CN110938682A - CYP2C19 gene polymorphism detection kit and application thereof - Google Patents
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Abstract
The invention discloses a CYP2C19 gene polymorphism detection kit and application thereof, wherein the kit comprises a forward and reverse primer, a wild type detection probe and a mutant type detection probe for detecting CYP2C19 x 2(G681A), a forward and reverse primer, a wild type detection probe and a mutant type detection probe for detecting CYP2C19 x 3(G636A), and a wild type quenching probe and a mutant type quenching probe shared by two sites, wherein the wild type detection probe and the mutant type detection probe are respectively marked with different fluorescent groups. The invention improves the specificity of probe combination by utilizing the locked nucleic acid technology, detects the CYP2C19 gene polymorphism by combining the multiple fluorescence PCR technology, has convenient and quick operation, easy interpretation of detection results, strong specificity and high accuracy and sensitivity, and provides an effective basis for clinical medication of clopidogrel.
Description
Technical Field
The invention relates to the field of gene detection, in particular to a CYP2C19 gene polymorphism detection kit and application thereof.
Background
Clopidogrel, a drug named boli, is a platelet aggregation inhibitor mediated by adenosine diphosphate receptors, can be used for preventing and treating complications caused by myocardial infarction, ischemic cerebral thrombosis, thromboangiitis obliterans, atherosclerosis and thromboembolism, can obviously reduce the incidence rate of stent thrombosis in the application after percutaneous coronary intervention, and is currently used as one of important drugs for standard antiplatelet therapy after percutaneous coronary intervention. However, patients on long-term treatment with clopidogrel sometimes experience a second occurrence of acute coronary syndrome, severe or even life threatening. A large number of studies prove that the clopidogrel-mediated platelet aggregation inhibition rate is caused by individual difference. Therefore, in recent years, studies on individual differences in clopidogrel response have been receiving increasing attention.
Clopidogrel as a prodrug has no anticoagulation effect, and can generate a platelet inhibition effect after being metabolized by Cytochrome P450 (CYP) and then converted into an active metabolite. In the action process of the clopidogrel in vivo, the clopidogrel is mainly metabolized by CYP2C19 and is greatly influenced by CYP2C19 gene polymorphism. According to the ability of CYP2C19 to metabolize S-MEIFENTOIN or other probe drugs, the genetically determined metabolic polymorphisms of CYP2C19 substrates have two phenotypes in the population, namely strong and weak metabolic types, and the corresponding individuals are classified into strong-metabolic-metabolites (EMs) and weak-metabolic-individuals (PMs), and the PMs have three genotypes (CYP2C19 x 2/. sup.2, CYP2C19 x 2/. sup.3 or CYP2C19 x 3/. sup.3) and two genotypes as homozygotes (CYP2C19 x 1/. sup.1) and heterozygotes (CYP2C19 x 1/. sup.2, 2C 19/. sup.3) as compared with the genotype analysis. 14% of Chinese population is CYP2C19 slow metabolism type, active metabolites generated by conventional dose of clopidogrel in slow metabolism type patients are reduced, platelet aggregation inhibition effect is reduced, and thrombosis forming risk is increased. Black box warning in clopidogrel manual modified by the U.S. food and drug administration in 2010: specifically, doctors are advised to carry out CYP2C19 genotyping detection on patients who are going to take clopidogrel, and personalized medication is carried out according to the genotypes of the patients. Therefore, CYP2C19 gene polymorphism detection has important significance for clinical clopidogrel administration.
At present, there are various methods for detecting polymorphism of CYP2C19 gene, mainly including a DNA direct sequencing method, a high resolution melting curve method and a liquid chip method. Although the direct DNA sequencing method is the most conventional detection method and is a gold standard, the steps are complicated and long in time consumption. The high resolution melting curve method has high requirements on instruments, and most real-time fluorescence quantitative PCR instruments cannot be applied. The liquid phase chip method has complex operation process, easy pollution and high false positive rate.
Disclosure of Invention
In view of the above, the invention provides a CYP2C19 gene polymorphism detection kit with convenient and rapid operation, good specificity and high sensitivity and application thereof.
The technical scheme of the invention is realized as follows:
in a first aspect, the present invention provides a primer probe composition for detecting a polymorphism of the CYP2C19 gene, which is used for detecting a gene polymorphism at the 681 th site and the 636 th site, comprising the following primers and probes:
primer probes for detecting CYP2C19 × 2 (G681A):
forward primer 1: 5'-AAATTTCCCCATCAAGATAT-3' (SEQ ID NO.1)
Reverse primer 1: 5'-TACGCAAGCAGTCACATAAC-3' (SEQ ID NO.2)
Wild-type detection probe 1: 5'-ATGACGTGATCTTGATACTATCATTGATTATTTCCCGG-3' (SEQ ID NO.3)
Mutant detection probe 1: 5'-TAGGTCAAGCGTAGCTGTAATTTGTTATGGGTTCCAA-3' (SEQ ID NO.4)
Primer probe for detecting CYP2C19 × 3 (G636A):
forward primer 2: 5'-ACTTTCATCCTGGGCTGTGC-3' (SEQ ID NO.5)
Reverse primer 2: 5'-TTGGGATATTCATTTCCTGTGC-3' (SEQ ID NO.6)
Wild-type detection probe 2: 5'-ATGACGTGATCTTGATATTGTAAGCACCCCCTGG-3' (SEQ ID NO.7)
Mutant detection probe 2: 5'-TAGGTCAAGCGTAGCTCTTGGCCTTACCTGGATT-3' (SEQ ID NO.8)
Wild type quenching probe shared by 681 and 636 sites: 5'-ATCAAGATCACGTCAT-3' (SEQ ID NO.9)
Mutant quenching probe shared by 681 and 636 sites: 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 technical means, it is preferable that the 37 th base of the wild-type detection probe 1 sequence is LAN-modified, the 36 th base of the mutant-type detection probe 1 sequence is LAN-modified, the 34 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.
In a second aspect, the present invention provides a use of the primer probe composition for detecting a polymorphism in the CYP2C19 gene according to the first aspect in the preparation of a reagent for detecting a polymorphism in the CYP2C19 gene.
In a third aspect, the present invention provides a CYP2C19 gene polymorphism detection kit, comprising the primer probe composition for detecting CYP2C19 gene polymorphism according to the first aspect of the present invention.
On the basis of the above technical solution, preferably, the CYP2C19 gene polymorphism detection kit further comprises a PCR reaction solution, and the PCR reaction solution comprises Tris-HCl buffer solution (ph8.3), Taq DNA polymerase, MgCl2KCl and dNTPs.
Further, preferably, the PCR reaction solution further comprises UNG enzyme and dUTP.
In a fourth aspect, the invention also provides a method for detecting the polymorphism of the CYP2C19 gene, wherein the kit for detecting the polymorphism of the CYP2C19 gene is adopted, the nucleic acid of a sample to be detected is used as a template, and fluorescent quantitative PCR is carried out to obtain the CYP2C19 genotype of the sample to be detected.
Compared with the prior art, the CYP2C19 gene polymorphism detection kit and the application thereof have the following beneficial effects:
(1) the kit provided by the invention can sensitively detect CYP2C19 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, the locked nucleic acid marker probe is designed and synthesized around two single nucleotide polymorphism sites of the CYP2C19 gene by utilizing the locked nucleic acid characteristic, and the complementary quenching probe is separately designed and synthesized, so that three genotypes of each site can be accurately distinguished, and the detection accuracy and sensitivity are greatly improved.
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 CYP2C19 x 2 locus in example 3.
FIG. 2 is a graph showing the amplification of the sample containing the CYP2C19 x 2 site heterozygous mutant in example 3.
FIG. 3 is a graph showing the amplification of a sample of homozygous mutant at CYP2C19 x 2 locus in example 3.
Fig. 4 is a graph showing the amplification of the sample homozygous for the wild type at CYP2C19 x 3 locus in example 3.
FIG. 5 is a graph showing the amplification of the sample containing the CYP2C19 x 3 site heterozygous mutant in example 3.
FIG. 6 is a graph showing the amplification of a sample of homozygous mutant at CYP2C19 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.
The experimental procedures in the following examples are conventional unless otherwise specified, and reference is made to the molecular cloning protocols (fourth edition). The primers and probes used in the examples were synthesized by the firm of Biotechnology engineering (Shanghai) Ltd.
Example 1 CYP2C19 Gene polymorphism detection kit
S1, design and synthesis of primer and probe
Primer probes for detecting CYP2C19 × 2 (G681A):
forward primer 1: 5'-AAATTTCCCCATCAAGATAT-3' the flow of the air in the air conditioner,
reverse primer 1: 5'-TACGCAAGCAGTCACATAAC-3' the flow of the air in the air conditioner,
wild-type detection probe 1: 5 '-FAM-ATGACGTGATCTTGATACTATCATTGATTATTTCCCGG-3', wherein the 37 th base of the sequence is modified with LAN,
mutant detection probe 1: 5 '-HEX-TAGGTCAAGCGTAGCTGTAATTTGTTATGGGTTCCAA-3', wherein the 36 th base of the sequence is modified by LAN;
primer probe for detecting CYP2C19 × 3 (G636A):
forward primer 2: 5'-ACTTTCATCCTGGGCTGTGC-3' the flow of the air in the air conditioner,
reverse primer 2: 5'-TTGGGATATTCATTTCCTGTGC-3' the flow of the air in the air conditioner,
wild-type detection probe 2: 5 '-FAM-ATGACGTGATCTTGATATTGTAAGCACCCCCTGG-3', wherein the 34 th base of the sequence is modified by LAN,
mutant detection probe 2: 5 '-HEX-TAGGTCAAGCGTAGCTCTTGGCCTTACCTGGATT-3', wherein the 34 th base of the sequence is modified by LAN;
wild-type quenching probes common to CYP2C19 x 2 and CYP2C19 x 3: 5 '-BHQ 1-ATCAAGATCACGTCAT-PO 4-3',
for detecting mutant quenching probes common to CYP2C19 x 2 and CYP2C19 x 3: 5 '-BHQ 1-AGCTACGCTTGACCTA-PO 4-3'.
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 0.01U/. mu.L and dUTP0.2mmol/L.
Example 2 performance verification of the kits of the invention
S1, artificial plasmid synthesis and extraction
Plasmids containing two polymorphic regions of the CYP2C19 gene are respectively synthesized by the company of Biotechnology engineering (Shanghai) and polymorphic variations (CYP2C19 x 2 and CYP2C19 x 3, G > A) are respectively introduced into the plasmids, and finally 2 wild-type plasmids and 2 mutant plasmids of the CYP2C19 gene are obtained. 4 artificial plasmids were purified using a plasmid extraction kit (Shanghai Biotech) according to the instructions. The purified plasmid was quantified using a NanoDrop 2000 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 double 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, and then respectively adding CYP2C19 x 2 mixed plasmid, CYP2C19 x 3 mixed plasmid and negative control 2 into the 3 stripsMu L, adding the same amount of template into two tubes in each tube, and finally supplementing ddH to 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 TE buffer solution without nucleic acid as a template are not linear and have no Ct, FAM and HEX signal amplification curves which take CYP2C19 x 2 mixed plasmid as the 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 CYP2C19 x 3 mixed plasmid as the template are S-shaped, the Ct value is less than or equal to 36, and the corresponding product can be effectively amplified. The kit can effectively and sensitively detect the polymorphism of the CYP2C19 gene.
Example 3 detection of blood sample Using the CYP2C19 polymorphism detection kit of the invention
S1, extracting genome DNA
In this example, 50 healthy human peripheral blood samples were collected, genomic DNA was extracted using a sermer feishil plasma sample genomic DNA extraction kit, and DNA was eluted using a TE buffer after extraction. The extracted and purified genomic DNA was quantified using a NanoDrop 2000 ultramicro spectrophotometer 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 described in the step S2 of the embodiment 2, wherein the CYP2C19 x 2 mixed plasmid and the CYP2C19 x 3 mixed plasmid are positive controls, and the TE buffer solution without nucleic acid 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 CYP2C19 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.
And (3) analyzing a detection result:
and (3) analyzing according to the detection result of the fluorescent quantitative PCR instrument: 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. The samples to be tested were obtained according to the criteria shown in table 2: fig. 1-3 show amplification graphs of CYP2C19 x 2 locus homozygous wild-type sample, heterozygous mutant sample, homozygous mutant sample, 39 CYP2C19 x 2 locus homozygous wild-type samples, 9 CYP2C19 x 2 locus heterozygous mutant samples, 2CYP2C19 x 2 locus homozygous mutant samples; fig. 4-6 show amplification graphs of CYP2C19 x 3 locus homozygous wild-type samples, heterozygous mutant samples, homozygous mutant samples, 32 CYP2C19 x 3 locus homozygous wild-type samples, 13 CYP2C19 x 3 locus heterozygous mutant samples, and 5 CYP2C19 x 3 locus homozygous mutant samples. The result is 100% consistent with the sequencing result, and the result of the kit for detecting the polymorphism of the CYP2C19 gene is reliable.
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 (8)
1. A primer probe composition for detecting CYP2C19 gene polymorphism is used for detecting gene polymorphism at 681 th site and 636 th site, and is characterized by comprising the following primers and probes:
primer probes for detecting CYP2C19 × 2 (G681A):
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 probe for detecting CYP2C19 × 3 (G636A):
the base sequence of the forward primer 2 is shown as SEQ ID 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 681 th and 636 th sites is shown in SEQ ID NO.9,
the base sequence of the mutant quenching probe shared by the 681 th site and the 636 th site is shown as 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.
2. The primer-probe composition for detecting a polymorphism of CYP2C19 gene 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 primer-probe composition for detecting a polymorphism of CYP2C19 gene according to claim 1, wherein: the 37 th base of the wild-type detection probe 1 sequence is LAN-modified, the 36 th base of the mutant-type detection probe 1 sequence is LAN-modified, the 34 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. Use of the primer probe composition for detecting polymorphism of CYP2C19 gene according to any one of claims 1 to 3, in the preparation of a reagent for detecting polymorphism of CYP2C19 gene.
5. A CYP2C19 gene polymorphism detection kit is characterized in that: comprising the primer-probe composition for detecting a polymorphism in CYP2C19 gene according to any one of claims 1 to 3.
6. The CYP2C19 gene polymorphism detection kit according to claim 5, wherein: also comprises a PCR reaction solution, wherein the PCR reaction solution comprises Tris-HCl buffer solution (pH8.3), Taq DNA polymerase and MgCl2KCl and dNTPs.
7. The CYP2C19 gene polymorphism detection kit according to claim 6, wherein: the PCR reaction solution further comprises UNG enzyme and dUTP.
8. A method for detecting polymorphism of CYP2C19 gene, which is characterized by comprising the steps of: the CYP2C19 gene polymorphism detection kit of claim 5, taking the nucleic acid of the sample to be tested as a template, and carrying out fluorescence quantitative PCR to obtain the CYP2C19 genotype of the sample to be tested.
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