CN113549686A - Detection kit for calcium ion antagonist metabolic marker, detection method and application thereof - Google Patents
Detection kit for calcium ion antagonist metabolic marker, detection method and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The invention discloses a detection kit for a calcium ion antagonist metabolic marker, a detection method and application thereof, wherein the kit comprises the following components: CYP3a5 x 3 amplification primer, CYP3a5 x 3 sequencing primer, NPPA (T2238C) amplification primer, NPPA (T2238C) sequencing primer, and positive control. The invention can quickly and effectively generate a large amount of amplification products at a constant temperature by amplifying CYP3A5 x 3 and NPPA (T2238C) in a plurality of RPA one-tube manner. The single-stranded DNA is captured specifically by the amino-labeled single-stranded DNA directly combined with the carboxyl modifier, and after washing, a sequencing primer and a sequencing raw material are added to perform pyrosequencing, so that the damage of a strong alkaline reagent to an amplified fragment is reduced, and the flow and time of sequencing treatment are simplified.
Description
Technical Field
The invention relates to a detection kit for a calcium ion antagonist metabolic marker, and a detection method and application thereof, and belongs to the field of gene detection.
Background
The antihypertensive drugs commonly used in clinical practice at present are of 5 types: diuretics, angiotensin converting enzyme inhibitors, angiotensin receptor blockers, beta-blockers, calcium antagonists. The drug is related to a plurality of drug metabolizing enzymes, transporters and receptors in the in vivo metabolic process, and the existence of gene polymorphism of the drugs finally causes obvious individual difference in the treatment effect, the generated poor development, the drug tolerance and the like of patients taking the same drug in the same way.
Calcium antagonists are chemical agents that lower blood pressure by blocking calcium channels. It can selectively inhibit Ca2+ from entering into cell via calcium channel on cell membrane, and has effects of dilating blood vessel, relieving negative muscle strength, relaxing smooth muscle of blood vessel, and reducing peripheral vascular resistance, thereby lowering blood pressure, but not reducing blood flow of brain, coronary artery and kidney. Nifedipine and amlodipine are commonly used calcium ion antagonists for treating hypertension. The research shows that the polymorphism of NPPA gene T2238C can affect the curative effect of diuretic and calcium ion antagonist. However, the gene has a low mutation frequency in Chinese population. The reduction amplitude of both systolic pressure and diastolic pressure of a patient with rs2238032G/T site GG type of CACNA1C gene in Chinese population at the end of 4 weeks is reported to be significantly lower than TT or GT type delta SBP4.0 +/-11.6 mmHg vs.19.9 +/-14.1 mmHg, and p is 0.007; delta DBP-2.2 +/-4.5 mmHg vs.9.9 +/-8.9 mmHg, p is 0.005), and the blood pressure of a GG type patient can reach the standard only by increasing the dose of amlodipine or adding other antihypertensive drugs.
The CYP3A family is a drug metabolism enzyme which mediates drug metabolism in human bodies to the maximum extent, and the oxidation and reduction reactions of more than 50% of clinical common drugs are catalyzed by CYP3A4 and CYP3A 5. Calcium antagonists are metabolized primarily by CYP3a5 enzymes. CYP3a5 expression and activity are highly polymorphic, with wide individual and ethnic differences. Wherein, CYP3A5 x 3(A6986G, intron3) is common, and Chinese people are about 65-73%. The CYP3A5 gene mutation is the main reason for the difference of enzyme activity, the CYP3A5 mutation enables the enzyme activity to be obviously reduced, the metabolism of a substrate drug is slowed down, and the drug action and the toxic and side reaction are obviously increased. Studies show that CYP3A5 x 3 genotype reduces amlodipine clearance rate and increases antihypertensive efficacy.
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 calcium ion antagonist metabolic marker based on multiple RPA amplification and optimized pyrosequencing technology, and a detection method and application thereof.
In order to achieve one of the above objects, the technical scheme of the detection kit for the calcium ion antagonist metabolic marker adopted by the invention is as follows:
the detection kit provided by the invention is used for designing specific amplification primers and sequencing primers aiming at polymorphism of CYP3A5 x 3 and NPPA (T2238C) genes, and comprises the following components: amplification reagent 1, amplification reagent 2, CYP3a5 × 3 sequencing primer, NPPA (T2238C) sequencing primer, positive control.
Preferably, the specific primers are designed as shown in the following table:
| primer name | SEQ ID | Sequence (5 'to 3') | Decoration |
| CYP3A5 x 3 pre-primer | 1 | CTTCCGCAGAGGAGGGTTT | |
| CYP3A5 x 3 rear primer | 2 | CAGACCCCCACATCATGCTG | |
| NPPA (T2238C) pre-primer | 3 | CGACCCTACAAAGGAAGCACTAA | |
| NPPA (T2238C) rear primer | 4 | CGACCCTACAAAGGAAGCACTAA | |
| CYP3A5 x 3 sequencing primer | 5 | AGCCTGGATGGGGAC | 5`NH2 C6 |
| NPPA (T2238C) sequencing primer | 6 | CCTTCTGCTGTCTCACTT | 5`NH2 C6 |
Preferably, the sequence of the specific primer group of CYP3A5 x 3 is shown in sequence tables SEQ ID NO 1-SEQ ID NO 2; the specific primer group sequence of the NPPA (T2238C) is shown in a sequence table SEQ ID NO: 3-SEQ ID NO: 4. More preferably, the amplification primers are all common amplification primers.
Preferably, the CYP3A5 x 3 sequencing primer and the NPPA (T2238C) 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 combination of agarose gel particles and amino-labeled DNA sequences, and serves as both a sequencing primer and a capture probe. After the primer is combined with the amplified single-stranded DNA, the primer can be directly used for sequencing reaction through simple washing.
More preferably, the sequencing primer is prepared by mixing a synthetic amino-labeled sequencing primer with carboxyl-modified agarose gel microparticles in a binding solution, washing to remove free sequencing primer, and storing in a storage solution at 2-8 ℃.
Preferably, the reagent 1 comprises: amplification buffer, 18mM magnesium acetate;
preferably, the reagent 2 comprises: CYP3a5 x 3 pre-primer, CYP3a5 x 3 post-primer, NPPA (T2238C) pre-primer, NPPA (T2238C) post-primer, dNTPS, strand displacement DNA polymerase, single-stranded DNA binding protein, recombinase binding single-stranded nucleic acid, trehalose; simultaneous amplification of CYP3a5 x 3/NPPA (T2238C) was performed under isothermal conditions.
More preferably, the final concentrations of the components of the reagent 2 are respectively as follows: CYP3A5 × 3 pre-primer (0.4uM), CYP3A5 × 3 post-primer (0.4uM), NPPA (T2238C) pre-primer (0.4uM), NPPA (T2238C) post-primer (0.4uM), dNTPS (0.3mM), strand displacement DNA polymerase (1.2 ng/. mu.L), single-stranded DNA binding protein (3.2 ng/. mu.L), recombinase binding to single-stranded nucleic acid (4.8 ng/. mu.L), trehalose (0.2%).
Preferably, the positive control comprises CYP3A5 x 3, NPPA (T2238C) 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 gene polymorphism related to calcium ion antagonist medication 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 multiple RPA amplification;
b. combining 10ul of reaction product with 3ul of sequencing primer;
c. pyrosequencing;
d. the genotypes of the CYP3a5 x 3 locus, the NPPA (T2238C) locus were determined.
Preferably, the reaction volume is 25ul, and the amplification conditions are: 25min at 40 ℃.
The invention also discloses a detection kit for the calcium ion antagonist metabolic marker and application of the method, and the detection kit is used for detecting CYP3A5 and NPPA (T2238C) gene polymorphism so as to guide calcium ion antagonist medication guidance 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: capable of binding to a single-stranded nucleic acid oligonucleotide primer), single-stranded DNA binding protein SSB), and strand-displacing DNA polymerase. 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 proceeds very quickly and detectable levels of amplification product are typically obtained within ten minutes (Piepenburg et al, 2006).
The invention can quickly and effectively generate a large amount of amplification products at a constant temperature by amplifying CYP3A5 x 3 and NPPA (T2238C) in a plurality of RPA one-tube manner. The single-stranded DNA is captured specifically by the amino-labeled single-stranded DNA directly combined with the carboxyl modifier, and after washing, a sequencing primer and a sequencing raw material are added to perform pyrosequencing, so that the damage of a strong alkaline reagent to an amplified fragment is reduced, and the flow and time of sequencing treatment are simplified.
Compared with the prior art, the kit provided by the invention detects the gene polymorphism related to the calcium ion antagonist by taking the multiple RPA amplification and optimized pyrophosphate sequencing technology as a combination, can simultaneously detect CYP3A5 x 3 and NPPA (T2238C) gene polymorphism, and provides a gene angle suggestion for clinical personalized medication.
Drawings
FIG. 1 is a diagram showing an example of the result of detecting CYP3A5 x 3 pyrophosphate according to the present invention;
FIG. 2 is an exemplary diagram of the NPPA (T2238C) pyrophosphate detection results provided by the present invention.
Detailed Description
The following examples are provided to further describe the detection kit for calcium antagonist metabolic markers, the detection method and the application thereof in detail and in full. 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 detection kit provided by the invention designs specific amplification primers and sequencing primers aiming at CYP3A5 x 3 and NPPA (T2238C) 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 NPPA gene sequence is as follows:
the sequence of CYP3A5 is as follows:
the primer sequences of this example are as follows:
(II) the detection kit of the embodiment comprises the following components:
(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 final concentration of each component of the reagent 2 is as follows: CYP3a5 x 3 rear primer (0.4uM), CYP3a5 x 3 rear primer (0.4uM), NPPA (T2238C) front primer (0.4uM), NPPA (T2238C) rear primer (0.4uM), 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%);
| composition (I) | Volume (ul) |
| Single-stranded nucleic acid binding recombinase at 100 ng/. mu.L | 1.2 |
| Single-stranded |
0.8 |
| Strand-displacing |
0.3 |
| dNTPs 25mM | 3 |
| CYP3A5 x 3 pre-primer 20. mu.M | 0.5 |
| CYP3A5 x 3 rear primer 20. mu.M | 0.5 |
| NPPA (T2238C) pre-primer 20. mu.M | 0.5 |
| NPPA (T2238C) rear primer 20. mu.M | 0.5 |
| Trehalose (20%) | 0.25 |
After the preparation is finished, 166.1 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)
①12.5mL of 1M Tris buffer pH 8
②50ul 10％
-20
(3) Sealing liquid
1) Taking 500ul of magnetic beads, magnetically attracting for 1min, and removing supernatant;
2) washing 1000ml 100MES solution for 1min, removing supernatant, and washing repeatedly for 2 times;
3) 500ul of each of 10mg/ml EDS and 10mg/ml NHS was added to 10ul of capture primer.
4) At 25 deg.C for 30min
5) Taking 1000ml of confining liquid, mixing uniformly, absorbing magnetism for 1min, and removing supernatant;
6) taking 1000ml TT Buffer, mixing uniformly, absorbing magnetism for 1min, and removing supernatant; washing was repeated 3 times;
7) add 1ml TE buffer to dissolve.
Example 2 detection of Pyrophosphoric acid
The apparatus used in the present invention is as follows: a PCR amplification instrument: ABI 2720PCR instrument;
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. Freeze-dry directly to reagent 2) add 440ul of reagent 1, vortex and mix well 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 |
| 40℃ | 25min。 | 1 |
(4) Pyrophosphoric acid sequencing
1) Adding 40 mu L of binding solution and 3ul of the binder of the agarose gel particles and the DNA sequence 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, oscillating at 1100rpm for 10min, and fully combining the sequencing primer with the single-stranded PCR product;
2) centrifuging at 7,000 Xg for 1min, and discarding the supernatant;
3) adding 150uL washing buffer solution into an EP tube, centrifuging at 7000g for 1min, and removing supernatant;
4) respectively adding 3uL sequencing enzyme and 3uL sequencing substrate into a sequencing tube;
5) 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;
6) sequencing was performed according to the instrument instructions, and the instrument assignment instructions were as follows.
(5) Interpretation of results
1) And (3) judging the effectiveness:
the blank control of the kit failed, and the positive control detected CYP3A5 x 3, NPPA (T2238C) type.
2) Criteria for determination of results
a.CYP3A5. multidot.3 DNA sequencing peak map,
the frequency of T is more than or equal to 90 percent, the frequency of C is more than or equal to 10 percent, namely TT (1/1) type;
frequency of 40% ≦ T ≦ 60%, frequency of 40% ≦ C ≦ 60%, which is CT (× 1/× 3) type;
the frequency of C is more than or equal to 90 percent, the frequency of T is more than or equal to 10 percent, and the model is CC (. about.3/. about.3);
in the DNA sequencing peak plot of NPPA (T2238C),
the frequency of T is larger than or equal to 90 percent, the frequency of C is smaller than or equal to 10 percent, and the model is NPPA (T2238C) AA;
the frequency of 40% to T is less than or equal to 60%, the frequency of 40% to C is less than or equal to 60%, and the NPPA (T2238C) AG type is obtained;
the frequency of C is larger than or equal to 90 percent, and the frequency of T is smaller than or equal to 10 percent, namely NPPA (T2238C) GG type;
example 3 correlation of Gene assay results with Metabolic Activity
The correlation of the gene assay results with metabolic activity is summarized:
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. A detection kit for calcium ion antagonist metabolic markers is used for detecting the gene polymorphism of metabolic related genes CYP3A5 x 3 and NPPA T2238C of a calcium ion antagonist, and comprises the following components: CYP3a5 x 3 amplification primer, CYP3a5 x 3 sequencing primer, NPPA T2238C amplification primer, NPPA T2238C sequencing primer, and positive control.
2. The detection kit for the calcium ion antagonist metabolic marker according to claim 1, wherein the CYP3A5 x 3 amplification primer is represented by SEQ ID NO: 1-2 of the sequence table.
3. The detection kit for the calcium ion antagonist metabolic marker according to claim 1, wherein the NPPA T2238C amplification primer is shown as SEQ ID NO. 3-4 of the sequence table.
4. The detection kit for the metabolic marker of the calcium ion antagonist according to claim 1, wherein the sequencing primer CYP3A5 x 3 is shown in sequence table SEQ ID NO. 5, and the sequencing primer NPPA T2238C is shown in sequence table SEQ ID NO. 6.
5. The kit for detecting the metabolic marker of the calcium ion antagonist according to claim 1, wherein the sequencing primer is a combination of agarose gel particles and amino-labeled DNA sequences.
6. The kit for detecting a metabolic marker for a calcium ion antagonist according to claim 1, wherein the kit further comprises an amplification buffer, 18mM magnesium acetate, dNTPS, strand displacement DNA polymerase, single-stranded DNA binding protein, recombinase binding to single-stranded nucleic acid, and trehalose.
7. The kit for detecting the metabolic marker of the calcium ion antagonist according to claim 6, wherein the final concentration of each component in the reagent 2 is as follows: 0.4uM for each primer before and after CYP3A5 x 3 amplification, 0.4uM for each primer before and after NPPA T2238C amplification, 0.3mM dNTPS, 1.2 ng/uL for strand-displacement DNA polymerase, 3.2 ng/uL for single-stranded DNA binding protein, 4.8 ng/uL for single-stranded nucleic acid binding recombinase, and 0.2% trehalose.
8. The kit for detecting a metabolic marker for a calcium antagonist according to claim 1, wherein the positive control comprises genomic DNA heterozygous for CYP3a5 x 3 and NPPA T2238C at a concentration of 20 ng/ul.
9. A detection method using the calcium ion antagonist metabolic marker detection kit according to any one of claims 1 to 8, wherein the detection method comprises the following steps:
a. amplifying the amplification reaction solution and 5ul of genome DNA to be detected by adopting multiple RPA amplification;
b. combining 10ul of reaction product with 3ul of sequencing primer;
c. pyrosequencing;
d. the genotypes of the CYP3a5 x 3 locus and the NPPA T2238C locus were determined.
10. Use of a detection kit for a calcium antagonist metabolic marker according to any one of claims 1 to 8, wherein the detection kit is used for in vitro detection of polymorphisms of CYP3A5 x 3 and NPPA T2238C genes in a sample to be detected.
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Cited By (2)
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| CN114150052A (en) * | 2021-10-29 | 2022-03-08 | 上海普然生物科技有限公司 | Detection kit for FVII gene polymorphism and methylation joint detection, detection method and application thereof |
| CN114182004A (en) * | 2021-10-29 | 2022-03-15 | 上海普然生物科技有限公司 | Detection kit for detecting sensitivity of temozolomide and detection method thereof |
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| CN114150052A (en) * | 2021-10-29 | 2022-03-08 | 上海普然生物科技有限公司 | Detection kit for FVII gene polymorphism and methylation joint detection, detection method and application thereof |
| CN114182004A (en) * | 2021-10-29 | 2022-03-15 | 上海普然生物科技有限公司 | Detection kit for detecting sensitivity of temozolomide and detection method thereof |
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