CN107955836A

CN107955836A - For the primer pair of lung cancer related gene SHOX2 DNA methylation assays, kit and method

Info

Publication number: CN107955836A
Application number: CN201711479687.1A
Authority: CN
Inventors: 韩林志; 肖芳
Original assignee: Individual
Current assignee: Individual
Priority date: 2017-12-29
Filing date: 2017-12-29
Publication date: 2018-04-24

Abstract

The present invention relates to a kind of primer pair for lung cancer related gene SHOX2 DNA methylation assays.The primer pair includes SHOX2 forward primers, reverse primer and detection probe, β Actin forward primers, reverse primer and detection probe.The present invention relates to a kind of kit for lung cancer related gene SHOX2 DNA methylation assays.The kit includes the PCR reaction solution containing above-mentioned primer and probe, it includes forward primer, reverse primer, detection probe, 10 × PCR buffer, dNTP, nuclease-free water and Ex Taq enzymes.The invention further relates to a kind of method for lung cancer related gene SHOX2 DNA methylation assays.Kit provided by the invention and its detection method have the advantages that testing result is accurate, detection flux is high, specificity is good, sensitivity is good, detection time is quick, easy to use and can effectively meet clinical requirement.

Description

For the primer pair of lung cancer related gene SHOX2 DNA methylation assays, kit and method

Technical field

The present invention relates to vitro diagnostic techniques field, particularly, is related to one kind and methylates for lung cancer related gene SHOX2 Primer pair, kit and the method for detection.

Background technology

Lung cancer is the cancer of Chinese incidence first place, although histology biopsy and cytology detection can make a definite diagnosis lung cancer, The state of an illness has been in relatively late more when it is detected, and recall rate is unsatisfactory.The short and small hox genes SHOX2 of people methylate and lung Cancer is closely related, and SHOX2 gene methylations are detected as solving the problems, such as that this provides a more effective option, multinomial clinic Experiment proves that it is used in combination with histology or cytology detection and improves diagnosis rate, is the effective tool that auxiliary makes a definite diagnosis lung cancer.

SHOX2 genes are the tumor suppressor genes closely related with lung cancer, and it is described that its promoter site CpG islands, which methylate, One of main mechanism of gene inactivation.The present invention is methylated by detecting SHOX2 gene promoter sub-portion CpG islands respectively, can be with The actual conditions that the lung cancer of more perfect assessment patient becomes and lapses on a molecular scale, effective guiding treatment.So as to avoid The mistaken diagnosis of lung cancer therapy now and crossing is treated, and is failed to pinpoint a disease in diagnosis.

DNA methylation refers to that under the action of dnmt rna the methyl (- CH3) of S-adenosylmethionine is covalently tied Close on 5 carbon atoms of cytimidine (C) base of DNA molecular, form 5-methylcytosine (5mC), and do not change DNA's Sequence.Numerous researchs show that the occurrence and development of SHOX2 gene promoter methylations and lung cancer are closely related, it is lung cancer spy Different molecular labeling.Therefore, the methylation state of lung cancer SHOX29 gene promoters, diagnosis, treatment, prognosis to lung cancer are detected Judge etc. is of great significance.

At present, tissue biopsy has larger wound for patients, as tumour has heterogeneity, produces false Positive findings.Sputum sample relative organization sample easily obtains, and noninvasive to patient.In addition, traditional methylation detecting method bag Include：Methylation status of PTEN promoter and bisulfite sequencing.But these methods are low and sensitive there are cumbersome, accuracy The shortcomings that property is not high, limits its extensive use in clinical labororatory.

The present invention uses fluorescent real time PCR technology, and patient's plasma free nucleic acid SHOX2 gene methylations are detected, After carrying out bisulfite processing to sample, nucleic acid is expanded by specific primer and probe, to reach quick The purpose of SHOX2 gene methylations is detected, it is final to provide effective information indirectly for human lung cancer early diagnosis, it is that human lung cancer is early found Early treatment provides a kind of easy-to-use method.

The content of the invention

In order to solve above-mentioned traditional methylation detecting method, there are the technology that cumbersome, accuracy are low and sensitiveness is not high Problem, the present invention provide that a kind of easy to operate, accuracy is high and sensitiveness is high and based on Fluorescence PCR assay to be used for lung cancer related Primer pair, kit and its method for gene SHOX2 DNA methylation assays.

The present invention provides a kind of primer pair for lung cancer related gene SHOX2 DNA methylation assays, including for SHOX2 The promoter region of gene and the specific primer and probe of β-Actin reference genes, it is as follows：

SHOX2 forward primers：5'-TTTTGGATAGTTAGGTAATTTTYGTT-3'(SEQ ID NO.1),

SHOX2 reverse primers：5'-ACGAACCCTTTAAACAACCAACATAA-3'(SEQ ID NO.2),

The first detection probes of SHOX2：5'FAM-AAACGCCTATACTCGTACG-3'MGB(SEQ ID NO.3)；

The second detection probes of SHOX2：5'VIC-CGATCGAACAAACG-3'MGB(SEQ ID NO.4)；

β-Actin forward primers：5'-GGTTAGGAAGGAGGTTGTTTGTTTT-3'(SEQ ID NO.5),

β-Actin reverse primers：5'-CCAAACTATAACCTCTACAACCTTCAAAA-3'(SEQ ID NO.6),

β-Actin detection probes：5'ROX-CCCATTAACTAAACACAACCT-3'MGB(SEQ ID NO.7).

Y is to annex base, i.e. Y=C/T.

Present invention also offers a kind of kit for lung cancer related gene SHOX2 DNA methylation assays, including containing such as The PCR reaction solution of above-mentioned primer and probe, the PCR reaction solution include：SHOX2 forward primers, SHOX2 reverse primers, SHOX2 First detection probe, the second detection probes of SHOX2, β-Actin forward primers, β-Actin reverse primers, β-Actin detections are visited Pin, 10 × PCR buffer, dNTP and nuclease-free water.

In a kind of preferred embodiment of the kit provided by the invention, the kit further includes Ex Taq enzymes.

In a kind of preferred embodiment of the kit provided by the invention, the component final concentration of the PCR reaction solution For：1 × PCR buffer, 0.4 μM of SHOX2 forward primer, 0.4 μM of SHOX2 reverse primer, 0.4 μM of SHOX2 first are detected Probe, 0.4 μM of second detection probe of SHOX2,0.4 μM of β-Actin forward primer, 0.4 μM of β-Actin reverse primer, 0.4 μM β-Actin detection probes, 0.25mM dNTP.

In a kind of preferred embodiment of the kit provided by the invention, the kit further includes positive reference substance And negative controls, the positive reference substance methylate standard items DNA for SHOX2, the negative controls are the non-mark that methylates Quasi- product DNA.

Wherein described SHOX2 methylate standard items DNA for normal human peripheral blood's genomic DNA through I methylases of Sss at Reason, can make the C in all CG sequences of genome methylate on C5 positions；The non-standard items DNA that methylates is outside normal person All blood genomic DNAs.

Present invention also offers a kind of method for lung cancer related gene SHOX2 DNA methylation assays, include the following steps：

Step 1：The DNA of sample extracting to be detected is taken, conversion processing is carried out to it, moulds of the DNA after conversion as PCR Plate；

Step 2：The above-mentioned kit for lung cancer related gene SHOX2 DNA methylation assays is provided, the template is carried out PCR amplification；

Step 3：Test sample to be checked is determined according to the relative fluorescence CT values of SHOX2 and β-Actin gene PCR amplifications This methyl rate, i.e., SHOX2/ β-Actin in the ratio divided by positive reference substance of SHOX2/ β-Actin in sample to be detected Ratio.

In a kind of preferred embodiment of the method provided by the invention, in the step 1 used by conversion processing Reagent is bisulfites or bisulfite.

In a kind of preferred embodiment of the method provided by the invention, pcr amplification reaction program in the step 2 For：95 DEG C of denaturation 5min；45cycles, 95 DEG C of 15sec, 60 DEG C of 30sec.

Compared to the prior art, primer pair, examination provided by the present invention for lung cancer related gene SHOX2 DNA methylation assays The beneficial effect of agent box and method is：

First, the primer and probe high by designing specificity, and it is configured to the reliable reagent of easy to use and testing result Box, the rational PCR reaction systems of the science of redesigning out so that the present invention has quick, high throughput, sensitive and specific good spy Point, realizes and the methylation of lung cancer SHOX2 genes quickly and is accurately measured, timely, effective to be carried out indirectly to lung cancer Diagnose and treat, reduce medical treatment cost, save social resources.

2nd, the quality of sample is controlled using gene β-actin as reference gene, it is contemplated that house-keeping gene may Situation about methylating, the position on no CpG islands is selected when designing internal control primer probe, is set for the sequence after its vulcanization Meter, ensures Quality Control of the house-keeping gene to sample.

3rd, after by carrying out bisulfite processing to sample, specific primer and probe expand nucleic acid, measure With the maximally related CpG sites of lung cancer tumor in the promoter region of SHOX2 genes, methylating for target gene is judged, and according to same Standard curve prepared by the positive sample and negative sample that the clinic of step processing determines, the methylation of preliminary judgement sample, Patients with lung cancer is evaluated in different times SHOX2 gene methylation degree；And the reference gene β-actin in sample are measured at the same time, Not only quality control can be carried out to sample, but also the methylation level of sample can be evaluated to a certain extent.The method is reachable It is final to provide effective information indirectly for human lung cancer early diagnosis to the purpose of quick detection SHOX2 gene methylations, it is human lung cancer It is early to find that early treatment provides a kind of easy-to-use method.

Brief description of the drawings

Fig. 1 is the amplification curve diagram of SHOX2 methylation positive concentration of specimens gradient dilutions；

Fig. 2 is the gDNA amplification curve diagrams of non-sulphite processing.

Embodiment

In order to make the purpose , technical scheme and advantage of the present invention be clearer, with reference to the accompanying drawings and embodiments, it is right The present invention is described in further detail.It should be appreciated that specific embodiment described herein is only to explain the present invention, and It is not used in the restriction present invention.

Embodiment 1：The preparation of kit

First, the design and synthesis of primer and probe

For the promoter region of SHOX2 genes in human genome and reference gene β-Actin (b-actin) (sequence referring to Mankind's whole genome sequence disclosed in ncbi database), use Primer Premier 3.0 and Methyl Primer Express v1.0 softwares, separately design a pair of of specific primer and probe.

Specific primer and probe sequence, it is as shown in the table：

Remarks：Y is degeneracy base, i.e. Y=C/T.

Wherein the first detection probes 5 ' of SHOX2 end is used as luminophore, the second detection probes 5 ' of SHOX2 using FAM marks End is used as luminophore as luminophore, the end of β-Actin detection probes 5 ' using VIC marks using ROX marks, and 3 ' ends make Quenching group is used as by the use of MGB.The probe of two SHOX2 detects 6 CPG islands jointly, adds the scope of DNA methylation assay.

2nd, reference substance selects

SHOX2 methylate standard items DNA for normal human peripheral blood's genomic DNA through I methylases of Sss handle, base can be made Methylate because organizing the C in all CG sequences on C5 positions；The non-standard items DNA that methylates is normal human peripheral blood's genome DNA。

3rd, PCR reaction solution forms

Including the PCR reaction solution containing above-mentioned specific primer and probe, the PCR reaction solution includes：

SHOX2 forward primers：5'-TTTTGGATAGTTAGGTAATTTTYGTT-3',

SHOX2 reverse primers：5'-ACGAACCCTTTAAACAACCAACATAA-3',

The first detection probes of SHOX2：5'FAM-AAACGCCTATACTCGTACG-3'MGB,

The second detection probes of SHOX2：5'VIC-CGATCGAACAAACG-3'MGB,

β-Actin forward primers：5'-GGTTAGGAAGGAGGTTGTTTGTTTT-3',

β-Actin reverse primers：5'-CCAAACTATAACCTCTACAACCTTCAAAA-3',

β-Actin detection probes：5'ROX-CCCATTAACTAAACACAACCT-3'MGB；

Y is to annex base, i.e. Y=C/T；

10 × PCR buffer,

DNTP and nuclease-free water.

Wherein, 10 × PCR buffer, dNTP and nuclease-free water are purchased from Dalian precious biology (precious biology (Dalian) Engineering Co., Ltd)；The nuclease-free water uses DEPC (Diethyl for (Nuclease-Free Water) Pyrocarbonate, pyrocarbonic acid diethyl ester) the treated and ultra-pure water through autoclave sterilization.

The PCR reaction solution component it is final concentration of：

1 × PCR buffer,

0.4 μM of (μm ol/L) SHOX2 forward primer,

0.4 μM of SHOX2 reverse primer,

0.4 μM of first detection probe of SHOX2,

0.4 μM of second detection probe of SHOX2；

0.4 μM of β-Actin forward primer,

0.4 μM of β-Actin reverse primer,

0.4 μM of β-Actin detection probe；

0.25mM(mmol/L)dNTP。

The kit further includes Ex Taq enzymes, purchased from the precious biology in Dalian (precious biology (Dalian) Engineering Co., Ltd)；Institute The activity for stating Ex Taq enzymes remains more preferable, and longer fragment can be effectively expanded relative to general T aq.

Embodiment 2：The method that lung cancer DNA methylation assay is carried out using mentioned reagent box

First, technical principle

The promoter region of SHOX2 genes and reference gene β-Actin (b-actin) separately design a pair in human genome The primer and probe of specific DNA methylation assay.Then sample of the amplification through sulphite conversion is removed with the primer and probe DNA, the methyl rate of sample to be tested is determined according to the relative fluorescence CT values of SHOX2 and β-Actin gene PCR amplifications, According to methyl rate indirectly come judge lung cancer change risk.

2nd, detection method

Wherein, reagent is bisulfites or bisulfite used by conversion processing, and other auxiliary (corresponding reagents Box is the EpiTect Fast DNA BisuLfite Kit purchased from German QIAGEN companies).

Wherein, pcr amplification reaction program is：95 DEG C of denaturation 5min；45cycles, 95 DEG C of 15sec, 60 DEG C of 30sec.

Specific detection method is as follows：

One) biological specimen is collected：

Selected from during in June, -2017 in January, 2017 in 33 non-small cell lung cancer phlegm of the refined hospital admission in Hunan Province Hunan Liquid, 30 normal person's sputums.

Two) tissue DNA is extracted：

The agent formulations used in sample tissue DNA are extracted below is selected from Hunan Honghao Genetic Biology Technology Co., Ltd 《Human peripheral genome DNA extracting reagent kit (filtration column method)》(the number of putting on record：The long tool in Hunan is for 20150166).

1) sample fetched is added into 2 times of volume sputum liquefaction agent, shaking liquefaction 30min；14 000r/min are centrifuged 5min。

2) precipitation 1.5mL deionizations shake, 12000rpm centrifugations 5min；Outwell top waste liquid, collecting pipe bottom precipitation.

3) repeat step 1), it is 2) that all sample process is complete.

4) 20 μ L Proteinase Ks, 250 μ L lysate ABL are sequentially added, vortex oscillation 10s, 65 DEG C of water-bath 15min, during which shake Swing mixing 2-3 times.

5) 250 μ L absolute ethyl alcohols, vortex oscillation 10sec, of short duration centrifugation 5sec are added after taking out.

6) adsorption column is inserted into collecting pipe, mixed liquor obtained by previous step is transferred in adsorption column.10,000rpm is centrifuged 1min。

7) waste liquid in collecting pipe is abandoned, adsorption column is reentered into collecting pipe；Add 500 μ L washing lotions I, 10,000rpm, centrifugation 1min。

8) waste liquid in collecting pipe is abandoned, adds 700 μ L washing lotions II, 10,000rpm, centrifugation 1min,.Abandon waste liquid.(washing lotion II makes Absolute ethyl alcohol need to be added before to 80%)

9) repeat step 8) once.

10) efflux is abandoned, adsorption column is turned back into collecting pipe, idle running centrifugation, 13,000rpm, 2min.

11) adsorption column is inserted into new EP pipes.Adding 30-100 μ L DNA lysates, (65 DEG C of preheatings can improve DNA Pick-up rate), it is stored at room temperature 5min.

12) again 10,000rpm, centrifuge 1min.Adsorption column is abandoned, EP liquid in pipe is DNA solution.2-8 DEG C of preservation, if It need to for a long time preserve, be placed in -20 DEG C or lower temperature.

Three) DNA conversion process：

1) by the DNA configuration sulphite transformation system configurations of extraction：

Component	Reaction volume (μ L)
		The DNA to be measured of extraction	10μL
Ultra-pure water (RNAse-free Water)	10μL
		Solution of sodium bisulfite (BisuLfite solution)	85μL
DNAprotect Buffer	35μL
		Amount to	140μL

2) operation architecture of sulphite conversion：

Reaction temperature	Reaction time
		95℃	5min
60℃	10min
		95℃	5min
60℃	10min
		20℃	∞

3) DNA is purified after sulphite conversion：

DNA purifying agent formulations used are selected from German QIAGEN companies after being converted below to sulphite EpiTect Fast DNA sodium hydrogensulfite kits.

A) product after sulfurous acid is converted is transferred in the EP pipes of 1.5ml；

B) being less than 100ng/ μ L DNA need to add in 1 μ L Carrier RNA to Buffer BL, if concentration is more than 100ng/ μ L Carrier RNA need not then be added；

C) plus 310 μ L Buffer BL are mixed；

D) add 250 μ L absolute ethyl alcohols again, mix 15sec；

E) mixture by more than is transferred to Filter column, 10000rpm centrifugations 1min；

F) filtrate is abandoned, adds 500 μ L Buffer BW, 10000rpm centrifugations 1min；

G) filtrate is abandoned, adds the static 15min of 500 μ L Buffer BD, 10000rpm centrifugations 1min；

H) filtrate is abandoned, adds 500 μ L Buffer BW, 10000rpm centrifugations 1min；

I) repeat step h)；

J) filtrate is abandoned, adds 250 μ L absolute ethyl alcohols, 10000rpm centrifugations 1min；

K) filtrate, then 12000rpm idle running 1min are abandoned；

L) plus 15 μ L Buffer EB room temperatures static 1min, 10000rpm centrifuge 1min；

M) DNA of collection is stored in -20 DEG C.

Four) PCR flows

1st, the PCR instrument device used (is purchased from Life Tech for 7500 type real-time fluorescence quantitative PCR systems of American AB I (applied biosystems companies), reaction system are 20 μ L；

2nd, the preparation of PCR reaction systems and condition, it is as shown in the table：

PCR reaction systems：

Component	Reaction volume (μ L)
		PCR reaction solution	18.3μL
Ex Taq enzymes	0.2μL
		DNA profiling after sulphite conversion	1.5μL

PCR response procedures：

Five) interpretation of result

With SHOX2/ in the ratio of SHOX2/ β-Actin in sample to be tested divided by positive control (methylate standard items DNA) The ratio of β-Actin, methylates ratio to calculate SHOX2.

Methyl rate calculation formula is borrows the methyl rate that the formula of relative quantification calculates sample to be detected, and formula is such as Shown in lower.

2^{- △ △ CT}=2^{Sample to be tested (SHOX2CT- β-Actin CT)-permethylated sample (SHOX2CT- β-Actin CT)}

Kit testing result meets Quality Control requirement, and sample is judged according to testing result.Pattern detection result is SHOX2 gene C t values≤35, β-actin≤33, then judge that sample is SHOX2 methylation positives；Pattern detection result is SHOX2 Gene C t values ＞ 35, β-actin≤33；Then sentence sample to methylate feminine gender for SHOX2, β-actin ＞ 33, then PCR reactions are invalid.

Six) lung cancer sputum and the distribution of normal sputum SHOX2 gene methylations

SHOX2 gene methylations do not detect in 30 normal populations, and detecting 22 in 33 peripheral blood of lung cancer patients (accounts for 66.7%, the P compared with normal group<0.001), the ratio that methylates of SHOX2 genes is raised with the exacerbation of the lung cancer course of disease, such as Shown in following table：

Lung cancer sputum and normal sputum SHOX2 gene methylation detection statistics

For specific experiment data based on the present invention referring to Fig. 1 and Fig. 2, wherein Fig. 1 is SOX2 methylation positive concentration of specimens The amplification curve diagram of gradient dilution：Fig. 2 is the gDNA amplification curve diagrams of non-sulphite processing.

Provided by the present invention for having for the primer pair of lung cancer related gene SHOX2 DNA methylation assays, kit and method Beneficial effect is：

The foregoing is merely the embodiment of the present invention, is not intended to limit the scope of the invention, every to utilize this hair The equivalent process transformation that bright description is made, is directly or indirectly used in other relevant technical fields, similarly wraps Include in the scope of patent protection of the present invention.

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Claims

1. a kind of primer pair for lung cancer related gene SHOX2 DNA methylation assays, it is characterised in that including for SHOX2 bases The promoter region of cause and the specific primer and probe of β-Actin reference genes, it is as follows：

SHOX2 forward primers：5'-TTTTGGATAGTTAGGTAATTTTYGTT-3',

SHOX2 reverse primers：5'-ACGAACCCTTTAAACAACCAACATAA-3',

The first detection probes of SHOX2：5'FAM-AAACGCCTATACTCGTACG-3'MGB；

The second detection probes of SHOX2：5'VIC-CGATCGAACAAACG-3'MGB；

β-Actin forward primers：5'-GGTTAGGAAGGAGGTTGTTTGTTTT-3',

β-Actin reverse primers：5'-CCAAACTATAACCTCTACAACCTTCAAAA-3',

β-Actin detection probes：5'ROX-CCCATTAACTAAACACAACCT-3'MGB.

2. a kind of kit for lung cancer related gene SHOX2 DNA methylation assays, it is characterised in that including being wanted containing such as right The PCR reaction solution of the specific primer and probe described in 1 is sought, the PCR reaction solution includes：SHOX2 forward primers, SHOX2 are anti- To primer, the first detection probes of SHOX2, the second detection probes of SHOX2, β-Actin forward primers, β-Actin reverse primers, β- Actin detection probes, 10 × PCR buffer, dNTP and nuclease-free water.

3. the kit according to claim 2 for lung cancer related gene SHOX2 DNA methylation assays, it is characterised in that The kit further includes Ex Taq enzymes.

4. the kit according to claim 2 for lung cancer related gene SHOX2 DNA methylation assays, it is characterised in that The component of the PCR reaction solution is final concentration of：1 × PCR buffer, 0.4 μM of SHOX2 forward primer, 0.4 μM of SHOX2 are reverse Primer, 0.4 μM of first detection probe of SHOX2,0.4 μM of second detection probe of SHOX2,0.4 μM of β-Actin forward primers, 0.4 μM β-Actin reverse primers, 0.4 μM of β-Actin detection probe, 0.25mM dNTP.

5. according to any kit for lung cancer related gene SHOX2 DNA methylation assays in claim 2-4, it is special Sign is, further includes positive reference substance and negative controls, and the positive reference substance methylates standard items DNA for SHOX2, described Negative controls are the non-standard items DNA that methylates.

A kind of 6. method for lung cancer related gene SHOX2 DNA methylation assays, it is characterised in that include the following steps：

Step 1：The DNA of sample extracting to be detected is taken, conversion processing is carried out to it, templates of the DNA after conversion as PCR；

Step 2：Kit as claimed in claim 5 is provided, PCR amplification is carried out to the template；

Step 3：Sample to be detected is determined according to the relative fluorescence CT values of SHOX2 and β-Actin gene PCR amplifications Methyl rate, i.e., in sample to be detected in the ratio divided by positive reference substance of SHOX2/ β-Actin SHOX2/ β-Actin ratio Value.

7. the method according to claim 6 for lung cancer related gene SHOX2 DNA methylation assays, it is characterised in that institute State in step 1 that reagent is bisulfites or bisulfite used by conversion processing.

8. the method according to claim 7 for lung cancer related gene SHOX2 DNA methylation assays, it is characterised in that institute Stating pcr amplification reaction program in step 2 is：95 DEG C of denaturation 5min；45cycles, 95 DEG C of 15sec, 60 DEG C of 30sec.