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.
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: 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.
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: 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.