MX2008001353A - Method for improving cell permeability to foreign particles - Google Patents

Abstract

The present invention provides a method for allowing foreign particles to penetrate, very efficiently, the cellwall, cell membrane, organelle membrane and/or nuclear membrane of a cell and hybridizing or binding to the complimentary target in the cell. The cells may be from a culture or from specimens obtained from a patient. The foreign particle can be a probe consisting of, for example, either individually or in any combination of two or more of the following:DNA, RNA, peptide nucleic acids (PNA), glycopeptides, lipopeptides, glycolipids or prions. The target is a cell, a cell component or, preferably, a pathogen or pathogen component. The pathogen can be, for example, bacteria, fungi, yeast or viruses.

Description

METHOD FOR IMPROVING CELL PERMEABILITY TO STRANGE PARTICLES Field of the Invention The present invention relates to compositions and methods for improving cellular permeability to foreign particles including the probes of the present invention. BACKGROUND Cells are the basic unit of all living organisms. The common attribute of almost all cells is that they are surrounded (or limited) by a cytoplasmic membrane. This membrane houses the internal contents of the cell and regulates the movement of substances inside and outside the cell. Only molecules that can diffuse through the membrane or are transported through it can move in and out of the cell. Some may pass through the lipid core of the membrane, but others must pass through the pores. Still other molecules must cross the membrane attached to vehicles in an energy-dependent manner. Also, the nucleus and other organelles have membranes to regulate the flow of molecules in and out of the organelle. Fixation is a chemical process that "places" cell molecules in place so that the cell or tissue can then be studied. Most agents used as fixatives (e.g., alcohols such as ethanol and aldehydes such as paraformaldehyde) function by crosslinking cellular molecules, especially proteins. This cross-linking process prevents degradation of the cellular structure. Several fixatives are more suitable for the preservation of different molecules and cell structures or for different detection methods. The fixator selected for any particular purpose will be determined by the nature of that purpose. Unfortunately, current fixation methods often hinder the subsequent ability of a researcher or physician to detect internal cellular components. In other words, just what prevents the degradation of the cell, the fixation, can also establish a barrier to the many types of research and diagnosis that depend on larger detection molecules. Because of this, efforts have been made to permeabilize the cells or create channels after fixation. Current methods of permeabilization of the cell membrane after fixation are not effective for all specimens, are very rigorous (therefore, destroy the structures to be studied) and / or require expensive equipment. For example, Hoffman, et al. (U.S. Patent No. 6,835,393) describes the use of polycarboxylic acid polymers and pH to interfere with cell membranes only for use in non-fixed samples. Conelly, et al. , (U.S. Patent Nos. 5,597,688 and 5,422,277) describe the use of a composition with 2,4-dinitrobenzene sulphonic acid, 2,4-dinitrobenzoic acid or 2-dinitrophenol for both cell membrane fixation and permeabilization but these compositions limit the choice of the researcher or doctor's fixator, therefore, they limit the necessary flexibility of the analysis. Commonly mechanical methods such as sonication, electroporation, etc. they commonly only act on non-fixed samples and require expensive equipment. In addition, research and diagnostic methods available from prior techniques for many cellular targets such as pathologies depend on microscopic evaluations, cellular morphological parameters, staining characteristics and the presence or absence of certain targets. However, many of these diagnostic methods are not completely accurate or sufficiently sensitive. What is needed are compositions and methods for improved permeability of cell membranes from specimens to foreign particles such as labeled detection molecules. In addition, what is needed are compositions and methods for the improved detection of cellular targets and pathogens.

SUMMARY OF THE INVENTION In one embodiment, the invention allows the detection of the target or target fragment, directly from the cells in a cell culture or specimen obtained from a patient, by in-situ hybridization. In a preferred embodiment, the cell is a pathogen. The method comprises several steps that are carried out, preferably, but not necessarily, in the order listed. A specimen of the culture or specimen is deposited on a slide. The sample is fixed on the slide either by heat or with a standard fixative. The fixative may be, for example, methanol, acetic acid methanol, acetone, formaldehyde or formalin. The fixed sample is treated with the IDF solutions. { see, Infra), colored or probed and observed. Alternatively, the specimen is mixed with IDF solution, incubated, then rubbed or otherwise placed on a glass slide, air dried and fixed. The IDF solution can comprise any combination of the following reagents: chaotropic salts (e.g., thiosulfate or guanidine hydrochloride), ionic detergents (eg, SDS) and / or non-ionic detergents (eg, IPGEL, deoxycholate, cholate or salts) biliary) or other reagents with similar properties, methanol and acetic acid. The concentration of each reagent in the IDF solution depends, for example, on the cell wall of the pathogen to be detected. Although the present invention is not limited by no theory or mechanism, it is believed that the IDF solution creates "channels" in the cell wall and / or membranes (cellular and nuclear) of the pathogen. These channels allow a probe to penetrate the cell wall and the cell membrane and enter the cytoplasm and / or the nucleus of a pathogen. The probe of the present invention may comprise DNA, RNA, APN, peptide, glycopeptide, lipoprotein or glycolipid or a mixture of any of the foregoing. The targets of the fixed cells in the sample are connected to a probe complex (the probe complex comprising target-specific binding agents) specific to the target under the conditions appropriate for hybridization or binding (eg: as described in U.S. Patent No. 6,165,723 to Shah and Harris, which is incorporated herein by reference). The unhybridized or unbound probe can then be rinsed from the sample. In one embodiment, the rinsed sample can then be stained with an appropriate contrast stain (e.g., Evans Blue, DAPI, potassium permanganate, etc.). The hybridized or bound probe complex is detected visually by, for example, microscope, the presence of the probe complex being an indication of the presence of the cell target. The method can be carried out with different hybridization buffers, the various non-limiting examples of which are described herein and in the U.S. Patent. No. 6,165,723 of Shah and Harris, which incorporated herein by reference. The hybridization buffer used is determined by the nature of the probe used. The method of the present invention is useful for detecting cells, cellular constituents and, preferably, pathogens in a specimen. In an exemplary manner, specific non-limiting probe complexes that are useful for detecting pathogens of the Microbacteria species are described herein. The methods of the present invention are useful, for example, in the detection of nucleic acids, peptides, glycopeptides, lipopeptides and glycolipids from a wide variety of specimens. Exemplary specimens include, for example, cells, cell types, tissues or a pathogen or pathogens of interest including or derived from, e.g., serum, plasma, sputum, urine, cerebral spinal fluid, tissues and breast milk. The compositions and methods of the present invention can be used in specimens of any organism including, but not limited to, mammals, reptiles, fish, birds, vegetables and insects. In one embodiment, the present invention contemplates a composition (IDF solution) for increasing the permeability of cell walls, cell membranes, organelle membranes and nuclear membranes, said composition comprising in one embodiment: GuSCN (guanidine thiocyanate), Tris-HCL , EDTA, IGEPAL (octylphenoxy) poly (ethyleneneoxy) ethanol), acetic acid, methanol, sodium cholate and sodium deoxycholate. The present invention further contemplates that the GuSCN is at a concentration of about 2.0 to 3.3 M; Tris-HCL is at a concentration of approximately 10 to 100 mM; Tris-HCL is at a pH of about 7.0 to 9.0; EDTA is at a concentration of approximately 5 to 50 mM; the IGEPAL is at a concentration of approximately 0.1 to 2.0 percent; acetic acid is found at a concentration of approximately 0.1 to 10.0 percent; the methanol is at a concentration of about 20 to 50 percent; Sodium cholate is at a concentration of approximately 0.02 to 2.5 percent and sodium deoxycholate is at a concentration of approximately 0.02 to 2.5 percent. In another embodiment, the GuSCN buffer is replaced with a GuHCL buffer between approximately 2 M to 6 M. Even in another embodiment, the IGEPAL is replaced with SDS between approximately 0.01% and 2.0%. In yet another mode the GuSCN is used in conjunction with GuHCL and / or IGEPAL is used in conjunction with SDS. In one embodiment, the present invention contemplates a method for coloring a target in a cell, comprising: a) contacting the cell with a composition comprising GuSCN (guanidine thiocyanate), Tris-HCL, EDTA, IGEPAL (octylphenoxy poly (ethyleneoxy) ethanol), acetic acid, methanol and sodium deoxycholate to create a permeabilized cell; b) contacting the permeabilized cell of step (a) with a specific binding agent to bind to said target, and; c) detecting said binding agent from step (b). In other aspects, the invention contemplates that the objective of the above method is selected from, for example, nucleic acids, peptide nucleic acids, peptides, glycoproteins, lipids, lipoproteins, viruses, prions and microplasma. In other embodiments, the present invention contemplates that the binding agent is selected from a group consisting of nucleic acids, peptide nucleic acids, peptides, lipoproteins, glycoproteins, antibodies or fragments of antibody and lipids. The binding agent of the present invention may additionally comprise a detection residue and the detection residue may be selected from a group comprising, for example, fluorescent labels, radioactive labels, dyes, colloidal metals, biotin / avidite, horseradish peroxidase, etc. In a preferred embodiment, the detection is by an antibody labeled with affinity for the target antigen. A binding agent comprising a detection residue is defined herein as a probe complex. In one embodiment, a clinical sample is treated with IDF solution in the tube, followed by boiling to release the nucleic acid in the solution. This technique is effective for targets such as microbacteria, fungi and yeasts that require mechanical lysis (e.g., by sonication) or prolonged incubations with enzymes to digest cell walls, for example. The target of interest can then be purified by (1) standard DNA purification techniques or (2) by intercalated hybridization using specific probes. The DNA and RNA of the purified target can then be amplified by PCR or RT-PCR respectively, if necessary, before detection. In a more preferred embodiment, the target is a nucleic acid of the microorganism Mycoba cterium tuberculosis and the binding agent is an oligonucleotide (or PNA probe) complementary to the nucleic acids of the microorganism Mycobacterium tuberculosis. In another aspect, the method also comprises background coloration to better highlight or visualize the detection residue. Background dyes and coloring techniques are known to those skilled in the art. DETAILED DESCRIPTION OF THE INVENTION The present invention is based on the discovery of an improved method to allow the probe to penetrate the cell wall and / or cell membrane of a cell (eg, a pathogen) to directly detect the presence of a target nucleic acid protein, peptide, lipopeptide, glycopeptide, lipid, etc., in cells of a culture or specimens obtained from a individual (eg, blood stained, biopsies, embedded paraffin tissues and markings), by in-situ hybridization. The method of the invention is particularly well suited to detect nucleotide sequences specific to pathogens that are found within, for example, sputum, whole blood, central spinal fluid (CSF), other bodily fluids or infected tissues. More specifically, new improvements to traditional methods of fixation / pretreatment are described that allow probes (eg, oligonucleotide probes) to penetrate into cells (eg, pathogens such as bacteria, viruses, fungi, yeast and protozoa), which they can be located either inside or outside infected host cells. In addition, a method with a contrast dye (e.g., DAPI, Evans Blue, potassium permanganate) after hybridization with fluorescent label probe allows the organisms that retain the hybridized probes to be easily visualized in culture or in clinical samples. The pre-treatment methods of hybridization in itself, the detection techniques and new and unique compositions of the present invention described herein allow the use of recombinant DNA, RNA, APN, peptide, glycoproteins, lipids and glycolipid probes in cells, mocroorganisms or sections of tissue and are compatible with routine microscopic examination performed in laboratories of bacteriology, parasitology, histology or pathology. The present invention applies, for example, a nucleic acid probe of predetermined nucleotide sequence to the sample (or tissue) cells and to the examination of the sample by, for example, microscopy, electron microscopy, flow cytometry or radioactive display ( eg, X-ray film, phosphorus visualization), to determine those cells (or tissues) within the population that contain specific targets (eg, nucleic acid sequences) of interest. Thus, in infected whole blood spots or in sections of tissue, pathogenic organisms such as bacteria, viruses, protozoa or fungi can be detected in infected cells. Such protocols provide very useful diagnostic and scientific information since the presence or absence of a specific nucleic acid can be correlated with one or more cells of observable structure and morphology, and in this way, provide a basis for medical diagnosis and prognosis. The method for detecting a target nucleic acid fragment directly from a specimen comprises the steps to be carried out, preferably, in the (s) order (s) listings. A specimen, usually obtained from an individual, is first deposited on a slide. The sample is fixed on the slide with fixative (e.g., methanol, acetic acid methanol fixative or an acetic acid formalin fixative). Once the sample is fixed, the sample cells are permeabilized with the compositions and methods of the present invention. Alternatively, the specimen is mixed with IDF solution in a tube, incubated and then deposited on a slide, dried by air and fixed. Next, the cells come into contact with a specific probe for a target under conditions suitable for hybridization. After a suitable period of hybridization, any unhybridized probe in the mixture is rinsed. In a preferred embodiment, the sample comes in contact with a contrast dye (e.g., DAPI, Evans Blue, potassium permanganate, etc.). Although the sample was stained with contrast dye, the probes that have hybridized to the target of the sample are visually detected by, for example, microscopy. The presence of probes within the sample is an indicator of the presence of the target fragment. The contrast of the sequence at the same time or sequentially with the hybridization analysis in si of the present invention improves the method allowing, for example, a clearer determination of the location of the target inside the sample. Such information helps, for example, to provide a clearer determination of background hybridization. This method is suitable for use with a specimen obtained from an individual. This includes, without limitation, whole blood, serum, plasma, sputum, urine, breast milk, brain spinal fluid and tissue. This method is also suitable for the detection of a pathogen or other target within the cells of an insect vector, insect cell, plant cells, fungus and bacteria. The purpose of fixing cells or tissues is to immobilize the cells and preserve the morphology of the cells or tissue in such a way that the constituents of the cell such as, for example, RNA are retained within the cell matrix during hybridization in itself. The preferred method therefore utilizes a fixative which is capable of preserving and retaining nucleic acids from the cell and at the same time crosslinking and / or precipitating the proteins in the cell matrix in such a way that the cell or tissue remains substantially in the open configuration for the cell. penetration of the probe and subsequent hybridization. In a preferred embodiment, the probes of the present invention comprise, for example, synthetically or biologically produced nucleic acids (DNA, RNA and equivalents); peptide nucleic acids (ANP; equivalents); peptides (and equivalents) that contain specific nucleic acid or peptide sequences that hybridize under stringent conditions to specific cellular targets. In another embodiment, the probes of the present invention comprise glycopeptides, lipopeptides and prions or prion-type molecules (or their equivalents) produced synthetically or biologically, which bind under strict conditions to specific targets within the cell. The probe complex is defined as a probe comprising a marker portion suitable for detection. If the probe is a nucleic acid, the marker portion adheres either to the 5 'end, the 3' end, internally, or in any combination thereof. The preferred marker portion is an identification mark such as radiolabel (e.g., p32, I125, H3), a bio-color label or a fluorescent label. Alternatively, the probe has a labeled polydeoxynucleotide fin that is used for detection of the probe complex. The probe complex may also comprise a plurality of different nucleic acid sequences, APNs, peptides, glycopeptides, lipopeptides or prions or any combination thereof comprising one or more labels with a marker portion. If more than one of the probe portions are marked it may be beneficial to mark each of the probe portions with a different marker portion.

The nucleotide sequence of an oligonucleotide probe is substantially complementary to at least a portion of the target nucleic acid. The target nucleic acid is either a nucleic acid naturally present within the fixed cell or tissue or, alternatively, that is not naturally present in the cell or tissue and is associated with an abnormal or pathological state. Each probe complex molecule is preferably comprised of a DNA or RNA fragment that varies in size from about 10-50 nucleotides. Peptide probes include, for example, antibodies and other molecules that are known to bind to a defined objective or range of objectives. Examples of probes without antibody include, for example, enzymes and enzyme substrates and their effector portions. Additionally, known drugs or chemicals can selectively bind target proteins (e.g., antibiotics can bind bacteria). Lipopeptides, for example, are very useful for detecting portions of lipid in a cell including specific organelles or portions of organelles and bacteria internalized in a cell. The glycopeptides, for example, interfere with the aggregation platelet and, therefore, can be used as necessary target molecules in the platelet function thus helping in the search and diagnosis of coagulation abnormalities. The prions, or portions thereof may be used, for example, as probes for neurological tissues. In the same way, prions can be targets in fixed samples. In a preferred embodiment, the probe is added to the sample in excess of the target (e.g., 10: 1, 100: 1 or 1000: 1). This is effected to handle the hybridization reaction efficiently and to promote a high rate of probe binding: obj ective. The probe complex (comprising, for example, DNA, RN and or APN) comes into contact with the sample target (e.g., nucleic acid) of the fixed sample, generally by adding a probe complex solution to the sample. Exemplary appropriate conditions for hybridization are solutions that provide the proper buffered environment. Some examples of suitable hybridization buffers are: 1) a buffer comprising between about 10% and 50% formamide, 2X.SSC (pH 7.4), and 1% NP40; 2) a buffer comprising between approximately 1.5 M and 4 M GuSCN buffer; 5 M buffer reserve GuSCN is produced from 5 M GuSCN, 100 mM Tris-HCl (pH 7.8), 40 mM EDTA, 1% NP40. This reserve buffer is diluted to the molarity of GuSCN indicated by adding lxTE pH 7.8 to produce the GuSCN buffer molarities referenced above. 3) a buffer comprising between about 2 to 6 M GuHCl buffer; 8 M buffer of GuHCl reserve are produced from 8 M GuHCl, 200 mM Tris-HCl, (pH 7.8), 40 mM EDTA, 1% NP40. The buffer buffer is diluted to the molarity of GuHCl indicated by adding lxTE pH7.8 to produce the molarities of GuHCl buffer referred to above. 4) a buffer comprising a mixture of formamide (20-50%) and GuSCN buffer. (e.g. 0.5 M to 3 M); 5) a damper comprising a mixture of GuSCN damper. (e.g. 0.5 M to 3 M) and GuHCL buffer (e.g. 1 M to 5 M). The specific composition and concentration of hybridization buffer vary with the type of probe or probe complex used. The composition and concentration of buffer is also used, depending on the Tm (melting point: the temperature at which double-stranded DNA separates forming two complementary single strands) from the probe, probe sequence, probe length and hybridization temperature and can be determined by one skilled in the art through the course of no more than routine experimentation.

The present invention is not limited to any particular hybridization temperature. However, it should be appreciated that the use of formamide in the hybridization buffer allows the hybridization to be carried out at a much lower temperature than standard hybridization protocols. For example, hybridization of an average probe complex specifically targeting (and not host cells) in aqueous hybridization buffer such as sodium chloride will generally require a temperature of about 60-65 ° C. The same hybridization carried out at about 42 ° C in the hybridization fluid 1) above, will provide equivalent specificity. Also, the use of GuSCN also allows hybridization to be performed at a much lower temperature than standard hybridization protocols. For example, in an average procedure, hybridization of the probe specifically to a target (and not to host cells) in aqueous hybridization buffer such as sodium chloride will require temperatures of about 60-65 ° C. However, the same hybridization carried out in the GuSCN or GuHCl hybridization buffer above at about 37 ° C will provide an equivalent hybridization specificity. After completing hybridization, the unhybridized probe is rinsed from the mixture, generally by applying a series of washes with a wash buffer. It is within the means of those skilled in the art to appropriately determine wash buffers and wash times. In a modality, the wash buffer comprises 0.3 M sodium chloride, 0.03 M sodium citrate, and 0.1% SDS. Another suitable wash buffer comprises buffered salt phosphate (PBS). After rinsing, the sample can be contrasted. In one embodiment, contrasting the background improves the visualization of the hybridized probes. Preferred contrasts are, for example, DAPI, Evans Blue and potassium permanganate. Other suitable contrasts are known to those of skill in the art. This coloring step is generally applied when the fluorescent labeled probe is used to detect nucleic acids, proteins, glycoproteins and lipoproteins that are specific for a target. Although they are of great help, contrasts are not required for the embodiments of the present invention. The probe is detected by means appropriate to the specific portion used to label the probe complex. The preferred method for detecting fluorescent labeled probes, for example, employs special green, red and blue microscope filters (i.e., fluorescent microscopy). Hybridized labeled radio probes can be detected by, for example, autoradiography and phosphorusvisualization. The probes labeled with biocolorant can be labeled by enzymatic detection systems and such detection systems are commercially available. The method described above allows the simultaneous detection of different pathogens in a single clinical sample by performing a reaction with a probe complex which is comprised of a plurality of different nucleic acid sequences, each labeled with a different marker portion. For simultaneous detection, the different oligonucleotide probes, which are specific for the different nucleotide acids of the different targets commonly present in the specimen, can be designed in such a way that the Tm values (melting point) of all the complex sequences of Probe are very similar. Then each specific oligonucleotide is labeled with a different detectable portion (e.g., different fluorescent portions). Hybridization is carried out with the multiple components of the probe complex. The hybridized sample is processed as described above and the sample is observed by appropriate means for the detection of different labeled oligonucleotides from the probe complex (eg, visualized using appropriate filters if different fluorescent portions are used) to detect which of the targets is found. present in the sample.

It will be recognized by professionals ordinarily skilled in this art that the new pre-treatment protocol for use with the in-hybridization protocol described herein is compatible with all previously known detection methods as well as those described herein. and it is not limited by the detection method used. The hybridization protocol itself has been rationalized in such a way that fewer manipulations are necessary and can therefore be carried out in a short period of time. The embodiments of the present invention also encompass equipment comprising the compositions of the present invention. When such compositions are provided in the form of equipment they will allow the practice of various modalities of the protocols presented herein including those that have been optimized for simplicity and compatibility with a wide variety of detection methods. It is also expected that such prepared kits containing specifically prepared reagents and probes are applicable in medical / diagnostic laboratories, where the ability to detect the presence or absence of specific nucleic acids will serve to positively or negatively identify pathological conditions characterized by the presence of the specific objectives. The diagnostic methods available from the technique Previous for many cellular pathologies depend on microscopic evaluations, cellular morphological parameters, coloration characteristics, and the presence or absence of certain targets. However, many of these diagnostic methods are not completely accurate or sufficiently sensitive. Hybridization in itself using the protocol and specific pathogen probes described above will allow a simpler and more precise identification of targets (including, but not limited to, pathogens) in the samples. The present invention provides a simple pre-treatment protocol for use in hybridization protocols in si tu that provide improved probe penetration into cells and, therefore, improve hybridization and detection characteristics compared to the protocols described above. Improvements include maximizing the sensitivity of the analysis by increasing the hybridization efficiency and detecting specific "signal". Although the present invention is not limited to any particular mechanism, it is believed that the increased sensitivity is due to improved hybridization due to enhanced probe penetration into the cells and, at the same time, to maximized retention of the target (eg. , nucleic acid sequences) in the cell or tissue and, maximizes the preservation of the other biochemical or morphological characteristics of the cell or tissue sample. EXPERIMENTS A preferred and non-limiting use of the above method is in the detection of Mycobacterium um tuberculosis from a culture or sputum. It will be understood and appreciated by one skilled in the art that the new methodology is equally applicable to a wide variety of other systems, cells, tissue cultures and tissues for specific nucleic acid hybridization (or detection of other cellular components of the target cells , tissues or pathogens) of interest with the preservation of cell integrity and compatible morphology. Example The patient's culture or sputum was stained on a glass plate and dried with air. The cultured cells were washed and concentrated by centrifugation. The washed cells were suspended in phosphate buffer with BSA. To render the cells inactive, the suspended cells were boiled for 15 minutes at 100 ° C. Sample Preparation Method 1 Sputum was processed either by 1) NACL / NaOH or 2) NACL / NaOH after boiling the processed sputum for 15 minutes at 100 ° C to make the sample inactive or 3) with a chaotropic solution such as a guanidine hydrochloride or thiosulfate (briefly, 2-3 volumes of 5 M were mixed GuSCN or 8 M GuHCL with sputum). The sample was incubated at 37 ° C for 20 minutes. The sample was centrifuged to granulate the cells. The cells were washed with phosphate buffer saline. The washed cells were suspended in phosphate buffer saline with 1% BSA or 4) a chaotropic solution such as guanidine hydrochloride or thiosulfate after boiling (same as in step 3, above) except that the cells suspended in buffered saline with 1% BSA are boiled for 15 minutes at 100 ° C to destroy Mycrobacteria. The prepared culture or sputum sample was stained on a glass slide and dried with air. The sample was fixed by methanol or acetic acid methanol or ethanol. The stain was treated with the IDF solution (as described by Supra) for 10 minutes. After 10 minutes the stain was washed 3 times with PBS and dried with air. Method of Preparation of Sample 2 An unprocessed volume of sputum from a patient was mixed with two volumes of IDF (Supra) solution in a tube and incubated at room temperature (20-25 ° C) for 15 minutes. The sputum-IDF mixture was stained on a glass slide, dried with air and fixed with methanol. The IDF treatment of the fixed spot before hybridization was omitted. Prior to hybridization the slide was washed with PBS three times.

Sample Preparation Method 3 The sputum-IDF mixture fixed with methanol was treated on a slide with 2% glutaraldehyde in PBS for 5 minutes at 20-25 ° C (room temperature), then rinsed with PBS three times and dried with air. The IDF treatment of the fixed spot before hybridization was omitted. Sample Preparation Method 4 An unprocessed sputum volume of a patient was mixed with two volumes of IDF (Supra) solution in a tube and incubated at about 20-25 ° C (room temperature) for 15 minutes. The sputum-IDF mixture was boiled for 15 minutes to release nucleic acids in the solution and at the same time make the sample non-infectious. The nucleic acids can be purified by standard techniques from the boiled sample or the target nucleic acid of interest can be selected by intercalated hybridization using specific probes and magnetic droplets as described by Shah et al. (Shah J. S., King, Liu J., Smith J., Serpe G. and Popoff S. (1997), Assay Improvements, Analysis Improvements) US Patent. No. 5,629,156.), Which is incorporated herein by reference). The purified target can be amplified by PCR (for a target DNA) or RT-PCR (for a target RNA). Sample Polling An oligonucleotide probe comprising one DNA sequence that hybridizes specifically to the ribosomal RNA S 23 of Mycobacterium tuberculosis as described by Shah, Nietupski and Liu (U.S. Patent No. 5,521,300) is preferably used in the detection of the presence of M. tuberculosis in cells. Examples of suitable probe complexes are: Pl. Probe TB 5 '-Rhodamine Green-AGA-ACA-CGC-CAC-TAT-TCA-CAC-GCG-CGT-ATG-C-3' [SEQ ID NO: 1] 66.5 c P2 - Tb-1 51-2c 5'-Rhodamine Green-TTC-GAG-GTT-AGA-TGC-CC-3 '[SEQ ID NO: 2] P3. Probe Mycobacterium 5'-Tamra-ATC GCC CGC ACG CTC ACÁ GTT AAG CCG TGA GAT TTC-3 ' [SEQ ID NO; 3] 68.7c P4 -Mycobacterium genus -54.1c 5'-Tamra-GCA-TTA-CCC-GCT-GGC-3 '[SEQ ID NO: 4] P5-Burkholderia probe 5' _FAM-CTT-GGC-TCT-AAT -ACA-GTC-GG-3 '[SEQ ID NO: 5] tm 52c PNA probe In one embodiment, this probe complex was contacted with nucleic acids from the fixed / pretreated sample in a 2.5 M hybridization buffer GuSCN, 50 mM Tris (pH 7.8), 20 mM EDTA and 1% NP40 at 37 ° C. In an alternative embodiment, this probe complex was contacted with the nucleic acids of the fixed sample in a 50% formamide hybridization buffer, 2X SSC (pH 7. 4), 20 mM EDTA, 1% NP40 at 42 ° C. Examples of oligonucleotide sequences suitable for use in alternative probe complexes for the detection of Mycobacteria species are: P2-Tb-1 51-2c 5'-Rhodamine Green-TTC-GAG-GTT-AGA-TGC-CC-3? [SEQ ID NO: 2] P3. Probe Mycobacterium 5'-Tamra-ATC GCC CGC ACG CTC AC GTT AAG CCG TGA GAT TTC-3 '[SEQ ID NO: 3] 68.7c P4-Mycobacterium genus -54.1c 5'-Tamra -GCA-TTA-CCC-GCT -GGC-3 '[SEQ ID NO: 4] SEQ ID Nos: 3 and 4 and their complements are suitable for the detection of Mycobacteria sp. SEQ ID Nos: 1 and 2 and their complements are suitable for the detection of M. tuberculosis. SEQ ID NO: 5 is suitable for the detection of Burkholderia sp. The ribosomal RNA sequence is selected for use in the detection of Mycobacteria pathogens due to the high abundance of rRNA in bacterial cells (1,000-10,000 copies). Preferably the oligonucleotide of the probe complex is a DNA with a sequence complementary to M. tuberculosis rRNA. The oligonucleotide is preferably labeled at the 3 'and 5' ends with fluorescein. It should be recognized that an RNA oligonucleotide probe can also be used.

As described above, the total amount of probe is a predetermined amount that must exceed the estimated amount of available rRNA that is believed to be within the sample (approximately 100: 1) to efficiently conduct the hybridization reaction and to promote a high probe tempering rate: objective. In quantitative terms, this requires the use of a probe comprising an oligonucleotide of 30 nucleotides in length at concentrations ranging from 1-10 μg / ml to produce reliable signal on the background. It should be appreciated that the use of GuSCN also allows hybridization at a much lower temperature than standard hybridization protocols. Hybridization of the probe specified specifically to a target (and not to host cells) in aqueous hybridization fluid such as sodium chloride will require a temperature of about 60-65 ° C. However, hybridization performed in the above GuSCN or GuHCl hybridization buffer at about 37 ° C ensures specificity. One of the advantages of the hybridization method in itself is that the relatively low number of cells comprising a sample and large quantities of identical samples can be processed for a short period of time. The unique method of in situ hybridization described is extremely simple. The methods of the present invention they can also be applied to any type of sample, including, without limitation, sections of tissue embedded with paraffin, and fixed samples of acetone. The results of these experiments show the detection of the target (pathogenic DNA) in the samples tested and no detection in the control samples. The detection of the target is consistently better in the samples treated with the IDF solutions of the present invention. The one skilled in the art will appreciate, understand and know that the IDF solutions of the present invention can be used in any situation that requires the effective entry of a probe (or other similar object) into a pathogenic cell (eg, located in a cell) or organelle, without excessive experimentation. It should be evident from the foregoing that the present invention provides compositions and methods for increasing cellular permeability, cell walls, cell membranes, organelles and organelle membranes to aid, for example, in the detection of cellular and / or pathogenic components.

Claims (28)

1. CLAIMS 1. A composition for increasing the permeability of cell walls, cell membranes and nuclear membranes, said composition comprising: GuSCN (guanidine thiocinate), Tris-HCL, EDTA, IGEPAL (octylphenoxy poly (ethyleneoxy) ethanol), acetic acid, methanol, sodium chloride and sodium deoxycholate.
2. 2. The composition of Claim 1, wherein said GuSCN is at a concentration of about 2.0 to 3.3 M. 3. The composition of Claim 1, wherein said Tris-HCL is at a concentration of about 10 to 100. mM. 4. The composition of Claim 1, wherein said Tris-HCL is at a pH of about 7.0 to 9. 0. The composition of Claim 1, wherein said EDTA is at a concentration of about 5 to 50 mM. 6. The composition of Claim 1, wherein said IGEPAL is at a concentration of about 0.1 to 2.0 percent. The composition of Claim 1, wherein said acetic acid is at a concentration of about 1.0 to 10 percent. 8. The composition of Claim 1, wherein said methanol is at a concentration of about 20 to 50 percent. 9. The composition of Claim 1, wherein said sodium cholate is at a concentration of about 0.02 to 2.5 percent. 10. The composition of Claim 1, wherein said sodium deoxycholate is at a concentration of about 0.02 to 2.5 percent. 11. A method for coloring a target in a cell, comprising: a) contacting the cell with a composition comprising GuSCN (guanidine thiocyanate), Tris-HCL, EDTA, IGEPAL (octylphenoxy poly (ethyleneoxy) ethanol), acetic acid, methanol, sodium chloride and sodium deoxycholate to create a permeabilized cell; b) contacting the permeabilized cell of step (a) with a specific binding agent to bind to said target, and; c) detecting said binding agent from step (b). The method of Claim 11, wherein said target is selected from a group consisting of nucleic acids, peptide nucleic acids, peptides, glycoproteins, lipids, lipoproteins, viruses and prions. 13. The method of Claim 11, wherein said binding agent is selected from the group consisting of nucleic acids, peptide nucleic acids, peptides, lipoproteins, glycoproteins and lipids. The method of Claim 11, wherein said binding agent further comprises a detection residue. The method of Claim 14, wherein the detection residue is selected from a group consisting of fluorescent labels, radioactive labels, dyes, colloidal metals, biotin / avidin peroxidase and radish. 16. The method of Claim 11, wherein said detection is effected by an antibody labeled with affinity for said binding agent. The composition of Claim 11, wherein said GuSCN is at a concentration of about 2.0 to 3.
3. 3 M. 18. The composition of Claim 11, wherein said Tris-HCL is at a concentration of about 10 to 100. mM. 19. The composition of Claim 11, wherein said Tris-HCL is at a pH of about 7.0 to 9.0. The composition of Claim 11, wherein said EDTA is at a concentration of about 5 to 50 mM. 21. The composition of Claim 11, wherein said IGEPAL is at a concentration of about 0.1 to 2.0 percent. 22. The composition of Claim 11, wherein said acetic acid is at a concentration of about 1.0 to 10 percent. 23. The composition of Claim 11, wherein said methanol is at a concentration of about 20 to 50 percent. 24. The composition of Claim 11, wherein said sodium cholate is at a concentration of about 0.02 to 2.5 percent. 25. The composition of Claim 11, wherein said sodium deoxycholate is at a concentration of about 0.02 to 2.5 percent. 26. The method of Claim 11, wherein said target is a nucleic acid of the microorganism Mycrobacterium tuberculosis. 27. The method of Claim 11, wherein said binding agent is an oligonucleotide complementary to a nucleic acid of the microorganism Mycrobacterium um tuberculosis. 28. The method of Claim 11, wherein said method additionally comprises background coloring.