Patent Application
20180275062
The present invention pertains generally to detection of bacteria. More specifically, the present invention pertains to the optical detection of bacteria using fluorescent tags. The invention is particularly, but not exclusively, useful for obtaining total bacteria counts, or total counts of a specific bacteria of interest, in a sample.
Total bacterial counts are often used to assess water quality for both recreation and drinking purposes (in water), as well to test for raw milk quality (for milk), aquaculture, etc. Currently, total bacterial counts are done using methods that incorporate standard plate count (SPC), which can take up to 48 hours. Alternatively, flow cytometers can be used to determine a total bacteria count, but flow cytometers are expensive and sophisticated devices.
In some instances, phages can be used to obtain total bacteria counts for a specific bacteria-of-interest. Phages are viruses whose hosts are bacterial cells. The phages identify their hosts through host cell-specific receptor molecules, which are located on the outside of the host cell. Once the phages find their specific receptors, they bind to the bacterial cell and inject their nucleic acid into the cell. The phage nucleic acid then takes over the host cell's machinery to make large amounts of phage components. The phage components are then assembled into new phages. The phages then direct production of an enzyme that breaks down the bacteria cell wall, which causes the bacteria to lyse, which further frees new phages. Phage lysis assays are known in the prior art for the detection and identification of various bacterial pathogens.
Antibodies have also been used to discriminate bacterial species. However there are a number of advantages to using phage-based detection schemes, as opposed to using antibody-based schemes. More specifically, antibodies are bare protein molecules. As such, they are potential food sources for bacteria. Bacterial “grazing’ of antibody-coated magnetic microparticles has been observed. Such grazing can result in false positives, when compared to a phage-based scheme. Other limitations of antibody-based immunoassays include antibody manufacturability and instability. To overcome these disadvantages, phages can be use in lieu of antibodies for the detection of bacteria.
As mentioned above, phages can also be used to detect and identify specific bacteria. This is because each bacterial species has at least one phage that will prey upon it. Phages can be readily obtained from phage libraries. One such library is the Felix D'Herelle Reference Center for Bacterial Viruses at Laval University in Quebec, Canada. This library has a collection of approximately 500 different species of phage. Phages are very robust and are not as sensitive to environmental conditions (pH, temperature, salinity, etc.) as antibodies are. Large quantities of phages can be easily cultured and purified. Additionally, the purified phage exhibits a long shelf life relative to antibodies.
In view of the above, it is an object of the present invention to provide a device and method for detecting total bacteria counts in a sample that minimizes false positives. Another object of the present invention is to provide a device and method for detecting total bacteria counts in a sample that is stable and that can easily be stored for extended periods of time prior to use. Still another object of the present invention is to provide an optical device and method for detecting bacteria that can use phages to determine a total count of a bacteria of interest that is known to react with the phage being used. Yet another object of the present invention to provide a device and method for detecting total bacteria count using fluorescent tags that is easy to manufacture, that is inexpensive, and that is easy to use by remote operators in the field.
A device and method for determining total bacteria count in a sample, with the sample containing eukaryotes and prokaryotes, can include the initial step of removing the larger eukaryotes from the sample. One way to remove the eukaryotes is by filtering the sample through a 0.45 μm pore size syringe filter. The syringe filter can be made of a low protein binding material, such as a polycarbonate (PC) or polyvinylidene (PVDF) material. Next, the sample can be tagged with a fluorescent tag and then filtered on an optical substrate for detection. The substrate is illuminated with a light source at a wavelength corresponding to an excitation wavelength for said fluorescent tag, to establish a test intensity for said sample. The test intensity can be compared to reference intensities, such as those embodied in calibration curves for example, to determine said total bacteria count.
In cases where a total bacteria count of a specific bacteria is desired, a phage that is known to react with the bacteria of interest can be selected. The phage can be fluorescently tagged and then mixed into the sample, and the resulting mixture is filtered on an optical substarte the can be illuminated with a light source at a wavelength corresponding to an excitation wavelength for the fluorescent tag, to establish a test intensity for the sample. The test intensity can be compared to reference intensities corresponding to determine said total bacteria count for that bacteria of interest. In several embodiments, the fluorescent tag can be SBYR® Gold cyanine dye. The bacteria count in the sample can also be optionally fixed by using a solution of 4% paraformaldehyde in phosphate buffer solution (PBS). The fixing allows for verification of the total bacteria count using alternative methods, such as optical microscopy, for example.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
The novel features of the present invention will be best understood from the accompanying drawings, taken in conjunction with the accompanying description, in which similarly-referenced characters refer to similarly-referenced parts, and in which:
Referring initially to
Once the sample has been filtered to substantially remove the eukaryotes, the resulting sample can be fluorescently tagged. As indicated by step 104 in
Referring again to
Referring now to
A collecting lens 24, which can be oriented 90° from the excitation light, can collect the fluorescence emitted by the SYBR® gold stained bacterial cells. Collecting lens 24 can include an emission filter 26 that blocks the excitation light and only lets the light due to the SYBR® gold fluorescence (λ>500 nm) hit the detector 28. The detector 28 can be a photodiode or a photomultiplier tube (PMT). When light hits the photocathode of the PMT or the photodiode, the light photons are converted into electricity that is then measured. The current is proportional to the intensity of fluorescent light that emits from sample 12, which is further proportional to the number of bacteria present in the sample. The intensity of the fluorescence is proportional to the number of total bacteria, a calibration curve 30 of reference intensities plotted against bacteria count, such as illustrated in
It some instances, a total bacteria count for a specific bacteria interest may be desired. In these instances, phages can be used. It has been shown in the prior art that phages 10 are able to inject their fluorescently labeled DNA/RNA into its host cell.
In several embodiments of the present invention, phages can be incorporated to obtain total bacteria counts for a specific bacteria of interest. Referring now to
Once the eukaryotes have been removed, and as indicated by step 404, a phage ΦA that is known to react with the bacteria species A of interest can be selected. The phage is fluorescently tagged. Next, and shown in step 406, fluorescently tagged phage ΦA can be added to the sample which is allowed to incubate. The phage-to-host ratio is such that multiple phages will inject their fluorescently labeled nucleic acid inside the bacterial host cell. After incubation (typically 10 minutes), the filtrate is filtered through a glass microfiber filter as shown by step 408 in
To prevent lysis of the infected bacteria and to preserve the sample for additional confirmation, and as indicated by step 410, the phage/host reaction can be quenched by the addition of 60 μL of 4% paraformaldehyde in PBS. As in the case of the total bacteria analysis without using phages (method 100), the paraformaldehyde can preserve the sample so that additional analysis of the sample can be done. Epifluorescent microscopy could be done to verify the counts. Raman microscopy can also be done, as it has been shown that each bacterial species has its own characteristic Raman signature.
Referring again to
The use of the terms “a” and “an” and “the” and similar references in the context of describing the invention (especially in the context of the following claims) is to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
This invention (Navy Case No. 101322) is assigned to the United States Government and is available for licensing for commercial purposes. Licensing and technical inquires may be directed to the Office of Research and Technical Applications, Space and Naval Warfare Systems Center, Pacific, Code 72120, San Diego, Calif. 92152; voice (619) 553-5118; email ssc pac T2®navy.mil.
