Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-11-28
2003-01-21
Smith, Lynette R. F. (Department: 1645)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S004000, C435S007100, C435S007210, C435S007350
Reexamination Certificate
active
06509166
ABSTRACT:
TECHNICAL FIELD
This invention relates to a method for detecting the presence or the absence of a microbial target analyte in a material sample; and enumerating the target analyte when it is found to be present in the sample. The material being analyzed can be a biological material; an environmental material; a food stuff material; or some other material which can harbor the target analyte. The target analyte is one or more specific living organisms such as microbes, bacteria, fungi, mycoplasma or protozoa which may be found in the material being tested; and the presence or absence of the target analyte or analytes is determined by causing selective metabolic reproduction of the target analyte or analytes in a suitable growth medium. The analysis can be performed in a gel or a semi-solid growth material which supports growth of the target analyte(s). In this manner, detectable target analyte colonies are formed in the medium. The colonies may be detected and counted by various techniques, including the use of detectable materials which will selectively bind to some portion of the target analyte(s).
BACKGROUND ART
The analysis of specimens for the presence or absence of target bacteria and other viable organisms such as fungi and protozoa, wherein the specimen is placed on a sterile growth medium, is well known. Various bacteria are usually differentiated by their appearance, their chemical reaction with growth or other medium, or by applying an immunological stain. Since bacterial colonies of different species appear similar, it is usually necessary to perform a number of tests on a number of colonies before one can be establish a correct identification. This is especially a problem if it is necessary to distinguish bacterial sub-species, for example, finding the location of a colony of
E. coli
serotype 0157:H7 within a mix of other, apparently identical
E. coli
colonies.
There are growth media with specialized characteristics which help in the rapid visual location of such organisms, but the more closely related the target organisms are to the background, the more difficult it is to distinguish them on chemical tests alone. It would be highly desirable if such colonies could be rapidly and specifically located, particularly if no additional operations were required by the technologist.
DISCLOSURE OF THE INVENTION
This invention relates to a method for detecting the presence or absence of one or more target microbial analytes in a sample. Detection of the target analyte is accomplished by growing the target analyte in a nutrient medium so as to form colonies of the target analyte in the medium, and then visually detecting and counting any resultant target analyte colonies which may form in the medium.
The analysis is performed in a medium which may also contain, or is combined with, one or more labeled analyte-specific materials (LASMs). The LASMs are homogeneously distributed throughout the medium. The LASMs include a binding element, which may be a protein, lectin or a nucleotide sequence that is specific to a binding site in or on the target analyte. The label may be a visible dye or a fluorophore which is attached to the binding element. The LASMs in the medium can diffuse and migrate through the medium to the target analyte colonies, and will bind to the individual organisms in the colonies thereby differentially highlighting the colonies relative to the remainder of the medium. The LASMs may either bind directly to the surface of the organism, or to a product of the organism, such as its capsular material or an internal molecular structure, so long as the LASM, being so bound, does not appreciably migrate away from the site. Because the binding of the LASMs locally depletes the LASM concentration in the regions of the medium occupied by the target analyte colonies, a “halo” of lower LASM concentration may form around each target analyte colony, and at the center of each halo, if one forms, will be a high intensity peak corresponding to the labeled colony. Any non-target organisms present in the medium will not bind the LASMs and will not show this characteristic high intensity peak.
A sample of a material to be analyzed is inoculated into the medium and spread out over the medium. If a quantitative analysis is desired, the amount of the sample inoculum will be volumetrically measured. As the organisms reproduce, colonies will form and will remain in a fixed location in or on the medium. The LASMs within the medium are sized so as to be able to migrate in or through the medium. Therefore, the LASMs in the region of the target analyte colonies are free to move through the medium and bind to the target organisms in the colonies, or to their products. This will cause the target organism colonies to show a higher label signal than surrounding areas in the medium. The reason for this is that the target organism colonies become LASM-enriched, and the surrounding areas become LASM-depleted. The immediately surrounding areas in the medium, which abut the target organism colonies, will tend to emit a weakened label signal. The net result is the formation of “bright” spots which may be surrounded by “dim” halos in the medium. In the event that there is no target analyte present in the sample, then the medium will retain a relatively even level of label signal emission, and will appear to be evenly “colored”.
The assay can be completed within about eighteen hours by visual inspection of the inoculated medium. Since one can know the volume of the sample inoculated into or spread onto the medium, one can quantify the amount of the target analyte per unit volume of the specimen sample by counting the number of highlighted colonies in the medium. This enables the use of the invention to obtain a quantitative analysis for target analyte. It is desirable that a portion of the medium which contains the LASM be protected against sample inoculation, thus allowing this portion of the medium to serve as a negative control.
Some samples, especially those from “dirty” sources, such as environmental samples or those containing a great deal of other material, may have a high level of intensity non-homogeniety, where there is a large variety of particles or other material which may in itself have some level of signal, and where these non-specific signals may be confused with those from the target analytes. This problem can be overcome, however, by making sequential images from the same areas in the medium, which images are separated from each other by suitable periods of time. Only those colonies formed from the target organism will increase in signal intensity by virtue of their continued growth. A comparison of successive images can separate these areas of increasing intensity from the surrounding medium. This method of kinetic analysis is the preferred method in all circumstances because this technique provides the greatest sensitivity, the best selectivity, and the fastest time to completion of the analysis.
In order to give the greatest signal
oise ratio, it is desirable that the medium be as thin as possible, i.e., about one millimeter. The concentration of the label should be such that the signal from it can just be detected from a non-inoculated area of the medium. For a visual examination, a useful signal would be one that is slightly visible using the naked eye under the appropriate lighting conditions and is not so marked so as to obscure the signal from the colonies. Fluorophores are suitable labels for the visual detection of the colonies. Note that two or more target organisms can be detected using this method if the labels of the LASMs for each target can be visually distinguished, for example, by color.
It is therefore an object of this invention to provide a method which can be used to assay a specimen sample for the presence or absence of a target analyte.
It is another object of this invention to provide a method of the character described wherein the sample is placed on a medium which is combined with a target analyte-specific labeled material that can migrate within the m
Ford Vanessa L
Jones William W.
Levine Robert A.
Smith Lynette R. F.
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