JP2514164B2 - Method for identifying and characterizing organisms - Google Patents

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION This invention relates to a method for rapidly and accurately characterizing and identifying organisms, including prokaryotic and eukaryotic organisms such as bacteria, plants and animals.

[0002]

Background of the Invention 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 kingdoms: plants (Plantae) and animals (Animalia). This classification is convenient for generally known organisms, but for organisms such as unicellular organisms (eg, flagellates, bacteria,
It is difficult for blue-green algae. This is because these are basically different from "plants" and "animals".

It has been proposed to simply divide an organism by the internal structure of its bacterium. In this way, all cellular organisms are prokaryotic or eukaryotic.
Or either. Prokaryotes are less complex than eukaryotes, they lack internal compartments by unit membrane organization and lack a clear nucleus.
Genetic information of prokaryotic cells is carried in the cytoplasm by double-stranded circular DNA. No other DNA is present in the cell (unless there are phage, bacterial viruses, and circular DNA plasmids capable of autonomous replication). 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 well-partitioned nuclei, as well as 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 nuclear cytoplasmic part, the glomerulus and the chloroplast.

However, the difference between prokaryotic and eukaryotic cells is disrupted when prokaryotic cells are compared for glomeruli and chloroplasts. These organelles are now considered to be derived from free-living prokaryotes, which enter into endosymiotic relationships with primitive eukaryotes. , Ultimately integrated with the structure of the host cell, making it impossible to be independent (see eg, Fox, GE et al. “Scienc.
e ''209; 457-463 (1980) p. 462; Stanier, RY et al. `` Th
e Microbial World "4th edition, published by Prentice-Hall,
1976, p.86). For example, DNA obtained from mouse L cell glomeruli carrying the ribosomal RNA gene region is Escherichia coli.
(Coli) ribosomal RNA has been shown to have significant sequence homology, strongly supporting the endosymbiotic model (Van Ett
en, RA et al, "Cell" 22: 157-170 (1980)). The nucleotide sequence of the 23S ribosomal DNA of corn (Zea mays) chloroplast is similar to that of Escherichia coli 23S ribosomal DNA.
It has also been shown to have 71% homology (Edwards, K.
& Kossel, H., "Nucleic Acids Research" 9: 2853-286
9 (1981)); Other related work (Bonen, L. & Gray, M.
W., 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 purposes of crossing and breeding, and so-called "higher" organisms or other This is the case when trying to accurately and reliably identify microorganisms that may infect the medium. 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, doctors or horticulturists should consider infectious organisms (parasites, fungi,
Bacteria) and Wills will be accurately identified. Correct identification of these organisms and the species of Weels is of particular importance.

This problem is best understood by explaining 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. Of bacterial species,
Expressing an accurate definition is
Difficult to define boundaries for strain diversity within a species (Buchanan, RE, International Bulletin
etin of Bacteriological Nomenclature and Taxonom
y ”, 15: 25-32 (1965)). The practical application of the species definition to the identification of unknown bacterial strains involves 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 Determinant Bacteriology (Buchanan, REand Gibbon
s, edited by NE, 1974, 8th edition, Willians & Wilkins
(Baltimore, Inc.) provides 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) “M
anual of Clinical Microbiology ", 3rd edition, American Society for Microbiology, Washington DC, 1980, pp. 1-6).

When used in bacteria, the term "species" generally resembles one another as distinct species of organic matter, with distinctive characteristics, and in terms of essential organic tissue characteristics. It has been defined as a group of organisms with sex. The problem with these definitions is that they are subjective (Brenner, ibid., P. 2). Species have also been defined based on criteria such as host range, pathogenicity, whether or not to produce gas during fermentation of certain sugars, and whether sugar fermentation is fast or slow.

[0009] In the 1960s, numerical bacterial taxonomy (also called computer taxonomy or genetic phenotypic taxonomy) became widely used. Numerical taxonomies are based on testing as much of an organism's potential 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 definition of 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. Hugh RH
RH) and Giliardi, GL (Manua)
l of Clinical Microbiology, 2nd Edition, American Microbial Society, Washington DC, 1974, p.250-269, which lists the minimal phenotypic traits as a means of using bacterial fractions to characterize bacterial species. There is. 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. This collation is necessary because each laboratory has different media and methods, and the strain is the standard for the species, 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. DNA-
Strains can be identified by 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 may be partially overcome by selecting phenotypic attributes that appear to be interrelated with the repair group, but when they are used alone, DNA-DNA
The definition of species by rematching still only applies indirectly.

Brenner, supra, page 3, the ideal means for identifying bacterial species is to isolate the gene and immediately determine the nucleic acid sequence in the strain to be of species-som of any known species.
It is said to be a “black box” that resembles mass spectrophotometric analysis in comparison to the standard pattern of Ething).

However, although Brenner is able to perform restriction endonuclease analysis to determine the general sequence of isolated genes, " Is unlikely to be available. " His words apply equally to every species of organism.

From this brief review of the prior art, unknown bacteria and other organisms have been identified and rapidly classified, especially pathogenic organisms or organisms that have available biochemical reactions. We conclude that there is now a need for a fast, accurate and reliable way to do this. The method must be universally and readily available in clinical laboratories, and must not depend on the number of trials conducted or the clinician's subjective prejudice, and can Do not rely on the trial or error trial or error method. 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.

[0014]

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

[0015] 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 clinical laboratories so that the cause of animal or plant diseases can be characterized and identified. It is to be.

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

[0018]

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

As a method of characterizing an unknown organism, a restriction endonuclease obtained from the organism, from a probe organism or from a ribosomal RNA information-containing nucleic acid derived from the organism that has hybridized or repaired. A method comprising comparing the chromatographic pattern of nuclease-digested DNA with an equivalent chromatographic pattern of at least two different known organism 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
If a species is a collection of individually separated strains that are associated with a common species-forming event, there should be objectively bounded species-similar strain-similarities, despite dispersal. , And the strains of the species are based on the inventor's recognition that they should have structural information that provides clues to their common source. Most of the past history of organisms remains in semantides, DNA and RNA (Zuckerkandle.E. And Pauling,
L, "Journal of Theoretical Biology" Volume 8: 357-36
6 (1965)), the inventor concluded that an experimental approach to harness the information contained in some conserved genes is to use rRNA. Ribosomal RNA has structural and functional roles in protein synthesis (Schaup, “Journal of Theoretical
Biology, 70: 215-224 (1978)), and general conclusions drawn from studies of rRNA-DNA hybridization indicate that the basic sequence of the ribosomal RNA gene is evolutionary compared to most other genes. It is said that it is less likely to undergo changes during the process and is more likely to be preserved (Moore, RL, "Current Topi
cs In Microbiology and Immunobiology '', 64, 105-1
28 (1974), Spring-Verlag, New York). For example, the primary structure of 16S type rRNA from many bacterial species was deduced from oligonucleotid analysis (Fox,
GE et al, International Journal of Systemat
ic Bacteriology, 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 Molecula
r Evolution ", 3: 63-77 (1974), but substantial differences between species can be used to generate a taxonomic classification table for bacteria (Fox, GE" Science "209: 457-. 463 (1980)).
Different strains of a bacterial species are not always identical, and restriction maps indicate that different EcoR I sites occur in the rRNA gene of two strains of E. coli (Boro.
s, IA et al, “Nucleic Acids Research” No. 6
Volume: 1817-1830 (1979)). Bacteria appear to share conserved rRNA gene sequences, and other sequences may vary (Fox, 1977, ibid.).

The present inventor has further found that restriction endonuclease digests of DNA are rRNAs that are similar in strains of certain organisms (eg bacteria) but different in strains of other organisms.
Having a combination of fragments containing gene sequences,
That is, despite the mutation of the strain, it has a high occurrence frequency,
It was also found that the specific combination of enzymes, of the restriction fragments with the least consensus genotype, determines the species. This is the essence of the present invention. The inventor intends to identify a ribosomal nucleic acid from an organism other than the organism to be identified whether it is prokaryotic or eukaryotic whether the method is the same or different in taxonomy (classical). It was also found to be general in that it can be applied to both prokaryotic and eukaryotic DNA using probes.

The present invention provides an objective method for defining an organism based on detecting a DNA fragment containing a ribosomal RNA gene sequence in a restriction endonuclease digest. The detection is performed by hybridizing or repairing the DNA fragment with a nucleic acid containing rRNA information from the probe organism.

"Organism" characterized by the process of the present invention (this term is taken to include "identify")
By definition, it means virtually all organisms, including DNA, by definition. It is useful to mention the traditional taxonomy for reference in this regard.

Plants and animals are included, eg Monella
In the (monera) kingdom, there are myxobacteria, spirochetes, and eubacteria.
a), the rickettsiae class of fission products (bacteria) and cyanobacteria: cyanophytha. In the plant kingdom, Euglenoids (Eugleno
phta), green algae: Chlorophyta, chlorophyceae and carophyceae
y-ceae), chrysophtas, xanthophyceae, chrysophyse
ae), Bacillariophyceae, Pyrrophyta Dinoflagerata
gellates), brown algae: phaeophyta; red algae: Rhodophta; slime molds: Myxomycophyta, myxomycete
s), acrasiaes, plasmodiole (pl)
asmodiophoreae), labyrinthuleae
Class: Yumycophyta: Fungi (Eumycophyta), phycomycetes, ascomycete
s), Basidomycetes class; moss, hepaticae, anthocerotae, musci class; vascular plant Tracheophita
yta), psilopsida, lycopsy
da), sphenopsida, pteopsida
ropsida), spermopsida (Spermopsida) subspecies, cycad (cycadae), zinc goe (ginkgoae), Konifere (conif
erae), gneteae and angiolospermae (a
The subclasses ngiospermae, dicotyledoneae, and monocotyloedoneae are described. In the animal kingdom, the protozoa sub-world, Protozoa protozoa
(protozoa), subplasma plasmodroma, flagellata, sarcodina and sporozoa classes; siliophora
Subphylum, ciliata; Subgenus of parasites, polyhera (porifera), calcarea, hexactinellida and desmospoguier (d)
esmospongiae); Mesozoan subdivision, Mesozoa phylum; Metazoan subdivision Radiata Division, coelenterata (coele)
nterata gate, hydrozoa, scifoph
ozoa), Utozoa class, Stenophora (ctenoph)
ora gate, tentaculata and nuda
a) class; protostomia division, platyhelmintes, tubellan
a), trematoda and cestoda
Rope; Nemertina Gate, Acantosephala (ac
anthocephala) Gate; aschelmintles
Gate, rotifera, gastrotrica (gastrotrich)
a), quinorhyncha, priapulid
a), nematoda and nematmorpha (nemat)
genus omorpha; Entoprocta phylum; ectoprocta phylum, Gymnolaemata
And the phylactolaemata class; the phoronida phylum; the braciopoda phyla,
Inarticulata and alticulata (a
rticulata); Molska mollusc (mollusca) phylum, amphineura, monoplaco
phora), gastropoda, scaphopoder
(scaphopoda), pelecypoda and cephalopoda (Sepunculida); sipunculi
da) Gate; Echiurida Gate, Annel
ida) gate, polychaeta, oligocator (oligo)
chaeta) and hirudinea classes; onychophora gate; tardigrada
Gate: pentastoimida Gate; arthropoda Gate; trylobita Gate,
Chelicerata submonium, xiphosu
ra), arachmida, pycnogo
mida) class, mandibulata submonium, krustae (crustacea), chilopoda (diplopoda), polopoda (pauropoda), symphyra
(symphyla) rope, corembola, protura (p
rotura, diplura, thysanur
a), ephemerida, odonata
, Orthoptera, dermapte
ra), embinia, plecopter
a), zoraptera, corrode
ntia), marlophaga, anoplura
ura), Chisas noptera (thysasnoptera), hemiputera (h
emiptera), Neuroptera, Coleoptera
insects of the order (coleoptera), hymenoptera, mecoptera, siphonaptera, diptera, trichoptera and lepidoptera; denterostomia
(Denterostomia) animal, chaetognath
a) Gate, echinodermata Gate, crinoidea, asterodea, asterodea, ophiuroidea, echinoidea, echinoidea,
And holoturoidea, Pogonophora
(pogonophora) gate, hemichordata gate,
Enteropneusta and pterobranchia classes; chordata gate, urochordata subgate, ascid ascid
iaciae), thaliaceae, larva
cea) class; cephalochordata submonium,
Vertebrata submonium, agnatha,
Chondrichthyes, osteichitis
chthyes), saccotage (saccopteiygii) subclass, crossopterygii and dipnoi
Eyes, amphibia, repitilia
, Aves and mammalia, Prototheria subclass, Theria subclass, Marsa pialis, marsupialis, Insectibola (insec)
tivora), dermoptera, chiroptera
ptera), primates, edentata,
Pholidota, lagomorpha
a), rodentia, cetaceae, carnivora, tubulidenta
ta), probosicdea, hirakoidea (hyr
acoidea, sirenia, perisodactyla
Sodactyla) and artiodactyla can be mentioned. 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. Nuclear DNA or organelle DNA when identified as a eukaryotic organism
Endonuclease digestion may be carried out in the same manner using (filamentous DNA or chloroplast DNA).

Briefly, rRNA gene sequences, maybe a subclass or subclass (infrasubspecif) subclass.
High molecular weight DNA and / or small circular DNA is isolated and identified from the organism for analysis of sequences that can be used to generate ic subdivisions. DNA
Are extracted by methods well known to those skilled in the art. Cut the DNA at a special site with a restriction enzyme to make a fragment. The fragments are sized according to size in a standard chromatographic system such as gel electrophoresis. The gel is stained and standardized for fragment size using a standard curve made of fragments of known size in a manner well known to those skilled in the art. The separated fragments were then spotted by Southern (Southern,
EM "Journal of Molecular Biology", 38: 503-517.
(1975), incorporated herein by reference), transferred to cellulose nitrite paper and covalently bonded thereto by heating. The fragment containing the rRNA gene sequence is then localized by its ability to hybridize to a nucleic acid probe containing the rRNA information. The nucleic acid probe is labeled with a non-radioactive substance or preferably with a radioactive substance. When labeled with a radioactive substance, the probe can be ribosomal RNA (rRNA) or can be complementary to ribosomal RNA, either synthesized by reverse transcription or contained on a clonal fragment that can be labeled by, for example, nick translation. Radiolabeled DNA (rRNAcDNA) may be used.

The probe is obtained from an arbitrarily chosen organism as will be seen later. Once hybridized,
Hybridized fragments can be detected by selectively detecting double-stranded nucleic acid (non-radiolabeled probe) or made visible, eg 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 readily compared to equivalent chromatographic patterns obtained from at least two, or a large number of known standard organisms, genera or species. After preliminary broad classification has already been performed (eg, using classical classification), comparison of the size of the nucleic acid fragments by visual inspection and matching with the appropriate chromatographic pattern allows detection of band intensities (hybrid The comparison can be carried out by the amount of chemical conversion or by combining these. Ideally, the comparison is done by a one-dimensional computer pattern recognizer, such as is 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,
The subtle differences are limited to subspecies differences due to strain variation, but the interspecies and genus differences (and even at higher levels of classification) are very large. 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.

“Probe organism” used in this study
(And from which the nucleic acid probe is obtained) may be any of the organisms listed above, either a eukaryotic organism or a prokaryotic organism. The only limitation is given by the fact that the rRNA-containing probe must maximally hybridize to the endonuclease digest of DNA of unknown organism.

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

DNA (digested with endonuclease)
There are four sources: 1) prokaryotic DNA, 2) eukaryotic glomerular DNA, 3) eukaryotic chloroplast DNA, 4) eukaryotic nuclear DNA.

The following hybridization table was thus created (Table 1).

[0034]

[Table 1]

The table shows that ribosomal RNA probes are capable of maximally hybridizing to DNA of unknown organisms. For example, to identify certain eukaryotic organisms, species-specific glomerular or chloroplast DNA is extracted,
It may be digested with an endonuclease, and this digest may be hybridized with a prokaryotic ribosomal RNA probe or a eukaryotic ribosomal probe extracted from an organelle. Similarly, to identify pronuclear organisms, species-specific cellular DNA is extracted, digested with endonucleases,
The digest may be hybridized with a prokaryotic ribosomal RNA probe or a eukaryotic ribosomal RNA probe extracted from an organelle. Eukaryotic organisms can also be identified by extracting and digesting species-specific nuclear DNA and hybridizing it with a non-organic eukaryotic ribosome probe. Eukaryotic cells could be defined by one or some combination of nuclei, glomeruli, or in some cases the chloroplast system. These cross-hybridizations were previously described in that the rRNA nucleic acid extracted from the eukaryotic organelle was
Nucleic-extracted eukaryotic DNA with a wide homology to NA nucleic acids
, And the homology with prokaryotic DNA is not so widespread.

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

The choice of pair of DNA to be digested 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, to detect and identify infectious agents,
For detection of prokaryotic species (eg bacteria) present in eukaryotic cells (eg animals or plants), choose a prokaryotic ribosomal probe and extract DNA from organelles
The processing may be performed under the condition that 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 a eukaryote (which is not infected by prokaryotic cells) is identified with a prokaryotic ribosomal probe, DNA from the organelle is isolated, for example, by separating the organelle from the nucleus and then extracting only the organelle's 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. Because this ribosomal probe does not hybridize well with DNA from prokaryotic cells.

Same world, same sub world, same department, same gate, same sub gate, same class, same sub class,
Alternatively, it is preferred to use pairs (DNA and ribosomal probes) from the same order, or the same family, or the same family, or the same genus. It is particularly preferred to use a prokaryotic ribosomal probe (eg a bacterial ribosomal probe) to hybridize to prokaryotic DNA. 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, and among them, E. coli is particularly 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 (for example, to identify a species of mouse, a ribosomal probe of mouse origin is used). Good to use).

The most sensitive and effective pairing system is the one in which the source of the ribosomal probe and the source of the restricted DNA are evolutionarily less distant or less dispersed.

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 (for example,
Drohan, W et al, "Biochem. Biophys. Acta", 521.
(1978), 1-15, hereby incorporated by reference). Since organelle DNA is small and circular, the spooling method helps separate acyclic nuclear DNA from circular, organelle-derived DNA. As a result, the unspooled material contains DNA from organelles that can be individually separated by density gradient centrifugation. As another method, the filament (or chloroplast)
The purified glomerular (or chloroplast) fraction, separated from the crushed cell mixture, is used for the preparation of organelle-derived DNA and the purified nuclear fraction is used for the preparation of nuclear DNA. (For example, "Nucleic Acids" by Bonen L. and Gray MW
Research "8: 319-335 (1980)).

Prokaryotic 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. are treated under well-known conditions designed to extract high molecular weight DNA. . For example, cells of an unknown organism are suspended in extraction buffer, lysozyme 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 digestion with protease, 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 is described in Davis, RW et al. “A Manual for Genetic E”.
ngineering, Advanced Bacterial Genetics "(hereafter referred to as" Davis "), Cold Spring Harbor Laboratory, Co.
ld Spring Harbor, New York, 1980, p.120-121.

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

Digestion of the extracted DNA is carried out 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
Eventually it will identify the organism. Because it is unlikely that the enzyme will cut DNA from its own source, the species). 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 possible restriction endonuclease enzymes are Bgl I, BamHI, 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. Normally, the DNA and endonuclease are incubated together in a suitable buffer for a suitable period of time (ranging from 1 to 48 hours, temperature range 25 ° C-65 ° C, preferably 37 ° C).

The resulting chromatographic pattern will depend on the type of one or more endonucleases 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 soaked in ethidium bromide solution and UV-probably exposed to visible marker fragments that may be added. Light-placed in a box.

After separating and visualizing, the DN
The A fragment is transferred to nitrocellulose filter paper by the Southern method ("Journal of Molecular Biology" 38: 503-517 (197
Five)). 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 digestion fragments bound to the filter paper is a nucleic acid probe, preferably derived 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, a notch translation labeled DNA probe containing ribosome information, a cloned DNA probe containing ribosome information or a probe Detectable labeled DNA that is complementary to ribosomal RNA from the airframe
(rRNA cDNA). 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 nucleotides 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., “Proceedin
gs of the National Academy of Sciences 70: 2238-
2242, 1973). Direct covalent mercuration of natural nucleic acids has been reported (Dale et al., "Biochemistry" 14: 244).
7-2457). Reannealing of Mercury Polymers
g) the nature is similar to that of the corresponding mercury-free polymer
(Dale and Ward, "Biochemistry" 14: 2458-2469)
. The metal-labeled probe is, for example, photo-acoustic spectroscopy, X-ray spectroscopy, for example X-ray fluorescence,
It can be detected by 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 collected using markers of known molecular weight. (For example, Perry, RP and Kelly, DE, “Sustainable Synthesis of 5S RNA When Production of 28S and 18S Ribosomal RNA Is Inhibited by Small Actinomycin D”, J. Ce.
ll. Physiol., 72: 235-246 (1968), incorporated herein by 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” 2
2: 157-170 (1980), or Edwards, K. et al., `` Nucleic
Acids Reserch ", 9: 2853-2869 (1981)).

If a radiolabeled ribosomal RNA probe is used, this is a probe which has been grown or cultivated in a nutrient containing a compound having suitable radioactivity, or in a culture medium containing such a compound. Separated from organisms. When the probe is complementary DNA (rRNAcDNA)
, This is made by reverse transcribing rRNA isolated from the probe organism in the presence of radioactive nucleoside triphosphates (eg ³² P-nucleosides or ³ H-nucleosides).

The labeled ribosomal probe may also be one obtained from a truncated translated DNA molecule, in particular a whole circular DNA from the organelle. Specifically, chloroplast or filamentous circular DNA is cut and translated in the presence of a radioactive label to obtain a labeled DNA ribosomal probe. The chloroplast-labeled probe hybridizes best with chloroplast DNA and the glomerular-labeled probe hybridizes
Will hybridize best with. The incision translation labeled ribosome probe of chloroplast (or glomerulus) is
It will hybridize second best with glomerular (or chloroplast) DNA. It will hybridize to the DNA of the whole plant (or animal), albeit in a less preferred form. The ribosome probe is a nucleus of eukaryotic cells, even if this method is not good in consideration of practicality.
It may also be obtained from DNA by truncation translation. A more useful approach to achieve this is to cut the rRNA gene from the eukaryotic nuclear DNA (with a restriction enzyme), split the fragment, identify the ribosomal gene sequence (by hybridization), and separate the ribosomal gene sequence. (By electrophoresis). The isolated rRNA sequence is then religated into a plasmid or vector, ³² P after transformation of an appropriate host.
-The cloning may be carried out in the containing medium. Another method is to grow a transformed host, then isolate the DNA,
Label it by truncation translation or separate the DNA, excise the rRNA sequence, and then label. The resulting ribosomal probe hybridizes in the same state as rRNA cDNA (see below).

A preferred nucleic acid probe is radiolabeled DNA complementary to rRNA from the probe organism. Specifically, rRNA separates ribosomes from probe organisms, separates, and the appropriate RNA is substantially purified as described above. Ribosomal RNA thus obtained
Is a ribosome-free carrier RNA (tRNA), or transducing RN.
It contains almost no other RNA such as A (mRNA). Prokaryotic rRNA usually contains three subgroups. So-called
5S, 16S, and 23S-fragments. Reverse transcription to cDNA is 3
It is done with a mixture of all species or else with a mixture of 16S and 23S fragments. Reverse transcription with only one of these components is possible in some situations, but less preferred. Eukaryotic rRNA is usually 2
Includes types of subgrooves, 18S and 28S. And reverse transcription into cDNA is performed by a mixture of 18S and 28S fragments, or each of these.

Pure containing almost no other types of RNA
The rRNA is incubated with a reverse transcriptase that can reverse transcribe to cDNA. More preferred in this case is incubation with reverse transcriptase from avian myeloblastosis virus (AMV) in the presence of primers such as calf thyroid DNA hydrolysate. The mixture must contain the appropriate deoxynucleoside triphosphates,
One at least of its nucleoside, for example by ³² P, are radioactively labeled. For example, deoxycytidine 5 ′-( ³² P), deoxythymidine 5′-
⁽³² P), deoxyadenine 5 '- ⁽³² P), or deoxyguanidine 5' - ⁽³² P) · triphosphates are used as radioactive nucleosides. 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. A radiolabeled cDNA probe in substantially pure form,
That is, there is no unlabeled molecule and cDNA complementary to other types of RNA.
Also, a radiolabeled cDNA probe that is free of protein substances and free of cellular components such as membranes and organelles constitutes one aspect of the present invention. Preferred probes are labeled prokaryotic rRNA cDNAs.
And most preferred is bacterial labeled rRNA cDNA.
The species of the probe is a bacterial microorganism such as Enterobacteriaceae or Brucella.
a), Bacillus, Pseudomona
s), Lactobacillus, Haemophillus, Microbacter
ium), Vibrio, Neisseria, species from 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.

It is important to recognize that the labeled cDNA probe must be a faithful copy of the rRNA, ie that the entire nucleotide sequence of the template rRNA is transcribed at every time synthesis takes place. 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: The cDNA must protect 100% of the labeled rRNA from ribonuclease digestion; and Labeled cDNA must anneal specifically to rRNA as evidenced by resistance to S1 nuclease.

Beljansky MM et al. “CRAcad.Sc
Paris "t286, Series D., p. 1825-1828 (1978), describes ³ H-radiolabeled cDNA derived from E. coli rRNA. The cDNA in this study was not prepared by reverse transcriptase in the presence of a primer as in the present invention, but was prepared by DNA polymerase I using rRNA previously divided with ribonuclease U ₂ as a template. is there. Beljansky et al.'S rRNA digestion product (R
NAse 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 Beljansky is known to accelerate the predominance of homopolymeric transcription over heteropolymeric transcription of rRNA (see Sarin PS et al., “Biochem. Biophys. Res. .Com
m., 59: 202-214 (1974)).

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

The next step in the process of the invention is the hybridization of isolated DNA digests from unknown organisms with unlabeled or (preferably) radiolabeled rRNA or cDNA probes. Hybridization was performed on paper containing covalently labeled DNA digests from unknown organisms.
It is carried out by contacting with a hybridization mixture containing a ribosomal probe. Incubation is carried out at elevated temperature (50-70 ° C) for an extended period of time, after which the filter paper is washed to remove unbound radioactivity (if necessary) and then air dried to prepare for detection. Another highly preferred hybridization, which is much faster than the above method, is the "Biochemistry" 16: Kohne, DE et al.
5329-5341 (1977), a room temperature phenol emulsion re-pairing method.

After hybridization, this method requires selective detection of appropriately hybridized fragments. This detection takes advantage of the fact that the hybridized fragment has a double-stranded structure, which allows a selective method (in the case of unlabeled probe), radio-autograph method or computerized or not. Well, 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 well known to the skilled person,
There is nothing more to say in this regard.

The final product of this method is a chromatographic band pattern with light and dark regions of varying intensity at specific locations. These positions can be easily matched to a particular fragment size (kilobase pair) by subjecting the marker to a marker such as EcoR I digested lambda bacteriophage DNA. 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 to almost infinite number of books listing the genus and species patterns of various organisms that can be defined. For example, it is estimated that there are about 100 pathologically relevant bacteria 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 types of endonuclease enzyme used, and is probably used for the particular organism used as the source of the radiolabeled probe (probe organism) and also for probe preparation. It will depend on the composition of the ribosomal RNA information (eg 5S, 16S or 23S subtypes of prokaryotic cells, 16S and 23S types only, etc.). Thus, the catalog may contain various enzyme-specific band patterns with recorded band sizes and relative intensities for each probe. As the concentration of bound DNA bound to the filter paper decreases, only the strongest bands become visible, and the size of these or these bands can identify the species. Each of these band patterns may comprise a band pattern obtained with the complete ribosomal RNA and / or a band pattern obtained with ribosomal RNA containing only certain subtypes. The above changes or permutations are, of course, all available to the library. Moreover, for eukaryotic organisms, the library may contain patterns of one type of DNA, or organelles and / or nuclear-DNA resulting from the use of 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 band patterns visually or by means of a one-dimensional computerized digital scanner programmed for pattern recognition. These computer scanners are time-of-sale.
sale) are well known to those skilled in the processing (commonly used "supermarket" checkout pattern readers). 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 represent the amount of hybridized bound DNA, so it can be used to estimate the extent of an organism, such as the presence of prokaryotic cells in eukaryotic cells. Can be used.

If the user wants to confirm the properties of a given organism and further identify, such a user can digest an unknown organism with a second, different endonuclease and obtain The resulting band pattern may be compared to the catalog band pattern of the organism for the second chosen endonuclease. 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 for Mycobacterium lepra (Mycob lepra).
Not possible for some microorganisms such as acteriun leprae) (pathogens of Ley's disease) 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. anthrac
is) (pathogen of anthrax). This method can eliminate these problems because it is based on the isolation of nucleic acids, which do not perform conventional bacterial isolation and characterization. It seems that this method can detect microorganisms that have not been formally described. Moreover, the method can distinguish between different strains, which is useful for epidemiological typing, for example in bacteriology. This method can be used in forensic laboratories to correctly and unambiguously identify plant or animal tissue in criminal investigations. It is also used by entomologists to quickly identify insect species when ascertaining the nature of crop damage.

Further, this method is applied to taxa other than subspecies (for example, plant root nitrogen enzyme gene; reference: Hennecke H 291 "Natu.
Re "354 (1981)), this methodology can be used to probe and identify genotypes of individual strains.

The method of the present 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. Detection and identification of bacterial DNA with pronuclear ribosome probes is highly selective and can be performed in animals (eg mammals).
It can be done even without the presence of 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 is the possibility, in addition to identifying the species and strain of the infecting microorganism, the possibility of discovering the presence of some unusual gene sequence in the microorganism. 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 hybridizing mixture,
It is possible to correctly determine the presence or absence of antibiotic resistance of the organism (one or a few bands appearing irregular).
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, use the first aliquot for identification, the second aliquot for the presence or absence of drug resistance sequences, and the third aliquot for the toxin gene. You can also test. Alternatively, as another method, a ribosomal probe labeled with one radionuclide (eg, ³² P) is added to an R-factor probe labeled with another radionuclide (eg, ³ H or ¹⁴ C) in a hybridization mixture. It could also be used. R in unknown DNA after hybridization
-The presence of factor DNA can be tested by scanning with two different scanners. One is for species and strain identification (eg ³² P), the other is for drug resistance etc. (eg ³ H or ¹⁴ C). In this way the experimenter is able to
Identification of genus and species, typing of strains, drug resistance, toxic production or other traits, or testing of taxa below species level detectable by labeled nucleic acid sequences or probes should all be done in a single experiment. it can.

Since the R-factor is ubiquitous and crosses the boundaries of the species, identification should be made in any bacterial genus or species.
It can be done with the same R-factor probe (see: Tomk
ins, LS et al. J. Inf. Dis., 141: 625-636 (198
1)).

In addition, 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 o
f Clinical Microbiology "3rd edition (published by Lennette,
All viruses listed in EH, Amer Soc Microb 1980, 774-778) can be identified. For example, picornaviridae, caliciviridae, reoviridae, togaviridae, ortho-maicoviride
myxoviridae), Paramaikoviride (paramyxoviridae)
, Rhabdoviridae, Retroviride
(retroviridae), arenaviride (arenaviridae), coronaviride (coronaviridae), buniaviride (bunyavi)
ridae), puboviride (parvoviridae), papovaviride (papovaviridae), adenoviride (adenoviridae),
Herpesviridae, Vidovilide (v
idoviridae) and poxyviridae etc.

1) When the virus genome is integrated into the host DNA (DNA virus such as papovaviride member, RNA virus such as retroviride member), high molecular weight DNA is extracted from a tissue and the restriction enzyme is used. Digest. 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 degree 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, using virus RNA, DN with reverse transcriptase.
A can be made and in the case of DNA viruses, for example, viral DNA labeled by truncation 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. Before centrifugation, cell RN
It is thought that the virus nucleic acid can be concentrated by removing A by spooling. It can also be used to determine if the Wiles genome has been integrated.

In order to hybridize a Weals 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 cloned clone virus sequence, which may be the entire genome or a part thereof.

The method described in Southern described above is useful for transferring large DNA fragments (about 0.5 kilopace or more) to nitrocellulose paper after alkaline denaturation. This method is useful in the case of DNA viruses,
In the case of Wheels it may not be useful. 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. Sc.
i "USA 77: 5201-5205 (1980)) is an RNA and small DN.
The A fragment can be used to efficiently transfer 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, DNA that is 100-2000 nucleotides
The fragments, and RNA migrate efficiently and remain on the nitrocellulose paper during hybridization. It is also useful for small ribosomal DNA fragments. Therefore, it is most preferable to divide the sample digested with the restriction enzyme,
That is, a part of the nucleic acid is denatured with glyoxal. The Southern and Thomas maneuvers will give the greatest amount of information.

2) In the case of DNA viruses, existing viruses can be identified by performing restriction analysis on double-stranded (DS) virus DNA. Single-stranded (SS) DNA viruses will have different length genomes. Hybridizing probes (sequence information can be converted to DS-DNA), patterns of hybridizing fragments and / or one or more 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 is used for cDNA synthesis.

3) In the case of RNA viruses, RNA is not digested by restriction endonucleases (sequence information is DS-D
It may have been converted to NA). The genomes of different RNA viruses have different sizes, and some RNA viruses have more than one molecule in their genome. 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 cD synthesized using virus RNA.
It is NA.

To search for infectious pathogens in a sample, nucleic acid is extracted from the sample, or the cells are first cultured in a medium or cells to increase the number of pathogens, or a concentration process such as centrifugation is used. Or try all the approaches directly.

From the present invention, it is easy to make a "kit" containing the necessary elements for carrying 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 to identify bacteria, (Preferably a nuclear rRNA cDNA). The labeled nucleic acid probe will be in lyophilized form or, if necessary, in a suitable buffer. One 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 standards during experiments,
If the user suspects that an unknown is actually of a given genus or species, he or she creates a known swath pattern and compares it to the unknown swath pattern. Good. 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 may include a brochure, or "catalog" or computer tape or disk, defined as a book or pamphlet, or a computer access number, which may be plant species, mammalian species, bacterial species, particularly disease species. It incorporates a chromatographic zone pattern of a group of different organisms such as bacteria, insect species, etc. of biological importance. 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 also includes a probe rR for probe synthesis in one container.
NA may be included, a radiolabeled deoxyribonucleoside triphosphate in another container, and a primer in another container. In this way users can create their own probe rRNA cDNA.

Finally, the kit may be a technique of the invention, such as buffers, growth media, enzymes, pipettes, plates, nucleic acids, nucleoside triphosphates, filter papers, gel raw materials, transfer materials, autoradiographic supplements. It may include all of the additional elements needed to do It also
It may also include an antibiotic resistance sequence probe, a Weals probe or a probe with other specific properties.

While the present invention has been generally described above,
The present invention may be better understood with 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. Bacterial high molecular weight DNA extraction Bacterial broth cultures were spun down and the cells washed with cold saline. Extraction buffer (0.15M sodium chloride, 0.1M sodium chloride) in a volume of about 10 times the gram weight of the packed cells.
It was suspended in EDTA, 0.03M Tris pH8.5). Lysozyme 10 mg / ml was added to give a final concentration of 0.5 mg / ml. The suspension was incubated at 37 ° C for 30 minutes. Cell disruption was performed by adding 25% SDS to a final concentration of 2.5% and raising the concentration to 60 ° C for 10 minutes. After cooling in a water bath, mercaptoethanol was added to a final concentration of 1%. Pronase (trade name) 20 mg / ml 0.02 M Tris buffer (pH 7.4) was pre-digested at 37 ° C. for 2 hours and then added to a final concentration of 1 mg / ml. The solution 37
Incubated at 18 ° C for 18 hours. Phenol was redistilled phenol 1 l, double distilled water 2.5 l, saturated Tris base 270 ml, mercaptoethanol 12 ml and final concentration 10
It was prepared by mixing EDTA in an amount of ⁻³ M and allowing the mixture to separate at 4 ° C. The phenol was added to the washing buffer (10 ^-1 M sodium chloride, 10 ^-3 M EDTA,
It was washed with 10 mM Tris pH 8.5). Then an equal volume of fresh buffer was added. Mercaptoethanol was added to a final concentration of 0.1%. The solutions were mixed and stored at 4 ° C. The prepared half volume of phenol and half volume of chloroform were added to the lysed cell solution. Shake this for about 10 minutes at 3400 xg
Spun for 15 minutes. The aqueous phase was removed with a 25 mg glass pipette. This extraction operation was repeated until almost no precipitate was found at the boundary. 1/9 volume of 2N sodium acetate (pH 5.5) was added to the aqueous phase. Two volumes of -20 ° C 95% ethyl alcohol were slowly poured along the wall of the flask. The tip of the Pasteur pipette was melted and closed and used to spool the precipitated DNA. High molecular weight DNA was dissolved in buffer (10 ^-3 M EDTA, 10 ^-2 M Tris pH 7.4). The DNA concentration is 260n using the conversion factor of 30 μg per unit of absorbance.
It was measured by the absorbance at m 2.

Restriction endonuclease digestion of DNA The EcoRI restriction endonuclease reaction was performed with 0.1 M Tris-
HCl pH 7.5, 0.05M NaCl, 0.005M MgCl ₂ , and 10
Performed in 0 μg / ml calf serum albumin. The EcoR I reaction mixture contains 5 units of enzyme per DNA / μg,
This was incubated at 37 ° C for 4 hours. PST I restriction endonuclease reaction is 0.006M Tris-HCl pH7.4,
0.05M sodium chloride, 0.006M magnesium chloride,
0.006M2-mercaptoethanol, and 100μg / ml
It was performed in calf serum albumin. The PST I reaction mixture contained 2 units of enzyme per DNA / μg, which was 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 EcoRI to create a marker band for fragment size determination. Usually 2 μg λ DNA
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 λDNA digests,
20 μl of 1 × EcoR I 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 are put into a per well and the surface is covered with agarose dissolved. Digested material is 0.02M sodium acetate, 0.002M
EDTA, 0.018M Tris base, and 0.028M Tris H
1% dye at 35V in 0.8% agarose containing Cl pH 8.05
Electrophoresis was performed until migration of 3 to 16 cm. The gel was then dipped in ethidium bromide (0.005 mg / ml) and placed in a UV light box to visualize the λ fragments. The DNA was transferred to nitrocellulose filter paper by the method of Southern described above. The gel was treated with denaturing solution (1.5 M sodium chloride, 0.5 M sodium hydroxide) on a shaking table for 20 minutes. Denatured solution is neutralized solution (3.0M sodium chloride, 0.5M Tris HCl
pH 7.5) and after 40 minutes the gel was checked with pH paper. After neutralization, the gel was treated with 6 × SSC buffer (SSC = 0.15M sodium chloride, 0.015M sodium citrate) for 10 minutes. Pass the gel and nitrocellulose paper, 6 × SS
By aspirating C with a bunch of paper towels for 15 hours
The DNA fragment was transferred from the gel to nitrocellulose paper. 3 mm
A filter was placed between two pieces of chromatograph paper, wrapped with an aluminum wheel with the shining side outward, and dried in a vacuum oven at 80 ° C. for 4 hours.

Synthesis of ³² P ribosomal RNA complementary DNA ( ³²
P-rRNA and DNA) ³² P-labelled DNA complementary to E. coli R-13 23S and 16S type ribosomal RNA was synthesized using reverse transcriptase from avian myeloblastosis virus (AMV). Reaction mixture is 5μ
l 0.2M dithiothreitol, 25 μl 1M Tris
pH 8.0, 8.3 μl 3M potassium chloride, 40 μl 0.1
M magnesium chloride, 70 μl actinomycin, 14
μl 0.04 M dATP, 14 μl 0.04 M dGDP, 14 μl
It contained 0.04 M dTTP, and 96.7 μl water. The following were added to a plastic tube: 137.5 μl reaction mix, 15 μl calf thoracic primer (10 mg / m 2
l), 7 μl H20, 3 μl rRNA (2.76 μg / μl using 40 μg / OD unit concentration), 40 μl deoxycytidine 5 ′-( ³² P) triphosphate (10 mCi / ml), and 13 μl AMV polymerase (6,900 units / μl). The fermentation reaction was carried out at 37 ° C for 1.5 hours. Then add the solution to 5
Extracted with ml of chloroform and prepared phenol. After centrifugation (JS 13,600 RPM), the aqueous phase was directly layered on a Sephadex (trade name) G-50 column (1.5 × 22 cm). A plastic 10 ml pipette was used as the column. One small glass bead was placed on the tip, a rubber tube equipped with a pinch fitting was attached, and degassed G-50 swollen by immersing it in 0.05% SDS overnight was added. The aqueous phase was poured directly along the wall into G-50 and then eluted with 0.05% SDS. 20 fractions of 0.5 ml were collected in plastic vials.
The tubes containing the peak fractions were found by Selenkoff counting, counting for 0.1 min for each sample using a ³ H-discriminator and recording the total count. The peak fractions were combined. Aliquots were added to Aquesol (trade name, commercially available) and CPM (counts per minute) of ³² P per ml was measured by scintillation counting.

Hybridization and Autoradiograph
The fragment containing the I over ribosomal RNA gene sequences, hybridization was liquid the DNA on the filters to ³² P- ^rRNAcDNA, and detected by autoradiography. Filter the hybridized mixture (3 x SSC, 0.1% SDS, 10
0 μg / ml denatured and sonicated dog DNA and Dynehardt's solution (0.2% calf serum albumin, respectively)
Ficoll, and polyvinylpyrrolidine))
It was immersed therein at 68 ° C. for 1 hour. ³² P rRNA cDNA was added to 4 × 10 ⁶
CPM / ml was added and the hybridization reaction was incubated at 68 ° C for 48 hours. Then filter 3 x SSC
And then 0.1% SDS for 15 minutes at 2 hours or until the cleaning solution contained about 3,000 cpm ³² P / ml. The filters were air dried, wrapped in plastic wrap and autoradiographed with Kodak X-OMATR film at -70 ° C for about 1 hour.

B. RRNA of mammalian experiment Mus musculus domesticus (mouse)
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. Remaining nucleic acid was collected by centrifugation, 50 mM MgCl ₂ and 100 mM Tris pH7.4.
Dissolved in the buffer solution. Concentration of DNAse (not including RNAse) 50
Added until μg / ml. The mixture was incubated at 37 ° C for 30 minutes. The RNA produced 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. Add the sample and switch the gradient to SW
It was spun at 40K rotor for 7 hours at 35K rpm. Fractions were collected by optical density. The 18S and 28S fractions were selected by comparison with known molecular weight markers. Relaxed hybridization conditions were used in all mammalian experiments. 54 ° C. Washing process is performed at 54 ℃, 0.05%
SDS was added and the mixture was washed 3 times with 3 x SSC for 15 minutes each three times.

Example 1 Bacterial species are restricted endonucleases of the ribosomal RNA gene
Determined by ase analysis. P. used in this example. Several strains of Aeruginosa have minimal phenotypic traits that identify the species (Hugh R.
H., et al. "Manual of Clinical Microbiology" 2nd
Edition, ASM, 1974, pp, 250-269) (Table 2). The other three Pseudomonas and two Acinetobactors
er) strains were selected to compare species and genera (Table 3).

[0085]

[Table 2]

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

[0087]

[Table 3]

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

The size of the fragments in the EcoR I digest (kilobase pair) was 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. anitra
tus) 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 3 fragments was not calculated); Luoffi (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 PSTI digest (kilobase pair) is shown below; Stowzeri 6.7, 6.1,
5.5; Fluorescence 10.0, 9.4, 7.8, 7.0;
P. Putida 10.5, 9.9, 6.8, 6.3, 4.4; Anitratus 36.0, 28.0, 20.5, 12.0, 10.0, 5.8, 3.7
, 2.6, 2.4; Luoffi 9.9, 8.7, 7.2, 5.7
, 4.0, 3.6, 3.2, 2.7.

P. Comparing hybridized restriction fragments from seven strains of Aeruginosa, this species shows that the 10.1, 9.4, 7.6 and 5.9 kilobase pairs (KBP)
, Which is defined by the EcoR I-specific combination of fragments containing the rRNA gene sequence (FIG. 1).

The 7.6 KBP EcoR I fragment appears in 4 out of 7 strains in this sample. A similar situation occurs in some phenotypic traits of species strains. Of fragments from 7 strains
The fact that the EcoRI combination is used to divide the strain into two groups is described in P. It draws the inference that there are two species with the minimal phenotypic trait of Aeruginosa. DNA
From the experimental results of digestion of E. coli with PST I (Fig. 2), EcoR I 7.
We conclude that strain variation exhibited by the 6 KBP fragment represents intra-species variation. Because one conserved combination of PST I fragments, 9.4, 7.1, 6.6 and 6.4
There is a KBP and it defines the species.
The 9.4 and 6.6 KBP PST I fragments are described in P. Appears in 6 out of 7 aeruginosa strains; 7.1 and 6.4 KB
The PPST I fragment appears in all of the sample strains. P
Mutations in the ST I fragment occur in strains that do not contain the EcoR I 7.6 KBP fragment; RH151 has 10.1 and 8.2 KBP fragments,
RH809 does not include 9.4KBP fragments, it has 6.0KBP fragments. And the type strain RH815 does not contain the 6.6KBP fragment. 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.

P. EcoR I in aeruginosa strains
Mutagenesis of the 7.6 KBP fragment may be envisaged by examining the hybridizing EcoR I fragment found in other Pseudomonas species type strains (FIG. 3). P. Stowelli, P. Fluorescence and P. Putida type strains do not contain the 7.6 KBP fragment but share an EcoR I fragment of the same size;
P. Aeruginosa and P. Stowelli 9.4 KBP
Fragment having P. Stowelli and P.P. Fluorescence has a 16KBP fragment, Fluorescence and P. Petida has 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 apply to PST I digests (Figure 4).

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 genus comrades are different. The two Acinetobacter species have a larger range of hybridizing fragment sizes than the four Pseudomonas species.

E. coli, Bacillus thuringiensis (Bac
illus thuringiensis) and B. Subtilis (B. subtili
where the enzyme cuts the rRNA gene, without the help of a restriction map such as that obtained in s), the number of copies per genome and the heterologous flanking flanks between multiple genes or heterogeneous genes. It is impossible to predict if there are regions). The E. coli rRNA cDNA probe may not hybridize to some restriction fragments containing the rRNA gene sequence, and if so, this may reflect an evolutionary distance or variance between the test organism and E. coli. are doing. 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 Comparison of restriction analysis with DNA-DNA liquid hybridization:
The strains used in this study are listed in Tables 4 and 5.

[0096]

[Table 4]

[0097]

[Table 5]

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

[0099]

[Table 6]

This data shows that there are two hybridization groups. Similar data can be found in B. Subtilis has been reported in the literature (Seki et al, "International
Journal of Systematic Bacteriology '' 25: 258-270 (1
975)). These two groups can be represented by RH3021 and RH2990. Restriction endonuclease analysis of the ribosomal RNA gene is performed. EcoR I digest (Figure 5)
Could be divided into two groups. The group 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). B. using EcoR I data Subtilis strain D
It can be placed at a suitable place in the NA-DNA hybridization group. According to the 70% rule for DNA-DNA hybridization,
B. Subtilis is actually two species. However, considering the PST I data (Figure 6), those groups can be thought of as two dispersive populations involved in common ancestor or speciation events. B. The conclusion that Subtilis is a species correlates with phenotypic data. The strains listed in Table 5 are Gordon R. "Th
e Genus Bacillus "Agriculture Handbook No.427 (US Department of Agriculture, Agricultural Research Services Washington D.
C.) p. 36-41 B. It is identified as subtilis. Restriction analysis can provide data comparable to DNA-DNA hybridization data, and by choosing the appropriate enzyme, restriction analysis can be well-defined species despite variance. RH3061 lost PST I site.
However, EcoR I data indicate that the strain is B. Suggests that it is a subtilis. The same can be concluded from the Bgl II data (Figure 7) and Sac I data (Figure 8).

Example 3 Stability patterns of restriction analysis and other Bacillus polymyxa experiments

[0102]

[Table 7]

B. Subtilis and B. Polymica, Ec
It can be distinguished by oR I data (Fig. 9), PST I data (Fig. 10), Bgl II data (Fig. 11 left) and Sac I data (Fig. 11 right). 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
B. NRRL from both culture collections Certainly, Polymica type strains 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 Swiss mouse, Mus musculus domesticus (M) , identifies bacterial species in mouse tissues without isolation.
us musculus domesticus) (inbred strain) to Streptococcus pneumoniae R
0.5 ml of a turbid suspension of H3077 (ATCC 6303) was inoculated intraperitoneally. When the mice were moribund, the heart, lungs and liver were removed. High molecular weight DNA was isolated from these tissues, S. New monnier
Isolation from RH3077 and Swiss mouse organs and manipulation of restriction endonuclease analysis of the rRNA gene was performed using EcoRI for digesting DNA. Filter 3 x SSC
2 × 15 minutes 0.3 × SSC and 0.05% SDS
Washed in. Autoradiography was performed for 48 hours. The data (Figure 12) showed that S. pneumoniae is defined by seven hybridizing fragments (17.0, 8.0
, 6.0, 4.0, 3.3, 2.6 and 1.8KBP). This bacterial cDNA probe contained two mouse DNA fragments (14.0 and 6.
(8KBP) is not so hybrid. The hybridizing fragment is S. cerevisiae in infected tissue. It informs the existence of Pneumonier. All seven bands are found in the heart DNA extract. Although less intense in liver DNA extracts, they are all visible by autoradiography. Only the 6.0KBP band appears in the 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 test the sensitivity of this assay, dilute bacterial DNA with mouse DNA,
It was subjected to electrophoresis. With 0.1 μg bacterial DNA, all seven bands were visible on the autoradiograph.
For 10 ^-3 μg bacterial DNA, 17.0, 8.0 and 6.0 KBP bands were observed. 10 ⁶ S. 5 × 10 ^-3 μ per Pneumonie cell
Using the number g DNA (Biochem Biophys Acta 2
6:68), 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 hybridizes with a mouse specific EcoR I fragment (see FIG. 9, fragments with 14.0 and 6.8 KBP). These fragments correspond to the EcoR I fragments detected by the mouse 18S and 28S type ribosomal RNA probes (FIG. 14, supra, shows that the 6.8KBP fragment contains the 28S type rRNA sequence).
Bacterial probes do not hybridize well with mammalian ribosomal RNA gene sequences, so the bands are less intense, the bacterial probe and nuclear mammalian RNA systems are less sensitive, and select against infected prokaryotic DNA. The sex appears clearly. Lane for bacterial probes
In an experiment hybridized to 10 μg / digested bacterial DNA, bacterial bands were clearly visible at 10 μg / lane even when clearly visible.
No hybridization to g-digested human or mouse DNA was found.

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

FIG. 13 shows that the genus of mammals is Mus musculus.
We show that this can be confirmed with the domesticus 18S and 28S rRNA probes, and that several species of Mus are distinguishable. In this figure, the enzyme is PST I and the objects and their respective bands are: 1. Mus musculus meus
lossinus) (mouse) 14.5, 13.5, 2.6 2. Mus musculus domesticus (mouse) 13.
5, 2.6 3. Canis familiaris (dog) 1
2.0 4. Cavia porcellus (guinea pig) 17.0, 14.0, 13.0, 8.8, 5.7, 4.7 and 3.0 and less than 1 band 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. Muscervicolo
r cervicolor) (mouse) 14.0, 2.7 11. Muscervicolor pa
peus) (mouse) 13.5, 2.6 12. Mus pahari (mouse) 13.0, 3.7 13. Mus cookii (mouse) 13.5, 2.
6

FIG. 14 shows that mouse and cat DNA is 28S type rRNA.
We show that they can be distinguished only by the 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) 6.8
KBP 2. Feliz catus (cat) 8.3KBP In Figure 15, the enzyme is Sac I and the objects and bands 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. Feliz 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

When comparing FIG. 15 (Sac I digestion) with the diagrams of other mammals, it can be seen that the hybridization pattern is enzyme specific.

FIG. 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 mulatta (Laser 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 (Langer monkey cultured cells) 14.0, 7.2, 5.
7 5. HeLa (cultured human cells)> 8.1, 6.6 6. J96 (cultured human 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] Pseudomonas aeruginosa
aeruginosa) showing EcoR I restriction endonuclease digestion of DNA isolated from this strain. E. coli as a probe 1
CDNAs for 6S and 23S ribosomal RNA (rRNA) were used.

[Figure 2] Pseudomonas aeruginosa (P. aeruginos)
a) PstI restriction endonuclease digestion of DNA isolated from the strain. E. coli 16S and 2 as probes
The cDNA for 3S rRNA was used.

FIG. 3 shows EcoR I restriction endonuclease digestion of DNA isolated from glucose-nonfermentative Gram-negative rod species. E. coli 16S and 23S rRNAs as probes
The cDNA for was used.

FIG. 4 shows Pst I restriction endonuclease digestion of DNA isolated from glucose-nonfermentative Gram-negative rod species. E. coli 16S and 23S rRNAs as probes
The cDNA for was used.

FIG. 5: Various Bacillus subtilis
is) shows EcoR I restriction endonuclease digestion of DNA isolated from the strain. 16S of E. coli as a probe and
The cDNA for 23S rRNA was used.

FIG. 6 shows Pst I data obtained with the same probe for the same strains 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 FIGS. 5-7.

FIG. 9: B. Subtilis and B. B.polymyx
Figure 7 shows EcoRI restriction endonuclease digestion of DNA isolated from a). 16S and 2 from E. coli as probes
The cDNA for 3S rRNA was used.

FIG. 10 shows Pst I data obtained using the same probe for the same strains as in FIG.

FIG. 11 shows Bgl II and Sac I data obtained with the same probe for the same strains as FIGS. 9 and 10.

Figure 12: 16S and 2 from E. coli as probes
Using cDNA for 3S-type rRNA, Ec
Streptococcus pneumoniae (S
detection of treptococcus pneumoniae).

[Figure 13] Mus musculus domesticus (Mus m
We show the identification of mouse species by comparing Pst I digestion of DNA isolated from mammalian tissues using cDNAs for 18S and 28S rRNAs from cytoplasmic ribosomes of usculus domesticus) (mouse).

[Figure 14] Mus Musculius Domesticus 28S type
Shows EcoR I digested DNA from mouse and cat tissues that hybridized with the rRNA cDNA 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 cultures hybridized with Mus musculus domesticus 18S and 28S rRNA cDNA probes.

Continuation of front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical indication C12R 1:40) (C12Q 1/68 (C12Q 1/68 C12R 1:12) C12R 1:12) (C12Q 1 / 68 (C12Q 1/68 C12R 1: 125) C12R 1: 125) (C12Q 1/68 (C12Q 1/68 C12R 1:19) C12R 1:19) (C12Q 1/68 (C12Q 1/68 C12R 1: 32) C12R 1:32) (C12Q 1/68 (C12Q 1/68 C12R 1: 385) C12R 1: 385) (C12Q 1/68 (C12Q 1/68 C12R 1:39) C12R 1:39) 9162-4B C12N 15/00 A

Claims (9)

(57) [Claims] 1. A kit for use in a method of identifying a species of an unknown organism, the method comprising: (1) restriction endonuclease digested DNA of an unknown organism.
To obtain a chromatographic pattern in which a ribosomal RNA information-containing nucleic acid of a known probe organism or derived from a known probe organism is hybridized, and (2) the chromatographic pattern and the limitation of a plurality of known organisms of different species. The endonuclease-digested DNA is compared with a plurality of additional chromatographic patterns obtained by hybridizing the ribosomal RNA information-containing nucleic acid of the known probe organism or derived from the known probe organism, and then (3) the unknown existence The unknown organism by detecting a combination of ribosomal RNA sequence-containing restriction fragment existing in the chromatographic pattern of the airframe, which is conserved among allogeneic strains but not among different species Is a method of identifying a species of: Has a carrier which is partitioned to be closely placed more containers; ribosomal R-derived or probe organism probe organism in its first primary container
NA information-containing nucleic acid is contained therein; and further, this kit is a catalog having a pattern consisting of ribosomal RNA sequence-containing restriction fragments which are substantially conserved among allogeneic strains but not among allogeneic strains. , A kit containing a catalog containing books, computer tapes, computer disks or computer access numbers. 2. The kit according to claim 1, further comprising a container containing one or more restriction endonucleases. 3. The kit according to claim 1, wherein the ribosomal RNA information-containing nucleic acid probe is detectably labeled. 4. The kit according to claim 1, wherein the ribosomal RNA information-containing nucleic acid probe is ribosomal RNA (rRNA). 5. The ribosomal RNA information-containing nucleic acid probe is a DNA (rRNAcD) complementary to ribosomal RNA.
NA)). 6. The kit according to claim 1, wherein the probe organism is a prokaryote. 7. The kit according to claim 1, wherein the probe organism is a eukaryote. 8. The kit according to claim 1, further comprising one or more containers containing a viral nucleic acid probe. 9. The kit according to claim 1, wherein the catalog further has a virus chromatograph pattern.