WO2017221960A1 - Oligonucléotide, sonde de détection d'aspergillus terreus, et méthode de détection d'aspergillus terreus - Google Patents

Oligonucléotide, sonde de détection d'aspergillus terreus, et méthode de détection d'aspergillus terreus Download PDF

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WO2017221960A1
WO2017221960A1 PCT/JP2017/022794 JP2017022794W WO2017221960A1 WO 2017221960 A1 WO2017221960 A1 WO 2017221960A1 JP 2017022794 W JP2017022794 W JP 2017022794W WO 2017221960 A1 WO2017221960 A1 WO 2017221960A1
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residue
oligonucleotide
base sequence
aspergillus terreus
probe
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Japanese (ja)
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▲そう▼明 村山
武 今西
文子 折田
育哉 伴
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学校法人日本大学
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • 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

Definitions

  • the present invention relates to an oligonucleotide, an Aspergillus terreus detection probe, and an Aspergillus terreus detection method.
  • Aspergillosis caused by Aspergillus spp. Is an invasive mycosis with a high infection frequency. Aspergillus spores are widely present in the environment and are not a problem for healthy individuals with immunity, but may cause opportunistic infections in patients with reduced immunity . Aspergillus spp. Are frequently detected from foliage plants in hospitals, water in vases, and air-conditioning outlets such as air conditioners, but the danger is not well known. Because the patient's immunity is originally reduced, the prognosis for aspergillosis is very poor and the fatality rate is high.
  • fungi are not successfully cultured, and even if they can be cultured, it is often difficult to identify the exact species. Even with biochemical methods, it is difficult to make a definitive diagnosis at the species level. Therefore, when mycosis is suspected, empirical treatment of antifungal drugs with few side effects is performed. If the bacterial species cannot be identified and the antifungal agent is not effective, symptoms often progress and fatal outcomes are often obtained. Since Aspergillus spp. Are difficult to cultivate depending on the specimen, it may be difficult to identify the exact species by culturing. Therefore, when aspergillosis is suspected, the exact bacterial species is not specified, and an antifungal agent applied to the genus Aspergillus is administered. Depending on the bacterial species, effective treatment is not performed and symptoms often progress.
  • Aspergillus terreus is a gold standard for antifungal drugs and has many primary resistant bacteria against amphotericin B that are effective against other Aspergillus species.
  • resistance to antifungal azole drugs is likely to be acquired as compared to other Aspergillus species. Therefore, infection by Aspergillus terreus is likely to become more serious than infection by other Aspergillus species.
  • the fatality rate is 90% or more. It is important to distinguish Aspergillus terreus from other Aspergillus species and provide effective treatment for Aspergillus terreus infection.
  • an effective method for discriminating and identifying Aspergillus terreus from other Aspergillus species has not been put into practical use, and it is extremely difficult to make a treatment policy.
  • Patent Document 1 discloses a nucleic acid probe for detecting Aspergillus species including Aspergillus terreus and other filamentous fungi.
  • a sequence specific to each bacterial species is found from the sequences of the internal transcription spacer region, 5.8S region, and 28S region of rDNA, and a probe for detecting each bacterial species is prepared.
  • Non-Patent Document 1 discloses an in-situ detection probe belonging to the genus Aspergillus using 2 ', 4'-BNA (LNA: locked nucleic acid).
  • the probe described in Patent Document 1 only confirms the specificity of the DNA extracted from each bacterial species to the PCR product, and it has not been confirmed whether the bacterial species can be accurately discriminated / identified in clinical specimens or the like.
  • the probe described in Non-Patent Document 1 is a universal detection probe for Aspergillus species, and Aspergillus cannot be distinguished at the species level.
  • an object of the present invention is to provide a technique capable of specifically detecting Aspergillus terreus.
  • the present invention is as follows.
  • Oligonucleotide selected from the group consisting of the following (i) to (iii): (I) an oligonucleotide having the base sequence set forth in SEQ ID NO: 15; (Ii) Specificity for ITS2 of Aspergillus tereus (internal transcription spacer region 2 of rDNA) consisting of a base sequence in which one or more residues are deleted, substituted or added in the base sequence of SEQ ID NO: 15 (Iii) an oligonucleotide having a base sequence complementary to the base sequence of the oligonucleotide of (i) or (ii).
  • the oligonucleotide according to (1) comprising the base sequence set forth in SEQ ID NO: 14 or 15 or a base sequence complementary to the base sequence.
  • oligonucleotide according to (3) comprising a base sequence selected from the group consisting of the following (a) to (c):
  • Base base configuration Column.
  • a probe for detecting Aspergillus terreus comprising the oligonucleotide according to any one of (1) to (4).
  • a method for detecting Aspergillus terreus comprising a step of hybridizing the probe for detecting Aspergillus terreus according to (5) to a subject.
  • Aspergillus terreus can be specifically detected in situ, Aspergillus terreus infection can be diagnosed quickly and accurately using a pathological tissue sample or the like.
  • the results of the ISH method using Ater01 to Ater03 for Aspergillus tereus and Aspergillus fumigatus cells are shown.
  • the results of the ISH method using Ater05 to Ater07 on Aspergillus terreus and Aspergillus fumigatus cells are shown.
  • the result of performing the ISH method on the kidney tissue specimen of Aspergillus tereus-infected mice is shown (200 times, bright field).
  • 18S rRNA gene probe of pan fungal probe was used
  • the result of performing the ISH method on the kidney tissue specimen of Aspergillus tereus-infected mice is shown (200 times, bright field).
  • Ater07 was used as a probe.
  • the result of performing the ISH method for the kidney tissue specimen of Aspergillus fumigatus-infected mice is shown (200 times, bright field).
  • an 18S rRNA gene probe, a panfungal probe was used.
  • the result of performing the ISH method for the kidney tissue specimen of Aspergillus fumigatus-infected mice is shown (200 times, bright field). Ater07 was used as a probe.
  • the result of having performed ISH method for the kidney tissue sample of an Aspergillus flavus-infected mouse is shown (200 times, bright field).
  • an 18S rRNA gene probe, a panfungal probe was used.
  • the result of performing the ISH method for the kidney tissue specimen of Aspergillus flavus-infected mice is shown (200 times, bright field). Ater07 was used as a probe.
  • the result of having performed ISH method for the kidney tissue sample of an Aspergillus nidulans infection mouse is shown (200 times, bright field).
  • an 18S rRNA gene probe of a panfungal probe was used.
  • the result of having performed ISH method for the kidney tissue sample of Aspergillus nidulans infection mouse is shown (200 times, bright field).
  • Ater07 was used as a probe.
  • the result of performing the ISH method for human clinical specimen 1 is shown (200 times). Ater07 was used as a probe.
  • the result of performing the ISH method for human clinical specimen 1 is shown (200 times).
  • Aspergillus fumigatus-specific Afut1 was used as a probe.
  • the result of performing the ISH method on human clinical specimen 2 is shown (200 times).
  • Ater07 was used as a probe.
  • the result of performing the ISH method on human clinical specimen 2 is shown (200 times).
  • Aspergillus fumigatus-specific Afut1 was used as a probe.
  • the result of performing the ISH method for human clinical specimen 1 is shown (40 times).
  • Ater05 was used as a probe.
  • the result of performing the ISH method for human clinical specimen 1 is shown (40 times).
  • Ater06 was used as a probe.
  • the result of performing the ISH method on human clinical specimen 2 is shown (200 times).
  • Ater05 was used as a probe.
  • the result of performing the ISH method on human clinical specimen 2 is shown (200 times).
  • Ater06 was used as a probe
  • the present invention provides an oligonucleotide selected from the group consisting of: (i) to (iii): (I) an oligonucleotide having the base sequence set forth in SEQ ID NO: 15; (Ii) an oligonucleotide having a nucleotide sequence of which one or more residues are deleted, substituted or added in the nucleotide sequence set forth in SEQ ID NO: 15 and having a specific binding ability to ITS2 of Aspergillus terreus; and (Iii) An oligonucleotide having a base sequence complementary to the base sequence of the oligonucleotide of (i) or (ii).
  • oligonucleotide is composed of only natural nucleotide residues, at least one residue containing nucleotide analog residues, and only nucleotide analog residues. It is meant to include everything.
  • the “residue” means a unit corresponding to a nucleotide in the case of DNA. That is, “residue” means a unit consisting of “base, sugar and phosphate” in the case of DNA or a unit corresponding thereto.
  • the “base” means a purine base or pyrimidine base such as adenine, guanine, thymine, cytosine, uracil.
  • the “base” means a base portion or a portion corresponding to the base portion of the residues consisting of “base, sugar and phosphate” in the case of DNA.
  • adenine residue”, “guanine residue”, “thymine residue”, “cytosine residue” and “uracil residue” are adenine, guanine, thymine, cytosine and uracil, respectively. Means residue.
  • nucleotide analog means a modified nucleotide containing a chemical modification at any position of the sugar moiety, or a monomer whose polymer exhibits a nucleic acid-like structure.
  • BNA BNA
  • ENA 2′-O, 4′-C-ethylene-crosslinked nucleic acid
  • examples of the latter include peptide nucleic acids (PNA), glycol nucleic acids (GNA), threose nucleic acids (TNA), enantiomer nucleotides (for example, ⁇ -L-deoxynucleotides instead of ⁇ -D-deoxynucleotides), etc. It has been.
  • nucleotide analog is not limited to these examples.
  • nucleotide analog may include a modification such as methylation in the base moiety.
  • nucleotide analog residue means a unit in which a unit corresponding to a nucleotide in the case of DNA is derived from a nucleotide analog.
  • the oligonucleotide of the present embodiment is an oligonucleotide selected from the group consisting of the following (i) to (iii): (I) an oligonucleotide having the base sequence set forth in SEQ ID NO: 15; (Ii) an oligonucleotide having a nucleotide sequence of which one or more residues are deleted, substituted or added in the nucleotide sequence set forth in SEQ ID NO: 15 and having a specific binding ability to ITS2 of Aspergillus terreus; and (Iii) An oligonucleotide having a base sequence complementary to the base sequence of the oligonucleotide of (i) or (ii).
  • the oligonucleotide of this embodiment is an oligonucleotide having the base sequence described in SEQ ID NO: 15 (AAGnnGCAAAnAAAnGCGnCG: n is a thymine residue or a uracil residue).
  • the base sequence described in SEQ ID NO: 15 is a sequence complementary to a part of the base sequence of ITS2 of Aspergillus terreus. Therefore, the oligonucleotide having the base sequence set forth in SEQ ID NO: 15 can hybridize to the sense strand of DNA encoding ITS2 of Aspergillus terreus and RNA having the ITS2 base sequence.
  • the base sequence described in SEQ ID NO: 15 is a base sequence specific to Aspergillus terreus, and other Aspergillus species and other fungal species are complementary bases having high similarity to this base sequence. It has no sequence in its genome. Therefore, the oligonucleotide consisting of the base sequence shown in SEQ ID NO: 15 does not hybridize to genomic DNA and RNA of other Aspergillus species and other fungal species. Therefore, the oligonucleotide having the base sequence set forth in SEQ ID NO: 15 specifically hybridizes to an RNA or ITS2 DNA region having the ITS2 base sequence of Aspergillus terreus.
  • the oligonucleotide of this embodiment has a nucleotide sequence in which one or more residues are deleted, substituted or added in the nucleotide sequence set forth in SEQ ID NO: 15, and has specific binding to ITS2 of Aspergillus terreus. It may be an oligonucleotide having a function.
  • the term “plurality” means 2 to 6, 2 to 5, 2 to 4, 2 to 3, or 2. As long as the specific binding ability of Aspergillus terreus to ITS2 is maintained, the position of the residue to be deleted, substituted or added is not particularly limited.
  • oligonucleotides include, for example, an oligonucleotide having a base sequence in which a plurality of residues located at the 5 ′ end and / or 3 ′ end of the base sequence shown in SEQ ID NO: 15 are deleted.
  • an oligonucleotide having a nucleotide sequence in which one, two, or three residues at the 3 'end of the nucleotide sequence set forth in SEQ ID NO: 15 are deleted is an example of the oligonucleotide of the present embodiment.
  • the base sequence described in SEQ ID NO: 14 is a base sequence in which three 3 'terminal residues of the base sequence described in SEQ ID NO: 15 are deleted.
  • the oligonucleotide having the base sequence set forth in SEQ ID NO: 14 is an example of the oligonucleotide of the present embodiment.
  • the oligonucleotide of this embodiment may be an oligonucleotide having a base sequence complementary to the base sequence of the oligonucleotide (i) or (ii).
  • the base sequence complementary to the base sequence of the oligonucleotide (i) is a sequence complementary to the base sequence described in SEQ ID NO: 15.
  • a base sequence complementary to the base sequence described in SEQ ID NO: 15 is shown in SEQ ID NO: 17 (CGACGCCAnnnnGCAACnn: n is a thymine residue or a uracil residue).
  • the base sequence described in SEQ ID NO: 17 is a sequence complementary to a part of the antisense strand of DNA encoding ITS2 of Aspergillus terreus. Therefore, the oligonucleotide consisting of the base sequence set forth in SEQ ID NO: 17 can hybridize to the antisense strand of DNA encoding ITS2 of Aspergillus terreus.
  • the base sequence described in SEQ ID NO: 17 is a base sequence specific to Aspergillus terreus, and other Aspergillus species and other fungal species are complementary bases having high similarity to this base sequence. It has no sequence in its genome.
  • the oligonucleotide consisting of the base sequence described in SEQ ID NO: 17 does not hybridize to genomic DNA and RNA of other Aspergillus species and other fungal species. Therefore, the oligonucleotide having the base sequence set forth in SEQ ID NO: 17 is an oligonucleotide that specifically hybridizes to the antisense strand of the ITS2 base sequence of Aspergillus terreus.
  • the oligonucleotide consisting of a base sequence complementary to the base sequence of the oligonucleotide of (ii) above is based on a base sequence in which one or more residues are deleted, substituted or added in the base sequence shown in SEQ ID NO: 17. And an oligonucleotide having specific binding ability to the antisense strand of the ITS2 nucleotide sequence of Aspergillus terreus.
  • Examples of such oligonucleotides include, for example, an oligonucleotide having a nucleotide sequence in which a plurality of residues located at the 5 ′ end and / or the 3 ′ end of the nucleotide sequence shown in SEQ ID NO: 17 are deleted. .
  • an oligonucleotide having a nucleotide sequence in which one, two, or three residues at the 5 'end of the nucleotide sequence set forth in SEQ ID NO: 17 are deleted is an example of the oligonucleotide of the present embodiment.
  • a base sequence complementary to the base sequence shown in SEQ ID NO: 14 is shown in SEQ ID NO: 16 (CGCAnnnnGnnACNAnn: n is a thymine residue or a uracil residue).
  • the oligonucleotide having the base sequence set forth in SEQ ID NO: 16 is an example of the oligonucleotide of the present embodiment.
  • all residues may be natural nucleotide residues, some residues may be nucleotide analog residues, and all residues are nucleotide analog residues. It may be.
  • the oligonucleotide of this embodiment contains a natural nucleotide residue, either a deoxyribonucleotide residue or a ribonucleotide residue may be sufficient as a natural nucleotide residue.
  • All natural nucleotide residues may be deoxyribonucleotide residues, and all natural nucleotide residues may be ribonucleotide residues.
  • the oligonucleotide of the present embodiment is an oligonucleotide having the base sequence shown in SEQ ID NO: 15, the residue at any position in the base sequence shown in SEQ ID NO: 15 is a ribonucleotide residue. Also good. The same applies to the oligonucleotide having the base sequence set forth in SEQ ID NO: 14, 16 or 17.
  • the base sequences described in SEQ ID NOs: 14 to 17 are all composed of deoxyribonucleotide residues, they are the base sequences described in SEQ ID NOs: 4, 5, 12, and 13, respectively.
  • the number of nucleotide analog residues is not particularly limited. For example, one nucleotide analog residue may be included, or two or more residues may be included. Moreover, all the residues may be nucleotide analog residues.
  • the oligonucleotide of this embodiment preferably contains at least one nucleotide analogue residue. For example, it can contain 5-15, preferably 7-12, more preferably 8-10 nucleotide analog residues. Nucleotide analogs such as BNA, PNA, GNA and TNA have high affinity for DNA and RNA of complementary strands and high thermal stability. In addition, it is not easily degraded by nucleases.
  • oligonucleotides containing nucleotide analog residues can be stably hybridized to the target sequence.
  • Residues other than nucleotide analog residues may be deoxyribonucleotide residues or ribonucleotide residues. All of the residues other than nucleotide analogues may be deoxyribonucleotide residues, or all may be ribonucleotide residues. Further, it may be composed of any number of deoxyribonucleotide residues and any number of ribonucleotide residues.
  • the residue at any position in the base sequence shown in SEQ ID NO: 15 is a nucleotide analog residue
  • the residue at any other position may be a ribonucleotide residue.
  • the type of nucleotide analog residue is not particularly limited.
  • the oligonucleotides of this embodiment can include nucleotide analog residues derived from the types of nucleotide analogs described above.
  • the nucleotide analog residue is preferably a BNA residue.
  • BNA has high binding affinity for complementary strand DNA and RNA, and also has high nuclease resistance. It is also easy to introduce a desired number of BNA residues at a desired position.
  • BNA is a cross-linked nucleic acid having a cross-linked structure in the sugar moiety, and so far 2 ′, 4′-BNA, 3′-amino-2 ′, 4′-BNA, 5′-amino-2 ′, 4′-BNA, 5′-amino-3 ′, 5′-BNA, 3′-amino-3 ′, 4′-BNA, 2 ′, 4′-BNA COC , 2 ′, 4′-BNA NC, etc.
  • 2 ′, 4′-BNA has a structure in which the oxygen atom at the 2 ′ site of ribose and the carbon atom at the 4 ′ site are bridged via a methylene group.
  • 3′-amino-2 ′, 4′-BNA has a structure in which the oxygen atom bonded to the carbon atom at the 3 ′ portion of 2 ′, 4′-BNA is substituted with —NH—.
  • 2 ′, 4′-BNA COC has a structure in which the oxygen atom at the 2 ′ site of ribose and the carbon atom at the 4 ′ site are bridged by —CH 2 OCH 2 —.
  • 2 ′, 4′-BNA NC has a structure in which the oxygen atom at the 2 ′ portion of ribose and the carbon atom at the 4 ′ portion are bridged by —NRCH 2 — (R is a functional group such as an alkyl group).
  • any of these BNAs can be used.
  • the BNA usable in the present embodiment is not limited to these, and known ones such as those described in International Publication No. 03/068795, International Publication No. 03/068794, International Publication No. 2005/021570, etc. BNA can be appropriately selected and used.
  • BNA may have a modification such as methylation at the base moiety.
  • BNA whose base moiety is methylated cytosine is generally used.
  • BNA include BNA having 5-methylcytosine in the base moiety.
  • the position of the nucleotide analog residue is not particularly limited.
  • the positions of nucleotide analog residues can include positions 1 to 4, 7, 8, 11, 11, and 15 to 18 in SEQ ID NO: 14.
  • the 1st position, the 3rd position, the 6th position, the 8th position, the 11th position, the 13th position, the 15th position, the 18th position, the 20th position and the 21st position in SEQ ID NO: 15 can be exemplified.
  • an oligonucleotide having a base sequence in which a uracil residue, an adenine residue at position 13, a thymine residue or uracil residue at position 15, and a guanine residue at position 18 are nucleotide analog residues is This is a preferred example of the oligonucleotide.
  • the 1st adenine residue, the 2nd adenine residue, the 3rd guanine residue, the 4th thymine residue or uracil residue, the 7th cytosine residue A thymine residue or uracil residue at position 11, a thymine residue or uracil residue at position 15, a guanine residue at position 16, a cytosine residue at position 17, and a guanine residue at position 18 are nucleotide analog residues
  • An oligonucleotide consisting of a base sequence is also a suitable example of the oligonucleotide of this embodiment.
  • a base wherein the adenine residue at position 13, the thymine or uracil residue at position 15, the guanine residue at position 18, the cytosine residue at position 20, and the guanine residue at position 21 are nucleotide analog residues
  • An oligonucleotide consisting of a sequence is also a suitable example of the oligonucleotide of the present embodiment.
  • the nucleotide analog residue is preferably a BNA residue.
  • the base moiety may be methylated.
  • a BNA residue having 5-methylcytosine in the base moiety can be used.
  • oligonucleotide in the base sequence shown in SEQ ID NO: 4, a 1-position adenine residue, a 4-position thymine residue, a 7-position cytosine residue, an 8-position adenine residue, Mention may be made of oligonucleotides consisting of a base sequence in which the thymine residue at position 11, the adenine residue at position 13, the thymine residue at position 15 and the guanine residue at position 18 are nucleotide analogue residues.
  • the 1st position adenine residue, the 2nd position adenine residue, the 3rd position guanine residue, the 4th position thymine residue, the 7th position cytosine residue, the 11th position also include oligonucleotides consisting of a base sequence in which the thymine residue, the thymine residue at position 15, the guanine residue at position 16, the cytosine residue at position 17, and the guanine residue at position 18 are nucleotide analogue residues. Can do.
  • An oligonucleotide consisting of a base sequence in which an adenine residue, a thymine residue at position 15, a guanine residue at position 18, a cytosine residue at position 20, and a guanine residue at position 21 are nucleotide analog residues is also provided.
  • the nucleotide analog residue is preferably a BNA residue.
  • the base moiety may be methylated as described above.
  • the oligonucleotide of this embodiment can be produced by a known nucleic acid synthesis method.
  • the oligonucleotide of this embodiment can be produced using a general nucleic acid automatic synthesizer by a general phosphoramidite method.
  • the nucleotide analog residue is a modified nucleotide such as BNA
  • an oligonucleotide having the modified nucleotide residue at the desired position is produced by incorporating the modified nucleotide at the desired position and performing nucleic acid synthesis. Can do.
  • the oligonucleotide of this embodiment can specifically hybridize to RNA having ITS2 nucleotide sequence of Aspergillus terreus and DNA sense strand or antisense strand encoding ITS2, the probe for detecting Aspergillus terreus Can be used as
  • the present invention provides an Aspergillus terreus detection probe comprising the oligonucleotide of the above embodiment.
  • a “probe” refers to a substance that specifically binds to a substance to be detected and enables the determination of the presence or absence of the substance to be detected in the subject.
  • the detection target is Aspergillus terreus, or its genomic DNA or its gene product.
  • the probe of this embodiment includes the oligonucleotide of the above-described embodiment.
  • the oligonucleotide of the above embodiment has a base sequence specific to ITS2 of Aspergillus tereus. Therefore, it can hybridize specifically to the sense strand or the antisense strand of RNA having ITS2 base sequence of Aspergillus terreus and DNA encoding ITS2. Therefore, if the oligonucleotide of the above-described embodiment is used, a probe that specifically detects only Aspergillus terreus can be created in distinction from other Aspergillus species.
  • the probe of this embodiment can contain an appropriate labeling substance in addition to the oligonucleotide of the above embodiment.
  • the labeling substance those generally used for nucleic acid probes can be used.
  • the labeling substance include fluorescent dyes, gold nanoparticles, biotin, antibodies, antigens, radioisotopes, chemiluminescent substances, enzymes, and the like.
  • fluorescent dyes examples include FAM (carboxyfluorescein), JOE (6-carboxy-4 ′, 5′-dichloro 2 ′, 7′-dimethoxyfluorescein), FITC (fluorescein isothiocyanate), TET (tetrachlorofluorescein), HEX (5′-hexachloro-fluorescein-CE phosphoramidite), Cy3, Cy5, Alexa568, Alexa647 and the like can be mentioned.
  • antigens examples include DIG (digoxigenin), FAM, FITC and the like.
  • the labeling substance can be bound to the oligonucleotide of the above embodiment using a known method according to the type of labeling substance.
  • the probe of this embodiment can be used for specifically detecting Aspergillus terreus.
  • the present invention provides a method for detecting Aspergillus terreus, comprising the step of hybridizing the probe of the above embodiment to a subject.
  • the hybridization method is not particularly limited.
  • a nucleic acid may be extracted from a clinical specimen or the like, and Southern hybridization or Northern hybridization may be performed.
  • the extracted nucleic acid may be subjected to PCR amplification using a universal primer capable of amplifying ITS2 of a fungal species, and Southern hybridization or the like may be performed on the obtained PCR product.
  • the probe of the above-described embodiment has a feature that Aspergillus terreus can be specifically detected even in the ISH method (in situ hybridization method). Therefore, the ISH method can be suitably used as a hybridization method.
  • the probe of the above-described embodiment is used as a probe. It has been found that the probe of the above embodiment can specifically detect Aspergillus terreus not only in nucleic acids extracted from tissues, cells, etc., and PCR amplified nucleic acids, but also in the ISH method. Therefore, if the probe of the above embodiment is used, Aspergillus terreus can be directly detected in a pathological tissue specimen or the like.
  • the ISH method can be performed by a conventional method.
  • a tissue section is prepared from a tissue collected from a patient suspected of Aspergillus terreus infection, subjected to protease treatment or the like in order to infiltrate the probe into the cell, and then the probe is hybridized. Then, the Aspergillus terreus can be detected on the tissue section by performing a visualization operation according to the labeling substance possessed by the probe.
  • the method of this embodiment is not limited to the method illustrated below.
  • the subject to be detected by Aspergillus terreus is a sample suspected of the presence of Aspergillus terreus.
  • the type of the sample is not particularly limited, and for example, a biological sample collected from a patient suspected of Aspergillus terreus infection can be used.
  • biological samples include tissue samples such as lung, bronchus, kidney, and skin, samples such as blood, sputum, saliva, urine, and stool, and bronchoalveolar lavage fluid (BAL).
  • BAL bronchoalveolar lavage fluid
  • the subject may be a culture of a biological sample as described above.
  • the subject is not limited to a biological sample, and may be a food sample, an environmental sample, or the like.
  • the subject preferably fixes tissue or cells.
  • the fixing method is not particularly limited, and a general tissue fixing method can be used.
  • the fixing solution include a fixing solution based on formalin or alcohol.
  • a fixative based on alcohol 95% ethanol is generally used.
  • the subject may be fixed on a slide glass or the like.
  • a formalin-fixed paraffin-embedded tissue section and the like can be mentioned.
  • the specimen may be pretreated before performing the hybridization reaction.
  • pretreatment include deparaffinization treatment, protease treatment, acetylation treatment, hydrochloric acid treatment, and surfactant treatment.
  • a deparaffinization process is performed as a pretreatment.
  • the deparaffinization treatment can be performed using xylene or the like.
  • protease treatment may be performed as pretreatment.
  • gentle protease treatment the probe can easily penetrate into the tissue without changing the localization of the bacteria.
  • the protein cross-linked to the target DNA is decomposed, and in the subsequent denaturation step, the DNA tends to become a single strand, and the probe can easily reach the target.
  • the protease used for the protease treatment those generally used in the ISH method can be used. Examples of the protease include protease K and pepsin.
  • acetylation treatment may be performed as pretreatment. By performing the acetylation treatment, electrostatic nonspecific binding of the probe to the tissue or the glass slide can be reduced. Furthermore, acetylation treatment is said to contribute to neutralization of positively charged molecules such as basic proteins.
  • the acetylation treatment can be performed with acetic acid, acetic anhydride triethanolamine solution, or the like.
  • hydrochloric acid treatment may be performed as pretreatment. Hydrochloric acid treatment removes basic proteins and exposes nucleic acids, increasing probe permeability and preventing non-specific reactions.
  • the subject may be subjected to pre-hybridization after appropriately performing the pretreatment as described above. By performing prehybridization, it can be expected to prevent background staining.
  • a prehybridization solution obtained by removing the probe from the hybridization solution can be used.
  • Prehybridization can be performed, for example, by dropping a prehybridization solution onto a specimen on a slide glass and allowing to stand at 40 to 60 ° C. for about 30 minutes to 2 hours.
  • the subject may be covered with a cover glass or parafilm.
  • Hybridization reaction is performed using the probe of the above-described embodiment. As other hybridization conditions, conditions normally used in the ISH method can be used.
  • the nucleic acid in the analyte is denatured so that it becomes a single strand. Denaturation can be carried out under conditions usually used in the ISH method. For example, the modification can be performed by heating at 80 to 100 ° C. for about 3 to 10 minutes.
  • the hybridization reaction can be performed by dropping a hybridization solution containing the probe of the above-described embodiment onto a specimen on a slide glass and allowing to stand at 40 to 60 ° C. for about 30 minutes to 15 hours. After dropping the hybridization solution, the subject may be covered with a cover glass, parafilm, or the like.
  • a hybridization solution can be prepared by mixing Denhardt's solution, formamide, dextran sulfate, SDS, EDTA, salt, buffer, and the like.
  • washing may be performed as necessary. By washing, non-specifically bound probes can be removed.
  • the specificity of washing can be adjusted according to the salt concentration of the washing solution and the washing temperature. In general, if the salt concentration of the washing solution is increased and the washing temperature is lowered, the specificity decreases. Addition of formamide to the washing solution reduces the specificity.
  • the label detection method can be appropriately selected according to the label used.
  • the label is 6-FAM
  • detection can be performed using an enzyme-linked anti-6-FAM antibody such as alkaline phosphatase.
  • enzyme-linked antibodies are commercially available for DIG, FITC, and the like, detection may be performed using these antibodies.
  • the label is a fluorescent dye
  • the label can be detected by observing with a fluorescence microscope.
  • the label is a radioisotope
  • the label can be detected by autoradiography or the like.
  • Aspergillus terreus can be specifically detected directly in a pathological tissue specimen or the like, so that even when culture is not possible or not cultivated, it can be performed quickly and accurately. Infections can be diagnosed. Retrospective analysis is also possible.
  • Table 3 shows the base sequences complementary to the probe.
  • Example 1 Detection by ISH method for cultured cells
  • Aspergillus terreus and Aspergillus fumigatus were tested for detection of Aspergillus terreus by the ISH method.
  • As the probes Ater01 to Ater03 and Ater05 to Ater07 were used.
  • the ISH method was performed according to the following procedure.
  • hydrochloric acid treatment was performed for 2 minutes at room temperature using 0.2N hydrochloric acid. Thereafter, the plate was washed with PBS for 2 minutes at room temperature, and then treated with 2 ⁇ g / mL protease K (PBS solution) at 37 ° C. for 5 minutes. After protease treatment, G-PBS (2 mg / mL glycine-containing PBS) was used for 10 minutes at room temperature, and then PBS was used for 2 minutes at room temperature.
  • PBS solution 2 ⁇ g / mL protease K
  • acetylation treatment was performed with 20% acetic acid at ice temperature for 15 seconds. After the acetylation treatment, the substrate was washed with PBS for 2 minutes at room temperature, and then the slide glass was lightly erected to drain water.
  • prehybridization was performed.
  • a box wetted with 50% formamide was preheated to 50 ° C., and a slide glass was placed in the box and allowed to stand for 1 hour for prehybridization.
  • the probe was prepared to be 100 ⁇ L with the hybridization solution.
  • a hybridization reaction was performed. First, the cover glass was removed with tweezers in warmed 2 ⁇ SSC (SSC: 0.03 M sodium citrate, 0.3 M sodium chloride), and the slide glass solution was cut. Thereafter, 100 ⁇ L of a hybridization solution containing the probe at a final concentration of 130 pmol / mL was dropped on a slide glass and covered with parafilm. The hybridization reaction was carried out by allowing it to stand at 50 ° C. overnight.
  • SSC 0.03 M sodium citrate, 0.3 M sodium chloride
  • the composition of the hybridization solution used in the above treatment is as follows. 1 ⁇ Denhardt's solution (Denhardt's solution: 1% Ficoll, 1% polyvinylpyrrolidone (P-5288) (SIGMA), 1% BSA (fraction V, SIGMA)) 50% formamide 10% dextran sulfate 0.25% SDS 600 mM NaCl 1 mM EDTA ⁇ 2Na pH 8.0 10 mM Tris pH 7.6
  • the slide glass was washed twice using 2 ⁇ SSC at 50 ° C. for 15 minutes under shaking at 77 rpm. Next, using 0.2 ⁇ SSC, washing was performed twice at 50 ° C. for 15 minutes under shaking at 77 rpm. Thereafter, the slide glass was rinsed with buffer 1 (100 mM Tris-HCl, 150 mM NaCl; pH 7.5).
  • the blocking treatment was performed by immersing the slide glass in the blocking solution and gently shaking at room temperature for 1 hour.
  • buffer 1 containing 1% blocking reagent (# 1096 176, Roche Life Science) was used.
  • the slide glass was taken out from the blocking solution, and alkaline phosphatase-labeled anti-FITC antibody (# 11, 426, 338, 910, Roche Life Science, 1.5 U / ml) was dropped onto the slide glass.
  • the slide glass was covered with parafilm and allowed to stand in a humid box at room temperature for 30 minutes. Thereafter, the slide glass was immersed in buffer 2 (buffer 1 containing 0.2% Tween 20), and washed 3 times with strong shaking for 15 minutes.
  • a color reaction was performed. First, the slide glass was rinsed with 0.1 M Tris pH 9.5. Next, the color developing solution was dropped on a slide glass, and a cover glass was placed thereon. The slide glass was placed in a wet box and a color reaction was performed at 37 ° C.
  • the coloring solution was prepared using Alkaline Phosphatase Conjugate Substrate Kit (# 170-6432, Bio-Rad). During the color development reaction, the color development reaction was performed for a maximum of one night while confirming the color development on the slide glass.
  • FIG. 2 shows the results of performing the ISH method using Ater05 to Ater07 as probes.
  • Ater01 to Ater03 color development was observed in both Aspergillus terreus and Aspergillus fumigatus cells. This result indicates that Ater01 to Ater03 hybridize not only to Aspergillus terreus DNA and RNA, but also to nucleic acids in Aspergillus fumigatus cells. Therefore, it was revealed that Ater01 to Ater03 cannot specifically detect Aspergillus terreus.
  • Ater01 to Ater03 are base sequences found by BLAST search as base sequences specific to Aspergillus terreus, and it was expected that Aspergillus terreus could be detected specifically. However, in the ISH method, nonspecific binding occurred, and Aspergillus terreus could not be specifically detected. This result shows that even if a base sequence specific to Aspergillus terreus is found by BLAST search or the like, it cannot always be used as a probe for the ISH method.
  • Ater05 to Ater07 color was observed in Aspergillus terreus cells, but no color was observed in Aspergillus fumigatus cells. This result indicates that Ater05 to Ater07 are probes that can specifically detect Aspergillus terreus. In addition, Ater06 and Ater07 had stronger coloring intensity in Aspergillus terreus cells than Ater05.
  • the hybridization reaction was carried out at 40 ° C., 55 ° C., and 60 ° C., but no change in specificity was observed from that at 50 ° C. (data not shown).
  • ISH ISH-infrared fluorescent in situ hybridization
  • Ater05 to Ater07 are probes that can specifically detect Aspergillus terreus cells by the ISH method.
  • Example 2 Detection by ISH method for infected animal tissues
  • Tissue samples were prepared from kidneys of Aspergillus terreus-infected mice and kidneys of Aspergillus fumigatus-infected mice, and detection of Aspergillus terreus was attempted by the ISH method.
  • the ISH method was performed in the same procedure as in Experimental Example 1. Ater07 was used as a probe. Ater07 was prepared to a final concentration of 130 pmol / mL and subjected to a hybridization reaction. As a positive control, the ISH method was also performed with an 18S probe (a panfungal probe targeting a fungal 18S rDNA sequence).
  • the 18S probe is a 568 bp probe DIG-labeled by PCR (Hanazawa® R, Murayama® SY, “Yamaguchi® H.“ In-situ detection ”of“ Aspergillus ”fumigatus.“ J ”Med® Microbiol. ), Adjusted to a final concentration of 1% and used.
  • FIGS. The results of the ISH method using Ater07 as a probe are shown in FIGS.
  • FIG. 3B With Ater07, color development was observed in kidney tissue sections of Aspergillus terreus-infected mice (FIG. 3B).
  • FIG. 4B no color development was observed in the kidney tissue section of Aspergillus fumigatus-infected mice (FIG. 4B).
  • FIG. 4A since color development was observed with the 18S probe, it was confirmed that the cells were in the tissue (FIG. 4A).
  • Ater07 is a probe that can be detected specifically in Aspergillus terreus even in tissue sections.
  • Example 1 Specimen 1 has been successfully cultured aspergillus terreus from the lung collected at necropsy and has been confirmed to be an aspergillus terreus-infected lung.
  • sample 2 Another clinical tissue sample is a mucous plug sample collected from a patient with allergic bronchopulmonary mycosis, which was provided by the National Hospital Organization Tokyo Hospital (hereinafter referred to as “Sample 2”). ). Specimen 2 has Aspergillus terreus cultured from the same patient's sputum.
  • the ISH method was performed in the same procedure as in Experimental Example 1. Ater05 to Ater07 were used as probes. Ater05 to Ater07 were prepared to a final concentration of 130 pmol / mL and subjected to a hybridization reaction. In addition, the ISH method was also performed with a probe Afut1 (245 bp, DIG labeling (Neuveglise C. Nucleic Acids Res 24: 1428-34, 1996) specific to Aspergillus fumigatus.
  • FIGS. 7 and 8 show the results of performing the ISH method using Ater07 as a probe.
  • 7 shows the sample 1
  • FIG. 8 shows the sample 2.
  • FIGS. 7 and 8 show the results of performing the ISH method using Ater07 as a probe.
  • FIGS. 7 and 8 show the results of performing the ISH method using Ater07 as a probe.
  • FIGS. 7 and 8 show the results of performing the ISH method using Ater07 as a probe.
  • FIGS. 7 and 8 shows the results of performing the ISH method using Ater07 as a probe.
  • Aft1 which is an Aspergillus fumigatus-specific probe
  • Ater07 can detect Aspergillus terreus specific even in human clinical specimens.
  • FIG. 9 the result of performing the ISH method using Ater05 or Ater06 as a probe is shown in FIG. 9 and FIG. 9 is for the sample 1 and FIG. 10 is for the sample 2.
  • FIG. 9 and FIG. 10 color development was also observed in both Sample 1 and Sample 2 for Ater05 and Ater06. From these results, it was confirmed that Ater05 and Ater06 can also specifically detect Aspergillus terreus in human clinical specimens. Note that Ater05 and Ater06 have lower color intensity than Ater07, and Ater07 is more suitable as a probe for the ISH method.
  • Ater05 to Ater07 are probes capable of specifically detecting Aspergillus terreus in clinical tissue samples by the ISH method.
  • Aspergillus terreus can be specifically detected in a pathological tissue specimen.
  • the present invention provides a rapid and accurate diagnostic method for Aspergillus terreus infection.

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Abstract

L'invention concerne un oligonucléotide sélectionné dans le groupe constitué par les oligonucléotides suivants (i) à (iii) : (i) un oligonucléotide qui comprend la séquence nucléotidique représentée par SEQ ID NO : 15; (ii) un oligonucléotide qui comprend une séquence nucléotidique produite par délétion, substitution ou addition d'un ou de plusieurs résidus au sein de la séquence nucléotidique représentée par SEQ ID NO : 15 et qui est apte à se lier spécifiquement à l'ITS2 d'Aspergillus terreus; et (iii) un oligonucléotide qui comprend une séquence nucléotidique complémentaire à la séquence nucléotidique de l'oligonucléotide (i) ou (ii).
PCT/JP2017/022794 2016-06-23 2017-06-21 Oligonucléotide, sonde de détection d'aspergillus terreus, et méthode de détection d'aspergillus terreus WO2017221960A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1266967A1 (fr) * 2001-06-15 2002-12-18 Gnosis Srl Procédé de production de pravastatine et de lovastatine
CN102031300A (zh) * 2010-08-18 2011-04-27 李国辉 一种检测土曲霉的dna探针、基因芯片及其应用
CN102321738A (zh) * 2011-07-29 2012-01-18 广州呼吸疾病研究所 检测致病曲霉菌的荧光定量pcr通用引物、检测探针和试剂盒

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1266967A1 (fr) * 2001-06-15 2002-12-18 Gnosis Srl Procédé de production de pravastatine et de lovastatine
CN102031300A (zh) * 2010-08-18 2011-04-27 李国辉 一种检测土曲霉的dna探针、基因芯片及其应用
CN102321738A (zh) * 2011-07-29 2012-01-18 广州呼吸疾病研究所 检测致病曲霉菌的荧光定量pcr通用引物、检测探针和试剂盒

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Title
MONTONE, KATHLEEN T. ET AL.: "In situ detection of aspergillus 18s ribosomal RNA sequences using a terminally biotinylated locked nucleic acids (LNA) probe", DIAGNOSTIC MOLECULAR PATHOLOGY, vol. 18, 2009, pages 239 - 242 *

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