Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1998-09-04
2002-02-05
Navarro, Mark (Department: 1645)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007200, C435S007320, C435S007900, C435S007920, C435S007940, C435S004000, C435S025000, C435S029000, C435S031000, C435S034000, C435S968000
Reexamination Certificate
active
06344332
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention is directed to the methods for rapid amplification and detection of actively respiring microorganisms. Such methods are useful in the detection of microbial contaminants in the environment, industrial systems, water purification systems, as well as clinical, food, cosmetic and pharmaceutical samples.
2. Description of the Background
Many U.S. industrial and medical business sectors are at risk for economic loss from microbial contamination. The presence of microorganisms can have a negative impact on business efficiency and productivity. Many bacterial species implicated in human disease as well as those non-pathogenic species that adversely impact industrial processes are ubiquitous in aqueous environments of all types including lakes, rivers, ponds, industrial process waters and even potable water supplies. The total potential economic loss to the U.S. gross domestic product due to microbial contamination has been estimated to be $1-$2 Trillion (THACO Corporation, Independent Market Research, 1993).
Three primary determinants for monitoring microorganism contamination in industrial processes are (i) determination of the quantity of microorganisms that are present, (ii) determination that there are no microorganisms present, and (iii) determination of the specific type (species) of microorganisms that are present.
The classical approach to determinants (i) and (ii) is to culture the sample in question in the presence of selective nutrients and microscopically examine a specimen after staining with specific reagents. Whereas this approach may be satisfactory for some definitive clinical examinations, it is necessary to provide rapid detection, enumeration and identification of microorganisms in industrial and other routine and non-routine medical examinations such as mass casualty or epidemic situations.
Modern methods for microorganism detection and enumeration have focused on the development of more sensitive methods of detecting microorganisms and to a lesser extent upon methods for amplification of the number of microorganisms present in the sample to be analyzed. Some of the technologies in current practice include DNA probes, RNA probes, ATP measurements, immunoassays, enzymatic assays, and respirometic measurements. Many of these tests do not rapidly detect less than 10
5
cfu/mL or still require complicated or lengthy amplification procedures to increase the concentration of the substrate being detected. Enhancement of the sensitivity of the detection system reduces the threshold concentration of microorganisms to be detected and consequently reduces the time required for amplification. These enhanced assay methods include fluorimetric, radiometric and photometric methods.
For instance, Schapp (U.S. Pat. No. 4,857,652) identified compounds that can be triggered by an activating agent to produce light. This luminescent reaction is used for ultra sensitive detection of phosphatase-linked antibodies and DNA probes. At least one such application of this technology has been commercialized as Photo Gene manufactured by Life Technologies, Inc. (Gaithersburg, Md.). Similarly, Abbas and Eden (U.S. Pat. No. 5,223,402) identify a method that uses 1,2-dioxetane chemiluminescent substrates linked to either alkaline phosphatase or &bgr;-D-galactosidase. Theoretically, their method can detect microorganism concentrations as low as 1-100 cfu/mL.
Another strategy for the enhancement of microbial detection is the utilization of fluorescence based detection systems. For example, Fleminger (Eur. J. Biochem. 125:609-15, 1982) used a fluorescent amino benzoyl group that was intra molecularly quenched by a nitrophenylalanyl group. In the presence of bacterial aminopeptidase P, the nitrophenylalanyl group is cleaved and the fluorescence of the sample increases proportionately. A wide variety of other enzymes have been assayed by similar procedures and include hydrolases, carboxypeptidases and endopeptidases. As is the case with the chemiluminescence based assays, fluorescence based assays are susceptible to interferences from chemical quenching agents typical in industrial process waters, require specialized equipment and operator training. Additionally, the fluorescent reagents themselves may be highly toxic and therefore unsuitable for some applications.
Although applicable in certain limited laboratory settings, chemiluminescent methods such as these are susceptible to interference from a variety of chemical quenching agents commonly found in industrial process waters, environmental water sources and biological matrices. Moreover, the methods, as taught in the above referenced patents, require specialized technical training of the user, specialized equipment, multiple steps in the conduct of the assay and enrichment of the microorganism concentration. Taken together, such considerations lengthen the total assay time, raise the capital costs and make this technology unsuitable for high volume, high throughput applications.
Species typing, referred to as determinant (iii) above, not only requires amplification of the microorganisms present, but also requires the selective detection of only the species of interest in the presence of background microflora. The classic approach to species typing is to selectively amplify the presence of the organism of interest through a pre-enrichment step followed by a selective enrichment step using a nutrient specific media followed finally by biochemical or serological confirmation. The time required for these procedures can be as long as six to seven days which is clearly outside the realm of practicality for use in industrial laboratories or high throughput clinical laboratories.
One strategy that has recently been commercialized is the GENE-TRAK™ calorimetric assay (GENE-TRAK Systems, Inc. Framingham, Mass.). This technology attempts to simultaneously exploit an amplification strategy and an enhancement of the detection system's sensitivity. The approach is an alternative to other approaches that use probes directed against chromosomal DNA. Instead, the GENE-TRAK system targets ribosomal RNA (rRNA) which is present in 1,000-10,000 copies per actively metabolizing cell. A unique homologous series of nucleotides, approximately 30 nucleotides in length and containing a poly-dA tail, is hybridized with the unique rRNA sequence in the target organism. This probe is referred to as the capture probe. A second unique probe of 35-40 nucleotides is labeled at the 3′ and the 5′ ends with fluorescein. This probe is the detector probe and binds to a region of the rRNA adjacent to the capture probe. After hybridization, bound complexes are captured on a solid support coated with poly-dT which hybridizes with the poly-dA tail of the capture probe. The rRNA-detector probe complex is detected with polyclonal anti-fluorescein antibody conjugated to horseradish peroxidase. This complex is then reacted with the enzyme substrate, hydrogen peroxide, in the presence of tetramethylbenzidine. The blue color that develops is proportional to the amount of rRNA captured.
Blackburn reviewed the development of rapid alternative methods for microorganism typing as it pertains to the food industry (C. de W. Blackburn, “Rapid and alternative methods for the detection of salmonellas in foods,” Journal of Applied Bacteriology, 75:199-214, 1993). Therein, Blackburn describes several techniques for detection of Salmonella that rely upon a selective pre-enrichment and enrichment approach to amplification, the best of which still required approximately six hours before detectable levels of Salmonella were present. Enhanced detection methods were also reviewed and included measurements of metabolism, immunoassays, fluorescent-antibody staining, enzyme immunoassay, immunosensors, bacteriophages and geneprobes. Analysis times could be reduced to as short as 20 minutes; the detection limits were about 10
5
cfu (Blackburn et al., “Separation and detection methods for salmonellas using immunomagnetic particles,
Baskar Padma
Ehrman Heller
Navarro Mark
Thaco Research, Ltd.
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