CN109321648A - MTHFR (C677T) genotype quick detection kit based on POCT mode - Google Patents
MTHFR (C677T) genotype quick detection kit based on POCT mode Download PDFInfo
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Abstract
The present invention provides a kind of MTHFR (C677T) genotype quick detection kit based on POCT mode, the kit contains fluorescence quantification PCR primer for detecting MTHFR (C677T) genotype and probe and cell pyrolysis liquid, the PCR primer sequence is as shown in SEQ ID NO.1-2, and the probe is as shown in SEQ ID NO.3-4.This kit can realize instant detection, it is purified without DNA, sample can be directly added into progress PCR reaction in reagent, it is particularly suitable for quick, the accurate detection of the lower sample of DNA content (such as mouth desquamated cells), Detection accuracy is up to 99% or more, detection sensitivity is high, can accurately detect down to 0.125ng genomic DNA；Entire detection is time-consuming short, and testing result can be obtained in 1 hour, can provide medication foundation to doctor in first time, reduce patient medication risk.
Description
Technical field
The present invention relates to molecular biology fields, specifically, being related to a kind of MTHFR (C677T) based on POCT mode
Genotype quick detection kit.
Background technique
Methylene tetrahydrofolate reductase gene (MTHFR, 5,10-methylenetetrahydrofolate
It reductase), is the key gene in methionine-folic acid metabolism system.
The major function of MTHFR is that catalysis 5,10- methylene tetrahydrofolate is converted into biologically active 5- methyl four
Hydrogen folic acid, the process are a rate-limiting steps of folic acid metabolism.Folic acid can not only be used to purine biosynthesis and phonetic by metabolism
Pyridine can also make nucleic acid and albumen methylate.Therefore, normal folic acid metabolism can not only maintain body cell growth and it is numerous
It grows, moreover it is possible to which gene expression is adjusted by methylation.Human body cannot synthesize folic acid as a kind of mammal itself, can only be from food
It is absorbed in object.The reduction of MTHFR activity will lead to albumen, and nucleic acid metabolism disorder, this will cause a series of diseases, such as folic acid generation
Thanking to the synthesis of DNA caused by exception and repairing to be obstructed will lead to the cell generation of dysfunction；The extremely caused methyl of folic acid metabolism
Change exception and also results in cell function exception；Homocysteine accumulation will cause cell abnormal, lead to Atherosclerosis
Change, and then induces coronary heart disease, the cardiovascular and cerebrovascular diseases such as cerebral infarction.In addition, the MTHFR activity reduction of pregnant woman not only may cause
The hypertensive disorder in pregnancy of pregnant woman itself and spontaneous abortion, it is also possible to lead to a series of neonatal diseases, such as nerve channel
Congenital malformation caused by defect, congenital heart disease, Down's syndrome, harelip etc..Therefore folic acid metabolism will will lead to extremely
Many adult and neonatal diseases, bring pain and financial burden, so needing to folic acid metabolism exception
People gives suitable medication guide to mitigate patient suffering.
MTHFR has the site multiple single nucleotide polymorphism (SNP), since SNP can change coding protein structure and then lead
MTHFR activity is caused to reduce, expression quantity is reduced and thermal stability reduces.It is to the most important SNP site of MTHFR activity influence
C677T(rs1801133).Therefore, the individual need of MTHFR (C677T) site mutation suitably increases folic acid intake
It can maintain the folic acid metabolism of human normal.The heterozygote in the site MTHFR (C677T) and the activity of no mutant homozygote are wild respectively
Raw homozygous 65% and 30%.Since the dosage of folic acid excessively also results in body metabolism burden and some body injuries,
So patient's dosage suggestion appropriate can just be given by needing to carry out people MTHFR (C677T) detection.According to statistics, in
The frequency of mutation of MTHFR (C677T) is 45.2% in state crowd.It, can be right by being detected to the site MTHFR (C677T)
Individual folic acid metabolism ability is assessed, and auxiliary doctor is to pregnant woman's expected date of childbirth newborn child's illness and adult folic acid metabolism risk
It is assessed, so that folic acid individuation be instructed to use.Therefore fast and accurately MTHFR (C677T) Genotyping is detected to disease
Individual human has great clinical meaning using folic acid.
Currently, the method for detection MTHFR (C677T) mainly has PCR sequencing PCR, gene chips and fluorescence quantitative PCR method.It surveys
The testing result of sequence method (such as patent CN105368933A) and gene chips (such as patent CN102533952A) has very high
Precision, but Large-Scale Precision Instrument and Equipment is needed, fixed laboratory and palpus professional operator, therefore, both detections
Method is at high cost, complex steps and time-consuming, is clinically difficult to be promoted.And the fluorescent PCR based on Taqman probe
Method (such as patent CN106434923A) is although have at low cost, and the time is short and the simple advantage of interpretation of result, since this is anti-
Answer system that can only detect DNA, so must be operated using multi-step multitube, it is also undesirable on clinical expansion.Therefore these
Conventional detection technique is all difficult to meet the domestic crowd for carrying MTHFR (C677T) mutated gene to mthfr gene detection fastly
Speed accurately requires.
Summary of the invention
The object of the present invention is to provide a set of for detecting the fluorescence quantification PCR primer pair of MTHFR (C677T) genotype
And probe combinations.
It is a further object of the present invention to provide a kind of, and MTHFR (C677T) genotype based on POCT mode quickly detects examination
Agent box.
In order to achieve the object of the present invention, it is fixed to provide a set of fluorescence for detecting MTHFR (C677T) genotype by the present invention
Measure PCR primer to and probe combinations, including upstream primer, downstream primer, wild-type probe and saltant type probe, their core
Nucleotide sequence difference is following (SEQ ID NO:1-4):
MTHFR (C677T) upstream primer: 5 '-CATCCCTATTGGCAGGTTAC-3 '；
MTHFR (C677T) downstream primer: 5 '-TGTGTCAGCCTCAAAGAAAA-3 '.
MTHFR (C677T) wild-type probe: 5 '-FAM-AATCGGCTCCCGCA-MGB-3 '；
MTHFR (C677T) saltant type probe: 5 '-Teaxs Red-AAATCGACTCCCGCA-MGB-3 '.
Preferably, the probe combinations include:
MTHFR (C677T) wild-type probe: 5 '-FAM-AATC+GGCTCCCGCA-MGB-3 '；
MTHFR (C677T) saltant type probe: 5 '-Teaxs Red-AAATCG+ACTCCCGCA-MGB-3 '.
Wherein, "+" indicates the single base on right side for LNA modification.
The probe need to be in 5 ' end connection reporter fluorescence groups, in 3 ' end connection fluorescent quenching groups.
Reporter fluorescence group and quenching group commonly used in the art can be selected in the reporter fluorescence group and quenching group.
In a specific embodiment of the invention, the reporter fluorescence group that the probe uses are as follows: wild type uses FAM
Label, saltant type are marked using Teaxs Red, and 3 ' ends of the probe are modified through MGB.
The present invention also provides the detection reagents or kit that contain the primer and probe.
MTHFR (C677T) genotype quick detection kit based on POCT mode that the present invention also provides a kind of, it is described
Kit at least contains the primer and probe and cell pyrolysis liquid；
Wherein, the cell pyrolysis liquid is made of lauryl sodium sulfate and Triton X-100.
Specifically with lauryl sodium sulfate 0.0005-0.015%w/v and Triton X-100 0.001-
0.03%w/v is as 200 × cell pyrolysis liquid mother liquor.In use, diluting in proportion.
Preferably, the cell pyrolysis liquid (200 × mother liquor) are as follows:
Lauryl sodium sulfate 0.009%w/v, Triton X-100 0.01%w/v；Or
Lauryl sodium sulfate 0.005%w/v, Triton X-100 0.02%w/v；Or
Lauryl sodium sulfate 0.005%w/v, Triton X-100 0.001%w/v；Or
Lauryl sodium sulfate 0.015%w/v, Triton X-100 0.003%w/v.
It further include dNTPs, Taq archaeal dna polymerase, Mg in kit of the present invention2+, PCR reaction buffer, standard male
At least one of property template, sampling rod (for example, disposable oral cavity swab), sample collection tube etc..
Preferably, the Taq archaeal dna polymerase is thermal starting Taq archaeal dna polymerase.
It will be appreciated by those skilled in the art that, when carrying out PCR reaction detection, being answered to obtain optimal detection effect
It is detected respectively in independent reaction system when for different test objects (different loci).
Each component is final concentration of in PCR reaction system matched with kit of the present invention: 0.5-1.5 × PCR is anti-
Answer buffer, Taq archaeal dna polymerase 0.04-0.1U/ μ L, dNTPs 0.1-0.5mM, 0.2-0.7 μM of upstream primer, downstream primer
0.2-0.7 μM, 0.2-0.7 μM of wild-type probe, 0.2-0.7 μM of saltant type probe, Mg2+1-2.5mM and 1 × cell pyrolysis liquid.
Preferably, in the PCR reaction system each component it is final concentration of: 1.1 × PCR reaction buffer, DNA polymerization
Enzyme 0.05U/ μ L, dNTPs 0.2mM, 0.4-0.6 μM of upstream primer, 0.4-0.6 μM of downstream primer, wild-type probe 0.4-0.6
μM, 0.4-0.6 μM of saltant type probe, Mg2+2.5mM and 1 × cell pyrolysis liquid.
Preferably, PCR response procedures matched with kit of the present invention are as follows: 95 DEG C of 5min；95 DEG C of 8s, 55 DEG C
35s, 50 circulations.
Kit of the invention is detected applied to actual sample, the detection sample can come from oral cavity wall, tongue
The positions such as head, palm, ear, the DNA that the cast-off cells from above-mentioned position release after lysate cracks are used equally for this
The genetic test of kit, since scraping oral cavity wall Sample Method is simple and efficient, and sample size is moderate, therefore chooses oral cavity
Sample is optimal.Sampling and load procedure can be completed in 20 seconds under normal circumstances.
(1) prepare before sampling
Patient must be gargled 2 times with clear water before sampling, and no less than 5 seconds every time, swallowed 2-3 times after gargling, avoided oral cavity as far as possible
Inner wall remains saliva.Sampling person must wearing gloves, mask.After above-mentioned be ready to complete, it can be sampled.
(2) sampling and sample-adding
Sampling tool is disposable oral cavity swab.Specific sampling process is as follows:
1) reaction solution and swab are taken out from kit and swab packaging bag respectively, then pulls out reagent plug (Fig. 1-a)；
2) swab end cap is removed, should pay attention to not contacting reagent plug (Fig. 1-b) in the process；
3) make cheek wall in the nearly 90 ° of contacts oral cavity of swab end, uniformly wipe 5 times (being up and down 1 time), dynamics is micro- with cheek
It is prominent to be advisable (Fig. 1-c)；
4) immediately by swab intercalation reaction liquid after sampling, pressing makes itself and the reagent seal of tube, is protected from light temporary (such as Fig. 1-
d)。
Analysis of test results: after reaction by analysis system can intuitively and accurately to testing result carry out analysis and
It interprets, can produce following three kinds of results altogether.
1. Wild homozygous
The testing result has been shown as in analysis system and only green fluorescence curve is exponentially increased trend, and
Have and only (cycle threshold refers to that the fluorescence signal in each reaction tube reaches and sets green fluorescence channel generation Ct value
Recurring number experienced when fixed threshold value).(Fig. 2)
2. mutated homozygous
The testing result has been shown as in analysis system and only red fluorescence curve is exponentially increased trend, and
Have and only red fluorescence channel generates Ct value.(Fig. 3)
3. heterozygous
The testing result shows as green fluorescence curve in analysis system and red fluorescence curve is exponentially increased
Gesture, and green fluorescence channel and red fluorescence channel generate Ct value.(Fig. 4)
By above-mentioned technical proposal, the present invention at least have following advantages and the utility model has the advantages that
(1) detection immediately can be achieved, purified without DNA, sample can be directly added into progress PCR reaction in reagent, 1 is small
When the interior detection information that corresponding gene loci can be obtained；Therefore medication foundation can be provided to doctor in first time, to avoid
Patient medication mistake.
(2) it joined cell pyrolysis liquid in reagent reaction system, Direct Pyrolysis cell and can release in nucleus
Sample is added in DNA, DNA extraction is reacted the stopped pipe in same branch Reagent Tube with PCR and carried out.
(3) this kit sample (DNA or Stomatocyte) Detection accuracy is up to 99% or more.
(4) detection method, sampling process is painless noninvasive, easy to operate, can be complete in single sampling sample-adding 20s
At.
(5) detection sensitivity is high, can accurately detect the genomic DNA down to 0.125ng.It is too long for the holding time
And the lower sample of the DNA contents such as buccal swab can be detected accurately.
(6) this kit can at least place 72h at normal temperature, and 2-8 DEG C can at least place 15 days, and -20 DEG C can at least put
It sets 12 months.
(7) present invention employs novel sample-addings to the mode of operation of one step of result, eliminates cumbersome intermediate ring
Section can go out testing result in 1 hour, solve emergency patients and treat urgent problems.Single step, single tube reagent, stopped pipe
A possibility that operating, considerably reducing environmental pollution.
(8) present invention has also matched intellectual analysis program, can directly automatically analyze to data, obtain at once
The genotype call results and medication guide of unbiasedness are reported, laboratory constraint is got rid of, to environment and operator without spy
It is different to require.
(9) common molecular beacon can be used in the probe that the present invention uses, and can also be used while having MGB
, but Taqman probe (minorgroovebinding) and the Taqman probe of LNA (locknucleicacid) dual modification
There are greater advantages compared with molecular beacon itself, Taqman probe not only greatly reduces background signal intensity, and further mentions
The high specificity of probe, sensitivity and accuracy.
(10) present invention additionally uses thermal starting enzyme, by optimizing reagent system, can contain oral cavity impurity and ring
The risk that border temperature change is brought.
Detailed description of the invention
Fig. 1 is sampling and the sample-adding operational flowchart of Stomatocyte in detection process.
Fig. 2 is MTHFR (C677T) Wild homozygous testing result figure.
Fig. 3 is MTHFR (C677T) mutated homozygous testing result figure.
Fig. 4 is MTHFR (C677T) heterozygous testing result figure.
Fig. 5-Figure 10 is respectively three kinds of three kinds of genotype Ct Data-Statistics figures of SLCO1B1 (T521C) reagent A group, B group genes
Type Ct Data-Statistics figure, three kinds of genotype Ct Data-Statistics figures of C group, A group three kinds of genotype end point fluorescence value (EPF) statistical charts, B groups
Three kinds of three kinds of genotype end point fluorescence value (EPF) statistical charts, C group genotype end point fluorescence value (EPF) statistical charts.Note: with it is right
It is compared according to group, corresponding each group of data difference is not significant (P > 0.05)." G " indicates that green fluorescence channel, " R " indicate red fluorescence
Channel.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, implementing
Example is according to conventional laboratory conditions or according to the condition of manufacturer's specification suggestion.Test material as used in the following examples
Material, is to be commercially available from conventional biochemical reagent company unless otherwise specified.
Embodiment 1 is used to detect the configuration of the PCR reaction solution of MTHFR (C677T) gene
The prepared PCR reaction solution of the present embodiment is 23.5 μ L systems, and raw material and its final concentration are as follows:
Archaeal dna polymerase 0.05U/ μ L；
dNTPs 0.2mM；
5 × reaction buffer 1.1 ×；
MgCl22.5mM；
Cell pyrolysis liquid 1 ×
0.5 μM of upstream primer；
0.5 μM of downstream primer；
0.5 μM of saltant type probe；
0.5 μM of wild-type probe；
Ultrapure water polishing is to 23.5 μ L.
Wherein, reaction buffer is Colorless Reaction Buffer；dNTPs,MgCl2、 5x Colorless
Reaction Buffer, archaeal dna polymerase are purchased from Shanghai Pu Luomaige biological products using GoTaq DNA Polymerase
Co., Ltd.
The cell pyrolysis liquid is made of lauryl sodium sulfate and Triton X-100；
Wherein, the concentration of lauryl sodium sulfate is 0.0005-0.015% (w/v)；Triton X-100
Concentration is 0.001-0.03% (w/v).
2 LNA of embodiment modifies probe and improves parting accuracy
LNA is a kind of oligonucleotide derivative, with DNA/RNA have similar structure, therefore can to DNA and RNA into
Row strong identification and combination.After LNA is for the modification of oligonucleotides, the thermal stability of primer or probe can be increased, improved
3~8 DEG C of its annealing temperature.The probe of this kit developing is modified using LNA, and through software prediction, the wild type after modification is visited
The Tm value of needle and saltant type probe in conjunction with template improves 4 DEG C or so.In order to sufficiently show LNA modification probe and without
LNA modifies the difference of probe, carries out following comparative's experiment, and PCR system is shown in Table 2-1, detects wild homozygote, heterozygote respectively
And no mutant homozygote, each genotype do three repetitions, response procedures are shown in Table 1.
Table 1
The primer and probe sequence are as follows:
Upstream primer: 5 '-CATCCCTATTGGCAGGTTAC-3 '；
Downstream primer: 5 '-TGTGTCAGCCTCAAAGAAAA-3 '；
The wild-type probe of LNA modification: 5 '-FAM-AATC+GGCTCCCGCA-MGB-3 '；The saltant type of LNA modification is visited
Needle: 5 '-Teaxs Red-AAATCG+ACTCCCGCA-MGB-3 '；
Wild-type probe without LNA modification:
5'-FAM-AATCGGCTCCCGCA-MGB-3'；
Saltant type probe without LNA modification:
5’-TeaxsRed-AAATCGACTCCCGCA-MGB-3’。
(note: "+" indicates right side base for LNA modification)
Table 2-1 PCR reaction system table
| Formula 1 | Formula 2 | Reactive component is dense eventually |
| 5×Promega Colourless Buffer | 5×Promega Colourless Buffer | 1.1× |
| dNTP(10mM) | dNTP(10mM) | 0.2mM |
| MgCl2(25mM) | MgCl2(25mM) | 2.5mM |
| Cell pyrolysis liquid (200 ×) | Cell pyrolysis liquid (200 ×) | 1× |
| Upstream primer (100 μM) | Upstream primer (100 μM) | 0.5μM |
| Downstream primer (100 μM) | Downstream primer (100 μM) | 0.5μM |
| Wild type LNA modifies probe | Wild type modifies probe without LNA | 0.5μM |
| Saltant type LNA modifies probe | Saltant type modifies probe without LNA | 0.5μM |
| Archaeal dna polymerase (5U/ μ L) | Archaeal dna polymerase (5U/ μ L) | 1.25U |
| DNA(10ng/μL) | DNA(10ng/μL) | 10ng |
| Ultrapure water is added to 23.5 μ L | Ultrapure water is added to 23.5 μ L | 23.5μL |
Experimental result:
Tri- kinds of genotype Ct Data-Statistics of table 2-2
It can be seen that from table 2-2 and meet the requirements through LNA modification probe groups to the Ct value of three kinds of genotype, and without
The probe groups of LNA modification may cause inspection not measure Ct value since EPF increment is too low.The experimental results showed that when probe is repaired through LNA
After decorations, be conducive to the combination of probe and target sequence.
The test of 3 accuracy of embodiment
This kit is used as amplified sample using mucous membrane of mouth cast-off cells, due to detected personnel's living habit and a
The diversity of body gene order may cause reagent and be interfered in parting, in order to verify the accuracy of this kit parting,
This experiment is tested (the wherein genotype of tester using the mucous membrane of mouth cast-off cells of three genotype difference personnel
Confirmed by PCR sequencing PCR), operating process, total sample number are 300 to operating method referring to Fig.1, agent prescription such as table 3-1 institute
Show.
Table 3-1 PCR reacts total system formula table
Experimental result is shown in Table 3-2:
The accuracy of tri- kinds of genotype of table 3-2 counts
It can be seen that in 300 test samples from table 3-2, the Detection accuracy of this kit can achieve
99.7%.Tentatively show in the case where being tested according to 1 operating process of attached drawing, which has very high accuracy, can
To reach 99% or more.
The test of 4 detection sensitivity of embodiment
In order to verify the sensitivity of MTHFR (C677T) genotype quick detection kit, we are by sample DNA (heterozygosis
Son) be added total amount respectively with: five concentration gradients of 1ng, 0.5ng, 0.25ng, 0.125ng and 0.1ng are added according to attached drawing 1
Sample detection, reagent are configured according to table 1.The above detection every time is at least repeated more than twice, and result is as shown in following table 4-1:
The testing result of table 4-1 difference sample concentration
| DNA additional amount | 1ng | 0.5ng | 0.25ng | 0.125ng | 0.1ng |
| Testing number | 100 | 100 | 100 | 100 | 100 |
| Positive exact figures | 100 | 100 | 100 | 100 | 99 |
| Accuracy rate | 100% | 100% | 100% | 100% | 99% |
By it is upper the results show that sample DNA concentration respectively with: 1ng, 0.5ng, 0.25ng, 0.125ng and 0.1ng five are dense
Degree gradient is detected with MTHFR (C677T) genotype detection kit, and accuracy rate reaches 100%, when DNA concentration is
When 0.1ng, accuracy 99%.In other words, MTHFR of the invention (C677T) genotype quick detection kit only needs
The DNA profiling amount of 35 copy numbers ensures that correct parting.
The detection of 5 anti-interference ability of embodiment
Since this kit is detected in such a way that mucous membrane of mouth cast-off cells directly expand, oral cavity feed
Whether residue afterwards can interfere reagent testing result, and urgent need is studied.This experiment is before sampling operation to being sampled personnel
(its genotype is confirmed through PCR sequencing PCR) is required, and sampling is as standard after 30min fasting before sampling, to eat sweet food, drink alkali
Property tea, eat acidic food, spicy food and drink after Chinese medicine that sampling immediately is as processing group, with same a collection of reagent to all samples
Product are detected respectively, wild homozygote 120 in every group of sample, and heterozygote 120, no mutant homozygote 60, agent prescription
As shown in table 3-1, testing result is as shown in table 5-1:
Table 5-1 reagent anti-interference ability accuracy test result
It is compared as can be seen from the above results with the detection accuracy of fasting sampling, the Detection accuracy of other experimental groups
Below 90%, show to eat up larger to the interference of detection after these foods, eating up should be laggard in 30min after these food
Capable sampling of gargling.Since this experiment sampling is limited, inspection should be still sampled after fasting 30min when carrying out clinical detection
It surveys.
Influence test of 6 environment of embodiment to detection kit
Whether testing result is influenced by environmental factor, we respectively routine office work area (gross area be 200 square metres,
It is encompassing to receive 50 people, their activity (arbitrarily can walk about or speak) is not limited, and environment temperature is 27 DEG C, humidity 77%) and
Grade of cleanliness is that 1,000,000 grades of toilet takes multiple subjects at random (its genotype is confirmed through PCR sequencing PCR)
Sample, each subject carry out repeating to test twice respectively in two regions, and agent prescription is referring to table 3-1, testing result statistics
As shown in Table 6-1.
The genotype call results accuracy of two sampling areas of table 6-1 counts
Result above, which can be seen that Office Area and the detection accuracy of clean area sampling, can reach 99% or more and standard
True rate only poor 0.4%, shows that environmental factor does not have larger impact to the result of detection.This time test is kit in hospital
Application portability provides data, to provide strong data supporting in the popularization of hospital and use later.
7 kit of the embodiment Detection of Stability that (room temperature, 2-8 DEG C, -20 DEG C) is placed under condition of different temperatures
This kit is based on POCT mode development, and the detection of hospital bed side, low-temperature storage and low-temperature operation are difficult to reality
It is existing, so after needing detection reagent to place certain time length at normal temperature, if adverse effect can be generated to testing result.It prepares big
Batch reagent is randomly divided into 3 temperature condition groups totally 24 processing groups, and grouping situation is shown in Table 7-1, wherein control group are as follows: with postponing
Upper machine testing (2-8 DEG C of preparation, the interior sampling loading coded program for completing 108 samples of 30min) immediately, each processing group is total
108 reagents, every kind genotype detection 36.After reagent processing to be done, is operated according to SOP, acquire the person's of being sampled (its
MTHFR (C677T) genotype is confirmed through PCR sequencing PCR) buccal sample, wild homozygote, no mutant homozygote, heterozygote respectively take 36
Branch.It is unified to carry out Fluorescence PCR according to table 1.
Table 7-1 reagent, which places condition and places duration, is grouped situation table
Experimental result:
Ct value and end point fluorescence value (EPF) mean value statistical result are shown in Fig. 5-Figure 10 respectively.
Three kinds of genotyping accuracy statistical results are shown in Table 7-2.
Tri- kinds of genotyping accuracy statistical forms of table 7-2
Above data show room temperature, 2-8 DEG C place, -20 DEG C after a long time can to Detection accuracy generate shadow
It rings.But it can be seen that each 108 number of cases evidence of processing group from table 7-2,1~7 component type accuracy of tri- temperature conditions of A/B/C is
100%, when to the 8th group of tri- temperature conditions of A/B/C, there is the situation of parting mistake.Illustrate to place reagent under normal temperature condition
At least 7 days, it will not influence the genotyping result of reagent；It is placed reagent at least 15 days under the conditions of 2-8 DEG C, will not influence point of reagent
Type result；It is placed reagent at least 12 months under the conditions of -20 DEG C, will not influence the genotyping result of reagent.In conclusion provable
MTHFR (C677T) reagent of this kit has good stability, is detected in hospital in use, of short duration (room temperature 5 days
It is interior) non-cryogenic environment place, will not influence parting accuracy, can satisfy POCT mode completely.
Embodiment 8 is used to detect the design of the primer and probe of MTHFR (C677T) genotype
After special primer and probe screening confirmation, concentration of the primer and probe in PCR reaction system need to be carried out excellent
Change (using Stomatocyte as template).The corresponding PCR reaction system of primed probe concentration is as shown in following table 8-1.
Table 8-1 primed probe concentration experiment PCR reacts total system formula table
Experimental result is as follows:
(1) primer concentration grads experiment (concentration one, concentration two, concentration three)
A.CT Data-Statistics are as follows
Table 8-2 CT Data-Statistics table
B.EPF Data-Statistics are as follows:
Tri- kinds of table 8-3, three kinds of concentration genotype EPF Data-Statistics table
C. heterozygous G/R ratio statistics is as follows:
Table 8-4 heterozygous G/R ratio
Note: " G/R ratio " is the ratio of green tunnel end points fluorescent value and red tunnel end points fluorescent value.
The reagent C T value configured by three concentration gradients of primer is but dense it was found that difference is unobvious between three
The extremely significant end point fluorescence value (EPF) higher than other two concentration of three genotype end point fluorescence values (EPF) of degree two.It can
A large amount of dimers can be formd between primer, affect the amplification efficiency of primer the reason is that concentration one is excessively high；Concentration three is very few, raw
It is inadequate at product amount.It can be seen that the ratio is close to 1 when primer concentration is concentration two by heterozygous G/R ratio.By
Known to this when upstream and downstream primer concentration is 0.6 μM, reaction system is optimal.
(2) concentration and probe concentration gradient experiment
In view of heterozygous G/R ratio is as far as possible close to 1, therefore concentration and probe concentration is adjusted.
A.CT Data-Statistics are as follows
Table 8-5 Ct Data-Statistics table
B.EPF Data-Statistics are as follows:
Tri- kinds of genotype EPF statistical forms of table 8-6
C. heterozygous G/R ratio statistics is as follows:
Table 8-7 heterozygous G/R ratio
The reagent C T value configured by three concentration gradients of primer is but dense it was found that difference is unobvious between three
The end point fluorescence value of degree two is significantly higher than the end point fluorescence value (p≤0.05) of concentration one and three.Illustrate in same probe concentration item
Under part, what concentration two expanded may participate in effective product amount highest of PCR reaction.Correctly matched amount is more for probe, and reagent is also
It is more stable.
It can be seen that by green red tunnel end points fluorescent value ratio (G/R ratio), the green red tunnel end points fluorescent value of concentration two
Ratio (G/R ratio) is closer to 1, as a result more acurrate reliable when illustrating to detect heterozygote genotype.
The reagent end point fluorescence value prepared by two concentration gradients of probe and green red channel fluorescence ratio (G/R
Ratio) it was found that, the extremely significant end point fluorescence value (p≤0.01) higher than concentration five of the end point fluorescence value of concentration four, concentration
Four green red channel fluorescence ratios (G/R ratio) are closer to 1, and kit sensitivity under the concentration is higher, testing result
It is more acurrate.
This illustration is to prove the spirit of reaction system used in MTHFR (C677T) loci gene type quick detection kit
Sensitivity is high, and stability is good, as a result accurately and reliably.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be modified or is improved, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to claimed model without departing from theon the basis of the spirit of the present invention
It encloses.
Sequence table
<110>Chongqing Jing Yin biotechnology Co., Ltd
<120>MTHFR (C677T) genotype quick detection kit based on POCT mode
<141> 2018-02-09
<160> 4
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<210> 1
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<210> 2
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<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
tgtgtcagcc tcaaagaaaa 20
<210> 3
<211> 14
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
aatcggctcc cgca 14
<210> 4
<211> 15
<212> DNA
<213>artificial sequence (Artificial Sequence)
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aaatcgactc ccgca 15
Claims (10)
1. a kind of primer pair for detecting MTHFR (C677T) gene pleiomorphism, which is characterized in that detection MTHFR (C677T) gene
Primer pair include:
MTHFR (C677T) upstream primer: 5 '-CATCCCTATTGGCAGGTTAC-3 '；
MTHFR (C677T) downstream primer: 5 '-TGTGTCAGCCTCAAAGAAAA-3 '.
2. with the matching used probe combinations of primer pair described in claim 1, which is characterized in that the probe combinations include:
MTHFR (C677T) wild-type probe: 5 '-FAM-AATCGGCTCCCGCA-MGB-3 '；
MTHFR (C677T) saltant type probe: 5 '-Teaxs Red-AAATCGACTCCCGCA-MGB-3 '.
3. probe combinations according to claim 2, which is characterized in that the probe combinations include:
MTHFR (C677T) wild-type probe: 5 '-FAM-AATC+GGCTCCCGCA-MGB-3 '；
MTHFR (C677T) saltant type probe: 5 '-Teaxs Red-AAATCG+ACTCCCGCA-MGB-3 '；
Wherein, "+" indicates the single base on right side for LNA modification.
4. detection reagent or kit containing probe combinations described in primer pair described in claim 1 and Claims 2 or 3.
5. a kind of PCR reaction solution for detecting MTHFR (C677T) gene pleiomorphism, which is characterized in that the PCR reaction solution includes
Primer pair described in claim 1 and probe combinations described in claim 2 or 3.
6. PCR reaction solution according to claim 5, which is characterized in that the PCR reaction solution further include archaeal dna polymerase,
DNTPs, buffer, MgCl2And cell pyrolysis liquid.
7. PCR reaction solution according to claim 6, which is characterized in that the final concentration of each component in the PCR reaction solution
Are as follows: 0.5-1.5 × PCR reaction buffer, archaeal dna polymerase 0.04-0.1U/ μ L, dNTPs 0.1-0.5mM, upstream primer 0.2-
0.7 μM, 0.2-0.7 μM of downstream primer, 0.2-0.7 μM of wild-type probe, 0.2-0.7 μM of saltant type probe, Mg2+1-2.5mM and 1
× cell pyrolysis liquid；
The cell pyrolysis liquid is made of lauryl sodium sulfate and Triton X-100；
Wherein, the concentration of lauryl sodium sulfate is 0.0005-0.015% (w/v)；The concentration of Triton X-100 is
0.001-0.03% (w/v).
8. PCR reaction solution according to claim 7, which is characterized in that the final concentration of each component in the PCR reaction system
Are as follows: 1.1 × PCR reaction buffer, archaeal dna polymerase 0.05U/ μ L, dNTPs 0.2mM, 0.4-0.6 μM of upstream primer, downstream are drawn
0.4-0.6 μM of object, 0.4-0.6 μM of wild-type probe, 0.4-0.6 μM of saltant type probe, Mg2+2.5mM and 1 × cell pyrolysis liquid.
9. MTHFR (C677T) genotype quick detection kit based on POCT mode, which is characterized in that the kit contains
It has the right to require any PCR reaction solution of 6-7.
10. kit according to claim 9, which is characterized in that when the kit carries out quantitative fluorescent PCR, work
Program are as follows: 95 DEG C of initial denaturation 5min；95 DEG C of denaturation 8s, 63 DEG C of annealing and extension 35s, recycle 50 times.
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Citations (3)
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|---|---|---|---|---|
| US20160273041A1 (en) * | 2013-07-31 | 2016-09-22 | Seegene, Inc. | Method for determining snp genotype |
| CN106434923A (en) * | 2016-09-28 | 2017-02-22 | 江苏睿玻生物科技有限公司 | MTHFR gene C677T non-invasive detection kit and method |
| CN107446998A (en) * | 2017-07-25 | 2017-12-08 | 重庆京因生物科技有限责任公司 | MTHFR C677T, rs1801133 single nucleotide polymorphisms quick detections primer, molecular beacon, kit and its detection method |
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2018
- 2018-02-14 CN CN201810152206.4A patent/CN109321648A/en active Pending
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|---|---|---|---|---|
| US20160273041A1 (en) * | 2013-07-31 | 2016-09-22 | Seegene, Inc. | Method for determining snp genotype |
| CN106434923A (en) * | 2016-09-28 | 2017-02-22 | 江苏睿玻生物科技有限公司 | MTHFR gene C677T non-invasive detection kit and method |
| CN107446998A (en) * | 2017-07-25 | 2017-12-08 | 重庆京因生物科技有限责任公司 | MTHFR C677T, rs1801133 single nucleotide polymorphisms quick detections primer, molecular beacon, kit and its detection method |
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| Title |
|---|
| ALEXANDRA BOZIKOVA等: "Ethnic Differences in the Association of Thrombophilic Polymorphisms With Obstetric Complications in Slovak and Roma (Gypsy) Populations", 《GENETIC TESTING AND MOLECULAR BIOMARKERS》 * |
| 阿周存等: "MTHFR基因677C/T多态性对***密度的影响", 《生殖与避孕》 * |
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Application publication date: 20190212 |