CN106636390A - Gene methylation detection method, detection kit and application thereof - Google Patents
Gene methylation detection method, detection kit and application thereof Download PDFInfo
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Abstract
The invention discloses a gene methylation detection method, a detection kit and an application thereof. Gene methylation detection sensitivity is improved by two strands of the same gene for the first time, namely, a positive-sense strand and antisense strand methylation fluorescent accumulating method. Compared with an existing methylation detection technology, the method for detecting gene methylation degrees by the two strands has the advantage of higher gene methylation detection sensitivity. The two strands of DNA (deoxyribonucleic acid) are sufficiently used, fluorescent signal values are increased, methylation fluorescence is double accumulated, the gene methylation detection sensitivity is improved, and the methylation degrees can be distinguished by a qualitative method. Besides, the detection process is reaction in closed pipes with the same hole, namely, methylation of the two strands can be simultaneously detected in one hole, operation is simple and rapid, and the possibility of pollution is decreased. Safety is achieved, the whole system is free from toxic and harmful substances, open pipes of PCR (polymerase chain reaction) products are omitted, and testers and environments are not harmed.
Description
Technical field
The invention belongs to the DNA methylation assay of biological field, more particularly to gene.
Background technology
The present invention relates to field of gene detection, more particularly to a kind of side that can improve gene methylation detection sensitivity
Method and its kit.DNA methylation is closely related with the generation of tumour development, and it is a tumorigenic earliest events, by
In CPG islands high methylation earlier than tumor proliferative, therefore detect some DNA sites methylate can as tumor markers,
It is a potential index of tumour early stage auxiliary diagnosis, ill prediction and curative effect evaluation.It is right that DNA methylation analysis generally needs
DNA carries out high temperature vulcanized conversion modification.After high temperature vulcanized conversion, double-stranded DNA can be dissociated into two it is not complementary single-stranded.Exist at present
It is existing also without the precedent for detecting the single-stranded aggregate level that methylates of same gene two simultaneously in existing DNA methylation assay technology
Some gene methylation detection techniques be both for one it is single-stranded enter performing PCR reaction, so as to detect gene methylation situation.At certain
In class lesion detection, the degree of DNA methylation is not it is obvious that the sensitivity of single-stranded detection gene methylation is relatively low, it is necessary to adopted
The degree of gene methylation is just can determine that with sizing technique, and then distinguishes cancer and non-cancer.Sizing technique analysis more trouble is complicated, far not
Such as qualitative method analysis simply.Single-stranded detection gene methylation does not take into full account the methylated aggregate level of two chains of gene.Example
Such as Application No. 201610264899.7《In a kind of human peripheral blood dissociative DNA septin9 gene methylations detection kit and
Detection method》.
The content of the invention
In view of this, one of purpose of the invention is to improve gene methylation detection sensitivity, can fully be detected
The aggregate level that methylates of two chains of gene.With reference gene as the accuracy and validity of criterion the inventive method.
The invention provides a kind of gene methylation detection method, the method is first against genes of interest purpose of design gene
The primer pair and genes of interest of positive-sense strand of methylating methylates the primer pair of antisense strand, and design testing goal gene methylation
Positive and negative adopted chain two probes, genes of interest methylate positive-sense strand primer pair be purpose gene methylation positive-sense strand just
Methylate the reverse primer of positive-sense strand to primer and genes of interest, and the methylate primer pair of antisense strand of genes of interest is purpose gene
The forward primer and genes of interest of antisense strand of methylating methylates the reverse primer of antisense strand, and genes of interest is methylated positive and negative
Two probes of adopted chain are respectively that genes of interest methylates positive-sense strand probe and genes of interest methylates antisense strand probe.Due to this
Method considers the methylated situation design relevant primer of two chains of gene and probe, can be detected using the primer and probe of design
The methylated integral level of genes of interest.With reference gene as the accuracy and validity of criterion the inventive method.
According to an aspect of the present invention, a kind of gene methylation detection method is additionally provided, genes of interest is septin9 bases
Cause, the septin9 methylation sites for promoter region CpG islands, sequence for transcription initiation site upstream -670～-
555bp areas；
According to an aspect of the present invention, a kind of gene methylation detection method is additionally provided,
Genes of interest septin9 methylates the forward primer sequence such as SEQ No.1 of positive-sense strand：
5’-TCGTTGTTTATTAGTTATTATG-3’
Genes of interest septin9 methylates the reverse primer sequences such as SEQ No.2 of positive-sense strand：
5’-TCCGAAATAATCCCATCCCAT-3’
Genes of interest septin9 methylates the forward primer sequence such as SEQ No.3 of antisense strand：
5’-TTCGAAATGATTTTATTTAGTTG-3’
Genes of interest septin9 methylates the reverse primer sequences such as SEQ No.4 of antisense strand：
5’-CGCTACCCACCAACCATC-3’；
According to an aspect of the present invention, a kind of gene methylation detection method is additionally provided,
Genes of interest septin9 methylates positive-sense strand probe sequence such as SEQ No.5：
5’-TAACCGCGAAATCCGACATA-3’
Genes of interest septin9 methylates antisense strand probe sequence such as SEQ No.6：
5’-TATACCGAACCCCGCGATCA-3’。
According to an aspect of the present invention, a kind of gene methylation detection method, genes of interest septin9 methyl are additionally provided
It is FAM to change positive-sense strand probe, the genes of interest septin9 antisense strand probe 5' ends reporter fluorescence group that methylates, genes of interest
Septin9 methylate positive-sense strand probe, genes of interest septin9 methylate antisense strand probe 3' ends quencher be BHQ1.
According to an aspect of the present invention, a kind of gene methylation detection method is additionally provided, is methylated using reference gene
Primer pair and reference gene methylate probe, reference gene is ACTB,
Reference gene primer sequence such as SEQ No.7：
ACTB-F:5 '-GAATTTGTGTTTGTTATTGTGTGTTGGGT-3 ',
Reference gene primer sequence such as SEQ No.8：
ACTB-R:5 '-CCTACTCCTCCCTTAAAAATTACAA-3 ',
Reference gene methylates probe sequence such as SEQ No.9：
ACTB-probe:5’-ACCACCACCCAACACACAATAACAAACACA-3’。
According to an aspect of the present invention, a kind of gene methylation detection method is additionally provided, method and step is：
A, DNA is extracted from sample to be tested,
B, the DNA to extracting carry out sulfuration conversion modification,
C, the DNA after modification is purified, obtains sample form,
D, the genes of interest the is methylated forward primer of positive-sense strand, genes of interest methylate reversely the drawing of positive-sense strand
The methylate forward primer sequence of antisense strand, the genes of interest of thing, genes of interest methylates reverse primer, the purpose base of antisense strand
Because methylating positive-sense strand probe and genes of interest methylates the methylated primer pair of antisense strand probe, reference gene and reference gene
Methylate probe and the sample form carry out pcr amplification reaction while being added in same hole,
Septin9 gene C P values in E, the positive and negative adopted double-strand fluorescence accumulative of calculating genes of interest；With reference gene as standard
Weigh the accuracy and validity of the inventive method.
According to an aspect of the present invention, a kind of gene methylation detection method, the end of pcr amplification reaction system are additionally provided
Concentration is consisted of：1～10 × PCR buffer solutions, 0.1～1mM dNTPs, 0.1～1uM genes of interest methylate the primer of positive-sense strand
Methylate primer pair, 0.05～2ng/ul template DNAs, 0.1～1uM genes of interest of antisense strand to, 0.1～1uM genes of interest
Methylate positive-sense strand probe, 0.1～1uM genes of interest methylate antisense strand probe, 0.01～0.10U/ul TaqDNA polymerization
Enzyme, 1～5mM magnesium chlorides, 0.1～1uM reference gene methylated primers pair, 0.1～1uM reference genes methylate probe.
According to an aspect of the present invention, a kind of gene methylation detection method is additionally provided, genes of interest is HOXA9 bases
Cause,
Genes of interest HOXA9 methylates the forward primer sequence such as SEQ No.10 of positive-sense strand：
5’-TTATTGTTTTGTTGGACGGGTACG-3’
Genes of interest HOXA9 methylates the reverse primer sequences such as SEQ No.11 of positive-sense strand：
5’-AATTTCATATAACAACTTAATAACACCG-3’
Genes of interest HOXA9 methylates the forward primer sequence such as SEQ No.12 of antisense strand：
5’-CGTTCGCGTTTTTATTGGTC-3’
Genes of interest HOXA9 methylates the reverse primer sequences such as SEQ No.13 of antisense strand：
5’-CGAACCATTAATAACGTACGAA-3’
Genes of interest HOXA9 methylates positive-sense strand probe sequence such as SEQ No.14：
5’-AAATTACCGACGCCCGCG-3’
Genes of interest HOXA9 methylates antisense strand probe sequence such as SEQ No.15：
5’-AAACGAACACGTAACGCG-3’；
According to an aspect of the present invention, a kind of carcinoma of the rectum detection method is additionally provided, is examined using above-mentioned gene methylation
Survey method testing goal gene methylation degree judges whether with the carcinoma of the rectum；
According to an aspect of the present invention, a kind of gene methylation detection kit is additionally provided, comprising genes of interest methyl
Change the methylate reverse primer of positive-sense strand, genes of interest of the forward primer of positive-sense strand, genes of interest to methylate the forward direction of antisense strand
The methylate reverse primer of antisense strand, genes of interest of primer, genes of interest methylates positive-sense strand probe and genes of interest methylates
Antisense strand probe；
According to an aspect of the present invention, a kind of gene methylation detection kit is additionally provided,
Genes of interest methylates the forward primer sequence such as SEQ No.1 of positive-sense strand：
5 '-TCGTTGTTTATTAGTTATTATG-3 ',
The genes of interest methylates the reverse primer sequences such as SEQ No.2 of positive-sense strand：
5 '-TCCGAAATAATCCCATCCCAT-3 ',
The genes of interest methylates the forward primer sequence such as SEQ No.3 of antisense strand：
5 '-TTCGAAATGATTTTATTTAGTTG-3 ',
The genes of interest methylates the reverse primer sequences such as SEQ No.4 of antisense strand：
5 '-CGCTACCCACCAACCATC-3 ',
The genes of interest methylates positive-sense strand probe sequence such as SEQ No.5：
5 '-TAACCGCGAAATCCGACATA-3 ',
The genes of interest methylates antisense strand probe sequence such as SEQ No.6：
5 '-TATACCGAACCCCGCGATCA-3 ', above-mentioned genes of interest is septin9.
According to an aspect of the present invention, a kind of gene methylation detection kit is additionally provided,
Genes of interest methylates the forward primer sequence such as SEQ No.10 of positive-sense strand：
5’-TTATTGTTTTGTTGGACGGGTACG-3’
Genes of interest methylates the reverse primer sequences such as SEQ No.11 of positive-sense strand：
5’-AATTTCATATAACAACTTAATAACACCG-3’
Genes of interest methylates the forward primer sequence such as SEQ No.12 of antisense strand：
5’-CGTTCGCGTTTTTATTGGTC-3’
Genes of interest methylates the reverse primer sequences such as SEQ No.13 of antisense strand：
5’-CGAACCATTAATAACGTACGAA-3’
Genes of interest methylates positive-sense strand probe sequence such as SEQ No.14：
5’-AAATTACCGACGCCCGCG-3’
Genes of interest methylates antisense strand probe sequence such as SEQ No.15：
5 '-AAACGAACACGTAACGCG-3 ', above-mentioned genes of interest is HOXA9.
According to certain aspects of the invention, a genoid methylation detection kit is additionally provided, genes of interest methylates
Positive-sense strand probe, the genes of interest antisense strand probe 5' ends reporter fluorescences group that methylates is FAM, and genes of interest methylates positive-sense strand
Probe, genes of interest methylate antisense strand probe 3' ends quencher be BHQ1；
According to an aspect of the present invention, a kind of gene methylation detection kit is additionally provided, it is characterised in that the examination
Agent box also methylates probe comprising the methylated primer pair of reference gene and reference gene, and the reference gene is ACTB,
Reference gene primer sequence such as SEQ No.7：
ACTB-F:5 '-GAATTTGTGTTTGTTATTGTGTGTTGGGT-3 ',
The reference gene primer sequence such as SEQ No.8：
ACTB-R:5 '-CCTACTCCTCCCTTAAAAATTACAA-3 ',
The reference gene methylates probe sequence such as SEQ No.9：
ACTB-probe:5’-ACCACCACCCAACACACAATAACAAACACA-3’
According to an aspect of the present invention, a kind of application of gene methylation detection kit, gene methylation are additionally provided
Detection kit is for carcinoma of the rectum detection.
The Integral Thought of the present invention is as follows：
The invention mainly solves the technical problem of a kind of method that can improve gene methylation detection sensitivity is provided,
The detection method makes full use of two chains of gene methylation, easy to operate, high specificity and detection sensitivity height.
One aspect of the present invention is：The sensitivity for improving gene methylation detection is detected using double-strand, this
Methylate the primer pair of antisense strand comprising the methylate primer pair and genes of interest of positive-sense strand of genes of interest in the method for invention, make
For the methylated universal primers pair of reference gene ACTB and probe of criterion, and testing goal gene methylation just,
Two probes of antisense strand.
One object of the present invention gene is septin9 genes；Septin9 methylation sites are the CpG islands of promoter region,
Sequence is -670～-555bp areas of transcription initiation site upstream.For the site, the genes of interest of present invention design methylates
The primer pair sequence of positive and negative adopted chain is as follows.
Genes of interest methylates the forward primer of positive-sense strand：5’-TCGTTGTTTATTAGTTATTATG-3’；
Genes of interest methylates the reverse primer of positive-sense strand：5’-TCCGAAATAATCCCATCCCAT-3’；
Genes of interest methylates the forward primer of antisense strand：5’-TTCGAAATGATTTTATTTAGTTG-3’；
Genes of interest methylates the reverse primer of antisense strand：5’-CGCTACCCACCAACCATC-3’；
The positive and negative adopted chain probe 5' ends reporter fluorescence group of the septin9 methylated genes of the present invention is FAM, septin9
The quencher at the positive and negative adopted chain probe 3' ends of methylated genes is BHQ1.For the detection of septin9 methylation sites design
Genes of interest methylate positive and negative adopted chain two probe sequences it is as follows.
Genes of interest methylates positive-sense strand probe sequence：5’-TAACCGCGAAATCCGACATA-3’；
Genes of interest methylates antisense strand probe sequence：5’-TATACCGAACCCCGCGATCA-3’；
The primer pair and probe of the internal reference ACTB methylated genes of the present invention, sequence is as follows：
ACTB-F:5’-GAATTTGTGTTTGTTATTGTGTGTTGGGT-3’
ACTB-R:5’-CCTACTCCTCCCTTAAAAATTACAA-3’
ACTB-probe:5’-ACCACCACCCAACACACAATAACAAACACA-3’
Method of the present invention also includes the conventional reagent of other PCR amplifications, mainly includes：Archaeal dna polymerase, Mg2+,
DNTPs, PCR buffer etc..The final concentration of the pcr amplification reaction system is consisted of：1～10 × PCR buffer solutions, 0.1～
Methylate positive-sense strand primer pair, 0.1～1uM septin9 of 1mM dNTPs, 0.1～1uM septin9 methylates antisense strand primer
, 0.1～1uM ACTB are methylated universal primer to, 0.05～2ng/ul template DNAs, 0.1～1uM septin9 methylate just
Adopted chain probe, 0.1～1uM septin9 methylate antisense strand probe, 0.1～1uM ACTB methylate general probe, 0.01～
0.10U/ul Taq DNA polymerases, 1～5mM magnesium chlorides.
A kind of detecting step of the present invention is as follows：
(1) DNA is extracted from sample to be tested.
(2) DNA after extraction is carried out into sulfuration conversion modification.
(3) DNA after modification is purified, obtains sample form.
(4) performing PCR amplification is entered to sample form, amplification condition is：95 DEG C of denaturations 20 minutes, PCR is expanded
The increasing stage, 95 DEG C of denaturation 20s, 56 DEG C of annealing 35s, and carry out 50 circulations.
(5) septin9 gene Cs P value and alone chain detection septin9 in positive and negative adopted double-strand fluorescence accumulative are compared
The CP values of gene, or compare the positive inspection of intestinal cancer sample septin9 gene methylations in double-strand detection method and single-stranded detection method
Go out rate and judge methylated detection level in two kinds of detection method sample forms.With the amplification curve of internal reference Gene A CTB and CP values
The accuracy and validity of judgment experiment.
It is true biological medicine that the DNA extracts reagents and DNA sulfurations conversion reagent that the present invention is used is Suzhou Industrial Park
The reagent of Co., Ltd's autonomous production, extracts and sulfiding reagent is for sale.
The present invention first using same gene two chains, i.e., positive and negative adopted chain methylate fluorescence accumulation method improve gene
DNA methylation assay sensitivity.Utilization double-strand detection gene methylation compared with existing DNA methylation assay technology, in the present invention
The method of degree has the advantages that higher gene methylation detection sensitivity, and the inventive method makes full use of two chains of DNA,
Increase fluorescence signal value, make the fluorescent dual accumulation that methylates, improve the detection sensitivity of gene methylation, can be in (95%) non-first
Methylating for genes of interest is detected under the background that base DNA is present, i.e., the side of present invention when methylate DNA content is only 5%
Method also can be detected accurately.The inventive method can distinguish methylation height using qualitative method, according to amplification curve and Cp values
Methylation height can be distinguished.In addition, utilization double-strand of the invention detection improves gene methylation detection sensitivity
Method have further the advantage that：(1) detection process is that stopped pipe reacts with hole, i.e., be detected simultaneously by two chains in a Kong Zhongke
Methylation status, it is simple to operate quick, reduce the possibility of pollution；(2) double reaction：Genes of interest is in reference gene
Different fluorescence channels, can be monitored and be assessed by reference gene to the accuracy of whole experiment and validity.(3) pacify
Entirely, whole system does not include poisonous and harmful substance, without the need for the open pipe of PCR primer, to testing crew and environment all non-hazardous.
The present invention has invented the kit of correlation according to the double-strand detection method, while PCR amplification conditions are optimized, further
Improve amplified reaction efficiency.
Description of the drawings
In order that the purpose of the present invention, technical scheme and beneficial effect are clearer, the present invention provides drawings described below and carries out
Explanation：
Fig. 1 is the principle schematic of the present invention；
Fig. 2 is the comparing result figure of the present invention and conventional method used in 1 intestinal cancer plasma sample；
Fig. 3 is the experimental result picture that the sensitivity that methylates is detected using the present invention；
Fig. 4 is the detection amplification figure using the present invention 1 lung cancer plasma sample (three multiple holes) of detection.
Specific embodiment
Below in conjunction with accompanying drawing, the preferred embodiments of the present invention are described in detail.It is unreceipted in preferred embodiment
The experimental technique of actual conditions, generally according to normal condition, or is carried out according to the condition proposed by manufacturer.
Embodiment 1
The technical scheme in the embodiment of the present invention will be clearly and completely described below, it is clear that described enforcement
Example is only a part of embodiment of the present invention, rather than the embodiment of whole.Based on the embodiment in the present invention, this area is common
All other embodiment that technical staff is obtained under the premise of creative work is not made, belongs to the model of present invention protection
Enclose.
Material：Methylation positive DNA, the moon that methylates that concentration is 2ng/ul of plasma sample to be checked, concentration for 2ng/ul
Property DNA.
Instrument：Lightcycler 480, rotation vortex mixer, water-bath, whirlpool concussion instrument, refrigerator.
Reagent：Archaeal dna polymerase (Roche Holding Ag), 10 × PCR Buffer (Roche Holding Ag), MgCl2(Roche Holding Ag),
DNTP (TaKaRa), purified water.
Primer and probe：The purity of all primers should reach electrophoresis level (PAGE) or HPLC levels, without miscellaneous band.All probes
5' ends reporter fluorescence group is FAM, and the quencher at 3' ends is BHQ1.The sequence of all primers and probe is by present invention research people
Member's autonomous Design, is then provided by supplier, and concentration is that 10 μm of ol/L are standby.
Septin9 methylates the forward primer sequence such as SEQ No.1 of positive-sense strand：
5’-TCGTTGTTTATTAGTTATTATG-3’
Septin9 methylates the reverse primer sequences such as SEQ No.2 of positive-sense strand：
5’-TCCGAAATAATCCCATCCCAT-3’
Septin9 methylates the forward primer sequence such as SEQ No.3 of antisense strand：
5’-TTCGAAATGATTTTATTTAGTTG-3’
Septin9 methylates the reverse primer sequences such as SEQ No.4 of antisense strand：
5’-CGCTACCCACCAACCATC-3’；
Septin9 methylates positive-sense strand probe sequence such as SEQ No.5：
5’-TAACCGCGAAATCCGACATA-3’
Septin9 methylates antisense strand probe sequence such as SEQ No.6：
5’-TATACCGAACCCCGCGATCA-3’。
Methylated probe using the methylated primer pair of reference gene and reference gene, reference gene is ACTB,
Reference gene primer sequence such as SEQ No.7：
ACTB-F:5 '-GAATTTGTGTTTGTTATTGTGTGTTGGGT-3 ',
Reference gene primer sequence such as SEQ No.8：
ACTB-R:5 '-CCTACTCCTCCCTTAAAAATTACAA-3 ',
Reference gene methylates probe sequence such as SEQ No.9：
ACTB-probe:5’-ACCACCACCCAACACACAATAACAAACACA-3’。
Sample to be tested is processed, the sample to be tested is human normal plasma or intestinal cancer blood plasma.
A. DNA is extracted from sample to be tested (Suzhou is true company).
B. the DNA to extracting carries out sulfuration conversion modification (Suzhou is true company).
C. the DNA after modification is purified, obtains sample form.
By septin9 positive-sense strands primer pair and positive-sense strand probe, the antisense strand primer pair and antisense strand probe of autonomous Design
And the methylated primer pair of DNA profiling, the reference gene and reference gene probe that methylates enters performing PCR while being added in same hole
Amplified reaction.
PCR reaction systems and condition
The final concentration of pcr amplification reaction system is consisted of：1 × PCR buffer solutions, 0.25mM dNTPs, 350nM septin9
Methylate positive-sense strand primer pair, 350nM septin9 methylates antisense strand primer pair, 0.05ng/ul template DNAs, 70nM
Methylate positive-sense strand probe, 70nM septin9 of septin9 methylates antisense strand probe, 1.5U Taq DNA polymerases, 2.5mM
Magnesium chloride.
The detailed process of pcr amplification reaction is：95 DEG C of denaturations 20 minutes, the PCR amplification stage, 95 DEG C
Denaturation 20s, 56 DEG C of annealing 35s, and carry out 50 circulations.If there is amplified signal, and S-type amplification curve, then it is positive sample,
The appearance that methylates is illustrated；If rising without amplification curve, illustrate for negative sample, that is, to methylate.The Cp values of amplification are little
It is as a result virtual value when equal to 40.
Collect BJ Union Hospital's plasma of colorectal cancer 87, human normal plasma 107.With above-mentioned double-strand detection method to blood
Slurry sample carries out septin9 gene methylation detections, is extracted with the plasma DNA of QIAGEN companies, vulcanizes purification kit
Single-stranded detection method detection septin9 gene methylations compare experiment.Statistic mixed-state result, the internal reference base of all plasma samples
Because ACTB has amplification and Cp values respectively less than 40, it was demonstrated that the experiment accurate and effective.Observation genes of interest passage, 87 colorectal cancers
Plasma sample, the detection of double-strand detection method it is positive for 67, positive coincidence rate is 77.01%；The positive of single-stranded detection method detection
For 59, positive coincidence rate is 67.82%.Negative sample is analyzed, 107 human normal plasma samples, double-strand detection method
In 9 testing results be the positive, negative match-rate is 91.59%；4 testing results are the positive, feminine gender symbol in single-stranded detection method
Conjunction rate is 96.26%.Fig. 2 is the comparing result of two kinds of detection methods of wherein 1 intestinal cancer plasma sample, as illustrated, double-strand
The Cp values (curve 1) of the septin9 gene methylations of detection method detection are 29.71, and fluorescent value is 33 or so；Single-stranded detection method inspection
The Cp values (curve 2) of the septin9 gene methylations for going out are 33.37, and fluorescent value is 18 or so.As can be seen here, double-strand detection method
1-2 higher than the Cp values of the septin9 gene methylations of single-stranded detection method detection is individual, and the fluorescent value of double-strand detection method detection is also
2 times of single-stranded detection method detection.
Methylation positive DNA and the negative DNA that methylates are diluted into same concentrations, i.e. 2ng/ul, respectively 95%,
85%th, 50%, 30% non-methylate DNA exist background under, with double-strand detection method detection level be 5%, 15%, 50%,
70% methylate DNA, the reference gene ACTB of all detection samples has amplification and Cp values respectively less than 40, it was demonstrated that the experiment is accurate
Truly have effect.And then observation genes of interest passage, statistic mixed-state result is as shown in figure 3, curve 1 is expansion that methylation content is 70%
Increase curve, CP values are 23.90；Curve 2 is the amplification curve that methylation content is 50%, and CP values are 25.23；Curve 3 is methyl
Change the amplification curve that content is 15%, CP values are 26.71；Curve 4 is the amplification curve that methylation content is 5%, and CP values are
27.28, can be obtained according to result, double-strand detection method can detect genes of interest under the background that (95%) non-methylate DNA is present
Methylate, DNA methylation assay sensitivity can be greatly improved.
When gene methylation degree is not high, when the methylated Cp values of genes of interest are more than 40 in sample, single-stranded detection is legal
Justice is feminine gender, and now double-strand detection method can make full use of two chains, accumulate the fluorescence that methylates, and increase methylated detection, from
And improve the methylated sensitivity of genes of interest and methylation positive recall rate.
Embodiment 2
Inventor has also done the double-strand detection of HOXA9 gene methylations.
The probe of positive-sense strand：AAATTACCGACGCCCGCG
The forward primer of positive-sense strand：TTATTGTTTTGTTGGACGGGTACG
The reverse primer of positive-sense strand：AATTTCATATAACAACTTAATAACACCG
The probe of antisense strand：AAACGAACACGTAACGCG
The forward primer of antisense strand：CGTTCGCGTTTTTATTGGTC
The reverse primer of antisense strand：CGAACCATTAATAACGTACGAA
20 lung cancer plasma samples of detection and 13 human normal plasma samples.Plasma sample is entered with above-mentioned double-strand detection method
Row HOXA9 gene methylations are detected.The reference gene ACTB of all plasma samples has amplification and Cp values are respectively less than 40, it was demonstrated that should
Experiment accurate and effective.Observation genes of interest passage, statistic mixed-state result, the reference gene ACTB of all plasma samples has amplification
And Cp values are respectively less than 40, it was demonstrated that the experiment accurate and effective.Observation genes of interest passage, 20 lung cancer plasma samples, double-strand detection
Method detection it is positive for 15, positive coincidence rate is 75%；Negative sample is analyzed, 13 human normal plasma samples are double
1 testing result is the positive in chain detection method, and negative match-rate is 92.31%.Fig. 4 is wherein 1 lung cancer plasma sample (three
Multiple holes) detection amplification figure.As can be seen here, double-strand detection method is applicable not only to the detection of septin9 gene methylations, right
The DNA methylation assay of lung cancer marker HOXA9 genes also has higher Sensitivity and specificity.
<110>Jiangsu is true biological medicine technology limited company
<120>Gene methylation detection method and detection kit and its application
<160> 15
<210> 1
<211> 22
<212> DNA
<213>Artificial sequence
<220>
<400> 1
tcgttgttta ttagttatta tg 22
<210> 2
<211> 22
<212> DNA
<213>Artificial sequence
<220>
<400> 2
tccgaaataa tcccatccc at 22
<210> 3
<211> 23
<212> DNA
<213>Artificial sequence
<220>
<400> 3
ttcgaaatga ttttatttag ttg 23
<210> 4
<211> 18
<212> DNA
<213>Artificial sequence
<220>
<400> 4
cgctacccac caaccatc 18
<210> 5
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<400> 5
taaccgcgaa atccgacata 20
<210> 6
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<400> 6
tataccgaac cccgcgatca 20
<210> 7
<211> 29
<212> DNA
<213>Artificial sequence
<220>
<400> 7
gaatttgtgt ttgttattgt gtgttgggt 29
<210> 8
<211>25
<212> DNA
<213>Artificial sequence
<220>
<400> 8
cctactcctc ccttaaaaat tacaa 25
<210> 9
<211> 30
<212> DNA
<213>Artificial sequence
<220>
<400> 9
accaccaccc aacacacaat aacaaacaca 30
<210> 10
<211> 24
<212> DNA
<213>Artificial sequence
<220>
<400> 10
ttattgtttt gttggacggg tacg 24
<210> 11
<211> 28
<212> DNA
<213>Artificial sequence
<220>
<400> 11
aatttcatat aacaacttaa taacaccg 28
<210> 12
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<400> 12
cgttcgcgtt tttattggtc 20
<210> 13
<211>22
<212> DNA
<213>Artificial sequence
<220>
<400> 13
cgaaccatta ataacgtacg aa 22
<210>14
<211> 18
<212> DNA
<213>Artificial sequence
<220>
<400> 14
aaattaccga cgcccgcg 18
<210>15
<211> 18
<212> DNA
<213>Artificial sequence
<220>
<400> 15
aaacgaacac gtaacgcg 18
Claims (16)
1. gene methylation detection method, it is characterised in that：For drawing for genes of interest purpose of design gene methylation positive-sense strand
Thing pair and genes of interest methylate the primer pair of antisense strand, and the two of the positive and negative adopted chain of design testing goal gene methylation
Bar probe, the genes of interest methylate positive-sense strand primer pair for purpose gene methylation positive-sense strand forward primer and purpose
The reverse primer of gene methylation positive-sense strand, the genes of interest methylate antisense strand primer pair be purpose gene methylation it is anti-
The forward primer and genes of interest of adopted chain methylates the reverse primer of antisense strand, the methylated positive and negative adopted chain of the genes of interest
Two probes be respectively that genes of interest methylates positive-sense strand probe and genes of interest methylates antisense strand probe.
2. gene methylation detection method according to claim 1, it is characterised in that described genes of interest is
Septin9 genes, the septin9 methylation sites for promoter region CpG islands, sequence for transcription initiation site upstream-
670～-555bp areas.
3. gene methylation detection method according to claim 2, it is characterised in that
The genes of interest methylates the forward primer sequence such as SEQ No.1 of positive-sense strand：
5’-TCGTTGTTTATTAGTTATTATG-3’
The genes of interest methylates the reverse primer sequences such as SEQ No.2 of positive-sense strand：
5’-TCCGAAATAATCCCATCCCAT-3’
The genes of interest methylates the forward primer sequence such as SEQ No.3 of antisense strand：
5’-TTCGAAATGATTTTATTTAGTTG-3’
The genes of interest methylates the reverse primer sequences such as SEQ No.4 of antisense strand：
5’-CGCTACCCACCAACCATC-3’。
4. gene methylation detection method according to claim 3, it is characterised in that
The genes of interest methylates positive-sense strand probe sequence such as SEQ No.5：
5’-TAACCGCGAAATCCGACATA-3’
The genes of interest methylates antisense strand probe sequence such as SEQ No.6：
5’-TATACCGAACCCCGCGATCA-3’。
5. gene methylation detection method according to claim 4, the genes of interest methylates positive-sense strand probe, purpose
Reporter fluorescences group in gene methylation antisense strand probe 5' ends is FAM, and the genes of interest methylates positive-sense strand probe, purpose base
Because the quencher at the antisense strand probe 3' ends that methylate is BHQ1.
6. gene methylation detection method according to claim 5, it is characterised in that draw using reference gene is methylated
Thing pair and reference gene methylate probe, and the reference gene is ACTB,
The reference gene primer sequence such as SEQ No.7：
ACTB-F:5 '-GAATTTGTGTTTGTTATTGTGTGTTGGGT-3 ',
The reference gene primer sequence such as SEQ No.8：
ACTB-R:5 '-CCTACTCCTCCCTTAAAAATTACAA-3 ',
The reference gene methylates probe sequence such as SEQ No.9：
ACTB-probe:5’-ACCACCACCCAACACACAATAACAAACACA-3’。
7. gene methylation detection method according to claim 6, it is characterised in that methods described step is：
A, DNA is extracted from sample to be tested,
B, the DNA to extracting carry out sulfuration conversion modification,
C, the DNA after modification is purified, obtains sample form,
D, the genes of interest the is methylated forward primer of positive-sense strand, genes of interest methylate reverse primer, the mesh of positive-sense strand
The forward primer of gene methylation antisense strand, the methylate reverse primer of antisense strand, genes of interest of genes of interest methylate just
Adopted chain probe and genes of interest methylate the methylated primer pair of antisense strand probe, reference gene and reference gene methylates probe
And the sample form is added in same hole and carries out pcr amplification reaction simultaneously.
Septin9 gene C P values in E, the positive and negative adopted double-strand fluorescence accumulative of calculating genes of interest.
8. gene methylation detection method according to claim 7, the final concentration composition of the pcr amplification reaction system
For：1～10 × PCR buffer solutions, 0.1～1mM dNTPs, 0.1～1uM genes of interest methylate positive-sense strand primer pair, 0.1～
The methylate primer pair of antisense strand, 0.05～2ng/ul template DNAs, 0.1～1uM genes of interest of 1uM genes of interest methylates just
Adopted chain probe, 0.1～1uM genes of interest methylate antisense strand probe, 0.01～0.10U/ul Taq DNA polymerases, 1～5mM
Magnesium chloride, 0.1～1uM reference gene methylated primers pair, 0.1～1uM reference genes methylate probe.
9. gene methylation detection method according to claim 1, it is characterised in that the genes of interest is HOXA9 bases
Cause,
The genes of interest methylates the forward primer sequence such as SEQ No.10 of positive-sense strand：
5’-TTATTGTTTTGTTGGACGGGTACG-3’
The genes of interest methylates the reverse primer sequences such as SEQ No.11 of positive-sense strand：
5’-AATTTCATATAACAACTTAATAACACCG-3’
The genes of interest methylates the forward primer sequence such as SEQ No.12 of antisense strand：
5’-CGTTCGCGTTTTTATTGGTC-3’
The genes of interest methylates the reverse primer sequences such as SEQ No.13 of antisense strand：
5’-CGAACCATTAATAACGTACGAA-3’
The genes of interest methylates positive-sense strand probe sequence such as SEQ No.14：
5’-AAATTACCGACGCCCGCG-3’
The genes of interest methylates antisense strand probe sequence such as SEQ No.15：
5’-AAACGAACACGTAACGCG-3’。
10. carcinoma of the rectum detection method, it is characterised in that usage right requires the method testing goal gene methylation described in 1～8
Degree judges whether with the carcinoma of the rectum.
11. gene methylation detection kits, it is characterised in that forward primer, the mesh of the positive-sense strand that methylates comprising genes of interest
The reverse primer of gene methylation positive-sense strand, the methylate forward primer of antisense strand, genes of interest of genes of interest methylate instead
The reverse primer of adopted chain, genes of interest methylate positive-sense strand probe and genes of interest methylates antisense strand probe.
12. gene methylation detection kits according to claim 11, it is characterised in that described genes of interest is
Septin9 genes,
The genes of interest methylates the forward primer sequence such as SEQ No.1 of positive-sense strand：
5’-TCGTTGTTTATTAGTTATTATG-3’
The genes of interest methylates the reverse primer sequences such as SEQ No.2 of positive-sense strand：
5’-TCCGAAATAATCCCATCCCAT-3’
The genes of interest methylates the forward primer sequence such as SEQ No.3 of antisense strand：
5’-TTCGAAATGATTTTATTTAGTTG-3’
The genes of interest methylates the reverse primer sequences such as SEQ No.4 of antisense strand：
5’-CGCTACCCACCAACCATC-3’；
The genes of interest methylates positive-sense strand probe sequence such as SEQ No.5：
5’-TAACCGCGAAATCCGACATA-3’
The genes of interest methylates antisense strand probe sequence such as SEQ No.6：
5’-TATACCGAACCCCGCGATCA-3’。
13. gene methylation detection kits according to claim 11, it is characterised in that the genes of interest is
HOXA9 genes,
The genes of interest methylates the forward primer sequence such as SEQ No.10 of positive-sense strand：
5’-TTATTGTTTTGTTGGACGGGTACG-3’
The genes of interest methylates the reverse primer sequences such as SEQ No.11 of positive-sense strand：
5’-AATTTCATATAACAACTTAATAACACCG-3’
The genes of interest methylates the forward primer sequence such as SEQ No.12 of antisense strand：
5’-CGTTCGCGTTTTTATTGGTC-3’
The genes of interest methylates the reverse primer sequences such as SEQ No.13 of antisense strand：
5’-CGAACCATTAATAACGTACGAA-3’
The genes of interest methylates positive-sense strand probe sequence such as SEQ No.14：
5’-AAATTACCGACGCCCGCG-3’
The genes of interest methylates antisense strand probe sequence such as SEQ No.15：
5’-AAACGAACACGTAACGCG-3’。
The 14. gene methylation detection kits according to claim 12 or 13, it is characterised in that the genes of interest first
Base positive-sense strand probe, the genes of interest antisense strand probe 5' ends reporter fluorescences group that methylates is FAM, the genes of interest methyl
It is BHQ1 to change the methylate quencher at antisense strand probe 3' ends of positive-sense strand probe, genes of interest.
15. gene methylation detection kits, it is characterised in that the kit also includes the methylated primer pair of reference gene
Methylate probe with reference gene, the reference gene is ACTB,
The reference gene primer sequence such as SEQ No.7：
ACTB-F:5 '-GAATTTGTGTTTGTTATTGTGTGTTGGGT-3 ',
The reference gene primer sequence such as SEQ No.8：
ACTB-R:5 '-CCTACTCCTCCCTTAAAAATTACAA-3 ',
The reference gene methylates probe sequence such as SEQ No.9：
ACTB-probe:5’-ACCACCACCCAACACACAATAACAAACACA-3’。
The application of the gene methylation detection kit described in 16. claims 12, it is characterised in that the gene methylation inspection
Test agent box is for carcinoma of the rectum detection.
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| CN112575082A (en) * | 2020-12-03 | 2021-03-30 | 广东辉锦创兴生物医学科技有限公司 | Fluorescent quantitative PCR detection kit for target gene methylation detection |
| CN113549694A (en) * | 2021-08-27 | 2021-10-26 | 深圳市优圣康生物科技有限公司 | Novel cervical cancer methylation gene detection method |
| CN116219020A (en) * | 2023-03-02 | 2023-06-06 | 北京和瑞精湛医学检验实验室有限公司 | Methylation reference gene and application thereof |
| CN116219020B (en) * | 2023-03-02 | 2023-11-07 | 北京和瑞精湛医学检验实验室有限公司 | Methylation reference gene and application thereof |
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