Chemistry: molecular biology and microbiology – Apparatus – Including measuring or testing
Reexamination Certificate
2000-05-03
2004-11-23
Fredman, Jeffrey (Department: 1634)
Chemistry: molecular biology and microbiology
Apparatus
Including measuring or testing
C435S006120, C435S091100, C435S091200, C536S023100, C536S024300
Reexamination Certificate
active
06821770
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to methods, compositions and devices for identifying microorganisms. More specifically, the invention relates to the use of a matrix of polynucleotide probes having specificity for ribosomal nucleic acids (rRNA and rDNA) that distinguish species or groups of organisms from each other based on molecular phylogeny.
BACKGROUND OF THE INVENTION
The procedures for detecting and identifying infectious organisms are some of the most critical tasks performed in the clinical laboratory. Whereas laboratory diagnoses of infectious diseases formerly were made by experienced technicians using visual inspection of stained clinical material, more rapid and objective results are obtainable using modern techniques. Immunoassays, including radioimmunoassays, enzyme-linked immunoassays, and latex agglutination and immunoblotting assays have developed into powerful diagnostic tools having utilities that are enhanced by the availability of monoclonal antibodies. More recent advances in signal and target amplification have introduced the era of molecular diagnostics based on the use of polynucleotide probes.
Indeed, clinical microbiologists now use an extensive array of techniques for identifying infectious organisms (see
Manual of Clinical Microbiology
Murray et al., eds., 6
th
edition, ASM Press (1995)). Automated substrate utilization systems typically rely on enzymatic reactions that release chromogenic of fluorogenic compounds, tetrazolium-based indicators of metabolic activity in the presence of different carbon sources, or detection of the acid products of metabolism. The patterns of positive and negative reactions with these substrates establish a biochemical profile that can be used to identify microorganisms isolated from clinical samples. The chromatographic profiles of the more than 300 fatty acids that contribute to the formation of lipids in bacteria and yeast have also been used to phenotype microorganisms. Despite the availability of these very powerful techniques, polynucleotide-based assays are rapidly gaining popularity in clinical laboratory practice.
The specificity of polynucleotide hybridization reactions, together with the extraordinary sensitivity afforded by nucleic acid amplification techniques, has made molecular diagnostics the method of choice for detecting and identifying microbes that are available in only very small quantities. Commonly used DNA probe hybridization formats include: solid phase hybridization, solution-phase hybridization and in situ hybridization. In solid phase hybridization methods, a sample containing microbial polynucleotides is immobilized to a solid support, denatured and then probed with a polynucleotide probe that harbors a detectable label. Unhybridized probe is removed from the system and specifically hybridized probe detected, for example by autoradiography or direct visual observation. In solution-phase hybridization procedures, the target polynucleotide and the labeled probe are free to interact in an aqueous hybridization buffer. Specifically hybridized probe is then detected as an indicator of the presence of target polynucleotides in the mixture. In situ hybridization using formalin-fixed tissue sections is used for obtaining information about the physical distribution and abundance of microorganisms. As they are conventionally practiced, molecular diagnostic assays are conducted to determine whether a particular species or group of organisms is present in a biological sample undergoing testing.
Bacteremia and fungemia are conditions marked by the presence of bacterial and fungal organisms in circulating blood. Sepsis refers to a severe bacterial infection that spreads in the blood throughout the entire body. In septicemia, the presence of microorganisms in the blood indicates that the host's immune system has failed to localize an infection. Organisms responsible for these conditions typically are identified after inoculating a “blood culture bottle” with a sample of patient blood, and then typing any microorganisms that are propagated. Mortality associated with bloodstream infections ranges from an estimated 20% to 50% (see
Clinics in Laboratory Medicine
14:9 (1994)). An estimated 50,000 deaths each year in the United States result from sepsis (
Vanderbilt Univ. Med. Center Reporter
1991 Mar. 1; 2(8):1,3). Thus, substantial attention has been devoted to the diagnosis and treatment of this syndrome.
Unfortunately, the blood culture methods that represent the “gold standard” for diagnosing septicemia have significant limitations (Weinstein,
Clin Infect Dis
23:40 (1996)). Indeed, no single medium is ideal for propagating all potential bloodstream organisms, some relevant microorganisms grow poorly in conventional media and systems, and positive results require hours to days of incubation. Each of these areas calls for improvement if diagnosis of septicemia is to become more rapid and accurate.
Molecular diagnostic methods employing collections of species-specific DNA probes have been used to identify microorganisms in blood cultures. According to one procedure, microorganisms present in growth-positive culture bottles were first isolated and then Gram-stained and analyzed for morphology (Davis et al.,
J. Clin Microbiol.
29:2193 (1991)). The appearance of the stained organisms determined which of several different polynucleotide probes were employed in a subsequent testing step. Instances wherein positive hybridization results were obtained yielded presumptive identifications. Identification of bacteria that yielded negative hybridization results were limited to observations made after Gram-staining and microscopic analysis. These investigations confirmed that DNA probes could be used for the rapid identification of bacteria taken directly from blood culture bottles, but still required evaluation of Gram-stained microorganisms by an experienced microbiologist. Importantly, polymicrobial bacteremias could not be analyzed in the procedure due to the lack of available probes.
In a study of several hundred positive blood cultures obtained from septicemia patients, Weinstein et al., in
Clin Infect Dis
24:584 (1997) concluded that the five most common pathogens were
Staphylococcus aureus, Escherichia coli
, coagulase-negative
staphylococci, Klebsiella pneumoniae
, and
Enterococcus
species. Yeasts were also common isolates from blood culture bottles and represented true fungemia when detected about 92% of the time.
Candida albicans
ranked among the top 10 microorganisms causing septicemia. Interestingly, about 91% of the instances of bacteremia and fungemia were unimicrobial while the remaining cases were associated with two or three organisms. The lowest associated mortality among the patients having positive blood cultures were associated with coagulase-negative
staphylococci
(5.5%) and the highest with yeasts and fungi (35.8%). Most important, septicemia-associated mortality was shown to increase in proportion to the duration of inappropriate antimicrobial therapy. This finding highlighted the importance of an early and proper clinical diagnosis.
The invention detailed below provides a rapid means for identifying microorganisms using techniques that are suited for automated analysis. The invented devices and methods can even be used to resolve the identity of microorganisms that are contained in a mixed population of microorganisms.
SUMMARY OF THE INVENTION
One aspect of the present invention relates to a device for hybridizing nucleic acids. The invented device includes a solid support and a plurality of addresses disposed on the solid support. Each of the addresses includes at least one probe that hybridizes ribosomal nucleic acids from at least one microbial species under high stringency hybridization conditions. The plurality of addresses includes a higher-order address, an intermediate-order address, and a lower-order address. The lower-order address hybridizes ribosomal nucleic acids from a subset of organisms having ribosomal nucleic acids that hybridize a
Fredman Jeffrey
Gen-Probe Incorporated
Gilly Michael J.
Goldberg Jeanine
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