CN107841540B - A kind of CYP2C9 and/or VKORC1 gene pleiomorphisms quick detection kit - Google Patents
A kind of CYP2C9 and/or VKORC1 gene pleiomorphisms quick detection kit Download PDFInfo
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Abstract
The invention discloses a kind of kit of detection CYP2C9 and/or VKORC1 gene pleiomorphisms, the kit contains following component：Thermal starting taq archaeal dna polymerases, blood Direct PCR buffer solution (10X), primer pair, probe pair and UNG enzymes；Simply, efficiently, safely：Fluorescent PCR amplification can directly be carried out to blood sample, need not move through nucleic acid extraction and purifying, reduce the risk of template cross contamination, avoid the use of the harmful reagents such as phenol；High sensitivity：It can accurately detect down to 2 μ l blood samples, required sample size is few, and specificity is good：Special primer and typing probes are designed for CYP2C9C430T, CYP2C9A1075C and VKORC1G 1639A, it can the corresponding polymorphism of specific amplification identification.
Description
Technical field
The invention belongs to molecular biology fields, are related to medicine and biotechnology, relate particularly to a kind of CYP2C9 and/
Or VKORC1 gene pleiomorphism quick detection kits.
Background technology
Warfarin (Warfarin) is anti-coagulants of new generation, by competing the effect of sex resistance vitamin K, inhibits liver cell
The synthesis of middle coagulation factor reduces the platelet agglutination of thrombin induction, to have anti-freezing and anti-platelet aggregation work(
Energy.
Warfarin is widely used in a variety of disease anticoagulant therapies by clinic as first oral anticoagulation, but magnificent method
The pharmacological action of woods is easily affected by many factors, and individual difference is big, and therapeutic window is narrow, the INR of effective dose 50 and median lethal dose
Level differs only by 1 times or so, even if the doses change of very little may lead to the adverse reactions such as bleeding, thus dosage adjustment is very
It is important.Clinical research proves that CYP2C9 and VKORC1 gene pleiomorphisms cause 35~50% patient to react in the presence of a warfarin
Body difference, this kind of crowd need lower initial dose.In August, 2007, the description of product of U.S. FDA approval update warfarin
Book, it is desirable that indicate the reaction that the hereditary difference of user will influence it to the drug in information warning, while reminding doctor such as
There are the variations of the two genes by fruit patient, and lower predose should be used in prescription warfarin.Therefore, patient is detected
The Clinical practice of the two gene polynorphisms situation energy scientific guidance warfarins.
Human cytochrome enzyme P450 (CYP2C9) is the main metabolic enzyme of warfarin, common allele type CYP2C9*2
(C430T), CYP2C9*3 (A1075C) can cause the activity of the enzyme to decline, when to make active constituents of medicine stop in blood
Between extend.And vitamin K epoxide reductase subunit 1 unit 1 (VKORC1) is the main function target spot of warfarin, SNP site
G-1639A, C1173T and G3739A variation can dramatically increase sensibility of the patient to warfarin.Wherein G-1639A is to grind at present
The major site studied carefully is about 7.59% in the distribution frequency of asian population.
Currently, for CYP2C9 and VKORC1 genetic polymorphism detections there are many ways to, mainly include DNA sequencing method,
Restriction fragment length polymorphism analysis method (PCR-PFLP), gene chips (Genechips), Luminex, high-resolution
Rate melting curve method (HRM), fluorescence quantitative PCR method etc..Wherein most common process is PCR sequencing PCR, and this method cost is few, flux is high,
But time-consuming and sensitivity is low for operation, is not suitable for clinical quickly detection；High-resolution melting curve method is more special to equipment requirement
Very, in clinical expansion, there are certain difficulties.And conventional fluorescent quantitative PCR method clinically applies relatively broad, but this method
Detection gene pleiomorphism is generally detected using Genotyping methods, and sample size and polymorphism is distributed in this method
It is certain to require, when sample size is few accurate parting cannot be carried out to sample gene pleiomorphism；It is carried in addition, blood sample need to usually pass through
It takes and purifies, obtain nucleic acid as fluorescent quantitative PCR template, although the purification that the nucleic acid purity of purification is higher, cumbersome
The not only possible cross contamination between causing sample of step, while it being also easy to cause the loss of nucleic acid, to increase follow-up fluorescent quantitation
The risk of PCR failures.Therefore need to establish it is a kind of quickly and effectively and can be used for directly detecting a small amount of blood sample CYP2C9 and/
Or the method for VKORC1 gene pleiomorphisms.
Invention content
Can be used for blood one of the objects of the present invention is to provide one kind, directly to detect CYP2C9 and/or VKORC1 genes more
The kit of state property, it is intended to when solving the prior art for blood progress fluorescent quantitative PCR, need in advance to carry out blood
The problem of cross contamination and toxic reagent use largely is lost so as to cause nucleic acid and increased to nucleic acid extraction purification process, this
Outside, also have the advantages that amplification efficiency and high sensitivity, specificity are good.
Realize that the technical solution of above-mentioned purpose is as follows：
A kind of kit of detection CYP2C9 and/or VKORC1 gene pleiomorphisms, the kit contain following component：Heat
Start taq archaeal dna polymerases, blood Direct PCR buffer solution (10X), primer pair, probe pair and UNG enzymes；
The primer pair is at least one set in following：For SEQ ID NO.1 and the SEQ ID of CYP2C9C430T
NO.4, SEQ ID NO.7 and SEQ the ID NO.10 for CYP2C9A1075C, for the SEQ ID of VKORC1G-1639A
NO.13 and SEQ ID NO.16；
The probe is selected from below for the saltant type probe of corresponding primer sites and at least one set of wild-type probe：
SEQ ID NO.22 and SEQ ID NO.19 for CYP2C9C430T, the SEQ ID NO.28 for CYP2C9A1075C and
SEQ ID NO.25, for SEQ ID NO.34 and SEQ the ID NO.31 of VKORC1G-1639A, the ends 5' of probe and the ends 3' mark
Note has fluorophor
The blood Direct PCR buffer solution (10X) includes：100±1mM Tris-HCL、500±5mM KCl、20±
1mM MgCl2, 50 ± 5% glycerine, 25 ± 1%DMSO, 0.5 ± 0.05%Tween-20, pH8.3 ± 0.1.
In one of the embodiments, the blood Direct PCR buffer solution (10X) include 100mM Tris-HCL,
500mM KCl、20mM MgCl2, 50% glycerine, 25%DMSO, 0.5%Tween-20, pH8.3.
Another object of the present invention also resides in the application method for providing mentioned reagent box, includes the following steps：
(1) PCR reaction systems are configured, each PCR system includes following component：Thermal starting Taq DNA enzymatics, blood are direct
PCR reaction solution (10X), primer pair, probe are to, dNTPs, UNG enzyme；And human blood sample to be measured is added.
(2) PCR system configured is put into fluorescence quantitative PCR instrument, carries out Real-time PCR detections；React item
Part is：
First stage：50 DEG C of UNG processing in 2 minutes；
Second stage：95 DEG C of 4 minutes pre-degenerations；
Phase III：95 DEG C of 15s, 60 DEG C of 35s, 5 cycles；
Fourth stage：95 DEG C of 15s, 60 DEG C of 35s, 40 cycles.
Signal collection：FAM and VIC signals are collected when 60 DEG C of fourth stage.
Detection kit and detection method of the present invention have at least following advantages：
(1) simple, efficient, safety：Blood Direct PCR buffer solution used can be such that archaeal dna polymerase mortifier in blood becomes
Property is (such as：Ferroheme, protein and fat etc.), the inhibiting effect to fluorescent PCR is reduced, DNA polymerase activity is enhanced, so as to
Fluorescent PCR amplification is directly carried out to blood sample, nucleic acid extraction and purifying is needed not move through, reduces template cross contamination
Risk avoids the use of the harmful reagents such as phenol；
(2) high sensitivity：It can accurately detect down to 2 μ l blood samples, required sample size is few；
(3) specificity is good：Special primer is designed for CYP2C9C430T, CYP2C9A1075C and VKORC1G-1639A
And typing probes, can specific amplification identify that corresponding polymorphism, testing result are consistent with goldstandard PCR sequencing PCR 100%；
(4) detection speed is fast：Entire detection process only needs 90min；
(5) it pollutes small：Detection process is stopped pipe reaction, and adds UNG enzymes, prevents PCR product from polluting.
Description of the drawings
Fig. 1 is the sites CYP2C9C430T homozygous wildtype (C/C) fluorescent quantitative pcr amplification curve graph；
Fig. 2 is the sites CYP2C9C430T homozygous mutant (T/T) fluorescent quantitative pcr amplification curve graph；
Fig. 3 is the sites CYP2C9A1075C homozygous wildtype (A/A) fluorescent quantitative pcr amplification curve graph；
Fig. 4 is the sites VKORC1G-1639A wild type (G/G) fluorescent quantitative pcr amplification curve graph；
Fig. 5 is the sites VKORC1G-1639A homozygous mutant (A/A) fluorescent quantitative pcr amplification curve graph；
Fig. 6 is that the fluorescent quantitative PCR of the sites CYP2C9C430T various concentration homozygous wildtype (C/C) standard items is bent
Line chart；
Fig. 7 be 3 different primers of embodiment and probe combinations to the sites CYP2C9C430T homozygous wildtype (C/C) sample into
Row parallel PCR amplification fluorescent amplification curve figure；
Fig. 8 be 3 different primers of embodiment and probe combinations to the sites CYP2C9C430T homozygous mutant (T/T) sample into
Row parallel PCR amplification fluorescent amplification curve figure；
Fig. 9 is the human blood sample of 3 separate sources of blood Direct PCR reaction solution and common PCR reaction liquid pair in embodiment 4
The fluorescent amplification curve figure of this progress parallel PCR amplification.
Specific implementation mode
In order to illustrate more clearly of the method for the present invention, the present invention is made with reference to preferred embodiments and drawings further
Explanation.It will be appreciated by those skilled in the art that specifically described content is illustrative and be not restrictive below, it should not be with
This is limited the scope of the invention.
Embodiment 1
An embodiment of the present invention provides a kind of examinations directly detecting CYP2C9 and/or VKORC1 gene pleiomorphisms for blood
Agent box, including following component：
Thermal starting taq archaeal dna polymerases, blood Direct PCR buffer solution (10X), primer pair, probe are to, dNTPs, UNG enzyme.
The primer pair be CYP2C9C430T primers, CYP2C9A1075C primers and VKORC1G-1639A primers, it is described
Primer is as shown in table 3：
3 gene primer sequence of table
The probe is described to for CYP2C9C430T probes, CYP2C9A1075C probes and VKORC1G-1639A probes
Specific probe sequence is as shown in table 4：The 5' of probe is marked with VIC or FAM, and 3' is marked with MGB.
4 gene probe sequence of table
The blood Direct PCR buffer solution (10X) is：100mM Tris-HCL、500mM KCl、20mM MgCl2, 50%
Glycerine, 25%DMSO, 0.5%Tween-20, pH8.3.
The application method of kit, includes the following steps：
(1) prepare human blood sample to be measured；
(2) each PCR system is 25 μ l total volumes：0.2 μ l of thermal starting Taq DNA enzymatics, 10X blood Direct PCRs buffering
17.7 μ l of liquid, 1 μ l of primer pair, 2 μ l of probe pair, 2 dNTPs μ l, 0.1 μ l of UNG enzymes, 2 μ l of blood sample.
(3) PCR system configured is put into fluorescence quantitative PCR instrument, carries out Real-time PCR detections；React item
Part is：
First stage：50 DEG C of UNG processing in 2 minutes；
Second stage：95 DEG C of 4 minutes pre-degenerations；
Phase III：95 DEG C of 15s, 60 DEG C of 35s, 5 cycles；
Fourth stage：95 DEG C of 15s, 60 DEG C of 35s, 40 cycles.
Signal collection：FAM and VIC signals are collected when 60 DEG C of fourth stage.
After completing above-mentioned PCR reactions, acquired results carry out result judgement by table 1：
1 result judgement of table
According to above-mentioned detection method, selection sequencing result be the sites CYP2C9C430T homozygous wildtype (C/C) sample,
The sites CYP2C9C430T homozygous mutant (T/T) sample, the sites CYP2C9A1075C homozygous wildtype (A/A) sample,
The sites VKORC1G-1639A homozygous wildtype (G/G) sample, the sites VKORC1G-1639A homozygous mutant (A/A) sample carry out
Detection.For testing result as shown in table 2 (1~sample of sample 5) and Fig. 1 to Fig. 5, there are apparent amplification curve and Ct in the only channels FAM Fig. 1
＜ 35；There are apparent amplification curve and Ct ＜ 35 in the only channels VIC Fig. 2；There are apparent amplification curve and Ct ＜ 35 in the only channels FAM Fig. 3；Figure
There are apparent amplification curve and Ct ＜ 35 in the 4 only channels FAM；There are apparent amplification curve and Ct ＜ 35 in the only channels VIC Fig. 5.1 knot of the table of comparisons
The kit testing result of fruit criterion, remaining 10 sample is as shown in table 2：
2 pattern detection result of table
| Sample | Kit testing result | Sequencing result |
| 1 | CYP2C9C430T(CC) | CYP2C9C430T(CC) |
| 2 | CYP2C9C430T(TT) | CYP2C9C430T(TT) |
| 3 | CYP2C9A1075C(AA) | CYP2C9A1075C(AA) |
| 4 | VKORC1G-1639A(GG) | VKORC1G-1639A(GG) |
| 5 | VKORC1G-1639A(AA) | VKORC1G-1639A(AA) |
| 6 | CYP2C9C430T(CC) | CYP2C9C430T(CC) |
| 7 | CYP2C9A1075C(AA) | CYP2C9A1075C(AA) |
| 8 | VKORC1G-1639A(GA) | VKORC1G-1639A(GA) |
| 9 | VKORC1G-1639A(GG) | VKORC1G-1639A(GG) |
| 10 | CYP2C9A1075C(AA) | CYP2C9A1075C(AA) |
| 11 | CYP2C9C430T(CC) | CYP2C9C430T(CC) |
| 12 | VKORC1G-1639A(GA) | VKORC1G-1639A(GA) |
| 13 | VKORC1G-1639A(GG) | VKORC1G-1639A(GG) |
| 14 | CYP2C9C430T(CC) | CYP2C9C430T(CC) |
| 15 | VKORC1G-1639A(GA) | VKORC1G-1639A(GA) |
Above-mentioned 15 fluorescents quantitative PCR detection result is consistent with sequencing result, shows provided in an embodiment of the present invention
It is reliable that detection kit for blood directly detects CYP2C9 and/or VKORC1 genetic polymorphism detection results, with direct Sequencing
Concordance rate reaches 100%, and detection method high sensitivity is in traditional sequencing methods, it is easy to operate quickly, be conducive to clinic
Detection and popularization.
Sensitivity of 2 kit of the present invention of embodiment for standard items
The present embodiment is by taking CYP2C9 gene C 430T site wild types (C/C) as an example, using various concentration standard items, respectively
It is 107copies/ul、106copies/ul、105copies/ul、104copies/ul、103Copies/ul carries out fluorescent quantitation
PCR amplification is tested.
The probe and primer of the kit that the present embodiment uses and the sites detection CYP2C9 gene C 430T in embodiment 1
Identical, other compositions are also identical.
The detection method that the present embodiment uses is same as Example 1.
It as a result as shown in Figure 6, can from the sites CYP2C9 gene C 430T wild type (C/C) standard items amplification curve
(it is respectively 10 to various concentration standard items7copies/ul、106copies/ul、105copies/ul、104copies/ul、
103Copies/ul) it is in parallel standard serpentine amplification curve.
The selection of embodiment 3CYP2C9 and/or VKORC1 genetic polymorphism detection specific primer sequence
By taking two polymorphic sites of CYP2C9 gene Cs 430T, A1075C as an example, the primer sequence of specificity is separately designed,
Using the complementary series forward or backwards of target sequence where the mutational site as template, it is directed to the wild of C430T, A1075C respectively
In type and saltant type design specific probe sequence, including the embodiment of the present invention 1 preferred specific primer and probe sequence with
And 2 alternative specific primers and probe sequence, as shown in Table 3, 4.Detection method is as described in Example 1.
3 primer sequence of table
4 specific probe sequence of table
The present invention first screens a series of primers and probe in development phase, then final through PCR conditions and system optimization
Screen that a group-specific is most strong, the optimal primer and probe of amplification efficiency.By taking the sites CYP2C9C430T as an example, screening is drawn
Object and probe are combined by table 5, and carry out fluorescent PCR augmentation detection to wild homozygous sample and mutant homozygous sample respectively.
5 primer and probe of table combines
Detection method used by the present embodiment is same as Example 1.
The primer and probe combined sorting result in the sites CYP2C9C430T see combined shown in Fig. 7 and Fig. 81 amplification curve
Maximum slope, in apparent serpentine curve and without non-specific amplification, and combine 2 and 3 have non-specific amplification and amplification efficiency compared with
It is low.
Embodiment 4
The blood Direct PCR buffer solution (10X) includes：100mM Tris-HCL、500mM KCl、20mM MgCl2、
50% glycerine, 25%DMSO, 0.5%Tween-20, pH8.3.It is described to include：100mM Tris-HCL、500mM KCl、
20mM MgCl2, pH8.3.By the blood Direct PCR buffer solution (10X) and regular-PCR buffer solution (10X) respectively to 3 parts not
Blood sample with source carries out fluorescent PCR augmentation detection.
Primer and probe used in the present embodiment is same as Example 1.
Detection method used by the present embodiment is same as Example 1.
Blood Direct PCR buffer solution (10X) provided in this embodiment and regular-PCR buffer solution (10X) are to 3 parts of separate sources
Blood sample carry out the fluorescent amplification curve figure of parallel PCR amplification and see Fig. 9, experimental result shows blood Direct PCR buffer solution
The fluorescent amplification curve inflection point and Exponential growth stage of (10X) are apparent, and regular-PCR buffer solution (10X) is bent without apparent " S " type amplification
Line.
Several embodiments of the invention above described embodiment only expresses, 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 the protection of the present invention
Range.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
Sequence table
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Claims (5)
1. a kind of kit of detection CYP2C9 and/or VKORC1 gene pleiomorphisms, which is characterized in that under the kit contains
Row component：Thermal starting taq archaeal dna polymerases, 10X blood Direct PCRs buffer solution, primer pair, probe pair and UNG enzymes；
The primer pair is at least one set in following：For SEQ ID NO.1 and the SEQ ID of CYP2C9 C430T
NO.4, SEQ ID NO.7 and SEQ the ID NO.10 for CYP2C9 A1075C, for the SEQ ID of VKORC1 G-1639A
NO.13 and SEQ ID NO.16；
The probe is selected from below for the saltant type probe of corresponding primer sites and at least one set of wild-type probe：For
SEQ ID NO.22 and SEQ ID NO.19 of CYP2C9 C430T, SEQ ID NO.28 and SEQ for CYP2C9 A1075C
ID NO.25, for SEQ ID NO.34 and SEQ the ID NO.31 of VKORC1 G-1639A, the ends 5' of probe and the ends 3' label
There is fluorophor；
The 10X blood Direct PCR buffer solution includes：100±1mM Tris-HCL、500±5mM KCl、20±1mM
MgCl2, 50 ± 5% glycerine, 25 ± 1%DMSO, 0.5 ± 0.05%Tween-20, pH8.3 ± 0.1.
2. kit according to claim 1, which is characterized in that the 10X blood Direct PCR buffer solution includes
100mM Tris-HCL、500mM KCl、20mM MgCl2, 50% glycerine, 25%DMSO, 0.5%Tween-20, pH8.3.
3. according to claim 1-2 any one of them kits, which is characterized in that the primer is for CYP2C9 C430T
SEQ ID NO.1 and SEQ ID NO.4, SEQ ID NO.7 and SEQ ID NO.10 and needle for CYP2C9 A1075C
To the SEQ ID NO.13 and SEQ ID NO.16 of VKORC1 G-1639A；
The probe is for SEQ ID NO.22 and SEQ ID NO.19 of CYP2C9 C430T, for CYP2C9 A1075C
SEQ ID NO.28 and SEQ ID NO.25, and SEQ ID NO.34 and the SEQ ID for VKORC1 G-1639A
NO.31。
4. according to claim 1-2 any one of them kits, which is characterized in that the fluorophor is：The 5' of probe is marked
Note has VIC or FAM, 3' to be marked with MGB.
5. the application method of kit described in claim 1-4, which is characterized in that including：
(1) PCR reaction systems are configured, each PCR system includes following component：Thermal starting Taq DNA enzymatics, 10X blood Direct PCRs
Buffer solution, primer pair, probe are to, dNTPs, UNG enzyme；And human blood sample to be measured is added；
(2) PCR system configured is put into fluorescence quantitative PCR instrument, carries out Real-time PCR detections；Reaction condition is：
First stage：50 DEG C of UNG processing in 2 minutes；
Second stage：95 DEG C of 4 minutes pre-degenerations；
Phase III：95 DEG C of 15s, 60 DEG C of 35s, 5 cycles；
Fourth stage：95 DEG C of 15s, 60 DEG C of 35s, 40 cycles；
Signal collection：FAM and VIC signals are collected when 60 DEG C of fourth stage.
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| CN108570498B (en) * | 2018-05-25 | 2021-11-23 | 山东维真生物科技有限公司 | Primer probe composition and kit for detecting human CYP2C9 and VKORC1 gene polymorphism and application |
| CN110760578B (en) * | 2019-11-27 | 2023-09-29 | 郑州安图生物工程股份有限公司 | Kit for detecting polymorphism of human CYP2C9 and VKORC1 genes |
| CN110819709A (en) * | 2019-12-16 | 2020-02-21 | 北京和合医学诊断技术股份有限公司 | Method for detecting CYP2C9 and VKORC1 gene polymorphism by fluorescent quantitative PCR (polymerase chain reaction) |
| CN111172253A (en) * | 2020-02-25 | 2020-05-19 | 上海未凡生物科技有限公司 | Kit and method for detecting CYP2C9 and VKORC1 gene polymorphism in one tube manner |
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| CN103710432A (en) * | 2013-10-31 | 2014-04-09 | 上海百傲科技股份有限公司 | Specific primer pair and specific probe for CYP2C9 (cytochrome P450 2C9) and (vitamin K epoxide reductase complex subunit 1) VKORC1 gene chip detection |
| CN105177116A (en) * | 2015-06-02 | 2015-12-23 | 武汉友芝友医疗科技有限公司 | Human CYP2C9 and VKORC1 genetic polymorphism detection kit |
| CN105002281A (en) * | 2015-07-27 | 2015-10-28 | 潮州凯普生物化学有限公司 | CYP2C9 and VKORC1 gene typing detection reagent kit |
| CN107287340A (en) * | 2017-08-15 | 2017-10-24 | 北京鑫诺美迪基因检测技术有限公司 | A kind of composition and its application for being used to detect CYP2C9 and VKORC1 gene pleiomorphisms |
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