Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2001-11-01
2004-08-24
Housel, James (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S004000, C435S005000, C435S007200, C422S082050, C436S164000, C436S172000, C436S536000, C436S538000
Reexamination Certificate
active
06780602
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to a method for the taxonomic identification of pathogenic microorganisms and the detection of their proteinaceous toxins.
Pathogenic microorganisms, particularly pathogenic bacteria which either occur naturally or which have acquired virulence factors, are responsible for many diseases which plague mankind. Many of these bacteria have been proposed as biowarfare agents. In addition, there is also the risk and likelihood that nonpathogenic microbes could also be used as pathogens after genetic manipulation (e.g.,
Escherichia coli
harboring the cholera toxin).
Typical pathogenic bacteria include those responsible for botulism, bubonic plague, cholera, diphtheria, dysentery, leprosy, meningitis, scarlet fever, syphilis and tuberculosis, to mention a few. During the last several decades, the public perception has been one of near indifference in industrialized nations, principally because of successes that have been achieved in combating these diseases using antibiotic therapy. However, bacteria are becoming alarmingly resistant to antibiotics. In addition, there have been recent revelations of new roles that bacteria perform in human diseases such as
Helicobacter pylori
as the causative agent of peptic ulcers,
Burkholderia cepacia
as a new pulmonary pathogen and
Chlamydia pneumoniae
as a possible trigger of coronary heart disease. Apart from those pathogens, various socioeconomic changes are similarly contributing to the worldwide rise in food-borne infections by bacteria such as
Escherichia coli
, Salmonella spp., Vibrio spp., and
Campylobacter jejuni.
Potential infections are also important considerations in battlefield medicine. A number of bacterial pathogens, including
Bacillis anthracis
and
Yersinia pestis
and their exotoxins, have been used as weapons. And there is always the risk that nonpathogenic microbes can be engineered to be pathogenic and employed as biowarfare agents.
Pathogenic microorganisms are also of concern to the livestock and poultry industries as well as in wildlife management. For example,
Brucella abortus
causes the spontaneous abortion of calves in cattle. Water supplies contaminated with exotoxin-producing microorganisms have been implicated in the deaths of bird, fish and mammal populations. More recently, mad cow disease has been traced to the oral transmission of a proteinaceous particle not retained by filters. Thus, there is clearly a need for rapid and inexpensive techniques to conduct field assays for toxic proteins and pathogenic microorganisms that plague animals as well as humans.
As a general proposition, bacterial contamination can be detected by ordinary light microscopy. This technique, however, is only of limited taxonomic value. The investigation and quantitation of areas greater than microns in size are difficult and time consuming. Many commercially available systems rely on the growth of cultures of bacteria to obtain sufficiently large samples (outgrowth) for the subsequent application of differential metabolic tests for species (genus) identification. However, techniques requiring bacterial outgrowth may fail to detect viable but nonculturable cells. To the contrary, the growth media employed may favor the growth of bacteria with specific phenotypes.
More sensitive and more rapid typing schemes are described in “Strategies to Accelerate the Applicability of Gene Amplification Protocols for Pathogen Detection in Meat and Meat Products” by S. Pillai and S. C. Ricke (
Crit. Rev. Microbiol.
21(4), 239-261 (1995)) and “Molecular Approaches for Environmental Monitoring of Microorganisms” by R. M. Atlas, G. Sayler, R. S. Burlage and A. K. Bej (
Biotechniques
12(5), 706-717 (1992)). Those techniques employ the polymerase chain reaction (PCR) for amplification of bacterial DNA or RNA, followed by nucleic acid sequencing to detect the presence of a particular bacterial species. Such general amplification and sequencing techniques require technical expertise and are not easily adaptable outside of specialized laboratory conditions. PCR-based techniques utilize the inference of microbial presence since these techniques provide only a positive analysis whenever an intact target nucleic acid sequence, not necessarily a microbe, is detected. PCR is also unable to detect the presence of toxic microbial proteins. Moreover, the detection of specific microorganisms in environmental samples is made difficult by the presence of materials that interfere with the effectual amplification of target DNA in ‘dirty’ samples.
Mass spectral analysis of volatile cell components (e.g., fatty acids) after sample lysis or pyrolysis has been used for the detection of bacteria and viruses. One description of the methods used to detect microorganisms with this method can be found in “Characterization of Microorganisms and Biomarker Development from Global ESI-MS/MS Analyses of Cell Lysates” by F. Xiang, G. A. Anderson, T. D. Veenstra, M. S. Lipton and R. D. Smith (
Anal. Chem.
72 (11), 2475-2481 (2000)). Unfortunately, identification of the analyte is unreliable as the compositions of a microbe's volatile components change depending upon different environmental growth conditions.
Another approach utilizes immunochemical capture as described in “The Use of Immunological Methods to Detect and Identify Bacteria in the Environment” by M. Schlotter, B. Assmus and A. Hartmann (
Biotech. Adv.
13, 75-80 (1995)), followed by optical detection of the captured cells. The most popular immunoassay method, enzyme-linked immunosorbent assay (ELISA), has a detection limit of several hundred cells. This is well below the ID
50
of extremely infectious bacteria such as
Shigella flexneri
. Piezoelectric detection techniques, such as those described by “Development of a Piezoelectric Immunosensor for the Detection of
Salmonella typhimurium
” by E. Prusak-Sochaczewski and J. H. T. Luong (
Enzyme Microb. Technol.
12: 173-177 (1990)) are even less sensitive having a detection limitation of about 5×10
5
cells. A recent report entitled “Biosensor Based on Force Microscope Technology” by D. R. Baselt, G. U. Lee and R. J. Colton (
Biosens. & Bioelectron.
13, 731-739 (1998)) describes the use of an atomic force microscope (AFM) to detect immunocaptured cells; this method has little utility outside a laboratory setting and when the sample volumes are large. Immunoassays are also presently used in the trace analysis of peptides and proteins.
Moreover, the prior art has made extensive use of immobilized antibodies in peptide/protein/microorganism capture. Those techniques likewise involve significant problems because the antibodies employed are very sensitive to variations in pH, ionic strength and temperature. Antibodies are susceptible to degradation by a host of proteolytic enzymes in “dirty” samples. In addition, the density of antibody molecules supported on surfaces (e.g., microwell plates or magnetic beads) is not as high as is frequently necessary. A good summary of the state of the art, still up-to-date, is “Microbial Detection” by N. Hobson, I. Tothill and A. Turner (
Biosens. & Bioelectron.
11, 455-477 (1996)).
Medical and military considerations call for better toxin and pathogen detection technologies. Real-time assessment of battlefield contamination by a remote sensing unit is necessary to permit and facilitate rapid diagnosis for administration of appropriate counter-measures. A microbe/toxic protein sensor useful in such situation requires the ability to globally discriminate between pathogens and non-pathogens. In addition, such techniques require high sensitivity when less than 100 cells are present and analysis that can be completed in the field in less than 15 minutes. Such techniques should be able to recognize pathogens and provide some assessment of strain virulence or toxigenicity.
To date, common approaches used for the identification of pathogenic microorganisms and their proteinaceous toxins have employed immunological methodologies. Immunological methods suffer from the se
Ellis, Jr. Walther R.
Lloyd Christopher R.
Powers Linda S.
Cornaby K. S.
Housel James
Lucas Zachariah
Microbiosystems, Limited Partnership
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