JP2016195565A - Method for detecting aromatic compound-decomposable bacteria belonging to family sphingomonadaceae and kit therefor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide: a method for detecting aromatic compound-decomposable bacteria belonging to family Sphingomonadaceae, the method being able to specifically detect aromatic compound-decomposable bacteria belonging to family Sphingomonadaceae even in a system in the presence of a plurality of microbial species and quantify the bacteria; and a kit used therefor.SOLUTION: The method for detecting aromatic compound-decomposable bacteria belonging to family Sphingomonadaceae comprises: a first step of obtaining amplified products by amplifying a nucleotide using a primer capable of amplifying a continuous 140 or more to 176 or less base sequence given in a base sequence of SEQ ID NO: 1 with the use of a DNA to be tested as a template; and a second step of detecting the amplified products.SELECTED DRAWING: None

Description

  The present invention relates to a method and kit for detecting an aromatic compound-degrading sphingomonadaceae bacterium.

  It is desired that wastewater from heavy chemical industries such as chemistry and steel is discharged into the natural environment in a state where organic substances are removed and the effects on humans and environmental organisms are sufficiently reduced. As a wastewater treatment method for removing organic substances, a biological treatment method using a combustion treatment or a compound-decomposable microorganism is used.

  As a method for obtaining a compound-degrading microorganism, a method (isolation culture method) is known in which microorganisms that degrade a target compound are accumulated and cultured, and the degrading microorganisms are identified, isolated, and purely cultured. As another method, in the case of a mixed microorganism system such as activated sludge, the target compound is given as a nutrient source at a low concentration, and the concentration is gradually increased to acclimate the activated sludge to the target compound, thereby reducing the resolution. There is a known method (acquisition method) called “acceleration”. For example, Patent Document 1 describes a method for acclimatizing activated sludge used for treating 1,4-dioxane, which is an organic substance, from wastewater.

  An organic compound contained in the wastewater is an aromatic compound. As a method for treating wastewater containing an aromatic compound, for example, there is a method using activated sludge conditioned using an aromatic compound as a nutrient source. At this time, it is desired to monitor and adjust the amount of aromatic compound-decomposing microorganisms in the activated sludge according to the amount of aromatic compound in the wastewater and the speed required for wastewater treatment. For that purpose, it becomes a problem to rapidly detect and quantify only the aromatic compound-decomposing microorganisms from the activated sludge in which a plurality of microbial species are present.

JP 2014-188506 A

  However, until now, it has been difficult to control the aromatic compound removal reaction in the wastewater because the specific detection and quantification of the aromatic compound-degrading microorganisms has not been possible from a system in which a plurality of microorganisms exist.

  In view of the above circumstances, the present invention provides a method for detecting an aromatic compound-degrading microorganism capable of specifically detecting and quantifying an aromatic compound-degrading microorganism even in a system in which a plurality of microorganism species exist, and therefore It aims at providing the kit used for.

  Based on the results of previous studies, the present inventors have confirmed that there is a specific Sphingomonasaceae bacterium having aromatic compound degradability in the activated sludge used for decomposing aromatic compounds. Revealed. Then, the inventors of the present invention have identified a specific region of the base sequence (SEQ ID NO: 9) of the 16S rRNA gene of the Sphingomonasaceae bacterium as a specific compound of the aromatic compound-degrading Sphingomonasaceae bacterium in a system in which a plurality of microbial species are present. The inventors have newly found that it is useful for detection and quantification, and have completed the present invention.

That is, the present invention relates to the following [1] to [7], for example.
[1] A method for detecting an aromatic compound-degrading sphingomonadaceae family bacterium, wherein the test DNA is used as a template, and the base sequence of SEQ ID NO: 1 is continuously 140 to 176 bases or less A method comprising a first step of amplifying nucleotides using a primer capable of amplifying the nucleotide sequence of, and obtaining an amplified product, and a second step of detecting the amplified product.
[2] The method according to [1], wherein the aromatic compound is (S) -2- (4-chlorophenyl) -3-methylbutanoic acid.
[3] In the second step, an amplification product is detected using a probe comprising a base sequence of 15 to 30 bases that hybridizes under stringent conditions with a nucleotide comprising the base sequence of SEQ ID NO: 2. The method according to [1] or [2].
[4] The method according to [3], wherein the probe comprises one base sequence selected from the base sequences described in SEQ ID NOs: 3 to 6.
[5] The primers used in the first step are a first primer containing the base sequence shown in SEQ ID NO: 7 and a second primer containing the base sequence shown in SEQ ID NO: 8, [1] to [ 4].
[6] A kit used for detection of an aromatic compound-degrading sphingomonadaceae family bacterium, comprising a first primer comprising the base sequence set forth in SEQ ID NO: 7, and the sequence set forth in SEQ ID NO: 8 A primer set consisting of a second primer containing a base sequence, and a probe containing a base sequence of 15 to 30 bases that hybridizes with a nucleotide containing the base sequence of SEQ ID NO: 2 under stringent conditions kit.
[7] The kit according to [6], wherein the probe comprises one base sequence selected from the base sequences described in SEQ ID NOs: 3 to 6.

  According to the present invention, a method for detecting an aromatic compound-degrading Sphingomonasaceae bacterium that can specifically detect and quantify an aromatic compound-degrading Sphingomonasaceae bacterium even in a system in which a plurality of microbial species are present, and Kits used for this purpose can be provided. Thereby, for example, in the waste water treatment, the aromatic compound removal reaction in the waste water can be controlled based on the detected amount of the aromatic compound-degrading Sphingomonasaceae family bacteria in the activated sludge.

  Hereinafter, a mode for carrying out the present invention (hereinafter referred to as “the present embodiment”) will be described in detail. In addition, this invention is not limited to the following embodiment.

<Method for detecting aromatic compound-degrading sphingomonadaceae family bacteria>
The method for detecting an aromatic compound-degrading Sphingomonadaceae bacterium according to this embodiment can amplify a base sequence of 140 to 176 bases in a sequence of SEQ ID NO: 1 using a test DNA as a template. It has the 1st process of amplifying a nucleotide using a primer and obtaining an amplification product, and the 2nd process of detecting an amplification product.

  As an aromatic compound which concerns on this embodiment, the aromatic compound represented by Formula (1) or Formula (2) is mentioned, for example.

In formula (1), R ¹ represents a hydrogen atom or a chlorine atom, and R ² represents an alkyl group having 1 to 3 carbon atoms or a hydroxyl group. The alkyl group may be a linear alkyl group or a branched alkyl group. Examples of the aromatic compound represented by the formula (1) include (S) -2- (4-chlorophenyl) -3-methylbutanoic acid, 2- (4-chlorophenyl) -3-methylbutanoic acid, 2-phenylbutane. Examples include acid, 2-hydroxy-2- (4-chlorophenyl) acetic acid, and 2-phenyl-3-methylbutanoic acid.

In Formula (2), R ³ represents a hydrogen atom, a hydroxymethyl group, a formyl group, or a carboxyl group, and R ⁴ and R ⁵ each independently represent a hydrogen atom or a hydroxyl group. Examples of the aromatic compound represented by the formula (2) include catechol, protocatechuic acid, protocatechualdehyde, benzoic acid, and benzaldehyde.

  Examples of the test DNA according to this embodiment include a plasmid, cDNA, and genomic DNA. These test DNAs may include DNA derived from a plurality of types of microorganisms, or may be DNA prepared from activated sludge. As the test DNA, DNA prepared from activated sludge is preferable from the viewpoint of controlling the aromatic compound removal reaction in the wastewater. As methods for preparing these test DNAs, methods well known to those skilled in the art can be used. As a method for preparing DNA from activated sludge, for example, a commercially available DNA extraction kit can be used.

[First step]
SEQ ID NO: 1 shows the 495th to 670th base sequence of SEQ ID NO: 9. SEQ ID NO: 9 shows the base sequence of the 16S rRNA gene of the aromatic compound-degrading sphingomonadaceae family bacterium. In the 16S rRNA gene, there are a conserved region containing a sequence common between species on the gene and nine variable regions (V1 to V9) containing sequences that differ depending on the species / genus. In SEQ ID NO: 9, the base sequence from the 506th to 567th is the V4 region.

  The base sequence described in SEQ ID NO: 1 is a region including the V4 region of the 16S rRNA gene. In the first step according to this embodiment, an amplification product is prepared by amplifying nucleotides using a primer capable of amplifying a base sequence of 140 to 176 bases in the base sequence of SEQ ID NO: 1. The amplification product may be prepared by amplifying nucleotides using a primer capable of amplifying a continuous base sequence of 150 to 176 bases, and a primer capable of amplifying a continuous base sequence of 160 to 176 bases May be prepared by amplifying nucleotides using. As long as it is an amplification product prepared by amplification with such a primer, it may be a base sequence having 1 to 2 nucleotides different from the corresponding region of the base sequence described in SEQ ID NO: 1. Base sequences having the same nucleotide may be used. By using the amplification product containing the above base sequence, the aromatic compound-degrading Sphingomonasaceae bacterium can be specifically detected in the subsequent second step.

  In the first step, the method for amplifying the nucleotide and obtaining the amplification product is not particularly limited, and methods well known to those skilled in the art can be used. Examples of such a method include a polymerase chain reaction method (PCR method), a real-time PCR method, and a LAMP method. Among these methods, the real-time PCR method is preferable because detection in the second step can be performed simultaneously with amplification, and amplification products can be quantified.

  The primer set used for obtaining the amplification product can be designed so that the region containing the V4 region of the 16S rRNA gene of the aromatic compound-degrading sphingomonadaceae bacterium can be amplified. The primer set includes a first primer (forward primer) that hybridizes to the antisense strand of DNA encoding 16S rRNA under stringent conditions and a second primer (reverse primer) that hybridizes to the sense strand under stringent conditions. There is a set consisting of primers.

In the present specification, “stringent conditions” means that a complementary strand of a nucleotide strand having homology to a target sequence preferentially hybridizes to the target sequence, and complementation of a nucleotide strand having no homology. It means conditions under which the strands do not substantially hybridize. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. Generally, stringent conditions are selected to be about 5 ° C. lower than the thermal melting temperature (Tm) for the specific sequence at a defined ionic strength and pH. Tm is the temperature at which 50% of the nucleotides complementary to the target sequence hybridize to the target sequence in equilibrium under a defined ionic strength, pH, and DNA concentration.
As the “primer or probe containing a base sequence that hybridizes under stringent conditions”, for example, a target nucleotide and a high ion concentration [for example, 6 × SSC (900 mM sodium chloride, 90 mM sodium citrate), etc. are used. It is done. ] At a temperature of 65 ° C. to form a DNA-DNA hybrid, and a low ion concentration [for example, 0.1 × SSC (15 mM sodium chloride, 1.5 mM sodium citrate) or the like is used. . ] Includes a primer or a probe that can maintain the hybrid even after washing at 65 ° C. for 30 minutes.

Examples of the first primer that hybridizes to the antisense strand of DNA encoding 16S rRNA under stringent conditions include, for example, differences from a base sequence of 18 or more bases in the DNA base sequence encoding 16S rRNA. A primer including a base sequence having 2 bases can be mentioned, and preferably a base sequence having a base number of 1 different from a continuous base sequence of 18 bases or more in the base sequence of DNA encoding 16S rRNA. More preferably, there can be mentioned a primer containing a base sequence of 18 or more bases in the base sequence of DNA encoding 16S rRNA.
Examples of the second primer that hybridizes under stringent conditions to the sense strand of DNA encoding 16S rRNA include, for example, consecutive 18 bases in a base sequence complementary to the base sequence of DNA encoding 16S rRNA. A primer including a base sequence having a base number of 2 different from the above base sequence can be mentioned, and preferably, 18 consecutive bases in a base sequence complementary to the base sequence of DNA encoding 16S rRNA A primer including a base sequence having a base number of 1 different from the above base sequence can be mentioned, and more preferably, a continuous 18 in the base sequence complementary to the base sequence of DNA encoding 16S rRNA. A primer including a base sequence of more than bases can be mentioned.

  The first and second primers are particularly limited as long as they are designed so as to amplify nucleotides having a base sequence of 140 to 176 bases in the base sequence of SEQ ID NO: 1. is not. The length of the primer is preferably 18 bases or more, more preferably 20 bases or more, from the viewpoint of obtaining sufficient specificity. The upper limit of the primer length is preferably 30 bases or less, more preferably 25 bases or less, and even more preferably 21 bases or less from the viewpoint of further improving the annealing efficiency. As a 1st primer, the primer containing the base sequence of sequence number 7 is mentioned, for example. As a 2nd primer, the primer containing the base sequence of sequence number 8 is mentioned, for example.

[Second step]
In the second step, the method for detecting the amplification product is not particularly limited, and methods well known to those skilled in the art can be used. Examples of the method for detecting the amplification product include an agarose gel electrophoresis method, a real-time PCR method, a sequence analysis method, and a Southern blotting method. Among these methods, a detection method using a probe is preferable from the viewpoint of excellent specificity. As a detection method using a probe, for example, a real-time PCR method or a Southern blotting method can be mentioned. Among them, a real-time PCR method or a Southern blotting method can be used. The PCR method is preferred.

  The probe used for detection is preferably an oligonucleotide that hybridizes under stringent conditions to the sense strand or antisense strand of the amplification product obtained in the first step. The length of the probe is preferably from 15 to 30 bases, more preferably from 15 to 25 bases, and even more preferably from 18 to 23 bases from the viewpoint of more specifically detecting the amplification product. A probe that hybridizes under stringent conditions with a nucleotide containing the base sequence shown in SEQ ID NO: 2 is preferred. As such a probe, for example, a probe containing the base sequences described in SEQ ID NOs: 3 to 6 can be mentioned.

  When the probe is used in the real-time PCR method, the probe is modified with a fluorescent substance at the 5 'end and a quencher for suppressing fluorescence emitted from the fluorescent substance at the 3' end. Examples of the fluorescent substance include FAM, TET, HEX, TAMRA, and Cyanine 5. Examples of the quencher include TAMRA, BHQ1, BHQ2, and BHQ3. The fluorescent substance and quencher used for probe modification can be appropriately selected according to the excitation wavelength and measurement wavelength of the real-time PCR apparatus to be used.

  The real-time PCR apparatus is not particularly limited as long as it includes a thermal cycler capable of amplifying DNA by PCR and a spectrofluorometer for detecting an amplification product. An example of the real-time PCR apparatus is StepOnePlus (manufactured by Applied Biosystems).

  The amplified sample detected in the second step can also be quantified. Therefore, the detection method of the present embodiment not only detects the aromatic compound-degrading Sphingomonasaceae bacteria from the test DNA, but also quantifies the amount of DNA derived from the aromatic compound-degrading Sphingomonasaceae bacteria contained in the test DNA. You can also. For example, when real-time PCR is used for detection of amplification products in the second step, the amount of test DNA can be quantified from a calibration curve obtained using a standard sample. The calibration curve can be created based on the Ct value indicating the number of cycles when the amplification product reaches a certain value and the initial template amount.

  In the detection method of this embodiment, when an amplification product is obtained from the test DNA in the first step and the amplification product can be detected in the second step, the test DNA has an aromatic compound degradability. It can be determined that DNA derived from bacteria belonging to the family Sphingomoadaceae is included.

<Aromatic Compound-Degrading Sphingomonasaceae Bacteria Kit>
The kit used for the detection of the aromatic compound-degrading Sphingomonasaceae bacteria of the present embodiment includes a first primer containing the base sequence set forth in SEQ ID NO: 7 and a second primer containing the base sequence set forth in SEQ ID NO: 8. A primer set comprising primers, and a probe comprising a base sequence of 15 to 30 bases that hybridizes with a nucleotide comprising the base sequence of SEQ ID NO: 2 under stringent conditions.

  As the first primer, the second primer, and the probe, the same primers as those used in the detection method described above can be used.

  In addition to the first primer, the second primer, and the probe containing the base sequence described in SEQ ID NO: 11, the kit of this embodiment may contain other reagents and the like as necessary. Examples of other reagents include DNA polymerase, deoxyribonucleotide mixture (dNTP Mix), buffer solution, sterilized water, and control DNA.

  When a buffer solution is included in the kit of this embodiment, examples of the buffer solution include a buffer solution used in a general PCR method.

Example 1. Cloning and sequencing of 16S rRNA gene of aromatic compound-degrading sphingomonadaceae bacteria
-LB liquid culture medium: 10 parts of LB Broth, 1.1G PER TABLE (manufactured by SIGMA) dissolved in 500 mL of ultrapure water and autoclaved.
-LB agar medium: LB Agar (manufactured by SIGMA) was dissolved at a ratio of 10 and sterilized by high-pressure steam, and dispensed into a plastic petri dish by about 25 mL each.
Template DNA extracted from a group of microorganisms having degradability of (S) -2- (4-chlorophenyl) -3-methylbutanoic acid (Compound 1)
-Forward primer for cloning (27f: SEQ ID NO: 10)
-Reverse primer for cloning (1492r: SEQ ID NO: 11)
Sequence analysis primer (M13F: SEQ ID NO: 12, M13R: SEQ ID NO: 13, 339F: SEQ ID NO: 14, 536R: SEQ ID NO: 15, 907F: SEQ ID NO: 16)

(Method)
25.0 μL of 2 × PCR buffer for KOD FX (Toyobo), 10.0 μL of dNTP mix (2 mM), 1.5 μL of cloning forward primer and cloning reverse primer (each 10 pmol / μL), 0.58 μL Template DNA, 10.4 μL of sterilized water, and 1.0 μL of DNA polymerase (KOD FX, 1 U / μL, manufactured by Toyobo Co., Ltd.) were added to the microtube and mixed. The microtube was subjected to a PCR apparatus, and template DNA amplification reaction was performed. The reaction is carried out at (1) 94 ° C for 2 minutes, (2) 98 ° C for 10 seconds, (3) 50 ° C for 30 seconds, (4) 68 ° C for 1.5 minutes, (2) to (4) This process was repeated 35 cycles. The amplified product after PCR was purified and used for cloning.

  Cloning was performed using the amplification product and Zero Blunt TOPO PCR Cloning Kit (manufactured by Invitrogen). 1 μL of pCRII-Blunt-TOPO, 1 μL of amplification product, 1 μL of Salt solution, and 3 μL of sterilized ultrapure water were mixed for 5 minutes at room temperature, and this was stored in ice. 2 μL of the above reaction solution and 50 μL of E. coli DH5α (manufactured by Takara) were mixed and allowed to stand in ice for 30 minutes. After standing, it was incubated at 42 ° C. for 30 seconds and stored in ice. After 450 μL of SOC medium was added to this reaction solution, the resulting mixture was cultured with shaking at 37 ° C., 200 rpm for 1 hour. The culture solution was applied to an LB plate (final concentration of kanamycin 50 μg / mL) and cultured (37 ° C., 20 hours).

  Twelve strains of the obtained colonies of E. coli DH5α were picked up, and the picked-up E. coli DH5α was cultured in 4 mL of LB medium (final concentration of kanamycin 50 μg / mL) at 37 ° C. for 16 hours. A plasmid solution was obtained by extracting a plasmid from the obtained Escherichia coli DH5α using a plasmid extraction apparatus (QIAcube, manufactured by QIAGEN).

  3.0 μL of the obtained plasmid solution, 5 U equivalent of EcoRI and 1 μL of 10 × H buffer were mixed, and a sterilized ultrapure water was added thereto to prepare a mixed solution with a volume of 10.0 μL. This mixture was incubated at 37 ° C. for 2 hours. After incubation, the EcoRI-treated sample was subjected to 1.0% agarose gel electrophoresis, and it was confirmed that the 16S rRNA gene fragment was excised by EcoRI.

  A plasmid solution equivalent to 150 ng, a sequence analysis primer (10 μM) 0.32 μL, and BigDye Terminator v3.1 (Applied Biosystems) 8.0 μL were mixed, and sterilized ultrapure water was added thereto to make the volume. A reaction solution of 20.0 μL was prepared. This reaction solution was subjected to an amplification reaction using a PCR apparatus. The reaction is carried out at (1) 96 ° C. for 1 minute, (2) 96 ° C. for 10 seconds, (3) 50 ° C. for 5 seconds, (4) 60 ° C. for 4 minutes, and steps (2) to (4) Was repeated 25 cycles. The obtained reaction solution was subjected to DNA sequence analysis, and the base sequence of the template DNA extracted from the group of microorganisms capable of decomposing Compound 1 was determined. The determined nucleotide sequence was determined at the genus level using RDP Classifier.

(result)
Of the 12 cases determined by sequence analysis, 7 cases were shown to be 16S rRNA genes derived from bacteria belonging to the family Sphingomonasaceae. The base sequence of the 16S rRNA gene at this time is shown in SEQ ID NO: 9. The Sphingomonasaceae bacterium having this base sequence is hereinafter referred to as Sphingomoadaceae bacterium NITE BP-02022. From these results, it was suggested that Sphingomoadaceae family bacteria NITE BP-02022 have a compound 1 resolution.

Example 2 Determination of the resolution of Compound 1 of the sphingomoadaceae family bacterium NITE BP-02022 (Method)
A glycerol stock of the sphingomonadaceae family NITE BP-0202 was inoculated into 4.0 mL of LB liquid medium and pre-cultured at 30 ° C. for 2 days. 4.0 mL of the preculture solution was added to 100 mL of LB medium, cultured at 30 ° C. for 2 days, then Compound 1 (final concentration 100 ppm) was added, and further cultured for 1 day.

  After completion of the culture, the culture solution was centrifuged at 6,000 × g and 4 ° C. for 5 minutes to recover the cells. The collected cells were washed with 20 mL of physiological saline, and then suspended in 50 mM sodium phosphate buffer (pH 7.5) so that the wet cell concentration was 100 mg / mL. 1.0 mL of the suspension and 1.0 mL of a phosphate buffer containing 2000 ppm of Compound 1 were added to a screw mouth test tube and reacted at 30 ° C. and 200 rpm for 3 days.

  200 μL of the reaction solution was sampled, the precipitate was removed by centrifugation, and the supernatant was collected. The collected supernatant was added to Amicon Ultra-0.5 Centrifugal Filter Device 100 K (manufactured by MILLIPORE), and ultrafiltration was performed by centrifuging at 14,000 × g for 10 minutes. The remaining amount of Compound 1 was detected from 100 μL of the obtained filtrate using liquid chromatography under the following conditions.

HPLC condition column: SUMPAX ODS A-211 (4.6 mmφ × 25 cm, 5 μm, manufactured by Sumika Chemical Analysis Co., Ltd.)
Mobile phase: A solution 0.01% TFA, B solution acetonitrile Elution method: Isocratic elution conditions: A solution (%): B solution (%)
0 min, 40:60
20 minutes, 40:60
Flow rate: 1.0 mL / min Column temperature: 40 ° C
Detection: 220nm

(result)
It was possible to decompose 1000 ppm of Compound 1 at a wet cell concentration of 50 mg / mL. From this, it was shown that Sphingomonadaceae family bacteria NITE BP-02022 has compound 1 resolution.

Example 3 Selection of primers and probes (materials)
-Forward primer shown in Table 1-Reverse primer shown in Table 2-Probe shown in Table 3-Template DNA (plasmid containing 16S rRNA gene having the base sequence of SEQ ID NO: 9)

(Method)
Using 19 primer / probe sets obtained by combining the primers and probes described in Tables 1 to 3, real-time PCR was performed using a plasmid containing a 16S rRNA gene having the base sequence represented by SEQ ID NO: 9 as a template DNA, It was examined whether or not the target gene fragment could be detected.

  10 μL of 2 × TaqMan fast Advanced Master Mix, 0.36 μL of forward primer (50 pmol / μL) and reverse primer (50 pmol / μL), 0.50 μL probe, 6.78 μL of sterile water, 2.00 μL of template DNA Added to the microtube and mixed. The microtube was subjected to a real-time PCR apparatus (StepOnePlus, Applied Biosystems) to carry out template DNA amplification reaction. The reaction was carried out at (1) 95 ° C. for 20 seconds, (2) 95 ° C. for 1 second, (3) 60 ° C. for 20 seconds, and steps (1) to (3) were repeated 50 cycles.

(result)
Table 4 shows the results. A plasmid (1 pg / μL) containing a 16S rRNA gene having the base sequence shown in SEQ ID NO: 9 was detected as ◯, and a plasmid that could not be detected as ×. Among the 19 primer / probe sets, 10 primer / probe sets (Nos. 10 to 19) were able to detect 1 pg / μL of the plasmid containing the 16S rRNA gene having the base sequence represented by SEQ ID NO: 9.

Example 4 Quantitative confirmation using primer / probe set (material)
・ Primer / probe sets 12 to 15 in Table 4

(Method)
As described in Example 3, except that a plasmid (final concentration 1, 0.1, 0.01, 0.001 pg / μL) containing a 16S rRNA gene having the base sequence shown in SEQ ID NO: 9 was used as a template DNA. The method was used for real-time PCR, and the R ² value of the calibration curve was calculated.

(result)
Table 5 shows the results. Regardless of which primer / probe set is used, the R ² value is 0.99 or more, and the primer / probe set of Nos. 12 to 15 is in the range of 1, 0.1, 0.01, 0.001 pg / μL. Showed that the quantitative property was good.

Example 5 FIG. Specificity test and quantitative test using one kind of plasmid (material)
-No. 12 primer / probe set in Table 4-Plasmid containing 16S rRNA gene derived from microorganisms described in Table 6

(Method)
Real-time PCR was performed by the method described in Example 3 to quantify amplification products, except that plasmid DNA containing 16S rRNA genes derived from various bacteria (final concentration 1 pg / μL) was used as template DNA.

(result)
Table 6 shows the results of quantification by real-time PCR. From this result, it was shown that the No. 12 primer / probe set specifically detects a plasmid containing a 16S rRNA gene derived from the sphingomodaaceae bacterium NITE BP-02022. The detection limit or lower is a concentration based on a calibration curve prepared using a plasmid (1, 0.1, 0.01, 0.001 pg / μL) containing a 16S rRNA gene having the base sequence represented by SEQ ID NO: 9. As a result, the value is 0.001 pg / μL or less.

Example 6 Specificity test and quantitative test using mixed plasmids (materials)
-Primer / probe set No. 12 or 15 in Table 4-Plasmid containing 16S rRNA gene derived from microorganisms described in Table 6

(Method)
Plasmids containing 16S rRNA gene from Sphingomoadaceae family bacteria NITE BP-02022 (final concentration 1 pg / μL, 0.1 pg / μL, 0.01 pg / μL or 0.001 pg / μL), and 16S rRNA genes derived from various bacteria Real-time PCR was performed by the method described in Example 3 to quantify the amplification products, except that the plasmid mixture was mixed with a mixture of plasmids containing each (final concentration of 1 pg / μL each) as template DNA.

(result)
Table 7 shows the results of quantification by real-time PCR. From this result, the primer / probe set of No. 12 or 15 can specifically detect and quantify a plasmid containing 16S rRNA gene derived from Sphingomonasaceae family bacteria NITE BP-02022 even in a system in which plural types of plasmids exist. Indicated.

Example 7 Specificity test using one type of genomic DNA (material)
-No. 12 primer / probe set in Table 4-Microorganism-derived genomic DNA described in Table 8

(Method)
Real-time PCR was performed by the method described in Example 3 except that genomic DNA derived from various bacteria (final concentration 1 ng / μL) was used as the template DNA.

(result)
Table 8 shows the bacteria from which the genomic DNA used in the test was derived and the detection results of those genomic DNA. According to the No. 12 primer / probe set, genomic DNA of the sphingomodaceae family bacterium NITE BP-02022 could be detected to a concentration of 0.001 ng / μL. On the other hand, genomic DNA derived from other bacteria was below the detection limit (0.001 ng / μL). From this result, it was shown that the primer / probe set of No. 12 can specifically detect the 16S rRNA gene of the Sphingomoadaceae family bacterium NITE BP-02022 even when genomic DNA is used.

Example 8 FIG. Specificity test and quantitative test using multiple types of genomic DNA (materials)
-No. 12 primer / probe set in Table 4-Microorganism-derived genomic DNA described in Table 9

(Method)
A mixture of genomic DNA (final concentration 100 pg / μL, 10 pg / μL, 1 pg / μL, or 0.1 pg / μL) derived from the sphingomonadaceae bacterium NITE BP-0202 and a mixture of genomic DNAs derived from various bacteria (final concentrations of 100 pg / μL, respectively) ) Was used as a template DNA, and real-time PCR was performed by the method described in Example 3 to quantify amplification products.

(result)
Table 10 shows the results of quantification by real-time PCR. It was shown that the No. 12 primer / probe set can specifically detect and quantify genomic DNA derived from the sphingomonadaceae family bacterium NITE BP-02022 even in a system in which multiple types of genomic DNA exist.

Claims (7)

A method for detecting an aromatic compound-degrading sphingomonadaceae bacterium, comprising:
A first step of amplifying a nucleotide using a test DNA as a template and a primer capable of amplifying a continuous base sequence of 140 to 176 bases of the base sequence of SEQ ID NO: 1 to obtain an amplified product; ,
A second step of detecting an amplification product.   The method according to claim 1, wherein the aromatic compound is (S) -2- (4-chlorophenyl) -3-methylbutanoic acid.   The amplification product is detected in the second step using a probe comprising a base sequence of 15 to 30 bases that hybridizes under stringent conditions with a nucleotide comprising the base sequence of SEQ ID NO: 2. The method according to 1 or 2.   The method according to claim 3, wherein the probe comprises one base sequence selected from the base sequences shown in SEQ ID NOs: 3 to 6.   5. The primer according to claim 1, wherein the primer used in the first step is a first primer containing the base sequence shown in SEQ ID NO: 7 and a second primer containing the base sequence shown in SEQ ID NO: 8. The method according to claim 1. A kit used for the detection of an aromatic compound-degrading sphingomonadaceae family bacterium,
A primer set consisting of a first primer containing the base sequence set forth in SEQ ID NO: 7 and a second primer containing the base sequence set forth in SEQ ID NO: 8;
A kit comprising a nucleotide comprising the nucleotide sequence of SEQ ID NO: 2 and a probe comprising a nucleotide sequence of 15 to 30 bases that hybridizes under stringent conditions.   The kit according to claim 6, wherein the probe comprises one base sequence selected from the base sequences shown in SEQ ID NOs: 3 to 6.