CN107236801A - The detection kit that a kind of step reverse transcription RT qPCR people MTHFR is expressed with MTRR gene pleiomorphisms - Google Patents
The detection kit that a kind of step reverse transcription RT qPCR people MTHFR is expressed with MTRR gene pleiomorphisms Download PDFInfo
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Abstract
The invention discloses the detection kit that a kind of step reverse transcription RT qPCR people MTHFR and MTRR gene pleiomorphisms are expressed, including MTHFR C677T reaction buffers, MTHFR A1298C reaction buffers, MTRR A66G reaction buffers, positive control, negative control, One step enzyme mixations.The present invention is based on the science of heredity of drug metabolism, on the basis of the advantage using real-time fluorescence PCR probe technique high sensitivity and high specific, for MTHFR and MTRR RNA sequence, directly the translation product for expressing region can be detected, compared with existing DNA real-time fluorescence PCRs method, closer to whether having the detection of marking protein (i.e. methylenetetrahydrofolate reductase and methionine synthetase reductase), testing result is with more specificity.
Description
Technical field
It is a kind of external diagnosis reagent the present invention relates to biology field, specifically refers to a kind of step reverse transcription RT-
The detection kit that qPCR people MTHFR is expressed with MTRR gene pleiomorphisms.
Background technology
Methylenetetrahydrofolate reductase (Methylenetetrahydrofolate reductase, MTHFR) is first sulphur
Key enzyme in propylhomoserin-folic acid metabolism system, with methionine synthetase reductase (Methionine synthase
Reductase, MTRR) together, maintain folic acid eubolism.In addition, MTHFR also participates in maintaining the normal Guang ammonia of homotype half in vivo
Sour water is put down.
Internal MTHFR and MTRR gene mutations can cause the folic acid metabolism key enzyme activity reduction that it is encoded, and cause folic acid
Dysbolism, causes folate level to reduce and hyperhomocysteinemiainjury (HCY).
For the people of MTHFR and MTRR gene unconventionalities, MTHFR and MTRR enzymatic activitys are substantially reduced, and cause folic acid metabolism to hinder
Hinder, cause the onset risk of the diseases such as neonate's NTD, Down's syndrome and harelip substantially to increase, this kind of people needs
More folic acid are supplemented, and expected effect is can be only achieved with longer cycle Supplement of folic acid.
In addition, hyperhomocysteinemiainjury (HCY) easily occur in MTHFR and MTRR gene unconventionalities, early stage carries out gene inspection
Survey is conducive to prevention hyperhomocysteinemiainjury (HCY) and cardiovascular and cerebrovascular disease.
Therefore, human body MTHFR and MTRR gene unconventionality are detected accurately and in time for instructing standby pregnant women how effective
Supplement of folic acid, prevents fetal anomaly, and have vital meaning in terms of the mankind prevent HCY, cardiovascular and cerebrovascular disease.
At present, the detection that human body MTHFR is expressed with MTRR gene pleiomorphisms and polymorphism has following several method：
1.DNA direct sequencings；2.DNA-PCR-SSCP (PCR-single-strand conformation polymorphism analysis)；3.DNA- high scores
Resolution melting curve analysis technology (HRM)；4.DNA- revert dot blot hybridizations；5.DNA- real-time fluorescence PCR methods；6.RNA- is glimmering in real time
Light PCR methods.
1.DNA direct sequencings
DNA direct sequencings are to carry out the most direct most accurate method of folic acid metabolism detection in Gene Mutation, are that detection is unknown
The poor mutator in ground key method.But it needs to expand sample, purified, sequence analysis, the required time is long, into
This height, the requirement to materials and technology is all higher, it is most important that limitation sensitivity is not high, has danger to environment and operator
Evil.Therefore there is certain limitation in clinical practice.
2.DNA-PCR-SSCP (PCR-single-strand conformation polymorphism analysis)
PCR-SSCP is a kind of classical detection method of gene mutation of comparison, is a kind of side of qualitative detection gene mutation
Method, can be detected to unknown mutation.With it is quick, easy, sensitive and economical the characteristics of, by the green grass or young crops of numerous researchers
Look at.But it also has certain defect, electrophoresis time is longer, complex operation step, can only carry out qualitative analysis, it is necessary to parallel mark
Quasi- control.
3.DNA- high-resolution melting curve analysis technologies (HRM)
DNA sequence dna melting curve using different length or different base compositions is different, and high-resolution is directly run after PCR
Melt and carry out sample mutation analysis, different mutational sites and different genotype can be distinguished, but HRM is higher to instrument requirements,
Used and limited by instrument.
4.DNA- revert dot blot hybridizations
Revert dot blot hybridization (RDB) is current domestic and international the most frequently used technology, and the advantage is that can be same on a hybond membrane
When a variety of point mutation of examination or genotype, its shortcoming is it is also obvious that due to needing carry out subsequent treatment of uncapping, operate numerous
Trivial, experimental period is long, and laboratory is also highly susceptible to the pollution of aerosol.
5.DNA- real-time fluorescence PCR methods
Real-time fluorescence PCR technology is to add fluorescence labeling probe or corresponding fluorescent dye on the basis of conventional PCR
To realize quantitatively or qualitatively function.It has broken away from gel electrophoresis technology, it is to avoid poisonous operation, easy to operate.Real-time fluorescence
PCR detects folic acid metabolism gene mutation, it is not necessary to examine the concrete content of genomic DNA, it is only necessary to detect whether sample has signal
, this causes whole process easier, and expense is lower, and high efficiency of the PCR reactions with nucleic acid amplification, even if only
Micro gene can also be detected, so that it has very high sensitivity.
Current majority MTHFR and MTRR genetic polymorphism detections reagent is detected have using real-time fluorescence PCR method
The advantage that sensitivity is high and specificity is good.But DNA- real-time fluorescence PCR methods are directed to MTHFR and MTRR DNA sequence dna, and
It is not the RNA for function, and DNA synthetic product not only includes the extron of translated region, in addition to non-coding
The intron sequences in area, therefore this method lacks the specificity of detection.
6.RNA- real-time fluorescence PCR methods
RNA- real-time fluorescence PCR methods are detected for RNA, and the detection currently for genomic expression RNA needs to lead to
It is cDNA that reverse transcription, which is crossed, by RNA reverse transcriptions, then carries out real-time PCR detection to cDNA.The operating method is cumbersome, it is necessary to two
Step reaction, while needing that follow-up real-time fluorescence PCR reaction could be carried out after being diluted cDNA, dilution is easily produced
Aerosol, causes the pollution in wide area in laboratory.
The content of the invention
In order to solve the above technical problems, the present invention is based on the science of heredity of drug metabolism, visited using real-time fluorescence PCR
, can be directly to table for MTHFR and MTRR RNA sequence on the basis of pin technology high sensitivity and the advantage of high specific
Translation product up to region is detected, compared with existing DNA- real-time fluorescence PCRs method, closer to whether having marking protein
The detection of (i.e. methylenetetrahydrofolate reductase and methionine synthetase reductase), testing result is with more specificity.
To achieve the above object, the invention provides a kind of step reverse transcription RT-qPCR people MTHFR and MTRR gene polymorphics
Property expression detection kit, including MTHFR-C677T reaction buffers, MTHFR-A1298C reaction buffers, MTRR-A66G
Reaction buffer, positive control, negative control, One step enzyme mixations.
It is preferred that, the detection kit includes 1 800 μ L MTHFR-C677T reaction buffers, 1 800 μ L
MTHFR-A1298C reaction buffers, 1 800 μ L MTRR-A66G reaction buffers, 1 50 μ L positive controls, 1 50 μ L are cloudy
Property control, 1 50 μ L One step enzyme mixation.
Further, the MTHFR-C677T reaction buffers include One step RT-qPCR reaction
Buffer, RT reinforcing agent, the specific upstream and downstream primer of dNTPs, C677T, C677T specific probes；
The MTHFR-A1298C reaction buffers strengthen including One step RT-qPCR reaction buffer, RT
Agent, the specific upstream and downstream primer of dNTPs, A1298C, A1298C specific probes；
The MTRR-A66G reaction buffers include One step RT-qPCR reaction buffer, RT reinforcing agent,
DNTPs, A66G specificity upstream and downstream primer, A66G specific probes.
Further, the specific upstream and downstream primers of the C677T are MTHFR-C677T-L, MTHFR-C677T-R；It is described
C677T specific probes are MTHFR-C677T-P-W, MTHFR-C677T-P-M.
Further, the specific upstream and downstream primers of the A1298C are MTHFR-A1298C-L, MTHFR-A1298C-R；Institute
A1298C specific probes are stated for MTHFR-A1298C-P-W, MTHFR-A1298C-P-M.(related base sequence is shown in Table 2)
Further, the specific upstream and downstream primers of the A66G are MTRR-A66G-L, MTRR-A66G-R；The A66G is special
Specific probes are MTRR-A66G-P-W, MTRR-A66G-P-M.(related base sequence is shown in Table 2)
Further, the positive control is human gene group DNA, mutant plasmid；The negative control is physiological saline.
Further, the One step enzyme mixations are Taq enzyme, M-MLV enzymes, Rnasin inhibitor, enzyme stabilization
The mixed liquor of agent.
The present invention also provides a kind of application method of above-mentioned detection kit, comprises the following steps：
(1) RNA is extracted；
(2) reagent prepares and is loaded；
(3) machine testing on, enters performing PCR amplification and fluorescent collecting；
(4) explanation of assay.
It is preferred that, PCR amplifications of the present invention and fluorescent collecting relative parameters setting are：
50 DEG C of 30min of reverse transcription；
95 DEG C of 5min of pre-degeneration；
PCR 95℃15s；60℃40s；Circulation 5 times；
95℃15s；60℃40s；Circulation 30 times.
The present invention be capable of qualitative detection folic acid metabolism related gene 3 SNP sites (mthfr gene C677T sites,
Mthfr gene A1298C sites and MTRR Gene A 66G sites) 6 kinds of genotype RNA, totally 3 by-reaction buffer solution is each anti-
Should pipe detect two kinds of genotype RNA, pass through two different fluorescence channels and read information.Genotype RNA detection cases by FAM,
HEX indicates that reference gene β-actin RNA are indicated by ROX, and the different genotype to mthfr gene and MTRR genes carries out spy
Opposite sex detection, the foundation of laboratory auxiliary diagnosis is provided for clinical examination.
The kit of the present invention extracts whole blood geneome RNA with the use of RNA extracts reagents (paramagnetic particle method).The present invention is optimal
The scheme of choosing is shown in Table 1 ingredients listed, and designed probe, primer and its sequence is shown in Table 2.
The most preferred composition composition table of the kit of the present invention of table 1
In above composition, One step RT-qPCR reaction buffer, RT reinforcing agents and the mixing of One step enzymes
Liquid three constitutes the one step qRT-qPCR system of the present invention.
The system is made up of enzyme system and buffer systems, is specifically included：
(1) enzyme system：
Taq enzyme, M-MLV enzymes, Rnasin inhibitor, enzyme stabilizers (50mM Tris-HclPH7.0,1mMEDTA,
1%Triton, 50% glycerine)；
(2) buffer systems：
One step RT-qPCR reaction buffer (20mM Tris-HclPH8.2,50mM potassium chloride, 1.5mM
Magnesium ion), RT reinforcing agents (5% formamide, 0.5%BSA).
The probe of the present invention of table 2, primer sequence table
In addition, the present invention uses Kingnova-ring probe designing techniques, probe used in detection is set to form stable ring
Structure：
MTHFR-C677T-P-W ring structures are as shown in Figure 1；
MTHFR-C677T-P-M ring structures are as shown in Figure 2；
MTHFR-A1298C-P-W ring structures are as shown in Figure 3；
MTHFR-A1298C-P-M ring structures are as shown in Figure 4；
MTRR-A66G-P-W ring structures are as shown in Figure 5；
MTRR-A66G-P-M ring structures are as shown in Figure 6.
The beneficial effects of the present invention are：
1. the present invention be used for detect folic acid metabolism related gene polymorphism, using ethylenediamine tetra-acetic acid (EDTA) anticoagulated whole blood as
Sample is detected, using RNA as detection target gene, foundation is provided for clinical individual Supplement of folic acid.
2. and existing people MTHFR is contrasted with MTRR gene pleiomorphism detecting methods, present invention detection folic acid metabolism dependency basis
Because of 6 kinds of 3 SNP sites in (mthfr gene site C677T, mthfr gene A1298C sites and MTRR Gene A 66G sites)
The RNA of genotype, to methylenetetrahydrofolate reductase and the enzyme activity defect of methionine synthetase reductase with more related
Property, testing result is with more specificity.
3. (it is cDNA by rna transcription with existing RNA detecting steps；By real-time fluorescence PCR technology for detection cDNA) compare.
The present invention realizes that real-time fluorescence method is directly detected by one step qRT-qPCR system transformation in an amplification pipe
3 SNP sites (mthfr gene C677T sites, mthfr gene A1298C sites and the MTRR genes of folic acid metabolism related gene
A66G sites) 6 kinds of genotype RNA, testing result is convenient and swift, while the processing that need not uncap, and is prevented effectively from laboratory dirty
Dye.
4. being compared with average probe, Kingnova-ring probe designing techniques, probe used in detection is set to form stabilization
Ring structures, are conducive to probe steady.
5. compared with average probe, Kingnova-ring probe designing technique combination LNA group labelling techniques, specifically
Property the SNP site that combines, can effectively distinguish homozygote and heterozygote, be not in non-specific amplification and false positive results, tool
There is detection specificity.
6. being compared with average probe, Kingnova-ring probe designing techniques are single structures, can preferably be combined
Target gene, with higher detection sensitivity.
Brief description of the drawings
Fig. 1 is MTHFR-C677T-P-W ring structures；
Fig. 2 is MTHFR-C677T-P-M ring structures；
Fig. 3 is MTHFR-A1298C-P-W ring structures；
Fig. 4 is MTHFR-A1298C-P-M ring structures；
Fig. 5 is MTRR-A66G-P-W ring structures；
Fig. 6 is MTRR-A66G-P-M ring structures；
Fig. 7-9 is positive control testing result amplification curve diagram, wherein：
Fig. 7 is the testing result amplification curve diagram of positive control MTHFR-677CT heterozygous genotypes-heterozygote；
Fig. 8 is the testing result amplification curve diagram of positive control MTHFR-1298AC heterozygous genotypes-heterozygote；
Fig. 9 is the testing result amplification curve diagram of positive control MTRR-66AG heterozygous genotypes-heterozygote；
Figure 10-12 is negative control testing result amplification curve diagram, wherein：
Figure 10 is the testing result amplification curve diagram of negative control MTHFR-677CC homozygous genotypes-wild type；
Figure 11 is the testing result amplification curve diagram of negative control MTHFR-1298AA homozygous genotypes-wild type；
Figure 12 is the testing result amplification curve diagram of negative control MTRR-66AA homozygous genotypes-wild type；
Figure 13-21 is sample to be tested testing result amplification curve diagram, wherein：
Figure 13 is the testing result amplification curve diagram of sample to be tested MTRR-677CC homozygous genotypes-wild type；
Figure 14 is the testing result amplification curve diagram of sample to be tested MTRR-677CT heterozygous genotypes-heterozygote；
Figure 15 is the testing result amplification curve diagram of sample to be tested MTRR-677TT homozygous genotypes-muton；
Figure 16 is the testing result amplification curve diagram of sample to be tested MTHFR-1298AA homozygous genotypes-wild type；
Figure 17 is the testing result amplification curve diagram of sample to be tested MTHFR-1298AC heterozygous genotypes-heterozygote；
Figure 18 is the testing result amplification curve diagram of sample to be tested MTHFR-1298CC homozygous genotypes-muton；
Figure 19 is the testing result amplification curve diagram of sample to be tested MTRR-66AA homozygous genotypes-wild type；
Figure 20 is the testing result amplification curve diagram of sample to be tested MTRR-66AG heterozygous genotypes-heterozygote；
Figure 21 is the testing result amplification curve diagram of sample to be tested MTRR-66GG homozygous genotypes-muton.
Embodiment
Embodiments of the invention are described below in detail, the example of the embodiment is shown in the drawings, wherein from beginning to end
Same or similar label represents same or similar element or the element with same or like function.Below with reference to attached
The embodiment of figure description is exemplary, it is intended to for explaining the present invention, and be not considered as limiting the invention.Embodiment
In unreceipted particular technique or condition person, according to the technology or condition described by document in the art or according to the description of product
Book is carried out.Agents useful for same or the unreceipted production firm person of instrument, being can be by the conventional products of acquisition purchased in market.
With reference to accompanying drawing 7-21, preferred forms of the invention are as follows：
1. sample
Clinical 10 parts of standby pregnant women blood sample, is respectively labeled as：Folic acid metabolism 01, folic acid metabolism 02, folic acid metabolism 03,
Folic acid metabolism 04, folic acid metabolism 05, folic acid metabolism 06, folic acid metabolism 07, folic acid metabolism 08, folic acid metabolism 09, folic acid metabolism 10,
1 part of positive control, 1 part of negative control.
2. reagent prepares (reagent area in preparation)
(1) corresponding reagent taken out from refrigerator in kit of the present invention for first 20 minutes is tested, balance to room temperature is vortexed mixed
After even, 10s is centrifuged in 2000rpm.
(2) every part of sample need carry out 3 pipe PCR reaction with detect 3 SNP sites (mthfr gene C677T sites,
Mthfr gene A1298C sites and MTRR Gene A 66G sites).
By anti-with portion sample MTHFR-C677T reaction buffers, MTHFR-A1298C reaction buffers, MTRR-A66G
The order of buffer solution is answered, MTHFR-C677T reaction buffers, the MTHFR- of 35 μ L/ pipes are added into corresponding PCR reaction tubes
A1298C reaction buffers, MTHFR-A66G reaction buffers.
It should be noted that the present invention needs sample quantity and reference substance quantity according to required for when time experiment to determine
The quantity that PCR reaction solutions are prepared.
(3) PCR reaction lids are covered, the PCR reaction tubes needed for experiment are transferred to sample area in preparation.
(4) remaining reagent puts back to -18 DEG C of refrigerator freezing preservations.
3. sample prepares (sample area in preparation)
(1) RNA is extracted：
The present invention supports the use RNA extracts kits (paramagnetic particle method) and carries out RNA extractions：Using in extracts kit specification
Defined DNA enzymatic processing method.
(2) RNA Concentration Testings：
Using micro-spectrophotometer, selection 260nm and 280nm wavelength detectings extract specimen dna concentration and purity, dense
Degree requires to be more than 30ng/ μ L.
Application claims purity OD260/OD280 be 1.90~2.00 between, if going beyond the scope needs to extract again to symbol
Close and require.
(3) it is loaded：Reference substance and sample should be entered performing PCR detection simultaneously by this experiment.
A. the PCR reaction tubes for having dispensed PCR reaction solutions are taken out, it is attached to remove tube wall using centrifugation slinger centrifugation 10s
Liquid.
B. press with portion sample MTHFR-C677T reaction buffers, MTHFR-A1298C reaction buffers, MTRR-A66G
Order, respectively to correspondence PCR reaction tubes in add 5 μ L RNA.
Experiment is both needed to while carrying out reference substance RNA detection every time.
C. PCR reaction lids are covered, centrifuge 10s to remove tube wall attaching liq.
D. the PCR reaction tubes for having added RNA templates are transferred to nucleic acid amplification area and carry out upper machine testing.
4. detection and analysis (nucleic acid amplification area)
(1) instrument is started shooting, and carries out instrument performance self-inspection.
(2) the PCR reaction tubes of this preparation block transitive are sampled, instrument sample groove relevant position is placed on, PCR reaction tubes are verified
Put order and record.
According to the form below sets instrument nucleic acid amplification and fluorescent collecting relevant parameter：
(3) response procedures are run.
(4) after program operation is finished, result is analyzed using software.
5. the explanation of assay
(1) three passages (FAM, HEX, ROX) of negative control reacting hole should without amplification curve, positive control reacting hole
Three passages (FAM, HEX, ROX) should have amplification curve, and otherwise this experiment is considered as invalid.The internal reference of sample to be tested reacting hole
Passage (ROX) should have amplification curve, and otherwise this experiment is considered as invalid.
(2) folic acid metabolism enzyme related gene polymorphism testing result is judged according to following standard：
(3) testing result
Fig. 7 is the testing result amplification curve diagram of positive control MTHFR-677CT heterozygous genotypes-heterozygote, wherein,
FAM channel C t value≤27, HEX channel C t value≤27, ROX channel C t value≤27；
Fig. 8 is the testing result amplification curve diagram of positive control MTHFR-1298AC heterozygous genotypes-heterozygote, wherein,
FAM channel C t value≤27, HEX channel C t value≤27, ROX channel C t value≤27；
Fig. 9 is the testing result amplification curve diagram of positive control MTRR-66AG heterozygous genotypes-heterozygote, wherein, FAM
Channel C t value≤27, HEX channel C t value≤27, ROX channel C t value≤27；
Figure 10 is the testing result amplification curve diagram of negative control MTHFR-677CC homozygous genotypes-wild type, wherein,
FAM channel C t values ＞ 27, HEX channel C t value≤27, ROX channel C t value≤27；
Figure 11 is the testing result amplification curve diagram of negative control MTHFR-1298AA homozygous genotypes-wild type, wherein,
FAM channel C t values ＞ 27, HEX channel C t value≤27, ROX channel C t value≤27；
Figure 12 is the testing result amplification curve diagram of negative control MTRR-66AA homozygous genotypes-wild type, wherein, FAM
Channel C t values ＞ 27, HEX channel C t value≤27, ROX channel C t value≤27；
Figure 13 is the testing result amplification curve diagram of sample to be tested MTRR-677CC homozygous genotypes-wild type, wherein,
FAM channel C t values ＞ 27, HEX channel C t value≤27, ROX channel C t value≤27；
Figure 14 is the testing result amplification curve diagram of sample to be tested MTRR-677CT heterozygous genotypes-heterozygote, wherein,
FAM channel C t value≤27, HEX channel C t value≤27, ROX channel C t value≤27；
Figure 15 is the testing result amplification curve diagram of sample to be tested MTRR-677TT homozygous genotypes-muton, wherein,
FAM channel C t value≤27, HEX channel C t values ＞ 27, ROX channel C t value≤27；
Figure 16 is the testing result amplification curve diagram of sample to be tested MTHFR-1298AA homozygous genotypes-wild type, wherein,
FAM channel C t values ＞ 27, HEX channel C t value≤27, ROX channel C t value≤27；
Figure 17 is the testing result amplification curve diagram of sample to be tested MTHFR-1298AC heterozygous genotypes-heterozygote, wherein,
FAM channel C t value≤27, HEX channel C t value≤27, ROX channel C t value≤27；
Figure 18 is the testing result amplification curve diagram of sample to be tested MTHFR-1298CC homozygous genotypes-muton, wherein,
FAM channel C t value≤27, HEX channel C t values ＞ 27, ROX channel C t value≤27；
Figure 19 is the testing result amplification curve diagram of sample to be tested MTRR-66AA homozygous genotypes-wild type, wherein, FAM
Channel C t values ＞ 27, HEX channel C t value≤27, ROX channel C t value≤27；
Figure 20 is the testing result amplification curve diagram of sample to be tested MTRR-66AG heterozygous genotypes-heterozygote, wherein, FAM
Channel C t value≤27, HEX channel C t value≤27, ROX channel C t value≤27；
Figure 21 is the testing result amplification curve diagram of sample to be tested MTRR-66GG homozygous genotypes-muton, wherein, FAM
Channel C t value≤27, HEX channel C t values ＞ 27, ROX channel C t value≤27.
(4) testing result explanation
| Gene | Site | Genotype |
| MTHFR | C677T | CC (normal) CT (poor) TT (risk) |
| MTHFR | A1298C | AA (normal) AC (poor) CC (risk) |
| MTRR | A66G | AA (normal) AG (poor) GG (risk) |
(5) folic acid subproposal
(6) prevention HCY and cardiovascular and cerebrovascular disease suggestion
Human body MTHFR and MTRR gene unconventionality can be detected accurately and in time using the present invention, for instructing standby pregnant woman
The how effective Supplement of folic acid of female, prevents fetal anomaly to have considerable meaning.
Although embodiments of the invention have been shown and described above, it is to be understood that above-described embodiment is example
Property, it is impossible to limitation of the present invention is interpreted as, one of ordinary skill in the art is not departing from the principle and objective of the present invention
In the case of above-described embodiment can be changed within the scope of the invention, change, replace and modification.
Sequence table
<110>Xiamen gold Nowe bio tech ltd
<120>The detection kit that a kind of step reverse transcription RT-qPCR people MTHFR is expressed with MTRR gene pleiomorphisms
<130> JNWS17001I
<160> 20
<170> PatentIn version 3.5
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cacagcaggg acaggc 16
<210> 14
<211> 21
<212> DNA
<213>It is artificial synthesized
<400> 14
ggctctaacc ttatcggatt c 21
<210> 15
<211> 14
<212> DNA
<213>It is artificial synthesized
<400> 15
tcgcagaaga aata 14
<210> 16
<211> 17
<212> DNA
<213>It is artificial synthesized
<400> 16
tgtgagaaaa aacacat 17
<210> 17
<211> 31
<212> DNA
<213>It is artificial synthesized
<400> 17
cagaagaaat gtgtgagcaa ttttttcaca c 31
<210> 18
<211> 19
<212> DNA
<213>It is artificial synthesized
<400> 18
ccgggacctg actgactac 19
<210> 19
<211> 19
<212> DNA
<213>It is artificial synthesized
<400> 19
cgacgtagca cagcttctc 19
<210> 20
<211> 19
<212> DNA
<213>It is artificial synthesized
<400> 20
cggctacagc ttcaccacc 19
Claims (10)
1. a kind of detection kit that step reverse transcription RT-qPCR people MTHFR is expressed with MTRR gene pleiomorphisms, its feature exists
In, including MTHFR-C677T reaction buffers, MTHFR-A1298C reaction buffers, MTRR-A66G reaction buffers, the positive
Control, negative control, One step enzyme mixations.
2. step reverse transcription RT-qPCR people MTHFR according to claim 1 is tried with the detection that MTRR gene pleiomorphisms are expressed
Agent box, it is characterised in that the detection kit includes 1 800 μ L MTHFR-C677T reaction buffers, 1 800 μ L
MTHFR-A1298C reaction buffers, 1 800 μ L MTRR-A66G reaction buffers, 1 50 μ L positive controls, 1 50 μ L are cloudy
Property control, 1 50 μ L One step enzyme mixation.
3. step reverse transcription RT-qPCR people MTHFR according to claim 1 is tried with the detection that MTRR gene pleiomorphisms are expressed
Agent box, it is characterised in that the MTHFR-C677T reaction buffers include One step RT-qPCR reaction
Buffer, RT reinforcing agent, the specific upstream and downstream primer of dNTPs, C677T, C677T specific probes；
The MTHFR-A1298C reaction buffers include One step RT-qPCR reaction buffer, RT reinforcing agent,
DNTPs, A1298C specificity upstream and downstream primer, A1298C specific probes；
The MTRR-A66G reaction buffers include One step RT-qPCR reaction buffer, RT reinforcing agent,
DNTPs, A66G specificity upstream and downstream primer, A66G specific probes.
4. step reverse transcription RT-qPCR people MTHFR according to claim 3 is tried with the detection that MTRR gene pleiomorphisms are expressed
Agent box, it is characterised in that the specific upstream and downstream primers of the C677T are MTHFR-C677T-L, MTHFR-C677T-R；It is described
C677T specific probes are MTHFR-C677T-P-W, MTHFR-C677T-P-M.
5. step reverse transcription RT-qPCR people MTHFR according to claim 3 is tried with the detection that MTRR gene pleiomorphisms are expressed
Agent box, it is characterised in that the specific upstream and downstream primers of the A1298C are MTHFR-A1298C-L, MTHFR-A1298C-R；Institute
A1298C specific probes are stated for MTHFR-A1298C-P-W, MTHFR-A1298C-P-M.
6. step reverse transcription RT-qPCR people MTHFR according to claim 3 is tried with the detection that MTRR gene pleiomorphisms are expressed
Agent box, it is characterised in that the specific upstream and downstream primers of the A66G are MTRR-A66G-L, MTRR-A66G-R；The A66G is special
Specific probes are MTRR-A66G-P-W, MTRR-A66G-P-M.
7. step reverse transcription RT-qPCR people MTHFR according to claim 1 is tried with the detection that MTRR gene pleiomorphisms are expressed
Agent box, it is characterised in that the positive control is human gene group DNA, mutant plasmid；The negative control is physiological saline.
8. step reverse transcription RT-qPCR people MTHFR according to claim 1 is tried with the detection that MTRR gene pleiomorphisms are expressed
Agent box, it is characterised in that the One step enzyme mixations are Taq enzyme, M-MLV enzymes, Rnasin inhibitor, enzyme stabilizers
Mixed liquor.
9. a kind of application method of any described detection kits of claim 1-8, comprises the following steps：
(1) RNA is extracted；
(2) reagent prepares and is loaded；
(3) machine testing on, enters performing PCR amplification and fluorescent collecting；
(4) explanation of assay.
10. the application method of detection kit according to claim 9, it is characterised in that the PCR amplifications and fluorescence are adopted
Collecting relative parameters setting is：
50 DEG C of 30min of reverse transcription；
95 DEG C of 5min of pre-degeneration；
PCR 95℃15s；60℃40s；Circulation 5 times；
95℃15s；60℃40s；Circulation 30 times.
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| CN201710463701.2A CN107236801A (en) | 2017-06-19 | 2017-06-19 | The detection kit that a kind of step reverse transcription RT qPCR people MTHFR is expressed with MTRR gene pleiomorphisms |
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| CN201710463701.2A CN107236801A (en) | 2017-06-19 | 2017-06-19 | The detection kit that a kind of step reverse transcription RT qPCR people MTHFR is expressed with MTRR gene pleiomorphisms |
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| CN201710463701.2A Withdrawn CN107236801A (en) | 2017-06-19 | 2017-06-19 | The detection kit that a kind of step reverse transcription RT qPCR people MTHFR is expressed with MTRR gene pleiomorphisms |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109055512A (en) * | 2018-10-07 | 2018-12-21 | 浙江数问生物技术有限公司 | A kind of kit and its detection method detecting people's MTHFR and MTRR gene pleiomorphism |
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| US20110212460A1 (en) * | 2002-02-22 | 2011-09-01 | Hogan Kirk J | Assay for nitrous oxide neurologic syndrome |
| CN105002275A (en) * | 2015-07-20 | 2015-10-28 | 武汉友芝友医疗科技有限公司 | Specific sequence specific primers-polymerase chain reaction primers and kit for human MTHFR and MTRR gene polymorphism detection |
| CN106591473A (en) * | 2017-01-20 | 2017-04-26 | 广州市达晖生物技术有限公司 | Human MTHFR and MTRR gene polymorphism detection primer, probe, test kit and method |
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2017
- 2017-06-19 CN CN201710463701.2A patent/CN107236801A/en not_active Withdrawn
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| US20110212460A1 (en) * | 2002-02-22 | 2011-09-01 | Hogan Kirk J | Assay for nitrous oxide neurologic syndrome |
| CN105002275A (en) * | 2015-07-20 | 2015-10-28 | 武汉友芝友医疗科技有限公司 | Specific sequence specific primers-polymerase chain reaction primers and kit for human MTHFR and MTRR gene polymorphism detection |
| CN106591473A (en) * | 2017-01-20 | 2017-04-26 | 广州市达晖生物技术有限公司 | Human MTHFR and MTRR gene polymorphism detection primer, probe, test kit and method |
Non-Patent Citations (2)
| Title |
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| BOYI YANG ET AL.: "Geographical Distribution of MTHFR C677T, A1298C and MTRR A66G Gene Polymorphisms in China: Findings from 15357 Adults of Han Nationality", 《PLOS ONE》 * |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109055512A (en) * | 2018-10-07 | 2018-12-21 | 浙江数问生物技术有限公司 | A kind of kit and its detection method detecting people's MTHFR and MTRR gene pleiomorphism |
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