JP4565100B2 - Polynucleotide for detecting microorganisms - Google Patents

Description

  The present invention relates to a polynucleotide for detecting a microorganism.

  Contamination of groundwater and soil with various organochlorine compounds, including tetrachlorethylene (PCE) and trichlorethylene (TCE), is a serious problem common to the world, and these substances are often taken up in the media on newspapers. There is a strong social demand for technological development to repair environmental pollution caused by

  Physicochemical methods and biological methods can be cited as the contaminated environment remediation techniques, but a bioenvironment remediation method (bioremediation) using microorganisms is particularly suitable for remediation of low concentration contamination. Bioremediation does not require soil excavation, is easy to repair the environment under the building, is low in cost and has a low environmental impact, and is expected to be put to practical use.

  Bioremediation is a method that enhances the ability of microorganisms to decompose and remove environmental pollutants by, for example, supplying various nutrients to microorganisms that naturally live in contaminated soil and groundwater. And a method of directly introducing a microorganism having the ability to decompose and remove environmental pollutants into the contaminated environment (bio augmentation: see, for example, Patent Document 1). In some cases, TCE-contaminated groundwater has been refurbished through biostimulation and bioaugmentation and has achieved excellent results.

When applying bioremediation, quickly determine whether the contaminated site can be biostimulated or whether bioaugmentation that introduces microorganisms capable of degrading contaminants from outside the system must be applied (For example, refer patent document 2).
JP 2003-154332 A Japanese Unexamined Patent Publication No. 2000-079000

  Therefore, an object of the present invention is to provide a polynucleotide that can determine which method should be applied in the application of bioremediation.

In order to achieve the above object, the polynucleotide of the present invention is any one of the following (1) to (4).
(1) A polynucleotide comprising any one of the nucleotide sequences of SEQ ID NOS: 1 to 17.
(2) A polynucleotide comprising a nucleotide sequence in which one to several bases of the polynucleotide of (1) are deleted, substituted or added, and complementary to the polynucleotide of (1) A polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a basic nucleotide sequence.
(3) A polynucleotide having a nucleotide sequence in which one to several bases of the polynucleotide of (1) are deleted, substituted or added, and the polynucleotide of (1) A polynucleotide having a homology of 90% or more.
(4) A polynucleotide comprising a base sequence complementary to the polynucleotide of any one of (1) to (3).

  As a result of intensive studies on the method for detecting anaerobic bacteria related to the degradation of PCE, the present inventors are rich in diversity in the 16S-23S Internal Transcribed Spacer (ITS) region of ribosomal DNA. It has been found that it has a unique base sequence. And it discovered that the said bacteria could be detected rapidly by utilizing this base sequence as a DNA probe, for example for gene detection techniques, such as a DNA chip, and reached the present invention.

  The nucleotide sequences of SEQ ID NOs: 1 to 17 in the polynucleotide of the present invention are ITS sequences of 17 types of anaerobic bacteria involved in PCE degradation described later, and are the sequences determined by the present inventors for the first time.

  If a polynucleotide comprising the polynucleotide of the present invention is used as a DNA probe, for example, at least 17 species of anaerobic PCE degradation-related bacteria in a contaminated environment can be rapidly detected. In addition, a DNA probe can be prepared from a known ITS sequence for the remaining 18 types of anaerobic PCE degradation-related bacteria currently reported, and if used together, all 18 types can be detected at once. It becomes possible. For example, if a DNA microarray is prepared using the DNA probe, the detection can be made simpler and quicker.

  Therefore, according to the polynucleotide of the present invention, it is possible to quickly detect bacteria related to PCE degradation in a contaminated environment and know the ability of the contaminated environment to remove PCE and its dechlorinated product. It is possible to easily determine whether biostimulation in mediation is possible.

The polynucleotide of the present invention may be a polynucleotide that is a part of the polynucleotides (1) to (4) above, for example, the polynucleotide of any one of (5) to (8) below: Also good.
(5) A polynucleotide comprising any one of the nucleotide sequences of SEQ ID NOS: 19 to 105.
(6) A polynucleotide having a nucleotide sequence in which one to several bases of the polynucleotide of (5) are deleted, substituted or added, and complementary to the polynucleotide of (5) A polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a basic nucleotide sequence.
(7) A polynucleotide having a base sequence in which one to several bases of the polynucleotide of (5) are deleted, substituted or added, and the polynucleotide of (1) A polynucleotide having a homology of 90% or more.
(8) A polynucleotide comprising a base sequence complementary to the polynucleotide of any one of (5) to (7).

The DNA probe of the present invention is a DNA probe for detecting any of the following bacteria A to Q, and is a DNA probe comprising the polynucleotide of the present invention.
A: Deharosupirirumu multi Tarnovo borane scan (Dehalospirillum multivorans)
B: Desulfitobacterium frappieri
C: Actinomycetales Sm-1 ( Rhodococcus sp. Sm-1)
D: Rhodococcus rhodococcus
E: key Santo Enterobacter flavus (Xanthobacter flavus)
F: Mycobacterium L1
G: Desulforobium norvegicum ( Desulfovibrio baculatus )
H: Desulfitobacterium dehalogenans
I: Desulfitobacterium hafniense
J: Clostridium formicoaceticum
K: Desulfuromonas chloroethenica
L: Acetobacterium woodii DSM 1030 ( Acetobacterium woodii DSM 1030)
M: Deharobakuta Resutorikutasu (Dehalobacter restrictus)
N: Desulfitobacterium sp. PCE1 ( Desulfitobacterium sp. Strain PCE1)
O: Desulfitobacterium frappieri TCE1
P: Acetobacterium woodii DSM 2396
Q: Desurufomoniru Tide Jay DCB-1 (Desulfomonile tiedjei DCB- 1)

  Specifically, one aspect of the DNA probe of the present invention is a DNA probe for detecting the bacterium of A, wherein the polynucleotide of the present invention is derived from any one of SEQ ID NO: 1 and SEQ ID NOs: 19 to 25 A DNA probe comprising: Here, the polynucleotide of the present invention derived from SEQ ID NO: 1 means the polynucleotide of any one of (1) to (4) wherein the SEQ ID NO: (1) is SEQ ID NO: 1.

  In another embodiment, the DNA probe of the present invention is a DNA probe for detecting the bacterium of B, which is a DNA comprising the polynucleotide of the present invention derived from any one of SEQ ID NO: 2 and SEQ ID NOs: 26 to 30 It is a probe.

  As another aspect, the DNA probe of the present invention is a DNA probe for detecting the C bacterium, and is a DNA comprising the polynucleotide of the present invention derived from any one of SEQ ID NO: 3 and SEQ ID NOs: 31 to 35 It is a probe.

  In another embodiment, the DNA probe of the present invention is a DNA probe for detecting the bacterium of D, which is a DNA comprising the polynucleotide of the present invention derived from any one of SEQ ID NO: 4 and SEQ ID NOs: 36 to 40 It is a probe.

  In another embodiment, the DNA probe of the present invention is a DNA probe for detecting the E bacterium, and is a DNA comprising the polynucleotide of the present invention derived from any one of SEQ ID NO: 5 and SEQ ID NOs: 41 to 45 It is a probe.

  In another embodiment, the DNA probe of the present invention is a DNA probe for detecting the F bacterium, which comprises the polynucleotide of the present invention derived from any one of SEQ ID NO: 6 and SEQ ID NOs: 46 to 48 It is a probe.

  In another aspect, the DNA probe of the present invention is a DNA probe for detecting the G bacterium described above, which is a DNA comprising the polynucleotide of the present invention derived from any one of SEQ ID NO: 7 and SEQ ID NOs: 49 to 53 It is a probe.

  In another embodiment, the DNA probe of the present invention is a DNA probe for detecting the aforementioned H bacterium, which is a DNA comprising the polynucleotide of the present invention derived from any one of SEQ ID NO: 8 and SEQ ID NOs: 54 to 57 It is a probe.

  In another embodiment, the DNA probe of the present invention is a DNA probe for detecting the bacterium of I above, and comprising the polynucleotide of the present invention derived from any one of SEQ ID NO: 9 and SEQ ID NOs: 58 to 62 It is a probe.

  In another embodiment, the DNA probe of the present invention is a DNA probe for detecting the above-mentioned bacteria of J, which is a DNA comprising the polynucleotide of the present invention derived from any one of SEQ ID NO: 10 and SEQ ID NOs: 63 to 68 It is a probe.

  In another embodiment, the DNA probe of the present invention is a DNA probe for detecting the aforementioned K bacteria, which is a DNA comprising the polynucleotide of the present invention derived from any one of SEQ ID NO: 11 and SEQ ID NOs: 69 to 74 It is a probe.

  In another embodiment, the DNA probe of the present invention is a DNA probe for detecting the L bacterium, and is a DNA comprising the polynucleotide of the present invention derived from any one of SEQ ID NO: 12 and SEQ ID NOs: 75 to 79 It is a probe.

  As another embodiment, the DNA probe of the present invention is a DNA probe for detecting the M bacteria, and is a DNA comprising the polynucleotide of the present invention derived from any one of SEQ ID NO: 13 and SEQ ID NOs: 80 to 86 It is a probe.

  In another aspect, the DNA probe of the present invention is a DNA probe for detecting the N bacteria, and is a DNA comprising the polynucleotide of the present invention derived from any one of SEQ ID NO: 14 and SEQ ID NOs: 87 to 91 It is a probe.

  In another embodiment, the DNA probe of the present invention is a DNA probe for detecting the O bacterium, and is a DNA comprising the polynucleotide of the present invention derived from any one of SEQ ID NO: 15 and SEQ ID NOs: 92 to 96 It is a probe.

  In another embodiment, the DNA probe of the present invention is a DNA probe for detecting the P bacterium, and is a DNA comprising the polynucleotide of the present invention derived from any one of SEQ ID NO: 16 and SEQ ID NOs: 97 to 99 It is a probe.

  In another embodiment, the DNA probe of the present invention is a DNA probe for detecting the Q bacterium, which comprises the polynucleotide of the present invention derived from any one of SEQ ID NO: 17 and SEQ ID NOs: 100 to 105 There is a probe.

  The DNA microarray of the present invention is a DNA microarray in which at least one DNA probe of the present invention is immobilized on a substrate. In the DNA microarray of the present invention, it is preferable that at least two or more of the DNA probes of the present invention are fixed and at least two or more of the A to Q bacteria can be detected simultaneously.

  The bacterial detection kit of the present invention is a kit for detecting at least one of the A to Q bacteria, and is detected by hybridizing to at least one DNA probe of the present invention and the DNA probe. A bacterial detection kit comprising a gene amplification primer and a gene amplification reagent for preparing a target to be prepared.

  In another aspect, the bacterial detection kit of the present invention is a kit for detecting at least one of the A to Q bacteria, and is hybridized with the DNA microarray of the present invention and the DNA microarray. A bacterial detection kit comprising a gene amplification primer and a gene amplification reagent for preparing a target to be detected.

  In the method for detecting a bacterium according to the present invention, a target is prepared from a nucleic acid prepared from a sample to be detected by a gene amplification method, and at least one of the target and the DNA probe of the present invention is hybridized to thereby detect the bacterium in the sample. It is a method of detection.

  In another aspect of the method for detecting a bacterium of the present invention, a target is prepared from a nucleic acid prepared from a sample to be detected by a gene amplification method, and the target and the DNA microarray of the present invention are hybridized to each other in the sample. This is a method for detecting bacteria.

  First, the base sequences of SEQ ID NOs: 1 to 17 constituting one of the polynucleotides of the present invention will be described. The base sequences of SEQ ID NOs: 1 to 17 are sequences corresponding to the ITS sequences of the bacteria A to Q, which are bacteria associated with anaerobic PCE degradation, respectively. The ITS sequence means a prokaryotic 16S-23S ITS sequence. Prokaryotic ribosomal RNA (rRNA) 16SrRNA, 23SrRNA and 5SrRNA are usually transcribed as a single transcription unit (operon), so the 16SrRNA gene and the 23SrRNA gene are arranged side by side on the genome. The region between the 16SrRNA gene and the 23SrRNA gene is a region called 16S-23S Internal Transcribed Spacer (ITS). The present inventors have determined for the first time the sequence of the ITS in the bacteria A to Q, and for the first time it has been found that with this ITS sequence, a DNA probe specific to each of the bacteria A to Q can be prepared. is there.

Therefore, the polynucleotide of the present invention includes any of the following polynucleotides (1) to (4).
(1) A polynucleotide comprising any one of the nucleotide sequences of SEQ ID NOS: 1 to 17.
(2) A polynucleotide comprising a nucleotide sequence in which one to several bases of the polynucleotide of (1) are deleted, substituted or added, and complementary to the polynucleotide of (1) A polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a basic nucleotide sequence.
(3) A polynucleotide having a nucleotide sequence in which one to several bases of the polynucleotide of (1) are deleted, substituted or added, and the polynucleotide of (1) A polynucleotide having a homology of 90% or more.
(4) A polynucleotide comprising a base sequence complementary to the polynucleotide of any one of (1) to (3).

The present inventors have also found that a part of the ITS sequence (SEQ ID NOs: 1 to 17) can be used as a DNA probe of the bacteria A to Q. Therefore, the polynucleotide of the present invention may be a polynucleotide that is a part of the polynucleotides (1) to (4) described above. For example, the polynucleotide of any one of (5) to (8) below: There may be.
(5) A polynucleotide comprising any one of the nucleotide sequences of SEQ ID NOS: 19 to 105.
(6) A polynucleotide having a nucleotide sequence in which one to several bases of the polynucleotide of (5) are deleted, substituted or added, and complementary to the polynucleotide of (5) A polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a basic nucleotide sequence.
(7) A polynucleotide having a base sequence in which one to several bases of the polynucleotide of (5) are deleted, substituted or added, and the polynucleotide of (5) A polynucleotide having a homology of 90% or more.
(8) A polynucleotide comprising a base sequence complementary to the polynucleotide of any one of (5) to (7).

  Here, the number of bases that can be deleted, substituted, or added is, for example, 1 to 6 by deletion / addition with respect to 40 bases, and preferably 1 to 3, more preferably 1 to 2 and 1 to 4 by substitution, preferably 1 to 2 and more preferably 1. Examples of the stringent conditions for hybridization include ± 10 ° C. of the Tm value of the base sequence represented by the SEQ ID NO. Moreover, as said homology, it is 90% or more, for example, 95% or more is preferable, More preferably, it is 97.5% or more.

  Next, the DNA probe of the present invention will be described. The DNA probe of the present invention is a DNA probe that can detect any of the bacteria A to Q, and is a DNA probe comprising the polynucleotide of the present invention. The DNA probe of the present invention is preferably derived from a part of the base sequence rather than the entire ITS sequence of bacteria A to Q (SEQ ID NOs: 1 to 17). This is because a DNA probe usually has a higher sequence specificity and a higher reliability as its length becomes shorter. On the other hand, the sequence itself needs to be unique to each of the bacteria. Therefore, the length of the DNA probe is not particularly limited. For example, the entire ITS sequence is from 10 bases to 40 to 80 bases.

  A specific example of a 40 base sequence that is a part of the entire ITS sequence (SEQ ID NOs: 1 to 17) of bacteria A to Q is a base sequence of SEQ ID NOs: 19 to 105, and SEQ ID NOs: 19 to 25 are sequences. SEQ ID NO: 26 to 30 corresponds to part of SEQ ID NO: 2, SEQ ID NO: 31 to 35 corresponds to part of SEQ ID NO: 3, SEQ ID NO: 36 to 40 corresponds to SEQ ID NO: 4 SEQ ID Nos. 41 to 45 correspond to a part of SEQ ID No. 5, SEQ ID Nos. 46 to 48 correspond to a part of SEQ ID No. 6, and SEQ ID Nos. 49 to 53 correspond to a part of SEQ ID No. 7. SEQ ID NOs: 54 to 57 correspond to a part of SEQ ID NO: 8, SEQ ID NOS: 58 to 62 correspond to a part of SEQ ID NO: 9, and SEQ ID NOS: 63 to 68 correspond to a part of SEQ ID NO: 10. Applicable, SEQ ID NO: 69 to 74 are part of SEQ ID NO: 11 Applicable, SEQ ID NOs: 75 to 79 correspond to a part of SEQ ID NO: 12, SEQ ID NOs: 80 to 86 correspond to a part of SEQ ID NO: 13, and correspond to a part of SEQ ID NO: 14 of SEQ ID NOs: 87 to 91 SEQ ID NOs: 92 to 96 correspond to part of SEQ ID NO: 15, SEQ ID NOs: 97 to 99 correspond to part of SEQ ID NO: 16, and SEQ ID NOs: 100 to 105 correspond to part of SEQ ID NO: 17.

At present, 18 types of anaerobic bacteria have been reported as anaerobic bacteria related to the degradation of PCE, including 17 types of bacteria from A to Q and R bacteria described below. The bacteria from A to Q are given the deposit numbers of the following biological resource preservation organizations ATCC or DSMZ.
R: Dehalococcoides ethenogenes 195
A: DSM 12446 B: DSM 13498 C: ATCC 51239 D: ATCC 21197 E: DSM 10330
F: DSM 6695 G: DSM 1741 H: DSM 9161 I: DSM 10644 J: ATCC 27076
K: DSM 12431 L: DSM 1030 M: DSM 9455 N: DSM 10344 O: DSM 12704
P: DSM 2396 Q: ATCC 49306
The genome sequence of the bacteria R was sequenced by Dr. Zinder of Cornell University. The present inventors have proposed a polynucleotide comprising the ITS sequence of the bacteria R (SEQ ID NO: 18) and a part thereof. It has been found for the first time that the above DNA probe is specific to the bacteria R and can be used as a DNA probe capable of detecting all of the 18 kinds of bacteria when used in combination with the DNA probe of the present invention.

Examples of the DNA probe for bacteria R include a DNA probe comprising any one of the polynucleotides (9) to (12) below.
(9) A polynucleotide comprising the base sequence of SEQ ID NO: 18 which is the ITS sequence of the bacteria R or a base sequence of 40 bases of any of SEQ ID NOs: 106 to 115 which is a part thereof.
(10) A polynucleotide comprising a nucleotide sequence in which one to several bases of the polynucleotide of (9) are deleted, substituted or added, and complementary to the polynucleotide of (9) A polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a basic nucleotide sequence.
(11) A polynucleotide having a base sequence in which one to several bases of the polynucleotide of (9) are deleted, substituted or added, and the polynucleotide of (9) A polynucleotide having a homology of 90% or more.
(12) A polynucleotide comprising a base sequence complementary to the polynucleotide of any one of (9) to (11).

  Next, the DNA microarray of the present invention will be described. The DNA microarray of the present invention is one in which at least one DNA probe of the present invention is immobilized on a substrate, and preferably two or more types of DNA probes of the present invention are immobilized and two or more types of bacteria are detected. More preferably, it is a DNA microarray on which DNA probes corresponding to the respective bacteria A to R are immobilized. If the DNA probe of the present invention corresponding to the 18 species is immobilized, 18 types of bacteria can be detected at a time. The DNA probes immobilized on the DNA microarray of the present invention preferably have a short length in order to suppress noise, and preferably have the same length in order to suppress cross-hybridization by keeping the Tm value constant. As the DNA probe of the present invention for detecting Q from the bacteria A, for example, a DNA probe derived from the nucleotide sequence of SEQ ID NOs: 19 to 105 can be used, and as a DNA probe for detecting the bacteria R, For example, a DNA probe derived from the nucleotide sequence of SEQ ID NOs: 106 to 115 can be used. The substrate is not particularly limited, and a commercially available DNA microarray substrate or the like can be used. A method for fixing the DNA probe on the substrate is not particularly limited, and a conventionally known method can be applied.

  Next, the bacterial detection kit of the present invention is a kit for detecting the A to R bacteria, and is detected by hybridizing to at least one DNA probe of the present invention and the DNA probe. A bacterial detection kit comprising a gene amplification primer and a gene amplification reagent for preparing a target.

  Using the detection kit of the present invention, for example, a nucleic acid is extracted from a sample for detecting bacteria, the nucleic acid is subjected to gene amplification using the gene amplification primer and the gene amplification reagent, and a target is prepared. Can be easily and rapidly detected to detect the A to R bacteria in the sample. The nucleic acid may be DNA or RNA, for example. The kit of the present invention may contain a reagent for nucleic acid extraction and a filter or a chip as necessary. The target is a sequence of bacteria in a sample and is a complementary sequence of the sequence of the DNA probe, and can be detected by hybridization of the target labeled with a detection method and the DNA probe. Become. The method of labeling the target is not particularly limited, and examples thereof include a method of labeling the gene amplification primer in advance.

  The gene amplification method is not particularly limited as long as it can amplify a region containing ITS which is the DNA probe of the present invention, and a conventionally known gene amplification method can be used. For example, when the PCR method is used as the gene amplification method, as the gene amplification primer, for example, a primer consisting of the nucleotide sequence of SEQ ID NO: 116 can be used as a sense primer, and an antisense primer other than the bacteria R can be used. Can use a primer consisting of the base sequence of SEQ ID NO: 117. For the bacteria R, a primer consisting of the base sequence of SEQ ID NO: 118 can be used. As the gene amplification reagent, a conventionally known reagent can be used, and examples thereof include a buffer, a polymerase, and a nucleotide.

  The method for hybridizing the target with the DNA probe of the present invention is not particularly limited, and conventionally known gene detection techniques such as Southern blot, DNA array, DNA microarray, and DNA chip can be used. Among these, it is preferable to use the microarray of the present invention, and it is more preferable to use the microarray of the present invention that can detect the 18 kinds of bacteria. Accordingly, a preferred embodiment of the kit of the present invention is a bacterium comprising the DNA microarray of the present invention and a gene amplification primer and a gene amplification reagent for preparing a target to be detected by hybridizing to the DNA microarray. This is a detection kit.

  Next, in the method for detecting a bacterium of the present invention, a target is prepared from a nucleic acid prepared from a sample to be detected by a gene amplification method, and the target and the DNA probe of the present invention are hybridized. To detect R bacteria. The hybridization method is not particularly limited, and conventionally known gene detection techniques such as Southern blot, DNA array, DNA microarray, and DNA chip can be used. Among these, the method using the DNA microarray of the present invention that can detect all the bacteria from A to R is preferable. The detection method of the present invention can be performed using, for example, the kit of the present invention described above.

  In general, as shown in FIG. 3A, for example, PCE is dechlorinated in this order from PCE to TCE, dichloroethylene (DCE), and vinyl chloride (VC), and the VC is decomposed into ethene or carbon dioxide. . FIG. 3B shows the degradation activity of the A to R bacteria against PCE and its dechlorinated product. As shown in FIG. 3B, the degradation activity of the A to R bacteria on PCE and its dechlorinated products varies. For example, the bacteria R dechlorinates PCE one by one up to ethene, but the bacteria M, A and G decompose PCE into cis-type DCE.

  Accordingly, if the A to R bacteria in the environment contaminated with PCE or TCE are detected using the polynucleotide of the present invention, the bacteria are detected based on the dechlorination ability of the bacteria against PCE and its dechlorinated product. It is possible to determine the PCE or TCE removal capability of the environment. The environment is not particularly limited, and examples thereof include contaminated soil, groundwater, pond, seawater and the like.

  In applying bioremediation, if it is possible to determine the ability to remove PCE and its dechlorinated product in a contaminated environment using the polynucleotide of the present invention as described above, for example, biostimulation is possible, or It is easy to determine whether or not For example, if bacteria R is detected, PCE can be made into ethene by the bacteria R, so that biostimulation that introduces nutrients that enhance the growth and activity of the bacteria R into the environment can be selected. Also, for example, when bacteria K and C are detected, biostimulation can be selected because PCE can be decomposed into carbon dioxide by the two kinds of bacteria.

  Furthermore, since it is considered that the decomposition of PCE is difficult under aerobic conditions, and normally 50 cm or less from the ground surface is considered to be anaerobic, for example, a PCE contaminated environment or a contaminated environment having a surface surface of 50 cm or less. It may be an extra cost to repair the anaerobic bacteria instead of the anaerobic bacteria originally available, but according to the polynucleotide of the present invention, the anaerobic bacteria can be easily and quickly added. Since it can be detected, for example, when a PCE-contaminated environment or a contaminated environment having a surface of 50 cm or less is repaired, it is possible to avoid the use of excess energy by utilizing aerobic bacteria.

  Examples of the present invention will be described below.

(Production of DNA probe)
A 40-base sequence was designed from the bacteria ITS R sequence (SEQ ID NOs: 1 to 18) and used as a DNA microarray DNA probe. The design of the DNA probe is a single strand of 40 bases, 48-50% GC content, no or little complementarity and no hits in the international database GenBank (at least 2 mispairs) Based on the above. As a result, 3 to 10 DNA probes were prepared for each of the bacteria (SEQ ID NOs: 19 to 115).

  Bacteria A probes A1 to A7 are the nucleotide sequences of SEQ ID NOs: 19 to 25, respectively, Bacteria B probes B1 to B5 are the nucleotide sequences of SEQ ID NOs: 26 to 30, respectively, and C5 is the base sequence of SEQ ID NOs: 31 to 35, the probes D1 to D5 of bacteria D are the base sequences of SEQ ID NOs: 36 to 40, respectively, and the probes E1 to E5 of bacteria E are the sequences respectively. The base sequences of numbers 41 to 45, the probes F1 to F3 of bacteria F are the base sequences of sequence numbers 46 to 48, respectively, and the probes G1 to G5 of bacteria G are the bases of sequences number 49 to 53, respectively. The probes H1 to H4 of bacteria H from SEQ ID NO: 54, respectively. 7, the probes I1 to I5 of bacteria I are the nucleotide sequences of SEQ ID NOs: 58 to 62, respectively, and the probes J1 to J6 of the bacteria J are the nucleotide sequences of SEQ ID NOs: 63 to 68, respectively. The probes K1 to K6 of the bacteria K are the base sequences of SEQ ID NOs: 69 to 74, the probes L1 to L5 of the bacteria L are the base sequences of the SEQ ID NOs: 75 to 79, respectively, and the probe M1 of the bacteria M To M7 are the base sequences of SEQ ID NOs: 80 to 86, the probes N1 to N5 of bacteria N are the base sequences of SEQ ID NOs: 87 to 91, respectively, and the probes O1 to O5 of bacteria O are respectively SEQ ID NOs: 92 to 96, the probes P1 to P3 of bacteria P are These are the base sequences of SEQ ID NOs: 97 to 99, the probes Q1 to Q6 of bacteria Q are the base sequences of SEQ ID NOs: 100 to 105, and the probes R1 to R10 of the bacteria R are respectively from SEQ ID NO: 106. 115 base sequences.

(Production of DNA microarray and confirmation of DNA probe specificity)
Next, the 97 types of DNA probes were custom-printed on Takara Hubble Slide using Affymetrix 417 Arrayer to prepare a DNA microarray. The target was prepared from each of the A to R bacteria, and the target was hybridized with the DNA microarray to confirm the specificity of the DNA probe.

  The target was prepared by amplifying the bacterial ITS region by PCR. In the PCR, a non-labeled primer consisting of the base sequence of SEQ ID NO: 116 is used as a sense primer, and a Cy3-labeled primer consisting of the base sequence of SEQ ID NO: 117 is used in addition to the bacterial R as an antisense primer, and the bacterial R A Cy3-labeled primer consisting of the base sequence of SEQ ID NO: 118 was used. PCR reaction conditions followed a standard protocol.

  The target amplification product of the PCR is desalted using Autoseq G-50 (Pharmacia) and suction-dried using SpeedVac (Savant). The final concentration is 5 × SSC, 0.2%. It was dissolved in a buffer of SDS, 50% formamide. The target lysate was boiled at 94 ° C. for 3 minutes and ice-cooled for at least 2 minutes, and then applied onto the DNA microarray. A cover glass was then placed over the DNA microarray and the so-prepared material was placed in a hybridization chamber set at 42 ° C. for at least 4 hours. The DNA microarray is then washed with 0.2 × SSC, 0.2% SDS for 5 minutes, 0.2 × SSC for 5 minutes, and 0.05 × SSC for several seconds and spin dried at 1,800 rpm. Scanning with Scanarry version 5 (Perkin Elmer Japan).

  A summary of the results is shown in FIG. FIG. 1 is a graph showing whether or not the ITS sequence target of 18 bacteria shown on the vertical axis and the 97 DNA probes shown on the horizontal axis were hybridized. This indicates that the DNA probe and the target are hybridized. The phylogenetic tree on the horizontal axis is prepared from the alignment of ITS sequences. As shown in FIG. 1, it was shown that the target prepared from each of the bacteria from A to R significantly hybridized only with the DNA probe of the bacteria from A to R without cross-hybridization.

  Detection was performed using an anaerobic enrichment culture sample provided by Dr. T.H. Lee (Korea) using a DNA microarray in which the following 96 types of DNA probes were arranged. First, DNA was extracted from the anaerobic enriched culture sample using FastPrep bead-beater and soil DNA extraction kit (Q Bionene) according to the instruction manual. A target was prepared from the DNA by PCR using the same primers as in Example 1, and hybridized with the DNA microarray under the same conditions as in Example 1.

  As a result, strong signals were detected for some probes of bacteria A, J, M, N and O, and weak signals were also detected for bacteria B and I probes. The result is shown in FIG. 2 and 3B show that the sample contains bacteria that convert PCE to cisDCE, which is consistent with the PCE / cisDCE analysis data of the enriched culture by Dr. TH Lee. It was.

  As described above, the polynucleotide of the present invention is useful for detecting bacteria associated with PCE degradation, and is useful, for example, in the field of repairing contaminated environments, particularly in the field of bioremediation.

FIG. 1 is a diagram showing a detection result by a DNA microarray in an example of the present invention. FIG. 2 is a diagram showing a detection result by a DNA microarray in another example of the present invention. FIG. 3 is a diagram illustrating the degradation activity of anaerobic PCE degradation-related bacteria according to the present invention.

SEQ ID NO: 116 sense primer 27F for PCR
SEQ ID NO: 117 antisense primer 132R for PCR
SEQ ID NO: 118 antisense primer 341R for PCR

Claims (6)

A DNA probe for detecting bacteria Dehalospirillum multivorans, comprising a polynucleotide represented by any one of the nucleotide sequences of SEQ ID NOS: 19 to 25 in the sequence listing and a nucleotide sequence complementary to the nucleotide sequence At least one of
DNA for detecting bacteria Desulfitobacterium frappieri, comprising a polynucleotide represented by any of the nucleotide sequences SEQ ID NO: 26 to 30 in the sequence listing and a nucleotide sequence complementary to the nucleotide sequence At least one of the probes;
Bacterium actinomycetales Sm-1 (Rhodococcus sp. Sm-1), comprising a polynucleotide represented by any of the nucleotide sequences of SEQ ID NOs: 31 to 35 in the sequence listing and a nucleotide sequence complementary to the nucleotide sequence At least one DNA probe for detecting Actinomycetales Sm-1 (Rhodococcussp. Sm-1)),
At least one of DNA probes for detecting Rhodococcus rhodococcus, comprising a polynucleotide represented by any one of the nucleotide sequences of SEQ ID NOS: 36 to 40 in the sequence listing and a nucleotide sequence complementary to the nucleotide sequence One,
A DNA probe for detecting bacterial Xanthobacter flavus, comprising a polynucleotide represented by any one of the nucleotide sequences of SEQ ID NOs: 41 to 45 in the sequence listing and a nucleotide sequence complementary to the nucleotide sequence At least one,
A DNA probe for detecting a bacterium Mycobacterium L1 (Mycobacterium L1), which comprises a polynucleotide represented by any one of the nucleotide sequences of SEQ ID NOs: 46 to 48 and a nucleotide sequence complementary to the nucleotide sequence At least one,
Desulfomicrobium norvegicum (Desulfomicrobium norvegicum) comprising a polynucleotide represented by any one of the nucleotide sequences of SEQ ID NOs: 49 to 53 and a nucleotide sequence complementary to the nucleotide sequence (Desulfovibrio baculatus)) at least one of the DNA probes,
DNA for detecting bacterial desulfitobacterium dehalogenans, comprising a polynucleotide represented by any of the nucleotide sequences SEQ ID NOs: 54 to 57 in the sequence listing and a nucleotide sequence complementary to the nucleotide sequence At least one of the probes;
DNA for detecting bacteria Desulfitobacterium hafniense, comprising a polynucleotide represented by any one of the nucleotide sequences of SEQ ID NOs: 58 to 62 and a nucleotide sequence complementary to the nucleotide sequence At least one of the probes;
A DNA probe for detecting bacterial Clostridium formicoaceticum, comprising a polynucleotide represented by any one of the nucleotide sequences of SEQ ID NOS: 63 to 68 in the sequence listing and a nucleotide sequence complementary to the nucleotide sequence At least one of
At least one of DNA probes for detecting bacteria Desulfuromonas chloroethenica, which comprises a polynucleotide represented by any one of the nucleotide sequences of SEQ ID NOS: 69 to 74 in the sequence listing and a nucleotide sequence complementary to the nucleotide sequence One,
In order to detect Bacterium acetobacterium woodi DSM 1030 (Acetobacterium woodii DSM 1030) comprising a polynucleotide represented by any one of the nucleotide sequences of SEQ ID NOs: 75 to 79 and a nucleotide sequence complementary to the nucleotide sequence At least one of the DNA probes of
At least one of the DNA probes for detecting bacteria Dehalobacter restrictus, comprising a polynucleotide represented by any one of the nucleotide sequences of SEQ ID NOs: 80 to 86 and a nucleotide sequence complementary to the nucleotide sequence One,
A bacterium Desulfitobacterium sp. PCE1 (Desulfitobacterium sp. Strain PCE1) comprising a polynucleotide represented by any one of the nucleotide sequences of SEQ ID NOs: 87 to 91 and a nucleotide sequence complementary to the nucleotide sequence, At least one of the DNA probes for detection;
In order to detect bacteria Desulfitobacterium frappieri TCE1 comprising a polynucleotide represented by any one of the nucleotide sequences SEQ ID NO: 92 to 96 in the sequence listing and a nucleotide sequence complementary to the nucleotide sequence At least one of the DNA probes of
In order to detect the bacterium Acetobacterium woodii DSM 2396 (Acetobacterium woodii DSM 2396) comprising a polynucleotide represented by any one of the nucleotide sequences SEQ ID NO: 97 to 99 in the sequence listing and a nucleotide sequence complementary to the nucleotide sequence At least one of the DNA probes of
Detects bacteria desulfomonile tiedjei DCB-1 comprising a polynucleotide represented by any of the nucleotide sequences of SEQ ID NOs: 100 to 105 in the sequence listing and a nucleotide sequence complementary to the nucleotide sequence. At least one of DNA probes for
A combination of DNA probes comprising A DNA microarray in which the combination of DNA probes according to claim 1 is fixed on a substrate. A kit for detecting bacteria, comprising: a combination of the DNA probes according to claim 1; a gene amplification primer and a gene amplification reagent for preparing a target to be detected by hybridizing to the DNA probe; Bacteria detection kit. A kit for detecting bacteria, comprising: the DNA microarray according to claim 2; and a gene amplification primer and a gene amplification reagent for preparing a target to be detected by hybridizing to the DNA probe. Bacteria detection kit. A method for detecting bacteria, comprising preparing a target from a nucleic acid prepared from a sample to be detected by a gene amplification method, and hybridizing the target and the combination of the DNA probes according to claim 1 to determine the bacteria in the sample. How to detect. A method for detecting bacteria, comprising preparing a target from a nucleic acid prepared from a sample to be detected by a gene amplification method, and detecting the bacteria in the sample by hybridizing the target and the DNA microarray according to claim 2. Method.

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