CN106367484A - Application of molecular marker to sepsis diagnosis - Google Patents
Application of molecular marker to sepsis diagnosis Download PDFInfo
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- CN106367484A CN106367484A CN201610753971.2A CN201610753971A CN106367484A CN 106367484 A CN106367484 A CN 106367484A CN 201610753971 A CN201610753971 A CN 201610753971A CN 106367484 A CN106367484 A CN 106367484A
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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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- 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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- 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
- C12Q2600/158—Expression markers
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- 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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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/70—Mechanisms involved in disease identification
- G01N2800/7095—Inflammation
Abstract
The invention discloses an application of a molecular marker to sepsis diagnosis, particularly an application of SMARCAD1 to the sepsis diagnosis. The expression of SMARCAD1 in a patient suffering from sepsis is down-regulated. According to the application, a possibility is provided for early diagnosis of the sepsis clinically, thereby reducing the death rate caused by severe sepsis or sepsis shock.
Description
Technical field
The invention belongs to biomedicine field, it is related to a kind of application in diagnosis of sepsis disease for molecular marker, specifically
This molecular marker is smarcad1.
Background technology
Sepsis refer to the systemic inflammatory response syndrome being caused by infection, be by microorganism (as antibacterial, funguses, virus,
Parasite etc.) invade human body and the fierce systemic inflammatory response that induces, and to organizing, there is abrasive pathophysiological process
One group of clinical manifestation, is usually secondary to serious disease, such as serious burn, multiple injury, surgical site infections etc..Severe sepsis are permissible
Lead to septic shock and multiple organ dysfunction, be non-cardiac disease main causes of death in current intensive care unit(ICU), about
28.3% to 41% sepsis patient is dead because of multiple organ dysfunction syndrome, and human health and economic development are constituted greatly
Threaten and challenge.
Pyemic pathogenic process is an extremely complex pathophysiological process, wherein excessive inflammatory reaction and compensatory
Property the generation of anti-inflammatory response, the disorder of blood coagulation system, disorderly, the immune activation of cell function and suppression and metabolism
Disorder of function etc. constitutes a complicated network system, is not independent between them, but connect each other, mutual shadow
Ring, promote the scope of pathological changes gradually to expand, the pathological process that lesion degree gradually increases.In these pathological changes pyemic
Cheng Zhong, the triggering of inflammatory mediator, enhancing and enlarge-effect are one of sepsis core mechanisms that sb.'s illness took a turn for the worse, therefore pyemic
Essence is generally considered to be excessive inflammatory reaction, and the disorder of cell function, the acceleration of blood coagulation system, metabolism and microcirculation
Deng the change of function both participated in pyemic pathological process, ultimately resulted in the aggravation of the sepsis state of an illness.
Definitely effective biomarker be there is no for diagnosis of sepsis and treatment, the biological mark clinically using at present
Note thing such as c reactive protein, the Sensitivity and Specificity such as Procalcitonin. is not all high, and cannot function as the potential target for the treatment of of sepsis
Point, new can be used to predict that the biological marker of sepsis prognosis and therapy target urgently find.
Content of the invention
In order to make up the deficiencies in the prior art, an object of the present invention, provide a kind of molecular diagnostic markers；
The second object of the present invention, provides a kind of product, in order to early diagnosiss sepsis.
To achieve these goals, the present invention adopts the following technical scheme that
The invention provides the application in the product preparing diagnosis of sepsis disease of smarcad1 gene and its expression product.
Further, described product is examined by the expression of smarcad1 gene and its expression product in detection sample
Disconnected sepsis.
Further, the method for expression of detection smarcad1 gene and its expression product includes: rt-pcr, fixed in real time
Amount pcr, immune detection, in situ hybridization or micro-array chip.
Further, the described primer at least including a pair of specific amplified smarcad1 gene with rt-pcr diagnosis of sepsis disease；
The described primer at least including a pair of specific amplified smarcad1 gene with real-time quantitative pcr diagnosis of sepsis disease；Described use immunity
Checkout and diagnosis sepsis include: the antibody being combined with smarcad1 protein-specific；Described use in situ hybridization diagnosis of sepsis disease bag
Include: the probe with the nucleic acid array hybridizing of smarcad1 gene；Described included with micro-array chip diagnosis of sepsis disease: protein chip
And gene chip；Wherein, protein chip includes the antibody being combined with smarcad1 protein-specific, gene chip include with
The probe of the nucleic acid array hybridizing of smarcad1 gene.
Further, described product includes chip and/or test kit；Wherein said chip includes gene chip, protein core
Piece；Described test kit includes gene detecting kit, protein immunization detection kit.
Further, described gene detecting kit at least includes the primer of a pair of specific amplification smarcad1 gene.?
In a specific embodiment of the present invention, the primer sequence of described use real-time quantitative pcr specific amplified smarcad1 gene is such as
Shown in seq id no.5 and seq id no.6.
The invention provides a kind of product of diagnosis of sepsis disease, described product can be by smarcad1 in detection sample
The expression of gene carrys out diagnosis of sepsis disease.
Further, product recited above includes chip or test kit.Wherein, described chip includes gene chip, albumen
Matter chip；Described test kit includes gene detecting kit and protein immunization detection kit.Described gene chip includes solid phase
Carrier and be fixed on oligonucleotide probe on solid phase carrier, described oligonucleotide probe is included for detecting smarcad1 base
Because transcriptional level is for the oligonucleotide probe of smarcad1 gene；Described protein chip includes solid phase carrier and fixation
The specific antibody of the smarcad1 albumen on solid phase carrier；Described gene detecting kit is included for detecting smarcad1
The reagent of gene transcription level；Described protein immunization detection kit includes the specific antibody of smarcad1 albumen.Wherein, institute
State gene chip to can be used for detecting multiple genes (for example, the multiple bases related to sepsis including smarcad1 gene
Cause) expression.Described protein chip can be used for detecting multiple protein including smarcad1 albumen (for example with
The related multiple protein of sepsis) expression.By detecting multiple and pyemic mark simultaneously, can significantly carry
The accuracy rate of high sepsis diagnosis.
Further, described gene detecting kit comprises sybr green polymerase chain reaction system, for amplification pipe
Family's gene and the primer pair of smarcad1 gene；Described sybr green polymerase chain reaction system comprises: pcr buffer,
Dntps, sybr green fluorescent dye.
Further, described house-keeping gene is gapdh, primer pair sequence such as the seq id no.3 and seq id of amplification gapdh
Shown in no.4, the primer pair sequence of described amplification c19orf48 gene is as shown in seq id no.5 and seq id no.6.
In the present invention, " antibody " covers monoclonal antibody, polyclonal antibody, multi-specificity antibody (such as bispecific
Antibody) and antibody fragment, combinatorial antibody etc., as long as they show desired biologic activity." monoclonal antibody " refers to
The antibody obtaining from the antibody of a group substantially homogeneity, that is, each antibody of composition colony is identical and/or combines same epitope, removes
Issuable during production monoclonal antibody may become external, such variant is typically with indivisible presence.Such list
Clonal antibody is typically include the antibody comprising the peptide sequence with reference to target, and wherein target Binding peptide sequence is by including
Comforming selects single target Binding peptide sequence to obtain in interior process in many peptide sequences.
Monoclonal antibody also includes " being fitted together to " antibody, wherein a part for heavy chain and/or light chain with derived from particular species
Or belong to that corresponding sequence in specific antibodies classification or the antibody of subclass is identical or homology, and the remainder of chain with derived from another
One species or belong to that corresponding sequence in another antibody isotype or the antibody of subclass is identical or homology, and the piece of this antibody-like
Section, as long as they show desired biologic activity.
" antibody or antibody fragment " refers to no matter derive from any species naturally occurring antibody, or by dna skill of recombinating
Art creates；No matter from serum, b cell, hybridoma, transfectoma, yeast or bacteria distribution antibody (such as igg, igm, iga,
Igd or ige) or fragment (such as fab, f (ab ')2, fv, disulphide connect fv, scfv, closure conformation multispecific resist
Body, the scfv of disulphide connection, double antibody).
In the present invention, term " probe " refers to be combined with the particular sequence of another molecule or subsequence or other parts
Molecule.Unless otherwise noted, term " probe " is often referred to match with another polynucleotide (often referred to as by complementary base
" target polynucleotide ") polynucleotide probes that combine.According to the preciseness of hybridization conditions, probe energy and lacking completely with this probe
The target polynucleotide of complementarity combines.Probe can make direct or indirect labelling, and its scope includes primer.Crossing system,
Include, but are not limited to: solution, solid phase, mixed phase or in situ hybridization algoscopy.
In the present invention, term " microarray " is that hybridised arrays original paper is ordered in substrate, and described hybridised arrays are former
Part such as polynucleotide probe (such as oligonucleotide) or bonding agent (such as antibody).Described substrate can be solid matrix, example
As, glass or silicon dioxide slide, pearl, fibre opticss binding agent or semi-solid matrix, such as nitrocellulose filter.Nucleotides sequence
Row can be any arrangement of dna, rna or therein.
Various probe arrays have been described above in the literature and can be used for detection in the context of the invention may be with herein
The related mark of described phenotype.For example, dna probe array chip or larger dna probe array chip (otherwise, Ke Yitong
Cross interrupt chip and obtain each individuality chip) be used for one embodiment of the invention.Dna probe array chip generally comprises glass
Glass chip, placed high density dna probe (short dna fragment) array thereon.These chips each can keep e.g., from about 6000
Ten thousand are used for identifying the dna of longer sample dna sequence (for example, from individual or colony, for example, comprising mark of interest)
Probe.Carried out by dna hybridization with the dna probe groups identification sample dna on chip glass.When dna sample and dna probe array
During hybridization, sample is incorporated into those complementary probes of sample dna sequence.More steady with those probes by evaluating individual sample dna
Admittedly hybridize it is possible to nucleotide sequence known to determining whether there is in sample, thereby determine that in nucleic acid find mark
Whether there is.Can also allow to distinguish single nucleotide by controlling hybridization conditions using this means, for example, for snp
Identification to carry out ash with the sample gene typing of one or more snp.Array provides one kind, and (or series winding) detection is multiple simultaneously
The convenience embodiment of polymorphic marker.
Above-mentioned probe has the base sequence with the specific base sequence complementary of target gene.Here, so-called " complementary ",
As long as hybridizing, can not be complete complementary.These polynucleotide are commonly angled relative to this specific base sequence to be had
More than 80%, preferably more than 90%, more preferably more than 95%, particularly preferred 100% homology.These probes can be dna,
Can also be rna, furthermore it is possible to be to pass through pna, lna, ena, gna, tna etc. manually in one part or whole nucleotide
The polynucleotide that replacement nucleic acid obtains.
In the context of the present invention, " smarcad1 gene " includes people's smarcad1 gene order and its variant.Described
" variant " of sequence is the sequence of biological activity, itself because in native sequences the insertion of one or more nucleotide, lack, repair
Decorations and/or replacement have the nucleotide sequences different from native sequences or wild-type sequence (or its complement).This variant is usual
It is less than 100% with the sequence identity of native sequences or its complement.However, the variant of biological activity generally and corresponding natural is deposited
Sequence or its complement have at least about 70% sequence identity, typically at least about 75%, more generally at least about 80%, very
To more generally at least about 85%, even more typically at least about 90% and even more typically at least about 95%, 96%, 97%, 98%
Or 99% sequence identity.The Variant nucleotide acid fragment of any length retains the biological activity of corresponding native sequences.Variant also wraps
The sequence including wherein at 5 ' or 3 ' ends or adding one or more nucleotide in native sequences or its complement.Variant also includes it
In some nucleotide be deleted and optionally by one or more difference nucleotide replace sequence.
Smarcad1 gene includes and smarcad1 gene in international at present public GenBank genebank
(nc_000004.12) dna sequence has more than 70% homology, and encodes the dna sequence of identical function protein；At this
In bright specific embodiments, the coded sequence of described smarcad1 gene is the dna sequence shown in seq id no.1.
The smarcad1 gene of the present invention can be natural or synthetic, or use can be expressed
The vector-transfected cell of the dna fragment of smarcad1 obtains.Carrier described in described carrier includes viral vector, eukaryotic expression carries
Body.
Viral vector can be any suitable carrier, including but not limited to retroviral vector, adenovirus vector, gland
Viral related viral vectors, herpesviruss (such as herpes simplex virus, vaccinia viruss and eb virus) carrier, alphavirus vectors.
Carrier for expression of eukaryon can be any suitable expression vector, including but not limited to pcmv-myc expression vector, pcdna3.0 table
Reach carrier, pcdna3.1 expression vector, pegfp expression vector, pef bos expression vector, ptet expression vector, ptre expression load
Body or engineered carrier on the basis of known expression vector, such as pbin438, pcambia1301 etc..
In the context of the present invention, smarcad1 gene expression product includes people's smarcad1 albumen and people
The partial peptide of smarcad1 albumen.The partial peptide of described smarcad1 albumen contains the functional domain related to sepsis.
" smarcad1 albumen " includes any function equivalent of smarcad1 albumen and smarcad1 albumen.Described work(
Smarcad1 albumen conservative variation's protein or its active fragment can be included by equivalent, or its reactive derivative, allelic variation
Body, natural mutation, induced mutants, the dna that can be hybridized with the dna of people smarcad1 under high or low stringent condition be compiled
The protein of code.In the specific embodiment of the present invention, described smarcad1 albumen is by seq id no.2 institute in sequence table
The protein of the aminoacid sequence composition showing.
It is known that, conventionally, the modification of one or more aminoacid in a protein does not interfere with the function of protein.
Those skilled in the art can approve change single amino acids or the aminoacid of little percentage ratio or the indivedual interpolations to aminoacid sequence,
Disappearance, insertion, replacement are conservative modifications, and wherein changing of protein produces the protein with identity function.Function phase is provided
As the Conservative substitution tables of aminoacid be well known in the art.
In the context of the present invention, " diagnosis of sepsis disease " both include judging experimenter whether suffered from sepsis, and
Including judge experimenter whether there is with pyemic risk.
In the present invention, term " sample ", can be blood, tissue, urine, serum, blood plasma, amniotic fluid, cerebrospinal fluid, Placenta Hominiss
Cell or tissue, endotheliocyte, leukocyte or mononuclear cell.Sample can derive from experimenter or object directly uses, or
Carry out pretreatment, such as by filtration, distillation, extraction, concentration, centrifugation, inactivate interference component, add the modes such as reagent to process, with
Some modes as described herein or known in the art modify the characteristic of sample.
In the present invention, term " experimenter " refers to any member of regnum animale, typically mammal.Term " suckling
Animal " refers to be categorized as any animal of mammal, including people, other High Primates, domestic animal and farming animals and zoo,
Motion or pet animals, such as Canis familiaris L., cat, cattle, horse, sheep, pig, goat, rabbit etc..Generally, mammal is people.
Advantages of the present invention and beneficial effect:
Present invention firstly discovers that the molecular marker smarcad1 gene related to sepsis, pyemic for studying
Pathogenesis have great importance.
The invention provides a kind of early diagnosiss product and means, by detecting the expression of smarcad1, to judge
Whether experimenter suffers from sepsis, thus reducing pyemic progression risk, controls conditions of patients.
Brief description
Fig. 1 shows using qpcr detection expression in sepsis patient for the smarcad1 gene.
Specific embodiment
The present invention is further detailed explanation with reference to the accompanying drawings and examples.Following examples are merely to illustrate this
Invention rather than restriction the scope of the present invention.The experimental technique of unreceipted actual conditions in embodiment, generally according to conventional strip
Part, such as sambrook et al., molecular cloning: laboratory manual (new york:cold spring harborlaboratory
Press, 1989) condition described in, or according to the condition proposed by manufacturer.
The embodiment 1 screening gene marker related to sepsis
1st, sample collection
Respectively collect 10 healthy human bloods and sepsis patient blood sample, the acquirement of above-mentioned all specimen is all by ethics
The agreement of committee.
2nd, the preparation of rna sample and quality analysiss
The preparation of 2.1rna sample
Rna extracts kit using promega company extracts total rna.Specifically comprise the following steps that
1) whole blood in take 1ml to be collected in test tube that heparin or edta were processed, puts in sterile centrifugation tube；
2) collect hemocyte: 3000rpm centrifugation 5min, carefully siphon away supernatant from sample top；
3) add 1ml hemocyte lysate, careful suction is put 4-5 time, resuspended precipitate；
4) 3000rpm centrifugation 5min；
5) repeat step 3) and 4) twice (totally three times)；
6) avoid cell pellet, carefully siphon away nearly all supernatant, only retain 100 μ l supernatant；Check bme
Through being added in rna lysate, then plus 175 μ l rna lysates are in cell, inhale and put resuspended and cell lysis；
7) add 350 μ l rna diluents, overturn mixing 3-4 time；
8) it is placed in 3min in 70 DEG C of water-baths；
9) 13000g centrifugation 10min under room temperature；
10) limpid lysate is transferred in a sterile centrifugation tube；
11) add 200 μ l 95% ethanol in cleared lysate, inhaled with liquid-transfering gun and put 3-4 time to mix；This is mixed
Thing is transferred in centrifugal column assembly, and 13000g is centrifuged 1min；
12) take down centrifugal column from centrifugal column assembly, discard the liquid in collecting pipe, centrifugal column is installed onto collecting pipe
On, check that rna wash solution uses ethanol dilution, plus 600 μ l rna cleaning mixture be in centrifugal column assembly,
13000g is centrifuged 1min；
13) discard the liquid in collecting pipe, centrifugal column is installed onto on collecting pipe, the dnase of fresh for 50 μ l preparation is incubated
Educate mixture to be applied directly on the film in centrifugal column；
14) it is incubated 15min under room temperature, add 200 μ l dna enzyme stop buffers (to confirm to have added second in centrifugal column
Alcohol), 13000g is centrifuged 1min；
15) add 600 μ l rna cleaning mixture (having added ethanol), 13000g, be centrifuged 1min；
16) empty collecting pipe, add 250 μ l rna cleaning mixture (having added ethanol), high speed centrifugation 2min into centrifugal column；
17) centrifugal column is transferred on eluting pipe from collecting pipe, add 100 μ l nuclease free water on film, by centrifugal column
Assembly is put centrifuge into and is made the lid facing outwards of eluting pipe, and 13000g is centrifuged 1min, abandons centrifugal column, covers and fill rna's
Eluting pipe, is stored in -70 DEG C.
The quality analysiss (nanodrop1000 spectrophotometer) of 2.2rna sample
Nanodrop1000 spectrophotometer detects rna sample, and the sample requirement of rna-seq sequencing: od260/od280 is
1.8-2.2.
The quality analysiss (agilent technologies 2100bioanalyzer) of 2.3rna sample
The rna of said extracted is entered row agarose gel electrophoresis, agilent technologies
2100bioanalyzer detects that rna sample quality, observation 28s rrna and 18s rrna master tape are obvious, no degrades, rna is complete
Sex index is qualified, concentration reach requirement meet rna-seq be sequenced cdna library construction requirement, can be used for library construction and
Sequencing.
3rd, high flux transcript profile sequencing
3.1rna-seq read positions
First by low-quality read remove obtain clean read, then utilize tophat v1.3.1 will clean fragment and
Ucsc h.sapiens reference gene group (hg19) is mated, the index building in advance of h.sapiens ucsc hg19 version
Download from tophat homepage, and as reference gene group, it is allowed to each read (is given tacit consent to when being mated with genome using tophat
To 20) there are multiple coupling sites, most 2 mispairing.Tophat sets up possible according to exon region and gt-ag shear signal
Shearing site storehouse, navigates on genome according to the read that these shearing site storehouses are not navigated to genome.We use
The system default parameter of tophat method.
3.2 transcript abundance assessments
By cufflinks v1.0.3 process, cufflinks v1.0.3 is by rna-seq piece for the read file matching
Hop count mesh is standardized calculating the relative abundance of transcript.Fpkm value refer to every 1,000,000 sequencing fragments in match specific
The segment number of the exon region of gene 1kb length.Calculate the confidence interval of fpkm estimated value by Bayesian inference method.
The gtf comment file of the reference that cufflinks uses downloads (homo_ from ensembl data base
sapiens.grch37.63.gtf).
The detection of 3.3 difference expression genes
The original document mated the ensembl gtf file of download and by tophat is transferred to cuffdiff,
Cuffdiff re-evaluates the gene expression abundance of the transcript listed in gtf file using original matching files, detects difference table
Reach.Only has q value ＜ 0.01 in cuffidff output, test display is considered as successfully more just differential expression.
4th, result
Rna-seq result shows, expression in sepsis patient blood for the smarcad1 gene is substantially less than Healthy People
In expression.
The differential expression of embodiment 2qpcr sequence verification smarcad1 gene
1st, the testing result according to high-flux sequence selects smarcad1 gene to carry out large sample qpcr checking.According to enforcement
Sample collection mode in example 1 selects sepsis patient blood and each 80 of healthy human blood.
2nd, rna extraction step is with embodiment 1.
3rd, reverse transcription: the reverse transcription reagent box using takara company is operated.Specifically comprise the following steps that
(1) take total rna 2 μ g to carry out reverse transcription, add oligo (dt) 2 μ l, fully mix；70 DEG C of water-baths；Vertical after 5min
I.e. ice bath 2-3min；
(2) 25 μ l reaction systems are built, including 5 × RT Buffer 5 μ l, dntp (2.5mm) 5 μ l, rnasin
40u/ μ l, m-mlv 200u/ μ l, benefit nuclease free water to 25 μ l；
After (3) 42 DEG C of water-bath 60min, 95 DEG C of water-bath 5min are to inactivate m-mlv；
(4) -20 DEG C store for future use.
4th, qpcr amplification
(1) design of primers
According to the coded sequence design qpcr amplimer of smarcad1 gene and gapdh gene in genebank, by upper
Hai Shenggong biotechnology Services Co., Ltd synthesizes.Concrete primer sequence is as follows:
Gapdh gene:
Forward primer is 5 '-ctctggtaaagtggatattgt-3 ' (seq id no.3)；
Reverse primer is 5 '-ggtggaatcatattggaaca-3 ' (seq id no.4).
Smarcad1 gene:
Forward primer is 5 '-tgatggaggatggctata-3 ' (seq id no.5)；
Reverse primer is 5 '-cggagttctgttatcttct-3 ' (seq id no.6).
(2) according to table 1 preparation pcr reaction system:
Wherein, sybr green polymerase chain reaction system is purchased from invitrogen company.
Table 1pcr reaction system
| Reagent | Volume |
| Forward primer | 1μl |
| Reverse primer | 1μl |
| Sybr green polymerase chain reaction | 12.5μl |
| Template | 2μl |
| Deionized water | Supply 25 μ l |
(3) pcr reaction condition: 95 DEG C of 10min, (95 DEG C of 15s, 60 DEG C of 60s) × 45 circulations.Using sybr green as
Fluorescent marker, carries out pcr reaction on light cycler fluorescent quantitation pcr instrument, true by melt curve analysis analysis and electrophoresis
Determine purpose band, δ δ ct method carries out relative quantification.
5th, statistical method
Experiment repeats to test using 3 times, and result data is all to be represented in the way of mean+SD, uses
Carrying out statistical analysiss, difference between the two is using t inspection it is believed that when p < has system when 0.05 for spss13.0 statistical software
Meaning learned by meter.
6th, result
Result as shown in figure 1, compared with the blood of Healthy People, expression in sepsis patient blood for the smarcad1 gene
Lower, difference has statistical significance (p < 0.05), consistent with rna-sep result.
The explanation of above-described embodiment is only intended to understand the method for the present invention and its core concept.It should be pointed out that for this
For the those of ordinary skill in field, under the premise without departing from the principles of the invention, some improvement can also be carried out to the present invention
And modification, these improve and modify also by the protection domain falling into the claims in the present invention.
Claims (10)
- The application in the product preparing diagnosis of sepsis disease of 1.smarcad1 gene and its expression product.
- 2. application according to claim 1 is it is characterised in that described product passes through smarcad1 gene in detection sample And its expression of expression product carrys out diagnosis of sepsis disease.
- 3. application according to claim 2 is it is characterised in that detect the expression water of smarcad1 gene and its expression product Flat method includes: rt-pcr, real-time quantitative pcr, immune detection, in situ hybridization or micro-array chip.
- 4. application according to claim 2 is it is characterised in that described product includes chip and/or test kit；Wherein said Chip includes gene chip, protein chip；Described test kit includes gene detecting kit, protein immunization detection kit.
- 5. application according to claim 4 is it is characterised in that described gene detecting kit at least includes a pair of specificity The primer of amplification smarcad1 gene.
- 6. application according to claim 5 is it is characterised in that the primer sequence of described specific amplification smarcad1 gene As shown in seq id no.5 and seq id no.6.
- 7. a kind of product of diagnosis of sepsis disease is it is characterised in that described product can be by smarcad1 base in detection sample The expression of cause and its expression product carrys out diagnosis of sepsis disease.
- 8. product according to claim 7 is it is characterised in that described product includes chip or test kit；Wherein, described Chip includes gene chip, protein chip；Described test kit includes gene detecting kit and protein immunization detection kit.
- 9. product according to claim 8 is it is characterised in that described gene detecting kit comprises sybr green polymerization Polymerase chain reaction system, for expanding house-keeping gene and the primer pair of smarcad1 gene；Described sybr green polymerase chain Formula reaction system comprises: pcr buffer, dntps, sybr green fluorescent dye.
- 10. product according to claim 9, it is characterised in that described house-keeping gene is gapdh, expands the primer of gapdh To sequence as shown in seq id no.3 and seq id no.4, the primer pair sequence such as seq id of described amplification smarcad1 gene Shown in no.5 and seq id no.6.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109022570A (en) * | 2018-09-14 | 2018-12-18 | 北京泱深生物信息技术有限公司 | Application of the non-coding RNA as sepsis diagnosis marker |
| CN109628584A (en) * | 2019-01-31 | 2019-04-16 | 泰山医学院 | One kind molecular marker relevant to pyemia occurrence and development and its application |
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| CN1522304A (en) * | 2001-06-29 | 2004-08-18 | 斯尔思实验室有限公司 | Use of a biochip for the diagnosis of sepsis or sepsis related syndrome |
| CN101622360A (en) * | 2005-12-15 | 2010-01-06 | 贝克顿迪金森公司 | Diagnosis of sepsis |
| CN105473743A (en) * | 2013-06-28 | 2016-04-06 | 睿智研究实验室私人有限公司 | Sepsis biomarkers and uses thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1522304A (en) * | 2001-06-29 | 2004-08-18 | 斯尔思实验室有限公司 | Use of a biochip for the diagnosis of sepsis or sepsis related syndrome |
| CN101622360A (en) * | 2005-12-15 | 2010-01-06 | 贝克顿迪金森公司 | Diagnosis of sepsis |
| CN105473743A (en) * | 2013-06-28 | 2016-04-06 | 睿智研究实验室私人有限公司 | Sepsis biomarkers and uses thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109022570A (en) * | 2018-09-14 | 2018-12-18 | 北京泱深生物信息技术有限公司 | Application of the non-coding RNA as sepsis diagnosis marker |
| CN109628584A (en) * | 2019-01-31 | 2019-04-16 | 泰山医学院 | One kind molecular marker relevant to pyemia occurrence and development and its application |
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