WO1992009704A1

WO1992009704A1 - Method for in situ detection and identification of nucleic acid sequences

Info

Publication number: WO1992009704A1
Application number: PCT/US1991/008760
Authority: WO
Inventors: Grant M. Westlake; David R. Scholl
Original assignee: Diagnostic Hybrids, Inc.
Priority date: 1990-11-29
Filing date: 1991-11-29
Publication date: 1992-06-11
Also published as: AU9137391A

Abstract

Method for the detection of a nucleic acid of known hybridization specificity in the cells of a cell culture, tissue section or direct specimen containing DNA and/or RNA by in situ hybridization analysis comprising: (a) contacting the cells with a solid support in the presence of an alcoholic alkaline solution which contains between about 50 and 90 percent by volume alcohol and has a concentration of between about 0.01M and about 0.5M alkali metal hydroxide, thereby affixing the cells to the solid support, rendering the cells permeable to nucleic acid probe for hybridization analysis, denaturing the DNA and any RNA containing secondary structure, and localizing the denatured DNA and/or RNA in its cellular environment, (b) reacting the cells affixed in step (a) with a hybridization probe having a nucleic acid sequence complementary to the nucleic acid of known hybridization specificity, and (c) analyzing the reaction product of step (b) for the formation of nucleic acid hybrids containing the hybridization probe.

Description

METHOD FOR IN SITU DETECTION AND

IDENTIFICATION OF NUCLEIC ACID SEQUENCES

BACKGROUND OF THE INVENTION

The present invention relates to an in situ hybridization method for detecting the presence of nucleic acids having a known hybridization specificity in cell cultures, tissue samples and direct specimens which is accurate, practical, relatively rapid and amenable to automation.

A critical factor in the clinical management of infectious diseases lies in the establishment of the identity of the pathogen responsible for the infection. Attending physicians necessarily place heavy reliance upon clinical microbiology laboratories to rapidly and reliably identify the pathogen so that a regimen of treatment can be initiated as soon as possible.

Several approaches have been used by clinical microbiology laboratories for the detection of pathogenic viruses. In one, a confluent culture of anchorage

dependent cells is inoculated with a clinical specimen suspected of containing a pathogenic virus. If present in the clinical specimen, the virus present will infect one or more cells of the culture and grow from cell to cell. Because virus infected cells develop characteristic

antigens on their surfaces, the infected cells can be detected by means of an immunoassay after allowing a sufficient period for growth. Conveniently, the anchorage dependent cells have been cultured in multiple well plates thus enabling clinical microbiology laboratories to

simultaneously analyze a plurality of specimens for antibody-antigen interaction.

Although the immunoassay technique offers convenience and efficiency, there are certain disadvantages. For instance, a mutation occurring in the reactive region of an antigen may alter the affinity of a specific antibody for that region, or the antigen may be repressed. In addition, for some pathogens reactive antibodies have not been found.

Accordingly, other diagnostic approaches have been proposed. Scholl et al., U.S. Patent No. 4,652,517 disclose a nucleic acid hybridization method for the detection and identification of unknown pathogens through the use of a porous, inert, positively charged solid support. According to one aspect of the invention disclosed and claimed therein, the nucleic acid of a specimen suspected of containing a pathogenic virus is liberated from the specimen in single stranded form and deposited on the solid support whereby the specimen nucleic acid migrates by capillary action and becomes affixed at areas on the support to which it has migrated. The resulting support carrying the fixed single stranded nucleic acid is hybridized with a probe having a nucleotide sequence complementary to that of a suspected pathogenic virus. The presence of hybrids between probe and immobilized nucleic acid thus reveals the presence of and identifies the pathogenic virus. In some instances, the nucleotide sequence is known. In other instances, while the nucleotide sequence has not been determined

hybridization specificity has been confirmed.

While the approach disclosed by Scholl et al. is a rapid, practical and accurate method for detecting pathogens directly from clinical specimens, this method is not easily amenable to automation. Users typically must handle each solid support individually. Thus, screening a single clinical specimen for a variety of pathogenic viruses can require the expenditure of a substantial amount of effort by users. In addition, the disclosed technique suffers from some sensitivity constraints and does not conveniently enable the user to determine the spatial relationship of positive cells in culture or the location of target nucleic acid within infected cells.

In contrast, in situ hybridization techniques have proven to be a valuable and highly sensitive method for the localization of specific cellular or chromosome nucleic acid sequences. In situ hybridization was originally described by Gall and Pardue, Proc Natl Acad Sci USA 63: 378-383 (1969) and involves the annealing of labeled polynucleotide probes to their complementary denatured sequences in cells and detection of the hybridized labeled probe. In situ hybridization is ideally suited to the detection of cellular nucleic acid sequences which are nonuniformly distributed in tissues and cells and has been used to detect target viral nucleic acid sequences in tissue sections and cell cultures.

Unfortunately, in situ hybridization protocols disclosed to date require a number of relatively time-consuming sample fixation steps. For instance. Van Prooijen-Knegt et al., Exp. Cell Res. 141:397-407, 1982, disclose a method for the preparation of glass slides for in situ hybridization analysis. The slides are cleaned by incubation overnight in a 10% solution of Extran MA01 (alkalisch, E. Merck, Darmstadt) in deionized water, rinsed with hot (60ºC) tap water and with deionized water, and dried at 80ºC. The slides are then incubated for 16 hours in a 2% (v/v) solution of 3-aminopropyltriethoxysilane in dry acetone, and rinsed in acetone and two changes of deionized water. In order to remove possibly present endogenous RNA, the slides with tissue section applied were treated with a solution of 100 μg RNase A plus 1 μg RNase T₁ per ml in 2×SSC for 2 hours at room temperature. A few drops of the RNase solution were layered over the preparation which was then covered with a cover glass and incubated in a moist chamber. After incubation, the slides were treated in 70% ethanol (twice), 90% ethanol (twice), and 100% ethanol to dehydrate the cells. Each alcohol dehydration was for 5 minutes after which the slides were air-dried. The DNA in the preparation was then denatured with freshly prepared 0.07 N NaOH for 3 minutes followed by rinses in 70% ethanol (twice) and 100% ethanol, for 1 minute each, and air-dried. After this pretreatment, hybridization was performed.

Landegent et al., Exp. Cell Res. 153 : 61-72 (1984) discloses an in situ hybridization protocol in which the slides were reported to have been prepared as outlined in Van Prooijen-Knegt et al., Exp. Cell Res. 141:397-407, 1982 with minor modifications. After RNase incubation (1 hour at 37ºC) to remove endogenous RNA, chromosomal DNA was denatured by incubation in 0.15N NaOH in 70% ethanol for 5 minutes. They report that alcoholic alkali was used instead of aqueous alkali (0.07N) to keep the DNA fixed during denaturation and that this step was critical to preservation of morphology. Slides were then dehydrated through an ethanol series, air-dried, treated with proteinase K (0.25 μg/ml, 2mM CaCl₂, 20 mM Tris-HCl, of pH 7.4) for 15 minutes at 37°C and dehydrated again. It was also reported that to obtain optimal results, the

concentration of proteinase K was sometimes slightly varied depending on the biological object under investigation.

Thus, while nucleic acid hybridization has been recognized and used in the detection of pathogenic viruses in clinical specimens, a need remains for a method for detecting pathogenic viruses which is rapid, accurate, and amenable to automation, and which enables the user to determine the spatial relationship of positive cells in a culture or the location of target nucleic acid within infected cells. SUMMARY OF THE INVENTION

Among the objects of the invention, therefore, is the provision of a practical, accurate and relatively rapid method for the in situ detection of a target nucleotide sequence in cell cultures, tissue samples and direct specimens; the provision of such a method which requires relatively few steps to affix the cells of the culture, tissue sample or direct specimen to a solid support, render the cells permeable to probe, and denature and affix the cellular nucleic acid within its cellular environment; the provision of such a method in which hybridization of probe to target is readily and conveniently detected; the provision of such a method which is amenable to automation; and, in the case of cell cultures, the provision of such a method in which the cells are affixed to the same solid support for in situ hybridization analysis as the solid support upon or in which the culture is grown.

Briefly, therefore, the present invention is directed to a method for the detection of a nucleic acid of known hybridization specificity in the cells of a cell culture, tissue section or direct specimen containing DNA and/or RNA by in situ hybridization analysis. The method comprises contacting the cells with a solid support in the presence of an alcoholic alkaline solution which contains between about 50 and 90 percent by volume alcohol and has a concentration of between about 0.01M and about 0.5M alkali metal hydroxide, thereby affixing the cells to the solid support, rendering the cells permeable to nucleic acid probe for hybridization analysis, denaturing the DNA and any RNA containing secondary structure, and localizing the denatured DNA and/or RNA in its cellular environment. The affixed cells are thereafter reacted with a hybridization probe having a nucleic acid sequence complementary to the nucleic acid of known hybridization specificity. The reaction product is then analyzed for the formation of nucleic acid hybrids containing the hybridization probe. The present invention is additionally directed to a method for screening a clinical specimen for a viral nucleic acid by in situ hybridization analysis. The method comprises growing a line of anchorage dependent cells on a solid support, contacting the anchorage dependent cells with the clinical specimen whereby viral nucleic acid present in the specimen infect the anchorage dependent cells, and amplifying the viral nucleic acid in any infected cells. The anchorage dependent cells are

thereafter contacted with an alcoholic alkaline solution which contains between about 50 and 90 percent by volume alcohol and has a concentration of between about 0.01M and about 0.5M alkali metal hydroxide, thereby affixing the anchorage dependent cells to the solid support, rendering the anchorage dependent cells permeable to nucleic acid probe for hybridization analysis, denaturing the DNA and any RNA containing secondary structure present in the anchorage dependent cells, and localizing the denatured DNA and/or RNA in its cellular environment. The affixed cells are thereafter reacted with a hybridization probe having a nucleic acid sequence complementary to the viral nucleic acid, and the reaction product thereof step is analyzed for the formation of nucleic acid hybrids containing the hybridization probe.

The present invention is further directed to a method for detection of a nucleic acid of known hybridization specificity suspected of being present in the cells of a cell culture, tissue section or direct specimen by in situ hybridization analysis. The method comprises affixing the cells to the surface of the well of a multiple well tissue culture plate by contacting the cells with the surfaces of the well in the presence of an alcoholic alkaline solution which contains between about 50 and 90 percent by volume alcohol and has a concentration of between about 0.01M and about 0.5M alkali metal hydroxide, thereby affixing the nucleic acid to the solid support, rendering the cell permeable to nucleic acid probe for hybridization analysis, denaturing the DNA and any RNA containing secondary structure present in the cell, and localizing the denatured DNA and/or RNA in its cellular environment. The affixed cells are reacted with a hybridization probe having a nucleic acid sequence complementary to the nucleic acid of known hybridization specificity, and the reaction product thereof is analyzed for the formation of nucleic acid hybrids containing the hybridization probe.

Other objects and features of the invention will be in part apparent and in part pointed out hereinafter. BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a perspective of a commercially available, prior-art, multiple well tissue culture plate.

Fig. 2 is a photograph of an infected cell monolayer after hybridization with labeled probe as

described in Example 2.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

As used herein, the term nucleic acid shall mean RNA or DNA, whether single or double stranded. The terms "affix" and "affixation" as used with respect to anchorage dependent cells shall mean causing the cells to adhere to a solid support with an affinity greater than that which is observed for anchorage dependent cells growing on a solid support surface of that type without modification. The terms "affix" and "affixation" as used with respect to suspension cells, tissue samples and direct specimens shall mean causing the suspension cells, tissue sample and direct specimen to adhere to a solid support. In accordance with the present invention, it has been discovered that clinical specimens such as blood, urine, sputum (e.g., throat swabs), vaginal swabs, or stool samples may be accurately and rapidly screened for the presence of pathogenic viruses by an in situ hybridization method which is amenable to automation. In one embodiment, the clinical specimen is contacted with an anchorage dependent cell culture being grown on a. solid support under conditions suitable for the infection of the cells by pathogenic virus(es) present in the specimen. Preferably, the anchorage dependent cells are substantially confluent at the time of contact and are selected from among African green monkey kidney cells (CV-1), human embryonic lung cells (HEL) isolated in 1980, human embryonic lung cells (MRC-5) isolated in 1966, human epithelial cells (Hep-2) isolated in 1952, or human embryonic lung cells (WI-38) isolated in 1952, all of which are commercially available from Whittaker M.A. Bioproducts (Walkersville, MD).

After a suitable period for viral amplification, e.g., about 24 hours for Herpes simplex virus, the cells are rapidly and reliably affixed to the solid support upon which they are being grown by contacting the cells with the fixative solution of the present invention. Significantly, such treatment additionally renders the cells permeable to a nucleic acid probe for hybridization analysis, denatures the cellular and viral deoxyribonucleic acid, eliminates potential secondary structure in ribonucleic acids, and affixes the treated nucleic acids to their localized cellular environment.

The fixative solution is an aqueous-based, alcoholic alkaline solution containing between about 50 and about 90% by volume, preferably between about 60 and about 80% by volume, and most preferably about 70% by volume ethanol or isopropanol. The fixative has a concentration of between about 0.01M to about 0.5M, preferably about 0.07M, alkali metal hydroxide, preferably potassium or sodium hydroxide, most preferably KOH.

Optionally, the fixative solution may

additionally comprise about 0.01M magnesium chloride

(MgCl₂) and/or ammonium acetate (CH₃COONH₄). Inclusion, of either composition, preferably magnesium chloride alone or in combination with the ammonium acetate, results in an improvement in the morphology of the fixed cells. That is, in some instances the cells are easier to visualize when magnesium chloride and/or ammonium acetate are used; without such compounds, in some instances the cells appear to shrink.

The solid support is glass, a plastic such as polystyrene or a methacrylate, or equivalent material. Significantly, it has been found, that the cells may be affixed to the solid support without the need for pretreatment; the cells are directly affixed onto the solid support surface as obtained from the manufacturer. For visual in situ examination, i.e., where the probe is enzyme, fluorescent or dye labeled, the solid support is preferably substantially transparent. However, for radioactive or rare earth labels, opaque solid supports may be used.

Conveniently, where a number of cell cultures are to be assayed using in situ hybridization techniques, a solid support which contains a plurality of wells, such as a multiple well tissue culture plate may advantageously be used for culturing and/or assaying each individual

culture. Particularly preferred for this purpose are clear, polystyrene microtiter dishes sold under the Falcon trademark (Becton Dickinson and Company; Lincoln Park, New Jersey).

Depicted in Fig. 1 is a typical commercially available multiple well tissue culture plate, designated generally by reference numeral 1. The tissue culture plate 1 has a lid 3 and bottom tray 5 which fits over and encloses the upper surface 6 of the bottom tray. The bottom tray has twenty-four wells 7 having cylindrical side walls 9 and flat bottoms (not shown). The bottom tray 5 has a base 11 having a length L of about 5 inches (127 mm) and a width W of about 3 1/3 inches (85 mm). The lid 3 has raised rings 13 on the inside face 15 which correspond in number to and contact each of the side walls 9 and thereby seal each of the wells 7 when the lid is placed over the upper surface of the bottom tray. Conveniently, tissue culture plates of the type depicted in Figure 1 are available with a standardized base but with different numbers of wells, e.g., 6, 24, and 96 wells. Such

standardization enables the user to acquire and utilize equipment designed to handle tissue culture plates of a predetermined size but which may vary in terms of the number of wells per plate as dictated by the application.

In addition, tissue culture plates of the type depicted in Figure 1 which contain live colonies of anchorage dependent cells are also commercially available from Diagnostic Hybrids, Inc. (Athens, Ohio). Such culture containing plates are particularly efficacious when used in accordance with the present invention.

The anchorage dependent cells are affixed to a solid support by contacting the cell culture being grown on the solid support with the fixative solution for a brief period, e.g., about 5 minutes. During this brief period, the cells are rendered permeable to single stranded nucleic acid probe for hybridization analysis, that is, the probe can thereafter diffuse through the cell wall and interact with the target nucleic acid. In the same step, the deoxyribonucleic acid and/or ribonucleic acid which has formed secondary structure in the affixed cells undergoes denaturation, and the nucleic acids are localized in the cellular environment, i.e., it becomes bound to a local component or structure in such a manner that it will not be washed away during in situ analysis. It is presently unknown whether the affixed nucleic acid is bound to a cellular component such as protein or to the solid support.

Tissue sections, direct specimens and suspension cell cultures may also be affixed to a solid support in accordance with a method of the present invention. In each instance, the tissue section, direct specimen or suspension cell culture is brought into contact with the solid support in the presence of the fixative solution for a brief period, again about 5 minutes. In the case of suspension cell cultures, the cells are preferably concentrated on the support by centrifugation prior to contact with the fixative solution. As described above with respect to anchorage dependent cells, the fixative will affix the cells of the tissue section, direct specimen or suspension cell culture to the solid support, render the cells

permeable to probe, denature dsDNA and potential RNA containing secondary structure, and localize the thus treated DNA and RNA in the cellular environment.

In a preferred embodiment of the present invention, the tissue section, direct specimen or cell culture is separately contacted with an ethanol solution of at least about 70% by volume, most preferably at least about 95% ethanol by volume, prior to use of the fixative. In addition to disinfecting the tissue section, direct specimen or cell culture, permeability of the cells to hybridization probe is improved.

The methods and fixative solution of the present invention are particularly useful for detecting the presence of unknown pathogens, particularly viruses in a clinical specimen. The clinical specimen is cultured in accordance with standard technique, e.g., the specimen is added to a well of a microtiter dish containing an appropriate growth medium and incubated for a period of time, e.g., incubated for 24 hours in a CO₂ incubator set at 5% CO₂. Preferably, the dish is briefly centrifuged, e.g., in a swinging bucket rotor for 10 minutes at 700×g, after the specimen is added to the well. Upon completion of incubation, the well is screened for high levels of cytopathic effect ("CPE") and/or cell toxicity, the culture fluid is aspirated from the well and the plate is then disinfected, preferably by being submerged in ethanol (e.g., 95% EtOH for 2 minutes at room temperature).

Upon removal of excess ethanol from the disinfected well, the fixative solution is added and the well is incubated for about 5 minutes at room temperature. After the fixative is aspirated from the well, in situ hybridization and probe detection is carried out in accordance with standard technique, i.e, reagent containing a nucleic acid probe is added to the well and the well is incubated at about 55ºC for about 30 minutes. The wells are then rinsed and analyzed for the presence of probe.

The selection of a probe is based on the principle that in any well defined species of pathogen, there are specific nucleotide sequences in the genetic material that are unique to that species. These unique sequences thus serve as a target for detection by hybridization with a labeled probe having a complementary sequence of nucleotides.

The probe may be labeled in accordance with any manner known in the art. For instance, the probe may be radioactively labeled with radioactive hydrogen (³H), phosphorous (³²P), sulfur (³⁵S), or iodine (¹²⁵I).

Alternatively, the probe may be chemically tagged with fluorescent or luminescent labels, rare earth metals biotin, enzymes, antibodies, haptens and the like. When radioactive labels are used, hybridization events can be detected by direct count of the support or by release of the labeled probe using denaturant. In the latter case, the labeled probe would be present in the supernatant and then transferred to a counting vial. In the case of nonradioactively labeled probes, hybridization events may be detected by microscopy or quantitative analysis. For example, enzyme labeled probe containing alkaline

phosphatase could be visualized by microscopy, quantitated by spectral analysis, or filmed and/or quantitated using chemiluminescent substrates.

Significantly, the method of the present invention is amenable to automation. In view of the relatively low number of reagents and steps necessary to culture, disinfect, fix, and detect the presence of target nucleic acid in accordance with the present invention, one or more of these steps may be carried out with the aid of a machine. Thus, the machine may even analyze each well of a multi-well microtiter dish for the presence of labeled probe.

The method of the present invention is broadly applicable to the affixation of any cell culture, tissue section or direct specimen for in situ hybridization analysis. Such analysis may be for the purpose of localization of virus infected cells, detection of gene amplified products, assay of gene expression in transfected cells, localization of gene expression to individual cells within a tissue, subcellular localization of mRNA or detection of DNA integrated into the genome of cells.

The following Examples will illustrate the invention. EXAMPLE 1

Multiple microtiter polystyrene plate wells containing tissue culture cells were inoculated with Herpes simplex virus (HSV). The virus amplifies by culture to a level of cytopathic effect (CPE) characteristic of 30-40 percent CPE within a 36 hour incubation period. After viral amplification, the viral-infected cell wells were treated with a particular fixative. Detection of HSV DNA was done with an enzyme-labeled HSV DNA probe. All

hybridization assays were done under identical hybridization conditions. Several replicates of uninfected negative cell wells were included in the analysis.

There were two means used to monitor the effect of a particular fixative after hybridization. First, cell wells were reacted with a soluble color substrate p-nitrophenylphosphate (pNPP). This substrate turns yellow in proportion to the amount of enzyme present in the cell well. The amount of color in the well is subsequently determined by the absorbance of the solution at 405nm. The greater the optical density of the solution, the greater the amount of enzyme present in the well.

The second means used to evaluate the effect of fixative on hybridization was to remove the soluble color substrate from each well, rinse the wells with a buffer, and then incubate the wells with an insoluble color generating system (BCIP/NBT) which caused a dye to deposit at the location where the enzyme was present. The insoluble color which formed has a purple cast when hybridization occurs in specific organelles, such as the nucleus of the cell. The nuclei of infected cells were identified by observing the purple dye in each nucleus with a microscope (40X magnification; See Figure 2).

The results are presented in Table 1 and demonstrate several points. First, the basal reagent of 70% basic ethanol (BE), i.e., a reagent which contains 70% ethanol and 0.07 M sodium hydroxide, provides fixation and hybridization reactivity comparable to either 60% or 80% ethanol, or 70% BE which is supplemented with ammonium acetate ("NH₄Ac") and/or magnesium chloride. Second, the use of 95% ethanol to fix the cells first followed by the combination reagent of 70% BE enhances reactivity approximately three-fold. Third, the use of 95% ethanol, followed first by HCl and subsequently by sodium hydroxide provides no reactivity at all. As a matter of fact, when these monolayers are inspected by microscopy it is apparent that the cells have not remained fixed to the polystyrene surface and thus were removed from the assay after the fixation step.

The results are tabulated according to the optical density values for positive and negative wells resulting from each of the reagent compositions tested. These results, and the reagents tested, are listed in Table 1 below.

TABLE 1

Eixative Composition Tested

70% BE, 10mM MgCl₂, 95% EtOH 95% EtOH

Infection 70% 60% 80% 70% 70%

Level BE* BE BE BE/NH₄Ac BE/MgCl₂ 10mM NH ₄AC 70% BE HCL NaOH

379

1032** 1054 997 1008 1017 1088 2230

±44 ±177 ±287 ±308 ±98 ±139 ±173 ±414

POSITIVE

90-100%

NEGATIVE

0% 439 345 438 332 378 365 537 327

96 ±198 ±114 ±98 ±67 ±111 ±46 ±197 ±

NET 39 723 1693 52 VALUE 593 709 559 676 6

*BE - Baste Ethanol (0.07M NaOH)

**ALL values are OD_405nm × 10 ^-3

EXAMPLE 2

The procedure of Example 1 was repeated except that the following four compositions were used: 70% ethanol alone; 70mM sodium hydroxide alone; 70% ethanol followed by 70mM sodium hydroxide; and a combination of 70% ethanol and 70mM sodium hydroxide (70% BE). Table 2 lists the positive and negative reactive values for these four conditions.

In summary, the data of Table 2 demonstrate the synergistic effect of the combination of alcohol and sodium hydroxide. All three other conditions showed limited reactivity above the reactivity of the negative control. In the 70% BE fixative, the cells remain attached to the polystyrene surface and thus remain reactive. In the case of the other three reagents, the cells detach from the polystyrene and thus do not provide any reactivity above the negative control. It appears critical to have the combination of alcohol and base in order for optimal fixation and hybridization reactivity. Treatment of the cell culture with 95% ethanol appears to enhance the fixation of the cells prior to the denaturation of the DNA. Subsequent treatment with 70% BE increases permeability in the cell and/or nuclear membrane for probe to pass, and effectively denatures the double stranded DNA to single stranded form. All of this occurs without significant loss of target DNA from the polystyrene surface.

Figure 2 is a photograph showing the infected cell monolayer after hybridization of cells which have been fixed with a reagent which contains the four component fixative (70% BE, 10mM MgCl₂, 10mM NH₄Ac). The photograph depicts four foci of virus infection on the cell monolayer as evidenced by the heavy staining appearing in four focal areas. The smallest stained area indicates the beginning of a HSV focus. The largest represents a focus of virus infection which is relatively mature, i.e., contains a large number of infected cells.

TABLE 2

Effect of Individual Versus

Combination of Reagents on Reactivity

Infection 70% 70mM 70% EtOH 70%

Level (% CPE) EtOH NaOH 70mM NaOH BE

POSITIVE

50% 626* 579 541 1480

±198 ±238 ±55 ±278

NEGATIVE

0% 466 403 445 549

±110 ±44 ±30 ±90

NET VALUE 160 176 96 931

*A11 values are OD_405nm × 10^-3 EXAMPLE 3

Efficiency of HSV Amplification in CVl Cells Cultured on Polystyrene Surface and on Glass Surface

A study was conducted to evaluate the use of a fixative of the present invention (70% ethanol; 0.07 M NaOH, 0.01M MgCl₂ and 0.01M NH₄Ac) on both polystyrene and glass. CVl cells were grown to confluency in polystyrene wells and on glass coverslips placed in individual wells. At three days post-seeding, cell wells were inoculated with HSVI(F) at different levels of virus, centrifuged for 40 min at 700×g for viral adsorption, and incubated at 37ºC, 5% CO₂. At 22 hours post-infection, the two plates, i.e., one with glass coverslips as monolayer support and the other with polystyrene as the support, were fixed and hybridized with a ¹²⁵I-labeled probe. To obtain a direct count of the ¹²⁵I-labeled probe to determine the amount of hybridization that occurred in each well, the ¹²⁵ι probe was released from its hybridized target by first treating the well with a hybrid releasing agent consisting of 1N sodium hydroxide. This treatment effectively denatured the hydrogen bonds which had formed in the hybrid, thus releasing the ¹²⁵I-labeled probe into the releasing agent supernatant. After five minutes of incubation in the solution, the supernatant was transferred to a counting tube and analyzed in a gamma counter.

Table 3 indicates that the hybridization reactivity was similar regardless of whether the cells were fixed to polystyrene or glass. TABLE 3

Comparison of polystyrene and glass

Culture Surface

HSV

Infectious Dose (pfu) Polystyrene Glass 500 3286 ± 151* 3276 ± 197

250 2259 ± 62 2340 ± 156

125 1335 ± 101 1459 ± 115

60 681 ± 135 828 ± 85

30 297 ± 43 509 ± 83

0 0 0 *values represent average of four values, in cpm.

EXAMPLE 4

Following the procedures of Examples 1 and 3 except as indicated, multiple microtiter plate wells containing tissue culture cells were inoculated with Herpes simplex virus (HSV). The virus amplifies by culture to a level of cytopathic effect (CPE) characteristic of 90-100 percent CPE within approximately a 36 hour incubation period. After viral amplification, the viral infected cell wells were disinfected by treating with 95% ethanol for 2 minutes. The 95% ethanol was removed and the cell wells were subsequently treated with a particular fixative. Detection of HSV DNA in individual wells was done with an 1²⁵I-labeled HSV DNA probe. All hybridization assays were done under identical hybridization conditions. Replicates of uninfected negative cell wells were included in the analysis.

The results of positive and negative infected wells, with their respective fixative reagent, are

presented in Table 4 below.

TABLE 4

FIXATIVE COMPOSITION TESTED

Infection Level

Reagent* Positive Negative

70% M/NaOH 1266 ± 27 0

70% M/KOH 1551 ± 110 0

70% E/NaOH 6188 ± 371 0

70% E/KOH 6774 ± 281 0

70% I/NaOH 5296 ± 331 0

70% I/KOH 6277 ± 467 0

100% B/NaOH 841 ± 49 3

* - M, methanol; E, ethanol; I, isopropanol; B, butanol

In view of the above, it will be seen that the several objects of the invention are achieved and other advantageous results obtained. As various changes could be made in the above products and methods without departing from the scope of the invention, it is intended that all matter contained in the above description or shown in the accompanying drawings shall be interpreted as illustrative and not in a limiting sense.

Claims

CLAIMS WHAT IS CLAIMED

1. A method for the detection of a nucleic acid of known hybridization specificity in the cells of a cell culture, tissue section or direct specimen containing DNA and/or RNA by in situ hybridization analysis comprising:

a) contacting the cells with a solid support in the presence of an alcoholic alkaline solution which contains between about 50 and 90 percent by volume alcohol and has a concentration of between about 0.01M and about 0.5M alkali metal hydroxide, thereby affixing the cells to the solid support, rendering the cells permeable to nucleic acid probe for hybridization analysis, denaturing the DNA and any RNA containing secondary structure, and localizing the denatured DNA and/or RNA in its cellular environment b) reacting the cells affixed in step (a) with a hybridization probe having a nucleic acid sequence complementary to the nucleic acid of known hybridization specificity, and

c) analyzing the reaction product of step (b) for the formation of nucleic acid hybrids containing the hybridization probe.

2. The method of claim 1 wherein the solid support is a multiple well tissue culture dish.

3. The method of claim 1 wherein the alcohol comprises ethanol or isopropanol.

4. The method of claim 1 wherein the alkali metal hydroxide is potassium hydroxide.

5. The method of claim 1 wherein the alcoholic alkaline solution has a concentration of between about 0 and about 0.01M magnesium chloride, between about 0 and about 0.01M ammonium acetate, between about 0.01M and 0.5M alkali metal hydroxide and the alcohol consists essentially of ethanol or isopropanol.

6. The method of claim 1 wherein the nucleic acid of known hybridization specificity is a viral nucleic acid.

7. The method of claim 1 wherein the cells are cells of an anchorage dependent cell culture.

8. The method of claim 1 wherein the hybridization probe is labeled.

9. The method of claim 1 wherein the hybridization probe is labeled with a radioisotope, a fluorescent or luminescent marker, a rare earth metal, biotin, an enzyme, an antibody or hapten.

10. A method for screening a clinical specimen for a viral nucleic acid by in situ hybridization analysis comprising:

a) growing a line of anchorage dependent cells on a solid support,

b) contacting the anchorage dependent cells with the clinical specimen whereby viral nucleic acid present in the specimen infect the anchorage dependent cells,

c) amplifying the viral nucleic acid in any cells infected in step (b),

d) contacting the anchorage dependent cells being grown on the solid support with an alcoholic alkaline solution which contains between about 50 and 90 percent by volume alcohol and has a concentration of between about 0.01M and about 0.5M alkali metal hydroxide, thereby affixing the anchorage dependent cells to the solid support, rendering the anchorage dependent cells permeable to nucleic acid probe for hybridization analysis, denaturing the DNA and any RNA containing secondary

structure present in the anchorage dependent cells, and localizing the denatured DNA and/or RNA in its cellular environment,

e) reacting the cells affixed in step (d) with a hybridization probe having a nucleic acid sequence

complementary to the viral nucleic acid, and

f) analyzing the reaction product of step (e) for the formation of nucleic acid hybrids containing the hybridization probe.

11. The method of claim 10 wherein the hybridization probe is labeled.

12. The method of claim 10 wherein the hybridization probe is labeled with a radioisotope, a fluorescent or luminescent marker, a rare earth metal, biotin, an enzyme or an antibody.

13. The method of claim 10 wherein the viral nucleic acid is amplified in step (c) by intracellular replication by the infected anchorage dependent cells.

14. The method of claim 10 wherein the solid support is a multiple well tissue culture dish.

15. The method of claim 10 wherein the alcoholcomprises ethanol or isopropanol.

16. The method of claim 10 wherein the alkali metal hydroxide is potassium hydroxide.

17. The method of claim 10 wherein the alcoholic alkaline solution has a concentration of between about 0 and 0.01 M magnesium chloride, between about 0 and 0.01 ammonium acetate, between about 0.01 and 0.5 M alkali metal hydroxide and the alcohol consists essentially of ethanol or isopropanol.

18. A method for detection of a nucleic acid of known hybridization specificity suspected of being present in the cells of a cell culture, tissue section or direct specimen by in situ hybridization analysis comprising:

a) affixing the cells to the surface of the well of a multiple well tissue culture plate by contacting the cells of the culture, tissue section or direct specimen with the surface of the well of a multiple well tissue culture plate in the presence of an alcoholic alkaline solution which contains between about 50 and 90 percent by volume alcohol and has a concentration of between about 0.01M and about 0.5M alkali metal hydroxide, thereby affixing the nucleic acid to the solid support, rendering the cell permeable to nucleic acid probe for hybridization analysis, denaturing the DNA and any RNA containing secondary structure present in the cell, and localizing the denatured nucleic DNA and/or RNA in its cellular environment,

b) reacting the affixed cell with a

hybridization probe having a nucleic acid sequence complementary to the nucleic acid of known hybridization specificity, and

19. The method of claim 18 wherein the hybridization probe is labeled.

20. The method of claim 18 wherein the hybridization probe is labeled with a radioisotope, a fluorescent or luminescent marker, a rare earth metal, biotin, an enzyme, an antibody or hapten.

21. The method of claim 18 wherein the nucleic acid of known hybridization specificity is viral nucleic acid.

22. The method of claim 18 wherein the alcohol comprises ethanol or isopropanol.

23. The method of claim 18 wherein the alkali metal hydroxide is potassium hydroxide.

24. The method of claim 18 wherein the alcoholic alkaline solution has a concentration of between about 0 and 0.01M magnesium chloride, between about 0 and about 0.01M ammonium acetate, between about 0.01 and about 0.5M alkali metal hydroxide and the alcohol consists essentially of ethanol or isopropanol.

25. The method of claim 18 wherein cells are affixed to the surfaces of a plurality of wells of the multiple well tissue culture plate in step (a).

26. A method as set forth in claim 18 wherein one or more of said contacting, treating or analyzing steps are carried out by a machine.

27. The method of claim 18 wherein cells are affixed to the surfaces of a plurality of wells of the multiple well tissue culture plate in step (a) and one or more of steps (a) and (b) and (c) is carried out by a machine.