed Logic Regression Models of 12 kinds of markers in inventive embodiments 4 Levy (ROC) curve graph；
Fig. 7 is that the tested of 6 kinds of Logic Regression Models for most having markers characteristic gene constructed is used in inventive embodiments 4 Person's operating characteristic (ROC) curve graph.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete Site preparation description.The following examples are only intended to illustrate the technical solution of the present invention more clearly, therefore is intended only as example, without It can be limited the scope of the invention with this.Experimental method in following embodiments is unless otherwise specified conventional side Method.Test material as used in the following examples is unless otherwise specified to be commercially available from routine biochemistry reagent shop.
The biomarker genes that the present invention uses are selected from the one or more of following gene: APC (Adenomatous Polypos is Coli)、DAPK(Death-Associated Protein Kinase)、FHIT(Fragile Histidine Triad)、FOXL2 (Forkhead Box L2)、HOXA9(Homeobox A9)、MGMT(O6-methylguanine-DNA Methyltra nsferase), P16 (=CNKN2A, cyclin dependent kinase inhibitor 2A), PTGER4 (Prostaglandin E Receptor 4)、RASSF1A(Ras association domain family 1A)、SHOX2 (Short stature home obox 2), SOX17 (SRY-related HMG-box 17) and ZAR1 (Zygote arrest 1)。
Term used herein " subject ", which refers to, suffers from or suspects the individual (preferably people) with certain disease, or Person, when predicting neurological susceptibility, " subject " may also comprise healthy individuals.The term usually can with " patient ", " detection pair As ", " treatment object " etc. be used interchangeably.
Term used herein " group " refers generally to healthy population.Referring to specified disease (such as Lung neoplasm and lung cancer) When, " group " may include the human body for not suffering from the specified disease but may suffering from Other diseases.Furthermore it is also possible to according to year Age, gender, health status, the features such as whether smoke only selected section individual as " group "." normal methyl group water in group It is flat " it can be obtained by being detected to enough individuals, or can be found in existing clinical literature.Some In the case of, which refers to no methylation.
Term used herein " Lung neoplasm and lung cancer status " includes neurological susceptibility and lung cancer of the subject to lung cancer In the presence of, progress, hypotype and/or by stages.It in some embodiments, can be according to the methyl of biomarker genes in subject Change level predicts the subject to the neurological susceptibility of lung cancer.In other embodiments, it can be marked according to biology in subject The methylation level of will object gene whether there is Lung neoplasm and lung cancer to identify in the subject；And if there is lung cancer Words identify its hypotype and/or by stages.Lung cancer hypotype may include gland cancer, squamous carcinoma, maxicell lung cancer and Small Cell Lung Cancer.Lung cancer point Phase may include I phase (IA, IB or IC), II phase, III phase and IV phase.In some embodiments, lung cancer is I phase lung cancer.One In a little embodiments, lung cancer is II phase lung cancer.In some embodiments, lung cancer is III phase lung cancer.In other embodiments In, lung cancer is IV phase lung cancer.
In the method for the invention, be also based on lung cancer arranges the treatment to subject by stages, it may for example comprise right Subject is more tested, carries out surgical operation, carries out drug therapy and do not take further action.At other In embodiment, method of the invention further include in subject after treating, again measure subject in APC, DAPK, FHIT, One or more of biology marks in FOXL2, HOXA9, MGMT, P16, PTGER4, RASSF1A, SHOX2, SOX17 and ZAR1 gene The methylation level of will object gene or its segment；And keeps measurement result associated with Lung neoplasm and lung cancer status, controlled with identification The change for whether leading to Lung neoplasm and lung cancer status in subject treated.In some embodiments, association is calculated by software classification Method carries out.
In the present invention, the detection to methylation level include DNA is extracted from biological sample, and with bisulfites at Reason then carries out pcr amplification reaction using the primer pair of methylation specific.Wherein bisulf iotate-treated causes DNA double chain point Unmethylated cytosine residues deamination, becomes uracil in son；And the cytosine residues to methylate remain unchanged.In this way, In subsequent pcr amplification reaction, the cytosine residues site to methylate in template is as the bird in cytosine residues and primer Adenine residues pairing, and unmethylated cytosine residues site is matched as uracil residues and the adenine residue in primer It is right.The present invention devises multiple primer pairs for every kind of biomarker genes, to detect target in every kind of biomarker genes The methylation level in region, wherein target region is respectively selected from sequence table in DNA shown in sequence 1-12 at least 15 continuous The segment of bases longs；And the nucleic acid sequence of primer pair is equivalent respectively, complementary or hybridization is in above-mentioned target region.It is provided by the invention Primer pair detects the methylation level of target region in biomarker genes using the methylation differential.When biomarker base When the target region of cause does not methylate, primer pair used cannot in pcr amplification reaction with the target region as template (through sulfurous Sour hydrogen salt processing) effectively pairing combination, it cannot (or few) generation amplified production；And work as the target gene of biomarker genes When being methylated, primer pair used can in pcr amplification reaction with the target region as template (at bisulfites Reason) effectively pairing combination, to generate amplified production.The difference of this amplified reaction can carry out process in amplified reaction Middle real-time monitoring, or can be judged by detection amplified production.The present invention passes through test of many times, has filtered out for biology Multiple primer pairs of marker gene, they are used alone or in combination, can help to identify Lung neoplasm and lung cancer in subject State.
The present invention often uses term " biomarker genes or its segment " when referring to detection methylation level, is Since in pcr amplification reaction, primer pair used can't distinguish whole gene or its segment in the selection of template, As long as the length of template is not less than the length in region to be amplified (in fact, extracting DNA and subsequent bisulfites In treatment process, it will usually cause gene break at different size of segment).
In some preferred embodiments, the present invention measures marker gene methylation, institute using HeavyMethyl method Closing primer has also been devised other than designing general T aqman primer.Closing primer is designed as and phase on nucleotide sequence It answers one section of template sequence in primer pair institute amplification region to match to combine.In addition, closing primer introduces chemical modification in 3 '-OH, Cause archaeal dna polymerase that can not expand, which is, for example, arm (C6Spacer), reversion between arm (C3Spacer), C6 between C3 3 ' ends (3 ' end of inverted), 3 ' phosphoric acid (3 ' P) etc..In embodiments of the present invention, the core of primer will be closed Nucleotide sequence is designed as combining with unmethylated template (handling through sulphite), without the template with methylation (through sulfurous Hydrochlorate processing) it combines.Thus, when closing in region corresponding to primer there is no methylating, can prevent accordingly to expand Reaction carries out, to improve the specificity of detection method.
In the methods of the invention, it is also preferable to include using fluorescence probe come real-time monitoring and/or quantitative pcr amplification reaction. It can be FAM, JOE, TET, HEX, Cy3, Texas Red, Rox or Cy5 that probe 5 ' used, which holds reporter fluorescence group,；3 ' end it is sudden The group that goes out is BHQ1, BHQ2, BHQ3, TAMRA, DABCYL or MGB.
It include detection biomarker genes to the detection of the methylation level of biomarker genes in the method for the present invention In with the presence or absence of methylation and the methylation is qualitatively and quantitatively detected.
The biological sample that the present invention uses, which is selected from, extracts from the fluid or tissue of subject, including blood, serum, blood plasma, Sputum, lymph, cerebrospinal fluid, hydrothorax, BAL fluid, urine and tissue biopsy etc..
In the method for the invention, it is also contemplated that the age of subject and cigarette smoking index SI, for predicting in subject Lung neoplasm and lung cancer status.
In some embodiments, method of the invention further includes providing the reading report or electronic report of lung cancer prediction Step, and optionally, report includes about lung cancer presence or absence in subject or its possibility or about subject's lung cancer Hierarchical risk prediction.
In some embodiments, method of the invention further includes that biomarker genes methylation is established for doctor relatively Horizontal report and by being reported as the transmission such as mailing, fax, mailbox.In one embodiment, it is passed by internet The data flow of the defeated report comprising biomarker genes methylation level.
In some embodiments, using the diagnosis of methylation level of the statistical method building based on biomarker genes Model, statistical method are selected from following methods: multiple linear regression, and look-up table, decision tree, support vector machines, Probit are returned, patrolled Collect recurrence, clustering, neighbor analysis, genetic algorithm, Bayes and non-bayes method etc..
In other embodiments, the prediction based on biomarker genes methylation level or diagnosis mould are provided Type.Model can be the form of software code, the form of computer-readable format or written explanation, for evaluating biomarker The opposite methylation level of gene.
New and important additional information can be obtained using method of the invention, doctor is assisted to suffer from the wind of lung cancer to patient Danger is classified and plans the diagnosis algorithm that next will be taken.Method provided by the invention similarly can also be used in assessment without disease Lung-cancer-risk in shape high-risk patient, and screening instruments are used as general groups.Consider that method of the invention can be faced Bed doctor is used as a part of other predictive and diagnostic index comprehensive assessments.
Method of the invention can be used for assessing existing chemotherapeutant and candidate chemical therapeutic agent and other types of cancer The treatment effect for the treatment of means.For example, biological sample can be obtained from subject before and after subject or during treatment Product, and by the detection for carrying out biomarker genes methylation level above, cancer in subject is identified by testing result The variation of state, so that it is determined that treatment effect.
Method of the invention can also be used to identify whether subject is potentially occurring cancer.It is tested being derived from any time Biomarker genes methylation relative level is detected in the biological sample of person, so that the biomarker of cancer feature will be directed toward Methylation level change is construed to be in progress towards generation cancer direction.
The combination of biomarker genes is provided in lung cancer development difference, middle prediction lung cancer to exist or detects by stages Sensitive, the special and accurate means of lung cancer.It can also be with the malignant tumour of patient to the evaluation of methylation level in biological sample The presence of preceding or preclinical illness has correlation.Therefore, disclosed method can be used for lung cancer in prediction or test sample In the presence of, the stage of lung cancer, the hypotype of lung cancer, benign or malignant, lung cancer the metastatic potential of lung cancer, superfluous life related with lung cancer The histological type of object, the Silent Neuritis of cancer or aggressiveness, and it is Cancer-Related with prevention, diagnosis, characterization and treatment Patients with Lung Other lung cancer features.
Method of the invention can also be used to assess the effect that drug candidate inhibits lung cancer, assess the effect of lung cancer therapy, prison The progress of lung cancer is surveyed, selection inhibits the medicament or therapy of lung cancer, monitors the treatment of patients with lung cancer, monitors the inhibition of lung cancer in patient Situation, and the methylation level by testing biomarker genes in animal after detection test compound exposure, to assess Test the carcinogenic potential of compound.
The present invention also provides the kits for detecting Lung neoplasm and lung cancer status.In some embodiments, the examination Agent box may include that DNA extracts reagent and bisulfite agent.DNA extract reagent may include lysis buffer, combination buffer, Cleaning buffer solution and elution buffer.Lysis buffer is usually inhibited by protein denaturant, detergent, pH buffer and nuclease Agent composition.Combination buffer is usually made of protein denaturant and pH buffer.Cleaning buffer solution be divided into cleaning buffer solution A and Cleaning buffer solution B: cleaning buffer solution A is made of protein denaturant, nucleic acid inhibitor, detergent, pH buffer and ethyl alcohol； Cleaning buffer solution B is made of nucleic acid inhibitor, pH buffer and ethyl alcohol.Elution buffer is usually by nucleic acid inhibitor and pH Buffer composition.Protein denaturant is selected from one of guanidinium isothiocyanate, guanidine hydrochloride and urea or a variety of；Detergent is selected from One of Tween20, IGEPAL CA-630, Triton X-100, NP-40 and SDS or a variety of；PH buffer be selected from Tris, One of boric acid, phosphate, MES and HEPES or a variety of；Nucleic acid inhibitor is selected from one of EDTA, EGTA and DEPC Or it is a variety of.Bisulfite agent includes bisulfite salt buffer and protection buffer.Wherein, it is sub- to be selected from weight for bisulfites One of sodium sulphate, sodium sulfite, sodium hydrogensulfite, ammonium bisulfite and ammonium sulfite are a variety of；Protect buffer by oxygen Free radical scavenger composition, oxygen free radical scavenger be selected from hydroquinone, vitamin E, vitamin e derivative, gallic acid, One or more of Trolox, trihydroxybenzoic acid and trihydroxybenzoic acid derivative.
Kit of the invention include for APC, DAPK, FHIT, FOXL2, HOXA9, MGMT, P16, PTGER4, One or more primer pairs for carrying out methylation status of PTEN promoter amplified reaction in RASSF1A, SHOX2, SOX17 and ZAR1 gene. These primer pairs detect the methylation of at least one section nucleotide sequence in corresponding gene target region nucleotide sequence respectively.
Kit of the invention may also include the closing primer and probe being used in combination with above-mentioned primer pair (above and below There is the explanation to these closing primer and probes).
In certain embodiments, kit, which may also include, extracts biological sample DNA using kit and with bisulfite The specification of salt reagent processing DNA.In other embodiments, kit further includes tested using reagent measuring in kit The specification of biomarker level in person.In other embodiment, kit include using the kit with determine by The specification of Lung neoplasm and lung cancer status in examination person.
The method that the present invention goes back Sustainable use kit detection biomarker genes or the methylation level of its segment, packet It includes following steps: the DNA in reagent extraction biological sample being extracted using DNA, obtained DNA will be extracted and tried using bisulfites Agent is handled；And using treated DNA as template, biomarker genes methyl is detected using provided primer pair Change horizontal.
Biomarker genes methylation level measurement method can be selected from one or more in following methods: real-time fluorescence PCR, digital pcr, bisulfite sequencing, methylation status of PTEN promoter, restriction endonuclease analysis, high-resolution solubility curve Technology, biochip technology and flight time mass spectrum.
Technical solution provided by the invention is described further combined with specific embodiments below.
Embodiment 1:DNA is extracted
DNA extracts reagent and is made of lysis buffer, combination buffer, cleaning buffer solution and elution buffer.Cracking is slow Fliud flushing is made of protein denaturant, detergent, pH buffer and nucleic acid inhibitor.Combination buffer is by protein denaturant and pH Buffer composition.Cleaning buffer solution is divided into cleaning buffer solution A and cleaning buffer solution B, cleaning buffer solution A by protein denaturant, Nucleic acid inhibitor, detergent, pH buffer and ethyl alcohol composition；Cleaning buffer solution B is by nucleic acid inhibitor, pH buffer and second Alcohol composition.Elution buffer is made of nucleic acid inhibitor and pH buffer.Wherein protein denaturant are as follows: guanidine hydrochloride；Detergent Are as follows: Tween20；PH buffer are as follows: Tris-HCl；Nucleic acid inhibitor are as follows: EDTA.
The present embodiment extracts plasma dna by taking Plasma of The Patients With Lung Cancer sample as an example.Extracting method includes the following steps:
(1) 1ml blood plasma is taken, the lysis buffer of same volume is added, Proteinase K and Carrier RNA is added, makes it Final concentration is respectively 100mg/L and 1 μ g/ml, and concussion mixes, 55 DEG C of incubation 30min；
(2) 100 μ l magnetic beads are added, add 2ml combination buffer, oscillation incubation 1 hour；
(3) with magnetic separator absorption magnetic bead (being purchased from Life technologies company, article No.: 37002D), supernatant is abandoned Solution；
(4) 1ml cleaning buffer solution A is added, magnetic bead, concussion cleaning 1min is resuspended；
(5) magnetic bead is adsorbed with magnetic separator, abandons supernatant；
(6) 1ml cleaning buffer solution B is added, magnetic bead, concussion cleaning 1min is resuspended；
(7) magnetic bead is adsorbed with magnetic separator, abandons supernatant solution；
(8) 10000rpm rapid centrifugation 1min adsorbs magnetic bead with magnetic separator, removes remaining supernatant solution；
(9) centrifuge tube equipped with magnetic bead is uncapped on the metal bath for being placed on 55 DEG C, dries 10min；
(10) 100 μ l elution buffers are added and magnetic bead is resuspended, be placed on 65 DEG C of metal baths, concussion elution 10min；
(11) magnetic bead is adsorbed with magnetic separator, takes out the buffer containing target DNA, quantitative DNA is marked；
(12) it is stand-by that the DNA after eluting deposits in 4 DEG C of refrigerators, or is stored in -20 DEG C of refrigerator long-term preservations.
Embodiment 2: bisulf iotate-treated DNA
Bisulf iotate-treated DNA is to be handled using bisulfite agent extracted DNA sample, sulfurous acid Hydrogen salt reagent is made of bisulfite salt buffer and protection buffer；Bisulfite salt buffer is sodium hydrogensulfite and water Mixed liquor；Protecting buffer is the mixed liquor of oxygen free radical scavenger hydroquinone and water.
The present embodiment extracts obtained DNA with embodiment 1 as process object, using bisulf iotate-treated DNA, specifically walks Suddenly include:
(1) it prepares bisulfite salt buffer: weighing 1g sodium hydrogensulfite powder, water is added to be configured to 3M buffer；
(2) it prepares protection buffer: weighing 1g hydroquinone reagent, water is added to be configured to 0.5M protection buffer；
(3) 100 μ l DNA solutions, 200 μ l bisulfite salt buffers and 50 μ l protection liquid are mixed, concussion mixing is equal It is even；
(4) thermal cycle: 95 DEG C of 5min, 80 DEG C of 60min, 4 DEG C of 10min is carried out；
(5) in the DNA solution after 1ml DNA combination buffer to be added to bisulf iotate-treated, 50 μ l magnetic are added Pearl, concussion are incubated for 1h；
(6) magnetic bead is adsorbed with magnetic separator, abandons supernatant solution；
(7) 0.5ml cleaning buffer solution A is added, magnetic bead, concussion cleaning 1min is resuspended；
(8) magnetic bead is adsorbed with magnetic separator, abandons supernatant；
(9) 0.5ml cleaning buffer solution B is added, magnetic bead, concussion cleaning 1min is resuspended；
(10) magnetic bead is adsorbed with magnetic separator, abandons supernatant solution；
(11) 10000rpm rapid centrifugation 1min adsorbs magnetic bead with magnetic separator, removes remaining supernatant solution；
(12) centrifuge tube equipped with magnetic bead is placed on 55 DEG C of metal bath, uncaps and dries 10min；
(13) 50 μ l elution buffers are added and magnetic bead is resuspended, be placed on 65 DEG C of metal baths, concussion elution 10min；
(14) magnetic bead is adsorbed with magnetic separator, takes out the buffer containing target DNA, quantitative DNA is marked.
Embodiment 3: real-time fluorescence PCR detection DNA methylation and primer sets verifying
The present embodiment detects the methylation level of biomarker genes by taking real-time fluorescence PCR as an example.Detecting gene is APC, DAPK, FHIT, FOXL2, HOXA9, MGMT, P16, PTGER4, RASSF1A, SHOX2, SOX17 and ZAR1 gene, it is interior Ginseng gene is ACTB.The present embodiment uses DNA of the embodiment 2 after bisulf iotate-treated to carry out real-time fluorescence for template PCR amplification.DNA sample, negative quality-control product, positive quality control product and no template control to be measured carry out 3 multiple holes detections.
For APC, DAPK, FHIT, FOXL2, HOXA9, MGMT, P16, PTGER4, RASSF1A, S HOX2, SOX17 and ZAR1 gene can design the combination of many set primer and probes, and there may be differences for the performance of every suit probe primer combination Not, so needing by being verified.In the present embodiment, with methylation and non-methylation DN A template to APC, DAPK, FHIT, FOXL2, HOXA9, MGMT, P16, PTGER4, RASSF1A, S HOX2, SOX17 and ZAR1 gene primer and probe into Row verifying.
Therefore, the application for APC, DAPK, FHIT, FOXL2, HOXA9, MGMT, P16, PTGER4, RASSF1A, SHOX2, SOX17 and ZAR1 gene devise a variety of primer and probes, can be equal to, are complementary to or hybridize respectively in SEQ ID At least 15 continuous nucleotides or its complementary series of sequence shown in NO:1-12；Made with methylation and non-methylated nucleic acid sequence For the validity of the designed primer and probe of template verifying.The application by real-time fluorescence PCR amplification filtered out with Under optimal primer sets and reference gene ACTB primer sets.
The first primer group (APC primer sets 1)
Primer 1:SEQ ID NO 13:5 '-TTATATGTCGGTTACGTGC-3 '
Primer 2: SEQ ID NO 14:5 '-ACCAAAACGCTCCCCATT-3 '
Close primer: 155 '-TGGTTATGTGTGTTTATATTTAGTTAATTGGT-C3-3 ' of SEQ ID NO
Probe: SEQ ID NO 16:5 '-JOE-CCCGTCGAAAACCCGCCGATTA-BHQ1-3 '
Second primer sets (APC primer sets 2)
Primer 1:SEQ ID NO 17:5 '-TTGCGGAGTGCGGGTC-3 '
Primer 2: SEQ ID NO 18:5 '-TCGACGAACTCCCGACG-3 '
Close primer: 195 '-AGTGTGGGTTGGGAAGTGGAGAGA-C3-3 ' of SEQ ID NO
Probe: SEQ ID NO 20:5 '-JOE-AAACGCCCTAATCCGCATCCAACGAA-BHQ1-3 '
Third primer sets (DAPK primer sets 1)
Primer 1:SEQ ID NO 21:5 '-TTTCGTTTAAAAGGCGGTAAGGAG-3 '
Primer 2: SEQ ID NO 22:5 '-AAACAAAATCCCCGACGTTAACTC-3 '
Close primer: SEQ ID NO 23:5 '-AGGTGGTAAGGAGTTGAGAGGTTGT-3'P-3 '
Probe: SEQ ID NO 24:5 '-FAM-TCCTCCTCACACTCCGAAACAACCTCTC-BHQ1-3 '
4th primer sets (DAPK primer sets 2)
Primer 1:SEQ ID NO 25:5 '-GTCGCGTAGAATTCGTAG-3 '
Primer 2: SEQ ID NO 26:5 '-ACCCTACAAACGAACTAAC-3 '
Close primer: SEQ ID NO 27:5 '-TGTAGAATTTGTAGTGTTGGTTTGG-3'P-3 '
Probe: SEQ ID NO 28:5 '-FAM-CCGAACTACCCTACCAAACCGA-BHQ1-3 '
5th primer sets (FHIT primer sets 1)
Primer 1:SEQ ID NO 29:5 '-ACGGGATAGAAGTTTATCGTTTC-3 '
Primer 2: SEQ ID NO 30:5 '-GCGAATCTAAATTTCCACG-3 '
Close primer: SEQ ID NO 31:5 '-AAATTTCCACACACATCAAATCATCACC-C3-3 '
Probe: SEQ ID NO 32:5 '-HEX-CCGAAACCCAATAAAACCGACGCCG-BHQ1-3 '
6th primer sets (FHIT primer sets 2)
Primer 1:SEQ ID NO 33:5 '-TGACGAATTATTTTAAAACGGC-3 '
Primer 2: SEQ ID NO 34:5 '-TCCTCCACCGTATTTATAC-3 '
Close primer: SEQ ID NO 35:5 '-TTAAAATGGTGGGTGTTGATTATATTTTTAGAG-C3-3 '
Probe: SEQ ID NO 36:5 '-HEX-AAACCACGCCAACGAATCCG-BHQ1-3 '
7th primer sets (FOXL2 primer sets 1)
Primer 1:SEQ ID NO 37:5 '-GGGTAAACGTAGGAAGCG-3 '
Primer 2: SEQ ID NO 38:5 '-GACTACTAACCCCTCTCG-3 '
Close primer: SEQ ID NO 39:5 '-TGTAGGAAGTGGGTTGGGTTAATTGTGGGT-3'P-3 '
Probe: SEQ ID NO 40:5 '-FAM-CCGCTAACGACGCCTCGATCG-BHQ1-3 '
8th primer sets (FOXL2 primer sets 2)
Primer 1:SEQ ID NO 41:5 '-CGGGAAGATTTCGGTTTG-3 '
Primer 2: SEQ ID NO 42:5 '-CCCCAAAACCTAAACTTAC-3 '
Close primer: SEQ ID NO 43:5 '-TTTTGGTTTGGAGCGTTTTGTGTTTTTGGGTTT-3'P-3 '
Probe: SEQ ID NO 44:5 '-FAM-CGCCGCCAACAAACCCGAAA-BHQ1-3 '
9th primer sets (FOXL2 primer sets 3)
Primer 1:SEQ ID NO 45:5 '-TAGGGTGTTTTCGATTGTC-3 '
Primer 2: SEQ ID NO 46:5 '-CCAACGTAAATAATCCGAA-3 '
Close primer: SEQ ID NO 47:5 '-TTTGATTGTTGGGTGGTGGGTGGAAGT-3'P-3 '
Probe: SEQ ID NO 48:5 '-FAM-CGCGCCGAAACTCTACAACCACTA-BHQ1-3 '
Tenth primer sets (HOXA9 primer sets 1)
Primer 1:SEQ ID NO 49:5 '-GGGTTTTAGTTAGGAGCGTATGT-3 '
Primer 2: SEQ ID NO 50:5 '-CCATCACCACCACCCCTACG-3 '
Close primer: SEQ ID NO 51:5 '-GAGTGTATGTATTTGTTGTTTGGTGTTGTTGTTG-C3-3 '
Probe: SEQ ID NO 52:5 '-TEXASRED-CGCCCGTAACGACGACGACGCCG-BHQ2-3 '
11st primer sets (HOXA9 primer sets 2)
Primer 1:SEQ ID NO 53:5 '-GATGGTGGTGGTATATCG-3 '
Primer 2: SEQ ID NO 54:5 '-ACGATATTTAACGCCTCG-3 '
Close primer: SEQ ID NO 55:5 '-GTGGTATATTGTAGTGGGTATAGTG-C3-3 '
Probe: SEQ ID NO 56:5 '-TEXASREDCGCGACGAACGCCAACGCTAT-BHQ2-3 '
12nd primer sets (MGMT primer sets 1)
Primer 1:SEQ ID NO 57:5 '-GTATCGTTTGCGATTTGGTGAGTG-3 '
Primer 2: SEQ ID NO 58:5 '-TACAAAACCACTCGAAACTACCACC-3 '
Close primer: SEQ ID NO 59:5 '-TGTTTGTGATTTGGTGAGTGTTTGGGTT-C3-3 '
Probe: SEQ ID NO 60:5 '-JOE-AAAACTCCGCACTCTTCCGAAAACGAAACG-BHQ1-3 '
Tenth three-primer group (MGMT primer sets 2)
Primer 1:SEQ ID NO 61:5 '-TGTTGGGATAGTTCGCGT-3 '
Primer 2: SEQ ID NO 62:5 '-CACTCTTCCGAAAACGAA-3 '
Close primer: SEQ ID NO 63:5 '-GGATAGTTTGTGTTTTTAGAATGTTTTGTG-C3-3 '
Probe: SEQ ID NO 64:5 '-JOE-CCCAAACACTCACCAAATCGCA-BHQ1-3 '
14th primer sets (P16 primer sets 1)
Primer 1:SEQ ID NO 65:5 '-TGGAGTTTTCGGTTGATTG-3 '
Primer 2: SEQ ID NO 66:5 '-ACAACGCCCGCACCT-3
Close primer: SEQ ID NO 67:5 '-TGGTTGATTGGTTGGTTATGGTTGT-C3-3 '
Probe: SEQ ID NO 68:5 '-TEXASRED-ACCCGACCCCGAACCGCG-BHQ1-3 '
15th primer sets (P16 primer sets 2)
Primer 1:SEQ ID NO 69:5 '-TCGAGTATTCGTTTACGGC-3 '
Primer 2: SEQ ID NO 70:5 '-TTTCTTCCTCCGATACTAACG-3
Close primer: SEQ ID NO 71:5 '-TTGTTTATGGTGTTTTTTTGTTTGGAAAGATATT-C3-3 '
Probe: SEQ ID NO 72:5 '-TEXASRED-CCTCCGACCCTATCCCTCAAATCCTCT-BHQ1- 3 '
16th primer sets (P16 primer sets 3)
Primer 1:SEQ ID NO 73:5 '-CGGATCGCGTGCGTTC-3 '
Primer 2: SEQ ID NO 74:5 '-CGAACCGCGACCGTAA-3
Close primer: SEQ ID NO 75:5 '-TGTGTGTTTGGTGGTTGTGGAGAG-C3-3 '
Probe: SEQ ID NO 76:5 '-TEXASRED-CCCGCCGCCCGCTACCTACTC-BHQ1-3 '
17th primer sets (PTGER4 primer sets 1)
Primer 1:SEQ ID NO 77:5 '-TTAGATATTTGGTGTTTTATCGATT-3 '
Primer 2: SEQ ID NO 78:5 '-AAAAACTAAAACCCGCGTACAT-3 '
Close primer: SEQ ID NO 79:5 '-TTTTATTGATTGGATTATTAATGTGATGGTGTATGTTG- 3'P- 3’
Probe: SEQ ID NO 80:5 '-JOE-ATAAACGACGTACGCCGTCACGTTAATA-BHQ1-3 '
18th primer sets (PTGER4 primer sets 2)
Primer 1:SEQ ID NO 81:5 '-TGGGTATTGTAGTCGCGAGTTATC-3 '
Primer 2: SEQ ID NO 82:5 '-CTACGTAAACAAACGATTAACG-3 '
Close primer: SEQ ID NO 83:5 '-TGTGAGTTATTGAGATTTATGTTGGGTAGTGT-C3-3 '
Probe: SEQ ID NO 84:5 '-JOE-CAATCTATACGTCCAACGTACTCTTTTACGCGCTA-BH Q1-3 '
19th primer sets (RASSF1A primer sets 1)
Primer 1:SEQ ID NO 85:5 '-GCGTTGAAGTCGGGGTTCG-3 '
Primer 2: SEQ ID NO 86:5 '-CCGATTAAACCCGTACTTC-3 '
Close primer: SEQ ID NO 87:5 '-TTGGGGTTTGTTTTGTGGTTTCGTTTGGTTTGT-C3-3 '
Probe: SEQ ID NO 88:5 '-JOE-CGCTAACAAACGCGAACCGA-BHQ1-3 '
20th primer sets (RASSF1A primer sets 2)
Primer 1:SEQ ID NO 89:5 '-GGGAGTTTGAGTTTATTGA-3 '
Primer 2: SEQ ID NO 90:5 '-GATACGCAACGCGTTAACACG-3 '
Close primer: SEQ ID NO 91:5 '-CACATTAACACACTCCAACCAAATACAACCCTT-C3- 3 '
Probe: SEQ ID NO 92:5 '-JOE-CGCCCAACGAATACCAACTCC-BHQ1-3 '
21st primer sets (SHOX2 primer sets 1)
Primer 1:SEQ ID NO 93:5 '-GTTCGTGCGATTTCGGTC-3 '
Primer 2: SEQ ID NO 94:5 '-TCGCTACCCCTAAACTCGA-3 '
Close primer: SEQ ID NO 95:5 '-TGATTTTGGTTGGGTAGGTGGGATG-C3-3 '
Probe: SEQ ID NO 96:5 '-FAM-CAACCAAATAATCTCCGTCCCGC-BHQ1-3 '
22nd primer sets (SHOX2 primer sets 2)
Primer 1:SEQ ID NO 97:5 '-GGCGGGCGAAAGTAATC-3 '
Primer 2: SEQ ID NO 98:5 '-CGAAAATCGCGAATATTCCG-3 '
Close primer: SEQ ID NO 99:5 '-ACAAATATTCCACTTAAACCTATTAATCTCTATAAATT AAACA-C3-3’
Probe: SEQ ID NO 100:5 '-FAM-AAAATCGAATCTACGTTTCCACGAAAA-BHQ1-3 '
20th three-primer group (SOX17 primer sets 1)
Primer 1:SEQ ID NO 101:5 '-GTTGCGTTAGTCGTTTGC-3 '
Primer 2: SEQ ID NO 102:5 '-GAACCAAAAACGAATCCCG-3 '
Close primer: SEQ ID NO 103:5 '-AGTTGTTTGTGTTTGTTTTTAGTTTATATTATG-C3-3 '
Probe: SEQ ID NO 104:5 '-FAM-ATCCGACGACCGATAAACGCTTTCAT-BHQ1-3 '
24th primer sets (SOX17 primer sets 2)
Primer 1:SEQ ID NO 105:5 '-ATGTTTCGAGGGTTGCGC-3 '
Primer 2: SEQ ID NO 106:5 '-CCGCAATATCACTAAACCG-3 '
Close primer: SEQ ID NO 107:5 '-TGAGGGTTGTGTGGGTTTTTTGGTTT-C3-3 '
Probe: SEQ ID NO 108:5 '-FAM-CGCGACAAACCAAAACACGAACGACGA-BHQ1-3 '
25th primer sets (ZAR1 primer sets 1)
Primer 1:SEQ ID NO 109:5 '-GTAGTGCGTTTATGGCG-3 '
Primer 2: SEQ ID NO 110:5 '-GCGAAATATAAATACCGATACG-3
Close primer: SEQ ID NO 111:5 '-GTGTTTATGGTGGTTTTGGGG-C3-3 '
Probe: SEQ ID NO 112:5 '-JOE-AACACGTAACCGTCCAACACCT-BHQ1-3 '
26th (ZAR1 primer sets 2)
Primer 1:SEQ ID NO 113:5 '-GTAGTTGGTAGTAGCGCG-3 '
Primer 2: SEQ ID NO 114:5 '-GCTCCCGCTAATAACTATC-3
Close primer: SEQ ID NO 115:5 '-GGTAGTAGTGTGGTAGGGGTT-C3-3 '
Probe: SEQ ID NO 116:5 '-JOE-TACTCGACGACCGTCAACCG-BHQ1-3 '
Reference gene ACTB primer and probe composition
Primer 1:SEQ ID NO 117:5 '-GTGATGGAGGAGGTTTAGTAAGT-3 '
Primer 2: SEQ ID NO 118:5 '-CCAATAAAACCTACTCCTCCCTT-3 '
Probe: SEQ ID NO 119:5 '-CY5-ACCACCACCCAACACACAATAACAAACACA-BHQ3- 3 '
Multiple groups primer and probe can distinguish methylation and non-methylation template, can be used as detection APC, DAPK, F respectively The primer of HIT, FOXL2, HOXA9, MGMT, P16, PTGER4, RASSF1A, SHOX2, SOX17 and ZAR1 gene methylation and Probe.Although different primer and probe combined effects are slightly different, the above primer and probe be respectively suitable for APC, The methylation of DAPK, FHIT, FOXL2, HOXA9, MGMT, P16, PTGER4, RASSF1A, SHOX2, SOX17 and ZAR1 gene Detection.The following table 1, table 2 and table 3 show methylation and the non-methylation mould combined using each primer and probe to said gene The testing result of plate (through bisulf iotate-treated).Obviously, the combination of designed each primer and probe is for the mould that methylates Plate is high degree of specificity.
Primer sets designed by table 1 are to one of the testing result of methylation and non-methylation template (Ct, mean value)
Two (Ct, mean values) of the primer sets designed by table 2 to the testing result of methylation and non-methylation template
Three (Ct, mean values) of the primer sets designed by table 3 to the testing result of methylation and non-methylation template
In addition, applicant further verifies primer and probe group as template using various cancers patient and Healthy People DNA The validity of conjunction.The plasma sample of 5 lung cancer, 3 liver cancer and 5 Healthy Peoples is extracted using the DNA extraction method of embodiment 1 DNA, the method for then using embodiment 2, using bisulf iotate-treated DNA profiling.Using above-mentioned multiple primer and probe groups, Carry out real-time fluorescence PCR experiment.The Ct value of the various marker genes of cancer sample and Healthy People sample is measured respectively, as a result It is shown in Table 4 and table 5.
Each primer sets of table 4 are to specified gene methylation water in known Lung neoplasm and lung cancer status individual (including healthy individuals) One of flat testing result
Each primer sets of table 5 are to specified gene methylation water in known Lung neoplasm and lung cancer status individual (including healthy individuals) The two of flat testing result
From APC, DAPK of the above cancer patient and healthy human sample, FHIT, FOXL2, HOXA9, MGMT, P16, PTGER4, RASSF1A, SHOX2, SOX17 and ZAR1 gene methylation detection Ct value can be seen that more than each primer and spy Needle combination has high degree of specificity amplification to lung cancer methylate DNA, and other cancers and Healthy People are big without expanding or expanding Ct value In 40.Although the primer and probe of difference group is combined to some differences of the amplification Ct value of lung cancer sample, it is all clearly distinguishable from it Its cancer and Healthy People sample, therefore above each primer sets are suitable for lung cancer detection.
Embodiment 4: kit detect Lung neoplasm, patients with lung cancer, benign conditions patients blood plasma sensitivity and specificity
It is determined using 348 samples for the patient for being determined as Lung neoplasm or lung cancer on pathology and on pathology For 123 samples (being shown in Table 6) of the patient of benign conditions, all samples are all always cured collected from PLA Navy Institute.Lung cancer sample include disease it is all by stages with common hypotype.Lung neoplasm and patients with lung cancer are all by iconography and pathology Learn what diagnosis was made a definite diagnosis, sample is based on international TNM stage standard by stages, and sample hypotype is true according to tissue biopsy and ImmunohistochemistryMethods Methods It is fixed.Benign sample includes the common type for the benign conditions seen in entirely research group.It is obtained completely in surgical site infections Clinicopathologia report, including patient age, smoking history, ethnic group, by stages, hypotype and to each sample encoded collect place.
The stages of lung cancer and other feature of the acquired sample object of table 6
DNA is extracted from sample with the DNA extraction method of embodiment 1, then uses the method bisulfite of embodiment 2 Salt treatment DNA profiling recycles the primer and probe provided in embodiment 3 combination (to adopt for each biomarker genes With primer sets 1) carry out real-time fluorescence PCR experiment, detection APC, DAPK, FHIT, FOXL2, HOXA9, MGMT, P16, PTGER4, RASSF1A, SHOX2, SOX17 and ZAR1 gene and reference gene ACTB, finally obtain Healthy People and patients with lung cancer sample is each The Ct value of gene.As described in example 3 above, the methylation level of each gene can be reflected by the Ct value.
Using commercially available software package (IBM SPSS Statistics 24 and MedCalc11.4.2.0, purchased from IBM and MedCalc descriptive statistic, Receiver Operating Characteristics' (ROC) curve and the figure exhibition of blood plasma biomarker level) are carried out Show.(ANOVA) is examined using nonparametric Kruskal-Wallis, statistics then has been determined using inspection after Dunn's Multiple range test Learn difference.All statistics is compared, value < 0.05 P is considered as statistically significant.
Using real-time fluorescence PCR measuring method, it is being determined as 348 patients of Lung neoplasm or lung cancer from pathology and is having In 123 individual blood plasma of benign lung illness, the methylation level of above-mentioned 12 kinds of marker genes is had detected.In order to help In the ability for determining that these biomarker genes distinguish symptom similar cancer and benign lung illness, all samples are from identical Clinical populations (based on there are pulmonary nodule carry out surgical operation patient) in obtain.Before any intervention and known disease All samples are just collected before state.Morbid state is then determined by the pathological examination of in vitro tissue.Using single Sample collection scheme collect blood plasma, monitor biddability.Which ensure that sample quality and eliminate in sample set appoint A possibility that collection, processing and biological bias.In this study without using normal healthy sample, the reason is that they are usually It is easier to be distinguished than benign conditions.These samples show the average patient age (60.3 in the individual for suffering from Lung neoplasm or lung cancer Year) it is higher than suffering from those of benign conditions (52.5 years old) individual, suffer from the average cigarette smoking index (312) in Lung neoplasm or the individual of lung cancer It is higher than suffering from those of benign conditions (98) individual, and all increase with the progress by stages of disease performance.Generally, lung cancer is sub- Distribution seen in all cases of lung cancer is similar in the distribution of type and crowd, wherein non-small cell lung cancer (including gland cancer, squamous carcinoma and big Cell cancer) ratio (85.92%) it is bigger than Small Cell Lung Cancer (table 6).Benign control in research represents common benign tuberculosis, Including benign protuberance, tumour.
For the methylation level detection data of every kind of biomarker, MedCalc11.4.2.0 software, selection are used 95% confidence interval generates ROC curve and its area under the curve (AUC) value.Relative to benign lung illness, 12 in lung cancer sample The AUC of kind biomarker genes methylation level is all larger than 0.7 (value < 0.05 P), (see Fig. 1 and table between 0.71-0.86 7)。
The area under the curve (AUC) of 7 12 kinds of marker gene Receiver Operating Characteristics (ROC) tracing analysis of table
In order to determine whether certain biomarker genes have different (the especially early stages) by stages of Lung neoplasm or lung cancer There is bigger discrimination, compares the methylation level (Fig. 2 and Fig. 3) of 12 kinds of biomarker genes in I phase and II phase (marker Detect most important period) discrimination in sample.For Lung neoplasm sample, both FOXL2 and MGMT have very high discrimination (value < 0.001 P) is then arranged in decreasing order as DAPK, HOXA9 and PTGER4 (P value 0.001 to 0.01), followed by APC, FHIT, RASSF1A and SOX17 (P value 0.01 to 0.05).For SHOX2 and ZAR1, do not have between Lung neoplasm and benign conditions Significant difference (value > 0.05 P).For I phase sample, FOXL2, HOXA9 and RASSF1A have very high discrimination (P value < 0.001) it, is then arranged in decreasing order as MGMT (P value 0.001 to 0.01), followed by APC, FHIT, P16, SHOX2, SOX17 (P Value 0.01 to 0.05).For DAPK, PTGER4 and ZAR1, there is no significant difference (P value between I phase cancer and benign conditions >0.05).For II phase sample, APC, DAPK and SHOX2 have very high discrimination (value < 0.001 P), followed by FHIT, PTGER4 and ZAR1 (P value 0.001 to 0.01), followed by FOXL2, MGMT, P16, RASSF1A (P value 0.01 to 0.05). HOXA9 and SOX17 does not have significant difference (value > 0.05 P).
The methylation level of above-mentioned biomarker genes is also evaluated from benign conditions and various hypotype lung cancer Whether statistical significant difference (Fig. 4 and Fig. 5) is had between sample.For gland cancer, DAPK, MGMT and RASSF1A have very high Discrimination (value < 0.001 P), is then arranged in decreasing order as HOXA9, SHOX2, FHIT, FOXL2, P16, PTGER4 and SOX17 (P Value 0.001 to 0.05).For squamous carcinoma, FHIT, FOXL2 and ZAR1 have very high discrimination (value < 0.001 P), then with drop Sequence is arranged as APC, DAPK, SHOX2, SOX17, HOXA9, MGMT and RASSF1A (P value 0.001 to 0.05).For maxicell Lung cancer, HOXA9, PTGER4, RASSF1A have very high discrimination (value < 0.001 P), be then arranged in decreasing order for MGMT, SHOX2, SOX17, ZAR1, APC, FHIT and P16 (P value 0.001 to 0.05).For Small Cell Lung Cancer, MGMT, SHOX2, SOX17 has very high discrimination (value < 0.001 P), is then arranged in decreasing order as DAPK, HOXA9, P16 and PTGER4 (P value 0.001 to 0.05).
For easy to operate and reduce cost, the methylation level of detection single biomarker gene is more better than detecting The methylation level of kind biomarker genes.It is apparent, however, that the methylation level of single biomarker gene may The intrinsic diversity information of complex disease can not be provided, so often also needing to construct the diagnostic model using more markers.It is more Marker diagnostic model needs to carry out using statistical analysis technique, constructs methylated genes mark by taking Logic Regression Models as an example below Will object diagnostic model detects lung cancer.
The training of Logic Regression Models carries out in the following way: sample being divided into case and control, then uses IBM 24 software of SPSS Statistics carrys out optimized regression coefficient.There is a regression coefficient for every kind of marker, in addition one Straggling parameter, so that likelihood of the Logic Regression Models for training data maximizes.
After training, regression coefficient set just defines Logic Regression Models.By by the methylation level of biomarker It is put into logistic regression equation, those skilled in the art can easily predict any new sample using such diagnostic model Tasting is set to a possibility that case or control.
The AUC that above-mentioned 12 kinds of marker genes obtain is all larger than 0.70, and next the present invention is combined with using logistic regression 12 kinds of marker genes, the AUC of generation are 0.978 (standard error: 0.00734；95%CI:0.946-0.993；P value: < 0.0001) (Fig. 6).In order to make method for monitoring and analyzing simpler, then the biggish 6 kinds of markers of AUC value are combined and are established Logic Regression Models.Obtained AUC value is 0.972 (standard error: 0.00876；95%CI:0.939-0.990；P value: < 0.0001) (Fig. 7) under 60% specificity, gives 99.0% sensitivity for the sample set.By in fixation The sensitivity of model is determined under specificity values and determines the specificity of model under fixed Sensitirity va1ue further to compare two kinds Model (see the table below 8 and table 9).Such as can choose when the sensitivity of method is greater than about 95%, sensitivity and specificity are total Greater than about 150%；Or when the specificity of method is greater than about 95%, sensitivity and specificity summation is greater than about 165%. In general, 12 kinds of markers are more a shade better than the sensitivity and specificity of the Logic Regression Models of 6 kinds of markers, but based on operation Program and cost consideration are analyzed, 6 kinds of marker combinations are also relatively good selection.
The Logic Regression Models of 85 kinds of table most characteristic marker genes and 12 kinds of marker genes are important special Sensitivity when property threshold value
The Logic Regression Models of 95 kinds of table most characteristic marker genes and 12 kinds of marker genes are important sensitive Spend specificity when threshold value
It should be pointed out that methylation level testing result provided in this embodiment is using for each biomarker What the primer sets 1 of gene obtained (for example, for apc gene, is drawn DAPK gene using DAPK using APC primer sets 1 Object group 1；And so on), but other primer sets provided by the invention are used, obtain similar testing result (data are not shown).
The above embodiments are merely illustrative of the technical solutions of the present invention, rather than its limitations；The ordinary skill people of this field Member is it is understood that be possible to modify the technical solutions described in the foregoing embodiments, or to some or all of Technical characteristic is equivalently replaced；And these are modified or replaceed, it does not separate the essence of the corresponding technical solution, and the present invention is each The range of embodiment technical solution should all cover in the range of description of the invention.
SEQUENCE LISTING
<110>Beijing Ai Kelun medical science and technology Co., Ltd
<120>for identifying kit and its application of Lung neoplasm and/or lung cancer status
<130> 3
<160> 119
<170> PatentIn version 3.3
<210> 1
<211> 300
<212> DNA
<213>people (Homo sapiens)
<400> 1
ttgctgggga ctggggccgc gagggcatac ccccgagggg tacggggcta gggctaggca 60
ggctgtgcgg ttgggcgggg ccctgtgccc cactgcggag tgcgggtcgg gaagcggaga 120
gagaagcagc tgtgtaatcc gctggatgcg gaccagggcg ctccccattc ccgtcgggag 180
cccgccgatt ggctgggtgt gggcgcacgt gaccgacatg tggctgtatt ggtgcagccc 240
gccagggtgt cactggagac agaatggagg tgctgccgga ctcggaaatg gggtaggtgc 300
<210> 2
<211> 414
<212> DNA
<213>people (Homo sapiens)
<400> 2
tagcaatgtg tcacaggtgg ggcgcccgcg ttccgggcgg acgcactggc tccccggccg 60
gcgtgggtgt ggggcgagtg ggtgtgtgcg gggtgtgcgc ggtagagcgc gccagcgagc 120
ccggagcgcg gagctgggag gagcagcgag cgccgcgcag aacccgcagc gccggcctgg 180
cagggcagct cggaggtggg tgggccgcgc cgccagcccg cttgcagggt ccccattggc 240
cgcctgccgg ccgccctccg cccaaaaggc ggcaaggagc cgagaggctg cttcggagtg 300
tgaggaggac agccggaccg agccaacgcc ggggactttg ttccctccgc ggaggggact 360
cggcaactcg cagcggcagg gtctggggcc ggcgcctggg agggatctgc gccc 414
<210> 3
<211> 371
<212> DNA
<213>people (Homo sapiens)
<400> 3
cggattttcg ctacttgtct atgaaactga cgaaccatcc caaaacggcg ggtgccgact 60
acacccccag agccaagagc tgtggcgtcc ccggacccgc tggcgtggcc ccgtgcctca 120
gtttccccaa tgtataaaca cggtggagga ggaacgggtt agggctaggg tcggtgcttg 180
ggaattgggg ggcccgagtc cccaccctga gaccctcgtg gggcggaaga gtactcggga 240
cagaagccca ccgccccgtg agcggcgccg gccccactgg gctccggggt gatgacctga 300
cgcgcgtgga aacccagacc cgcgccccag aaacagtatt ccacttgggc ttgcctcccc 360
gcccctacct t 371
<210> 4
<211> 475
<212> DNA
<213>people (Homo sapiens)
<400> 4
tgcggggaaa ggttttttaa aactcggttt catactatta ttattactaa ggacaaccgg 60
gcaggctgag gtcccaacgt ggatgatccg agttggcctc gcgccggggc tctgcagcca 120
ctgccctgtg cgctcagcac ctctgggggc gatcagggcc cctgcgcttc cgcccgccgc 180
ccggcagtcg agagcaccct gtgcccagac tggccgactc attctccccc gaattttgtt 240
tagagctggc aagggggact tagctcgcgc cccaagacct gggcttgcag cgccgccaac 300
aggcccgggg acacgaggcg ctccaggccg gggtcttccc ggctgctggc ccctctcgct 360
ccccacccgc tggcggcgcc tcggtcgccc gcaattgacc caacccgctt cctgcgtttg 420
cccctcaggt ttcccgtttc tccacaaagg cctaggggag cctcgcccac aggct 475
<210> 5
<211> 353
<212> DNA
<213>people (Homo sapiens)
<400> 5
aggtttaatg ccataaggcc ggctggaggg caagcccgcg aaggagagcg caccgggcgt 60
gggctccagc caggagcgca tgtacctgcc gtccggcgcc gccgccgcca cgggcgcctg 120
ggggtgcacg taggggtggt ggtgatggtg gtggtacacc gcagcgggta cagcgttggc 180
gcccgccgcg tgcactgggt tccacgaggc gccaaacacc gtcgccttgg actggaagct 240
gcacgggctg aagtcggggt gctcggccag cgtcgccgcc tgccggggag gctggcccag 300
ggtccccggc gcatagcggc caacgctcag ctcatccgcg gcgtcggcgc cca 353
<210> 6
<211> 414
<212> DNA
<213>people (Homo sapiens)
<400> 6
gtgcccctcg gccccgcccc cgcgccccgg atatgctggg acagcccgcg cccctagaac 60
gctttgcgtc ccgacgcccg caggtcctcg cggtgcgcac cgtttgcgac ttggtgagtg 120
tctgggtcgc ctcgctcccg gaagagtgcg gagctctccc tcgggacggt ggcagcctcg 180
agtggtcctg caggcgccct cacttcgccg tcgggtgtgg ggccgccctg acccccaccc 240
atcccgggcg agctccaggt gcgccccaag tgcctcccag gtgttgccca gcctttcccc 300
gggcctgggg ttcctggact aggctgcgct gcagtgactg tggactggcg tgtggcgggg 360
gtcgtggcag cccctgcctt acctctaggt gccagcccca ggcccgggcc ccgg 414
<210> 7
<211> 408
<212> DNA
<213>people (Homo sapiens)
<400> 7
atcggcctcc gaccgtaact attcggtgcg ttgggcagcg cccccgcctc cagcagcgcc 60
cgcacctcct ctacccgacc ccgggccgcg gccgtggcca gccagtcagc cgaaggctcc 120
atgctgctcc ccgccgccgg ctccatgctg ctccccgccg cccgctgcct gctctccccc 180
tctccgcagc cgccgagcgc acgcggtccg ccccaccctc tggtgaccag ccagcccctc 240
ctctttcttc ctccggtgct ggcggaagag ccccctccga ccctgtccct caaatcctct 300
ggagggaccg cggtatcttt ccaggcaagg ggacgccgtg agcgagtgct cggaggaggt 360
gctattaact ccgagcactt agcgaatgtg gcacccctga agtcgccc 408
<210> 8
<211> 450
<212> DNA
<213>people (Homo sapiens)
<400> 8
cttcttcagc ctgtccggcc tcagcatcat ctgcgccatg agtgtcgagc gctacctggc 60
catcaaccat gcctatttct acagccacta cgtggacaag cgattggcgg gcctcacgct 120
ctttgcagtc tatgcgtcca acgtgctctt ttgcgcgctg cccaacatgg gtctcggtag 180
ctcgcggctg cagtacccag acacctggtg cttcatcgac tggaccacca acgtgacggc 240
gcacgccgcc tactcctaca tgtacgcggg cttcagctcc ttcctcattc tcgccaccgt 300
cctctgcaac gtgcttgtgt gcggcgcgct gctccgcatg caccgccagt tcatgcgccg 360
cacctcgctg ggcaccgagc agcaccacgc ggccgcggcc gcctcggttg cctcccgggg 420
ccaccccgct gcctccccag ccttgccgcg 450
<210> 9
<211> 443
<212> DNA
<213>people (Homo sapiens)
<400> 9
ctgcgagagc gcgcccagcc ccgccttcgg gccccacagt ccctgcaccc aggtttccat 60
tgcgcggctc tcctcagctc cttcccgccg cccagtctgg atcctggggg aggcgctgaa 120
gtcggggccc gccctgtggc cccgcccggc ccgcgcttgc tagcgcccaa agccagcgaa 180
gcacgggccc aaccgggcca tgtcggggga gcctgagctc attgagctgc gggagctggc 240
acccgctggg cgcgctggga agggccgcac ccggctggag cgtgccaacg cgctgcgcat 300
cgcgcggggc accgcgtgca accccacacg gcagctggtc cctggccgtg gccaccgctt 360
ccagcccgcg gggcccgcca cgcacacgtg gtgcgacctc tgtggcgact tcatctgggg 420
cgtcgtgcgc aaaggcctgc agt 443
<210> 10
<211> 490
<212> DNA
<213>people (Homo sapiens)
<400> 10
tggctctctg cctaccgcaa acttgctggt ctaatttagg aacaattggg ccgaaaggta 60
tcagcgagag caacagaccc cggtgttgtg ccgcacaggg agccgcatcc gcagacgccc 120
ctcgctgccc ctgggctcgg gccaaaccct gcataaggtc ccctggacag ccaggtaatc 180
tccgtcccgc ctgcccgacc ggggtcgcac gagcacaggc gcccacgcca tgttggctgc 240
ccaaagggct cgccgcccaa gccgggccag aaggcaggag gcggaaaacc agcctccggt 300
ggcgggcgaa agcaaccgct ctttctgttc tctcttcgcc ctccctcgtg gaaacgcaga 360
ctcgacccta aacgcttaac ccacagagat caacaggttc aagcggaata ttcgcgatcc 420
tcggtttcta ttggttgctc aaagcctttt catgcaacca gcagctcgga tgtttaataa 480
aatatgaatt 490
<210> 11
<211> 528
<212> DNA
<213>people (Homo sapiens)
<400> 11
aaaaggcccc gcggcccagg ggcgcccgca gtgtcactag gccggctggg ggccctgggt 60
acgctgtaga ccagaccgcg acaggccaga acacgggcgg cggcttcggg ccgggagacc 120
cgcgcagccc tcggggcatc tcagtgcctc actccccacc ccctcccccg ggtcggggga 180
ggcggcgcgt ccggcggagg gttgagggga gcggggcagg cctggagcgc catgagcagc 240
ccggatgcgg gatacgccag tgacgaccag agccagaccc agagcgcgct gcccgcggtg 300
atggccgggc tgggcccctg cccctgggcc gagtcgctga gccccatcgg ggacatgaag 360
gtgaagggcg aggcgccggc gaacagcgga gcaccggccg gggccgcggg ccgagccaag 420
ggcgagtccc gtatccggcg gccgatgaac gctttcatgg tgtgggctaa ggacgagcgc 480
aagcggctgg cgcagcagaa tccagacctg cacaacgccg agttgagc 528
<210> 12
<211> 404
<212> DNA
<213>people (Homo sapiens)
<400> 12
gcctatttag ggtgcggcgg cgggcgggag cagtgcgccc atggcggccc tgggggacga 60
ggtgctggac ggttacgtgt tcccggcgtg ccccccctgc tcgtaccggt acccataccc 120
cgcggccacc aagggcaagg gcgcggcggg cggcagctgg cagcagcgcg gcaggggctg 180
ccttcccgcc tcctccccct gctcggcggg cgcggcctcg ttgtccttcc cgggctgcgg 240
gcggctgacg gccgccgagt acttcgacag ctaccagcgg gagcggctca tggctctcct 300
ggcgcaggtg gggccgggtc tcgggccgcg cgcccgcagg gccggcagct gcgacgtggc 360
ggtgcaggtg agcccgcgca tcgacgccgc ggtacagtgc tcgc 404
<210> 13
<211> 19
<212> DNA
<213>artificial sequence
<400> 13
ttatatgtcg gttacgtgc 19
<210> 14
<211> 18
<212> DNA
<213>artificial sequence
<400> 14
accaaaacgc tccccatt 18
<210> 15
<211> 32
<212> DNA
<213>artificial sequence
<400> 15
tggttatgtg tgtttatatt tagttaattg gt 32
<210> 16
<211> 22
<212> DNA
<213>artificial sequence
<400> 16
cccgtcgaaa acccgccgat ta 22
<210> 17
<211> 16
<212> DNA
<213>artificial sequence
<400> 17
ttgcggagtg cgggtc 16
<210> 18
<211> 17
<212> DNA
<213>artificial sequence
<400> 18
tcgacgaact cccgacg 17
<210> 19
<211> 24
<212> DNA
<213>artificial sequence
<400> 19
agtgtgggtt gggaagtgga gaga 24
<210> 20
<211> 26
<212> DNA
<213>artificial sequence
<400> 20
aaacgcccta atccgcatcc aacgaa 26
<210> 21
<211> 24
<212> DNA
<213>artificial sequence
<400> 21
tttcgtttaa aaggcggtaa ggag 24
<210> 22
<211> 24
<212> DNA
<213>artificial sequence
<400> 22
aaacaaaatc cccgacgtta actc 24
<210> 23
<211> 25
<212> DNA
<213>artificial sequence
<400> 23
aggtggtaag gagttgagag gttgt 25
<210> 24
<211> 28
<212> DNA
<213>artificial sequence
<400> 24
tcctcctcac actccgaaac aacctctc 28
<210> 25
<211> 18
<212> DNA
<213>artificial sequence
<400> 25
gtcgcgtaga attcgtag 18
<210> 26
<211> 19
<212> DNA
<213>artificial sequence
<400> 26
accctacaaa cgaactaac 19
<210> 27
<211> 25
<212> DNA
<213>artificial sequence
<400> 27
tgtagaattt gtagtgttgg tttgg 25
<210> 28
<211> 22
<212> DNA
<213>artificial sequence
<400> 28
ccgaactacc ctaccaaacc ga 22
<210> 29
<211> 23
<212> DNA
<213>artificial sequence
<400> 29
acgggataga agtttatcgt ttc 23
<210> 30
<211> 19
<212> DNA
<213>artificial sequence
<400> 30
gcgaatctaa atttccacg 19
<210> 31
<211> 28
<212> DNA
<213>artificial sequence
<400> 31
aaatttccac acacatcaaa tcatcacc 28
<210> 32
<211> 25
<212> DNA
<213>artificial sequence
<400> 32
ccgaaaccca ataaaaccga cgccg 25
<210> 33
<211> 22
<212> DNA
<213>artificial sequence
<400> 33
tgacgaatta ttttaaaacg gc 22
<210> 34
<211> 19
<212> DNA
<213>artificial sequence
<400> 34
tcctccaccg tatttatac 19
<210> 35
<211> 33
<212> DNA
<213>artificial sequence
<400> 35
ttaaaatggt gggtgttgat tatattttta gag 33
<210> 36
<211> 20
<212> DNA
<213>artificial sequence
<400> 36
aaaccacgcc aacgaatccg 20
<210> 37
<211> 18
<212> DNA
<213>artificial sequence
<400> 37
gggtaaacgt aggaagcg 18
<210> 38
<211> 18
<212> DNA
<213>artificial sequence
<400> 38
gactactaac ccctctcg 18
<210> 39
<211> 30
<212> DNA
<213>artificial sequence
<400> 39
tgtaggaagt gggttgggtt aattgtgggt 30
<210> 40
<211> 21
<212> DNA
<213>artificial sequence
<400> 40
ccgctaacga cgcctcgatc g 21
<210> 41
<211> 18
<212> DNA
<213>artificial sequence
<400> 41
cgggaagatt tcggtttg 18
<210> 42
<211> 19
<212> DNA
<213>artificial sequence
<400> 42
ccccaaaacc taaacttac 19
<210> 43
<211> 33
<212> DNA
<213>artificial sequence
<400> 43
ttttggtttg gagcgttttg tgtttttggg ttt 33
<210> 44
<211> 20
<212> DNA
<213>artificial sequence
<400> 44
cgccgccaac aaacccgaaa 20
<210> 45
<211> 19
<212> DNA
<213>artificial sequence
<400> 45
tagggtgttt tcgattgtc 19
<210> 46
<211> 19
<212> DNA
<213>artificial sequence
<400> 46
ccaacgtaaa taatccgaa 19
<210> 47
<211> 27
<212> DNA
<213>artificial sequence
<400> 47
tttgattgtt gggtggtggg tggaagt 27
<210> 48
<211> 24
<212> DNA
<213>artificial sequence
<400> 48
cgcgccgaaa ctctacaacc acta 24
<210> 49
<211> 23
<212> DNA
<213>artificial sequence
<400> 49
gggttttagt taggagcgta tgt 23
<210> 50
<211> 20
<212> DNA
<213>artificial sequence
<400> 50
ccatcaccac cacccctacg 20
<210> 51
<211> 34
<212> DNA
<213>artificial sequence
<400> 51
gagtgtatgt atttgttgtt tggtgttgtt gttg 34
<210> 52
<211> 23
<212> DNA
<213>artificial sequence
<400> 52
cgcccgtaac gacgacgacg ccg 23
<210> 53
<211> 18
<212> DNA
<213>artificial sequence
<400> 53
gatggtggtg gtatatcg 18
<210> 54
<211> 18
<212> DNA
<213>artificial sequence
<400> 54
acgatattta acgcctcg 18
<210> 55
<211> 25
<212> DNA
<213>artificial sequence
<400> 55
gtggtatatt gtagtgggta tagtg 25
<210> 56
<211> 21
<212> DNA
<213>artificial sequence
<400> 56
cgcgacgaac gccaacgcta t 21
<210> 57
<211> 24
<212> DNA
<213>artificial sequence
<400> 57
gtatcgtttg cgatttggtg agtg 24
<210> 58
<211> 25
<212> DNA
<213>artificial sequence
<400> 58
tacaaaacca ctcgaaacta ccacc 25
<210> 59
<211> 28
<212> DNA
<213>artificial sequence
<400> 59
tgtttgtgat ttggtgagtg tttgggtt 28
<210> 60
<211> 30
<212> DNA
<213>artificial sequence
<400> 60
aaaactccgc actcttccga aaacgaaacg 30
<210> 61
<211> 18
<212> DNA
<213>artificial sequence
<400> 61
tgttgggata gttcgcgt 18
<210> 62
<211> 18
<212> DNA
<213>artificial sequence
<400> 62
cactcttccg aaaacgaa 18
<210> 63
<211> 30
<212> DNA
<213>artificial sequence
<400> 63
ggatagtttg tgtttttaga atgttttgtg 30
<210> 64
<211> 22
<212> DNA
<213>artificial sequence
<400> 64
cccaaacact caccaaatcg ca 22
<210> 65
<211> 19
<212> DNA
<213>artificial sequence
<400> 65
tggagttttc ggttgattg 19
<210> 66
<211> 15
<212> DNA
<213>artificial sequence
<400> 66
acaacgcccg cacct 15
<210> 67
<211> 25
<212> DNA
<213>artificial sequence
<400> 67
tggttgattg gttggttatg gttgt 25
<210> 68
<211> 18
<212> DNA
<213>artificial sequence
<400> 68
acccgacccc gaaccgcg 18
<210> 69
<211> 19
<212> DNA
<213>artificial sequence
<400> 69
tcgagtattc gtttacggc 19
<210> 70
<211> 21
<212> DNA
<213>artificial sequence
<400> 70
tttcttcctc cgatactaac g 21
<210> 71
<211> 34
<212> DNA
<213>artificial sequence
<400> 71
ttgtttatgg tgtttttttg tttggaaaga tatt 34
<210> 72
<211> 27
<212> DNA
<213>artificial sequence
<400> 72
cctccgaccc tatccctcaa atcctct 27
<210> 73
<211> 16
<212> DNA
<213>artificial sequence
<400> 73
cggatcgcgt gcgttc 16
<210> 74
<211> 16
<212> DNA
<213>artificial sequence
<400> 74
cgaaccgcga ccgtaa 16
<210> 75
<211> 24
<212> DNA
<213>artificial sequence
<400> 75
tgtgtgtttg gtggttgtgg agag 24
<210> 76
<211> 21
<212> DNA
<213>artificial sequence
<400> 76
cccgccgccc gctacctact c 21
<210> 77
<211> 25
<212> DNA
<213>artificial sequence
<400> 77
ttagatattt ggtgttttat cgatt 25
<210> 78
<211> 22
<212> DNA
<213>artificial sequence
<400> 78
aaaaactaaa acccgcgtac at 22
<210> 79
<211> 38
<212> DNA
<213>artificial sequence
<400> 79
ttttattgat tggattatta atgtgatggt gtatgttg 38
<210> 80
<211> 28
<212> DNA
<213>artificial sequence
<400> 80
ataaacgacg tacgccgtca cgttaata 28
<210> 81
<211> 24
<212> DNA
<213>artificial sequence
<400> 81
tgggtattgt agtcgcgagt tatc 24
<210> 82
<211> 22
<212> DNA
<213>artificial sequence
<400> 82
ctacgtaaac aaacgattaa cg 22
<210> 83
<211> 32
<212> DNA
<213>artificial sequence
<400> 83
tgtgagttat tgagatttat gttgggtagt gt 32
<210> 84
<211> 35
<212> DNA
<213>artificial sequence
<400> 84
caatctatac gtccaacgta ctcttttacg cgcta 35
<210> 85
<211> 19
<212> DNA
<213>artificial sequence
<400> 85
gcgttgaagt cggggttcg 19
<210> 86
<211> 19
<212> DNA
<213>artificial sequence
<400> 86
ccgattaaac ccgtacttc 19
<210> 87
<211> 33
<212> DNA
<213>artificial sequence
<400> 87
ttggggtttg ttttgtggtt tcgtttggtt tgt 33
<210> 88
<211> 20
<212> DNA
<213>artificial sequence
<400> 88
cgctaacaaa cgcgaaccga 20
<210> 89
<211> 19
<212> DNA
<213>artificial sequence
<400> 89
gggagtttga gtttattga 19
<210> 90
<211> 21
<212> DNA
<213>artificial sequence
<400> 90
gatacgcaac gcgttaacac g 21
<210> 91
<211> 33
<212> DNA
<213>artificial sequence
<400> 91
cacattaaca cactccaacc aaatacaacc ctt 33
<210> 92
<211> 21
<212> DNA
<213>artificial sequence
<400> 92
cgcccaacga ataccaactc c 21
<210> 93
<211> 18
<212> DNA
<213>artificial sequence
<400> 93
gttcgtgcga tttcggtc 18
<210> 94
<211> 19
<212> DNA
<213>artificial sequence
<400> 94
tcgctacccc taaactcga 19
<210> 95
<211> 25
<212> DNA
<213>artificial sequence
<400> 95
tgattttggt tgggtaggtg ggatg 25
<210> 96
<211> 23
<212> DNA
<213>artificial sequence
<400> 96
caaccaaata atctccgtcc cgc 23
<210> 97
<211> 17
<212> DNA
<213>artificial sequence
<400> 97
ggcgggcgaa agtaatc 17
<210> 98
<211> 20
<212> DNA
<213>artificial sequence
<400> 98
cgaaaatcgc gaatattccg 20
<210> 99
<211> 43
<212> DNA
<213>artificial sequence
<400> 99
acaaatattc cacttaaacc tattaatctc tataaattaa aca 43
<210> 100
<211> 27
<212> DNA
<213>artificial sequence
<400> 100
aaaatcgaat ctacgtttcc acgaaaa 27
<210> 101
<211> 18
<212> DNA
<213>artificial sequence
<400> 101
gttgcgttag tcgtttgc 18
<210> 102
<211> 19
<212> DNA
<213>artificial sequence
<400> 102
gaaccaaaaa cgaatcccg 19
<210> 103
<211> 33
<212> DNA
<213>artificial sequence
<400> 103
agttgtttgt gtttgttttt agtttatatt atg 33
<210> 104
<211> 26
<212> DNA
<213>artificial sequence
<400> 104
atccgacgac cgataaacgc tttcat 26
<210> 105
<211> 18
<212> DNA
<213>artificial sequence
<400> 105
atgtttcgag ggttgcgc 18
<210> 106
<211> 19
<212> DNA
<213>artificial sequence
<400> 106
ccgcaatatc actaaaccg 19
<210> 107
<211> 26
<212> DNA
<213>artificial sequence
<400> 107
tgagggttgt gtgggttttt tggttt 26
<210> 108
<211> 27
<212> DNA
<213>artificial sequence
<400> 108
cgcgacaaac caaaacacga acgacga 27
<210> 109
<211> 17
<212> DNA
<213>artificial sequence
<400> 109
gtagtgcgtt tatggcg 17
<210> 110
<211> 22
<212> DNA
<213>artificial sequence
<400> 110
gcgaaatata aataccgata cg 22
<210> 111
<211> 21
<212> DNA
<213>artificial sequence
<400> 111
gtgtttatgg tggttttggg g 21
<210> 112
<211> 22
<212> DNA
<213>artificial sequence
<400> 112
aacacgtaac cgtccaacac ct 22
<210> 113
<211> 18
<212> DNA
<213>artificial sequence
<400> 113
gtagttggta gtagcgcg 18
<210> 114
<211> 19
<212> DNA
<213>artificial sequence
<400> 114
gctcccgcta ataactatc 19
<210> 115
<211> 21
<212> DNA
<213>artificial sequence
<400> 115
ggtagtagtg tggtaggggt t 21
<210> 116
<211> 20
<212> DNA
<213>artificial sequence
<400> 116
tactcgacga ccgtcaaccg 20
<210> 117
<211> 23
<212> DNA
<213>artificial sequence
<400> 117
gtgatggagg aggtttagta agt 23
<210> 118
<211> 23
<212> DNA
<213>artificial sequence
<400> 118
ccaataaaac ctactcctcc ctt 23
<210> 119
<211> 30
<212> DNA
<213>artificial sequence
<400> 119
accaccaccc aacacacaat aacaaacaca 30

Claims (10)

1. a kind of for identifying the kit of Lung neoplasm and/or lung cancer status, it is characterised in that:
The kit includes for detecting the first of biomarker genes or respective segments in the biological sample from subject The primer pair of baseization level；
The primer pair is used for carry out PCR amplification as template through the biomarker genes of bisulf iotate-treated or its segment Reaction；
The biomarker genes be selected from APC, DAPK, FHIT, FOXL2, HOXA9, MGMT, P16, PTGER4, RASSF1A, One of SHOX2, SOX17 and ZAR1 or a variety of.
2. according to claim 1 for identifying the kit of Lung neoplasm and/or lung cancer status, it is characterised in that:
The biomarker genes be selected from APC, DAPK, FHIT, FOXL2, HOXA9, MGMT, P16, PTGER4, RASSF1A, 2 kinds in SHOX2, SOX17 and ZAR1 or two or more；
Preferably, the biomarker genes be selected from APC, DAPK, FHIT, FOXL2, HOXA9, MGMT, P16, PTGER4, 5 kinds or 5 kinds or more in RASSF1A, SHOX2, SOX17 and ZAR1；
Preferably, the biomarker genes of the Lung neoplasm are FOXL2 and/or P16；
Preferably, the lung cancer status is lung cancer I phase or II phase, and the biomarker genes are HOXA9 and/or RASSF1A；
Preferably, the lung cancer status is gland cancer, and the biomarker genes are DAPK and/or MGMT；
Preferably, the lung cancer status is squamous carcinoma, and the biomarker genes are FHIT and/or ZAR1；
Preferably, the lung cancer status is maxicell lung cancer, and the biomarker genes are HOXA9 and/or PTGER4；
Preferably, the lung cancer status is Small Cell Lung Cancer, and the biomarker genes are SHOX2 and/or SOX17.
3. according to claim 1 for identifying the kit of Lung neoplasm and/or lung cancer status, it is characterised in that:
The primer pair includes the first primer group, the second primer sets, third primer sets, the 4th primer sets, the 5th primer sets, the 6th Primer sets, the 7th primer sets, the 8th primer sets, the 9th primer sets, the tenth primer sets, the 11st primer sets, the 12nd primer sets, Tenth three-primer group, the 14th primer sets, the 15th primer sets, the 16th primer sets, the 17th primer sets, the 18th primer Group, the 19th primer sets, the 20th primer sets, the 21st primer sets, the 22nd primer sets, the 20th three-primer group, It is one or more groups of in 24 primer sets, the 25th primer sets and the 26th primer sets；Each primer sets include upper Swim primer, downstream primer, closing primer and probe；
Wherein, the first primer group includes the primer that DNA shown in sequence 13-16 is formed in sequence table；Second primer Group includes the primer of the composition of DNA shown in sequence 17-20 in sequence table；The third primer sets include sequence 21- in sequence table The primer of the composition of DNA shown in 24；4th primer sets include the primer that DNA shown in sequence 25-28 is formed in sequence table； 5th primer sets include the primer that DNA shown in sequence 29-32 is formed in sequence table；6th primer sets include sequence The primer that DNA shown in sequence 33-36 is formed in table；7th primer sets include DNA shown in sequence 37-40 in sequence table The primer of composition；8th primer sets include the primer that DNA shown in sequence 41-44 is formed in sequence table；Described 9th draws Object group includes the primer that DNA shown in sequence 45-48 is formed in sequence table；Tenth primer sets include sequence in sequence table The primer of the composition of DNA shown in 49-52；11st primer sets include in sequence table DNA shown in sequence 53-56 form Primer；12nd primer sets include the primer that DNA shown in sequence 57-60 is formed in sequence table；Tenth three-primer Group includes the primer of the composition of DNA shown in sequence 61-64 in sequence table；14th primer sets include sequence in sequence table The primer of the composition of DNA shown in 65-68；15th primer sets include in sequence table DNA shown in sequence 69-72 form Primer；16th primer sets include the primer that DNA shown in sequence 73-76 is formed in sequence table；17th primer Group includes the primer of the composition of DNA shown in sequence 77-80 in sequence table；18th primer sets include sequence in sequence table The primer of the composition of DNA shown in 81-84；19th primer sets include in sequence table DNA shown in sequence 85-88 form Primer；20th primer sets include the primer that DNA shown in sequence 89-92 is formed in sequence table；Described 21st draws Object group includes the primer that DNA shown in sequence 93-96 is formed in sequence table；22nd primer sets include sequence in sequence table Arrange the primer of the composition of DNA shown in 97-100；The 20th three-primer group includes in sequence table shown in sequence 101-104 The primer of DNA composition；24th primer sets include the primer that DNA shown in sequence 105-108 is formed in sequence table；Institute Stating the 25th primer sets includes the primer that DNA shown in sequence 109-112 is formed in sequence table；26th primer sets Primer including the composition of DNA shown in sequence 113-116 in sequence table.
4. according to claim 1 for identifying the kit of Lung neoplasm and/or lung cancer status, it is characterised in that:
The kit further includes reference gene ACTB primer sets, and the reference gene ACTB primer sets are used for the biological sample ACTB gene or its segment in product after bisulf iotate-treated are that template carries out pcr amplification reaction；
The reference gene ACTB primer sets include the primer that DNA shown in sequence 117-119 is formed in sequence table；
Preferably, the kit further includes DNA extraction reagent and bisulfite agent, the bisulfite agent include Sodium hydrogensulfite；
Preferably, the kit further includes illustrating the kit application method and being carried out with logistic regression to testing result The specification of processing.
5. application of the described in any item kits of claim 1-4 in preparation identification Lung neoplasm and/or lung cancer status product.
6. application according to claim 5, it is characterised in that:
The Lung neoplasm and/or lung cancer include Lung neoplasm and/or lung cancer susceptibility, the presence of lung cancer, progress, hypotype and/or point Phase.
7. it is a kind of using claim 1-5 described in any item kits detection APC, DAPK, FHIT, FOXL2, HOXA9, The methylation of one of MGMT, P16, PTGER4, RASSF1A, SHOX2, SOX17 and ZAR1 or several genes and its segment Method, which is characterized in that comprising steps of
S1: the biological sample of subject is acquired；
S2: the methylation level of biomarker genes in the biological sample is detected using the kit；Wherein, the life Object marker gene includes APC, DAPK, FHIT, FOXL2, HOXA9, MGMT, P16, PTGER4, RASSF1A, SHOX2, SOX17 With one of ZAR1 or a variety of；
S3: the methylation level that will test is compared to the normal methyl group level of biomarker genes corresponding in group.
8. according to the method described in claim 7, it is characterized by:
It further include repeating step S1 and S2, the methylation water that then will be obtained twice after the subject receives medical treatment Flat testing result is compared, with the situation of change of Lung neoplasm and/or lung cancer in the determination subject.
9. according to the method described in claim 7, it is characterized by:
In S1, the biological sample is selected from blood, serum, blood plasma, sputum, lymph, cerebrospinal fluid, hydrothorax, the bronchus of subject One of bronchoalveolar lavage fluid, urine and tissue biopsy are a variety of.
10. according to the method described in claim 7, it is characterized by:
In S2, include the steps that the methylation level for detecting target region in the biomarker genes, the target region difference For the nucleotide sequence or its complementary series of at least 15 bases longs in the biomarker genes.
CN201810886500.8A 2018-08-06 2018-08-06 For identifying kit and its application of Lung neoplasm and/or lung cancer status Pending CN109022567A (en)

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CN110387421A (en) * 2019-08-28 2019-10-29 深圳市新合生物医疗科技有限公司 DNA methylation qPCR kit and application method for lung cancer detection
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
CN111310792A (en) * 2020-01-17 2020-06-19 华南农业大学 Decision tree-based drug sensitivity experiment result identification method and system
CN111363812A (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 agent and kit based on DMRTA2 gene
CN111363811A (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 agent and kit based on FOXD3 gene
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CN114645087A (en) * 2020-12-17 2022-06-21 广州市基准医疗有限责任公司 Methylated molecular markers for detecting benign and malignant lung nodules or combination and application thereof
CN114645043A (en) * 2020-12-17 2022-06-21 广州市基准医疗有限责任公司 Methylated molecular marker combination for detecting benign and malignant lung nodules and application
CN114645043B (en) * 2020-12-17 2022-12-09 广州市基准医疗有限责任公司 Methylated molecular marker combination for detecting benign and malignant lung nodules and application
CN112941180A (en) * 2021-02-25 2021-06-11 浙江大学医学院附属妇产科医院 Group of lung cancer DNA methylation molecular markers and application thereof in preparation of lung cancer early diagnosis kit
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CN113234821A (en) * 2021-04-29 2021-08-10 北京艾克伦医疗科技有限公司 Method and kit for identifying esophageal cancer status
CN113278692A (en) * 2021-05-19 2021-08-20 北京艾克伦医疗科技有限公司 Method and kit for identifying pulmonary nodule status
CN113186293A (en) * 2021-06-04 2021-07-30 杭州圣庭医疗科技有限公司 Nucleic acid composition, kit and detection method for detecting lung cancer related gene methylation
WO2024001668A1 (en) * 2022-06-28 2024-01-04 广州市基准医疗有限责任公司 Methylation molecular marker for detecting benign and malignant pulmonary nodules and use thereof

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Application publication date: 20181218