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WO2002040713A1 - Acides nucleiques permettant de detecter des champignons appartenant au genre candida et methode permettant de detection des champignons appartenant au genre candida a l'aide de ces acides nucleiques - Google Patents

Acides nucleiques permettant de detecter des champignons appartenant au genre candida et methode permettant de detection des champignons appartenant au genre candida a l'aide de ces acides nucleiques Download PDF

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WO2002040713A1
WO2002040713A1 PCT/JP2001/009938 JP0109938W WO0240713A1 WO 2002040713 A1 WO2002040713 A1 WO 2002040713A1 JP 0109938 W JP0109938 W JP 0109938W WO 0240713 A1 WO0240713 A1 WO 0240713A1
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nucleic acid
base sequence
seq
candida
detecting
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Japanese (ja)
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Koji Yokoyama
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SSP Co Ltd
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SSP Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
    • C07K14/40—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts from Candida
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae

Definitions

  • 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.
  • the main pathogens of deep mycosis include Candida (hereinafter abbreviated as G.) genus fungi, Aspergilus (hereinafter abbreviated as ⁇ ⁇ ) genus fungi, and Cryptococcus (hereinafter abbreviated as Cr.).
  • G. genus fungi
  • ⁇ ⁇ genus fungi
  • Cr. Cryptococcus
  • 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.
  • 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.
  • 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.
  • the present inventors found specific sequences for each type of Candida fungus and designed primers specific to each species based on the sequences. .
  • primers specific to Candida albicans SEQ ID NOs: 1 and 6 in the Sequence Listing
  • primers specific to Candida glabrata SEQ ID NOs: 2 and 7, as primers specific to Candida parapsilosis SEQ ID Nos.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the present invention provides the above-mentioned method for detecting a Candida fungus using each of the nucleic acids for detection.
  • 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.
  • 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.
  • 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)
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 ).
  • the kit is provided with a 10-fold concentration of X10 PGR buffer (or 10X PGR buffer), which is used after dilution).
  • X10 PGR buffer or 10X PGR buffer
  • 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
  • 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.
  • a nucleic acid having the nucleotide sequence of SEQ ID NO: 1 including the above-described portions and variants thereof.
  • SEQ ID NO: 1 May be simply referred to as “SEQ ID NO: 1” for convenience)
  • SEQ ID NO: 6 including the above-described portions and variants
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • SEQ ID NO: 2 (including the above-mentioned portions and variants) is used as the forward primer
  • SEQ ID NO: 7 including the above-mentioned portions and variants
  • SEQ ID NO: 2 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.
  • 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.
  • the base sequence described above may include substitution, deletion, insertion or addition of a base group.
  • 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.
  • 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.
  • SEQ ID NO: 3 (including the above-mentioned portions and variants) is used as a forward primer
  • SEQ ID NO: 8 is used as a reverse primer. It is preferable to use them.
  • SEQ ID NO: 3 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • SEQ ID NO: 4 (including the above-mentioned portions and variants) is used as the forward primer
  • SEQ ID NO: 9 including the above-described portions and variants
  • SEQ ID NO: 4 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • SEQ ID NO: 5 (including the above-mentioned portions and variants) is used as the forward primer
  • SEQ ID NO: 10 including the above-described portions and variants
  • SEQ ID NO: 5 was used as the forward primer
  • SEQ ID NO: 10 was used as the reverse primer
  • the mitochondrial cytochrome b gene of Candida doubriniensis was present in the test sample.
  • 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.
  • 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.
  • the nucleic acid of the present invention 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the reagent kit 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.
  • 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.
  • nucleic acid synthases eg, DNA polymerase, RNA polymerase, reverse transcriptase, etc.
  • dATP xyliponucle
  • the nucleic acid of the present invention can be bound to a solid phase carrier and used as a capture probe.
  • a sandwich assay may be performed using a combination of a capture probe and a labeled probe.
  • 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.
  • nucleic acid having a sequence represented by SEQ ID NOS: 10 to 10 in the sequence listing was chemically synthesized.
  • 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
  • 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.
  • 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.
  • the total volume was made up to 50 I with water.
  • the reaction conditions were as follows.
  • 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 2 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 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 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 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.

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Abstract

L'invention concerne des acides nucléiques permettant de détecter selon l'espèce cinq champignons appartenant au genre Candida (c'est-à-dire, C. albicans, C. glabrata, C. parapsilosis, C. tropicalis et C. dubliniensis), parmi les champignons responsables de mycoses profondes. Les acides nucléiques permettant de détecter les C. albicans présentent au moins 15 bases consécutives provenant du 3'-terminal d'une séquence de base représentée par SEQ ID NO:1 ou 6; les acides nucléiques permettant de détecter les C. glabrata présentent au moins 15 bases consécutives provenant du 3'-terminal d'une séquence de base représentée par SEQ ID NO:2 ou 7; les acides nucléiques permettant de détecter les C. parapsilosis présentent au moins 15 bases consécutives provenant du 3'-terminal d'une séquence de base représentée par SEQ ID NO:3 ou 8; les acides nucléiques permettant de détecter les C. tropicalis présentent au moins 15 bases consécutives provenant du 3'-terminal d'une séquence de base représentée par SEQ ID NO:4 ou 9; les acides nucléiques permettant de détecter les C. dubliniensis présentent au moins 15 bases consécutives provenant du 3'-terminal d'une séquence de base représentée par SEQ ID NO:5 ou 10.
PCT/JP2001/009938 2000-11-14 2001-11-14 Acides nucleiques permettant de detecter des champignons appartenant au genre candida et methode permettant de detection des champignons appartenant au genre candida a l'aide de ces acides nucleiques Ceased WO2002040713A1 (fr)

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JP2000-347184 2000-11-14
JP2000347184A JP2002142775A (ja) 2000-11-14 2000-11-14 カンジダ属真菌検出用核酸及びそれを用いたカンジダ属真菌の検出方法

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AU2006292174B2 (en) * 2005-09-20 2012-02-23 Advandx, Inc. Reagents, methods and kits for classification of fungi and direction of anti-fungal therapy
AU2009256585B2 (en) * 2008-06-13 2015-02-19 National University Of Ireland, Galway EIF2 gamma gene as a diagnostic target for the identification of fungal and yeast species
IES20090467A2 (en) * 2008-06-13 2010-03-31 Nat Univ Ireland Ace2 as a target gene for the molecular identification of yeast and fungal species.
IES20090464A2 (en) * 2008-06-13 2010-03-03 Nat Univ Ireland SWI5 gene as a diagnostic target for the identification of fungal and yeast species

Non-Patent Citations (3)

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Title
KOJI YOKOYAMA ET AL.: "Identification and phylogenetic relationship of the most common pathogenic candida species inferred from mitochondrial cytochrome b gene sequences", JOURNAL OF CLINICAL MICROBIOLOGY, vol. 38, no. 12, December 2000 (2000-12-01), pages 4503 - 4510, XP002908211 *
LI WANG ET AL.: "Mitochondrial cytochrome b gene analysis of aspergillus fumigatus and related species", JOURNAL OF CLINICAL MICROBIOLOGY, vol. 38, no. 4, April 2000 (2000-04-01), pages 1352 - 1358, XP002908210 *
SWARAJIT KUMAR BISWAS ET AL.: "Identification of candida dubliniensis based on the specific amplification of mitochondrial cytochrome b gene", JPN. J. MED. MYCOL., vol. 42, no. 2, April 2001 (2001-04-01), pages 95 - 98, XP002908212 *

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