JP4375607B2 - Oligonucleotides specific for Clostridium bifermentans DPH-1 strain and use thereof - Google Patents

Description

【図面の簡単な説明】
【図１】 MY培地の組成を示す図である。
【図２】 16SrDNAの増幅に用いたプライマーの塩基配列を示す図である。
【図３】 16SrDNA増幅のためのPCR条件を示す図である。
【図４】 DPH-1株に特異的な４つのプライマーの塩基配列を示す図である。
【図５】プライマー評価のためのPCR条件を示す図である。
【図６】定量的PCRの反応条件を示す図である。
【図７】 C.paraputrificum strain M21用の培地組成を示す図である。
【図８】 Bacillus sp.用の培地組成を示す図である。
【図９】大腸菌（E.coli）用の培地組成を示す図である。
【図１０】プライマーセット（1130F＋1294R）による16SrDNAの増幅を解析するための電気泳動の結果を示す写真である。
【図１１】リアルタイム定量的PCRによる標準曲線の一例を示す図である。
【図１２】プライマーセット(1130F+1294R)のDPH-1株に対する特異性を調べた結果を示す図である。
【図１３】 DPH-1株と他の微生物とを混合したサンプルからDNAを抽出し、プライマーセット(1130F+1294R)を用いて定量的PCRを行なった結果を示す図である。
【図１４】 FISH法に用いた３つのプローブを構成するオリゴヌクレオチドの塩基配列を示す図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an oligonucleotide specific for a Clostridium bifermentans DPH-1 strain, a detection method for a Clostridium bifermentans DPH-1 strain, a quantification method, and the like.
[0002]
[Prior art]
In recent years, there has been a problem that groundwater, soil, and the like are contaminated with heavy metals and organic compounds. Of particular concern is contamination by organochlorine compounds such as trichlorethylene (TCE) and tetrachloroethylene (PCE), which are toxic and pointed out as hazardous substances by the Water Pollution Control Act. These organochlorine compounds have been used as solvents useful in various industries for more than 30 years as cleaning solvents for cleaning, degreasing cleaning of metal parts, raw materials for products, and the like. To date, PCE and TCE are known to have the following toxicities. Acute toxicity includes central nervous system depression, skin disorders, kidney and liver disorders, etc. Chronic toxicities when a small amount is ingested for a long time include finger paralysis, respiratory and heart damage, visual and auditory disorders, etc. . Carcinogenicity has also been pointed out, and reported cancer involvement includes liver cancer, esophageal cancer, cervical cancer, non-Hodgkin lymphoma, and the like. It has also been found that PCE and TCE are decomposed into cis-1,2-dichloroethylene (cDCE) by the action of microorganisms when leached into soil.
[0003]
There are two types of PCE artificial purification methods: physicochemical methods and biological methods. In physicochemical methods, “soil gas suction technology” and “groundwater pumping aeration technology” that take advantage of PCE's tendency to volatilize have been developed and put into practical use. As biological methods, bioremediation for purifying contaminated environments using microorganisms and phytoremediation using plants are considered. The former bioremediation basically covers organic pollutants that can be decomposed and converted, but if the metal itself must be removed, such as heavy metals, it must be extracted from the soil and concentrated. Must not. In such a case, the latter phytoremediation can be used. Heavy metals and other substances concentrated in plants by phytoremediation must be burned, removed, and finally disposed of.
[0004]
Bioremediation is classified into three methods according to the form of microbial utilization. The first method is called biostimulation, and it is already inhabited in the contaminated site by adding inorganic nutrients such as phosphorus and nitrogen, substrates serving as energy sources, and blowing in oxygen. It activates and purifies the degradation ability of the pollutant-degrading microorganisms. The second method is called bioaugmentation, and is a method in which purified microorganisms that have been cultured purely or a group of decomposed microorganisms that have been cultured in culture are introduced from the outside for purification ([Non-Patent Document 1]). reference). The third method is called natural attenuaion, which monitors the extent to which it is decomposed by letting it be decomposed by the natural purification action. Such bioremediation techniques are currently classified into the following four types according to the process used.
● Solid phase bioremediation
A method to excise and pile up contaminated soil, ventilate air or oxygen, add water and nutrients, and increase the activity of aerobic microorganisms in the soil to purify it. Represented by biopile effective for purification of oil pollution.
● Slurry phase baioremediation
A method in which water is added to contaminated soil to form a slurry, which is transferred into a reaction layer, decomposed microorganisms and nutrients are added, and the mixture is stirred and mixed. Suitable when the pollutant is persistent and high in concentration.
● In situ bioremediation
A method to increase the activity of microorganisms by injecting nutrients such as nitrogen and phosphorus, organic matter, oxygen, and if necessary, decomposing microorganisms into soil contaminated with organic substances.
● Bioreactor
A method of pumping contaminated groundwater and treating it on the ground using a reactor. Applicable to wastewater and exhaust gas treatment.
[0005]
In order to perform bioremediation efficiently, it is necessary to know the actual condition of the contamination site (the extent of contamination, the amount and concentration of contaminants, and the presence or absence of degrading bacteria), and decide the treatment method based on the results. There is a need. For this reason, there is a strong demand for the establishment of a detection and quantification method for degrading bacteria that degrade PCE and other harmful substances.
[0006]
At present, DNA and the like can be extracted and amplified from all microorganisms in the environment, and molecular biological techniques are used for analyzing microbial communities. The basis is the analysis of 16S ribosomal DNA (16SrDNA). By determining the sequence of 16S rDNA, information on each species can be obtained, so it has become a powerful document for biological system evolution and classification research and has been compiled into a database (Ribosomal Database Project II: http: // rdp. cme.msu.edu.html /). The analysis of microbial communities using these molecular biological methods can be broadly classified into two purposes: phylogenetic analysis using PCR (Polymerase Chain Reaction) and detection of specific species based on genetic analysis.
The former method using phylogenetic analysis is a method in which gene fragments of mixed microbial communities in environmental samples are amplified and separated, and then the microbial strains existing in the environment are inferred from the cloned base sequences. PCR-DGGE (Denatureing Gragient Gel Electrophoresis) method in which gel is subjected to electrophoresis with a denaturant gradient and separated, T-RFLP (Terminal-Restrictuin) which detects PCR fragments by restriction enzyme treatment Fragment Length Polymorphisms) method, and quantitative PCR method for imparting quantification to PCR reaction itself (see, for example, [Non-Patent Document 2] etc.).
Methods based on the latter gene analysis include a FISH (Fluorescent in situ Hybridization) method in which a specific species is detected using an oligonucleotide specific to the gene sequence of each microorganism. This method is a method in which a microorganism is labeled with a fluorescent dye without observing the detection and detection without extracting DNA from the microorganism. It is superior to the method using the PCR method in that microorganisms can be observed without being affected by DNA extraction or PCR amplification efficiency.
[0007]
Conventionally, Clostridium bifermentans strain DPH-1 (Clostridium bifermentans strain DPH-1) is known as one of the powerful microorganisms that have a function of degrading PCE (see [Non-patent Document 3]). ).
[0008]
[Non-Patent Document 1]
Nobuo Kaki, Kazuya Watanabe, Safety assessment of oil pollution bioremediation, Journal of Biotechnology, 80, 527-529 (2002)
[Non-Patent Document 2]
Katsuji Tani, Masahiro Muneta, Kanji Nakamura, Katsutoshi Shibuya, and Masao Nasu Monitoring of Ralstonia eutropha KT1 in Groundwater in an Experimental Bioaugmentation Field by In Situ PCR Appl.Envrion.Microbiol., 68,412-416,2002
[Non-Patent Document 3]
Chang Y. C. Hatsu, K. Takamizawa, Isolation and characterization of a tetrachloroethylene dechlorination bacterium, Clostridium bifermentans DPH-1. , J. Biosci. Bioeng, 89, 489-491, 2000
[0009]
[Problems to be solved by the invention]
The present invention establishes a method for more easily detecting and quantifying the Clostridium bifermentans DPH-1 strain by using an oligonucleotide specific for the aforementioned Clostridium bifermentans DPH-1 strain. It is an object to do.
[0010]
[Means for Solving the Problems]
  In order to solve the above problems, the inventions described in the following (1) to (5) are configured.
(1) Sequence listingSEQ ID NO: 1 and SEQ ID NO: 3The nucleotide sequence described inConsist ofAnd an oligonucleotide capable of selectively amplifying a specific region of 16S rDNA of Clostridium bifermentans DPH-1 strain by functioning as a primer in the PCR methodset.
(2) A method for detecting Clostridium bifermentans DPH-1 strain present in a specimen,
  The oligonucleotide according to (1)setA first step of trying to amplify DNA present in the specimen by functioning as a primer in the PCR method;
And a second step of determining that the Clostridium bifermentans DPH-1 strain is present in the sample when the DNA concentration in the sample increases in the first step. A method for detecting the Clostridium bifermentans DPH-1 strain.
(3) A method for quantifying Clostridium bifermentans DPH-1 strain present in a specimen,
  The oligonucleotide according to (1)setA first step of predetermining the correlation between the initial DNA concentration of the DPH-1 strain and the number of cycles when the threshold value is exceeded by functioning as a primer in a quantitative PCR method,
  Microbial DNA present in the sample is amplified by quantitative PCR using the primers used in the first step, and the number of cycles when the amount of amplified DNA exceeds a specific threshold is determined. A second step;
  The initial DNA concentration of the DPH-1 strain present in the sample is determined in the second step based on the correlation between the initial DNA concentration determined in the first step and the number of cycles when the threshold is exceeded. And a third step of calculating back from the number of cycles performed, and a method for quantifying the Clostridium bifermentans DPH-1 strain.
(4) of the sequence listingSEQ ID NO: 8The nucleotide sequence described inConsist ofIn addition, an oligonucleotide capable of selectively hybridizing to a specific region of 16S rDNA of Clostridium bifermentans DPH-1 strain by functioning as a probe in the FISH method.
(5) A method for detecting Clostridium bifermentans DPH-1 strain present in a specimen,
  A first step of attempting to hybridize with DNA present in the specimen by causing the oligonucleotide according to (4) to function as a probe in the FISH method;
  A second step of determining that the Clostridium bifermentans DPH-1 strain is present in the sample when hybridization with DNA present in the sample is confirmed in the first step; A method for detecting a Clostridium bifermentans DPH-1 strain characterized by comprising:
[0011]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, embodiments of the present invention are roughly divided into two sections: “1. Analysis by real-time quantitative PCR method” and “2. Detection of Clostridium bifermentans strain DPH-1 by FISH method”. Explained.
[0012]
1. Analysis by real-time quantitative PCR
[Principle of PCR method]
First, the principle of the PCR method and quantitative PCR method will be briefly described.
The PCR (Polymerase Chain Reaction) method is a technique for amplifying a specific DNA sequence in a large amount in a short time. The principle is that denaturation of double-stranded DNA becomes single-strand, annealing that binds an oligonucleotide primer having a sequence complementary to single-stranded DNA, and elongation that performs DNA elongation reaction. A reaction consisting of three steps of extension (extension) is defined as one cycle, and several tens of cycles are performed to amplify the target DNA sequence.
Theoretically, since one copy of a gene doubles in one cycle, it becomes 2n copies in n cycles. However, in reality, the amplification efficiency is poor at the beginning of the cycle and the PCR reaction efficiency decreases (plateau effect) due to enzyme deactivation or substrate consumption, so the amplified copy number is lower than the theoretical value and the initial template amount is I, the product When the amplification factor of E is E and the number of cycles is n, the following [Equation 1] is obtained.
y = I × Eⁿ(1 ≦ E ≦ 2) ・ ・ ・ ・ ・ ・ [1 set]
There are two analysis methods in the quantitative PCR method. The first is to analyze the amount of reaction product during the exponential increase period, using the feature that the reaction product increases exponentially to a certain amount in the PCR reaction and then reaches a plateau. How to calculate The second method is to determine the number of PCR cycles (Ct) in which the reaction product amount exceeds a certain threshold (threshold) by monitoring the reaction product in real time. In either analysis method, it is necessary to perform PCR by changing the amount of DNA at a known concentration, analyze the reaction product at each cycle number, and determine a quantitative PCR cycle number range from its kinetics. . Based on the result, the abundance of the target gene in an unknown sample is estimated.
[0013]
[DNA extraction from co-test strains]
Extraction of DNA from Clostridium bifermentans DPH-1 strain (hereinafter sometimes simply referred to as DPH-1 strain) was performed as follows.
Place 95 ml of MY medium in a 125 ml vial, and add vitamin solution (ρ-aminobenzoic acid 0.1 mg / 100 ml and biotin 0.0001 mg / 100 ml) and iron solution (iron sulfate heptahydrate 0.02 g / l) each. % (v / v) was added, and 3% (v / v) of the DPH-1 strain culture broth confirmed to have been degraded by PCE was inoculated to a total volume of 100 ml, and left to stand in a 37 ° C incubator for 1 day. . The composition of the MY medium is as shown in FIG.
The prepared culture solution (100 ml) was centrifuged (8,000 rpm, 10 min, 4 ° C.), and the cells were pelleted. After adding 6 ml of TE buffer (Tris 10 mM, EDTA 2NA 1 mM, pH 8.0) and resuspending, freeze-and-thaw cycle (freeze at -80 ° C and immediately at 50 ° C to increase the permeability of the cell membrane. Melting) was repeated 5 times. 10 mg of lysozyme (Lysozyme) was added and stirred gently (it is better not to use a vortex mixer. The same applies to the following stirring steps). 750 μl of 10% SDS and 75 μl of 20 mg / ml protenase K were added, stirred and incubated at 37 ° C. for 2 hours. 2.7 ml of 5M NaCl and 2.25 ml of CTAB / NaCl (270 mM CTAB (cetyltrimethylammonium bromide), 70 mM NaCl) were added, stirred, and then incubated at 65 ° C. for 20 minutes. The reaction solution was allowed to stand at room temperature, an equivalent amount of CIA (Chloroform: Isoamyl alcohol = 24: 1) was added, and the mixture was gently stirred for 1 hour. Centrifugation (8,000 rpm, 10 min, 4 ° C.) was performed, and the supernatant was separated into another centrifuge tube. The same amount of PCI (natural Phenol: Chloroform: Isoamyl alchol = 25: 24: 1) as the collected supernatant was added and stirred, and the supernatant was separated into another centrifuge tube. To the collected supernatant Xml, 10% (v / v) of 0. Xml of 3M sodium acetate (pH 5.2) was added and stirred, and then an equal amount of isoamyl alcohol was added and left at room temperature for 10 minutes. Centrifugation (12,000 rpm, 10 min, 4 ° C.) was performed, and the supernatant was removed. 3 ml of cooled 70% ethanol was added and gently stirred to wash the inside of the centrifuge tube. Centrifugation (12,000 rpm, 2 min, 4 ° C.) was performed, and the supernatant was removed. The pellet was dried using a desiccator, 1.5 ml of TE buffer was added, and the mixture was allowed to stand overnight at 4 ° C. to dissolve.
[0014]
[Confirmation of extracted DNA by electrophoresis]
Electrophoresis sample containing 1 μl of extracted DNA (DNA loading buffer (-XC: xylene cyanol) 2 μl, ddH₂After preparing an electrophoresis agarose gel (0.7% agarose, 0.5 μl / ml ethidium bromide) in an electrophoresis tank, 10 μl of λEcoT14Idigest as a molecular weight marker and 12 μl of a sample were placed in a well. Electrophoresis was performed at 100 V for 30 minutes, and image analysis was performed using UV transilluminator FASIII (Toyobo Co., Ltd.).
[0015]
[RNA treatment]
Since the obtained DNA contained RNA, RNA was removed. 1% (v / v) of 10 mg / ml RNase A was added to the DNA obtained by the above operation and incubated at 37 ° C. for 1 hour. Thereafter, an equal amount of PCI was added and gently stirred for 5-10 minutes. Centrifugation (13,000 rpm, 5 min, 4 ° C.) was performed, and the aqueous phase was separated into a new Eppendorf tube. Add 0.1Xml of 3M sodium acetate equivalent to 10% (v / v) to isocratic water phase and add isoamyl alcohol in an amount equal to it, and let stand at room temperature for 2 minutes, then centrifuge (13,000rpm , 5 min, 4 ° C.), and the supernatant was removed with a micropipette. After adding 1 ml of 70% cold ethanol and stirring so as to wash the inside of the Eppendorf tube, the mixture was centrifuged (13,000 rpm, 2 min, 4 ° C.), and the supernatant was removed with a micropipette. The obtained pellets were dried with a freeze centrifugal dryer, an equal amount of TE buffer was added, and the mixture was allowed to stand overnight to be dissolved.
[0016]
[PEG precipitation]
Polyethylene glycol (PEG) is a high-molecular ether alcohol having a structure of H- (O-CH2-CH2) n-OH, and there are those having various molecular weights depending on the degree of polymerization n. PEG is thought to promote precipitation of DNA molecules by precipitating DNA hydration water and causing precipitation. At this time, RNA is more hydrophilic than DNA by the hydroxyl group at the 2-position of ribose compared to DNA. Therefore, aggregation does not occur easily and precipitation does not occur, so that RNA can be removed together with the solution.
Add an equal amount of PEG solution (13% polyethylene glycol (PEG8000; average molecular weight 7,000-9,000), 1.6M NaCl) to Rnase-treated DNA sample, stir well, and stand overnight at 4 ° C in ice did. Thereafter, the mixture was centrifuged (13,000 rpm, 2 min, 4 ° C.), and the supernatant was removed with a micropipettor. After adding 1 ml of 70% cold ethanol and stirring so as to wash the inside of the Eppendorf tube, the mixture was centrifuged (13,000 rpm, 2 min, 4 ° C.), and the supernatant was removed with a micropipette. The obtained pellets were dried with a freeze centrifugal dryer, an equal amount of TE buffer was added, and the mixture was allowed to stand overnight to be dissolved.
[0017]
[Measurement of DNA concentration]
When measuring the concentration of a nucleic acid solution (here, DNA), since the amount of sample to be measured is extremely small, a sample solution diluted to an appropriate concentration is measured as a sample solution. Usually, a nucleic acid solution is measured with a spectrophotometer at a short wavelength of 260 nm. At this time, since protein contamination in the solution cannot be ignored, it is necessary to measure the absorbance at 280 nm at the same time. Measured O.D.₂₆₀ (assuming q) O.D.₂₈₀(p) can be used to confirm the contamination of protein and phenol in the solution by the following [formula 2]. This is disclosed, for example, in the document “Hiroki Nakayama, Bio-Experiment Illustrated 1, 1995, Shujunsha”.
p / (r-q) = 1.8 to 2.0 ... [2 formulas]
(If 1.8 ≦, protein and phenol mixed)
In addition, since the extinction coefficient varies depending on the properties of the nucleic acid used as the sample, it is necessary to consider an extinction coefficient suitable for the properties of the nucleic acid in the sample. In the case of double-stranded DNA, the extinction coefficient is 0.05, and the concentration unit at that time is μg / μl. Therefore, the nucleic acid concentration is obtained by [Formula 3].
Nucleic acid concentration (μg / μl) = O.D. Obtained₂₆₀× Dilution factor × 0.05 [3 formulas]
Using a quartz cell for nucleic acid measurement (capacity 400 μl) and TE buffer, calibration was performed at a wavelength of 260 nm using Ultrospec 2000 (Pharmacia Biotech). Immediately after calibration, the wavelength was changed to 280 nm, and the absorbance at that time was confirmed. A DNA solution from which RNA has been removed by RNase treatment and PEG precipitation is diluted 50-fold, 100-fold, and 200-fold with TE buffer.₂₆₀, Immediately change the wavelength to 280 nm, O.D.₂₈₀Was measured. Measured O.D.₂₆₀ (assuming q) O.D.₂₈₀From the ratio of (p), the contamination of protein and phenol in the solution was confirmed, and the nucleic acid concentration was calculated.
[0018]
[Amplification of 16S rDNA]
16S rDNA was amplified using a DNA solution from which RNA was removed by Rnase treatment and PEG precipitation treatment as a template. 2 μl each of bacterial forward primer 27F and universe reverse primer 1525R adjusted to 4 μM, ddH₂A reaction system was prepared by adding 20.5 μl of O and 0.5 μl of template to a total volume of 25 μl. The base sequences of the primers used at this time are shown in FIG. 2 (SEQ ID NO: 5 and SEQ ID NO: 6). In the prepared reaction system, 25 μl of Ex Taq premix (TAKARA) was added as a DNA polymerase and PCR was performed. The PCR reaction conditions at this time are as shown in FIG. After completion of the reaction, the PCR product was purified using a spin column for nucleic acid purification. The purified PCR product was subjected to electrophoresis (provided that -BPB was used instead of -XC as the DNA loading buffer), and it was confirmed that the target gene was amplified.
[0019]
[Determination of nucleotide sequence]
In the present embodiment, the autosequencer ABI310 (Applied Biosystems) is used for determining the base sequence. PCR product 3 μl, 10 × sequencing primer 1 μl, Dye solution 1 μl, Diluent (1.5M Tris pH 8.8 267 μl, 3M MgCl₂ 3 μl, H₂O 730 μl) 3.5 μl, dimethylsulfonic acid (DMSO) 1 μl, ddH₂10.5 μl of O was mixed to prepare a reaction system with a total volume of 20 μl, and a sequence reaction was performed. The sequence reaction product was purified using a sequence reaction purification spin column in order to remove excess salts and dyes. The sequence reaction product was lyophilized using a freeze centrifugal dryer. 12 μl of Template Supplession Reagent (TSR) was added and shaken for 1 hour. Thereafter, denaturation was performed at 95 ° C. for 2 minutes, and then the sample was placed in a 0.5 ml sequencing tube, and the base sequence was determined with an autosequencer ABI310.
[0020]
[Primer design]
The base sequence obtained by the auto sequencer is the CHIMERA CHECK of Ribosomal Database Project (HYPERLINK http://www.cme.msu.edu/RDP/) http://www.cme.msu.edu/RDP/) After that, multiple alignment was performed by BLAST of National Center for Biotechnology Information (NCBI: http://www.nibi.nlm.nih.gov/), and 16S rDNA sequences of related species were selected from the results. The obtained sequence was subjected to multiple alignment by DNASIS, Seq pop and Clastal W1.6.1. The alignment sequence was visually observed, and the length of the primer and the Tm (Melting Temperature) value were noted where the different base sequences differed from the target strain and related species, and several regions of about 20 mer were selected. The optimal primer region was searched from the 16S rDNA sequence using Oligo 4.0-s. At that time, attention was paid to the Tm value and the presence or absence of a hairpin structure. In real-time quantitative PCR, it is assumed that the amplification product is amplified and increased in full length during the extension reaction, so the length of the amplified product is so that it can be completely amplified within the extension reaction time. We also paid attention to the length of the amplified product so that it was 200 bp or less. Finally, primers were designed by comprehensively evaluating the results of multiple alignments using DNASIS and NCBI BLAST, visualizing alignment sequences, and searching for optimal primer regions using Oligo 4.0-s.
[0021]
[Primer evaluation]
The designed primers were evaluated using the extracted and purified DNA of the DPH-1 strain. There are four types of designed primers, 1130F (forward), 1226R (reverse), 1294R (reverse), and 1211F (forward), and their base sequences are as shown in FIG. 4 (SEQ ID NO: 1 to SEQ ID NO: 4). ). PCR was performed using primer combinations of 1130F + 1294R, 1211F + 1294R, and 1130F + 1230R. The PCR reaction conditions at this time are as shown in FIG. After completion of the reaction, electrophoresis was performed and image analysis was performed with a UV transilluminator FASIII. The amplification products in each primer set were compared, and each primer was evaluated. After primer evaluation, DNA extraction from E. coli JM109, Bacillus sp., Methanothermobacter sp. RHT-3, C. paraputrificum, Clostridium sp. RHT-4 using the same procedure as for DPH-1 strain -Purification was performed and PCR was performed with each primer set. After completion of the reaction, electrophoresis was performed and image analysis was performed with a UV transilluminator FASIII.
[0022]
[Detection of DPH-1 by real-time quantitative PCR]
In the present embodiment, real-time quantitative PCR was performed using ABI7700 (Applied Biosystems). SYBR Green 1 was used as the detection dye. SYBR Green 1 is a substance that has the property of binding to double-stranded DNA and emitting fluorescence.
The principle of the assay using SYBR Green 1 is that it first binds to a double-stranded DNA as a template and emits fluorescence. Next, when double-stranded DNA becomes single-stranded DNA due to denaturation, SYBR Green 1 is removed from the DNA and fluorescence is attenuated. PCR products are amplified through the Annealing and extension reactions, and after the extension reaction, SYBR Green 1 binds to double-stranded DNA again, increasing the overall fluorescence.
In the ABI7700, fluorescence is recorded by a CCD camera after the reaction of each cycle is completed and plotted to quantify amplification products in real time.
[0023]
[Detection of DPH-1 strain in pure culture, preparation of standard curve]
DNA was extracted and purified from purely cultured Clostridium bifermentans strain DPH-1, and the DNA concentration was measured. A standard curve was prepared using this DNA as a sample of known concentration. For quantitative PCR, qPCR^TMMastermix for Sybr^TMGreen1 (EUROGENTEC) was used. ddH₂6.25 μl of O, 2.5 μl of 1130F and 1294R, 0.75 μl of SYBR Green1, 0.5 μl of template, 2 × reaction buffer (dNTPs (including dUTP), Hot Goldstar DNA polymerase, Uracil-N-Glycosylase 5 mM MgCl₂ 12.5 μl of ROX) was added to prepare a reaction system with a total volume of 25 μl. The prepared reaction solution was transferred to a 96-well plate, and real-time quantitative PCR was performed using ABI7700 to analyze the data. The reaction conditions for quantitative PCR at this time are as shown in FIG.
[0024]
[Primer evaluation under real-time quantitative PCR conditions]
10 ng each of C.bifermentans DPH-1, C. paraputrificum, Clostridium sp. RHT-4, E. coli, and Bacillus sp. Genomes were prepared, and real-time quantitative PCR was performed to evaluate primer specificity. .
In addition, many kinds of microorganisms live in the environment, live while coexisting, and rarely live alone. Therefore, in order to investigate how the symbiotic relationship with other microorganisms affects the results of quantitative PCR, C. bifermentans DPH-1 strain and one other microorganism culture medium are mixed together and DNA Extraction was performed. Then, real-time quantitative PCR was performed and the effect was examined. The number of each microorganism was confirmed by the plate count method using the media shown in FIG. 7 (C. paraputrificum strain M21), FIG. 8 (Bacillus sp.), And FIG. 9 (E. coli).
[0025]
〔result〕
(1) Since oligonucleotide primers and probes are required for the FISH method and real-time quantitative PCR, it is necessary to determine a region where these act specifically on the DPH-1 strain.
Therefore, in this embodiment, first, all 16S rDNAs were examined, and then a region specific to Clostridium bacteria was examined. In the present embodiment, mainly focusing on the expression region (V region: variable regions, existing from V1 to V10) in the 16S rDNA sequence, among them, V2, which is said to have many sequences specific to the bacterial genus, and bacterial species Specific regions were examined by targeting two regions V339 (339-539, E. coli numbering), which are said to have many specific sequences. As a result, several regions were obtained that were speculated to be specific for Clostridium bifermentans strain DPH-1. Based on the obtained regions, primer regions capable of amplifying these regions were examined. As a result, it was possible to design four primers that appeared to be specific for the DPH-1 strain (SEQ ID NO: 1 to SEQ ID NO: 4). As an evaluation of the designed primer, PCR was performed using a test strain, Gram-negative bacteria and other Gram-positive bacteria using a primer set (1130F + 1294R). The analysis result by electrophoresis of 16S rDNA amplified by this PCR is shown in FIG. In the photograph shown in FIG. 10, the numbers 1 to 6 and the symbol M attached to the upper side are 1: DPH-1 strain, 2: C. praputrificum, 3: Methanothermobacter sp. RHT-3, 4: Clostridium sp. RHT-4, 5: E. coli, 6: Bacillus sp., M: 100 bp marker. From this result, it was confirmed that a band of the desired size (about 150 bp) was amplified in the DPH-1 strain. Moreover, since amplification was not seen in other microorganisms, it was found that the designed primer was specific to the DPH-1 strain.
[0026]
(2) As an example of a primer set, a primer curve (1130F + 1294R) was used and a standard curve was drawn by real-time quantitative PCR. An example of the obtained standard curve is shown in FIG. The standard curve formula is
Ct = −2.969 (LogC₀) +22.428 r²= 0.9961
C₀: Initial DNA concentration
Ct: Number of cycles when the threshold is exceeded
It became. This makes it possible to amplify the DNA present in the sample using the 130F and 1294R primer sets, and to quantify the DPH-1 strain using SYBR Green 1 and the 1130F and 1294R primer sets. It turned out to be.
[0027]
That is, in the present embodiment, it was possible to establish a method for quantifying the abundance of the DPH-1 strain present in a specimen by the following steps (a) to (c).
(A) Initial DNA concentration C of DPH-1 strain by real-time quantitative PCR₀And the number of cycles Ct when the threshold value is exceeded are determined in advance in the form of a formula or graph indicating a standard curve. At this time, in the PCR reaction, an oligonucleotide having a base sequence described in any one of SEQ ID NO: 1 to SEQ ID NO: 4 that functions as a primer for specifically amplifying a specific region of 16S rDNA of the DPH-1 strain is used. As such primers, for example, the four primers (1130F, 1226R, 1294R, 1211F) described in FIG. 4 can be used.
(B) Microbial DNA present in the sample is amplified by real-time quantitative PCR using the primers used in step (a). Then, the number of cycles Ct when the amount of amplified DNA exceeds a specific threshold is determined.
(C) Initial DNA concentration C of DPH-1 strain present in the sample₀C determined in (a)₀Based on the correlation between Ct and Ct (graphs and formulas showing a standard curve), it is calculated by calculating back from the cycle number Ct determined in (b).
[0028]
(3) As an example of the primer set, the results of examining the specificity of the primer set (1130F + 1294R) for the DPH-1 strain are shown in FIG.
The DPH-1 strain was able to quantify 9.73 ng out of 10 ng, and in addition, only a small amount of Clostridium sp. RHT-4 was detected, and the others were not detected. .bifermentans DPH-1 strain was found to be detectable.
[0029]
(4) FIG. 13 shows the results obtained by extracting DNA from a sample in which the DPH-1 strain and other microorganisms were mixed, and performing quantitative PCR using the primer set (1130F + 1294R). In all cases where C. paraputrificum, Clostridium sp. RHT-4, E. coli, and Bacillus sp. Were mixed, DNA was detected. In addition, with the exception of C. paraputrificum, the level was almost the same as that of the control DPH-1 strain. Therefore, even if other microorganisms coexist, the PCR is performed using the four primers (SEQ ID NO: 1 to SEQ ID NO: 4) designed in the present embodiment, so that they are present in the specimen. It was confirmed that the DPH-1 strain could be specifically detected.
[0030]
That is, in the present embodiment, it was possible to establish a method for detecting the DPH-1 strain present in a specimen by the following steps (a) to (b).
(A) The oligonucleotide having the base sequence described in any one of SEQ ID NO: 1 to SEQ ID NO: 4 is caused to function as a primer in the PCR method to try to amplify DNA present in the sample.
(B) When the DNA concentration in the sample is increased by the operation of (a), it is determined that the Clostridial bifermentans DPH-1 strain is present in the sample.
[0031]
2. Detection of Clostridium bifermentans DPH-1 by FISH method
Hereinafter, a method for detecting the DPH-1 strain by using an oligonucleotide that specifically acts on a specific region of the DNA of the DPH-1 strain as a probe in the FISH method will be described.
[0032]
[Principle of FISH method]
The FISH (Fluorescence In Situ Hybridization) method is a method in which a specific gene sequence is used as a target and single-stranded DNA labeled with a fluorescent dye or the like is hybridized to a complementary site and directly detected under a microscope. This method is essentially a technique developed to determine the chromosomal locus. In recent years, it is used not only for determining loci but also for analyzing the localization of DNA and RNA in tissues such as bacteria. The FISH method was performed according to the method of R. I. Amann. This method is described in, for example, the literature `` Amann, RI: in situ identification of micro-organisms by whole cell hybridization with rRNA-targeted nucleic acid probe.Molecular microbial ecology manua InMOLECULAR ECOLOGY MANUAL.Edited by ADLAkkermans, JD van Elsas & RJ de Brujin Kluwer Academic Publishers, London (1995) ".
[0033]
[About immobilization of the test strain]
<Reagent>
・ 1 × Phosphate buffered saline (PBS)
3 x PBS to 1 x PBS ddH₂Dilute with O (sterilized).
3 x PBS
390 mM NaCl
30mM Na₂HPO_Four
pH 7.2
・ 4% paraformaldehyde fixative
(Pre-adjusted 20% paraformaldehyde (can be stored for 1 month, refrigerated) ddH₂Dilute to 4% with O (sterilized). Also, 20% paraformaldehyde is ddH to powder (paraformaldehyde) in the draft₂O was added and dissolved by warming at 50-60 ° C. with a few drops of 10M NaOH. )
300 μl of a fresh culture solution of DPH-1 strain cultured in MY medium was transferred to a 1.5 ml Eppendorf tube, 3 times the amount of 4% paraformaldehyde was added, and the mixture was allowed to stand overnight at 4 ° C. to be immobilized. The immobilized cells were centrifuged (8000 rpm, 15 min, 4 ° C.), and the supernatant was removed with a micropipette. 500 μl of 1 × PBS was added to the immobilized cells, resuspended, centrifuged (8000 rpm, 10 min, 4 ° C.), and the supernatant was removed with a micropipette. 10 μl of 1 × PBS was added, and the freeze-and-thaw cycle was repeated 5 times to improve cell wall permeability. An equal amount of ethanol (ice-cooled) was added and stirred well using a vortex mixer. This was used as a cell immobilization solution and stored at −20 ° C. until use.
[0034]
[Adjustment of slide glass for hybridization]
<Reagent>
・ Slide coating aqueous solution
0.1% gelatin
0.01% potassium chromium (III) sulfate
Prepare a 10% potassium chromium sulfate aqueous solution and dilute it before use.
Immerse the slide glass for hybridization with 8 holes (φ8mm) in the washing solution for 1 hour (alkali treatment), and wash the surface of the slide glass, then ddH₂Rinse with O and air dry. Using a water bath, the slide glass was immersed in an aqueous slide coating solution that had been set to 70 ° C. in advance, and gelatin coating was performed. Then it was air-dried.
[0035]
[Fixation of cells on gelatin-coated slide glass]
3 μl of the preserved bacterial cell fixing solution was added to a well of a glass slide coated with gelatin, and air-dried. After air drying, the cells were immersed for 3 minutes in the order of 50%, 80%, and 99.5% ethanol to dehydrate and fix the immobilized cells, and air-dried again.
[0036]
[Hybridization]
<Reagent>
・ Hybridization buffer
2 x Hybridization buffer (pH 7.2)
1.8M NaCl
40 mM Tris-HCl
1% SDS
x% formaldehyde
As for the hybridization buffer, prepare 2 × hybridization buffer prepared in advance as described above, and each final concentration is 1 × hybridization buffer, 0.1% SDS, x% formaldehyde ( x: eg, 5% in the case of the bacterial probe EUB338 and 35% in the case of the archaeal probe ARC915, and used as a hybridization buffer.
<Operation>
A sealed container soaked with the hybridization buffer was previously set to 62 ° C. using a water bath, and this was used as a constant temperature chamber. 9.0 μl of a hybridization buffer containing 1.0 μl of a probe adjusted to 50 ng / μl (see FIG. 14) was dropped on a slide glass on which the cells were immobilized, and moved quickly to a constant temperature chamber at 62 ° C. for 2 hours. Hybridization was performed. After this operation, all operations were performed with light shielding. After completion of the hybridization, the slide glass was taken out from the constant temperature chamber, and the probe was washed from the slide glass with a hybridization buffer preliminarily warmed at 62 ° C. to stop the hybridization. This operation was repeated once for double staining. At that time, attention was paid to the Td (dissolved temperature) value, and hybridization was performed from the one having a high Td value. The slide glass was immersed in a centrifuge tube containing 50 ml of hybridization buffer and incubated at 62 ° C. for 20 minutes. Then ddH₂The glass slide was washed with O to remove excess salt. The slide glass is air-dried, glycerol and PBS mixed at a ratio of 1: 1 and 0.1% (w / v) paraphenylenediamine is added dropwise to the slide glass well. Observation was performed with a confocal laser scanning microscope (CLSM).
[0037]
[Optimization of hybridization conditions]
In order to determine the optimum formaldehyde concentration, hybridization was performed by changing the formaldehyde concentration in the hybridization buffer to 0%, 5%, and 10%. Observation was performed using a confocal laser microscope, and the obtained images were used to evaluate the hybridization efficiency at each formaldehyde concentration.
[0038]
[Evaluation of hybridization probe]
In order to evaluate the probe designed in this experiment, hybridization was performed using the Gram-negative bacterium E. coli JM109 strain, the Gram-positive bacterium Bacillus sp., And Clostridium sp. RHT-4.
A bacterial cell immobilization solution was prepared using the above bacterial cells in the same procedure as described above, and the bacterial cells were dehydrated and fixed in the wells of gelatin-coated slide glass. Hybridization was performed by changing the formaldehyde concentration in the hybridization buffer. And it observed using the confocal laser microscope, and hybridization efficiency was evaluated using the obtained image.
[0039]
〔result〕
In the present embodiment, two probes (16SC135 and DPH302) that can specifically hybridize to a specific region of 16S rDNA of the DPH-1 strain were designed. Then, the FISH method was carried out using 16SC135 (SEQ ID NO: 7), DPH302 (SEQ ID NO: 8) and universal probe UNI1392 (SEQ ID NO: 9), which are oligonucleotide probes specific to the DPH-1 strain, and a confocal laser Bacteria were observed using a microscope. The base sequences of the oligonucleotides constituting these three probes are as shown in FIG. 16SC135 and DPH302 were labeled with FITC, and UNI-1392 was labeled with carboxyl rhodamine.
First, the optimal formaldehyde concentration of the probe was examined using the DPH-1 strain. The FISH method was performed by changing the formaldehyde concentration to 0%, 5%, and 10%. Then, double staining with UNI1392 was performed and the optimum formaldehyde concentration was determined to be 5%. The sensitivity of the probe was higher with DPH302 than with 16SC135. Next, in order to examine the specificity of the probe, FISH method was performed using Clostridium sp. RHT-4, E. coli JM109 strain and Bacillus sp. As a result, it was found that DPH302 hybridizes to DPH-1 among the test strains used this time and does not hybridize to other bacteria.
[0040]
That is, in the present embodiment, it was possible to establish a method for detecting the DPH-1 strain present in a specimen by the following steps (a) to (b).
(A) An oligonucleotide having the base sequence shown in SEQ ID NO: 7 or 8 in the sequence listing is caused to function as a probe in the FISH method, and hybridization with DNA present in the sample is attempted. As such a probe, for example, two probes (16SC135, DPH302) described in FIG. 14 can be used.
(B) When hybridization with DNA in the sample is confirmed by the operation of (a), it is determined that the Clostridium bifermentans DPH-1 strain is present in the sample.
[0041]
【The invention's effect】
As described above, according to the present invention, the Clostridium bifermentans DPH-1 strain can be more easily detected by using an oligonucleotide specific for the Clostridium bifermentans DPH-1 strain. The method and quantification method could be established.
[0042]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 is a diagram showing the composition of a MY medium.
FIG. 2 is a diagram showing the base sequence of primers used for 16SrDNA amplification.
FIG. 3 shows PCR conditions for 16S rDNA amplification.
FIG. 4 is a diagram showing the base sequences of four primers specific to the DPH-1 strain.
FIG. 5 is a diagram showing PCR conditions for primer evaluation.
FIG. 6 is a diagram showing reaction conditions for quantitative PCR.
FIG. 7 is a view showing a medium composition for C. paraputrificum strain M21.
FIG. 8 is a diagram showing a medium composition for Bacillus sp.
FIG. 9 is a diagram showing a medium composition for E. coli.
FIG. 10 is a photograph showing a result of electrophoresis for analyzing amplification of 16S rDNA by a primer set (1130F + 1294R).
FIG. 11 shows an example of a standard curve obtained by real-time quantitative PCR.
FIG. 12 shows the results of examining the specificity of the primer set (1130F + 1294R) for the DPH-1 strain.
FIG. 13 is a view showing the results of quantitative PCR using a primer set (1130F + 1294R) after extracting DNA from a sample obtained by mixing DPH-1 strain and other microorganisms.
FIG. 14 is a view showing the base sequences of oligonucleotides constituting three probes used in the FISH method.

Claims (5)

With the base sequence described in SEQ ID NO: 1 and SEQ ID NO: 3 of the sequence listing, thereby selectively amplify a specific region of 16SrDNA of Clostridium bi Fell Men chest DPH-1 strain by functioning as primers in the PCR method Oligonucleotide set . A method for detecting Clostridium bifermentans DPH-1 strain present in a specimen comprising:
A first step of trying to amplify DNA present in a specimen by causing the oligonucleotide set according to claim 1 to function as a primer in a PCR method;
And a second step of determining that the Clostridium bifermentans DPH-1 strain is present in the sample when the DNA concentration in the sample increases in the first step. A method for detecting the Clostridium bifermentans DPH-1 strain. A method for quantifying Clostridium bifermentans DPH-1 strain present in a specimen,
First, a correlation between the initial DNA concentration of the DPH-1 strain and the number of cycles when the threshold is exceeded is determined in advance by causing the oligonucleotide set according to claim 1 to function as a primer in a quantitative PCR method. And the steps
Microbial DNA present in the sample is amplified by quantitative PCR using the primers used in the first step, and the number of cycles when the amount of amplified DNA exceeds a specific threshold is determined. A second step;
The initial DNA concentration of the DPH-1 strain present in the sample is determined in the second step based on the correlation between the initial DNA concentration determined in the first step and the number of cycles when the threshold is exceeded. And a third step of calculating back from the number of cycles performed, and a method for quantifying the Clostridium bifermentans DPH-1 strain. Oligo consisting of the nucleotide sequence set forth in SEQ ID NO: 8 in the Sequence Listing and capable of selectively hybridizing to a specific region of 16S rDNA of Clostridium bifermentans DPH-1 strain by functioning as a probe in the FISH method nucleotide. A method for detecting Clostridium bifermentans DPH-1 strain present in a specimen comprising:
A first step of attempting hybridization with DNA present in a specimen by causing the oligonucleotide according to claim 4 to function as a probe in the FISH method;
A second step of determining that the Clostridium bifermentans DPH-1 strain is present in the sample when hybridization with DNA present in the sample is confirmed in the first step; A method for detecting a Clostridium bifermentans DPH-1 strain characterized by comprising:

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