Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-04-04
2003-10-07
Horlick, Kenneth R. (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S005000, C435S029000, C435S034000, C435S039000, C435S804000, C435S822000, C435S196000, C536S024300, C536S024320
Reexamination Certificate
active
06630302
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the identification of species, and in particular, methods and compositions for distinguishing between bacterial and fungal species and determining the identity of bacterial and fungal pathogens in biological samples.
BACKGROUND
The detection and identification of microorganisms recovered from clinical specimens or environmental sources is an important aspect of clinical microbiology, as this information is important to physicians in making decisions related to methods of treatment. In order that a particular microorganism is identified correctly and consistently, regardless of the source or the laboratory identifying the organism, reproducible systems for identifying microorganisms are critical. As stated by Finegold, “The primary purpose of nomenclature of microorganisms is to permit us to know as exactly as possible what another clinician, microbiologist, epidemiologist, or author is referring to when describing an organism responsible for infection of an individual or outbreak” (S. Finegold, “Introduction to summary of current nomenclature, taxonomy, and classification of various microbial agents,”
Clin. Infect. Dis.,
16:597 [1993]).
Classification, nomenclature, and identification are three separate, but interrelated aspects of taxonomy. Classification is the arranging of organisms into taxonomic groups (i.e., taxa) on the basis of similarities or relationships. A multitude of prokaryotic organisms has been identified, with great diversity in their types, and many more organisms being characterized and classified on a regular basis.
Classification has been used to organize the seemingly chaotic array of individual bacteria into an orderly framework. Through use of a classification framework, a new isolate can be more easily be characterized by comparison with known organisms. The choice of criteria for placement into groups is currently somewhat arbitrary, although most classifications are based on phylogenetic relationships. An example of the arbitrariness of bacterial classification is reflected in the genetic definition of a “species” as being strains of bacteria that exhibit 70% DNA relatedness, with 5% or less divergence within related sequences (Baron et al., “Classification and identification of bacteria,” in
Manual of Clinical Microbiology,
Murray et al. (eds.), ASM Press, Washington, D.C., pp. 249-264 [1995]).
Generally, identification of a bacterium is based on its overall morphological and biochemical patterns observed in culture. Indeed, this is the primary technique employed today in clinical laboratories. Of course, this approach is flawed by the fact that diverse organisms can have similar morphologies and/or biochemical requirements. Moreover, numerous organisms associated with disease may not be cultured in vitro. Indeed, some do not grow well in traditional in vivo culture systems, such as cell cultures or embryonated eggs, nor in vitro such as various nutrient agars and broths.
What is needed is a more defined system for speciation, and in particular, speciation of bacteria and fungi. Such an approach should be amenable to automation, permitting the approach to be used routinely in a clinical laboratory.
SUMMARY OF THE INVENTION
The present invention relates to the identification of microbial species, and in particular, methods and compositions for determining the species for an unknown bacterium (or fungus) in a sample. The methods and compositions of the present invention permit distinguishing between bacterial species (or between fungal species) and determining the identity of bacterial (or fungal) pathogens in biological samples. The present invention contemplates a method of speciation that does not require the sequencing of nucleic acid from biological samples. Instead, the method is based on detection of heretofore unknown uniquely conserved portions of ribosomal nucleic acid, such portions being conveniently revealed by restriction digestion of DNA encoding ribosomal nucleic acid, i.e. rRNA genes.
In one embodiment of the method of the present invention for speciation, the present invention contemplates analysis of one or more so-called Ribosomal operons (“rrn”) of a clinical isolate, the operon comprising three genes often arranged in the order 16S-23S-5S for prokaryotes (and 18S-5.8S-25S for eukaryotes), with “spacer” DNA separating each gene (hereinafter represented by: 5′-16S -spacer-23S-spacer-5S-3′). The present invention contemplates that the analysis of at least one of these operons in an unknown bacterial or fungal species (when evaluated for the “signature band sets” of a particular species, the signature bands and methods for determining signature bands herein described) allows for accurate speciation.
It is not intended that the present invention be limited by the technique by which the operons are analyzed. In one embodiment, primers directed to these sequences can be employed in an amplification reaction (such as PCR). On the other hand, these conserved sequences can conveniently be analyzed with restriction enzymes. Specifically, the present invention contemplates digesting bacterial or fungal DNA with one or more restriction enzymes which will produce a piece of nucleic acid which is within (or bounded by) the 5′ and 3′ ends of the operon. The resulting digestion product will be conserved for any given species and can serve as a “signature” for that particular species (other species having one or more signature bands of a different size).
Specific embodiments of such a method include (but are not limited to) digestion with one or more restriction enzymes so as to produce any one of the following digestion products:
5′-16S-spacer-23S-spacer-5S-3′,
5′-16S-spacer-23S-spacer-3′,
5′-16S-spacer-23S-3′,
5′-16S-spacer-3′,
5′-16S-3′,
5′-spacer-23S-spacer-5S-3′,
5′-23S-spacer-5S-3′,
5′-spacer-5S-3′,
5′-5S-3′,
5′-23S-3′
5′-spacer-23S-spacer-3′, or
5′-spacer-23S-3′
The present invention also contemplates a host of variations on the above digestion products by cleaving in the middle of genes and/or in the middle of spacer regions. However, for the convenience of detecting such digestion products by gel electrophoresis, it is preferred that the digestion product (due to the relatively limited resolution level of gel electrophoresis) be at least 200 bp in size (and more preferably between 400 and 3000 bp in size).
In one embodiment, the present invention contemplates digestion of such DNA with restriction enzymes that cut only once in the DNA encoding 16S ribosomal RNA and only once in the DNA encoding 23S ribosomal RNA. In a preferred embodiment, the present invention contemplates digestion of bacterial DNA using a single restriction enzyme which cuts only once in the DNA encoding 16S ribosomal RNA and only once in the DNA encoding 23S ribosomal RNA.
In one embodiment, the present invention contemplates a method for bacterial speciation, comprising: i) isolation of bacterial DNA from a sample, said DNA comprising DNA encoding 16S and 23S rRNA; ii) digestion of said isolated DNA with one or more restriction enzymes under conditions such that restriction fragments are produced, said restriction fragments comprising a first digestion product of said DNA encoding 16S and 23S rRNA, said first digestion product comprising at least a portion of said DNA encoding 16S rRNA and at least a portion of said DNA encoding 23S rRNA; iii) separation of said restriction fragments (e.g. by gel electrophoresis), iv) detection of said first digestion product.
In another embodiment, the present invention contemplates a method for bacterial speciation, comprising: i) isolation of bacterial DNA from a sample; said DNA comprising DNA encoding 16S and 23S rRNA; ii) digestion of said isolated DNA with one or more restriction enzymes under conditions such that restriction fragments are produced, said restriction fragments compr
Horlick Kenneth R.
The Trustees of Boston University
Weingarten Schurgin, Gagnebin & Lebovici LLP
Wilder Cynthia
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