JP2005211004A - Nucleic acid fragment for detecting chlorinated ethane-decomposing bacterium, detection method and method for decomposing chlorinated ethane - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new and useful nucleic acid fragment capable of being utilized for detection of chlorinated ethane-decomposing bacterium and preferentially hybridized with 16SrRNA or rDNA of chlorinated ethane-decomposing bacterium and a labeled probe for detection of chlorinated ethane-decomposing bacterium composed of the nucleic acid fragment and to provide a chlorinated ethane-decomposing bacterium-detecting method, a chlorinated ethane-decomposing bacterium-monitoring method and a chlorinated ethane-decomposing method by each using the nucleic acid fragment or labeled probe. <P>SOLUTION: A nucleic acid fragment for detection of the chlorinated ethane-decomposing bacterium, composed of 18-23 nucleotides preferentially hybridized with 16SrRNA or rDNA of the chlorinated ethane-decomposing bacterium and having a specific base sequence, a base sequence having ≥90% homology with the specific base sequence or a base sequence which is complementary to the specific base sequence and the labeled probe and provides the chlorinated ethane-decomposing bacterium-detecting method, the chlorinated ethane-monitoring method and a chlorinated ethane-decomposing method by each using the nucleic acid fragment or the labeled probe. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a nucleic acid fragment preferentially hybridizing to 16S rRNA or rDNA of chlorinated ethane-degrading bacteria, a labeled probe for detecting chlorinated ethane-degrading bacteria comprising this nucleic acid fragment, and chlorination using these nucleic acid fragments or labeled probes. The present invention relates to a method for detecting and monitoring ethane-degrading bacteria, and a method for degrading chlorinated ethane.

  It is known that chlorinated ethane-degrading bacteria having extremely high chlorinated ethane decomposing activity exist in soil or groundwater contaminated with chlorinated ethane (Non-patent Document 1). By using chlorinated ethane-degrading bacteria present in such contaminated soil, it is conceivable to dechlorinate chlorinated ethane and purify the contaminated soil. If these bacteria are not present in the contaminated soil or groundwater, it may be possible to purify by adding chlorinated ethane-degrading bacteria collected elsewhere. However, even if such chlorinated ethane-degrading bacteria are present, it is difficult to detect or collect them, and such bacteria can be grown and dechlorinated at the contaminated site to obtain a good treatment effect. There is no guarantee. For this reason, when purifying soil or groundwater contaminated with chlorinated ethane using chlorinated ethane-decomposing bacteria, a method for determining in advance whether or not dechlorination is effectively performed is desired.

Non-Patent Document 1 reports a chlorinated ethane-degrading bacterium ( Dehalobacter sp str.TCA1) having a high chlorinated ethane degrading activity, and the 16S rDNA classifies the bacterium as belonging to the dehalobacter restrictus . . However, it does not show a method for detecting this bacterium, or a nucleic acid fragment used as a primer therefor, nor a method for obtaining or growing the bacterium. Therefore, it is unclear whether soil or groundwater contaminated with chlorinated ethane can be purified using the above chlorinated ethane-degrading bacteria.

Non-patent document 2 shows that a bacterium having a degrading activity on trichlorethylene ( Dehalobacter restrictus str. TEA) degrades trichlorethylene with high activity. PCE-RDase (reductive degradation of trichlorethylene) produced by it is shown. When the substrate-degrading activity of the enzyme -Pca-A) on trichloroethylene is defined as 100, the substrate-degrading activity on trichloroethane is 1.4 ± 0.1, indicating that the degradation activity on trichloroethane is extremely low. Even this is a high decomposition activity to trichlorethylene bacteria, not be said high decomposition activity against trichloroethane, also in the bacterium belonging to the closely including Dehalobacter restrictus, and high decomposition activity to trichlorethylene bacteria, trichloroethane It is shown that there are bacteria with high degrading activity against. Since these bacteria belong to each other, they are difficult to separate, and it is difficult to detect in a short time by a simple method whether or not there is a bacterium having a high degradation activity against trichloroethane.
Science Vol298 1 NOVEMBER 2002, p. 1023-1025 Applied and Environ. Microbiology Vol 69 No.8 pp4628-4638

  An object of the present invention is a nucleic acid fragment that can be used for detection of chlorinated ethane-degrading bacteria, and a novel and useful nucleic acid fragment that preferentially hybridizes to 16S rRNA or rDNA of chlorinated ethane-degrading bacteria, The present invention proposes a labeled probe for detecting chlorinated ethane-decomposing bacteria comprising fragments, a method for detecting and monitoring chlorinated ethane-degrading bacteria using these nucleic acid fragments or labeled probes, and a method for degrading chlorinated ethane.

The present invention relates to a nucleic acid fragment that preferentially hybridizes to 16S rRNA or rDNA of the following chlorinated ethane-degrading bacteria, a labeled probe for detecting chlorinated ethane-degrading bacteria comprising this nucleic acid fragment, and chlorine using these nucleic acid fragments or labeled probes A method for detecting and monitoring chloroethane-degrading bacteria, and a method for degrading chlorinated ethane.
(1) A nucleic acid fragment consisting of 18 to 23 nucleotides that preferentially hybridizes to 16S rRNA or rDNA of a chlorinated ethane-degrading bacterium, comprising any one of SEQ ID NOS: 1 to 17 and 90% of these nucleotide sequences A nucleic acid fragment having a base sequence having the above homology or a base sequence complementary to these base sequences.
(2) a nucleic acid fragment consisting of 10 to 50 nucleotides preferentially hybridizing to 16S rRNA or rDNA of a chlorinated ethane-degrading bacterium, wherein at least 10 consecutive base sequences are any of SEQ ID NOs: 1 to 17 A nucleic acid fragment having the same base sequence as the base sequence or a base sequence complementary to these base sequences.
(3) The nucleic acid fragment according to (1) or (2) above, which is used for detection of chlorinated ethane-degrading bacteria.
(4) A labeled probe for detecting chlorinated ethane-degrading bacteria, wherein the nucleic acid fragment according to any one of (1) to (3) is labeled with a labeling substance.
(5) Chlorinated ethane for detecting a synthesized DNA fragment by performing PCR (polymerase chain reaction) using the nucleic acid fragment according to any one of (1) to (3) as a primer and the nucleic acid in the sample as a template. Method for detecting degrading bacteria.
(6) Chlorine to be detected by using the labeled probe for detecting chlorinated ethane-degrading bacteria described in (4) above and a sample or a nucleic acid prepared from the sample for RNA or DNA hybridization, and then using the label as an index Of detecting ethane-degrading bacteria.
(7) The method for detecting ethane-degrading bacteria described in the above (5) or (6) is performed using groundwater or soil as a sample, and groundwater, soil, or a culture solution inoculated with them is detected. A chlorinated ethane decomposition method that is introduced into soil or groundwater contaminated with chlorinated ethane.
(8) The method for detecting ethane-degrading bacteria described in (5) or (6) above is carried out, and the chlorinated ethane-degrading bacteria detected in soil or groundwater contaminated with chlorinated ethane or the introduced chlorinated ethane degrading A method for monitoring chlorinated ethane-degrading bacteria by quantifying the number of bacteria.

  As a result of investigating the reason why the treatment effect is not always good in the method of purifying soil or groundwater contaminated with chlorinated ethane using chlorinated ethane-degrading bacteria, chlorinated ethane decomposition which dechlorinates chlorinated ethane The treatment effect is good when bacteria are present at the treatment site, and the treatment effect cannot be expected when bacteria are not present. Therefore, it is possible to determine whether or not the treatment can be performed satisfactorily by examining the soil and groundwater at the target site and confirming whether or not chlorinated ethane-degrading bacteria are present. If chlorinated ethane-degrading bacteria are not present in the contaminated soil or groundwater as a result of the survey, chlorinated ethane-degrading bacteria collected elsewhere can be added and purified. Detection of degrading bacteria is required. By utilizing the nucleic acid fragment of the present invention, chlorinated ethane-degrading bacteria can be detected, and the above-described determination becomes possible.

  The nucleic acid fragment of the present invention is a nucleic acid fragment consisting of 18 to 23 nucleotides that preferentially hybridizes to 16S rRNA or rDNA of a chlorinated ethane-degrading bacterium, and the nucleotide sequence of any one of SEQ ID NOs: 1 to 17 in the Sequence Listing, Preferentially hybridizes to a nucleic acid fragment having a base sequence having 90% or more homology with these base sequences, a nucleic acid fragment having a base sequence complementary to these base sequences, or 16S rRNA or rDNA of chlorinated ethane-degrading bacteria. , Preferably a nucleic acid fragment consisting of 15 to 35 nucleotides, wherein at least 10 consecutive base sequences are the same base sequence as any one of SEQ ID NOs: 1 to 17 or a base complementary to these base sequences Nucleic acid fragments that are sequences (hereinafter, including the nucleic acid fragments of the respective SEQ ID NOs and variants thereof) Te is a nucleic acid that fragment etc.) of each of SEQ ID NO. Nucleic acid fragments having the same base sequence as 10 or more consecutive base sequences starting from an arbitrary position of the base sequences of SEQ ID NOs: 1 to 17 or a base sequence complementary to this base sequence are also included. A base may be bound upstream and / or downstream of the same base sequence as or a base sequence complementary to this base sequence.

  Nucleic acid fragments of the present invention, that is, the base sequences of SEQ ID NOs: 1 to 17, base sequences having 90% or more homology with these base sequences, base sequences complementary to these base sequences, and at least 10 consecutive sequences A nucleic acid fragment having the same base sequence as any one of SEQ ID NOS: 1 to 17 or a nucleic acid fragment having a base sequence complementary to these base sequences can be easily chemically synthesized by a known method. However, it may be excised from the nucleic acid of bacteria such as chlorinated ethane-degrading bacteria (KWI-A1) obtained in Examples described later.

  The nucleic acid fragment of the present invention utilizes a portion specific to 16S rRNA or rDNA of a chlorinated ethane-degrading bacterium after determination of the base sequence of 16S rDNA of the chlorinated ethane-degrading bacterium. Designed. The nucleic acid fragment having the nucleotide sequence of SEQ ID NO: 1 to 17 of the present invention is actually a portion of 16S rDNA unique to the chlorinated ethane-degrading bacterium (KWI-A1) of the example having 16 S rDNA of the nucleotide sequence of SEQ ID NO: 18. It is designed as a nucleic acid fragment of a base sequence. These nucleic acid fragments and the like of SEQ ID NOs: 1 to 17 are shown in FIG. 1 as positions in the base sequence of SEQ ID NO: 18 in Examples described later. The nucleic acid fragments and the like of the present invention are actually designed using the base sequence of a specific chlorinated ethane-degrading bacterium, but bacteria having a slightly different base sequence are also detected by PCR.

As described above, the nucleic acid fragment and the like of the present invention are designed using the specific portion of the 16S rDNA of chlorinated ethane-degrading bacteria after determining the nucleotide sequence of chlorinated ethane-degrading bacteria. To preferentially hybridize. For this reason, the nucleic acid fragment of the present invention can be used to detect chlorinated ethane-degrading bacteria with high accuracy and easily by PCR using primers or hybridization as described later. Can do. Specific examples of the chlorinated ethane-degrading bacterium detected here include chlorinated ethane-degrading bacteria belonging to the genus Dehalobacter, and the chlorinated ethane-degrading bacterium of the example having 16S rRNA having the base sequence of SEQ ID NO: 18 ( This includes KWI-A1) and chlorinated ethane-degrading bacteria ( Dehalobacter sp str.TCA1) shown in Non-Patent Document 1. Although it is possible to decompose chlorinated ethane without separating them, the chlorinated ethane-degrading bacteria (KWI-A1) of the examples may be isolated.

Since the nucleic acid fragment of the present invention has a base sequence similar to the rDNA of a bacterium having a degrading activity against trichlorethylene in Non-Patent Document 2 ( Dehalobacter restrictus str. TEA), if this bacterium is present, its 16S rRNA or rDNA Hybridize to. However, chlorinated ethane-degrading bacteria exist in soil or groundwater that is contaminated with chlorinated ethane, does not contain trichlorethylene, and undergoes chlorinated ethane decomposition, but non-patent document 2 does not have trichlorethylene-degrading bacteria. So the bacteria will never be detected. Both chlorinated ethane-degrading bacteria and chlorinated ethylene-degrading bacteria are detected, but chlorinated ethane-degrading bacteria (KWI-A1) have a chlorinated ethane-degrading gene having the nucleotide sequence of SEQ ID NO: 19 as a chromosome. Therefore, the degradation gene can be detected by PCR or the like, or can be separated by culturing in a medium containing chlorinated ethane.

  Specific examples of chlorinated ethane decomposed (dechlorinated) by chlorinated ethane-degrading bacteria include 1,1,1-trichloroethane, 1,1-dichloroethane, and their dechlorinated intermediates. It is done. The chlorinated ethane-degrading bacterium to be detected in the present invention is a bacterium having the activity of degrading these chlorinated ethanes, and inhabits soil or groundwater contaminated with these chlorinated ethanes. In a system where chlorinated ethane-degrading bacteria detected by the present invention are present, these chlorinated ethanes can be decomposed. When chlorinated ethane-decomposing bacteria, chlorinated ethylene-decomposing bacteria and other bacteria are detected, chlorinated ethane can be decomposed without separation, but they may be separated.

  The labeled probe for detecting chlorinated ethane-degrading bacteria of the present invention is a probe obtained by labeling the nucleic acid fragment of the present invention with a labeling substance such as a radioactive element, a fluorescent substance, a chemical substance, an antigen, an antibody or an enzyme. Conventionally used labeling substances can be used as such labeling substances. Specific examples include radioactive elements such as 32P; fluorescent substances such as FITC (Fluorescence isothiocyanate) and rhodamine; haptens such as dicoxygenin; alkaline phosphatase And enzymes such as peroxidase; biochemical substances such as biotin. These labeling substances can be introduced into the nucleic acid fragment by a known method. The labeled probe for detecting chlorinated ethane-degrading bacteria of the present invention is hybridized with a sample to detect the presence or absence of chlorinated ethane-degrading bacteria, and then the labeled probe is hybridized by detecting using the labeling substance as an index. Chlorinated ethane-degrading bacteria can be detected with specific high precision and easily.

  Any of the nucleic acid fragments having the nucleotide sequences of SEQ ID NOs: 1 to 17 of the present invention can be used as a PCR primer, or labeled with a labeling substance and used as a probe for detecting bacteria. In any case, the above nucleic acid fragments or the like may be used alone or in combination, or may be used in combination with the above nucleic acid fragments and other nucleic acid fragments. Among the above nucleic acid fragments and the like, the nucleic acid fragments of SEQ ID NOs: 1 to 4 and 7 to 17 are preferably used as primers, and the nucleic acid fragments of SEQ ID NOs: 5 to 6 are preferably used as probes. Absent. When the nucleic acid fragment of the present invention is used in combination with another nucleic acid fragment, for example, when the nucleic acid fragment of the present invention is used as one primer, another known nucleic acid fragment such as a universal primer is used as the other primer. be able to.

  The method for detecting a chlorinated ethane-degrading bacterium of the present invention is a method for detecting a chlorinated ethane-degrading bacterium using the nucleic acid fragment of the present invention. That is, PCR is performed using the nucleic acid fragment of the present invention as a primer and a nucleic acid prepared from a sample to be detected for the presence or absence of chlorinated ethane-degrading bacteria as a template. If DNA of the expected size is synthesized, It can be judged that chlorinated ethane-degrading bacteria existed. In this case, a highly active chlorinated ethane-degrading bacterium having a base sequence of SEQ ID NO: 18 or a base sequence having 90% or more homology with 16S rRNA or rDNA can be detected.

  PCR can be performed by a known method, or can be performed using a commercially available PCR kit. PCR usually uses two types of primers, Upper Primer and Lower Primer, and the nucleic acid fragment of the present invention can be used as one or both of the primers. The detection accuracy can be further increased by performing detection a plurality of times using a plurality of nucleic acid fragments of different types as primers. Although block PCR can be used for semi-quantitative detection, Real-Time PCR is preferably used for detection with higher quantitativeness.

  In the method for detecting a chlorinated ethane-decomposing bacterium according to the present invention, the chlorinated ethane-decomposing bacterium can be detected using the labeled probe for detecting a chlorinated ethane-decomposing bacterium according to the present invention. In this method, the labeled probe for detecting chlorinated ethane-degrading bacteria of the present invention is contacted with a sample to be detected for the presence or absence of chlorinated ethane-degrading bacteria or a nucleic acid prepared from this sample, and RNA or DNA hybridization is performed. This is a method for detecting chlorinated ethane-degrading bacteria using a label as an indicator. Hybridization can be performed by a method similar to the conventional method. Although detection using such a label as an index can be performed during PCR, it may be performed independently of PCR. For example, when performing Real-Time PCR, it is preferable to use a nucleic acid fragment labeled with a labeling substance as a probe because the amplification state in the PCR process can be detected.

  Detection after hybridization can be performed by a known method according to the type of the labeling substance. For example, when labeled with a radioactive element, it can be detected by measuring the radioactivity by a known method. Moreover, when it labels with a fluorescent substance, it can detect by measuring a light quantity by a well-known method. Moreover, when label | marking with an enzyme, it can detect by measuring an enzyme activity by a well-known method. When labeled with a chemical substance, it can be detected by analyzing the chemical substance. Further, when labeled with an antigen or an antibody, it can be detected by reacting with the antigen or antibody using an antibody or antigen that specifically reacts with the labeled antigen or antibody, and measuring the reaction product by a known method.

  As described above, the nucleic acid fragments of SEQ ID NOs: 1 to 17 can be used as a primer or a probe, and a primer and a probe can be used in combination. A nucleic acid fragment or the like simply used as a probe may have a labeling substance attached to either the 5 ′ end or the 3 ′ end. However, a nucleic acid fragment or the like that also serves as a primer has a labeling substance attached to the 5 ′ end. By leaving it in a free state, extension of the nucleic acid toward the 3 ′ end can be allowed. When a labeling substance is attached to the 5 ′ end of a nucleic acid fragment or the like used exclusively for the probe, the extension of the nucleic acid toward the 3 ′ end can be prevented by performing PCR by blocking the 3 ′ end with phosphoric acid or the like. it can. Nucleic acid fragments etc. that are used exclusively for probes by blocking the 3 ′ end are detected by hybridizing to single-stranded nucleic acids, but they are flipped off as the nucleic acids are extended from the primers by the progress of PCR. It is detected by hybridizing again to the nucleic acid that has become a single strand as it progresses. Nucleic acid fragments and the like used exclusively for primers can be used without labeling.

  By detecting chlorinated ethane-degrading bacteria using the method described above, dechlorination is performed well when chlorinated ethane-degrading bacteria are used to purify soil or groundwater contaminated with chlorinated ethane. It can be determined in advance whether or not. Such a determination can be made only by detecting the base sequence of the nucleic acid of bacteria contained in the sample collected from the contaminated place or other places. Operations such as culture, separation, and identification of chlorinated ethane-degrading bacteria are not possible. It is unnecessary and accurate determination can be made in a short time. When chlorinated ethane-decomposing bacteria cannot be detected, it is possible to take measures such as adding chlorinated ethane-degrading bacteria detected elsewhere.

  The method for decomposing chlorinated ethane of the present invention is carried out by detecting the chlorinated ethane-decomposing bacteria of the present invention using groundwater or soil as a sample, and inoculating the groundwater, soil, or them in which chlorinated ethane-degrading bacteria are detected. Introduce culture fluid (hereinafter collectively referred to as chlorinated ethane-degrading bacteria detected substances) into soil or groundwater contaminated with chlorinated ethane (hereinafter collectively referred to as contaminated environment) Thus, chlorinated ethane is decomposed. Since the chlorinated ethane-degrading bacteria are anaerobic bacteria, the chlorinated ethane-degrading bacteria can be grown and decomposed by contacting them with chlorinated ethane in an anaerobic state.

  The detected chlorinated ethane-degrading bacteria introduced into the contaminated environment may be detected (collected) anywhere. For example, groundwater, soil, or a culture solution inoculated with chlorinated ethane-degrading bacteria in a place not contaminated with chlorinated ethane can be introduced into soil or groundwater contaminated with chlorinated ethane. It is also possible to introduce groundwater, soil or culture medium inoculated with chlorinated ethane-degrading bacteria in a place contaminated with chlorinated ethane into the same area contaminated with chlorinated ethane. It can also be installed in another location that is not in the same area.

  As a method of introducing the chlorinated ethane-decomposing bacteria detection substance into the contaminated environment, a method of spraying the chlorinated ethane-decomposing bacteria detection substance etc. on the contaminated soil surface, a method of injecting into the soil from the injection pipe (injection well), For example, it can be injected into the groundwater source. The introduction point can be introduced not only in a contaminated place, but also upstream of the contaminated environment. In either case, the chlorinated ethane-degrading bacteria are introduced so as to be in uniform contact with the chlorinated ethane contaminated site. When degrading chlorinated ethane, it may be sufficient to introduce groundwater, soil, or culture medium inoculated with chlorinated ethane-degrading bacteria into the contaminated environment. It can also be introduced. In addition, when it cannot be completely decomposed by one introduction, the introduction can be repeated. Further, a flocculant can be added to the chlorinated ethane-degrading bacteria detection substance to cause aggregation, or can be introduced after being supported on a carrier. By decomposing chlorinated ethane in this way, a contaminated environment contaminated with chlorinated ethane can be purified.

  In a contaminated environment contaminated with chlorinated ethane, the number of chlorinated ethane-degrading bacteria can be quantified using a labeled probe for detecting chlorinated ethane-degrading bacteria. As a result, the number of chlorinated ethane-decomposing bacteria detected in the contaminated environment or the introduced chlorinated ethane-degrading bacteria can be monitored, and the change of the chlorinated ethane-decomposing bacteria in the contaminated environment can be followed. By simultaneously monitoring chlorinated ethane-degrading bacteria and monitoring chlorinated ethane as a pollutant, the progress of environmental purification by chlorinated ethane decomposition can be investigated.

The nucleic acid fragments of the present invention are novel and useful. Since the nucleic acid fragment of the present invention has a specific base sequence and hybridizes preferentially to 16S rRNA or rDNA of chlorinated ethane-degrading bacteria, it can be used for detection of chlorinated ethane-degrading bacteria.
Since the nucleic acid fragment for detecting a chlorinated ethane-degrading bacterium of the present invention comprises the above-mentioned nucleic acid fragment or the like, the chlorinated ethane-degrading bacterium can be specifically detected with high precision and easily by using this nucleic acid fragment. it can.
Since the labeled probe for detecting chlorinated ethane-degrading bacteria of the present invention labels the nucleic acid fragment etc., it is possible to detect chlorinated ethane-degrading bacteria specifically with high accuracy and easily using this label as an index. Can do.
The detection method and monitoring method of the chlorinated ethane-degrading bacterium of the present invention uses the above-mentioned nucleic acid fragment or the like or a labeled probe, so that the chlorinated ethane-degrading bacterium can be specifically detected with high precision and easily. Can do.
The method for degrading chlorinated ethane of the present invention is to introduce groundwater, soil or a culture solution inoculated with chlorinated ethane-degrading bacteria in the above detection method into soil or groundwater contaminated with chlorinated ethane. Since chlorinated ethane is decomposed, the chlorinated ethane can be easily and efficiently decomposed to purify the environment.

  Next, examples of the present invention will be described.

  Sampling the groundwater from a total of four locations, points A and B where 1,1,1-trichloroethane is dechlorinated to 1,1-dichloroethane and chloroethane, and points C and D where no dechlorination is occurring DNA was extracted from 100 mL of this groundwater as follows.

(1) DNA extraction:
After filtering 100 mL of groundwater with a filter having a pore size of 0.2 μm, this filter was put into a 2 mL tube. Further, 1 mL of Zirconia / Silica Beads (diameter 0.1 mm) and Extraction Buffer (100 mM Tris-HCl [pH 8.0], 100 mM sodium EDTA [pH 8.0], 100 mM sodium phosphate [pH 8.0], 1.5 M NaCl) are added to the tube. And processed for 2 minutes with a bead beater. Next, freeze-thaw was repeated three times, 10 μL of Proteinase K (10 mg / ml) was added, and the mixture was incubated at 37 ° C. for 30 minutes. To this solution, 250 μL of 10% SDS solution was added and kept at 65 ° C. for 2 hours, and then the above-mentioned Bead Beater treatment was performed again. Thereafter, the mixture was centrifuged at 8000 × g for 10 minutes at room temperature, and the supernatant was collected. The supernatant was extracted with chloroform, and after adding an equal volume of isopropanol, the mixture was allowed to stand at room temperature for 60 minutes and centrifuged at 8000 × g at room temperature for 20 minutes to precipitate DNA. The precipitate was washed with 70% ethanol, dried, and then dissolved in 50 μL of sterile distilled water. Using this extracted DNA solution, a PCR reaction was carried out as follows to investigate whether chlorinated ethane-degrading bacteria were present.

(2) Amplification of 16S rDNA by PCR:
16SrDNA was amplified by PCR using 1 μL of the extracted DNA solution obtained in (1) as a template. The total volume of the PCR amplification reaction solution was 100 μL, and 2.5 U Ex Taq DNA polymerase (Takara Shuzo) and 200 μM dNTP were used. As shown in Table 1, as a primer pair, any one of KWI-Deb1 to KWI-Deb2 and KWI-Deb7 to KWI-Deb11 is designated as Upper Primer, and Bact1492 (5′-ACGG C / T TACCTTTGTTAGACTT- 3 ′) are 7 primer pairs with Lower Primer, and Bact0011 (5′-GTTTGATCCTGCTCAG-3 ′) of SEQ ID NO: 21 is Upper Primer, and KWI-Deb3 to KWI-Deb4 and KWI-Db12 to KWI-Deb17 10 pmols of 8 primer pairs each having one of complementary base sequences as a lower primer were used. Other reaction solution compositions followed the manual attached to the PCR kit. The PCR reaction was pre-heating; 94 ° C., 2 minutes, followed by 1st stage; 94 ° C., 20 seconds, 2nd stage; 55 ° C., 30 seconds, 3rd stage; 72 ° C., 2 minutes, 30 cycles, Post extension: 72 ° C., 7 minutes.

  2 μL of the PCR reaction solution was subjected to agarose electrophoresis, and if a DNA fragment of the expected size was synthesized, it was judged that chlorinated ethane-degrading bacteria were present. The results are shown in Table 1.

○：ＤＮＡの合成が観察された
×：ＤＮＡの合成が観察されない

○: DNA synthesis observed x: DNA synthesis not observed

  The base sequences of KWI-Deb1 to KWI-Deb17 in Table 1 and Example 2 are as shown in Table 2. The base sequence of 16S rDNA of the chlorinated ethane-degrading bacterium (KWI-A1) detected in the present invention is as shown in SEQ ID NO: 18, and the arrangement of KWI-Deb1 to KWI-Deb17 in this base sequence is shown in FIG. In addition, the nucleotide sequence of the chlorinated ethane degrading gene of the chlorinated ethane degrading bacterium (KWI-A1) detected in the present invention is shown in SEQ ID NO: 19.

  From the results in Table 1, at points A and B where dechlorination was observed, exceptions in which DNA synthesis was not observed by PCR and electrophoresis were observed three times out of 30, but most DNA synthesis was observed. It was done. On the other hand, no DNA synthesis was observed at points C and D where no dechlorination was observed. From this result, it was shown that chlorinated ethane-degrading bacteria always exist at the point where dechlorination reaction occurs, and these can be monitored.

(1) Detection by Light Cycler:
The extracted DNA solution of Example 1 was further accurately detected and quantified by Real-Time PCR using a Light Cycler manufactured by Roche Diagnostics. At that time, KWI-Deb1 was used as the upper primer, and an oligonucleotide complementary to KWI-Deb4 was used as the lower primer. As hybridization probes, those in which the 3 ′ end of KWI-Deb5 is labeled with FITC (Fluorescence isothiocyanate) and the 3 ′ end of KWI-Deb6 is phosphorylated and the 5 ′ end is labeled with FITC. used. The PCR reaction was performed using the Light Cycler DNA Master Hybridization Probes Kit (trademark) according to the manual. The reaction conditions are as shown in Tables 3 to 6.

  As a result, the target DNA was PCR-synthesized in the groundwater at points A and B, and the number of chlorinated ethane-degrading bacteria could be quantified. However, at points C and D, the target DNA was not synthesized and was below the lower limit of quantification, so it was determined that there were no chlorinated ethane-degrading bacteria. These results also indicate that the primers shown in Table 2 can also be used as hybridization probes.

  100 ml of groundwater contaminated with 1,1,1-trichloroethane (TCA) was placed in a 150 ml vial, and after adding a nutrient such as citric acid, it was sealed with a butyl rubber stopper and sealed with an aluminum cap. This vial was periodically sampled while standing at 30 ° C., and the concentration of ethanes in the vial was measured and the number of chlorinated ethane-degrading bacteria was quantified. In addition, 10 days after plugging, the bacterial suspension in which the chlorinated ethane-degrading bacterial gene was detected was inoculated so that the final concentration of the chlorinated ethane-degrading bacterial gene was 10 6 copies / ml. . The result of the concentration of ethanes is shown in FIG. 2, and the result of the number of decomposing bacteria is shown in Table 7.

  As shown in FIG. 2, when a bacterial suspension in which a chlorinated ethane degrading gene was detected was added 10 days later, the chlorinated ethane degradation became prominent about 19 days after the experiment started, and 1,1-dichloroethane (DCA) Began to be detected. Thereafter, DCA was also decomposed and completely converted to monochloroethane (CA) in about 60 days. In addition, the number of decomposing bacteria increased to 10 8 power / ml.

  It is used to detect and monitor chlorinated ethane-degrading bacteria in soil or groundwater contaminated with chlorinated ethane, and to purify the environment by degrading chlorinated ethane.

It is an arrangement plan of nucleic acid fragments. 10 is a graph showing the results of Example 3.

Claims (8)

A nucleic acid fragment consisting of 18 to 23 nucleotides preferentially hybridizing to 16S rRNA or rDNA of a chlorinated ethane-degrading bacterium, comprising any one of SEQ ID NOs: 1 to 17, and 90% or more homology with these nucleotide sequences Or a nucleic acid fragment having a base sequence complementary to these base sequences. A nucleic acid fragment consisting of 10 to 50 nucleotides preferentially hybridizing to 16S rRNA or rDNA of a chlorinated ethane-degrading bacterium, wherein at least 10 consecutive base sequences are any one of SEQ ID NOs: 1 to 17 A nucleic acid fragment having the same base sequence or a base sequence complementary to these base sequences. The nucleic acid fragment according to claim 1 or 2, which is used for detection of chlorinated ethane-degrading bacteria. A labeled probe for detecting chlorinated ethane-degrading bacteria, wherein the nucleic acid fragment according to any one of claims 1 to 3 is labeled with a labeling substance. A method for detecting a chlorinated ethane-degrading bacterium, which detects a synthesized DNA fragment by performing PCR (polymerase chain reaction) using the nucleic acid fragment according to any one of claims 1 to 3 as a primer and the nucleic acid in a sample as a template. A chlorinated ethane-degrading bacterium that detects a chlorinated ethane-degrading bacterium according to claim 4 after contacting the sample or a nucleic acid prepared from the sample with RNA or DNA hybridization and then detecting using the label as an indicator Detection method. 7. The method for detecting ethane-degrading bacteria according to claim 5 or 6 is carried out using groundwater or soil as a sample, and the groundwater, soil or culture solution inoculated with chlorinated ethane-degrading bacteria is contaminated with chlorinated ethane. Of degrading chlorinated ethane introduced into soil or groundwater. The method for detecting ethane-degrading bacteria according to claim 5 or 6 is carried out, and the number of chlorinated ethane-degrading bacteria detected in soil or groundwater contaminated with chlorinated ethane or introduced chlorinated ethane-degrading bacteria is quantified. To monitor chlorinated ethane-degrading bacteria.

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