CN107828869A - A kind of kit of molecular beacon probe method detection mankind's mthfr gene polymorphism, method and its application - Google Patents

A kind of kit of molecular beacon probe method detection mankind's mthfr gene polymorphism, method and its application Download PDF

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Publication number
CN107828869A
CN107828869A CN201711138721.9A CN201711138721A CN107828869A CN 107828869 A CN107828869 A CN 107828869A CN 201711138721 A CN201711138721 A CN 201711138721A CN 107828869 A CN107828869 A CN 107828869A
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China
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mthfr gene
primer
gene polymorphism
molecular beacon
probe
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任绪义
张锋
曹子豪
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Beijing Dean Medical Laboratory Laboratory Co Ltd
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Beijing Dean Medical Laboratory Laboratory Co Ltd
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

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Abstract

The invention provides a kind of primer that mankind's mthfr gene polymorphism is detected using molecular beacon probe method, following nucleotide sequence is included：Expand mthfr gene rs1801133 (c.677C>T) primer pair of pleomorphism site and the probe of detection.The invention also discloses kit of the above-mentioned primer and probe in detection amplification mthfr gene polymorphism, method and its application.As a result accurately the present invention, can be easy to interpretation with quick detection mthfr gene rs1801133 polymorphisms.Compared with PCR sequencing PCR, this method need not carry out the subsequent treatment of a series of complex, PCR amplifications and the synchronous progress of detection to PCR primer, whole detection process need not uncap operation, the risk that PCR primer pollutes is reduced, detection time is substantially reduced, reduces testing cost.

Description

A kind of kit of molecular beacon probe method detection mankind's mthfr gene polymorphism, side Method and its application
Technical field
The invention belongs to technical field of gene detection, and in particular to a kind of molecular beacons detection mankind mthfr gene is polymorphic The kit of property, method and its application.
Background technology
Mthfr gene is methylenetetrahydrofolate reductase protein coding gene, is folic acid metabolism and methionine metabolism In key enzyme.MTHFR albumen can make 5,10-CH2-THFA be reduced to 5-methyltetrahydrofolate, so as to be used as first The indirect donor of base participates in internal purine, the synthesis of pyrimidine and DNA, RNA, protein methylates, while remains normal in vivo Homocysteine level.Normal MTHFR activity could maintain the validity of folic acid methionine cycle, ensure DNA's What is synthesized and methylate is normally carried out.If MTHFR enzymatic activitys change, cause 5-methyltetrahydrofolate dyspoiesis, draw Rise DNA synthesis and aberrant methylation, hyperhomocysteinemiainjury and cause the generation of a variety of genetic diseases.
Research finds that the polymorphism in the sites of MTHFR 677 is to influence a key factor of the enzymatic activity, causes enzymatic activity Decline with heat endurance.MTHFR C677T are a common thermo-labile missense mutation, and the born of the same parents in its No. 677 codon are phonetic Pyridine (C) is replaced by thymidine (T).If its MTHFR activity is 100% when carrying 677CC genotype with individual, CT genes are carried The activity of type is then the 71% of CC, and genotype is only the 34% of TT types.It is reported that MTHFR C677T sites TT genotype The Hcy levels of body can raise about 25% compared with CC or CT genotype.Low plasma folic acid concentration and vitamin intake deficiency can aggravate T etc. Position gene-correlation risk.
, can be down to the heredity directly found in terms of detected person's folic acid metabolism by mthfr gene C677T site primers Defect (i.e. folic acid Utilization ability), so as to suggest more accurately replenishers according to risk height (level of activity of associated metabolic enzyme) Amount.
Mainly there are quantitative fluorescent PCR (qPCR), PCR- restriction fragments length currently for the method for polymorphic position point analysis Spend polymorphism (PCR-Restriction Length Polymorphism, PCR-RFLP), amplification refractory mutation system-PCR (Amplification refractory mutation system PCR, ARMS-PCR) etc., but because these methods are grasped It is more to make step, wastes time and energy, or needs PCR to post-process, is also easy to produce pollution, therefore is not suitable for crowd and quickly examines on a large scale Survey.PCR sequencing PCR is the goldstandard of nucleic acid sequencing, although accurately, complex for operation step and cost is higher.The present invention, by non- Symmetrical PCR, molecular beacons technology and solubility curve analytical technology, a kind of fast and accurately methods of genotyping and examination is invented Agent box.
The content of the invention
The technical problems to be solved by the invention are to provide a kind of examination of molecular beacons detection mankind mthfr gene polymorphism Agent box, method and its application.
In order to solve the above technical problems, the technical solution adopted by the present invention is as follows：
A kind of primer of molecular beacon probe method detection mankind's mthfr gene polymorphism, including following nucleotide sequence：
(1) primer pair of mthfr gene rs1801133 pleomorphism sites, its nucleotide sequence such as SEQ ID NO. are expanded： 1 and SEQ ID NO.：Shown in 2；
(2) it is used for the probe sequence for detecting mthfr gene rs1801133 pleomorphism sites, its nucleotide sequence such as SEQ IDNO.：Shown in 3,5 end mark FAM, 3 end mark DABSYL of its middle probe.
Above-mentioned primer pair and fluorescence probe in one-time detection to being used in conjunction with.
The primer and probe of above-mentioned molecular beacon probe method detection mankind's mthfr gene polymorphism is in detection mthfr gene Applying within protection scope of the present invention in polymorphism.
A kind of molecular beacon probe method is used for the kit for detecting mankind's mthfr gene polymorphism, and the kit includes power Profit requires the primer and probe and 2x PCR reaction mixtures described in 1, positive criteria product.
A kind of molecular beacon probe method is used for the method for detecting mankind's mthfr gene polymorphism, comprises the following steps：
(1) human peripheral genomic DNA is extracted；
(2) respectively using the human peripheral genomic DNA of extraction and positive criteria product as drawing in template, claim 1 and 2 Thing, probe, PCR reaction mixtures, carry out real-time fluorescence quantitative PCR reaction；
(3) solubility curve genotype judgement is carried out to rs1801133 sites according to the fluorescence signal value detected, wherein glimmering Optical channel selects FAM passages,
Further, in the step (2), the amplification system of real-time fluorescence quantitative PCR reaction is：Cumulative volume 20 μ L, 2x PCR reaction mixtures 10uL, SEQ ID NO.：1 0.0 2uM, SEQ ID NO.：2,0.2uM, SEQ ID NO.：3 0.2uM, Template DNA 50ng.
Further, in the step (2), the amplification program in real-time fluorescence quantitative PCR reaction is：95 DEG C, 2min； 95 DEG C, 5s, 60 DEG C, 45s, collect fluorescence, 50 circulations；Solubility curve analysis program is：95℃1min；45 DEG C 2 minutes, 45- 90 DEG C, continuously collect fluorescence.
The invention provides a kind of molecular beacon probe method detection mankind's mthfr gene polymorphism primer and kit, Realize and quick, simple detection is carried out to mthfr gene pleomorphism site.Testing result of the present invention is accurate, is easy to interpretation.With PCR sequencing PCR is compared, and this method need not carry out the subsequent treatment of a series of complex to PCR primer, and PCR expands and detected same stepping OK, whole detection process need not uncap operation, reduce the risk that PCR primer pollutes, substantially reduce detection time, drop Low testing cost.
Brief description of the drawings
Fig. 1 is that testing result corresponding to different genotype illustrates.
Embodiment
According to following embodiments, the present invention may be better understood.It is however, as it will be easily appreciated by one skilled in the art that real Apply the content described by example and be merely to illustrate the present invention, without should be also without limitation on sheet described in detail in claims Invention.
Embodiment 1：
Step 1：DNA is extracted
200 μ L peripheral bloods are taken, it is (new purchased from Hangzhou to carry out DNA extractions according to Whole Blood Genomic DNA extracts kit specification Prompt bio tech ltd), extraction DNA is used for subsequent experimental or deposits in -20 DEG C.
Step 2：PCR is expanded and detection
(1) primer pair of mthfr gene rs1801133 pleomorphism sites, its nucleotide sequence such as SEQ ID NO. are expanded： 1 and SEQ ID NO.：Shown in 2；
(2) it is used for the molecular beacon sequences for detecting mthfr gene rs1801133 pleomorphism sites, its nucleotide sequence is such as SEQID NO.：Shown in 3,5 end mark FAM, 3 end mark DABSYL of its middle probe.
In 0.2mL, reaction system is formulated as follows：Cumulative volume 20 μ L, 2x PCR reaction mixtures 10uL, SEQ ID NO.：1 0.02uM, SEQ ID NO.：2,0.2uM, SEQ ID NO.：3 0.02uM, template DNA 50ng., add water to complement to The μ L of cumulative volume 20, while set up positive criteria product and negative control.Real-time fluorescence quantitative PCR reaction in amplification program be：95 DEG C, 2min；95 DEG C, 5s, 60 DEG C, 45s, collect fluorescence, 50 circulations；Solubility curve analysis program is：95℃1min；45℃2 Minute, 50-90 DEG C, continuously collect fluorescence.
Step 3：Interpretation of result
After reaction terminates, carry software using instrument and carry out data analysis.Solubility curve genotyping program is run, is entered Row genotype interpretation.
Step 4：As a result count
The genotype results statistics of 10 samples is as follows：
It is described above, it is preferred embodiments of the present invention, and the limitation that non-invention is any formal or substantial, should Point out, for those skilled in the art, under the premise of the scope of the present invention is not departed from, it can also be modified And improvement, these are improved and supplement should also be considered as protection scope of the present invention.
Sequence table
<110>Beijing Dean Laboratory of medical test Co., Ltd
<120>A kind of kit of molecular beacon probe method detection mankind's mthfr gene polymorphism, method and its application
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
ctcgccttga acaggtggag 20
<210> 2
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
cggaagaatg tgtcagcctc a 21
<210> 3
<211> 37
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
ccggatctgt ctgcgggagc cgatttcatc gatccgg 37
<210> 4
<211> 181
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
ctcgccttga acaggtggag gccagcctct cctgactgtc atccctattg gcaggttacc 60
ccaaaggcca ccccgaagca gggagctttg aggctgacct gaagcacttg aaggagaagg 120
tgtctgcggg agccgatttc atcatcacgc agcttttctt tgaggctgac acattcttcc 180
g 181
<210> 5
<211> 181
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
ctcgccttga acaggtggag gccagcctct cctgactgtc atccctattg gcaggttacc 60
ccaaaggcca ccccgaagca gggagctttg aggctgacct gaagcacttg aaggagaagg 120
tgtctgcggg agtcgatttc atcatcacgc agcttttctt tgaggctgac acattcttcc 180
g 181

Claims (6)

1. a kind of primer of molecular beacon probe method detection mankind's mthfr gene polymorphism, it is characterised in that including following nucleic acid Sequence：
(1) primer pair of mthfr gene rs1801133 pleomorphism sites, its nucleotide sequence such as SEQ ID NO. are expanded：1 He SEQ ID NO.：Shown in 2；
(2) it is used for the probe sequence for detecting mthfr gene rs1801133 pleomorphism sites, its nucleotide sequence such as SEQ ID NO.：Shown in 3,5 end mark FAM, 3 end mark DABSYL of its middle probe.
Above-mentioned primer pair and fluorescence probe in one-time detection to being used in conjunction with.
2. a kind of primer of molecular beacon probe method detection mankind's mthfr gene polymorphism is in mthfr gene polymorphism is detected Application.
3. a kind of molecular beacon probe method is used for the kit for detecting mankind's mthfr gene polymorphism, it is characterised in that the reagent Box includes the primer and probe described in claim 1, and 2x PCR reaction mixtures, positive criteria product.
4. a kind of molecular beacon probe method is used for the method for detecting mankind's mthfr gene polymorphism, it is characterised in that including as follows Step：
(1) human peripheral genomic DNA is extracted；
(2) respectively using the human peripheral genomic DNA of extraction and positive criteria product as the primer in template, claim 1 and 2, Probe, PCR reaction mixtures, carry out real-time fluorescence quantitative PCR reaction；
(3) solubility curve genotype judgement is carried out to rs1801133 sites according to the fluorescence signal value detected, wherein fluorescence leads to Road selects FAM passages.
5. molecular beacon probe method according to claim 4 is used for the method for detecting mankind's mthfr gene polymorphism, it is special Sign is, in the step (2), the amplification system of real-time fluorescence quantitative PCR reaction is：Cumulative volume 20 μ L, 2x PCR reactions are mixed Close liquid 10uL, SEQ ID NO.：1 0.02uM, SEQ ID NO.：2,0.2uM, SEQ ID NO.：3 0.2uM, template DNA 50ng。
6. molecular beacon probe method according to claim 5 is used for the method for detecting mankind's mthfr gene polymorphism, it is special Sign is, in described step (2), the amplification program in real-time fluorescence quantitative PCR reaction is：95 DEG C, 2min；95 DEG C, 5s, 60 DEG C, 45s, collect fluorescence, 50 circulations；Solubility curve analysis program is：95℃1min；45 DEG C 2 minutes, it is 45-90 DEG C, continuous to receive Collect fluorescence.
CN201711138721.9A 2017-11-16 2017-11-16 A kind of kit of molecular beacon probe method detection mankind's mthfr gene polymorphism, method and its application Pending CN107828869A (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108949966A (en) * 2018-08-24 2018-12-07 山东德诺生物科技有限公司 For detecting the primed probe group and its application of rs1801133
CN110283899A (en) * 2019-06-18 2019-09-27 深圳友一生物科技有限公司 A kind of primer, probe and its kit detecting people's MTHFR and MTRR gene mutation
CN112301113A (en) * 2019-07-29 2021-02-02 上海利康精准医疗技术有限公司 Probe, primer and kit for detecting methylenetetrahydrofolate reductase gene polymorphism

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020037507A1 (en) * 1999-12-16 2002-03-28 Walkerpeach Cindy R. Compositions, methods and kits for allele discrimination
CN106957903A (en) * 2016-11-01 2017-07-18 上海泽因生物科技有限公司 One kind detection folic acid metabolism key gene pleomorphism site genotyping kit and its detection method

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020037507A1 (en) * 1999-12-16 2002-03-28 Walkerpeach Cindy R. Compositions, methods and kits for allele discrimination
CN106957903A (en) * 2016-11-01 2017-07-18 上海泽因生物科技有限公司 One kind detection folic acid metabolism key gene pleomorphism site genotyping kit and its detection method

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
周国华: "分子信标探针技术", 《SNP检测技术与个体化药物治疗》 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108949966A (en) * 2018-08-24 2018-12-07 山东德诺生物科技有限公司 For detecting the primed probe group and its application of rs1801133
CN110283899A (en) * 2019-06-18 2019-09-27 深圳友一生物科技有限公司 A kind of primer, probe and its kit detecting people's MTHFR and MTRR gene mutation
CN110283899B (en) * 2019-06-18 2021-09-07 深圳友一生物科技有限公司 Primer, probe and kit for detecting human MTHFR and MTRR gene mutation
CN112301113A (en) * 2019-07-29 2021-02-02 上海利康精准医疗技术有限公司 Probe, primer and kit for detecting methylenetetrahydrofolate reductase gene polymorphism

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