CN110283899A - A kind of primer, probe and its kit detecting people's MTHFR and MTRR gene mutation - Google Patents
A kind of primer, probe and its kit detecting people's MTHFR and MTRR gene mutation Download PDFInfo
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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
- 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
Abstract
The invention discloses a kind of primer, probe and its kits for detecting people's MTHFR and MTRR gene mutation, belong to technical field of gene detection.The present invention provides primer and probe SEQ ID NO:1~SEQ ID NO:9 of detection people MTHFR C677T and A1298C gene mutation and MTRR A66G gene mutation；People MTHFR C677T and A1298C gene mutation and MTRR A66G gene mutation are detected by asymmetric PCR and TaqMan probe melting curve analysis technology, for judging the folic acid Utilization ability of individual, other clinical detection results of association and clinical symptoms can auxiliary diagnosis clinical individual folic acid supplementary behaviors.
Description
Technical field
The invention belongs to technical field of gene detection, in particular to a kind of to detect drawing for people's MTHFR and MTRR gene mutation
Object, probe and its kit.
Background technique
Folic acid is called Vitamin B9, is a kind of water soluble vitamin, by pteridine, p-aminobenzoic acid and Pidolidone group
At.Folic acid is methyl donor important in human body, participates in the methylation of DNA synthesis, duplication and reparation and genome and protein
Regulation.The internal Newborn Birth-defects disease relationship such as folic acid deficiency and neural tube closure defect is close, while also will increase the heart
The risk of a variety of diseases such as vascular diseases, neurodegenerative disease, diabetes, kidney trouble, osteoporosis.Internal folic acid lacks
It is weary to be environment and gene influences each other result.Human body cannot synthesize folic acid, and external source intake is the unique channel that human body obtains folic acid,
Therefore also folic acid deficiency is to lead to a reason of internal folic acid deficiency in diet.On the other hand, on folic acid one carbon metabolism access
Key gene mutation causes folic acid Utilization ability absorbed to diet low.In addition, having the excessive supplement folic acid of display now
It may promote cancer development.Therefore it needs to carry out individuation to the folic acid Utilization ability of individual to assess to take the photograph to meet individual folic acid
The reasonable level taken avoids folic acid deficiency or long-term supplement excessive.
5,10-CH2-THFA reductase (5,10-Methylene tetrahydrofolate reductase,
MTHFR) be folic acid one carbon metabolism approach key enzyme, catalysis generate human body in most important indirect methyl donor 5- methyl tetrahydro
Folic acid.It has been found that there are 30 several genes to be mutated by MTHFR, wherein influencing maximum and most commonly C677T to enzymatic activity
(rs1801133) and A1298C (rs1801131).The former betides the catalyst structure domain of MTHFR prediction, influences on enzymatic activity
Greatly, researches show that maximums, and enzyme activity can be made to reduce about 70%；The latter betides the Regulatory domain of MTHFR prediction, to enzymatic activity shadow
Sound it is relatively small, mainly with the former and act synergistically.
((Methionine syntesis reductas, MTRR/MSR) can be for methionine synthetase reductase
It is catalyzed S-adenosylmethionine under NADPH existence condition and cobalamin (II) is allowed to re-form Mecobalamin element (III), makes first sulphur
Propylhomoserin synzyme (Methionine synthase, MTR/MS) catalysis in 5-methyltetrahydrofolate-cobalamin-first sulphur ammonia
The one carbon metabolism circulation of acid can be normally carried out.MTRR A66G (rs1801394) is the most common mutation on MTRR gene, is drawn
The 22nd amino acid of protein for playing MTRR gene coding becomes methionine from isoleucine, influences the enzyme activity of MTRR to shadow
5-methyltetrahydrofolate-cobalamin-methionine one carbon metabolism circulation is rung, is especially cooperateed with MTHFR C677T mutation
When effect.
It is existing to be used substantially for people MTHFR and MTRR gene mutation detection kit based on TaqMan hydrolysis probes
Fluorescence PCR method, although compared to the methods of PCR-RELF, direct Sequencing with have detect it is quick, easy to operate and be not easy
The advantages of cross contamination, but it is limited to fluorescent PCR instrument sense channel, it is only capable of detection single site mutation (MTHFR C677T), no
Associated gene mutation site can be covered comprehensively, it can not the individual folic acid Utilization ability of Comprehensive assessment.In addition, due to being single alkali
The difference of base, therefore no matter Standard PCR or ARMS PCR is used non-specific amplification curve (there are non-specific Ct) easily occur, because
This passes through frequently with secondary calculating result (△ Ct) testing result and needs to limit the pcr template concentration of addition, additional to increase
Add detection operation and result interpretation workload.In conclusion current urgent need, which establishes one kind, only needs single PCR reaction tube and complete area
The saltant type and wild type of only single base difference is divided to cover MTHFR C677T (rs1801133), A1298C simultaneously
(rs1801131) and the detection method of MTRR A66G (rs1801394) three kinds of gene mutations.
Summary of the invention
To overcome above-mentioned shortcoming and deficiency existing in the prior art, the primary purpose of the present invention is that providing a kind of detection
Primer, the probe of people's MTHFR and MTRR gene mutation.Above-mentioned primer, probe are based on improvement asymmetric PCR and TaqMan
The primer and probe of probe melting curve analysis detection people's MTHFR and MTRR gene mutation.
The present invention is to provide the kit comprising above-mentioned primer and probe in a further object, by adjusting the primer and spy
It is high special that the dosage of needle, the composition of PCR buffer and dosage, the dosage of magnesium ion, the dosage of Taq enzyme have the kit
Property, the feature of high accuracy and high sensitivity.
The purpose of the invention is achieved by the following technical solution:
A kind of primer, probe detecting people's MTHFR and MTRR gene mutation, including following nucleotide sequence (5 ' -3 ')
Primer and probe:
MTHFR C677T primer pair:
Primer 1 is SEQ ID NO:1, and primer 2 is SEQ ID NO:2；
MTHFR C677T probe:
Probe 1:SEQ ID NO:7；
MTHFR A1298C primer pair:
Primer 3 is SEQ ID NO:3, and primer 4 is SEQ ID NO:4；
MTHFR A1298C probe:
Probe 2:SEQ ID NO:8；
MTRR A66G primer pair:
Primer 5 is SEQ ID NO:5, and primer 6 is SEQ ID NO:6；
MTRR A66G probe:
Probe 3:SEQ ID NO:9.
Primer pair described above is characterized in that two primer inequalities of each primer pair (NO:1~6 SEQ ID) make
With progress asymmetric pcr generates a large amount of single stranded DNAs so as to hybridize with probe and carry out melting curve analysis, primer 1/3/5
Dosage is 50nM~200nM/ reaction, and primer 2/4/6 dosage is 400nM~2000nM/ reaction；Probe dosage be 400nM~
1600nM/ reaction；Since primer Tm is to influence a key factor of PCR efficiency and use concentration directly proportional to primer,
Therefore the practical Tm value of the few primer of dosage (primer 1/3/5) is lower than software prediction, to effectively improve asymmetric pcr efficiency,
In design of primers, the Tm value of the few primer of dosage (primer 1/3/5) software prediction is than an other primer (primer 2/4/6)
Tm value is 4 DEG C high~and 10 DEG C.
Software used is preferably Primer Premier5.0 when the software prediction；
Preferably, the Tm value of the few primer of dosage (primer 1/3/5) is higher than the Tm value of an other primer (primer 2/4/6)
5 DEG C~8 DEG C.
The nucleotide sequence 5 ' of the probe holds mark fluorescent group, 3 ' end label quenching groups；Wherein fluorophor is most
Big wavelength of transmitted light be 510nM~518nM, 538nm~560nM, 563nM~583nM, 600nM~670nM and 705nM~
It is mutually different between one of 715nM and probe；Meanwhile according to selected fluorophor emission maximum optical wavelength range, quench
Group absorptions optical wavelength range of going out 380nM~550nM, 470nM~560nM, 480nM~580nM, 550nM~650nM and
It is selected in 620nM~730nM.
In the label of above-mentioned probe, the quenching group be ECLIPSE, DABCYL, TAMRA, BHQ1, MGB, BHQ2,
One or both of BHQ3；When quenching group is ECLIPSE, the fluorophor that uses is 6-FAM, TET, HEX, JOE,
Any one in VIC, TAMRA, NED, ROX, Texas Red, LC Red610；When quenching group be DABCYL, TAMRA,
When BHQ1/MGB, the fluorophor used is any one in 6-FAM, TET, HEX, JOE, VIC；When quenching group is
When BHQ2/MGB, the fluorophor used is HEX, VIC, Cy3, Cy3.5, TAMRA, NED, ROX, Texas Red, LC
Red610, LC Red640, any one in Cy5, Cy5.5, Quasar670；When quenching group is BHQ3, what is used is glimmering
Light group is LC Red640, Cy5, Cy5.5, in Quasar670, Quasar705, LC Red705, Alexa Fluor680
Any one
Preferably, the fluorophor in probe and quenching group combination, including fluorophor 6-FAM and quenching group
BHQ1/MGB, fluorophor HEX, JOE, VIC and quenching group BHQ1/MGB, fluorophor NED and quenching group BHQ2/MGB,
Fluorophor ROX and quenching group BHQ2, fluorophor Cy5 and quenching group BHQ3, fluorophor Quasar705, LC
Red705, Alexa Fluor680 and quenching group BHQ3.
A kind of kit detecting people's MTHFR and MTRR gene mutation, including above-mentioned detection people's MTHFR and MTRR gene
Primer, the probe (NO:1~9 SEQ ID) of mutation.
The kit of detection people's MTHFR and MTRR gene mutation, including reaction solution, enzyme, positive quality control product and blank
Quality-control product；
The reaction solution includes: primer and probe (NO:1~9 SEQ ID), PCR buffer, dN (U) TP and magnesium ion；
Preferably, two primer inequalities of each primer pair (NO:1~6 SEQ ID) use, and 1/3/5 dosage of primer is
50nM~200nM/ reaction, primer 2/4/6 dosage are 400nM~2000nM/ reaction；Probe dosage is that 400nM~1600nM/ is anti-
It answers；
Preferably, PCR buffer includes: the KCl of Tris-HCl (pH8.7), 40mM~50mM/ reaction of 10mM/ reaction
With (the NH of 1mM~20mM/ reaction4)2SO4。
It is highly preferred that PCR buffer also includes: 1%~10% glycerol (v/v) of reaction/, 0.2%~2%/reaction
One or several kinds among the glycine betaine of DTT (v/v), 10mM~50mM/ reaction and the proline of 10mM~50mM/ reaction.
Preferably, dN (U) TP includes: dATP, 0.2mM of 0.1mM~0.3mM/ reaction~0.6mM/ reaction dUTP,
The dGTP of dCTP and 0.1mM~0.3mM/ reaction of 0.1mM~0.3mM/ reaction.
Preferably, magnesium ion concentration is 1.5mM~4.5mM/ reaction.
Enzyme described above includes: Taq archaeal dna polymerase, uracil-DNA glycosylase and enzyme save buffer；
Preferably, Taq archaeal dna polymerase has thermal starting performance, and concentration is 1.25U~5U/ reaction；Uracil-DNA sugar
Base enzyme concentration is 0.5U~3.0U/ reaction；Enzyme save buffer include: 20mM Tris-HCl (pH9.0), 100mM KCL,
1mM DTT, 0.1mM EDTA, 0.5%NP40 (v/v), 0.5% polysorbas20 (v/v), 50% glycerol (v/v).
Positive quality control product described above be genotype be MTHFR C677T, people's base of MTHFR A1298A and MTRR A66A
Because a group DNA, the artificial constructed MTHFR C1298C plasmid comprising amplification target fragment include amplification target fragment with artificial constructed
MTRR G66G plasmid mixture；Preferably, human genome concentration is 5~10ng/ μ L, plasmid concentration in positive quality control product
It is 1.0~5.0 × 106copies/mL；
Preferably, the described artificial constructed MTHFR C1298C plasmid comprising amplification target fragment, for by MTHFR
C1298C plasmid aim sequence SEQ ID NO:10 is inserted into construct in empty carrier and obtain；
Preferably, the described artificial constructed MTRR G66G plasmid comprising amplification target fragment, for by MTHFR C1298C
Plasmid aim sequence SEQ ID NO:11 is inserted into construct in empty carrier and obtain；
The empty carrier is preferably pUC57；
Blank quality-control product described above is purified water.
The kit of above-mentioned detection people MTHFR and MTRR gene mutation, preferred detecting step and result interpretation method are such as
Under:
1) nucleic acid extraction: extracting sample to be tested DNA using commercialization paramagnetic particle method or centrifugal column method nucleic acid extraction kit, to
Surveying sample type includes but is not limited to peripheral blood, exuviation cell, tissue；
Particularly, kit blank quality-control product component is needed referring to sample to be tested, synchronous to carry out nucleic acid extraction operation, thus right
Entire detection process quality is monitored with pollution condition；
2) PCR reaction solution is prepared:
The kit of cryo-conservation is taken out sufficiently to be mixed and of short duration centrifugation after candidate agent box each component melts；
It is 22 μ L/ reaction by reaction solution dosage, enzyme dosage is that the ratio of 1 μ L/ reaction prepares PCR reaction solution, and PCR is reacted
Liquid, which is reacted after being sufficiently mixed by 23 μ L/, is dispensed into each PCR reaction tube；Particularly, it to prevent packing to be lost, needs according to be measured
Sample size suitably increases the quantity of PCR reaction solution preparation；
3) PCR reaction is detected with melting curve:
By DNA to be measured by 2 μ L/ reaction be added to PCR reaction tube, closed PCR reaction tube and it is of short duration centrifugation be placed on it is glimmering
In light PCR instrument；
By the good PCR response procedures of following condition setting:
Sense channel: channel one (FAM), channel two (VIC/HEX) and channel three (ROX)；
React total volume: 25 μ L；
Reaction condition: 50 DEG C of 1~3min of reaction carry out 1 circulation, and 95 DEG C of 3~15min of reaction carry out 1 and recycle, and 95 DEG C
55~65 DEG C of 35~60sec of reaction carry out 50 circulations to 1~10sec of reaction altogether again, and 95 DEG C of 1~5min of reaction carry out 1 circulation,
40~55 DEG C of 1~5min of reaction carry out 1 circulation, are then uniformly to slowly warm up to 80 DEG C and period acquires each PCR reaction tube
Interior each sense channel fluorescence signal value (Cooling rate and signal acquisition frequency use instrument default parameters), 37 DEG C of reaction 10sec
Carry out 1 circulation；
4) result interpretation: after to previous step, on the matched analysis software of fluorescent PCR instrument carry out interpretation of result with
Interpretation；
Quality control: positive quality control product is in each sense channel there are two melting peakss and its Tm value is (65 in target zone
± 2 DEG C and 71 ± 2 DEG C)；Negative quality-control product is showed no melting peakss in each sense channel.Meet conditions above and then illustrates this detection
Procedure quality is qualified, anti-regular unqualified.
As a result interpretation: sample to be tested must (65 ± 2 DEG C and 71 ± 2 DEG C) appearance within the scope of each sense channel target Tm value
At least one melting peaks then illustrates that the pattern detection result is qualified.It is on the contrary then illustrate the pattern detection fail, need to be to the sample weight
Sample quality is analyzed in new detection.Under pattern detection result acceptance condition, according to the form below 1 carries out result interpretation.
Table 1 is testing result interpretation explanation.
The present invention has the following advantages and effects with respect to the prior art:
Present invention employs TaqMan probe melting curve analysis technologies, on the one hand using TaqMan probe hybridization state with
Mismatch site is influenced on hybridization stability and (is embodied in Tm variation) by molten in the fluorescence signal difference and sequence of free state
The accurate parting to single base mutation is realized in solution curve analysis, avoids the conventional non-spy easily occurred using TaqMan hydrolysis probes
Xor signal and thus cause must it is increased operation (such as sample in fact concentration control and result interpretation need testing result secondary
Calculate etc.), the different fluorophor label of TaqMan probe is on the other hand utilized and increases detection flux, to realize to more
Quick, easy, the intuitive and accurate parting of a SNP.
The present invention is buffered by the primer and probe of optimization design, the primer optimized and revised, probe, enzyme, magnesium ion, PCR
The dosage of liquid improves entire kit assay performance.It compares by detection clinical sample and with its Sanger sequencing result,
Accuracy and specificity are 100%；It is single with single locus by the human gene group DNA of the various concentration of different genotype
Genotype is minimum detection limit not less than 95% recall rate concentration, determines that lowest detection is limited to 0.5ng/ μ L；By being repeated 20 times
High, medium and low three concentration is detected, genotype is the human genome of MTHFR C677T, MTHFR A1298A and MTRR A66A
DNA, the artificial constructed MTHFR C1298C plasmid comprising amplification target fragment are with artificial constructed comprising expanding target fragment
The Cv of the mixture of MTRR G66G plasmid, each Tm value is respectively less than 5%.
The present invention detects people MTHFR C677T and A1298C gene mutation by TaqMan probe melting curve analysis technology
With MTRR A66G gene mutation, for judge individual folic acid Utilization ability, other clinical detection results of association and
Clinical symptoms can auxiliary diagnosis clinical individual folic acid supplementary behavior.
Detailed description of the invention
Fig. 1 is the testing result of detection kit positive quality control product provided by the invention and blank quality-control product component.
Specific embodiment
In order to enable those skilled in the art to better understand the solution of the present invention, below with reference to examples and drawings to this hair
Bright to be described in further detail, embodiments of the present invention are not limited thereto.
The present invention provides a kind of primer, probe and its kits for detecting people's MTHFR and MTRR gene mutation.
A kind of kit production and assembly for detecting people's MTHFR and MTRR gene mutation of embodiment 1:
Primer, probe specifically:
MTHFR C677T primer 1 (SEQ ID NO:1)；
MTHFR C677T primer 2 (SEQ ID NO:2)；
MTHFR C677T probe 1 (nucleotides sequence is classified as SEQ ID NO:7), ROX-
TGTCTGCGGGAGCCGATTTCATCATC-BHQ2；
MTHFR A1298C primer 3 (SEQ ID NO:3)；
MTHFR A1298C primer 4 (SEQ ID NO:4)；
MTHFR A1298C probe 2 (nucleotides sequence is classified as SEQ ID NO:8), FAM-CCAGTGAAGAAAGTGTCTTT-
MGB；
MTRR A66G primer 5 (SEQ ID NO:5)；
MTRR A66G primer 6 (SEQ ID NO:6)；
MTRR A66G probe 3 (nucleotides sequence is classified as SEQ ID NO:9), VIC-CAGAAGAAATGTGTGAGCAAG-
MGB。
Kit includes:
Reaction solution (form be shown in Table 2 in detail), enzyme (composition is shown in Table 3 in detail), positive quality control product (composition is shown in Table 4 in detail) and sky
White quality-control product (composition is shown in Table 4 in detail).
Kit production and assembly:
Kit each component respectively correspond by 2~table of table, 4 compositing formula pole total amount be added and after mixing, by kit
Person-portion number specification dispenses to corresponding component and saves pipe, and wherein reaction solution is added the damage of 5% volume (rounding up) by compliance marker
Pre- allowance is consumed, enzyme packing specification is added the pre- allowance of loss of 10% volume (rounding up), positive quality control product point by compliance marker
Dress specification is 15 μ L/ pipe, and blank quality-control product packing specification is 1.5mL/ pipe.Finally by reaction solution, enzyme, positive quality control product, sky
It is kit finished product that white quality-control product and the assembling of kit operation instructions, which are placed interior with kit package box,.
Table 2
Table 3
| Enzyme composition | Dosage (μ L/ reaction) |
| 5U/ μ L Taq archaeal dna polymerase | 0.5 |
| 5U/ μ L uracil-DNA glycosylase | 0.5 |
| Always | 1.0 |
Table 4
Artificial constructed MTHFR C1298C plasmid described in ※, for by MTHFR C1298C plasmid aim sequence SEQ ID
NO:10 is inserted into construct in pUC57 carrier and obtain；The artificial constructed MTRR G66G plasmid, for by MTHFR C1298C plasmid
Aim sequence SEQ ID NO:11 is inserted into construct in pUC57 carrier and obtain.
The kit provided by the invention of embodiment 2 is used for the detection of clinical sample
30 clinical sample detections are carried out using 48 person-portion specification kits of the embodiment of the present invention 1, kit forms are shown in
Table 5.
Table 5
| Reagent constituents | Component specification | Number of components |
| Reaction solution | 1109 μ L/ pipe | 1 pipe |
| Enzyme | 53 μ L/ pipe | 1 pipe |
| Positive quality control product | 15 μ L/ pipe | 1 pipe |
| Blank quality-control product | 1.5mL/ pipe | 1 pipe |
1. nucleic acid extraction
200 μ L whole blood samples, 200 μ L blank quality-control products are taken, (Tiangeng is raw using paramagnetic particle method poba gene group extracts kit
Change scientific and technological (Beijing) Co., Ltd, DP329) Yu Zidong instrument for extracting nucleic acid (TIANGEN Biotech (Beijing) Co., Ltd., TGuide
S32 nucleic acid extraction) is carried out, extracts and is carried out according to extracts kit specification and extraction apparatus specification, the DNA after extraction is placed in low
Temperature preservation is to be checked, and 2 DEG C~8 DEG C can be reserved for 3 months, and -18 DEG C or less can be reserved for 2 years, and -70 DEG C or less can long-term preservation.
Kit blank quality-control product component is needed referring to sample to be tested, synchronous to carry out nucleic acid extraction operation, thus to entire inspection
Procedure quality is surveyed to be monitored with pollution condition.
2. kit detects
2.1.PCR reaction solution is prepared:
The kit of cryo-conservation is taken out sufficiently to be mixed and of short duration centrifugation after candidate agent box each component melts.
It is 22 μ L/ reaction by reaction solution dosage, enzyme dosage is that the ratio of 1 μ L/ reaction prepares PCR reaction solution, and PCR is reacted
Liquid, which is reacted after being sufficiently mixed by 23 μ L/, is dispensed into each PCR reaction tube.Particularly, it to prevent packing to be lost, needs according to be measured
Sample size suitably increases the quantity of PCR reaction solution preparation.
2.2.PCR reaction is detected with melting curve:
By DNA to be measured by 2 μ L/ reaction be added to PCR reaction tube, closed PCR reaction tube and it is of short duration centrifugation be placed on it is glimmering
In light PCR instrument.
By the good PCR response procedures of following condition setting:
Sense channel: channel one (FAM), channel two (VIC/HEX) and channel three (ROX).
React total volume: 25 μ L.
Reaction condition: (being shown in Table 6)
Table 6
2.3. result interpretation:
Quality control:
There are two melting peakss and its Tm value (65 ± 2 DEG C and 71 in target zone in each sense channel for positive quality control product
±2℃)；Negative quality-control product is showed no melting peakss in each sense channel.Meet conditions above and then illustrates this detection process quality
Qualification, it is anti-regular unqualified.
As a result interpretation:
Sample to be tested (65 ± 2 DEG C and 71 ± 2 DEG C) must occur at least one within the scope of each sense channel target Tm value
Melting peakss then illustrate that the pattern detection result is qualified.It is on the contrary then illustrate the pattern detection fail, which need to be detected again or
Sample quality is analyzed.Under pattern detection result acceptance condition, result interpretation is carried out by table 1.
3.Sanger sequencing detection
Above-mentioned PCR product entrusts Sangon Biotech (Shanghai) Co., Ltd.) limited liability company's progress Sanger sequencing detection, respectively
Use 10 μM of MTHFR C677T primers 1 (SEQ ID NO:1), 10 μM of MTHFR A1298C primers 3 (SEQ ID NO:3), 10
μM MTRR A66G primer 5 (SEQ ID NO:5) is sequenced as sequencing primer.
4. testing result compares
The coincidence rate of kit test result and Sanger sequencing assay result is 100%, and accuracy and specificity are
100%, there is no false retrieval, missing inspection.Table 7 summarizes for testing result.
Table 7
The above description is only a preferred embodiment of the present invention, is merely used to help understand the contents of the present invention and is not used to limit
The system present invention, for those skilled in the art, the present invention can have various modifications and variations.It is all to utilize description of the invention
And converted on an equal basis made by accompanying drawing content, it is used in relevant technical field, it should be within scope of patent protection of the invention.
Sequence table
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aacggccgct ggtgagggcc tgcagacctt ccttgcaaat acatctttgt tcttgggagc 240
gggagggcag aagaagtttg catgcttgtg gttgacctgg gaggagtcag gggcagaatt 300
tacaggaatg gcctcctggg catgtggtgg cactgccctc tgtcaggagt gtgccctgac 360
ctctgggcac ccctctgcca ggggcaattc ctcttcccct gcctttgggg agctgaagga 420
ctactacctc ttctacctga agagcaagtc ccccaaggag gagctgctga agatgtgggg 480
ggaggagctg accagtgaag caagtgtctt tgaagtcttc gttctttacc tctcgggaga 540
accaaaccgg aatggtcaca aagtgagtga tgctggagtg gggaccctgg ttcatcccct 600
gcccctggcc tgaccccagc tgcaggccag gctgcggggc tgtgacttcc ccatcctgtg 660
ccctcccctc catgctgtgg acatggcaaa gggagaaggg taagttggga gacctccacc 720
tggaagggct tagggaggca aagacaggct gggtctttgt tgggggccgt gagagggact 780
cagggtgcca aacctgatgg tcgccccagc cagctcaccg tctctcccag gtgacttgcc 840
tgccctggaa cgatgagccc ctggcggctg agaccagcct gctgaaggag gagctgctgc 900
gggtgaaccg ccagggcatc ctcaccatca actcacagcc caacatcaac gggaagccgt 960
cctccgaccc catcgtgggc tggggcccca gcgggggcta t 1001
<210> 11
<211> 1001
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
taggaaacac agattcaagc ccaagtagtt tcgagccgat catctgattt ctgagccatg 60
gaattagagt ttcattcgta cactctcctt aatttgatga attcttattt aggctcattt 120
gagattagtg ctgaaaacaa atttagaaaa ggccatttca tattatgtgt gggtattgtt 180
gcattgtttc ttaaagattg agggagaatt aatatcttta ggttgttact gcttcattaa 240
aaagaggatc ttttttcccc catttttcag tttcactgtt acatgccttg aagtgatgag 300
gaggtttctg ttactatatg ctacacagca gggacaggca aaggccatcg cagaagaaat 360
gtgtgagcaa gctgtggtac atggattttc tgcagatctt cactgtatta gtgaatccga 420
taaggttaga gccgttacag tggattttac cgttttgtgc tttgaagaat tttggttggg 480
aagtgatatt tatgaaacaa aaggacacta ataccaccac atagtctttg ttttttaaca 540
gaaatgtgtt tgttcaatgg tatagtaaga tatcaccagc atttttttaa tatagaagtg 600
tgtagttgaa ttagactaaa tggtctcaaa aattgaaaca agtaacaact ccttaactgt 660
aaatgacaga agtagcatat gctgtgtctg tttcacaggg tggctgtgag aacaagtgag 720
atgtgtgttt gcctgctcag gtgtgctttc cacctcttga atctcagtgt cccacttttt 780
tccccaaaag aaaggccttg ccgtgcgtgt tagagttcct cctgatgaag gaccgcctag 840
attcttttgt gaaatagaat ttaaagtagt agtgtaagga tctggaaaaa actcggctgc 900
ttcttcaggg actagttgtt tatgttcagt ctctgcagaa gttcctacct tagggtatat 960
gcaactttac catcttgtgt tcttttattt agtttgctgc c 1001
Claims (10)
1. a kind of primer, probe for detecting people's MTHFR and MTRR gene mutation, it is characterised in that: including following nucleotide sequence
Primer pair and probe:
MTHFR C677T primer pair:
Primer 1 is SEQ ID NO:1, and primer 2 is SEQ ID NO:2；
MTHFR C677T probe:
Probe 1:SEQ ID NO:7；
MTHFR A1298C primer pair:
Primer 3 is SEQ ID NO:3, and primer 4 is SEQ ID NO:4；
MTHFR A1298C probe:
Probe 2:SEQ ID NO:8；
MTRR A66G primer pair:
Primer 5 is SEQ ID NO:5, and primer 6 is SEQ ID NO:6；
MTRR A66G probe:
Probe 3:SEQ ID NO:9.
2. primer, the probe of detection people's MTHFR and MTRR gene mutation according to claim 1, it is characterised in that:
Two primer inequalities of each primer pair use in the primer pair, and 1/3/5 dosage of primer is that 50nM~200nM/ is anti-
It answers, primer 2/4/6 dosage is 400nM~2000nM/ reaction；Probe dosage is 400nM~1600nM/ reaction；In design of primers
When, the Tm value of 1/3/5 software prediction of primer is 4 DEG C higher than the Tm value of primer 2/4/6~and 10 DEG C.
3. primer, the probe of detection people's MTHFR and MTRR gene mutation according to claim 1, it is characterised in that: described
The nucleotide sequence 5 ' of probe holds mark fluorescent group, 3 ' end label quenching groups；Wherein fluorophor emission maximum optical wavelength
Simultaneously for one of 510nM~518nM, 538nm~560nM, 563nM~583nM, 600nM~670nM and 705nM~715nM
And it is mutually different between probe；Meanwhile according to selected fluorophor emission maximum optical wavelength range, quenching group absorbs optical wavelength
Range carries out in 380nM~550nM, 470nM~560nM, 480nM~580nM, 550nM~650nM and 620nM~730nM
Selection.
4. detecting primer, the probe of people's MTHFR and MTRR gene mutation according to claim 3, it is characterised in that: described to quench
The group that goes out is one or both of ECLIPSE, DABCYL, TAMRA, BHQ1, MGB, BHQ2, BHQ3；When quenching group is
When ECLIPSE, the fluorophor used is 6-FAM, TET, HEX, JOE, VIC, TAMRA, NED, ROX, Texas Red, LC
Any one in Red610；When quenching group is DABCYL, TAMRA, BHQ1/MGB, the fluorophor that uses be 6-FAM,
Any one in TET, HEX, JOE, VIC；When quenching group is BHQ2/MGB, the fluorophor that uses be HEX, VIC,
Cy3, Cy3.5, TAMRA, in NED, ROX, Texas Red, LC Red610, LC Red640, Cy5, Cy5.5, Quasar670
Any one；When quenching group is BHQ3, the fluorophor that uses is LC Red640, Cy5, Cy5.5, Quasar670,
Quasar705, LC Red705, any one in Alexa Fluor680.
5. a kind of kit for detecting people's MTHFR and MTRR gene mutation, it is characterised in that: including any one of Claims 1 to 4
Primer, the probe of detection people's MTHFR and MTRR gene mutation.
6. the kit of detection people's MTHFR and MTRR gene mutation according to claim 5, it is characterised in that: including anti-
Answer liquid, enzyme, positive quality control product and blank quality-control product；
The reaction solution includes: the described in any item primer and probes of Claims 1 to 4, PCR buffer, dN (U) TP and magnesium from
Son.
7. the kit of detection people's MTHFR and MTRR gene mutation according to claim 6, it is characterised in that: described to draw
Two primer inequalities of each primer pair of object use, and 1/3/5 dosage of primer is 50nM~200nM/ reaction, primer 2/4/6
Dosage is 400nM~2000nM/ reaction；The probe dosage is 400nM~1600nM/ reaction；
The PCR buffer includes: Tris-HCl, 40mM of the pH8.7 of 10mM/ reaction~50mM/ reaction KCl and 1mM~
(the NH of 20mM/ reaction4)2SO4；
DN (U) TP includes: dATP, 0.2mM of 0.1mM~0.3mM/ reaction~0.6mM/ reaction dUTP, 0.1mM~
The dGTP of dCTP and 0.1mM~0.3mM/ reaction of 0.3mM/ reaction；
The concentration of the magnesium ion is 1.5mM~4.5mM/ reaction.
8. the kit of detection people's MTHFR and MTRR gene mutation according to claim 7, it is characterised in that: the PCR
Buffer also includes: volumetric concentration 1%~10%/reaction glycerol, volumetric concentration 0.2%~2%/reaction DTT, 10mM~
One or several kinds among the glycine betaine of 50mM/ reaction and the proline of 10mM~50mM/ reaction.
9. the kit of detection people's MTHFR and MTRR gene mutation according to claim 6, it is characterised in that: the enzyme
Include: Taq archaeal dna polymerase, uracil-DNA glycosylase and enzyme save buffer；
The Taq archaeal dna polymerase concentration is 1.25U~5U/ reaction；Uracil-DNA glycosylase concentration is 0.5U~3.0U/
Reaction；Enzyme saves buffer: the Tris-HCl of 20mM pH9.0,100mM KCL, 1mM DTT, 0.1mM EDTA, volume
Percentage composition 0.5%NP40,0.5% polysorbas20 of volumn concentration, 50% glycerol of volumn concentration.
10. the kit of detection people's MTHFR and MTRR gene mutation according to claim 6, it is characterised in that: the sun
Property quality-control product be genotype be MTHFR C677T, human gene group DNA, the artificial constructed packet of MTHFR A1298A and MTRR A66A
The MTHFR C1298C plasmid of the target fragment containing amplification is mixed with the artificial constructed MTRR G66G plasmid comprising amplification target fragment
Close object；Human genome concentration is 5~10ng/ μ L in positive quality control product, and plasmid concentration is 1.0~5.0 × 106copies/mL；
The blank quality-control product is purified water.
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| CN114350791A (en) * | 2022-01-26 | 2022-04-15 | 上海宝藤生物医药科技股份有限公司 | Primer probe combination for detecting polymorphism of folate metabolism related gene and application thereof |
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