JP2011045297A - Primer, primer set, eikelboom type 021n detection method and biological treatment method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To accurately detect and determine filamentous bulking-causing bacteria to suppress proliferation of the bacteria and to prevent solid-liquid separation trouble. <P>SOLUTION: There is provided a biological treatment method performing a step of detecting Eikelboom type 021N in the sludge by using a forward primer and a reverse primer having a specific base sequence to amplify the nucleic acids of Eikelboom type 021N. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a primer or primer set used for nucleic acid amplification, and further relates to a method for detecting Eikeboom type 021N and a biological treatment method.

Conventionally, sludge containing bacteria that can be used for biological water treatment, such as activated sludge, has been widely used for sewage treatment, wastewater treatment in food factories, chemical factories, and the like.
In such a biological treatment method, sewage or industrial wastewater is introduced into the biological treatment tank as treated water and brought into contact with activated sludge (hereinafter also simply referred to as “sludge”) contained in the biological treatment tank. A treatment target material is decomposed, and treated water in which the treatment target material is reduced is caused to flow down from the biological treatment tank.
The treated water after such a biological treatment process is carried out in a biological treatment tank often contains the sludge as a floating solid (hereinafter also referred to as “SS” (suspended solid)). In order to remove sludge, conventionally, the treated water flowed down from the biological treatment tank is introduced into a settling tank to precipitate and separate the sludge, or the treated water is subjected to membrane separation with an ultrafiltration membrane or the like. Has been done.

In such a solid-liquid separation step, it is conventionally known that a solid-liquid separation failure is caused by bulking due to filamentous bacteria.
For example, in a sedimentation tank, (1) filamentous bulking caused by filamentous bacteria such as Eikeboom type021N and Sphaerotilus natans , (2) floating sludge and septic sludge, and (3) disintegrated sludge with deteriorated cohesiveness It is known that a solid-liquid separation failure is caused (see Non-Patent Document 1 below).

Twenty or more kinds of filamentous bacteria appearing in general biological treatment methods are known, but among them, there are not many that cause solid-liquid separation troubles that cause problems in sedimentation tanks, Eikeboom type021N, Sphaerotilus natans , Microthrix paravicella and the like.
Since the cause of its appearance varies depending on the type, identification of the filamentous bacteria is necessary for the countermeasure against the solid-liquid separation disorder (filamentous bulking) caused by the filamentous bacteria (see Non-Patent Document 2 below).

Conventionally, the identification of filamentous bacteria has been mainly carried out using a phase contrast microscope based on the classification and identification method modified by Jenkins, Richard et al. Based on the morphological features and methods proposed by Eikebroom.
However, this method requires skill, and it is difficult to accurately identify even an expert even if the feature is difficult to understand or the form may change depending on the appearance environment.

With the recent development of molecular biological techniques, for example, specific bacterial species constituting sludge can be shortened by the polymerase chain reaction (hereinafter also referred to as “PCR”) or the fluorescence in situ hybridization (hereinafter also referred to as “FISH”) method. It is possible to accurately detect the amount of time and to quantify the quantity.
For example, Non-Patent Document 3 listed below describes a PCR primer for detection and quantification of Eikebroom type 021N, which is one of the main causative bacteria of solid-liquid separation (filamentous bulking).
Further, for example, the following Non-Patent Document 4 describes the detection of Eikeboom type 021N by the FISH method.

"Microbiology for the latest environmental purification" Kohei Inamori, Kodansha Scientific, December 10, 2008, first edition issued “Biological Diagnosis of Color Illustrated Wastewater Treatment” by Nishihara Environmental Technology, Industrial Water Research Committee, November 11, 2008 H. Vervaeren, K.M. De Wilde, J. et al. Matthys, N.M. Boon, L.M. Raskin, W.M. Verstraete (2005) Appl. Microbiol Biotechnol. 68: 695-704 T.A. Kanagawa, Y .; Kamagata, S .; Aruga, T .; Kohno, M .; Horn, M.M. Wagner (2000) Appl. Environ. Microbiol. 66: 5043-5052

  The present inventor tried to detect Eikeboom type021N from activated sludge using the PCR primers described in Non-Patent Document 3 above. As a result, in addition to the bacterium, a nucleic acid highly homologous to a bacterium belonging to the genus Acinetobacter was also found. It has been amplified and found to have a problem with its accuracy.

That is, it is difficult to say that the primer described in Non-Patent Document 3 can accurately detect or quantify Eikeboom type021N, and conventionally, a primer suitable for the detection and quantification of Eikeboom type021N has been provided. Have the problem of not.
Therefore, it is difficult to improve the accuracy in the Eikeboom type 021N detection method, and it is difficult to take measures against solid-liquid separation failures in the biological treatment method.

  An object of the present invention is to provide a primer and a primer set suitable for detection of Eikeboom type021N, and to improve the detection accuracy of Eikeboom type021N, and to prevent solid-liquid separation failure in a biological treatment method.

  As a result of intensive studies to solve the above problems, the present inventors have found a primer suitable for the detection of Eikeboom type 021N and have completed the present invention.

  That is, the present invention relating to a primer is used for amplification of a nucleic acid of Eikeboom type 021N and is characterized by having any one of the nucleotide sequences of SEQ ID NOs: 1 to 4.

  In addition, the present invention relating to the primer set is used for amplification of the nucleic acid of Eikeboom type021N, and has a forward primer having the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3 and the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4. The reverse primer which is used is used.

  And this invention which concerns on the detection method of Eikeboom type021N WHEREIN: The forward primer which has the base sequence of sequence number 1 or sequence number 3, and the reverse primer which has the base sequence of sequence number 2 or sequence number 4 It is characterized by detecting Eikeboom type 021N in a sample by amplifying the nucleic acid of Eikeboom type021N.

  Furthermore, the present invention relating to a biological treatment method includes a biological treatment step for biologically treating water to be treated using sludge, and a solid-liquid separation for removing the sludge by solid-liquid separation of the treated water after the biological treatment step. A biological treatment method in which a separation step is performed, and a forward primer having a base sequence of SEQ ID NO: 1 or SEQ ID NO: 3 and a reverse primer having a base sequence of SEQ ID NO: 2 or SEQ ID NO: 4 A step of detecting Eikeboom type021N in the sludge by amplifying the nucleic acid of Eikeboom type021N using the method is further performed.

According to the present invention, it is possible to accurately detect Eikebroom type021N, which is one of the main causative bacteria of solid-liquid separation trouble (filamentous bulking), which is a problem in biological treatment methods.
Therefore, the biological treatment conditions (apparatus operating conditions and the like) can be changed so that the growth of the bacteria can be suppressed, and the occurrence of solid-liquid separation failure can be suppressed.

An agarose gel electrophoresis photograph of a PCR product using a conventional primer. The agarose gel electrophoresis photograph of the PCR product using the primer of this invention.

  About the embodiment of the present invention, sewage or industrial wastewater is introduced into a biological treatment tank as treated water, and a biological treatment process for decomposing the treatment target substance by contacting with the activated sludge accommodated in the biological treatment tank; An example of a biological treatment method will be described in which the treated water after the biological treatment step is introduced into a sedimentation tank to precipitate the activated sludge and the solid-liquid separation step of allowing the supernatant liquid to flow down from the precipitation tank.

In the biological treatment method of the present embodiment, the step of sampling the sludge in the biological treatment tank or the sedimentation tank and detecting Eikelboom type021N by PCR on the sludge is performed separately.
The detection of Eikebroom type021N by PCR has an advantage that the filamentous bulking-causing bacteria can be detected and quantified accurately and easily without using a skilled engineer, such as a detection method based on morphological characteristics.
And the tendency of the increase / decrease of Eikeboom type021N can be grasped | ascertained regularly by detecting Eikeboom type021N in sludge.
That is, it becomes possible to accurately detect and quantify the filamentous bulking-causing bacteria, thereby suppressing the growth of the bacteria and preventing solid-liquid separation troubles.

Possible causes of the increase in Eikeboom type 021N include inflow of septic water, insufficient dissolved oxygen concentration in the aeration tank, insufficient nutrient (especially nitrogen), and high load operation.
If the abundance ratio of Eikeboom type 021N is approximately 1% or more of the total number of bacteria and this bacterium is considered to be the main cause of filamentous bulking, the above causes may be investigated and improved first. It can be cited as a countermeasure.
In addition, by providing an anaerobic or anoxic tank so that the hydrodynamic residence time (HRT) is 2 to 3 hours, or by performing an intermittent aeration operation, the filamentous bulking-causing bacterium Eikeboom type021N is reduced. And the solid-liquid separation trouble in the precipitation tank can be suppressed.
If you want to improve the settleability of activated sludge as soon as possible, it can be improved by killing the bacteria by introducing sodium hypochlorite or a commercial bulking preparation.

The detection of the Eikeboom type 021N can be performed by real-time PCR or the like.
As this real-time PCR, a method of intercalating a fluorescent dye or a method using a probe to which a fluorescent dye is bound may be employed.
Further, for example, this real-time PCR can be performed using a real-time PCR apparatus such as Applied Biosystems 7300 (Applied Biosystems Japan Co., Ltd.).

In this real-time PCR, Eik021Nf1 (SEQ ID NO: 1 5′-ATAGAGATCCGGAAGGAACATCAGT-3 ′) or Eik021Nf2 (SEQ ID NO: 5′-ATGCATAGAGATCGGGAAGGAAC-3 ′) designed by the present inventor was used as a forward primer, and a reverse primer It is important to use Eik021Nr1 (SEQ ID NO: 5'-TAATGCGTTAGCTGCACCAC-3 ') or Eik021Nr2 (SEQ ID NO: 5'-AATGCGGTTAGCTGCACCAC-3') designed by the present inventors.
By using the primer (primer set), it is possible to carry out amplification of DNA having high homology with the DNA of Eikeboom type 021N while suppressing amplification of other nucleic acids.
Therefore, the detection and quantification of Eikeboom type 021N can be performed with higher accuracy than in the case of using a conventional primer.

  EXAMPLES Next, although an Example is given and this invention is demonstrated in more detail, this invention is not limited to these.

(Method of verification)
1) Detection of Eikeboom type 021N using an existing PCR primer . PCR primer "21Nf"(5'-CGTAGGCGGTTTAAGTC (A / G) GAT-3 ') and "21Nr"(5'-CCGACGGCTGTGTATGATGAT-3) and group 21 Eikboom type021N devised by Vervaeren et al. In each case, Eikeboom type 021N was detected from the sludge used in the biological treatment of sewage.

PCR was performed under the following conditions.
That is, from 1 ng of DNA purified from sludge in a sewage treatment plant using Fast DNA SPIN kit for SOI (manufactured by Qbiogene), AmpliTaq Gold PCR Master Mix (Applied Biosystems Japan Co., Ltd.) and 0.25 μM of each PCR primer It carried out on reaction conditions shown in Table 1 using the comprised reaction liquid.

2) PCR primer design database (DDBJ (DNA Data Bank of Japan): http://www.ddbj.nig.ac.jp/Welcome-j.html, NCBI ( Nation) for Biotechnology): http://www.ncbi.nlm.nih.gov/) A new PCR primer was designed based on the 16S rRNA gene sequence of Eikeboom type021N registered in http://www.ncbi.nlm.nih.gov/).
PCR primer design was performed using Primer BLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastDescAd).

3) Verification of novel PCR primers Using the four primer sets shown in Table 3, the 16S rRNA gene of Eikeloom type021N was amplified using DNA as a template.
Subsequently, after cloning each amplification product, the nucleotide sequence is determined, homology analysis is performed with the known bacterial 16S rRNA gene registered in the database, and the amplification product is the 16S rRNA gene of Eikeboom type021N, that is, the purpose and It was confirmed that only the genes to be amplified were amplified, and the genes of non-target bacteria were not amplified.
That is, PCR conditions in Table 2 were prepared using a reaction solution composed of 1 ng of DNA purified in 1) above, AmpliTaq Gold PCR Master Mix (Applied Biosystems Japan Co., Ltd.) and 0.25 μM of each PCR primer set. PCR was performed.

Subsequently, these amplification products were purified using a QIAquick PCR Purification Kit (manufactured by Qiagen), and then cloned into a pCR4-TOPO vector using a TOPO TA cloning kit (manufactured by Invitrogen) to transform Escherichia coli.
Twelve obtained transformants were arbitrarily selected, and the recombinant plasmid was purified using QIAquick Spin Miniprep Kit (manufactured by Invitrogen Corporation).
Using these plasmids as templates, the base sequence of the PCR product was determined using BigDye Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems Japan Co., Ltd.).
The determined base sequence is the 16S rRNA gene of an existing bacterium registered on a database (DDBJ (DNA Data Bank of Japan): http://www.ddbj.nig.ac.jp/Welcome-j.html). By determining the base sequence and homology, it was estimated from which bacterial species the amplified PCR product was derived.

  Examination of quantitative PCR conditions using 4 sets of new primer sets shown in Table 3, and homology analysis with 16S rRNA gene of known bacteria of PCR product when quantitative PCR was performed under optimum conditions, and amplification product was Eikeboom type021N It was confirmed that it was a 16S rRNA gene, that is, only the target gene was amplified, and the gene of the non-target bacteria was not amplified.

4) Examination and verification of quantitative PCR conditions using a new PCR primer Reaction solution composed of 1 ng of DNA purified in 1) above, 300 nM Forward primer and Reverse primer, and QuantTect SYBR Green PCR Kit (manufactured by Qiagen Co., Ltd.) Quantitative PCR was performed under the conditions shown in Table 4.
The PCR reaction was performed using an Applied Biosystems 7300 real-time PCR system (manufactured by Applied Biosystems Japan Co., Ltd.).
Furthermore, the PCR product performed with each primer set was purified and cloned by the same method as in 3) above, and 24 transformants obtained were arbitrarily selected and the nucleotide sequence was determined.
The determined nucleotide sequence was determined to be homologous with a known bacterial 16S rRNA gene on the database in the same manner as in 3) above.

5) Quantification of Eikboom type 021N in Sewage Treatment Plant Activated Sludge by Quantitative PCR Using Novel PCR Primer Using the four sets of primer sets shown in Table 3, detection of Eikeboom type 021N in activated sludge from sewage treatment plant Quantification was performed.
Specifically, 1 ng of the DNA purified in the above (Step 1), 300 nM Forward primer, Reverse primer and Quantitect SYBR Green PCR Kit (manufactured by Qiagen) were used for quantitative PCR under the conditions shown in Table 4. Went.
The PCR reaction was performed using an Applied Biosystems 7300 real-time PCR system (manufactured by Applied Biosystems Japan Co., Ltd.).

(inspection result)
1) Detection of Eikebell type 021N using existing PCR primers FIG. 1 shows the results of agarose gel electrophoresis of PCR amplification products.
lane 1 is a 100 bp molecular weight marker, lane 2 is a negative control PCR amplification product containing no DNA, and lane 3 is a PCR amplification product of DNA purified from activated sludge in a sewage treatment plant.
In PCR using DNA purified from activated sludge from a sewage treatment plant as a template, an expected amplification product (arrow) of about 200 bp was obtained.
Therefore, in order to verify whether or not only the 16S rRNA gene of the desired Eikeboom type 021N was amplified by this PCR, the PCR product was cloned and its nucleotide sequence was determined.
The determined base sequence is homologous to an existing base sequence registered on a database (DDBJ (DNA Data Bank of Japan): http://www.ddbj.nig.ac.jp/Welcome-j.html). Was used to estimate from which bacterial species the amplified PCR product was derived.
Table 5 shows the results of the homology search.

As shown in Table 5, only 11 clones of the target Eikeboom type021N among the 11 PCR product clones were amplified, and the other 16S rRNA genes from other bacteria were amplified.
From the above results, the above-mentioned H.P. The accuracy of PCR primers devised by Vervaeren et al. Was found to be low and not suitable for accurate detection and quantification of Eikeboom type 021N.

2) Design of PCR primers for detection and quantification of Eikeboom type 021N The accuracy of the PCR primer devised by Vervaeren et al. Was found to be low, and it was found that there was a risk of detecting and quantifying bacteria other than Eikeboom type021N by mistake.
Therefore, databases (DDBJ (DNA Data Bank of Japan): http://www.ddbj.nig.ac.jp/Welcome-j.html and NCBI (National Center for Biotechnology): http: //www.nw. The new PCR primers shown in Table 6 were designed based on the sequence of the 16S rRNA gene of Eikeboom type021N registered in .nih.gov /).

3) Verification of novel PCR primers FIG. 2 shows an agarose gel electrophoresis photograph of PCR products amplified using the four PCR primer sets shown in Table 3 above.
lane 1 is a 100 bp molecular weight marker, and lane 2 is No. 1 primer set, lane 3 is No. 2 primer set, lane 4 is No. 3 primer set, lane 5 is No. The PCR amplification product when PCR is performed using the 4 primer set is shown.
In all PCRs, the expected amplification product (arrow) of approximately 170 bp was obtained.

Next, in order to verify that only the 16S rRNA gene of the desired Eikeboom type021N was amplified by PCR using these PCR primers, the PCR product was cloned and its nucleotide sequence was determined.
The determined base sequence is homologous to an existing base sequence registered on a database (DDBJ (DNA Data Bank of Japan): http://www.ddbj.nig.ac.jp/Welcome-j.html). By determining the sex, it was estimated from which bacterial species the amplified PCR product was derived.
Table 7 shows the results of the homology search.

As shown in Table 7, it was confirmed that all 8 PCR product clones were amplified only for the 16S rRNA gene of the bacteria having 98% or more homology with the target Eikeboom type021N (Thiotrix eikeboomii). .
From the above results, it was verified that the primer (primer set) according to the present invention is a highly accurate primer capable of detecting only the target bacteria.

4) the presence or absence of the appearance of the negative control when performing the quantitative PCR and the standard sample as a template under the conditions shown in the condition examination of quantitative PCR using a novel PCR primers and verification table 8, R ² value of the calibration curve, the amplification efficiency Table 9 shows the results of the lower limit of quantification.

As shown in this table, amplification of the negative control was not observed in quantitative PCR using all primer sets, the R ² value of the calibration curve was 0.99 or more, and the amplification efficiency was 90% or more. It was.
The lower limit of quantification was 12.8 copies / reaction and a small amount of 16S rRNA gene of the bacterium could be detected.

Furthermore, by obtaining the homology of the base sequence of the quantitative PCR product with the existing base sequence registered on the database (DDBJ (DNA Data Bank of Japan)), the amplified PCR product is derived from which bacterial species. Estimated what to do.
The results are shown in Tables 10 to 13.

As shown in Tables 10 to 13, all quantitative PCR products subjected to homology analysis have 97% or more homology with the target Eikelboom type021N (Thiotrix eikeboomii), and only the bacterial 16S rRNA gene is amplified. Confirmed that.
From the above results, it was confirmed that the primer of the present invention was a highly accurate primer useful for quantification of Eikeboom type 021N.

5) Quantification of Eikeroom type 021N in MLSS (Mixed liquid Suspended Solid) of Sewage Treatment Plant by Quantitative PCR Using Novel PCR Primer Among the four sets of new PCR primer sets shown in Table 3, No. Using the primer set of 1 (Eik021Nf1 / Eik021Nr1), the abundance of Eikeboom type 021N in the MLSS of the sewage treatment plant was determined under the conditions of Table 4.
The results are shown in Table 14.

  From the specific examples as described above, it can be seen that according to the present invention, a primer (primer set) suitable for the detection and quantification of Eikeboom type021N, which is a causative bacterium of filamentous bulking, can be provided.

Claims (4)

  A primer which is used for amplification of a nucleic acid of Eikeboom type021N and has any one of the nucleotide sequences of SEQ ID NOs: 1 to 4.   A forward primer having a base sequence of SEQ ID NO: 1 or SEQ ID NO: 3 and a reverse primer having a base sequence of SEQ ID NO: 2 or SEQ ID NO: 4 are used for the amplification of a nucleic acid of Eikeboom type021N A primer set characterized by   In a sample by amplifying a nucleic acid of Eikeboom type021N using a forward primer having the base sequence of SEQ ID NO: 1 or SEQ ID NO: 3 and a reverse primer having the base sequence of SEQ ID NO: 2 or SEQ ID NO: 4 A method for detecting Eikeboom type021N, comprising: detecting Eikeboom type021N. A biological treatment method in which a biological treatment process for biologically treating water to be treated using sludge and a solid-liquid separation process for solid-liquid separation of the treated water after the biological treatment process and removing the sludge are performed. There,
The sludge is obtained by amplifying the nucleic acid of Eikeboom type021N using a forward primer having the base sequence of SEQ ID NO: 1 or SEQ ID NO: 3 and a reverse primer having the base sequence of SEQ ID NO: 2 or SEQ ID NO: 4. A biological treatment method, wherein the step of detecting Eikeboom type 021N is further performed.