Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Reexamination Certificate
2000-04-13
2002-08-20
Gitomer, Ralph (Department: 1627)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
C435S039000
Reexamination Certificate
active
06436661
ABSTRACT:
BACKGROUND OF THE INVENTION
Detection of bacteria is important in a variety of industries, including the food and beverage industry. For example, the need to screen food and water for pathogenic bacteria is crucial to ensuring consumer safety. The determination of levels of certain families of bacteria is a commonly used approach to estimating the shelf life and microbial acceptability of food products and hygienic status of the processing equipment and raw materials used in their manufacture. The diagnosis of microbial infections also relies on the detection of the causative organism(s).
There are many methods known for detecting bacteria. For example, bacteriophage, which are viruses that infect bacteria, may be employed. The presence of the bacteriophage, the infected bacteria, or the lack thereof, may be detected. Typically, a target bacteria is detected by infecting the bacteria with a bacteriophage (BP) specific to the bacteria, inactivating the excess BP, and then manipulating the BP-infected bacteria in some manner to detect the presence or absence of the BP as an indirect indication of whether or not the sample originally contained the target bacteria. Bacterial “helper cells” can be used to amplify the number of BP-infected bacteria and thereby enhance, e.g., make more rapid, the assay method. A common detection method in the final stages of such an assay is to incubate the bacterial helper cells with the BP-infected bacteria and either observe changes in solution turbidity or, alternatively, observe BP plaque formation on an appropriate growth medium.
For example, U.S. patent application Ser. No. 09/434,586 (Wicks et al.) describes devices and methods for the detection of bacteria in a sample. Briefly, a sample containing suspect (target) bacteria is infected with a BP specific to the suspect bacteria, the excess BP is inactivated with an antiviral agent, and the BP-infected bacteria are added to bacterial helper cells to amplify the BP and to produce a signal that can be detected visually or with an instrument. For example, the BP can be detected by incubating the helper cells on agar and counting the number of BP plaques that are formed.
It would be very useful in such assay methods to employ enzyme substrates (ES) as indicators for detecting the presence of BP (and, thus, indirectly the presence or absence of target bacteria). Utilizing ES indicators could lead to significant advantages over trying to observe changes in solution turbidity or counting plaque formation. The use of ES indicators could lead to more convenient, more rapid, and less expensive assay methods. However, the use of traditional soluble ES indicators is generally not possible in such assay methods. The soluble ES would undesirably react with enzyme within the intact bacteria cells of both non-target bacteria and, if used, bacterial helper cells and thereby produce unacceptable levels of background signal.
SUMMARY OF THE INVENTION
The present invention solves the problem of the prior art by utilizing enzyme substrates as indicators that have been bonded (i.e., immobilized) to an insoluble solid support. The use of an immobilized enzyme substrate prevents the enzyme substrate from crossing a bacteria cell wall to react with enzyme within intact bacteria cells. As a result, the enzyme substrate can only react with an enzyme released from a lysed bacteria cell.
Thus, the present invention provides a method of detecting (identifying and/or quantifying) a target bacteriophage. The method includes: combining bacteria and a sample of interest to form a reaction mixture; incubating the reaction mixture under conditions effective for any target bacteriophage present in the sample of interest to lyse the bacteria and release enzyme; adding an immobilized enzyme substrate to the reaction mixture; and monitoring the reaction mixture for a detectable signal produced from interaction between the immobilized enzyme substrate and any released enzyme present. Adding the immobilized enzyme substrate to the reaction mixture can occur before or after incubating the reaction mixture. This method can involve a qualitative or quantitative determination of bacteriophage in a sample. For a quantitative determination, the reaction mixture is plated out on an appropriate growth medium, and the areas emitting the detectable signal are counted.
Alternatively, there is provided a method of detecting (identifying and/or quantifying) target bacteria. The method includes: combining bacteriophage and a sample of interest to form a reaction mixture; incubating the reaction mixture under conditions effective for the bacteriophage to lyse any target bacteria present in the sample of interest and release enzyme; adding an immobilized enzyme substrate to the reaction mixture; and monitoring the reaction mixture for a detectable signal produced from interaction between the immobilized enzyme substrate and any released enzyme present. Adding the immobilized enzyme substrate to the reaction mixture can occur before or after incubating the reaction mixture. This method can involve a qualitative or quantitative determination of bacteria in a sample.
In a preferred embodiment, the present invention provides a method of detecting target bacteria that involves: combining bacteriophage and a sample of interest to form a reaction mixture; allowing the bacteriophage to infect any target bacteria present in the sample of interest; adding an antiviral agent to inactivate any extracellular bacteriophage; adding bacterial helper cells to the reaction mixture; adding an immobilized enzyme substrate to the reaction mixture; incubating the reaction mixture under conditions effective for the bacteriophage to lyse any target bacteria present and the bacterial helper cells and release enzyme; and monitoring the reaction mixture for a detectable signal produced from interaction between the immobilized enzyme substrate and any released enzyme present. This method is preferably used for the quantitative determination of bacteria in a sample, although it can also involve a qualitative determination.
The present invention also provides an immobilized enzyme substrate that includes a porous solid support and an enzyme substrate covalently bonded thereto.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
The present invention provides a method of detecting the presence or absence of bacteriophage or bacteria using an immobilized enzyme substrate (i.e., enzyme reactant). Thus, the present invention provides a method that uses enzyme activity for the detection of bacteriophage, which provides an indirect method for the detection of bacteria, or for the detection of bacteria directly.
The enzyme substrate preferably includes a detectable label that, upon contact with enzyme present in the sample to be tested, produces a change, for example, in the spectral properties of the enzyme substrate or its reaction products resulting from the enzyme reaction. This change is used for the determination of the enzyme activity, and hence, the presence or absence of bacteriophage, and hence bacteria, or bacteria directly. Preferably, the change is a spectral change in the fluorescence radiation of the enzyme substrate, although other spectral changes can be used such as changes in absorption or excitation, for example.
The enzyme substrate can be immobilized on a variety of solid supports. Preferably, it is a porous support, although other supports can be used such as an optical fiber, as disclosed in U.S. Pat. No. 5,238,809 (Wolfbeis). In this latter embodiment, the enzyme substrate is attached to the end of an optical fiber and a photodetector for subsequent signal evaluation is provided, which will measure the signal, e.g., fluorescent light, emitted by the enzyme substrate or its reaction products, upon reaction with an enzyme. Suitable photodetectors are photomultipliers, phototransistors and photodiodes. Preferably, the optical fiber is a single fiber, but it may also be configured as a multi-fiber bundle.
Solid Support
Acceptable supports for use in the present invention
Adams Carl A.
Krejcarek Gary E.
Wicks James H.
3M Innovative Properties Company
Gitomer Ralph
Gram Christopher D.
Rogers James A.
Sprague Robert W.
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