JP2007174902A - Method for discriminating fungi, discrimination kit and oligonucleotide - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for accurately, rapidly, simply and inexpensively discriminating whether or not the high-order taxonomical group to which the fungi belong is Zygomycetes of the order Mucorales or Basidiomycetes in discrimination of the fungi. <P>SOLUTION: A primer set for the Zygomycetes of the order Mucorales or a primer set for the Basidiomycetes is utilized to carry out a PCR reaction (polymerase chain reaction) in chromosomal DNAs prepared from a pure fungal strain. Thereby, the high-order taxonomical group to which the fungal strain belongs can be discriminated. When a mixed sample of the chromosomal DNAs is subjected to the PCR reaction, the chromosomal DNAs derived from the Zygomycetes of the order Mucorales or the Basidiomycetes and contained therein can be detected. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for differentiating fungi at the gate-eye level. More specifically, the present invention relates to a method for detecting and discriminating fungal zygomycetes and basidiomycetes at a gene level quickly, simply, reliably and inexpensively.

  Fungi are broadly classified into four families: basidiomycetes, ascomycetes, zygomycetes, and fungi (Non-Patent Document 1). This classification is a classification method that depends on the sexual reproduction of fungi and relies heavily on comparative morphological similarity. In addition, molecular phylogenetic evidence from the base sequence of the 18S rRNA gene generally supports this gate-level classification.

  The classification of organisms has a hierarchical structure, and in the main class, from the top, it is the world, gate, class, eye, family, genus, and species (Non-Patent Document 2).

  There is also a method of classifying fungi as molds, mushrooms, and yeasts for the sake of appearance and convenience, and they are traditionally used because they are intuitive. However, if we classify fungi in terms of bioevolution, fungi that fall into the mold category are distributed in all four gates, and fungi that fall into the mushroom category are distributed in both basidiomycetes and ascomycetes. Yeast was only ascomycetes before 1967, but has been distributed to both basidiomycetes and ascomycetes since then. Although the method of classifying fungi such as mold, mushroom, and yeast is intuitively easy to understand, it has no meaning in terms of evolutionary and biological properties, so it has been adopted taxonomically. Not.

  Conventionally, fungi other than yeasts have been classified and differentiated by using a method based on morphological observation (morphological method) in all classification classes. However, since this method requires considerable skill and time for observation, it has not been an industrially easy technique.

  In the classification and identification of bacteria, in addition to morphological observation, biochemical tests such as saccharide assimilation are also used, and these techniques have been adopted for yeast. However, inspections using these have many inspection items and are cumbersome and time-consuming. Nevertheless, it is difficult to obtain accurate and objective results. These techniques were not applied to mold and mushrooms, and morphological observation was still the basis for classification and identification.

  In recent years, taxonomic reorganization of bacteria has been carried out mainly by the method using the base sequence of 16S rRNA gene, and fungi have been somewhat delayed, and classification using the base sequence of genes such as ribosomal RNA has been performed. It has come to be. Reflecting this, a method (sequence method) using a gene base sequence similar to that for identifying a bacterium is becoming common.

  Recognizing fungi according to the classification based on phylogenetic evolution is important for understanding the essence of the fungi and for industrial convenience. For example, in the field of pathogenic fungi, Candida albicans and Cryptococcus neoformans are both important pathogenic yeasts, while the former is an ascomycete (ascomycete-type yeast) whereas the latter Is a basidiomycete (basidiomycetous yeast), and there are differences in the polysaccharide composition of the cell wall. Since the presence or absence of a cell wall is the biggest difference between cells of animals such as humans and cells of pathogenic fungi, antifungal agents targeting them exist. An antifungal agent whose mechanism of action is the inhibition of β-1,3-D-glucan synthesis in the cell wall is a basidiomycetous yeast, although it exhibits antifungal activity against the Candida albicans There may be a difference that it does not show antifungal activity against certain Cryptococcus neoformans. In addition, when fungus is the main causative fungus in zygomycosis and antifungal agents that are effective against cryptococcosis may not work, it is important to determine whether basidiomycetes or fungus zygomycetes There is.

  Thus, there are many cases where discrimination up to the gate level is sufficient without detailed identification to the genus and species of fungi.

  As described above, in order to carry out the conventional morphological method, the skill of the operator is required, and there is a disadvantage that it takes a long time for culturing and morphological observation. If the sex reproductive organ was not formed, there was a fault that it did not show any power.

  On the other hand, in order to perform the sequencing method, the worker is required to have basic molecular biological knowledge and skills, but the operation itself is easy and no special skill is required. However, since a DNA sequencer (base sequence decoding device) is indispensable, initial cost and running cost are high, and users who can use it are limited.

  Both morphological methods and sequences can be applied for the purpose of differentiating fungi to higher taxon levels from the gate to the eye level, but for each method in terms of speed, accuracy, economy and simplicity. If it is possible to obtain necessary information by a method that is superior to the other, it is desirable.

  In recent years, a method has been reported in which a gene of a specific fungus is rapidly and specifically detected by a gene amplification technique such as a PCR method, and this is used for differentiation and detection of the fungus. In this method, it is only necessary to introduce an inexpensive gene amplification device (PCR thermal cycler, etc.). However, reports on the differentiation of fungi by this method so far are limited to the target fungus Ganoderma boninnense. (Patent Document 1), Candida genus, Cryptococcus genus, and Aspergillus genus, which are only part of each species (Patent Document 2), Candida (Candia) genus Those that are only species (Patent Document 3), those that are limited to a part of the genus Candida (Candia), Aspergillus genus, Cryptococcus (Patent Document 4), and the target range is unclear (Patent Document 5), those limited to the genus Rhizoctonia (Patent Document 6), and limited to some mycorrhizal fungi such as matsutake among basidiomycetes (Patent Document 7) and some of the genus Candida, those limited to Aspergillus fumigatus and Pneumocystis carinii (Patent Document 8), etc., and fungi Until now, there has been no idea and method for differentiating and detecting fungi at the higher-level taxonomic group, that is, at the gate-eye level.

The medical care listed in the above example by providing means that can accurately and quickly, inexpensively and easily identify and detect higher taxonomic groups at the gate-eye level rather than limited genus and species of fungi It can be used not only in the field, but also in the field of environmental analysis and quality control such as mycoflora analysis.
Japanese Patent Laid-Open No. 2001-314199, “Detection Method and Primer for Detection of Oil Palm Disease Ganoderma boninense” JP 2002-142774 A "Nucleic acid for fungus detection and fungus detection method using the same" JP 2002-142775 A "Candida fungus detection nucleic acid and Candida fungus detection method using the same" JP 2004-201636 A “Method for detecting pathogenic fungal gene and oligonucleotide for fungal species identification” Japanese Unexamined Patent Publication No. 2004-201637 “Detection of Pathogenic Fungal Genes and Oligonucleotides Used Therefor” Japanese Patent Laid-Open No. 10-234381 “Nucleic Acid Sequence for Detection of Rhizoctonia spp.” Japanese Patent Laid-Open No. 5-252999 "DNA probe for detecting mycorrhizal fungi" Japanese Patent Application Laid-Open No. 8-89254 “Oligonucleotide for fungus detection and fungus species identification” Kirk, PM et al., Ainsworth &Bisby's Dictionary of the Fungi, 9th Ed., 2001, CAB Publishing Ryuichi Yasugi et al., Iwanami Biology Dictionary 4th edition, 1996, Iwanami Shoten

In order to objectively identify and detect fungi with high accuracy, it is desirable to perform comparison and detection at the gene level, but for this purpose, the base sequence of the gene is determined and the homology search against the base sequence database is performed. It was essential to do. However, the base sequence can be determined by pure DNA extraction after the isolation and culture of the fungus, DNA fragment amplification by means of gene amplification in the region such as 18S rRNA gene, cycle sequencing reaction for base sequencing and DNA sequencer After decoding the base sequence and determining the base sequence, it was necessary to perform a search using a homology search means such as BLAST against a base sequence database such as an international base sequence database. This method (sequencing method) requires about 3 hours for the PCR reaction for DNA fragment amplification, about 3 hours for the cycle sequencing reaction as a preparation step for base sequence decoding, and about 3 hours for the base sequence decoding. It was not possible to meet the demand for promptness of receiving fungal specimens and producing results in the evening.
In addition, in the sequencing method, it is necessary to apply the same protocol to a wide range of fungi, so oligonucleotide primers used for PCR reactions are those that can amplify genes of fungi in general and are not selective . Therefore, the fungal specimen must be pure, and it has not been possible to detect whether the fungus is present in the mixed system.
The object of the present invention is to utilize the difference in the base sequence of the 18S rRNA gene of fungi contained in a specimen, and by using a PCR reaction, the sequencing method can be performed with higher accuracy, speed, and convenience than with the morphological method. It is to provide a method for discriminating fungal zygomycetes and basidiomycetes more quickly, simply and inexpensively than by the method described above.

  In order to solve the above problems, the present inventors have made various studies on the base sequence of the 18S rRNA gene throughout the fungi, and among the fungi, the base sequence common to the fungus zygomycetes and the common basidiomycetes are common. The base sequence was found to exist.

The present invention according to claim 1 made based on the above knowledge is a method for differentiating fungi, comprising preparing DNA of fungi to be differentiated, using the prepared DNA as a template, described in SEQ ID NO: 1. Amplification of nucleic acid using the oligonucleotide of No. 2 as a forward primer and the oligonucleotide of SEQ ID NO: 2 as a reverse primer, and when a specific amplification product is observed, the fungus is identified as a mold fungus It is a method to do.
The present invention according to claim 2 is a method for differentiating fungi, comprising preparing fungal DNA to be differentiated, using the prepared DNA as a template, and using the oligonucleotide of SEQ ID NO: 3 as a forward primer. In addition, nucleic acid amplification using the oligonucleotide represented by SEQ ID NO: 4 as a reverse primer is performed, and when a specific amplification product is observed, the fungus is identified as a basidiomycete.
In addition, the present invention described in claim 3 is a kit for differentiating the fungal zygomycetes comprising at least a primer set consisting of the oligonucleotide described in SEQ ID NO: 1 and the oligonucleotide described in SEQ ID NO: 2.
The present invention described in claim 4 is a kit for differentiating basidiomycetes comprising at least a primer set comprising the oligonucleotide described in SEQ ID NO: 3 and the oligonucleotide described in SEQ ID NO: 4.
The invention according to claim 5 is the oligonucleotide according to SEQ ID NO: 1.
The invention according to claim 6 is the oligonucleotide according to SEQ ID NO: 2.
The invention according to claim 7 is the oligonucleotide according to SEQ ID NO: 3.
The invention according to claim 8 is the oligonucleotide according to SEQ ID NO: 4.

  According to the present invention, there is provided a differentiation method capable of differentiating and detecting fungal zygomycetes and basidiomycetes more accurately, quickly and simply than by the morphological method, more quickly, simply and cheaply than by the sequencing method. The

  In the method for distinguishing fungi of the present invention, the method for preparing DNA of fungi to be identified is not particularly limited, and can be prepared by a method known per se. Of course, you may prepare using a commercially available DNA extraction kit.

  The forward primer and reverse primer used for nucleic acid amplification may be chemically synthesized by a method known per se, or directly cut out from the fungal gene from which the design originated using a restriction enzyme or the like. It is also possible to clone these genes into plasmids such as E. coli, collect the plasmids after bacterial growth, excise them and use them. The DNA polymerase used for nucleic acid amplification and reaction conditions are not particularly limited. Since the oligonucleotide used as a primer in the present invention has a length sufficient to use various nucleic acid amplification methods known per se, it is possible to accurately distinguish fungi from the gate to the eye level in a short time. it can. When nucleic acid amplification is performed in three steps of denaturation / annealing / extension, the temperature cycle condition is, for example, 90 ° C. to 96 ° C. for 1 second to 60 seconds → 50 ° C. to 70 ° C. for 1 second to 120 seconds → 65 ° C. to Conditions such as 1 to 120 seconds at 75 ° C. can be listed as standard conditions, but are not limited to these conditions. The presence or absence of an amplification product may be performed by electrophoresis using, for example, Agilent 2100 Bioanalyzer (Agilent Technologies), but is not limited to this method.

  The oligonucleotide described in SEQ ID NO: 1 (5'-tcagttatgatctac-3 ') and the oligonucleotide described in SEQ ID NO: 2 (5'-rttygactacggacg-3') are respectively Saccharomyces cerevisiae (registration number: J01353). ) Corresponding to pos.106-120 and pos.637-623 of 18S rRNA gene. When nucleic acid amplification is performed using the oligonucleotide shown in SEQ ID NO: 1 as a forward primer and the oligonucleotide shown in SEQ ID NO: 2 as a reverse primer, the amplified DNA is specific only when the fungus to be differentiated is a mold fungus A fragment (a single amplification product of interest: the same applies hereinafter) can be obtained. For example, the fungus to be identified is 524 bp for Absidia corymbifera, 536 bp for Mucor hiemalis, 536 bp for Mucor racemosus, Rhizopus oryzae (ae) ) 534bp, Rhizopus stolonifer 533bp, Rhizomucor miehei 515bp, Circinomucor circinelloides 534bp, and Rhizopus oligosporus oligo sporus In the case of Mucor plumbeus, a specific amplified DNA fragment of 537 bp can be obtained. Since there are some differences in the base sequence and base sequence length depending on the strain, if a specific amplified DNA fragment of about 500 to 550 bp is obtained, it is possible to determine that the fungus to be differentiated is a mold fungus.

  The oligonucleotide described in SEQ ID NO: 3 (5'-aggattgacagattgata-3 ') and the oligonucleotide described in SEQ ID NO: 4 (5'-tarcgacrggcggtgtgtac-3') are respectively Saccharomyces cerevisiae (registration number: J01353). ) Corresponding to pos.1223-1240 and pos.1646-1627 of 18S rRNA gene. When nucleic acid amplification is performed using the oligonucleotide shown in SEQ ID NO: 3 as a forward primer and the oligonucleotide shown in SEQ ID NO: 4 as a reverse primer, a specific amplified DNA fragment is obtained only when the fungus to be differentiated is a basidiomycete. Obtainable. For example, when the fungus to be differentiated is the basidiomycete Pleurotus tuberregium, it is 424 bp, and when the base sequence of the 18S rRNA gene is Moniliella acetoabutans described in SEQ ID NO: 5, 441 bp, SEQ ID NO: 6 447 bp for Moniliella suaveolens described in, 424 bp for Sporotrichum pruinosum described in SEQ ID NO: 7, 427 bp for Wallemia sebi described in SEQ ID NO: 8, Agaricus bisporus A specific amplified DNA fragment of 423 bp can be obtained in the case of (Agaricus bisporus) and 423 bp in the case of Filobabasidiella neoformans. Since there are some differences in the base sequence and base sequence length depending on the strain, if a specific amplified DNA fragment of about 400 to 450 bp is obtained, the fungus to be identified can be determined to be a basidiomycete.

  Further, according to the present invention, in the detection of fungi, it is possible to accurately, quickly, conveniently, and inexpensively detect that the prepared chromosomal DNA contains chromosomal DNA derived from the fungus zygomycetes and / or basidiomycetes. Can do. That is, according to the present invention, it is possible to specifically distinguish the fungal zygomycetes and basidiomycetes, so in the mixed sample of chromosomal DNA of a plurality of types of fungi, the chromosomes derived from the fungus zygomycetes and / or basidiomycetes It can be determined whether or not DNA is contained. The mixed sample of chromosomal DNA of multiple types of fungi may be prepared from a sample containing multiple types of fungi using chromosomal DNA preparation means, or chromosomal DNA extraction means from a purely isolated strain alone. What was prepared by mixing the chromosomal DNA extracted using it may be used. For the preparation of chromosomal DNA, a commercially available kit such as “Gen Toru-kun” (Takara Bio) can be used. In addition, the PCR reaction can be performed using “PCR beads” (Amersham Bioscience) or the like.

  The present invention also provides a fungal discrimination kit. This kit comprises at least a forward primer and a reverse primer for nucleic acid amplification, but may further contain DNA polymerase, dATP, dCTP, dTTP, dGTP solutions, buffers, etc. .

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated further in detail, this invention is limited to the following description and is not interpreted.

Example: Microbial flora test example in oolong tea production process Microorganisms were separated from tea leaf samples collected from each process of oolong tea production. For isolation of mold and yeast, chloramphenicol-added PDA medium (hereinafter referred to as PDA medium) was cultured at 25 ° C. for 7 days. Growing colonies were isolated and subjected to pure culture to obtain 8 differential samples. For each differential sample, chromosomal DNA was prepared from colonies purified using a commercially available kit. Using this DNA as a template, a primer set for discriminating mold molds consisting of the oligonucleotide shown in SEQ ID NO: 1 and the oligonucleotide shown in SEQ ID NO: 2 (used as a mixture of four kinds of oligonucleotides), and SEQ ID NO: 3 A PCR reaction was performed according to a general method using a basidiomycete discrimination primer set consisting of the oligonucleotide described and the oligonucleotide described in SEQ ID NO: 4 (used as a mixture of four kinds of oligonucleotides). When the reaction product was confirmed by electrophoresis, in the PCR reaction using the primer set for distinguishing fungal zygomycetes, specific amplification of a DNA fragment of about 530 bp was observed in samples 4 and 6, as shown in FIG. It was. In the PCR reaction using the basidiomycete discrimination primer set, specific amplification of a DNA fragment of about 400 bp was observed in sample 1 and sample 5, as shown in FIG. Therefore, it was found that the fungi of sample 1 and sample 5 were basidiomycetes, the fungi of samples 4 and 6 were fungus-zygous fungi, and the fungi of other samples were not basidiomycetes or fungi-zygous fungi. According to the analysis of the 28S rDNA D2 region sequence, the fungus of sample 1 is Pezzizales (basidiomycetes), the fungus of sample 4 is Mucor genus (mushroom zygomycetes), sample 5 It was confirmed that the fungi were Polyporales (Basidiomycetes), and that the fungus of Sample 6 was Rhizopus genus (Coleoptera zygomycetes).

Reference example:
FIG. 3 is a table describing typical fungal species of fungi.
FIG. 4 is a view showing a region of an 18S rRNA gene containing a base sequence specific to the fungus zygomycete. In the figure, (A) is an ascomycete, (B) is a basidiomycete, (C) is a camellia, (Zm) is a fungus zygomycete, (ZZ) is a zygomycetes zygomycete other than the fungus, (ZT ) Is a Tricomikes zygomycetes (the same applies to FIGS. 5 to 7). The boxed region for the fungus zygomycete is the primer region consisting of the oligonucleotide described in SEQ ID NO: 1. This region corresponds to 106 → 120 of the base sequence of the 18S rRNA gene of Saccharomyces cerevisiae (registration number: J01353). The underlined portion of Saccharomyces cerevisiae indicates the nucleotide sequence corresponding to the primer position.
FIG. 5 is a diagram showing other regions of the 18S rRNA gene containing a base sequence specific to the fungus zygomycete. The boxed portion for the fungus zygomycete is the primer region consisting of the oligonucleotide described in SEQ ID NO: 2. This region corresponds to 637 → 623 of the base sequence of the 18S rRNA gene of Saccharomyces cerevisiae (registration number: J01353). The underlined portion of Saccharomyces cerevisiae indicates the nucleotide sequence corresponding to the primer position.
FIG. 6 is a diagram showing an 18S rRNA gene region containing a base sequence specific to all basidiomycetes. The boxed portion for basidiomycetes is a primer region consisting of the oligonucleotide described in SEQ ID NO: 3. This region corresponds to 1223 → 1240 of the base sequence of the 18S rRNA gene of Saccharomyces cerevisiae (registration number: J01353). The underlined portion of Saccharomyces cerevisiae indicates the nucleotide sequence corresponding to the primer position.
FIG. 7 shows other regions of the 18S rRNA gene containing a base sequence specific to all basidiomycetes. The boxed portion for basidiomycetes is a primer region consisting of the oligonucleotide described in SEQ ID NO: 4. This region corresponds to 1646 → 1627 of the base sequence of the 18S rRNA gene of Saccharomyces cerevisiae (registration number: J01353). The underlined portion of Saccharomyces cerevisiae indicates the nucleotide sequence corresponding to the primer position.

  The present invention provides a differentiation method capable of differentiating and detecting fungal zygomycetes and basidiomycetes more accurately, quickly and simply than by the morphological method, more quickly, simply and inexpensively than by the sequencing method. Has industrial applicability.

Results of PCR reaction using primer set for fungus zygomycetes Results of PCR reaction using basidiomycete primer sets Table with representative fungal species Diagram showing the position where the nucleotide sequence of SEQ ID NO: 1 is given in the region where the nucleotide sequence is highly conserved among the fungal 18S rRNA gene nucleotide sequences Diagram showing the position where a base sequence complementary to SEQ ID NO: 2 is given in a region where the base sequence is highly conserved in the fungal zygomycete, among multiple aligned sequences of 18S rRNA gene base sequences of fungi The figure which shows the position which gives the base sequence of SEQ ID NO: 3 in the region where the base sequence is highly conserved in basidiomycetes in the multiple alignment sequence of 18S rRNA gene base sequence Diagram showing the position where a base sequence complementary to SEQ ID NO: 4 is provided in a region where the base sequence is highly conserved in basidiomycetes among multiple aligned sequences of 18S rRNA gene base sequences of fungi

Claims (8)

  A method for distinguishing fungi, comprising preparing DNA of fungi to be differentiated, using the prepared DNA as a template, the oligonucleotide set forth in SEQ ID NO: 1 as a forward primer, and the oligonucleotide set forth in SEQ ID NO: 2 A method of performing nucleic acid amplification using a reverse primer and discriminating that the fungus is a fungus zygote when a specific amplification product is observed.   A method for distinguishing fungi, comprising preparing DNA of fungi to be differentiated, using the prepared DNA as a template, the oligonucleotide set forth in SEQ ID NO: 3 as a forward primer, and the oligonucleotide set forth in SEQ ID NO: 4 A method of differentiating a fungus from basidiomycetes when a specific amplification product is observed after performing nucleic acid amplification using a reverse primer.   A kit for discriminating fungiform zygomycetes comprising at least a primer set comprising the oligonucleotide set forth in SEQ ID NO: 1 and the oligonucleotide set forth in SEQ ID NO: 2.   A kit for distinguishing basidiomycetes comprising at least a primer set comprising the oligonucleotide described in SEQ ID NO: 3 and the oligonucleotide described in SEQ ID NO: 4.   The oligonucleotide set forth in SEQ ID NO: 1.   The oligonucleotide set forth in SEQ ID NO: 2.   The oligonucleotide set forth in SEQ ID NO: 3.   The oligonucleotide set forth in SEQ ID NO: 4.