CN110129427A - ACE insertion mutation type probe, ACE deficient mutant probe, kit and its application - Google Patents
ACE insertion mutation type probe, ACE deficient mutant probe, kit and its application Download PDFInfo
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- CN110129427A CN110129427A CN201910298977.9A CN201910298977A CN110129427A CN 110129427 A CN110129427 A CN 110129427A CN 201910298977 A CN201910298977 A CN 201910298977A CN 110129427 A CN110129427 A CN 110129427A
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Abstract
The invention belongs to molecular biology and gene engineering technology field, are related to a kind of ACE insertion mutation type probe.Identify to the ACE insertion mutation type probe specificity that the ACE gene of insertion mutation occurs for the 16th introne；For the nucleotide sequence of the ACE insertion mutation type probe as shown in SEQ ID NO:3, the SEQ ID NO:3 is 5'-CCAAACTTCACAGTCTCT-3'.ACE insertion mutation type probe provided by the invention can specifically identify that the ACE gene of insertion mutation occurs for the 16th introne, can be used for detecting or whether screening ACE gene occurs the 16th introne and insertion mutation occurs.
Description
Technical field
The invention belongs to molecular biology and gene engineering technology field, more particularly, to a kind of ACE insertion mutation type
Probe, ACE deficient mutant probe, the kit including ACE insertion mutation type probe and ACE deficient mutant probe and
Their application.
Background technique
Angiotensin converting enzyme (ACE) is also known as kininaseⅡ or peptidyl-carboxypeptidase, belongs to endothelial cellular membrane and combines
Amino acid is cut to from two sections of transformation by the C-terminal of peptide, peptide chain C-terminal dipeptide residue can be made to hydrolyze by enzyme.Major function has catalysis blood
Angiotensin I is converted into angiotensinⅡ；Inactivate bradykinin.ACE becomes treatment hypertension, mental and physical efforts because of both functions
The promising target of the diseases such as failure, diabetes B and diabetic nephropathy.Pril drug is actually angiotensins conversion
Enzyme inhibitor treats disease by inhibiting ACE activity, has reversing hypertension left ventricular hypertrophy, delays left ventricular hypertrophy
The effects of development.
ACE gene is located at chromosome 17q23, gene length 21kb, contains 26 exons and 25 intrones.ACE gene tool
There is polymorphism, the octapeptide Ang that inactive decapeptide Ang I can be made to be converted to tool height vasoactive, Aldosterone Secretion can be stimulated
II, and bradykinin is promoted to degrade.There is inserting for one section of Alu repeated sequence in 16th introne of the discovery ACE gene such as nineteen ninety Rig
Enter (insertion is abbreviated as I)/missing (deletion is abbreviated as D).ACE gene includes three kinds of genotype accordingly, respectively
Are as follows: ACE gene insertion mutation type (abbreviation ACE I/I), ACE gene insertion/deletion saltant type (abbreviation ACE I/D) and ACE
Deletion mutant type (abbreviation ACE D/D).The frequency of D allele appearance is respectively in white people, black race and Asian
56.2%, 60.3% and 39.0%.
ACE gene polynorphisms can influence the level of blood plasma Angiotensin-Converting invertase (ACE), ACE D/D type individual
The activity of Plasma ACE increases, and the decline of ACE activity becomes apparent from after enalapril treatment；In the hypertensive patient just controlled, Fu Xinpu
Benefit enhances the efficacy of antihypertensive treatment of ACE D/D type patient；In the patient of hypertensive patients left ventricular hypertrophy and diastolic filling obstacle
In, heart function improves degree better than ACE I/D and ACE I/I type after ACE D/D type patient takes enalapril and lisinopril
Patient；Decreased renal function becomes apparent from when ACE I/I type patient applies lisinopril or captopril.
Summary of the invention
The object of the present invention is to provide a kind of ACE insertion mutation type probe, ACE deficient mutant probe, kit and its
Using selecting suitable pril drug to provide guidance for patient.
To achieve the goals above, the present invention provides a kind of ACE insertion mutation type probe, and the ACE insertion mutation type is visited
Needle specifically identifies that the ACE gene of insertion mutation occurs for the 16th introne；
As shown in SEQ ID NO:3, the SEQ ID NO:3 is the nucleotide sequence of the ACE insertion mutation type probe
5'-CCAAACTTCACAGTCTCT-3'。
Specifically, the end 5' and/or the end 3' of the ACE insertion mutation type probe have fluorescent marker.Such as ACE insertion is prominent
There is FAM or VIC to mark at the end 5' of modification probe, and the end 3' of ACE insertion mutation type probe is marked with MGB.
The present invention also provides a kind of ACE deficient mutant probe, is identified to the ACE deficient mutant probe specificity
The ACE gene of 16 intrones generation deletion mutation；
As shown in SEQ ID NO:4, the SEQ ID NO:4 is the nucleotide sequence of the ACE deficient mutant probe
5'-AACATTCTCATCCACGC-3'。
Specifically, the end 5' and/or the end 3' of the ACE deficient mutant probe have fluorescent marker.Such as ACE missing is prominent
There is FAM or VIC to mark at the end 5' of modification probe, and the end 3' of ACE deficient mutant probe is marked with MGB.
The present invention also provides a kind of kit, the kit includes:
Primer；
Above-mentioned ACE insertion mutation type probe；And
Above-mentioned ACE deficient mutant probe；
Wherein, ACE gene is expanded to the primer specificity, the primer includes forward primer and reverse primer；
For the nucleotide sequence of the forward primer as shown in SEQ ID NO:1, SEQ ID NO:1 is 5'-
AGATGTAGGGTATGCAGTTTCTGC-3'；
For the nucleotide sequence of the reverse primer as shown in SEQ ID NO:2, SEQ ID NO:2 is 5'-
CCAGAGATTGATTGATGAGAGCG-3'。
Specifically, the kit further includes positive reference substance, and the positive reference substance includes prominent with above-mentioned ACE insertion
The ACE genetic fragment and the ACE in conjunction with above-mentioned ACE deficient mutant probe specificity that modification probe specificity combines
Genetic fragment.
Specifically, the kit further includes positive reference substance, and the positive reference substance includes that the insertion of ACE gene loci is prominent
Modification plasmid and ACE gene loci deficient mutant plasmid；
The ACE gene loci insertion mutation type plasmid has in conjunction with above-mentioned ACE insertion mutation type probe specificity
ACE genetic fragment；
The ACE gene loci deficient mutant plasmid has in conjunction with above-mentioned ACE deficient mutant probe specificity
ACE genetic fragment.
More specifically, the ACE genetic fragment in conjunction with above-mentioned ACE insertion mutation type probe specificity is by above-mentioned
ACE insertion mutation type probe carries out expanding resulting product；
More specifically, the ACE genetic fragment in conjunction with above-mentioned ACE deficient mutant probe specificity is by above-mentioned
ACE deficient mutant probe carries out expanding resulting product.
More specifically, positive reference substance includes positive reference substance one, positive reference substance two and positive reference substance three；Its
In, positive reference substance one is above-mentioned ACE gene loci insertion mutation type plasmid；Positive reference substance two is above-mentioned ACE gene
The mixture of site insertion mutation type plasmid and above-mentioned ACE gene loci deficient mutant plasmid；Positive reference substance three is upper
The ACE gene loci deficient mutant plasmid stated.
More specifically, in positive reference substance two, above-mentioned ACE gene loci insertion mutation type plasmid and above-mentioned ACE
The mass ratio of gene loci deficient mutant plasmid is 1:1.
The present invention also provides a kind of ACE gene loci insertion mutation type plasmid, the ACE gene loci insertion mutation type matter
Grain has ACE genetic fragment, and the ACE genetic fragment is to carry out expanding resulting production by above-mentioned ACE insertion mutation type probe
Object.
The present invention also provides a kind of ACE gene loci deficient mutant plasmid, the ACE gene loci deficient mutant matter
Grain has ACE genetic fragment, and the ACE genetic fragment is to carry out expanding resulting production by above-mentioned ACE deficient mutant probe
Object.
More specifically, the kit further includes at least one of following components:
Negative controls；
Buffer；
dNTP；
Archaeal dna polymerase；And
Aqua sterilisa.
More specifically, the negative control can be aqua sterilisa.
More specifically, the archaeal dna polymerase is HS Taq enzyme.
The present invention also provides above-mentioned ACE insertion mutation type probes, above-mentioned ACE deficient mutant probe and above-mentioned
The application in the preparation of preparation detection and/or screening ACE genotype of at least one of kit.
ACE insertion mutation type probe provided by the invention can specifically identify that insertion mutation occurs for the 16th introne
ACE gene, can be used for detect or screening ACE gene whether occur the 16th introne occur insertion mutation.
ACE deficient mutant probe provided by the invention can specifically identify that deletion mutation occurs for the 16th introne
ACE gene, can be used for detect or screening ACE gene whether occur the 16th introne occur deletion mutation.
Kit provided by the invention, convenient for whether detection or screening ACE gene are occurred the 16th introne and be inserted into
And/or deletion mutation, have the characteristics that easy to operate, detection is quick, high, the specific good and sensitivity of accuracy is good.
The present invention selects suitable pril drug to provide guidance for patient.
Other features and advantages of the present invention will then part of the detailed description can be specified.
Detailed description of the invention
Exemplary embodiment of the invention is described in more detail in conjunction with the accompanying drawings, it is of the invention above-mentioned and its
Its purpose, feature and advantage will be apparent.
Fig. 1 shows the amplification curve of the quantitative fluorescent PCR of sample one, and wherein top curve indicates VIC label, and lower section is bent
Line indicates FAM label.
Fig. 2 shows the amplification curves of the quantitative fluorescent PCR of sample two, wherein two curves of top indicate VIC label,
Two curves of lower section indicate FAM label.
Fig. 3 shows the amplification curve of the quantitative fluorescent PCR of sample three, and wherein top curve indicates VIC label, and lower section is bent
Line indicates FAM label.
Specific embodiment
The preferred embodiment of the present invention is described in more detail below.Although the following describe preferred implementations of the invention
Mode, however, it is to be appreciated that may be realized in various forms the present invention without that should be limited by the embodiments set forth herein.It is real
The person that is not specified actual conditions in example is applied, is all carried out according to conventional conditions or manufacturer's recommended conditions.Agents useful for same or instrument are not
Production firm person is indicated, is the conventional products that can be obtained by commercially available purchase.
Embodiment 1
One, the design and synthesis of primer and probe
For the ACE gene loci in human genome, (sequence is referring to mankind's full-length genome sequence disclosed in ncbi database
Column), using Primer Premier 5.0 and Methyl Primer Express v1.0 software, separately design drawing for specificity
Object and probe, middle probe are marked with fluorescein, and see Table 1 for details.
1 primer and probe of table
Two, reference substance selects
Positive reference substance one is ACE gene loci insertion mutation type plasmid.
Positive reference substance two is that ACE gene loci insertion mutation type plasmid and deficient mutant plasmid 1:1 in mass ratio are mixed
It closes.
Positive reference substance three is ACE gene loci deficient mutant plasmid.
Blank control product are nuclease free distilled water or aqua sterilisa.
Three, PCR system forms
PCR reaction solution includes: that the forward primer of ACE, the reverse primer of ACE, ACE insertion mutation type probe, ACE missing are prominent
Modification probe, 1 × PCR buffer, dNTP, HS Taq enzyme and nuclease free distilled water.
Wherein, PCR buffer, dNTP, HS Taq enzyme are purchased from the precious biology in Dalian (precious biology (Dalian) engineering have
Limit company)；The nuclease free distilled water is ultrapure water processed and through autoclave sterilization with DEPC；HS Taq enzyme is tool
There is the archaeal dna polymerase of high sensitivity, the high specific specially developed is thermophilic archaeal dna polymerase product.
PCR reaction solution is 20 μ L, and the final concentration of each component is as follows:
1×PCR buffer；
The forward primer of 0.25 μM of ACE；
The reverse primer of 0.25 μM of ACE；
0.25 μM of ACE insertion mutation type probe；
0.25 μM of ACE deficient mutant probe；
The HS Taq enzyme of 1U；
The dNTP of 0.25mM (mmol/L)；And
The nuclease-free water of surplus.
Embodiment 2
One, biological sample
Biomaterial of the present invention is all from Yancheng City third the People's Hospital.For during the 9-12 month in 2018 in salt
The anticoagulation sample of 200 patients of city third the People's Hospital detection.
Two, the DNA for taking sample extracting to be detected, as pcr template.
For DNA extraction kit purchased from precious biological (Dalian) Engineering Co., Ltd, operating procedure is as follows:
1) sterile 1.5mL centrifuge tube is taken, 200 μ L whole bloods are added.
2) add 750 μ L cell pyrolysis liquids, be mixed by inversion 5-6 times, be stored at room temperature 10min.
3) 10000rpm is centrifuged 2min；Waste liquid is abandoned, the precipitating in centrifuge tube is retained.
4) 25 μ L Proteinase Ks, 200 μ L lysate A are sequentially added into precipitating, vortex oscillation 20 seconds, 65 DEG C of water-baths
15min, oscillation mixes 2-3 times during water-bath.
5) after water-bath, 200 μ L dehydrated alcohols of addition, vortex oscillation 10 seconds.
6) adsorption column is inserted into new centrifuge tube, the resulting mixed liquor of step 5) is transferred in adsorption column；10000rp
It is centrifuged 1min, the liquid in adsorption column enters in centrifuge tube, this is waste liquid.
7) waste liquid in centrifuge tube is discarded, 500 μ L eluents I are added into adsorption column, 10000rpm is centrifuged 1min.
8) waste liquid in centrifuge tube is discarded, 700 μ L eluent II, 10000rpm are added into adsorption column and are centrifuged 1min, abandon useless
Liquid.
9) it is primary to repeat step 8).
10) waste liquid in centrifuge tube is discarded, adsorption column is turned back into centrifuge tube, 15000rpm is centrifuged 2min.
11) adsorption column is inserted into new centrifuge tube；50-100 μ L DNA lysate is added, and (65 DEG C of preheatings can be improved
DNA pick-up rate), it is stored at room temperature 5min.
12) 10000rpm is centrifuged 1min, discards adsorption column, and centrifugation liquid in pipe is DNA solution, can be used as subsequent PCR
The DNA profiling of reaction, -20 DEG C save backup.
By the PCR reaction solution and DNA profiling mixing in the kit.
(PCR pipe number=sample of every detection site is transferred in 8 connecting legs from 18.5 μ L PCR reaction solutions are taken out in kit
Number+3 positive controls of+1 blank control).
1.5 μ L are taken to add in PCR reaction solution from sample to be examined DNA (or reference substance).
After oscillation mixes, it is centrifuged 10s, moves it to amplification region.
Four, the condition of PCR reaction
According to 95 DEG C of initial denaturation 4min first, then 95 DEG C of denaturation 1min, 54 DEG C are annealed 40 seconds, 75 DEG C of extension 1min, and 35
The operating process of a circulation carries out PCR reaction.
Five, result judgement
In situation as defined in meeting in the positive reference substance and blank control product (negative controls) of the kit, according to
Fluorescent amplification curve obtains CT value and carries out result judgement, obtains for the corresponding ACE genotype of pril drug personalized medicine.
See Table 2 for details for the relationship of positive reference substance ACE genotype.
2 positive reference substance of table and negative controls, as shown in table 2:
| Detection site | Positive control one | Positive control two | Positive control three | Negative controls |
| ACE gene | Insertion mutation (I/I type) | Heterozygous mutant (I/D type) | Deletion mutation (D/D type) | Aqua sterilisa |
Blank control result is negative (value >=32 No Ct or Ct).
See Table 3 for details for the judgement result of quantitative fluorescent PCR and ACE genotype.
The judgement result of 3 quantitative fluorescent PCR of table and ACE genotype
This one, sample two of sampling and sample three carry out quantitative fluorescent PCR, and according to the result of table 3 to sample one, sample
Sheet two and the ACE genotype of sample three are determined.
The result of the quantitative fluorescent PCR of sample one is referring to Figure 1.Fig. 1 shows the amplification of the quantitative fluorescent PCR of sample one
Curve, wherein top curve indicates VIC label, and lower curve indicates FAM label.As shown in Figure 1, sample one is that ACE insertion is prominent
Modification (ACE I/I type).
The result of the quantitative fluorescent PCR of sample two refers to Fig. 2.Fig. 2 shows the amplifications of the quantitative fluorescent PCR of sample two
Curve, wherein two curves of top indicate that VIC label, two curves of lower section indicate FAM label.As shown in Figure 2, sample two
For ACE insertion/deletion saltant type (ACE I/D type).
The result of the quantitative fluorescent PCR of sample three refers to Fig. 3.Fig. 3 shows the amplification of the quantitative fluorescent PCR of sample three
Curve, wherein top curve indicates VIC label, and lower curve indicates FAM label.From the figure 3, it may be seen that sample three is that ACE missing is prominent
Modification (ACE D/D type).
Six, kit test result is instructing the application in pril drug medication
ACE genotype is obtained according to the result judgement in quantitative fluorescent PCR.ACE genotype and pril drug individuation
See Table 4 for details for medication relationship.
The medication relationship of table 4 ACE genotype and pril drug
Various embodiments of the present invention are described above, above description is exemplary, and non-exclusive, and
It is not limited to disclosed each embodiment.Without departing from the scope and spirit of illustrated each embodiment, for this skill
Many modifications and changes are obvious for the those of ordinary skill in art field.
Sequence table
<110>Jiangsu medical profession institute
<120>ACE insertion mutation type probe, ACE deficient mutant probe, kit and its application
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 1
agatgtaggg tatgcagttt ctgc 24
<210> 2
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 2
ccagagattg attgatgaga gcg 23
<210> 3
<211> 18
<212> DNA
<213> Artificial Sequence
<400> 3
ccaaacttca cagtctct 18
<210> 4
<211> 17
<212> DNA
<213> Artificial Sequence
<400> 4
aacattctca tccacgc 17
Claims (9)
1. a kind of ACE insertion mutation type probe, which is characterized in that identify the 16th to the ACE insertion mutation type probe specificity
The ACE gene of introne generation insertion mutation；
For the nucleotide sequence of the ACE insertion mutation type probe as shown in SEQ ID NO:3, the SEQ ID NO:3 is 5'-
CCAAACTTCACAGTCTCT-3'。
2. ACE insertion mutation type probe according to claim 1, which is characterized in that the ACE insertion mutation type probe
The end 5' and/or the end 3' have fluorescent marker.
3. a kind of ACE deficient mutant probe, which is characterized in that identify the 16th to the ACE deficient mutant probe specificity
The ACE gene of introne generation deletion mutation；
For the nucleotide sequence of the ACE deficient mutant probe as shown in SEQ ID NO:4, the SEQ ID NO:4 is 5'-
AACATTCTCATCCACGC-3'。
4. ACE deficient mutant probe according to claim 3, which is characterized in that the ACE deficient mutant probe
The end 5' and/or the end 3' have fluorescent marker.
5. a kind of kit, which is characterized in that the kit includes:
Primer；
ACE insertion mutation type probe of any of claims 1 or 2；And
ACE deficient mutant probe described in claim 3 or 4；
Wherein, ACE gene is expanded to the primer specificity, the primer includes forward primer and reverse primer；
For the nucleotide sequence of the forward primer as shown in SEQ ID NO:1, SEQ ID NO:1 is 5'-
AGATGTAGGGTATGCAGTTTCTGC-3'；
For the nucleotide sequence of the reverse primer as shown in SEQ ID NO:2, SEQ ID NO:2 is 5'-
CCAGAGATTGATTGATGAGAGCG-3'。
6. kit according to claim 5, which is characterized in that the kit further includes positive reference substance, the sun
Property reference substance include ACE genetic fragment in conjunction with ACE insertion mutation type probe specificity of any of claims 1 or 2 and
ACE genetic fragment in conjunction with ACE deficient mutant probe specificity described in claim 3 or 4.
7. kit according to claim 5, which is characterized in that the kit further includes positive reference substance, the sun
Property reference substance includes ACE gene loci insertion mutation type plasmid and ACE gene loci deficient mutant plasmid；
The ACE gene loci insertion mutation type plasmid has and ACE insertion mutation type probe specificity described in claim 1
In conjunction with ACE genetic fragment；
The ACE gene loci deficient mutant plasmid has and ACE deficient mutant probe specificity as claimed in claim 3
In conjunction with ACE genetic fragment.
8. detecting the kit of ACE genotype according to any one of claim 5-7, which is characterized in that the examination
Agent box further includes at least one of following components:
Negative controls；
Buffer；
dNTP；
Archaeal dna polymerase；And
Aqua sterilisa.
9. ACE deficient mutant described in ACE insertion mutation type probe of any of claims 1 or 2, claim 3 or 4 is visited
At least one of kit described in any one of needle and claim 5-8 is in preparation detection and/or screening ACE base
Because of the application in the preparation of type.
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| CN201910298977.9A CN110129427A (en) | 2019-04-15 | 2019-04-15 | ACE insertion mutation type probe, ACE deficient mutant probe, kit and its application |
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| CN201910298977.9A CN110129427A (en) | 2019-04-15 | 2019-04-15 | ACE insertion mutation type probe, ACE deficient mutant probe, kit and its application |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106367479A (en) * | 2016-08-25 | 2017-02-01 | 杭州百迈生物股份有限公司 | Detection composition for guiding hypertension medication, applications of detection composition, kit and detection method |
| CN108410966A (en) * | 2018-03-06 | 2018-08-17 | 深圳美因医学检验实验室 | The method be inserted into and lacked for detecting ACE genes |
| CN109355368A (en) * | 2018-10-22 | 2019-02-19 | 江苏美因康生物科技有限公司 | A kind of kit and method of quick detection hypertension individuation medication gene pleiomorphism |
-
2019
- 2019-04-15 CN CN201910298977.9A patent/CN110129427A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106367479A (en) * | 2016-08-25 | 2017-02-01 | 杭州百迈生物股份有限公司 | Detection composition for guiding hypertension medication, applications of detection composition, kit and detection method |
| CN108690876A (en) * | 2016-08-25 | 2018-10-23 | 杭州百迈生物股份有限公司 | Detect primer, probe and application, kit and the detection method of ACE gene pleiomorphisms |
| CN108410966A (en) * | 2018-03-06 | 2018-08-17 | 深圳美因医学检验实验室 | The method be inserted into and lacked for detecting ACE genes |
| CN109355368A (en) * | 2018-10-22 | 2019-02-19 | 江苏美因康生物科技有限公司 | A kind of kit and method of quick detection hypertension individuation medication gene pleiomorphism |
Non-Patent Citations (2)
| Title |
|---|
| W. KOCH, ET AL.: "Genotyping of the angiotensin I-converting enzyme gene insertion/deletion polymorphism by the TaqMan method", 《CLIN. CHEM.》 * |
| 杨杰书: "第四章 心内科常用药物治疗", 《临床心血管疾病综合治疗学》 * |
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Application publication date: 20190816 |