CN109182474A - A kind of CYP2D6 detection in Gene Mutation primer and kit - Google Patents
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Abstract
The invention discloses a kind of CYP2D6 detection in Gene Mutation primers, including the upstream primer as shown in SEQ ID No.1, probe shown in downstream primer and SEQ ID No.3 shown in SEQ ID No.2.The invention also discloses the detection primers to prepare purposes and a kind of CYP2D6 gene mutation detection kit in CYP2D6 detection in Gene Mutation reagent.Detection primer provided by the invention may be implemented in same pipe PCR reaction while detecting wild type, heterozygous mutant and the homozygous mutation in the site CYP2D6*10 (rs1065852 C100T), have many advantages, such as that easy to operate, high sensitivity, specificity are high and at low cost.
Description
Technical field
The invention belongs to detection in Gene Mutation technical fields, and in particular to a kind of CYP2D6 detection in Gene Mutation primer and examination
Agent box.
Background technique
Cytochrome P450 (CYP450) enzyme system belongs to monooxygenase, is a kind of using ferroheme as the b race cell color of prothetic group
Plain superfamily gene metabolic enzyme is named because the absorption peak of its reduction-state is at 450nm.It is catalyzed many and drug metabolism and conjunction
At the related reaction of glutathione (Cho).
CYP2D6 is a member in CYP450 family, is a kind of enzyme that important participation exogenous material is metabolized in vivo,
It is mainly distributed on liver, accounts for the 4% of liver CYP450 enzyme total amount, participates in being metabolized clinically 25% common drug, is at first
It is found to have the drug metabolic enzyme of gene pleiomorphism.CYP2D6 gene is the gene with complete function, coding
The gene of CYP2D6 is located at 22q13.2, chain with CYP2D7 and CYP2D8P pseudogene.CYP2D6 gene overall length is 7kb, contains 9
A exon and 8 intrones, 1 491bp of encoding base sequences overall length encode 497 amino acid, relative molecular mass 51
000.The main reason for CYP2D6 gene mutates is that the base of encoding gene is subjected to displacement, and is inserted into or lacks and makes coding
Sequence changes, so that enzymatic activity and quantity be made to change, and individual is caused to have notable difference to the metabolism of drug.
Research confirms that CYP2D6*10 (rs1065852C100T) mutation is the main variants of asian population, and frequency occurs
Rate be 38%~70%, also, to metastatic breast cancer the study found that have the homozygous patient of CYP2D6*10/*10 with
Other CYP2D6 genotype are compared, and tamoxifen (TAM) active metabolite concentration significantly reduces in blood plasma.Clinically CYP2D6*
10/*10 genotype patient is not suitable for treating using TAM, or biggish dosage is needed just to can achieve effective blood medicine
Concentration, but this may cause more serious adverse reaction, increase the risk that patient uses drug, accordingly, it is determined that CYP2D6 base
Because type has directive significance to patient with breast cancer's medication.
Summary of the invention
Based on this, a kind of CYP2D6 gene is provided it is an object of the invention to overcome above-mentioned the deficiencies in the prior art place
Abrupt climatic change primer has easy to operate, high sensitivity, specificity high and cost when being used for CYP2D6 detection in Gene Mutation
Low advantage.
To achieve the above object, the technical solution adopted by the present invention are as follows: a kind of CYP2D6 detection in Gene Mutation primer, including
The upstream primer as shown in SEQ ID No.1, probe shown in downstream primer and SEQ ID No.3 shown in SEQ ID No.2；
The end of probe 5 ' is connected with fluorescent reporter group, and 3 ' ends are connected with fluorescent quenching group.
SEQ ID No.1:5 '-TTGGTAGTGAGGCAGGTAT-3 '；
SEQ ID No.2:5 '-TGGTCGAAGCAGTATGGT-3 '；
SEQ ID No.3:5 '-GCTGGGCTGCACGCTACCCACCAGGCCCCCTGCCA
CTG-3’。
Preferably, the fluorescent reporter group is ROX, and the fluorescent quenching group is BHQ2.
Preferably, the work of the detection primer is final concentration of: upstream primer 0.025- shown in SEQ ID No.1
Probe shown in downstream primer 0.25-1.5umol/L and SEQ ID No.3 shown in 0.15umol/L, SEQ ID No.2
0.15-0.3umol/L。
The present invention also provides the detection primers to prepare the purposes in CYP2D6 detection in Gene Mutation reagent.
Detection primer of the present invention can be used for preparing the reagent of detection CYP2D6 gene mutation, while realize CYP2D6*10
(rs1065852C100T) detection of site wild type, heterozygous mutant and homozygous mutation has patient with breast cancer's medication important
Directive significance.
The present invention also provides a kind of CYP2D6 gene mutation detection kits, include the detection primer.
Preferably, the detection kit can also include following components: PCR buffer, Mg2+, dNTPs and DNA polymerization
Enzyme；The PCR buffer includes Tris-HCl and KCl.
Preferably, the pH value of the PCR buffer is 8.0-9.0.
Preferably, the Mg2+For MgCl2。
The dNTPs includes dATP, dGTP, dTTP and dCTP, and the concentration phase of described dATP, dGTP, dTTP and dCTP
Together.
Preferably, the kit is final concentration of when each component reaction when for detecting: Tris-HCl 2-
20mmol/L、KCl 10-100nmol/L、Mg2+1.875-4.375mmol/L, dNTPs 0.125-0.4375mmol/L, upstream
Primer 0.025-0.15umol/L, downstream primer 0.25-1.5umol/L, probe 0.15-0.3umol/L and archaeal dna polymerase
0.025-0.125U/ul。
Preferably, the kit is final concentration of when each component reaction when for detecting: Tris-HCl 10mmol/
L、KCl 50nmol/L、Mg2+2.5mmol/L, dNTPs 0.3125mmol/L, upstream primer 0.125umol/L, downstream primer
1.25umol/L, probe 0.15umol/L and archaeal dna polymerase 0.05U/ul.
Preferably, the detection program of the kit are as follows: 95 DEG C of 3min；10 circulations: 95 DEG C of 15s, 62 DEG C of 30s, 72 DEG C
30s；40 circulations: 95 DEG C of 15s, 54 DEG C of 30s (collecting fluorescence), 72 DEG C of 30s；1 circulation: 95 DEG C of 3min, 55 DEG C of 2min；1
Circulation: 55 DEG C of 1min, 85 DEG C of 15s (collecting fluorescence, 0.06 DEG C/s of heating rate).
Preferably, result judgment criteria of kit when for detecting are as follows:
(1) CYP2D6 gene wild type: Ct value is less than 30；Tm value: 74.48 ± 0.68 DEG C, unimodal；
(2) CYP2D6 genetic heterozygosis saltant type: Ct value is less than 30；Tm value: (66.88 ± 0.75 DEG C)/(74.48 ± 0.68
DEG C), it is bimodal；
(3) CYP2D6 homozygous mutation type: Ct value is less than 30；Tm value: 66.88 ± 0.75 DEG C, unimodal.
Compared with the existing technology, the invention has the benefit that the present invention uses Fluorescence PCR assay, asymmetric PCR technology
And melting curve analysis technology is, it can be achieved that detect CYP2D6*10 (rs1065852 C100T) simultaneously in same pipe PCR reaction
Site wild type, heterozygous mutant and homozygous mutation have many advantages, such as that easy to operate, high sensitivity, specificity are high and at low cost, right
Patient with breast cancer's medication has important directive significance.
Detailed description of the invention
Fig. 1 is testing result figure of the kit of the present invention to No. 1 sample CYP2D6 gene；
Fig. 2 is the sequencing result figure of No. 1 sample CYP2D6 gene；
Fig. 3 is testing result figure of the kit of the present invention to No. 2 sample CYP2D6 genes；
Fig. 4 is the sequencing result figure of No. 2 sample CYP2D6 genes；
Fig. 5 is testing result figure of the kit of the present invention to No. 3 sample CYP2D6 genes；
Fig. 6 is the sequencing result figure of No. 3 sample CYP2D6 genes.
Specific embodiment
It to facilitate the understanding of the present invention, below will be to invention is more fully described.But the present invention can be to be permitted
Mostly different form is realized, however it is not limited to embodiment described herein.On the contrary, purpose of providing these embodiments is makes
It is more thorough and comprehensive to the understanding of the disclosure.
Embodiment 1
A kind of embodiment of CYP2D6 gene mutation detection kit of the present invention, on as shown in SEQ ID No.1
Swim probe shown in downstream primer shown in primer, SEQ ID No.2 and SEQ ID No.3；The end of probe 5 ' is connected with glimmering
Light reporter group ROX, 3 ' ends are connected with fluorescent quenching group BHQ2.
SEQ ID No.1:5 '-TTGGTAGTGAGGCAGGTAT-3 '；
SEQ ID No.2:5 '-TGGTCGAAGCAGTATGGT-3 '；
SEQ ID No.3:5 ' ROX-GCTGGGCTGCACGCTACCCACCAGGCCCCCTG
CCACTG-BHQ2 3’。
The primer is synthesized by biotech firm, and by specification is configured.
Embodiment 2
The present embodiment detects CYP2D6 gene using kit described in embodiment 1.
1, reaction system configures
Reaction system is configured by component shown in table 1 and component final concentration:
1 reaction system of table
The DNA profiling is the DNA extracted from patient peripheral's haemocyte.
2, response procedures
PCR response procedures are as shown in table 2:
2 response procedures of table
3, result judgment criteria
Kit of the present invention can be used for detecting the wild type in the site CYP2D6*10 (rs1065852 C100T), heterozygous mutant
And homozygous mutation.Testing result judgment criteria is as follows:
(1) CYP2D6 gene wild type: Ct value is less than 30；Tm value: 74.48 ± 0.68 DEG C, unimodal；
(2) CYP2D6 genetic heterozygosis saltant type: Ct value is less than 30；Tm value: (66.88 ± 0.75 DEG C)/(74.48 ± 0.68
DEG C), it is bimodal；
(3) CYP2D6 homozygous mutation type: Ct value is less than 30；Tm value: 66.88 ± 0.75 DEG C, unimodal.
4, testing result
3 clinical samples are detected, as a result as follows:
Amplification curve is smooth as shown in Figure 1: for No. 1 pattern detection result, is in " S " type；CT value is 15.62；Tm value: 74.33
DEG C, it is unimodal；It is judged as CYP2D6 gene wild type (CC), testing result and generation sequencing result meet (Fig. 2)；
No. 2 pattern detection results are as shown in Figure 3: amplification curve is smooth, is in " S " type；CT value is 15.76；Tm value: 66.68
DEG C/74.34 DEG C, it is bimodal；It is judged as CYP2D6 genetic heterozygosis saltant type (CT), testing result and generation sequencing result meet (figure
4)；
No. 3 pattern detection results are as shown in Figure 5: amplification curve is smooth, is in " S " type；CT value is 15.96；Tm value: 66.93
DEG C, it is unimodal；It is judged as that CYP2D6 homozygous mutation type (TT), testing result and generation sequencing result meet (Fig. 6).
Embodiment 3
The present embodiment detects the CYP2D6 gene of 15 clinical samples.Person under test's peripheral blood sample is taken, DNA is extracted
It afterwards using kit described in embodiment 1, is detected according to detection method described in embodiment 2, while generation survey is carried out to sample
Sequence.Testing result is as shown in table 3:
3 testing result of table
The above results show that kit of the present invention has good accuracy, detection when for CYP2D6 genetic test
As a result completely the same with sequencing result.
4 Mg of table2+Final concentration optimizing reaction system
The response procedures of 5 reaction system optimization process of table
Embodiment 4
The present embodiment studies Mg in reaction system2+Influence of the final concentration to testing result.
The final concentration of PCR reaction system other components carries out different Mg with table 5 with table 4, response procedures2+The inspection of final concentration
It surveys.Following Mg is respectively configured2+The reaction system of final concentration: 1.875mmol/L, 2.5mmol/L, 3.125mmol/L,
3.75mmol/L and 4.375mmol/L.The result shows that working as Mg2+Final concentration in the reaction system is in 1.875~4.375mmol/
Detection is able to achieve when L, wherein work as Mg2+When final concentration of 2.5mmol/L in the reaction system, testing result is best.
Embodiment 5
The present embodiment studies influence of the dNTPs final concentration to testing result in reaction system.
The final concentration of PCR reaction system other components carries out different dNTPs final concentrations with table 5 with table 6, response procedures
Detection.Be respectively configured the reaction system of following dNTPs final concentration: 0.125mmol/L, 0.1875mmol/L, 0.25mmol/L,
0.3125mmol/L, 0.375mmol/L and 0.4375mmol/L.The result shows that when the final concentration of dNTPs in the reaction system exists
Detection is able to achieve when 0.125~0.4375mmol/L, wherein when dNTPs in the reaction system final concentration of
When 0.3125mmol/L, testing result is best.
6 dNTPs final concentration optimizing reaction system of table
7 upstream and downstream primer ratio optimization reaction system of table
Embodiment 6
The present embodiment studies influence of the upstream and downstream primer ratio to testing result in reaction system.
It is whole to carry out different upstream and downstream primers with table 5 with table 7, response procedures for the final concentration of PCR reaction system other components
The detection of concentration ratio.The reaction system (upstream primer: downstream primer) of following upstream and downstream primer ratio is respectively configured: (0.5:
0.5) umol/L, (0.25:0.5) umol/L, (0.167:0.5) umol/L, (0.125:0.5) umol/L, (0.1:0.5)
Umol/L, (0.05:0.5) umol/L, (0.025:0.5) umol/L and (0.0125:0.5) umol/L.The result shows that when it is upper,
The final concentration of downstream primer in the reaction system is able to achieve detection in (0.1:0.5)-(0.025:0.5) umol/L, wherein
As upstream and downstream primer final concentration of (0.05:0.5) umol/L in the reaction system, testing result is best.
Embodiment 7
The present embodiment studies influence of the upstream and downstream primer final concentration to testing result in reaction system.
It is whole to carry out different upstream and downstream primers with table 5 with table 8, response procedures for the final concentration of PCR reaction system other components
The detection of concentration.The reaction system (upstream primer: downstream primer) of following upstream and downstream primer final concentration is respectively configured: (0.025:
0.25) umol/L, (0.05:0.5) umol/L, (0.075:0.75) umol/L, (0.1:1.0) umol/L, (0.125:1.25)
Umol/L and (0.15:1.5) umol/L.The result shows that when the final concentration of upstream and downstream primer in the reaction system (0.025:
0.25) detection-(0.15:1.5) is able to achieve when umol/L, wherein when upstream and downstream primer in the reaction system final concentration of
When (0.1:1.0) umol/L, testing result is best.
Concentration optimization reaction system in 8 upstream and downstream primer of table
Embodiment 8
The present embodiment studies influence of the reaction system middle probe final concentration to testing result.
The final concentration of PCR reaction system other components carries out the inspection of different probe final concentration with table 5 with table 9, response procedures
It surveys.The reaction system of following probe final concentration: 0.05umol/L, 0.1umol/L, 0.15umol/L, 0.2umol/ is respectively configured
L, 0.25umol/L and 0.3umol/L.The result shows that when the final concentration of probe in the reaction system is in 0.15-0.3umol/L
It is able to achieve detection, wherein as probe final concentration of 0.2umol/L in the reaction system, testing result is best.
9 probe final concentration optimizing reaction system of table
Embodiment 9
The present embodiment studies upstream and downstream primer and influence of the probe final concentration ratio to testing result in reaction system.
The final concentration of PCR reaction system other components with table 10, response procedures with table 5, carry out different upstream and downstream primers with
The detection of probe final concentration ratio.The reaction system that following upstream and downstream primer and probe final concentration ratio is respectively configured (above has and draws
Object: downstream primer: probe): (0.1:1.0:0.15) umol/L, (0.1:1.0:0.2) umol/L, (0.125:1.25:0.15)
Umol/L, (0.125:1.25:0.2) umol/L, (0.15:1.5:0.15) umol/L and (0.15:1.5:0.2) umol/L.Knot
Fruit shows the inspection when upstream and downstream primer in reaction system and probe final concentration ratio are (0.125:1.25:0.15) umol/L
It is best to survey result.
10 upstream and downstream primer of table and probe final concentration optimizing reaction system
11 archaeal dna polymerase final concentration optimizing reaction system of table
Embodiment 10
The present embodiment studies influence of the archaeal dna polymerase final concentration to testing result in reaction system.
It is dense eventually to carry out different archaeal dna polymerases with table 5 with table 11, response procedures for the final concentration of PCR reaction system other components
The detection of degree.Be respectively configured the reaction system of following archaeal dna polymerase final concentration: 0.025U/ul, 0.05U/ul, 0.075U/ul,
0.1U/ul and 0.125U/ul.The result shows that when the final concentration of archaeal dna polymerase in the reaction system is in 0.025-0.125U/ul
Shi Jun is able to achieve detection, wherein as archaeal dna polymerase final concentration of 0.05U/ul in the reaction system, testing result is best.
Embodiment 11
The present embodiment studies influence of step 2 annealing temperature to testing result in response procedures.
With table 5, reaction system carries out different step 2 in response procedures and anneals temperature other steps of PCR response procedures with table 1
The detection of degree.It is respectively set the response procedures of 2 annealing temperature of following steps: 64 DEG C, 62 DEG C, 60 DEG C, 58 DEG C and 56 DEG C.As a result table
It is bright, detection is able to achieve when step 2 annealing temperature is at 56~64 DEG C, wherein when step 2 annealing temperature is 62 DEG C, detection
As a result best.
Embodiment 12
The present embodiment studies influence of step 3 annealing temperature to testing result in response procedures.
With table 12, reaction system carries out different step 3 in response procedures and anneals temperature other steps of PCR response procedures with table 1
The detection of degree.It is respectively set the response procedures of 3 annealing temperature of following steps: 51 DEG C, 52 DEG C, 53 DEG C, 54 DEG C and 55 DEG C.As a result table
It is bright, detection is able to achieve when step 3 annealing temperature is at 51~55 DEG C, wherein when step 3 annealing temperature is 54 DEG C, detection
As a result best.
Step 3 annealing temperature optimizes in 12 PCR response procedures of table
Embodiment 13
The present embodiment studies influence of step 4 annealing temperature to testing result in response procedures.
Other response procedures carry out the inspection of 4 annealing temperature of different step in response procedures with table 1 with table 13, reaction system
It surveys.It is respectively set the response procedures of 4 annealing temperature of following steps: 48 DEG C, 50 DEG C, 52 DEG C, 55 DEG C and 58 DEG C.The result shows that when
Step 4 annealing temperature is able to achieve detection at 48~58 DEG C, wherein when step 4 annealing temperature is 55 DEG C, testing result is most
It is good.
Step 4 annealing temperature optimizes in 13 PCR response procedures of table
Therefore, comprehensive analysis embodiment 4 is to embodiment 13 as a result, the optimal final concentration of each component of PCR reaction system
Such as table 1, PCR reacts optimization routines such as table 2.
In addition, the present invention is to judge genotype by the Tm value of solubility curve, thus whether Tm value is stable to this kit
Performance be the most key element.By optimizing experiment discovery above, fixed in 10*PCR Buffer (buffer) ingredient
Under the conditions of, influencing maximum three agent formulations to solubility curve Tm value in reaction system is respectively: magnesium ion, dNTPs and DNA
Polymerase.Therefore, magnesium ion, dNTPs and archaeal dna polymerase should be paid special attention to before preparation of reagents.Furthermore only operation error,
System amplification fails caused by the problems such as PCR ingredient is problematic or detecting instrument is faulty, otherwise amplification curve of the invention
S type curve can be amplified, i.e., amplification curve of the invention can be used as " internal reference " and no longer need one pipe of more increases
" internal reference " of family's gene as amplification system.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than protects to the present invention
The limitation of range is protected, although the invention is described in detail with reference to the preferred embodiments, those skilled in the art should
Understand, it can be with modification or equivalent replacement of the technical solution of the present invention are made, without departing from the essence of technical solution of the present invention
And range.
SEQUENCE LISTING
<110>Jiangmen city healthcare hospital for women & children
<120>a kind of CYP2D6 detection in Gene Mutation primer and kit
<130> 2018.8.23
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 19
<212> DNA
<213>artificial synthesized
<400> 1
ttggtagtga ggcaggtat 19
<210> 2
<211> 18
<212> DNA
<213>artificial synthesized
<400> 2
tggtcgaagc agtatggt 18
<210> 3
<211> 38
<212> DNA
<213>artificial synthesized
<400> 3
gctgggctgc acgctaccca ccaggccccc tgccactg 38
Claims (10)
1. a kind of CYP2D6 detection in Gene Mutation primer, which is characterized in that including the upstream primer as shown in SEQ ID No.1,
Probe shown in downstream primer shown in SEQ ID No.2 and SEQ ID No.3；
The end of probe 5 ' is connected with fluorescent reporter group, and 3 ' ends are connected with fluorescent quenching group.
2. detection primer according to claim 1, which is characterized in that the fluorescent reporter group is ROX, and the fluorescence is sudden
The group that goes out is BHQ2.
3. detection primer according to claim 1, which is characterized in that the work of the detection primer is final concentration of: SEQ
Downstream primer 0.25-1.5umol/L shown in upstream primer 0.025-0.15umol/L, SEQ ID No.2 shown in ID No.1
With probe 0.15-0.3umol/L shown in SEQ ID No.3.
4. described in any item detection primers are preparing the use in CYP2D6 detection in Gene Mutation reagent according to claim 1~3
On the way.
5. a kind of CYP2D6 gene mutation detection kit, which is characterized in that include the described in any item inspections of claims 1 to 3
Survey primer.
6. detection kit according to claim 5, which is characterized in that can also include following components: PCR buffer,
Mg2+, dNTPs and archaeal dna polymerase；The PCR buffer includes Tris-HCl and KCl.
7. detection kit according to claim 6, which is characterized in that final concentration of when each component reacts: Tris-
HCl 2-20mmol/L、KCl 10-100nmol/L、Mg2+1.875-4.375mmol/L、dNTPs 0.125-0.4375mmol/
L, upstream primer 0.025-0.15umol/L, downstream primer 0.25-1.5umol/L, probe 0.15-0.3umol/L and DNA polymerization
Enzyme 0.025-0.125U/ul.
8. detection kit according to claim 7, which is characterized in that final concentration of when each component reacts: Tris-
HCl 10mmol/L、KCl 50nmol/L、Mg2+2.5mmol/L, dNTPs 0.3125mmol/L, upstream primer 0.125umol/
L, downstream primer 1.25umol/L, probe 0.15umol/L and archaeal dna polymerase 0.05U/ul.
9. according to the described in any item detection kits of claim 5~8, which is characterized in that the detection program of the kit
Are as follows: 95 DEG C of 3min；10 circulations: 95 DEG C of 15s, 62 DEG C of 30s, 72 DEG C of 30s；40 circulations: 95 DEG C of 15s, 54 DEG C of 30s (are collected glimmering
Light), 72 DEG C of 30s；1 circulation: 95 DEG C of 3min, 55 DEG C of 2min；1 circulation: 55 DEG C of 1min, 90 DEG C of 15s (collect fluorescence, heating
0.06 DEG C/s of rate).
10. according to the described in any item detection kits of claim 5~8, which is characterized in that the kit is for detecting
When result judgment criteria are as follows:
(1) CYP2D6 gene wild type: Ct value is less than 30；Tm value: 74.48 ± 0.68 DEG C, unimodal；
(2) CYP2D6 genetic heterozygosis saltant type: Ct value is less than 30；Tm value: (66.88 ± 0.75 DEG C)/(74.48 ± 0.68 DEG C),
It is bimodal；
(3) CYP2D6 homozygous mutation type: Ct value is less than 30；Tm value: 66.88 ± 0.75 DEG C, unimodal.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112695097A (en) * | 2021-03-10 | 2021-04-23 | 厦门大学 | CYP2D6 x 10 genetic polymorphism detection kit for distinguishing CYP2D7P and CYP2D8P |
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