CN107523612A - Primer sets, kit and method for the detection of children's safety medication related gene - Google Patents
Primer sets, kit and method for the detection of children's safety medication related gene Download PDFInfo
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Abstract
The present invention relates to a kind of primer sets for the detection of children's safety medication related gene.The primer sets include the specific primer and probe for CYP2C19*2, CYP2C19*3, CYP3A4*1G, CYP2C9*3, CYP2D6*10 and CYP3A5*3 gene loci.The present invention relates to a kind of kit for the detection of children's safety medication related gene.The kit includes PCR reaction solutions, PCR buffer, dNTP, nuclease-free water and HS Taq enzymes containing above-mentioned primer and probe.The invention further relates to a kind of method for the detection of children's safety medication related gene.Kit provided by the invention and its detection method have that simple to operate, detection is quick, accuracy is high, specificity is good, sensitivity is good, easy to use and the advantages of can effectively meet clinical requirement.
Description
Technical field
The present invention relates to vitro diagnostic techniques field, particularly, is related to a kind of children's safety medication related gene that is used for and examines
Primer sets, kit and the method for survey.
Background technology
Medicine first has to be inactivated, while also to promote it certainly as exogenous active material when entering in body
Eliminate in vivo.Internal medicine mainly loses pharmacological activity through bioconversion in liver and minority is activated, and it is high to be changed into polarity
Water soluble metabolites is further excreted.And the bioconversion of medicine then depends on the catalysis of drug metabolic enzyme.
Drug metabolic enzyme include participate in I phase metabolism cytochrome oxidase CYP450 superfamilies (CYP1A1/2,1B1,
2A6,2B6,2C8,2C9,2C19,2D6,2E1,3A4/5/7), acetaldehyde dehydrogenase (ALDH), alcohol dehydrogenase (ADH), dihydro it is phonetic
Mutually the N- acetyltransferases (NAT1/2) of metabolism, uridine triphosphate glucuronyl- turn pyridine dehydrogenase (DPD) etc. with participating in II
Move enzyme (UGT), thiopurine methyltransferase (TPMT) etc..Wherein cytochrome oxidase CYP450 is one group of 26S Proteasome Structure and Function phase
The isodynamic enzyme of the superfamily gene code of pass, the metabolism of most of endogenous materials (such as aliphatic acid, vitamin, cholic acid) is participated in,
The removing toxic substances of exogenous material (such as medicine), the activation of procarcinogen (such as aromatic substance), is played important in drug metabolism
Effect.
The genetic polymorphism of many drug metabolic enzymes has significant functional meaning, causes metabolism of the crowd to its substrate to go out
Now significant individual difference, Common drugs metabolic enzyme and metabolic drug, referring to table 1.
The metabolism substrate of the important CYP450 enzymes of table 1
The genetic polymorphism of drug metabolic enzyme can cause the pharmacotoxicological effect of the medicine as its substrate to strengthen or extend；Hair
Raw adverse drug reaction or adverse reaction aggravate；Prodrug metabolism can not be made to generate activated product and produce pharmacological action, medicine has
Imitating dosage reduces；Medicine improves via the metabolic rate compensatory of other metabolic pathways；Drug interaction strengthens.
Wherein, CYP2C19, CYP2C9, CYP3A4, CYP2D6 and CYP3A5 gene that the present invention studies have obvious lose
Polymorphism is passed, important middle effect is played in drug metabolism, is related to three major types children's common drug：That is children's antipyretic-antalgic class
Medicine, children's digestive system similar drug, children's classes of anti-infective medicine (referring to table 2).Therefore, by CYP2C19*2,
The genetic polymorphism detection in CYP2C19*3, CYP3A4*1G, CYP2C9*3, CYP2D6*10 and CYP3A5*3 site, it can carry out
The auxiliary of clinical application scheme, foundation, the adverse reaction of prophylactic agent etc. are provided for clinical children's safety medication.
The children medicine related to CYP450 enzymes of table 2
At present frequently with genetic polymorphism detection technology mainly include PCR sequencing PCR and fluorescence method.It is numerous operation to be present in PCR sequencing PCR
The trivial, shortcoming such as time-consuming, sensitivity is low, limits its quick and precisely examination in clinical labororatory.The purpose of the present invention is to carry
Have for one kind and improve Quality Control, detection is fast and accurately used for CYP2C19*2/*3, CYP2C9*3, CYP2D6*10, CYP3A4*
The kit of 1G and CYP3A5*3 genetic polymorphism detections.The present invention uses TAQMAN allele technology and Fluorescence PCR assay
The detection kit being combined, suitable for clinical expansion and industrialization.
The content of the invention
In order to which clinical application adverse reaction easily occur in children when solving the diagnosis of traditional clinical experience medical mode and medication
Technical problem, the present invention provide it is a kind of it is simple to operate, detection is quick, accuracy is high and what specificity was good is used for children's safety medication
Primer sets, kit and its method for related gene detection.
The invention provides a kind of primer sets for the detection of children's safety medication related gene, including for CYP2C19*
2nd, the specific primer and probe of CYP2C19*3, CYP3A4*1G, CYP2C9*3, CYP2D6*10 and CYP3A5*3 gene loci,
It is as follows：
CYP2C19*2 forward primers：
5'-CTTAGATATGCAATAATTTTCCCACTATCA-3'(SEQ ID NO.1)；
CYP2C19*2 reverse primers：
5'-CCAAAATATCACTTTCCATAAAAGCAAGG-3'(SEQ ID NO.2)；
CYP2C19*3 forward primers：
5'-TGAATGAAAACATCAGGATTGTAAGC-3'(SEQ ID NO.3)；
CYP2C19*3 reverse primers：
5'-AAGTGGTTTCTCAGGAAGCAAAAA-3'(SEQ ID NO.4)；
CYP3A4*1G forward primers：
5'-GGTTTCACCTCCTCCCTCCTT-3'(SEQ ID NO.5)；
CYP3A4*1G reverse primers：
5'-GAAATTGATGCAGTTTTACCCAATAA-3'(SEQ ID NO.6)；
CYP2C9*3 forward primers：
5'-CCTGCATGCAAGACAGGAGC-3'(SEQ ID NO.7)；
CYP2C9*3 reverse primers：
5'-GCAGGCTGGTGGGGAGA-3'(SEQ ID NO.8)；
CYP2D6*10 forward primers：
5'-CCATTTGGTAGTGAGGCAGGTA-3'(SEQ ID NO.9)；
CYP2D6*10 reverse primers：
5'-TGGAAGTCCACATGCAGCAG-3'(SEQ ID NO.10)；
CYP3A5*3 forward primers：
5'-CTCTACTGTCATTTCTAACCATAATCTCTTTA-3'(SEQ ID NO.11)；
CYP3A5*3 reverse primers：
5'-CTTCATATGATGAAGGGTAATGTGGT-3'(SEQ ID NO.12)；
CYP2C19*2 wild-type probes：
5'FAM-ATTTCCCGGGAACC-MGB-3'(SEQ ID NO.13)；
CYP2C19*2 saltant type probes：
5'VIC-ATTTCCCAGGAACCCAT-MGB-3'(SEQ ID NO.14)；
CYP2C19*3 wild-type probes：
5'FAM-CCCCCTGGATCCAGGTA-MGB-3'(SEQ ID NO.15)；
CYP2C19*3 saltant type probes：
5'VIC-CCCCCTGAATCCAGGT-MGB-3'(SEQ ID NO.16)；
CYP3A4*1G saltant type probes：
5'FAM-TCCATGTATCATCCACT-MGB-3'(SEQ ID NO.17)；
CYP3A4*1G wild-type probes：
5'VIC-TCCATGTACCATCCAC-MGB-3'(SEQ ID NO.18)；
CYP2C9*3 wild-type probes：
5'FAM-TCCAGAGATACATTGAC-MGB-3'(SEQ ID NO.19)；
CYP2C9*3 saltant type probes：
5'VIC-TCCAGAGATACCTTGAC-MGB-3'(SEQ ID NO.20)；
CYP2D6*10 wild-type probes：
5'FAM-CTACCCACCAGGCC-MGB-3'(SEQ ID NO.21)；
CYP2D6*10 saltant type probes：
5'VIC-CACGCTACTCACCA-MGB-3'(SEQ ID NO.22)；
CYP3A5*3 saltant type probes：
5'FAM-TCTTTCAGTATCTCTTCC-MGB-3'(SEQ ID NO.23)；
CYP3A5*3 wild-type probes：
5'VIC-TCTTTCAATATCTCTTCC-MGB-3'(SEQ ID NO.24)。
Present invention also offers a kind of kit for the detection of children's safety medication related gene, including contain such as right
It is required that the PCR reaction solutions of the specific primer and probe described in 1, the PCR reaction solutions be respectively CYP2C19*2PCR reaction solutions,
CYP2C19*3PCR reaction solutions, CYP3A4*1G PCR reaction solutions, CYP2C9*3PCR reaction solutions, CYP2D6*10PCR reaction solutions and
CYP3A5*3PCR reaction solutions, wherein
CYP2C19*2PCR reaction solutions include：
CYP2C19*2 forward primers, CYP2C19*2 reverse primers, CYP2C19*2 wild-type probes and CYP2C19*2 dash forward
Modification probe；
CYP2C19*3PCR reaction solutions include：
CYP2C19*3 forward primers, CYP2C19*3 reverse primers, CYP2C19*3 wild-type probes and CYP2C19*3 dash forward
Modification probe；
CYP3A4*1G PCR reaction solutions include：
CYP3A4*1G forward primers, CYP3A4*1G reverse primers, CYP3A4*1G saltant types probe and CYP3A4*1G are wild
Raw type probe；
CYP2C9*3PCR reaction solutions include：
CYP2C9*3 forward primers, CYP2C9*3 reverse primers, CYP2C9*3 wild-type probes and CYP2C9*3 saltant types
Probe；
CYP2D6*10PCR reaction solutions include：
CYP2D6*10 forward primers, CYP2D6*10 reverse primers, CYP2D6*10 wild-type probes and CYP2D6*10 dash forward
Modification probe；
CYP3A5*3PCR reaction solutions include：
CYP3A5*3 forward primers, CYP3A5*3 reverse primers, CYP3A5*3 saltant types probe and CYP3A5*3 wild types
Probe；
Above-mentioned PCR reaction solutions also include PCR buffer, dNTP, HS Taq enzymes and nuclease-free water.
In a kind of preferred embodiment of the kit provided by the invention, the composition final concentration of the PCR reaction solutions
For：1 × PCR buffer, 0.1~0.3 μM of each specific primer and probe, 0.25mM dNTP, 1U HS Taq enzymes.
In a kind of preferred embodiment of the kit provided by the invention, in addition to positive reference substance one, the positive are right
According to product two and positive reference substance three；
The positive reference substance one be CYP2C19*2, CYP2C19*3, CYP3A4*1G, CYP2C9*3, CYP2D6*10 and
Six gene loci wild plasmid mixtures of CYP3A5*3；
The positive reference substance two be CYP2C19*2, CYP2C19*3, CYP3A4*1G, CYP2C9*3, CYP2D6*10 and
Six gene loci wild types of CYP3A5*3 press 1 with saltant type：Plasmid mixture of 1 quantity than heterozygosis；
The positive reference substance three be CYP2C19*2, CYP2C19*3, CYP3A4*1G, CYP2C9*3, CYP2D6*10 and
Six gene point mutation type plasmid mixtures of CYP3A5*3.
In a kind of preferred embodiment of the kit provided by the invention, in addition to blank control product, the blank
Reference substance is nuclease-free water.
Present invention also offers a kind of method for the detection of children's safety medication related gene, comprise the following steps：
Step 1：The DNA of sample extracting to be detected is taken, as pcr template；
Step 2：Kit as claimed in claim 5 is provided, takes out appropriate PCR reaction solutions, by the PCR reaction solutions
Mixed with the appropriate pcr template；
Step 3：Carry out pcr amplification reaction：95 DEG C of denaturation 5min；40cycles, 95 DEG C of 15sec, 60 DEG C of 30sec, collection
Fluorescence；
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, obtain the genotype of children's safety medication related gene.
Compared to prior art, primer sets, reagent provided by the present invention for the detection of children's safety medication related gene
The beneficial effect of box and method is：By designing the high primer of specificity and TAQMAN-MGB fluorescent detection probes and passing through inspection
The release and intensity for surveying fluorescence detect gene pleiomorphism, are reconfigured to the reliable reagent of easy to use and testing result in addition
Box, design scientific and reasonable PCR reaction systems so that the present invention has that simple to operate, detection is quick, accuracy is high and special
The characteristics of property is good, realizes the quick and Accurate Determining to children's safety medication related gene polymorphism, normal to reduce clinical children
See the adverse reaction of medication, reduce medical treatment cost, save social resources.
Brief description of the drawings
Fig. 1 is the PCR amplification curve diagrams of clinical sample CYP2C19*2 wild types；
Fig. 2 is the PCR amplification curve diagrams of clinical sample CYP2C19*2 heterozygous；
Fig. 3 is the PCR amplification curve diagrams of clinical sample CYP2C19*2 saltant types；
Fig. 4 is the PCR amplification curve diagrams of clinical sample CYP2C19*3 wild types；
Fig. 5 is the PCR amplification curve diagrams of clinical sample CYP2C19*3 heterozygous；
Fig. 6 is the PCR amplification curve diagrams of clinical sample CYP2C19*3 saltant types；
Fig. 7 is the PCR amplification curve diagrams of clinical sample CYP2C9*3 wild types；
Fig. 8 is the PCR amplification curve diagrams of clinical sample CYP2C9*3 heterozygous；
Fig. 9 is the PCR amplification curve diagrams of clinical sample CYP2C9*3 saltant types；
Figure 10 is the PCR amplification curve diagrams of clinical sample CYP2D6*10 wild types；
Figure 11 is the PCR amplification curve diagrams of clinical sample CYP2D6*10 heterozygous；
Figure 12 is the PCR amplification curve diagrams of clinical sample CYP2D6*10 saltant types；
Figure 13 is the PCR amplification curve diagrams of clinical sample CYP3A4*1G wild types；
Figure 14 is the PCR amplification curve diagrams of clinical sample CYP3A4*1G heterozygous；
Figure 15 is the PCR amplification curve diagrams of clinical sample CYP3A4*1G saltant types；
Figure 16 is the PCR amplification curve diagrams of clinical sample CYP3A5*3 wild types；
Figure 17 is the PCR amplification curve diagrams of clinical sample CYP3A5*3 heterozygous；
Figure 18 is the PCR amplification curve diagrams of clinical sample CYP3A5*3 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 CYP2C19*2, CYP2C19*3, CYP3A4*1G, CYP2C9*3, CYP2D6*10 in human genome and
CYP3A5*3 gene locis (sequence is referring to mankind's whole genome sequence disclosed in ncbi database), use Primer Premier
3.0 and Methyl Primer Express v1.0 softwares, separately design the probe of specific primer and MGB marks.
Specific primer and probe sequence, it is as shown in the table：
2nd, reference substance selects
Positive reference substance one be CYP2C19*2, CYP2C19*3, CYP3A4*1G, CYP2C9*3, CYP2D6*10 and
Six gene loci wild plasmid mixtures of CYP3A5*3；
Positive reference substance two be CYP2C19*2, CYP2C19*3, CYP3A4*1G, CYP2C9*3, CYP2D6*10 and
Six gene loci wild types of CYP3A5*3 press 1 with saltant type：Plasmid mixture of 1 quantity than heterozygosis；
Positive reference substance three be CYP2C19*2, CYP2C19*3, CYP3A4*1G, CYP2C9*3, CYP2D6*10 and
Six gene point mutation type plasmid mixtures of CYP3A5*3 '；
Blank control product are nuclease-free water.
3rd, PCR reaction solutions form
Including the PCR reaction solutions containing above-mentioned specific primer and probe, the PCR reaction solutions are respectively CYP2C19*
2PCR reaction solutions, CYP2C19*3PCR reaction solutions, CYP3A4*1G PCR reaction solutions, CYP2C9*3PCR reaction solutions, CYP2D6*
10PCR reaction solutions and CYP3A5*3PCR reaction solutions, wherein
CYP2C19*2PCR reaction solutions include：
CYP2C19*2 forward primers, CYP2C19*2 reverse primers, CYP2C19*2 wild-type probes and CYP2C19*2 dash forward
Modification probe；
CYP2C19*3PCR reaction solutions include：
CYP2C19*3 forward primers, CYP2C19*3 reverse primers, CYP2C19*3 wild-type probes and CYP2C19*3 dash forward
Modification probe；
CYP3A4*1G PCR reaction solutions include：
CYP3A4*1G forward primers, CYP3A4*1G reverse primers, CYP3A4*1G saltant types probe and CYP3A4*1G are wild
Raw type probe；
CYP2C9*3PCR reaction solutions include：
CYP2C9*3 forward primers, CYP2C9*3 reverse primers, CYP2C9*3 wild-type probes and CYP2C9*3 saltant types
Probe；
CYP2D6*10PCR reaction solutions include：
CYP2D6*10 forward primers, CYP2D6*10 reverse primers, CYP2D6*10 wild-type probes and CYP2D6*10 dash forward
Modification probe；
CYP3A5*3PCR reaction solutions include：
CYP3A5*3 forward primers, CYP3A5*3 reverse primers, CYP3A5*3 saltant types probe and CYP3A5*3 wild types
Probe；
Above-mentioned PCR reaction solutions also include PCR buffer, dNTP, HS Taq enzymes and nuclease-free water；
Wherein, it is (precious to be purchased from the precious biology in Dalian for 10 × PCR buffer, dNTP, HS Taq enzyme and nuclease-free water
Biological (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) it is to be directed to general T aq archaeal dna polymerase high sensitivities, the situation of non-specific band is also easy to produce, specially
The thermophilic archaeal dna polymerase product of high specific that door is developed,.
The PCR reaction solutions composition it is final concentration of：
1×PCR buffer
0.2 μM of each specific primer and probe
1U HS Taq enzymes
0.25mM (mmol/L) dNTP
System (is supplemented to 18.5ul) by nuclease free appropriate amount of water.
Embodiment 2：The method that the detection of children's safety medication related gene is carried out using mentioned reagent box
First, biological specimen
Biomaterial of the present invention is all from the refined medical test institute in Hunan.For during the 6-12 months in 2016 in the refined doctor in Hunan
Learn 500 remaining crowd's anticoagulations of inspection institute's detection.
2nd, the DNA of sample extracting to be detected is taken, as pcr template：
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, vortex oscillation 10s, 65 DEG C of water-bath 15min, is during which shaken
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, adds 700uL washing lotions II, 10,000rpm, 1min, centrifugation.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；Adsorption column is abandoned, 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, take out appropriate PCR reaction solutions, by the PCR reaction solutions and the appropriate pcr template
Mix：
Connecting leg (PCR pipe number=the sample number+1 per detection site of requirement PCR reaction solutions 8 is taken out from kit
+ 3 positive controls of blank control).
After PCR reaction solution room temperatures are melted, brief centrifugation, lid is opened.
1.5 μ L are taken to add in PCR reaction solutions from sample DNA to be checked (or reference substance).
After vibration mixes, brief centrifugation 10s, amplification region is moved it to.
4th, pcr amplification reaction is carried out：95 DEG C of denaturation 5min；40cycles, 95 DEG C of 15sec, 60 DEG C of 30sec, collection are glimmering
Light.
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 20ul；
PCR reaction conditions are as shown in the table：
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, obtain the genotype of children's safety medication related gene.
As a result availability deciding, it is as shown in the table：
Blank control result is negative (No Ct or Ct value >=38).
PCR result judgements, it is as shown in the table：
Based on each gene loci testing result of clinical sample of the present invention, referring to Fig. 1-Figure 18, wherein Fig. 1 is clinical sample
The PCR amplification curve diagrams of CYP2C19*2 wild types；Fig. 2 is the PCR amplification curve diagrams of clinical sample CYP2C19*2 heterozygous；Figure
3 be the PCR amplification curve diagrams of clinical sample CYP2C19*2 saltant types；Fig. 4 is that the PCR of clinical sample CYP2C19*3 wild types expands
Increase curve map；Fig. 5 is the PCR amplification curve diagrams of clinical sample CYP2C19*3 heterozygous；Fig. 6 is that clinical sample CYP2C19*3 dashes forward
The PCR amplification curve diagrams of modification；Fig. 7 is the PCR amplification curve diagrams of clinical sample CYP2C9*3 wild types；Fig. 8 is clinical sample
The PCR amplification curve diagrams of CYP2C9*3 heterozygous；Fig. 9 is the PCR amplification curve diagrams of clinical sample CYP2C9*3 saltant types；Figure 10
For the PCR amplification curve diagrams of clinical sample CYP2D6*10 wild types；Figure 11 is that the PCR of clinical sample CYP2D6*10 heterozygous expands
Increase curve map；Figure 12 is the PCR amplification curve diagrams of clinical sample CYP2D6*10 saltant types；Figure 13 is clinical sample CYP3A4*1G
The PCR amplification curve diagrams of wild type；Figure 14 is the PCR amplification curve diagrams of clinical sample CYP3A4*1G heterozygous；Figure 15 is clinic
The PCR amplification curve diagrams of sample CYP3A4*1G saltant types；Figure 16 is the PCR amplification curves of clinical sample CYP3A5*3 wild types
Figure；Figure 17 is the PCR amplification curve diagrams of clinical sample CYP3A5*3 heterozygous；Figure 18 is clinical sample CYP3A5*3 saltant types
PCR amplification curve diagrams.
Provided by the present invention for the beneficial effect of the primer sets of children's safety medication related gene detection, kit and method
Fruit is：By designing the high primer of specificity and TAQMAN-MGB fluorescent detection probes and by detecting the release of fluorescence and strong
Spend to detect gene pleiomorphism, be reconfigured to the reliable kit of easy to use and testing result in addition, design scientific and reasonable
PCR reaction systems so that the present invention has the characteristics of simple to operate, detection is quick, accuracy is high and specificity is good, realizes pair
The quick and Accurate Determining of children's safety medication related gene polymorphism, to reduce the adverse reaction of the common medication of clinical children,
Medical treatment cost is reduced, saves social resources.
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 table
<110>Han Linzhi
<120>Primer sets, kit and method for the detection of children's safety medication related gene
<130> 2017
<160> 24
<170> SIPOSequenceListing 1.0
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<211> 30
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<213>Artificial sequence (Artificial Sequence)
<400> 1
cttagatatg caataatttt cccactatca 30
<210> 2
<211> 29
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
ccaaaatatc actttccata aaagcaagg 29
<210> 3
<211> 26
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
tgaatgaaaa catcaggatt gtaagc 26
<210> 4
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
aagtggtttc tcaggaagca aaaa 24
<210> 5
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
ggtttcacct cctccctcct t 21
<210> 6
<211> 26
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 6
gaaattgatg cagttttacc caataa 26
<210> 7
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 7
cctgcatgca agacaggagc 20
<210> 8
<211> 17
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 8
gcaggctggt ggggaga 17
<210> 9
<211> 22
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 9
ccatttggta gtgaggcagg ta 22
<210> 10
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 10
tggaagtcca catgcagcag 20
<210> 11
<211> 32
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 11
ctctactgtc atttctaacc ataatctctt ta 32
<210> 12
<211> 26
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 12
cttcatatga tgaagggtaa tgtggt 26
<210> 13
<211> 14
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 13
atttcccggg aacc 14
<210> 14
<211> 17
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 14
atttcccagg aacccat 17
<210> 15
<211> 17
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 15
ccccctggat ccaggta 17
<210> 16
<211> 16
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 16
ccccctgaat ccaggt 16
<210> 17
<211> 17
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 17
tccatgtatc atccact 17
<210> 18
<211> 16
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 18
tccatgtacc atccac 16
<210> 19
<211> 17
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 19
tccagagata cattgac 17
<210> 20
<211> 17
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 20
tccagagata ccttgac 17
<210> 21
<211> 14
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 21
ctacccacca ggcc 14
<210> 22
<211> 14
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 22
cacgctactc acca 14
<210> 23
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 23
tctttcagta tctcttcc 18
<210> 24
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 24
tctttcaata tctcttcc 18
Claims (6)
1. it is a kind of for children's safety medication related gene detection primer sets, it is characterised in that including for CYP2C19*2,
The specific primer and probe of CYP2C19*3, CYP3A4*1G, CYP2C9*3, CYP2D6*10 and CYP3A5*3 gene loci, such as
Under：
CYP2C19*2 forward primers：
5′-CTTAGATATGCAATAATTTTCCCACTATCA-3′；
CYP2C19*2 reverse primers：
5′-CCAAAATATCACTTTCCATAAAAGCAAGG-3′；
CYP2C19*3 forward primers：
5′-TGAATGAAAACATCAGGATTGTAAGC-3′；
CYP2C19*3 reverse primers：
5′-AAGTGGTTTCTCAGGAAGCAAAAA-3′；
CYP3A4*1G forward primers：
5′-GGTTTCACCTCCTCCCTCCTT-3′；
CYP3A4*1G reverse primers：
5′-GAAATTGATGCAGTTTTACCCAATAA-3′；
CYP2C9*3 forward primers：
5′-CCTGCATGCAAGACAGGAGC-3′；
CYP2C9*3 reverse primers：
5′-GCAGGCTGGTGGGGAGA-3′；
CYP2D6*10 forward primers：
5′-CCATTTGGTAGTGAGGCAGGTA-3′；
CYP2D6*10 reverse primers：
5′-TGGAAGTCCACATGCAGCAG-3′；
CYP3A5*3 forward primers：
5′-CTCTACTGTCATTTCTAACCATAATCTCTTTA-3′；
CYP3A5*3 reverse primers：
5′-CTTCATATGATGAAGGGTAATGTGGT-3′；
CYP2C19*2 wild-type probes：
5′FAM-ATTTCCCGGGAACC-MGB-3′；
CYP2C19*2 saltant type probes：
5′VIC-ATTTCCCAGGAACCCAT-MGB-3′；
CYP2C19*3 wild-type probes：
5′FAM-CCCCCTGGATCCAGGTA-MGB-3′；
CYP2C19*3 saltant type probes：
5′VIC-CCCCCTGAATCCAGGT-MGB-3′；
CYP3A4*1G saltant type probes：
5′FAM-TCCATGTATCATCCACT-MGB-3′；
CYP3A4*1G wild-type probes：
5′VIC-TCCATGTACCATCCAC-MGB-3′；
CYP2C9*3 wild-type probes：
5′FAM-TCCAGAGATACATTGAC-MGB-3′；
CYP2C9*3 saltant type probes：
5′VIC-TCCAGAGATACCTTGAC-MGB-3′；
CYP2D6*10 wild-type probes：
5′FAM-CTACCCACCAGGCC-MGB-3′；
CYP2D6*10 saltant type probes：
5′VIC-CACGCTACTCACCA-MGB-3′；
CYP3A5*3 saltant type probes：
5′FAM-TCTTTCAGTATCTCTTCC-MGB-3′；
CYP3A5*3 wild-type probes：
5′VIC-TCTTTCAATATCTCTTCC-MGB-3′。
2. a kind of kit for the detection of children's safety medication related gene, it is characterised in that including containing such as claim 1
Described specific primer and the PCR reaction solutions of probe, the PCR reaction solutions be respectively CYP2C19*2 PCR reaction solutions,
CYP2C19*3 PCR reaction solutions, CYP3A4*1G PCR reaction solutions, CYP2C9*3 PCR reaction solutions, CYP2D6*10 PCR reactions
Liquid and CYP3A5*3 PCR reaction solutions, wherein
CYP2C19*2 PCR reaction solutions include：
CYP2C19*2 forward primers, CYP2C19*2 reverse primers, CYP2C19*2 wild-type probes and CYP2C19*2 saltant types
Probe；
CYP2C19*3PCR reaction solutions include：
CYP2C19*3 forward primers, CYP2C19*3 reverse primers, CYP2C19*3 wild-type probes and CYP2C19*3 saltant types
Probe；
CYP3A4*1G PCR reaction solutions include：
CYP3A4*1G forward primers, CYP3A4*1G reverse primers, CYP3A4*1G saltant types probe and CYP3A4*1G wild types
Probe；
CYP2C9*3 PCR reaction solutions include：
CYP2C9*3 forward primers, CYP2C9*3 reverse primers, CYP2C9*3 wild-type probes and CYP2C9*3 saltant type probes；
CYP2D6*10 PCR reaction solutions include：
CYP2D6*10 forward primers, CYP2D6*10 reverse primers, CYP2D6*10 wild-type probes and CYP2D6*10 saltant types
Probe；
CYP3A5*3 PCR reaction solutions include：
CYP3A5*3 forward primers, CYP3A5*3 reverse primers, CYP3A5*3 saltant types probe and CYP3A5*3 wild-type probes；
Above-mentioned PCR reaction solutions also include PCR buffer, dNTP, HS Taq enzymes and nuclease-free water.
3. the kit according to claim 2 for the detection of children's safety medication related gene, it is characterised in that described
The composition of PCR reaction solutions is final concentration of：1 × PCR buffer, 0.1 μM -0.3 μM of each specific primer and probe, 0.25mM
DNTP, 1U HS Taq enzymes.
4. according to any described kit for being used for children's safety medication related gene and detecting, its feature in claim 2-3
It is, in addition to positive reference substance one, positive reference substance two and positive reference substance three；
The positive reference substance one be CYP2C19*2, CYP2C19*3, CYP3A4*1G, CYP2C9*3, CYP2D6*10 and
Six gene loci wild plasmid mixtures of CYP3A5*3；
The positive reference substance two be CYP2C19*2, CYP2C19*3, CYP3A4*1G, CYP2C9*3, CYP2D6*10 and
Six gene loci wild types of CYP3A5*3 press 1 with saltant type：Plasmid mixture of 1 quantity than heterozygosis；
The positive reference substance three be CYP2C19*2, CYP2C19*3, CYP3A4*1G, CYP2C9*3, CYP2D6*10 and
Six gene point mutation type plasmid mixtures of CYP3A5*3.
5. the kit according to claim 4 for the detection of children's safety medication related gene, it is characterised in that also wrap
Blank control product are included, the blank control product are nuclease-free water.
A kind of 6. method for the detection of children's safety medication related gene, it is characterised in that comprise the following steps：
Step 1：The DNA of sample extracting to be detected is taken, as pcr template；
Step 2：Kit as claimed in claim 5 is provided, takes out appropriate PCR reaction solutions, by the PCR reaction solutions with fitting
The pcr template is measured to mix；
Step 3：Carry out pcr amplification reaction：95 DEG C of denaturation 5min；40cycles, 95 DEG C of 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, obtain the genotype of children's safety medication related gene.
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| CN110157798A (en) * | 2019-06-27 | 2019-08-23 | 中南大学湘雅医院 | A kind of primer combination of probe instructing diabetic individual medication gene loci polymorphism and its application and kit |
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| CN110931080A (en) * | 2019-12-12 | 2020-03-27 | 爱尔生基因医学科技有限公司 | Gene chip information processing system and method for detecting safe medication of children |
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| CN110157798A (en) * | 2019-06-27 | 2019-08-23 | 中南大学湘雅医院 | A kind of primer combination of probe instructing diabetic individual medication gene loci polymorphism and its application and kit |
| CN110157799A (en) * | 2019-06-27 | 2019-08-23 | 中南大学 | Primed probe group, kit and method for instructing diabetic individual medication related gene polymorphism to detect |
| CN110157798B (en) * | 2019-06-27 | 2023-04-18 | 中南大学湘雅医院 | Primer probe combination for guiding polymorphism of gene locus of diabetes personalized medicine, application and kit thereof |
| CN110931080A (en) * | 2019-12-12 | 2020-03-27 | 爱尔生基因医学科技有限公司 | Gene chip information processing system and method for detecting safe medication of children |
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Application publication date: 20171229 |