WO1992015883A1

WO1992015883A1 - Improved assays including colored organic compounds

Info

Publication number: WO1992015883A1
Application number: PCT/US1992/001617
Authority: WO
Inventors: Kimberly A. Foster; Donna Bottari; Susan Garramone
Original assignee: Amoco Corporation
Priority date: 1991-03-08
Filing date: 1992-03-04
Publication date: 1992-09-17

Abstract

The disclosure relates to the inclusion of one or more colored organic compounds in reaction reagents to be used in an assay for the detection of microorganisms. The colored organic compounds can be acid/base indicator compounds, or colored organic compounds having a color which is not pH sensitive.

Description

IMPROVED ASSAYS IMGLDDINQ COLORED ORGANIC COMPOUNDS

Background of the Invention

Assays for the detection of microorganisms, viruses or components thereof typically require the sequential addition of a variety of reaction reagents followed by incubation for predetermined periods of time under defined conditions. In many instances, the sample which is to be tested is of limited supply and difficult or expensive to obtain. Similarly, reaction reagents can be very expensive. In addition, some assays can take several weeks to carry out, especially if the assay involves a culture phase.

Thus, it is extremely important that such assays be conducted in a careful manner so that the expense, and time involved in repeating an aborted experiment is avoided.

Summary of the Invention

The subject invention relates to assays for the detection of microorganisms (e.g., bacteria, protozoan and viruses) or components thereof. More specifically, the invention relates to an improvement in such assays comprising the inclusion in the assay of one or more colored organic compounds.

In one embodiment of the method of the present invention, one or more colored organic compounds is used in an assay for the detection of a microorganism. A liquid sample to be assayed for the presence of a microorganism is treated with a lysis or disruption solution to lyse or disrupt any microorganism which may be present in the sample. The lysis or disruption solution contains a colored organic compound. Following lysis or disruption, a detection solution is added. The addition of the detection solution results in a color change which is either based on a pH change in the solution or on the inclusion of a second colored organic compound in the detection solution. In preferred embodiments, the improved assay is useful for the detection of Gram-positive or Gram-negative bacterial species. Such bacteria are first enriched in a sample by growth in selective media. A series of reagents are added subsequently to both lyse the bacteria and to detect components thereof. Colored organic compounds are included in these subsequently added reagents.

When included in a reaction reagent, colored organic compounds serve several useful functions. For example, the inclusion of such compounds in a reaction reagent produces a color in the reaction mixture which is useful as an indicator to insure not only that each reaction reagent has been added, but that each is added in the proper sequence. In addition, the inclusion of colored organic compounds provides a visual indication that proper mixing has taken place in a reaction. For example, in an acid extraction step, it is important to know that the solution has been thoroughly mixed to insure that extraction yield is optimized. Furthermore, because there can be a wide range of variability with respect to the buffering capacity of a particular sample, the use of a colored organic compound which is also an acid/base indicator provides a convenient method for determining when the sample has reached the desired pH range.

Detailed Description of the Invention

The subject invention is based on the recognition that colored organic compounds can be used as indicators in an assay for the detection of microorganisms. Typically, such assays employ antibodies or nucleic acid sequences which are specifically reactive with the microorganism, virus, or component thereof which is to be detected. Colored organic compounds can be broadly classified into two groups: those whose color is pH dependent (acid/base indicators) and those whose color does not change with variations in pH. As discussed below, both types are useful in the method of the claimed invention.

Acid/Base Indicators

Acid/base indicator compounds are well known in the art. They are highly colored organic molecules possessing acidic or basic functional groups. Variations in pH cause the acid/base indicator molecule to lose or gain a proton and, because substantial structural rearrangement accompanies the proton-transfer process, a dramatic color change occurs. A list of acid/base indicator compounds is presented in Table 1, along with the colors of the acid and base forms of each indicator and the pH value at which one or the other form is sufficiently predominant to impart its color to the solution. Most of the indicators listed in Table 1 possess acid and base forms which are both colored. If the total concentration of a two-color indicator is increased, the individual concentrations of the acid and base forms will increase proportionally, and the pH transition interval should remain unchanged, even though the color intensities are increased.

Several of the acid/base indicators included in Table 1 are one-color indicators. Phenolphthalein is a familiar example of such an indicator. It has a colorless, acid form and a pink-colored base form in equilibrium with each other at any given pH.

Table l pH Non-Sensitive Colored Organic Compounds

Colored organic compounds which do not change color in response to pH changes in a solution are also useful as indicator compounds as described herein. More specifically, such colored compounds can be included in sequentially added reaction reagents to induce a color change resulting from the cumulative effect of the compounds on the absorbance characteristics of the reaction mixture. For example, if a reaction reagent containing a blue organic compound is added to a reaction reagent containing a yellow organic compound, the resulting mixture will absorb light in the green range of the visible spectrum and the reaction mixture will appear as a green solution. The term "colored organic compound^M has been used throughout this application in preference to the term "dye". A dye is a colored organic compound which is used to impart color to an object or fabric. Thus, a dye must remain fast (i.e. remain attached to the object or fabric during washing or cleaning) . To be useful in connection with this invention, the colored organic compound need not be color fast, although it can be. Such colored organic compounds are well known to those skilled in the art. One skilled in the art will recognize that an acid/base indicator compound can be used as an indicator in a reaction mixture under conditions which will not result in a color change based on pH changes. For example, the acid form of phenol red, which is predominant at pH 6.7, is yellow in color. The base form of phenol red, which is predominant at pH 8.4, is red in color. In a reaction wherein the pH remains relatively constant at approximately 6.7, the presence of phenol red in the solution will result in a yellow appearance. The subsequent addition of a red solution, without any appreciable change in the pH of the mixture, will result in a green appearance of the solution due to the cumulative effect of the dyes in the solution. Thus, the pH sensitivity of an acid/base indicator compound need not be utilized in every instance in which the compound is used as an indicator.

Detection of Microorganisms Generally

The method of this invention can be used for the detection of any microorganism. It is preferred that the sample to be tested be a liquid sample. The liquid sample can be treated to promote the reproduction of any microorganism in the sample, if necessary, to bring the concentration of the microorganism up to a level which can be detected. This concentration varies depending upon the sensitivity of the detection system employed. Typically the microorganism is lysed (e.g. in the case of bacteria) or disrupted (e.g. in the case of viruses) to provide greater exposure of the components thereof. The particular lysis or disruption reagent is selected depending upon the particular microorganism of interest. For example, in the exemplification which follows, the lysis of Gram-negative and Gram-positive bacteria is discussed. Those skilled in the art are familiar with the wide range of lysis and disruption reagents which are appropriate for the treatment of a selected microorganism. The lysis or disruption reagent includes a colored organic compound for use as an indicator. In a first embodiment, the colored organic compound is not an acid/base indicator. Thus, its color is not affected by changes in the pH of the solution. In a second embodiment, the colored organic compound is an acid/base indicator.

Following lysis, a detection solution is added to the mixture. This solution contains a detection molecule which binds specifically to a component of the microorganism. Such detection molecules include, for example, antibodies and nucleic acids. This solution may also contain, if necessary, other components necessary to establish conditions appropriate for the binding of the detection molecule to the component of the microorganism.

The addition of this reagent results in a change in the color of the reaction mixture. In the first embodiment, the color change is based on the inclusion of a colored organic compound in the detection solution. In the second embodiment, the color change is based on a change in pH caused by the addition of the detection solution which causes a change in the color of the acid base indicator.

Detection of Gram-Negative Bacteria

In a preferred embodiment for the detection of Gram- negative bacteria in a sample (e.g. a food sample) , the presence of such bacteria are enriched by growth in selective media. The use of selective media for enrichment purposes is a procedure well known to those skilled in the art. This enrichment step increases the number of cells representing the particular Gram-negative bacteria of interest in the sample thereby raising the number to detectable levels. As will be recognized by those skilled in the art, the number of cells necessary for detection depends upon the sensitivity of the particular detection method to be employed.

An acid/base indicator having a colored basic form which predominates at a pH above about 8, and a basic solution are added as a mixture to the enriched Gram- negative bacteria. The addition of the basic solution raises the pH of the resulting mixture to a pH equal to or greater than the pH at which the basic form of the acid/base indicator predominates. A preferred acid/base indicator is thymophthalein, the basic form of which is predominant at a pH of 10.6 or greater. At the resulting pH, Gram-negative bacteria present in the sample are lysed.

A detection solution is then added to the mixture of lysed Gram-negative bacteria. The detection solution contains a detection molecule which binds to a component of the Gram-negative bacteria. The detection solution also contains a colored organic compound which alters the color of the solution containing the lysed bacteria so that the resulting solution is visually distinguishable from the solution prior to the addition of the detection solution. The detection solution also contains any other components which may be necessary to establish conditions appropriate for the binding of the detection molecule to the component of the Gram-negative bacteria for which it is specific. The detection molecule can by any molecule which binds specifically to a component of the Gram-negative bacteria. Such detection molecules include, for example, antibodies or antigen-binding fragments thereof, and nucleic acids. In a preferred embodiment, the detection molecule is an oligonucleotide which binds to ribosomal RNA from the Gram- negative bacteria. The binding of the detection molecule to the component of the bacteria for which it is specific can be detected by any of a variety of conventional methods which are well known to those skilled in the art.

Detection of Gram-Positive Bacteria

Gram-positive bacteria can be detected in a similar manner. As described above, the presence of Gram-positive bacteria in a sample is enriched by growth in selective media. However, because of the more complex nature of the cell wall of Gram-positive bacteria, enzymatic digestion steps are included to effect lysis. A solution containing a first proteolytic enzyme (e.g. lysozyme) is added to the enriched mixture of cells. This proteolytic solution contains a first colored organic compound which imparts a color to the solution.

Following incubation with the proteolytic enzyme, a lysis reagent containing a second proteolytic enzyme (e.g. proteinase K) and a second colored organic compound is added. The addition of this lysis reagent results in a visually detectable color change in the solution. Addition of the lysis reagent results in the lysis of the Gram- positive cells. To the solution of lysed cells a detection solution is added. As described above, in connection with the discussion relating to Gram-negative cells, the detection solution contains a detection molecule which binds to a component of the Gram-positive bacteria. The detection solution also contains a third colored organic compound which alters the color of the solution so that the color of the solution following the addition of the detection solution is visually distinguishable from the solution prior to the addition of the detection solution. The detection solution also contains other components, as necessary, to establish conditions appropriate for the binding of the detection molecule to the component of the Gram-positive bacteria for which it is specific.

In preferred embodiments, the detection molecule is an oligonucleotide which binds specifically to ribosomal RNA from Gram-positive bacteria.

EXEMPLIFICATION

The exemplification describes detection of the presence of Salmonella or Listeria in a sample using DNA hybridization technology with a non-radioactive labeling and detection system. The hybridization and detection technology is generic in that, given the appropriate enrichment and sample preparation procedures for a particular organism, and the appropriate specific DNA probes, the basic assay system can be applied to the analysis of a wide variety of potential target bacteria from both food and clinical speicimens. The assay requires little equipment and can be completed in approximately two and one-half hours to three hours, depending on the organism under analysis and the extent of sample preparation required. In general, for food applications, the assay is performed after two days of broth culture enrichment of the sample.

The assay employs synthetic oligonucleotide DNA probes directed against ribosomal RNA (rRNA) of the target organism. This approach is an alternative to that of using probes directed against chromosomal DNA targets, and offers the distinct advantage of increased sensitivity due to the fact that rRNA, as an integral part of the bacterial ribosome, is present in multiple (1,000 - 10,000) copies per cell. The number of ribosomes present per cell is dependent on the growth state of the bacterial culture; maximum numbers are present in actively replicating (i.e., logarithmic phase) bacteria. Thus, enrichment protocols used in conjunction with the assay must be designed such that target organisms are in logarithmic or early stationary phase at the time of assay in order to achieve maximum sensitivity.

A second advantage of the rRNA approach is that a vast amount of nucleic acid sequence information is available for various rRNA species, including those of the Gram- negative enteric bacteria. This makes direct sequence comparison between the rRNA of target and genetically related competitor organisms possible, with specific design of probes following from this analysis. Even though rRNA sequences are among the most highly conserved (or invariant) nucleic acid sequences in bacteria, some degree of sequence divergence exists even between bacteria that are genetically related to a high degree (e.g., E. coli vs. Salmonella.. These differences can be exploited to design and chemically synthesize DNA probes homologous to the unique sequences of a particular rRNA molecule. The probes used in the assay are designed in this way. Each probe is a synthetic oligodeoxyribonucleic acid of approximately 35 to 40 nucleotides in lenthe homologous to unique regions of either the 16S or 23S rRNA of the target organism. Each assay employs multiple probes for reasons discussed below.

The assay can be divided into four phases for purposes of discussion. These are: a) sample treatment and lysis, b) hybridization, c) hybrid capture, and d) detection. It should be emphasized that the basic test format is generic, but minor variations in test chemistry and protocol may exist for different applications.

In the sample treatment and lysis phase, broth culture enrichments of test samples are treated to lyse target organisms and release rRNA. For Gram-negative bacteria, this will usually involve a simple treatment with alkali. For Gram-positive bacteria, a chemical lysis/denaturation step will usually be preceeded by an enzymatic treatment to digest the more complex Gram-positive cell wall (e.g., lysozyme, etc.). In assays in which an alkaline denaturation is used, this step will be followed by a chemical neutralization. Incubation times for this phase of the test are about 5 and 30 minutes for Gram-negative and Gram-positive target bacteria, respectively. The hybridization phase includes the addition of specific DNA probes. In each assay, two probes are used, each with a distinct function. Both probes are homologous to unique rRNA sequences of the target organism, and the two probes hybridize to adjacent regions on the same target rRNA molecule. The capture probe is approximately 30 nucleotides in length and contains a poly-dA (polydeσxyadenylic) tail of more than 100 nucleotides attached to the 3' end of the probe sequence. The purpose of the poly-dA tail is to allow capture of formed hybrid molecules onto a solid support. This will be discussed in more detail below. The detector probe is about 35 to 40 nucleotides in length and is labeled at both the 3' and 5' ends with fluorescein, which is used as a componenet of the detection system of the assay. During the hybridization phase, both the capture and the detection probe hybridize to separate regions a rRNA species.

In the capture phase, hybrids formed as described above are captured on a solid support. The solid support is a plastic (polystyrene) "dipstick" coated with poly-dT (polydeoxythymidylic acid) , a homopolymer of thymidine residues. The poly-dT molecule is homologous to the poly- dA tail of the capture probe, and upon introduction of the coated dipstick into the hybridization cocktail, hybridization occurs between the poly-dA and poly-dT molecules, binding the target/probe hybrids to the solid support. The detector probe is indued in the formed complex, since it has hybridized to a contiguous region of the same rRNA target molecule to which the capture probe has hybridized. The capture reaction requires l hour at 65 degrees C for most assays.

The detection phase is a variation of the standard enzymatic/colorimetric approach commonly used in enzyme immunoassay. The rRNA/probe complex captured on the dipstick is first reacted with a polyclonal anti- fluorescein antibody (anti-FI) conjugated to the enzyme horseradish peroxidase (HRP) . This conjugate binds to the fluorescein molecules attached to the detector probe. The complex is then reacted with a substrate of HRP, hydrogen peroxide, in the presence of a chromogen (tetramethylbenzidine, TMB) , and a blue color develops in proportion to the amount of enzyme conjugate bound to the complex and thus also in proportion to the amount of target rRNA captured. The reaction is stopped with sulfuric acid, changing the developed color from blue to yellow. The intensity of the color is measured by determining the absorbance at 450 nm. Absorbance in excess of a cutoff value indicates a positive result and thus the presence of the target organism in the test sample. The following exemplification relates to the detection of Salmonella and Listeria in an assay including colored organic compounds.

Salmonella Assay

This example describes an assay for the detection of Salmonella in a food sample. Food which can be tested using the assay include, for example, red meat, poultry, seafood and raw milk. Any Salmonella in the sample are enriched using a selective growth procedure prior to the detection phase of the assay. For example, a food sample is homogenized in 9 volumes lactose broth and incubated for 22-24 hours at 35 degrees C. Following, this growth phase, 1 ml of the culture is transferred to 10 ml of tetrathionate broth (TT) . Another 1 ml of the culture is transferred to 10 ml of selenite cysteine broth (SC) . The TT and SC cultures are incubated for 16-18 hours at 35 degrees C. Following this growth period, 1 ml of each of TT and SC cultures is used to inoculate separate 20 ml cultures of Gram negative broth (GN) . The GN cultures are incubated for 6 hours at 35 degrees C. A 0.25 ml sample of each GN culture is transferred to a test tube, and 0.1 ml of lysis solution is added to each. The lysis solution contains the acid/base indicator thymolphthalein at a concentration of 0.05%. In its acid form, which predominates at pH 9.3, thymolphthalein is colorless. In its base form, which predominates at pH 10.6, thymolphthalein is blue in color. The culture and lysis solution are thoroughly mixed and incubated at room temperature for 5 minutes. The resulting solution containing lysed cells is blue in color.

To the resulting blue solution, 0.2 ml of hybridization solution is added. The hybridization solution is a phosphate buffered solution having a pH of about 8-9 which contains the capture and detection probes. The solution also contains other components, the inclusion of which establishes an environment appropriate for hybridization of complementary nucleic acids in the reaction mixture. The hybridization solution also contains 0.025% cresol red. Cresol red is a colored organic compound having a color which is not affected by pH changes in solution. With the addition of the hybridization solution, the pH of the mixture changes from 10.6 or greater to approximately 8-9. This results in a change in color of the thymolphthalein from blue to colorless. Thus, the color of the reaction mixture following this addition of the hybridization solution is red due to the presence of the cresol red. The resulting mixture is incubated at 65 degrees C for approximately 15 minutes to allow the capture and detection probes to hybridize to any Salmonella rRNA which may be present in the sample. Following the 15 minute incubation, the dipstick having attached d(T) oligonucleotides is immersed in the reaction mixture. This mixture is incubated at 65 degrees C for approximately l hour. Specific hybridization is detected as described above. Listeria Assay

The detection of Listeria in a food product such as red meat, poultry and seafood can be accomplished in a similar manner. Approximately 25 grams of the food product to be tested is homogenized in 225 ml of phosphate-buffered Listeria enrichment broth (PEB) . This mixture is incubated for approximately 24 hours (+/- 4 hours) at 35 degrees C. A sterile cotton swab is dipped into the culture and used to deposit culture onto the entire surface of an LPM agar plate. The LPM plate is incubated for approximately 24 hours (+/- 2 hours) at 35 degrees C.

Using another sterile cotton swab, growth is removed from the LPM plate and resuspended in 1 ml of phosphate- buffered saline (PBSO in a sterile, capped tube by swirling the swab vigorously in PBS for 5 seconds. A 0.5 ml sample from this suspension is added to a test tube to be analyzed for the presence of nucleic acids from Listeria.

To the 0.5 ml sample, 0.10 ml of reconstituted pre- treatment reagent containing the enzyme lysozyme is added, the mixture is shaken for approximately 5 seconds and the mixture is incubated at 37 degrees C for approximately 15 minutes. The pretreatment reagent contains 0.0075% bromophenol blue and the reaction mixture is purple in color following the addition of this reagent. While maintaining the temperature at 37 degrees C, 0.10 ml of reconstituted lysis reagent which contains proteinase K is added to each sample. The samples are shaken by hand for approximately 5 seconds to insure mixing. The tubes are incubated for an additional 15 minutes at 37 degrees C. The reconsituted lysis reagent contains 0.05% brilliant yellow. Due to the cumulative effect of the blue and yellow dyes, the solution exhibits an absorption maximum within the green portion of the visible spectrum. If the resulting solution is not green in color, this indicates that the solutions were not added in the proper order or that a component has been omitted.

After the 15 minute incubation with the lysis reagent, 0.10 ml of Listeria probe solution is added to the mixture. In addition to the capture and detection probe, this solution contains 0.10% cresol red. The resulting mixture is a reddish-brown, or rust color. A dipstick is then immersed in the reaction mixture. This reaction mixture is incubated for approximately 1 hour at 65 degrees C. Following the incubation at 65 degrees C, the dipsticks are removed from the reaction mixture and washed to remove non-specifically bound nucleic acid. Specific hybridization of Literia rRNA to the dipsticks is detected as described above.

Equivalents

Those skilled in the art will know, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. These and all other equivalents are intended to be encompassed by the following claims.

Claims

1. An assay for the detection of a microorganism in a liquid sample, comprising: a) providing a liquid sample; b) adding to the liquid sample a lysis or disruption solution which results in lysis or disruption of the microorganism in the liquid sample, the lysis or disruption solution containing a first colored organic compound; c) adding to the product of step b) a detection solution containing a detection molecule which binds to a component of the microorganism, the detection solution containing a second colored organic compound.

2. An assay for the detection of a microorganism in a liquid sample, comprising: a) providing a liquid sample; b) adding to the liquid sample a lysis or disruption solution which results in lysis or disruption of the microorganism in the liquid sample, the lysis or disruption solution containing an acid/base indicator; c) adding to the product of step b) a detection solution containing a detection molecule which binds to a component of the microorganism, the addition of the detection solution resulting in a pH change which alters the color of the acid/base indicator.

3. An assay for the detection of Gram-negative bacteria in a sample, comprising: a) enriching for the presence of the Gram-negative bacteria by growth in selective culture media; b) adding to the product of step a) a basic solution containing an acid/base indicator having a colored basic form which predominates at a pH above about 8.0, the resulting mixture having a pH equal to or greater than the pH at which the basic form of the acid/base indicator predominates; c) adding to the mixture from step b) a detection solution containing a detection molecule which binds to a component of the Gram-negative bacteria, the detection solution containing a colored organic compound which alters the color of the mixture of step c) so that it is visually distinguishable from the mixture produced by step b) ; and d) detecting binding of the detection molecule to the component of the Gram-negative bacteria.

4. A method of Claim 3, wherein the acid/base indicator is thymolphthalein.

5. A method of Claim 4, wherein the component of the Gram-negative bacteria is a nucleic acid and the detection molecule is an oligonucleotide.

6. A method of Claim 5, wherein the nucleic acid is a ribosomal RNA molecule.

7. A method of Claim 6, wherein the Gram-negative bacteria is of the genus Salmonella.

8. A method for the detection of a Gram-positive bacteria, comprising: a) enriching for the presence of the Gram-positive bacteria by growth in selective media; b) adding a mixture including a first proteolytic enzyme which degrades Gram-positive cell wall components and a first colored organic compound; c) adding to the product of step b) a lysis reagent which contains a second proteolytic enzyme which degrades Gram-positive cell wall components and a second colored organic compound which alters the color of the mixture of step c) so that it is visually distinguishable from the mixture produced by step b) ; d) adding to the mixture of step c) a detection solution which contains a detection molecule which binds to a component of the Gram-positive bacteria and a third colored organic compound which alters the color of the mixture of step d) so that is visually distinguishable from the mixture produced by step c) ; and e) detecting binding of the detection molecule to the component of the Gram-positive bacteria.

9. A method of Claim 8, wherein the component of the Gram-positive bacteria is a nucleic acid and the detection molecule is an oligonucleotide.

10. A method of Claim 9, wherein the nucleic acid is a ribosomal RNA molecule.

11. A method of Claim 10, wherein the Gram-negative bacteria is of the genus Listeria.