JPH1066579A - Oligonucleotides for detecting phenol-degrading bacteria and uses thereof - Google Patents

Abstract

(57) [Problem] To provide a novel means for detecting phenol-degrading bacteria contained in a soil sample or the like. SOLUTION: In any one of the sequences (A), (B) and (C), an oligonucleotide containing a sufficient number of consecutive bases, and a probe and primer for detecting a phenol-degrading bacterium comprising the oligonucleotide; A method for detecting phenol-degrading bacteria using these. Embedded image [Effect] It is possible to easily and accurately determine whether or not phenol-degrading bacteria are contained in a soil sample or the like with high sensitivity.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a reagent and a method for detecting phenol-degrading bacteria.

[0002]

2. Description of the Related Art In a method of decomposing phenol in the environment by mixing phenol-decomposing bacteria into the environment such as soil and wastewater, it is necessary to grasp the behavior and fate of the phenol-degrading bacteria in the environment being treated. is there. Examples of a method for measuring the presence or absence and the number of a specific microorganism in a sample at a genetic level include a method of detecting DNA derived from the microorganism with a probe specific to the microorganism, a method of detecting DNA specific to the microorganism,
Is known by the polymerase chain reaction (PCR) method.

[0003] In these methods, a probe for detecting DNA specific to the microorganism to be detected and a primer for amplifying DNA specific to the microorganism to be detected are required. Since phenol-degrading bacteria have a phenol hydroxylase gene, it is possible to detect phenol-degrading bacteria by detecting the gene. However, no suitable probe is known for detecting the phenol hydroxylase gene.

[0004]

Accordingly, the present invention provides a method for detecting a phenol-degrading bacterium in a sample by detecting the phenol hydroxylase gene of the phenol-degrading bacterium, and a probe and a primer therefor. is there.

[0005]

In order to solve the above problems, the present invention provides the following sequences (A), (B) and (C):

Embedded image

[0006] In any one of the above, an oligonucleotide containing a base sequence having a length equal to or longer than the minimum base sequence length necessary for performing specific and effective hybridization to a detection target, or the oligonucleotide. To provide nucleotides complementary to This oligonucleotide preferably contains 10 or more consecutive nucleotides in the sequence (A), (B) or (C). This oligonucleotide is useful as a probe for detecting a phenol hydroxylase gene of a phenol-degrading bacterium and as a primer for a chain extension reaction for amplifying the gene.

[0007] The present invention also provides a probe for detecting a phenol-degrading bacterium, comprising the above-mentioned oligonucleotide. The probe is optionally labeled. The present invention further provides a primer comprising one or more of the above oligonucleotides for specifically amplifying the phenol hydroxylase gene of phenol-degrading bacteria or a part thereof. The present invention further provides a kit for detecting a phenol-degrading bacterium, comprising the probe and / or the primer.

The present invention also provides a method for detecting phenol-degrading bacteria, comprising: (1) extracting DNA from a test sample;
(2) placing the extracted DNA and the probe under conditions that allow hybridization, and (3) assaying whether hybridization has occurred. The present invention also provides a method for detecting a phenol-degrading bacterium, which comprises (1) extracting DNA from a test sample, (2) performing an amplification reaction using the primers, and (3) testing whether amplification has occurred. And a method characterized by the above. The detection in the above (3) can also be performed using the aforementioned probe. The present invention also provides a method for detecting a phenol-degrading bacterium, wherein (1) a test sample is incubated with a phenol-containing medium,
(2) placing the DNA of the grown microorganism and the probe under conditions that allow hybridization, and (3) assaying whether hybridization has occurred. Amplification can also be performed using the primers before the hybridization in the above (2). The assay of (3) can also be performed using the probe.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION It is preferable that a probe and a primer for detecting phenol-degrading bacteria are specific to phenol-degrading bacteria and can detect a wide range of phenol-degrading bacteria in common. Therefore, in the present invention, it is a part of the phenol hydroxylase gene specifically possessed by various phenol-degrading bacteria, and is well conserved among phenol hydroxylase genes derived from various phenol-degrading bacteria (the homology is low). High) region. As the base sequence of the phenol hydroxylase gene, Bio-Dafbase GENETYX-
Pseudomonas putida strain P35X, CF600 strain and H strain recorded in CDMEBL (September 1995)
The following three types of sequences (A), (B) and (C) were selected as base sequences of a portion having high homology between these sequences and the sequences derived from Pseudomonas piqueti PKO1 strain. .

[0010]

Embedded image

When used as a probe or primer, the oligonucleotide of the present invention needs to specifically hybridize to the phenol hydroxylase gene, and to hybridize in an effectively bound state. For that purpose, it is necessary that any one of the above three types of sequences preferably comprises 10 or more consecutive nucleotides. This is because if the length of the oligonucleotide is further shortened, it becomes difficult to specifically hybridize to the phenol hydroxylase gene. The oligonucleotide of the present invention may have at least 10 consecutive nucleotides in any of the sequences (A), (B) and (C), and one or both ends of the base sequence thus selected. May be extended by binding to other mono- or oligonucleotides, or may be modified by other methods.

The oligonucleotide of the present invention may also have a nucleotide complementary to 10 or more consecutive nucleotides as defined above. The present invention also provides a probe for detecting a phenol hydroxylase gene, comprising the above-mentioned oligonucleotide. Although it depends on the detection method, it is preferable to be labeled in order to function as a probe. P as a sign
^32, radiolabeled by S ³⁵ and the like, labeling with a fluorescent substance such as fluorescein, peroxidase, etc. labeled with an enzyme such as alkaline phosphatase may be used labels are commonly used to label the oligonucleotide.

[0013] The present invention also provides a primer for specifically amplifying the phenol hydroxylase gene of a phenol-degrading bacterium. The primer needs to be composed of a pair of oligonucleotides defining both ends of the sequence to be amplified, but is composed of a pair of oligonucleotides composed of one of the oligonucleotides of the present invention defined above and one universal primer. May be. Alternatively, the primer may be composed of two oligonucleotides comprising 10 or more consecutive nucleotides in any two of the sequences (A), (B) and (C). These primers extend the chain of the sequence to be amplified, so-called PCR method,
It can be used in amplification methods such as LCR method and TAS method. These amplification methods themselves do not constitute the features of the present invention, and the application of the primers of the present invention is not limited to a specific amplification method.

The present invention also provides a method for detecting a phenol-degrading bacterium using the above primer or probe. The detection method of the present invention can be applied to various samples expected to contain phenol-degrading bacteria, and the type of the sample is not particularly limited. However, when phenol-degrading bacteria are artificially added to the environment such as soil and wastewater to decompose phenol, it is necessary to understand the behavior and fate of the phenol-degrading bacteria. Especially useful for According to one embodiment of the method for detecting a phenol-degrading bacterium of the present invention, an amplification reaction is performed using a primer capable of specifically amplifying a phenol hydroxylase gene, and it is determined whether amplification has occurred.

DNA for PCR amplification from microorganisms in soil
Have been described (for example, Seiya Tsushima, Soil and Microorganisms, Vol. 46, p. 33-40, 1995; Jizhong
Zhou et al., Applied and Environmental Microbiology, Vo
l.62 (2), 1996). Therefore, in the method of the present invention,
Known DNA extraction methods or other suitable DNA extraction methods can be used. Amplification of the extracted DNA can be performed by any amplification method, for example, a PCR method.

The product from the amplification operation is subjected to, for example, electrophoresis, for example, agarose gel electrophoresis, and the presence or absence of the amplified DNA is detected by a conventional method, for example, staining with ethidium bromide, and then detecting by UV irradiation. Can be. Alternatively, it can be detected by the probe of the present invention. According to the method of the present invention, amplification does not occur when phenol-degrading bacteria are not present in a test sample.For example, a detection method that does not involve separation of amplification products, such as a blotting method or a reverse blotting method, should be used. Can also.

According to another aspect of the method of the present invention, phenol-degrading bacteria can be detected by growing the microorganism itself without using the specific amplification as described above. For example, if a test sample is incubated in a medium that supports the growth of microorganisms, and if a phenol-degrading bacterium is present in the test sample, the sample will grow in the medium. Whether or not a phenol hydroxylase gene is present in DNA can be determined using the probe of the present invention. Alternatively, the test sample can be applied directly or after enrichment culture as described above, applied to a solid medium, and the generated colonies can be detected by the colony hybridization method using the probe of the present invention. Colony hybridization can be performed using a well-known technique.

According to the present invention, various phenol-degrading bacteria can be detected.
765 (Pseu)
domesticus pickettii B-1 (FER
MBP-5288) and Japanese Patent Application No. 8-100466.
Burkholderia sp. (Burkholde
ria sp. ) N16-1 (FERM BP-550)
4) Burkholderia Cepacia
eria sepacia) N15-2 (FERM B)
P-5503) and Burkholderia cepacia N15
-1 (FERMBP-5502) and the like.

[0019]

Next, the present invention will be described more specifically with reference to examples. Embodiment 1 FIG . Oligonucleotide Synthesis Oligonucleotides having the same sequences as the above-mentioned sequences (A) to (C) were chemically synthesized. Chemical synthesis was performed using the DNA synthesizer 392.
Using the type A (manufactured by Applied Biosystems), a phosphoramidite method was used. Purification of the synthesized oligonucleotide was performed by high-speed chromatography using a C18 reverse-phase column.

Embodiment 2 FIG. Preparation of Specimens Four phenol-degrading bacteria (Birkholderia N1) obtained by enrichment culture in a phenol medium (Table 1) were used.
6-1 (FERM BP-5504), Burkholderia N15-1 (FERM BP-5501), Burkholderia N15-2 (FERM BP-5502), and Pseudomonas Picetti B1 (FERM BP-).
5288)) was used. Each strain was inoculated into an appropriate medium and cultured overnight at 30 ° C. under aerobic conditions.

[0021]

[Table 1]

On the other hand, soil samples were prepared at soil points A, B, C, and D, and enrichment culture was performed from those soils using a phenol medium. A phenol-degrading bacteria group was obtained from point A and point B, and no phenol-degrading bacteria group was obtained from point C and point D. The obtained phenol-degrading bacteria groups were collected at points A and B, and the soil from points C and D was incubated with a nutrient broth medium (manufactured by Oxoid) to collect the obtained bacterial groups.

Further, 1 g of soil at points A, B, C and D
Was placed in a 100 ml sterile Erlenmeyer flask, 10 g of physiological saline was added, and the mixture was shaken at 100 rpm for 20 minutes.
The supernatant was collected after allowing the soil to sediment by standing still for minutes. The four strains of phenol-degrading bacteria, and the phenol-enriched culture and soil extract at points A, B, C, and D were each diluted with 10 mM Tris-HCl buffer (pH 7.5),
After a heat treatment at 95 ° C. for 10 minutes, these were centrifuged, and the supernatant was used as a sample.

Embodiment 3 FIG. Amplification by PCR 10 µl of the bacterial sample prepared in Example 2 was used for 10 µl of 10 × reaction buffer, 10 µl of dNTP solution, 13 µl of primer C (forward) (60 pmol), and primer B
(Reverse) 13 μl (60 pmol), 0.5 μl of Taq polymerase (Takara Shuzo), and 43.5 μ of sterile distilled water
1 and mixed. The heat denaturation step lasts 1 minute at 95 ° C, then the binding step lasts 1 minute at 55 ° C, and the extension step lasts 72 minutes.
A cycle of 1 minute and 30 seconds at ° C was performed for 30 cycles.

The PCR products were detected by agarose gel electrophoresis as follows. Agarose gel has a gel concentration of 6
%, 2.5 mM EDTA and 0.5 μg / ml
The sample was electrophoresed together with a molecular weight marker in a 50 mM Tris-borate buffer containing ethidium bromide, and was detected by irradiation with 260 nm UV light. The PCR product was detected at around 200 bp by comparing the relative mobility with the molecular weight marker. The results are shown in Table 2 below.

[0026]

[Table 2]

Embodiment 4 FIG. Preparation of Probe A labeled probe was prepared using the oligonucleotide prepared in Example 1 with a DIG oligonucleotide 3 'end labeling kit (Boehringer). That is, after reacting 60 µl of the reaction solution shown in Table 3 at 37 ° C for 15 minutes, 8 µl of 0.2 M EDTA, 0.4 mg / ml glycogen, 10 µl of LiCl, and 300 µl of ethanol were added, and the mixture was added at -80 ° C for 1 hour. I left it. The precipitate was collected by centrifugation, washed with 70% ethanol, dried, and then 500 μl of 10 mM Tris-HCl buffer pH 8, 1 mM
Dissolved in EDTA.

[0028]

[Table 3]

Embodiment 5 FIG. Colony hybridization
Bacterial samples 100μl prepared in emissions Example 2 was sprayed on an agar medium, and cultured at 30 ° C., where colonies formed was about 1mm in diameter, a nylon film on the agar surface (Hybond -N, Amersham ) And gently pressed to transfer the colonies on the agar to a nylon membrane. This nylon membrane was placed on a filter paper soaked in a 0.5 M sodium hydroxide solution for 10 minutes. Then, the filter paper was immersed in 0.5 M Tris-HCl buffer (pH 7.5) for 5 minutes, and the filter paper immersed in 1.5 M sodium chloride / 0.5 M Tris-HCl buffer (pH 7.5) for 5 minutes. 2 × SSC (SSC = 0.15M
Sodium chloride / 15 mM trisodium citrate solution, pH
After placing on filter paper soaked in 7) for 5 minutes, it was placed on dry filter paper and air-dried.

After irradiating the nylon membrane with UV light of 260 nm for 5 minutes, the membrane was immersed in a 14 U / ml pronase solution (Wako) to remove the bacterial residue remaining on the nylon membrane, and then immersed in 40 nm.
C. and left for 20 hours. Next, it was immersed in a 0.5 M Tris-HCl buffer (pH 7.5) for 15 minutes, washed, and immersed in a hybridization solution at 65 ° C. for 4 hours to perform prehybridization. The composition of the hybridization solution was 5 × SSC, 1% blocking reagent (Boehringer), 0.1% lauroyl sarcosine, 0.02
% Sodium dodecyl sulfate.

Next, of the probes prepared in Example 4, the probe represented by the formula B was immersed in a hybridization solution to which a concentration of 25 ng / ml was added, and left at 65 ° C. for 20 hours. Then, containing 0.1% sodium dodecyl sulfate 2
Wash twice with XSSC at 65 ° C. for 5 minutes, and wash with 0.1 × SSC containing 0.1% sodium dodecyl sulfate for 5 minutes.
Washed twice at ° C. After washing with 0.1 M Tris-HCl buffer (pH 7.5) containing 0.15 mM sodium chloride for 15 minutes, the plate was immersed in 0.15 U / ml anti-DIG-AP antibody (manufactured by Boehringer) for 30 minutes to bind the antigen-antibody. And then washed twice with 0.1 M Tris-HCl buffer (pH 7.5) containing 0.15 mM sodium chloride for 15 minutes. And 0.
Immerse in a Tris-HCl buffer (pH 9.5) containing 5 mg / ml nitrotetrazolium, 0.2 mg / ml 5-bromo-4-chloro-3-indolyl phosphate, 50 mM magnesium chloride, and 0.1 M sodium chloride, and place in a dark place. At room temperature for 20 hours, and a black spot coloring in the form of a nylon film was detected. The results are shown in Table 4 below.

[0032]

[Table 4]

As is evident from the above results, both the method of specifically amplifying the phenol hydroxylase gene using the primer of the present invention and the method of detecting the phenol hydroxylase gene using the probe of the present invention. It is possible to accurately determine whether or not phenol-degrading bacteria are present in a test sample.

Continuation of the front page (51) Int.Cl. ⁶ Identification number Agency reference number FI Technical display location C12R 1:38) (C12N 1/20 C12R 1:40) (72) Inventor Osamu Asami Nagakute-cho, Aichi-gun, Aichi Prefecture (1) Inside the Toyota Central Research Institute, Co., Ltd. (72) Inventor Hidehiko Sugiyama In the 41st Toyota Central Research Institute, Inc. (72) Inventor Kenro Tokuhiro No. 41, Toyota Chuo R & D Laboratories Co., Ltd. (72) Inventor Koichi Numata 1-98, Horakai, Midori-ku, Nagoya-shi, Aichi Oriental Building 202

Claims (12)

[Claims] 1. The following sequences (A), (B) and (C): In any of the above, an oligonucleotide containing a nucleotide sequence having a length equal to or longer than the minimum nucleotide sequence length necessary for performing specific and effective hybridization for the detection target, or for the oligonucleotide Complementary oligonucleotide. 2. The oligonucleotide according to claim 1, wherein the minimum base sequence is a sequence of 10 consecutive bases in any of the sequences (A), (B) and (C). 3. A probe for detecting a phenol-degrading bacterium, comprising the oligonucleotide according to claim 1 or 2, which is optionally labeled. 4. The oligonucleotide 1 according to claim 1,
Or a primer for specifically amplifying a phenol hydroxylase gene or a part thereof, comprising a plurality of types. 5. A kit for detecting phenol-degrading bacteria, comprising the probe according to claim 2 and / or the primer according to claim 3. 6. A method for detecting a phenol-degrading bacterium, comprising: (1) extracting DNA from a test sample; and (2) subjecting the extracted DNA and the probe according to claim 3 to hybridization conditions. Put, and (3)
A method for testing whether hybridization has occurred. 7. A method for detecting a phenol-degrading bacterium, comprising: (1) extracting DNA from a test sample; (2) performing an amplification reaction using the primer according to claim 4; and (3) determining whether amplification has occurred. Testing the presence or absence of the method. 8. The test in (3) above,
The method according to claim 7, wherein the method according to claim 3 is used to determine whether amplification of NA has occurred. 9. A method for detecting a phenol-degrading bacterium, comprising: (1) incubating a test sample with a phenol-containing medium; and (2) subjecting the grown microorganism DNA and the probe according to claim 2 to hybridization conditions. put,
And (3) a method for testing whether hybridization has occurred. 10. The DNA using the primer according to claim 4 prior to the (2) hybridization.
The method according to claim 9, wherein the amplification is performed. 11. The method according to claim 9, wherein the assay of (3) is performed using the primer of claim 3. 12. The detection target is Pseudomonus pickettii.
B-1 (FERM BP-5288), Burkholderia sp. (Burkholderia sp.) N16
-1 (FERM BP-5504), Burkholderia cepacia
a) N15-2 (FERM BP-5503) or Burkholderia cepacia N15-1 (FERM BP-
The method according to any one of claims 6 to 11, wherein the method is (5502).