JP5779440B2 - Method for detecting benzene-degrading bacteria - Google Patents

Description

The present invention relates to a method for detecting an Azoarcus sp . DN11 strain that degrades benzene under both aerobic and anaerobic conditions using a benzene monooxygenase gene as an index.

  Many petroleum components including benzene are known to be decomposed in an aerobic environment by indigenous bacteria that live in the ground. A biostimulation method that activates petroleum-degrading bacteria by supplying oxygen and nutrient salts into the soil has been put to practical use as a purification technique for petroleum-contaminated ground that uses this property. However, since soil and groundwater contaminated with petroleum components such as benzene are usually in an anaerobic environment with anoxic conditions, supply of air (oxygen) to form an aerobic environment suitable for the growth of petroleum degrading bacteria. is necessary. For this reason, purification using aerobic microorganisms requires air supply facilities and a large amount of energy investment, resulting in high costs.

On the other hand, the Azoarcus sp. DN11 strain is a facultative anaerobic denitrifying bacterium characterized by its ability to degrade benzene both aerobically and anaerobically. This is the second example (Non-Patent Document 1). Therefore, by supplying the Azoarcus sp . DN11 strain to the benzene-contaminated site, bioaugmentation that purifies the anaerobic state without changing the redox potential in the ground is possible. Bioaugmentation cleans soil contamination by artificially culturing microorganisms collected at other locations and injecting them into certain sections of the contamination site when microorganisms that degrade the contamination do not exist in the soil to be purified. When implementing this method, it is necessary to monitor specific microorganisms introduced at the time of purification and confirm the purification effect and safety.

  In general, as a method for confirming the progress of bioremediation at a benzene-contaminated site, benzene concentration tracing, measurement of the total number of bacteria, measurement of the number of viable bacteria (plate method, MPN method) and the like are known. However, the trace of benzene concentration is indistinguishable from physical reduction such as microbial degradation, diffusion dilution, and volatilization, the count of total bacteria is indistinguishable from viable and dead, and the count of viable bacteria is 1 There was a problem of spending more than a week.

As another method for confirming the progress of bioremediation, a method for detecting the base sequence of a 16S rRNA gene of a microorganism by a PCR method is also known. A specific sequence was also found in the 16S rRNA gene in the Azoarcus sp . DN11 strain, but the 16S rRNA gene is possessed by all prokaryotes, and the sequence difference between closely related species is particularly small. There was a question about its specificity (Non-patent Document 2). In addition, in order to detect specific microorganisms using the 16S rRNA gene as a target, it has been necessary to perform complicated operations such as cloning and sequencing the fragments amplified by PCR one by one.

Moreover, in order to detect and monitor pollutant-purifying microorganisms, there is also a method using as an index a gene involved in the purification, an aromatic ring hydroxylase dioxygenase gene involved in aromatic compound purification (Patent Document 1), cyanide The report has already been made on the rhodanase gene (Patent Document 2) involved in compound purification. There have been many reports on genes related to aerobic benzene degradation so far, and the Azoarcus sp . DN11 strain can also aerobically degrade benzene. However, there has been no attempt to identify an aerobic benzene-degrading gene possessed by Azoarcus sp . DN11 strain and use it for detection of the strain as an index.

JP-A-9-234070 JP2008-283890

Kasai Y et al., Appl. Environ. Microbiol. 2006, 72, p.3586-3592 Kasai Y et al., Environ. Sci. Technol. 2007, 41, p.6222-6227

An object of the present invention is to provide a means for accurately and rapidly detecting and quantifying Azoarcus sp. DN11 strain capable of degrading benzene under both aerobic and anaerobic conditions from a complex biological system.

As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that a universal primer for an aerobic benzene degrading gene (Baldwin BR et al. Appl. Environ. Micobiol. 2003, 69, p.3350-3358). ) Was used to carry out PCR, cloning, and sequence analysis from the genomic DNA of Azoarcus sp . DN11 strain, and it was revealed that the benzene monooxygenase gene shown in SEQ ID NO: 1 was retained. When BLASTN and BLASTP analyzes were performed on the gene information, a region specific to DN11 strain was found. Furthermore, when a primer and a probe were designed based on the sequence of the specific region and real time PCR was performed using them, it was confirmed that the DN11 strain could be accurately detected and quantified. The present invention has been completed based on such findings.

That is, the present invention includes the following inventions.
(1) A method for detecting Azoarcus sp. DN11 strain, using DNA extracted from an environmental sample as a template, and (i) all or part of the region from position 219 to 248 in the base sequence shown in SEQ ID NO: 1. A primer set comprising a sense primer comprising a base sequence and (ii) an antisense primer comprising a complementary sequence of all or part of the base sequence of positions 339 to 368 in the base sequence represented by SEQ ID NO: 1. The method as described above, comprising the steps of PCR amplification of the benzene monooxygenase gene using and detecting the amplified DNA fragment.
(2) The sense primer according to (1), wherein the sense primer is an oligonucleotide having a base sequence represented by SEQ ID NO: 2, and the antisense primer is an oligonucleotide having a base sequence represented by SEQ ID NO: 3. Method.

(3) The detection of the DNA fragment is carried out by hybridization with a probe comprising an oligonucleotide consisting of the whole or a part of the region from position 282 to 311 in the nucleotide sequence shown in SEQ ID NO: 1 or a partial nucleotide sequence thereof or a complementary sequence thereof. The method according to (1) or (2).
(4) The method according to (3), wherein the oligonucleotide is an oligonucleotide having the base sequence represented by SEQ ID NO: 4.
(5) The method according to any one of (1) to (4), wherein the DNA fragment is detected quantitatively by real-time PCR.

(6) A method of monitoring the Azoarcus sp. DN11 strain by quantifying the number of Azoarcus sp . DN11 strains in the benzene-contaminated site using the method described in (5).
(7) A method for quantifying the number of Azoarcus sp . DN11 strains in a benzene-contaminated site using the method described in (5) and evaluating the degree of purification or repair of the benzene-contaminated site by the Azoarcus sp. DN11 strain.
(8) A primer set for detection of Azoarcus sp. DN11 strain, comprising: (i) a sense primer comprising all or part of the base sequence of positions 219 to 248 in the base sequence represented by SEQ ID NO: 1, ii) The above primer set comprising an anti-primer consisting of a complementary sequence of all or part of the region of positions 339 to 368 in the base sequence shown in SEQ ID NO: 1.

(9) The sense primer is an oligonucleotide having a base sequence represented by SEQ ID NO: 2, and the antisense primer is an oligonucleotide having a base sequence represented by SEQ ID NO: 3, Primer set.
(10) A probe for detection of Azoarcus sp. DN11 strain, which comprises an oligonucleotide consisting of all or a part of the region of positions 282 to 311 in the nucleotide sequence shown in SEQ ID NO: 1 or a complementary sequence thereof Above probe.
(11) The probe according to (10), wherein the oligonucleotide is an oligonucleotide having the base sequence represented by SEQ ID NO: 4.
(12) A kit for detecting Azoarcus sp. DN11 strain, comprising the primer set according to (8) or (9) and the probe according to (10) or (11).

According to the present invention, there is provided a method for accurately and rapidly detecting and quantifying Azoarcus sp. DN11 strain capable of degrading benzene under both aerobic and anaerobic conditions using a benzene monooxygenase gene as an index. Since there is no other microbial homologue having high homology with the base sequence of the benzene monooxygenase gene of the Azoarcus sp . DN11 strain, by using the method of the present invention, the Azoarcus sp . Can be specifically detected even in natural flora containing a large number of. In addition, by using the method of the present invention, in conducting bioaugmentation with Azoarcus sp . DN11 strain on benzene contaminated sites, confirmation of growth and activity of Azoarcus sp. DN11 strain, monitoring of purification treatment status, etc. Is possible.

The amino acid sequence of benzene monooxygenase of Azoarcus sp. DN11 strain, the base sequence encoding the amino acid sequence, and the design regions of primers and probes on each sequence are shown.

The detection method of the benzene-degrading bacterium Azoarcus sp . DN11 strain (hereinafter sometimes simply referred to as “DN11 strain”) of the present invention uses DNA extracted from an environmental sample as a template, and benzene present in the DN11 strain genome. It includes a step of PCR amplification of a partial base sequence of the benzene monooxygenase gene using a base sequence of a specific region on the monooxygenase gene as a primer, and a step of detecting the amplified DNA fragment.

  The above benzene monooxygenase gene is a gene involved in aerobic degradation of benzene having the base sequence shown in SEQ ID NO: 1, and is characterized by a large base sequence difference from related species compared to the 16S rRNA gene. (BLASTN search: http://blast.ddbj.nig.ac.jp/top-j.html).

  First, the primer used in the amplification step is designed in a specific region of the benzene monooxygenase gene, and consists of all or part of the base sequence of the region. The specific region of the benzene monooxygenase gene is the region at positions 219 to 248 and 339 to 368 in the base sequence shown in SEQ ID NO: 1, and is present at positions 73 to 82 in the amino acid sequence shown in SEQ ID NO: 5, respectively. Trp-Ile-Val-Ala-Gln-Phe-Glu-Arg-Ala-Leu (SEQ ID NO: 6), Tyr-Trp-Arg-Pro-Thr-Val-Trp-Trp-Asn present at positions 113-122 -Pro (SEQ ID NO: 7) is encoded.

  When a partial base sequence of the above region is used as a primer, the length is not particularly limited, but is about 15 to 25 bases, preferably about 20 to 25 bases.

  In PCR amplification, the above primers are combined and used as a primer set. For the sense primer, a base sequence encoding an amino acid sequence represented by Trp-Ile-Val-Ala-Gln-Phe-Glu-Arg-Ala-Leu (SEQ ID NO: 6) located upstream is used, The antisense primer uses a base sequence encoding the amino acid sequence represented by Tyr-Trp-Arg-Pro-Thr-Val-Trp-Trp-Asn-Pro (SEQ ID NO: 7) located on the downstream side. The base sequences encoding the amino acid sequences 6 and 7 may be used as primers as they are, or primers may be designed based on them.

  As a preferred example of the sense primer used in the method of the present invention, an oligonucleotide consisting of 5′-TGGATTGTGGCGCAGTTCGAACGCGCCCTG-3 ′ (SEQ ID NO: 2), and as a preferred example of the antisense primer, 5′-CGGATTCCACCATACCGTCGGGCGCCAGTA-3 ′ (SEQ ID NO: 3) An oligonucleotide consisting of These oligonucleotides may be modified (deletion, substitution, insertion, or addition) in the sequence as long as they have substantially the same function. The number of oligonucleotides to be deleted is not particularly limited, but is usually 5 or less, preferably 3 or less, and more preferably 1. The nucleotide addition may be on the 3 'side or 5' side.

  A partial base sequence of benzene monooxygenase encoding the amino acid sequence sandwiched between the amino acid sequence of SEQ ID NO: 6 and the amino acid sequence of SEQ ID NO: 7 on benzene monooxygenase by performing PCR using the above-described sense primer and antisense primer in combination Can be amplified.

  The sense primer and antisense primer used in the method of the present invention are usually prepared by methods known in the art as oligonucleotide synthesis methods, for example, phosphotriester method, phosphodiester method, ethyl phosphoramidite method, etc. It can be synthesized using an automatic DNA synthesizer (for example, Model 394 manufactured by Applied Biosystems).

  The environmental sample to be detected is not particularly limited as long as it may be contaminated with benzene. However, soil contaminated with benzene, river bottom soil, groundwater, stored water, seawater, river water, Examples include water and activated sludge collected from wastewater treatment facilities (treatment facilities such as sewage, sewage, factory wastewater, and agricultural wastewater), soils around the wastewater treatment facilities, soils in shallow water and tidal flats, and soils in rivers and lakes. It is done.

  For extraction of DNA from environmental samples, any method known to those skilled in the art as nucleic acid extraction methods from microbial cultures may be used, for example, extraction with phenol / chloroform or extraction using a commercially available DNA extraction reagent. It can be carried out. Alternatively, the extraction may be performed using a commercially available column kit (QIAGEN QIAamp (registered trademark) DNA Blood Midi Kit, QIAGEN DNeasy (registered trademark) Tissue Kit, etc.).

  In the method of the present invention, PCR can be performed according to a conventional method. For example, in the presence of template DNA, the above-described sense primer and antisense primer, 4 types of bases (dNTP), and heat-resistant DNA polymerase, denaturation, annealing and extension are usually performed for 20 to 40 cycles. Do. Denaturation is a process for dissociating double-stranded DNA into single strands, and is usually performed at 94 to 96 ° C. for 15 seconds to 5 minutes. Annealing is a process of annealing a primer complementary to a single-stranded template DNA at a temperature below the Tm value set according to the primer used, for example, 50 to 65 ° C., for 30 seconds to 2 minutes. Perform the process. The extension reaction is a reaction for extending a primer along the template DNA, and is usually treated at 72 ° C. for 30 seconds to 10 minutes. The Tm value can be appropriately set by those skilled in the art based on the base sequence of the primer used. In the detection method of the present invention, the annealing temperature is preferably a Tm value of −5 ° C. or higher in order to guarantee specific binding of the primer. Such a series of PCR operations can be performed using a commercially available PCR kit or PCR apparatus according to the operating instructions. For example, GeneAmp PCR System 9700 (Applied Biosystems), GeneAmp PCR System 9600 (Applied Biosystems), or the like can be used as the PCR apparatus. In order to facilitate the detection of the PCR amplification product, a labeling substance may be added to the primer in advance, or a modified nucleotide (eg, digoxigenin (DIG) labeled nucleotide) may be used as a substrate during PCR.

Next, DNA fragments amplified by PCR are detected. The Azoarcus sp. DN11 strain in the sample can be detected and quantified based on the detection result of the DNA fragment (presence / absence of amplification, amount of amplification). "Detecting the Azoarcus sp. DN11 strain" as used herein, not only to determine whether there is Azoarcus sp. DN11 strain in environmental samples, Azoarcus sp. DN11 strain in environmental samples It also includes determining how much is present (ie quantitative detection).

  In the present invention, a DNA fragment (PCR product) amplified by PCR can be detected by a method commonly practiced in the art. Examples of the PCR product detection method include an electrophoresis method using an agarose gel or the like, a hybridization method using a labeled probe, a PCR-ELISA method, and the like, and a hybridization method using a labeled probe is preferable.

  As the probe, an oligonucleotide that can hybridize to the base sequence of the DNA fragment amplified by the above primer set and has a length of at least 10 bases, preferably 20 bases or more, more preferably 25 bases or more may be designed. . Specifically, a region at positions 282 to 311 in the base sequence shown in SEQ ID NO: 1 (Glu-Gln-Phe-Leu-Ala-Asp-Leu present at positions 94 to 103 in the amino acid sequence shown in SEQ ID NO: 5). Oligonucleotides consisting of all or part of the base sequence of -Asn-Glu-Thr (SEQ ID NO: 8) or a complementary sequence thereof can be used as a probe. A preferred example of the probe used in the method of the present invention is an oligonucleotide comprising 5′-GAACAGTTCCTGGCAGACCTCAATGAAACT-3 ′ (SEQ ID NO: 4), but the sequence is modified within the range having substantially the same function ( Deletions, substitutions, insertions or additions) may be added. The number of oligonucleotides to be deleted or the like is not particularly limited, but is usually 5 or less, preferably 3 or less, and more preferably 1. The nucleotide addition may be on the 3 ′ side or the 5 ′ side. In addition, as a probe labeling substance, a fluorescent substance, a radioisotope, a chemiluminescent substance, an enzyme, biotin, or the like well known in the art can be used.

  Examples of the hybridization method using a labeled probe include dot hybridization, real-time PCR, FISH method (fluorescence in situ hybridization method), and direct detection or quantitative detection is possible.

  Detection with a labeled probe can also be performed by a microarray method in which a detection target is fixed on a substrate such as a slide glass, silicon, or nylon film.

Real-time PCR is preferable in that the amplified DNA fragment can be detected quantitatively. Real-time PCR is a technique in which PCR is performed using a real-time PCR apparatus, and amplification by PCR is measured over time to quantify DNA as a template based on the amplification rate.
In real-time PCR, PCR amplification products are obtained by fluorescence, and fluorescence detection includes a method using an intercalator and a method using a fluorescently labeled probe (TaqMan method), both of which can be carried out according to conventional methods. In the intercalator method, when a reagent that binds to double-stranded DNA and emits fluorescence (SYBR Green I) is added to the PCR reaction system and PCR is performed, SYBR Green I binds to double-stranded DNA. By measuring the intensity of emitted fluorescence, the amount of amplified product can be monitored for each cycle. In the TaqMan method, PCR is performed by adding an oligonucleotide probe in which the 5 ′ end is modified with a fluorescent substance (such as FAM) and the 3 ′ end is modified with a quencher substance (such as TAMRA) to the PCR reaction system.

The present invention also provides a method for monitoring the Azoarcus sp. DN11 strain by quantifying the number of Azoarcus sp . DN11 strains in the benzene-contaminated site using the detection method described above, and the detection method described above. by quantifying Azoarcus sp. DN11 strain number of bacteria in the benzene contaminated sites, provides a method for evaluating the degree of purification or repair benzene contaminated sites by Azoarcus sp. DN11 strain. For example, by quantifying the number of DN11 strains present in or introduced into the benzene-contaminated site using the detection method described above, the behavior of the DN11 strain at the benzene-contaminated site and the total microbial community The ratio (dominance) to the can be monitored. If the amount of DN11 strain introduced into the benzene-contaminated site is adjusted based on this monitoring result (change in DN11 strain, etc.), more efficient bioremediation becomes possible. In addition, by simultaneously monitoring DN11 strain and benzene as a pollutant, it is possible to investigate the progress of purification or repair of benzene-contaminated sites by introducing DN11 strain.

  The detection method of the present invention may further include a step of determining the base sequence of the benzene monooxygenase gene product PCR-amplified as described above. The base sequence of the amplified DNA fragment can be determined by a known base sequence determination method such as dideoxy method. The base sequence of the benzene monooxygenase gene differs between homologous species or strains, and when compared between the same species, the difference is significantly higher than the base sequence of the 16S rRNA gene. There is no need to determine a long base sequence using a plurality of primers unlike a gene.

The present invention also provides a primer (primer set) and a probe that are used in the detection method of Azoarcus sp. DN11 strain. These primers (primer sets) and probes are as described above.

  The primer set and probe can also be made into a kit. The kit of the present invention only needs to include the above primer set and probe, and if necessary, a DNA extraction reagent, a PCR reagent such as a PCR buffer or a DNA polymerase, or a PCR that serves as a positive control for the reaction. A DNA solution containing an amplification region, a solid phase (such as a microplate), and instructions may be included.

Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.
Example 1
Azoarcus sp. DN11 strain is mixed with arbitrary groundwater, and four samples with different mixing ratios of Azoarcus sp. DN11 strain (see Table 3 below) with respect to the total number of bacteria: groundwater without mixing DN11 strain, 10 ⁵ Groundwater mixed with cells / ml, groundwater mixed with 10 ⁶ cells / ml of DN11 strain, and pure culture solution of DN11 strain were prepared. DNA was extracted from 10 ml of these samples according to the Current Protocols in Microbiology technique.

For the extracted DNA, the sense primer bmo-Fw (5'-TGGATTGTGGCGCAGTTCGAACGCGCCCTG-3 ': SEQ ID NO: 2) and the antisense primer bmo-Rv (5') were prepared based on the nucleotide sequence of the primer design region shown in FIG. -CGGATTCCACCATACCGTCGGGCGCCAGTA-3 ': SEQ ID NO: 3), and probe bmo-p (5'-GAACAGTTCCTGGCAGACCTCAATGAAACT-3': SEQ ID NO: 4), further modified with the fluorescent substance FAM on the 3 'side and the quencher substance TAMRA on the 5' side Used to perform a quantitative real-time PCR amplification test for absolute quantification of the copy number of the benzene monooxygenase gene. For creating the absolute quantification of the calibration curve, using what has been cloned nucleotide sequence (SEQ ID NO: 1) in pGEM-T-Easy (Promega) plasmids in Figure 1, at the same time of 10 ⁴ -10 ⁹ copies Subjected to quantitative real-time PCR.

The composition of the PCR reaction solution is as shown in Table 1 below. In the PCR reaction, first, the reaction solution was subjected to an initial denaturation reaction at 96 ° C. for 10 minutes. Subsequently, a series of 40 cycles of a denaturation reaction of 96 ° C. for 30 seconds, an annealing reaction of 65 ° C. for 30 seconds, and an extension reaction of 72 ° C. for 30 seconds was performed. The real-time PCR reaction apparatus used ABI 7500Fast, and the amplification signal was detected by the TaqMan Probe ^™ method.

For comparison, quantitative real-time PCR was performed to quantify the specific region (arz) of the conventional 16S rRNA gene and the 16S rRNA gene region (universal) universal to prokaryotes. For the former PCR, primers of arz-Fw (5′-CGTTCGGAGAGGTAACTCACT-3 ′: SEQ ID NO: 9) and arz-Rv (5′-CCGCATCTCTGCAGGATT-3 ′: SEQ ID NO: 10) were used, and for the latter PCR , Univ261-Fw (5′-AGCTAGTTGGTGGGGT-3 ′: SEQ ID NO: 11) and Univ342-Rv (5′-CTGCTGCSYCCCGTAG-3 ′: SEQ ID NO: 12) primers were used. To create a calibration curve for absolute quantification, the entire length of 16S rRNA gene of DN11 strain was cloned into pGEM-T-Easy (Promega) plasmid, and 10 ⁴ to 10 ⁹ copies were simultaneously subjected to quantitative real-time PCR. Provided.

  The reaction composition of quantitative real-time PCR is as shown in Table 2 below. In the PCR reaction, first, the reaction solution was subjected to an initial denaturation reaction at 96 ° C. for 10 minutes. Subsequently, a series of 40 cycles of a denaturation reaction of 96 ° C. for 30 seconds, an annealing reaction of 50 ° C. for 30 seconds, and an extension reaction of 72 ° C. for 30 seconds were performed. This amplification signal was detected by the intercalator method using SYBR Green I.

The number of gene amplification copies from each sample is shown in Table 3 below.

Although the DN11 strain / total bacterial count ratio (Table 3, third column from the left) and bmo copy number / 16S rRNA (universal) copy number ratio (Table 3, rightmost column) are almost identical On the other hand, the ratio of conventional 16S (arz) copy number / 16S rRNA (universal) copy number (Table 3, second column from the right) is higher than that, and detection is also high from the system that does not mix DN11 strain The value was confirmed. This is probably because the design position of the arz primer is on the 16S rRNA gene, and there is no significant difference in sequence with related species, so it was specifically lacked and Azoarcus bacteria other than the DN11 strain were counted. As described above, by performing PCR using the primer and probe on the benzene monooxygenase gene designed in the present invention, the Azoarcus DN11 strain can be detected more specifically, and reliable monitoring can be performed even in a mixed system. Indicated.

  It can be used to monitor the purification treatment of benzene-contaminated soil, drainage or groundwater.

Claims (8)

A method for detecting Azoarcus sp. DN11 strain, comprising a DNA extracted from an environmental sample as a template, a sense primer comprising the base sequence represented by SEQ ID NO: 2 and an antisense primer comprising the base sequence represented by SEQ ID NO: 3 The method comprising PCR amplification of a benzene monooxygenase gene using a primer set comprising: and detecting the amplified DNA fragment. The method according to claim 1 , wherein the detection of the DNA fragment is performed by hybridization with a probe comprising an oligonucleotide having the base sequence shown in SEQ ID NO: 4 . The method according to claim 1 or 2 , wherein the DNA fragment is detected quantitatively by real-time PCR. A method for quantifying the number of Azoarcus sp. DN11 strains in a benzene-contaminated site using the method according to claim 3 and monitoring the Azoarcus sp . DN11 strain. A method for quantifying the number of Azoarcus sp. DN11 strains in a benzene-contaminated site using the method according to claim 3 and evaluating the degree of purification or repair of the benzene-contaminated site by the Azoarcus sp . DN11 strain. A primer set for detection of Azoarcus sp. DN11 strain , comprising a sense primer consisting of the base sequence shown in SEQ ID NO: 2 and an anti-primer consisting of the base sequence shown in SEQ ID NO: 3 . A probe for detection of Azoarcus sp. DN11 strain , comprising an oligonucleotide having the base sequence represented by SEQ ID NO: 4 . A kit for detecting Azoarcus sp. DN11 strain, comprising the primer set according to claim 6 and the probe according to claim 7 .

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