CN111500707A - Method for synchronously detecting polymorphism of two SNP sites of CYP3A4 gene - Google Patents

Abstract

The invention provides a primer group and a detection method for synchronously detecting polymorphism of two SNP sites of CYP3A4 gene, which are beneficial to improving detection flux. The invention firstly provides a primer group for synchronously detecting the polymorphism of two SNP sites of the CYP3A4 gene, which comprises two groups of primer pairs: the nucleotide sequence of the upstream primer of the first group of primer pairs is shown by SEQ ID NO.1, and the nucleotide sequence of the downstream primer is shown by SEQ ID NO. 2; the nucleotide sequence of the upstream primer of the second group of primer pairs is shown by SEQ ID NO.3, and the nucleotide sequence of the downstream primer is shown by SEQ ID NO. 4. When the primer group is used for carrying out multiplex PCR amplification reaction, the CYP3A4 gene x 1B (rs2740574) and x 1G (rs2242480) SNP sites are simultaneously amplified in one amplification system, so that the detection flux can be improved to the greatest extent.

Description

Method for synchronously detecting polymorphism of two SNP sites of CYP3A4 gene

Technical Field

The invention relates to the technical field of gene detection, in particular to a primer group and a detection method for synchronously detecting polymorphism of two SNP sites of CYP3A4 gene.

Background

The cytochrome P450 enzyme system exists in the smooth endoplasmic reticulum of various histiocytes, is a membrane protein with mixed functions, and belongs to an oxidase super family. The P450 enzyme system not only participates in the metabolic process of endogenous substances such as cholic acid, fatty acid, steroid and prostaglandins, but also influences the in vivo process of exogenous substances such as drugs, exotoxin and carcinogenic factors. At present, 12P 450 enzyme subtypes are reported in human bodies, and the metabolic enzymes of CYP1, CYP2 and CYP3 have the strongest action. CYP3A accounts for 25% of the total amount of the P450 enzyme system in human liver, and is distributed with emphasis on liver cells or columnar jejunal villous epithelial cells. CYP3A4 is one of four genotypes of CYP3A, and fentanyl is a metabolite thereof.

Fentanyl is widely applied to the field of labor analgesia since the artificial synthesis of the 20 th 60 th, but the clinical findings show that the fentanyl with the same dosage has obvious difference in analgesic effect and adverse reactions such as nausea and vomiting, respiratory depression and the like. It has been shown that the individual variability of fentanyl in the analgesic effect on puerperal women is closely related to the Single Nucleotide Polymorphism (SNP) of cytochrome P450CYP3A4 x 1G (rs 2242480). CYP3A 4X 1G (rs2242480) is found for the first time in Japan, the gene is located on chromosome 7 q22.1, and has a length of about 27kbp, and the gene structure comprises 13 exons and 12 introns. It is a mutation with functional significance, and the distribution rate in Chinese population is 22.1%. The CYP3A4 activity is reduced due to CYP3A4 x 1G (rs2242480) gene mutation, the metabolism of fentanyl is reduced, and the analgesic effect is enhanced. Research shows that compared with GA and AA types, GG type has weakened analgesic effect of fentanyl. The polymorphism of CYP3A 4X 1G (rs2242480) gene has certain influence on individualized medication of a patient applying fentanyl delivery analgesia, the CYP3A 4X 1G (rs2242480) SNP investigation can be clinically carried out on a puerperal before the delivery analgesia, and the dose of fentanyl required is determined according to different types so as to improve the analgesia effect, so that the aim of better protecting the health of a newborn and the puerperal is finally fulfilled.

Cyclosporine is mainly used for the anti-rejection reaction of kidney. The drug application has a narrow therapeutic index and shows large pharmacokinetic difference among individuals. This difference is related to the activity and expression difference of CYP3a4 × 1B (rs2740574) in humans. The CYP3A4 x 1B (rs2740574) mononucleotide polymorphism site is positioned on the promoter of the gene, and the mutation of the site can influence the expression of the protein. The research shows that the mutant CYP3A4 has enhanced mRNA transcription level and enhanced enzyme activity under the regulation of calcineurin; patients after kidney transplantation surgery carrying CYP3a4 x 1B (rs2740574) wild type AA (./1) cyclosporine had a lower metabolic capacity in vivo than the homozygous mutant, requiring significantly higher doses than the heterozygous mutant AG (./1 x 1B) and the homozygous mutant GG (/ 1B). Therefore, CYP3A4 x 1B (rs2740574) site mutation investigation can be carried out on the patient before operation, and the anti-rejection reaction after the kidney transplantation can be guided according to different genotypes.

PCR (Polymerase Chain Reaction) has been widely used in medicine, genetics, microbiology, and even throughout life sciences. Currently, PCR detection tests can be designed for the sites of 1G (rs2242480) and 1B (rs2740574) of CYP3a4 gene, respectively, to detect the mutation of the gene. Because each PCR reaction only aims at one SNP site, the detection flux is low. Multiplex PCR is a novel amplification technique developed on the basis of conventional PCR, i.e., two or more pairs of primers can be added into a reaction system to simultaneously amplify a plurality of nucleic acid fragments. The multiplex PCR has important application in microbe, genetic disease and tumor pharmacogenomics.

Disclosure of Invention

In order to solve the above-mentioned drawbacks, the present invention provides a primer set and a detection method for synchronously detecting polymorphisms at two SNP sites of the CYP3a4 gene, which are beneficial to improving the detection throughput.

In order to achieve the purpose, the invention is realized by the following technical scheme:

firstly, a primer group for synchronously detecting the polymorphism of two SNP sites of the CYP3A4 gene is provided, which comprises two groups of primer pairs: the nucleotide sequence of the upstream primer of the first group of primer pairs is shown by SEQ ID NO.1, and the nucleotide sequence of the downstream primer is shown by SEQ ID NO. 2; the nucleotide sequence of the upstream primer of the second group of primer pairs is shown by SEQ ID NO.3, and the nucleotide sequence of the downstream primer is shown by SEQ ID NO. 4.

The primer group is specially designed for gene specific amplification of CYP3A4 gene site 1B (rs2740574) and 1G (rs2242480), the first group of primer pairs are primer pairs for detecting CYP3A4 gene 1B (rs2740574), the second group of primer pairs are primer pairs for detecting CYP3A4 gene 1G (rs2242480), and the multiple PCR technology can simultaneously, synchronously, efficiently, specifically and accurately amplify specific gene segments of the gene at the site, and detect the gene polymorphism thereof by sequencing. When the primer set is used for carrying out a multiplex PCR amplification reaction, CYP3A4 gene x 1B (rs2740574) and x 1G (rs2242480) sites are simultaneously amplified in one amplification system, namely, two SNP sites including x 1B (rs2740574) and x 1G (rs2242480) are simultaneously amplified. The detection flux can be improved to the maximum extent.

In general, when the upstream primer SEQ ID NO.1 and the downstream primer SEQ ID NO.2 are used for amplifying CYP3A4 gene x 1B (rs2740574), the fragment length of the corresponding amplification product is 792 bp; when the upstream primer SEQ ID NO.3 and the downstream primer SEQ ID NO.4 are used for amplifying CYP3A4 gene x 1G (rs2242480), the fragment length of the corresponding amplification product is 399 bp. The DNA fragment was then recovered by cutting and sequenced.

Multiple tests prove that the primer pair provided by the invention has better specificity and amplification accuracy, can be applied to the preparation of a kit for synchronously detecting the polymorphism of two SNP sites of the CYP3A4 gene, and can quickly and accurately obtain the result of the polymorphism of the two SNP sites of the CYP3A4 gene after a sample is collected and tested by preparing a finished product kit.

The kit mainly comprises the primer group provided by the invention, wherein the final concentration of the primer is preferably 20-300 nM. Other PCR reagents may be selected according to conventional techniques, for example, a preferred embodiment further comprises a DNA polymerase, a PCR buffer, a mixture of 4 kinds of dNTPs (deoxyribose nucleotide triphosphates), and ultrapure water. Wherein the amount of DNA polymerase is 0.5-5U, and the final concentration of each dNTP is 50-500. mu.M.

The DNA polymerase can be Taq, KOD FX or other DNA polymerase, so the PCR buffer is a concentrated buffer corresponding to the selected DNA polymerase, and the specific concentration degree can be 2 ×, 5 × or 10 ×.

For example, when KOD FX is selected as the DNA polymerase and 2 × is selected as the buffer concentrate, the amount of each component in the PCR system may be 0.5-2. mu.l of the DNA polymerase, 20-30. mu.l of the PCR buffer, 5-30. mu.l of a mixture of dNTPs, 2-15. mu.l of a mixture of 2 primer pairs, 5-1000 ng of DNA, and an amount of ultrapure water to replenish water to 50. mu.l, and may be other volume sizes prepared in the same ratio.

When the finished product kit is manufactured, a reagent for extracting the DNA of a sample or a professional DNA extraction kit can be selectively prepared according to actual needs; the DNA of the sample to be detected can be obtained more conveniently and quickly, and the convenience and the rapidity of the detection finished product kit are enhanced. The sample to be tested can be any blood, cell, tissue or buccal swab sample containing genomic DNA.

On the basis of the primer set provided by the invention, the invention further provides a detection method for synchronously detecting the polymorphism of two SNP sites of the CYP3A4 gene, and the primer pair is adopted for PCR detection.

According to a preferred embodiment, the detection method comprises the following specific steps:

(1) extracting genome DNA from a sample to be detected as an amplification template;

(2) preparing a multiplex PCR reaction system comprising the primer group and the amplification template;

(3) performing multiple PCR amplification reaction on the multiple PCR reaction system to obtain a PCR product;

(4) and (3) determining two SNP site polymorphisms of the CYP3A4 gene according to the PCR product.

Wherein the most preferable reaction conditions for the PCR amplification reaction are: 92-96 ℃: 1-10 min; 93-98.5 ℃: 5-40 s; 51-68 ℃: 10-60 s; 67-72 ℃: 10 s-5 min; 25-45 cycles in total; 68-72 ℃: 0-30 min.

On the basis of the method and the conditions, the detection method can quickly, effectively and conveniently simultaneously and synchronously obtain the site mutation conditions of CYP3A4 gene x 1B (rs2740574) and x 1G (rs2242480) of the sample to be detected, and can be used for non-diagnosis purposes.

According to a preferred embodiment of the present invention, the step (4) of determining gene polymorphism comprises: electrophoretically detecting the PCR product to verify the amplified fragment size of the PCR product; and (5) carrying out sequence determination on the PCR product after the PCR product is verified to be correct so as to obtain the mutation conditions of CYP3A4 gene x 1B (rs2740574) and x 1G (rs2242480) sites of the sample to be tested. In detail, the PCR amplified fragment can be detected by agarose gel electrophoresis or polyacrylamide gel electrophoresis.

Compared with the prior art, the invention at least has the following beneficial effects: (1) and (3) improving the detection flux: the common PCR only aims at one SNP site per reaction, the multiplex PCR can simultaneously detect more than 2 SNP sites, and the CYP3A4 gene x 1B (rs2740574) and x 1G (rs2242480) sites can be detected by one reaction. Therefore, more than 90 samples can be detected simultaneously, the detection efficiency is improved, and the cost is greatly saved. (2) The cost is reduced: the invention can reduce the PCR reaction system from 2 systems/procedures to 1 system/procedure, thereby reducing the use amount of reagents and consumables such as DNA polymerase, dNTP and the like and reducing the detection cost.

Drawings

FIG. 1 shows the result of agarose gel electrophoresis detection according to an embodiment of the present invention;

fig. 2 is a portion of the PCR product sequencing results provided by an embodiment of the present invention for a mutation at the 1B (rs2740574) site;

fig. 3 shows the mutation part of the site of 1G (rs2242480) in the PCR product sequence determination result provided by an embodiment of the present invention.

Detailed Description

To further illustrate the present invention, reference is made to the following examples. Specifically, the reagents used in the implementation of the invention are all commercial products, and the databases used in the implementation of the invention are all public online databases. The following examples are illustrative only and are not to be construed as limiting the invention.

Example 1

Designing and synthesizing a primer group, comprising the following steps:

step 1.1: based on CYP3A4 gene x 1B (rs2740574) and x 1G (rs2242480) loci, an upstream primer and a downstream primer of a specific amplification gene SNP locus mutation region and a corresponding sequencing primer are designed.

For designing the primers, Primer Quest and Primer Premier 5.0 are adopted to design the primers and analyze the mismatch of the dimer and the stem loop, the primers are designed at two ends containing mutation sites, and the annealing temperatures of 2 pairs of primers are basically kept consistent.

The primer set provided in this example covers the region of mutations at CYP3a4 gene x 1B (rs2740574) and x 1G (rs2242480) sites. Because the small sequence change can cause the amplification efficiency of the primers to be obviously reduced and the specificity to be poor, multiple PCR primer sets are respectively designed aiming at different SNP sites, and after the screening of a pre-experiment, the length of a product fragment and the site inclusion condition are integrated, and the primer set with the best amplification effect shown in the following table 1 is selected.

TABLE 1

Step 1.2: the primer set designed in 1.1 was synthesized.

Example 2

The method for extracting the genome DNA from the sample to be detected comprises the following steps:

step 2.1: mouth-shed cells were collected with a mouth swab or fresh peripheral blood samples were collected with a blood collection tube.

In this embodiment, the sample source is a normal human.

And 2.2, extracting the genomic DNA from the sample by adopting a Tiangen buccal swab genomic DNA extraction kit (DP322) or a blood/cell/tissue genomic DNA extraction kit (DP304), measuring the concentration and purity of the DNA by adopting NP80-touch (IMP L EN in Germany), and storing the genomic DNA.

Example 3

A multiplex PCR detection method for synchronously detecting CYP3A4 gene x 1B (rs2740574) and x 1G (rs2242480) loci, comprising the following steps:

step 3.1: and (3) taking the genome DNA obtained in the step 2.2 as an amplification template, and adopting the primer group synthesized in the step 1.2 to prepare a multiple PCR reaction system.

In this example, a multiplex PCR amplification system was prepared by using DNA polymerase and buffer as basic raw materials in KOD FX enzyme system (cat. KFX-101) manufactured by Toyobo, Inc., and adjusting the primer concentration, dNTP concentration, buffer concentration and enzyme amount based on the amplification system in the enzyme system specification, and the specific composition of this reaction system is shown in Table 2 below. Of course, the equal scale enlargement/reduction of the reaction system is within the protection scope of the embodiment of the invention; the amplification can also be achieved by replacing other DNA polymerase systems and adjusting the appropriate proportion.

TABLE 2

Reagent composition	Volume of
		2×PCR buffer for FX	25μl
2mM dNTP	8μl
		Primer Mix	5μl
KOD FX(1U/μi)	1μl
		DNA	1μl
Water (W)	Adding water to 50 μ l

The primers were mixed equimolar, the primer concentration being 10 micromolar; the amount of the DNA template can be adjusted, and 20ng of genomic DNA can be used in this example.

Step 3.2: the PCR apparatus was programmed according to the multiplex PCR reaction conditions shown in Table 3 below.

TABLE 3

Step 3.3: and (3) carrying out multiple PCR amplification reaction on the multiple PCR reaction system prepared in the step (3.1) by using a programmed PCR instrument to obtain a PCR product.

Example 4

Electrophoresis detection, comprising the following steps:

step 4.1: the PCR product obtained in step 3.3 was examined by agarose gel electrophoresis to verify the size of the PCR product fragment.

The detection results are shown in fig. 1, JYK, ZK, L XJ and the like shown in fig. 1 are mainly used for distinguishing different samples to be detected, the left-most column of fig. 1 shows a ruler bar, the right-most column shows electrophoresis results of PCR products of a blank control group, and the middle columns show electrophoresis results of PCR products of different samples.

According to the comparison between the position of each product bright band and the left scale bar, the amplification product of which SNP site corresponds to each product bright band can be identified. For example, 2 bright bands from top to bottom are typically PCR amplification products corresponding to the sites of CYP3a4 gene x 1B (rs2740574) and x 1G (rs2242480), respectively.

Referring to fig. 1, it can be seen from the electrophoresis results of the blank set that the environmental factors have no adverse effect on the electrophoresis detection results of the sample to be detected. According to the electrophoresis result of each sample to be detected, 2 bright bands respectively correspond to the PCR amplification products of CYP3A4 gene x 1B (rs2740574) and x 1G (rs2242480) sites, and the number of the bright bands is consistent with the theory; the 2 bright bands are clear and have obvious intervals, no overlapping and no smear exist among different bright bands, and the bright band effect is good. Thus, it can be shown that when the PCR amplification primer set designed in step 1.1 is used for PCR amplification, only the expected target product is generated, but no other irrelevant product is generated, and the design of the primer set is reasonable.

Step 4.2: after the size of the PCR product fragment is verified to be correct, the sequence of the PCR product can be determined.

Example 5

Sequence determination comprising the steps of:

step 5.1: and (4) after the sizes of the PCR product fragments are verified to be correct in the step 4.2, sending the PCR product obtained in the step 3.3 to a sequencing company for sequence determination to obtain a sequencing result in the format of ab 1.

Step 5.2: the sequencing results obtained in step 5.1 were analyzed by Chromas sequence analysis software to obtain mutations at the sites of CYP3a4 gene x 1B (rs2740574) and x 1G (rs 2242480).

The partial sequencing results are shown in FIGS. 2-3.

Referring to fig. 2, fig. 2 shows the nucleotide base sequence at and upstream and downstream of the site of CYP3a4 gene x 1B (rs 2740574). Referring to the box line in fig. 2, the genotype at the site of CYP3a4 gene × 1B (rs2740574) is known as AA.

Referring to fig. 3, fig. 3 shows the nucleotide base sequence at and upstream and downstream of the site of CYP3a4 gene x 1G (rs 2242480). Referring to the box line in fig. 3, the genotype at the site of CYP3a4 gene x 1G (rs2242480) is known as GA type.

Example 6: preparation of the kit

Based on the above experimental results, the present embodiment provides a preferred kit for simultaneously detecting polymorphisms at the CYP3a4 gene x 1B (rs2740574) and x 1G (rs2242480) sites, the kit comprising the following reagents:

1. multiplex PCR primer set: the first set of primer pairs: f upstream primer (10. mu.M), R downstream primer (10. mu.M); a second set of primer pairs: f upstream primer (10. mu.M), R downstream primer (10. mu.M);

2、KOD FX(1U/μl)；

3、2×PCR buffer for FX：

4、dNTP：

5. a sample DNA extraction reagent;

6. sample DNA collection kits (e.g., containing buccal swabs and swab kits);

7. ultrapure water.

The reagent is reasonably placed in the kit, the instructions of the kit are put in, the instructions comprise the step of collecting the DNA of the sample to be detected, the collected sample is put in a storage box, the step of extracting the DNA is carried out, and finally the multiple PCR amplification process is carried out according to the detection method.

It should be noted that, in this document, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other similar elements in a process, method, article, or apparatus that comprises the element.

The above-mentioned embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements made to the technical solution of the present invention by those skilled in the art without departing from the spirit of the present invention shall fall within the protection scope defined by the claims of the present invention.

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Claims (10)

1. A primer group for synchronously detecting the polymorphism of two SNP sites of a CYP3A4 gene, which is characterized by comprising two groups of primer pairs: the nucleotide sequence of the upstream primer of the first group of primer pairs is shown by SEQ ID NO.1, and the nucleotide sequence of the downstream primer is shown by SEQ ID NO. 2; the nucleotide sequence of the upstream primer of the second group of primer pairs is shown by SEQ ID NO.3, and the nucleotide sequence of the downstream primer is shown by SEQ ID NO. 4.

2. The primer set of claim 1, wherein said SNP sites are x 1B (rs2740574) and x 1G (rs2242480) sites of the CYP3A4 gene; the first set of primer pairs is a primer pair for detecting CYP3a4 gene x 1B (rs2740574), and the second set of primer pairs is a primer pair for detecting CYP3a4 gene x 1G (rs 2242480).

3. Use of the primer set according to claim 1 or 2 in the preparation of a kit for synchronously detecting polymorphisms of two SNP sites of the CYP3A4 gene.

4. A kit for synchronously detecting the polymorphism of two SNP sites of a CYP3A4 gene, which is characterized by comprising the primer set according to claim 1 or 2.

5. The kit according to claim 4, further comprising a DNA polymerase, a PCR buffer, a mixture of 4 dNTPs, and ultrapure water.

6. The kit according to claim 5, wherein the DNA polymerase is used in an amount of 0.5 to 5U, each dNTP is used at a final concentration of 50 to 500. mu.M, and the primer is used at a final concentration of 20 to 300 nM.

7. The kit according to any one of claims 4 to 6, further comprising a test sample DNA extraction reagent or DNA extraction kit.

8. A detection method for synchronously detecting the polymorphism of two SNP sites of a CYP3A4 gene is characterized in that the primer group of claim 1 or 2 is adopted to carry out multiplex PCR detection on a sample to be detected.

9. The detection method according to claim 8, characterized by comprising the steps of:

(1) extracting genome DNA from a sample to be detected as an amplification template;

(2) preparing a multiplex PCR reaction system comprising the primer group and the amplification template;

(3) performing multiple PCR amplification reaction on the multiple PCR reaction system to obtain a PCR product;

(4) and (3) determining two SNP site polymorphisms of the CYP3A4 gene according to the PCR product.

10. The detection method according to claim 9, wherein the reaction conditions of the PCR amplification reaction are: 92-96 ℃: 1-10 min; 93-98.5 ℃: 5-40 s; 51-68 ℃: 10-60 s; 67-72 ℃: 10 s-5 min; 25-45 cycles in total; 68-72 ℃: 0-30 min.

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