JP2001346589A - Polynucleotide specific to hads-degrading microorganism and method for detecting and quantitatively determining hads-degrading microorganism using the polynucleotide - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for rapidly, specifically and simply detecting an HADS(hydroxylaminedisulfonic acid)-degrading microorganism. SOLUTION: This is a method for detecting and quantitatively determining an HARDS-degrading microorganism using a DNA encoding 16S rRNA region specific to the HADS-degrading microorganism.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a polynucleotide specific to HADS (hydroxylamine disulfonic acid) -degrading bacteria. The present invention also provides a method for rapidly, specifically and easily detecting and quantifying HADS-degrading bacteria using the polynucleotide.

[0002]

2. Description of the Related Art Conventionally, a sulfur-containing nitrogen compound (hereinafter referred to as an NS compound), which is a hardly decomposable COD component in wastewater, is
Although treated by a chemical method, the present inventors have found that HADS, which is a kind of NS compound, can be biologically decomposed by activated sludge.
No. 3395.

[0003] However, the treatment is unstable, and it is expected that the cause is related to the number of HADS-degrading bacteria in the sludge. Therefore, it is necessary to know the priority and the growth status of the degrading bacteria in the sludge. There is.

For this purpose, HADS-degrading bacteria in activated sludge must be specifically detected and measured.
Since there is no method for selectively growing only S-degrading bacteria, so far, colonies have been formed on a plate medium prepared with a nutrient medium capable of growing as many microorganisms as possible, and after isolation and cultivation, HADS-degrading activity was determined. And HADS-degrading bacteria in the sludge were measured by measuring the number of colonies having HADS-degrading activity with respect to all the colonies that appeared.

However, in this method, the isolation and
Since it takes a lot of time to measure the cultivation / decomposition activity, it is impossible to treat many samples. Therefore, there has been a demand for a method for simply and quickly detecting and counting the number of HADS-degrading bacteria in a mixed bacteria group such as activated sludge.

[0006]

SUMMARY OF THE INVENTION The present invention has been made to solve the above problems in the prior art,
An object is to provide a method for rapidly and specifically detecting and quantifying HADS-degrading bacteria.

[0007]

The present invention provides a method for specifically detecting or quantifying a HADS-degrading bacterium, which comprises solving a HADS-degrading bacterium contained in a nucleic acid encoding 16S rRNA. And a step of specifically detecting or quantifying HADS-degrading bacteria by detecting or quantifying the amplified region.

Further, the present invention relates to a method for specifically detecting or quantifying a HADS-degrading bacterium, wherein a region specific to the HADS-degrading bacterium contained in a nucleic acid encoding 16S rRNA is hybridized to the HADS-degrading bacterium. And a step of specifically detecting or quantifying HADS-degrading bacteria by detecting or quantifying the hybridized region.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, non-HADS-degrading bacteria are mixed using a region (SEQ ID NO: 1) specific to HADS-degrading bacteria in a DNA region encoding 16S rRNA (hereinafter referred to as rDNA region). The present invention provides a method for specifically detecting HADS-degrading bacteria from a sample.

[0010] As used herein, the term "HADS-degrading bacterium" refers to any microorganism (bacterium, fungus, etc.) capable of degrading HADS, and includes Roseobacter species.

As described in detail in Example 2, the above-mentioned region specific to HADS-degrading bacteria is a representative HADS-degrading bacterium in activated sludge, and the Roseobacter sp. SH134 isolated by the present inventors. -8 strains and non-HA
By comparing the base sequences of the rDNA regions of DS-degrading bacteria (E. coli, Bacillus, Pseudomonas, etc.), the present inventors have found that they are specific to HADS-degrading bacteria.

Using the above-mentioned region found by the present inventors, Ruege, which is taxonomically related to HADS-degrading bacteria, can be used.
ria algicola, Paracoccus a
minophilus and Paracoccus a
HADS-degrading bacteria can be specifically detected and quantified from a sample containing M. minovorans. Therefore, if the region is used, HADS-degrading bacteria can be specifically detected and quantified from any sample.

Therefore, the term “specifically detecting or quantifying HADS-degrading bacteria” as used herein refers to any non-HAD-degrading bacteria.
This means detecting or quantifying only HADS-degrading bacteria from any sample containing S-degrading bacteria.

More specifically, the present invention relates to a primer of SEQ ID NO: 2 (hereinafter referred to as primer 1) and a primer of SEQ ID NO: 3.
Using primers (hereinafter referred to as primer 2)
Provided is a method for specifically detecting HADS-degrading bacteria from a sample containing non-HADS-degrading bacteria by amplifying an rDNA region specific to ADS-degrading bacteria.

In order to amplify rDNA specific to HADS-degrading bacteria using the primer pair, the following operation is typically performed.

First, a sample from which HADS-degrading bacteria are to be detected,
For example, nucleic acids are extracted from activated sludge. As a method for extracting nucleic acids from bacteria, any known method including phenol extraction can be used.

Next, a polymerase chain reaction (hereinafter referred to as PCR) is carried out using the extracted nucleic acid as a template and a primer pair consisting of primer 1 and primer 2. It is sufficient that the DNA polymerase used in the reaction has heat resistance at a temperature of 90 ° C. or higher. The temperature conditions in the PCR reaction are 90-98 ° C. in a heat denaturation reaction for converting double-stranded DNA into a single strand, and 57-67 ° C. in an annealing reaction in which primers are hybridized to template DNA. 70-72 ° C for long reaction. A target gene can be amplified by performing several tens of cycles in which these three reactions are defined as one cycle. After the PCR reaction, the amplified nucleic acid product is detected and quantified using an appropriate nucleic acid detection means including gel electrophoresis and microarray. If the amplified nucleic acid product is detected at the designed length,
It can be determined that HADS-degrading bacteria are present in the sample.

In addition, a PCR method using primers 1 and 2 and MPN (most)
The number of HADS-degrading bacteria can also be measured by the MPN-PCR method in combination with the probe number (most probable number) method. In the MPN-PCR method,
The extracted nucleic acid is diluted, PCR is performed under certain conditions, and P
The number of bacteria is estimated from the dilution limit amplified by the CR method.

Since the above operation is an outline of an extremely general operation, the nucleic acid extraction method, the PCR modification method (such as nested PCR), and the nucleic acid detection method use various modified methods. It can be used in combination.

As described above, according to the present invention, HADS
The HADS-degrading bacteria can be specifically detected and quantified using the nucleotide sequence specific to the degrading bacteria.

As is clear from the above description, the method of the present invention is completely different from the conventional method in that HADS-degrading bacteria are detected using a nucleic acid sequence.

The subject of the present invention resides in the rapid and specific detection of HADS-degrading bacteria using a polynucleotide specific for HADS-degrading bacteria.
It is not limited to the use of ~ 2.

Accordingly, from the nucleotide sequence of the DNA encoding the rRNA of the HADS-degrading bacterium discovered by the present inventors, a suitable polynucleotide other than primers 1 and 2, that is, a nucleotide sequence specifically found in the HADS-degrading bacterium, It should be noted that a method for specifically detecting HADS-degrading bacteria by the same operation using the primers and the primers is also included in the present invention.

Such a base sequence can be easily selected based on the base sequence of DNA (SEQ ID NO: 1) encoding rRNA specific to HADS-degrading bacteria specified by the present inventors. Would.

The present invention further provides a method for visualizing and detecting HADS-degrading bacteria by using probe 1 (SEQ ID NO: 4) and probe 2 (SEQ ID NO: 3) having a base sequence specific to HADS-degrading bacteria. Also provide. The probe was designed to have a sequence having at least three or more mismatches with the corresponding region of the non-HADS-degrading bacteria among the rDNA regions specific to the HADS-degrading bacteria. And selectively hybridize to said rDNA region.

In order to detect HADS-degrading bacteria using the probe, the following operation is typically performed.

First, a sample in which a HADS-degrading bacterium is to be detected is immobilized. Probes 1-2 labeled with a detectable label are hybridized to the immobilized HADS-degrading bacterium, followed by washing. 2 is hybridized to the chromosomal DNA of HADS-degrading bacteria.

The most preferable probe detection method is the FISH method frequently used in gene mapping. FI
When the SH method is used, the probe is labeled with a fluorescent substance such as fluorescein isothiocyanate, tetramethylrhodamine isothiocyanate (hereinafter referred to as TRITC), Cy3, and the like, and hybridization of the probe is detected by a fluorescence microscope.

As a method other than the FISH method, a nucleic acid detection means including a hybridization on a membrane such as dot hybridization with RNA / DNA extracted from a sample or a microarray method can be used. A detectable substance such as an isotope can be used by binding to a probe. If the probe binds only to a region specific to the HADS-degrading bacterium found by the discoverer, the probe may have another base added to the 5 ′ or 3 ′ end of sequences 2 and 3. You may.

Hereinafter, the present invention will be described in more detail by way of examples.

[Example 1] In this experimental example, a DNA sequence specific to HADS-degrading bacteria, which was found by comparing the rDNA region of HADS-degrading bacteria with the rDNA region of non-HADS-degrading bacteria, will be described.

16S rRNA is an RNA that constitutes a subunit of rRNA together with a plurality of proteins.
Since the primary structure of 16S rRNA, that is, the nucleotide sequence, differs depending on the species, the difference in the base sequence of DNA encoding 16S rRNA (hereinafter, referred to as rDNA) is used as a marker for specifically detecting microorganisms.

In order to utilize rDNA as a marker for specifically detecting a HADS-degrading bacterium, the present inventors examined the rDNA base sequence of the HADS-degrading bacterium and examined the sequence specific to the HADS-degrading bacterium. Was found.

Specifically, non-HADS degrading bacteria (HAD) obtained from databases such as DDBJ and Genbank
S-degrading bacteria) (see Table 1 below) and rDNA regions of Escherichia coli, Bacillus, Pseudomonas, etc. resident in activated sludge, and HADS-degrading bacteria SH134-8 analyzed by the present inventors. Based on the rDNA region of the rDNA region of the strain (SEQ ID NO: 1), the desired HA was obtained by multiple alignment using a gene sequence analysis program.
A nucleotide sequence specific to DS-degrading bacteria was searched.

Based on the rDNA region specific to HADS-degrading bacteria revealed by the search,
Primers for amplifying and detecting a base sequence specific to S-decomposing bacteria were designed. The following Sequence Listing shows Primer 1 (SEQ ID NO: 2) and Primer 2 (SEQ ID NO: 3) designed to detect HADS-degrading bacteria.

Further, based on the rDNA region, non-H
Probe 1 (complementary to the HADS-degrading bacteria-specific base sequence) has at least three or more mismatches compared to the ADS-degrading bacteria, and has a sequence that can bind only to the rDNA region of HADS-degrading bacteria. SEQ ID NO: 4) and probe 2 (SEQ ID NO: 3) were designed.

The thus designed primers and probes were synthesized using an oligonucleotide synthesizer. Alternatively, it can be prepared by enzymatically excising from the gene of HADS-degrading strain SH134-8.

As described above, the primers 1 and 2 and the probe 1
~ 2 are paired with rDNA specific to HADS-degrading bacteria, so that using the primers and / or probes,
From the group of bacteria resident in the mixed bacteria group such as activated sludge, H
ADS-degrading bacteria can be specifically detected and quantified.

The length (base length) of a nucleic acid product obtained by amplifying the gene of a HADS-degrading bacterium using the above primers 1 and 2 is about 800 bp.
It is.

[Example 2] In this example, the HADS-degrading bacteria and activated sludge containing Escherichia coli and various microorganisms similar in classification to the HADS-degrading bacteria shown in Table 1 below were designed in Example 1. A PCR method using the synthesized primers 1 and 2 is performed, and it is shown that the primers 1 and 2 can specifically detect and quantify only HADS-degrading bacteria.

(1) Extraction of Genomic DNA from Test Strain The examination of the specific detection conditions for the HADS-degrading strain SH134-8 was carried out by examining the HADS-degrading strain SH134-8 and HAD
Bacteria similar in classification to S-degrading bacteria, Escherichia coli as a representative bacterium, and activated sludge were used. Table 1 shows the samples used.

[0042]

[Table 1]

The samples in Table 1 were prepared by using TE buffer (pH
8.0) Suspension in 1 mL, Proteinase K
(20 mg / mL) 10 μL and 10% SDS 1
0 μL was added and incubated at 37 ° C. for 1 hour.
Then, 140 μL of 5M NaCl and 10% C
After adding 114 μL of TAB (in 0.7 M NaCl) and incubating at 65 ° C. for 10 minutes, phenol extraction and ethanol precipitation were performed.

The obtained precipitate was dissolved in 50 μL of distilled water, subjected to RNase treatment at 37 ° C. for 1 hour, and further subjected to phenol extraction and ethanol precipitation to reduce the DNA to 50 μL.
It was dissolved in L of distilled water and used as a template DNA in a PCR reaction. The activated sludge was subjected to floc dispersion by ultrasonic waves (using Olympus Bioruptor) and used as a sample.

(2) Preparation of Primer Primers were prepared by the method described in Example 1. (3) PCR beads (Pharmacia) were used for the reaction. 1 μL each of 12.5 μM of primer 1 and primer 2 was added to the PCR beads,
10 μL of NA and 13 μL of distilled water were added to prepare a total of 25 μL of the reaction solution. This was carried out under the following temperature conditions: heat denaturation: 95 ° C., 1 minute Annealing: 67 ° C., 1 minute Chain length reaction: 74 ° C., 1 minute, and this cycle was repeated 30 times.
R was performed. Before starting the cycling reaction, 5 ° C
After completion of the cycle reaction, a heat treatment was performed at 72 ° C. for 10 minutes. The PCR device was manufactured by PerkinElmer D
An NA thermal cycler was used.

By these operations, in the reaction system containing the HADS-degrading bacterium, a DNA fragment of about 800 bp is amplified by amplification from the combination of the primers 1 and 2.

(4) Detection 4 μL of the reaction solution after the completion of PCR was electrophoresed on a 1.5% (w / v) agarose gel. At the same time, λDNA / HindIII digest was also run as a molecular weight marker. After the electrophoresis, ethidium bromide solution (about 0.5 μg /
mL) for 15 minutes, and then observed under ultraviolet irradiation.
Photographs were taken. The length (base length) of the amplification product was estimated from the relative value to the mobility of the molecular weight marker.

(5) Results The results of the electrophoresis are shown in FIG. The genomic DNA extracted from each sample was adjusted to a concentration of about 50 μg / mL, and 10 μL thereof was used for a PCR reaction. From FIG. 1, when the genomic DNA of the SH134-8 strain was used as a template, a band was detected at a position of about 800 bp.
When the genomic DNA derived from activated sludge was used as a template, no band was detected. From this, by using the primers having the sequences of SEQ ID NOs: 2 and 3 and performing PCR under the conditions of the above (3),
It was revealed that the SH134-8 strain can be specifically detected.

Example 3 In this example, the number of HADS-degrading bacteria was measured by combining the PCR using the primers 1 and 2 with the MPN method.

Activated sludge (MLSS 11.5 g / L) 1
0, 10¹, 10², 10 ³, 10⁴, 1
0⁵, 10⁶, 10⁷HADS-degrading bacteria SH134-
After adding 8 strains and suspending well, TE buffer was added.
(PH 8.0) and washed three times by centrifugation. Then ultrasonic
Flock is crushed by processing, and the same as in Example 2
DNA was extracted by the method and dissolved in 50 μL of distilled water.

These are represented by 10 ⁻¹ , 10 ⁻² ,
10 ^-3 , 10 ^-4 , 10 ^-5 , 10 ^-6 , and 10 ^-7 dilutions were used as template DNA in a PCR reaction. Primers, PCR, detection, etc. were performed in the same manner as in Example 2. However, in PCR, the number of cycles is 6
It was set to 0 times.

The PCR reaction was performed for three samples, and the number of bacteria was estimated from the most probable numerical table of the three methods. MP
The results of N-PCR and the relationship between the number of inoculated cells and the number of detected cells are shown in Table 2 and FIG. 2, respectively. The SH134-8 strain was 1
The number of bacteria was determined assuming that one copy of 6S rRNA was present.

[0053]

[Table 2]

As shown in Table 2, SH134-
Even when 8 strains were not added, SH134-8 strain of 2 × 10 ⁵ cells per 1 g (dry weight) of the activated sludge was detected from the activated sludge used. From Table 2, the detection rate (M
(The number of bacteria detected by the PN-PCR method / the number of bacteria added) was about 10 to 20%. Therefore, the sludge used originally contained about 1-2 × 10 ⁶ per 1 g (dry weight) of activated sludge.
It can be assumed that the SH134-8 cell line is present.

Further, from FIG. 2, it was found that when the SH134-8 strain added per 1 g (dry weight) of the activated sludge was 9 × 10 ⁶ cells or more, the number of bacteria added and the number of detections were almost correlated. It can be seen that detection by the PCR method has a quantitative property.

In FIG. 2, the number of added bacteria was 9 ×
For 10 ^6, Nanoha number detection is constant, as described above, the sludge was used originally, since SH134-8 strain activated sludge 1 g (dry weight) per about 1 to 2 × 10 ⁶ cells were present It is.

As described above, by using the method of this example, the number of SH134-8 strains can be measured for a sample whose number is unknown.

Example 4 In this example, the FISH method using fluorescently labeled probes 1 and 2 was used for HADS-degrading bacteria and HADS-degrading bacteria shown in Table 3 which are similar in classification to HADS-degrading bacteria (Table 3). Fluorescence in situ hybridization method) shows that probes 1-2 can specifically detect and quantify only HADS-degrading bacteria.

[0059]

[Table 3]

(1) Preparation of Fluorescent Probe In this example, each probe 1 designed and synthesized in Experimental Example 1 was used.
2 was labeled with TRITC (tetramethylrhodamine isothiocyanate) at the 5 ′ end. In order to examine the specificity of the prepared probe, an oligonucleotide EUB338 (Amann, RI, e, having the sequence of SEQ ID NO: 5) used for detecting ordinary bacteria is used.
t al. 1990 J.C. Bacteriol.
172: 762-770) with the 5 ′ end labeled with TRITC.

(2) FISH method In this example, a 4% paraformaldehyde solution which does not emit fluorescence by itself was treated at 4 ° C. for 5 hours to fix HADS-degrading bacteria.

The hybridization with the probe
The washing was performed at 46 ° C. for 3 hours, and the washing was performed at 48 ° C. for 20 minutes.
The composition of the solution used for hybridization was 5.26 g of sodium chloride, 0.2 mL of 5% SDS, and 1 M
2 mL of Tris-HCl (pH 7.2) and 10 mL of formamide dissolved in distilled water were used as 100 mL.

Thereafter, in order to detect all microorganisms, D
Staining with API was performed. Hybridization,
The sample subjected to DAPI staining was observed with a fluorescence microscope and photographed. Thereafter, the result of detection by the fluorescent substance is compared with the result of DAPI staining by image processing or the like.
It was possible to detect only the target HADS-degrading bacteria by judging that those which were not stained with the above were contaminants.

Table 4 shows the results.

[0065]

[Table 4]

In probe 2, the HADS-degrading bacterium SH134 was used.
Fluorescence was observed only in -8, and no fluorescence was observed in other strains. In addition, EU, which is a normal probe for detecting bacteria,
In B338, fluorescence was observed in all strains.
From these, HADS-degrading bacteria SH13
It became clear that the 4-8 strain can be specifically detected.

According to this embodiment, the probes 1 and 2 are HA
It became clear that only DS-degrading bacteria could be specifically detected and quantified.

[Example 5] In this example, the FISH method (fluorescence in s
HADS-degrading bacteria were detected from the activated sludge by an itu hybridization method).

(1) Preparation of Fluorescent Probe In this example, each probe 1 designed and synthesized in Example 1 was used.
2 was labeled with TRITC (tetramethylrhodamine isothiocyanate) at the 5 ′ end.

(2) FISH method HADS-degrading strain SH143-8 was mixed with activated sludge so as to be 0 to 100% of the total number of bacteria, and a floc dispersed by ultrasonic treatment was used as a sample. . After the sample was immobilized in the same manner as in Example 4, the HADS-degrading bacterium SH134-8 in the sludge was detected by the probe 2. In addition, the thing which fluorescence was seen by DAPI staining was made into the total number of bacteria, and the ratio of the strain fluoresced by the probe 2 was plotted on the graph.

As a result, no bacteria were detected at the inoculation rate of 0%, but as the inoculation rate of the SH134-8 strain increased, the number of bacteria detected by fluorescence increased. It was found that the ratio (occupancy) of the detected strains to showed a substantially proportional relationship (FIG. 3).

In addition, Ruege, a fungus similar in classification,
Similarly, a positive correlation was found between the inoculation rate and the occupancy rate under the mixing conditions with ria algicola IAM14591.

As described above, according to this embodiment, the probe 2
The HADS degrading bacteria S in the mixed bacteria group such as sludge
It became clear that H134-8 can be specifically detected and measured.

The probe 1 can be used in the same manner.

According to the method of this example, the presence or absence of HADS-degrading bacteria in a mixed bacterium such as activated sludge can be confirmed quickly, specifically and easily, and at the same time, the ratio of HADS-degrading bacteria to all bacteria can be determined. You can figure out.

[0076]

According to the present invention, HADS-degrading bacteria can be detected, quantified quickly, specifically and easily.

[0077]

[Sequence List] SEQUENCE LISTING <110> Mitsubishi Heavy Industries <120> Method for detecting and determining HADS degrading microorganisms <160> 1 <170> PatentIn Ver. 2.0 <210> 1 <211> 1430 <212> DNA <213> Roseobacter sp. <400> 1 tagagtttga tcctggctca gaacgaacgc tggcggcagg cctaacacat gcaagtcgag 60 cgcgcccttc ggggtgagcg gcggacgggt gagtaacgcg tgggaacgtg ccctcttctg 120 cggaatagcc actggaaacg gtgagtaata ccgcatacgc ccttcggggg aaagatttat 180 cggaggagga tcggcccgcg ttggattagg tagttggtgg ggtaatggcc taccaagccg 240 acgatccata gctggtttta gaggatgatc agccacactg ggactgagac acggcccaga 300 ctcctacggg aggcagcagt ggggaatctt agacaatggg cgcaagcctg atctagccat 360 gccgcgtgag tgacgaaggc cctagggtcg taaagctctt tcgcctgtga tgataatgac 420 agtagcaggt aaagaaaccc cggctaactc cgtgccagca gccgcggtaa tacggagggg 480 gttagcgttg ttcggaatta ctgggcgtaa agcgcgcgta ggcggactgg aaagttgggg 540 gtgaaatccc ggggctcaac cccggaactg cctccaaaac tctcagtctg gagttcgaga 600 gaggtgagtg gaattccgag tgtagaggtg aaattcgtag atattcggag gaacaccagt 660 ggcgaaggcg gctcactggc tcgatactga cgctgaggtg cgaaagtgtg gggagcaaac 720 aggattagat accctggtag tccacaccgt aaacgatgaa tgccagtcgt cggcaagcat 780 gcttgtcggt gacacaccta acggattaag cattccgcct ggggagtacg gccgcaaggt 840 taaaactcaa aggaattgac gggggcccgc acaagcggtg gagcatgtgg tttaattcga 900 agcaacgcgc agaaccttac caacccttga catggatatc gcgggaccag agatggttct 960 ttcagttcgg ctggatatca cacaggtgct gcatggctgt cgtcagctcg tgtcgtgaga 1020 tgttcggtta agtccggcaa cgagcgcaac ccacatcccc agttgccagc aggtcatgct 1080 gggcactctg tggaaactgc ccgtgataag cgggaggaag gtgtggatga cgtcaagtcc 1140 tcatggccct tacgggttgg gctacacacg tgctacaatg gtggtgacaa tgggttaatc 1200 cccaaaagcc atctcagttc ggattggggt ctgcaactcg accccatgaa gtcggaatcg 1260 ctagtaatcg cgtaacagca tgacgcggtg aatacgttcc cgggccttgt acacaccgcc 1320 cgtcacacca tgggagttgg gtctacccga cggccgtgcg ctaaccctta cgggaggcag 1380 cggaccacgg taggctcagc gactggggtg aagtcgtaac aaggtagcca 1430 <210> 2 <211> 19 <212 > DNA <213> Roseobacter sp. <400> 2 cttctgcgga atagccact 19 <210> 3 <211 > 16 <212> DNA <213> Roseobacter sp. <400> 3 tggtcccgcg atatcc 16 <210> 4 <211> 16 <212> DNA <213> Roseobacter sp. <400> 4 uggucccgcg auaucc 16 <210> 5 <211 > 18 <212> DNA <213> Artificial Sequence <400> 5 gctgcctccc gtaggagt 18

[Brief description of the drawings]

FIG. 1. HADS-degrading bacterium SH13 by the method of the present invention.
The photograph which shows the electrophoresis which demonstrates that the 4-8 strain can be specifically amplified.

FIG. 2 is a diagram showing the relationship between the number of inoculated cells and the number of detected cells by MPN-PCR.

FIG. 3 is a diagram showing specific detection and quantification of HADS-degrading bacteria in activated sludge by FISH.

 ──────────────────────────────────────────────────続 き Continued on the front page F term (reference) 4B024 AA11 AA17 CA09 GA27 HA12 4B063 QA01 QA18 QQ06 QQ50 QR08 QR32 QR55 QR62 QS25 QS34 QX01

Claims (6)

[Claims] 1. The following group: a polynucleotide having the nucleotide sequence of SEQ ID NO. 1, a polynucleotide having the nucleotide sequence of SEQ ID NO. 2, a polynucleotide having the nucleotide sequence of SEQ ID NO. 3, and having a nucleotide sequence of SEQ ID NO. A polynucleotide selected from polynucleotides. 2. Use of a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 2 and a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 3 as primers. 3. Use of a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 3 and a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 4 as probes. 4. A method for specifically detecting or quantifying a HADS-degrading bacterium, wherein the method comprises the steps of: HAD contained in a nucleic acid encoding 16S rRNA;
Amplifying a region specific to S-decomposing bacteria, and detecting or quantifying the amplified region to obtain H
Specifically detecting or quantifying ADS-degrading bacteria. 5. The region according to claim 4, wherein the region is amplified using a primer pair consisting of a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 2 and a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 3. Method. 6. A method for specifically detecting or quantifying a HADS-degrading bacterium, wherein the HAD contained in a nucleic acid encoding 16S rRNA is provided.
A method comprising the steps of: hybridizing a region specific to S-degrading bacteria to HADS-degrading bacteria; and detecting or quantifying the hybridized region to specifically detect or quantify HADS-degrading bacteria.