CN102965447A - 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 - Google Patents
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 Download PDFInfo
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Abstract
The invention discloses an in-situ hybridization assay kit comprising a hybridization probe and a marker. The invention further discloses a method for in-situ hybridization assay of APOE-4 gene mRNA (messenger ribonucleic acid) which is closely related to pathologic evolution in the early stage of Alzheimer's disease (AD) by using the kit, and the method comprises the following steps of: (1) enabling RNA (ribonucleic acid) to be assayed in a substrate to be in contact with the hybridization probe under the condition that the hybridization probe and a target sequence can form a stable hybridcomplex so as to form the hybridcomplex; and (2) assaying the hybridcomplex. According to the kit and the assay method, disclosed by the invention, the expression function of an APOE-4 gene can be assayed on the mRNA level, the assay can be earlier than existing clinical biochemical assay indexes and imaging medical examination, the AD lesion early-stage mRNA level screening can be truly realized, and the purposes of preventive diagnosis and treatment can be further achieved. Simultaneously, the assay method disclosed by the invention is simple, convenient, low in cost and convenient to popularize and apply in hospitals.
Description
Technical field
The present invention relates to field of biological detection, more particularly, relate to change with senile dementia (AD) pathology mrna expression in early stage the correlation detection technology of (Pathologic process).
Background technology
Alzheimer's disease (Alzheimer ' s disease-, AD) be modal dull-witted type, account for 60% ~ 80% of all dementias, also be one of disease that disability rate is high and burden is large.By 2006, human first killer's deaths from heart disease rate 11.5%, the second killer's palsy mortality ratio that descended than 2000 descended 18.1%, but the mortality ratio of alzheimer's disease has risen 47.1%, becomes the elderly's the 5th cause of death.Along with the increase of global population size and predicted life, alzheimer's disease has become the world health problem, and according to the prediction in future, prevalence of dementia nearly doubled in per 20 years, 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.It is not balanced that various countries' patients with Alzheimer disease is counted increase.The dull-witted number prediction of developed country increases with 100% ratio, and the developing countries such as China/India and South Asia thereof and West Pacific Ocean neighbouring country will increase with the ratio above 300%, and the AD number of China then will increase with 336% ratio.China is the maximum country of the aging radix of world population, ends for the end of the year 2008, China more than 60 years old elderly population account for 12% of total population near 1.69 hundred million.Trans-Provincial/Municipal (Beijing/Shanghai/Chengdu/Xi'an) epidemiology survey result according to the leader of BJ Union Hospital in 2005 estimates that over-65s the elderly AD morbidity is 4.8%, and increases with age.Therefore, China never is the low country of sending out of an AD, and is on the contrary, but the AD number is at most and the fastest country of rate of growth in the world.Therefore the swift and violent disease burden that increases will produce material impact to socio-economic development and the family life of China.
AD is the disease that disability rate is high and burden is heavy in the world.The estimation about global disease burden according to 2003 annual World Health Organization reports, more than 60 years old among the elderly dull-witted causing disabled account for 11.2%, far above palsy (9.5%), skeletal muscle obstacle (8.9%), cardiovascular diseases (5%) and various types of cancer (2.4%).Estimate that according to the common recognition of an interdisciplinary expert group of the U.S. except Spinal injury and terminal cancer, the AD weighting that disables is significantly higher than any other healthy state.In the U.S., the cost that AD population has surpassed 5,000,000, AD surpasses 1,000 hundred million dollars, estimates that the year two thousand fifty AD patient number will rise to 1,300 ten thousand people in the geometricprogression mode, through adjusting, will spend 140,000,000,000 dollars every year.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 carrying out property cognitive decrease, typical case's pathological change is senile plaque (sen ile plaque, SP), neurofibrillary tangles (neurona l fibrillary tang les, NFT) and neuron loss, in addition with granulovacuolar degeneration (granulov acuolar degeneration, GD), Hirano body and cerebrovascular change.After the cause of disease and pathogenetic research through many decades relevant AD, manyly do not understand although the cause of disease of AD also had, its pathology and physiological mechanism have been had certainly and comprehensively be familiar with, and therefrom found the stigmata that some are important.Modern molecular genetics is meaning a great aspect the pathogenesis that helps human knowledge AD, finds that the euchromosome sudden change can cause the increasing expression of A β 42 peptides.These genes comprise with relevant No. 21 karyomit(e)s of onset Alzheimer disease morning on amyloid precursor protein (APP), presenilin 1(PS1 on No. 14 karyomit(e)) and the presenilin 2(PS2 on No. 1 karyomit(e)) sudden change, apo E (APOE) gene on the 19 good karyomit(e)s that stigmata also comprises and late onset Alzheimer disease is relevant, scientist is after investigating more than 600 North America people, found the onset relation of APOE-4 transgenation and AD, a kind of enzyme that this Gene Handling produces, can help brain to decompose unnecessary cholesterol, in their research, the APOE-4 gene produces the people of variation in those bodies, the risk of not only suffering from senile dementia increases, and the cholesterol in the brain also can increase.Before this, other scientists found once that the gene of APOE-4 produced variation, also can increase the risk that the people suffers from senile dementia.This gene plays a role in the transportation of body's cholesterol.Switzerland scientist's recent studies on then shows, CYP46 and APOE-4 gene all produce the patient of variation, suffer from the risk of senile dementia than 10 times of normal people height nearly, and the content of beta-amyloid protein is also the highest in this class brain in patients.
Senile dementia is a kind of of Alzheimer's disease (being degenerative brain disorder), generally 65 years old with sequela, the state of an illness can slowly increase the weight of, cardinal symptom be the memory impaired.The definite cause of disease of senile dementia it be not immediately clear, finds that beta in the brain in patients-starch protein content can increase unusually but dissect research.Some scientists infer that accordingly beta-starch albumen may disturb the electric signal transmission between brain cell, final trigger cell mortality.The APOE-4 gene of finding further shows, perhaps has certain association between the gathering of cholesterol and beta-starch albumen, and the metabolic deficiency of body's cholesterol might be " arch-criminal " who causes senile dementia.
The biomarker of AD can help clinical diagnosis well, finds to have good consistence with pathology.New diagnosis definition and standard are recommended by the U.S. state-run ageing research institute in 2010 and alzheimer's disease federation, emphasize memory impairment is combined with biomarker.AD is the whole clinical course of disease, has comprised dull-witted early stage of AD and dull-witted phase AD.Then point out that existing early stage pathology physiology changes to the very long stage between the appearance cognitive symptom the earliest the clinical prodromal stage of AD.
Current, the AD medicine of clinical use is many, but all lacks the curative effect that clinical meaning is arranged.Dementia is serious Cognitive function damage, is progressivity and non-reversibility, realize that stable disease or alleviation are extremely difficult by drug intervention.Therefore, dementia being implemented prevention should be an important clinical study direction.Up-to-date AD research report shows, as " baby " this generation, will have 1,000 3 hundred 50 ten thousand people to become the AD dementia patients in the U.S. to the year two thousand fifty.If supposing to have a kind of intervention can be morbidity delay 5-10 or ahead of time 5-10 prediction and the examination of AD dementia, AD dementia patients number will reduce 80% so, and the expectation medical expense that is used for AD will greatly reduce.Therefore, seek effective AD screening instruments, preclinical phase AD patient is reached the early screening that AD high risk population does not occur, carry out as early as possible therapeutic intervention in the crowd, the final incidence that reduces AD has important clinical and significance of times.Develop as early as possible and be applied to the horizontal kit for screening of AD pathology mRNA in early stage, this will change the diagnosis and treatment pattern after present AD clinically occurs, the diagnosis and treatment of AD are improved, advance to pathology in earlier stage, accomplish early stage intervention and the treatment of preventative (preventiveing treatment of disease), and further accomplish regulation and control and the gene early treatment of mRNA level, AD is eliminated in bud.
The inventor is in the middle discovery that studies for a long period of time, and the major cause that causes the senile dementia treatment poor effect is to accomplish real early diagnosis.This problem intends adopting the nucleic acid hybridization in situ technology, seek the closely-related CYP46 gene one-level functional transcription product-mRNA that falls ill with AD, observe it and express variation patient AD, high risk population, normal control crowd, its clinical application meaning and value of statistical study, and cultivate, develop and can be applicable to clinical early screening kit, be used for preclinical phase AD patient and high risk population's early screening, accomplish preventative diagnosis and treatment, carry out as early as possible therapeutic intervention, 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 prevention senile dementia, makes great progress.So far, we might do more accurate early screening and diagnosis at the one-level functional transcription product (mRNA level) of gene, in the gene physiopathology differentiation early stage of senile dementia, just can accomplish early prediction and examination.The present invention adopts the nucleic acid hybridization in situ technology, selects many group clinical samples (patients with Alzheimer disease, high risk population, normal control), and the early warning of APOE-4 gene and senile dementia is detected analysis.
The mRNA of APOE-4 gene has very important clinical diagnosis meaning in earlier stage as early screening senile dementia pathology.The mRNA of APOE-4 gene is over-expression in senile dementia early stage and pathological process.It do in examination in senile dementia pathology early stage, and the detection index of senile dementia early prevention and the recurrence early warning after the treatment very important clinical meaning is also arranged.
The contriver is in long-term research, drawn a kind of new concept, the clinical diagnosis and treatment pattern of clinical major disease must change, can not only stop present treating the disease affected (diagnosis and treatment after the 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, the contriver is bold in innovation theoretical and technical in the horizontal kit for screening of the mRNA of development and production major disease and medicine.Particularly screen clinical samples (normal population, high risk population, Disease), broken through healthy tissues and diseased tissue consistency research and development thinking relatively, seek 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, striven for time and the space of diagnosis of disease, reach the prevention major disease.
Detection technique and test kit according to existing documents and materials APOE-4 gene mRNA level have no report.
The inventor is organizing (patients with Alzheimer disease, high risk population, normal control people) example, detect with hybridization in situ technique, the result shows the overexpressions of above senile dementia APOE-4 gene, and the high risk population has and expresses in various degree 15-25%, and the normal people zero expresses.Show that the APOE-4 gene is the important symbol thing of senile dementia pathology examination in early stage.
Hybridization in situ technique (in situ hybridization) is that molecular biology and Cytochemical Technique are combined, take the nucleic acid molecule of mark as probe, in the technology of histocyte in situ detection specific nucleic acid molecule.Its principle is to make the nucleic acid strand (being probe) that contains distinguished sequence, process mark, under optimum conditions with histocyte in the 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 the properties of nucleic acids difference.For the ease of spike, probe must with certain means mark in addition, be beneficial to later detection.Marker commonly used comprises radionuclide and non-radioactive marker's two large classes.Isotopic label commonly used has
3H,
35S,
125I and
32P.Although isotopic label has the advantages such as susceptibility is high, back end is comparatively clear, because radio isotope all can damage human and environment, the trend that is replaced by heterotope is arranged recently.At present the most frequently used in the heterotope marker have three kinds of vitamin H, digoxin and fluoresceins.The method that detects these markers all is extremely sensitive.
Can be divided into DNA-DNA again, RNA-DNA, RNA-RNA hybridization according to used probe and the difference that will detect nucleic acid.No matter but the hybridization of any form, all must be through five large processes, namely histiocytic fixing, prehybridization, hybridization, flushing and demonstration.The present invention adopts the Crossing system of RNA-RNA, synthetic probe (mRNA) and the said target mrna that detects are the principles that adopts base complementrity (hybridization is complementary), simultaneously through long-time research and observation, start and termination place residue on the not impact of result that detects (because, the mRNA sequence that the contriver adopts all surpasses more than the 600bp, and for long base sequence, surpass more than the 1000bp, we can select the CDS of this gene to make probe, if the base sequence length of CDS also surpasses more than the 1000bp, whether we can select wherein that one section base sequence comes synthesising probing needle, have identical base sequence fragment to exist in the body but will analyze through Blast).
Original intention of the present invention is to want to change at present the clinically diagnosis and treatment pattern of major disease, become preventative preventiveing treatment of disease from treating the disease affected, reach preventative diagnosis and treatment, present medical imaging means and numerous biochemical marker can't be detected senile dementia pathology differentiation mRNA level in early stage quantification change technology, done the technological breakthrough of novelty, the horizontal examination technology of the front mRNA of change of senile dementia is provided.Make the technology that has had clinically a new senile dementia pathology to develop the real early screening of mRNA level in early stage, for the early prevention of clinical senile dementia with treatment is raced against time and the space.
In sum, purpose of the present invention at first provides 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 the mentioned reagent box to be used for developing with senile dementia pathology the in situ hybridization detection method of the APOE-4 gene in early stage.
Summary of the invention
For realizing purpose of the present invention, technical scheme of the present invention is as follows:
The present invention at first provides a kind of hybridization in situ detection kit, and it comprises hybridization probe and marker, wherein, described hybridization probe is the complementary sequence of sequence shown in the sequence table SEQ ID NO.1, APOE-4 gene 19bp length, 600bp carries out reverse complemental hybridization with the target fragment in the body.
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.
Senile dementia pathology differentiation gene screening test kit in early stage using value of the present invention is, can in the mRNA level, develop the examination in early stage and the judge of senile dementia curative effect after treatment to senile dementia pathology early.The APOE-4 gene is a kind of senile early ageing gene, and its expression changes the possibility that shows senile dementia Pathologic in early stage, and the prompting clinician gets involved prevention and treatment early.
The present invention also provides a kind of detection method of APOE-4 gene hybridization in situ, may further comprise the steps:
(1) described hybridization probe and target sequence can form under the condition of stablizing hybridization complex in the above, and RNA to be measured in the substrate is contacted with hybridization probe, form hybridization complex; With
(2) detect described hybridization complex.
Detection method of the present invention, wherein preferably, the described condition of stablizing hybridization complex that forms is: the temperature of nucleic acid hybridization is 42 ℃; 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 leucocyte sample or other organ-tissue cell or samples of CSF.More preferably be that described blood preparation or other organ-tissue cell specimen are from patients with Alzheimer disease, high risk population, normal population.
Detection method of the present invention, wherein preferably, described high risk population, senile dementia build up a family fortune well family, through the treatment after care of patients with senile dementia.
Detection kit of the present invention is to adopt nucleic acid hybridization technique and groupization immunization method to combine, take the APOE-4 gene as detected object, synthesising probing needle is the RNA sequence of APOE-4 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 APOE-4 gene.Judge the expression amount of above gene according to immunohistochemical methods colour developing after the hybridization, normal people APOE-4 genetic expression is low or do not express, and does not namely develop the color, and the APOE-4 gene has aobvious difference Patients With Senile Dementia and normal people, the expression amount of this gene is all higher than normal people expression amount, highly colour developing.
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 the molecular biology insider all knows, and concrete operation step is to carry out quantitative analysis, report the test under sample disposal, prehybridization, hybridization, immunohistochemical staining, the mirror, 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, and is automatically rear fixing;
4). instrument discards liquid automatically, automatically prehybridization (42 ℃);
5). instrument discards liquid automatically, automatically cleans;
6). instrument discards liquid automatically, automatically hybridization (42 ℃);
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 the mounting microscopy.
The scheme of a preferred embodiment of the present invention is: the nucleic acid probe that synthesizes take the APOE-4 gene as the goal gene digoxigenin labeled (cDNA of digoxigenin labeled, RNA and oligonucleotide probe, not only probe has a biotin labeling advantage, also having overcome biotin labeled probe is organized Endogenous Biotin to do the shortcomings such as sorrow in the crossover process in position), this hybridization probe and the leukocytic RNA nucleic acid to be measured of blood of human body are hybridized, method with immunohistochemical methods develops the color again, under light microscopic, observe existence and the location of 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 commonly used at present, the method is by the APOE-4 gene expression amount in the detection substrate cell, be used for determining whether the differentiation of senile dementia pathology occurs, express or do not express because the APOE-4 gene is low in the normal people, if APOE-4 genetic expression height illustrates that the senile dementia occurrence risk is very high, and the patient effect is passed judgment on after the treatment, thereby obtain senile dementia examination and the diagnostic message in early stage, help the clinician to get involved early.A test kit can many person-portions use or person-portion use.
As mentioned above, when the expression that detects the APOE-4 gene was higher than normal control, then measurable experimenter's senile dementia pathology developed and occurs.
The present invention has following beneficial effect:
Clinical meaning of the present invention is generation, the development trend that more early stage tracking detects the generation of senile dementia pathology and Pathologic process person in middle and old age dementia disease.The present invention can detect the APOE-4 gene unconventionality expression at gene level, also do not form before the senile dementia, can accomplish early the information acquisition of above abnormal gene expression, develop the High risk group in early stage for clinical senile dementia pathology, efficacy determination after real early screening and the treatment.So just might implement early screening, early prevention, the early treatment of senile dementia, might from the source, thoroughly effect a radical cure this foul disease of senile dementia.
In addition, characteristics highly sensitive, high specificity that test kit provided by the invention has, simultaneously, detection method of the present invention is convenient and simple for operation, can be widely used and promoted in Municipal Hospitals.
Description of drawings
Fig. 1 is APOE-4 gene hybridization in situ implementing procedure figure of the present invention.
Fig. 2 is embodiment of the invention Patients With Senile Dementia APOE-4 overexpression picture.
Fig. 3 is that normal people APOE-4 expresses picture in the embodiment of the invention.
Fig. 4 is that high risk population APOE-4 expresses picture in the embodiment of the invention.
Embodiment
Below in conjunction with embodiment, be described more specifically content of the present invention.Should be appreciated that the following examples are used for explanation and non-limiting content of the present invention, any pro forma change or accommodation will fall into protection scope of the present invention.
Prepare the in situ hybridization test kit of present embodiment according to ordinary method, this test kit comprises hybridization probe, marker, the specification sheets take the APOE-4 gene as the testing goal gene design, wherein:
The probe mark thing of present embodiment is selected digoxin.
The test kit hybridization solution forms:
| Digestive system | 100 μ L/ |
1 pipe/box | Colourless transparent liquid |
| Protection liquid | 100 μ L/ |
1 pipe/box | Colourless transparent liquid |
| Prehybridization solution | 1300 μ L/ pipe | 2 pipe/boxes | Colourless transparent liquid |
| The justice hybridization solution | 10 μ L/ |
1 pipe/box | Colourless transparent liquid |
| The antisense hybridization solution | 10 μ L/ |
1 pipe/box | Colourless transparent liquid |
| Confining liquid | 1000 μ L/ |
1 pipe/box | Colourless transparent liquid |
| The alkaline |
1 μ L/ |
1 pipe/box | Colourless transparent liquid |
| Developer A | 175 μ L/ |
1 pipe/box | Yellow liquid |
| Developer B | 320 μ L/ |
1 pipe/box | Colourless transparent liquid |
| The damping fluid I | The 90mL/ |
1 bottle/box | Light yellow or colourless transparent liquid |
| The damping fluid II | The 80mL/ |
1 bottle/box | Light yellow or colourless transparent liquid |
| The damping fluid III | The 20mL/ bottle | 3 bottle/boxes | Light yellow or colourless transparent liquid |
| The damping fluid IV | The 90mL/ |
1 bottle/box | Light yellow or colourless transparent liquid |
| Stationary liquid | The 90mL/ |
1 bottle/box | Colourless transparent liquid |
| The positive control sample | 6/box |
The reagent preparation working concentration
1). 10 * damping fluid I is diluted to 1 * damping fluid I with tri-distilled water by 1:10;
2). 20 * damping fluid II is diluted to 2 * damping fluid II with tri-distilled water by 1:10;
Be diluted to 0.2 * damping fluid II by 1:100; Be diluted to 0.1 * damping fluid II by 1:200;
3). 10 * damping fluid III is diluted to 1 * damping fluid III with tri-distilled water by 1:10;
4) .10 * damping fluid IV with tri-distilled water by 1:10 be diluted to * the damping fluid IV (get 1#, 2#, each 10mL of 3#, add water to 100mL both can).
Embodiment 2
Use the nucleic acid hybridization in situ detection method to the implementation process of patients with Alzheimer disease () genetic expression:
1). get two of samples to be measured;
2). add Digestive system (Digestive system 100 μ L add 1 * damping fluid I 99.9ml, are working concentration) 50 ml in glass jar, 37 ℃ of water-bath preheating 10min put 16 slides into, process 12 min, use 1 * damping fluid I to wash 5min again for 37 ℃;
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 the above process of 5min(and is all carried out at glass jar), take out slide, allow 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 ℃ of constant water bath box more than the 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 ℃ of constant water bath box;
7). take out slide, discard cover glass, slide is put into glass jar:
In 42 ℃ of constant water bath box, wash twice with 2 * damping fluid II, each 15min;
In 42 ℃ of constant water bath box, wash once each 15min with 0.2 * damping fluid II;
In 42 ℃ of constant water bath box, wash twice with 0.1 * damping fluid II, each 15min;
8). wash 30s with 1 * damping fluid III, take out slide, seasoning;
9). slide is put into moisture preservation box, add 0.5% confining liquid (the 1ml confining liquid adds 5ml 1 * damping fluid III), 100 μ L/ sheets, cover tightly moisture preservation box, at room temperature act on 30min.(this step need not 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 enzyme antibody, to wherein adding 1.8ml 1 * 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 need not 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 lucifuge 16h is to more than the 18h;
14). wash 5min with tri-distilled water, seasoning, (add with glycerine 10% 1 * damping fluid I mixing) mounting microscopy.
Nucleic acid hybridization in situ detection method of the present invention is with the goal gene digoxigenin labeled, become the RNA nucleic acid probe, the RNA nucleic acid to be measured of probe and human leukocytes is hybridized, method with immunohistochemical methods develops the color again, therefore under light microscopic, observe existence and the location of mRNA, according to the cell count of dyeing, judge the expression amount of goal gene.
20 of Patients With Senile Dementias, the crowd is 20 more than 55 years old, 20 of Normal groups.Take out all people's to be checked peripheral blood 3-5 milliliter (separation white corpuscle) and do in situ hybridization.The result represents that all patients with Alzheimer disease CYP46 genes have overexpression, and cell dyeing is dark; Normal group CYP46 gene is not expressed, and cell dye-free concrete outcome is seen Fig. 2, Fig. 3, Fig. 4.
SEQUENCE LISTING
＜110〉Rui bends biotechnology (Shanghai) Co., Ltd.
＜120〉senile dementia pathology early stage the horizontal in situ hybridization kit for screening of APOE-4 gene mRNA and screening method and
Use
<130> 。
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 19
<212> DNA
<213> Homo sapiens
<400> 1
cggccgcgca cgtcctcca 19
Claims (10)
1. a hybridization in situ detection kit comprises hybridization probe and marker, it is characterized in that, described hybridization probe is the complementary sequence of sequence shown in the sequence table SEQ ID NO.1.
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. APOE-4 gene hybridization in situ detection method is characterized in that the method may further comprise the 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 the 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 described condition of stablizing hybridization complex that forms is: the temperature of nucleic acid hybridization is 42 ℃; 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 people's blood leucocyte and cerebrospinal fluid cell specimen.
9. detection method as claimed in claim 6, it is characterized in that, described sample is patients with Alzheimer disease, high risk population, normal control crowd more than 55 years old, comprise high risk population's pathology Pathologic in early stage clinically, and the APOE-4 gene mRNA expression changes in the recurrence Pathologic process after the patients with Alzheimer disease treatment.
10.APOE-4 the application of gene in preparation detection senile dementia in situ hybridization test kit.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112362876A (en) * | 2020-08-06 | 2021-02-12 | 武汉天德生物科技有限公司 | Colloidal gold test strip for detecting early senile dementia and preparation method thereof |
| CN112540178A (en) * | 2020-08-06 | 2021-03-23 | 武汉天德生物科技有限公司 | Immunohistochemical kit for detecting early senile dementia and using method thereof |
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| CN112362876B (en) * | 2020-08-06 | 2023-12-15 | 武汉天德生物科技有限公司 | Colloidal gold test strip for detecting early senile dementia and preparation method thereof |
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