WO2010041444A1 - Method for detecting integron, method for detecting integron pollution in the environment, and primer pair - Google Patents

Method for detecting integron, method for detecting integron pollution in the environment, and primer pair Download PDF

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Publication number
WO2010041444A1
WO2010041444A1 PCT/JP2009/005222 JP2009005222W WO2010041444A1 WO 2010041444 A1 WO2010041444 A1 WO 2010041444A1 JP 2009005222 W JP2009005222 W JP 2009005222W WO 2010041444 A1 WO2010041444 A1 WO 2010041444A1
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integron
class
nucleic acid
present
integrons
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PCT/JP2009/005222
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French (fr)
Japanese (ja)
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石川秀
上野嘉之
山澤哲
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鹿島建設株式会社
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria

Definitions

  • the present invention relates to a primer pair capable of simultaneously amplifying a sequence of a predetermined region present in each of a plurality of classes of integron among class 1 to class 4 integrons, and a procedure for performing nucleic acid amplification using the primer pair.
  • the present invention relates to a method, an integron contamination detection method for detecting the state of integron contamination in an environment from an environmental sample by the above procedure.
  • the resistance of pathogenic microorganisms such as bacteria to drugs such as antibiotics is called drug resistance.
  • drug resistance The resistance of pathogenic microorganisms such as bacteria to drugs such as antibiotics is called drug resistance.
  • drug-resistant bacteria makes it difficult to treat infections.
  • multidrug-resistant bacteria having resistance to a plurality of drugs are emerging and spreading, and are becoming important as causative bacteria for intractable infections and hospital infections.
  • the appearance of multidrug-resistant bacteria has also been reported in food poisoning-causing bacteria.
  • Integron is known to be one of the mechanisms of bacterial drug resistance, especially multidrug resistance.
  • Integron is a DNA region having a structure into which an exogenous gene cassette can be inserted. Generally, it includes (1) a region encoding integrase, (2) a region encoding one or more acquired gene cassettes, (3) a promoter for expressing the acquired gene, and the above three sequence regions.
  • Integrase is an enzyme that inserts a gene cassette downstream of a region encoding integrase. When a cassette of drug resistance gene is inserted by the action of integrase, the bacteria acquire drug resistance. Integron is a gene structure that can efficiently incorporate a plurality of drug resistance genes. When a plurality of drug resistance gene cassettes are accumulated in integron, the bacterium becomes multidrug resistant.
  • integron is thought to have no ability to move on the gene itself.
  • integron is usually present on a plasmid or transposon, the same fungus as the acquired gene (drug resistance gene, etc.) There is a possibility of horizontal transmission between species and even across bacterial species.
  • integrons are classified into class 1 to class 5 based on the homology of the region encoding integrase.
  • Patent Document 1 discloses a specific and universal method for rapidly detecting and identifying common bacterial pathogens and antibiotic resistance genes from clinical specimens for routine diagnosis in a microbiology laboratory. Specific probes and amplification primers have been described.
  • Non-Patent Document 1 describes an overview of integron, and Non-Patent Documents 2 to 3 describe a common sequence of integrase.
  • Non-Patent Document 4 describes known primers used in Examples described later. JP 2007-125032 A Dider Mazel, “Integrons: agents of bacterial evolution”, Naturevol.4: 608-620 (2006) S. E.
  • Integron is widely present in the environment, and is known to be detected in environments where chemical exposure is frequent or in sewage treatment facilities. Integron has the potential to be propagated horizontally between the same bacterial species and even across bacterial species.
  • the main object of the present invention is to provide a means for detecting integron existing in the environment simply, inexpensively and with high accuracy.
  • the present inventors can simultaneously amplify a sequence of a predetermined region respectively present in a plurality of classes of integron.
  • the inventors successfully succeeded in simultaneously amplifying the sequences of predetermined regions respectively present in class 1 to class 4 integrons.
  • the present application includes a primer pair capable of simultaneously amplifying a sequence of a predetermined region present in each of a plurality of classes of integron among class 1 to class 4 integrons, and a procedure for performing nucleic acid amplification using the primer pair.
  • An integron detection method is provided.
  • Integralon can be detected easily, inexpensively and with high accuracy from the environment by performing nucleic acid amplification using this primer pair under predetermined conditions and detecting the presence or absence of amplification of DNA fragments and the like.
  • the present invention is a means for simultaneously detecting a plurality of classes of integrons instead of specifically detecting a specific class or a specific type of integron by a nucleic acid amplification method. Therefore, for example, by detecting the integron from the environmental sample using the means according to the present invention, the contamination state of the integron in the environment can be detected.
  • the present invention is also effective as a means for detecting an unknown / unidentified integron.
  • the primer pair according to the present invention is effective for detecting not only a specific class or a specific type of integron but also all integron. Therefore, by employing the means according to the present invention, there is a possibility that an unknown / unidentified class or an unknown / unidentified type of integron can be detected.
  • the present invention makes it possible to detect integrons existing in the environment, etc. easily, inexpensively and with relatively high accuracy.
  • the primer pairs according to the present invention include all primer pairs capable of simultaneously amplifying sequences of predetermined regions respectively present in a plurality of classes of integrons among class 1 to class 4 integrons.
  • a primer pair that can simultaneously amplify a sequence of a predetermined region present in each of class 1 to class 4 integrons is suitable.
  • the primer pair according to the present invention may be designed based on, for example, a common sequence of class 1 to class 4 integrons.
  • the primer pair according to the present invention may be a primer pair that can simultaneously amplify a sequence of a predetermined region present in each of a plurality of classes of integron among class 1 to class 4 integrons. That is, it does not have to be completely homologous to the sequence of a predetermined region in all classes of integrons, and may partially include sequences that are not homologous to all classes or any class of integrons.
  • a DNA fragment in which a part of the sequence is substituted with a base that does not specifically bind to four types of normal bases may be used.
  • the four types of normal bases refer to adenine, guanine, cytosine, and thymine.
  • the base that does not specifically bind to the four types of bases is not particularly limited.
  • inosine can be used as such a base.
  • a preferred primer pair according to the present invention is a DNA fragment in which the forward primer includes at least any sequence of SEQ ID NOs: 1 to 4, and the reverse primer includes at least any sequence of SEQ ID NOs: 5 to 8.
  • a primer pair that is a fragment is a fragment.
  • the primer according to the present invention includes all nucleic acid fragments that hybridize with the above-described sequences under stringent conditions.
  • Stringent conditions can be obtained from known techniques based on the length of each primer, Tm value, and the like.
  • the integron detection method according to the present invention includes all methods including procedures for performing nucleic acid amplification using one or more primer pairs according to the present invention.
  • the nucleic acid amplification means for example, a method using PCR (polymerase chain reaction, hereinafter the same), quantitative PCR, or the like can be employed.
  • quantitative PCR or the like can be employed.
  • real-time PCR can be employed as the quantitative PCR.
  • This method can be used as a means for detecting whether a known integron (for example, a class 1 to class 4 known integron) is present in a sample or the like.
  • a known integron for example, a class 1 to class 4 known integron
  • This method can also be used as a means for searching for an unknown integron (for example, an unknown / unidentified integron that does not belong to class 1 to class 5).
  • an unknown integron for example, an unknown / unidentified integron that does not belong to class 1 to class 5.
  • the primer pair according to the present invention is effective for the detection of integrons in general. Therefore, by employing the means according to the present invention, there is a possibility that an unknown / unidentified class or type of integron can be detected.
  • the setting conditions for nucleic acid amplification may be based on known knowledge, and may be set as appropriate according to, for example, the Tm value of the primer and the length (bp) of the amplified portion. Further, only one pair of primers may be used, or two or more pairs may be used as a cocktail.
  • each primer When a cocktail primer pair is used, each primer may be added in an equal amount, or may be added with a concentration difference of 10 to 100 times for each primer, depending on the purpose and the like.
  • the composition ratio of class 1 integrons is often larger than that of other classes. In that case, for example, a primer pair that easily amplifies an integron other than class 1 is added 10 to 100 times higher than the concentration of a primer pair that easily amplifies a class 1 integron. There is a possibility that it can be detected.
  • the PCR product is obtained with a substantially single band (substantially the same molecular weight). Therefore, for example, by performing nucleic acid amplification by quantitative PCR, integrons in a sample can be quantitatively detected and evaluated.
  • Quantitative PCR can be performed by a known method. For example, real-time PCR is performed by changing the template concentration using a DNA template (positive control) amplified with the primer pair according to the present invention, an amplification curve at each concentration is obtained, and a calibration curve is obtained based on the threshold value and the concentration. create. At the same time, quantitative PCR is performed using the sample as a template to obtain an amplification curve, and the integron content concentration is estimated based on the calibration curve from the threshold.
  • the PCR product obtained by performing nucleic acid amplification according to the present invention is analyzed by the DGGE method (Degrading Grand Gel Electrophoresis; denaturing gradient gel electrophoresis, the same applies hereinafter) to construct the PCR product.
  • DGGE method Degrading Grand Gel Electrophoresis; denaturing gradient gel electrophoresis, the same applies hereinafter
  • Analysis by the DGGE method can be performed by a known method. For example, a sequence of about 40 base pairs rich in guanine or cytosine (hereinafter referred to as “GC clamp”) is added to the 5 ′ end of one primer, and PCR is performed. Then, by electrophoresis of the PCR product by the DGGE method, a plurality of nucleic acids constituting the PCR product are separated as respective bands in the gel. Cut out the band from the gel, extract the nucleic acid by known means, perform sequencing, and compare with the sequence of the same region in the known integron to obtain knowledge about the integron class, origin, species, variation, etc. Can do.
  • GC clamp a sequence of about 40 base pairs rich in guanine or cytosine
  • the integron contamination detection method according to the present invention includes all methods including a procedure for nucleic acid amplification using one or more primer pairs according to the present invention to detect integron from an environmental sample.
  • detecting the state of integron contamination in the environment is effective as a means of evaluating the risk of multidrug-resistant bacteria widely spreading in the environment including human living areas.
  • the contamination state of the integron in the environment can be detected.
  • nucleic acid amplification may be performed by quantitative PCR. As a result, it is possible to quantitatively detect and evaluate the state of Integron contamination in the environment.
  • the PCR product obtained when the nucleic acid amplification according to the present invention is performed may be analyzed by the DGGE method.
  • knowledge about each class, origin, species, variation, etc. of integron present in the environmental sample can be obtained, so that more detailed integrons contamination status can be detected and evaluated.
  • the present invention encompasses all nucleic acids obtained by performing nucleic acid amplification using the above-described primer pairs according to the present invention.
  • the primer pair according to the present invention it is possible to simultaneously amplify the sequences of predetermined regions (sequences of almost the same region / length) existing in each of a plurality of integrons, including known and unknown ones. .
  • the presence of integron and the class / origin of integron are obtained by hybridizing each nucleic acid obtained in the present invention with a nucleic acid in an environmental sample as a probe (detector).
  • a probe detector
  • knowledge on species and variations can be obtained relatively easily and inexpensively.
  • a well-known technique can be employ
  • the present invention provides, for example, a nucleotide sequence substantially the same as the full length or partial length of one or more kinds of nucleic acids obtained by performing nucleic acid amplification using the primer pair according to the present invention or a complementary sequence thereof.
  • the single-stranded nucleic acid can be applied to a DNA chip (DNA microarray) by immobilizing the single-stranded nucleic acid on a substrate or the like.
  • the DNA having the full length of the region amplified by the nucleic acid amplification according to the present invention or the partial length sequence thereof is arranged side by side on a substrate, etc.
  • the class, species, variation, etc. of integron contained in the sample can be detected and identified in one or a few experiments.
  • a well-known means can be employ
  • the nucleic acid according to the present invention may be used as a fluorescent probe.
  • a fluorescent probe For example, both single-stranded nucleic acids having a base sequence substantially the same as the partial length of a nucleic acid (PCR product internal region) obtained by performing nucleic acid amplification using the primer pair according to the present invention or a complementary sequence thereof.
  • a fluorescent reporter and a quencher excitation energy absorber
  • quantitative PCR and the like can be performed with higher accuracy.
  • a well-known technique is employable about the fluorescent substance used for a fluorescent reporter, a quencher, the modification method of a nucleic acid, a probe design method, etc.
  • the nucleic acid according to the present invention may be a nucleic acid in a region amplified by performing nucleic acid amplification using the primer pair according to the present invention, and the region in the integron of each known and unknown class / various / variation Includes all sequences.
  • the nucleic acids according to the present invention include not only nucleic acids such as DNA and RNA themselves, but also nucleic acids obtained by subjecting them to predetermined modifications according to the purpose and application.
  • a single-stranded nucleic acid having a base sequence substantially the same as the full length or partial length of SEQ ID NO: 9 or 22 or a complementary sequence thereof detects a class 1 integron or a known or unknown integron of the same class.
  • a probe for this purpose it can be used for biosensors, DNA chips, fluorescent probes, and the like.
  • a single-stranded nucleic acid having a base sequence substantially the same as the full length or partial length of the base sequence of SEQ ID NO: 10, 20, or 21 or a complementary sequence thereof is a class 3 integron or a known or unknown class of the same class.
  • a probe for detecting integron it can be used for biosensors, DNA chips, fluorescent probes, and the like.
  • a single-stranded nucleic acid having a base sequence substantially the same as the full length or partial length of any one of SEQ ID NOs: 11 to 19 or a complementary sequence thereof is used as a probe for detecting an unknown integron. It can be used for sensors, DNA chips, fluorescent probes, and the like.
  • nucleic acid according to the present invention is not limited to a narrow range only when used in applications such as biosensors, DNA chips, and fluorescent probes.
  • Example 1 an integron detection from an environmental sample was attempted using the primer according to the present invention.
  • PCR was performed on the template DNA.
  • primers according to the present invention primers of SEQ ID NOs: 1 to 8 were simultaneously added to the PCR cocktail.
  • known class 1 to class 4 integron detection primers were added to the PCR cocktail (see Non-Patent Document 2).
  • the composition of the PCR cocktail is shown in Table 1, and the temperature cycle of PCR is shown in Table 2.
  • Table 1 “Gene Amp dNTP MIX”, “Gene Amp 10 ⁇ PCR Gold Buffer”, “AmpliTaq Gold DNA” “polymerase” is manufactured by Applied Biosystems.
  • PCR products were separated by agarose gel electrophoresis, stained with ethidium bromide, and visualized using a transilluminator.
  • FIG. 1 is a drawing-substituting photograph showing a PCR pattern when PCR is performed using a primer according to the present invention.
  • lane 1 is a size marker (100 to 1,000 bp)
  • lane 2 is a size marker (500 to 5,000 bp)
  • lane 3 is a PCR using a known class 1 integron detection primer.
  • Lane 4 shows the PCR pattern when amplified using a known class 2 integron detection primer
  • Lane 5 shows the PCR pattern when amplified using a known class 3 integron detection primer
  • Lane 6 shows Each of the PCR patterns when amplified using the primers of SEQ ID NOs: 1 to 4 at a time is shown.
  • the arrow in a figure represents the position of the band expected to be detected based on the primer design.
  • Example 2 the PCR product amplified with the primer according to the present invention in Example 1 was treated with a restriction enzyme, and it was examined which class of integron was amplified from its digestion pattern.
  • the PCR product was purified according to the attached protocol using “illustra GFX PCR and Gel Band Purification Kit (manufactured by GE Healthcare Bioscience Co., Ltd., hereinafter the same)”.
  • the final elution scale was 20 ⁇ L.
  • the purified product was digested at 37 ° C. for 1 hour using three types of restriction enzymes BciVI, EciI, and PvuII.
  • the digests were separated by agarose gel electrophoresis, stained with ethidium bromide, and visualized using a transilluminator.
  • FIG. 2 is a restriction enzyme map of a PCR product obtained when PCR is performed using the primer according to the present invention.
  • FIG. 3 is a drawing-substituting photograph showing a digestion pattern when a PCR product obtained using the primer according to the present invention is treated with a restriction enzyme.
  • lane 1 is the size marker (100 to 1,000 bp)
  • lane 2 is the PCR product obtained in Example 1 (without restriction enzyme; 480 bp)
  • lane 3 is the size marker (100 to 1,000 bp). 000 bp)
  • lane 4 represents the digestion pattern when the PCR product obtained in Example 1 was treated with a restriction enzyme.
  • the band indicated by arrow A is a fragment derived from class 2 integron
  • the band indicated by arrow b is a fragment derived from class 3 integron
  • the band indicated by arrow c is derived from class 4 integron. It is presumed that the fragment indicated by arrow D and the band indicated by arrow D (around 160 bp) is a fragment derived from class 1 integron.
  • This result shows that all the integrons of class 1 to class 4 were amplified when PCR was performed using the primer according to the present invention. That is, this experimental result shows that a plurality of different classes of integrons can be detected simultaneously by performing PCR using the primer according to the present invention.
  • Example 3 an integron detection from an environmental sample other than sewage sludge was attempted using the primer according to the present invention.
  • septic tank sludge was used as an environmental sample. Centrifuge 1-2 ml of septic tank sludge (10,000 rpm, 5 minutes), and prepare a template DNA sample from the sedimented fraction using “EZNA Soil DNA Isolation Kit”. PCR was performed according to the procedure, and PCR products were separated by agarose gel electrophoresis, stained with ethidium bromide, and visualized using a transilluminator.
  • FIG. 4 is a drawing-substituting photograph showing a PCR pattern when PCR is performed using septic tank sludge as an environmental sample. Each description in the figure is the same as that in FIG.
  • a substantially single band is detected at a position (around 480 bp) where detection is expected based on the primer design, as in FIG. It was.
  • the PCR product detected as a single band was treated with a restriction enzyme in the same manner as in Example 2 to examine which class of integron was amplified from the digestion pattern.
  • FIG. 5 is a drawing-substituting photograph showing a digestion pattern when a PCR product obtained by using septic tank sludge as an environmental sample is treated with a restriction enzyme. Each description in the figure is the same as that in FIG.
  • Example 4 clonal analysis was performed on PCR products derived from sewage sludge and septic tank sludge, and the diversity of integron classes in the sample was investigated.
  • each PCR product was purified using “illustra GFX PCR and Gel Band Purification Kit” and incorporated into a cloning vector to prepare a library. Each obtained clone was then sequenced and collated with the sequence data in the GenBank database.
  • intelon present in sludge was mainly class 1 in both sewage sludge and septic tank sludge.
  • class 1 integrons accounted for the majority (94%) of clones whose base sequences could be decoded.
  • 6 clones of the class 3 integron sequence were confirmed.
  • the partial sequence of class 1 integron obtained as a PCR product is shown in SEQ ID NO: 9, and the partial sequence of class 3 integron is shown in 10.
  • the unknown sequence A has 65.5% homology with the sequence of the integrase region of class 1 integron (accession number: EU675686) present on the plasmid of E. coli. Admitted.
  • the unknown sequence C was confirmed to have 66.7% homology with the sequence of the integron integrase region of “Xanthomonas 83campestris” (accession number: AF324443) and the integration of class 1 integron of “Shigella flexneri”. A homology of 65.0% was confirmed with the lyase region (accession number: AY574195).
  • Example 5 the PCR product derived from sewage sludge and septic tank sludge was analyzed by the DGGE method.
  • each primer was prepared by adding a 40 bp GC clamp upstream of the forward primer (SEQ ID NOs: 1 to 4) on the 5 'side.
  • a template DNA sample was prepared from sewage sludge or septic tank sludge by the same method as in Example 1 and PCR was performed in the same procedure and conditions as in Example 1.
  • Each primer of SEQ ID NOs: 1 to 4 with a GC clamp added as a forward primer and each primer of SEQ ID NOs: 5 to 8 as a reverse primer were simultaneously added to the PCR cocktail.
  • DGGE analysis was performed using the PCR product.
  • an 8% polyacrylamide gel prepared by adjusting the concentration gradient of the DNA denaturant to 25 to 65% (7M urea, 40% formamide (deionized) as 100%) was used.
  • the electrophoresis tank was kept at 60 ° C., and electrophoresis was performed at an electrophoresis voltage of 100 V for 12 hours. After electrophoresis, the gel was stained with SYBR Gold (registered trademark, manufactured by Molecular Probe) and observed.
  • DNA base sequence was determined.
  • the DNA was collected by permeating the excised gel into 100 ⁇ L of TE buffer overnight to elute the DNA.
  • the base sequence was determined by the same method as in Example 4.
  • FIG. 6 is an electrophoretogram showing the results of DGGE analysis of PCR products derived from sewage sludge and septic tank sludge.
  • lane 1 shows the result when sewage sludge is used as a sample
  • lane 2 shows the result when septic tank sludge is used as a sample.
  • “25% to 65%” represents the concentration gradient of the denaturant
  • symbols “A” to “G” represent the positions of the bands.
  • DNA in the “c” and “d” bands in lane 1 and the DNA in the “e”, “f” and “g” bands in lane 2 are class 1 integrons, and these Were all the same base sequence.
  • the DNA sequence (partial length sequence of class 1 integron) obtained from these bands is shown in SEQ ID NO: 22.
  • Example 6 examined the selectivity of the detection performance for each class of integron for the primer pair according to the present invention.
  • integrase region DNAs of class 1 and class 2 integrons were prepared.
  • a template DNA sample is prepared using sewage sludge in the same manner as in Example 1, etc., and PCR is performed using a known class 1 integron detection primer or a known class 2 integron detection primer. It was purified using illustra GFX PCR and Gel Band Purification Kit.
  • FIG. 7 is a drawing-substituting photograph showing a PCR pattern when PCR is performed using the integrase region of class 1 integron as a template DNA
  • FIG. 8 is a diagram showing PCR using the integrase region of class 2 integron as template DNA. It is a drawing substitute photograph which shows the PCR pattern at the time of performing.
  • lane 1 is amplified using a size marker (100 to 1,000 bp)
  • lane 2 is amplified using a size marker (500 to 5,000 bp)
  • lane 3 is amplified using a known class 1 integron detection primer.
  • the PCR pattern is lane 4, the PCR pattern when amplified using two types of primers SEQ ID Nos. 1 and 2 and the reverse using two types of primers SEQ ID Nos. 5 and 6, and lane 5 is the sequence number SEQ ID No.
  • the PCR pattern in the case of amplification using two kinds of primers 3 and 4 and two kinds of primers SEQ ID Nos. 7 and 8 in the reverse, lane 6 forward, four kinds of primers SEQ ID Nos. 1-4, and reverse PCR patterns when amplified using four types of primers of SEQ ID NOs: 5 to 8 Representing, respectively.
  • lane 3 represents a PCR pattern when amplified using a known class 2 integron detection primer.
  • the other lanes are the same as in FIG. 8.
  • the asterisk indicates the expected band position when amplified using known class 1 or class 2 integron detection primers, and the arrow indicates when amplified using the primers of SEQ ID NOs: 1-8. Each band position is represented.
  • the primer pairs according to the present invention can detect a plurality of different classes of integrons simultaneously by using them alone or in combination.
  • Example 7 the detection sensitivity when detecting integron from an environmental sample using the primer pair according to the present invention was examined.
  • Sewage sludge-derived DNA, septic tank sludge-derived DNA, and class 1 and class 2 integron integrase region DNAs were prepared in the same manner as in Examples 1, 3, and 6, respectively.
  • Each DNA was adjusted to 10 pg, 1 pg, 100 fg, 10 fg, 1 fg, and 100 ag, and PCR was performed in the same manner as in Example 1. These PCR products were separated by agarose gel electrophoresis, stained with ethidium bromide, and visualized using a transilluminator.
  • amplification of the target band could be confirmed when template DNA was added in an amount of 100 fg or more in any sample. Therefore, if at least 100 fg of DNA can be obtained from an environmental sample, integron can be detected according to the present invention.
  • integron is widely present in the environment, and is known to be detected in environments where chemical exposure is frequent or in sewage treatment facilities. Therefore, for example, drug-resistant gene clusters may be imparted to various bacteria by integrons that remain without being decomposed even in sewage treatment, or unknown multi-drug resistant pathogens may appear. Therefore, in order to prevent such a risk, a means for detecting the contamination status of integron in the environment is required.
  • the present invention since a plurality of types of integrons in the environment can be detected simultaneously by a single detection operation, a wide range of inspections and risk assessments in the environment are possible.
  • the present invention can be applied to inspecting the state of contamination of integrons in various wastewaters and the environment, evaluating the safety of compost, and detecting drug-resistant bacteria in air conditioning equipment.
  • FIG. 1 the drawing substitute photograph which shows the PCR pattern at the time of performing PCR using the primer which concerns on this invention in Example 1.
  • FIG. 2 The restriction enzyme map of the PCR product obtained when PCR is performed using the primer according to the present invention.
  • FIG. 2 the drawing substitute photograph which shows the digestion pattern at the time of carrying out the restriction enzyme process of the product of PCR performed using the primer which concerns on this invention.
  • Example 3 the drawing substitute photograph which shows the PCR pattern at the time of performing PCR using septic tank sludge for an environmental sample.
  • Example 3 the drawing substitute photograph which shows the digestion pattern at the time of carrying out the restriction enzyme process of the PCR product performed using the septic tank sludge for an environmental sample.
  • Example 5 the electrophoresis photograph which shows the result of having performed the DGGE analysis about the PCR product derived from a sewage sludge and a septic tank sludge.
  • FIG. 5 is a drawing-substituting photograph showing a PCR pattern when PCR was performed using the integrase region of class 1 integron as a template DNA in Example 6.
  • FIG. 6 a drawing-substituting photograph showing a PCR pattern when PCR was performed using the integrase region of class 2 integron as template DNA.

Abstract

Provided is a simple, inexpensive and highly precise means for detecting integron present in the environment. Also provided is a primer pair which enables simultaneous amplification of sequences of given regions present in a plurality of classes of integron within class 1 to class 4 integron. By amplifying nucleic acid under given conditions using this primer pair, and detecting the presence or absence of amplification of DNA fragments, integron can be detected simply, inexpensively and highly precisely in the environment, etc. By detecting integron in environmental samples using the above means, the status of integron pollution in the environment can be detected. The above means is also effective for detecting unknown or unidentified integrons.

Description

インテグロン検出方法、環境中におけるインテグロン汚染検出方法、並びにプライマー対Integron Detection Method, Integron Contamination Detection Method in the Environment, and Primer Pair
　本発明は、クラス１～クラス４のインテグロンのうち、複数のクラスのインテグロンにそれぞれ存在する所定領域の配列を同時に増幅しうるプライマー対、該プライマー対を用いて核酸増幅を行う手順を含むインテグロン検出方法、環境中におけるインテグロンの汚染状況を環境試料から前記手順により検出するインテグロン汚染検出方法などに関する。 The present invention relates to a primer pair capable of simultaneously amplifying a sequence of a predetermined region present in each of a plurality of classes of integron among class 1 to class 4 integrons, and a procedure for performing nucleic acid amplification using the primer pair. The present invention relates to a method, an integron contamination detection method for detecting the state of integron contamination in an environment from an environmental sample by the above procedure.
　細菌などの病原性微生物が抗生物質などの薬剤に対して抵抗力を持つことを薬剤耐性という。薬剤耐性菌の出現は、感染症などの治療を困難なものにしている。また、近年、複数の薬剤に対し抵抗性を有する多剤耐性菌も出現・拡散しつつあり、難治性感染症や院内感染症の原因菌として重要になっている。その他、食中毒起因菌などでも、多剤耐性菌の出現が報告されている。 The resistance of pathogenic microorganisms such as bacteria to drugs such as antibiotics is called drug resistance. The emergence of drug-resistant bacteria makes it difficult to treat infections. In recent years, multidrug-resistant bacteria having resistance to a plurality of drugs are emerging and spreading, and are becoming important as causative bacteria for intractable infections and hospital infections. In addition, the appearance of multidrug-resistant bacteria has also been reported in food poisoning-causing bacteria.
　細菌の薬剤耐性化機構、特に多剤耐性化機構の一つとして、インテグロンの存在が知られている。 Integron is known to be one of the mechanisms of bacterial drug resistance, especially multidrug resistance.
　インテグロンは、外因性遺伝子カセットを挿入可能な構造を有するＤＮＡ領域である。一般的に、（１）インテグラーゼをコードする領域、（２）獲得した一又は複数の遺伝子カセットをコードする領域、（３）獲得遺伝子を発現させるプロモーター、以上３つの配列領域を含む。 Integron is a DNA region having a structure into which an exogenous gene cassette can be inserted. Generally, it includes (1) a region encoding integrase, (2) a region encoding one or more acquired gene cassettes, (3) a promoter for expressing the acquired gene, and the above three sequence regions.
　インテグラーゼは、インテグラーゼをコードする領域の下流に遺伝子カセットを挿入する酵素である。インテグラーゼの働きで薬剤耐性遺伝子のカセットが挿入されると、その細菌は薬剤耐性を獲得する。また、インテグロンは、複数の薬剤耐性遺伝子を効率よく取り込むことができる遺伝子構造であり、複数の薬剤耐性遺伝子のカセットがインテグロンに集積されると、その細菌は多剤耐性化する。 Integrase is an enzyme that inserts a gene cassette downstream of a region encoding integrase. When a cassette of drug resistance gene is inserted by the action of integrase, the bacteria acquire drug resistance. Integron is a gene structure that can efficiently incorporate a plurality of drug resistance genes. When a plurality of drug resistance gene cassettes are accumulated in integron, the bacterium becomes multidrug resistant.
　インテグロンは、トランスポゾンと違い、その構造自体が遺伝子上を移動する能力はないと考えられているが、通常、プラスミドやトランスポゾン上に存在するため、獲得遺伝子（薬剤耐性遺伝子など）とともに、同一の菌種間、さらには菌種を越えて、水平伝播される可能性を有している。 Unlike transposon, integron is thought to have no ability to move on the gene itself. However, since integron is usually present on a plasmid or transposon, the same fungus as the acquired gene (drug resistance gene, etc.) There is a possibility of horizontal transmission between species and even across bacterial species.
　現在のところ、インテグロンは、インテグラーゼをコードする領域の相同性に基づき、クラス１からクラス５に分類されている。 At present, integrons are classified into class 1 to class 5 based on the homology of the region encoding integrase.
　これらのインテグロンは、環境中にも広く存在し、また、薬剤暴露の頻繁な環境や、下水処理施設などで検出されることが知られている。 These integrons are widely present in the environment, and are known to be detected in environments where chemical exposure is frequent and sewage treatment facilities.
　本発明の関連文献として、例えば、特許文献１には、微生物検査室における日常的診断用の臨床検体からの通常の細菌病原体および抗生物質耐性遺伝子を迅速に検出及び同定するための特異的および普遍的プローブおよび増幅プライマーについて、記載されている。非特許文献１には、インテグロンの概要が、非特許文献２～３には、インテグラーゼの共通配列などが記載されている。なお、非特許文献４には、後述の実施例で用いた既知のプライマーが記載されている。
特開２００７－１２５０３２号公報 Dider Mazel, "Integrons: agents of bacterial evolution", Naturevol.4 :608-620 (2006) S. E. Nunes-Duby, et al "Similarities and differences among 105members of the Int family of site-specific recombinases", Nucleic AcidResearch, 1998, Vol. 26, No.2, 391-406 F. Drouin, et al, "The IntI-Like Tyrosine Recombinase of Shewanella oneidensisIs Active as Integron Integrase", Journal of Bacteriology, Mar. 2002, Vol.184,No.6 p1811-1815 Xu, H. et al, (2007), J. Bacteriol., 189, 6276-6283 As a related document of the present invention, for example, Patent Document 1 discloses a specific and universal method for rapidly detecting and identifying common bacterial pathogens and antibiotic resistance genes from clinical specimens for routine diagnosis in a microbiology laboratory. Specific probes and amplification primers have been described. Non-Patent Document 1 describes an overview of integron, and Non-Patent Documents 2 to 3 describe a common sequence of integrase. Non-Patent Document 4 describes known primers used in Examples described later.
JP 2007-125032 A Dider Mazel, "Integrons: agents of bacterial evolution", Naturevol.4: 608-620 (2006) S. E. Nunes-Duby, et al "Similarities and differences among 105members of the Int family of site-specific recombinases", Nucleic AcidResearch, 1998, Vol. 26, No.2, 391-406 F. Drouin, et al, "The IntI-Like Tyrosine Recombinase of Shewanella oneidensis Is Active as Integron Integrase", Journal of Bacteriology, Mar. 2002, Vol.184, No.6 p1811-1815 Xu, H. et al, (2007), J. Bacteriol., 189, 6276-6283
　上述の通り、インテグロンは、環境中にも広く存在し、また、薬剤暴露の頻繁な環境や、下水処理施設などで検出されることが知られている。また、インテグロンは、同一の菌種間、さらには菌種を越えて、水平伝播される可能性を有している。 As described above, integron is widely present in the environment, and is known to be detected in environments where chemical exposure is frequent or in sewage treatment facilities. Integron has the potential to be propagated horizontally between the same bacterial species and even across bacterial species.
　そのため、例えば、環境中で多剤耐性遺伝子カセットを有するインテグロンが出現し、それらのインテグロンが菌種を超えて伝播された場合、新しい多剤耐性の病原菌が出現し、かつ人間の生活領域を含む環境中に広く拡散する可能性がある。従って、そのようなリスクを未然に防止するために、環境中におけるインテグロンの汚染状況を検出する手段が必要である。 Therefore, for example, if integrons with multidrug resistance gene cassettes appear in the environment, and those integrons are transmitted across species, new multidrug resistant pathogens emerge and include human living areas Can spread widely in the environment. Therefore, in order to prevent such a risk, a means for detecting the contamination status of integron in the environment is required.
　一方、上述の通り、インテグロンは、現在のところ、クラス１からクラス５まで分類され、同定されているものだけでも多種類ある。それに対し、所定の環境中に含まれる多種多様なインテグロンの存在の有無を、簡易、低廉かつ高精度に検出する手段はほとんど知られていない。 On the other hand, as described above, there are currently many types of integrons that have been classified and identified from class 1 to class 5. On the other hand, few means are known for simply, inexpensively and accurately detecting the presence or absence of a wide variety of integrons contained in a given environment.
　そこで、本発明は、環境中に存在するインテグロンを簡易、低廉かつ高精度に検出する手段を提供することなどを主な目的とする。 Therefore, the main object of the present invention is to provide a means for detecting integron existing in the environment simply, inexpensively and with high accuracy.
　本発明者らは、特定のクラス又は特定の種類のインテグロンだけでなく、複数のクラスのインテグロンを同時に検出する手段として、複数のクラスのインテグロンにそれぞれ存在する所定領域の配列を同時に増幅しうるプライマー対を作製し、かつ、それらのプライマー対を用いて核酸増幅を行うことにより、クラス１～クラス４のインテグロンにそれぞれ存在する所定領域の配列を同時に増幅させることに成功した。 As a means for simultaneously detecting not only a specific class or a specific type of integron but also a plurality of classes of integrons, the present inventors can simultaneously amplify a sequence of a predetermined region respectively present in a plurality of classes of integron. By creating nucleic acid pairs and performing nucleic acid amplification using these primer pairs, the inventors successfully succeeded in simultaneously amplifying the sequences of predetermined regions respectively present in class 1 to class 4 integrons.
　そこで、本願では、クラス１～クラス４のインテグロンのうち、複数のクラスのインテグロンにそれぞれ存在する所定領域の配列を同時に増幅しうるプライマー対、並びに該プライマー対を用いて核酸増幅を行う手順を含むインテグロン検出方法を提供する。 Therefore, the present application includes a primer pair capable of simultaneously amplifying a sequence of a predetermined region present in each of a plurality of classes of integron among class 1 to class 4 integrons, and a procedure for performing nucleic acid amplification using the primer pair. An integron detection method is provided.
　このプライマー対を用いて所定条件で核酸増幅を行い、ＤＮＡ断片などの増幅の有無を検出することにより、環境中などから、簡易、低廉かつ高精度にインテグロンを検出できる。 Integralon can be detected easily, inexpensively and with high accuracy from the environment by performing nucleic acid amplification using this primer pair under predetermined conditions and detecting the presence or absence of amplification of DNA fragments and the like.
　本発明は、特定のクラス又は特定の種類のインテグロンを特異的に核酸増幅法で検出するのではなく、複数のクラスのインテグロンを同時に検出する手段である。従って、例えば、本発明に係る手段を用いて環境試料からインテグロンを検出することにより、環境中におけるインテグロンの汚染状況を検出できる。 The present invention is a means for simultaneously detecting a plurality of classes of integrons instead of specifically detecting a specific class or a specific type of integron by a nucleic acid amplification method. Therefore, for example, by detecting the integron from the environmental sample using the means according to the present invention, the contamination state of the integron in the environment can be detected.
　また、本発明は、未知・未同定のインテグロンを検出する手段としても有効である。本発明に係るプライマー対は、特定のクラス又は特定の種類のインテグロンだけでなく、インテグロン全般の検出に有効である。従って、本発明に係る手段を採用することにより、未知・未同定のクラスや未知・未同定の種類のインテグロンを検出できる可能性がある。 The present invention is also effective as a means for detecting an unknown / unidentified integron. The primer pair according to the present invention is effective for detecting not only a specific class or a specific type of integron but also all integron. Therefore, by employing the means according to the present invention, there is a possibility that an unknown / unidentified class or an unknown / unidentified type of integron can be detected.
　本発明により、環境中などに存在するインテグロンを簡易、低廉かつ比較的高精度に検出できる。 The present invention makes it possible to detect integrons existing in the environment, etc. easily, inexpensively and with relatively high accuracy.
　＜本発明に係るプライマー対＞
　本発明に係るプライマー対は、クラス１～クラス４のインテグロンのうち、複数のクラスのインテグロンにそれぞれ存在する所定領域の配列を同時に増幅しうるプライマー対を全て包含する。例えば、クラス１～クラス４のインテグロンにそれぞれ存在する所定領域の配列を同時に増幅しうるプライマー対が好適である。 <Primer pair according to the present invention>
The primer pairs according to the present invention include all primer pairs capable of simultaneously amplifying sequences of predetermined regions respectively present in a plurality of classes of integrons among class 1 to class 4 integrons. For example, a primer pair that can simultaneously amplify a sequence of a predetermined region present in each of class 1 to class 4 integrons is suitable.
　本発明に係るプライマー対は、例えば、クラス１～クラス４のインテグロンの共通配列に基づいて設計してもよい。但し、本発明に係るプライマー対は、クラス１～クラス４のインテグロンのうち、複数のクラスのインテグロンにそれぞれ存在する所定領域の配列を同時に増幅しうるプライマー対であればよい。即ち、全クラスのインテグロンにおける所定領域の配列と完全に相同的である必要はなく、また、全クラス又はいずれかのクラスのインテグロンと非相同的な配列を一部に含んでいてもよい。 The primer pair according to the present invention may be designed based on, for example, a common sequence of class 1 to class 4 integrons. However, the primer pair according to the present invention may be a primer pair that can simultaneously amplify a sequence of a predetermined region present in each of a plurality of classes of integron among class 1 to class 4 integrons. That is, it does not have to be completely homologous to the sequence of a predetermined region in all classes of integrons, and may partially include sequences that are not homologous to all classes or any class of integrons.
　その他、配列の一部を４種類の通常の塩基と特異的に結合しない塩基に置換したＤＮＡ断片を用いてもよい。ここで、４種類の通常の塩基とは、アデニン、グアニン、シトシン、チミンをいう。 In addition, a DNA fragment in which a part of the sequence is substituted with a base that does not specifically bind to four types of normal bases may be used. Here, the four types of normal bases refer to adenine, guanine, cytosine, and thymine.
　４種類の塩基と特異的に結合しない塩基は、特に限定されない。例えば、そのような塩基として、イノシンを用いることができる。 The base that does not specifically bind to the four types of bases is not particularly limited. For example, inosine can be used as such a base.
　本発明に係る好適なプライマー対は、フォワードのプライマーが配列番号１～４のいずれかの配列を少なくとも含むＤＮＡ断片であり、リバースのプライマーが配列番号５～８のいずれかの配列を少なくとも含むＤＮＡ断片であるプライマー対である。 A preferred primer pair according to the present invention is a DNA fragment in which the forward primer includes at least any sequence of SEQ ID NOs: 1 to 4, and the reverse primer includes at least any sequence of SEQ ID NOs: 5 to 8. A primer pair that is a fragment.
　本発明に係るプライマーは、上述の各配列とストリンジェントな条件下でハイブリダイズする核酸断片も全て包含する。ストリンジェントな条件は、各プライマーの長さ、Ｔｍ値などに基づき、公知技術より取得できる。 The primer according to the present invention includes all nucleic acid fragments that hybridize with the above-described sequences under stringent conditions. Stringent conditions can be obtained from known techniques based on the length of each primer, Tm value, and the like.
　＜本発明に係るインテグロン検出方法＞
　本発明に係るインテグロン検出方法は、一又は二以上の本発明に係るプライマー対を用いて核酸増幅を行う手順を含むものを全て包含する。核酸増幅手段としては、例えば、ＰＣＲ（ポリメラーゼ連鎖反応、以下同じ）、定量ＰＣＲを用いた方法などを採用できる。定量ＰＣＲとしては、例えば、リアルタイムＰＣＲなどを採用できる。 <Integron detection method according to the present invention>
The integron detection method according to the present invention includes all methods including procedures for performing nucleic acid amplification using one or more primer pairs according to the present invention. As the nucleic acid amplification means, for example, a method using PCR (polymerase chain reaction, hereinafter the same), quantitative PCR, or the like can be employed. For example, real-time PCR can be employed as the quantitative PCR.
　この方法は、既知のインテグロン（例えば、クラス１～クラス４の既知のインテグロン）が試料中などに存在するかどうかを検出する手段として用いることができる。 This method can be used as a means for detecting whether a known integron (for example, a class 1 to class 4 known integron) is present in a sample or the like.
　また、この方法は、未知のインテグロン（例えば、クラス１～クラス５に属しない未知・未同定のインテグロン）を探索する手段としても用いることができる。 This method can also be used as a means for searching for an unknown integron (for example, an unknown / unidentified integron that does not belong to class 1 to class 5).
　上述の通り、本発明に係るプライマー対は、インテグロン全般の検出に有効である。従って、本発明に係る手段を採用することにより、未知・未同定のクラスや種類のインテグロンを検出できる可能性がある。 As described above, the primer pair according to the present invention is effective for the detection of integrons in general. Therefore, by employing the means according to the present invention, there is a possibility that an unknown / unidentified class or type of integron can be detected.
　なお、核酸増幅の設定条件などは、公知の知見に基づき行えばよく、例えば、プライマーのＴｍ値、増幅部分の長さ（ｂｐ）に応じて、適宜設定すればよい。また、プライマーは、一対のみを用いてもよく、二以上の対をカクテルして用いてもよい。 The setting conditions for nucleic acid amplification may be based on known knowledge, and may be set as appropriate according to, for example, the Tm value of the primer and the length (bp) of the amplified portion. Further, only one pair of primers may be used, or two or more pairs may be used as a cocktail.
　カクテルのプライマー対を用いる場合、各プライマーを等量ずつ添加してもよいし、目的などに応じて、プライマーごとに例えば１０～１００倍の濃度差をつけて添加してもよい。一般的に、環境中では、クラス１インテグロンの構成比が他のクラスと比較して大きい場合が多い。その場合、例えば、クラス１以外のインテグロンを増幅させやすいプライマー対を、クラス１インテグロンを増幅させやすいプライマー対の濃度よりも１０～１００倍多く添加することにより、クラス１以外のインテグロンを高感度に検出できる可能性がある。 When a cocktail primer pair is used, each primer may be added in an equal amount, or may be added with a concentration difference of 10 to 100 times for each primer, depending on the purpose and the like. Generally, in the environment, the composition ratio of class 1 integrons is often larger than that of other classes. In that case, for example, a primer pair that easily amplifies an integron other than class 1 is added 10 to 100 times higher than the concentration of a primer pair that easily amplifies a class 1 integron. There is a possibility that it can be detected.
　本発明に係るプライマー対を用いて核酸増幅を行った場合、そのＰＣＲ産物は、ほぼ単一のバンド（ほぼ同一の分子量）で得られる。従って、例えば、核酸増幅を定量ＰＣＲにより行うことにより、試料中のインテグロンを定量的に検出・評価できる。 When nucleic acid amplification is performed using the primer pair according to the present invention, the PCR product is obtained with a substantially single band (substantially the same molecular weight). Therefore, for example, by performing nucleic acid amplification by quantitative PCR, integrons in a sample can be quantitatively detected and evaluated.
　定量ＰＣＲは、公知の方法により、行うことができる。例えば、本発明に係るプライマー対で増幅するＤＮＡ鋳型（ポジティブコントロール）を用いて鋳型の濃度を振ってリアルタイムＰＣＲを行い、各濃度における増幅曲線を取得し、その閾値と濃度に基づいて検量線を作成する。同時に、試料を鋳型として定量ＰＣＲを行って、増幅曲線を取得し、その閾値から検量線に基づき、インテグロンの含有濃度を推定する。 Quantitative PCR can be performed by a known method. For example, real-time PCR is performed by changing the template concentration using a DNA template (positive control) amplified with the primer pair according to the present invention, an amplification curve at each concentration is obtained, and a calibration curve is obtained based on the threshold value and the concentration. create. At the same time, quantitative PCR is performed using the sample as a template to obtain an amplification curve, and the integron content concentration is estimated based on the calibration curve from the threshold.
　また、本発明に係る核酸増幅を行った場合に得られるＰＣＲ産物を、ＤＧＧＥ法（Ｄｅｎａｔｕｒｉｎｇ　Ｇｒａｎｄｉｅｎｔ　Ｇｅｌ　Ｅｌｅｃｔｒｏｐｈｏｒｅｓｉｓ；変性剤濃度勾配ゲル電気泳動法、以下同じ）で解析することにより、そのＰＣＲ産物を構成する複数の核酸をそれぞれ分離・同定できる。 In addition, the PCR product obtained by performing nucleic acid amplification according to the present invention is analyzed by the DGGE method (Degrading Grand Gel Electrophoresis; denaturing gradient gel electrophoresis, the same applies hereinafter) to construct the PCR product. A plurality of nucleic acids can be separated and identified.
　ＤＧＧＥ法による解析は、公知の方法により、行うことができる。例えば、グアニン又はシトシンが豊富な４０塩基対程度の配列（以下、「ＧＣクランプ」とする。）を一方のプライマーの５’末端に付加し、ＰＣＲを行う。そして、そのＰＣＲ産物をＤＧＧＥ法により電気泳動することにより、ゲル内で、そのＰＣＲ産物を構成する複数の核酸がそれぞれのバンドとして分離される。ゲル内からそのバンドを切り出し、公知手段により、核酸を抽出、シーケンシングを行い、既知のインテグロンにおける同一領域の配列と比較することにより、インテグロンのクラス・由来・種・バリエーションなどに関する知見を得ることができる。 Analysis by the DGGE method can be performed by a known method. For example, a sequence of about 40 base pairs rich in guanine or cytosine (hereinafter referred to as “GC clamp”) is added to the 5 ′ end of one primer, and PCR is performed. Then, by electrophoresis of the PCR product by the DGGE method, a plurality of nucleic acids constituting the PCR product are separated as respective bands in the gel. Cut out the band from the gel, extract the nucleic acid by known means, perform sequencing, and compare with the sequence of the same region in the known integron to obtain knowledge about the integron class, origin, species, variation, etc. Can do.
　＜本発明に係るインテグロン汚染検出方法＞
　本発明に係るインテグロン汚染検出方法は、一又は二以上の本発明に係るプライマー対を用いて核酸増幅を行い、環境試料からインテグロンを検出する手順を含むものを全て包含する。 <Integron contamination detection method according to the present invention>
The integron contamination detection method according to the present invention includes all methods including a procedure for nucleic acid amplification using one or more primer pairs according to the present invention to detect integron from an environmental sample.
　上述の通り、環境中におけるインテグロンの汚染状況を検出することは、多剤耐性菌が人間の生活領域を含む環境中に広く拡散するリスクを評価する手段として有効である。本発明に係る手段を用いて環境試料からインテグロンを検出することにより、環境中におけるインテグロンの汚染状況を検出できる。 As described above, detecting the state of integron contamination in the environment is effective as a means of evaluating the risk of multidrug-resistant bacteria widely spreading in the environment including human living areas. By detecting the integron from the environmental sample using the means according to the present invention, the contamination state of the integron in the environment can be detected.
　また、例えば、核酸増幅を定量ＰＣＲにより行ってもよい。これにより、環境中などにおけるインテグロンの汚染状況を定量的に検出・評価できる。 For example, nucleic acid amplification may be performed by quantitative PCR. As a result, it is possible to quantitatively detect and evaluate the state of Integron contamination in the environment.
　その他、本発明に係る核酸増幅を行った場合に得られるＰＣＲ産物を、ＤＧＧＥ法で解析してもよい。これにより、環境試料中に存在するインテグロンのそれぞれのクラス・由来・種・バリエーションなどに関する知見を得ることができるため、より詳細なインテグロンの汚染状況を検出・評価できる。 In addition, the PCR product obtained when the nucleic acid amplification according to the present invention is performed may be analyzed by the DGGE method. As a result, knowledge about each class, origin, species, variation, etc. of integron present in the environmental sample can be obtained, so that more detailed integrons contamination status can be detected and evaluated.
　＜本発明に係る核酸増幅により得られた核酸などについて＞
　本発明は、上述の本発明に係るプライマー対を用いて核酸増幅を行うことにより得られた核酸をすべて包含する。 <Nucleic acids obtained by nucleic acid amplification according to the present invention>
The present invention encompasses all nucleic acids obtained by performing nucleic acid amplification using the above-described primer pairs according to the present invention.
　上述の通り、本発明に係るプライマー対を用いることにより、既知及び未知のものを含め、複数のインテグロンにそれぞれ存在する所定領域の配列（ほぼ同一の領域・長さの配列）を同時に増幅しうる。 As described above, by using the primer pair according to the present invention, it is possible to simultaneously amplify the sequences of predetermined regions (sequences of almost the same region / length) existing in each of a plurality of integrons, including known and unknown ones. .
　従って、例えば、バイオセンサーなどにおいて、本発明で得られた種々の核酸をプローブ（検出子）として、環境試料中などの核酸とそれぞれハイブリダイゼーションさせることにより、インテグロンの存在やインテグロンのクラス・由来・種・バリエーションなどに関する知見を比較的簡易かつ低廉に取得できる可能性がある。なお、バイオセンサーの構成は公知技術を採用できる。 Therefore, for example, in a biosensor, the presence of integron and the class / origin of integron are obtained by hybridizing each nucleic acid obtained in the present invention with a nucleic acid in an environmental sample as a probe (detector). There is a possibility that knowledge on species and variations can be obtained relatively easily and inexpensively. In addition, a well-known technique can be employ | adopted for the structure of a biosensor.
　また、本発明は、例えば、本発明に係るプライマー対を用いて核酸増幅を行うことにより得られた一種又は複数種の核酸の全長又は部分長と実質的に同一の塩基配列又はその相補配列を有する一本鎖核酸を、それぞれ基板上などに固定化することにより、ＤＮＡチップ（ＤＮＡマイクロアレイ）にも適用できる。 In addition, the present invention provides, for example, a nucleotide sequence substantially the same as the full length or partial length of one or more kinds of nucleic acids obtained by performing nucleic acid amplification using the primer pair according to the present invention or a complementary sequence thereof. The single-stranded nucleic acid can be applied to a DNA chip (DNA microarray) by immobilizing the single-stranded nucleic acid on a substrate or the like.
　例えば、各クラス・各種・各バリエーションのインテグロンの配列のうち、本発明に係る核酸増幅により増幅される領域の全長又はその部分長の配列を有するＤＮＡを基板上などに並べて配置することにより、環境試料中に含有したインテグロンのクラス・種・バリエーションなどを一回又は少数回の実験で検出・同定できる可能性がある。なお、ＤＮＡチップの基板などに核酸プローブを固定化などする方法は、公知手段を採用できる。 For example, among the sequences of integron of each class / various / variation, the DNA having the full length of the region amplified by the nucleic acid amplification according to the present invention or the partial length sequence thereof is arranged side by side on a substrate, etc. There is a possibility that the class, species, variation, etc. of integron contained in the sample can be detected and identified in one or a few experiments. In addition, a well-known means can be employ | adopted for the method of immobilizing a nucleic acid probe etc. on the board | substrate of a DNA chip.
　その他、本発明に係る核酸を蛍光プローブなどとして用いてもよい。例えば、本発明に係るプライマー対を用いて核酸増幅を行うことにより得られた核酸の部分長（ＰＣＲ産物内部領域）と実質的に同一の塩基配列又はその相補配列を有する一本鎖核酸の両末端をそれぞれ蛍光レポーター及びクエンチャー（励起エネルギー吸収剤）で修飾し、それを蛍光プローブとして用いることにより、より高精度に定量ＰＣＲなどを行うことができる。なお、蛍光レポーターに用いる蛍光物質、クエンチャー、核酸の修飾方法、プローブ設計方法などについては、公知技術を採用できる。 In addition, the nucleic acid according to the present invention may be used as a fluorescent probe. For example, both single-stranded nucleic acids having a base sequence substantially the same as the partial length of a nucleic acid (PCR product internal region) obtained by performing nucleic acid amplification using the primer pair according to the present invention or a complementary sequence thereof. By modifying each end with a fluorescent reporter and a quencher (excitation energy absorber) and using them as fluorescent probes, quantitative PCR and the like can be performed with higher accuracy. In addition, a well-known technique is employable about the fluorescent substance used for a fluorescent reporter, a quencher, the modification method of a nucleic acid, a probe design method, etc.
　本発明に係る核酸は、本発明に係るプライマー対を用いて核酸増幅を行うことにより増幅される領域の核酸であればよく、既知及び未知の各クラス・各種・各バリエーションのインテグロンにおける該領域の配列をすべて包含する。また、本発明に係る核酸には、ＤＮＡ、ＲＮＡなどの核酸自体だけでなく、目的・用途などに応じてそれらに所定の修飾を施した核酸も広く包含される。 The nucleic acid according to the present invention may be a nucleic acid in a region amplified by performing nucleic acid amplification using the primer pair according to the present invention, and the region in the integron of each known and unknown class / various / variation Includes all sequences. In addition, the nucleic acids according to the present invention include not only nucleic acids such as DNA and RNA themselves, but also nucleic acids obtained by subjecting them to predetermined modifications according to the purpose and application.
　例えば、配列番号９又は２２の塩基配列の全長又は部分長と実質的に同一の塩基配列又はその相補配列を有する一本鎖核酸は、クラス１インテグロン又は同クラスの既知又は未知のインテグロンを検出するためのプローブとして、バイオセンサー、ＤＮＡチップ、蛍光プローブなどに利用できる。 For example, a single-stranded nucleic acid having a base sequence substantially the same as the full length or partial length of SEQ ID NO: 9 or 22 or a complementary sequence thereof, detects a class 1 integron or a known or unknown integron of the same class. As a probe for this purpose, it can be used for biosensors, DNA chips, fluorescent probes, and the like.
　例えば、配列番号１０、２０、又は、２１の塩基配列の全長又は部分長と実質的に同一の塩基配列又はその相補配列を有する一本鎖核酸は、クラス３インテグロン又は同クラスの既知又は未知のインテグロンを検出するためのプローブとして、バイオセンサー、ＤＮＡチップ、蛍光プローブなどに利用できる。 For example, a single-stranded nucleic acid having a base sequence substantially the same as the full length or partial length of the base sequence of SEQ ID NO: 10, 20, or 21 or a complementary sequence thereof is a class 3 integron or a known or unknown class of the same class. As a probe for detecting integron, it can be used for biosensors, DNA chips, fluorescent probes, and the like.
　例えば、配列番号１１～１９のいずれかの塩基配列の全長又は部分長と実質的に同一の塩基配列又はその相補配列を有する一本鎖核酸は、未知のインテグロンを検出するためのプローブとして、バイオセンサー、ＤＮＡチップ、蛍光プローブなどに利用できる。 For example, a single-stranded nucleic acid having a base sequence substantially the same as the full length or partial length of any one of SEQ ID NOs: 11 to 19 or a complementary sequence thereof is used as a probe for detecting an unknown integron. It can be used for sensors, DNA chips, fluorescent probes, and the like.
　なお、本発明に係る核酸は、バイオセンサー、ＤＮＡチップ、蛍光プローブなどの用途で用いる場合のみに狭く限定されない。 Note that the nucleic acid according to the present invention is not limited to a narrow range only when used in applications such as biosensors, DNA chips, and fluorescent probes.
　実施例１では、本発明に係るプライマーを用いて、環境試料からのインテグロン検出を試みた。 In Example 1, an integron detection from an environmental sample was attempted using the primer according to the present invention.
　まず、下水汚泥１～２ｍＬを遠心（１０，０００ｒｐｍ、５分間）し、「Ｅ．Ｚ．Ｎ．Ａ．　Ｓｏｉｌ　ＤＮＡ　Ｉｓｏｌａｔｉｏｎ　Ｋｉｔ（Ｏｍｅｇａ　Ｂｉｏ－Ｔｅｋ，　Ｉｎｃ．製、以下同じ）」を用いて、付属プロトコルに従い、沈降画分から鋳型ＤＮＡ試料を調製した。 First, 1 to 2 mL of sewage sludge was centrifuged (10,000 rpm, 5 minutes), and “EZNAA Sol DNA Isolation Kit (Omega Bio-Tek, Inc., the same applies hereinafter)” was used. A template DNA sample was prepared from the precipitated fraction according to the attached protocol.
　次に、鋳型ＤＮＡについて、ＰＣＲを行った。本発明に係るプライマーとして、配列番号１～８のプライマーを同時にＰＣＲカクテルに添加した。また、対照として、既知のクラス１～クラス４のインテグロン検出用プライマーを、それぞれ、ＰＣＲカクテルに添加した（非特許文献２参照）。 Next, PCR was performed on the template DNA. As primers according to the present invention, primers of SEQ ID NOs: 1 to 8 were simultaneously added to the PCR cocktail. As a control, known class 1 to class 4 integron detection primers were added to the PCR cocktail (see Non-Patent Document 2).
　ＰＣＲカクテルの組成を表１に、ＰＣＲの温度サイクルを表２に示す。なお、表１中、「Gene Amp dNTP MIX」、「Gene Amp 10× PCR Gold Buffer」、「AmpliTaq Gold DNA
polymerase」はApplied Biosystems社製である。
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000002
 The composition of the PCR cocktail is shown in Table 1, and the temperature cycle of PCR is shown in Table 2. In Table 1, “Gene Amp dNTP MIX”, “Gene Amp 10 × PCR Gold Buffer”, “AmpliTaq Gold DNA”
“polymerase” is manufactured by Applied Biosystems.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000002
　次に、ＰＣＲ産物を、アガロースゲル電気泳動により分離し、エチジウムブロマイドを用いて染色し、トランスイルミネーターを用いて可視化した。 Next, PCR products were separated by agarose gel electrophoresis, stained with ethidium bromide, and visualized using a transilluminator.
　結果を図１に示す。 The results are shown in FIG.
　図１は、本発明に係るプライマーを用いてＰＣＲを行った際のＰＣＲパターンを示す図面代用写真である。 FIG. 1 is a drawing-substituting photograph showing a PCR pattern when PCR is performed using a primer according to the present invention.
　図中、レーン１はサイズマーカー（１００～１，０００ｂｐ）を、レーン２はサイズマーカー（５００～５，０００ｂｐ）を、レーン３は既知のクラス１インテグロン検出用プライマーを用いて増幅した場合におけるＰＣＲパターンを、レーン４は既知のクラス２インテグロン検出用プライマーを用いて増幅した場合におけるＰＣＲパターンを、レーン５は既知のクラス３インテグロン検出用プライマーを用いて増幅した場合におけるＰＣＲパターンを、レーン６は配列番号１～４のプライマーを一度に用いて増幅した場合におけるＰＣＲパターンを、それぞれ表す。なお、図中の矢印は、プライマー設計に基づき、検出が予想されるバンドの位置を表す。 In the figure, lane 1 is a size marker (100 to 1,000 bp), lane 2 is a size marker (500 to 5,000 bp), and lane 3 is a PCR using a known class 1 integron detection primer. Lane 4 shows the PCR pattern when amplified using a known class 2 integron detection primer, Lane 5 shows the PCR pattern when amplified using a known class 3 integron detection primer, and Lane 6 shows Each of the PCR patterns when amplified using the primers of SEQ ID NOs: 1 to 4 at a time is shown. In addition, the arrow in a figure represents the position of the band expected to be detected based on the primer design.
　図１に示す通り、レーン３～レーン５では、予想された位置にバンドが検出された。この結果は、実験に用いた下水汚泥に、クラス１、クラス２、クラス３のインテグロンが含まれていたことを示す。なお、クラス１及びクラス２のインテグロンについては、シーケンシングを行い、塩基配列を確認した。その他、レーン５では、予想された位置以外のバンドも検出された。これは、クラス３インテグロン検出用のプライマーを用いた場合の副産物であると推測する。 As shown in FIG. 1, in lanes 3 to 5, bands were detected at the expected positions. This result shows that the sewage sludge used in the experiment contained class 1, class 2, and class 3 integrons. For class 1 and class 2 integrons, sequencing was performed and the nucleotide sequence was confirmed. In addition, in lane 5, bands other than the expected position were also detected. This is presumed to be a byproduct of using a primer for detecting class 3 integron.
　一方、図１のレーン６に示す通り、本発明に係るプライマーを用いて増幅した場合、プライマー設計に基づき検出が予想される位置（４８０ｂｐ付近）に、ほぼ単一のバンドが検出された。 On the other hand, as shown in lane 6 of FIG. 1, when amplification was performed using the primer according to the present invention, a substantially single band was detected at a position where detection was expected based on the primer design (around 480 bp).
　実施例２では、実施例１において本発明に係るプライマーで増幅したＰＣＲ産物を制限酵素で処理し、その消化パターンからどのクラスのインテグロンが増幅されたか、検討を行った。 In Example 2, the PCR product amplified with the primer according to the present invention in Example 1 was treated with a restriction enzyme, and it was examined which class of integron was amplified from its digestion pattern.
　まず、上記ＰＣＲ産物を、「ｉｌｌｕｓｔｒａ　ＧＦＸ　ＰＣＲ　ａｎｄ　Ｇｅｌ　Ｂａｎｄ　Ｐｕｒｉｆｉｃａｔｉｏｎ　Ｋｉｔ（ＧＥヘルスケア　バイオサイエンス株式会社製、以下同じ）」を用いて、付属プロトコルに従い、精製した。最終の溶出スケールは２０μＬとした。 First, the PCR product was purified according to the attached protocol using “illustra GFX PCR and Gel Band Purification Kit (manufactured by GE Healthcare Bioscience Co., Ltd., hereinafter the same)”. The final elution scale was 20 μL.
　次に、ＢｃｉＶＩ、ＥｃｉＩ、ＰｖｕＩＩの３種類の制限酵素を用いて、３７℃、１時間、その精製物を消化した。そして、その消化物を、アガロースゲル電気泳動により分離し、エチジウムブロマイドを用いて染色し、トランスイルミネーターを用いて可視化した。 Next, the purified product was digested at 37 ° C. for 1 hour using three types of restriction enzymes BciVI, EciI, and PvuII. The digests were separated by agarose gel electrophoresis, stained with ethidium bromide, and visualized using a transilluminator.
　ここで、制限酵素処理を行った場合における予想されるバンドの位置について、図２を用いて以下説明する。図２は、本発明に係るプライマーを用いてＰＣＲを行った場合に得られるＰＣＲ産物の制限酵素地図である。 Here, the expected band position when the restriction enzyme treatment is performed will be described below with reference to FIG. FIG. 2 is a restriction enzyme map of a PCR product obtained when PCR is performed using the primer according to the present invention.
　上述の通り、試料中にインテグロンが含まれている場合、ＰＣＲを行うと、プライマー設計に基づき、４８０ｂｐ付近にバンドが検出される。そのＰＣＲ産物について制限酵素処理を行うと、図２に示す通り、クラス１インテグロンが増幅された場合にはそれぞれ１１２、９５、１５８、１１７ｂｐに４つのバンドが、クラス２インテグロンが増幅された場合には４８２ｂｐの単一のバンドが、クラス３インテグロンが増幅された場合にはそれぞれ１１２、３７０ｂｐの２つのバンドが、クラス４インテグロンが増幅された場合にはそれぞれ２１０、２７５ｂｐの２つのバンドが、検出されることが予想される。 As described above, when integron is contained in the sample, when PCR is performed, a band is detected in the vicinity of 480 bp based on the primer design. When restriction enzyme treatment is performed on the PCR product, as shown in FIG. 2, when class 1 integron is amplified, four bands are respectively obtained at 112, 95, 158, and 117 bp, and when class 2 integron is amplified. A single band of 482 bp, two bands of 112 and 370 bp, respectively, when the class 3 integron is amplified, and two bands of 210 and 275 bp when the class 4 integron is amplified, respectively. It is expected that
　結果を図３に示す。 The results are shown in FIG.
　図３は本発明に係るプライマーを用いて行ったＰＣＲ産物を制限酵素処理した場合における消化パターンを示す図面代用写真である。 FIG. 3 is a drawing-substituting photograph showing a digestion pattern when a PCR product obtained using the primer according to the present invention is treated with a restriction enzyme.
　図中、レーン１はサイズマーカー（１００～１，０００ｂｐ）を、レーン２は実施例１で得たＰＣＲ産物（制限酵素未処理のもの；４８０ｂｐ）を、レーン３はサイズマーカー（１００～１，０００ｂｐ）を、レーン４は実施例１で得たＰＣＲ産物を制限酵素処理した場合における消化パターンを、それぞれ表す。 In the figure, lane 1 is the size marker (100 to 1,000 bp), lane 2 is the PCR product obtained in Example 1 (without restriction enzyme; 480 bp), and lane 3 is the size marker (100 to 1,000 bp). 000 bp), and lane 4 represents the digestion pattern when the PCR product obtained in Example 1 was treated with a restriction enzyme.
　図３のレーン４では、少なくとも６本の明瞭なバンドが検出された。 3 In lane 4 of FIG. 3, at least 6 distinct bands were detected.
　そのうち、矢印イで示すバンド（４８０ｂｐ付近）はクラス２インテグロン由来の断片、矢印ロで示すバンド（３７０ｂｐ付近）はクラス３インテグロン由来の断片、矢印ハで示すバンド（２１０ｂｐ付近）はクラス４インテグロン由来の断片、矢印ニで示すバンド（１６０ｂｐ付近）はクラス１インテグロン由来の断片であると推測する。 Of these, the band indicated by arrow A (near 480 bp) is a fragment derived from class 2 integron, the band indicated by arrow b (near 370 bp) is a fragment derived from class 3 integron, and the band indicated by arrow c (near 210 bp) is derived from class 4 integron. It is presumed that the fragment indicated by arrow D and the band indicated by arrow D (around 160 bp) is a fragment derived from class 1 integron.
　この結果は、本発明に係るプライマーを用いてＰＣＲを行った場合、クラス１～クラス４の全てのインテグロンが増幅されたことを示す。即ち、本実験結果は、本発明に係るプライマーを用いてＰＣＲを行うことにより、複数の異なるクラスのインテグロンを同時に検出できることを示す。 This result shows that all the integrons of class 1 to class 4 were amplified when PCR was performed using the primer according to the present invention. That is, this experimental result shows that a plurality of different classes of integrons can be detected simultaneously by performing PCR using the primer according to the present invention.
　実施例３では、本発明に係るプライマーを用いて、下水汚泥以外の環境試料からのインテグロン検出を試みた。 In Example 3, an integron detection from an environmental sample other than sewage sludge was attempted using the primer according to the present invention.
　本実施例では、環境試料に浄化槽汚泥を用いた。浄化槽汚泥１～２ｍＬを遠心（１０，０００ｒｐｍ、５分間）し、「Ｅ．Ｚ．Ｎ．Ａ．　Ｓｏｉｌ　ＤＮＡ　Ｉｓｏｌａｔｉｏｎ　Ｋｉｔ」を用いて沈降画分から鋳型ＤＮＡ試料を調製し、実施例１と同様の手順でＰＣＲを行い、ＰＣＲ産物を、アガロースゲル電気泳動により分離し、エチジウムブロマイドを用いて染色し、トランスイルミネーターを用いて可視化した。 In this example, septic tank sludge was used as an environmental sample. Centrifuge 1-2 ml of septic tank sludge (10,000 rpm, 5 minutes), and prepare a template DNA sample from the sedimented fraction using “EZNA Soil DNA Isolation Kit”. PCR was performed according to the procedure, and PCR products were separated by agarose gel electrophoresis, stained with ethidium bromide, and visualized using a transilluminator.
　結果を図４に示す。 The results are shown in FIG.
　図４は、環境試料に浄化槽汚泥を用いてＰＣＲを行った際のＰＣＲパターンを示す図面代用写真である。図中の各記載は図１のものと同様である。 FIG. 4 is a drawing-substituting photograph showing a PCR pattern when PCR is performed using septic tank sludge as an environmental sample. Each description in the figure is the same as that in FIG.
　図４のレーン６に示す通り、本発明に係るプライマーを用いて増幅した場合、図１と同様、プライマー設計に基づき検出が予想される位置（４８０ｂｐ付近）に、ほぼ単一のバンドが検出された。 As shown in lane 6 of FIG. 4, when amplified using the primer according to the present invention, a substantially single band is detected at a position (around 480 bp) where detection is expected based on the primer design, as in FIG. It was.
　続いて、単一のバンドとして検出されたＰＣＲ産物を実施例２と同様の手順で制限酵素処理し、その消化パターンからどのクラスのインテグロンが増幅されたか、検討を行った。 Subsequently, the PCR product detected as a single band was treated with a restriction enzyme in the same manner as in Example 2 to examine which class of integron was amplified from the digestion pattern.
　結果を図５に示す。 The results are shown in FIG.
　図５は環境試料に浄化槽汚泥を用いて行ったＰＣＲ産物を制限酵素処理した場合における消化パターンを示す図面代用写真である。図中の各記載は図３のものと同様である。 FIG. 5 is a drawing-substituting photograph showing a digestion pattern when a PCR product obtained by using septic tank sludge as an environmental sample is treated with a restriction enzyme. Each description in the figure is the same as that in FIG.
　図５のレーン４では、少なくとも６本の明瞭なバンドが検出された。この結果は、図３と同様、本発明に係るプライマーを用いてＰＣＲを行った場合、クラス１～クラス４の全てのインテグロンが増幅されたことを示す。即ち、本実験結果は、本発明に係るプライマーを用いてＰＣＲを行うことにより、下水汚泥以外の環境試料についても、複数の異なるクラスのインテグロンを同時に検出できることを示す。 In lane 4 of FIG. 5, at least 6 distinct bands were detected. This result shows that all of the class 1 to class 4 integrons were amplified when PCR was performed using the primer according to the present invention, as in FIG. That is, this experimental result shows that a plurality of different classes of integrons can be simultaneously detected from environmental samples other than sewage sludge by performing PCR using the primer according to the present invention.
　実施例４では、下水汚泥及び浄化槽汚泥由来のＰＣＲ産物についてクローン解析を行い、該試料中のインテグロンのクラスの多様性を調査した。 In Example 4, clonal analysis was performed on PCR products derived from sewage sludge and septic tank sludge, and the diversity of integron classes in the sample was investigated.
　まず、実施例１などと同様に、下水汚泥及び浄化槽汚泥１～２ｍＬをそれぞれ遠心（１０，０００ｒｐｍ、５分間）し、「Ｅ．Ｚ．Ｎ．Ａ．　Ｓｏｉｌ　ＤＮＡ　Ｉｓｏｌａｔｉｏｎ　Ｋｉｔ」を用いて、付属プロトコルに従い、沈降画分から鋳型ＤＮＡ試料を調製した。 First, in the same manner as in Example 1 or the like, 1 to 2 mL of sewage sludge and septic tank sludge are each centrifuged (10,000 rpm, 5 minutes) and attached using “EZNA Soil DNA Isolation Kit”. A template DNA sample was prepared from the sedimented fraction according to the protocol.
　次に、各ＰＣＲ産物を「ｉｌｌｕｓｔｒａ　ＧＦＸ　ＰＣＲ　ａｎｄ　Ｇｅｌ　Ｂａｎｄ　Ｐｕｒｉｆｉｃａｔｉｏｎ　Ｋｉｔ」を用いて精製し、クローニングベクターに組み込み、ライブラリーを作製した。そして、得られた各クローンについて、シーケンシングを行い、ＧｅｎＢａｎｋデータベースの配列データと照合した。 Next, each PCR product was purified using “illustra GFX PCR and Gel Band Purification Kit” and incorporated into a cloning vector to prepare a library. Each obtained clone was then sequenced and collated with the sequence data in the GenBank database.
　結果を表３に示す。
Figure JPOXMLDOC01-appb-T000003
 The results are shown in Table 3.
Figure JPOXMLDOC01-appb-T000003
　表３に示す通り、クローン解析の結果、下水汚泥及び浄化槽汚泥のいずれにおいても、汚泥中に存在するインテグロンは、主にクラス１のものであった。特に、浄化槽汚泥では、塩基配列が解読できたクローンのうち、その大半（９４％）をクラス１インテグロンが占めた。また、下水汚泥では、クラス３インテグロンの配列が６クローン確認された。ＰＣＲ産物として得られたクラス１インテグロンの部分配列を配列番号９に、クラス３インテグロンの部分配列を１０に、それぞれ示す。 As shown in Table 3, as a result of clonal analysis, intelon present in sludge was mainly class 1 in both sewage sludge and septic tank sludge. In particular, in septic tank sludge, class 1 integrons accounted for the majority (94%) of clones whose base sequences could be decoded. In the sewage sludge, 6 clones of the class 3 integron sequence were confirmed. The partial sequence of class 1 integron obtained as a PCR product is shown in SEQ ID NO: 9, and the partial sequence of class 3 integron is shown in 10.
　ＢＬＡＳＴを用いて相同性検索を行った結果、塩基配列を決定した領域（４００～５００塩基）の配列とＧｅｎＢａｎｋデータベースの配列データとの間で有意な相同性を示さなかったクローンが、下水汚泥由来のＰＣＲ産物中には１種類３１クローン（未知配列Ａ）、浄化槽汚泥由来のＰＣＲ産物中には２種類５クローン（未知配列Ｂ、Ｃ）存在した。ＰＣＲ産物として得られた未知配列Ａの配列を配列番号１１～１６に（６つのバリエーション）、未知配列Ｂの配列を配列番号１７及び１８に（２つのバリエーション）、未知配列Ｃの配列を配列番号１９に、それぞれ示す。 As a result of homology search using BLAST, clones that showed no significant homology between the sequence of the region where the nucleotide sequence was determined (400 to 500 nucleotides) and the sequence data of the GenBank database were derived from sewage sludge In one PCR product, 31 clones (unknown sequence A) were present, and in the PCR product derived from septic tank sludge, two clones (unknown sequences B and C) were present. The sequence of unknown sequence A obtained as a PCR product is represented by SEQ ID NOs: 11 to 16 (6 variations), the sequence of unknown sequence B is represented by SEQ ID NOs: 17 and 18 (2 variations), and the sequence of unknown sequence C is represented by SEQ ID NO: 19, respectively.
　ＦＡＳＴＡを用いて再度相同性検索を行った結果、未知配列Ａは、大腸菌のプラスミド上に存在するクラス１インテグロンのインテグラーゼ領域の配列（アクセッション番号：ＥＵ６７５６８６）と６５．５％の相同性が認められた。 As a result of homology search again using FASTA, the unknown sequence A has 65.5% homology with the sequence of the integrase region of class 1 integron (accession number: EU675686) present on the plasmid of E. coli. Admitted.
　同様に、未知配列Ｂは「Serratia marcescens」のクラス３インテグロンのインテグラーゼ領域の配列（アクセッション番号：ＡＦ４１６２９７）と６７．６％の相同性が認められた。 Similarly, the unknown sequence B was found to have 67.6% homology with the sequence of the integrase region of the class 3 integron of “Serratia marcescens” (accession number: AF416297).
　同様に、未知配列Ｃは「Xanthomonas campestris」のインテグロンのインテグラーゼ領域の配列（アクセッション番号：ＡＦ３２４４８３）と６６．７％の相同性が確認されたほか、「Shigella flexneri」のクラス１インテグロンのインテグラーゼ領域（アクセッション番号：ＡＹ５７４１９５）とも６５．０％の相同性が確認された。 Similarly, the unknown sequence C was confirmed to have 66.7% homology with the sequence of the integron integrase region of “Xanthomonas ８３campestris” (accession number: AF324443) and the integration of class 1 integron of “Shigella flexneri”. A homology of 65.0% was confirmed with the lyase region (accession number: AY574195).
　これらの結果は、Ａ、Ｂ、Ｃの各未知配列がインテグロン類由来のバンドであることを示唆する。即ち、本発明が、新規・未知なインテグロンの検出にも有効であることを示唆する。 These results suggest that each unknown sequence of A, B, and C is a band derived from integrons. That is, it is suggested that the present invention is also effective for detection of new and unknown integrons.
　実施例５では、下水汚泥及び浄化槽汚泥由来のＰＣＲ産物について、ＤＧＧＥ法による解析を試みた。 In Example 5, the PCR product derived from sewage sludge and septic tank sludge was analyzed by the DGGE method.
　まず、フォワードのプライマー（配列番号１～４）の５’側上流に４０ｂｐのＧＣクランプを付加したプライマーをそれぞれ作製した。 First, each primer was prepared by adding a 40 bp GC clamp upstream of the forward primer (SEQ ID NOs: 1 to 4) on the 5 'side.
　次に、下水汚泥又は浄化槽汚泥から実施例１などと同様の方法で鋳型ＤＮＡ試料を調製し、実施例１などと同様の手順・条件でＰＣＲを行った。フォワードのプライマーとしてＧＣクランプを付加した配列番号１～４の各プライマーを、リバースのプライマーとして配列番号５～８の各プライマーを、同時にＰＣＲカクテルに添加した。 Next, a template DNA sample was prepared from sewage sludge or septic tank sludge by the same method as in Example 1 and PCR was performed in the same procedure and conditions as in Example 1. Each primer of SEQ ID NOs: 1 to 4 with a GC clamp added as a forward primer and each primer of SEQ ID NOs: 5 to 8 as a reverse primer were simultaneously added to the PCR cocktail.
　次に、そのＰＣＲ産物を用いて、ＤＧＧＥ解析を行った。ゲルには、ＤＮＡ変性剤の濃度勾配を２５～６５％（７Ｍ尿素、４０％ホルムアミド（脱イオン）を１００％とする。）に調製した８％ポリアクリルアミドゲルを用いた。泳動槽を６０℃に保温し、泳動電圧１００Ｖで１２時間電気泳動を行った。泳動後、ＳＹＢＲ　Ｇｏｌｄ（登録商標、Ｍｏｌｅｃｕｌａｒ　Ｐｒｏｂｅ社製）でゲルを染色し、観察した。 Next, DGGE analysis was performed using the PCR product. As the gel, an 8% polyacrylamide gel prepared by adjusting the concentration gradient of the DNA denaturant to 25 to 65% (7M urea, 40% formamide (deionized) as 100%) was used. The electrophoresis tank was kept at 60 ° C., and electrophoresis was performed at an electrophoresis voltage of 100 V for 12 hours. After electrophoresis, the gel was stained with SYBR Gold (registered trademark, manufactured by Molecular Probe) and observed.
　また、ゲル中のバンドを観察した後、それらのバンドを切り出し、ＤＮＡの塩基配列を決定した。ＤＮＡの回収は、切り出したゲルをＴＥ緩衝液１００μＬの中に一晩浸透し、ＤＮＡを溶出させて行った。塩基配列の決定方法は、実施例４と同様の方法で行った。 Further, after observing bands in the gel, these bands were cut out and the DNA base sequence was determined. The DNA was collected by permeating the excised gel into 100 μL of TE buffer overnight to elute the DNA. The base sequence was determined by the same method as in Example 4.
　結果を図６に示す。図６は、下水汚泥及び浄化槽汚泥由来のＰＣＲ産物について、ＤＧＧＥ解析を行った結果を示す電気泳動写真である。図中、レーン１は下水汚泥をサンプルに用いた場合の結果を、レーン２は浄化槽汚泥をサンプルに用いた場合の結果をそれぞれ表わす。「２５％～６５％」は変性剤濃度勾配を、符号「イ」～「ト」は、バンドの位置をそれぞれ表わす。 The results are shown in FIG. FIG. 6 is an electrophoretogram showing the results of DGGE analysis of PCR products derived from sewage sludge and septic tank sludge. In the figure, lane 1 shows the result when sewage sludge is used as a sample, and lane 2 shows the result when septic tank sludge is used as a sample. “25% to 65%” represents the concentration gradient of the denaturant, and symbols “A” to “G” represent the positions of the bands.
　図６に示す通り、レーン１（下水汚泥）では「イ」～「ニ」の４つのバンドが、レーン２（浄化槽汚泥）では「ホ」～「ト」の３つのバンドがそれぞれ観察された。 As shown in Fig. 6, four bands from "I" to "D" were observed in lane 1 (sewage sludge), and three bands from "E" to "G" were observed in lane 2 (septic tank sludge).
　各バンドについて、塩基配列を解読した結果、「イ」、「ロ」のバンド中のＤＮＡがクラス３インテグロンのであること、及び、判読した４８２塩基中８塩基で両者の配列が相違することが分かった。「イ」のバンドから得られたＤＮＡの配列（クラス３インテグロンの部分長配列）を配列番号２０に、「ロ」のバンドから得られたＤＮＡの配列（同じく、クラス３インテグロンの部分長配列）を配列番号２１に、それぞれ示す。 As a result of decoding the base sequence for each band, it was found that the DNA in the bands “I” and “B” is a class 3 integron, and that the sequences of both were different at 8 bases in the 482 bases read. It was. The DNA sequence obtained from the band “A” (partial length sequence of class 3 integron) is shown in SEQ ID NO: 20, and the sequence of DNA obtained from the band “B” (also a partial length sequence of class 3 integron) Are shown in SEQ ID NO: 21, respectively.
　また、レーン１の「ハ」、「ニ」のバンド中のＤＮＡ、及び、レーン２の「ホ」、「ヘ」、「ト」のバンド中のＤＮＡがクラス１インテグロンであること、及び、これらがすべて同じ塩基配列であることが分かった。これらのバンドから得られたＤＮＡの配列（クラス１インテグロンの部分長配列）を配列番号２２に示す。 In addition, the DNA in the “c” and “d” bands in lane 1 and the DNA in the “e”, “f” and “g” bands in lane 2 are class 1 integrons, and these Were all the same base sequence. The DNA sequence (partial length sequence of class 1 integron) obtained from these bands is shown in SEQ ID NO: 22.
　以上の結果は、本発明に係るＰＣＲ－ＤＧＧＥ法を用いることにより、インテグロンの多様性を可視化、プロファイリングできることを示す。 The above results indicate that the diversity of integron can be visualized and profiled by using the PCR-DGGE method according to the present invention.
　実施例６では、本発明に係るプライマー対について、各クラスのインテグロンに対する検出性能の選択性について、検討を行った。 Example 6 examined the selectivity of the detection performance for each class of integron for the primer pair according to the present invention.
　まず、クラス１及びクラス２インテグロンのインテグラーゼ領域のＤＮＡを調製した。下水汚泥を用いて実施例１などと同様の方法で鋳型ＤＮＡ試料を調製し、既知のクラス１インテグロン検出用プライマー又は既知のクラス２インテグロン検出用プライマーを用いてＰＣＲを行い、そのＰＣＲ産物を「ｉｌｌｕｓｔｒａ　ＧＦＸ　ＰＣＲ　ａｎｄ　Ｇｅｌ　Ｂａｎｄ　Ｐｕｒｉｆｉｃａｔｉｏｎ　Ｋｉｔ」を用いて精製した。 First, integrase region DNAs of class 1 and class 2 integrons were prepared. A template DNA sample is prepared using sewage sludge in the same manner as in Example 1, etc., and PCR is performed using a known class 1 integron detection primer or a known class 2 integron detection primer. It was purified using illustra GFX PCR and Gel Band Purification Kit.
　次に、（ａ）フォワードに配列番号１及び２の二種類のプライマー、リバースに配列番号５及び６の二種類のプライマー、（ｂ）フォワードに配列番号３及び４の二種類のプライマー、リバースに配列番号７及び８の二種類のプライマー、（ｃ）フォワードに配列番号１～４の四種類のプライマー、リバースに配列番号５～８の四種類のプライマー、のいずれかを用いて、下水汚泥由来のＤＮＡを鋳型として、実施例１などと同様の方法により、ＰＣＲを行った。 Next, (a) two types of primers of SEQ ID NOS: 1 and 2 in the forward direction, two types of primers of SEQ ID NOS: 5 and 6 in the reverse direction, (b) two types of primers in SEQ ID NOS: 3 and 4 in the forward direction, reverse From either sewage sludge using two kinds of primers of SEQ ID NOs: 7 and 8, (c) four kinds of primers of SEQ ID NOs: 1 to 4 for forward, and four kinds of primers of SEQ ID NOs: 5 to 8 for reverse PCR was performed in the same manner as in Example 1 using the DNA of as a template.
　結果を図７及び図８に示す。それぞれ、図７はクラス１インテグロンのインテグラーゼ領域を鋳型ＤＮＡに用いてＰＣＲを行った際のＰＣＲパターンを示す図面代用写真、図８はクラス２インテグロンのインテグラーゼ領域を鋳型ＤＮＡに用いてＰＣＲを行った際のＰＣＲパターンを示す図面代用写真である。 Results are shown in FIG. 7 and FIG. FIG. 7 is a drawing-substituting photograph showing a PCR pattern when PCR is performed using the integrase region of class 1 integron as a template DNA, and FIG. 8 is a diagram showing PCR using the integrase region of class 2 integron as template DNA. It is a drawing substitute photograph which shows the PCR pattern at the time of performing.
　図７中、レーン１はサイズマーカー（１００～１，０００ｂｐ）を、レーン２はサイズマーカー（５００～５，０００ｂｐ）を、レーン３は既知のクラス１インテグロン検出用プライマーを用いて増幅した場合におけるＰＣＲパターンを、レーン４はフォワードに配列番号１及び２の二種類のプライマー、リバースに配列番号５及び６の二種類のプライマーを用いて増幅した場合におけるＰＣＲパターンを、レーン５はフォワードに配列番号３及び４の二種類のプライマー、リバースに配列番号７及び８の二種類のプライマーを用いて増幅した場合におけるＰＣＲパターンを、レーン６はフォワードに配列番号１～４の四種類のプライマー、リバースに配列番号５～８の四種類のプライマーを用いて増幅した場合におけるＰＣＲパターンを、それぞれ表わす。 In FIG. 7, lane 1 is amplified using a size marker (100 to 1,000 bp), lane 2 is amplified using a size marker (500 to 5,000 bp), and lane 3 is amplified using a known class 1 integron detection primer. The PCR pattern is lane 4, the PCR pattern when amplified using two types of primers SEQ ID Nos. 1 and 2 and the reverse using two types of primers SEQ ID Nos. 5 and 6, and lane 5 is the sequence number SEQ ID No. The PCR pattern in the case of amplification using two kinds of primers 3 and 4 and two kinds of primers SEQ ID Nos. 7 and 8 in the reverse, lane 6 forward, four kinds of primers SEQ ID Nos. 1-4, and reverse PCR patterns when amplified using four types of primers of SEQ ID NOs: 5 to 8 Representing, respectively.
　図８中、レーン３は既知のクラス２インテグロン検出用プライマーを用いて増幅した場合におけるＰＣＲパターンを表わす。他のレーンは図７と同様である。 In FIG. 8, lane 3 represents a PCR pattern when amplified using a known class 2 integron detection primer. The other lanes are the same as in FIG.
　両図中、星印は既知のクラス１又はクラス２インテグロン検出用プライマーを用いて増幅した場合における予想されるバンドの位置を、矢印は配列番号１～８のプライマーを用いて増幅した場合に予想されるバンドの位置を、それぞれ表わす。 In both figures, the asterisk indicates the expected band position when amplified using known class 1 or class 2 integron detection primers, and the arrow indicates when amplified using the primers of SEQ ID NOs: 1-8. Each band position is represented.
　図７では、フォワードに配列番号１及び２の二種類のプライマー、リバースに配列番号５及び６の二種類のプライマーを用いてＰＣＲを行った場合、クラス１インテグロンを検出できたが（レーン４）、フォワードに配列番号３及び４の二種類のプライマー、リバースに配列番号７及び８の二種類のプライマーを用いてＰＣＲを行った場合、クラス１インテグロンを検出できなかった（レーン５）。 In FIG. 7, when PCR was performed using two types of primers of SEQ ID NOs: 1 and 2 in the forward direction and two types of primers of SEQ ID NOs: 5 and 6 in the reverse direction, class 1 integrons could be detected (lane 4). When PCR was performed using the two types of primers of SEQ ID NOs: 3 and 4 in the forward direction and the two types of primers of SEQ ID NOs: 7 and 8 in the reverse direction, class 1 integron could not be detected (lane 5).
　一方、図８では、フォワードに配列番号１及び２の二種類のプライマー、リバースに配列番号５及び６の二種類のプライマーを用いてＰＣＲを行った場合、及び、フォワードに配列番号３及び４の二種類のプライマー、リバースに配列番号７及び８の二種類のプライマーを用いてＰＣＲを行った場合、いずれも、クラス２インテグロンを検出できた（レーン４、レーン５）。 On the other hand, in FIG. 8, when PCR was performed using two types of primers of SEQ ID NOS: 1 and 2 in the forward direction and two types of primers of SEQ ID NOS: 5 and 6 in the reverse direction, When PCR was performed using two kinds of primers and two kinds of primers of SEQ ID NOs: 7 and 8 in reverse, class 2 integron could be detected (lane 4, lane 5).
　以上より、本発明に係るプライマー対は、単独で又は複数を混合して用いることにより、複数の異なるクラスのインテグロンを同時に検出できる。 As described above, the primer pairs according to the present invention can detect a plurality of different classes of integrons simultaneously by using them alone or in combination.
　実施例７では、本発明に係るプライマー対を用いて環境試料中からインテグロンを検出する際における検出感度について、検討した。 In Example 7, the detection sensitivity when detecting integron from an environmental sample using the primer pair according to the present invention was examined.
　下水汚泥由来のＤＮＡ、浄化槽汚泥由来のＤＮＡ、及び、クラス１及びクラス２インテグロンのインテグラーゼ領域のＤＮＡを、それぞれ実施例１、３、６と同様の方法で調製した。 Sewage sludge-derived DNA, septic tank sludge-derived DNA, and class 1 and class 2 integron integrase region DNAs were prepared in the same manner as in Examples 1, 3, and 6, respectively.
　各ＤＮＡをそれぞれ１０ｐｇ、１ｐｇ、１００ｆｇ、１０ｆｇ、１ｆｇ、１００ａｇになるように調整し、実施例１と同様の方法でＰＣＲを行った。そして、それらのＰＣＲ産物を、アガロースゲル電気泳動により分離し、エチジウムブロマイドを用いて染色し、トランスイルミネーターを用いて可視化した。 Each DNA was adjusted to 10 pg, 1 pg, 100 fg, 10 fg, 1 fg, and 100 ag, and PCR was performed in the same manner as in Example 1. These PCR products were separated by agarose gel electrophoresis, stained with ethidium bromide, and visualized using a transilluminator.
　その結果、いずれのサンプルでＰＣＲを行った場合でも、鋳型ＤＮＡを１００ｆｇ以上添加した場合に目的のバンドの増幅を確認できた。従って、環境試料から少なくとも１００ｆｇのＤＮＡを取得できる場合、本発明に基づいて、インテグロンを検出可能である。 As a result, amplification of the target band could be confirmed when template DNA was added in an amount of 100 fg or more in any sample. Therefore, if at least 100 fg of DNA can be obtained from an environmental sample, integron can be detected according to the present invention.
　上述の通り、インテグロンは、環境中にも広く存在し、また、薬剤暴露の頻繁な環境や、下水処理施設などで検出されることが知られている。そのため、例えば、下水処理などでも分解されずに残存したインテグロンによって薬剤耐性遺伝子クラスターが様々な細菌に付与されたり、未知の多剤耐性病原菌が出現したりする可能性がある。従って、そのようなリスクを未然に防止するために、環境中におけるインテグロンの汚染状況を検出する手段が必要である。 As described above, integron is widely present in the environment, and is known to be detected in environments where chemical exposure is frequent or in sewage treatment facilities. Therefore, for example, drug-resistant gene clusters may be imparted to various bacteria by integrons that remain without being decomposed even in sewage treatment, or unknown multi-drug resistant pathogens may appear. Therefore, in order to prevent such a risk, a means for detecting the contamination status of integron in the environment is required.
　本発明では、一度の検出操作で、環境中の複数種類のインテグロンを同時に検出できるため、環境中における広汎な検査、リスク評価などが可能である。例えば、各種廃水や環境中におけるインテグロンの汚染状況の検査、堆肥などの安全性の評価、空調設備中の薬剤耐性菌の検出などに適用できる。 In the present invention, since a plurality of types of integrons in the environment can be detected simultaneously by a single detection operation, a wide range of inspections and risk assessments in the environment are possible. For example, the present invention can be applied to inspecting the state of contamination of integrons in various wastewaters and the environment, evaluating the safety of compost, and detecting drug-resistant bacteria in air conditioning equipment.
実施例１において、本発明に係るプライマーを用いてＰＣＲを行った際のＰＣＲパターンを示す図面代用写真。In FIG. 1, the drawing substitute photograph which shows the PCR pattern at the time of performing PCR using the primer which concerns on this invention in Example 1. FIG. 本発明に係るプライマーを用いてＰＣＲを行った場合に得られるＰＣＲ産物の制限酵素地図。The restriction enzyme map of the PCR product obtained when PCR is performed using the primer according to the present invention. 実施例２において、本発明に係るプライマーを用いて行ったＰＣＲの産物を制限酵素処理した場合における消化パターンを示す図面代用写真。In FIG. 2, the drawing substitute photograph which shows the digestion pattern at the time of carrying out the restriction enzyme process of the product of PCR performed using the primer which concerns on this invention. 実施例３において、環境試料に浄化槽汚泥を用いてＰＣＲを行った際のＰＣＲパターンを示す図面代用写真。In Example 3, the drawing substitute photograph which shows the PCR pattern at the time of performing PCR using septic tank sludge for an environmental sample. 実施例３において、環境試料に浄化槽汚泥を用いて行ったＰＣＲ産物を制限酵素処理した場合における消化パターンを示す図面代用写真。In Example 3, the drawing substitute photograph which shows the digestion pattern at the time of carrying out the restriction enzyme process of the PCR product performed using the septic tank sludge for an environmental sample. 実施例５において、下水汚泥及び浄化槽汚泥由来のＰＣＲ産物について、ＤＧＧＥ解析を行った結果を示す電気泳動写真。In Example 5, the electrophoresis photograph which shows the result of having performed the DGGE analysis about the PCR product derived from a sewage sludge and a septic tank sludge. 実施例６において、クラス１インテグロンのインテグラーゼ領域を鋳型ＤＮＡに用いてＰＣＲを行った際のＰＣＲパターンを示す図面代用写真。FIG. 5 is a drawing-substituting photograph showing a PCR pattern when PCR was performed using the integrase region of class 1 integron as a template DNA in Example 6. 実施例６において、クラス２インテグロンのインテグラーゼ領域を鋳型ＤＮＡに用いてＰＣＲを行った際のＰＣＲパターンを示す図面代用写真。In FIG. 6, a drawing-substituting photograph showing a PCR pattern when PCR was performed using the integrase region of class 2 integron as template DNA.

Claims (8)

  1. 　クラス１～クラス４のインテグロンのうち、複数のクラスのインテグロンにそれぞれ存在する所定領域の配列を同時に増幅しうる一又は二以上のプライマー対を用いて核酸増幅を行う手順を含むインテグロン検出方法。 An integron detection method comprising a step of performing nucleic acid amplification using one or two or more primer pairs capable of simultaneously amplifying sequences of predetermined regions respectively present in a plurality of classes of integron among class 1 to class 4 integrons.
  2. 　環境中におけるインテグロンの汚染状況を検出するインテグロン汚染検出方法であって、
    　クラス１～クラス４のインテグロンのうち、複数のクラスのインテグロンにそれぞれ存在する所定領域の配列を同時に増幅しうる一又は二以上のプライマー対を用いて核酸増幅を行い、環境試料からインテグロンを検出する手順を含むインテグロン汚染検出方法。     An integron contamination detection method for detecting the state of integron contamination in the environment,
    Among class 1 to class 4 integrons, nucleic acid amplification is performed using one or more primer pairs capable of simultaneously amplifying sequences of predetermined regions respectively present in a plurality of classes of integrons, and integrons are detected from environmental samples Integron contamination detection method including procedures.
  3. 　前記核酸増幅を定量ＰＣＲにより行う請求項２記載のインテグロン汚染検出方法。 The integron contamination detection method according to claim 2, wherein the nucleic acid amplification is performed by quantitative PCR.
  4. 　前記増幅した核酸をＤＧＧＥ法により解析する手順を含む請求項２記載のインテグロン汚染検出方法。 3. The integron contamination detection method according to claim 2, comprising a procedure for analyzing the amplified nucleic acid by a DGGE method.
  5. 　フォワードのプライマーに配列番号１～４のいずれかの配列を少なくとも含むＤＮＡ断片を、リバースのプライマーに配列番号５～８のいずれかの配列を少なくとも含むＤＮＡ断片を、それぞれ一対又は二以上の対で用いて核酸増幅を行う請求項２記載のインテグロン汚染検出方法。 A DNA fragment containing at least one of the sequences of SEQ ID NOs: 1 to 4 in the forward primer, and a DNA fragment containing at least any of the sequences of SEQ ID NOs: 5 to 8 in the reverse primer, respectively, in pairs or in pairs The integron contamination detection method according to claim 2, wherein nucleic acid amplification is performed using the method.
  6. 　クラス１～クラス４のインテグロンのうち、複数のクラスのインテグロンにそれぞれ存在する所定領域の配列を同時に増幅しうるプライマー対。 Primer pairs that can simultaneously amplify sequences of a predetermined region present in each of multiple classes of integron among class 1 to class 4 integrons.
  7. 　請求項６記載のプライマー対を用いて核酸増幅を行うことにより得られた核酸。 A nucleic acid obtained by performing nucleic acid amplification using the primer pair according to claim 6.
  8. 　請求項７記載の核酸の全長又は部分長と実質的に同一の塩基配列又はその相補配列を有する一本鎖核酸が少なくとも固定化されたＤＮＡチップ。 A DNA chip on which at least a single-stranded nucleic acid having a base sequence substantially the same as the full length or partial length of the nucleic acid according to claim 7 or a complementary sequence thereof is immobilized.
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* Cited by examiner, † Cited by third party
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