CN113512585A

CN113512585A - Rapid reaction kit for aspergillin dosage prediction and detection method and application thereof

Info

Publication number: CN113512585A
Application number: CN202110799434.2A
Authority: CN
Inventors: 张玉; 曾芳; 周涛; 龚卫静
Original assignee: Hunan Feisite Precision Medical Technology Co ltd
Current assignee: Hunan Feisite Precision Medical Technology Co ltd
Priority date: 2021-06-17
Filing date: 2021-07-15
Publication date: 2021-10-19

Abstract

The invention discloses a rapid reaction kit for the dosage prediction of sertraline and a detection method and application thereof, wherein the detection kit is used for detecting the gene polymorphism of two genes of a sertraline metabolism marker CYP2C 19681G & gt A and CYP2C 196 19636G & gt A, and the kit comprises the following components: CYP2C 19681G > A amplification primer, CYP2C 19681G > A sequencing primer, CYP2C 19636G > A amplification primer, CYP2C 19636G > A sequencing primer and positive control. The invention adopts multiple RPA amplification and optimized pyrosequencing technology to detect gene polymorphism related to the prediction of the curative effect of the sertraline medicament by combining, and the kit can simultaneously detect CYP2C19(681G > A) and CYP2C19(636G > A) gene polymorphism and provides a suggestion of gene angle for clinical personalized medication.

Description

Rapid reaction kit for aspergillin dosage prediction and detection method and application thereof

Technical Field

The invention relates to a rapid reaction kit for aspergillin dosage prediction, and a detection method and application thereof, and belongs to the field of gene detection.

Background

Trarsine is a selective 5-hydroxytryptamine reuptake inhibitor (SSRIs) that increases serotonergic activity by decreasing presynaptic 5-hydroxytryptamine reuptake. Sertraline is a common antidepressant used for various indications in pediatric patients. The metabolism of sertraline is mainly by cytochrome P4502C 19(CYP2C19) enzymes, whereas other enzymes (e.g. cytochrome P4502D 6) contribute to a lesser extent. Genetic variation in CYP2C19 results in individual variation in CYP2C19 function. PM individuals may experience toxic side effects when conventional doses of drugs are used, and clinical pharmacogenetics administration association (CPIC) guidelines recommend a 50% reduction in the initial sertraline dose in PM individuals to be considered. EM and Intermediaries (IM) individuals may be given conventional doses.

The genetic variation of CYP2C19 can cause individual variation of enzyme activity, which causes 4 phenotypes in the population, including Ultrafast Metabolisers (UM), fast metabolisers (EM), Intermediate Metabolisers (IM) and slow metabolisers (PM). CYP2C19 x 2(rs4244285, c.681G > A) and CYP2C19 x 3(rs4986893, c.636G > A) are the 2 major alleles present in the Chinese population that result in deficiency of the CYP2C19 enzyme. CYP2C19 x 2 resulted in splicing deletion, CYP2C19 x 3 was a stop codon mutation. EM individuals carry only CYP2C19 x 1 alleles, IM individuals carry CYP2C19 x 2 or CYP2C19 x 3 heterozygous genotypes; PM individuals included CYP2C19 x 2/' 2, CYP2C19 x 2/' 3 and CYP2C19 x 3/' 3 genotypes. 75-85% of PM in the eastern population is caused by CYP2C19 x 2, and about 20-25% of PM is caused by CYP2C19 x 3. The CYP2C19 enzyme has gene polymorphism, which can cause different metabolic capacities to the sertraline among individuals and races, so that the blood concentration of the drug presents obvious individual difference.

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 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, there is a need to establish a simple, rapid, efficient, and inexpensive method for detecting gene polymorphisms.

Disclosure of Invention

Aiming at the problems in the prior art, the invention aims to obtain a quick reaction kit for the dose prediction of the aspergillin, and a detection method and application thereof.

In order to realize one of the above purposes, the technical scheme of the rapid reaction kit for predicting the dose of the trastuzumab adopted by the invention is as follows:

the rapid reaction kit provided by the invention designs specific amplification primers and sequencing primers aiming at polymorphism of CYP2C19(681G A) and CYP2C19(636G A), and comprises the following components: amplification reaction liquid, CYP2C19(681G > A) sequencing primer, CYP2C19(636G > A) 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 CYP2C19(681G > A) is shown in sequence tables SEQ ID NO 1-SEQ ID NO 2; the sequence of the specific primer group of CYP2C19(636G > A) is shown in sequence tables SEQ ID NO. 3-SEQ ID NO. 4.

Preferably, the CYP2C19(681G > A) sequencing primer and the CYP2C19(636G > A) 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 CYP2C19(681G > A) sequencing primer is a sequence to be detected by CYP2C19(681G > A), and is shown as SEQ ID NO:7 in the sequence table; the sequencing region corresponding to the CYP2C19(636G > A) sequencing primer is a to-be-detected sequence of CYP2C19(636G > A), and is shown as a sequence table SEQ ID NO: 8.

Preferably, CYP2C19(681G > A) and CYP2C19(636G > A) share a dispense instruction as set forth in SEQ ID NO:9 of the sequence Listing.

Preferably, the amplification reaction solution comprises CYP2C19(681G > A) and CYP2C19(636G > A) specific amplification primers, and further comprises a blood sample direct-amplification PCR premix (2X) and trehalose;

more preferably, the final concentrations of each component of the reaction solution are CYP2C19(681G > A) pre-primer (0.2uM), CYP2C19(681G > A) post-primer (0.2uM), CYP2C19(636G > A) pre-primer (0.25uM), CYP2C19(636G > A) 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 CYP2C19 x 2 x 3 type genomic DNA with the concentration of 20ng/ul, provides a reference for type determination of an unknown sample, 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 performed for 35 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 the prediction of the dosage of the sertraline 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-calandria, and sequentially adding dATP, dTTP, dGTP, dCTP, CYP2C19(681G > A) sequencing primer, CYP2C19(636G > A) sequencing primer and ddATP from one round and smooth end to the flat end; lightly knocking the bottom of the calandria against the tabletop to enable the bases to be flatly paved at the bottom of the calandria;

h. pyrosequencing;

i. the genotype of CYP2C19 was determined.

The invention also discloses a rapid reaction kit for the dosage prediction of the sertraline and application of the method, wherein the detection kit is used for detecting CYP2C19(681G > A) and CYP2C19(636G > A) so as to guide the dosage prediction of the sertraline from a gene level.

The rapid amplification method of CYP2C19 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 a 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 CYP2C19(681G > A) and CYP2C19(636G > A) at two sites, and pyrosequencing at the two sites is carried out in one reaction. The sequencing is carried out by adding CYP2C19(681G > A) sequencing primer and sequencing raw material to carry out pyrosequencing, and adding ddATP into the last base to terminate the sequencing reaction. Then adding CYP2C19(636G > A) 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.

Compared with the prior art, the invention detects gene polymorphism related to the prediction of the dosage of the sertraline by taking blood direct amplification, rapid amplification and optimized pyrosequencing technology as a combination, and provides a gene angle suggestion for clinical personalized medication of the sertraline.

Drawings

FIG. 1 is a graph showing the effect of electrophoresis on different volumes of EDTA anticoagulated whole blood provided by the present invention;

FIG. 2 is a schematic structural view of a PCR reaction tube provided in the present invention;

FIG. 3 is an exemplary illustration of the CYP2C19(681GG)/CYP2C19(636GG) type sequencing results provided by the present invention;

FIG. 4 is an exemplary graph of the sequencing results of CYP2C19(681GA)/CYP2C19(636GA) type provided by the present invention;

FIG. 5 is an exemplary illustration of the sequencing results of CYP2C19(681GG)/CYP2C19(636AA) type provided by the present invention;

FIG. 6 is an exemplary graph of the CYP2C19(681AA)/CYP2C19(636GG) type sequencing results provided by the present invention;

FIG. 7 is an exemplary graph of the CYP2C19(681GG)/CYP2C19(636AG) type sequencing results provided by the present invention;

FIG. 8 is an exemplary diagram of the sequencing result of CYP2C19(681GA)/CYP2C19(636GG) type provided by the present invention.

Detailed Description

The following examples are provided to further detail and fully illustrate the rapid response kit for predicting the dose of traselin, 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 rapid reaction kit provided by the invention designs specific amplification primers and sequencing primers aiming at CYP2C19(681G > A) and CYP2C19(636G > A) for amplification and pyrosequencing detection. The design of primers based on the rapid amplification technology is one of the keys of the invention, and the amplification length is controlled to be 60-120bp as much as possible in order to ensure the amplification speed and the detection sensitivity. 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 single-person preparation system of the PCR reaction solution of the detection kit of the embodiment is as follows:

the final concentrations of the components of the PCR reaction solution are respectively as follows: CYP2C19 (681G)>A) The pre-primer (0.2uM), CYP2C19 (681G)>A) Rear primer (0.2uM), CYP2C19 (636G)>A) The primer (0.25uM), CYP2C19 (636G)>A) 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
		CYP2C19(681G>A) Front primer (10. mu.M)	0.4
CYP2C19(681G>A) Rear primer (10. mu.M)	0.4
		CYP2C19(636G>A) Front primer (10. mu.M)	0.5
CYP2C19(636G>A) Rear primer (10. mu.M)	0.5
		Trehalose (20%)	0.2

And (5) subpackaging 400 ul/tube after configuration.

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. 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 structure of the PCR reaction tube is schematically shown in FIG. 2.

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 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) add 150uL of wash buffer to EP tube and centrifuge at 7,000 × g for 1 min;

5) 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;

6) taking an 8-calandria, and sequentially adding dATP, dTTP, dGTP, dCTP, CYP2C19(681G > A) sequencing primer, CYP2C19(636G > A) sequencing primer and ddATP from one round and smooth end to the flat end; lightly knocking the bottom of the calandria against the tabletop to enable the bases to be flatly paved at the bottom of the calandria;

7) pyrosequencing; the sequencing results are shown in FIGS. 3-8.

(5) Interpretation of results

1) And (3) judging the effectiveness:

the blank control of the kit failed, and the positive control detected CYP2C19 x 2 x 3.

2) Criteria for determination of results

In the DNA sequencing peak map of CYP2C19(681G > A),

the frequency of G is not less than 90 percent, the frequency of A is not less than 10 percent, and the model is 681 GG;

the frequency of 40% G is equal to or less than 60%, the frequency of 40% A is equal to or less than 60%, and the result is 681GA type;

the frequency of A is not less than 90 percent, the frequency of G is not less than 10 percent, and the model is 681 AA;

b. CyP2C19(636G > A) in a DNA sequencing peak plot,

the frequency of G is not less than 90 percent, the frequency of A is not less than 10 percent, and the model is 636 GG;

the frequency of 40% G is equal to or less than 60%, the frequency of 40% A is equal to or less than 60%, and this is 636GA type;

the frequency of A is equal to or greater than 90%, the frequency of G is equal to or less than 10%, and the formula is 636AA

(6) Medication guide

The dosage regimen for drug use in populations of different metabolic capacities was adjusted according to guidelines issued by the united states Clinical Pharmacogenomics Implementation Consortium (CPIC) as follows:

example 3 amplification efficiency at different volumes of EDTA-anticoagulated Whole blood

Blood Direct PCR Master Mix (2X) containing Blood-resistant Hemotaq^TMDNA polymerase and anti-depressants exhibit superior resistance to various PCR inhibitors such as heme in whole blood. Adding EDTA anticoagulant whole blood in 5%, 10%, 20%, 30%, 40% and 50% of PCR reaction liquid. The maximum added whole blood sample volume was tested. The specific results of the test are shown in FIG. 1, and the test shows that 20% volume of EDTA anticoagulated whole blood has no influence on the amplification efficiency.

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. The rapid reaction kit for the dosage prediction of the sertraline is characterized in that the detection kit is used for detecting the gene polymorphism of two genes of a metabolic marker CYP2C 19681G > A and CYP2C 19636G > A of the sertraline, and the kit comprises the following components: CYP2C 19681G > A amplification primer, CYP2C 19681G > A sequencing primer, CYP2C 19636G > A amplification primer, CYP2C 19636G > A sequencing primer and positive control.

2. The rapid reaction kit for dosage prediction of sertraline according to claim 1, wherein the CYP2C 19681G > A amplification primers are represented by SEQ ID NO: 1-2 of the sequence Listing.

3. The rapid reaction kit for dosage prediction of sertraline according to claim 1, wherein the CYP2C 19636G > A amplification primers are represented by SEQ ID NO 3-4 of the sequence Listing.

4. The rapid reaction kit for dosage prediction of sertraline according to claim 1, wherein the CYP2C 19681G > A sequencing primer is shown in sequence table SEQ ID NO:5, and CYP2C 196 19636G > A sequencing primer is shown in sequence table SEQ ID NO: 6.

5. The rapid reaction kit for the dosage prediction of sertraline according to claim 1, wherein said sequencing primer is a conjugate of agarose gel particles and amino-labeled DNA sequences.

6. The rapid response kit for the dosage prediction of sertraline according to claim 1, wherein said kit further comprises a blood sample direct amplification PCR premix and trehalose.

7. A rapid response kit for the dosage prediction of sertraline according to claim 6, wherein the final concentration of each component in the kit is: CYP2C19(681G > A) pre-primer (0.2uM), CYP2C19(681G > A) post-primer (0.2uM), CYP2C19(636G > A) pre-primer (0.25uM), CYP2C19(636G > A) post-primer (0.25uM), PCR premix (1X), trehalose (0.2%).

8. The rapid response kit for the dosage prediction of sertraline according to claim 1, wherein said positive control comprises CYP2C 19681G > A, CYP2C 19636G > a heterozygous genomic DNA at a concentration of 20 ng/ul.

9. A detection kit adopting the voriconazole metabolic marker as defined in 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 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, CYP2C19(681G > A) sequencing primer, CYP2C19(636G > A) sequencing primer and ddATP from one round and smooth end to the flat end;

g. pyrosequencing;

h. and determining the genotypes of the CYP2C19(681G > A) site and the CYP2C19(636G > A) site tamoxifen site.

10. The application of the voriconazole metabolic marker detection kit as claimed in any one of claims 1-8, wherein the detection kit is used for in vitro detection of CYP2C 19681G > A and CYP2C 19636G > A gene polymorphisms in a sample to be detected.