CN110373462A - The method of target gene methylation level in Diagnosis of Bladder system and detection urine - Google Patents

The method of target gene methylation level in Diagnosis of Bladder system and detection urine Download PDF

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CN110373462A
CN110373462A CN201910492668.5A CN201910492668A CN110373462A CN 110373462 A CN110373462 A CN 110373462A CN 201910492668 A CN201910492668 A CN 201910492668A CN 110373462 A CN110373462 A CN 110373462A
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methylation level
methylation
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韩君
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Guangying Medical Technology (shanghai) Co Ltd
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers

Abstract

The invention discloses a kind of non-invasive detection methods of bladder cancer.The early screening of bladder cancer may be implemented in the non-invasive detection methods of bladder cancer of the invention, the parting and/or prognosis prediction of the risk assessment of bladder cancer, bladder cancer occur for subject, sensitivity, specificity and the accuracy for improving diagnosis realize the effect of noninvasive, convenient, efficient quick diagnosis bladder cancer.The present invention also provides a kind of Diagnosis of Bladder systems.The present invention also provides a kind of methods of target gene methylation level in detection urine.

Description

The method of target gene methylation level in Diagnosis of Bladder system and detection urine
Technical field
The invention belongs to field of biological medicine.In particular it relates to the non-invasive detection methods of bladder cancer.More Body, it the present invention relates to the use of the bladder cancer non-invasive detection methods of the DNA methylation biomarker of bladder cancer.
Background technique
Bladder cancer is a kind of common and high lethality rate malignant tumour.According to statistics, annual about 429800 of the whole world is new Increase case and 165100 deaths.Traditional bladder cancer inspection is cystoscopy and cytology biopsy, but cystoscopy It looks into and belongs to invasive inspection, it is costly and certain pain can be brought to patient, and its result may be by operator's subjective judgement And bring certain error；And cytology biopsy then haves the defects that sensitivity is low (34% or so).Therefore, Non-invasive detection becomes One of the important development direction of bladder cancer detection.
DNA methylation is important epigenetic modification relevant to many genes activity and bioprocess.The frequent table of DNA The progress for being genetically changed and can lead to cancer is seen, and includes that bladder cancer is associated with many cancers.DNA methylation detection advantage it One is that it not only can also carry out diagnosing tumor using tumor tissues using body fluids such as blood, saliva, urine etc., real It is existing noninvasive painless.The study found that DNA methylation relevant to bladder cancer can be used for the diagnosis of bladder cancer, and it can be used as bladder cancer The target spot for the treatment of.
High-resolution solubility curve polymerase chain reaction (HRM-PCR) is a kind of not need PCR product subsequent operation, simple The technology of single, sensitive, quick and inexpensive detection gene methylation level.This method designs near methylation decorating site Real-time quantitative PCR primer carries out specific PCR amplification to from the correlation zone of the DNA modified through bisulfite.MS-HRM tool There is hypersensitivity, the sequence difference that as little as 1 base changes in amplified production can be distinguished.
It still needs to carry out extensive evaluation to the DNA methylation biomarker of bladder cancer at present, it is a kind of efficiently and accurate to develop Bladder cancer non-invasive detection methods.
Summary of the invention
The present inventor recognizes one group of methyl-sensitive gene by extensive research screening, i.e. HOXA9, ONECUT2, PCDH17, PENK, TWIST1, VIM and ZNF154 gene.In bladder cancer patients, in specific site height occurs for said gene Methylation.Therefore, pass through the first of detection HOXA9, ONECUT2, PCDH17, PENK, TWIST1, VIM and ZNF154 assortment of genes Baseization is horizontal, can carry out the detection and/or diagnosis of bladder cancer, and bladder cancer occurs for early screening, subject including bladder cancer Risk assessment, bladder cancer parting and/or prognosis prediction.
The purpose of the present invention is to provide a kind of non-invasive detection methods of bladder cancer.In addition, the present invention also aims to A kind of method detecting target gene methylation level in urine is provided.
On the one hand, the present invention provides a kind of non-invasive detection methods of bladder cancer, comprising:
1) urine sample is provided, DNA in urine is extracted；
2) DNA extracted in step 1) is subjected to bisulfite conversion processing；
3) measure target gene methylation level, the target gene include: HOXA9, ONECUT2, PCDH17, PENK, TWIST1, VIM and ZNF154 detect that then methylation level is denoted as 1 for methylation, and methylation is not detected, and then methylation level is remembered It is -1；
4) the target gene methylation level that step 3) obtains is brought into risk score model, obtains G value,
Wherein, the risk score model are as follows: G=(m1 × HOXA9 methylation level)+(m2 × ONECUT2 methylation water It is flat)+(m3 × PCDH17 methylation level)+(m4 × PENK methylation level)+(m5 × TWIST1 methylation level)+(m6 × VIM methylation level)+(m7 × ZNF154 methylation level),
Wherein, m1 be 1.99~39.964, m2 be 1.128~161.832, m3 be 1.51~114, m4 be 0.146~ 43.883, m5 be 0.246~5.374, m6 be 0.042~3.929, m7 be 0.033~2.862；
5) according to G value, bladder cancer testing result is obtained,
Wherein, if G value is higher than setting value, it is judged as high-risk-type bladder cancer；If G value is equal to or less than setting value, Then it is judged as low risk bladder cancer.
According to a specific embodiment, urine sample is urinary precipitation sample, such as arena.
According to a specific embodiment, in step 1), EDTA solution is prestored in the collecting pipe of urine sample, is urinated collecting It is mixed after liquid.This facilitates the preservation of urine sample, reduces the DNA degradation during long-term urine saves, and then improve urine The extraction yield of DNA.
According to a specific embodiment, in step 3), target gene methylation level is by high-resolution solubility curve Polymerase chain reaction (HRM-PCR), methylation status of PTEN promoter (MS-PCR), M-MLPA, pyrosequencing, MALDI-TOF measurement 's.
According to a preferred embodiment, target gene methylation level is measured by HRM-PCR.Utilize HRM- PCR, which carries out DNA methylation assay, has the advantages that easy to operate, inexpensive, low false positive rate, low jamming rate, is widely portable to section Grind experiment and clinical detection.
Risk score model according to a specific embodiment, in step 4) are as follows: G=(39.964 × HOXA9 methylation It is horizontal)+(161.832 × ONECUT2 methylation level)+(114 × PCDH17 methylation level)+(43.883 × PENK methyl Change horizontal)+(5.374 × TWIST1 methylation level)+(3.929 × VIM methylation level)+(2.862 × ZNF154 methylation It is horizontal), also, the setting value in step 5) is -143.844.
Risk score model according to a specific embodiment, in step 4) are as follows: (1.99 × HOXA9's G=methylates water It is flat)+(1.128 × ONECUT2 methylation level)+(1.51 × PCDH17 methylation level)+(0.146 × PENK methylation water It is flat)+(0.246 × TWIST1 methylation level)+(0.042 × VIM methylation level)+(0.033 × ZNF154 methylation water It is flat), also, the setting value in step 5) is -0.473.
Risk score model according to a preferred embodiment, in step 4) are as follows: G=(8.919 × HOXA9 methylation It is horizontal)+(13.513 × ONECUT2 methylation level)+(13.119 × PCDH17 methylation level)+(2.534 × PENK methyl Change horizontal)+(1.151 × TWIST1 methylation level)+(0.405 × VIM methylation level)+(0.306 × ZNF154 methylation It is horizontal), also, the setting value in step 5) is -13.709.
The present invention provides a simple noninvasive detection method for bladder cancer, is very suitable for the diagnosis of bladder cancer.
On the other hand, the present invention provides a kind of Diagnosis of Bladder system, including data obtaining module, computing module and diagnosis Module,
Wherein, data obtaining module be used for executes acquisition subject's detection information operation, the detection information include by The methylation level of target gene in examination person's sample, the target gene include: HOXA9, ONECUT2, PCDH17, PENK, TWIST1, VIM and ZNF154, wherein detect that then methylation level is denoted as 1 for methylation, methylation is not detected and then methylates Level is denoted as -1；
Computing module, which is used to execute, substitutes into risk score model for the methylation level of target gene, calculates the operation of G value, Risk score model are as follows: G=(m1 × HOXA9 methylation level)+(m2 × ONECUT2 methylation level)+(m3 × PCDH17 first Baseization is horizontal)+(m4 × PENK methylation level)+(m5 × TWIST1 methylation level)+(m6 × VIM methylation level)+(m7 × ZNF154 methylation level), wherein it be 1.128~161.832, m3 is 1.51~114, m4 that m1, which is 1.99~39.964, m2, It is 0.246~5.374, m6 for 0.146~43.883, m5 be 0.042~3.929, m7 is 0.033~2.862；
Diagnostic module is for executing the operation for judging subject's health status according to G value, wherein if G value is higher than setting Value, then judge the subject for high-risk-type bladder cancer；If G value is equal to or less than setting value, judge that the subject is Low risk bladder cancer.
Risk score model according to a specific embodiment, in computing module are as follows: G=(39.964 × HOXA9 methyl Change horizontal)+(161.832 × ONECUT2 methylation level)+(114 × PCDH17 methylation level)+(43.883 × PENK first Baseization is horizontal)+(5.374 × TWIST1 methylation level)+(3.929 × VIM methylation level)+(2.862 × ZNF154 methyl Change horizontal), and the setting value in diagnostic module is -143.844.
Risk score model according to a specific embodiment, in computing module are as follows: G=(1.99 × HOXA9 methylation It is horizontal)+(1.128 × ONECUT2 methylation level)+(1.51 × PCDH17 methylation level)+(0.146 × PENK methylation It is horizontal)+(0.246 × TWIST1 methylation level)+(0.042 × VIM methylation level)+(0.033 × ZNF154 methylation water It is flat), and the setting value in diagnostic module is -0.473.
Risk score model according to a preferred embodiment, in computing module are as follows: G=(8.919 × HOXA9 methyl Change horizontal)+(13.513 × ONECUT2 methylation level)+(13.119 × PCDH17 methylation level)+(2.534 × PENK first Baseization is horizontal)+(1.151 × TWIST1 methylation level)+(0.405 × VIM methylation level)+(0.306 × ZNF154 methyl Change horizontal), and the setting value in diagnostic module is -13.709.
According to a preferred embodiment, Diagnosis of Bladder system further includes result output module, and the result exports mould Block is for exporting the judgement that diagnostic module obtains.
On the other hand, the present invention provides a kind of method for detecting target gene methylation level in urine, the purpose base Because being selected from: HOXA9, ONECUT2, PCDH17, PENK, TWIST1, VIM and ZNF154 the described method comprises the following steps:
1) centrifugal treating urine sample extracts DNA；
2) DNA extracted in step 1) is subjected to bisulfite conversion processing；
3) methylation level of target gene is measured,
Wherein, it is measured using primer pair 1~7 selected from the following: nucleotide sequence such as SEQ ID NO:1 and SEQ ID The primer pair 2, nucleotides sequence as shown in SEQ ID NO:3 and SEQ ID NO:4 of primer pair 1, nucleotide sequence shown in NO:2 Arrange primer pair 3, nucleotide sequence such as SEQ ID NO:7 and SEQ ID NO as shown in SEQ ID NO:5 and SEQ ID NO:6: The primer pair 5, nucleotide sequence as shown in SEQ ID NO:9 and SEQ ID NO:10 of primer pair 4, nucleotide sequence shown in 8 The primer pair 6 as shown in SEQ ID NO:11 and SEQ ID NO:12 and nucleotide sequence such as SEQ ID NO:13 and SEQ Primer pair 7 shown in ID NO:14.
According to a specific embodiment, urine sample is urinary precipitation sample, such as arena.
According to a specific embodiment, in step 1), EDTA solution is prestored in the collecting pipe of urine sample, is urinated collecting It is mixed after liquid.This facilitates the preservation of urine sample, reduces the DNA degradation during long-term urine saves, and then improve urine The extraction yield of DNA.
According to a specific embodiment, the volume of urine sample is 20ml or more, preferably midstream urine.
According to a specific embodiment, in step 3), the methylation level of target gene be dissolved by high-resolution it is bent Line polymerase chain reaction (HRM-PCR), methylation status of PTEN promoter (MS-PCR), M-MLPA, pyrosequencing, MALDI-TOF are surveyed Fixed.
According to a preferred embodiment, the methylation level of target gene is measured by HRM-PCR.Utilize HRM- PCR, which carries out DNA methylation assay, has the advantages that easy to operate, inexpensive, low false positive rate, low jamming rate, is widely portable to section Grind experiment and clinical detection.
The early screening of bladder cancer may be implemented in the non-invasive detection methods of bladder cancer of the invention, bladder cancer occurs for subject Risk assessment, bladder cancer parting and/or prognosis prediction, improve sensitivity, specificity and the accuracy of diagnosis, realize Noninvasive, convenient, efficient, quick diagnosis bladder cancer effect.
(positive predictive value 100%, negative predictive value 98%) is predicted by accurate risk assessment, wing of the invention Acceptance of the non-invasive detection methods of Guang cancer in the unobvious crowd of asymptomatic or symptom is high, can be used for routine physical examination, reduces The unnecessary invasive inspection of low risk (i.e. low-risk) patient.
In addition, primer specificity designed by the present inventor is strong, expanding effect and detection efficiency, the inspection of acquisition are improved Surveying result has high confidence level.
The method of target gene methylation level is easy to operate in detection urine of the invention, saves detection time, knot Fruit is clearly reliable.
Detailed description of the invention
Fig. 1 is the experiment flow schematic diagram for detecting target gene methylation level in urine sample.
Fig. 2 is the ratio of bladder cancer patients (44) and non-bladder cancer patients (37) DNA methylation probability in 81 samples Compared with.Statistical significance examines (student ' s t-test) to determine by student t, wherein *, * *, * * * respectively represent P < 0.05, P < 0.01、P<0.001。
Fig. 3 is using risk score model of the invention to 81 sample queues (44 bladder cancers and 37 normal controls) Carry out Receiver Operating Characteristics (ROC) curve of bladder cancer prediction, wherein abscissa is specificity, and ordinate is sensitivity.
Fig. 4 be using risk score model of the invention to cancer gene group map (TCGA) queue (412 bladder cancers and 21 normal controls) carry out bladder cancer prediction Receiver Operating Characteristics (ROC) curve, wherein abscissa be specificity, indulge sit It is designated as sensitivity.
Fig. 5 is that each coefficient takes the subject when upper limit value of its numberical range to work in risk score model of the invention Indicatrix (ROC).
Fig. 6 is that each coefficient takes the subject when lower limit value of its numberical range to work in risk score model of the invention Indicatrix (ROC).
Specific embodiment
The present invention is described in further detail with reference to the accompanying drawings and examples.Following embodiment is merely to illustrate this It invents rather than limits the scope of the invention.Test method without specific conditions in embodiment, usually according to conventional strip Part, or according to the normal condition proposed by manufacturer.
" marker " described in this specification refers to specific biological characteristic, biochemical characteristics or aspect Molecular indicator can be used for determining the serious journey presence or absence of specified disease or situation and/or specified disease or situation Degree.
" high (degree) methylation " described in this specification refers to that there are high methylations, hydroxyl by CpG in a gene order Methylation, aldehyde methylation or carboxy methylation modification.
As known to those skilled in the art, DNA is after carrying out bisulfite conversion processing, wherein the born of the same parents not methylated Pyrimidine is converted into uracil, and the cytimidine to methylate remains unchanged.
" sample " described in this specification or " sample " include (related indication if any disease in the urological system from any individual Subject) in obtain, be suitable for methylation state of DNA detection substance.It is mixed in the urine of bladder cancer and falls off on a small quantity Tumour cell.Therefore, sample is preferably urine sample or the urine sample (especially urinary precipitation) by processing.
Embodiment 1
Sample treatment (urine sample)
1) in the urine collection tube for prestoring 600ul EDTA solution, collect 30ml urine, after mixing at 3000g from The heart 10 minutes；
2) urine DNA is extracted using Qiagen kit；
3) EZ Gold methylating reagent box is used, bisulfite conversion is carried out to urine DNA.
Urine preservation condition: when the holding time was less than 48 hours, 4 DEG C are stored in；Holding time is more than 48 hours, then saves In -80 DEG C.
Embodiment 2
It is horizontal that HRM-PCR detects gene methylation
High solubility curve polymerase chain reaction (HRM-PCR) experiment is following to be carried out:
1) reagent needed for preparing HRM-PCR (1 reaction):
A.5ul 2X Zymo premix；
B.0.5ul primer, primer sequence is as shown in NO:1~14 SEQ ID；
C.0.5ul LC GreenPlusSaturated fluorescence dyestuff；
D.3ul water；
E.1ul sample, the sample are handled according to the method for embodiment 1；
2) HRM-PCR measurement is carried out, gene methylation level is obtained, reaction step and condition are as follows:
A. pre-activate 10 minutes at 95.0 DEG C；
B. it is denaturalized 15 seconds at 95.0 DEG C, anneals 30 seconds at 60.0 DEG C, extend 15 seconds at 72.0 DEG C, carry out 50 with this A circulation；
C. it according to following reaction condition, obtains HRM curve: temperature being risen to 95.0 from 72.0 DEG C with 1.6 DEG C/sec of rate DEG C, and kept for 15 seconds；60.0 DEG C are cooled to again with 0.025 DEG C/sec of rate；Then with 0.025 DEG C/sec of rate from 60.0 DEG C 95.0 DEG C are risen to, is kept for 15 seconds, and be cooled to 60.0 DEG C with 1.6 DEG C/sec of rate.
Embodiment 3
The building of bladder cancer risk score model
1. sample collection
Collect 81 mankind's urine samples, including 44 bladder cancer patients and 37 normal controls.
2. model construction
To the methylation water of 7 selected genes (HOXA9, ONECUT2, PCDH17, PENK, TWIST1, VIM and ZNF154) Flat to carry out single argument and multivariable logistic regression analysis, building includes the detection model of 7 genes.Using the method for embodiment 2, The methylation level of 7 genes in each sample is obtained respectively, and the operating process of test experience is as shown in Figure 1.Firstly, carrying out single Variable logistic regression analysis.As shown in Fig. 2, 7 selected genes are all significant related to bladder cancer.Then multivariable is carried out to patrol Regression analysis is collected, the regression coefficient of 7 kinds of genes is obtained.
All statistical analysis use (the Statistical Product and Service of SPSS 19.0 Solutions, IBM Corporation, Armonk, New York, the U.S.) and (the R Foundation for of R 3.4.2 editions Statistical Computing, Vienna, Austria) it carries out.
Based on the methylation level of this 7 genes, creation can be used for bladder cancer detection/diagnosis risk score model: G= (8.919 × HOXA9 methylation level)+(13.513 × ONECUT2 methylation level)+(13.119 × PCDH17 methylation water It is flat)+(2.534 × PENK methylation level)+(1.151 × TWIST1 methylation level)+(0.405 × VIM methylation level)+ (0.306 × ZNF154 methylation level).Wherein, the methyl of the gene methylation then gene is detected using HRM-PCR method Change level is denoted as 1, and the methylation level that the gene methylation then gene is not detected is denoted as -1.
3. result
When G value is higher than -13.709, it is judged as high-risk-type bladder cancer (high risk)；When G value is equal to or less than -13.709 When, it is judged as low risk bladder cancer (low-risk).As shown in figure 3, above-mentioned risk score model carries out the special of Diagnosis of Bladder Property up to 94.6%, sensitivity shows to make bladder cancer detection/diagnosis with good instruction up to 75.0%, AUC value 89.4% With.
Embodiment 4
The verifying of bladder cancer risk score model
Utilize the methylation sample information in cancer gene group map (Cancer Genome Atlas, TCGA) database (412 bladder cancers and 21 normal controls) carry out individual authentication to the risk score model in embodiment 3.When G value be higher than- When 13.709, it is judged as high-risk-type bladder cancer (high risk)；When G value is equal to or less than -13.709, it is judged as low risk bladder Cancer (low-risk).
As a result as shown in figure 4, the specificity of bladder cancer detection is up to 91.2%, sensitivity is up to 73.4%, AUC value 85.1%, show that risk score model shows in bladder cancer detection and stablizes.
Embodiment 5
Coefficient range in bladder cancer risk score model
The present inventor, which also studies, compares influence of the coefficient value to model validation in risk score model.According to risk Rating Model: G=(8.919 × HOXA9 methylation level)+(13.513 × ONECUT2 methylation level)+(13.119 × PCDH17 methylation level)+(2.534 × PENK methylation level)+(1.151 × TWIST1 methylation level)+(0.405 × VIM methylation level)+(0.306 × ZNF154 methylation level), 95% confidence of each gene methylation horizontal coefficients is provided Section is listed in table 1.
Table 1.
Coefficient (95% confidence interval coefficient range)
HOXA9 8.919 (1.99~39.964)
ONECUT2 13.513 (1.128~161.832)
PCDH17 13.119 (1.51~114)
PENK 2.534 (0.146~43.883)
TWIST1 1.151 (0.246~5.374)
VIM 0.405 (0.042~3.929)
ZNF154 0.306 (0.033~2.862)
1) when each coefficient takes range higher limit, risk score model are as follows: G=(39.964 × HOXA9 methylation level)+ (161.832 × ONECUT2 methylation level)+(114 × PCDH17 methylation level)+(43.883 × PENK methylation level) + (5.374 × TWIST1 methylation level)+(3.929 × VIM methylation level)+(2.862 × ZNF154 methylation level).
Using above-mentioned risk score model (coefficient capping value), to 7 genes in above-mentioned 81 urine samples (HOXA9, ONECUT2, PCDH17, PENK, TWIST1, VIM and ZNF154) methylation level carry out data statistic analysis.When G value is high When -143.844, it is judged as high-risk-type bladder cancer (high risk)；When G value is equal to or less than -143.844, it is judged as low danger Type bladder cancer (low-risk).As a result as shown in figure 5, the risk score model carries out the specificity of Diagnosis of Bladder up to 94.6%, Sensitivity is up to 75.0%, AUC value 89.3%.
2) when each coefficient takes lower range limit, risk score model are as follows: G=(1.99 × HOXA9 methylation level)+ (1.128 × ONECUT2 methylation level)+(1.51 × PCDH17 methylation level)+(0.146 × PENK methylation level)+ (0.246 × TWIST1 methylation level)+(0.042 × VIM methylation level)+(0.033 × ZNF154 methylation level).
Using above-mentioned risk score model (coefficient removes limit value), to 7 genes in above-mentioned 81 urine samples (HOXA9, ONECUT2, PCDH17, PENK, TWIST1, VIM and ZNF154) methylation level carry out data statistic analysis.When G value is high When -0.473, it is judged as high-risk-type bladder cancer (high risk)；When G value is equal to or less than -0.473, it is judged as low risk wing Guang cancer (low-risk).As a result sensitive as shown in fig. 6, the risk score model carries out the specific up to 94.6% of Diagnosis of Bladder Degree is up to 75.0%, AUC value 89.6%.
Therefore, risk score model can be with are as follows: and G=(m1 × HOXA9 methylation level)+(m2 × ONECUT2 methylates water It is flat)+(m3 × PCDH17 methylation level)+(m4 × PENK methylation level)+(m5 × TWIST1 methylation level)+(m6 × VIM methylation level)+(m7 × ZNF154 methylation level), wherein m1 be 1.99~39.964, m2 be 1.128~ 161.832, m3 be 1.51~114, m4 be 0.146~43.883, m5 be 0.246~5.374, m6 be 0.042~3.929, m7 It is 0.033~2.862.
Embodiment 6
The preparation of kit
Reagent preparation box includes: primer sequence as shown in Table 2, and 2 × ZymoTaq qPCR Premix (is purchased from Zymo Research company), LC GreenPlus(being purchased from BioFire Diagnostics, Inc., salt lake city, the Utah State, the U.S.), The pure water of RNase-free and DNase-free.
Table 2.
Using the above-mentioned kit prepared, using HRM-PCR method, the methylation level of testing goal gene is obtained Highly sensitive, specificity and accuracy testing result.
The explanation of above-described embodiment is only intended to understand method and its core concept of the invention.It should be pointed out that for this For the those of ordinary skill in field, without departing from the principle of the present invention, several improvement can also be carried out to the present invention And modification, these improvement and modification will also be fallen into the protection scope of the claim of this application.
Sequence table
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Claims (10)

1. a kind of Diagnosis of Bladder system, including data obtaining module, computing module and diagnostic module,
Wherein, data obtaining module is used to execute the operation for obtaining subject's detection information, and the detection information includes subject The methylation level of target gene in sample, the target gene include: HOXA9, ONECUT2, PCDH17, PENK, TWIST1, VIM and ZNF154, wherein detect that then methylation level is denoted as 1 for methylation, methylation is not detected, and then methylation level is remembered It is -1；
Computing module, which is used to execute, substitutes into risk score model for the methylation level of target gene, calculates the operation of G value, risk Rating Model are as follows: G=(m1 × HOXA9 methylation level)+(m2 × ONECUT2 methylation level)+(m3 × PCDH17 methylation It is horizontal)+(m4 × PENK methylation level)+(m5 × TWIST1 methylation level)+(m6 × VIM methylation level)+(m7 × ZNF154 methylation level), wherein it be 1.128~161.832, m3 is that 1.51~114, m4 is that m1, which is 1.99~39.964, m2, It is 0.042~3.929, m7 is 0.033~2.862 that 0.146~43.883, m5, which are 0.246~5.374, m6,；
Diagnostic module is for executing the operation for judging subject's health status according to G value, wherein if G value is higher than setting value, Judge the subject for high-risk-type bladder cancer；If G value is equal to or less than setting value, judge the subject for low risk Bladder cancer.
2. system according to claim 1, wherein the risk score model in the computing module are as follows: G=(39.964 × HOXA9 methylation level)+(161.832 × ONECUT2 methylation level)+(114 × PCDH17 methylation level)+ (43.883 × PENK methylation level)+(5.374 × TWIST1 methylation level)+(3.929 × VIM methylation level)+ (2.862 × ZNF154 methylation level), also, the setting value in the diagnostic module is -143.844.
3. system according to claim 1, wherein the risk score model in the computing module are as follows: G=(1.99 × HOXA9 methylation level)+(1.128 × ONECUT2 methylation level)+(1.51 × PCDH17 methylation level)+(0.146 × PENK methylation level)+(0.246 × TWIST1 methylation level)+(0.042 × VIM methylation level)+(0.033 × ZNF154 methylation level), also, the setting value in the diagnostic module is -0.473.
4. system according to claim 1, wherein the risk score model in the computing module are as follows: G=(8.919 × HOXA9 methylation level)+(13.513 × ONECUT2 methylation level)+(13.119 × PCDH17 methylation level)+ (2.534 × PENK methylation level)+(1.151 × TWIST1 methylation level)+(0.405 × VIM methylation level)+ (0.306 × ZNF154 methylation level), also, the setting value in the diagnostic module is -13.709.
5. system according to any one of claims 1 to 4, wherein described the system also includes result output module As a result output module is for exporting the judgement that the diagnostic module obtains.
6. the method for target gene methylation level, the target gene are selected from a kind of detection urine: HOXA9, ONECUT2, PCDH17, PENK, TWIST1, VIM and ZNF154 the described method comprises the following steps:
1) centrifugal treating urine sample extracts DNA；
2) DNA extracted in step 1) is subjected to bisulfite conversion processing；
3) methylation level of target gene is measured,
Wherein, it is measured using primer pair 1~7 selected from the following: nucleotide sequence such as SEQ ID NO:1 and SEQ ID NO: Primer pair 2, the nucleotide sequence as shown in SEQ ID NO:3 and SEQ ID NO:4 of primer pair 1, nucleotide sequence shown in 2 are such as Such as SEQ ID NO:7 and SEQ ID NO:8 institute of primer pair 3, nucleotide sequence shown in SEQ ID NO:5 and SEQ ID NO:6 The primer pair 4 shown, nucleotide sequence primer pair 5, nucleotide sequence as shown in SEQ ID NO:9 and SEQ ID NO:10 are such as Primer pair 6 shown in SEQ ID NO:11 and SEQ ID NO:12 and nucleotide sequence such as SEQ ID NO:13 and SEQ ID Primer pair 7 shown in NO:14.
7. according to the method described in claim 6, wherein, the volume of the urine sample is 20ml or more, and being midstream urine.
8. according to the method described in claim 6, it is molten wherein, in step 1), in the collecting pipe of the urine sample to prestore EDTA Liquid mixes after collecting urine.
9. according to the method described in claim 6, wherein, in step 3), the methylation level of the target gene is to pass through height Solubility curve polymerase chain reaction HRM-PCR, methylation status of PTEN promoter, M-MLPA, pyrosequencing, MALDI-TOF is differentiated to survey Fixed.
10. the method according to any one of claim 6~9, wherein in step 3), the methylation of the target gene Level is measured by HRM-PCR.
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Application publication date: 20191025