CN104711328A - Alzheimer disease early-stage lesion mRNA level in-situ hybridization detection kit, screening method, and applications thereof - Google Patents
Alzheimer disease early-stage lesion mRNA level in-situ hybridization detection kit, screening method, and applications thereof Download PDFInfo
- Publication number
- CN104711328A CN104711328A CN201310669556.5A CN201310669556A CN104711328A CN 104711328 A CN104711328 A CN 104711328A CN 201310669556 A CN201310669556 A CN 201310669556A CN 104711328 A CN104711328 A CN 104711328A
- Authority
- CN
- China
- Prior art keywords
- hybridization
- test kit
- probe
- early
- situ
- 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
- C12Q1/6813—Hybridisation assays
- C12Q1/6841—In situ hybridisation
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Pathology (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, and further discloses a method utilizing the in-situ hybridization detection kit to detect the PIRB gene mRNA, which is closely related with the Alzheimer disease (AD) early-stage pathologic evolution. The method comprises the following steps: (1) under a condition that a hybridization probe and a target sequence can form a stable hybridization complex, contacting RNA to be detected in a substrate with a hybridization probe so as to form a hybridization complex; (2) detecting the obtained hybridization complex. The provided kit and detection method can detect the expression function of PIRB gene in the mRNA level, can detect the cancer in an earlier stage compared with the medical imaging technology and conventional clinical biochemical index detection, can achieve real mRNA level screening in the early-stage lesion of AD so as to prevent and treat AD in advance, moreover, have the advantages of simpleness, convenience, and low cost, and thus are convenient to promote and use in hospitals.
Description
Technical field
The present invention relates to field of biological detection, more particularly, relate to the correlation detection technology changing (Pathologic process) with senile dementia (AD) pathology mrna expression in early stage.
Background technology
Alzheimer's disease (Alzheimer ' s disease-, AD) be modal dementia type, accounting for 60% ~ 80% of all dementias, is also one of disease that disability rate is high and burden is large.By 2006, the mankind first killer deaths from heart disease rate have dropped 11.5% than 2000, and second killer's palsy mortality ratio have dropped 18.1%, but the mortality ratio of alzheimer's disease rises 47.1%, became the 5th cause of death of the elderly.Along with the increase of global population size and predicted life, alzheimer's disease has become world health problem, and according to the prediction in future, the every Two decades years of prevalence of dementia nearly doubles, and will reach 6,570 ten thousand to the year two thousand thirty, will reach 100,000,000 1,540 ten thousand to the year two thousand fifty.Various countries' patients with Alzheimer disease number increase is not balanced.The dull-witted number prediction of developed country increases with the ratio of 100%, and China/India and the developing country such as South Asia and neighbouring country of West Pacific Ocean thereof will increase with the ratio more than 300%, and the ratio with 336% then increases by the AD number of China.China is the maximum country of the aging radix of world population, ends for the end of the year 2008, and China's more than 60 years old elderly population, close to 1.69 hundred million, account for 12% of total population.A Trans-Provincial/Municipal (Beijing/Shanghai/Chengdu/Xi'an) epidemiology survey result according to BJ Union Hospital leader in 2005 is estimated, over-65s the elderly AD morbidity is 4.8%, and increases with age.Therefore, China is never an AD low country, and just the opposite, but AD number is at most and the fastest country of rate of growth in the world.Therefore the swift and violent Disease Spectrum increased will produce material impact to the socio-economic development of China and family life.
AD is the high and disease that burden is heavy of disability rate in the world.According to the estimation about global disease burden of 2003 annual World Health Organization reports, in more than 60 years old the elderlys, dull-witted causing disabled accounts for 11.2%, far above palsy (9.5%), skeletal muscle disorder (8.9%), cardiovascular diseases (5%) and various types of cancer (2.4%).Common recognition according to an interdisciplinary expert group of the U.S. is estimated, except Spinal injury and terminal cancer, the AD weighting that disables is significantly higher than any other healthy state.In the U.S., AD population more than the cost of 5,000,000, AD more than 1,000 hundred million dollars, estimate that the year two thousand fifty AD patient numbers will rise to 1,300 ten thousand people in geometricprogression mode, through adjusting, every year by cost 140,000,000,000 dollars.AD has become great public health problem, and is defined as the research emphasis of the public health problem coming years.
AD is a kind of common nervous system degenerative disease, clinical manifestation is Progressive symmetric erythrokeratodermia cognitive decrease, typical pathologic changes into senile plaque (sen ile plaque, SP), neurofibrillary tangles (neurona l fibrillary tang les, and neuron loss NFT), in addition with granulovacuolar degeneration (granulov acuolar degeneration, GD), Hirano body and cerebrovascular change.After many decades is about the cause of disease of AD and pathogenetic research, manyly not understand although also have the cause of disease of AD, had certainly its pathology and physiological mechanism and comprehensive understanding, and therefrom found some important stigmatas.Modern molecular genetics means a great in the pathogenesis helping human knowledge AD, finds that euchromosome sudden change can cause the increasing expression of A β 42 peptide.These genes comprise the amyloid precursor protein (APP) on No. 21 karyomit(e)s relevant to Early onset alzheimer's disease, the presenilin 1(PS1 on No. 14 karyomit(e)s) and No. 1 karyomit(e) on presenilin 2(PS2) sudden change, the apo E (APOE) on the 19 good karyomit(e)s that stigmata also comprises and late onset Alzheimer disease is correlated with and No. 10 chromosome mutations recently found.By beta amyloid peptide (A β) or cerebrospinal fluid (CSF) the A β level of positron emission tomography (PET) video picture; By the outstanding exception that fluorodeoxyglucose-PET and functional MRI show; The N euron loss that in CSF, phosphorylation microtubule-associated protein (p-tau) level and MRI volume reflect.The biomarker of AD can help clinical diagnosis well, finds that there is good consistence with pathology.New diagnosis definition and standard are recommended by US National ageing research institutes in 2010 and alzheimer's disease federation, emphasize memory impairment to be combined with biomarker.AD is the whole clinical course of disease, includes AD pre-dementia and dull-witted phase AD.The clinical prodromal stage of AD then points out that existing early stage pathology physiology changes to the stage very long between appearance cognitive symptom the earliest.
Current, the AD medicine of Clinical practice is many, but all lacks the curative effect having clinical meaning.Dementia is serious Cognitive function damage, in progressivity with non-reversibility, will realize stable disease or alleviation is extremely difficult by drug intervention.Therefore, implementing prevention to dementia should be an important clinical study direction.Up-to-date AD research report shows, as " baby " this generation, 1,000 3 hundred 50 ten thousand people will be had to the year two thousand fifty to become AD dementia patients in the U.S..If suppose have a kind of intervention can be the delayed onset 5-10 of AD dementia or do sth. in advance 5-10 prediction and examination, so AD dementia patients number will reduce 80%, and the expectation medical expense for AD will greatly reduce.Therefore, find effective AD screening instruments, to preclinical phase AD patient and the early screening that AD high risk population does not occur, in crowd, carry out therapeutic intervention as early as possible, the final incidence reducing AD has important clinical and significance of times.Develop as early as possible and be applied to AD pathology mRNA level in-site kit for screening in early stage, this diagnosis and treatment pattern that will change after current AD generation clinically, the diagnosis and treatment of AD are improved, advances to pathology in earlier stage, accomplish early stage intervention and the treatment of preventative (preventiveing treatment of disease), and accomplish regulation and control and the gene early treatment of mRNA level in-site further, AD is eliminated in bud.
The present inventor finds in studying for a long period of time, causes the major cause of senile dementia treatment poor effect to be to accomplish real early diagnosis.This problem is intended adopting nucleic acid hybridization in situ technology, find and to fall ill closely-related PIRB gene one-level functional transcription product-mRNA with AD, observe it and express change patient AD, high risk population, normal control population, its clinical application meaning and value of statistical study, and cultivate, develop and can be applicable to clinical early screening kit, for the early screening of preclinical phase AD patient and high risk population, accomplish preventative diagnosis and treatment, carry out therapeutic intervention as early as possible, reduce the final incidence of AD.
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 and treatment senile dementia and prevent senile dementia, makes great progress.So far, we likely do more accurate early screening and diagnosis on the one-level functional transcription product (mRNA level in-site) of gene, develop early stage, just can accomplish early prediction and examination in the gene physiopathology of senile dementia.The present invention adopts nucleic acid hybridization in situ technology, selects to organize clinical samples (patients with Alzheimer disease, high risk population, normal control) more, carries out detection analyze the early warning of PIRB gene and senile dementia.
The mRNA of PIRB gene has very important clinical diagnosis meaning as early screening senile dementia pathology early stage.MRNA over-expression in senile dementia early stage and pathological process of PIRB gene.It makes in the Testing index of the examination in early stage of senile dementia pathology and senile dementia early prevention and the recurrence early warning after treating also has very important clinical meaning.
Contriver is in long-term research, draw a kind of new concept, the clinical diagnosis and treatment pattern of clinical major disease must change, only can not stop present treating the disease affected (diagnosis and treatment after morbidity), accomplish preventative diagnosis and treatment, accomplish treating the disease affected, only in this way could reduce the M & M of major disease, reduce social cost and medical treatment cost.Therefore, contriver in the mRNA level in-site kit for screening and medicine of development and production major disease, theoretical and be technically all bold in innovation.Particularly screen clinical samples (normal population, high risk population, Disease), breach the consistency Research idea that healthy tissues compares with diseased tissue, find and develop pathology phase premessenger RNA level, develop closely related with disease early gene physiopathology, and the extremely important target of clinical meaning, disease is clinically formed the preventative diagnosis and treatment that rear diagnosis and treatment pattern becomes disease, has striven for the Time and place of diagnosis of disease, reached prevention major disease.
Report is had no according to the detection technique of existing documents and materials PIRB gene mRNA levels and test kit.
The present inventor is to (patients with Alzheimer disease, high risk population, normal control people) routine group, detect with hybridization in situ technique, result shows all overexpressions of above senile dementia PIRB gene, and high risk population has and expresses 15-25% in various degree, and normal people is zero expression.Show that PIRB gene is the important symbol thing of senile dementia pathology examination in early stage.
Hybridization in situ technique (in situ hybridization) is combined with Cytochemical Technique by molecular biology, with the nucleic acid molecule marked for probe, in the technology of histocyte in situ detection specific nucleic acid molecules.Its principle makes containing distinguished sequence, the single nucleic acid strands (i.e. probe) passing through mark, hybridize with the complementary nucleic acid strand in histocyte and target nucleic acid under optimum conditions, with radioautograph or immunocytochemistry, label probe is detected again, thus show special DNA or RNA molecule at cell in-situ.
The probe of in situ hybridization is that the unknown but nucleic acid molecule that molecule is known of the molecule of known array or sequence is (though this molecule full sequence indefinite, 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 be marked by certain means, is 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 replaced by heterotope recently.The most frequently used at present in non-isotopic labels have vitamin H, digoxin and fluorescein three kinds.The method detecting these markers is all extremely sensitive.
Can be divided into DNA-DNA again according to probe used and the difference that will detect nucleic acid, RNA-DNA, RNA-RNA are hybridized.No matter but the hybridization of any form, all have to pass through five large processes, namely histiocytic fixing, prehybridization, hybridization, flushing and display.The present invention adopts the Crossing system of RNA-RNA, the probe (mRNA) of synthesis and the said target mrna of detection are the principles adopting base complementrity (hybridize complementary), simultaneously through long-time research and observation, start and termination place residue on detect result do not affect (because, the mRNA sequence that contriver adopts is all more than more than 600bp, and for longer base sequence, more than more than 1000bp, we can select the CDS of this gene to make probe, if the base sequence length of CDS is also more than more than 1000bp, we can select wherein one section of base sequence to carry out synthesising probing needle, but whether have identical base sequence fragment exist) if will analyze in body through Blast.
Original intention of the present invention wants to change at present the diagnosis and treatment pattern of major disease clinically, become preventative from treating the disease affected to preventive treatment of disease, reach preventative diagnosis and treatment, current medical imaging means and numerous biochemical marker cannot be detected senile dementia pathology develops mRNA level in-site quantification change in early stage technology, do the technological breakthrough of novelty, before senile dementia is provided, become mRNA level in-site examination technology.Make to have had clinically a new senile dementia pathology to develop the technology of the real early screening of mRNA level in-site in early stage, for clinical senile dementia early prevention and treatment is raced against time and space.
In sum, 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 developing the in situ hybridization detection method of the PIRB gene in early stage with senile dementia pathology.
Summary of the invention
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, it be 2792bp, CDS is from 82 that described hybridization probe has sequence table SEQ ID NO:XM_005277228(full length gene respectively ... 1977bp, is positioned at karyomit(e) 19q13.4 " on; this probe is one section in this gene C DS; the long 480bp of probe, carries out reverse complemental hybridization with the target fragment in body, and described hybridization probe sequence is as shown in SEQ ID NO.1.
A preferred version of test kit of the present invention is, 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 also comprises hybridization solution.
A preferred version of test kit of the present invention also comprises toughener.
A preferred version of test kit of the present invention also comprises developer.
Senile dementia pathology of the present invention develops gene screening test kit using value in earlier stage and is, in mRNA level in-site, can develop the examination in early stage and the judge of senile dementia curative effect after treatment early to senile dementia pathology.PIRB gene is a kind of closely related gene of morbidity of senile dementia, and its expression change shows the possibility of senile dementia Pathologic in early stage, and prompting clinician gets involved prevention and therapy early.
The present invention also provides a kind of detection method of PIRB gene hybridization in situ, comprises the following steps:
(1) under hybridization probe recited above and target sequence can form the condition of stable hybridization complex, RNA to be measured in substrate is contacted with hybridization probe, form hybridization complex; With
(2) described hybridization complex is detected.
Detection method of the present invention, is wherein preferably, and the described condition forming stable hybridization complex 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, is wherein preferably, and described substrate selects blood leucocyte sample or other organ-tissue cell or the samples of CSF of people.More preferably, described blood preparation or other organ-tissue cell specimen from patients with Alzheimer disease, high risk population, normal population.
Detection method of the present invention, is wherein preferably, described high risk population, senile dementia build up a family fortune well race, through treatment after care of patients with senile dementia.
Detection kit of the present invention adopts nucleic acid hybridization technique and groupization immunization method to combine, with PIRB gene for detected object, synthesising probing needle is the RNA sequence of PIRB gene, and the substrate of detection is the expression amount of the RNA of blood of human body sample white corpuscle or histocyte and cerebrospinal fluid.The display packing of hybridization in situ technique can provide sxemiquantitative or the quantitative expression deciding degree of PIRB gene.The expression amount of above gene is judged according to immunohistochemical visualization after hybridization, normal people PIRB genetic expression is low or do not express, and does not namely develop the color, and PIRB gene has at Patients With Senile Dementia and normal people and shows difference, the expression amount of this gene is all higher than normal people expression amount, highly develops the color.
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 carries out quantitative analysis, report the test under sample disposal, prehybridization, hybridization, immunohistochemical staining, mirror, and the concrete steps of wherein hybridizing 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, fixing automatically;
4). instrument discards liquid automatically, automatic 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: the nucleic acid probe digoxigenin labeled (cDNA of digoxigenin labeled of gene chemical synthesis for the purpose of PIRB gene, RNA and oligonucleotide probe, not only probe has biotin labeling advantage, also overcoming biotin labeled probe is organized Endogenous Biotin to do the shortcomings such as sorrow in crossover process in position), this hybridization probe and the leukocytic RNA nucleic acid to be measured of blood of human body are 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 goal gene.
The inventive method is nucleic acid hybridization in situ technology conventional at present, the method is by the PIRB gene expression amount in detection substrate cell, be used for determining that senile dementia pathology develops whether to occur, because the low expression or do not express in normal people of PIRB gene, if PIRB genetic expression height, illustrates that senile dementia occurrence risk is very high, and after treatment, patient effect is passed judgment on, thus obtain senile dementia early stage examination and diagnostic message, help clinician get involved early.A test kit can many person-portions use or person-portion use.
As mentioned above, when the expression of PIRB gene being detected higher than normal control, then measurable experimenter's senile dementia pathology develops and occurs.
The present invention has following beneficial effect:
Clinical meaning of the present invention is generation, the development trend of the generation of more early stage tracing detection senile dementia pathology and Pathologic process person in middle and old age dementia.The present invention can detect PIRB gene unconventionality expression on gene level, also before not forming senile dementia, the information acquisition of above abnormal gene expression can be accomplished early, High risk group in early stage is developed, efficacy determination after a real early screening and treatment to clinical senile dementia pathology.So just likely implement early screening, early prevention, the early treatment of senile dementia, likely from source, thoroughly effect a radical cure this foul disease of senile dementia.
In addition, test kit provided by the invention has feature that is highly sensitive, high specificity, and meanwhile, detection method of the present invention is convenient and simple for operation, can be widely used and promoted in Municipal Hospitals.
Accompanying drawing explanation
Fig. 1 is PIRB gene hybridization in situ implementing procedure figure of the present invention;
Fig. 2 is that in the embodiment of the present invention, normal people PIRB expresses picture;
Fig. 3 is that in the embodiment of the present invention, high risk population PIRB expresses picture;
Fig. 4 is embodiment of the present invention Patients With Senile Dementia PIRB overexpression picture.
Embodiment
Below in conjunction with embodiment, further illustrate content of the present invention.Should be appreciated that, the following examples for illustration of and non-limiting content of the present invention, any pro forma change or flexible will fall into protection scope of the present invention.
Embodiment 1
Conventionally prepare the in situ hybridization test kit of the present embodiment, it is hybridization probe, marker, the specification sheets of testing goal gene design that this test kit comprises with gene, wherein:
The probe mark thing optionally Gaoxin of the present embodiment.
Test kit hybridization solution forms:
/ box colourless transparent liquid managed by Digestive system 100 μ L/ pipe 1
/ box colourless transparent liquid managed by protection liquid 100 μ L/ pipe 1
/ box colourless transparent liquid managed by prehybridization solution 1300 μ L/ pipe 2
/ box colourless transparent liquid managed by justice hybridization solution 10 μ L/ pipe 1
/ box colourless transparent liquid managed by antisense hybridization liquid 10 μ L/ pipe 1
/ box colourless transparent liquid managed by confining liquid 1000 μ L/ pipe 1
/ box colourless transparent liquid managed by alkaline phosphatase antibodies 1 μ L/ pipe 1
/ box yellow liquid managed by developer A 175 μ L/ pipe 1
/ box colourless transparent liquid managed by developer B 320 μ L/ pipe 1
Light yellow or the colourless transparent liquid of damping fluid I 90mL/ bottle 1 bottle/box
Light yellow or the colourless transparent liquid of damping fluid II 80mL/ bottle 1 bottle/box
Light yellow or the colourless transparent liquid of damping fluid III 20mL/ bottle 3 bottle/box
Light yellow or the colourless transparent liquid of damping fluid IV 90mL/ bottle 1 bottle/box
Stationary liquid 90mL/ bottle 1 bottle/box colourless transparent liquid
Positive control sample 6/box.
Reagent preparation working concentration
1). 10 × damping fluid I tri-distilled water is diluted to 1 × damping fluid I by 1:10;
2). 20 × damping fluid II tri-distilled water is diluted to 2 × damping fluid II by 1:10;
0.2 × damping fluid II is diluted to by 1:100; 0.1 × damping fluid II is diluted to by 1:200;
3). 10 × damping fluid III tri-distilled water is diluted to 1 × damping fluid III by 1:10;
4) .10 × damping fluid IV with tri-distilled water by 1:10 be diluted to × damping fluid IV (get 1#, each 10mL of 2#, 3#, add water to 100mL both can).
Embodiment 2
Application nucleic acid hybridization in situ detection method is to the implementation process of patients with Alzheimer disease PS-genetic expression:
1). get sample to be measured two;
2). in glass jar, add Digestive system (Digestive system 100 μ L adds 1 × damping fluid I 99.9ml, is working concentration) 50 ml, 37 DEG C of water-bath preheating 10min, put 16 slides into, 37 DEG C of process 12 min, then use 1 × damping fluid I to wash 5min;
3). (protection liquid 1ml adds 1 × damping fluid to the protection liquid with 0.2%
, 99ml is working concentration) and wash 10min, tri-distilled water is washed more than 5min(process and is all carried out at glass jar), take out slide, allow its seasoning;
4). slide is put into moisture preservation box, and add prehybridization solution 25 μ L/ sheet (being added in the place of cell), covered, covers tightly moisture preservation box, is placed on more than 3h in 42 DEG C of constant water bath box;
5). take out slide, discard cover glass, slide is put into glass jar, respectively wash 2min with the ethanol of 70%, 90%, 95%, take out, seasoning;
6). slide is put into moisture preservation box, and one adds just hybridization solution 25 μ L/ sheet, and another adds antisense hybridization liquid 25 μ L/ sheet, 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:
Twice is washed with 2 × damping fluid II, each 15min in 42 DEG C of constant water bath box;
Wash once with 0.2 × damping fluid II in 42 DEG C of constant water bath box, each 15min;
Twice is washed with 0.1 × damping fluid II, each 15min in 42 DEG C of constant water bath box;
8). wash 30s with 1 × damping fluid III, take out slide, seasoning;
9). slide is put into moisture preservation box, adds 0.5% confining liquid (1ml confining liquid adds 5ml 1 × damping fluid III) 100 μ L/ sheet, cover tightly moisture preservation box, at room temperature act on 30min.(this step does not need to add cover glass);
10). take out slide, wash 30s with 1 × damping fluid III, seasoning;
11). slide is put into moisture preservation box, add X-AP antibody and (get a pipe alkaline phosphatase antibodies, add 1.8ml 1 × damping fluid III wherein) 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, each 15min;
13). wash 2min with 1 × damping fluid IV, add developer (developer A73.3 μ L, developer B157.5 μ L is added in 30mL 1 × damping fluid IV, mixing), room temperature more than lucifuge 16h to 18h;
14). wash 5min with tri-distilled water, seasoning, (1 × damping fluid I adding 10% with glycerine mixes) 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 mRNA, according to the cell count of dyeing, judge the expression amount of goal gene.
Patients With Senile Dementia 20, more than 55 years old crowd 20, Normal group 20.The peripheral blood 3-5 milliliter (separation white corpuscle) taking out all people to be checked does in situ hybridization.Result represents, all patients with Alzheimer disease PIRB genes have overexpression, and cell dyeing is dark; Normal group PIRB gene is not expressed, and cell does not dye; The low expression of high risk population, the cell concrete outcome that dyes on a small quantity is shown in Fig. 2, Fig. 3, Fig. 4.
SEQUENCE LISTING
<110> Rui Qu biotechnology (Shanghai) Co., Ltd.
<120> senile dementia pathology mRNA level in-site in situ hybridization kit for screening in early stage and screening method and application
<130> 、
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 480
<212> DNA
<213> Homo sapiens
<400> 1
ccactggaac ccaagaacaa ggccagattc tccatcccat ccatgacaga gcaccatgcg 60
gggagatacc gctgccacta ttacagctct gcaggctggt cagagcccag cgaccccctg 120
gagctggtga tgacaggatt ctacaacaaa cccaccctct cagccctgcc cagccctgtg 180
gtggcctcag gggggaatat gaccctccga tgtggctcac agaagggata tcaccatttt 240
gttctgatga aggaaggaga acaccagctc ccccggaccc tggactcaca gcagctccac 300
agtggggggt tccaggccct gttccctgtg ggccccgtga accccagcca caggtggagg 360
ttcacatgct attactatta tatgaacacc ccccaggtgt ggtcccaccc cagtgacccc 420
ctggagattc tgccctcagg cgtgtctagg aagccctccc tcctgaccct gcagggccct 480
Claims (10)
1. a hybridization in situ detection kit, comprises hybridization probe and marker, it is characterized in that, described hybridization probe sequence is as shown in SEQ ID NO.1.
2. test kit as claimed in claim 1, it 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, it is characterized in that, this test kit also comprises hybridization solution.
4. test kit as claimed in claim 1, it is characterized in that, this test kit also comprises synergistic agent.
5. test kit as claimed in claim 1, it is characterized in that, this test kit also comprises developer.
6. a PIRB gene hybridization in situ detection method, is characterized in that, the method comprises the following steps:
(1) under hybridization probe according to claim 1 and target sequence can form the condition of stable hybridization complex, RNA to be measured in substrate is contacted with hybridization probe, form hybridization complex; With
(2) described hybridization complex is detected.
7. detection method as claimed in claim 6, it is characterized in that, the described condition forming stable hybridization complex 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, it is characterized in that, described substrate selects blood leucocyte and the cerebrospinal fluid cell specimen of people.
9. detection method as claimed in claim 6, it is characterized in that, described sample is patients with Alzheimer disease, high risk population more than 55 years old, normal control population.
10.PIRB gene detects the application in senile dementia pathology in situ hybridization in early stage test kit in preparation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310669556.5A CN104711328A (en) | 2013-12-11 | 2013-12-11 | Alzheimer disease early-stage lesion mRNA level in-situ hybridization detection kit, screening method, and applications thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310669556.5A CN104711328A (en) | 2013-12-11 | 2013-12-11 | Alzheimer disease early-stage lesion mRNA level in-situ hybridization detection kit, screening method, and applications thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104711328A true CN104711328A (en) | 2015-06-17 |
Family
ID=53411046
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310669556.5A Pending CN104711328A (en) | 2013-12-11 | 2013-12-11 | Alzheimer disease early-stage lesion mRNA level in-situ hybridization detection kit, screening method, and applications thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104711328A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021048258A1 (en) * | 2019-09-12 | 2021-03-18 | Universiteit Gent | Means to detect whether acute hepatopancreatic necrosis disease-causing vibrio parahaemolyticus is virulent or non-virulent |
-
2013
- 2013-12-11 CN CN201310669556.5A patent/CN104711328A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021048258A1 (en) * | 2019-09-12 | 2021-03-18 | Universiteit Gent | Means to detect whether acute hepatopancreatic necrosis disease-causing vibrio parahaemolyticus is virulent or non-virulent |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| ES2700877T3 (en) | Phosphodiesterase 4D7 as a marker of prostate cancer | |
| CN105483218B (en) | Refining piRNA markers of detection and/or prediction male reproductive function obstacle or combinations thereof and its 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 | |
| CN104711328A (en) | Alzheimer disease early-stage lesion mRNA level in-situ hybridization detection kit, screening method, and applications thereof | |
| CN104711334A (en) | Alzheimer disease early-stage lesion PS-1 gene mRNA level in-situ hybridization detection kit | |
| CN104711320A (en) | Alzheimer disease early-stage lesion MS4A4 gene mRNA level in-situ hybridization detection kit | |
| CN109182490A (en) | LRSAM1 gene SNP mutational site serotype specific primer and its application in coronary disease disease forecasting | |
| CN104711319A (en) | Alzheimer disease early-stage lesion PS-2 gene mRNA level in-situ hybridization detection kit | |
| CN104711332A (en) | Alzheimer disease early-stage lesion CYP46 gene mRNA level in-situ hybridization detection kit | |
| CN104711323A (en) | Alzheimer disease early-stage lesion EPHA1 gene mRNA level in-situ hybridization detection kit | |
| CN103045729A (en) | In-situ hybridization screening kit for mRNA (messenger Ribonucleic Acid) level of gene CD2AP of prophase lesion of Alzheimer's disease (AD) and screening method and application thereof | |
| CN104711330A (en) | Alzheimer disease early-stage lesion CR-1 gene mRNA level in-situ hybridization detection kit | |
| CN104711318A (en) | Alzheimer disease early-stage lesion PICALM gene mRNA level in-situ hybridization detection kit | |
| CN104711324A (en) | Alzheimer disease early-stage lesion TAU gene mRNA level in-situ hybridization detection kit | |
| CN102943126A (en) | In-situ hybridization screening kit and method for early-stage Alzheimer's disease (AD) amyloid precursor protein (APP) gene mRNA level and application of kit and method | |
| CN103045730A (en) | In-situ hybridization screening kit for mRNA (messenger Ribonucleic Acid) level of gene CD33 of prophase lesion of Alzheimer's disease (AD) and screening method and application thereof | |
| CN101429550B (en) | Synthetic detection kit for hybridization in situ and detection method | |
| CN103045728A (en) | In-situ hybridization screening kit for mRNA (messenger Ribonucleic Acid) level of gene CLUSTERIN of prophase lesion of Alzheimer's disease (AD) and screening method and application thereof | |
| CN102965446A (en) | Alzheimer's disease (AD) lesion early-stage ALPHA-2 gene mRNA (messenger ribonucleic acid) level in-situ hybridization screening kit, as well as screening method and application thereof | |
| CN104711331A (en) | Cancer pathologic evolution early-stage Gankyrin gene mRNA level in-situ hybridization detection kit, detection method and applications thereof | |
| CN104711336A (en) | Solid tumor pathologic evolution early-stage GADD45G gene mRNA level in-situ hybridization detection kit, detection method and applications thereof | |
| 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 | |
| CN102533990B (en) | In situ hybridization detection kit for mRNA level at precancerous stage of breast cancer, detection method and application thereof | |
| CN109295197A (en) | BSND gene SNP mutational site serotype specific primer and its application in coronary disease disease forecasting | |
| CN104131064A (en) | In-situ hybridization detection kit of miR-378 in early stage of pathologic evolution of gastric cancer, detection method and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20150617 |