CN106521014A - Hereditary cardiovascular and cerebrovascular disease diagnostic reagent kit - Google Patents
Hereditary cardiovascular and cerebrovascular disease diagnostic reagent kit Download PDFInfo
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- CN106521014A CN106521014A CN201611256840.XA CN201611256840A CN106521014A CN 106521014 A CN106521014 A CN 106521014A CN 201611256840 A CN201611256840 A CN 201611256840A CN 106521014 A CN106521014 A CN 106521014A
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Abstract
The invention provides a hereditary cardiovascular and cerebrovascular disease diagnostic reagent kit. According to the technical scheme, through search of OMIM and related literature, a brand-new cardiovascular and cerebrovascular related pathogenic gene combination is designed. The combination comprises 229 genes, coding regions and flank +/-30bp regions of the 229 genes can be captured based on the hybrid capture principle, then next generation sequencing is conducted on the captured target regions, standard information analysis and variation interpretation are conducted on sequencing results, analysis and interpretation are conducted on abnormal variation detected through sequencing, pathogenic mutations of a subject are determined, accurate diagnosis and targeted treatment of a cerebrovascular disease patient are achieved, and the disease is prevented from relapsing in the family. According to the human genome HG10, exon sequences and the flank +/-30bp regions of the 229 genes are selected to design a probe combination, and the coverage degree of a probe in the combination to a targeted region reaches 99%.
Description
Technical field
The present invention relates to technical field of molecular biology, further to examination heredity cardiovascular and cerebrovascular disease related gene
The detection kit of mutation and its medical science unscrambling data storehouse, and in particular to a kind of heredity cardiovascular and cerebrovascular disease diagnostic kit.
Background technology
The cerebral tissue change that cerebrovascular disease refers to cerebrovascular insult caused by the various causes of disease and causes, clinical manifestation is various brains
Dysfunction, is the second largest cause of death and the modal reason that disables, brings huge spirit pressure to personal family and society
Power and heavy financial burden.Increasing research shows that inherent cause is the important pathogenesis of cerebrovascular disease, by right
Hereditary cerebral vasculopathy patient gene detects, can clarify a diagnosis, patients with cerebrovascular disease early stage individuation prevented and treated and is provided with strong evidence
According to.Two generation sequencing technologies (NGS) are high with its flux in recent years, low cost, can detect mutational range it is wide the characteristics of, have developed into
Important clinical gene analytical technology, and combining target areas captured technology (target region capture
Technology target zone) can be further enriched with, detection efficiency reduces cost is improved, can currently without a kind of product or technology
To cover whole cerebrovascular disease Disease-causing genes comprehensively.
The content of the invention
It is contemplated that for the technological deficiency of prior art, there is provided a kind of heredity cardiovascular and cerebrovascular disease diagnostic reagent
Box, to solve the skill that heredity cardiovascular and cerebrovascular disease diagnostic method in prior art more limits to, lacks molecular biology foundation
Art problem.
The invention solves the problems that another technical problem be diagnosis side in prior art for heredity cardiovascular and cerebrovascular disease
Method accuracy is relatively low.
The invention solves the problems that another technical problem be when using significant gene as heredity cardiovascular and cerebrovascular disease diagnose
During foundation, due to such diseases range relatively wide, parting complexity, therefore significant gene is chaotic with disease corresponding relation.
To realize above technical purpose, the present invention is employed the following technical solutions：
A kind of heredity cardiovascular and cerebrovascular disease diagnostic kit, the kit detect 229 genes as shown in the table
Catastrophe：
.In above technical scheme, the mutation is the conventional mutational site in this area, has been preferably comprised indel and has dashed forward
Become site (sequence that insertion or disappearance cause changes insertion or deletion, indel), nonsense mutation site, splicing
Region mutation site or missense mutation site.
Preferably, the diagnostic kit includes the primer of 229 genes of the above, the amplification scope of the primer includes
Whole exon regions of 229 genes of the above.
Preferably, the kit is included for expanding the multiple PCR primer of the exon region, and it is used for two
The reagent of generation sequencing.
Preferably, the kit include by extract in detection object genomic DNA make be available for be sequenced library
Reagent.
Preferably, the kit includes dNTP solution, archaeal dna polymerase, for the reagent of isolated or purified nucleic acid, sun
Property control or negative control.
Preferably, the heredity cardiovascular and cerebrovascular disease is cerebral amyloid angiopathy, the detection object of the kit
For the catastrophe of app gene.
Preferably, the heredity cardiovascular and cerebrovascular disease is cerebrotendinous xanthomatosis, the detection object of the kit is
The catastrophe of CYP27A1 genes.
Preferably, the heredity cardiovascular and cerebrovascular disease is white matter ablative leukoencephalopathy, the detection of the kit is right
As the catastrophe for EIF2B1 genes.
Preferably, the heredity cardiovascular and cerebrovascular disease is periventrcular nodositas dystopy, the detection of the kit is right
As the catastrophe for ERMARD genes.
Preferably, the heredity cardiovascular and cerebrovascular disease is thrombasthenia, the detection object of the kit is
The catastrophe of ITGA2B genes.
The catastrophe of 229 gene extron subregions of the present invention can be used to do heredity cardiovascular and cerebrovascular disease
Go out auxiliary diagnosis, the conventional diagnostic method of heredity cardiovascular and cerebrovascular disease includes morphology, cytogenetics and flow cyctometry,
The method of the invention preferably makes diagnosis to a series of heredity cardiovascular and cerebrovascular diseases from molecular biology angle.
The present invention provides the multiple PCR primer of one group of detection correlating markings gene extron subregion, the expansion of the primer
Increasing scope includes the exon region of 229 significant genes.The multiple PCR primer provided using the present invention, was surveyed with reference to two generations
Sequence technology, can detect the whole exon regions in presently disclosed 229 significant genes.
Two wherein described generation sequencing reagents are reagent commonly used in the art, gained sequence are entered as long as disclosure satisfy that
The requirement of two generation of row sequencing.The preparation method of reagent thereof is this area customary preparation methods, preferably commercially available.
Detection kit of the present invention more preferably also includes that the genomic DNA that will be extracted in detection object is made and is available for surveying
The reagent in the library that sequence is used, this kind of reagent is reagent commonly used in the art, as long as disclosure satisfy that multiplex PCR to genome
The requirement of DNA mass.The preparation method of reagent thereof is this area customary preparation methods, preferably commercially available.
Detection kit of the present invention preferably also includes：DNTP solution, archaeal dna polymerase, for isolated or purified core
Acid is Backman magnetic beads.
Tissue of the sample of the present invention for detection object, as long as the gene of detection object can be extracted from detection sample
Group DNA.The detection sample is preferably one or more in marrow, blood, solid tumor sample, it is therefore preferable to marrow
Sample.
A kind of using method of kit of the present invention, which comprises the following steps：
(1) marrow of detection object is extracted using detection kit of the present invention, genomic DNA is extracted；
(2) exon region of 229 significant genes described in invention in genomic DNA is entered with multiple PCR primer
Row amplification；
(3) fragment of amplification gained in step (2) is made the library for being available for Ion Torrent microarray datasets to be sequenced；
(4) gained DNA library in step (3) is sequenced, obtains lower machine data；
(5) to (4) in lower machine data carry out bioinformatic analysis, then understood using medical science unscrambling data storehouse
Make diagnosis.
The concrete operation method of each step refers to kit specification above.
Reagent and raw material used by the present invention is commercially available.
The invention provides a kind of heredity cardiovascular and cerebrovascular disease diagnostic kit, the technical scheme by OMIM and
The lookup of pertinent literature, devises a brand-new cardiovascular and cerebrovascular related genes combination, and the combination includes 229 genes,
The code area of this 229 genes and flank ± 30bp regions can be captured based on hybrid capture principle, subsequently to capture
Target area carries out the sequencing of two generations and information analysis and variation deciphering is standardized to sequencing result, by sequencing detection
Abnormal variation is analyzed deciphering, specifies the pathogenic mutation of person under inspection, realizes Accurate Diagnosis, specific aim to patients with cerebrovascular disease
Treatment and prevent disease from recurring in family.According to human genome HG19, the extron sequence of above-mentioned 229 genes is chosen
Row and flank ± 30bp regions design probe combinations, this combination middle probe is to target area coverage up to 99%.
The invention discloses one group of sequencing kit for being used for examination heredity cardiovascular and cerebrovascular disease associated gene mutation, with
And follow-up medical science unscrambling data storehouse.Heredity cardiovascular and cerebrovascular disease related gene group includes ABCA1, ABCC2, ABCC6 etc. 229
Individual gene.Sequencing kit includes the multiple of the 229 gene whole exon amplifications related to heredity cardiovascular and cerebrovascular disease
PCR primer.The kit of the examination heredity cardiovascular and cerebrovascular disease associated gene mutation that the present invention is provided have detection efficiency it is high,
Mutator broad covered area, the advantages of recall rate is high, the medical science for being given understand report with accurately, authority the characteristics of.
Specific embodiment
Hereinafter the specific embodiment to the present invention is described in detail.In order to avoid excessive unnecessary details,
Will not be described in detail to belonging to known structure or function in following examples.In addition to being defined, institute in following examples
Technology and scientific terminology are with the identical meanings being commonly understood by with those skilled in the art of the invention.
In Baijing Tiantan Hospital, cardiovascular and cerebrovascular associated department in January, 2016 was examined in December, 2016
In disconnected patient, according to following standard, we enter Line Continuity and enter group (85).
Inclusion criteria is：Routine blood test, biochemistry detection and iconography auxiliary examination can not specify the patient of kinds of Diseases.Experiment
Group and feature are as shown in table 1：
1 experimental subjects of table and its Clinical symptoms
Sample collection：3ml venous blood.
Sample process：Sample genome utilizes Tiangeng DNA extraction kit (article No.：DP316) it is stripped, concrete operations
Method is referring to kit operation instructions.
Library construction：The library construction Kit produced using KAPA companies by the DNA fragmentation that extraction is obtained, experimental implementation
Operation instructions of the method referring to KAPA companies kit.Genomic DNA is expanded with multiple PCR primer, concrete operations
The operation instructions of the PCR amplification kits of method Jian Jitai company production.
Hybrid capture：The library for having expanded, hybrid capture is carried out using the probe that Agilent company customizes.
High-flux sequence：The sequencing library for building is carried out into high-flux sequence on Illumina companies platform, is sequenced
Method refer to the operation instructions of Illumina companies high-flux sequence instrument and its corollary equipment.
Bioinformatic analysis：Using Cutadapt (https://pypi.python.org/pypi/cutadapt) and
FastQC (www.bioinformatics.babraham.ac.uk/projects/fastqc/) filters low-quality sequencing piece
Section, compares software (http using bwa://bio-bwa.sourceforge.net/bwa.shtml) by qualified sequencing fragment
Mankind's reference gene group hg19 is compared, with GATK software (http://www.broadinstitute.org/gatk/) analysis
Aligned sequences obtain SNP and Indel mutation, by SNP and Indel mutation using ANNOVAR (http://
Annovar.openbioinformatics.org/en/latest/) software is annotated, and notes content includes mutation in gene
Position in group, the gene of association, gene extron numbering, nucleotide level variation, corresponding protein level variation, mutation
In dbSNP (http://www.ncbi.nlm.nih.gov/snp/), 1000Genomes database (http://
Www.1000genomes.org/), esp6500 databases (http://evs.gs.washington.edu/EVS/),
PoliPhen2(http://genetics.bwh.harvard.edu/phh2/)、sift(http://sift.jcvi.org/)、
MutationTaster(http://www.mutationtaster.org/), GERP function prediction results.And hgmd
(http://www.hgmd.cf.ac.uk/ac/index.php) disease site database.Further use above principle screening
Doubtful pathogenic sites.
Medical science is understood：Molecular pathology is made to the genic mutation type that bioinformatic analysis draw with omim database to examine
It is disconnected.The results are shown in Table 2 and table 3.
2 cerebrovascular disease type of table and gene number
3 genopathy corresponding relation of table
Show Jing after statistics：(being summarized according to above table) as can be seen here, the method being sequenced using two generations to one is
Row cardiovascular and cerebrovascular disease carries out auxiliary diagnosis, and with diagnosing, accurate, parting is clear, obtain the features such as containing much information.
Above embodiments of the invention are described in detail, but the content have been only presently preferred embodiments of the present invention,
Not to limit the present invention.All any modification, equivalent and improvement made in the application range of the present invention etc., all should
It is included within protection scope of the present invention.
Claims (10)
1. a kind of heredity cardiovascular and cerebrovascular disease diagnostic kit, it is characterised in that the kit detects as shown in the table 229
The catastrophe of individual gene：
2. a kind of heredity cardiovascular and cerebrovascular disease diagnostic kit according to claim 1, it is characterised in that the diagnosis
Kit includes the primer of 229 genes of the above, and the amplification scope of the primer includes whole extrons of 229 genes of the above
Region.
3. a kind of heredity cardiovascular and cerebrovascular disease diagnostic kit according to claim 2, it is characterised in that the reagent
Box is included for expanding the multiple PCR primer of the exon region, and for the reagent of two generations sequencing.
4. a kind of heredity cardiovascular and cerebrovascular disease diagnostic kit according to claim 2, it is characterised in that the reagent
Box include by extract in detection object genomic DNA make be available for be sequenced library reagent.
5. a kind of heredity cardiovascular and cerebrovascular disease diagnostic kit according to claim 2, it is characterised in that the reagent
Box includes dNTP solution, archaeal dna polymerase, for the reagent of isolated or purified nucleic acid, positive control or negative control.
6. a kind of heredity cardiovascular and cerebrovascular disease diagnostic kit according to claim 1, it is characterised in that the heredity
Property cardiovascular and cerebrovascular disease be cerebral amyloid angiopathy, the catastrophe of the detection object of the kit for app gene.
7. a kind of heredity cardiovascular and cerebrovascular disease diagnostic kit according to claim 1, it is characterised in that the heredity
Property cardiovascular and cerebrovascular disease be cerebrotendinous xanthomatosis, the catastrophe of the detection object of the kit for CYP27A1 genes.
8. a kind of heredity cardiovascular and cerebrovascular disease diagnostic kit according to claim 1, it is characterised in that the heredity
Property cardiovascular and cerebrovascular disease be white matter ablative leukoencephalopathy, the catastrophe of the detection object of the kit for EIF2B1 genes.
9. a kind of heredity cardiovascular and cerebrovascular disease diagnostic kit according to claim 1, it is characterised in that the heredity
Property cardiovascular and cerebrovascular disease be periventrcular nodositas dystopy, the catastrophe of the detection object of the kit for ERMARD genes.
10. a kind of heredity cardiovascular and cerebrovascular disease diagnostic kit according to claim 1, it is characterised in that the heredity
Property cardiovascular and cerebrovascular disease be thrombasthenia, the catastrophe of the detection object of the kit for ITGA2B genes.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201611256840.XA CN106521014A (en) | 2016-12-30 | 2016-12-30 | Hereditary cardiovascular and cerebrovascular disease diagnostic reagent kit |
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| CN201611256840.XA CN106521014A (en) | 2016-12-30 | 2016-12-30 | Hereditary cardiovascular and cerebrovascular disease diagnostic reagent kit |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108179148A (en) * | 2018-02-11 | 2018-06-19 | 北京乐普基因科技股份有限公司 | A kind of probe for detecting genetic cardiomyopathies and its application |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103981273A (en) * | 2014-05-30 | 2014-08-13 | 复旦大学附属肿瘤医院 | Mutant gene group for mammary cancer risk assessment and detection kit thereof |
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- 2016-12-30 CN CN201611256840.XA patent/CN106521014A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103981273A (en) * | 2014-05-30 | 2014-08-13 | 复旦大学附属肿瘤医院 | Mutant gene group for mammary cancer risk assessment and detection kit thereof |
Non-Patent Citations (8)
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108179148A (en) * | 2018-02-11 | 2018-06-19 | 北京乐普基因科技股份有限公司 | A kind of probe for detecting genetic cardiomyopathies and its application |
| CN108179148B (en) * | 2018-02-11 | 2024-02-13 | 北京爱普益医学检验中心有限公司 | Probe for detecting hereditary cardiomyopathy and application thereof |
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Application publication date: 20170322 |