Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Reexamination Certificate
2002-01-22
2004-06-15
Saucier, Sandra (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
C435S049000
Reexamination Certificate
active
06750006
ABSTRACT:
This invention relates to a method and apparatus for sensing the presence of microbes (bacteria, fungi, protozoa, rickettsiae and or other microorganisms) and spores on non-living surfaces, in air and in liquids.
BACKGROUND OF THE INVENTION
It is of course elementary that all microbial cells produce energy for their cellular activity through respiration. As cellular respiration occurs in living cells, pyridine nucleotides are reduced, flavins are oxidized, and other coenzymes and metabolites are produced. Alternatively, spores are found to be abundant with a calcium dipicolinic acid complex (a fluorescent compound otherwise rare in nature). The oxidation state of pyridine nucleotides, flavins and other cofactors, and/or the presence of calcium dipicolinate, can be simultaneously elucidated by concurrent excitation of each component with the appropriate electromagnetic radiation followed by detection of the characteristic radiation emitted by these individual fluorophores. Simultaneous excitation of a sample with multiple energies characteristic of the excitation for fluorescent cellular and endospore components with the subsequent collection and detection of emitted and reflected/scattered light energies (both associated with and independent of the fluorophores, respectively) is fundamental for the detection of microbes in a sample or on a non-living surface by the method described herein.
The detection of respiring cells in real world samples is made more reliable by the aforementioned method for two reasons. First, the simultaneous excitation of microbes by multiple excitation energies and ensuing coincident detection of numerous fluorescence signals reduces the chance of interference, as the probability of an interference source duplicating the characteristics of numerous fluorophores is extremely small. Second, the relative quantities of the intrinsic metabolites, and thus of the resulting fluorescent signals, have been found to fall within defined physiological ranges. Analysis of the signals is achieved with a method capable of two things: (1) separating the detected fluorescent signals originating from any microbes present from interferences or background signals and or scattered excitation signals, and (2) a requirement that the intensities of the signals from microbial metabolites, microbial components and spore components fall within physiological ranges. Thus, the basis for the detection of microbes in a sample is comprised of the following steps: first, excitation of a sample simultaneously with multiple excitation energies characteristic of cellular metabolites, microbial components, and spore components; second, the subsequent collection of the numerous individual fluorescence signals (associated with the maxima and minima of the emissions of these excited metabolites); and finally, analysis of the collected signals with a method capable of removing background fluorescence and comparing the relative signal magnitudes of metabolites to known physiological ranges.
Long-established technologies and methods used for microbial detection rely upon detection of products resulting from metabolic reactions, immunological capture or the amplification of expected nucleotide sequences. Since this invention employs detection of multiple intrinsic fluorophores from microbes, coupled with an analysis of the relative amount of signals due to these fluorophores, it can not only determine the presence of microbes, but is also capable of differentiating between viable cells, non-viable cells and spores. This method and apparatus uses no reagents, requires no physical contact with the sample, and delivers ‘real-time’ results.
There are other microbial detection methods that utilize fluorescence. Many of the flow cytometry methodologies rely on the fluorescence of dye molecules conjugated to immunological proteins targeted to the analyte of interest. An example of this can be found in U.S. Pat. No. 4,745,285 (to Recktenwald, et al.). Other fluorescence methods use added fluorescent metabolic dyes or dye conjugates (as in U.S. Pat. No. 4,900,934 to Peeters, et al.).
Some of the fluorescence-based microbe detection methodologies utilize intrinsic cellular fluorophores. One method (U.S. Pat. No. 5,424,959 to Reyes, et al.) simply compares the fluorescence spectra of the sample with a library of spectra. The method described in U.S. Pat. No. 5,474,910 to Alfano, compares the fluorescence of a sample surface to that of a clean surface. A popular intrinsic fluorophore used in microbial detection methods is the reduced pyridine nucleotide NADH. In U.S. Pat. No. 5,701,012 to Ho, NADH fluorescence is detected in a forced airstream containing the sample and compared to a blank. Alternatively, the ratio of NADH fluorescence to either the scattered excitation signal or other fluorescence emissions is used in U.S. patents to Powers (U.S. Pat. Nos. 5,760,406 and 5,968,766).
In U.S. Pat. Nos. 5,760,406 and 5,968,766, which issued Jun. 02, 1998 and Oct. 19, 1999, respectively, and which are incorporated herein by reference, there is disclosed a method and apparatus for the detection of microbes on non-living surfaces and samples. The sample to be examined is excited with electromagnetic radiation (1) having a wavelength greater than 350 nm causing the excitation of pyridine nucleotides present in microbial cells, and (2) having a wavelength below 340 nm as a measure of other characteristics of the environment. The ratios of the microbial pyridine nucleotide fluorescence emission (resultant from the excitation at the different wavelengths) to the reflected excitation signals are calculated and compared, as the basis for both the detection and quantitation of microbes present on the sample. This invention is able to locate and quantitate microbes on non-living surfaces, including meats.
Whereas the aforementioned patents to Powers depend upon ratio fluorescence for the detection of a single metabolite, the present invention utilizes excitation of one or more fluorophores coupled with an algorithm that subtracts the detected signals due to the scattered/reflected excitation energies. This difference in design and methodology makes the current invention better able to detect and quantitate microbes on non-living surfaces, in liquids and in air relative to other fluorescence methods. The current invention is superior in its detection of microbes as the detection of multiple intrinsic fluorophores reduces the probability of false positive results due to background interferences. The detection of microbes with the foregoing method and apparatus will have uses in biowarfare agent detection, cell sorting, medical diagnostics, sterilization verification, water quality testing, food production and preparation safety, and emergency response teams tasked with the detection, decontamination and protection of public infrastructure facilities.
With recent announcements of bacterial contamination in foodstuffs (meats, poultry, seafood, juices, fruits and vegetables), there has been a need to provide a method and apparatus that can be used to detect such microbial contamination in foods, on foods and on food preparation surfaces. This method and apparatus, as an object of the invention, should be operated inexpensively and rapidly in, for example, meat and poultry production facilities.
It is yet another object of the invention to provide a method and apparatus for use in the detection of microbial contamination on foods in which the fluorescence of pyridine nucleotides, flavins and other cofactors and spore components are excited by electromagnetic radiation to distinguish the metabolic reactions and spore components of microbes from the tissue of foodstuffs, allowing microbial contamination on foods to be determined without contact with said food.
It is accordingly an object of the invention to provide a method and apparatus that can be used in the detection of microbial contamination on non-living surfaces, in liquids and air. As a specific object of the invention, the method and apparatus can be used to find mic
Lloyd Christopher R.
Powers Linda S.
Cornaby K. S.
Microbiosystems, Limited Partnership
Saucier Sandra
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