CN104774957A

CN104774957A - Method for diagnosing methylation of human SHOX2 gene and human RASSF1A gene and kit thereof

Info

Publication number: CN104774957A
Application number: CN201510203539.1A
Authority: CN
Inventors: 王方金; 陈静文; 姚见儿
Original assignee: Shanghai Tou Jing Life Science Limited-Liability Co
Current assignee: Shanghai Jiayu Life Technology Co.,Ltd.
Priority date: 2015-04-24
Filing date: 2015-04-24
Publication date: 2015-07-15
Anticipated expiration: 2035-04-24
Also published as: CN104774957B

Abstract

The invention relates to a method for diagnosing methylation of human SHOX2 gene and human RASSF1A gene and a kit thereof and firstly discloses a method for cooperatively detecting methylation DNA of two lung cancer markers to diagnose lung cancer and improve lung cancer detection rate. The lung cancer markers comprise human SHOX2 gene and human RASSF1A gene. The invention also provides an optimized reagent for detecting methylation DNA of SHOX2 gene and human RASSF1A gene and a detection method thereof.

Description

The method of diagnosis people's SHOX2 gene and people RASSF1A gene methylation and test kit

Technical field

The invention belongs to gene diagnosis field, more specifically, the present invention relates to a kind of method and the test kit of diagnosing people SHOX2 gene and people RASSF1A gene methylation.

Background technology

Lung cancer has become one of human cancer main causes of death.Be the cancer that sickness rate is the highest at lung cancer in China, and M & M increase rapidly.The incidence of lung cancer and mortality ratio are the highest in all tumours, but lung cancer is not but maximum tumour of diagnosis, in the U.S., breast tumor and tumor of prostate have higher diagnosis, reason is can early diagnosis thus can early treatment, substantially increase 5 years survival rates (being respectively 89 and 99%), and 5 of lung cancer years survival rate ability 15%.

In clinical practice, lung cancer early diagnosis is difficult point always, and the effective treatment of early discovery to cancer patients of cancer is very important.The current diagnosis Main Basis clinical symptom to cancer, iconography detection and histopathological examination etc., but there have many cancer clinical symptoms to occur to be more late, and living body sampling detects also difficulty, has had a strong impact on the early diagnosis of cancer and the prognosis of patient.Relative to biopsy, the samples such as bronchoalveolar lavage fluid, blood plasma, sputum easily obtain, and also do not have wound to person under inspection.At present, in the circulating of periphery, the DNA of same gene abnormal methylation only accounts for the small part of all DNA, and about 0.1% ~ 1%, only there is small difference in these non-methylate DNAs and methylate DNA, needs to detect abnormal methylate DNA from " background " of high complexity.The fragment that DNA in addition in circulating degrades usually (being generally base pair more than tens to one hundred), so need special extracting and detection technique could obtain higher sensitivity.

Along with technical fast development, the tumor markers diagnosing tumor developed into after diagnostic imaging, pathological diagnosis treats new field, creates great effect to the diagnosis of tumour, monitoring and treatment.Tumor markers can detect in body fluid or tissue, can reflect that the existence of tumour, differentiation degree, prognosis are estimated and judge result for the treatment of etc.The early stage of lung cancer is owing to often being discovered by doctor and patient hardly without characteristic symptom, also early discovery and early stage etiologic diagnosis is difficult to the diagnostic method of routine, in addition some tumor markers only can not be able to be made a definite diagnosis as the primary dcreening operation of lung cancer or auxiliary diagnosis prompting, and therefore the early diagnosis of lung cancer is comparatively difficult.At present, along with gene diagnosis technology development, bring hope to the early diagnosis of lung cancer, one of them field is exactly that methylate DNA detects.DNA methylation almost has generation in all tumours, becomes a reliable target of diagnosing tumor.DNA methylation is a tumorigenic earliest events, just can be detected before disease is made a definite diagnosis, and is early diagnosis of tumor, risk prediction, clinical disease course monitoring and the potential index of curative effect evaluation.

DNA methylation is as a kind of novel molecular mark, and the importance in diagnosing tumor comes into one's own day by day, and its advantage is mainly: one, in neoplastic process, the occurrence frequency of promotor supermethylation is very high, even higher than transgenation, is wherein no lack of and forms relevant important gene with tumour; Its two, methylating is that early stage critical event occurs tumour; Its three, DNA methylation stable existence, detects by PCR scale effect.Therefore, DNA methylation assay has potential using value to early diagnosis of tumor.Recently research both at home and abroad finds that the contents level of the methylate DNA of some genes in blood plasma is used as early diagnosis, susceptibility is better than existing protein sera mark, that wherein relatively gives prominence to has cyclin-dependent kinase inhibitor 2A gene p16 (p16) relevant to the early stage of lung cancer, the genes such as H-cadherin (CDH13).Smoking is the main reason of lung cancer, has found several gene unconventionality often occurred in Human Lung Cancer in the respiratory tract of normal smoking population.Wherein, studying more is p16 Methylation of Gene.When cast-off cells in normal smoking population sputum specimen are detected, find that p16 Methylation of Gene frequency is higher, then do not find this change in normal non-smokers' sputum specimen, illustrate that this gene alteration may be closely related with early stage respiratory tract neoplasms generating process.In sputum, related gene promoter methylation increases along with tumour danger and increases, and CDKN2A/p16 and/or mgmt gene can detect before diagnose at clear and definite lung squamous cancer 3 years in phlegm.At present, the cell in a lot of research detection blood, sputum, bronchoalveolar lavage fluid or methylation state of DNA is had, to finding the mark for lung cancer early diagnosis.

Although had been found that the DNA methylation of some genes and the dependency of lung cancer in prior art, but still need in the art to study the genes involved that can be applied to pulmonary cancer diagnosis effectively further, and develop the detection reagent with higher detection accuracy.

Summary of the invention

The object of the present invention is to provide a kind of method and the test kit of diagnosing people SHOX2 gene and people RASSF1A gene methylation.

In a first aspect of the present invention, provide a kind of test kit for detection of lung cancer, described test kit comprises: the detection reagent of the DNA methylation of specific detection people SHOX2 gene; With the detection reagent of the DNA methylation of specific detection people RASSF1A gene.

In a preference, described DNA methylation sequence contains CpG dinucleotides structure, and the C base of CpG dinucleotides structure is with methyl group.

In another preference, in test kit, the detection reagent of the DNA methylation of described specific detection people SHOX2 gene is primer for people SHOX2 gene and probe; Preferably, described primer and probe are for the sequence (SEQ ID NO:4) of SEQ ID NO:3 in people SHOX2 gene after hydrosulphite or heavy hydrosulphite or hydrazonium salt modification; Or the detection reagent of the DNA methylation of described specific detection people RASSF1A gene is primer for people RASSF1A gene and probe; Preferably, described primer and probe are for the sequence (SEQ ID NO:2) of SEQ ID NO:1 in people RASSF1A gene after hydrosulphite or heavy hydrosulphite or hydrazonium salt modification.

In another preference, in test kit, the detection reagent of the DNA methylation of described specific detection people SHOX2 gene is: the primer shown in SEQ ID NO:14 and SEQ ID NO:18 and the probe shown in SEQID NO:25; Or the detection reagent of the DNA methylation of described specific detection people RASSF1A gene is: the primer shown in SEQ ID NO:7 and SEQ ID NO:10 and the probe shown in SEQ ID NO:22.

In another preference, in test kit, also comprise in described test kit: the detection reagent of specific detection reference gene; Preferably, the detection reagent of described specific detection reference gene is primer for people β-actin gene and probe; More preferably, described primer and probe are for the sequence (SEQ ID NO:6) of SEQ ID NO:5 in people β-actin gene after hydrosulphite or heavy hydrosulphite or hydrazonium salt modification; More preferably, the detection reagent of the DNA methylation of described specific detection reference gene is: the primer shown in SEQ IDNO:19 and SEQ ID NO:21 and the probe shown in SEQ ID NO:26.

In another preference, described reference gene is used to indicate the quality of DNA extraction and modification.

In another preference, in test kit, the probe for each gene is connected with specific fluorophor; Preferably, for the probe mark VIC fluorescein of people SHOX2 gene, for the probe mark FAM fluorescein of people RASSF1A gene, for the probe mark CY5 fluorescein of people β-actin gene.

In another preference, in this test kit, for the primer of people SHOX2 gene and the ratio (primer: probe) of probe be: 0.2:0.06 (such as, designing PCR amplification system primer final concentration in test kit: probe final concentration=0.2uM:0.06uM); Or in this test kit, for the primer of people RASSF1A gene and the ratio (primer: probe) of probe be: 0.2:0.06 (such as, designing PCR amplification system primer final concentration in test kit: probe final concentration=0.2uM:0.06uM); Or in this test kit, for the primer of people β-actin gene and the ratio (primer: probe) of probe be: 0.67:0.02 (such as, designing PCR amplification system primer final concentration in test kit: probe final concentration=0.67uM:0.02uM).

In another preference, in this test kit, also include, but is not limited to: archaeal dna polymerase (as Taq enzyme), dNTPs, Mg ²⁺ion or comprise archaeal dna polymerase (as Taq enzyme), dNTPs, Mg ²⁺the PCR system of ion; Preferably, describedly archaeal dna polymerase, dNTPs, Mg is comprised ²⁺in the PCR system of ion, described Mg ²⁺ionic concn is 3.5 ± 0.5mM (preferably 3.5 ± 0.3mM); Or described dNTPs concentration is 0.25 ± 0.3mM (preferably 0.25 ± 0.1mM); Or described archaeal dna polymerase is 1.5 ± 0.2U (preferably 1.5 ± 0.1U).Preferably, also comprise in described test kit: hydrosulphite or heavily hydrosulphite or hydrazonium salt.

In another preference, also include, but is not limited in described test kit: quality control product, negative control and/or specification sheets.

In another aspect of this invention, the purposes of the reagent of the DNA methylation of specific detection people SHOX2 gene and people RASSF1A gene is provided, for the preparation of the test kit of detection of lung cancer.

In a preference, the detection reagent of the DNA methylation of described specific detection people SHOX2 gene is primer for people SHOX2 gene and probe; Preferably, described primer and probe are for the sequence (SEQ ID NO:4) of SEQ ID NO:3 in people SHOX2 gene after hydrosulphite or heavy hydrosulphite or hydrazonium salt modification; More preferably, the detection reagent of the DNA methylation of described specific detection people SHOX2 gene is: the primer shown in SEQ ID NO:14 and SEQ ID NO:18 and the probe shown in SEQ IDNO:25; Or the detection reagent of the DNA methylation of described specific detection people RASSF1A gene is primer for people RASSF1A gene and probe; Preferably, described primer and probe are for the sequence (SEQ ID NO:2) of SEQ ID NO:1 in people RASSF1A gene after hydrosulphite or heavy hydrosulphite or hydrazonium salt modification; More preferably, the detection reagent of the DNA methylation of described specific detection people RASSF1A gene is: the primer shown in SEQ ID NO:7 and SEQ ID NO:10 and the probe shown in SEQ ID NO:22.

In another aspect of this invention, detect the method for the DNA methylation of people SHOX2 gene and people RASSF1A gene with providing a kind of external nondiagnostic, described method comprises:

(1) testing sample carried out hydrosulphite or weigh hydrosulphite or hydrazonium salt process, obtaining modified testing sample;

(2) utilize the foregoing modified testing sample of test kit to step (1) for detection of lung cancer to detect, obtain the DNA methylation situation of people SHOX2 gene and the DNA methylation situation of people RASSF1A gene.

In a preference, if the DNA methylation of people SHOX2 gene DNA methylation positive and/or people RASSF1A gene is positive, then show that the supplier of testing sample is for High Risk of Lung Cancer or patients with lung cancer.

In another preference, in a reaction tubes, carry out multiple nucleic acid fragment detection; And in a reaction tubes, carry out the detection of multiple wave band fluorescent signal simultaneously, distinguish different DNA fragmentations by different fluorescent signals.Or, the detection of different IPs acid fragment can be carried out in different reaction tubess; And in a reaction tubes, carry out the detection of single wave band fluorescent signal simultaneously, distinguish different DNA fragmentations by fluorescent signal.

In another preference, described sample of nucleic acid derives from: the nucleic acid of the sample extraction containing cell, as bronchoalveolar lavage fluid, takes from the tissue of diseased region, hydrothorax, sputum etc.; Or containing deriving from the sample of nucleic acid of cell, as blood plasma, serum etc.

Other side of the present invention, due to disclosure herein, is apparent to those skilled in the art.

Accompanying drawing explanation

Fig. 1, people SHOX2 gene and people RASSF1A gene methylation DNA detection specificity.

Fig. 2, people SHOX2 gene and people RASSF1A gene methylation DNA detection sensitivity (VIC signal), numeral cell count in figure.

Fig. 3, people SHOX2 gene and people RASSF1A gene methylation DNA detection sensitivity (FAM signal), numeral cell count in figure.

Embodiment

The present inventor, through deep research, discloses a kind of method that methylate DNA by cooperation detection two kinds of lung cancer markers improves lung cancer recall rate first, and described lung cancer marker is people SHOX2 gene and people RASSF1A gene.The present inventor also optimizes pcr amplification reaction program, further increases amplification efficiency.The present inventor also optimizes the reagent, the detection method that detect people's SHOX2 gene and people RASSF1A gene methylation DNA.Complete the present invention on this basis.

As used herein, " sample to be tested ", " testing sample " or " determined nucleic acid (as DNA) sample " refer to sample of nucleic acid to be detected, wherein containing a kind of nucleic acid or multiple nucleic acids, need to understand wherein whether there is target nucleic acid.In the present invention, described sample to be tested can be: bronchoalveolar lavage fluid, takes from the tissue of diseased region, hydrothorax, sputum etc.; Or containing deriving from the sample of nucleic acid of cell, as blood plasma, serum etc.

As used herein, " target nucleic acid " refers to interested nucleic acid fragment, and it is people SHOX2 gene and people RASSF1A gene methylation DNA specific fragment.

As used herein, " probe " refers to a kind of single-chain nucleic acid with known nucleotide sequence, and it has the nucleotide sequence structure substantially complementary with target nucleic acid, can form double-strand with " target nucleic acid ".Described " probe " can carry marker.Such as, marker can be connected to 5 ' end or the 3 ' end of probe.

People SHOX2 (short stature homeobox 2) gene is a member of short and small hox genes family, and its nucleotide sequence can be substantially identical with the sequence of GenBank accession number: NC_000003.Its gene expression regulation and allelotaxis closely related.Research finds, SHOX2 gene is expressed in mesoderm and ectoderm at embryonic stage, plays an important role to bone, heart and neural growth.SHOX2 gene is abnormal expression in multiple noumenal tumour, as lung cancer, mammary cancer, kidney etc.

People RASSF1A (Ras-association domain family 1A, RASSFlA) gene is Ras relevant range family lA gene, and its nucleotide sequence can be substantially identical with the sequence of GenBank accession number: DQ444319.The target gene of RASSFlA regulation and control relates to the aspects of contents such as genetic transcription, signal transduction, cytoskeleton, cell cycle, cell adhesion and apoptosis, has biological action widely.And RASSFlA is as a kind of novel tumour suppressor gene, its tumour occur and evolution in effect particularly important.Think at present, RASSFlA genetic expression inactivation is mainly relevant with the hyper-methylation of promoter region exception, loss of heterozygosity and chromosome deletion.

The present inventor is surprised to find that, in clinical patients with lung cancer, in the tumor specimen of SHOX2 gene methylation DNA feminine gender, it is positive that most sample there will be RASSF1A gene methylation DNA, joint-detection people SHOX2 gene and people RASSF1A gene methylation DNA more individually detect two kinds of genes, and the accuracy rate (recall rate) of lung cancer detection can significantly improve.

Based on the above-mentioned new discovery of the present inventor, provide the purposes of the reagent of the DNA methylation of specific detection people SHOX2 gene and people RASSF1A gene, for the preparation of the test kit of detection of lung cancer.

Further, present inventor has performed screening and repetition test, provide primer and probe that a class is particularly suitable for people SHOX2 gene and the detection of people RASSF1A gene methylation DNA specific fragment.The selection in gene methylation site is directly relevant to clinical detection sensitivity, and generally all at the promoter region of gene, but the methylation sites that much gene is relevant to tumour is also likely positioned at First Intron and First Exon region.Through a large amount of experiments with compare, the present inventor have chosen people SHOX2 gene and people RASSF1A gene and specifically to methylate region, as corresponded to the SEQ ID NO:3 sequence of people SHOX2 gene, or corresponds to the SEQ ID NO:1 sequence of RASSF1A gene.On this basis, the present inventor have also been devised the primer of high special to amplify different object fragments, and identifies with the probe of high special.

The successful design of primed probe is an extremely important ring for real-time PCR detection, sulphite impels " C " in DNA chain to be converted into " U " after modifying, cause GC content to reduce, occur in the sequence long continuous " T " after PCR is reacted, easily cause DNA chain break.In addition, the loss of DNA can also be caused in purge process.The present inventor finds, when the length of PCR primer is greater than 300bp, being just difficult to the DNA after with sulphite modification for template increases, and the length of 200bp is proper; Modify with Standard PC R the GC content reducing template DNA unlike, sulphite, make to occur in sequence long continuous " T ", cause being difficult to select that there is suitable Tm value and stable primer; On the other hand, in order to distinguish the DNA that sulphite is modified and do not had sulphite moditied processing and not sulphite modification completely, needing primer to have " C " of sufficient amount, each of which increases the difficulty selecting to stablize primer.For these difficulties, the present inventor is on the basis that sufficient sequence is studied, select to optimize suitable section, devise amplimer and the probe of high special, establish a kind of highly sensitive people SHOX2 gene and people RASSF1A gene methylation DNA method for detecting specificity and test kit.

As optimal way of the present invention, in method of the present invention or test kit, also apply the quality that internal reference indicates DNA extraction and modification.Described internal reference is such as preferably β-actin.

In method of the present invention, need the DNA to extracting to carry out chemically modified, hydrosulphite or heavily hydrosulphite or hydrazonium salt are modified and all be can be applicable to this kind of chemically modified.In research process, the present inventor is by sulphite sequence measurement, and the sulphite comparing QIAGEN company and ZYMO company modifies test kit, on transformation efficiency and the rate of recovery, the two does not have significant difference, from cost consideration, selects the sulphite of ZYMO company to modify test kit.

According to the needs of clinical detection, utilizing real-time fluorescence PCR technology to carry out, people SHOX2 gene and people RASSF1A gene methylation DNA specific fragment detect is comparatively preferred.When applying real-time fluorescence PCR and detecting, described probe is connected with the fluorophor being suitable for screening different genes methylated DNA fragments.The front end of probe is marked with fluorophor, and the other end is marked with quenching group, and wherein quenching group can the fluorescence that sends of quenching fluorescence group.When pcr amplification reaction carries out, utilize that the forward of polysaccharase is exo-acting to be cut off the base with fluorophor, the fluorophor after free no longer by the impact of quenching group, can launch the fluorescent signal of certain wavelength under the effect of exciting light.Along with the continuous accumulation of PCR primer, fluorescent signal constantly strengthens, thus the existence of special methylate DNA can be detected.

As optimal way of the present invention, for convenience of Clinical practice, when detecting, adopt the three kinds of specific probes adding three kinds of different fluorophor marks in same reaction tubes (corresponding to people SHOX2 gene, people RASSF1A gene and β-actin gene), there is situation in what in a reaction tubes, indicate 3 kinds of object methylated DNA fragments simultaneously.

As optimal way of the present invention, for convenience of Clinical practice, the fluorophor of detection probes mark can be VIC, ROX, FAM, Cy5, HEX, TET, JOE, NED etc.; Quenching group can be TAMRA, BHQ, MGB, Dabcyl.To be applicable to the conventional hyperchannel PCR detection system of current clinical detection, realize carrying out multicolor fluorescence detection in a reaction tubes.

In optimal ways more of the present invention, the present inventor also optimizes pcr amplification reaction, further increases amplification efficiency.Comprise consumption, the dNTPs consumption of optimizing enzyme, the concentration of primed probe in PCR system, after PCR is reacted, Ct value Relatively centralized, reaches ideal effect.Should be understood that when not optimizing can actualizing technology effect, and after carrying out the optimization of pcr amplification program and amplification system, then effect is even more ideal.

Detection reagent of the present invention is placed in test kit, thus is convenient to people's application.Except primer and probe reagent, also can comprise in test kit: hydrosulphite or heavily hydrosulphite or hydrazonium salt, PCR reaction solution, quality control product, negative control and/or working instructions.

Method of the present invention and test kit are particularly suitable for lung cancer auxiliary diagnosis and early diagnosis, the feature such as have highly sensitive, high specific, easy and simple to handle, sense cycle is short.Effectively can improve Detection accuracy, and reduce the false negative of result.

Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, conveniently condition such as J. Pehanorm Brooker etc. is write usually, Molecular Cloning: A Laboratory guide, the third edition, Science Press, the condition described in 2002, or according to the condition that manufacturer advises.

The selection of embodiment 1, detection target gene

Methylate DNA has obvious advantage as detection target, and compare protein-based mark, DNA can increase, and is easy to detect; Compared with sudden change class mark, methylate DNA is all positioned at the privileged site of gene, generally in promoter region, makes detection become easier and convenient.In order to complete the present invention, contriver has consulted lot of documents, in the gene relevant to lung cancer of report, select representational p16, H-cadherin (CDH13), SHOX2, HOXA9, RAR β, RASSF1A detection gene alternatively, study each gene methylation site distribution situation, the primed probe that design detects is respectively used to detect.Each gene test primed probe is as follows:

The detection primer of p16 and probe are:

P16 primers F: ACGTCGTGAGCGAGTGTTC (SEQ ID NO:27);

P16 primer R:TACCAACGCTAACTCTAACGAA (SEQ ID NO:28);

P16 probe: ROX-CTTCCGACTAATACCCCCGAAA-BHQ (SEQ ID NO:29);

Detection primer and the probe of H-cadherin (CDH13) are:

Primers F: GAAAATATGTTTAGTGTAGTCGCGT (SEQ ID NO:30);

Primer R:ACGCACAAAACGAACGAAAT (SEQ ID NO:31);

Probe: CY5-TTTTATCCGACTAGAAACGCCCG-BHQ (SEQ ID NO:32);

The detection primer of SHOX2 and probe are:

Primers F: TTGTTTTTGGGTTCGGGTT (SEQ ID NO:12)

Primer R:CATAACGTAAACGCCTATACTCG (SEQ ID NO:16)

Probe: VIC-ATCGAACAAACGAAACGAAAATTACC-BHQ (SEQ ID NO:24)

The detection primer of HOXA9 and probe are:

Primers F: GGTATATCGTAGCGGGTATAGC (SEQ ID NO:33);

Primer R:AACTTCCAATCCAAAACGACG (SEQ ID NO:34);

Probe: FAM-TTCGTCGCGTGTATTGGGTTTTAC-BHQ (SEQ ID NO:35);

The detection primer of RAR β and probe are:

Primers F: CGAGAACGCGAGCGATTC (SEQ ID NO:36);

Primer R:AACCTTCCGAATACGTTCCG (SEQ ID NO:37);

Probe: VIC-TCCTACCCCGACGATACCCAAAC-BHQ (SEQ ID NO:38);

The detection primer of RASSF1A and probe are:

Primers F: CGGGGTTCGTTTTGTGGTTTC (SEQ ID NO:7)

Primer R:CCGATTAAATCCGTACTTCGC (SEQ ID NO:10)

Probe: FAM-TCGCGTTTGTTAGCGTTTAAAGT-BHQ (SEQ ID NO:22)

Experimentation:

1, DNA is extracted

Collect and make a definite diagnosis the sample of patients with lung cancer and non-tumour patient sample, isolated cell, for TIANGEN Biotech's genome DNA extracting reagent kit (DP304), extract DNA.

2, DNA modification

Illustrate that sulphite is modified with ZYMO RESEARCH biotech firm test kit EZ DNA Methylation-DirectTMKIT (D5020).

3, amplification and detection

Following preparation 10 × primed probe mix.

Following preparation reaction system (20 μ L):

Pcr amplification program is as follows:

First stage: 95 DEG C, 10min, 1 circulation;

Subordinate phase: 95 DEG C, 15sec, 60 DEG C of 30sec, 5 circulations;

Phase III: 95 DEG C, 15sec, 57 DEG C of 30sec, 40 circulations;

Signal collection: collect FAM, VIC, ROX, CY5 signal during the phase III 60 DEG C.

4, detected result

According to each amplification situation, detected result is in table 1.

Table 1

p16

CDH13

SHOX2

HOXA9

RARβ

RASSF1A

Tumor specimen 1

-

+

-

+

Tumor specimen 2

+

-

+

Tumor specimen 3

-

Tumor specimen 4

+

-

+

Tumor specimen 5

+

Tumor specimen 6

-

+

-

Tumor specimen 7

-

+

-

+

Tumor specimen 8

-

Tumor specimen 9

+

-

+

-

Tumor specimen 10

+

Tumor specimen 11

-

+

Tumor specimen 12

+

-

+

Tumor specimen 13

-

+

Tumor specimen 14

+

-

+

-

Tumor specimen 15

+

-

+

Tumor specimen 16

+

-

+

Tumor specimen 17

+

-

Tumor specimen 18

-

+

Tumor specimen 19

+

-

+

Tumor specimen 20

+

-

+

Non-tumor specimen 1

-

Non-tumor specimen 2

-

Non-tumor specimen 3

-

+

-

Non-tumor specimen 4

-

Non-tumor specimen 5

-

Note: "+" is positive for methylate DNA detects, "-" is negative for methylate DNA detects

As can be seen from the above results, when detecting simultaneously, SHOX2 methylation positive rate is the highest, CDH13, HOXA9, RAR β gene pairs improves system Positive rate and does not act synergistically, wherein RAR β has one to detect positive in non-tumor specimen, affect detected result on the contrary with its joint-detection, reduce Detection accuracy; RASSF1A is test positive in sample 11,16, and sample 11 and sample 16 are SHOX2 DNA methylation assay feminine gender, and therefore, the positive rate that RASSF1A gene pairs improves system detection has synergy.Also there is similar collaborative use (p16 detects the positive to sample 16, and SHOX2 detects negative) in p16, but its synergy is remarkable not as RASSF1A.Therefore select RASSF1A and SHOX2 gene methylation to carry out cooperation detection.

For this reason, contriver expands sensing range, have collected 50 routine tumor samples and 5 routine non-tumor samples (some experimental data is shown in embodiment 7), whether checking RASSF1A and SHOX2 cooperation detection contributes to the raising of Positive rate further, contributes to the accuracy improving diagnosis.Found that, methylating at 4 routine SHOX2 presents in negative tumor sample, and 3 routine RASSF1A present the positive.This more determines RASSF1A and SHOX2 cooperation detection and contributes to improving Positive rate.

According to above result, contriver determines to select people SHOX2 and people RASSF1A alternatively to detect gene.

Embodiment 2, primer and probe design

In order to optimize the detection reagent being applicable to detect people SHOX2 gene and people RASSF1A gene methylation DNA simultaneously, the present inventor conducts in-depth research for each gene order, after repeatedly studying comparison, the amplification region sequence of selected each gene, as shown in table 2.

Table 2

According to the determined region of table 2, the present inventor further optimization design Auele Specific Primer and detection probes.Designed primer and detection probes as shown in table 3.All primers and probe synthesize by Sangon Biotech (Shanghai) Co., Ltd..

Table 3

Embodiment 3, different primers probe combinations are tested

DNA extraction, sulphite modify, detection system preparation, pcr amplification program with embodiment 1, amplification object be modify after sample DNA and contrast DNA (DNA fragmentation of SHOX2, RASS1FA).After experiment end of run, analytical procedure is as follows:

(1) determine whether experiment is credible:

A () CY5 has signal, and CY5 signal Ct value >=12, then realize credible;

If Ct value < 12, point out the DNA added excessive, do again after answering dilution DNA;

If b () is without CY5 signal, points out the DNA added to contain PCR inhibitor or DNA process failure, need again to extract DNA and sulfiting;

Wherein, positive quality control product reaction tubes FAM, VIC and CY5 all should have signal, and negative quality control product should without FAM and VIC amplified signal, without CY5 signal.

(2) reference of non-selected correction fluorescence is confirmed, arrange and analyze FAM fluorescent signal, needing to select positive quality control product reaction tubes and example reaction pipe simultaneously, according to the flex point of actual amplification curve situation determination amplification curve, adjustment baseline position, obtains the Ct value of FAM fluorescent signal.

A the amplification curve of () FAM fluorescent signal is smooth ' S ' shape, and Ct value < 35, prompter RASSF1A gene methylation is positive;

B () Ct value >=35, prompter RASSF1A gene methylation feminine gender or methylation are lower than minimum detectability.

(3) reference of non-selected correction fluorescence is confirmed, arrange and analyze VIC fluorescent signal, needing to select positive quality control product reaction tubes and example reaction pipe simultaneously, according to the flex point of actual amplification curve situation determination amplification curve, adjustment baseline position, obtains the Ct value of VIC fluorescent signal.

A the amplification curve of () VIC fluorescent signal is smooth ' S ' shape, and Ct value < 32, prompter SHOX2 gene methylation is positive;

B () Ct value >=32, prompter SHOX2 gene methylation feminine gender or methylation are lower than minimum detectability.

Result decision method is as table 4.

Table 4

Each primed probe combination and detected result (CT value) are as table 5.

Table 5

Group	Primed probe combines	500 cell CT value/5000 cell CT values
			Group 1	RASS F1，RASS R1，RASS P1	23/19.8
Group 2	RASS F1，RASS R1，RASS P2	23.8/20.1
			Group 3	RASS F1，RASS R2，RASS P1	24.2/21.6
Group 4	RASS F1，RASS R2，RASS P2	23.5/20.8
			Group 5	RASS F2，RASS R1，RASS P1	24.1/21.5
Group 6	RASS F2，RASS R1，RASS P2	23.8/22.5
			Group 7	RASS F2，RASS R2，RASS P1	24.6/20.9
Group 8	RASS F2，RASS R2，RASS P2	24.3/21.5
			Group 9	RASS F3，RASS R1，RASS P1	23.5/20.2
Group 10	RASS F3，RASS R1，RASS P2	24/20.5
			Group 11	RASS F3，RASS R2，RASS P1	23.6/20
Group 12	RASS F3，RASS R2，RASS P2	24.8/21
			Group 13	SH F-n1，SH R-n1，SH P-n3.1	23.6/19.5
Group 14	SH F-n1，SH R-n1，SH P-n3.2	24.5/20.5
			Group 15	SH F-n1，SH R-n2，SH P-n3.1	24.5/21
Group 16	SH F-n1，SH R-n2，SH P-n3.2	24.1/20.5
			Group 17	SH F-n1，SH R-n3，SH P-n3.1	23.7/20.6
Group 18	SH F-n1，SH R-n3，SH P-n3.2	24.1/21.5

Group 19	SH F-n2，SH R-n1，SH P-n3.1	23.3/21.4
			Group 20	SH F-n2，SH R-n1，SH P-n3.2	23.3/22.5
Group 21	SH F-n2，SH R-n2，SH P-n3.1	22.8/21.4
			Group 22	SH F-n2，SH R-n2，SH P-n3.2	23.5/21.5
Group 23	SH F-n2，SH R-n3，SH P-n3.1	23.5/20.8
			Group 24	SH F-n2，SH R-n3，SH P-n3.2	24.1/21.2
Group 25	SH F-n3，SH R-n1，SH P-n3.1	23.8/22.1
			Group 26	SH F-n3，SH R-n1，SH P-n3.2	23.5/22.5
Group 27	SH F-n3，SH R-n2，SH P-n3.1	23.5/21.8
			Group 28	SH F-n3，SH R-n2，SH P-n3.2	22.8/22.2
Group 29	SH F-n3，SH R-n3，SH P-n3.1	23.1/21.5
			Group 30	SH F-n3，SH R-n3，SH P-n3.2	22.1/19.5
Group 31	SH F-n4，SH R-n1，SH P-n3.1	24.5/22.8
			Group 32	SH F-n4，SH R-n1，SH P-n3.2	23.6/22.2
Group 33	SH F-n4，SH R-n2，SH P-n3.1	24.8/23.1
			Group 34	SH F-n4，SH R-n2，SH P-n3.2	23.8/22.5
Group 35	SH F-n4，SH R-n3，SH P-n3.1	24.2/22.8
			Group 36	SH F-n4，SH R-n3，SH P-n3.2	24.6/23.5
Group 37	AC-F，AC-R1，AC-P	21.7/19.8
			Group 38	AC-F，AC-R2，AC-P	21.3/19.0

According to each group of amplification curve and CT value, preferred primer probe sequence is as table 6.

Table 6

Embodiment 4, detection system optimizing research

Object: the primed probe concentration in optimizing reaction system, hot start Taq polymerase add-on, dNTPs, Mg ²⁺ionic concn, and then SSR-PCR optimization, improve detection sensitivity and specificity.

1, SHOX primed probe ratio screening

Object: the ratio of SHOX primer and probe and amount are screened.

Primed probe ratio screening concentration is: SHOX (primer: probe) final concentration 0.1uM:0.03uM, 0.2uM:0.06uM, 0.3uM:0.1uM, 3 concentration are screened.

Wild type human genome after the SHOX2 reference product of 50 copy/μ L after modifying using sulphite, the process of 500 copy/μ L is as DNA profiling to be checked.

Conclusion: SHOX primed probe concentration ratio screening experiment result show, SHOX2 primer: when concentration and probe concentration is 0.2uM:0.06uM sensitivity and specificity best.

2, RASSF1A primed probe ratio screening

Object: the ratio of RASS primer and probe and amount are screened.

Primed probe ratio screening concentration is: RASS (primer: probe) final concentration 0.1uM:0.03uM, 0.2uM:0.06uM, 0.3uM:0.1uM, 3 concentration are screened.

Wild type human genome after the RASSF1A reference product of 50 copy/μ L after modifying using sulphite, the process of 500 copy/μ L is as DNA profiling to be checked.

Conclusion: RASSF1A primed probe concentration ratio screening experiment result show, RASSF1A primer: when concentration and probe concentration is 0.2uM:0.06uM sensitivity and specificity best.

3, β-actin primed probe ratio screening

Object: screening AC primed probe ratio.

β-actin weighs in PCR as internal reference 3, the effect that DNA extraction efficiency and sulphite are modified can be indicated, in reaction system, resource had better not be striven with index S HOX, RASS, primed probe amplification efficiency comparatively high 1 about the Ct of two indices of AC design own, so AC concentration in screening is on the low side, and screening to be combined with RASS, SHOX 2 pairs of primers.So by AC primer: concentration and probe concentration first prepares 0.2uM:0.06uM.Screening concentration is respectively the original content of 1/3 original content, 1/2 original content and preparation.

Conclusion: as can be seen from experimental result, the amplification efficiency reducing AC concentration declines 1 about Ct, does not affect result and judges; After reducing AC primed probe concentration, RASSF1A and SHOX2 amplification efficiency increases to some extent, so AC concentration reduces the amplification efficiency that can better improve test kit.Determine that AC primed probe ratio is: 0.67uM:0.02uM.

4, Mg ²⁺ionic concn is optimized

Object: to Mg in system ²⁺ionic concn is optimized, screening Mg ²⁺ionic concn is 2.5mM (carrying in 10 × buffer without the need to adding), 3.5mM, 4.5mM, 5.5mM, the Mg of 25mM ²⁺ion add-on is respectively 0 μ L, 0.8 μ L, 1.6 μ L, 2.4 μ L.

Wild type human genome after RASSF1A and the SHOX2 reference product of 50 copy/μ L after modifying using sulphite, the process of 500 copy/μ L is as DNA profiling to be checked.

Conclusion: Mg ²⁺but ionic concn increase can improve amplification efficiency also can be more and more serious non-specific, considers 3.5mM Mg ²⁺ionic concn amplification meets the requirements most.

5, dNTP concentration optimization

Object: be optimized dNTP concentration in system, the dNTP solution of screening dNTP concentration 0.15mM, 0.2mM, 0.25mM, 0.3mM 2.5mM concentration adds 1.2 μ L, 1.6 μ L, 2 μ L, 2.4 μ L respectively.

Conclusion: the amplification efficiency of detection system increases along with dNTPs concentration and improves, and reach capacity when dNTPs concentration reaches 0.25mM state.Determine that dNTPs concentration is 0.25mM.

6, Taq polymerase concentration is optimized

Experiment purpose: the amount adding Taq polysaccharase by adjustment adjusts the enzyme concn in PCR reaction system, and then SSR-PCR optimization.

The enzyme amount of screening is that the enzyme amount that 0.5U, 1U, 1.5U, 2U add in system is respectively: 0.1 μ L, 0.2 μ L, 0.3 μ L, 0.4 μ L.

Conclusion: as can be seen from experimental result, enzyme concn increases amplification efficiency also along with increase, and when the usage quantity of enzyme reaches 1.5U unit, amplification efficiency no longer improves, and the enzyme add-on determined is 1.5U.

Embodiment 5, specificity experiments

Collect clinical sample, get rid of lung cancer and be diagnosed as congenital dysplastic bronchoalveolar lavage fluid sample 8 example of the infectious diseases such as pneumonia, pulmonary abscess, pulmonary tuberculosis, bronchiectasis, pulmonary aspergillosis, hydatid cyst of lung and lung, centrifugal separating cell.

The modification of DNA extraction, sulphite, examination and analysb process are with embodiment 3, and the preferred version of embodiment 3 (table 6) selected by primer, probe.

Detected result as shown in Figure 1.Positive quality control PC and 8 non-tumour patient sample, all can detect CY5 signal (instruction β-actin gene), except people SHOX2 gene in positive control reaction tubes and the people RASSF1A gene methylation DNA detection positive, in all the other sample 1 ~ 8 reaction tubess, people VIC signal (assignor SHOX2 gene) and FAM signal (assignor RASSF1A gene) methylate DNA detect and are feminine gender, illustrate that test kit specificity is good, detection system non-false positive phenomenon of the present invention.

Embodiment 6, sensitivity for analysis are tested

The lung cancer cell line HCC827 cell counting of cultivating, gets 50,100,1000,10000 cells, adds 1ml respectively and take from the peripheral blood of Healthy People, as the simulated samples of sensitivity experiment.The modification of DNA extraction, sulphite, examination and analysb process, with embodiment 3, select preferred primer, the probe sequence of embodiment 3 (table 6).

Detected result is as shown in Fig. 2 (VIC signal), Fig. 3 (FAM signal), 50 HCC827 cell specimens, people SHOX2 gene and the people RASSF1A gene methylation DNA detection positive, show that reaction system sensitivity for analysis of the present invention all can reach 50 cells/reaction.

Embodiment 7, clinical samples detect

Collect bronchoalveolar lavage fluid sample totally 25 examples that certain Cancer Hospital clinical definite is lung cancer and non-lung cancer patient, centrifugal separating cell.The modification of DNA extraction, sulphite, examination and analysb process, with embodiment 3, select preferred primer, the probe sequence of embodiment 3 (table 6).

Detected result is as shown in table 7.

Table 7

FAM channel C t >=35, or VIC channel C t >=32, all indicate with "/".

Table 7 result shows, in 25 routine samples, 20 examples are lung cancer specimen, detects positive 19 examples of methylate DNA, undetected 1 example.In positive test symbol, positive 16 examples of people SHOX2 gene methylation DNA detection, positive 12 examples of people RASSF1A gene methylation DNA detection, it is positive that 3 routine SHOX2 methylate DNAs detect negative RA SSF1A gene methylation DNA detection, the Detection accuracy of single inspection SHOX2 gene methylation DNA is 80%, after people SHOX2 and people RASSF1A gene methylation DNA joint-detection, positive accuracy rate improves 15%, substantially increase Detection accuracy, again prove that RASSF1A is to the synergy of SHOX2 gene in lung cancer methylate DNA detects.In 5 routine non-lung cancer patient samples, do not detect the positive, illustrate that specificity is good.

The test kit of embodiment 8, detection of lung cancer

Test kit is provided, comprising:

PCR reaction solution: the amplimer of 0.2uM RASSF1A gene is to (SEQ ID NO:14, SEQID NO:18), the detection probes (SEQ ID NO:25) of 0.06uM RASSF1A, the amplimer of 0.2uM SHOX2 gene is to (SEQ ID NO:7, SEQ ID NO:10), the detection probes (SEQ ID NO:22) of 0.06uM SHOX2, and the amplimer of 0.67uM β-actin gene is to the detection probes (SEQ ID NO:26) of (SEQ ID NO:19, SEQ ID NO:21) and 0.02uM-actin gene; 3.5mMMgCl ₂, 0.25mM dNTP;

Polysaccharase: 200U Taqman polysaccharase;

Positive quality control product (500 cell/μ L, the DNA for extracting after nonsmall-cell lung cancer HCC827 clone counting);

Negative control: aqua sterilisa.

DNA modification liquid: 3M sodium bisulfite (pH5.0).

The all documents mentioned in the present invention are quoted as a reference all in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.

Claims

1. for a test kit for detection of lung cancer, it is characterized in that, described test kit comprises:

The detection reagent of the DNA methylation of specific detection people SHOX2 gene; With

The detection reagent of the DNA methylation of specific detection people RASSF1A gene.

2. test kit as claimed in claim 1, is characterized in that, the detection reagent of the DNA methylation of described specific detection people SHOX2 gene is primer for people SHOX2 gene and probe; Preferably, described primer and probe are for the sequence of SEQ ID NO:3 in people SHOX2 gene after hydrosulphite or heavy hydrosulphite or hydrazonium salt modification; Or

The detection reagent of the DNA methylation of described specific detection people RASSF1A gene is primer for people RASSF1A gene and probe; Preferably, described primer and probe are for the sequence of SEQ ID NO:1 in people RASSF1A gene after hydrosulphite or heavy hydrosulphite or hydrazonium salt modification.

3. test kit as claimed in claim 2, it is characterized in that, the detection reagent of the DNA methylation of described specific detection people SHOX2 gene is: the primer shown in SEQ ID NO:14 and SEQ ID NO:18 and the probe shown in SEQ ID NO:25; Or

The detection reagent of the DNA methylation of described specific detection people RASSF1A gene is: the primer shown in SEQ IDNO:7 and SEQ ID NO:10 and the probe shown in SEQ ID NO:22.

4. test kit as claimed in claim 1, is characterized in that, also comprise in described test kit:

The detection reagent of specific detection reference gene; Preferably, the detection reagent of described specific detection reference gene is primer for people β-actin gene and probe; More preferably, described primer and probe are for the sequence of SEQ ID NO:5 in people β-actin gene after hydrosulphite or heavy hydrosulphite or hydrazonium salt modification; More preferably, the detection reagent of the DNA methylation of described specific detection reference gene is: the primer shown in SEQ ID NO:19 and SEQ ID NO:21 and the probe shown in SEQ ID NO:26.

5. the test kit as described in as arbitrary in claim 2-4, it is characterized in that, the probe for each gene is connected with specific fluorophor; Preferably, for the probe mark VIC fluorescein of people SHOX2 gene, for the probe mark FAM fluorescein of people RASSF1A gene, for the probe mark CY5 fluorescein of people β-actin gene.

6. the test kit as described in as arbitrary in claim 2-4, is characterized in that, in this test kit, be: 0.2:0.06 for the primer of people SHOX2 gene and the ratio of probe; Or

In this test kit, for the primer of people RASSF1A gene and the ratio of probe be: 0.2:0.06; Or

In this test kit, for the primer of people β-actin gene and the ratio of probe be: 0.67:0.02.

7. the arbitrary described test kit of claim 2-4, is characterized in that, in this test kit, also comprise: archaeal dna polymerase, dNTPs, Mg ²⁺ion or comprise archaeal dna polymerase, dNTPs, Mg ²⁺the PCR system of ion;

Preferably, describedly archaeal dna polymerase, dNTPs, Mg is comprised ²⁺in the PCR system of ion, described Mg ²⁺ionic concn is 3.5 ± 0.5mM; Or described dNTPs concentration is 0.25 ± 0.3mM; Or described archaeal dna polymerase is 1.5 ± 0.2U;

Preferably, also comprise in described test kit: hydrosulphite or heavily hydrosulphite or hydrazonium salt.

8. the purposes of the reagent of the DNA methylation of specific detection people SHOX2 gene and people RASSF1A gene, for the preparation of the test kit of detection of lung cancer.

9. purposes as claimed in claim 8, is characterized in that, the detection reagent of the DNA methylation of described specific detection people SHOX2 gene is primer for people SHOX2 gene and probe; Preferably, described primer and probe are for the sequence of SEQ ID NO:3 in people SHOX2 gene after hydrosulphite or heavy hydrosulphite or hydrazonium salt modification; More preferably, the detection reagent of the DNA methylation of described specific detection people SHOX2 gene is: the primer shown in SEQ ID NO:14 and SEQ ID NO:18 and the probe shown in SEQID NO:25; Or

The detection reagent of the DNA methylation of described specific detection people RASSF1A gene is primer for people RASSF1A gene and probe; Preferably, described primer and probe are for the sequence of SEQ ID NO:1 in people RASSF1A gene after hydrosulphite or heavy hydrosulphite or hydrazonium salt modification; More preferably, the detection reagent of the DNA methylation of described specific detection people RASSF1A gene is: the primer shown in SEQ IDNO:7 and SEQ ID NO:10 and the probe shown in SEQ ID NO:22.

10. external nondiagnostic ground detects a method for the DNA methylation of people's SHOX2 gene and people RASSF1A gene, and it is characterized in that, described method comprises:

(2) utilize the arbitrary described modified testing sample of test kit to step (1) for detection of lung cancer of claim 1-7 to detect, obtain the DNA methylation situation of people SHOX2 gene and the DNA methylation situation of people RASSF1A gene.