CN112941182B

CN112941182B - Gene detection method for medicine for treating rheumatic immune diseases based on nucleic acid mass spectrometry and application of gene detection method

Info

Publication number: CN112941182B
Application number: CN202110266947.7A
Authority: CN
Inventors: 王应东; 王丽男; 胥慧; 刘世霆; 郑萍; 莫立乾; 赵进军; 林静丽; 李诗濛; 任用
Original assignee: Jiangsu Xiansheng Diagnostic Technology Co ltd; Nanjing Xiansheng Diagnostic Technology Co ltd; Nanjing Xiansheng Medical Laboratory Co ltd
Current assignee: Jiangsu Xiansheng Diagnostic Technology Co ltd; Nanjing Xiansheng Diagnostic Technology Co ltd; Nanjing Xiansheng Medical Laboratory Co ltd
Priority date: 2021-03-11
Filing date: 2021-03-11
Publication date: 2021-09-21
Anticipated expiration: 2041-03-11
Also published as: CN112941182A

Abstract

The invention provides a gene detection method for drugs for rheumatic immune diseases (particularly rheumatoid diseases) based on nucleic acid mass spectrometry and application thereof. The primer group for optimizing and detecting the SNP sites related to the rheumatoid immunity medication based on the MassArray nucleic acid mass spectrum platform and the detection system thereof can be used for accurately typing the point mutation related to the rheumatoid immunity medication, and have the advantages of strong accuracy, high sensitivity, good repeatability, low cost, short detection period, intuitive result and the like.

Description

Gene detection method for medicine for treating rheumatic immune diseases based on nucleic acid mass spectrometry and application of gene detection method

Technical Field

The invention relates to the field of gene detection, in particular to a nucleic acid mass spectrum-based gene detection method for rheumatism medicines (especially rheumatoid diseases) and application thereof.

Background

The most common clinical rheumatic diseases include rheumatoid arthritis, systemic lupus erythematosus, ankylosing spondylitis, gout, etc. Among them, Rheumatoid Arthritis (RA) is a chronic systemic disease mainly of inflammatory synovitis, the etiology of which is unknown. It is characterized by multiple joints, symmetry and invasive arthritis of small joints of hands and feet, and joint deformity and function loss caused by the fact that the external organs of joints are affected by serum rheumatoid factor positivity. The rheumatism immune disease is a disease which is persistent and repeated, most patients need to live with the disease for a long time, most of the diseases have no radical treatment method at present, and the treatment aims at controlling the disease condition through medicines, maintaining clinical remission and improving the life quality. Unfortunately, the rate of disease control and remission remains low, even when regularly treated under the guidelines of standard drug regimens.

In clinic, in the treatment process of the rheumatic immune disease, the gene polymorphism of a patient with the rheumatic immune disease can cause individual difference of drug curative effect and adverse reaction, and an individualized administration scheme can be formulated through screening of the genes of the patient, so that the curative effect is improved, and the adverse reaction is reduced.

Although the prior art has the detection related to the individualized medication aiming at the rheumatic immune diseases, different detection methods are different in weight and difficult to meet the actual clinical requirements. At present, the detection related to the individualized administration of the rheumatic immune diseases (particularly the rheumatoid immune diseases) in the market cannot accurately and comprehensively evaluate the effectiveness and the safety in treatment.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The technical problem to be solved by the invention is to find a method suitable for detecting genes related to the medicines for the rheumatic immune diseases (particularly rheumatoid diseases), so that the invention has the following aims:

the first purpose of the invention is to provide a primer group for detecting related SNP sites of related genes of rheumatoid immune diseases.

The second purpose of the invention is to provide the application of the primer group of the SNP locus related to the detection of the related genes of the rheumatoid immune diseases in the detection of the related genes of the rheumatoid immune diseases.

The third purpose of the invention is to provide a method for detecting the drug-related gene of the rheumatic immune disease.

In order to achieve the purpose, the invention provides the following technical scheme:

the invention provides a primer group for detecting drug-related SNP sites of rheumatic immune diseases, which aims at the following SNP sites: rs10306114, rs1042597, rs1045642, rs1050828, rs1050829, rs10509681, rs1051266, rs1057910, rs1061631, rs1065852, rs10919563, rs11188072, rs1128503, rs1142345, rs11572080, rs11678615, rs116855232, rs11706052, rs12041331, rs 12212212257, rs12248560, rs1229984, rs1495741, rs 4104105, rs16947, 1695, rs17602729, rs1799724, rs1799971, rs1800462, rs1800629, rs 1801131131133, rs 1801271279, rs 1801281281281280, rs2032582, rs 7662188, rs2231142, rs2234693, rs 27404097574, rs289714, rs 321321321321971003, rs 18056375637563756375637563756379, rs 4473563756375637563756379, rs 44729, rs 4473563756375637563756375637563756375637563756379, rs 375637563756375637563756375637563756375637563756375637563756379, rs 375637563756375637563756375637563756375637563756375637563756375637569, rs 375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563745, rs 37563756375637563756375637563756375637563756375637563756375637563756375637567, rs 375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563745, rs 37563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637567, and 1.

In some embodiments, the primer components are grouped into 4 groups as follows:

the first group comprises primer groups for detecting rs41303343, rs10306114, rs5918, rs2231142, rs671, rs1799724, rs854555, rs1051266, rs1061631, rs11572080, rs17602729, rs4846051, rs662, rs3213422, rs1042597, rs4986893, rs1495741, rs1801133, rs 0821059, rs7301582, rs983332, rs1695, rs2740574, rs1229984, rs10509681, rs 4101544105 and rs 730012;

the second group comprises primer groups for detecting rs361525, rs289714, rs7254579, rs762551, rs9340799, rs1800629, rs12041331, rs7046653, rs1057910, rs1045642, rs11188072, rs1050828, rs4673993, rs6138150, rs776746, rs12248560, rs34650714, rs1801279, rs11678615, rs3794271, rs6028945, rs11706052, rs116855232, rs 464646437, rs1801280 and rs 927135;

the third group comprises primer groups for detecting rs10919563, rs2187668, rs3131003, rs75995567, rs3758149, rs3731722, rs35167514, rs1799971, rs4244285, rs7439366, rs12208357, rs1142345, rs2234693, rs2032582 and rs 1800462;

the fourth group comprises primer groups for detecting rs16947, rs1065852, rs1801131, rs5030865, rs9923231, rs1128503, rs3397, rs4802101 and rs 7574865.

In some embodiments, the primer sequences are shown as SEQ ID nos. 1-154, respectively, or the primer sequences are at least 85% identical to SEQ ID nos. 1-154, respectively.

In some embodiments, the primer set further comprises an extension primer, wherein the extension primer sequence is shown in SEQ ID NO.155-231, or the extension primer sequence has at least 85% identity to SEQ ID NO. 155-231.

In some preferred embodiments, the primer set sequences are grouped as follows:

a first group: the primer sequences are shown as SEQ ID NO.1-54 and SEQ ID NO. 155-206;

second group: the primer sequences are shown as SEQ ID NO.55-106 and SEQ ID NO. 182-207;

third group: the primer sequences are shown as SEQ ID NO.107-136 and SEQ ID NO. 208-222;

and a fourth group: the primer sequences are shown as SEQ ID NO.137-154 and SEQ ID NO. 223-231.

The invention provides application of the primer group in preparation of related products of drugs for detecting rheumatic immune diseases.

The invention also provides application of the primer group in nucleic acid mass spectrometry detection.

The invention also provides a composition/product/kit for detecting the related genes of the drugs for the rheumatic immune diseases, which is characterized in that the composition/product/kit comprises the primer group.

In some embodiments, the compositions/products/kits further comprise conventional reagents and/or software for detecting SNP sites.

In some embodiments, the software is MassARRAY software.

The invention also provides a gene detection method for the medicine for the rheumatic immune diseases, which comprises the step of detecting the nucleotide sequence of the SNP locus in the genome of a sample to be detected by applying the primer group.

In some embodiments, the detection is based on the MassARRAY platform.

The invention has the beneficial technical effects that:

1) the invention screens the related SNP loci for Chinese population and rheumatism immune disease (especially rheumatoid immune disease), and can realize comprehensive and effective detection of related genes of rheumatoid drug.

2) According to the invention, through large sample testing and screening, the obtained primer sequence can accurately type the sample and has high specificity through adjusting and optimizing the primer sequence, the primer targeting segment, the extension primer direction and the like; in addition, by grouping optimization, the mutual interference among primers in each group is ensured. The optimization meets the requirements of mass spectrum detection technology, and the rapid and effective detection of the drug use of the MassARRAY system on the rheumatic immune diseases (especially rheumatoid immune diseases) of the sample to be detected is realized.

3) The detection system has the advantages of low cost, short period, simple operation, wide popularization in the market and high commercial return.

Drawings

Fig. 1 is a cluster diagram of sites before rs4802101 site optimization provided in embodiment 2 of the present invention;

fig. 2 is a cluster diagram of the site optimized by the rs4802101 site provided in embodiment 2 of the present invention;

fig. 3 is a cluster diagram of sites before rs10509681 site optimization provided in embodiment 2 of the present invention;

fig. 4 is a clustering diagram of the site optimized by the rs10509681 site provided in embodiment 2 of the present invention;

fig. 5 is a cluster map of sites before rs1229984 site optimization provided in embodiment 2 of the present invention;

fig. 6 is a cluster map of the optimized locus of rs1229984 locus provided in embodiment 2 of the present invention;

fig. 7 is a cluster map of sites before rs116855232 site optimization provided in embodiment 2 of the present invention;

fig. 8 is a cluster map of sites after rs116855232 site optimization provided in embodiment 2 of the present invention;

FIG. 9 is a peak diagram of site before optimization of site rs10919563 provided in embodiment 2 of the present invention;

FIG. 10 is a peak diagram of the site optimized by the locus rs10919563 provided in embodiment 2 of the present invention;

fig. 11 is a peak diagram of sites before optimization of the rs1128503 site provided in embodiment 2 of the present invention;

fig. 12 is a peak diagram of the site optimized by the rs1128503 site provided in embodiment 2 of the present invention;

FIG. 13 is a cluster map of the rs1801131 locus after merging as provided in example 2 of the present invention;

FIG. 14 is a cluster map of the rs1801131 locus after merging provided in example 2 of the present invention;

FIG. 15 is a cluster map of the rs7574865 site after merger provided in example 2 of the present invention;

FIG. 16 is a cluster map of the rs7574865 site after merger of holes provided in example 2 of the present invention;

fig. 17 is a cluster map of the rs9923231 site after merging provided in example 2 of the present invention;

fig. 18 is a cluster map of the rs9923231 site after merging provided in example 2 of the present invention;

FIG. 19 is a graph showing a peak at the rs7574865 site and a sequencing result provided in example 3 of the present invention;

FIG. 20 is a graph of a peak at rs9923231 site and a sequencing result provided in example 3 of the present invention;

FIG. 21 is a graph showing a peak at the rs11706052 site and a sequencing result in example 3 of the present invention;

FIG. 22 is a graph showing a peak at the rs1128503 locus and a sequencing result obtained in example 3 of the present invention;

FIG. 23 is a graph of peaks and sequencing results of the rs10306114 site provided in example 3 of the present invention.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The invention is further described by the accompanying drawings and the following examples, which are intended to illustrate specific embodiments of the invention and are not to be construed as limiting the scope of the invention in any way.

The main reagent information used in the examples of the present invention is as follows:

TABLE 1 Primary reagent information

The main instrument information used in the embodiment of the invention is as follows:

TABLE 2 Main Instrument information

Example 1 demonstration and screening of assay sites

The genotype characteristics of Chinese population are comprehensively considered, corresponding polymorphic sites of the medicine for treating the rheumatic immune diseases (particularly rheumatoid diseases) are selected, and feasible corresponding SNP sites are determined as follows: rs10306114, rs1042597, rs1045642, rs1050828, rs1050829, rs10509681, rs1051266, rs1057910, rs1061631, rs1065852, rs10919563, rs11188072, rs1128503, rs1142345, rs11572080, rs11678615, rs116855232, rs11706052, rs12041331, rs 12212212257, rs12248560, rs1229984, rs1495741, rs 4104105, rs16947, 1695, rs17602729, rs1799724, rs1799971, rs1800462, rs1800629, rs 1801131131133, rs 1801271279, rs 1801281281281280, rs2032582, rs 7662188, rs2231142, rs2234693, rs 27404097574, rs289714, rs 321321321321971003, rs 18056375637563756375637563756379, rs 4473563756375637563756379, rs 44729, rs 4473563756375637563756375637563756375637563756379, rs 375637563756375637563756375637563756375637563756375637563756379, rs 375637563756375637563756375637563756375637563756375637563756375637569, rs 375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563745, rs 37563756375637563756375637563756375637563756375637563756375637563756375637567, rs 375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563745, rs 37563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637567, and 1.

Example 2 design of primers and establishment of reaction System

Since MassARRAY detection is a reaction based on multiplex PCR amplification, the primer combination must avoid the problems of cross amplification, preferential amplification and non-specificity (D.van den Boom et al/International Journal of Mass Spectrometry, 238 (2004); 173-.

Firstly, related parameters are adjusted through MassARRAY website primer Design software (Assay Design Suite), PCR of 77 sites and preliminary Design of primers of UEP are completed, designed primers and parameter files are exported, and the primers are synthesized. And preparing an amplification primer MIX and an extension primer MIX according to a primer configuration table, and finely adjusting the extension primer MIX until the requirements are met. Primer testing and optimization were then performed. The method comprises the following specific steps:

(1) genomic DNA samples were diluted to 10 ng/. mu.L and PCR reaction MIX (hereinafter, single sample size) was prepared as follows

TABLE 3 PCR reaction System

Membrane sealed, 2272g centrifuged for 1 min, and the plate was placed on a PCR instrument for the following thermal cycling:

TABLE 4 PCR amplification reaction conditions

(2) Shrimp alkaline phosphatase digestion (SAP)

The PCR plate was removed, centrifuged at 2272g for 1 min, and the SAP reaction was prepared as follows (single sample size:

TABLE 5 SAP reaction System

Add 2. mu.L of SAP mix to each reaction well, seal the membrane, centrifuge at 2272g for 1 minute, place the plate on a PCR instrument for the following thermal cycles:

TABLE 6 enzymatic purification conditions

Temperature (. degree.C.)	Time
		37	40min
85	5min
		10	Heat preservation

(3) Single base Extension (EXT)

Taking out the PCR plate, centrifuging for 1 minute at 2272g, and preparing an EXT reaction system according to the following table, wherein the extended Primer Mix is a mixture of two groups of extension primers with different sites (the following is a single sample amount):

TABLE 7 EXT reaction System

Add 2. mu.L of extension mix to each reaction well, seal the membrane, centrifuge at 2272g for 1 minute, place the plate on a PCR instrument for the following thermal cycles:

TABLE 8 EXT reaction conditions

(4) Resin desalination

A. Spreading clean Resin (Resin) on the sample plate hole, and air-drying for at least 10 min;

B. taking out the sample plate, and centrifuging for 1 minute by using a plate centrifuge 2272 g;

C. adding 16 mu L of water into each hole with the sample of the sample plate, and sealing the plate;

D. gently overturning the sample plate in a volleyball manner, putting the sample plate on the sample plate with the resin, and then overturning the sample plate together with the sample plate (the two quick plates cannot move horizontally in the process), so that the resin falls into the holes;

e.2272g centrifuge for 5 minutes.

(5) Dispensing spotting

Samples were spotted using MassARRAY CPM onto the corresponding SpectroCHIP (chip).

(6)MALDI-TOF

Data were obtained using MALDI-TOF (matrix assisted laser desorption ionization-time of flight) mass spectrometer.

It should be noted that the specificity of the MassARRAY assay is that a fragment containing a target SNP site is amplified by PCR reaction, then the base of the SNP site is extended by the extension primer, and the SNP site information is judged by the molecular weight of the product. The invention can detect information of a plurality of SNP sites in a reaction system, which requires that PCR reaction and extension reaction of each SNP site can not have obvious interference.

Target region adjustment, UEP primer adjustment: the primers of the rs4802101, rs10509681, rs1229984, rs116855232, rs10919563 and rs1128503 sites are optimized as an example:

no call appears at the rs4802101 site, and after a PCR primer (the sequence of an upstream primer before modification is ACGTTGGATGTCGAGACCATCCTGGCCCAC, and the sequence of a downstream primer before modification is ACGTTGGATGAGCTGGGATTAAAAGTACCC) and a UEP primer (the sequence of the UEP before modification is CCATCTGTACTAAAAACACAAAAATTA) are redesigned, the modified PCR primer and the UEP primer are tested according to the steps, so that the test effect of the modified PCR primer and the UEP primer is better, and the no call phenomenon is improved. No call appears in the 40 samples, the specific clustering peak graph before the primer is changed is shown in figure 1, and the specific clustering peak graph after the primer is changed is shown in figure 2. It can be seen from the figure that the clusters before changing the primers are all no calls, and the clusters after changing are normal and all successfully report the locus genotypes, which indicates that the primers after changing are superior to the primers before changing and meet the detection requirements of the project.

No call appears at the rs10509681 site, and after the PCR primers are redesigned (the sequence of the upstream primer before the change is ACGTTGGATGTGGCATTACTGACTTCCGTG, and the sequence of the downstream primer before the change is ACGTTGGATGCAGGGCACAACCATAATGGC), the test is carried out according to the steps, so that the test effect of the changed PCR primers is better, and the no call phenomenon is improved. No call appears in the 40 samples, the specific clustering peak graph before the primer is changed is shown in FIG. 3, and the specific clustering peak graph after the primer is changed is shown in FIG. 4. It can be seen from the figure that the clusters before changing the primers are all no calls, and the clusters after changing are normal and all successfully report the locus genotypes, which indicates that the primers after changing are superior to the primers before changing and meet the detection requirements of the project.

No call appears at the rs1229984 site, and after the PCR primers (the sequence of the upstream primer before the change is ACGTTGGATGAGGTTGCCACTAACCACGTG and the sequence of the downstream primer before the change is ACGTTGGATGTGAATCTGAACAGCTTCTCT) and the UEP primers (the sequence of the UEP primers before the change is GACAGATTCCTACAGC) are redesigned, the modified PCR primers and the UEP primers are tested according to the steps, so that the test effect is better, and the no call phenomenon is improved. No call appears in the 40 samples, the specific clustering peak graph before the primer is changed is shown in FIG. 5, and the specific clustering peak graph after the primer is changed is shown in FIG. 6. It can be seen from the figure that the clusters before changing the primers are all no calls, and the clusters after changing are normal and all successfully report the locus genotypes, which indicates that the primers after changing are superior to the primers before changing and meet the detection requirements of the project.

No call appears at the rs116855232 site, and after the PCR primers (the sequence of the upstream primer before the change is ACGTTGGATGCCTTTGTATCCCACCAGATG, and the sequence of the downstream primer before the change is ACGTTGGATGGAACTACCTCCCCTGGACCA) and the UEP primers (the sequence of the UEP primers before the change is GCCTTGTTCTTTTAAACAAC) are redesigned, the modified PCR primers and the UEP primers are tested according to the steps, so that the test effect is better, and the no call phenomenon is improved. No call appears in the 40 samples, the specific clustering peak graph before the primer is changed is shown in FIG. 7, and the specific clustering peak graph after the primer is changed is shown in FIG. 8. It can be seen from the figure that the clusters before changing the primers are all no calls, and the clusters after changing are normal and all successfully report the locus genotypes, which indicates that the primers after changing are superior to the primers before changing and meet the detection requirements of the project.

The rs10919563 site shows a lower peak, and the modified PCR primer is tested according to the steps after the PCR primer is redesigned (the sequence of the forward primer is ACGTTGGATGGAGAAGGGATCCCAGACCAA before modification and the sequence of the forward primer is ACGTTGGATGTCATTTGCATGTTTACAGTAT before modification), so that the test effect of the modified PCR primer is better, and the lower peak is improved. No peak reduction was observed in the 40 samples tested, the peak pattern of the site before optimization is shown in FIG. 9, and the peak pattern of the site after primer optimization is changed is shown in FIG. 10. It can be seen from the figure that the peak appearance of the site is lower before the primer is changed, and the changed clusters are normal and successfully report the site genotype, which indicates that the changed primer is superior to the primer before the change and meets the detection requirement of the project.

The rs1128503 locus has a peak deviation phenomenon, and after the UEP primer is redesigned (the sequence of the primer before the change is ACTCTGCACCTTCAGGTTCAG), the test is carried out according to the steps, so that the test effect of the UEP primer after the change is better, and the peak deviation phenomenon is improved. No obvious peak bias phenomenon appears in the tests of 40 samples, the peak pattern of the site before optimization is shown in FIG. 11, and the peak pattern of the site after primer optimization is changed is shown in FIG. 12. The peak deviation phenomenon of the site before changing the primer can be seen from the figure, and the changed cluster is normal and successfully reports the site genotype, which shows that the changed primer is superior to the primer before changing and meets the detection requirement of the project.

Inter-primer and well alignment: taking rs1801131, rs7574865, rs9923231 site optimization primers as an example.

The rs1801131, rs7574865 and rs9923231 sites are in front of the hole, the rs9923231 is in the well1, and the rs1801131 and rs7574865 are in the well 2; all after the optimization adjustment are in well 4. By clustering 40 samples, the rs1801131 site clustering graph 13, the rs7574865 site clustering graph 14 and the rs9923231 site clustering graph 15 can be seen to meet the requirements of the project after merging.

In summary, optimal PCR amplification primers and single base extension (UEP) primers were obtained, and the specific primer sequences are shown in tables 9 and 10.

TABLE 9 PCR amplification primer sequence information

TABLE 10 UEP primer sequences

Example 3 clinical results experiments (comparison with one generation sequencing)

After the optimal reaction system is confirmed, a series of verification experiments are carried out, including the experiment verification of accuracy, repeatability and one-time experiment success rate. The specific verification scheme is as follows:

(1) accuracy experiment verification scheme: at least 5 samples of 77 sites were selected for Sanger sequencing and compared to the results of the Sanger sequencing and MassARRAY.

And if the consistency is required to be more than 95%, the verification is passed.

(2) Protocol for validation of the repeatability experiment: 5 clinical specimens were picked and tested in 2 replicates per specimen.

The consistency of the result of 2 tests is 100%.

The specific verification process is as follows: first, amplification primer MIX and extension primer MIX were prepared according to the system addition table provided in example 1 of the present invention. Then, the results were analyzed by PCR amplification, shrimp alkaline phosphatase consumption, single-base extension, resin desalting, and MassARRAY CPM spotting, according to the procedures described in example 1. The accuracy and repeatability results are shown in the table below.

TABLE 11 verification of accuracy (partial sites as examples)

The comparison between the MassARRAY result and the Sanger result of at least 5 clinical samples shows that the accuracy of the system verification experiment is 100%. Partial sites rs7574865, rs9923231, rs11706052, rs1128503 and rs10306114 are selected for result display (fig. 19, fig. 20, fig. 21, fig. 22 and fig. 23), and the result of the drawing can also show that the accuracy of the invention is 100%.

Finally, it should be noted that: the above embodiments are only used for illustrating the technical solutions of the present application, and not for limiting the same; although the present application has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present application.

Sequence listing

<110> Nanjing Xian Sound medical inspection Co., Ltd

Jiangsu Xiansheng Diagnostic Technology Co., Ltd.

Nanjing Xiansheng Diagnostic Technology Co., Ltd.

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acgttggatg ttgtgtctgc gtctgtgttg 30

Claims

1. A primer group for detecting drug-related SNP sites of rheumatic immune diseases is characterized in that the primer group aims at the following sites: rs10306114, rs1042597, rs1045642, rs1050828, rs1050829, rs10509681, rs1051266, rs1057910, rs1061631, rs1065852, rs10919563, rs11188072, rs1128503, rs1142345, rs11572080, rs11678615, rs116855232, rs11706052, rs12041331, rs 08312212257, rs12248560, rs1229984, rs1495741, rs 4104105, rs16947, 1695, rs17602729, rs1799724, rs1799971, rs1800462, rs1800629, rs 1801131131133, rs 1801271279, rs 1801281281280, rs2032582, rs 7662188, rs2231142, rs2234693, rs 27404097574, rs289714, rs 3213213213213219, rs 1805637563756375637563756379, rs 447356375637563756379, rs 4473563756375637563756379, rs 3756375637563756375637563756375637563756379, rs 37563756375637563756375637563756375637563756375637563756379, rs 37563756375637563756375637563756375637563756375637563756375637563756375637563756379, rs 3756375637563756375637563756375637563756375637563756375637563745, rs 375637563756375637563756375637563756375637563756375637563756375637563756375637563745, rs 3756375637563756375637563756375637563756375637563745, rs 37563756375637563745, rs 375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563745, rs 3756375637563756375637563745, rs 37563756375637563756375637563756375637563756375637563756375637563756375637563756375637563745, rs 37563756375637563745, rs 37563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563745, rs 37563756375637563756375637563756375637563756375637563745, rs 37563756375637563745, rs 375637563745, rs 37563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563745, rs 3756375637563756375637563745, rs 375637563756375637563756375637563756375637563745, rs 3756375637563745, rs 3745, rs 375637563745, rs 3745, rs 375637563745;

the primer sequences are respectively shown in SEQ ID NO. 1-154;

the primer group further comprises an extension primer, and the sequence of the extension primer is shown in SEQ ID NO. 155-231.

2. The primer set of claim 1, wherein: the primer groups are further divided into the following 4 groups:

3. Use of the primer set of any one of claims 1-2 in the preparation of a drug-related product for detecting rheumatoid diseases.

4. A composition for detecting genes related to drugs for rheumatism, wherein the composition comprises the primer set of any one of claims 1 to 2.

5. A product for detecting genes related to drugs for rheumatism, which comprises the primer set of any one of claims 1 to 2.

6. A kit for detecting genes related to drugs for rheumatism, which comprises the primer set of any one of claims 1 to 2.