CN113736874A

CN113736874A - Gene polymorphism detection kit for salbutamol metabolic marker, detection method and application thereof

Info

Publication number: CN113736874A
Application number: CN202111059790.7A
Authority: CN
Inventors: 孙悦; 刘丹; 周虹桥
Original assignee: Shanghai Puran Biotechnology Co ltd
Current assignee: Shanghai Puran Biotechnology Co ltd
Priority date: 2021-09-10
Filing date: 2021-09-10
Publication date: 2021-12-03

Abstract

The invention discloses a gene polymorphism detection kit of a salbutamol metabolic marker, a detection method and application thereof, wherein the salbutamol metabolic marker is the gene polymorphism of ADRB2A46G, the kit designs specific amplification primers and sequencing primers aiming at the gene polymorphism of ADRB2A46G, and the kit comprises the following components: sample processing solution, magnetic beads, amplification reagent 1, amplification reagent 2, ADRB2A46G sequencing primer and positive control. On one hand, the invention adopts a mode of extracting and amplifying the same tube, thereby avoiding the risks of extracting multiple tube moving and losing nucleic acid; on the other hand, the rapid constant temperature amplification is carried out by adding an anti-inhibitor; the invention aims to detect the gene polymorphism of salbutamol curative effect by taking constant temperature PCR and pyrophosphoric acid detection as a combination, and provides a gene angle suggestion for clinical personalized medication.

Description

Gene polymorphism detection kit for salbutamol metabolic marker, detection method and application thereof

Technical Field

The invention relates to a gene polymorphism detection kit for a salbutamol metabolic marker, a detection method and application thereof, belonging to the field of gene detection.

Background

Salbutamol (salbutamol), a short-acting beta 2 adrenergic receptor agonist, is used as an antiasthmatic, and can effectively inhibit the release of allergic substances such as histamine and the like and prevent bronchospasm. The addition of trace amount of salbutamol in the feed for livestock can increase lean meat amount and meat change rate of livestock and reduce fat, but the toxicity is far higher than that of ractopamine with the same function. It is suitable for treating bronchial asthma, asthmatic bronchitis, bronchospasm, and emphysema. The effect of the salbutamol treatment on part of patients is not ideal, the acute attack still occurs to the patients, and the different responses of the patients to the drug treatment cannot be simply considered to be caused by different severity degrees of diseases or different drug compliance. The reason for the difference in therapeutic effect of salbutamol is related to the beta 2-AR gene polymorphism of patients.

ADRB2 protein is a typical coupled G protein membrane receptor, and can activate intracellular adenylate cyclase to cause a series of downstream signal reactions, and generate effects such as bronchiectasis and the like. The coding region of ADRB2 (beta 2 adrenergic receptor) has 9 genetic polymorphism sites, wherein, the nucleotide polymorphism of 4 sites causes the change of amino acid sequence, which respectively causes the change of Argl6 → Gly, Gln27 → Glu, Va134 → Met, Thrl64 → Ile of the coded amino acids at 16 th, 27 th, 34 th and 164 th positions, wherein, the polymorphism of the 46 th position (rs1042713, 16 th position of a peptide chain) of the nucleotide sequence causes the change of the function of the beta 2-adrenergic receptor. The beta 2-receptor agonist is used as a medicine for treating asthma, can selectively act on a beta 2-adrenergic receptor (coupled G protein membrane receptor), activates Adenylate Cyclase (AC), increases cAMP in cells, further phosphorylates protein kinase by cAMP, promotes substrate phosphorylation, relaxes smooth muscle, and relieves symptoms such as bronchospasm. Therefore, the ADRB2 gene polymorphism affects the tolerance of the organism to drugs to a certain extent. The genetic polymorphism at amino acid 16 of ADRB2 is related to the receptor down-regulation and desensitization mediated by beta 2-receptor agonist. Endogenous catecholamines promote the down regulation of receptors of homozygous Glyl6 and Glu27, the 16-site polymorphism plays a leading role in drug responsiveness is different, compared with the GG genotype, asthma children carrying the AA genotype have lower response to salbutamol or salmeterol, and the use of long-acting beta 2 receptor agonists can increase the risk of asthma exacerbation. The clinical efficacy of short-acting beta 2 receptor agonists is affected by the polymorphism of the beta 2-AR gene, and it is therefore recommended that beta 2 receptor agonist therapy be avoided for patients with A/G genotype asthma.

At present, there are many methods for detecting gene polymorphism, and fluorescence PCR is mainly used. Mainly comprises a high-resolution melting curve method, a taqman fluorescence probe method and an allele specific amplification method. The high-resolution melting curve method has simple steps, but has low specificity and higher requirements on instruments and equipment; the allele specific amplification method adopts ARMS primers to carry out specific amplification, and has simple operation method, but strict detection condition requirements, and easy occurrence of primer mismatching in actual operation to generate false positive. the taqman fluorescence probe method has higher test cost. The invention establishes a simple, quick and effective pyrophosphoric acid sequencing method for detecting gene polymorphism with low price and high specificity.

At present, the blood DNA is obtained mainly by the traditional column extraction and the magnetic bead extraction, and the two methods both take longer time and are relatively complicated to operate. CN201610022581.8 proposes a real-time fluorescent quantitative PCR method for extracting nucleic acid and amplifying by magnetic beads in one tube, adding lysis solution mixed with magnetic beads and a sample to be detected into a PCR amplification tube, mixing uniformly, standing, performing magnetic attraction, sucking out mixed solution, and washing the obtained magnetic beads once; and adding the prepared PCR reaction solution into the PCR amplification tube to perform real-time fluorescent quantitative PCR reaction on the target nucleic acid. The invention also requires washing of the magnetic beads. Therefore, there is an urgent need to establish a simple, rapid, effective, inexpensive, and highly specific pyrosequencing method for detecting gene polymorphisms.

Disclosure of Invention

Aiming at the problems in the prior art, the invention aims to obtain a gene polymorphism detection kit for a salbutamol metabolic marker, and a detection method and application thereof.

In order to achieve one of the above objects, the present invention adopts a technical solution of a kit for detecting gene polymorphism of a salbutamol metabolic marker, which comprises:

the gene polymorphism detection kit for the salbutamol curative effect prediction is used for designing a specific amplification primer and a sequencing primer aiming at the polymorphism of ADRB2(A46G), and comprises the following components: sample treatment solution, magnetic beads, amplification reagent 1, amplification reagent 2, ADRB2(A46G) sequencing primer, and positive control.

Specifically, the specific primer sequences are shown in the following table 1:

preferably, the ADRB2(A46G) amplification primer is shown as a sequence table SEQ ID NO: 1-2.

Preferably, the ADRB2(A46G) sequencing primer is shown as a sequence table SEQ ID NO. 3.

Preferably, the sequencing region corresponding to the ADRB2(A46G) sequencing primer is an ADRB2(A46G) to-be-detected sequence, which is shown as a sequence table SEQ ID NO. 4; preferably, the ADRB2(A46G) allocation instruction is shown in SEQ ID NO:5 of the sequence Listing.

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 sample treatment solution, which does not contain a guanidine salt, is used to lyse the sample under alkaline conditions and with a surfactant. Comprises 0.1-0.6% of lithium dodecyl sulfate, 0.1-0.5% of triton X-100, 2-50mg/mL of sodium hydroxide, 5-15% of trehalose, 3-7 mmol/L of BSA, 20-80mM of Tris-HCl and 100mM of NaCl, wherein the pH value is 8.5-9.5.

More preferably, the sample treatment solution comprises 0.3-0.5% of lithium dodecyl sulfate, 0.2-0.4% of Triton X-100, 10-20mg/mL of sodium hydroxide, 8-12mM of betaine, 8-12% of trehalose, 4-6 mM of BSA, 50mM of Tris-HCl, 100mM of NaCl, and pH 9.

Preferably, the magnetic beads are carboxyl magnetic beads, the particle size is 600mm, the suspension property is good, the magnetic property is strong, and the adsorption capacity is large. The concentration of magnetic beads in the reaction solution is 0.2mg/25 mu L, and the DNA adsorption amount in the blood sample is 30 ng-260 ng, which completely meets the DNA amount required by amplification.

Preferably, the amplification reagent 1 comprises: amplification buffer, 15mM magnesium acetate;

preferably, the amplification reagent 2 comprises: ADRB2(A46G) pre-primer (0.32uM), ADRB2(A46G) post-primer (0.32uM), dNTPS (0.3mM), strand displacement DNA polymerase (1.2 ng/. mu.L), single-stranded DNA binding protein (3.2 ng/. mu.L), recombinase binding single-stranded nucleic acid (4.8 ng/. mu.L).

More preferably, the amplification reagent 2 comprises: trehalose (0.2%), 10mM manganese acetate, 0.1M sorbitol, 5ug/mL BSA. Trehalose has nonspecific protection effect on bioactive substances, can improve the thermal stability of DNA polymerase, reduce the melting temperature of a DNA template, and reduce the secondary structure formed by self-complementary pairing of a G-C rich region, thereby improving the specificity of PCR reaction. Sorbitol and manganese acetate have the stabilizing effect of the PCR premix, and have the stabilizing effect in the freeze drying process. The bovine serum albumin can improve the amplification efficiency of the PCR reaction and reduce the influence of PCR inhibitors in the system on the reaction.

Preferably, the reaction volume is 25ul, and the reaction conditions are as follows: 30min at 42 ℃.

Preferably, the positive control comprises ADRB2(A46G) heterozygous genomic DNA at a concentration of 20 ng/ul. The positive control corresponds to the heterozygosis of the detected gene locus, provides reference for the type determination of an unknown sample, and simultaneously performs quality control on the effectiveness of the reaction solution.

The invention also discloses a gene polymorphism detection method for salbutamol curative effect prediction by adopting the kit, which comprises the following steps:

a. mixing 100ul of sample treatment solution, 4ul of magnetic beads and 30ul of EDTA (ethylene diamine tetraacetic acid) anticoagulated whole blood sample, and standing at room temperature for 5 min;

b. placing the PCR amplification tube on a magnetic frame, and sucking out the mixed solution from the opposite side of the magnetic beads after the magnetic beads are completely adsorbed to one side;

c. adding the prepared PCR reaction solution into the PCR amplification tube obtained in the step b), fully and uniformly mixing magnetic beads and the PCR reaction solution, centrifuging, and carrying out constant-temperature reaction;

d. binding the binding solution (containing the microbeads) with the amplification product;

e. treating the denatured liquid to obtain a single-chain product;

f. adding a washing buffer solution for rinsing;

g. adding a sequencing enzyme and a sequencing substrate to each sequencing tube;

h. taking an 8-row tube, and sequentially adding sequencing primers dATP, dTTP, dGTP, dCTP and ADRB2(A46G) 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;

i. and (4) pyrosequencing.

The invention also discloses an application of the gene polymorphism detection kit for salbutamol curative effect prediction, and the detection kit is used for detecting ADRB2(A46G) so as to reflect the salbutamol curative effect from a gene level and further give a gene level suggestion for guiding the use of salbutamol.

Compared with the prior art, the invention adopts the sample processing liquid to rapidly release the DNA from the sample, the sample amount is 30ul of whole blood, and compared with a one-step cracking method and direct amplification (about 2-10ul), enough genome DNA can be obtained and adsorbed on the magnetic beads for subsequent analysis. Most of the inhibitor can be removed by removing the sample and lysis mix. The DNA template was amplified by isothermal PCR at the ADRB2(A46G) gene locus to produce large amounts of biotin-labeled single-stranded DNA. The biotin-labeled single-stranded DNA is combined with streptavidin, and a sequencing primer and a sequencing raw material are added after washing to perform pyrosequencing, so that the sequencing process and time are simplified. The invention uses rapid DNA preparation, constant temperature PCR amplification and pyrosequencing technology as combination to detect the gene polymorphism of salbutamol curative effect, and provides gene angle suggestion for clinical personalized medication.

The rapid amplification method is optimized mainly from two aspects, on one hand, the mode of extracting and amplifying the same tube is adopted, and the risks of extracting multiple tube moving and losing nucleic acid are avoided; on the other hand, the rapid constant temperature amplification is carried out by adding an anti-inhibitor; the invention aims to obtain a gene polymorphism detection kit for salbutamol curative effect prediction based on constant temperature PCR and pyrophosphoric acid detection, and a detection method and application thereof.

Drawings

FIG. 1 is a diagram illustrating the detection result of ADRB2(A46G) AA pyrogen provided by the present invention;

FIG. 2 is a diagram showing an example of the detection result of ADRB2(A46G) GA-type pyrophosphate provided by the present invention;

FIG. 3 is an exemplary graph of ADRB2(A46G) GG type pyrophosphate detection results provided by the present invention.

Detailed Description

The following examples are provided to further describe the gene polymorphism detection kit of the salbutamol metabolic marker of the present invention, the detection method thereof 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

(I) design of specific primers

The kit of the invention designs specific amplification primers and sequencing primers aiming at ADRB2(A46G) gene polymorphism and is used for pyrophosphate PCR detection. Gene polymorphism sequences are based on published sequences in Genebank, and primer sequences are shown in the following table.

(II) kit composition

The detection kit comprises the components shown in the following table 2:

TABLE 2 kit component table

(III) the sample treatment solution preparation system comprises the following steps:

sample treatment solution containing 0.4% lithium lauryl sulfate, 0.3% Triton X-100, 15mg/mL sodium hydroxide, 10mM betaine, 10% trehalose, 5mM BSA, 50mM Tris-HCl, 100mM NaCl, pH 9.

(IV) the single-person configuration system of the detection kit amplification reagent 1 of the embodiment is as follows:

composition (I)	Volume (ul)
		Amplification buffer	24
200mM magnesium acetate	1

(V) the detection kit amplification reagent 2 of the present embodiment is configured as follows in a single-person configuration system:

ADRB2(A46G) pre-primer (0.32uM), ADRB2(A46G) post-primer (0.32uM), 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%), 10mM manganese acetate, 0.1M sorbitol, 5ug/mL BSA. The PCR system is shown in Table 3:

TABLE 3 PCR systems

Composition (I)	Volume (ul)
		Recombinase binding single-stranded nucleic acid (100 ng/. mu.L)	1.2
Single-stranded DNA binding protein (100 ng/. mu.L)	0.8
		Strand Displacement DNA polymerase (100 ng/. mu.L)	0.3
dNTPs(25mM)	0.3
		ADRB2(A46G) Pre-primer (20. mu.M)	0.4
ADRB2(A46G) rear primer (20. mu.M)	0.4
		Trehalose (20%)	0.25
1M manganese acetate	0.25
		10M sorbitol	0.25
5mg/mL BSA	0.25

After the preparation is finished, 96.8 ul/tube is subpackaged and freeze-dried.

Example 2 kit detection procedure

The apparatus used in the present invention is as follows: thermostats, pyrosequencing instruments (Wuhan Firster Biotech, Inc.).

1) Taking 30 mu L of EDTA anticoagulated whole blood sample in a PCR amplification tube;

2) adding 100 μ L sample treatment solution and 4ul magnetic beads, and standing for 5 min;

3) placing the PCR amplification tube on a magnetic frame, and sucking out the mixed solution from the opposite side of the magnetic beads after the magnetic beads are completely adsorbed to one side;

4) adding the amplification reagent 1 into the amplification reagent 2 dry powder, and fully dissolving and uniformly mixing;

5) adding 25ul of prepared PCR reaction solution into the PCR amplification tube obtained in the step 4), fully and uniformly mixing magnetic beads and the PCR reaction solution, centrifuging, and carrying out constant-temperature reaction.

6) And (3) amplifying by adopting a PCR instrument, wherein the reaction system is 25 mu L, and the amplification conditions are as follows:

temperature of amplification	Time	Number of cycles
			42℃	25min		1

7) Adding 40 mu L of binding solution and 3ul of agarose gel particles into a PCR reaction tube, adding 20 mu L of PCR product into the PCR reaction tube, placing the PCR reaction tube on a table type oscillator, and oscillating at 1100rpm for 10min to ensure that the microbeads and the PCR product are fully bound;

8) centrifuging at 7,000 Xg for 1min, and discarding the supernatant;

9) adding 22uL of diluted working solution of the denatured liquid, standing for 5min, centrifuging for 1min at 7,000 Xg, and collecting by an EP tube to obtain a single-chain product.

10) To the EP tube, 150uL of washing buffer was added, and centrifuged at 7,000 Xg for 1 min. (repeat 3 times)

11) 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.

12) Respectively adding 3uL sequencing enzyme and 3uL sequencing substrate into a sequencing tube;

13) taking an 8-row pipe, and adding dATP, dTTP, dGTP, dCTP, ADRB2(A46G) sequencing primer and ddATP in sequence from the round end to the flat end. Lightly knocking the bottom of the calandria against the tabletop to enable the bases to be flatly paved at the bottom of the calandria.

14) Pyrosequencing; the sequencing results are shown in FIGS. 1 to 3.

15) Interpretation of results

1) And (3) judging the effectiveness:

the blank control of the kit does not pass, and the detection result of the positive control is ADRB2(A46G) hybrid mutant.

2) Criteria for determination of results

The DNA sequencing peak plot of ADRB2(a46G) was reverse sequencing;

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

the frequency of 40% to T is less than or equal to 60%, and the frequency of 40% to C is less than or equal to 60%, which is GA type;

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

16) correlation table of gene detection result and salbutamol curative effect prediction resistance

Fifth, the performance test result of the kit

5.1. Specificity of

The specific sample (including non-human DNA template and dilution of amplification product of different sites or homologous sites of the same human gene) is detected, and the result is negative.

5.2. Accuracy of

The detection of reference substances (including ADRB2(A46G) wild, heterozygous and mutant genotypes) of different genotypes in the kit range can detect the corresponding genotypes.

5.3. Minimum detection limit

The minimum detection limit should not be higher than 2 ng/ul.

5.4. Repeatability of

In the detection kit, each reference substance is subjected to 10 times of detection, the results are corresponding mutation types, and the Coefficient of Variation (CV) of the Ct value of a corresponding detection channel is less than or equal to 5.0%.

Finally, it must be said here that: the above embodiments are only used for further detailed description of the technical solutions of the present invention, and should not be understood as limiting the scope of the present invention, and the insubstantial modifications and adaptations made by those skilled in the art according to the above descriptions of the present invention are within the scope of the present invention.

Sequence listing

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Claims

1. A kit for detecting the gene polymorphism of a salbutamol metabolic marker is the gene polymorphism of ADRB2A46G, and the kit is used for designing specific amplification primers and sequencing primers aiming at the gene polymorphism of ADRB2A46G and comprises the following components: sample processing solution, magnetic beads, amplification reagent 1, amplification reagent 2, ADRB2A46G sequencing primer and positive control.

2. The kit for detecting the gene polymorphism of a salbutamol metabolic marker according to claim 1, wherein the ADRB2A46G amplification primer is shown as a sequence table SEQ ID NO. 1-2.

3. The kit for detecting the gene polymorphism of a salbutamol metabolic marker according to claim 1, wherein the ADRB2A46G sequencing primer is shown as a sequence table SEQ ID NO. 3.

4. The kit for detecting the genetic polymorphism of a salbutamol metabolic marker according to claim 1, wherein the ADRB2A46G assignment instruction is as shown in SEQ ID NO. 5 of the sequence Listing.

5. The kit for detecting gene polymorphism of a salbutamol metabolic marker according to claim 1, wherein the sample treatment solution comprises 0.1% -0.6% of lithium dodecyl sulfate, 0.1-0.5% of triton X-100, 2-50mg/mL of sodium hydroxide, 5-15% of trehalose, 3-7 mmol/L of BSA, 20-80mM Tris-HCl, 100mM NaCl, and 8.5-9.5 of pH.

6. The kit for detecting gene polymorphism of a salbutamol metabolic marker according to claim 1, wherein the magnetic beads are carboxyl magnetic beads, and the particle size is 600 mm.

7. The kit for detecting gene polymorphism of a salbutamol metabolic marker according to claim 1, wherein the amplification reagent 1 comprises: amplification buffer and 15mM magnesium acetate.

8. The kit for detecting gene polymorphism of a salbutamol metabolic marker according to claim 1, wherein the amplification reagent 2 comprises: ADRB2A46G front primer 0.32uM, ADRB2A46G rear primer 0.32uM, dNTPS 0.3mM, strand displacement DNA polymerase 1.2 ng/. mu.L, single-stranded DNA binding protein 3.2 ng/. mu.L, and single-stranded nucleic acid binding recombinase 4.8 ng/. mu.L.

9. A detection method using the kit for detecting gene polymorphism of a salbutamol metabolic marker according to any one of claims 1 to 8, characterized in that the detection method comprises the steps of:

d. combining the binding solution containing the microbeads with the amplification product;

e. treating the denatured liquid to obtain a single-chain product;

f. adding a washing buffer solution for rinsing;

h. taking an 8-row pipe, and sequentially adding dATP, dTTP, dGTP, dCTP and ADRB2A46G sequencing primers from one round smooth end to the flat end;

i. and (4) pyrosequencing.

10. The use of the kit and the method for detecting the gene polymorphism of the salbutamol metabolic marker according to any one of claims 1 to 9, wherein the kit and the method are used for detecting the gene polymorphism of ADRB2A 46G.