JP5568233B2 - Mold detection probe, mold detection DNA chip, mold detection method, mold life / death discrimination method, and mold identification method - Google Patents

Description

  The present invention relates to a mold detection probe, a mold detection DNA chip, a mold detection method, a mold life / death discrimination method, and a mold identification method.

  The following methods are known as conventional techniques for (I) life / death determination and (II) mold type identification for molds present indoors and in food.

(I) Life / death determination (a) Culture method The survival rate is determined by culturing, and life / death determination is performed. Specifically, a method for visually determining the growth of colonies by adjusting fungi collected from a specimen by appropriate means to a predetermined number of bacteria and then culturing for about several days to one week using an agar medium or the like.
Alternatively, after inoculating the collected fungi in a chromogenic medium mixed with chromogenic enzyme, it is cultured for several days to one week in an incubator such as an incubator maintained at a predetermined temperature, and the chromogenic enzyme is decomposed with β-galactosidase enzyme. A method of color development and interpretation.
Either method requires several days to about one week of culture.

(B) Rapid method 1) A fluorescent staining reagent, for example, acridine orange, stains and emits light if the detection target microorganism is dead, and does not emit light because it is discharged outside the bacteria if it is alive. Can be used to distinguish between life and death. Some reagents, such as CFDA, stain live bacteria. Also, as in the fluorescein diacetate (FDA) / ethidium bromide (EB) staining method, live bacteria decompose FDA due to esterase activity and emit green fluorescence, and dead bacteria have no esterase activity. Not seen, stained with EB and emitting orange fluorescence. In addition, a method using a fluorescent staining reagent composed of fluorescein or a derivative thereof and propidium iodide has been proposed (see Patent Document 1). Furthermore, a method for measuring a fluorescent staining reagent by irradiating a pulsed beam (see Patent Document 2) has also been proposed.

2) Determination from RNA / DNA ratio (see Patent Document 3)
According to this method, cell viability is discriminated based on the magnitude of the RNA / DNA value determined by measurement of quantitative reverse transcription PCR (quantitative RT-PCR) of RNA extracted from a specimen and quantitative PCR of DNA.

(II) Identification of mold species (a) Culture method In a closed container (such as a desiccator with an inner diameter of 20 cm or more) in which a salt saturated solution such as distilled water or barium chloride (relative humidity 90% / 25 ° C) is placed at the bottom, This is a method in which a sample such as food is put, covered, and cultured in an incubator at 20 ° C. or 25 ° C. Observe daily to examine the type, number and growth status of the fungus that has grown.
Furthermore, after adjusting the number of fungi collected from the specimen appropriately by means, it is cultured for several days to one week using an agar medium or the like, and colony growth is visually determined.

(B) Rapid inspection method It has been pointed out that mold inspection by culturing requires several days to one week of culturing and is not suitable for quality control that requires rapidity. In addition, direct microscopic analysis from mold-contaminated foods is limited to foods that can be applied and is not suitable for samples with low amounts of contaminating bacteria, so mold inherent components, metabolic activity, immunological specificity, electrical Attempts to quantitatively or qualitatively analyze the presence of fungi in a short time by detecting these characteristics and genes have been developed, and rapid test methods exemplified below have been developed. Some of them are commercially available as test kits, but their use conditions are limited, so it is necessary to use them in consideration of their characteristics.

1) Measuring method of β-glucan By measuring the amount of (1,3) -β-D-glucan in a mold sample by a colorimetric method or a turbidimetric method, a method for estimating the amount of fungi (mold, yeast). is there. Detection kits and analyzers are commercially available (for example, “β-glucan test Wako” manufactured by Wako Pure Chemical Industries, Ltd. and “Toxinometer MT-5500”).

2) Chitin analysis method In this method, glucosamine or chitosan in the mold cell membrane is hydrolyzed, then measured by high performance liquid chromatography or gas chromatography, and the mold contamination level is estimated from the value.

3) ATP measurement method This is a method for detecting the presence of microorganisms by measuring microorganism-derived ATP present in food. Kits using the fluorescent substance luciferin as an index have been applied to the measurement of bacteria and mold. It is necessary to use it in consideration that it is difficult to distinguish it from ATP derived from biological components and bacteria. Detection kits and analyzers are commercially available (for example, “Unilite” manufactured by Biotraces, UK, “Clean Trace”, “Lumitester C-110”, “Lucifer Series” manufactured by Kikkoman).

4) Immunological methods Analysis by fluorescent antibody method or enzyme antibody method has been attempted. At the time when the standardization of these substances was studied in Japan, the reliability of the measurement method using antigen-antibody reaction was low and the value of instrumental analysis was absolute. It became possible to produce a large quantity with high purity. However, since standardization has not been studied, it is difficult to set as a standard method.
An immunological technique (ELISA method rapid quantification kit) is commercially available for mold venom.

5) DNA chip for fungal detection A DNA chip (also called a DNA microarray) has a large number of DNA fragments arranged at high density on a substrate such as plastic or glass in order to measure the gene expression level in cells. Analytical instrument. By using this DNA chip, it is possible to examine tens to tens of thousands of gene expressions at once. For example, although the number of human genes is said to be 30,000 to 40,000, all these gene fragments can be fixed on a single substrate, and a gene fragment called a probe and a cell called a target The amount of gene expression in a cell can be measured by contacting and hybridizing with a messenger RNA (mRNA) extracted from the above and labeled with a labeling compound.
The conventional method for designing a probe for a DNA chip used for mold detection is to design a probe using information on a base sequence derived from or including a spacer region (ITS) of a mold ribosome, A method for identifying the type of mold using it has been proposed. For example, Patent Document 4 describes that the nucleotide sequence of the ITS region is used as a nucleic acid probe used in a microarray for detecting and identifying mites and molds that cause house dust allergy. In addition, it is described that the base sequence of the ITS region is used as a probe for detecting a microarray used in a mold detection method. In addition, the pharmacopeia of Japan recommends testing the base sequence of ITS.
JP-A-9-275998 Japanese Patent Laid-Open No. 11-178568 JP 2001-299399 A JP 2008-35773 A Japanese Patent Laid-Open No. 2008-5760

However, the above-described conventional techniques have the following problems.
(I) About (a) culture method which is a prior art of life-and-death determination, since about 1 week and time are required for culture, rapid determination cannot be made. It is not suitable for tests that require quickness such as indoors where people live and foods that people eat.

  (I) (b) Rapid method 1) which is a conventional technique for life / death determination The method using a fluorescent staining reagent is quick in measuring life / death discrimination using each reagent. Because it requires high technology, it is not an easy inspection method. This method is difficult for non-experts to carry out, and it is difficult to obtain quantitativeness and reproducibility. For example, in an extreme case, live bacteria that should not fluoresce with acridine orange may fluoresce.

  (I) (b) Rapid method 2), which is a conventional technique for life / death determination, is based on the RNA / DNA ratio method. In order to amplify each of RNA and DNA from a specimen by the PCR method, it takes about 2 days and special time. Requires equipment, high expertise. At least quantification of both RNA and DNA is required, and it is difficult to say that it is a simple test method.

(II) The (a) culture method for identifying mold species has a problem of lack of rapidity. It has been pointed out that mold inspection by culture takes several days to one week, and is not suitable for quality control that requires rapidity.
In addition, direct spectroscopic methods from mold-contaminated foods are difficult to apply to samples with limited applicable foods and a small amount of contaminating bacteria, and require high expertise. Furthermore, the identification of mold requires experience and expertise.

(II) Among the (b) rapid test methods for the identification of mold species, 1) β-glucan measurement methods include commercially available kits, which are easy to measure. However, this method can estimate the total amount of mold, but cannot identify the mold species.
In addition, (b) Among the rapid test methods, 2) chitin analysis methods use high-performance liquid chromatography or gas chromatography, and therefore require special expertise and pre-treatment. I can not say. Although this method can estimate the total amount of mold, it cannot identify mold species.
In addition, (b) among the rapid test methods, 3) ATP measurement methods are commercially available kits and are simple, but it is difficult to distinguish mold from other microorganisms, and it is possible to estimate the total amount of microorganisms However, mold species cannot be identified.
In addition, among (b) rapid test methods, 4) immunological methods are available for commercial molds for detection of mold toxins, but are commercialized for mold detection and mold species identification. Not.

(B) Among the rapid testing methods, 5) the DNA chip for fungal detection can quickly and easily identify the presence or absence of mold in the specimen and the type of the mold, and even without advanced specialized knowledge and technology Accurate discrimination is possible.
However, in the prior art disclosed in Patent Documents 4 and 5, when designing a probe for a DNA chip, it is derived from a region containing or containing a mold ribosome spacer region (ITS). The probe is designed using the information of the base sequence to be processed. At present, the amount of base sequence data in the ITS region is small, so that there is a problem that it is difficult to design and produce a mold detection probe.
Moreover, in the prior arts disclosed in Patent Documents 4 and 5, since the base sequence of the probe to be used requires about 40 to 50 bases, it is difficult to produce a probe or a DNA chip. There's a problem.
Furthermore, in the hybridization using a probe having a base sequence derived from ITS disclosed in Patent Documents 4 and 5, cross-hybridization may occur, and several probes are often required for the same species. There is a problem that the measurement accuracy of mold species identification using a DNA chip cannot be obtained sufficiently.
Further, the base sequences of the probes disclosed in Patent Documents 4 and 5 are derived from ITS of mold ribosomes. Since this ITS is not involved in protein synthesis via mRNA specific to live bacteria, it is unsuitable for determining the viability of fungi in a specimen based on the RNA amount of the specimen.

  The present invention has been made in view of the above-described circumstances, and can determine whether or not mold is present indoors or in food and identify mold species individually or simultaneously, and with high accuracy by a simple measurement method. An object is to provide a DNA chip that can be discriminated and identified.

  In order to achieve the above object, the present invention provides a mold detecting probe comprising any one base sequence selected from the group consisting of the base sequences of SEQ ID NOs: 1 to 34 or substantially the same base sequences. I will provide a.

The present invention also provides
(1) A mold detection probe (A) comprising the base sequence of SEQ ID NO: 1 or a base sequence substantially the same as that for detection of Absidia coerulea,
(2) Mold detection probe (A) comprising the base sequence of SEQ ID NO: 2 or substantially the same base sequence as a target for detection of Absidia corymbifera,
(3) A fungus detection probe (c) consisting of the base sequence of SEQ ID NO: 3 or a base sequence substantially the same as that to detect Absidia glauca, and the base sequence of SEQ ID NO: 4 or substantially the same A mold detecting probe comprising the same base sequence (D),
(4) A fungus detection probe (e) consisting of the base sequence of SEQ ID NO: 5 or substantially the same base sequence as that for detection of Absidia repens, and the base sequence of SEQ ID NO: 6 or substantially the same Mold detection probe (mosquito) consisting of the same base sequence,
(5) Alternaria alternata (Alternaria alternata) detection target mold detection probe consisting of the base sequence of SEQ ID NO: 7 or substantially the same base sequence (ki),
(6) A mold detection probe comprising a base sequence of SEQ ID NO: 8 or a base sequence substantially the same as that to detect Aspergillus nidulans;
(7) A fungus detection probe consisting of a base sequence of SEQ ID NO: 9 or a base sequence substantially the same as that to detect Aspergillus niger, a base sequence of SEQ ID NO: 10 or substantially the same A mold detection probe (co) comprising the same base sequence, a base sequence of SEQ ID NO: 11 or a mold detection probe (sa) comprising substantially the same base sequence, a base sequence of SEQ ID NO: 12 or substantially the same A fungus detection probe consisting of the same base sequence (si), and a fungus detection probe consisting of the base sequence of SEQ ID NO: 13 or substantially the same base sequence (s);
(8) A mold detection probe (C) comprising the base sequence of SEQ ID NO: 14 or substantially the same base sequence as that for detection of Aspergillus versicolor;
(9) Mold detection probe (So) consisting of the nucleotide sequence of SEQ ID NO: 15 or a nucleotide sequence substantially the same as Candida albicans to be detected;
(10) Mold detection probe (ta) consisting of the base sequence of SEQ ID NO: 16 or substantially the same base sequence as that for detection of Cladosporium cladosporioides, the base sequence of SEQ ID NO: 17 or A fungus detection probe consisting of substantially the same base sequence (H), and a fungus detection probe consisting of the base sequence of SEQ ID NO: 18 or substantially the same base sequence (tu),
(11) For mold detection comprising the base sequence of SEQ ID NO: 19 which is a detection target of Cladosporium cladosporioides and Cladosporium sphaerospermum or a base sequence substantially identical thereto A probe (G), and a fungus detection probe (G) consisting of the nucleotide sequence of SEQ ID NO: 20 or substantially the same nucleotide sequence thereof,
(12) Mold detection probe (na) consisting of the base sequence of SEQ ID NO: 21 or substantially the same base sequence as that for detection of Cladosporium langeronii, and the base sequence of SEQ ID NO: 22 or Mold detection probe consisting of substantially the same base sequence (D),
(13) A fungus detection probe (nu) comprising the nucleotide sequence of SEQ ID NO: 23 or substantially the same nucleotide sequence as that for detection of Eurotium repens,
(14) a fungus detection probe (ne) comprising the base sequence of SEQ ID NO: 24 or a base sequence substantially the same as that to be detected as Fusarium culmorum;
(15) A fungus detection probe comprising a base sequence of SEQ ID NO: 25 or a base sequence substantially the same as that to detect Fusarium oxysporum (no),
(16) A mold detection probe (c) comprising the base sequence of SEQ ID NO: 26 or substantially the same base sequence thereof as a detection target for Mucor racemosus,
(17) A fungus detection probe (h) consisting of the base sequence of SEQ ID NO: 27 or a base sequence substantially the same as that to detect Paecilomyces tenuipes,
(18) Mold detection probe (F) comprising the base sequence of SEQ ID NO: 28 or a base sequence substantially the same as that to be detected in the genus Forma (Phoma),
(19) Mold detection probe (F) comprising the nucleotide sequence of SEQ ID NO: 29 or a nucleotide sequence substantially the same as that to be detected as a genus Rhizopus,
(20) A fungus detection probe (e) consisting of the base sequence of SEQ ID NO: 30 or substantially the same base sequence as that for detection of Stachybotrys chartarum, and the base sequence of SEQ ID NO: 31 or substantially the same Mold detection probe (ma) consisting of the same base sequence,
(21) a fungus detection probe (mi) comprising the nucleotide sequence of SEQ ID NO: 32 or a nucleotide sequence substantially identical to the nucleotide sequence of SEQ ID NO: 32 for detecting Trichoderma viride;
(22) A fungus detection probe (mu) comprising the base sequence of SEQ ID NO: 33 or substantially the same base sequence as that targeted for detection of the genus Wallemia, and the base sequence of SEQ ID NO: 34 or substantially the same Mold detection probe (me) consisting of the same base sequence,
A mold detecting probe selected from the group consisting of:

  The present invention also provides a mold detecting DNA chip in which one or more of the aforementioned mold detecting probes according to the present invention are fixed to a substrate.

  The present invention also provides a mold detecting DNA chip in which two or more of the mold detecting probes (a) to (me) described above are fixed in a mixed state on a substrate.

  In addition, the present invention provides mold detection in which two or more of the aforementioned mold detection probes (a) to (me) are fixed to a substrate in a state where the mold detection probes are separated from each other and arranged. A DNA chip for use is provided.

  The mold detecting DNA chip of the present invention may have a cascade structure in which the same or different mold detecting probe is connected to the tip of the mold detecting probe having one end fixed to the substrate.

The present invention also uses the above-described DNA chip for mold detection according to the present invention,
Producing a detection target labeled with a labeling compound from a nucleic acid in a test sample;
Next, the step of hybridizing the detection target to the mold detection probe of the mold detection DNA chip;
Next, whether or not the mold to be detected by the mold detection probe is present in the test sample by washing the mold detection DNA chip and detecting the labeled compound on the washed DNA chip. The present invention provides a method for detecting molds comprising the step of examining

The present invention also uses the above-described DNA chip for mold detection according to the present invention,
Producing a detection target labeled with a labeling compound from a nucleic acid in a test sample;
Next, the step of hybridizing the detection target to the mold detection probe of the mold detection DNA chip;
Next, the mold-detecting DNA chip is washed, and the labeled compound is detected from the washed DNA chip. When the detected amount of the labeled compound relative to the mold cell is larger than the reference value, When the mold present in the sample is alive and the detected amount of the labeled compound relative to the amount of mold is less than the reference value, the mold is alive or dead having a step of determining that the mold present in the sample to be tested is dead Provide a method of discrimination.

The present invention also uses the above-described DNA chip for mold detection according to the present invention,
Producing a detection target labeled with a labeling compound from a nucleic acid in a test sample;
Hybridizing the detection target to the mold detection probe of the mold detection DNA chip;
Next, the mold detecting DNA chip is washed, and the labeled compound is detected on the washed DNA chip, so that mold in the sample to be inspected is fixed on the DNA chip where the labeled compound is detected. Provided is a mold identification method comprising the step of identifying a mold species as a mold to be detected by a mold detection probe.

The present invention also uses the above-described DNA chip for mold detection according to the present invention,
Producing a detection target labeled with a labeling compound from a nucleic acid in a test sample;
Hybridizing the detection target to the mold detection probe of the mold detection DNA chip;
Next, the mold detecting DNA chip is washed, and the labeled compound is detected on the washed DNA chip, so that mold in the sample to be inspected is fixed on the DNA chip where the labeled compound is detected. The mold detection probe identifies the mold species as the mold to be detected, and
When the detected amount of the labeled compound is larger than the reference value, mold present in the sample to be inspected is alive, and when the detected amount of the labeled compound is smaller than the reference value, Provided is a method for detecting mold, which comprises a step of discriminating that an existing mold is dead.

The fungus detection probe according to the present invention comprises the nucleotide sequence of SEQ ID NOs: 1 to 34 derived from the 18S rDNA nucleotide sequence of mold ribosome in which data such as nucleotide sequences are relatively abundant, or a nucleotide sequence substantially the same as that Since it has any one base sequence selected from the group consisting of sequences, it is possible to easily design and produce appropriate mold detection probes corresponding to various mold species.
The mold detection probe according to the present invention hybridizes with a high probability to a detection target in a test sample containing a mold nucleic acid to be detected, while having a relatively short base sequence of 30 or less bases. Therefore, the probe can be easily designed and manufactured, and a DNA chip having the probe fixed on the substrate can be easily manufactured. Furthermore, since it has a relatively short base sequence, various changes, for example, a multistage cascade structure in which one end of the same or different mold detection probe is connected to the tip of the first stage mold detection probe. Etc. can also be performed relatively easily.
In addition, since the mold detection probe according to the present invention hybridizes with a detection target in a test sample containing a mold nucleic acid to be detected with high probability, a cross-over occurs when hybridizing with the detection target. The probability of occurrence of hybrid is low, and mold can be detected with high accuracy.

Since the mold detecting DNA chip according to the present invention is the above-described mold detecting probe according to the present invention fixed on a substrate, the mold nucleic acid to be detected is a relatively short base sequence having 30 or fewer bases. Can be hybridized with a high probability with a target for detection in a test sample containing a DNA chip, which facilitates the manufacture of a DNA chip including probe design and production.
The mold detection DNA chip according to the present invention has a mold detection probe that hybridizes with a high probability to a detection target in a test sample containing a mold nucleic acid to be detected. When hybridizing, the probability of occurrence of cross-hybrid is low, and mold can be detected with high accuracy.
The mold chip for mold detection according to the present invention is obtained by fixing a probe having a base sequence derived from the base sequence of 18S rDNA of a mold ribosome to a substrate, and thus has a probe having a base sequence derived from ITS. Compared with the DNA chip, the variation in the amount of nucleic acid hybridized to the probe is increased due to the difference in the survival / killing of the mold to be detected. Therefore, it is possible to determine whether the mold to be detected is viable or not by examining the nucleic acid amount it can.
Further, in the mold chip for mold detection according to the present invention, two or more of the mold detection probes according to the present invention are fixed to the substrate in a state where the mold fixing probe fixing regions are separated and arranged. In this configuration, the mold species can be identified by examining which fixed region of the mold-derived nucleic acid hybridizes with the mold-derived nucleic acid present in the test sample. Since the probability of occurrence is low, mold species can be identified with high accuracy.
In addition, in the mold chip for mold detection according to the present invention, if the structure has a cascade structure in which the same or different mold detection probe is connected to the tip of the mold detection probe having one end fixed to the substrate, Molds can be detected with higher accuracy, and mutant and similar species that cannot be detected with one probe can be detected.

The mold detection method according to the present invention uses the aforementioned mold detection DNA chip according to the present invention, hybridizes a detection target and a probe in a labeled test sample, and hybridizes to the probe after washing. By detecting the detection target based on the label, a specific mold can be detected with higher accuracy than when a DNA chip having a conventional ITS-derived probe is used.
In addition, the mold viability determination method according to the present invention uses the above-described mold detection DNA chip according to the present invention to hybridize a detection target and a probe in a labeled test sample, and to the probe after washing. The hybridized detection target is detected based on the label, and the viability of the fungus in the test sample can be determined by the level of the amount detected relative to the fungal cell mass. Highly accurate and simple.
In addition, the mold identification method according to the present invention is obtained by fixing two or more types of mold detection probes according to the present invention to a substrate in a state where the mold detection probes are separated from each other and arranged. Using a DNA chip, the detection target in the labeled test sample is hybridized with the probe, and after washing, the detection target hybridized to the probe is detected based on the label. The mold species can be identified as the mold to be detected by the mold detection probe fixed to the area where the labeled compound is detected on the DNA chip, so that the mold species can be identified quickly and accurately. This can be done easily.
In addition, since the mold identification method can identify mold life and death as well as mold species, it is possible to simultaneously determine mold life and death and mold species identification with a single DNA chip.

(Mold detection probe)
The mold detection probe according to the present invention is a probe containing a polynucleotide that can specifically hybridize to the genome region of the mold to be detected. The length of the mold detection probe is not particularly limited, but is, for example, 10 to 200 nucleotides, preferably 20 to 100 nucleotides, and more preferably 20 to 50 nucleotides. The probe length may be the same or different between the plurality of probes.

  The polynucleotide of the probe is not particularly limited, and DNA, RNA, or a conventionally known derivative or analog thereof can be used. The method for producing the mold detection probe is not particularly limited, and for example, it may be produced by chemical synthesis by a conventionally known method, or may be produced enzymatically in vivo or in vitro.

  In the present invention, since the mold detection probe is used as a DNA chip (microarray), the mold detection probe may be modified to be fixed to the substrate of the DNA chip. The modification can be selected from conventionally known modifications depending on the type of the DNA chip substrate to be used, and examples thereof include C (3-7) amination at the 5 'end or 3' end of the probe polynucleotide.

The mold detection probe sequence may be a conventionally known sequence specific to the mold to be detected, or may be designed based on the mold genome sequence. The method for designing the probe sequence is not particularly limited. For example, a conventionally known algorithm or software can be used.
For example, it is described in the following paper as a known sequence.
(1) Zhihong Wu, Yoshihiko Tsumura et al, 18S rRNA Gene Variation among Common Airborne Fungi, and Development of Specific Oligonucleotide Probes for the Detection of Fungal Isolates, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Vol. 69, No. 9, p. 5389− 5397 (2003)
(2) WILLEM JG MELCHERS, PAUL E VERWEIJ, General Primer-Mediated PCR for Detection of Aspergillus Species, JOURNAL OF CLINICAL MICROBIOLOGY, Vol. 32, No. 7, p. 1710-1717 (1994)

Examples of the software include ARB (The ARB project at Technical University of Munich-the official ARB homepage, http://www.arb-home.de).
The software ARB is also described in the following references.
(1) Michael Mayl, BjoErn Nonhoffl et al., ARB: a software environment for sequence data, Nucleic Acids Research, Vol.32, No.4, pp.1363-1371 (2004)
(2) Park Sakaki, Kikuharu Ito, ARB; rRNA database processing and probe design, Intestinal Bacteriology Journal, Vol.19 pp.53-60 (2005)

  In a preferred embodiment of the present invention, the mold detection probe comprises the nucleotide sequence of SEQ ID NOs: 1 to 34 or a nucleotide sequence substantially the same as that derived from the nucleotide sequence of 18S rDNA of the mold ribosome to be detected. It has any one base sequence selected from the group.

  In the present invention, the “substantially identical base sequence” comprises a base sequence in which one or several nucleic acids are deleted, substituted, inserted or added in the base sequences of SEQ ID NOs: 1 to 34, It refers to a base sequence that can hybridize with DNA to which a mold detection probe consisting of the corresponding base sequence of SEQ ID NOs: 1 to 34 hybridizes.

Preferred embodiments of the mold detection probe according to the present invention are exemplified below.
(1) As a mold detection probe for detecting Absidia coerulea, a mold detection probe (A) having the base sequence of SEQ ID NO: 1 or a base sequence substantially the same as that is included.

(2) The mold detection probe (a) having the base sequence of SEQ ID NO: 2 or a base sequence substantially the same as the base for detection of mold that detects Absidia corymbifera (Absidia corymbifera).

(3) As a mold detection probe for detecting Absidia glauca, a mold detection probe (c) consisting of the base sequence of SEQ ID NO: 3 or substantially the same base sequence, and SEQ ID NO: 4 Or a mold detection probe (D) comprising the substantially same base sequence.

(4) As a mold detection probe for detecting Absidia repens, a mold detection probe (e) consisting of the base sequence of SEQ ID NO: 5 or substantially the same base sequence, and SEQ ID NO: 6 Or a fungus detection probe (fungi) consisting of a substantially identical base sequence.

(5) Alternaria As a mold detection probe for detecting alternata alternata, a mold detection probe (ki) consisting of the base sequence of SEQ ID NO: 7 or a base sequence substantially the same as that is included.

(6) As a mold detection probe for detecting Aspergillus nidulans, there is a mold detection probe (C) consisting of the base sequence of SEQ ID NO: 8 or a base sequence substantially the same as that.

(7) As a fungus detection probe for detecting Aspergillus niger, a fungus detection probe consisting of the base sequence of SEQ ID NO: 9 or a base sequence substantially the same as that (SEQ ID NO: 10), A fungus detection probe (co) comprising a base sequence or a base sequence substantially the same as that, a fungus detection probe comprising a base sequence of SEQ ID NO: 11 or a base sequence substantially the same (sa), SEQ ID NO: 12 Examples include a fungus detection probe (si) comprising a base sequence or a base sequence substantially identical thereto, and a fungus detection probe (su) comprising a base sequence of SEQ ID NO: 13 or a base sequence substantially identical thereto.

(8) As a mold detection probe for detecting Aspergillus versicolor, a mold detection probe (C) consisting of the base sequence of SEQ ID NO: 14 or a base sequence substantially the same as that is included. .

(9) Candida albicans As a detection target for molds, a mold detection probe (So) consisting of the base sequence of SEQ ID NO: 15 or a base sequence substantially the same as that is included.

(10) As a mold detection probe for detecting Cladosporium cladosporioides, a mold detection probe comprising a base sequence of SEQ ID NO: 16 or a base sequence substantially identical thereto (Table) A fungus detection probe (h) consisting of the base sequence of SEQ ID NO: 17 or a base sequence substantially the same as that, and a fungus detection probe (tu) consisting of the base sequence of SEQ ID NO: 18 or a base sequence substantially the same as it Is mentioned.

(11) As a probe for detecting mold that detects both cladosporium cladosporioides and cladosporium sphaerospermum, the base sequence of SEQ ID NO: 19 or substantially the same Examples include a mold detection probe (te) comprising the same base sequence, and a mold detection probe (g) comprising the base sequence of SEQ ID NO: 20 or a base sequence substantially identical thereto.

(12) As a mold detection probe for detecting Cladosporium langeronii, a mold detection probe (na) consisting of the base sequence of SEQ ID NO: 21 or a base sequence substantially identical thereto, and a sequence And a mold detection probe (d) comprising the base sequence of No. 22 or a base sequence substantially the same as the base sequence.

(13) As a mold detection probe for detecting Eurotium repens, a mold detection probe (nu) having the base sequence of SEQ ID NO: 23 or a base sequence substantially the same as that is included.

(14) As a mold detection probe for detecting Fusarium culmorum, a mold detection probe (ne) consisting of the base sequence of SEQ ID NO: 24 or a base sequence substantially the same as that is included.

(15) Examples of the mold detection probe that detects Fusarium oxysporum include the base sequence of SEQ ID NO: 25 or a mold detection probe (no) consisting essentially of the same base sequence.

(16) As a mold detection probe for detecting Mucor racemosus, a mold detection probe (C) having the base sequence of SEQ ID NO: 26 or a base sequence substantially the same as that is included.

(17) As a mold detection probe for detecting Paecilomyces tenuipes, a mold detection probe (h) consisting of the base sequence of SEQ ID NO: 27 or a base sequence substantially identical thereto.

(18) As a mold detection probe whose detection target is the genus Forma, a mold detection probe (F) comprising the base sequence of SEQ ID NO: 28 or a base sequence substantially the same as the base sequence is included.

(19) Examples of the mold detection probe targeting the genus Rhizopus include the mold detection probe (f) consisting of the base sequence of SEQ ID NO: 29 or a base sequence substantially identical thereto.

(20) As a mold detection probe for detecting Stachybotrys chartarum, a mold detection probe (e) consisting of the base sequence of SEQ ID NO: 30 or a base sequence substantially identical thereto, and SEQ ID NO: 31 Or a mold detecting probe (m) comprising the substantially same base sequence.

(21) As a mold detection probe for detecting Trichoderma viride, a mold detection probe (mi) having the base sequence of SEQ ID NO: 32 or a base sequence substantially the same as that is included.

(22) As a mold detection probe whose detection target is the genus Wallemia, a mold detection probe (mu) comprising the base sequence of SEQ ID NO: 33 or a base sequence substantially identical thereto, and SEQ ID NO: 34 Examples include a mold detection probe (me) having a base sequence or a base sequence substantially identical to the base sequence.

The mold detection probes of (a) to (me) described above are the nucleotide sequences of SEQ ID NOs: 1 to 34 derived from the 18S rDNA nucleotide sequence of mold ribosomes that have relatively abundant data such as nucleotide sequences and the like. Since it has any one base sequence selected from the group consisting of substantially the same base sequences, it is easy to design and prepare appropriate mold detection probes corresponding to various mold species. Can do.
In addition, since the mold detection probe has a relatively short base sequence of 30 or less bases, it hybridizes with a high probability to a detection target in a test sample containing a mold nucleic acid to be detected, The probe can be easily designed and manufactured, and a DNA chip having the probe fixed on the substrate can be easily manufactured. Furthermore, since it has a relatively short base sequence, various changes, for example, a multistage cascade structure in which one end of the same or different mold detection probe is connected to the tip of the first stage mold detection probe. Etc. can also be performed relatively easily.
In addition, since the mold detection probe hybridizes with a detection target in a test sample containing a mold nucleic acid to be detected with high probability, cross-hybridization occurs when hybridizing with the detection target. The probability is low and mold can be detected with high accuracy.

(DNA chip for mold detection)
The mold detecting DNA chip according to the present invention is a DNA chip formed by fixing one or more of the above-described mold detecting probes according to the present invention to a substrate.
As a preferred embodiment of the mold detecting DNA chip according to the present invention, two or more, preferably all, of the mold detecting probes (a) to (me) described above are fixed to each mold detecting probe. Examples include a mold detecting DNA chip that is fixed to a substrate in a state where regions are divided and arranged.

  The arrangement method of the fixed region on the substrate is not particularly limited as long as it can clearly recognize where the mold detection probes (a) to (me) are fixed. For example, the fixed spots of the mold detection probes (A) to (M) can be formed in a straight line and arranged in parallel at intervals, and circular fixed spots can be regularly arranged vertically and horizontally.

  The substrate used for the mold detecting DNA chip according to the present invention is not particularly limited, and various substrates known in the art can be used. Examples of the material of the substrate include inorganic materials such as glass and ceramics; polyethylene, polypropylene, cyclic polyolefin, polyisobutylene, polyethylene terephthalate, unsaturated polyester, fluorine-containing resin, polyvinyl chloride, polyvinylidene chloride, polyvinyl acetate, polyvinyl Alcohol, polyvinyl acetal, acrylic resin, polyacrylonitrile, polystyrene, acetal resin, polycarbonate, polyamide, phenol resin, urea resin, epoxy resin, melamine resin, styrene / acrylonitrile copolymer, acrylonitrile / butadiene styrene copolymer, polyphenylene oxide and Examples thereof include organic materials such as polysulfone. The shape of the substrate is not limited to a rectangle, a square, a circle, or an ellipse.

  The method for producing the mold detecting DNA chip according to the present invention by fixing the mold detecting probe to the substrate can be carried out using a known method in the production of the DNA chip, and is not particularly limited. For example, a method can be used in which a mold detection probe prepared in advance is spotted on a substrate using a spotter. In addition, examples of the spot method in this case include a pin method based on mechanical contact of the pin tip on the substrate, an ink jet method using an ink jet printer, a capillary method using a capillary tube, and the like, but is not particularly limited thereto. As another preferred manufacturing method, a method of artificially synthesizing the mold detection probe on a substrate using a photolithography technique and a solid-phase reaction chemistry technique may be employed.

Since this mold detecting DNA chip has the above-described mold detecting probes (a) to (me) immobilized on a substrate, the mold nucleic acid to be detected is a relatively short base sequence having 30 or fewer bases. Can be hybridized with a high probability with a target for detection in a test sample containing a DNA chip, which facilitates the manufacture of a DNA chip including probe design and production.
In addition, this DNA chip has a mold detection probe (a) to (me) that hybridizes with a high probability to a detection target in a test sample containing mold nucleic acid to be detected. When it hybridizes, the probability of cross-hybridization is low, and mold can be detected with high accuracy.
In addition, this mold detecting DNA chip is formed by fixing probes (a) to (me) having a base sequence derived from the base sequence of 18S rDNA of mold ribosome to the substrate. Compared to a conventional DNA chip having a probe, the variation in the amount of nucleic acid that hybridizes to the probe is increased due to the difference in the survival / killing of the mold to be detected. Can be determined.
Further, in this mold detection DNA chip, two or more of the mold detection probes (a) to (me) are fixed to the substrate in a state where the mold detection probe fixing regions are separated and arranged. The mold species can be identified by examining which fixed region of the mold-derived nucleic acid hybridizes with the mold-derived nucleic acid present in the test sample. Therefore, mold species can be identified with high accuracy.
In addition, in this mold detecting DNA chip, if a structure having a cascade structure in which the same or different mold detecting probes are connected to the tip of the mold detecting probe having one end fixed to the substrate, a specific mold is more It can be detected with high accuracy, and it is also possible to detect mutant species and similar species that cannot be detected with a single probe.

(Funge detection method)
The mold detection method of the present invention uses the aforementioned mold detection DNA chip,
Producing a detection target labeled with a labeling compound from a nucleic acid in a test sample;
Next, the step of hybridizing the detection target to the mold detection probe of the mold detection DNA chip;
Next, whether or not the mold to be detected by the mold detection probe is present in the test sample by washing the mold detection DNA chip and detecting the labeled compound on the washed DNA chip. It is characterized by examining.

  In the present invention, the “label compound” is not particularly limited, and examples thereof include a luminescent label and a radioactive label compound. Examples of the luminescent label include a fluorescent label, a bioluminescent label, a chemiluminescent label, and a colorimetric label, and a fluorescent label compound such as fluorescein, phosphor, rhodamine, and polymethine dye derivative is preferable.

  Examples of commercially available fluorescent labeling compounds include fluorescence such as fluoreprime (FluorePrime, Amersham Pharmacia), fluoredite (Fluoredite, Millipore), FAM (ABI), Cy3 and Cy5 (Amersham Pharmacia), etc. Phosphoramidites are mentioned.

When the fluorescent labeling compound is used as the labeling compound, the detection target labeled with the fluorescent labeling compound is hybridized to the mold detection probe of the mold detection DNA chip, and after washing, the mold detection DNA chip is set in the scanner. And read the fluorescence intensity. The presence or absence of mold in the test sample can be determined by irradiating the fluorescent labeling compound used with excitation light having a predetermined wavelength and measuring the fluorescence intensity of the specific wavelength emitted from the fluorescent labeling compound.
The mold detection method of the present invention can detect a specific mold with higher accuracy than when a conventional DNA chip having an ITS-derived probe is used.

(Method for distinguishing mold life and death)
The mold detecting DNA chip according to the present invention is obtained by fixing the mold detecting probes (a) to (me) having a base sequence derived from the base sequence of 18S rDNA of mold ribosome to a substrate. Compared to a conventional DNA chip having a probe with a base sequence derived from, the variation in the amount of nucleic acid hybridized to the probe is increased due to the difference in the survival / killing of mold to be detected. Can determine the life and death of mold. When RNA is extracted from living mold and dead mold with the same amount of bacteria, and the amount of each RNA is compared, it can be seen that the amount of RNA in the living mold is much higher than in the case of death . For this reason, it is possible to easily determine whether the fungus to be detected is viable or not based on the RNA amount or the detected amount of the labeled compound after hybridization of the mold-detecting DNA chip.

That is, the mold life / death discrimination method according to the present invention uses the above-described mold detection DNA chip according to the present invention,
Producing a detection target labeled with a labeling compound from a nucleic acid in a test sample;
Next, the step of hybridizing the detection target to the mold detection probe of the mold detection DNA chip;
Next, the mold-detecting DNA chip is washed, and the labeled compound is detected from the washed DNA chip. When the detected amount of the labeled compound relative to the mold cell is larger than the reference value, When the mold present in the sample is alive and the amount of the labeled compound detected relative to the amount of mold is less than the reference value, it is determined that the mold present in the test sample is dead. And

  The mold viability determination method according to the present invention uses the mold detection DNA chip according to the present invention, hybridizes a detection target and a probe in a labeled test sample, and hybridizes to the probe after washing. Since the target is detected based on the label, the viability of the mold in the test sample can be discriminated by the level of the amount detected against the fungal cell mass. Can be done.

(How to identify mold)
In the mold identification method according to the present invention, two or more of the mold detection probes (a) to (me) are fixed to a substrate in a state where the mold detection probes are separated from each other and arranged. Using a DNA chip
Producing a detection target labeled with a labeling compound from a nucleic acid in a test sample;
Hybridizing the detection target to the mold detection probe of the mold detection DNA chip;
Next, the mold detecting DNA chip is washed, and the labeled compound is detected on the washed DNA chip, so that mold in the sample to be inspected is fixed on the DNA chip where the labeled compound is detected. It is characterized by having a step of identifying the mold species as a mold to be detected by the mold detection probe.

  The mold detection DNA chip used in this mold identification method is a mold detection probe (A) to (M) according to the present invention. It is fixed to the substrate in a state where the regions are divided and arranged, so that it can be seen which mold detection probe is fixed to a specific fixed region. By using this DNA chip, the detection target in the labeled test sample and the probe are hybridized, and after washing, the detection target hybridized to the probe is detected based on the label. By examining whether the detection target is hybridized to the mold detection probe, the mold in the sample to be inspected is any mold to be detected by the mold detection probe (A) to (M) Can be identified.

  The mold identification method according to the present invention uses a DNA chip in which two or more types of mold detection probes are fixed to a substrate in a state where the mold detection probes are separated from each other. The detection target in the test sample and the probe are hybridized, the detection target hybridized to the probe after washing is detected based on the label, and the mold in the test sample becomes a labeled compound of the DNA chip. Since the mold species can be identified as the mold to be detected by the mold detection probe fixed in the region where the mold is detected, the mold species can be identified quickly, accurately and easily. it can.

(Simultaneous measurement of mold life / death discrimination and mold species identification)
In the above-described mold identification method, the mold detection DNA chip is detected by washing the mold detecting DNA chip and detecting the labeled compound on the washed DNA chip. The mold compound is detected as the mold to be detected by the mold detection probe fixed to the region, and at the same time, the labeled compound is detected from the washed DNA chip, When the detected amount of the labeled compound relative to the body weight is greater than the reference value, the mold present in the sample to be inspected is alive, and when the detected amount of the labeled compound relative to the fungus body mass is less than the reference value By performing the step of discriminating that the mold present in the sample to be inspected is dead, it is possible to simultaneously measure the viability of the mold and the identification of the mold species.
Thus, since the mold identification method can identify mold life and death as well as mold species, it is possible to simultaneously perform mold life / death distinction and mold species identification with a single DNA chip. it can.

  The contents described in the above-described embodiments are merely examples, and various modifications can be made without departing from the gist of the present invention.

(Design of mold detection probe)
No. in Table 1 34 mold detection probes having the base sequences shown in SEQ ID NOs: 1 to 34 were designed as mold detection probes targeting 1 to 22 molds.

[Example 1]
<Determination of mold life / death by hybridization using DNA chip>
The following describes RNA extraction, quantification of RNA amount, and measurement using a DNA chip (microarray), but the present invention is not particularly limited to the kit, DNA chip, and protocol used here, and generally used RNA extraction, The kit includes a kit for the purpose of quantification of RNA amount and measurement using a DNA chip (microarray), a DNA chip, and a protocol attached to them.

(A) RNA extraction procedure No. 1 in Table 1 No. 7, Aspergillus niger, No. 7 About 10 Cladosporium cladosporioides (Cladosporium cladosporioides), the same amount of viable and dead bacteria was collected, frozen and crushed with liquid nitrogen, respectively, and Fungal RNA Isolation kit V-Spin Column E. Z. N. Using an A extraction kit (Omega Bio-tek), RNA was extracted and purified according to the protocol attached to the kit.

  Specifically, first, frozen and ground mold cells are collected in a 1.5 mL microtube, and 500 μL of Buffer RB of the extraction kit described above is added and suspended vigorously. The suspension is poured into a 2 mL collection tube set with a column (Homogenizer Colmn, green) attached to the kit. The collection tube is centrifuged at 13000 × g for 5 minutes at room temperature. Transfer the liquid collected in the collection tube to a new 1.5 mL tube, add 0.5 equivalent of absolute ethanol and stir.

  Next, the entire amount of the agitated liquid is added to a 2 mL collection tube on which a column (HiBind RNA Colum, red) attached to the kit is set. Centrifuge at room temperature for 10,000 seconds at 10,000 xg, discard the liquid collected at the bottom of the collection tube, add 500 mL of RNA Wash Buffer I supplied with the kit to the column, and centrifuge again at room temperature at 10,000 xg for 30 seconds. To wash the column.

  Further, 700 μL of RNA Wash Buffer II (diluted with ethanol) is added to the column, and the column is washed by centrifugation at room temperature, 10000 × g for 30 seconds, and centrifugation again after adding 500 μL of RNA Wash Buffer II. Centrifuge at full speed for 1 minute at room temperature to completely remove the liquid from the column.

  Finally, the above-mentioned column is reset in a 1.5 mL tube, and 50 μL of DEPC (Diethyl Pyrocarbonate) water attached to the kit is added. Centrifuge for 1 minute at full speed at room temperature and elute the RNA from the column with DEPC water to the bottom of the tube.

(B) Measurement of RNA amount For quantification of RNA, a spectrophotometer (nanodrop; trade name NanoDrop ND-1000, manufactured by Thermo Science) was used. Using 1 μL of the solution from which RNA was eluted, the measurement was performed under the conditions described in Table 2. Table 3 shows the measurement results.

  As shown in Table 3, the RNA concentration of live bacteria was 10 times or more higher than the RNA concentration of dead bacteria in both bacteria (No. 7, No. 10).

(C) Measurement with DNA chip (c-1) rRNA labeling treatment rRNA labeling treatment and subsequent hybridization were performed using a fluorescent labeling reagent kit (trade name: LabelIT miRNA Labeling Kit, manufactured by Miras). Specifically, as shown in Table 4, first, an RNA sample (equivalent amount of live and dead bacteria), Nuclease free water, and 10 × Labeling Buffer M and LabelIT CyDye attached to the kit are added to make 100 μL, and labeling is performed. It was set as the reaction liquid. These reaction solutions were put in a tube, set in an aluminum block thermostat set at 37 ° C., and labeled for 1 hour.
Then, 10 μL of 10 × STOP Reagent was added to each tube solution and mixed well to stop the labeling reaction.

Next, the labeling reaction solution was purified. A labeling reaction solution is injected into a column of a micro-con (having a function of capturing rRNA of a certain molecular weight or more depending on the roughness of the eyes by a membrane filter of the column), and 20 ° C. at 14000 × g. Centrifugation for 1 min, add 100 μL of Nuclease free water to the column, centrifuge at 14000 × g for 25 min at room temperature, add 100 μL of Nuclease free water to the column again, and purify by centrifugation at 14,000 × g for 30 min. It was.
Thereafter, the purified labeling reaction solution in the column was taken out by setting the column in a 15 mL tube in reverse and centrifuging at 1000 × g for 1 minute at room temperature.
Further, in order to keep the amount of the labeling reaction solution constant, the concentration was adjusted to 9.3 μL using a vacuum concentrator.

(C-2) Hybridization and fluorescence measurement First, 0.3 μL of HybridBuffer A attached to the above-described kit (trade name: LabelIT miRNA Labeling Kit, manufactured by Miras) was added to the labeling reaction solution and held at 65 ° C. for 3 minutes. . Furthermore, 30.3 μL of HybridBuffer B was added and stirred, and kept at 32 ° C. for 5 minutes to obtain a hybridization reaction solution.

  DNA chips are synthesized by synthesizing the base sequences of SEQ ID NOs: 9, 11, 12, 13, 16, 18 and 20 on a DNA chip substrate manufactured by Toray Industries, Inc. using photolithography technology and solid phase reaction chemistry technology. Produced.

  Next, a reaction solution of each mold live or dead was injected into the DNA chip. Next, the DNA chip was set in a dedicated chamber, and hybridization was performed for 16 hours while rotating at 32 ° C. and 250 rpm.

Furthermore, after the hybridization reaction, the excess reaction solution was removed, the probe on the chip was washed, and probe fluorescence detection (fluorescence intensity measurement) was performed with a scanner.
Specifically, the rack in which the chip is set in the primary cleaning solution at 30 ° C. shown in Table 5 is shaken, washed for 5 minutes, similarly immersed in the secondary cleaning solution at 30 ° C. for 10 minutes, and the tertiary cleaning solution at 30 ° C. Wash for 5 minutes. In the ozone-free booth, after the completion of the third cleaning, the cleaning liquid in the rack was wiped off, and moisture was blown away at once with a spin dryer before the chips were dried.

  For measurement of the DNA chip after hybridization, a scanner (trade name: ProScanArray HT, manufactured by Perkin Elmer) was used. First, the DNA chip was set in a scanner, and the fluorescence intensity indicating the result of rRNA hybridization between each probe and mold was measured.

(C-3) Measurement results FIG. 1 shows the measurement intensity ratio of live and dead bacteria of Aspergillus niger.
In addition, FIG. 2 shows the measured intensity ratio of viable and dead bacteria of Cladosporium cladosporioides.
From the results of FIG. 1 and FIG. 2, using a DNA chip to which a mold detection probe consisting of the base sequences of SEQ ID NOs: 9, 11, 12, 13, 16, 18, and 20 according to the present invention is immobilized, SEQ ID NOs: 9, 11, Aspergillus niger and Cladosporium cladosporioides RNA, which are detection targets of the mold detection probe consisting of 12, 13 base sequences, could be detected.
Moreover, in all of the mold detection probes comprising the nucleotide sequences of SEQ ID NOs: 9, 11, 12, 13, 16, 18, and 20, when the viable bacteria and dead bacteria have the same amount, the measurement intensity of dead bacteria is increased. On the other hand, the measured intensity of viable bacteria was nearly 10 times higher. With this difference in intensity, it is possible to distinguish between viable and dead bacteria, and it has been demonstrated that the use of this DNA chip makes it possible to determine whether mold is alive or dead.

[Example 2]
<Identification of mold species using DNA chip (1)>
Using Aspergillus niger 2 strains (referred to as A and B), a probe consisting of SEQ ID NOs: 9, 11, 12, and 13 for detecting Aspergillus niger and SEQ ID NOs: 16, 18, and 20 for other fungi Hybridization was performed with a DNA chip having a probe made of The operation procedure is the same as that of the first embodiment.

  The result is shown in FIG. From the result of FIG. 3, the fluorescence intensity was detected with the probes consisting of SEQ ID NOs: 9, 11, 12, and 13 for detecting Aspergillus niger, but the fluorescence intensity was not detected with the other mold probes. From this, it was demonstrated that selective discrimination between Aspergillus niger and other mold species is possible with the mold detection probe according to the present invention.

[Example 3]
<Identification of mold species using DNA chip (2)>
Using the two strains of Cladosporium cladosporioides (denoted as A and B), a probe comprising SEQ ID NOs: 16, 18, and 20 for detection of Cladosporium cladosporioides and other mold sequences Hybridization was performed with a DNA chip in which probes consisting of Nos. 9, 11, 12, and 13 were fixed to a substrate. The operation procedure is the same as that of the first embodiment.

  The result is shown in FIG. From the result of FIG. 4, the fluorescence intensity was detected with the probes consisting of SEQ ID NOs: 16, 18, and 20 for cladsporium clad sporoid detection, but the fluorescence intensity was not detected with the other mold probes. From this, it was demonstrated that the selective detection between cladosporium cladosporoid and other mold species is possible with the fungus detection probe according to the present invention.

2 is a graph showing the measured intensity ratio of live and dead Aspergillus niger measured in Example 1. FIG. It is a graph which shows the measurement intensity | strength ratio of the living microbe of the cladosporoid (Cladosporium cladosporioides) measured in Example 1, and a dead microbe. 3 is a graph showing hybridization results of Aspergillus niger measured in Example 2. FIG. It is a graph which shows the hybridization result of Cladosporium cladosporioides (Cladosporium cladosporioides) measured in Example 3.

Claims (6)

One or two or more kinds, DNA for mold detection made by fixing the substrate of any one of the nucleotide sequences comprising a mold detection probe selected from the group consisting of the nucleotide sequence of SEQ ID NO: 1-34 Use the tip
Producing a detection target labeled with a labeling compound from a nucleic acid in a test sample;
Next, the step of hybridizing the detection target to the mold detection probe of the mold detection DNA chip;
Next, the mold-detecting DNA chip is washed, and the labeled compound is detected from the washed DNA chip. When the detected amount of the labeled compound relative to the mold cell is larger than the reference value, When the mold present in the sample is alive and the detected amount of the labeled compound relative to the amount of mold is less than the reference value, the mold is alive or dead having a step of determining that the mold present in the sample to be tested is dead How to determine. (1) Absidia Koerurea (Absidia coerulea) mildew detection probe consisting of a nucleotide sequence of SEQ ID NO: 1 to be detected (A),
(2) Absidia Korinbifera (Absidia corymbifera) The consisting of the nucleotide sequence of SEQ ID NO: 2 to be detected mold detection probe (b),
(3) Absidia glauca (Absidia glauca) the detection target to SEQ ID NO: Mold detection probe consisting of three base sequence (c), and molds detection probe consisting of a nucleotide sequence of SEQ ID NO: 4 (d),
(4) Absidia Repensu (Absidia repens) a detection target to base sequence consists of a sequence fungus detection probe of SEQ ID NO: 5 (e), and molds detection probe consisting of a nucleotide sequence of SEQ ID NO: 6 (f),
(5) Alternaria Arutanata (Alternaria alternata) mildew detection probe consisting of a nucleotide sequence of SEQ ID NO: 7 to be detected (key),
(6) Mold detection probe consisting of a nucleotide sequence of SEQ ID NO: 8 to be detected Aspergillus nidulans (Aspergillus nidulans) (h),
(7) Aspergillus niger (Aspergillus niger) a detection target comprising the nucleotide sequence of SEQ ID NO: 9, mold detection probe (Ke), molds detection probe consisting of a nucleotide sequence of SEQ ID NO: 10 (co), SEQ ID NO: 11 base sequence consists of a sequence fungus detection probe (Sa), probe mold detection comprising the nucleotide sequence of SEQ ID NO: 12 (shea), and molds detection probe consisting of a nucleotide sequence of SEQ ID NO: 13 (scan),
(8) Aspergillus Var Shi color (Aspergillus versicolor) a consisting of the nucleotide sequence of SEQ ID NO: 14 to be detected mold detection probe (Se),
(9) Candida albicans (Candida albicans) and consisting of the nucleotide sequence of SEQ ID NO: 15 to be detected mold detection probe (SEO),
(10) Cladosporium class dos POI Lloyd (Cladosporium Cladosporioides) mildew detection probe consisting of a nucleotide sequence of SEQ ID NO: 16 to be detected (data), molds detection probe consisting of a nucleotide sequence of SEQ ID NO: 17 (H ), and comprising the nucleotide sequence of SEQ ID NO: 18 mold detection probe (Tsu),
(11) Cladosporium class dos POI Lloyd (Cladosporium Cladosporioides) and Cladosporium spheroplasts Cum arm (Cladosporium Sphaerospermum) mildew detection probe consisting of a nucleotide sequence of SEQ ID NO: 19 to be detected (Te), and SEQ ID NO: mold detection probe consisting of a nucleotide sequence of 20 (g),
(12) Cladosporium Rangeroni (Cladosporium Langeronii) mildew detection probe consisting of a nucleotide sequence of SEQ ID NO: 21 to be detected (Na), and molds detection probe consisting of a nucleotide sequence of SEQ ID NO: 22 (d) ,
(13) Yurochiumu Repensu (Eurotium repens) mildew detection probe consisting of a nucleotide sequence of SEQ ID NO: 23 to be detected (j),
(14) Fusarium Karumoramu (Fusarium culmorum) a consisting of the nucleotide sequence of SEQ ID NO: 24 to be detected mold detection probe (ne),
(15) Fusarium oxysporum (Fusarium oxysporum) and consisting of the nucleotide sequence of SEQ ID NO: 25 to be detected mold detection probe (Bruno),
(16) Mucor Rasemosasu (Mucor racemosus) mildew detection probe consisting of a nucleotide sequence of SEQ ID NO: 26 to be detected (c),
(17) Paecilomyces Tenuipesu (Paecilomyces tenuipes) a consisting of the nucleotide sequence of SEQ ID NO: 27 to be detected mold detection probe (arsenide),
(18) former (Phoma) genus mildew detection probe consisting of a nucleotide sequence of SEQ ID NO: 28 to be detected (off),
(19) Rhizopus (Rhizopus) genus mildew detection probe consisting of a nucleotide sequence of SEQ ID NO: 29 to be detected (f),
(20) Stachybotrys Charutoramu (Stachybotrys chartarum) the base sequence consists of a sequence fungus detection probe (e) of SEQ ID NO: 30 to be detected, and molds detection probe consisting of a nucleotide sequence of SEQ ID NO: 31 (Ma),
(21) Trichoderma viride (Trichoderma viride) and consisting of the nucleotide sequence of SEQ ID NO: 32 to be detected mold detection probe (Mi),
(22) Waremia (Wallemia) genus detection target to the nucleotide sequence of SEQ ID NO: 33 Mold detection probe (beam), and molds detection probe consisting of a nucleotide sequence of SEQ ID NO: 34 (main),
Using a mold detecting DNA chip formed by fixing one or more kinds of mold detecting probes selected from the group consisting of
Producing a detection target labeled with a labeling compound from a nucleic acid in a test sample;
Next, the step of hybridizing the detection target to the mold detection probe of the mold detection DNA chip;
Next, the mold-detecting DNA chip is washed, and the labeled compound is detected from the washed DNA chip. When the detected amount of the labeled compound relative to the mold cell is larger than the reference value, When the mold present in the sample is alive and the detected amount of the labeled compound relative to the amount of mold is less than the reference value, the mold is alive or dead having a step of determining that the mold present in the sample to be tested is dead How to determine.   The mold viability determination method according to claim 2, wherein a mold detection DNA chip is used, wherein two or more of the mold detection probes (a) to (me) are fixed in a mixed state on a substrate.   A mold detection DNA chip formed by fixing two or more of the mold detection probes (A) to (M) on a substrate in a state where the mold detection probe fixing regions are separated and arranged is used. The mold life / death determination method according to claim 2. The mold detection DNA chip having a cascade structure in which the same or different mold detection probes are connected to the tip of the mold detection probe, one end of which is fixed to a substrate, according to any one of claims 1 to 4. The mold life / death discrimination method described. (1) Absidia Koerurea (Absidia coerulea) mildew detection probe consisting of a nucleotide sequence of SEQ ID NO: 1 to be detected (A),
(2) Absidia Korinbifera (Absidia corymbifera) The consisting of the nucleotide sequence of SEQ ID NO: 2 to be detected mold detection probe (b),
(3) Absidia glauca (Absidia glauca) the detection target to SEQ ID NO: Mold detection probe consisting of three base sequence (c), and molds detection probe consisting of a nucleotide sequence of SEQ ID NO: 4 (d),
(4) Absidia Repensu (Absidia repens) a detection target to base sequence consists of a sequence fungus detection probe of SEQ ID NO: 5 (e), and molds detection probe consisting of a nucleotide sequence of SEQ ID NO: 6 (f),
(5) Alternaria Arutanata (Alternaria alternata) mildew detection probe consisting of a nucleotide sequence of SEQ ID NO: 7 to be detected (key),
(6) Mold detection probe consisting of a nucleotide sequence of SEQ ID NO: 8 to be detected Aspergillus nidulans (Aspergillus nidulans) (h),
(7) Aspergillus niger (Aspergillus niger) a detection target comprising the nucleotide sequence of SEQ ID NO: 9, mold detection probe (Ke), molds detection probe consisting of a nucleotide sequence of SEQ ID NO: 10 (co), SEQ ID NO: 11 base sequence consists of a sequence fungus detection probe (Sa), probe mold detection comprising the nucleotide sequence of SEQ ID NO: 12 (shea), and molds detection probe consisting of a nucleotide sequence of SEQ ID NO: 13 (scan),
(8) Aspergillus Var Shi color (Aspergillus versicolor) a consisting of the nucleotide sequence of SEQ ID NO: 14 to be detected mold detection probe (Se),
(9) Candida albicans (Candida albicans) and consisting of the nucleotide sequence of SEQ ID NO: 15 to be detected mold detection probe (SEO),
(10) Cladosporium class dos POI Lloyd (Cladosporium Cladosporioides) mildew detection probe consisting of a nucleotide sequence of SEQ ID NO: 16 to be detected (data), molds detection probe consisting of a nucleotide sequence of SEQ ID NO: 17 (H ), and comprising the nucleotide sequence of SEQ ID NO: 18 mold detection probe (Tsu),
(11) Cladosporium class dos POI Lloyd (Cladosporium Cladosporioides) and Cladosporium spheroplasts Cum arm (Cladosporium Sphaerospermum) mildew detection probe consisting of a nucleotide sequence of SEQ ID NO: 19 to be detected (Te), and SEQ ID NO: mold detection probe consisting of a nucleotide sequence of 20 (g),
(12) Cladosporium Rangeroni (Cladosporium Langeronii) mildew detection probe consisting of a nucleotide sequence of SEQ ID NO: 21 to be detected (Na), and molds detection probe consisting of a nucleotide sequence of SEQ ID NO: 22 (d) ,
(13) Yurochiumu Repensu (Eurotium repens) mildew detection probe consisting of a nucleotide sequence of SEQ ID NO: 23 to be detected (j),
(14) Fusarium Karumoramu (Fusarium culmorum) a consisting of the nucleotide sequence of SEQ ID NO: 24 to be detected mold detection probe (ne),
(15) Fusarium oxysporum (Fusarium oxysporum) and consisting of the nucleotide sequence of SEQ ID NO: 25 to be detected mold detection probe (Bruno),
(16) Mucor Rasemosasu (Mucor racemosus) mildew detection probe consisting of a nucleotide sequence of SEQ ID NO: 26 to be detected (c),
(17) Paecilomyces Tenuipesu (Paecilomyces tenuipes) a consisting of the nucleotide sequence of SEQ ID NO: 27 to be detected mold detection probe (arsenide),
(18) former (Phoma) genus mildew detection probe consisting of a nucleotide sequence of SEQ ID NO: 28 to be detected (off),
(19) Rhizopus (Rhizopus) genus mildew detection probe consisting of a nucleotide sequence of SEQ ID NO: 29 to be detected (f),
(20) Stachybotrys Charutoramu (Stachybotrys chartarum) the base sequence consists of a sequence fungus detection probe (e) of SEQ ID NO: 30 to be detected, and molds detection probe consisting of a nucleotide sequence of SEQ ID NO: 31 (Ma),
(21) Trichoderma viride (Trichoderma viride) and consisting of the nucleotide sequence of SEQ ID NO: 32 to be detected mold detection probe (Mi),
(22) Waremia (Wallemia) genus detection target to the nucleotide sequence of SEQ ID NO: 33 Mold detection probe (beam), and molds detection probe consisting of a nucleotide sequence of SEQ ID NO: 34 (main),
Using a mold detection DNA chip in which two or more types of mold detection probes selected from the group consisting of are fixed to a substrate in a state where the mold detection probe fixing regions are separated and arranged,
Producing a detection target labeled with a labeling compound from a nucleic acid in a test sample;
Hybridizing the detection target to the mold detection probe of the mold detection DNA chip;
Next, the mold detecting DNA chip is washed, and the labeled compound is detected on the washed DNA chip, so that mold in the sample to be inspected is fixed on the DNA chip where the labeled compound is detected. The mold detection probe identifies the mold species as the mold to be detected, and
When the detected amount of the labeled compound is larger than the reference value, mold present in the sample to be inspected is alive, and when the detected amount of the labeled compound is smaller than the reference value, A method for detecting mold, comprising a step of determining that existing mold is dead.

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