CN104141000A - MRNA level in-situ hybridization detection kit of KLF4 gene in earlier stage of pathologic evolution of human cardiovascular and cerebtovascular disease, detection method and application - Google Patents
MRNA level in-situ hybridization detection kit of KLF4 gene in earlier stage of pathologic evolution of human cardiovascular and cerebtovascular disease, detection method and application Download PDFInfo
- Publication number
- CN104141000A CN104141000A CN201310167923.1A CN201310167923A CN104141000A CN 104141000 A CN104141000 A CN 104141000A CN 201310167923 A CN201310167923 A CN 201310167923A CN 104141000 A CN104141000 A CN 104141000A
- Authority
- CN
- China
- Prior art keywords
- hybridization
- cardiovascular
- detection method
- detection
- test kit
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- 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
- C12Q1/6804—Nucleic acid analysis using immunogens
-
- 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
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Analytical Chemistry (AREA)
- Immunology (AREA)
- Genetics & Genomics (AREA)
- Pathology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses an in-situ hybridization detection kit which comprises a hybridization probe and a marker. The invention also discloses a method for in-situ hybridization detection of vascular endothelial cell's genetic factor (KLF4) gene's mRNA which is closely related with pathologic evolution of cardiovascular and cerebtovascular vascular endothelial injury in the earlier stage by the use of the kit. The method comprises the following steps: (1) under the condition that a hybridization probe and a target sequence can form a stable hybrid complex, RNA to be tested in a substrate is contacted with the hybridization probe so as to form a hybrid complex; and (2) the hybrid complex is detected. According to the detection kit and the detection method, gene expression quantity can be detected at the RNA level, the index of the detection method is ealier than present clinical biochemical detection index (protein detection index), and genuine RNA level screening of the earlier stage of cardiovascular and cerebtovascular disease can be realized. Meanwhile, the detection method provided by the invention is simple and convenient; cost is low; and the method is convenient for popularization and application in district-level hospitals.
Description
Technical field
The present invention relates to field of biological detection, more particularly, relate to the correlation detection technology of mrna expression change (Pathologic process) in developing with people's cardiovascular and cerebrovascular disease reason.
Background technology
The data providing according to domestic and international authoritative institution, the newly-increased number more than 300 ten thousand of the annual cardiovascular diseases of China, death toll nearly 2,500,000, patient more than 900 ten thousand, the annual newly-increased cardiovascular patient more than 1,200 ten thousand in the whole world, and death toll approaches more than 1,000 ten thousand, patient approximately has more than 100,000,000 ten thousand, according to authoritative institution of world prediction, to the year two thousand thirty above number will double, this is one group of fearful numeral.Cardiovascular diseases diagnosis and treatment cost is more and more higher, by patient's year medical expense 100,000, (poor area may be higher, developed regions may exceed 200,000 far away), more than 900 ten thousand patients, annual cost is 1.8 trillion Renminbi, deduction cost 35% approximately more than 6 hundred billion, approximately has 1.2 trillion Renminbi to consume in vain every year.And mortality is very high, in all diseases, the mortality ratio of cardiovascular and cerebrovascular disease remains first.Therefore, existing clinical cardiovascular and cerebrovascular disease diagnosis and treatment pattern must change, and innovative point of the present invention is to accomplish in advance preventative examination, then gets involved in time preventative regulation and control and prophylactic treatment, accomplishes preventiveing treatment of disease of gene level cardiovascular and cerebrovascular disease.
The inventor is in the middle discovery that studies for a long period of time, and the major reason that causes cardiovascular and cerebrovascular disease mortality ratio not fallen is to accomplish real early stage diagnosis and treatment.Diagnose according to existing laboratory biochemistry (cardiovascular and cerebrovascular damage involved enzyme class must detect) index, major part all rests on cardiovascular and cerebrovascular disease and forms the observation of curative effect after rear detection and sick sending out.
Along with molecular biotechnology is day by day perfect, functional genomics, the deep expansion of the research such as disease genomics, in order to seek more early stage examination cardiovascular and cerebrovascular disease, treatment cardiovascular and cerebrovascular disease and prevention cardiovascular and cerebrovascular disease, makes great progress.So far, we likely do more accurate early screening and diagnosis on the one-level functional transcription product (mRNA level or microRNA) of gene, before cardio cerebrovascular affection early stage or cardiovascular and cerebrovascular endothelial cell damage, just can accomplish early prediction and examination.The present invention adopts nucleic acid hybridization in situ technology, selects many group clinical samples, and Patients with geriatric cardiovascular and cerebrovascular diseases, high risk population (three high crowds), normal control, detect analysis to the early warning of KLF4 gene mRNA and various cardio cerebrovascular affections.
Contriver is in long-term research, draw a kind of new concept, the clinical diagnosis and treatment pattern of cardiovascular and cerebrovascular disease and other clinical major disease must change, can not only stop present treating the disease affected (diagnosis and treatment after morbidity), accomplish preventative diagnosis and treatment, accomplish to preventive treatment of disease, only in this way could reduce the M & M of major disease, reduce social cost and medical treatment cost.Therefore, contriver, in the rna level kit for screening and medicine of development and production major disease, innovates theoretical and technical.Particularly screen clinical samples (normal population, high risk population, cardiovascular and cerebrovascular patient), break through the consistency research and development thinking of healthy tissues and pathological tissues comparison, find and develop the cardio cerebrovascular affection rna level in early stage, develop closely related with cardiovascular and cerebrovascular disease early gene physiopathology, and the extremely important target of clinical meaning, the early prevention diagnosis and treatment that cardiovascular and cerebrovascular disease are clinically sent out diagnosis and treatment pattern rear and become, strive for time and the space of cardiovascular and cerebrovascular disease diagnosis and treatment, reached prevention cardiovascular and cerebrovascular disease and occur.The inventor under study for action KLF4 gene is an important gene that prevents that vascularization from blocking.If be responsible for the shortage of the gene KLF4 that regulates vascular endothelial cell, will more easily form harmful plaque and fatty deposits, KLF4 deficiency makes blood vessel more easily form patch.Patch accumulation (being atherosclerosis) narrows blood vessel, lays the first stone for thrombosis causes heart disease and stroke outbreak.On the contrary, the KLF4 of enough levels exempts from protection lining blood vessels can trigger and forms patch and the toxin of grumeleuse and the infringement of other objectionable impuritiess.A basic problem about vascular health has been answered in research, has found that KLF4 is the master regulation device of the main function of endotheliocyte.These genic levels change because of human body diseases, therefore as target or feasible therapeutic strategy.
At present the research of KLF4 gene is all adopted to high flux gene chip analytical technology, and these methods are used for scientific research aspect, be not suitable with clinical application, and detect RNA, than genetic analysis, more scientific (DNA analysis major part is in the presentation of susceptibility, mRNA is functional embodiment), than analysis of protein more reliable (inventor research just Double Labelling Technique find that mRNA and albumen transcribes sometimes asynchronously, have mRNA to transcribe, but sometimes there is no protein expression).Have no report according to detection technique and the test kit of existing documents and materials KLF4 gene mRNA level.
Hybridization in situ technique (in situ hybridization) is that molecular biology and Cytochemical Technique are combined, taking the nucleic acid molecule of mark as probe, in the technology of histocyte in situ detection specific nucleic acid molecule.Its principle is the nucleic acid strand (being probe) that makes to contain distinguished sequence, passes through mark, under optimum conditions with histocyte in complementary nucleic acid strand be that target nucleic acid is hybridized, with radioautograph or immunocytochemistry, label probe is surveyed again, thereby shown special DNA or RNA molecule at cell in-situ.
The probe of in situ hybridization is the molecule of known array or sequence the unknown but known nucleic acid molecule (though indefinite this molecule full sequence of molecule, but known its for what target molecule), the kind of probe can be divided into again DNA probe, cDNA probe, cRNA probe and synthetic oligonucleotide probe by properties of nucleic acids difference.For the ease of spike, probe must, with certain means mark in addition, be beneficial to later detection.Conventional marker comprises radionuclide and the large class of non-radioactive marker two.Conventional isotopic label has
3h,
35s,
125i and
32p.The advantages such as susceptibility is high although isotopic label has, back end is comparatively clear, because radio isotope all can damage human and environment, have the trend being replaced by heterotope recently.At present the most frequently used in heterotope marker have three kinds of vitamin H, digoxin and fluoresceins.The method that detects these markers is all extremely sensitive.
Can be divided into again DNA-DNA, RNA-DNA, RNA-RNA hybridization according to probe used and the difference that will detect nucleic acid.No matter but the hybridization of any form, all must be through five large processes, i.e. histiocytic fixing, prehybridization, hybridization, flushing and demonstration.The present invention adopts the Crossing system of RNA-RNA, synthetic probe (RNA) and the target RNA detecting are the principles that adopts base complementrity (hybridization is complementary), simultaneously through long-time research with observe in situ hybridization process, start and termination place residue does not affect the result detecting.
In view of the diagnosis of cardiovascular and cerebrovascular disease clinically at present (biochemical indicator thing is all the diagnosis after most of cardiovascular and cerebrovascular disease occur) is diagnosis in late period, treatment is also treatment of late stage, the treatment pattern that causes mortality ratio not fallen.Original intention of the present invention is to want to change at present the diagnosis and treatment pattern of major disease clinically, becomes preventative preventiveing treatment of disease from treating the disease affected, reaches preventative diagnosis and treatment.Adopt nucleic acid hybridization in situ technology and ImmunohistochemistryMethods Methods to combine to detect cardiovascular and cerebrovascular endothelial cell damage to become mrna expression sick early stage and change, done the technological breakthrough of novelty, the horizontal examination technology of the cardiovascular and cerebrovascular damage early stage mRNA of pathology is provided.Make to have had clinically the technology of a new real early screening of cardiovascular and cerebrovascular damage pathology mRNA level in early stage, for the diagnosis and treatment of clinical cardiovascular and cerebrovascular endothelial cell damage are raced against time and space.
Summary of the invention
First object of the present invention is to provide a kind of hybridization in situ detection kit, and it comprises in situ hybridization detection probes and marker.
Secondly, the present invention also will provide mentioned reagent box for the in situ hybridization detection method relevant to early warning after various cardiovascular and cerebrovascular endothelial injury pathology examinations in early stage and patient treatment.
For realizing object of the present invention, technical scheme of the present invention is as follows: first the present invention provides a kind of hybridization in situ detection kit, it comprises hybridization probe and marker, wherein, described hybridization probe is the complementary sequence of sequence shown in sequence table SEQ ID NO.1, sequence number: NM_004235.4, and sequence length is 2949bp, CDS:595 ... 2034bp, is positioned at karyomit(e) 9q31 " on.(probe sequence of the present invention is that in KLF4 gene C DS one section is from 901...1261bp). in the technical problem of probe mark, repeat specification once, the sequence of probe is the hybridization complementary base sequence of KLF4 gene mRNA, and the process of probe mark is exactly the complementary process of a hybridization)
A preferred version of test kit of the present invention is that described marker is selected from radioactive substance, chemoluminescence or substance that show color, vitamin H, metal king crab, fluorescein, enzyme and nano material.
A preferred version of test kit of the present invention is also to comprise hybridization solution.
A preferred version of test kit of the present invention is also to comprise toughener.
A preferred version of test kit of the present invention is also to comprise developer.
Canceration KLF4 kit for screening in early stage using value of the present invention is, to various cardiovascular and cerebrovascular endothelial injury pathology examinations in early stage, especially the examination in early stage of heart stalk, and cardiovascular and cerebrovascular damages disease and sends out rear or the rear course of disease judge for the treatment of and early warning, further coordinates clinical treatment.
The present invention also provides a kind of detection method of KLF4 in situ hybridization, comprises the following steps:
(1) described hybridization probe and target sequence can form under the condition of stablizing hybridization complex in the above, RNA to be measured in substrate contacted to formation hybridization complex with hybridization probe; With
(2) detect described hybridization complex.
Detection method of the present invention, wherein preferably, the condition that hybridization complex is stablized in described forming is: the temperature of nucleic acid hybridization is 42 DEG C; The time of nucleic acid hybridization is 16-24 hour.
Detection method of the present invention, wherein preferably, described substrate is selected people's blood preparation.More preferably, described blood preparation is from various cardiovascular and cerebrovascular endothelial cell damage patients in early stage, various cardiovascular diseases high risk population, healthy normal population.
Detection method of the present invention, wherein preferably, described cardiovascular and cerebrovascular endothelial cell damage high risk population, cardiovascular diseases build up a family fortune well family, various cardiovascular and cerebrovascular patient (particularly the heart obstructs early stage patient).
Detection kit of the present invention is to adopt nucleic acid hybridization technique and groupization immunization method to combine, and taking KLF4 as detected object, synthesising probing needle is the complementary sequence of KLF4 sequence, and the substrate of detection is the expression amount of KLF4 gene mRNA in blood of human body.The display packing of hybridization in situ technique can provide sxemiquantitative or the quantitative expression deciding degree of KLF4.Judge the expression amount of above mRNA according to immunohistochemical methods colour developing after hybridization, normal people KLF4 high expression level, cell develops the color in a large number; KLF4 is low expression 6-16% in cardiovascular and cerebrovascular endothelial cell damage patient; High risk population has a certain amount of expression 18-28%, and normal control crowd relatively has and showing difference, and the expression amount of this gene is all lower than normal people expression amount.
The component of diagnostic kit of the present invention is by hybridization probe, hybridization solution, developer, the compositions such as synergistic agent.The nucleic acid hybridization principle of this test kit is that molecular biology insider all knows, and concrete operation step is under sample disposal, prehybridization, hybridization, immunohistochemical staining, mirror, to carry out quantitative analysis, report the test, and wherein the concrete steps of hybridization comprise:
1). sample to be measured is put into reactive tank;
2). instrument discards liquid automatically, automatically adds Digestive system;
3). instrument discards liquid automatically, automatically rear fixing;
4). instrument discards liquid automatically, automatically prehybridization (42 DEG C);
5). instrument discards liquid automatically, automatically cleans;
6). instrument discards liquid automatically, automatically hybridization (42 DEG C);
7). instrument discards liquid automatically, automatically cleans;
8). instrument discards liquid automatically, and automatic and DIG antibody is cultivated (room temperature);
9). instrument discards liquid automatically, automatically cleans colour developing;
10). take out mounting microscopy.
The scheme of a preferred embodiment of the present invention is: with the synthetic digoxigenin labeled for nucleic acid probe of KLF4 (cDNA, RNA and the oligonucleotide probe of digoxigenin labeled, not only probe has a biotin labeling advantage, also overcome biotin labeled probe and in crossover process, organized the shortcomings such as the dry sorrow of Endogenous Biotin in position), mRNA nucleic acid to be measured in this hybridization probe and blood of human body is hybridized, develop the color by the method for immunohistochemical methods again, in existence and the location of light Microscopic observation mRNA, according to the cell count of dyeing, judge the expression amount of object mRNA.
The inventive method is current conventional nucleic acid hybridization in situ technology, the method is by the KLF4 expression amount in detection substrate cell, be used for determining the mRNA variable quantity of various cardiovascular and cerebrovascular endotheliocyte Pathologics early stage (particularly heart stalk is early stage), the various cardio cerebrovascular affections of early warning (myocardial infarction) whether occur and various cardiovascular and cerebrovascular disease patient treatment after prognosis prediction.Because KLF4 is high expression level in normal people, if KLF4 expression amount reduces, illustrate that cardiovascular and cerebrovascular endothelial cell damage pathologic process starts, illustrate that cardiovascular and cerebrovascular damage occurs, or the variation of cardiovascular and cerebrovascular damage patient cardiovascular and cerebrovascular pathology mechanism after treatment and curative effect judge, thereby the early diagnosis information of acquisition cardiovascular and cerebrovascular endothelial cell damage.
A test kit can many person-portions use or person-portion use.
The present invention has following beneficial effect: clinical meaning of the present invention is to follow the tracks of more in early days the variation that detects KLF4 expression amount in (special heart stalk, the apoplexy) generation of various cardio cerebrovascular affections and Pathologic process, and the various cardiovascular and cerebrovascular disease of early warning occur, development trend.Diagnostic kit of the present invention is from other detects with the inspection of cardiovascular diseases mark and has and showing different clinically.The present invention can be at mRNA level detection KLF4 unconventionality expression, before cardiovascular diseases biochemical indicator (existing clinical indices) does not produce extremely, also before there is not heart stalk, can accomplish early the information acquisition of above abnormal gene expression, give real early warning of clinical cardiovascular and cerebrovascular endothelial cell damage patient.So just likely implement early screening, early prevention, the early treatment of cardiovascular and cerebrovascular disease damage, likely from source, thoroughly effect a radical cure the lethality of various cardiovascular diseasess.
In addition, feature highly sensitive, high specificity that test kit provided by the invention has, meanwhile, detection method of the present invention is convenient and simple for operation, can be widely used and promoted in Municipal Hospitals.
Brief description of the drawings
Fig. 1 is KLF4 hybridization in situ technique schema of the present invention (nucleic acid hybridization in situ techniqueflow chart)
Fig. 2 is the low expression picture of embodiment of the present invention Myocardial damage patient KLF4.
Fig. 3 is that in the embodiment of the present invention, high risk population KLF4 expresses picture.
Fig. 4 is normal people KLF4 high expression level picture in the embodiment of the present invention.
Embodiment
Below in conjunction with embodiment, be described more specifically content of the present invention.Should be appreciated that, the following examples are non-limiting content of the present invention for explanation, and any pro forma change or accommodation will fall into protection scope of the present invention.
Embodiment 1
Prepare the in situ hybridization test kit of the present embodiment according to ordinary method, this test kit comprises hybridization probe, marker, the specification sheets with KLF4 design, wherein: the probe mark thing of the present embodiment is selected digoxin.
Test kit hybridization solution composition:
Alkaline phosphatase
Positive control mark
This 6/box
Reagent preparation working concentration
1). by 10 × damping fluid I with tri-distilled water by being diluted to 1 × damping fluid I at 1: 10;
2). by 20 × damping fluid II with tri-distilled water by being diluted to 2 × damping fluid II at 1: 10;
By being diluted to 0.2 × damping fluid II at 1: 100; By being diluted to 0.1 × damping fluid II at 1: 200;
3). by 10 × damping fluid III with tri-distilled water by being diluted to 1 × damping fluid III at 1: 10;
4) .10 × damping fluid IV with tri-distilled water by 1: 10 be diluted to × damping fluid IV (get 1#, 2#, the each 10mL of 3#, add water to 100mL both can).
Embodiment 2
The implementation process of application nucleic acid hybridization in situ detection method to each group of blood preparation KLF4 expression amount:
1). get two of samples to be measured;
2). in glass jar, add Digestive system (Digestive system 100 μ L add 1 × damping fluid I99.9ml, are working concentration) 50ml, 37 DEG C of water-bath preheating 10min, put 16 slides into, process 12min, then use 1 × damping fluid I to wash 5min for 37 DEG C;
3). protection liquid with 0.2% (protection liquid 1ml adds 1 × damping fluid I, and 99ml is working concentration) is washed 10min, and tri-distilled water is washed 5min (above process is all carried out at glass jar), takes out slide, allows its seasoning;
4). slide is put into moisture preservation box, add prehybridization solution 25 μ L/ sheets (being added in the place of cell), covered, covers tightly moisture preservation box, is placed in 42 DEG C of constant water bath box more than 3h;
5). take out slide, discard cover glass, slide is put into glass jar, the ethanol with 70%, 90%, 95% is respectively washed 2min, takes out seasoning;
6). slide is put into moisture preservation box, and one adds just hybridization solution 25 μ L/ sheets, and another adds antisense hybridization solution 25 μ L/ sheets, and covered, covers tightly moisture preservation box, is placed on 16-24h in 42 DEG C of constant water bath box;
7). take out slide, discard cover glass, slide is put into glass jar:
In 42 DEG C of constant water bath box, wash twice with 2 × damping fluid II, each 15min;
In 42 DEG C of constant water bath box, wash once each 15min with 0.2 × damping fluid II;
In 42 DEG C of constant water bath box, wash twice with 0.1 × damping fluid II, each 15min;
8). with 1 × damping fluid, III washes 30s, takes out slide, seasoning;
9). slide is put into moisture preservation box, add 0.5% confining liquid (1ml confining liquid adds 5ml1 × damping fluid III), 100 μ L/ sheets, cover tightly moisture preservation box, at room temperature act on 30min.(this step does not need to add cover glass);
10). take out slide, with 1 × damping fluid, III washes 30s, seasoning;
11). slide is put into moisture preservation box, add X-AP antibody and (get a pipe alkaline phosphatase enzyme antibody, add wherein 1.8ml1 × damping fluid III) 100 μ L/ sheets, cover tightly moisture preservation box and at room temperature act on 30min, time can not be long, otherwise can produce false positive (this step does not need to add cover glass);
12). take out slide, wash 3 times with 1 × damping fluid III, at every turn 15min;
13). with 1 × damping fluid, IV washes 2min, adds developer (developer A73.3 μ L, developer B157.5 μ L is added in 30mL1 × damping fluid IV, mixes), and room temperature lucifuge 16h is to more than 18h;
14). wash 5min with tri-distilled water, seasoning, (add with glycerine 10% 1 × damping fluid I mix) mounting microscopy.
Nucleic acid hybridization in situ detection method of the present invention is by goal gene digoxigenin labeled, become RNA nucleic acid probe, the RNA nucleic acid to be measured of probe and human leukocytes is hybridized, develop the color by the method for immunohistochemical methods again, therefore in existence and the location of light Microscopic observation RNA, according to the cell count of dyeing, judge the expression amount of object RNA.
Patients with geriatric cardiovascular and cerebrovascular diseases 20 examples, high risk population (three high crowds) 20 examples, 20 of Normal groups.The peripheral blood 3-5 milliliter (separation white corpuscle) of taking out all people to be checked does in situ hybridization.Result represents, all cardiovascular patient KLF4 expression amounts are low, and cell dyeing is shallow; High risk population expresses slightly and reduces, decimal dyeing; Normal group KLF4 expression amount is high, the dyeing of cell great majority, and concrete outcome is shown in Fig. 2, Fig. 3, Fig. 4.
Claims (10)
1. a hybridization in situ detection kit, comprises hybridization probe and marker, it is characterized in that, described hybridization probe is divided into shown in sequence table SEQ ID NO.1 the complementary sequence of a section in CDS in sequence, from 901....1261bp.
2. test kit as claimed in claim 1, is characterized in that, described marker is selected from radioactive substance, chemoluminescence or substance that show color, vitamin H, metal king crab, fluorescein, enzyme and nano material.
3. test kit as claimed in claim 1, is characterized in that, this test kit also comprises hybridization solution.
4. test kit as claimed in claim 1, is characterized in that, this test kit also comprises synergistic agent.
5. test kit as claimed in claim 1, is characterized in that, this test kit also comprises developer.
6. a KLF4 gene hybridization in situ detection method, is characterized in that, the method comprises the following steps:
(1) can form under the condition of stablizing hybridization complex at hybridization probe claimed in claim 1 and target sequence, RNA to be measured in substrate is contacted with hybridization probe, form hybridization complex; With
(2) detect described hybridization complex.
7. detection method as claimed in claim 6, is characterized in that, the condition that hybridization complex is stablized in described forming is: the temperature of nucleic acid hybridization is 42 DEG C; The time of nucleic acid hybridization is 16-24 hour.
8. detection method as claimed in claim 6, is characterized in that, described substrate is selected the mRNA sample in human blood.
9. detection method as claimed in claim 8, is characterized in that, described blood mRNA composition is selected from cardiovascular and cerebrovascular diseases, high risk population's (hypertension, hyperlipidemia, hyperglycemia and heart stalk are in earlier stage), normal people's sample.
10. detection method as claimed in claim 9, is characterized in that, described sample comprises in various cardiovascular high risk population's Pathologic process in early stage clinically, and the curative effect judge in Pathologic process after treatment of various cardiovascular and cerebrovascular disease.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310167923.1A CN104141000A (en) | 2013-05-09 | 2013-05-09 | MRNA level in-situ hybridization detection kit of KLF4 gene in earlier stage of pathologic evolution of human cardiovascular and cerebtovascular disease, detection method and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310167923.1A CN104141000A (en) | 2013-05-09 | 2013-05-09 | MRNA level in-situ hybridization detection kit of KLF4 gene in earlier stage of pathologic evolution of human cardiovascular and cerebtovascular disease, detection method and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104141000A true CN104141000A (en) | 2014-11-12 |
Family
ID=51850341
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310167923.1A Pending CN104141000A (en) | 2013-05-09 | 2013-05-09 | MRNA level in-situ hybridization detection kit of KLF4 gene in earlier stage of pathologic evolution of human cardiovascular and cerebtovascular disease, detection method and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104141000A (en) |
-
2013
- 2013-05-09 CN CN201310167923.1A patent/CN104141000A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101429550B (en) | Synthetic detection kit for hybridization in situ and detection method | |
| CN104141000A (en) | MRNA level in-situ hybridization detection kit of KLF4 gene in earlier stage of pathologic evolution of human cardiovascular and cerebtovascular disease, detection method and application | |
| CN102559887A (en) | Messenger ribonucleic acid (mRNA) level in-situ hybridization detection kit for JAGGED1 in early stage of pathological change of breast cancer bone metastasis, and detection method and application | |
| CN102965447A (en) | Alzheimer's disease (AD) lesion early-stage APOE-4 gene mRNA (messenger ribonucleic acid) level in-situ hybridization screening kit, as well as screening method and application thereof | |
| CN104131064A (en) | In-situ hybridization detection kit of miR-378 in early stage of pathologic evolution of gastric cancer, detection method and application | |
| CN102586419A (en) | MRNA level in-situ hybridization detection kit for beta-catenin of liver cancer prophase as well as detection method and application of mRNA level in-situ hybridization detection kit | |
| CN104711331A (en) | Cancer pathologic evolution early-stage Gankyrin gene mRNA level in-situ hybridization detection kit, detection method and applications thereof | |
| CN102628078A (en) | MicroRNA-155 level in-situ hybridization detection kit for early stage of various cancer lesions, detection method and application | |
| CN104711336A (en) | Solid tumor pathologic evolution early-stage GADD45G gene mRNA level in-situ hybridization detection kit, detection method and applications thereof | |
| CN104140999A (en) | MRNA level in-situ hybridization detection kit of AE1 gene in earlier stage of pathologic evolution of stomach cancer, detection method and application | |
| CN104131065A (en) | In-situ hybridization detection kit of AFPR gene in early stage of pathologic evolution of hepatic carcinoma, detection method and application | |
| CN102605060A (en) | In-situ hybridization assay kit for premalignant lung cancer 14-3-3thta level as well as assay method and application | |
| CN102559884A (en) | LET-7micro ribose nucleic acid (MIRNA) level in-situ hybridization detection kit for pathologic evolution early stage of various cancer, and detection method and application | |
| CN104711328A (en) | Alzheimer disease early-stage lesion mRNA level in-situ hybridization detection kit, screening method, and applications thereof | |
| CN102533989B (en) | In-situ hybridization detection kit and detection method for liver cancer precancer NOTCH1 on mRNA (messenger ribonucleic acid) level, and applications thereof | |
| CN104711320A (en) | Alzheimer disease early-stage lesion MS4A4 gene mRNA level in-situ hybridization detection kit | |
| CN102605056A (en) | In-situ hybridization assay kit for mRNA level of premalignant pancreatic cancer ATDC (telangiectasia group D associated protein) gene as well as assay method and application | |
| CN104141001A (en) | MRNA in-situ hybridization detection kit of Evi-1 in earlier stage of pathologic evolution of acute leukemia, detection method and application | |
| CN104711334A (en) | Alzheimer disease early-stage lesion PS-1 gene mRNA level in-situ hybridization detection kit | |
| CN104140998A (en) | In-situ hybridization detection kit of miR-886-3P level in earlier stage of pathologic evolution of small cell lung cancer, detection method and application | |
| CN102533976A (en) | Kit and method for detecting horizontal in situ hybridization of MICRORNA-21 at the early pathological evolution stage of a variety of cancers and application thereof | |
| CN102453768A (en) | In-situ hybridization detection kit for mRNA level in early stage of brain glioma neoplasia, detection method and application | |
| CN104711327A (en) | Cancer pathologic evolution early-stage GPR116 gene mRNA level in-situ hybridization detection kit, detection method and applications thereof | |
| CN104711335A (en) | Cancer metastasis pathologic evolution early-stage HOPX gene mRNA level in-situ hybridization detection kit, detection method and applications thereof | |
| CN104711326A (en) | Multi-cancer pathologic evolution early-stage Bmi1 gene mRNA level in-situ hybridization detection kit, detection method and applications thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| DD01 | Delivery of document by public notice |
Addressee: JIAXING RUIKANG BIOLOGICAL TECHNOLOGY CO., LTD. Document name: Notification of Publication of the Application for Invention |
|
| DD01 | Delivery of document by public notice |
Addressee: Patent manager of Rui Xing Biotechnology (Shanghai) Co., Ltd. Document name: Notification that Application Deemed not to be Proposed |
|
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20141112 |