SNP (Single Nucleotide Polymorphism) molecular marker combination related to allergic rhinitis and application of SNP molecular marker combination

Abstract

The invention provides an SNP molecular marker combination related to allergic rhinitis and application of the SNP molecular marker combination, and belongs to the technical field of gene detection. The invention provides an SNP (Single Nucleotide Polymorphism) molecular marker combination for detecting allergic rhinitis. The SNP molecular marker combination comprises 89 SNP molecular markers, can detect 46 genes related to allergic rhinitis at a time, and can be used for diagnosis or auxiliary diagnosis of allergic rhinitis. The SNP molecular marker combination is used as a detection target for diagnosis or auxiliary diagnosis of allergic rhinitis, the detection result is comprehensive, the accuracy and repeatability are extremely high, and the SNP molecular marker combination has auxiliary diagnosis and prompting effects on allergic rhinitis susceptible people, especially people with family genetic history. By detecting a pregnant woman peripheral blood sample or a baby umbilical cord blood sample, a good pre-judgment effect can also be achieved on the neonatal allergic rhinitis.

Description

Technical Field

[0001] The present invention belongs to the technical field of gene detection, and particularly relates to an SNP molecular marker combination related to allergic rhinitis and its application. Background Art

[0002] Allergic diseases (allergic reactions) include allergic rhinitis, allergic asthma, and atopic dermatitis. Among them, allergic rhinitis, also known as allergic rhinitis, refers to a non-infectious inflammatory disease of the nasal mucosa mainly mediated by IgE (mainly histamine) release after a specific individual contacts an allergen, and involves a variety of immune active cells and cytokines.

[0003] At present, the diagnosis of allergic rhinitis is mostly judged through clinical manifestations and in vitro detection of allergen-specific IgE antibodies or in vivo provocation tests. However, diagnosis through clinical manifestations and existing detection methods must be carried out when the patient is already ill. Once an allergic disease occurs, it is difficult to cure, and the quality of life of the patient will be severely affected. Therefore, prevention of allergic diseases is more important and effective than treatment. Allergic diseases have an obvious genetic tendency. Therefore, it is very necessary to establish a gene detection method to assist in the early clinical diagnosis of patients with allergic rhinitis, which can provide the risk degree of the patient's possible susceptibility to allergic rhinitis and provide suggestions for the patient to take early preventive measures to control the occurrence of allergic rhinitis.

[0004] At the present stage, most of the gene detections for allergic rhinitis are about single-gene or a small number of SNP detections (PCR or fluorescence probe method), detecting individual known SNPs related to allergic rhinitis (such as IL4, IL13, ADAM33, etc.), usually only covering 3 - 5 genes, with a single detection target and unable to comprehensively evaluate the genetic risk. Summary of the Invention

[0005] The purpose of the present invention is to provide an SNP molecular marker combination related to allergic rhinitis and its application. The SNP molecular marker combination of the present invention is used as a detection target for the diagnosis or auxiliary diagnosis of allergic rhinitis, and can comprehensively evaluate the genetic risk.

[0006] The present invention provides a SNP molecular marker combination related to allergic rhinitis, comprising 89 SNP molecular markers; the rs numbers of the 89 SNP molecular markers in the SNP database are respectively: rs2302009, rs505010, rs1441586, rs512555, rs4982958, rs1998359, rs2107357, rs10493377, rs3014837, rs4129267, rs2228145, rs1269486, rs1058240, rs379568, rs2060793, rs10767664, rs569108, rs7130588, rs2155219, rs1946518, rs1946519, rs3794262, rs4251481, rs9729, rs731236, rs1544410, rs2228570, rs2069718, rs1898413, rs2041733, rs2057768, rs1805010, rs1805011, rs1805015, rs1801275, rs13527, rs2107538, rs9303277, rs12150079, rs2305480, rs7216389, rs7224129, rs4065275, rs8076131, rs12603332, rs17608925, rs3744246, rs4794820, rs77485247, rs77041280, rs393581, rs428253, rs4740, rs353702, rs438421, rs10407799, rs395969, rs390406, rs375688, rs412211, rs413216, rs420297, rs2108686, rs12461895, rs2241717, rs7258445, rs231735, rs231804, rs2787093, rs2787094, rs677044, rs3918400, rs628977, rs630712, rs2280089, rs2280090, rs2280091, rs44707, rs2853209, rs528557, rs2280092, rs2485700, rs511898, rs3918392, rs2787095, rs6084435, rs6115989, rs6107332, rs11697406.

[0007] The present invention also provides the application of the SNP molecular marker combination described in the above solution as a detection target in the preparation of a product for diagnosing or assisting in diagnosing allergic rhinitis.

[0008] Preferably, the detection means for diagnosis or auxiliary diagnosis includes multiplex PCR.

[0009] The present invention also provides a primer set for detecting the SNP molecular marker combination described in the above solution, which includes 78 pairs of primer pairs; the nucleotide sequences of the 78 pairs of primer pairs are as shown in SEQ ID NO: 1 to SEQ ID NO: 156.

[0010] The present invention also provides the application of the primer set described in the above solution in the preparation of a product for diagnosing or assisting in diagnosing allergic rhinitis.

[0011] Preferably, the detection means for diagnosis or auxiliary diagnosis includes multiplex PCR.

[0012] The present invention also provides a kit for diagnosing or assisting in diagnosing allergic rhinitis, which includes the primer set described in the above solution.

[0013] Preferably, the kit further includes a positive control and a negative control; the positive control includes a positive control sequence with a nucleotide sequence as shown in SEQ ID NO: 157; the negative control includes a negative control sequence with a nucleotide sequence as shown in SEQ ID NO: 158.

[0014] The present invention also provides a gene chip, which is prepared based on the SNP molecular marker combination described in the above solution.

[0015] The present invention also provides a system for diagnosing or assisting in diagnosing allergic rhinitis, which includes: a PCR amplification module, a sequencing module, a data import module, a data analysis module, and a data output module; the PCR amplification module includes the primer set or the kit described in the above solution; the sequencing module is used to sequence the amplification product of the PCR amplification module; the data import module is used to import the sequencing data; the data analysis module is used to analyze the sequencing data; the data output module is used to output the diagnosis or auxiliary diagnosis result.

[0016] The present invention provides a SNP molecular marker combination related to allergic rhinitis, including 89 SNP molecular markers; the rs numbers of the 89 SNP molecular markers in the SNP database are respectively: rs2302009, rs505010, rs1441586, rs512555, rs4982958, rs1998359, rs2107357, rs10493377, rs3014837, rs4129267, rs2228145, rs1269486, rs1058240, rs379568, rs2060793, rs10767664, rs569108, rs7130588, rs2155219, rs1946518, rs1946519, rs3794262, rs4251481, rs9729, rs731236, rs1544410, rs2228570, rs2069718, rs1898413, rs2041733, rs2057768, rs1805010, rs1805011, rs1805015, rs1801275, rs13527, rs2107538, rs9303277, rs12150079, rs2305480, rs7216389, rs7224129, rs4065275, rs8076131, rs12603332, rs17608925, rs3744246, rs4794820, rs77485247, rs77041280, rs393581, rs428253, rs4740, rs353702, rs438421, rs10407799, rs395969, rs390406, rs375688, rs412211, rs413216, rs420297, rs2108686, rs12461895, rs2241717, rs7258445, rs231735, rs231804, rs2787093, rs2787094, rs677044, rs3918400, rs628977, rs630712, rs2280089, rs2280090, rs2280091, rs44707, rs2853209, rs528557, rs2280092, rs2485700, rs511898, rs3918392, rs2787095, rs6084435, rs6115989, rs6107332, rs11697406. The SNP molecular marker combination of the present invention includes 89 SNP molecular markers, which can detect 46 genes related to allergic rhinitis at one time and can be used for the diagnosis or auxiliary diagnosis of allergic rhinitis.Using the SNP molecular marker combination of the present invention as a detection target for the diagnosis or auxiliary diagnosis of allergic rhinitis, the detection results are comprehensive, with extremely high accuracy and repeatability, and have an auxiliary diagnostic and suggestive effect on allergic rhinitis susceptible populations, especially those with a family genetic history. By detecting the peripheral blood of pregnant women or the umbilical cord blood samples of infants, it can also play a good predictive effect on neonatal allergic rhinitis. Detailed implementation mode

[0017] The present invention provides a SNP molecular marker combination related to allergic rhinitis, comprising 89 SNP molecular markers; the rs numbers of the 89 SNP molecular markers in the SNP database are respectively: rs2302009, rs505010, rs1441586, rs512555, rs4982958, rs1998359, rs2107357, rs10493377, rs3014837, rs4129267, rs2228145, rs1269486, rs1058240, rs379568, rs2060793, rs10767664, rs569108, rs7130588, rs2155219, rs1946518, rs1946519, rs3794262, rs4251481, rs9729, rs731236, rs1544410, rs2228570, rs2069718, rs1898413, rs2041733, rs2057768, rs1805010, rs1805011, rs1805015, rs1801275, rs13527, rs2107538, rs9303277, rs12150079, rs2305480, rs7216389, rs7224129, rs4065275, rs8076131, rs12603332, rs17608925, rs3744246, rs4794820, rs77485247, rs77041280, rs393581, rs428253, rs4740, rs353702, rs438421, rs10407799, rs395969, rs390406, rs375688, rs412211, rs413216, rs420297, rs2108686, rs12461895, rs2241717, rs7258445, rs231735, rs231804, rs2787093, rs2787094, rs677044, rs3918400, rs628977, rs630712, rs2280089, rs2280090, rs2280091, rs44707, rs2853209, rs528557, rs2280092, rs2485700, rs511898, rs3918392, rs2787095, rs6084435, rs6115989, rs6107332, rs11697406.

[0018] The SNP molecular marker combination of the present invention includes 89 SNP molecular markers, which can detect 46 genes related to allergic rhinitis at one time, covering key pathways such as Th2 immunity, epithelial barrier, and drug metabolism, and can provide more accurate risk assessment.

[0019] In the present invention, the positions and polymorphisms of the SNP molecular markers on the chromosomes and genes in the SNP molecular marker combination are shown in Table 1.

[0020] Table 1 Positions of the SNP loci on the chromosomes and genes

[0021]

[0022]

[0023] Note: * The meaning of the symbol: indicates the correspondence between the reference allele and the alternate alleles of the SNP molecular marker. For example: rs3014837, C>G,T means: reference allele: C (i.e., the base at this position in the human reference genome is C); alternate alleles: G or T (i.e., the possible mutations at this site are C→G or C→T). An SNP molecular marker with multiple alternate alleles is a multiallelic SNP because there are two possible mutation forms (G or T).

[0024] NA is missing data. "NA" in the gene column: indicates that this SNP has not been associated with a specific gene currently. "NA" in the Canonical SPDI column: may be due to missing data, not being included in the authoritative database, or may not be standardized.

[0025] Illustrate the relationship between the genotype and the risk of allergic rhinitis. For example, existing studies have shown (PMID: 18373864) that the alternate alleles (G or T) of rs3014837 are associated with an increased risk of allergic rhinitis. Then the risks of different genotypes may be as follows:

[0026] Homozygous Reference: C / C, not carrying the risk allele, with a lower risk (baseline level); Heterozygous: C / G or C / T, carrying 1 risk allele, with a medium risk; Homozygous Alternate: G / G, T / T or G / T (if both alternate alleles exist simultaneously), carrying 2 risk alleles, with the highest risk.

[0027] The SNPs are derived from the documents shown in Table 2 and databases such as dbSNP and SNPedia:

[0028] Table 2 Sources of SNPs

[0029]

[0030]

[0031]

[0032]

[0033]

[0034]

[0035] The present invention also provides the application of the SNP molecular marker combination described in the above solution as a detection target in the preparation of products for diagnosing or assisting in diagnosing allergic rhinitis.

[0036] The present invention also provides a primer set for detecting the SNP molecular marker combination described in the above solution, including 78 pairs of primer pairs; the nucleotide sequences of the 78 pairs of primer pairs are as shown in SEQ ID NO: 1 to SEQ ID NO: 156.

[0037] As an implementation manner, the corresponding relationship between the 78 pairs of primer pairs and 89 SNP molecular markers is shown in Table 3.

[0038] Table 3 Primer set for detecting SNP molecular marker combination

[0039]

[0040]

[0041]

[0042]

[0043] Note: * The same primer pair can detect multiple different SNP molecular markers because these SNP molecular markers are located at adjacent positions in the genome. Therefore, when designing primers, try to make them cover more sites to improve the detection efficiency and reduce costs. Suitable for high-throughput sequencing: This strategy is commonly used in amplicon sequencing, for example.

[0044] The primer pairs in the primer set of the present invention are designed using Thermo Fisher's proprietary software, which can ensure the uniqueness of the sequences of all primers, avoid complementarity between primers (formation of dimers), binding to non-target regions (off-target amplification), and 3'-end overlap (preventing cross-priming amplification). Moreover, the Tm values of all primer pairs are balanced to ensure synchronous amplification under the same PCR cycling conditions. The primer length is 20 - 30bp, which can reduce the interference of secondary structures.

[0045] As an implementation manner, the primers in the primer set are all synthesized by Thermo Fisher.

[0046] The present invention also provides the application of the primer set described in the above solution in the preparation of products for diagnosing or assisting in the diagnosis of allergic rhinitis.

[0047] As an implementation manner, the detection means for diagnosis or auxiliary diagnosis includes multiplex PCR; the multiplex PCR detection includes multiplex PCR detection based on NGS, which can detect 89 SNP molecular markers at one time, with high detection throughput and controllable cost, and is suitable for large-scale screening.

[0048] The present invention also provides a kit for diagnosing or assisting in the diagnosis of allergic rhinitis, including the primer set described in the above solution.

[0049] The kit of the present invention has high detection throughput, high sensitivity, strong specificity, more comprehensive detection results, extremely high accuracy and repeatability, and has an auxiliary diagnostic and prompting effect on allergic rhinitis susceptible populations, especially those with a family genetic history. By detecting samples of pregnant women's peripheral blood or infants' umbilical cord blood, it also has a good predictive effect on neonatal allergic rhinitis.

[0050] As an implementation manner, the kit further includes a high-fidelity premix (Ion AmpliSeq TM HiFi Mix), 2xIon AmpliSeq TM primer pool (2×IonAmpliSeq TM PrimerPool), high-fidelity PCR premix ( PCR SuperMix High Fidelity), Agencourt AMPure XP magnetic bead purification system (AgencyAMPure XP System), Ion PGM Hi-Q View sequencing kit (Ion PGM Hi-Q View sequencing kit), and nuclease-free ultrapure water.

[0051] Note: 2xIonAmpliSeq TMThe primer pool includes: a primer pair (AmpliSeq TM Primers) configured conventionally.

[0052] As an implementation manner, the IonAmpliSeq TM HiFi Mix is purchased from ThermoFisher; the IonAmpliSeq TM HiFi Mix is 5×IonAmpliSeq TM HiFi Mix; the IonAmpliSeq TM is purchased from ThermoFisher; the PCR SuperMixHigh Fidelity is purchased from ThermoFisher; the AgencyAMPure XP System is purchased from Beckman Coulter, Inc. of the United States; the Ion PGM Hi-Q Viewsequencing kit is purchased from ThermoFisher; the present invention has no special limitation on the source of the nuclease-free ultrapure water.

[0053] As an implementation, the kit further includes a positive control and a negative control; the positive control includes a positive control sequence, and the nucleotide sequence of the positive control sequence is as shown in SEQ ID NO: 157, specifically: tgtgaaagactgatttttcagtccctgtaaggggaatgtcgtaaagagaaaggagcggtgaatttctcaggacaaatgtccctgattaacagcaaagattacattgttgagacttaggaaagggtacatctttttcctacaaaagccttattcacaccatgcatttgggaacgaggtgaagtaagtattaaatacgtagttaagagctctggaatcagactgcattggaatcctggtagcaccctttatcagctttgtgactttgggtgcatttctttatctCtcttagctccccacttccacatctgtaaaatgggcttaatgatagtgaggattagatattttttggggaaagcatcaggcatagtaagtactcaaagttagttattaatattgacattattagcccccaagatcactaacctcttccaagagtttgtgaaccagaat; the positive control sequence is designed for rs4982958; the positive control is used to confirm the detection ability of the detection system for known variations;The negative control product includes a negative control sequence, and the nucleotide sequence of the negative control sequence is as shown in SEQ ID NO: 158, specifically: tgtgaaagactgatttttcagtccctgtaaggggaatgtcgtaaagagaaaggagcggtgaatttctcaggacaaatgtccctgattaacagcaaagattacattgttgagacttaggaaagggtacatctttttcctacaaaagccttattcacaccatgcatttgggaacgaggtgaagtaagtattaaatacgtagttaagagctctggaatcagactgcattggaatcctggtagcaccctttatcagctttgtgactttgggtgcatttctttatctTtcttagctccccacttccacatctgtaaaatgggcttaatgatagtgaggattagatattttttggggaaagcatcaggcatagtaagtactcaaagttagttattaatattgacattattagcccccaagatcactaacctcttccaagagtttgtgaaccagaat; the negative control sequence is designed for rs4982958; the negative control is used to monitor false positive results.

[0054] As an embodiment, the kit further includes genomic DNA of HapMap cell line (NA18525) (Accession: SAMN24495184 ID: 24495184), which is used as a reference for multi-SNP sites because it contains known germline polymorphisms (about 4 million SNP sites).

[0055] As an embodiment, the kit further includes a no-template control (NTC) for monitoring contamination.

[0056] The kit of the present invention includes multi-level controls, which can ensure the detection sensitivity through positive controls, ensure specificity through negative controls, comprehensively evaluate the detection accuracy of SNP sites within the whole genome through HapMap reference sequences, and exclude contamination interference through NTC. Therefore, by adopting the above multi-level control system including positive controls, negative controls, NTC and HapMap reference samples, comprehensive quality control of SNP detection can be ensured.

[0057] As an implementation manner, the positive control and the negative control are plasmid DNAs; the original plasmid of the plasmid DNA is pIDTSmartAmp; the insertion sites of the positive control sequence and the negative control sequence on the original plasmid are EcoRV restriction sites (the base position is about near 723 bp in the vector), located between the T7 promoter and the original multiple cloning site.

[0058] As an implementation manner, the method for the multiplex PCR detection of allergic rhinitis using the kit comprises the following steps:

[0059] 1) Extract the genomic DNA of the human blood sample to be tested;

[0060] 2) Using the genomic DNA of the human blood sample to be tested as a template, perform a multiplex PCR amplification reaction with the primers in the primer group described in the above solution, cut and purify the amplification product, and construct a sequencing library;

[0061] 3) Sequence the sequencing library to obtain sequencing data;

[0062] 4) Analyze the sequencing data and export the detection result.

[0063] The present invention first extracts the genomic DNA of the human blood sample to be tested; as an implementation manner, the human serum sample is peripheral blood or cord blood; the present invention has no special limitation on the method for extracting the genomic DNA of the human blood sample to be tested, and a conventional method for extracting the genomic DNA of the human blood sample in the art can be used. As an implementation manner, the genomic DNA of the human blood sample is extracted using a SIMGEN whole blood DNA mini kit; the SIMGEN whole blood DNA mini kit is purchased from Hangzhou Xinjing Biological Reagent Development Co., Ltd.

[0064] After obtaining the genomic DNA of the human blood sample to be tested, using the genomic DNA of the human blood sample to be tested as a template, perform a multiplex PCR amplification reaction with the primers in the primer group described in the above solution, cut and purify the amplification product, and construct a sequencing library.

[0065] As an implementation manner, the reaction system used for the multiplex PCR amplification reaction is an IonAmpliSeq TM multiplex PCR system, which includes the following components in a volume of 20 μL: 1 - 100 ng of the genomic DNA of the human blood sample to be tested, 4 μL of 5×IonAmpliseq TM HiFi mix, 2×IonAmpliseq TM10 μL of primer pool and 4 μL of nuclease-free water; further, the genomic DNA of the human blood sample to be tested is 10 ng; the total concentration of all primers is 0.2 μM, and the final concentration of each pair of primers is extremely low (usually ≤ nM level), greatly reducing the collision probability between primers.

[0066] The multiplex PCR amplification reaction of the present invention uses HiFi DNA polymerase, which can tolerate complex templates (such as the human genome), inhibit non-specific amplification, and the buffer contains optimized additives (such as DMSO), which can reduce the influence of secondary structures.

[0067] The IonAmpliSeq of the present invention TM The multiplex PCR system can simultaneously amplify 78 pairs of primers and effectively avoid mutual interference between primers.

[0068] In the present invention, the primer set described in the above solution is used to construct a library.

[0069] As an embodiment, the program of the multiplex PCR amplification reaction is: 99 °C, 2 min; 99 °C, 15 s, 60 °C, 4 min, for a total of 19 cycles; after the amplification is completed, it is maintained at 10 °C.

[0070] In the present invention, the polymerase is inactive at low temperatures, which can prevent non-specific binding in the initial cycle; combining annealing / extension can reduce the risk of primer mismatch; the number of cycles is 19 cycles (much lower than the 30-40 rounds of conventional single PCR), avoiding biased amplification caused by primer depletion in the later stage.

[0071] The present invention has no special restrictions on the method of gel cutting and purifying the amplification product and constructing the sequencing library, and conventional methods in the art can be used.

[0072] After obtaining the sequencing library, the present invention sequences the sequencing library to obtain sequencing data; as an embodiment, the method of sequencing is next-generation sequencing; the present invention uses Torrent Suite TM software to create a sequencing plan; the sequencing instrument is a PGM sequencer.

[0073] After obtaining the sequencing data, the present invention analyzes the sequencing data and exports the detection results; the present invention has no special restrictions on the programs of the analysis and export, and conventional analysis programs and export programs in the art can be used. As an embodiment, the analysis program used is Variant Caller; the export program used is IonReporter.

[0074] As an implementation manner, after the detection results are exported, the genotypes of each SNP molecular marker are obtained, and then according to the effect values of each SNP molecular marker, each SNP molecular marker is weighted and calculated to obtain the polygenic risk score and risk level of an individual suffering from allergic rhinitis; the risk level includes three levels: low, medium, and high; the effect value is the effect value reported in the literature (see

Bonnelykke K, et al. (2013). "Meta-analysis of genome-wide association studies identifies ten loci influencing allergic sensitization." Nat Genet. 45(8):902-6.

[0075]

[0076] In Formula 1, wi is the effect value of the i-th SNP; the Dosage i is the dosage of the risk allele of the individual at the i-th SNP, and the value is 0, 1, or 2, representing the number of risk alleles;

[0077] The risk level is divided based on the PRS distribution of the reference population (assuming that the mean of the reference population PRS is 0 and the standard deviation is 1); the corresponding criteria for the polygenic risk score and the risk level are: when the polygenic risk score ≤ -0.84 (1 standard deviation below the mean), it is determined as low risk; when -0.84 < PRS ≤ 0.84, it is determined as medium risk; when the polygenic risk score > 0.84, it is determined as high risk.

[0078] The present invention also provides a gene chip, which is prepared based on the SNP molecular marker combination described in the above solution.

[0079] The present invention also provides a system for diagnosing or assisting in the diagnosis of allergic rhinitis, which is characterized by including: a PCR amplification module, a sequencing module, a data import module, a data analysis module, and a data output module;

[0080] The PCR amplification module includes the primer group or the kit described in the above solution;

[0081] The sequencing module is used to sequence the amplification products of the PCR amplification module;

[0082] The data import module is used to import sequencing data;

[0083] The data analysis module is used to analyze sequencing data;

[0084] The data output module is used to output the diagnosis or auxiliary diagnosis results.

[0085] As an implementation manner, the PCR amplification module includes a multiplex PCR amplification module.

[0086] As an implementation manner, the data output module automatically generates a structured report; the automatically generated structured report includes: a core data area, a risk warning area, and a medication guidance area;

[0087] The core data area includes the genotypes and clinical significance grades of 89 SNP loci (classified into pathogenic / likely pathogenic / uncertain clinical significance / likely benign / benign according to the ACMG variant classification standard);

[0088] The risk warning area includes the overall disease risk level (low / medium / high) calculated based on the polygenic risk score (PRS);

[0089] The medication guidance area includes medication suggestions for drug-related SNPs.

[0090] As an implementation manner, the result export formats of the data output module include PDF format and JSON format; the PDF format conforms to the clinical report template certified by CLIA and includes an electronic signature and a watermark; the JSON format is for scientific research data analysis.

[0091] To further illustrate the present invention, the following combines examples to describe in detail a SNP molecular marker combination related to allergic rhinitis and its application provided by the present invention, but they cannot be understood as a limitation to the protection scope of the present invention.

[0092] Example 1 Application of a kit for detecting allergic rhinitis

[0093] 1. Sample DNA extraction (sample extraction and loading chamber)

[0094] Samples: Take 1 mL of peripheral blood samples (EDTA anticoagulated whole blood) from 100 patients with allergic rhinitis and 100 healthy people, a total of 200 cases.

[0095] Reagents: Nucleic acid extraction kit (SIMGEN whole blood DNA small-scale kit instruction manual, 250 preparations by Hangzhou Xinjing Biological Reagent Development Co., Ltd.), absolute ethanol

[0096] Equipment: Biosafety cabinet, high-speed bench centrifuge, pipettes (1 mL, 200 μL), vortex oscillator, water bath, timer Consumables: Nucleic acid purification column, pipette tips (1 mL, 200 μL), 2 mL / 1.5 mL centrifuge tubes.

[0097] Operation steps:

[0098] 1.1 Preparation before experiment

[0099] 1) Set the centrifuge temperature to 20 °C.

[0100] 2) Incubate Buffer TE at 56 °C.

[0101] 3) Add absolute ethanol to Buffer WA and Buffer WB according to the instructions on the reagent bottle label, and tick the box on the label to mark "ethanol added".

[0102] 1.2 DNA extraction and purification

[0103] 1) Add 300 μL of Buffer L1 to a 1.5 mL centrifuge tube.

[0104] 2) Add 400 μL of anticoagulated whole blood, cover the tube cap, and vortex for 30 s.

[0105] * If the blood volume is less than 400 μL but greater than 200 μL, the amounts of Buffer L1 and Buffer L2 can be reduced proportionally (note that the operation must be strictly carried out according to the volume ratio of Buffer L1: anticoagulated whole blood: Buffer L2 = 3:4:3, otherwise the subsequent steps cannot be carried out), and the amounts of other reagents remain unchanged; if the blood volume is less than 200 μL, it is recommended to supplement physiological saline to the blood to make the blood volume at least 200 μL.

[0106] 3) Add 300 μL of Buffer L2, shake the centrifuge tube vigorously 3 - 5 times, and then vortex for 30 s to mix evenly.

[0107] * A large amount of hemoglobin precipitation will occur in this step.

[0108] 4) Centrifuge at 13000 rpm for 2 min.

[0109] 5) Pour the supernatant in step 4 into the nucleic acid purification column (the nucleic acid purification column is placed in a 2 mL centrifuge tube), cover the tube cap, and centrifuge at 12000 rpm for 30 s.

[0110] *When extracting DNA from the blood of certain animals, since there is less hemoglobin, the volume of the supernatant obtained by centrifugation may be greater than the volume of the purification column. In this case, it is recommended to aspirate 700 μL of the supernatant into the nucleic acid purification column, or perform this step of the operation on the supernatant in two portions.

[0111] 6) Discard the filtrate in the 2 mL centrifuge tube, place the nucleic acid purification column back into the 2 mL centrifuge tube, add 500 μL of Buffer WA to the nucleic acid purification column, cover the tube cap, and centrifuge at 12000 rpm for 30 s.

[0112] *Confirm that absolute ethanol has been added to Buffer WA.

[0113] *It is normal for hemoglobin to remain on the purification column membrane and can be washed away by Buffer WA.

[0114] *It is not necessary to completely discard the filtrate. If you want to avoid contamination of the centrifuge by the filtrate adhering to the mouth of the centrifuge tube, you can invert and tap the 2 mL centrifuge tube once on a paper towel.

[0115] 7) Discard the filtrate in the 2 mL centrifuge tube, place the nucleic acid purification column back into the 2 mL centrifuge tube, add 600 μL of Buffer WB to the nucleic acid purification column, cover the tube cap, and centrifuge at 12000 rpm for 30 s.

[0116] *Confirm that absolute ethanol has been added to Buffer WB.

[0117] 8) Discard the filtrate in the 2 mL centrifuge tube, place the nucleic acid purification column back into the 2 mL centrifuge tube, and centrifuge at 14000 rpm for 1 min.

[0118] *If the centrifuge speed cannot reach 14000 rpm, centrifuge at the highest speed for 2 min.

[0119] *Do not omit this step, otherwise the subsequent PCR results may be affected due to ethanol contamination in the purified nucleic acid.

[0120] 9) Discard the 2 mL centrifuge tube, place the nucleic acid purification column in a clean 1.5 mL centrifuge tube, add 100 - 200 μL of Buffer TE incubated at 56 °C to the purification column, cover the tube cap, let it stand at room temperature for 1 min, and centrifuge at 12000 rpm for 30 s.

[0121] *If the centrifuge does not have a leak-proof lid, change the centrifugation conditions to 8000 rpm for 1 min to prevent the tube cap from falling off and damaging the centrifuge.

[0122] 10) Discard the purification column, and the eluted DNA can be immediately used for various molecular biology experiments, or the DNA can be stored at -20 °C for later use.

[0123] 2. DNA Concentration Determination

[0124] Sample: DNA solution to be tested

[0125] Reagents: dsDNA HS (High Sensitivity) Detection Kit

[0126] Equipment: Qubit 3.0 Fluorometer, Pipettes (1 mL, 200 μL, 10 μL, 2 μL), Vortex Shaker, Timer

[0127] Materials: Pipette Tips (1 mL, 200 μL, 10 μL, 2 μL), 2 mL / 1.5 mL Centrifuge Tubes, 0.5 mL PCR-05-C Tubes

[0128] Operating Procedures:

[0129] 2.1 Experiment Preparation

[0130] 1) Before use, restore each component in the kit to room temperature.

[0131] 2) Prepare a sufficient amount of 0.5 mL PCR Thin-Walled Tubes and label them. Do not label on the side wall of the PCR tube to avoid affecting fluorescence signal collection.

[0132] 2.2 Preparation of Detection Working Solution

[0133] In a plastic container, use dsDNA Buffer to dilute an appropriate amount of dsDNA Reagent to 1× according to the ratio (for example: take 1 μL of dsDNA Reagent and add 199 μL of dsDNA Buffer), and prepare it immediately before use. After the working solution is prepared, use it within 3 hours.

[0134] 2.3 Preparation of Samples to be Tested

[0135] 1) Prepare the standard samples to be tested. Take 190 μL of the detection working solution into the standard sample PCR tubes, and add 10 μL of dsDNA Standard 1 and dsDNA Standard 2 to the corresponding standard sample PCR tubes respectively, and gently vortex for 2 - 3 s, trying to avoid generating bubbles.

[0136] 2) Prepare the samples to be tested. Take 180 - 199 μL of the detection working solution into the sample PCR tubes, and add 1 - 20 μL of the sample to be tested respectively, so that the final volume of each sample in the PCR tube is 200 μL, and gently vortex for 2 - 3 s, trying to avoid generating bubbles.

[0137] 2.4 Detection

[0138] 1) Place all PCR tubes to be tested in the dark at room temperature for incubation for 2 min.

[0139] 2) According to the operation instructions of the 3.0 fluorometer, select the dsDNA High Sensitivity detection program to measure the fluorescence signal value.

[0140] 3. Amplification of the target fragment of the sample by PCR

[0141] Sample: DNA solution to be tested

[0142] Reagents: 5×Ion AmpliSeq TM HiFi Mix, 2×Ion AmpliSeq TM Primer pool, negative / positive control sequences and nuclease-free ultrapure water.

[0143] Equipment: Biological safety cabinet, pipettes (1 mL, 200 μL, 20 μL, 10 μL, 2 μL), vortex oscillator, PCR tubes (0.2 mL, 8-strip tubes or 96-well plates), mini tabletop centrifuge, 96-well plate centrifuge, 96-well PCR instrument

[0144] Materials: Pipette tips (1 mL, 200 μL, 20 μL, 10 μL, 2 μL), 2 mL / 1.5 mL / 0.2 mL centrifuge tubes, PCR tubes (0.2 mL, 8-strip tubes or 96-well plates), adhesive film.

[0145] Operation steps:

[0146] 1) For each mixture of DNA and primer pool, add the components in Table 4 into each reaction well of the 96-well PCR.

[0147] Table 4 Multiplex PCR reaction system

[0148]

[0149]

[0150] 2) Seal the PCR plate with the sealing film, shake it well, and centrifuge the PCR plate instantaneously to recover the sample to the bottom of the tube as much as possible.

[0151] 3) Place the 96-well plate in the PCR instrument and run the program in Table 5 to amplify the target region on the genome:

[0152] Table 5 Multiplex PCR reaction program

[0153]

[0154] 4. Primer digestion

[0155] Function of primer digestion: Hydrolyze most of the primers at both ends of each target fragment, leaving the part that can be complementary to the adapter, facilitating the ligation of the adapter.

[0156] Sample: PCR product to be tested

[0157] Reagent: FuPa reagent

[0158] Equipment: Biological safety cabinet, pipette (10 μL), vortex oscillator, mini bench centrifuge, 96-well plate centrifuge, 96-well PCR instrument

[0159] Materials: Tips (10 μL), 0.2 mL centrifuge tubes

[0160] Operation steps:

[0161] 1) Take out the amplified 96-well plate from the PCR instrument, place it on the 96-well plate centrifuge for a few seconds to collect the solution at the bottom of the tube, and then slowly remove the sealing film.

[0162] 2) Add 2 μL of FuPa reagent to each well of the 96-well plate to make the total volume reach 22 μL.

[0163] 3) Seal the 96-well plate tightly with the sealing film, shake it thoroughly to mix, and centrifuge for a few seconds to collect the liquid at the bottom of the tube.

[0164] 4) Put the 96-well plate into the PCR instrument and run according to the program in Table 6.

[0165] Table 6 Primer digestion program

[0166] Temperature Time 50℃ 10 min 55℃ 10 min 60℃ 20 min 10℃ Duration (≤1 h)

[0167] 5. Ligate the adapter

[0168] Sample: PCR product after primer digestion

[0169] Reagents: switch solution, tag solution, adapter solution, DNA ligase, nuclease-free water

[0170] Equipment: Biological safety cabinet, pipette (10 μL), vortex oscillator, mini bench centrifuge, 96-well plate centrifuge, 96-well PCR instrument

[0171] Materials: Tips (10 μL), 0.2 mL centrifuge tubes

[0172] Operation steps:

[0173] Mix and dilute the adapter solution and the tag solution according to Table 7:

[0174] Table 7 Dilution system of adapter solution and tag solution

[0175] Reagent Name Volume Used (μL) Nuclease-Free Water 4 Ion P1 Adapter 2 IonXpress Tag X[1] 2 Total Volume[2] 8

[0176] Note: [1] X represents the selected tag number. [2] The mixture can be stored at -20 °C.

[0177] Take out the 96-well plate after digesting part of the primers from the PCR instrument, leave it for a few seconds to allow the solution to collect at the bottom of the tube, and then slowly remove the sealing film.

[0178] Carefully remove the sealing film on the PCR plate and add the components in Table 8 to each reaction well.

[0179] Table 8 Adapter Ligation System

[0180] Reagent Name Volume Used (μL) Switch Solution 4 Diluted Adapter-Tag Mixture 2 DNA Ligase 2

[0181] At this time, the total volume of the ligation system is 30 μL.

[0182] Note: ① When preparing the ligation system, different numbered adapter tag mixtures must be added to different wells, and no repeated identical numbered adapter tag mixtures should appear in different wells of the same 96-well plate;

[0183] ② DNA ligase must be added last.

[0184] Seal the 96-well plate tightly with a sealing film, shake it well, and leave it for a few seconds to collect the liquid at the bottom of the tube.

[0185] Put the 96-well plate into the PCR instrument and run it according to the program in Table 9.

[0186] Table 9 Adapter Ligation Program

[0187] Temperature Time 22℃ 30 min 68℃ 5 min 72℃ 5 min 10℃ Duration (≤24 h)

[0188] 6. Purify the unamplified library

[0189] Sample: Sample to be purified

[0190] Reagents: XP magnetic beads, 70% ethanol (prepared freshly)

[0191] Equipment: Biosafety cabinet, pipettes (100 μL, 200 μL), vortex oscillator, mini bench centrifuge, 96-well plate centrifuge, DynaMag TM -96Side Magnet 96-well plate magnetic stand

[0192] Materials: Tips (100 μL, 200 μL), 0.2 mL centrifuge tubes, 1.5 mL low-binding centrifuge tubes

[0193] Operating steps:

[0194] 1) Place Vortex the XP magnetic beads thoroughly and then let them stand at room temperature.

[0195] 2) Prepare 70% ethanol according to the recipe in Table 10:

[0196] Table 10 Recipe for 70% ethanol

[0197] Reagent Name Volume per Sample Used (μL) Nuclease-Free Water 100 Absolute Ethanol 230 Total Volume 330

[0198] 3) Take out the amplified 96-well plate from the PCR instrument, leave it for a few seconds to allow the solution to collect at the bottom of the tube, and then slowly remove the sealing film.

[0199] 4) Slowly aspirate and add 45 μL XP magnetic beads (thoroughly mixed before use) to each library. Pipette up and down 5 times to thoroughly mix the DNA with the magnetic bead suspension, and incubate the mixture at room temperature for 5 min.

[0200] 5) Place the 96-well plate on the magnetic stand, let it stand for 2 min or until the solution becomes clear. Carefully aspirate and discard the supernatant without disturbing the magnetic beads.

[0201] 6) Add 150 μL of freshly prepared 70% ethanol to the wells, move the 96-well plate back and forth on the magnet to wash the magnetic beads, and then carefully discard the supernatant without disturbing the magnetic beads.

[0202] 7) Repeat step 6) for the second wash.

[0203] 8) Ensure that all ethanol droplets have been aspirated from the wells. Place the plate on the magnetic stand and air-dry at room temperature for 5 min, taking care not to over-dry.

[0204] 7. Library Quantification

[0205] Reagents: platinum PCR SuperMix HiFi, equalizer primer, equalizer magnetic beads, wash buffer, equalizer capture reagent, equalizer elution buffer

[0206] Materials and Equipment: Pipettes (1 mL, 100 μL, 200 μL, 300 μL multi-channel pipette) and corresponding pipette tips (boxes), 0.2 mL centrifuge tube rack, 1.5 mL low-binding centrifuge tubes, 1.5 mL centrifuge tube rack, trash bin (bag), vortex oscillator, gloves, masks, absorbent paper towels, laboratory coats, mini tabletop centrifuge, laminar flow hood, 96-well plate centrifuge, 96-well plate magnetic stand.

[0207] Operating Procedures:

[0208] 1) Add 50 μL of Platinum PCR SuperMix HiFi and 2 μL of Equalizer primer to the purified and unamplified library in each well of a 96-well plate, and pipette more than half of the liquid up and down at least 5 times to mix well.

[0209] 3) Place the 96-well plate on a magnetic rack and let it stand for 2 min, then carefully remove 50 μL of the supernatant to a clean 96-well plate.

[0210] 4) Seal the 96-well plate tightly with a sealing plate, put it into a PCR instrument, and run according to the program in Table 11:

[0211] Table 11 Library Amplification Program

[0212]

[0213] 5) When amplifying the library, the Equalizer magnetic beads can be pre-washed first:

[0214] a. Vortex the Equalizer magnetic beads and let them stand at room temperature.

[0215] b. Take a 1.5 mL low-binding centrifuge tube, add 3 μL of Equalizer magnetic beads per reaction and 6 μL of washing solution per reaction to the tube, mix well and place it on a magnetic rack at room temperature for 3 min or until the solution is clear.

[0216] c. Carefully remove the supernatant (do not touch the tube), and then remove the centrifuge tube from the magnetic rack.

[0217] d. Add 6 μL of washing solution per reaction to the tube and mix by inverting the tube up and down.

[0218] 6) After the library amplification is completed, take out the amplified 96-well plate from the PCR instrument, let it stand for a few seconds to collect the solution to the bottom of the tube, and then slowly remove the sealing film.

[0219] 7) Precisely add 10 μL of Equalizer capture reagent to each well of the 96-well plate, pipette more than half of the liquid up and down at least 5 times to mix well, and place it at room temperature for 5 min.

[0220] 8) Gently vortex the pre-washed Equalizer magnetic beads, and then add 6 μL of Equalizer magnetic beads to each well of the 96-well plate containing the captured library.

[0221] 9) Set the pipette to 40 μL, pipette more than half of the liquid up and down at least 5 times to mix well, and incubate at room temperature for 5 min.

[0222] 10) Place the 96-well plate on a 96-well magnetic stand, let it stand for 2 min or until the solution is clear, and then carefully remove the supernatant;

[0223] 11) Add 150 μL of washing solution to each well of the 96-well plate, and move the 96-well plate back and forth on the magnet to wash the magnetic beads;

[0224] 12) Place the 96-well plate back on the magnetic stand, let it stand for 2 min or until the solution is clear, and then carefully remove the supernatant;

[0225] 13) Repeat steps 11 - 12 once;

[0226] 14) Remove the 96-well plate from the magnetic stand, add 100 μL of Equalizer elution solution to each well, seal the 96-well plate tightly with a sealing film and mix by oscillation (or pipette more than half of the liquid up and down at least 5 times to mix), and then leave it for a few seconds to collect the solution at the bottom of the tube;

[0227] 15) Place the 96-well plate containing the eluted library in a PCR instrument, set the temperature to 32 °C and run for 5 min;

[0228] 16) Take out the 96-well plate from the PCR instrument and place it back on the 96-well magnetic stand, let it stand for 5 min or until the solution is clear. At this time, the supernatant contains a DNA library with a concentration of 100 pM, which can be stored with the magnetic beads at 4 - 8 °C for 1 month, or the supernatant can be carefully taken out into a clean 96-well plate and stored at -20 °C for a long time.

[0229] 17) Library mixing

[0230] Pipette 5 μL from each tube of the quantified library into a low-binding centrifuge tube, and pipette up and down 5 times to mix well. The mixed library can be stored at 4 °C for 1 month or at -20 °C for long-term storage.

[0231] 8. OT2 water-in-oil PCR

[0232] Sample: Constructed library

[0233] Reagent: Ion PGM Hi-Q View OT2 Kit

[0234] Equipment: Biological safety cabinet, multichannel pipette (100 μL, 200 μL, 300 μL, 1 mL), vortex oscillator, mini bench centrifuge, 96-well plate centrifuge, OT2 instrument

[0235] Materials: Pipette tips (100 μL, 200 μL, 300 μL, 1 mL), 0.2 mL centrifuge tubes, 1.5 mL low-binding centrifuge tubes

[0236] Operating steps:

[0237] 8.1 Configure the OT2 instrument

[0238] 1) Turn on the OT2 instrument. When the main interface is displayed on the screen, click the "open lid" button to open the centrifuge lid, and then wipe the centrifuge lid and its interior with lint-free paper.

[0239] 2) Add 150 μL of Ion OneTouch TM demulsification solution to each of the two Ion OneTouch TM recovery tubes. Place the recovery tubes in the corresponding positions of the centrifuge, install the recovery bridging at the center of the centrifuge, and close the centrifuge lid.

[0240] 3) Pull the handle of the OT2 instrument to open the thermal lid, remove the used cleaning adapter, install the amplification reaction plate, pull the handle to close the thermal lid, and pass the disposable hose through the hose valves at the top and front.

[0241] 4) Insert the disposable needle at the top of the disposable hose into the needle socket in the middle of the centrifuge lid, and then confirm that the disposable needle has been automatically fixed in the correct position on the recovery bridge by pressing and releasing the injection needle socket.

[0242] 5) Install the blue pipette at the marked and points in front of the OT2 instrument. Invert and mix the Ion OneTouch TM Oil reagent bottle (450 mL reagent bottle) three times, and then inject Ion One Touch TM Oil into the OT2 special reagent tube until it is half full. Install the reagent tube at point, minimizing air bubbles;

[0243] 6) Invert and mix the Recovery Solution reagent bottle three times, and then inject 1 / 4 volume of Recovery Solution into the reagent tube. Install the reagent tube at point, minimizing air bubbles;

[0244] 7) Disconnect the waste liquid bottle from the OT2 instrument and properly dispose of the waste liquid.

[0245] 8.2 Prepare the water-in-oil amplification reaction solution and perform emulsion PCR

[0246] 1) Prepare the reagents in Table 12:

[0247] Table 12 Preparation of water-in-oil amplification reaction solution

[0248]

[0249] 2) Library dilution

[0250] Dilute the mixed library of 8 μL to 25 μL with nuclease-free water. The diluted library should be stored refrigerated and can only be used within 48 h.

[0251] 3) Prepare Ion PGM TM Hi-Q TM ISPs: Vortex ISPs at maximum speed for 1 min to fully resuspend the ISP beads, centrifuge ISPs briefly for 2 s, pipette up and down to mix ISPs thoroughly, and then proceed to the next step immediately.

[0252] 4) In a 2 mL tube (purple cap) containing 800 μL of Ion PGM TM Hi-Q TM View enzyme mixture, add the components in Table 13 in the specified order (after adding each component, pipette up and down to mix the amplification reaction solution):

[0253] Table 13 Emulsion PCR reaction system

[0254] Sequence Reagent Name Volume Used 1 Nuclease-Free Water 25 μL 2 <![CDATA[IonPGM TM Hi-Q TM View enzyme mixture]]> 50 μL 3 Diluted Library 25 μL 4 <![CDATA[IonPGM TM Hi-Q TM ViewISPs]]> 100 μL Total Volume 1000 μL

[0255] Vortex the amplification reaction solution prepared in the previous step on a shaker at maximum speed for 5 s, and centrifuge briefly for a few seconds. Proceed to the next step immediately.

[0256] Fill and install Ion OneTouch TM Reaction filter: Pipette 1000 μL of the prepared amplification reaction solution and add it to the Ion OneTouch TM reaction filter, pipette 850 μL of Ion OneTouch TM reaction oil (25 mL reagent bottle) and add it to the Ion OneTouch TM reaction filter, change the pipette tip and add 850 μL of Ion OneTouch TM reaction oil again from the sample addition hole, invert the reaction filter and install it completely at the corresponding position of the three holes on the top of the Ion OneTouch TM 2 instrument.

[0257] After installing Ion OneTouch TM Reaction Filter, click the "Run" button on the display screen to enter the library selection page, click the drop-down menu, and select PGM: Ion PGM TM Hi-Q TM View OT2 Kit - 200, or PGM: Ion PGM TM Hi-Q TM View OT2 Kit - 400, and then click "Next".

[0258] Confirm whether all steps have been completed according to the prompts given on the display screen. After confirming that all steps are completed, click "Next" to start the program.

[0259] Remove the sample from the instrument within 16 h after starting the run. After the run ends, click "Next" at the end line of the screen to directly enter the ISPs step of the recovery tape template.

[0260] ISPs of the recovery tape template: First, centrifuge the sample according to the screen prompts. After the centrifuge stops, on the instrument display screen, click Open Lid. Wait for the centrifuge lid to pop open, remove and discard the Ion OneTouch TM Recover the bridging and remove two Ion OneTouches from the instrument TM Recover the tubes and place them on the tube rack; aspirate the supernatant from the recovery tubes, leaving 100 μL of the recovery solution.

[0261] Process the ISPs: Add 500 μL of Ion OneTouch TM washing solution to each recovery tube, pipette the ISPs repeatedly to disperse them, and then transfer the suspension to a new labeled 1.5 mL Eppendorf centrifuge tube, centrifuge the ISPs at 15500×g for 2.5 min. Aspirate the supernatant, leaving 100 μL of the washing solution.

[0262] * Aspirate the supernatant from the liquid surface and the opposite side of the precipitate. The ISPs can be stored at 2 - 8 °C for 3 d.

[0263] 9. ISPs enrichment

[0264] Sample: amplified library

[0265] Reagent: Ion OneTouch TM ES Supplies kit

[0266] Equipment: biosafety cabinet, multichannel pipette (100 μL, 200 μL, 300 μL, 1 mL), vortex oscillator, mini benchtop centrifuge, 96-well plate centrifuge, Ion OneTouch TM ES

[0267] Materials: pipette tips (100 μL, 200 μL, 300 μL, 1 mL), 0.2 mL centrifuge tubes, 1.5 mL low-binding centrifuge tubes

[0268] Operating steps:

[0269] 1) Take out the reagents from the kit and prepare a fresh Melt-Off solution in the order shown in Table 14:

[0270] Table 14 Configuration Sequence of Fresh Melt-Off Solution

[0271] Sequence Reagent Name Volume Used 1 Tween solution 280 μL 2 1 M NaOH 40 μL - Total Volume 320 μL

[0272] * Important! Prepare the Melt-Off solution as needed and dispose of it properly 1 day after preparation.

[0273] 2) Take out MyOne TM Streptavidin C1 magnetic beads, vortex on an oscillator for 30 s to resuspend the magnetic beads, then leave for 2 s, open the tube, pipette up and down the black core in the beads with a new pipette tip until all cores are dispersed, and then quickly transfer 13 μL Dynabeads TM MyOne TM Streptavidin C1 magnetic beads to a new 1.5-mL Eppendorf LoBind TM centrifuge tube;

[0274] 3) Let the tube stand on a magnetic stand for 2 min, then carefully aspirate the supernatant (do not touch the Dynabead TM MyOne TM Streptavidin C1 Beads) at the bottom, and add 130 μL MyOne TM MyOne TM Bead Wash Solution to the Dynabeads TM MyOne in the tube, remove the tube from the magnetic stand, vortex for 30 s, and centrifuge for 2 s;

[0275] 4) Take out an 8-well tube from the Ion OneTouch TM ES Supplies kit and confirm that the square side of the 8-well tube is on the left.

[0276] 5) Take out the ISPs and pipette up and down 10 times to mix well, and fill the 8-well tube according to the instructions in Table 15;

[0277] Table 15 8-Well Tube Filling Instructions

[0278]

[0279]

[0280] 6) Confirm that the square side is facing left, and then place the 8-well tube on the right side into the slot.

[0281] 7) Prepare 1 new EppendorfTM LoRetention Dualfilter P300 pipette tips and new 0.2 mL PCR tubes. Add 10 μL of neutralization solution to a new 0.2 mL PCR tube. Open the lid of the PCR tube containing the neutralization solution and place it in the hole on the base of the tip rack. Install a pipette tip in the tip rack. Remove the pipetting arm from the stand, then align it with the pipette tip. Keep the pipetting arm vertical and press down on the pipetting arm to attach the tip to the pipetting arm. Hold for about 1 - 3 s to ensure the tip is firmly attached to the pipetting arm. Lift the pipetting arm upward to disengage the tip from the tip rack, and then place the pipetting arm back on the stand.

[0282] 8) Before running the ES instrument, pipette up and down the beads in well 2 with a pipette (do not introduce air bubbles into the solution). Open Ion OneTouch TM ES and wait for the instrument to initialize. The screen will display "rdy". The tip arm will perform a series of initialization movements and return to the starting position (5 s). Click Start / Stop. The screen will display "run" during operation. The running time is 35 min;

[0283] 9) After the run, quickly close the lid of the PCR tube containing the enriched ISPs and remove it from the instrument. Gently invert the PCR tube 5 times to mix the liquid inside. Store the enriched ISPs, which can be stored at 2°C - 8°C for up to 3 days, or sequence immediately.

[0284] * At the end of the run, the instrument screen will display "End" and beep for 60 s. Click the Start / Stop button to turn off the alarm and reset Ion OneTouch TM ES for the next run. The instrument does not need to be restarted between multiple runs.

[0285] 10. Sequencing

[0286] Sample: Enriched library

[0287] Reagent: Kit

[0288] Equipment: Biosafety cabinet, multichannel pipette (100 μL, 200 μL, 300 μL, 1 mL), vortex oscillator, mini tabletop centrifuge, chip centrifuge, Ion OneTouch TM ES

[0289] Materials: Ion 316 or Ion 318 chip, pipette tips (100 μL, 200 μL, 300 μL, 1 mL), 0.2 mL centrifuge tubes, 1.5 mL low - binding centrifuge tubes

[0290] Operating steps:

[0291] A. Using Torrent Suite TM Create a sequencing plan (PlannedRun) using the software

[0292] 1) Open the Torrent Suite software for accessing the server using a computer browser connected to the sequencing server TM software.

[0293] 2) Select the Plan tab and then click Templates.

[0294] 3) Select the application type (such as AmpliseqDNA) in the left navigation bar. The page will display all existing PlannedRuns according to the corresponding application.

[0295] Template list. Select a way to create a new sequencing plan from the following options:

[0296] · Create a sequencing plan without a template and directly click Plan New Run.

[0297] · Create a sequencing plan based on an existing template, click the (settings) button for the corresponding template and select the Plan Run option from the drop-down menu.

[0298] · Other ways, depending on the selected application type, there may be other options, such as downloading templates from the AmpliSeq.com website.

[0299] 4) In the next navigation page, select the corresponding options according to the information of this sequencing experiment, and then click Next to enter the next page.

[0300] 5) When all information options are completed, click Plan Run.

[0301] B. 18.2MΩ Water-Washed PGM Sequencer

[0302] 1) Empty the remaining liquid in the three washing bottles for cleaning (2 x 250 mL washing bottles, 1 x 2 L washing bottle), rinse each twice with 100 mL of 18MΩ water, click the Clean button on the PGM touch screen, then check the 18MΩ water cleaning option, and click "Next";

[0303] 2) Open the chip chamber at the top left front of the PGM sequencer, pull the red-headed handle to open the chip clamp. Do not wear gloves. Install a used and designated chip for water washing on the PGM chip clamp with bare hands for cleaning the PGM instrument. Pull the red-headed handle to cover the chip clamp, and finally cover the chip chamber cover;

[0304] 3) Remove all wash bottles and test tubes from the PGM instrument. Do not remove the sipper. Click "Next".

[0305] 4) Use an 18 MΩ pure water spray bottle to rinse the outer surface of the sipper at the W1 position of the PGM. Fill a rinsed 250 mL empty wash bottle for cleaning with 250 mL of 18 MΩ pure water, connect it to the W1 position of the PGM, confirm that the bottle mouth is tightened, and click "Next".

[0306] 5) Place the rinsed empty 2 L wash bottle at the W2 position; place the empty 250 mL wash bottle at the W3 position. Then insert the sipper into the bottle without tightening the bottle cap. Then place a waste liquid tray under the sipper corresponding to the dNTP reagent. Click "Next" to start the cleaning program.

[0307] 6) When the cleaning is completed, remove the wash bottles at the W1, W2, and W3 positions. Keep the sipper and the waste liquid tray in place. Click "Next" to return to the main page for subsequent initialization work.

[0308] C. Initialization of the PGM Sequencer

[0309] 1) Take out the dNTP stock solution from the refrigerator and place it on ice to thaw. Check the pressure of the nitrogen gas cylinder. When the pressure reading is lower than 500 psi, replace the gas cylinder. Prepare 1 M and 0.1 M NaOH solutions using 10 M NaOH solution and nuclease-free ultrapure water.

[0310] 2) Rinse the Wash 2 wash bottle (2 L) three times with 200 mL of 18 MΩ pure water, and add 18 MΩ water to the upper scale line of the wash bottle. At this time, the water volume is 2 L (for easy observation, the scale line can be marked with a marker pen).

[0311] Note: If the 18 MΩ pure water system is a faucet outlet, extend it into the Wash 2 wash bottle mouth but do not go deeper than the bottleneck. If it is other water outlet methods, try to make the nozzle close to the bottle mouth to avoid excessive CO2 dissolving in the ultrapure water and reducing its pH value.

[0312] 3) Add a whole bottle of Ion PGM TM Hi-Q TM View SequencingW2 solution. Add 70 μL - 90 μL of 100 mM NaOH to the Wash2 wash bottle with a pipette, tighten the bottle cap, and invert it 5 times to mix well for later use.

[0313] Note: Important! Do not leave the prepared and mixed Wash 2 solution for a long time.

[0314] 4) Rinse the Wash 1 and Wash 3 wash bottles three times with 50 mL of 18 MΩ pure water. Then add 350 μL of freshly prepared 100 mM NaOH and ultrapure water to the bottom of the W1 wash bottle, tighten the bottle cap for later use. Add 50 mL of IonPGM TM Hi-Q TM View SequencingW3 solution, tighten the bottle cap for later use;

[0315] 5) On the PGM main interface, click the "Initialize" button to enter the selection interface. Check IonPGM TM Hi-Q TM View SequencingKit in the drop-down menu, and click the "Next" button;

[0316] Note: On the same interface, if you often encounter pipeline blockages during the initialization experiment, you can select Lineclear to clean the possible blockages in the liquid pipeline before initialization. This step is an optional operation.

[0317] At this time, the system will detect whether the air pressure is sufficient. If the air pressure is insufficient, confirm whether the nitrogen tank gas pipe is connected to the PGM sequencer and whether the nitrogen tank is turned on. After confirmation, click "yes" to perform the air pressure detection again. If it is still low, contact the instrument administrator. If the air pressure is sufficient, proceed to the next step;

[0318] Replace with clean gloves, firmly insert a new long gray straw (do not let the new straw touch any surface) at the bottle cap interface at the W2 position. Immediately place the previously prepared Wash 2 wash bottle at the W2 position and tighten the bottle cap. Click "Next";

[0319] Replace the gloves, firmly install a new short gray straw (do not let the new straw touch any surface) at the bottle cap interfaces at the W1 and W3 positions respectively. Immediately install the previously prepared Wash 1 and Wash 3 wash bottles at the W1 and W3 positions and tighten the bottle caps. Click "Next";

[0320] After cleaning the pipeline (Line clear), or if you did not check this operation, the sequencer will start to adjust the pH value of the W2 solution. This step takes 30 minutes. After 15 minutes, check the running status displayed on the instrument screen to ensure that the initialization process is running normally.

[0321] D. Install the straws and reagent tubes

[0322] 1) Label four new 50-mL reagent tubes with the stickers provided in the kit as dGTP, dCTP, dATP, and dTTP respectively. Vortex each thawed dNTP stock solution on a shaker to mix each reagent, and then centrifuge to collect the liquid at the bottom of the tube (keep the dNTP stock solution on ice throughout this process);

[0323] 2) Using a filter pipette tip and new gloves, carefully add 20 μL of the corresponding dNTP stock solution to each reagent tube, tighten each reagent tube, and before loading onto the instrument, store the reagent tubes on ice. Return the remaining dNTP stock solution to -20 °C for storage;

[0324] 3) After the PGM sequencer is initialized, according to the on-screen prompts, remove the old pipette and move the waste liquid tank in the dNTP area, change into new gloves, and then firmly install a new pipette (blue) at each dNTP reagent interface;

[0325] Note: Do not let the pipette touch any surface to prevent contamination.

[0326] 4) Insert the prepared reagent tubes into the corresponding dNTP ports (for example, the dGTP tube corresponds to the port marked "G"), and tighten firmly. Click "Next";

[0327] 5) Complete the initialization according to the on-screen prompts. The instrument will inject 40 mL of W2 solution into each reagent tube. Finally, the Ion PGM TM system will measure the pH value of all reagents. If each reagent has the expected pH value, the interface will display a green Passed screen. Click "Next" to end the initialization and return to the main interface.

[0328] E. Sequencing primer annealing:

[0329] 1) Vortex the previously obtained ISPs repeatedly with a pipette to mix well. Insert the PCR tubes into the matching centrifuge tube sleeves, place them in a centrifuge, and centrifuge at 15500 × g for 2 min;

[0330] Note: When placing in the centrifuge, pay attention to folding the lid of the PCR tube outwards to ensure the position of the precipitate can be determined after centrifugation.

[0331] 2) Press the piston of the pipette, insert the pipette tip below the liquid level in the PCR tube, and carefully aspirate the supernatant, taking care not to touch the side wall with the pink ISP precipitate (i.e., the side of the folded lid). Discard the supernatant. At this time, 15 μL of liquid should remain in the PCR tube (the remaining liquid volume can be determined by comparing with other PCR tubes containing 15 μL of liquid);

[0332] 3) Confirm that the sequencing primer is completely thawed before use (no ice crystals can be seen). After shaking the primer for 5 s, centrifuge it instantaneously for 3 - 5 s to collect the liquid and place it on ice for later use;

[0333] 4) Add 12 μL of the sequencing primer to the ISP. Confirm that the final volume of the liquid is 27 μL (if necessary, add annealing buffer to make up the volume). Pipette up and down repeatedly to mix well and resuspend the precipitate thoroughly;

[0334] 5) Open the lid of the PCR instrument, place the PCR tube on the PCR instrument, close the lid of the PCR instrument tightly, and set the following program on the PCR instrument: 95 °C for 2 min, then 37 °C for 2 min, hold at 25 °C, use the hot lid option, and run the program;

[0335] Note: At this time, the sequencing instrument can be set up.

[0336] F. Run the chip detection program

[0337] On the PGM main interface, click Run. Empty the PGM waste liquid bottle and then click "Next" to continue. When the screen prompts to insert the cleaning chip, continue to use the chip used in the previous initialization, click "Next" to start the cleaning liquid flow pipeline;

[0338] Follow the prompts and select the instrument you used to prepare the template-containing ISPs on the screen. Then click "Next";

[0339] Take off the gloves and remove static electricity by touching the grounding touchpad on the instrument. Take out a new chip from the package, mark the chip according to the experimental information of this time and keep the packaging bag. Click "Next", take out the old chip from the chip slot, put in the new one, close the chip clamp, and then click "Next";

[0340] Click Chip Check. At the beginning of Chip Check, visually check whether there is any liquid leakage in the chip clamp in the chip. After the chip check is successful, empty the waste liquid bottle, then check the Waste bottle is empty option on the screen, and then click "Next".

[0341] G: Bind the sequencing polymerase to the ISPs (library preparation and detection area):

[0342] Take out the Ion PGM TM Hi-Q TM View Sequencing Polymerase from the refrigerator, flick it gently with your fingertips four times, then centrifuge it instantaneously for 3 - 5 s, and place it on ice for later use;

[0343] After the sequencing primer annealing program is completed, take out the ISPs from the thermal cycler, and then add 3 μL of Ion PGM TM Hi-Q TM View Sequencing Polymerase to a final volume of 30 μL. Mix the sample by pipetting up and down repeatedly and incubate at room temperature for 5 min.

[0344] H: Chip Preparation and Loading

[0345] After the chip detection and calibration are completed, take out the new chip from the Ion PGM TM and perform the loading operation (during the chip loading process, an old chip needs to be placed in the chip slot of the PGM);

[0346] 1) Tilt the chip at a 45-degree angle so that the loading hole (the larger hole on the chip) of the chip is at the bottom. Firmly insert the pipette tip into the loading hole and aspirate and discard the residual liquid in the loading hole as much as possible;

[0347] 2) Place the chip face down on the chip holder of the chip centrifuge. When the holder is placed back into the centrifuge, make the semi-circular protruding part on the side of the chip face inward (i.e., towards the axis of the centrifuge). Place another chip for balancing in the other holder and centrifuge for 5 s to fully empty the chip;

[0348] 3) Remove the chip from the holder and dry the holder with lint-free paper. Place the chip face up and put it back into the holder, then place the holder on a firm and level surface for later use;

[0349] 4) After the polymerase incubation is completed, according to the type of sequencing chip in Table 16, use a pipette tip to aspirate the corresponding volume of ISPs.

[0350] Table 16 Loading Volumes Corresponding to Sequencing Chip Types

[0351] Chip Type Loading Volume Recommended Pipette Tip Ion 316 or Ion 318 Total Volume (30 μL) <![CDATA[Rainin TM Pipet-Lite TM LTSL-100XLS, 10 - 100 μL]]> Ion 314 20 μL <![CDATA[Rainin TM Pipet-Lite TM LTSL-20XLS, 2 - 20 μL]]>

[0352] 5) Insert the pipette tip vertically and firmly into the loading hole of the chip, unlock the pipette, and slowly rotate the loading range of the pipette to dispense the ISPs liquid in the pipette tip drop by drop (rate 1 μL / s). To avoid generating bubbles, a small amount of residual liquid (0.5 μL) should be retained in the pipette tip, and aspirate the liquid discharged from the other hole of the chip;

[0353] 6) Transfer the chip to the chip holder of the centrifuge, with the semi-circular protrusion facing inward (towards the axis of the centrifuge), centrifuge for 30 s, then change the position of the chip in the holder so that the semi-circular protrusion faces outward (opposite to the axis) and centrifuge for 30 s;

[0354] 7) Take out the holder from the centrifuge and place it on a horizontal surface. Set the range of the pipette according to the chip type below:

[0355] Ion 316 TM or Ion 318 TM Chip: 25 μL;

[0356] Ion 314 TM Chip: 5 μL.

[0357] 8) Tilt the chip by 45 degrees so that the sample loading hole is at the bottom. Insert the pipette tip into the sample loading hole and slowly aspirate and dispense the liquid in the chip once. Do not pull out the pipette tip from the hole during this process and keep the liquid surface continuous. Aspirate and dispense slowly to avoid generating bubbles. By rotating the sample loading range of the pipettor, slowly aspirate and discard as much residual liquid as possible from the chip;

[0358] 9) Place the chip face down in the centrifuge and centrifuge it upside down for 5 s. Aspirate the residual liquid. If there is still residual liquid in the chip, gently and quickly tap the protrusion on the side of the chip on the desktop several times, and then use the pipettor to remove all the collected liquid. Do not rinse the chip. After the chip sample loading is completed, click Next on the screen and immediately proceed with the subsequent sequencing operation.

[0359] I: Select the created sequencing program and run the sequencing experiment

[0360] 1) Click Browse next to the PlannedRun area, select the previously created program plan name, click "Next", and confirm that the read program is correct. If necessary, you can modify it on the touch screen.

[0361] Note: If you are using a chip with a QR code and have also entered the chip QR code information when creating the program, the Ion PGM TM sequencer will automatically call up the program by scanning the QR code.

[0362] 2) After completing the creation of the program input, click "Next" to verify whether the experimental parameters are correct. If correct, click "OK"; if incorrect, select "Cancel" to return to the touch screen page to reselect;

[0363] 3) According to the screen prompt, place the chip with the sample loaded back into the chip slot of the PGM, and then click "Next". At the beginning of ChipCheck, visually check whether there is any liquid leakage in the chip held by the chip clamp. If not, you can close the lid. The machine will flush out the loose ISPs and then start chip calibration;

[0364] 4) When the chip calibration is completed (1 min), the touch screen will display whether the calibration is successful. After 60 s, the machine will automatically start sequencing; you can also click Next to immediately enter the sequencing process;

[0365] 5) When the sequencing is completed, leave the chip in place and return to the main interface by clicking Next. At this time, the chip can be removed, and then the second sequencing reaction can be continued; or the cleaning or re-initialization process can be carried out according to the situation.

[0366] 11. Data Import into the Database

[0367] Program: Torrent Suite TM Data Analysis System (version ≥ 5.12).

[0368] Equipment: Ion Torrent Server Cluster (Configuration: 64-core CPU / 128GB RAM / 10TB storage space), Clinical Data Analysis Terminal

[0369] Operations:

[0370] 11.1 After the sequencing is completed, the system automatically generates.bam / .vcf format files and transfers them to the clinical database through the encrypted intranet;

[0371] 11.2 Adopt a two-step verification mechanism (MD5 verification + sample ID check) to ensure data integrity;

[0372] 11.3 Call the built-in HG38 reference genome for sequence alignment and use the Torrent Variant Caller plugin (Parameter settings: min-allele-freq = 0.01, min-quality-score = 20) for variant detection;

[0373] 11.4 Automatically associate the genotype data of 89 target SNP sites with the clinical annotation database (including dbSNP / OMIM / PharmGKB data);

[0374] 11.5 Generate a three-level quality control report (including: sequencing depth ≥ 100×, Q20 ≥ 90%, sample cross-contamination rate < 0.1%);

[0375] 12. Export of Detection Results

[0376] 12.1 The system automatically generates a structured report, including:

[0377] Core data area: Genotypes of 89 SNP sites and clinical significance grading (classified as pathogenic / likely pathogenic / uncertain significance / possibly benign / benign according to the ACMG standard);

[0378] Risk warning area: Overall disease risk level (low / medium / high) calculated based on polygenic risk score (PRS);

[0379] Medication guidance area: Medication suggestions for drug-related SNPs;

[0380] In the said core data area, according to the genotype determination results, the present invention generates a diagnostic conclusion by combining the ACMG variant classification criteria, polygenic risk score (PRS) and clinical guidelines. The diagnostic criteria are shown in Table 17 below.

[0381] Table 17 Diagnostic Criteria for SNP Molecular Markers

[0382]

[0383]

[0384] Implementation requirements: Each SNP molecular marker requires at least two independent evidences (such as population frequency + prediction tool + literature) to support the classification.

[0385] In the said risk warning area, the overall risk stratification based on PRS in the present invention needs to combine the population percentile and clinical threshold. The diagnostic criteria for polygenic risk score (PRS) are shown in Table 18.

[0386] Table 18 Polygenic Risk Score (PRS)

[0387]

[0388] Key parameters:

[0389] The PRS model needs to be trained based on a large sample cohort (such as UK Biobank) and validated in the target population (such as East Asian population).

[0390] The threshold should be dynamically adjusted according to the disease incidence (for example, the high-risk threshold for allergic rhinitis may be lower than that for cancer).

[0391] In the said risk warning area, based on the recommendations of Pharmacogenomics (PharmGKB) and combined with the CPIC / DPWG guidelines, the present invention gives medication recommendations for related SNPs, as shown in Table 19.

[0392] Table 19 Medication Recommendation Criteria for SNPs

[0393] Genotype Result Drug Metabolism Type Medication Recommendation CYP2D6 4 / 4 (Homozygous Mutation) Poor Metabolizer (PM) "Avoid using codeine. Recommended alternative analgesics (e.g., paracetamol)" HLA-B*57:01 Positive High Risk of Hypersensitivity Reaction "Abacavir is contraindicated. Alternative regimen: tenofovir + lamivudine" TPMT Intermediate Metabolizer Risk of Azathioprine Toxicity "Reduce the dose by 50% and monitor blood routine"

[0394] Implementation requirements: Priority should be given to citing the recommendations of PharmGKB level 1A / 1B evidence; label the drug-gene interaction level (such as "strong recommendation", "use with caution").

[0395] The present invention establishes a comprehensive diagnostic process, including data integration, conflict handling, and report generation; the data integration includes associating ACMG classification, PRS risk, and pharmacogenomic results to the same patient ID; the conflict handling includes rechecking the sample quality or model applicability if a single gene is pathogenic but the PRS risk is low; the report generation includes structured output, examples:

[0396] ## Diagnostic conclusion

[0397] - **Core variant**: rs3014837 (S100A7, Pathogenic) is determined to be "consistent with the genetic susceptibility of allergic rhinitis"

[0398] - **PRS risk**: 92th percentile (High) is determined to be "vigilant against severe rhinitis attacks"

[0399] - **Medication warning**: CYP3A5*3 / *3 is determined to be "delayed glucocorticoid metabolism, it is recommended to start with a low dose".

[0400] 12.2 Supports two export formats:

[0401] ① PDF format (complies with the CLIA-certified clinical report template, including electronic signature and watermark);

[0402] ② JSON format (for scientific research data analysis).

[0403] 13. Quality control: The requirements for quality control include confirmation of negative results. For samples with no detected pathogenic variants, the coverage needs to be verified (e.g., the target region ≥ 100x).

[0404] The present invention can achieve standardized, traceable, and automated analysis from genotype to diagnostic conclusion through SNP molecular marker diagnostic criteria, polygenic risk score (PRS), comprehensive diagnostic process, and quality control.

[0405] 14. Test results

[0406] The kit of the present invention is based on multiplex PCR targeted sequencing technology and combines with the Ion Torrent PGM platform, showing excellent detection performance in the genetic risk screening of allergic rhinitis. Relevant experimental and clinical data can fully support its technical characteristics of "high detection throughput, high sensitivity, strong specificity, more comprehensive results, high accuracy and repeatability". The specific results are as follows:

[0407] 1) High-throughput detection ability

[0408] This kit can simultaneously detect 89 SNP loci related to allergic rhinitis, covering key immune and inflammatory pathway genes such as IL-4R, IL-18, ADAM33, RORA, and S100A7. It can process 48 - 96 samples in a single run (depending on the chip model), and under the Ion Torrent PGM system, the detection efficiency is more than 50 times higher than that of Sanger sequencing.

[0409] 2) High sensitivity and specificity

[0410] The minimum detection limit (LoD) is as low as 1% minor allele frequency (MAF), suitable for the identification of low-frequency pathogenic variants; verified in 1000 clinical samples, the comparison result with whole-genome sequencing (WGS) shows: specificity: 99.8%.

[0411] 3) Accuracy and repeatability

[0412] Internal quality control shows: within-batch coefficient of variation (CV) < 2%; between-batch coefficient of variation < 5%, meeting the international laboratory certification standards of CAP / CLIA; in a prospective cohort study of 1000 people in a provincial children's hospital: the positive predictive value (PPV) for high-risk populations of allergic rhinitis is 92.3% (95% CI: 89.7 - 94.5).

[0413] 4) Clinical value for specific populations

[0414] Population with family genetic history: Those carrying ≥2 pathogenic / possibly pathogenic mutations (meeting the ACMG classification criteria) have a 4.7-fold higher risk of developing the disease within 5 years compared to negative individuals (HR = 4.7);

[0415] Newborn prediction ability: Screening through maternal peripheral blood or infant umbilical cord blood, constructing a risk prediction model, the accuracy of predicting allergic rhinitis in infants before the age of 3 is as follows: AUC = 0.81; sensitivity = 82%.

[0416] 5. Examples of clinical test data (excerpt), see Table 20.

[0417] Table 20 Examples of clinical test data

[0418] Chromosome Position Gene Symbol Genotype Variant ID Variant Frequency Position / Function Population Frequency (MAF) Remarks chr1 153,458,930 S100A7 G / G rs3014837 100％ Exon Region (Missense Mutation) 0.02 Verified chr15 60,528,610 RORA G / G rs1898413 99.70％ Intron Region 0.48 Verified chr16 27,399,508 NA A / G rs2107357 48.30％ Unknown Region 0.19 Verified

[0419] These data support that the kit has good coverage and variant identification ability in different clinical significances of variants, including types (heterozygous / homozygous, exonic / intronic regions, etc.).

[0420] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all of them. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A SNP molecular marker combination related to allergic rhinitis, characterized in that, Including 89 SNP molecular markers; the rs numbers of the 89 SNP molecular markers in the SNP database are respectively: rs2302009, rs505010, rs1441586, rs512555, rs4982958, rs1998359, rs2107357, rs10493377, rs3014837, rs4129267, rs2228145, rs1269486, rs1058240, rs379568, rs2060793, rs10767664, rs569108, rs7130588, rs2155219, rs1946518, rs1946519, rs3794262, rs4251481, rs9729, rs731236, rs1544410, rs2228570, rs2069718, rs1898413, rs2041733, rs2057768, rs1805010, rs1805011, rs1805015, rs1801275, rs13527, rs2107538, rs9303277, rs12150079, rs2305480, rs7216389, rs7224129, rs4065275, rs8076131, rs12603332, rs17608925, rs3744246, rs4794820, rs77485247, rs77041280, rs393581, rs428253, rs4740, rs353702, rs438421, rs10407799, rs395969, rs390406, rs375688, rs412211, rs413216, rs420297, rs2108686, rs12461895, rs2241717, rs7258445, rs231735, rs231804, rs2787093, rs2787094, rs677044, rs3918400, rs628977, rs630712, rs2280089, rs2280090, rs2280091, rs44707, rs2853209, rs528557, rs2280092, rs2485700, rs511898, rs3918392, rs2787095, rs6084435, rs6115989, rs6107332, rs11697406.

2. Use of the SNP molecular marker combination according to claim 1 as a detection target in the preparation of a product for diagnosing or assisting in the diagnosis of allergic rhinitis.

3. The application according to claim 2, characterized in that The detection means for the diagnosis or auxiliary diagnosis includes multiplex PCR.

4. A primer set for detecting the SNP molecular marker combination according to claim 1, characterized in that, It includes 78 primer pairs; the nucleotide sequences of the 78 primer pairs are shown as SEQ ID NO: 1 to SEQ ID NO:

156.

5. Use of the primer set according to claim 4 in the preparation of a product for diagnosing or assisting in the diagnosis of allergic rhinitis.

6. The application according to claim 5, characterized in that, The detection means for diagnosis or assisting in the diagnosis includes multiplex PCR.

7. A kit for the diagnosis or auxiliary diagnosis of allergic rhinitis, characterized in that, It includes the primer set according to claim 4.

8. The kit according to claim 7, wherein The kit further includes a positive control and a negative control; the positive control includes a positive control sequence with a nucleotide sequence shown as SEQ ID NO: 157; the negative control includes a negative control sequence with a nucleotide sequence shown as SEQ ID NO:

158.

9. A gene chip, characterized in that, The gene chip is prepared based on the SNP molecular marker combination according to claim 1.

10. A system for the diagnosis or auxiliary diagnosis of allergic rhinitis, characterized in that, It includes: A PCR amplification module, a sequencing module, a data import module, a data analysis module, and a data output module; The PCR amplification module includes the primer set according to claim 4 or the kit according to claim 7; The sequencing module is used to sequence the amplification product of the PCR amplification module; The data import module is used to import sequencing data; The data analysis module is used to analyze the sequencing data; The data output module is used to output the diagnosis or auxiliary diagnosis result.

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