Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Reexamination Certificate
1999-08-13
2002-04-30
Stucker, Jeffrey (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
C435S007100, C435S007320, C435S007350, C435S007720, C435S007940, C435S007950, C435S030000, C435S038000, C435S039000, C435S173400, C435S244000, C436S501000, C436S536000, C436S539000, C530S388100, C530S389100
Reexamination Certificate
active
06379918
ABSTRACT:
TECHNICAL FIELD
The present invention relates generally to compositions, formulae, devices, and methods for detecting and identifying microorganisms and, more particularly, to compositions, formulae, devices, and methods for detecting microorganisms by exposing highly conserved antigenic sites of target microorganisms without inactivating the microorganisms' ability to grow to sufficient levels to be detected by an antibody linked detection system.
BACKGROUND OF THE INVENTION
Microbial diseases have long been a major health concern worldwide. Significant increase in the frequency and severity of outbreaks have occurred throughout the world. New pathogenic bacteria, such as
E. coli
0157:H7, have been identified. Furthermore, previously recognized pathogenic genera have mutated to form drug resistant highly infectious strains such as
Salmonella typhimirium
DT 104. A key feature in the prevention of such diseases is early diagnosis. Epidemiologists must look for microbial contamination in the environment as well as in food products to find the effective disease prevention strategies.
One example is the outbreak in 1992 of Enterohemorrhagic
E. coli
(EHEC) in the Pacific Northwest of the United States due to contaminated ground beef. EHEC is a relatively “newly discovered” pathogen. EHEC was first isolated in 1975, and it was not until 1982 that
E. coli
0157:H7 was associated with two food related outbreaks of hemorrhagic colitis in the United States. The reported incidence of
E. coli
0157
:H7 cases is increasing. Typically,
E. coli
strains are harmless commensals, but a few strains are pathogenic. EHEC is particularly virulent and can trigger deadly complications, including severe abdominal cramps and acute renal failure in children as well as cardiovascular and central nervous system problems.
As another example, Salmonella is the leading cause (more than 50%) of total bacterial foodborne disease outbreaks, according to the United States Centers for Disease Control (CDC) surveillance of foodborne diseases. More than 40,000 cases per year were reported to the CDC during the period 1988-1992. Salmonella can infect a broad variety of warm- and cold blooded animals, and can survive for long periods of time outside a host.
In a further example,
Salmonella typhimurium
DT 104 was first identified in the United Kingdom in the early 1990s. It is a highly adapted drug resistant strain of Salmonella known for its virulence. Resultingly, significant clinical interest has surrounded this serotype.
S. typhimurium
DT 104 contains core cell wall antigen epitopes that are highly conserved among the genus Salmonella.
Listeria, a genus of gram positive bacteria, is widely distributed in nature, having been isolated from soil, water, vegetation and many animal species. The detection frequency for Listeria in the agricultural environment appears to be increasing. For specific outbreaks of listeriosis, estimates place mortality at 30% to 40% of affected patients, however, little is known of the minimum infective dose. One particularly troublesome aspect of Listeria control in foods is that Listeria can grow at temperatures as low as −0.4° C. and as high as 44° C. These factors all contribute to the increasing significance of Listeria as a food pathogen.
Campylobacter jejuni
and coli have recently been identified as the lead causes of enteritis, especially from poultry sources. This has led to an increased need to discriminate these two species from several other Campylobacter species which are not human pathogens. This requires the differential selection of more specific cell wall membrane antigen epitopes.
The ability to monitor potential environmental and food sources of microbial contamination quickly and easily, but with very high specificity, would reduce the risk of human infection. Therefore, an analytical method which affords high specificity, preferably combined with a device able to assay for microorganisms, including bacteria, yeasts, molds, fungi, parasites and viruses, that requires no special or technical equipment, can be performed in the field and does not require special skills would be useful. In the case of foodborne bacterial contamination, four of the major disease-related organisms are Salmonella, Listeria, EHEC and Campylobacter.
While there are a number of Salmonella, Listeria, and EHEC detection methods presently available, trained laboratory technicians and a minimum of 2-5 days are required to obtain test results by the standard cultural methods of analysis. New, more rapid methods are based on such techniques as enzyme linked immunoassay EIA), DNA hybridization, immunodiffusion, or growth/metabolism measurements. While taking much less time than the cultural methods, these rapid tests still require skilled technical training, a functional laboratory, and specialized equipment. These tests generally take a total of two or more days, including considerable hands-on time. Campylobacter detection methodology to date is technically intensive requiring fastidious media and environmental conditions, in addition to well-trained analysts.
Another recent technology in the diagnostic field involves lateral flow immunoassays. Such tests have been developed for the detection of human chorionic gonadotropin (hCG), and applied to pregnancy testing. Typically, a monoclonal or polyclonal antibody is immobilized in a discrete band near the distal end of a solid carrier strip, called the detection zone. Another amount of antibody is labeled with a detection reagent such as an inorganic sol or dyed polystyrene particle. This labeled antibody is reversibly fixed near the proximal end of the carrier strip. Upon hydration of the proximal end with a sample fluid potentially containing the antigen, the antigen reacts with the labeled antibody and the complex passes through the zone of immobilized antibody, forming a sandwich upon reacting with the immobilized antibody. The capture of the chromogenic reagent-antigen complex causes the formation of a visible signal in the detection zone.
Two major challenges must be addressed to distinguish pathogenic bacteria, as opposed to distinguishing hormones or other soluble molecular targets. These challenges are the need to detect all of the strains of a pathogenic microorganism in the presence of numerous antigenically related organisms, with a low tolerance for false positive results and a very low, preferably zero, tolerance for false negatives. The second challenge is the physical size and heterogeneity of the microorganism itself. A typical clinical diagnostic test, such as a test for hCG in urine, is focused on detecting a single, small, unique entity (ie., a hormone) in a well characterized matrix (e.g., urine). Furthermore, the structure of the analyte (hCG) is defined and uniform in size and composition.
Pathogen detection, for example, a test for Salmonella, must distinguish a particular pathogenic strain from nonpathogenic strains of similar microorganisms, such as Citrobacter spp. and Enterobacter spp. In contrast to the well-defined small size and structure of most hormones or marker proteins, microorganisms are very large, their surfaces are heterogeneous containing many distinct antigen epitopes that can undergo changes, such as the phase-switching of Salmonella flagella.
In addition, the cell wall membrane of many microorganisms contain antigen epitopes, such as lipopolysaccharides, which are repeated with a high degree of consistency within a given genus. These antigen epitopes serve as highly desirable targets for reaction with complimentary specific antibodies which, in turn, provides a method of high accuracy with low false positives. The ability to isolate and bind to antibodies reacting with these highly conserved antigen epitopes is difficult, however, because they can be sterically hindered by O-antigen polysaccharide chains. They are generally inaccessible because of a phenomenon known as steric interference. This steric interference is provided by the surface antigen epitopes of the microorganism. Exampl
Feldsine Philip T.
Kerr David E.
Mui Linda
Zhu Ping
Biocontrol Systems Inc.
SEED Intellectual Property Law Group PLLC
Stucker Jeffrey
Winkler Ulrike
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