CN110577993A

CN110577993A - Primer group, probe group, kit and application for detecting allergy-related gene mutation

Info

Publication number: CN110577993A
Application number: CN201910940379.7A
Authority: CN
Inventors: 张惠丹; 戴敬; 赵捷; 赵洪玉
Original assignee: Suzhou Qiankang Gene Co Ltd
Current assignee: Suzhou Qiankang Gene Co Ltd
Priority date: 2019-09-30
Filing date: 2019-09-30
Publication date: 2019-12-17

Abstract

The invention discloses a primer group, a probe group, a kit and application for detecting allergy-related gene mutation. The primer group of the invention comprises primer sequences respectively shown as SEQ ID NO. 1-60. The probe sequences of the probe set are respectively shown in SEQ ID NO. 61-90. The invention provides a set of amplification primers, a probe and a kit which can detect 25 genes related to allergy, and a pair of upstream and downstream primers are designed for gene amplification aiming at each related gene; aiming at each detection site, designing a corresponding probe for single base amplification; the invention adopts a multiple PCR nucleic acid mass spectrum flight technology to quickly, accurately and sensitively detect the risk sites of 25 genes related to allergy, can detect the polymorphism of 30 SNP sites of 25 genes related to allergy at one time, quickly, efficiently, accurately and high cost performance, and achieves the effect of allergy risk reminding.

Description

primer group, probe group, kit and application for detecting allergy-related gene mutation

Technical Field

the invention relates to the technical field of molecular biology, in particular to a primer group, a probe group and a kit for detecting 25 gene mutations related to allergy, which are used for a method for performing gene detection by a multiple PCR flight nucleic acid mass spectrometry technology.

Background

allergy is a common autoimmune disease and has a great influence on the daily life of people. For patients allergic to pollen, the allergic symptoms are affected in the spring and autumn every year, and the symptoms such as red and swollen skin, pruritus, cough and sneeze are shown. Food allergy is very easy to cause serious anaphylactic reaction, and threatens the life safety of patients, so food allergy patients need to pay special attention to diet in daily life. For infants, both food allergy (peanuts, milk, wheat gluten, fish and shrimp, peaches, nut food, etc.) and environmental allergy (pollen, dust mites, natural latex, pet hair, etc.) seriously threaten the health and development of children. In addition, allergy also has occupational tendency, for example, medical staff is easy to be allergic to natural latex, and the health of the medical staff is seriously influenced while the work of the medical staff is interfered.

Clinically, most common allergen detection methods are skin tests, and partial allergens can be used for detecting the content of IgE in blood. However, these tests are usually carried out after the patient has developed a severe allergic reaction, which seriously affects the life health of the person. Especially, in infants who have difficulty in clearly expressing their own symptoms, allergy seriously impairs the health and development of children. However, in recent years, with the completion of the human genome project and the widespread use of second-generation sequencing, scientists have been able to use large samples for META analysis to investigate the relationship between allergy expression and genetic information polymorphism, making it possible to predict allergy risk in advance.

the flight technology of the multiple PCR nucleic acid mass spectrometry is accurate and rapid in detection, high in cost performance under the condition of detecting the same quantity of SNP, and more suitable for comprehensive allergy risk screening. The technique comprises the following advantages: all experimental operations are completed in one EP tube, so that the possibility of sample pollution is greatly reduced; can carry out up to 40 times of PCR reaction and genotype detection; means such as fluorescence labeling and gel electrophoresis are not involved; the genotype of the target fragment is detected according to the difference of the motion tracks of the charge-to-mass ratio of the amplification product in an electric field, and the detection sensitivity is high and quick.

however, this method has high requirements for primer specificity and primer combination. On the basis of ensuring that a single tube carries out the most multiple reactions, 3 primers with the total base number not more than 80bp are reasonably designed for each SNP locus, so that the primers with lower specificity are prevented from generating non-target products and cross amplification, the primers are prevented from forming hairpin structures and the like, and the difficulty in carrying out the multi-locus polymorphism detection is solved.

disclosure of Invention

The main purpose of the present invention is to provide a primer set and a probe set for detecting allergy-related gene mutation, so as to overcome the defects in the prior art.

Another main object of the present invention is to provide a kit for detecting allergy-associated gene mutation.

the invention also aims to provide the application of the kit in detecting the allergy-related gene mutation.

another object of the present invention is to provide a method for detecting allergy-associated gene mutation.

in order to achieve the purpose, the technical scheme adopted by the invention comprises the following steps:

the embodiment of the invention provides a primer group for detecting allergy-related gene mutation, wherein the allergy-related genes respectively comprise ADRB2, BDNF, CRHR1, CRHR2, FCER1A, FLG-AS1, GSDMB, HLA-DQB1, IL1R1, IL10, IL13, IL18, IL18R1, IL33, LRRC3C, MS4A2, PTGDR, RAD50, RTEL1, SERPINB7, STAT6, TMEM232, TNF and TSLP, and the primer group comprises:

The SNP locus of the ADRB2 gene is rs1042713, the primer pair comprises a first primer and a second primer, the sequence of the first primer is shown as SEQ ID NO.1, and the sequence of the second primer is shown as SEQ ID NO. 2;

The SNP locus of the BDNF gene is rs10767664, the primer pair of the BDNF gene comprises a third primer and a fourth primer, the sequence of the third primer is shown as SEQ ID NO.3, and the sequence of the fourth primer is shown as SEQ ID NO. 4;

the SNP locus of the CRHR1 gene is rs1876828, the primer pair comprises a fifth primer and a sixth primer, the sequence of the fifth primer is shown as SEQ ID NO.5, and the sequence of the sixth primer is shown as SEQ ID NO. 6;

The SNP locus of the CRHR2 gene is rs7793837, the primer pair comprises a seventh primer and an eighth primer, the sequence of the seventh primer is shown as SEQ ID NO.7, and the sequence of the eighth primer is shown as SEQ ID NO. 8;

the SNP loci of the FCER1A gene are rs7793837, rs2251746 and rs2427827, wherein a primer pair corresponding to rs7793837 comprises a ninth primer and a tenth primer, the sequence of the ninth primer is shown as SEQ ID No.9, and the sequence of the tenth primer is shown as SEQ ID No. 10; the primer pair corresponding to rs2251746 comprises an eleventh primer and a twelfth primer, wherein the sequence of the eleventh primer is shown as SEQ ID NO.11, and the sequence of the twelfth primer is shown as SEQ ID NO. 12; the primer pair corresponding to rs2427827 comprises a thirteenth primer and a fourteenth primer, the sequence of the thirteenth primer is shown as SEQ ID NO.13, and the sequence of the fourteenth primer is shown as SEQ ID NO. 14;

The SNP locus of the FLG gene is rs61816761, the primer pair comprises a fifteenth primer and a sixteenth primer, the sequence of the fifteenth primer is shown as SEQ ID NO.15, and the sequence of the sixteenth primer is shown as SEQ ID NO. 16;

the SNP locus of the FLG-AS1 gene is rs3126085, the primer pair comprises a seventeenth primer and an eighteenth primer, the sequence of the seventeenth primer is shown AS SEQ ID NO.17, and the sequence of the eighteenth primer is shown AS SEQ ID NO. 18;

the SNP locus of the GSDMB gene is rs2305480, the primer pair comprises a nineteenth primer and a twentieth primer, the sequence of the nineteenth primer is shown as SEQ ID NO.19, and the sequence of the twentieth primer is shown as SEQ ID NO. 20;

The SNP locus of the HLA-DQB1 gene is rs1063355, the primer pair comprises a twenty-first primer and a twenty-second primer, the sequence of the twenty-first primer is shown as SEQ ID NO.21, and the sequence of the twenty-second primer is shown as SEQ ID NO. 22;

The SNP locus of the IL1R1 gene is rs1558641, the primer pair comprises a twenty-third primer and a twenty-fourth primer, the sequence of the twenty-third primer is shown in SEQ ID NO.23, and the sequence of the twenty-fourth primer is shown in SEQ ID NO. 24;

the SNP locus of the IL10 gene is rs1800871, the primer pair comprises a twenty-fifth primer and a twenty-sixth primer, the sequence of the twenty-fifth primer is shown as SEQ ID NO.25, and the sequence of the twenty-sixth primer is shown as SEQ ID NO. 26;

The SNP loci of the IL13 gene are rs1800925 and rs20541, wherein a primer pair corresponding to rs1800871 comprises a twenty-seventh primer and a twenty-eighth primer, the sequence of the twenty-seventh primer is shown as SEQ ID NO.27, and the sequence of the twenty-eighth primer is shown as SEQ ID NO. 28; the primer pair corresponding to rs20541 comprises a twenty-ninth primer and a thirtieth primer, the sequence of the twenty-ninth primer is shown as SEQ ID NO.29, and the sequence of the thirtieth primer is shown as SEQ ID NO. 30;

The SNP loci of the IL18 gene are rs1946518 and rs187238, wherein a primer pair corresponding to rs1946518 comprises a thirty-first primer and a thirty-second primer, the sequence of the thirty-first primer is shown as SEQ ID NO.31, and the sequence of the thirty-second primer is shown as SEQ ID NO. 32; the primer pair corresponding to rs187238 comprises a thirty-third primer and a thirty-fourth primer, and the sequence of the thirty-third primer is shown as SEQ ID NO.33, and the sequence of the thirty-fourth primer is shown as SEQ ID NO. 34;

The SNP locus of the IL18R1 gene is rs13015714, the primer pair comprises a thirty-fifth primer and a thirty-sixth primer, the sequence of the thirty-fifth primer is shown in SEQ ID NO.35, and the sequence of the thirty-sixth primer is shown in SEQ ID NO. 36;

the SNP locus of the IL33 gene is rs928413, the primer pair comprises a thirty-seventh primer and a thirty-eighth primer, the sequence of the thirty-seventh primer is shown as SEQ ID NO.37, and the sequence of the thirty-eighth primer is shown as SEQ ID NO. 38;

the SNP locus of the LRRC3C gene is rs4794820, the primer pair comprises a thirty-ninth primer and a forty-fourth primer, the sequence of the thirty-ninth primer is shown as SEQ ID NO.39, and the sequence of the forty-fourth primer is shown as SEQ ID NO. 40;

the SNP locus of the MS4A2 gene is rs569108, the primer pair comprises a fortieth primer and a fortieth primer, the sequence of the fortieth primer is shown as SEQ ID NO.41, and the sequence of the fortieth primer is shown as SEQ ID NO. 42;

The SNP locus of the PTGDR gene is rs8004654, the primer pair comprises a forty-third primer and a forty-fourth primer, the sequence of the forty-third primer is shown as SEQ ID NO.43, and the sequence of the forty-fourth primer is shown as SEQ ID NO. 44;

the SNP locus of the RAD50 gene is rs6871536, the primer pair comprises a forty-fifth primer and a forty-sixth primer, the sequence of the forty-fifth primer is shown as SEQ ID NO.45, and the sequence of the forty-sixth primer is shown as SEQ ID NO. 46;

the SNP locus of the RTEL1 gene is rs6010620, the primer pair comprises a forty-seventh primer and a forty-eighth primer, the sequence of the forty-seventh primer is shown as SEQ ID NO.47, and the sequence of the forty-eighth primer is shown as SEQ ID NO. 48;

The SNP locus of the SERPINB7 gene is rs12964116, the primer pair comprises a forty-ninth primer and a fifty-fifth primer, the sequence of the forty-ninth primer is shown as SEQ ID NO.49, and the sequence of the fifty-fifth primer is shown as SEQ ID NO. 50;

the SNP loci of the STAT6 gene are rs1059513 and rs324015, wherein a primer pair corresponding to rs1059513 comprises a fifty-first primer and a fifty-second primer, the sequence of the fifty-first primer is shown as SEQ ID NO.51, and the sequence of the fifty-second primer is shown as SEQ ID NO. 52; the primer pair corresponding to rs324015 comprises a fifty-third primer and a fifty-fourth primer, the sequence of the fifty-third primer is shown as SEQ ID NO.53, and the sequence of the fifty-fourth primer is shown as SEQ ID NO. 54;

the SNP locus of the TMEM232 gene is rs7701890, the primer pair comprises a fifty-fifth primer and a fifty-sixth primer, the sequence of the fifty-fifth primer is shown as SEQ ID NO.55, and the sequence of the fifty-sixth primer is shown as SEQ ID NO. 56;

The SNP locus of the TNF gene is rs1800630, the primer pair comprises a fifty-seventh primer and a fifty-eighth primer, the sequence of the fifty-seventh primer is shown as SEQ ID NO.57, and the sequence of the fifty-eighth primer is shown as SEQ ID NO. 58;

The SNP locus of the TSLP gene is rs1898671, the primer pair comprises a fifty-ninth primer and a sixty primer, the sequence of the fifty-ninth primer is shown as SEQ ID NO.59, and the sequence of the sixty primer is shown as SEQ ID NO. 60.

The embodiment of the invention also provides a probe set for detecting the mutation of an allergy-related gene, wherein the allergy-related gene respectively comprises ADRB2, BDNF, CRHR1, CRHR2, FCER1A, FLG-AS1, GSDMB, HLA-DQB1, IL1R1, IL10, IL13, IL18, IL18R1, IL33, LRRC3C, MS4A2, PTGDR, RAD50, RTEL1, SERPINB7, STAT6, TMEM232, TNF and TSLP, and the probe set comprises:

A first probe for detecting SNP locus rs1042713 of ADRB2 gene, the sequence of which is shown in SEQ ID NO. 61;

a second probe for detecting SNP locus rs10767664 of the BDNF gene, the sequence of which is shown in SEQ ID NO. 62;

a third probe for detecting the SNP locus rs1876828 of the CRHR1 gene, wherein the sequence of the third probe is shown as SEQ ID NO. 63;

a fourth probe for detecting SNP locus rs7793837 of CRHR2 gene, the sequence of which is shown in SEQ ID NO. 64;

Detecting a fifth probe of the SNP locus rs2427837, a sixth probe of the SNP locus rs2251746 and a seventh probe of the SNP locus rs2427827 of the FCER1A gene, wherein the sequences of the probes are respectively shown as SEQ ID NO.65, SEQ ID NO.66 and SEQ ID NO. 67;

an eighth probe for detecting the SNP locus rs61816761 of the FLG gene, wherein the sequence of the eighth probe is shown as SEQ ID NO. 68;

A ninth probe for detecting SNP locus rs3126085 of FLG-AS1 gene, the sequence of which is shown in SEQ ID NO. 69;

a tenth probe for detecting SNP locus rs2305480 of the GSDMB gene, the sequence of which is shown as SEQ ID NO. 70;

an eleventh probe for detecting the SNP locus rs1063355 of the HLA-DQB1 gene, wherein the sequence of the eleventh probe is shown as SEQ ID NO. 71;

A twelfth probe for detecting the SNP locus rs1558641 of the IL1R1 gene, wherein the sequence of the twelfth probe is shown as SEQ ID NO. 72;

a thirteenth probe for detecting the SNP locus rs1800871 of the IL10 gene, wherein the sequence of the thirteenth probe is shown as SEQ ID NO. 73;

detecting a fourteenth probe of an SNP locus rs1800925 and a fifteenth probe of an SNP locus rs20541 of the IL13 gene, wherein the sequences of the fourteenth probe and the fifteenth probe are respectively shown as SEQ ID NO.74 and SEQ ID NO. 75;

a sixteenth probe for detecting the SNP locus rs1946518 and a seventeenth probe for detecting the SNP locus rs187238 of the IL18 gene, wherein the sequences of the sixteenth probe and the seventeenth probe are respectively shown as SEQ ID NO.76 and SEQ ID NO. 77;

an eighteenth probe for detecting the SNP locus rs13015714 of the IL18R1 gene, the sequence of which is shown in SEQ ID NO. 78;

A nineteenth probe for detecting the SNP locus rs928413 of the IL33 gene, the sequence of which is shown as SEQ ID NO. 79;

a twentieth probe for detecting the SNP locus rs4794820 of the LRRC3C gene, wherein the sequence of the twentieth probe is shown as SEQ ID NO. 80;

a twenty-first probe for detecting SNP locus rs569108 of MS4A2 gene, the sequence of which is shown as SEQ ID NO. 81;

a twenty-second probe for detecting the SNP locus rs8004654 of the PTGDR gene, wherein the sequence of the twenty-second probe is shown as SEQ ID NO. 82;

A twenty-third probe for detecting the SNP locus rs6871536 of the RAD50 gene, wherein the sequence of the twenty-third probe is shown as SEQ ID NO. 83;

a twenty-fourth probe for detecting the SNP locus rs6010620 of the RTEL1 gene, wherein the sequence of the twenty-fourth probe is shown as SEQ ID NO. 84;

A twenty-fifth probe for detecting the SNP locus rs12964116 of the SERPINB7 gene, wherein the sequence of the twenty-fifth probe is shown as SEQ ID NO. 85;

the twenty-sixth probe for detecting the SNP locus rs1059513 and the twenty-seventh probe for detecting the SNP locus rs324015 of the STAT6 gene have sequences respectively shown as SEQ ID NO.86 and SEQ ID NO. 87;

A twenty-eighth probe for detecting SNP locus rs7701890 of the TMEM232 gene, wherein the sequence of the twenty-eighth probe is shown as SEQ ID NO. 88;

A twenty-ninth probe for detecting SNP locus rs1800630 of TNF gene, the sequence of which is shown in SEQ ID NO. 89;

The thirtieth probe for detecting the SNP locus rs1898671 of the TSLP gene has a sequence shown in SEQ ID NO. 90.

the embodiment of the invention also provides a kit for detecting allergy-related gene mutation, which comprises at least one primer group and at least one probe group, wherein one primer group is the primer group, and one probe group is the probe group.

the embodiment of the invention also provides application of the kit in preparation of products for detecting allergy-related gene mutation.

Further, the method for detecting the allergy-associated gene mutation by using the product comprises the following steps:

Providing a DNA sample to be detected;

mixing the DNA sample to be detected with a conventional component of the PCR amplification detection of the kit to form a mixed solution;

performing PCR amplification on the mixed solution by using a primer group and a probe group contained in the kit by adopting a multiple PCR nucleic acid mass spectrometry flight technology;

And detecting the PCR amplification product, and judging whether the DNA sample contains the mutated allergy-related gene.

The embodiment of the invention also provides a product containing the kit, which is applied to a method for detecting allergy-related gene mutation, and the method comprises the following steps:

Providing a DNA sample to be detected;

performing PCR amplification on the mixed solution by using a primer group and a probe group contained in the kit by adopting a multiplex PCR technology;

And (3) separating the probe extension products based on a flight mass spectrum platform, and analyzing whether the DNA sample contains the mutated allergy-related gene.

Compared with the prior art, the invention has the advantages that:

1) The invention provides a set of amplification primers, probes and a kit capable of detecting 25 genes related to allergy, improper SNP is screened out, SNP sites in strong linkage disequilibrium with the screened-out sites are introduced, design and experimental verification are carried out again until proper primers and combinations thereof are optimized, and a pair of upstream and downstream primers are designed for gene amplification aiming at each related gene; aiming at each detection site, designing a corresponding probe for single base amplification;

2) the invention adopts a multiple PCR nucleic acid mass spectrum flight technology, rapidly, accurately and sensitively detects the risk sites of 25 genes related to allergy, can detect the polymorphism of 30 SNP sites of 25 genes related to allergy, which is supported by literature or a database, at one time, rapidly, efficiently, accurately and at high cost performance, and achieves the effect of allergy risk reminding.

drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments described in the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a diagram illustrating the detection result of the allergy-associated 25 gene 30 site in an exemplary embodiment of the present invention.

fig. 2 is a schematic diagram of the detection result of rs1042713 in an exemplary embodiment of the invention.

Fig. 3 is a schematic diagram of a detection result of rs928413 in an exemplary embodiment of the invention.

Detailed Description

The technical solution of the present invention will be explained in more detail below. It is to be understood, however, that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with one another to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

the present invention will be further described with reference to specific examples, which are intended for illustrative purposes only and are not intended to be limiting.

before describing the examples, it is necessary to provide some remarks:

The reagent of different manufacturers and different batches can cause the difference of experimental results, and belongs to the normal phenomenon.

In small-scale experiments, in order to ensure the repeatability among parallel experiments, the reagent is recommended to be prepared, fully mixed and subpackaged so as to ensure the uniformity of the reagent in each experiment.

Generally, the risk of allergy detected is as follows:

food allergy: peanut, fish and shrimp, milk, peach, nut, wheat gluten; the related SNP loci comprise rs12964116, rs12123821, rs12964116, rs61816761, rs12123821, rs12964116, rs13015714 and rs 1059513.

environmental allergy: pollen, dust mites, natural latex, pet dander; the related SNP loci comprise rs2155219, rs928413, rs2155219, rs1898671, rs1800925, rs1946518 and rs 7775228.

allergic constitution: asthma, allergic cough, dermatitis, rubella, allergic rhinitis; the related SNP loci comprise rs6871536, rs928413, rs1558641, rs187238, rs10767664, rs20541, rs2305480, rs7701890, rs6010620, rs20541, rs3126085, rs1800871, rs1800630, rs2427827, rs8004654, rs2251746, rs1059513, rs1800925, rs20541, rs569108, rs2155219, rs7775228, rs 47820, rs 242787837, rs2251746, rs 7837, rs2251746, rs1063355, rs1042713, rs 77937 and rs 1876828.

The invention relates to a multiplex PCR nucleic acid mass spectrum flight technology, which comprises a set of amplification primers and probes capable of detecting 25 genes (ADRB 2, BDNF, CRHR1, CRHR2, FCER1A, FLG-AS1, GSDMB, HLA-DQB1, IL1R1, IL10, IL13, IL18, IL18R1, IL33, LRRC3C, MS4A2, PTGDR, RAD50, RTEL1, SERPINB7, STAT6, TMEM232 and TSLP) related to allergy. The invention comprises the following steps: aiming at each involved gene, a pair of upstream and downstream primers are designed for gene amplification; aiming at each detection site, a corresponding probe is designed for single base amplification.

An aspect of an embodiment of the present invention provides a primer set for detecting a mutation in an allergy-related gene, wherein the allergy-related gene includes ADRB2, BDNF, CRHR1, CRHR2, FCER1A, FLG-AS1, GSDMB, HLA-DQB1, IL1R1, IL10, IL13, IL18, IL18R1, IL33, LRRC3C, MS4a2, PTGDR, RAD50, RTEL1, SERPINB7, STAT6, TMEM232, and TSLP, respectively, the primer set including:

the SNP locus of the ADRB2 gene is rs1042713, the primer pair comprises a first primer and a second primer, the sequence of the first primer is shown as SEQ ID No.1 (namely, an upstream primer, the specific sequence is ACGTTGGATGGAACGGCTTCTTG), and the sequence of the second primer is shown as SEQ ID No.2 (namely, a downstream primer, the specific sequence is ACGTTGGATGTTGGCGCTGTGATGAC);

the SNP locus of the BDNF gene is rs10767664, the primer pair comprises a third primer and a fourth primer, the sequence of the third primer is shown as SEQ ID NO.3 (namely an upstream primer, the specific sequence is ACGTTGGATGTTCCCTTTTGTCGG), and the sequence of the fourth primer is shown as SEQ ID NO.4 (namely a downstream primer, the specific sequence is ACGTTGGATGGGTAGGAACTAGC);

The SNP locus of the CRHR1 gene is rs1876828, the primer pair comprises a fifth primer and a sixth primer, the sequence of the fifth primer is shown as SEQ ID No.5 (namely, an upstream primer, the specific sequence is ACGTTGGATGCAAAGATGCTCTGG), and the sequence of the sixth primer is shown as SEQ ID No.6 (namely, a downstream primer, the specific sequence is ACGTTGGATGGATGTAGAGCCCTCC);

The SNP locus of the CRHR2 gene is rs7793837, the primer pair comprises a seventh primer and an eighth primer, the sequence of the seventh primer is shown as SEQ ID No.7 (namely, an upstream primer, the specific sequence is ACGATGAGAGGGAACATCTTGAC), and the sequence of the eighth primer is shown as SEQ ID No.8 (namely, a downstream primer, the specific sequence is ACGTTGTTCTTTTCAGTGTCCACTGC);

the SNP loci of the FCER1A gene are rs7793837, rs2251746 and rs2427827, wherein the primer pair corresponding to rs7793837 comprises a ninth primer and a tenth primer, the sequence of the ninth primer is shown as SEQ ID No.9 (namely, the upstream primer, the specific sequence is ACGTTGGATGTCTCTCTGCTACCTG), and the sequence of the tenth primer is shown as SEQ ID No.10 (namely, the downstream primer, the specific sequence is ACGTTGGATGGTGAGCCTGTTGTTG); the primer pair corresponding to rs2251746 comprises an eleventh primer and a twelfth primer, wherein the sequence of the eleventh primer is shown as SEQ ID No.11 (i.e. the upstream primer, the specific sequence is ACGTTGGATGCGCATATGTTTGGTA), and the sequence of the twelfth primer is shown as SEQ ID No.12 (i.e. the downstream primer, the specific sequence is ACGTTGGATGCCACATAACCTGGTC); the primer pair corresponding to rs2427827 comprises a thirteenth primer and a fourteenth primer, the sequence of the thirteenth primer is shown as SEQ ID No.13 (i.e. the upstream primer, the specific sequence is ACGTTGGATGTAAACCAGTGGGCAC), and the sequence of the fourteenth primer is shown as SEQ ID No.14 (i.e. the downstream primer, the specific sequence is ACGTTGGATGTTTTCATGGCTCCGTG);

the SNP locus of the FLG gene is rs61816761, the primer pair comprises a fifteenth primer and a sixteenth primer, the sequence of the fifteenth primer is shown as SEQ ID NO.15 (namely, an upstream primer, the specific sequence is ACGTTGGATGACGAATGGTGTCTG), and the sequence of the sixteenth primer is shown as SEQ ID NO.16 (namely, a downstream primer, the specific sequence is ACGTGTCTGGACGTTCAGGGTCTTC);

the SNP locus of the FLG-AS1 gene is rs3126085, the primer pair comprises a seventeenth primer and an eighteenth primer, the sequence of the seventeenth primer is shown AS SEQ ID No.17 (namely, an upstream primer, the specific sequence is ACGTTGGATGTCTCAAGCAGAACTC), and the sequence of the eighteenth primer is shown AS SEQ ID No.18 (namely, a downstream primer, the specific sequence is ACGTTGGATGGCCAAGATTCCAGTTC);

the SNP locus of the GSDMB gene is rs2305480, the primer pair comprises a nineteenth primer and a twentieth primer, the sequence of the nineteenth primer is shown as SEQ ID No.19 (namely, an upstream primer, the specific sequence is ACGTTGGATGGTCCAGCTTTTGCAC), and the sequence of the twentieth primer is shown as SEQ ID No.20 (namely, a downstream primer, the specific sequence is ACGTTGGATGATCTGCTGTGAGAGC);

the SNP locus of the HLA-DQB1 gene is rs1063355, the primer pair comprises a twenty-first primer and a twenty-second primer, the sequence of the twenty-first primer is shown as SEQ ID No.21 (namely, an upstream primer, the specific sequence is ACGTTGGATGGCTTTCAGTCAAGG), and the sequence of the twenty-second primer is shown as SEQ ID No.22 (namely, a downstream primer, the specific sequence is ACGTTGGATGAGACTGCTCAAGCAC);

the SNP locus of the IL1R1 gene is rs1558641, the primer pair comprises a twenty-third primer and a twenty-fourth primer, the sequence of the twenty-third primer is shown as SEQ ID No.23 (namely, an upstream primer, the specific sequence is ACGTTGGATGCTTACTCCATGGAAG), and the sequence of the twenty-fourth primer is shown as SEQ ID No.24 (namely, a downstream primer, the specific sequence is ACGTTGGATGAAGGCATAGGGCTCTC);

the SNP locus of the IL10 gene is rs1800871, the primer pair comprises a twenty-fifth primer and a twenty-sixth primer, the sequence of the twenty-fifth primer is shown as SEQ ID No.25 (namely, an upstream primer, the specific sequence is ACGTTGGATGAGTGGAAAGTACTGC), and the sequence of the twenty-sixth primer is shown as SEQ ID No.26 (namely, a downstream primer, the specific sequence is ACGTTGGATGTGGAGGTGTACAGGG);

The SNP loci of the IL13 gene are rs1800925 and rs20541, wherein a primer pair corresponding to rs1800871 comprises a twenty-seventh primer and a twenty-eighth primer, the sequence of the twenty-seventh primer is shown as SEQ ID No.27 (namely an upstream primer, the specific sequence is ACGTTGGATGCCCAGTTTGTAACCTG), and the sequence of the twenty-eighth primer is shown as SEQ ID No.28 (namely a downstream primer, the specific sequence is ACGTTGGATGATTTTACCCCCTAACC); the primer pair corresponding to rs20541 comprises a twenty-ninth primer and a thirty-ninth primer, the sequence of the twenty-ninth primer is shown as SEQ ID No.29 (namely, the upstream primer, the specific sequence is ACGTTGGATGCAACCCACAGCAATG), and the sequence of the thirtieth primer is shown as SEQ ID No.30 (namely, the downstream primer, the specific sequence is ACGTTGGATGACTCTGCAGGATCACC);

The SNP loci of the IL18 gene are rs1946518 and rs187238, wherein a primer pair corresponding to rs1946518 comprises a thirty-first primer and a thirty-second primer, the sequence of the thirty-first primer is shown as SEQ ID NO.31 (namely an upstream primer, the specific sequence is ACGTTGGATGCCCAAGCTCGGTGTC), and the sequence of the thirty-second primer is shown as SEQ ID NO.32 (namely a downstream primer, the specific sequence is ACGTTGGATGCTCTCCAAGCTTATC); the primer pair corresponding to rs187238 comprises a thirty-third primer and a thirty-fourth primer, the sequence of the thirty-third primer is shown as SEQ ID No.33 (i.e. the upstream primer, the specific sequence is ACGTTGGATGTTCCAGGGCATGGTC), and the sequence of the thirty-fourth primer is shown as SEQ ID No.34 (i.e. the downstream primer, the specific sequence is ACGTTGGATGCTAGGACTAAGGAGC);

The SNP locus of the IL18R1 gene is rs13015714, the primer pair comprises a thirty-fifth primer and a thirty-sixth primer, the sequence of the thirty-fifth primer is shown as SEQ ID No.35 (namely, an upstream primer, the specific sequence is ACGTTGGATGCATTTTGCCGCTATG), and the sequence of the thirty-sixth primer is shown as SEQ ID No.36 (namely, a downstream primer, the specific sequence is ACGTTGGATGGGATGTTCACTATAGG);

the SNP locus of the IL33 gene is rs928413, the primer pair comprises a thirty-seventh primer and a thirty-eighth primer, the sequence of the thirty-seventh primer is shown as SEQ ID NO.37 (namely, an upstream primer, the specific sequence is ACGTTGATGTSTGAAGCACTGA), and the sequence of the thirty-eighth primer is shown as SEQ ID NO.38 (namely, a downstream primer, the specific sequence is ACGTTGGARTCTTCAKCCACAACACC);

the SNP locus of the LRRC3C gene is rs4794820, the primer pair comprises a thirty-ninth primer and a forty-fourth primer, the sequence of the thirty-ninth primer is shown as SEQ ID No.39 (namely, an upstream primer, the specific sequence is ACGTTGGATGCAGAGATGGCTTAC), and the sequence of the forty-fourth primer is shown as SEQ ID No.40 (namely, a downstream primer, the specific sequence is ACGTTGGATGACCACATTACGTGTG);

the SNP locus of the MS4A2 gene is rs569108, the primer pair comprises a fortieth primer and a fortieth primer, the sequence of the fortieth primer is shown as SEQ ID No.41 (namely, an upstream primer, the specific sequence is ACGTTGGATGTATCAGGTAGGATCG), and the sequence of the fortieth primer is shown as SEQ ID No.42 (namely, a downstream primer, the specific sequence is ACGTTGGATGTAGCCCCTTAAGACC);

The SNP locus of the PTGDR gene is rs8004654, the primer pair comprises a forty-third primer and a forty-fourth primer, the sequence of the forty-third primer is shown as SEQ ID No.43 (namely, an upstream primer, the specific sequence is ACGTTGCCTCGCCTATGCAATG), and the sequence of the forty-fourth primer is shown as SEQ ID No.44 (namely, a downstream primer, the specific sequence is ACGTTGGATGTCGAAAGCGAAGAGG);

the SNP locus of the RAD50 gene is rs6871536, the primer pair comprises a forty-fifth primer and a forty-sixth primer, the sequence of the forty-fifth primer is shown as SEQ ID No.45 (namely an upstream primer, the specific sequence is ACGTTGGATGGGAGGAGAACGG), and the sequence of the forty-sixth primer is shown as SEQ ID No.46 (namely a downstream primer, the specific sequence is ACGTTGGATGCACAGTGGATAAACCC);

the SNP locus of the RTEL1 gene is rs6010620, the primer pair comprises a forty-seventh primer and a forty-eighth primer, the sequence of the forty-seventh primer is shown as SEQ ID No.47 (namely, an upstream primer, the specific sequence is ACGTTGGATGGCCTAGAGCTAGCG), and the sequence of the forty-eighth primer is shown as SEQ ID No.48 (namely, a downstream primer, the specific sequence is ACGTTGGATGCCATCTCAGCAACC);

the SNP locus of the SERPINB7 gene is rs12964116, the primer pair comprises a forty-ninth primer and a fifty-fifth primer, the sequence of the forty-ninth primer is shown as SEQ ID No.49 (namely, an upstream primer, the specific sequence is ACGTTGGATGCAACCACCTGATAC), and the sequence of the fifty-fifth primer is shown as SEQ ID No.50 (namely, a downstream primer, the specific sequence is ACGTTGGATACTGTCCAAAGGTGC);

the SNP sites of the STAT6 gene are rs1059513 and rs324015, wherein a primer pair corresponding to rs1059513 comprises a fifty-first primer and a fifty-second primer, the sequence of the fifty-first primer is shown as SEQ ID NO.51 (namely, an upstream primer, the specific sequence is ATTGATGACCAAGGTCTTGTTAAGG), and the sequence of the fifty-second primer is shown as SEQ ID NO.52 (namely, a downstream primer, the specific sequence is ACGTTGGATGAGCTGTGTGTGGTCA); the primer pair corresponding to rs324015 comprises a fifty-third primer and a fifty-fourth primer, and the sequence of the fifty-third primer is shown as SEQ ID No.53 (i.e. the upstream primer, the specific sequence is ACGTTGGATGGTTGTCAGCAAAGC), and the sequence of the fifty-fourth primer is shown as SEQ ID No.54 (i.e. the downstream primer, the specific sequence is ACGTTGGATGAGCCCCAAATTCCTC);

the SNP locus of the TMEM232 gene is rs7701890, the primer pair comprises a fifty-fifth primer and a fifty-sixth primer, the sequence of the fifty-fifth primer is shown as SEQ ID NO.55 (namely, the upstream primer, the specific sequence is ACGTTGCCAATGAAGCTACTGGG), and the sequence of the fifty-sixth primer is shown as SEQ ID NO.56 (namely, the downstream primer, the specific sequence is ACGTTGGATGCAAACTGGAAAACCC);

The SNP locus of the TNF gene is rs1800630, the primer pair comprises a fifty-seventh primer and a fifty-eighth primer, the sequence of the fifty-seventh primer is shown as SEQ ID NO.57 (namely, the upstream primer, the specific sequence is ACGTTGGATGCTATGGAAGGAGTG), and the sequence of the fifty-eighth primer is shown as SEQ ID NO.58 (namely, the downstream primer, the specific sequence is ACGTTGGATGTCGGCTTCCAAGGATC);

The SNP locus of the TSLP gene is rs1898671, the primer pair comprises a fifty-ninth primer and a sixty primer, the sequence of the fifty-ninth primer is shown as SEQ ID No.59 (namely, the upstream primer, the specific sequence is ACGTTGGATGTGGCATTTTGTCAAG), and the sequence of the sixty primer is shown as SEQ ID No.60 (namely, the downstream primer, the specific sequence is ACGTTGGATGCAATAGTCAGAGTAAC).

yet another aspect of the embodiments of the present invention provides a probe set for detecting a mutation of an allergy-associated gene, wherein the allergy-associated gene includes ADRB2, BDNF, CRHR1, CRHR2, FCER1A, FLG-AS1, GSDMB, HLA-DQB1, IL1R1, IL10, IL13, IL18, IL18R1, IL33, LRRC3C, MS4a2, PTGDR, RAD50, RTEL1, SERPINB7, STAT6, TMEM232, TNF, and TSLP, respectively, the probe set including:

The sequence of the first probe for detecting the SNP locus rs1042713 of the ADRB2 gene is shown as SEQ ID NO.61, and the specific sequence is CCGGCGCATTTC;

A second probe for detecting SNP locus rs10767664 of the BDNF gene, the sequence of the second probe is shown as SEQ ID NO.62, and the specific sequence is ggatGCTTACATGTTTTT;

a third probe for detecting SNP locus rs1876828 of CRHR1 gene, the sequence of which is shown as SEQ ID NO.63, and the specific sequence is aGCCTCTCTCCCTG;

a fourth probe for detecting SNP locus rs7793837 of CRHR2 gene, the sequence of which is shown as SEQ ID NO.64, and the specific sequence is ggGGTTTTGTGCGTTAG;

The sequences of a fifth probe for detecting the SNP locus rs2427837, a sixth probe for detecting the SNP locus rs2251746 and a seventh probe for detecting the SNP locus rs2427827 of the FCER1A gene are respectively shown as SEQ ID No.65, SEQ ID No.66 and SEQ ID No.67, wherein the specific sequence shown as SEQ ID No.65 is ctACTTCTCCCCATGATG, SEQ, the specific sequence shown as cGTTGCTGCTGTTTTCTGC, SEQ and ID No.67 is aTGATGGGCCAGATATGA;

An eighth probe for detecting the SNP locus rs61816761 of the FLG gene has a sequence shown as SEQ ID No.68, and the specific sequence is tttCTTGCCTGTGTCTC;

A ninth probe for detecting SNP locus rs3126085 of FLG-AS1 gene, the sequence of which is shown in SEQ ID NO.69, the specific sequence is gaATTTGGCTTGTAAGT;

a tenth probe for detecting SNP locus rs2305480 of the GSDMB gene, the sequence of which is shown as SEQ ID NO.70, and the specific sequence is GGAGCTCACGAGGAC;

an eleventh probe for detecting the SNP locus rs1063355 of the HLA-DQB1 gene, wherein the sequence of the eleventh probe is shown as SEQ ID NO.71, and the specific sequence is ccCAAGAAGCACATGAAAA;

a twelfth probe for detecting the SNP locus rs1558641 of the IL1R1 gene, wherein the sequence of the twelfth probe is shown as SEQ ID NO.72, and the specific sequence is CCTGGAAGAAGGAGGA;

a thirteenth probe for detecting the SNP locus rs1800871 of the IL10 gene, wherein the sequence of the thirteenth probe is shown as SEQ ID NO.73, and the specific sequence is ttgCCCTTCAGGTGATGTAA;

The sequences of a fourteenth probe for detecting the SNP locus rs1800925 and a fifteenth probe for detecting the SNP locus rs20541 of the IL13 gene are respectively shown as SEQ ID NO.74 and SEQ ID NO.75, wherein the specific sequence shown as SEQ ID NO.74 is gggaTTTTTCGCGGAC, SEQ ID NO.75, and the specific sequence is GGAGGACTTCTAGGAAAA;

the sequences of the sixteenth probe for detecting the SNP locus rs1946518 and the seventeenth probe for detecting the SNP locus rs187238 of the IL18 gene are shown as SEQ ID No.76 and SEQ ID No.77 respectively, wherein the specific sequence shown as SEQ ID No.76 is gTGCAGGTGTAATTATTA, SEQ ID No.77, and the specific sequence is tccTGATATCTCATGAAAT;

an eighteenth probe for detecting the SNP locus rs13015714 of the IL18R1 gene has a sequence shown as SEQ ID No.78, and the specific sequence is ccttTGGCTGTTATTTAAC;

a nineteenth probe for detecting the SNP locus rs928413 of the IL33 gene, wherein the sequence of the nineteenth probe is shown as SEQ ID NO.79, and the specific sequence is MTGCCTTGGCCYGCTCT;

A twentieth probe for detecting the SNP locus rs4794820 of the LRRC3C gene, wherein the sequence of the twentieth probe is shown as SEQ ID NO.80, and the specific sequence is ccTCCTCAAGGTTTGTTG;

the twenty-first probe for detecting the SNP locus rs569108 of the MS4A2 gene has a sequence shown as SEQ ID NO.81, and the specific sequence is cgTGGAACCCGG;

a twenty-second probe for detecting the SNP locus rs8004654 of the PTGDR gene, wherein the sequence of the twenty-second probe is shown as SEQ ID NO.82, and the specific sequence is ACGTGAGTTATCTTTACC;

a twenty-third probe for detecting the SNP locus rs6871536 of the RAD50 gene, wherein the sequence of the twenty-third probe is shown as SEQ ID NO.83, and the specific sequence is GCGGACATCTTTGTATA;

a twenty-fourth probe for detecting the SNP locus rs6010620 of the RTEL1 gene, wherein the sequence of the twenty-fourth probe is shown as SEQ ID NO.84, and the specific sequence is TTACCTGGGAAGC;

a twenty-fifth probe for detecting the SNP locus rs12964116 of the SERPINB7 gene, wherein the sequence of the twenty-fifth probe is shown as SEQ ID NO.85, and the specific sequence is GGGATGGCCCCTA;

the sequences of the twenty-sixth probe for detecting the SNP locus rs1059513 and the twenty-seventh probe for detecting the SNP locus rs324015 of the STAT6 gene are respectively shown as SEQ ID NO.86 and SEQ ID NO.87, wherein the specific sequence shown as SEQ ID NO.86 is CTGCTTTTAGATTTATT, SEQ ID NO.87, and the specific sequence is CGAACTCAGCCCT;

a twenty-eighth probe for detecting SNP locus rs7701890 of the TMEM232 gene, wherein the sequence of the twenty-eighth probe is shown as SEQ ID NO.88, and the specific sequence is CATACCTACTAAAT;

A twenty-ninth probe for detecting SNP locus rs1800630 of TNF gene, the sequence of the twenty-ninth probe is shown as SEQ ID NO.89, and the specific sequence is ATGGCCCTCTTCATTAAG;

the thirtieth probe for detecting the SNP locus rs1898671 of the TSLP gene has a sequence shown as SEQ ID No.90, and the specific sequence is AGACCGCGTTATG.

In another aspect of the embodiments of the present invention, a kit for detecting allergy-associated gene mutation is further provided, which includes at least one primer set and at least one probe set, where one primer set is the aforementioned primer set and one probe set is the aforementioned probe set.

further, the kit also comprises a conventional component for PCR amplification detection, wherein the conventional component for PCR amplification detection comprises buffer solution for PCR reaction, deoxynucleotide triphosphate mixed solution (dNTPs), DNA polymerase and the like.

The invention also provides application of the kit in preparation of products for detecting allergy-related gene mutation.

providing a DNA sample to be detected;

The source of the DNA sample to be detected includes but is not limited to any one or the combination of more than two of detection materials which can obtain complete genome DNA, such as oral mucosa, blood, saliva, hair follicle and the like of a subject, and the method is convenient for the subject to sample and does not need blood drawing.

Another aspect of the embodiments of the present invention also provides a product comprising the aforementioned kit, which is applied to a method for detecting allergy-associated gene mutation, the method comprising:

providing a DNA sample to be detected;

the technical solutions of the present invention are further explained below with reference to some preferred embodiments, but the experimental conditions and the setting parameters should not be construed as limitations of the basic technical solutions of the present invention. And the scope of the present invention is not limited to the following examples.

example 1

the 25 genes and corresponding gene amplification primers and probes involved in this example are shown in the following table:

The method for detecting the allergy-associated gene mutation in the embodiment specifically comprises the following steps:

1. Extraction of genomic DNA

putting a cotton swab which is scraped through oral mucosa for 3-4 times into a 1.5mL centrifugal tube, adding 1mL PBS buffer solution, and repeatedly squeezing and stirring to ensure that cells fall off. 10000r/min for 3min, discarding the supernatant, and adding 100. mu.l TE buffer.

② shaking at high speed for 5-10s, suspending and precipitating, boiling with boiling water for 10min, then ice-cooling for 3-5min, shaking at high speed for 5-10s again, centrifuging for 3min at 10000/min, and storing the supernatant at 4 ℃ for later use.

2. Amplification of a fragment of interest

PCR reactions two PCR amplifications and one single-stranded DNA digestion were performed in a Proflex PCR instrument using 384-well plates.

5 mul PCR reaction system, which contains 20-50ng DNA template, 0.5U DNA polymerase, 0.5pmol each amplification primer and 0.25 mul mol/L dNTP. The PCR reaction process is as follows: pre-denaturation at 95 ℃ for 2min, (denaturation at 95 ℃ for 30s, annealing at 56 ℃ for 30s, and extension at 72 ℃ for 60s) are cycled for 45 times, and extension at 72 ℃ for 5 min.

② digestion of SAP. After the PCR reaction was completed, the mixture was centrifuged at 4000rpm for 5min, 2ul of iplex-SAP reaction solution was added, and digestion was performed in a Proflex PCR apparatus. The digestion conditions were: digesting at 37 deg.C for 40min, digesting at 85 deg.C for 5min, centrifuging at 4000rpm for 5min, and storing at 4 deg.C for use.

extension of single base. Mu.l of an iplex-EXT reaction containing 0.94. mu. mol/L of probe Mix, 0.2. mu.l of gold buffer, 0.2. mu.l of stop Mix, and 0.041U of DNA polymerase were added. Centrifuge at 4000rpm for 5 min. The PCR reaction process is as follows: pre-denaturation at 95 ℃ for 30s, (denaturation at 95 ℃ for 5s, annealing at 56 ℃ for 5s, and extension at 80 ℃ for 5s) for 40 external cycles, and extension at 72 ℃ for 3min, and storing at 4 ℃ for later use.

3. resin purification: add 16. mu.L of purified water and 6mg of clean resin to each well of 384-well plate, pour the dried resin into the extension product, rotate vertically at low speed for 30min to bring the resin into full contact with the reactants, and centrifuge to sink the resin to the bottom of the well.

4. Co-crystallization and detection of sample analytes: the purified product was transferred to 384-well SpectroCHIP chips and tested on the machine. The SpectroCHIP chip is analyzed by MALDI-TOF-MS, and the detection result is typed and output by Typer software. The detection results are shown in FIG. 1, and the sample points of different genotypes are separated from each other and are not intersected with each other; while the peak patterns and separations for the different typing in FIGS. 2 and 3 (partial site illustration) are good. Wherein the abscissa in fig. 1-3 is molecular mass (daltons) and the ordinate is signal intensity.

5. suggesting analysis of the detection results

FIG. 1 shows that the peak positions of 30 SNP sites of 25 genes detected are obviously separated and do not overlap with each other, and the primer related to the application can efficiently amplify the target fragment. FIGS. 2 and 3 show that the probe of the present invention can efficiently and precisely extend only one base. And the extension products are different in charge-to-mass ratio, so that under the action of an electric field and a magnetic field after the machine is operated, the different extension products fall on different positions and are detected by a mass spectrometer after moving for the same time and different tracks, and the peak positions on the atlas are obviously separated and are not mutually overlapped.

in summary, according to the above technical scheme, the invention provides a set of amplification primers, a probe and a kit capable of detecting allergy-related 25 genes, and a pair of upstream and downstream primers are designed for gene amplification for each gene involved; aiming at each detection site, designing a corresponding probe for single base amplification; the invention adopts a multiple PCR nucleic acid mass spectrum flight technology, rapidly, accurately and sensitively detects the risk sites of 25 genes related to allergy, can detect the polymorphism of 30 SNP sites of 25 genes related to allergy at one time, rapidly, efficiently, accurately and at high cost performance, and achieves the effect of allergy risk reminding.

The aspects, embodiments, features and examples of the present invention should be considered as illustrative in all respects and not intended to be limiting of the invention, the scope of which is defined only by the claims. Other embodiments, modifications, and uses will be apparent to those skilled in the art without departing from the spirit and scope of the claimed invention.

the use of headings and chapters in this disclosure is not meant to limit the disclosure; each section may apply to any aspect, embodiment, or feature of the disclosure.

Throughout this specification, where a composition is described as having, containing, or comprising specific components or where a process is described as having, containing, or comprising specific process steps, it is contemplated that the composition of the present teachings also consist essentially of, or consist of, the recited components, and the process of the present teachings also consist essentially of, or consist of, the recited process steps.

It should be understood that the order of steps or the order in which particular actions are performed is not critical, so long as the teachings of the invention remain operable. Further, two or more steps or actions may be performed simultaneously.

While the invention has been described with reference to illustrative embodiments, it will be understood by those skilled in the art that various other changes, omissions and/or additions may be made and substantial equivalents may be substituted for elements thereof without departing from the spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from its scope. Therefore, it is intended that the invention not be limited to the particular embodiment disclosed for carrying out this invention, but that the invention will include all embodiments falling within the scope of the appended claims.

sequence listing

<110> Suzhou Qiankang Gene Co., Ltd

<160> 90

<170> SIPOSequenceListing 1.0

<210> 1

<211> 23

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 1

acgttggatg gaacggcttc ttg 23

<210> 2

<211> 26

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 2

acgttggatg ttggcgctgt gatgac 26

<210> 3

<211> 24

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 3

acgttggatg ttcccttttg tcgg 24

<210> 4

<211> 23

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 4

acgttggatg ggtaggaact agc 23

<210> 5

<211> 24

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 5

acgttggatg caaagatgct ctgg 24

<210> 6

<211> 25

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 6

acgttggatg gatgtagagc cctcc 25

<210> 7

<211> 23

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 7

acgatgagag ggaacatctt gac 23

<210> 8

<211> 26

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 8

acgttgttct tttcagtgtc cactgc 26

<210> 9

<211> 25

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 9

acgttggatg tctctctgct acctg 25

<210> 10

<211> 25

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 10

acgttggatg gtgagcctgt tgttg 25

<210> 11

<211> 25

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 11

acgttggatg cgcatatgtt tggta 25

<210> 12

<211> 25

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 12

acgttggatg ccacataacc tggtc 25

<210> 13

<211> 25

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 13

acgttggatg taaaccagtg ggcac 25

<210> 14

<211> 26

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 14

acgttggatg ttttcatggc tccgtg 26

<210> 15

<211> 24

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 15

acgttggatg acgaatggtg tctg 24

<210> 16

<211> 25

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 16

acgtgtctgg acgttcaggg tcttc 25

<210> 17

<211> 25

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 17

acgttggatg tctcaagcag aactc 25

<210> 18

<211> 26

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 18

acgttggatg gccaagattc cagttc 26

<210> 19

<211> 25

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 19

acgttggatg gtccagcttt tgcac 25

<210> 20

<211> 25

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 20

acgttggatg atctgctgtg agagc 25

<210> 21

<211> 24

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 21

acgttggatg gctttcagtc aagg 24

<210> 22

<211> 25

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 22

acgttggatg agactgctca agcac 25

<210> 23

<211> 25

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 23

acgttggatg cttactccat ggaag 25

<210> 24

<211> 26

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 24

acgttggatg aaggcatagg gctctc 26

<210> 25

<211> 25

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 25

acgttggatg agtggaaagt actgc 25

<210> 26

<211> 25

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 26

acgttggatg tggaggtgta caggg 25

<210> 27

<211> 26

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 27

acgttggatg cccagtttgt aacctg 26

<210> 28

<211> 26

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 28

acgttggatg attttacccc ctaacc 26

<210> 29

<211> 25

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 29

acgttggatg caacccacag caatg 25

<210> 30

<211> 26

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 30

acgttggatg actctgcagg atcacc 26

<210> 31

<211> 25

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 31

acgttggatg cccaagctcg gtgtc 25

<210> 32

<211> 25

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 32

acgttggatg ctctccaagc ttatc 25

<210> 33

<211> 25

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 33

acgttggatg ttccagggca tggtc 25

<210> 34

<211> 25

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 34

acgttggatg ctaggactaa ggagc 25

<210> 35

<211> 25

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 35

acgttggatg cattttgccg ctatg 25

<210> 36

<211> 26

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 36

acgttggatg ggatgttcac tatagg 26

<210> 37

<211> 22

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 37

acgttgatgt stgaagcact ga 22

<210> 38

<211> 26

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 38

acgttggart cttcakccac aacacc 26

<210> 39

<211> 24

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 39

acgttggatg cagagatggc ttac 24

<210> 40

<211> 25

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 40

acgttggatg accacattac gtgtg 25

<210> 41

<211> 25

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 41

acgttggatg tatcaggtag gatcg 25

<210> 42

<211> 25

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 42

acgttggatg tagcccctta agacc 25

<210> 43

<211> 22

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 43

acgttgcctc gcctatgcaa tg 22

<210> 44

<211> 25

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 44

acgttggatg tcgaaagcga agagg 25

<210> 45

<211> 22

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 45

acgttggatg ggaggagaac gg 22

<210> 46

<211> 26

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 46

acgttggatg cacagtggat aaaccc 26

<210> 47

<211> 24

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 47

acgttggatg gcctagagct agcg 24

<210> 48

<211> 24

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 48

acgttggatg ccatctcagc aacc 24

<210> 49

<211> 24

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 49

acgttggatg caaccacctg atac 24

<210> 50

<211> 24

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 50

acgttggata ctgtccaaag gtgc 24

<210> 51

<211> 25

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 51

attgatgacc aaggtcttgt taagg 25

<210> 52

<211> 25

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 52

acgttggatg agctgtgtgt ggtca 25

<210> 53

<211> 24

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 53

acgttggatg gttgtcagca aagc 24

<210> 54

<211> 25

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 54

acgttggatg agccccaaat tcctc 25

<210> 55

<211> 23

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 55

acgttgccaa tgaagctact ggg 23

<210> 56

<211> 25

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 56

acgttggatg caaactggaa aaccc 25

<210> 57

<211> 24

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 57

acgttggatg ctatggaagg agtg 24

<210> 58

<211> 26

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 58

acgttggatg tcggcttcca aggatc 26

<210> 59

<211> 25

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 59

acgttggatg tggcattttg tcaag 25

<210> 60

<211> 26

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 60

acgttggatg caatagtcag agtaac 26

<210> 61

<211> 12

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 61

ccggcgcatt tc 12

<210> 62

<211> 18

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 62

ggatgcttac atgttttt 18

<210> 63

<211> 14

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 63

agcctctctc cctg 14

<210> 64

<211> 17

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 64

ggggttttgt gcgttag 17

<210> 65

<211> 18

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 65

ctacttctcc ccatgatg 18

<210> 66

<211> 19

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 66

cgttgctgct gttttctgc 19

<210> 67

<211> 18

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 67

atgatgggcc agatatga 18

<210> 68

<211> 17

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 68

tttcttgcct gtgtctc 17

<210> 69

<211> 17

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 69

gaatttggct tgtaagt 17

<210> 70

<211> 15

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 70

ggagctcacg aggac 15

<210> 71

<211> 19

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 71

cccaagaagc acatgaaaa 19

<210> 72

<211> 16

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 72

cctggaagaa ggagga 16

<210> 73

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 73

ttgcccttca ggtgatgtaa 20

<210> 74

<211> 16

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 74

gggatttttc gcggac 16

<210> 75

<211> 18

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 75

ggaggacttc taggaaaa 18

<210> 76

<211> 18

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 76

gtgcaggtgt aattatta 18

<210> 77

<211> 19

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 77

tcctgatatc tcatgaaat 19

<210> 78

<211> 19

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 78

cctttggctg ttatttaac 19

<210> 79

<211> 17

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 79

mtgccttggc cygctct 17

<210> 80

<211> 18

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 80

cctcctcaag gtttgttg 18

<210> 81

<211> 12

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 81

cgtggaaccc gg 12

<210> 82

<211> 18

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 82

acgtgagtta tctttacc 18

<210> 83

<211> 17

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 83

gcggacatct ttgtata 17

<210> 84

<211> 13

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 84

ttacctggga agc 13

<210> 85

<211> 13

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 85

gggatggccc cta 13

<210> 86

<211> 17

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 86

ctgcttttag atttatt 17

<210> 87

<211> 13

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 87

cgaactcagc cct 13

<210> 88

<211> 14

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 88

catacctact aaat 14

<210> 89

<211> 18

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 89

atggccctct tcattaag 18

<210> 90

<211> 13

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 90

agaccgcgtt atg 13

Claims

1. A primer set for detecting a mutation in an allergy-associated gene, wherein the allergy-associated gene comprises ADRB2, BDNF, CRHR1, CRHR2, FCER1A, FLG-AS1, GSDMB, HLA-DQB1, IL1R1, IL10, IL13, IL18, IL18R1, IL33, LRRC3C, MS4a2, PTGDR, RAD50, RTEL1, SERPINB7, STAT6, TMEM232, TNF and TSLP, respectively, wherein the primer set comprises:

the SNP locus of the IL1R1 gene is rs1558641, the primer pair comprises a twenty-third primer and a twenty-fourth primer, the sequence of the twenty-third primer is shown as SEQ ID NO.23, and the sequence of the twenty-fourth primer is shown as SEQ ID NO. 24;

the SNP loci of the IL13 gene are rs1800925 and rs20541, wherein a primer pair corresponding to rs1800871 comprises a twenty-seventh primer and a twenty-eighth primer, the sequence of the twenty-seventh primer is shown as SEQ ID NO.27, and the sequence of the twenty-eighth primer is shown as SEQ ID NO. 28; the primer pair corresponding to rs20541 comprises a twenty-ninth primer and a thirty-ninth primer, the sequence of the twenty-ninth primer is shown as SEQ ID NO.29, and the sequence of the thirty-eighth primer is shown as SEQ ID NO. 30;

the SNP locus of the IL18R1 gene is rs13015714, the primer pair comprises a thirty-fifth primer and a thirty-sixth primer, the sequence of the thirty-fifth primer is shown as SEQ ID NO.35, and the sequence of the thirty-sixth primer is shown as SEQ ID NO. 36;

2. a probe set for detecting mutation of allergy-related genes, wherein the allergy-related genes respectively comprise ADRB2, BDNF, CRHR1, CRHR2, FCER1A, FLG-AS1, GSDMB, HLA-DQB1, IL1R1, IL10, IL13, IL18, IL18R1, IL33, LRRC3C, MS4a2, PTGDR, RAD50, RTEL1, SERPINB7, STAT6, TMEM232, TNF and TSLP, the probe set comprising:

3. a kit for detecting allergy-associated gene mutation, comprising at least one primer set and at least one probe set, wherein one primer set is the primer set according to claim 1, and one probe set is the probe set according to claim 2.

4. the kit of claim 3, further comprising a conventional component of a PCR amplification assay.

5. The kit of claim 4, wherein: the conventional components for PCR amplification detection comprise buffer solution for PCR reaction, base triphosphate deoxynucleotide mixed solution and DNA polymerase.

6. Use of the kit according to any one of claims 3 to 5 for the preparation of a product for the detection of allergy-associated gene mutations.

7. The use according to claim 6, wherein the method for detecting allergy-associated gene mutations using said product comprises:

providing a DNA sample to be detected;

8. Use according to claim 7, characterized in that: the DNA sample to be detected is derived from any one or the combination of more than two of oral mucosa, blood, saliva and hair follicle of a subject.

9. a product comprising the kit of any one of claims 3-5 for use in a method of detecting allergy-associated gene mutations, the method comprising:

providing a DNA sample to be detected;

10. The product of claim 9, wherein: the DNA sample to be detected is derived from any one or the combination of more than two of oral mucosa, blood, saliva and hair follicle of a subject.