CN113755574A - Detection kit and detection method for methylprednisolone metabolic marker - Google Patents
Detection kit and detection method for methylprednisolone metabolic marker Download PDFInfo
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Abstract
The invention discloses a detection kit and a detection method of methylprednisolone metabolic markers, wherein the detection kit is used for designing specific amplification primers and sequencing primers aiming at polymorphism of PAI-1(4G/5G) and ABCB1(C3435T), and comprises the following components: sample processing liquid, magnetic beads, amplification reagent 1, amplification reagent 2, PAI-1(4G/5G), ABCB1(C3435T) sequencing primer and positive control. The rapid amplification method is optimized mainly from three aspects, and on one hand, the method adopts a mode of extracting and amplifying the same tube, so that the risks of extracting multiple tube moving and losing nucleic acid are avoided; on the other hand, the rapid constant temperature amplification is carried out by adding an anti-inhibitor; in the third aspect, double PCR amplification of PAI-1(4G-5G) and ABCB1(3435T > C) at two sites was used, and pyrosequencing was performed at two sites in a single reaction.
Description
Technical Field
The invention relates to a detection kit for methylprednisolone metabolic markers and a detection method thereof, belonging to the field of gene detection.
Background
Methylprednisolone (methylprednisolone sodium succinate) is a glucocorticoid with immune mechanism action, anti-inflammation and anti-inflammation, and a high-concentration solution is suitable for treating certain endocrine diseases and treating a plurality of non-infectious inflammatory diseases and allergic inflammation, and is used for treating pathological changes requiring strong and rapid hormone action. Glucocorticoid shock therapy can be used for treating dermatoses such as systemic lupus erythematosus, severe seborrheic dermatitis, severe psoriasis, mycosis fungoides, and urticaria. But the reasonable and effective use of hormone therapy is very important for femoral head necrosis possibly caused by hormone shock therapy or long-term use. The existence of the gene polymorphism causes different effects and reactions of the same medicament on different crowds and individuals, and causes serious adverse reactions of part of patients.
The drug-resistant gene PAI-1(4G-5G) of methylprednisolone is an effector molecule of plasminogen activator inhibitor PAI-1, PAI-1 is a main inhibitor in a plasma fibrinolysis system, the activity of the fibrinolysis system can be controlled and reduced, and the plasma fibrinolysis activity can be maintained only by maintaining the dynamic balance of the fibrinolysis system through the regulation effect of PAI-1. Therefore, thrombus formation is easily induced by abnormal PAI-1 levels in the blood circulation. The femoral head itself has a limited blood supply and the risk of necrosis is greatly increased if there is a risk of thrombus. PAI-1(4G-5G) gene mutation, reduced plasma fibrinolytic activity, thrombosis, reduced femoral head blood supply, increased risk of femoral head necrosis, and imbalance between coagulation system and fibrinolysis system are important reasons for the occurrence of non-traumatic ONFH. Non-invasive ONFH patients PAI-1mRNA and protein are highly expressed, and it is thought that bone marrow adipocytes might be involved in the development of non-invasive ONFH by increasing PAI-1 expression.
ABCB1(3435T > C) is the 3435 site of the multi-resistant gene ABCB1 and is a glucocorticoid drug transporter. The SNP site of the single nucleotide polymorphism, ABCB1 is located on the long arm of chromosome 7 and contains 29 exons, and the SNP site of the single nucleotide polymorphism is located at the position of exon 26C 3435T and is most widely researched. In the Caco-2 cell line, the penetration of the hormone into cells is limited by P.gp, and the concentration of the hormone in tissues or cells is reduced due to the knockout or the expression of ABCB1 gene. And the increase of the expression of P.gp can reduce the accumulation of hormone in cells to reduce the incidence rate of SONFH. The ABCB1(3435T > C) gene polymorphism and glucocorticoid-induced femoral head necrosis research shows that the C allele increases the correlation of glucocorticoid-induced femoral head necrosis. The drug resistance sites PAI-1(4G-5G) and ABCB1(3435T > C) of methylprednisolone, which is a chemical drug, are detected, so that accurate medication guidance is provided for patients treated by methylprednisolone, and the risk of femoral head necrosis is avoided.
At present, there are many methods for detecting gene polymorphism, and fluorescence PCR is mainly used. Mainly comprises a high-resolution melting curve method, a taqman fluorescence probe method and an allele specific amplification method. The high-resolution melting curve method has simple steps, but has low specificity and higher requirements on instruments and equipment; the allele specific amplification method adopts ARMS primers to carry out specific amplification, and has simple operation method, but strict detection condition requirements, and easy occurrence of primer mismatching in actual operation to generate false positive. the taqman fluorescence probe method has higher test cost. At present, the blood DNA is obtained mainly by the traditional column extraction and the magnetic bead extraction, and the two methods both take longer time and are relatively complicated to operate. CN201610022581.8 proposes a real-time fluorescent quantitative PCR method for extracting nucleic acid and amplifying by magnetic beads in one tube, adding lysis solution mixed with magnetic beads and a sample to be detected into a PCR amplification tube, mixing uniformly, standing, performing magnetic attraction, sucking out mixed solution, and washing the obtained magnetic beads once; and adding the prepared PCR reaction solution into the PCR amplification tube to perform real-time fluorescent quantitative PCR reaction on the target nucleic acid. The invention also requires washing of the magnetic beads. Therefore, it is necessary to provide a gene polymorphism detection kit which can simplify the detection steps and improve the detection efficiency.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to obtain a detection kit for a methylprednisolone metabolic marker and a detection method thereof.
In order to realize one of the above purposes, the technical scheme of the detection kit for the methylprednisolone metabolic marker adopted by the invention is as follows:
the methylprednisolone adverse reaction risk assessment gene polymorphism detection kit is used for designing specific amplification primers and sequencing primers aiming at the polymorphism of PAI-1(4G/5G) and ABCB1(C3435T), and comprises the following components: sample processing liquid, magnetic beads, amplification reagent 1, amplification reagent 2, PAI-1(4G/5G), ABCB1(C3435T) sequencing primer and positive control.
Specifically, the specific primer sequences are shown in the following table 1:
preferably, the sequence of the specific primer group of the PAI-1(4G/5G) is shown in a sequence table SEQ ID NO. 1-SEQ ID NO. 2; the sequence of the specific primer group of the ABCB1(C3435T) is shown as SEQ ID NO. 3-SEQ ID NO. 4 of the sequence table.
Preferably, the PAI-1(4G/5G) sequencing primer and the ABCB1(C3435T) sequencing primer are respectively shown as SEQ ID NO: 5-SEQ ID NO:6 of the sequence table.
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. Binding to DNA is more stable than DNA/DNA binding.
Preferably, the sequencing region corresponding to the PAI-1(4G/5G) sequencing primer is a to-be-detected PAI-1(4G/5G) sequence, and is shown as a sequence table SEQ ID NO. 7; the sequencing region corresponding to the sequencing primer of ABCB1(C3435T) is the sequence to be detected of ABCB1(C3435T), and is shown as the sequence table SEQ ID NO: 8.
Preferably, PAI-1(4G/5G) and ABCB1(C3435T) share a common assignment instruction as set forth in SEQ ID NO:9 of the sequence Listing. "ddC" in SEQ ID NO 9 indicates that the PAI-1(4G/5G) sequencing reaction adds the last base ddCTP, which can terminate the sequencing reaction. The "-" in SEQ ID NO 9 indicates that the addition of the reagent was suspended for 3 min. During this pause ABCB1(C3435T) sequencing primers were added.
Preferably, the sample processing solution does not contain guanidine salt, and the sample is cracked by alkaline conditions and a surfactant, and comprises 0.1-0.6% of lithium dodecyl sulfate, 0.1-0.5% of triton X-100, 2-50mg/mL of sodium hydroxide, 5-15% of trehalose, 3-7 mmol/L of BSA, 20-80mM Tris-HCl and 100mM NaCl, wherein the pH value is 8.5-9.5.
More preferably, the sample treatment solution comprises 0.3-0.5% of lithium dodecyl sulfate, 0.2-0.4% of Triton X-100, 10-20mg/mL of sodium hydroxide, 8-12mM of betaine, 8-12% of trehalose, 4-6 mM of BSA, 50mM of Tris-HCl, 100mM of NaCl, and pH 9.
Preferably, the magnetic beads are carboxyl magnetic beads, the particle size is 600mm, the suspension property is good, the magnetic property is strong, and the adsorption capacity is large. The concentration of magnetic beads in the reaction solution is 0.2mg/25 mu L, and the DNA adsorption amount in the blood sample is 30 ng-260 ng, which completely meets the DNA amount required by amplification.
Preferably, the amplification reagent 1 comprises: amplification buffer, 15mM magnesium acetate;
preferably, the amplification reagent 2 comprises: PAI-1(4G/5G) pre-primer (0.32uM), PAI-1(4G/5G) (0.32uM), ABCB1(C3435T) pre-primer (0.32uM), ABCB1(C3435T) (0.32uM) dNTPS (0.3mM), strand displacement DNA polymerase (1.2 ng/. mu.L), single-stranded DNA binding protein (3.2 ng/. mu.L), single-stranded nucleic acid binding recombinase (4.8 ng/. mu.L).
More preferably, the amplification reagent 2 comprises: trehalose (0.2%), 10mM manganese acetate, 0.1M sorbitol, 5ug/mL BSA. Trehalose has nonspecific protection effect on bioactive substances, can improve the thermal stability of DNA polymerase, reduce the melting temperature of a DNA template, and reduce the secondary structure formed by self-complementary pairing of a G-C rich region, thereby improving the specificity of PCR reaction. Sorbitol and manganese acetate have the stabilizing effect of the PCR premix, and have the stabilizing effect in the freeze drying process. The bovine serum albumin can improve the amplification efficiency of the PCR reaction and reduce the influence of PCR inhibitors in the system on the reaction.
Preferably, the reaction volume is 25ul, and the reaction conditions are as follows: 30min at 42 ℃.
Preferably, the positive control comprises PAI-1(4G/5G), ABCB1(C3435T) heterozygote 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 gene polymorphism detection method for methylprednisolone adverse reaction risk assessment by adopting the kit, which comprises the following steps:
a. mixing 100ul of sample treatment solution, 4ul of magnetic beads and 30ul of EDTA (ethylene diamine tetraacetic acid) anticoagulated whole blood sample, and standing at room temperature for 5 min;
b. placing the PCR amplification tube on a magnetic frame, and sucking out the mixed solution from the opposite side of the magnetic beads after the magnetic beads are completely adsorbed to one side;
c. adding the prepared PCR reaction solution into the PCR amplification tube obtained in the step b), fully and uniformly mixing magnetic beads and the PCR reaction solution, centrifuging, and carrying out constant-temperature reaction;
d. binding the binding solution (containing the microbeads) with the amplification product;
e. treating the denatured liquid to obtain a single-chain product;
f. adding a washing buffer solution for rinsing;
g. adding a sequencing enzyme and a sequencing substrate to each sequencing tube;
h. taking an 8-row pipe, sequentially adding dATP, dTTP, dGTP, dCTP and PAI-1 from one round smooth end to the flat end
(4G/5G), ABCB1(C3435T) sequencing primer, ddCTP; lightly knocking the bottom of the calandria against the tabletop to enable the bases to be flatly paved at the bottom of the calandria;
i. and (4) pyrosequencing.
The invention also discloses application of the gene polymorphism detection kit for adverse reaction risk assessment of methylprednisolone, and the detection kit is used for detecting PAI-1(4G/5G) and ABCB1(C3435T) so as to reflect adverse reaction risk of methylprednisolone from a gene level and further give gene level suggestions for guiding methylprednisolone dosage.
The rapid amplification method is optimized mainly from three aspects, and on one hand, the method adopts a mode of extracting and amplifying the same tube, so that the risks of extracting multiple tube moving and losing nucleic acid are avoided; on the other hand, the rapid constant temperature amplification is carried out by adding an anti-inhibitor; in the third aspect, double PCR amplification of PAI-1(4G-5G) and ABCB1(3435T > C) at two sites was used, and pyrosequencing was performed at two sites in a single reaction. The sequencing is carried out by firstly adding a PAI-1(4G-5G) sequencing primer and sequencing raw materials to carry out pyrosequencing, and adding ddNTP to the last base to terminate the sequencing reaction. Then adding ABCB1(3435T > C) sequencing primer and corresponding dNTP for sequencing. The sequencing of two sites is carried out in sequence by one treatment, so that the operation time is reduced and the sequencing flux is improved. The invention aims to obtain a gene polymorphism detection kit for assessing adverse reaction risk of methylprednisolone based on constant temperature PCR and pyrophosphoric acid detection, and a detection method and application thereof.
Compared with the prior art, the invention adopts the sample processing liquid to rapidly release the DNA from the sample, the sample amount is 30ul of whole blood, and compared with a one-step cracking method and direct amplification (about 2-10ul), enough genome DNA can be obtained and adsorbed on the magnetic beads for subsequent analysis. Most of the inhibitor can be removed by removing the sample and lysis mix. The DNA template is used for amplifying gene sites of PAI-1(4G/5G) and ABCB1(C3435T) by isothermal PCR to generate a large amount of biotin-labeled single-stranded DNA. The biotin-labeled single-stranded DNA is combined with streptavidin, and a sequencing primer and a sequencing raw material are added after washing to perform pyrosequencing, so that the sequencing process and time are simplified. The invention uses rapid DNA preparation, constant temperature PCR amplification and pyrosequencing technology as a combination to detect the gene polymorphism of methylprednisolone adverse reaction risk assessment, and provides a gene angle suggestion for clinical personalized medication.
Drawings
FIG. 1 is a diagram showing an example of the detection results of pyrophosphoric acid from PAI-1(4G/5G) and ABCB1(3435CT) according to the present invention;
FIG. 2 is a diagram showing an example of the detection results of PAI-1(4G/4G) and ABCB1(3435TT) pyrophosphate provided by the present invention;
FIG. 3 is an exemplary diagram of PAI-1(5G/5G) and ABCB1(3435CC) pyrophosphate detection results provided by the present invention.
Detailed Description
The detection kit for methylprednisolone metabolic marker and the detection method thereof provided by the invention are further detailed and completely described below by combining with the embodiment. 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.
(I) design of specific primers
The kit of the invention designs specific amplification primers and sequencing primers aiming at the gene polymorphism of PAI-1(4G/5G) and ABCB1(C3435T) for detecting pyrophosphate PCR. Gene polymorphism sequences are based on published sequences in Genebank, and primer sequences are shown in Table 1 below.
TABLE 1 primer sequence Listing
(II) kit composition
The detection kit comprises the components shown in the following table 2:
TABLE 2 kit component table
| Serial number | Composition of | Indicating the amount of filling |
| 1 | Sample treatment liquid | 2000 uL X1 tube |
| 2 | Magnetic bead | 80 μ L X1 tube |
| 3 | Amplification reagent 1 | 500 μ L X1 tube |
| 4 | Amplification reagent 2 | Dry powder |
| 5 | PAI-1(4G/5G) sequencing primer | 70ul X1 tube |
| 6 | ABCB1(C3435T) sequencing primer | 70ul X1 tube |
| 7 | Positive control | 50 μ L X1 tube |
(III) the sample treatment solution preparation system comprises the following steps:
sample treatment solution containing 0.4% lithium lauryl sulfate, 0.3% Triton X-100, 15mg/mL sodium hydroxide, 10mM betaine, 10% trehalose, 5mM BSA, 50mM Tris-HCl, 100mM NaCl, pH 9.
(IV) the single-person configuration system of the detection kit amplification reagent 1 of the embodiment is as follows:
(V) the detection kit amplification reagent 2 of the present embodiment is configured as follows in a single-person configuration system:
PAI-1(4G/5G) pre-primer (0.32uM), PAI-1(4G/5G) (0.32uM), ABCB1(C3435T) pre-primer (0.32uM), ABCB1(C3435T) (0.32uM) dNTPS (0.3mM), strand displacement DNA polymerase (1.2 ng/. mu.L), single stranded DNA binding protein (3.2 ng/. mu.L), single stranded nucleic acid binding recombinase (4.8 ng/. mu.L), trehalose (0.2%), 10mM manganese acetate, 0.1M sorbitol, 5ug/mL BSA. The PCR system is shown in Table 3:
TABLE 3 PCR systems
| Composition (I) | Volume (ul) |
| Recombinase binding single-stranded nucleic acid (100 ng/. mu.L) | 1.2 |
| Single-stranded DNA binding protein (100 ng/. mu.L) | 0.8 |
| Strand Displacement DNA polymerase (100 ng/. mu.L) | 0.3 |
| dNTPs(25mM) | 0.3 |
| PAI-1(4G/5G) Pre-primer (20. mu.M) | 0.4 |
| PAI-1(4G/5G) rear primer (20. mu.M) | 0.4 |
| ABCB1(C3435T) front primer (20. mu.M) | 0.4 |
| ABCB1(C3435T) rear primer (20. mu.M) | 0.4 |
| Trehalose (20%) | 0.25 |
| 1M manganese acetate | 0.25 |
| 10M sorbitol | 0.25 |
| 5mg/mL BSA | 0.25 |
After the preparation is finished, 98 ul/tube is subpackaged and freeze-dried.
Example 2 kit detection procedure
The apparatus used in the present invention is as follows: thermostats, pyrosequencing instruments (Wuhan Firster Biotech, Inc.).
1) Taking 30 mu L of EDTA anticoagulated whole blood sample in a PCR amplification tube;
2) adding 100 μ L sample treatment solution and 4ul magnetic beads, and standing for 5 min;
3) placing the PCR amplification tube on a magnetic frame, and sucking out the mixed solution from the opposite side of the magnetic beads after the magnetic beads are completely adsorbed to one side;
4) adding the amplification reagent 1 into the amplification reagent 2 dry powder, and fully dissolving and uniformly mixing;
5) adding 25ul of prepared PCR reaction liquid into the PCR amplification tube obtained in the step 4), fully and uniformly mixing magnetic beads and the PCR reaction liquid, centrifuging, and carrying out constant-temperature reaction;
6) 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 |
| 42℃ | 30min | 1 |
7) Adding 40 mu L of binding solution and 3ul of agarose gel particles into a PCR reaction tube, adding 20 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;
8) centrifuging at 7,000 Xg for 1min, and discarding the supernatant;
9) adding 22uL of diluted working solution of the denatured liquid, standing for 5min, centrifuging for 1min at 7,000 Xg, and collecting by an EP tube to obtain a single-chain product;
10) to the EP tube, 150uL of washing buffer was added, and centrifuged at 7,000 Xg for 1 min. (repeat 3 times);
11) transferring the single-stranded product in the EP tube to a sequencing tube, and adding 3uL sequencing enzyme and 3uL sequencing substrate to each sequencing tube;
12) respectively adding 3uL sequencing enzyme and 3uL sequencing substrate into a sequencing tube;
13) taking an 8-calandria, and sequentially adding dATP, dTTP, dGTP, dCTP, PAI-1(4G/5G) sequencing primer, ABCB1(C3435T) sequencing primer and ddATP from one round 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;
14) pyrosequencing; the sequencing results are shown in FIGS. 1 to 3.
15) Interpretation of results
1) And (3) judging the effectiveness:
the blank control of the kit does not pass, and the detection result of the positive control is PAI-1(4G/5G) and ABCB1(C3435T) hybrid mutant.
2) Criteria for determination of results
In the DNA sequencing peak diagram of PAI-1(4G/5G),
the height of G is 4 times of T, namely type 4G/4G;
the height of G is 4.5 times of T, namely type 4G/5G;
the height of G is 5 times of T, namely 5G/5G;
in the DNA sequencing peak map of ABCB1(C3435T),
the frequency of C is not less than 90 percent, the frequency of T is not less than 10 percent, and the model is CC;
the frequency of 40% to C is less than or equal to 60%, and the frequency of 40% to T is less than or equal to 60%, which is CT type;
the frequency of T is not less than 90 percent, the frequency of C is not less than 10 percent, and the model is TT;
16) the correlation table of the gene detection result and the adverse reaction risk of methylprednisolone is as follows:
fifth, the performance test result of the kit
5.1. Specificity of
The result of the detection of 16 specific samples (including non-human DNA templates and dilutions of amplification products of different sites or homologous sites of the same human gene) is negative.
5.2. Accuracy of
When 12 reference products (including three genotypes of PAI-1(4G/5G) and ABCB1(C3435T) wild, heterozygous and mutant) with different genotypes in the kit range are detected, the corresponding genotypes can be detected.
5.3. Minimum detection limit
The minimum detection limit should not be higher than 2 ng/ul.
5.4. Repeatability of
In the detection kit, each reference substance is subjected to 10 times of detection, the results are corresponding mutation types, and the Coefficient of Variation (CV) of the Ct value of a corresponding detection channel is less than or equal to 5.0%.
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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<220>
<221> unsure
<222> (1)..(15)
<400> 6
cctttgctgc cctca 15
<210> 7
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> unsure
<222> (1)..(19)
<400> 7
cgtrggggag tcagccgtg 19
<210> 8
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> unsure
<222> (1)..(27)
<400> 8
cdatctcttc ctgtgacacc acccggc 27
<210> 9
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> unsure
<222> (1)..(20)
<400> 9
gcgtaggagd dtgcgtatct 20
Claims (10)
1. A methylprednisolone metabolic marker detection kit is characterized in that the gene polymorphism detection kit is used for designing specific amplification primers and sequencing primers aiming at polymorphism of PAI-1 and ABCB1, and the kit comprises the following components: sample processing fluid, magnetic beads, amplification reagent 1, amplification reagent 2, PAI-1 and ABCB1 sequencing primers, and positive controls.
2. The methylprednisolone metabolic marker detection kit according to claim 1, wherein the sequence of the specific primer group of PAI-1 is shown as SEQ ID NO 1-SEQ ID NO 2 of the sequence list; the sequence of the specific primer group of the ABCB1 is shown in a sequence table SEQ ID NO. 3-SEQ ID NO. 4.
3. The methylprednisolone metabolic marker detection kit as claimed in claim 1, wherein said PAI-1 sequencing primer and ABCB1 sequencing primer are respectively shown as SEQ ID NO 5-SEQ ID NO 6 of sequence Listing.
4. The methylprednisolone metabolic marker detection kit as claimed in claim 1, wherein the PAI-1 and ABCB1 share one assignment instruction as shown in SEQ ID NO. 9 of the sequence list.
5. The methylprednisolone metabolic marker detection kit as claimed in claim 1, wherein said sample treatment solution comprises 0.1% -0.6% of lithium dodecyl sulfate, 0.1-0.5% of triton X-100, 2-50mg/mL of sodium hydroxide, 5-15% of trehalose, 3-7 mmol/L of BSA, 20-80mM Tris-HCl and 100mM NaCl, and the pH is 8.5-9.5.
6. The methylprednisolone metabolic marker detection kit as claimed in claim 1, wherein said amplification reagent 1 comprises: amplification buffer, 15mM magnesium acetate.
7. The methylprednisolone metabolic marker detection kit as claimed in claim 1, wherein said amplification reagent 2 comprises: PAI-1 pre-primer 0.32uM, PAI-10.32 uM, ABCB1 pre-primer 0.32uM, ABCB 10.32uM, dNTPS 0.3mM, strand displacement DNA polymerase 1.2 ng/. mu.L, single-stranded DNA binding protein 3.2 ng/. mu.L, and single-stranded nucleic acid binding recombinase 4.8 ng/. mu.L.
8. The methylprednisolone metabolic marker detection kit as claimed in claim 1, wherein said positive control comprises PAI-1 and ABCB1 heterozygous genomic DNA with a concentration of 20 ng/ul.
9. The detection method of the methylprednisolone metabolic marker detection kit as claimed in any one of claims 1-8, wherein the detection method comprises the following steps:
a. mixing 100ul of sample treatment solution, 4ul of magnetic beads and 30ul of EDTA (ethylene diamine tetraacetic acid) anticoagulated whole blood sample, and standing at room temperature for 5 min;
b. placing the PCR amplification tube on a magnetic frame, and sucking out the mixed solution from the opposite side of the magnetic beads after the magnetic beads are completely adsorbed to one side;
c. adding the prepared PCR reaction solution into the PCR amplification tube obtained in the step b), fully and uniformly mixing magnetic beads and the PCR reaction solution, centrifuging, and carrying out constant-temperature reaction;
d. combining the binding solution containing the microbeads with the amplification product;
e. treating the denatured liquid to obtain a single-chain product;
f. adding a washing buffer solution for rinsing;
g. adding a sequencing enzyme and a sequencing substrate to each sequencing tube;
h. taking an 8-row pipe, and sequentially adding dATP, dTTP, dGTP, dCTP, PAI-1, ABCB1 sequencing primer and ddCTP from one round smooth end to the flat end;
i. and (4) pyrosequencing.
10. The methylprednisolone metabolic marker detection kit and the application of the detection method thereof as claimed in any one of claims 1-9, wherein the detection kit detects PAI-1 and ABCB1 gene polymorphisms to reflect methylprednisolone adverse reaction risk at a gene level.
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| CN202111048800.7A CN113755574A (en) | 2021-09-08 | 2021-09-08 | Detection kit and detection method for methylprednisolone metabolic marker |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113667743A (en) * | 2021-09-09 | 2021-11-19 | 菲思特(上海)生物科技有限公司 | Detection kit for budesonide metabolic marker and detection method and application thereof |
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2021
- 2021-09-08 CN CN202111048800.7A patent/CN113755574A/en not_active Withdrawn
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113667743A (en) * | 2021-09-09 | 2021-11-19 | 菲思特(上海)生物科技有限公司 | Detection kit for budesonide metabolic marker and detection method and application thereof |
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