CN107090517A - For primer sets, kit and the method for instructing dexmedetomidine hydrochloride medication related gene polymorphism to detect - Google Patents
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Abstract
The present invention relates to a kind of primer sets for being used to instruct dexmedetomidine hydrochloride medication related gene polymorphism to detect.The primer sets include CYP2A6*1 forward primers and reverse primer, CYP2A6*4 reverse primers, CYP2A6*1 detection probes and CYP2A6*4 detection probes.The invention further relates to a kind of kit, the kit includes PCR reaction solutions, and PCR reaction solutions include CYP2A6*1 forward primers, CYP2A6*1 reverse primers, CYP2A6*4 reverse primers, CYP2A6*1 detection probes, CYP2A6*4 detection probes, 10 × PCRbuffer, dNTPS, HS Taq enzyme etc..The kit and its detection method that the present invention is provided have quick simple to operate, detection, accuracy height, specific good, sensitivity high, and can effectively meet the advantage of clinical requirement.
Description
Technical field
The present invention relates to vitro diagnostic techniques field, particularly, it is related to a kind of for instructing dexmedetomidine hydrochloride medication
Primer sets, kit and the method for related gene polymorphism detection.
Background technology
Dexmedetomidine hydrochloride is a kind of relative selectivity α2Receptor agonism medicine, have calmness, antianxiety, hypnosis, analgesia and
Sympathetic block is acted on.Dexmedetomidine almost completely by bioconversion, is seldom discharged with original shape from urine and excrement.It is biological
Conversion includes the metabolism of direct glucuronidation and cytochrome P 450 mediated.The main metabolic pathway of Dexmedetomidine is：Directly
Connect N- glucuronides and be melted into nonactive metabolite；Fat hydroxylization effect (mainly being mediated by CYP2A6) produces the right U.S. of 3- hydroxyls
Hold in the palm fixed miaow, 3- hydroxyl Dexmedetomidine glucosiduronic acids and 3- carboxyl Dexmedetomidines；Dexmedetomidine N-, which methylates, produces 3- hydroxyls
Base N- methyl Dexmedetomidine, 3- carboxyls N- methyl Dexmedetomidine and N- methyl O- glucosiduronic acid Dexmedetomidines.
On CYP2A6 research discovery, the nearly 6kb of CYP2A6 full length genes, including 9 extrons, positioned at 19q12-
Between 19q13.2, it and CYP2A7, CYP2A13 and two CYP2A7 pseudogenes are located at 350kb gene cluster together
It is interior.In CYP2A6 multiple allele, the allele with normal enzymatic activity is CYP2A6*1, and CYP2A6*4 equipotentials
The azymia activity of gene code.CYP2A6*4 allele is the overall missing of CYP2A6 genes, and the deletion segment comes from height
3 ' end regions of the similar CYP2A6 and CYP2A7 genes of degree do not wait exchange, cause a deletion allele, its exchange area
Domain is located on the 8th introne or the 9th extron.
CYP2A6*4 genes are high polymorphisms, about 40 allele variants.These allele are homozygosis
The CYP2A6 of son lacks activity.CYP2A6*4 gene frequencies are 15%, Finland artificial 1.0%, Spain in Chinese population
For 0.5%, CYP2A6*4 allele be facilitate asian population PM phenotypes (CYP2A6 Gene Partials lack or all missing facilitates
PM phenotypes) major allele.Therefore administration when, should give the patient containing CYP2A6*4 allele leave it is enough
Notice, to prevent the generation of adverse reaction.
The method of conventional detection gene pleiomorphism has DNA direct sequencings, RFLP point at present
Analysis method (PCR-PFLP), high-resolution solubility curve (HRM), genetic chip, Luminex, fluorescence quantitative PCR method etc..Survey
Sequence technology is the goldstandard of generally acknowledged detection gene mutation, but its equipment cost height, detection cycle length, detection flux are low, detection
Sensitivity is low, technical operation to experimenter requires the reasons such as height, result judgement complex steps, it is more difficult to form commercialized examine
Stopping pregnancy product；Restriction fragment length polymorphism analysis method detection sensitivity equally not high, complex operation step, the result of detection is still
The checking again of progress generation PCR sequencing PCR is needed, especially easily causes the cross pollution of PCR primer easily to go out when sample size is more
Existing digestion is insufficient or digestion excessively false negative or false positive results occurs, therefore can not also be applied to clinically.Chip is examined
Survey because the accuracy and repeatability of its testing result are poor, the shortcomings of experimental period is long, be also unsuitable for developing clinical detection reagent
Box.The detection method of quantitative fluorescent PCR possesses that cost is low, sensitivity is high, specificity good, the advantages of as a result reproducible.
The content of the invention
It is an object of the invention to provide a kind of primer for instructing dexmedetomidine hydrochloride medication related gene polymorphism to detect
Group, kit and method, have the advantages that simple to operate, detection is quick, accuracy is high and specific good.
The present invention provides a kind of primer sets for being used to instruct dexmedetomidine hydrochloride medication related gene polymorphism to detect, institute
Stating primer sets includes specific primer and probe, as follows：
CYP2A6*1 forward primers：
5’-AAAATGGGCATGAACGCCC-3’；
CYP2A6*1 reverse primers：
5’-GAGGGGCGCAGCTAAGAC-3’；
CYP2A6*4 reverse primers：
5’-CGGAAGAGGCGGGTATAAGAA-3’；
CYP2A6*1 detection probes：
5’FAM-CTTTCCGCCATCCT-3’；
CYP2A6*4 detection probes：
5’VIC-CTTTCCCGCATCTT-3’。
The present invention also provides a kind of kit for being used to instruct dexmedetomidine hydrochloride medication related gene polymorphism to detect,
Including the PCR reaction solutions containing specific primer as described above and probe, it is positive that the PCR reaction solutions include CYP2A6*1
Primer, CYP2A6*1 reverse primers, CYP2A6*4 reverse primers, CYP2A6*1 detection probes, CYP2A6*4 detection probes, 10 ×
PCRbuffer, dNTPS, HS-Taq enzyme and nuclease-free water.
In the preferred embodiment of the kit one that the present invention is provided, the composition of the PCR reaction solutions is final concentration of：1
× PCR buffer, 0.1~0.3 μM of CYP2A6*1 forward primers, 0.1~0.3 μM of CYP2A6*1 reverse primers, 0.1~0.3
μM CYP2A6*4 reverse primers, 0.1~0.3 μM of CYP2A6*1 detection probes, 0.1~0.3 μM of CYP2A6*4 detection probe,
0.2~0.3mM dNTPS, 1U HS-Taq enzymes.
In the preferred embodiment of the kit one that the present invention is provided, in addition to positive reference substance one, positive reference substance
Two and positive reference substance three；
The positive reference substance one is 30ng/ul CYP2A6*4 gene loci wild plasmids；
The positive reference substance two presses 1 for 30ng/ul CYP2A6*4 gene locis wild type with saltant type：1 quantity ratio
The plasmid of heterozygosis；
The positive reference substance three is 30ng/ul CYP2A6*4 gene point mutation type plasmids.
In the preferred embodiment of the kit one that the present invention is provided, in addition to blank control product, the blank control
Product are nuclease-free water.
The present invention also provides a kind of method for instructing dexmedetomidine hydrochloride medication related gene polymorphism to detect polymorphism,
Comprise the following steps：
Step one：The DNA of sample extracting to be detected is taken, pcr template is used as；
Step 2：Kit as described above is provided, appropriate PCR reaction solutions are taken out, by the PCR reaction solutions and in right amount
The pcr template is mixed；
Step 3：Carry out pcr amplification reaction：25 DEG C of UNG ferment treatments 10min；95 DEG C of denaturation 5min；40cycles, 95 DEG C
15sec, 60 DEG C of 30sec；
Step 4：PCR result judgements：Meet defined situation in the positive reference substance and blank control product of the kit
Under, CT values are obtained according to fluorescent amplification curve and carry out result judgement, the genotype of sample to be tested is obtained.
Compared to prior art, provided by the present invention for instructing dexmedetomidine hydrochloride medication related gene polymorphism to examine
The beneficial effect of the primer sets of survey, kit and method is：The primer and TAQMAN-MGB fluorescence high by designing specificity
Detection probe simultaneously detects gene pleiomorphism by the release and intensity for detecting fluorescence, obtains CYP2A6*4 genotype, to
During medicine, the patient containing CYP2A6*4 allele is given more notices, to prevent the generation of adverse reaction；In addition it is reconfigured at
Into the reliable kit of easy to use and testing result, scientific and reasonable PCR reaction systems are designed so that the present invention has behaviour
Make the characteristics of simple, detection is quick, accuracy is high and specific good.
Brief description of the drawings
Fig. 1 is the PCR amplification curve diagrams of clinical sample CYP2A6*4 wild types；
Fig. 2 is the PCR amplification curve diagrams of clinical sample CYP2A6*4 heterozygous；
Fig. 3 is the PCR amplification curve diagrams of clinical sample CYP2A6*4 saltant types.
Embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, below in conjunction with the accompanying drawings and embodiment, it is right
The present invention is described in further detail.It should be appreciated that specific embodiment described herein is only to explain the present invention, and
It is not used in the restriction present invention.
Embodiment 1：The preparation of kit
First, the design and synthesis of primer and probe
For CYP2A6*4 gene locis in human genome, (sequence is referring to mankind's full-length genome sequence disclosed in ncbi database
Row), using Primer Premier 3.0 and Methyl Primer Express v1.0 softwares, separately design specific primer
And the probe of MGB marks.
Specific primer and probe sequence, as shown in Table 1：
Table one, specific primer and probe sequence
2nd, reference substance is selected
Positive reference substance one is 30ng/ul CYP2A6*4 gene loci wild plasmids；
Positive reference substance two presses 1 for 30ng/ul CYP2A6*4 gene locis wild type with saltant type：1 quantity compares heterozygosis
Plasmid；
Positive reference substance three is 30ng/ul CYP2A6*4 gene point mutation type plasmids；
Blank control product are nuclease-free water.
3rd, PCR reaction solutions are constituted
The PCR reaction solutions include CYP2A6*1 forward primers, CYP2A6*1 reverse primers, CYP2A6*4 reverse primers,
CYP2A6*1 detection probes, CYP2A6*4 detection probes, 10 × PCRbuffer, dNTPS, HS-Taq enzyme and nuclease-free water.
Wherein, described 10 × PCR buffer, dNTP and nuclease-free water are purchased from precious biological (the precious biology (Dalian) in Dalian
Engineering Co., Ltd)；The nuclease-free water is that (Nuclease-Free Water) uses DEPC (Diethyl
Pyrocarbonate, pyrocarbonic acid diethyl ester) the treated and ultra-pure water through autoclave sterilization；HS-Taq enzymes (TaKaRa Taq
Hot Start Version, precious biological purchased from Dalian) it is high for the sensitivity of general T aq archaeal dna polymerases, it is also easy to produce non-specific
The situation of property band, the thermophilic archaeal dna polymerase product of high specific specially developed.
The PCR reaction solutions composition it is final concentration of：1 × PCR buffer, 0.1~0.3 μM of CYP2A6*1 forward direction is drawn
Thing, 0.1~0.3 μM of CYP2A6*1 reverse primers, 0.1~0.3 μM of CYP2A6*4 reverse primers, 0.1~0.3 μM of CYP2A6*
1 detection probe, 0.1~0.3 μM of CYP2A6*4 detection probe, 0.2~0.3mM dNTPS, 1U HS-Taq enzymes, nuclease free
System (is supplemented to 18.5ul) by appropriate amount of water.
Embodiment 2：The method that CYP2A6*4 gene pleiomorphisms are detected using mentioned reagent box
First, biological specimen
Biomaterial of the present invention is all from the refined medical test institute in Hunan.In the refined doctor in Hunan during for the 6-12 months in 2016
Learn 500 remaining crowd's anticoagulations of inspection institute's detection.
2nd, the DNA of sample extracting to be detected is taken, pcr template is used as：
DNA extraction kit is the extracts kit of independent research《Nucleic acid extraction or purified reagent》Extracted and (put on record
Number：The long tool in Hunan is for 20150166).
1) take sterile 1.5mL EP to manage, add 250uL whole bloods；
2) add 750ul cell pyrolysis liquids, overturn and mix 5-6 times, be stored at room temperature 10min；
3) 12000rpm centrifuges 1.5min；Outwell top waste liquid, collecting pipe bottom precipitation；
4) 20uL Proteinase Ks, 250uL lysate ABL are sequentially added, during which vortex oscillation 10s, 65 DEG C of water-bath 15min shake
Swing mixing 2-3 times；
5) take out, add 250uL absolute ethyl alcohols, vortex oscillation 10s, of short duration centrifugation 5s；
6) adsorption column is inserted in collecting pipe, mixed liquor obtained by previous step is transferred in adsorption column；10,000rpm is centrifuged
1min；
7) waste liquid in collecting pipe is abandoned, adsorption column is reentered into collecting pipe；Add 500uL washing lotions I, 10,000rpm, 1min
Centrifugation；
8) waste liquid in collecting pipe is abandoned, 700uL washing lotions II, 10,000rpm, 1min, centrifugation is added.Abandon waste liquid；(washing lotion II makes
Absolute ethyl alcohol need to be added before to 80%)
9) repeat step 8 is once；
10) efflux is abandoned, adsorption column is turned back into collecting pipe, idle running centrifugation, 13,000rpm, 2min；
11) adsorption column is inserted into new EP pipes；Adding 30-100uL DNA lysates, (65 DEG C of preheatings can improve DNA
Pick-up rate), it is stored at room temperature 5min；
12) 10,000rpm, 1min, centrifugation, abandon adsorption column, EP liquid in pipe is DNA solution.2-8 DEG C of preservation, if needing
Long-term preserve please be placed in -20 DEG C or lower temperature.
The 3rd, the kit is provided, appropriate PCR reaction solutions are taken out, by the PCR reaction solutions and the appropriate pcr template
Mix：
Requirement PCR reaction solutions connecting leg (PCR pipe number=sky of sample number+1 per detection site is taken out from kit
+ 3 positive controls are compareed in vain)；
After PCR reaction solution room temperatures are melted, brief centrifugation opens lid；
1.5 μ L are taken to add in PCR reaction solutions from sample DNA to be checked (or reference substance)；
After vibration is mixed, brief centrifugation 10s moves it to amplification region.
4th, pcr amplification reaction is carried out：25 DEG C of UNG ferment treatments 10min；95 DEG C of denaturation 5min；40cycles, 95 DEG C
15sec, 60 DEG C of 30sec.
The PCR instrument device used is ABI 7500 or day FQD-96A fluorescent quantitative poly chain reaction (PCR) inspection is won in Hangzhou
Examining system, reaction system is 20ul；
PCR reaction conditions are as shown in Table 2：
Table two, pcr amplification reaction condition
5th, PCR result judgements：In the case of as defined in meeting in the positive reference substance and blank control product of the kit,
CT values are obtained according to fluorescent amplification curve and carry out result judgement, CYP2A6*4 genotype is obtained.
As a result availability deciding, as shown in Table 3：
Table three
Blank control result is negative (No Ct or Ct value >=38).
PCR result judgements, as shown in Table 4：
Table four
Based on each gene loci testing result of clinical sample of the present invention, referring to Fig. 1-Fig. 3, wherein Fig. 1 is clinical sample
The PCR amplification curve diagrams of CYP2A6*4 wild types, Fig. 2 is the PCR amplification curve diagrams of clinical sample CYP2A6*4 heterozygous, Fig. 3
For the PCR amplification curve diagrams of clinical sample CYP2A6*4 saltant types.
Embodiment 3：CYP2A6*4 genotype medication guide
CYP2A6*4 changes enzymatic activity or directly results in the forfeiture of enzymatic activity by influenceing the expression of CYP2A6 enzyme contents.
Therefore in administration, the patient containing CYP2A6*4 allele should be given with reference to clinic and leave enough notices, to prevent not
The generation of good reaction.CYP2A6*4 genotype is drawn by the result judgement (table four) in quantitative fluorescent PCR.Doctor according to
CYP2A6 genotype and phenotype refer to table five to deserved relation, with reference to the pathophysiological features, drug combination, clinic of patient
The other factors such as performance carry out comprehensive descision to determine final therapeutic regimen.
Table five
Primer sets, reagent provided by the present invention for instructing the detection of dexmedetomidine hydrochloride medication related gene polymorphism
The beneficial effect of box and method is：By designing the high primer of specificity and TAQMAN-MGB fluorescent detection probes and by inspection
The release and intensity for surveying fluorescence detect gene pleiomorphism, obtain CYP2A6*4 genotype, in administration, give containing
The more notices of patient of CYP2A6*4 allele, to prevent the generation of adverse reaction；In addition be reconfigured to it is easy to use and
The reliable kit of testing result, designs scientific and reasonable PCR reaction systems so that the present invention has simple to operate, detection
Quickly, the characteristics of accuracy is high and specific good.
Embodiments of the invention are the foregoing is only, are not intended to limit the scope of the invention, it is every to utilize this hair
The equivalent flow conversion that bright description is made, or other related technical fields are directly or indirectly used in, similarly wrap
Include in the scope of patent protection of the present invention.
SEQUENCE LISTING
<110>Han Linzhi
<120>For primer sets, kit and the method for instructing dexmedetomidine hydrochloride medication related gene polymorphism to detect
<130> 2017
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 19
<212> DNA
<213>Artificial sequence（Artificial Sequence）
<400> 1
aaaatgggca tgaacgccc 19
<210> 2
<211> 18
<212> DNA
<213>Artificial sequence（Artificial Sequence）
<400> 2
gaggggcgca gctaagac 18
<210> 3
<211> 21
<212> DNA
<213>Artificial sequence（Artificial Sequence）
<400> 3
cggaagaggc gggtataaga a 21
<210> 4
<211> 14
<212> DNA
<213>Artificial sequence（Artificial Sequence）
<400> 4
ctttccgcca tcct 14
<210> 5
<211> 14
<212> DNA
<213>Artificial sequence（Artificial Sequence）
<400> 5
ctttcccgca tctt 14
Claims (6)
1. a kind of primer sets for being used to instruct dexmedetomidine hydrochloride medication related gene polymorphism to detect, it is characterised in that institute
Stating primer sets includes specific primer and probe, as follows：
CYP2A6*1 forward primers：
5’-AAAATGGGCATGAACGCCC-3’；
CYP2A6*1 reverse primers：
5’-GAGGGGCGCAGCTAAGAC-3’；
CYP2A6*4 reverse primers：
5’-CGGAAGAGGCGGGTATAAGAA-3’；
CYP2A6*1 detection probes：
5’FAM-CTTTCCGCCATCCT-3’；
CYP2A6*4 detection probes：
5’VIC-CTTTCCCGCATCTT-3’。
2. a kind of kit for being used to instruct dexmedetomidine hydrochloride medication related gene polymorphism to detect, it is characterised in that bag
The PCR reaction solutions containing specific primer as claimed in claim 1 and probe are included, the PCR reaction solutions include CYP2A6*1
Forward primer, CYP2A6*1 reverse primers, CYP2A6*4 reverse primers, CYP2A6*1 detection probes, CYP2A6*4 detection probes,
10 × PCRbuffer, dNTPS, HS-Taq enzyme and nuclease-free water.
3. the reagent according to claim 2 for being used to instruct dexmedetomidine hydrochloride medication related gene polymorphism to detect
Box, it is characterised in that the composition of the PCR reaction solutions is final concentration of：1 × PCR buffer, 0.1~0.3 μM of CYP2A6*1 is just
To primer, 0.1~0.3 μM of CYP2A6*1 reverse primer, 0.1~0.3 μM of CYP2A6*4 reverse primer, 0.1~0.3 μM
CYP2A6*1 detection probes, 0.1~0.3 μM of CYP2A6*4 detection probe, 0.2~0.3mM dNTPS, 1U HS-Taq enzymes.
4. the examination for being used to instruct dexmedetomidine hydrochloride medication related gene polymorphism to detect according to Claims 2 or 3
Agent box, it is characterised in that also including positive reference substance one, positive reference substance two and positive reference substance three；
The positive reference substance one is 30ng/ul CYP2A6*4 gene loci wild plasmids；
The positive reference substance two presses 1 for 30ng/ul CYP2A6*4 gene locis wild type with saltant type：1 quantity compares heterozygosis
Plasmid；
The positive reference substance three is 30ng/ul CYP2A6*4 gene point mutation type plasmids.
5. the reagent according to claim 4 for being used to instruct dexmedetomidine hydrochloride medication related gene polymorphism to detect
Box, it is characterised in that also including blank control product, the blank control product are nuclease-free water.
6. a kind of method for instructing dexmedetomidine hydrochloride medication related gene polymorphism to detect polymorphism, it is characterised in that bag
Include following steps：
Step one：The DNA of sample extracting to be detected is taken, pcr template is used as；
Step 2：Kit as claimed in claim 5 is provided, appropriate PCR reaction solutions are taken out, by the PCR reaction solutions with fitting
The pcr template is measured to mix；
Step 3：Carry out pcr amplification reaction：25 DEG C of UNG ferment treatments 10min；95 DEG C of denaturation 5min；40cycles, 95 DEG C
15sec, 60 DEG C of 30sec；
Step 4：PCR result judgements：In the case of as defined in meeting in the positive reference substance and blank control product of the kit,
CT values are obtained according to fluorescent amplification curve and carry out result judgement, the genotype of sample to be tested is obtained.
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