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 PDF

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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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赵秀侠
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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
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups

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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

A kind of molecular marker of childrens respiratory tract syncytial virus infection and its application
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.
CN201811607574.XA 2018-12-27 2018-12-27 A kind of molecular marker of childrens respiratory tract syncytial virus infection and its application Pending CN109504767A (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
KATHRYN M. DUPNIK等: "Transcriptional Changes That Characterize the Immune Reactions of Leprosy", 《THE JOURNAL OF INFECTIOUS DISEASES》 *

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