CN109371123B - Probe set and kit for detecting pathogenic gene of autoinflammatory disease - Google Patents
Probe set and kit for detecting pathogenic gene of autoinflammatory disease Download PDFInfo
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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Abstract
The invention provides a probe set and a kit for detecting pathogenic genes of autoinflammatory diseases, wherein the probe set comprises a probe capable of specifically capturing pathogenic genes affecting inflammatory corpuscle deficiency, non-inflammatory corpuscle deficiency and interferon pathway deficiency related autoinflammatory disease pathogenic genes at the same time. The invention provides a probe set and a kit for detecting pathogenic genes of an autoinflammatory disease, which can detect 50 known autoinflammatory disease pathogenic genes simultaneously, obtain the probe set for detecting the autoinflammatory disease pathogenic genes based on screening genes with strong pertinence and stability and reliability, and can accelerate the diagnosis period of the autoinflammatory disease by combining a high-throughput sequencing method, improve the diagnosis efficiency, diagnose a plurality of new disease types, especially detect children patients with the autoinflammatory disease, and achieve the effects of not missing diagnosis, having less relative analysis data amount and shortening the detection period.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a probe set and a kit for detecting a pathogenic gene of an autoinflammatory disease.
Background
Autoinflammatory disorders (AIDs) are a group of inherited inflammatory disorders that are characterized by fever, rash, joint pain, arthritis, ocular lesions, and the like, often accompanied by elevated levels of inflammatory proteins. Autoinflammatory diseases are a large group of primary immunodeficiency diseases, 50 pathogenic genes are discovered by 2018, and new genes are continuously discovered.
The second Generation of high throughput Sequencing (NGS) is called high throughput Sequencing technology, and the core idea of the second Generation of Sequencing technology is Sequencing by Synthesis (Sequencing by Synthesis), i.e. determining the sequence of DNA by capturing the marker of the newly synthesized end, and the existing technology platforms mainly include Roche/454FLX, Life Tech semiconductor Sequencing technology, Illumina/solvent Genome Analyzer and Applied Biosystems SOLID system. Based on the sequencing method of Sanger and the like, by technical innovation, four different kinds of dNTP are marked by fluorescence with different colors, when a DNA polymerase synthesizes a complementary strand, different fluorescence can be released when adding one kind of dNTP, and the sequence information of the DNA to be detected is obtained by processing the captured fluorescence signal through specific computer software.
The method has the advantages of rapidness, accuracy and low cost aiming at the second generation high-throughput sequencing technology, can simultaneously detect various types of mutation of a plurality of genes, and is widely applied to genetic defect etiology detection and molecular genetics diagnosis, however, no high-throughput capture probe or kit specially aiming at self-inflammatory disease related gene detection exists so far, so that the progress of the disease related field is seriously lagged, the diagnosis rate is low, genetic consultation and necessary prenatal intervention measures cannot be implemented, huge pain and heavy economic burden are brought to patients and families, and the great improvement of the overall population quality of China is also seriously hindered.
Disclosure of Invention
The invention provides a probe set and a kit for detecting pathogenic genes of autoinflammatory diseases, aiming at the technical problems, and can simultaneously detect 50 known autoinflammatory disease related genes. The sensitivity and specificity of detecting the pathogenic genes of the inflammatory diseases are improved, the detection time is shortened, and the effects of not missing detection and diagnosis, relatively less analysis data amount and shortening the detection period can be achieved.
In order to achieve the purpose, the invention adopts the technical scheme that:
a probe set for detecting a causative gene of an auto inflammatory disease, the probe set comprising a probe capable of specifically capturing a causative gene affecting an inflammatory corpuscle deficiency, a causative gene not affecting an inflammatory corpuscle deficiency, and an causative gene of an auto inflammatory disease associated with a defect in an interferon pathway at the same time.
Preferably, the disease-causing genes affecting the inflammatory corpuscle defect include MEFV, MVK, NLRP3, NLRP12, NLRC4, PLCG2, NLRP 1;
the non-inflammatory body deficiency disease-causing gene comprises TNFRSF1A, IL10, IL10RA, IL10RB, PSTPIP1, NOD2, ADAM17, LPIN2, IL1RN, IL36RN, IL1RN, SLC29A3, CARD14, SH3BP2, COPA, OTULIN, TNFAIP3, ADA2, APIS3, LACC1, HPS1, HPS4, HPS6, GUCY2C, COL7A1, FERMT1, GREAD 2, SHARPIN, RNF31, RBCK 1;
the interferon pathway defect related autoinflammatory disease pathogenic genes include PSMB8, TMEM173, TREX1, RNASEH2B, RNASEH2A, RNASEH2C, SAMHD1, ADAR, IFIH1, ISG15, ACP5, POLA1, DDX 58.
Preferably, the probe for capturing and detecting the ADAR gene comprises a probe with a base sequence shown as SEQ ID No. 1; the probe for capturing and detecting the COPA gene comprises a probe with a base sequence shown as SEQ ID No. 2; the probe for capturing and detecting the NLRP3 gene comprises a probe with a base sequence shown as SEQ ID No. 3; the probe for capturing and detecting the SLC29A3 gene comprises a probe with a base sequence shown as SEQ ID No. 4; the probe for capturing and detecting the HPS1 gene comprises a probe with a base sequence shown as SEQ ID No. 5; the probe for capturing and detecting the HPS6 gene comprises a probe with a base sequence shown as SEQ ID No. 6; the probe for capturing and detecting the RNASEH2C gene comprises a probe with a base sequence shown as SEQ ID No. 7; the probe of TNFRSF1A gene includes the probe whose base sequence is shown in SEQ ID No. 8; the probe for capturing and detecting the GUCY2C gene comprises a probe with a base sequence shown as SEQ ID No. 9; the probe for capturing and detecting the MVK gene comprises a probe with a base sequence shown as SEQ ID No. 10; the probe for capturing and detecting the LACC1 gene comprises a probe with a base sequence shown as SEQ ID No. 11; the probe for capturing and detecting the RNASEH2B gene comprises a probe with a base sequence shown as SEQ ID No. 12; the probe for capturing and detecting the RNF31 gene comprises a probe with a base sequence shown in SEQ ID No. 13; the probe for capturing and detecting the PSTPIP1 gene comprises a probe with a base sequence shown as SEQ ID No. 14; the probe for capturing and detecting MEFV gene comprises a probe with a base sequence shown as SEQ ID No. 15; the probe for capturing and detecting the NOD2 gene comprises a probe with a base sequence shown as SEQ ID No. 16; the probe for capturing and detecting the PLCG2 gene comprises a probe with a base sequence shown as SEQ ID No. 17; the probe for capturing and detecting the NLRP1 gene comprises a probe with a base sequence shown as SEQ ID No. 18; the probe for capturing and detecting the CARD14 gene comprises a probe with a base sequence shown as SEQ ID No. 19; the probe for capturing and detecting the LPIN2 gene comprises a probe with a base sequence shown as SEQ ID No. 20; the probe for capturing and detecting the ACP5 gene comprises a probe with a base sequence shown as SEQ ID No. 21; the probe for capturing and detecting the RNASEH2A gene comprises a probe with a base sequence shown as SEQ ID No. 22; the probe for capturing and detecting the ADGRE2 gene comprises a probe with a base sequence shown as SEQ ID No. 23; the probe for capturing and detecting the NLRP12 gene comprises a probe with a base sequence shown as SEQ ID No. 24; the probe for capturing and detecting the ADAM17 gene comprises a probe with a base sequence shown as SEQ ID No. 25; the probe for capturing and detecting the NLRC4 gene comprises a probe with a base sequence shown as SEQ ID No. 26; the probe for capturing and detecting the IL36RN gene comprises a probe with a base sequence shown as SEQ ID No. 27; the probe for capturing and detecting the IL1RN gene comprises a probe with a base sequence shown as SEQ ID No. 28; the probe for capturing and detecting the IFIH1 gene comprises a probe with a base sequence shown as SEQ ID No. 29; the probe for capturing and detecting the AP1S3 gene comprises a probe with a base sequence shown as SEQ ID No. 30; the probe for capturing and detecting the RBCK1 gene comprises a probe with a base sequence shown as SEQ ID No. 31; the probe for capturing and detecting the FERMT1 gene comprises a probe with a base sequence shown as SEQ ID No. 32; the probe for capturing and detecting SAMHD1 gene comprises a probe with a base sequence shown as SEQ ID No. 33; the probe for capturing and detecting the ADA2 gene comprises a probe with a base sequence shown as SEQ ID No. 34; the probe for capturing and detecting the USP18 gene comprises a probe with a base sequence shown as SEQ ID No. 35; the probe for capturing and detecting the HPS4 gene comprises a probe with a base sequence shown as SEQ ID No. 36; the probe for capturing and detecting the TREX1 gene comprises a probe with a base sequence shown as SEQ ID No. 37; the probe for capturing and detecting the COL7A1 gene comprises a probe with a base sequence shown as SEQ ID No. 38; the probe for capturing and detecting the SH3BP2 gene comprises a probe with a base sequence shown as SEQ ID No. 39; the probe for capturing and detecting the OTULIN gene comprises a probe with a base sequence shown as SEQ ID No. 40; the probe for capturing and detecting the TMEM173 gene comprises a probe with a base sequence shown as SEQ ID No. 41; the probe for capturing and detecting the PSMB8 gene comprises a probe with a base sequence shown as SEQ ID No. 42; the probe for capturing and detecting the TNFAIP3 gene comprises a probe with a base sequence shown as SEQ ID No. 43; the probe of the SHARPIN gene comprises a probe with a base sequence shown as SEQ ID No. 44; the probe for capturing and detecting the DDX58 gene comprises a probe with a base sequence shown as SEQ ID No. 45; the probe for capturing and detecting the POLA1 gene comprises a probe with a base sequence shown as SEQ ID No. 46; the probe for capturing and detecting the IL10 gene comprises a probe with a base sequence shown as SEQ ID No. 47; the probe for capturing and detecting the IL10RA gene comprises a probe with a base sequence shown as SEQ ID No. 48; the probe for capturing and detecting the IL10RB gene comprises a probe with a base sequence shown as SEQ ID No. 49; the probe for capturing and detecting the ISG15 gene comprises a probe with a base sequence shown as SEQ ID No. 50.
A kit for detecting a pathogenic gene of an auto-inflammatory disease comprises the probe set for detecting the pathogenic gene of the auto-inflammatory disease.
Preferably, the kit further comprises an enrichment buffer, wherein the enrichment buffer comprises human cot-1 DNA; salmon sperm DNA and primer pairs; the sequences of the primer pair are shown as SEQ ID No.51 and SEQ ID No. 52.
Preferably, the kit further comprises a hybridization buffer, wherein the hybridization buffer comprises SSPE buffer, a dengue solution, EDTA and SDS.
Preferably, the kit further comprises a binding buffer solution, a rinsing solution, a NaOH solution, a Tris-HCl buffer solution, a PCR reaction solution and a TE buffer solution;
the binding buffer comprises NaCl, Tris-HCl and EDTA; the rinse liquid comprises a first rinse liquid and a second rinse liquid, and the first rinse liquid and the second rinse liquid both comprise SSC and SDS; the PCR reaction solution comprises: dNTPs, PCR amplification primers, Phusion buffer, Hotstart Phusion enzyme, DMSO and dH2O。
Preferably, the sequences of the PCR amplification primers are shown as SEQ ID No.53 and SEQ ID No. 54.
Preferably, the probe set for detecting the auto-inflammatory disease pathogenic gene is a liquid phase capture probe.
Compared with the prior art, the invention has the advantages and positive effects that:
the invention provides a probe group and a kit for detecting pathogenic genes of an autoinflammatory disease, which can detect 50 known autoinflammatory disease pathogenic genes simultaneously, obtain the probe group for detecting the autoinflammatory disease pathogenic genes based on screening genes with strong pertinence and stability and reliability, and can accelerate the diagnosis period of the autoinflammatory disease by combining a high-throughput sequencing method, improve the diagnosis efficiency, diagnose a plurality of new disease species, especially detect children patients with the autoinflammatory disease, and achieve the effects of not missing diagnosis, having less relative analysis data quantity and shortening the examination period; the probe set and the kit have the advantages of high efficiency, accuracy, rapidness and low price, are used for detecting the genes related to the pathogenesis of the autoinflammatory disease and diagnosing molecular genetics, and have great significance.
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Some specific embodiments of the invention will be described in detail hereinafter, by way of illustration and not limitation, with reference to the accompanying drawings. The same reference numbers in the drawings identify the same or similar elements or components. Those skilled in the art will appreciate that the drawings are not necessarily drawn to scale. In the drawings:
FIG. 1 is a genomic diagram of the detection result of the kit for infant patient numbered C150710C01201 in example 3 of the present invention;
FIG. 2 is a graph of the sequencing analysis of Sanger in infant patient No. C150710C01201 according to example 3 of the present invention;
FIG. 3 is a graph of the parent Sanger sequencing analysis of infant patient No. C150710C01201 in example 3 of the present invention;
FIG. 4 is a graph of a parent Sanger sequencing analysis of infant numbered C150710C01201 according to example 3 of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. 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.
It should be emphasized that the probes included in the 50 genes listed in the present invention are 1 probe in each gene, and may include other probes that can be directly obtained by conventional techniques based on different regions of the genome sequence. For the present invention, other probes that can be directly synthesized based on the methods known in the art from the above 50 genes are all within the scope of the present invention.
The embodiment of the invention provides a probe set for detecting a pathogenic gene of an autoinflammatory disease, which comprises a pathogenic gene capable of specifically capturing and influencing an inflammatory corpuscle defect, a non-inflammatory corpuscle defect and an interferon pathway defect related autoinflammatory disease.
In an alternative embodiment, the disease-causing genes affecting inflammasome defects include MEFV, MVK, NLRP3, NLRP12, NLRC4, PLCG2, NLRP 1;
the non-inflammatory body deficiency disease-causing gene comprises TNFRSF1A, IL10, IL10RA, IL10RB, PSTPIP1, NOD2, ADAM17, LPIN2, IL1RN, IL36RN, IL1RN, SLC29A3, CARD14, SH3BP2, COPA, OTULIN, TNFAIP3, ADA2, APIS3, LACC1, HPS1, HPS4, HPS6, GUCY2C, COL7A1, FERMT1, GREAD 2, SHARPIN, RNF31, RBCK 1;
the interferon pathway defect related autoinflammatory disease pathogenic genes include PSMB8, TMEM173, TREX1, RNASEH2B, RNASEH2A, RNASEH2C, SAMHD1, ADAR, IFIH1, ISG15, ACP5, POLA1, DDX 58.
Wherein, specifically, the MEFV gene is familial Mediterranean fever pathogenic gene, the MVK gene is high IgD syndrome pathogenic gene, the NLRP3 gene is CAPS syndrome pathogenic gene, the TNFRSF1A gene is tumor necrosis factor receptor related periodic thermal syndrome pathogenic gene, the TNFRSF1A gene is tumor necrosis factor receptor related periodic thermal syndrome pathogenic gene, IL10, IL10RA, IL10RB gene is inflammatory bowel disease pathogenic gene of early onset, PSTPIP1 gene is pyogenic aseptic arthritis pyoderma gangrenosum acne syndrome pathogenic gene, NOD2 gene is Blau syndrome pathogenic gene, LPIN2 gene is Majeed syndrome and chronic recurrent multifocal myelitis and congenital erythropoiesis block anemia pathogenic gene, IL1RN gene is DIRA pathogenic gene, PSTPIP2 gene is autoinflammatory bone disease pathogenic gene, SH3BP2 gene is familial giant maxillofacial disease pathogenic gene, PSMB8 gene is chronic atypical neutrophilic dermatitis with lipodystrophy pathogenic gene.
The genes are all known autoinflammatory disease pathogenic genes in the field, 50 known autoinflammatory disease pathogenic genes with strong pertinence, stability and reliability are reasonably screened, a more reasonable probe set for detecting the autoinflammatory disease pathogenic genes is obtained, the diagnosis period of autoinflammatory diseases can be accelerated by combining a high-throughput sequencing method, the diagnosis efficiency is improved, and a plurality of new disease types can be diagnosed.
The method for obtaining the pathogenic gene for detecting the autoinflammatory disease provided by the invention comprises the following steps:
step 1: designing a corresponding capture probe aiming at a pathogenic gene of the autoinflammatory disease;
step 2: constructing a whole genome library and capturing the pathogenic gene DNA of the autoinflammatory disease by using the capture probe designed in the step 1;
and step 3: sequencing the captured and enriched DNA on a computer, and performing bioinformatics analysis, wherein the specific steps comprise: extracting 3-5ug of DNA from human blood, breaking and amplifying the DNA to construct a whole genome library, and mixing the capture probe of the autoinflammatory disease related gene with the whole genome library of a sample to be detected; hybridizing a gene fragment related to the inflammatory disease of a sample to be detected to a probe, and then adsorbing the gene fragment on a magnetic bead through the combination of the biotin and the streptavidin modified magnetic bead; the DNA fragments of the non-target area can be washed away through elution treatment, so that the disease gene fragments are enriched, the genes related to the inflammatory diseases of the individuals are captured (a plurality of samples can be captured in a mixed mode at the same time), then a new generation sequencer Illumina HiSeq 2000 is used for high-throughput sequencing and analyzing and finding out all mutation information of the genes related to the inflammatory diseases of the individuals, and therefore the variation condition of the genes related to the inflammatory diseases of the individuals is obtained, and the purpose of accurate gene diagnosis is achieved.
In an alternative embodiment, the probe for capturing and detecting the ADAR gene comprises a probe with a base sequence shown in SEQ ID No. 1; the probe for capturing and detecting the COPA gene comprises a probe with a base sequence shown as SEQ ID No. 2; the probe for capturing and detecting the NLRP3 gene comprises a probe with a base sequence shown as SEQ ID No. 3; the probe for capturing and detecting the SLC29A3 gene comprises a probe with a base sequence shown as SEQ ID No. 4; the probe for capturing and detecting the HPS1 gene comprises a probe with a base sequence shown as SEQ ID No. 5; the probe for capturing and detecting the HPS6 gene comprises a probe with a base sequence shown as SEQ ID No. 6; the probe for capturing and detecting the RNASEH2C gene comprises a probe with a base sequence shown as SEQ ID No. 7; the probe of TNFRSF1A gene includes the probe whose base sequence is shown in SEQ ID No. 8; the probe for capturing and detecting the GUCY2C gene comprises a probe with a base sequence shown as SEQ ID No. 9; the probe for capturing and detecting the MVK gene comprises a probe with a base sequence shown as SEQ ID No. 10; the probe for capturing and detecting the LACC1 gene comprises a probe with a base sequence shown as SEQ ID No. 11; the probe for capturing and detecting the RNASEH2B gene comprises a probe with a base sequence shown as SEQ ID No. 12; the probe for capturing and detecting the RNF31 gene comprises a probe with a base sequence shown as SEQ ID No. 13; the probe for capturing and detecting the PSTPIP1 gene comprises a probe with a base sequence shown as SEQ ID No. 14; the probe for capturing and detecting MEFV gene comprises a probe with a base sequence shown as SEQ ID No. 15; the probe for capturing and detecting the NOD2 gene comprises a probe with a base sequence shown as SEQ ID No. 16; the probe for capturing and detecting the PLCG2 gene comprises a probe with a base sequence shown as SEQ ID No. 17; the probe for capturing and detecting the NLRP1 gene comprises a probe with a base sequence shown as SEQ ID No. 18; the probe for capturing and detecting the CARD14 gene comprises a probe with a base sequence shown as SEQ ID No. 19; the probe for capturing and detecting the LPIN2 gene comprises a probe with a base sequence shown as SEQ ID No. 20; the probe for capturing and detecting the ACP5 gene comprises a probe with a base sequence shown as SEQ ID No. 21; the probe for capturing and detecting the RNASEH2A gene comprises a probe with a base sequence shown as SEQ ID No. 22; the probe for capturing and detecting the ADGRE2 gene comprises a probe with a base sequence shown in SEQ ID No. 23; the probe for capturing and detecting the NLRP12 gene comprises a probe with a base sequence shown as SEQ ID No. 24; the probe for capturing and detecting the ADAM17 gene comprises a probe with a base sequence shown as SEQ ID No. 25; the probe for capturing and detecting the NLRC4 gene comprises a probe with a base sequence shown as SEQ ID No. 26; the probe for capturing and detecting the IL36RN gene comprises a probe with a base sequence shown in SEQ ID No. 27; the probe for capturing and detecting the IL1RN gene comprises a probe with a base sequence shown as SEQ ID No. 28; the probe for capturing and detecting the IFIH1 gene comprises a probe with a base sequence shown as SEQ ID No. 29; the probe for capturing and detecting the AP1S3 gene comprises a probe with a base sequence shown as SEQ ID No. 30; the probe for capturing and detecting the RBCK1 gene comprises a probe with a base sequence shown as SEQ ID No. 31; the probe for capturing and detecting the FERMT1 gene comprises a probe with a base sequence shown as SEQ ID No. 32; the probe for capturing and detecting SAMHD1 gene comprises a probe with a base sequence shown as SEQ ID No. 33; the probe for capturing and detecting the ADA2 gene comprises a probe with a base sequence shown as SEQ ID No. 34; the probe for capturing and detecting the USP18 gene comprises a probe with a base sequence shown as SEQ ID No. 35; the probe for capturing and detecting the HPS4 gene comprises a probe with a base sequence shown as SEQ ID No. 36; the probe for capturing and detecting the TREX1 gene comprises a probe with a base sequence shown as SEQ ID No. 37; the probe for capturing and detecting the COL7A1 gene comprises a probe with a base sequence shown as SEQ ID No. 38; the probe for capturing and detecting the SH3BP2 gene comprises a probe with a base sequence shown in SEQ ID No. 39; the probe for capturing and detecting the OTULIN gene comprises a probe with a base sequence shown as SEQ ID No. 40; the probe for capturing and detecting the TMEM173 gene comprises a probe with a base sequence shown as SEQ ID No. 41; the probe for capturing and detecting the PSMB8 gene comprises a probe with a base sequence shown as SEQ ID No. 42; the probe for capturing and detecting the TNFAIP3 gene comprises a probe with a base sequence shown as SEQ ID No. 43; the probe of SHARPIN gene comprises a probe with a base sequence shown in SEQ ID No. 44; the probe for capturing and detecting the DDX58 gene comprises a probe with a base sequence shown as SEQ ID No. 45; the probe for capturing and detecting the POLA1 gene comprises a probe with a base sequence shown as SEQ ID No. 46; the probe for capturing and detecting the IL10 gene comprises a probe with a base sequence shown as SEQ ID No. 47; the probe for capturing and detecting the IL10RA gene comprises a probe with a base sequence shown as SEQ ID No. 48; the probe for capturing and detecting the IL10RB gene comprises a probe with a base sequence shown as SEQ ID No. 49; the probe for capturing and detecting the ISG15 gene comprises a probe with a base sequence shown as SEQ ID No. 50.
In the above, the design and preparation of the probe set for detecting the causative gene of the autoinflammatory disease comprise the steps of:
the method comprises the following steps: detection of acquisition of causative genes of autoinflammatory diseases: as in steps 1-3 above;
step two: designing and preparing a probe: based on the genomic sequence of the autoinflammatory disease-associated gene, probes designed in the past were stacked in order along the respective sequences. Each probe sequence employed in the present invention was 78 in length. The base sequence is shown in SEQ ID No. 1-50. The capture probe of this example was formed by labeling the synthesized probe with biotin.
Specific operations are (see also the method in patent WO 2013/003585): the above probes were synthesized by methods known in the art and mixed homogeneously in a total volume of 1.2ml of dH2In O, 15ul of the samples were taken and subjected to PCR amplification in three tubes using a universal PCR primer (5 '-terminal sequence GACTACATGGGACAT, 3' -terminal sequence GGAACCTACGACGTA), wherein primer GACTACATGGGACAT was a biotin-labeled primer.
PCR amplification System: 5ul of the probe solution; forward primer (25uM), 2 ul; reverse primer (25uM), 2 ul; MgCl2(50mM), 4 ul; 10X Platinum Taq polymerase buffer (from Life Technologies), 5 ul; dNTPs (10 mM each), 4 ul; platinum Taq polymerase (5U/ul, from Life Technologies), 1 ul; h2O, 27 ul; the total volume was 50 ul.
Amplification conditions: 30s at 98 ℃; (98 ℃, 30s, 60 ℃, 25s, 72 ℃, 45s)35 cycles; 72 ℃ for 5 min.
After the PCR product was purified using MinElute PCR purification kit (from Life Technologies), 500ng of PCR product was bound using MyOne streptavidin magnetic beads (from Invitrogen). Then adding alkaline NaOH to denature and elute the complementary strand without biotin; the whole magnetic beads were then washed with a 100 ℃ formamide liquid to separate the probes from the magnetic beads. The biotin-labeled probe set of this example was obtained after ethanol precipitation.
In an alternative embodiment, the probe set for detecting the causative gene of the auto-inflammatory disease is labeled with biotin.
The present embodiment also provides a kit for detecting an auto-inflammatory disease-causing gene, including the probe set for detecting an auto-inflammatory disease-causing gene described above. The kit for the pathogenic gene of the autoinflammatory disease detects the mutation of the pathogenic gene of the autoinflammatory disease to carry out molecular genetic diagnosis or disease risk prediction of the subject.
In an optional embodiment, the kit further comprises an enrichment buffer, wherein the enrichment buffer comprises human cot-1 DNA; salmon sperm DNA and primer pairs; the sequences of the primer pair are shown as SEQ ID No.51 and SEQ ID No. 52.
In an alternative embodiment, the kit further comprises a hybridization buffer, wherein the hybridization buffer comprises an SSPE buffer, a Dermax solution, EDTA and SDS.
In an optional embodiment, the kit further comprises a combination buffer solution, a rinsing solution, a NaOH solution, a Tris-HCl buffer solution, a PCR reaction solution and a TE buffer solution;
the binding buffer comprises NaCl, Tris-HCl and EDTA; the rinse liquid comprises a first rinse liquid and a second rinse liquid, and the first rinse liquid and the second rinse liquid both comprise SSC and SDS; the PCR reaction solution comprises: dNTPs, PCR amplification primers, Phusion buffer, Hotstart Phusion enzyme, DMSO and dH2O。
In an alternative embodiment, the sequences of the PCR amplification primers are shown as SEQ ID No.53 and SEQ ID No. 54.
In a preferred embodiment, the binding buffer comprises 1M NaCl, 10mM Tris-HCl (pH7.5), and 1mM EDTA; the first rinse comprises 1-fold concentration of SSC and 0.1% SDS and the second rinse comprises 0.1-fold concentration of SSC and 0.1% SDS; the content of the NaOH solution is 0.1M; the pH of the Tris-HCl buffer solution is 7.5, and the content of the Tris-HCl buffer solution is 1M; the PCR reaction solution included 2ul dNTPs, 0.5ul PCR amplification upstream primer (50pmol), 0.5ul PCR amplification downstream primer (50pmol), 20ul 5-fold concentration Phusion buffer, 1ul Hotstart Phusion enzyme (purchased from New England Biolabs), 5ul DMSO and 51ul dH2O。
Specifically, the kit for detecting the pathogenic gene of the autoinflammatory disease comprises 10 tubes of solutions:
tube 1: 160ul of the probe set of the present invention described above, at a concentration of 150ng/ul,
tube 2: the concentration of the above enrichment buffer, 208ul,
tube 3: the hybridization buffer, 800ul,
tube 4: 3.2ml of the above-mentioned binding buffer,
tube 5: 9ml of the first rinsing solution was added thereto,
tube 6: 45ml of the above-mentioned second rinsing liquid,
and (7) a tube: the above 0.1M NaOH solution, 1ml,
tube 8: 1.2ml of the above-mentioned 1M Tris-HCl buffer (pH7.5),
tube 9: the PCR reaction solution was purified by, 580ul,
tube 10: TE buffer as above, 800 ul.
In an alternative embodiment, the probe set for detecting the auto-inflammatory disease pathogenic gene is a liquid phase capture probe.
In order to more clearly and specifically describe the probe set and the kit for detecting the causative gene of the auto-inflammatory disease provided in the embodiments of the present invention, the following description will be made with reference to specific embodiments.
Example 1:
a probe set for detecting a causative gene of an auto-inflammatory disease, the probe set comprising probes capable of simultaneously capturing specifically 50 genes: ADAR, COPA, NLRP3, SLC29A3, HPS1, HPS6, RNASEH2C, TNFRSF1A, GUCY2C, MVK, LACC1, RNASEH2B, RNF31, PSTPIP1, MEFV, NOD2, PLCG2, NLRP1, CARD1, LPIN 1, ACP 1, RNASEH 21, ADGRE 1, NLRP1, ADAM1, NLRC 1, IL 68536, IL 11, IFIH1, AP1S 1, RBCK1, FERMT1, SAMHD1, ADA 1, USP1, HPS1, TREX1, COL7A1, SH 31, TNFRIN 1, TMSHELL 1, PSMIM 1, SAMHD1, PSMAFI 1, and AIARIL 68510.
The probes for capturing and detecting each gene comprise probes with base sequences shown in SEQ ID No. 1-50.
Example 2:
a kit for detecting a pathogenic gene of an autoinflammatory disease, comprising the following components: the probe set obtained in example 1 (160ul, 150ng/ul), enrichment buffer (208ul), hybridization buffer (800ul), binding buffer (3.2ml), first rinse (9ml), second rinse (45ml), NaOH solution (0.1M, 1ml), Tris-HCl buffer (1M, pH7.5, 1.2ml), PCR reaction solution (580ul), TE buffer (800 ul). Wherein the buffer solution comprises the following components:
1. enrichment buffer (per 20 ul):
human cot-1DNA (purchased from Invitrogen), 7 ul; salmon sperm DNA (purchased from Invitrogen), 3 ul;
the specific blocking primers shown as SEQ ID No.51 and SEQ ID No.52, total 10ul, each primer concentration of 1 nmol/ul:
2. hybridization buffer: 5-fold concentration of SSPE, 5-fold concentration of Denhardt's solution, 5mM EDTA, 0.1% SDS;
3. binding buffer: 1M NaCl, 10mM Tris-HCl (pH7.5), 1mM EDTA
4. First rinsing liquid: SSC solution at 1-fold concentration, 0.1% SDS
5. Second rinsing liquid: 0.1-fold concentration of SSC solution, 0.1% SDS;
6. PCR reaction solution: 2ul dNTPs (10 mM each), 0.5ul primer 1 shown in SEQ ID No.49, 0.5ul primer 2 shown in SEQ ID No.50, 20ul Phusion buffer solution with 5 times concentration, 1ul Hotstart Phusion enzyme (from New England Biolabs), 5ul DMSO, 51ul dH2O; indicates a thio modification in the middle;
some commonly used solution formulations or sources are:
SSPE buffer at 20-fold concentration, purchased from AMRESCO corporation; 50 times the concentration of Denhardt's solution, available from USB; EDTA solution: 0.5M, pH8.0, available from Mediatech; phusion buffer at 5-fold concentration, available from New England Biolab;
SSC solution: NaCl 175g and trisodium citrate 88g, pH 7.4, dH2O constant volume is 1 liter;
TE buffer solution: 10mM Tris-HCl, 1mM EDTA, adjusted pH to 8.0, water to 500 ml.
The use method of the kit for detecting the pathogenic gene of the autoinflammatory disease comprises the following steps:
step 1: DNA extraction and Whole genome library preparation:
extracting DNA from peripheral blood using Qiagen DNA mini kit (250) (purchased from Qiagen) kit or similar products; the sample quality is detected by using a spectrophotometer and agarose gel electrophoresis, and the electrophoresis band of the complete genome DNA is usually not less than 20 kb. Adjusting the concentration of DNA qualified by quality inspection to 75 ng/mu l, randomly breaking and amplifying the total amount of 3-5 mu g of DNA, and thus establishing a whole genome library;
step 2: specific capture and sequencing of target gene fragments:
(1) the following mixed system was prepared: taking 1ug of the constructed whole genome library of step 1, 13ul of enrichment buffer, and 5ul of the probe set of the present invention of example 1 (5474 oligonucleotide probes, the base sequence of which is shown in SEQ ID Nos. 1-46); placing on a PCR instrument: 95 deg.C, 7min, then 65 deg.C, 2 min;
(2) adding 23ul of hybridization buffer solution preheated at 65 ℃ into the mixed solution obtained in the step (1), and then hybridizing the mixture on a PCR instrument at 65 ℃ for 22 hours to obtain an enrichment system mixture;
(3) vortex MyOne C1 streptavidin magnetic beads (purchased from Invitrogen) to fully suspend the magnetic beads, centrifuge the magnetic beads for a short time to the bottom of the tube, and take 50ul MyOne C1 streptavidin magnetic beads into a new centrifuge tube of 1.5 ml;
(4) vortexing a 1.5ml centrifuge tube containing 50ul of MyOne C1 streptavidin magnetic beads for at least 5s to fully suspend the magnetic beads, placing the centrifuge tube on a magnetic rack to keep static for one minute after short-time centrifugation (without rotating the centrifuge tube), and carefully absorbing and removing supernatant;
(5) taking down the centrifuge tube, adding 50ul of binding buffer solution with the concentration of 1 time, rotating the nest and shaking for at least 5s, placing the centrifuge tube on a magnetic frame for standing for one minute after short-time centrifugation, carefully absorbing and removing supernatant, and repeating the operation for three times;
(6) taking down the centrifuge tube, adding 100ul of 2-fold concentration binding buffer solution, shaking the rotary nest for at least 5s, centrifuging for a short time, and placing the centrifuge tube on a magnetic frame for standing for one minute;
(7) adding the enrichment system mixture obtained in the step (2) into the centrifugal tube in the step (6), shaking the rotary nest for at least 5s (without centrifugation), and placing the rotary nest on a rotary instrument to rotate for 1 hour (60 revolutions per minute) at room temperature;
(8) then, washing the magnetic beads in the step (7) once for 15 minutes at room temperature by using the first rinsing liquid, and then washing for 3 times by using the second rinsing liquid at 65 ℃ for 15 minutes each time;
(9) eluting the magnetic beads in the step (8) with 0.1M NaOH at room temperature for 10 minutes, then carrying out vortex oscillation on the eluent for at least 5 seconds, placing the eluent on a magnetic frame for standing for one minute after short-time centrifugation, and then transferring the supernatant into a clean centrifugal tube containing 70ul of Tris-HCl buffer solution (1M, pH 7.5);
(10) the DNA solution obtained in step (9)) was purified using Qiagen MinElute Column (available from Qiagen), which was referred to the Experimental manual of Qiagen MinElute Kit;
(11) the purified DNA was finally amplified by PCR for 15 cycles;
PCR reaction solution: 2ul dNTPs (10 mM each), 0.5ul primer 1(50pmol) shown in SEQ ID No.49, 0.5ul primer 2(50pmol) shown in SEQ ID No.50, 20ul of 5 fold concentration Phusion buffer (from New England Biolabs), 1ul of hotspot Phusion enzyme (from New England Biolabs), 5ul DMSO, 51ul dH2O; indicates a thio modification in the middle. PCR program 98 ℃ 30s (1 cycle); 98 ℃, 25s,65 ℃, 30s,72 ℃, 30s (15 cycles); 72 ℃ for 5min (1cycle))；
(12) Purifying the PCR product of step (11) by using an Agencourt AMPure XP nucleic acid purification kit (purchased from Beckman Coulter) according to an instruction manual;
(13) sequencing the DNA product obtained in the step (12) on an Illumina Nexseq 500 sequencer;
and step 3: and (3) bioinformatics analysis process and result output:
(1) SNP analysis procedure
Firstly, obtaining an original short sequence;
removing joints, low-quality data and the like in the sequencing data;
(iii) positioning the short sequence at the position corresponding to the human MHC3.6M genome data using the SOAPaligner software, using the parameters: soap2.20-a-b-t-v 3-l 42-s 63-m 100-x 400, wherein the number of sequence mismatches is3 l;
counting sequencing result information, short sequence quantity, target area coverage size, average sequencing depth and the like;
the SOAPsnp is used for finding out the genotype of the locus in the target region, and the used parameters are as follows: sopsnp-i-d-o-r 0.00005-e 0.0001-M-T-u-L-s-2-T;
filtering SNPs with low quality value (quality value > 20) and low coverage (depth > 10);
the SNP is annotated by using information of CCDS, a human genome database (NCBI 37.2) and dbSNP (v138), and the generation gene, coordinates, mRNA locus, amino acid change, SNP function (missense mutation/nonsense mutation/variable shearing locus), SIFT prediction SNP influence protein function prediction and the like of a mutation locus are determined;
selecting SNPs which are common to the disease samples and do not exist in the normal group as candidate SNPs according to the information of the disease samples and the normal samples, and removing SNPs appearing in dbSNP, HAPMAP, 1000 human MHC3.6M genome and other exon sequencing items from the candidate SNPs. Meanwhile, filtering SNPs with SIFT prediction without influence on protein function as candidate SNPs related to the final disease;
(2) InDel analysis procedure
Alignment of linker-removed sequences and low-quality sequencing data to the human MHC3.6M genome using a Burrows-Wheeler Aligner (BWA) using the parameters: bwa aln-L-L31-i 10-k 2-t 7-e 40;
finding out the insertion/deletion (InDel) information contained in the sequence by using the GATK software;
annotating InDel with information of CCDS, human genome database (NCBI 37.2) and dbSNP (v138), and determining the change of gene, coordinate, mRNA site and coding region sequence of mutation site, influence on amino acid and InDel function (amino acid insertion/amino acid deletion/frame shift mutation);
and outputting a basic statistical report of the sequencing data and a data analysis result.
Example 3:
the kit effect detection experiment for detecting the pathogenic gene of the autoinflammatory disease comprises the following steps:
the result of detecting 3 samples by using the kit of the embodiment 2 of the invention proves that the capture rate of the target pathogenic/susceptible gene is satisfactory, more than 50% of original short sequences can be compared back to the reference sequence of the target region, the average effective sequencing data volume of the target region reaches 200Mb, and the average sequencing depth of the target region is 300X and is far higher than the general genetic disease diagnosis requirement (generally 100X).
1. The number of the infant is C150710C01201, the male is 4 years old, the detection result is shown in figure 1, the gene TNFRSF1A is mutated by using the kit of the invention. The mutation is generated at the chr12-6442930, namely, the exon3 generates deletion mutation c.295T > A, the disease phenotype is periodic fever, and the autosomal dominant inheritance is realized.
TABLE 1 detection result of infant patient number C150710C01201
The results of the family analysis by one generation Sanger validated sequencing are shown in table 2, the curves shown in figures 2, 3 and 4, with the patient's c.295t > a mutation being from his mother.
TABLE 2Sanger verified sequencing family analysis results
The patient detects 1 heterozygous mutation of the TNFRSF1A gene from mother, the mutation site is reported in literature, and the detection result shows that the patient has periodic fever and is autosomal dominant inheritance. The experimental results show that the probe combination kit can be applied to molecular genetic diagnosis of individual patients, can be used for general survey of high-incidence population with self-inflammation, screening high-risk population with disease incidence, providing reference for corresponding genetic counseling and the like, and accords with the development trend of accurate medical treatment. The probe and the kit for detecting the genes related to the autoinflammatory diseases have the characteristics of simple and convenient operation, low cost, good specificity, high sensitivity and the like.
Sequence listing
<110> Beijing coordination of Chinese academy of medical sciences
<120> Probe set and kit for detecting auto-inflammatory disease pathogenic gene
<130> CC18K10284CCN
<141> 2018-12-24
<160> 54
<170> SIPOSequenceListing 1.0
<210> 1
<211> 78
<212> DNA
<213> Artificial Sequence
<400> 1
cagagataaa agttcttttc ctcctggggt ttgctaatcc agttcccata gcccatatcc 60
ttcaggcctt ttttgaag 78
<210> 2
<211> 78
<212> DNA
<213> Artificial Sequence
<400> 2
catatggtga ctgacccatg cacacaaagg gggccttagc gaaactgcag aggactgatc 60
cttaaaccaa tcacatct 78
<210> 3
<211> 78
<212> DNA
<213> Artificial Sequence
<400> 3
caagcacccg ctgcaagctg gccaggtacc tggaggacct ggaggatgtg gacttgaaga 60
aatttaagat gcacttag 78
<210> 4
<211> 78
<212> DNA
<213> Artificial Sequence
<400> 4
ggcagcggcg gcgtggcgca gcggcgacag taagtgcggg ccggctcggg ctcttccggc 60
tacggtcccg gccgcccc 78
<210> 5
<211> 78
<212> DNA
<213> Artificial Sequence
<400> 5
gggatacggg aggcctccca gaggcgccgg gccagctggc cggcctgctg caccagcagg 60
tcagtgggga tgacagac 78
<210> 6
<211> 78
<212> DNA
<213> Artificial Sequence
<400> 6
ggacctgagc gccttcggcg gcgcggcgcg gctccgggag ctggtggccg gggactcagc 60
ggtccgagtc cgtggcag 78
<210> 7
<211> 78
<212> DNA
<213> Artificial Sequence
<400> 7
ctggtttact gctgtgaagg gatcgcagct ttgaatttca agctctggtt ctcagtcctc 60
gggcacctgt gcgtgaat 78
<210> 8
<211> 78
<212> DNA
<213> Artificial Sequence
<400> 8
agactaaagc gcctcctcga tgtcctccag gcagcccagc aggtccatgt cgcggagcac 60
gcgtcccagc agctccag 78
<210> 9
<211> 78
<212> DNA
<213> Artificial Sequence
<400> 9
gtgctctcct tgtctgtggt attcagctgc aagtattcca gagtgccttt tttatagctg 60
gctacccgtc tgggtttt 78
<210> 10
<211> 78
<212> DNA
<213> Artificial Sequence
<400> 10
tttccctttt aggattccca ggagccatgt tgtcagaagt cctactggtg tctgctccgg 60
ggaaagtcat ccttcatg 78
<210> 11
<211> 78
<212> DNA
<213> Artificial Sequence
<400> 11
ctgttttgat tgatcttttt ggtttgaaat tgaactctca aaaaaactgc catcagacat 60
tactgaagac tttgaatg 78
<210> 12
<211> 78
<212> DNA
<213> Artificial Sequence
<400> 12
gccgctggcg tggactgcgg ggacggggtt ggcgcccggc agcacgtgtt cctggtttca 60
ggtaaacacg cgcgcccg 78
<210> 13
<211> 78
<212> DNA
<213> Artificial Sequence
<400> 13
tggtggcccg cgaggagctg gcgagcgccc tgaggaggga ttccgggcag gcgttttccc 60
tggagcagct ccggccgc 78
<210> 14
<211> 78
<212> DNA
<213> Artificial Sequence
<400> 14
aatgacagat gccctagacc tgggtaagcc cctccagaat gacttgctag tgcggggtgg 60
ggagtctggc atctctgg 78
<210> 15
<211> 78
<212> DNA
<213> Artificial Sequence
<400> 15
ctgaccaccc actggacaga tagtcagagg agctgtgttc ttccctccat cacgtgtccc 60
agggctgaag ataggttg 78
<210> 16
<211> 78
<212> DNA
<213> Artificial Sequence
<400> 16
gcctgctccc ccagcctaat gggctttgat gggggaagag ggtggttcag cctctcacga 60
tgaggaggaa agagcaag 78
<210> 17
<211> 78
<212> DNA
<213> Artificial Sequence
<400> 17
ccttgcggaa tatgagaaga gccagatcaa gagagccctg gagctgggga cggtgatgac 60
tgtgttcagc ttccgcaa 78
<210> 18
<211> 78
<212> DNA
<213> Artificial Sequence
<400> 18
agtggcagga gtcccttttt gctgcccttc tcccagagtt ccataatgag gtgaggatgg 60
gtctccttca gggcttgg 78
<210> 19
<211> 78
<212> DNA
<213> Artificial Sequence
<400> 19
tgtgccgcag ggactccgca ctcacggcac tggacgagga gacactgtgg gagatgatgg 60
agagccaccg ccacagga 78
<210> 20
<211> 78
<212> DNA
<213> Artificial Sequence
<400> 20
cagctgcctt cccttgctgt ggggaggggg accaagccct gcccacccac tgaggtgccg 60
cctcaagaca ggtcatcc 78
<210> 21
<211> 78
<212> DNA
<213> Artificial Sequence
<400> 21
gctcgcctcg gcagcctggt cttaaagagg gacttgcccg aggcctcgat gtaagtgaca 60
gtcatctctt tggagctg 78
<210> 22
<211> 78
<212> DNA
<213> Artificial Sequence
<400> 22
gcgagctgga gagagacaat acaggccgct gtcgcctgag ttcgcctgtg cccgcggtgt 60
gccgcaagga gccttgcg 78
<210> 23
<211> 78
<212> DNA
<213> Artificial Sequence
<400> 23
ctcaaagatt gttcagattt tccacgggca aagagggaag atcttattca gaagattttt 60
ctagttaacc tgaactct 78
<210> 24
<211> 78
<212> DNA
<213> Artificial Sequence
<400> 24
agagccagca gataggacca ttcagcagcc aatgtccaaa taaggttttg ttactcgaag 60
cgctgccaac ctactgtg 78
<210> 25
<211> 78
<212> DNA
<213> Artificial Sequence
<400> 25
ctggtgaccg gatggtccgt gagatcctca aatgacttgg cagctgtgct gctatttggg 60
aaggggtcct tctcaaac 78
<210> 26
<211> 78
<212> DNA
<213> Artificial Sequence
<400> 26
tgccacccaa caagcctagc ttcttgcaga aaagttaact tggataacac ttggctaagt 60
tttctgacta atgctgga 78
<210> 27
<211> 78
<212> DNA
<213> Artificial Sequence
<400> 27
taggggagtc tacaccctgt ggagctcaag atggtcctga gtggggcgct gtgcttccgg 60
tgagtgtatg aggccctg 78
<210> 28
<211> 78
<212> DNA
<213> Artificial Sequence
<400> 28
ggaggaggag aaggtgaaga caatgctgac tcaaagggta aattattttt aggatccaag 60
tttgaaaaca attttagg 78
<210> 29
<211> 78
<212> DNA
<213> Artificial Sequence
<400> 29
acagcattct gaatagtcaa gattgggaaa tgtgataggt aattctaccc actttttgta 60
ttgtttcttt gttgaatt 78
<210> 30
<211> 78
<212> DNA
<213> Artificial Sequence
<400> 30
ctggagtctt caagtagatt tccagttaaa atgtaggctt gttcatgtat tcttccattg 60
tctgaaagcg aacaataa 78
<210> 31
<211> 78
<212> DNA
<213> Artificial Sequence
<400> 31
cccggaggta gcatttccca ggaggcacgg tcccccccag ggggatgggc acagccacgc 60
cagatggacg agaagacc 78
<210> 32
<211> 78
<212> DNA
<213> Artificial Sequence
<400> 32
ccgccggtca atttgtggaa caagtcctca tcgagtgttt cattctggtc cttggagcgg 60
gtggacaaga aaatgtag 78
<210> 33
<211> 78
<212> DNA
<213> Artificial Sequence
<400> 33
gctggactct gcttttggat gcttctcgga ggcgagttgg attttggact gaagtactgt 60
cgttccattc cttttttt 78
<210> 34
<211> 78
<212> DNA
<213> Artificial Sequence
<400> 34
atcccatctc ttcttccaga tttccatgaa agtatttttc tcactctcca acagggtact 60
gtacctgcag gaagagga 78
<210> 35
<211> 78
<212> DNA
<213> Artificial Sequence
<400> 35
cgtttgggct cctgaggcaa atctgtcagt ccatcctggc tgagtcctcg cagtccccgg 60
cagatcttga agaaaaga 78
<210> 36
<211> 78
<212> DNA
<213> Artificial Sequence
<400> 36
atacagctgg tcgcaggtgc aggtccagtg tttagatcca aatggaaaac agcagcttct 60
aagaggtaca actgttgg 78
<210> 37
<211> 78
<212> DNA
<213> Artificial Sequence
<400> 37
ggcctgagat gtgcttctgc ccacccccta ccccactccc tccccttcgg atcttaacac 60
tgggcactca cacaccca 78
<210> 38
<211> 78
<212> DNA
<213> Artificial Sequence
<400> 38
gggcctcagt cctgggcagt acctggtgag gacaggttgg aaacggtcgt cagccatctg 60
accttccccg gagacgct 78
<210> 39
<211> 78
<212> DNA
<213> Artificial Sequence
<400> 39
cctgggctgg tggcccctga gccgcatggc ctccctgggc cccaggacac cggccccgag 60
caggtcacga ggacggag 78
<210> 40
<211> 78
<212> DNA
<213> Artificial Sequence
<400> 40
cccgaagcgt ggccaggcgc gagctgcgcc gagaagatgc agtgcccggc cgagcaatga 60
gtcgggggac tatgcccc 78
<210> 41
<211> 78
<212> DNA
<213> Artificial Sequence
<400> 41
actgatgagg agctcaggct cttgggacat cgtggaggta ctgggcaccg ctgaggtctt 60
caagctgccc acagtaac 78
<210> 42
<211> 78
<212> DNA
<213> Artificial Sequence
<400> 42
cccagctgcc accaccacca ttattgattg gcttcccggt actggtgcag caggtcactg 60
acatctgtac tttctact 78
<210> 43
<211> 78
<212> DNA
<213> Artificial Sequence
<400> 43
agctgtgaag atacgggaga gaactccaga agacattttt aaacctacta atgggatcat 60
tcatcatttt aaaaccat 78
<210> 44
<211> 78
<212> DNA
<213> Artificial Sequence
<400> 44
cctctgggtg ctacacatct cacagccagg gcggtctggg gcattgatga aggtgcagga 60
aggacaggac cagctggg 78
<210> 45
<211> 78
<212> DNA
<213> Artificial Sequence
<400> 45
atttctgctg gatcaaatgg tatcttctca aaatgaaagt ccttccactt cgagtacagt 60
gtctgaactc cagttgca 78
<210> 46
<211> 78
<212> DNA
<213> Artificial Sequence
<400> 46
ccagttttgg gctggttggc gcggaatcgg gagattcggg accatggcac ctgtgcacgg 60
cgacgactgt gagatagg 78
<210> 47
<211> 78
<212> DNA
<213> Artificial Sequence
<400> 47
agtcgccacc ctgatgtctc agtttcgtat cttcattgtc atgtaggctt ctatgtagtt 60
gatgaagatg tcaaactc 78
<210> 48
<211> 78
<212> DNA
<213> Artificial Sequence
<400> 48
ctccggcccc ggacgatgcg gcgcgcccag gatgctgccg tgcctcgtag tgctgctggc 60
ggcgctcctc agcctccg 78
<210> 49
<211> 78
<212> DNA
<213> Artificial Sequence
<400> 49
gtgtgcttgg aggaagccgc ggaaccccca gcgtccgtcc atggcgtgga gccttgggag 60
ctggctgggt ggctgcct 78
<210> 50
<211> 78
<212> DNA
<213> Artificial Sequence
<400> 50
agcgaactca tctttgccag tacaggagct tgtgccgtgg cccacagccc acagcccaca 60
gccatggtaa ggcagatg 78
<210> 51
<211> 50
<212> DNA
<213> Artificial Sequence
<400> 51
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct 50
<210> 52
<211> 50
<212> DNA
<213> Artificial Sequence
<400> 52
caagcagaag acggcatacg agatcggtct cggcattcct gctgaaccgc 50
<210> 53
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 53
aatgatacgg cgaccaccga g 21
<210> 54
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 54
caagcagaag acggcatacg a 21
Claims (8)
1. A probe set for detecting a causative gene of an auto-inflammatory disease, characterized in that: the probe set comprises a probe which can simultaneously and specifically capture pathogenic genes affecting inflammatory corpuscle deficiency, non-inflammatory corpuscle deficiency pathogenic genes and interferon pathway deficiency-related autoinflammatory disease pathogenic genes;
the pathogenic genes affecting the inflammatory body defect comprise MEFV, MVK, NLRP3, NLRP12, NLRC4, PLCG2 and NLRP 1;
the non-inflammatory body deficiency disease-causing genes comprise TNFRSF1A, IL10, IL10RA, IL10RB, PSTPIP1, NOD2, ADAM17, LPIN2, IL1RN, IL36RN, SLC29A3, CARD14, SH3BP2, COPA, OTULIN, TNFAIP3, ADA2, APIS3, LACC1, HPS1, HPS4, HPS6, CYGU 2C, COL7A1, FERMT1, ADGRE2, SHARPPIN, RNF31, RBCK 1;
the interferon pathway defect related autoinflammatory disease pathogenic gene comprises PSMB8, TMEM173, TREX1, RNASEH2B, RNASEH2A, RNASEH2C, SAMHD1, ADAR, IFIH1, ISG15, ACP5, POLA1 and DDX5, and the probe for capturing and detecting the ADAR gene comprises a probe with a base sequence shown as SEQ ID No. 1; the probe for capturing and detecting the COPA gene comprises a probe with a base sequence shown as SEQ ID No. 2; the probe for capturing and detecting the NLRP3 gene comprises a probe with a base sequence shown as SEQ ID No. 3; the probe for capturing and detecting the SLC29A3 gene comprises a probe with a base sequence shown as SEQ ID No. 4; the probe for capturing and detecting the HPS1 gene comprises a probe with a base sequence shown as SEQ ID No. 5; the probe for capturing and detecting the HPS6 gene comprises a probe with a base sequence shown as SEQ ID No. 6; the probe for capturing and detecting the RNASEH2C gene comprises a probe with a base sequence shown as SEQ ID No. 7; the probe of TNFRSF1A gene includes the probe whose base sequence is shown in SEQ ID No. 8; the probe for capturing and detecting the GUCY2C gene comprises a probe with a base sequence shown as SEQ ID No. 9; the probe for capturing and detecting the MVK gene comprises a probe with a base sequence shown as SEQ ID No. 10; the probe for capturing and detecting the LACC1 gene comprises a probe with a base sequence shown as SEQ ID No. 11; the probe for capturing and detecting the RNASEH2B gene comprises a probe with a base sequence shown as SEQ ID No. 12; the probe for capturing and detecting the RNF31 gene comprises a probe with a base sequence shown as SEQ ID No. 13; the probe for capturing and detecting the PSTPIP1 gene comprises a probe with a base sequence shown as SEQ ID No. 14; the probe for capturing and detecting MEFV gene comprises a probe with a base sequence shown in SEQ ID No. 15; the probe for capturing and detecting the NOD2 gene comprises a probe with a base sequence shown as SEQ ID No. 16; the probe for capturing and detecting the PLCG2 gene comprises a probe with a base sequence shown as SEQ ID No. 17; the probe for capturing and detecting the NLRP1 gene comprises a probe with a base sequence shown as SEQ ID No. 18; the probe for capturing and detecting the CARD14 gene comprises a probe with a base sequence shown as SEQ ID No. 19; the probe for capturing and detecting the LPIN2 gene comprises a probe with a base sequence shown as SEQ ID No. 20; the probe for capturing and detecting the ACP5 gene comprises a probe with a base sequence shown as SEQ ID No. 21; the probe for capturing and detecting the RNASEH2A gene comprises a probe with a base sequence shown as SEQ ID No. 22; the probe for capturing and detecting the ADGRE2 gene comprises a probe with a base sequence shown as SEQ ID No. 23; the probe for capturing and detecting the NLRP12 gene comprises a probe with a base sequence shown as SEQ ID No. 24; the probe for capturing and detecting the ADAM17 gene comprises a probe with a base sequence shown as SEQ ID No. 25; the probe for capturing and detecting the NLRC4 gene comprises a probe with a base sequence shown as SEQ ID No. 26; the probe for capturing and detecting the IL36RN gene comprises a probe with a base sequence shown in SEQ ID No. 27; the probe for capturing and detecting the IL1RN gene comprises a probe with a base sequence shown as SEQ ID No. 28; the probe for capturing and detecting the IFIH1 gene comprises a probe with a base sequence shown as SEQ ID No. 29; the probe for capturing and detecting the AP1S3 gene comprises a probe with a base sequence shown as SEQ ID No. 30; the probe for capturing and detecting the RBCK1 gene comprises a probe with a base sequence shown as SEQ ID No. 31; the probe for capturing and detecting the FERMT1 gene comprises a probe with a base sequence shown as SEQ ID No. 32; the probe for capturing and detecting SAMHD1 gene comprises a probe with a base sequence shown as SEQ ID No. 33; the probe for capturing and detecting the ADA2 gene comprises a probe with a base sequence shown as SEQ ID No. 34; the probe for capturing and detecting the USP18 gene comprises a probe with a base sequence shown as SEQ ID No. 35; the probe for capturing and detecting the HPS4 gene comprises a probe with a base sequence shown as SEQ ID No. 36; the probe for capturing and detecting the TREX1 gene comprises a probe with a base sequence shown as SEQ ID No. 37; the probe for capturing and detecting the COL7A1 gene comprises a probe with a base sequence shown as SEQ ID No. 38; the probe for capturing and detecting the SH3BP2 gene comprises a probe with a base sequence shown as SEQ ID No. 39; the probe for capturing and detecting the OTULIN gene comprises a probe with a base sequence shown as SEQ ID No. 40; the probe for capturing and detecting the TMEM173 gene comprises a probe with a base sequence shown as SEQ ID No. 41; the probe for capturing and detecting the PSMB8 gene comprises a probe with a base sequence shown as SEQ ID No. 42; the probe for capturing and detecting the TNFAIP3 gene comprises a probe with a base sequence shown as SEQ ID No. 43; the probe of the SHARPIN gene comprises a probe with a base sequence shown as SEQ ID No. 44; the probe for capturing and detecting the DDX58 gene comprises a probe with a base sequence shown as SEQ ID No. 45; the probe for capturing and detecting the POLA1 gene comprises a probe with a base sequence shown as SEQ ID No. 46; the probe for capturing and detecting the IL10 gene comprises a probe with a base sequence shown as SEQ ID No. 47; the probe for capturing and detecting the IL10RA gene comprises a probe with a base sequence shown as SEQ ID No. 48; the probe for capturing and detecting the IL10RB gene comprises a probe with a base sequence shown as SEQ ID No. 49; the probe for capturing and detecting the ISG15 gene comprises a probe with a base sequence shown as SEQ ID No. 50.
2. A kit for detecting a causative gene of an auto-inflammatory disease, characterized in that: a probe set for detecting a causative gene of an auto-inflammatory disease according to claim 1.
3. The kit for detecting a causative gene of an auto-inflammatory disease according to claim 2, which is characterized in that: further comprising an enrichment buffer comprising human cot-1 DNA; salmon sperm DNA and primer pairs; the sequences of the primer pair are shown as SEQ ID No.51 and SEQ ID No. 52.
4. The kit for detecting a causative gene of an auto-inflammatory disease according to claim 3, which is characterized in that: further included are hybridization-containing buffers including SSPE buffer, Dermart solution, EDTA and SDS.
5. The kit for detecting a causative gene of an auto-inflammatory disease according to claim 4, which is characterized in that: further comprises a combination buffer solution, a rinsing solution, a NaOH solution, a Tris-HCl buffer solution, a PCR reaction solution and a TE buffer solution.
6. The kit for detecting a causative gene of an auto-inflammatory disease according to claim 5, which is characterized in that: the binding buffer comprises NaCl, Tris-HCl and EDTA; the rinse liquid comprises a first rinse liquid and a second rinse liquid, and the first rinse liquid and the second rinse liquid both comprise SSC and SDS; the PCR reaction solution comprises: dNTPs, PCR amplification primers, Phusion buffer, hotspot Phusion enzyme, DMSO, and dH 20.
7. The kit for detecting a causative gene of an auto-inflammatory disease according to claim 6, which is characterized in that: the sequences of the PCR amplification primers are shown as SEQ ID No.53 and SEQ ID No. 54.
8. The kit for detecting a causative gene of an auto-inflammatory disease according to claim 2, which is characterized in that: the probe set for detecting the pathogenic gene of the autoinflammatory disease is a liquid phase capture probe.
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| CN110512004B (en) * | 2019-10-11 | 2020-11-24 | 新乡医学院 | Molecular marker for detecting esophageal cancer cell migration and invasion capacity and application thereof |
| CN112725368B (en) * | 2019-10-28 | 2023-05-09 | 深圳市儿童医院 | Mutant gene of autoinflammatory disease related gene TNFAIP3 and application thereof |
| CN110734970A (en) * | 2019-10-29 | 2020-01-31 | 苏州乾康基因有限公司 | auto-inflammatory monogenic disease detection primer group and method |
| CN112553324A (en) * | 2020-12-28 | 2021-03-26 | 济南艾迪康医学检验中心有限公司 | Primer, kit and method for detecting TREX1 gene mutation |
| CN113151447A (en) * | 2021-05-08 | 2021-07-23 | 深圳市儿童医院 | Capture probe and kit for primary atopic disease related gene and application thereof |
| CN114369658B (en) * | 2022-02-18 | 2024-04-16 | 中国医学科学院北京协和医院 | Application of mutant form of NLRP3 related autoinflammatory disease related gene NLRP3 |
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| CN104603289A (en) * | 2012-06-15 | 2015-05-06 | 哈里·斯泰利 | Methods of detecting diseases or conditions |
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