CN104711326A - Multi-cancer pathologic evolution early-stage Bmi1 gene mRNA level in-situ hybridization detection kit, detection method and applications thereof - Google Patents
Multi-cancer pathologic evolution early-stage Bmi1 gene mRNA level in-situ hybridization detection kit, detection method and applications thereof Download PDFInfo
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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 Bmi1 gene RNA expression change, which is closely related with the pathologic evolution in the early stage of multiple cancers. 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 quantity of Bmi1 gene in the RNA level, can detect the cancers in an earlier stage compared with the medical imaging technology and conventional clinical biochemical index detection, can achieve real RNA level screening in the early stage of cancers, moreover, have the advantages of simpleness, convenience, and low cost, and thus are convenient to promote and use in county/district hospitals.
Description
Technical field
The present invention relates to field of biological detection, more particularly, relate to the correlation detection technology changing (in Pathologic process) with cancer Pathologic mrna expression in early stage.
Background technology
According to the data that domestic and international authoritative institution provides, the newly-increased number of the annual cancer of China reaches 3,120,000, death toll nearly 2,600,000, patient is more than more than 700 ten thousand, the annual newly-increased cancer patients 8,000,000 in the whole world, death toll is close to more than 800 ten thousand, and patient about has more than 8,400 ten thousand people, to double to the above number of the year two thousand twenty, this is one group of fearful numeral.Cancer diagnosis and treatment cost is more and more higher, (poor regional possible higher by the year medical expense 200,000 of cancer patients, developed regions may exceed 200,000 far away), more than 700 ten thousand patients, annual cost is 1.4 trillion Renminbi, deduction cost 35% about 400,000,000,000, about has 1,000,000,000,000 Renminbi to consume in vain every year.And cancer patients's major part can be dead soon after the treatment.Therefore, existing clinical cancer diagnosis and treatment pattern must change, innovative point of the present invention accomplishes preventative examination in advance, the mrna expression amount of examination Bmi1 gene before knurl block does not occur, to expression amount reduction person, the preventative regulation and control of timely intervention and prophylactic treatment, accomplish preventiveing treatment of disease of gene level cancer.
It is failure that eight units such as U.S. sanitary research institute, cancer research institute, Disease Control and Prevention Centers in 2005 have done in anticancer Great War, conclusion is that cancer mortality does not reduce, and it lists and causes several factors of anticancer Great War failure to be: 1. tumour cell heterogeneity (polymorphism); 2. tumor cell drug resistance; 3. Anti-Cancer Drug Design thinking imperfection (animal model designs not science) etc.Meanwhile, the measure of answering re-examine existing diagnosis and treatment cancer is also proposed in this report.
The present inventor finds in studying for a long period of time, and the major reason causing cancer mortality not fallen to accomplish real early diagnosis.Diagnosing cancer is carried out according to existing clinical medicine image (B ultrasonic, CT, Magnetic resonance imaging etc.) and with other biochemistry (cancer antigen, carcinomebryonic antigen, carbohydrate hormone, the cytolemma factor, nuclear factor, cell streaming technology) index, all that tumour forms rear diagnosis, the former will learn change or existing occupying lesion in a organized way, and the latter's major part is the marker that tumour forms rear secreted, release or tumour.Traditional clinical idea thinks that occupancy cancer block is the diagnosis belonging to early-stage cancer under 2 centimeters, this concept is worth conscientiously discussing, it is rigorous not that less than 2 centimeters early stage these of cancer block genus define science, analyze from cytology angle, the lump of 1 centimeter about has 100,000,000 tumour cells, its three-dimensional cell superposition number of the lump of 2 centimeters is far above 200,000,000 tumour cells, produce and formed the cancer block of 2 centimeters from multiple pre-cancerous to mono-clonal cancer cells, its Pathologic process is quite long, it may be 5 years or 10 years, or even more than 10 years (except special case), what be difficult to confirmation is in this Pathologic process, lump is the unique spot of cancer and independent focus, possible cancer cells moves to other tissue or organ growth already.Clinical study confirms already, once while forming lump, other cancer cells moves to other position clonal growth by different approaches, once after excision primary tumor, other organ recurrence stove or multiple cancer block stove are successively formed.Therefore, define whether in early days with the cancerous swelling piece size of less than 2 centimeters clinically, rigorous not (some case, when finding primary lesion, find metastatic lesion simultaneously, not in the content of our statement), be at this moment late diagnosis and treatment of late stage in fact, this is the true cause causing cancer mortality not fallen.
Along with molecular biotechnology is day by day perfect, functional genomics, the deep expansion of the research such as cancer gene group, in order to seek more early stage diagnosing cancer, Therapeutic cancer and preventing cancer, 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 or microRNA) of gene, form (mono-clonal) front, just can accomplish early prediction and examination at multiple pre-cancerous or cancer cells.
Grind and confirm that Bmi1 is a new proto-oncogene, express at various cancerous tissue height, it is the factor that cancer occurs and shifts, the high expression level of Bmi1 and the process of various cancer, recurrence and prognosis significant correlation, indicating Bmi1 is the marker that a potential various cancerous lesions early stage and prognosis have extremely important meaning.
More the present invention selects to organize clinical samples (cancer patient, high risk population, normal control) and adopts nucleic acid hybridization in situ technology and the early warning of ImmunohistochemistryMethods Methods to Bmi1 gene mRNA mammary cancer to carry out detection analysis.The present inventor finds that Bmi1 gene mRNA has obvious expression amount to change patient with breast cancer, cancer high risk population, normal control people under study for action, using Bmi1 gene mRNA as various canceration pathological change screening indexes in early stage, there is very important clinical diagnosis meaning.The Preventive early warning done in after multiple pre-cancerous examination and cancer therapy 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 cancer and other clinical major disease must change, only can not stop existing treating the disease affected (diagnosis and treatment after morbidity), accomplish preventative diagnosis and treatment, accomplish to preventive treatment of disease, only in this way could reduce the M & M of major disease, reduce social cost and medical treatment cost.Therefore, contriver in the rna level kit for screening and medicine of development and production major disease, theoretical and technically all innovate.Particularly screen clinical samples (normal population, cancer high risk population, tumour patient), breach the consistency Research idea that healthy tissues compares with tumor tissues, the rna level become before finding and develop cancer, develop closely related with cancer early gene physiopathology, and the extremely important target of clinical meaning (disease gene), tumour is clinically formed the preventative diagnosis and treatment that rear diagnosis and treatment pattern brings up to tumour, has striven for the Time and place of tumor diagnosis and treatment, reached preventing cancer.
At present, the analytical technologies such as the Northern hybridization of Bmi1 expression profiling method, gene chip, real-time fluorescence quantitative PCR and Solexa order-checking, and these methods are used for scientific research aspect, be not suitable with clinical application, and detect mRNA more more scientific than DNA analysis (DNA analysis major part in the presentation of susceptibility, mRNA is the embodiment of DNA function), more reliable than analysis of protein (contriver finds mRNA and protein transcription and expresses on space-time in the research of employing Double Labelling Technique, sometimes asynchronous).The detection technique of hybridization in situ technique and groupization immunization method detection Bmi1 gene mRNA levels expression amount and test kit is adopted to have no report according to existing documents and materials.
The present inventor is in the requirement for innovations, devise (patient with breast cancer, high risk population, normal control) different pieces of information example group, detect with hybridization in situ technique, result shows above cancer patient Bmi1 gene mRNA all high expression levels, high risk population has and expresses 10-25% in various degree, and normal control is all zero expression.Show that Bmi1 gene mRNA is the important symbol thing of multiple pre-cancerous examination.
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, and the probe (RNA) of synthesis and the target RNA detected are the principles adopting base complementrity (hybridize complementary), simultaneously through long-time research with observe, start and termination place residue does not affect the result detected.
In view of the diagnosis (medical imaging and biochemical indicator thing are all the diagnosis after tumour is formed) of current multiple pre-cancerous is clinically late diagnosis, treatment is also treatment of late stage, is also the treatment pattern causing mortality ratio not fallen.Original intention of the present invention wants to improve 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, become mRNA level in-site before current medical imaging means and numerous biochemical marker cannot be detected cancer and quantize change technology, do the technological breakthrough of novelty, before cancer is provided, become mRNA level in-site examination technology.Become the technology of the real early screening of mRNA level in-site before making to have had clinically a new cancer, the diagnosis and treatment for clinical cancer are 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 the in situ hybridization detection method that kinds cancer reason develops examination in early stage and after treating, transfer early warning is relevant.
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, described hybridization probe sequence is as shown in SEQ ID NO.1, sequence table SEQ ID NO:JF271685, nucleotide sequence length is 791bp, CDS sequence is 31...633bp, and probe total length 480bp to take from CDS one section.
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.
Mammary cancer Pathologic of the present invention Bmi1 gene mRNA in early stage kit for screening using value is, to the examination in early stage of cancer Pathologic, and Preventive, diffusion prediction occurring after canceration or after treatment, coordinate clinical treatment further.
The present invention also provides a kind of detection method of Bmi1 in situ hybridization, 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 specimen of people.More preferably, described blood preparation or other organ-tissue cell specimen are from kinds cancer patient, high risk population, healthy normal population.
Detection kit of the present invention adopts nucleic acid hybridization technique and groupization immunization method to combine, with Bmi1 gene mRNA for detected object, synthesising probing needle is Bmi1 gene recombination complementary sequence, and the substrate of detection is the expression amount of blood of human body sample white corpuscle or histiocytic Bmi1 gene mRNA.The display packing of hybridization in situ technique can provide sxemiquantitative or the quantitative expression deciding degree of Bmi1 gene mRNA.Judge the expression amount of above mRNA according to immunohistochemical visualization after hybridization, normal people Bmi1 zero expresses, and cell does not develop the color; The low expression of high risk population, a small amount of cell dyeing; Cancer patient high expression level, cell dyes in a large number.
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: with the nucleic acid probe digoxigenin labeled (cDNA of digoxigenin labeled of Bmi1 gene mRNA synthesis, 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 RNA, according to the cell count of dyeing, judge the expression amount of object RNA.
The inventive method is nucleic acid hybridization in situ technology conventional at present, the method is by the Bmi1 gene mRNA expression amount in detection substrate cell, be used for determining the mRNA variable quantity in kinds cancer Pathologic early stage, whether early warning cancer occurs and the prediction of whether Preventive after cancer patients's treatment.Because Bmi1 gene mRNA is expressed normal people zero, Bmi1 gene is similar to proto-oncogene carcinogenesis and short cancer metastasis effect, if expression amount increase illustrate cancer occurred or risk of developing cancer high, get involved early period regulation and treatment early clinically, suppress the expression of Bmi1 gene, keep cancer suppressor gene and oncogene physiological equilibrium, preventing cancer occurs.
A test kit can many person-portions use or person-portion use.
The present invention has following beneficial effect:
Clinical meaning of the present invention is the change of Bmi1 gene mRNA expression amount in more early stage tracing detection cancer pathology evolution process, and early warning cancer occurs, development trend.Diagnostic kit of the present invention is with other detects and cancer markers clinically, and medical imaging inspection has and shows difference.The present invention can detect the unconventionality expression of Bmi1 gene in mRNA level in-site, before medical imaging inspection does not find the recurrence of occupancy carninomatosis stove, before cancer biochemical indicator does not produce extremely, also before not forming tumour, the information acquisition of Bmi1 abnormal gene expression can be accomplished early, to the early warning that clinical cancer patient one is real, so just likely implement the early screening of cancer, early prevention, early treatment, likely from source, thoroughly effect a radical cure cancer cachexia.
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 that illustration is implemented in Bmi1 gene mRNA in situ hybridization of the present invention;
Fig. 2 is cancer patient Bmi1 gene mRNA expression picture in the embodiment of the present invention;
Fig. 3 is high risk population Bmi1 gene mRNA expression picture in the embodiment of the present invention;
Fig. 4 is normal people Bmi1 gene mRNA expression picture in the embodiment of the present invention.
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, this test kit comprises with hybridization probe, marker, the specification sheets of the design of Bmi1 gene mRNA, 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 each group of blood preparation Bmi1 gene mRNA expression amount:
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 RNA, according to the cell count of dyeing, judge the expression amount of object RNA.
Cancer patient 20, high risk population 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 cancer patients Bmi1 gene mRNAs zero are expressed, cell dye-free; High risk population expresses and slightly reduces, and decimal dyes; Normal group Bmi1 gene mRNA high expression level, cell dyes in a large number, and concrete outcome is shown in Fig. 2, Fig. 3, Fig. 4.
| Cancer occurrence numbers | Expression amount % | High-risk number | Expression amount % | Normal number | Expression amount % |
| 1 | 76 | 1 | 18 | 1 | 0 |
| 2 | 78 | 2 | 20 | 2 | 0 |
| 3 | 70 | 3 | 16 | 3 | 0 |
| 4 | 82 | 4 | 14 | 4 | 0 |
| 5 | 76 | 5 | 22 | 5 | 0 |
| 6 | 78 | 6 | 18 | 6 | 0 |
| 7 | 68 | 7 | 22 | 7 | 0 |
| 8 | 80 | 8 | 20 | 8 | 0 |
| 9 | 76 | 9 | 18 | 9 | 0 |
| 10 | 72 | 10 | 16 | 10 | 0 |
| 11 | 80 | 11 | 0 | 11 | 0 |
| 12 | 66 | 12 | 16 | 12 | 0 |
| 13 | 70 | 13 | 14 | 13 | 0 |
| 14 | 78 | 14 | 20 | 14 | 0 |
| 15 | 76 | 15 | 18 | 15 | 0 |
| 16 | 70 | 16 | 12 | 16 | 0 |
| 17 | 72 | 17 | 20 | 17 | 0 |
| 18 | 78 | 18 | 18 | 18 | 0 |
| 19 | 80 | 19 | 12 | 19 | 0 |
| 20 | 82 | 20 | 10 | 20 | 0 |
(note: various cancer patients; High risk population has selected 30 years lengths of smoking and slow liver liver cirrhosis; Normal company employee, clinical health check-up normal people).
SEQUENCE LISTING
<110> Rui Qu biotechnology (Shanghai) Co., Ltd.
The hybridization in situ detection kit of <120> kinds cancer Pathologic Bmi1 gene mRNA levels in early stage and detection method and application
<130> 、
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 480
<212> DNA
<213> Homo sapiens
<400> 1
gaagatgacg aaggccctcc ttatagcctg gaaaaaatga cagatcttgt agccgtttgg 60
gatgttgctt taagtgatgg agttcataag attgaatttg aacatgggac cacatcaggc 120
aaaagagtgg tatacgtaga tgggaaggaa gagataagaa aagaatggat gttcaaatta 180
gtgggcaaag aaaccttctg tgttggagcc gcaaagacaa aagcaaccat aaatatagat 240
gctgtcagtg gttttgctta tgaatatact ctggaaatta atgggaagag tctcaagaag 300
tatatggaga gcagatcaaa aaccaccaac acttgggtac tgcagttgga taatgaggac 360
tttagagttg tgttggaaaa agacactatg gatgtctggt gcaatggtaa aaagatggag 420
acggcaggcg agtttgtaga tgatgggact gaaactcact tcagtattgg gaaccatgac 480
Claims (9)
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 Bmi1 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, is characterized in that, described substrate choosing is human peripheral blood white corpuscle sample.
9.Bmi1 gene detects the application in kinds cancer Pathologic in situ hybridization in early stage test kit in preparation.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310669554.6A CN104711326A (en) | 2013-12-11 | 2013-12-11 | Multi-cancer pathologic evolution early-stage Bmi1 gene mRNA level in-situ hybridization detection kit, detection method and applications thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN201310669554.6A CN104711326A (en) | 2013-12-11 | 2013-12-11 | Multi-cancer pathologic evolution early-stage Bmi1 gene mRNA level in-situ hybridization detection kit, detection method and applications thereof |
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