WO2002040663A1 - Nucleic acids for detecting fungi and method of detecting fungi by using the same - Google Patents

Abstract

Nucleic acids to be used in detecting fungi causative of deep-seated fungal infections, in particular, fungi belonging to the genera Candida and Cryptococcus. These nucleic acids are nucleic acid mixtures for detecting fungi which comprise at least two nucleic acids selected from among nucleic acids having a plural number of base sequences each consisting of at least 15 consecutive bases from the 3'-end of at least two base sequences represented by SEQ ID NOS:1, 2, 3 and 4.

Description

 Specification

 Nucleic acid for detecting fungi and method for detecting fungi using the same

 Technical field

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

 Background art

 The main pathogens of deep mycosis are Candida (hereinafter abbreviated as G.) genus fungi, Aspergillus (hereinafter abbreviated as A.) genus fungi, and Cryptococcus (abbreviated as Gr. Below). ) Genus fungi are known. Major pathogens of the genus Candida include Candida albicans (G. albicans), Candida albicans 'G. glabrata', Candida albicans 'G. tropical is', and Candida albicans (G. parapsi losis) Candida albicans 'G. dubl iniensis and other major pathogens of the genus Cryptococcus include Cryptococcus neoformans (Gr. Neoformans). The main pathogens of the genus Aspergillus include A. fumigatus, A. flavus, A. niger, and the two gar groups, Aspergillus drans (A. n). idurans) and Aspergillus' terreus (A. terreus), and it is desired to rapidly detect, classify, and identify these strains.

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 bacteria are identified early and appropriate treatment is performed. It is necessary to. Diagnosis of deep mycosis is based on the blood culture method, but there are problems with detection sensitivity. Antibody-based diagnostics have been marketed so far, but antibody-based diagnosis is not useful for immunodeficient patients. Under these circumstances, in recent years, methods for directly detecting antigens and other bacterial cell components have been developed. For example, methods have been developed to detect mannan, D-arabinitol, glucan, etc. to diagnose infection. On the other hand, with the development of molecular biology, methods of extracting DNA of various pathogens such as Mycobacterium tuberculosis and Chlamydia and diagnosing them by detecting nucleic acids by a so-called PCR method have been developed. Ribosome RN also in the field of deep mycosis Methods for detecting and diagnosing A (Clinical Pathology, Vol. 43, Supplement, pp. 119, 1995) have been proposed. The present inventors have previously focused on the gene of cytochrome b present in the mitochondria of fungi causing these various mycosis, and provided a nucleic acid used for detecting a fungus of the genus Aspergillus. A simple, rapid, specific, and sensitive method for the detection of fungi causing deep mycosis, and a method for the detection of fungi of the genus Aspergillus by using E. coli (Materials and methods for detecting fungi, W098 / 10073) .

 Disclosure of the invention

 An object of the present invention is to provide a nucleic acid used for detecting fungi causing deep mycosis, particularly fungi of the genus Candida and Cryptococcus, and to use the nucleic acid in a simple, rapid, specific and specific manner. It is an object of the present invention to provide a highly sensitive method for detecting fungi of deep mycosis, particularly fungi of the genus Candida and cryptococci.

The present inventors have conducted various studies on the genes of various fungal fungi, especially Candida fungi and Cryptococcus fungi, and as a result, the fungi causing these various fungal diseases have been identified. A part of the cytochrome b gene present in mitochondria was amplified and its sequence was successfully decoded. As a result of further studies based on the sequences, the present inventors found specific sequences for each type of Candida fungus, and amplified a part of the cytochrome b gene of each species based on them. A universal primer mixture has been designed to be able to do so. By performing a nucleic acid amplification reaction using these primer mixtures, a primer extension product can be obtained even if the species of the causative microorganism of deep mycosis present in the sample has not been identified. That is, the present invention relates to the present invention, wherein each of a plurality of base sequences represented by SEQ ID NOS: 1, 2, 3 or 4, each showing a plurality of base sequences (provided that thymine at any position is substituted with peracil. Or at least two or more of its complementary base sequences, each consisting of at least 15 bases continuous from the 3 ′ end, or hybridizing with each base sequence under stringent conditions. Provided is a nucleic acid mixture for detecting a fungus, comprising a mixture of at least two or more nucleic acids having a base sequence. Further, the present invention relates to at least 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 peracyl). Out of two or more kinds, a base sequence consisting of at least 15 bases continuous from the 3 ′ end or a mixture of the base sequence and a nucleic acid having a base sequence that hybridizes under stringent conditions. It is used as a primer on one side and has at least two or more of a plurality of base sequences represented by SEQ ID NO: 3 or 4 in the sequence listing (though thymine at any position may be substituted with peracyl). 'A nucleic acid amplification method using a nucleotide sequence comprising at least 15 nucleotides continuous from the terminal or a mixture of nucleic acids having a nucleotide sequence that hybridizes with the nucleotide sequence under stringent conditions as a reverse primer. Provided is a method for detecting a fungus, comprising amplifying a test nucleic acid and detecting an amplification product. Further, 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. Further, the present invention provides a fungal detection probe obtained by labeling the nucleic acid mixture of the present invention. Further, the present invention provides a capture probe for detecting a fungus, wherein the nucleic acid mixture of the present invention is bound to a carrier. Furthermore, the present invention provides the use of the nucleic acid mixture of the present invention for producing a probe or primer for detecting a fungus.

 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, it has become possible to detect Candida fungi and Cryptococcus fungi quickly, easily and specifically and with high sensitivity, which have conventionally required classification <time for identification. 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 fungi having different nucleotide sequences by a single amplification reaction. In addition, the sensitivity and specificity can be further increased by detecting the product amplified by the primer with a probe, and its clinical significance is great.

 BEST MODE FOR CARRYING OUT THE INVENTION

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 substituted with peracil. Or at least two of its complementary base sequences Among them, those having a base sequence consisting of at least 15 bases continuous from at least the 3 'end or a base sequence that hybridizes with the base sequence under stringent conditions. As is evident from the fact that thymine may be substituted for peracil, the nucleic acids 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 peracyl) A nucleotide sequence consisting of at least 15 bases continuous from the terminal is preferable, and a nucleic acid mixture represented by SEQ ID NO: 1, 2, 3, or 4 and having a plurality of nucleotide sequences is particularly preferable. However, the nucleic acid mixture for detecting a fungus of the present invention comprises a nucleotide sequence represented by SEQ ID NO: 1, 2, 3 or 4 in the sequence listing (however, thymine at any position may be substituted with peracyl) or its complement. A basic sequence consisting of at least 15 bases continuous from at least its 3 'end under stringent conditions, the base sequence described above may be substituted with at least 3 bases. It may include deletions, insertions or additions. Here, “under stringent conditions” means that under the annealing conditions in ordinary PCR as described in the Examples below, the target nucleic acid hybridizes to type II and functions as a primer. (Note that the buffer commonly used for PCR is PCR buffer (final concentration of 50 mM KG I, 10 mM Tris-HCl (pH 8.4-9. at 25 ° C), 1.5 mM MgG I ₂ ) .Kit usually comes with a 10-fold concentration of X10 PGR buffer (or 10X PGR buffer), which is diluted. Use). In addition, the nucleotide sequence represented by SEQ ID NO: 1, 2, 3 or 4 (however, thymine at any position may be substituted with peracyl) or at least the 3 ' A nucleic acid mixture having a base sequence consisting of at least 15 bases consecutive from the end and a sequence in which only one or two bases are substituted (excluding those in which the 3 'end is substituted) is usually used for the purpose of the present invention. Can be used for

 The nucleic acid of the present invention can be easily produced by chemical synthesis using a commercially available DNA synthesizer or the like.

SEQ ID NOs: 1 to 4 indicate multiple bases with one letter, such as m, r, and w, respectively. Therefore, one sequence number indicates multiple sequences. The nucleic acid of the present invention is a mixture of nucleic acids having at least two or more, and preferably all of these plural nucleotide sequences (including the above-mentioned portions and variants). By using such a mixture, various mutant strains of fungi can be detected.

 The nucleic acid mixture of the present invention may comprise a region within the mitochondrial cytochrome b gene of a fungus, particularly a fungus belonging to the genus Candida, Cryptococcus, Aspergillus or Penicillium, preferably a fungus belonging to the genus Candida or Cryptococcus. It is what hybridizes. Preferred fungal species that can be detected using the nucleic acid mixture of the present invention include Candida 'grabrata, Candida parapsilosis, Candida tropicalis, Candida' albicans, Cryptococcus' neoformans and Candida doubryensis But are not limited to these. 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 doubriniensis.

 The mitochondrial cytochrome b gene is located in mitochondria, is difficult to recombine due to cytoplasmic inheritance, and is thought to undergo base changes in proportion to evolution. Therefore, it is suitable for detecting various fungi. Further, as will be described in more detail later, the region 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, It has the advantage that it is easy to distinguish between DNA types within a species. Therefore, the species or strain of fungi can be identified by amplifying the test nucleic acid using the nucleic acid of the present invention as a primer and determining the base sequence of the amplification product.

As described above, the nucleic acid mixture of the present invention hybridizes with a region in the cytochrome b gene of the fungal mitochondria, so that the fungus can be detected by using the nucleic acid mixture of the present invention as a probe or a primer for a nucleic acid amplification method. it can. 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 invention is After the fungal gene in the test sample and the labeled probe are hybridized, the bound product of the fungal gene and the labeled probe or the unbound labeled probe is detected by an appropriate detection method suitable for the labeling agent. Labeling agents used for probes are well known in this field, and include fluorescent labels, radioactive labels, enzyme labels, biotin and the like. The hybridization of the sample, i.e., the fungal gene in the test sample, to the probe is performed by pretreating and purifying the sample by a usual method to obtain a test sample, and mixing it with a labeled nucleic acid mixture as a probe, and then room temperature. It can be carried out by treating at 10 to 70 ° C for 10 minutes to 48 hours. 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, thereby obtaining a cytochrome of a Candida fungus and a Cryptococcus fungus. A fungus can be detected by specifically amplifying only the b gene fragment and measuring the amplified 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.Any method using a short-chain oligonucleic acid as a primer to initiate gene synthesis is 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 carried out by fragmenting the amplified cytochrome b gene fragment with a restriction enzyme and comparing the generated patterns of the fragment. The restriction enzymes used here are used alone or 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 a normal 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 clear band for detection. The primers are also used in the amplification reaction during the amplification reaction. By using a liponucleic acid mixture (ATP, UTP, GTP, CTP) as a raw material for DNA or RNA synthesis, amplification products can be directly detected with high sensitivity. Alternatively, by using a labeled nucleic acid mixture obtained by labeling the nucleic acid of the present invention as a primer, the 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, in which case the nucleic acid to be tested can be quantified. Therefore, “detection” as used herein includes quantitative detection.

 When used as a primer, a nucleic acid mixture having the nucleotide sequence shown in SEQ ID NO: 1 and SEQ ID NO: 2 (including the above-mentioned part and mutant thereof, the same applies hereinafter) is preferably used as a fore-side primer, The nucleic acid mixture having the nucleotide sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4 is preferably used as a reverse primer. It is preferable to use a combination of these forward primer and linear 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. Also, species can be identified by determining the base sequence of the obtained amplification product. The reagent kit for detecting a fungus of the present invention comprises the nucleic acid mixture of the present invention or a labeled nucleic acid obtained by labeling the nucleic acid mixture with an appropriate labeling agent. The reagent kit of the present invention may contain the above-described nucleic acid, and may contain a labeling 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 in only its means, and is essentially the same for detecting 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. Can be used as a reagent kit.

The reagent kit of the present invention when the nucleic acid mixture in the reagent kit is used as a probe May contain, in addition to the nucleic acid mixture for detecting Candida fungi and Cryptococcus fungi or the labeled nucleic acid mixture for detecting Candida fungi and Cryptococcus fungus of the present invention, a labeling detection reagent, a buffer, and the like. Good. When the nucleic acid mixture in the reagent kit is used as a primer, the reagent kit of the present invention is 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 detection. In addition to nucleic acid mixtures for use, nucleic acid synthases (eg, DNA polymerase, RNA polymerase, reverse transcriptase, etc.), mixtures of deoxyribonucleotides (dATP, dTTP, dGTP, dGTP), and mixtures of ribonucleotides (ATP, UTP) , GTP, CTP), restriction enzymes, buffers and the like. Specific examples of these kits include, for example, those obtained by adding the nucleic acid mixture of the present invention to commercially available PCR kits described in the following Examples. Further, the nucleic acid mixture of the present invention can be used as a capture probe by binding to a solid phase carrier. In this case, the sandwich assay may be performed by combining two of the capture probe and the labeled probe. 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, hybridized, and then captured with an avidin-bound carrier. In the sandwich assay, if the nucleic acid mixture of the present invention is used for either one, specific measurement of the nucleic acid mixture of the present invention becomes possible, and there is no problem even if the specificity of the other nucleic acid mixture is slightly lower. . The binding of the nucleic acid to the solid phase carrier can be easily performed by a well-known technique widely used in the field of DNA chips and the like.

Example

 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 cytochrome b gene amplification reaction) The cells of Candida albicans (Gandida albicans IF 40009) obtained by culturing in a port dextrose liquid medium were treated with 75% ethanol and sterilized. The cells were obtained by centrifugation at 1500 g for 10 minutes, and about 40 ml of extraction buffer (0.9 M sorbitol, 10 mM EDTA, 10 mM Tris-HCl buffer, pH 7.1) was added, and the mixture was shaken well. Thereafter, the supernatant was discarded by centrifugation at 1500 g for 10 minutes, 30 ml of the same buffer was added. Similarly, the supernatant was discarded after centrifugation, and 9 ml of the same buffer was added. To this was added 1 ml of Zymolyase (Seikagaku Kogyo Co., Ltd. cell wall lysing enzyme, 10 mg / ml) dissolved in the same buffer, and the mixture was incubated at 37 ° C. for 1 hour and then treated with an ultrasonic washer for 1 minute. This was centrifuged at 1500 g for 10 minutes to obtain a supernatant, and further centrifuged at 20000 g for 15 minutes to obtain a precipitate. The precipitate was washed with an extraction buffer and centrifuged again at 20000 g for 15 minutes to obtain a mitochondrial fraction. To this mitochondrial fraction was added 0.5 tnl of 1 mg / ml protease K (Protease), and the mixture was incubated at 37 ° C for 1 hour. Add 1 ml of a mixture of phenol, black form and isoamyl alcohol (25: 24: 1), shake, and remove the upper layer by centrifugation at 1500 g for 10 minutes. Add 1 ml of water-saturated phenol and shake. After mixing, the upper layer was removed by centrifugation at 1500 g for 10 minutes. To this, 0.1 ml of a 3 M sodium acetate and 0.1 M magnesium chloride solution was added, 3 ml of ethanol was added, and the mixture was allowed to stand at -20 ° C. This was centrifuged at 1500 g for 10 minutes to obtain a precipitate.This precipitate was washed with 75% ethanol at -20 ° C. After centrifugation at 1500 g for 10 minutes, the supernatant was discarded, nucleic acid was obtained, and after drying,- Stored at 20 ° C.

 Using this nucleic acid as a mirror, using the nucleic acid mixture 2 and the nucleic acid mixture 4 obtained in Example 1 as a primer and using TaKaRa PGR Amplification Kit (R011) of Takara Shuzo Co., Ltd. Using (MIR-D30), PCR 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 PGR buffer (kit reagent) 5 I

PGR dNTP mixed solution (kit reagent) 4 I

 Taq DNA synthase (kit reagent) 1 I (2.5U)

Nucleic acid mixture 2 1 1

Nucleic acid mixture 4 1 1 Mitochondrial DNA 1 1

The total volume was made up to 50 I with water.

 The reaction conditions were as follows.

Thermal denaturation: 94 ° C, I min

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. Using a DNA sequencer (ABI Prism 377) manufactured by PerkinElmer and analyzed according to the Dye Terminator method according to the operation guide of the company. Nucleic acid mixture 2 was used on the forward side and nucleic acid mixture 4 was used on the reverse side as primers for base 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, using the nucleic acid mixture 1 and the nucleic acid mixture 3 obtained in Example 1, a part of the cytochrome b gene was amplified from Candida glabrata (Candida glabrata IFM 46843) by a PCR reaction, The 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 (Amplification reaction of Candida parapsilosis cytochrome b gene) Using the nucleic acid mixture 1 and the nucleic acid mixture 3 obtained in Example 1 in the same manner as in Example 2, Candida parapsilosis was used. losis 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 as 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, a part of the cytochrome b gene from Candida tropical is (FM 46816) was subjected to a PCR reaction. It was amplified and its nucleotide sequence was determined. Base sequence solution Nucleic acid mixture 1 was used on the forward side and nucleic acid mixture 3 was used on the reverse side as the primer for analysis. The nucleotide sequence is shown in SEQ ID NO: 8 in the sequence listing.

Example 6 (Amplification reaction of Candida doubriniensis cytochrome b gene) Using the nucleic acid mixture 2 and nucleic acid mixture 4 obtained in Example 1 in the same manner as in Example 2, Candida doubriniensis (Candida doubriniensis) was used. A part of the cytochrome b gene from Candida dubl iniensis IFM 48313) was amplified by PCR, and its nucleotide sequence was determined. The nucleotide sequence analysis primer used was a nucleic acid mixture 2 on the forward side and a nucleic acid mixture 4 on the reverse side. The nucleotide sequence is shown in SEQ ID NO: 9 in the sequence listing.

Example II (Cryptococcus neoformans cytochrome b gene amplification reaction)

 Using the nucleic acid mixture 2 and the nucleic acid mixture 4 obtained in Example 1 in the same manner as in Example 2, a portion of the cytochrome b gene is amplified from Cryptococcus neoformans (Gryptococcus neoformans IFM 46 138) by PCR. Then, its nucleotide sequence was determined. For the primer for nucleotide sequence analysis, the nucleic acid mixture 2 was used on the forward side, and the nucleic acid mixture 4 was used on the reverse side. The nucleotide sequence is shown as SEQ ID NO: 10 in the sequence listing.

Claims

The scope of the claims  1. A plurality of base sequences represented by SEQ ID NO: 1 (however, thymine at any position may be substituted with peracyl) or at least two or more of its complementary base sequences, each having a 3'-terminal A nucleic acid mixture for detecting a fungus, comprising a mixture of at least two or more nucleic acids having a base sequence consisting of at least 15 bases continuous from or a base sequence that hybridizes with each base sequence under stringent conditions. 2. The nucleic acid mixture contains at least two or more 3's of each of a plurality of base sequences represented by SEQ ID NO: 1 in the sequence listing (though thymine at any position may be substituted with peracyl). 2. The nucleic acid mixture for detecting a fungus according to claim 1, which has a base sequence consisting of at least 15 bases continuous from the terminal.  3. The fungal detection according to claim 2 or 3, wherein the nucleic acid mixture has a plurality of base sequences represented by SEQ ID NO: 1 in the sequence listing (though thymine at any position may be substituted with peracyl). For nucleic acid mixtures.  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 Benicillium.  5. The nucleic acid mixture for detecting a fungus according to claim 4, wherein the fungus is Candida 'Grabrata, Candida' parapsilosis, Candida tropicalis, Candida albicans, Cryptococcus neoformans or Candida doubryensis. 6. The nucleic acid mixture for detecting a fungus according to claim 5, wherein the fungus is Candida 'Grabrata, Candida' parapsilosis or Candida tropicalis. 7. The nucleic acid mixture for detecting a fungus according to any one of claims 1 to 6, which is a forward primer in a nucleic acid amplification method (provided that the primer has a complementary sequence of SEQ ID NO: 1 or a partial base sequence thereof). Excluded). 8. A plurality of nucleotide sequences represented by SEQ ID NO: 2 (thymine at any position may be substituted with peracyl) or at least two or more complementary nucleotide sequences thereof at the 3 'end Or a base sequence consisting of at least 15 bases continuous from or a string that hybridizes under stringent conditions with each base sequence. A nucleic acid mixture for detecting a fungus, comprising a mixture of at least two or more nucleic acids. 9. The nucleic acid mixture contains 3 'of each of at least two or more of a plurality of base sequences represented by SEQ ID NO: 2 in the sequence listing (though thymine at any position may be substituted with peracyl). 9. The nucleic acid mixture for detecting a fungus according to claim 8, which has a base sequence consisting of at least 15 bases continuous from the terminal.  0. The fungal detection according to claim 8 or 9, 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 peracyl). For nucleic acid mixtures.  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 fungus for detecting fungi according to claim 11, wherein the fungus is Candida albicans, Candida doubriniensis, Cryptococcus neoformans, Candida 'grabrata, candida.no' rapsilosis or Candida'tropicalis. Nucleic acid mixture.  13. The nucleic acid mixture for detecting a fungus according to claim 12, wherein the fungus is Candida albicans, Candida doubriniensis 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 the nucleic acid mixture has a complementary strand of SEQ ID NO: 2 or a partial base sequence thereof. except for) .  15. Multiple base sequences represented by SEQ ID NO: 3 (thymine at any position may be substituted with peracyl) or at least two or more of its complementary base sequences at the 3 'end A nucleic acid mixture for detecting a fungus, comprising a mixture of at least two or more nucleic acids having a base sequence consisting of at least 15 bases contiguous with or a base sequence that hybridizes with each base sequence under stringent conditions. 16. The nucleic acid mixture contains at least two 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 peracyl). 'Base sequence consisting of at least 15 bases consecutive from the end 16. The nucleic acid mixture for detecting a fungus according to claim 15, comprising:  17. The nucleic acid mixture according to claim 15 or 16, 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 peracyl). A nucleic acid mixture for detecting fungi. 18. The fungus belongs to the genus Candida, Cryptococcus, Aspergillus or Penicillium (however, excluding Candida albicans, Cryptococcus ■ neoformans and Candida doubryensis). Claims 15 to 1 7. A nucleic acid mixture for detecting fungi according to 7.  19. The nucleic acid mixture for detecting a fungus according to claim 18, wherein the fungus is Candida 'grabrata, 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 strand of SEQ ID NO: 3 or a partial base sequence thereof). Excluded).  21. A plurality of base sequences represented by SEQ ID NO: 4 (however, thymine at any position may be substituted with peracyl) or at least a complementary base sequence thereof At least two or more nucleic acids, each having at least 15 bases consecutive from the 3 ′ end or having a base sequence that hybridizes with each base sequence under stringent conditions, A nucleic acid mixture for detecting fungi, comprising the mixture. 22. The nucleic acid mixture is continuous from 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 peracyl). 3. The nucleic acid mixture for detecting a fungus according to claim 2, which has a nucleotide sequence of at least 15 bases.  23. The nucleic acid mixture according to claim 21 or 22, wherein the nucleic acid mixture has a plurality of base sequences represented by SEQ ID NO: 4 in the sequence listing (though thymine at any position may be substituted with peracyl). A nucleic acid mixture for detecting fungi.  24. The nucleic acid mixture for detecting a fungus according to any one of claims 21 to 23, wherein the fungus belongs to the genus Candida, Cryptococcus, Aspergillus or Penicillium. 25. The fungi are Candida albicans, Candida doubriniensis, 25. The nucleic acid mixture for detecting a fungus according to claim 24, which is Cryptococcus 'Neoformans, Candida' glabra, Candida parapsilosis or Candida tropicalis.  26. The nucleic acid mixture for detecting a fungus according to claim 25, wherein the fungus is Candida albicans, Candida doubriniensis or Cryptococcus neoformans.  27. The nucleic acid mixture for detecting a fungus 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 complementary sequence of SEQ ID NO: 4 or a partial sequence thereof is Except those that have). 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 obtained. A method for detecting a fungus, comprising detecting.  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 A method for identifying a fungus, comprising determining  30. A kit for detecting a fungus, comprising the nucleic acid mixture according to any one of claims 1 to 29.  31. A labeled probe for detecting a fungus, which is obtained by labeling the nucleic acid mixture according to any one of claims 1 to 27. 32. A capture probe for detecting a fungus, comprising the nucleic acid mixture according to any one of claims 1 to 27 bound to a carrier.  33. Use of the nucleic acid mixture according to any one of claims 1 to 27 for producing a probe or primer for detecting a fungus.