JPH0824584B2 - Method for identifying and characterizing organisms - Google Patents

Description

FIELD OF THE INVENTION The present invention provides a method for rapidly and accurately characterizing and identifying organisms, including prokaryotic and eukaryotic organisms such as bacteria, plants and animals. It is about.

[Conventional technology]

The classification of living organisms has traditionally been done more or less voluntarily and along some artificial line. For example, the world of life is divided into two worlds: plants (Plan
tae) and animals (Animalia). This classification is convenient for generally known organisms, but difficult for organisms such as single-celled organisms (eg, green caterpillars, bacteria, cyanobacteria). This is because these are basically different from "plants" and "animals".

It was proposed to simply divide the organism by the internal structure of the bacterium. In this way, all cellular organisms are either prokaryotic or eukaryotic. Prokaryotes are eukaryotes (e
They are less complex than ukaryotes), they lack internal divisions by unit membrane tissue and lack a clear nucleus. The genetic information of prokaryotic cells is carried in the cytoplasm on double-stranded circular DNA. No other DNA is present in the cell (phage, bacterial viruses, and autonomously replicating circular
Except if the DNA plasmid is present). On the other hand, eukaryotes have a wide variety of unit membrane tissues that serve to separate many functional components into specialized and isolated regions. For example, genetic information (DNA) is found in clearly demarcated nuclei, and also in organelles glomeruli (mitochondria) and (in photosynthetic organisms) chloroplasts. The replication, transcription and translation of the eukaryotic genome occurs in two or three distinct sites within the cell, the nucleocytoplasmic part, the glomerulus and the chloroplast.

However, the difference between prokaryotes and eukaryotes is disrupted when performing glomerular and chloroplast comparisons in prokaryotic cells.
These organelles are today free-living (free-
is believed to be derived from prokaryotic organisms, which enter into endosymiotic relationships with primitive eukaryotes and are ultimately closely integrated with the structure of the host cell. , Independent existence has become impossible (reference example, Fox, GB et al. “Science” 209; 457-463).
(1980) p.462; Stanier, RY et al. `` The Microbial Wo
rld "4th edition, published by Prentice-Hall, 1976, p.86). For example, the DNA obtained from mouse L cell filaments that carry the ribosomal RNA gene region is Escherichia coli ribosomal R.
It has been shown to show significant sequence homology with NA, strongly supporting the endosymbiotic model (Van Etten, RA et al, “Ce
ll "22: 157-170 (1980)). Also, corn (Zea
The nucleotide sequence of 23S ribosomal RNA of chloroplasts has also been shown to have 71% homology to Escherichia coli 23S ribosomal RNA (Edwards, k. & Kossel, H., “Nu
cleic Acids Research ”9: 2853-2869 (1981); Other related research (Bonen, L. & Gray, MW, ibid. 8: 319-335).
(1980)). It further supports the general idea.

In this model, eukaryotic cells are phylogenetic "chimeras" with organelles that are clearly prokaryotic in nature. The “pronuclear-eukaryotic” dichotomy also has drawbacks, even as a broad class of methods.

The classification of organisms is more than a scientific task when attempting to identify plants or animals for the purpose of crossing and breeding, and to infect so-called "higher" organisms or other vehicles. This is the case when attempting to accurately and reliably identify a microorganism that may do so. For example, plant growers, livestock farmers or fish farmers will want to have a quick and reliable way to identify heterologous and atypical strains. Veterinarians, physicians or horticulturists will want to accurately identify infectious organisms (parasites, fungi, bacteria, etc.) and viruses in test plants and animal tissues. Correct identification of these organisms and the species of Weels is of particular importance.

This problem is best seen when describing the identification of bacteria. The name of a bacterial species usually refers to many strains, and one strain is considered to be a population derived from one cell. Strains of a species have similar combinations of attributes that help define that species. Expressing an accurate definition of a bacterial species is difficult because it is necessary to demarcate the diversity of strains within a species in order to establish the boundaries of the species (Buchanan, RE, “International Bulletin of Ba
cteriogical Nomenclature and Taxonomy, '' 15: 25−32
(1965)). To actually apply the definition of species to the identification of unknown bacterial strains, the selection of appropriate probes such as substrates and conditions to detect phenotypic attributes, and radioactively labeled DNA from the same species. is necessary. It is necessary to use a screening method to temporarily confirm the strain so that an appropriate probe for identifying the strain can be selected. The challenge is to define the boundaries of the species exactly, and preferably to do this using standard probes that provide species-specific information. By doing so, species definition can be applied directly and equally to the identification of unknown strains.

Vergie's Manual of Determinating Bacteriology (Buchanan, RBand Gibbo-ns, NB editor, 1974, 8th edition, Willians & Wil-kins, Baltimore)
Shows the most easy-to-understand bacterial taxonomy, especially nomenclature, basic strains, and related literature. However, this is only the starting point for species identification. This is especially because it is obsolete and space limited so the description of the species is too simple. (Reference example: Brenner DJ) "Manual of Clinic
al Microbiology ", 3rd edition, American Society for Microbiology, Washington DC, 1980, pp. 1-6).

The term "seed" when used in bacteria refers to different types of organic matter, with distinctive characteristics,
It has been generally defined as a group of organisms that have similarities to each other in the characteristics of the essential organism organization. The problem with these definitions is that they are subjective (Brenner, ibid., P2). Also, the species is simply a range of hosts,
It was sometimes defined based on criteria such as pathogenicity, whether or not to produce gas during fermentation of certain sugars, and whether sugar fermentation was fast or slow.

In the 1960s, the numerical bacterial taxonomy (also called computer taxonomy or genetic phenotypic taxonomy) became widely used. Mathematical taxonomy is the heredity of an organism (po
based on testing as many tentials as possible. By classifying on the basis of a number of characteristics, groups of strains with a certain degree of similarity can be formed and these can be considered as species. However,
This method of defining a species is not directly and practically applicable to the identification of unknown strains, as a test that is valid for characterizing one species may not be useful for the next. Even if these attributes are used for identification of unknown strains and can be partially overcome by choosing attributes that appear to be species-specific, this type of definition has an indirect application. (Brenner, ibid., P.2-
6). Moreover, the general method has several problems when it is used as the sole basis for defining species, among them the number and nature of the tests to be used and how well the tests are evaluated. What should be done to reflect the relevance, how much similarity should be chosen, and whether the same similarity criteria can be applied to all groups. "Manual of Clinical Mi" by Hugh, RH and Giliardi, GL
crobiology "Second Edition, American Microbial Association, Washington D.
C., 1974, p.250-269, lists the minimal phenotypic traits as a means of defining bacterial species using genomic fractions. By studying multiple and randomly selected strain samples from within a single species, it is possible to select and define the attributes that are the most highly conserved or common to so many strains. it can. The use of minimal properties is an advance, starting with screening methods to tentatively identify strains and allowing the selection of appropriate additional media. The known attributes conserved in the species are then examined, expecting the strain to have most of the minimal characteristics. Some of the minimal characteristics do not appear in all strains of the species. Related concepts include comparative studies of species types, neotypes, or established control strains of the genus. Since each laboratory has different media and methods,
This matching is necessary and the strain is the standard for this species
But not the method.

A molecular-level approach to bacterial classification is to compare two genomes by DNA-DNA reassociation. The genetic definition of a species includes the assumption that the strains of the species are more than 70% related. Strains can be identified by DNA-DNA repairing only if the radiolabeled DNA probe and the unknown DNA are of the same species. However, the practical application of this 70% species definition is limited by the choice of the appropriate probe. this is,
Some may be overcome by selecting phenotypic attributes that appear to be interrelated with the repair group, but when used alone, the definition of species by DNA-DNA repairing is After all, it is only applied indirectly.

Brenner, supra, page 3, the ideal means of identifying bacterial species is to isolate the gene and immediately insert the nucleic acid sequence into the strain of interest to any known species-something.
It is said to be a "black box" similar to mass spectrophotometric analysis compared to the standard pattern of.

However, although Brenner can perform restriction endonuclease analysis to determine the general sequence of isolated genes, "We have a suitable black box, especially one suitable for use in clinical laboratories. It's unlikely to come in. " His words apply equally to every species of organism.

From this brief reexamination of the prior art, to identify unknown bacteria and other organisms, to quickly classify them, and to identify in particular pathogenic organisms or organisms that have available biochemical reactions. We conclude that a fast, accurate and reliable method is now needed. The method must be universally and readily available in the clinical laboratory, must not depend on the number of trials conducted, the clinician's subjective prejudice, and must Do not rely on the trial or error method of trial or error. In addition, it helps identify and distinguish the genus and species of any living organisms, by veterinarians, plant growers, toxicologists, animal keepers, entomologists, and in other relevant areas where such identification is required. It is also necessary to have a method that can be used easily and reliably.

[Problems to be Solved by the Invention]

It is therefore an object of the present invention to provide a rapid, accurate and reliable method for the objective identification of organisms, especially, but not exclusively, microorganisms.

Another object of the present invention is to provide a method for identifying an organism such as a bacterium by utilizing the genome of the organism.

Another object of the present invention is to provide a method for characterizing and identifying species and genera of pathogenic organisms in a clinical laboratory so that the cause of animal or plant diseases can be characterized and identified. is there.

Another object of the present invention is to provide various organisms useful in the taxonomy described above.

[Means for solving the problem]

These and other objects of the present invention have been achieved by providing the following methods, as will be more readily found below.

A restriction endonuclease digestion obtained from the organism, from the probe organism, or from the organism, or hybridized or repaired with a ribosomal RNA information-containing nucleic acid, as a method of characterizing an unknown organism. DNA
The method of claim 1, wherein the chromatographic pattern is compared to an equivalent chromatographic pattern of at least two different known organic species.

Another object of the present invention has been achieved by providing the following method.

A method of diagnosing a pathogenic organism infection in a sample, the method comprising identifying an organism in the sample by the method described above.

The present invention provides strain-sharing similarities that objectively delimit species, despite dispersal, if the species is a collection of individually separated strains that are associated with a common speciation event. Based on the inventor's recognition that the strains of a species should have structural information that provides clues to their common source. The history of organisms is the most abundant in history, semantides, DN
Since it remains in A and RNA (Zuckerkandle.E. And Pauling, L, "Journal of Theoretical Biology" 8: 357-366 (1965)), the inventor has provided information on some conserved genes. It was concluded that the experimental approach utilized was to use rRNA. Ribosomal RNA has structural and functional roles in protein synthesis (Schaup, “Journal
of Theoretical Biology '' 70: 215-224 (197
8)), and general conclusions drawn from rRNA-DNA hybridization studies indicate that the basic sequence of the ribosomal RNA gene is less likely to undergo evolutionary changes and more conserved than most other genes. It seems to be rumor (Moor
e, RL, "Current Topics In Microbiology and Imm"
unobiology '', 64, 105-128 (1974), Spring-Verlag
Company, New York). For example, the primary structure of 16S-type rRNA from many bacterial species was deduced from oligonucleotid analysis (Fox, GB et al, "International J
ournal of Systematic Bacteriology ", Volume 27: 44-57
(1977)). There are negligible differences in the 16S oligomer catalogs of several strains of E. coli (Uchida T. et al;
"Journal of Molecular Evolution", Volume 3: 63-77 (197
4)), but substantial differences between species can be used to generate a bacterial phylogenetic taxonomy (Fox, GB “Science” 209).
Volume: 457-463 (1980). A strain of one bacterial species is
It is not always the same, and the restriction maps show different Ec
It has been shown that the oR I site occurs in the rRNA gene of two strains of Escherichia coli (Boros, IA et al, “Nucleic Acids”).
Research ", Volume 6: 1817-1830 (1979)). Bacteria preserved
It appears to share the rRNA gene sequence, and other sequences may vary (Fox, 1977, ibid.).

The inventors have further shown that restriction endonuclease digests of DNA combine fragments containing rRNA gene sequences that are similar in strains of certain organisms (eg, bacteria) but different in strains of other organisms. In other words, it was also found that, despite the mutation of the strain, a specific combination of enzymes, which has a high frequency of occurrence and a restriction fragment with a minimum congenotype trait, determines the species. This is the essence of the present invention. The inventor intends to identify this method whether it is the same or different in taxonomy (classical) regardless of whether it is prokaryotic or eukaryotic. Pronuclear and eukaryotic DN with probe
It was also found to be general in that it could be applied to both.

The present invention provides an objective method of defining an organism based on detecting DNA fragments containing ribosomal RNA gene sequences in restriction endonuclease digests. The detection is
This is done by hybridizing or repairing the DNA fragment with a nucleic acid containing rRNA information from the probe organism.

By the term "organism" (which term is construed to include "identify"), which is characterized by the process of the present invention, is meant virtually any organism that includes DNA by definition. It is useful to mention the traditional taxonomy for reference in this regard.

Includes plants and animals, eg monera
In the kingdom, myxobacteria, spirochetes, eubacacteri
a), a fragmented product of the rickettsiae class (bacteria)
And cyanobacteria: cyanophytha. Euglenoides (Eu
glenophta), green algae: Chlorophyt
a), chlorophyceae and carophyceae, chrysopht
a) Xanthophyceae, chrysophyseae, bacilla bacilla
riophyceae); pyrrophytas Dinoflagellates, brown algae: phaeophyta; red algae: Rhodop
hta); slime mold: Myxomycophyt
a), myxomycetes, acracie
siaes), Plasmodiophorea
e), Labyrinthuleae class; Yumycophyta: Fungi (Eumycophyta), Phycomyceta (p
hycomycetes), ascomycetes, bassidomycetes class; bryophytes, hepattiers (hep)
aticae), antocerote (anthocerotae), musushi (mu)
sci); vascular plant Tracheophyt
a), psilopsida, lycop
syda), sphenopsida, sphenopsida, pteropsids, spermopsida subclass, cycadae, ginkgoae,
Coniferae, gneteae and angiospermae classes, dicotyledoneae, monocotyloed
oneae) subclass is described. In the animal kingdom, there are sub-Protozoa, Protozoa (protozoa), Subplasmas plasmodroma, Flagellata,
Sarcodina and sporozo
a) Class: Ciliophora subphylum, ciliata class; Subterranean subdivision, Polyhera (Spongea p
orifera, calcarea, hexactinellida and desmospoguier
pongiae); Mesozoa subdivision, Mesozoa phylum; Metazoa subdivision Radiata division, coelentata
erata gate, hydrozoa, sifozoa (scy)
phozoa), Utozoa class, Stenophora (ct)
enophora, tentaculata and nuda; the protostomia division, platyhelmintes, platyhelmintes, tubellana, trematoda and cestoda; nemertina ，
Acanthocephala gate; aschelmintles gate, rotifera, gastrotricha, quinorhynch
a), priapulida, nematoda
oda) and the nematomorpha genus; the entoprocta phylum; ectopr
octa), Gymnolaemata and phylactolaemata; Fulonida (p
horonida Gate; braciopoda Gate, inarticulata and articulata
culata); Morusca mollusca, amphineura, monoplacophora (monopl)
acophora), gastropoda, scaphopoda, pelecypoda
And the cephalopoda class; sipunculida gate; echiurida gate,
Annelida Gate, Polychaeta,
Oligochaeta and hirdinea (hir
udinea); onychophora gate; tardigrada gate; pentastoimida
astoimida gate, arthropoda gate; trylobita gate, chericerata
ta) Amon, Kifosura, arakimida
chmida), pycnogomida class, subdivision of mandibulata, crustacea,
Chilopoda, diplopod
a), polopoda, symphyl
a) rope, collembola, protu
ra), diplura, thysanur
a), ephemerida, odonat
a), orthoptera, or dermaptera
maptera), embinia, precoptera (p
lecoptera), zoraptera, corrodentia, mallophaga, anoplura, chisasnopter
a), hemiptera, neuroptera
tera), Coleoptera (coleptera), Hymenoptera (h
ymenoptera, mecoptera, siphonaptera, diptera, trichoptera and lepidopter
a) Insects of the eye; animals of the Denterostomia division, the gate of chaetognatha, the gate of echinodermata, the crinoide
a), asterodea, ophiuroidea, echinoidea, and echinoidea, holoturoidea, Pogonophora phylum, hegoncordata (hemichordata)
Gate, enteropneusta and pterobranchia class; cordata (chordat)
a) Gate, sub-gate of urochordata, ascidiaciae, thaliaceae,
Larvacea class; ceph allocol data (ceph
submonium alochordata, submonium vertebrata, agnatha, chondrich
thyes), ostechichthyes, saccopteiygii subclass, crossopte
rygii and dipnoi eyes, amphibia, repitilia, aves
s) and mammalia ropes, protothelia (p
subclass of rototheria, subclass of theria, marsupialis, insectivora
a), dermoptera, chiroptera (chi
roptera), primates, edenta
ata), pholidota, lagomorpha (la)
gomorpha, rodentia, setase (ce)
taceae), carnivora, tubulidentata, probosicdes,
Hiracoidea, Sirenia,
Mention may be made of the order Perissodactyla and Artiodactyla. It is known that there are still families, tribes, genera, species and subspecies under the eyes, and taxa, strains or individuals outside the subspecies. In addition, there are cell taxa, and strains or individuals. Moreover, cultured cells (plants or animals) can be identified in the same way as viruses. These classifications are used in this application for descriptive purposes only and not as a limitation.
The organism to be identified may be known or unknown, but is most commonly unknown.

Functionally, it is convenient for the purposes of the present invention to divide all organisms into eukaryotic and prokaryotic populations. When identified as a prokaryotic organism, the DNA that is subject to endonuclease digestion is present in the cell or in an uncompartmented chromosome. When identified as a eukaryotic organism, nuclear DNA or DNA within organelles (filament DNA)
Alternatively, chloroplast DNA) may be used to similarly perform endonuclease digestion.

Briefly, rRNA gene sequences, maybe subclass-specific or subclass specific (infrasubspecific subdivi).
High molecular weight DNA and / or small circular DNA is isolated and identified from the organism in order to analyze sequences that can be used to generate the sion). DNA is extracted by methods well known to those skilled in the art. The DNA is cut into a fragment by a restriction enzyme at a special site. The fragments are sized according to size in a standard chromatographic system such as gel electrophoresis. The gel is stained by methods well known to those skilled in the art,
Standardize for fragment size using a standard curve made of fragments of known size. Then, the separated fragments were spotted by Southern (EM, Jou
rnal of Molecular Biology ", 38: 503-517 (1975),
(Herein incorporated by reference) to a nitrite cellulose paper and covalently bound thereto by heating. rR
The fragment containing the NA gene sequence is then localized by its ability to hybridize to a nucleic acid probe containing rRNA information. The nucleic acid probe is labeled with a non-radioactive substance or preferably with a radioactive substance.
When labeling with radioactive material, the probe is ribosomal RN
A (rRNA), can be synthesized by reverse transcription,
Ribosomal RNA contained on a clonal fragment that can be labeled, for example, by nick translation
It may be a radiolabeled DNA (rRNAcDNA) complementary to.

The probe is obtained from an arbitrarily chosen organism as will be seen later. Once hybridization has taken place, the hybridized fragments can be detected by selectively detecting double-stranded nucleic acid (non-radioactively labeled probe) or made visible, for example by radioautograph. (Radiolabeled probe). The size of each hybridized fragment is determined from the migration distance using the standard curve described above. The amount of hybridisation, the hybridisation pattern, and the size of the hybridised fragments are used individually or in combination to identify organisms.

The pattern resulting from this hybridization can be easily compared to equivalent chromatographic patterns obtained from at least two, or a large number of known standard organisms, genera or species. Bandwidth (hybrid hybrid It is possible to make a comparison by the amount of chemical conversion or by combining these. Ideally, the comparison should be done with a one-dimensional computer pattern recognizer, such as used in point-of-sale processing.

When the inventor uses the above method, the chromatographic patterns of organisms of the same species are very similar and the slight difference is only the difference between the subspecies due to the change of strain, but the difference between the species and The differences between genera (and even at higher levels of taxonomy) are enormous.

Mutations in enzyme-specific fragments between strains of a given species can also be used to type strains for various purposes, eg in the case of bacteria, epidemiological purposes. In fact, restriction enzymes can be chosen that can distinguish between strains within a species.

The "probe organism" (and from which the nucleic acid probe is obtained) used in this study may be any of the organisms listed above, either a eukaryotic organism or a pronuclear organism. The only limitation is that the rRNA-containing probe is the DNA of an unknown organism.
It must be maximally hybridized with the endonuclease digestion of.

Ribosomal RNA information-There are 4 types of probes: 1) Prokaryotic ribosomal probe (especially rR obtained from bacteria)
NA), 2) eukaryotic glomerular ribosome probe, 3) eukaryotic chloroplast ribosome probe, 4) eukaryotic non-organic ribosome probe.

Source of DNA (digested with endonuclease) is 4
There are several types: 1) prokaryotic DNA, 2) eukaryotic glomerular DNA, 3) eukaryotic chloroplast.
DNA, 4) Eukaryotic nuclear DNA.

Thus the following hybridization table was created (first
table).

The table shows the DNA of an organism whose ribosomal RNA probe is unknown.
And shows that it can hybridize to the maximum. For example, to identify a eukaryotic organism,
Species-specific glomerular or chloroplast DNA is extracted, digested with endonuclease, and the digest is pronuclear ribosome
It may be hybridized with an RNA probe or a eukaryotic ribosome probe extracted from an organelle. Similarly, to identify pronuclear organisms, species-specific cellular DNA was extracted, digested with endonucleases, and digests were extracted from prokaryotic ribosomal DNA probes or organelles of eukaryotic ribosomes. It may be hybridized with an RNA probe.
Eukaryotic organisms can also be identified by extracting and digesting species-specific nuclear DNA and hybridizing it with non-organorganic eukaryotic ribosome probes. Eukaryotic cells could be defined by one or some combination of nuclei, glomeruli, or in some cases the chloroplast system. These cross-hybridizations are
RRNA nucleic acid extracted from eukaryotic organelles has a wide homology with prokaryotic rRNA nucleic acid, but nuclear-extracted eukaryotic DNA and prokaryotic DNA
And is based on the fact that the homology does not exist so widely.

In summary, because the genetic code is ubiquitous, all cells (eukaryotic or prokaryotic) require ribosomes to translate the coding information into proteins, and ribosomal RNA is highly Because it is preserved, the cross-
There is sufficient homology between the hybridizing elements ("+" in Table 1) to ensure that this method is effective.

The choice of pair of DNA to digest and its associated ribosomal probes is optional and will depend on the organism to be identified. That is, it will depend on the question asked. For example, when detecting a prokaryotic cell species (for example, bacteria) present in a eukaryotic cell (for example, an animal or a plant) for the purpose of detecting and identifying an infectious substance, a prokaryotic ribosomal probe is selected, The derived DNA may be treated under the condition that it is not extracted or the minimum amount is extracted. Using this approach, one can be confident that the interference between organelle-derived DNA and pronuclear DNA is minimal. When identifying a eukaryote, which is not infected by prokaryotic cells, with a prokaryotic ribosomal probe, for example, separating the organelle from the nucleus and then
DNA derived from organelles, such as extracting only DNA
It is best to maximize the concentration of. If it is desired to identify prokaryotic infected eukaryotic organisms, it is best to use non-organ, non-prokaryotic ribosomal probes. This ribosomal probe does not hybridize sufficiently with DNA from prokaryotic cells.

Same world, same subworld, same department, same gate, same subgate, same class, same subclass, or same eye,
Or a pair from the same family, the same family, or the same genus (DNA
And ribosomal probes) are preferably used. Prokaryotic ribosomal probes (eg bacterial ribosomal probes) to hybridize with prokaryotic DNA
Is particularly preferably used. In this way, genus, species and strains of pronuclear organisms could be detected, quantified and identified. One of the most preferred pronuclear ribosome probes is obtained from bacteria, among which:
E. coli is preferable because it is easy to use and easy to obtain. Ribosomal probes obtained from E. coli can be used to identify any organism, especially all prokaryotic organisms, and are most preferred for the identification of any genus, species and strains of bacteria. Another, particularly preferred embodiment is to identify eukaryotic organisms of the same family using eukaryotic ribosomal probes from a given family (e.g. to identify mammalian organisms). Using a mammalian ribosomal probe). Most preferred is the use of ribosomal probes and DNA obtained from organisms of the same subfamily and / or order and / or family (e.g. to identify certain species of mouse, ribosomal probes derived from mouse Good to use).

The most sensitive and effective pairing system is the one with less evolutionary distance or less variance between the source of the ribosomal probe and the source of the restricted DNA.

The individual steps involved in this technique are generally known to those skilled in the art. From now on they will be referred to both eukaryotic cells and prokaryotic cells (where applicable) or, where technically different, for each type of cell separately.

The first step is the extraction of DNA from unknown organisms. Nuclear DNA from eukaryotic cells can be selectively extracted by standard methods known to those of skill in the art (eg Drohan, We.
Tal, "Biochem. Biophys. Acta", 521 (1978), 1-1.
5, inserted here as a reference). Organ DN
Spooling because A is small and circular
The method serves to separate non-circular nuclear DNA from circular, organelle-derived DNA. As a result, the non-spooled material contains DNA from organelles, which can be individually separated by density gradient centrifugation. Alternatively, the glomeruli (or chloroplasts) are separated from the mixture of crushed cells and the purified fraction of glomeruli (or chloroplasts) is used to prepare organelle-derived DNA. The prepared nuclear fraction is used for preparation of nuclear DNA. (Eg Bonen
L. and Gray MW "Nucleic Acids Research" 8: 319-
335 (1980)).

Pronuclear DNA extraction is also well known to those skilled in the art. For example, unknown bacteria present in media such as industrial fermentation suspensions, agar, plant or animal tissues or samples etc.
Process under well known conditions designed to extract high molecular weight DNA. For example, cells of an unknown organism are suspended in extraction buffer, ribozyme is added to it and the suspension is incubated. Cell disruption can be further promoted by the addition of detergents and / or increased temperature. After protease digestion, chloroform / phenol extraction and ethanol precipitation are performed to complete DNA extraction. Another extraction method, which is much faster than phenol / chloroform extraction, is the rapid separation of DNA using ethanol precipitation. This method is preferably used to separate DNA directly from colonies or small volumes of liquid medium. This method
Davis, RW et al. `` A Manual for Genetic Engineeri
ng, Advanced Bacterial Genetics "(hereafter" Davis "), Cold Spring Harbor Laboratory, Cold Spring
Harbor, New York, 1980, p.120-121.

DNA (prokaryotic or eukaryotic (nuclear or non-nuclear))
Is dissolved in physiological buffer for the next step.

Digestion of the extracted DNA is performed with a restriction endonuclease enzyme. Any restriction endonuclease enzyme can be used, with the proviso that it is, of course, not from the same species of organism as the one to be identified.
Because, if this condition is violated, the DNA will remain completely intact (this will eventually identify the organism, because the enzyme is its own origin). It is unlikely to cleave the DNA obtained from the seed). Since the organism species to be characterized will be unknown, a minimum amount of trial and error will be required to obtain a fragment digest, which will be routinely performed by a skilled person without undue experimentation. It is a work that can be performed. Examples of potential restriction endonuclease enzymes are Bgl I, Ba
mH 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 and others. Davis, Id., P. 228-230. Mixtures of one or more endonucleases can also be used for digestion. Usually, the DNA and endonuclease are incubated in a suitable buffer for a suitable time (in the range of 1 to 48 hours, the temperature range is 25 ° C to 65 ° C, preferably 37 ° C).

The resulting chromatographic pattern will depend on the type of endonuclease (s) used and will be specific to the endonuclease. Therefore, it is necessary to pay attention to which enzyme (one kind or several kinds) was used for digestion. This is because the comparative patterns used in the catalog must be made with the same enzyme or enzymes.

After endonuclease digestion, the incubation mixture containing fragments of different sizes is separated by suitable chromatographic methods. Any method that allows the nucleic acid digests to be separated by size and allows subsequent hybridization with nucleic acid probes can be used. The gel electrophoresis method is preferable, and the agarose gel electrophoresis method is particularly preferable. In this device, the DNA digest is usually electrophoresed in a suitable buffer and the gel is usually immersed in an ethidium bromide solution, possibly with a UV- Light-placed in a box.

After separation and visualization, the DNA fragment was transferred to nitrocellulose filter paper by the Southern method (see "Jour.
nal of Molecular Biology "38: 503-517 (1975)).
This transfer can be done after the denaturation and neutralization steps and is usually transferred from the gel to the filter paper over a long period of time (about 10-20 hours) or by applying electrical force. Devices that facilitate transfer from gel to filter paper are commercially available. The received nitrocellulose filter paper is then heated at elevated temperature (60-80 ° C) for several hours to bind the DNA to the filter paper. The probe utilized to hybridize the DNA digest fragments attached to the filter paper is a nucleic acid probe, preferably one obtained from a given well-known organism. It is either detectably labeled or unlabeled, but it is preferably detectably labeled. In this case, the nucleic acid probe is a detectable labeled ribosomal DNA (rRNA) obtained from such an organism, an incision translation labeled DNA probe containing ribosome information, a clone DNA probe containing ribosome information or a probe Detectable labeled DNA (rRNAcD that is complementary to ribosomal DNA from the airframe)
NA). Depending on the choice of combination, the ribosomal probe may be from a prokaryotic cell or a eukaryotic cell (cytoplasm-derived or organelle-derived). Most preferably, the detectable label is a radioactive label such as radioactive phosphorus (eg ³² P, ³ H or ¹⁴ C). The nucleic acid probe may be labeled with a metal atom. For example, uridine and cytidine nucleotinodes can form covalently bound mercury derivatives. Nucleoside triphosphate bound to mercury is a good substrate for many nucleic acid polymerases, including reverse transcriptase (Dale et al., “Proceedings of the N
`` ational Academy of Sciences '' 70: 2238-2242,197.
3). Direct covalent mercuration of natural nucleic acids has been reported (Dale et al., "Biochemistry" 14: 2447-245.
7). The reannealing properties of mercurized polymers are similar to those of corresponding mercury-free polymers (Dale
and Ward, "Biochemistry" 14: 2458-2469). Metal-labeled probes are, for example, light-acoustic spectroscopy,
It can be detected by X-ray spectroscopy, for example X-ray fluorescence, X-ray absorption or photon spectroscopy.

Separation of rRNA from eukaryotic and prokaryotic cells is well known to those of skill in the art. For example, to prepare rRNA from eukaryotic cytoplasmic ribosomes, RNA can be extracted from whole cells or ribosomes and separated by sucrose gradient centrifugation and the 18S and 28S fractions are collected using markers of known molecular weight. Can be (eg Perry,
RP and Kelly, DE, 28S and 18S ribosomal RNA
Synthesis of 5S RNA when production of Escherichia coli is inhibited by a small amount of actinomycin D "J. Cell. Physiol., 72: 235-24.
6 (1968), listed here as a reference). As a corollary, organelle-derived rRNA is isolated and purified from organelle fractions in a similar manner (eg, Van
Etten RA et al., "Cell" 22: 157-170 (1980), or Edwards, K. et al., "Nucleic Acids Reserch", 9: 285.
3-2869 (1981)).

If a radiolabeled ribosomal RNA probe is used, it may be derived from a probe organism grown or cultivated in nutrients containing compounds with suitable radioactivity, or in culture media containing such compounds. To be separated. If the probe is complementary DNA (rRNA cDNA), it reverse transcribes rRNA isolated from the probe organism in the presence of radioactive nucleoside triphosphates (e.g. ³² P-nucleosides or ³ H-nucleosides). Made by.

Labeled ribosome probes may also be obtained from truncated translated DNA molecules, especially whole circular DNA from organelles. Specifically, circular DNA of chloroplast or glomerulus
Is transcribed in the presence of a radioactive label to give a labeled DNA ribosomal probe. The chloroplast labeled probe will hybridize best with chloroplast DNA and the glomerular labeled probe will hybridize best with glomerular DNA. The chloroplast (or glomerular) incision translation labeled ribosome probe will hybridize second best with the glomerular (or chloroplast) DNA. It will hybridize to the DNA of the whole plant (or animal), albeit in a less preferred form. The ribosomal probe may be obtained from the nuclear DNA of a eukaryotic cell by incision translation, even if this method is not good in consideration of practicality. A more useful approach to achieve this is to convert rRN from eukaryotic nuclear DNA.
Cut the A gene (by restriction enzyme), divide the fragment,
Identifying the ribosomal gene sequences (by hybridization) and separating the ribosomal gene sequences (by electrophoresis). The isolated rRNA sequences can then be religated into a plasmid or vector and cloned in a ³² P-containing medium after transformation of a suitable host. Another method involves growing a transformed host and then isolating the DNA and labeling it by truncation translation, or isolating the DNA and excising the rRNA sequence and then labeling. The resulting ribosomal probe hybridizes in the same state as rRNA cDNA (see below).

A preferred nucleic acid probe is rRNA from a probe organism.
Is a radiolabeled DNA complementary to. Specifically, rRN
A separates ribosomes from the probe organism, separates them, and the appropriate RNA is substantially purified as described above. The ribosomal RNA thus obtained does not contain a ribosome and is a carrying RNA (tRNA). Or, it contains almost no other RNA such as transfer RNA (mRNA). Prokaryotic rRNA usually contains three subgroups. So-called 5S, 16S, and 23S-fragments. Reverse transcription to cDNA is performed with a mixture of all three species, or with a mixture of 16S and 23S fragments. Reverse transcription of only one of these components is possible in some situations, but less preferred. Eukaryotic rRNA usually contains two subgroups, the 18S and 28S types. And reverse transcription into cDNA is performed by a mixture of 18S and 28S fragments, or each of these.

Pure rRNA containing almost no other types of RNA was labeled with cDN
Incubate with a reverse transcriptase that can reverse transcribe to A. More preferred in this case is calf thyroid DN
Incubation with reverse transcriptase from avian myeloblastosis virus (AMV) in the presence of primers such as A hydrolyzate. The mixture must contain a suitable deoxynucleoside triphosphate, at least one of the nucleosides being, for example, by ³² P,
It is radioactively labeled. For example, deoxycytidine 5 ′-( ³² P), deoxythymidine 5 ′-( ³² P), deoxyadenine 5 ′-( ³² P), or deoxyguanidine 5 ′-( ³² P) triphosphate is used as the radioactive nucleoside. To be After incubation for 30 minutes to 5 hours at 25 ° C to 40 ° C, extraction with chloroform and phenol, centrifugation and chromatography, the radiolabeled fractions are combined and used as a cDNA probe. Substantially pure form of radiolabeled cDNA probe, i.e., no unlabeled molecule, no cDNA complementary to other forms of RNA, no proteinaceous material, no cellular components such as membranes, organelles, etc. Not radiolabeled
The cDNA probe also constitutes one aspect of the present invention. Preferred probes are prokaryotic labeled rRNA cDNAs, most preferably bacterial labeled rRNA cDNAs. The species of the probe is a bacterial microorganism such as Enterobacteriacea (Enterobactacea).
eriaceae), Brucella, Bacillus
us), Pseudomonas, Lactobacillus, Haemophillu
s), Microbacterium, Vibrio, Neisseria, species in the Bactroides family, and other anaerobic groups such as Legionella. Although the pronuclear cell example in this application is limited to the use of E. coli as a bacterial pronuclear probe organism, it is by no means limited to this microorganism. The use of radiolabeled cDNA as a probe is preferred over the use of radiolabeled ribosomal RNA because of its greater stability during DNA hybridization.

That the labeled cDNA probe must be a faithful copy of the rRNA, i.e. the template rR at any time when synthesis takes place.
It is important to recognize that the entire nucleotide sequence of NA is transcribed. The use of primers is essential in this respect. That the cDNA is a faithful copy can be evidenced by the fact that it has two properties after hybridization: 1. The cDNA must protect 100% of the labeled rRNA from ribonuclease digestion; and 2. Labeled cDNA must specifically anneal to rRNA as evidenced by resistance to S1 nuclease.

Beljansky MM et al. "CRAcad.Sc Paris" t28
6, Series D., 1825-1828 (1978), describes ³ H-radiolabeled cDNA derived from E. coli rRNA. The cDNA in this study was not prepared with reverse transcriptase in the presence of a primer as in the present invention, but was prepared with DNA polymerase I using rRNA previously divided with ribonuclease U ₂ as a template. is there. Berjansky et al.'S rRNA digestion product (by RNAse U ₂ ) has a different base ratio than the original rRNA, indicating that bases and / or short fragments have been lost. The cDNA thus obtained is not a faithful copy. Moreover, the use of DNA polymerase I used by Belgiansky is known to accelerate the predominance of homopolymeric transcription over heteropolymeric transcription of rRNA (see Sarin PS
et al., "Biochem. Biophys. Res. Comm.", 59: 202-214.
(1974)).

Taken together, the "ribosome" probe is
a) from genomic DNA containing the rRNA gene, by cloning and / or truncation translation, b) from the ribosomal RNA itself, or c) from rRNA cDNA, by reverse transcription of rRNA.

The next step in the process of the present invention is to separate DNA from unknown organisms.
Hybridization of the digest with unlabeled or (preferably) radiolabeled rRNA or cDNA probes. Hybridization is performed by contacting a paper containing covalently labeled DNA digests from unknown organisms with a hybridization mixture containing ribosomal probes. Incubate under heating (50-70
(° C.) for an extended period of time, after which the filter paper is washed to remove unbound radioactivity (if required), then air dried and ready for detection. Another highly preferred hybridization, which is much faster than the above method, is described in Kohne, DE et al., "Biochemistry" 16: 5329-5.
341 (1977). Room temperature phenol emulsion re-pairing method.

After hybridization, this method requires selective detection of properly hybridized fragments. This detection is
Taking advantage of the fact that the hybridized fragment has a double-stranded structure, a selective method (in the case of an unlabeled probe), a radio-autograph method, or a computerized method may be used. This can be done by any suitable radiation scanner method (in the case of labeled probes) which will increase the detection speed. These methods are familiar to the skilled person and will not be described further in this respect.

The end product of this method is a chromatographic band pattern with light and dark regions of varying intensity at specific locations. These positions are EcoR I digested λ bacteriophage DN
By subjecting to a marker separation method such as A, it is possible to easily match a specific fragment size (kilobase pair). In this way, the relative positions of the bands and the absolute size of each band can be easily ascertained. The banding pattern of the unknown organism is compared to the banding pattern found in the catalog or library. The catalog or library may consist of at least two, almost infinite, books listing determinable patterns of various organism genera and species. For example, it is estimated that there are about 100 recently pathologically relevant pathogens that cause human illness, so a standard catalog of pathogenic bacteria would contain 50-150 such patterns. A catalog of bacterial strain types for epidemiological judging systems may also be included.

The banding pattern depends on the type or group of endonuclease enzymes used, probably on the particular organism used as the source of the radiolabeled probe (probe organism), and also on the ribosome used to prepare the probe. Composition of RNA information (eg 5S, 16S or 23S of prokaryotic cells)
Type subtype, 16S and 23S only, etc.). Thus, the catalog shows for each probe a variety of enzymes with recorded band sizes and relative intensities.
It may contain specific band patterns. As the concentration of bound DNA bound to the filter paper decreases, only the strongest bands become visible, and the size of this or these bands can identify the species. Each one of these band patterns may include a band pattern obtained with complete ribosomal DNA and / or a band pattern obtained with ribosomal DNA containing only certain subtypes. The above changes or permutations are, of course, all available to the library. Moreover, for eukaryotic organisms, the library is of one type
It may include patterns generated by using DNA, or organelle and / or nucleus-DNA combinations.
The pattern of each DNA digest will depend on the probe composition. The catalog is organized so that if one or more strains or species are present in the extracted sample and detected by the probe, the pattern resulting therefrom can be interpreted.

The user can compare the resulting swath patterns visually or with a one-dimensional computerized digital scanner programmed for pattern recognition. These computer scanners are well known to those skilled in the art of time-of-sale processing (a commonly used "supermarket" checkout pattern reader). Ideally, the library or catalog should be in computer memory with the relative banding patterns of multiple organisms and the absolute value of the molecular weight or size of the fragments. If so, the catalog comparison is performed by matching the unknown pattern with one of the patterns present in the library by one or both of the stored information elements (relative pattern and / or absolute size element). The intensity of each band when compared to the standard can also express the amount of hybridized bound DNA, so it can be used to estimate the degree of presence of organisms, for example the degree of prokaryotic cells in eukaryotic cells. Can be used.

If the user confirms the nature of the given organism,
If further identification is desired, such a user would digest an unknown organism with a second different endonuclease and the resulting banding pattern would be the case for the second chosen endonuclease. Compare with the catalog band pattern of the aircraft. This process can be repeated as many times as necessary to make an accurate identification. However, it is usually sufficient in most cases to perform a single analysis with a single probe.

The invention and its variants have numerous applications. The present invention may be used by plant growers or animal keepers to correctly identify their objects, and in clinical and microbiology laboratories, bacteria present in any medium, including eukaryotic cells, parasites. It may be used to identify insects or fungi. In this latter case, this method is used as a standard microbial assay. This is because there is no need for microbial isolation and growth. In vitro growth and characterization is currently underway at Mycobacteriupra.
n leprae) (pathogen of Ley's disease) is not possible and some microorganisms such as essential intracellular bacteria (eg rickettcher, gramimidia, etc.) are not possible on standard medium Or very dangerous, if not impossible (eg B. anthracis (pathogen of anthrax). This method is based on the isolation of nucleic acids, which is based on conventional bacterial isolation and characterization. It is possible to eliminate these problems, since this method is able to detect microorganisms not previously formally described. This is useful for epidemiological typing in, for example, bacteriology This method can be used in forensic laboratories to correctly and unambiguously identify plant or animal tissue in criminal investigations. If you ascertain the nature of the damage, also used for entomologist to identify on whether the fast species of insects.

Furthermore, this method is applied to taxa other than subspecies (eg, plant root nitrogen enzyme gene; see: Hennecke H291 “Nature” 354 (198
Combined with the identification of 1)), this methodology can be used to investigate and identify genotypes of individual strains.

The method of the invention is preferably used for identification of microorganisms wherever they are found. These microorganisms will be found in physiological as well as non-physiological substances. They will be found in industrial growth media, culture broth, etc. and will be concentrated, for example by centrifugation. It is preferred that the microorganism be found in physiological media, most preferably it is found in the infected animal source. In this latter embodiment, the method is used to diagnose bacterial infections in animals, especially humans.
The detection and identification of bacterial DNA with pronuclear ribosome probes is highly selective and can be performed without hindrance even in the presence of animal (eg mammalian) DNA. If a pronuclear ribosome probe is used, one can choose conditions that minimize hybridization to the glomerular DNA or subtract the glomerular bands from the pattern. In this way, this technique can be used in clinical laboratories, fungal depositary institutions, industrial fermentation laboratories and the like.

Of particular interest, in addition to identifying the species and strain of the infecting organism, is the possibility of discovering the presence of some unusual gene sequence in the organism. For example, the presence of antibiotic resistance sequences found on transmissible plasmid R factors that mediate drug resistance can be detected. By adding labeled factor R DNA or clone-labeled antibiotic resistance sequences to the hybridisation mixture, one can correctly determine whether or not the organism is resistant to antibiotics (one or more non-normal bands appear). Alternatively, the filter paper once hybridized in the presence of the added antibiotic resistance sequence probe (one or several kinds) may be rehybridized. Alternatively, divide the unknown DNA into aliquots and use the first aliquot for identification, the second aliquot for the presence or absence of the drug resistance sequence, and the third aliquot for the toxin gene. You can also test. Alternatively, one radionuclide (eg ³²
A ribosomal probe labeled with P) could also be used in the hybridization mixture with the addition of an R-factor probe labeled with another radionuclide (eg ³ H or ¹⁴ C). After hybridization, the presence of R-factor DNA in the unknown DNA can be tested by scanning with two scanners. One is for species and strain identification (eg ³² P), the other for drug resistance etc. (eg ³ H or ¹⁴ C). In this way, experimenters can identify genera and species, type strains, detect drug resistance, toxic production or other characteristics, or labeled nucleic acid sequences or probes without having to isolate and characterize the microorganism. All tests for sub-species taxa can be done in one experiment.

Since the R-factor is universal and crosses species boundaries,
Identification can be done with the same R-factor probe in any bacterial genus or species (see: Tomkins, LSeta.
lJInf.Dis., 141: 625-636 (1981)).

Furthermore, the presence of viruses or viral-related sequences in eukaryotic or prokaryotic cells can also be detected and identified by the method of the invention. "Manual of Clinical
Microbiology "Third Edition (Published by Lennette, EH, Amer Soc
All viruses listed in Microb 1980, 774-778) can be identified. For example, picornaviridae, caliciviri
dae), reoviridae, togaviride (t
ogaviridae), orthomyxovirid
ae), Paramaikoviride (paramyxoviridae), Rhabdoviride (rhabdoviridae), Retroviride (retro)
viridae), arenaviride (arenaviridae), coronaviride (coronaviridae), buniaviride (bunyavi)
ridae), pavoviride (parvoviridae), papovaviride (papovaviridae), adenoviride (adenoviri)
dae), herpesviridae, vidoviridae and poxviri
dae) etc.

1) When the viral genome is integrated into the host DNA (DNA viruses such as papovaviride member, R
NA viruses, such as retroviride members, extract high molecular weight DNA from tissues and digest with restriction enzymes. The overall procedure is similar to that for bacteria. The choice of viral probe will again depend on the task sought and will depend on the degree of homology between the "probe virus" and the viral related sequence to be detected. To obtain convenient sequence homology, it will be necessary for the probe and tissue sequences to be related to the same family or species of Weels. In addition to the extent of conserved sequences, the Willes probe may or may not hybridize to the Wills related sequences of the host DNA, depending on the conditions of hybridization, for example, stringent or relaxed conditions. . The result of the hybridization will be a single band or band pattern indicating the presence of viral sequences integrated into the host DNA. This information helps predict carcinogenesis. Any of the labeled complementary nucleic acid probes containing the cloned viral sequences can be used as the probe. In the case of RNA viruses, for example, viral RNA can be used to produce DNA with reverse transcriptase, and in the case of DNA viruses, for example, viral DNA labeled by cut translation can be used. Again, multiple probes can be used, especially those with different labels.

The same general characteristics apply equally to DNA and RNA viruses. The viruses genome is relatively small and the precipitated nucleic acids are preferably collected by centrifugation. All operations may be performed using all nucleic acids, or various operations may be performed separately. It is thought that the virus nucleic acid can be concentrated by removing cellular DNA by spooling on the day before centrifugation. It can also be used to determine if the Wiles genome has been integrated.

To hybridize the Wheels probe,
It is necessary, and at least most preferred that the probe is of the same family, genus or species as the unknown organism. The reaction conditions, stringent or relaxing, determine whether a given probe will hybridize to a less related genome. The probe may be a labeled cloned viral sequence, the complete genome or a part thereof.

The method described in Southern, supra, is useful for transferring large DNA fragments (about 0.5 kilopace or more) to nitrocellulose paper after alkaline denaturation. This method is useful for DNA viruses and may not be useful for RNA viruses. RNA can be transferred to activated cellulose paper (diazobenzyloxymethyl paper) and covalently attached and used for RNA viruses. Thomas's modification of the Southern method (Thomas, P., "Proc. Nat. Acad. Sci" USA 77: 5201-520.
5 (1980)) can be used to efficiently transfer RNA and small DNA fragments to nitrocellulose paper for hybridization. RNA and small DNA fragments are denatured with glyoxal and dimethylsulfoxide and electrophoresed in an agarose gel. With this operation, 100 ~ 2
DNA fragments that are 000 nucleotides, and RNA migrate efficiently and remain on nitrocellulose paper during hybridization. It is also useful for small ribosomal DNA fragments. Therefore, it is most preferable to divide a sample digested with a restriction enzyme and denature part of the nucleic acid with glyoxal. Southern and Thomas maneuvers will give the greatest amount of information.

2) In the case of DNA viruses, double-stranded (DS) viruses DNA
The existing viruses can be identified by performing a restriction analysis with. Single-stranded (SS) DNA viruses will have different length genomes. A probe forming a hybrid (sequence information can be converted into DS-DNA),
Pattern of hybridized fragments and / or 1
Alternatively, multiple sizes can be used to identify viruses. Again, there are numerous ways to obtain complementary nucleic acid probes. For example, in the case of DS-DNA, truncated translation can be used, and in the case of SS-DNA, DNA polymerase
Used for cDNA synthesis.

3) In the case of RNA viruses, RNA is not digested by restriction endonucleases (sequence information may have been converted to DS-DNA). The genomes of different RNA viruses have different sizes, one in some RNA viruses.
There are more molecules than molecules. As a result, RNA viruses can be identified along the base sequence detected by a certain probe or a coalescing probe. One example of a probe is cDNA synthesized using viral RNA.

When looking for infectious pathogens in a sample, extract the nucleic acid from the sample, or increase the number of pathogens by first culturing in a medium or cells to increase the number of pathogens, or use a concentration process such as centrifugation. You can search directly by trying the approach of.

From the present invention, it is easy to make a "kit" containing the necessary elements to carry out the method. Such a kit would consist of a carrier which is partitioned to tightly pack one or more containers, such as test tubes or vials, therein. One of the containers is a non-labeled nucleic acid probe or a detectably labeled nucleic acid probe, such as radiolabeled cDNA for ribosomal RNA from an organism probe (in the case of a kit for identifying bacteria, (Preferably a nuclear rRNA cDNA). The labeled nucleic acid probe will be in lyophilized form or, if necessary, in a suitable buffer. 1
Or more containers will contain one or more endonuclease enzymes utilized in the digestion of DNA from unknown organisms. The enzymes are present alone or as a mixture in lyophilized form or in a suitable buffer solution. Ideally, the enzymes used in the kit are those for which catalogs are prepared. But nothing prevents users from creating their own comparison standard during experimentation, so if a user suspects that some unknown is actually of a given genus or species. For example, the person can create a known swath pattern and compare it to the unknown swath pattern. Thus, the kit may include all the elements necessary to carry out this subprocess. These elements include one or more known organisms (such as bacteria) or DNA isolated from known organisms. In addition, the kit is
Broadly include booklets, or "catalogs" or computer tapes or discs, or computer access numbers, etc., defined as books or pamphlets, which include plant species, mammalian species, bacterial species, particularly pathologically important bacteria. , A group of various organisms, such as insect species, etc. In this way, the user need only prepare a banding pattern of unknown organisms and compare this visually (or computer) with the pattern in the catalog. The kit may also include probe rRNA for probe synthesis in one container, radiolabeled deoxyribonucleoside triphosphate in another container, and a primer in another container. good. In this way, users can create their own probe rRNA cDNA.

Finally, the kit consists of buffer, growth medium, enzyme, pipette, plate, nucleic acid, nucleoside triphosphate, filter paper,
It may include all of the additional elements necessary to practice the techniques of the present invention, such as gel raw materials, transfer materials, autoradiographic supplements. It may also include antibiotic resistance sequence probes, viral probes or probes with other specific properties.

Although the present invention has been generally described above, it may be better understood by reference to certain embodiments. It should be noted that the examples are described merely for the purpose of explanation, and are not intended to limit the present invention unless otherwise specified.

Materials and Methods A. Bacteria High molecular weight DNA extraction Bacterial broth cultures were spun down and cells were washed with cold saline.
About the gram weight of the packed cells
10 times volume of extraction buffer (0.15M sodium chloride, 0.1
It was suspended in M EDTA, 0.03 M Tris pH 8.5). Lysozyme
10 mg / ml was added to give a final concentration of 0.5 mg / ml. Suspension
The liquid was incubated at 37 ° C for 30 minutes. 25 cell destruction
Add% SDS to a final concentration of 2.5% and adjust the concentration for 10 minutes.
This was done by raising the temperature to 60 ° C. Cooling in water bath
Then, add mercaptoethanol to a final concentration of 1%.
added. Pronase 20 mg / ml 0.02 M Tris buffer (pH 7.
4) was pre-digested at 37 ° C for 2 hours, then to a final concentration of 1 mg / m
I added it to be l. Ink the solution at 37 ℃ for 18 hours
Was added. Phenol is redistilled phenol 1
, 2.5 l of double distilled water, 270 ml of saturated Tris base, Merca
12 ml of ethanol and final concentration of 10^-3Amount to be M
Mixing EDTA and allowing the mixture to separate at 4 ° C
Was prepared by. Wash the phenol with the washing buffer (10^-1
M sodium chloride, 10^-3M EDTA, 10 mM Tris pH8.5)
washed. Then an equal volume of fresh buffer was added. Mercap
Ethanol was added to a final concentration of 0.1%. Melting
The liquids were mixed and stored at 4 ° C. Half volume of prepared phenol
The volume and half volume of chloroform were added to the lysed cell solution. this
Was shaken for about 10 minutes and spun down at 3400 xg for 15 minutes. Water phase
Was removed with a 25 mg glass pipette. Almost settled on the border
This extraction operation was repeated until nothing was left. 2 of 1/9 capacity
N sodium acetate (pH 5.5) was added to the aqueous phase. Double capacity
-20 ℃ 95% Ethyl alcohol along the wall of the flask
I poured it slowly. Melt the tip of the Pasteur pipette
Closed and used to spool the precipitated DNA. High molecular
DNA was dissolved in buffer (10^-3M EDTA, 10^-2M bird
PH 7.4). As for the DNA concentration, the conversion factor is 1 unit of absorbance.
About 30 μg, and measured by absorbance at 260 nm
did.

Restriction endonuclease digestion of DNA The EcoR I restriction endonuclease reaction is 0.1M Tris-
HCl pH 7.5, 0.05M NaCl, 0.005M MgCl ₂ , and 100 μg /
performed in ml calf serum albumin. The EcoR I reaction mixture contained 5 units of enzyme per DNA / μg, which was
Incubated at 4 ° C for 4 hours. The PST I restriction endonuclease reaction was performed in 0.006M Tris-HCl pH7.4, 0.05M sodium chloride, 0.006M magnesium chloride, 0.006M2-mercaptoethanol, and 100 μg / ml bovine serum albumin. PST I reaction mixture is 2 per DNA / μg
Units of enzyme were included and incubated at 37 ° C for 4 hours. Usually, 10 μg of DNA was digested to a final volume of 40 μl. A 10 × buffer was added. Sterile distilled water was added according to the amount of DNA dissolved. λ-Bacteriophage DNA was restricted with EcoR I to create a marker band for fragment size determination. Usually 2 μg λ DNA was digested with 20 units of EcoR I to a final volume of 20 μl.

Gel Electrophoresis and DNA Transfer DNA digests were spiked with up to about 20% glycerol and bromophenol blue dye. For λ DNA digests, 20 μl of 1 × EcoRI buffer was added to each 20 μl reaction mixture. Normally, 15 μl of 75% glycerol and 5 μl of 0.5% bromophenol dye were added to each 40 μl reaction mixture.

 10 μg of digested bacterial DNA and 2 μg of digested λDNA
With agarose melted in a per well
Cover the surface. Digested material 0.02M sodium acetate, 0.002M
EDTA, 0.018M Tris base, and 0.028M Tris HCl pH 8.
Pigment swims 13 to 16 cm at 35 V in 0.8% agarose containing 05
Electrophoresis was performed until it moved. Then the gel
Immerse in Umpromide (0.005mg / ml) and see the λ fragment
I put it in a UV light box to make it look like. DNA above Southern
It was transferred to nitrocellulose filter paper by the method described in 1. Gel vibrates
Denaturing solution (1.5M sodium chloride, 0.5M water for 20 minutes on the table
Sodium oxide). Add the denaturing solution to the neutralizing solution (3.
Placed in 0M sodium chloride, 0.5M Tris HCl pH7.5)
Well, 40 minutes later, the gel was checked with pH paper. After neutralization,
The gel was washed with 6 × SSC buffer (SSC = 0.15M sodium chloride, 0.0
15 M sodium citrate) for 10 minutes. A gel,
Through nitrocellulose paper, pass 6 × SSC onto paper tao
DNA fragments from the gel by aspirating for 15 hours with a bundle of
Transferred to nitrocellulose paper. 3mm chromatograph paper 2
Put a filter between the sheets, aluminum wheel, shining
Wrap side up and dry in a vacuum oven at 80 ° C for 4 hours
did.³² Synthesis of P ribosomal RNA complementary DNA (³²P-rRNA cDNA) Complementary to E. coli R-13 23S and 16S type ribosomal RNA
Typical³²Avian myeloblastosis virus (AMV)
Was synthesized using the reverse transcriptase from The reaction mixture is 5
μl 0.2M dithiothreitol, 25 μl 1M Tris pH 8.
0, 8.3μl 3M potassium chloride, 40μl 0.1M magnesium chloride
Sium, 70 μl actinomycin, 14 μl 0.04M dA
TP, 14 μl 0.04M dGDP, 14 μl 0.04M dTTP, and
It contained 96.7 μl of water. Made of plastic
Added to tubes: 137.5 μl reaction mix, 15 μl pups
Beef thoracic primer (10 mg / ml), 7 μl H20, 3 μl
rRNA (2.76 μg / μl using 40 μg / OD unit concentration)
, 40 μl of deoxycytidine 5 ′-(³²P) Triphosphoric acid
(10 mCi / ml), and 13 μl of AMV polymerase (6,900
Unit / μl). The fermentation reaction was carried out at 37 ° C for 1.5 hours. So
After that, add the solution to each 5 ml of chloroform and preparative solution.
It was extracted with enol. After centrifugation (JS13,600RPM), water
Phase Direct Sephadex G-50 column (1.5 x 22 cm)
Layered on top. Column with a plastic 10 ml pipette
Used as. Place a small glass bead on the tip,
Rubber tube with pinch fittings is attached and 0.05% SDS overnight
The degassed G-50 which had been swollen by immersing in water was added. Water phase along the wall
Directly into G-50 and then eluted with 0.05% SDS.
Was. 20 fractions of 0.5 ml each with a plastic
Collected in Al. The tube containing the peak fraction is
³Count for 0.1 minutes for each sample using the H-discriminator, and
Discovered by the Serenkov method of recording counts. Pee
The fractions were united. Aliquot of aquezole
In addition to (available commercially),³²P CPM (per minute
Was measured by a scintillation counter.

Hybridization and Autoradiography Fragments containing ribosomal RNA gene sequences were detected by autoradiography, in which the DNA on the filter was hybridized to ³² P-rRNA cDNA.

The filter was mixed with a hybridized mixture (3 x SSC, 0.1
The samples were soaked in 68% SDS, 100 μg / ml denatured and sonicated dog DNA and Dynehardt's solution (0.2% calf serum albumin, Ficoll, and polyvinylpyrrolidine, respectively) at 68 ° C. for 1 hour. ³² PrRNA cDNA was added at a rate of 4 × 10 ⁶ CPM / ml, and the hybridization reaction solution was added to 68
Incubated at 48 ° C for 48 hours. The filters were then washed with 3 × SSC, then 0.1% SDS at 15 minute intervals for 2 hours or until the cleaning solution contained approximately 3,000 cpm ³² P / ml. The filter was air dried, wrapped in plastic wrap and autoradiographed with Kodak X-OMATR film at 70 ° C for about 1 hour.

B. Mammal experiment rR of Mus musculus domesticus (mouse)
NA probes were synthesized from 18S and 28S and 28S only rRNA. Nucleic acid was extracted from mouse liver and precipitated. High molecular weight DNA was spooled and removed. The remaining nucleic acid was collected by centrifugation and dissolved in a buffer containing 50 mM MgCl ₂ and 100 mM Tris pH 7.4. Concentration of DNAse (not including RNAse) 50 μg /
Add to ml. The mixture was incubated at 37 ° C for 30 minutes. The generated RNA was re-extracted, ethanol-precipitated, and dissolved in 1 mM sodium phosphate buffer pH 6.8. A 5-20% sucrose gradient solution of 0.1 M Tris pH 7.4 and 0.01 M EDTA was prepared. A sample was added and the gradient was spun at 35K rpm for 7 hours on a SW40 rotor. Fractions were collected by optical density. By comparison with known molecular weight markers
The 18S and 28S fractions were chosen.

Relaxed hybridization conditions were used in all mammalian experiments. 54 ° C. The washing treatment was performed at 54 ° C., 0.05% SDS was added, and the mixture was washed with 3 × SSC for 15 minutes three times separately.

Example 1 Bacterial species are defined by restriction endonuclease analysis of the ribosomal RNA gene.

Several strains of P. aeruginosa used in this example have minimal phenotypic characteristics that identify the species (Hugh R.
H., et al. "Manual of Clinical Microbiology" 2nd
Ed., ASM, 1974, pp, 250-269) (Table 2). The other three Pseudomonas and two Acinetobac
ter) and compared species and genera (3rd
table).

The strains used for comparison of Pseudomonas and Acinetobacter species are listed in Table 3.

The Acinetobacter species were chosen for genus comparison because they share certain attributes with many Pseudomonas species.

The size of the fragments in the EcoR I digest (kilobase pair) is P.
P. stutzeri 16.0, 12.0, 9.4; P. fluorescens 16.0, 10.0, 8.6, 7.8,
7.0; P. putida 24.0, 15.0, 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 (the size of the smallest three fragments was not calculated); A. lwoffii 12.
0, 10.0, 9.1, 7.0, 6.4, 5.7, 5.5, 5.3, 4.8, 4.4,
3.6, 3.2, 2.9 (the size of the smallest 3 fragments was not calculated). The size of the fragments in the PST I digest (kilobase pair) is shown below; P. stowzeri 6.7, 6.1, 5.5;
P. Fluorescence 10.0, 9.4, 7.8, 7.0; P. Petida 10.
5, 9.9, 6.8, 6.3, 4.4; A. Anitratus 36.0, 28.0, 2
0.5, 12.0, 10.0, 5.8, 3.7, 2.6, 2.4; A. Luoffi 9.
9, 8.7, 7.2, 5.7, 4.0, 3.6, 3.2, 2.7.

Comparing the hybridized restriction fragments obtained from the seven strains of P. aeruginosa, this species shows 10.1, 9.
We come to the conclusion that 4,7.6 and 5.9 kilobase pairs (KBP) are defined by the EcoR I-specific combination of fragments containing the rRNA gene sequence (FIG. 1).

The 7.6KBP EcoR I fragment appears in 4 out of 7 strains in this sample. A similar situation occurs in some phenotypic traits of species strains. The fact that the EcoR I combination of fragments from 7 strains is used to divide the strains into 2 groups has the two phenotypic characteristics of P. aeruginosa.
Draws the inference that there are two species. From the results of the experiment in which DNA was digested with PST I (Fig. 2), it is concluded that the mutation of the strain indicated by the EcoR I7.6KBP fragment represents an intraspecific mutation. Because there is one conserved combination of PST I fragments, 9.4, 7.1, 6.6 and 6.4KBP, which defines its species. 9.4 and 6.6 KBP PST
The I fragment appears in 6 of the 7 strains of P. aeruginosa; the 7.1 and 6.4 KBP PST I fragments appear in all of the sampled strains. Mutation of PST I fragment is EcoR I7.6KB
Occurs on strains without P fragment; RH151 10.1 and 8.2KB
It has P fragment, RH809 does not contain 9.4KBP fragment, 6.0KBP
Have fragments. And the type strain RH815 is 6.6KBP
Does not include fragments. The pattern of hybridized fragments supports the conclusion that enzyme-specific conservative combinations can be used for species determination. Strains of one species probably have multiple fragments as conserved combinations. Fragment mutations in some strains do not interfere with identification and prove useful in epidemiological studies.

Mutagenesis of the EcoR I7.6 KBP fragment in P. aeruginosa strains may be considered prospectively by examining the hybridizing EcoR I fragment found in other Pseudomonas sp. Type strains (Figure 3). Type strains of P. stowelli, P. fluorescens and P. putida do not contain the 7.6 KBP fragment, but share an EcoR I fragment of the same size; P. aeruginosa and P. stoweeri have Having P. Stowelli and P.
Fluorescence has a 16KBP fragment, P. Fluorescence and P. putida carry a 10KBP fragment. In general, the fragment size is unique to each of the four Pseudomonas species type strains; and each species type strain has different size range fragments. These general explanations also apply to PST I digests (4th
Figure).

When comparing the respective fragment patterns of the four Pseudomonas species and the two Acinetobacter species or one strain thereof, it can be concluded that the species of each genus are similar, but the comrades are different. The two Acinetobacter species have a larger range of hybridizing fragment sizes than the four Pseudomonas species.

E. coli, Bacillus thuringiensis
ingiensis) and B. subtilis, without the help of restriction maps, where the enzyme cuts the rRNA gene, the number of copies per genome and the number of multiple genes or heterogeneous genes. It is impossible to predict whether or not there are heterologous flanking regions (fianking regions). E. coli rRNA cDNA probe is rRNA
It may not hybridize with some restriction fragments containing the gene sequence, and if so, this reflects an evolutionary distance or variance between the test organism and E. coli. The conservative nature of rRNA can be used to argue that this is not the case. However, this is a minor problem compared to the advantage of having a standard probe that is equally applicable to any unknown species.

Example 2 Restriction analysis compared to DNA-DNA liquid hybridization: The strains used in this study are listed in Tables 4 and 5.

High molecular weight DNA was isolated from each strain. Liquid DNA-DNA hybridization data were collected using RH3021 and RH2990 labeled DNA. The results are shown in Table 6.

This data indicates that there are two hybridized groups. Similar data were reported in the literature for B. subtilis (Seki et al, "International Journal of
Systematic Bacteriology "25: 258-270 (1975). These two groups can be represented by RH3021 and RH2990. A restriction endonuclease analysis of the ribosomal RNA gene is performed. The EcoR I digest (Fig. 5) could be divided into two groups. The herd represented by RH3021 has two strongly hybridizing fragments (2.15 and 2.1KBP). The group represented by RH2990 has two strongly hybridizing fragments (2.6 and 2.5KBP). The EcoR I data was used to DNA-DNA the B. subtilis strain.
It can be placed in any suitable place in the hybridisation group. DNA
-According to the 70% rule for DNA hybridization, B. subtilis is actually two species. However, considering the PST I data (Fig. 6), those groups can be considered as two dispersed populations involved in common ancestral or speciation events. The conclusion that B. subtilis is a species correlates with phenotypic data. The strains listed in Table 5 are the strains of "The Genus Bacil" by Gordon RE et al.
lus ”Agriculture Handbook No.427 (US Department of Agriculture,
Agricultural Research Service Washington DC) Identified as B. subtilis in p36-41. Restriction analysis can provide data comparable to DNA-DNA hybridization data, and by selecting the appropriate enzyme, restriction analysis can be well defined despite variance.
RH3061 lost PST I site. However, EcoRI data suggests that the strain is B. subtilis. The same can be concluded from the Bgl II data (Fig. 7) and Sac I data (Fig. 8).

Example 3 Stability patterns of restriction analysis and other Bacillus polymyxa experiments B. subtilis and B. polymica can be distinguished by EcoR I data (Fig. 9), PST I data (Fig. 10), Bal II data (Fig. 11 left) and Sac I data (Fig. 11 right). it can. From the large difference in the PST I band pattern, it can be concluded that Bacillus polymica is in the wrong genus. Both species produce spores, but they are phenotypically dissimilar. ATCC and NRRL
It is certain that the B. polymica type strains from both culture collections have the same banding pattern. However, the important data is that asporic mutants can be identified. If they were unable to sporulate, identifying Bacillus species would be very difficult and probably impossible.

Example 4 Identification of bacterial species in mouse tissues without isolation Swiss mice, Mus, Mus musculs domesticus (inbred strain) to Streptococcus pneumonia
e) 0.5 ml of a turbid suspension of RH3077 (ATCC6303) was intraperitoneally inoculated. When the mice were moribund, the heart, lungs and liver were removed. High molecular weight DNA was isolated from these tissues, S. pneumoniae RH3077 and Swiss mouse organs, and engineered for restriction endonuclease analysis of the rRNA gene with EcoR I to digest the DNA. Filter 3 × SS
Besides washing with C, it was washed with 0.3 × SSC and 0.05% SDS for 2 × 15 minutes. Autoradiography was performed for 48 hours. The data (Figure 12) showed that S. pneumoniae was defined by seven hybridizing fragments (17.0, 8.0,
6.0, 4.0, 3.3, 2.6 and 1.8KBP). This bacterial cDNA probe does not hybridize well with the two mouse DNA fragments (14.0 and 6.8KBP). The hybridizing fragment signals the presence of S. pneumoniae in the infected tissue. All seven bands are found in the heart DNA extract. Although less intense in liver DNA extracts,
All can be seen by autoradiography. Only 6.0KBP band appears in lung DNA extract. The low number of bacteria in the lungs may be explained by the fact that the mice have sepsis rather than pneumonia. The lungs showed no solidification at necropsy. To examine the sensitivity of this assay, bacterial DNA was diluted with mouse DNA and electrophoresed. With 0.1 μg bacterial DNA, all seven bands were visible on the autoradiograph. 10 ^-3 μg bacteria
In DNA, 17.0, 8.0 and 6.0 KBP bands were found. 10 ⁶ S.
Using the number of 5 × 10 ⁻³ μg DNA per P. pneumoniae (Biochem Biophys Acta 26:68), 10 ⁻¹ μg corresponds to 2 × 10 ⁷ cells. This method is useful for diagnosing infectious diseases at this sensitivity level.

This example also shows that the bacterial probe is a mouse-specific EcoR
It is shown to hybridize with the I fragment (see FIG. 9, fragments with 14.0 and 6.8 KBP). These fragments correspond to the EcoR I fragments detected by mouse 18S and 28S ribosomal RNA probes (Fig. 14, above, 6.8K).
It is shown that the BP fragment contains 28S type rRNA sequence). The bacterial probe does not hybridize well with the mammalian ribosomal RNA gene sequences, so the bands are less intense,
Bacterial probe and nuclear mammalian RNA systems are less sensitive and show a clear selectivity for infected prokaryotic DNA. 10 bacterial probes per lane
In an experiment hybridized to μg digested bacterial DNA, no hybridization was found to 10 μg digested human or mouse DNA per lane when the bacterial bands were clearly visible.

Examples 5-8 Mammalian Experiments These examples demonstrate that the concept of confirming organisms by rRNA restriction analysis has been successfully applied to complex eukaryotic organisms as well as bacteria. is there.

Figure 13 shows that the genera of mammals can be confirmed by Mus Mus musculus domesticus 18S and 28S rRNA probes,
And some species of Muss are distinguishable. In this figure, the enzyme is PST I and the objects and their respective bands are as follows:

1. Mus musculus m
elossinus) (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 below 3.0 bands 5. Cricetulus griseus
(Hamster) 25.0, 4.7 6. Homo sapiens (Human) 15.0, 5.
7 7. Felis catus (cat) 20.0, 9.7 8. Ratus norvegicus (rat) 12.5 9. Mus musculus domesticus (mouse) 13.
5, 2.6 10. Muscervicolor
cervicolor) (mouse) 14.0, 2.7 11. Muscervicolor pape
eus (mouse) 13.5, 2.6 12. Mus. Mus pahari (mouse) 13.0, 3.7 13. Mus cookii (mouse) 13.5,
2.6 Figure 14 shows that mouse and cat DNA can be distinguished only by 28S type rRNA cDNA, and that the pattern of hybridized bands depends on the composition of the probe sequence.
In Figure 14, the enzyme is EcoR I and the objects and bands are as follows.

1. Mus musculus domesticus (mouse) 6.8K
BP 2. Feliz catus (cat) 8.3KBP In Figure 15, the enzyme is Sac I and the target and band are as follows.

1. Erythrocebus patas (Patus 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. Ferris Catus (cat) 9.5, 5.3, 4.0, <3.0, <3.
0 5. Homo sapiens (human) 10.5, <3.0 6. Macaca mulatta (Laser monkey)
9.8, <3.0 Comparing Figure 15 (Sac I digestion) to that of other mammals, it can be seen that the hybridization pattern is enzyme specific.

Figure 16 shows that primates animals are distinguishable. Cultured cells have a band in common with the tissue of their original species, and different human cell cultures can be distinguished by the addition or lack of bands. In this figure, the enzyme is EcoR I and the objects and bands are:

1. Erythrocebas Patas (Patus monkey)> 22.0, 11.0,
7.6, 2.6 2. Macaca Murata (Laser monkey) 22.0, 11.5, 7.6 3. Homo sapiens (human)> 22.0, 22.0, 16.0, 8.
1,6.6 4.M241 / 88 (Langer monkey cultured cells) 14.0, 7.2, 5.7 5.HeLa (human cultured cells)> 8.1, 6.6 6.J96 (human cultured cells)> 22.0, 22.0, 16.0, 11.0, 8.
1, 6.6 7. AO (human cultured cells) 22.0, 16.0, 8.1, 6.6 8.X-381 (Laser monkey) 22.0, 11.5, 7.6 Now that the present invention has been sufficiently described, those skilled in the art can understand the present invention or Obviously, many modifications and substitutions can be made over a wide range without distorting the principle or purpose of the embodiment.

[Brief description of drawings]

Figure 1 shows Pseudomonas aeruginosa.
S. aeruginosa) DNA isolated from an EcoR I restriction endonuclease digestion. E. coli as a probe
CDNA for 16S and 23S ribosomal RNA (rRNA)
Was used. Figure 2 shows P. aerugino.
sa) shows Pst I restriction endonuclease digestion of DNA isolated from the strain. E. coli 16S and 23S as probes
The cDNA for the type rRNA was used. FIG. 3 shows EcoR I restriction endonuclease digestion of DNA isolated from glucose-nonfermentative Gram-negative rod species. CDNAs for Escherichia coli 16S type and 23S type rRNA were used as probes. FIG. 4 shows Pst I restriction endonuclease digestion of DNA isolated from glucose-nonfermentative Gram-negative rod species. CDNAs for Escherichia coli 16S type and 23S type rRNA were used as probes. Figure 5 shows various Bacillus subtilis.
lis) shows EcoR I restriction endonuclease digestion of DNA isolated from the strain. E. coli 16S and 2 as probes
The cDNA for 3S rRNA was used. FIG. 6 shows Pst I data obtained by using the same probe for the same strain as in FIG. FIG. 7 shows Bgl II data obtained with the same probe for the same strains as FIGS. 5 and 6. FIG. 8 shows Sac I data obtained using the same probe for the same strains as in FIG. 5-7. Figure 9 shows B. subtilis and B. polymyxa
3 shows EcoRI restriction endonuclease digestion of DNA isolated from. 16S and 23S from E. coli as probes
The cDNA for rRNA was used. FIG. 10 shows Pst I data obtained using the same probe for the same strain as in FIG. FIG. 11 shows Bgl II and Sac I data obtained with the same probe against the same strains as FIGS. 9 and 10. Fig. 12 shows 16S type and 23S type from E. coli as probes
EcoR I from infected mouse tissues using cDNA for rRNA
Streptococcus pneumoniae (Strep
tococcus pneumoniae) is shown. Figure 13 shows Mus musculus domesticus (Mus mu
Figure 3 shows the identification of mouse species by comparing Pst I digestion of DNA isolated from mammalian tissue using cDNA for 18S and 28S rRNA from cytoplasmic ribosomes of sculus domesticus) (mouse). Figure 14 shows Mus musculus domesticus 28S type rRN.
Shows EcoR I digested DNA from mouse and cat tissues that hybridized with the AcDNA probe. FIG. 15 shows Sac I digested DNA from mammalian tissue hybridized with Mus musculus domesticus 18S and 28S rRNA cDNA probes. FIG. 16 shows EcoR I digested DNA from mammalian tissue and cell culture hybridized with Mus musculus domesticus 18S and 28S rRNA cDNA probes.

Continuation of front page (56) References Molec. gen. Genet. 179, p. 539-545 (1980) Molec. gen. Genet. 180, p. 475-477 (1980) Molec. gen. Genet. 182, p. 502-504 (1981) Int. J. Syst. Bacteri ol. 28 (2), P. 154-168 (1978) Int. J. Syst. Bacteri ol. 30 (1), P. 7-27 (1980) Int. J. Syst. Bacteri ol. 30 (1), P. 106-122 (1980) Int. J. Syst. Bacteri ol. 30 (3), P. 547-556 (1980) Cytogenet. Cell. Gen et. 19, P.I. 256 ~ 261 (1977)

Claims (8)

[Claims] 1. A faithful copy of ribosomal RNA (rRNA), detectably labeled and in substantially pure form, useful in hybridization reactions for the detection or identification of pronuclear organisms. DNA (rRNAcDNA) that is complementary to pronuclear ribosomal RNA. 2. The rRNA cDNA according to claim 1, which is radiolabeled or metal-labeled. [Claim 3] The first nuclear rRNA is bacterial rRNA.
Item rRNA cDNA. 4. The rRNA cDNA according to claim 1, which is substantially free of cDNA complementary to RNA other than rRNA. 5. The first claim, which is substantially free of cellular components.
Item rRNA cDNA. 6. The rRNA cDNA according to claim 1, which is obtained by reverse transcription of rRNA in the presence of a primer. 7. The rRNA cDNA according to claim 6, obtained by using a DNA hydrolyzate primer. 8. The rRNA of claim 6 obtained by reverse transcription with reverse transcription oxygen from avian myeloblastosis virus.
cDNA.