CN110938688A

CN110938688A - CYP2C9 gene polymorphism detection kit and application thereof

Info

Publication number: CN110938688A
Application number: CN201911395731.XA
Authority: CN
Inventors: 赵平锋
Original assignee: Wuhan Optical Valley United Medical Laboratory Co Ltd
Current assignee: Wuhan Optical Valley United Medical Laboratory Co Ltd
Priority date: 2019-12-30
Filing date: 2019-12-30
Publication date: 2020-03-31

Abstract

The invention discloses a CYP2C9 gene polymorphism detection kit and application thereof, the kit comprises a forward and reverse primer, a wild type detection probe and a mutant type detection probe for detecting CYP2C9 x 2(C430T), a forward and reverse primer, a wild type detection probe and a mutant type detection probe for detecting CYP2C9 x 3(A1075C), and a wild type quenching probe and a mutant type quenching probe which are shared by the two sites, wherein the wild type detection probe and the mutant type detection probe are respectively marked with different fluorescent groups, and the CYP2C9 gene typing is judged according to the starting condition of a fluorescent signal of fluorescent quantitative PCR reaction. The invention further improves the specificity of probe combination by utilizing the locked nucleic acid technology, detects the CYP2C9 gene polymorphism by combining the multiple fluorescence PCR technology, has convenient and quick operation, easy interpretation of the detection result, strong detection specificity and high accuracy.

Description

CYP2C9 gene polymorphism detection kit and application thereof

Technical Field

The invention relates to the field of gene detection, in particular to a CYP2C9 gene polymorphism detection kit and application thereof.

Background

Cytochrome P450 superfamily (CYP) is a kind of monooxygenase mainly existing in liver and intestinal tract, is positioned on endoplasmic reticulum of cells, catalyzes the metabolism of various endogenous and exogenous substances (including most clinical drugs), and is a main enzyme for metabolizing drugs in human body. The P450 enzyme can transfer electrons to oxidize heterologous substances through iron ions in the non-covalently bound heme, so that the water solubility of the heterologous substances is enhanced, and the heterologous substances are easier to discharge out of a body. There are several subfamilies of CYP, of which CYP2C9(Cytochrome P4502C 9) is an important member in the second subfamily and accounts for 20% of the total amount of liver microsome P450 protein. CYP2C9 is capable of hydrozymerizing many drugs of different natures, primarily acidic substrates. Statistically, about 16% of the clinical drugs currently are metabolized by CYP2C 9.

Warfarin is a vitamin K antagonist widely applied clinically and is mainly used for the primary and secondary prevention of venous thromboembolic diseases, the prevention of atrial fibrillation thromboembolism, valvular diseases, thrombosis in heart cavities and the like. Because the warfarin treatment window is narrow, the dosage difference of the medicines among individuals is large and can reach 10-20 times, the medicines have slow effect and failure, and the INR value needs to be frequently detected and the dosage needs to be adjusted, so that the treatment effect and the bleeding risk are difficult to grasp, and the clinical use of the warfarin is greatly limited. The CYP2C9 gene x 2 and x 3 allelic gene type is closely related to the anticoagulation effect of warfarin, and the genotype is a key variable for determining the best anticoagulation effect of warfarin. The united states drug administration (FDA) modified the instructions for warfarin use as early as 2007, and the black box warned that CYP2C9 gene polymorphisms affected warfarin anticoagulation, suggesting that physicians genotype patients prior to warfarin use. The FDA revised warfarin prescription information in 2010, providing a recommended dosage schedule based on CYP2C9 genotyping. The CYP2C9 gene has genetic polymorphism, the subtypes are divided into 1, 2 and 3, the most common allele is named CYP2C9 1 (wild type), and the most closely related to Asian population is the A/C polymorphism at the c.1075 nucleotide of CYP2C9 gene. The polymorphic site is C (CYP2C9 x 3 type), the enzyme activity is reduced by 80% compared with the wild type, and CYP2C9 x 2 type allele (C430T) is rarely found in Chinese. The CYP2C9 gene polymorphism detection has important significance for warfarin medication.

The existing nucleotide primer and method for detecting CYP2C9 gene polymorphism have low accuracy and specificity, are complex in steps and long in time consumption, and are difficult to be widely applied clinically.

Disclosure of Invention

In view of this, the invention provides a CYP2C9 gene polymorphism detection kit with convenient and rapid operation, high specificity and high accuracy and application thereof.

The technical scheme of the invention is realized as follows:

in a first aspect, the invention provides a CYP2C9 gene polymorphism detection kit, comprising a primer probe composition, a PCR reaction solution, a positive control and a negative control, wherein the primer probe composition comprises a primer probe for detecting a 430 th site and a primer probe for detecting a1075 th site, and specifically comprises the following components:

primer probes for detecting CYP2C9 × 2 (C430T):

forward primer 1: 5'-GGATCTCCCTCCTAGTTTCG-3' (SEQ ID NO.1)

Reverse primer 1: 5'-AACCAGGACTCATAATGAAAG-3' (SEQ ID NO.2)

Wild-type detection probe 1: 5'-ATGACGTGATCTTGATAGAGGAGCATTGAGGACC-3' (SEQ ID NO.3)

Mutant detection probe 1: 5'-TAGGTCAAGCGTAGCTGGCTTCCTCTTGAACACA-3' (SEQ ID NO.4)

Primer probes for detecting CYP2C9 × 3(a 1075C):

forward primer 2: 5'-GACAGGAGCCACATGCCCTAC-3' (SEQ ID NO.5)

Reverse primer 2: 5'-GCCCCAAACTGGAAACAAGA-3' (SEQ ID NO.6)

Wild-type detection probe 2: 5'-ATGACGTGATCTTGATCGAGGTCCAGAGATACA-3' (SEQ ID NO.7)

Mutant detection probe 2: 5'-TAGGTCAAGCGTAGCTGGTGGGGAGAAGGTCAAG-3' (SEQ ID NO.8)

Wild-type quenching probe shared by sites 430 and 1075: 5'-ATCAAGATCACGTCAT-3' (SEQ ID NO.9)

Mutant quenching probe shared by sites 430 and 1075: 5'-AGCTACGCTTGACCTA-3' (SEQ ID NO.10)

The detection method comprises the following steps that 5 ' ends of a wild type detection probe 1 and a wild type detection probe 2 are connected with a first fluorescent group, 5 ' ends of a mutant type detection probe 1 and a mutant type detection probe 2 are connected with a second fluorescent group, 5 ' ends of a wild type quenching probe are connected with a first quenching group capable of quenching a fluorescent signal emitted by the first fluorescent group, 5 ' ends of a mutant type quenching probe are connected with a second quenching group capable of quenching a fluorescent signal emitted by the second fluorescent group, and 3 ' ends of the wild type quenching probe and the mutant type quenching probe are connected with a phosphate group.

On the basis of the above technical scheme, preferably, the first fluorophore is a fluorophore FAM, the second fluorophore is a fluorophore HEX, and both the first quencher and the second quencher are BHQ 1.

In addition to the above, it is preferable that the 34 th base of the wild-type detection probe 1 sequence is LAN-modified, the 34 th base of the mutant-type detection probe 1 sequence is LAN-modified, the 33 th base of the wild-type detection probe 2 sequence is LAN-modified, and the 34 th base of the mutant-type detection probe 2 sequence is LAN-modified.

On the basis of the above technical scheme, preferably, the PCR reaction solution comprises Tris-HCl buffer solution (pH8.3), Taq DNA polymerase, MgCl₂KCl and dNTPs.

Based on the above technical solution, preferably, the PCR reaction solution further comprises UNG enzyme and dUTP.

On the basis of the technical scheme, preferably, the positive control product comprises a CYP2C9 gene 430 th site wild-type plasmid, a CYP2C9 gene 430 th site mutant-type plasmid, a CYP2C9 gene 1075 th site wild-type plasmid and a CYP2C9 gene 1075 th site mutant-type plasmid, and the negative control product is DEPC water.

In a second aspect, the present invention also provides a method for detecting a polymorphism in CYP2C9 gene, comprising the steps of:

s1, extracting the genome DNA of the peripheral blood of the subject as a sample to be detected;

s2, performing fluorescent quantitative PCR by using the CYP2C9 gene polymorphism detection kit of the first aspect of the invention respectively using a sample to be detected, a positive control and a negative control as templates, and collecting two fluorescent signals;

s3, after the PCR amplification reaction is finished, judging the CYP2C9 genotype of the subject according to the line-starting condition of the collected fluorescent signal.

Compared with the prior art, the human CYP2C9 gene mutation detection kit and the application thereof have the following beneficial effects:

(1) the kit provided by the invention can sensitively detect CYP2C9 gene polymorphism, can distinguish different genotypes in one-time reaction, is convenient and quick to operate, has low requirements on instruments, and simultaneously reduces the processes of glue preparation, electrophoresis, sequencing and the like due to direct tube closing operation, thereby greatly reducing the probability of cross contamination and enabling the result to be more accurate;

(2) according to the invention, two different fluorescent groups are marked on the wild type detection probe and the mutant type detection probe, and the simultaneous detection of the single nucleotide polymorphism sites in one tube is realized by utilizing multiple fluorescence PCR, so that the detection efficiency is improved, the detection result is accurate and easy to observe;

(3) according to the invention, by utilizing the characteristic of locked nucleic acid, specific locked nucleic acid marker probes are designed and synthesized around two single nucleotide polymorphism sites of CYP2C9 gene, and complementary quenching probes are separately designed and synthesized, so that three genotypes of each site can be accurately distinguished, and the detection accuracy and sensitivity are greatly improved;

(4) the PCR reaction solution of the kit comprises UNG enzyme and dUTP, the UNG enzyme is added before PCR amplification reaction to destroy the PCR product containing dU in the reaction solution, but does not act on natural nucleic acid, and then the UNG enzyme is inactivated and amplified, so that the pollution of aerosol formed by the amplified product in a laboratory can be effectively prevented, and the occurrence of false positive is avoided.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a graph showing the amplification of a sample homozygous for the wild type at CYP2C9 x 2 locus in example 3.

FIG. 2 is a graph showing the amplification of the sample containing the CYP2C9 x 2 site heterozygous mutant in example 3.

FIG. 3 is a graph showing the amplification of a sample of homozygous mutant at CYP2C9 x 2 locus in example 3.

Fig. 4 is a graph showing the amplification of the sample homozygous for the wild type at CYP2C9 x 3 locus in example 3.

FIG. 5 is a graph showing the amplification of the sample containing the CYP2C9 x 3 site heterozygous mutant in example 3.

FIG. 6 is a graph showing the amplification of a sample of homozygous mutant at CYP2C9 x 3 locus in example 3.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.

Example 1 CYP2C9 Gene polymorphism detection kit

S1, design and synthesis of primer and probe

Primer probes for detecting CYP2C9 × 2 (C430T):

forward primer 1: 5'-GGATCTCCCTCCTAGTTTCG-3' the flow of the air in the air conditioner,

reverse primer 1: 5'-AACCAGGACTCATAATGAAAG-3' the flow of the air in the air conditioner,

wild-type detection probe 1: 5 '-FAM-ATGACGTGATCTTGATAGAGGAGCATTGAGGACC-3', wherein the 34 th base of the sequence is modified by LAN,

mutant detection probe 1: 5 '-HEX-TAGGTCAAGCGTAGCTGGCTTCCTCTTGAACACA-3', wherein the 34 th base of the sequence is modified by LAN;

primer probes for detecting CYP2C9 × 3(a 1075C):

forward primer 2: 5'-GACAGGAGCCACATGCCCTAC-3' the flow of the air in the air conditioner,

reverse primer 2: 5'-GCCCCAAACTGGAAACAAGA-3' the flow of the air in the air conditioner,

wild-type detection probe 2: 5 '-FAM-ATGACGTGATCTTGATCGAGGTCCAGAGATACA-3', wherein the 33 rd base of the sequence is modified by LAN,

mutant detection probe 2: 5 '-HEX-TAGGTCAAGCGTAGCTGGTGGGGAGAAGGTCAAG-3', wherein the 34 th base of the sequence is modified by LAN;

wild-type quenching probes common to CYP2C9 x 2 and CYP2C9 x 3: 5 '-BHQ 1-ATCAAGATCACGTCAT-PO 4-3',

for detecting mutant quenching probes common to CYP2C9 x 2 and CYP2C9 x 3: 5 '-BHQ 1-AGCTACGCTTGACCTA-PO 4-3'.

All of the primers and probes described above were synthesized by Competition Biotechnology (Shanghai) Ltd.

S2 and kit composition

Wherein the concentration of each primer is 0.4 mu mol/L, the concentration of each probe is 0.6 mu mol/L, and the 2 XPCR reaction solution comprises: Tris-HCl buffer (pH8.3)20mmol/L, MgCl₂4mmol/L, KCl 100mmol/L, dNTPs 2mmol/L, TaqDNA polymerase 0.08U/. mu.L, UNG enzyme 0.02U/. mu.L and dUTP0.3mmol/L.

Example 2 performance verification of the kits of the invention

S1, artificial plasmid synthesis and extraction

Plasmids containing two polymorphic regions of the CYP2C9 gene are respectively synthesized by the company Limited in the Biotechnology engineering (Shanghai), and polymorphic variations CYP2C9 x 2(430C > T) and CYP2C9 x 3(1075A > C) are respectively introduced into the plasmids, so that 2 wild-type plasmids and 2 mutant plasmids of the CYP2C9 gene are finally obtained. 4 artificial plasmids were purified using a plasmid extraction kit (Shanghai Biotech) according to the instructions. The purified plasmid was quantified using a NanoDrop2000 ultramicro spectrophotometer and diluted to 1000 copies/. mu.L with deionized water for subsequent real-time fluorescent quantitative PCR detection. Simultaneously, each mutant plasmid and wild plasmid are mixed according to the proportion of 1:100, and the content of the mutant plasmid in the finally obtained mixed plasmid is 1 percent.

S2 fluorescent quantitative PCR verification

Taking 3 duplex PCR reaction strips respectively filled with 12.5 mu L reaction reagent, placing the reaction strips at room temperature for dissolving, centrifuging at 5000r/min for several seconds, depositing the reaction reagent to the bottom of a tube, then respectively adding CYP2C 9X 2 mixed plasmid, CYP2C 9X 3 mixed plasmid and negative control 2 mu L into the 3 reaction strips, adding the same equivalent template into two tubes in each tube, and finally supplementing ddH into each tube₂O to 25. mu.L.

Setting PCR reaction conditions on a PCR instrument according to the conditions shown in Table 1, selecting a FAM channel to detect a wild type, selecting a HEX channel to detect a mutant type, and completing the fluorescent quantitative PCR reaction.

TABLE 1 fluorescent quantitative PCR reaction conditions

And observing a PCR reaction result, wherein negative controls FAM and HEX which take DEPC water as a template are not drawn and have no Ct, FAM and HEX signal amplification curves which take CYP2C9 x 2 mixed plasmid as a template are S-shaped, the Ct value is less than or equal to 36, a corresponding product can be effectively amplified, FAM and HEX signal amplification curves which take CYP2C9 x 3 mixed plasmid as a template are S-shaped, the Ct value is less than or equal to 36, and a corresponding product can be effectively amplified. The kit can effectively and sensitively detect the polymorphism of the CYP2C9 gene.

Example 3 detection of blood sample Using the CYP2C9 polymorphism detection kit of the invention

S1, extraction and purification of genome DNA

In this example, 100 healthy human peripheral blood samples were collected, and genomic DNA was extracted and purified using a sermer feishil plasma sample genomic DNA extraction kit. The purified genomic DNA was quantified using a NanoDrop2000 ultraspectrophotometer and diluted to a concentration of 10 ng/. mu.L with deionized water.

S2 fluorescent quantitative PCR

Taking 2 mu L of genome DNA of a sample to be detected, and carrying out fluorescent quantitative PCR detection on the sample according to the same operation method of the step S2 in the embodiment 2, wherein the CYP2C 9X 2 mixed plasmid and the CYP2C 9X 3 mixed plasmid are positive controls, and DEPC water is a negative control; and simultaneously detecting the DNA by using a DNA direct sequencing method.

The determination standard for detecting the genotype of the sample to be detected by the fluorescence quantitative PCR method is shown in Table 2.

TABLE 2 determination criteria for CYP2C9 Gene polymorphism

Note that "+" represents the deta Rn endpoint fluorescence value >150,000 and the amplification curve is S-shaped; "-" indicates that the deta Rn endpoint fluorescence value is <150,000, or the amplification curve is non-S-shaped. Negative control FAM and HEX signals are not broken and have no Ct, positive control FAM and HEX signal amplification curves are S-shaped, and Ct values are less than or equal to 36, so that the system performance is good, and the result is effective.

And (3) analyzing a detection result: fig. 1-3 show amplification graphs of CYP2C9 x 2 locus homozygous wild type sample, heterozygous mutant sample, homozygous mutant sample, 68 cases of CYP2C9 x 2 locus homozygous wild type sample, 23 cases of heterozygous mutant sample, 9 cases of homozygous mutant sample; fig. 4-6 show amplification graphs of CYP2C9 x 3 locus homozygous wild-type sample, heterozygous mutant sample, homozygous mutant sample, 75 cases of CYP2C9 x 3 locus homozygous wild-type sample, 19 cases of CYP2C9 x 3 locus heterozygous mutant sample, and 6 cases of CYP2C9 x 3 locus homozygous mutant sample. The result is 100% consistent with the sequencing result, and the result of the kit for detecting the polymorphism of the CYP2C9 gene is reliable and high in accuracy.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

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Claims

1. A CYP2C9 gene polymorphism detection kit comprises a primer probe composition, PCR reaction liquid, a positive control product and a negative control product, and is characterized in that the primer probe composition comprises a primer probe for detecting a 430 th site and a primer probe for detecting a1075 th site, and the specific steps are as follows:

primer probes for detecting CYP2C9 × 2 (C430T):

the base sequence of the forward primer 1 is shown as SEQ ID NO.1,

the base sequence of the reverse primer 1 is shown as SEQ ID NO.2,

the base sequence of the wild type detection probe 1 is shown as SEQ ID NO.3,

the base sequence of the mutant detection probe 1 is shown as SEQ ID NO. 4;

primer probes for detecting CYP2C9 × 3(a 1075C):

the base sequence of the forward primer 2 is shown as SEQID NO.5,

the base sequence of the reverse primer 2 is shown as SEQ ID NO.6,

the base sequence of the wild type detection probe 2 is shown as SEQ ID NO.7,

the base sequence of the mutant detection probe 2 is shown as SEQ ID NO. 8;

the base sequence of the wild type quenching probe shared by the 430 th site and the 1075 th site is shown as SEQ ID NO.9,

the base sequence of the mutant quenching probe shared by the 430 th site and the 1075 th site is shown as SEQ ID NO. 10;

2. The CYP2C9 gene polymorphism detection kit according to claim 1, wherein: the first fluorescent group is a fluorescent group FAM, the second fluorescent group is a fluorescent group HEX, and the first quenching group and the second quenching group are both BHQ 1.

3. The CYP2C9 gene polymorphism detection kit according to claim 1, wherein: the 34 th base of the wild-type detection probe 1 sequence is LAN-modified, the 34 th base of the mutant-type detection probe 1 sequence is LAN-modified, the 33 th base of the wild-type detection probe 2 sequence is LAN-modified, and the 34 th base of the mutant-type detection probe 2 sequence is LAN-modified.

4. The CYP2C9 gene polymorphism detection kit according to claim 1, wherein: what is needed isThe PCR reaction solution comprises Tris-HCl buffer solution (pH8.3), Taq DNA polymerase and MgCl₂KCl and dNTPs.

5. The CYP2C9 gene polymorphism detection kit according to claim 1, wherein: the PCR reaction solution further comprises UNG enzyme and dUTP.

6. The CYP2C9 gene polymorphism detection kit according to claim 1, wherein: the positive control product comprises a CYP2C9 gene 430 th site wild-type plasmid, a CYP2C9 gene 430 th site mutant-type plasmid, a CYP2C9 gene 1075 th site wild-type plasmid and a CYP2C9 gene 1075 th site mutant-type plasmid, and the negative control product is DEPC water.

7. A method for detecting a polymorphism of CYP2C9 gene, comprising the steps of:

s2, adopting the CYP2C9 gene polymorphism detection kit of claim 1, respectively taking a sample to be detected, a positive control and a negative control as templates, carrying out fluorescence quantitative PCR, and collecting two fluorescence signals;