CN108949955A - Fluorescent quantitation method detects kit and its application of FMR1 gene promoter region methylation - Google Patents
Fluorescent quantitation method detects kit and its application of FMR1 gene promoter region methylation Download PDFInfo
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Abstract
The present invention relates to the kits of fluorescent quantitation method detection FMR1 gene promoter region methylation, the kit includes the following PCR primer and probe for detecting the methylation of FMR1 gene promoter region, and there is design in primer and probe is the base being designed for FMR1 gene methylation site.Relative to PCR capillary electrophoresis method, fluorescence quantifying PCR method is detected kit of the present invention for FMR1 methylation, is not limited by (CGG) n repeat number number is excessive, therefore do not allow to easily cause and fail to pinpoint a disease in diagnosis；And relative to MS-PCR, fluorescence quantifying PCR method on probe in addition to the specificity for methylation sites detection, also also increasing the specificity to methylation sites detection on primer.
Description
Technical field
The present invention relates to molecular diagnosis field, in particular to fluorescent quantitation method detects FMR1 gene promoter region methyl
The kit of change and its application.
Background technique
Fragile X mental retardation (Fragile X Syndrome, FXS) be only second to Down syndrome disease incidence second it is high
Heredity feeblemindedness disease is incomplete dominance X linked genetic diseases.Fragile X mental retardation feeblemindedness gene I (Fragile X
Mental Retardation I, FMR1) be located at chromosome x q27.3, in genome sequence about 38Kb, by 17 exons and
16 introne compositions.The molecular genetics basis of FXS is that the number of the end FMR1 gene 5' noncoding region (CGG) n is different, is divided into
Normally, premutation, full mutation.(CGG) length of n repetitive sequence has polymorphism in crowd.In FXS patient, (CGG) n
230 times, even up to thousands of times are repeated more than, this seed type is known as full mutation.Full mutation be frequently accompanied by the duplicate block (CGG) n and
The high methylation of FMR1 gene promoter region.Another situation is that FXS patient inherits abnormal FRM1 from its mother
Allele, their mother or carry it is similar it is abnormal repeat, but due to women X chromosome it is random inactivate without
It shows abnormal phenotype or its FMR1 equipotential is only interim form, referred to as premutation.(CGG) n repeat number of premutation is 53-
Between 230, small premutation does not have abnormal methylation；Big premutation will appear partial methylation.
FXS clinical phenotypes include feeblemindedness, attention disorders, have self-closing disease tendency.Typical patient also has after puberty
There are the corporal characteristics such as prominent high forehead, big ear and lower jaw, big testis, range of motion be excessive.Due in preadolescence illness youngster
Virgin clinical manifestation is without specificity, so medical diagnosis on disease and screening are mainly according to laboratory diagnostic method.Currently used molecule
Diagnostic method has PCR capillary electrophoresis, gene with methylation specific PCR (MS-PCR).PCR capillary electrophoresis is for FMR1
(CGG) n repeat region of gene designs suitable primer, expands the repeat region, amplified fragments size will be repeated by (CGG) n
Number determines, the size of PCR product is distinguished by Capillary Electrophoresis, extrapolates (CGG) n repeat number according to the size.PCR capillary
The advantages of electrophoresis tube method is that normal and small premutation is particularly effective.But big full mutation amplification is just difficult, may lead
Cause is failed to pinpoint a disease in diagnosis.
MS-PCR method is the promoter region that can cause FMR1 gene for the full mutation of FMR1 gene and big premutation
The situation that methylates realizes diagnosis.But MS-PCR finally needs to detect whether that there are methyl by electrophoretic
Change, it is difficult to do the screening of great amount of samples.And the specificity of PCR primer is depended merely in MS-PCR only to distinguish methylation and non-methyl
The DNA of change state, it is specific and bad.
Summary of the invention
The present invention provides a kind of kit of fluorescent quantitation method detection FMR1 gene promoter region methylation, the examination
Agent box includes the following PCR primer and probe for detecting the methylation of FMR1 gene promoter region: upstream primer:Downstream primer:And spy
Needle sequence:Wherein tiltedly bold-faced base is for FMR1 gene methyl
Change the base that site is designed.
In one embodiment, 5 ' ends of the probe sequence have fluorescent reporter group VIC, and 3 ' ends are quenched with fluorescence
Go out group MGB.
In one embodiment, the amplification reference gene behaviour Actb gene；Actb upstream region of gene primer:
GTGTATAGGAGAGTATTAGGAAGTGGT, Actb downstream of gene primer: CCACTAAACCTCCATTCAACTAACC；And Actb
The sequence of gene probe are as follows: AAAACAAAAACTCCAACCCCACCC.
In one embodiment, the end of Actb gene probe 5 ' has fluorescence radiation group VIC, and 3 ' ends have fluorescence
Quenching group MGB.
In one embodiment, application of the mentioned reagent box in fragile X mental retardation detection is provided.
The present invention is directed to the defect of Capillary Electrophoresis and MS-PCR, and fluorescence quantifying PCR method has been used to detect FMR1 gene
The state of methylation.Relative to PCR capillary electrophoresis method, fluorescence quantifying PCR method is detected for FMR1 methylation, no
It is limited by (CGG) n repeat number number is excessive, therefore is not allowed to easily cause and fail to pinpoint a disease in diagnosis；And relative to MS-PCR, quantitative fluorescent PCR side
Method on probe in addition to, with the specificity for methylation sites detection, also also increasing and detecting to methylation sites on primer
Specificity, in the specificity of DNA methylation assay be better than MS-PCR；And fluorescent quantitation method is no longer needed to through electrophoresis
It is online observation as a result, a possibility that decreasing pollution.Primer and probe of the invention all cannot with do not methylate
Sequence reacts.Primer and probe of the present invention includes the site to methylate ingenious in designly, so that greatly reducing non-spy
Anisotropic phenomenon.
Detailed description of the invention
It in order to more clearly explain the technical solutions in the embodiments of the present application, below will be to needed in the embodiment
Attached drawing is briefly described, it should be apparent that, the accompanying drawings in the following description is only some embodiments as described in this application, right
For those of ordinary skill in the art, without creative efforts, it can also be obtained according to these attached drawings
Its attached drawing.
Fig. 1 is the fluorescent quantitation result exhibition of fragile X positive sample (Fig. 1 a) and negative sample (Fig. 1 b) that the present invention detects
Diagram, wherein short scatterplot lines indicate FMR1 gene methylation probe；Long scatterplot line indicates reference gene Actb probe；With
Fig. 2 is the fluorescent quantitation result display diagram with general primer and probe in detecting negative sample, and short dash line is to be directed to
The primer of FMR1 gene methylation, probe design, short scatterplot lines indicate FMR1 gene methylation probe, and short dash line part has non-
The reaction of specificity plays peak, and long scatterplot line indicates reference gene Actb probe.
Specific embodiment
In order to make art technology field personnel more fully understand the technical solution in the application, below in conjunction with following reality
Applying example, the invention will be further described, it is clear that described embodiments are only a part of embodiments of the present application, rather than complete
The embodiment in portion.Based on the embodiment in the application, those of ordinary skill in the art are without making creative work
All other embodiment obtained, shall fall within the protection scope of the present application.
One .FMR1 gene of embodiment carries out the primer and probe of qPCR method detection methylation
To overcome the defects of MS-PCR method mentioned above, the present invention provides detected based on fluorescent quantitation method
The kit of FMR1 gene promoter region methylation.Primer involved in the present invention, probe sequence such as the following table 1.
Table 1.FMR1 gene carries out the primer and probe of qPCR method detection methylation
Wherein the base of underscore overstriking is designed for FMR1 gene methylation site, and qPCR primer size is
143bp.Its middle probe 5 ' end has fluorescent reporter group FAM, and 3 ' ends have fluorescent quenching group MGB.
In upstream primer in CGIn downstream primer in GCBoth for the sequence to be methylated by sub-
It is still maintained after sulfuric acid salt treatmentOrThe characteristics of design；If there is no methylation, by sulphite
The sequence of this position has reformed into T after reason.Therefore this primer and probe cannot all react with the sequence not methylated.
Primer and probe of the present invention includes the site of methylation ingenious in designly, so that greatly reducing non-specific phenomenon；
If but methylation sites position is bad, even if primer, probe include methylation sites, just there is non-specific phenomenon.
In order to monitor whether qPCR reaction system works normally, also while the Actb gene of reference gene people joined,
The primer of Actb gene, probe such as the following table 2.
The primer and probe of 2. reference gene Actb gene of table progress qPCR method detection
| Title | Sequence (5 ' -3 ') | Sequence number | Tm value |
| Actb-F (upstream primer) | GTGTATAGGAGAGTATTAGGAAGTGGT | SEQ ID NO:4 | 58.1 |
| Actb-R (downstream primer) | CCACTAAACCTCCATTCAACTAACC | SEQ ID NO:5 | 58.3 |
| Actb-P (probe) | AAAACAAAAACTCCAACCCCACCC | SEQ ID NO:6 | 63.3 |
Its middle probe 5 ' end has fluorescent reporter group VIC, and 3 ' ends have fluorescent quenching group MGB.
The application of two, of embodiment kit of the present invention
Present invention specimen types detected are the clinical sample of periphery whole blood, and blood sample is after extracting genomic DNA, first
PCR+ capillary electrophoresis confirms that examined samples method is that fragile X is positive and fragile X is negative.
The DNA of extraction, with the amount of 400ng, with the sulphite treatment kits of Jiangsu Jing Shan Biotechnology Co., Ltd
The DNA after conversion is handled and recycles, the DNA after elution is 60 μ l.By following quantitative fluorescent PCR reaction system into
Row reaction, qPCR reaction system uses the product of U.S. Promega company, such as table 3, reaction condition such as table 4.
Table 3.qPCR method detects the reaction system of FMR1 gene promoter region methylation
| Title | Volume (20 μ l reaction system) | Final concentration |
| 5×buffer | 4 | 1× |
| dNTP | 1 | 0.4mM |
| MgCl2 | 1 | 3mM |
| GoTaq thermal starting enzyme | 0.5 | 2.5U |
| Two downstream of gene primers | 2 | 0.4 μM every kind |
| Two gene probes | 2 | 0.15 μM every kind |
| It is nucleic acid-templated after sulphite conversion | 2 | |
| Moisturizing | 7.5 |
Table 4.qPCR reaction condition
On quantitative fluorescent PCR instrument ABI7500, the detection FMR1 promoter methylation positive and negative fluorescent quantitation are anti-
Answer curve graph respectively referring to Fig. 1 a and 1b, it can be clearly it can be seen that for primer and probe of the invention, substantially from Fig. 1 b
It is not non-specific.Fig. 2 is the light quantitative reaction with above the same terms and the primer and probe that is typically designed for negative sample
Curve graph, wherein increasing the probability of false positive there are stronger non-specific phenomenon；Short dash line is for FMR1 gene methylation
Primer, probe design, short dash line part have it is nonspecific react peak, long scatterplot line expression reference gene Actb probe.
Theoretically normal person does not methylate, therefore should not play peak.
10 fragile X positive samples and 20 fragile X negative samples confirmed by PCR+ Capillary Electrophoresis are taken, with this
Invention the method is detected, and positive and negative findings are 100% identical.In addition it is crisp to detect that a column clinical symptoms are shown as
Property X, but Standard PCR+Capillary Electrophoresis electrophoresis method is detected as feminine gender, is detected as FMR1 gene promoter methylation with the present invention
It is positive.Pass through Southern Blot hybridization check below, is confirmed as fragile X positive sample, but number of repetition > 400 (CGG) n
Secondary repetition, except PCR+ capillary electrophoresis detection range, therefore by PCR+ capillary electrophoresis method missing inspection.
It should be understood that the present invention disclosed is not limited only to specific method, scheme and the substance of description, because these
It is alterable.It will also be understood that purpose of the terminology used here just for the sake of the specific embodiment scheme of description, rather than
It is intended to limit the scope of the invention, the scope of the present invention is limited solely by the attached claims.
Those skilled in the art, which will also be appreciated that or be able to confirm that, uses no more than routine experiment, institute herein
The many equivalents for the specific embodiment of the invention stated.These equivalents are also contained in the attached claims.
Sequence table
<110>the emerging biomedical Science and Technology Ltd. of Shunde District of Foshan City brightness brocade wound
<120>kit of fluorescent quantitation method detection FMR1 gene promoter region methylation and its application
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
tgatttttta cgttattgag t 21
<210> 2
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
gaatcgaaaa acaaacgc 18
<210> 3
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
cactacctcg cgaaaaccaa 20
<210> 4
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
gtgtatagga gagtattagg aagtggt 27
<210> 5
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
ccactaaacc tccattcaac taacc 25
<210> 6
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
aaaacaaaaa ctccaacccc accc 24
Claims (5)
1. the kit that fluorescent quantitation method detects the methylation of FMR1 gene promoter region, which is characterized in that the kit
Following PCR primer and probe including detection FMR1 gene promoter region methylation:
Upstream primer: TGATTTTTTAGTTATTGAGT；
Downstream primer: GAATCAAAAACAAACC；With
Probe sequence: CACTACCTCCAAAACCAA；Wherein tiltedly bold-faced base is for FMR1 gene methylation site
The base being designed.
2. kit according to claim 1,5 ' ends of the probe sequence have fluorescent reporter group VIC, 3 ' ends
With fluorescent quenching group MGB.
3. kit according to claim 1, the amplification reference gene behaviour Actb gene；On Actb gene
Swim primer: GTGTATAGGAGAGTATTAGGAAGTGGT, Actb downstream of gene primer:
CCACTAAACCTCCATTCAACTAACC；With the sequence of Actb gene probe are as follows: AAAACAAAAACTCCAACCCCACCC.
4. kit according to claim 3, the Actb gene probe 5 ', which is held, has fluorescence radiation group VIC, and 3 '
End has fluorescent quenching group MGB.
5. application of any kit of claim 1-4 in fragile X mental retardation detection.
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| CN201810874061.9A CN108949955A (en) | 2018-08-03 | 2018-08-03 | Fluorescent quantitation method detects kit and its application of FMR1 gene promoter region methylation |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112608991A (en) * | 2020-12-21 | 2021-04-06 | 广东省妇幼保健院 | qMS-PCR kit for detecting Prader-Willi syndrome/Angelman syndrome |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112608991A (en) * | 2020-12-21 | 2021-04-06 | 广东省妇幼保健院 | qMS-PCR kit for detecting Prader-Willi syndrome/Angelman syndrome |
| CN112608991B (en) * | 2020-12-21 | 2023-12-12 | 广东省妇幼保健院 | qMS-PCR kit for detecting Prader-Willi syndrome/Angelman syndrome |
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Address after: 528318 Xinji International Creative Park, Block C 201, 68 Renmin West Road, Dongchung Community Resident Committee, Longjiang Town, Shunde District, Foshan City, Guangdong Province Applicant after: Guangdong Huijin Chuangxing Biomedical Technology Co.,Ltd. Address before: 528318 Xinji International Creative Park, Block C 201, 68 Renmin West Road, Dongchung Community Resident Committee, Longjiang Town, Shunde District, Foshan City, Guangdong Province Applicant before: FOSHAN SHUNDE HUIJIN CHUANGXING BIOMEDICAL TECHNOLOGY CO.,LTD. |
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