JPH09121868A - Oligonucleotide for detecting methane-assimilating bacterium having decomposing ability for organic chlorine compound and detection using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an oligonucleotide capable of rapidly and specifically detecting and counting a methane-assimilating bacterium capable of decomposing organic chlorine compounds such as trichloroethylene. SOLUTION: This oligonucleotide is capable of specifically hybridizing at least a part of a sequence site having the homology of a soluble methane monooxygenase gene (mmoX gene) respectively possessed by three strains of a Methylocystis sp. M strain, a Methylosinus trichosporium OB3b strain and a Methylococcus capsulatus Bath strain, e.g. at least a part of a gene sequence of formulas I and II. The oligonucleotide of polymerase chain reactional(PCR) grade is obtained by chemical synthesis.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for counting and monitoring methane-utilizing bacteria degrading organochlorine compounds such as trichlorethylene, and an oligonucleotide used for this method.

[0002]

2. Description of the Related Art Organochlorine compounds such as trichlorethylene are frequently used as cleaning agents in metal factories and the like, and in recent years, pollution of groundwater and soil has become a serious social problem. Several methods using microorganisms have been reported as effective and efficient methods for removing and detoxifying organochlorine compounds, and among them, methane represented by methylocinus bacteria described in JP-A-2-92274. It is known that assimilating bacteria have a superior ability to decompose organochlorine compounds as compared with other microorganisms. Utilizing this ability of methane-utilizing bacteria, in-situ bioremediation technology for purifying contaminated soil and groundwater by multiplying indigenous methane-utilizing bacteria in soil and groundwater is currently being put into practical use. It is expected to be a new technology that can clean up a wide range of pollution because it does not cost the cost of excavation and extraction. In this technology, it is important to properly grasp the growth state of methane-utilizing bacteria in soil and groundwater and set the optimum operating conditions accordingly. Conventionally, in-situ bioremediation technology is used to measure the number of methane-utilizing bacteria in on-site soil and groundwater during purification operation.
MPN (Most assimilation), which measures the number of methanotrophs depending on the presence or absence of growth, by collecting on-site groundwater, diluting it in several stages, and feeding the diluted solution with nutrient salts, oxygen, and methane gas to carry out culture. The Probable Number method is commonly used.

[0003]

However, this method requires 3 to 4 weeks for the growth of the bacteria, and in order to control the purification operation conditions according to the growth state of the bacteria, it is necessary to obtain a measured value. There was a problem that it took too long. Also,
Methane-utilizing bacteria are classified into type I, type II, and type X depending on the cell membrane structure and metabolic pathway classification. Of these, soluble methane has the ability to decompose organochlorine compounds such as trichlorethylene. Monooxygenase gene (mm
The type II methane-assimilating bacterium that cannot decompose organochlorine compounds is also measured because all the bacteria that grow with methane are measured by the MPN method. There was a problem.

As a means for solving the above problems,
Currently, measurement methods using fluorescent antibodies and gene detection methods are being studied worldwide. However, since the fluorescent antibody method is an antigen-antibody reaction on the surface of bacterial cells, there is no direct correlation with the presence or absence of the soluble methane monooxygenase gene, and in both cases of fluorescent antibody and gene detection method, a single strain or A specific detection method for the genus to which the bacterium belongs, that is, a method for specifically detecting all the bacteria having a soluble methane monooxygenase gene, has not been obtained. In addition, the number of bacteria present in the environmental water is usually 10 ³ to 10 ⁸ cells / ml in total, and the nucleic acid concentration is too low when directly extracted, resulting in low yield during extraction and purification, However, there was a problem in that the quantification and reliability were not sufficient when detecting the methanotrophic bacteria. That is, an object of the present invention is to provide a new method for rapidly and specifically detecting and counting methane-assimilating bacteria that decompose organic chlorine compounds.

[0005]

[Means for Solving the Problems] The inventors of the present invention have made earnest studies on a method for detecting and enumerating a group of methane-assimilating bacteria capable of solving the above problems and decomposing organochlorine compounds with high sensitivity. As a result, the present invention was devised. That is, the present invention searches the gene sequence of the soluble methane monooxygenase gene, which is a gene specific to a group of methane-assimilating bacteria having an organochlorine compound-degrading activity, and finds an oligonucleotide sequence with high conservation between genera. This makes it possible to detect low-concentration bacteria by functioning as a primer for chain length reaction, selectively amplifying the target nucleotide sequence site, and collecting and concentrating the sample before the extraction of nucleic acid from the sample. In addition, the methane-assimilating bacterium concentration in the original sample is quantified by gradually reducing the amount of the nucleic acid extract added to the chain length reaction.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION Oligonucleotides used as primers are Methylocystis sp.
) M strain, methylosis sinasis trichosporium (Methy
losinus trichosporium) OB3b strain and Methylococcus capsulatus Bat
It is an oligonucleotide that specifically hybridizes (anneals) to at least a part of the sequence site having homology with the soluble methane monooxygenase gene (mmoX gene) of each of the 3 h strains.
It is generally said that 70% or more homology between the two sequences is required for the efficient binding of the primer and the target nucleotide sequence. In the present invention, a nucleotide sequence site having a homology of 80% or more among the genes of the three strains was searched for as a target. As an example, the oligonucleotide is at least part of the following gene sequence: (5 ')-CGCTGTGGAAGGGCATGAAGCG- (3') ... (SEQ ID NO: 1) (5 ')-GCTCGACCTTGAACTTGGAGCC- (3') ... (SEQ ID NO: 2)

Generally, a sequence of 10 bases or more is used. This nucleotide sequence has 100% homology between the genes of the three strains. At present, the gene sequence of the mmoX gene has been determined only in the above-mentioned 3 strains, and the 3 strains belong to different genera and are of type II and X.
Since almost all types of methane-assimilating bacteria are covered, the above-mentioned oligonucleotide sequences conserved in these 3 strains are not isolated even in methane-assimilating bacteria having an organochlorine compound degrading activity. As a result of the investigation, it was possible to detect methane-assimilating bacteria having an activity of decomposing organic chlorine compounds in groundwater in various places.

Here, gene amplification is performed based on the Polymerase Chain Reaction (hereinafter referred to as PCR method) developed by Saiki et al. In the case of detecting a specific gene region (mmoX gene in the present invention), this method targets a nucleotide sequence of a part of the region and recognizes the + strand at one end and the − strand at the other end. To prepare two oligonucleotides which hybridize with each other. The sample nucleic acid is separated into single strands by thermal denaturation, the oligonucleotide is made to function as a primer for the template-dependent nucleotide polymerization reaction, the generated double-stranded nucleotides are separated again into single strands, and the same reaction is performed again. Wake up. By repeating this series of operations, the number of copies in the region between the two primers increases to a detectable amount.

As the sample, ground water, surface water or the like is used. In particular, groundwater contains almost no suspensions of bacteria or less, so the filtration and collection described below is easy. The total number of bacteria contained in environmental water such as groundwater is 10 ³ ~
Since it is 10 ⁸ cells / ml and the nucleic acid concentration is too low when extracted as it is, 100 ml to 1000 ml is previously prepared by using a micromembrane filter having a pore size of 0.45 μm or less.
The sample water is filtered to collect methane-utilizing bacteria on the filter. By cutting this filter with a razor blade and filling it in a microtube and performing the nucleic acid extraction operation, the total amount of the nucleic acid extract can be suppressed to 1 ml. By this operation, the concentration of the target nucleic acid extract can be increased to 100 to 1000 times that in the case where no filtration is performed. By increasing the concentration in this way, it is possible to reduce the proportion of nucleic acids that cannot be recovered due to adsorption to microtubes or aggregation / precipitation during precipitation purification operation to a negligible level. It is possible to reduce the error in the measured value due to the operation. Further, by increasing the usage amount of the sample water by 100 to 1000 times, it is possible to reduce the error due to the bacterial concentration distribution of the sample water itself.

The presence or absence of the target nucleotide amplified by the PCR reaction is examined by subjecting the reaction solution after the PCR reaction to agarose gel electrophoresis as it is, and then staining the agarose gel with an ethidium bromide solution to observe luminescence under ultraviolet light. By doing so, it is determined whether or not the nucleotide of the desired length is present. The minimum concentration of methane-utilizing bacteria in groundwater detected by this method is 10 ⁴ to 10 ⁵ cells / ml in the usual method, and in the case of 100-fold concentration by using the above-mentioned concentration method, 10 ²
The detection limit improves up to -10 ³ cells / ml.

This detection limit depends on extraction conditions, purification conditions, PC
Although it changes about 1000 times depending on the R reaction condition and the detection condition, an almost constant detection limit can be obtained by setting a constant condition. Utilizing this property, the amount of nucleic acid extract added to the PCR reaction system is gradually reduced, and the approximate concentration of methanotrophic bacteria in the original sample is determined by the amount up to which the PCR product was detected. You can ask. In this case, the Methylocystis sp. M strain, the Methylosinus trichosporium OB3b strain and the Methylococcus capsartus (Methylococcus) strains whose bacterial concentration is known as an external standard are used.
It is desirable to conduct a similar dilution detection test in parallel using a suspension of any of the three strains of Capsulatus) Bath and to quantify the bacterial concentration by comparison with the results. Further, as a more quantitative method, PCR is carried out by using 3 or 5 PCR solutions at each dilution step using equal amounts of a 10-fold diluted nucleic acid extract solution.
By comparing the CR product detection probability with the MPN table,
It is also possible to determine the bacterial concentration in the sample.

The above-mentioned counting method does not give an absolute value like the observation of the total number of bacteria by a microscope, since the measured value depends on the fluctuation of the detection efficiency. However, in-situ bioremediation technology is considered to be sufficiently effective as a means for understanding the fluctuation state of the number of methane-utilizing bacteria in groundwater. In addition, in-situ bioremediation technology is used to collect on-site soil during purification operation and to collect methane-utilizing bacteria in groundwater instead of groundwater, in order to collect soil at the target depth. It is not realistic because excavation work is required for each sampling.

[0013]

The present invention will be described in detail based on the following examples, but the present invention is not limited to the following examples. Example 1 The following test was conducted in order to evaluate the effect of reducing the measurement error caused by microfilter filtration. Preparation of Specimen Methylocystis sp. M strain is NMS
A medium (ATCC media 1306) was used to dilute to 10 ⁴ cells / ml, and this was used as a sample. Preparation of nucleic acid extract was performed under two extraction conditions. In one system, sample 100
ml is filtered through a micropore membrane filter with a pore size of 0.45 μm, the filter is cut and filled in a microtube, 500 μl of ultrapure water is added and ultrasonically crushed, and proteinase K solution is added as it is and in a hot water bath. 60 ℃,
After reacting for 60 minutes, it was heated at 100 ° C. for 10 minutes. The supernatant of this solution was collected to obtain a nucleic acid extract. In the other system, no filter filtration was performed, and 1 ml of the sample was subjected to the same ultrasonic disruption, enzyme addition, and heating, and the supernatant was used as a nucleic acid extract. The total amount of the nucleic acid extract was adjusted to 1 ml in any system. In addition, an extraction liquid was prepared for each three lines.

Primer Methylocystis sp. Strain M, Methylosinus trichosporium
porium) OB3b strain and Methylococcus capsulatus Bath strain 3 isolates were examined for homology of the soluble methane monooxygenase gene (mmoX) sequence, and the oligonucleotides having the sequences shown below were used as primers. (5 ′)-CGCTGTGGAAGGGCATGAAGCG- (3 ′) ………… (1) (5 ′)-GCTCGACCTTGAACTTGGAGCC- (3 ′) ………… (2) In addition, the same applies even if each phase trapping sequence of the above sequence is used. Are getting the results. The chemical synthesis of the primer was commissioned to a private contracting synthesis institution, and a PCR grade product was purchased.

PCR reaction 0.5 μl of the nucleic acid extract of the system collected by filtration and 50 μl of the nucleic acid extract of the system directly extracted were used so that the amount of nucleic acid added during the PCR reaction was theoretically equal. . 45.5 μl of ultrapure water was added to the PCR reaction system of the system collected by filtration, and then 10 × reaction buffer 10 μm was added to both systems in the same manner.
l, dNTP mixed solution 10 μl, primer (1) 13 μl
1, 13 μl of primer (2), and 0.5 μl of thermostable DNA polymerase were added, and mineral oil was overlaid to prepare a reaction solution. Using three extracts of each series, three PCR reaction solutions were prepared. The reaction conditions are as follows. Thermal denaturation: 94 ° C., 1 minute Annealing: 50 ° C., 1 minute Polymerization reaction: 72 ° C., 2 minutes The process from thermal denaturation to annealing to the polymerization reaction is 1
The cycle was repeated 30 times.

Detection In order to detect the amplified nucleotide fragment from the reaction solution, agarose gel electrophoresis was performed as follows.
The agarose gel had a gel concentration of 1% (w / v). The electrophoresis conditions were a constant voltage of 100 V and the amount of the PCR reaction solution injected at 30 minutes into the gel was 5 μl per sample. After the electrophoresis was completed, the gel was immersed in a 1% ethidium bromide solution for staining, and the red light emission upon irradiation with 250 nm ultraviolet light was photographed to detect the nucleotide fragment.
In addition to the reaction solution, molecular weight markers are also electrophoresed at the same time,
The length of the nucleotide fragment was calculated by comparing the relative mobilities.

Results FIG. 1 shows PCR of filtered and direct extracted samples.
The electrophoresis result of the reaction solution is shown. In the figure, M is a molecular weight marker, 1, 2, and 3 are filtered and extracted samples, and 4, 5, and 6 are directly extracted samples. Although the theoretical amount of the added nucleic acid extract is equal, clear electrophoretic bands are observed in all of 1, 2, and 3, whereas electrophoretic bands are weak in 4, 5, and 6, and particularly in 6, Almost never detected. From this figure, it was shown that the work error at the time of extraction of low-concentration methane-utilizing bacteria can be reduced by collecting the sample by filtration. Also, in the subsequent dilution test (Example 3), reproducibility was confirmed in the detection limit of the methane-utilizing bacteria collected by filtration.

Example 2 In order to confirm that the primer used in Example 1 is common to general methane-utilizing bacteria having organochlorine compound degrading activity and selective to other bacteria,
The following tests were carried out using various standard strains and unidentified strains isolated from groundwater and soil at organochlorine compound contaminated sites in various parts of Japan. Preparation of Specimens Each strain shown in Table 1 was treated with NMS medium (A
It was diluted to 10 ⁴ cells / ml using TCC media 1306) and used as a sample. The strains of M-6.5 or less in the table are unidentified methane-assimilating bacteria isolated by the inventors from organochlorine compound contaminated sites in various parts of Japan, and C-hex at the bottom.
The only one is a heterotrophic bacterial enrichment culture that was cultivated according to the heterotrophic bacterial culture method (water supply test method ¹⁹⁹³ ) using groundwater as the seed source.

Test Method In the same manner as in Example 1, 100 ml of the sample was filtered to extract nucleic acid, and 0.5 μl of the supernatant was used as a nucleic acid extract for PCR reaction. The detection method is also Example 1
The same as above. Results are shown in Table-1. The detected strain was previously investigated using headspace gas chromatography (JIS-K0125). It was completely the same as the methane-utilizing strain with positive activity for degrading organic chlorine compounds (trichloroethylene) under aerobic conditions. Therefore, it can be said that the oligonucleotide of the present invention, that is, the primer, selectively reacts only with methane-assimilating bacteria having an organochlorine compound degrading activity. Similar results were also obtained when each phase-trapping chain sequence of the used primer sequence was used as a primer.

[0020]

[Table 1]

Example 3 The results of the measurement of the number of methane-utilizing bacteria in the on-site groundwater during the purification operation by the in-situ bioremediation technique are shown below. Preparation of samples 10-20 underground contaminated with trichlorethylene
An in-situ bioremediation test was carried out to inject indigenous methane-utilizing bacteria by injecting water in which methane, oxygen and nutrients were dissolved into the aquifer of m. Groundwater in the aquifer at this time was collected daily and used as it was. In addition, Methylocys
tis sp.) M strain (hereinafter also referred to as M strain) was diluted to a concentration of 10 ⁴ cells / ml using on-site groundwater that had been sterilized by filtration, and this was used as a standard sample.

Preparation of nucleic acid extract As in Example 1, 100 ml of a sample was filtered through a micropore membrane filter having a pore size of 0.45 μm, the filter was cut and filled in a microtube, and then ultrapure water was 500 μm.
1 and sonicate, add proteinase K solution as it is, and react in a water bath at 60 ° C. for 60 minutes, then 100
Heated at 10 ° C for 10 minutes. The supernatant of this solution was collected to obtain a nucleic acid extract. PCR reaction The same primers as in Example 1 were used. Nucleic acid extract is 1-fold according to the expected concentration of methane-utilizing bacteria
1000 times, 10 times to 10000 times or 100 times
Diluted in 4 steps every 10 times up to 100,000 times, and 50 μl of each of the diluted solutions was used as a sample for PCR reaction. The PCR reaction and detection conditions were the same as in Example 1.

Results FIG. 2 shows an example of the results of electrophoresis. In the figure, M is a molecular weight marker, 1 is a 1-fold dilution of a standard M strain sample (10 ⁴ cells / ml), 2 and 3 and 4 are the same 10, 100 and 1000-fold dilutions, and 5 is a 10-fold dilution of the on-site groundwater sample. Diluent, 6, 7,
Similarly, 8 is a 100, 1000, 10000-fold diluted solution. The electrophoretic band was detected up to 3, but not detected in 4. From this, the bacterial concentration in the sample was 10 ⁴ cells.
1 / ml, it is detected up to 100-fold dilution, but 1
It can be seen that it is not detected at 000 times. Also 5, 6,
The electrophoretic band was detected in 7 but not in 8. From this, it can be seen that the on-site groundwater sample can be detected up to 1000 times dilution. Therefore, the concentration of methane-utilizing bacteria having an organochlorine compound-decomposing activity in this groundwater sample is estimated to be 10 times that of the standard M strain sample (10 ⁴ cells / ml), and is estimated to be 10 ⁵ cells / ml. Although bands were observed at positions other than the target electrophoretic band in 5, 6, 7, and 8, it was confirmed by Southern hybridization that only the band at the target position was soluble methane monooxygenase. It was shown to be the nucleotide of the gene.

FIG. 3 shows daily changes in the concentration of methane-utilizing bacteria in groundwater measured by various methods. During the in-situ bioremediation operation in which methane, oxygen, and nutrients are injected, the number of methane-assimilating bacteria measured by the method of the present invention is always about 10 of the measurement results by the MPN method and the soluble methane monooxygenase ⁺ MPN method. A double measurement was shown. The soluble methane monooxygenase ⁺ MPN method is used herein to measure the trichloroethylene-degrading activity of methane-assimilating bacteria grown by the MPN method using the above headspace gas chromatography, and the growing bacterial cells confirmed to have the activity. It is a method of measuring only as +1. According to this method, cells having no organochlorine compound degrading activity among the cells grown by the MPN method can be excluded and counted. The counting result according to the present invention is one of the counting results by the soluble methane monooxygenase ⁺ MPN method.
The reason for becoming 0 times is that the method according to the present invention measures the total number of bacteria, whereas the soluble methane monooxygenase ^- M
The counting method based on the PN method is considered appropriate because it counts the number of viable bacteria. The time required for measurement is MPN method,
Both the soluble methane monooxygenase ^- MPN method required 4 weeks, while the method according to the present invention obtained the measurement result in 10 hours.

In FIG. 3, the injection of methane, oxygen and nutrients was stopped 20 days after the start of operation. Under this condition, the count value by the MPN method does not decrease so much, whereas the count result by the present invention and the count result by the soluble methane monooxygenase ⁺ MPN method decrease in parallel. This means that the MPN method also counts methane-assimilating bacteria that do not decompose organochlorine compounds, and therefore reflects the decrease in the number of methane-assimilating bacteria that can decompose organochlorine compounds in the counting results. It is thought that it is not done. That is, it can be said that the present invention has shown a method for rapidly and specifically detecting and counting methane-assimilating bacteria that decompose organic chlorine compounds.

[0026]

INDUSTRIAL APPLICABILITY As described above, according to the present invention, it is possible to rapidly and specifically detect and count methane-assimilating bacteria that decompose organic chlorine compounds. INDUSTRIAL APPLICABILITY The present invention is widely used as a monitoring technique for restoration of soils contaminated with organic chlorine compounds and groundwater.

[0027]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 22 Sequence type: Nucleic acid Number of strands: 1 Strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Characteristic symbols: primer bind Features were determined Method: E sequence CGCTGTGGAA GGGCATGAAG CG 22

SEQ ID NO: 2 Sequence length: 22 Sequence type: Nucleic acid Number of strands: 1 Strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence characteristics Characteristic symbols: primer bind Characteristics The method of determining: E sequence GCTCGACCTT GAACTTGGAG CC 22

[Brief description of the drawings]

FIG. 1 is an electrophoretic photograph showing the results of PCR reaction of the present invention using a filtered and direct extraction sample of a standard sample.

FIG. 2 is an electrophoretic photograph showing the results of performing PCR reaction of the present invention by serially diluting a standard sample and a test sample.

FIG. 3 is a graph comparing the measurement results of the number of methane-utilizing bacteria in groundwater in a contaminated site by the PCR reaction method of the present invention, the MPN method, and the soluble methane monooxygenase ⁺ MPN method.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location C12R 1:01) (C12Q 1/68 C12R 1:01) (72) Inventor Tatsuo Shimomura Fujisawa, Kanagawa Prefecture 4-2-1 Ichimoto Fujisawa Ebara Research Institute Co., Ltd.

Claims (5)

[Claims] 1. A methylocystis sp.
) M strain, methylosis sinasis trichosporium (Methy
losinus trichosporium) OB3b strain and Methylococcus capsulatus Bat
An oligonucleotide that specifically hybridizes to at least a part of a sequence site having homology with the soluble methane monooxygenase gene (mmoX gene) of each of the 3 h strains. 2. An oligonucleotide which is at least a part of the following gene sequence. (5 ')-CGCTGTGGAAGGGCATGAAGCG- (3') ... (SEQ ID NO: 1) (5 ')-GCTCGACCTTGAACTTGGAGCC- (3') ... (SEQ ID NO: 2) 3. A method for detecting a bacterium having a target nucleotide sequence by amplifying the target nucleotide sequence by PCR and using the amount of the amplified nucleotide as a guideline, wherein the target nucleotide sequence is methylocystis genus (Methylococcus).
ystis sp.) M strain, Methylosinus trichosporium OB3b strain and Methylococcus capsulat
us) A sequence site having homology to the soluble methane monooxygenase gene (mmoX gene) possessed by each of the 3 strains of Bath, wherein the oligonucleotide according to claim 1 or 2 is used as a primer for PCR. , Method for detecting methanotrophic bacteria having degradability of organic chlorine compounds. 4. A nucleic acid component-containing liquid is prepared by (a) filtering a predetermined amount of test water to collect bacterial cells on a filter, and (b) extracting the nucleic acid component of the collected bacterial cells. (C) The method for detecting a methane-assimilating bacterium capable of degrading an organochlorine compound according to claim 3, wherein a predetermined amount of the nucleic acid component-containing liquid is used as a test liquid for PCR. 5. The detection method according to claim 3, wherein: (a) a predetermined amount of a suspension of a standard strain of a methane-assimilating bacterium capable of degrading an organochlorine compound and a predetermined amount of test water are filtered. And (b) extracting the nucleic acid components of the bacterial cells collected by the respective filters to prepare nucleic acid component-containing liquids, and (c) the respective nucleic acid component-containing liquids. PCR is carried out under the same conditions by using the stepwise changed amount of the above as the test liquid for PCR, and (d) the amount of the nucleic acid component-containing liquid used is compared with the amplified nucleotide amount, respectively. Method for measuring the concentration of methane-utilizing bacteria having the ability to decompose organochlorine compounds.