Primer combination, detection reagent, detection kit and application thereof

Abstract

The invention relates to the field of gene detection, in particular to a primer combination, a detection reagent, a detection kit and application thereof. The invention provides an amplification primer group. The amplification primer group has a nucleotide sequence which is randomly shown as SEQ ID NO: 1 to SEQ ID NO: 26. The invention provides a specific primer group for detecting an antithrombotic individualized medication gene based on an Autof Gene platform and application of the specific primer group. By using the provided specific primer group, the genotypes of 13 SNP loci can be synchronously detected in a reaction system, and the specific primer group has the advantages of high sensitivity, strong specificity, easy result recognition and high accuracy, and provides more reasonable medication guidance for antithrombotic individualized medication.

Description

Technical Field

[0001] The present invention relates to the field of gene detection, and in particular to a primer combination, a detection reagent, a detection kit and applications thereof. Background Art

[0002] MALDI-TOF MS (matrix-assisted laser desorption / ionization time offlight massspectrometry) technology is a new soft ionization organic mass spectrometry technology developed in recent decades. It breaks the tradition that mass spectrometry can only analyze small molecules, and makes it possible to study biological macromolecules such as nucleic acids and proteins by mass spectrometry. It has greatly promoted the development of genomics and proteomics, and brought revolutionary development to the biological and medical fields.

[0003] Autof Gene is a nucleic acid mass spectrometer launched by Antu Experimental Instrument (Zhengzhou) Co., Ltd. Based on the AutofGene platform, using single base extension technology and MALDI-TOF MS technology, it can realize the detection of multiple amplification reaction genotypes. Its main principle is to first amplify the target fragment containing the SNP to be tested by PCR primers, add shrimp alkaline phosphatase (SAP) after PCR to remove dNTP in the reaction solution, and then add extension primers and ddNTP and other related components for single base extension. The extension primer is designed to be located one base before the mutation site, and one base can be extended at the mutation site. Different SNP templates can obtain different extension bases. Then the prepared sample analyte and the matrix are co-crystallized on the target plate for mass spectrometry detection. Since the molecular weight of each extension product is different, it is possible to check whether the detection peak appears at each molecular weight position, and then determine the SNP typing of the sample.

[0004] With the arrival of an aging society in China, on the one hand, the incidence of vascular embolic diseases has been rising year by year; on the other hand, cardiovascular and cerebrovascular interventional treatments and other surgeries have developed rapidly in my country, and anticoagulant, antiplatelet and lipid-lowering treatments are followed up simultaneously after stent surgery, which has promoted the further expansion of my country's anti-thrombotic drug market.

[0005] Aspirin, clopidogrel, and ticagrelor are commonly used antiplatelet drugs in clinical practice. Clopidogrel is used to prevent and treat heart, brain, and other arterial circulatory disorders caused by high platelet aggregation, such as recent stroke, myocardial infarction, and confirmed peripheral arterial disease. Aspirin can prevent the formation of blood clots and is clinically used to prevent the formation of blood clots after transient ischemic attacks, myocardial infarction, artificial heart valves, venous fistulas, or other surgeries. Ticagrelor is a new type of antiplatelet drug in the same class as clopidogrel. It is used to treat patients with acute coronary syndrome or a history of myocardial infarction and high risk factors for atherothrombotic events. The most common adverse reaction is bleeding. Warfarin is a common anticoagulant drug in clinical practice that can inhibit the synthesis of vitamin K-dependent coagulation factors.

[0006] In recent years, pharmacogenomics (PGx) has developed rapidly as a powerful means to achieve personalized drug delivery. It mainly studies the relationship between human genome information and drug response, and uses genomic information to interpret the causes of individual differences in drugs (sensitivity, metabolism, and adverse reactions). Different patients carry different single nucleotide polymorphisms and respond differently to drugs. The efficacy and side effects of the same drug in different patients also vary greatly. Therefore, it is very necessary to conduct genetic testing to guide precise medication, which can not only reduce the economic burden of patients, improve treatment effects, but also reduce the side effects of drugs on patients.

[0007] Although the second generation sequencing has a large throughput, the experimental process and data analysis cycle are long, the cost is high, and the technical threshold for operators is high; although the fluorescence quantitative PCR has a short experimental cycle, the throughput is small; Sanger sequencing technology can only detect one SNP site in one reaction well, and the experimental cost is high; the gene chip platform has a small throughput, low sensitivity, and relatively high cost. Therefore, it is necessary to develop a detection solution for the polymorphism of personalized drug gene sites that is simple to operate, low cost, high throughput, high sensitivity, and easy to distinguish. Summary of the invention

[0008] In view of this, the present invention provides a primer combination, a detection reagent, a detection kit and its application. The present invention provides a specific primer set for detecting anti-thrombotic personalized drug gene detection based on the Autof Gene platform and its application. Using the provided specific primer set, the genotype detection of 13 SNP sites can be simultaneously achieved in one reaction system, which has high sensitivity, strong specificity, easy to identify results, high accuracy, and provides more reasonable medication guidance for anti-thrombotic personalized medication.

[0009] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0010] The present invention provides an amplification primer set having:

[0011] (1) a nucleotide sequence as shown in any of SEQ ID NO: 1 to SEQ ID NO: 26; and / or

[0012] (2) a nucleotide sequence obtained by modifying, replacing, deleting and / or adding one or more bases to the nucleotide sequence shown in (1); and / or

[0013] (3) a sequence having at least 80% homology with the nucleotide sequence shown in (1); and / or

[0014] (4) A complementary sequence of the sequence shown in (1), (2) or (3).

[0015] In some embodiments of the present invention, the amplification primer set includes one or more of the following combinations:

[0016] Combination X:

[0017] (5), the upstream primer has the nucleotide sequence shown in SEQ ID NO: (2X-1); and

[0018] (6), the downstream primer has the nucleotide sequence shown in SEQ ID NO: (2X); or

[0019] (7) A nucleotide sequence obtained by substituting, deleting or adding one or more bases to the nucleotide sequence shown in (5) or (6), and having the same or similar function as the nucleotide sequence shown in (5) or (6); or

[0020] (8) a nucleotide sequence having at least 80% homology to the nucleotide sequence shown in (5) or (6);

[0021] Wherein, X is any integer selected from 1 to 13.

[0022] The present invention also provides an extension primer set, which has:

[0023] (9) A nucleotide sequence as shown in any of SEQ ID NO:27 to SEQ ID NO:39;

[0024] (10) A nucleotide sequence obtained by modifying, replacing, deleting and / or adding one or more bases of the nucleotide sequence shown in (9);

[0025] (11) a sequence having at least 80% homology to the nucleotide sequence shown in (9);

[0026] (12) A complementary sequence of the sequence shown in (9), (10) or (11).

[0027] The present invention also provides a primer combination, including: the above-mentioned amplification primer set and the above-mentioned extension primer set.

[0028] The present invention also provides a detection reagent, comprising: the above-mentioned amplification primer set, the above-mentioned extension primer set and / or the above-mentioned primer combination and an acceptable auxiliary agent.

[0029] In some embodiments of the present invention, in the above detection reagent, the auxiliary agent includes: one or more of: PCR reaction mixture, PCR enzyme mixture, SAP reaction mixture, SAP enzyme mixture, extension reaction mixture and extension enzyme mixture.

[0030] The present invention also provides a detection kit, comprising: the above-mentioned amplification primer set, the above-mentioned extension primer set, the above-mentioned primer combination and / or the above-mentioned detection reagent and an acceptable device and / or carrier.

[0031] The present invention also provides the use of the above-mentioned amplification primer set, the above-mentioned extension primer set, the above-mentioned primer combination, the above-mentioned detection reagent and / or the above-mentioned detection kit in screening anti-thrombotic drugs.

[0032] The present invention also provides the use of the above-mentioned amplification primer set, the above-mentioned extension primer set, the above-mentioned primer combination, the above-mentioned detection reagent and / or the above-mentioned detection kit in detecting anti-thrombotic personalized medication genes and / or gene site polymorphisms.

[0033] In some embodiments of the present invention, in the above application, the gene includes: wild type and / or mutant type of one or more of VKORC1, CYP2C9, CYP4F2, CYP2C19, SLCO1B1, LTC4S, GP1BA, PTGS1 and ITGB3.

[0034] In some embodiments of the present invention, in the above application, the gene locus includes: a SNP locus; the SNP locus includes: one or more of rs9934438, rs9923231, rs1799853, rs1057910, rs2108622, rs4244285, rs4986893, rs12248560, rs4149056, rs730012, rs6065, rs10306114 and rs5918.

[0035] The present invention also provides a method for detecting polymorphism of anti-thrombotic personalized medication gene loci, which comprises taking the above primer combination to amplify the sample to be tested, SAP purification, single base extension, and detection to obtain the test result.

[0036] In some embodiments of the present invention, in the above detection method, in the primer combination, the final concentration of the amplification primer set is 0.5 μM; and the final concentration of the extension primer set is 5-10 μM.

[0037] In some embodiments of the present invention, in the above detection method, the amplification conditions include:

[0038]

[0039] The beneficial effects of the present invention include:

[0040] (1) This application can simultaneously detect mutant and wild-type genes of VKORC1, CYP2C9, CYP4F2, CYP2C19, SLCO1B1, LTC4S, GP1BA, PTGS1, and ITGB3 in whole blood samples with good specificity, and can provide individualized guidance for antithrombotic medication.

[0041] (2) The present invention has been verified to have a detection rate of 82.3% for primer combination 1, 95.0% for primer combination 2, and 100% for primer combination 3 in 29 samples. Primer combination 3 is the optimal combination, which can not only accurately detect all sites, but also has an accuracy rate of 100%.

[0042] (3) The product of the present invention is used to perform genotype detection on the sample to be tested, with simple operation steps, short detection cycle, high throughput, low requirements on operators, and high detection efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0044] Figure 1 Primer combination 2 is shown to detect sample 8063;

[0045] Figure 2 Primer combination 2 is shown to detect sample 8063;

[0046] Figure 3 Primer combination 2 is shown to detect sample 8063;

[0047] Figure 4 Primer combination 2 is shown to detect sample 8063;

[0048] Figure 5 Primer combination 1 is shown to detect sample 8064;

[0049] Figure 6 Primer combination 3 is shown to detect sample 8063;

[0050] Figure 7 Primer combination 3 is shown to detect sample 8063;

[0051] Figure 8Primer combination 3 is shown to detect sample 8063;

[0052] Fig. 9 Primer combination 3 is shown to detect sample 8063;

[0053] Fig.10 Primer combination 3 is shown for detecting sample 8064. DETAILED DESCRIPTION

[0054] The invention discloses a primer combination, a detection reagent, a detection kit and applications thereof.

[0055] It should be understood that the expression "one or more of..." includes each of the items recited after the expression individually and in various different combinations of two or more of the recited items, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited items should be understood to have the same meaning, unless otherwise understood from the context.

[0056] The use of the terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.

[0057] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the present invention remains operable. In addition, two or more steps or actions may be performed simultaneously.

[0058] The use of any and all examples or exemplary language, such as "for example" or "including", herein is intended only to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.

[0059] In addition, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

[0060] The present invention firstly amplifies the target fragment containing the SNP to be detected by PCR primers, adds shrimp alkaline phosphatase (SAP) to remove dNTP in the reaction solution after PCR, then adds SNP extension primers and ddNTP and other related components to the reaction solution in the previous step to carry out a single base extension reaction, and determines the genotype of each SNP site according to the molecular weight of one base after extension.

[0061] In Examples 1 to 5 of the present invention, all the raw materials and reagents used can be purchased from the market.

[0062] The present invention will be further described below in conjunction with embodiments:

[0063] Example 1 Selection of SNP sites

[0064] The inventors screened out 9 genes and 13 loci for personalized anti-thrombotic medication based on market research, literature search and reference to the PharmGKB database, FDA database and CPIC guidelines. The 9 genes are VKORC1, CYP2C9, CYP4F2, CYP2C19, SLCO1B1, LTC4S, GP1BA, PTGS1, and ITGB3, and the 13 loci are rs9934438, rs9923231, rs1799853, rs1057910, rs2108622, rs4244285, rs4986893, rs12248560, rs4149056, rs730012, rs6065, rs10306114, and rs5918.

[0065] Example 2 Preparation of DNA samples

[0066] Genomic DNA was extracted using a commercial blood extraction kit (DP304). Human genomic DNA was extracted from 200 μL of whole blood from each of the 29 patients and eluted with 100 μL of TE.

[0067] Example 3 Selection of amplification primers and extension primers

[0068] Multiplex PCR experiments are not simply mixing multiple pairs of specific primers into a reaction system. It is like a "leg-binding race". The amplification conditions between multiple targets are incompatible, and each target requires other primers to cooperate. We designed three sets of amplification primers and extension primers, and selected the primer combination that met the requirements and achieved 100% accuracy.

[0069] Table 1

[0070]

[0071]

[0072]

[0073]

[0074] Example 4 Mass spectrometry detection

[0075] The reagent information used is as follows:

[0076] Table 2

[0077]

[0078] The instrument information used is as follows:

[0079] Table 3

[0080]

[0081] A) Multiplex PCR reaction

[0082] According to the grouping in Example 3, the amplification primers and extension primers of each group are mixed in equal moles to form multiple PCR amplification primers and multiple extension primers. The final concentration of each amplification primer in the mixed solution is 0.5 μM, and the final concentration of each extension primer in the mixed solution is 5-10 μM.

[0083] PCR reaction system:

[0084] Table 4

[0085]

[0086] Add 3 μL of PCR amplification reaction mixture to each well of the 96-well plate, and then add 2 μL of DNA sample. Seal with PCR sealing film to prevent reagent evaporation. Centrifuge the 96-well plate with PCR sealing film at 2000 rpm for 1 minute and then perform PCR amplification reaction. The reaction procedure is as follows:

[0087] Table 5

[0088]

[0089] After the reaction was completed, the 96-well plate was taken out and centrifuged at 2000 rpm for 1 min.

[0090] SAP purification

[0091] SAP purification system:

[0092] Table 6

[0093]

[0094] Add 2 μL of SAP reaction purification mixture to each well of the 96-well plate after the above amplification, and seal it tightly with PCR sealing film to prevent the reagent from evaporating. Centrifuge the 96-well plate with PCR sealing film at 2000 rpm for 1 minute and then perform SAP purification reaction. The reaction procedure is as follows:

[0095] Table 7

[0096]

[0097] After purification, take out the 96 / 384-well plate and centrifuge at 2000 rpm for 1 min.

[0098] Single base extension

[0099] Single base extension system:

[0100] Table 8

[0101]

[0102] Add 2 μL of the single-base extension reaction mixture to each well of the purified 96-well plate and seal it with PCR sealing film to prevent the reagent from evaporating. Centrifuge the 96-well plate with PCR sealing film at 2000 rpm for 1 minute and then perform single-base extension. The reaction procedure is as follows:

[0103] Table 9

[0104]

[0105] After the reaction is completed, take out the 96 / 384-well plate.

[0106] The resin was added to the above product and rotated on a rotating mixer for 40 minutes for desalting and purification. The product was then spotted onto a specimen plate using an automatic spotter and detected using a time-of-flight mass spectrometry detection system (Autof Gene) to automatically obtain the results.

[0107] 29 samples were selected to cover the genotypes of all sites to be tested. The above method was used for testing and verification with the gold standard Sanger sequencing method (commissioned by a third-party testing agency (General Bio (Anhui) Co., Ltd.)). The results of primer combination 3 were 100% consistent with the Sanger sequencing method. There were sites that were not extended in the results of primer combination 1 and primer combination 2. The first group of amplification primers and extension primers detected rs730012 without extension. The second group of amplification primers and extension primers detected sample 8063, and rs12248560, rs4986893, rs730012, and rs4244285 were not extended.

[0108] The results are as follows:

[0109] Sample 8063 was tested for rs12248560 using primer combination 2. Figure 1 ;

[0110] Sample 8063 was tested for rs4986893 using primer combination 2. Figure 2 ;

[0111] Sample 8063 was tested for rs730012 using primer combination 2. Figure 3 ;

[0112] Sample 8063 was tested for rs4244285 using primer combination 2. Figure 4 ;

[0113] Sample 8064 was tested for rs730012 using primer combination 1. Figure 5 ;

[0114] Sample 8063 was tested for rs12248560 using primer combination 3. Figure 6 ;

[0115] Sample 8063 was tested for rs4986893 using primer combination 3. Figure 7 ;

[0116] Sample 8063 was tested for rs730012 using primer combination 3. Figure 8 ;

[0117] Sample 8063 was tested for rs4244285 using primer combination 3. Fig. 9 ;

[0118] Sample 8064 was tested for rs730012 using primer combination 3. Fig.10 ;

[0119] Figures 1 to 4 For primer combination 2, sample 8063 was tested. Figure 5 Sample 8064 was tested with primer combination 1, and the results showed only UEP peaks without extension, that is, no results were detected. Figures 6 to 9 For primer combination 3, sample 8063 was tested. Fig.10 Sample 8064 was tested with primer combination 3, and the results were all normal, that is, the test results were normal and completely consistent with the results of the Sanger sequencing method. Therefore, primer combination 3 is the best combination for testing personalized anti-thrombotic drugs.

[0120] Example 5

[0121] Optimized PCR program:

[0122] Table 10

[0123]

[0124] Test results:

[0125] Table 11

[0126]

[0127] As shown in Table 11, based on the Autof Gene platform, 13 loci of 9 genes for personalized antithrombotic medication were detected in 29 samples using primer combination 3. Compared with the gold standard Sanger sequencing method, the accuracy rate was 100%.

[0128] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An amplification primer set, characterized in that: It has: (1) a nucleotide sequence as shown in any of SEQ ID NO: 1 to SEQ ID NO: 26; and / or (2) a nucleotide sequence obtained by modifying, replacing, deleting and / or adding one or more bases to the nucleotide sequence shown in (1); and / or (3) a sequence having at least 80% homology with the nucleotide sequence shown in (1); and / or (4) A complementary sequence of the sequence shown in (1), (2) or (3).

2. The amplification primer set according to claim 1, characterized in that: The amplification primer set includes one or more of the following combinations: Combination X: (5), the upstream primer has the nucleotide sequence shown in SEQ ID NO: (2X-1); and (6), the downstream primer has the nucleotide sequence shown in SEQ ID NO: (2X); or (7) A nucleotide sequence obtained by substituting, deleting or adding one or more bases to the nucleotide sequence shown in (5) or (6), and having the same or similar function as the nucleotide sequence shown in (5) or (6); or (8) a nucleotide sequence having at least 80% homology to the nucleotide sequence shown in (5) or (6); Wherein, X is any integer selected from 1 to 13.

3. An extension primer set, characterized in that: It has: (9) A nucleotide sequence as shown in any of SEQ ID NO:27 to SEQ ID NO:39; (10) A nucleotide sequence obtained by modifying, replacing, deleting and / or adding one or more bases of the nucleotide sequence shown in (9); (11) a sequence having at least 80% homology to the nucleotide sequence shown in (9); (12) A complementary sequence of the sequence shown in (9), (10) or (11).

4. A primer combination, characterized in that: include: The amplification primer set according to claim 1 or 2 and the extension primer set according to claim 3.

5. A detection reagent, characterized in that: include: The amplification primer set according to claim 1 or 2, the extension primer set according to claim 3 and / or the primer combination according to claim 4 and an acceptable auxiliary agent.

6. A detection kit, characterized in that: include: The amplification primer set according to claim 1 or 2, the extension primer set according to claim 3, the primer combination according to claim 4 and / or the detection reagent according to claim 5 and an acceptable device and / or carrier.

7. Use of the amplification primer set according to claim 1 or 2, the extension primer set according to claim 3, the primer combination according to claim 4, the detection reagent according to claim 5 and / or the detection kit according to claim 6 in screening antithrombotic drugs.

8. Use of the amplification primer set according to claim 1 or 2, the extension primer set according to claim 3, the primer combination according to claim 4, the detection reagent according to claim 5 and / or the detection kit according to claim 6 in detecting polymorphisms of anti-thrombotic personalized medication genes and / or gene loci.

9. The use according to claim 8, characterized in that The genes include: wild type and / or mutant type of one or more of VKORC1, CYP2C9, CYP4F2, CYP2C19, SLCO1B1, LTC4S, GP1BA, PTGS1 and ITGB3.

10. The use according to claim 8 or 9, characterized in that: The gene loci include: SNP loci; the SNP loci include: one or more of rs9934438, rs9923231, rs1799853, rs1057910, rs2108622, rs4244285, rs4986893, rs12248560, rs4149056, rs730012, rs6065, rs10306114 and rs5918.

11. A method for detecting polymorphism of gene loci for personalized antithrombotic medication, characterized in that: The primer combination as claimed in claim 4 is used to amplify the sample to be tested, perform SAP purification, single base extension, and detect to obtain the test result.

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