CN109504767A - A kind of molecular marker of childrens respiratory tract syncytial virus infection and its application - Google Patents
A kind of molecular marker of childrens respiratory tract syncytial virus infection and its application Download PDFInfo
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- CN109504767A CN109504767A CN201811607574.XA CN201811607574A CN109504767A CN 109504767 A CN109504767 A CN 109504767A CN 201811607574 A CN201811607574 A CN 201811607574A CN 109504767 A CN109504767 A CN 109504767A
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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Abstract
The invention discloses a kind of molecular marker of childrens respiratory tract syncytial virus infection and its applications, the present invention has found that H2AFJ and RGL4 express up-regulation in the child patient of respiratory syncytial virus infection using the method for high-flux sequence for the first time, may determine that whether subject suffers from respiratory syncytial virus infection disease by detecting H2AFJ and RGL4 expression.
Description
Technical field
The invention belongs to biomedicine field, be related to a kind of childrens respiratory tract syncytial virus infection molecular marker and its
Using.
Background technique
Respiratory Syncytial Virus(RSV) (Respiratory syncytial virus, RSV) is to infect the important respiratory tract of the mankind
Virus, main infection children, old man and hypoimmunity crowd can lead to the serious breathing including pneumonia and bronchitis
Tract disease is one of the main reason for leading to death of child in world wide.Although the vaccine and therapeutic antibodies for RSV are ground
Study carefully and achieve certain progress, but there is no the vaccine and efficient antiviral intervention means of commercialization at present.Study virus host because
Son facilitates the new antiviral target spot of discovery, is research hotspot and emphasis in current area.
How to influence the existing certain understanding of host immune defenses in course of infection on RSV at present, but exists to host factor
Effect study in rsv infection is less, especially lacks and replicates recognizing comprehensively for related host factor to RSV at the genomic level
Know.The interaction for understanding virus and host is the basis for going deep into understanding RSV pathogenic mechanism.
With the innovation of biology barrier means, the appearance for the cutting edge technologies methods such as group is learned is comprehensive, system grinds
Study carefully the virus host factor and provides technological means.With the appearance of next-generation sequencing technologies, and develop on this basis
Transcript profile sequencing technologies RNAseq is also used for virus-host's repercussion study, and finds virus infection in transcript profile level
The host gene of differentiation expression, the adjustment mechanism to construct RSV- host's interactive network, illustrating rsv infection provide afterwards
Theoretical basis also provides research direction for the relevant biomarker of discovery rsv infection and new antiviral target spot.
Summary of the invention
In order to make up for the deficiencies of the prior art, the purpose of the present invention is to provide can be used for judging that it is sick that childrens respiratory tract closes packet
The molecular marker of poison infection, to realize the early diagnosis of childrens respiratory tract syncytial virus infection.
To achieve the goals above, the present invention adopts the following technical scheme:
The reagent that the first aspect of the present invention provides detection molecules marker closes born of the same parents' disease in preparation diagnosis childrens respiratory tract
Application in poison infection product, the molecular marker are H2AFJ.
Further, the molecular marker further includes RGL4.
Further, the reagent is selected from:
The probe of gene described in specific recognition;Or
The primer of gene described in specific amplification;Or
Specifically bind the bonding agent of the albumen of the gene coding.
Further, the primer sequence of specific amplification H2AFJ is as shown in NO.1~2 SEQ ID, specific amplification RGL4's
Primer sequence is as shown in NO.3~4 SEQ ID.
The second aspect of the present invention provides a kind of product, and the product includes dividing described in detection first aspect present invention
The reagent of sub- marker expression level.
Further, the product includes chip, kit, nucleic acid film item.
Further, the chip includes genetic chip, protein-chip, and wherein genetic chip includes being directed to molecular marker
Specific primer or probe, protein-chip include specific binding molecules marker coding albumen antibody or ligand.
Further, the kit includes gene detecting kit, protein immunization detection kit, wherein the gene
Detection kit includes the specific primer, probe or genetic chip of detection molecules marker;The protein immunization detection reagent
Box includes antibody, ligand or the protein-chip of the albumen of specific binding molecules marker coding.
The third aspect of the present invention provides the application of product described in second aspect of the present invention, is used to prepare diagnosis children
The tool of respiratory syncytial virus infection disease.
The fourth aspect of the present invention provides molecular marker described in first aspect present invention and exhales in building prediction children
Inhale the application in the computation model of road syncytial virus infection.
The advantages of the present invention:
Present invention firstly discovers that relevant to childrens respiratory tract syncytial virus infection molecular marker, the molecular marker
Object is H2AFJ and/or RGL4;By the expression of said gene in detection subject's blood, childrens respiratory tract may be implemented
The early diagnosis of syncytial virus disease, to carry out early intervention treatment.
Detailed description of the invention
Fig. 1 is expression figure of the molecular marker in respiratory syncytial virus infection children.
Specific embodiment
The present invention after extensive and in-depth study, is screened by high throughput sequencing technologies in Respiratory Syncytial Virus(RSV)
The gene of differential expression is presented in the child patient of infection, inquires into its relationship between respiratory syncytial virus infection, is children
The early detection of respiratory syncytial virus infection finds better approaches and methods, while being respiratory syncytial virus infection disease
Treatment new target spot is provided.By screening, present invention firstly discovers that in the child patient of respiratory syncytial virus infection
The expression of H2AFJ and RGL4 significantly raises, and H2AFJ and RGL4 is prompted to can be used as Testing index examining applied to rsv infection
It is disconnected.
" molecular marker " or " biomarker ", which refers to, has specific biological characteristic, biochemical characteristics or side
The molecular indicator in face can be used for determining presence or absence of the tight of specified disease or situation and/or specified disease or situation
Weight degree.
In the present invention, " marker " refers to parameter (i.e. " biomarker ") relevant to one or more biomolecule,
Such as natural or artificial synthesized generation nucleic acid (i.e. genes of individuals, and coding and noncoding DNA and RNA) and albumen (such as
Peptide, polypeptide)." marker " in the present invention further includes that finger can be by considering the expression number from two or more unlike signal objects
According to the single parameter for calculating or otherwise obtaining.
In the present invention, the marker is H2AFJ and RGL4 gene and its encoded albumen, H2AFJ and RGL4 packet
Include wild type, saltant type or its segment.ID of the H2AFJ gene in current international public nucleic acid database GeneBank be
ID of 55766, the RGL4 genes in current international public nucleic acid database GeneBank is 266747.
As non-limiting embodiment, a kind of nucleotide sequence of representative H2AFJ gene is such as international at present public
In nucleic acid database GeneBank shown in H2AFJ gene (NM_177925.3);A kind of nucleotides sequence of representative RGL4 gene
Column are as shown in RGL4 gene (NM_001329424.1) in current international public nucleic acid database GeneBank.
In the present invention, it can use the expression of any method known in the art measurement gene or albumen.This field
It will be appreciated by the skilled person that the means of measurement gene or protein expression are not importances of the invention.
Gene marker of the invention can be used multiple nucleic acids technology known to persons of ordinary skill in the art and be examined
It surveys, these technologies include but is not limited to: nucleic acid sequencing, nucleic acid hybridization, nucleic acid amplification technologies and protein immunization detection technology.
The exemplary, non-limitative example of Nucleic acid sequencing techniques includes but is not limited to chain terminator (Sanger) sequencing and dye
Expect terminator sequencing.Those skilled in the art it will be recognized that due to RNA in cell less stable and in an experiment
It is more vulnerable to nuclease attack, therefore usually by RNA reverse transcription at DNA before sequencing.
The present invention simultaneously can expand nucleic acid (for example, ncRNA) before detection or with detection.Nucleic acid amplification technologies
Exemplary, non-limitative example include but is not limited to: polymerase chain reaction (PCR), reverse transcriptase polymerase chain reaction (RT-
PCR), the amplification (TMA) of transcriptive intermediate, ligase chain reaction (LCR), strand displacement amplification (SDA) and based on nucleic acid sequence
It expands (NASBA).Those skilled in the art will be it will be recognized that certain amplification techniques (for example, PCR) needs will before amplification
RNA reverse transcription is at DNA (for example, RT-PCR), and other amplification techniques then direct cloning RNA (for example, TMA and NASBA).
The polymerase chain reaction of commonly referred to as PCR uses denaturation, the annealing and primer extend of primer pair and opposite strand
Multiple circulations, exponentially increase target nucleic acid sequence copy number;The amplification of the transcriptive intermediate of TMA is (substantial constant
Temperature, multiple copies of target nucleic acid sequence are autocatalytically synthesized under conditions of ionic strength and pH, wherein target sequence is more
A RNA copy autocatalytically generates other copy;The ligase chain reaction of LCR uses miscellaneous with the adjacent area of target nucleic acid
The two groups of complementary DNA oligonucleotides handed over;Other amplification methods include for example: the commonly referred to as expansion based on nucleic acid sequence of NASBA
Increase;Use the amplification of rna replicon enzyme (commonly referred to as Q β replicase) amplification probe molecule itself;Amplification method based on transcription;
And the sequence amplification of self―sustaining.
Protein immunization technology includes but is not limited to sandwich immunoassay, such as sandwich ELISA, wherein using identification biology
Two kinds of antibody of different epitopes carry out the detection of the biomarker on marker;Radiommunoassay (RIA), directly, indirectly
Or comparison enzyme linked immunosorbent assay (ELISA) (ELISA), enzyme immunoassay (EIA) (EIA), fluorescence immunoassay (FIA), immunoblotting,
Immuno-precipitation and immunoassays based on any particle are (as used gold particle, Argent grain or latex particle, magnetic-particle or amount
Sub- point).Immunization for example can be implemented in the form of microtiter plate or item.
It can be used in the detection of molecular marker of the invention any directly (as used sensor chip) or indirect
Method.
In the present invention, the bonding agent for specifically binding H2AFJ, RGL4 albumen includes but is not limited to H2AFJ, RGL4 albumen
Receptor, the agglutinin in conjunction with H2AFJ, RGL4 albumen, the antibody for H2AFJ, RGL4 albumen, be directed to H2AFJ, RGL4 albumen
Peptide antibody, the agent of bispecific dual combination or bispecific antibody form.
The present invention provides the products of detection H2AFJ and/or RGL4 expression, and the product includes but is not limited to core
Piece, nucleic acid film item or kit.Wherein chip includes: solid phase carrier;And the orderly widow being fixed on the solid phase carrier
Nucleotide probe or small peptide, the oligonucleotide probe some or all of specifically correspond to shown in H2AFJ, RGL4 sequence
Column, the small peptide specifically bind the encoded albumen of H2AFJ, RGL4.
Nucleic acid film item includes substrate and the oligonucleotide probe that is fixed in the substrate;The substrate can be any suitable
In the substrate of immobilized oligonucleotide probe, for example, it is nylon membrane, nitrocellulose filter, polypropylene screen, sheet glass, silica gel chip, micro-
Contracting magnetic bead etc..
In the present invention, kit includes the reagent and one or more objects selected from the group below for detecting H2AFJ, RGL4
Matter: container, operation instructions, positive control, negative control object, buffer, auxiliary agent or solvent.
In kit of the invention can also have kit operation instructions, be described how using kit into
Row detection, and how to be judged using child patient of the testing result to respiratory syncytial virus infection, to therapeutic scheme into
Row selection.
The component of kit can pack in the form of aqueous medium or in the form of freeze-drying.Container appropriate in kit
It include typically at least a kind of bottle, test tube, flask, PET bottle, syringe or other containers, wherein a kind of component can be placed, and
And preferably, suitably equal part can be carried out.There are more than one group timesharing in kit, generally also will include in kit
Second, third or other additional containers, wherein being positioned separately additional component.However, the component of various combination can be wrapped
It is contained in a bottle.Kit of the invention will include generally also a kind of container for being used to accommodate reactant, seal to be used for
Commercial distribution.This container may include the plastic containers of injection molding or blowing mould, wherein can retain required bottle.
Statistical method
In the present invention, experiment is at least tested using 3 repetitions, and result data is all in a manner of mean+SD
Indicate, come using SPSS18.0 statistical software for statistical analysis, difference between the two is using t inspection, it is believed that when P <
There is statistical significance when 0.05.
The present invention is described in further detail with reference to the accompanying drawings and examples.Following embodiment is merely to illustrate this
It invents rather than limits the scope of the invention.Test method without specific conditions in embodiment, usually according to conventional strip
Part, or according to the normal condition proposed by manufacturer.
Embodiment 1 screens gene marker relevant to childrens respiratory tract syncytial virus infection
1, sample collection
The blood sample for collecting 5 normal child's blood and childrens respiratory tract syncytial virus infection patient respectively, writes sample exactly
The acquirement of situations such as this title, number, Date of Sampling, sample process process, above-mentioned all samples pass through Ethics Committee
Agree to.
2, the preparation and quality analysis of RNA sample
2.1 extract total serum IgE using the RNA extracts kit of Promega company.Specific step is as follows:
1) it takes 1ml to collect the whole blood in heparin or the processed test tube of EDTA, puts into sterile centrifugation tube, 3000rpm
It is centrifuged 5min, carefully siphons away supernatant at the top of sample;
2) plus 1ml haemocyte lysate, careful inhale are put 4-5 times, sediment are resuspended, 3000rpm is centrifuged 5min;
3) step 2) is repeated twice;
4) cell precipitate is avoided, nearly all supernatant is carefully siphoned away, only retains 100 μ l supernatants;Plus 175 μ l then
For RNA lysate into cell, resuspension and lytic cell are put in suction;
5) plus 350 μ l RNA dilutions, reverse mixing 3-4 times are placed in 3min in 70 DEG C of water-baths;
6) 13000g is centrifuged 10min at room temperature;
7) limpid lysate is transferred in a sterile centrifugation tube, 200 μ l, 95% second is added into cleared lysate
Alcohol puts 3-4 times with liquid-transfering gun suction with mixing;This mixture is transferred in centrifugal column assembly, 13000g is centrifuged 1min;
8) plus 600 μ l RNA cleaning solutions are in centrifugal column assembly, and 13000g is centrifuged 1min;
9) liquid in collecting pipe is discarded, the freshly prepared DNase mixtures incubated of 50 μ l is applied directly in centrifugal column
Film on;
10) it is incubated for 15min at room temperature, 200 μ l DNA enzymatic stop buffers, 13000g centrifugation are added into centrifugal column
1min;
11) 600 μ l RNA cleaning solutions are added, 13000g is centrifuged 1min;
12) collecting pipe is emptied, 250 μ l RNA cleaning solutions, high speed centrifugation 2min are added into centrifugal column;
13) plus 100 μ l nuclease-free waters, 13000g are centrifuged 1min, abandon centrifugal column, cover the elution pipe for filling RNA, deposit
In -70 DEG C.
The quality analysis of 2.2RNA sample
The RNA of said extracted is subjected to agarose gel electrophoresis, using Nanodrop2000 to the concentration of mentioned RNA and pure
Degree is detected, and agarose gel electrophoresis detects RNA integrality, and Agilent2100 measures RIN value.Single requirement for construction data base RNA is total
5 μ g are measured, concentration >=200ng/ μ L, OD260/280 is between 1.8~2.2.
3, construction cDNA library
The rRNA in total serum IgE is removed using Ribo-Zero kit, utilizes Illumina TruseqTM RNA
Sample Prep Kit carries out the building of cDNA library, and concrete operations by specification carries out.
4, upper machine sequencing
CDNA library is sequenced using Illumina X-ten microarray dataset, concrete operations by specification carries out.
5, high-throughput transcript profile sequencing data analysis
Bioinformatic analysis is carried out to sequencing result, carries out the positioning of RNA-seq read using TopHat v1.3.1, it will
Valid data comparison is examined on genome to ginseng, is analyzed using metaMA packet, and p value merging method therefor is in meta analysis
inverse normal method.When FDR<0.05, | Combined.ES | when>0.8, it is believed that the expression of gene significant difference.
6, result
Sequencing result is shown, compared with the expression of gene in the blood of normal child, H2AFJ and RGL4 are in respiratory tract
Expression dramatically increases in the child patient of syncytial virus infection.
The differential expression of embodiment 2QPCR sequence verification gene
1, large sample QPCR verifying is carried out to difference expression gene according to the testing result of high-flux sequence.According to embodiment
Sample collection mode in 1 selects child patient blood 30 and normal child's blood 25 of respiratory syncytial virus infection.
2, RNA extraction step is the same as embodiment 1
3, reverse transcription:
(1) reverse transcription reaction:
Using 25 μ l reaction systems, each sample takes 1 μ g total serum IgE as template ribonucleic acid, is separately added into PCR pipe following
Component: DEPC water, 5 × RT Buffer, 10mM dNTP, 0.1mM DTT, 30 μM of Oligo dT, 200U/ μ l M-MLV,
Template ribonucleic acid.42 DEG C of incubation 1h, 72 DEG C of 10min, of short duration centrifugation.
(2) design of primers
QPCR amplimer is designed according to the coded sequence of H2AFJ in Genbank and RGL4 gene and GAPDH gene, by
Shanghai Sangon Biological Engineering Technology And Service Co., Ltd's synthesis.Specific primer sequence is as follows:
H2AFJ gene:
Forward primer is 5 '-GTGTTGGAGTACCTTACG-3 ' (SEQ ID NO.1);
Reverse primer is 5 '-ATTATCCTGGTCTTCTTGTT-3 ' (SEQ ID NO.2);
RGL4 gene:
Forward primer is 5 '-TCACCTATTTCACAAGAATGGA-3 ' (SEQ ID NO.3);
Reverse primer is 5 '-TGAGACGATCCTGGACAT-3 ' (SEQ ID NO.4);
GAPDH gene:
Forward primer is 5 '-TTTAACTCTGGTAAAGTGGATAT-3 ' (SEQ ID NO.5);
Reverse primer is 5 '-GGTGGAATCATATTGGAACA-3 ' (SEQ ID NO.6).
(3) QPCR amplification is examined:
Using 25 μ l reaction systems, 3 parallel pipes are arranged in each sample, and all amplified reactions are repeated three times above to protect
Demonstrate,prove the reliability of result.
Following reaction system: 12.5 μ l of SYBR Green polymerase chain reaction system is prepared, forward and reverse primer (5 μM) is each
1 μ l, template cDNA 2.0 μ l, no 8.5 μ l of enzyme water.Operations are carried out on ice.
Amplification program are as follows: 95 DEG C of 60s, (95 DEG C of 15s, 60 DEG C of 15s, 72 DEG C of 45s) × 35 circulations.
Using SYBR Green as fluorescent marker, it is anti-that PCR is carried out on Light Cycler fluorescence real-time quantitative PCR instrument
It answers, determines that purpose band, Δ Δ CT method carry out relative quantification by melt curve analysis analysis and electrophoresis.
4, result
As a result as shown in Figure 1, compared with normal child, the blood of children of H2AFJ and RGL4 in respiratory syncytial virus infection
Expression in liquid is significantly raised, and difference has statistical significance (P < 0.05), consistent with high-flux sequence result.
The explanation of above-described embodiment is only intended to understand method and its core concept of the invention.It should be pointed out that for this
For the those of ordinary skill in field, without departing from the principle of the present invention, several improvement can also be carried out to the present invention
And modification, these improvement and modification will also be fallen into the protection scope of the claims in the present invention.
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Claims (10)
1. application of the reagent of detection molecules marker in preparation diagnosis childrens respiratory tract syncytial virus infection product, feature
It is, the molecular marker is H2AFJ.
2. application according to claim 1, which is characterized in that the molecular marker further includes RGL4.
3. application according to claim 1 or 2, which is characterized in that the reagent is selected from:
The probe of gene described in specific recognition;Or
The primer of gene described in specific amplification;Or
Specifically bind the bonding agent of the albumen of the gene coding.
4. application according to claim 3, which is characterized in that the primer sequence of specific amplification H2AFJ such as SEQ ID
Shown in NO.1~2, the primer sequence of specific amplification RGL4 is as shown in NO.3~4 SEQ ID.
5. a kind of product, which is characterized in that the product includes molecular marker expression described in detection claims 1 or 22
Horizontal reagent.
6. product according to claim 5, which is characterized in that the product includes chip, kit, nucleic acid film item.
7. product according to claim 6, which is characterized in that the chip includes genetic chip, protein-chip, wherein
Genetic chip includes the specific primer or probe for molecular marker, and protein-chip includes specific binding molecules mark
The antibody or ligand of the albumen of object coding.
8. product according to claim 6, which is characterized in that the kit includes that gene detecting kit, albumen are exempted from
Epidemic disease detection kit, wherein the gene detecting kit includes the specific primer, probe or gene of detection molecules marker
Chip;The protein immunization detection kit includes antibody, ligand or the egg of the albumen of specific binding molecules marker coding
White matter chip.
9. the application of the described in any item products of claim 5-8, which is characterized in that be used to prepare diagnosis childrens respiratory tract and close born of the same parents
The tool of virus infection.
10. molecular marker described in claims 1 or 2 is in the calculating mould of building prediction childrens respiratory tract syncytial virus infection
Application in type.
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Citations (3)
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CN104059995A (en) * | 2014-06-06 | 2014-09-24 | 深圳市疾病预防控制中心 | Kit and method for simultaneously detecting respiratory syncytial virus, adenovirus and human bocavirus |
CN105018488A (en) * | 2015-08-03 | 2015-11-04 | 博奥生物集团有限公司 | Kit used for detecting respiratory viruses and application thereof |
CN105018648A (en) * | 2015-08-03 | 2015-11-04 | 博奥生物集团有限公司 | Kit for detecting respiratory viruses and application thereof |
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2018
- 2018-12-27 CN CN201811607574.XA patent/CN109504767A/en active Pending
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Publication number | Priority date | Publication date | Assignee | Title |
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CN104059995A (en) * | 2014-06-06 | 2014-09-24 | 深圳市疾病预防控制中心 | Kit and method for simultaneously detecting respiratory syncytial virus, adenovirus and human bocavirus |
CN105018488A (en) * | 2015-08-03 | 2015-11-04 | 博奥生物集团有限公司 | Kit used for detecting respiratory viruses and application thereof |
CN105018648A (en) * | 2015-08-03 | 2015-11-04 | 博奥生物集团有限公司 | Kit for detecting respiratory viruses and application thereof |
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Title |
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