CN108929906A - For detecting the primed probe group and its application of rs1799889 - Google Patents
For detecting the primed probe group and its application of rs1799889 Download PDFInfo
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Abstract
The invention discloses a kind of for detecting the primed probe group and its application of rs1799889.The present invention protects a kind of primed probe group first, is made of primers F, primer R and probe P；Primers F is single strand dna shown in the sequence 1 of sequence table；Primer R is single strand dna shown in the sequence 2 of sequence table；Probe P is the single strand dna that an end has fluorescent quenching group with fluorophor and another end, and the partial nucleotide in DNA molecular is lock nucleic acid, and the nucleotides sequence of DNA molecular is classified as shown in the sequence 3 of sequence table.The present invention can be used for detecting rs1799889, judge genotype of the sample to be tested based on the site, to instruct to use medication, have great application and popularization value.
Description
Technical field
The present invention relates to a kind of for detecting the primed probe group and its application of rs1799889.
Background technique
Fibrinolytic system composition includes activation and the inhibition ingredient of the plasminogen and plasminogen of direct thrombolysis.Plasminogen
Activator includes t-PA, urokinase, the factor, high-molecular-weight kininogen and kallikrein etc. in contact activation system；Fibrinolytic suppression
Object processed includes the mortifier PAI and plasmin inhibitor of tissue plasminogen activator (t-PA).Fibrinolysis activation of zymogen
Object inhibitor 1(Plasminogen activator inhibitor-1, PAI-1) be fibrinolytic system main regulatory factors, with
After activator of plasminogen combines, makes its fast deactivation and play anti-fibrinolysis activity, fibrin degradation can be reduced, cause fiber
Albumen aggregation, keeps the dynamic equilibrium of fibrinolytic system and blood coagulation system in human normal blood.T-PA and PAI-1 mutually restricts tune
It is whole and maintain normal plasma fibrinolytic.
In blood circulation, once coagulation system activation, fibrin is formed, then plasma fibrinolysis system, which is transferred, activates,
Fibrinolysin is become by t-PA activation plasminogen, makes fibrin degradation, and plays thrombolytic effect.But t-
The effect of PA will be directly affected fibrinolytic system by the control and adjusting of its mortifier PAI, the latter's concentration or active height
Activity.One of PAI activity or horizontal excessively high the main reason for being thrombosis, too low then easy cause bleeding.
PAI-1 concentration, which increases, can be used as the recurrent prediction index of myocardial infarction.- 1 concentration of plasma PAI is increased with cerebral infarction again
The danger level of hair, which increases, to be positively correlated.- 1 concentration of plasma PAI increases the prediction index that can be used as Recurrent Cerebral Infarction.Cerebral infarction is sent out again
Accompanied with hypertension person is significantly more than first cerebral infarction the dead, and its blood PAI-1 activity and fibrin level are significantly higher than first cerebral infarction
The dead.
Studies have shown that the insertion/deletion of one guanine in people's PAI-1 gene promoter area is to -1 activity of plasma PAI
Have critically important influence, this site 4G/5G polymorphism is not only related with PAI-1 activity in blood plasma, also with many artery and vein thrombus
Property disease is related.The insertion mutation of guanine affects the activity of PAI-1, improves its inhibiting effect to plasminogen, into
And the balance of blood coagulation and haemolysis in blood is influenced, cause a series of potential diseases.5G/5G mutant homozygous genotype crowd is compared with 4G/
5G heterozygous genotypes crowd and the wild homozygous genotype crowd of 4G/4G, it is higher to suffer from cerebral infarction probability.Base is carried out for the SNP site
Because of detection, clinical cerebral infarction and the disease treatment and medication of hypertensive patient can be instructed, there is great application value.
Summary of the invention
The object of the present invention is to provide a kind of for detecting the primed probe group and its application of rs1799889.
The present invention protects a kind of primed probe group first, is made of primers F, primer R and probe P；
Primers F is following (a1) or (a2):
(a1) single strand dna shown in the sequence 1 of sequence table；
(a2) have by sequence 1 by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 1 identical
The DNA molecular of function；
Primer R is following (b1) or (b2):
(b1) single strand dna shown in the sequence 2 of sequence table；
(b2) have by sequence 2 by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 2 identical
The DNA molecular of function；
Probe P is the single strand dna that an end has fluorescent quenching group with fluorophor and another end, and
Partial nucleotide in DNA molecular is lock nucleic acid, and the nucleotides sequence of DNA molecular is classified as following (c1) or (c2):
(c1) shown in the sequence 3 of sequence table；
(c2) by sequence 3 by the substitution and/or deletion and/or addition of one or several nucleotide.
Probe P is 5 ' ends with FAM fluorophor and 3 ' ends have the single stranded DNA point of BHQ1 fluorescent quenching group
Son.
Specifically, the nucleotide sequence of the probe P is as shown in the sequence 3 of sequence table, and 1-4 nucleotide and
23-25 nucleotide are lock nucleic acid.
In the primed probe group, the mol ratio of primers F, primer R, probe P are as follows: 10:2.5:15.
The present invention also protects the primed probe group preparing the application in the kit for detecting rs1799889.
The present invention also protects a kind of for detecting the kit of rs1799889, including the primed probe group.
The present invention also protects component first and component second preparing the application in the kit for detecting rs1799889；Group
Part first is the primed probe group；Component second is that reagent or reagent combine, and the function of component second is to be used for blood sample preparation
The template samples of quantitative fluorescent PCR.Component second includes lysate.The lysate is the NaCl aqueous solution of 1.5-2 g/100ml,
The concretely NaCl aqueous solution of 1.7 g/100ml.Component second further includes saving liquid.Save the preparation method of liquid: 1. by 25 bodies
Product part FG buffer and the mixing of 4 parts by volume Proteinase K Solutions, obtain mixed liquor；2. 1. mixed liquor that 1 parts by volume step is obtained
With 5 parts by volume ddH2O mixing obtains saving liquid.
The present invention also protects a kind of for detecting the kit of rs1799889, including component first and component second；Component first is
The primed probe group；Component second is that reagent or reagent combine, and the function of component second is fixed for fluorescence with blood sample preparation
Measure the template samples of PCR.Component second includes lysate.The lysate is the NaCl aqueous solution of 1.5-2 g/100ml, specifically may be used
For the NaCl aqueous solution of 1.7 g/100ml.Component second further includes saving liquid.Save the preparation method of liquid: 1. by 25 parts by volume FG
Buffer and the mixing of 4 parts by volume Proteinase K Solutions, obtain mixed liquor；2. 1. mixed liquor and 5 bodies that 1 parts by volume step is obtained
Product part ddH2O mixing obtains saving liquid.
The present invention also protects a kind of kit, including lysate.The function of the kit is with blood sample preparation for glimmering
The template samples of Fluorescent Quantitative PCR.The lysate is the NaCl aqueous solution of 1.5-2 g/100ml, concretely 1.7 g/100ml
NaCl aqueous solution.The kit further includes saving liquid.Save the preparation method of liquid: 1. by 25 parts by volume FG buffers and 4
The mixing of parts by volume Proteinase K Solution, obtains mixed liquor；2. 1. mixed liquor and 5 parts by volume ddH that 1 parts by volume step is obtained2O
Mixing obtains saving liquid.
A kind of method that the present invention also protects template samples with blood sample preparation for quantitative fluorescent PCR, including such as
Lower step:
(1) anticoagulation is taken, the lysate is added, is mixed by inversion, supernatant is abandoned in centrifugation；
(2) after completing step (1), the preservation liquid, 60-70 DEG C first (concretely 65 DEG C) water-bath, then 80-90 DEG C of (tool is added
Body can be 85 DEG C) water-bath, it is then centrifuged for, when use takes supernatant, as template samples.
The method of the template samples with blood sample preparation for quantitative fluorescent PCR, specifically comprises the following steps:
(1) 65 μ L EDTA anticoagulations are taken, lysate described in 500 μ L of addition is mixed by inversion, 12000rpm centrifugation 4min, in abandoning
Clearly；
(2) it after completing step (1), is added and saves liquid described in 120 μ L, first 65 DEG C of water-bath 10min, then 85 DEG C of water-bath 10min, then
12000rpm is centrifuged 4min, is subsequently placed in 4 DEG C and saves backup, and when use takes supernatant, as template samples.
The present invention also protects specific probe carrying out the application in fluorescence quantitative PCR detection；The specific probe be with
The molecular beacon of lock nucleic acid modification.
The present invention also protects a kind of specific probe for fluorescence quantitative PCR detection, for the molecule modified with lock nucleic acid
Beacon.
Any description above rs1799889 is the 36th core of nucleotide shown in the sequence 4 of sequence table in human gene group DNA
Thuja acid.
Any description above rs1799889 is sequence in 1 gene of plasminogen activator inhibitor activity in human genome
36th nucleotide of nucleotide shown in the sequence 4 of list.
The present invention is directed to detect most important single nucleotide polymorphism position in 1 gene of plasminogen activator inhibitor activity
The Genotyping of point determines phlebothrombosis high risk.
Primed probe group provided by the invention using molecular beacon as probe, and introduces several lock nucleic acids.Molecule
Beacon is a kind of fluorescence probe, by ring-shaped area and stem district's groups at the 5' end mark fluorophor in stem area, the end 3' is marked
Remember quencher, is in hairpin structure when closure, the fluorescence that fluorophor issues is quenched group absorptions, whole not issue fluorescence letter
Number.In the presence of target sequence, in conjunction with target sequence, stem part is forced to open ring portion, fluorophor far from quenching group,
Issue fluorescence.Δ Tm value very little between nucleotide chain with mononucleotide difference, general detection means are difficult to differentiate between.Lock nucleic acid
It is a kind of double-ring oligonucleotide derivative, base pair complementarity principle can be followed in conjunction with nucleic acid, the β-D- in structure
2'-O and 4'-C of ribofuranose act on forming Oxymethylene bridge, sulphur methylene bridge or amine methylene bridge by shrink, and even
It is connected into annular, this annular bridge has locked the N configuration of furanose C3'- inner mold, reduces the flexibility of ribose structure, increases
The stability of phosphate backbone partial structurtes.In LNA/DNA heteroduplex, one LNA monomer of every increase, Tm improves 2-6 DEG C.
The application method of primed probe group provided by the invention: template samples are prepared with blood sample, then use primer
Probe groups carry out unbalanced fluorescent quantitative PCR, judge genotype by observing melting curve.Imbalance amplification: it is added
The excessive and reversed primer of probe, a small amount of primer in the same direction with probe and suitable probe P, after amplification, due to
Side primer is present in excess, and the template strand that participation amplification obtains is extra far more than the chain obtained with other side primer amplification
The not formed double-strand of template strand, the individualism in system.
The method of template samples provided by the invention with blood sample preparation for quantitative fluorescent PCR, it is easy to operate,
Product may be implemented to carry out effective fluorescent quantitative PCR as template.
Provided by the present invention for the specific probe of fluorescence quantitative PCR detection, for the molecular beacon modified with lock nucleic acid.
The Tm value of nucleotide combination double-strand can be significantly improved due to introducing lock nucleic acid.By controlling the temperature change of melting curve,
To control the closure of molecular beacon, analyzes to obtain fluorescence curve by data and change corresponding peak value map and distinguish open country well
The mutation chain of raw chain and unit point.In addition, will appear two peaks in the peak value map of melting curve for heterozygous sample
Value, avoids general PCR and expands the cumbersome of definitive result twice.
The present invention can be used for detecting rs1799889, judge genotype of the sample to be tested based on the site, to judge vein
Thrombus high risk, guidance medication, has great application and popularization value.
Detailed description of the invention
Fig. 1 is the melting curve of exemplary sample (4G/4G is homozygous).
Fig. 2 is the melting curve of exemplary sample (5G/5G is homozygous).
Fig. 3 is the melting curve (4G/5G heterozygous) of exemplary sample.
Fig. 4 is the melting curve of exemplary sample (4G/4G is homozygous).
Fig. 5 is the melting curve of exemplary sample (5G/5G is homozygous).
Fig. 6 is the melting curve (4G/5G heterozygous) of exemplary sample.
Fig. 7 is the melting curve of exemplary sample (4G/4G is homozygous).
Fig. 8 is the melting curve of exemplary sample (5G/5G is homozygous).
Fig. 9 is the melting curve (4G/5G heterozygous) of exemplary sample.
Specific embodiment
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified certainly
What routine biochemistry reagent shop was commercially available.Quantitative test in following embodiment is respectively provided with three repeated experiments, as a result makes even
Mean value.Unless otherwise specified, each nucleotide in primer, probe and target sequence is the nucleotide on conventional meaning.
Embodiment 1, the design of primed probe group for detecting rs1799889
By largely designing, preliminary experiment, effect compare, obtain one group for detecting the primed probe group of rs1799889.
Primers F: 5'- CCTCAGGGGCACAGAGAGAGTC -3'；
Primer R:5'- GTCTTTCCCTCATCCCTGCCAT -3'；
Probe P:5'-FAM-ACTCCCCCAAGTGTCCAGACTCAGT-BHQ1-3'。
Primers F is single strand dna shown in the sequence 1 of sequence table.Primer R is single-stranded shown in the sequence 2 of sequence table
DNA molecular.Probe P is 5 ' ends with FAM fluorophor and 3 ' ends have the single stranded DNA point of BHQ1 fluorescent quenching group
Son, the nucleotide sequence of DNA molecular is as shown in the sequence 3 of sequence table, nucleotide (i.e. the 1st, the 2nd, the of underscore mark
3, the 4th, the 23rd, the 24th and the 25th) it is lock nucleic acid (LNA).
The target sequence of primers F and primer R are as shown in the sequence 4 of sequence table.
The application of embodiment 2, primed probe group for detecting rs1799889
One, blood sample processing (processing method of single blood sample)
1,65 μ L EDTA anticoagulations are taken, 500 μ L lysates are added, are mixed by inversion, 12000rpm is centrifuged 4min, abandons supernatant.
Lysate are as follows: 1.7g/100ml NaCl aqueous solution.
2, after completing step 1,120 μ L is added and save liquid, first 65 DEG C of water-bath 10min, then 85 DEG C of water-bath 10min, then
12000rpm is centrifuged 4min, is subsequently placed in 4 DEG C and saves backup, and when use takes supernatant, as template samples.
It saves the preparation method of liquid: 1. 25 parts by volume FG buffers and 4 parts by volume Proteinase K Solutions being mixed, are mixed
Close liquid；2. 1. mixed liquor and 5 parts by volume ddH that 1 parts by volume step is obtained2O mixing obtains saving liquid.
FG buffer: the Buffer FG in poba gene group DNA extraction system (0.1-20ml) (DP319) kit.Egg
White enzyme K solution: the Proteinase K in poba gene group DNA extraction system (0.1-20ml) (DP319) kit, concentration are
20mg/ml.The producer of poba gene group DNA extraction system (0.1-20ml) (DP319) is that Tiangeng biochemical technology (Beijing) is limited
Company.
Two, the amplification of PCR and the acquisition of fluorescence signal
1, it takes EP to manage, each component is added, prepare reaction system.
Reaction system: 7.5 μ L 2 × Reaction Buffer, 0.12 μ L Taq archaeal dna polymerase solution (Taq containing 0.6U
Archaeal dna polymerase), 0.09 μ L dNTP solution (in dNTP solution contain dATP, dCTP and dGTP, dATP, dCTP and dGTP exist
Concentration in dNTP solution is 33mmol/L), (in dUTP solution, the concentration of dUTP is 100mmol/ to 0.06 μ L dUTP solution
L), 0.06 μ L UDG enzyme solutions (enzyme of UDG containing 0.12U), 3 μ L 5 × PCR Enhancer, 0.3 μ L primers F solution, 0.3 μ L draw
DdH is added in the template samples that object R solution, 0.3 μ L probe P solution, 1 μ L step 1 obtain2O to 15 μ L.In reaction system, draw
The concentration of object F is 10 μM, and the concentration of primer R is 2.5M, and the concentration of probe P is 15 μM.
Buffer: Fei Peng Biological Co., Ltd. of 2 × Reaction.Taq archaeal dna polymerase solution, that is, AnstartTaq
DNA Polymerase, 5U/ μ L, Fei Peng Biological Co., Ltd..UDG enzyme solutions, that is, Uracil-DNA Glycosylase,
2U/ μ L, Fei Peng Biological Co., Ltd..5 × PCR Enhancer: Tiangeng biochemical technology Co., Ltd, article No. RP202.
2, the EP pipe for taking into step 1, is added 25 μ L paraffin.
3, the EP pipe for taking into step 2, carries out quantitative fluorescent PCR.
①50℃ 2min,95℃ 10min；
2. 95 DEG C of 15s, 55 DEG C of 15s, 72 DEG C of 20s, 50 circulations；
③95℃ 5min,20℃ 5min；45 DEG C of 1s, it is then heated up for interval up to 75 DEG C (every since 45 DEG C with 0.3 DEG C
A temperature acquisition fluorescence obtains melting curve) and then 75 DEG C of 15s.
If being shown as unimodal in melting curve, and the corresponding Tm value of peak value is 59.20 DEG C -60.30 DEG C, sample is based on
The genotype of rs1799889 is that 4G/4G is homozygous.
If being shown as unimodal in melting curve, and the corresponding Tm value of peak value is 63.40 DEG C -64.50 DEG C, sample is based on
The genotype of rs1799889 is that 5G/5G is homozygous.
If being shown as bimodal in melting curve, and the peak value at two peaks respectively corresponds 59.20 DEG C -60.30 DEG C and 63.40
DEG C -64.50 DEG C, genotype of the sample based on rs1799889 is 4G/5G heterozygous.
To volunteer's EDTA anticoagulation of 500 informed consents, then detected according to above-mentioned steps, 195
The genotype of volunteer is that 4G/4G is homozygous, genotype of 77 volunteers are that 5G/5G is homozygous, gene of 228 volunteers
Type is 4G/5G heterozygous.It extracts the anticoagulant genomic DNA of EDTA and carries out sequence verification, sequencing result shows above-mentioned steps
The accuracy rate identified is 100%.
In volunteer's EDTA anticoagulation of 500 informed consents, the result is shown in Figure 1s of 3 exemplary samples, Fig. 2 and
Fig. 3.It is shown as unimodal in the melting curve of exemplary sample in Fig. 1, the corresponding Tm value of peak value is 59.88 DEG C, the volunteer's
Genotype is that 4G/4G is homozygous.It is shown as unimodal in the melting curve of exemplary sample in Fig. 2, the corresponding Tm value of peak value is
63.93 DEG C, the genotype of the volunteer is that 5G/5G is homozygous.It is shown as bimodal in the melting curve of exemplary sample in Fig. 3,
The corresponding Tm value of peak value is respectively 59.88 DEG C and 63.93 DEG C, and the genotype of the volunteer is 4G/5G heterozygous.
Comparative example 1,
One, blood sample is handled
With the step of embodiment 2 one.
Two, the amplification of PCR and the acquisition of fluorescence signal
1, it takes EP to manage, each component is added, prepare reaction system.
Replace probe P with probe DP, other with the step of embodiment 2 two 1.
Probe DP:5'-FAM- ACTCCCCCAAGTGTCCAGACTCAGT-BHQ1-3'.
2, the EP pipe for taking into step 1, is added 25 μ L paraffin.
3, the EP pipe for taking into step 2, carries out quantitative fluorescent PCR.
Response procedures with the step of embodiment 2 one 3.
To 500 parts of EDTA anticoagulations in embodiment 2, then detected according to above-mentioned steps.
The sample standard deviation of each genotype is shown without effective peak.
The result of 3 exemplary samples is shown in Fig. 4 (4G/4G is homozygous), Fig. 5 (5G/5G is homozygous) and Fig. 6 (4G/5G heterozygosis
Type).
Comparative example 2,
One, blood sample is handled
With the step of embodiment 2 one.
Two, the amplification of PCR and the acquisition of fluorescence signal
1, it takes EP to manage, each component is added, prepare reaction system.
Replace primers F with primer DF, and replace primer R with primer DR, other with the step of embodiment 2 two 1.
DF:5'- CTCAGGGGCACAGAGAGAGTCT'；
DR:5'- TCTTTCCCTCATCCCTGCCATG3'.
2, the EP pipe for taking into step 1, is added 25 μ L paraffin.
3, the EP pipe for taking into step 2, carries out quantitative fluorescent PCR.
Response procedures with the step of embodiment 2 one 3.
To 500 parts of EDTA anticoagulations in embodiment 2, then detected according to above-mentioned steps.
The sample standard deviation of each genotype is shown without effective peak.
The result of 3 exemplary samples is shown in Fig. 7 (4G/4G), Fig. 8 (5G/5G), Fig. 9 (4G/5G).
Sequence table
<110>Shandong De-Nol Biotechnology Co., Ltd
<120>for detecting the primed probe group and its application of rs1799889
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
cctcaggggc acagagagag tc 22
<210> 2
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
gtctttccct catccctgcc at 22
<210> 3
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
actcccccaa gtgtccagac tcagt 25
<210> 4
<211> 91
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<222> (36)
<223> y=a or ga
<400> 4
cctcaggggc acagagagag tctggacacg tggggagtca gccgtgtatc atcggaggcg 60
gccgggcaca tggcagggat gagggaaaga c 91
Claims (10)
1. primed probe group is made of primers F, primer R and probe P；
Primers F is following (a1) or (a2):
(a1) single strand dna shown in the sequence 1 of sequence table；
(a2) have by sequence 1 by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 1 identical
The DNA molecular of function；
Primer R is following (b1) or (b2):
(b1) single strand dna shown in the sequence 2 of sequence table；
(b2) have by sequence 2 by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 2 identical
The DNA molecular of function；
Probe P is the single strand dna that an end has fluorescent quenching group with fluorophor and another end, and
Partial nucleotide in DNA molecular is lock nucleic acid, and the nucleotides sequence of DNA molecular is classified as following (c1) or (c2):
(c1) shown in the sequence 3 of sequence table；
(c2) by sequence 3 by the substitution and/or deletion and/or addition of one or several nucleotide.
2. primed probe group as described in claim 1, it is characterised in that: the nucleotide sequence such as sequence table of the probe P
Shown in sequence 3, and 1-4 nucleotide and 23-25 nucleotide are lock nucleic acid.
3. primed probe group as claimed in claim 1 or 2 is preparing the application in the kit for detecting rs1799889.
4. a kind of for detecting the kit of rs1799889, including primed probe group as claimed in claim 1 or 2.
5. component first and component second are preparing the application in the kit for detecting rs1799889；Component first is claim 1
Or 2 primed probe group；Component second is that reagent or reagent combine, and the function of component second is to be used for fluorescence with blood sample preparation
The template samples of quantitative PCR.
6. a kind of for detecting the kit of rs1799889, including component first and component second；Component first is claims 1 or 2 institute
State primed probe group；Component second is that reagent or reagent combine, and the function of component second is to be used for fluorescent quantitation with blood sample preparation
The template samples of PCR.
7. a kind of kit of the template samples with blood sample preparation for quantitative fluorescent PCR, including lysate；The cracking
Liquid is the NaCl aqueous solution that the lysate is 1.5-2 g/100ml.
8. a kind of method of the template samples with blood sample preparation for quantitative fluorescent PCR, includes the following steps:
(1) anticoagulation is taken, lysate described in claim 7 is added, is mixed by inversion, supernatant is abandoned in centrifugation；
(2) it after completing step (1), is added and saves liquid, first 60-70 DEG C of water-bath, then 80-90 DEG C of water-bath is then centrifuged for, and when use takes
Supernatant, as template samples.
9. specific probe is carrying out the application in fluorescence quantitative PCR detection；The specific probe is point modified with lock nucleic acid
Sub- beacon.
10. a kind of specific probe for fluorescence quantitative PCR detection, for the molecular beacon modified with lock nucleic acid.
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