JPH04126100A - Identification of an organism and method of a characterization - Google Patents

Abstract

PURPOSE: To quickly and accurately identify an organism by hybridization of a specified probe with a fragment containing the ribosome RNA information of a prokaryote.

CONSTITUTION: First, a ribosome RNA information-contg. probe A such as prokaryotic or eukaryotic mitochondria, capable of maximal hybridization with the DNA endonuclease digested product of a specimen organism, is obtained by extraction of prokaryotic and eukaryotic organisms. Secondly, a restriction endonuclease is added to a DNA obtained through extraction from the specimen organism and allowed to act at 25-65°C for 1-48 h to obtain a digested product B. Thirdly, the component B is isolated to obtain a DNA fragment C with a desired molecular weight. Fourthly, the component C is hybridized at 50-70°C with a labeled probe D obtained by labeling the component A with e.g. ³²P to obtain a hybrid E. Subsequently, the component E is subjected to radio-autography to detect the aimed organisms including prokaryotic and eukaryotic organisms.

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 Animalia.

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.
yotis). prophyte (prok)
aryotes) are eukaryotes (eukaryotes)
They are uncomplex, have no internal division by unit membranes, and lack a distinct nucleus. Genetic information in pronuclear cells is transported to the cytoplasm on double-stranded circular DNA.

No other DNA is present in the cell (except in the presence of phages, bacterial viruses, and circular DNA plasmids capable of autonomous replication). 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 nucleocytoplasmic part, 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) is thought to be derived from prokaryotes, which enter into an endosymbiotic relationship with primitive eukaryotes.
Ultimately, it is so tightly integrated with the structure of the host cell that it cannot exist independently (see examples, FOX, G
,E,et al,r 5science J 2
09; 457-463 (1980) p, 462; 5t
anier, R.Y., et al.

rThe Microbial World J 4th edition, published by Prentice-Hal 1, 1976,
p. 86). For example, DNA obtained from the mouse L cell glomerulus, which carries the liposomal RNA gene region, was obtained from Escherichia coli (Bs
Cherichia coli) liposomal RNA was demonstrated to exhibit significant sequence homology, strongly supporting the endosymbiosis model (Van Etten, R,A.

at al, rcell J 22:157-170 (
1980)). Also, Zea may be corny.
s) It has also been shown that the nucleotide sequence to type 233 liposomal DNA of chloroplasts has 71% homology with the type 233 liposomal DNA of E. coli (Edwards,
& Kossel, H, 5earch to rNucleic 8cids R' 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 infecting so-called "higher" organisms or other vectors. This is the case when attempting to accurately and reliably identify microorganisms that may cause

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 must identify infectious organisms (parasites, fungi, bacteria, etc.) in test plants and animal tissues.
and will want to accurately identify the virus. 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 as it is necessary to demarcate the diversity of strains within a species in order to establish species boundaries (Buchanan, R.E. rlntern
ational Bulletin of Bacter
iological Nomenclature an
dTaxonomyJ, 15:25-32 (196
5)) 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 the selection of dissipative substances from conspecifics. DNA labeled with is required. It is necessary to use screening methods to temporarily confirm the strain so that appropriate probes for identifying the strain can be selected. The challenge is to precisely delimit species, and preferably to do this using standard probes that give species-specific information. Species definition is then directly and equally applicable to the identification of unknown strains.

Buchanan, R, tl', an
d Gibbons, N.E., ed., 1974.
8th edition, Willians & Wil-kins
(Baltimore, Inc.) provides the easiest to understand bacterial taxonomy, especially nomenclature, basic bacterial strains, and related literature. However, this is only a starting point for species identification. This is especially true because it is outdated and too easy to describe spatially limited species.

(Reference example: Brenner 〇, J (Brenner D,
J) rManual of C1nical M
"Icrobiology" 3rd edition.

American Society for Microbiology, Washington D.C., 1980, 1-
(page 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 part of an organism. The problem with these definitions is that they are subjective (Brenner, ibid., p. 2). Also, species simply refers to host range and pathogenicity.

It was sometimes defined based on criteria such as whether gas is produced or not during the fermentation of sugar, and whether sugar fermentation is fast or slow.

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 many potentials 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 appear to be species-specific, as these attributes can be used to confirm unknown strains, this method of defining species indirectly (Bren
ner, ibid., p. 2-6). Moreover, the target method presents several problems when used as the sole basis for determining species, including the number and nature of tests to be used, how to use the tests, etc. How and how much similarity should be selected to reflect relatedness? Whether the same similarity criterion can be applied to all groups, etc.
, H (Hugh, R, H) and Giliage G, L
(Giliardi, G, L) rManual o
f Clinica1 MicroC11nica1 2nd
Edition, American Society for Microbiology, Washington, D.C. [”, 197
4. p. 250-269 lists minimal phenotypic traits as a means of determining bacterial species 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 from a screening method to tentatively identify the strain, 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 strain is the standard for the species.
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. A bacterial strain can be identified by DNA-DNA repairing only if the radiolabeled mRNA 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 isolated genes and restriction endonucleases for general sequencing.

However, it is unlikely that a suitable black box would be available, especially for use in open-air laboratories. ” he admits. His words apply equally to both species of organisms.

From this brief review of the prior art, we will identify and rapidly categorize unknown spores and other organisms, particularly identifying pathogenic organisms or organisms producing biochemical reactions that may be exploited. We conclude that there is now a need for a fast, accurate, and reliable way to do this. The method must be universally and easily available in clinical laboratories.

Depending on the number of trials conducted and the subjective bias of the clinician, it is also possible that past, accidental or consequential Pg methods (Tri
There is no point in depending on the error method). Additionally, it is useful for the identification and differentiation of R. spp. of any living organism, and is useful for veterinarians, plant cultivators, toxicologists, animal breeders, entomologists, and others for whom such identification is necessary. It is also necessary that the method be easily and reliably usable in the relevant field.

[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.

[Means to solve the problem]

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

As a method of characterizing an unknown organism, liposomal RN^ information from or derived from the probe organism obtained from the organism - a restriction endonuclease hybridized or re-paired with a supposed nucleic acid. -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 a pathogenic organism infection 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 the strains of a species must have structural information that can be used as clues to find their common roots. The past history of an organism is most often expressed as a semantic
s), DNA and RNA (Z)
uckerkandle, E.

and Pauling, L., Journal o.
f Theoretical Biology J Vol. 8: 357-366 (19135)), the inventors concluded that an experimental approach to exploiting the information contained in some conserved genes is to use rRNA. Liposomal RNA has structural and functional roles in protein synthesis.
ret-+cal Biology J Volume 70: 21
5-224 (1978)), rRNA-DNA
The general conclusion from hybridization studies is that the basic sequences of liposomal RNA genes are less likely to change during evolution and are likely to be more conserved than most other genes (Moore et al. , R.L., rcurrent Topics InMicr
obiology and immunobiolog
y”, vol. 64, 105-128 <1974>,
Spring-Verlag, =y-:l-ri).

For example, the primary structure of 16S rRNA from numerous bacterial species was deduced from oligonucleotide analysis (Fox, G.E., et al., rlnt
ernationalJournal of Syst
Bacteriology, Volume 27: 44-57 (1977)). 1 of several strains of E. coli
There are negligible differences in the type 63 oligomer catalog (UchiclaT, at al; rJour
nal of Molecular Evolution
3: 63-77 (1974)), but substantial differences between species can be used to create bacterial phylogenetic classification tables (FOX, G, E, Science J.
209:457-463 (1980)).

Different strains of one bacterial species are not necessarily identical, and restriction enzyme maps show that different EcoRl sites occur in the rRNA genes of two strains of E. coli (13oros, 1.^, at al. Nuc
leicacids Re5earcJ5th earcJ
17-1830 (1979)). Bacteria appear to share a conserved rRN octogen 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 taxonomy (classical);
It has also been found to be general in that it can be applied to both prokaryotic and eukaryotic DNA, using liposomal nucleic acid probes from organisms other than those to be identified, regardless of their eukaryotic nature.

The present invention provides an objective method for defining an organism based on detecting DNA fragments containing liposomal 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, we mean 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, Rickettsia + -
(rickettsiae) 1liil fission products (bacteria) and blue-green algae: cyanophyta
ytha). The plant kingdom includes the euglenophta, the green algae Chlorophyta, the ch 1orophyceae and the charophyceae w! , chrysophta, xanthophyceae, chrysophyseae, bacillariophyceae; pyrrophyta, Dinofragerata lagellates), brown algae: phaeophyta; red algae: Rhodophyta; Slime mold: Myxomycophyta,
Myxomycetes.

acrasiaes, plasmodiophoreae,
Love Rintari Z (labyrinthuleae) I
liiiI; Euphycophyta: Fungus @ (Eumy
cophyta), phycomyceta (phycomyc)
etes), ascomyceta (ascomycet)
es) * Hasidomyceta (hasidomycet-
es) Class; Hepaticae
, anthocerotae,
Musk (musci) iml: Vascular plants Tracheophyta, Psilopsida, Lycocida (L)
ycopsyda).

Sphenopsida, Pte-ropsida, Spermopsida
permops 1da) Legumes.

Cycadae, Zinc Cochin Axis
nkgoae), ]nihere (coniferae)
, Angiospermae lil, Dicotyledoneae, and mon-ocotylodeoneae are described. In the animal kingdom, there are subkingdom Protozoa and phylum Protozoa.

Plasmoclroma Ximen, Flagellata, Sarcodina (s)
arcodina) and sporozo
a) Class; Ciliophora (cili.

phora) Western Phylum, Class Ciliata; Subdivision Parazoa, Polyhera (Phylum Porifera
ra), calcarea iil, hexactinellida and desmospongiae
iii; Mesozoa, Phylum Mesozoa; Subdivision Metazoa, Division Radiata, Corentellata (c
oelenterata), Hydrocya
zoa), cyphoz-oa
, authozoa, stenophora (
cte-nophora), tentaculata (ten
taculata) and class nuda; division protostomia, phylum Platyhelmintes.

tubellana, ) tremato-da and cestoda
oda); phylum Nemertina (nemert 1na);
phylum acanthocephala; phylum aschelmintles;
rotifera, gastrotrifolia (g
astrotr 1ch-a), kino
rhyncha), briaprada <priapulid
a), genus Nematoda (ne+natoda) and nematomorpha; phylum entoprocta; ectobrocta (
ectoprocta), Gymno-mata (gymno-mata)
laemata) and phylactreta (phyl
actolaemata) i! ; 7 Oronida (ph
phylum oroni-da; braciopoda
oda) phylum, inarticulata
a) and class articulata; phylum Mollusca (mo I] usca), T
amph 1neura, monoplacophora, gastropod-a, scaphopoda, pe
lecypoda) and cepha
lop.

class da); phylum sipunculida; phylum echiuricla, phylum annelida.

polychaeta, oligoch-aeta and hiruclinea 1! I; Onychophora (
phylum onychophora; Tardeigrada (tar)
phylum digrada; penta-stoimida
phylum stoimida; phylum arthropoda;
phylum trylobita, chel 1cerata, bean quantity, xiphosura, arachimida
a-chmicla), Pycnogomida (pycn
ogomida) w4. mandebrata (mandi)
bulata) beans, crustacea (crustacea)
), chilopoda, diplopoda, paur
opoda), symphyla, colembola, protula (
protura), diplura
, thysanura, ephemer 1da.

Odonata, Ordobutella
hoptera).

dermaptera, x embiania plecopte
ra), zoraptera, corrodentia, mallophaga, anopl
ura), thysasnopter
a), Hemibutera, Neuroptera, Coleoptera, Hymenobtera (h)
ymenoptera).

mecoptera, siphonaptera, diptera
tera), )trichopte
ra) and lepidoptera
Insects of the order; Denterostomia
chaetogna, an animal of the mia) division
thal), echinoderma
ta) phylum, crinoidea, asterodea, ophiuroidea, echinoidea
hinoi-dea), and hololoidea (hol
oturoidea) 1jA.

phylum pogonophora, phylum hemichordata, phylum enteropneusta and phylum pterobranchia, phylum chordata, uroc
hordata) bean amount, ascidia
ciae).

taliaceae, class I arvacea; cephalocordata (c
ephalochorclata) bean amount.

vertebrata bean quantity, agn-atha, :] ndrikichi'L (c
hondrichthyes), osteochites (ost.
eachthyes), sir/sacco
ptei-ygii) type reticulum, crosobuteri (cros
sopterygii) and dipnoi (dipno
i) Order, amphi-bia, repitilia, av
es) J and mammalia*, protocilia (pr.

totheria type net, terrier type net, Marsupialis (marsupialis), insectivora (insectivora), dermoptera (dermoptera), chiroptera (ch
1roptera), Burimatis (pr imat)
es), edentata, folidota, labomorph 7
(Iagomorpha), odentia (ro
dentia).

cetaceae, carnivora
nivora).

tubuliclentata
, probosicdea ,
Hyracoidea.

sirenia, bellisodactyla (pe)
r 1ssoda-ctyla) and artiodactyla orders. 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 this application for descriptive purposes only and are not to be used in a limiting manner. The organism identified may be known or unknown, but most commonly unknown.

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

Briefly, rRNA gene sequences, subspecies classification or subspecies subclassification (inf rasubsp)
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 ecific 5ubdivisions. 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 (Southi, B, M, rJou
RNA of Molecular Biology
J, 38:503-517 (1975), incorporated herein 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 liposomal RN^ (rRNA), synthesized by reverse transcription, or synthesized by, for example, nick translation (nick translation).
A radiolabeled D complementary to the liposomal RNA contained on the cloned fragment can be labeled by
It may also be NA (rRNAcDN^).

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-radioactively labeled probes) or visualized, e.g. by radioautograph methods. (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 would be made by a -dimensional computer pattern recognition device, such as that used in a paint-of-5ale process.

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 (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 to the unknown organism's DNA.

There are four types of liposomal RNA information-containing probes: l) pronuclear liposome probes (particularly rRNA obtained from bacteria), 2) eukaryotic chloroplast liposome probes, 3) eukaryotic chloroplast liposome probes, and 4) true chloroplast liposome probes. Nuclear non-organelle posome probe.

There are also four sources of DNA (digested by endonucleases): l) prokaryotic DNA, 2) eukaryotic glomerular DNA, 3)
) eukaryotic chloroplast DNA, 4) eukaryotic cell nucleus ON^.

The following hybrid representative was thus created (Table 1).

Table 1 Eu, ``' Mitochondria + + Bu, chloroplast + + + Note (1) Bu, = eukaryotic cell (2) = less effective hybrid Postscript Example 4
The reference table shows that liposomal RNA probes are available for unknown organisms [
It shows that it can hybridize maximally with 1llA. For example, to identify eukaryotic organisms consisting of
It may be digested with an endonuclease and the digest hybridized with a prokaryotic liposomal RNA probe or a eukaryotic liposome probe extracted from the organelle. Similarly, to identify prokaryotic organisms, one can extract species-specific cellular DNA, digest it with endonucleases, and combine the digest with prokaryotic liposomal RNA probes or eukaryotic liposomes 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 liposome probes.

Eukaryotic cells may be defined by one or some combination of the following: a nucleus, a medulla, or in some cases a chloroplast. These cross-hybridizations are possible because, as previously mentioned, rRNA nucleic acids extracted from eukaryotic organelles are linked to pronuclear rRN
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 liposomes to translate the coded information into protein, and because liposomal 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 liposome 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 (e.g. animals or plants), pronuclear liposome probes should be selected and processed under conditions such that no or minimal organelle-derived DNA is extracted. Bye. Using this approach, one can be confident that there is minimal interference between organelle-derived DNA and pronuclear DNA. When eukaryotic species (which are not infected by prokaryotic cells) are identified with prokaryotic liposome probes, the organelle-derived ON It is best to maximize the concentration of If it is desired to identify a eukaryotic organism that has been infected by a prokaryotic organism, it is best to use liposome probes derived from non-organelle, non-prokaryotic cells. This is because this liposome probe does not hybridize well with DNA from pronuclear cells.

the same kingdom, or the same sub-kingdom, or the same division, or the same phylum, or the same plane, or the same class, or the same type network, or the same order;
or a pair from the same family, or the same tribe, or the same genus (DN
A # and liposome probes) are preferably used. It is particularly preferred to use prokaryotic liposome probes (eg bacterial liposome probes) to hybridize with pronuclear ON^. In this way, genera, species and strains of prokaryotic organisms could be detected, quantified and identified. One of the most preferred pronuclear liposome probes is derived from bacteria, particularly from Escherichia coli due to its ease of use and availability. Liposomal 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 liposome probes from a given family of to identify eukaryotic organisms of the same family (e.g. mammalian to identify mammalian organisms). (using liposome probes). Most preferred is the use of liposome probes and DNA from organisms of the same subfamily and/or order and/or tribe (e.g., when identifying a species of mouse, a liposome probe of mouse origin is used). ).

The most sensitive and effective pairing systems are those in which the a site of the liposome 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 DN from unknown organisms.

Nuclear ON^ 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.

^ctaJ, 52H1978), 1-15,
(It is included as a reference in Neeko). organelle D
Since NA is small and circular, it is called spooling (spoo).
ling) 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. Another method is to use globules (or chloroplasts)
are separated from a mixture of crushed cells, and the purified filamentous (or chloroplast) fraction is used for the preparation of organelle-derived DNA, and the purified nuclear fraction is used for the preparation of nuclear DNA. (For example, Bonen L.

ancl Gray M,llll rNucle
ic 8cids Re5earcJ 8:
319-335 (1980)).

Pronuclear []NA 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 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.

After protease digestion, loloform/phenol extraction and ethanol precipitation are performed to complete DNA extraction. Another extraction method that is much faster than phenol/chloroform extraction is the use of ethanol precipitation to rapidly separate DNA. This method is preferred when DNA is isolated directly from colonies or small volumes of liquid culture.

This method is described by Davis.

R, W, et al. ``＾Manual for
GeneticBngineering, ^dvanc
ed Bacterial Genetics” (hereinafter referred to as “Davis”), Co1d SpringH
Arbor Research Institute, Co1d Spring Har
bor, New York, 1980. p, 12
0-121.

DNA (of prokaryotic or eukaryotic cells (nuclear or non-nuclear))
) is dissolved in physiological M equilibration solution for the next step.

Digestion of the extracted ON 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, Bam) I I.

EcoRL Pst I, Hind m, Bal
L Hga I.

Sal I, Xbar, Sac I, 5st
-T, Bcl I, Xh.

1, Kpn I, Pvu I[, Sau ma, and others.

See also Davis, ibid., p. 228-230. Mixtures of one or more endonucleases can also be used in the digestion process. 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 inkinivation 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. With this device, the DNA digest is usually suitable! l [? Subjected to electrophoresis in liquid, the gel is typically soaked in ethidium bromide solution and placed in a UV-light box to visualize standard marker fragments that may be added.

After separation and visualization, the DNA fragments were transferred to nitrocellulose filter paper by the Southern method (rJo
Urnal of Molecular Biolog
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,
Nucleic acid probes can include detectably labeled liposomal DNA (rRNA) obtained from such organisms, truncated translated labeled DNA probes containing liposome information, cloned DNA probes containing liposome information or liposomal RN from the probe organism. Either a detectably labeled DNA bRNA cDNA ^ that is complementary to ^).

Depending on the choice of combination, the liposome 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 (
For example, "P, 3H or 14C). Nucleic acid probes may be labeled with metal atoms. For example, uridine and cytidine nucleotides can form covalently bound mercury derivatives. It is a good substrate for many nucleic acid polymerases, including reverse transcriptase (Date et al., rProceedi
ngs of the National＾cademy
of 5sciences”70: 223g224.
2.1973). Direct covalent mercurylation of natural nucleic acids has been reported (Dale et al, +'r
Biochemistry J 14: 2447
-2457). Re-annealing of mercurylated polymers
nnealing properties are similar to those of the corresponding mercury-free polymers (Dale and Ward).

rBiochemistry J 14: 245B-
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.

Isolation of rRNA from eukaryotic and prokaryotic cells is well known to those skilled in the art. For example, to prepare rRNA from eukaryotic cytoplasmic liposomes, RNA can be extracted from whole cells or liposomes, separated by sucrose gradient centrifugation, and type 18S and type 2BS fractions collected using markers of known molecular weight. (e.g. Perry, R,P.

and elly, D.B., “28S and 18
Actinomycin D produces a small amount of S liposomal RNA.
Sustained synthesis of 5S RNA when inhibited by
J, Ce1l.

Physiol...? 2:235-246 (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
Btten R,A.

et al., rcellJ 22:157-17
0 (1980), or Edwards, at
al,, rNucleic Ac1ds Re5er
ch J.

9:2853-2869 (1981)).

If radiolabeled liposomal 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 (rRNAcDNA^),
It is made by reverse transcription of rRNA isolated from the probe organism in the presence of radioactive nucleoside triphosphates (eg 32P-nucleosides or 3H-nucleosides).

Labeled liposomal probes may also be obtained from cut translational DNA molecules, particularly whole circular DNA from organelles. Specifically, the annular D of the chloroplast or the thread body
The NA is cleaved and translated in the presence of a radioactive label, resulting in a labeled ON^liposome probe. Chloroplast-labeled probes will hybridize best with chloroplast DNA, and thread-labeled probes will hybridize best with thread-plast DNA. Chloroplast (or chloroplast) incision translation-labeled liposome 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. Liposome 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 accomplishing this is to excise the rRNA gene from the eukaryotic nuclear DNA (by restriction enzymes), separate the fragments, identify the liposome gene sequence (by hybridization), and isolate the liposome gene sequence. (by electrophoresis). The isolated rRNA is then recombined into the plasmid or vector and transformed into a suitable host.
formation) and then cloning may be performed in a Q2p-containing medium. Another method is to propagate the transformed host, then isolate the DNA and label it by truncating translation, or isolate the DNA, excise the rRNA sequence, and then label it with MA.

The obtained liposome probe hybridizes with rRNA cDN^ in the same conditions (see below).

Preferred nucleic acid probes are rRN from the probe organism.
This is radiolabeled DNA complementary to A. Specifically, the rRNA separates the liposome from the probe organism;
Individually separate and appropriate RNA substantially purified as described above. The liposomal RNA thus obtained does not contain liposomes and is a carrier RNA (rRNA). It also contains almost no other RNA, such as transfer RN^ (+II RNA). 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
and 2BS types. Reverse transcription into cDNA is then performed 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 (^MV) 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 e.g.
It is radiolabeled by 2P. For example, deoxyadene 5'-(32P), deoxythymidine 5
'(32P), deoxyadenine 5'-(''P)
, or deoxyguanidine 5'-(32P) triphosphate is used as the M-paired nucleoside. 30 minutes~
After incubation for 5 hours at 25-40°C, extraction with chloroform and phenol, centrifugation and chromatography, the radiolabeled fractions are combined into a cDNA probe. Radiolabeled cDNA blobs in substantially pure form, ie, 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 and organelles. Non-radiolabeled cDNA DNA blocks also constitute an aspect of the invention. A preferred probe is a labeled rRNA cDNA of a prokaryotic cell;
Most preferred is bacterial labeled rRNA cDNA. The species of the probe is a bacterial microorganism, such as Enterobacter 1aceae.
), Brucella, Bacillus
illus), Pseudomonas (Pseudom.

nas), Lactobacillus (Lactobacillus)
s), Hae+nophilus
), Microbacterium
m), Vibrio, Nei
sseria), Bactroides (Bactro 1)
Species included in the family Des) and other anaerobic groups, such as Legionella, 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 liposomal 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 rRNA, ie, the entire nucleotide sequence of the template rRNA is transcribed at every time synthesis is performed.

The use of brimers 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: The L 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.

Perjansky M, M, et al. C, R, Acad
, 5cParis J t286. Series D,
, p. 1B25-1828 (1978), E. coli r
3H-radiolabeled cDNA that can be made into RNA has been described. The cDNA used in this study was not prepared using reverse transcriptase in the presence of primer as in the present invention, but was prepared using DNA polymerase ■ using rRNA that had been previously cleaved using ribonuclease U2 as a template. . r of Perjansky et al.
The RNA digestion products (by RNAse L12) have different base ratios than the initial rRNA, indicating that bases and/or short fragments have been lost. The cDN^ obtained in this way is not a faithful copy. Moreover, the use of DNA polymerase ■, as used by Perujansky, is known to accelerate the predominance of homopolymerized transcription over heteropolymerized transcription of rRNA (see, 5.
n P,S, et al,, rBiochem,
Biophys, Res.

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

To summarize, the “liposome” probe is: a)
From genomic DNA containing the rRNA gene, by cloning and/or truncated translation, b) from the liposome RN^ itself, or c) from rRNAc, DNA, by reverse transcription of the 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 involves using a paper containing a covalently labeled DNA digest from an unknown organism.
This is done by contacting the liposome with a hybridization mixture containing the 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 sludge (if necessary).
It is then air dried and prepared for detection. Another highly preferred hybridization, which is much faster than the above method, is Kohne, D,E.
rBiochemistry J 16:532
This is a room temperature phenol emulsion resealing method described as a reference in 9-5341 (1977).

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 can be performed by selective methods (in the case of non-mated probes), by radioautograph methods, or by computerized methods. This can also be carried out by suitable radiological scanner techniques (in the case of labeled probes), which may 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 BcoRI-digested λ bacteriophage D
By subjecting it to marker separation methods such as NA,
Can be easily matched to a specific fragment size (K4 base pairs). In this way, both the relative position of the bands to each other and the absolute size of the bands 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 reliable 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 broom 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).
Additionally, liposome RN was used for probe preparation.
A composition of information (e.g. 5S, 165 or 2 of pronuclear cells)
3S type subtype or only 16S and 23S types, etc.)
It will depend on.

Thus, the catalog may contain a variety of enzyme-specific band patterns, with band sizes and relative intensities recorded for each probe. Bonded ON to filter paper
As the concentration of ^ decreases, only the strongest bands become visible and species can be identified by the size of this or these bands. Each one of these band patterns may each include a band pattern obtained with complete liposomal RNA and/or a band pattern obtained with liposome RN^ containing only subtypes consisting of. All variations or permutations of the above may, of course, be utilized in the library. Moreover, for eukaryotic organisms, the library may contain patterns generated by using one type of DNA, or combinations of organelle and/or nuclear DNA. The catalog will depend on the probe composition.The catalog will be organized in such a way that if one or more strains or species are present in the extracted sample and detected by the probe, then the resulting pattern can be interpreted. There is.

A user can compare the resulting band patterns visually or by a one-dimensional computer-based digital scanner programmed for pattern recognition. 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 the multiband when compared to the standard can also indicate the amount of bound DNA that has become hybridized and can therefore be used to estimate the extent of the presence of organisms, such as the presence of prokaryotic cells in eukaryotic cells. It can be used to

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 can be compared to the organism's catalog band pattern for the second endonuclease of choice. 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, or in clinical and microbiology laboratories, to identify bacteria present in any medium, including eukaryotic cells. May be used to identify parasites 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 leprae (Mycobacterium leprae).
This is not possible for some microorganisms, such as Bac-teriun Ieprae (the causative agent of leprosy)
Some microorganisms, such as obligate intracellular bacteria (e.g. Rickettsiae, Gramidi T., etc.), are either impossible to grow on standard media or are very dangerous (e.g. B. , anthracis) (causative bacterium of anthraxiasis). Since the nature is based on the isolation of nucleic acids, which does not involve traditional bacterial isolation and characterization, these problems can be eliminated. This method has hitherto It may be possible to detect microorganisms that have not been formally described.In addition, their nature makes it possible to distinguish between different strains of a species, which is useful, for example, in epidemiological typification in bacteriology. It can be used in forensic laboratories for the correct and unambiguous identification of plant or animal tissue in criminal investigations, and for entomologists to quickly identify insect species when establishing the nature of crop damage. is also used.

Furthermore, this method can be applied to taxa other than subspecies (e.g. plant root nitrogen enzyme genes: reference: Hennecke H291rN).
By combining this with the identification of ``Cure'' 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 may be found in industrial growth media, culture broths, etc., and may be concentrated, for example, by centrifugation. 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 illegal is an animal;
Particularly preferred is its use in diagnosing bacterial infections in humans. Bacterial DNA with pronuclear liposome probes
The detection and identification of is highly selective and can be carried out without hindrance even in the presence of animal (eg mammalian) ONs. If pronuclear liposome probes are used, choose conditions that minimize hybridization with glomerular 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 the messenger plasmid R-factor that mediates 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 separated into coats, with the first aliquot used for identification, the second aliquot for the presence or absence of drug resistance sequences, and the third aliquot for the presence or absence of toxin genes. It can also be tested in such a way that it can be used for Alternatively, a liposome probe that has been aiaylated with a radionuclide (e.g. 32P) can be used in a hybridization mixture by adding an R-factor probe that has been MAmylated with another radionuclide (e.g. 3H or l4c). You can do it. After hybridization, the presence of R-factor DNA in the unknown DNA can be tested by scanning with two types of scanners. One for species and strain identification (e.g. “P”) and the other for drug resistance etc. (e.g. 3H or 14C).
). In this way, experimenters can identify genera and species, type strains, detect drug resistance, toxin production or other properties, 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, Dis,,
141:625-636 (1981)).

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 (Publisher Lennette,
E.H.Amer Sac Microb 1980
774-778) can be identified. For example, picornaviriclae, calicivir-idae, leoviride (
reov i r 1dae), togaviride (tog
aviridae), orthomycoviridae (ort.
h.

myxoviridae), rosemycoviridae (pa.
ramyxov 1-ridae), love doviride (
rhabdoviridae), retroviridae (r
etrov ir 1dae), arenaviride (ar
enav ir 1 dae), corona viride (cor
onaviridae) bunyav
ir 1dae), parvov
iridae), papovaviride (papovavir)
idae) adenoviridae
), herpesviridae
, vidoviridae and poxviridae, etc.

1) If the viral genome is integrated into the host DNA (DNA viruses such as Vapoviride member, RNA viruses such as Retroviride member), high molecular weight DNA is extracted from the tissue and digested with restriction enzymes. The overall procedure is similar to that for bacteria.

The choice of viral probe again depends on the question being asked, and on the degree of homology between the "blob virus" and the viral-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 a virus germ lobe hybridizes or not to virus-associated sequences in host DNA depends on the hybridization conditions, e.g., whether stringent or relaxed conditions. 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. Any labeled complementary nucleic acid probe containing a cloned virus sequence can be used as a probe.

In the case of RN^ virus, for example, virus RN^
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 and precipitated nucleic acids are preferably 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 RN^ prior to centrifugation. This can also be used to check whether a virus has been installed.

In order to hybridize the Virus gellobe, 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, stringency or laxity, determine whether a given probe will hybridize to a less closely related genome. The probe may be a labeled cloned virus sequence, or it may be the complete genome or a portion thereof.

The method described by Southern, supra, is useful for transferring large DNA fragments (about 0.5 kilobases or larger) to nitrocellulose paper after alkaline denaturation. This method is useful in the case of ON^ viruses, but may not be useful in the case of RN^ viruses. RNA can be transferred to activated cellulose paper (diazobenzyloxymethyl paper) and covalently bound, and used for RN^virus. Thortus's modification of the saunaan method (Thomas, P., rProc
, Nat, ^cad, 5ciJ [IS^77: 52
01-5205 (1980)) can be used to efficiently transfer RNA and small DNA fragments to nitrocellulose paper for hybridization. RNA
The # and small DN^ fragments are denatured with glyoxal and dimethyl sulfoxide and subjected to electrophoresis in an agarose gel. With this operation, DNA fragments of 100 to 2000 nucleotides and RNA are efficiently moved,
The hybrid pores remain on the nitrocellulose paper. This is also useful for small liposomal 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. Probes that form hybrids (sequence information is from DS-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 OS-DNA, truncated translation can be used, and in the case of 5S-DNA, DNA polymerases are used for cDNA.

3) In the case of RNA viruses, 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 liposomal RNA from an organic probe (in the case of a bacterial identification kit). , prokaryotic cell rRNA cDNA (more preferably). 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 catalogs are 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. Additionally, the kit may include a brochure, or a "catalogue" broadly defined as a book or brochure, or a computer tape or disk, or a convenience access number, etc., which may contain plant species, apocampal species, bacterial species, etc. Contains chromatographic band patterns of various organisms of groups such as species, especially pathologically important bacteria, insect species, etc. In this way, the user only has to prepare a band pattern of an unknown organism and visually (or computationally) compare it with the patterns in the catalog.

The kit also includes probe rRNA for probe synthesis in one container, radiolabeled deoxyliponucleozide triphosphate in another container, and primers in another container. It's okay to stay. In this way users can create their own probes to rRNA cDNA.

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, Wills gellobes or other probes with 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 an extraction buffer (0.15M sodium chloride, 0.IM εDTA, 0.03M) pH 8.5 in a ml volume approximately 10 times the duram weight of the packed cells. Ta. Lysozyme 10mg/m
f to a maximum concentration of 0.5 mg/rn1. I added it so that The suspension was incubated at 37°C for 30 minutes. Cell disruption was performed by adding 25% SOS 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■20rng/mf
o, 02M) pre-digested in Lys buffer (pH 7,4) for 2 hours at 37°C, then at a final concentration of 1■/ml! I added it so that it becomes . The solution was incubated at 37°C for 18 hours. Phenol was prepared by mixing redistilled phenol 11, 2.5 L of double-distilled water, 270 L saturated Tris base, 12 mercaptoethanol, and an amount of EDTA to give a final concentration of 10 M, and the mixture was heated at 4°C. It was prepared by separation. The phenol was washed with a washing buffer (1[)-'M chloride) IJum, 10-'M
Washed with EDTA, 10mM), pH 8.5). 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 listen for about 10 minutes and then centrifuged at 3400×g for 15 minutes. The aqueous phase was removed with a 25 mg 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 -20"C 95% ethyl alcohol were slowly poured along the walls of the flask.

Melt the tip of the bathtub rubipette to close it and precipitate the DNA.
used to spool. High molecular weight DNA was dissolved in buffer (10-3M EDTA, 10-2M
) 'J SuI]87.4). DNA m& uses 30 με for 1 unit of absorbance as a conversion factor, and
Measured by absorbance at r+n+.

Restriction Endonuclease Digestion of DNA The BcoRI restriction endonuclease reaction was performed using 0.1M Tris-HClp)17,5.0.05M NaC! !
, 0.005M MgCj! 2, and 100μ
g/-conidal serum albumin. The pcoR1 reaction mixture contained 5 units of enzyme per ON^/μg and was incubated for 4 hours at 37°C. PST
I restriction endonuclease reaction: 0.006M) Lis-HCl pH 7,4,0, 05M sodium chloride, 0.006M magnesium chloride, 0.006M2
- mercaptoethanol, and 100 μg/- conidal serum albumin.

The PST I reaction mixture contained 2 units of enzyme per μg DNA and was incubated for 4 hours at 37°C. Typically, 10 Mg of DNA was digested to a final volume of 40 μp. A 10x buffer was added. DN sterile distilled water
＾Added according to the amount dissolved. λ-bacteriophage DNA
to create marker bands for fragment size determination,
Restricted by EcoRI. Normally 2μgλDNA is 20
Digested with 1 unit of ECORI to a final volume of 20 μm.

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

Digested bacterial DNA 10Mg and digested λDN^
Place 2 μg in a per well and cover the surface with melted agarose. Digested product was dissolved in 0.02M sodium acetate, 0.002M EDTA, 0.018
M ) Lis base, and 0.028 M ) Lis ) In
I! 3 in 0.8% agarose containing pH 8.05.
Electrophoresis was performed at 5 V until the dye migrated 13 to 16 cm. The gel was then treated with ethidium bromide (0,0
05■/if) and placed in a UV light box to visualize the λ fragment. DNA was transferred to nitrocellulose filter paper using Southern's method as described above. Gel was placed on a shaking table for 2
Denaturing solution (1,5M sodium chloride, 0.5M
treated with sodium hydroxide). Neutralize the denaturing solution (
3.0M Sodium Chloride, 0.5M) Lis 1lcf
pH 7.5) and after 40 minutes, the gel was
(I checked it on paper.

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

Synthesis of 32P liposomal RNA complementarity ON (32Pr
RNA and DNA) 32p-labeled DNA complementary to E. coli R-1323S and 16S type liposome RN8 was synthesized using reverse transcriptase from avian myeloblastosis virus (AMV).

The reaction mixture consisted of 5 μm of 0.2 M dithiothreitol, 25 μl of IM)
M potassium chloride, 40 μl of 0. LM Chloride Mac 2,
column, 70 μl actinomycin, 14 μl 0.
04M dATP, 14μ! 0. (14M dG
OP, contained 14 μl of 0.04 M clTTP S and 96.7 μl of water.

The following were added to the plastic tube: 137,
5 μl reaction mixture, 15 μm conidial thymus primer (
10 μm/rd), 7 μl H2O, 3 μm rRNA (
4D μg Using a 100 unit concentration, 2,76 μg
/μl), 40μ! deoxycytidine 5'-
(“P) triphosphate (10mCi/mi), and 13μ
m AMV polymerase (6,900 units/μl). The fermentation reaction was carried out at 37°C for 1.5 hours. The solution was then extracted with 5 portions of chloroform and prepared phenol. After centrifugation (JS 13,60ORPM)
, transfer the aqueous phase directly to a Sephadex G-50 column (1,5
x22cm). plastic 10rd
A pipette was used as a column. small glass peas 1
Degassed G-50 was placed at the tip, a rubber tube with a pinch fitting was attached, and the degassed G-50 was swollen by soaking in 0.05% SO3 overnight.
added. Pour the aqueous phase directly into G-50 along the wall,
It was then eluted with 0.05% SO3. Twenty 0,5- fractions were collected in plastic vials.

Tubes containing peak fractions were discovered using the Shichinkoff counting method, using a 3H-discriminator to count each sample for 0.1 min and recording the total count. Peak fractions were combined.

Add an aliquot to Esol (commercially available) and add 1
CPM (counts per minute) of 32p per - was measured by a scintillation counter.

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

Filter the hybridization mixture (3 x 5SC10
, 1% SOS, 100 μg/- of denatured and sonicated canine DNA and Dinehardt's solution (0.0 μg/- each).
2% conidic serum albumin, Ficoll
), and polyvinylpyrrolidine)) for 1 hour at 68°C. "P rRNA cDNA 4X10'C
PM/ml! 68% of the hybridization reaction solution.
Incubate for 48 hours at °C. The filter was then washed with 3X SSC and then 0.1% SO for 2 hours at 15 minute intervals or the cleaning solution was about 3.000 cpm.”
P/ml.

Washed until it contained. Air dry the filter, wrap it in plastic wrap, and place it in the Kodak X-OMATR
Autoradiography was performed on the film at -70°C for about 1 hour.

B. Mammalian experiment Mus musculus domesticus (mouse) rR
NA probes were synthesized from 18S and 28S 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 diluted with 50mM MgCf2.
and 100mM) dissolved in a buffer of Lis pH 7.4. DNA5e (without RNAse) at a concentration of 50μ
g/rnf. The mixture was incubated at 37°C for 30 minutes. The generated RNA was re-extracted, ethanol precipitated, and added to 1mM sodium phosphate buffer pH 6,
8. 0. IMF squirrel pH 7, 4 and 0.
A 5-20% sucrose gradient solution of 01MEDT^ 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
I chose SJ and 2BS fraction.

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% SDS.

Example 1 Several strains of P. aeruginosa used in this example have minimal phenotypic characteristics to identify the species (Hugh
R,H,, et al, rManual of C
l1nica] Microbiology J 2nd edition, ^
SM, 1974, pp, 250-269) (
Table 2). Three other Pseudomonas and two cinetobacter strains were selected and their species and genera were compared (Table 3).

Table 2: Minimum table of P. aeruginosa for comparison of bacterial strains Strains used for comparison of Pseudomonas and Acinetobacter species are listed in Table 3.

Table 3 below for comparison of species and genera, Types, Neotai P, aeruginosa P, 5 tutzeri P, f Iuorescens P, putida A, anltratLIs 815 10145 10332 Type 2601 17588
Neotype 818 13523 100
38 Neotype 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 EcoRI digest is P, 5 tutzeri 16.
0.12.0.9.4; P, Fluorescence (P
, f 1uorescens) 16.0. 10.0
．． 8.6,? , 8.7.0; P, putida (P, p
utida) 24.0.15.0.10.0.8.9
;A, anitratus (^, 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. Ruoffi (
^, 1woffii) 12.0.10.0.9.1.
7.0.6.4.5.7.5.5.5J, 4.8.4.
4.3.6.3.2.2.9 (the sizes of the three smallest fragments were not calculated). The sizes (in kilobase pairs) of the fragments in the PST I digest are as follows: P, Stoutzer 6,7.6,1.5.5; .7.0, P
, putida 10.5.9.9.6.8.6.3.4.4
, A. Anitratus 36, 0.28.0.20.5
．． 12.0.10.0.5.8.3.7.2.6.2.
4; A, 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 seven strains of A. aeruginosa shows that this species
．． 9.4.7.6 and 5.9 kilobase pairs (KBP)
(
Figure 1).

The 7.6 KBP EcoRl fragment is present in 4 out of 7 strains in this sample. A similar situation occurs in some phenotypic characteristics of strains of species. The fact that the BCOR1 combination of fragments from 7 strains is used to divide the strains into two groups draws the inference that there are two species with minimal phenotypic characteristics of P. aeruginosa.

The experimental results of digesting DN8 with PSTl (Fig. 2) lead to the conclusion that the strain mutations indicated by the EcoRI 7.6 KBP fragment result in mutations within the strain. Because one conserved combination of PST l fragments, 9.4,? , 1.6.6 and 6.4KBP, which define the species. PSTl fragments of 9.4 and 6.6 KBP are found in 6 of 7 strains of P. aeruginosa; 7.1 and 6.
The 4 KBP PST I fragment is present in all of the sampled strains. Mutations in the PST l fragment were carried out using EcoRI
7.6 Occurs in strains that do not contain the KBP fragment; R
I1151 has 10.1 and 8.2 KBP fragments,
RH809 does not contain 9.4KBP fragment, 6.0K
It has a BP fragment. and the type strain RH815
does not contain the 6.6KBP 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.

EcoRl 7.6 in P. aeruginosa strains
Mutation of the KBP fragment may be viewed in perspective by testing hybridizing EcoRI 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, P Stoulaeri and P. fluorescens have a 16 KBP fragment, P. fluorescens and P. putida have a 10 KB fragment.
It has a P fragment. In general, the size of the fragments is unique to the type strains of each of the four Pseudomonas species: and the type strains of each species each have a different size range of fragments. These shooting explanations also apply to the PST I digest (Figure 4).

When comparing the fragment patterns of each of the four Pseudomonas species and the two Annetobacter 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. shuringiensis and B. subtilis, it is difficult to determine where enzymes cut rRNA genes, the number of copies per genome, and the number of copies between multiple genes or heterogeneous genes. has a non-homologous flanking part (f
It is impossible to predict whether there will be ranking regions. The E. coli rRNAcD'NA probe may not hybridize to some restriction fragments containing rRNA gene sequences, and if so, this may indicate that there is evolutionary distance or dispersion between the test organism and E. coli. is reflected. 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 Type Table B-354 (NR3-231) B-356 (NR3-238) NR3-265 NRS-659 NR3-73O NR3-737 NRS-741 NR3-773 NR3-1106 High molecular weight DNA is extracted from each strain. Separated. RH30
2] and liquid D using labeled DNA of RH2990.
NA-ON^ Hybridization data were collected. The results are shown in Table 6.

Table 6 Labeled ON^ 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
atic Bacteriology” 25: 25
B-270 (1975)). These two groups are RH3021
and RH2990.

Restriction endonuclease analysis of liposomal RNA genes is performed. The EcoRl digest (Figure 5) could be divided into two groups. The group represented by RII3021 has two strongly hybridizing fragments (2.1
5 and 2.1 KBP). The group represented by RH2990 has two strongly hybridizing fragments (2,6 and 2.5 KBP). The EcoRl data can be used to place the B. subtilis strain in the appropriate place in the DNA-0N^ hybridization group. According to the 70% DNA-DNA hybridization rule, B. subtilis is actually two species. However, considering the PST I data (Fig. 6), the groups can be considered as two dispersed populations 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 determine species despite dispersion. R
H3061 lost the PST l site. However, the EcoRl data suggest that the strain is B. subtilis. The same can be concluded from the BgA II data (Fig. 7) and the Sac I data (Fig. 8).

Example 3 Table 7 B, Subtilis and B, Polymica Neotie B, 5u
btilis 3021 6051B, poly
myxa 3074 842 neotype neotype B, polymyxa RS neotype B, subtilis and B, bolymica, EcoRl data (Fig. 9), PST l data (Fig. 10) Bgl
They can be distinguished by IIl data (11th Em left) and Sacl data (11th Father).

Due to the large differences in PST I band patterns, it can be concluded that Banrus 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 Vanrus species if they were unable to produce spores.

Example 4 Identification of Bacterial Species in Mouse Tissues Without Separation
usculus domesticus) (inbred strain)
eptoco-ccus pneumoniae) R
0.5m of mixed S suspension of H3077 (ATC Ro6303)
f' was inoculated intraperitoneally. When the mice were near death, their hearts, lungs, and livers were removed. 1EcoR to isolate high molecular weight DNA from these tissues, S. pneumoniae RH3077 and Swiss mouse organs and digest the DNA.
Restriction endonuclease analysis of rRNA genes was performed using I. In addition to washing the filter with 3X SSC, 2 x 15 min of 0.3X SSC and 0.05%
Washed with SO3. Autoradiography was performed for 48 hours. The data (Figure 12) are 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 KBF). The hybridized fragments are S, 2 in the infected tissue.

-It announces Monier's presence. All seven bands are found in cardiac mRNA extracts. 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 superficialization at autopsy.

To test the sensitivity of this assay, bacterial DNA was added to mouse D.
It was diluted with NA and subjected to electrophoresis. 0.1 μg bacterial DN
Using A, all seven bands could be seen on the traradiograph. For 10-3 μg bacterial DNA, 17
．． 0.8.0 and 6.0 KBP bands were seen.

106S, 5 X 10-3 per cell of Pneumoniae
Using the number μgDNA (Biochem B
iophysActa 26:68), 1o-'μg
corresponds to 2×107 cells. This level of sensitivity makes the latter method useful for diagnosing infectious diseases.

This example also shows that the bacterial probe is mouse-specific Eco.
This shows that it hybridizes with the Rl fragment (No. 9
(see figure, fragments with 14.0 and 6.8 KBP) These fragments are mouse 18S and 2BS type liposomal RN.
Corresponds to the EcoRl fragment detected by the A probe (Fig. 14, above, the 6.8 KBF fragment is 283 type rR
(indicates that it contains an NA sequence). Bacterial probes hybridize poorly with mammalian liposomal RN^ gene sequences, so the bands are less intense, and bacterial probe and nuclear mammalian RNA systems are less sensitive and less sensitive to the DNA of infected prokaryotic cells. Selectivity is clearly visible. 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 melonuinus (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 (mouth avia porcellus)
us) (guinea pig) 17.0.14.0.13.0
．． 8, 8.5.7.4.7 and 1 band below 3.0 5, Crisetulu griseus (Cricetulu
s griseus) (hamster) 25.0.4.
76, Ho(--Sapiens)
) (Human) 15.0.5.7 7, Felis catus
(Cat) 20.0.9.7 8. Ratus norveg
icus) (rat) 12.5 9, Mus muschinilus domesticus (mouse) 1
3.5.2.6 10 Mus Serpicolo Serpicolo (MIJS)
cervicolor cervicolor) (
7us) 14.0.2.7 11. Muscerν Babeus
ic.

Jar papeus) (Mouse>13.5.2.6
12. Mus pahari (Mus pahari)
Mouse) 13.0.3.7 13. Mus cookii
(Mouse) 13.5.2.6 Figure 14 shows that mouse and cat DNA are 2BS type rRNA.
We show that cDNA alone is distinguishable and that the pattern of noibrid bands depends on the composition of the probe sequence. In FIG. 14, the enzyme is EcoRl, and the objects and bands are as follows.

1. Mus musculus domesticus (mouse) i
3,3KBP 2, Fueris catas (cat) 8.3KBP In Figure 15, the enzyme is Sac I, and the objects and bands are as follows.

1. Erythrocebu patus
s patas) (batas monkey) 8.5.3.7, <
3.02, Mouse norvegicus (rat) 25.0.
9.5.3.6 , <3.0 3, Mus-Mus cow Yurus 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. .

6. 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 EcoRi and the objects and bands are as follows.

1. Erythrocebus Patas (Patas m) >22.0
．． 11.0.7.6.2.6 2.7 Kaka Muratsuta (Lezas 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, HaLa (human cultured cells)>8.1.6.6
6, J96 (human cultured cells)>22.0.22.0
．． 16.0゜11.0.8.1, 6.6 7, AO (human cultured cells) 22.0.16.0.8
, 1.6.68, X-381 (Lazas Monkey) 22.0
,]1.5.7. 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. cDNAs for E. coli 16S type and 23S type liposomal RNA (rRNA) were used as probes. Figure 2 shows Pseudomonas aeruginosa (P, ae
pst of DNA isolated from S. ruginosa) strain
1 shows restriction endonuclease digestion. cD for E. coli 16S type and 23S type rRNA as probes
NA was used. FIG. 3 shows EcoRI restriction endonuclease digestion of DNA isolated from glucose-nonfermenting Gram 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 BcoRl restriction endonuclease digestion of DNA isolated from various Bacillus subtilis strains. cDNAs for Escherichia coli 16S type and 23S type rRNA were used as probes. FIG. 6 shows Pst I data obtained using the same probe for the same strain as in FIG. 5. FIG. 7 shows Bgl It data obtained using the same probe against the same strain as in FIGS. 5 and 6. FIG. 8 shows SaCl data obtained using the same probes for the same strains as in FIGS. 5-7. Figure 9 shows B, subtilis and B, polymica (B, p
Figure 3 shows EcoRl restriction endonuclease digestion of DNA isolated from S. olymyxa). cDNAs for 16S and 23S rRNA from E. coli as probes
A was used. Figure 1O shows Pstl data obtained using the same probe for the same strain as in Figure 9. Figure 11 shows Bgl II and Sac obtained using the same probe for the same strain as in Figures 9 and 10.
l data is shown. Figure 12 shows that Streptococcus pn.
Fig. 2 shows the detection of C. eumoniae). Figure 13 shows Mus musculus domesticus (Mu
s musculus domesticus) (
Type 18S and 2B from cytoplasmic liposomes of
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.
Figure 2 shows Sac I digested DNA from mammalian tissue hyflitted with type 28 and type 28S rRNA cDNA probes. ! 161m is Mus musculus domesticus 18
Eco from mammalian tissue and cell culture hybridized with S-type and 28S-type rRNA cDN^ probes.
RI digested DNA is shown. that's all

Claims (1)

[Claims] 1. Existing in eukaryotes or characterized by selectively hybridizing a fragment containing ribosol RNA information of a prokaryote with a detectably labeled hybridization probe containing prokaryotic rRNA information A method for detecting prokaryotes that coexist with this.