WO2002040713A1 - Nucleic acids for detecting fungi belonging to the genus candida and method of detecting fungi belonging to the genus candida by using the same - Google Patents

Abstract

Nucleic acids whereby five fungi belonging to the genus Candida (i.e., C. albicans, C. glabrata, C. parapsilosis, C. tropicalis and C. dubliniensis), among fungi causative of deep-seated mycosis, can be species-specifically detected are disclosed. Namely, nucleic acids for detecting C. albicans having at least 15 consecutive bases from the 3'-end of a base sequence represented by SEQ ID NO:1 or 6; nucleic acids for detecting C. glabrata having at least 15 consecutive bases from the 3'-end of a base sequence represented by SEQ ID NO:2 or 7; nucleic acids for detecting C. parapsilosis having at least 15 consecutive bases from the 3'-end of a base sequence represented by SEQ ID NO:3 or 8; nucleic acids for detecting C. tropicali having at least 15 consecutive bases from the 3'-end of a base sequence represented by SEQ ID NO:4 or 9; nucleic acids for detecting C. dubliniensis having at least 15 consecutive bases from the 3'-end of a base sequence represented by SEQ ID NO:5 or 10.

Description

 Specification

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

 Technical field

 The present invention relates to a nucleic acid for distinguishing and detecting five Candida fungi and a method for distinguishing and detecting five Candida fungi using the nucleic acid.

 Background art

 The main pathogens of deep mycosis include Candida (hereinafter abbreviated as G.) genus fungi, Aspergilus (hereinafter abbreviated as Α ·) genus fungi, and Cryptococcus (hereinafter abbreviated as Cr.). Abbreviated fungi are known. Major pathogens of the genus Candida include C. albicans, G. glabrata, C. tropicalis, and C. parapsi. losis) Cryptococcus neoformans (Gr. neoformans) is known as a major causative agent of the genus Cryptococcus, such as G. dubl iniensis. The main pathogens of the genus Aspergillus include Aspergillus' Fumigatus, A. flavus, A. niger, and the two gar groups, A. nigerans. nidurans) and Aspergillus' Teleus (A. terreus), and it is desired to rapidly detect, classify, and identify these bacterial species.

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, DNA of various pathogens such as Mycobacterium tuberculosis and Chlamydia has been extracted, and nucleic acids have been detected by the so-called PCR method. Therefore, a method of diagnosis has been developed. In the field of deep mycosis, methods for detecting and diagnosing ribosomal RNA (Clinical Pathology, Vol. 43, Vol. 11, pp. 119, 1995) have been proposed. The present inventors have previously focused on the gene for cytochrome b present in the mitochondria of fungi that cause these various mycosis, provided nucleic acids used for detecting Aspergillus fungi, and further using the same. A simple, rapid, specific and highly sensitive method for the detection of fungal pathogens of deep mycosis, as well as a method for detecting fungi of the genus Aspergillus, has been developed (materials and methods for detecting fungi, W098 / 10073).

 Disclosure of the invention

 An object of the present invention is to provide five fungi belonging to the genus Candida among the causative fungi of deep mycosis, namely, Candida albicans, Candida, glabrata, Candida parapsilosis, Candida tropicalis and Candida * doubrinien. To provide a nucleic acid capable of detecting cis in a species-specific manner, and to provide a simple, rapid, specific and highly sensitive method for detecting these causative bacteria of deep mycosis by using the nucleic acid. It is.

The present inventors have conducted various studies on the genes of various causative fungi, especially of the fungi of the genus Candida, and found that cytochromes present in the mitochondria of fungi that cause these various fungal diseases We amplified part of the gene b and succeeded in decoding its sequence. As a result of further studies based on the sequences, the present inventors found specific sequences for each type of Candida fungus and designed primers specific to each species based on the sequences. . For example, as primers specific to Candida albicans, SEQ ID NOs: 1 and 6 in the Sequence Listing, as primers specific to Candida glabrata, SEQ ID NOs: 2 and 7, as primers specific to Candida parapsilosis SEQ ID Nos. 3 and 8 in the Sequence Listing, SEQ ID Nos. 4 and 9 in the Sequence Listing as primers specific for Candida tropicalis, and SEQ ID Nos. 5 and 10 in the Sequence Listing as primers specific to Candida doubliniensis And so on. Based on these, a simple, rapid, specific and highly sensitive detection of fungi of the genus Candida was obtained, a nucleic acid was obtained for classification and identification, and a detection method using the nucleic acid was established, thereby completing the present invention. Was. That is, the present invention relates to the base sequence represented by SEQ ID NO: 1 or 6 in the sequence listing (however, thymine at any position may be substituted with peracyl) or its complementary base sequence from the 3 ′ end. It is intended to provide a nucleic acid for detecting Candida albicans having a nucleotide sequence consisting of at least 15 consecutive nucleotides or a nucleotide sequence which hybridizes with the nucleotide sequence under stringent conditions. In addition, the present invention relates to the nucleotide sequence represented by SEQ ID NO: 2 or 7 in the sequence listing (though thymine at any position may be substituted with peracyl) or its complementary 3′-terminal in the nucleotide sequence. And a nucleic acid for detecting Candida glabrata having a base sequence consisting of at least 15 bases and a base sequence that hybridizes with the base sequence under stringent conditions. Furthermore, the present invention relates to a nucleotide sequence represented by SEQ ID NO: 3 or 8 in the Sequence Listing (however, thymine at any position may be substituted with peracyl) or a nucleotide sequence complementary thereto. 'A nucleic acid for detecting Candida' parapsilosis having a nucleotide sequence consisting of at least 15 bases continuous from the terminus or a nucleotide sequence hybridizing under stringent conditions with the nucleotide sequence is provided. Furthermore, the present invention relates to a nucleotide sequence represented by SEQ ID NO: 4 or 9 in the sequence listing (though thymine at any position may be substituted with peracyl) or its complementary 3′-terminal in the nucleotide sequence. The present invention provides a nucleic acid for detecting Candida tropicalis having a nucleotide sequence consisting of at least 15 consecutive nucleotides or a nucleotide sequence which hybridizes with the nucleotide sequence under stringent conditions. Furthermore, the present invention relates to a nucleotide sequence represented by SEQ ID NO: 5 or 10 in the sequence listing (though thymine at any position may be substituted with peracyl) or a 3 ′ nucleotide in its complementary nucleotide sequence. Provided is a nucleic acid for detecting Candida doubliniensis having a base sequence consisting of at least 15 bases continuous from the terminal or a base sequence that hybridizes with the base sequence under stringent conditions. Further, the present invention provides the above-mentioned method for detecting a Candida fungus using each of the nucleic acids for detection. Furthermore, the present invention provides the above-mentioned reagent kits for detecting Candida fungi, each containing the above-described nucleic acids for detection. Furthermore, the present invention provides a labeling probe obtained by labeling the nucleic acid of the present invention. Further, the present invention provides a capture probe obtained by binding the nucleic acid of the present invention to a carrier. Further, the present invention provides the probe or probe according to the present invention. Provide use for producing a limer.

 According to the present invention, a nucleic acid capable of detecting Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis and Candida doubriniensis in a species-specific manner, and species-specific fungi of these Candida fungi using the same. Detection methods were provided. According to the present invention, it has become possible to quickly, easily and specifically detect and classify and identify fungi of the genus Candida, which conventionally took a long time to classify and identify. The nucleic acid of the present invention can be used as a primer for an amplification reaction or as a probe for direct detection. The sensitivity and specificity can be further increased by detecting the product amplified by the primer with a probe, and its clinical significance is significant.

 BRIEF DESCRIPTION OF THE FIGURES

 FIG. 2 is a schematic diagram showing an electrophoresis pattern of an amplification product amplified by PCR using each nucleic acid of the present invention in an example of the present invention.

Lane 1: Size marker (100, 200, 300, 400, 500, 600, 700, 900, 1000, 15000)

Lane 2: Candida tropicalis IFM 46816

Lane 3: Candida tropicalis IFM 48776

Lane 4: Candida parapsilosis IFM 46829

Lane 5: Candida parapsilosis IFM 5464

Lane 6: Candida Doubriniensis IFM 48184

Lane 7: Candida doubryensis IFM 48313

Lane 8: Candida albicans IFM 48311

Lane 9: Candida albicans IFM 5728

Lane 10: Candida Grabrata IFM 46843

Lane 11: Candida Grabrata IFM 5768

 BEST MODE FOR CARRYING OUT THE INVENTION

As described above, the nucleic acid for detecting Candida albicans of the present invention has a nucleotide sequence represented by SEQ ID NO: 1 or 6 (provided that thymine at any position is replaced with peracyl). Or a base sequence consisting of at least 15 bases continuous from the 3 'end of the complementary base sequence or a base sequence that hybridizes with the base sequence under stringent conditions It is. As is clear from the fact that thymine may be substituted with peracyl, the nucleic acid of the present invention may be DNA or RNA. The nucleic acid for detecting Candida albicans of the present invention includes the 3 ′ terminal in the base sequence represented by SEQ ID NO: 1 or 6 in the sequence listing (though thymine at any position may be substituted with peracyl). It is preferable to have a base sequence consisting of at least 15 bases that are continuous with the base sequence, and particularly preferable to have a base sequence represented by SEQ ID NO: 1 or 6. However, the nucleic acid for detecting Candida albicans of the present invention may be a nucleotide sequence represented by SEQ ID NO: 1 or 6 in the sequence listing (though thymine at any position may be substituted with peracyl) or its complementary sequence. If the base sequence hybridizes under stringent conditions with a base sequence consisting of at least 15 bases continuous from the 3 'end, a substitution, deletion, insertion or It may include additions. Here, the term "stringent conditions" means that the primers hybridize to the type III target nucleic acid and function as primers under ordinary PCR annealing conditions as described in the Examples below. I do. (Note that the buffer commonly used in PCR is PGR buffer (final concentration of 50 mM KG I, 10 mM Tris-HC I (pH 8.4-9.0 at 25 ° C.) C), 1.5 mM MgG I ₂ ). Usually, the kit is provided with a 10-fold concentration of X10 PGR buffer (or 10X PGR buffer), which is used after dilution). Of the nucleotide sequence represented by SEQ ID NO: 1 or 6 in the sequence listing (though thymine at any position may be substituted with peracyl) or its complementary nucleotide sequence, at least A nucleic acid mixture having a base sequence consisting of 15 bases and a sequence in which only one or two bases have been substituted (excluding those in which the 3 ′ end has been substituted) can usually be used for the purpose of the present invention ( The same applies to nucleic acids having the sequence shown in other SEQ ID NOs for detecting Candida of other species, which will be described below.) The nucleotide sequences represented by SEQ ID NO: 1 and SEQ ID NO: 6 correspond to the 16n1 :: 45 nt region and 242n1 :: 270 in the mitochondria and cytochrome b gene determined by Candida albicans. It hybridizes with each of the nt regions.

The above-described nucleic acid for detecting Candida albicans of the present invention can be used as a probe by binding a labeling agent, as described later, or can be used as a primer in a nucleic acid amplification method such as PCR. When used as a primer, a nucleic acid having the nucleotide sequence of SEQ ID NO: 1 (including the above-described portions and variants thereof. Unless otherwise confused, "a nucleic acid having the nucleotide sequence of SEQ ID NO: 1" May be simply referred to as “SEQ ID NO: 1” for convenience))) as the forward primer, and SEQ ID NO: 6 (including the above-described portions and variants) as the reverse primer. It is preferable to use them in combination. When SEQ ID NO: 1 was used as the forward primer and SEQ ID NO: 6 was used as the source / side primer, if the Candida albicans mitochondrial cytochrome b gene was present in the test sample, A nucleic acid with a length of 255 base pairs (SEQ ID NO: 11) is amplified. In this case, amplification does not occur even if genes of other species of Candida are present. Therefore, species can be identified by examining the presence or absence of amplification. The nucleic acid for detecting Candida glabrata of the present invention comprises a base sequence represented by SEQ ID NO: 2 or 7 in the sequence listing (however, thymine at any position may be substituted with peracil). It has a base sequence consisting of at least 15 bases continuous from the 3 'end of the sequence or a base sequence that hybridizes with the base sequence under stringent conditions. As is clear from the fact that thymine may be substituted with peracyl, the nucleic acid of the present invention may be DNA or RNA. The nucleic acid for detecting Candida glabrata of the present invention includes a nucleotide sequence represented by SEQ ID NO: 2 or 7 in the sequence listing (though thymine at any position may be substituted with peracyl) from the 3 'end. Those having a base sequence of at least 15 consecutive bases are preferable, and those having the base sequence of SEQ ID NO: 2 or 7 are particularly preferable. However, the nucleic acid for detecting Candida 'glabrata of the present invention may be a nucleotide sequence represented by SEQ ID NO: 2 or 7 in the sequence listing (however, thymine at any position may be substituted with peracyl) or its complementary sequence. Among the base sequence, a base sequence consisting of at least 15 bases continuous from the 3 'end and a string The base sequence described above may include substitution, deletion, insertion or addition of a base as long as it hybridizes under the above conditions. Here, the “stringent conditions” are as described above. The nucleotide sequences represented by SEQ ID NO: 2 and SEQ ID NO: 7 hybridize with the 24n1: to 48nt region and 210n1: to 230nt region in the mitochondria and cytochrome b gene determined by Candida gravulata. It is a thing.

 The above-described nucleic acid for detecting Candida glabrata of the present invention can be used as a probe by binding a labeling agent, as described later, or can be used as a primer in a nucleic acid amplification method such as PCR. When used as a primer, SEQ ID NO: 2 (including the above-mentioned portions and variants) is used as the forward primer, and SEQ ID NO: 7 (including the above-mentioned portions and variants) is used as the reverse primer. Are preferably used in combination. When SEQ ID NO: 2 is used as the forward primer and SEQ ID NO: 7 is used as the reverse primer, if the mitochondrial cytochrome b gene of Candida glabrata is present in the test sample, the length is 205 base pairs. Is amplified (SEQ ID NO: 12). In this case, amplification does not occur even if genes of other species of Candida are present. Therefore, species can be identified by examining the presence or absence of amplification.

The nucleic acid for detecting Candida parapsilosis of the present invention comprises a nucleotide sequence represented by SEQ ID NO: 3 or 8 in the sequence listing (however, thymine at any position may be substituted with peracyl) or a nucleotide sequence complementary thereto. A nucleotide sequence consisting of at least 15 nucleotides continuous from the 3 ′ end of the above or a nucleotide sequence hybridizing with the nucleotide sequence under stringent conditions. As is clear from the fact that thymine may be substituted with peracyl, the nucleic acid of the present invention may be DNA or RNA. The nucleic acid for detecting Candida parapsilosis of the present invention may be a nucleotide sequence represented by SEQ ID NO: 3 or 8 in the sequence listing (though thymine at any position may be substituted with peracyl). It is preferable to have a base sequence of at least 15 bases, and particularly preferable to have a base sequence represented by SEQ ID NO: 3 or 8. However, Candida parapsilosis of the present invention The nucleic acid for detecting nucleic acid may be a nucleotide sequence represented by SEQ ID NO: 3 or 8 in the sequence listing (though thymine at any position may be substituted with peracyl) or its complementary nucleotide sequence. As long as it hybridizes under stringent conditions to a base sequence consisting of at least 15 bases continuous from the above, the base sequence described above may include substitution, deletion, insertion or addition of a base group. Here, the “stringent conditions” are as described above. The nucleotide sequences shown in SEQ ID NO: 3 and SEQ ID NO: 8 hybridize with the mitochondria determined by Candida parapsilosis, the region of 46 n1: to 68 nt and the region of 366 n1: to 395 nt in the cytochrome b gene, respectively. It is.

 As described below, the Candida parapsilosis detection nucleic acid of the present invention can be used as a probe by binding a labeling agent, or can be used as a primer in a nucleic acid amplification method such as PCR. When used as a primer, SEQ ID NO: 3 (including the above-mentioned portions and variants) is used as a forward primer, and SEQ ID NO: 8 (including the above-described portions and variants) is used as a reverse primer. It is preferable to use them. When SEQ ID NO: 3 was used as the forward primer and SEQ ID NO: 8 was used as the reverse primer, if the mitochondrial cytochrome b gene of Candida parapsilosis was present in the test sample, 355 base pairs long Nucleic acid (SEQ ID NO: 13) is amplified. In this case, amplification does not occur even if genes of other species of Candida are present. Therefore, species can be identified by examining the presence or absence of amplification.

The nucleic acid for detecting Candida tropicalis of the present invention is a nucleotide sequence represented by SEQ ID NO: 4 or 9 in the sequence listing (however, thymine at any position may be substituted with peracyl) or a nucleotide sequence complementary thereto. It has a base sequence consisting of at least 15 bases continuous from the 3 ′ end or a base sequence that hybridizes with the base sequence under stringent conditions. As is clear from the fact that thymine may be substituted with peracyl, the nucleic acid of the present invention may be DNA or RNA. The nucleic acid for detecting Candida tropicalis of the present invention includes a nucleotide sequence represented by SEQ ID NO: 4 or 9 in the sequence listing (provided that thymine at any position is substituted with peracyl). Preferably have a base sequence consisting of at least 15 bases continuous from the 3 ′ end, and particularly preferably have a base sequence represented by SEQ ID NO: 4 or 9. However, the nucleic acid for detecting Candida tropicalis of the present invention may be a nucleotide sequence represented by SEQ ID NO: 4 or 9 in the sequence listing (however, thymine at any position may be substituted with peracyl) or its complement. If the nucleotide sequence hybridizes with a nucleotide sequence consisting of at least 15 nucleotides continuous from the 3 'end under stringent conditions, the above nucleotide sequence may be replaced or deleted with a nucleotide. , Insertions or additions. Here, the “stringent conditions” are as described above. The nucleotide sequences shown in SEQ ID NO: 4 and SEQ ID NO: 9 were hybridized with the -13n1: to 10 nt region and the 363 nt to 392 nt region in the mitochondria and cytochrome b gene determined by Candida tropicalis, respectively. That is what you do.

 As described below, the nucleic acid for detecting Candida tropicalis of the present invention can be used as a probe by combining a labeling agent, or can be used as a primer in a nucleic acid amplification method such as PCR. When used as a primer, SEQ ID NO: 4 (including the above-mentioned portions and variants) is used as the forward primer, and SEQ ID NO: 9 (including the above-described portions and variants) is used as the reverse primer. Are preferably used in combination. When SEQ ID NO: 4 is used as the forward primer and SEQ ID NO: 9 is used as the reverse primer, if the Candida tropicalis mitochondrial cytochrome b gene is present in the test sample, 405 base pairs A nucleic acid of length (SEQ ID NO: 14) is amplified. In this case, amplification does not occur even if genes of other species of Candida are present.

The nucleic acid for detecting Candida doubryensis of the present invention comprises a nucleotide sequence represented by SEQ ID NO: 5 or 10 in the sequence listing (provided that thymine at any position may be substituted with peracyl) or its complement. A base sequence consisting of at least 15 bases continuous from the 3 'end in a typical base sequence 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 acid of the present invention may be DNA or RNA. There may be. The nucleic acid for detecting Candida doubryensis of the present invention includes a nucleotide sequence represented by SEQ ID NO: 5 or 10 in the sequence listing (though thymine at any position may be replaced with peracyl). 'It is preferable to have a base sequence consisting of at least 15 bases continuous from the terminus, and particularly preferable to have a base sequence represented by SEQ ID NO: 5 or 10. However, the nucleic acid for detecting Candida dough Brignensis of the present invention has a nucleotide sequence represented by SEQ ID NO: 5 or 10 in the sequence listing (however, thymine at any position may be substituted with peracyl) or its complement. If the base sequence hybridizes under stringent conditions with a base sequence consisting of at least 15 bases continuous from the 3 'end of the basic base sequence, base substitution, deletion, It may include insertions or additions. Here, the “stringent conditions” are as described above. The nucleotide sequences shown in SEQ ID NO: 5 and SEQ ID NO: 10 are the mitochondria determined by Candida's dubliniensis, the region of 61 nt: to 90 nt and the region of 336: to 366 nt in the cytochrome b gene, respectively. And hybridize.

 As described below, the nucleic acid for detecting Candida doubryensis of the present invention can be used as a probe by binding a labeling agent, or can be used as a primer in a nucleic acid amplification method such as PCR. When used as a primer, SEQ ID NO: 5 (including the above-mentioned portions and variants) is used as the forward primer, and SEQ ID NO: 10 (including the above-described portions and variants) is used as the primer. It is preferable to use them in combination as primers on the upstream side. When SEQ ID NO: 5 was used as the forward primer and SEQ ID NO: 10 was used as the reverse primer, the mitochondrial cytochrome b gene of Candida doubriniensis was present in the test sample. For example, a nucleic acid having a length of 305 base pairs (SEQ ID NO: 15) is amplified. In this case, amplification does not occur even if genes of other species of Candida are present.

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

As described above, each of the nucleic acids of the present invention is a mitochondria of each of the above Candida fungi. Therefore, each Candida fungus can be detected by using each nucleic acid of the present invention as a probe or a primer for a nucleic acid amplification method.

 When the nucleic acid of the present invention is used as a probe, it is preferable to use a nucleic acid labeled with a labeling agent. In this case, the labeled nucleic acid of the present invention is used as a probe, and the Candida fungus gene in the test sample is hybridized with the labeled probe, and then a conjugated or unbound labeled probe of the Candida fungal gene and the 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, and biotin. 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 the usual method to obtain a test sample, and mixing it with a labeled nucleic acid as a probe, and then 70 ° from room temperature. This can be done by treating with C for 10 minutes to 48 hours. The sample may be subjected to an amplification reaction in advance using the nucleic acid of the present invention as a primer.

When each of the nucleic acids of the present invention is used as a primer for a nucleic acid amplification method, an elongation reaction is performed with a DNA polymerase or the like using each of the nucleic acids of the present invention as a primer to amplify the gene, whereby the cytochrome b gene of each of the above described fungi of the genus A. Fungi can be detected by specifically amplifying only the fragment and measuring the amplification product. In this case, a labeled nucleic acid obtained by labeling the nucleic acid 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 here include a PCR method.However, the method is 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. The gene amplification method described above can also be used. The method for detecting a fungus of the present invention can be carried out by detecting an amplification product re-amplified by the above method, ie, a cytochrome b gene fragment. Alternatively, it can be performed by fragmenting the amplified cytochrome b gene fragment with a restriction enzyme and comparing the production patterns of the fragment. The restriction enzymes used here are used alone or in combination. Amplification product Amplified cytochrome b gene fragment, or further fragmented with restriction enzymes The means for detecting the gene fragment is not particularly limited, and a normal gene detection method (eg, electrophoresis) 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. During the amplification reaction, primers can be used as a starting material for DNA or RNA synthesis, using a mixture of deoxyliponucleic acid (dATP, dTTP, dGTP, dCTP) or ribonucleic acid (ATP, UTP, GTP, CTP) labeled with an appropriate labeling agent By using it, it is possible to detect amplification products directly with high sensitivity. Alternatively, by using a labeled nucleic acid 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 nucleic acid to be tested can be quantified. Therefore, “detection” in the present specification includes quantitative detection.

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

In the case where the nucleic acid in the reagent kit is used as a probe, the reagent kit of the present invention can be used for detecting a label other than the nucleic acid for detecting a fungus of the present invention or the nucleic acid for detecting a fungus of the present invention. It may contain reagents, restriction enzymes, buffers and the like. The reagent kit of the present invention when the nucleic acid in the reagent kit is used as a primer In addition to the nucleic acids for detecting fungi of the genus Genus of the present invention or the labeled nucleic acids for detecting a fungus of the genus Candida, nucleic acid synthases (eg, DNA polymerase, RNA polymerase, reverse transcriptase, etc.), It may contain a xyliponucleotide mixture (dATP, dTTP, dGTP, dCTP), a ribonucleotide mixture (ATP, UTP, GTP, CTP), a buffer, or 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 of the present invention can be bound to a solid phase carrier and used as a capture probe. In this case, a sandwich assay may be performed using a combination of a capture probe and a labeled probe. There is also a method of labeling and capturing a target nucleic acid. There is also a method of labeling nucleic acids with biotin, hybridization, and capturing with an avidin-bound carrier. In the San German Chiassy, if the nucleic acid of the present invention is used for either one, specific measurement of the nucleic acid of the present invention becomes possible, and there is no problem even if the specificity of the other nucleic acid is slightly low. 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 acids)

 A nucleic acid having a sequence represented by SEQ ID NOS: 10 to 10 in the sequence listing was chemically synthesized. Hereinafter, various nucleic acids represented by SEQ ID NOs: 1 to 10 in the sequence listing are referred to as nucleic acids 1 to 10, respectively. Example 2 (Identification of Candida albicans by PCR)

 The cells of Candida albicans (Gandida albicans IFM5728 and 48311) obtained by culturing in a potato dextrose liquid medium were treated with 75% ethanol and sterilized. The cells were obtained by centrifugation at 1500 g for 10 minutes, and approximately 40 ml of extraction buffer (0.

9M Sorbi! Add 10 mM MEDTA, 10 mM Tris-HCl buffer ρΗ7.1), shake well, discard the supernatant by centrifugation at 1500 g for 10 minutes, add 30 ml of the same buffer, and centrifuge similarly. Thereafter, the supernatant was discarded, and 9 ml of the same buffer was added. Same as this 1 ml of Zymolyase (Seikagaku Corporation cell wall lysing enzyme, lOmg / ml) dissolved in the buffer was added, the mixture was kept at 37 ° C for 1 hour, and then treated with an ultrasonic washing machine 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, 0.5 ml of Img / ml proteinase K (Protease K) was added, and the mixture was incubated at 37 ° C for 1 hour. Add 1 ml of a mixture of phenol, chloroform, and isoamyl alcohol (25: 24: 1), shake, and remove the upper layer by centrifugation at 1500 g for 10 minutes. Add water-saturated phenol to this. After adding 1 ml and shaking, 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. overnight. This was centrifuged at 1500 g for 10 minutes to obtain a precipitate, and the 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 dried. , Stored at -20 ° C.

 This nucleic acid was converted into type III, and the nucleic acid 1 and nucleic acid 6 obtained in Example 1 were used as primers using TaKaRa PGR Amplification Kit (R011) of Takara Shuzo Co., Ltd., and a DNA amplification device of Sanyo (MIR-D30) was used. ) Was used to perform a PCR reaction in the following manner 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 synthetase (reagent kit Bok) 1 I ⁽² .5U)

Nucleic acid 1 1 1

Nucleic acid 6 1 ^ 1

Mitochondrial DNA \ μ I

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

Polymerization reaction: 72 ° C, 2 minutes Number of cycles: 30

 The amplified DNA was detected by electrophoresis (Fig. 1, lanes 8, 9). When the length of the detected nucleic acid fragment was calculated, it was identified as about 255 base pairs.

Example 3 (PCR identification of Candida glabra)

 In the same manner as in Example 2, using nucleic acid 2 and nucleic acid 7 obtained in Example 1, a portion of the cytochrome b gene was amplified from Candida glabrata (Candida glabrata IFM 5768 and 46843) by PCR, The amplified DNA was detected by electrophoresis (Fig. 1, lanes 10 and 11). When the length of the detected nucleic acid fragment was calculated, it was identified as about 205 base pairs.

Example 4 (Identification of Candida parapsilosis by PCR)

 In the same manner as in Example 2, a portion of the cytochrome b gene was amplified by PCR from Candida parapsilosis (Candida parapsilosis IFM 5464 and 46829) using nucleic acid 3 and nucleic acid 8 obtained in Example 1. Then, the amplified DNA was detected by electrophoresis (Fig. 1, lanes 4, 5). When the length of the detected nucleic acid fragment was calculated, it was identified as about 355 base pairs.

Example 5 (Identification of Candida 'tropicalis by PCR)

 In the same manner as in Example 2, a part of the cytochrome b gene was amplified by PCR from Candida tropical is (Candida tropical is IFM 46816 and 48776) using nucleic acid 4 and nucleic acid 9 obtained in Example 1. The amplified DNA was detected by electrophoresis (Figure 1, lanes 2, 3). When the length of the detected nucleic acid fragment was calculated, it was identified as about 405 base pairs.

Example 6 Identification of Candida albicans by PCR

 In the same manner as in Example 2, a part of the cytochrome b gene was subjected to PCR from Candida dubl iniensis (Candida dubl iniensis IFM 48184 and 48313) using nucleic acid 4 and nucleic acid 9 obtained in Example 1. The amplified DNA was detected in the reaction, and the amplified DNA was detected by electrophoresis (Fig. 1, lane 6, f). When the length of the detected nucleic acid fragment was calculated, it was identified as about 305 base pairs.

Claims

The scope of the claims  1. The nucleotide sequence represented by SEQ ID NO: 1 in the sequence listing (though thymine at any position may be substituted with peracyl) or its complementary nucleotide sequence, at least 15 nucleotides continuous from the 3 'end Or a nucleic acid for detecting Candida albicans having a nucleotide sequence that hybridizes with said nucleotide sequence under stringent conditions.  2. It has a base sequence consisting of at least 15 bases continuous from the 3 'end in the base sequence represented by SEQ ID NO: 1 in the sequence listing (though thymine at any position may be substituted with peracyl) The nucleic acid according to claim 1. 3. The nucleic acid according to claim 2, which has a base sequence represented by SEQ ID NO: 1 in the sequence listing (provided that thymine at any position may be substituted with peracyl).  4. The nucleic acid according to claim 2 or 3, which is a foreside primer in a nucleic acid amplification method.  5. The nucleotide sequence represented by SEQ ID NO: 2 in the sequence listing (though thymine at any position may be substituted with peracyl) or its complementary nucleotide sequence, at least 15 nucleotides continuous from the 3 'end Or a nucleic acid for Candida glabrata detection having a nucleotide sequence that hybridizes with the nucleotide sequence under stringent conditions.  6. It has a base sequence consisting of at least 15 bases continuous from the 3 'end in the base sequence represented by SEQ ID NO: 2 in the sequence listing (though thymine at any position may be substituted with peracyl) The nucleic acid according to claim 5.  7. The nucleic acid according to claim 6, which has a base sequence represented by SEQ ID NO: 2 in the sequence listing.  8. The nucleic acid according to claim 6 or 7, which is a forward primer in a nucleic acid amplification method. 9. At least 15 bases consecutive from the 3 'end in the base sequence represented by SEQ ID NO: 3 in the sequence listing (though thymine at any position may be substituted with peracyl) or its complementary base sequence Or a Candida parapsilo having a base sequence that hybridizes with the base sequence under stringent conditions. Cis detection nucleic acid.  10. It has a base sequence consisting of at least 15 bases continuous from the 3 'end in the base sequence represented by SEQ ID NO: 3 in the sequence listing (though thymine at any position may be substituted with peracyl) The nucleic acid according to claim 9. 11. The nucleic acid according to claim 10, which has a base sequence represented by SEQ ID NO: 3 in the sequence listing.  12. The nucleic acid according to claim 10 or 11, which is a forward primer in a nucleic acid amplification method.  1 3. The nucleotide sequence represented by SEQ ID NO: 4 in the sequence listing (though the thymine at any position may be replaced with peracyl) or at least one of the nucleotides complementary to the 3 'terminal in its complementary nucleotide sequence A nucleic acid for detecting Candida tropicalis having a base sequence consisting of 5 bases or a base sequence that hybridizes with the base sequence under stringent conditions.  4. A base sequence comprising at least 15 bases continuous from the 3 'end in the base sequence represented by SEQ ID NO: 4 in the sequence listing (though thymine at any position may be substituted with peracyl) Item 13. The nucleic acid according to Item 13.  15. The nucleic acid according to claim 14, which has a base sequence represented by SEQ ID NO: 4 in the sequence listing.  16. The nucleic acid according to claim 14 or 15, which is a forward primer in a nucleic acid amplification method.  17. The nucleotide sequence shown by SEQ ID NO: 5 in the sequence listing (though the thymine at any position may be replaced with peracyl) or at least one continuous nucleotide from the 3 'terminal in its complementary nucleotide sequence A nucleic acid for detecting Candida doublingensis having a base sequence consisting of 5 bases or a base sequence that hybridizes with the base sequence under stringent conditions.  18. It has a base sequence consisting of at least 15 bases continuous from the 3 'end in the base sequence represented by SEQ ID NO: 5 in the sequence list (though thymine at any position may be substituted with peracyl) A nucleic acid according to claim 17.  19. The nucleic acid according to claim 18, which has a base sequence represented by SEQ ID NO: 5 in the sequence listing. 20. The claim 18 or 19 which is a forward primer in a nucleic acid amplification method. A nucleic acid as described.  21. The nucleotide sequence represented by SEQ ID NO: 6 in the sequence listing (though the thymine at any position may be replaced with peracyl) or at least one nucleotide from the 3 'terminal in its complementary nucleotide sequence A nucleic acid for detecting Candida albicans having a base sequence consisting of 5 bases or a base sequence that hybridizes with the base sequence under stringent conditions.  22. Has a nucleotide sequence consisting of at least 15 nucleotides continuous from the 3 'end in the nucleotide sequence shown by SEQ ID NO: 6 in the sequence listing (though thymine at any position may be substituted with peracyl) The nucleic acid according to claim 21. 23. The nucleic acid according to claim 22, having a base sequence represented by SEQ ID NO: 6 in the sequence listing (though thymine at any position may be substituted with peracyl). 24. The nucleic acid according to claim 22 or 23, which is a reverse-side primer in a nucleic acid amplification method.  25. The base sequence represented by SEQ ID NO: 7 in the sequence listing (though thymine at any position may be substituted with peracyl) or at least one continuous sequence from the 3 'terminal in its complementary base sequence A nucleic acid for detecting Candida glabra having a base sequence consisting of 5 bases or a base sequence that hybridizes with the base sequence under stringent conditions.  26. Has a nucleotide sequence consisting of at least 15 nucleotides continuous from the 3 'end in the nucleotide sequence shown by SEQ ID NO: 7 in the Sequence Listing (though thymine at any position may be substituted with peracyl) 26. The nucleic acid according to claim 25.  27. The nucleic acid according to claim 26, having the base sequence of SEQ ID NO: 7 in the sequence listing. 28. The nucleic acid according to claim 26 or 27, which is a reverse primer in a nucleic acid amplification method. 29. The base sequence represented by SEQ ID NO: 8 in the sequence listing (though thymine at any position may be substituted with peracyl) or its complementary base sequence, at least one of which is continuous from the 3 'terminal. Candida parapsilo having a base sequence consisting of 5 bases or a base sequence that hybridizes with the base sequence under stringent conditions —Nucleic acids for cis detection.  30. It has a nucleotide sequence consisting of at least 15 nucleotides continuous from the 3 'end in the nucleotide sequence represented by SEQ ID NO: 8 in the sequence listing (though thymine at any position may be substituted with peracyl) 29. The nucleic acid according to claim 29. 31. The nucleic acid according to claim 30, having the base sequence of SEQ ID NO: 8 in the sequence listing. 32. The nucleic acid according to claim 30, which is a reverse primer in a nucleic acid amplification method.  33. The nucleotide sequence represented by SEQ ID NO: 9 in the sequence listing (though thymine at any position may be substituted with peracyl) or its complementary nucleotide sequence, at least one of which is continuous from the 3 'terminal. A nucleic acid for detecting Candida tropicalis having a base sequence consisting of 5 bases or a base sequence that hybridizes with the base sequence under stringent conditions.  34. Has a nucleotide sequence consisting of at least 15 nucleotides continuous from the 3 'end in the nucleotide sequence shown by SEQ ID NO: 9 in the sequence listing (though thymine at any position may be substituted with peracyl) 34. The nucleic acid according to claim 33. ·  35. The nucleic acid according to claim 34, having the base sequence of SEQ ID NO: 9 in the sequence listing. 36. The nucleic acid according to claim 34 or 35, which is a forward primer in a nucleic acid amplification method.  37. The base sequence represented by SEQ ID NO: 10 in the sequence listing (though thymine at any position may be replaced with peracyl) or its complementary base sequence at least at the 3 'end A base sequence consisting of 15 bases or a string with the base sequence; i: a nucleic acid for detection of Candida albicans having a base sequence that hybridizes under simple conditions.  38. A base sequence consisting of at least 15 bases continuous from the 3 'end in the base sequence represented by SEQ ID NO: 10 in the sequence listing (thymine at any position may be substituted with peracyl) The nucleic acid according to claim 3, comprising: 39. The nucleic acid according to claim 38, which has a base sequence represented by SEQ ID NO: 10 in the sequence listing. 40. The nucleic acid according to claim 38 or 39, which is a reverse-side primer in a nucleic acid amplification method.  41. A method for detecting Candida albicans using the nucleic acid for detecting Candida albicans according to any one of claims 1 to 4 and 21 to 24 as a primer or a probe.  42. A method for detecting Candida * glabrata, wherein the nucleic acid for detecting Candida glabrata according to any one of claims 5 to 8 and 25 to 28 is used as a primer or a probe. 3. A method for detecting Candida and parapsilosis, wherein the nucleic acid for detecting Candida parapsilosis according to any one of claims 9 to 12 and 29 to 32 is used as a primer or a probe.  44. A method for detecting Candida tropicalis, wherein the nucleic acid for detecting Candida tropicalis according to any one of claims 13 to 16 and claim 33 to 36 is used as a primer or a probe. 45. Detection of Candida doubliniensis using the nucleic acid for detecting Candida doubuliniensis according to any one of claims 17 to 20 and 37 to 40 as a primer or a probe Method.  46. A method for detecting C. albicans, comprising performing a nucleic acid amplification method using the forward primer according to claim 4 and the reverse primer according to claim 24, and detecting an amplification product.  47. A method for detecting a signal labler, comprising performing a nucleic acid amplification method using the forward primer according to claim 8 and the reverse primer according to claim 28, and detecting an amplification product.  48. A method for detecting Candida parapsilosis, comprising performing a nucleic acid amplification method using the forward primer according to claim 12 and the reverse primer according to claim 32 to detect an amplification product. . 49. The method includes performing a nucleic acid amplification method using the forward primer according to claim 16 and the reverse primer according to claim 36, and detecting an amplification product. Detection method for Candida and tropicalis.  50. A Candida dough, which comprises performing a nucleic acid amplification method using the foreside primer according to claim 20 and the re / side side primer according to claim 40 to detect an amplification product. How to detect Blinensis. 51. The reagent kit for detecting Candida albicans comprising the nucleic acid for detecting Candida albicans according to any one of claims 1 to 4 and 21 to 24.  52. A reagent kit for detecting Candida's gravrata, comprising the nucleic acid for detecting Candida and the glabrata according to any one of claims 5 to 8 and 25 to 28.  53. A reagent kit for detecting forceps parapsilosis comprising the nucleic acid for detecting forceps parapsilosis according to any one of claims 9 to 12 and claims 29 to 32.  54. A reagent kit for detecting Candida's tropicalis comprising the nucleic acid for detecting Candida's tropicalis according to any one of claims 13 to 16 and 33 to 36.  55. A reagent kit for detecting Candida's doubriniensis comprising the nucleic acid for detecting Candida's doubriniensis according to any one of claims 17 to 20 and 37 to 40. 56. A labeled probe obtained by labeling the nucleic acid according to any one of claims 1 to 40.  57. A capture probe comprising the nucleic acid according to any one of claims 1 to 40 bound to a carrier.  58. Use of the nucleic acid according to any one of claims 1 to 40 for producing a probe or a primer.