CN109295219B - The biomarker and its detection kit of the congenital fibrinogen defect of one group of detection - Google Patents
The biomarker and its detection kit of the congenital fibrinogen defect of one group of detection Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The present invention provides the biomarkers and its detection kit of the congenital fibrinogen defect of one group of detection, are related to technical field of molecular biology.The biomarker for detecting congenital fibrinogen defect is mutated gene group, the set including FGA, FGB, FGG mutated gene of totally 3 genes, and exon region carries 201 mutational sites altogether.The kit for detecting congenital fibrinogen defect includes primer for expanding mutated gene in the mutated gene group of congenital fibrinogen defect.The kit has many advantages, such as that detection efficiency height, mutated gene broad covered area, recall rate are high, and it is more accurate, authoritative to interpret report using medicine given by the detection kit.
Description
Technical field
The present invention relates to technical field of molecular biology, and in particular to the life of the congenital fibrinogen defect of one group of detection
Substance markers-mutated gene group and its detection kit.
Background technique
Fibrinogen (fibrinogen, Fg) is a kind of macromolecular glycoprotein containing 2964 amino acid, by A α, B
Beta, gamma, 3 polypeptide chains be connected with interchain disulfide bond, that is, A α Cys28, γ Cys8 with Cys9 constitute symmetry dimer, more than 3
Peptide chain is encoded by 3 independent gene FGA, FGB, FGG respectively.
Congenital fibrinogen defect is a kind of
The orphan disease of feature, clinical manifestation are mainly petechiae, ecchymosis or different degrees of bleeding, and laboratory, which checks, to be can be found that
There is prothrombin time (PT), activated partial thromboplastin time in the antigen of fibrinogen or active exception, majority
(aPTT) extend simultaneously with thrombin time (TT), but the performance for also there are some patientss not have obvious bleeding is even shown as
The symptom of thrombus.So far, at home, congenital fibrinogen defect still lacks understanding, and diagnosis is low, and laboratory checks
Project is not perfect, is often obscured with other deficiencies of coagulation factors either hemorrhagic disease, makes congenital fibrinogen defect
It is constantly in the state underestimated.Conventional diagnostic method has thrombin time test, fibrinogen level detection and fiber
Proteinogen Activity determination and thrombus hemostasis laboratory inspection etc..But in the conventional detection in laboratory, fibrin
Former detection project is not very perfect, and the cumbersome complexity of above-mentioned detection method, and there are still very big mistaken diagnosis or
The possibility failed to pinpoint a disease in diagnosis.
In recent years, it makes remarkable progress for congenital fibrinogen defect molecular etiology basic research, at present
The Disease-causing gene for relating generally to congenital fibrinogen defect known has tri- genes of FGA, FGB, FGG, and 1981 years,
Higgins etc. first reported the gene defect of FGA, and Arg16His mutation occurs, generates dysfibrinogenemia.Mesh
Before, FGA gene mutation type has reported 83 kinds, and 75 kinds of the gene defect of FGB, the mutation type of FGG gene has 43 kinds.The secret victory of Zhao
Hereditary dysfibrinogenemia man caused by 7 fibrinogen γ chain Arg275 site mutations is disclosed Deng (2017)
The genotype and Analysis of clinical features of system have found Arg275His and Arg275Cys two in 15 Arg275 mutation patients altogether
Kind mutation type.The existing FGA mutation announced includes Gly13Arg, Arg16His, Argl6Cys, Arg35His,
Cys36Arg etc., FGB are mutated including Asn190Ser etc., and FGG mutation includes Asp185Asn, Arg275His, Arg275Cys,
Arg301His etc..
Quickly and effectively disease can be diagnosed by genetic test and make parting, wherein the reality of molecular biology
Proved recipe method mainly has nest-type PRC, generation sequencing etc..The mutant nucleotide sequence that PCR method cannot intuitively be had, the side of generation sequencing
Method is influenced by sequencing throughput, can not disposably detect whole exon regions of multiple genes, and the sequencing of two generations is used as one
The high-throughput detection method of kind, disposably can accurately detect the exon and correlated series of multiple genes, make precisely to diagnose
Congenital fibrinogen defect is possibly realized.Since Fg gene mutation site is numerous, and existing known mutational site is less, is
Make congenital fibrinogen dcc gene diagnosis more comprehensively, covering surface is wider, provide more mutational sites and its
Detection kit becomes inevitable trend.
Summary of the invention
The purpose of the present invention is to provide the biomarker of the congenital fibrinogen defect of one group of detection and its detection examinations
Agent box, overcomes problems of the prior art, improves the accuracy of the diagnosis to congenital fibrinogen defect.
One aspect of the present invention provides the biomarker of the congenital fibrinogen defect of one group of detection, the biology mark
It is denoted as mutated gene group, the mutated gene group includes the set of FGA, FGB, FGG mutated gene of totally 3 genes；
The exon region of the mutated gene carries 201 mutational sites altogether；
201 mutational sites are as shown in table 1:
The biomarker that another aspect of the present invention provides a kind of congenital fibrinogen defect is congenital in preparation detection
Application in the kit of property fibrinogen defect.
The kit includes the reagent for detecting 201 above-mentioned mutational sites.
Another aspect of the present invention additionally provides a kind of kit for detecting congenital fibrinogen defect, the reagent
Box includes: the reagent for detecting 201 above-mentioned mutational sites.
The detection reagent includes as shown in table 2 for expanding the PCR primer in 201 above-mentioned mutational sites:
The detection kit further includes the reagent that genomic DNA is made to the library for sequencing, this kind of reagent is
Reagent commonly used in the art, as long as can satisfy requirement of the PCR amplification to genomic DNA quality.The reagent preparation
Method is this field customary preparation methods, preferably commercially available, such as: the fast run-up library kit (Ion of DNA was sequenced in two generations
Torrent), two generations sequencing rapid DNA is built library kit (Illumina), and NGS builds library kit (Illumina) etc..
The kit further includes being expanded for the reagent that two generations were sequenced using amplimer provided by the invention
The individual chip length arrived is 275bp or so, in conjunction with two generation sequencing technologies, is able to detect in mutated gene group of the present invention
Whole exon regions.
Wherein the reagent for the sequencing of two generations is reagent commonly used in the art, as long as can satisfy to gained sequence
Column carry out the requirement of two generations sequencing.The preparation method of reagent thereof be this field customary preparation methods, it is preferably commercially available can
, such as: two generation sequencing library quantification kits, sequencing reagent kit etc..
The kit further includes dNTP solution, and archaeal dna polymerase is positive right for the reagent of isolated or purified nucleic acid
According to and/or negative control.
Reagent for isolated or purified nucleic acid can be Beckman magnetic bead.
Preferably, the positive control is the positive control of this field routine, preferably comprising of the present invention prominent
Become the genomic DNA stock solution of mutant gene sequence or the matter comprising mutant gene sequence in corresponding mutated gene group in gene group
Grain is used as positive control.
Preferably, the negative control is the negative control of this field routine, preferably confirmed derived from Healthy People
Wild type gene group DNA sequence dna storing liquid not comprising mutant gene sequence.
Sample used is the tissue of test object, as long as the genomic DNA of test object can be extracted from detection sample
?.
Preferably, detection sample used be one or more of blood, marrow, solid tissue sample, it is highly preferred that
Detection sample is blood.
A kind of application method of kit of the present invention comprising following steps:
(1) using the peripheral blood of detection kit of the present invention extracting test object, genomic DNA is extracted；
(2) with PCR primer to 3 described in invention in genomic DNA congenital fibrinogen defect related genes
Exon region is expanded；
(3) library for the sequencing of Ion Torrent microarray dataset is made in the resulting segment of amplification in step (2)；
(4) gained DNA library in step (3) is sequenced, obtains lower machine data；
(5) bioinformatic analysis is carried out to the lower machine data in (4), is then interpreted using medicine unscrambling data library
Make diagnosis.
Reagent used in the present invention and raw material are commercially available.
Medicine unscrambling data library used is as follows:
(1) FGA gene mutation known to is related to congenital fibrinogen defect, and congenital fibrinogen defect is usual
For the incomplete recessive hereditary disease of autosome, there is consanguineous mating history, patient has different degrees of bleeding all the life more.Laboratory
It checks that plasma fibrinogen level reduces, is often lower than the 50% of normal value, blood FIB (fiber egg when afibrinogenemia
White original) detection it is micro, often < 20mg/dL, platelet counts and function are normal, APTT (activated partial thromboplastin time), PT
(plasma prothrombin time), TT (blood plasma hemase time) extend, and can be entered normal blood plasma and fibrinogen is corrected,
Patient also may occur in which that the textural anomaly of fibrinogen causes its function to change, and women is common, and about half patient is asymptomatic, can go out
Existing hyporrhea tendency and thrombosis；
(2) FGB gene mutation known to is related to congenital fibrinogen defect, and congenital fibrinogen defect is usual
For the incomplete recessive hereditary disease of autosome, there is consanguineous mating history, patient has different degrees of bleeding all the life more.Laboratory
It checks that plasma fibrinogen level reduces, is often lower than the 50% of normal value, blood FIB detection is micro- when afibrinogenemia
Amount, often < 20mg/dL, platelet counts and function are normal, and APTT, PT, TT extend, and can be entered normal blood plasma and fiber
Proteinogen is corrected, and patient also may occur in which that the textural anomaly of fibrinogen causes its function to change, and women is common, about half patient
It is asymptomatic, it may occur in which hyporrhea tendency and thrombosis；
(3) FGG gene mutation known to is related to congenital fibrinogen defect, and congenital fibrinogen defect is usual
For the incomplete recessive hereditary disease of autosome, there is consanguineous mating history, patient has different degrees of bleeding all the life more.Laboratory
It checks that plasma fibrinogen level reduces, is often lower than the 50% of normal value, blood FIB detection is micro- when afibrinogenemia
Amount, often < 20mg/dL, platelet counts and function are normal, and APTT, PT, TT extend, and can be entered normal blood plasma and fiber
Proteinogen is corrected, and patient also may occur in which that the textural anomaly of fibrinogen causes its function to change, and women is common, about half patient
It is asymptomatic, it may occur in which hyporrhea tendency and thrombosis.
Compared with prior art, positive and beneficial effect of the invention is:
The mutated gene group of the congenital fibrinogen defect of one group of detection provided by the invention and its detection kit, can
For the mutated gene group of the congenital fibrinogen defect of screening, in conjunction with high throughput sequencing technologies, may be implemented raw in molecule
Object level diagnoses congenital fibrinogen defect, makes especially for the subsequent orientation treatment of disease and effect
Anticipation has special advantage.Detection kit provided by the invention has detection efficiency height, mutated gene broad covered area, detection
It is more accurate, authoritative to interpret report using medicine given by the detection kit for the advantages that rate is high.
Specific embodiment
The explanation of following embodiment is merely used to help understand method and its core concept of the invention.It should be pointed out that pair
For those skilled in the art, without departing from the principle of the present invention, the present invention can also be carried out
Some improvements and modifications, these improvements and modifications also fall within the scope of protection of the claims of the present invention.To disclosed implementation
The following the description of example, enables those skilled in the art to implement or use the present invention.Various modifications to these embodiments
It will be readily apparent to those skilled in the art, the general principles defined herein can not depart from this
In the case where the spirit or scope of invention, realize in other embodiments.Therefore, the present invention is not intended to be limited to illustrated herein
These embodiments in, but can be applied to meet broader model consistent with the principles and novel features disclosed in this article
It encloses.Although can be used in implementation or test of the invention and heretofore described similar or of equal value any method and material
Material, preferred method and material are enumerated by place herein.
Unless otherwise defined, all technical and scientific terms used herein have and the technical field of the invention
The normally understood identical meaning of those of ordinary skill.
In the examples where no specific technique or condition is specified, described technology or conditions according to the literature in the art
(such as with reference to J. Pehanorm Brooker etc. write, " Molecular Cloning:A Laboratory guide " that Huang Peitang etc. is translated, the third edition, Science Press) or
Person carries out according to product description.
The screening of the congenital fibrinogen defect mutated gene group of 1 Chinese population of embodiment
Material and method:
In blood disease hospital, the Chinese Academy of Medical Sciences and coordinate magnificent medical diagnosis center in January, 2017 in July, 2018
In the patient of diagnosis, according to following standard, carries out continuity and enter group (23).
Inclusion criteria are as follows: the patient of fibrinogen defect is diagnosed as through clinical symptoms and laboratory inspection, and
Screening needs to carry out genetic test to carry out auxiliary and be confirmed whether it is the case of congenital fibrinogen defect, 23 patients'
Diagnostic result is as shown in table 3.
3 patient's diagnostic result list of table:
Collection 3mL peripheral blood in patients extraction genomic DNA is to be measured, which passes through Chinese Academy of Medical Sciences's blood disease hospital
Ethics Committee's approval.
1. sample process: genome (is purchased from Tiangeng biochemical technology Co., Ltd, goods using DNA extraction kit in sample
Number: it DP318-03) is stripped, concrete operation method: (1) pre-processing: 500ul whole blood, sample being 1. added in the EP pipe of 1.5mL
The cell pyrolysis liquid CL of 800ul is added in product, is mixed by inversion, 10000rpm is centrifuged 1min；2. sucking supernatant, it is heavy to leave nucleus
It forms sediment；3. into the nucleus precipitating being collected into plus 200 μ l buffer GS, oscillation are mixed to thorough.(2) it lytic cell: is 1. added
20 μ l Proteinase K solution mix；2. plus 200 μ l buffer GB, be sufficiently mixed by inversion, be mixed by inversion therebetween for several times, 56
DEG C place 10min, solution strain it is limpid.(3) DNA precipitating and column absorption is crossed: 1. plus 200 μ l dehydrated alcohols, sufficiently reverse immediately
It mixes；2. previous step acquired solution and flocculent deposit are added in adsorption column CB3,12000rpm is centrifuged 30s, by adsorption column CB3
It is put into new collecting pipe.(4) deproteinized, rinsing: 500 μ l buffer GD, 12000rpm being 1. added into adsorption column CB3 and are centrifuged 30s,
Adsorption column CB3 is put into new collecting pipe；2. 600 μ l rinsing liquid PW, 12000rpm centrifugation 30s are added into adsorption column CB3, will inhale
Attached column CB3 is put into new collecting pipe；3. repetitive operation step 7；4. 12000rpm is centrifuged 2min, adsorption column CB3 is transferred to 1.5mLEP
Adsorption column CB3 is uncapped to be placed in Biohazard Safety Equipment and be blown several minutes by Guan Zhong, dries rinsing liquid remaining in adsorbent material.(5)
DNA elution: being 1. vacantly added dropwise the elution buffer TB that 40 μ l are preheated in advance to adsorbed film middle position, be placed at room temperature for 2-5min,
12,000rpm centrifugation 2min, solution is collected into centrifuge tube；2. measuring concentration.
2. the building in library: the part of the exons coding district and flank of choosing testing gene FGA, FGB and FGG includes
Subregion, using Ion amplicon sequencing library building kit 2.0-LV96 (be purchased from Thermo Fisher company, article No.:
4475345) multiple PCR method is carried out to genomic DNA and builds library, the specific steps are as follows: (1) target of PCR amplification genomic DNA
Region: being 1. added pure water into DNA sample according to concentration and be diluted to 5ng/ul, including pool1 and pool2, establishes reaction respectively
System:
2. covering 1400rpm brief centrifugation, MixMate 2000r 5s is mixed, and 1400rpm brief centrifugation carries out PCR, instead
Answer condition as follows:
(2) digestion: pool1 reaction solution is transferred in pool2-PCR pipe, the FuPa of 1.9 μ l is added by centrifugation PCR pipe
Reagent is mixed after sealing, and centrifugation, soft be vortexed are centrifuged again, and endonuclease reaction condition is as follows:
| Temperature | Time |
| 50℃ | 20min |
| 55℃ | 20min |
| 60℃ | 20min |
| 10℃ | 1h |
(3) it expands effective library: 1. dissolving Switch Sloution, respective volume is added into each digestion products
The Switch Solution and Barcode diluted, component are as follows:
| Component | Volume |
| Switch Solution | 4μl |
| Barcode | 2μl |
| Total volume | 28ul |
2. the DNA ligase of 2 μ l is added in every hole, reaction condition is as follows after mixing:
| Temperature | Time |
| 22℃ | 30min |
| 72℃ | 10min |
| 10℃ | 1h |
(4) purify the library not expanded: 1. 45 μ l AMPure Beads, vortex 5min are added in every hole in DNA library
(1800rpm) is placed at room temperature for 5min, moves to experimental plate on magnetic frame after centrifugation, puts to clarifying and then discarding supernatant, is added
The alcohol of 150 μ l fresh 70%, rotation Ep pipe two enclose or move left and right 96 orifice plates, discard alcohol；2. air-drying magnetic after coming again
Pearl；EP pipe is removed, the Low TE that 100 μ l are added disperses magnetic bead, and soft be vortexed mixes；3. being placed at room temperature for 2min to move back to magnetic frame
On, library is shifted after clarifying into clean EP pipe.
3. prepared by template: specific to walk with reference to the operation instructions of Ion Torrent sequenator corollary equipment One Touch
Rapid as follows: (1) dilute library and quantitative: the library gradient that 1. will be built up is centrifuged after sufficient vortex to diluting 200 times；2. standard
For 10 times of gradient dilutions of product to 6.8pM, 0.68pM, 0.068pM, 7500 instrument reaction system of ABI is as follows:
| Ion AmpliSeqTMDNA library | |
| 2×Master Mix | 5μl |
| 20×TaqMan | 0.5μl |
| Sample (standard items) | 4.5μl |
Reaction condition is as follows:
(2) Water-In-Oil PCR amplification: 1. the library sufficient vortex for being diluted to 200 times is mixed, reaction system is as follows:
*: library applied sample amount is calculated according to qRT-pcr quantitative result, is adjusted in the total applied sample amount of 700~750pmol
It is whole.
2. selecting Proton:Ion PITMHi-QTMOT2 200Kit program runs Water-In-Oil PCR reaction；3. removing oily packet
Recovery Tube after water PCR reaction, discards the Recovery solution on upper layer, and microballon is resuspended, and prepares Melt-off solution,
System is as follows:
| Sequentially | Component | Volume |
| 1 | Tween solution | 280μl |
| 2 | 1M NaOH | 40μl |
| Total volume | 320μl |
(3) it enrichment with magnetic bead: is vortexedMyOneTM130 μ L-Ion are added in Streptavidin C1Beads
PITMMyOneTMBeads Capture Solution is enriched with.(4) One touch and ES: 1. will corresponding agent transfer
Dedicated 8 connecting leg of ES carries out ES reaction, and sequence is as follows:
| 8 platoon holes number | Reagent |
| 1 | The positive ISP template of 100 μ l |
| 2 | 130μl-MyOneTMBeads eluent |
| 3 | 300μl Ion PITMES Wash Solution |
| 4 | 300μl Ion PITMES Wash Solution |
| 5 | 300μl Ion PITMES Wash Solution |
| 6 | It is empty |
| 7 | 300 μ l-Melt-off solution |
| 8 | It is empty |
2. being centrifuged the ISP 5min 15500g after ES, abandons supernatant and only stay 10ul that microballon is resuspended；After cleaning and being centrifuged ES
ISP 5min 15500g abandons supernatant and leaves and takes 10ul resuspension magnetic bead, moisturizing to 100ul, and pressure-vaccum mixes 10 times.
4. sequencing reaction: referring to high-flux sequence instrument Ion Torrent operation instructions, the specific steps are as follows: (1) prepare
Sample: 1. vortex Ion PI is sequencedTMControl Ion SphereTMParticles 5s is centrifuged 2s, after adding 4 μ l to ES
Soft to be vortexed in re-suspension liquid, 15500rpm is centrifuged 3min；2. discarding supernatant, 10 μ l are stayed, 15 μ l-Ion PITM are then added
Annealing Buffer adds 20ul Sequencing Primer, soft to be vortexed, and is centrifuged 2s；Sequencing primer annealing is anti-
It answers: 95 DEG C, 2min；37 DEG C, 2min；25 DEG C of maintenances；(2) be centrifuged PCR pipe, be added 10ul Loading Buffer, mix and from
The heart, total 55ul；(3) washing of Ion-Proton sequenator, chlorine are washed and are initialized；(4) Loading chip；(5) machine is sequenced on.
5. bioinformatic analysis: using Ion Report software (v4.6, Thermo Fisher, Carlsbad, CA,
USA) filter low-quality sequencing fragment, and by qualified sequencing fragment compare to the mankind with reference to genome hg19 (http: //
Hgdownload.cse.ucsc.edu/downloads.html), using Torrent Variant Caller (v4.6.0.7)
Subprogram detects mutational site, and software parameter uses the setting of default.This software has been optimized for processing Ion
The distinctive type of error of Torrent microarray dataset.To the mutation found out include SNP and Indel using ANNOVAR (http: //
Annovar.openbioinformatics.org/en/latest/) software is annotated, and notes content includes mutation in gene
Position in group, associated gene, gene extron number, nucleotide level variation, corresponding protein level variation, and should
Mutation is in dbSNP database (http://www.ncbi.nlm.nih.gov/snp/), thousand human genome databases
1000Genomes (http://www.1000genomes.org/), exon group integrated database ExAC (http: //
) and human mutation database HGMD (http://www.hgmd.cf.ac.uk/ exac.broadinstitute.org/
Ac/index.php the annotation in), PolyPhen (http://genetics.bwh.harvard.edu/pph2/) and SIFT
(http://sift.jcvi.org/) function prediction result etc..
Further use following principle screening pathogenic mutation site: (1) according to genomic locations and type mutation where,
Filter out the mutation not having an impact to protein product sequence；(2) 1000Genomes data and ExAC data are utilized, annotation is every
Ratio of a mutation in crowd, is not considered as polymorphic site if ratio is less than or equal to 1%；(3) human gene is retrieved
Whether mutation database, it is on the books in HGMD to inquire a mutation, appears in the correlations such as congenital fibrinogen defect
In disease；(4) predict whether the mutation influences protein function using protein function forecasting software PolyPhen-2 and SIFT；
(5) if after (2) (3) (4), mutation meet " non-polymorphic ", " congenital fibrinogen defect was recorded " and
At least two in " influence protein function " this three, will be judged to may be related to disease.If although a mutation is unsatisfactory for
(5) but there is record in HGMD, be then read the mutation for interrogatory, if a last mutation is defined in the literature
It is recorded as highly relevant with disease, is then directly judged to hot spot mutation.
6. statistical analysis: counting the ratio that each gene carries pathogenic mutation, filter out the highest gene of mutation rate, use
Software be Excel.
7. medicine is interpreted: making molecular disease to the genic mutation type that bioinformatic analysis obtains with medicine unscrambling data library
Reason diagnosis.Medicine unscrambling data library used is as follows:
(1) FGA gene mutation known to is related to congenital fibrinogen defect, and congenital fibrinogen defect is usual
For the incomplete recessive hereditary disease of autosome, there is consanguineous mating history more.Patient has different degrees of bleeding all the life.Laboratory
It checks that plasma fibrinogen level reduces, is often lower than the 50% of normal value, blood FIB detection is micro- when afibrinogenemia
Amount, often < 20mg/dL, platelet counts and function are normal, and APTT, PT, TT extend, and can be entered normal blood plasma and fiber
Proteinogen is corrected.Patient also may occur in which that the textural anomaly of fibrinogen causes its function to change, and women is common, about half patient
It is asymptomatic, it may occur in which hyporrhea tendency and thrombosis.
(2) FGB gene mutation known to is related to congenital fibrinogen defect, and congenital fibrinogen defect is usual
For the incomplete recessive hereditary disease of autosome, there is consanguineous mating history more.Patient has different degrees of bleeding all the life.Laboratory
It checks that plasma fibrinogen level reduces, is often lower than the 50% of normal value, blood FIB detection is micro- when afibrinogenemia
Amount, often < 20mg/dL, platelet counts and function are normal, and APTT, PT, TT extend, and can be entered normal blood plasma and fiber
Proteinogen is corrected.Patient also may occur in which that the textural anomaly of fibrinogen causes its function to change, and women is common, about half patient
It is asymptomatic, it may occur in which hyporrhea tendency and thrombosis.
(3) FGG gene mutation known to is related to congenital fibrinogen defect, and congenital fibrinogen defect is usual
For the incomplete recessive hereditary disease of autosome, there is consanguineous mating history more.Patient has different degrees of bleeding all the life.Laboratory
It checks that plasma fibrinogen level reduces, is often lower than the 50% of normal value, blood FIB detection is micro- when afibrinogenemia
Amount, often < 20mg/dL, platelet counts and function are normal, and APTT, PT, TT extend, and can be entered normal blood plasma and fiber
Proteinogen is corrected.Patient also may occur in which that the textural anomaly of fibrinogen causes its function to change, and women is common, about half patient
It is asymptomatic, it may occur in which hyporrhea tendency and thrombosis.
It is as shown in table 1 to the selection result of the congenital fibrinogen defect mutated gene group of Chinese population, congenital fibre
Fibrillarin original defect mutated gene group includes the set of FGA, FGB and FGG mutated gene of totally 3 genes, exon region
201 mutational sites are carried altogether, can be used for detecting the related disease of congenital fibrinogen defect.
Primer needed for embodiment 2 synthesizes detection kit
Design of primers: being directed to mutational site of the present invention, is expanded using Ion AmpliSeq Designer software design
Required multiple PCR primer finally found that primer sequence primer combination as shown in Table 2 can balance, efficiently amplify institute
There is mutational site.
Primer synthesis: designed primer is sent to Synesis Company and is synthesized.
Embodiment 3 detects clinical congenital fibrinogen defect patient's sample using kit described in embodiment 2
(1) peripheral blood for collecting 3mL patient described in embodiment 1 (is purchased from Tiangeng biochemistry section using DNA extraction kit
Skill Co., Ltd, article No.: DP318-03) genomic DNA is extracted, mutated gene sequence in mutated gene group of the present invention will be included
The genomic DNA stock solution of column will be derived from the confirmed genome not comprising mutant gene sequence of Healthy People as positive control
DNA sequence dna storing liquid is as negative control；
(2) multiple PCR primer in the kit described in embodiment 2 expands genomic DNA, PCR amplification it is anti-
It answers system and response procedures is conventional method in that art；
(3) the resulting segment of amplification in step (2) is surveyed using two generations sequencing Ion amplicon with the method in embodiment 1
Sequence library construction Kit 2.0-LV96 (is purchased from Thermo Fisher company, article No.: 4475345) is made for Ion
The library of Torrent microarray dataset sequencing；
(4) with the method in embodiment 1 to gained DNA library in step (3) after Water-In-Oil is handled in Ion Torrent
High-flux sequence is carried out on platform, obtains lower machine data；
(5) bioinformatic analysis is carried out to the lower machine data in (4), is then interpreted using medicine unscrambling data library
Make diagnosis.The positive rate of 3 genes detected by 23 patients is as shown in table 4 below.
The congenital fibrinogen defect related gene positive rate of table 4:
After counting the result shows that: detect to be congenital fibrinogen defect in 23 patients, wherein there is carrying
FGA gene mutation patient 13, FGB gene mutation patient 7 is carried, carries FGG gene mutation patient 3, and obtain
Conclusion is consistent with clinical last diagnostic.It can be seen that can be effectively to congenital fibrin using the detection kit
Former defect patient diagnoses, have the characteristics that diagnosis quickly, it is accurate, contained much information.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Within mind and principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (6)
1. a kind of kit for detecting relevant 201 mutational sites of congenital fibrinogen defect, which is characterized in that described
Kit includes the reagent for detecting 201 mutational sites；
201 mutational sites are located at FGA, FGB, FGG totally 3 genes；201 mutational sites are as follows:
The reagent include for and meanwhile expand the PCR primer in 201 above-mentioned mutational sites:
2. kit according to claim 1, which is characterized in that the kit further includes that genomic DNA is made
For the reagent in the library of sequencing.
3. kit according to claim 1, which is characterized in that the kit further includes the examination for the sequencing of two generations
Agent.
4. kit according to claim 1, which is characterized in that the kit further include dNTP, archaeal dna polymerase,
Reagent, positive control and/or negative control for isolated or purified nucleic acid.
5. kit according to any one of claims 1-4, which is characterized in that detection sample used is blood, bone
Marrow or solid tissue sample.
6. kit according to any one of claims 1-4, which is characterized in that detection sample used is blood.
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| 一个遗传性无纤维蛋白原血症家系的基因缺陷分析;谢燕燕;《中国优秀硕士学位论文全文数据库 医药卫生科技辑》;20170215(第02期);E65-589 |
| 一例低纤维蛋白原血症家系基因缺陷的研究;张洋;《中国优秀硕士学位论文全文数据库 医药卫生科技辑》;20140515(第05期);E065-225 |
| 遗传性异常纤维蛋白原血症FGA与FGG基因突变研究;闫婕;《中国优秀硕士学位论文全文数据库 医药卫生科技辑》;20170215(第02期);E062-698 |
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Address after: 301617 Block C, Tuanbo football field, West Tuanbo new city, Jinghai District, Jinghai County, Tianjin Patentee after: Tianjin Jiankang Huamei Medical Diagnosis Technology Co.,Ltd. Address before: 301617 Block C, Tuanbo football field, West District, Tuanbo new city, Jinghai District, Tianjin (cancelled) Patentee before: TIANJIN SINO-US DIAGNOSTICS MEDICAL DIAGNOSIS TECHNOLOGY Co.,Ltd. |