JPH04126082A - Method for identification and characterization of organism - Google Patents

Abstract

PURPOSE: To quickly and accurately identify an organism by hybridization of a digested product from the restriction enzyme treatment of the DNA of a specimen with a probe containing eukaryotic or prokaryotic organism-derived ribosome RNA information.

CONSTITUTION: First, a digested product A is obtained by reaction at 25-65°C of a restriction enzyme such as BamH1 with a DNA obtained by subjecting eukaryotic or prokaryotic cells to extraction with e.g. chloroform. Whereas, a probe B containing labeled ribosome RNA information is obtained by labeling, with e.g. radioactive ³²P, a nucleic acid probe afforded through extraction from desired cells (e.g. prokaryotic ribosome probe as bacteria-derived rRNA). Secondly, the components A and B are brought into contact with each other to obtain a reaction product C through hybridization at 50-70°C. Subsequently, the component C is subjected to chromatography, and the resulting band pattern is detected by e.g. radio-autography to identify the aimed desired organism.

COPYRIGHT: (C)1992,JPO

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application] 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 related to.

[Conventional technology]

Classification of living organisms has traditionally been more or less arbitrary and along somewhat artificial lines. For example, the living world is divided into two kingdoms: Plants (Pla
ntae) and animals (Anima Iia).

Although this classification is convenient for commonly known organisms, it becomes difficult for organisms such as unicellular organisms (eg, green flagellates, bacteria, and blue-green algae). This is because these are fundamentally different from "plants" and "animals."

It was proposed to divide organisms simply by their bacterial internal structure. In this way, all cellular organisms are either prokaryotic or eukaryotic.
ryotis). prokaryotes (pro
karyotes) are eukaryotes (eukaryotes)
) are uncomplicated; they lack internal division by unit membranes and lack a distinct nucleus. Genetic information in pronuclear cells is carried to the cytoplasm on double-stranded DNA. No other DNA exists in the cell (phage, bacterial viruses, and true DNA that can replicate autonomously).
(except when plasmids are present). On the other hand, eukaryotes have a wide variety of unit membrane tissues, which serve to separate many functional components into specialized and isolated regions.

For example, genetic information (DNA) is found in the well-defined nucleus and also in the organelles mitochondria and (in photosynthetic organisms) chloroplasts. Replication, transcription, and translation of the eukaryotic genome occur at two or three distinct sites within the cell.

Occurs in the nucleocytoplasm, globules, and chloroplasts.

However, the difference between prokaryotes and eukaryotes is broken down when comparing globules and chloroplasts in prokaryotes. These organelles are now free living.
-1iving) prokaryotes, which enter into an endosymbiotic relationship with primitive eukaryotes, ultimately forming a close relationship with the structure of the host cell. (Reference examples: Fax,
G, E, et al, [5science J 20
9; 457-463 (1980) p, 462; 5ta
n+er, R, Y, et al.

rThe Microbial World J
4th edition, published by Prenshi1ce-Hal1,
1976, p. 86). For example, DNA obtained from mouse L cell globules, which carry the ribosomal RNA gene region, has been shown to exhibit significant sequence homology with Escherichia coli ribosomal RN^, strongly supporting the endosymbiosis model ( Van Etten,
RlA.

et al, rcellJ 22:157-170 (
1980)). Also, corn and lea may
s) It has also been shown that the nucleotide sequence of the 235 type ribosomal DNA of chloroplasts has 71% homology with the 233 type ribosomal DNA of E. coli (Edwards,
In, & Kossel, H,, rNucleic^c
ids Re5earch J 9:2853-2869
(1981)): Other related studies (Bonen,
L., & Gray, M. W., supra. 8:319-33.
5 (1980)). Furthermore, it supports the general concept.

In this model, eukaryotic cells are phylogenetic "chimeras" with organelle parts that are clearly prokaryotic in nature.
It is. The "prokaryotic-eukaryotic" dichotomy also has shortcomings even as a broad classification method.

The classification of organisms becomes more than a scientific problem when one seeks to identify plants or animals for purposes of hybridization and breeding, and when so-called higher j
This is the case when attempting to accurately and reliably identify microorganisms that may infect organisms or other media. For example, plant growers, livestock keepers, or fish keepers would want to have a quick and reliable way to identify heterologous and atypical strains. A veterinarian, physician, or horticulturist will want to accurately identify infectious organisms (parasites, fungi, bacteria, etc.) and viruses in test plants and animal tissues.

Correct identification of the species of these organisms and viruses is of particular importance.

This problem is best illustrated when discussing the identification of bacteria. The names of bacterial species usually refer to many strains; a strain is thought of as a population derived from a single cell. Strains of a species have a similar combination of attributes that help define the species. Expressing precise definitions of bacterial species is difficult because establishing species boundaries requires demarcating the diversity of strains within a species.Buchanan, R.E. rlntern
at+onal Bulletinof Bact
eriological Nomenclature
andTaxonomyJ, 15:25-3
2 (1965)) The practical application of the species definition to the identification of unknown bacterial strains requires the selection of appropriate probes, such as substrates and conditions for detecting phenotypic attributes, and radioactivity from conspecifics. DNA labeled with is required. It is necessary to use screening methods to temporarily confirm the strain so that appropriate probes can be selected to identify the strain. The challenge is to precisely delimit species, and preferably to do this using standard probes that provide species-specific information.

Species definition is then directly and equally applicable to the identification of unknown strains.

Buchanan's Manual of Deterministic Bacteriology (Buchanan, R.B. and
Gibb.

ns, N.E., editor, 1974. 8th edition, Will
lians & Witkins, Baltimore) is the easiest to understand bacterial taxonomy, especially nomenclature, basic strains,
Related literature etc. are shown. However, this is only a starting point for species identification. because,
This is especially true because it is outdated, space-limited, and the description of species is too simple.

(Reference example: Brenner D, J (Brenner D,
J) rManual of C11nical M
icrobiology J 3rd edition, American Society for Microbiology, 3rd edition, D, C, 1980, pages l-6).

When applied to bacteria, the term ``species'' refers to different types of organisms that have distinguishing characteristics and that generally have close similarities to each other in essential organic organizational characteristics. It has been defined as a group of organisms. The problem with these definitions is that they are subjective (Brenner, ibid., p. 2). Species have also been defined simply on the basis of host range, pathogenic sugar species that produce gas during sugar fermentation, and whether they are fast or slow sugar fermenters.

In the 1960s, numerical bacterial classification (also called computer classification or genetic phenotyping) became widely used. Numerical taxonomy is based on the genetic potential of an organism (
It is based on testing as much potential as possible. By classifying strains based on a number of characteristics, it is possible to form groups of strains that have a certain degree of similarity, and these can be considered as species.

However, this method of species definition is not directly and practically applicable to the identification of unknown strains, since tests that are useful for characterizing one species may not be useful for the next species. Even though this can be partially overcome by choosing attributes that are likely to be species-specific, as these attributes can be used for confirmation of unknown strains, this type of definition method has indirect applications. (Br
Enner, ibid., p. 2-6). Moreover, common methods suffer from a number of problems when used as the sole basis for establishing species, including the number and nature of tests that should be used, and how well they should be used. How and how much similarity should be selected to reflect the relevance that should be evaluated, and whether the same similarity criterion can be applied to all groups. Hugh R0H (Hugh, RlH) and Giriage G, L
(Giliarcli, G, L) rManual
of (:lin+caMicrob+ology
J 2nd edition, American Society for Microbiology, Washington D.C., 19
74, p. 250-269, lists minimal phenotypic traits as a means of determining species of bacteria using fractions of the genome.

By studying a large and randomly selected sample of strains from one species, we select the attributes that are most highly conserved or common to a large number of strains;
Species can be defined.

The use of minimal characteristics is an advance, starting with a screening method to tentatively identify the strain and allowing suitable additional media to be selected. The known attributes conserved in the species are then examined, with the expectation that the strain will have most of the minimal characteristics. Some of the minimal characteristics are not present in all strains of the species. A related idea is the comparative study of species types, neotypes or confirmed control strains of the genus. This verification is necessary because each laboratory uses different media and methods, and the strains are the seeds! Semi(st)
and andard), not a method.

A molecular-level approach to bacterial classification involves combining two genomes with DNA-DNA reassociation.
). The genetic definition of a species includes the assumption that strains of a species are more than 70% related. Strains can be identified by DNA-DNA repairing only if the radiolabeled DNA probe and the unknown DNA belong to the same species. However, the practical application of this 70% species-unique definition is limited by the need to select appropriate probes. This may be partially overcome by selecting phenotypic attributes that appear to correlate with the repairing group, but if these are used alone, the The definition is still only applied indirectly.

Brenner, op.
It is described as a "black box" analogous to mass spectrophotometry analysis, which is compared to a standard pattern (ething).

However, Brenner says that although restriction endonuclease analysis can be done to determine the general sequence of isolated genes, ``we do not have a suitable black box, especially one suitable for use in a clinical laboratory, at hand.''"Idon't think I'll get in," he admits. His words apply equally to all species of organisms.

This brief review of the prior art provides useful information for identifying unknown bacteria and other organisms, quickly classifying them, and in particular for identifying pathogenic organisms or organisms that produce exploitable biochemical reactions. It is concluded that a fast, accurate and reliable method is now needed. The method must be universally and easily available in clinical laboratories, must not depend on the number of tests performed or the subjective biases of clinicians, and must not depend on historical or Target or inevitable trial and error method (T
do not rely on real and error methods. Furthermore, it is useful for the identification and differentiation of genera and species of any living organism, and is useful by veterinarians, plant growers, toxicologists, animal breeders, entomologists, and in other related fields where such identification is necessary. It is also necessary that the method be easy and reliable to use.

[Problem to be solved by the invention]

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

Another object of the present invention is to provide a method for identifying organisms, such as bacteria, using the organism's genome.

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 causes of diseases in animals or plants can be characterized and identified. be.

Another object of the present invention is to provide a variety of products useful for the classification methods described above.

SUMMARY OF THE INVENTION These and other objects of the invention were achieved by providing the following method, as will be more readily apparent below.

As a method of characterizing an unknown organism, ribosomal RNA information obtained from the organism, from the probe organism, or derived from the organism, containing nucleic acids and hybridization sequences are re-paired with restriction endonucleases. Digestion DN
A method comprising comparing the chromatographic pattern of A with equivalent chromatographic patterns of at least two different known organic volumes.

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

A method for diagnosing infection with a pathogenic organism in a sample comprising identifying the organism in the sample by the method described above.

The invention proposes that if a species is a collection of individually isolated strains related to a common speciation event, then the similarities shared by strains objectively delimit the species, despite dispersal. This is based on the inventor's recognition that there must be a certain gender, and that the strains of a species must have structural information that can provide clues to their common roots. The past history of an organism is most often described in semantics.
s), remains in DNA and RNA (2u
kerkandle, E.

and Pauling, L., Journal o.
f Theoretical Biology J Volume 8:
357-366 (1965)), the inventors concluded that an experimental approach to exploiting the information contained in some conserved genes is to use rRNA. Ribosomal RNA has structural and functional roles in protein synthesis.
Retical Biology J Volume 70: 215
-224 (1978)) and the general conclusion from rRNA-DNA hybridization studies is that the basic sequence of ribosomal RNA genes is less susceptible to change during evolution than most other genes; (Moore, R.L., author.
rCurrent Topics InMicrob
"Iology and Immunobiology"
, Volume 64, 10512B (1974), Spri
ng-Verlag, 2U-York).

For example, the primary structure of 16S rRNA from numerous bacterial species was deduced from oligonucleotide analysis (FOX, G.E. et al., rlr+
cerna + anal Journal of Sys
tematic Bacteriology”, No. 27
Volume: 44-57 (1977)). There are negligible differences in the type 163 oligomer catalogs of several E. coli strains (Uchida T, et al.;
Journal of Molecular E
volut+on” Volume 3: 63-77 (1974)
), but substantial differences between species can be used to create bacterial phylogenetic taxonomies (Fax, G, E, r
Scienca J 209 Volume 2 457-46
3 (1980)).

Different strains of one bacterial species are not necessarily identical, and restriction enzyme maps show that different EcoRI sites occur in the rRNA genes of two strains of E. coli (Boros, 1.^, et al. N
ucleic Acids Re5earch” Volume 6:
1817-1830 (1979)). Bacteria are preserved rR
They appear to share the NA gene sequence, and other sequences can vary (FOX, 1977, supra).

The inventors further show that restriction endonuclease digests of DNA contain combinations of fragments containing rRNA gene sequences that are similar in strains of one species of organism (e.g., bacteria) but different in strains of other species of organisms. We also found that, despite strain variation, a specific combination of enzymes of restriction fragments with a high frequency of occurrence and minimal common genotypic characteristics determines the species. This is the essence of the invention. The inventors believe that this method can be applied to pronuclear cells, whether they are the same or different in the taxonomy (classical).
It has also been found to be general in that it can be applied to both prokaryotic and eukaryotic DNA, using ribosomal nucleic acid probes from organisms other than those to be identified, regardless of their eukaryotic nature.

The present invention provides an objective method for identifying organisms based on detecting DNA fragments containing ribosomal RNA gene sequences in restriction endonuclease digests. Detection is performed 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 interpreted to include "identifying") characterized by the process of the present invention is meant virtually any organism that by definition contains DNA. In this regard, it is useful to refer to traditional classification tables for reference.

Includes plants and animals, such as Monera
a) Mycobacterium (myxobacterium)
ia).

spirochetes, eubacteria, rickettsii
? - (rickettsiae) ail fission products (bacteria) and blue-green algae: cyanophyta
ytha). In the plant kingdom, there are euglenoids (Euglenophta), green algae: Chlorophyta, and RiD.
D 7 ch 1 orophyceae and charophyceae x w
4. chrysophata, xanthophyceae, chrysophyseae, bacillus 1-ariophyc
eae) class; pyrrophyta Dei/7 Dinoflagellates
), brown algae: phaeophyta
); Red algae: Rhodophyta
; slime mold, Myxomycophyta (Myxomycop
hyta), myxomyceta
es) acrasiaes, plasmodiophoreae,
labyrinthuleae
w4; Eumycophita: Fungi
yta), phycomyceta
es), ascomycetes
), bas idomycet-es
)*;Ii liverwort, hepaticae
, Anthocerotae ,
Musci 194; vascular plants Tracheophyta, Psi 1aps 1da, Lycopsyda.

sphenopsida, pteropsida, sp.
ermopsida) subfamily.

cycadae, zinc colloid ink
goae), ]nihere (coniferae),
Gnetae and Angyrospermae (
angiospermae) i, decotyledonae (di
cotyledoneae), monocotyloedoefu (
The subclass (manocotyloedonae) is described. Animalia Niha, subkingdom Protozoa, phylum Protozoa.

Subphylum Plasmodroma, order Age I la ta, Sarcodina and Sporosa.
ozoa) a; Ciliophora (cil+.

phora) subphylum, syriata (cil + aja) w4
; subdivision Parazoa, Polyhera (phylum Porifera)
a), calcarea w4. Class hexactinellida and desmospongiae wll
subkingdom Mesozoa, phylum Mesozoa; subdivision Metazoa Division Radiata, coele
phylum hydrozoa
, cyphozOa, Udosia (
authozoa) class, Stenophora (ctenop
hora), tentaculat
a) and nuda I: protostomy (pr
otostomia) Division Platyhelminthes Phylum platyhelmintes.

tubellana, tremato-da and ce
i! : Nemertina (nemert 1)
na) phylum, acanthocephala
-ala) phylum; T shell mink (aschelmint)
les), rotifera, gastrotr 1ch-a, kinorhyncha, pri
apu 11da), nematoda
) and nematomorph 7 (nematomorpha
); phylum encoprocta; phylum ectOprOcta, gymnolaemata and phylactolaemata @; phylum phoronida; phylum br.
phylum aciopoda, inarti
culata) and alticulata (articulata)
ata) ill: Mollusca mollusc (mollusca)
usca), phylum alIlphine
ura), monobutyrophora (monoplacoph)
ora), gastropoda,
scaphopoda, pelecypoda and cephalop.

da) class; sipunculida
Phylum: Ech iurida, Annelida.

polychaeta, oligochaeta and hirdinea (h
irudinea) 1lIl; Onychophora (ony
chophora) phylum; tar-di
grada; phylum Pentastomida;
oimida) phylum; Arthroboda (arthropo)
da) phylum; phylum trylobita, subphylum chelicerata, xiphosura, arachimida
hmida), Pycnocomida (pycnogom)
+da) class, 7 mandibula
ta) Aphylum, Crustacea, Chilopoda, Diploboda (d
iplopoda), pauropoda
a), symphyla w4. colembola, brothula (pr)
otura), diplura, thysanura, Emerida (ep
hemer 1da).

odonata, ordobutera
thoptera).

Dermaptera, embiania.

Plecoptera, zoraptera, corro
dentia), ? Mallophaga (mallophaga)
ga), T noplura, thysasnoptera, hemip-tera, neurolobutera (ne
uroptera), Coleobtera (co 1e)
optera), Hymenoptera (hymenoptera)
tera).

Mecoptera, Siphonaptera, Diptera
era), ) trichoptera
a) and insects of the order Lepidoptera;
ia) Division animals, chaetognat
ha) phylum, Echinodermata (ech+nodermat
a) phylum, tarnoidea,
asterodea, ophiuro+dea, ech+noidea, and holoturoidea ill.

phylum ρogonophora, phylum hemichordata, classes enteropneusta and pterobranchia; phylum chordata, uroD Jl/data (
urochordata) subphylum, Acideaceae (asc)
idiaciae).

Thaliaceae, class Larvacea; subphylum Cephalochordata.

subphylum vertebrata, agnatha, :lndrikichix (cho
ndrichthyes), osteochthys (ostei
chthyes), saccopte
iygii) subclass, crossopteridi
erygii) and diρnoi orders,
Anchobia (amphibia), Lepicillia (
repitilia), Aves and Mammalia w4. Protothyria (pr.

totheria) subclass, theria subclass, marsupialis,
InsectlVOra, DermobuteraJ, Baby Lobterra
ch 1roptera), Primatis<pr
+ma ces), edentata
, Funari Dota (pholidoLa) ,
lagomorph 7, rotent + a, cet
aceae), carnivora (carnivora)
.

tubulidentata,
Probosicdea 7 (probosicdea),
Hyracoidea (hyraco 1dea).

sirenia (sirenia), perisodakunara (p.
Mention may be made of the orders er+5sodactyla and art+odactyla. There are still families, tribes, genera, species, and even subspecies under the eyes.
It is known that there are taxa, strains, or individuals outside the subspecies. Additionally, there are cell taxa and strains or individuals. Moreover, cultured cells (plant or animal) can also be identified as well as viruses. These classifications are used in these eight applications for descriptive purposes only and are not intended to be used in a limiting manner. The organism identified may be known or unknown, but most commonly unknown.

Functionally, for the purposes of the present invention, it is convenient to divide all organisms into eukaryotic and prokaryotic cell groups. Once the organism has been identified, the DNA that is subjected to endonuclease digestion is that which is present within the cell or within uncompartmentalized chromosomes. If identified as a eukaryotic organism, nuclear DNA or organelle DNA (
Endonuclease digestion may be performed in the same manner using phloemone DNA or chloroplast DNA.

Briefly, rRNA gene sequences, classification below the multispecies level, or subspecies subclassification (infrasubspecies)
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 create the cific 5ubdivision). DNA is extracted by methods well known to those skilled in the art. DNA is cut into fragments by restriction enzymes at specific sites. The fragments are separated by size using standard chromatographic systems such as gel electrophoresis. The gel is stained and normalized for fragment size using a standard curve made of fragments of known size, using methods well known to those skilled in the art.

The separated fragments are then processed into Southern
spot method (Souti 廂E, M, rJourn
al of MolecularB+ology J
, 38:503-517 (1975), herein incorporated by reference), to cellulose nitrite paper and covalently bonded thereto by heating. rRNA
Fragments containing gene sequences are then localized by their ability to hybridize with nucleic acid probes containing rRNA information. Nucleic acid probes are labeled with non-radioactive substances or, preferably, with radioactive substances. When labeled with a radioactive substance, the probe may be ribosomal RNA (rRNA), synthesized by reverse transcription or, for example, by nick translation (nick trans).
Radioactive m-embrittleable DNA (rRNAcDNA) complementary to ribosomal RNA may be contained on the cloned fragments that can be labeled by cation).

Probes are obtained from randomly chosen organisms as will be seen below. Once hybridization has occurred, the hybridized fragments can be detected by selectively detecting double-stranded nucleic acids (non-radioactive 8i recognition probes) or visually, e.g. by radiotography. Make visible (radiolabeled probe). The size of each hybridized fragment is determined from the migration distance using the previously described standard curve. The amount of hybridization, the pattern of hybridization, and the size of the hybridized fragments can be used individually or in combination to identify an organism.

The pattern resulting from this hybridization can be readily compared to equivalent chromatographic patterns obtained from at least two, or even many, known standard organisms, genera, or species. After a preliminary broad classification has already been carried out (e.g. using classical classification methods), the band intensities (hybridization Comparisons can be made by (amounts) or by a combination of these. Ideally, the comparison should be made with a -dimensional computer pattern Di device, such as that used in paint-of-5ale processing.

The inventors believe that when using the above method, the chromatographic patterns of organisms of the same species are very similar, and slight differences are limited to differences between subspecies due to changes in bacterial strains, but differences between species and The differences in physical quantities (and even at higher levels of classification) are enormous.

The variation of enzyme-specific fragments between strains of a species can be exploited for various purposes, e.g.
Strains can also be typed for epidemiological purposes.

In fact, restriction enzymes can be chosen that can distinguish strains within a species.

The "probe organism" used in this study (and from which the nucleic acid probe is obtained) can be any of the organisms listed above, and can be eukaryotic or prokaryotic. The only limitation is given by the fact that the rRNA-containing probe must hybridize maximally with the endonuclease digest of [lNA] of the unknown organism.

There are four types of ribosomal RNA information-containing probes: 1) pronuclear ribosome probes (particularly rRNA obtained from bacteria), 2) eukaryotic globulular ribosome probes, 3) eukaryotic chloroplast ribosome probes, and 4) true nuclear non-
Organelle ribosome probe.

There are also four sources of DNA (digested by endonucleases): 1) prokaryotic ON^, 2) eukaryotic glomerular DNA, and 3)
Eukaryotic chloroplast ON^, 4) Eukaryotic cell nuclear DNA 0 Thus, the following hybrid representatives were created (Table 1).

Table 1 Hybrid Representative Notes (1) Eu, - Eukaryotic cells (2) - Smaller/'% hybridization The reference table for Example 4 below shows that ribosomal RNA probes are used in unknown organisms [
It shows that it can hybridize maximally with lNA. For example, to identify eukaryotic organisms consisting of
It may be digested with an endonuclease and the digest hybridized with a prokaryotic ribosomal RNA probe or a eukaryotic ribosomal probe extracted from the organelle. Similarly, to identify prokaryotic organisms, species-specific cellular DNA can be extracted, digested with endonucleases, and the digest can be used with prokaryotic ribosomal RNA probes or eukaryotic ribosomes extracted from organelles. 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-organelle eukaryotic ribosomal probes.

Eukaryotic cells may be defined by one or some combination of the following: a nucleus, a medulla, or in some cases a leaf margin system. These cross-hybridizations, as previously described, indicate that rRNA nucleic acids extracted from eukaryotic organelles are linked to pronuclear rRNs.
Although it has broad homology with A nucleic acids, nuclear-extracted eukaryotic DNA
This is based on the fact that such homology does not exist extensively between A and pronuclear DNA.

In summary, because the genetic code is universal, because all cells (eukaryotic or prokaryotic) require ribosomes to translate the coded information into proteins, and because ribosomal RNA is highly Because it is preserved, the crossover
Sufficient homology exists between the hybridizing elements ("+" in Table 1) to ensure that the method is effective.

The choice of the DNA to be digested and the associated ribosomal probe pair is arbitrary and will depend on the organism to be identified. That would depend on the question being asked. For example, eukaryotic cells (
When detecting pronuclear cell species (e.g. bacteria) present in animals or plants, select pronuclear ribosome probes and process under conditions where organelle-derived DNA is either not extracted or extracted in minimal amounts. do it. Using this approach, one can be confident that there is minimal interference between organelle-derived DNA and pronuclear []NA. When eukaryotic species (which are not infected by prokaryotic cells) are identified with prokaryotic ribosome probes, organelle-derived DNA can be isolated, for example by separating the organelle from the nucleus and then extracting only the organelle DNA. It is best to maximize the concentration of If you want to identify a eukaryotic organism that has been infected by a prokaryotic organism,
It is best to use ribosomal probes derived from non-organelle, non-pronuclear cells. This is because this ribosomal probe does not hybridize well with DNA from pronuclear cells.

the same kingdom, or the same subkingdom, or the same division, or the same phylum, or the same western phylum, or the same class, or the same subclass, or the same order;
or a pair from the same family, or the same tribe, or the same genus (DN
A and ribosome probes) are preferably used.

It is particularly preferred to use prokaryotic 11a IJ bosomal probes (eg, bacterial ribosomal probes) to hybridize with pronuclear DNA. In this way, genera, species and strains of prokaryotic organisms could be detected, quantified and identified. One of the most preferred pronuclear ribosome probes is derived from bacteria, particularly from Escherichia coli due to its ease of use and availability. Ribosomal probes obtained from E. coli can be used for the identification of any organism, especially all prokaryotic organisms, and are most preferred for the identification of all genera, species and strains of bacteria. Another particularly preferred embodiment is to use eukaryotic ribosome probes from a given family of to identify eukaryotic organisms of the same family (e.g. mammalian to identify mammalian organisms). (using ribosome probes). Most preferred is to use DI4A with a ribosomal probe obtained from an organism of the same subfamily and/or order and/or tribe (e.g., to identify a species of mouse, use a ribosomal probe derived from mouse). ).

The most sensitive and effective pairing systems are those in which the sources of the ribosomal probe and the source of the restriction DNA are evolutionarily less distant or less dispersed.

The individual steps involved in this technique are generally known to those skilled in the art. They will now be described with reference to both eukaryotic and prokaryotic cells (where applicable), or, where there are any technical differences, separately for each type of cell.

The first step is the extraction of ONs from unknown organisms.

Nuclear DNA from eukaryotic cells can be selectively extracted by standard methods known to those skilled in the art (e.g. Dr.
ohan, W et al, rBiochem
, Biophys.

ActaJ, 52H1978), 1-15,
(Inserted here as a reference). organelle D
Since NA is small and circular, it is called spooling (spoo).
l ing) method helps to separate non-circular nucleated DNA from circular, organelle-derived DNA. As a result, the unspooled material contains organelle-derived DNA, which can be individually separated by density gradient centrifugation. Alternatively, the globules (or chloroplasts) can be separated from the crushed cell mixture and the purified globules (or chloroplasts) fraction can be isolated from organelle-derived DNA.
The purified nuclear fraction is used for the preparation of nuclear DNA. (For example, Bonen L.

and Gray M, II rNucleic
Ac+ds Re5earcJ 8:31
9-335 (1980)).

Pronuclear DNA extraction is also well known to those skilled in the art.

For example, unknown bacteria present in a medium such as an industrial fermentation suspension, an agar plate, a plant or animal tissue or sample, etc. are treated under well-known conditions designed to extract high molecular weight DNA. . For example, cells of an unknown organism are suspended in an extraction buffer, lysozyme is added thereto, and the suspension is incubated. Cell disruption can be further promoted by adding detergents and/or increasing temperature.

Protea-1! ! After the reaction, chloroform/phenol extraction and ethanol precipitation are performed to complete the DNA extraction.

Another extraction method, much faster than phenol/chloroform extraction, uses ethanol precipitation to quickly separate DNA. This method is preferably used to isolate DNA directly from colonies or small volumes of liquid culture. This method is described by Davis.

Ro W, et at, author “＾ Manua
l for Genetic Engineering
g, Advanced Bacterial Gen
etics” (hereinafter referred to as “Davis”), Co1
d Spring Harbor Research Institute, Co1d S
pring Harbor, 2, - York, 1
980. p, 120-121.

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

Digestion of the extracted DNA is performed with restriction endonuclease enzymes. Any restriction endonuclease enzyme can be used, provided, of course, that it is not obtained from the same species of organism as that to be ascertained.

This is because if this condition is violated, the DNA will remain completely intact (this would eventually identify the organism, since the enzyme is its own origin). (This is because it is not expected to cut the DNA obtained from the seeds.) Since the organism species to be characterized may be unknown, obtaining a digest of fragments imposes a minimal amount of trial and error, which can be routinely performed by one skilled in the art without unnecessary experimentation. It is a work that can be carried out.

An example of a possible restriction endonuclease enzyme is Bg
l I, BamHI.

EcoRI, Pst I, Hind [I, B
al1. HgaI.

Sal I, Xba I, Sac I, Ss
t I, Bcl I, Xh.

r, Kpn I, Pvu II, Sau I[I
a, others.

See also Davis, ibid., p. 228-230. Mixtures of one or more endonucleases can also be used for digestion. Typically, the DNA and endonuclease are incubated together in a suitable buffer for a suitable period of time (within the range of 1 to 48 hours, at a temperature range of 25°C to 65°C, preferably 3°C).
Incubate at 7°C).

The resulting chromatographic pattern was
or more will depend on the type of endonuclease and will be endonuclease specific.

Therefore, care must be taken as to which enzyme (one or several) is used for digestion. This is because the comparison patterns used in the catalog must be made using the same enzyme or enzymes.

After endonuclease digestion, the incubation mixture containing fragments of various sizes is separated by a suitable loftography method. Any method that allows for size separation of nucleic acid digests and subsequent hybridization with nucleic acid probes can be used. Preferred is gel electrophoresis, particularly preferred is agarose gel electrophoresis. In this device, the DNA digest is subjected to electrophoresis, usually in a suitable buffer, and the gel is usually soaked in an ethidium bromide solution and possibly UV-treated to visualize the added standard marker fragments. Placed in a light box.

After separation and visualization, the DNA fragments were transferred to nitrocellulose filter paper by the Southern method (rJo
urnal of Molecular B+oloH
yj 38:503517 (1975)). This transfer can take place after a denaturation and neutralization step, and is usually transferred from the gel to the filter paper over an extended period of time (approximately 10-20 hours) or by the application of electrical force. Equipment that facilitates transfer from gel to filter paper is commercially available. The received nitrocellulose filter paper is then heated at high temperature (60-80°C) for several hours to bind the DNA to the filter paper. The probes utilized for hybridization of the digested DNA fragments bound to the filter paper are nucleic acid probes, preferably derived from a given well-known organism. It can be either detectably labeled or unlabeled, with detectably labeled being preferred. In this case, the nucleic acid probe is a detectably labeled ribosomal DNA (rRNA) obtained from such an organism, 'J
Either a truncated translated labeled DNA probe containing bosomal information, a cloned DNA probe containing ribosomal information, or a detectably labeled DNA that is complementary to ribosomal RNA from the probe organism (rRNAcDNA). Depending on the choice of combination, the ribosomal probes can be from prokaryotic or eukaryotic cells (cytoplasmic or organelle-derived). Most preferably, the detectable label is a radioactive label such as radioactive phosphorus (eg 32P, 3H or 14C). Nucleic acid probes may be labeled with metal atoms. For example, uridine and cytidine nucleotides can form covalent mercury derivatives. Nucleosides combined with mercury
Triphosphate is a good substrate for many nucleic acid polymerases, including reverse transcriptase (Dale et at., rPro
ceedings of the National^c
``ademy of 5 sciences'' 70°223g
-2242, 1973). Direct covalent mercurylation of natural nucleic acids has been reported (Dale et al.,
rBiochemistry J 14: 244
7-2457). Re-annealing (re) of mercurylated polymers
The annealing properties are similar to those of the corresponding mercury-free polymers (Dale and Ward).

rBioche+n1stry” 14: 2458-
2469) o Metal-labeled probes can be used for example in photo-acoustic spectroscopy, X-ray spectroscopy, e.g.
Detection can be by radiation fluorescence, X-ray absorption or photon spectroscopy.

Separation of rRN from eukaryotic and prokaryotic cells is well known to those skilled in the art. For example, to prepare rRNA from eukaryotic cytoplasmic ribosomes, RNA can be extracted from whole cells or ribosomes, separated by sucrose gradient centrifugation, and type 185 and 28S fractions collected using markers of known molecular weight. (e.g. Perry, R,P.

and Kelly, D.E., “28S and 18
S Sustained synthesis of 5S RNA when ribosomal RNA production is inhibited by small amounts of actinomycin D
J, Ce1l.

Physiol, 72:235-246 (1968)
, listed here as a reference). As a corollary, organelle-derived rRNA is isolated and purified from organelle fractions in a similar manner (e.g. Van
Etten R,A.

at al., rcelJ 22:157-170
(1980), or Bdwards, K. et.
al,, rNucleic Ac1ds Re5er
ch J.

9° 2853-2869 (1981)).

If radiolabeled ribosomal RNA probes are used, they can be obtained from probe organisms grown or cultured in nutrients containing appropriate radioactive compounds, or in culture media containing such compounds. Separated. When the probe is complementary DNA (rRNA cDNA)
, which is the rRN isolated from the probe organism.
It is produced by reverse transcription of A in the presence of radioactive nucleoside/triphosphate (for example, 32P-nucleoside or H'H-nucleoside).

Labeled ribosomal probes may also be obtained from truncated translated DNA molecules, particularly whole circular DNA from organelles. Specifically, the annular D of the chloroplast or the thread body
NA is cleaved and translated in the presence of a radioactive label, resulting in a labeled DNA IJ bosome probe. Chloroplast-labeled probes hybridize best with chloroplast DNA, and globulin-labeled probes hybridize best with chloroplast DNA.
It would hybridize best with A. Chloroplast (or chloroplast) incision translation-labeled ribosome probes will hybridize second best to globulin (or chloroplast) DNA. It will also hybridize with whole plant (or animal) DNA, although in a less favorable manner. Ribosome probes may also be obtained from the nuclear DNA of eukaryotic cells by truncated translation, although this method cannot be said to be good from a practical standpoint. A more useful approach to achieving this is
The rRNA gene is excised from the nuclear DNA of eukaryotic cells (by restriction enzymes), the fragments are separated, the ribosomal gene sequence is identified (by hybridization), and the ribosomal gene sequence is separated (by electrophoresis). The isolated rRNA sequences are then recombined into a plasmid or vector and transformed into a suitable host.
nsformat 1on) and then cloning can be performed in a 32P-containing medium. Another method is to propagate the transformed host, then isolate the DNA and label it by cleavage translation, or isolate the DNA and label it with rR
The NA sequence is excised and then labeled.

The obtained ribosomal probe is the same as rRNA cDNA.
hybridize in the state (see below).

Preferred nucleic acid probes are rRN from the probe organism.
This is radiolabeled DNA complementary to A. Specifically, the rRNA separates the ribosome from the probe organism;
Individually separate and appropriate RNA substantially purified as described above. The ribosomal RNA thus obtained is free of ribosomes and contains little other RNA such as transport RNA (tRNA) or transfer RNA (mRNA). Prokaryotic cell rRNA normally contains three subgroups. The so-called 5S and 16S.

and 23S-fragment. Reverse transcription to cDNA is performed with a mixture of all three or alternatively with a mixture of 16S and 23S fragments. Although it is possible to perform reverse transcription using only one of these components, it is not very preferable.

Eukaryotic rRNAs usually fall into two subgroups, 18S
Including type and 28S type. Reverse transcription into cDNA is then carried out with a mixture of 18S and 28S fragments or each of these.

Pure rRNA, containing little other types of RNA, is incubated with reverse transcriptase, which can be reverse transcribed into cDNA. More preferred in this case is incubation with reverse transcriptase from avian myeloblastosis virus (AMV) in the presence of a primer such as conidic thyroid DNA hydrolyzate. The mixture must contain suitable deoxynucleoside triphosphates, at least one of the nucleosides being radiolabeled, for example with 32P. For example, deoxycytidine 5'-("P), deoxythymidine 5'("P), deoxyadenine 5'-("P
), or deoxyguanidine 5'-(”P)・
Triphosphate is used as a radioactive nucleoside. After incubation for 30 minutes to 5 hours at 25°C-40°C, extraction with chloroform and phenol, centrifugation and chromatography, the radiolabeled fractions are combined into a cDNA probe. Radioactive i-embrittled cDNA probes in substantially pure form, i.e., free of unlabeled molecules, free of cDNA complementary to other types of RNA, free of proteinaceous material, and free of cellular components such as membranes, organelles, etc. Radiolabeled cDNA probes that do not contain radiolabels also constitute an aspect of the invention. Preferred probes are labeled rRNA cDNAs of prokaryotic cells, most preferably labeled rRNA cDNAs of bacteria. The species of probe is a bacterial microorganism, such as Enterobacter lacea (Enterobacter lacea).
e), Pulsella (3ruce l la), Bacillus (
Bacillus 1us), Pseudomonas (Pseudomonas)
domo-nas), Lactobacillus (Lactobacillus)
cillus), Haemophilus () Iaemoρh
illus), Microbacterium (M 1croba
cterium), Vibrio, Neisseria, Batatroides (B.
species in the family (Leg 1des), and other anaerobic groups, such as Legionella (Leg 1one I).
la) etc. are used. Although the examples of prokaryotic cells in this application are limited to the use of E. coli as the bacterial prokaryotic probe organism, they are by no means limited to this microorganism.

The use of radiolabeled cDNA as a probe
It is preferred over the use of radiolabeled ribosomal RN^ due to its greater stability during hybridization of NA.

It is important to recognize that the labeled cDNA probe must be a faithful copy of the rRN, ie, the entire nucleotide sequence of the template rRNA is transcribed at every time synthesis is performed.

The use of primers is essential in this regard. That the cDNA is a faithful copy can be evidenced by the fact that after hybridization it has two properties: 1. The cDNA must be 100% protected from ribonuclease digestion to labeled rRN: And 2
, the labeled cDNA must anneal specifically to rRNA, as evidenced by resistance to S1 nuclease.

rC, R, ^cad, 5c of Perujansky M1 et al.
Paris J t286. Series D, p.
1825-1828 (1978) describes 3H-radiolabeled cDNA derived from E. coli rRNA. Note that cDNA in this study was not prepared with reverse transcriptase in the presence of primers as in the present invention, but was prepared with DNA polymerase I using rRNA pre-cleaved with ribonuclease U2 as a template. . The rRNA digestion products (by RNAse lJ2) of Perjansky et al.
It has a different base ratio than A, 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 Perujansky
It is known that the predominance of homopolymerized transcription over heteropolymerized transcription of A.
S', et al, rBiochem, Bioph
ys, Res.

Comm, J, 59:202-214 (1974
)).

To summarize, the “ribosome” probe is: a)
b) from the IJ bosomal RNA itself, or c) from the rRNAcON by cloning and/or truncated translation from genomic DNA containing the rRNA gene.
obtained from A by reverse transcription of rRNA.

The next step in the process of the present invention is to isolate 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 a covalently tiled DNA digest from an unknown organism with a hybridization mixture containing a ribosomal probe. Incubation is performed 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).
, then air dried and prepared for detection. one more,
A highly preferred hybridization, which is much faster than the above methods, is the method described by Kohne, D.
B) et al.'s rBiochemistryj 16:
5329-5341 (1977) as a reference, a room temperature phenol emulsion resealing method.

After hybridization, this method requires selective detection of properly hybridized fragments.

This detection takes advantage of the double-stranded structure of the hybridized fragments and may be performed by selective methods (in the case of unlabeled probes), by radioautograph methods, or by computerized or non-computerized methods. , can be carried out by suitable radiation scanner methods (in the case of labeled probes), which would increase the speed of detection. These methods are well known to those skilled in the art and will not be described further at this point.

The end product of this method is a chromatographic band pattern with bright and dark regions of varying intensity at specific locations.

These positions were determined by EcoRI-digested λ bacteriophage D
By subjecting it to marker separation methods such as NA,
Can be easily matched to a specific fragment size (kilobase pairs). In this way, both the relative position of the bands to each other and the absolute size of each band can be easily ascertained. Compare the unknown organism's band pattern to band patterns in a catalog or library. A catalog or library may consist of books listing patterns of at least two, but almost an infinite number, of ascertainable genera and species of different organisms. For example, the number of pathologically relevant patterns that cause human disease is estimated to be around 100, so it is estimated that a standard catalog of pathogenic bacteria would contain between 50 and 150 such patterns. A catalog of bacterial strain types for epidemiological determination systems may also be included.

The band pattern depends on the type or types of endonuclease enzyme used, and possibly on the particular organism used as the source of the radiolabeled probe (probe organism).
and the ribosomal RN used for probe preparation.
^ Composition of information (e.g. 5S, 16S or 23 of pronuclear cells)
It will depend on whether it is a type S subtype or only 16S and 23S types, etc.).

Thus, the catalog may contain a variety of enzyme-specific band patterns, with band sizes and relative intensities recorded for each probe. Binding DN bound to filter paper
As the concentration of A decreases, only the strongest bands become visible and species can be identified by the size of this or these bands. Each of these band patterns may each include a band pattern obtained with complete ribosomal RNA and/or a band pattern obtained with ribosomal RNA comprising only subtypes. All variations or permutations of the above may, of course, be utilized in the library. Moreover, for eukaryotic organisms, the library IJ- may contain patterns generated by using one type of DNA or a combination of organelle and/or nuclear DNA. The pattern of each DNA digest will depend on 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 resulting pattern 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 Di. These computer scanners have a time-of-5
ale) processes (commonly utilized "supermarket" checkout pattern readers) are well known to those skilled in the art. Ideally, a library or catalog should be stored in computer memory with the relative band patterns of multiple organisms and the absolute molecular weights or sizes of the fragments. Catalog comparison is then performed by matching the unknown pattern to 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 a standard can also indicate the amount of hybridized bound DNA and can therefore be used to estimate the extent of the presence of organisms, e.g., the presence of prokaryotic cells in eukaryotic cells. It can be used for.

If a user wishes to confirm the nature of a given organism and further identify it, such user may digest the unknown organism with a second, different endonuclease and use the resulting band. The pattern may be compared to the organism's catalog band pattern for the second selected endonuclease. This process can be repeated as many times as necessary to achieve accurate identification. However, a single analysis with a single probe is usually sufficient in most cases.

The invention and its variations have countless applications. The present invention may be used by plant growers or animal breeders to properly identify their subjects, and in clinical and microbiology laboratories, to identify bacterial, parasitic, and other organisms present in any medium, including eukaryotic cells. May be used to identify insects or fungi. In this latter case, the method is used as a standard microbial assay. This is because there is no need to isolate and propagate microorganisms. In vitro growth and characterization is currently being performed on Mycobacterium lebra (lJyc
obac-teriun 1 eprae) (the causative agent of leprosy), and obligate intracellular bacteria (e.g. Rickettsiae, Gramidi T.
Some microorganisms such as B.
B. anthracis (causative bacterium of anthracis). The nature is based on the isolation of nucleic acids, which eliminates these problems because it does not involve traditional bacterial isolation and characterization. This method appears to be able to detect microorganisms that have not previously been formally described. Furthermore, the method can distinguish between different strains of a species, which is useful for example in epidemiological typification 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 determining the nature of crop damage.

Furthermore, this method can be applied to taxa other than subspecies (e.g. plant root nitrogen enzyme genes; see: ) Iennecke H291r.
Nature J 354 (1981)), this methodology can be used to genotype and identify individual strains.

The method of the invention is preferably used for the identification of microorganisms wherever they are found.

These microorganisms may be found in both physiological and non-physiological materials. They are found in industrial growth media, culture broths etc. and are a
It will be shrunk. Preferably, the microorganism is found in a physiological medium, and most preferably, it is found in an infected animal source. In this latter example, the final law is an animal,
Particularly preferred is its use in diagnosing bacterial infections in humans. Bacterial DNA with pronuclear ribosome probes
The detection and identification of is highly selective and can be carried out without hindrance even in the presence of animal (eg mammalian) DNA. If pronuclear ribosome probes are used, choose conditions that minimize hybridization with globulin DNA;
Bands of threads can be subtracted from the pattern.

This technique can thus be used in clinical laboratories, fungi depositories, industrial fermentation laboratories, etc.

What is particularly interesting is that in addition to identifying the species and strain of the infecting microorganism, it is possible to discover the presence of some special gene sequences within 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 R-factor DNA or clonally labeled antibiotic resistance sequences to the hybridization mixture, the presence or absence of antibiotic resistance in the organism can be correctly determined (one or more bands that are not genuine will be present). .

Alternatively, filter paper that has been hybridized once may be rehybridized in the presence of added antibiotic resistance sequence probe(s). Alternatively, the unknown DNA can be divided into aliquots, with a first aliquot used for identification, a second aliquot for the presence or absence of drug resistance sequences, and a third aliquot for the toxin gene. It can also be tested by using it. Alternatively, a ribosomal probe labeled with a radionuclide (e.g. 32P) can be used in a hybridization mixture with the addition of an R-factor probe labeled with another radionuclide (e.g. 3H or 14C). You can do it. Unknown DN after hybridization
The presence of R-factor DNA in A can be tested by scanning with two types of scanners. One for species and strain identification (eg "P") and the other for drug resistance etc. (eg H or "C"). In this way, experimenters can identify genera and species, type strains, detect drug resistance, toxin production or other characteristics, or label nucleic acid sequences or probes without having to isolate and characterize microorganisms. All tests for taxa below the species level can be performed in one experiment.

Since R-factor is universal and crosses species boundaries, identification can be performed with the same R-factor probe in any bacterial genus or species (see: Tomkins,
L, S, etal, J, Inf, Di
s,, 141: 625636 (198
1) ).

Furthermore, the presence of viruses or virus-related sequences in eukaryotic or prokaryotic cells can also be detected and identified in conjunction with the methods of the invention.

rManual of C11nical Micro
biology J 3rd edition (published by ennette,
E.H.Amer Soc Microb 1980
774-778) can be identified. For example, picornaviridae, caliciviridae, reoviridae, tocaviridae,
gaviridae), orthomycoviridae (or
Th.

myxoviridae), rosemycoviridae (pa.
ramyxov 1-ridae), love doviride (
rhabdoviridae), retroviridae (r
etroiiridae), arenaviridae (arenviridae)
av i r 1 dae), corona viride (coro
nav ir 1dae) bunya viride (bunya
viridae), parvovir
idae), babobaviride (papovavir ir)
1dae) adenov ir 1d
ae), herpesvirida
e), vidoviridae and poxviridae, etc.

1) When the virus is integrated into the host DNA (ON^ virus, e.g., baboba virus de member, RN^ virus, e.g., retroviride member)
extracts high molecular weight DNA from tissue and digests it with restriction enzymes. The overall procedure is similar to that for bacteria. The choice of viral probe will again depend on the question being asked, and will depend on the degree of homology between the "blob virus" and the virus-related sequences to be detected.

In order to obtain favorable sequence homology, it will be necessary that the probe and tissue sequences are related to the same family or species of virus. In addition to the degree of sequence conservation, whether the virus germ lobe hybridizes with virus-associated sequences in host 11JA depends on the hybridization conditions, e.g., whether stringent or relaxed conditions are used. Dew. The result of hybridization will be a band or band pattern indicating that there is a viral genome integrated into the host DNA. This information is useful for predicting carcinogenesis. The probe can be any labeled complementary nucleic acid probe that includes a cloned viral sequence.

In the case of RNA viruses, for example, viral RNA
DNA can be made with reverse transcriptase using
In the case of viruses, for example, viral DNA labeled by cleavage translation can be used. Here again, multiple probes, especially differently labeled probes, can be used.

The same general characteristics apply equally to DNA and RN^ viruses. Viral genomes are relatively small;
Preferably, the precipitated nucleic acids are collected by centrifugation. All operations may be performed using the entire nucleic acid, or various operations may be performed separately. It is believed that viral nucleic acids can be concentrated by spooling away cellular RNA prior to centrifugation. This can also be used to check whether a virus genome has been integrated.

In order to hybridize a viral probe, it is necessary, or at least most preferred, that the probe be of the same family, genus or species as the unknown organism. Reaction conditions, stringent or relaxed, determine whether a given probe will hybridize to a less related genome. The probe may be a labeled clone virus sequence, or it may be the complete genome or a portion thereof.

The method described in Southern, supra, is useful for transferring large DNA fragments (about 0.5 kP or larger) to nitrocellulose paper after alkaline denaturation. This method is useful in the case of DNA viruses, but may not be useful in the case of RNA viruses. RNA can be transferred to activated cellulose paper (diazobenzyloxymethyl paper) and covalently bound and used for RNA viruses. Thomas' modification of Southern's method (Thomas, P., rProc
, Nat, Acad, Sci” USA 77: 5
201-5205 (1980)) It, RNA Oyohi small size DNA fragment for hybridization, 2) O
It can be used for efficient transfer to cellulose paper.

RNA and small DNA fragments are denatured with glyoxal and dimethyl sulfoxide and subjected to electrophoresis in an agarose gel. With this operation, 1o.

DNA fragment that is ~2000 nucleotides, and RN
A efficiently migrates and remains on the nitrocellulose paper during hybrid death. This is also useful for small ribosomal DNA fragments. Therefore, the most preferable method is to divide a specimen digested with restriction enzymes and denature some of the nucleic acids with glyoxal. Southern and Thomas manipulations will give the greatest amount of information.

2> In the case of DNA viruses, the viruses present can be identified by performing restriction analysis on double-stranded (O3) viral DNA. Single-stranded (SS) DNA
Viruses may have genomes of varying lengths. Probe that forms a hybrid (sequence information is 03-DN
A), the pattern and/or size of the hybridized fragments can be used to identify the virus.

Again, there are numerous ways to obtain complementary nucleic acid probes. For example, in the case of 03-DNA, truncated translation can be used, and in the case of 5S-ON, [INA polymerase is used for c[INA synthesis.

3) In the case of RN^ virus, RNA is not digested by restriction endonucleases (sequence information is available at 03-
(It may have been converted into DNA.) Different RNA virus genomes have different sizes, and some RNA virus genomes have more than one molecule. Thereby, RNA viruses can be identified along the base sequence detected by the probe or the combined probe. One example of a probe is cDNA synthesized using viral RNA.

When searching for infectious pathogens in a specimen, the number of pathogens can be increased by extracting nucleic acids from the specimen, or by first culturing them in culture media or cells, or by using concentration processes such as centrifugation, or all of the following. You can search directly by trying this approach.

From the present invention, it is easy to create "kits" containing the necessary elements to carry out the method. Such a kit would consist of a carrier compartmented so that one or more containers, such as test tubes or vials, can be tightly packed therein. One of the containers contains a detectably labeled nucleic acid probe, such as an unlabeled nucleic acid probe or a radiolabeled cDNA for ribosomal RNA from an organic probe (in the case of a kit for identifying bacteria, the (more preferably nuclear cell rRNA cDNA). Labeled nucleic acid probe is
It will be in lyophilized form or, if necessary, in a suitable buffer. One or more containers contain D from unknown organisms.
It will contain one or more endonuclease enzymes used to digest NA. These enzymes alone
or as a mixture, either in lyophilized form or in a solution in a suitable buffer. Ideally, the enzymes used in the kit are those for which a catalog is available. However, there is nothing to prevent users from creating their own standards of comparison when experimenting, so if a user doubts that the unknown is actually of a given genus or species. , the person can create a band pattern of the known object and compare it with the band pattern of the unknown object.

Thus, the kit may contain all the elements necessary to carry out this sub-process. These elements are 1
These include known organisms (such as bacteria) or DNA isolated from known organisms. In addition, the kit may include a brochure, or a "catalog" broadly defined as a book or brochure, or a computer tape or disk, or a computer access number, etc., which may contain plant species, mammal species, bacterial species, particularly diseased species. Contains chromatographic band patterns of various organisms of the following groups, such as biologically important bacteria, insect species, etc. In this way, the user can simply prepare a band pattern for an unknown organism and visually (or in a combinator) compare it with patterns in the catalog.

The kit may also contain probe rRNA for probe synthesis in one container, radiolabeled deoxyribonucleozide triphosphate in another container, and primers in another container. good. In this way the user can generate his or her own probe rRNA cDNA.
can be made.

Finally, the kit includes materials for carrying out the technique of the invention, such as buffers, growth media, enzymes, pipettes, plates, nucleic acids, nucleozide triphosphates, filter paper, gel materials, transfer materials, and autoradiography supplies. It may also include all of the additional elements necessary. It may also contain antibiotic resistance sequence probes, viral probes or probes with other specific properties.

Although the invention has been described generally, it may be better understood by reference to certain embodiments. It should be noted that the examples are provided solely for illustrative purposes and are not intended to limit the invention unless otherwise specified.

Materials and Methods A. Extraction of Bacterial High Molecular Weight DNA Bacterial broth cultures were spun down and cells washed with cold saline. The cells were suspended in a volume of extraction buffer (0.15M sodium chloride, 0.1M BDTA, 0.03M), pH 8.5, approximately 10 times the duram weight of the packed cells. Lysozyme 10■/l11
1 was added to give a final concentration of 0.5μ/IR1. The suspension was incubated at 37°C for 30 minutes. Cell disruption was performed by adding 25% SO3 to a final concentration of 2.5% and increasing the concentration to 60°C for 10 minutes.

After cooling in a water bath, add mercaptoethanol to a final concentration of 1%.
I added it so that it becomes . Pronase 020mg/mlO,
02M) Lys buffer (pH 7.4) was predigested for 2 hours at 37°C and then added to a final concentration of 1/d. The solution was incubated at 37°C for 18 hours. Phenol was prepared by mixing redistilled phenol 11 with redistilled water 2.5f, saturated Tris base 270-, mercaptoethanol 12- and an amount of EDTA to give a final concentration of 10-3M, and the mixture was heated at 4°C. It was prepared by separation. Use that phenol for cleaning! Solution (10-'M chloride) IJum, 10-3M BD
TA, lomM)7. pH 8,5) Tewashi ivy. Then an equal volume of fresh buffer was added. Mercaptoethanol was added to a maximum concentration of 0.1%.

The solution was mixed and stored at 4°C. Half volume of prepared phenol and half volume of chloroform were added to the lysed cell solution. This was left to stand for about 10 minutes and then centrifuged at 3400Xg for 15 minutes. The aqueous phase was removed with a 25-hole glass pipette. This extraction operation was repeated until there was almost no precipitate at the border. 1/9 volume of 2N sodium acetate (pH 5.5) was added to the aqueous phase. Two volumes of 120 tons of 95% ethyl alcohol were slowly poured along the walls of the flask. The tip of a Pasteur pipette was melted closed and used to spool the precipitated ON^. High molecular weight ON^ was dissolved in buffer (10-'M BDTA, 10'M Tris pH
7,4). DNA 711 degrees was measured by absorbance at 260 nm using 30 Mg per unit of absorbance as a conversion factor.

Restriction Endonuclease Digestion of DNA The BcoRI restriction endonuclease reaction was performed in 0.1M Tris-HCl pH 7,5,0.05M NaCl
, 0,005 M MgCl2, and 100 μg/calf serum albumin. The EcoRI reaction mixture contained 5 units of enzyme per μg of DNA;
This was incubated at 37°C for 4 hours. PST I
The restriction endonuclease reaction was performed using 0.006M)
HCl1 pH7,4,0,05M sodium chloride,
Performed in 0.006M magnesium chloride, 0.006M 2-mercaptoethanol, and 100 μg/− conidal serum albumin.

PST [The reaction mixture contained 2 units of enzyme per μg of ON8 and was incubated for 4 hours at 37°C.

Typically, 10 Mg of DNA was digested to a final volume of 40 μβ. 10 times the concentration of buffer was added. Sterile distilled water to DNA
It was added according to the amount dissolved. λ-bacteriophage DNA is used to create marker bands for fragment size determination.
Restricted by coRl. Typically 2 μg λ DNA was digested with 20 units of EcoRI to a final volume of 20 μl.

Gel Electrophoresis and DNA Transfer DNA digests were supplemented with up to approximately 20% glycerol and bromophenol blue dye. For λ DNA digests, 20 μl of lXBCORI buffer was added to each 20M1 reaction mixture. Usually 15 μl of 75% glycerol
and 5 μl of 0.5% bromophenol dye each at 40 M
1 was added to the reaction mixture.

Digested bacterial DNA 10Mg and digested λ[]N
Place 2 μg of A into a per well and cover the surface with melted agarose. Digested product 0.02M
Sodium acetate, 0.002M BDTA, 0.
018M) lis base, and 0.028M) lis
3 in 0.8% agarose containing HCf pH 8.05.
Electrophoresis was performed at 5V until the dye migrated between 13 and 16 cITl. The gel was then treated with ethidium bromide (0
,005 mg/if) and placed in a UV light box to visualize the λ fragments. [InA was transferred to nitrocellulose filter paper using Southern's method as described above. The gel was treated with denaturing solution (1,5M sodium chloride, 0.5M sodium hydroxide) for 20 minutes on a shaking table. Add the denaturing solution to the neutralizing solution (3.0M sodium chloride, 0.5M).
After 40 minutes, the gel was checked with pH paper.

After neutralization, apply 6xSSC to the gel! Solution solution (SSC=0.15M
Sodium chloride, 0.015M sodium citrate) for 10 minutes. ON^ fragments were transferred from the gel to the nitrocellulose paper by suctioning 6X SSC through the gel and nitrocellulose paper for 15 hours east of a paper towel. The filter was placed between two sheets of 3M chromatography paper, wrapped in aluminum foil with the shiny side facing out, and dried in a vacuum oven at 80° C. for 4 hours.

'32P ribosomal RNA complementary synthesis to ON ('P-
rRNA and DNA) Escherichia coli R-1323S and 16s type ribosomal RNA
32P-labeled DNA complementary to was synthesized using reverse transcriptase from avian myeloblastosis virus (AMV).

The reaction mixture consisted of 5 μl of 0.2 M dithiothreitol, 25 μl of IM) pH 8, 0, 8, 3 μl of 3 M potassium chloride, 40 μl of 0.1 M magnesium chloride, 70 μl of actinomycin, ■ 4 μl of 0.0
4M dATP, 14 μl of 0.04M clGD
P, 14 μl of (LO4M dTTP, and 96
．． It contained 7 μl of water.

The following were added to the plastic tube: 137,
5 μl reaction mixture, 15 μl conidial thymus primer (
10■/a11), 7μl H2O, 3μl rRN
A (using a concentration of 40 p g/00 units, 2.7
6 μg/μl), 40 μl of deoxycytidine 5
'-(''P) triphosphate (10 mCi/wd, and 13 μ4217) AMV polymerase (6,90 O units/μm). The fermentation reaction was carried out at 37° C. for 1.5 hours. The solution was then mixed with 5 rR1 Extracted with chloroform and prepared phenol. After centrifugation (JS 13,6
0 ORPM), the aqueous phase was directly layered onto a Sephadex ■G-50 column (1.5 x 22 cm). A plastic 10-pipette was used as a column. A small glass bead was placed on the tip, a rubber tube with pinch fittings was attached, and degassed G-50, swollen by soaking in 0.05% SO3 overnight, was added. The aqueous phase was poured directly into the G-50 along the wall and then eluted with 0.05% SO8. 0,
Twenty 5-fraction fractions were collected in plastic vials. The tube containing the peak fraction was
- Discovered using the selenium core counting method, which uses a discriminator to count each sample for 0.1 minutes and record the total count. Peak fractions were combined.

Add the finished coat to Aquesil@ (commercially available and can be done by hand),
The CPM (counts per minute) of 32P per it' was measured by a scintillation counter.

Hybridization and autoradiography The fragments containing the ribosomal RNA gene sequences were added to the DNA on the filter.
A was hybridized to P-rRNA cDNA and detected by autoradiography.

Filter the hybridization mixture (3 x 5SC10
, 1% SO8, too μg/mf denatured and sonicated canine DNA and Dynhardt's solution (0.2 μg/mf each)
% conidic serum albumin, Ficoll
, and polyvinylpyrrolidine) at 68°C.
Soaked for an hour. "P rRNA cDNA 4XlO'CP
M/m1. Add the hybridization reaction solution at 68℃
and incubated for 48 hours. The filter was then washed with 3X SSC and then with 0.1% SOS for 2 hours at 15 minute intervals or the cleaning solution was washed at approximately 3.000 cpm ”P/
a+j! Washed until it contained. Air dry the filter, wrap it in plastic wrap, and place it in Kodak X-O.
Autoradiography was performed on MATR film at -70°C for about 1 hour.

B, @Mammalian experiment Mus muschinilus domesticus (mouse) 0) r
RNA probes were synthesized from L8S and 2BS type and 28S type only rRNA. Nucleic acids were extracted and precipitated from mouse liver. High molecular weight DNA was spooled and removed. The remaining nucleic acids were collected by centrifugation and collected at 50mM MgCj!
2 and 100mM) Lys was dissolved in pH 7.4 buffer. DNA5e (without RNAse) at a concentration of 5
It was added until it became 0 μg/d. The mixture was incubated at 37°C for 30 minutes. The generated RNA was re-extracted, ethanol precipitated, and added to 101M sodium phosphate buffer pH 6.
, 8. 0.1M) Squirrel pH7,4 and 0
．． A 5-20% sucrose gradient solution of 0 IMεOT^ was prepared.

Add the sample and run the gradient for 7 hours on a 5W40 rotor for 35 minutes.
Rotated at K rpm. Fractions were collected by optical density. By comparison with known molecular weight markers 18
The S and 28S fractions were chosen.

relaxed in all mammalian experiments
Hybridization conditions were used. 54℃. The cleaning process is 54
℃ and washed three times for 15 minutes each in 3X SSC with 0.05% SO3.

Example 1 Several strains of P. aeruginosa used in this example have minimal phenotypic characteristics to identify the species (Hugh
R, )! ,, et al., rManual of
Clinica1MicroC11nica1 2nd edition,
^SM, 1974, pp, 250269) (
Table 2). Three other Pseudomonas and two cinetobacter strains were selected and their species and genera were compared (Table 3).

Table 2 Comparison of Bacterial Strains Minimum Table of P. 7 P. aeruginosa Bacterial strains used for comparison of Pseudomonas and Acinetobacter species are listed in Table 3.

Table 3 in the margin below for comparison of species and genera, Types, Neotai P, aeruginosa P, 5 tutzeri P, f 1uorescens P, putida ^, anitratus 815 10145 10332 Type 2601 1758g
Neotype 818 13523 100
38 Neodive 827 12633
Neotype 2208 19
The genus Acinetobacter spp. 606 was chosen for comparison because they share certain attributes with many Pseudomonas species.

The size of the fragments (in kilobase pairs) in the BcoRI digest is P, 5 tutzeri 16.0
．． 12.0.9.4; IP, Fluorescence (P
, f 1uorescens) 16.0. 10.0
．． 8.6.7.8,? , 0 ; P, putida (P, p
utida) 24.0.15.0,■0.0.8.9
;A, anitratus
20.0.15.0.12.5.9.8.7.8.6
, 1.5, 2.4.8.3.8.2.8 (the sizes of the three smallest fragments were not calculated); A, Rouault/
7 I (A, 1woffii) 12.0, l010
．． 9.1.7, 0.6.4.5.7.5.5.5.3.
4.8.4.4.3.6.3.2.2.9 (the sizes of the three smallest fragments were not calculated). PST I
The sizes of the fragments in the digest (in kilobase pairs) are:
P, Stouzeri 6.7.6.1.5.5; P
, Fluorescence 10.0.9.4.7.8.7
．． 0, P, putida 10.5.9.9.6.8.6
．． 3.4.4; A, Anitratus 36.0.28.
0.20.5.12.0, ■0.0.5.8.3.7.
2.6.2.4; A. Ruoffi 9.9.8.7
．． 7.2.5.7.4.0.3, 6.3.2.2.7゜P, a comparison of hybridized restriction fragments obtained from strain 711 of A. aeruginosa indicates that this species
1.9.4.7.6 and 5.9 kilobase pair (KBP
), it is determined by the EcoRI-specific combination of fragments containing the rRNA gene sequence (FIG. 1).

The 7.6KBPεcoRI fragment was found in 4 out of 7 strains in this sample. A similar situation occurs in some phenotypic characteristics of strains of species. Fragments from 7 strains [:
The fact that the coRI combination is used to separate the strains into two groups draws the inference that there are two species with minimal phenotypic characteristics of P. aeruginosa. D
From the experimental results (Fig. 2) in which NA was digested with PST l,
This leads to the conclusion that the strain variation exhibited by the fEcoR[7,6 KBP fragment may lead to intraspecific variation. Because one conserved combination of PST I fragments, 9.
There are 4.7, 1.6.6 and 6.4KBP, which define the species. 9.4 and 6.6
The PST I fragment of KBP is found in 6 of 7 strains of P. aeruginosa; 7.1 and 6.4 KB
The P PST I fragment is present in all of the sampled strains. Mutation of PST I fragment is EcoRr 7.6
It occurs in strains that do not contain the KBP fragment. RH151
has BP fragments at 10.1 and 8.2, and RH809
does not contain the 9.4 KBP fragment and has a 6.0 KBP fragment. And the type strain R)1815 is 6.6
Contains no KBP fragment. The pattern of hybridized fragments supports the conclusion that enzyme-specific conserved combinations can be used to determine species.

A strain of one species probably has many fragments in conserved combinations. Even if some strains have fragment mutations, this does not hinder identification and can be said to prove useful for epidemiological research.

EcoR in P. ruginosa strains [7,6KB
Mutation of the P fragment may be viewed prospectively by testing hybridizing EcoR1 fragments found in type strains of other Pseudomonas species (Figure 3). P., Stoulaeri, P.;

The type strains of fluorescens and P. putida are 7.
6KBP fragment, but share the same size EcoRI fragment; P. aeruginosa and P.
, Stoulaeri has a 9.4 KBP fragment and P, Stoulaeri and P, Fluorescence I;! 16 KBP fragments 'f- and P, fluorescens and P, putida have 10 BP fragments. Generally, the size of the fragments is unique to the type strain of each of the four Pseudomonas species; and the type strain of each species has a different size range of fragments. These general descriptions are in PS
The same can be said for the Tf digest (Figure 4).

When comparing the fragment patterns of each 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 that the genera are different. The two Acinetobacter species have a larger range of hybridizing fragment sizes compared to the four Pseudomonas species.

Escherichia coli, Bacillus thuringinensis
Without the help of restriction enzyme maps, such as those available for B. sthuringiensis and B. subtilis, it is difficult to determine where the enzyme cuts the rRNA gene, the number of copies per genome, and the number of genes or genes. It is impossible to predict whether there will be non-homologous flanking regions between homogeneous genes. Escherichia coli rRNAcD
The NA probe may not hybridize to some restriction fragments containing rRNA gene sequences, and if so, this may reflect evolutionary distance or dispersion between the test organism and E. coli. There is. The conserved nature of rRNA can be used to argue that this is not the case. However, this is a small problem compared to the advantage of having standard probes that are equally applicable to any unknown species.

Example 2 Comparison of restriction analysis and DNA-DNA liquid hybridization: The strains used in this study are listed in Tables 4 and 5.

Table 4B Neotypic strains of S. subtilis and lower analogues (j
strain number B of each type strain (unior synonyms), 5ubtilis 302
1 6051 Neotype B, unif
lagellatus 2990 15134
Type B, amyloliquafaciens
3061 23350 Dive Table B-354 (NR3-231) B-356 (NR5-238) NR3-265 NR3-659 NR3-73O NR3-737 NR3-741 NR3-773 NR3-1106 High molecular weight ON^ indicates each strain separated from. RM30
Liquid D using labeled ON^ of 21 and RH2990.
NA-DNA hybridization data were collected.

The results are shown in Table 6.

Table 6 Labeled DNA probes and B. subtilis strains to D
% Hybridization between NA This data shows that there are two hybridization groups. Similar data have been reported in the literature for B. subtilis (Seki et al.
-ational Journal of System
matic Bacteriology J25: 2
58-270 (1975)). These two groups are RH302
1 and R) 12990. Restriction endonuclease analysis of the ribosomal ARN^ gene is performed. The EcoRI digest (Figure 5) could be divided into two groups. The group represented by RH3021 has two strongly hybridizing fragments (2.
15 and 2.1 KBP). The group represented by R112990 consists of two strongly hybridizing fragments (2,
6 and 2.5 KBF). The BcoRl data can be used to place the B. subtilis strain in its proper place in the DNA-DNA hybridization group. DNA-DNA
According to the 70% hybridization rule, B. subtilis is actually two species. However, PST l
Considering the data (Figure 6), we can define these groups as
They can be thought of as two dispersed populations that were involved in a common ancestry or speciation event.

B. The conclusion that S. subtilis is one species correlates with phenotypic data. The bacterial strains listed in Table 5 are the Genus Bacillus by Gorton R, E et al.
”Agriculture Handbook No.
427 (U.S. Department of Agriculture, Agricultural Research Service
Washington D, C,) p36-41 identified B. subtilis. Restriction analysis can provide data comparable to DNA-DNA hybridization data, and by choosing appropriate enzymes, restriction analysis can adequately define species despite dispersion. R
H3061 is PST [Lost the site. However, the EcoRl data suggest that the strain is B. subtilis. Same thing with egI! II data (7th
Fig. 8) and Sacl data (Fig. 8).

Example 3 Table 7 B, subtilis strain B, 5ubtilis 3021 6051B,
polymyxa 3074 842 net type neotype B, poly+nyxa NR3-1105 net type B, subtilis and B, polymyxa, EcoRl data (Fig. 9), PST l data (Fig. 10) Bgl
IIl data (Figure 11 left) and Sacl data (
(Figure 11 right).

From the large differences in PST l-band patterns, it can be concluded that Bacillus polymica is in the wrong genus. Although both species produce spores, they are phenotypically dissimilar. It is certain that the type strains of B. polymica in both ATCC and NRRL culture collections have the same band pattern. However, the important data is that aporous mutants can be identified. It would be very difficult, and perhaps impossible, to identify Bacillus species if they were unable to produce spores.

Example 4 Identification of Bacterial Species in Mouse Tissues Without Isolation The Swiss mouse, Mus m
usculus domesticus (inbred strain) and Streptococcus pneumoniae (Streptococcus pneumoniae).
ptoc.

ccus pneumoniae) RH3077(A
A turbid suspension of TCC6303) was inoculated intraperitoneally. When the mice were near death, their hearts, lungs, and livers were removed. High molecular weight DNA was isolated from these tissues, S. pneumoniae RH3077 and Swiss mouse organs;
Using EcaRI to digest the DNA, rRNA
Restriction endonuclease analysis of the genes was performed.

In addition to washing the filter 3x SSC for 2x 10 min.
Washed with 0.3 x ssc g and 0.05% SO3. Autoradiography was performed for 48 hours. Data (first
Figure 2) is S.

showed that Pneumoniae is defined by seven hybridization fragments (17.0.8.0.6.0.4
．． 0.3.3.2.6 and 1.8KBP). This bacterial cDNA probe consists of two mouse DNA fragments (14,
0 and 6.8 KBP). Hybridized fragments signal the presence of S. pneumoniae in infected tissues. Heart W [lNA
All seven bands are found in the extract. Their intensity is lower in liver DNA extracts, but they can all be seen by autoradiography.

Only the 6.0 KBP band is found in the lung DNA extract.

The low number of bacteria in the lungs could be explained by the fact that the mice had sepsis rather than pneumonia. The lungs showed no hardening at autopsy.

To test the sensitivity of this assay, bacterial DNA was transformed into mouse D
It was diluted with NA and subjected to electrophoresis. 0.1 μg bacterial DN
Using A, all seven bands could be seen in the autoradiograph. For 10-3 μg bacterial DNA, 17
BP bands were observed at , 0.8.0 and 6.0.

10'S, 5XLO-3μg per Pneumoniae cell
Using the number DNA (Biochem Bio
physActa 26:68), 10-'μg is 2
Corresponds to X10' cells. This level of sensitivity is useful for diagnosing infectious diseases.

This example also shows that the bacterial probe is mouse-specific.
This shows that it hybridizes with the RI fragment (No. 9).
(see figure, fragments with 14.0 and 6.8 KBP) These fragments are mouse 18S and 28S ribosomal RNs.
Corresponds to the EcoRI fragment detected by the A probe (Fig. 14, above, the 6.8 KBP fragment is 285 type rR
(indicates that it contains an NA sequence). Bacterial probes do not hybridize very well to mammalian ribosomal RNA gene sequences, so the bands are less intense, and bacterial probe and nuclear complement RNA systems are less sensitive and less sensitive to the DNA of the infected prokaryotic cell. The selectivity for 10μ of bacterial probe per lane
In experiments with hybridization to g-digested bacterial DNA, no hybridization was found to 10 μg digested human or mouse DNA per rune, even when bacterial bands were clearly visible.

Examples 5-8 Mammalian Experiments These examples illustrate that the concept of identifying organisms by rRNA restriction analysis can be successfully applied not only to bacteria but also to complex eukaryotic organisms. be.

Figure 13 shows that the mammalian genus can be confirmed with Mus musculus domesticus type 18S and 28S rRNA probes and that several species of Mus can be distinguished. In this figure, the enzyme is PST I and the objects and their respective bands are as follows.

1. Mus musculus melosinus (Mus mu
sculus melossinus) (mouse) 1
4.5.13.5.2.62, Mus musculus domesticus (mouse) 13.5.2,6 3, Canis familialis
iaris) (dog) 12.0 4, Cavia porcellus
us) (guinea pig) 17.0.14.0.13.0
．． 8.8.5.7.4.7 and 1 band below 3.05, Crisetulu griseus
s grrs-eus) (hamster) 25.0.4
．． 76, Homo sapiens (Homo 5apiens)
) (Human) 15.0.5.7 7, Felis catus
(Cat) 20.0.9.7 8. Ratus norveg
icus) (rat) 12.5 9, Mus musculus domesticus (mouse) 1
3.5.2.6 10. Musce Serpicolo Serpicolo
rvicolor cervicolor) (mouse) 14.0.2.7 11, Muscerv cervicolor 1 Babeus (Muscerv
ic.

1or papeus) (mouse) 13.5.2.6
12. Mus pahari (mouse) 13.0.3.7 13. Mus cookiei
(Mouse) 13.5.2.6 Figure 14 shows that mouse and cat ON^ have 28S type rRNA.
cON and that the pattern of hybridized bands depends on the composition of the probe sequence. In Figure 14, the enzyme is BcoRI,
The objects and bands are as follows.

1. Mus musculus domesticus (mouse) 6
．． 8 BP 2, Felis Catas (cat) 8.3 KBP In Figure 15, the enzyme is Sacl, and the objects and bands are as follows.

1. Erythrocebus batas (Snake rythrocebu)
s patas) (batas monkey) 8.5.3. T, <
3.02, Rattus norvegicus (rat) 25.0.
9.5.3.6, <3.0 3, Mus musculus domesticus (mouse) 6
．． 8, <3.0 4, Felis Catas (Cat) 9.5.5.3.4.0
, <3.0, <3.0 5, Homo sapiens (human) 10.5, <3.06,
Macaca mulatta
(Lazas monkey) 9.8, <3.0 When comparing Figure 15 (Sac I digestion) with other mammalian figures, it can be seen that the hybridization pattern is enzyme specific.

Figure 16 shows that primates can be distinguished. Cultured cells have bands in common with the tissues of their original species, and different human cultured cells can be distinguished by the addition or absence of bands. In this figure, the enzyme is EcoRl and the objects and bands are as follows.

1. Erythrocebus Patas (Patas m) >22.0
,■1.0,? , 6.2.6 2.7 Kaka Muratsuta (Lezas Monkey>22.0, ■1.
5.7.6 3. Homo sapiens (human) >22.0.22.0,
■6.0.8.1.6.6 4, M 241/88 (Langer monkey cultured cells)
14.0.7.2.5.7 5, HeLa (human cultured cells) >13.1.6.6
6, J96 (human cultured cells)>22.0.22.0
．． 16.0.11.0.8.1.6.6 7, An (human cultured cells) 22.0.16.0.8
．． l, 6.68, X-381 (Lazas Monkey) 22
．． 0.11.5.7. Having thus fully described the invention, it will be apparent to those skilled in the art that many modifications and substitutions may be made thereto to a wide extent without departing from the principle or purpose of the invention or embodiments thereof.

[Brief explanation of drawings]

Figure 1 shows Pseudomonas aeruginosa (Pseud
omonas aeruginosa) strain of BcoRl restriction endonuclease. cDNAs for E. coli type 163 and 23S ribosomal RNA (rRNA) were used as probes. Figure 2 shows Pseudomonas aeruginosa (P, ae
rug 1nosa) Pst of DNA isolated from the strain
1 shows restriction endonuclease digestion. c for E. coli 16S type and 23S type rRNA as probes.
DNA was used. FIG. 3 shows EcoR restriction endonuclease digestion of ON isolated from a glucose-nonfermenting Gram-negative bacillus species. E. coli type 16S and 2 as probes.
cDNA for 3S type rRNA was used. Figure 4 shows Pstl restriction endonuclease digestion of DNA isolated from glucose-nonfermenting Gram-negative bacillus species. E. coli type 16S and 2 as probes.
cDNA for 3S type rRNA was used. FIG. 5 shows EcoRI restriction endonuclease digestion of DNA isolated from various Bacillus subtilis strains. cITNA for E. coli 16S type and 23S type rRNA was used as a probe. Figure 6 shows Pst I data obtained using the same probe against the same strain as in Figure 5. FIG. 7 shows BgI n data obtained using the same probe against the same strain as in FIGS. 5 and 6. Figure 8 shows Sac I data obtained using the same probe for the same strain as in Figures 5-7. Figure 9 shows B, subtilis and B, polymica (B, p
Figure 3 shows BcoRl restriction endonuclease digestion of DNA isolated from S. olymyxa. cDNAs for 16S and 23S rRNA from E. coli as probes
A was used. FIG. 10 shows Pst I data obtained using the same probe for the same strain as in FIG. 9. FIG. 11 shows Bgl I [and Sac
I data is shown. FIG. 12 shows that Streptococcus pneumoniae (Streptococcus pn.
Fig. 2 shows the detection of C. eumoniae). Figure 13 shows Mus musculus domesticus (Mu
s musculus domesticus) (
Type 18S and 28 from cytoplasmic ribosomes of mouse)
Identification of mouse species by comparing Pst I digestion of DNA isolated from mammalian tissues using cDNA for S-type rRNA is shown. Figure 14 is Mus musculus domesticus 28S.
ECORI-digested DNA from mouse and cat tissues hybridized with type rRNA cDNA probes. Figure 15 shows Mus musculus domesticus 18S.
Sac I digested DNA from mammalian tissues hybridized with type 28S and type 28S rRNA cDNA probes. Figure 16 shows Mus musculus domesticus 18S.
BcoRI-digested DNA from mammalian tissue and cell culture hybridized with type and type 28S rRNA cDNA^ probes. that's all

Claims (1)

Claims: 1. DNA (rRNAc) that is a faithful copy of ribosomal RNA (rRNA) and is detectably labeled and complementary to pronuclear ribosomal RNA in substantially pure form;
DNA). 2. The rRNA cDNA of claim 1, which is radiolabeled or metal-labeled. 3. Claim 1, wherein the pronuclear rRNA is bacterial rRNA
rRNA cDNA of term. 4. The rRNA cDNA according to claim 1, which does not substantially contain cDNA complementary to RNA other than rRNA. 5. rR according to claim 1, which does not substantially contain cellular components.
NAcDNA. 6. The rRNA cDNA of claim 1 obtained by reverse transcription rRNA in the presence of primers. 7. The rRNA cDNA of claim 6 obtained by using a DNA hydrolyzate primer. 8. Reverse transcription from avian myeloblastosis virus The rRNAcD of claim 6 obtained by reverse transcription with oxygen
N.A. 9. A kit for use in identifying a species of an unknown organism comprising a carrier tightly partitioned into one or more containers, the first container containing a probe organism or a probe. Organism-derived ribosome R
NA information-containing nucleic acids; the kit also includes a catalog having hybridized or repaired chromatographic band patterns of at least two different known organism species; A kit comprising at least two different known species of organisms or DNA derived therefrom.