CN107828870A - One kind detects MTHFR and MTRR gene pleiomorphisms kit and method simultaneously using molecular beacon probe melting curve method - Google Patents
One kind detects MTHFR and MTRR gene pleiomorphisms kit and method simultaneously using molecular beacon probe melting curve method Download PDFInfo
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- CN107828870A CN107828870A CN201711138722.3A CN201711138722A CN107828870A CN 107828870 A CN107828870 A CN 107828870A CN 201711138722 A CN201711138722 A CN 201711138722A CN 107828870 A CN107828870 A CN 107828870A
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- 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
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- C12Q1/6858—Allele-specific amplification
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- 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
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/16—Primer sets for multiplex assays
Abstract
The present invention relates to application of the molecular beacon probe melting curve technology in terms of genetic polymorphism detection, more particularly to method that is a kind of while detecting MTHFR and MTRR gene pleiomorphisms, a kind of amplimer of MTHFR and MTRR genes, molecular beacon probe and the kit containing them are further related to.The method that the technology of the present invention provides is simple to operate quick, and specificity is good, high sensitivity, accuracy are high, flux is high, testing cost is low, has broad application prospects.And the technology of the present invention method PCR amplifications and the synchronous progress of detection, whole detection process need not carry out operation of uncapping, and while testing cost is saved, substantially reduce detection cycle, the efficiency of detection is improved, reduces the risk that PCR primer pollution causes false positive in addition.
Description
Technical field
The invention belongs to technical field of gene detection, and in particular to one kind utilizes molecular beacon probe melting curve method simultaneously
Detect MTHFR and MTRR gene pleiomorphisms kit and method.
Background technology
Folic acid is one kind of B family vitamin, the synthesis of other important compounds such as DNA, RNA, protein is participated in, in machine
Played an important role in body metabolic process, be usually used in maternal weight gain supplement, so as to prevent plasma homocysteine and nerve
The diseases such as defective tube.At present, it turned out that closely related gene has methylenetetrahydrofolate reductase with folic acid metabolism
(MTHFR) and methionine synthesizes reductase (MTRR) gene.MTHFR and MTRR is the key enzyme in folic acid metabolism system, its
The change of gene structure, the reduction of folic acid metabolism key enzyme activity can be caused, influence the biosynthesis of nucleic acid.Folic acid deficiency and its generation
It is that one of Etiological that NTD (NTD) occurs occurs for fetus to thank to obstacle.
Methylenetetrahydrofolate reductase (methylene tetrahydrofolate reductase, MTHFR) is in leaf
Acid metabolic, DNA methylate with DNA synthesis etc. play an important role, its gene pleiomorphism can cause folic acid metabolism key enzyme activity
Property reduce, cause folate metabolism disorder, so as to trigger a variety of diseases, wherein with caused by hyperhomocysteinemiainjury brain soldier
In and neonate's defect it is the most serious.MTHFR activity may largely be influenceed by gene pleiomorphism, so as to shadow
The effect of ringing generation and the medicine of disease.Mthfr gene C677T and A1298C are two common pleomorphism sites, there is research
The change of MTHFR enzymatic activitys can be caused by showing the gene pleiomorphism in the site, may cause larger individual occur between patient
Difference.The people of MTHFR gene unconventionalities, influences folic acid eubolism, and the effect of the medicine such as methotrexate (MTX), 5-FU and poison pair
Effect, may cause hyperhomocysteinemiainjury, cause blood coagulation tendency to increase, headstroke, coronary heart disease and venous blood occurs
The risk of bolt also increases.Detection mthfr gene C677T polymorphism can be used in clinical guidance 5-FU classes, methotrexate (MTX) etc.
Body medication and Women of Childbearing Age folic acid supplement.
Methionine synthesis reductase (methionine synthase reductase, MTRR) is that vitamin B12 relies on
Property enzyme, plays a significant role during methyl turns to Met again in homocysteine.Research is found, if MTRR genes the 66th
Bit codon is undergone mutation, it will is caused enzymatic activity to change, is caused internal folic acid deficiency or homocysteine (Hcy)
Level rise, and then induce a variety of diseases, such as nerve channel disease, angiocardiopathy.
Detected by MTHFR the and MTRR gene loci polymorphism related to folic acid metabolism, can be from molecular level
Utilization ability of the individual to folic acid is assessed, so as to carry out individual character according to risk height (level of activity of associated metabolic enzyme)
Change medication and guidance.
Currently for gene polymorphism sites analysis method have a lot, as fluorescence quantitative PCR method, gene chips, directly
Meet PCR sequencing PCR, pyrosequencing method, high-resolution melting curve method, PCR- RFLPs (PCR-
Restriction Length Polymorphism, PCR-RFLP), amplification refractory mutation system-PCR (Amplification
Refractory mutation system PCR, ARMS-PCR) etc..Conventional fluorescent quantitative PCR method is clinically using more
Extensively, but this method detection gene pleiomorphism typically detected using Genotyping methods, this method to sample size with
And polymorphism distribution there are certain requirements, sample size can not accurately be detected when few to sample gene pleiomorphism.Gene chips
It is higher to instrument requirements and costly, it is unfavorable for large-scale promotion.Although PCR sequencing PCR is the gold mark of genetic polymorphism detection
Standard, but step is more and cumbersome, and cycle length, workload is big, and cost is higher.High-resolution melting curve method is by instrument temperature control
System, false positive are high.PCR-RFLP method complex operations, the cross pollution of PCR primer is easily caused when sample is more and digestion easily occurs
Excessively there is the result of false negative or false positive in insufficient or digestion, and reliability is low.
The content of the invention
The defects of in order to overcome above-mentioned prior art, the invention provides one kind using asymmetric multiplex PCR, molecular beacon
Probe and melting curve technology for detection MTHFR (rs1801131 and rs1801133) and MTRR (rs1801394) gene pleiomorphism
Kit and method.With quick, easy, high sensitivity, high specific, testing result accurately and reliably, simple to operate, flux
High, the advantages that cost is low, pollution-free.
In order to realize foregoing invention purpose, this invention takes following technical scheme：
A kind of primer of molecular beacon probe melting curve method detection people's MTHFR and MTRR gene pleiomorphism, including it is as follows
Nucleotide sequence：
(1) primer pair of mthfr gene (rs1801133, C677T) pleomorphism site, its nucleotide sequence such as SEQ are expanded
Shown in ID NO.1 and SEQ ID NO.2；
(2) it is used for the molecular beacon probe sequence for detecting mthfr gene (rs1801133, C677T) pleomorphism site, its
Nucleotide sequence as shown in SEQ ID NO.3, quench by 5 ' end mark fluorophor CY5 of its middle probe, 3 ' end mark fluorescence
Go out group DABSYL；
(3) primer pair of mthfr gene (rs1801131,1298A/C) pleomorphism site is expanded, its nucleotide sequence is such as
Shown in SEQ ID NO.4 and SEQ ID NO.5；
(4) it is used for the molecular beacon probe sequence for detecting mthfr gene (rs1801131,1298A/C) pleomorphism site,
Its nucleotide sequence is as shown in SEQ ID NO.6,5 ' end mark fluorophor HEX of its middle probe, 3 ' end mark fluorescence
Quenching group DABSYL；
(5) primer pair of MTRR genes (rs1801394,66A/G) pleomorphism site, its nucleotide sequence such as SEQ are expanded
Shown in ID NO.7 and SEQ ID NO.8；
(6) it is used for the molecular beacon probe sequence for detecting MTRR genes (rs1801394,66A/G) pleomorphism site, its core
Nucleotide sequence is as shown in SEQ ID NO.9,5 ' end mark fluorophor FAM of its middle probe, 3 ' end mark fluorescent quenchings
Group DABSYL；
Above-mentioned primer pair and fluorescence probe are used in conjunction with one-time detection.
Present invention also offers one kind to utilize molecular beacon probe melting curve method detection people's MTHFR and MTRR gene polymorphic
The kit of property, including primer and molecular beacon probe described in claim 1.
Preferably, the kit also includes 2 x PCR reaction mixtures, positive control, negative control and blank pair
According to；Contain 2 x Taq DNA Polymerase, 2 x PCR buffer solutions, 2 x in the 2 x PCR reaction mixtures
dNTPs；Contain 6 kinds of DNAs in the positive control, wherein respectively containing in SNP site in 6 kinds of DNAs
Rs1801131 A types gene and c-type gene, rs1801133 c-type gene and T-shaped gene, rs1801394 A types gene and
G type genes；The negative control and blank control are distilled water.
It is used to detect MTHFR and MTRR gene pleiomorphisms present invention also offers a kind of molecular beacon probe melting curve method
Method, comprise the following steps：
(1) people (peripheral blood or mouth desquamated cells) genomic DNA is extracted；
(2) using the human gene group DNA extracted as template, the primer pair in claim 1 and 2, probe, 2 x are utilized
PCR reaction mixtures, real-time fluorescence quantitative PCR reaction is carried out, wherein, MTHFR the and MTRR gene pleiomorphisms of each sample
Detection carries out real-time fluorescence quantitative PCR reaction in same pipe.
Further, the quantitative fluorescent PCR reaction system is：
Further, the amplification program of the quantitative fluorescent PCR reaction is：95 DEG C of pre-degeneration 2min, after pre-degeneration according to
Following procedure carries out 30 circulations：95 DEG C, 15s, 60 DEG C, 30s, collect fluorescence signal；Then melted according to following procedure
Tracing analysis：95 DEG C of pre-degeneration 1min；45 DEG C of 1s, 85 DEG C are then warming up to from 45 DEG C with 0.04 DEG C/s speed and collects melting
Curve data, then judge rs1801133, rs1801131 and rs1801394 loci gene type according to specific melting peakss, wherein
Fluorescence channel selects CY5, HEX and FAM passage respectively.
The present invention people's MTHFR and MTRR genetic polymorphism detection kit using asymmetric multiple fluorescence quantitative PCR,
Molecular beacon probe and melting curve technology, establish in same reaction tube while add 3 pairs of specific gene amplimers
And the molecular beacon probe of 35 ' end mark difference fluorophors, according to different genes amplified fragments and molecular beacon probe
It is incorporated in the multiple gene polymorphism sites of melting curve peak analysis and genotype that different fluorescence channels produce diverse location, energy
Fast and effeciently detect multiple gene polymorphism sites.Other the present inventor's MTHFR and MTRR genetic polymorphism detection kit
Using primer amplified purpose template and molecular beacon probe specific detection purpose template method, to specific detection
Same gene different genes polymorphism provides dual guarantee, while adds negative control and blank control, farthest reduces
False positive produces, and more accurately, stably can carry out polymorphic detection to sample.
The kit of the present invention has advantages below：
1. flux is high：93 samples can once be detected.
2. high sensitivity：As little as 1ng genomic DNA can accurately be detected；For the holding time is long and oral cavity
The very low sample standard deviation of these extracting concentrations of swab can be detected accurately.
3. high specificity：Specific primer is designed for MTHFR and MTRR genotype and specific molecular beacon is visited
Pin, can be identified with specific amplification corresponding to gene pleiomorphism.
4. synchronously being expanded and being detected using quantitative fluorescent PCR and melting curve technology, add without carrying out follow-up uncap
Sample and post-processing, operation is simpler, avoids false positive caused by Aerosol Pollution of uncapping.
5. three kinds of gene polynorphisms, easy to operate, visual result, a mesh can be detected simultaneously in same reaction tube
So.
6. it is applied to a variety of sample types：The present invention can not only detect the whole blood DNA of conventional EDTA anti-freezings, moreover it is possible to detect
Ropy buccal swab DNA is extracted, can also directly detect the leucocyte DNA after whole blood cells splitting erythrocyte, detection knot
The fruit degree of accuracy is high.
7. cost is low, molecular beacon probe melting curve method, the high-resolution of melting curve, molecular beacon can not only be retained
The high sensitivity and high specific of probe, and can also be cost-effective.
8. detection speed is fast, whole detection process only needs 60 minutes.
9. safety：Whole kit does not include poisonous and harmful substance, to operating personnel and environment non-hazardous.
In a word, MTHFR and MTRR genetic polymorphism detections kit of the present invention is applied to clinical multiple types pattern
This detection, there is flux is high, and high sensitivity, high specificity, experimental period is short, simple to operate, safety and environmental protection, and cost is low etc.
Remarkable advantage.
Can quickly be realized to mankind MTHFR (rs1801131 and rs1801133) using the detection method of the present invention and
The detection of MTRR (rs1801394) loci polymorphism, doctor can be aided in pregnant perinatal period NTD, the high blood of gestation
The onset risk of the pressure disease such as disease is assessed, and effectively instructs clinical 5-FU classes personalized medicine, can accomplish early detection,
Early stage corrects high Hcy mass formed by blood stasis；Meanwhile provide gene foundation for Women of Childbearing Age Supplement of folic acid.
Brief description of the drawings
Fig. 1 is the homozygous wild pattern detection results of MTHFR (rs1801133,677C/T) C/C；
Fig. 2 is MTHFR (rs1801133,677C/T) T/T homozygous mutation pattern detection results；
Fig. 3 is MTHFR (rs1801133,677C/T) C/T heterozygous mutant pattern detection results；
Fig. 4 is the homozygous wild pattern detection results of MTHFR (rs1801131,1298A/C) A/A；
Fig. 5 is MTHFR (rs1801131,1298A/C) A/C heterozygous mutant pattern detection results；
Fig. 6 is the homozygous wild pattern detection results of MTRR (rs1801394,66A/G) A/A；
Fig. 7 is MTRR (rs1801394,66A/G) A/G heterozygous mutant pattern detection results；
Fig. 8 is negative control/blank control testing result.
Embodiment
In order that technical problems, technical solutions and advantageous effects to be solved by the present invention are more clearly understood, below in conjunction with
Drawings and Examples, the present invention will be described in further detail.But illustrated embodiment is not as a limitation of the invention.
One aspect of the present invention provides while detects the specific primer of mankind's MTHFR and MTRR gene pleiomorphism
With molecular beacon probe sequence, wherein, the nucleotide sequence such as SEQ ID of specific primer and the molecular beacon probe sequence
Shown in No.1-9.
Wherein, SEQ ID NO.1 and SEQ ID NO.2, SEQ ID NO.4 and SEQ ID NO.5, SEQ ID NO.7 and
SEQ ID NO.8 are common PCR primers, and amplification respectively includes MTHFR 677, MTHFR 1298, the gene pleiomorphisms of MTRR 66
The DNA fragmentation in site；Wherein SEQ ID NO.3, SEQ ID NO.6, SEQ ID NO.9 are molecular beacon probe, for respectively
Specifically bind containing MTHFR 677, MTHFR 1298,66 3 locus specificities of MTRR DNA fragmentation.3 molecule letters
Probe is marked respectively in 5 ' end mark fluorophor CY5, HEX and FAM, while in its 3 ' end mark fluorescent quenching group
DABSYL。
It is used to detect the detection kit of mankind's MTHFR and MTRR gene pleiomorphism simultaneously present invention also offers a kind of,
Contain the specific primer described in claim 1 and molecular beacon probe sequence in the kit.
The detection kit of the present invention also includes positive control, negative control and blank control, sets positive control and sky
White control can monitor being normally carried out for real-time fluorescence quantitative PCR reaction, and positive control is for monitoring fluorescent quantitative PCR
It is no normal, blank control be used to monitor in reaction solution process for preparation whether no cross contamination, set negative control to monitor from core
Acid extracts being normally carried out for quantitative fluorescent PCR course of reaction, ensures no cross contamination during nucleic acid extraction；The positive
Contain two kinds of polymorphism MTHFR 677 of mthfr gene, the MTHFR 1298 being related in the present invention, MTRR genes one in comparison liquid
Kind of polymorphism MTRR66 6 kinds of DNA mixed liquors, the plasmid can be plasmid well known to those skilled in the art, this 6 germplasm
Grain concentration is identical, is 103copies/μl；The negative control and blank control are distilled water, and negative control need to be used as sample
This progress nucleic acid extraction.
Another technical scheme of the present invention is method that is a kind of while detecting mankind's MTHFR and MTRR gene pleiomorphism,
The method comprises the following steps：
(1) people (peripheral blood or mouth desquamated cells) genomic DNA is extracted；
(2) using the specific primer to, molecular beacon probe, 2 x PCR reaction buffers carry out quantitative fluorescent PCR
Reaction, wherein, the mthfr gene polymorphism and MTRR genetic polymorphism detections of each sample are carried out in same reaction tube
PCR reacts.
By taking 20 μ l reaction systems as an example, it is dense eventually that in obtained mixture each component is added after detected sample into reaction tube
Degree and content are as follows：
Above-mentioned system is only illustration, can proportionally expand or shrink the volume of mixture and its each in actual applications
Constituent content.
Specifically, the program of the quantitative fluorescent PCR reaction is：95 DEG C of pre-degeneration 2min, according to following journey after pre-degeneration
Sequence carries out 30 circulations：95 DEG C, 15s, 60 DEG C, 30s, collect fluorescence signal；Then melting curve point is carried out according to following procedure
Analysis：95 DEG C of pre-degeneration 1min；45 DEG C of 1s, 85 DEG C of collection melting curve numbers are then warming up to from 45 DEG C with 0.04 DEG C/s speed
According to.
After above-mentioned reaction terminates, result judgement is carried out by table 1：
The result judgement of table 1.
The detection method of the present invention has the advantages that flux height, high sensitivity, high specificity, result accurately and reliably, simultaneously
The detection method is simple to operate quick, and detection, and result interpretation ocular and clear can be completed in 60 minutes, very clear.
The present invention is expanded on further below by way of specific embodiment.
Embodiment 1
MTHFR the and MTRR genetic polymorphism detection kits of the present invention are prepared, are comprised the following steps：
1. primer and probe synthesis：
Design and synthesize 3 couples of specific primer SEQ ID NO:1、SEQ ID NO:2；SEQ IDNO:4、SEQ ID NO:
5； SEQ ID NO:7、SEQ ID NO:8；3 molecular beacon probe SEQ ID NO:3、SEQ ID NO:6；SEQ ID NO:
9, and in SEQ ID NO:35 ' end mark CY5 fluorophors, 3 ' the not luminous quenching groups of end mark DABSYL, SEQ ID
NO:65 ' end mark HEX fluorophors, 3 ' the not luminous quenching groups of end mark DABSYL, SEQ ID NO:95 ' end marks
FAM fluorophors, 3 ' the not luminous quenching groups of end mark DABSYL.Primer, probe are prepared into 100 μM of mother liquor respectively to store up
Deposit.
2. preparing positive control, 6 kinds of DNAs are contained in the positive control, contain this hair respectively in these DNAs
MTHFR 677C that bright kit is related to, MTHFR 677T, MTHFR 1298A, MTHFR 1298C, MTHFR 66A, MTHFR
6 kinds of different polymorphic DNA fragments of 66G genes, the selection of the plasmid and to be designed as those skilled in the art known are
103copies/μl。
3.PCR reaction solutions are prepared：Different system PCR reaction solutions are carried out according to the following table to prepare
| 2 x PCR reaction mixtures | 10μl |
| SEQ ID NO.1 | 20~100nmol/L |
| SEQ ID NO.2 | 100~500nmol/L |
| SEQ ID NO.3 | 20~100nmol/L |
| SEQ ID NO.4 | 50~200nmol/L |
| SEQ ID NO.5 | 250~1000nmol/L |
| SEQ ID NO.6 | 20~100nmol/L |
| SEQ ID NO.7 | 50~200nmol/L |
| SEQ ID NO.8 | 250~1000nmol/L |
| SEQ ID NO.9 | 20~100nmol/L |
| Add distilled water extremely | 18μl |
4. assemble kit：In kit comprising 1 pipe detection MTHFR and MTRR genetic polymorphism detection PCR reaction solutions, 1
Pipe negative control (distilled water), 1 pipe blank control (distilled water) and 1 pipe positive control；Made according to each composition of PCR reaction systems
Dosage, calculates 20 person-portions and each composition usage amount of 96 person-portions, two kinds of specifications, composition and is assembled in each pipe of reagent preparation box.
Embodiment 2：
People's MTHFR and MTRR the genetic polymorphism detection kit prepared with embodiment 1 detects to testing sample.
The anticoagulant whole blood sample of 20 folate metabolism disorders is collected in the present embodiment, genomic DNA is extracted therefrom, with reality
Apply the MTHFR and MTRR of the people's MTHFR and MTRR genetic polymorphism detection kit detection measuring samples obtained in example 1 base
Because of polymorphic implementations.
1：The extraction (Self-made reagent) of people's Whole Blood Genomic DNA
1) the μ l of whole blood 200 are taken, add 20 μ L Proteinase K (20mg/ml) solution, are mixed, add 300 μ l cracking
Liquid BL, fully shaking mix, 56 DEG C of water-bath 10min.
2) 260 μ l absolute ethyl alcohols are added, it is fully reverse to mix, now it is possible that flocculent deposit.
3) previous step resulting solution and flocculent deposit all add in an adsorption column to (adsorption column is put into 2ml collecting pipes
In), 12,000rpm centrifugation 30sec, the waste liquid in collecting pipe is outwelled, adsorption column is put into collecting pipe.
4) 500 μ l lavation buffer solutions W1 (please first checked whether before use and added absolute ethyl alcohol) are added into adsorption column,
12,000rpm centrifugation 30sec, outwell the waste liquid in collecting pipe, adsorption column are put into collecting pipe.
5) 600 μ l lavation buffer solutions W2 (please first checked whether before use and added absolute ethyl alcohol) are added into adsorption column,
12,000rpm centrifugation 30sec, outwell the waste liquid in collecting pipe, adsorption column are put into collecting pipe.
6) 600 μ l lavation buffer solutions W3 (please first checked whether before use and added absolute ethyl alcohol) are added into adsorption column,
12,000rpm centrifugation 30sec, outwell the waste liquid in collecting pipe, adsorption column are put into collecting pipe.
7) 14,000rpm centrifuges 1min, outwells waste liquid.Adsorption column is placed in into room temperature to place several minutes, thoroughly to dry suction
Remaining rinsing liquid in enclosure material.
8) adsorption column is transferred in 1.5ml centrifuge tubes, 100 μ l elution buffers is vacantly added dropwise to adsorbed film centre position
TE, room temperature are placed 2-5min, 12,000rpm centrifugation 1min, solution are collected into centrifuge tube.
9) 2 μ l resulting solution Nanodrop spectrophotometric determination DNA concentrations are taken, sample DNA is then diluted to 2
~50ng/ μ l, take 2 μ l to add into embodiment 1 in obtained kit respectively and carry out the PCR reactions of next step.Extraction
DNA does not have to that -20 DEG C should be deposited in for a long time.
Note：Negative control need to be used as sample to carry out above nucleic acid extraction step.
2：Asymmetric multiple fluorescence quantitative PCR amplification and detection
The embodiment 1 for taking 2 μ l to be added separately to 18 μ l successively the DNA sample after being diluted in step 1 and negative control
In the detection reaction system of kit, make reaction system cumulative volume be 20 μ l, after instantaneous low-speed centrifugal, reaction plate is put into glimmering
In Fluorescent Quantitative PCR instrument, amplified reaction is carried out after PCR response procedures are set as described below：95 DEG C, 2min；95 DEG C, 15s, 60
DEG C, 30s, 30 circulations；CY5, HEX and FAM fluorescence signal are collected after each circulation.Then melted according to following procedure
Solution curve is analyzed：95 DEG C of pre-degeneration 1min；45 DEG C of 1s, 85 DEG C are then warming up to from 45 DEG C with 0.04 DEG C/s speed and is collected and is melted
Solution curve data, the genotype in rs1801133, rs1801131 and rs1801394 site is then judged according to specific melting peakss.
3. the Analysis of test results of sample：
Homozygous wild 2, the samples of MTHFR 677C/C, one of testing result is as shown in Figure 1；
Homozygous wild 3, the samples of MTHFR 677T/T, one of testing result is as shown in Figure 2；
2, MTHFR 677C/T heterozygous mutants sample, one of testing result is as shown in Figure 3；
Homozygous wild 4, the samples of MTHFR 1298A/A, one of testing result is as shown in Figure 4；
3, MTHFR 1298A/C heterozygous mutants sample, one of testing result is as shown in Figure 5；
Homozygous wild 4, the samples of MTRR 66A/A, one of testing result is as shown in Figure 6；
2, MTRR 66A/G heterozygous mutants sample, one of testing result is as shown in Figure 7；
Negative control and blank control, one of testing result are as shown in Figure 8.
Above-mentioned 20 sample fluorescence quantitative PCR detection results and direct Sequencing result are completely the same.Result above shows this
The testing result that the detection kit that inventive embodiments provide is used for people's MTHFR and MTRR gene pleiomorphism is reliable, and the present invention
Detection method high sensitivity is simple to operate quick in traditional sequencing methods, beneficial to large-scale promotion.
Embodiment described above is only to absolutely prove preferred embodiment that is of the invention and being lifted, protection model of the invention
Enclose not limited to this.The equivalent substitute or conversion that those skilled in the art are made on the basis of the present invention, in the present invention
Protection domain within.
Sequence table
<110>Shenyang Dean Co., Ltd of medical test institute
<120>One kind using molecular beacon probe melting curve method simultaneously detect MTHFR and MTRR gene pleiomorphisms kit and
Method
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ctcgccttga acaggtggag 20
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cggaagaatg tgtcagcctc a 21
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ccggatctgt ctgcgggagc cgatttcatc gatccgg 37
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cccgatgatc gcagaagaaa tatgtgagca agcatcggg 39
Claims (8)
- A kind of 1. primer of molecular beacon probe melting curve method detection MTHFR and MTRR gene pleiomorphisms, it is characterised in that bag Include following nucleotide sequence：(1) primer pair of mthfr gene (rs1801133, C677T) pleomorphism site, its nucleotide sequence such as SEQ ID are expanded Shown in NO.1 and SEQ ID NO.2；(2) it is used for the molecular beacon probe sequence for detecting mthfr gene (rs1801133, C677T) pleomorphism site, its nucleosides Acid sequence is as shown in SEQ ID NO.3,5 ' end mark fluorophor CY5 of its middle probe, 3 ' end mark fluorescent quenching bases Group DABSYL；(3) primer pair of mthfr gene (rs1801131,1298A/C) pleomorphism site, its nucleotide sequence such as SEQ are expanded Shown in ID NO.4 and SEQ ID NO.5；(4) it is used for the molecular beacon probe sequence for detecting mthfr gene (rs1801131,1298A/C) pleomorphism site, its core Nucleotide sequence is as shown in SEQ ID NO.6,5 ' end mark fluorophor HEX of its middle probe, 3 ' end mark fluorescent quenchings Group DABSYL；(5) primer pair of MTRR genes (rs1801394,66A/G) pleomorphism site, its nucleotide sequence such as SEQ ID are expanded Shown in NO.7 and SEQ ID NO.8；(6) it is used for the molecular beacon probe sequence for detecting MTRR genes (rs1801394,66A/G) pleomorphism site, its nucleotides Sequence is as shown in SEQ ID NO.9,5 ' end mark fluorophor FAM of its middle probe, 3 ' end mark fluorescent quenching groups DABSYL；Above-mentioned primer pair and fluorescence probe are used in conjunction with one-time detection.
- 2. a kind of molecular beacon probe melting curve method is used for the kit for detecting people's MTHFR and MTRR gene pleiomorphism, it is special Sign is that the kit includes the primer and molecular beacon probe described in claim 1.
- 3. a kind of molecular beacon probe melting curve method according to claim 2 is used to detect people's MTHFR and MTRR gene The kit of polymorphism, it is characterised in that the kit also includes 2x PCR reaction mixtures, positive control, negative control And blank control.
- 4. a kind of molecular beacon probe melting curve method according to claim 3 is used to detect people's MTHFR and MTRR gene The kit of polymorphism, in the 2x PCR reaction mixtures containing 2x Taq DNA Polymerase, 2x PCR buffer solutions, 2x dNTPs；Contain 6 kinds of DNAs in the positive control, wherein respectively containing in SNP site in 6 kinds of DNAs Rs1801131 A types gene and c-type gene, rs1801133 c-type gene and T-shaped gene, rs1801394 A types gene and G Type gene；The negative control and blank control are distilled water.
- 5. people MTHFR according to claim 2 and MTRR gene pleiomorphisms detection kit, it is characterised in that described Primer SEQ ID NO.1, SEQ ID NO.4 and SEQ ID NO.7 work final concentration be respectively 20~100nmol/L, 50~ 200nmol/L, 50~200nmol/L；Primer SEQ ID NO.2, SEQ ID NO.5 and SEQ ID NO.8 work final concentration point Wei not 100~500nmol/L；250~1000nmol/L, 250~1000nmol/L；Molecular beacon probe SEQ ID NO.3, SEQ ID NO.6 and SEQ ID NO.9 work final concentrations are 20~100nmol/L.
- 6. a kind of molecular beacon probe melting curve method is used for the method for detecting MTHFR and MTRR gene pleiomorphisms, its feature exists In comprising the following steps：(1) people (peripheral blood or mouth desquamated cells) genomic DNA is extracted；(2) using the human gene group DNA extracted as template, reacted using the primer pair in claim 1 and 2, probe, 2x PCR Mixed liquor, real-time fluorescence quantitative PCR reaction is carried out, wherein, MTHFR the and MTRR genetic polymorphism detections of each sample are same Real-time fluorescence quantitative PCR reaction is carried out in pipe.
- 7. a kind of molecular beacon probe melting curve method according to claim 6 is more for detecting MTHFR and MTRR genes The method of state property, it is characterised in that the quantitative fluorescent PCR reaction system is：。
- 8. a kind of molecular beacon probe melting curve method according to claim 6 or 7 is used to detect MTHFR and MTRR genes The method of polymorphism, it is characterised in that the amplification program of quantitative fluorescent PCR reaction is：95 DEG C of pre-degeneration 2min, pre-degeneration Afterwards 30 circulations are carried out according to following procedure：95 DEG C, 15s, 60 DEG C, 30s, collect fluorescence signal；Then enter according to following procedure Row melting curve analysis：95 DEG C of pre-degeneration 1min；45 DEG C of 1s, 85 DEG C of collections are then warming up to from 45 DEG C with 0.04 DEG C/s speed Melting curve data, then judge rs1801133, rs1801131 and rs1801394 loci gene type according to specific melting peakss, Wherein fluorescence channel selects CY5, HEX and FAM passage respectively.
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| CN108220407A (en) * | 2018-03-29 | 2018-06-29 | 深圳鼎新融合科技有限公司 | Detect primer pair, probe and the kit of mankind's GDF5, COL5A1, SOD2, CRP gene pleiomorphism |
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