CN102533989B - In-situ hybridization detection kit and detection method for liver cancer precancer NOTCH1 on mRNA (messenger ribonucleic acid) level, and applications thereof - Google Patents
In-situ hybridization detection kit and detection method for liver cancer precancer NOTCH1 on mRNA (messenger ribonucleic acid) level, 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. The invention also discloses a method for in-situ hybridization detection of mRNA (messenger ribonucleic acid) of signal conducting channel (Notch1) gene closely related to early-stage pathologic evolution of liver cancer by using the kit. The method comprises the following steps of: (1) contacting the RNA in a substrate to be detected with a hybridization probe under the condition that the hybridization probe and a target sequence can form a stable hybridization complex, so as to form the hybridization complex; and (2) detecting the hybridization complex. By adopting the kit and detection method, the expression amount of the NOTCH1 gene can be detected on the mRNA level, the detection time is earlier than that of imaging medicine and the traditional clinical biochemical detection indexes, and the mRNA-level screening and examination at the precancerous stage is truly realized; and at the same time, the detection method is simple, convenient, low in cost and convenient for popularization and application in county and district-level hospitals.
Description
Technical field
The present invention relates to field of biological detection, more particularly, relate to cancer before the mrna expression that becomes change the correlation detection technology of (Pathologic process).
Background technology
The data that provides according to domestic and international authoritative institution, the newly-increased number 2,600,000 of the annual cancer of China, death toll nearly 2,100,000, the patient more than 700 ten thousand, the annual newly-increased cancer patients 8,000,000 in the whole world, death toll is near 8,000,000, and the patient approximately 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 area may be higher by cancer patients's year medical expense 200,000, developed regions may exceed 200,000 far away), more than 700 ten thousand patients, annual cost is 1.4 trillion Renminbi, deduction cost 35% approximately 400,000,000,000 approximately has 1,000,000,000,000 Renminbi to consume in vain every year.And the cancer patients is most of can be dead soon after treatment.Therefore, existing clinical cancer diagnosis and treatment pattern must change, and innovative point of the present invention is to accomplish in advance preventative examination, then in time gets involved preventative regulation and control and prophylactic treatment, accomplishes preventiveing treatment of disease of gene level cancer.
An annual report has been done by eight 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 lists and causes several factors of anticancer Great War failure to be: 1. tumour cell heterogeneous (polymorphism); 2. tumor cell drug resistance; 3. cancer therapy drug mentality of designing imperfection (animal model designs not science) etc.Simultaneously, also propose to answer the measure of the existing diagnosis and treatment cancer of re-examine in this report.
The inventor is in the middle discovery that studies for a long period of time, and causing the major reason that cancer mortality is not fallen is to accomplish real early diagnosis.Come diagnosing cancer 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 in a organized way and change or existing occupying lesion, latter's major part be tumour form rear secreted, discharge or the marker 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 concept is worth conscientiously discussing, it is rigorous not that 2 centimeters early stage these of following cancer piece genus define science, analyze from the cytology angle, the lump of 1 centimeter approximately has 100,000,000 tumour cells, its three-dimensional cell stack number of the lump of 2 centimeters is far above 200,000,000 tumour cells, produce and form the cancer piece of 2 centimeters to the mono-clonal cancer cells early stage from canceration, 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 confirm 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, in case when forming lump, other cancer cells moves to other position clonal growth by different approaches, in case after the excision primary tumor, other organ recurrence kitchen range 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 not (some case, when finding primary lesion, find simultaneously metastatic lesion, not in the content of our statement), be at this moment diagnosis in late period and treatment of late stage in fact, this is the true cause that causes 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, 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, and are front 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 (hepatocarcinoma patient, high risk population, normal control), and the early warning of NOTCH1 gene and liver cancer is detected analysis.
The property sent out hepatocellular carcinoma (Hepatocellular carcinoma, HCC) is common malignant tumour, occupies the 3rd in global mortality of malignant tumors, causes that the hundreds of thousands of them is dead every year.China is the hepatocellular carcinoma district occurred frequently, and sickness rate has risen to the second of malignant tumour, and annual death rate is positioned at the 2nd～3 of mortality of malignant tumors, and its sickness rate has the trend that increases in recent years.Liver cancer has now become one of malignant tumour of serious harm people's life safety.The treatment of liver cancer at present mainly contains excision, chemotherapy, radiotherapy etc.To belong to late period when going to a doctor due to patient much more, though above-mentioned methods for the treatment of has obtained very large progress, advanced liver cancer patient's prognosis is extreme difference still.Therefore, the mechanism of illustrating tumour is for preventing the generation of tumour from the source, and then finally to defeat tumour be very important, and for this reason, the scientist in the whole world is carrying out unremitting effort.The Notch1 gene discovery is in 1919, the transmembrane receptor that is comprised of 2753 amino acid of encoding.In recent years, the homolgous molecule of having cloned a series of Notch1 acceptors from mammal comprises Notch11～4 and 5 kind of part, is respectively DLL1, DLL3, DLL4, Jagged1 and Jagged2.After the Notch1 binding of receptor and ligand, acceptor discharges its born of the same parents' internal area NICD(Notch1 Intracellular Domain), NICD induces transcribing of target gene after being transported in core.The Notch1 signal path is not only closely related with the propagation of cell, differentiation, with the genesis of tumour, close contacting is arranged.1991, Notch1 was out identified in mankind T lymphoblast leukemia, pointed out itself and Tumor-assaciated, and research is subsequently found, chromosome translocation t(7; 9) (q34; Q34.3) cause being positioned at the Notch1l gene break on karyomit(e) No. 9, the Notch1 extracellular region is lost, and its intracellular region obtains the composition activity and has the induced pain effect.But Notch1 plays the part of oncogene or cancer suppressor gene role actually in the process of tumor development.It works in tumorigenic that (a bit) link.At present all still without final conclusion.β-catenin plays the core molecularity at the Wnt path, tumour is formed, breaks up, infiltrates and shifts play an important role.Think at present, Notch1 and Wnt signal path connect each other and coordinated balance, exist cross-talk (cross-talking) between them.The researchist uses tissue array technology, liver cancer sample to large sample carries out Notch1, the detection of Jagged1 and β-catenin, observe and analyze their differential expression, inquire into the effect of Notch1 signal path in liver cancer, inquire into the mechanism of liver cancer, further understand the effect of Notch1/Jagged1 in hepatocellular carcinoma.Simultaneously the clinical pathologic characteristic of its positive expression and hepatocellular carcinoma is analyzed, to finding more information.In addition, they collect fresh hepatocellular carcinoma and cancer beside organism's specimens from pri and culture hepatocyte cancer strain HepG2 and normal cell lines of human liver strain LO2, use the method for immunohistochemical methods and RT-PCR, the further expression of inspection Notch1/Jagged1 and β-catenin mRNA, express by research β-catenin the dependency of expressing with Notch1/Jagged1 simultaneously, contact in the discussion hepatocellular carcinoma between Notch1 signal path and Wnt signal path, being intended to provides relevant theoretical foundation for the mechanism of further illustrating liver cancer.The paraffin embedding sample of 711 routine excision cases is collected in research altogether, comprising 339 routine hepatocellular carcinomas, 22 routine Hepatocholangiocarcinomas, 82 routine metastatic carcinomas, the 174 other hepatic tissues of routine cancer, 94 routine normal liver tissues.Use tissue array technology that above-mentioned preparation of specimen is become 13 organization chip wax stones, detect Notch1/Jagged1 and β-catenin antibody at the expression of various cases by immunohistochemistry technology, analyze contacting between the clinical pathologic characteristic of the positive expression of antibody and hepatocellular carcinoma.The RNA that extracts simultaneously 37 routine fresh hepatocellular carcinomas, the 37 other hepatic tissues of routine cancer and hepatocellular carcinoma strain HepG2, normal cell lines of human liver strain LO2 carries out RT-PCR, further detects the expression of Notch1/Jagged1 and β-catenin mRNA.Result of study shows: (1) Notch1/Jagged1 is low the expression in hepatocellular carcinoma, high expression level in nonneoplastic tissue.The positive correlation of Notch1/Jagged1 expression level significance.Prompting Notch1/Jagged1 may interact in hepatocellular carcinoma, plays cancer suppressing action.(2) expression and the age, serum concentration of AFP, tumour cell differentiation degree significant correlation of Notch1 in hepatocellular carcinoma; The expression of Jagged1 and tumour cell differentiation degree significant correlation.In hepatocellular carcinoma, Notch1/Jagged1 is at the good cell high expression level of differentiation, and the poor cell of differentiation hangs down expresses; Prompting Notch1/Jagged1 signal path may play to the differentiation of tumour cell important regulating and controlling effect.(3) expression of Notch1/Jagged1 and TTPV form significant correlation, send out relevant in prompting Notch11/Jagged1 and liver cancer.(4) kytoplasm/nuclear expression of β-catenin high expression level in hepatocellular carcinoma, the low expression in nonneoplastic tissue; Kytoplasm/nuclear expression of β-catenin is low at the good cell expressing of differentiation, and the cell expressing of differentiation difference is high.Prompting β-catenin plays a part short cancer in the carcinogenesis of human of hepatocellular carcinoma.(5) there is the significance negative correlation in the expression level of Notch11 and β-catenin, points out their mutually antagonism, regulation and control persons of the other side each other in hepatocellular carcinoma.(6) sxemiquantitative RT-PCR result shows that in hepatocellular carcinoma, Notch1/Jagged1 expresses low, β-catenin expresses high, synchronize with their protein expression situation, the expression of prompting Notch1/Jagged1 in hepatocellular carcinoma descends, may not that transcriptional level occurs, but be caused by other reason.
The mRNA of NOTCH1 there is very important clinical diagnosis meaning in earlier stage as the early screening hepatocarcinogenesis.The mRNA of NOTCH1 is low the expression in hepatocarcinogenesis early stage and Carcinogenesis.He do in examination in liver cancer canceration early stage, and the Preventive early warning of Liver Cancer Operation very important clinical meaning is also arranged.
The contriver is in long-term research, drawn a kind of innovative idea, the clinical diagnosis and treatment pattern of cancer and other clinical major disease must change, can not only stop present treating the disease affected (diagnosis and treatment after morbidity), accomplish preventative diagnosis and treatment, accomplish 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 innovates 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, cancer high risk population, tumour patient), broken through healthy tissues and tumor tissues consistency research and development thinking relatively, seek and develop the mRNA level that becomes before cancer, develop closely related with cancer early gene physiopathology, and the extremely important target of clinical meaning, tumour is clinically formed the preventative diagnosis and treatment that rear diagnosis and treatment pattern becomes tumour, striven for time and the space of tumor diagnosis and treatment, reach preventing cancer.
At present high flux gene chip and proteinogram analytical technology are all adopted in the research of NOTCH1 gene, and these methods are multiplex in the scientific research aspect, be not suitable with clinical application, more scientific (the DNA analysis major part is on the presentation of susceptibility than genetic analysis and detect mRNA, mRNA is functional thing), than analysis of protein more reliable (mRNA and albumen are transcribed sometimes asynchronous).Have no report according to detection technique and the test kit of existing documents and materials Notch1 gene mRNA level.
The inventor is in the requirement for the novelty invention, designed (liver cancer patient, high risk population, normal control people) different pieces of information example group, detect with hybridization in situ technique, result shows the low expression of above hepatocarcinoma patient NOTCH1 gene, the high risk population has the low 15-25% of expression in various degree, and the normal people is high expression level.The important symbol thing that shows NOTCH1 gene liver cancer canceration 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 the large class of non-radioactive marker two.Isotopic label commonly used has
3H,
35S,
125I and
32P.The advantages such as susceptibility is high although isotopic label has, back end is comparatively clear all can damage human and environment due to radio isotope, and 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 is all extremely sensitive.
Can be divided into DNA-DNA again, RNA-DNA, RNA-RNA hybridization according to probe used and the difference that will detect nucleic acid.No matter but the hybridization of any form, all must be through five large processes, 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, more than the mRNA sequence that the contriver adopts all surpasses 600bp).
In view of the diagnosis of cancer clinically (medical imaging and biochemical indicator thing are all the diagnosis after tumour forms) at present is the diagnosis in late period, treatment is also treatment of late stage, the treatment pattern that causes mortality ratio not fallen.Original intention of the present invention is to want to change at present the diagnosis and treatment pattern of major disease clinically, 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 cancer and quantize the change technology, done the technological breakthrough of novelty, the horizontal examination technology of the front mRNA of change of cancer is provided.Making has had a front 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
At first purpose 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 the mentioned reagent box to be used for and liver cancer canceration examination in early stage and postoperative recurrence, in situ hybridization detection method that the transfer early warning is relevant.
For realizing purpose of the present invention, technical scheme of the present invention is as follows:
At first the present invention provides a kind of hybridization in situ detection kit, it comprises hybridization probe and marker, wherein, described hybridization probe is sequence as shown in SEQ ID NO.1, is one section base sequence in the NOTCH1 gene order, from 2000 to 2800bp, NOTCH1 sequence number NM_017617, sequence as shown in SEQ ID NO.2, the nucleotide sequence length of gene is 9309bp, CDS is 1 ... 7668bp is positioned at karyomit(e) 9q34.3 " on.If (gene order is oversize in the probe mark process, surpass more than 1000bp, we use the sequence of CDS and come designing probe, if the sequence of CDS also surpasses more than 1000bp, can adopt one section of the centre base sequence of gene to come synthesising probing needle, base sequence is no less than 500bp, will do sequential detection after probe is synthetic, and function be carried out the analysis of clinical meaning).
A preferred version of test kit of the present invention is that described marker is selected from radioactive substance, chemoluminescence or substance that show color, vitamin H, metal king crab, fluorescein, enzyme and nano material.
A preferred version of test kit of the present invention is also to comprise hybridization solution.
A preferred version of test kit of the present invention is also to comprise toughener.
A preferred version of test kit of the present invention is also to comprise developer.
Canceration of the present invention NOTCH1 gene screening in early stage test kit using value is, can be in the mRNA level, to hepatocarcinogenesis examination in early stage, and after canceration or postoperative recurrence, transfer, diffusion generation early warning, further coordinate clinical treatment.
The present invention also provides a kind of detection method of NOTCH1 gene hybridization in situ, comprises the following 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 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 specimen.More preferably that described blood preparation or other organ-tissue cell specimen are from liver cancer patient, liver cancer high risk population, Healthy People.
Detection method of the present invention, wherein preferably, described cancer high risk population, cancer build up a family fortune well family, liver cancer patient.
Detection kit of the present invention is to adopt nucleic acid hybridization technique and groupization immunization method to combine, take the one-level functional transcription product mRNA of NOTCH1 gene as detected object, synthesising probing needle is the RNA sequence of NOTCH1 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 sxemiquantitative or the quantitative expression deciding degree of NOTCH1 gene.Judge the expression amount of above gene according to immunohistochemical methods colour developing after hybridization, normal people NOTCH1 gene high expression, i.e. a large amount of colour developings, the NOTCH1 gene is in the low expression of cancer patient, high risk population's (hbv-liver cirrhosis) also has low expression in various degree, with the normal people, aobvious difference is arranged, the expression amount of this gene of liver cancer patient is all lower than normal people expression amount.
The component of diagnostic kit of the present invention is by hybridization probe, hybridization solution, developer, the compositions such as synergistic agent.The nucleic acid hybridization principle of this test kit is that 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, 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 NOTCH1 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 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, observe existence and the location of mRNA under light microscopic, cell count according to dyeing, the expression amount of judgement goal gene.
The inventive method is nucleic acid hybridization in situ technology commonly used at present, the method is by the NOTCH1 gene expression amount in the detection substrate cell, be used for determining the canceration mRNA variable quantity in early stage, whether the early warning hepatocarcinogenesis occurs and the whether prediction of Preventive of liver cancer patient postoperative.Because NOTCH1 gene high expression level in the normal people, if the NOTCH1 gene expression amount is low, the risk of suffering from cancer is described, illustrate that canceration occurs, or cancer patient postoperative Preventive, thereby the diagnostic message of acquisition cancer.A test kit can many person-portions use or person-portion use.
As mentioned above, during lower than normal control, measurable experimenter occurs for the hepatocarcinogenesis pathologic process when the expression amount that the NOTCH1 gene detected.
The present invention has following beneficial effect:
Clinical meaning of the present invention is to follow the tracks of more in early days the variation that detects NOTCH1 gene mRNA expression amount in hepatocarcinogenesis generation and Pathologic process, and early warning liver cancer occurs, development trend.Diagnostic kit of the present invention and other detection and cancer markers clinically, and the medical imaging inspection has aobvious difference.The present invention can be at the one-level function product mRNA of genetic transcription level detection NOTCH1 gene unconventionality expression, before the recurrence of occupancy carninomatosis kitchen range is not found in the medical imaging inspection, before the cancer biochemical indicator does not produce extremely, before also not forming tumour, can accomplish early the information acquisition of above abnormal gene expression, predict early for the rear transfer and relapse of real early diagnosis of clinical cancer sufferer and treatment.So just might implement early screening, early prevention, the early treatment of cancer, might thoroughly effect a radical cure the liver cancer foul disease from the source.
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 NOTCH1 gene hybridization in situ techniqueflow chart of the present invention.
Fig. 2 is that in the embodiment of the present invention, hepatocarcinoma patient NOTCH1 expresses the reduction picture.
Fig. 3 is high-order crowd (liver cirrhosis) picture.
Fig. 4 is that in the embodiment of the present invention, normal people NOTCH1 expresses picture.
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, and any pro forma change or accommodation will fall into protection scope of the present invention.
The in situ hybridization test kit for preparing the present embodiment according to ordinary method, this test kit comprise hybridization probe, marker, the specification sheets take the NOTCH1 gene as the testing goal gene design, wherein:
The probe mark thing of the 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/ pipes | 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 each organized the implementation process of blood preparation NOTCH1 gene expression amount:
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, then use 1 * damping fluid I to wash 5min 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 the above process of 5min(and all carried out at glass jar), the taking-up slide allows its seasoning;
4). slide is put into moisture preservation box, add prehybridization solution 25 μ L/ sheets (being added in the place of cell), covered covers tightly moisture preservation box, is placed in 42 ℃ of constant water bath box more than 3h;
5). take out slide, discard cover glass, slide is put into glass jar, the ethanol with 70%, 90%, 95% is respectively washed 2min, takes out seasoning;
6). slide is put into moisture preservation box, and one adds just hybridization solution 25 μ L/ sheets, and another adds antisense hybridization solution 25 μ L/ sheets, and covered covers tightly moisture preservation box, is placed on 16-24h in 42 ℃ of constant water bath box;
7). take out slide, discard cover glass, slide is put into glass jar:
Wash twice with 2 * damping fluid II in 42 ℃ of constant water bath box, each 15min;
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 in 42 ℃ of constant water bath box, 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, add wherein 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 18h;
14). wash 5min with tri-distilled water, seasoning, (adding 1 * damping fluid I mixing of 10% with glycerine) 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 observe existence and the location of mRNA under light microscopic, according to the cell count of dyeing, the expression amount of judgement goal gene.
20 of hepatocarcinoma patients, 20 of high risk population's (liver cirrhosis), 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.Result represents, all cancer patients NOTCH1 genetic expressions reduce, the shallow or dye-free of cell dyeing; High risk population and family members Shi Renyuan express and reduce, decimal dyeing; Normal group NOTCH1 genetic expression is high, and the most dyeing of cell concrete outcome is seen Fig. 2, Fig. 3, Fig. 4.
| The liver cancer number | Expression amount % | High-risk number (liver cirrhosis) | Expression amount % | Normal number | |
| 1 | 4 | 1 | 21 | 1 | 84 |
| 2 | 2 | 2 | 26 | 2 | 65 |
| 3 | 4 | 3 | 22 | 3 | 76 |
| 4 | 6 | 4 | 20 | 4 | 70 |
| 5 | 2 | 5 | 19 | 5 | 58 |
| 6 | 2 | 6 | 18 | 6 | 82 |
| 7 | 3 | 7 | 20 | 7 | 81 |
| 8 | 3 | 8 | 23 | 8 | 77 |
| 9 | 8 | 9 | 19 | 9 | 86 |
| 10 | 3 | 10 | 23 | 10 | 90 |
| 12 | 2 | 12 | 22 | 12 | 88 |
| 13 | 0 | 13 | 25 | 13 | 90 |
| 14 | 2 | 14 | 19 | 14 | 84 |
| 15 | 3 | 15 | 12 | 15 | 78 |
| 16 | 8 | 16 | 14 | 16 | 70 |
| 17 | 0 | 17 | 18 | 17 | 84 |
| 18 | 2 | 18 | 19 | 18 | 78 |
| 19 | 0 | 19 | 16 | 19 | 92 |
| 20 | 2 | 20 | 18 | 20 | 86 |
SEQ ID NO.1(probe sequence)
2001 gccgggctac acagggagca tgtgtaacat caacatcgat
2041 gagtgtgcgg gcaacccctg ccacaacggg ggcacctgcg aggacggcat caatggcttc
2101 acctgccgct gccccgaggg ctaccacgac cccacctgcc tgtctgaggt caatgagtgc
2161 aacagcaacc cctgcgtcca cggggcctgc cgggacagcc tcaacgggta caagtgcgac
2221 tgtgaccctg ggtggagtgg gaccaactgt gacatcaaca acaatgagtg tgaatccaac
2281 ccttgtgtca acggcggcac ctgcaaagac atgaccagtg gctacgtgtg cacctgccgg
2341 gagggcttca gcggtcccaa ctgccagacc aacatcaacg agtgtgcgtc caacccatgt
2401 ctgaaccagg gcacgtgtat tgacgacgtt gccgggtaca agtgcaactg cctgctgccc
2461 tacacaggtg ccacgtgtga ggtggtgctg gccccgtgtg cccccagccc ctgcagaaac
2521 ggcggggagt gcaggcaatc cgaggactat gagagcttct cctgtgtctg ccccacgggc
2581 tggcaagggc agacctgtga ggtcgacatc aacgagtgcg ttctgagccc gtgccggcac
2641 ggcgcatcct gccagaacac ccacggcggc taccgctgcc actgccaggc cggctacagt
2701 gggcgcaact gcgagaccga catcgacgac tgccggccca acccgtgtca caacgggggc
2761 tcctgcacag acggcatcaa cacggccttc tgcgactgcc
SEQ ID NO.2 NOTCH1 gene order and number: NM_017617
1 atgccgccgc tcctggcgcc cctgctctgc ctggcgctgc tgcccgcgct cgccgcacga
61 ggcccgcgat gctcccagcc cggtgagacc tgcctgaatg gcgggaagtg tgaagcggcc
121 aatggcacgg aggcctgcgt ctgtggcggg gccttcgtgg gcccgcgatg ccaggacccc
181 aacccgtgcc tcagcacccc ctgcaagaac gccgggacat gccacgtggt ggaccgcaga
241 ggcgtggcag actatgcctg cagctgtgcc ctgggcttct ctgggcccct ctgcctgaca
301 cccctggaca atgcctgcct caccaacccc tgccgcaacg ggggcacctg cgacctgctc
361 acgctgacgg agtacaagtg ccgctgcccg cccggctggt cagggaaatc gtgccagcag
421 gctgacccgt gcgcctccaa cccctgcgcc aacggtggcc agtgcctgcc cttcgaggcc
481 tcctacatct gccactgccc acccagcttc catggcccca cctgccggca ggatgtcaac
541 gagtgtggcc agaagcccgg gctttgccgc cacggaggca cctgccacaa cgaggtcggc
601 tcctaccgct gcgtctgccg cgccacccac actggcccca actgcgagcg gccctacgtg
661 ccctgcagcc cctcgccctg ccagaacggg ggcacctgcc gccccacggg cgacgtcacc
721 cacgagtgtg cctgcctgcc aggcttcacc ggccagaact gtgaggaaaa tatcgacgat
781 tgtccaggaa acaactgcaa gaacgggggt gcctgtgtgg acggcgtgaa cacctacaac
841 tgccgctgcc cgccagagtg gacaggtcag tactgtaccg aggatgtgga cgagtgccag
901 ctgatgccaa atgcctgcca gaacggcggg acctgccaca acacccacgg tggctacaac
961 tgcgtgtgtg tcaacggctg gactggtgag gactgcagcg agaacattga tgactgtgcc
1021 agcgccgcct gcttccacgg cgccacctgc catgaccgtg tggcctcctt ctactgcgag
1081 tgtccccatg gccgcacagg tctgctgtgc cacctcaacg acgcatgcat cagcaacccc
1141 tgtaacgagg gctccaactg cgacaccaac cctgtcaatg gcaaggccat ctgcacctgc
1201 ccctcggggt acacgggccc ggcctgcagc caggacgtgg atgagtgctc gctgggtgcc
1261 aacccctgcg agcatgcggg caagtgcatc aacacgctgg gctccttcga gtgccagtgt
1321 ctgcagggct acacgggccc ccgatgcgag atcgacgtca acgagtgcgt ctcgaacccg
1381 tgccagaacg acgccacctg cctggaccag attggggagt tccagtgcat ctgcatgccc
1441 ggctacgagg gtgtgcactg cgaggtcaac acagacgagt gtgccagcag cccctgcctg
1501 cacaatggcc gctgcctgga caagatcaat gagttccagt gcgagtgccc cacgggcttc
1561 actgggcatc tgtgccagta cgatgtggac gagtgtgcca gcaccccctg caagaatggt
1621 gccaagtgcc tggacggacc caacacttac acctgtgtgt gcacggaagg gtacacgggg
1681 acgcactgcg aggtggacat cgatgagtgc gaccccgacc cctgccacta cggctcctgc
1741 aaggacggcg tcgccacctt cacctgcctc tgccgcccag gctacacggg ccaccactgc
1801 gagaccaaca tcaacgagtg ctccagccag ccctgccgcc acgggggcac ctgccaggac
1861 cgcgacaacg cctacctctg cttctgcctg aaggggacca caggacccaa ctgcgagatc
1921 aacctggatg actgtgccag cagcccctgc gactcgggca cctgtctgga caagatcgat
1981 ggctacgagt gtgcctgtga gccgggctac acagggagca tgtgtaacat caacatcgat
2041 gagtgtgcgg gcaacccctg ccacaacggg ggcacctgcg aggacggcat caatggcttc
2101 acctgccgct gccccgaggg ctaccacgac cccacctgcc tgtctgaggt caatgagtgc
2161 aacagcaacc cctgcgtcca cggggcctgc cgggacagcc tcaacgggta caagtgcgac
2221 tgtgaccctg ggtggagtgg gaccaactgt gacatcaaca acaatgagtg tgaatccaac
2281 ccttgtgtca acggcggcac ctgcaaagac atgaccagtg gctacgtgtg cacctgccgg
2341 gagggcttca gcggtcccaa ctgccagacc aacatcaacg agtgtgcgtc caacccatgt
2401 ctgaaccagg gcacgtgtat tgacgacgtt gccgggtaca agtgcaactg cctgctgccc
2461 tacacaggtg ccacgtgtga ggtggtgctg gccccgtgtg cccccagccc ctgcagaaac
2521 ggcggggagt gcaggcaatc cgaggactat gagagcttct cctgtgtctg ccccacgggc
2581 tggcaagggc agacctgtga ggtcgacatc aacgagtgcg ttctgagccc gtgccggcac
2641 ggcgcatcct gccagaacac ccacggcggc taccgctgcc actgccaggc cggctacagt
2701 gggcgcaact gcgagaccga catcgacgac tgccggccca acccgtgtca caacgggggc
2761 tcctgcacag acggcatcaa cacggccttc tgcgactgcc tgcccggctt ccggggcact
2821 ttctgtgagg aggacatcaa cgagtgtgcc agtgacccct gccgcaacgg ggccaactgc
2881 acggactgcg tggacagcta cacgtgcacc tgccccgcag gcttcagcgg gatccactgt
2941 gagaacaaca cgcctgactg cacagagagc tcctgcttca acggtggcac ctgcgtggac
3001 ggcatcaact cgttcacctg cctgtgtcca cccggcttca cgggcagcta ctgccagcac
3061 gatgtcaatg agtgcgactc acagccctgc ctgcatggcg gcacctgtca ggacggctgc
3121 ggctcctaca ggtgcacctg cccccagggc tacactggcc ccaactgcca gaaccttgtg
3181 cactggtgtg actcctcgcc ctgcaagaac ggcggcaaat gctggcagac ccacacccag
3241 taccgctgcg agtgccccag cggctggacc ggcctttact gcgacgtgcc cagcgtgtcc
3301 tgtgaggtgg ctgcgcagcg acaaggtgtt gacgttgccc gcctgtgcca gcatggaggg
3361 ctctgtgtgg acgcgggcaa cacgcaccac tgccgctgcc aggcgggcta cacaggcagc
3421 tactgtgagg acctggtgga cgagtgctca cccagcccct gccagaacgg ggccacctgc
3481 acggactacc tgggcggcta ctcctgcaag tgcgtggccg gctaccacgg ggtgaactgc
3541 tctgaggaga tcgacgagtg cctctcccac ccctgccaga acgggggcac ctgcctcgac
3601 ctccccaaca cctacaagtg ctcctgccca cggggcactc agggtgtgca ctgtgagatc
3661 aacgtggacg actgcaatcc ccccgttgac cccgtgtccc ggagccccaa gtgctttaac
3721 aacggcacct gcgtggacca ggtgggcggc tacagctgca cctgcccgcc gggcttcgtg
3781 ggtgagcgct gtgaggggga tgtcaacgag tgcctgtcca atccctgcga cgcccgtggc
3841 acccagaact gcgtgcagcg cgtcaatgac ttccactgcg agtgccgtgc tggtcacacc
3901 gggcgccgct gcgagtccgt catcaatggc tgcaaaggca agccctgcaa gaatgggggc
3961 acctgcgccg tggcctccaa caccgcccgc gggttcatct gcaagtgccc tgcgggcttc
4021 gagggcgcca cgtgtgagaa tgacgctcgt acctgcggca gcctgcgctg cctcaacggc
4081 ggcacatgca tctccggccc gcgcagcccc acctgcctgt gcctgggccc cttcacgggc
4141 cccgaatgcc agttcccggc cagcagcccc tgcctgggcg gcaacccctg ctacaaccag
4201 gggacctgtg agcccacatc cgagagcccc ttctaccgtt gcctgtgccc cgccaaattc
4261 aacgggctct tgtgccacat cctggactac agcttcgggg gtggggccgg gcgcgacatc
4321 cccccgccgc tgatcgagga ggcgtgcgag ctgcccgagt gccaggagga cgcgggcaac
4381 aaggtctgca gcctgcagtg caacaaccac gcgtgcggct gggacggcgg tgactgctcc
4441 ctcaacttca atgacccctg gaagaactgc acgcagtctc tgcagtgctg gaagtacttc
4501 agtgacggcc actgtgacag ccagtgcaac tcagccggct gcctcttcga cggctttgac
4561 tgccagcgtg cggaaggcca gtgcaacccc ctgtacgacc agtactgcaa ggaccacttc
4621 agcgacgggc actgcgacca gggctgcaac agcgcggagt gcgagtggga cgggctggac
4681 tgtgcggagc atgtacccga gaggctggcg gccggcacgc tggtggtggt ggtgctgatg
4741 ccgccggagc agctgcgcaa cagctccttc cacttcctgc gggagctcag ccgcgtgctg
4801 cacaccaacg tggtcttcaa gcgtgacgca cacggccagc agatgatctt cccctactac
4861 ggccgcgagg aggagctgcg caagcacccc atcaagcgtg ccgccgaggg ctgggccgca
4921 cctgacgccc tgctgggcca ggtgaaggcc tcgctgctcc ctggtggcag cgagggtggg
4981 cggcggcgga gggagctgga ccccatggac gtccgcggct ccatcgtcta cctggagatt
5041 gacaaccggc agtgtgtgca ggcctcctcg cagtgcttcc agagtgccac cgacgtggcc
5101 gcattcctgg gagcgctcgc ctcgctgggc agcctcaaca tcccctacaa gatcgaggcc
5161 gtgcagagtg agaccgtgga gccgcccccg ccggcgcagc tgcacttcat gtacgtggcg
5221 gcggccgcct ttgtgcttct gttcttcgtg ggctgcgggg tgctgctgtc ccgcaagcgc
5281 cggcggcagc atggccagct ctggttccct gagggcttca aagtgtctga ggccagcaag
5341 aagaagcggc gggagcccct cggcgaggac tccgtgggcc tcaagcccct gaagaacgct
5401 tcagacggtg ccctcatgga cgacaaccag aatgagtggg gggacgagga cctggagacc
5461 aagaagttcc ggttcgagga gcccgtggtt ctgcctgacc tggacgacca gacagaccac
5521 cggcagtgga ctcagcagca cctggatgcc gctgacctgc gcatgtctgc catggccccc
5581 acaccgcccc agggtgaggt tgacgccgac tgcatggacg tcaatgtccg cgggcctgat
5641 ggcttcaccc cgctcatgat cgcctcctgc agcgggggcg gcctggagac gggcaacagc
5701 gaggaagagg aggacgcgcc ggccgtcatc tccgacttca tctaccaggg cgccagcctg
5761 cacaaccaga cagaccgcac gggcgagacc gccttgcacc tggccgcccg ctactcacgc
5821 tctgatgccg ccaagcgcct gctggaggcc agcgcagatg ccaacatcca ggacaacatg
5881 ggccgcaccc cgctgcatgc ggctgtgtct gccgacgcac aaggtgtctt ccagatcctg
5941 atccggaacc gagccacaga cctggatgcc cgcatgcatg atggcacgac gccactgatc
6001 ctggctgccc gcctggccgt ggagggcatg ctggaggacc tcatcaactc acacgccgac
6061 gtcaacgccg tagatgacct gggcaagtcc gccctgcact gggccgccgc cgtgaacaat
6121 gtggatgccg cagttgtgct cctgaagaac ggggctaaca aagatatgca gaacaacagg
6181 gaggagacac ccctgtttct ggccgcccgg gagggcagct acgagaccgc caaggtgctg
6241 ctggaccact ttgccaaccg ggacatcacg gatcatatgg accgcctgcc gcgcgacatc
6301 gcacaggagc gcatgcatca cgacatcgtg aggctgctgg acgagtacaa cctggtgcgc
6361 agcccgcagc tgcacggagc cccgctgggg ggcacgccca ccctgtcgcc cccgctctgc
6421 tcgcccaacg gctacctggg cagcctcaag cccggcgtgc agggcaagaa ggtccgcaag
6481 cccagcagca aaggcctggc ctgtggaagc aaggaggcca aggacctcaa ggcacggagg
6541 aagaagtccc aggacggcaa gggctgcctg ctggacagct ccggcatgct ctcgcccgtg
6601 gactccctgg agtcacccca tggctacctg tcagacgtgg cctcgccgcc actgctgccc
6661 tccccgttcc agcagtctcc gtccgtgccc ctcaaccacc tgcctgggat gcccgacacc
6721 cacctgggca tcgggcacct gaacgtggcg gccaagcccg agatggcggc gctgggtggg
6781 ggcggccggc tggcctttga gactggccca cctcgtctct cccacctgcc tgtggcctct
6841 ggcaccagca ccgtcctggg ctccagcagc ggaggggccc tgaatttcac tgtgggcggg
6901 tccaccagtt tgaatggtca atgcgagtgg ctgtcccggc tgcagagcgg catggtgccg
6961 aaccaataca accctctgcg ggggagtgtg gcaccaggcc ccctgagcac acaggccccc
7021 tccctgcagc atggcatggt aggcccgctg cacagtagcc ttgctgccag cgccctgtcc
7081 cagatgatga gctaccaggg cctgcccagc acccggctgg ccacccagcc tcacctggtg
7141 cagacccagc aggtgcagcc acaaaactta cagatgcagc agcagaacct gcagccagca
7201 aacatccagc agcagcaaag cctgcagccg ccaccaccac caccacagcc gcaccttggc
7261 gtgagctcag cagccagcgg ccacctgggc cggagcttcc tgagtggaga gccgagccag
7321 gcagacgtgc agccactggg ccccagcagc ctggcggtgc acactattct gccccaggag
7381 agccccgccc tgcccacgtc gctgccatcc tcgctggtcc cacccgtgac cgcagcccag
7441 ttcctgacgc ccccctcgca gcacagctac tcctcgcctg tggacaacac ccccagccac
7501 cagctacagg tgcctgagca ccccttcctc accccgtccc ctgagtcccc tgaccagtgg
7561 tccagctcgt ccccgcattc caacgtctcc gactggtccg agggcgtctc cagccctccc
7621 accagcatgc agtcccagat cgcccgcatt ccggaggcct tcaagtaaac ggcgcgcccc
7681 acgagacccc ggcttccttt cccaagcctt cgggcgtctg tgtgcgctct gtggatgcca
7741 gggccgacca gaggagcctt tttaaaacac atgtttttat acaaaataag aacgaggatt
7801 ttaatttttt ttagtattta tttatgtact tttattttac acagaaacac tgccttttta
7861 tttatatgta ctgttttatc tggccccagg tagaaacttt tatctattct gagaaaacaa
7921 gcaagttctg agagccaggg ttttcctacg taggatgaaa agattcttct gtgtttataa
7981 aatataaaca aagattcatg atttataaat gccatttatt tattgattcc ttttttcaaa
8041 atccaaaaag aaatgatgtt ggagaaggga agttgaacga gcatagtcca aaaagctcct
8101 ggggcgtcca ggccgcgccc tttccccgac gcccacccaa ccccaagcca gcccggccgc
8161 tccaccagca tcacctgcct gttaggagaa gctgcatcca gaggcaaacg gaggcaaagc
8221 tggctcacct tccgcacgcg gattaatttg catctgaaat aggaaacaag tgaaagcata
8281 tgggttagat gttgccatgt gttttagatg gtttcttgca agcatgcttg tgaaaatgtg
8341 ttctcggagt gtgtatgcca agagtgcacc catggtacca atcatgaatc tttgtttcag
8401 gttcagtatt atgtagttgt tcgttggtta tacaagttct tggtccctcc agaaccaccc
8461 cggccccctg cccgttcttg aaatgtaggc atcatgcatg tcaaacatga gatgtgtgga
8521 ctgtggcact tgcctgggtc acacacggag gcatcctacc cttttctggg gaaagacact
8581 gcctgggctg accccggtgg cggccccagc acctcagcct gcacagtgtc ccccaggttc
8641 cgaagaagat gctccagcaa cacagcctgg gccccagctc gcgggacccg accccccgtg
8701 ggctcccgtg ttttgtagga gacttgccag agccgggcac attgagctgt gcaacgccgt
8761 gggctgcgtc ctttggtcct gtccccgcag ccctggcagg gggcatgcgg tcgggcaggg
8821 gctggaggga ggcgggggct gcccttgggc cacccctcct agtttgggag gagcagattt
8881 ttgcaatacc aagtatagcc tatggcagaa aaaatgtctg taaatatgtt tttaaaggtg
8941 gattttgttt aaaaaatctt aatgaatgag tctgttgtgt gtcatgccag tgagggacgt
9001 cagacttggc tcagctcggg gagccttagc cgcccatgca ctggggacgc tccgctgccg
9061 tgccgcctgc actcctcagg gcagcctccc ccggctctac gggggccgcg tggtgccatc
9121 cccagggggc atgaccagat gcgtcccaag atgttgattt ttactgtgtt ttataaaata
9181 gagtgtagtt tacagaaaaa gactttaaaa gtgatctaca tgaggaactg tagatgatgt
9241 atttttttca tcttttttgt taactgattt gcaataaaaa tgatactgat ggtgaaaaaa
9301 aaaaaaaaa
Claims (4)
1. a hybridization in situ detection kit, comprise hybridization probe and marker, it is characterized in that, described hybridization probe is the sequence shown in 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 developer.
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