JP2003038199A - Method for molecular genetically analyzing and evaluating polluted environment and environmental sample - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new method for molecular genetic detection and determination which can be carried out with a non-RI method and applied to the whole microorganisms and a specific microorganism in the environment, and to provide a method for analyzing and evaluating a polluted environment and an environmental sample by using that method. SOLUTION: A method for detecting and measuring a nucleic acid of a specific functional microorganism in an environmental sample with a non-RI method, and analyzing the priority of the specific functional microorganism in the environment. The method comprises (1) a process in which a target nucleic acid is extracted from a microorganism-containing specimen taken out from the environment, and purifying and storing the targeting nucleic acid, (2) a process in which a sequence specific to the specific functional microorganism is selected, and a non-IR labeled nucleic acid probe specific to the specific functional microorganism is prepared, (3) a process in which the nucleic acid obtained in the process (1) is fixed on a base board, the probe prepared in the process (2) is applied on the treated base board, and thereby hybridization is performed, followed by washing, (4) a process in which the signal attributable to the hybridized non-IR labeled nucleic acid probe is analyzed after visualization, and the quantitative value is obtained, and (5) a process in which the obtained quantitative value is compared with the quantitative values determined on whole organisms and on each domain of the living world, and the priority of the specific functional microorganism is calculated.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention generally relates to a method for analyzing and evaluating a polluted environment, and specifically, for diagnosing a polluted environment of toxic chemicals, particularly a petroleum polluted environment, and repairing a bioenvironment by microorganisms in the polluted environment ( Bioremediation) is a method of molecular genetic analysis / evaluation, and a method of molecular genetically detecting / quantifying and analyzing harmful microorganisms such as useful substance-producing microorganisms or pathogenic microorganisms, and these microorganisms. The present invention relates to a method for analyzing and evaluating an environment including an environmental sample.

[0002]

2. Description of the Related Art Up to now, as a method for evaluating a polluted environment,
Qualitative and quantitative analysis of harmful chemical substances in the environment using analytical equipment that combines various fractionation methods such as gas chromatography and high performance liquid chromatography with various elemental analysis methods, mass spectrometry methods, optical detection methods, etc. The method is widely used. In addition, COD that measures the oxygen demand of polluted environmental samples
Law and BOD method are also available. However, a simple and reliable molecular genetic evaluation method for specific microorganisms (groups) present in environmental samples, not limited to contaminated environments, is in the process of development, and the dominance analysis of specific microorganisms (group) is underway. There is no established method to enable the. For some environmental pollutants such as petroleum components, various treatment methods and treatment techniques have been developed by elucidating the physicochemical factors that influence natural purification.

However, there is no simple and highly accurate method for analyzing and evaluating the composition and variation of the microbial community in nature, which is responsible for natural purification, and the characteristics of pollutant-degrading bacteria that dominate the polluted environment. It is difficult to evaluate the effectiveness of technology and its universality.

[0004] In carrying out environmental evaluation for microorganisms in this contaminated environment, there are a plate culture / counting method using an agar plate medium and an MPN (most probable number) culture / counting method using a liquid medium. . However, the above-mentioned conventional culture / counting method has a drawback that it takes a lot of time and labor and requires a long culture time to detect the specific degrading microorganism.

In addition, the number of microorganisms inhabiting the natural world that can be detected by these conventional separation / culturing methods is extremely small. That is, a direct microscopic counting method (for example, JE that stains with a fluorescent DNA stain and counts under a microscope).
Hobbie, RJ Daley, and S. Jasper: Appl. Environ. M
icrobiol. 33: 1225-1228, 1977 and KG Porter and Y.
S. Feig: Limnol. Oceanogr. 25: 943-948, 1980), the ratio of microorganisms that can be separated and cultivated is considered to be less than 1% (RI Amann,
W. Ludwig and KH. Schleifer: Phylogenetic Identif
ication and In Situ Detection of Individual Microb
ial Cell without Cultivation, Microbial. Rev., 59,
143-169, 1995). Therefore, there is a major drawback that it is difficult to analyze the composition of the whole microbial community inhabiting the habitat, the fluctuation of the community, the behavior of specific microorganisms, and the like.

Therefore, in recent years, molecular genetic detection and quantification have been attempted for all microorganisms or specific microorganisms by using DNA probes specific thereto and independent of conventional separation / culture methods. For example, a molecular level detection and quantification method by a blot hybridization method using an RI-labeled oligonucleotide probe has been reported (SJ Giovannoni, TB Britschg.
i, CL Moyer, KG Field: Genitic diversity in
Srgasso Sea bacterioplankton. Nature, 345: 60-63, 1
990). In addition, a probe release curve analysis method required for hybridization has been reported using RI-labeled oligonucleotide probes (D.
AE Zheng, DA Stahl, and L. Raskin: Character
ization of Universal Samll-Subunit rRNA Hybridizat
ion Probes for Quantitative Molecular Microbial Ec
ology Studies. Applied and Environmental Microbiol
ogy, 62: 4504-4513, 1996).

However, since the conventional method is premised on the use of an RI-labeled probe, the use of a special RI-handling facility is indispensable for its detection and quantification, and for this reason, it is monitored in an ordinary laboratory. Therefore, it is difficult to make a kit of this monitoring method and to develop the technology for its automation.

[0008] As another molecular genetic detection and quantification method, a quantitative PCR which enables a target gene region of a specific microorganism to be amplified and quantitatively analyzed by using a DNA primer specific to the specific microorganism. There are methods (for example,
LG Lee, CR Connell and W. Bloch: Nucleic Aci
ds Research, 21, 3761-3766, 1993). However, with this method, it is difficult to obtain a quantitative value of the total microbial community in the environment, and there is a large technical limitation that the ratio (dominance) of a specific microorganism to all the microorganisms cannot be analyzed.

The FISH method has been used to detect a specific microorganism at the cellular level regardless of the separation / culture method (RI Amann et al .: 1995, supra). Furthermore, the FISH-DC method is effective for analyzing the proportion of specific microorganisms in the total community at the cellular level in water environment samples (A. Maruyama and M. Sunamura: Simultaneous dire
ct counting of total and specific microbial cells
in seawater, using adeep-sea microbe as biomarker.
Applied and Environmental Microbiology, 66: 2211-
2215, 2000). However, in order to detect and identify microbial cells with low metabolic activity that make up the majority in the environment, some kind of signal amplification means must be used in combination, and there are still major technical limitations at this time (A. Maruyama and M. Sunamur
a: 2000, supra).

The microbial diversity and microbial community structure in the natural environment are analyzed by conventional separation and culture methods, and in addition to direct nucleic acid extraction of environmental microbial samples, PCR, cloning, nucleotide sequencing and molecular analysis are conducted. It is gradually being elucidated using a series of methods called systematic analysis (for example, T.
M. Schmidt, EF DeLong and NR Pace: Analysis
of a marine picoplankton community by 16S rRNA ge
ne cloning and sequencing, J. Bacteriol., 14, 4371
-4378, 1991).

From the analysis using these methods, it is becoming clear that a special microbial group, which has been difficult to separate and culture by the conventional method, appears in the environment polluted with petroleum or the like. For example, the aromatic hydrocarbon degrading bacterium Cyclocla
sticus pugetii on the US West Coast (SE Dyksterhouse,
JP Gray, RP Herwig, JC Lara and JT Sta
ley, Cycloclasticus pugetii gen. nov., sp. nov., a
n aromatic hydrocarbon-degrading bacterium from ma
rine sediments, Int. J. Syst. Bacteriol., 45, 116-1
23, 1995) and the aliphatic hydrocarbon-degrading bacterium Alcanivorax borkumensis, which was found in the coast of the Sea of Japan, and the North Sea (MM Yakimov, PN Golyshin, S. Lang, ER Moo
re, WR Abraham, H. Lunsdorf and KN Timmis: A
lcanivorax borkumensis gen. nov., sp. nov., a ne
w, hydrocarbon-degrading and surfactant-producing
marine bacterium, Int. J. Syst. Bacteriol., 48, 33
9-348, 1998), the coast of the Seto Inland Sea, the coast of the Sea of Japan, etc. (Kishira and Harayama: Biotechnology Society, Proceedings, P.291, 1998)
Have been found in. Among them, a method in which the above-mentioned total cell count method and MPN culture counting method were combined to further improve its accuracy was used to measure the Cycloc
The dominance analysis of lasticus pugetii has already been carried out (Japanese Patent Application No. 11-237818). However, there is no case where the above-mentioned dominance analysis of microbial groups was carried out by a molecular genetic method regardless of the use of RI.

[0012]

DISCLOSURE OF THE INVENTION The present invention is directed to both all microorganisms inhabiting the natural environment and specific microorganisms growing therein, and its molecular genetic detection and quantification are carried out.
Regarding a new method that can be performed by the Non-RI method (method that does not use radioactive isotope) without using PCR method and radioactive isotope, and analysis and evaluation method of polluted environment using the method It is an object of the present invention to provide a method for monitoring and monitoring an environment, particularly an oil-polluted environment, and for analyzing and evaluating a bio-environmental repair (bioremediation) process by microorganisms in the polluted environment by molecular genetics. Another object of the present invention is to provide a method for molecular genetically detecting and quantifying harmful microorganisms such as useful substance-producing microorganisms and pathogenic microorganisms in a natural environment or an artificial environment. further,
In a natural environment or an artificial environment, specific genetic microorganisms that produce useful substances such as enzymes, and dominance of specific functional microorganisms such as pathogenic microorganisms are analyzed by molecular genetics, and environmental samples collected from the environment are analyzed. It is also included in the object of the present invention to provide a method for analyzing / evaluating usefulness and harmfulness. Further, the nucleic acid probe disclosed in the present specification, which is specific to the newly developed petroleum-degrading bacterium Alcanivorax borkumensis, is used for detection / quantification of the petroleum-degrading bacterium, and analysis / evaluation of an oil-saturated environment and environmental samples thereby. It is provided by the present invention as a useful tool for.

[0013]

[Means for Solving the Problems] Environmental pollutants such as petroleum that have flowed out from an artificially controlled environment are gradually decomposed by microbial activities in the natural world and are naturally purified over time. If it is a persistent substance such as PCB, it will take a long time to decompose, but if the contamination affects the growth of general microorganisms, microorganisms specific to the contaminated environment ( It is thought that the group) will dominate.

For example, in the ocean, one of the field microbial communities
It is said that hydrocarbon degrading bacteria are widely distributed at a level of less than%, but the ratio is often high in petroleum-polluted areas.
It is said to be over 10% (RM Atlas: Petro
leum biodegradation and oil spill bioremediation,
Marine Pollution Bulltetin, 31, 178-182, 1995, R.
M. Atlas and R. Bartha: Hydrocarbon Biodegradation
and Oil Spill Bioremediation. In: Advances in Mic
robiolbial Ecology, Ed. KC Marshall, PlenumPres
S., New York, 12, 287-338, 1992), and the proportion of degrading bacteria in the entire microbial community is considered to reflect the degree of oil pollution (RM Atlas and R. Bartha: 1992, supra).

In addition, the number of PAHs-degrading bacteria in harbor sediments contaminated with creosote (containing approximately 85% of polycyclic aromatic hydrocarbons [PAHs]) discharged from wood processing facilities was investigated. (AD Geiselbrecht et al: Enumeration
and Phylogenetic Analysisof Polycyclic Aromatic Hy
drocarbon-Degrading Mairne Bacteria from PugetSoun
d Sediment, Appl. Environ. Microbiol., 62, 3344-33
49, 1996). According to it, the number of PAHs degrading bacteria, which was counted by MPN culture counting method, was 10 ⁴ to 10 ⁷ MPN / at the contaminated site.
g (dry weight), 10 ³ to 10 ⁴ MPN / g (dry weight) at the non-contaminated site. On the other hand, the total number of bacteria in the sediment is 10 ⁹ cells /
It was said that there was almost no difference between the contaminated site and the non-contaminated site at about g (dry weight). That is, in the creosote-contaminated site, PAHs degrading bacteria were
Not only was it 1000 times more common, but its proportion in the total microbial community had also increased to about 1%. In this way, it has been found that the proportion of the pollutant-decomposing bacteria group in the microbial community is increasing, reflecting the pollution of the environment,
The predominance of contaminant-degrading bacteria is considered to be a good indicator of the polluted environment.

However, the number of petroleum-degrading bacteria and the number of PAHs-degrading bacteria reported above are those detected and counted by the plate culture method or the culture method by the MPN method which requires a great deal of labor and time in any case. Also, as mentioned above, the number of microorganisms that can be separated and cultured is usually 1% or less of the total microbial community,
There is a drawback that the number of degrading microorganisms concentrated in the environmental microbial group is not sufficiently reflected in the counting by the culture method. further,
As in the method according to the present invention, the enumeration or quantification of specific degrading bacteria (microorganisms of a specific genus or microorganisms having genetic information that can be phylogenetically specified) in the concentration of environmental microbial groups has not yet been made.

Therefore, if it becomes possible to easily and quickly monitor the behavior of a specific microorganism that serves as an indicator of the environment containing these degrading bacteria, and the proportion (dominance) of it in the total microbial community. It is considered that the degree of pollution, the degree of restoration of the polluted environment, and the degree of restoration of the polluted environment can be diagnosed with high accuracy and at an early stage. To that end, it replaces the conventional separation and culture method, which has a large limit in conducting analysis of whole microbial communities and specific functional microorganisms,
Development of detection and quantification methods for specific microorganisms in environmental samples is essential. Furthermore, these methods need to be applicable to the diagnosis of various polluted environments and can be easily used. Based on these facts, the following steps were taken to develop a new method that meets these needs.

First, two types of petroleum degrading bacteria differing in petroleum component degrading characteristics, namely aliphatic hydrocarbon degrading bacteria and aromatic hydrocarbon degrading, which have been revealed to predominantly appear in actual oil turbid environments, are decomposed. Bacteria were selected and a nucleic acid probe specific to each was developed.

The aliphatic hydrocarbon-degrading bacteria selected here are
It decomposes aliphatic hydrocarbons but does not decompose aromatic hydrocarbons and sugars such as glucose. On the other hand, aromatic hydrocarbon-decomposing bacteria decompose difficult-to-decompose polycyclic aromatic hydrocarbons, but do not utilize aliphatic hydrocarbons or sugars such as glucose. Therefore, the dominance of these two petroleum degrading bacteria is considered to fluctuate depending on the hydrocarbon component and concentration in the spilled petroleum. It is possible to analyze and evaluate the natural purification process of pollutants and the bio-environmental restoration process, such as the ratio, concentration and fate of hydrocarbon components in the environment, by quantitatively understanding and analyzing the microflora at the nucleic acid level. It is supposed to be.

[0020] Next, the inventors of the present invention have established a No. 1 method for molecular genetic detection and quantification of all microorganisms and specific microorganisms in the environment.
It was the first successful development of the n-RI method. That is, the conditions of the hybridization and washing processes, which are indispensable when using a new probe, are examined by a method that does not use radioisotopes, and the most important hybridization process also uses a fluorescent or chemiluminescent label. The detection process can be performed by fluorescence detection method or chemiluminescence detection method. As a result, we succeeded in developing a relative quantification method of microbial nucleic acid in the environment by Non-RI method without using radioisotope throughout the whole process.

Since various contaminants are contained in the environmental sample, and it is difficult to set an internal standard, nucleic acid, especially RNA
Since is a component that is susceptible to enzymatic degradation, it is difficult to accurately estimate the efficiency of nucleic acid extraction and purification from environmental microorganisms. Therefore, even if a highly purified nucleic acid sample can be quantified, there is a large limit to the accurate quantification (absolute quantification) of the total nucleic acid content present in the used environmental sample. Therefore, the target sequence and the reference sequence are selected from among the nucleic acid molecules (the same molecule or multiple molecules may behave) that exhibit the same behavior during extraction and purification, and their abundance ratios are calculated from their abundances to calculate the relative amounts It is considered most effective to evaluate the abundance of microorganisms in environmental samples. This analysis method is called a relative molecular quantification method.

The principle of such a relative molecular quantification method is premised on hybridization by a plurality of probes in the same sample, and a quantification value of hybridization of a specific microbial probe (which detects a target sequence) is a universal probe common to all organisms. Corrected by the quantitative value of hybridization (detecting the reference sequence) to obtain an accurate relative value. In practice, standardization is performed by using the average value of the values obtained for various standard strains as reference data. It can be carried out. In this standardization, the existing
Although the method presented by the RI method can be used, the novelty of the present invention lies in the fact that all processes are first realized by the Non-RI method. In addition, this analysis method also has a great feature and advantage in that the PCR method is not used at all.

Immediately after the oil spill accident in 1997, an on-site microbial sample that had been concentrated, collected and frozen at the site of contamination was analyzed using this newly developed method. As a result, the predominance of the aliphatic hydrocarbon-decomposing bacteria and the aromatic hydrocarbon-decomposing bacteria in the total microbial group concentration could be estimated. In addition, the nucleic acid probe newly developed for the aliphatic hydrocarbon-degrading bacterium also enabled detection and quantification at the cell level by the fluorescence in situ hybridization (FISH) method.

As described above, the Non-RI analysis method according to the present invention is effective for the molecular genetic diagnosis of the actual oil pollution environment and the analysis / evaluation of the bio-environment restoration process. In addition, the analysis method is highly convenient in terms of kitting and automation, and further, this series of methods is used for the evaluation of polluted environments and environmental samples contaminated by harmful chemical substances other than petroleum. It has the advantage that it is extremely useful for the analysis and evaluation of the bio-environmental repair process. Also,
It is also extremely useful for detecting / quantifying, analyzing / evaluating harmful microorganisms such as useful substance-producing microorganisms and pathogenic microorganisms without depending on the separation / culture method.

The present invention has been completed based on these findings. That is, the gist of the present invention is as follows. 1. Nucleic acid of a specific functional microorganism in a sample obtained from a natural environment or an artificial environment is detected and quantified by the Non-RI method (method without using radioisotope), and the predominance of the specific functional microorganism in the environment is detected. The method comprises the following steps: 1) a step of extracting, purifying, and storing a target nucleic acid from a microorganism-containing sample collected from a natural environment or an artificial environment, 2) a target of a specific functional microorganism A nucleic acid sequence specific to the specific functional microorganism is selected from the nucleic acid base sequence information, and an oligonucleotide having the sequence is synthesized and labeled with a Non-RI label to be specific to the specific functional microorganism. Non-RI
Step of producing labeled nucleic acid probe, 3) Immobilize the target nucleic acid extracted and purified in 1) on a hybridization substrate, and contact this with a Non-RI labeled nucleic acid probe specific to a specific functional microorganism. After performing a hybridization treatment with the above, a step of performing a washing treatment, 4) a step of visualizing and analyzing a signal derived from the hybridized Non-RI labeled nucleic acid probe and obtaining a quantitative value thereof, 5) a determined quantitative value And a method of calculating the predominance of a microorganism having a specific function by comparing with a quantitative value measured for all organisms and each domain of the organism kingdom.

2. 3. The method according to 1 above, wherein the washing treatment of 3) is performed at an optimum washing temperature obtained from a release curve of a probe prepared using a Non-RI labeled nucleic acid probe specific to a specific functional microorganism. . 3. The non-RI labeled nucleic acid probe is fluorescently labeled or chemiluminescently labeled, 1 or 2 above
The method described in. 4. 4. The method according to any one of 1 to 3 above, wherein the specific functional microorganism is a microorganism that decomposes a specific chemical substance. 5. 5. The method according to 4 above, wherein the specific chemical substance is a harmful chemical substance.

6. 5. The method according to 4 above, wherein the specific chemical substance is petroleum and petroleum components. 7. 4. The method according to any one of 1 to 3 above, wherein the specific functional microorganism is a specific useful substance-producing microorganism. 8. 4. The method according to any one of 1 to 3 above, wherein the specific functional microorganism is a specific harmful microorganism including a pathogenic microorganism. 9. By using the method according to any one of 1 to 8 above, the microbial community function of the environment is analyzed by analyzing the dominance of specific function microorganisms in a natural environment or an artificial environment.
How to evaluate. 10. Using the method according to any one of 1 to 8 above,
How to analyze and evaluate the polluted environment.

11. A method for analyzing and evaluating an environment contaminated with harmful chemicals using the method described in 4 or 5 above. 12. A method for analyzing and evaluating an oil spill environment using the method described in 6 above. 13. An RNA having a base length of 10-50 bp, which has a part of the nucleotide sequence of SEQ ID NO: 5 or a part of the corresponding riboxynucleotide sequence and can specifically hybridize with a nucleic acid derived from a petroleum-degrading bacterium of the Alcanivorax family or genus. Or a DNA probe. 14. DNA or RNA having the nucleotide sequence of SEQ ID NO: 1 or the corresponding riboxynucleotide sequence capable of specifically hybridizing with a nucleic acid derived from a petroleum-degrading bacterium of the Alcanivorax family or genus and detecting or quantifying it probe. 15. Alcivorax family or genus petroleum-degrading bacteria are Alc
13 which is an anivorax borkumensis or a closely related species
Or the DNA or RNA probe according to 14.

16. Non-RI specific to a specific functional microorganism
As the labeled nucleic acid probe, the DNA or RNA probe described in 13 above, which is capable of specifically hybridizing with a nucleic acid derived from a petroleum-degrading bacterium of the Alcanivorax family or genus, or the nucleotide sequence described in SEQ ID NO: 1 or a riboxynucleotide corresponding thereto. A DNA or RNA probe having a sequence, and / or a part of the nucleotide sequence of SEQ ID NO: 6 or a corresponding riboxynucleotide sequence capable of specifically hybridizing with a nucleic acid derived from a petroleum-degrading bacterium of the genus Cycloclasticus , An RNA or DNA probe having a base length of 10-50 bp, or a DN having the base sequence described in SEQ ID NOs: 2 to 4 or a ribonucleotide sequence corresponding thereto
A nucleic acid probe selected from A or RNA probes is used, and according to the method described in 1 above, Al concentrated in environmental microorganisms is collected.
canivorax family or genus and / or Cycloclastic
Analysis of the oil spill environment by analyzing the dominance of petroleum-degrading bacteria belonging to us
How to evaluate.

17. Cycloclasticu, an aliphatic hydrocarbon-degrading bacterium Alcanivorax borkumensis or its related species, and / or an aromatic hydrocarbon-degrading bacterium concentrated in environmental microbes
17. The method according to 16 above, which comprises analyzing the dominance of S. pugetii or its related species.

18. The DNA or RNA probe according to 13 above, which has a base length of 10- having a part of the base sequence of SEQ ID NO: 6 or a ribonucleotide sequence corresponding thereto which can specifically hybridize with a nucleic acid derived from a petroleum-degrading bacterium of the genus Cycloclasticus A 50-bp DNA or RNA probe, and / or one or more nucleic acid probes selected from DNA or RNA probes having the nucleotide sequences shown in SEQ ID NOS: 1 to 4 or a ribonucleotide sequence corresponding thereto are used as Non-RI. A kit for molecular genetic analysis / evaluation of a contaminated environment and an environmental sample, which contains a Non-RI labeled probe obtained by labeling.

[0032]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention is 1) targeted by a microorganism-containing sample collected from a natural environment or an artificial environment.
Step of extracting, purifying, and storing nucleic acids such as S rRNA, 2) selecting a sequence specific to the specific functional microorganism from the base sequence information such as 16S rDNA that is the target of the specific functional microorganism,
An oligonucleotide having the sequence is synthesized, labeled with a Non-RI label, and labeled with a No.
Step of preparing n-RI-labeled nucleic acid probe, 3) Immobilize the target nucleic acid such as 16S rRNA extracted and purified in 1) on a hybridization substrate, and prepare the specific functional microorganism prepared in 2) Of non-RI-labeled nucleic acid probe that is specific for the above, and after the hybridization treatment, a washing treatment, 4) hybridized Non-RI
The step of visualizing and analyzing the signal derived from the RI-labeled nucleic acid probe to determine its quantitative value, 5) using the determined quantitative value, and comparing it with the quantitative value of the reference sequence measured for all organisms and each domain of the living kingdom Then, the nucleic acid of the specific function microorganism is detected and quantified by the Non-RI method (method not using radioactive isotope) from the natural environment or the artificial environment including the step of calculating the degree of predominance of the specific function microorganism. A method for analyzing the dominance of specific functional microorganisms in the environment is provided.

In this method, measuring the abundance of communities of all organisms and domains of the living world is useful for knowing the size of the entire microbial community (size of the population) in the target environmental sample. It is indispensable, and has a very important meaning in the process of estimating the predominance of the specific functional microorganism in the group concentration.

The term "dominance" refers to the ratio of the specific microorganism of interest to all the microorganisms in the sample or the microorganisms belonging to each domain. Here, "natural environment" means the biosphere on the earth, such as the oceans, lakes and marshes, hydrospheres such as rivers, soil and land underground, geosphere such as submarine underground, atmospheres such as the earth's surface layer, and animal and plant bodies. It refers to the entire environment on the earth where microorganisms inhabit such as the carcass.

The term "artificial environment" means an environment that is artificially controlled to some extent, for example, an environment in a laboratory flask or a verification test device, food, brewing, pharmaceuticals, etc. Environment in manufacturing tanks such as fermentation tanks used in the field of chemistry and biotechnology, or tap water, water for domestic and industrial use,
Artificial production of water, such as cooling water, circulating water, drainage, sewage,
It refers to the environment related to processing, storage, transportation, use, and disposal, and refers to any artificially controlled environment in which microorganisms may be present or contaminated.

The "specific functional microorganism" refers to a microorganism whose function and characteristics are specified by culturing, genetic analysis and the like among microorganisms inhabiting the natural environment, and examples thereof include petroleum and petroleum components. Microorganisms that decompose specific chemical substances, trichlorethylene, and PCBs, organic chlorine compounds such as dioxins, environmental hormone substances (alkylphenol, bisphenol A, phthalate ester, etc.), organic mercury cyanide compounds, organic tin compounds, and other harmful chemical substances A microorganism that decomposes can be exemplified. Also, chitinase, lipase, cellulase, xylanase, microorganisms that produce useful substances such as useful enzymes such as lignin-degrading enzymes and various antibiotics, Escherichia coli containing Escherichia coli O157 strain, cholera, and pathogenic microorganisms such as anthrax, and Examples of the microorganisms having a specific function include, but are not limited to, a sulfate-reducing bacterium that causes corrosion of a tank and an offensive odor by producing hydrogen sulfide.

The term "closely related species" cannot be assigned to a known species based on its phenotypic traits and homology of gene sequences, but it is necessary to establish a new species by distinguishing it from the characteristics of the known species. Species that do not reach the target range, and have very similar phenotypic traits among known species or species, and have close phylogenetic positions. When used herein, the homology is judged to be 90% or higher, preferably 94% or higher, more preferably 94% or higher, as judged from the homology of 16S rDNA widely used as an indicator of systematics of organisms. Indicates 96% or more. Examples of closely related species of Alcanivorax borkumensis include Alcanivorax sp.ST-T1, Alcanivorax sp.WF-1, and Alca.
nivorax sp. Shm-2, Alcanivorax sp.K3-3, Alcanivorax
sp.K2-1, Alcanivorax sp.TE-9, fFundlbacter jadens
Is is mentioned. Cycloclasticus sp.M4-6 and Cycloclasticus s are related species of Cycloclasticus pugetii.
p.E16-S, Cycloclasticus sp.W, Cycloclasticus sp.
G, Cycloclasticus sp.N3-PA321, Cycloclasticus sp.
(Cycloclasticus oligotrophus) and the like (these rDNA sequences are registered in the Japan DNA Data Bank (DDBJ)). However, it is not limited to the examples listed here.

The "microorganism-containing sample collected from a natural environment or an artificial environment" is a natural environment sample itself such as seawater, lake water, river water, bottom mud, sediment, soil, minerals, groundwater, pore water, animals and plants. , Or the above-mentioned artificial environmental samples themselves, and when there are few microorganisms in their environment,
It means a sample in which microorganisms in the environment are concentrated by filtration or centrifugation. For example, using a filter such as a flat membrane filter or a hollow fiber membrane filter having a pore size of 0.2 micrometer smaller than the size of most common microorganisms, microorganisms in environmental water are concentrated on the filter by a filtration operation, and the concentrate Can be used as a sample. Also,
Using a flat membrane filter with a pore size of 0.2 micrometer, for example, using a Millipore tangential flow filter, a liquid sample is filtered in a horizontal direction rather than in a vertical direction for filtration and concentration, and the concentrated solution can be used as a sample. . In addition, the sample can be directly subjected to high-speed centrifugation, for example, about 8,000 xg.
By subjecting to centrifugation for 10 minutes or more with the above centrifugal force, microorganisms can be precipitated to obtain a concentrated sample, which can be used as a nucleic acid extraction sample.

[0039] A known method (for example, MG Murray and WF Tho) can be used to extract nucleic acid from the above-mentioned microorganism-containing sample.
mpson, Nucleic Acids Research 8: 4321-4325, 1980
Method described in 1) can be used. For soil and sediment samples, purification methods using hydroxyapatite (eg KJ Purdy, DB Nedwell,
TM Embley and S. Takii: Use of 16S rRNA-targete
d oligonucleotide probes to Investigate the occurr
ence and selection of sulfate-reducing bacteria In
response to nutrient addition to sediment slurry
microcosms from aJapanese estuary, FEMS Microbiol.
Ecol., 24, 221-234, 1997) is also useful. In particular,
When the analysis target is RNA such as 16S rRNA, a commercially available RNA extraction kit, for example, Qiagen RNeasy kit, Stratagene RN
A RT-PCR Miniprep kit, Clontech NucleoSpin RNA ki
t, Ambion RNAqueous kit, etc. can be used.
Further, when a sample contains a large amount of contaminants, several nucleic acid extraction techniques can be used together to improve the degree of purification of the nucleic acid extract. This degree of purification can be easily confirmed, for example, by measuring an absorption spectrum around a wavelength of 220 to 400 nm using a spectrophotometer and comparing the absorption spectrum with a pure RNA or DNA standard sample. In any case, it is necessary to pay close attention during the extraction and purification operations in order to prevent contamination of microorganisms, nucleolytic enzymes and contaminants.

"Preparing a non-RI labeled nucleic acid probe" means that an oligonucleotide having a sequence selected as a probe and having a length of about 10 to 50 bases is subjected to RI for fluorescence detection, chemiluminescence detection, chemiluminescence detection, or the like. It means that a Non-RI labeled nucleic acid probe is prepared by performing labeling so as to allow analysis by a detection method that is not used. Here, the oligonucleotide can be synthesized by a known method, for example, the phosphoramide method or the triester method. Or DNA
You may synthesize | combine with an automatic synthesizer. It is also known to those skilled in the art that the oligonucleotide may be directly labeled with a fluorescent dye, or may be labeled with a peptide substance such as digoxigenin and indirectly labeled via a substance that binds to the peptide substance. is there. Fluorescent dyes include rhodamine, fluoroscein-isothiocyanate (FITC), LuciferYellow CH, rhodamine 123 (Rho
damine 123), Pyronin Y, Propidium iodide, Ethidium homodimer, Carboxyfluorescein diacetate (CFDA), Fluorescein diacetate (FD)
A), carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM), 5-cyano-2,30 ditolyltetrazolium (CTC), tetramethylrhodamine isothiocyanate (TRITC), sulforfodamine 101 acid chloride ( Sulforhodamine 101 acid chloride; Texas Red), Cy3, Cy5, Cy7, 2-hydroxy-3-
Naphtholic acid-2'-phenylanilide phosphate (2-hy
droxy-3-naphtoic acid-2'-phenylanilide phosphate;
HNPP) and the like can be exemplified, but not limited thereto, and any fluorescent substance known to those skilled in the art may be used. Alternatively, a method in which an oligonucleotide is labeled with an antigen substance and chemiluminescence or fluorescence (also called chemifluorescence) generated by enzymatic decomposition of a substrate is detected using an enzyme immunological method can be used. As a label for using the indirect labeling and the enzyme immunological method, biotin / avidin, digoxigenin / anti-digoxigenin system, etc. can be used, but not limited thereto.

In any of the above cases, there is no or weak background fluorescence or luminescence derived from a substrate such as a membrane filter used for hybridization and a glass slide treated with an organic film, and nonspecific to the substrate. It is preferred to use fluorescent dyes with no or very little binding or protein-protein binding or enzyme-substrate reaction systems. For example, when performing fluorescence detection using a membrane filter as a basis for hybridization, it is preferable to use a fluorescent dye such as Cy 5, and when performing chemiluminescence detection in the same manner, for example, labeling with digoxigenin (DIG) and the like. However, a chemiluminescence system (for example, a kit manufactured by Roche) using CDP-Star as a substrate for alkaline phosphatase may be used by using an alkaline phosphatase-labeled anti-digoxigenin antibody.

In order to "determine the release curve of a probe by the Non-RI method", a nucleic acid probe labeled with an antigenic substance such as the fluorescent dye or digoxigenin or a peptidic substance prepared above is used, and it is complementary to the probe sequence. Hybridization is performed on a nucleic acid extracted sample of a microorganism having a different rRNA sequence, for example, on a hybridization substrate such as a positively charged nylon membrane filter or a slide glass coated with an organic film such as poly-L-lysine. Next, Zheng et al. (DAE Zheng et al .: 1996, supra).
According to the method performed by using the RI-labeled nucleic acid probe, the fluorescence amount and the chemiluminescence amount are measured. That is, a few degrees
Each time the temperature is raised, the washing solution is exchanged every time the temperature is raised, and the washing process is performed.Fluorescence or chemiluminescence derived from the Non-RI labeled nucleic acid probe released in the washing solution at each washing process. Is measured and the measured value is plotted against the washing temperature to obtain a release curve. Finally, in the obtained S-shaped release curve, the temperature near the inflection point seen on the low temperature side is set as the optimum probe cleaning temperature.

In order to "immobilize nucleic acid on a substrate for hybridization", first, for example, a positively charged nylon membrane filter or a slide glass treated with an organic coating such as poly-L-lysine is used. Drop a fixed amount of nucleic acid sample on the substrate for hybridization,
Adhere nucleic acids. Next, if a filter is used as a base, a UV irradiation device such as a UV crosslinker (St
Ratagene) or the like to perform UV irradiation treatment or to perform alkaline solution treatment to strongly adsorb or bind the nucleic acid on the filter, thereby immobilizing the nucleic acid on the hybridization substrate. be able to. When using slide glass as a base,
After forming a film on the glass surface with a substance that mediates the adsorption of nucleic acids between the substrate and the nucleic acids (such as poly-L-lysine), drop the nucleic acid sample on top of it to adsorb the nucleic acids It can be immobilized on a substrate for hybridization.

In order to "perform the hybridization treatment and washing treatment by the Non-RI method", the following steps are performed. The nucleic acid sample immobilized on the substrate by the above method is first mixed with a hybridization solution (for example, Raskin L, SJ,
Rittmann BE, Stahl DA: Group-Specific 16S rRNA Hy
bridization Probes to Describe Natural Communities
of Methanogens. Applied and Environmental Microbi
ology, 60: 1232-1240, 1994) for 30 minutes at an arbitrary temperature, for example 35 ° C. (prehybridization process). Next, the above-mentioned hybridization solution to which the above-mentioned Non-RI labeled nucleic acid probe has been added is brought into contact overnight at an arbitrary temperature, for example, 35 ° C. (hybridization process). In these steps,
The latter process is essential. Finally, the immobilized nucleic acid sample after hybridization is washed with a washing solution (e.g. D.
AE Zheng et al .: 1996 [solution described above],
The probe used is contacted twice for 30 minutes each at the optimum cleaning temperature determined by the above method (cleaning process).

"The fluorescent or chemiluminescent signal signal derived from the hybridized Non-RI labeled nucleic acid probe is visualized and analyzed to determine its quantitative value" is performed according to the following procedure. DNA that has hybridized to the nucleic acid sample immobilized on the substrate by complementary binding of nucleic acids after the above hybridization and washing treatment
Fluorescent or chemiluminescent signal derived from the probe, using an apparatus and conditions depending on the type of signal derived from the labeled probe, that is, in the case of a fluorescent signal, a fluorescence image analyzer such as FluoroImager or Stor
m, Typhoon (above, Amersham Pharmacia Biotech)
Or FX Pro (manufactured by BioRad) and cooled CCD camera, in case of chemiluminescent signal, Photomul or cooled CCD camera,
A device such as a photon counting camera is used to visualize the image and analyze it to obtain the signal intensity as a quantitative value.

"All organisms" means Archaea,
It means the sum total of all three domains of the living kingdom such as bacteria (Bacteria) and Eucarya (Eucarya), and "calculates the dominance of specific functional microorganisms for all organisms or each domain of the living kingdom" means all three domains above. As a denominator, the total of quantitative values measured for the target or the quantitative value measured for the domain to which the target specific functional microorganism belongs,
A quantitative value measured for a specific functional microorganism is calculated as a molecule. However, in practice, it is necessary to make some corrections as described below.

For example, in the case of targeting 16S rRNA, even if the target nucleotide sequence regions of two kinds of probes exist in a ratio of 1: 1 in one molecule of rRNA, they are affected by the three-dimensional structure of rRNA and the like. The quantitative values are not always the same. In such a case, it is necessary to make correction so that a quantitative value proportional to the abundance ratio of the target base sequence region can be obtained.

Further, unless the extraction / purification efficiency is accurately estimated, the absolute quantification of the target base sequence region specific to the specific functional microorganism is impossible, but the accurate estimation of the extraction / purification efficiency is as described above. Currently not possible. Therefore, it is necessary to relatively evaluate the abundance of the target base sequence region based on the ratio of the target base sequence region to the base sequence region (universal region) common to all of the above three domains.

In the case of rRNA, the abundance per cell is considered to vary depending on the cell type as well as the cell activity. Furthermore, it is considered that the ratio of the region specific to each domain to the universal region is different depending on the type even within the same domain due to the three-dimensional structure of rRNA. Therefore, it is necessary to measure the variation in the quantitative value based on the difference between these types in advance using some typical standard strains, obtain the average value, and perform the calculation correction based on this.

Finally, it is necessary to subtract the quantitative value of the background, which is provided by the hybridization substrate itself and the nucleic acid probe non-specifically adsorbed thereto, from the measured value. In making corrections in calculating the dominance of these specific functional microorganisms, the following formula 1 (SJ Giovannoni et al .: 1
990, supra).

[0051]

[Equation 1]

In this calculation, Amann was used to detect and quantify each domain of bacteria, archaea and eucarya.
(RI Amann et al .: 1995, supra), DNA probes such as Euba338, Arch 915, Euka 502 can be used. In addition, targeting all living organisms common areas
For DNA probes, Univ 1390 (DAE Zheng et al .:
1996, supra) can be used. To detect and quantify a microorganism with a specific function, the gene nucleotide sequence region unique to that microorganism, such as 16S rDNA, 18S rDNA, 23S rDNA, gy
A base sequence region specific to the species may be selected from gene base sequence information such as rB, and a probe containing the sequence may be synthesized. The Non-RI labeled nucleic acid probe can be prepared by the method described above.

By using the above method to analyze the degree of predominance of a specific function microorganism predominant in a natural environment or an artificial environment, the microbial community function of that environment can be evaluated. Here, the "microbial community function" refers to the overall function performed by a plurality of types of microorganisms inhabiting the target environment. The term "type of microorganism" as used herein is preferably a species that is an ordinary classification unit, but may be an experimental functional unit, for example, one functional unit such as a petroleum-degrading bacterial group or a PCB-degrading bacterial group. Therefore, the function of only one specific function microorganism is not called the microbial community function.

For example, regarding the decomposition of petroleum, in addition to microorganisms that decompose aliphatic hydrocarbons and microorganisms that decompose aromatic hydrocarbons, many microorganisms are involved in the process of decomposing intermediate metabolites of these decompositions to carbon dioxide. It is thought that these microorganisms also coexist with microorganisms that supply trace essential nutrients such as vitamins, etc., and multiple microorganisms cooperate to contribute to mineralization and detoxification of petroleum. Can be considered as being. In this case, when a certain degradation product can be a growth inhibitor, coexistence of the degradation product with an available microorganism is considered to increase the degradation efficiency, and a high degradation function is expressed in the entire microbial community. Will be.

Specifically, one or more kinds of specific functional microorganisms in the microbial community, such as aerobic hydrocarbon-decomposing bacteria, PCB-decomposing bacteria, protein-degrading bacteria, glucose-assimilating bacteria, etc., by the method described above, and Anaerobic sulfate-reducing bacteria, methanogenic bacteria, etc., and harmful microorganisms such as pathogenic bacteria, etc. Priorities of useful substance-producing bacteria that produce various enzymes or antibiotics, etc. have been investigated, and by examining the change over time,
It is possible to analyze the transition of each specific functional microorganism.
As a result, for example, in the coastal surface water, if the predominance of sulfate-reducing bacteria increased at a certain time, the microbial community at that time could be evaluated as potentially having a high sulfate-reducing function. It can be presumed that they were transferred from the normal habitat due to the storm of the bottom mud caused by typhoons and crustal activities and the inflow of anaerobic drainage before the collection.

The polluted environment can be analyzed and evaluated by using the above method. For example, if the target specific functional microorganism is a heterotrophic bacterium that predominantly predominantly exists in the wastewater of a pulp mill, if its predominance increases at a certain time, the environment will change to this wastewater. If it can be judged that there is a high possibility that it is contaminated, and if the change in the degree of dominance is monitored over a long period of time and the periodicity and seasonality of the transition are grasped, the change is sudden. It is possible to evaluate whether there is it and whether the load is artificial. Further, it is possible to estimate the collection location, season / season of environmental samples containing useful substance-producing microorganisms, and the generation location, season / season of harmful microorganisms.

Furthermore, by using the above method, it is possible to analyze and evaluate an environmental sample contaminated with a harmful chemical substance and an environmental sample which is considered to contain a specific microorganism. For example, if the target specific microorganism is a PCB-degrading bacterium, if its predominance increases at a certain time, it can be determined that the environment is likely to be PCB-contaminated, and the microbial community is It can be determined that the PCB decomposition function is increasing as a whole. Furthermore, if the change in the degree of dominance is monitored for a long period of time and the periodicity and seasonality of the transition are grasped, whether the change is sudden or not, and the load is artificial due to the inflow of factory wastewater. Can be estimated.

Further, the oil turbid environment can be analyzed and evaluated by using the above method. For example, if the target specific functional microorganism is derived from a petroleum component, for example, a tetradecane or anthracene degrading bacterium, if its predominance increases at a certain time, its environment is Among the petroleum components, saturated hydrocarbons such as n-alkanes, and in the latter case aromatic hydrocarbons, it can be considered highly likely that they are contaminated, respectively, and the microbial community as a whole has the function of degrading the petroleum components. It can be determined that it is increasing. In addition, by investigating changes in the dominance of specific functional microorganisms, it is possible to analyze and evaluate the degree of petroleum pollution or other pollution and the bioremediation process. Furthermore, if the change in the degree of dominance is monitored over a long period of time and the periodicity and seasonality of the transition are grasped, whether the change is sudden or not and the load may cause a ship accident or the like. It is possible to estimate whether the cause is artificial.

Focusing on the aliphatic hydrocarbon-decomposing bacteria known to be distributed in many sea areas as the specific function microorganisms, the aliphatic hydrocarbon-decomposing bacteria GR211 purely separated from the coastal area of Shikoku through enrichment culture -P1 strain (patent deposit number FERM
BP-7788: Transferred to international deposit dated November 5, 2001)
Has a part of the nucleotide sequence of 16S rDNA (SEQ ID NO: 5 in the Sequence Listing) of Alcanivorax and is a petroleum-degrading bacterium of the genus Alcanivorax, especially Alcanivorax bo
The nucleotide sequence region of SEQ ID NO: 1 (base number 207 in SEQ ID NO: 5) that specifically hybridizes with a nucleic acid derived from rkumensis and its related species and can detect or quantify it.
~ 226 areas. A numbering system showing the position from the 5'end in the nucleotide sequence of Escherichia coli 16S rDNA [HF Noller and CR Woese, Science, 212:
403-411, 1981], a region corresponding to the base number 222 to 241) and a DNA having a base length of 10 to 50 bp, preferably a base length of 15 to 25, including all or part of the base sequence of this region.
It is advisable to design probes, or RNA probes having ribonucleotide sequences corresponding to them. The following probes can be mentioned as an example. (1) 5'-C
GA CGC GAG CTC ATC CAT CA-3 '(Albo 222, 20 mer)
(SEQ ID NO: 1) 3'-GCT GCG CTC GAG TAG GTA GT-5 '
(SEQ ID NO: 7) When these probes are synthesized and labeled with Non-RI, that is, with a fluorescent dye or an antigenic substance, the method described above may be used.

When attention is paid to aromatic hydrocarbon-degrading bacteria whose distribution is confirmed in a plurality of sea areas as the specific functional microorganism, DNA probes and RNA probes having the following base sequences can be used. (1) 5'-GGAAACCCGCCCAACAGT-3 '(Cypug829-846 ^* , 18m
er: a region of nucleotide numbers 823 to 840 in SEQ ID NO: 6) (SEQ ID NO: 2) 3'-CCTTTGGGCGGGTTGTCA-5 '(SEQ ID NO: 8) (2) 5'-TGCACCACTAAGCGGAAACC-3' (Cypug840-859 ^* , 2
0mer: a region of base numbers 834 to 853 in SEQ ID NO: 6)
(SEQ ID NO: 3) 3'-ACGTGGTGATTCGCCTTTGG-5 '(SEQ ID NO: 9) (3) 5'-TGCACCACTAAGCGGAAACCCGCCCAACAGT-3' (Cypug
829-859 ^* , 31mer: nucleotide numbers 823 to 85 in SEQ ID NO: 6
(Region 3) (SEQ ID NO: 4) (Note that ^* (number) indicates the position (numbering system) from the 5'end in the 16S rDNA nucleotide sequence of Escherichia coli (Noller HF and CR Woese, 1981. Sc.
ience, 212: 403-411). The base sequence of the probe of (3) is a sequence in which the base sequences of the probes of (1) and (2) are adjacent to each other, but in this case, the probe may self-bind, so caution is required.

These probes were used in the coastal waters of Mikuni Town, Fukui Prefecture, where the bow sank and a large amount of oil spilled after the oil spill in the Sea of Japan due to the Russian Tankana Khotka on January 2, 1997. A portion of the 16S rDNA nucleotide sequence (SEQ ID NO: 6 in the Sequence Listing) derived from a petroleum degrading bacterium found in a sample collected on January 15, 1997, and is an aromatic hydrocarbon degrading bacterium of the Cycloclasticus genus, especially Cycloclasticus pugetii
And its relatives.

In addition to these nucleic acid probes for detecting specific functional microorganisms, a nucleic acid probe for detecting the domain to which the specific functional microorganism belongs and a universal probe for all living organisms containing the same are used to detect the target environment. By applying the above-mentioned molecular genetic detection and quantification method (relative molecular quantification method) to microorganism-containing samples, Alca
Aliphatic hydrocarbon-degrading bacteria of the genus nivorax, especially Alcanivorax
borkumensis and its relatives, and Cycloclasticus
Aromatic hydrocarbon degrading bacteria of the genus, especially Cycloclasticus puget
It is possible to accurately measure, at the molecular level, the degree of predominance of ii and its related species in the bacterial domains to which the entire microbial community and / or both belong. Furthermore, by analyzing the temporal and / or spatial changes in this dominance,
It is possible to analyze and evaluate the state of natural environment and artificial environment, especially the state of oil pollution environment contaminated with petroleum from a microbiological viewpoint.

According to the method of the present invention, an environment contaminated with harmful chemical substances such as petroleum, microorganisms inhabiting the environment, conventional separation / culture methods, RI (radioisotope) and PCR methods are used. It can be easily and quickly evaluated by molecular genetics without using it. In addition, the method of the present invention,
In particular, it can be applied to the detection / quantification of specific functional microorganisms such as useful enzyme-producing microorganisms producing useful substances such as antibiotics, and screening. Furthermore, the method of the present invention, detection and quantification of specific functional microorganisms such as harmful microorganisms such as pathogens,
It can also be applied to screening. It is therefore possible and advantageous to provide the kit with the reagents and equipment necessary for the method of the invention and to automate the method of the invention. Further, the method of the present invention comprises
It is extremely useful as a diagnostic tool for evaluation of the environment contaminated with harmful chemical substances, analysis of bioenvironmental repair (bioremediation) process, and evaluation of repair effect. Further, the present invention is included in a natural environment or an artificial environment,
It is useful for detecting, quantifying, and screening microorganisms that produce useful substances and microorganisms with specific functions such as harmful microorganisms such as pathogenic microorganisms. Therefore, the present invention is extremely useful in the analysis / evaluation of the environmental sample containing such a specific functional microorganism.

Further, the DNA probe disclosed in the present invention is
It is capable of specifically identifying a specific aliphatic hydrocarbon-degrading bacterium that has been reported to appear in many sea areas in recent years, and is extremely useful for its detection and quantification.

[0065]

[Example 1]

(1) Development of DNA probe for detection of Alcanivorax borkumensis The GR211-P1 strain of aliphatic hydrocarbon degrading strain (patent deposit number FERM BP-7788: Heisei 13) successfully isolated purely from a sample collected from the coast of Shikoku through enrichment culture. 16S rDNA nucleotide sequence information (SEQ ID NO: 5) transferred to the international deposit on November 5, 2014
From among, Stahl and Amann (Development and Applic
ation of Nucleic Acid Probes. In: Nucleic Acid Tec
hniques in Bacterial Systematics. Ed .: E. Stackebr
andt and M. Goodfellow, John Wiley and Sons, Chiche
ster, pp. 205-248, 1991), a sequence specific to this fungal species is selected from the region where the disorder due to the higher order structure is not likely to be seen, and an oligonucleotide having the sequence is synthesized. , Its 5'end is FITC, TRITC or Cy 5
Of the non-RI-labeled DNA probe (hereinafter referred to as Albo 22
2) is produced.

The sequence of this probe is 100 with the sequence of Alcanivorax borkumensis SK2 (reference strain; MM Yakimov et al .: 1998, supra) and its related species Fundibacter jadensis T9 (reference strain) on the international nucleotide sequence database. Alcanivorax b was confirmed to show% homology.
It was shown to be effective as a DNA probe for detecting orkumensis or Alcanivorax strains. This relative Fund
ibacter jadensis (A. Bruns and L. Berthe-Corti: Fu
ndibacter jadensis gen. nov., sp. nov., a newsligh
tly halophilic bacterium, isolated from intertidal
sediment, Int. J. Syst. Bacteriol., 49, 441-448,
1999) is based on the molecular phylogeny of 16S rDNA sequences in Alcani.
vorax borkumensis considered to be in the same strain group, and its r
Since the total DNA nucleotide sequence has a high homology of about 98% with the rDNA nucleotide sequence of Alcanivorax borkumensis, it is highly likely that it will be included in the Alcanivorax genus that has been proposed in the near future. Judged

In using the DNA probe shown in the present invention, Alcanivorax borkumensis DSM11573 (standard strain) is used as a target microorganism, Pseudomonas aeruginosa IFO12689.
(Standard strains) and other target microorganisms, Maruyama and Sun
amura (Simultaneous direct counting of total and s
pecific microbial cells in seawater, using a deep-
sea microbe as biomarker. Applied and Environmenta
l Microbiology, 66: 2211-2215, 2000) and actually confirmed its effectiveness by the FISH method. The hybridization was carried out at 43 ° C in the presence of 20% formamide, and the washing treatment was carried out at 44 ° C.

(2) DNA probe for detecting Cycloclasticus pugeti As a DNA probe for detecting Cycloclasticus pugetii which is another target microorganism, Cyclopug 829-84
6 (SEQ ID NO: 2: sometimes referred to as Cypug829 hereinafter) was used. The array of this probe is located in the coastal waters of Mikuni Town, Fukui Prefecture, where a large amount of oil spilled on the bow after the oil spill in the Sea of Japan caused by the Russian Tankanakhodka on January 2, 1997. From the sample collected on January 15th,
Cycloclasticus pugetii 16S was obtained by direct extraction of DNA from a growth-positive front sample in a serial dilution culture count (MPN) sample of petroleum-degrading bacteria using C heavy oil as the sole carbon source, and its sequencing. It was found as a sequence specific to Cycloclasticus pugetii from rDNA nucleotide sequence information.

[Example 2] Using these DNA probes, Cycloclasticus p
The results of the dominance analysis of ugetii and Alcanivorax borkumensis are shown below.

1) Sampling and storage of samples After the oil spill accident in the Sea of Japan due to the Russian Tankana Khotka on January 2, 1997, a large amount of oil spilled due to the bottom of the bow. We used a sample that had been frozen and preserved on January 15, 1997 in the coastal waters of Mikuni Town, Fukui Prefecture, where it washed ashore. That is, about 3 oil-saturated seawater collected locally
The liter was immediately filtered and concentrated on the filter using a hollow fiber membrane filter HF400 (manufactured by Millipore) having a pore size of 0.2 micrometer, and the whole filter was frozen and stored.

2) Cell disruption, RNA extraction, storage The obtained environmental microorganism-enriched sample was used together with the hollow fiber membrane filter.
Transfer to a 12 ml rigid glass tube and use Qiagen RNeasy
The RLT buffer contained in the Midi Kit was added. Glass beads (GLA manufactured by Sigma) that have been sterilized by dry heat at 180 ° C for 1 hour or more.
RLT buffer using SS BEADS 106microns and finer)
After adding 1.5 g to 1.5 ml, the cells were disrupted by a reciprocal shaking treatment for 5 minutes with a cell disruptor (MSK Cell Homogenizer manufactured by B. Braun). This process
Monitoring was performed with an electronic thermometer, and liquid carbon dioxide was sprayed to maintain the temperature of the sample at about 5 ° C. In addition, this treatment time is longer than that of Gram-negative bacteria because the cell wall is harder and Bacillus subti belongs to the Gram-positive bacteria that live on land.
Using lis IFO13719 cells, observe the state of cell disruption under a microscope, and analyze the nucleic acid eluted in the buffer solution by agarose gel electrophoresis. The time that does not cause the change was selected as the processing condition. From the obtained cell disruption sample, Qiagen's RNea
RNA was extracted and purified using sy Midi Kit and 50-100 each
Each aliquot was divided into aliquots and stored at -80 ° C.

3) Measurement of total RNA amount Before the hybridization analysis, the total RNA amount in the sample was measured by RiboGreen RNA Quantitation Kit (Molecular Prob
es) and a fluorescence spectrophotometer RF-5300PC (Shimadzu) with an excitation wavelength of 480 nm, a fluorescence wavelength of 520 nm, and a bandwidth of 5
It was measured under the setting condition of nm.

4) Hybridization by Non-RI method Hybridization by the Non-RI method using a fluorescent probe was carried out as follows. First, the frozen sample is stored in a buffer solution (Dimethyl pyrocarbonate (DMP
C) Treated water, 20xSSC [0.15 M NaCl and 0.015 M sodi
um citrate] and formaldehyde at a ratio of 5: 3: 2), and the nucleic acid in the sample is adsorbed on a positive charge nylon membrane filter (Roche Diagnostics), and UV Stratalinker (Stratagene (Manufactured by the company) was immobilized on the filter. Next, hybridization solution (Raskin L, SJ, Rittmann
BE, Stahl DA: Group-Specific 16S rRNA Hybridization
Probes to Describe Natural Communities of Methano
ges. Applied and Environmental Microbiology, 60: 1
232-1240, 1994) and pre-hybridization was performed at 35 ° C. for 30 minutes. Then, using the above-mentioned hybridization solution to which the Cy 5 labeled DNA probe was added so that the final concentration was 10 pmol / ml, after overnight hybridization at 35 ° C., the filter was washed with a solution (1% Sodium 1x SSC with Dodecyl Sulfate [0.1
5 M NaCl and 0.015 M sodium citrate]) for a total of 2 times for 30 minutes at the optimum washing temperature of each probe determined by the following method.

5) Measurement of probe release curve by Non-RI method and determination of optimum washing temperature The optimum washing temperature is Zheng et al. (DAE Zheng et al.
l .: 1996, supra), and determined by the Non-RI method.
That is, each standard strain (Cycloclasticus pugetii A
TCC51542 and Alcanivorax borkumensis DSM11573) nucleic acid extraction samples were immobilized on the membrane filter by Dod blot (adding about 2 μg RNA per dot),
Each DNA probe was hybridized by the method described above, and hybridized at 35 ° C. overnight.
Then, the membrane filter was cut into 8 dots, and the fragment was transferred to a 5 ml plastic tube containing 2 ml of the washing solution. Next, wash temperature from 35 ℃ to 80 ℃ 3 ℃
Each time, the temperature was raised, and each time the temperature was raised, a new washing solution was exchanged for a washing treatment. The amount of the fluorescent signal of the probe released in each of the obtained washing solutions was measured with a fluorescence spectrophotometer RF-5300PC (manufactured by Shimadzu Corporation) (measurement conditions were excitation wavelength 640 nm, fluorescence wavelength 662). nm and band width 5 nm). Finally, the release curve of each probe with respect to the washing temperature was obtained from the amount of fluorescent signal in the washing solution at each temperature. The optimum washing temperature determined as described above using the release curve was 4 for the detection probe of Alcanivorax borkumensis.
7 ° C, 41 with probe for detection of Cycloclasticus pugetii
C was determined. The release curve obtained for the probe for detecting Alcanivorax borkumensis (Albo222) is shown in Fig. 1, and the probe for detecting Cycloclasticus pugetii (Cypug829).
The release curve obtained for the above is shown as FIG.

6) Visualization and Quantitative Analysis of Fluorescent Signal The sample subjected to the hybridization with the Cy5 labeled DNA probe and the washing treatment described above was fixed to the filter with a fluorescent image analyzer STORM (Amersham Pharmacia Biotech). The fluorescence signal of the DNA probe hybridized to the denatured nucleic acid is excited by the red laser diode (output 5 mW, wavelength 635 nm),
Detection was performed using a corresponding fluorescence filter (650 nm long pass filter), and the fluorescence signal was visualized as a dot image image. In addition, IPLab (Scanaly
tics) was used to analyze the fluorescence image to obtain a quantitative value. That is, subtract the background signal from the image image of the signal of the sample to be measured, and use the calibration curve obtained from the image image of the serially diluted sample of the 16S rRNA standard sample of E. coli that had been blotted on the same membrane. Then, the true quantitative value of the fluorescence signal derived from the sample was obtained.

7) Analysis of dominance of specific microorganisms The dominance of target microorganisms with respect to all organisms and bacterial domains was calculated by the above-mentioned formula 1 proposed by the RI method. (Correction of measured value) Here, the correction value used for calculating the dominance of various domains was obtained using the following standard strains.

Bacteria domain Escherichia coli DSM30083 Psychrobacter pacificensis IFO16279 Cycloclasticus pugetii ATCC51542 Alcanivorax borkumensis DSM11 583 cerevisiae IFO10217 For extraction of nucleic acids from the standard strains used for these corrections, RN
Using an easy Maxi kit (manufactured by Qiagen), 200 μl of each was divided into small aliquots and stored at -80 ° C.

Next, the total RNA quantification,
Hybridization, washing treatment, acquisition of dot image images, quantitative analysis of images, etc. were performed. As a probe for hybridization at the domain level, Euba338 (bacterial domain) and Euka 50 described in Amman et al. (RI Amann et al .: 1995, supra) are used.
2 (Eucariah domain) and Arch 915 (Archia domain) were used. In addition, as a DNA probe for the common region of all organisms, Univ 1390 (DAE Zheng et al .: 1996,
The above) was used.

Cycloclasticus pugetii and Alcani, which are target functional microorganisms in the bacterial domain
For vorax borkumensis, each of the species-specific DNA probes Cypug 829 and Albo 222 described above were used. The measurement results are shown in Table 1. In Table 1, sample 1 is a sample taken in the fishing port of Yasshima, Mikuni Town (Stn.A), and sample 2 is a sample taken in the reef area (Stn.B) near the bottom of the bow on the northern coast of Yasshima, Mikuni Town. .

[0080]

[Table 1]

As shown in Table 1, in Sample 1, the bacterial domain was about 45.2% (61.4% when the whole was 100%), the Eucalyptus domain was about 19.9%, and the Archea domain. Was found to be about 8.5% (total 73.6%). In Sample 2, the bacterial domain was about 53.3% (59% based on 100% total), the Eucalyptus domain was about 24.0%, and the Archea domain was about 1%.
3.3% (total 90.6%) was found.

In Table 1, the numerical value in parentheses of the bacterial domain indicates the ratio of Cypug829 and Albo222 to Euba338 which represents the dominance of the entire domain. That is,
Cycloclasticus, an aromatic hydrocarbon-degrading bacterium that is a target microorganism belonging to this domain in the bacterial domain of Sample 1
23.6% of pugetii and 4.4% of Alcanivorax borkumensis, another target microorganism that decomposes aliphatic hydrocarbons.
Indicates the degree of dominance. Similarly, in the bacterial domain of sample 2, Cycloclasticus pugetii, which is a target microorganism belonging to this domain, is 24.5%, and another target microorganism, an aliphatic hydrocarbon-degrading bacterium, Alcanivo.
rax borkumensis shows a dominance of 6.5%.

As described above, the nucleic acid extraction sample of the microorganism in the contaminated environment as shown in the present method is used to quantify the predominance of the specific functional microorganism by the Non-RI method to determine the actual contaminated environment as a molecule. It was revealed that it is possible to analyze, evaluate, and diagnose genetically.

[0084]

Industrial Applicability According to the present invention, a microorganism having a specific function (for example, a petroleum-degrading bacterium, a useful substance-producing microorganism, a pathogenic microorganism, a sulfate-reducing bacterium, etc.) that appears in a natural environment or an artificial environment (for example, a petroleum-polluting environment) ) Of non-RI without using the conventional labor-intensive and time-consuming culture counting method.
And (method not using RI) were provided for quantitatively analyzing the molecular genetics. Such an analysis of the abundance cannot be carried out by the PCR method at present, and the abundance analysis by the Non-RI method according to the present invention is extremely effective for the analysis / evaluation of the microbial community contained in the environment. Is a technique.

The nucleotide sequence of the nucleic acid probe specific to the petroleum-degrading bacterium Alcanivorax borkumensis newly developed by the method of the present invention can be applied to natural petroleum-degrading microorganisms having the ability to dominate widely in oil pollution environments in coastal areas. It comes from. Therefore, the base sequence of this probe is under petroleum processing conditions such as bio-restoration process of the petroleum-degrading microorganisms in the natural world and oil spill environment, as well as under outdoor bio-environment restoration technology demonstration tests and laboratory experimental conditions. It is extremely useful for elucidating behavior and as a diagnostic tool for oil spill environment and as a diagnostic tool for evaluating the repair effect of oil spill environment by biomedation.

Further, according to the present invention, typical aliphatic hydrocarbon decomposing bacteria known to be widely distributed in coastal areas and
/ Or Dominance analysis of typical aromatic hydrocarbon degrading bacteria was realized by molecular genetic techniques. Therefore, the present invention is useful for biologically monitoring the residual state and decomposition process of petroleum components in petroleum-polluted waters, and spraying these nutrients with nutrients, etc. It is effective for the development of environmental remediation technology (bioremediation technology) that promotes the microbial decomposition of petroleum on site, and is extremely convenient. Furthermore, the present invention is extremely useful for detecting, quantifying, and screening for specific functional microorganisms such as harmful microorganisms such as useful substance-producing microorganisms and pathogenic microorganisms, and also for analyzing and evaluating environmental samples containing such specific functional microorganisms. Be beneficial.

[0087]

[Sequence list]                           SEQUENCE LISTING    <110> National Institute of Advanced Industrial Science and Technology    <120> A method for molecular genetic analysis of polluted environments a nd environmental samples.    <130> 331-01425    <140> <141>    <150> JP 2000/341765 <151> 2000-11-09    <160> 9    <170> PatentIn Ver. 2.0    <210> 1 <211> 20 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: synthetic DNA    <400> 1 cgacgcgagc tcatccatca 20       <210> 2 <211> 18 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: synthetic DNA    <400> 2 ggaaacccgc ccaacagt 18    <210> 3 <211> 20 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: synthetic DNA    <400> 3 tgcaccacta agcggaaacc 20    <210> 4 <211> 31 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: synthetic DNA    <400> 4 tgcaccacta agcggaaacc cgcccaacag t 31    <210> 5 <211> 1494 <212> 16S rDNA <213> GR211-P1 (FERM BP-7788)    <400> 5 ttatcatggc tcagattgaa cgctggcggc aggcctaaca catgcaagtc gagcggaaac 60 gatcctagct tgctaggagg cgtcgagcgg cggacgggtg agtaacacgt gagaatctgc 120 ccattagagg gggataacct ggggaaaccc aggctaatac cgcataatcc ctacggggga 180 aagcagggga tcttcggacc ttgtgctgat ggatgagctc gcgtcggatt agcttgttgg 240 tgaggtaatg gctcaccaag gcgacgatcc gtagctggtc ttagaggatg atcagccaca 300 ccgggactga gacacggccc ggactcctac gggaggcagc agtggggaat cttggacaat 360 gggggcaacc ctgatccagc catgccgcgt gtgtgaagaa ggccttcggg ttgtaaagca 420 ctttcagtag ggaggaaggc ttatccttaa tacggatgag tacttgacgt tacctacaga 480 agaagcaccg gctaatttcg tgccagcagc cgcggtaata cgaaaggtgc gagcgttaat 540 cggaattact gggcgtaaag cgcgcgtagg cggtttgcta agtcagatgt gaaagccccg 600 ggctcaacct gggaactgca tttgaaactg gcaggctaga atgcagtaga gggaggtgga 660 atttccggtg tagcggtgaa atgcgtagag atcggaagga acaccagtgg cgaaggcggc 720 ctcctggact gacattgacg ctgaggtgcg aaagcgtggg gagcaaacag gattagatac 780 cctggtagtc cacgccgtaa acgatgtcta ctagtcgttg ggaatcttag tattcttggt 840 gacgaagtta acgcgataag tagaccgcct ggggagtacg gccgcaaggt taaaactcaa 900 atgaattgac gggggcccgc acaagcggtg gagcatgtgg tttaattcga tgcaacgcga 960 agaaccttac caggccttga catccttgga actttctaga gatagattgg tgccttcggg 1020 agccaagtga caggtgctgc atggctgtcg tcagctcgtg tcgtgagatg ttgggttaag 1080 tcccgtaacg agcgcaaccc ttgtccctag ttgccagcac gtaatggtgg gaactctagg 1140 gagactgccg gtgacaaacc ggaggaaggt ggggacgacg tcaagtcatc atggccctta 1200 cggcctgggc tacacacgtg ctacaatggg cagtacagag ggcagcaaag tcgcgaggcc 1260 aagcaaatcc cttaaaactg ttcgtagtcc ggattggagt ctgcaactcg actccatgaa 1320 gtcggaatcg ctagtaatcg cggatcagaa tgccgcggtg aatacgttcc cgggccttgt 1380 a cacaccgcc cgtcacacca tgggagtgga ttgcaccaga agtggatagt ctaaccttcg 1440 ggaggacgtt caccacggtg tggttcatga ctggggtgaa gtcgtaacaa ggta 1494    <210> 6 <211> 1532 <212> DNA <213> Cycloclasticus pugetii    <220> <221> rRNA <222> (1) .. (1532)    <400> 6 agagtttgat catggctcag attgaacgct ggcggcatgc ctaacacatg caagtcgaac 60 gga aacagaa tgcagcttgc tagcaggcgg tcgagtggcg gacgggtgag ttatgcatag 120 gaatc cgccc gatagtgggg gacaacctcc tgaaaacgct gctaataccg cataatcccg 180 cgggggc aaa gacggggacc ttcgggcctt gctctaatgg atgagcctac aggggattag 240 gtagttggt g aggtaacggc tcaccaaggc aacgatccct agctggtttg agaggatgat 300 cagccacact gggactgaga cacggcccag actcctacgg gaggcagcag tggggaatat 360 tgcacaatgg ag gaaactct gatgcagcaa tgccgcgtgt gtgaagaagg ccttagggtt 420 gtaaagcact ttca gtaggg aggaaaagtt taagggtaat aacccttagg ccctgacgtt 480 acctacagaa gaagca ccgg ctaactccgt gccagcagcc gcggtaatac ggagggtgca 540 agcgttaatc ggaattac tg ggcgtaaagc gcgcgcaggc ggttaaacaa gtcagatgtg 600 aaagccccgg gctcaacctg  ggaactgcat ttgaaactgg ctagctagag tgtggtagag 660 gagagtggaa tttcaggtgt a gcggtgaaa tgcgtagata tctgaaggaa caccagtggc 720 gaaggcggct ctctggacca aca ctgacgc tgaggtgcga aagcgtgggt agcaaacggg 780 attagatacc ccggtagtcc acgcc gtaaa cgatgtcaac taactgttgg gcgggtttcc 840 gcttagtggt gcastaacgc aataagt tga ccgcctgggg agtacggccg caaggctaaa 900 actcaaatga attgacgggg gcccgcaca a gcggtggagc atgtggttta attcgatgca 960 acgcgaagaa ccttacctac ccttgacata cagagaactt tctagagata gattggtgcc 1020 ttcgggaact ctgatacagg tgctgcatgg c tgtcgtcag ctcgtgtcgt gagatgttgg 1080 gttaagtccc gtaacgagcg caacccttat cc ttagttgc taccatttag ttgggcactc 1140 taaggagact gccggtgata aaccggagga agg tggggac gacgtcaagt catcatggcc 1200 cttatgggta gggctacaca cgtgctacaa tggc cggtac agagggccgc aaactcgcga 1260 gagtaagcta atcccttaaa gccggtccta gtccg gattg cagtctgcaa ctcgactgca 1320 tgaagctgga atcgctagta atcgcggatc agaatg ccgc ggtgaattcg ttcccgggcc 1380 ttgtacacac cgcccgtcac accatgggag tgggttg caa aagaagtggg taggctaacc 1440 ttcgggaggc cgctcaccac tttgtgattc atgactgg gg tgaagtcgta acaaggtagc 1500 cctaggggaa cctggggctg gatcacctcc tt 1532    <210> 7 <211> 20 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: synthetic DNA    <400> 7 gctgcgctcg agtaggtagt 20    <210> 8 <211> 20 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: synthetic DNA    <400> 8 cctttgggcg ggttgtca 18    <210> 9 <211> 20 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: synthetic DNA    <400> 9 acgtggtgat tcgcctttgg 20

[Brief description of drawings]

Figure 1: Alcanivorax borkumensis detection probe (Alb
The release curve obtained for o222) is shown.

[Figure 2] Cycloclasticus pugetii detection probe (Cypu
g829) shows the release curve obtained.

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Keiko Kitamura             1-1-1 Higashi 1-1-1 Tsukuba City, Ibaraki Prefecture             National Institute of Advanced Industrial Science and Technology Tsukuba Center (72) Inventor Tomonari Sunamura             3-8-11-4C, Abiko City, Chiba Prefecture (72) Inventor Ryuichiro Kurane             1-1-1 Higashi 1-1-1 Tsukuba City, Ibaraki Prefecture             National Institute of Advanced Industrial Science and Technology Tsukuba Center F-term (reference) 4B024 AA11 CA09 HA12                 4B063 QA18 QQ06 QQ42 QR55 QS34                       QX02

Claims (18)

[Claims] 1. A nucleic acid of a specific functional microorganism in a sample obtained from a natural environment or an artificial environment is detected and quantified by the Non-RI method (method not using a radioactive isotope), and the specific function in the environment is detected. A method for analyzing the dominance of microorganisms,
The following steps: 1) A step of extracting, purifying, and storing a target nucleic acid from a microorganism-containing sample collected from a natural environment or an artificial environment, 2) From the target nucleic acid nucleotide sequence information of a specific functional microorganism A nucleic acid sequence specific to the specific functional microorganism is selected, an oligonucleotide having the sequence is synthesized, and the oligonucleotide is labeled with a Non-RI label to give a non-RI specific to the specific functional microorganism.
Step of producing labeled nucleic acid probe, 3) Immobilize the target nucleic acid extracted and purified in 1) on a hybridization substrate, and contact this with a Non-RI labeled nucleic acid probe specific to a specific functional microorganism. After performing a hybridization treatment with the above, a step of performing a washing treatment, 4) a step of visualizing and analyzing a signal derived from the hybridized Non-RI labeled nucleic acid probe and obtaining a quantitative value thereof, 5) a determined quantitative value And a method of calculating the predominance of a microorganism having a specific function by comparing with a quantitative value measured for all organisms and each domain of the organism kingdom. 2. The washing treatment of 3) is performed at an optimum washing temperature obtained from a release curve of a probe prepared using a Non-RI labeled nucleic acid probe specific to a specific functional microorganism. The method of claim 1. 3. The method according to claim 1, wherein the Non-RI labeled nucleic acid probe is fluorescently labeled or chemiluminescently labeled. 4. The method according to claim 1, wherein the specific functional microorganism is a microorganism that decomposes a specific chemical substance. 5. The method according to claim 4, wherein the specific chemical substance is a harmful chemical substance. 6. The method according to claim 4, characterized in that the specific chemicals are petroleum and petroleum components. 7. The method according to claim 1, wherein the specific functional microorganism is a specific useful substance-producing microorganism. 8. The method according to claim 1, wherein the specific functional microorganism is a specific harmful microorganism including a pathogenic microorganism. 9. The method according to any one of claims 1 to 8 is used to analyze the predominance of a specific functional microorganism in a natural environment or an artificial environment to determine the microbial community function of that environment. How to analyze and evaluate. 10. A method for analyzing and evaluating a polluted environment using the method according to claim 1. 11. A method for analyzing / evaluating an environment contaminated with a harmful chemical substance by using the method according to claim 4. 12. A method for analyzing and evaluating an oil spill environment using the method according to claim 6. 13. Alcani having a part of the nucleotide sequence of SEQ ID NO: 5 or a corresponding riboxynucleotide sequence,
RNA or DN having a base length of 10-50 bp, which can specifically hybridize with a nucleic acid derived from a petroleum-degrading bacterium of the vorax family or genus
A probe. 14. A nucleotide sequence according to SEQ ID NO: 1 or a corresponding riboxynucleotide sequence capable of specifically hybridizing with a nucleic acid derived from a petroleum-degrading bacterium of the Alcanivorax family or genus and detecting or quantifying it. Have, DN
A or RNA probe. 15. The DNA or RNA probe according to claim 13 or 14, wherein the petroleum-degrading bacterium of the Alcanivorax family or genus is Alcanivorax borkumensis or a related species thereof. 16. The DNA or RNA probe according to claim 13, which can specifically hybridize with a nucleic acid derived from a petroleum-degrading bacterium of the Alcanivorax family or genus as a Non-RI labeled nucleic acid probe specific to a specific functional microorganism. Alternatively, a DNA or RNA probe having the nucleotide sequence shown in SEQ ID NO: 1 or a riboxynucleotide sequence corresponding thereto and / or a nucleic acid derived from a petroleum-degrading bacterium of the genus Cycloclasticus can be specifically hybridized, Having an RNA or DNA probe with a base length of 10-50 bp, which has a part of the base sequence or the corresponding riboxynucleotide sequence, or has the base sequence described in SEQ ID NOs: 2 to 4 or the corresponding ribonucleotide sequence DNA
Alternatively, a nucleic acid probe selected from RNA probes is used, and by the method according to claim 1,
Alcanivorax family or genus and / or Cycloclast
A method to analyze and evaluate the oil spill environment by analyzing the dominance of petroleum degrading bacteria of the genus icus. 17. An aliphatic hydrocarbon degrading bacterium, Alcanivorax borkumensis or a closely related species thereof, and / or an aromatic hydrocarbon degrading bacterium, Cycloclasticus puge concentrated in environmental microbial groups.
The method according to claim 16, characterized by analyzing the dominance of tii or its related species. 18. The DNA or RNA probe according to claim 13, which is capable of specifically hybridizing with a nucleic acid derived from a petroleum-degrading bacterium of the genus Cycloclasticus, or a part of the nucleotide sequence of SEQ ID NO: 6 or a ribonucleotide sequence corresponding thereto. A DNA or RNA probe having a base length of 10-50 bp, and /
Alternatively, Non-RI obtained by labeling Non-RI with one or more nucleic acid probes selected from DNA or RNA probes having the nucleotide sequences shown in SEQ ID NOs: 1 to 4 or ribonucleotide sequences corresponding thereto A kit for molecular genetic analysis and evaluation of contaminated environments and environmental samples, which contains a labeled probe.