Primer composition and kit for detecting safe medication gene of children

Abstract

The invention discloses a primer composition and a kit for gene detection of safe medication of children, and belongs to the technical field of gene detection. The primer composition disclosed by the invention is used for detecting safe medication genes of children; the primer composition comprises 56 amplification primers and 28 extension primers, the sequences of the amplification primers are as shown in SEQ ID NO. 1 to 56, and the sequences of the extension primers are as shown in SEQ ID NO. 57 to 84. The kit can detect different genotypes of 28 common sites related to clinical safe medication genes for children through one hole, and is high in sensitivity, strong in specificity, high in accuracy, simple and convenient to operate, low in cost, high in throughput, rapid in detection, automatic in result interpretation and easy to clinically popularize and apply.

Description

Technical Field

[0001] The present invention relates to the technical field of gene detection, and in particular to a primer composition and a kit for gene detection of safe drug use in children. Background Art

[0002] The information disclosed in the background of the invention is only intended to enhance the understanding of the overall background of the invention and should not be necessarily regarded as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] The lack of preparations suitable for children, the lack of information on children's drug instructions, the lack of pediatric drug guidelines and standards, and the insufficient supply of clinical drugs are the four major problems facing children's drug use in medical institutions. Due to the lack of dosage forms and specifications suitable for children's drugs, pediatric clinics have to manually divide the doses for young children. The lack of children's drugs and the abuse of adult drugs have led to the fact that the safe use of drugs for children has become a social problem.

[0004] Pharmacogenomics is the science of studying the effects of drug-related genes and their product polymorphisms on drug efficacy. Pharmacogenomic research can provide rich information for clinical precision treatment. Compared with adults, children have greater individual differences at different ages, which is not only reflected in changes in height and weight, but also in the significant differences in pharmacokinetic and pharmacodynamics due to the different development levels of various systems in children at different ages. Genetic variations in drug metabolism, transport and drug target genes in the body and changes in their expression levels can lead to individual differences in drug responsiveness by affecting the concentration and sensitivity of drugs in the body. In recent years, with the development of human genomics, the field of pharmacogenomics has developed rapidly, and more and more pharmacogenomic biomarkers and their detection methods have emerged. Detection of drug-metabolizing enzymes and drug target genes can guide clinicians to select appropriate drugs and dosages for specific patients, realize individualized medication, thereby improving the effectiveness and safety of drug treatment and preventing the occurrence of serious adverse drug reactions.

[0005] At present, the main methods and products for detecting genes for safe drug use in children include fluorescent quantitative melting curve method, reverse dot hybridization method, Sanger sequencing method, and high-throughput sequencing method. The single-well detection throughput of the fluorescent quantitative combined with melting curve method is limited, and it can only detect known mutations at a few sites. Specific primers need to be designed for each mutant genotype at each site; covering common sites requires multiple wells and multiple detections in each well, which requires high requirements for multiple probe design and high technical difficulty. The detection probes are expensive, and the results are manually interpreted and prone to misjudgment; the reverse dot hybridization system requires probes to be designed for each genotype at each site, which is expensive, and the resolution depends on the effectiveness of the probes. It is prone to non-specificity, and the detection process is long, the operation is complicated, the system is open, and it is very easy to contaminate between samples; Sanger sequencing is the gold standard for sequences, but the process is long, the operation is complicated, and each well needs to be manually interpreted. For one site, covering common sites requires multi-well testing, which is costly and has low throughput. Although high-throughput sequencing can cover multiple gene sites at one time, its instruments and detection reagents are expensive, and when the number of test samples is small, it will cause waste, resulting in higher costs. In addition, the system requires a high amount of nucleic acid samples (more than 50ng), and the amount of newborn heel blood samples is small and the concentration is low. Screening tests for multiple projects are often required, so it is difficult to obtain enough nucleic acid templates. The high-throughput sequencing detection process takes up to 3-5 days, the operation is complicated, and professional bioinformatics analysts are required to analyze the data. The results are difficult to interpret, so it cannot be promoted and applied on a large scale as a screening project in local small and medium-sized hospitals at all levels across the country. Summary of the invention

[0006] In view of this, the present invention provides a primer composition and a kit for genetic detection of safe drug use in children. The present invention screens out 37 drugs commonly used by children (esomeprazole, omeprazole, lansoprazole, rabeprazole, pantoprazole, diazepam, oseltamivir, ibuprofen, acetaminophen, codeine, acetylcysteine, dextromethorphan, dexamethasone, fexofenadine, salbutamol, salmeterol, prednisone, prednisolone, fluticasone propionate, triamcinolone acetonide, budesonide , desloratadine, montelukast, amikacin, amoxicillin, clavulanic acid, voriconazole, peginterferon α-2a, peginterferon α-2b, ribavirin, kanamycin, streptomycin, gentamicin, ceftriaxone, cefotaxime, azithromycin, metronidazole) and their related gene loci, with comprehensive detection sites and accurate results. MALDI-TOFMS technology is used for detection, which has the advantages of multiple detection sites, accuracy, high throughput, low cost and short reporting cycle.

[0007] In a first aspect, the present invention provides a primer composition, wherein the primer composition is used for genetic testing of safe medication for children;

[0008] The primer composition includes 56 amplification primers and 28 extension primers. The sequences of the amplification primers are shown in SEQ ID NOs. 1 to 56, and the sequences of the extension primers are shown in SEQ ID NOs. 57 to 84.

[0009] Preferably, at the rs16947 site of the CYP2D6 gene, the sequence of the amplification primer is SEQ ID No.1-2, and the sequence of the extension primer is SEQ ID No.57; at the rs1065852 site of the CYP2D6 gene, the sequence of the amplification primer is SEQ ID No.3-4, and the sequence of the extension primer is SEQ ID No.58; at the rs1045642 site of the ABCB1 gene, the sequence of the amplification primer is SEQ ID No.5-6, and the sequence of the extension primer is SEQ ID No.59; at the rs12248560 site of the CYP2C19 gene, the sequence of the amplification primer is SEQ ID No.7-8, and the sequence of the extension primer is SEQ ID No.60; at the rs6092 site of the SERPINE1 gene, the sequence of the amplification primer is SEQ ID No.9-10, and the sequence of the extension primer is SEQ ID No.61; at the rs13120400 site of the ABCG2 gene, the sequence of the amplification primer is SEQ ID No.11-12, and the sequence of the extension primer is SEQ ID No.62; rs1799853 site of CYP2C9 gene, the sequences of its amplification primers are SEQ ID No.13-14, and the sequence of its extension primer is SEQ ID No.63; rs10929303 site of UGT1A gene, the sequences of its amplification primers are SEQ ID No.15-16, and the sequence of its extension primer is SEQ ID No.64; rs4986893 site of CYP2C19 gene, the sequences of its amplification primers are SEQ ID No.17-18, and the sequence of its extension primer is SEQ ID No.65; rs267606619 site of MT-RNR1 gene, the sequences of its amplification primers are SEQ ID No.19-20, and the sequence of its extension primer is SEQ ID No.66; rs4244285 site of CYP2C19 gene, the sequences of its amplification primers are SEQ ID No.21-22, and the sequence of its extension primer is SEQ ID No.67; rs11322783 site of IFNL4 gene, the sequences of its amplification primers are SEQ ID Nos.23-24, and the sequence of its extension primer is SEQ ID No.68; rs1057910 site of CYP2C9 gene, the sequences of its amplification primers are SEQ ID Nos.25-26, and the sequence of its extension primer is SEQ ID No.69; rs901865 site of HRH1 gene, the sequences of its amplification primers are SEQ ID Nos.27-28, and the sequence of its extension primer is SEQ ID No.70; rs11568482 site of SLC22A8 gene, the sequences of its amplification primers are SEQ ID Nos.29-30, and the sequence of its extension primer is SEQ ID No.71; MT-RNR1 gene rs267606617 site, the sequence of its amplification primers is SEQ ID No.31-32, and the sequence of its extension primer is SEQ ID No.72; IFNL3 gene rs8099917 site, the sequence of its amplification primers is SEQ ID No.33-34, and the sequence of its extension primer is SEQ ID No.73; ABCB1 gene rs2032582 site, the sequence of its amplification primers is SEQ ID No.35-36, and the sequence of its extension primer is SEQ ID No.74; GLCCI1 gene rs37973 site, the sequence of its amplification primers is SEQ ID No.37-38, and the sequence of its extension primer is SEQ ID No.75; OPRM1 gene rs1799971 site, the sequence of its amplification primers is SEQ ID No.39-40, and the sequence of its extension primer is SEQ ID No.76; ADRB2 gene rs1042713 site, the sequence of its amplification primers is SEQ ID No. ID No.41-42, the sequence of the extension primer is SEQ ID No.77; the ITPA gene rs1127354 site, the sequence of its amplification primer is SEQ ID No.43-44, the sequence of the extension primer is SEQ ID No.78; the CYP2D6 gene rs3892097 site, the sequence of its amplification primer is SEQ ID No.45-46, the sequence of the extension primer is SEQ ID No.79; the CYP2A6 gene rs28399433 site, the sequence of its amplification primer is SEQ ID No.47-48, the sequence of the extension primer is SEQ ID No.80; the IFNL4 gene rs12979860 site, the sequence of its amplification primer is SEQ ID No.49-50, the sequence of the extension primer is SEQ ID No.81; the TOLLIP gene rs3750920 site, the sequence of its amplification primer is SEQ ID No.51-52, the sequence of the extension primer is SEQ ID No.82; HLA-DQB1 gene rs9274407 site, the sequence of its amplification primers is SEQ ID No.53-54, and the sequence of its extension primer is SEQ ID No.83; ABCC1 gene rs119774 site, the sequence of its amplification primers is SEQ ID No.55-56, and the sequence of its extension primer is SEQ ID No.84.

[0010] In a second aspect, the present invention provides the use of the above primer combination in the preparation of a kit for detecting or guiding safe medication for children.

[0011] In a third aspect, the present invention provides a kit for detecting genes for safe medication for children, the kit comprising the above-mentioned primer combination.

[0012] In a fourth aspect, the present invention provides a kit for guiding children in safe use of medications, the kit comprising the above-mentioned primer composition.

[0013] Preferably, the kit further comprises a multiplex PCR reaction reagent, a single base extension reaction reagent and a dNTP removal mixture reaction reagent.

[0014] Preferably, the multiple PCR reaction reagent includes a PCR reaction amplification primer mixture, and the PCR reaction amplification primer mixture includes amplification primers with nucleotide sequences shown in SEQ ID NOs.1 to 56; the single base extension reaction reagent includes a single base extension reaction primer premix, and the single base extension reaction primer premix includes extension primers with nucleotide sequences shown in SEQ ID NOs.57 to 84.

[0015] Furthermore, in the PCR reaction amplification primer mixture, the concentration of each amplification primer is 0.5 μM.

[0016] Furthermore, in the single base extension reaction primer premix, the concentration of each extension primer is as follows:

[0017] Extension primer sequence Concentration (μM) Extension primer sequence Concentration (μM) SEQ ID NO.57 2.613 SEQ ID NO.71 19.113 SEQ ID NO.58 7.320 SEQ ID NO.72 11.273 SEQ ID NO.59 5.793 SEQ ID NO.73 11.533 SEQ ID NO.60 15.153 SEQ ID NO.74 12.787 SEQ ID NO.61 4.980 SEQ ID NO.75 13.800 SEQ ID NO.62 3.600 SEQ ID NO.76 9.767 SEQ ID NO.63 5.480 SEQ ID NO.77 26.013 SEQ ID NO.64 8.313 SEQ ID NO.78 17.160 SEQ ID NO.65 7.027 SEQ ID NO.79 27.173 SEQ ID NO.66 6.627 SEQ ID NO.80 29.207 SEQ ID NO.67 4.207 SEQ ID NO.81 20.107 SEQ ID NO.68 4.440 SEQ ID NO.82 32.607 SEQ ID NO.69 17.827 SEQ ID NO.83 22.080 SEQ ID NO.70 10.233 SEQ ID NO.84 25.873

[0018] In a fifth aspect, the present invention provides a detection method based on the above kit for non-diagnostic purposes, comprising the following steps:

[0019] (1) Multiplex PCR: Using amplification primers with sequences as shown in SEQ ID NOs. 1 to 56, a total of 28 pediatric safe drug gene polymorphic sites are simultaneously amplified to obtain PCR products containing 28 gene polymorphic sites:

[0020] (2) SAP digestion: Use shrimp alkaline phosphatase (SAP) to digest and remove the remaining dNTPs in the reaction system to prevent interference with the next base extension reaction;

[0021] (3) Single base extension reaction: adding an extension primer with a sequence as shown in SEQ ID NO. 57 to 84 to perform a single base extension reaction, using a modified dideoxy triphosphate nucleoside (ddNTP) as a reaction substrate, so that the extension primer extends one base at a specific single nucleotide site and then terminates the reaction;

[0022] (4) Resin desalting and purification: using cation exchange resin to adsorb salt ions in the system and purify the extension reaction products;

[0023] (5) Mass spectrometry detection: The purified product is detected using a nucleic acid mass spectrometer;

[0024] (6) Interpretation of results

[0025] In a sixth aspect, the present invention provides application of the above detection method in guiding children's safe use of medicines.

[0026] Compared with the prior art, the present invention has achieved the following beneficial effects:

[0027] The present invention provides a method for detecting gene polymorphisms for guiding safe medication for children by combining multiple PCR technology, single base extension technology and mass spectrometry detection technology. The method covers as comprehensive as possible gene loci for children's medication, including 28 pairs of amplification primers designed for 28 loci corresponding to 20 genes corresponding to 37 drugs, which can specifically amplify 28 common loci regions of genes for safe medication for children; and also includes 28 single base extension primers. The kit of the present invention can realize 1-hole detection of different genotypes of 28 common loci related to clinical genes for safe medication for children, with high sensitivity, strong specificity and high accuracy, and is easy to operate, low-cost, high-throughput, fast detection, automatic interpretation of results, and easy clinical promotion and application. The present invention can be applied to gene detection for safe medication for children, providing a reliable detection system and kit for safe medication for children, with important clinical application value and good market application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The drawings constituting part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their description are used to explain the present invention and do not constitute an improper limitation of the present invention. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0029] Figure 1 This is a diagram of the mass spectrometry detection results of Example 3 of the present invention.

[0030] Figure 2 This is a representative mass spectrometry result diagram of Example 4 of the present invention. DETAILED DESCRIPTION

[0031] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0032] The technical solution of the present invention is further described below in conjunction with specific embodiments.

[0033] Example 1

[0034] This example screened out 37 drugs that are commonly used by children (esomeprazole, omeprazole, lansoprazole, rabeprazole, pantoprazole, diazepam, oseltamivir, ibuprofen, acetaminophen, codeine, acetylcysteine, dextromethorphan, dexamethasone, fexofenadine, salbutamol, salmeterol, prednisone, prednisolone, fluticasone propionate, triamcinolone acetonide, budesonide, desloratadine, montelukast, amikacin, a Moxicillin, clavulanic acid, voriconazole, peginterferon α-2a, peginterferon α-2b, ribavirin, kanamycin, streptomycin, gentamicin, ceftriaxone, cefotaxime, azithromycin, metronidazole) and their related gene loci (20 genes and 28 loci), provided primers used for genetic testing of safe drug use in children, as shown in Table 1, all of which were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0035] Table 1 Sites and corresponding amplification primers and extension primer sequences

[0036]

[0037]

[0038]

[0039] Example 2

[0040] This embodiment provides a kit for genetic testing of safe medication for children.

[0041] (1) PCR reaction amplification primer mixture: 100 μM specific primers of the sequences shown in SEQ ID NOs. 1 to 56, 1 μL each, 144 μL ddH2O, a total of 200 μL.

[0042] (2) PCR reaction system: 2 μL of PCR premix; 1 μL of deionized water; 1 μL of PCR amplification primer mixture; 1 μL of DNA template; 5 μL in total. The PCR premix contains PCR enzyme, PCR buffer, and dNTP mixture.

[0043] (3) Shrimp alkaline phosphatase (SAP) digestion reaction system: 0.3 μL SAP enzyme, 0.17 μL SAP buffer, 1.53 μL deionized water, a total of 2 μL. The SAP enzyme includes shrimp alkaline phosphatase and glycerol; the SAP buffer includes water, Tris-HCl, and MgCl2.

[0044] (4) Single base extension reaction primer premix:

[0045] The initial concentration of the extension primers used in the single-base extension reaction primer mixture was 500 μM.

[0046] Table 2 Composition of primer mixture for single base extension reaction

[0047]

[0048]

[0049] (5) Single base extension reaction system: E-ddNTPmix 1μL; MPE buffer 1.4μL; MPE enzyme 0.6μL; single base extension reaction primer premix 1μL; total 4μL. Among them, E-ddNTPmix contains ddATP, ddTTP, ddGTP, ddCTP; MPE enzyme contains high-specificity amplification polymerase, water and Tris-HCl; MPE buffer contains water, Tris-HCl and MgCl2.

[0050] (6) Desalting resin: includes cation exchange resin powder for removing salt ions from the elongation reaction solution.

[0051] Example 3

[0052] This embodiment provides a method for detecting genes for safe medication for children based on the kit of Example 2.

[0053] 1. Preparation of human gDNA nucleic acid template: Use Tiangen Dried Blood Spot Genomic DNA Extraction Kit to extract DNA from dried blood spot samples. The extraction must be performed strictly in accordance with the instructions of the kit. The extracted DNA samples can be stored at -18℃ or below for 12 months and at -70℃ or below for 2 years.

[0054] 2. Use a set of optimized amplification primers and a set of optimized single-base extension primers to detect common loci of genes for safe medication in children, specifically including the following steps:

[0055] (1) Multiplex PCR: Aliquot 4 μL of PCR mix (2 μL of PCR premix, 1 μL of deionized water, and 1 μL of PCR amplification primer mixture) into each PCR reaction tube, add 1 μL of the extracted DNA template (10 ng in total), vortex gently to mix, and centrifuge at 1000 rpm for 1 min.

[0056] PCR reaction program: heated lid 105°C;

[0057] 95℃15min;

[0058] 95°C for 15 s, 59°C for 30 s, 72°C for 30 s; 40 cycles in total;

[0059] 60℃10min;

[0060] 4℃Hold.

[0061] (2) SAP digestion: Use shrimp alkaline phosphatase (SAP) to remove the remaining dNTPs in the reaction system to prevent interference with the next base extension reaction.

[0062] Heat cover 105℃;

[0063] 37℃40min;

[0064] 85℃5min; 40 cycles in total;

[0065] 4℃Hold.

[0066] (3) Single base extension reaction: using modified dideoxynucleoside triphosphate (ddNTP) as a reaction substrate, the extension primer is extended by one base at a specific single nucleotide site and then the reaction is terminated. The reaction system is shown in Example 2.

[0067] The reaction procedure is as follows:

[0068]

[0069] (4) Resin desalting and purification: Add 14 μL of ultrapure water to each reaction well. Gently flip the eight-tube containing the resin over and place it on the sample plate, ensuring that the resin holes are aligned with each hole of the sample. Then tap the resin tube to allow the resin to fall into the holes of the sample plate. Use a palm centrifuge to centrifuge instantly to prevent the resin from adhering to the tube wall. Place the sample plate with the resin in a flip mixer and mix at 20 rpm for 30 minutes. After mixing, centrifuge the sample at 2000 rpm for 1 minute, and the supernatant is tested.

[0070] (5) Mass spectrometry detection: Take out the target plate, add 0.5-1 μL of the purified supernatant in step (4), dry naturally and crystallize, and then load it onto the machine (time-of-flight mass spectrometry detection system YS EXT 7900MD) for detection. The results are analyzed using MALDI-TOF MS technology. The detection results are as follows: Figure 1 As shown, it can be seen that all sites have stable peaks and relatively high signal values.

[0071] Example 4

[0072] This example provides a method for testing the accuracy of genetic testing for safe medication use in children using the method of Example 3.

[0073] Test plan: Using 10 clinical samples, the method of the present invention was used for comparative research with Sanger sequencing.

[0074] The specific test process is as follows: prepare the system required for the reaction according to Example 2 of the present invention, and then perform multiple PCR amplification, shrimp alkaline phosphatase digestion, single base extension, resin desalting purification and mass spectrometry detection according to the operating steps in Example 3, and then analyze the results. The accuracy results are shown in Table 3, and the representative mass spectrometry detection results are shown in Figure 3. Figure 2 shown.

[0075] Table 3 Accuracy test results

[0076]

[0077] Comparison of nucleic acid mass spectrometry results of 10 samples with Sanger results showed that the accuracy of the system verification experiment of the present invention was 100%.

[0078] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A primer composition, characterized in that The primer composition is used for gene detection of safe drug use in children; the primer composition includes 56 amplification primers and 28 extension primers, the sequences of the amplification primers are shown in SEQ ID NOs.1 to 56, and the sequences of the extension primers are shown in SEQ ID NOs.57 to 84.

2. Use of the primer composition as claimed in claim 1 in preparing a kit for detecting or guiding safe medication for children.

3. A kit for detecting genes for safe drug use in children, characterized in that: The kit comprises the primer combination according to claim 1.

4. A kit for guiding children to use medicines safely, characterized in that: The kit comprises the primer combination according to claim 1.

5. The kit according to claim 4, characterized in that The kit also includes a multiplex PCR reaction reagent, a single base extension reaction reagent and a dNTP removal mixture reaction reagent.

6. The kit according to claim 5, characterized in that The multiple PCR reaction reagent includes a PCR reaction amplification primer mixture, and the PCR reaction amplification primer mixture includes amplification primers with nucleotide sequences shown in SEQ ID NOs.1 to 56; the single base extension reaction reagent includes a single base extension reaction primer premix, and the single base extension reaction primer premix includes extension primers with nucleotide sequences shown in SEQ ID NOs.57 to 84.

7. The kit according to claim 6, characterized in that In the PCR reaction amplification primer mixture, the concentration of each amplification primer is 0.5 μM.

8. The kit according to claim 6, characterized in that In the single base extension reaction primer premix, the concentration of each extension primer is as follows:

9. A detection method based on the kit according to any one of claims 3 to 8, for non-diagnostic purposes, characterized in that: The steps include: (1) Multiplex PCR: Using amplification primers with sequences as shown in SEQ ID NOs. 1 to 56, a total of 28 pediatric safe drug gene polymorphic sites are simultaneously amplified to obtain PCR products containing 28 gene polymorphic sites: (2) SAP digestion: Use shrimp alkaline phosphatase to remove the remaining dNTPs in the reaction system to prevent interference with the next base extension reaction; (3) Single base extension reaction: adding an extension primer with a sequence as shown in SEQ ID NO. 57 to 84 to perform a single base extension reaction, using a modified ddNTP as a reaction substrate, so that the extension primer extends one base at a specific single nucleotide site and then terminates the reaction; (4) Resin desalting and purification: The extension reaction product is purified by using a cation exchange resin to adsorb the salt ions in the system; (5) Mass spectrometry detection: The purified product is detected using a nucleic acid mass spectrometer; (6) Interpretation of results 10. Use of the detection method as claimed in claim 9 in guiding children to use medicines safely.

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