CN113462758A - Detection kit for cisplatin metabolic marker, detection method and application thereof - Google Patents
Detection kit for cisplatin metabolic marker, detection method and application thereof Download PDFInfo
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Abstract
The invention discloses a detection kit for cis-platinum metabolic markers, a detection method and application thereof, wherein the kit is used for detecting gene polymorphisms of cis-platinum metabolic markers TPMT 3C A719G and XPC 2815A, and comprises the following components: TPMT 3C A719G amplification primer, TPMT 3C A719G sequencing primer, XPC 2815A amplification primer, XPC 2815A sequencing primer and positive control. The invention uses multiple RPA amplification and optimized pyrosequencing technology as a combination to detect the gene polymorphism related to cisplatin adverse reaction prediction, and the kit can simultaneously detect the TPMT 3C (A719G) and XPC (C2815A) gene polymorphism cisplatin drugs and provides a gene angle suggestion for clinical personalized medication.
Description
Technical Field
The invention relates to a detection kit for cisplatin metabolic markers, a detection method and application thereof, and belongs to the field of gene detection.
Background
Cisplatin has the characteristics of wide anticancer spectrum, strong action, synergistic action with various antitumor drugs, no cross drug resistance and the like, and is one of the most commonly used drugs in the current combined chemotherapy. Similar to bifunctional alkylating agents, DNA replication processes can be inhibited. Cisplatin is widely used in the treatment of various solid tumors, and ototoxicity is one of the main adverse reactions. The incidence of cisplatin-induced ototoxicity in pediatric patients is as high as 61%, mostly bilateral hearing loss, and often results in irreversible hearing loss. The clinical curative effect and the adverse drug reaction of the cisplatin drugs have great individual difference, so that how to reasonably, individually and safely use the thiopurine drugs for treatment has very important clinical significance.
The human XPC gene is located on chromosome 3p25 and comprises 16 exons and 15 introns, is 33.5 kb in length and encodes an XPC protein comprising 940 amino acid residues. The protein has the main functions of combining with an expression product of RAD23B gene to form an XPC-RAD23B compound capable of identifying a DNA damage part, guiding a II type transcription repair factor compound to be combined with the DNA damage part, and further activating downstream repair protein to complete the repair of damaged DNA. In the cisplatin-mediated signal transduction initiation process, the XPC protein can play a repairing role by activating the p53 cancer suppressor gene and inducing cell cycle arrest. A defect in the XPC gene can reduce the responsiveness of the p53 oncogene to cisplatin treatment and the activity of Caspase 3 (Caspase-3).
TPMT can inhibit cell death caused by cisplatin by promoting metabolism of cisplatin-purine complex and reducing cross-linking with DNA. The TPMT low enzymatic activity allele can increase the risk of cisplatin induced ototoxicity, and the TPMT mutant allele predicts 96 percent of positive predictive value of cisplatin induced hearing loss. The FDA approved cisplatin modification instructions in 2011, and medication safety information of TPMT genetic variation and cisplatin-induced ototoxicity in children was added. Children carrying the TPMT mutant allele are advised to switch to other platinum chemotherapeutic agents such as carboplatin, which are equally effective.
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 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 cisplatin metabolic marker and a detection method and application thereof based on multiple RPA amplification and pyrosequencing technologies.
In order to realize one of the above objects, the technical scheme of the detection kit for cisplatin 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 polymorphism of two genes, namely TPMT 3C (A719G) and XPC (C2815A), and comprises the following components: reagent 1-reagent 5.
Preferably, the specific primers are designed as shown in the following table:
preferably, the sequence of the specific primer group of the TPMT 3C (A719G) is shown in a sequence table SEQ ID NO. 1-SEQ ID NO. 2; the sequence of the specific primer group of the XPC (C2815A) is shown in a sequence table SEQ ID NO. 3-SEQ ID NO. 4.
Preferably, the TPMT 3C (A719G) sequencing primer and the XPC (C2815A) 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 TPMT 3C (A719G) sequencing primer is a sequence to be detected of the TPMT 3C (A719G), and is shown as a sequence table SEQ ID NO. 7; the sequencing region corresponding to the XPC (C2815A) sequencing primer is an XPC (C2815A) to-be-detected sequence, which is shown as a sequence table SEQ ID NO. 8.
Preferably, TPMT 3C (A719G) and XPC (C2815A) share one assignment instruction as shown in SEQ ID NO 9 of the sequence Listing. "ddG" in SEQ ID NO 9 indicates that the last base ddGTP was added in the sequencing reaction of TPMT 3C (A719G), and the addition of this base can terminate the sequencing reaction. The "-" in SEQ ID NO 9 indicates that the addition of the reagent was suspended for 3 min. XPC (C2815A) sequencing primers were added during this pause.
Preferably, the reagent 1 comprises: amplification buffer, 18mM magnesium acetate;
preferably, the reagent 2 comprises: TPMT 3C (a719G) rear primer (0.32uM), TPMT 3C (a719G) rear primer (0.32uM), XPC (C2815A) front primer (0.32uM), XPC (C2815A) rear 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%); simultaneous amplification of TPMT × 3C (a719G)/XPC (C2815A) can be performed under isothermal conditions.
Preferably, the reaction volume is 25ul, and the reaction conditions are as follows: 25min at 40 ℃.
Preferably, the positive control comprises 20ng/ul of genomic DNA heterozygous for TPMT 3C (A719G) and XPC (C2815A). 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 cisplatin adverse reaction related gene polymorphism detection method 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. binding the binding solution (containing the microbeads) with the amplification product;
c. treating the denatured liquid to obtain a single-chain product;
d. adding a washing buffer solution for rinsing;
e. adding a sequencing enzyme and a sequencing substrate to each sequencing tube;
f. taking an 8-calandria, and sequentially adding dATP, dTTP, dGTP, dCTP, TPMT 3C (A719G) sequencing primer, XPC (C2815A) sequencing primer and ddGTP from one round smooth end to a 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;
g. pyrosequencing;
h. determining the genotypes of the TPMT 3C (A719G) site and the XPC (C2815A) site cis-platinum site.
The invention also discloses a kit for adverse reaction of cisplatin and application of the method, wherein the detection kit is used for detecting TPMT 3C (A719G) and XPC (C2815A) so as to guide the prediction of adverse reaction of cisplatin from a gene level.
The invention amplifies TPMT 3C (A719G) and XPC (C2815A) in one tube by Recombinase Polymerase Amplification (RPA), and captures target single-stranded DNA by streptavidin. After rinsing the product, TPMT 3C (A719G) sequencing primer and sequencing material are added to perform pyrosequencing, and ddGTP is added to the last base to terminate the sequencing reaction. Then XPC (C2815A) sequencing primer and corresponding dNTP are added 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.
Compared with the prior art, the invention uses the multiple RPA amplification and optimized pyrosequencing technology as a combination to detect the gene polymorphism related to cisplatin adverse reaction prediction, and the kit can simultaneously detect the TPMT 3C (A719G) and XPC (C2815A) gene polymorphism cisplatin drugs and provides gene angle suggestions for clinical personalized medication.
Drawings
FIG. 1 is a graph showing the result of the type TPMT 3C (A719G) AA/XPC (C2815A) CC sequencing provided by the present invention;
FIG. 2 is a graph showing an example of the sequencing results of the type TPMT 3C (A719G) AG/XPC (C2815A) CA provided by the present invention;
fig. 3 is an exemplary graph of the AA type sequencing results of TPMT × 3C (a719G) GG/XPC (C2815A) provided by the present invention.
Detailed Description
The following examples are provided to further detail and fully illustrate the detection kit for cisplatin metabolic marker, the detection method and the application thereof. 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 TPMT 3C (A719G) and XPC (C2815A) 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:
(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: TPMT 3C (a719G) front primer (0.32uM), TPMT 3C (a719G) rear primer (0.32uM), XPC (C2815A) front primer (0.32uM), XPC (C2815A) rear 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%);
| 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 |
| TPMT 3C (A719G) Pre-primer (20. mu.M) | 0.4 |
| TPMT 3C (A719G) rear primer (20. mu.M) | 0.4 |
| XPC (C2815A) pre-primer (20. mu.M) | 0.4 |
| XPC (C2815A) rear primer (20. mu.M) | 0.4 |
| Trehalose (20%) | 0.25 |
After the preparation is finished, 98 ul/tube is subpackaged and freeze-dried.
Example 2 detection of Pyrophosphoric acid
The apparatus used in the present invention is as follows: thermostats, pyrosequencing instruments (Wuhan Firster 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 |
| 40℃ | 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) centrifuging at 7,000 Xg for 1min, and discarding the supernatant;
3) 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.
4) To the EP tube, 150uL of washing buffer was added, and centrifuged at 7,000 Xg for 1 min. (repeat 3 times)
5) The single stranded product from the EP tube was transferred to sequencing tubes and 3uL of sequencing enzyme and 3uL of sequencing substrate was added to each sequencing tube.
6) Respectively adding 3uL sequencing enzyme and 3uL sequencing substrate into a sequencing tube;
7) taking an 8-calandria, and sequentially adding dATP, dTTP, dGTP, dCTP, TPMT 3C (A719G) sequencing primer, XPC (C2815A) sequencing primer and ddGTP from one round smooth end to a 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;
8) pyrosequencing; the sequencing 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 TPMT × 3C (a719G) or XPC (C2815A) types.
2) Criteria for determination of results
In the DNA sequencing peak plot of tpmt x 3C (a719G),
the frequency of A is not less than 90 percent, the frequency of G is not less than 10 percent, and the product is GG type;
the frequency of 40% to A is 60% and the frequency of 40% to G is 60%, which is AG type;
the frequency of G is not less than 90 percent, the frequency of A is not less than 10 percent, and the product is GG type;
XPC (C2815A) DNA sequencing peak map,
the frequency of C is not less than 90 percent, the frequency of A 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 A is less than or equal to 60%, which is CT type;
the frequency of A is not less than 90 percent, the frequency of C is not less than 10 percent, and the product is AA type
Example 3 correlation of Gene detection results with cisplatin adverse reaction prediction
Correspondence between gene locus and drug
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 of the cisplatin metabolic marker is used for detecting the gene polymorphism of two genes of the cisplatin metabolic marker TPMT 3C A719G and XPC 2815A, and comprises the following components: TPMT 3C A719G amplification primer, TPMT 3C A719G sequencing primer, XPC 2815A amplification primer, XPC 2815A sequencing primer and positive control.
2. The cisplatin metabolic marker detection kit as claimed in claim 1, wherein the TPMT 3C A719G amplification primer is represented by SEQ ID NO 1-2 of sequence Listing.
3. The detection kit for the cisplatin metabolic marker as claimed in claim 1, wherein the XPC C2815A amplification primer is shown in sequence table SEQ ID NO. 3-4.
4. The cisplatin metabolic marker detection kit as claimed in claim 1, wherein the TPMT 3C A719G sequencing primer is shown as SEQ ID NO. 5 of sequence Listing, and the XPC 2815A sequencing primer is shown as SEQ ID NO. 6 of sequence Listing.
5. The cisplatin metabolic marker detection kit as claimed in claim 1, wherein the sequencing primer is a conjugate of agarose gel particles and amino-labeled DNA sequence.
6. The cisplatin metabolic marker detection kit as claimed in claim 1, wherein the kit further comprises amplification buffer, 18mM magnesium acetate, dNTPS, strand displacement DNA polymerase, single-stranded DNA binding protein, recombinase binding single-stranded nucleic acid, and trehalose.
7. The cisplatin metabolic marker detection kit as claimed in claim 6, wherein the final concentration of each component in the kit is: 0.32uM of each primer before and after TPMT 3C A719G amplification, 0.32uM of each primer before and after XPC 2815A amplification, 0.3mM dNTPS, 1.2 ng/muL of strand displacement DNA polymerase, 3.2 ng/muL of single-stranded DNA binding protein, 4.8 ng/muL of recombinase for binding single-stranded nucleic acid and 0.2% trehalose.
8. The cisplatin metabolic marker detection kit as claimed in claim 1, wherein the positive control comprises 20ng/ul TPMT × 3C A719G, XPC 2815A heterozygote genomic DNA.
9. A detection kit for cisplatin metabolic marker as defined in any of claims 1-8, 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. binding the binding solution (containing the microbeads) with the amplification product;
c. treating the denatured liquid to obtain a single-chain product;
d. adding a washing buffer solution for rinsing;
e. adding a sequencing enzyme and a sequencing substrate to each sequencing tube;
f. taking an 8-calandria, and sequentially adding dATP, dTTP, dGTP, dCTP, TPMT 3C (A719G) sequencing primer, XPC (C2815A) sequencing primer and ddGTP from one round smooth end to a flat end;
g. pyrosequencing;
h. determining the genotypes of the TPMT 3C (A719G) site and the XPC (C2815A) site cis-platinum site.
10. The use of the cisplatin metabolic marker detection kit as claimed in claim 1, wherein said detection kit is used for in vitro detection of TPMT × 3C A719G and XPC 2815A gene polymorphism in a sample to be tested.
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