CN108977523A - For detecting primer pair, probe and the kit of SLCO1B1 521T > C gene pleiomorphism - Google Patents
For detecting primer pair, probe and the kit of SLCO1B1 521T > C gene pleiomorphism Download PDFInfo
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Abstract
Primer pair, probe and the kit that the invention discloses a kind of for detecting SLCO1B1 521T > C gene pleiomorphism；The kit includes: for base sequence primer as shown in SEQ ID No.1, SEQ ID No.2 of 521 site SLCO1B1 design；Wherein, 5 ' end bases of the base sequence have amido modified group；For base sequence fluorescence probe as shown in SEQ ID No.3, SEQ ID No.4 of 521 site SLCO1B1 design；Wherein, a base in the base sequence has amido modified group.Compared with prior art, kit of the present invention uses amino group Mdification primer and probe, in addition further defines detection time, the time required to can significantly shortening detection, reduce non-specific amplification, it can detecte SNPs and point mutation, reduce design difficulty and testing cost, and do not influence diagnostic accuracy, realize method of the single reaction pipe without detection mutation of uncapping, Aerosol Pollution is avoided to greatest extent, facilitates clinical use, reduces error caused by operating.
Description
Technical field
The present invention relates to technical field of molecular biology, more particularly to one kind is for detecting SLCO1B1 521T > C gene
Primer pair, probe and the kit of polymorphism.
Background technique
Polymerase chain reaction (PCR) is a kind of for amplifying the Protocols in Molecular Biology for expanding specific DNA fragmentation.It is glimmering
Light PCR (qPCR) detection, is the real-time detection technology in round pcr base growth, whole using fluorescence signal accumulation real-time monitoring
A PCR process carries out quantitative analysis to unknown template finally by standard curve or accumulates detection target base according to fluorescence signal
Cause.The technology realizes leap of the PCR from qualitative to quantitative, and compared with Standard PCR, it has specificity stronger, effective
The features such as solving PCR pollution problem, high degree of automation, to being widely used at the beginning of 21 century.
Although PCR detection method has the characteristics that quick, it is such as cultivated, state at present relative to other inspections
Inside and outside outpatient service detection have increasing need for faster going out as a result, and each circulation of current round pcr is needed to heat and be cooled down, reach quick
The method of PCR purpose is concentrated mainly on shortening and heats cooling time, and shortening chronergy is limited, since conventional primer probe designs
By sequence-specific, a certain range of Tm value is needed, causes PCR reaction itself to need 75 seconds or so the time of each circulation, is
Shortening detection time, can only reduce recurring number, lead to fast PCR sensitivity decrease.
Single nucleotide polymorphism (single nucleotide polymorphisms, SNPs), is primarily referred to as in genome
DNA sequence polymorphism caused by a single nucleotide variation in level, SNPs have known as third generation genetic marker
Property, hereditability, detectability, for the positioning of disease gene, clone and identification, SNPs have an effect on body physiological characteristic,
Medicament metabolism ability, pathological conditions are usually used in the neurological susceptibility studied or diagnosed the illness, personalized medicine；Such as by SLCO1B1 base
Because coding organic anion transport polypeptide 1B1 (OATP1B1, also known as OATP-C, OATP2 or LST1), liver cell intake and
Remove endogenous and exogenous material such as: statins, thyroxine, non-binding type bilirubin, bile acid, Repaglinide,
In enalaprilat, Temocapril, Olmesartan, Valsartan, methotrexate (MTX) and Irinotecan active metabolite SN-38 etc.
It plays a significant role.
SLCO1B1 gene the 5th exon 521T > C (rs4149056, Val174Ala) polymorphism is xanthous main
Hereditary variation, gene frequency 10~15%, its intake energy to substrate can be significantly reduced by carrying SLCO1B1521C homozygote
Power rises the blood concentration of the statins such as Pravastatin, Atorvastatin and rosuvastatin, so as to lead to liver
The serious adverse reactions such as function reduction and rhabdomyolysis.For reduce statins serious adverse reaction occurrence risk,
Suggest in " drug metabolic enzyme and drug target technique of gene detection guide (tentative) " " clinically according to SLCO1B1 gene
Type selection statins are treated."
The method for being used to detect the site SLCO1B1 gene 521T > C at this stage mainly has: 1) PCR-SSCP method；2) heterologous
RNA polymerase method (HA)；3) Mutant-enriched PCR (mutant-enriched PCR)；4) allele-specific few nucleosides
Acid analysis method (allele-specific oligonucleotide, ASO)；5) DNA chip technology (DNA chip)；6) it is mutated
Amplification system (amplification refractory mutation system, ARMS)；7) the methods of pyrosequencing.
PCR-SSCP method, Heteroduplex analysis, Mutant-enriched PCR etc. need first step PCR anti-in the above method
Test tube cap is opened after answering, reaction product is easy to cause to pollute air, is formed aerosol, is led to false positive results；And it is sequenced
Method needs to configure expensive tester, and test agent is expensive, it is difficult to and it is universal, while overlapping and height of the PCR sequencing PCR by wave crest
Degree is judged, causes to be not easy to detect that low-copy is mutated in height copy wild type signal background；Before chip method needs to carry out
PCR amplification is easy to produce Aerosol Pollution, and detection principle is mutant probe and wild-type probe fluorescent value comparing calculation, and is surveyed
Sequence method, which equally has, is not easy the problem of detecting low-copy mutation in height copy wild type signal background；ARMS-PCR method is ancient
Allusion quotation method is two sets of reaction systems of design, is judged by the Ct value difference in two peaceful systems of method is different, and reaction system needs anti-
Multiple optimization, while two reaction tubes are needed, or need to design scorpion type primer, annular region and linear areas are needed using high
Expensive space modification, while designing many and diverse, needs to verify the primed probe of multiple sequences, and causing to research and develop feasibility reduces, inspection
Survey cost increase；Conventional method primer and probe length is long simultaneously, needs longer annealing (annealing) and extends
(Extension) time causes detection time long, and the defect and design difficulty of these technologies influence clinical use range and clinic
It promotes, needs a kind of new PCR detection method.
Summary of the invention
The primer pair that the purpose of the present invention is to provide a kind of for detecting SLCO1B1 521T > C gene pleiomorphism, probe
And kit, so that time-consuming, non-specific amplification is bright for SLCO1B1 521T > C genetic polymorphism detection in the prior art for solution
The problems such as aobvious.
To achieve the goals above, The technical solution adopted by the invention is as follows:
In a first aspect, the present invention provide it is a kind of for detecting the primer pair of SLCO1B1 521T > C gene pleiomorphism, including
Have: for base sequence primer as shown in SEQ ID No.1, SEQ ID No.2 of 521 site SLCO1B1 design；Wherein,
5 ' end bases of the base sequence have amido modified group.
Second aspect, the present invention provide a kind of for detecting the preparation of the primer pair of SLCO1B1 521T > C gene pleiomorphism
Method includes the following steps: to design base sequence such as SEQ ID No.1, SEQ ID No.2 institute for 521 site SLCO1B1
The primer shown, and modified with 5 ' end bases of the amino group to the base sequence.
The third aspect, the present invention provide a kind of for detecting the probe of SLCO1B1 521T > C gene pleiomorphism, include:
For base sequence fluorescence probe as shown in SEQ ID No.3, SEQ ID No.4 of 521 site SLCO1B1 design；Its
In, a base in the base sequence has amido modified group.
The fluorescence probe of the base sequence such as SEQ ID No.3 in one of the embodiments, 5 ' ends start third
A base has amido modified group；The fluorescence probe of the base sequence such as SEQ ID No.4,5 ' ends start the 6th alkali
Base has amido modified group.
Fourth aspect, the present invention provide a kind of for detecting the preparation side of the probe of SLCO1B1 521T > C gene pleiomorphism
Method, including visited for 521 site SLCO1B1 design base sequence fluorescence as shown in SEQ ID No.3, SEQ ID No.4
Needle, and modify with amino group a base in the base sequence.
The site of the modification includes: 5 ' ends of base sequence shown in SEQ ID No.3 in one of the embodiments,
5 ' the ends for starting base sequence shown in third base and/or SEQ ID No.4 start the 6th base.
5th aspect, the present invention provide it is a kind of for detecting the kit of SLCO1B1 521T > C gene pleiomorphism, including
Have:
For base sequence primer as shown in SEQ ID No.1, SEQ ID No.2 of 521 site SLCO1B1 design；
Wherein, 5 ' end bases of the base sequence have amido modified group；
For base sequence fluorescence as shown in SEQ ID No.3, SEQ ID No.4 of 521 site SLCO1B1 design
Probe；Wherein, a base in the base sequence has amido modified group.
The fluorescence probe of the base sequence such as SEQ ID No.3 in one of the embodiments, 5 ' ends start third
A base has amido modified group；The fluorescence probe of the base sequence such as SEQ ID No.4,5 ' ends start the 6th alkali
Base has amido modified group.
6th aspect, the present invention provide a kind of fluorescence PCR method for detecting SLCO1B1 521T > C gene pleiomorphism, including
The step of PCR reaction system, setting reaction condition, is prepared in primer pair, fluorescence probe selection, wherein the primer pair is using upper
The primer pair stated, the fluorescence probe use above-mentioned fluorescence probe.
In one of the embodiments, in the step of setting reaction condition, reaction temperature is set as 60-63.2 DEG C.
Compared with prior art, the invention has the following advantages:
Kit of the present invention, for 521 site of the SLCO1B1 gene design 20bp especially primer of 15-20bp below
Sequence and the 20bp especially probe sequence of 15-20bp below, by many experiments from numerous primer sequences, probe sequence
The sequence of high specific has been filtered out in column as primer pair, probe, and the primer pair filtered out is modified using amino group
And probe, it is significant to shorten detection required time, non-specific amplification is reduced, primer, probe design difficulty and testing cost are reduced,
Diagnostic accuracy is not influenced, realizes that single reaction pipe closes lid and can detect 521 site C/C, C/T, T/T base of SLCO1B1 gene simultaneously
Because of type, Aerosol Pollution is avoided to greatest extent, facilitates clinical use, reduces error caused by operating.
Primer of the invention uses amino group modification length for the 5 ' of 20bp especially 15-20bp primer below
End, reaches 60 DEG C or so to improve primer Tm, institute's expanding fragment length can be realized specific inspection in the case where being shorter than 60bp
It surveys, high-precision detection, and shortens detection time.
Fluorescence probe of the invention is 20bp especially 15-20bp probe below using amino group modification length
One base reaches 68-70 DEG C to improve probe Tm value, this design ensure that probe when 60 DEG C of annealing extend probe it is miscellaneous
It hands over efficiency very low, along with allele has two probes, guarantees 100% homotactic probe prior to other one in reaction always
There is item the probe of a base to hybridize to template, and the hybridization of the homotactic probe of Reverse transcriptase non-100% is finally reached spy
The purpose of opposite sex detection SNPs.
Detection method of the invention, based on the above-mentioned restriction to primer pair, probe, compared with the existing detection method, denaturation
Time only needs 2-10s, and annealing extension of time only needs 2-30s, and entire testing process shortens 20-35min；Further, it reacts
The shortening of time also further shortens non-100% identical sequence probes hybridization time, so that the mismatch probe of inefficiency is miscellaneous
The amount of hybridization of friendship more reduces, and is finally reached the purpose of specific detection point mutation.
Detailed description of the invention
Fig. 1 is temperature gradient PCR programme diagram: instrument is Biorad company CFX nucleic acid amplification PCR instrument；
Fig. 2 is detection PCR amplification program figure: instrument is Life Tech (applied biosystems) ABI7500
Nucleic acid amplification equipment；
Fig. 3 is detection PCR amplification program figure: Biorad company CFX nucleic acid amplification PCR instrument.
Specific embodiment
In order to be more clearly understood that technology contents of the invention, spy lifts following embodiment and is described in detail.It should be understood that this
It is a little that examples are only for illustrating the present invention and not for limiting the scope of the present invention.Actual conditions are not specified in the following example
Experimental method, usually according to normal condition, for example (,) Sambrook et al., molecular cloning: laboratory manual (New York:
ColdSpring Harbor Laboratory Press, 1989) condition described in, or according to item proposed by manufacturer
Part.Used various common chemical reagent, are commercial product in embodiment.
Unless otherwise defined, all technical and scientific terms used in the present invention and belong to technical field of the invention
The normally understood meaning of technical staff it is identical.Term used in the description of the invention is intended merely to describe specific reality
The purpose for applying example is not used in the limitation present invention.Term "and/or" used in the present invention includes one or more relevant institutes
Any and all combinations of list of items.
Embodiment 1, primer pair for detecting SLCO1B1 521T > C gene pleiomorphism and preparation method thereof
The present embodiment devises primer pair for 521 site SLCO1B1.
According to rs4149056 sequence (SEQ ID No.5) design primer pair, primer pair sequence is as follows:
Forward primer (SEQ ID No.1): 5 '(amino group)-GGTCATACATGTGGAT3'；
Reverse primer (SEQ ID No.2): 5 '(amino group)-TCTCCCCTATTCCAC 3’。
In the present embodiment, the preparation of primer includes: to design base sequence such as SEQ ID for 521 site SLCO1B1
Primer shown in No.1, SEQ ID No.2, and with amino group to the 5 ' of base sequence end bases modified to get.
During preparing above-mentioned primer pair, using ABI company primer-design software primer express respectively to ammonia
The Tm value of the base sequence after base sequence, amino group modification before base base group modification is tested, as a result as follows:
Before modification: the Tm value of forward primer base sequence is 37.9 DEG C, and the Tm value of reverse primer base sequence is 40.8 DEG C；
After modification: the Tm value of forward primer base sequence is 58.2 DEG C, and the Tm value of reverse primer base sequence is 59.7
℃；
The Tm value of base sequence is lower than conventional fluorescent PCR primer temperature before modifying, by 5 ' the first base amino bases of end
Tm value after group's modification is obviously improved.
The primer pair commission Guangzhou Ai Ji biotechnology that the present invention is used to detect SLCO1B1 521T > C gene pleiomorphism has
The synthesis of limit company.
Embodiment 2, probe for detecting SLCO1B1 521T > C gene pleiomorphism and preparation method thereof
The present embodiment devises fluorescence probe for 521 site SLCO1B1.
Segment design wild-type probe and saltant type probe are mutated according to rs4149056 sequence (SEQ ID No.5):
Wild-type probe (SEQ ID No.3):
5 ' FAM-TAT (amino group) G-TThe end-GTTCATGGGTAATATGC-BHQ1 3 ', 5 ' starts third base
(T) it is modified using amino group, it is SNP site that 5 ' ends, which start the 5th base T, is wild type.
Saltant type probe (SEQ ID No.4):
5’Cy5-FAM-TATG-C- G (amino group) TTCATGGGTAATATGC-BHQ2 3 '；
5 ' ends are started the 6th base (G) and are modified using amino group, and it is the site SNP that 5 ' ends, which start the 5th base C,
For anomaly.
The preparation of above-mentioned fluorescence probe include: for 521 site SLCO1B1 design base sequence such as SEQ ID No.3,
Fluorescence probe shown in SEQ ID No.4, and with amino group modify a base in the base sequence to get.
During preparing above-mentioned probe, using ABI company primer-design software primer express respectively to amino base
The Tm value of the base sequence after base sequence, amino group modification before group's modification is tested, as a result as follows:
Before modification: the Tm value of wild-type probe base sequence is 51.5 DEG C, and the Tm value of saltant type probe base sequence is
56.3℃；
After modification: the Tm value of wild-type probe base sequence is 69.2 DEG C, and the Tm value of saltant type probe base sequence is
71.6℃。
The probe commission Guangzhou Ai Ji biotechnology that the present invention is used to detect SLCO1B1 521T > C gene pleiomorphism is limited
Company's synthesis.
Embodiment 3, the kit for detecting SLCO1B1 521T > C gene pleiomorphism
The present embodiment devises the reagent containing 2 probe of 1 primer pair of embodiment and embodiment for 521 site SLCO1B1
Box.
Embodiment 4, building plasmid
Construct 521 site SNPs wild type of SLCO1B1 gene and mutant plasmids:
NCBI-dbSNPs is retrieved, is found rs4149056 sequence (referring to SEQ ID No.6), the auspicious brilliant biological section in commission Shanghai
Skill Co., Ltd constructs wild plasmid and mutant plasmids, and wild plasmid sequence is as shown in SEQ ID No.6, saltant type matter
Grain sequence is as shown in SEQ ID No.7.
Embodiment 5, the optimization of the reaction temperature of kit for detecting SLCO1B1 521T > C gene pleiomorphism
In order to verify primer, probe and find optimal reaction temperature, temperature gradient PCR test is carried out, Bole is used
(Biorad) CFX fluorescent PCR instrument carries out temperature gradient fluorescent PCR.
1, reaction system is as follows:
End reaction system 25ul.
Reaction solution needed for proportionally preparing 48 reactions, dispenses 48 PCR 200ul white centrifuge tube, adds DNA
Template.
Wherein, DNA profiling (WT is wild plasmid, MT is mutant plasmids) is referring to such as the following table 1:
Table 1
| Temperature | 01 | 02 | 03 | 04 | 05 | 06 | |
| A | 68.0℃ | WT-107 | WT-105 | WT-104 | WT-103 | MT-107 | Blank control |
| B | 67.6℃ | WT-107 | WT-105 | WT-104 | WT-103 | MT-107 | Blank control |
| C | 66.7℃ | WT-107 | WT-105 | WT-104 | WT-103 | MT-107 | Blank control |
| D | 65.1℃ | WT-107 | WT-105 | WT-104 | WT-103 | MT-107 | Blank control |
| E | 63.2℃ | WT-107 | WT-105 | WT-104 | WT-103 | MT-107 | Blank control |
| F | 61.6℃ | WT-107 | WT-105 | WT-104 | WT-103 | MT-107 | Blank control |
| G | 60.6℃ | WT-107 | WT-105 | WT-104 | WT-103 | MT-107 | Blank control |
| H | 60.0℃ | WT-107 | WT-105 | WT-104 | WT-103 | MT-107 | Blank control |
2, response procedures such as Fig. 1.
95 degree of 3 minutes -1 circulations
95 degree 2 seconds, 60~68 degree temperature gradient 30 seconds -45 recycle
3, reaction result
Reaction result refers to table 2, as can be seen from Table 2: can detecte out wild-type template at 60-63.2 DEG C, and is mutated
Pattern plate has no fluorescence, and optimum temperature is 60 DEG C, and all temperature do not detect saltant type template, thus substantially determines reaction temperature
Degree and condition.
Table 2
Embodiment 6 carries out wild-type template and saltant type template detection using the kit that embodiment 3 provides
1, ABI7500 fluorescent PCR amplification instrument
(1) reaction system is as follows:
Wherein, DNA profiling (WT is wild plasmid, MT is mutant plasmids) is shown in Table 3:
Table 3
| 7 | 8 | 9 | 10 | |
| A | MT-103 | MT-107 | WT-103 | WT-107 |
| B | MT-103 | MT-107 | WT-103 | WT-107 |
| C | MT-103 | MT-107 | WT-103 | WT-107 |
| D | MT-103 | MT-107 | WT-103 | WT-107 |
| E | MT-103 | MT-107 | WT-103 | WT-107 |
| F | MT-103 | MT-107 | WT-103 | WT-107 |
| G | MT-103 | MT-107 | WT-103 | WT-107 |
| H | Blank | Blank | Blank | Blank |
(2) response procedures such as Fig. 2,45 circulation total time-consuming 67 minutes.
95 degree of 5 minutes -1 circulations
95 degree 10 seconds, 60 degree 30 seconds -45 circulation
(3) reaction result
Please result refer to table 4, as seen from Table 4: the method for the present invention can effectively detect high copy number and low copy number
Wild type and saltant type SLCO1B1 plasmid, and do not detect non-specific signals.
Table 4
When using ABI7500 fluorescent PCR amplification instrument, denaturation temperature is 95 DEG C, denaturation time is 10 seconds, elongating temperature of annealing
For 60 DEG C, annealing extension of time be 30 seconds, each recycle ratio Standard PCR (denaturation time 15 seconds, annealing extension of time 60 seconds) contracting
Short reaction time 35 seconds, 35-45 circulation is carried out, total time at least shortens 20 minutes.
2, Bio-Rad CFX96Touch real-time fluorescent PCR amplification instrument
(1) reaction system is as follows:
Wherein, DNA profiling (WT is wild type, MT is mutability plasmid) is shown in Table 5:
Table 5
| 7 | 8 | 9 | 10 | |
| A | MT-103 | MT-107 | WT-103 | WT-107 |
| B | MT-103 | MT-107 | WT-103 | WT-107 |
| C | MT-103 | MT-107 | WT-103 | WT-107 |
| D | MT-103 | MT-107 | WT-103 | WT-107 |
| E | MT-103 | MT-107 | WT-103 | WT-107 |
| F | MT-103 | MT-107 | WT-103 | WT-107 |
| G | MT-103 | MT-107 | WT-103 | WT-107 |
| H | Blank | Blank | Blank | Blank |
(2) response procedures such as Fig. 3,50 circulation total time-consuming 45 minutes.
95 degree 3 points -1 circulations
It 95 DEG C, 2 seconds, 60 DEG C, recycles within 10 seconds 50
(3) reaction result
6 please be the results are shown in Table, as seen from Table 6: kit of the present invention can also effectively detect height on the instrument of Bio-Rad
The wild type and saltant type SLCO1B1 plasmid of copy number and low copy number, and without non-specific performance signals.
Table 6
When using Bio-Rad CFX96Touch real-time fluorescent PCR amplification instrument, denaturation temperature is 95 DEG C, denaturation time 2
Second, annealing elongating temperature is 60 DEG C, annealing extension of time is 10 seconds, and each circulation shortens the time 58 seconds, carries out 35-45 and follows
Ring, total time at least shorten 35 minutes than Standard PCR (denaturation time 15 seconds, annealing extension of time 60 seconds).
In the above embodiment of the present invention, as a result interpretation foundation are as follows: SLCO1B1 gene C 677T wild-type probe fluorescence report
Group is FAM fluorophor, saltant type probes report group is Cy5 fluorophor, and wild-type homozygote (T/T) can only detect
FAM fluorescence, mutant homozygote (C/C) can only detect Cy5 fluorescence, heterozygous (C/T) can detect simultaneously FAM fluorescence and
Cy5 fluorescence.
In the above embodiment of the present invention, after tested, wild-type probe and 100% homotactic hybridization temperature and 1 mispairing
Hybridization temperature differs 17.1 DEG C；Anomaly probe and 100% homotactic hybridization temperature and 1 mismatch hybridization temperature difference 18.9
℃.Thus wild-type probe and saltant type template hybridization temperature: t=69.2-17.1=52.1 DEG C of Tm- Δ can be calculated, than moving back
60 DEG C of fiery elongating temperature is 7.9 DEG C low；Saltant type probe and wild-type template hybridization temperature Tm- Δ t=71.2-18.9=52.3
DEG C, than 60 DEG C of elongating temperature low 7.7 DEG C of annealing.
The above embodiment of the present invention is limited using the design of unconventional taqman probe and primer design method and time
PCR method processed, quick and convenient effectively low cost detection SNPs, detection method realization detect simultaneously in single PCR reaction tube
521 site C/C, C/T, T/T genotype of SLCO1B1 gene facilitates clinical manipulation, reduces Aerosol Pollution, keeps away to greatest extent
Exempt from false positive and false negative.This method is directed to diagnostic purpose flexible Application, while can be used for other purposes: scientific research, genetic analysis
Deng.
The above embodiment of the present invention, using the primer of amino group modification, probe, and length is in 20bp hereinafter, amplification
Product total length is less than 60bp, the time required to thus reducing amplification, realizes fast PCR purpose, probe Tm temperature is about 70 DEG C of left sides
The right side, and the Tm value after one base of mispairing is lower than annealing elongating temperature, this design ensure that probe when 60 DEG C of annealing extend
The hybridization efficiency of probe is very low, along with allele has two probes, guarantees that 100% homotactic probe is excellent in reaction always
Probe prior to other one with a base hybridizes to template, the hybridization of the homotactic probe of Reverse transcriptase non-100%,
It is finally reached the purpose of specific detection SNPs, and artificially to shorten non-100% identical sequence probes miscellaneous for time restriction PCR program
The time is handed over to be finally reached the mesh of specific detection point mutation so that the amount of hybridization that the mismatch probe of inefficiency hybridizes more reduces
's.
Comparative example
This comparative example is the comparative example of 6 part 1 of embodiment (using the part of ABI7500 fluorescent PCR amplification instrument), is
The effect of verifying different loci base modification, devises same SEQ ID NO1, SEQ ID NO2, SEQ ID NO3, SEQ ID
NO3 sequence is identical, and the different multiple groups primer and probe of decorating site compares, and only one group of discovery can detect fluorescence, right
It is only that in the primer and probe of kit that amino group decorating site is different than place, specific as follows:
Contrast groups forward primer (SEQ ID No.8): 5 ' GG(amino group)-TCATACATGTGGAT 3'；
Contrast groups reverse primer (SEQ ID No.9): 5 ' TCT(amino group)-CCCCTATTCCAC 3’。
Contrast groups wild-type probe (SEQ ID No.10):
5 ' FAM-TA (amino group) TG-TThe end-GTTCATGGGTAATATGC-BHQ1 3 ', 5 ' starts second base
(A) it is modified using amino group, it is SNP site that 5 ' ends, which start the 5th base,.
Contrast groups saltant type probe (SEQ ID No.11):
5 ' Cy5-TAT (amino group) G-C-GTTCATGGGTAATATGC-BHQ2 3'；
Buffer and reaction system are identical as experimental group, and response procedures use equal conditions.
Testing result: 1, ABI7500 fluorescent PCR amplification instrument
(1) reaction system is as follows:
Wherein, DNA profiling (WT is wild plasmid, MT is mutant plasmids) is shown in Table 7:
Table 7
(2) response procedures such as Fig. 2,45 circulation total time-consuming 67 minutes.
95 degree of 5 minutes -1 circulations
95 degree 10 seconds, 60 degree 30 seconds -45 circulation
(3) reaction result
Please result refer to table 8, as seen from Table 8: the method for the present invention (positive control) surveys high copy number and low copy number
Wild type and saltant type SLCO1B1 plasmid can detect fluorescence signal, and change amido modified position primed probe inspection
It surveys high copy number template Ct value and moves to right 9~13 or so, low copy number template fails to detect fluorescence signal, shows that sensitivity is dropped
It is low.
Table 8
Gene tester of the invention is also applied for the detection of non-mutation gene, and reaction time range and temperature be more at this time
Extensively, 90-96 DEG C of denaturation temperature range, reaction of degeneration (RD) time range 2-15 seconds or longer, extension temperature range of annealing was in 51-
65 DEG C, the time about 2-60 seconds or longer.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art
It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to protection of the invention
Range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Sequence table
<110>Dongguan Bo Sheng Biotechnology Co., Ltd
<120>for detecting SLCO1B1 521T>primer pair, probe and the kit of C gene pleiomorphism
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 16
<212> DNA
<213> Artificial Sequence
<400> 1
ggtcatacat gtggat 16
<210> 2
<211> 15
<212> DNA
<213> Artificial Sequence
<400> 2
tctcccctat tccac 15
<210> 3
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 3
tatgtgttca tgggtaatat gc 22
<210> 4
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 4
tatgcgttca tgggtaatat gc 22
<210> 5
<211> 1001
<212> DNA
<213> Homo sapiens
<400> 5
taatctccac atgccaagag tggggccagg tagagataat tgaattttgg gggcggtttc 60
ccccatactg ttctcgtggt aatgaagaag tctcacaaga tctgatgatt ttataaatga 120
gagttcccct gcaaaagctc tcttgcctgc caccatgtaa gacatggctt tgctcttcct 180
tcatcttccg ccatgattgt gaggcccccc agccatgagg aactatgagt ccattagacc 240
cttttccttt ataaattacc cagtctcagg tatgtattta ttagcagcat aagaatggac 300
taatacacca tattgtcaaa gtttgcaaag tgaatataaa ttacttgtac ttgtaaatta 360
aaaaaaaata agtagaataa ttaagagttt acaagtagtt aaatttgtaa tagaaatgct 420
aaaattaatg tttaaaatga aacactctct tatctacata ggttgtttaa aggaatctgg 480
gtcatacatg tggatatatg ygttcatggg taatatgctt cgtggaatag gggagactcc 540
catagtacca ttggggcttt cttacattga tgatttcgct aaagaaggac attcttcttt 600
gtatttaggt aatgtacaca aaatattaaa ttgtatgatc actttccctt tgtctacttt 660
tgaaatagta gagttactaa acttattatt ttacctatta gaacatatat ttgggtatat 720
gtattgtatc atatttcttt taaaaacatg gtgaataaga accatgcatt cttggcatct 780
agtaaaattg ctttataata ttttcaggta tattgaatgc aatagcaatg attggtccaa 840
tcattggctt taccctggga tctctgtttt ctaaaatgta cgtggatatt ggatatgtag 900
atctaagtaa gtacaaccag aacaaggtac catgataacg tctttctaag cacacatgcg 960
aaaaacattt tttcaaataa ctgaattcac tctttcaata g 1001
<210> 6
<211> 2988
<212> DNA
<213> Artificial Sequence
<400> 6
tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60
cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120
ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180
accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240
attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300
tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360
tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt agaactcggt acgcgcggat 420
cttccagaga ttaagtagtt aaatttgtaa tagaaatgct aaaattaatg tttaaaatga 480
aacactctct tatctacata ggttgtttaa aggaatctgg gtcatacatg tggatatatg 540
tgttcatggg taatatgctt cgtggaatag gggagactcc catagtacca ttggggcttt 600
cttacattga tgatttcgct aaagaaggac attcttcttt gtatttaggt aatgtacaca 660
aaatattaaa ttgtatgatc actttccctt tgtctacttt tgaaatagta gagttactaa 720
acttataatc gtcgaacggc aggcgtgcaa acttggcgta atcatggtca tagctgtttc 780
ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt 840
gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc 900
ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg 960
ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct 1020
cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca 1080
cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga 1140
accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc 1200
acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg 1260
cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat 1320
acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt 1380
atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc 1440
agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg 1500
acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg 1560
gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg 1620
gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg 1680
gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca 1740
gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga 1800
acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga 1860
tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt 1920
ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt 1980
catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat 2040
ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag 2100
caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct 2160
ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt 2220
tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg 2280
cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca 2340
aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt 2400
tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat 2460
gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac 2520
cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa 2580
aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt 2640
tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt 2700
tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa 2760
gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt 2820
atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa 2880
taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta 2940
tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtc 2988
<210> 7
<211> 2988
<212> DNA
<213> Artificial Sequence
<400> 7
tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60
cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120
ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180
accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240
attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300
tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360
tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt agaactcggt acgcgcggat 420
cttccagaga ttaagtagtt aaatttgtaa tagaaatgct aaaattaatg tttaaaatga 480
aacactctct tatctacata ggttgtttaa aggaatctgg gtcatacatg tggatatatg 540
cgttcatggg taatatgctt cgtggaatag gggagactcc catagtacca ttggggcttt 600
cttacattga tgatttcgct aaagaaggac attcttcttt gtatttaggt aatgtacaca 660
aaatattaaa ttgtatgatc actttccctt tgtctacttt tgaaatagta gagttactaa 720
acttataatc gtcgaacggc aggcgtgcaa acttggcgta atcatggtca tagctgtttc 780
ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt 840
gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc 900
ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg 960
ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct 1020
cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca 1080
cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga 1140
accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc 1200
acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg 1260
cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat 1320
acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt 1380
atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc 1440
agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg 1500
acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg 1560
gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg 1620
gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg 1680
gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca 1740
gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga 1800
acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga 1860
tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt 1920
ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt 1980
catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat 2040
ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag 2100
caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct 2160
ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt 2220
tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg 2280
cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca 2340
aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt 2400
tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat 2460
gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac 2520
cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa 2580
aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt 2640
tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt 2700
tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa 2760
gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt 2820
atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa 2880
taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta 2940
tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtc 2988
Claims (10)
1. the primer pair for detecting SLCO1B1 521T > C gene pleiomorphism, it is characterised in that: the sequence of primer pair such as SEQ
Shown in ID NO:1 and 2, the primer is to design for 521 site SLCO1B1, and 5 ' ends of the primer are repaired with amino
Adorn group.
2. a kind of according to claim 1 for detecting the preparation side of the primer pair of SLCO1B1 521T > C gene pleiomorphism
Method, which comprises the steps of: design base sequence such as SEQ ID No.1, SEQ for 521 site SLCO1B1
Primer shown in ID No.2, and modified with 5 ' end bases of the amino group to the base sequence.
3. the probe for detecting SLCO1B1 521T > C gene pleiomorphism, which is characterized in that include: for SLCO1B1
Base sequence fluorescence probe as shown in SEQ ID No.3, SEQ ID No.4 of 521 sites design；Wherein, the base sequence
A base in column has amido modified group.
4. according to claim 3 for detecting the probe of SLCO1B1 521T > C gene pleiomorphism, the alkali of the probe
For basic sequence as shown in SEQ ID NO:3 and 4, wherein 5 ' the ends of SEQ ID NO:3, which start third base, has amido modified base
Group；, its 5 ' end of SEQ ID NO:4, which starts the 6th base, has amido modified group.
5. a kind of according to claim 3 or 4 for detecting the preparation of the probe of SLCO1B1 521T > C gene pleiomorphism
Method, which comprises the steps of: design base sequence such as SEQ ID No.3, SEQ for 521 site SLCO1B1
Fluorescence probe shown in ID No.4, and modify with amino group a base in the base sequence.
6. it is according to claim 5 for detecting the preparation method of the probe of SLCO1B1 521T > C gene pleiomorphism,
Be characterized in that, the site of the modification include: base sequence shown in SEQ ID No.3 5 ' end start third bases and/or
5 ' ends of base sequence shown in SEQ ID No.4 start the 6th base.
7. the kit for detecting SLCO1B1 521T > C gene pleiomorphism, which is characterized in that include:
For base sequence primer as shown in SEQ ID No.1, SEQ ID No.2 of 521 site SLCO1B1 design；Its
In, 5 ' end bases of the base sequence have amido modified group；
For base sequence fluorescence probe as shown in SEQ ID No.3, SEQ ID No.4 of 521 site SLCO1B1 design；
Wherein, a base in the base sequence has amido modified group.
8. according to claim 7 for detecting the kit of SLCO1B1 521T > C gene pleiomorphism, which is characterized in that
The fluorescence probe of the base sequence such as SEQ ID No.3,5 ' ends, which start third base, has amido modified group；It is described
The fluorescence probe of base sequence such as SEQ ID No.4,5 ' ends, which start the 6th base, has amido modified group.
9. a kind of fluorescence PCR method for detecting SLCO1B1 521T > C gene pleiomorphism, including the selection of primer pair, fluorescence probe,
The step of preparing PCR reaction system, setting reaction condition, which is characterized in that the primer pair is drawn using described in claim 1
Object pair, the fluorescence probe is using fluorescence probe described in claim 3 or 4.
10. the fluorescence PCR method of detection SLCO1B1 521T > C gene pleiomorphism according to claim 9, feature exist
In in, the setting reaction condition the step of, reaction temperature is set as 60-63.2 DEG C.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113584146A (en) * | 2021-06-15 | 2021-11-02 | 湖南菲思特精准医疗科技有限公司 | Detection kit for statin metabolic marker, detection method and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104846085A (en) * | 2015-04-30 | 2015-08-19 | 武汉友芝友医疗科技有限公司 | Human SLCO1B1 and ApoE (apolipoprotein E) gene polymorphism detection kit |
| CN105803099A (en) * | 2016-05-16 | 2016-07-27 | 钟诗龙 | Kit for simultaneously detecting SLCO1B1, APOE and LDLR gene multisite mutation |
| CN107099602A (en) * | 2017-05-27 | 2017-08-29 | 宁波美晶医疗技术有限公司 | It is a kind of at the same detect statins metabolic gene multisite mutation kit |
| CN107267645A (en) * | 2017-07-31 | 2017-10-20 | 广州德臻生物技术有限公司 | Primer pair, probe and kit for detecting mthfr gene polymorphism |
-
2018
- 2018-04-28 CN CN201810404538.7A patent/CN108977523A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104846085A (en) * | 2015-04-30 | 2015-08-19 | 武汉友芝友医疗科技有限公司 | Human SLCO1B1 and ApoE (apolipoprotein E) gene polymorphism detection kit |
| CN105803099A (en) * | 2016-05-16 | 2016-07-27 | 钟诗龙 | Kit for simultaneously detecting SLCO1B1, APOE and LDLR gene multisite mutation |
| CN107099602A (en) * | 2017-05-27 | 2017-08-29 | 宁波美晶医疗技术有限公司 | It is a kind of at the same detect statins metabolic gene multisite mutation kit |
| CN107267645A (en) * | 2017-07-31 | 2017-10-20 | 广州德臻生物技术有限公司 | Primer pair, probe and kit for detecting mthfr gene polymorphism |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113584146A (en) * | 2021-06-15 | 2021-11-02 | 湖南菲思特精准医疗科技有限公司 | Detection kit for statin metabolic marker, detection method and application thereof |
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