JP6916482B2 - Aspergillus teres detection probe and Aspergillus teres detection method - Google Patents

Description

The present invention relates to oligonucleotides, probes for detecting Aspergillus terreus, and methods for detecting Aspergillus terreus.

Currently, 300 million people worldwide are infected with invasive mycoses annually, and about 1.35 million people die annually. Among mycoses, aspergillosis caused by Aspergillus spp. Is a highly infected and invasive mycosis. Aspergillus spores are widely present in the environment, and there is no problem if they are healthy people with immunity, but in patients with weakened immunity, they cause opportunistic infections. .. Aspergillus spp. Are frequently detected in foliage plants in hospitals, water in vases, and air-conditioning outlets such as air conditioners, but the danger is not known at present. The prognosis for aspergillosis is extremely poor and the case fatality rate is high because the patient's immune system is naturally weakened.

Fungi are often unsuccessful in culturing, and even if they can be cultivated, it is often difficult to identify the exact species. Even if biochemical methods are used, it is difficult to make a definitive diagnosis at the bacterial species level. Therefore, when mycosis is suspected, an antifungal drug with few side effects is administered as a prophylactic administration. If antifungal drugs are not effective due to lack of bacterial species identification, symptoms often progress and have fatal outcomes. Aspergillus spp. Is difficult to culture, so it is difficult to culture and identify the exact bacterial species. Therefore, in general, when aspergillosis is suspected, an antifungal drug having a wide antibacterial spectrum is administered without accurately identifying the bacterial species. As a result, effective treatment is not performed and symptoms often progress.

Among Aspergillus spp., Aspergillus terreus has many primary resistant bacteria to amphotericin B, which is a gold standard for mycosis and an effective antifungal drug against other Aspergillus spp. It is also reported that resistance to azole antifungal drugs is more likely to be acquired than other Aspergillus spp. Perhaps because of this, infection with Aspergillus terreus is more likely to be more severe than infection with other Aspergillus spp. In the case of disseminated infection, the case fatality rate is reported to be 90% or more. It is important to distinguish Aspergillus terreus from other Aspergillus spp. And provide effective treatment for Aspergillus terreus infection. However, an effective method for distinguishing Aspergillus tereus from other Aspergillus spp. And identifying it has not been put into practical use, and it is extremely difficult to formulate a treatment policy.

In general, the PCR method is considered to have good sensitivity as a method for identifying pathogens. However, it is not approved in Japan. The causes are 1) mycosis caused by saprophytic bacteria, it is difficult to prevent the problem of contamination, and 2) it is difficult to adapt unless it is a sterile sample such as blood or cerebrospinal fluid. Be done. On the other hand, Patent Document 1 discloses a nucleic acid probe for detecting Aspergillus spp. Species including Aspergillus terreus and other filamentous fungi. In Patent Document 1, 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 produced.

On the other hand, in recent years, artificial nucleic acids such as BNA (bridged nucleic acid) and PNA (peptide nucleic acid) have been developed. These artificial nucleic acids have a higher binding affinity to RNA or DNA complementary strands than natural nucleic acids, and also show high resistance to nucleic acid degrading enzymes. It is also applied to probes for detecting fungal species. For example, Non-Patent Document 1 discloses a probe for detecting in situ of the genus Aspergillus using 2', 4'-BNA (LNA: locked nucleic acids).

Japanese Unexamined Patent Publication No. 2010-42005

Montone KT, Feldman MD., Diagn Mol Pathol. 2009 Dec; 18 (4): 239-42.

However, the probe described in Patent Document 1 has only confirmed the specificity of the DNA extracted from each bacterial species with respect to the PCR product, and has not confirmed whether the bacterial species can be accurately identified and identified from clinical specimens and the like. .. On the other hand, the probe described in Non-Patent Document 1 is a universal detection probe for Aspergillus spp., And Aspergillus spp. Cannot be discriminated at the bacterial species level.

Therefore, an object of the present invention is to provide a technique capable of specifically detecting Aspergillus terreus.

The present invention is as follows.
(1) Oligonucleotides selected from the group consisting of the following (i) to (iii):
(I) Oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 15;
(Ii) The nucleotide sequence shown in SEQ ID NO: 15 consists of a nucleotide sequence in which one or more residues are deleted, substituted or added, and is specific to Aspergillus tereus ITS2 (internal transcription spacer region 2 of rDNA). Oligonucleotides capable of binding; and oligonucleotides consisting of a base sequence complementary to the base sequence of the oligonucleotide of (iii) (i) or (ii).
(2) The oligonucleotide according to (1), which comprises the nucleotide sequence of SEQ ID NO: 14 or 15 or a nucleotide sequence complementary to the nucleotide sequence.
(3) The oligonucleotide according to (1) or (2), which comprises at least one BNA residue.
(4) The oligonucleotide according to (3), which consists of a base sequence selected from the group consisting of the following (a) to (c):
(A) In the base sequence shown in SEQ ID NO: 14, the adenine residue at the 1-position, the timine residue or the uracil residue at the 4-position, the citocin residue at the 7-position, the adenine residue at the 8-position, and the timine residue at the 11-position. The base sequence in which the group or uracil residue, the adenine residue at the 13th position, the timine residue or the uracil residue at the 15th position, and the guanine residue at the 18th position are BNA residues;
(B) In the base sequence shown in SEQ ID NO: 14, adenine residue at position 1, adenine residue at position 2, guanine residue at position 3, timine residue or uracil residue at position 4, and cytosine residue at position 7. The group, the timine or uracil residue at the 11th position, the timine or uracil residue at the 15th position, the guanine residue at the 16th position, the cytosine residue at the 17th position, and the guanine residue at the 18th position are BNA residues. There is a base sequence; and (c) in the base sequence shown in SEQ ID NO: 15, the 1-position adenine residue, the 3-position guanine residue, the 6-position guanine residue, the 8-position adenine residue, and the 11-position Chimin residue or uracil residue, adenine residue at position 13, timine residue or uracil residue at position 15, guanine residue at position 18, citocin residue at position 20, and guanine residue at position 21 are BNA residues. The base sequence that is the group.
(5) A probe for detecting Aspergillus terreus, which comprises the oligonucleotide according to any one of (1) to (4).
(6) A method for detecting Aspergillus terreus, which comprises a step of hybridizing the probe for detecting Aspergillus terreus according to (5) with a subject.

According to the present invention, since Aspergillus terreus can be specifically detected in situ, Aspergillus terreus infection can be diagnosed quickly and accurately using a histopathological specimen or the like.

The results of the ISH method using Ater01 to Ater03 on Aspergillus tereus and Aspergillus fumigatus cells are shown. The results of the ISH method using Aspergillus tereus and Aspergillus fumigatus cells using Ater05 to Ater07 are shown. The results of the ISH method on kidney tissue samples of Aspergillus terreus-infected mice are shown (200x, bright field). In (a), the panfungal probe 18S rRNA gene probe was used as a positive control, and in (b), Ater07 was used as a probe. The results of the ISH method on kidney tissue samples of Aspergillus-Fumigatus-infected mice are shown (200x, bright field). In (a), the panfungal probe 18S rRNA gene probe was used as a positive control, and in (b), Ater07 was used as a probe. The results of the ISH method on kidney tissue samples of Aspergillus flavus-infected mice are shown (200x, bright field). In (a), the panfungal probe 18S rRNA gene probe was used as a positive control, and in (b), Ater07 was used as a probe. The results of the ISH method on kidney tissue samples of Aspergillus nidurance-infected mice are shown (200x, bright field). (A) used the 18S rRNA gene probe of the panfungal probe as a positive control, and (b) used Ater07 as the probe. The result of performing the ISH method on human clinical sample 1 is shown (200 times). Aspergillus fumigatus-specific Afut1 was used as a probe in (a) as Ater07 and in (b) as a negative control. The result of performing the ISH method on human clinical sample 2 is shown (200 times). Aspergillus fumigatus-specific Afut1 was used as a probe in (a) as Ater07 and in (b) as a negative control. The result of performing the ISH method on human clinical sample 1 is shown (40 times). Ater05 was used as a probe in (a), and Ater06 was used as a probe in (b). The result of performing the ISH method on human clinical sample 2 is shown (200 times). Ater05 was used as a probe in (a), and Ater06 was used as a probe in (b).

[Oligonucleotide]
In one embodiment, the present invention provides an oligonucleotide selected from the group consisting of the following (i) to (iii):
(I) Oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 15;
(Ii) An oligonucleotide consisting of a nucleotide sequence in which one or more residues are deleted, substituted or added in the nucleotide sequence shown in SEQ ID NO: 15 and having a specific binding ability to ITS2 of Aspergillus tereus; (Iii) An oligonucleotide consisting of a base sequence complementary to the base sequence of the oligonucleotide of (i) or (ii).

As used herein, an "oligonucleotide" is composed of only native nucleotide residues, one in which at least one residue contains a nucleotide analog residue, and one composed of only nucleotide analog residues. It means to include everything. Further, the “residue” means a unit corresponding to a nucleotide in the case of DNA. That is, the "residue" means a unit composed of "base, sugar and phosphoric acid" in the case of DNA or a unit corresponding thereto. Further, the "base" means a purine base such as adenine, guanine, thymine, cytosine, and uracil, or a pyrimidine base. That is, in the present specification, the "base" means a base portion of a residue consisting of "base, sugar and phosphoric acid" in the case of DNA or a portion corresponding thereto. Further, the "adenine residue", "guanine residue", "thymine residue", "cytosine residue" and "uracil residue" have base portions of adenine, guanine, thymine, cytosine and uracil, respectively. Means a residue.

As used herein, the term "nucleotide analog" means a modified nucleotide containing a chemical modification at any position of the sugar moiety, or a monomer in which a polymer thereof exhibits a nucleic acid-like structure. As examples of the former, BNA, ENA (2'-O, 4'-C-ethylene-crosslinked nucleic acid) and the like are known. Further, as an example of the latter, peptide nucleic acid (PNA), glycol nucleic acid (GNA), threose nucleic acid (TNA), mirror image nucleotide (for example, β-L-deoxynucleotide instead of β-D-deoxynucleotide) and the like are known. Has been done. The "nucleotide analog" is not limited to these examples. Further, the nucleotide analog may include a modification such as methylation in the base portion. As used herein, the term "nucleotide analog residue" means a unit in which the unit corresponding to a nucleotide in the case of DNA is derived from a nucleotide analog.

The oligonucleotide of this embodiment is an oligonucleotide selected from the group consisting of the following (i) to (iii):
(I) Oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 15;
(Ii) An oligonucleotide consisting of a nucleotide sequence in which one or more residues are deleted, substituted or added in the nucleotide sequence shown in SEQ ID NO: 15 and having a specific binding ability to ITS2 of Aspergillus tereus; (Iii) An oligonucleotide consisting of a base sequence complementary to the base sequence of the oligonucleotide of (i) or (ii).

The oligonucleotide of this embodiment is an oligonucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 15 (AAGnnGCAAanAAAanGCGnCG: n is a thymine residue or a uracil residue). The base sequence shown in SEQ ID NO: 15 is a sequence complementary to a part of the base sequence of ITS2 of Aspergillus terreus. Therefore, the oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 15 can hybridize to the sense strand of the DNA encoding ITS2 of Aspergillus terreus and the RNA having the ITS2 nucleotide sequence. On the other hand, the nucleotide sequence shown in SEQ ID NO: 15 is a nucleotide sequence specific to Aspergillus terreus, and other Aspergillus genus strains and other fungal species have complementary nucleotides having high similarity to this nucleotide sequence. It does not have a sequence in its genome. Therefore, the oligonucleotide consisting of the nucleotide sequence of SEQ ID NO: 15 does not hybridize to the genomic DNA and RNA of other Aspergillus spp. And other fungal species. Therefore, the oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 15 specifically hybridizes to the RNA or ITS2DNA region having the ITS2 nucleotide sequence of Aspergillus terreus.

In addition, the oligonucleotide of this embodiment consists of a base sequence in which one or more residues are deleted, substituted or added in the base sequence shown in SEQ ID NO: 15, and is a specific binding of Aspergillus terreus to ITS2. It may be an oligonucleotide having a function. In the present specification, the term "plurality" means 6, 5, 4, 3, or 2. The position of the residue to be deleted, substituted or added is not particularly limited as long as the specific binding ability of Aspergillus terreus to ITS2 is maintained. Examples of such oligonucleotides include oligonucleotides consisting of a base sequence in which a plurality of residues located at the 5'end and / or 3'end of the base sequence set forth in SEQ ID NO: 15 are deleted. .. For example, an oligonucleotide consisting of a base sequence in which the residue at the 3'end of the base sequence shown in SEQ ID NO: 15 is deleted by 1, 2, or 3 is an example of the oligonucleotide of the present embodiment. The nucleotide sequence shown in SEQ ID NO: 14 (AAGnnGCAAanAAanGCG: n is a thymine residue or a uracil residue) is a nucleotide sequence in which three 3'terminal residues of the nucleotide sequence shown in SEQ ID NO: 15 are deleted. The oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 14 is an example of the oligonucleotide of this embodiment.

Further, the oligonucleotide of the present embodiment may be an oligonucleotide having a base sequence complementary to the base sequence of the oligonucleotide of (i) or (ii) above. The base sequence complementary to the base sequence of the oligonucleotide of (i) above is a sequence complementary to the base sequence shown in SEQ ID NO: 15. A base sequence complementary to the base sequence shown in SEQ ID NO: 15 is shown in SEQ ID NO: 17 (CGACGCannAnnnGCAACnn: n is a thymine residue or a uracil residue). The nucleotide sequence shown in SEQ ID NO: 17 is a sequence complementary to a part of the antisense strand of the DNA encoding ITS2 of Aspergillus terreus. Therefore, the oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 17 can hybridize to the antisense strand of the DNA encoding ITS2 of Aspergillus terreus. On the other hand, the nucleotide sequence shown in SEQ ID NO: 17 is a nucleotide sequence specific to Aspergillus terreus, and other Aspergillus genus strains and other fungal species have complementary nucleotides having high similarity to this nucleotide sequence. It does not have a sequence in its genome. Therefore, the oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 17 does not hybridize to the genomic DNA and RNA of other Aspergillus spp. And other fungal species. Therefore, the oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 17 is an oligonucleotide that specifically hybridizes to the antisense strand of the ITS2 nucleotide sequence of Aspergillus terreus.

The oligonucleotide consisting of the nucleotide sequence complementary to the nucleotide sequence of the oligonucleotide (ii) is derived from the nucleotide sequence in which one or more residues are deleted, substituted or added in the nucleotide sequence shown in SEQ ID NO: 17. It is an oligonucleotide that has the ability to specifically bind to the antisense strand of the ITS2 base sequence of Aspergillus tereus. Examples of such an oligonucleotide include, for example, an oligonucleotide consisting of a base sequence in which a plurality of residues located at the 5'end and / or 3'end of the base sequence set forth in SEQ ID NO: 17 are deleted. .. For example, an oligonucleotide consisting of a base sequence in which the residue at the 5'end of the base sequence shown in SEQ ID NO: 17 is deleted by 1, 2, or 3 is an example of the oligonucleotide of the present embodiment. As an example, a base sequence complementary to the base sequence shown in SEQ ID NO: 14 is shown in SEQ ID NO: 16 (CGCAnnnAnnnGCAACnn: n is a thymine residue or a uracil residue). The oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 16 is an example of the oligonucleotide of this embodiment.

In 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. When the oligonucleotide of the present embodiment contains a natural nucleotide residue, the natural nucleotide residue may be either a deoxyribonucleotide residue or a ribonucleotide residue. All natural nucleotide residues may be deoxyribonucleotide residues, and all natural nucleotide residues may be ribonucleotide residues. Further, it may be composed of an arbitrary number of deoxyribonucleotide residues and an arbitrary number of ribonucleotide residues. For example, when the oligonucleotide of the present embodiment is an oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 15, the residue at an arbitrary position in the nucleotide sequence shown in SEQ ID NO: 15 is a ribonucleotide residue. May be good. The same applies to the oligonucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 14, 16 or 17. When all the base sequences shown in SEQ ID NOs: 14 to 17 are composed of deoxyribonucleotide residues, the base sequences are the base sequences shown in SEQ ID NOs: 4, 5, 12 and 13, respectively.

When the oligonucleotide of the present embodiment contains nucleotide analog residues, the number of nucleotide analog residues is not particularly limited. For example, it may contain one nucleotide analog residue or may contain two or more residues. In addition, all residues may be nucleotide analog residues. The oligonucleotide of this embodiment preferably contains at least one nucleotide analog residue. For example, it can contain 5 to 15, preferably 7 to 12, more preferably 8 to 10 nucleotide analog residues. Nucleotide analogs such as BNA, PNA, GNA, and TNA have high affinity for complementary strands of DNA and RNA, and also have high thermal stability. It is also less susceptible to degradation by nucleases. Therefore, the oligonucleotide containing the nucleotide analog residue can stably hybridize to the target sequence. The residue other than the nucleotide analog residue may be either a deoxyribonucleotide residue or a ribonucleotide residue. All residues other than nucleotide analogs may be deoxyribonucleotide residues, or all may be ribonucleotide residues. Further, it may be composed of an arbitrary number of deoxyribonucleotide residues and an arbitrary number of ribonucleotide residues. For example, when the oligonucleotide residue of the present embodiment is an oligonucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 15, the residue at an arbitrary position in the nucleotide sequence set forth in SEQ ID NO: 15 is a nucleotide analog residue. And the residue at any other position may be a ribonucleotide residue. The same applies to the oligonucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 14, 16 or 17.

When the oligonucleotide of the present embodiment contains a nucleotide analog residue, the type of the nucleotide analog residue is not particularly limited. For example, the oligonucleotide of this embodiment can contain nucleotide analog residues derived from the above types of nucleotide analogs. In the oligonucleotide of this embodiment, the nucleotide analog residue is preferably a BNA residue. BNA has a high binding affinity for complementary strands of DNA and RNA, and also has high nuclease resistance. In addition, it is easy to introduce a desired number of BNA residues at a desired position.

BNA is a crosslinked nucleic acid having a crosslinked 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. Are known. For example, 2', 4'-BNA has a structure in which an oxygen atom at the 2'site of ribose and a carbon atom at the 4'site are crosslinked via a methylene group. Further, 3'-amino-2'and 4'-BNA have a structure in which the oxygen atom bonded to the carbon atom at the 3'site of 2', 4'-BNA is replaced with -NH-. Further, the 2', 4'-BNA ^COC has a structure in which an oxygen atom at the 2'site of ribose and a carbon atom at the 4'site are _{crosslinked with −CH 2} OCH _{2 −.} Further, 2 ', ^{4'-BNA NC} is 2 ribose' oxygen atom and the 4 'site carbon atoms sites, -NRCH ₂ - a (R is a functional group such as an alkyl group) crosslinked with structure Have. In this embodiment, any of these BNAs can be used. Further, the BNA that can be used in the present embodiment is not limited to these, and is known, for example, those described in International Publication No. 03/068795, International Publication No. 03/068794, and International Publication No. 2005/021570. BNA can be appropriately selected and used.

The BNA may have a modification such as methylation at the base portion. For example, BNA or the like whose base portion is methylated cytosine is generally used. Examples of such BNA include BNA having 5-methylcytosine as a base moiety.

When the oligonucleotide of this embodiment contains a nucleotide analog residue, the position of the nucleotide analog residue is not particularly limited. For example, as the position of the nucleotide analog residue, the 1st to 4th position, the 7th position, the 8th position, the 11th position, the 13th position, the 15th to 18th position and the like in SEQ ID NO: 14 can be mentioned. Further, the 1st, 3rd, 6th, 8th, 11th, 13th, 15th, 18th, 20th, 21st and the like in SEQ ID NO: 15 can be mentioned.

For example, in the nucleotide sequence shown in SEQ ID NO: 14, the 1-position adenine residue, the 4-position thymine residue or uracil residue, the 7-position citocin residue, the 8-position adenine residue, and the 11-position thymine residue. Alternatively, an oligonucleotide consisting of a base sequence in which the uracil residue, the adenine residue at the 13th position, the thymine residue or the uracil residue at the 15th position, and the guanine residue at the 18th position are nucleotide analog residues is the present embodiment. Is a good example of the oligonucleotide of. Further, in the nucleotide sequence shown in SEQ ID NO: 14, the adenine residue at the 1-position, the adenine residue at the 2-position, the guanine residue at the 3-position, the thymine residue or uracil residue at the 4-position, and the cytosine residue at the 7-position. , 11th thymine or uracil residue, 15th thymine or uracil residue, 16th guanine residue, 17th citosine residue, and 18th guanine residue are nucleotide sequence residues. An oligonucleotide consisting of a base sequence is also a good example of the oligonucleotide of this embodiment. Further, in the nucleotide sequence shown in SEQ ID NO: 15, the 1-position adenine residue, the 3-position guanine residue, the 6-position guanine residue, the 8-position adenine residue, the 11-position timine residue or the uracil residue , Adenine residue at position 13, timine or uracil residue at position 15, guanine residue at position 18, guanine residue at position 20, and guanine residue at position 21 are nucleotide sequence residues. Sequence-based oligonucleotides are also a good example of the oligonucleotides of this embodiment. In the above example, the nucleotide analog residue is preferably a BNA residue. When a BNA residue is used, the base portion may be methylated. For example, for the cytosine residue, a BNA residue having 5-methylcytosine as a base moiety or the like can be used.

As an example of the above-mentioned oligonucleotide, in the nucleotide sequence shown in SEQ ID NO: 4, the adenine residue at the 1-position, the thymine residue at the 4-position, the cytosine residue at the 7-position, and the adenine residue at the 8-position, Examples thereof include an oligonucleotide consisting of a base sequence in which the thymine residue at position 11 and the adenine residue at position 13 and the thymine residue at position 15 and the guanine residue at position 18 are nucleotide analog residues. Further, in the nucleotide sequence shown in SEQ ID NO: 4, the 1-position adenine residue, the 2-position adenine residue, the 3-position guanine residue, the 4-position thymine residue, the 7-position cytosine residue, and the 11-position Also include an oligonucleotide consisting of a base sequence in which the thymine residue, the thymine residue at the 15th position, the guanine residue at the 16th position, the cytosine residue at the 17th position, and the guanine residue at the 18th position are nucleotide analog residues. Can be done. Further, in the nucleotide sequence shown in SEQ ID NO: 5, the 1-position adenine residue, the 3-position guanine residue, the 6-position guanine residue, the 8-position adenine residue, the 11-position thymine residue, and the 13-position Also, an oligonucleotide consisting of a base sequence in which the adenine residue, the thymine residue at the 15th position, the guanine residue at the 18th position, the cytosine residue at the 20th position, and the guanine residue at the 21st position are nucleotide analog residues. Can be mentioned. Also in these examples, the nucleotide analog residue is preferably a BNA residue. When the BNA residue is used, the base portion may be methylated as described above.

The oligonucleotide of this embodiment can be produced by a known nucleic acid synthesis method. For example, the oligonucleotide of the present embodiment can be produced by using a nucleic acid automatic synthesizer or the like by a general phosphoramidite method. When the oligonucleotide of the present embodiment contains a nucleotide analog residue, it can also be produced by a known method. For example, if the nucleotide analog residue is a modified nucleotide such as BNA, an oligonucleotide having the modified nucleotide residue at the desired position can be produced by incorporating the modified nucleotide at a desired position and performing nucleic acid synthesis. Can be done.

Since the oligonucleotide of this embodiment can specifically hybridize to the sense strand or antisense strand of RNA having the ITS2 base sequence of Aspergillus terreus and DNA encoding ITS2, a probe for detecting Aspergillus terreus. Can be used as.

[Probe for detecting Aspergillus terreus]
In one embodiment, the present invention provides a probe for detecting Aspergillus terreus, which comprises the oligonucleotide of the above embodiment.

As used herein, the term "probe" refers to a substance that specifically binds to a substance to be detected and enables determination of the presence or absence of the substance to be detected in a subject. In the probe of this embodiment, the detection target is Aspergillus terreus, 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 terreus. Therefore, it can specifically hybridize to the sense strand or antisense strand of RNA having the ITS2 base sequence of Aspergillus terreus and DNA encoding ITS2. Therefore, by using the oligonucleotide of the above-described embodiment, it is possible to prepare a probe that specifically detects only Aspergillus teres, distinguishing it from other Aspergillus spp.

The probe of this embodiment can contain an appropriate labeling substance in addition to the oligonucleotide of the above embodiment. As the labeling substance, those generally used for nucleic acid probes can be used. For example, examples of the labeling substance include fluorescent dyes, gold nanoparticles, biotins, antibodies, antigens, radioisotopes, chemical illuminants, enzymes and the like. Examples of fluorescent dyes include FAM (carboxyfluorescein), JOE (6-carboxy-4', 5'-dichloro2', 7'-dimethoxyfluorescein), FITC (fluorescein isothiocyanate), TET (tetrachlorofluorescein), HEX (5'-hexachloro-fluorescein-CE phosphoromidite), Cy3, Cy5, Alexa568, Alexa647 and the like can be mentioned. Further, examples of the antigen include DIG (digoxigenin), FAM, FITC and the like. The labeling substance can be bound to the oligonucleotide of the above embodiment by using a known method depending on the type of the labeling substance.

The probe of this embodiment can be used to specifically detect Aspergillus terreus.

[How to detect Aspergillus tereus]
In one embodiment, the present invention provides a method for detecting Aspergillus terreus, which comprises the step of hybridizing the probe of the above embodiment to a subject.

In the method of the present embodiment, the hybridization method is not particularly limited. For example, nucleic acid may be extracted from a clinical sample or the like and subjected to Southern hybridization or Northern hybridization. Further, the extracted nucleic acid may be PCR-amplified 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.

On the other hand, the probe of the above-described embodiment has a feature that Aspergillus tereus can be specifically detected even in the ISH method (in situ hybridization method). Therefore, the ISH method can be preferably used as the hybridization method.

In the method of this embodiment, the probe of the above-described embodiment is used as the probe. It was found that the probe of the above embodiment can specifically detect Aspergillus terreus not only in nucleic acids extracted from tissues, cells and the like and nucleic acids amplified by PCR, but also in the ISH method. Therefore, by using the probe of the above embodiment, Aspergillus terreus can be directly detected in a pathological tissue sample or the like.

In the method of this embodiment, the ISH method can be performed by a conventional method. For example, a tissue section is prepared from a tissue collected from a patient suspected of having Aspergillus terreus infection, subjected to protease treatment or the like in order to allow the probe to penetrate into the cell, and then the probe is hybridized. Then, by performing a visualization operation according to the labeling substance possessed by the probe, Aspergillus terreus can be detected on the tissue section. Hereinafter, an example of the method of the present embodiment will be described, but the method of the present embodiment is not limited to the method exemplified below.

In the method of the present embodiment, the subject to be detected by Aspergillus terreus is a sample in which the presence of Aspergillus terreus is suspected. The type of sample is not particularly limited, and for example, a biological sample collected from a patient suspected of having Aspergillus terreus infection can be used. Examples of such biological samples include tissue samples such as lungs, bronchi, kidneys and skin, samples such as blood, sputum, saliva, urine and stool, and bronchoalveolar lavage fluid (BAL). Further, the subject may be a culture of a biological sample as described above. Further, the subject is not limited to a biological sample, but may be a food sample, an environmental sample, or the like.

It is preferable that the subject is fixed with tissue or cells. The fixing method is not particularly limited, and a general tissue fixing method can be used. Examples of the fixative include formalin, alcohol-based fixatives, and the like. As the alcohol-based fixing solution, 95% ethanol is generally used. Further, the subject may be fixed on a slide glass or the like. Preferable examples of the subject in the method of the present embodiment include formalin-fixed paraffin-embedded tissue sections.

In addition, the subject may be pretreated before the hybridization reaction is carried out. Examples of the pretreatment include deparaffinization treatment, protease treatment, acetylation treatment, hydrochloric acid treatment, surfactant treatment and the like.

When the subject is a paraffin-embedded tissue section or the like, a deparaffin treatment may be performed as a pretreatment. The deparaffinizing treatment can be performed using xylene or the like.

Moreover, you may perform a protease treatment as a pretreatment. By mildly performing protease treatment, the probe can easily penetrate into the tissue without changing the localization of the bacterium. In addition, the protein cross-linked to the target DNA is degraded, and the DNA tends to become single-stranded in the subsequent denaturation step, facilitating the reach of the probe to the target. As the protease used for the protease treatment, those generally used in the ISH method can be used. Examples of proteases include protease K and pepsin.

Further, an acetylation treatment may be performed as a pretreatment. Acetylation treatment can reduce the electrostatic non-specific binding of the probe to tissue and glass slides. Furthermore, the acetylation treatment is said to contribute to the neutralization of positively charged molecules such as basic proteins. Acetylation can be carried out with acetic acid, acetic anhydride triethanolamine solution or the like.

Further, hydrochloric acid treatment may be performed as a pretreatment. Hydrochloric acid treatment removes basic proteins and exposes nucleic acids to increase probe permeability and prevent non-specific reactions.

The subject may be prehybridized after performing the above-mentioned pretreatment as appropriate. Prehybridization can be expected to prevent background staining. When prehybridization is performed, the prehybridization solution obtained by removing the probe from the hybridization solution or the like can be used. Prehybridization can be performed, for example, by dropping the prehybridization solution onto a subject on a slide glass and allowing it to stand at 40 to 60 ° C. for about 30 minutes to 2 hours. After dropping the prehybridization solution, the subject may be coated with a cover glass, parafilm, or the like.

The hybridization reaction is carried out using the probe of the above-described embodiment. As other hybridization conditions and the like, conditions and the like usually used in the ISH method can be used.

Generally, before the hybridization reaction between the probe and the subject is performed, the nucleic acid in the subject is denatured so as to become a single strand. The modification can be carried out under the conditions usually used in the ISH method. For example, denaturation can be performed by heating at 80 to 100 ° C. for about 3 to 10 minutes.

The hybridization reaction can be carried out by dropping a hybridization solution containing the probe of the above-described embodiment onto a subject on a slide glass and allowing it to stand at 40 to 60 ° C. for about 30 minutes to 15 hours. After dropping the hybridization solution, the subject may be coated with a cover glass, parafilm, or the like.

As the hybridization solution, one generally used in the ISH method may be used. For example, a hybridization solution can be prepared by mixing Denhardt solution, formamide, dextran sulfate, SDS, EDTA, salt, buffer and the like.

After the hybridization reaction, washing may be performed if necessary. Washing can remove non-specifically bound probes. For washing, the specificity can be adjusted by the salt concentration of the washing liquid, the washing temperature, and the like. Generally, the specificity decreases when the salt concentration of the cleaning solution is increased and the cleaning temperature is decreased. Adding formamide to the cleaning solution reduces specificity.

After the hybridization reaction, appropriate washing is performed, and then the label of the probe hybridizing to the target sequence is detected. The method for detecting the label can be appropriately selected depending on the label to be used. For example, when the label is 6-FAM, detection can be performed using an enzyme-bound anti-6-FAM antibody such as alkaline phosphatase. In addition, since enzyme-binding antibodies are commercially available for DIG, FITC, and the like, detection may be performed using these antibodies. When the label is a fluorescent dye, the label can be detected by observing with a fluorescence microscope. When the label is a radioisotope, the label can be detected by autoradiography or the like.

According to the method of the present embodiment, Aspergillus terreus can be directly detected in a histopathological specimen or the like, so that Aspergillus terreus can be quickly and accurately detected even when culturing is not possible or not culturing. Infection can be diagnosed. In addition, retrospective analysis is also possible.

Hereinafter, the present invention will be described with reference to Experimental Examples, but the present invention is not limited to the following Experimental Examples.

[Making a probe for detecting Aspergillus terreus]
In Aspergillus varieties including Aspergillus tereus and Aspergillus humigatus, BLAST search of 18S-28S rDNA sequences including ITS region was performed, and Aspergillus tereus-specific sequences were searched.

As a result, five types of sequences shown in Table 1 were selected as candidate sequences for the probe for detecting Aspergillus terreus.

The nucleotide sequence in Table 1 above was confirmed by BLAST search to have no similarity to other fungal species other than Aspergillus spp.

Based on the base sequence of Table 1 above, 6 types of probes shown in Table 2 were designed. In the base sequence shown in Table 2, the residues shown in bold underline are BNA residues. The BNA used for the synthesis of the probe was 2', 4'-BNA ^NC (N-Me) (the oxygen atom at the 2'site of ribose and the carbon atom at the 4'site were -NRCH _2- (R is a methyl group). ) Bridged). As the BNA having cytosine as a base, one in which the 5-position of cytosine was methylated was used.

The base sequence complementary to the probe is shown in Table 3.

Based on the sequences shown in Table 2, 6 kinds of oligonucleotides were synthesized and the 5'ends were labeled with 6-FAM to prepare probes Ater01-03 and Ater05-07.

[Experimental example 1: Detection by ISH method for cultured cells]
Aspergillus tereus was attempted to be detected by the ISH method in cultured cells of Aspergillus tereus and Aspergillus humigatus. As the probe, Ater01 to Ater03 and Ater05 to Ater07 were used. The ISH method was carried out in the following procedure.

(Preprocessing)
First, paraffin-embedded sections of cultured Aspergillus tereus and Aspergillus humigatus cells were prepared and fixed on a slide glass. Next, deparaffin treatment was performed using xylene. Specifically, a slide glass on which bacterial cells were fixed was placed in a doze containing xylene and heated at 50 ° C. for 30 minutes. During that time, the slide glass was moved up and down with tweezers as appropriate. Next, the slide glass was placed in another doze containing new xylene and allowed to stand at room temperature for 10 minutes. Then, it was immersed in 100%, 90%, 80%, and 70% ethanol for 2 minutes each to make it hydrophilic. After the hydrophilization treatment, the slide glass was immersed in distilled water and allowed to stand at room temperature for 10 minutes.

Next, hydrochloric acid treatment was carried out at room temperature for 2 minutes using 0.2 N hydrochloric acid. Then, the washing treatment was carried out using PBS at room temperature for 2 minutes, and then the protease treatment was carried out at 37 ° C. for 5 minutes using 2 μg / mL proteinase K (PBS solution). After the protease treatment, a washing treatment was carried out using G-PBS (PBS containing 2 mg / mL glycine) at room temperature for 10 minutes, and then using PBS for 2 minutes at room temperature.

Next, acetylation treatment was performed with 20% acetic acid at ice temperature for 15 seconds. After the acetylation treatment, the washing treatment was carried out using PBS at room temperature for 2 minutes, and then the slide glass was lightly erected to drain water.

(In situ hybridization)
100 μL of the hybridization solution preheated to 50 ° C. was dropped onto the cells on the slide glass, and the cover glass was placed on the cells. Then, the slide glass was heated at 94 ° C. for 10 minutes on a hot plate to perform denaturation treatment. Then, the slide glass was rapidly cooled on an aluminum plate on ice.

Next, prehybridization was performed. The box moistened with 50% formamide was preheated to 50 ° C., a slide glass was placed in the box, and the mixture was allowed to stand for 1 hour to perform prehybridization.

The probe was prepared to 100 μL in the hybridization solution.

Next, a hybridization reaction was performed. First, the cover glass was removed with tweezers in a heated 2 × SSC (SSC: 0.03 M sodium citrate, 0.3 M sodium chloride), and the liquid of the slide glass was drained. Then, 100 μL of the hybridization solution containing the probe at a final concentration of 130 pmol / mL was dropped onto a slide glass and covered with parafilm. The hybridization reaction was carried out by allowing the mixture to stand at 50 ° C. overnight.

The composition of the hybridization solution used in the above treatment is as follows.
1 x Denhardt solution (Denhardt 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

(Detection reaction)
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, two washes were performed at 50 ° C. for 15 minutes under shaking at 77 rpm. Then, the slide glass was rinsed with buffer 1 (100 mM Tris-HCl, 150 mM NaCl; pH 7.5).

Next, a blocking process was performed. The blocking treatment was carried out by immersing the slide glass in the blocking solution and gently shaking it at room temperature for 1 hour.

As the blocking solution, buffer 1 containing a 1% blocking reagent (# 1096 176, Roche Life Sciences) was used.

The slide glass was removed from the blocking solution, and an alkaline phosphatase-labeled anti-FITC antibody (# 11 426 338 910, Roche Life Sciences, 1.5 U / ml) was added dropwise onto the slide glass. The slide glass was covered with parafilm and allowed to stand in a wet box for 30 minutes at room temperature. Then, the slide glass was immersed in buffer 2 (buffer 1 containing 0.2% Tween 20), and washed three times with strong shaking for 15 minutes.

Next, a color reaction was carried out. First, the slide glass was rinsed at 0.1 M Tris pH 9.5. Next, the coloring liquid was dropped onto the slide glass, and the cover glass was placed on the slide glass. The slide glass was placed in a wet box and a color reaction was carried out at 37 ° C. The coloring liquid was prepared using Alkaline Phosphatase Conjugate Substrate Kit (# 170-6432, Bio-Rad). During the color development reaction, the color development reaction was carried out for a maximum of one night while confirming the color development on the slide glass.

(result)
The results of the ISH method using Ater01 to Ater03 as probes are shown in FIG. Further, FIG. 2 shows the results of the ISH method using Ater05 to Ater07 as the probe.

As shown in FIG. 1, in Ater01 to Ater03, color development was observed in both Aspergillus tereus and Aspergillus humigatus cells. This result indicates that Ater01 to Ater03 hybridize not only to Aspergillus terreus DNA and RNA, but also to nucleic acids in Aspergillus humigatus. Therefore, it was clarified that Aspergillus tereus could not be specifically detected in Ater01 to Ater03.

Ater01 to Ater03 are base sequences found by BLAST search as a base sequence specific to Aspergillus terreus, and it was expected that Aspergillus tereus could be specifically detected. However, the ISH method did not allow specific detection of Aspergillus terreus due to non-specific binding. This result indicates 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.

On the other hand, as shown in FIG. 2, in Ater05 to Ater07, color development was observed in Aspergillus terreus cells, but no color development was observed in Aspergillus humigatus cells. This result indicates that Ater05-5 to Ater07 are probes capable of specifically detecting Aspergillus terreus. In addition, Ater06 and Ater07 had stronger color development intensity in Aspergillus terreus cells than Ater05.

The hybridization reaction was also carried out at 40 ° C., 55 ° C., and 60 ° C., but no change in specificity was observed as compared with the case of 50 ° C. (data not shown).

In addition, other filamentous fungal cells (Aspergillus flavus), Aspergillus niger, Candida albicans (Candida albicans), Fusarium, which are found clinically using Ater05 to Ater07. The ISH method was also performed on solani, Pseudallescheria boydi, Rhizopus oryzae, and Penicillium, but no color was observed in these cells (data). ).

From the above results, it was confirmed that Ater05-5 to Ater07 are probes capable of specifically detecting Aspergillus terreus cells by the ISH method.

[Experimental example 2: Detection by ISH method for infected animal tissues]
Tissue specimens were prepared from the kidneys of Aspergillus terreus-infected mice and the kidneys of Aspergillus humigatus-infected mice, and the detection of Aspergillus terreus was attempted by the ISH method.

The ISH method was carried out in the same procedure as in Experimental Example 1. Ater07 was used as the probe. Ater07 was prepared to a final concentration of 130 pmol / mL and subjected to a hybridization reaction. In addition, as a positive control, the ISH method was also performed with an 18S probe (a pan-fungal probe targeting the 18S rDNA sequence of the fungus). The 18S probe is a DIG-labeled 568 bp probe by the PCR method (Hanazawa R, Murayama SY, Yamaguchi H. In-situ detection of Aspergillus fumigatus. J Med Microbiol. 2000 Mar; 49 (3): 285-90. ), The final concentration was adjusted to 1% and used.

The results of the ISH method using Ater07 as a probe are shown in FIGS. 3 to 6. As shown in FIG. 3, in Ater07, color development was observed in the kidney tissue section of Aspergillus terreus-infected mice. On the other hand, as shown in FIG. 4, no color development was observed in the kidney tissue section of Aspergillus fumigatus-infected mice (Fig. 4, right). Since color development was observed with the 18S probe, it was confirmed that the bacterial cells were present in the tissue (Fig. 4, left).

In addition, Aspergillus flavus and Aspergillus nidurance, which are closely related species of Aspergillus teleus, were also subjected to the ISH method using Ater07 for the kidney tissue of infected mice. As a result, as shown in FIGS. 5 and 6, no color development was observed in the kidney tissue sections of these infected mice. Since color development was observed with the 18S probe, it was confirmed that the bacterial cells were present in the tissue (Fig. 5, left, Fig. 6 left).

From the above results, it was confirmed that Ater07 is a probe that can be specifically detected in Aspergillus terreus even in a tissue section.

[Experimental example 3: Detection by ISH method for human clinical specimens]
We attempted to detect Aspergillus terreus by the ISH method in two types of clinical tissue specimens. One of the clinical tissue samples is a lung tissue sample collected from a patient with invasive pulmonary aspergillosis, and was provided by Kinki University (hereinafter referred to as "Sample 1"). Specimen 1 has succeeded in culturing Aspergillus terreus from the lung collected at the time of autopsy, and it has been confirmed that it is an Aspergillus terreus-infected lung. Another clinical tissue sample is a mucous embolus sample collected from a patient with allergic bronchopulmonary mycosis, which was provided by the National Hospital Organization Tokyo National Hospital (hereinafter referred to as "Sample 2"). ). Aspergillus tereus is cultured from the sputum of the same patient in Specimen 2.

The ISH method was carried out in the same procedure as in Experimental Example 1. As a probe, Ater05 to Ater07 were used. Ater05 to Ater07 were prepared at a final concentration of 130 pmol / mL, and a hybridization reaction was carried out. The ISH method was also performed on the probe Aft1 (245 bp, DIG label (Neuveglise C. Nucleic Acids Res 24: 1428-34, 1996)) specific for Aspergillus fumigatus.

The results of the ISH method using Ater07 as a probe are shown in FIGS. 7 and 8. FIG. 7 is for sample 1 and FIG. 8 is for sample 2. As shown in FIGS. 7 and 8, in Ater07, color development was observed in both the sample 1 and the sample 2. On the other hand, when Afut1, which is an Aspergillus-Fumigatus-specific probe, was used, no color development was confirmed in any of the samples. The Kinki University sample was collected in 1998, and it was shown that specific detection of Aspergillus terreus is possible even in a sample almost 30 years ago.

From the above results, it was confirmed that Ater07 is capable of Aspergillus terreus-specific detection even in human clinical specimens.

The results of the ISH method using Ater05 or Ater06 as the probe are shown in FIGS. 9 and 10. FIG. 9 is for sample 1 and FIG. 10 is for sample 2. As shown in FIGS. 9 and 10, color development was also observed in both Specimen 1 and Specimen 2 in Ater05 and Ater06. From these results, it was confirmed that Ater05 and Ater06 are also capable of specific detection of Aspergillus terreus in human clinical specimens. The color development intensity of Ater05 and Ater06 is weaker than that of Ater07, and Ater07 is more suitable as a probe for the ISH method.

From the above results, it was shown that Ater05-5 to Ater07 are probes capable of specifically detecting Aspergillus terreus even in clinical tissue samples by the ISH method.

According to the oligonucleotide, probe, and detection method of the present invention, Aspergillus tereus can be specifically detected in a histopathological specimen. The present invention provides a rapid and accurate method for diagnosing Aspergillus terreus infection.

Claims (2)

Aspergillus terreus detection probe containing an oligonucleotide selected from the group consisting of the following (i) and (ii) :
(I) An oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 14, which is an adenine residue at the 1-position, an adenine residue at the 2-position, a guanine residue at the 3-position, a thymine residue at the 4-position, or a uracil residue. , 7-position thymine residue, 11-position thymine residue or uracil residue, 15-position thymine residue or uracil residue, 16-position guanine residue, 17-position citocin residue, and 18-position guanine residue An oligonucleotide whose group is a BNA residue; and
(Ii) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 15, at position 1 adenine residue, position 3 guanine residue, position 6 guanine residue, position 8 adenine residue, and position 11. Thymine residue or uracil residue, adenine residue at position 13, thymine residue or uracil residue at position 15, guanine residue at position 18, guanine residue at position 20, and guanine residue at position 21 are BNA residues. The group, the oligonucleotide . A method for detecting Aspergillus terreus, which comprises a step of hybridizing the probe for detecting Aspergillus terreus according to claim 1 with a subject.

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