1. A detection kit for statin therapeutic effect prediction is characterized in that the kit is used for detecting the gene polymorphism of two genes, namely, an ApoE T388C gene and an ApoE C526T gene, related to the statin therapeutic effect, and comprises the following components: an ApoE T388C amplification primer, an ApoE T388C sequencing primer, an ApoE C526T amplification primer, an ApoE C526T sequencing primer, and a positive control.

2. The detection kit for statin therapeutic effect prediction according to claim 1, wherein the ApoE T388C amplification primer is represented by SEQ ID NO 1-2 of the sequence Listing.

3. The detection kit for statin therapeutic effect prediction according to claim 1, wherein the ApoE C526T amplification primer is represented by SEQ ID NO. 3-4 of the sequence list.

4. The detection kit for statin therapeutic effect prediction according to claim 1, wherein the ApoE T388C sequencing primer is shown in SEQ ID NO. 5 of the sequence table, and the ApoE C526T sequencing primer is shown in SEQ ID NO. 6 of the sequence table.

5. The detection kit for statin therapeutic effect prediction according to claim 1, wherein the sequencing primer is a conjugate of agarose gel particles and amino-labeled DNA sequences.

6. A test kit for use in the prediction of statin therapeutic effect according to claim 1, wherein the kit further comprises amplification buffer, 18mM magnesium acetate, dNTPS, strand displacement DNA polymerase, single stranded DNA binding protein, recombinase binding to single stranded nucleic acid, and trehalose.

7. A test kit for use in the prediction of statin therapeutic effect according to claim 6, wherein the final concentrations of the components in the kit are: 0.32uM for each primer before and after amplification of ApoE T388C, 0.32uM for each primer before and after amplification of ApoE C526T, 0.3mM of dNTPS, 1.2 ng/uL of strand displacement DNA polymerase, 3.2 ng/uL of single-stranded DNA binding protein, 4.8 ng/uL of recombinase for binding single-stranded nucleic acid, and 0.2% of trehalose.

8. A test kit for use in the prediction of statin therapeutic effect according to claim 1, wherein the positive control comprises ApoE T388C and ApoE C526T heterozygous genomic DNA at a concentration of 20 ng/ul.

9. A detection method using the detection kit for statin therapeutic effect prediction according to any one of claims 1 to 8, characterized in that the detection method comprises the following steps:

a. amplifying the amplification reaction solution and 5ul of genome DNA to be detected by adopting multiple RPA amplification;

b. combining 10ul of reaction product with 3ul of sequencing primer;

c. pyrosequencing;

d. the genotype of ApoE T388C site and ApoE C526T site was determined.

10. Use of a test kit according to any one of claims 1 to 8 for the prediction of statin therapeutic effect, wherein the test kit is used for in vitro detection of polymorphisms of ApoE T388C and ApoE C526T in a sample to be tested.

Background

According to epidemiological investigation in China, the total prevalence rate of dyslipidemia of Chinese adults is 40.40% in 2012, and is greatly increased in 10 years ago, and the increase of the serum Total Cholesterol (TC) level can increase about 920 ten thousand of future cardiovascular disease events in China. Statins significantly reduce the risk of cardiovascular events in both the primary and secondary prevention of ASCVD, and are the most important drugs for the prevention and treatment of these diseases. However, although different statins are similar in chemical structure, they differ from individual to individual in pharmacokinetics, interaction with other drugs, efficacy of the drug, and adverse effects caused by statins, and these differences are due in part to inheritance and variation of common drug genes. Apolipoprotein E (ApoE) is an apolipoprotein present in chylomicrons and intermediate density lipoproteins, is mainly produced by the liver and macrophages, and is involved in the transport, storage and excretion of blood lipids.

Apolipoprotein E (ApoE) is involved in the regulation of lipid metabolism in the body through various pathways and is an important intrinsic factor affecting the blood lipid level of the body. ApoE polymorphisms are considered to be susceptibility candidate genes for hyperlipoproteinemia and atherosclerotic vascular disease. The human ApoE gene is located on chromosome 19, has 4 exons and 3 introns, mainly has two single nucleotide polymorphisms 526C > T and 388T > C, and can form 3 haplotypes, namely ApoE3(388T-526C), ApoE2(388T-526T) and ApoE4 (388C-526C). The ApoE4 carrier is reported in the literature to be at a 40% higher risk of developing coronary heart disease, and statins are often not good or effective in treating ApoE4 carrier, but have the strongest lipid-lowering effect on ApoE2 carrier. Currently, the FDA has listed APOE2 as a biomarker associated with the drug response of pravastatin.

At present, there are many methods for detecting gene polymorphism, such as direct sequencing, chip method, high-resolution melting curve method, allele-specific amplification method, taqman fluorescence probe method, etc. The sequencing method and the chip method have the disadvantages of complicated operation steps, long detection period and easy pollution of amplification products; the high-resolution melting curve method has simple steps, low specificity and higher requirements on instruments and equipment; the allele specific amplification method adopts ARMS primers for specific amplification, the design of the primers is difficult to optimize, and the detection condition is strict. The Taqman fluorescent probe method has high test cost and low amplification flux for a plurality of genes. Therefore, it is necessary to establish a simple, rapid, efficient, inexpensive, and highly specific method for detecting gene polymorphisms.

Disclosure of Invention

Aiming at the problems in the prior art, the invention aims to obtain a detection kit for statin curative effect prediction and a detection method and application thereof based on the detection kit for statin curative effect prediction and the detection method and application thereof.

In order to achieve one of the above objects, the invention adopts a detection kit for statin therapeutic effect prediction, a detection method and an application technical scheme as follows:

the detection kit provided by the invention is used for designing specific amplification primers and sequencing primers aiming at polymorphisms of two genes, namely ApoE (T388C) and ApoE (C526T), and comprises the following components: reagent 1, reagent 2, ApoE (T388C) sequencing primer, ApoE (C526T) sequencing primer, positive control.

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

preferably, the sequence of the specific primer group of the ApoE (T388C) is shown in the sequence table SEQ ID NO. 1-SEQ ID NO. 2; the sequence of the specific primer group of the ApoE (C526T) is shown in a sequence table SEQ ID NO. 3-SEQ ID NO. 4.

Preferably, the ApoE (T388C) sequencing primer and the ApoE (C526T) 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.

More preferably, the sequencing primer is a combination of agarose gel particles and amino-labeled PNA sequence, the combination is more stable and specific than the combination of DNA/DNA and is not influenced by the salt ion concentration, and the combination is combined with the DNA and is higher than the capture of the DNA/DNA. Can be used as a sequencing primer and a capture probe, and can be directly used for sequencing reaction after being combined with an amplified product and simply washed.

Preferably, the sequencing region corresponding to the ApoE (T388C) sequencing primer is an ApoE (T388C) to-be-detected sequence, and is shown as a sequence table SEQ ID NO. 7; the sequencing region corresponding to the ApoE (C526T) sequencing primer is an ApoE (C526T) to-be-detected sequence, and is shown as a sequence table SEQ ID NO. 9.

Preferably, the assignment instruction of the ApoE (T388C) to be detected sequence corresponding to the ApoE (T388C) is shown as the sequence table SEQ ID NO. 8; the distribution instruction of the to-be-detected sequence ApoE (C526T) corresponding to ApoE (C526T) is shown in a sequence table SEQ ID NO: 10.

Preferably, the reagent 1 comprises: amplification buffer, 18mM magnesium acetate;

preferably, the reagent 2 comprises: ApoE (T388C) pre-primer, ApoE (T388C) post-primer, ApoE (C526T) pre-primer, ApoE (C526T) post-primer, dNTPS, strand-displacing DNA polymerase, single-stranded DNA binding protein, recombinase which binds single-stranded nucleic acid, trehalose; the simultaneous amplification of ApoE (T388C)/ApoE (C526T) can be carried out under isothermal conditions.

More preferably, the concentrations of the components of the reagent 2 are respectively as follows: ApoE (T388C) pre-primer (0.32uM), ApoE (T388C) post-primer (0.32uM), ApoE (C526T) pre-primer (0.32uM), ApoE (C526T) 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), single-stranded nucleic acid binding recombinase (4.8 ng/. mu.L), trehalose (0.2%).

Preferably, the positive control comprises ApoE (T388C) and ApoE (C526T) heterozygotic genomic DNA at a concentration of 20 ng/ul. The positive control corresponds to the heterozygosis of the detected gene locus, provides reference for the type determination of an unknown sample, and simultaneously performs quality control on the effectiveness of the reaction solution.

The invention also discloses a method for detecting ondansetron medication-related gene polymorphism by adopting the kit, which comprises the following steps:

a. amplifying the amplification reaction solution and 5ul of genome DNA to be detected by adopting multiple RPA amplification;

b. combining 10ul of reaction product with 3ul of sequencing primer;

c. to each sequencing tube were added a sequencing enzyme and a sequencing substrate.

d. A dNTP calandria is taken, and dATP alpha S, dTTP, dGTP and dCTP are sequentially added from the 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.

e. Pyrosequencing;

f. determining the genotypes of ApoE (T388C) site and ApoE (C526T) site and statin drug site.

Preferably, the reaction volume is 25ul, and the reaction conditions are as follows: 25min at 40 ℃.

The invention also discloses a detection kit for statin curative effect prediction and application of the method, wherein the detection kit is used for detecting ApoE (T388C) and ApoE (C526T) so as to guide statin curative effect prediction from a gene level.

Recombinase Polymerase Amplification (RPA), is known as a nucleic acid detection technique that can replace PCR. RPA technology relies primarily on three enzymes: recombinases that bind single-stranded nucleic acids (oligonucleotide primers), single-stranded DNA binding proteins (SSBs), and strand-displacing DNA polymerases. The mixture of these three enzymes is also active at ambient temperature, with an optimum reaction temperature around 37 ℃. The recombinase, in combination with the primer, forms a protein-DNA complex that is able to search for homologous sequences in double-stranded DNA. Once the primers locate the homologous sequences, strand exchange reaction formation occurs and DNA synthesis is initiated, and the target region on the template is exponentially amplified. The replaced DNA strand binds to SSB, preventing further replacement. In this system, a single synthesis event is initiated by two opposing primers. The entire process is performed very quickly and detectable levels of amplification product are typically obtained within twenty minutes.

Compared with the prior art, the invention can rapidly and effectively generate a large amount of amplification products at a constant temperature by amplifying ApoE (T388C) and ApoE (C526T) through multiple RPA tubes. The kit can detect ApoE (T388C) and ApoE (C526T) gene polymorphism drugs simultaneously, and provides a gene angle suggestion for clinical personalized medication.

Drawings

FIG. 1 is a diagram showing an example of sequencing results of ApoE (T388C) TT genotype provided by the present invention;

FIG. 2 is a diagram showing an example of the sequencing results of the genotype of ApoE (T388C) CT provided by the present invention;

FIG. 3 is a diagram showing an example of the sequencing results of the ApoE (T388C) CC genotype provided by the present invention;

FIG. 4 is an exemplary illustration of the sequencing results for ApoE (C526T) CC genotype provided by the present invention;

FIG. 5 is a diagram showing an example of sequencing results of ApoE (C526T) TT genotype provided by the present invention;

FIG. 6 is an exemplary illustration of the sequencing results of the ApoE (C526T) CT genotype provided by the present invention.

Detailed Description

The following examples are provided to further describe the test kit for statin therapeutic effect prediction, the test method and the applications thereof 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 detection kit provided by the invention designs specific amplification primers and sequencing primers aiming at ApoE (T388C) and ApoE (C526T), and is used for isothermal amplification and pyrosequencing detection. The design of the primer based on the recombinase polymerase amplification technology is one of the keys of the invention, and the primer design of the technology cannot be carried out by auxiliary software and only depends on manual design. In order to ensure the amplification speed and the detection sensitivity, the length of the primer should be controlled to be 30-35 bp, the non-specific amplification is increased easily to cause false positive if the primer is designed to be too short, and the amplification cannot be performed easily if the primer is designed to be too long. Gene polymorphism sequences are subject to published sequences in Genebank.

The 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 concentration of each component of the reagent 2 is as follows: ApoE (T388C) pre-primer (0.32uM), ApoE (T388C) post-primer (0.32uM), ApoE (C526T) pre-primer (0.32uM), ApoE (C526T) 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), single-stranded nucleic acid binding recombinase (4.8 ng/. mu.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(2.5mM)	3
		ApoE (T388C) Pre-primer (20. mu.M)	0.4
ApoE (T388C) rear primer (20. mu.M)	0.4
		ApoE (C526T) Pre-primer (20. mu.M)	0.4
ApoE (C526T) rear primer (20. mu.M)	0.4
		Trehalose (20%)	0.25

After the preparation is finished, 158 ul/tube is subpackaged and freeze-dried.

(V) the preparation process of the sequencing primer of the detection kit of the embodiment is as follows:

(1) preparation of MES solution

①100mM MES,pH 4.8(100mL)：

②2.13g MES(2-[N-morpholino]ethane sulfonic acid,MW 213.25).

③pH 4.8

(2) TT Buffer (50mL) configuration

①12.5mL of 1M Tris buffer pH 8

②50ul 10％

(3) Sequencing primer preparation

Firstly, taking 500ul of magnetic beads, carrying out magnetic attraction for 1min, and removing supernatant;

② taking 1000ml 100MES solution to wash and attract magnetism for 1min, removing supernatant, and repeatedly washing for 2 times;

③ taking 500ul of each of 10mg/ml EDS and 10mg/ml NHS, and adding 10ul of capture primer.

Fourthly, the temperature of the housewares is 25 ℃ for 30min

Fifthly, taking 1000ml of confining liquid, mixing uniformly, absorbing magnetism for 1min, and removing supernatant;

sixthly, taking 1000ml of TT Buffer, mixing uniformly, absorbing magnetism for 1min, and removing supernatant; washing was repeated 3 times;

seventhly, adding 1ml of TE buffer solution for dissolving.

Example 2 detection of Pyrophosphoric acid

The apparatus used in the present invention is as follows: a thermostat;

pyrophosphoric acid sequencer: wuhan Firstet Biotech, Inc.

(1) Reagent preparation (reagent preparation Chamber)

The reagent was removed in advance and reagent 1 was vortexed for 15 seconds and centrifuged at low speed until use. 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) centrifuging at 7,000 Xg for 1min, and discarding the supernatant;

4) adding 150uL washing buffer solution into an EP tube, centrifuging at 7000g for 1min, and removing supernatant;

5) respectively adding 3uL sequencing enzyme and 3uL sequencing substrate into a sequencing tube;

6) a dNTP comb was loaded with 20. mu.l ATP. alpha.S, 20. mu.l dTTP, 20. mu.l dGTP, and 20. mu.l dCTP sequentially from the round end to the blunt end. Lightly knocking the bottom of the calandria against the tabletop to enable the bases to be flatly paved at the bottom of the calandria;

7) pyrosequencing results are shown in FIGS. 1 to 6.

(5) Interpretation of results

1) And (3) judging the effectiveness:

the blank control of the kit fails, and the detection results of the positive control are ApoE (T388C) and ApoE (C526T) types.

2) Criteria for determination of results

ApoE (T388C) in the DNA sequencing peak plot,

the frequency of T is not less than 90 percent, the frequency of C is not less than 10 percent, and the model is TT;

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 CT type;

the frequency of C is not less than 90 percent, the frequency of T is not less than 10 percent, and the model is CC;

ApoE (C526T) in the DNA sequencing peak map,

the frequency of T is not less than 90 percent, the frequency of C is not less than 10 percent, and the model is TT;

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 CT type;

the frequency of C is not less than 90 percent, the frequency of T is not less than 10 percent, namely the type CC

Example 3 correlation of Gene test results with drug efficacy and cardiovascular disease

Apolipoprotein E is a very important protein in the human body and has a close correlation with lipoprotein metabolism. Such as enzymes involved in the activation of hydrolyzed fats, immunomodulation and regeneration of neural tissue, thereby affecting the occurrence, progression and prognosis of a number of diseases.

Correspondence between phenotype and gene locus

Phenotype of gene	ApoE type	Genotype of corresponding site
			E2	ε2/ε2	388T/T +526T/T type
E2	ε2/ε3	388T/T +526T/C type
			E3	ε2/ε4	388T/C +526T/C type
E3	ε3/ε3	388T/T +526C/C type
			E4	ε3/ε4	388T/C +526C/C type
E4	ε4/ε4	388C/C +526C/C type

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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