JP2004261124A - Primer for detecting methanogen, detecting method using the same, etc. - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for exhaustively detecting methanogens in an environmental specimen such as methane fermentation sludge and provide a primer for the detection method. <P>SOLUTION: The method for the detection of methanogens in a nucleic acid specimen derived from methanogens in an environmental specimen comprises a PCR method using either one of the following primer sets consisting of (a) an oligonucleotide having the 1st specific base sequence or a base sequence essentially homologous to the 1st sequence and an oligonucleotide having the 2nd specific base sequence or a base sequence essentially homologous to the 2nd sequence and (b) an oligonucleotide having the 3rd specific base sequence or a base sequence essentially homologous to the 3rd sequence and an oligonucleotide having the 4th specific base sequence or a base sequence essentially homologous to the 4th sequence, and the detection of the amplification product obtained by the PCR. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

なお、上記ＰＣＲ反応液組成において、各プライマーは１ｐｍｏｌずつ入っている。

なお、上記ＰＣＲ産物は、ＰＣＲ反応後の液をアガロースゲルからＤＮＡを抽出後、精製したものを用いた。
〔シークエンスＰＣＲ反応液〕
下記組成で全体量が２０μになるように調製した。

〔ＰＣＲ反応条件〕
９６℃３０ｓｅｃ−（９６℃１０ｓｅｃ−５０℃５ｓｅｃ−６０℃３ｍｉｎ）×３０ｃｙｃｌｅ−（９６℃１０ｓｅｃ−６０℃１０ｓｅｃ−７２℃１ｍｉｎ）×１５ｃｙｃｌｅ−４℃∞
〔ＤＧＧＥ泳動条件〕
使用ゲル：１０％ ポリアクリルアミドゲル
変性濃度勾配：２０−７０％
２００Ｖ定電圧で３時間から３時間半泳動
なお、ＤＧＧＥの泳動後は、Ｖｉｓｔｒａ ｇｒｅｅｎ ｎｕｃｌｅｉｃ ａｃｉｄで染色し、ＦｌｕｏｒＩｍａｇｅｒ５９５によりバンドを検出した。
【００６９】
【実施例４】
メタン生成細菌の群集構造の解析
上述したように、メタン発酵汚泥中の全メタン生成細菌に由来する１６Ｓ ｒＤＮＡ遺伝子のＰＣＲ増幅産物をＤＧＧＥのゲル上に展開することにより、当該メタン生成細菌の群集構造を映像的に捕らえた基礎データが得られる。そして、この種の指標データを特定のメタン発酵槽について定期的に記録していくことで、その発酵槽内のメタン生成細菌群の種類と菌数の変遷を追跡することができる。そのような追跡から、メタン生成細菌群の種類と菌数の変遷と、実際に観測される発酵槽の状態変化との相関関係が明らかになる。
【００７０】
従って、本発明によるＤＧＧＥの泳動結果は、メタン発酵槽の状態の善し悪しを診断し、その運転条件等を制御するための目安を与える指標データとして利用できる。すなわち、この種の映像データは、発酵槽中の分解活性を左右するメタン生成細菌の群集構造の変遷を早期に且つ直接的に写し取った指標データである。このような指標データを取得し、検査することによれば、細菌の群集構造の変遷に追従して変化するであろう発酵槽の実際の状態変化（例えばメタンガス発生量の変動）を観測してから判断するよりも、迅速かつ的確な状況判断ができるようになる。例えば、発酵槽の運転状況が理想的な状況から逸脱する場合に追跡して得られたＤＧＧＥの泳動結果をその状態悪化を示す指標データとして記録しておき、その後、ＤＧＧＥの泳動結果を検査する際には、その指標データに照らし合わせることで、発酵槽の状態悪化を事前に予測し、対処することができる。
【００７１】
本実施例では、一定期間内において、メタン発酵槽内からのメタンガス発生量を測定すると共に、そのメタン発酵槽から定期的に採取したＤＮＡ試料についてＰＣＲ−ＤＧＧＥで得られる泳動バンドを記録した。
【００７２】
図４は、メタンガスの発生量と対応する泳動写真とを対比して示す。同図で示されるようにメタンガス発生量が変動する発酵槽では、各泳動写真中に現れるバンドの数や太さにも経時的に変化する傾向が見られ、このように細菌の生息状況の経時的な変化が可視的に把握されることが分かった。
【００７３】
図４のデータにおいては、例えば、メタンガス発生が比較的不安定なＩ期（１２／１０〜２／２５）、それが比較的安定なＩＩ期（３／４〜５／１３）、ガス量の急激な上昇を伴うＩＩＩ期（５／２０〜）が見られるが、これらガス発生量の変遷を各時期の泳動結果に照らしてみると、それぞれの時期に特有の形態を持つ泳動バンドが出現していることが分かった。
【００７４】
【発明の効果】
以上詳細に述べたように、本発明によれば、環境試料中に存在するメタン生成細菌を網羅的に検出することができ、必要であれば、そのメタン生成細菌群集構造についての指標データを得ることができる。
【００７５】
【配列表】

【図面の簡単な説明】
【図１】図１は、メタン生成細菌の１６Ｓ ｒＤＮＡ塩基配列解析の結果を示す。
【図２】図２は、メタン生成細菌の系統樹を示す。
【図３】図３は、実施例によるＤＧＧＥの泳動写真の一例を示す。
【図４】図４は、メタンガスの発生量とそれに対応する泳動バンドとを経時的に対比して示す。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a method for detecting methanogenic bacteria present in methane fermentation-treated sludge such as organic waste and wastewater and primers used therefor, and more specifically, a comprehensive and comprehensive description of the methanogenic bacterial community structure. The present invention relates to a detection method and the like useful for understanding.
[0002]
[Prior art]
Conventionally, aerobic biological treatment methods are often used to treat wastewater containing organic pollutants, but this method consumes a lot of energy and disposal of excess sludge is a major problem. . On the other hand, an anaerobic treatment method (methane fermentation) has been frequently used for treating wastewater or organic sludge containing a high concentration of organic pollutants. This method has the advantages that energy consumption can be saved because aeration power is not required, treatment cost is low because the amount of surplus sludge is small, and methane gas useful as energy can be recovered.
[0003]
Methane fermentation involves the following three steps: (1) the process of solubilizing high molecular organic matter, (2) the process of acid fermentation of solubilized organic matter, and (3) the methane production process. Is a complex biological reaction system.
[0004]
In particular, when the organic matter load increases, accumulation of organic acids occurs in the methane fermentation sludge, and the stability of biogas generation is lost. That is, in methane fermentation, the activity or concentration of the microorganisms involved in methane production is an important factor that determines the final rate and amount of methane generation.
[0005]
For example, in order to quantify the methane production activity in the above-mentioned environmental sample of a biological reaction system, an attempt has been made to measure the fluorescence intensity of the coenzyme F420 having autofluorescence possessed by the methane-producing bacteria. However, the types of methanogens having F420 are limited, and there is a problem that the methanogens cannot always exhibit the overall methanogenic activity and are susceptible to contaminants.
[0006]
For the measurement of the number of methanogenic bacteria, an MPN method or the like based on culture is used. However, since the methanogenic bacteria grow slowly, it takes one month to count. Moreover, it is said that the ratio of bacteria capable of growing on an agar medium is only 1% or less of the total bacteria present in the case of a sludge sample or the like. Such non-cultivable bacteria, if present, cannot be detected.
[0007]
On the other hand, in recent years, methods for detecting microorganisms using molecular biological techniques have been developed. For example, an oligonucleotide having a sequence complementary to a specific portion of a target bacterial gene sequence is prepared, and a DNA probe to which a fluorescent dye is attached is hybridized in a cell by a FISH (fluorescent in situ hybridization) method ( According to Non-Patent Document 1) or a PCR (polymerase chain reaction) method using a probe specific to a bacterial gene, a target bacterium can be detected from an environmental sample without culturing.
[0008]
For example, in the FISH method, target bacteria can be detected for each dye in the same field of view by attaching two or three types of fluorescent dyes to different DNA probes and hybridizing them to the same sample. However, for quantification, it is necessary to count a plurality of visual fields using a fluorescence microscope and obtain an average value, and it is a problem that increasing the types of target bacteria requires much time and labor.
[0009]
Regarding the PCR method, the DNA in the sample is extracted, and the 16S rRNA gene is selectively selected by the PCR method using oligonucleotide primers having a sequence complementary to the 16S rRNA gene that is commonly conserved among methanogens. There is known a detection method for amplifying the DNA (Patent Document 1).
[0010]
Furthermore, as one of the techniques for analyzing PCR products, denaturing gradient gel electrophoresis (hereinafter also referred to as "DGGE") capable of separating double-stranded DNAs of the same length due to differences in their GC contents. Is known (Non-Patent Document 2).
[0011]
[Non-patent document 1]
R. I. Amman, L .; Krumholz and D.K. A. Stahl: Fluorescent-oligonucleotide probing of whole cells for determine, phylogenetic and environmental studies in microbiology. Bacteriol. , 172, 762-770, 1990
[Non-patent document 2]
G. FIG. Muizer, E .; C. De Waal, and A. G. FIG. Uitterlinden: Profiling of complex microbiological populations by dedenaturing gradient gel electrophoresis analysis of polymerization chain reaction-amplification-amplification-amplification-amplification-amplification-amplification-amplification-amplification-amplification-amplification-amplification-amplification-aid. , Vol. 59, No. 3, p. 69-700, 1993
[Patent Document 1]
JP 2001-327290 A
[0012]
[Problems to be solved by the invention]
Meanwhile, for diagnosis of environmental samples such as organic wastes and wastewater, etc., microbial community structure analysis for examining genes derived from the microbial communities present therein has become promising. When performing this type of analysis on methane fermented sludge, it is necessary to detect the methanogenic bacteria by applying the above-described gene detection method to the collected sludge sample.
[0013]
However, when examining the methanogenic bacterial community structure in methane fermentation sludge, simply applying the above-mentioned conventional detection method has the following inconveniences.
In applying a detection method based on a molecular biological technique, conventional oligonucleotide primers for detecting methanogenic bacteria can detect only a specific genus and / or group. No one with the desired specificity that could fully cover the bacterial population has been obtained.
[0014]
The oligonucleotide primers described in Patent Document 1 are said to be capable of detecting all methanogens. However, using these primers, it is necessary to successfully analyze the methane bacteria community structure mixed in the sludge. It has not been investigated at all whether the process can be performed, for example, whether an amplification product suitable for applying the denaturing gradient gel electrophoresis can be obtained.
[0015]
Therefore, while providing comprehensive detection of methanogenic bacteria from environmental samples such as methane fermentation-treated sludge, a quick and simple detection method capable of comprehensively grasping the status of methanogenic bacteria, and primers suitable for the method are provided. There is a need to
[0016]
An object of the present invention is to provide a method for exhaustively detecting methanogenic bacteria from an environmental sample, and a primer and the like for the method.
A further object of the present invention is to provide a detection method capable of comprehensively detecting methanogenic bacteria from an environmental sample and comprehensively understanding the community structure of the methanogenic bacteria, and a method of obtaining such index data. It is in.
[0017]
[Means for Solving the Problems]
The present inventors have conducted intensive studies to solve the above problems, and as a result, a primer capable of comprehensively detecting genes derived from methane-producing bacteria present in methane fermentation-treated sludge of organic waste and wastewater. Heading, the present invention has been completed.
[0018]
That is, the present invention provides a primer set comprising a set of oligonucleotides described in (a) or (b) below:
(A) an oligonucleotide having a nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence substantially homologous thereto, and an oligonucleotide having a nucleotide sequence of SEQ ID NO: 2 or a nucleotide sequence substantially homologous thereto; or
(B) an oligonucleotide having the nucleotide sequence of SEQ ID NO: 3 or a nucleotide sequence substantially homologous thereto, and an oligonucleotide having the nucleotide sequence of SEQ ID NO: 4 or a nucleotide sequence substantially homologous thereto.
[0019]
The present invention also relates to a method for detecting a methane-producing bacterium, wherein a PCR method is performed on a nucleic acid sample derived from a methane-producing bacterium in an environmental sample using any of the primer sets described above. A method comprising detecting an amplification product obtained by the method.
[0020]
In a preferred embodiment of the detection method according to the present invention, the first PCR method is performed on a nucleic acid sample derived from a methanogen in an environmental sample using the primer set described in (a) above, and The above method, further comprising performing a second PCR method on the amplification product obtained by the first PCR method using the primer set described in the above (b).
[0021]
In addition, the present inventors applied a denaturant gradient gel electrophoresis (DGGE) to a nucleic acid fragment group selectively amplified by using the primers, thereby obtaining a comprehensive and comprehensive view of the community structure of methanogens. Revealed that it can be caught.
[0022]
That is, in the method for detecting a methane-producing bacterium according to the present invention, when a PCR method is performed using the primer set described in the above (b), the amplification product obtained by the PCR method is subjected to denaturant concentration gradient gel electrophoresis. Preferably, the method further comprises subjecting
[0023]
Further, the present invention comprises using the distribution of electrophoretic bands obtained by the electrophoresis by the denaturing agent concentration gradient gel electrophoresis as index data for the methanogenic bacterial community structure present in the environmental sample. A method for evaluating a sample is also provided.
[0024]
A preferred environmental sample to which the method of the present invention is applied is methane fermentation sludge.
[0025]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, embodiments of the present invention will be described in detail.
The present invention selects the presence of a 16S rDNA gene derived from methane-producing bacteria in a microbial reaction system environmental sample such as methane fermented sludge (hereinafter, also simply referred to as “environmental sample” or “sludge sample”) through PCR. This is a method for detecting methanogenic bacteria, which relies on the detection of the methanogenic bacteria.
[0026]
In the present detection method, the primer used in the PCR method can anneal to the 16S rDNA gene derived from a methanogen such as dominant in methane fermentation sludge under a predetermined PCR condition, but under the same conditions. Below, it is designed to have a base sequence that does not anneal to 16S rDNA genes from eubacteria and non-methanogens, and has global specificity for methanogens.
[0027]
As such a PCR primer, a primer pair consisting of an oligonucleotide having the nucleotide sequence of SEQ ID NO: 1 and an oligonucleotide having the nucleotide sequence of SEQ ID NO: 2, or an oligonucleotide having the nucleotide sequence of SEQ ID NO: 3 and SEQ ID NO: 4 Examples of the primer pairs include oligonucleotides having the following base sequences, but are not limited thereto. Primers that can be used in the present detection method also include a primer pair consisting of an oligonucleotide having a base sequence substantially homologous to the base sequences listed above, depending on the base length, PCR conditions, and the like.
[0028]
As described above, PCR using the primers of the present invention having the desired specificity results in the desired nucleic acid amplification in a sample in which at least one methanogen is present, but the methanogen is substantially A significant nucleic acid amplification will not occur in a sample that is not present. Therefore, in the PCR method using the primer of the present invention, exhaustive detection of a group of methanogenic bacteria present in an environmental sample is possible only by checking whether or not an expected nucleic acid is amplified. Thus, according to the present invention, a method for detecting a group of methanogenic bacteria by a simple PCR method in a short time is provided.
[0029]
In order to confirm that the amplification product obtained by the PCR method is the target nucleic acid fragment, electrophoresis of the amplification product may be performed, or the nucleic acid region between the used primers, that is, the nucleic acid to be amplified Detection may be performed using a DNA probe or the like complementary to the region.
[0030]
In addition, the amount of the amplified target nucleic acid correlates with the number of methanogens inhabiting the environmental sample (or taking into account its 16S rDNA gene copy number). That is, based on the band width (brightness or concentration) of the target nucleic acid obtained by electrophoresis of these amplification products, or on the basis of easily quantified detection by competitive PCR or the like, various types of methane contained in a certain environmental sample can be used. It is possible to know the total viable count of produced bacteria. Any known means known in the art can be used for the detection and quantification of these amplification products.
[0031]
First embodiment
The first embodiment of the detection method of the present invention uses a primer specific to a group of methanogens as described above, and amplifies a region close to the “full-length sequence” of 16S rDNA of the methanogen using the primers. Based on the first PCR method designed to be used.
[0032]
According to this embodiment, the obtained amplification products can provide 16S rDNA gene information on a scale sufficient to be useful for phylogenetic analysis when they are cloned, so that identification of methanogenic bacterial species and the like can be easily performed. Bring the advantage of becoming. For example, a known gene cloning method using Escherichia coli with the obtained amplified nucleic acid as a sample (Hiroki Nakayama and Takato Nishikata, Bio-Experiment Illustrated (2) Basics of Gene Analysis, Shujunsha, pp. 69-88, 1995) can be performed to perform a detailed analysis based on the cloned nucleic acid.
[0033]
The primer that can be used in the first embodiment is, for example, a pair of oligonucleotides consisting of the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2. According to this primer set, in the full length of 16S rDNA (about 1500 bases), typically, about 85 to 1350 base region corresponding to the base position counted from the 5 'end side is used. A 1265 base fragment is amplified.
[0034]
Second embodiment
The second embodiment of the detection method of the present invention uses primers specific to a group of methanogens as described above, and amplifies a “partial sequence” in the 16S rDNA nucleotide sequence of the methanogen by the primers. This is based on the second PCR method designed to perform That is, according to this embodiment, the obtained amplification product is obtained as an amplified nucleic acid having a fragment length to which the PCR-DGGE (denaturing gradient gel electrophoresis) method can be preferably applied. Such a preferred fragment length is around 200 nucleotides.
[0035]
The primer that can be used in the second embodiment is, for example, a pair of oligonucleotides of each base sequence of SEQ ID NO: 3 and SEQ ID NO: 4. When this primer set is used, typically, a fragment of about 200 bases corresponding to about 1200 to about 1400 at the base position counted from the 5 'end in the 16S rDNA base sequence is amplified.
[0036]
By the way, in PCR-DGGE, by subjecting a PCR product to electrophoresis (DGGE) in a gel in which a concentration gradient of a DNA denaturing agent is formed, a DNA fragment of the same length that a band is overlapped in normal electrophoresis is obtained. It is a known means that can be separated. According to the PCR-DGGE method, as a PCR product consisting of a PCR-amplified nucleic acid fragment having the same base length starts to migrate in a double-stranded form and migrates in a direction in which the concentration gradient of the DNA denaturant increases, individual DNA Since fragments can be dissociated at different denaturant concentrations depending on the intrinsic GC content, differences in base sequence appear as differences in migration distance. Since the DGGE conditions depend on the GC content of the amplified nucleic acid, a CG clamp sequence for providing an appropriate migration distance may be added to the end of the primer as needed.
[0037]
The DGGE separation of the PCR amplified 16S rDNA fragment group forms a plurality of bands in a manner reflecting the difference in the nucleotide sequence of each 16S rDNA fragment. These bands separated by DGGE indicate the presence of different methanogenic bacteria, as the differences strongly correlate with the phylogenetic positioning of such bacteria.
[0038]
Thus, the distribution of bands appearing in DGGE indicates the relative abundance (diversity) of various methanogens in the methanogen cluster concentration, and the thickness (brightness or concentration) of each band is determined by each band. The relative abundance (the number of bacteria) of the same bacterial group corresponding to the above. In this manner, the present detection method provides a comprehensive band of basic data on the structure of the methanogenic community as a visible band distribution, that is, an index in which both the type and the number of methanogens are copied. Data can be presented.
[0039]
Third embodiment
A third embodiment of the detection method of the present invention comprises a first PCR method for amplifying a region close to the full-length sequence of 16S rDNA of a methanogen, and a second PCR for amplifying a partial sequence region on the 16S rDNA. It is a method that combines the law and the law.
[0040]
According to this aspect, for a sample in which nucleic acids of various microorganisms derived from a sludge sample are mixed, the target nucleic acid is concentrated by performing exhaustive detection of a group of methanogenic bacteria by the first PCR method. A second PCR method is further performed on the concentrated product. By performing such a two-step PCR, the specificity to the methane-producing bacteria is increased, and more accurate detection becomes possible. In order to carry out this embodiment, the primers used in the second PCR method are designed by selecting a specific base sequence by targeting a region in the amplified nucleic acid obtained by the first PCR method. Need to be
[0041]
In the third embodiment, also, by separating the amplification product by the second PCR method with DEGE, index data on the methanogen group can be obtained.
Further, by utilizing the third aspect, it is possible to provide a method for analyzing a methanogenic bacterial community as described below.
[0042]
The long 16S rDNA fragment amplified by the first PCR method was cloned and its nucleotide sequence was determined and analyzed. Further, each of the cloned 16S rDNA fragments was individually subjected to the second PCR method and the DEGE method. Obtain a single band from the cloned sample. At this time, the DEEG electrophoresis of each cloned sample is performed simultaneously with the electrophoresis of the non-cloned DNA sample derived from the sample sludge, so that a single band derived from each cloned sample is obtained from a plurality of bands derived from the sludge sample. , Which band is supported. Then, since the 16S rDNA of each cloned sample can identify the type of bacteria by nucleotide sequence analysis when cloned as a long 16S rDNA fragment as described above, thus, the bacterial group constituting each band can be identified. A phylogenetic classification is also possible. In this way, it is also possible to analyze and evaluate basic data that is an indicator of the structure of the methanogenic bacterial community.
[0043]
The primers that can be used in the third embodiment include a primer set using a set of oligonucleotides consisting of each nucleotide sequence of SEQ ID NO: 3 and SEQ ID NO: 2, and each nucleotide sequence of SEQ ID NO: 3 and SEQ ID NO: 4. And a set of oligonucleotides consisting of
[0044]
As a method for extracting and purifying a nucleic acid sample from a sludge sample to apply the present detection method, any known means can be used.
The PCR conditions vary depending on the test sample and the like, and in any case, can be appropriately selected based on common general knowledge in the technical field or empirical rules that can be recognized by those skilled in the art. Preferred PCR conditions are, for example, those described in the following Examples.
[0045]
As used herein, the term "community structure of methanogens" refers to the state of existence that is grasped by the classification and abundance of methanogens that inhabit environmental samples such as methane fermentation sludge. Specifically, it is composed of a relatively small number of members, but it is defined by the taxonomic position and the number of bacteria of each group of methanogens that are phylogenetically diverse. Further, in the present invention, it corresponds to the distribution of the migration band group appearing on the electrophoresis gel (variation in migration distance and thickness of each migration band).
[0046]
The term "environmental sample" or "sludge sample" referred to in the present specification means an organic matter-containing sample having a microbial reaction system such as methane fermentation sludge which is expected to purify or assimilate organic matter, Typically, a BOD of about 2000 mg / L to about 10,000 mg / L and / or a COD of about 6000 mg / L to about 30,000 mg / L._Cr(Oxygen consumption by potassium bichromate) and a sludge sample under high-temperature anaerobic conditions suitable for methane-producing bacteria to perform methane fermentation. The term “nucleic acid sample” refers to a sample containing DNA and / or RNA derived from microorganisms present in the environmental sample and prepared so as to be used for PCR. In particular, 16S rRNA derived from microorganisms is used for PCR. Is a sample that can be used as a template for
[0047]
The expression "base sequences are substantially homologous" as used herein for an oligonucleotide means that the oligonucleotide has homology to a fragment length that can function as a PCR primer or the like.
[0048]
For example, the primer does not necessarily need to be designed from a site having 100% homology depending on the purpose of use and conditions, and may differ by several bases near the 5 ′ end of the primer in the target region. . Even if such a primer is used, the target DNA fragment can be appropriately amplified by examining the annealing temperature and the like. That is, if high specificity is required in practicing the present invention, completely homologous sequence sites (typically, the nucleotide sequences of SEQ ID NOs: 1 to 4) are used and only those sequences are used. It is sufficient to select PCR conditions that do not anneal. On the other hand, if relatively low specificity conditions are allowed, a PCR that uses a sequence several nucleotides different from the base sequence of SEQ ID NOs: 1 to 4 and still anneals is used. What is necessary is just to select conditions.
[0049]
Whether or not the primers are substantially homologous or complementary can be determined, for example, by the above-mentioned probe check program published on the Internet (The RDP-II: Ribosomal Database Project, Maidak BL, Cole JR, Lilburn TG). , Parker CT Jr, Saxman PR, Farris RJ, Garrity GM, Olsen GJ, Schmidt TM, Tiedje JM., Nucleic Acids Res 2001 Jan Jan; 29 (1);
[0050]
Embodiment 1
Primer design
In order to search for a consensus sequence found on 16S rDNA of methanogens, the 16S rDNA nucleotide sequences of various taxonomic methanogens were analyzed from a phylogenetic standpoint.
[0051]
FIG. 1 shows the results of 16S rDNA nucleotide sequence analysis of methanogenic bacteria, in which the nucleotide sequences of the consensus regions found are listed.
As shown in the figure, as a result of the analysis, four target regions named M85F, AP5F, AP7R, and M1350R were selected in order from the 5 'end of the 16S rDNA, and a consensus sequence in each of these regions (see FIG. (The four base sequences arranged at the bottom of the sequence). The primers actually synthesized include a set of oligonucleotides complementary to the M85F and M1350R regions, respectively (described in SEQ ID NO: 1 and SEQ ID NO: 2, respectively), and a pair of oligonucleotides complementary to the AP5F and AP7R regions, respectively. Set of oligonucleotides (described in SEQ ID NO: 3 and SEQ ID NO: 4, respectively). For example, each nucleotide position of the primersMethanobacterium formulamicumThe base number in the 16S rDNA base sequence (1476 bp; GenBank accession number M36508) is as follows.
SEQ ID NO: 1 (M85F): 86-103
SEQ ID NO: 2 (M1350R): 1332-1349
SEQ ID NO: 3 (AP5F-GC): 1167-1188
SEQ ID NO: 4 (AP7R): 1329-1349
[0052]
FIG. 2 shows a phylogenetic tree of methanogenic bacteria. As is apparent from the figure, the primer design is based on 16S rDNA nucleotide sequence information obtained from the details of the phylogenetic tree and from a wide variety of methanogens. In this way, it was intended to have a global specificity covering all species, strains or species levels that could be classified as methanogens.
[0053]
According to a first embodiment, from methane fermentation sludge 16S rDNA Fragment detection and cloning
A DNA sample is extracted and purified from a test sample collected from methane fermentation sludge by QIAamp DNA Tool Mini Kit, and a primer set consisting of a set of base sequences of SEQ ID NO: 1 and SEQ ID NO: 2 is directly applied to the DNA sample. The 16S rDNA gene in the DNA sample was subjected to PCR amplification (reaction conditions: 94 ° C for 3 min- (94 ° C for 1 min, 60 ° C for 1 min, 72 ° C for 2 min) x 30 cycle- 72 ° C for 3min-4 ° C). After electrophoresis of the amplified fragment on an agarose gel, a band containing the DNA fragment of the target gene was cut out. The excised DNA fragment was purified, and TA cloning was performed using pT7 Blue T-Vector.
[0054]
Cloning was performed in E. coli DH5α, and color selection was performed using a selective medium containing ampicillin, IPTG, and X-gal. A white colony formed on the selection medium was cultured overnight at 37 ° C. in a liquid LB medium containing ampicillin, and then transformed with QIAGEN QIAprep spin Miniprep Kit. A plasmid was extracted from E. coli DH5α. Using this plasmid as a template, the ABI DNA sequencing Kit (BioDye^TM  After PCR by Terminator Cycle Sequencing Ready Reaction, an excess Dye terminator was removed using Edge Bio Systems (PERFORMA DTR Gel Filtration Cartridges), and a capillary sequencer was used.
[0055]
The cloned sequences incorporated into these plasmids are representative of the target 16S rDNA fragment derived from the same kind of bacteria, as exemplified in Example 3 below.
[0056]
Using a primer set consisting of a set of base sequences of SEQ ID NO: 1 and SEQ ID NO: 2, a DNA sample extracted from a test sample was used as a template to amplify by PCR, and the amplified fragment was subjected to electrophoresis on agarose gel. As a result, a band having a high intensity level was detected at a position showing the same length as the expected length of the amplified fragment of about 1265 bases. In addition, although non-specific amplification was hardly observed, some DNA samples in which about two bands with extremely low intensity levels were detected were also found.
[0057]
As a result of phylogenetic analysis of the obtained cloned sequence, the base sequences were all sequences belonging to Archaea, and no sequence belonging to Bacteria was detected so far. The nucleic acids belonging to Archaea detected so far are as follows:Methanoculleus , MethanospirillumOrMethanosarcina , MethanobacteriumClassified and sequences belonging to a number of other Uncultured archaeons were also positioned at least in clusters belonging to methanogenic bacteria.
[0058]
In this way, the 16S rDNA gene derived from various methanogens in the test sample is specifically and comprehensively obtained by the PCR method using the primer set consisting of a set of base sequences of SEQ ID NO: 1 and SEQ ID NO: 2. Amplified and identified.
[0059]
Embodiment 2
According to the second embodiment PCR-DGGE analysis
A DNA sample is extracted and purified from a test sample collected from methane fermentation sludge by QIAamp DNA Tool Mini Kit, and a primer set consisting of a set of base sequences of SEQ ID NO: 3 and SEQ ID NO: 4 is directly applied to the DNA sample. The 16S rDNA gene in the DNA sample was PCR-amplified (reaction conditions: 94 ° C. for 3 min- (94 ° C. for 1 min, 60 ° C. for 1 min, 72 ° C. for 2 min) × 30 cycle-72 ° C. for 3 min-4 ° C.). The product was electrophoresed by the DGGE method. FIG. 3 shows an example of the DGGE migration result.
[0060]
It should be noted that PARCH340f and PARCH519r (Ovreas L, Forney L, Daae FL, Torsvik V: Distribtion of edacton ionica bacterioplankton internatio tank erroneo prékété s ｉ ＤＮＡ ＤＮＡ ＤＮＡ ＤＮＡ ＤＮＡ ＤＮＡ ＤＮＡ ＤＮＡ ＤＮＡ ＤＮＡ ＤＮＡ ＤＮＡ ＤＮＡ ＤＮＡ ＤＮＡ ＤＮＡ ＤＮＡ ＤＮＡ ＤＮＡ ＤＮＡ 、 な お ＤＮＡ な お な お な お) PCR amplification was performed using gel electrophoresis of PCR-amplified gene fragments coding for 16S rRNA, Applied Environmental Microbiology, 63 (9), 3367-3373, 1997). No amplified fragment as described above was obtained. On the other hand, it was obtained by PCR (reaction conditions: 94 ° C. 3 min- (94 ° C. 1 min, 60 ° C. 1 min, 72 ° C. 2 min) × 30 cycle-72 ° C. 3 min) using the primer sets of SEQ ID Nos. 3 and 4. Electrophoresis of the PCR product on an agarose gel revealed a single band with a high intensity level near the expected amplified fragment length of about 200 bases (not shown).
[0061]
In the results of the above-mentioned PCR product electrophoresed by the DGGE method (see, for example, FIG. 3), a large difference was observed in the number of bands between DNA samples from fermenters under different operating conditions, and DNA samples from which a relatively large number of bands were obtained were obtained. And a DNA sample from which only a small number of bands were obtained. The separation between the bands was good.
[0062]
Embodiment 3
Two steps according to the third embodiment PCR-DGGE analysis
As described in Example 1 above, the PCR of the second embodiment was performed using the plasmid into which the 16S rDNA long chain fragment obtained by the PCR of the first embodiment was incorporated as a template. As a result, a short chain portion was selectively amplified from the cloned 16S rDNA long chain inserted in each plasmid to obtain a clone-derived PCR product (reaction conditions: 94 ° C. 3 min- (94 ° C. 1 min, 60 ° C. 1 min, 60 ° C.) 1 min, 72 ° C. 2 min) × 30 cycle-72 ° C. 3 min-4 ° C.). Next, the clone-derived PCR product was subjected to electrophoresis.
[0063]
As a result of subjecting the clone-derived PCR product to agarose gel electrophoresis, a single band having a high intensity level was observed around the expected amplified fragment length, and the clone-derived PCR product was analyzed by DGGE. One band was seen.
[0064]
When the DGGE of the clone-derived PCR product is electrophoresed, the PCR product obtained by using the primer set of the second embodiment also directly on the DNA sample derived from the methane fermentation sludge sample along with the clone-derived PCR product (Hereinafter referred to as “hybrid PCR product”). In this way, it was possible to determine which of the multiple bands actually obtained from the hybrid PCR product the clone-derived PCR product corresponds to.
[0065]
From the results of sequencing and phylogenetic analysis of each 16S rDNA long chain fragment, it was found that bands detected at the same position had almost the same sequence of methanogenic bacteria. In addition, it was found that bands detected at different positions had almost different sequences of methanogens. In this manner, the association between the DGGE results of the cloned DNA samples derived from the respective methanogen groups and the bands separated by DGGE of the mixed DNA samples derived from the methane fermented sludge sample is not problematic. Could be done.
[0066]
In addition, from the results of DGGE on the cloned sample performed as described above, methane generation that could not be detected by DGGE electrophoresis on the hybrid PCR product derived from methane fermentation sludge using the same primer set could not be detected. Bacteria could also be detected.
[0067]
As in this example, the PCR according to the first embodiment, in which the 16S rDNA long chain of the methanogen can be comprehensively acquired and phylogenetic analysis can be efficiently applied, and the base sequence information on the 16S rDNA short chain By combining with the PCR-DGGE of the second embodiment, which can detect the distribution of the migration band reflecting the difference of the above, the type and the number of the methanogenic bacteria in the environmental sample can be clarified.
[0068]
In addition, the various conditions regarding PCR and electrophoresis applied in the above-mentioned Example are as follows.

In the PCR reaction solution composition, each primer contains 1 pmol.

The PCR product used was a solution obtained by extracting DNA from an agarose gel and purifying the solution after the PCR reaction.
[Sequence PCR reaction solution]
The following composition was prepared so that the total amount was 20 μm.

[PCR reaction conditions]
96 ° C 30sec- (96 ° C 10sec-50 ° C 5sec-60 ° C 3min) x 30cycle- (96 ° C 10sec-60 ° C 10sec-72 ° C 1min) x 15 cycle-4 ° C
[DGGE electrophoresis conditions]
Gel used: 10% polyacrylamide gel
Denaturing concentration gradient: 20-70%
Electrophoresis at 200V constant voltage for 3 to 3 1/2 hours
After DGGE electrophoresis, staining was performed with Vista green nucleic acid, and a band was detected with FluorImager 595.
[0069]
Embodiment 4
Analysis of community structure of methanogens
As described above, by developing the PCR amplification product of the 16S rDNA gene derived from all methane-producing bacteria in methane-fermented sludge on a DGGE gel, the basic data obtained by visualizing the community structure of the methane-producing bacteria is obtained. Is obtained. Then, by periodically recording this kind of index data for a specific methane fermentation tank, it is possible to track changes in the type and the number of methanogens in the fermentation tank. Such tracing reveals a correlation between changes in the type and number of methanogens and the observed changes in fermenter conditions.
[0070]
Therefore, the DGGE electrophoresis results according to the present invention can be used as index data for diagnosing the state of the methane fermentation tank and for providing a guide for controlling the operating conditions and the like. That is, this kind of video data is index data that quickly and directly captures changes in the community structure of methanogens that affect the decomposition activity in the fermenter. By acquiring and inspecting such index data, it is possible to observe changes in the actual state of the fermenter (for example, fluctuations in the amount of methane gas generated) that will change following changes in the bacterial community structure. This makes it possible to make quick and accurate situation judgments rather than making judgments from the beginning. For example, when the operation state of the fermenter deviates from an ideal state, the DGGE electrophoresis result obtained by tracking is recorded as index data indicating the deterioration of the state, and then the DGGE electrophoresis result is inspected. At this time, by referring to the index data, deterioration of the state of the fermenter can be predicted in advance, and a countermeasure can be taken.
[0071]
In this example, the amount of methane gas generated in the methane fermentation tank was measured within a certain period, and the migration band obtained by PCR-DGGE was recorded for a DNA sample periodically collected from the methane fermentation tank.
[0072]
FIG. 4 shows the amount of generated methane gas and the corresponding electrophoretic photograph in comparison. As shown in the figure, in the fermenter where the amount of methane gas generated fluctuates, the number and thickness of the bands appearing in each electrophoresis photograph also tend to change over time. It was found that the visual change was visually grasped.
[0073]
In the data of FIG. 4, for example, phase I (12/10 to 2/25) in which methane gas generation is relatively unstable, phase II (3/4 to 5/13) in which it is relatively stable, Stage III (from 5/20) with a sharp rise is seen. When the changes in the amount of generated gas are compared with the migration results at each stage, migration bands having a morphology unique to each stage appear. I knew it was.
[0074]
【The invention's effect】
As described above in detail, according to the present invention, it is possible to comprehensively detect methanogens present in an environmental sample, and obtain index data on the methanogen community structure if necessary. be able to.
[0075]
[Sequence list]

[Brief description of the drawings]
FIG. 1 shows the results of 16S rDNA nucleotide sequence analysis of methanogens.
FIG. 2 shows a phylogenetic tree of methanogenic bacteria.
FIG. 3 shows an example of a DGGE electrophoresis photograph according to an example.
FIG. 4 shows the amount of generated methane gas and the corresponding migrating band over time.

Claims (6)

A primer set comprising a set of oligonucleotides according to (a) or (b) below:
(A) an oligonucleotide having the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence substantially homologous thereto, and an oligonucleotide having a nucleotide sequence of SEQ ID NO: 2 or a nucleotide sequence substantially homologous thereto; or (b) SEQ ID NO: An oligonucleotide having the nucleotide sequence of SEQ ID NO: 3 or a nucleotide sequence substantially homologous thereto, and an oligonucleotide having the nucleotide sequence of SEQ ID NO: 4 or a nucleotide sequence substantially homologous thereto. A method for detecting a methanogen, wherein a PCR method is performed on a nucleic acid sample derived from a methanogen in an environmental sample using any of the primer sets according to claim 1, wherein the PCR is performed. A method comprising detecting an amplification product obtained by the method. A first PCR method is performed on a nucleic acid sample derived from a methanogen in an environmental sample using the primer set according to claim 1 (a), and obtained by the first PCR method. The method according to claim 2, further comprising performing a second PCR method on the obtained amplification product using the primer set according to claim 1 (b). The method according to claim 2, further comprising performing a PCR method using the primer set according to claim 1 (b), and then subjecting the amplification product obtained by the PCR method to denaturing gradient gel electrophoresis. 3. The method according to 3. A method for evaluating an environmental sample, comprising using a distribution of electrophoretic bands obtained by the electrophoresis according to the method according to claim 4 as index data for a community structure of methanogens present in the environmental sample. The method according to any one of claims 2 to 5, wherein the environmental sample is methane fermentation sludge.

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