CN109694912A

CN109694912A - The nucleic acid compositions and its kit and detection method of application, the detection methylation of methylation sites

Info

Publication number: CN109694912A
Application number: CN201910150962.8A
Authority: CN
Inventors: 阙艳鹏; 钱纯亘; 郑刚; 胡鹍辉
Original assignee: Shenzhen Yhlo Biotech Co Ltd
Current assignee: Shenzhen Yhlo Biotech Co Ltd
Priority date: 2019-02-28
Filing date: 2019-02-28
Publication date: 2019-04-30
Anticipated expiration: 2039-02-28
Also published as: CN109694912B

Abstract

The present invention relates to a kind of application of methylation sites, the nucleic acid compositions of detection methylation and its kit and detection methods.Application of the methylation sites as biomarker in preparation lung cancer detection reagent, lung cancer detection kit or lung cancer detection device, the methylation sites are chr7:19730212.The detection kit researched and developed using above-mentioned methylation sites as biomarker is lower to the false positive rate of lung cancer detection.

Description

The applications of methylation sites, the nucleic acid compositions of detection methylation and its kit and Detection method

Technical field

The present invention relates to molecular biology field, application, detection more particularly to a kind of methylation sites methylate Nucleic acid compositions and its kit and detection method.

Background technique

Lung cancer is that morbidity and mortality growth is most fast, to one of population health and the maximum malignant tumour of life threat. Many countries all report that the morbidity and mortality of lung cancer obviously increase in the past 50 years, and male lung cancer morbidity and mortality are equal First of all malignant tumours is accounted for, women disease incidence accounts for second, and the death rate accounts for second.The cause of disease of lung cancer is still endless so far Complete clear, great mass of data shows that long-term a large amount of smokings have very close relationship with lung cancer.In addition, atmosphere pollution and Carcinogen in flue dust may also lead to the generation of lung cancer.

Pulmonary cancer diagnosis mainly has three classes: x-ray inspection and fiberoptic bronchoscopy.X-ray inspection is by perspective or positive side Position X rabat detection, to find the shade of lung, but the early carcinoma for diameter less than 2cm shows and often has any problem.Carcinomebryonic antigen examination It tests since specificity, sensibility are limited, it is also little to the diagnostic value of early-stage cases.Although fiberoptic bronchoscopy accuracy Height, but fiberoptic bronchoscopy has certain pain, and patient is difficult to receive.

DNA methylation refers under the action of DNA methylation transferase, in the cytimidine 5 ' of genome CpG dinucleotides Carbon potential covalently bonded unifies a methyl group.DNA methylation is one of DNA modification, is the important component of epigenetics. DNA methylation can cause the change of chromatin Structure, DNA conformation, DNA stability and DNA and protein interaction mode, from And control gene expression.The change of methylation state of DNA is to cause a major reason of lung cancer.By detecting lung cancer dependency basis The methylation of cause can be used in lung cancer early screening and diagnosis.However, the examination of the methylation of traditional detection lung cancer related gene The false positive rate of agent box is higher, is unfavorable for the accurate detection of lung cancer.

Summary of the invention

Based on this, it is necessary to provide a kind of application of methylation sites as biomarker, be based on the methylation sites The false positive rate that the detection kit of exploitation detects lung cancer is lower.

In addition, there is a need to provide the nucleic acid compositions and its kit and detection method of a kind of detection methylation.

Methylation sites are as biomarker in preparation lung cancer detection reagent, lung cancer detection kit or lung cancer detection dress Application in setting, which is characterized in that the methylation sites are chr7:19730212.

By having carried out a large amount of exploration in terms of the biomarker of lung cancer, it has been unexpectedly found that being located at No. 7 dye The methylation catastrophe and lung cancer in the 19730212nd site of colour solid have high correlation, therefore, can be as biology mark Will object is applied in preparation lung cancer detection reagent, lung cancer detection kit or lung cancer detection device.Experiment proves that by above-mentioned first Base site is lower than 5% to the false positive rate of lung cancer detection as the detection kit that biomarker is researched and developed, and is conducive to lung cancer Accurate detection.

A kind of nucleic acid compositions of detection methylation, comprising:

Amplimer pair, the amplimer to for target gene No. 7 chromosome on the 19730022nd~ The design of 19730301 site areas；And

DNA methylation assay probe, the DNA methylation assay probe is for the 19730212nd site on No. 7 chromosome The target gene design to methylate.

The sequence of the forward primer of the amplimer pair such as SEQ ID No.1 in one of the embodiments,.

The sequence of the reverse primer of the amplimer pair is as shown in SEQ ID No.2 in one of the embodiments,.

The sequence of the DNA methylation assay probe is as shown in SEQ ID No.3 in one of the embodiments,.

It in one of the embodiments, further include non-DNA methylation assay probe, the non-DNA methylation assay probe is directed to institute State the target gene design that the 19730212nd site does not methylate on No. 7 chromosome.

The DNA methylation assay probe is respectively connected with glimmering with the non-DNA methylation assay probe in one of the embodiments, The fluorophor of light group, the fluorophor of the DNA methylation assay probe and the non-DNA methylation assay probe is not Together.

The sequence of the non-DNA methylation assay probe is as shown in SEQ ID No.4 in one of the embodiments,.

A kind of kit of detection methylation, the nucleic acid compositions including above-mentioned detection methylation.

A kind of detection method of methylation, includes the following steps:

Extract the target gene in sample to be tested；

Sulphite processing is carried out to the target gene；And

Using nucleic acid compositions, to sulphite, treated that the target gene carries out fluorescent quantitative PCR reaction, Tested and analyzed according to reaction result, wherein the nucleic acid compositions include amplimer to and DNA methylation assay probe, institute Amplimer is stated to design for the 19730022nd~19730301 site areas on No. 7 chromosome of target gene, it is described The target gene that DNA methylation assay probe methylates for the 19730212nd site on No. 7 chromosome is set Meter.

Detailed description of the invention

Fig. 1 is the amplification curve of positive quality control product；

Fig. 2 is the amplification curve of negative quality-control product；

Fig. 3 is the amplification curve of blank quality-control product.

Specific embodiment

In order to make the foregoing objectives, features and advantages of the present invention clearer and more comprehensible, combined with specific embodiments below and Specific embodiments of the present invention will be described in detail for attached drawing.Be explained in the following description many details in order to Fully understand the present invention.But the invention can be embodied in many other ways as described herein, art technology Personnel can do similar improvement without violating the connotation of the present invention, therefore the present invention is not by following public specific implementation Limitation.

The application of one embodiment, methylation sites are as biomarker in preparation lung cancer detection reagent, lung cancer detection Application in kit or lung cancer detection device, methylation sites chr7:19730212.That is on No. seven chromosome 19730212 sites.The methylation status of the target gene of sample to be tested is able to detect by the methylation sites.

This research has been surprisingly found that the methylation catastrophe of the chr7:19730212 in lung cancer patient and Healthy People, and there are bright The methylation frequency of mutation of aobvious difference, the chr7:19730212 of lung cancer patient significantly increases.Therefore, chr7:19730212 energy Enough biomarkers as the detection early stage of lung cancer, for lung cancer early diagnosis, predicted treatment, course of disease detection or monitoring recurrence Deng.

Refer to can be with tagging system, organ, tissue and eucaryotic cell structure or function for biomarker in one of the embodiments, The biochemical indicator of change or the change that may occur of energy.

In a specific example, sample to be tested contains CTC cell.Target gene is CtDNA, and methylation sites are located at On No. seven chromosome of CTC cell on the 19730212nd site.It should be noted that above-mentioned methylation sites are not limited to use in inspection The methylation status for surveying CTC cell, also can detecte the methylation status of other cells, such as can detecte in cancerous tissue slice Methylation status.

CTC (Circulating Tumor Cell, circulating tumor cell) is that all kinds of tumours that are present in peripheral blood are thin The general designation of born of the same parents.Studies have shown that CTC is present in peripheral blood with different shape, existing free single CTC also has aggregation agglomerating Cell mass (CTM Circulating Tumor Microemboli).Tumour cell is during entering Peripheral Circulation Epithelial-mesenchymal transformation (EMT Epithelial Mesenchymal Transition) can occur, therefore form the cell class of CTC Type includes that epithelial cell phenotype, interstitial cell phenotype, epithelial cell with interstitial cell mix phenotype.CtDNA(circulating Tumor DNA, Circulating tumor DNA) it is distinctive DNA sequence dna in CTC cell.The study found that the change of detection CtDNA can be passed through Change, observes the development of tumour, infantile tumour is diagnosed, tracking and monitoring is carried out to oncotherapy effect.

By having carried out a large amount of exploration in terms of the biomarker of lung cancer, it has been unexpectedly found that being located at No. 7 dye The methylation catastrophe and lung cancer in the 19730212nd site of colour solid have high correlation, therefore, can be as biology mark Will object is applied in preparation lung cancer detection reagent, lung cancer detection kit or lung cancer detection device.Experiment proves that by above-mentioned first Base site is lower than 5% to the false positive rate of the detection of lung cancer as the detection kit that biomarker is researched and developed, and is conducive to lung The accurate detection of cancer.

The lung cancer detection kit that above-mentioned methylation sites are researched and developed as biomarker only passes through detection chr7: 19730212 methylation status, can be obtained have to lung cancer detection it is compared with high specific and lower false positive rate as a result, behaviour Make simply and conveniently.

One embodiment detection methylation nucleic acid compositions, including amplimer to and DNA methylation assay probe, expand Increase primer pair for the 19730022nd~19730301 site areas design on No. 7 chromosome of target gene, methylation inspection The target gene design that probing needle methylates for the 19730212nd site on No. 7 chromosome.

In a specific example, target gene CtDNA.The 19730022nd on No. 7 chromosome of target gene~ The sequence of 19730301 site areas is as shown in SEQ ID No.5.Specifically, the sequence as shown in SEQ ID No.5 are as follows: 5 '- GTCATGGTGGACGGATCACAAGGTCAGGAGATCGAGACCATCCTGGCTAACACGGTGAAACCCCGTCTCTACTAAA AATACAAAAAATTAGCCGGGCGTGGTGGCAGGCGCCTATGATCCCAGCTATTCCGGAGGCTGAGGCAGGAGAATGG CGTGAACCTGGGAGGCGAGGCTTGCAGTGAGCCAAGATCGCGCCACTGCATTCCAGCCTGGGTGACGGAGCAAGAC TCCGTCTCAAAAAAGAAAAAAAAAAAAAAGAACACAAAGGGATAAAGCAATT-3’。

In a specific example, the sequence of the forward primer of amplimer pair is as shown in SEQ ID No.1.Specifically, The sequence as shown in SEQ ID No.1 are as follows: 5 '-GGAGAATGGCGTGAACCTG-3 '.

In a specific example, the sequence of the reverse primer of amplimer pair is as shown in SEQ ID No.2.Specifically, The sequence as shown in SEQ ID No.2 are as follows: 5 '-TGCTTTATCCCTTTGTGTTC-3 '.

In a specific example, the sequence of DNA methylation assay probe is as shown in SEQ ID No.3.Specifically, such as SEQ Sequence shown in ID No.3 are as follows: 5 '-TCGGTTGTAGCgCGGTGACGTAAGGT-3 '.

Nucleic acid compositions further include non-DNA methylation assay probe in one of the embodiments,.Non- DNA methylation assay probe The target gene design not methylated for the 19730212nd site on No. 7 chromosome.It is non-in a specific example The sequence of DNA methylation assay probe is as shown in SEQ ID No.4.The sequence as shown in SEQ ID No.4 is 5 '- TCGGTTGTAGCaCGGTGACGTAAGGT-3’。

Further, DNA methylation assay probe and non-DNA methylation assay probe are respectively connected with fluorophor, DNA methylation assay The fluorophor of probe is different from the fluorophor of non-DNA methylation assay probe.By double fluorescence signals, detection can be improved Specificity and accuracy reduce false positive rate.

Fluorophor is selected from one of FAM, VIC, FITC and HEX in one of the embodiments,.

DNA methylation assay probe and non-DNA methylation assay probe are respectively connected with quencher in one of the embodiments,. Further, quencher is selected from one of TAMRA, BHQ-1 and Dabcyl.

5 ' ends of DNA methylation assay probe are all connected with 5 ' ends of non-DNA methylation assay probe in one of the embodiments, There is fluorophor, 3 ' ends of DNA methylation assay probe are respectively connected with quencher with 3 ' ends of non-DNA methylation assay probe.

In a specific example, the fluorophor of 5 ' end connections of DNA methylation assay probe is FAM.Non- DNA methylation assay The fluorophor of 5 ' end connections of probe is VIC.3 ' ends of DNA methylation assay probe and 3 ' ends of non-DNA methylation assay probe are equal Being connected with quencher is TAMRA.

The nucleic acid compositions of above-mentioned detection methylation, including amplimer to and DNA methylation assay probe, amplimer pair It is designed for the 19730022nd~19730301 site areas on No. 7 chromosome of target gene, DNA methylation assay probe needle To the target gene design that the 19730212nd site on No. 7 chromosome methylates, so that using above-mentioned nucleic acid compositions False positive rate is lower when to lung cancer detection, and accuracy is higher.Experiment proves that the lung cancer detection containing above-mentioned nucleic acid compositions is tried Agent box is lower than 5% to the false positive rate of the detection of lung cancer, and specificity is not less than 95%, is conducive to the accurately and effectively inspection of lung cancer It surveys.

The kit of the detection methylation of one embodiment, the Nucleic acid combinations of the detection methylation including above embodiment Object.

Kit further includes positive quality control product, negative quality-control product, archaeal dna polymerase and PCR anti-in one of the embodiments, Answer at least one of liquid.

Positive quality control product is peripheral blood or the tumour III phase patient of tumour III phase patient in one of the embodiments, CTC cell in peripheral blood.Further, positive quality control product is peripheral blood or the lung cancer III phase patient of lung cancer III phase patient CTC cell in peripheral blood.

Negative quality-control product is the peripheral blood or swollen of normal person's physical examination serum, tumour I phase patient in one of the embodiments, CTC cell in the peripheral blood of tumor I phase patient.Further, negative quality-control product is the peripheral blood or lung cancer I of lung cancer I phase patient CTC cell in the peripheral blood of phase patient.

In a specific example, archaeal dna polymerase is Taq archaeal dna polymerase.

PCR reaction solution includes Tris buffer, dNTP and Mg in one of the embodiments,²⁺.Further, PCR reacts Liquid includes the Mg of Tris buffer, the dNTP of 0.1mmol/L~0.5mmol/L and 3.1mmol/L~3.9mmol/L²⁺.Wherein, DNTP is deoxyribonucleoside triphosphate, including dATP, dGTP, dTTP, dCTP.

Kit further includes that CtDNA extracts reagent in one of the embodiments,.Further, CtDNA extracts reagent packet Include cell pyrolysis liquid, adsorbent, eluant, eluent.

Wherein, cell pyrolysis liquid is used for CTC cell cracking, with released dna.Further, cell pyrolysis liquid is to be purchased from life The cell pyrolysis liquid of work bioengineering (Shanghai) limited liability company.

Adsorbent is used to adsorb the DNA of CTC cell release.Further, adsorbent is magnetic bead.It should be noted that inhaling Attached dose is not limited to magnetic bead, can also be other adsorbents, such as can be nano-pore adsorption microspheres.

Eluant, eluent from magnetic bead for eluting DNA.Further, eluant, eluent is organic solvent.Further, Eluant, eluent includes at least one of ethyl alcohol and chloroform.It should be noted that eluant, eluent is not limited to the above-mentioned reagent pointed out, may be used also Think other organic solvents, such as can be phenol.

It further includes impurity removal reagents and redissolution reagent that CtDNA, which extracts reagent to include, in one of the embodiments,.

Wherein, protein, grease, RNA, carbohydrate, the inorganic salts in DNA that impurity removal reagents are discharged for CTC cell etc. are miscellaneous Matter.Further, impurity removal reagents are selected from least one of dehydrated alcohol and chloroform.

Reagent is redissolved to be used to melt the DNA under elution again.Further, redissolving reagent is sterile TE solution or sterile double steamings Water.It should be noted that redissolve reagent be not limited to it is above-mentioned point out reagent, reagents can also be redissolved for other, such as can be nothing Bacterium dezymotizes water.

Kit further includes sulphite conversion reagent in one of the embodiments,.Sulphite conversion reagent is used for It is uracil by the 5 ' Cytosines not methylated in testing gene DNA, and the 5 ' cytimidines to methylate are not sent out It is raw to change, finally obtain Bis-DNA.By that can convert the cytimidine (C) in testing gene DNA to uracil (U), and 5- first The probability that base cytosine deamination is converted into uracil is extremely low, and therefore, the born of the same parents in DNA sample after bisulf iotate-treated are phonetic Pyridine can only come as 5-methylcytosine.After DNA methylation, since the presence of 5-methylcytosine has no effect on matching for base It is right, therefore can be lost compared to traditional polymerase chain reaction (polymerase chain reaction PCR) amplification procedure Methylation information is lost, DNA methylation information can effectively be retained by sulphite conversion.

The kit of above-mentioned detection methylation includes the nucleic acid compositions of above embodiment, can not only be to lung cancer mid-term And advanced stage is detected, and can also carry out screening and diagnosis to lung cancer early stage, detection false positive rate is lower, and accuracy is higher, special It is anisotropic stronger；Meanwhile the course of disease of patients with lung cancer can also be detected using mentioned reagent box, give patients with lung cancer anaphase Genotype reference is provided, can be used in the assessment of lung cancer therapy effect.

As the high speed development of completion and high throughput sequencing technologies is sequenced in human genome, gene screening becomes pulmonary cancer diagnosis Direction.First generation DNA sequencing technology is that the chain that 1975 Nian Yousang lattice (Sanger) and Cauer gloomy (Coulson) are started is whole The only chemical method (chain solution) that method and 1976-1977 are invented by Maxime (Maxam) and gilbert (Gilbert) Integrated application.And in 1977, first genome sequence is determined.The central principle of this method is: due to ddNTP 2 ' and 3 ' not hydroxyls, are separately added into certain proportion with labelled with radioisotope in the synthetic reaction system of DNA DdNTP, by the DNA sequence dna that can determine testing molecule after gel electrophoresis and autoradiograph according to the position of electrophoresis band. The shortcomings that sequencing cost of first generation sequencing technologies is high, and the aspects such as the time is long, and flux is low are sequenced in individual gene, has seriously affected it Really large-scale application.It needs first to be handled with a kind of polymerase and single-stranded collection hop protein in second generation sequencing technologies, before sequencing Magnetic bead is then placed on a kind of PTP plate by the magnetic bead with DNA.On this plate it is special there are many diameter be about 44 μm Aperture, each aperture be only capable of accommodating a magnetic bead, the position of each magnetic bead is fixed by this method, to be surveyed with pyrophosphoric acid The sequencing of sequence method.A kind of magnetic bead more smaller than hole diameter on PTP plate is put into aperture, sequencing reaction is started.If dNTP energy It is matched with sequence to be measured, then can discharge pyrophosphoric acid group in post synthesis, the pyrophosphoric acid group of release can be with the ATP in reaction system Sulfurylase reaction generates ATP.The ATP and luciferase of generation aoxidize that rout up the fluorescein molecule in sequencing reaction glimmering jointly The fluorescence of light, sending is recorded by the CCD camera of the PTP plate other side, is carried out optical signal prosessing finally by computer and is obtained Final sequencing result.Second generation sequencing technologies are difficult to be detected for gene methylation.

Therefore, one embodiment of this research also provides a kind of detection method of methylation, can be to the methyl of target gene Change the detection for carrying out the diagnosing and treating of non-disease, by the detection of the methylation status to target gene, with can be more preferable Ground development inhibits the drug of cytogene methylation.Specifically, above-mentioned detection method includes the following steps S110~S130:

S110, the target gene for extracting sample to be tested.

Sample to be tested is the blood product of experiment in one of the embodiments,.

Sample to be tested contains CTC cell, target gene CtDNA in one of the embodiments,.

In a specific example, the method for extracting the target gene of sample to be tested are as follows: using the examination of above embodiment CtDNA extracts the DNA that reagent extracts circulating tumor cell in agent box.It should be noted that being not limited to using above embodiment Kit in CtDNA extract reagent, can also with commercially available CtDNA extraction reagent.

It further include the circulating tumor cell obtained in sample to be tested in one of the embodiments, before S110.

The method for obtaining the circulating tumor cell in sample to be tested in one of the embodiments, is using micro-fluidic chip Circulating tumor cell is separated from sample to be tested.It should be noted that the method for obtaining the circulating tumor cell in sample to be tested It is not limited to the above-mentioned method pointed out, can also be other methods, such as can be sorted for fluidic cell thin to select circulating tumor Born of the same parents.

S120, sulphite processing is carried out to target gene.

The step of carrying out sulphite processing to target gene in one of the embodiments, includes: by 22 μ of μ L~28 L Target gene mixed with the aqueous solution of the NaOH of 22 μ L of μ L~28,2.9mol/L, in 40 DEG C~44 DEG C incubation 28min~ 32min；The quinhydrones that 13 μ of μ L~17 L are added continues to be incubated for；It is in faint yellow for being incubated for Incubating Solution, and it is mixed that solution of sodium bisulfite is added It is even so that total volume be 300 μ L, be added 150 μ L paraffin oil or vaseline, be protected from light water-bath 15h~17h in 48 DEG C~52 DEG C, obtain To sulphite treated DNA.Wherein, solution of sodium bisulfite is the Asia of pH4.9~5.1,3.4mol/L~3.8mol/L The aqueous solution of sodium bisulfate.

S130, fluorescent quantitative PCR reaction is carried out to sulphite treated target gene using nucleic acid compositions, Tested and analyzed according to reaction result, wherein nucleic acid compositions include amplimer to and DNA methylation assay probe, amplification draw Object is designed for the 19730022nd~19730301 site areas on No. 7 chromosome of target gene, and DNA methylation assay is visited The target gene design that needle methylates for the 19730212nd site on No. 7 chromosome.

Nucleic acid compositions further include non-DNA methylation assay probe in one of the embodiments,.Non- DNA methylation assay probe The target gene design not methylated for the 19730212nd site on No. 7 chromosome.

In a specific example, the sequence of non-DNA methylation assay probe is as shown in SEQ ID No.4.Such as SEQ ID Sequence shown in No.4 is 5 '-TCGGTTGTAGCaCGGTGACGTAAGGT-3 '.Further, the 5 ' of DNA methylation assay probe The fluorophor of end connection is FAM.The fluorophor of 5 ' end connections of non-DNA methylation assay probe is VIC.DNA methylation assay is visited It is TAMRA that 3 ' ends of needle, which are respectively connected with quencher with 3 ' ends of non-DNA methylation assay probe,.

Carrying out the reaction system of fluorescent quantitative PCR reaction in one of the embodiments, includes 0.1ng/L~1ng/ The sample to be tested of L, 0.1 μm of ol/L~2 μm ol/L amplimer to, the Taq DNA polymerase of 1.3U~1.7U, 0.1mmol/L The Mg of the dNTP and 3.1mmol/L~3.9mmol/L of~0.5mmol/L²⁺, 0.1 μm of ol/L~2 μm ol/L DNA methylation assay visit Needle.Further, reaction system further includes the non-DNA methylation assay probe of 0.1 μm of ol/L~2 μm ol/L.

The response procedures of fluorescent quantitative PCR reaction are carried out in one of the embodiments, are as follows: 94 DEG C~95 DEG C pre- changes Property 2min~5min, 1 circulation；94 DEG C~95 DEG C denaturation 15s~20s, 53 DEG C~58 DEG C annealing 15s~20s, 68 DEG C~72 DEG C Extend 15s~20s, totally 50~60 circulations, circulation terminates to collect first order fluorescence signal every time.

One embodiment wherein, the step of being tested and analyzed according to reaction result include: by determining methylation inspection The size of the Ct value of fluorescence channel where probing needle, to determine the methylation of target gene in sample to be tested.Specifically, first The Ct value of fluorescence channel is smaller where base detection probe, and the circulating tumor cell to methylate in sample to be tested is more.At one In specific example, the Ct value of FAM fluorescence channel is smaller, and the circulating tumor cell to methylate in sample to be tested is more.

Further, the step of being tested and analyzed according to reaction result further include: by determining that non-DNA methylation assay is visited The size of the Ct value of fluorescence channel where needle, to determine the non-methylation of target gene in sample to be tested.Specifically, non-first The Ct value of fluorescence channel is bigger where base detection probe, and the circulating tumor cell of non-methylation is more in sample to be tested.One In a specific example, the Ct value of VIC fluorescence channel is bigger, and the circulating tumor cell of non-methylation is more in sample to be tested.

In one of the embodiments, the S140 the step of before, further include the steps that preparing positive quality control product: taking lung cancer The tumor tissue section of III phase patient carries out originally culture, and it is 10 that digestion, which is diluted to cell density, after its proliferation⁵A/mL~ 10⁸The cell suspension of a/mL.

In one of the embodiments, the S140 the step of before, further include the steps that preparing negative quality-control product: taking lung cancer The tumor tissue section of I phase patient carries out originally culture, and it is 10 that digestion, which is diluted to cell density, after its proliferation⁵A/mL~10⁸ The cell suspension of a/mL.

The platform of fluorescence quantitative PCR detection includes conventional fluorogenic quantitative detection platform, example in one of the embodiments, As the fluorogenic quantitative detection platform of Roche company, the fluorogenic quantitative detection platform of Bio-Rad company, AgiIent company fluorescence Quantitative detection platform, ABI company fluorogenic quantitative detection platform.

The detection method of above-mentioned methylation, is capable of the methylation of accurate testing goal gene, detection false positive rate compared with Low, accuracy is higher, and specificity is stronger, can be used in the treatment that non-disease is carried out to the methylation status of gene and diagnostic Detection, can be applied to develop in the drug for inhibiting target gene methylation.

Further, the sample to be tested of above embodiment is blood sample, 10mL whole blood in general initial stage cancer patient There was only about 10 CTC cells in the inside.In the case where CTC cell concentration is very little, it is difficult to control the CTC cell quantity of sample to be tested. Above embodiment passes through the copy number that double fluorescence channels detect DNA, to replace the quantity of detection cell.What DNA detected Quantity is more, and Ct value is smaller, and the CTC cell represented in sample to be tested is more, easy to operate, as a result accurately.

The following are specific embodiment parts:

Embodiment 1

Extract the DNA of CTC cell in sample to be tested

(1) CTC cell is filtered out from sample to be tested by Hylo-Dva280 model micro-fluidic chip, sample to be tested is Whole blood.

(2) the limited public affairs of raw work bioengineering (Shanghai) share (are purchased from using the paramagnetic particle method DNA extraction kit of commercialization Department) and the DNA in CTC cell is extracted according to the operation instruction of the kit, concrete operations are as follows:

(a) the CTC cell of collection is fitted into sample cell, is diluted to 50 μ L with the buffer solution with salt equilibrium function, The cell pyrolysis liquid of 430 μ L is added, concussion is resuspended, and 65 DEG C of constant temperature water baths place 5min.After being cooled to room temperature, it is added 20 μ L's RNaseA places 2min after mixing by vortex oscillator.The magnetic bead dilution of 400 μ L and the magnetic of 10 μ L are added into sample cell Pearl oyster liquid is shaken after mixing 20s by vortex oscillator, stands 1min.

(b) sample cell is placed in 30s on magnetic separtor, after magnetic bead component is adsorbed to tube wall completely, inhales and abandon supernatant, And sample cell is taken out from magnetic separator, 400 μ L are added into sample cell for cleaning the buffer solution of magnetic bead, are shaken by vortex After swinging device mixing 10s, sample cell is reapposed in 30s on magnetic separator, after magnetic bead component is drawn to tube wall completely, is discarded Clear component, and sample cell is taken out from magnetic separator.

(c) 700 μ L are added into sample cell, the ethanol water that mass percentage is 70%, are gently blown with pipettor It beats after mixing.Sample cell is placed in 30s on magnetic frame, after being adsorbed to tube wall completely to magnetic bead component, is taken from magnetic frame Sample cell out is inhaled and abandons supernatant.And the ethanol water cleaning step that mass percentage is 70% is repeated once.By sample Pipe is uncapped, and dry 10min is placed in 25 DEG C or so of bellows, until without any residual liquid in sample cell.

(d) the TE solution of 100 μ L, 65 DEG C of heating water bath 7min are added into sample cell.In heating process, mix 4 times.It takes It sample cell and is placed on magnetic frame out, observes the careful Aspirate supernatant after all magnetic bead components are all adsorbed on tube wall To new centrifuge tube, the DNA of CTC cell in sample to be tested is obtained, DNA as to be processed.

Embodiment 2

The DNA methylation processing of CTC cell in sample to be tested

(1) compound concentration is the aqueous solution of sodium bisulfite of 3.6mol/L, and is titrated to the NaOH aqueous solution of 3mol/L PH5.0。

(2) it takes the DNA to be processed of 25 μ L, is added the NaOH aqueous solution of the 3mol/L of 2.5 μ L, after 42 DEG C of water-bath 30min, add Enter the quinhydrones of 15 μ L, water-bath is kept, to solution at faint yellow.Above-mentioned aqueous solution of sodium bisulfite is added, makes overall solution volume 300μL.EP pipe is protected from light, and 150 μ L vaseline are added after being slowly mixed by inversion.It is wrapped up with tinfoil to be protected from light, 50 DEG C of water-bath 16h are obtained To methylation treated DNA, i.e., DNA to be measured.

Embodiment 3

Detect the kit of methylation

The kit includes nucleic acid compositions, positive quality control product, negative quality-control product, blank quality-control product, archaeal dna polymerase, PCR Reaction solution；

Nucleic acid compositions are as shown in table 1, and nucleic acid compositions include amplimer to, DNA methylation assay probe and non-methylation Detection probe, amplimer are set to for the 19730022nd~19730301 site areas on No. 7 chromosome of target gene Meter, the target gene design that DNA methylation assay probe methylates for the 19730212nd site on No. 7 chromosome, non-first The target gene design that base detection probe does not methylate for the 19730212nd site on No. 7 chromosome, purpose base Because of CtDNA, in target gene on No. 7 chromosome the 19730022nd~19730301 site areas sequence such as SEQ ID Shown in No.5, specifically, the sequence as shown in SEQ ID No.5 are as follows: 5 '-GTCATGGTGGACGGATCACAAGGTCAGGAGA TCGAGACCATCCTGGCTAACACGGTGAAACCCCGTCTCTACTAAAAATACAAAAAATTAGCCGGGCGTGGTGGCAG GCGCCTATGATCCCAGCTATTCCGGAGGCTGAGGCAGGAGAATGGCGTGAACCTGGGAGGCGAGGCTTGCAGTGAG CCAAGATCGCGCCACTGCATTCCAGCCTGGGTGACGGAGCAAGACTCCGTCTCAAAAAAGAAAAAAAAAAAAAAGA ACACAAAGGGATAAAGCAATT-3'；

The preparation process of positive quality control product are as follows: the tumor tissues vivisection for taking lung cancer III phase patient, in DMEM culture medium In 37 DEG C, 5%CO in (being purchased from Hyclone company)₂Concentrations of cells incubator (is purchased from Thermo Fisher Scientific) Originally culture is carried out, 48h is so that after cell Proliferation for culture, the pancreas egg for being 2.5% with the mass percentage that ultrapure water is diluted to White enzyme solutions digestion is diluted to the cell suspension that cell density is 10^5/mL, 1mL cell suspension is taken, according to the behaviour of embodiment 1 It is handled, obtained DNA to be processed.The calibration object dilution for finally containing heat-inactivated cow's serum with 1mL (is purchased from Life science company), DNA to be processed is diluted, positive quality control product is obtained；

The preparation process of negative quality-control product are as follows: the tumour vivisection for taking lung cancer I phase patient (is purchased from DMEM culture medium Hyclone company) in 37 DEG C, 5%CO₂Concentrations of cells incubator (being purchased from Thermo Fisher Scientific) carries out former It is commissioned to train feeding, for culture 48h so that after cell Proliferation, the trypsase for being 2.5% with the mass percentage that ultrapure water is diluted to is molten Liquid digestion is diluted to the cell suspension that cell density is 10^5/mL, takes 1mL cell suspension, carries out according to the operation of embodiment 1 Processing, obtained DNA to be processed, the calibration object dilution for finally containing heat-inactivated cow's serum with 1mL (are purchased from life Science company), DNA to be processed is diluted, negative quality-control product is obtained；

Blank quality-control product are as follows: the calibration object dilution (being purchased from life science company) containing newborn bovine serum；

Archaeal dna polymerase is Taq archaeal dna polymerase.

PCR reaction solution includes Tris buffer, dNTP and Mg²⁺。

1 nucleic acid compositions of table

Embodiment 4

Fluorescent quantitation is carried out to positive quality control product, negative quality-control product, blank quality-control product respectively using the kit of embodiment 3 PCR detection, obtains the Ct value of each sample to be tested.Measurement result is detailed in Fig. 1~3, and in Fig. 1~3, " Cycle Number " expression is followed Number of rings, " Δ Rn " indicate that fluorescent value, " FAM " indicate the channel FAM, and " VIC " indicates the channel VIC.Fig. 1 is the amplification of positive quality control product Curve, Ct value are；Fig. 2 is the amplification curve of negative quality-control product, and Ct value is 42；Fig. 3 is the amplification curve of blank quality-control product.

Wherein, reaction system are as follows: the sample to be tested of 0.5ng/L, the amplimer of 1 μm of ol/L are poly- to, the Taq DNA of 1.5U The Mg of synthase, the dNTP of 0.3mmol/L and 3.5mmol/L²⁺, the DNA methylation assay probe of 1 μm of ol/L, 1 μm of ol/L non-methyl Change detection probe；Sample to be tested is positive quality control product, negative quality-control product or blank quality-control product；

Response procedures are as follows: 94 DEG C of initial denaturation 5min, 1 circulation；94 DEG C of denaturation 17s, 55 DEG C of annealing 15s, 70 DEG C extend 20s, first order fluorescence signal is collected in totally 60 circulations, every time circulation end；

Sense channel is the channel FAM and the channel VIC.

From Fig. 1~3 as can be seen that amplification curve of the blank quality-control product in the channel FAM and the channel VIC is straight line, and it is empty Ct value of the white quality-control product in two channels is UNDET.Negative quality-control product has amplification curve, and negative Quality Control in the channel VIC The Ct value of product is 40~60.Positive quality control product has amplification curve in the channel FAM, and the Ct value of positive quality control product is 20~30.

Embodiment 5

(1) sample to be tested for taking 15mL carries out CTC cell screening by Hylo-Dva280 model micro-fluidic chip.It is to be measured Sample is respectively 20 lung cancer I phase patient blood samples, 20 lung cancer II phase patient blood samples, 20 lung cancer III phase patients Blood sample, 20 lung cancer IV phase patient blood samples (having airway wall and pathological examination), 20 healthy human blood's samples, 20 Example pure water sample.

(2) referring to the operation of embodiment 1, raw work bioengineering (is purchased from using the paramagnetic particle method DNA extraction kit of commercialization (Shanghai) limited liability company) and the DNA in CTC cell is extracted according to the operation instruction of the kit, concrete operations are as follows:

(a) the CTC cell of collection is added in sample cell, 430 μ L cell pyrolysis liquids, concussion weight is added into sample cell It is outstanding, sample tube cover is closed, 65 DEG C of constant temperature place 5min.It is cooled to room temperature, the RNaseA of 20 μ L is added, passes through vortex oscillator 2min is placed after mixing.The magnetic bead dilution of 400 μ L and the magnetic bead mother liquor of 10 μ L are added into sample cell, passes through vortex oscillator After concussion mixes 20s, 1min is stood.

(b) it by sample cell as 30s on magnetic separtor, after magnetic bead component is drawn to tube wall completely, inhales and abandons supernatant, and Sample cell is taken out from magnetic separator, and the buffer solution for being used to clean magnetic bead of 400 μ L is added into sample cell, is shaken by vortex After swinging device mixing 10s, sample cell is reapposed in 30s on magnetic separator, after magnetic bead component is drawn to tube wall completely, is discarded Clear component, and sample cell is taken out from magnetic separator.

(c) 700 μ L are added into sample cell, the ethyl alcohol water mixed solution that mass percentage is 70%, it is light with pipettor After mixing is played in featheriness.Sample cell is placed in 30s on magnetic frame, after being adsorbed to tube wall completely to magnetic bead component, from magnetic frame Upper taking-up sample cell is inhaled and abandons supernatant.And organic solution cleaning step is repeated once.Sample cell is uncapped, 25 DEG C of left sides are placed on In right bellows, dry 10min, until without any residual liquid in sample cell.

(d) the TE solution of 75 μ L, 65 DEG C of heating water bath 7min are added into sample cell.In heating process, mix 4 times.It takes It sample cell and is placed on magnetic frame, is observed after all magnetic bead components are all adsorbed on tube wall out, Aspirate supernatant is to new Centrifuge tube, obtain the DNA of CTC cell in sample to be tested, DNA as to be processed.

(3) methylation processing is carried out to DNA to be processed according to the operation of embodiment 2, obtains DNA to be measured.

(4) fluorescence quantitative PCR detection, reaction system and response procedures are carried out to DNA to be measured using the kit of embodiment 1 Referring to embodiment 4, each sample to be tested is counted in the Ct value in the channel FAM and the channel VIC, see Table 2 for details for measurement result~and 3.

Ct value of each sample to be tested of table 2 in the channel FAM

Ct value	0~15	16~30	30~45	45~60	UNDET	It amounts to
							IV phase sample (example)	16	3	1	0	0	20
III phase sample (example)	8	7	3	2	0	20
							II phase sample (example)	5	6	8	1	0	20
I phase sample (example)	1	0	6	11	2	20
							Normal person's sample (example)	0	0	0	1	19	20
Pure water sample (example)	0	0	0	0	20	20

Ct value of each sample to be tested of table 3 in the channel VIC

Ct value	0~15	16~30	30~45	45~60	UNDET	It amounts to
							IV phase sample (example)	2	1	4	8	5	20
III phase sample (example)	2	3	9	4	2	20
							II phase sample (example)	1	1	13	2	3	20
I phase sample (example)	8	7	3	2	1	20
							Normal person's sample (example)	0	0	0	3	17	20
Pure water sample (example)	0	0	0	0	20	20

From table 2~3 as can be seen that I~IV phase sample case quantity that Ct value is 0~15 in the channel FAM successively increases, Illustrate that the methylation of CTC cell in lung cancer mid-term and advanced stage sample is higher.And IV phase sample Ct value in the channel VIC is 45 ~60 case quantity is greater than I phase sample, it may be possible to since the CTC cell in IV phase sample is more than I phase sample.

Embodiment 6

(1) according to the CTC cell in operation 50 samples to be tested of screening of (1) the step of embodiment 5,50 samples to be tested The patient blood sample and 20 healthy human blood's samples for being diagnosed as lung cancer III phase and IV phase including 30.

(2) referring to the operation of embodiment 1, extracted using the paramagnetic particle method blood DNA extracts kit of Shanghai Sangon Biotech Company etc. The DNA in CTC cell is measured, DNA to be processed is obtained.

(4) fluorescence quantitative PCR detection, reaction system and response procedures are carried out to DNA to be measured using the kit of embodiment 1 Referring to embodiment 4.See Table 4 for details for measurement result.Calculate susceptibility, specificity and false positive:

Sensitivity=testing result is the lung cancer sample number of positive lung cancer sample number/total；

Specificity=testing result is the normal sample number of negative lung cancer sample number/total；

False positive rate=testing result is the normal sample number of positive lung cancer sample number/total.

The testing result of 4 50 samples to be tested of table

	Patient's sample	Healthy People sample
			Positive (a)	29	1
Negative (a)	1	19
			Sensitivity (%)	96.7%	/
Specific (%)	/	95%
			False sun forthright (%)	/	5%

From table 4, it can be seen that the methylation status of the kit of above embodiment CTC cell in detection sample to be tested When, sensitivity is up to 96.7%, and specificity is up to 95%, and false positive rate is down to 5%.

To sum up, the lung cancer detection kit containing above-mentioned nucleic acid compositions of above embodiment is able to detect sample to be tested CTC cell methylation status, and the performance indicators such as detection sensitivity, specificity are good.Meanwhile the kit only needs Whole blood sample can be detected, and does not need body fluid biopsy or histotomy detection, can be used in the early screening and diagnosis to lung cancer, Treatment in patients with lung cancer effect can be disclosed, provides genotype reference for treatment in patients with lung cancer.

Each technical characteristic of embodiment described above can be combined arbitrarily, for simplicity of description, not to above-mentioned reality It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited In contradiction, all should be considered as described in this specification.

The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to protection of the invention Range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.

Sequence table

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ccccgtctct actaaaaata caaaaaatta gccgggcgtg gtggcaggcg cctatgatcc 120

cagctattcc ggaggctgag gcaggagaat ggcgtgaacc tgggaggcga ggcttgcagt 180

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aagaaaaaaa aaaaaaagaa cacaaaggga taaagcaatt 280

Claims

1. methylation sites are as biomarker in preparation lung cancer detection reagent, lung cancer detection kit or lung cancer detection device In application, which is characterized in that the methylation sites be chr7:19730212.

2. a kind of nucleic acid compositions of detection methylation characterized by comprising

Amplimer pair, the amplimer is to for the on No. 7 chromosome of target gene the 19730022nd~19730301 Site areas design；And

DNA methylation assay probe, the DNA methylation assay probe occur for the 19730212nd site on No. 7 chromosome The target gene design of methylation.

3. the nucleic acid compositions of detection methylation according to claim 2, which is characterized in that the amplimer pair is just To the sequence such as SEQ ID No.1 of primer.

4. it is according to claim 2 detection methylation nucleic acid compositions, which is characterized in that the amplimer pair it is anti- To primer sequence as shown in SEQ ID No.2.

5. the nucleic acid compositions of detection methylation according to claim 2, which is characterized in that the DNA methylation assay probe Sequence as shown in SEQ ID No.3.

6. according to the nucleic acid compositions of the described in any item detection methylations of claim 2~5, which is characterized in that further include non- DNA methylation assay probe, the non-DNA methylation assay probe do not occur for the 19730212nd site on No. 7 chromosome The target gene design of methylation.

7. the nucleic acid compositions of detection methylation according to claim 6, which is characterized in that the DNA methylation assay probe Be respectively connected with fluorophor with the non-DNA methylation assay probe, the fluorophor of the DNA methylation assay probe with it is described The fluorophor of non-DNA methylation assay probe is different.

8. the nucleic acid compositions of detection methylation according to claim 6, which is characterized in that the non-DNA methylation assay is visited The sequence of needle is as shown in SEQ ID No.4.

9. a kind of kit of detection methylation, which is characterized in that including the described in any item detection methyl of claim 2~8 The nucleic acid compositions of change.

10. a kind of detection method of methylation, which comprises the steps of:

Extract the target gene in sample to be tested；

Sulphite processing is carried out to the target gene；And

Using nucleic acid compositions, to sulphite, treated that the target gene carries out fluorescent quantitative PCR reaction, according to Reaction result is tested and analyzed, wherein the nucleic acid compositions include amplimer to and DNA methylation assay probe, the expansion Increase primer pair for the design of the 19730022nd~19730301 site areas, the methyl on No. 7 chromosome of target gene Change the target gene design that detection probe methylates for the 19730212nd site on No. 7 chromosome.