Nucleic acid combination for detecting SNP (Single Nucleotide Polymorphism) site combination of novel coronavirus-resistant drug gene, detection product and application of nucleic acid combination and detection product

Abstract

The invention discloses a nucleic acid combination for detecting an SNP site combination of an anti-novel coronavirus drug gene, a detection product and application of the nucleic acid combination and the detection product, and relates to the technical field of novel coronavirus drug gene detection. The nucleic acid composition and the detection product provided by the invention have extremely high detection success rate and detection accuracy, and the detection result is reliable. The method is suitable for pharmacogenomics research and formulation of clinical multiple precise medication implementation schemes, and therefore the basis value is provided for precise medication of new crown patients.

Description

Technical Field

[0001] This invention relates to the field of novel coronavirus drug gene detection technology, and more specifically, to a nucleic acid combination for detecting SNP site combinations of anti-novel coronavirus drug genes, a detection product, and its application. Background Technology

[0002] In recent years, with the global spread of the novel coronavirus (SARS-CoV-2), and due to the lack of specific treatments for COVID-19, many off-label drugs have been urgently used for COVID-19 treatment. Although drug efficacy and toxicity have been optimized, life-threatening severe adverse drug reactions due to drug interactions and other factors still occur frequently. As a platform for precision medicine, pharmacogenomics aims to reveal the impact of gene variations related to drug metabolism and transport on drug response and efficacy. Therefore, pharmacogenomics testing has become a key technology for avoiding adverse drug reactions and optimizing drug efficacy. Recent studies have found that multiple pharmacogenomics variations have a significant impact on the efficacy and safety of commonly used COVID-19 treatments, including antiviral small molecule drugs, hormonal drugs, immunomodulatory drugs, and nonsteroidal anti-inflammatory drugs, as well as vaccine safety.

[0003] Therefore, it is necessary to establish an economical and convenient gene detection method to detect gene-related variants of multiple drugs associated with COVID-19, providing feasibility for clinical application. Currently, single-gene detection kits for single drugs related to COVID-19 treatment, including those for hormone drugs, immunomodulatory drugs, and nonsteroidal anti-inflammatory drugs, are available. However, these tests mainly use quantitative real-time PCR or first-generation sequencing to detect gene loci related to drug efficacy and side effects, resulting in low detection efficiency and throughput. They cannot simultaneously detect multiple gene loci related to the efficacy and side effects of multiple drugs being taken. Furthermore, no kits for multi-gene locus detection of pharmacogenomic drugs targeting the novel coronavirus have yet been developed.

[0004] As a crucial tool for multiplex gene detection, nucleic acid mass spectrometry based on matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) can accurately detect and quantify molecules in the 4500-9000 Da mass range, enabling flexible target gene assays and rapid result output. Nucleic acid mass spectrometry has significantly advanced the development of genomics. Currently, it is becoming a key tool for promoting precision medicine in clinical practice due to its unique advantages. The basic principle of nucleic acid mass spectrometry involves mixing the sample with a matrix, introducing the sample, using laser desorption / ionization as the ion source, and performing mass analysis using time-of-flight methods.

[0005] Studies have shown that nucleic acid mass spectrometry (NMS) detection technology offers medium throughput, high flexibility, high accuracy, and low cost. Furthermore, NMS-based gene combination detection technology is suitable for pharmacogenetic research and the development of multiple precision medicine strategies in clinical settings. Nucleic acid mass spectrometry has been extensively studied and proven to have broad application prospects in areas such as deafness gene detection, liquid biopsy, and tumor susceptibility gene polymorphism detection. Combining high sensitivity, high specificity, high accuracy, medium-to-high throughput, and low cost, and capable of detecting various types of gene alterations including single nucleotide variants, insertions / deletions, copy number variations, tandem repeats, and structural variations, this technology holds significant promise for clinical gene testing.

[0006] Developing new methods for detecting multiple gene variant sites with high specificity and sensitivity based on nucleic acid mass spectrometry is a very active research and application exploration direction in the field of pharmacogenomics. However, research and application of screening multiple gene sites related to the efficacy and side effects of COVID-19 treatment drugs and using nucleic acid mass spectrometry for detection have not been reported at home and abroad.

[0007] In view of this, the present invention is proposed. Summary of the Invention

[0008] The purpose of this invention is to provide a nucleic acid combination for detecting SNP site combinations of anti-novel coronavirus drug genes, a detection product, and its application, so as to achieve detection of anti-novel coronavirus drug gene sites with high specificity and sensitivity and low cost.

[0009] This invention is implemented as follows: In a first aspect, the present invention provides a nucleic acid combination for detecting SNP site combinations of genes containing anti-novel coronavirus drugs. The SNP locus combination is selected from at least two SNP loci from the following SNP loci: rs4910008, rs12746200, rs79085477, rs11265618, rs429358, rs1902023, rs2032582, rs1805034, rs7412, rs12083537, rs1042640, rs11052877, rs776746, rs4553808, rs1057910, rs212091, rs2854117, and rs396991; Nucleic acid combinations include amplification primers and extension primers for detecting SNP sites; The nucleotide sequences of the primers for amplifying the SNP sites rs4910008, rs12746200, rs79085477, rs11265618, rs429358, rs1902023, rs2032582, rs1805034, rs7412, rs12083537, rs1042640, rs11052877, rs776746, rs4553808, rs1057910, rs212091, rs2854117, and rs396991 are shown in SEQ ID NO: 1-36, respectively. The nucleotide sequences of the extension primers for detecting the SNP sites rs4910008, rs12746200, rs79085477, rs11265618, rs429358, rs1902023, rs2032582, rs1805034, rs7412, rs12083537, rs1042640, rs11052877, rs776746, rs4553808, rs1057910, rs212091, rs2854117, and rs396991 are shown in SEQ ID NO: 37-54.

[0010] Secondly, the present invention also provides a detection product for SNP site combinations of anti-novel coronavirus drug genes, comprising: the above-mentioned nucleic acid combination for detecting SNP site combinations of anti-novel coronavirus drug genes, wherein the detection product is selected from reagents, kits, chips or detectors.

[0011] Thirdly, the present invention also provides the application of nucleic acid combinations for detecting SNP site combinations of anti-novel coronavirus drug genes in the preparation of detection products for SNP site combinations of anti-novel coronavirus drug genes, the application including the following detection methods: Mix the DNA from the sample to be tested with the amplification primers in the nucleic acid combination, and perform a PCR reaction in the amplification system; process the PCR product with SAP. Then, the SAP-treated product was subjected to a single-base extension reaction using extension primers; the extension reaction product was then analyzed.

[0012] The present invention has the following beneficial effects: This invention provides a nucleic acid combination for detecting SNP sites in genes of anti-novel coronavirus drugs (ritonavir, dexamethasone, tocilizumab, ibuprofen, and acetaminophen). The targeted SNP sites are SNP sites that are clearly associated with the efficacy and side effects of COVID-19 drugs, screened from international authoritative pharmacogenomics databases and research literature.

[0013] By combining various SNP sites, the detection of each SNP site does not interfere with each other, and multiple sites can be detected efficiently. The nucleic acid combinations and detection products provided by this invention have extremely high detection success rates and accuracy, and the detection results are reliable.

[0014] The detection products provided by this invention are particularly suitable for nucleic acid mass spectrometry detection, featuring medium throughput, high flexibility, high accuracy, smaller biological sample volume, and low cost. Furthermore, the gene combination detection technology based on nucleic acid mass spectrometry is applicable to pharmacogenomics research and the formulation of clinical multiple precision drug implementation plans. Therefore, this invention provides valuable evidence for precision drug treatment for COVID-19 patients. Attached Figure Description

[0015] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 The MassARRAY test results for 6 SNP sites in 100 patients are shown in the figure. Figure 2 The MassARRAY test results for 6 SNP sites in 100 patients are shown in the figure. Figure 3 The MassARRAY test results for 6 SNP sites in 100 patients are shown in the figure. Figure 4 The MassARRAY detection results and first-generation sequencing results for the rs2032582 site in patients O228 and O320; Figure 5 The images show the MassARRAY detection results and first-generation sequencing results for the rs2032582 site in patients O434 and O364. Figure 6 The images show the MassARRAY detection results and first-generation sequencing results for the rs2032582 site in patients O498 and O914. Detailed Implementation

[0017] Reference will now be made to detailed embodiments of the present invention, one or more of which are described below. Each example is provided for explanation and not for limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the invention without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment may be used in another embodiment to produce further embodiments.

[0018] SNP stands for Single Nucleotide Polymorphism, which is the presence of two or more different base types at a specific position in the genomic DNA sequence.

[0019] In a first aspect, the present invention provides a nucleic acid combination for detecting SNP site combinations of genes containing anti-novel coronavirus drugs. The SNP locus combination is selected from at least two SNP loci from the following SNP loci: rs4910008, rs12746200, rs79085477, rs11265618, rs429358, rs1902023, rs2032582, rs1805034, rs7412, rs12083537, rs1042640, rs11052877, rs776746, rs4553808, rs1057910, rs212091, rs2854117, and rs396991; Nucleic acid combinations include amplification primers and extension primers for detecting SNP sites; The nucleotide sequences of the primers for amplifying the SNP sites rs4910008, rs12746200, rs79085477, rs11265618, rs429358, rs1902023, rs2032582, rs1805034, rs7412, rs12083537, rs1042640, rs11052877, rs776746, rs4553808, rs1057910, rs212091, rs2854117, and rs396991 are shown in SEQ ID NO: 1-36, respectively. The nucleotide sequences of the extension primers for detecting the SNP sites rs4910008, rs12746200, rs79085477, rs11265618, rs429358, rs1902023, rs2032582, rs1805034, rs7412, rs12083537, rs1042640, rs11052877, rs776746, rs4553808, rs1057910, rs212091, rs2854117, and rs396991 are shown in SEQ ID NO: 37-54.

[0020] The COVID-19 drug gene locus primer set and nucleic acid mass spectrometry-based detection method provided in this invention can be used for precision medication of COVID-19 patients. This invention, referring to previous evidence from the PharmGKB and CPIC databases for COVID-19 related drugs, as well as relevant research literature, screened gene loci associated with the efficacy and adverse drug reactions of five commonly used COVID-19 drugs (ritonavir, dexamethasone, tocilizumab, ibuprofen, and acetaminophen); primer pairs were designed for the corresponding gene mutation sites. By detecting SNP sites of anti-COVID-19 drug genes in 100 previously diagnosed COVID-19 patients, the success rate for all 18 SNP sites was 100%; the p-values ​​were all greater than 0.05, indicating that the detection results were reliable.

[0021] The specific sequence list is as follows:

[0022] The ACGTTGGATG sequence in the primers above is a "universal primer sequence" used to improve amplification specificity and efficiency.

[0023] In one embodiment, the SNP site combination is selected, for example, from: the combination of rs4910008 and rs12746200, the combination of rs4553808 and rs79085477, the combination of rs4553808, rs79085477 and rs11052877, the combination of rs4553808, rs79085477, rs11052877 and rs396991, and rs4553808... Combinations of rs79085477, rs11052877, rs396991 and rs4910008; combinations of rs4553808, rs79085477, rs11052877, rs396991, rs4910008 and rs12083537; rs4553808, rs79085477, rs11052877, rs396991, rs491000 8. Combinations of rs12083537 and rs11265618, rs4553808, rs79085477, rs11052877, rs396991, rs4910008, rs12083537, rs11265618 and rs1057910, rs4553808, rs79085477, rs11052877, rs396991, rs49 Combinations of 10008, rs12083537, rs11265618, rs1057910 and rs12746200, and combinations of rs4553808, rs79085477, rs11052877, rs396991, rs4910008, rs12083537, rs11265618, rs1057910, rs12746200 and rs1805034.

[0024] In one implementation, the SNP site combinations are selected from the following 18 combinations: rs4910008, rs12746200, rs79085477, rs11265618, rs429358, rs1902023, rs2032582, rs1805034, rs7412, rs12083537, rs1042640, rs11052877, rs776746, rs4553808, rs1057910, rs212091, rs2854117, and rs396991.

[0025] For 100 samples, the detection success rate of nucleic acid mass spectrometry for the above 18 SNP site combinations was 100%, with P values ​​all greater than 0.05, indicating that the detection results are reliable. Nucleic acid mass spectrometry validation of the designed primers using 100 samples showed a satisfactory detection rate. First-generation sequencing validation of the samples yielded results consistent with the nucleic acid mass spectrometry detection, demonstrating the advantage of high detection accuracy.

[0026] Secondly, the present invention also provides a detection product for SNP site combinations of anti-novel coronavirus drug genes, comprising: the above-mentioned nucleic acid combination for detecting SNP site combinations of anti-novel coronavirus drug genes, wherein the detection product is selected from reagents, kits, chips or detectors.

[0027] The reagent may be in the form of a liquid or a solid, especially a lyophilized form.

[0028] In a preferred embodiment of the present invention, when the detection product is a kit, it further includes at least one of the following components: negative control, positive control, PCR buffer, and Mg. 2+ dNTP premix, DNA polymerase, shrimp alkaline phosphatase reaction components, and single base extension reaction components.

[0029] DNA polymerase, preferably a hot-start DNA polymerase, such as Tth DNA polymerase, Taq DNA polymerase, etc.

[0030] The PCR buffer includes buffer system reagents, including but not limited to the PB series, Tris series, etc. In one embodiment, the PCR buffer also includes a lyophilization protectant, such as at least one of mannitol, trehalose, dextran, gelatin, hydrogenated maltose, and sucrose.

[0031] In one embodiment, an anti-PCR inhibitor factor, such as spermidine, trehalose, betaine, etc., is also added to the PCR buffer.

[0032] The detection products (such as kits) provided by the present invention may optionally include any reagents and / or consumables acceptable in the art for PCR reactions or for preparing PCR reaction systems. Specific embodiments may include, but are not limited to, one or more of blank controls, calibrators, and PCR reaction containers.

[0033] Thirdly, the present invention also provides the application of nucleic acid combinations for detecting SNP site combinations of anti-novel coronavirus drug genes in the preparation of detection products for SNP site combinations of anti-novel coronavirus drug genes, the application including the following detection methods: Mix the DNA from the sample to be tested with the amplification primers in the nucleic acid combination, and perform a PCR reaction in the amplification system; process the PCR product with SAP. Then, the SAP-treated product was subjected to a single-base extension reaction using extension primers. The extended reaction product was analyzed.

[0034] After the PCR reaction, a large number of residual nucleotides (dNTPs) remain in the system. These must be degraded using shrimp alkaline phosphatase (SAP), otherwise they will interfere with subsequent extension reactions.

[0035] The extension primer provided by this invention is an oligonucleotide primer designed to be located immediately next to the SNP site (different from the SNP site by one base). Its 3' end points exactly to the SNP base to be detected.

[0036] During the extension reaction, dideoxynucleotides (ddNTPs) are added. Unlike the dNTPs used in regular PCR, ddNTPs lack a 3'-OH group in their ribose. Once incorporated into the DNA strand, the synthesis reaction terminates immediately (hence the name "single-base extension").

[0037] In a preferred embodiment of the present invention, the final concentration of the amplification primers in the amplification system is 0.5 μM. High amplification efficiency is achieved under this final concentration condition.

[0038] In a preferred embodiment of the present invention, during the single-base extension reaction, the final concentration of the extension primer in the single-base extension reaction system is 2.5 μM. Under this final concentration condition, a high extension efficiency is achieved.

[0039] In a preferred embodiment of the present invention, the PCR amplification program during the PCR reaction includes: 95-100℃, 1-5 min; 95-96℃, 30 s, 60℃±0.5℃, 30 s, 72℃, 60 s, 40-45 cycles; 72℃, 1-10 min.

[0040] In a preferred embodiment of the present invention, the procedure for the single-base extension reaction includes: 94-95℃, 30s; 94-95℃, 5s; 52℃±0.5℃, 5s; 80℃±0.5℃, 5s; 40 cycles; 72℃, 1-10min.

[0041] In a preferred embodiment of the present invention, the method for detecting the extension reaction product is selected from: nucleic acid mass spectrometry; In a preferred embodiment of the present invention, the nucleic acid mass spectrometry is matrix-assisted laser desorption / ionization time-of-flight mass spectrometry.

[0042] The fluorescent reporter group is selected from any one of 5-FAM, 6-FAM, HEX, TET, VIC, JOE, Cy3, Cy3.5, NED, TAMRA, ROX, TexasRed, Cy5, Cy5.5 and Quasar670.

[0043] The aforementioned fluorescence polarization detection includes the following method: In one embodiment, the 5' end of the extension primer is labeled with a fluorescent group. During the reaction, polymerase incorporates fluorescently labeled ddNTPs (each ddNTP is labeled with a different colored fluorescent dye) that match the template. After the reaction is complete, the primer is excited with polarized light of a specific wavelength. Small, rapidly rotating molecules (such as undoped fluorescent ddNTPs) emit depolarized light; while large, slowly rotating molecules (such as primers doped with a ddNTP) emit highly polarized light. By measuring the degree of polarization of the emitted light, it can be determined whether an extension reaction has occurred. Furthermore, by detecting the wavelength (color) of the emitted light, it can be determined which ddNTP was incorporated.

[0044] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0045] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0046] Example 1 This embodiment provides a kit for combining SNP sites in genes used to treat drugs against the novel coronavirus.

[0047] Based on previous evidence from the PharmGKB and CPIC databases for COVID-19 related drugs, as well as relevant research literature, single nucleotide polymorphism (SNP) gene loci associated with the efficacy and adverse drug reactions of five commonly used COVID-19 drugs were screened.

[0048] The kit includes 18 primer sets, each containing a pair of amplification primers and an extension primer. This application designs primer sets for the genes and corresponding SNP sites of five anti-novel coronavirus drugs (ritonavir, dexamethasone, tocilizumab, ibuprofen, and acetaminophen). The specific information of the 18 primer sets is shown in Table 1.

[0049] Table 1 Primer sequence information for 18 primer sets

[0050] The ACGTTGGATG sequences in Table 1 are "universal primer sequences" used to improve amplification specificity and efficiency.

[0051] This invention selected 100 previously diagnosed COVID-19 patients and used the above-mentioned kit to detect 18 loci. The gene detection results, success rate, and accuracy are shown in Table 2 below. As shown in Table 2, the success rate (call rate) for detecting the above-mentioned COVID-19 drug genes and corresponding SNP loci was 100%; the P-values ​​were all greater than 0.05, indicating that the detection results were reliable.

[0052] Table 2. Statistical table of gene detection results, success rate, and accuracy for 18 SNP loci.

[0053] HWE is the Hardy-Weinberg balance number.

[0054] Example 2 This embodiment provides a method for detecting SNP sites in COVID-19 drug gene based on multiplex PCR time-of-flight mass spectrometry, which includes the following steps: I. DNA Extraction 1. Extracting DNA from blood samples DNA was extracted from peripheral venous whole blood of enrolled patients using an automated nucleic acid extraction method. The experimental steps are as follows: Take 250 μl of whole blood into a 1.5 ml centrifuge tube, add 20 μl of proteinase K and 300 μl of buffer GHL, vortex to mix, and lyse at 75 °C for 15 min, inverting the tube 3 times during this period, 3-5 times each time.

[0055] Remove the pre-packaged deep-well plate, invert it several times to mix, resuspend the magnetic beads, and gently shake the plate to collect the reagents and magnetic beads at the bottom (or use a plate centrifuge at 500 rpm for 1 min). Before use, carefully remove the aluminum foil sealing film to avoid vibration and prevent liquid splashing. Place the deep-well plate steadily into the automated nucleic acid extractor, and then insert the stirring sleeve into the slot.

[0056] Take 500 μl of the processed sample and add it to columns 2 and 8 of the pre-sealed plate. Place the 96-well plate on the deep well plate base of the 32-channel automated nucleic acid extractor and insert the magnetic rod sleeve into the magnetic rod sleeve holder slot of the instrument.

[0057] Set the program for the nucleic acid extractor according to Table 3, and then click "Run" to start the experiment. After the automated program finishes, extract the DNA nucleic acid from columns 6 and 12.

[0058] Table 3. Peripheral blood DNA extraction procedure using an automated nucleic acid extractor.

[0059] 2. DNA concentration determination The extracted DNA should have an OD 260 / 280 between 1.6 and 1.8, and a concentration greater than 50 ng / μL. Add 2 μL of pure water to the measurement well of the Simpinano micro-detector, zero the instrument, and blot the well dry with absorbent paper. Then, add 2 μL of sample DNA to each well and record the DNA concentration and 260 / 280 ratio. Remove and discard the DNA sample. Blot the well dry with absorbent paper each time you change the liquid.

[0060] 3. DNA template dilution The final DNA concentration should ideally be 10-20 ng / μL before genotyping. If the concentration is higher than this, dilute empirically by approximately 4-fold (measured concentration * 0.1 * 4 = X μL). After dilution, add X μL of ddH2O to each of the labeled 1.5 ml eight-tube strips, then add 4 μL of DNA sample sequentially. Store at 4°C for later use.

[0061] II. Primer Design Before primer design, the target SNP combination needs to be determined. SNP site information can be found in public databases. The PharmGKB database contains pharmacogenomics evidence for SNP sites (https: / / www.pharmgkb.org / ); the NCBI-dbSNP database contains gene frequencies in East Asian populations (https: / / www.ncbi.nlm.nih.gov / snp / ).

[0062] Access Agena Bioscience's assay design software online (https: / / support.agenabio.com / s / online-tools) and upload the rs number of the target SNP combination. Specify the iPLEX reuse level: select "Low Reuse iPLEX Preset" when the target SNP combination has fewer than 12 sites, "Medium Reuse iPLEX Preset" when the target SNP combination has between 13 and 24 sites, and "High Reuse iPLEX Preset" when the target SNP combination has more than 24 sites. During assay design, the "High Reuse iPLEX Preset" setting can be extended from the default 36 SNPs to 40 SNPs. The program will not accept more than 40 SNPs entered in the "High Reuse iPLEX Preset" box.

[0063] After specifying all design options, click the "Start Run" button to automatically run each step of the design process: 1) Retrieving and formatting SNP sequences; 2) Identifying proximal SNPs in the region to be amplified that may interfere with primer binding; 3) Finding the optimal primer positions; 4) Designing detection methods by combining compatible SNPs into groups suitable for multiplex reactions; 5) Validating the experiment by checking that all different combinations of multiplex primers do not amplify unwanted regions in the genome. The final primer design for the target SNP combination is then obtained.

[0064] III. Preparation of PCR primers and extension primers The quantity of PCR primers is 25 nmol, and the quantity of extension primers is 100 nmol. All primers should be desalted to remove small molecule impurities and delivered in lyophilized form. Centrifuge the lyophilized PCR primer powder at 12,000 rpm for 5 min using a low-temperature high-speed centrifuge. The lyophilized PCR primers (forward and reverse) should be diluted to 100 μM, and the extension primers should be diluted to 500 μM with ultrapure water. They should be stored at -20°C.

[0065] The ratio of forward and reverse primers can be adjusted appropriately based on the heterozygous amplification of the loci. It is recommended that the maximum concentration ratio not exceed 1:3. The initial volume is 0.5 μl (100 μM) of forward / reverse primers for each locus in a 100 μl system. The amount of extension primers should be adjusted according to the peak height observed in the mass spectrometry of the locus, maintaining a peak intensity of 30-60.

[0066] IV. Multiplex Polymerase Chain Reaction (PCR) 1. Prepare the PCR primer mixture. Take fixed amounts of 100 μM forward and reverse primers for multiplex PCR and mix them with ultrapure water. Dilute all primers to the final working concentration for multiplex PCR reaction to 0.5 μM and incubate at room temperature for several hours or at 4°C overnight.

[0067] 2. Place the 384-well plate on ice and use Agena “Complete PCR Reagent Set”. The reagent components are shown in Table 2. After the PCR reaction working solution is prepared, it needs to be vortexed on a vortex mixer for 10 seconds and then centrifuged on a handheld centrifuge for 10 seconds.

[0068] Table 4 PCR reaction working solution

[0069] 3. Add 3 μl of PCR reaction working solution to the wells of an empty 384-well plate.

[0070] 4. Add 2 μl of 10 ng / μl DNA to each reaction.

[0071] 5. After adding the samples, seal the PCR plate with the PCR plate membrane. Place the sealed 384-well PCR plate on a vortex mixer to mix well, and then centrifuge at 2000 rpm for 1 min in a plate centrifuge.

[0072] 6. Place the 384-well plate containing the PCR amplification system into a 384-well PCR instrument. Set the PCR instrument parameters as shown in Table 5, and start the PCR program. PCR products can be stored at 4°C for later use.

[0073] Table 5. PCR amplification program parameter settings

[0074] V. Shrimp alkaline phosphatase (SAP) digestion SAP is used to neutralize any remaining dNTPs in the PCR reaction. SAP cleaves a phosphate group from an unbound dNTP, preventing it from being added as nucleotides in the iPLEX extension reaction.

[0075] According to Table 6, the SAP working solution was prepared on ice using the provided reagents. After the SAP working solution was prepared, it was vortexed for 10 seconds on a vortex shaker and then centrifuged briefly for 10 seconds on a mini centrifuge.

[0076] Table 6. SAP Reaction Working Fluid

[0077] 2. Place the amplified PCR plate into a plate centrifuge and centrifuge at 2000 rpm for 1 min.

[0078] 3. Use a pipette to add 2 μl of SAP reaction working solution to each well of the analytical plate and mix with the PCR products.

[0079] 4. After adding the samples, seal the PCR plate with the PCR plate membrane and centrifuge at 2000 rpm for 1 min in a plate centrifuge.

[0080] 5. Place the 384-well plate into a 384-well PCR instrument. The PCR instrument parameters are shown in Table 7. Start the shrimp digestion reaction program.

[0081] Table 7. SAP Digestion Reaction Program Parameter Settings

[0082] VI. Single-base extension (iPLEX) 1. Prepare the iPLEX working solution on ice according to the components in Table 8. After preparation, vortex it on a vortex shaker for 10 seconds, and then centrifuge it briefly on a handheld centrifuge for 10 seconds.

[0083] Table 8. iPLEX working solution

[0084] 2. Add 2 μL of the final iPLEX working solution to each reaction well. After adding the samples, seal the PCR plate with a membrane and centrifuge at 2000 rpm for 1 min to allow the reagents to aggregate together.

[0085] 3. Place the 384-well plate into the PCR instrument. The PCR instrument parameters are shown in Table 9. Start the single-base extension program.

[0086] Table 9. Parameter settings for single-base extension reaction program

[0087] 4. After single-base extension, add 16 µl of ddH2O to each well in a clean bench, for a total volume of 25 µl. After sample addition, seal the PCR plate with a PCR membrane and centrifuge at 2000 rpm for 1 min. Transfer the 384-well plate to the amplification product analysis area.

[0088] VII. Mass Spectrometry Flight Detection Select appropriate combinations of pharmacogenomic multiplexing sites and use the MassARRAY platform to perform pharmacogenotyping on patients. The experimental procedures are as follows: 1. Open the sample cell of the DP-TOF mass spectrometer, carefully peel off the film from the 384-well plate, place the 384-well plate into the holder of the mass spectrometer, and align the I / A sensor with the lower left corner of the user's head. Remove the chip tray from the mass spectrometer, carefully remove the numbered SpectroCHIPs chip with tweezers, place it in the tray, and close the sample cell. During operation, the DP-TOF mass spectrometer automatically desaltes the sample using resin.

[0089] 2. Prepare templates for nucleic acid mass spectrometry detection before use. Import the Assay Design file using AssayEditor, create the template using Typer software, and create the template using PlateManager software.

[0090] The parameter settings for the DP-TOF mass spectrometer are as follows: Accelerate Voltage:20kV、Mass Range:4000 10000Da, laser wavelength: 337nm, number of pulses: 30.

[0091] 3. Confirm that the CPM deck is ready; confirm that the Run Setup settings in SpectroACQUIRE are complete; in the toolbar at the top of the window, click start chip prep module to begin running.

[0092] 4. Before generating the report, the results need to be checked. For loci not identified by the instrument, they need to be manually identified by combining peak patterns and Call Cluster Plot. The Traffic Light Pane displays the 384 Wells sample panel; the Cluster Plot Pane displays the Cluster Plot panel; the Details Pane, Display the Detailed Plane displays the Peak Plot Details panel; and the Plate Data Pane displays the Plate Data panel. Click on each Well in the Traffic Light sample: Conservative indicates the result is correct; Moderate indicates the result is moderate and reasonable; Aggressive indicates the result needs to be challenged and verified; Low Probability indicates a low probability and needs to be corrected; No-Alleles indicates no alleles and needs to be retested; User Call indicates the result modified by the user. For questionable loci, right-click, select Change Call in the dialog box, and manually judge based on the peak shape and the region position in the Call Cluster Plot to select the correct allele.

[0093] Patient verification This application collected samples from 100 patients who had previously been diagnosed with COVID-19, and performed MassARRAY gene testing on all 100 samples using the method described above.

[0094] The MassARRAY assay of 100 samples in this application confirmed that the primer combination and kit design were reasonable and the detection rate was qualified (Table 10); the first-generation sequencing of 20 samples in this application was used to verify the results (Table 11), which were consistent with the MassARRAY assay results and had high accuracy.

[0095] Table 10. MassARRAY's detection results for 18 SNP sites in 100 previously diagnosed COVID-19 patients.

[0096] Table 11. Sequencing results of first-generation sequencing of 18 SNP sites in 20 samples.

[0097] The MassARRAY test results for 18 SNP sites in 100 patients are shown in the figure below. Figure 1 , Figure 2and Figure 3 As shown, Figure 1 In the middle, "Other (68)" refers to genotypes other than C, T, CT, and TC at this SNP locus. The results showed that the kit provided by this invention could detect 100% of the 18 SNP loci in 100 patients.

[0098] Taking patients numbered O228, O320, O434, O364, O498, and O914 (6 of the 20 patients who underwent first-generation sequencing validation) as examples, the peak diagrams of MassARRAY detection results for the rs2032582 gene locus and the peak diagram results of first-generation sequencing validation are shown. Figure 4 , Figure 5 , Figure 6 The results showed that the MassARRAY test results were consistent with the first-generation sequencing results.

[0099] TT: Single peak Mass 7648.8; CC: Single peak Mass 7568.9; AA: Single peak Mass 7593; CT: Double peak Mass 7568.9 and 7648.8; AC: Double peak Mass 7568.9 and 7593; AT: Double peak Mass 7593 and 7648.8.

[0100] In summary, the detection method using the above primer combination combined with nucleic acid mass spectrometry can simultaneously and efficiently detect multiple SNP sites of COVID-19 drug genes with a success rate of 100% and high accuracy.

[0101] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A nucleic acid combination for detecting SNP site combinations of genes against novel coronavirus drugs, characterized in that, The SNP site combination is selected from at least two SNP sites from the following: rs4910008, rs12746200, rs79085477, rs11265618, rs429358, rs1902023, rs2032582, rs1805034, rs7412, rs12083537, rs1042640, rs11052877, rs776746, rs4553808, rs1057910, rs212091, rs2854117, and rs396991; The nucleic acid combination includes amplification primers and extension primers for detecting the SNP site; The nucleotide sequences of the primers for amplifying the SNP sites rs4910008, rs12746200, rs79085477, rs11265618, rs429358, rs1902023, rs2032582, rs1805034, rs7412, rs12083537, rs1042640, rs11052877, rs776746, rs4553808, rs1057910, rs212091, rs2854117, and rs396991 are shown in SEQ ID NO: 1-36, respectively. The nucleotide sequences of the extension primers for detecting the SNP sites rs4910008, rs12746200, rs79085477, rs11265618, rs429358, rs1902023, rs2032582, rs1805034, rs7412, rs12083537, rs1042640, rs11052877, rs776746, rs4553808, rs1057910, rs212091, rs2854117, and rs396991 are shown in SEQ ID NO: 37-54.

2. The nucleic acid combination for detecting SNP site combinations of anti-novel coronavirus drug genes according to claim 1, characterized in that, The SNP locus combinations were selected from the following SNP loci: rs4910008, rs12746200, rs79085477, rs11265618, rs429358, rs1902023, rs2032582, rs1805034, rs7412, rs12083537, rs1042640, rs11052877, rs776746, rs4553808, rs1057910, rs212091, rs2854117, and rs396991.

3. A detection product for SNP site combinations of genes used in drugs against the novel coronavirus, characterized in that, It includes: a nucleic acid combination for detecting SNP site combinations of anti-novel coronavirus drug genes as described in any one of claims 1-2, wherein the detection product is selected from reagents, kits, chips or detectors.

4. The detection product for SNP site combinations of anti-novel coronavirus drug genes according to claim 3, characterized in that, When the detection product is a kit, it also includes at least one of the following components: negative control, positive control, PCR buffer, Mg2+, dNTP premix, DNA polymerase, shrimp alkaline phosphatase reaction component, and single base extension reaction component.

5. The application of the nucleic acid combination for detecting SNP site combinations of anti-novel coronavirus drug genes as described in any one of claims 1-2 in the preparation of a detection product for SNP site combinations of anti-novel coronavirus drug genes, characterized in that, The application includes the following detection methods: The DNA of the sample to be tested is mixed with the amplification primers in the nucleic acid combination, and a PCR reaction is performed in the amplification system; the PCR product is then treated with SAP. Then, the SAP-treated product was subjected to a single-base extension reaction using extension primers. The extended reaction product was analyzed.

6. The application according to claim 5, characterized in that, The final concentration of the amplification primers in the amplification system is 0.5 μM.

7. The application according to claim 5, characterized in that, During the single-base extension reaction, the final concentration of the extension primer in the single-base extension reaction system is 2.5 μM.

8. The application according to claim 5, characterized in that, The PCR amplification program for the PCR reaction includes: 95-100℃, 1-5 min; 95-96℃, 30 s, 60℃±0.5℃, 30 s, 72℃, 60 s, 40-45 cycles; 72℃, 1-10 min.

9. The application according to claim 5, characterized in that, The procedure for the monobasic extension reaction includes: 94-95℃, 30s; 94-95℃, 5s; 52℃±0.5℃, 5s; 80℃±0.5℃, 5s; 40 cycles; 72℃, 1-10min.

10. The application according to claim 5, characterized in that, The method for detecting the extended reaction products is selected from: nucleic acid mass spectrometry; Preferably, the nucleic acid mass spectrometry is matrix-assisted laser desorption / ionization time-of-flight mass spectrometry.

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