JP2002142774A - Nucleic acid for detecting fungi and method for detecting fungi using the same - Google Patents

Abstract

(57) [Problem] To provide a nucleic acid used for detecting a causative fungus of deep mycosis, particularly a fungus of the genus Candida and a bacterium of the genus Cryptococcus. SOLUTION: Each of a plurality of base sequences represented by SEQ ID NO: 1, 2, 3 or 4 showing a plurality of base sequences, each consisting of at least two or more kinds, each consisting of at least 15 bases continuous from its 3 'end Provided is a nucleic acid mixture for detecting fungi, comprising a mixture of at least two or more nucleic acids each having a base sequence.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a nucleic acid mixture for detecting a fungus and a method for detecting a fungus using the mixture.

[0002]

2. Description of the Related Art As a major pathogen of deep mycosis,
Fungi of the genus Candida (hereinafter abbreviated as C.), fungi of the genus Aspergillus (hereinafter abbreviated as A.), fungi of the genus Cryptococcus (hereinafter abbreviated as Cr.) And the like are known. Major pathogens of the genus Candida include C. albicans, C. glabrata, C. tropicalis, C. tropicalis, C. parapsilosis and C. parabranosis. (C. dubliniensis) and the like are known as major pathogens of the genus Cryptococcus, such as Cryptococcus neoformans. The main pathogenic bacteria of the genus Aspergillus include Aspergillus fumigatus (A. fumigatus), Aspergillus flavus (A. flavus), and Aspergillus niger (A. nige).
r) and the Niger group, Aspergillus nidurans (A. nidurans), Aspergillus teleus (A. te)
rreus), and it is desired to rapidly detect, classify, and identify these bacterial species.

[0003] In recent years, deep mycosis has increased as an opportunistic infection in immunodeficient patients, but those patients often have serious underlying illness, and the pathogenic bacterium was clarified at an early stage to make an accurate determination. It is necessary to treat. Diagnosis of deep mycosis is based on the blood culture method, but has problems in detection sensitivity and the like. Although diagnostics using antibodies have been marketed so far,
Diagnosis with antibodies does not help immunocompromised patients.
Under such circumstances, in recent years, methods for directly detecting antigens and other bacterial cell components have been developed. For example, a method for detecting infection by detecting mannan, D-arabinitol, glucan, and the like has been developed. On the other hand, with the development of molecular biology, various pathogens such as Mycobacterium tuberculosis and Chlamydia
Methods for diagnosing by extracting NA and detecting nucleic acids by the so-called PCR method have been developed. Methods for Detecting and Diagnosing Ribosomal RNA in the Field of Deep Mycosis (Clinical Pathology, Vol. 43, 119, 1995)
Year) is presented. The present inventors previously focused on the gene of cytochrome b present in the mitochondria of fungi that cause these various mycosis, provided a nucleic acid used for detecting a fungus of the genus Aspergillus, and further using it. A simple, rapid, specific and highly sensitive method for the detection of fungi causing deep mycosis, as well as a method for detecting fungi of the genus Aspergillus, has been developed (materials and methods for detecting fungi, WO98 / 10073).

[0004]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a nucleic acid used for detecting a causative fungus of deep mycosis, in particular, a fungus of the genus Candida and a genus of Cryptococcus. It is an object of the present invention to provide a simple, rapid, specific, and sensitive method for detecting fungi causing deep mycosis, particularly fungi of the genus Candida and cryptococcus.

[0005]

Means for Solving the Problems The present inventors have conducted various studies on the genes of various causative fungi of deep mycosis, in particular, the genes of Candida fungi and Cryptococcus fungi. A part of the cytochrome b gene present in the fungal mitochondria causing mycosis was successfully amplified and its sequence was successfully decoded. The present inventors have further studied based on the sequence, and as a result, have found specific sequences for each kind of Candida fungi, and based on them, amplify a part of the cytochrome b gene of each species. A universal primer mixture has been designed to be able to do so. By performing a nucleic acid amplification reaction using these primer mixtures, primer extension products can be obtained even if the species of causative bacteria of deep mycosis present in the specimen has not been identified.

[0006] That is, the present invention relates to a method for preparing a plurality of base sequences, each having a plurality of base sequences represented by SEQ ID NOs: 1, 2, 3, or 4, wherein thymine at any position is substituted with uracil. Or a base sequence consisting of at least two or more of its complementary base sequences, each consisting of at least 15 consecutive bases from its 3 ′ end, or a base sequence that hybridizes with each base sequence under stringent conditions A nucleic acid mixture for detecting a fungus, comprising a mixture of at least two or more nucleic acids, comprising: In addition, the present invention relates to at least two or more of a plurality of base sequences represented by SEQ ID NOS: 1 and 2 in the sequence listing (though thymine at any position may be substituted with uracil). A base sequence consisting of 15 bases continuous from the 3 'end or a mixture of nucleic acids having a base sequence that hybridizes with the base sequence under stringent conditions was used as a forward primer and represented by SEQ ID NO: 3 or 4 in the sequence listing. At least two or more of a plurality of base sequences (though thymine at any position may be substituted with uracil)
Amplifying a test nucleic acid by a nucleic acid amplification method using a base sequence consisting of at least 15 bases continuous from the 3 'end or a mixture of nucleic acids having a base sequence that hybridizes with the base sequence under stringent conditions as a reverse primer And a method for detecting a fungus, comprising detecting an amplification product. Furthermore, the present invention provides a method for identifying a fungus, comprising determining the base sequence of the amplification product. The present invention further provides a kit for detecting a fungus containing the nucleic acid of the present invention.

[0007]

As described above, the nucleic acid mixture for detecting a fungus of the present invention comprises a plurality of base sequences each represented by SEQ ID NO: 1, 2, 3 or 4 (provided that thymine at any position is Uracil) or a base sequence consisting of at least 15 bases continuous from at least the 3 'end of at least two or more of its complementary base sequences or under stringent conditions with the base sequence. It has a hybridizing base sequence.
As is clear from the fact that thymine may be substituted for uracil, the nucleic acid of the present invention may be DNA or RNA. As the nucleic acid mixture of the present invention, at least 3 ′ of a plurality of base sequences represented by SEQ ID NOs: 1, 2, 3 or 4 (provided that thymine at any position may be substituted with uracil) A base sequence consisting of at least 15 bases continuous from the terminal is preferred, and a nucleic acid mixture having a plurality of base sequences each represented by SEQ ID NO: 1, 2, 3 or 4 is particularly preferred. However, the nucleic acid mixture for detecting a fungus of the present invention has a nucleotide sequence represented by SEQ ID NO: 1, 2, 3 or 4 in the Sequence Listing (though thymine at any position may be substituted with uracil) or its complement. If the base sequence hybridizes under stringent conditions with a base sequence consisting of at least 15 bases continuous from at least its 3 ′ end, the above-described base sequence may be substituted with a base, deleted, It may include insertions or additions. Here, “under stringent conditions” means that it hybridizes to a target nucleic acid serving as a template and functions as a primer under annealing conditions in ordinary PCR as described in Examples below. The nucleic acid of the present invention is commercially available DN
It can be easily manufactured by chemical synthesis using an A synthesizer or the like.

[0008] SEQ ID Nos. 1 to 4 include m, r, w
Thus, one sequence number indicates a plurality of sequences because a single character includes a symbol indicating a plurality of bases.
The nucleic acid of the present invention comprises at least two or more of these multiple base sequences (including the above-described portions and variants),
Preferably, it is a mixture of nucleic acids having all kinds of base sequences. By using such a mixture, various mutant strains of fungi can be detected.

The nucleic acid mixture according to the invention hybridizes with a region in the mitochondrial cytochrome b gene of a fungus, in particular a fungus belonging to the genus Candida, Cryptococcus, Aspergillus or Penicillium, preferably a fungus belonging to the genus Candida or Cryptococcus. Is what you do. Preferred fungal species that can be detected using the nucleic acid mixtures of the present invention include Candida glabrata, Candida
Parapsilosis, Candida tropicalis, Candida albicans, Cryptococcus neoformans and Candida dubriniensis can include, but are not limited to. However, SEQ ID NO: 3 (including the above-mentioned portions and variants) cannot be used for the detection of Candida albicans, Cryptococcus neoformans and Candida dubriniensis.

The mitochondrial cytochrome b gene is
It is located in mitochondria, DNA recombination is difficult due to cytoplasmic inheritance, and it is considered that base changes occur in proportion to evolution. Therefore, it is suitable for detecting various fungi. As will be described in more detail later, the region to be amplified when the nucleic acid combination of the present invention is used as a primer in a nucleic acid amplification method is a relatively variable portion, and it is easy to distinguish species. Has the advantage that it can be easily distinguished by DNA type. Therefore, it is possible to identify a fungal species or strain by amplifying a test nucleic acid using the nucleic acid of the present invention as a primer and determining the base sequence of the amplified product.

As described above, the nucleic acid mixture of the present invention hybridizes with the region in the cytochrome b gene of fungal mitochondria, and therefore, by using the nucleic acid mixture of the present invention as a probe or a primer for a nucleic acid amplification method,
Fungi can be detected.

When the nucleic acid mixture of the present invention is used as a probe, it is preferable to use a nucleic acid mixture labeled with a labeling agent. In this case, the labeled nucleic acid mixture of the present invention is used as a probe, and after a fungal gene in a test sample and the labeled probe are hybridized, a conjugated product of the fungal gene and the labeled probe or an unbound labeled probe is labeled. Detection is carried out by an appropriate detection method suitable for the agent. Labeling agents used for probes are well known in this field, and include fluorescent labels, radioactive labels, enzyme labels, biotin, and the like. Specimen, that is, hybridization between the fungal gene in the test sample and the probe, pretreatment of the specimen in the usual manner,
The purified sample is used as a test sample, and the mixture is mixed with a labeled nucleic acid mixture as a probe.
It can be implemented by performing time processing. The sample may be subjected to an amplification reaction in advance using the nucleic acid mixture of the present invention as a primer.

When the nucleic acid mixture of the present invention is used as a primer for a nucleic acid amplification method, the nucleic acid mixture of the present invention is used as a primer to carry out an elongation reaction with a DNA polymerase or the like to amplify the gene to thereby produce cytochromes of Candida fungi and cryptococcus fungi. Fungi can be detected by specifically amplifying only the b gene fragment and measuring the amplification product. In this case, a labeled nucleic acid obtained by labeling the nucleic acid mixture of the present invention with a labeling agent as described above may be used as a primer. Specific examples of the gene amplification method used herein include a PCR method, but are not particularly limited to this method, and any method using a short-chain oligonucleic acid as a primer to initiate gene synthesis can be used. Can also be used. The method for detecting a fungus of the present invention can be performed by detecting an amplification product amplified by the above method, ie, a cytochrome b gene fragment. Alternatively, it can also be performed by fragmenting the amplified cytochrome b gene fragment with a restriction enzyme and comparing the production patterns of the fragment. The restriction enzyme used here is single,
Alternatively, they are used in combination. Means for detecting a cytochrome b gene fragment amplified with an amplification product or a gene fragment further fragmented with a restriction enzyme is not particularly limited, and an ordinary gene detection method (for example, an electrophoresis method) can be used. The amplification product is fractionated by, for example, electrophoresis and can be easily detected as a specific and sufficiently sharp band for detection. Primers also use a deoxyribonucleic acid mixture (dATP, dTTP, dGTP, dCTP) or ribonucleic acid mixture (ATP, UTP, GTP, CTP) labeled with an appropriate labeling agent as a raw material for DNA or RNA synthesis during amplification reaction By doing so, it is possible to directly detect the amplification product with high sensitivity. Alternatively, by using a labeled nucleic acid mixture obtained by labeling the nucleic acid of the present invention as a primer, an amplified product can be directly detected with high sensitivity. In such a case, the labeling agent used for labeling is preferably a radioactive substance, a fluorescent substance, or the like. It can also be used as a primer for so-called real-time detection PCR, and in this case, the amount of the test nucleic acid can be determined. Therefore, "detection" in the present specification includes quantitative detection.

When used as a primer, SEQ ID NO: 1
And a nucleic acid mixture having the base sequence shown in SEQ ID NO: 2 (including the above-mentioned part and mutants, the same applies hereinafter) is preferably used as a forward primer, and the base shown in SEQ ID NO: 3 and SEQ ID NO: 4 The nucleic acid mixture having a sequence is preferably used as a reverse primer. It is preferable to use a combination of these forward primer and reverse primer, whereby a 396 bp region is amplified as specifically described in the Examples below. By separating and detecting the obtained amplification product by various electrophoresis methods or the like, it is possible to determine whether a fungus is present in the sample. In addition, species can be identified by determining the base sequence of the obtained amplification product.

The reagent kit for detecting fungi of the present invention is characterized by containing the nucleic acid mixture of the present invention or a labeled nucleic acid obtained by labeling this nucleic acid mixture with a suitable labeling agent. The reagent kit of the present invention may contain the above-described nucleic acid, and may contain a label detection reagent, a buffer, and the like as other components. The nucleic acid in the reagent kit of the present invention can be used as a primer or a probe. Whether a nucleic acid is used as a primer or a probe is different only in its means, and is essentially the same with respect to detection of fungi. When the nucleic acid is used as a probe, the reagent kit is used as a reagent kit for detecting a fungus by the method using the above-described probe, and when used as a primer, for detecting a fungus by the nucleic acid amplification method described above. It can be used as a reagent kit.

When the nucleic acid mixture in the reagent kit is used as a probe, the reagent kit of the present invention comprises a nucleic acid mixture for detecting a Candida fungus and a Cryptococcus fungus of the present invention or a labeled Candida fungus and a Cryptococcus fungus for detection. In addition to the nucleic acid mixture, a label detection reagent,
It may contain a buffer or the like. When the nucleic acid mixture in the reagent kit is used as a primer, the reagent kit of the present invention is different from the nucleic acid mixture for detecting Candida fungi and Cryptococcus fungi of the present invention or the labeled nucleic acid mixture for detecting Candida fungus and Cryptococcus fungus of the present invention. In addition, nucleic acid synthases (eg, DNA polymerase, RN
A polymerase, reverse transcriptase, etc.), deoxyribonucleotide mixtures (dATP, dTTP, dGTP, dCTP), ribonucleotide mixtures (ATP, UTP, GTP, CTP), restriction enzymes, buffers and the like. Further, the nucleic acid mixture of the present invention can be bound to a solid phase carrier and used as a capture probe. In this case, the capture probe and labeled probe
The sandwich assay may be performed in combination of the two.
There is also a method of labeling and capturing a target nucleic acid. Furthermore, there is a method in which the nucleic acid mixture is labeled with biotin, and after hybridization, captured with an avidin-bound carrier. In the sandwich assay, when the nucleic acid mixture of the present invention is used for one of the two, specific measurement can be performed with the nucleic acid mixture of the present invention, and there is no problem even if the specificity of the other nucleic acid mixture is slightly lower.

[0017]

EXAMPLES Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.

Example 1 (Synthesis of Various Nucleic Acid Mixtures) A nucleic acid mixture having the sequences represented by SEQ ID NOs: 1 to 4 in the sequence listing was synthesized by chemical synthesis. Hereinafter, the various nucleic acid mixtures represented by SEQ ID NOs: 1 to 4 in the sequence listing are referred to as nucleic acid mixtures 1 to 4, respectively.

Example 2 (Candida albicans chito)
Chromium b gene amplification reaction) Candida obtained by culturing in potato dextrose liquid medium
・ Albicans (Candida albicans IFM 40009)
Was treated with 75% ethanol and sterilized. 1500g for 10 minutes
The cells were obtained by centrifugation, and about 40 ml of extraction buffer
(0.9 M sorbitol, 10 mM EDTA, 10 mM
HCl (pH 7.1), shake well, and add 1500
g, discard the supernatant by centrifugation for 10 minutes, and add 30 ml of the same buffer
And centrifugation, discard the supernatant, and add 9 ml of the same buffer.
The liquid was added. Add Zymolyase (raw) dissolved in the same buffer
1 ml of cell wall lytic enzyme (10 mg / ml, manufactured by Chemical Industry Co., Ltd.)
Incubate for 1 minute in an ultrasonic cleaner after keeping the temperature at ℃ for 1 hour.
Was. This was centrifuged at 1500 g for 10 minutes to obtain a supernatant.
Further, the mixture was centrifuged at 20,000 g for 15 minutes to obtain a precipitate. this
The precipitate is washed with an extraction buffer, and again at 20000 g for 15 minutes.
After centrifugation, a mitochondrial fraction was obtained. This mitocon
1 mg / ml Protease K in Doria fraction
0.5 ml was added, and the mixture was kept at 37 ° C for 1 hour. This is phenol,
A mixed solution of chloroform and isoamyl alcohol (25:24:
Add 1ml of 1), shake and mix at 1500g for 10 minutes
Separate the upper layer and add 1 ml of water-saturated phenol to it.
After shaking, remove the upper layer by centrifugation at 1500g for 10 minutes.
Was. 0.1 ml of 3M sodium acetate, 0.1M magnesium chloride
Add nesium solution, add 3 ml of ethanol, -20 ° C
Left overnight. This is centrifuged at 1500 g for 10 minutes to settle.
And the precipitate was washed with 75% ethanol at -20 ° C.
After centrifugation at 500 g for 10 minutes, the supernatant was discarded to obtain the nucleic acid, which was then dried.
After drying, it was stored at -20 ° C.

Using this nucleic acid as a template, and using the nucleic acid mixture 2 and the nucleic acid mixture 4 obtained in Example 1 as primers, using a TaKaRa PCR Amplification Kit (R011) of Takara Shuzo Co., Ltd., a Sanyo DNA amplification device (MIR- Using D30), a PCR reaction was performed by the following method to amplify a part of the cytochrome b gene. The composition of the reaction solution was as follows.

X10 PCR buffer (kit reagent) 5 μl PCR dNTP mixture (kit reagent) 4 μl Taq DNA synthase (kit reagent) 1 μl (2.5 U) nucleic acid mixture 2 1 μl nucleic acid mixture 4 1 μl mitochondrial DNA 1 μl in water To a total volume of 50 μl.

The reaction conditions were as follows. Thermal denaturation: 94 ° C, 1 minute Annealing: 50 ° C, 1 minute Extension reaction: 72 ° C, 2 minutes Number of cycles: 30

The amplified DNA was separated and purified by agarose gel electrophoresis, and the nucleotide sequence was analyzed by the following method.
PerkinElmer DNA sequencer (ABI Prism
377) and analyzed by the die terminator method according to the company's operation guide. Nucleic acid mixture 2 was used on the forward side and nucleic acid mixture 4 was used on the reverse side as primers for nucleotide sequence analysis. The determined nucleotide sequence is shown in SEQ ID NO: 5 in the sequence listing.

Example 3 (Candida glabrata cytochrome b gene amplification reaction) In the same manner as in Example 2, the nucleic acid mixture 1 and the nucleic acid mixture 3 obtained in Example 1 were used to prepare Candida glabrata (Ca
ndida glabrata IFM 46843), a part of the cytochrome b gene was amplified by PCR, and its nucleotide sequence was determined. Nucleic acid mixture 1 was used on the forward side and nucleic acid mixture 3 was used on the reverse side as primers for nucleotide sequence analysis. The nucleotide sequence is shown in SEQ ID NO: 6 in the sequence listing.

Example 4 (Candida parapsilosis Cytochrome b Gene Amplification Reaction) In the same manner as in Example 2, using the nucleic acid mixture 1 and the nucleic acid mixture 3 obtained in Example 1, Candida parapsilosis IFM 46829), a part of the cytochrome b gene was amplified by PCR, and its nucleotide sequence was determined. Nucleic acid mixture 1 was used on the forward side and nucleic acid mixture 3 was used on the reverse side as primers for nucleotide sequence analysis. The nucleotide sequence is shown in SEQ ID NO: 7 in the sequence listing.

Example 5 (Amplification reaction of Candida tropicalis cytochrome b gene) In the same manner as in Example 2, using the nucleic acid mixture 1 and the nucleic acid mixture 3 obtained in Example 1, Candida tropicalis (Ca
ndida tropicalis IFM 46816), a part of the cytochrome b gene was amplified by PCR, and its nucleotide sequence was determined. Nucleic acid mixture 1 was used on the forward side and nucleic acid mixture 3 was used on the reverse side as primers for nucleotide sequence analysis. The nucleotide sequence is shown in SEQ ID NO: 8 in the sequence listing.

Example 6 (Candida dubriniensis cytochrome b gene amplification reaction) In the same manner as in Example 2, using the nucleic acid mixture 2 and the nucleic acid mixture 4 obtained in Example 1, Candida dubriniensis (Candida dubliniensis IFM 48313), a part of the cytochrome b gene was amplified by PCR, and its nucleotide sequence was determined. Nucleic acid mixture 2 was used on the forward side and nucleic acid mixture 4 was used on the reverse side as primers for nucleotide sequence analysis. The nucleotide sequence is shown in SEQ ID NO: 9 in the sequence listing.

Example 7 (Cryptococcus neoformans cytochrome b gene amplification reaction) In the same manner as in Example 2, using the nucleic acid mixture 2 and the nucleic acid mixture 4 obtained in Example 1, Cryptococcus neoformans (Cryptococcus neoformans IFM 46138), a part of the cytochrome b gene was amplified by PCR,
The nucleotide sequence was determined. Nucleic acid mixture 2 was used on the forward side and nucleic acid mixture 4 was used on the reverse side as primers for nucleotide sequence analysis. The nucleotide sequence is represented by SEQ ID NO: 1 in the sequence listing.
0.

[0029]

Industrial Applicability According to the present invention, a fungal nucleic acid for use in detecting various fungi and a method for detecting a fungus using the nucleic acid are provided. According to the present invention, Candida fungi and Cryptococcus fungi, which conventionally took a long time to classify and identify, can be detected quickly, easily and specifically with high sensitivity. The nucleic acid mixture of the present invention can be used as a primer for an amplification reaction or as a probe for direct detection. By using the nucleic acid mixture of the present invention as a primer for an amplification reaction, it has become possible to detect a plurality of types of fungi having different nucleotide sequences by a single amplification reaction. Further, the sensitivity and specificity can be further increased by detecting the product amplified by the primer with the probe, and its clinical significance is great.

[0030]

[Sequence List] SEQUENCE LISTING <110> SSP CO., LTD. <120> Oligonucleotides for detecting fungi and method for detecting fun gi using the same <130> 00679 <160> 10

<210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide used as a forward primer for amplifying a region in mitochondrial cytochrome b gene of fungi <400> 1 tgrggwgcwa cwgttattac ta twenty two

<210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide used as a forward primer for amplifying a region in mitochondrial cytochrome b gene of fungi <400> 2 gttattacta ayttattmtc 20

<210> 3 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide used as a reverse primer for amplifying a region in mitochondrial cytochrome b gene of fungi <400> 3 ggwatagmws ktaawayagc ata twenty three

<210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide used as a reverse primer for amplifying a region in mitochondrial cytochrome b gene of fungi <400> 4 agcacgtarw attgcgtaga atgg twenty four

[0035] <210> 5 <211> 396 <212> DNA <213> Candida albicans <400> 5 aatttattat cagctatacc tttcataggt aatgatatcg taccatttat atgaggaggt 60 ttctctgtta gtaaccctac tatacaacgg ttcttcgcat tgcatttctt attacctttc 120 atacttgctg cattagtatg tatgcacttg atggccttac atgtacatgg ttcatctaac 180 cctgtaggta ttactggtaa tattgataga ttgccaatgc atccttactt catatttaaa 240 gacttaatta ctgtctttgt attcttatta atatttagtt tattcgtatt ctattcacct 300 aatacattag gacatcctga taactatata ccaggtaacc ctatggtaac acctccttca 360 attgtaccag aatgatactt attaccattc tacgca 396

[0036] <210> 6 <211> 396 <212> DNA <213> Candida glabrata <400> 6 aatttattct cagctattcc tttcgttggt caagatattg tacaatgatt atgaggaggt 60 ttctcagtat ctaatcctac tattcaaaga ttctttgcat tacattattt agtaccattt 120 attattgctg ctttagtagt aatgcatttt atggctttac atgtacatgg ttcatctaat 180 cctttaggta ttacaggtaa tatggataga attggaatgc atggttattt catttttaaa 240 gatttaatta ctgtttttgt attcttaatt ttcttctcat tatttgtatt cttctcacct 300 aatactttag gacatcctga taattatatt cctggtaatc ctttagtaac accagcatct 360 attgtacctg aatgatattt attaccattt tatgct 396

[0037] <210> 7 <211> 396 <212> DNA <213> Candida parapsilosis <400> 7 aacttattat ctgctatacc attcattggt aaagatattg ttccgtttat ttgaggaggt 60 ttcagtgtaa gtaaccctac aattcaaaga ttctttgctc ttcacttctt attaccattc 120 attttagctg ctttagtatg tatgcattta atggcattac atgttaatgg ttcatctaac 180 ccattaggta ttacaggtaa cgttgataga ttaccaatgc atccttactt tatttttaaa 240 gatttagtaa ctgtatttgt attcttatta gtatttagtt tatttgtatt ctattctcca 300 aatacattag gtcaccctga taactatatt cctggggaccc ctttagttac tcctccttct 360 attgttccag agtgatatct attacctttc tatgct 396

[0038] <210> 8 <211> 396 <212> DNA <213> Candida tropicalis <400> 8 aacctactgt cagccattcc attcattggt aacgatatcg ttccctttat ttgaggtggt 60 ttctcagtaa gtaaccctac tatccaacgg ttctttgcct tacacttcct tctaccattc 120 atcttagctg cccttgtatg tatgcattta atggcattac atgtaaatgg atcatctaac 180 cctgttggta tcacaggtaa catcgaccga ttaccaatgc atccttactt catcttcaaa 240 gatctagtaa cagtcttcgt attcatcctt atattcagcc tgtttgtgtt ctatagccct 300 aacacgctag gacacccaga taactacatc ccaggtaacc ctatggtaac acctccttca 360 atcgtacctg agtgatacct attaccattc tacgca 396

[0039] <210> 9 <211> 396 <212> DNA <213> Candida dubliniensis <400> 9 aatctattat cagctatacc ttttatcggt aatgatatag taccctttat atgaggaggt 60 ttctctgtaa gtaatcctac aatacagcgt ttctttgcat tacatttctt attacctttt 120 atattagcag cattagtatg tatgcactta atggcattac atgtaaatgg ttcatcaaac 180 cctgtaggta taacaggtaa tatagataga ttaccaatgc atccttattt catatttaaa 240 gatcttataa ctgtatttgt attcttatta atatttagtt tatttgtatt ttattcacct 300 aatacattag gtcatcctga taattatata ccaggaaacc caatggtgac acctccatca 360 attgtaccag aatgatatct attaccattc tacgca 396

[0040] <210> 10 <211> 396 <212> DNA <213> Cryptococcus neoformans <400> 10 aacctgctat ctgctattcc ttggattggt acagacttta cacagtttgt ttggggtgga 60 ttctctgtta ataatgccac acttaaccga ttcttttcac ttcactatct actaccattc 120 attctagcag ctctagctct agtacacatg ctaacactac acacacatgg tagctcaaac 180 cctgaaggta ttagctcaaa tgcagaaaag gcaccaatgc acccatactt tatctttaag 240 gatctagtaa caattatcct tttcttcatt gctctaacag ctctagtaat gtatgcacct 300 aacatgctag gacacagtga caactacatt cctgctaacc caatgcaaac accaccttcg 360 attgtaccgg aatggtatct tctaccattc tacgca 396

Claims (30)

[Claims] Claims 1. A plurality of base sequences represented by SEQ ID NO: 1 (though thymine at any position may be substituted with uracil) or at least two or more of its complementary base sequences. A nucleic acid mixture for detecting fungi comprising a mixture of at least two or more nucleic acids having a base sequence consisting of 15 bases consecutive from the 3 ′ end or a base sequence hybridizing under stringent conditions to each base sequence. 2. The nucleic acid mixture is represented by SEQ ID NO: 1 in the sequence listing.
At least two nucleotide sequences represented by the following formulas (however, thymine at any position may be substituted with uracil)
2. The nucleic acid mixture for detecting a fungus according to claim 1, wherein the nucleic acid mixture has a base sequence consisting of at least 15 bases continuous from at least the 3 'end of each of at least three species. 3. The nucleic acid mixture according to SEQ ID NO: 1 in the sequence listing.
The nucleic acid mixture for detecting a fungus according to claim 2 or 3, wherein the nucleic acid mixture has a plurality of base sequences represented by the following formulas (however, thymine at any position may be substituted with uracil). 4. The nucleic acid mixture for detecting a fungus according to claim 1, wherein the fungus belongs to the genus Candida, the genus Cryptococcus, the genus Aspergillus, or the genus Penicillium. 5. The fungus, wherein the fungus is Candida glabrata,
The nucleic acid mixture for detecting a fungus according to claim 4, which is Candida parapsilosis, Candida tropicalis, Candida albicans, Cryptococcus neoformans or Candida dubriniensis. 6. The fungus, wherein the fungus is Candida glabrata,
The nucleic acid mixture for detecting a fungus according to claim 5, which is Candida parapsilosis or Candida tropicalis. 7. The nucleic acid mixture for fungal detection according to claim 1, which is a forward primer in a nucleic acid amplification method, provided that the nucleic acid mixture has a complementary sequence of SEQ ID NO: 1 or a partial base sequence thereof. except for). 8. A plurality of base sequences represented by SEQ ID NO: 2 (though thymine at any position may be substituted with uracil) or at least two or more of its complementary base sequences. A nucleic acid mixture for detecting fungi comprising a mixture of at least two or more nucleic acids having a base sequence consisting of 15 bases consecutive from the 3 ′ end or a base sequence hybridizing under stringent conditions to each base sequence. 9. The nucleic acid mixture according to SEQ ID NO: 2 in the sequence listing.
At least two nucleotide sequences represented by the following formulas (however, thymine at any position may be substituted with uracil)
9. The nucleic acid mixture for detecting a fungus according to claim 8, which has a nucleotide sequence consisting of at least 15 consecutive bases from at least the 3 'end of each of at least three species. 10. The nucleic acid mixture according to claim 8, wherein the nucleic acid mixture has a plurality of base sequences represented by SEQ ID NO: 2 in the sequence listing (though thymine at any position may be substituted with uracil). A nucleic acid mixture for detecting fungi. 11. The nucleic acid mixture for detecting a fungus according to claim 8, wherein the fungus belongs to the genus Candida, the genus Cryptococcus, the genus Aspergillus or the genus Penicillium. 12. The nucleic acid mixture according to claim 11, wherein the fungus is Candida albicans, Candida dubriniensis, Cryptococcus neoformans, Candida glabrata, Candida parapsilosis or Candida tropicalis. 13. The nucleic acid mixture for detecting a fungus according to claim 12, wherein the fungus is Candida albicans, Candida dubriniensis or Cryptococcus neoformans. 14. The nucleic acid mixture for fungal detection according to any one of claims 8 to 13, which is a forward primer in a nucleic acid amplification method (provided that it has a complementary sequence of SEQ ID NO: 2 or a partial base sequence thereof). except for). 15. At least two or more of a plurality of base sequences represented by SEQ ID NO: 3 (though thymine at any position may be substituted with uracil) or a complementary base sequence thereof. A nucleic acid mixture for detecting fungi comprising a mixture of at least two or more nucleic acids having a base sequence consisting of 15 bases consecutive from the 3 ′ end or a base sequence hybridizing under stringent conditions to each base sequence. 16. The nucleic acid mixture comprises at least two or more of each of a plurality of base sequences represented by SEQ ID NO: 3 in the sequence listing (though thymine at any position may be substituted with uracil). 16. The nucleic acid mixture for detecting a fungus according to claim 15, which has a base sequence consisting of at least 15 consecutive bases from its 3 'end. 17. The nucleic acid mixture according to claim 15, wherein the nucleic acid mixture has a plurality of base sequences represented by SEQ ID NO: 3 in the sequence listing (though thymine at any position may be substituted with uracil). A nucleic acid mixture for detecting fungi. 18. The fungus according to claim 15, wherein the fungus belongs to the genus Candida, Cryptococcus, Aspergillus or Penicillium (excluding Candida albicans, Cryptococcus neoformans and Candida dubriniensis). Nucleic acid mixture for detection. 19. The nucleic acid mixture for detecting a fungus according to claim 18, wherein the fungus is Candida glabrata, Candida parapsilosis or Candida tropicalis. 20. The fungal detection nucleic acid according to any one of claims 15 to 19, which is a reverse primer in a nucleic acid amplification method (provided that the nucleic acid has a complementary sequence of SEQ ID NO: 3 or a partial base sequence thereof). except). 21. At least each of a plurality of base sequences represented by SEQ ID NO: 4 (provided that thymine at any position may be substituted with uracil) or a base sequence complementary to at least two of them. A nucleic acid mixture for detecting fungi comprising a mixture of at least two or more nucleic acids having a base sequence consisting of 15 bases consecutive from the 3 ′ end or a base sequence hybridizing under stringent conditions to each base sequence. 22. The nucleic acid mixture comprises at least the 3 ′ end of each of a plurality of base sequences represented by SEQ ID NO: 4 in the sequence listing (though thymine at any position may be substituted with uracil). 22. The nucleic acid mixture for detecting a fungus according to claim 21, which has a base sequence consisting of 15 bases continuous from the base. 23. The nucleic acid mixture according to claim 21, wherein the nucleic acid mixture has a plurality of base sequences represented by SEQ ID NO: 4 in the sequence listing (provided that thymine at any position may be substituted with uracil). A nucleic acid mixture for detecting fungi. 24. The nucleic acid for detecting a fungus according to claim 21, wherein the fungus belongs to the genus Candida, the genus Cryptococcus, the genus Aspergillus or the genus Penicillium. 25. The nucleic acid according to claim 24, wherein the fungus is Candida albicans, Candida dubriniensis, Cryptococcus neoformans, Candida glabrata, Candida parapsilosis or Candida tropicalis. 26. The nucleic acid for detecting a fungus according to claim 25, wherein the fungus is Candida albicans, Candida dubriniensis or Cryptococcus neoformans. 27. The fungal detection nucleic acid according to any one of claims 21 to 26, wherein the nucleic acid is a reverse primer in a nucleic acid amplification method, provided that the nucleic acid has a complementary sequence of SEQ ID NO: 4 or a partial base sequence thereof. except). 28. A test nucleic acid is amplified by a nucleic acid amplification method using a combination of the forward primer according to claim 7 or 14 and the reverse primer according to claim 20 or 27, and an amplification product is detected. A method for detecting a fungus, comprising: 29. A test nucleic acid is amplified by a nucleic acid amplification method using a combination of the forward primer according to claim 7 or 14 and the reverse primer according to claim 20 or 27, and the base sequence of the amplification product is determined. Including determining
A method for identifying fungi. A kit for detecting a fungus, comprising the nucleic acid according to any one of claims 1 to 29.