Data processing: measuring – calibrating – or testing – Measurement system in a specific environment – Biological or biochemical
Reexamination Certificate
2001-08-31
2004-09-28
Martinell, James (Department: 1631)
Data processing: measuring, calibrating, or testing
Measurement system in a specific environment
Biological or biochemical
C435S006120
Reexamination Certificate
active
06799122
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates generally to the fields of population and molecular genetics. In particular, it relates to a method for identifying polymorphic markers in a population.
2. Related Art
As a general rule, the taxonomic classification of species is generally reserved for organisms that are genetically similar and capable of mating productively. Since bacteria are asexual organisms, species generally refers to populations that share genetic and biochemical similarity. Despite the fact that species of bacteria share similarity, significant diversity can be observed when comparing different populations of a given species. To illustrate, the gut bacterium
Escherichia coli
consists of approximately 170 different serotypes.
One of the most important tasks of a clinical or industrial microbiologist is the precise determination of what microorganism, if any, is present in a sample. Using some commonly known and simple techniques, the microbiologist can generally deduce the species of the unknown microorganism relatively quickly. However, subspecies or actual strain determination of the microorganism present in the sample frequently requires sophisticated methods of genetic or biochemical analysis. This, of course, translates into higher costs and a slower turnaround time.
Determination of a specific strain of bacteria rather than the mere species that is present in a sample is particularly important to the food industry. For example, of the approximately 170 strains of
E. coli
, only about 30 of them are pathogenic to humans. Depending on the pathogenic potential of strains or subspecies, processors may often elect to dump a batch contaminated with the species rather than invest time and effort in determining the precise strain or subspecies classification. This is because of the aforementioned costs associated with deducing the actual strain to determine if it is in fact pathogenic. The obvious problem with such “dumping” is that it also has costs associated with it, namely lost revenues. Therefore, it is desirable to have some method of quickly identifying what strain of bacteria may be present in a sample. In order to develop diagnostic tools for the rapid identification of bacterial strains, it is first necessary to identify genetic markers which are characteristic of problematic and less problematic strains.
In addition to the practical application of strain-level classification, understanding genetic characteristics of populations of bacteria is also important for creating safer food environments. Alteration of the genome by gene acquisition, deletion, and mutation, along with new routes of transmission into the food chain, and the selective pressures that are imposed in food production environments, are the elements that drive evolution and emergence of foodborne pathogens. Thus, it increasingly important that new methods are devised for understanding how pathogenic and spoilage organisms enter the food supply, how different populations of pathogenic organisms are effected by selective pressures in food production environments, and how this relates to characteristics that confer increased virulence, spoilage, and/or transmissibility on certain populations. Several molecular genetic approaches have been developed to provide high-resolution information about populations, including random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), octamer-based genome scanning (OGBS), and multi-locus sequencing. Each of these approaches suffers from the fact that they provide only limited coverage of the genome in a single experiment and must therefore be performed in a plurality of intentions to increase genome coverage, particularly in the case of closely related strains. The present invention overcomes this limitation by allowing for coverage of the entire genome in a single experiment and by determination of genetic segments that are specific to relevant populations.
Another bacterial of particular interest to the food industry is
Listeria monocytogenes
. Although several serotypes of
Listeria monocytogenes
strains are found in foods and in the environment, most human infections (>95%) are caused by only three serotypes, 1/2a, 1/2b and 4b. These strains belong to two major genetic groups, one of which includes serotype 1/2a while 1/2b and 4b belong to the other group. Most molecular genetic and immunologic studies have used strains from the first genetic group, including 1/2a (strains 10403s, EGD, NCTC7973 Mack) and 1/2c (strains LO28). Strains representing the other group have largely been omitted from molecular genetic studies. However, strains from this group, especially strains of serotype 4b, may be of the most significance to the food industry and public health.
Strains of serotype 4b account not only for a substantial fraction (ca. 40%) of sporadic infections but also for almost all of the common-source outbreaks of listeriosis that have been studied, including the 1985 Jalisco cheese out break in Los Angeles and the latest multi-state outbreak in the United States traced to contaminated hot dogs. There is a need for a relatively quick, simple, and inexpensive method for determining unique DNA sequence information for rapidly distinguishing among different subpopulations of
L. monocytogenes
isolates. Such tests are crucial for high-throughput analyses necessary for epidemiological studies and risk assessment studies.
Listeria monocytogenes
is a ubiquitous gram-positive organism that can cause life-threatening infections ranging from meningitis, septicemia, and fetal death. Although the incidence of listeriosis is low, the associated morbidity can be quite high, particularly in pregnant women and immunocompromised individuals (Gellin and Broome, 1989).
L. monocytogenes
is well known for its robust physiological characteristics and is one of few pathogenic bacteria capable of growth at refrigeration temperatures, under conditions of low pH, and/or high osmolarity (Farber and Brown, 1990; Farber and Pterkin, 1991; Kroll and Patchett, 1992; Miller 1992; Wilkins et al. 1972). Kroll and Patchett, 1992).
L. monocytogenes
can grow in several types of cultured cells and is capable of intracellular growth and spread to adjacent host cells through the use of host cell cytoskeletal components (Galliard et al. 1987; Portnoy et al. 1988; Tilney and Portnoy, 1989; Mounier et al. 1990). Genetic analysis of virulence in
L. monocytogenes
has identified several loci that contribute directly to the series of events that occur during host cell invasion (reviewed in Portnoy et al. 1992, Sheehan et al. 1994). These virulence genes include adhesins, a cytolytic toxin, an actin polymerizing protein and phospholipases, that function in host cell entry, vacuole escape, replication, and spread to adjacent host cells respectively.
Several signals, such as temperature and carbohydrates seem to control regulation of the virulence genes (Leimeister-Wachter et al. 1992; Park and Kroll, 1993) and recent evidence suggests that these are separate pathways that govern expression of the virulence genes (Renzoni et al. 1997). Thus, the virulence gene regulator, called PrfA, may couple transcription of the virulence genes to a variety of cues that could signal entry into a host.
L. monocytogenes
strains display serotypic differences in somatic (numbered) and flagellar (lettered) antigens (Seelinger and Hoehne, 1979). Although 13 different serotypes of
L. monocytogenes
are found in foods and in the environment (Farber and Pterkin, 1991), most clinical isolates are of only 3 serotypes, 1/2a, 1/2b and 4b (Schuchat et al. 1991), suggesting that these serotypes may be particularly virulent for humans or are better able to survive the necessary hurdles for transmission and establishment of infection.
Several studies have been conducted to examine genetic relationships among
L. monocytogenes
strains. One of the most significant was an early study using Multi-Locus Enzyme Electrophoresis (MLEE), which identified 45 di
Conagra Grocery Products Company
Martinell James
Nutter & McClennen & Fish LLP
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