CN113667752A

CN113667752A - Detection kit for daunorubicin metabolic marker and detection method and application thereof

Info

Publication number: CN113667752A
Application number: CN202111055354.2A
Authority: CN
Inventors: 刘丹; 孙悦; 陈立波
Original assignee: Fist Shanghai Biotechnology Co ltd
Current assignee: Fist Shanghai Biotechnology Co ltd
Priority date: 2021-09-09
Filing date: 2021-09-09
Publication date: 2021-11-19

Abstract

The invention discloses a detection kit for a daunorubicin metabolic marker, a detection method and application thereof, wherein the detection kit is used for detecting the polymorphism of two genes of the daunorubicin metabolic marker SLC28A3 and UGT1A6 x 4, the kit designs a specific amplification primer and a sequencing primer according to the polymorphism of the two genes of SLC28A3 and UGT1A6 x 4, and the kit comprises the following components: amplification reaction solution, SLC28A3 sequencing primer, UGT1a6 × 4 sequencing primer and positive control. The invention uses the technologies of blood direct amplification, rapid amplification and optimized pyrosequencing as a combination to detect gene polymorphism related to daunorubicin curative effect and adverse reaction prediction, and provides gene angle suggestions for clinical personalized daunorubicin administration.

Description

Detection kit for daunorubicin metabolic marker and detection method and application thereof

Technical Field

The invention relates to a detection kit for daunorubicin metabolic markers, a detection method and application thereof, and belongs to the field of gene detection.

Background

Anthracyclines are chemical substances with antitumor activity produced by microorganisms, including doxorubicin (doxorubicin), epirubicin (epirubicin), daunorubicin, aclarubicin and the like, widely used for treating hematological malignancies and solid tumors, including acute leukemia, lymphoma, breast cancer, ovarian cancer, gastric cancer, soft tissue sarcoma and the like, and have the action mechanism that by embedding a DNA molecule, non-specifically inserted between adjacent base pairs, the DNA molecule is locally uncoiled and binds to a ribose-phosphate backbone, which can interfere with the reconnection of a broken DNA double strand by topoisomerase II, thereby hindering DNA replication, transcription and RNA synthesis processes. Anthracyclines are mainly hydroxylated or reduced in vivo by metabolic enzymes in the liver, such as carbonyl reductases and aldo-keto reductases, to form the corresponding secondary alcohol metabolites. Researches find that various efflux and uptake transporters such as P-glycoprotein (P-gp), multidrug resistance related protein (MRP) and cation transporter (OCT) are involved in the transportation of anthracycline drugs in cells, and influence the distribution of the drugs in vivo. The adverse reactions of the anthracycline are mainly cardiotoxicity, bone marrow suppression, gastrointestinal tract reaction and the like, wherein the cardiotoxicity is the most serious adverse reaction of the anthracycline, and the generation mechanism of the cardiotoxicity is not very clear at present. The chronic and delayed cardiotoxicity of anthracyclines is positively correlated with their cumulative dose, with greater doses of the drug causing more severe cardiac damage. In clinical application, the Anthracycline Cardiotoxicity (ACT) has great individual difference, the low dose anthracycline can cause cardiotoxicity, some patients treated by the low dose adriamycin have abnormal cardiac function in long-term follow-up, and the cardiac damage can be observed in other patients before the maximum cumulative dose of the anthracycline is reached. Anthracyclines are metabolized in vivo by a variety of metabolic enzymes, and their efflux and uptake in cells are also affected by a variety of transporters, and polymorphisms exist in the genes encoding these metabolic enzymes and transporters. The gene polymorphism of metabolic enzyme and transporter can influence the absorption, distribution, metabolism and excretion of the anthracycline in vivo, thereby influencing the pharmacokinetics and pharmacodynamics of the anthracycline in vivo and the difference of adverse reactions.

Glucuronic acid transferase 1A6, UGT is one of the most important enzymes for biphasic metabolism in vivo, and can catalyze glucuronic acid to perform glucuronic acid binding reaction with endogenous or exogenous chemical substances. UGT is mainly present in the endoplasmic reticulum membrane and the nuclear membrane of hepatocytes, and other tissues such as kidney and intestine are distributed to different extents. Glucuronidation is an important detoxification pathway in the body, and affects the decomposition, metabolism and excretion of many drugs and poisons. Although doxorubicin and daunorubicin parent drugs do not undergo glucuronidation, their metabolites can be metabolized by UGT to produce glucuronidated products. Therefore UGT gene mutations alter their glucuronidation levels, leading to the accumulation of toxic anthracycline metabolites.

SLC28A3(hCNT3) is expressed in various tissues throughout the body, including the heart. SLC28A3 selectively transports pyrimidines and purines as well as certain anticancer drugs such as gemcitabine and fludarabine. Recent studies have found that SLC28a3 can also transport some anthracyclines (e.g., doxorubicin and daunorubicin) into cells. The SLC28A3 mutation may reduce the expression of SCL28A3 mRNA. In prior studies on the effect of metabolic enzyme and transporter gene polymorphisms on ACT, it has been concluded and relatively high-grade evidence indicates that patients with UGT1a6 mutations have a higher risk of ACT, while patients with SLC28a38 mutations have a lower risk of ACT, and therefore it is recommended that pediatric cancer patients be tested at the SLC28A3 and UGT1a6 gene sites prior to doxorubicin or daunorubicin treatment.

At present, methods for detecting gene polymorphism mainly include a direct sequencing method, a chip method, a high-resolution melting curve method, an allele specific amplification method, a taqman fluorescence probe method and the like. 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, the allele specific amplification method and the Taqman fluorescent probe method have the advantages of simple steps, low specificity, higher requirements on instruments and equipment or high test cost. Therefore, there is a need to establish a simple, rapid, efficient, and inexpensive method for detecting gene polymorphisms.

Disclosure of Invention

In view of the above problems in the prior art, the present invention aims to obtain a detection kit for daunorubicin metabolic markers, and a detection method and application thereof.

In order to achieve one of the above objects, the technical solution of the detection kit for daunorubicin metabolic markers adopted by the present 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 two genes, namely SLC28A3(C1381T) and UGT1A6 x 4(G > T), and comprises the following components: amplification reaction solution, SLC28A3(C1381T) sequencing primer, UGT1a6 × 4(G > T) sequencing primer, and positive control.

Preferably, the specific primers are designed as shown in the following table:

preferably, the sequence of the specific primer group of the SLC28A3(C1381T) is shown as the sequence tables SEQ ID NO. 1-SEQ ID NO. 2; the sequence of the specific primer group of UGT1A 6X 4(G > T) is shown in sequence tables SEQ ID NO. 3-SEQ ID NO. 4.

Preferably, the SLC28A3(C1381T) sequencing primer and the UGT1A 6X 4(G > T) 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. The binding to DNA is more stable than the binding of DNA/DNA

Preferably, the sequencing region corresponding to the sequencing primer of the SLC28A3(C1381T) is the sequence to be detected of the SLC28A3(C1381T), and is shown as the sequence table SEQ ID NO. 7; the sequencing region corresponding to the UGT1A 6X 4(G > T) sequencing primer is a sequence to be detected in UGT1A 6X 4(G > T), and is shown in a sequence table SEQ ID NO. 8.

Preferably, SLC28A3(C1381T) and UGT1A 6X 4(G > T) share a common assignment command as shown in SEQ ID NO:9 of the sequence Listing.

Preferably, the amplification reaction solution comprises SLC28A3(C1381T) and UGT1A6 x 4(G > T) specific amplification primers, and further comprises blood sample PCR premix (2X) and trehalose.

More preferably, the concentrations of the components of the reaction solution are SLC28A3(C1381T) pre-primer (0.2uM), SLC28A3(C1381T) post-primer (0.2uM), UGT1a6 x 4(G > T) pre-primer (0.25uM), UGT1a6 x 4(G > T) post-primer (0.25uM), PCR premix (1 x), trehalose (0.2%);

preferably, the PCR premix is Blood Direct PCR Master Mix (2X) and contains Blood-resistant Hemotaq^TMDNA polymerase exhibits an extremely high resistance to various PCR inhibitors such as hemoglobin in whole blood.

Preferably, to achieve the highest detection sensitivity, the maximum amount of blood added to a 20. mu.l PCR amplification system can be up to 4. mu.l, i.e., 20% by volume.

Preferably, the positive control comprises genome DNA of SLC28A3-1381C/T type and UGT1A6 x 1/x 4 type at a concentration of 20ng/ul, provides a reference for type determination of unknown samples, and controls the effectiveness of the reaction solution.

Preferably, the reaction volume is 20ul, and the reaction conditions are as follows: the pre-denaturation temperature was set to 95 ℃, the pre-denaturation time was set to 5min, the denaturation temperature was set to 95 ℃, the denaturation time was set to 0s, the annealing extension temperature was set to 58 ℃, the annealing extension time was set to 0s, and amplification was carried out for 40 cycles.

Preferably, the PCR tube sealing film used for amplification has a concave design matched with the heating column at the PCR reaction hole, the thickness of the concave design is 85 microns, the attaching degree is high, and the heat transfer is fast. More preferably, the PCR tube sealing film is permeable, and the product recovery can be performed by using a pipette tip or a probe.

The invention also discloses a gene polymorphism detection method related to daunorubicin curative effect and adverse reaction prediction by adopting the kit, which comprises the following steps:

a) uniformly mixing the amplification reaction solution with 4ul of EDTA (ethylene diamine tetraacetic acid) anticoagulated whole blood to be detected to perform PCR amplification;

b) mixing the binding solution containing streptavidin labeled microbeads with the amplification product;

c) adding a washing buffer solution for rinsing;

d) treating the denatured liquid to obtain a single-chain product;

e) adding a washing buffer solution for rinsing;

f) adding a sequencing enzyme and a sequencing substrate to each sequencing tube;

g) taking an 8-row pipe, and sequentially adding dATP, dTTP, dGTP, dCTP, SLC28A3(C1381T) sequencing primer, UGT1A6 x 4(G > T) sequencing primer and ddGTP 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;

h) pyrosequencing;

i) and determining the genotype.

The invention also discloses a kit and an application of the method for predicting the curative effect and adverse reaction of daunorubicin, wherein the kit is used for detecting SLC28A3(C1381T) and UGT1A 6X 4(G > T) so as to predict the curative effect and adverse reaction of daunorubicin from a gene level.

Compared with the prior art, the method provided by the invention uses the technologies of direct blood amplification, rapid amplification and optimized pyrosequencing as a combination to detect gene polymorphisms related to daunorubicin curative effect and adverse reaction prediction, and provides a gene angle suggestion for clinical personalized daunorubicin administration.

The rapid amplification method is optimized mainly from three aspects, on one hand, a blood direct amplification mode is adopted, the step of nucleic acid extraction is omitted, and only the sample and other components necessary for PCR are added into a reaction tube and mixed uniformly. On the other hand, the heating module of the PCR instrument is designed to be composed of a heating base and a heating column, the periphery of the heating column is connected with the base, the middle of the heating column corresponds to the reaction hole, the heating module extends into the PCR reaction tube during PCR amplification, so that the reaction liquid is dispersed between the heating column and the wall of the PCR tube, and the temperature is changed simultaneously from the middle and the periphery, thereby remarkably improving the heat transfer efficiency, reducing the temperature change difference of each part of the reaction liquid, improving the temperature consistency and the temperature change speed of the whole reaction liquid, and providing another key element for the rapid amplification of the PCR. In the third aspect, double PCR is adopted to amplify two sites of SLC28A3(C1381T) and UGT1A6 x 4(G > T), and pyrosequencing is carried out on the two sites in one reaction. The sequencing is carried out by firstly adding a sequencing primer of SLC28A3(C1381T) and sequencing raw materials to carry out pyrosequencing, and adding ddNTP into the last base to terminate the sequencing reaction. UGT1A6 × 4(G > T) sequencing primers and corresponding dNTPs 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.

Drawings

FIG. 1 is a schematic structural view of a PCR reaction tube provided in the present invention;

FIG. 2 is an exemplary graph of the results of SLC28A3(1381C/C), UGT1A6 x 1/' 1 type sequencing;

FIG. 3 is an exemplary graph of the results of SLC28A3(1381C/T), UGT1A6 x 1/x 4 type sequencing;

FIG. 4 is an exemplary graph of the sequencing results for SLC28A3(1381T/T), UGT1A6 x 4/. x 4 types.

Detailed Description

The following embodiments are provided to further describe the kit for detecting daunorubicin metabolic markers, the method for detecting daunorubicin metabolic markers, and the applications of the kit for detecting daunorubicin metabolic markers 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 of the invention designs specific amplification primers and sequencing primers aiming at SLC28A3(C1381T) and UGT1A6 x 4(G > T) for amplification and pyrosequencing detection. The design of primers based on rapid amplification technology is one of the keys of the invention. 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:

serial number	Composition of	Indicating the amount of filling
			1	Amplification primer, blood sample direct PCR premixed solution and trehalose	400 uL X1 tube
2	SLC28A3(C1381T) sequencing primer	70ul X1 tube
			3	UGT1A6*4(G>T) sequencing primer	70ul X1 tube
4	Positive control	50 μ L X1 tube

(III) the single-person preparation system of the PCR reaction solution of the detection kit of the embodiment is as follows:

PCR reaction solutionThe concentrations of the components are respectively as follows: SLC28A3(C1381T) pre-primer (0.2uM), SLC28A3(C1381T) post-primer (0.2uM), UGT1A6 x 4 (G)>T) pre-primer (0.25uM), UGT1A6 x 4 (G)>T) rear primer (0.25uM), PCR premix (1X), trehalose (0.2%); wherein Easy-Load^TMBlood Direct PCR Master Mix (2X) was purchased from Shanghai Yu Bo Biotech, Inc.

Composition (I)	Volume (ul)
		Blood Direct PCR Master Mix(2×)	10
Nuclease-Free Water	4
		SLC28A3(C1381T) Pre-primer (10. mu.M)	0.4
SLC28A3(C1381T) rear primer (10. mu.M)	0.4
		UGT1A6*4(G>T) front primer (10. mu.M)	0.5
UGT1A6*4(G>T) rear primer (10. mu.M)	0.5
		Trehalose (20%)	0.2

And (5) subpackaging 200 ul/tube after configuration.

Example 2 detection of Pyrophosphoric acid

The apparatus used in the present invention is as follows: an amplification apparatus, a pyrophosphate sequencer (Wuhan Firster Biotech, Inc.).

(1) Reagent preparation (reagent preparation Chamber)

The reagents were removed in advance, and the PCR reaction solution was vortexed and shaken for 15 seconds and centrifuged at a low speed for use. 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 16. mu.L/tube.

(2) Application of sample detection (sample preparation room)

Adding EDTA (ethylene diamine tetraacetic acid) anticoagulated whole blood, a positive control and a blank control into a PCR reaction tube according to the sample adding amount of 4 mu L, covering a tube cover tightly, carrying out low-speed centrifugation 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 20 mu L, and the amplification conditions are as follows:

(4) pyrophosphoric acid sequencing

1) Adding 40 μ L of the binding solution and 3ul of agarose gel particles into a PCR reaction tube (shown in FIG. 1), adding 10 μ L of PCR product, placing on a bench-top shaker, and shaking at 1100rpm for 10min to fully bind the beads and the PCR product; the PCR tube sealing film adopted by amplification has a concave design matched with the heating column at the PCR reaction hole, the thickness of the concave design is 85 micrometers, the attaching degree is high, and the heat transfer is fast. The PCR tube sealing film has penetrability, and can be penetrated by a pipette tip or a probe for product recovery.

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.

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) Taking an 8-calandria, and sequentially adding dATP, dTTP, dGTP, dCTP, SLC28A3(C1381T) sequencing primer, UGT1A6 x 4(G > T) 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;

7) pyrosequencing; the sequencing results are shown in FIGS. 2-4.

(5) 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 SLC28A3-1381C/T,

UGT1a6 × 1/' 4 type.

2) Criteria for determination of results

SLC28A3(C1381T) is a reverse sequencing map in a DNA sequencing peak map,

the frequency of G is not less than 90 percent, the frequency of A is not less than 10 percent, and the model is 1381 CC;

the frequency of 40% G is less than or equal to 60%, the frequency of 40% A is less than or equal to 60%, and the model is 1381 CT;

the frequency of A is not less than 90 percent, the frequency of G is not less than 10 percent, and the model is 1381 TT;

UGT1A6X 4(G > T) in a DNA sequencing peak map,

the frequency of G is not less than 90 percent, the frequency of T is not less than 10 percent, and the product is GG type;

the GT type is obtained when the frequency of G is less than or equal to 40% and less than or equal to 60%, and the frequency of T is less than or equal to 40% and less than or equal to 60%;

t has a frequency of 90% or more and G has a frequency of 10% or less, i.e., TT type

(6) Medication guide

The dosage regimen of the drugs used by the population with different metabolic capacities is as follows:

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

1. A detection kit for a daunorubicin metabolic marker is used for detecting the polymorphism of two genes of the daunorubicin metabolic marker SLC28A3 and UGT1A6 x 4, and the kit is used for designing a specific amplification primer and a sequencing primer aiming at the polymorphism of two genes of SLC28A3 and UGT1A6 x 4, and comprises the following components: amplification reaction solution, SLC28A3 sequencing primer, UGT1a6 × 4 sequencing primer and positive control.

2. The kit for detecting a daunorubicin metabolic marker according to claim 1, wherein the sequence of the specific primer set of SLC28A3 is shown in sequence tables SEQ ID NO 1-SEQ ID NO 2; the sequence of the specific primer group of UGT1A6 is shown in sequence tables SEQ ID NO. 3-SEQ ID NO. 4.

3. The kit for detecting a daunorubicin metabolic marker according to claim 1, wherein the SLC28A3 sequencing primer and UGT1A 6X 4 sequencing primer are respectively shown as SEQ ID NO 5-SEQ ID NO 6 of the sequence list.

4. The test kit for a daunorubicin metabolic marker according to claim 1, wherein SLC28A3 and UGT1A6 x 4 share a dispensing instruction as shown in sequence Listing SEQ ID NO 9.

5. The detection kit for a daunorubicin metabolic marker according to claim 1, wherein said amplification reaction solution comprises SLC28A3 and UGT1a6 x 4 specific amplification primers, blood sample PCR pre-mix and trehalose.

6. The test kit for a daunorubicin metabolic marker according to claim 1, wherein said positive control comprises SLC28A3-1381C/T type, UGT1A6 x 1/' 4 type genomic DNA at a concentration of 20 ng/ul.

7. The detection kit for a daunorubicin metabolic marker according to claim 1, wherein said PCR tube is a thin film sealed PCR tube.

8. A method for detecting a daunorubicin metabolic marker using any one of claims 1 to 7, comprising the steps of:

c) adding a washing buffer solution for rinsing;

d) treating the denatured liquid to obtain a single-chain product;

e) adding a washing buffer solution for rinsing;

g) taking an 8-row pipe, and sequentially adding dATP, dTTP, dGTP, dCTP, SLC28A3(C1381T) sequencing primer, UGT1A6 x 4(G > T) sequencing primer and ddGTP from one round smooth end to the flat end;

h) and (4) pyrosequencing.

9. The method for detecting a daunorubicin metabolic marker according to claim 8, wherein the amplification reaction volume is 20ul, and the reaction conditions are: the pre-denaturation temperature was set to 95 ℃, the pre-denaturation time was set to 5min, the denaturation temperature was set to 95 ℃, the denaturation time was set to 0s, the annealing extension temperature was set to 58 ℃, the annealing extension time was set to 0s, and amplification was carried out for 40 cycles.

10. The use of the kit and the method for detecting a daunorubicin metabolic marker as claimed in any one of claims 1 to 9, wherein the kit and the method for detecting the same detect SLC28A3 and UGT1a6 x 4, so as to predict daunorubicin therapeutic effect and adverse reaction from a gene level.