CN113549685A - Detection kit for celecoxib metabolic marker and detection method and application thereof - Google Patents

Detection kit for celecoxib metabolic marker and detection method and application thereof Download PDF

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CN113549685A
CN113549685A CN202110639950.9A CN202110639950A CN113549685A CN 113549685 A CN113549685 A CN 113549685A CN 202110639950 A CN202110639950 A CN 202110639950A CN 113549685 A CN113549685 A CN 113549685A
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celecoxib
cyp2c9
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陈立波
刘丹
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Hunan Feisite Precision Medical Technology Co ltd
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Abstract

The invention discloses a celecoxib metabolic marker detection kit, a detection method and application thereof, wherein the detection kit is used for detecting the gene polymorphism of a celecoxib metabolic marker gene locus CYP2C9 x 3, and comprises a CYP2C9 x 3 amplification primer, a CYP2C9 x 3 sequencing primer and a positive control. The invention uses RPA amplification and optimized pyrosequencing technology as a combination to detect celecoxib adverse reaction related gene polymorphism, and the kit can simultaneously detect CYP2C9 x 3 gene polymorphism celecoxib adverse reaction prediction and provide gene angle suggestions for clinical personalized medication.

Description

Detection kit for celecoxib metabolic marker and detection method and application thereof
Technical Field
The invention relates to a detection kit for a celecoxib metabolic marker, and a detection method and application thereof, and belongs to the field of gene detection.
Background
Celecoxib is a coxib non-steroidal anti-inflammatory drug, has antipyretic, analgesic and anti-inflammatory effects by specifically inhibiting cyclooxygenase-2, and has adverse reactions related to cardiovascular system, gastrointestinal tract, central nervous system and respiratory system, such as hypertension, dyspepsia, headache and the like. Celecoxib is metabolized in the liver primarily by CYP2C 9. Patients carrying a CYP2C9 low enzyme activity genotype are advised to reduce the dosage of celecoxib and thereby reduce the risk of developing adverse drug reactions.
Cytochrome oxidase P450 is mainly present in liver and intestinal tract, and plays an important role in drug metabolism. CYP2C9 is an important member of cytochrome oxidase P450 family, and accounts for 20% of the total amount of liver microsome P450 protein. The gene is located at 10q24, and the coded protein can hydroxylate and metabolize a plurality of medicines with different properties, mainly acidic substrates. CYP2C9 participates in the hydroxylation metabolism of various drugs such as anticoagulants, anticonvulsants, hypoglycemic drugs, nonsteroidal antipyretic analgesic and anti-inflammatory drugs, antihypertensive drugs and diuretic drugs, wherein warfarin, tolbutamide and phenytoin are all drugs with narrow therapeutic indexes. Changes in CYP2C9 activity can lead to large changes in the in vivo concentration of these drugs and even to the occurrence of serious adverse drug reactions. CYP2C9 x 3(A1075C, Ile359Leu) causes the reduction of CYP2C9 enzyme activity, and CYP2C9 x 3 homozygote individual enzyme activity is only 4-6% of wild type homozygote genotype individuals (carrying CYP2C9 x 1 or Arg144/Ile359 allele) at the site. The frequency of CYPC2C9 × 3 was 3%. CYP2C9 genetic polymorphism causes variation of enzyme activity, thereby causing phenomena of drug metabolism ethnicity and individual difference.
At present, there are many methods for detecting gene polymorphism, such as direct sequencing, chip method, high-resolution melting curve method, allele-specific amplification method, taqman fluorescence probe method, etc. The sequencing method and the chip method have the disadvantages of complicated operation steps, long detection period and easy pollution of amplification products; the high-resolution melting curve method has simple steps, low specificity and higher requirements on instruments and equipment; the allele specific amplification method adopts ARMS primers for specific amplification, the design of the primers is difficult to optimize, and the detection condition is strict. The Taqman fluorescent probe method has high test cost and low amplification flux for a plurality of genes. Therefore, it is necessary to establish a simple, rapid, efficient, inexpensive, and highly specific method for detecting gene polymorphisms.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to obtain a detection kit for a celecoxib metabolic marker based on RPA amplification and pyrosequencing technology, and a detection method and application thereof.
In order to realize one of the above objects, the technical scheme of the detection kit for celecoxib metabolic markers adopted by the invention is as follows:
the kit provided by the invention is used for designing specific amplification primers and sequencing primers aiming at the polymorphism of CYP2C9 x 3 gene, and comprises the following components: amplification reaction solution, CYP2C9 × 3 sequencing primer, and positive control.
Preferably, the specific primers are designed as shown in the following table:
Figure BDA0003107190580000021
preferably, the sequence of the specific primer group of CYP2C9 x 3 is shown in sequence tables SEQ ID NO. 1-SEQ ID NO. 2.
Preferably, the CYP2C9 x 3 sequencing primer is shown in a sequence table SEQ ID NO. 3.
More preferably, the sequencing primer is a nucleic acid analogue, the skeleton of which is a peptide bond rather than a phosphodiester bond, and the peptide bond skeleton is connected with a corresponding base. The structure has stable biological properties, and is not easy to degrade by protease or nuclease.
More preferably, the sequencing primer is a combination of agarose gel particles and amino-labeled PNA sequence, the combination is more stable and specific than the combination of DNA/DNA and is not influenced by the salt ion concentration, and the combination is combined with the DNA and is higher than the capture of the DNA/DNA. Can be used as a sequencing primer and a capture probe, and can be directly used for sequencing reaction after being combined with an amplified product and simply washed.
Preferably, the sequencing region corresponding to the CYP2C 9X 3 sequencing primer is a sequence to be detected by CYP2C 9X 3, and is shown in a sequence table SEQ ID NO: 4.
Preferably, the assignment instruction of the sequence to be detected by CYP2C 9X 3 corresponding to CYP2C 9X 3 is shown in the sequence table SEQ ID NO: 5.
Preferably, the reagent 1 comprises: amplification buffer, 18mM magnesium acetate;
preferably, the reagent 2 comprises: CYP2C9 × 3 pre-primer, CYP2C9 × 3 post-primer, dNTPS, strand displacement DNA polymerase, single-stranded DNA binding protein, recombinase for binding single-stranded nucleic acid, and trehalose; CYP2C9 x 3 amplification can be performed under isothermal conditions.
More preferably, the concentrations of the components of the reagent 2 are respectively as follows: CYP2C9 × 3 pre-primer (0.32uM), CYP2C9 × 3 post-primer (0.32uM), dNTPS (0.3mM), strand displacement DNA polymerase (1.2 ng/. mu.L), single-stranded DNA binding protein (3.2 ng/. mu.L), recombinase binding single-stranded nucleic acid (4.8 ng/. mu.L), trehalose (0.2%).
Preferably, the positive control comprises CYP2C9 x 3 hybrid genomic DNA at a concentration of 20 ng/ul. The positive control corresponds to the heterozygosis of the detected gene locus, provides reference for the type determination of an unknown sample, and simultaneously performs quality control on the effectiveness of the reaction solution.
The invention also discloses a method for detecting the gene polymorphism of the celecoxib adverse reaction by adopting the kit, which comprises the following steps:
a. amplifying the amplification reaction solution and 5ul of genome DNA to be detected by adopting RPA amplification;
b. combining 10ul of reaction product with 3ul of sequencing primer;
c. adding a sequencing enzyme and a sequencing substrate to each sequencing tube;
d. taking a dNTP calandria, and sequentially adding dATP alpha S, dTTP, dGTP and dCTP from the round and smooth end to the flat end;
lightly knocking the bottom of the calandria against the tabletop to enable the bases to be flatly paved at the bottom of the calandria;
e. pyrosequencing;
f. determining the genotype of the celecoxib site at the CYP2C9 x 3 site.
Preferably, the reaction volume is 25ul, and the reaction conditions are as follows: 25min at 39 ℃.
The invention also discloses a kit for celecoxib adverse reaction prediction and application of the method, wherein the detection kit is used for detecting CYP2C9 x 3 so as to guide celecoxib adverse reaction prediction from a gene level.
Recombinase Polymerase Amplification (RPA), is known as a nucleic acid detection technique that can replace PCR. RPA technology relies primarily on three enzymes: recombinases that bind single-stranded nucleic acids (oligonucleotide primers), single-stranded DNA binding proteins (SSBs), and strand-displacing DNA polymerases. The mixture of these three enzymes is also active at ambient temperature, with an optimum reaction temperature around 37 ℃. The recombinase, in combination with the primer, forms a protein-DNA complex that is able to search for homologous sequences in double-stranded DNA. Once the primers locate the homologous sequences, strand exchange reaction formation occurs and DNA synthesis is initiated, and the target region on the template is exponentially amplified. The replaced DNA strand binds to SSB, preventing further replacement. In this system, a single synthesis event is initiated by two opposing primers. The entire process is performed very quickly and detectable levels of amplification product are typically obtained within twenty minutes.
Compared with the prior art, the invention can rapidly and effectively generate a large amount of amplification products at a constant temperature by amplifying CYP2C9 x 3 through RPA. Specifically capturing single-stranded DNA through an amino-labeled single-stranded DNA analogue directly combined with carboxyl modifier, washing, adding a sequencing primer to anneal with template DNA, and adding a sequencing raw material to perform pyrosequencing. The invention uses RPA amplification and optimized pyrosequencing technology as a combination to detect celecoxib adverse reaction related gene polymorphism, and the kit can simultaneously detect CYP2C9 x 3 gene polymorphism celecoxib adverse reaction prediction and provide gene angle suggestions for clinical personalized medication.
Drawings
Fig. 1 is an exemplary graph of sequencing results for CYP2C9 × 1 types provided by the present invention;
fig. 2 is an exemplary graph of sequencing results for CYP2C9 × 1 × 3 types provided by the present invention;
fig. 3 is an exemplary graph of sequencing results for CYP2C9 × 3 types provided by the present invention;
FIG. 4 is a graph of the relationship between celecoxib plasma levels and the CYP2C9 gene locus provided by the present invention.
Detailed Description
The following embodiments are provided to further describe the celecoxib metabolic marker detection kit, the detection method and the application thereof in detail and completely. The following examples are illustrative only and are not to be construed as limiting the invention.
The experimental procedures in the following examples are conventional unless otherwise specified. The experimental materials used in the following examples were all commercially available unless otherwise specified.
Example 1 preparation of kit
The kit provided by the invention designs specific amplification primers and sequencing primers aiming at CYP2C9 x 3, and is used for isothermal amplification and pyrosequencing detection. The design of the primer based on the recombinase polymerase amplification technology is one of the keys of the invention, and the primer design of the technology cannot be carried out by auxiliary software and only depends on manual design. In order to ensure the amplification speed and the detection sensitivity, the length of the primer should be controlled to be 30-35 bp, the non-specific amplification is increased easily to cause false positive if the primer is designed to be too short, and the amplification cannot be performed easily if the primer is designed to be too long. Gene polymorphism sequences are subject to published sequences in Genebank.
The primer sequences of this example are as follows:
Figure BDA0003107190580000041
(II) the detection kit of the embodiment comprises the following components:
Figure BDA0003107190580000042
Figure BDA0003107190580000051
(III) the detection kit reagent 1 of the embodiment is prepared by the following single-person preparation system:
composition (I) Volume (ul)
Amplification buffer 18.8
300mM magnesium acetate 1.2
(IV) the detection kit reagent 2 of the embodiment is configured by the following single-person system:
the concentration of each component of the reagent 2 is as follows: CYP2C9 × 3 pre-primer (0.32uM), CYP2C9 × 3 post-primer (0.32uM), dNTPS (0.3mM), strand displacement DNA polymerase (1.2ng/μ L), single stranded DNA binding protein (3.2ng/μ L), recombinase binding single stranded nucleic acid (4.8ng/μ L), trehalose (0.2%);
Figure BDA0003107190580000052
after the preparation is finished, 98 ul/tube is subpackaged and freeze-dried.
(V) the preparation process of the sequencing primer of the detection kit of the embodiment is as follows:
(1) preparation of MES solution
①100mM MES,pH 4.8(100mL)：
②2.13g MES(2-[N-morpholino]ethane sulfonic acid,MW 213.25).
③pH 4.8
(2) TT Buffer (50mL) configuration
①12.5mL of 1M Tris buffer pH 8
②50ul 10％
Figure BDA0003107190580000053
-20
Figure BDA0003107190580000061
(3) Sequencing primer preparation
Firstly, taking 500ul of magnetic beads, carrying out magnetic attraction for 1min, and removing supernatant;
② taking 1000ml 100MES solution to wash and attract magnetism for 1min, removing supernatant, and repeatedly washing for 2 times;
③ taking 500ul of each of 10mg/ml EDS and 10mg/ml NHS, and adding 10ul of capture primer.
Fourthly, the temperature of the housewares is 25 ℃ for 30min
Fifthly, taking 1000ml of confining liquid, mixing uniformly, absorbing magnetism for 1min, and removing supernatant;
sixthly, taking 1000ml of TT Buffer, mixing uniformly, absorbing magnetism for 1min, and removing supernatant; washing was repeated 3 times;
seventhly, adding 1ml of TE buffer solution for dissolving.
Example 2 detection of Pyrophosphoric acid
The apparatus used in the present invention is as follows: a thermostat;
pyrophosphoric acid sequencer: wuhan Firstet Biotech, Inc.
(1) Reagent preparation (reagent preparation Chamber)
The reagent was removed in advance and reagent 1 was vortexed for 15 seconds and centrifuged at low speed until use. 440ul of reagent 1 was added directly to reagent 2 (lyophilized) and mixed well by vortexing for 15 seconds. And determining the reaction number N, wherein N is the number of samples to be detected (N), the number of quality control products (1) and a blank control. It is recommended that positive control and blank control analyses be performed simultaneously for each PCR experiment. Then, the reaction solution was dispensed into a PCR reaction tube at a volume of 20. mu.L/tube.
(2) Application of sample detection (sample preparation room)
Adding the sample DNA, the positive control and the blank control into a PCR reaction tube according to the sample adding amount of 5 mu L, covering the tube cover tightly, centrifuging at low speed for 15 seconds to completely throw liquid on the tube wall to the tube bottom, and then immediately carrying out PCR amplification reaction.
(3) PCR amplification (between amplifications)
And (3) amplifying by adopting a PCR instrument, wherein the reaction system is 25 mu L, and the amplification conditions are as follows:
temperature of amplification Time Number of cycles
39℃ 25min 1
(4) Pyrophosphoric acid sequencing
1) Adding 40 mu L of binding solution and 3ul of agarose gel particles into a PCR reaction tube, adding 10 mu L of PCR product into the PCR reaction tube, placing the PCR reaction tube on a table type oscillator, and oscillating at 1100rpm for 10min to ensure that the microbeads and the PCR product are fully bound;
2) adding 150uL washing buffer solution into an EP tube, centrifuging at 7000g for 1min, and removing supernatant;
3) respectively adding 3uL sequencing enzyme and 3uL sequencing substrate into a sequencing tube;
4) a dNTP comb was loaded with 20. mu.l ATP. alpha.S, 20. mu.l dTTP, 20. mu.l dGTP, and 20. mu.l dCTP sequentially from the round end to the blunt end. Lightly knocking the bottom of the calandria against the tabletop to enable the bases to be flatly paved at the bottom of the calandria;
5) pyrosequencing results are shown in FIGS. 1 to 3.
(5) Interpretation of results
1) And (3) judging the effectiveness:
the blank control of the kit failed, and the positive control detected CYP2C9 x 1 x 3.
2) Criteria for determination of results
In the DNA sequencing peak map of CYP2C9 x 3,
the frequency of A is greater than or equal to 90 percent, the frequency of C is less than or equal to 10 percent, and the model is 1 x 1;
frequency of 40% ≦ a ≦ 60%, frequency of 40% ≦ C ≦ 60%, i.e., type 1 × 3;
the frequency of C is equal to or greater than 90%, the frequency of A is equal to or less than 10%, and the type is 3 x 3;
example 3 correlation of Gene test results with adverse drug reactions and cardiovascular diseases
A panel of 139 subjects with different CYP2C9 genotypes, 96 CYP2C9 x 1, 34 CYP2C9 x 1 x 3, and 9 CYP2C9 x 3. Each subject received a single oral dose of 200 mg celecoxib and 240 ml water. The subjects remained in a fasting state for 4h prior to dosing. Venous blood samples were collected at 0.5, 1, 2, 3, 4, 6, 8, 10, 12, 24, 36, 48 and 96h post-dose, respectively. The plasma concentrations of celecoxib and its carboxylic acid metabolites were determined using a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) system. The results show that celecoxib plasma concentrations were higher in the carrier of the CYP2C9 x 3 allele than in the CYP2C9 x 1 allele. The relationship between celecoxib blood concentration and CYP2C9 gene locus is shown in figure 4.
Finally, it must be said here that: the above embodiments are only used for further detailed description of the technical solutions of the present invention, and should not be understood as limiting the scope of the present invention, and the insubstantial modifications and adaptations made by those skilled in the art according to the above descriptions of the present invention are within the scope of the present invention.
Sequence listing
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Claims (10)

1. The detection kit for the celecoxib metabolic marker is characterized by detecting the gene polymorphism of a celecoxib metabolic marker gene locus CYP2C9 x 3, and comprises a CYP2C9 x 3 amplification primer, a CYP2C9 x 3 sequencing primer and a positive control.
2. The celecoxib metabolic marker detection kit according to claim 1, wherein the CYP2C9 x 3 amplification primer is shown in a sequence table SEQ ID NO 1-2.
3. The celecoxib metabolic marker detection kit according to claim 1, wherein the CYP2C9 x 3 sequencing primer is shown in a sequence table SEQ ID NO. 3.
4. The celecoxib metabolic marker detection kit according to claim 1, wherein the sequencing primer is a conjugate of agarose gel particles and amino-labeled DNA sequences.
5. The celecoxib metabolic marker detection kit according to claim 1, wherein the kit further comprises an amplification buffer, 18mM magnesium acetate, dNTPS, a strand displacement DNA polymerase, a single-stranded DNA binding protein, a single-stranded nucleic acid binding recombinase and trehalose.
6. The celecoxib metabolic marker detection kit according to claim 5, wherein the final concentration of each component in the kit is as follows: before and after amplification, 0.32uM of each primer, 0.3mM of dNTPS, 1.2 ng/mu L of strand displacement DNA polymerase, 3.2 ng/mu L of single-strand DNA binding protein, 4.8 ng/mu L of recombinase for binding single-strand nucleic acid and 0.2% of trehalose.
7. The celecoxib metabolic marker detection kit according to claim 1, wherein the sequencing region corresponding to the CYP2C9 x 3 sequencing primer is a sequence to be detected in CYP2C9 x 3, and is shown in a sequence table SEQ ID NO. 4.
8. The celecoxib metabolic marker detection kit according to claim 1, wherein the CYP2C9 x 3 test sequence corresponds to the CYP2C9 x 3 assignment instruction shown in the sequence table SEQ ID NO. 5.
9. A method for detecting the celecoxib metabolic marker by using the celecoxib metabolic marker detection kit according to any one of claims 1-8, wherein the method comprises the following steps:
a. amplifying the amplification reaction solution and 5ul of genome DNA to be detected by adopting RPA amplification;
b. combining 10ul of reaction product with 3ul of sequencing primer;
c. adding a sequencing enzyme and a sequencing substrate to each sequencing tube;
d. taking a dNTP calandria, and sequentially adding dATP alpha S, dTTP, dGTP and dCTP from the round and smooth end to the flat end;
e. pyrosequencing;
f. determining the genotype of the celecoxib site at the CYP2C9 x 3 site.
10. The use of the celecoxib metabolic marker detection kit according to any one of claims 1-8, wherein the detection kit is used for in vitro detection of the CYP2C9 x 3 gene polymorphism in a sample to be detected.
CN202110639950.9A 2021-06-09 2021-06-09 Detection kit for celecoxib metabolic marker and detection method and application thereof Pending CN113549685A (en)

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Application publication date: 20211026