CN111607644A - Method for detecting CYP2C19 genotyping and kit thereof - Google Patents
Method for detecting CYP2C19 genotyping and kit thereof Download PDFInfo
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Abstract
The invention discloses a method for detecting CYP2C19 genotyping for non-disease treatment and diagnosis purposes, which comprises the following steps: the first step, taking DNA of a sample to be detected as a template, and carrying out fluorescent quantitative PCR amplification to obtain a PCR amplification product; secondly, detecting the fluorescence signal of the amplification product; thirdly, judging whether the sample contains CYP2C19 x 3 genotype or not according to the detection result; wherein, the reaction system for PCR amplification contains a specific primer pair for amplifying CYP2C19 x 3 gene and a fluorescent probe for detecting CYP2C19 x 3 gene. The invention also discloses a primer pair and a fluorescent probe for detecting CYP2C19 genotyping. The method has high specificity and high detection result accuracy.
Description
Technical Field
The invention belongs to the technical field of biological engineering, and particularly relates to a method for detecting CYP2C19 genotyping and a kit thereof.
Background
CYP2C19 is an important member of the second subfamily of CYP450 enzymes. A protein consisting of 490 amino acids located on chromosome 10 is expressed in many tissues of an organism, mainly in liver tissues, and at least 10% of drugs act on it in clinical practice. CYP2C19 has many SNP sites, and at present, at least 10 mutations among the 25 mutant alleles of CYP2C19 have been found to cause changes in enzyme activity, with the slow metabolic form being dominated by CYP2C19 x 2, CYP2C19 x 3, and the fast metabolic form being dominated by CYP2C19 x 17.
Asians and Pacific island residents have higher probability of non-functional mutations of CYP2C19, the mutations may influence the treatment of related drugs, such as antiplatelet drug chlorpyrrolidine for treating coronary atherosclerotic heart disease, the anti-platelet drug chlorpyrrolidine has no activity per se, the antiplatelet effect is required to be exerted after the organism absorbs and transforms, and polymorphism of related genes such as CYP2C19 among different individuals causes difference of functional protein level, thereby influencing the active metabolic level of chlorpyrrolidine. Therefore, accurate determination of the genotype of CYP2C19 is of great importance for the amount of drug used and the treatment of disease.
At present, there are many methods for detecting CYP2C19 genotyping, and the conventional methods include: (1) the direct sequencing method comprises the steps of extracting human peripheral venous blood, extracting genome DNA, amplifying and then carrying out direct DNA sequencing so as to judge the genotype; the method can directly display the position and type of the known mutation site. However, the method has long detection period, low flux and high cost. (2) RFLP method (restriction fragment length polymorphism) is used for detecting the size of a specific DNA fragment formed after the restriction enzyme digestion of DNA. However, the SNP site of this method must contain an enzyme cleavage site, and is highly restricted. (3) The PCR-SSCP method is a novel technique for screening mutations created by Orita et al, Japan, 1989. It is a simple, rapid and economical point mutation screening means. The basic principle of the PCR-SSCP technology is that DNA fragments amplified by PCR are denatured into single-stranded DNA, the single-stranded DNA forms different three-dimensional conformations when electrophoresed in neutral polyacrylamide gel, the conformation directly influences the swimming speed, and the nucleotide sequence of the single-stranded DNA with the same length only has the difference of single base, so that the three-dimensional conformations can be different, the swimming speed is different, and different swimming bands are generated. The DNA fragment amplified by PCR is denatured into single strand, electrophoresed in neutral gel without denaturant, and the presence of base substitution can be presumed by comparing the displacement of the electrophoretic band with the normal one. The nature of the base substitution must be determined by DNA sequencing. Therefore, PCR-SSCP detection is a common means of mutation screening prior to sequencing. However, the method has high technical requirements and low detectable rate. (4) The HRM (high resolution Melt) method, i.e., the high resolution melting curve, is the latest SNP and mutation research tool which has been raised abroad in recent years, is a simple, rapid and low-cost detection technology after PCR amplification, and can be used for high-throughput mutation scanning and genotyping. The technology can be carried out only by adding a double-stranded DNA binding dye on the basis of a standard PCR reagent without a sequence specific probe, and the genotype of a sample can be analyzed by directly running a high-resolution melting curve after the PCR is finished. However, the method has complicated instruments and equipment and is inconvenient to use. (5) The SNaPshot genotyping technology is a genotyping technology based on the principle of fluorescence labeling single-base extension chain termination reaction developed by ABI company in America, and mainly aims at medium and small flux SNP genotyping projects. However, this method is expensive and complicated in operation.
QPCR (Real-time Quantitative PCR detection System), namely a Real-time fluorescent Quantitative nucleic acid amplification detection System. The technology can be combined with a TaqMan probe to quickly detect the mutation of a certain nucleic acid site in DNA. The TaqMan fluorescent probe is an oligonucleotide probe, the 5 'end of the TaqMan fluorescent probe is connected with a fluorescent group, the 3' end of the TaqMan fluorescent probe is connected with a quenching group, the TaqMan fluorescent probe can be specifically combined with a template, a complete probe fluorescent signal is absorbed by the quenching group, the probe is subjected to enzyme digestion hydrolysis by the exonuclease activity of Taq enzyme along with the PCR amplification, the fluorescent group is released, and therefore the fluorescent signal is detected.
Disclosure of Invention
The invention designs specific TaqMan probes and primers aiming at mutation sites of 2 and 3 of CYP2C19, and can simultaneously determine a plurality of samples by matching with a real-time fluorescent quantitative PCR technology, thereby rapidly and accurately determining the SNP genotype. Accordingly, it is a first object of the present invention to provide a method for detecting CYP2C19 genotyping for non-disease therapeutic and diagnostic purposes. The second purpose of the invention is to provide a primer pair and a fluorescent probe for detecting CYP2C19 genotyping. The third purpose of the invention is to provide a kit for detecting CYP2C19 genotyping. The fourth purpose of the invention is to provide an application of the kit for detecting CYP2C19 genotyping in detecting CYP2C19 genotyping for non-disease treatment and diagnosis purposes.
In order to achieve the purpose, the invention adopts the following technical scheme:
as a first aspect of the present invention, a method for detecting CYP2C19 genotyping for non-disease therapeutic and diagnostic purposes, comprising the steps of:
the first step, taking DNA of a sample to be detected as a template, and carrying out fluorescent quantitative PCR amplification to obtain a PCR amplification product;
secondly, detecting the fluorescence signal of the amplification product;
thirdly, judging whether the sample contains CYP2C19 x 2 or CYP2C19 x 3 genotype or not according to the detection result;
wherein, the reaction system for PCR amplification contains a specific primer pair for amplifying CYP2C19 x 3 gene and a fluorescent probe for detecting CYP2C19 x 3 gene,
the nucleotide sequence of the specific primer pair for amplifying the CYP2C19 x 3 gene is shown as SEQ ID NO: 5 and SEQ ID NO: 6 is shown in the specification;
the nucleotide sequence of the fluorescent probe for detecting CYP2C19 x 3 gene is shown as SEQ ID NO: 7 and SEQ ID NO: shown in fig. 8.
According to the invention, the conditions for the PCR amplification are as follows:
the reaction system is 20 μ L: 2 × HieffTM qPCR TaqMan Probe Master Mix, 10 μ L; primers were 10. mu.M each, 0.4. mu.L; probe 10. mu.M, 0.2. mu.L; 10ng of human genome; the balance of sterile ultrapure water;
and (3) PCR reaction conditions: pre-denaturation, 95 ℃, 1min,1 cycle; denaturation, 95 ℃ for 10 s; annealing and extending for 30s at 60 ℃; 40 cycles.
As a second aspect of the present invention, a primer set and a fluorescent probe for detecting CYP2C19 genotyping, comprising a specific primer set for amplifying the CYP2C19 x 3 gene and a fluorescent probe for detecting the CYP2C19 x 3 gene,
the nucleotide sequence of the specific primer pair for amplifying the CYP2C19 x 3 gene is shown as SEQ ID NO: 5 and SEQ ID NO: 6 is shown in the specification;
the nucleotide sequence of the fluorescent probe for detecting CYP2C19 x 3 gene is shown as SEQ ID NO: 7 and SEQ ID NO: shown in fig. 8.
As a third aspect of the invention, a kit for detecting CYP2C19 genotyping, said kit comprising the above-mentioned primer pair for detecting CYP2C19 genotyping and a fluorescent probe.
As a fourth aspect of the present invention, a kit for detecting CYP2C19 genotyping for use in detecting CYP2C19 genotyping for non-disease therapeutic and diagnostic purposes.
The invention has the beneficial effects that:
1. the invention can perform detection of medium density at one time by using a TaqMan qPCR method, and has short time and simple operation.
2. The probe and the primer designed by the invention have high specificity and high detection result accuracy.
3. Compared with other methods, the method has lower cost.
Drawings
Fig. 1 shows the results of SNP typing at CYP2C19 × 2 sites of 96 samples.
Fig. 2 shows the results of SNP typing at the CYP2C19 × 3 sites of 48 samples.
Detailed Description
The present invention will be further described with reference to the following examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not specified, in the following examples are generally conducted under conventional conditions or conditions provided by manufacturers.
Example 1 design of primer pairs and probes
1. Search of CYP2C19 SNP site sequence
In this example, the SNP sites of CYP2C19 were searched on dbSNP database, and the sites x 2 and x 3 were selected for primer and probe design. The website of the dbSNP database in this embodiment is: http:// www.bioinfo.org.cn/relative/dbSNP% 20 Home% 20Page.
2. Primer pairs and probes were designed according to the primer probe design principle, as shown in Table 1.
TABLE 1 primer pairs and probes
Wherein, the amplification length of CYP2C19 x 2 is 244 bp; CYP2C19 x 3 amplified length 235 bp.
3. qPCR amplification system
The probes synthesized at each SNP site were mixed into 10uM TaqMan Probe mix, and each site was reacted in a 20. mu.L system according to the formulation in Table 2.
TABLE 2 qPCR amplification System
4. qPCR amplification procedure
The qPCR reaction amplification procedure is shown in table 3.
TABLE 3 qPCR reaction amplification procedure
Example 2 practical verification
(1) 96 samples of 50 GG type, 36 GA type and 8 AA type determined by first-generation sequencing were subjected to typing test, and the negative control group was water, 2 groups. The test results are shown in fig. 1.
The results show that: the CYP2C19 x 2 sites in these samples were 50 homozygous GG, 8 homozygous AA, and 36 heterozygous GA, which corresponded well to the first-generation sequencing results.
(2) 48 samples (i.e., 26 samples of GG type, 16 samples of GA type, and 4 samples of AA type, respectively, the negative control group being water, and 2 samples) with known typing results were subjected to typing tests, and the test results are shown in FIG. 2.
The results show that: the CYP2C19 x 3 sites in these samples were 26 homozygous GG, 4 homozygous AA, and 16 heterozygous GA, consistent with the theoretical results.
And (4) conclusion:
genotyping was performed using the primer set and probe of example 1 with 100% accuracy; the test results were negative for other non-CYP 2C19 x 2 and CYP2C19 x 3 nucleic acid samples, and example 2 was quality tested for two sites, and thus the specificity was good.
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are given by way of illustration of the principles of the present invention, and that various changes and modifications may be made without departing from the spirit and scope of the invention as defined by the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.
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Claims (5)
1. A method for testing CYP2C19 genotyping for non-disease therapeutic and diagnostic purposes comprising the steps of:
the first step, taking DNA of a sample to be detected as a template, and carrying out fluorescent quantitative PCR amplification to obtain a PCR amplification product;
secondly, detecting the fluorescence signal of the amplification product;
thirdly, judging whether the sample contains CYP2C19 x 3 genotype or not according to the detection result;
wherein, the reaction system for PCR amplification contains a specific primer pair for amplifying CYP2C19 x 3 gene and a fluorescent probe for detecting CYP2C19 x 3 gene,
the nucleotide sequence of the specific primer pair for amplifying the CYP2C19 x 3 gene is shown as SEQ ID NO: 5 and SEQ ID NO: 6 is shown in the specification;
the nucleotide sequence of the fluorescent probe for detecting CYP2C19 x 3 gene is shown as SEQ ID NO: 7 and SEQ ID NO: shown in fig. 8.
2. The method for testing CYP2C19 genotyping for non-disease therapeutic and diagnostic purposes according to claim 1, wherein said PCR amplification conditions are as follows:
the reaction system is 20 μ L: 2 × HieffTM qPCR TaqMan Probe Master Mix, 10 μ L; primers were 10. mu.M each, 0.4. mu.L; probe 10. mu.M, 0.2. mu.L; 10ng of human genome; the balance of sterile ultrapure water;
and (3) PCR reaction conditions: pre-denaturation, 95 ℃, 1min,1 cycle; denaturation, 95 ℃ for 10 s; annealing and extending for 30s at 60 ℃; 40 cycles.
3. A primer pair and a fluorescent probe for detecting CYP2C19 genotyping are characterized by comprising a specific primer pair for amplifying CYP2C19 x 3 gene and a fluorescent probe for detecting CYP2C19 x 3 gene,
the nucleotide sequence of the specific primer pair for amplifying the CYP2C19 x 3 gene is shown as SEQ ID NO: 5 and SEQ ID NO: 6 is shown in the specification;
the nucleotide sequence of the fluorescent probe for detecting CYP2C19 x 3 gene is shown as SEQ ID NO: 7 and SEQ ID NO: shown in fig. 8.
4. A kit for detecting CYP2C19 genotyping, wherein said kit comprises the primer pair for detecting CYP2C19 genotyping of claim 3 and a fluorescent probe.
5. Use of the kit for detecting CYP2C19 genotyping according to claim 4 for detecting CYP2C19 genotyping for non-disease therapeutic and diagnostic purposes.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105506118A (en) * | 2016-01-08 | 2016-04-20 | 智海生物工程（北京）股份有限公司 | Primer pair used for detecting CYP2C19 genotypes, fluorescent probe, reagent kit and method |
| CN106755535A (en) * | 2017-03-01 | 2017-05-31 | 上海荻硕贝肯医学检验所有限公司 | Primer, probe, kit and method for detecting mankind's CYP2C19 gene pleiomorphisms |
| CN110387408A (en) * | 2019-08-29 | 2019-10-29 | 无锡市申瑞生物制品有限公司 | For detecting primer combination of probe object, reagent, kit and the detection method of people's CYP2C19 Genotyping |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105506118A (en) * | 2016-01-08 | 2016-04-20 | 智海生物工程（北京）股份有限公司 | Primer pair used for detecting CYP2C19 genotypes, fluorescent probe, reagent kit and method |
| CN106755535A (en) * | 2017-03-01 | 2017-05-31 | 上海荻硕贝肯医学检验所有限公司 | Primer, probe, kit and method for detecting mankind's CYP2C19 gene pleiomorphisms |
| CN110387408A (en) * | 2019-08-29 | 2019-10-29 | 无锡市申瑞生物制品有限公司 | For detecting primer combination of probe object, reagent, kit and the detection method of people's CYP2C19 Genotyping |
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