CN109371123A - For detecting the probe groups and kit of auto-inflammatory disease Disease-causing gene - Google Patents
For detecting the probe groups and kit of auto-inflammatory disease Disease-causing gene Download PDFInfo
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- CN109371123A CN109371123A CN201811584606.9A CN201811584606A CN109371123A CN 109371123 A CN109371123 A CN 109371123A CN 201811584606 A CN201811584606 A CN 201811584606A CN 109371123 A CN109371123 A CN 109371123A
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Abstract
The present invention propose it is a kind of for detecting the probe groups and kit of auto-inflammatory disease Disease-causing gene, the probe groups include can specificity capture simultaneously influence the probe of the small volume defect Disease-causing gene of inflammation, noninflammatory small volume defect Disease-causing gene, interferon access defect correlation auto-inflammatory disease Disease-causing gene.The present invention provides a kind of for detecting the probe groups and kit of auto-inflammatory disease Disease-causing gene, auto-inflammatory disease Disease-causing gene known to 50 kinds can be detected simultaneously, it is with strong points based on screening, reliable and stable gene obtains the probe groups for detecting auto-inflammatory disease Disease-causing gene, in conjunction with the method for high-flux sequence, it can accelerate the interval between diagnosis to auto-inflammatory disease, improve diagnosis efficiency, diagnosable several new diseases, it is detected especially for auto-inflammatory disease infant, it can achieve neither missing inspection to fail to pinpoint a disease in diagnosis, relation analysis data volume is less again, shorten the effect of inspection cycle.
Description
Technical field
The invention belongs to biomedicine technical fields, more particularly to one kind is for detecting auto-inflammatory disease Disease-causing gene
Probe groups and kit.
Background technique
Auto-inflammatory disease (autoinflammatory disorder, AIDs) is one group of heredity inflammation disease, with
Fever, fash, arthralgia, arthritis, Eye disease etc. are prominent symptom, are often accompanied by the raising of inflammatory protein.Auto-inflammatory
Disease is the major class in primary immunodeficiency disease, and the Disease-causing gene by the end of discovery in 2018 has 50 kinds, new gene
In constantly discovering.
Second generation high throughput sequencing technologies (Next Generation Sequencing, NGS) are called high-flux sequence skill
Art, the core concept of second generation sequencing technologies are that (Sequencing by Synthesis) is sequenced in synthesis, that is, pass through capture
The sequence of newly synthesized end marked to determine DNA, existing technology platform mainly includes Roche/454FLX, Life
Semiconductor sequencing technologies, Illumina/Solexa Genome Analyzer and the Applied Biosystems of Tech
SOLID system.On the basis of the sequencing approaches such as Sanger, by technological innovation, with four kinds of fluorescent marker of different colours
Different dNTP, it is every to add a kind of dNTP and release different fluorescence when archaeal dna polymerase synthesizes complementary strand, according to catching
The fluorescence signal and the specific software processing of process caught, to obtain the sequence information of DNA to be measured.
Have for second generation high throughput sequencing technologies (next-generation sequencing technology) fast
Speed, accurate, low cost advantage, can simultaneously be mutated the various types of multiple genes and detect, be widely used in
The cause of disease detection and molecular genetics diagnosis of genetic defect, however up to the present, there has been no specifically for auto-inflammatory always
Property disease related gene detection high-throughput capture probe or kit examined so that the progress of this disease related fields seriously lags
Disconnected rate is low, and genetic counselling and necessary antenatal intervening measure can not be implemented, not only huge to patient and its family's bring
Painful and heavy financial burden also seriously hinders increasing substantially for China's entirety population quality.
Summary of the invention
The present invention for the above technical issues, proposes a kind of for detecting the probe of auto-inflammatory disease Disease-causing gene
Group and kit can detect auto-inflammatory disease related gene known to 50 kinds simultaneously.Improve detection auto-inflammatory disease
The sensitivity and specificity of Disease-causing gene, shorten detection time, can achieve neither missing inspection and fail to pinpoint a disease in diagnosis, and relation analysis data volume
It is less, shorten the effect of inspection cycle.
In order to achieve the above object, the technical solution adopted by the present invention are as follows:
It is a kind of for detecting the probe groups of auto-inflammatory disease Disease-causing gene, the probe groups include can specificity simultaneously
Capture influences itself related inflammation of the small volume defect Disease-causing gene of inflammation, noninflammatory small volume defect Disease-causing gene, interferon access defect
The probe of disease property disease Disease-causing gene.
Preferably, the small volume defect Disease-causing gene of influence inflammation include MEFV, MVK, NLRP3, NLRP12, NLRC4,
PLCG2,NLRP1；
The noninflammatory small volume defect Disease-causing gene include 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、ADGRE2、SHARPIN、
RNF31,RBCK1；
The interferon access defect correlation auto-inflammatory disease Disease-causing gene include PSMB8, TMEM173, TREX1,
RNASEH2B、RNASEH2A、RNASEH2C、SAMHD1、ADAR、IFIH1、ISG15、ACP5、POLA1、DDX58。
Preferably, the probe of capture and detection ADAR gene includes base sequence probe as shown in SEQ ID No.1；
The probe of capture and detection COPA gene includes base sequence probe as shown in SEQ ID No.2；Capture and detection NLRP3 base
The probe of cause includes base sequence probe as shown in SEQ ID No.3；The probe of capture and detection SLC29A3 gene includes alkali
Basic sequence probe as shown in SEQ ID No.4；The probe of capture and detection HPS1 gene includes base sequence such as SEQ ID
Probe shown in No.5；The probe of capture and detection HPS6 gene includes base sequence probe as shown in SEQ ID No.6；It catches
The probe for obtaining and detecting RNASEH2C gene includes base sequence probe as shown in SEQ ID No.7；TNFRSF1A gene
Probe includes base sequence probe as shown in SEQ ID No.8；The probe of capture and detection GUCY2C gene includes base sequence
Arrange the probe as shown in SEQ ID No.9；The probe of capture and detection mvk gene includes base sequence such as SEQ ID No.10 institute
The probe shown；The probe of capture and detection LACC1 gene includes base sequence probe as shown in SEQ ID No.11；Capture and
The probe for detecting RNASEH2B gene includes base sequence probe as shown in SEQ ID No.12；Capture and detection RNF31 base
The probe of cause includes base sequence probe as shown in SEQ ID No.13；Capture and the probe for detecting PSTPIP1 gene include
Base sequence probe as shown in SEQ ID No.14；The probe of capture and detection MEFV gene includes base sequence such as SEQ ID
Probe shown in No.15；The probe of capture and detection NOD2 gene includes base sequence probe as shown in SEQ ID No.16；
The probe of capture and detection PLCG2 gene includes base sequence probe as shown in SEQ ID No.17；Capture and detection NLRP1
The probe of gene includes base sequence probe as shown in SEQ ID No.18；Capture and the probe for detecting CARD14 gene include
Base sequence probe as shown in SEQ ID No.19；The probe of capture and detection LPIN2 gene includes base sequence such as SEQ
Probe shown in ID No.20；The probe of capture and detection ACP5 gene includes base sequence spy as shown in SEQ ID No.21
Needle；The probe of capture and detection RNASEH2A gene includes base sequence probe as shown in SEQ ID No.22；Capture and inspection
The probe for surveying ADGRE2 gene includes base sequence probe as shown in SEQ ID No.23；Capture and detect NLRP12 gene
Probe includes base sequence probe as shown in SEQ ID No.24；The probe of capture and detection ADAM17 gene includes base sequence
Arrange the probe as shown in SEQ ID No.25；The probe of capture and detection NLRC4 gene includes base sequence such as SEQ ID
Probe shown in No.26；The probe of capture and detection IL36RN gene includes base sequence spy as shown in SEQ ID No.27
Needle；The probe of capture and detection IL1RN gene includes base sequence probe as shown in SEQ ID No.28；Capture and detection
The probe of IFIH1 gene includes base sequence probe as shown in SEQ ID No.29；The probe of capture and detection AP1S3 gene
Including base sequence probe as shown in SEQ ID No.30；Capture and detection RBCK1 gene probe include base sequence such as
Probe shown in SEQ ID No.31；The probe of capture and detection FERMT1 gene includes base sequence such as SEQ ID No.32 institute
The probe shown；The probe of capture and detection SAMHD1 gene includes base sequence probe as shown in SEQ ID No.33；Capture
Probe with detection ADA2 gene includes base sequence probe as shown in SEQ ID No.34；Capture and detection USP18 gene
Probe include base sequence probe as shown in SEQ ID No.35；The probe of capture and detection HPS4 gene includes base sequence
Arrange the probe as shown in SEQ ID No.36；The probe of capture and detection TREX1 gene includes base sequence such as SEQ ID
Probe shown in No.37；The probe of capture and detection COL7A1 gene includes base sequence spy as shown in SEQ ID No.38
Needle；The probe of capture and detection SH3BP2 gene includes base sequence probe as shown in SEQ ID No.39；Capture and detection
The probe of OTULIN gene includes base sequence probe as shown in SEQ ID No.40；Capture and detect TMEM173 gene
Probe includes base sequence probe as shown in SEQ ID No.41；The probe of capture and detection PSMB8 gene includes base sequence
Arrange the probe as shown in SEQ ID No.42；The probe of capture and detection TNFAIP3 gene includes base sequence such as SEQ ID
Probe shown in No.43；The probe of SHARPIN gene includes base sequence probe as shown in SEQ ID No.44；Capture and
The probe for detecting DDX58 gene includes base sequence probe as shown in SEQ ID No.45；Capture and detect POLA1 gene
Probe includes base sequence probe as shown in SEQ ID No.46；The probe of capture and detection IL10 gene includes base sequence
The probe as shown in SEQ ID No.47；The probe of capture and detection IL10RA gene includes base sequence such as SEQ ID No.48
Shown in probe；The probe of capture and detection IL10RB gene includes base sequence probe as shown in SEQ ID No.49；It catches
The probe for obtaining and detecting ISG15 gene includes base sequence probe as shown in SEQ ID No.50.
It is a kind of for detecting the kit of auto-inflammatory disease Disease-causing gene, including above-mentioned for detecting auto-inflammatory
The probe groups of property disease Disease-causing gene.
Preferably, further comprising harvesting buffer, the harvesting buffer includes people cot-1DNA；Salmon essence
DNA and primer pair；The sequence of the primer pair is as shown in SEQ ID No.51 and SEQ ID No.52.
Preferably, further comprising containing hybridization buffer, the hybridization buffer includes SSPE buffer, steps on Hart
Solution, EDTA and SDS.
Preferably, it is anti-to further comprise combination buffer, rinsing liquid, NaOH solution, Tris-HCl buffer, PCR
Answer liquid and TE buffer；
The combination buffer includes NaCl, Tris-HCl and EDTA；The rinsing liquid includes the first rinsing liquid and second
Rinsing liquid, first rinsing liquid and the second rinsing liquid include SSC and SDS；The PCR reaction solution includes: that dNTPs, PCR expand
Increase primer, Phusion buffer, Hotstart Phusion enzyme, DMSO and dH2O。
Preferably, the sequence of the PCR amplification primer is as shown in SEQ ID No.53 and SEQ ID No.54.
Preferably, the probe groups for detecting auto-inflammatory disease Disease-causing gene are liquid phase capture probe.
Compared with prior art, the advantages and positive effects of the present invention are:
The present invention provides a kind of for detecting the probe groups and kit of auto-inflammatory disease Disease-causing gene, can examine simultaneously
Auto-inflammatory disease Disease-causing gene known to 50 kinds is surveyed, is obtained based on strong points, reliable and stable gene is screened for detecting
The probe groups of auto-inflammatory disease Disease-causing gene can accelerate in conjunction with the method for high-flux sequence to auto-inflammatory disease
Interval between diagnosis, improves diagnosis efficiency, and diagnosable several new diseases are examined especially for auto-inflammatory disease infant
It surveys, can achieve neither missing inspection and fail to pinpoint a disease in diagnosis, and relation analysis data volume is less, shorten the effect of inspection cycle；Probe groups of the present invention
With kit have the advantages that efficiently, it is accurate, quickly, low price, for auto-inflammatory disease pathogenic related gene detection and
Molecular genetics diagnosis, meaning are very great.
Detailed description of the invention
Some specific embodiments of the present invention is described in detail by way of example and not limitation with reference to the accompanying drawings hereinafter.
Identical appended drawing reference denotes same or similar part or part in attached drawing.It should be appreciated by those skilled in the art that these
What attached drawing was not necessarily drawn to scale.In attached drawing:
Fig. 1 is 3 number C150710C01201 infant kit test result gene group picture of the embodiment of the present invention；
Fig. 2 is 3 number C150710C01201 infant Sanger sequencing analysis curve graph of the embodiment of the present invention；
Fig. 3 is father's Sanger sequencing analysis curve graph of 3 number C150710C01201 infant of the embodiment of the present invention；
Fig. 4 is mother's Sanger sequencing analysis curve graph of 3 number C150710C01201 infant of the embodiment of the present invention.
Specific embodiment
Below technical solution in the embodiment of the present invention describe clear and completely, it is clear that described implementation
Example is only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, this field is common
Technical staff's every other embodiment obtained without making creative work belongs to the model that the present invention protects
It encloses.
The probe, it is emphasized that included in 50 genes cited by the present invention is additionally needed, in respectively each gene
1 probe, may also include based on genome sequence different zones use conventional technical means can be directly obtained its
His probe.For the present invention, it by above-mentioned 50 genes enumerated, can directly be synthesized based on approach well known
Other probes, are within the scope of the invention.
The embodiment of the invention provides a kind of for detecting the probe groups of auto-inflammatory disease Disease-causing gene, the probe
Group includes specific capture influencing the small volume defect Disease-causing gene of inflammation, noninflammatory small volume defect Disease-causing gene, interferon simultaneously
Access defect correlation auto-inflammatory disease Disease-causing gene.
In an alternative embodiment, the small volume defect Disease-causing gene of influence inflammation include MEFV, MVK, NLRP3,
NLRP12,NLRC4,PLCG2,NLRP1；
The noninflammatory small volume defect Disease-causing gene include 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、ADGRE2、SHARPIN、
RNF31,RBCK1；
The interferon access defect correlation auto-inflammatory disease Disease-causing gene include PSMB8, TMEM173, TREX1,
RNASEH2B、RNASEH2A、RNASEH2C、SAMHD1、ADAR、IFIH1、ISG15、ACP5、POLA1、DDX58。
Wherein specifically, the MEFV gene is familial Mediterranean fever Disease-causing gene, mvk gene is high IgD syndrome
Disease-causing gene, NLRP3 gene are CAPS syndrome Disease-causing gene, and TNFRSF1A gene is Tumor Necrosis Factor Receptors relevant week
Phase hot syndrome Disease-causing gene, TNFRSF1A gene are the relevant periodic fever syndrome Disease-causing gene of Tumor Necrosis Factor Receptors,
IL10, IL10RA, IL10RB gene are the inflammatory bowel disease Disease-causing gene of early stage onset, and PSTPIP1 gene is suppurative sterile
Property arthritis pyoderma gangrene acne syndrome Disease-causing gene, NOD2 gene be Blau syndrome Disease-causing gene, LPIN2 gene
For Majeed syndrome and the multifocal myelitis of chronic recurrent and congenital Dyserythropoiesis anaemia Disease-causing gene,
IL1RN gene is DIRA Disease-causing gene, and PSTPIP2 gene is auto-inflammatory osteopathy Disease-causing gene, and SH3BP2 gene is family
Property cherubism Disease-causing gene, PSMB8 gene is that chronic atypical neutrophil leucocyte dermatitis is caused a disease base with lipodystrophy
Cause.
Said gene be in art technology known auto-inflammatory disease Disease-causing gene, the present invention rationally screen needle
Auto-inflammatory disease Disease-causing gene known to strong to property, reliable and stable 50 kinds obtains more reasonable for detecting itself
The probe groups of diseases associated with inflammation Disease-causing gene can accelerate the diagnosis to auto-inflammatory disease in conjunction with the method for high-flux sequence
Period improves diagnosis efficiency, diagnosable several new diseases.
The wherein acquisition methods of detection auto-inflammatory disease Disease-causing gene provided by the present invention, comprising the following steps:
Step 1: designing corresponding capture probe for auto-inflammatory disease Disease-causing gene；
Step 2: simultaneously being caused a disease using the capture probe designed in step 1 to auto-inflammatory disease in building full-length genome library
Gene DNA is captured；
Step 3: machine on the DNA after capture and enrichment is sequenced, carry out bioinformatic analysis, specific steps include: from
3-5ug DNA is extracted in blood of human body, and is interrupted, is expanded, to construct full-length genome library, utilizes of the invention itself
The full-length genome library mixing of the capture probe and sample to be tested of diseases associated with inflammation related gene；Sample to be tested auto-inflammatory disease
Sick related gene segment is hybridised on probe, and then is combined and be adsorbed to by the magnetic bead that biotin and Streptavidin are modified
On magnetic bead；The DNA fragmentation of nontarget area can be washed off by elution processing, to be enriched with disease gene segment, thus
Auto-inflammatory disease related gene has been captured out (can simultaneously the multiple samples of hybrid acquisition), has recycled new-generation sequencing instrument
Illumina HiSeq 2000 carries out high-flux sequence and analyzes all abrupt informations for finding out disease related gene, thus
To the variation situation of individual auto-inflammatory disease related gene, to achieve the purpose that make accurate gene diagnosis.
In an alternative embodiment, the probe of capture and detection ADAR gene includes base sequence such as SEQ ID No.1 institute
The probe shown；The probe of capture and detection COPA gene includes base sequence probe as shown in SEQ ID No.2；Capture and inspection
The probe for surveying NLRP3 gene includes base sequence probe as shown in SEQ ID No.3；Capture and detect SLC29A3 gene
Probe includes base sequence probe as shown in SEQ ID No.4；The probe of capture and detection HPS1 gene includes base sequence
The probe as shown in SEQ ID No.5；The probe of capture and detection HPS6 gene includes base sequence as shown in SEQ ID No.6
Probe；The probe of capture and detection RNASEH2C gene includes base sequence probe as shown in SEQ ID No.7；
The probe of TNFRSF1A gene includes base sequence probe as shown in SEQ ID No.8；Capture and detect GUCY2C gene
Probe includes base sequence probe as shown in SEQ ID No.9；Capture and detection mvk gene probe include base sequence such as
Probe shown in SEQ ID No.10；The probe of capture and detection LACC1 gene includes base sequence such as SEQ ID No.11 institute
The probe shown；The probe of capture and detection RNASEH2B gene includes base sequence probe as shown in SEQ ID No.12；It catches
The probe for obtaining and detecting RNF31 gene includes base sequence probe as shown in SEQ ID No.13；Capture and detection PSTPIP1
The probe of gene includes base sequence probe as shown in SEQ ID No.14；The probe of capture and detection MEFV gene includes alkali
Basic sequence probe as shown in SEQ ID No.15；The probe of capture and detection NOD2 gene includes base sequence such as SEQ ID
Probe shown in No.16；The probe of capture and detection PLCG2 gene includes base sequence spy as shown in SEQ ID No.17
Needle；The probe of capture and detection NLRP1 gene includes base sequence probe as shown in SEQ ID No.18；Capture and detection
The probe of CARD14 gene includes base sequence probe as shown in SEQ ID No.19；The spy of capture and detection LPIN2 gene
Needle includes base sequence probe as shown in SEQ ID No.20；Capture and detection ACP5 gene probe include base sequence such as
Probe shown in SEQ ID No.21；The probe of capture and detection RNASEH2A gene includes base sequence such as SEQ ID No.22
Shown in probe；The probe of capture and detection ADGRE2 gene includes base sequence probe as shown in SEQ ID No.23；It catches
The probe for obtaining and detecting NLRP12 gene includes base sequence probe as shown in SEQ ID No.24；Capture and detection ADAM17
The probe of gene includes base sequence probe as shown in SEQ ID No.25；Capture and the probe for detecting NLRC4 gene include
Base sequence probe as shown in SEQ ID No.26；The probe of capture and detection IL36RN gene includes base sequence such as SEQ
Probe shown in ID No.27；The probe of capture and detection IL1RN gene includes base sequence as shown in SEQ ID No.28
Probe；The probe of capture and detection IFIH1 gene includes base sequence probe as shown in SEQ ID No.29；Capture and detection
The probe of AP1S3 gene includes base sequence probe as shown in SEQ ID No.30；The probe of capture and detection RBCK1 gene
Including base sequence probe as shown in SEQ ID No.31；Capture and detection FERMT1 gene probe include base sequence such as
Probe shown in SEQ ID No.32；The probe of capture and detection SAMHD1 gene includes base sequence such as SEQ ID No.33 institute
The probe shown；The probe of capture and detection ADA2 gene includes base sequence probe as shown in SEQ ID No.34；Capture and
The probe for detecting USP18 gene includes base sequence probe as shown in SEQ ID No.35；Capture and detect HPS4 gene
Probe includes base sequence probe as shown in SEQ ID No.36；The probe of capture and detection TREX1 gene includes base sequence
Arrange the probe as shown in SEQ ID No.37；The probe of capture and detection COL7A1 gene includes base sequence such as SEQ ID
Probe shown in No.38；The probe of capture and detection SH3BP2 gene includes base sequence spy as shown in SEQ ID No.39
Needle；The probe of capture and detection OTULIN gene includes base sequence probe as shown in SEQ ID No.40；Capture and detection
The probe of TMEM173 gene includes base sequence probe as shown in SEQ ID No.41；The spy of capture and detection PSMB8 gene
Needle includes base sequence probe as shown in SEQ ID No.42；The probe of capture and detection TNFAIP3 gene includes base sequence
Arrange the probe as shown in SEQ ID No.43；The probe of SHARPIN gene includes base sequence as shown in SEQ ID No.44
Probe；The probe of capture and detection DDX58 gene includes base sequence probe as shown in SEQ ID No.45；Capture and detection
The probe of POLA1 gene includes base sequence probe as shown in SEQ ID No.46；The probe of capture and detection IL10 gene
Including base sequence probe as shown in SEQ ID No.47；Capture and detection IL10RA gene probe include base sequence such as
Probe shown in SEQ ID No.48；The probe of capture and detection IL10RB gene includes base sequence such as SEQ ID No.49 institute
The probe shown；The probe of capture and detection ISG15 gene includes base sequence probe as shown in SEQ ID No.50.
Among the above, the probe groups for detecting auto-inflammatory disease Disease-causing gene are designed and prepared including following
Step:
Step 1: the acquisition of detection auto-inflammatory disease Disease-causing gene: such as the content of above-mentioned steps 1-3；
Step 2: the design and preparation of probe: according to the genome sequence of above-mentioned auto-inflammatory disease related gene, edge
Each sequence stack gradually over design probe.Every probe sequence length that the present invention uses is 78.Base sequence such as SEQ
Shown in ID No.1-50.The probe of above-mentioned synthesis is subjected to biotin labeling, forms the capture probe of the present embodiment.
Concrete operations are (see also the method in patent WO2013/003585): being synthesized using approach well known
Above-mentioned probe is evenly mixed in the dH of total volume 1.2ml2In O, taking wherein 15ul using general PCR primer, (5 ' terminal sequences are
The sequence that GACTACATGGGACAT, 3 ' are held is GGAACCTACGACGTA), point three pipes carry out PCR amplification, wherein primer
GACTACATGGGACAT is the primer with biotin labeling.
PCR amplification system: above-mentioned probe solution, 5ul；Forward primer (25uM), 2ul；Reverse primer (25uM), 2ul；
MgCl2(50mM), 4ul；10x Platinum Taq polymerase buffer (is purchased from Life Technologies), 5ul；dNTPs
(every kind of 10mM), 4ul；Platinum Taq polymerase (5U/ul is purchased from Life Technologies), 1ul；H2O, 27ul；
Total volume 50ul.
Amplification condition: 98 DEG C, 30s；(98 DEG C, 30s, 60 DEG C, 25s, 72 DEG C, 45s) 35 circulations；72 DEG C, 5min.
After purification with MinElute PCR purification kit (being purchased from Life Technologies) by PCR product, it takes
500ng is combined PCR product with MyOne streptavidin magnetic bead (being purchased from Invitrogen).Then alkalinity NaOH is added
By the complementary strand denaturation of not biotin, elute；Then by 100 DEG C of the formamide liquid scrubbing of entire magnetic bead, make to visit
Needle is separated from magnetic bead.With the probe groups of the present embodiment for obtaining biotin labeling after ethanol precipitation.
In an alternative embodiment, the probe groups for detecting auto-inflammatory disease Disease-causing gene are using biotin
Label.
The present embodiment also provide it is a kind of for detecting the kit of auto-inflammatory disease Disease-causing gene, including above-mentioned use
In the probe groups of detection auto-inflammatory disease Disease-causing gene.The kit for auto-inflammatory disease Disease-causing gene is logical
It crosses and detects the above-mentioned mutation for being used to detect auto-inflammatory disease Disease-causing gene, to carry out the molecular genetics diagnosis by inspection individual
Or onset risk prediction.
In an alternative embodiment, further comprise harvesting buffer, the harvesting buffer includes people cot-1DNA；
Salmon sperm dna and primer pair；The sequence of the primer pair is as shown in SEQ ID No.51 and SEQ ID No.52.
In an alternative embodiment, further comprise containing hybridization buffer, the hybridization buffer includes SSPE buffering
Liquid, denhardt solution, EDTA and SDS.
In an alternative embodiment, further comprise combination buffer, rinsing liquid, NaOH solution, Tris-HCl buffering
Liquid, PCR reaction solution and TE buffer；
The combination buffer includes NaCl, Tris-HCl and EDTA；The rinsing liquid includes the first rinsing liquid and second
Rinsing liquid, first rinsing liquid and the second rinsing liquid include SSC and SDS；The PCR reaction solution includes: that dNTPs, PCR expand
Increase primer, Phusion buffer, Hotstart Phusion enzyme, DMSO and dH2O。
In an alternative embodiment, the sequence of the PCR amplification primer such as SEQ ID No.53 and SEQ ID No.54 institute
Show.
In a preferred embodiment, the combination buffer includes 1M NaCl, 10mM Tris-HCl (pH7.5) and 1mM
EDTA；First rinsing liquid includes the SSC and 0.1%SDS of 1 times of concentration, and second rinsing liquid includes 0.1 times of concentration
SSC and 0.1%SDS；The NaOH solution content is 0.1M；The pH of Tris-HCl buffer is 7.5 and content is 1M；It is described
PCR reaction solution includes 2ul dNTPs, 0.5ulPCR amplification upstream primer (50pmol), 0.5ulPCR amplification downstream primer
The Phusion buffer, 1ul Hotstart Phusion enzyme of 5 times of concentration of (50pmol), 20ul (are purchased from New England
Biolabs), 5ul DMSO and 51ul dH2O。
Specifically, the kit for detecting auto-inflammatory disease Disease-causing gene includes 10 pipe solution:
Pipe 1: probe groups described in aforementioned present invention, 160ul, concentration 150ng/ul,
Pipe 2: above-mentioned harvesting buffer, 208ul,
Pipe 3: above-mentioned hybridization buffer, 800ul,
Pipe 4: above-mentioned combination buffer, 3.2ml,
Pipe 5: above-mentioned first rinsing liquid, 9ml,
Pipe 6: above-mentioned second rinsing liquid, 45ml,
Pipe 7: above-mentioned 0.1M NaOH solution, 1ml,
Pipe 8: above-mentioned 1M Tris-HCl buffer (pH 7.5), 1.2ml,
Pipe 9: above-mentioned PCR reaction solution, 580ul,
Pipe 10: above-mentioned TE buffer, 800ul.
It is described for detecting the probe groups of auto-inflammatory disease Disease-causing gene as liquid phase capture in an alternative embodiment
Probe.
It a kind of causes a disease for detecting auto-inflammatory disease to become apparent to introduce the embodiment of the present invention in detail and provide
The probe groups and kit of gene, are described below in conjunction with specific embodiment.
Embodiment 1:
It is a kind of for detecting the probe groups of auto-inflammatory disease Disease-causing gene, the probe groups include can specificity simultaneously
Capture the probe of following 50 genes: ADAR, COPA, NLRP3, SLC29A3, HPS1, HPS6, RNASEH2C, TNFRSF1A,
GUCY2C、MVK、LACC1、RNASEH2B、RNF31、PSTPIP1、MEFV、NOD2、PLCG2、NLRP1、CARD14、LPIN2、
ACP5、RNASEH2A、ADGRE2、NLRP12、ADAM17、NLRC4、IL36RN、IL1RN、IFIH1、AP1S3、RBCK1、
FERMT1、SAMHD1、ADA2、USP18、HPS4、TREX1、COL7A1、SH3BP2、OTULIN、TMEM173、PSMB8、
TNFAIP3、SHARPIN、DDX58、POLA1、IL10、IL10RA、IL10RB、ISG15。
Capturing and detect probe included by above-mentioned each gene includes base sequence as shown in No.1~50 SEQ ID
Probe.
Embodiment 2:
It is a kind of for detecting the kit of auto-inflammatory disease Disease-causing gene, including following components: embodiment 1 is obtained
Probe groups (160ul, 150ng/ul), harvesting buffer (208ul), hybridization buffer (800ul), combination buffer
(3.2ml), the first rinsing liquid (9ml), the second rinsing liquid (45ml), NaOH solution (0.1M, 1ml), Tris-HCl buffer
(1M, pH 7.5,1.2ml), PCR reaction solution (580ul), TE buffer (800ul).Wherein each buffer composition is as follows:
1, harvesting buffer (every 20ul):
People cot-1DNA (is purchased from Invitrogen), 7ul；Salmon sperm dna (is purchased from Invitrogen), 3ul；
The specificity as shown in SEQ ID No.51 and SEQ ID No.52 closes primer, total 10ul, and every kind of primer concentration is
1nmol/ul:
2, hybridization buffer: the SSPE of 5 times of concentration, the Denhardt solution of 5 times of concentration, 5mM EDTA, 0.1%SDS；
3, combination buffer: 1M NaCl, 10mM Tris-HCl (pH 7.5), 1mM EDTA
4, the first rinsing liquid: the SSC solution of 1 times of concentration, 0.1%SDS
5, the second rinsing liquid: the SSC solution of 0.1 times of concentration, 0.1%SDS；
6, PCR reaction solution: 2ul dNTPs (every kind of 10mM), the 0.5ul primer 1 as shown in SEQ ID No.49,0.5ul is such as
Primer 2 shown in SEQ ID No.50, the Phusion buffer of 5 times of concentration of 20ul, 1ul Hotstart Phusion enzyme (are purchased from
New England Biolabs), 5ul DMSO, 51ul dH2O；* indicating intermediate has thio-modification；
Some common solution formulas or source are as follows:
The SSPE buffer of 20 times of concentration is purchased from AMRESCO company；The Denhardt solution of 50 times of concentration, it is public purchased from USB
Department；EDTA solution: 0.5M, pH8.0 are purchased from Mediatech company；The Phusion buffer of 5 times of concentration is purchased from New
England Biolab company；
SSC solution: NaCl 175g, trisodium citrate 88g adjust pH to 7.4, dH2O is settled to 1 liter；
TE buffer: 10mM Tris-HCl, 1mM EDTA adjusts pH to 8.0, and water is settled to 500ml.
The application method of the kit for being used to detect auto-inflammatory disease Disease-causing gene are as follows:
Step 1:DNA is extracted and the preparation of full-length genome library:
It is extracted in peripheral blood using Qiagen DNA mini kit (250) (being purchased from Qiagen) kit or similar products
DNA；Quantitative with spectrophotometer, agarose gel electrophoresis detects sample quality, and complete genome dna electrophoresis band is usual
20kb should be not less than.Concentration is adjusted to 75ng/ μ l by the DNA of quality inspection qualification, and total amount 3-5 μ g DNA is interrupted at random, expanded, thus
Establish full-length genome library；
Step 2: the specificity capture and sequencing of target gene segment:
(1) it prepares following mixed system: taking the full-length genome library of 1ug step 1 built, 13ul harvesting buffer,
The probe groups of the present invention of 5ul embodiment 1 (5474 oligonucleotide probes, base sequence is as shown in SEQ ID No.1-46)；It sets
In in PCR instrument: 95 DEG C, 7min, 65 DEG C later, 2min；
(2) take 65 DEG C preheating hybridization buffer 23ul be added in the mixed liquor that above-mentioned steps (1) obtain, then in
Hybridize 22 hours for 65 DEG C in PCR instrument, obtains enrichment system mixture；
(3) magnetic bead is made sufficiently to suspend MyOne C1 Streptavidin MagneSphere (being purchased from Invitrogen) whirlpool concussion, it is short
Temporarily centrifugation, is centrifuged magnetic bead to bottom of the tube, takes 50ul MyOne C1 Streptavidin MagneSphere to the centrifuge tube of new 1.5ml；
(4) the 1.5ml centrifuge tube whirlpool equipped with 50ul MyOne C1 Streptavidin MagneSphere is shaken at least 5s, makes magnetic
Pearl sufficiently suspends, and is put on magnetic frame and remain stationary one minute (should not rotating centrifugal pipe) after of short duration centrifugation, and careful inhale abandons supernatant；
(5) centrifuge tube is removed, the combination buffer of 1 times of concentration of 50ul is added, rotation nest shakes at least 5s, after of short duration centrifugation
It is put on magnetic frame static one minute, careful inhale abandons supernatant, in triplicate；
(6) centrifuge tube is removed, the combination buffer of 2 times of concentration of 100ul is added, rotation nest shakes at least 5s, of short duration centrifugation
After be put on magnetic frame static one minute；
(7) the enrichment system mixture that step (2) obtains is added in the centrifuge tube of rapid (6), rotation nest shakes at least 5s
(not having to centrifugation) is placed in room temperature on gyroscope and rotates 1 hour (60 revs/min)；
(8) then, the magnetic bead using the first rinsing liquid room temperature cleaning step (7) is primary, and 15 minutes, then again with the second drift
Washing lotion is cleaned 3 times, 65 DEG C, every time 15 minutes；
(9) magnetic bead of step (8) is eluted 10 minutes with 0.1M NaOH at room temperature, then by eluent whirlpool shake to
Few 5s is put on magnetic frame static one minute after of short duration centrifugation, is then transferred to supernatant and buffers containing 70ul Tris-HCl
In the clean centrifuge tube of liquid (1M, pH 7.5)；
(10) using Qiagen MinElute Column (be purchased from Qiagen) to step (9)) obtained DNA solution carries out
Purifying, Laboratory Manual of the purification step referring to Qiagen MinElute Kit；
(11) DNA purified finally expands 15 circulations by PCR；
PCR reaction solution: 2ul dNTPs (every kind of 10mM), 0.5ul such as SEQ ID No.49 primer 1 (50pmol), 0.5ul
The Phusion buffer of the primer 2 (50pmol) as shown in SEQ ID No.50,5 times of concentration of 20ul (is purchased from New England
Biolabs), 1ul Hotstart Phusion enzyme (being purchased from New England Biolabs), 5ul DMSO, 51ul dH2O；*
Indicating intermediate has thio-modification.PCR program: 98 DEG C, 30s (1cycle)；98 DEG C, 25s, 65 DEG C, 30s, 72 DEG C, 30s
(15cycles)；72 DEG C, 5min (1cycle)；
(12) using Agencourt AMPure XP Nucleic acid purification kits (be purchased from Beckman Coulter), according to making
With the PCR product of handbook purification step (11)；
(13) DNA product that step (12) obtains is sequenced on 500 sequenator of Illumina Nexseq；
Step 3: bioinformatic analysis process and result output:
(1) snp analysis process
1. obtaining original short sequence；
2. removing connector and the low quality data etc. in sequencing data；
3. short sequence is navigated on the corresponding position of people's MHC3.6M genomic data with SOAPaligner software, it is used
To parameter: soap2.20-a-b-t-v 3-l 42-s 63-m 100-x 400, wherein sequence mismatch number is 3l；
4. counting sequencing result information, short sequence quantity, target area covering size, average sequencing depth etc.；
5. SOAPsnp is used to find out the genotype in site, used parameter: soapsnp-i-d-o-r in target area
0.00005-e 0.0001-M-t-u-L-s-2–T；
6. filtering the SNP of low quality value (mass value >=20) and low cover degree (depth >=10)；
7. being annotated using CCDS, human genome database (NCBI 37.2), dbSNP (v138) information to SNP, really
Determine gene, coordinate, the site mRNA, the amino acid change, (missense mutation/nonsense mutation/variable of SNP function of mutational site generation
Shearing site), SIFT prediction SNP influence protein function prediction etc.；
8. selecting and being not present common to disease sample and in normal group according to disease sample and normal specimens information
SNP as candidate SNPs, got rid of in candidate SNPs dbSNP, HAPMAP, 1000 people MHC3.6M genomes, other
The SNP occurred in exon sequencing project.Meanwhile SNPs of the SIFT prediction on protein function without influence is filtered out as last disease
The relevant candidate SNP s of disease；
(2) InDel analysis process
1. removal joint sequence and low-quality sequencing data are compared with Burrows-Wheeler Aligner (BWA)
Onto people's MHC3.6M genome, used parameter: bwa aln-L-l 31-i 10-k 2-t 7-e 40；
2. with GATK software find out sequence contained in insertion/deletion (InDel) information；
3. InDel is annotated using CCDS, human genome database (NCBI 37.2), dbSNP (v138) information,
Determine gene, coordinate, the site mRNA, the change of Coding region sequence, the influence to amino acid, InDel that mutational site occurs
Function (amino acid insertion/amino acid deletions/frameshift mutation)；
Output sequencer data basic statistics report and data analysis result.
Embodiment 3:
Kit effect detection for detecting auto-inflammatory disease Disease-causing gene is tested:
Detect 3 samples using kit described in the embodiment of the present invention 2, as a result confirm purpose cause a disease/tumor susceptibility gene catches
It is satisfied to obtain rate, 50% or more original short sequence can be compared back the reference sequences of target area, and being averaged for target area has
Effect sequencing data amount reaches 200Mb, and the average sequencing depth of target area is 300X, significantly larger than general diagnosis of genetic disorders
It is required that (generally 100X).
1, infant number C150710C01201, male, 4 years old, is detected, testing result is such as using kit of the invention
Shown in Fig. 1, mutate for gene TNFRSF1A.It mutates in the position chr12-6442930*, i.e. it is prominent that missing occurs for exon3
Become c.295T > A, disease phenotype is periodic fever, autosomal dominant inheritance.
1 infant number C150710C01201 testing result of table
Sequencing pedigree analysis the results are shown in Table 2 data are verified by generation Sanger, the curve as shown in Fig. 2,3 and 4 is suffered from
Person c.295T > A mutation come from its mother.
Table 2Sanger verifying sequencing pedigree analysis result
Patient detects that TNFRSF1A gene has 1 heterozygous mutant, come from mother, this mutational site it has been reported that
Testing result shows that patient is periodic fever, autosomal dominant inheritance.There is above-mentioned experimental result can be shown that, spy of the invention
Needle composite reagent box can be applied to the molecular genetics diagnosis of individual patients, while it is general to can be used for auto-inflammatory group of people at high risk
It looks into, screening morbidity people at highest risk and provides reference for corresponding genetic counselling etc., meet the development trend of accurate medical treatment.The present invention
The probe for being used to detect auto-inflammatory disease related gene and kit provided has easy to operate, low in cost, spy
The features such as opposite sex is good, high sensitivity.
Sequence table
<110>Chinese Academy of Medical Sciences Beijing consonance doctor
<120>for detecting the probe groups and kit of auto-inflammatory disease Disease-causing 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 (10)
1. a kind of for detecting the probe groups of auto-inflammatory disease Disease-causing gene, it is characterised in that: the probe groups include energy
Specificity capture simultaneously influences the small volume defect Disease-causing gene of inflammation, noninflammatory small volume defect Disease-causing gene, interferon access defect
The probe of related auto-inflammatory disease Disease-causing gene.
2. according to claim 1 for detecting the probe groups of auto-inflammatory disease Disease-causing gene, it is characterised in that:
The small volume defect Disease-causing gene of influence inflammation includes MEFV, MVK, NLRP3, NLRP12, NLRC4, PLCG2, NLRP1；
The noninflammatory small volume defect Disease-causing gene include 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、ADGRE2、SHARPIN、RNF31、
RBCK1；
The interferon access defect correlation auto-inflammatory disease Disease-causing gene include PSMB8, TMEM173, TREX1,
RNASEH2B、RNASEH2A、RNASEH2C、SAMHD1、ADAR、IFIH1、ISG15、ACP5、POLA1、DDX58。
3. according to claim 2 for detecting the probe groups of auto-inflammatory disease Disease-causing gene, it is characterised in that: catch
The probe for obtaining and detecting ADAR gene includes base sequence probe as shown in SEQ ID No.1；Capture and detection COPA gene
Probe include base sequence probe as shown in SEQ ID No.2；The probe of capture and detection NLRP3 gene includes base sequence
Arrange the probe as shown in SEQ ID No.3；The probe of capture and detection SLC29A3 gene includes base sequence such as SEQ ID
Probe shown in No.4；The probe of capture and detection HPS1 gene includes base sequence probe as shown in SEQ ID No.5；It catches
The probe for obtaining and detecting HPS6 gene includes base sequence probe as shown in SEQ ID No.6；Capture and detection RNASEH2C
The probe of gene includes base sequence probe as shown in SEQ ID No.7；The probe of TNFRSF1A gene includes base sequence
The probe as shown in SEQ ID No.8；The probe of capture and detection GUCY2C gene includes base sequence such as SEQ ID No.9 institute
The probe shown；The probe of capture and detection mvk gene includes base sequence probe as shown in SEQ ID No.10；Capture and inspection
The probe for surveying LACC1 gene includes base sequence probe as shown in SEQ ID No.11；Capture and detection RNASEH2B gene
Probe include base sequence probe as shown in SEQ ID No.12；The probe of capture and detection RNF31 gene includes base
Sequence probe as shown in SEQ ID No.13；The probe of capture and detection PSTPIP1 gene includes base sequence such as SEQ ID
Probe shown in No.14；The probe of capture and detection MEFV gene includes base sequence probe as shown in SEQ ID No.15；
The probe of capture and detection NOD2 gene includes base sequence probe as shown in SEQ ID No.16；Capture and detection PLCG2
The probe of gene includes base sequence probe as shown in SEQ ID No.17；Capture and the probe for detecting NLRP1 gene include
Base sequence probe as shown in SEQ ID No.18；The probe of capture and detection CARD14 gene includes base sequence such as SEQ
Probe shown in ID No.19；The probe of capture and detection LPIN2 gene includes base sequence as shown in SEQ ID No.20
Probe；The probe of capture and detection ACP5 gene includes base sequence probe as shown in SEQ ID No.21；Capture and detection
The probe of RNASEH2A gene includes base sequence probe as shown in SEQ ID No.22；Capture and detect ADGRE2 gene
Probe includes base sequence probe as shown in SEQ ID No.23；The probe of capture and detection NLRP12 gene includes base sequence
Arrange the probe as shown in SEQ ID No.24；The probe of capture and detection ADAM17 gene includes base sequence such as SEQ ID
Probe shown in No.25；The probe of capture and detection NLRC4 gene includes base sequence spy as shown in SEQ ID No.26
Needle；The probe of capture and detection IL36RN gene includes base sequence probe as shown in SEQ ID No.27；Capture and detection
The probe of IL1RN gene includes base sequence probe as shown in SEQ ID No.28；The probe of capture and detection IFIH1 gene
Including base sequence probe as shown in SEQ ID No.29；Capture and detection AP1S3 gene probe include base sequence such as
Probe shown in SEQ ID No.30；The probe of capture and detection RBCK1 gene includes base sequence such as SEQ ID No.31 institute
The probe shown；The probe of capture and detection FERMT1 gene includes base sequence probe as shown in SEQ ID No.32；Capture
Probe with detection SAMHD1 gene includes base sequence probe as shown in SEQ ID No.33；Capture and detection ADA2 gene
Probe include base sequence probe as shown in SEQ ID No.34；The probe of capture and detection USP18 gene includes base
Sequence probe as shown in SEQ ID No.35；The probe of capture and detection HPS4 gene includes base sequence such as SEQ ID
Probe shown in No.36；The probe of capture and detection TREX1 gene includes base sequence spy as shown in SEQ ID No.37
Needle；The probe of capture and detection COL7A1 gene includes base sequence probe as shown in SEQ ID No.38；Capture and detection
The probe of SH3BP2 gene includes base sequence probe as shown in SEQ ID No.39；The spy of capture and detection OTULIN gene
Needle includes base sequence probe as shown in SEQ ID No.40；The probe of capture and detection TMEM173 gene includes base sequence
Arrange the probe as shown in SEQ ID No.41；The probe of capture and detection PSMB8 gene includes base sequence such as SEQ ID
Probe shown in No.42；The probe of capture and detection TNFAIP3 gene includes base sequence spy as shown in SEQ ID No.43
Needle；The probe of SHARPIN gene includes base sequence probe as shown in SEQ ID No.44；Capture and detection DDX58 gene
Probe include base sequence probe as shown in SEQ ID No.45；The probe of capture and detection POLA1 gene includes base
Sequence probe as shown in SEQ ID No.46；The probe of capture and detection IL10 gene includes base sequence such as SEQ ID
Probe shown in No.47；The probe of capture and detection IL10RA gene includes base sequence spy as shown in SEQ ID No.48
Needle；The probe of capture and detection IL10RB gene includes base sequence probe as shown in SEQ ID No.49；Capture and detection
The probe of ISG15 gene includes base sequence probe as shown in SEQ ID No.50.
4. a kind of for detecting the kit of auto-inflammatory disease Disease-causing gene, it is characterised in that: including claims 1 to 3
For detecting the probe groups of auto-inflammatory disease Disease-causing gene described in any one.
5. according to claim 4 for detecting the kit of auto-inflammatory disease Disease-causing gene, it is characterised in that: into
One step further includes harvesting buffer, and the harvesting buffer includes people cot-1DNA；Salmon sperm dna and primer pair；The primer
Pair sequence as shown in SEQ ID No.51 and SEQ ID No.52.
6. according to claim 5 for detecting the kit of auto-inflammatory disease Disease-causing gene, it is characterised in that: into
One step further includes containing hybridization buffer, and the hybridization buffer includes SSPE buffer, denhardt solution, EDTA and SDS.
7. according to claim 6 for detecting the kit of auto-inflammatory disease Disease-causing gene, it is characterised in that: into
One step further includes combination buffer, rinsing liquid, NaOH solution, Tris-HCl buffer, PCR reaction solution and TE buffer.
8. according to claim 7 for detecting the kit of auto-inflammatory disease Disease-causing gene, it is characterised in that: institute
Stating combination buffer includes NaCl, Tris-HCl and EDTA；The rinsing liquid includes the first rinsing liquid and the second rinsing liquid, described
First rinsing liquid and the second rinsing liquid include SSC and SDS；The PCR reaction solution includes: dNTPs, PCR amplification primer,
Phusion buffer, Hotstart Phusion enzyme, DMSO and dH2O。
9. according to claim 8 for detecting the kit of auto-inflammatory disease Disease-causing gene, it is characterised in that: institute
The sequence of PCR amplification primer is stated as shown in SEQ ID No.53 and SEQ ID No.54.
10. according to claim 4 for detecting the kit of auto-inflammatory disease Disease-causing gene, it is characterised in that:
The probe groups for detecting auto-inflammatory disease Disease-causing gene are liquid phase capture probe.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN201811584606.9A CN109371123B (en) | 2018-12-24 | 2018-12-24 | Probe set and kit for detecting pathogenic gene of autoinflammatory disease |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201811584606.9A CN109371123B (en) | 2018-12-24 | 2018-12-24 | Probe set and kit for detecting pathogenic gene of autoinflammatory disease |
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| Publication Number | Publication Date |
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| CN109371123A true CN109371123A (en) | 2019-02-22 |
| CN109371123B CN109371123B (en) | 2022-06-17 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN201811584606.9A Active CN109371123B (en) | 2018-12-24 | 2018-12-24 | Probe set and kit for detecting pathogenic gene of autoinflammatory disease |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110512004A (en) * | 2019-10-11 | 2019-11-29 | 新乡医学院 | It is a kind of to be migrated and the molecular marker of invasive ability and its application for detecting esophageal cancer cell |
| 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 |
| CN112725368A (en) * | 2019-10-28 | 2021-04-30 | 深圳市儿童医院 | Mutant gene of autoinflammatory disease-related gene TNFAIP3 and application thereof |
| 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 |
| CN114369658A (en) * | 2022-02-18 | 2022-04-19 | 中国医学科学院北京协和医院 | Application of mutant form of NLRP 3-related autoinflammatory disease-related gene NLRP3 |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110512004A (en) * | 2019-10-11 | 2019-11-29 | 新乡医学院 | It is a kind of to be migrated and the molecular marker of invasive ability and its application for detecting esophageal cancer cell |
| CN112725368A (en) * | 2019-10-28 | 2021-04-30 | 深圳市儿童医院 | Mutant gene of autoinflammatory disease-related gene TNFAIP3 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 |
| CN114369658A (en) * | 2022-02-18 | 2022-04-19 | 中国医学科学院北京协和医院 | Application of mutant form of NLRP 3-related autoinflammatory disease-related gene NLRP3 |
| 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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| CN109371123B (en) | 2022-06-17 |
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