CN110387417A - The method of target gene methylation level in Diagnosis of Bladder system, reagent combination and detection urine - Google Patents
The method of target gene methylation level in Diagnosis of Bladder system, reagent combination and detection urine Download PDFInfo
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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；A kind of reagent combination, for detecting the methylation level of target gene in urine；A kind of method of target gene methylation level in detection urine.
Description
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
With 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, PCDH17,
POU4F2 and ONECUT2 gene.In bladder cancer patients, in specific site high methylation occurs for said gene.Therefore, pass through
Detect HOXA9, PCDH17, POU4F2 and ONECUT2 assortment of genes methylation level, can carry out bladder cancer detection and/
Or the parting and/or prognosis of the risk assessment of bladder cancer, bladder cancer occur for diagnosis, early screening, subject including bladder cancer
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, PCDH17, POU4F2 and
ONECUT2 detects that then methylation level is denoted as 1 for methylation, and methylation is not detected, and then methylation level is denoted as -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=(6.096 × HOXA9 methylation level)+(3.517 × PCDH17 first
Baseization is horizontal)+(4.582 × POU4F2 methylation level)+(5.268 × ONECUT2 methylation level)；
5) according to G value, bladder cancer testing result is obtained,
Wherein, when G value is higher than setting value, it is judged as high-risk-type bladder cancer；When G value is equal to or less than setting value, sentence
Break as low risk bladder cancer.
According to a specific embodiment, in step 5), setting value is -1.893.
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 are surveyed
Fixed.
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.
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, PCDH17, POU4F2 and
ONECUT2, wherein detect that then methylation level is denoted as 1 for methylation, methylation is not detected, and then methylation 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=(6.096 × HOXA9 methylation level)+(3.517 × PCDH17 methylation level)+(4.582
× POU4F2 methylation level)+(5.268 × ONECUT2 methylation level)；
Diagnostic module is for executing the operation for judging subject's health status according to G value, wherein when G value is higher than setting value
When, then it is judged as high-risk-type bladder cancer；When G value is equal to or less than setting value, then it is judged as low risk bladder cancer.
According to a specific embodiment, in diagnostic module, setting value is -1.893.
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 reagent combination, including 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 the primer pair 3 as shown in SEQ ID NO:5 and SEQ ID NO:6 and nucleotide sequence such as SEQ ID NO:7 and SEQ
Primer pair 4 shown in ID NO:8, the reagent set share the methylation level of the target gene in detection urine, the purpose
Gene includes: HOXA9, PCDH17, POU4F2 and ONECUT2.
According to a specific embodiment, reagent combination further includes selected from LC GreenPlus、LC Green、Eva Green
Or the saturated fluorescence dyestuff of SYTO 9.
Preferably, reagent combination includes LC GreenPlusSaturated fluorescence dyestuff.It is demonstrated experimentally that LC GreenPlusIt is saturated glimmering
The sensibility of photoinitiator dye is higher, and detection sensitivity can be improved.
According to a specific embodiment, reagent combination is that bladder cancer occurs for the early screening of bladder cancer, subject
Risk assessment, the parting of bladder cancer and/or the kit of prognosis prediction.
According to a specific embodiment, mentioned reagent box includes the container and operation instruction for accommodating each reagent
Book, wherein indicating test experience step and result judgement standard etc..
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, PCDH17, POU4F2 and ONECUT2 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) it is combined using mentioned reagent, measures the methylation level of target gene.
According to a specific embodiment, the volume of urine specimen is 20ml or more, preferably midstream urine.
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), 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
Measurement.
According to a preferred embodiment, the methylation level of target gene is measured by HRM-PCR.It utilizes
HRM-PCR, which carries out DNA methylation assay, has the advantages that easy to operate, inexpensive, low false positive rate, low jamming rate, is broadly applicable
In scientific 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.
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.
Reagent combination/kit of the invention has the advantages that easy to operate, saving diagnosis/detection time, result are clear
It is clear reliable.Using reagent combination/kit of the invention, the effect of noninvasive, convenient, efficient quick diagnosis bladder cancer may be implemented
Fruit.
Reagent set of the invention closes strong applicability, is widely portable to scientific experiment and clinical detection.Also, due to this
Reagent combination/kit of invention is suitable for Non-invasive detection, and the acceptance of the unobvious crowd of asymptomatic or symptom is high, can be used for often
Regulator inspection.
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.
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 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.
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:1~8 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, carried out with this
50 circulations；
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
Single argument and is carried out more to the methylation level of 4 selected genes (HOXA9, PCDH17, POU4F2 and ONECUT2)
Variable logistic regression analysis, building include the detection model of 4 genes.Using the method for embodiment 2, each sample is obtained respectively
In 4 genes methylation level, the operating process of test experience is as shown in Figure 1.Firstly, carrying out single argument logistic regression point
Analysis.4 selected genes are all significant related to bladder cancer.Then multivariable logistic regression analysis is carried out, 4 kinds of genes are obtained
Regression coefficient.
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 4 genes, creation can be used for bladder cancer detection/diagnosis risk score model: G=
(6.096 × HOXA9 methylation level)+(3.517 × PCDH17 methylation level)+(4.582 × POU4F2 methylation level)+
(5.268 × ONECUT2 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 -1.893, it is judged as high-risk-type bladder cancer (high risk)；When G value is equal to or less than -1.893,
It is judged as low risk bladder cancer (low-risk).As shown in Fig. 2, the specificity that above-mentioned risk score model carries out Diagnosis of Bladder reaches
73.2%, sensitivity shows there is good indicative function to bladder cancer detection/diagnosis up to 90.5%, AUC value 0.871.
Embodiment 4
The preparation of kit
Reagent preparation box includes: primer sequence as shown in Table 1, 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 1.
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.
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Claims (10)
1. a kind of Diagnosis of Bladder system, including data obtaining module, computing module and diagnostic module,
Wherein, the 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, PCDH17, POU4F2 and ONECUT2,
Wherein, detect that then methylation level is denoted as 1 for methylation, methylation is not detected, and then methylation level is denoted as -1；
The 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=(6.096 × HOXA9 methylation level)+(3.517 × PCDH17 methylation level)+(4.582 ×
POU4F2 methylation level)+(5.268 × ONECUT2 methylation level)；
The diagnostic module is for executing the operation for judging subject's health status according to G value, wherein when G value is higher than setting value
When, then it is judged as high-risk-type bladder cancer；When G value is equal to or less than setting value, then it is judged as low risk bladder cancer.
2. system according to claim 1, wherein in the diagnostic module, setting value is -1.893.
3. system according to claim 1 or 2, wherein the Diagnosis of Bladder system further includes result output module, institute
Result output module is stated for exporting the judgement that diagnostic module obtains.
4. a kind of reagent combination, including nucleotide sequence primer pair 1, nucleosides as shown in SEQ ID NO:1 and SEQ ID NO:2
Acid sequence primer pair 2, nucleotide sequence such as SEQ ID NO:5 and SEQ ID as shown in SEQ ID NO:3 and SEQ ID NO:4
The primer pair 4 as shown in SEQ ID NO:7 and SEQ ID NO:8 of primer pair 3 and nucleotide sequence shown in NO:6, it is described
Reagent set share in detection urine in target gene methylation level, the target gene include: HOXA9, PCDH17,
POU4F2 and ONECUT2.
5. reagent combination according to claim 4, the reagent combination includes LC GreenPlusSaturated fluorescence dyestuff.
6. reagent according to claim 4 or 5 combination, the reagent combination is the early screening, tested for bladder cancer
Risk assessment, the parting of bladder cancer and/or the kit of prognosis prediction of person's generation bladder cancer.
7. the method for target gene methylation level, the target gene are selected from a kind of detection urine: HOXA9, PCDH17,
POU4F2 and ONECUT2, 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) it is combined using reagent described in any one of claim 6~8, measures the methylation level of target gene.
8. according to the method described in claim 7, wherein, in step 1), EDTA solution, In are prestored in the collecting pipe of urine sample
It is mixed after collecting urine.This facilitates the preservation of urine sample, reduces the DNA degradation during long-term urine saves, Jin Erti
The extraction yield of high urine DNA.
9. method according to claim 7 or 8, wherein in step 3), the methylation level of target gene is to pass through high score
Distinguish solubility curve polymerase chain reaction (HRM-PCR), methylation status of PTEN promoter (MS-PCR), M-MLPA, pyrosequencing,
MALDI-TOF measurement.
10. method according to claim 7 or 8, wherein in step 3), the methylation level of target gene is to pass through
HRM-PCR measurement.
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