CA1214980A - Method for identifying and characterizing organisms - Google Patents

Abstract

Abstract Method for Identifying and Characterizing Organisms A method of characterizing and/or identifying an unknown organism which comprises comparing the chromatographic pattern of restriction endonuclease-digested DNA from the organism, which digested DNA has been reassociated or hybridized with ribosomal RNA-information containing nucleic acid from a probe organism, with equivalent chromatographic patterns of at least two known different known organism species,

Description

BACKGROUND OF THE INVENTION

Field of the Invention The present invention relates to a method for the rapid and accurate characterization and identification of organisms, including prokaryotic and eukaryotic organisms, such as bacteria, plants, and animals.

Description of the Prior Art The classification of living organisms has traditionally been done along more or less arbitrary and somewhat artificial lines. For example, the living world has been divided into two kingdoms: Plantae (plants) and Animalia (animals). This classification works well for generally familiar organisms, but becomes difficult for such organisms as unicellular ones (e.g., green flagellates, bacteria, blue green algae)~ since these differ in fundamental ways from the "plants" and !'animals".
It has been suggested to simply divide organisms with respect to the internal architecture of the cell. In thi~ scheme, all cellular organisms are either pro}caryotic or eulcaryotic. Prokaryotes are less cornple~ than eukaryotes, they lack internal compartmentaliæation by unit membrane systems, and lack a defined nucleusO Prokaryotic genetic information is carried in the cytoplasm on douhle-stranded, circular DNA; no other DNA is present in cells (except for the possible presence of phage, bacterial viruses, and circular DNA plasmids, capable of autonomous replication~. Eukaryotes on the other hand have a multiplicity of unit membrane systems which serve to segregate many of the functional cornponents into speeialized and isolated regions. For example, genetic information (~NA) can be found in a well-compartmentalized nucleus and also in organelles:
mitochondria and (in photosynthetic organisms) chloroplasts. Tne replication, transcription, and translation of the eukaryotic genome occurs at either two or three distinct sites within the cell: in the nucleocytoplasmic region, in the mitochondrion and in the ehloroplast.
The differenees between prokaryotes and eukaryotes, howeover, brea]cs down when a comparison of mitocllondria and chloroplasts is carried Ol1t with prokaryotes: these organelles are today considered to have been derived from free-living prokaryotes, which entered into an endosymbiotie relation with primitive eukaryotes, and eventually became closely integrated witll the machirlery of the host cell and incapable of independent existence (see e.g., Fox, G.E., _ t al, Science 209:457-463 (1980), at 462; Stanier, R.Y. et al, "The Microbial World", Fourth Edition, ~rentice-Hall, Inc. 1976~ at p. 86). For example, it has been demonstrated that DNA from mouse L cell mitochondria carrying the ribosomal P~ gene region exhibits notable sequence homologies to Escherichia coli ribosomal RN~, thus providing strong support for the endosymbiotic model (Van Etten, R.A., et al, Cell, 22:157-170 (1980)). It has also been shown that the nucleotide sequence of ~3S ribosomal DNA from ~ chloroplast has 71~ homology with 23S ribosomal DNA from E. coli (Edwards, K. and Kossel, E~., Nucleic Acids Research, 9:2853-2869 (1981)); other related work (Bonen, L. and Gray, M.W., i_ , 3:319-335 (1930)) also further supports the general concept.
In this model the eukaryotic cell is a phylogenetic "chimera" with organelle components that are clearly prokaryotic in nature. The "prokaryotic-eukcaryotic" dichotomy then, also has drawbacks, even as a broad classification method.
Where classification of organisms becomes more than a scientific exercise is in the identification of plants and animals for hybridi2ation and breediny purposes, and in the accurate and reliable identification of microorganisms which may infect so-called "higher" organisms or other media. For example, the plant-breeder, cattle breeder, or fish breeder may wish to have a quick and reliable means of identifying dif~erent species and strains of their subjects. The veterinarian, physician/ or horticulturist May wish to have an accurate identiication of any inEectious organisms (parasites, fun(~i, bacteria, etc.) and viruses present in examined plant or animal tissues.
The correct identification of species of these organisms and viruses is of particular importance.
The problem can best be illustrated by referring to the identification of bacteria, ~ames of bacterial species usually represent many strains, and a strain is considered to be a population derived from a single cell. Strains of a species have similar sets of attributes which serve to define the species. Precise definitions of bacterial species are diEficult to express because subjective limits to strain diversity withill species are required to define species boundaries. (Buchanan, R. E., International Bulletin o Bacteriological Nomenclature and Taxonomy, 15:25-32 (1965)). The practical application of definitions of species to the identification of an un~nown bacterial strain requires the selection of relevant probes, such as substrates and conditions to detect phenotypic attributes, and radioactively-labeled DNA frorn the same species. It is necessary to use a screeniny procedure to presumptively identify t~le strain so that the appropriate probe can be selected to identify the strain. The challenge is to precisely define the boundaries of species, preferably in terms of a standard probe which reveals species-specific information, so that definitions o~ species can be directly and equally applied to the identification of unknown strains.
Bergey's Manual of Determinative Bacteriology (~uchanan, R. E. and Gibbons, N. Æ., Editors, 1974, 8th Edition, The Williams & Wilkins Company, Baltimore) provides the most comprehensive treatment of bacterial classification particularly for nomenclature, type strains, pertinent literature, and the like. It is, however, only a starting point for the identification of any species since, inter alia, it is normally out of date, and is limited ln space to describing species quite briefly. (See for example Brenner, D.J. "Manual of Clinical Microbiology", 3rd Edition, American Society of Microblology, Washington, D.C. 1980, pages The term "species", as applied to bacteria, has been defined as a distinct kind of organism, having certain distinguishiny features, and as a yroup of organisms which generally bear a close resemblance to one another in the more essential features of their organization. The problem with these definitions is that they are subjective; Brenner, supra, at page 2.
Species have also been defined solely on the basis of criteria such as l)ost range, pathogenicity, ability or inability to produce gas in the fermentation of a given sugar, and rapid or delayed fermentation of sugars.
In the 1960's, numerical bacterial taxonomy (also called computer or phenetic taxonomy) became wi.dely used. ~umerical taxonomy is based on an examination of as much of the organism's genetic potential as possible. By classifying on the basis of a large number oE characteristics, it is possible to form groups of strains with a stated degree of similarity and consider them species. Tests which are valuable for the characterization of one species, however, may not be useful for the next, so this means to define species is not directly and practically applicable to the identification of unknown strains. Although this may be overco,ne in part by selecting attributes which seem to be species specific, when these attributes are used to identify unknown strains, the species definition is applied indirectly, See for example Brenner, supra at pages 2-6. The general method, furtllermore, suffers from several problems when it is used as the sole basis for deflning a species, among them the number and nature of the tests to be used~
whether the tests should be weighted and how, what level of similarity should be chosen to reflect relatedness, whether the same level of similarities is applicable to all groups, etc.
Hugh R.H. and Giliardi, G.L. "Manual of Clinical Microbiology", 2nd Edition, American Society for Microbiology, Washington, D.C., 1974, pages 250-269, list minimal phenotypic characters as a means to define bacterial species that rnakes use of fractions of genomes. By studying a large, randomly selected sample of strains of a species, the attributes most highly conserved or common to a vast majority of the strains can be selected to define the species. The use of minimal characters is progressive and begins with a screening procedure to presumptively identify a strain, so that the appropriate additional media can be selected. Then the known conserved attributes of the species are studied ~ith the expectation that the strain will have most of the millirnal characters. Some o~ the minimal characters do not occur in all strains oE the species. A related concept is the comparative study oE the type, the neo-type, or a recognized reference strain of the species. This control is _9_ necessary because media and procedures may differ among laboratories, and it is the strain, not the procedure, that is the s~andard for the species.
A molecular approach to bacteriai classiEication is to compare two genomes by DNA-D~A reassociation. A
genetic definition of species includes the provision that strains o-E species are 706 or more related. With DNA-DNA reassociation a strain can be identified only if the radioactively labeled DNA probe and unknown DNA
are from the same species. The practical application of this 70% species definition however is limited by selection of an appropriate probe. This may be overcome in part by selecting phenotypic attributes which seem to correlate with the reassociation group, but when these are used alone the D~A-DNA reassociation species definition is also applied indirectly 9renner, su~ at page 3, states that tlle ideal means of identifying bacterial species would be a 'black box' which would separate genes, and instantly compare the nucleic acid sequences in a given strain with a standard pattern for every known species - something akin to mass spectrophotometric analysisO .
Brenner, however, concedes that although restriction -endonuclease analysis can be done to determine common sequences in isolated genes, "we are not at all close to having an appropriate black box, especially one suited for clinical laboratory use". E~is words could be equally applied to any species of organisms.
This brief review of the prior art leads to the conclusion that there presently exists a need for a rapid, accurate, and reliable means for identifying un~nown bacteria and other organisms, and to quickly classify the same, especially to iclentify the organism of a disease, or of a desirable biochemical reaction. The method should be generally and readily useful in clinical laboratories, should not be dependent on the number of tests done, on the subjective prejudices of the clinician, nor the fortuitous or unfortuitous trial and error methods of the past. Further, a need also exists for a method useful for identifying and distinguishing genera and species of any living organism, which can be readily and reliably used by veterinarians, plant-breeders, toxicologists, animal breeders, entomologists and in other related areas, where such identification is necessary.

SUMMARY OF TIlE INVENTION
It is therefore an object of the invention to provide a quick, reliable and accurate method of objectively identifying or~3anisms, especially - but not limited to - rnicroor~3anisms.
Yet another object of the invention is to provide a method of identifying orgarlisms such as bacteria which utilizes the organisms' genome.
Another object of the invention is to provide a method o characterizing and identifying species and genera of pathogenic organisms in the clinical laboratory, so as to provide the capability of characterizing and identifying the cause of any given animal.or plant disease.
Still another object of the invention is to provide various products useful in the aforementioned methodologies.
These and other objects of the invention, as will hereinafter become more readily apparent, have been attained by providing:
A method o~ characterizing an unknown organism which comprises comparincJ the chromatographic pattern of restriction endonuclease-digested D~A from said organism, which digested DNA has been hybridized or reassociated with ribosomal ~lA information-containing nucleic acid from or derived from a probe organism, with equivalent chromatographic patterns of at least two known different organism species.
Still another object of the invention has been attained by providing:
A method of diagnosing a pathogenic orc3allism inection in a sample which comprises identifying the organism in said sample by the aforementioned method~

iJ

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BR~EF DrscRIpTIoN OF THE DRAWINGS
FIGURE 1 shows the EcoR I restriction endonuclease digest of DNA isolated from strains of Pseudomonas aeruginosa, using cDNA to 16S and 23S ribosomal RNA
(rRNA) of E. coli as the probe.
FIGURE 2 shows the Pst I restriction endonuclease digest of DNA isolated from strains of P. aeruginosa, using cDNA to 16S and 23S rRNA of E. coli as the probe.
FIGURE 3 shows the EcoR I res-triction endonuclease digest of DNA isolated from species of glucose-nonfermenting, gram~negative rods, using cDNA to 16S and 23S rRNA of E. coli as the probe.
FIGURE 4 shows the Pst I restriction endonuclease digest of DNA isolated from species of glucose-non-fermenting, gram-negative rods using cDNA to 16S and 23S
rRNA of E. coli as the probe.
FIGURE 5 shows the EcoR I restriction endonuclease digest of DNA isolated frorn various Bacillus subtilis strains, using cDNA to ]6S and 23S rRNA of E. coli as the probe.
FIGURE 6 shows the Pst I data for the same strains as in FIGURE 5, with the same probe.
FIGURE 7 shows the Bgl II data for the same strains as in FIGURES 5 and 6, ~ith the same probe.
FIGURE 8 sllowS the Sac I data for the same strains as in FIG~RES 5-7, with the same probe.

FIGUl~E 9 shows the EcoR I restriction endonuclease digest of ~NA isolated from B. subtilis and B. polymyxa, using cDNA to 16S and 23S rRNA from E. coli as the probe.
FIG~RE 10 shows the Pst I data for the same strains as in FIGURE 9 with the same probe.
FIGURE 11 shows the Bgl II and Sac I data for the same strains as in FIG~RES 9 and 10, with the same probe.
FIGURE 12 shows the detection of Streptococcus pneumoniae in EcoR I digested DNA Erom infected mouse tissues using cDNA from 16S and 23S rRNA from E. coli as the probeO
FIGURE 13 shows the identification of a mouse species by comparing Pst I digests of DNA isolated from mammalian tissues, using cDNA to 18S and 28S rRNA from cytoplasmic ribosomes of Mus musculus domesticus (mouse).
FIGURE 14 shows the EcoR I digested DNA from mouse and cat tissues hybridized with Mus musculus domesticus 28S rRNA cDNA probe.
FIGURE 15 shows Sac I digested DNA from mammalian tissues hybridized with ~us musculus domesticus 18S and 28S rRNA cDNA probe.
FIG~R~ 16 shows EcoR I di~ested DNA from mammalian tissues and cell cultures hybridized with Mus musculus domesticus 18S and 28S rRNA cDNA probe.

DESCRIPTION OF T~IE PREFER~ED EMBODIMENTS
This invention is based on the inventor's realization that, if species are discrete clusters of strains related to a comrnoll speciation event, there should be, despite divergence, a likeness shared by strains that objectively defir-es the species boundary;
strains of species should contain structural information which is a clue to their common origin. Since the greatest amount of an oryanism's past history survives in semantides, DNA and RNA,(Zuckerkandl, E._ and Pauling, L., Journal of Theoretical Biology 8:357-366 (1965)), the inventor concluded that an experimental approach which makes use of information contained in a few conserved genes is the use of rRNA. Ribosomal RNA ~as a structural and functional role in protein synthesis (Schaup, Journal of Theoretical Biology, 70:215-224 (1978)), and the general conclusion from rRNA-DNA hybridization studies, is that the base sequences of ribosomal RNA genes are less likely to chanqe, or are more conserved during evolution, than are the majority of other genes (Moore, R.I.. Current Topics In ~icrobiology and Immunobiology, Vol. 64:105-128 (1974), Springer-Verlag, New Yor]c). For example, the primary struc-ture o~ 16S rR~A from a number of bacterial species has been inferred from oligonucleotide analysis (Fox, G.~., et al, Interna~ional Journal of Systernatic Bacteriology, 27:~4-57 (1977)).

There are negligible differences in the 16S oligomer catalo~s of several strains of E.coli ( chida, T. et al, Journal of Molecular Evolution, 3:63-77 (1974)); the substan-~ial differences among species, however, can be used for a scheme of bacterial phylogeny ( o_ G.E., Science, 209:457-4G3 (1980)). Di~eren-t strains of a bacterial species are not necessarily identical;
restriction enzyme maps show tha-t differen-t EcoR I sites occur in rRNA genes in two strains o~ E.Coli (Boros, I.A.
et al, Nucleic Acids Research 6:1~17--1830 (1979)).
Bacteria appear to share conserved rRN~ gene sequences and the other sequences are variable (Fox, 1977, supra.
The present inventor further discovered that restriction endonuclease digests of DNA have sets o~
fragments containing rRNA gene sequences -that are similar in strains of a species of organism (e.g., bacteria), but different in strains o~ other species of the organism;
i.e., despite strain variation, enzyme specific sets of restriction fragments with high ~requencies of occurrence, and minimal common genotypic characters, define the species. This is the essence of the invention. The present inventor also discovered that -the method is general, in that it is applicable to both eukaryotic and prokaryotic DNA, using a ribosomal nucleic acid probe from any organism, prokaryotic or eukaryotic, oE ~he same or different (classic) taxonomic classification than the organism being identified.

-15a-The present invention ofers an ohjec-tive method of defining organisms based on detecting DNA ~ragments J
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contaillin~ ri~osornal RNA gene sequences in restriction endonuclease digests. The detection is carried out by hybridizing or reassociating DNA fragments ~ith nueleie acid containing rRNA information from a probe organism.
By the "organism" which can be charaeterized (which term is meant to include "identified") by the process of the invention, it is meant to include virtually any organism which, by definition, contains DMA. In this respect it is useful to refer to a classical taxonomie scheme as a point of reference.
All organisms belonging to the Kingdoms Monera, Plantae and Animalia are included. For example, among those of the Kingdom Monera can be mentioned the Schi20mycetes (Bacteria) of the classes myxobacteria, spirochetes, eubacteria, rickettsiae, and the cyanopytha (blue green algae). Among those of the Kingdom Plantae can be mentioned the Division Euglenophyta (Euglenoids), Division Chlorophyta (green-algae) elasses ehlorophyceae and charophyeeae; Division Chrysophyta, elasses xanthophyeeae, chrysophyseae, bacillariophyceae; Division Pyrrophyta (Dinoflagellates); Division Phaeophyta (~rown algae); Division Rhoclophyta (Red algae)j Division Myxomycophyta (slime molds), elasses myxomycetes, acrasiae, plasmodiophoreae, labyrinthuleae; Division Eumycophyta (true fungi), elasses phycolllycetes, ascomyeetes, and basidomycetes; Division ~ryophyLa, classes hepaticae, anthocerotae, and musci; Division Tracheophyta (Vascular plants), subdivisions psilopsida, lycopsyda, sphenopsida, pteropsida, spermopsiaa classes cycadae, ginkgoae, coni~erae, gneteae and angiospermae subclasses dicotyledoneae, monocotyledoneaeO Among those o~ the Kingdom Animalia can be mentioned the Subkingdom Protozoa, Phylum Protozoa (~cellular animals) subphylum plasmodroma, classes flagellata, sarcodina and sporozoa;
subphylum ciliophora, class ciliata; the Subkingdom Parazoa, Phylum pori~era (Sponges), classes calcarea, he~actinellida, and desrnospongiae; the Sub~ingdom ~esozoa, phylurn mesozoa; the Subkingdom Metazoa, Section Radi.ata, Phylum coelenterata, classes hydrozoa, scyphozoa, anthozoa, Phylum ctenophora, classes tentaculata and nuda; Section Protostomia Phylum platyhelmintes (flatworms) classes turbellana, trematoda, and cestoda; Phylum nemertina; Phylum acanthocephala;
phylum aschelmintles, classes roti~era, gastrotricha, ~inorhyncha, priapulida, nematoda and nematomorpha;
Phylum entoprocta; Phylurn ectoprocta, classes gymnolaemata and phylactolaemata; Phylum phoronida;
Phylum brachiopoda, classes inarticulata and articulata;
Phylum mollusca (molluscs) classes ainphineura, monoplacophora, gastropoda, scaphopoda, pelecypoda, an.d cephalopoda; Phylum sipunculida; Phylum echiurida; phylum annelida, classes polychaeta, oligochaeta and hirudinea, Phylum onycl~ophora; Phylum i:ardigrada; phylum pentastomida; phylum arthropoda, subphylum trylobita, subphylum chelicerata classes xiphosura, arachmida, pycnogomida, subphylum mandibulata classes crustacea, chilopoda, diplopoda, pauropoda, symphyla, insecta of the orders collembola, protura, diplura, thysanura, ephemerida, odonata, orthoptera, dermaptera, embiania, plecoptera, zoraptera, corrodentia, mallophaga, anoplura, thysanoptera, hemiptera, neuroptera, coleoptera, hymenoptera, mecoptera, siphonaptera, diptera, triehoptera and lepidoptera; those of the Seetion Deuterostomia, phylum chaetognatha, phylum echinodermata, elasses crinoideal asterordea, ophiuroidea, echinoidea, and ho].oturoidea, phylum pogonophora; phylum hemichordata, elasses enteropneusta, and pterobranchia;
phylum chordata, subphylum urochordata, classes aseidiaciae, thaliaceae, larvacea; subphylum cephalochordatar subphylum vertebrata, classes a~natha, ehondrichthyes, osteiehthyes (subclass saccopteiygii orders crossopterygii and dipnoi), amphibia, reptilia, aves and mammalia, subelass prototheria, subclass theria, orders marsupialia, inseetivora, dermoptera, ehiroptera, primates, edentatar pholidota, lagomorpha, rodentia, eetaeeae, carnivora, tubulidentata, probosicdea, hyraeoidea, sirenia, perissodactyla and artiodactyla. It is understood that beyond the order, the organisms are still classified accordin~ to their families, tribes~
genus and species, and even subspecies, infrasubspecific taxons, and strains or individuals. In addition cell cultures (plant or animal), as well as viruses can also be identified. These classifications are used in this application for illustrative purposes only, and are not to be taken as exclusive. The organism is either known or unkno-~n, most commonly the organism is an unknown being identified.
Functionally, for the purposes of this invention, it is convenient to divide all organisms into the eukaryotes and the prokaryotes. When identifying a prokaryotic organism, the DNA to be endonuclease-digested is that present in the cells or in the non-compartmentalized chromosomes. When identifying a eulcaryotic organism one may either use the nuclear DNA or the organelle DNA
(mitochondrial DN~ or chloroplast DNA), and endonuclease-digest the same.
Briefly, high molecular weight DNA and/or small circular DNAs are isolated ~rom the or~anism to be identified in order to analyze the rRNA gene sequences and possibly sequences that could be used to create a taxon below the rank of species or infrasubspecific subdivisions. The DNA's are extracted by methods which are well known to the art. The DNA's are cut at specific sites into fragments, by restriction endonucleases. The fragments are separated according to size by a standard chromatographic system, such as gel electrophoresis. The yels are stained, as is otherwise well known in the art, and stanc-~arc3ized as to the Erayrnent sizes usiny standard curves constructed t~ith fragments of kno~n siæes. The separated frac31nents are then transferred to cellulose nitrate paper by the Southern blot technique (Southern, E.~., Journal of Molecular Biology, 38:503-517 (1975), herein incor~orated by reference), and covalently bound thereto by heating. The fragments containing the rRNA
gene sequences are then located, by their capacity to hybridize with a nucleic acid probe containing rRNA
information. The nucleic acid probe can either be non-radioactively labeled or (preferably) radioactively labeled. When radioactively labeled, the probe can be ribosomal ~NA (rRNA), or radioactively labeled DNA which is complementary to ribosomal RNA (rRNAcDNA), either synthesized by reverse transcription or contained on a cloned fragment, which can be labeled, for example, by nick translation.
The probe is derived from an arbitrarily chosèn organism, see in~ra. Once hybridization has ~occured, the -hybridized ~rayments are detected by selectively detecting double stranded nucleic acid (non-radiolabeled probe), or visualized ~y, e.g. autora~iograplly (radiolal~eled probe). The size oE eclcil Eragment whici has been hybridized is determilled from the distance traveled ~sing stan~ard curves as described previously.
The amount of hybridization, the pattern of hybridization, and the size of the hybridized fragments can be used individually or in conjunction to identify the organism.
The pattern that emerges from this hybridization can be readily compared to e~uivalent chromatographic patterns derived from at least two and up to a multiplicity of known, standard organisms, genera or species. After a preliminary broad classification has already been carried out (using, for example, classical taxonomy), the comparison can be either by visual inspection and matching of appropriate chromatographic patterns, by co-mparison of nucleic acid fragment sizes, by band intensity (amount of hybridization) or by any cornbination thereof. Ideally, the comparison is carried out with a one-dimensional computer-based pattern recognition system, such as those used in point-of-sale transactions.
The present inventor discovered that when using the aforementioned method, the chromatographic patterns for organisms of the same species are substantially similar, with minor variations allowed for intraspecies differences due to strain variations/ whereas differences between species, and differences between qenera (and higher classificatiolls) are maximal.
The use o~ enzyrne-speci~ic fragment variations in strains of a species even permits the typing of strains for various purposes; e.g. in the case oE bacteria, for epidemiological purposes. In fact, restriction enzymes can be chosen for their ability to distinguish strains within species.
The "probe organism" used in the present invention, and from which is obtained the nucleic acid probe, can also be any of the aforementioned organisms; it can be either eukaryotic or prokaryotic. The only limitation is given by the fact that the rRNA-containing probe should hybridize maximally with the endonuclease digest of the unknown organism's DNA.
There are four types of ribosomal RNA information-containing probes: 1) prokaryotic ribosomal probe (especially bacterial-derived rRNA), 2) eukaryotic mitochondrial ribosomal probe, 3) eukaryotic chloroplast ribosomal probe, and 4) eukaryotic non-organelle ribosomal probe. There are also four sources of DNA (to be endonuclease di~ested): 1) prokaryotic cellular DNA,

2) eukaryotic mitochondrial DNA, 3) eukaryotic chloroplast DNA, and 4) eukaryotic nuclear DNA. The following hybridization Table can thus be constructed (Table 1).

Table 1 .
Hybridizati Table .
Ribosomal Probe Unkncwn organis~
DNA Prokaryotic Eukaryotic Mito- Chloro- Non-chondrial plast organelle Prokaryotic ~ + +

Eu.(l) Mitochondria + + -~ _ Eu. Chloroplast + ~ +

EU; Nuc_ear (2) _ _ +
-(2) = refers -to less effective hybridization, see Example 4, infra.
The Table shows which ribosomal RNA probes can be maximally hybridized with which unknown DNA. For example, one can identify a eukaryotic or~anism by extracting species specific mitochondrial or chloroplast DNA, endonuclease-digesting it and hybridizing the digest with either a prokaryotic ribosomal Rl~A probe, or with an organelle derived eukaryotic ribosomal probe. In the same manner, one can identify a prokaryotic organism by extracting species-speci~ic cellular DNA, endonuclease-digesting it, and hybridizing tile digest with either a prokaryotic ribosornal RNA probe, or an organelle-derived eukaryotic ribosomal RNA probe. Also, one can identify a eukaryotic organism by extracting and di~estin~ species-. ,"~. -c~

L ?~ t !~

-2~-specific nuclear DNA, and hybridizing it with a non-or~anelle derived eukaryotic ribosornal probe. Eukaryotes could be defined by one or any combination of the nuclear, mitochondria, or in some cases chloroplast systems. These cross~hybridizations are based on the previously mentioned fact that rRNA nucleic acid derived from eukaryotic organelles has extensive homology with prokaryotic rRNA nucleic acid, hut that the same homologies are not preserlt to such extent between nuclear-derived eukaryotie DN~ and prokaryotic DNA.
In essence, hecause the genetic code is universal, because all cells (eukaryotic or prokaryotie) require ribosomes to translate the information of the eode into protein, and because ribosomal RNA is highly conserved, there are su~ficient homologies between the cross-hybridizing elements ("+" on Table 1) to assure that tne method will work as indieated.
The choice of any pair of DNA to be digested and accornpanying ribosomal probe is arbitrary, and will depend on the or~anism being identified, i.e. it will depend on the question asked. For examplel in detecting a prokaryotic speeies (e.g. bacteria) present in a eukaryotic cell (e.g. animal or plant) for purposes Orc deteeting and identiEying an infeetinc3 agent, one may choose a prokaryotie ribosomal probe and work under eonditions where organelle-derived ~NA is not extraeted or, only minimally extracted. In this manner one assures that interference between organelle-derived DNA and prokaryotic DJ~ is mini~al. In identifyinc3 a eukaryotic species (which is not infected with a prokaryote) with a prokaryotic ribosomal probe, it is best to maximize the concentration of organelle derivecl DNA, as for example by separating organelles from nuclei, and then extracting only organelle DNA If one wishes to identify a eukaryo~ic organisrn which has been infected with a prokaryotic organism, it is best to use a non-organelle, non-prokaryotic derivecl ribosomal probe since it will not hybridize well with the DNA from the prokaryote.
It is preierred to use a pair ( DNA and ribosomal probe) fro;n the same cingdom, or same subkingdom, or same section, or same phylum, or same subphylum, or same class, or same subclass, or same order, or same family or sa)ne tribe or same genus. It is particularly preferred to use prokaryotic ribosomal probe (e.g. bacterial ribosomal probe) to hybridize with prokaryotic DNA. In this manner one could detect, quantify, and identify genera, species, and strains o~ prokaryotic organlsms.
One of the most pre~erred prokaryotic ribosomal probes is derived from bacteria and further, because of the ease and availability, from E. coli. The ribosolnal probe from -. coli can be used to identify any organism, especially any prokaryotic organism, most preferably any bacterial , ,. . ~ ~

genera, species or strains, Another particularly preferred embodiment is to use eukaryotic ribosomal probe derived from a given class to identify eukaryotic organisms of the same class (e.g. mammalian ribosomal probe to identify mammalian organisms). Most preferred is to use ribGsomal probe and DNA
from the same subclass and/or order and/or family of organisms, (e.~. if identifying a species of mouse, it is preferred to use mouse-deri~ed ribosomal probe.
The most sensitive and useful pair systems are those where there is less evolutionary distance or diversity between the source of the ribosomal probe and restricted DNA.
The individual steps involved in the technique are generally known in the art. ~hey will be described herein--after broadly with reference to both eukaryotic and prokaryotic cells when applicable, or specifically for each type of cell is some difference in technique exists.

The first step is extraction of the DNA from the unknown organism. Nuclear DNA from eukaryotic cells can be selectively extracted by standard methodology well known to the art ~see for example Drohan, W. et al,Biochem. Biophys. Acta 521 (197~3 1-15)because organelle DNA is small and circular, spooling techniques serve to separate the non-circular nuclear DNA from the circular.

~ i3 organelle-derived DNA. ~s a corollary, the non-spooled material contains the organelle-derive(3 D~A which can separately be isolated by density gradient centrifuyation. ~lternatively mitochondria (or chloroplasts) are separated from a mixture of disrupted cells; the purified mitochondrial (or chloroplast) fraction is used for the preparation of organelle -derived DNA while the purified nuclear fraction is used to prepare nuclear DNA. (see for example Bonen L. and Gray, M.W., Nucleic Acids Research _:319-335 (1980)).
Prokaryotic DNA extraction is also well known in the art. Thus, for example, an unknown bacterium present in any medium, such as an industrial fermentation suspension, agar medium, plant or animal tissue or sample or the like, is trea~ed under well known conditions designed to extract high molecular weight DNA
therefrom. For example, cells of the unknown organism can be suspended in extraction buffer, lysozyme added thereto, and the suspension incubated. Cell disruption can be further accelerated by addition of detergents, and/or by increase in temperature. Protease digestion followed by chloroform/ phenol extraction and ethanol precipitation can be used to finalize the extraction of DNA. An alternative method of extraction, which is much faster than phenol/chloro~orm extraction, is rapid isolation of DNA using ethanol precipitaton~ This method - 2~ -is preferably used to isolate DNA directly from colonies or small, liquid cultures. ~he method is described in Davis, R.W.
et al: "A Manual for Genetic Engineering, Advanced Bacterial Genetics", (hereinafter "Davis") Cold Spring Harbor I,aboratory, Cold Spring, Harbor, New York, 1980, pp. 120-121.
The DNA (prokaryotic or eukaryotic (nuclear or non-nuclear)) is dissolved in physiological buffer for the next step.
Digestion of extracted DNA is carried out with restr:iction endonuclease enzymes. Any restriction endonuclease enzyme can be used, provided of course that it is not from the same organism species as -that being identified, since otherwise, the DNA may remain intact. (This may, in any event, identify the organism, since the enzymes are not expected to cut DNA
from the species of their origin). Since the organism species being characterized may be unknown, obtaining a digest of fragments may entail a minimum amount of trial and error which can routinely be carried out by those skilled in the art without undue experimentation. Examples of possible restriction endo-nuclease enzymes are Bgl I, BamH I, EcoR I, Pst I, Hind III, Bal I, Hga I, Sal I, Xba I, Sac I, Sst I, Bcl I, Xho I, Kpn I, Pvu II, Sau IIIa, or the like~ See also Davis, ~E~ra, at pp 228-230. ~ mixture of one or more -2'3-endonucleases can also be used for the digestion.
Normally, DNA and endonuclease are incubated together in an appropriate buffer for an ap~ropriate period of tirne (ranging from 1 to 48 hours, at temperatures ranging from 25C-65C, preferably 37C).
The resulting chromatoc3raphic pattern will depend on the type or types of endonucleases utilized, and will be endonuclease-specific. It is therefore necessary to note which enzyme or enzymes have been used for the digestion since the comparative patterns used in the catalog should have been prepared using the same enzyme or enzymes.
After endonuclease digestion, the incubation mixture, which contains fragments of varying sizes, is separated -thereinto by an appropriate chromatographic method. Any method which is capable of separating nucleic acid digests according to size, and which allows the eventual hybridization with the nucleic acid probe, can be used. Preferred is gel electropnoresis, most preferred is agarose gel electrophoresis. -In this system~
the DN~ digests are normally electrophoresed in an appropriate buffer, the gels are normally immersed in an etllidiurn bromide solution, and placed on a ~V-light box to visualize standard marker fragments ~hich may have been added.
After separation and visualization, the DNA
fraglnents are transferred onto nitrocellulose filter paper by the method of So~hern (Journal of Molecular Biology, 38:503-517 (1975)). The transfer can be carried out after denaturation and neutralization s~eps, and is usually done or long periods of time (approxima-tely 10-20 hours) or, alternatively by means o~ an electrically driven transfer from gel -to paper. In.struments used to accelerate the transfer from gel to paper are commercially available. ~he receiving nitrocellulose Eilter papers are then normally baked at high temperatures (60-80C) for several hours, to bi~d the DNA
to the filter.
The probe utilized for the hybridization of the paper-bound DNA digest fragments is a nucleic acid probe preferably from a given well-defined organism. It may be detectably labeled or non-labeled, preferably detectably labeled. In such case, it is ei-ther de~ectably labeled ribosomal RNP~ (rRNA) from such organism, nick-translated labeled DNA probes containing ribosomal information, cloned DNA probes containing ribosomal information, or detectably labeled DNA which is complementary to the ribosomal RN~ from the probe organism (rRNAcD~A).
Depending on the choice of pair, the ribosomal probe may be from a prokaryo-te, or from a eukaryote (cytoplasm-derived, or organelle derived). Most preferably, the det~ctable label i~ a radioactive label such as radioactive phosphorus (e.g., 32p, 3H or 14C). The nucleic acid probe may also be labeled with metal -30a-atoms. For example, uridine and cytidine nucleotides can forrn covalent mercury derivatives. Mercura-ted nucleoside triphosphates are good substra~es for many nucleic acid polymerases, includiny reverse transcrip-tase (Dale et al, Proceedings of the National Academy of Sciences 70:2238-2242, 1973). Direct covalent mercuration of natural nucle]c acids has been described. (Dale et al, Biochemistry 14:2447-2457). Reannealing properties of mercurated polymers resemble those of the corresponding nonmercurated polymers (Dale and Ward, Biochemistry 14:2458-2469). Metal labelled probes can be de-tec-ted, for example, by photo-acoustic spectroscopy, x-ray spectroscopy, e.g., x-ray fluorescence, x-ray absorbance, or photon spectroscopy.

isolation of rRNA from eukaryotes or prokaryotes is well known in the art. For example, to prepare rRNA from eukaryotic cytoplaslnic ribosomes, RNA can be extracted from whole cells or ribosomes, separated by sucrose gradient centrifugation, and the 18S and 2gS fractions can be collected using known molecular weight markers.
(See for e~ample Perr~, R.P. and D. E. Kelley, "Persistent Synthesis of 5S'RNA when Production of 28S
and 18S Ribosomal RNA is Inhibited by Low Doses of Actinomycin D", J. Cell. Physiol. 72:235-2~6 (1~68), herein incorporated by reference). As a corollary, organelle-derived rRNA is isolated and purified from the organelle fractions in the same manner (see e.g. Van Etten, R.A. r et al, Cell 22:157-170 (1980), or Edwards, K. et al, Nucleic Acids Research 9:2853-2869 (1981)).

-If radioactively labeled ribosomal R~A probe isused, the same is isolated from the probe organism after growth or cultivation of the organism with nutrients or in culture media containing appropriately radioactive compounds. When the probe is complementary DNA
(rRNAcDNA), the same is prepared by reverse transcribing isolated rRNA from the probe organism, in the presence of radioactive nucleoside triphosphates (e.g.r32p_ nucleosides or 311-nucleosides).
The labeled ribosomal probe may also be a nic]c-translated DNA molecule, especially one obtained from ... ,, . ,~ , ~ . .

organelle-derived whole circular DNA. In this embodiment, chloroplast or mitochondrial circular DMA is nick-translated in the presence o~ radiolabel, and a labeled DNA ribosomal probe is thereby obtained. The chloroplast labeled probe will h~bridize best with chloroplast D`.~A, and the mitochondrial labeled probe will hybridize best with mitocnondrial DNA. The chloroplast (or mitochondrial) nick-translated labeled ribosomal probe will hybridize second best with mitochondrial (or chloroplast) DNA; it will also hybridize, albeit in less favorable fashion~ with whole plant (Gr animal) DNA. The ribosomal probe may also be obtained from eukaryotic nuclear DNA by nick-translation, although practical considerations would rule against this mode. A more useful approach in this embodiment is to cut out rRNA
genes from the nuclear eukaryotic DNA (by restriction enzymes), separate the fragments, identify the ribosomal gene sequences (as by hybridization), and isolate the ribosomal gene sequences (as by electrophoresis). Th isolated rR~A sequences may then be recombined into a plasrnid or vector, and after trans~ormation of an appropriate host, cloned in 32P-containing medià.
A1-ternatively, the transformed host is grown, and the DN~
is then isolated and labeled by nick-translation; or the DNA is isolated, the rRI~A sequences are cut out and then labeled. The resulting ribosomal probe will hybridize in the same instances as r~NAcD~A (see infra).
The preferred nucleic acid probe is radioactively labeled DNA complementary to rRNA from the probe organism. In this embodimellt, rRNA is purified frol-n the probe or~anism by isolatiny the ribosomes and separating and substantially purifying therefrom -the appropriate RNA
as described supra. The riboso-nal ~NA is thus ribosome-free and is also substantially free of other RNA's such as transfer RNA (tXNA) or messenger RNA (mRNA).
Prokaryotic rRNA normally contains three subspecies: the so-called 5S, 16S and 23S fragments. The reverse transcription into cDNA can be carried out with a mixture of all three, or alternatively, with a mixture of 16S and 23S fragments. It is less preferred to carry out the reverse transcription with only one of the components, althou~h under certain conditions this may be feasible.
Eukaryotic rRNA normally contains two subspecies: 18S and 28S, and the reverse transcription into cDNA can be carried out with a mixture of 18S and 2~S fragments or with each.
The pure rRNA, substantially free of other types of RNA, is incubated with any reverse transcriptase capable of reverse transcribing it into cDNA, preferably with reverse transcriptase from avian myeloblastosis virus (AL~V) in the presence of a primer such as calf thymus DNA
hydrolysate. The mixture should contain appropriate ~ J

-.3'1 -deo~ynucleoside triphosphates, wherein at least one of said nucleosides is radioactively labeled, for example with 32p. For example, deoxycy~idine 5,_~32p)l deoxythymidine 5~ _(32p), deoxyadenine 5l_(32p), or deoxyguanidine 5'-3~) triphosphates can be used as the radioactive nucleosides~ After incubation, from 30 minutes to 5 hours at 25C-40C, extraction with chloroEorm and phenol, and centrifugation as well as chromatography, the radioactively labeled fractions are pooled, and constitute the cDNA probe. The radioactively labeled cD~A probe in substantially purified for~, i.e., free of non-labeled molecules, free of cDNA ~hich is complementary to other types of RNA, free of proteinaceous materials as well as free of cellular components such as membranes, organelles and the like, also constitutes an aspect of the present invention. A
preferred probe is prokaryotic labelled rRNAcDNA, most preferred bein~ the bacterial labelled rRNAc~NA. The probe species can be any bacterial microorganism, such as those of the family Enterobacteriaceae, Brucella, Bacillus, Pseudomonas, Lactobacillus, Haemophilus, ~ . . _ . .
Micobacterium, Vibrio, Neisseria, Bactroides and other anaerobic groups, Legionella, and the like. Althouyh the prokaryotic examples in the present application are limited to the use of E.coli as a bacterial prokaryotic probe organism, this aspect of the invention is by no means limited to this microor~anism. The use of cDNA in radioactively labeled form as the probe is preferred to the use of radioactively labeled ribosomal ~A because DNA has greater stability during hybridi~ationO
It is important to recognize that the labeled cDNA
probe should be a faithful copy of the rRNA, i.e~ be one wherein all nucleotide sequences of the template rRNA are transcrihed each time the synthesis is carried out. The use o~ a primer is essential in this respect. That the cDNA is a faithful copy can be demonstrated by -the fact that it should have two properties following hybridization 1. The cDNA should protect 100% of labeled rRNA
from ribonuclease digestion; an~
2. The labeled cDNA should specifically anneal to the r~NA as shown by resistance to Sl nuclease.
Beljanski M.M. et al, C.R~ Acad. Sc Paris t 28~, Serie D. p. 1825-1828 (1978), describe 3H radioactively labeled cDNA derived from E.coli rRNA. The cDNA in this work was not prepared with reverse transcriptase in the presence of a primer as in -the present invention, but was prepared with a DNA polymerase I, usiny as a template rRNA which had been pre-cleaved using ribonuclease U2~
The rRNA digestion product (with RNAse U2~ of Beljanski et al has a different base ratio than the initial rRNA, indicating a loss of bases and/or loss of short fragmentsO ~hus the cDNA obtained therefrom is not a faithful copy. In addition, the use of DNA polymerase I
used by Beljanski is known to favor predominance of homopoly-meric over heteropolymeric transcription of rRNA (see Sarin, P.S., et al, Biochem. Biophys. Res. Comm~ 202-214 (1974)).
In sum, the "ribosomal" probe can be seen as being derived a) from genome DNA containing rRNA genes, by cloning and/or nick-translation, b) from ribosomal RNA itself or c) from rRNAcDNA by reverse transcription of rRNA.
The next step in the process of the invention is the hybridization of the separated D~TA digest from the unknown organism with the unlabeled or (preferably) radioactively labeled rRNA or cDNA probe. Hybridization is carried out by contacting the paper containing covalently labeled DNA
digest from -the unknown, with a hybridization mix containing the ribosomal probe. Incubation is carried out at elevated temperatures (50-70C) for long periods of time, filter papers are then washed to remove unbound radioactivity (if needed~, air dried and readied for detection. An alternative, highly preferred hybridization, which is much more rapid than the one described above, is the room temperature phenol emulsion reassociation technique o~ Kohne, D.E. et_al, Biochemistry, 16:5329-5341 (1977).

After hybridization,, the technique requires selective detection of the appropriately hybridized fragments. This detection can be carried out by taking advantage of the double strandedness of the hybridized fragments and using a selective method therefor (for non-labeled probe)l or by autoradiography or by an appropriate radiation scanner which may or may not be computerized, and which may increase the speed of detection (for labeled probe). These techniques are wel7 known to those skilled in the art and will not be further described at this point.
The end product of the technique is a chromatographic band pattern having light and dark regions of various inten-sities at speci~ic locations. These locations can be readily matched to specific ~ragment sizes (in kilobase pairs) by introduction into the separation technique of a marker, such as EcoR I digested ~ bacteriophage DNA. In this manner, both the relative position of the bands to each other, as well as the absolute size of each band can be readily ascertained.
The band pattern for the unknown is then compared with band patterns present in a catalog or library. The catalog or library can cons:ist of a book containing patterns for at least two, and up to a virtually unlimited number of defined dif~erent organisms ?

~3~-genera and species. For example, the number of pathologically relevant bacteria that cause human disease is estimated to be about lO0, so it is estimated that a standard catalog of pathogenic bacteria would contain anywhere between 50 and 150 such patterns. A catalog of types of bac-terial strains for epiderniological typing systems can also be included.
The band patterns will depend on the type or types of endonuclease enzymes used, possibly on the particular organism used as the source for the radioactively labeled probe (the probe organism), and on the composition of the ribosomal RNA inforn~ation utilized to prepare the probe (e.g. containing either prokaryotic 5S, 16S or 23S
subtypes, or only 16S and 23S, or the like). Thus, the catalog may, for each probe, contain a variety of enzyme-specific band patterns, with the size of each band listed, and with the relative intensity noted. As the concentration of the bound DNA bound to the filter decreases, only the most intense bands can be seen, and the size of this band or bands can thus identify species. Each one of these band patterns may in turn comprise a band pattern obtained for complete ribosomal RN~ and/or a band pattern obtained for ribosomal RNA
containing only certain subtypes~ Any variation or permutation of the above can of course be used for the library. Additionally, for a eukaryotic organism the library may contain patterns that result from the use of one type of DNA or any combination of organelle and/or nuclear DNA. The pattern for each DN~ digest will depend on the probe composition. The catalog may be arranged so that if more than one strain or species is present in the extracted sample and detected by the probe~ the resulting pattern can be interpreted.
A user can either compare the obtained band pattern visually, or by aid of a one-dimensional, computer assisted, digital scanner proyrammed ~or recognition of patterns. These computer scanners are well known in the art of the time-of-sale transactions tthe commonly utilized "supermarket" check-out pattern readers~.
Ideally, the library or catalog is present in a computer memory both in terms of the relative band patterns for a plurality of organisms, and in terms of the absolute values of molecular weight or size of the fragments~ The catalog comparison then consists of matching the unknown pattern with one of the patterns present in the library by means of either one or both of the stored information elements trelative patterns and/or absolute size elements). The intensity of each band when compared to a standard can also reveal the amount of bound DNA
hybridized, and thus can be used to estimate the extent of the presence of a an organism, for example a prokaryote in a eukaryote.

--~0--If a user wishes to further confirm the nature and identi~ication of a given organism, such user can diyest the unknown with a second, different endonuclease, and compare the resulting band pattern to catalog band patterns of the organism ~or the second chosen endonuclease. This process can be repeated as many times as necessary to get an accurate identification.
Normally, however, a single analysis with a single probe would be sufficient in most instances.
The present invention and its variations can be used for a myriad of applications. It may be used by plant or animal breeders to correc-tly identify their subjects, or it may be used by clinical and microbiological laboratories to identify bacteria, parasites or fungi present in any medium, including in eukaryotic cells. In this latter use, the method is preferred to the standard microbiological assays, since it does not require isolation and growth of the microbes. In vitro growth and characterization is now either impossible for some microorganisms such as Mycobacterium leprae (agent of leprosy), impossible on standard media for soMe microorganisms such as the obligate intracellular bacteria (e.g. rickettsia, chlamydia, etc)/ or highly dallgerous (e.g. B. anthracis (agen~ of anthrax)). The present method depends on the isolation of nucleic acid and avoids these problems since it avoids conventional 4~-bacterial isolation and characterizationO The method is expected to detect microoryanisms that have not yet been conventionally described. In addition, the present method allows distinguishing differen~ strains of species, and this can be useful, for example, for epidemiological typing in bacteriology. The method can be used by forensic laboratories to correctly and unambiguously identify plant or animal tissues in criminal investigations~ It can also be used by entomologists to quickly identify insect species, when ascertaining the nature of crop infestations.
In addition, upon the conjunction of the method with the identification of infrasubspecific taxons (such as e.g., nitrogenase genes in plant roots, see Hennecke, H.
291 Nature 354 (1931)), the methodology can be utilized to search for and identify the genotypes of individual strains.
The method of this invention is preferably used for the identification of microorganisms wherever they may be found. These microorganisms may be found in physiological as well as non-physiological materials.
They may be found in industrial growth media, culture broths, or the like, and may be concentrated for example by centrifugation. Preferably, the microorganisms are found in physiological media, most preferably they are found in animal sources infected there~ith. In this -~2-latter embodiment, the method is used to diagnose hacterial infections in animals, most preferably in hu~nans. The ~etection ancl identification oE bacterial DNA with a prokaryotic ribosomal probe is highly selective and occurs without hindrance, even in the presence of animal, (e.g., mammalian) DNA. If a prokaryotic ribosomal probe is used, conditions can be selected which minimize hybridization with mitochondrial DNA, or mitochondrial bands can be subtracted from the pattern. The technique can thus be used in clinical laboratories, bacterial depositories, industrial fermentation laboratories, and the like.
Of particular interest is the possibility of detecting, in addition to the species and strain identity of the infectin~ microorganism, the presence, in the microorganism of any specific genetic sequences. For example, it is possible to detect the presence of antibiotic resistance sequences found on R factors, transmissible plasmids mediating drug resistance. One can add labeled R-factor DNA or cloned laheled antibiotlc resistance sequences to the hybridization mixture in order to correctly determine the antibiotic resistance of the organism, (an extra band or bands would appear), or one can rehyhridize the once hybridized filter in the presence of added antibiotic resistance sequence probe or probes. Alternatively one t could separate the unknown DN~ into aliquots, and test the first aliquot for identification, the second for the presence of d~uy resistance sequences, the third for toxin genes, etc. Alterna-tively, one could use ribosomal probe labeled with one radionuclide (e.g. 32p) in a hybridization mixture with added R-factor probe labeled with a different radionuclide (e.g. 3H or l~C). After hybridization, the presence of R-factor DNA in the unknown DNA can be tested by scanning with two different scanners: One for species and strain iden-tification (e.g. 32p)~ the other for drug resistance, or the like (e.g. 3H or 14C). In this manner the lab can/ without isolating and characteri~ing the microorganism, identify the genus and species, type the strain and test for drug resistance, possible toxin production or any other character or taxon belo-~ the rank of species that can be detected with a labeled nucleic acid sequence or probe, all in one experiment.
The R-factors are universal and cross species boundaries, so that identification can be carried out in any bacterial genus or species with the same R-factor probe (see To~kins, L.S., et al, J. Inf. Dis., 141:625-636 (19~1)).
In addition, the presence of viruses or virus-related sequences in eul~aryotes or prokaryotes can also be detected and identified in conjunction with the method -~4-of the invention: Any of the viruses described in "Manual of Clinical Microbioloyy", 3d edition, edited by Lennette, E.~., Ar,ler. Soc. Microb., 1980, 77~-778 can be iden-tified, eq. picornaviridae, caliciviridae, reoviridae, togaviridae, orthomyxoviridae, paramy~oviridae, rhabdoviridae, retroviridae, arenaviridae, coronaviridae, bunyaviridae, parvoviridae, papovaviridaet adenoviridae, herpesviridae, vidoviridae and poxviridaeO 1) When the viral genome is integrated into host DNA ~as with DNA viruses, for example members of Papovaviridae, and RNA viruses, for example, members of Retroviridae), high molecular weight DNA is extracted from the tissue and digested with restriction endonucleases The overall procedure is the same as used for bacteria. The choice of a viral probe again depends on the question asked, and on the extent of homology between the "probe virus" and the viral related sequences to be detected. In order to have suitable sequence homology/ it may be necessary that the probe and tissue sequences are related to the same familyr genus, or species of virus. I~l addition to the extent of conserved sequences, whether or not a viral probe hybridizes to viral related sequences in host DNA may be determined by the hybridization conditions, which can be stringent or relaxed. The result of the hybridization will be a band or a pattern of bands showing that there are viral ~p~

sequences incorporate~ into the host DI~A. This information may be useful in helping to predict the occurrence of cancer. Tlle probe can be any labelled complementary nucleic acid probe including cloned viral sequences~ E`or RNA viruses, for example viral ~NA can be used to make a DN~ with reverse transcriptase; for DNA
viruses, for example, viral DNA labelled by nick translation can be used. Again multiple probes can be used, especially with different labels.
Same general features apply equally to DNA and RNA
viruses4 Viral genomes are relatively small, so the precipitated nucleic acid is preferably collec-ted by centri~ugation; all of the procedures can use the total nucleic acid or the various procedures can be run separately. It is expected that viral nucleic acid can be concentrated by spooling cellular DNA to remove it before centrifugation. This can also be used to determine if the viral genome is integrated.
For the viral probe to hybridize, it may be necessary and at least most preferred that the probe be from the same family, genus, or species as the unknown, ~eaction conditions, stringent or relaxed, may determine whether or not a given probe hybridizes a distantly related genome. The probe may be cloned viral se~uences that are labeled, or may be the complete genome or a portion of it.

The technique described by Southern, supra is useful for the transfer of large DNA fragments (greater than about 0.5 kilobases) to nitrocellulose paper after alkali denaturation. This techni~ue might be useful for DNA
viruses but not for RNA viruses. RNA has been transferred and covalently coupled to activated cellulose paper (diazobenzyloxymethyl-paper), and this can be used for RNA virusesO The modification of the outhern technique by Thomas (Thomas, P., Proc. Nat. Acad. SciD, USA, 77; 5201-5205 (19~0)) can be used for the efficient transfer of ~NA, and small D.~ fragments to nitrocellulose paper for hybridization. RNA and sMall DNA fragments are denatured with glyoxal and dimethyl sulfoxide, and electrophoresed in agarose gel. This procedure trans-fers D~A fragments between 100 and 2000 nucleotides and RNA efficiently, and they are retained on the nitrocellulose paper during hybridization. This is useful for small ribosomal DNA fragments as well. So it is most preferred to divide the restriction-enzyme di~ested specimen and denature the nucleic acid in one portion with glyoxal. The Southern and Thomas procedures would yield a maximum amount of information~
2) For DI~A viruses, restriction analysis can be carried out with double stranded (DS) viral DNA's to identify viruses present. Single-stranded ~SS) DNA
viruses will have different genome lengthsO The probe ~ J ~

(the sequence information could be converted to DS DNA) that hybridizes, the ~lybridized fragment pattern and/or the sizes or size can be used to identify viruses. There are again a number o~ ways to obtain complementary nucleic acid probes. ~or example, ~or DS DNA nick-translation can be used; for SS DNA, DNA polymerase can be used to synthesize a cDNA.

3) For ~NA viruses, RNA is not digested by restriction endonucleases (the se~uence information could be converted to DS DNA). The genomes of diferent ~A
viruses are of different sizes, and some RNA viruses have more than 1 molecule in their genome. This, along with the base sequences detected by certain probes or pooled probes allows the RNA viruses to be identified. An example o a probe would be cDNA synthesized using viral RNA.
When searching for infectious agents in specimens it is possible to search directly by ex-tracting nucleic acid from the specimen, or by culturing first in media or cells to increase the number of agents, or by using a concentration step such as centrifugation or trying all approaches.
The present invention lends itself readily to the preparation of "kits" containing the elements necessary to carry out the process. Such a kit may comprise a carrier being compartmentalized to receive in close confinement therein one or more container means, such as tubes or vials. One of said container means may contain unlabeled or detectably labeled nucleic acid probe, such as for example the radioactively labeled cDNA to riboso~al RNA from the organism probe, (preferably prokaryotic rRNAcDNA in the case of a kit to identify bacteria). The labeled nucleic acid probe may be present in lyophilized form, or in an appropriate buffer as necessary. One or more container means may con-tain one or more endonuclease enzymes to be utilized in digesting the DNA from the unknown organism. These enzymes may be present by themselves or in admixturesl in lyophilized form or in appropriate buffers. Ideally, the enzymes utilized in the kit are those for which corresponding catalogs have been prepared. ~othing stops the user, however, from preparing his or her own coinparative standard at the moment of experiment. Thus, if a user suspects that an unknown is in fact of a given genus or species, he or she may prepare the band pattern for the known and compare it with the band pattern for the un-known. The kit may thus also contain all of the elements necessary in order to carry out this sub-process. These elements may include one or more known organisms, (such as bacteria), or isolated DNA from known or~anisms. In addition, the kit may also contain a "catalog", defined broadly as a booklet, or book, or pamphlet, or computer '~

_L~9_ tape or disk, or computer access nulnber, or the like, haviny tlle chromatographic band patterns ~or a variety of organisms of a certain group, such as plant species, mammal species, microbe species, especially pathologic-ally ilnportant bacteria, insect species or the like. In this mode, a ~ser would only need to prepare the band pattern for the unknown organism, and then visually (or by col-nputer) com~are the obtained pattern with the patterns in the catalog The kit may also contain in one container probe rR~A for probe synthesis, in ano-ther container radiolabeled deoxyribonucleoside triphosphate, and in another container primer. In this Inanner the user can prepare his or her own probe rR~AcDNA.
Finally, the kit may contain all of the additional elements necessary to carry out the technique of the invention, such as buffers, growth media, enzymes, pipettes, plates, nucleic acids, nucleoside triphosphates, filter paper, gel materials, transfer materials, autoradiography supplies, and the like. It may also contain antibiotic resistance sequence probes, viral probes, or other speci~ic character probes.
Having now yenerally described this invention, the same will be better understood by reference to certain specific examples which are included herein for purposes of illustration only and are not intended to be liniting of tne invention, unless specified.

~50--MAT~I~IALS AND METHODS
_ _ A. Bacterial Extraction of ~ligh_Molecular_~eight D~A
sacterial broth cultures were centrifuged and the cells were washed with cold saline. The cells were suspended in a volume measured in rnl of extraction buffer (0.15 M sodium chloride, 0.l M EDT~, 0.03 M tris pll 8.5) approximately l0 times the ~ram weight of the packed cells. Lysozyme at l0 mg/ml was added to 0O5 mg/ml inal concentration. The suspension was incubated at 37C for 30 minutes. Cell disruption was completed by the addition of 25% SDS to 2.5% final concentration, and raising the temperature to 60C for l0 minutes. After cooling in a tap water bath, mercaptoethanol was added to l~ final concentration. Pronase~ at 20 rng/~l in 0.02 M
tris pH 7.4 was predigested at 37C for 2 hours and then added to l mg/ml final concentration. The solution was incubated at 37C for 18 hours. Phenol was prepared by mixing one liter redistilled phenol, 2.5 liters double distilled water, 270 ml saturated Tris base, 12 ml mercaptoethanol, and EDTA to l0 3M final concentration and allowing the mixture to separate at 4C. The phenol was washed with wash buffer (l0 l~1 sodium chloride, l0 3M
EDTA, l0~nM tris p~ ~.5). Then an e-~ual volume of resh bu~er was added. Mercaptoethanol ~as added to 0.l~
inal concentration. The solution was mixed and stored -51~

at 4C. One llalf volume prepared phenol and one half volume chloroform was added to the lysed cell solution.
This was shaken for approximately 10 min~ltes and centrifuged at 3,400 x g Eor 15 minutes. The aqueous phase was removed with an inverted 25 ml glass pipette.
The extraction procecJure was repeated until there was little precipitate at the interface. One-ninth volume 2 N sodium acetate pE~ 5.5 was added to the aqueous phase.
Two times volume of 95~ ethyl alcohol at -20C was poured slowly down the side of the flask. The end of a Pasteur pipette was rnelted close and used to spool tlle precipi-tated DNA. High molecular weight DNA was dissolved in buffer (10 3EDTA, 10 2M tris pH 7.4). The concentration of DNA was determined by absorbance at 260 nm using 30 micrograms per absorbance unit as conversion factor.

Restriction Endonuclea e Digestion of D~JA.
EcoR I restriction endonuclease reactions were performed in 0.1 M tris-HCl pH 7.5, 0.05 M NaCl, 0.005 M
M~C12, and 100 micrograms per ml bovine serum albumin.
EcoR I reaction mixtures contained 5 units of enzyme per microgram of DNA, and were incubated four hours at 37C. PST I restriction endonuclease reactions were performed in 0.006 M tris-HCl pH 7 4, 0.05 M sodi~m chloride, 0.006 M maynesi~m chloride, 0.006 M 2-mercaptoethdnol, and 100 microyrams per ml of bovine -52~

serum albumin. PST I reaction mixtures contained 2 units of en~yme per microcJrarn DNA1 and were incubated four hours at 37C. ~sually lO micrograms DNA was digested in a final volume of 40 microliters. Ten times concentration buffers were added. Sterile distilled water was added depending on the volume of DNAo A ~acteriophage DNA was restricted with EcoR I to provide marker bands for fragment size determinations. Usually 2 micrograms ~ DNA was digested with 20 units Eco~ I in a ~inal volume of 20 microliters.

el Electrophoresis and DNA Transfer.
DNA digests were supplemented with glycerol, to about 20%, and bromophenol blue trac]cing dye. In the case of ~ DNA digests, 20 microliters of lx EcoR I buffer was added to each 20 microliter reaction mixture.
Usually 15 microliters 75~ glycerol and 5 rnicroliters 0.5~ bromophenol blue were added to each 40 microliter reaction mixture.
10 micrograms digested bacterial DNA or 2 micrograms digested ~DNA were loaded per well and overlaid with molten agarose. Digests were electrophoresed in 0.8%
agarose wlth 0.02 M sodium acetate, 0.002 ~I EDTA~ 0.018 M
tris base, and 0.028 M tris HC1 pH 8.05 at 35 V until the dye migrated 13 to 16 cm. Gels were then immersed in ethidium bromide ~0.005 mg/rnl) and placed on a UV-light - ~ ` \

box to visualize the ~ Era(3ments. DNA was transferred to nitrocellulose filter paper by the method of Southern, supra. Gels were treated with denaturing solution (1.5 M
sodium chloride, 0.5 M sodium hydroxide) on a rocker table for 20 min. Denaturing solution was replaced with neutralization solution (3 0 M sodium chloride, 0.5 M
tris HCl, pH 75), and after 40 minutes the gels were checked with pH paper. Following neutralization, the gels were treated with 6 x SSC buf~er (SSC - 0.15 M
sodium chloride, 0.015 M sodium citrate) for 10 minutes. DNA fragments were transferred from the gel to the nitrocellulose paper by drawing 6 X SSC through the gel and nitrocellulose paper with a stack of paper towels for 15 hours. Filters were placed between two sheets of 3 MM chromatography paper, wrapped in aluminum foil, shiny side out, and dried in a vacuum oven at 80C for 4 hours.

Synthesis of 32p ribosomal R~A Complementary DNA (32P
rRNA cDNA).
32P-labeled D~A complementary to E. coli R-13 23S
and 16S ribosomal RNA was synthesi~ed using reverse transcriptase from avian myeloblastosis virus (AMV). The reaction mixture contained 5 microliters 0.2 M
dithiothreithol, 25 microliters 1 M tris pH 8.0, 8.3 microliters 3 M potassium chloride, 40 microliters 0.1 M

~.

magnesium chloride, 70 micrograms actinomycin, 14 microliters 0.04 M dATP, 14 microliters 0.04 M dGDP, 14 microliters 0.04 M dTTP and 96.7 microliters H2O. The following were added to a plastic tube: 137.5 microliters reaction mixture, 15 microliters calf thymus primer (10 mg/ml), 7 microliters H 20, 3 microliters rRNA tUsing 40 micrograms/OD unit concentration, is 2.76 micrograms/microliters), 40 microliters deoxycitydine 5'-(32p) triphosphate (10 mCi/ml); and 13 microliters AMV
polymerase (6,900 units ~1. The enzymatic reaction was incubated 1.5 hours at 37C. Then the solution was extracted in 5 ml each of chloroform and prepared phenol. After centrifugation (JS 13,600 RP~I), the aqueous phase was layered directly on a Sephadex~ G-50 column (1.5 x 22 cm~. A plastic 10 ml pipette was used for the column. A small glass bead was placed in the tip, rubber tubing with a pinch clamp was attached, and degassed G-50 swelled in 0.05% SDS overnight was added.
The aqueous phase was allowed to Elow directly into the G-50 and was then eluted with 0.05~ SDS. 20 fractions at 0.5 ml each were collected in plastic vials. Tubes containing peak fractions were detected by Cerenkov counting using a 3H discriminator, counting for 0.1 min.
per sample and recording total counts. Peak fractiorls were pooled. Aliquots were added to Aquesol~
(commercially available), and the CPM of 32p per ml was determined by scintillation counting.

Hybridization and Autoradioqraphy.
Fragments containing ribosomal RNA gene sequences were detected by autoradiography after hybridization of the DNA on the -filters to P~rRNA cDNA. Filters were soaked in hybridization mix (3 x SSC, 0.1% SDS, 100 micrograms/ml denatured and sonicated canine DNAI and Deinhart's solution (0.2% each of bovine serum albumen, Ficoll, and polyvinyl pyrrolidine)), for 1 hour at 68C. P rRNA CD~A was added at

4 x 106 CPM/ml, and the hybridization reaction was incubated at 68C for 48 hours. Filters were then washed in 3 x SSC, and 0.1% SDS at 15 min. intervals for 2 hours or until the wash solution contained about 3,000 cpm 3 P per ml. Filters were air dried, wrapped in plastic wrap and autoradiographed approximately 1 hour with KODAK X-OMAT R (trada mark) film at B. Mammalian_experiments. Mus musculus domesticus (mouse) rRNA probes were synthesized from 18S and 28S, and only 28S rRNA. Nucleic acid was extracted from mouse liver and precipitated. High molecular weight DNA was spoo~ed and removed. The remaining nucleic acid was collected by centrifugation and dissolved in buffer, 50 mM mgC12 and 100 mM
Tris pH 7.4~ DNAse (RNAse free) was added to a concentration of 50 ~g/ml. The mixture was ~, ;i.
., ,0~

3~

incubat~d at 37C for 30 min. The resulting RNA was re-extracted, etllanol precipitated9 and disolved in lmM
sodium phosphate buffer pH 6.8. ~ 5 to 20~ sucrose gradient in O.lr~ Tris pH 7.4 and O.OlM EDTA was prepared. The sample was added and the gradients spun in an SW40 rotor 7 hr. at 35K RPM. Fractions were collected by optical density. The 18S and 2~S fractions were selected by comparison to known molecular weight markers.
For all of the mammalian experirnen~s relaxed hybridization conditions were used, 54C. The washing procedure, carried out at 54C, was 3 separate washes with 3xSSC with 0.05% SDS for 15 min. each.

E~AMPLE 1 Bacterial Species are Defined by Restriction Endonuclease Analysis of Ribosomal RNA Genes The several strains of P. aeruginosa used in this example have the minimal phenotypic characters which identify the species (Hugh R.~l., et al, in: Manual of Clinlcal Microbiology, 2d Ed. ASM, 1974, pp. 250-269).
(Table 2). Strains of three other Pseudomonas and two Acinetobacter species were selected to compare species and genera (Table 3).

Corresponding strain numbers oE isolates with the minimal phenotypic characters of P. Aeruginosa f or the compa r i son of S tra i ns .

ATCC

810 ~689 815 101~5 .

Strains used for comparlson of Pseudomonas and Acinetobacter species are listed in Table 3.

Correspondiny strain numbers of type, neotype and reference strains for the o~nparison of species and genera Species RH ATCC NC~rC Strain status P. aeruyinosa 815 10145 10332 type P. stutzeri 260:L 17588 neotype P. f luorescens 818 13525 10038 neotyr~e P. pu~ida 827 12633 neotype A. anitratus 2208 19606 type A. lwoffii 462 7976 reference Acinetobacter species were selected for comparison of yenera because they share certain a-ttributes with many Pseudomonas species.
The sizes (kilobase pairs~ of fragments in EcoR I
digests are: P. stutzeri 16.0, 12.0, 9.4; P. fluorescens 16.0, 10.0, 8~6, 7.8, 7/0; P. putida 24.0, 15.û, 10.0, 8.9; A. anitratus 20.0, 15.0, 12.5, 9.8, 7.8, 6.1, 5.2, 4.8, 3.8, 2.8 (size of the smallest 3 fragments not calculated); A. lwoffi 12~0, 10.0, 9.1, 7.0, 6.4, 5.7,

5~5, 5.3, 4.8, 4.4, 3.6, 3.2, 209 (size of the smallest 3 fragments not calculated). The sizes (kilobase pairs) of fragments in PST I digests are; P. stutzeri 6.7, 6.1, 5.5; P. fluorescens 10.0, 9.4, 7.8, 7.0; P. putida 10.5, 9~9, 6.8, 6.3, 4.4; A. anitratus 36.0, 28.0, 20.5, 12.0, 10.0, 5.8, 3,7, 2.6, 2.4; A. lwoffi 9.9, 8.7, 7.2, 5.7, 4.0, 3.6, 3.2, 2.7.
Comparison of the hybridized restriction fragments from the seven strains of P. aeruginosa leads to the conclusion tllat this species can be defined by an EcoR I
specific set of fragments containing rR~A ~3e~ne sequellces, 10.1, 9.4, 7.6, and 5.9 kilobase pairs (~sP) (EIGt~RE
1). The 7.6 KBP EcoR I fragment occurs in 4 of the 7 strains in ti~is sample. An analogous situation occurs amon~ certain phenotypic characters of strains of species. The fact that the EcoR I sets of ~ra~men~s from the 7 strains can be used to separate the strains into two groups prompts speculation that there ~nay be two species with the minimal phenotypic characters of PO
aeruginosa. The results of experiments in ~hich DNA was diyested with Pst I (FIGURE 2) lead to the conclusion that the strain variation shown by the EcoR I 7.6 KBP
fragment represents variation within the species, since there is a single conserved set of PST I fragments, 9.~, 7.1, 6.6, and 6.4 KBP, that define the species. The 9.4 and 6.6 KBP Pst I fragments occur in 6 of the 7 strains of P. aeruginosa; the 7.1 and 6.4 KBP PST I fragments occur in all of the strains sa!npled. PST I fragment variation occurs in strains that do not contain an EcoR I
7.6 KBP fragment; RH 151 has 10~1 and 8.2 KBP fragments, RH 809 does not contain a 9.4 KBP fragment and has a 6.0 KBP fragment, and RH 815; the type strain, does not contain a 6.6 KBP fragment. The patterns of hybridized fragments support the conclusion that enzyme specific, conserved sets can be used to define species. Strains of a species probably have a majority of the fragments in the conserved set. The occurrence of fragment variations in solne strains does not prevellt identification and may prove useful in epidemiolo~ical studies.
The occurrence of variation, EcoR I 7.6 KBP fra~ment J

in P. aeruyinosa strains, may be put into perspective by examining hybridized EcoR I fragments found in the type strains of other Pseudononas species (FIGURE 3). The type strains of P stut~erl, P. fluorescens, and P.
putida do no-t contain a 7.6 Ksp fragment, but do have EcoR I fragments of the same size in common; P.
aeruginosa and P. stutzeri each have a 9.4 KBP fragment, P. stutzeri and P. fluorescens each have a 16 ~BP
Eragment, and P. fluorescens and P. putida each have a 10 hBP fragment. In general, the sizes of the fragments are unique in the type strains of each of the 4 2seudomonas species; and the type s-train of each species has a different size range of fragments. I'hese general comments are also true for the PST I digests (FIGURE 4).
When the fragment patterns of one strain of each of the 4 Pseudomonas and 2 Acinetobacter species are compared, it can be concluded that the species of each genus are similar, but the genera differ. The 2 Acinetobacter species have a greater range of hybridized fragment sizes than do the 4 Pseudomonas species.
~ ithout the aid of restriction enzyme maps such as those available for E. coli, Bacillus thuringiensis and B

.
subtilis, it is not possible to predict where en~umes cut rRNA genes the number of copies per geno,ne, whether there are neterologous flanking regions between genes, or gene het~rogenei-ty. The ~.. coll rRNAcDMA probe Inay f~il to hybridize with some restriction fra(3ments containing rRNA

&~

gene sequences, and iE so, this re~lects the evolutionary distance or diversity between the test organism and E.
coli. The conserved nature of rRNA can be used to argue that this is not the case. However, this is a minor problem compared to the advanta~Je of havincJ a standard probe that can be equally applied to a by unknown species.

Comparison of Restriction Analysis with DNA-DNA
Llquid Hybridization-The strains used in this study are listed in Tables4 and 5, Table 4.
Corresponding Strain ~umbers oE Neotype strains of B. subtilis and type strains of junior synonyms Species RH ATCC Strain status B. subtllis 3021 G051 neotype ~. uniElagellatus 2990 15134 t~pe B. amyloliquafaciens 3061 23350 type . . . _ ~62-TABLE 5. Corres~ondinc3 strain number of strains of B. Subtilis _ _ RH NRRL ATCC
__ .
3063L-354(NRS-231) 6633 3064B-356(NRS-238) 7067 , Higll molecular weight DNA was isolated from each of the strains. Liquid DNA-DNA hybridization data was collected using RH 3021 and RH 2990 labeled DNAs and results are shown in Table 6.

Percent hybridization between labeled DNA probe and DNA from strains of B. subtilis . _ .. . . .
Labeled ~NA
probe RH 3063 RH306~ RH3066 RH3067 RH30~;3 RH 3065 _ RH 3069 111 3070 RH 3071 RH 3072 RH 3021 R~l 2990 RH 2990 100 -~ 17 100 20 100 ~H 3021 11 ~1 29~0 70 The data shows there are two hybridization groups.
Similar data is reported in the literature for B.
subtilis (Seki et al, International Journal of Systematic Bact:eriology, 25:258-270 (1975)). These two groups can be represented by RH 3021 and RH 2990. When restriction endonuclease analysis of ribosomal ~NA genes is carried out, the EcoR I digests (FIGURE 5) can be separated into two yroups~ The group represented by REI 3021 has two intensely hybridized fragments ( 2.15 and 2.1 KBP). The group represented by RH 2990 has two intensely hybridized

6~-fragments (206 and 2.5 KBP). The EcoR I data can be used to place B. s~btiiis strains in appropriate DNA-DNA
hybridization groups. According to the D~ DNA
hybridization 70~ rule, B. subtilis is actually two species. ~lowever, when the PST I data (FIGURE 6) is considered, it is possible to think of the groups as two divergent populations related to a common ancestor or speciation eventO The conclusion that B. sub-tilis is one species correlates with phenotypic data. The strains listed in Table 5 are identified as B. subtilis in Gordon, R.E et al "The Genus Bacillus", A~riculture Handbook No. 427, A~ricultural Research Service, U. S.
Dept. of Agriculture, Wasnington~ D.C. pp. 36-41.
Restriction analysis can provide data which is comparable to DNA-DNA hybridization data, or by selecting the proper enzyme~ restriction analysis can adequately de~ine species despite divergence. ~ 3061 has lost PST I
sites. Howev~r~ the EcoR I data suggests that the strain is B. subtilis. The same is concluded from the Bgl II
data (FIGURE 7) and Sac I data (FIGURE 3)c ~XAMPL~ 3 Stability of the Restriction Analysis Pattern_and Other Bacillus polymyxa Ex~eriments Neotype strains of B. Subtilis and 2. Polymyxa ~ies KH A~CC NRRL Comments B~ sub ilis 30216051 neo-type B. ~oly~xa 3074842 neotype B. polymyxa 3033 as~orogenous mutant derived from R~ 3074 B. polyl~xa 3062 NRS-1105 neotype Bo polymyxa 3073 asporogenous mutant derived from NRS-1105 B. subtilis and B._polymyxa can be distinguished by EcoR
I data (FIGURE 9~, PST I data (FIGURE 10), Bgl II data (FIGUR~ 11, left) and Sac I da-ta (FIGURE 11, right). It can be concluded from the major di-fferences in the P~T I
band patterns, that 2acillus polymyxa is in the wrong ~3enus. While botll species produce spores, they are not phenotypically similar. It is reassuring that the type strain of B. polylnyxa from both culture collections, ATCC
and NRRL, have the same band patterns. The important data, however, is that the asporogenous mutants can be identified~ It is very difficult, perhaps impossible, to i~entify Bacillus species if they fail to form spores.

-~6-EXAi~PL~ 4 Identification of a Bacterial Species In Mouse Tissue rWithout Isolation _ A Swiss mouse~ Mus musculus domesticus (inbred -strain), was inoculated intraperitoneally with 0.5 rnl of a turbid susoension of Stre~tococcus pneumoniae RH 3077 __ (ATCC 6303). When the mouse became moribund, the heart, lungs, and liver were removed. E~igh molecular weight DNA
was isolated from these tissues, S. pneumoniae ~ 3077l and Swiss mouse organs, and the procedure Eor restriction endonuclease analysis of rR~A genes was carried out using ~coR I to digest the DNAs. In addition to washing the Eilters in 3 x SSC, they were washed for 2 x 15 minutes periods in 0.3 x SSC and 0.05% SDS. Autoadiography was carried out for 48 hr. The data (FIG~RE 12), shows that S. pneumoniae can be defined by seven hybridized fragments (17.0, ~.0, 6.0, 4.0, 3.3, 2.6 and 1.8 KBP).
The bacterlal cDNA probe hybridizes poorly to two l~ouse DN~ fragments (14.0 and 6~8 KBP). Hybridized fragments signal the presence of S. pneumoniae in the in~ected tissues. All seven bands can be seen in the heart DN~
extract. They are less intense in the liver DNA extract, but all can be seen in the autoradiograph. Only tlle 6.0 KBP band appears in the lung DNA extract. The lesser number of bacteria in the luncJs can be explained by the mouse havin-3 septicemia rather than pneumonia. The lungs s;lowed IlO consolidation at autopsy. In order to determirle the sensitivity of the assay, bacterial DMA was diluted with mouse D;~A and electrophoresed. All seven bands can be seen in the autoradioc3raph when Ool microyralns of bacterial DNA is used. The 17.0, 8.0 and 6.0 KBP bands can be seen with 10 3~g of bacterial DNA.
If the figure of 5 X 10 3~g DNA per 106 S. pneumoniae cells is used (Biochim Biophys Acta 26:68), 10 l~g is equivalent to 2 X 107 cells~ The present technique is thus useful for diagnosing infections at this level of sensitivity.
This Example also demonstrates that the bacterial probe hybridizes with mouse-speci-fic EcoR I fragments ~see FIGURE 9, fragments having 14.0 and 6~8 KBP). These fragments correspond to EcoR I fragments detected by mouse 18S and 28S ribosomal RNA probe. (Figure 1-~ infra shows that the 6.8 KBP fragment contains the 28S rX~A
sequences). The bacterial probe does not hybridize as well to mammalian ribosomal RNA gene sequences, so the bands are less intense, the system of bacterial probe and nuclear mammalian DN~ is less sensitive, and selectivity for DNA Erom infecting prokaryotes is clearly demonstrated. In experiments where bacterial probe was hybridi~ed to 10 ~g di9ested bacterial DNA per lane, no hybridization to 10 ~g di~ested human or mouse DNA per lane was detected on the autoradiographs when -the bacterial bands were clearly seen.

-6~-Examples 5-8 Mammalian ex~eriments These examples illustrates that the concept of rRNA
restriction analysis to identify organisms can be successfully applied not only to bacteria but to complex, eukaryotic organisms.
Figure 13 shows that mammalian genera can be recognized with Mus musculus domesticus 18S and 23S rRNA
probe, and that several species of Mu_ can be distinguished. In this Figure the enzyme is PST I, and the subjects and corresponding bands are as follows:
1. Mus musculus melossinus (mouse) 14.5, 13.5, 2.6 2. Mus musculus domesticus (mouse) 13.5, 2.6 3. Canis familiaris (dog) 12.0 4. Cavia porcellus (guinea pig) 17.0, 14.0, 13.0, 8.8, 5.7, 4.7 and one band less than 3.0 5. Cricetulus griseus (hamster) 25.0, 4.7 6. Homo sapiens (human) 15.0, 5.7

7. _elis catus (cat) 20.0, 9.7

8. ~atus norvegicus (rat) 12.5

9. _us musculus domesticus (mouse) 13.5, 2.6

10. Mus cervicolor cervicolor (mouse) 14.0, 2.7

11. Mus cervicolor papeus (mouse) 13.5, 2.6

12. Mus pahari (mouse) 13.0, 3.7

13. Mus cookii (mouse) 13.5, 2.6 -t~

Figure 14 shows that mouse and cat DNA can be distinguished by the 28S rRNA cDNA alone, and that the pattern of hybridized bands is dependent on the composition of the probe sequences. In Figure 14 the enzyme is EcoR I, and the subjects and bands are as follows:
1. Mus musculus domesticus (mouse) 6.8 KsP
2. Felis catus (cat) 8.3 KBP

In Figure 15 the enzyme is Sac I, and the subjects and bands are as follows:
1. Erythrocebus patas (patas monkey) 8.5, 3.7, ~ 3.0 2. Ratus norvegicus (rat) 25.0, 9.5, 3.6 < 3.0 3~ Mus musculus domesticus (mouse) 6.8, < 3.0 4. Felis catus (cat) 9.5, 5.3, 4.0, < 3.0, < 3.0 5. Homo sapiens (human) 10.5, < 3.0 6. _ aca mulatta (rhesus monkey) 9.8, < 3.0 When Figure 15 (Sac I digests) is compared to the other mammalian figures i.t can be seen that the hybridized pattern is enzyme speclfic.
Figure 16 shows that primates can be distinguished. Cell cultures have bands in common with tissue from the species of origin, and different human cell cultures can be distinguished by additions and dele~ions of bands. In this figure, the enzyme is Eco~
I, and the subjects and bands are as follows:

1. Erythrocebus patas (patas monkey) > 22.0, 11.0, 7~6, 2 ~

t,t 2. Macaca mulatta (rhesus monkey) 22.0, 11,5, 7,6 3~ Homo sapiens (human) > 22.0, 22.0, 16.0, 8.1, 6~6 4. M 241/88 (langur monkey cell culture) 14.0, 7.2, 5.7 5. HeLa (human cell culture) 8.1, 6.6 6. J96 (humarl cell culture) > 22.0, 22.0, 16.0, 11.0, ~.1, 6.6 7. AO (human cell culture1 22.0, 16.0, 8.1, 6.6 8. X-381 (rhesus rnonkey) 22.0, 11~5, 7.6 Having now ~ully described this invention, it will be apparent to one of ordinary skill in the art that many variations and permutations can be carried within a wide range of equivalents without effecting the spirit or scope of the invention, or of any embodiments thereofO

Claims (79)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A method of characterizing an unknown organism, which comprises the steps of:

a) comparing the chromatographic pattern of restriction endonuclease-digested DNA from said unknown organism, which digested DNA has been hybridized or re-associated with ribosomal RNA information-containing nucleic acid from or derived from a known probe organism, with at least two equivalent chromatographic patterns; each one of said equivalent chromatographic patterns defining a known different organism species, and b) establishing the species of said unknown organism by means of a conserved set of ribosomal RNA

sequence-containing restriction fragments present in said chromatographic pattern of said unknown organism.

2. The method of claim 1, wherein said ribosomal RNA

information-containing nucleic acid is detectably labeled.

3. The method of claim 2, wherein said ribosomal RNA

information-containing nucleic acid is radiolabeled.

4. The method of claims 1, 2 or 3, wherein said ribosomal RNA information-containing nucleic acid probe is ribosomal RNA.

5. The method of claims 1, 2 or 3, wherein said ribosomal RNA information containing nucleic acid probe is DNA complementary to ribosomal RNA.

6. The method of claims 1, 2 or 3, wherein said ribosomal RNA information-containing nucleic acid probe is DNA obtained by nicktranslating or cloning DNA complement-ary to ribosomal RNA,

7. the method of claim 1, wherein said unknown organism being characterized is a cell or cells of a strain of an in vitro culture.

8. The method of claim l, wherein said unknown organism being characterized and said probe organism are both from the same taxonomic group selected from the group consisting of a kingdom, subkingdom, division, subdivision, phylum, subphylum, class, subclass, order, family, tribe and genus.

9. The method of claim l, wherein said unknown organism being characterized and said probe organism are both prokaryotic.

10. The method of claim 1, wherein said unknown organism being characterized and said probe organism are both prokaryotic.

11. The method of claim l, wherein said unknown organism being characterized is eukaryotic and said probe organism is prokaryotic.

12. the method of claim l, wherein said unknown organism being characterized is prokaryotic and said probe organism is eukaryotic.

13. The method of claim10, wherein said unknown prokaryo-tic organism being characterized is selectively being detected while present in or associated with a eukaryotic organism.

14. The method of claim 12, wherein said unknown prokaryo-tic organism being characterized is selectively being detected while present in or associated with a eukaryotic organism.

15. the method of claims 13 or 14, wherein said unknown prokaryotic organism is a bacterium.

16. the method of claims 13 or 14, which comprises:
extracting the total DNA of said prokaryotic and said eukaryotic organisms, and selectively detecting the ribosomal RNA information present in the DNA of said prokaryotic organism, in the presence of ribosomal RNA information present in the DNA of said eukaryotic organism.

17. The method of claim 9, wherein the DNA from said unknown eukaryotic organism being characterized is nuclear DNA and the ribosomal RNA information-containing nucleic acid from said eukaryotic probe organism is not derived from mitochondria or chloroplasts.

18. The method of claim 9, wherein the DNA from said unknown eukaryotic organism being characterized is mitochondria dria DNA and the ribosomal RNA information-containing nucleic acid from said eukaryotic probe organism is derived from mitochondria or chloroplasts.

19. the method of claim 9, wherein DNA from said unknown eukaryotic organism being characterized is chloroplast DNA
and the ribosomal RNA information-containing nucleic acid from said eukaryotic probe organism is derived from mitochondria or chloroplasts.

20. The method of claim 11, wherein said DNA from said unknown eukaryotic organism being characterized is derived from mitochondrial DNA.

21. the method of claim 11, wherein said DNA from said unknown eukaryotic organism being characterized is derived from chloroplast DNA.

22. the method of claim 12, wherein said ribosomal RNA
information-containing nucleic acid probe is derived from mitochondria or from chloroplasts.

23. the method of claim 9, which further comprises identifying in said unknown eukaryotic organism being charac-terized a nucleic acid sequence or sequences creating a taxon below the rank of species or an infra subspecific subdivision.

24. The method of claim 23, wherein said nucleic acid sequence or sequences comprises a virus or virus-derived DNA.

25. A method of identifying an unknown bacterial strain present in a sample, which comprises the steps of:
a) comparing the chromatographic pattern of restric-tion endonuclease digested DNA from said strain, which digested DNA has been hybridized or reassociated with ribosomal RNA
information-containing nucleic acid from or derived from a known probe bacterium, with at least two equivalent chromato-graphic patterns; each one of said equivalent chromatographic patterns defining a known different bacterial species; and b) establishing the species of said unknown bacterial strain by means of a conserved set of ribosomal RNA
sequence-containing restriction fragments present in said chromatographic pattern of said unknown bacterial strain.

26. the method of claim 25, wherein said unknown bacterium is present in a fermentation medium or in a secretion or excretion product.

27. The method of claim 25, wherein said unknown bacterium is present in or associated with eukaryotic tissue.

28. the method of claim 26, which comprises:
extracting the total DNA of said bacterium and said eukaryotic tissue, and selectively detecting the ribosomal RNA information present in the DNA of said bacterium, in the presence of the ribosomal RNA information present in the DNA in said eukaryotic tissue.

29. The method of claim 27, wherein said bacterium is present in or associated with animal or plant cells.

30. The method of claim 29, wherein said bacterium is present in or associated with human cells, or associated with plant root cells.

31. The method of claim 25, wherein said ribosomal information-containing nucleic acid from said probe bacterium is detectably labeled.

32. the method of claim 31, wherein said label is a radiolabel.

33. the method of claim 31, wherein said nucleic acid from said probe bacterium is ribosomal RNA.

34. The method of claim 31, wherein said nucleic acid from said probe bacterium is complementary DNA to ribosomal RNA.

35. The method of claim 25, wherein said unknown bacterium is pathogenic towards plants or animals.

36. The method of claims 26, 27, or 30, wherein said unknown bacterium is pathogenic towards plants or animals.

37, the method of claim 25, which further comprises detecting in said bacterial strain for the presence of a nucleic acid sequence or sequences creating a taxon below the rank of species or an infra subspecific subdivision.

38. The method of claim 37, wherein said nucleic acid sequence or sequences are all or part of a bacteriophage genome.

39. The method of claim 37, wherein said nucleic acid sequence or sequences are all or part of an extrachromosomal genetic element, a plasmid, or a episome.

40. the method of claim 37, wherein said sequence or sequences code for an R-factor or for an antibiotic resistance factor.

41. The method of claim 25, wherein said chromatographic patterns of known bacteria are present in a catalogue containing patterns for at least two different bacteria.

42, Detectably labeled DNA complementary to prokaryotic ribosomal RNA (rRNA cDNA) in substantially pure form, wherein sand rRNA cDNA is a faithful copy of said ribosomal RNA (rRNA).

43. the rRNA of claim 42, wherein said prokaryotic RNA
is bacterial rRNA.

44. The rRNA cDNA of claim 42, which is substantially free of cDNA which is complementary to types of RNA other than rRNA.

45. The rRNA cDNA of claim 42, which is substantially free of cellular components.

46. the rRNA cDNA of claim 42, which has been obtained by reverse transcribing rRNA in the presence of a primer.

47. the rRNA cDNA of claim 46, which has been obtained by using a DNA hydrolysate primer.

48. the rRNA cDNA of claim 46, which has been obtained by reverse transcribing with reverse transcriptase from avian myeloblastosis virus.

49. the rKNA cDNA of claim 42, which is radiolabeled.

50. The rRNA cDNA of claim 49, which is labeled with 32p, 14C, or 3H.

51. A kit comprising a carrier being compartmentalized to receive in close confinement therein one or more container means, wherein a first container means contains ribosomal RNA
(rRNA) information-containing nucleic acid from or derived from a probe organism; and wherein said kit also contains a catalogue having chromatographic band patterns of rRNA
information-containing restriction fragments for at least two known different organism species, or said kit contains at least two known different organisms species, or DNA derived therefrom.

52. the kit of claim 51, which also comprises a container means containing one or more restriction endonuclease enzymes.

53. The kit of claims 51 or 52, wherein said ribosomal RNA information-containing nucleic acid probe is detectably labeled.

54. The kit of claim 51, wherein said ribosomal RNA

information-containing nucleic acid probe is ribosomal RNA
(rRNA).

55. the kit of claim 52, wherein said ribosomal RNA
information-containing nucleic acid probe is ribosomal RNA
(rRNA).

56. The kit of claim 51, wherein said ribosomal RNA
information-containing nucleic acid probe is DNA complementary to ribosomal RNA (rRNA cDNA).

57. The kit of claim 55, which also comprises a third container means containing one or more detectable labeled deoxynucleoside triphosphates.

58. The kit of claim 51, wherein said probe organism is a prokaryote.

59. The kit of claim 51, wherein said probe organism is a eukaryote.

60. The kit of claim 58, wherein said prokaryote is a bacterium.

61. The kit of claim 59, wherein said nucleic acid is a probe of said eukaryote is derived from an organelle thereof.

62. The kit of claim 56, wherein said rRNA cDNA is labeled with 32p, 14C, 3H.

63. The kit of claim 54, wherein rRNA cDNA is a faith-ful copy of the rRNA it is derived from.

64. The kit of claims 51,or 52, which also comprises one or more container means containing viral nucleic acid probes.

65. The kit of claims 51 or 52, wherein said catalogue is a book, a computer tape, a computer disc, or a computer memory.

66. The kit of claims 51 or 52, wherein said catalogue also includes viral chromatographic patterns.

67. A kit comprising a carrier being compartmentalized to receive in close confinement therein one or more container means, wherein a first container means contains ribosomal RNA
information-containing nucleic acid from or derived from a probe organism, said ribosomal RNA information-containing nucleic acid being in detectably labeled form; and wherein said kit also comprises a second container means containing one or more restriction endonuclease enzymes.

68. The kit of claim 67, wherein said ribosomal RNA
information-containing nucleic acid probe is ribosomal RNA
(rRNA).

69. The kit of claim 67, wherein said ribosomal RNA
information-containing nucleic acid is DNA complementary ribosomal RNA (rRNA cDNA).

70. The kit of claim 67, which also comprises a container means containing one or more detectably labeled deoxinucleoside thiphosphates.

71. The kit of claim 67, wherein said probe organism is a prokaryote.

72. The kit of claim 67, wherein said probe organism is a eukaryote.

73. The kit of claim 71, wherein said prokaryote is a bacterium.

74. The kit of claim 72, wherein said nucleic acid probe of said eukaryote is derived from an organelle thereof.

75. The kit of claim 69, wherein said rRNA cDNA is labeled with 32p, 14C or 3H.

76. The kit of claim 69, wherein said rRNA cDNA is a faithful copy of the rRNA it is derived from.

77. The kit of claim 67, which also comprises one or more container means containing viral nucleic acid probes.

78. The kit of claims 63 or 67, wherein said rRNA
cDNA is derived from the rRNA of a prokaryotic probe organism.

79. A method for detecting a prokaryotic organism while in the presence of or associated with a eukaryotic organism which comprises:
selectively hydridizing ribosomal RNA sequences of said prokaryotic organism with a detectably labelled prokaryotic rRNA information containing hydridization probe.