CN102453769A - Leukemia lesion prophase mRNA level in-situ hybridization detection kit, detection method and application - Google Patents
Leukemia lesion prophase mRNA level in-situ hybridization detection kit, detection method and application Download PDFInfo
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Abstract
The invention discloses an in situ hybridization detection kit, which comprises a hybridization probe and a marker. Also discloses a method for detecting mRNA of a transcription factor (DJ-1) gene closely related to the pathological evolution of the leukemia lesion prophase by using the kit through in situ hybridization, which comprises the following steps: (1) under the condition that the hybridization probe and the target sequence can form a stable hybridization complex, contacting RNA to be detected in a substrate with the hybridization probe to form a hybridization complex; and (2) detecting the hybridization complex. The kit and the detection method can detect the expression quantity of the DJ-1 gene on the mRNA level, the existing clinical biochemical detection indexes are earlier, the real mRNA level screening of leukemia lesion at the early stage can be realized, and meanwhile, the detection method is simple and convenient, has low cost and is convenient to popularize and apply in county and district hospitals.
Description
Technical field
The present invention relates to field of biological detection, more particularly, relate to express the correlation detection technology that changes (pathology evolution process) with white blood disease pathology mRNA in early stage.
Background technology
White blood disease is the malignant disease of hemopoietic system; Being commonly called as " leukemia " />, is one of ten big malignant tumours occurred frequently through the country, and its characteristics are the generation neoplasm of leukemia cell in marrow or other hemopoietic tissues of a certain type in the hemopoietic tissue; And each internal organs, tissue in the infiltration body; Cause the normal hematopoiesis cell to be suppressed, produce various symptoms, clinical manifestation is characteristics with heating, hemorrhage, anaemia, liver, spleen, the pouring enlargement of currying favour.White blood disease generally is divided into acute and chronic by the natural course of disease and the inmature degree of cell, be divided into types such as granulocyte, lymphocyte, monocyte by cell type, and clinical manifestation respectively has similarities and differences part.Can be through Chinese medicine and chemotherapy, major part can reach alleviation, but also bone marrow transplantation treatment, the healing even a part can be survived lastingly.China leukaemic is about 3～4 people/100,000 populations, and is the highest with leukemic sickness rate in children's's the malignant tumour.According to investigations, China < sickness rate of 10 years old childhood lymphoblastic leukemia is 2.2810 ten thousand, no matter what age all can fall ill, man's sickness rate is higher than the women.All can fall ill throughout the year, the rural area is more than the city.The ratio of national white blood disease incidence such as the U.S., Japan, Britain and death toll is also on the rise over surplus in the of nearly ten year, and the whole world has 240,000 acute leukemic patients approximately.Acute leukemia belongs to categories such as traditional Chinese medicine " consumptive disease ", " blood trouble ", " seasonal febrile diseases ".The definite cause of disease of human leukemia is not bright so far.Several factors is considered to take place relevant with white blood disease.Virus possibly be primary factor, and ionizing rays, chemical toxicant or medicine, inherited genetic factors etc. are still arranged in addition.1, it is historical that the viral etiological study of viral human leukemia has the many decades moon, but have only adult t HTLV to be caused by virus certainly so far.Other quasi-leukemias also do not have its viral factor of method proof, do not have infectivity; 2, the ionizing rays ionizing rays causes white blood disease effectiveness, and its effectiveness is relevant with irradiated site with the radiation dose size, and once the irradiation of heavy dose or multiple low dose all causes white blood disease effectiveness; 3, it is more sure that chemical substance benzene causes white blood disease effectiveness.It is main with anxious grain and erythroleukemia that benzene causes acute leukemia; 4, some white blood disease of inherited genetic factors is sent out relevant with inherited genetic factors; 5. topmost paathogenic factor is for a long time, accumulates and use the pollution of agricultural chemicals to human habitat.Acute leukemia divide clinically acute myelocytic leukemia (acute myeloblastic leukia, aml) and acute lymphoblastic leukemia (acute lymphocytic leukia, all).White blood disease is referred to as leukemia clinically, and is similar with cancer.
An annual report has been done by eight tame units such as U.S. sanitary research institute in 2005, cancer research institute, Disease Control and Prevention Center; Anticancer Great War to initiation in 1972 is looked back; Report thinks that the mankind are failures in anticancer Great War; Conclusion is that cancer mortality does not reduce, and it is enumerated out and causes the Several Factors of anticancer Great War failure to be: 1. tumour cell heterogeneous (polymorphum); 2. tumor cell drug resistance; 3. cancer therapy drug mentality of designing imperfection (animal model designs not science) etc.Simultaneously, also propose to examine closely again the measure of existing diagnosis and treatment cancer in this report.
The inventor is in the middle discovery that studies for a long period of time, and the major reason that causes cancer mortality not fallen is to accomplish real early diagnosis.Come diagnosing cancer according to existing clinical medicine image (B ultrasonic, CT, zeugmatography etc.) and with other biochemistry (cancer antigen, CEACAMS, carbohydrate hormone, the cytolemma factor, the nucleus factor, cell streaming technology) index; All be that tumour forms the back diagnosis; The former will learn in a organized way and change or existing occupying lesion, latter's major part be tumour form the back secreted, discharge or the affinity tag of tumour.The clinical idea of tradition thinks that occupancy cancer piece is the diagnosis that belongs to early-stage cancer under 2 centimeters; This notion is worth conscientiously discussing, and it is rigorous inadequately that 2 centimeters early stage these of following cancer piece genus define science, analyzes from the cytology angle; 1 centimeter lump has 100,000,000 tumour cells approximately; Its three-dimensional cell stack number of 2 centimeters lump is far above 200,000,000 tumour cells, produces from canceration early stage to the mono-clonal cancer cells and forms 2 centimeters cancer piece, and its pathology evolution process is quite long; Possibly be (except the special case) more than 5 years or 10 years or even 10 years; What be difficult to confirm is in this pathology evolution process, and lump is unique spot of cancer and independent focus, and possible cancer cells is moved to other tissue or organ growth already.Clinical study confirms already, in case when forming lump, other cancer cells is moved to other position clonal growth through different approaches, in case behind the excision primary tumor, other organ recurrent foci or multiple cancer piece kitchen range successively form.Therefore; Whether define in early days with the cancerous swelling piece size below 2 centimeters clinically, rigorous inadequately (some case is when finding primary lesion; Find metastatic lesion simultaneously; Not in the content of our statement), at this moment be diagnosis in late period and treatment of late stage in fact, this is the true cause that causes cancer mortality not fallen.Leukemic clinical diagnosis and treatment also is so, in case find to be late period all, does not do Bone Marrow Stem Cells Transplantation, and most of being difficult to cured, and the type number of joining of Bone Marrow Stem Cells Transplantation is less than the white blood disease number of the infected far away.
Along with molecular biotechnology is perfect day by day, functional genomics, the deep expansion of research such as cancer genomics in order to seek more early stage diagnosing cancer, treatment cancer and preventing cancer, makes great progress.So far, we might do more accurate early screening and diagnosis on the one-level functional transcription product (mRNA level) of gene, preceding in canceration early stage or cancer cells formation (mono-clonal), just can accomplish early prediction and examination.The present invention adopts the nucleic acid hybridization in situ technology, selects many group clinical samples (leukaemic, high risk population, normal control), and DJ-1 gene and leukemic early warning are carried out check and analysis.
The researchist through detecting patient's sample of bone marrow, finds two transcription factors (TWIST and DJ-1) abnormal expression in the marrow abnormality proliferation syndrome.And utilize the Nucleotide perturbation technique to reduce the TWIST and the DJ-1 of unconventionality expression, and the expression that can increase cancer suppressor gene p53 significantly, thus the cancer cells generation apoptosis of abnormality proliferation impelled greatly.Marrow abnormality proliferation syndrome abbreviates " MDS " as, be called as " preleukemia ", be one group and originate from multi-functional hemopoietic stem cell clone property myeloproliferative diseases. in the pastThought in the past that this disease was prone to send out the elderly over fifty years old, but in recent years, this disease has the trend that increases, and the case of in the teenager, finding is quite a few.Find that through clinical observation this disease might not all can develop into white blood disease, if the diagnosis in early stage in time, take the efficacious therapy means, patient's curative ratio is higher.Utilize gene chip and proteomic image technology, through found behind the sample of bone marrow of analyzing nearly hundred patients DJ-1 have preventative diagnosis and early stage PCI " preleukemia " the important clinical meaning arranged
The mRNA of DJ-1 there is very important clinical diagnosis meaning in earlier stage as early screening white blood disease pathology.The mRNA of DJ-1 the preleukemia and pathological process in over-expression.It do in examination in white blood disease pathology early stage, and leukemia treating after the recurrence early warning very important clinical meaning is also arranged.
The contriver is in long term studies; Drawn a kind of new concept, the clinical diagnosis and treatment pattern of cancer and other clinical major disease must change, and can not only stop present treating the disease affected (morbidity back diagnosis and treatment); 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 mRNA and medicine of exploitation and production major disease.Particularly screen clinical samples (normal population, high risk population, leukaemic); Broken through healthy tissues and tumor tissues consistency research and development thinking relatively; Seek and develop the mRNA level that becomes before the white blood disease; Closely related with the differentiation of white blood disease early gene physiopathology, and the very important target of clinical meaning, the preventative diagnosis and treatment of cancer (white blood disease) can diagnosis and treatment pattern after cancer forms clinically be become; Strive for the time and the space of white blood disease diagnosis and treatment, reached the prevention white blood disease.
At present high flux gene chip technology and proteinogram technology are all adopted in the research of DJ-1 gene, and these methods are used for the scientific research aspect more, the incompatibility clinical application, particularly personalized China's present stage condition that is applied in is still immature.Detection technique and test kit according to existing literature data DJ-1 gene mRNA level do not appear in the newspapers.
The inventor is in the requirement to the novelty invention; Designed different pieces of information example group (leukaemic, high risk population, normal control people) example group; Detect with hybridization in situ technique; The result shows the over-expressess of above leukemia patient DJ-1 gene, and the high risk population has and expresses 15-25% in various degree, and the normal people zero expresses.Show that the DJ-1 gene is the important symbol thing of white blood disease pathology examination in early stage.
Hybridization in situ technique (in situ hybridization) combines molecular biology and cytochemistry technology, is probe with the nucleic acid molecule of mark, 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 molecule or sequence the unknown but the known nucleic acid molecule of molecule (though indeterminate this molecule full sequence of known array; But what target molecule known its is directed against), the kind of probe can be divided into dna probe, cDNA probe, cRNA probe and synthetic oligonucleotide probe again by the properties of nucleic acids difference.For the ease of spike, probe must be used certain means mark in addition, is beneficial to later detection.Affinity tag commonly used comprises radionuclide and non-radioactive marker's two big classes.Isotopic label commonly used has
3H,
35S,
125I with
32P.Advantages such as susceptibility is high though isotopic label has, the back of the body end is comparatively clear because ri all can damage human and environment, have by the substituted trend of heterotope recently.At present the most frequently used in the heterotope affinity tag have three kinds of vitamin H, digoxin and resorcinolphthaleins.The method that detects these affinity tags all is a sensitive extremely.
According to used probe and to detect nucleic acid difference can be divided into DNA-DNA again, RNA-DNA, RNA-RNA hybridization.No matter but the hybridization of any form, all must be through five big processes, promptly histiocytic fixing, prehybridization, hybridization, flushing and demonstration.The present invention adopts the hybridization mode 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 the result not influence (because mRNA sequence that contriver adopt all surpass 600bp more than) of residue to detecting.
In view of the diagnosis of cancer clinically (medical imaging and biochemical indicator thing all are the diagnosis after tumour forms) at present is the diagnosis in late period, treatment also is a 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; 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 become the mRNA level before the cancer and quantize changes technology, do the technological breakthrough of novelty, provide that to become the horizontal examination of mRNA before the cancer technological.Making has had a preceding technology that becomes the real early screening of mRNA level of new cancer clinically, for the diagnosis and treatment of clinical cancer are raced against time and the space.
Summary of the invention
The object of the invention at first provides a kind of hybridization in situ detection kit, and it comprises in situ hybridization detection probes and affinity tag.
Secondly, the present invention also will provide the mentioned reagent box to be used for taking place, recurring with white blood disease the in situ hybridization detection method of relevant DJ-1 gene.
For realizing the object of the invention, technical scheme of the present invention is following:
The present invention at first provides a kind of hybridization in situ detection kit, and it comprises hybridization probe and affinity tag, and wherein, described hybridization probe is a DJ-1 gene base sequence, i.e. sequence shown in the SEQ ID NO.1, and the nucleotide sequence length of DJ-1 gene is 822bp.If (gene order is oversize in the probe mark process; Surpass more than the 1000bp, we use the sequence of CDS and come designing probe, if the sequence of CDS also surpasses more than the 1000bp; Can adopt one section base sequence in centre of gene to come synthesising probing needle; Base sequence is no less than 500bp, will do sequential detection after probe is synthetic, and function is carried out the analysis of clinical meaning).
A preferred version of test kit of the present invention is that described affinity tag is selected from radioactive substance, chemoluminescence or substance that show color, vitamin H, metal king crab, resorcinolphthalein, 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 of the present invention DJ-1 gene screening in early stage test kit using value is, can be at mRNA
Level, the early warning of early examination in white blood disease pathology early stage and white blood disease being recurred after treating.The DJ-1 gene is a kind of similar oncogene, its high expression level show have become before the white blood disease maybe and treatment after might recur, the prompting clinician gets involved diagnosis and treatment early.
The present invention also provides a kind of detection method of DJ-1 gene hybridization in situ, may further comprise the steps:
(1) described in the above hybridization probe and target sequence can form under the condition of stablizing hybridization complex, and RNA to be measured in the substrate is contacted with hybridization probe, form hybridization complex; With
(2) detect said 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 specimen for use.More preferably be that described blood preparation or other organ-tissue cell specimen are from leukaemic, high risk population, normal control.
Detection method of the present invention, wherein preferably, described white blood disease high risk population, patient's recurrence and leukaemic after treatment.
Detection kit of the present invention is to adopt nucleic acid hybridization technique and groupization immunization method to combine; With the DJ-1 gene is detected object; Synthesising probing needle is the RNA sequence of DJ-1 gene, and the substrate of detection is the expression amount of blood of human body sample white corpuscle or histiocytic RNA.The display packing of hybridization in situ technique can provide the sxemiquantitative or the quantitative expression deciding degree of DJ-1 gene.Judge above expression of gene amount according to the immunohistochemical methods colour developing of hybridization back; Normal people DJ-1 genetic expression is low or do not express, and does not promptly develop the color, and the DJ-1 gene has apparent difference leukemia patient and normal people; This expression of gene amount is all higher than normal people expression amount, and the high risk population has low the expression.
The component of diagnostic kit of the present invention is by hybridization probe, hybridization solution, developer, compositions such as synergistic agent.The nucleic acid hybridization principle of this test kit is that the molecular biology insider all knows, and the concrete operations step is to carry out quantitative analysis, result's report 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, adds Digestive system automatically;
3). instrument discards liquid automatically, and the back is fixing automatically;
4). instrument discards liquid automatically, automatically prehybridization (42 ℃);
5). instrument discards liquid automatically, cleans automatically;
6). instrument discards liquid automatically, automatically hybridization (42 ℃);
7). instrument discards liquid automatically, cleans automatically;
8). instrument discards liquid automatically, and automatic and DIG antibody is cultivated (room temperature);
9). instrument discards liquid automatically, cleans colour developing automatically;
10). take out the mounting microscopy.
The scheme of a preferred embodiment of the present invention is: with the DJ-1 gene is that goal gene synthetic nucleic probe is with 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 the endogenous vitamin H to do 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,, under light microscopic, observe existence and the location of mRNA again with the method colour developing of immunohistochemical methods; According to painted cell count, judge the expression amount of goal gene.
The inventive method is a nucleic acid hybridization in situ technology at present commonly used, and this method is through detecting the DJ-1 gene expression amount in the substrate cell, be used for confirming white blood disease whether take place and treat after whether recur; Express or do not express because the DJ-1 gene is low in the normal people; If DJ-1 genetic expression height explains that white blood disease pathology risk is very high, having reached treatment back patient possibly recur; Thereby obtain white blood disease the pathology 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 stated, when detecting the DJ-1 expression of gene and be higher than normal control, then measurable experimenter is taken place for the white blood disease pathology.
The present invention has following beneficial effect:
Clinical meaning of the present invention is more early stage generation, the development trend that detects white blood disease pathology in generation of white blood disease pathology and the pathology evolution process of following the tracks of.Diagnostic kit of the present invention is with other detects mark (cancer antigen, CEACAMS, carbohydrate hormone, the cytolemma factor, the nucleus factor) and medical imaging inspection and has apparent different clinically.The present invention can detect the DJ-1 gene unconventionality expression on gene level; Before occupancy carninomatosis kitchen range is not found in the medical imaging inspection; Before the cancer biochemical indicator did not produce unusually, the white blood disease morbidity can be accomplished the information acquisition of above abnormal gene expression in earlier stage early; Give clinical white blood disease pathology excessive risk crowd in early stage, a real early screening and the prediction early of treating the back recurrence.So just might implement early screening, early prevention, the early treatment of breast cancer, might from the source, thoroughly effect a radical cure the white blood disease foul disease.
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 a DJ-1 gene hybridization in situ techniqueflow chart of the present invention.
Fig. 2 is a leukemia patient DJ-1 overexpression picture in the embodiment of the invention.
Fig. 3 is that normal people DJ-1 expresses picture in the embodiment of the invention.
Fig. 4 is that high risk population DJ-1 expresses picture.
Embodiment
Below in conjunction with embodiment, content of the present invention is described more specifically.Should be appreciated that following embodiment is 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 with the DJ-1 gene being hybridization probe, affinity tag, the specification sheets of testing goal gene design, wherein:
The probe mark thing of present embodiment is selected digoxin for use.
The test kit hybridization solution is formed:
| 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 implementation process of nucleic acid hybridization in situ detection method to each group blood preparation DJ-1 gene 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 I 99.9ml, are working concentration) 50 ml, 37 ℃ of water-bath preheating 10min put 16 slides into, handle 12 min, use 1 * damping fluid I to wash 5min again for 37 ℃;
3). the 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, lets its seasoning;
4). slide is put into the box of preserving moisture, add prehybridization solution 25 μ L/ sheets (being added in the place of cell), covered, the lid box of tightly preserving moisture is placed in 42 ℃ of constant water bath box more than the 3h;
5). take out slide, discard deckglass, slide is put into glass jar, the ethanol with 70%, 90%, 95% is respectively washed 2min, takes out seasoning;
6). slide is put into the box of preserving moisture, and one adds just hybridization solution 25 μ L/ sheets, and another adds antisense hybridization solution 25 μ L/ sheets, and covered is covered the box of tightly preserving moisture, and is placed on 16-24h in 42 ℃ of constant water bath box;
7). take out slide, discard deckglass, slide is put into glass jar:
In 42 ℃ of constant water bath box, wash twice, each 15min with 2 * damping fluid II;
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, each 15min with 0.1 * damping fluid II;
8). wash 30s with 1 * damping fluid III, take out slide, seasoning;
9). slide is put into the box of preserving moisture, add 0.5% confining liquid (the 1ml confining liquid adds 5ml 1 * damping fluid III), 100 μ L/ sheets, cover the box of tightly preserving moisture, at room temperature act on 30min.(this step need not add deckglass);
10). take out slide, wash 30s with 1 * damping fluid III, seasoning;
11). slide is put into the box of preserving moisture; Add X-AP antibody (getting a pipe alkaline phosphatase enzyme antibody) 100 μ L/ sheets, cover the box of tightly preserving moisture and at room temperature act on 30min to wherein adding 1.8ml 1 * damping fluid III; Time can not be long, otherwise can produce false positive (this step need not add deckglass);
12). take out slide, wash 3 times, each 15min with 1 * damping fluid III;
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 used digoxigenin labeled with goal gene; Become the RNA nucleic probe; The RNA nucleic acid to be measured of probe and human leukocytes is hybridized,, therefore under light microscopic, observe existence and the location of mRNA again with the method colour developing of immunohistochemical methods; According to painted cell count, judge the expression amount of goal gene.
20 of white blood disease people, 20 of high risk population, 20 of normal control groups.Take out all people's to be checked peripheral blood 3-5 milliliter (separation white corpuscle) and do in situ hybridization.The result representes that all white blood disease disease patient DJ-1 genes have over-expresses, and cell dyeing is dark; The high risk population is low to express; Normal control group DJ-1 gene is not expressed, and cell dye-free concrete outcome is seen Fig. 2, Fig. 3, Fig. 4.
| The white blood disease number | Expression amount % | High-risk number | Expression amount % | Normal number | |
| 1 | 90 | 1 | ?16 | 1 | ? 0 |
| 2 | ?82 | 2 | ?25 | 2 | ? 2 |
| 3 | ?62 | 3 | ?15 | 3 | ? 0 |
| 4 | ?74 | 4 | ?16 | 4 | ? 0 |
| 5 | ?68 | 5 | ?19 | 5 | ? 0 |
| 6 | ?70 | 6 | ?17 | 6 | ? 0 |
| 7 | ?62 | 7 | ?19 | 7 | ? 3 |
| 8 | ?90 | 8 | ?15 | 8 | ? 0 |
| 9 | ?60 | 9 | ?20 | 9 | ? 0 |
| ? 10 | ?68 | ? 10 | ?18 | ? 10 | ? 0 |
| ? 12 | ?58 | ? 12 | ?15 | ? 12 | ? 0 |
| ? 13 | ?66 | ? 13 | 8 | ? 13 | ? 0 |
| ? 14 | ?56 | ? 14 | ?22 | ? 14 | ? 0 |
| ? 15 | ?70 | ? 15 | ?19 | ? 15 | ? 2 |
| ? 16 | ?92 | ? 16 | ?19 | ? 16 | ? 0 |
| ? 17 | ?80 | ? 17 | ?18 | ? 17 | ? 0 |
| ? 18 | ?68 | ? 18 | ?18 | ? 18 | ? 0 |
| ? 19 | ?72 | ? 19 | 12 | ? 19 | ? 0 |
| ? 20 | ?64 | ? 20 | ? ?15 | ? 20 | ? 0 |
。
SEQ ID NO:1 probe sequence
1?ataaaaatgg?cttccaaaag?agctctggtc?atcctggcta?aaggagcaga?ggaaatggag
61?acggtcatcc?ctgtagatgt?catgaggcga?gctgggatta?aggtcaccgt?tgcaggcctg
121?gctggaaaag?acccagtaca?gtgtagccgt?gatgtggtca?tttgtcctga?tgccagcctt
181?gaagatgcaa?aaaaagaggg?accatatgat?gtggtggttc?taccaggagg?taatctgggc
241?gcacagaatt?tatctgagtc?tgctgctgtg?aaggagatac?tgaaggagca?ggaaaaccgg
301?aagggcctga?tagccgccat?ctgtgcaggt?cctactgctc?tgttggctca?tgaaataggt
361?tttggaagta?aagttacaac?acaccctctt?gctaaagaca?aaatgatgaa?tggaggtcat
421?tacacctact?ctgagaatcg?tgtggaaaaa?gacggcctga?ttcttacaag?ccgggggcct
481?gggaccagct?tcgagtttgc?gcttgcaatt?gttgaagccc?tgaatggcaa?ggaggtggcg
541?gctcaagtga?aggctccact?tgttcttaaa?gactagagca?gcgaactgcg?acgatcactt
601?agagaaacag?gccgttagga?atccattctc?actgtgttcg?ctctaaacaa?aacagtggta
661?ggttaatgtg?ttcagaagtc?gctgtcctta?ctacttttgc?ggaagtatgg?aagtcacaac
721?tacacagaga?tttctcagcc?tacaaattgt?gtctatacat?ttctaagcct?tgtttgcaga
781?ataaacaggg?catttagcaa?actaaaaaaa?aaaaaaaaaa?aa
Claims (10)
1. a hybridization in situ detection kit comprises hybridization probe and affinity tag, it is characterized in that, described hybridization probe is the sequence shown in the sequence table SEQ ID NO.1.
2. test kit as claimed in claim 1 is characterized in that, described affinity tag is selected from radioactive substance, chemoluminescence or substance that show color, vitamin H, metal king crab, resorcinolphthalein, 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. DJ-1 gene hybridization in situ detection method is characterized in that this method may further comprise the steps:
(1) can form under the condition of stablizing hybridization complex at described hybridization probe of claim 1 and target sequence, RNA to be measured in the substrate is contacted with hybridization probe, form hybridization complex; With
(2) detect said 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 sample for use.
9. detection method as claimed in claim 8 is characterized in that, described blood leucocyte sample is selected from leukaemic, high risk population, normal control sample.
10.DJ-1 gene detects the application in the white blood disease pathology in situ hybridization test kit in preparation.
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| WO2010132440A2 (en) * | 2009-05-11 | 2010-11-18 | Cytotech Labs, Llc | Methods for treatment of oncological disorders using epimetabolic shifters, multidimensional intracellular molecules, or environmental influencers |
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| WO2010132440A2 (en) * | 2009-05-11 | 2010-11-18 | Cytotech Labs, Llc | Methods for treatment of oncological disorders using epimetabolic shifters, multidimensional intracellular molecules, or environmental influencers |
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| REN,H.等: "NM_001123377", 《GENBANK》, 18 December 2011 (2011-12-18) * |
| REN,H.等: "NM_007262", 《GENBANK》, 18 December 2011 (2011-12-18) * |
| 刘航: "iASPP对白血病细胞的增殖凋亡的作用及DJ-1基因在急性白血病中的表达及其对白血病细胞增殖凋亡的影响", 《北京协和医院/中国医学科学院博士研究生学位论文》, 15 July 2009 (2009-07-15), pages 1 - 1 * |
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