CN1083113A - The rapid detection of biological polymer in the stained specimens - Google Patents

Abstract

The invention provides the method that adopts novel hybridization in situ technique to detect the existence of the biological polymer in prestained sample.But the biological polymer of this method analysis of cells, especially RNA and DNA.Novel in situ hybridization program can be finished by a step or two steps (fix afterwards earlier and hybridize).The inventive method can be used to detect the existence of microorganism (as bacterium, virus, fungi) or cytogene (as oncogene, tumor-inhibiting factor or growth-stimulating factor) in prestained cell or tissue.

Description

The rapid detection of biological polymer in the stained specimens

The present invention relates to the in situ hybridization field.More specifically, relating to the detection of use in situ hybridization analysis means lacks to the biological polymer (biopolymer) of the cell of 1 copy in a staining cell.

In situ hybridization provides the technology of the biological polymer such as nucleic acid (DNA and RNA) and protein in unicellular level evaluation and quantitative tissue.This hybridization technique can detect the existence or the disappearance of specific gene in the tissue on unicellular level.The in situ hybridization program can also be used in the expression of unicellular level detection gene product.

By using special nucleic acid (RNA or DNA) probe, can detect that infect and the genetic marker other diseases state.The feature of some genetic diseases is to exist some in normal cell and non-existent gene.The feature of some other paathogenic factor is the translation product (that is, polypeptide or protein) of expressing some DNA that do not express or RNA in normal cell.The feature of some morbid state is certain gene of disappearance or gene fragment, or the disappearance of the expression of gene product or protein or change.

Prior art can detect these signs, but time-consuming relatively and sensitivity is limited.The basis of hybridization technique is the pairing of single stranded DNA or RNA and complementary nucleic acid chains (i.e. hybridization) ability.This hybridization makes the development of specific probe can differentiate special gene (DNA), or the existence of polynucleotide order, perhaps identifies these gene transcription (mRNA) and expression.

The solution hybridization method need to destroy cell and also isolates nucleic acid from cell before carrying out hybridization, thereby sacrificed the three-dimensional resolving power and the sensitivity of cell integrity, detection.In situ hybridization can detect RNA or DNA sequence in individual cells.In situ hybridization around getting rid of with RNA other cell external sources and DNA to particular target gene, nucleic acid or proteinic diluting effect, thereby have sensitivity than solution hybridization Fa Genggao.In situ hybridization can also detect multiple material simultaneously, i.e. gene in individual cells, nucleic acid or protein, thus can identify the cell of specific express cell gene or virus order.In addition, because the in situ hybridization analysis can operate and quantitative analysis unicellular, so only need MIN sample and reagent.

Before the present invention, the in situ hybridization program can only detect in each cell greater than 10 nucleic acid that copy.Such program needed operate to the time above 14 days at least 8 hours.In situ hybridization program in the past can not be quantitative, can not carry out multiple detection simultaneously.Nearest for some time, at two pendent United States Patent (USP) SN.784,690(1991 applied for October 28) and SN.668,751(1991 applied for March 13) in disclosed in situ hybridization program, with time decreased to being less than 4 hours, and sensitivity brought up to single each cell of copy.

The laboratory method of traditional discriminating bacterium relies on the large program and uses easy or special microbiological assay (as Gram dyeing) very much, in the growth of multiple common (or special) substrate and use specific biochemical test subsequently.In recent years, a megatrend that occurs in the microbial taxonomy is, uses direct characterization of molecules, i.e. DNA based composition, and DNA/DNA hybridization, gene sequencing etc. are come defining classification unit.These methods make the research of bacterium evolution aspect that change take place, still, and in given environment or sample, identifying the particular organisms contribution seldom.Therefore, separate pure growth and carry out the method that culture is identified, remain unchanged substantially with traditional program.

Having begun to attempt development Rapid identification method of microorganism recently learns.One of them makes and is to use the dna probe special to the 16SrRNA of biology.But, there is the defective on the method in prior art, there is not the sensitivity of enough detection 16S DNA, promptly detect few level (S.Jinks-Robertson et al. to 7 copy 16S DNA, In Cellular and Molecular Biology, 2: 1367-1385(1987)).Use the advantage of 16S DNA to be now to have a large amount of base sequence data messages.About 1000 kinds of 16SDNA order has been made into file and has been used for the research that bacterial system takes place.These base data have comprised a large amount of bacteriums data of (comprising pathogenic agent).From technical prospect, the fragment of 16S DNA can be as molecular probe, and the species that are used for any bacterium are identified, no matter are that live, quick growth, still organism dead, dormancy.

Used a large amount of different dyeing processs in cytology and histological sample, for example, but Pan Pani Laplace dyeing (papanicolaou stain) is a kind of staining that is used for the cell that obtains from cervical samples (cervical specimen).Usually, only prepare a microscope slide and with its dyeing from a patient.If, for example, on painted microscope slide, there are some to be suspect to be the cell that human papillomavirus (HPV) infects, prior art does not provide a kind of removal cover glass (coverslip) and analyzes the method for these suspicious cells with in-situ hybridization method.Correspondingly, must obtain extra sample, but in sampling subsequently, exist potential to miss the possibility of focus.

Also there is defective in prior art, promptly the in situ hybridization program can not be used for detecting DNA or the mRNA or the cytogene of stained specimens bacterium or virus.Using the form that hybridization in situ technique detects DNA or RNA and mRNA, identify bacterium or virus strains and cytogene then in painted histology or cytological sample, is the technical problem that this technical field midium or long term has pressed for and expected to solve.

In concrete an enforcement of the present invention, provide a kind of detection method that biological polymer exists in having the prestain sample of basic intact cell film.This " two steps " method has comprised the existence of measuring the biological polymer of cell.At first, sample can be by adding heat setting or fixing with the medium that contains certain precipitation agent and/or linking agent.Subsequently, the fixed cell contacts with hybridization solution.Hybridization solution contains denaturing agent, the hybrid stability agent, and buffer reagent, selective membrane pore former and at least a probe, probe have and the basic at least complementary nucleotide sequence of detected specific target nucleotide.Then, cell sample is incubated with hybridization solution in the presence of at least a detectable marker.This method can detect few to each cell of single biopolymer.

In another embodiment of the present invention, a kind of " step " method is provided, detect the existence of the biological polymer in the prestained sample with basic intact cell film.This method has comprised the existence of measuring the biological polymer of cell.At first, sample with contain denaturing agent, the hybrid stability agent, buffer reagent, the medium of film pore former and at least a probe contacts.Best, medium contains fixing agent.Sample contacts under hybridization conditions.Subsequently, cell sample is incubated with medium in the presence of at least a detectable marker.Detect duplex or triplex by the certification mark thing, and be not required to be the non-specific combination of blocking-up probe and carry out the prehybridization step, enter thereby be convenient to probe.This method can detect the target biological polymer of the every cell of single copy.

In another embodiment of the present invention, provide one to be used for determining the test kit (kit) that whether exists at prestained sample biological polymer with basic intact cell film.This test kit can be used to measure the biological polymer of cell.Test kit contains hybridization solution, and this hybridization solution contains denaturing agent, the hybrid stability agent, and buffer reagent and film pore former preferably also contain fixing agent.Test kit can also at random contain a kind of probe of selection, thereby this probe can form hybridization complex with suspicious biological polymer hybridization.In addition, thus this test kit can contain the equipment that described suspicious specimen is contacted the probe of the equipment that forms hybridization complex and measuring mark with probe.

But Fig. 1 has shown in using the painted cervical cell of Pan Pani Laplace staining in advance, detects the result of HPC.

But Fig. 2 has shown in using the painted cell of Pan Pani Laplace staining in advance, detects and distinguish the result of 16 types and 18 type HPV simultaneously.

Fig. 3 has shown in using the painted cervical tissue of phenodin and eosin in advance, detects the result of HPV.

Fig. 4 has shown in using the painted cell of Diff-Quick method in advance, detects the result of chromosomal DNA.

New method of the present invention can be for detection of a large amount of different microorganisms, and these microorganisms cause the reason of numerous disease physiological status just. For example, method of the present invention can be used for detecting and cause such as septicemia, sexually transmitted disease, the microorganism of the disease physiological status of diarrhoea and breathing problem and so on.

New method of the present invention can be used for detecting a large amount of different bacterium, virus and fungies. The representational example of the bacterium that can be detected by the inventive method comprises: streptococcus (Strepococ-cus), staphylococcus (Staphylococcus), Clostridium (Clostridium), Bacillus (Bacillus), pseudomonas (Pseudomonas), Salmonella (Salmonella), klebsiella (Kleb-siella), class (plan) Bacillus (Bacteroides), Escherichia coli (Escherichia coli), Neisseria gonorrhoeae (Neisseria gonrrhea) and chlamydiaceae (Chlamydia). The representational example of fungi comprises reads Coccus (Candida), Cryptococcus neoformans (Cryptococ-cus neformans), Blastomyces dermatitidis (Blastomyces dermatitides), Histoplasma capsulatum (Histoplasma capsulatum), posadasis spheriforme (Coccidioides immi-tis) and Paracoccidioides brasiliensis (Paracoccidioides brasiliensis). The representational example of detectable virus comprises: HPV, herpes simplex virus type 2, hepatitis viruse, HIV, influenza virus, parainfluenza virus and rotavirus.

Spirochetal representational example comprises: Spirochaeta pallida (Treponema pal-lidum), Borrelia burgdoyferi (Borrelia burgdorferi) and Leptospira (Lep-tospira). Protozoic representational example comprises: entamoeba historlytica (Entamoe-ba histolytic), colon basket worm (Balantidium coli), giardia lamblia (Giardia lambli-a), Leishmania tropica, Plasmodium (Plasmodium), the species of trichomonas vaginalis (Tri-chomonas vaginalis) and Trypanosoma (Trypanosoma).

Except detecting microorganism, the inventive method also be used for detecting with quantitative prestained Mammals sample in cytogene.The representational example of such cytogene comprises: oncogene, tumor suppressor gene and growth-stimulating factor gene.Preferably, such oncogene is neu(c-erb-B-2), c-ras, c-myc, and c-myb.Equally, the inventive method can be used for detecting and quantitative tumor suppressor gene and stimulating growth factor gene.The example of tumor suppressor gene comprises P53 and retinoblastoma gene.The representational example of somatomedin comprises: G CFS-granulocyte/scavenger cell (CSF-GM), transforming growth factor (TGF-α) and Urogastron (EGF).Those skilled in the art will appreciate that, other cell biological polymkeric substance adopts the inventive method to detect from prestained sample and in addition quantitative.

Any is arranged is special expectation, and promptly a large amount of different cell sample and tissue samples can be used for technology described in the invention.These cell samples comprise following representational example: cervical cell, medullary cell, liver cell, celiolymph cell, hemocyte, oral mucosa cell, pneumonocyte and skin cells.The representational example of tissue sample comprises: lymph node tissue, breast tissue, cervical tissue, colon, prostatitis (gland) tissue, heart tissue and cerebral tissue.In addition, can also detect body fluid and secretory product, as urine, ight soil, phlegm.

The cell and the tissue sample that are used for the inventive method can be painted with any staining commonly used in cytology or the histology.But the example of representational staining comprises Pan Pani Laplace staining, Wright staining and phenodin/eosin staining, and Diff-Quick staining.

Make the cell/tissue sample

The first step of in situ hybridization program can be being deposited on the solid carrier sample.Constituting of sample contains cell or part cell, perhaps arbitrary material of being made up of cell or part cell.Cell can be that live or dead, as long as the target biological polymer, i.e. DNA or mRNA change basically or do not destroy, can be detected.Sample must contain complete substantially cytolemma.Though must all membrane structures not be kept perfectly, thereby film must fully be preserved and can keep target biological polymer and detection probes can be introduced the biological polymer site.

The type that sample deposition is depended on cell or tissue in the technology of solid aunt loading.They comprise, for example, and the cell centrifugation of the standard section of tissue or smear or single-cell suspension liquid.

The solid carrier of many types can both be used for the present invention.The carrier that can use includes but is not limited to: glass, Scotland band (Scotch tape) (3M), nylon, Gene Screen Plus(New England Nuclear) and cellulose nitrate.Best is microslide.Using these carriers and the program of deposited samples in the above, is clearly for those skilled in the art.The material of selection carrier depends on the program and the employed quant program of observation of cell.Some filtering material and non-homogeneous thickness, therefore, pucker ﹠ bloat is inhomogeneous in the hybridization program in position.In addition, the carrier of some autofluorescence can disturb determining of low-level fluorescence.As the solid carrier, microslide is most preferred, because it has high signal/noise ratio and can treatedly keep tissue better.

Cell preparation in the one step method

In a step in situ hybridization program of the present invention, tissue sample is broken into single-cell suspension liquid with physics, chemistry or enzyme process.

Single solution is added on cell/tissue (hereinafter referred is made " sample ").Solution contains following: a kind of gentle fixing agent that can not add that adds, a kind of chaotropic reagent (chaotrope) or other denaturing agent, a kind of synthetic oligonucleotide probe (RNA of mark or dna probe in advance) and/or antibody probe, salt, washing agent, buffer reagent and encapsulant (blocking agent).Insulation in this solution was carried out 5-30 minute at 40 ℃-60 ℃.

In a step method program, and though sample be suspend or on the solid carrier, the hybridization program is carried out with single hybridization solution.The fixing agent that at random, can contain a kind of gentleness in the solution of same energy fixed cell.Immobilization is to finish in the solution same with hybridization and with hybridization.Fixing agent is a kind of found to the analyzed optimal fixing agent of specific cell type (for example, bone marrow and peripheral blood have a kind of optimal fixing agent, contain countless distinct cell types although be somebody's turn to do " tissue ").Fixing agent can be the combination of precipitation fixing agent (as ethanol) and cross linking fixative (as aldehyde), the wherein concentration of cross linking fixative very low (being less than 10%).The concentration and the type of precipitation fixing agent and cross linking fixative are variable, depend on the tight degree (strin-gency) and the desired hybridization temperature of probe and needed probe.

The selection of fixing agent and immobilization program can influence cellular constituent and cellular form; Such influence can be tissue-specific.Preferably, the precipitation fixing agent has following feature: by precipitating action fixed cell composition; The result is a reversible, and cellular form is retained, and the antigenicity of desired cellular constituent is retained, and nucleic acid is retained in the appropriate location in the cell; Can not be formed bifilar or three strands of hybridization chains to such an extent as to nucleic acid does not modify; To such an extent as to the target sequence hybridization that cellular constituent is not affected and suppresses nucleic acid and all existence.Preferably, being used for fixing agent of the present invention is selected from as follows: acetate, ethanol-acetate, methyl alcohol and methyl alcohol-acetone.The fixing agent of implementing the best of a step procedure comprises 10-40% ethanol, 10-40% methyl alcohol, 10-40% acetone or its combination.These fixing agents can keep cellular form well, and protection antigen is also kept its acceptability (accessibility) and had high hybridization efficiency.Typically, solution contains 1-40% ethanol and 5% formalin.

Fixed cell/tissue in two step methods

After staying in solution in the solid carrier or with them sample deposition, fixed sample.Fixing agent is selected from: precipitate fixing agent or cross linking fixative or their combination arbitrarily; Fixing agent can be the aqueous solution or non-aqueous solution.Fixing agent is selected from the group that following material is formed: formaldehyde solution, ethanol, salts solution, mercury chloride, sodium-chlor, sodium sulfate, potassium bichromate, potassiumphosphate, brometo de amonio, calcium chloride, sodium acetate, lithium chloride, cesium acetate, lime acetate or magnesium acetate, saltpetre, potassium bichromate, Sodium chromate, sodium iodide, sodium iodate, Sulfothiorine, picric acid, acetate, paraformaldehyde, sodium hydroxide, acetone, chloroform, glycerine and thymol.

Preferably, fixing agent contains by precipitating action with cellular constituent fixed reagent and have following feature: the result is a reversible, and cellular form is retained, and the antigenicity of desired cellular constituent is retained, and nucleic acid is retained in the appropriate location in the cell; To such an extent as to being modified, nucleic acid can not form hybridization chains bifilar or three strands; To such an extent as to cellular constituent is not affected the target sequence hybridization that suppresses nucleic acid and all existence.Preferably, be used for fixing agent of the present invention and be selected from the group that following material is formed: acetate, ethanol-acetate, methyl alcohol and methyl alcohol-acetone, these fixing agents can be satisfied with hybridization efficiently and protect cellular form well.

Fixing agent simultaneously can contain a kind of compound, and this compound is by with the crosslinked together and fixed cell component of these materials (as glutaraldehyde or formaldehyde).Linking agent " glues " generally more, and causes cell and film component to be fixed or to cling, thereby has kept the feature of above mentioned fixing agent.During use, linking agent is most preferably less than the 10%(volume/volume, V/V).

Linking agent is in protection during ultrastructure, usually because form reticulation with nucleic acid and wherein antigen coated, causes them can not be approaching for probe and antibody, thereby fallen closely the efficient of hybridization.Some linking agent is covalent modification nucleic acid also, thereby stoped the formation of later hybridization chain.The example of linking agent comprises paraformaldehyde, formaldehyde, the storage of dimethylsilserimidate and ethyl dimethylamino-propyl carbodiimide diimine (ethyldimethylamino-propylcarbodimide) cell/tissue

After fixing, slide and other solid carriers that contain sample can at room temperature be preserved 6 months through dry air, can preserve in cold (4 ℃) 70% aqueous ethanolic solution 6-12 month.If sample is handled under the condition of no RNA enzyme, sample can dewater in gradient ethanol and at room temperature preserve at least 12 months so.

Prehybridization is handled

According to the present invention, without any need for formal prehybridization step.Block the non-specific combination of probe and make things convenient for entering of probe in hybridization solution, to finish.If want to do of short duration hybridization (being less than 30 minutes), microscope slide can be heated to hybridization temperature in advance before adding hybridization solution so.

Hybridization

Nucleic acid hybridization is such process, wherein two or more existence or synthetic naturally DNA, RNA, dual or three remirrors or the opposite strand of oligonucleotide multimer Nucleotide or their any combination are discerned mutually, and by forming the spontaneous or inductive chemical bond of certain form, be generally hydrogen bond, thereby combine.The bonded degree can be controlled, and depends on the type of the nucleic acid that converges, by " correctly " bonded program of the nucleic acid decision that converges normally and by normal " correctly " bonded program that forms chemical bond and the decision of paired chemical rule.For example, if two chains are in chain length, formed 9 correct pairings at 10 o'clock in 10, this combination is said to be tool 90% homology so.

The nucleus order can detect with the method for molecular hybridization.Probe in some way " mark " thus allow to detect any complementary nucleus order that in individual cells, exists.

In the present invention, term " hybridization " means the combination of antibody to target antigen equally.

In a step crossover program, the hybridization mixing solutions contains denaturing agent, is generally methane amide, but other chaotropic reagent, NaI for example, urea, thiocyanic acid (salt), guanidine, trichoroacetic acid(TCA) (salt), tetramethyl-amine and perchlorate also can both act on.In addition, several precipitations and/or cross linking fixative also have gentle sex change characteristic; These character can by with main denaturing agent with add and or cooperative form be used.The difference that the hybridization mixed solution constitutes can allow only to form RNA-RNA or RNA-DNA hybridization chain selectively.This point can be by regulating denaturing agent concentration and salt concn (mainly being the metallic cation and the ammonium ion of monovalent I main group) and hybridization temperature reach.Allow like this probe optionally or with cell RNA or and DNA, perhaps simultaneously with RNA and DNA(with different probes) hybridize.This allows that also probe be may reside in and is pre-mixed in the solution, thereby the optimum condition that produces signal and maximizes noise reduction can be provided when the cell/tissue form " is fixed " on optimum ground at the same time.

The hybridization solution composition

Hybridization solution can typically contain: chaotropic denaturing agent, buffer reagent, pore former, hybrid stability agent.Chaotropic denaturing agent comprises: methane amide, urea, thiocyanic acid (salt), guanidine, trichoroacetic acid(TCA) (salt), tetramethyl-amine, perchlorate and sodium iodide.Anyly maintain the pH value at least that the buffer reagent between the 7.0-8.0 can use.

Pore former is, for example, such as Brij35, Brij58, sodium laurylsulfonate, tween (Tween), the stain remover of CHAPS or TritonX-100 and so on.According to the choice of location pore former of target biological polymer, so that probe is by cytoplasmic membrane, nuclear membrane or cell compartment structure.For example, 0.05%Brij35 or 0.1%TritonX-100 allow probe by cytoplasmic membrane, and can not pass through nuclear membrane.On the contrary, Sodium desoxycholate allows probe to pass through nuclear membrane.Therefore, for being confined to, hybridization so just should avoid using the nuclear membrane pore former at the target biological polymer in the tenuigenin.When the target biological polymer was positioned at tenuigenin, this optionally ubcellular distributed, and by the hybridization of elimination probe and complementary nucleus order, thereby had improved specificity and the sensitivity analyzed.Reagent except stain remover, also has (pore-forming) function as fixing agent.

Contain the hybrid stability agent in hybridization solution, the cationic salts of monovalence and divalence for example is to promote the complementary formation of hydrogen bond in proper order between probe and its target biological polymer.Preferably, working concentration is the sodium-chlor of 0.15M to 1M.In order to prevent the non-specific combination of nucleic acid probe, in hybridization solution, add and the irrelevant nucleic acid of target biological polymer.The representative example of hybrid stability agent has: sodium-chlor, lithium chloride, Manganous chloride tetrahydrate and ferric sulfate.

Preparation is used for the cell of flow cytometry

When cell was analyzed with flow cytometry (flow cytomety), whole procedure is in the solution at sample operated.In brief, cell suspension " in the phosphate buffered saline(PBS) (PBS), centrifugation then.Fixing agent or the hybridization solution that contains fixing agent are added on cell precipitation.Behind resuspending, cell is incubated by the mode of describing below.Centrifugal, after resuspending and the washing, the cell resuspending is in mounting medium (mounting mediun) and analyze on flow cytometer.

Probe type

Probe is defined as hereditary material DNA, RNA or oligonucleotide or contain DNA or the polynucleotide of RNA and antibody.DNA or RNA can be by bases adenine, uridylic, and thymus pyrimidine, guanine, any natural or artificial derivative of cytosine(Cyt) or they is formed.Probe can by with the combining of complementary or mirror image target cell genetic sequence, and form double-stranded or three chain hybrids.Take place in conjunction with chemical bond, usually by forming hydrogen bond by one or more types.Combination degree refers to allow the number of mispairing in combination or crossover process.Probe is relevant with the complementary degree same in conjunction with program and probe and target sequence of the target cell order of naturally occurring or synthetic.The size of probe is adjusted to such degree: form stable hybridization chain on desired mispairing level; Typically, in order to detect the probe that single base mispairing need be about the 12-50 base.Bigger probe (from 50 bases to tens thousand of bases) more uses when the mispairing level of being everlasting is represented with the full per-cent of the similarity of probe and target genetic sequence.The size of probe also can change, thereby allows or prevent that probe from entering or being incorporated into the different zones of genetic material or cell.Similarly, the type of probe (for example, with RNA to DNA) can reach these purposes.The size of probe influence equally the speed that probe spreads, and the possibility or the like that finds the target counterpart of cell.Typically, when nucleotide sequence is target, in hybridization, use double-stranded DNA (dsDNA), single stranded DNA (ssDNA) or RNA.

The preparation of probe

Can be used for the method that RNA of the present invention or dna probe can know according to the those of skill in the art in present technique field and be prepared, perhaps can buy.Rna probe can be according to Green et al.(1981) method described in 32: 681 of Cell is prepared.Dna probe can be prepared according to the method that present technique field those of skill in the art know, for example Rigby et al.(1977) J.Mol.Biol.113: 237 methods of describing.The oligonucleotide probe of synthetic can be as Wallace, et al(1979) Nucleic Acids Research6: 3543 described like that or as the manufacturer (as ABI) of nucleic acid synthesizer advises, be prepared.Being used for probe of the present invention also can be according to two pendent patent application U.S.SN784, and 690(1991 applied for October 28) and US.SN668,751(1991 applied for March 13) in disclosed method be prepared.

Nucleic acid probe can be prepared with the various methods that well known to a person skilled in the art.The double-stranded DNA order (ds DNA) of purifying can be carried out the not damaged mark with nick-translation or random primer extension method.Double-chain probe and the hybridization ability that is fixed on intracellular nucleic acid, because complementary chain hybridization mutually earlier before hybridizing with nucleus in the solution, and descend to some extent.Single stranded DNA (ss DNA) probe is not limited, and can pass through synthetic oligonucleotide, by using single stranded phage M13 or its plasmid derivative thing, perhaps makes by reverse transcription purified RNA template.In hybridization, use the single stranded RNA probe may provide than using strand or the bigger signal/noise ratio of double chain DNA probe.No matter use dsDNA in hybridization, three strands of ssDNA or ssRNA be probe circlewise, and the means that crossbred forms that detect must be arranged.The means that the detection crossbred forms have been utilized the probe with certain detectable marker " mark ".

Antibody probe is known for those skilled in the art.Term " antibody probe " refers to the special and bonded antibody with it to arbitrary target antigen.Such target antigen can be a polypeptide, protein, and hydrocarbon polymer or any other biological polymer are for these material antibody combinations specifically.

The preparation of antibody probe

For the special antibody probe of antigen (for example viral or its special decision family, the polypeptide of different sources and protein, hydrocarbon polymer part and a large amount of different biological polymer) is known for a person skilled in the art.The method for preparing such antibody is known for a person skilled in the art equally.

In brief, polyclonal antibody can be by preparing with the antigen-immunized animal host.Preferably, antigen is pressed weekly subcutaneous injection at interval and is given the host, is using for the last time weekly dosage after January subsequently, uses booster dose one time.Then, results serum, therefrom precipitating antibody and can be detected ground mark with technology well known by persons skilled in the art quilt.

MONOCLONAL ANTIBODIES SPECIFIC FOR can be according to any method well known by persons skilled in the art.Method with the spleen group born of the same parents of myeloma cell and immune donor are merged has proved a successful method, and it can produce the hybridoma successive clone of inheritance stability, thereby can produce in a large number the monoclonal antibody at target antigen (as tumour and virus).For example, MONOCLONAL ANTIBODIES SPECIFIC FOR can be according to U.S. Patent No. 4,172, the 124 disclosed methods at Koprowski etc., or according to the U.S. Patent No. 4,196,265 of Koprowski etc.The program of traget antibody is known to these those skilled in the art.

Detection system

Before adding hybridization, probe can be detected ground mark.Perhaps, can select detectable marker, after hybridization is finished, add, thereby combine with the hybridization product.In order to be used for enforcement of the present invention, probe can carry out mark with any detectable group.Such labelling groups can be any have can be detected physics or the material of chemical property.Such detectable has fully developed in the immunoassay field, and generally, most of any markers that can be used for these methods can both be applied to the present invention.Useful especially is the enzymic activity group, for example enzyme (referring to Clin.Chem., 22: 1243(1976)); Enzyme substrates (referring to British Pat.Spec.1,548,741), coenzyme (referring to U.S.Patents Nos.4,230,797 and 4,238,565); Enzyme inhibitors (referring to U.S.Patent No.4,134,792); Fluorescent agent; (referring to Chin.Chem., 25: 353(1979)); Chromophoric group; Luminous agent, as chemoluminescence agent (chemiluminescers) and luminescent biological agent (bioluminescers(is referring to Clin.Chem., 25: 512(1979); The part of specific combination; With immediate interactional counterpart; And radio isotope, as ³H, ³⁵S, ³²P, ¹²⁵I and ¹⁴C.

Biotin labeled Nucleotide can pass through nick translation, and enzymatic method or chemical method participate in DNA or RNA.Can use avidin/streptavidin in conjunction with biotinylated probes after hybridization, fluorescence, enzymatic or colloidal gold conjugate detects.But the fluorescent derivative or the vitamin H analogue of also available other fluorescent chemicals immunodetection of nucleic acid carry out mark.With histone HI with radioactivity or fluorescence, enzyme (alkaline phosphatase and peroxidase), or the nucleic acid of strand combination (ssB) protein-crosslinking also can use.In order to increase the sensitivity that detects colloidal gold or peroxidase product, can use some increase that utilizes silver-colored solution or amplification procedures.

Can also use a kind of indirect immunocytochemistry program (Rudkin and Stollar(1977) Nature 265: 472; Van Prooijen, et al.(1982) Exp.Cell, Res, 141: 397). in this program, by (poly(Ra)-poly(dT) produce polyclonal antibody to animal injection poly (Ra)-poly (dT) at RNA-DNA hybridization chain.Dna probe and cell are carried out in situ hybridization, then, detect the hybridization chain by being incubated with antibody at RNA-DNA hybridization chain.

The size of probe and concentration

Its rate of diffusion of the effect length of probe, the stability of hybridization chain formation speed and hybridization chain.According to the present invention, little probe (15-100 base) produces sensitive, quick and the most stable system.The mixture that has prepared short probe (15-100 base), they have covered the total length of detected target biological polymer.For example:, thereby cover all zones of target biological polymer fully if long 1000 bases of target biological polymer are used the probe of about 40 " different " long 25 bases so in hybridization solution.

Some parameters of the concentration affects in situ hybridization reaction of probe.Can use high density to increase diffusion process, reduce the time of hybridization, and make all cell orders saturated.For reaction is carried out fast, the hybridization solution middle probe is preferably 0.01-100 μ g/ml from concentration.Best probe working concentration is 2.5 μ g/ml.

Hybridization solution and temperature

In a preferred embodiment, the hybridization solution of one step in-situ hybridization method is made up of following: 25% methane amide, 5 * SSC, 15 * Ficoll/PVP, 0.4M guanidinium isothiocyanate, about 50mM sodium phosphate (pH7.4), 50mM DTT, about 1mg/ml salmon sperm dna, 5%TritonX-100,50mMEDTA and 21%PEG.The oligonucleotide of synthetic is added in this solution.Probe size is no less than the 15-20 base, and preferably about 25 bases are with photobiotin (Photobitin ^TM) mark.The suitableeest best hybridization temperature is 40-45 ℃.Yet operable temperature can be from 15-80 ℃.

In a step method and two step methods, the probe of hybridization in the mixed solution can be before hybridization mark.Probe can be a kind of in aforementioned many types.If probe Photobiotin ^TMMark, hybrid can be by using and fluorescence molecule (FITC for example, rhodamine, Texas Red ^TM) link coupled streptavidin/avidin (S/A); Perhaps with enzyme link coupled S/A; Perhaps use the S/A of the heavy metal mark such as colloidal gold and detect.Particularly, after hybridization, contain the solution of streptavidin conjugate, can directly join in the hybridization mixture on the cell.Cell is incubated 5-30 minute in 45 ℃ in this solution.Yet also can use longer hybridization time and higher or lower temperature.The time of hybridization can change, and depends on the composition of hybridizing mixed solution, and this mixed solution contains fixing agent (type of precipitation agent and/or linking agent and concentration), buffer reagent, pore former, denaturing agent and hybrid stability agent.Similar with it, temperature also can change as described.Perhaps, probe can be directly with fluorescence dye or molecule [pontamine sky-blue (Pontamine Sky Blue for example ^TM)] mark, promptly by nucleic acid probe and dyestuff are incubated together (1: 10, weight: weight) thus make dyestuff combination/embedding probe.Then probe precipitates from this liquid of dyestuff, and unnecessary not combination dye is by removing with 70% washing with alcohol repeatedly.Probe is direct covalent labeling also, promptly by duplex molecule (RNA-RNA, RNA-DNA, or DNA-DNA) is incubated and mark with the marker that can be covalently bonded in nucleic acid.Under the condition that reaction is carried out, after being incubated, chain is separated, and the separated chain of each bar all can be used as probe.Typical concentration at this liquid middle probe is 2.5 μ g/ml, although also can use the concentration of 0.01-100 μ g/ml.Concentration and probe concentration can influence reaction kinetics and meeting impact analysis sensitivity and signal/noise ratio.

If probe directly detects with the enzyme labelling substance markers or with detection system enzymatic or secondary, this reaction can be carried out before or after any washing step so.With sample after the suitable reducing of this enzyme is incubated, microscope slide is incubated with the substrate of this enzyme then under the manufacturers of this enzyme or supplier's defined terms.

Cell can be deposited on the microscope slide perhaps centrifugation after fixing/hybridization/detection reaction.Then, unconjugated probe is with 0.1 * SSC and 0.1%Triton X-100 ^TMThereby solution washing is flush away from cell once.Each slide (is per 100,000 isolated cells or every about 1cm ²Tissue block) can use the washings of total amount as 1-200ml.Hybrid stability agent/denaturing agent and or the concentration and the type of hole agent can change, depend on cell one type, the type of probe and acceptable hybridization mispairing degree.

The result who obtains with a step or two step methods

When cell deposition was on microscope slide, if use the fluorescent probe of direct or indirect mark, the result can observe by the operation fluorescent microscope so.Perhaps, the result also can analyze on based on the imaging analysis system of fluorescence automatically.

When cell was retained in the solution, the result can draw by the fluorescence volume that uses flow cytometer to write down each cell, and fluorescence volume has been represented the hybridization amount of each cell.Perhaps, resultant signal in a cell sample can use such as the microtiter plate reader of liquid flashing counting device (being used for radiogram) or chemiluminescent/fluorescence at the equipment of marker and draw.

The speed of in situ hybridization, sensitivity and quantitative

In the concrete enforcement of a step method, the present invention only need just can finish in 5 minutes, and sensitivity reaches each cell less to a cell biological polymer molecule.Speed and sensitivity are from following at least four factors: (1) cellular constituent irreversibly is not deposited in or is fixed on the nucleic acid; (2) if fixing, then should fixedly be the specific tissue that is suitable for using most; (3) the reaction kinetics process is carried out sooner under the temperature of high concentration and probe concentration and rising; (4) probe is built into and is convenient to pass in and out fast cytolemma and cell component.

When using radioactive probe, can estimate the mRNA or the DNA copy number of each cell by the numbers of particles on the cell.When adopting fluorescence or enzyme process to detect, can estimate the copy number that number goes out mRNA or DNA to relative estimation fluorescence or that precipitate painted product.Usually, taking pictures in craft, after film and comparison photograph, is than being easier to draw about copy number purpose.To the quantitative analysis of the signal of the radioactive or fluorescence mentioned after the in situ hybridization, can use imaging analysis system (as Meridian ACAS570 workstation) to carry out automatically.

The sensitivity of hybridization in situ technique of the present invention allows the genetic sequence by the single copy that exists in visual and the detection single cell of taking a picture.

For example (but being not limited thereto), when each was about the single fluorophore of one on the probe band of 30 bases, the genetic sequence that is about 6,000 bases so can be detected reliably by using standard fluorescence amount micro mirror observations.When 4 fluorophores are attached to a probe, be about 1500 bases specific genetic sequence disappearance or exist just can be by using the soaking time of mentioning herein, and detected reliably.In addition, when the probe that uses at the respective sequence of " meaningful " and " antisense " chain of double-strandednucleic acid, use the soaking time of mentioning to detect the existence or the disappearance of the order that is as short as about 750 base pairs herein, perhaps, people can use the detection of imaging analysis system few to the 75-150 base pair.

Detect three kinds of mRNA simultaneously

Manual can both detect with the different substances in the like cell (mRNA, DNA and protein) simultaneously with a step in situ hybridization program.It can be realized with in two kinds of methods any.Multiple probe all is added in the hybridization solution together, and every kind of probe contains a kind of distinguished marker (for example, detecting luminous fluorescent marker " A " " B " and " C " of wavelength in difference respectively).Perhaps, also can once hybridize earlier and detect,, carry out another time hybridization then residual unreacted probe and marker flush away with a kind of probe and marker.This process can indiscriminately ad. as one wishes repeat repeatedly.Perhaps, probe A, B and C contain same marker.

When detecting DNA and RNA simultaneously, the type of selecting probe is most important.When the cellular targets biological polymer is RNA, can in analysis, use the ssDNA probe of antisense.If the cell target DNA is a biological polymer to be detected, in analysis, can use the single stranded RNA probe of sense strand.This probe is selected, and adds the selection and the concentration of the composition of fixing/hybridization solution, can be so that only form RNA-DNA hybridization.Therefore, probe can only be incorporated into desired target cell biological polymer; Basically prevent other nucleic acid disturbance reponse analyses.

A step hybridization in situ technique that is used to detect DNA or mRNA can provide with the form of test kit (kit).Such test kit contains following material:

1. the solution that contains fixing/hybridization mixed solution and one or more label probes.Preferably, this solution contains the 50mM guanidinium isothiocyanate, the 25-40% methane amide, 21%PEG, 0.4M DTT, 15 * Ficoll/PVP, 50mM EDTA, the 1mg/ml salmon sperm dna, 50mM Tris-acetate (pH7-8), the synthetic oligonucleotide of directly using report molecule (reporter molecule) mark of about 5%Triton X-100 and about 0.06 μ g/ μ l.Solution and probe should have can be measured predetermined and identification feature and with the reactivity of cell and target sequence.

2. carry out the instrument and the explanation of in situ hybridization reaction of the present invention.Perhaps, test kit also can contain:

The secondary report system that detects, it can react with probe or probe-target hybrid.

3. can directly use or fully dilute the spissated storage solution of back formation washings.

4. be essential or useful any machinery part to implementing the present invention, for example solid carrier (as slide) with the equipment that cell fixation is carried in described aunt, is perhaps assisted the equipment of insulation sample or washing sample.

5. be used to write down the result's who uses analysis of the present invention and obtain photographic plat or sensitive emulsion.

The another kind of type of test kit can contain the probe solution that is bundled into capsule shape by liposome or microballoon.

Provide the following example for purposes of illustration, and do not take pains in limiting to the present invention by any way.In all embodiment, be by weight for all per-cents of solid, be by volumes for all per-cents of liquid, all temperature all are centigradetemperatures, unless dated especially.The document of all references is all expressed together with the reference form.

Embodiment 1

But detect the HPV that uses in advance on the painted smear of Pan Pani Laplace by in situ hybridization with dna probe

The preparation of cell

To place on the conveying medium with the cervical cell that Uterine neck bush obtains.After waiting to transport to the laboratory, with cell vortex concussion 1-3 second, centrifugal 10 minutes then at 1500rpm.The supernatant liquor that inclines, with the cell resuspending in the 1ml conveying medium.Cell is directly put on the microscope slide of handling through organosilicon by desired density.Then, but with the Pan Pani Laplace dyeing procedure of standard with cell dyeing.

With microscope slide in water logging 5 times, dyeing is 10 seconds in Gills prescription #2; Then in deionized water, soak 10 times.In 95% ethanol, soak 10 times.Dyeing is one minute in OG-50.In 95% ethanol, soak 10 times.In EA-50, dyed 2 minutes.In 95% ethanol, decolour, soak 10 times.Freezing microscope slide one end Dropwise 5,0 μ lGEL/ slide glass (mount) (Biomeda Corp., Foster City, CA-Catalog M01) in standard.Cover glass with 24 * 50mm covers and placed 10 minutes.

Before the hybridization, microscope slide is heavily steaming H in position ₂Soak until the cover glass landing about 10 minutes usually among the O.Microscope slide is at fresh H ₂Soaked again among the O 5 minutes.Then at gradient ethanolic soln (50%, 70% ﹠amp; 95%) dehydration in.

The preparation probe

The positive control probe contains human body α-kinetochore repetition DNA, known it can with the hybridization of everyone Autosome.Negative probe is known as NR, is the nitrogen reductase gene development of finding from bacterium and coming, and known not with eukaryotic cell in nucleic acid hybridization.The order of HPV16 type and HPV18 type obtains from the order of publishing, and can pass through the genetic sequence database, Genbank, and (Genetic Sequence Data Bank, Genbank Version69.0) obtains 69.0 versions.

Probe title seat title fluorescent marker molecular probe share

The catalogue # of company

HPV16 PPH16 rhodamine derivative L-20

HPV18 PPH18 rhodamine derivative L-20

Design 500 kinds of different probes (each 250 kinds on 16 types and 18 types), length is 25 bases and synthetic.

The DNA sequence preparation that the synthetic oligonucleotide of several length 25 bases is listed from below.

Table 1

The chromosomal loci title fluorescent marker that the probe title detects

α-kinetochore tumor-necrosis factor glycoproteins X HUMSATA fluorescein

α-kinetochore tumor-necrosis factor glycoproteins Y HUMSATB fluorescein

Synthetic and the mark of probe

Synthetic oligodeoxynucleotide (Applied Biosystems DNA SynthesizerModel 380B, and use the A.B.I reagent of recommending), and in the end the stage with amino hexyl phosphoric acid ester connector attached to 5 ' end.Then with 5 '-amino hexyl oligodeoxynucleotide and Molecular Probes, fluorescence rhodamine derivative (catalog number (Cat.No.) #L-20) coupling of InC., and carry out purifying at baseline 810 chromatographic working stations by Waters HPLC.

Hybridization

In order to carry out the hybridization program, 50 μ l hybridization mixed solution (is contained 22%PEG, 30% methane amide, 5 * SSC, 0.3mg/ml salmon sperm dna, 15 * Ficoll/PVP, 0.4M guanidinium isothiocyanate, 50mM DTT, 5%Triton X-100,50mM EDTA, 7.5% polysorbas20,50mM Na ₃PO ₄) and the probe of 0.02 μ g/ μ l be added on microscope slide.Add that cover glass is heated to 95 ℃ with microscope slide, 5 minutes, let alone to be cooled to 42 ℃ and this temperature insulation 25 minutes.

Washing

After hybridization, microscope slide is placed Coplin jar (coplin jar), in cylinder, add the 100ml washings.Washings contains 0.1 * SCC and 0.4M guanidinium isothiocyanate and 0.1%Triton X-100.Rock washings up to the cover glass landing, in solution, kept 4 minutes then.This washings is removed and is added second washings of 0.1 * SCC and 0.1%Triton.Rocked liquid 1 minute, and inclined, repeated washing is 5 times then.After washing, add the anti-decolourant of 8 μ l/Hoechst counterstain.Microscope slide is added a cover cover glass and is observed under fluorescent microscope.

Fluoroscopic examination

On the Olympus BH10 microscope of band fluorescent functional, carry out photomicrograph, adopt the Ektachrome EES-135(PS800/1600 of Kodak) film.Exposure, button places the 1600ASA shelves.Use 50 second time shutter always, thereby can between all Photomicrographs that obtain, directly compare.

The result

Fig. 1 has shown the typical consequence when HPV probe (using the rhodamine mark) and positive control probe (using the FITC mark) join same hybridization mixed solution together.It is HPV that the nuclear of less cell is become the nuclear of positive (being green in the photochrome) bigger cell by the positive control probe, male (being glassy yellow in the photochrome).

The program of embodiment may be modified as following form:

(1) synthesize 400 different probes (200 are used for 16 types, and 200 are used for 18 types), each probe design is that 30 bases are long.But, except the preparation with these 400 corresponding probes of different oligonucleotide (these oligonucleotide are configured for the probe of a chain of two any target sequences in the HPV target altogether), people can also prepare 400 kinds of other oligonucleotide probes that are used for the second chain of two HPV targets.Consider their distributions on the HPV Genome Atlas, the probe that is used for article one chain will be in " different phase " with respect to the probe of second chain, therefore, half of the probe of each article one chain (15 Nucleotide) will be complementary to half of a second chain probe on nucleotide sequence, and the probe of this article one chain second half (15 Nucleotide) will be complementary to another second chain probe half.Probe is staggered mutually means because the part very short (15 Nucleotide) that overlaps, so the probe of article one chain will be main not can with the probe hybridization of second chain.On the other hand, compare, can detect the hybridization of about twice like this with only using situation corresponding to the probe of a chain.

(2) probe is made the thiophosphatephosphorothioate oligonucleotide, each 30 polymer (30-mer) has 4 sulphur atoms.Applied Biosystem(ABI is adopted in preparation) dna synthesizer, model 380B also uses the ABI reagent of recommending.It is as follows that sulphur atom distributes: first is positioned at the 5' end of probe, and second (begins number from the 5' end) between the 7th and the 8th nucleosides, the 3rd between the 22nd and 23 nucleosides, the 4th between the 29th and the 30th nucleosides.In the following manner, then with the sulphur atom of the oligonucleotide of poly sulphur and the coupling of fluorescence dye iodo-acid amide fluorescein (can adjusted volume and synthesize less amount): the dried oligonucleotide of 200 μ g be dissolved in the 250mM Tris damping fluid (pH7.4) of 100 μ l, thereby forms first solution.1mg iodo-acid amide and the dried dimethyl formamide of 100 μ l (being 100%DMF) are mixed in second solution.Two kinds of solution are mixed and shake and spend the night.After incubated overnight, the oligonucleotide of mark precipitates with ethanol and 3M sodium acetate.This raw product follow upper prop in PD-10 layer pole to remove the free dyestuff.Collect required part then.Liquid is removed in a vacuum.Raw product is then used the HPLC(high performance liquid chromatography) carry out purifying.

(3) negative and positive control probe set by step (1) and (2) make up similarly.

(4) hybridization mixed solution can be amended as follows: replace 21%PEG with 1.5%PEG, replace 21% methane amide with 30% methane amide, the adding 10%DMSO(10%, v/v) and 5%(v/v) vitamin-E.Equally, not that 50 μ l hybridization mixed solution is added on microscope slide, but 40 μ l mixed solutions are added 5 μ l squalenes and 5 μ l pyrrolidone, then these blended 50 μ l solution are added on microscope slide.Can in per 100 μ l mixtures, add 5 μ l1MDTT and 5 μ l Proteinase K (1mg/ml) solution equally, carry out hybridization then, for example, carry out 5 minutes, then carry out 5 minutes at 95 ℃ at 45 ℃.Can in the hybridization mixed solution, add about 0.5% or 0.10% aurin tricarboxylic acid equally.

(5) be different from the anti-decolourant/Hoechst of 8 μ l is added on microscope slide, the following solution of 8 μ l is added on microscope slide: 9 parts of volume solution A add the solution B of a volume.Wherein solution A is to be dissolved in 50%(v/v) glycerine and 50%(v/v) 1 * PBS(0.136M NaCl, 0.003M KCl, 0.008M Na ₂HPO ₄, 0.001M KH ₂PO ₄) 0.01%1,4-diphenylamine (anti-decolourant) adds nuclear staining agent Hoechst(#33258; 1 μ g/ml) add 0.0025%Evans Blue, and solution B is a dodecyl ethanol.

Embodiment 2

But detect the chlamydia trachomatis (Chlamydia trachomatous) use in advance on the painted smear of Pan Pani Laplace and the DNA and the mRNA of gonococcus (Neisse-ria gonorrhea) by in situ hybridization with dna probe

Cell preparation

To place on the conveying medium with the cervical cell that Uterine neck bush obtains.After waiting to transport to the laboratory, with cell vortex concussion 1-3 second, centrifugal 10 minutes then at 1500rpm.The supernatant liquor that inclines, with the cell resuspending in the 1ml conveying medium.Cell is directly put on the microscope slide of handling through organosilicon by desired density.Then, but cell dye with the Pan Pani Laplace dyeing procedure of standard, as described in Example 1.

The preparation of probe

Positive probe preparation is as described in the embodiment 1.

The plasmid of-7.4kb is in the genomic general integral part of CT and each genome 10 copies to be arranged approximately, and its sequence can be passed through Genetic Sequence Data Bank, Genbank(69.0 version) obtain.7.4kb total length be divided into 296 different oligonucleotide probes in proper order, each contains 25 bases.These 296 oligomers are synthetic and mark by the carrying out of describing below.

To GC, three repetition DNA elements can pass through Genetic Sequence Data Bank, Genbank(69.0 version) and obtained.

These are cut into discrete oligonucleotide probe in proper order, and each contains 25 bases, and synthesize and mark by the carrying out of describing below.

Table 2

Probe title seat title

C.T. CHTP

G.C. NGORPTA

NGORPTB

NGORPTC

Table 3

Fluorescent marker molecular probe catalogue #

Fluorescein F-143

Rhodamine derivative L-20

The red T-1905 of Texas

Coumarin derivatives D-1412

Synthetic and the mark of probe

Synthetic oligodeoxynucleotide (Applied Biosystems DNA Synthesizer Model 380B, and use the A.B.I reagent of recommending), and in the end the stage with amino hexyl phosphoric acid ester connector attached to the 5' end.Then with amino hexyl oligodeoxynucleotide of 5'-and Molecular Probes, fluorescence rhodamine derivative (catalog number (Cat.No.) #L-20) coupling of InC., and carry out purifying at baseline 810 chromatographic working stations by Waters HPLC.

In order to detect two kinds of organisms simultaneously, probe GC and CT can be with different fluorescent-substance markers.For example, GC can be with fluorescein-labelled and CT with rhodamine derivative mark.Above alternative fluorescence dye (subsidiary catalog number) is listed in.

Hybridization

In order to carry out the hybridization program, 50 μ l hybridization mixed solution (is contained 30%PEG, 30% methane amide, 5 * SSC, 0.3mg/ml salmon sperm dna, 15 * Ficoll/PVP, 0.4M guanidinium isothiocyanate, 50mM DTT, 5%TritonX-100,50mM EDTA, 50mM Na ₃PO ₄) and the probe of 0.02 μ g/ μ l be added on microscope slide.Add cover glass and when DNA is target, microscope slide is heated to 95 ℃ and reaches 5 minutes, let alone to be cooled to 42 ℃ and be incubated 25 minutes in this temperature.If mRNA is a target, then save 95 ℃ of heating stepses.

Washing

After hybridization, microscope slide is placed Coplin jar (coplin jar), in cylinder, add the 100ml washings.Washings contains 0.1 * SCC and 0.4M guanidinium isothiocyanate and 0.1%TritonX-100.Rock washings up to the cover glass landing, in solution, kept 4 minutes then.This washings is removed and is added second washings of 0.1 * SCC and 0.1%Triton.Rock liquid 1 minute, and inclined, and then repeat back once washing 5 times.After washing, add the anti-decolourant of 25 μ l/Hoechst counterstain.Microscope slide is added a cover cover glass and is observed under fluorescent microscope.

Fluoroscopic examination

On the Olympus BH10 microscope of band fluorescent functional, carry out photomicrograph, adopt the Ektachrome EES-135(PS800/1600 of Kodak) film.Exposure, button places the 1600ASA shelves.Use 50 second time shutter always, thereby can between all Photomicrographs that obtain, directly compare.Perhaps, the operator can use commercially available low light brightness monochrome and color video camera, thus not only can be on TV screen observations but also can make permanent record with the form of photochrome.

When using CT and GC specific probe respectively, CT and GC organism are dyed the tangible positive.

Embodiment 3

But use ⅡXing Paozhenbingdu DNA or mRNA on the painted smear of Pan Pani Laplace in advance by the in situ hybridization detection with dna probe

The preparation of cell

As embodiment 1, prepare prestained cervical cell.

The positive control probe

The positive control probe is as described in the embodiment 1.

The special order of ⅡXing Paozhenbingdu (HSV-II)

The order of HSV-II is by Genetic Sequence Data Bank, Genbank(69.0 version) obtain.The very early time of HSV-II is divided into different oligonucleotide with the order of early gene, and each contains 25 bases.These oligomers are by 1 described synthesizing and mark of embodiment.

Hybridization, washing and fluoroscopic examination

Hybridization, washing and fluoroscopic examination are all by embodiment 1 described carrying out.

When using HSV-II probe, the cell that contains the HSV-II is dyed the bright positive.

Embodiment 4

But detect in advance with the HPV in the painted cell of Pan Pani Laplace staining by in situ hybridization and detect and distinguish HPV16 and HPV18 simultaneously with dna probe

The preparation of cell

Cultivate C-33A(ATCC HTB#31), Ca Ski(ATCC CRL #1550) and the Hela cell, use trypsin treatment, and, directly put on the microscope slide that organosilicon was handled by required density by 1: 1: 1 mixed.Then but cell is with the dyeing of standard Pan Pani Laplace dyeing procedure and prepare to carry out in situ hybridization, such shown in embodiment 1.

The preparation of probe

Positive control probe preparation is as described in the embodiment 1.

The order of HPV16 type and HPV18 type obtains from the order of publishing, and by Genetic Sequence Data Bank, Genbank(69.0 version) obtain, as described in example 1 above.

Synthetic and the mark of probe

It is described to press embodiment 1, synthetic mark oligodeoxynucleotide.Fluorescein (catalog number (Cat.No.) F-143) is invested the HPV16 probe and rhodamine derivative (L-20) is invested the HPV18 probe.

Hybridization, washing and fluoroscopic examination

As embodiment 1, hybridize washing and fluoroscopic examination step.

The result

Fig. 2 has shown three kinds of different cells in single photo.HPV-16 probe with the FITC mark produces positive signal (bright spot in the nuclear) in Ca Ski cell, be positioned at the upper left corner of " 2A " photo; And produce positive signal (bright spot in the nuclear) with the HPV-18 probe of rhodamine mark, be positioned at the lower right corner of same " 2A " photo.The cell that is positioned at photo central authorities is the C-33A cell, is correct feminine gender." 2B " is three kinds of photos that nuclear Hoechst redyes.It shows that this analysis system can detect and distinguish two types of HPV, i.e. HPV16 type and HPV18 type simultaneously in prestained cell.

Embodiment 5

But detect in advance with the single copy HPV in the painted cell of Pan Pani Laplace staining by in situ hybridization with dna probe

Cell preparation

Cultivate C-33A(ATCC HTB#31), Ca Ski(ATCC CRL #1550) and SiHa(ATCC HTB #35) clone, use trypsin treatment, directly put on the microscope slide that organosilicon was handled by required density.Then but cell is with the dyeing of standard Pan Pani Laplace dyeing procedure and prepare to carry out in situ hybridization, such shown in embodiment 1.

The probe preparation

The order of HPV16 type and positive control probe are pressed embodiment 1 described preparation.

Synthetic and the mark of probe

It is described to press embodiment 1, synthetic and mark oligodeoxynucleotide probe.

Hybridization, washing and fluoroscopic examination

As embodiment 1, hybridize, washing and fluoroscopic examination step.

Because unique copy of the dna integration of HPV16 type is only arranged, therefore in the SiHa cell, has only a bright spot in the SiHa cell.Ca Ski has some bright spots and C-33A does not have.

Embodiment 6

Use phenodin and eosin (H﹠amp with dna probe in advance by the in situ hybridization detection; E) HPV in the painted cervical tissue section

Tissue preparation

The available following mode of organizing of paraffin-embedded formaldehyde fixed is handled to remove cover glass and to use H﹠amp; E dyeing:

1. dimethylbenzene changes three times ... each 2 minutes

2. dehydrated alcohol ... soak ten times

3.95% ethanol changes three times ... soak ten times at every turn

4. tap water ... rinsing can be trickled reposefully until water

5. phenodin-Delafield, Ehrlich's, or Harris, acetate do not contained ... 10-15 minute

6. tap water changes twice ... soak ten times at every turn.

7. be dissolved in 70% alcoholic acid, 1% hydrochloric acid ... soak 5-10 time

8. flowing water ... thorough washing

9. ammoniacal liquor (0.24%) or Quilonum Retard (0.5%) ... 30 seconds

10. tap water changes twice ... soak ten times at every turn

11. eosin ... soak 10-20 time

Perhaps eosin one flame is red ... 1-3 minute

12.95% ethanol changes twice ... soak 10 times at every turn

13. dehydrated alcohol changes three times ... soak 10 times at every turn

14. dimethylbenzene changes three times ... soak 10 times at every turn

15.95% ethanol changes twice ... soak 10 times at every turn

16.70% ethanol changes twice ... soak 10 times at every turn

17.50% ethanol changes twice ... soak 10 times at every turn

18. redistilled water changes twice ... soak 10 times at every turn

19. by following described hybridization

The preparation of probe

As described in example 1 above, obtain the order of HPV16 type and HPV18 type.

Synthetic and the mark of probe

It is described to press embodiment 1, synthesizes the oligodeoxynucleotide probe with mark.

Hybridization, washing and glimmering big gun detect

As embodiment 1, hybridize washing and fluoroscopic examination step.

The result

Fig. 3 has shown and has used phenodin and eosin (H﹠amp in advance; E) detected result of HPV16 type and 18 types in the painted cervical tissue.Fig. 3 A is the H﹠amp before hybridization; The photo of the painted cervical tissue of E (ratio of enlargement=10X).Fig. 3 B is the Hoechst photo of redying nuclear (ratio of enlargement=40X), and the photo of Fig. 3 C to be the HPV that obtains after hybridizing with the mixed solution that contains HPV be positive painted cell (ratio of enlargement=40X).Though all cells obviously is positive when redying with Hoechst, having only small amounts of cells is obviously to the HPV male.Negative control probe is appropriate feminine gender.

Embodiment 7

Detect multidrug resistant gene and the ErbB gene of using in advance in phenodin and the painted tissue sample of eosin by in situ hybridization with dna probe

Cell preparation

After examination of living tissue human body uterine neck or breast, obtain tissue, fixing in formaldehyde, use H﹠amp; E dyeing is analyzed then.

The preparation of probe

The positive control probe contains the probe of describing among the embodiment 1.

C-erb-B-2 and MDR1 order obtains from the order of publishing, and can be by Gen-bank(69.0 version sheet) obtain.

Table 4

Probe title seat title fluorescent marker

C-erb-B-2 HUMERB2R lists among the embodiment 2

Any fluorescence dye of MDR1 HUMMDR1

Synthetic and the mark of probe

It is described to press embodiment 1, synthetic mark oligodeoxynucleotide probe.

Hybridization, washing and fluoroscopic examination

As embodiment 1, hybridize washing and fluoroscopic examination step.

The cell that contains a plurality of c-erb-B-2 or MDR1 gene copy has than much bigger cell and/or the much better than cytoplasmic dyeing of normal cell (cell that promptly has the single-gene copy).In addition, the power of signal can be carried out with the imaging analysis system of any model quantitatively, as Meridian Instructions, and the ACAS 570 that Okemos, Michigan are produced.

Embodiment 8

Detect the chromosomal DNA of using in advance in the painted sample of Diff-Quick method by the in situ hybridization program with dna probe

Cell preparation

To place on the conveying medium with the cervical cell that Uterine neck bush obtains, after waiting to transport to the laboratory, with cell vortex concussion 1-3 second, centrifugal 10 minutes then at 1500rpm.The supernatant liquor that inclines, with the cell resuspending in the 1ml conveying medium.Cell is directly put in the loading of handling through organosilicon by desired density.Then, according to supplier's suggestion the Diff-Quick method of cell with standard dyeed.Cover glass is removed as embodiment 1.

Synthetic and the mark of probe

It is described to press embodiment 1, synthetic and mark oligodeoxynucleotide probe.

Hybridization, washing and fluoroscopic examination

As embodiment 1, hybridize washing and fluoroscopic examination step.

The result

Fig. 4 has shown the result (4C amplifies 10 times, and 4D amplifies 40 times) who obtains in the painted cervical cell of Diff-Quick method, and then with the result who contains positive contrast probe hybridization (4A amplifies 10 times, and 4B amplifies 10 times).Bright positive signal can clearly see in the nuclear of these cells, and this shows that the DNA in probe and these the prestained cells hybridizes.

Embodiment 9

But detect the Trichomonas vaginalis of using in advance on the painted smear of Pan Pani Laplace (Trichomonas vaginalis), DNA or the mRNA of Candida (Candida) species and Treponoma palladium (Treponema pallidum) by in situ hybridization with dna probe

Cell preparation

The cervical cell that preparation obtains with Uterine neck bush, and dye as embodiment 1.The vagina focus is scraped thing place clean slide glass by standard method.

The positive control probe

The preparation of positive control probe as described in example 1 above.

Trichomonas vaginalis Trichomonas vaginalis) and Treponoma palladium (Treponema pallidum) Candida albicans Candida albicans),

Candida albicans, the order of Trichomonas vaginalis and Treponema pal-lidum obtains by the order of Sequence Data Bank or publication.Order is divided into discrete oligonucleotide probe, and each contains 25 bases.These oligomers are by 1 described synthesizing and mark of embodiment.

Hybridization, washing and fluoroscopic examination

As embodiment 1, hybridize washing and detection step.

When use respectively design be used to detect the specific probe of these microorganisms the time, microorganism Candida, Trichomonas and Treponema are dyed the bright positive.

All patents mentioned in this manual and publication have shown all standards of the those skilled in the art under the present invention.All patents and publication are introduced with the reference form herein, and its degree all shows especially and individually as each independent publication, introduces with the reference form.

Those skilled in the art will readily appreciate that the present invention is suitable for realizing goal of the invention very much, result and benefit that acquisition is mentioned, and other intrinsic things.Component described herein, method, program and technology be the representative of preferable concrete enforcement at present, is intended for the model, rather than be used for limiting the scope of the invention.Those skilled in the art can change and are used for other aspects it, but this does not still break away from purport of the present invention, and in appended claim institute restricted portion.

Claims (107)

1. Thereby 1, a kind of by detecting the existence whether method that the cell biological polymkeric substance detects biological polymer in prestained sample, wherein sample has complete substantially cytolemma, it is characterized in that, contains following steps:

   With the fixing described sample of mounting medium, this mounting medium contains at least a reagent that is selected from the group of being made up of precipitation agent and linking agent;

   Described fixed sample is contacted with hybridization solution, and hybridization solution contains denaturing agent, the hybrid stability agent, and buffer reagent, selective membrane pore former and at least a probe, described contact is carried out under hybridization conditions;

   Detect hybridization by the method that detects described marker and form thing.
2. 2, the method for claim 1 is characterized in that, described hybridization forms thing and is selected from duplex or triplex.
3. 3, the method for claim 1 is characterized in that, the nucleotide sequence of described probe is complementary to particular target nucleotide sequence to be detected at least substantially.
4. 4, method as claimed in claim 3 is characterized in that, about 75 bases of described target nucleotide order.
5. 5, the method for claim 1 is characterized in that, described sample contains microorganism.
6. 6, method as claimed in claim 5 is characterized in that, described microorganism is selected from bacterium, virus and fungi.
7. 7, the method for claim 1 is characterized in that, described sample contains eukaryotic cell.
8. 8, method as claimed in claim 7 is characterized in that, described eukaryotic cell is people's cell.
9. 9, the method for claim 1 is characterized in that, described sample contains cytogene.
10. 10, method as claimed in claim 9 is characterized in that, described cytogene is selected from oncogene, the tumor suppression group and the stimulating growth factor.
11. 11, method as claimed in claim 10 is characterized in that, described oncogene is selected from: c-erb-B-2, c-myc, c-myb and c-ras.
12. 12, method as claimed in claim 10 is characterized in that, described tumor suppressor gene is selected from P-53 and retinoblastoma gene.
13. 13, method as claimed in claim 10 is characterized in that, the described stimulating growth factor is selected from: α-Zhuan Huashengchangyinzi, Urogastron and colony-stimulating (growth) factor-granulocyte/scavenger cell.
14. 14, method as claimed in claim 6, it is characterized in that, in described prestained sample, described bacterium is selected from: streptococcus (Streptococcus), Staphylococcus (Staphylococcus), genus clostridium (Clostridium), Bacillus (Bacillus), Rhodopseudomonas (Pseudomonas), salmonella (Salmonella), klebsiella (Klebsiella), class (plan) Bacillaceae (Bacteroides), intestinal bacteria (Escherichia coli), gonococcus (Neisseria gonorrhea), and chlamydiaceae (Chlamydia).
15. 15, method as claimed in claim 6 is characterized in that, described bacterium is selected from: gonococcus (Neisseria gonorrhoea) and chlamydia trachomatis (Chlamydia trachomatous).
16. 16, method as claimed in claim 6, it is characterized in that, described fungi is selected from: read Coccus (Candida), Cryptococcus neoformans (Cryptococcus neoformans), Blastomyces dermatitidis (Blastomyces dermatitides), Histoplasma capsulatum (Histoplasma capsulatum), thick ball spore bacterium (Coccidioides immitis) and Paracoccidioides brasiliensis (Paracoccidioides brasiliensis).
17. 17, the method for claim 1 is characterized in that, described prestained sample is cell sample or tissue sample.
18. 18, method as claimed in claim 17 is characterized in that, described cell sample is selected from: cervical cell, medullary cell, liver cell, celiolymph cell, hemocyte, oral mucosa cell, pneumonocyte and skin cells.
19. 19, method as claimed in claim 17 is characterized in that, described tissue sample is selected from: lymph node tissue, breast tissue, cervical tissue, colon, prostatitis (gland) tissue, heart tissue and cerebral tissue.
20. 20, the method for claim 1 is characterized in that, described prestained sample is painted with following staining: but Pan Pani Laplace staining, Wright staining, phenodin and eosin staining and Diff-Quick staining.
21. 21, method as claimed in claim 6 is characterized in that, the described virus in described prestained sample is selected from: human papillomavirus, herpes simplex virus type 2, hepatitis virus, HIV (human immunodeficiency virus), influenza virus, parainfluenza virus and rotavirus.
22. 22, method as claimed in claim 14 is characterized in that, described biological polymer is mRNA.
23. 23, method as claimed in claim 14 is characterized in that, described biological polymer is DNA.
24. 24, method as claimed in claim 14 is characterized in that, described biological polymer is 16SrRNA.
25. 25, method as claimed in claim 14 is characterized in that, described biological polymer is selected from: mRNA, DNA, synthetic biological polymer and 16SrRNA.
26. 26, method as claimed in claim 14 is characterized in that, described biological polymer is the biological polymer of synthetic.
27. 27, method as claimed in claim 21 is characterized in that, described biological polymer is mRNA.
28. 28, method as claimed in claim 21 is characterized in that, described biological polymer is the synthetic biological polymer.
29. 29, method as claimed in claim 21 is characterized in that, described biological polymer is DNA.
30. 30, method as claimed in claim 21 is characterized in that, described biological polymer is 16SrRNA.
31. 31, method as claimed in claim 21 is characterized in that, described biological polymer is selected from: mRNA, DNA, synthetic biological polymer and 16SrRNA.
32. 32, method as claimed in claim 14 is characterized in that, described sample is a cervical cell.
33. 33, method as claimed in claim 21 is characterized in that, described sample is a cervical cell.
34. 34, method as claimed in claim 14 is characterized in that, but described staining is a Pan Pani Laplace staining.
35. 35, method as claimed in claim 21 is characterized in that, described staining is a Pan Pani Laplace staining.
36. 36, the method for claim 1 is characterized in that, described marker is attached to described probe.
37. 37, the method for claim 1 is characterized in that, described marker adds after hybridization forms again.
38. 38, the method for claim 1 is characterized in that, described marker is selected from: radioactively labelled substance, fluorescent agent, chemoluminescence agent and enzyme labelling thing.
39. 39, the method for claim 1 is characterized in that, described marker is the binding substances of avidin and streptavidin.
40. 40, the method for claim 1 is characterized in that, described fixing agent is selected from: ethanol, methyl alcohol, acetone and formaldehyde.
41. 41, the method for claim 1, it is characterized in that, described linking agent is selected from: paraformaldehyde, formaldehyde, dimethylsilserimidate and ethyl dimethylamino-propyl carbodiimide diimine (ethyldimethylamino-propylcarbodimide).
42. 42, the method for claim 1 is characterized in that, described denaturing agent is selected from: methane amide, urea, sodium iodide, thiocyanic acid (salt), guanidine, perchlorate, trichoroacetic acid(TCA) (salt), tetramethyl-amine.
43. 43, the method for claim 1 is characterized in that, described hybrid stability agent is selected from: sodium-chlor, lithium chloride, Manganous chloride tetrahydrate and ferric sulfate.
44. 44, the method for claim 1 is characterized in that, described pore former is selected from: Brij35 tween, Brij58, TritonX-100, CHAPS ^TM, Septochol (salt) and dodecyl sodium sulfonate (salt).
45. 45, the method for claim 1 is characterized in that, analyzes two kinds of biological polymers in same sample at least simultaneously.
46. 46, the method for claim 1 is characterized in that, described temperature is 15-85 ℃.
47. 47, the method for claim 1 is characterized in that, described temperature is 40-45 ℃.
48. 48, the method for claim 1 is characterized in that, the described sample that is fixed contacted about 5 minutes with described hybridization medium (liquid)-Yue 240 minutes.
49. 49, the method for claim 1 is characterized in that, described method was finished within about 5 minutes.
50. 50, the method for claim 1 is characterized in that, described probe comprises the mixture at the short probe in a plurality of zones of target polymkeric substance.
51. Thereby 51, a kind of existence whether method by analysis of cells biological polymer detection biological polymer in prestained sample, this sample has complete substantially cytolemma, it is characterized in that, contains following steps:

    Described sample is contacted with medium, and this medium contains denaturing agent, the hybrid stability agent, and buffer reagent, film pore former and at least a probe, described contact is carried out under hybridization conditions and when having a kind of probe at least;

    Detect hybridization by the method that detects described marker and form thing,

    And saved hybridization step, prehybridization is in order to stop the non-specific combination of described probe, and is convenient to entering at described sample and probe before described medium contacts.
52. 52, method as claimed in claim 51 is characterized in that, described medium also contains fixing agent.
53. 53, method as claimed in claim 51 is characterized in that, described sample comprises microorganism.
54. 54, method as claimed in claim 51 is characterized in that, it is duplex and triplex that described hybridization forms thing.
55. 55, method as claimed in claim 51 is characterized in that, the nucleotide sequence of described probe is complementary to particular target nucleotide sequence to be detected at least substantially.
56. 56, method as claimed in claim 55 is characterized in that, about 75 bases of described target nucleotide order.
57. 57, method as claimed in claim 53 is characterized in that, described microorganism is selected from bacterium, virus and fungi.
58. 58, method as claimed in claim 51 is characterized in that, described sample contains eukaryotic cell.
59. 59, method as claimed in claim 58 is characterized in that, described eukaryotic cell is people's cell.
60. 60, method as claimed in claim 51 is characterized in that, described sample contains cytogene.
61. 61, method as claimed in claim 60 is characterized in that, described cytogene is selected from oncogene, the tumor suppressor gene and the stimulating growth factor.
62. 62, method as claimed in claim 61 is characterized in that, described oncogene is selected from: c-erb-B-2, c-myc, c-myb and c-ras.
63. 63, method as claimed in claim 61 is characterized in that, described tumor suppressor gene is selected from P-53 and retinoblastoma gene.
64. 64, method as claimed in claim 61 is characterized in that, the described stimulating growth factor is selected from: α-Zhuan Huashengchangyinzi, Urogastron and colony-stimulating (growth) factor-granulocyte/scavenger cell.
65. 65, method as claimed in claim 57, it is characterized in that, be selected from the described bacterium described in the described prestained sample: streptococcus (Streptococcus), Staphylococcus (Staphylococcus), genus clostridium (Clostridium), Bacillus (Bacillus), Rhodopseudomonas (Pseudomonas), salmonella (Salmonella), klebsiella (Klebsiella), class (plan) Bacillaceae (Bac-teroides), intestinal bacteria (Escherichia coli), gonococcus (Neisseria gonorrhea), and chlamydiaceae (Chlamydia).
66. 66, method as claimed in claim 57, it is characterized in that, described fungi is selected from: read Coccus (Candida), Cryptococcus neoformans (Cryptococcus neoformans), Blastomyces dermatitidis (Blastomyces dermatitides), Histoplasma capsulatum (Histo-plasma capsulatum), thick ball spore bacterium (Coccidioides immitis) and Paracoccidioides brasiliensis (Paracoccidioides brasiliensis).
67. 67, method as claimed in claim 51 is characterized in that, described prestained sample is cell sample or tissue sample.
68. As the described method of claim 67, it is characterized in that 68, described cell sample is selected from: cervical cell, medullary cell, liver cell, celiolymph cell, hemocyte, oral mucosa cell, pneumonocyte and skin cells.
69. As the described method of claim 67, it is characterized in that 69, described tissue sample is selected from: lymph node tissue, breast tissue, cervical tissue, colon (colon) tissue, prostatitis (gland) tissue, heart tissue and cerebral tissue.
70. 70, method as claimed in claim 51 is characterized in that, described prestained sample is painted with following staining: but Pan Pani Laplace staining, Wright staining, phenodin and eosin staining and Diff-Quick staining.
71. 71, method as claimed in claim 57 is characterized in that, the described virus in the painted sample of described pre-gram is selected from: human papillomavirus, herpes simplex virus type 2, hepatitis virus, HIV (human immunodeficiency virus), influenza virus, parainfluenza virus and rotavirus.
72. As the described method of claim 65, it is characterized in that 72, described biological polymer is mRNA.
73. As the described method of claim 65, it is characterized in that 73, described biological polymer is DNA.
74. As the described method of claim 65, it is characterized in that 74, described biological polymer is 16SrRNA.
75. As the described method of claim 65, it is characterized in that 75, described biological polymer is selected from: mRNA, synthetic biological polymer, DNA and 16SrRNA.
76. As the described method of claim 65, it is characterized in that 76, described biological polymer is the biological polymer of synthetic.
77. As the described method of claim 71, it is characterized in that 77, described biological polymer is mRNA.
78. As the described method of claim 71, it is characterized in that 78, described biological polymer is DNA.
79. As the described method of claim 71, it is characterized in that 79, described biological polymer is 16SrRNA.
80. As the described method of claim 71, it is characterized in that 80, described biological polymer is the synthetic biological polymer.
81. As the described method of claim 71, it is characterized in that 81, described biological polymer is selected from: mRNA, DNA, 16SrRNA and synthetic biological polymer.
82. As the described method of claim 65, it is characterized in that 82, described sample is a cervical cell.
83. As the described method of claim 71, it is characterized in that 83, described sample is a cervical cell.
84. 84, as the described method of claim 65, it is characterized in that, but described staining is a Pan Pani Laplace staining.
85. As the described method of claim 71, it is characterized in that 85, described staining is a Pan Pani Laplace staining.
86. 86, method as claimed in claim 51 is characterized in that, described marker is attached to described probe.
87. 87, method as claimed in claim 51 is characterized in that, described marker adds after hybridization forms again.
88. 88, method as claimed in claim 51 is characterized in that, described marker is selected from: radioactively labelled substance, fluorescent agent, chemoluminescence agent and enzyme labelling thing.
89. 89, method as claimed in claim 51 is characterized in that, described marker is the binding substances of avidin and streptavidin.
90. 90, method as claimed in claim 52 is characterized in that, described fixing agent is selected from: ethanol, methyl alcohol, acetone and formaldehyde.
91. 91, method as claimed in claim 51 is characterized in that, described denaturing agent is selected from: methane amide, urea, sodium iodide, thiocyanic acid (salt), guanidine, perchlorate, trichoroacetic acid(TCA) (salt) and tetramethyl-amine.
92. 92, method as claimed in claim 51 is characterized in that, described hybrid stability agent is selected from: sodium-chlor, lithium chloride, Manganous chloride tetrahydrate and ferric sulfate.
93. 93, method as claimed in claim 51 is characterized in that, described pore former is selected from: Brij35, tween, Brij58, TritonX-100, CHAPS ^TM, Septochol (salt) and dodecyl sodium sulfonate (salt).
94. 94, method as claimed in claim 51 is characterized in that, analyzes two kinds of biological polymers in same sample at least simultaneously.
95. 95, method as claimed in claim 51 is characterized in that, described method can detect few target biological polymer to single copy in each cell.
96. 96, method as claimed in claim 51 is characterized in that, described hybridization conditions comprises that temperature is 15-85 ℃.
97. 97, method as claimed in claim 51 is characterized in that, described hybridization conditions comprises that temperature is 40-45 ℃.
98. 98, method as claimed in claim 51 is characterized in that, described method was finished within about 5 minutes.
99. 99, method as claimed in claim 51 is characterized in that, described probe comprises the mixture at the short probe in a plurality of zones of target polymkeric substance.
100. Thereby 100, a kind of existence whether test kit by analysis of cells target biological polymer detection of biological polymkeric substance in prestained sample, this sample has complete substantially cytolemma, it is characterized in that, and test kit contains:

     Hybridization solution, this hybridization solution contains denaturing agent, hybrid stability agent, buffer reagent and film pore former.
101. 101, as the described test kit of claim 100, it is characterized in that, also contain:

     Probe, thus described probe can form hybridization complex with described target biological polymer hybridization.
102. 102, as the described test kit of claim 100, it is characterized in that, also contain,

     Be used for thereby described suspicious specimen is contacted the equipment that forms described hybridization complex with described probe; With

     Be used to measure the equipment whether described probe exists.
103. 103, as the described test kit of claim 100, it is characterized in that, also contain,

     Can detect the detectable marker that hybridization forms thing.
104. As the described test kit of claim 103, it is characterized in that 104, the detection that described hybridization forms thing is quantitative.
105. 105,, it is characterized in that described detectable is the marker that releases energy as the described test kit of claim 103.
106. 106, method as claimed in claim 51 is characterized in that, contact is carried out in solution.
107. 107, method as claimed in claim 51 is characterized in that, detects by flow cytometry.

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