JP2019033704A - Oligonucleotide probe for yeast detection, microarray for yeast detection, and kit for yeast detection - Google Patents

Abstract

To provide methods enabling accurate and specific detection of wide variety of yeasts, in a test to confirm the presence or absence of yeasts.SOLUTION: Provided is an oligonucleotide probe for yeast detection in which 51 kinds of yeasts comprising at least either following oligonucleotide probe of (a) or (b) are arranged in one microarray to enable detection. (a) Each oligonucleotide probe consisting of a particular nucleic acid sequence, and (b) each oligonucleotide probe having at least 90% identity to the base sequence of each oligonucleotide probe of the (a) above.SELECTED DRAWING: None

Description

  The present invention relates to an oligonucleotide effective for the detection and identification of fungi that are considered problematic in foods and the environment.

In recent years, in food production sites, clinical sites, agricultural sites, cultural property protection environments, etc., we check for the presence of fungi such as molds and yeasts to confirm their safety and soundness, and prevent their reproduction It is important.
In such examination of fungi, identification methods using genes are performed. That is, DNA is extracted from fungi collected from the environment, the target region is amplified by PCR (polymerase chain reaction) method, etc., and the amplification product is analyzed to identify the fungus in the environment. ing. As a method for analyzing an amplification product, a method for analyzing the size of the amplification product by electrophoresis, a method using a microarray in which probes that bind complementarily to the amplification product are arranged, and the like have been proposed (Patent Documents 1 and 2). reference).

Japanese Patent No. 5670623 Japanese Patent No. 5196848 Japanese Patent No. 5328343 Japanese Patent No. 5317430

The present inventors have so far developed a mold inspection technique in an indoor environment, and various kinds of oligonucleotide probes arranged on a substrate capable of detecting mold floating in the air or adhering to the environment. A microarray is being created.
By the way, yeast belonging to the same fungus as mold is collected from the environment together with mold by a collecting method such as wiping, and the yeast gene is amplified simultaneously with the mold gene in the DNA amplification step. However, while mold is detected by the microarray, since an oligonucleotide probe capable of detecting this is not arranged in the microarray, yeast gene amplification products are actually obtained in the mold inspection process. Nevertheless, no yeast was detected.

Here, depending on the type of yeast, there are those that cause problems such as deterioration of food and candidiasis in humans, so it is beneficial to make yeast detectable.
Examples of techniques for detecting yeast include oligonucleotides for detecting microorganisms described in Patent Document 3 and probe sets described in Patent Document 4. Using these techniques, it is possible to detect specific yeast.

However, for many yeasts, oligonucleotide probes for detecting them have not been developed, and it has been impossible to detect them.
Therefore, the present inventors have intensively studied, organized the reporting status of harmfulness of a large number of yeasts, and developed oligonucleotide probes (52 probes) that can detect 51 types of yeast. Succeeded.

That is, the yeast according to SEQ ID NOs: 1 to 12 is a kind that adversely affects food, agriculture, etc., and oligonucleotide probes that can detect these (especially those selected from the ITS region of yeast DNA) I never found it before.
Moreover, since the yeast which concerns on sequence number 13-31 is a yeast which may cause a deterioration phenomenon, in order to grasp | ascertain the distribution, it made it detectable. No oligonucleotide probe has been found so far that can detect them, particularly those selected from the ITS region of yeast DNA.

  Here, in the microarray for mold inspection that the inventors have already developed, molds of the same genus and subgenus can be detected at once. The reason for this is that similar molds cause similar damage as a result of similar properties such as growth environment (temperature, humidity, water activity) and characteristics (heat resistance, chilling, toxin production). Because. Even if yeast is of a type that has not been reported to be harmful at present, there is a potential risk of causing similar damage if the properties are similar. Therefore, the present inventors have made it possible to detect the yeasts according to SEQ ID NOs: 13 to 31 as such potentially dangerous yeasts.

In addition, the yeasts according to SEQ ID NOs: 32 to 52 have been reported to be harmful in food and medical fields, and there have already been reports on oligonucleotide probes that can detect them.
However, there has been no report on a unique oligonucleotide probe that can be detected at the same time by arranging 51 types of yeast targeted by the present invention in one microarray.

  Of the specific target regions in each DNA of a plurality of yeasts, a probe is selected by selecting a base sequence that specifically binds only to each yeast and does not bind to other types of yeast. Theoretically, each of the plurality of yeasts can be specifically detected by each probe. However, actually, even a probe obtained in this way does not always function effectively. For this reason, in order to create each probe, it is necessary to confirm the effect of the probe by experiment and to find a probe that functions effectively, which requires a lot of repetition of trial and error.

  The present invention has been made in view of the above circumstances, and an oligonucleotide probe for yeast detection, a microarray for yeast detection, and a kit for yeast detection capable of specifically detecting a wide range of types of yeast quickly and accurately. The purpose is to provide.

In order to achieve the above object, the oligonucleotide probe for yeast detection of the present invention is an oligonucleotide probe for yeast detection comprising at least one of the following oligonucleotide probes (a) or (b).
(A) Each oligonucleotide probe consisting of the base sequence shown in SEQ ID NO: 1 to 12 (b) Each oligonucleotide probe having 90% or more identity to the base sequence of each oligonucleotide probe of (a) above

In addition, the yeast array for microarray of the present invention has a configuration in which at least the above-described oligonucleotide probe is arranged on a substrate.
In addition, the yeast detection kit of the present invention includes a microarray in which at least the above-described oligonucleotide probe is arranged on a substrate, a primer and a sequence consisting of a base sequence shown in SEQ ID NO: 53 for amplifying the ITS region of yeast DNA. And a solution containing a primer set consisting of a primer consisting of the base sequence shown in No. 54.

  According to the present invention, it is possible to specifically detect a wide variety of types of yeast quickly and accurately.

It is a figure which shows the base sequence of the oligonucleotide probe (sequence number 1-12) for yeast detection which concerns on embodiment of this invention. It is a figure which shows the base sequence of the oligonucleotide probe (sequence number 13-31) for yeast detection which concerns on embodiment of this invention. It is a figure which shows the base sequence of the oligonucleotide probe (sequence number 32-52) for yeast detection which concerns on embodiment of this invention. It is a figure which shows the base sequence of the primer set for amplifying the target area | region of the target yeast detected by the oligonucleotide probe for yeast detection which concerns on embodiment of this invention. It is a figure which shows the fluorescence intensity (S / N ratio value) separately detected in Test 1 about the oligonucleotide probe (sequence number 1-12) for yeast detection which concerns on embodiment of this invention. It is a figure which shows the fluorescence intensity (S / N ratio value) separately detected in Test 1 about the oligonucleotide probe (sequence number 13-31) for yeast detection which concerns on embodiment of this invention. It is a figure which shows the fluorescence intensity (S / N ratio value) separately detected in Test 1 about the oligonucleotide probe (sequence number 32-52) for yeast which concerns on embodiment of this invention. It is a figure which shows the yeast, strain number, and corresponding sequence number which were mixed in the test 2 (samples 1-4) about the oligonucleotide probe for yeast detection which concerns on embodiment of this invention. It is a figure which shows the yeast mixed in Test 2 (samples 5-8), a strain number, and a corresponding sequence number about the oligonucleotide probe for yeast detection which concerns on embodiment of this invention. It is a figure which shows the yeast, strain number, and corresponding sequence number which were mixed in the test 2 (samples 9-12) about the oligonucleotide probe for yeast detection which concerns on embodiment of this invention. It is a figure which shows the fluorescence intensity (S / N ratio value) detected by mixing in Test 2 about the oligonucleotide probe (sequence number 1-18) for yeast which concerns on embodiment of this invention. It is a figure which shows the fluorescence intensity (S / N ratio value) detected by mixing in the test 2 about the oligonucleotide probe (sequence number 19-36) for yeast which concerns on embodiment of this invention. It is a figure which shows the fluorescence intensity (S / N ratio value) detected by mixing in Test 2 about the oligonucleotide probe (sequence number 37-52) for yeast which concerns on embodiment of this invention.

  Hereinafter, an embodiment of the oligonucleotide probe for yeast detection, microarray for yeast detection, and kit for yeast detection of the present invention will be described in detail. However, the present invention is not limited to the specific contents of the following embodiment and examples described later.

[Oligonucleotide probe for yeast detection]
The oligonucleotide probe for yeast detection of this embodiment is a nucleic acid fragment consisting of a base sequence specified by SEQ ID NOs: 1 to 52, respectively.
First, the yeasts according to SEQ ID NOs: 1 to 12 are of a type that causes a deterioration phenomenon that adversely affects food, agriculture and the like.
Specifically, Kazachstania exigua detected by the probe of SEQ ID NO: 1 causes off-flavor (ethanol odor), expansion (gas generation due to spoilage), and foreign matter generation (spots).
Hanseniaspora osmophila detected by the probe of SEQ ID NO: 2 has a foul odor and is symbiotic with Drosophila sp. There is a problem of being involved in.

  Moreover, Hansenia spora occidentalis (Hanseniaspora occidentalis), Hanseniaspora valbyensis (Clavispora lusitaniae) (Clavispora lusitaniae), Lachansea thermostolera (Lachthera thermostolera)・ Lentinas (Zygotorulaspora florentinus) also has a problem of being involved in yeast contamination mediated by fly body surfaces because it is symbiotic with Drosophila sp.

Debaryomyces carsonii detected by the probe of SEQ ID NO: 8 produces an off-flavor.
Saccharomycopsis capsularis detected by the probe of SEQ ID NO: 9 causes foreign matter generation (black spots).
Torulaspora globosa and Torulaspora delbrueckii detected by the probes of SEQ ID NOs: 10 and 11, respectively, are spoilage yeasts for preserved foods that can be stored for a long time with high sugar or high salt.
Eremothecium ashbyi detected by the probe of SEQ ID NO: 12 causes soybean seed blight disease.

Therefore, the yeast probe for detecting yeast according to the present embodiment includes at least one of the following oligonucleotide probes (a) or (b), two or more of these, or all of them: (Hereinafter, it may be referred to as an A group probe).
(A) Each oligonucleotide probe consisting of the base sequence shown in SEQ ID NO: 1 to 12 (b) Each oligonucleotide probe having 90% or more identity to the base sequence of each oligonucleotide probe of (a) above According to the yeast probe for yeast detection of this embodiment, it is possible to detect a yeast that causes a deterioration phenomenon that adversely affects food, agriculture, etc., which could not be detected so far.

Moreover, the yeast which concerns on sequence number 13-31 is a kind which has the possibility of causing a deterioration phenomenon.
Specifically, Barnettozyma pratensis detected by the probe of SEQ ID NO: 13, Candida berthetii detected by the probe of SEQ ID NO: 14, Candida detected by the probe of SEQ ID NO: 15 Detected by Candida maltosa, Citeromyces matritensis detected by the probe of SEQ ID NO: 16, Issatchenkia occidentalis detected by the probe of SEQ ID NO: 17, probe of SEQ ID NO: 18 Kazachstania unispora, Kregervanrija fluxuum detected by the probe of SEQ ID NO: 19, Lindnera satu detected by the probe of SEQ ID NO: 20. Nuss (Lindnera saturnus), Roderomaisesu-Erongisuporasu (Lodderomyces elongisporus) detected by the probe of SEQ ID NO: 21, a Nakaseomaisesu-Derufenshisu detected by the probe of SEQ ID NO: 22 (Nakaseomyces delphensis).

  Further, Naumovia castellii detected by the probe of SEQ ID NO: 23, Paffymyces thermotolerans detected by the probe of SEQ ID NO: 24, Pichia farinosa detected by the probe of SEQ ID NO: 25 ( Pichia farinosa), Pichia fermentans detected by the probe of SEQ ID NO: 26, Saturnispora ahearnii detected by the probe of SEQ ID NO: 27, and Tetrapicis polara detected by the probe of SEQ ID NO: 28 • Tetrapisispora phaffii, Vanderwaltozyma polyspora detected by the probe of SEQ ID NO: 29, Wicker Hammyces ciferii detected by the probe of SEQ ID NO: 30 amomyces ciferrii), Zygoascus hellenicus detected by the probe of SEQ ID NO: 31.

Therefore, the yeast probe for detecting yeast according to the present embodiment includes at least one of the following oligonucleotide probes (c) or (d), two or more of these, or all of them: (Hereinafter, it may be referred to as a B group probe). Moreover, it is also preferable to set it as a set of probes of A group and B group.
(C) Each oligonucleotide probe comprising the nucleotide sequence shown in SEQ ID NOs: 13 to 31 (d) Each oligonucleotide probe having 90% or more identity to the nucleotide sequence of each oligonucleotide probe in (c) above According to the yeast probe for detecting a yeast of this embodiment, it is possible to detect a potentially dangerous yeast that could not be detected so far.

In addition, the yeasts according to SEQ ID NOs: 32 to 38 have many of bad smell (ethanol odor), swelling (gas generation due to spoilage), foreign matter generation (spots), and spoilage of stored food (high sugar / high salt). Wake up.
Specifically, Candida krusei, Zygosaccharomyces bailii, Zygosaccharomyces bisporus, and Zygosaccharomyces charis (Zygosaccharomyces) melis (Zygosaccharomyces acomis) are detected by the probes of SEQ ID NOs: 32-35, respectively. mellis) can cause all the above problems.
Moreover, Saccharomyces cerevisiae (Saccharomyces cerevisiae) detected by the probes of SEQ ID NOs: 36 and 37, respectively, causes off-flavor (ethanol odor), swelling (gas generation due to spoilage), and generation of foreign matter (spots).
In addition, Debaryomyces hansenii detected by the probe of SEQ ID NO: 38 causes off-flavors, foreign matter formation (spots), and spoilage of stored food (high sugar / high salt).

Furthermore, the yeast according to SEQ ID NOs: 39 to 42 causes mycosis (candidiasis) and the like.
Specifically, Candida parapsilosis and Candida tropicalis detected by the probes of SEQ ID NOs: 39 and 40, respectively, cause foreign body formation (spots) and mycosis (candidiasis). Candida glabrata and Pichia guilliermondii detected by the probes of SEQ ID NOs: 41 and 42, respectively, cause mycosis (candidiasis).

Moreover, the yeast which concerns on sequence number 43-48 raise | generates a strange odor and a foreign material production | generation.
Specifically, Rhodotorula glutinis, Rhodotorula minuta, Rhodotorula mucilaginosa, which are detected by the probes of SEQ ID NOs: 43 to 45, respectively, have off-flavors and foreign matter production (red) Cause spots). In addition, Brettanomyces custersianus, Brettanomyces naardenensis, and Dekkera bruxellensis detected by the probes of SEQ ID NOs: 46 to 48, respectively, are beer spoilage. It is yeast and causes off-flavor and foreign matter generation (turbidity).

Furthermore, the yeasts according to SEQ ID NOs: 49 to 52 cause various problems.
Specifically, Yarrowia lipolytica detected by the probe of SEQ ID NO: 49 causes an odor. Malassezia furfur detected by the probe of SEQ ID NO: 50 is lipid auxotrophic and is present in human fingers. Trichosporon cutaneum detected by the probe of SEQ ID NO: 51 is an infectious disease-causing bacterium. Cryptococcus laurentii detected by the probe of SEQ ID NO: 52 has a lipolytic activity and has a property of growing at a low temperature.

Therefore, the yeast probe for detecting yeast according to the present embodiment includes at least one of the following oligonucleotide probes (e) or (f), two or more of these, or all of them: (Hereinafter, it may be referred to as a C group probe). It is also preferable to use a probe set of two or more groups of A group, B group, and C group.
(E) Each oligonucleotide probe consisting of the base sequences shown in SEQ ID NOs: 32-38 (f) Each oligonucleotide probe having 90% or more identity to the base sequence of each oligonucleotide probe of (e) above

Moreover, the oligonucleotide probe for yeast detection of this embodiment is an oligonucleotide probe for yeast detection containing at least any one of these oligonucleotide probes of the following (g) or (h), these two or more, or all of these. (Hereinafter, it may be referred to as a D group probe). It is also preferable to use a probe set of two or more groups of A group, B group, C group, and D group.
(G) Each oligonucleotide probe consisting of the base sequences shown in SEQ ID NOs: 39 to 42 (h) Each oligonucleotide probe having 90% or more identity to the base sequence of each oligonucleotide probe of (g) above

Furthermore, the oligonucleotide probe for yeast detection of this embodiment includes at least one of the following (i) or (j) oligonucleotide probes, two or more of these, or all of them: (Hereinafter, it may be referred to as an E group probe). It is also preferable to use a probe set of two or more groups of A group, B group, C group, D group, and E group.
(I) Each oligonucleotide probe comprising the nucleotide sequence shown in SEQ ID NOs: 43 to 48 (j) Each oligonucleotide probe having 90% or more identity to the nucleotide sequence of each oligonucleotide probe of (i) above

Moreover, the oligonucleotide probe for yeast detection of this embodiment contains at least one of the following (k) or (l) oligonucleotide probes, two or more of these, or all of them: (Hereinafter, it may be referred to as an F group probe). It is also preferable to use a probe set of two or more groups of A group, B group, C group, D group, E group, and F group.
(K) Each oligonucleotide probe comprising the nucleotide sequence shown in SEQ ID NOs: 49 to 52 (l) Each oligonucleotide probe having 90% or more identity to the nucleotide sequence of each oligonucleotide probe of (k) above

Furthermore, the oligonucleotide probe for yeast detection of this embodiment includes at least one of the following (m) or (n) oligonucleotide probes, two or more of these, or all of them: It is also preferable to use a probe (hereinafter sometimes referred to as a G group probe). It is also preferable to use a probe set of two or more groups of A group, B group, and G group.
(M) Each oligonucleotide probe comprising the nucleotide sequence shown in SEQ ID NOs: 32-52 (n) Each oligonucleotide probe having 90% or more identity to the nucleotide sequence of each oligonucleotide probe of (m) above

  The probe of SEQ ID NO: 36 for detecting Saccharomyces cerevisiae was selected from the β tubulin gene, and the other probes of SEQ ID NOs: 1-35 and 37-52 were The rDNA gene is selected from the ITS region.

[Probe synthesis]
The yeast probe for detecting yeast of this embodiment has a length of 25 to 39 bases, and can be synthesized by a general DNA synthesizer. As the probes consisting of the base sequences shown in SEQ ID NOs: 1 to 52 used in Examples described later, those synthesized by a DNA synthesizer were used. In addition, the primer which consists of a base sequence shown to sequence number 53-56 used for PCR in the Example is also 19-26 bases in length, and what was synthesize | combined with the DNA synthesizer was used.

  Further, the probe consisting of the base sequence shown in SEQ ID NO: 1 to 52 in this embodiment is not limited to the sequence itself, and one or several bases are deleted, substituted or substituted in the base sequence shown in SEQ ID NO: 1 to 52. Added probes can be used. A probe capable of hybridizing under stringent conditions to a nucleic acid fragment consisting of a base sequence complementary to the base sequences shown in SEQ ID NOs: 1 to 52 can also be used. Moreover, the probe which has a base sequence complementary to the probe which consists of these probes and the base sequence shown to sequence number 1-52 can also be used.

  Stringent conditions refer to conditions in which specific hybrids are formed and non-specific hybrids are not formed. For example, a DNA having high homology (with a homology of 90% or more, preferably 95% or more) with respect to the DNA consisting of the base sequence shown in SEQ ID NOs: 1 to 52 consists of the base sequence shown in SEQ ID NOs: 1 to 52 The conditions for hybridizing with DNA each consisting of a base sequence complementary to DNA can be mentioned. Usually, it means a case where hybridization occurs at a temperature about 5 ° C. to about 30 ° C., preferably about 10 ° C. to about 25 ° C. lower than the melting temperature (Tm) of the complete hybrid. For stringent conditions, see J.M. Sambrook et al., Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989), especially in section 11.45 “Conditions for Hybridization”.

[Microarray for yeast detection]
The yeast array for detecting yeast according to this embodiment is characterized in that the above-described oligonucleotide probe for detecting yeast is arranged on a substrate.
This yeast detection microarray can be produced by an existing general method using probes having the nucleotide sequences shown in SEQ ID NOs: 1 to 52.
For example, when creating an affixed DNA chip as this microarray for yeast detection, the probe can be prepared by immobilizing probes on a glass substrate with a DNA spotter and forming spots corresponding to each probe. it can. When a synthetic DNA chip is produced, it can be produced by synthesizing a single-stranded oligo DNA having the above sequence on a glass substrate by a photolithography technique. Furthermore, the substrate is not limited to glass, and a plastic substrate, a silicon wafer, or the like can also be used. Further, the shape of the substrate is not limited to a flat plate shape, and may be various three-dimensional shapes, and a substrate having a functional group introduced so that a chemical reaction can be performed on the surface can be used. .

  The yeast array microarray for use in this embodiment may have all or part of the probes having the base sequences shown in SEQ ID NOs: 1 to 52 immobilized thereon. In addition, the probe may be fixed for each set of probes in the various groups.

[Method of detecting yeast]
The method for detecting yeast using the oligonucleotide probe for yeast detection and the microarray for yeast detection of the present embodiment can be, for example, as follows.
That is, it can include (1) collecting yeast, (2) extracting DNA, (3) amplifying the target region, and (4) confirming the amplified product.

(1) Yeast collection First, yeast is collected from the environment. For example, samples are collected by wiping specimens from food factory walls, floors, doors, windows, manufacturing equipment (instruments, kitchen knives, bats), tanks, workers' hands, work clothes, and the like. Next, the obtained specimen is frozen with liquid nitrogen or the like to disrupt yeast cells.

(2) Extraction of DNA Next, genomic DNA is extracted from the obtained disrupted yeast cells. Genomic DNA can be extracted by a general method such as a method using a CTAB method (Cetyl trimethyl ammonium bromide) or a method using a DNA extraction apparatus.

(3) Amplification of target region Next, the target region in the extracted genomic DNA is amplified. That is, a DNA fragment containing a target region in genomic DNA is amplified. The method for amplifying the target region is not particularly limited, but the PCR method can be suitably used. In the PCR method, a target region is amplified using a PCR reaction solution containing a primer set for amplifying the target region. A general thermal cycler or the like can be used as the PCR apparatus.
In this embodiment, the target region of each yeast can be suitably amplified by performing PCR under the following reaction conditions, for example.
(A) 95 ° C. 10 minutes, (b) 95 ° C. (DNA denaturation step) 30 seconds, (c) 56 ° C. (annealing step) 30 seconds, (d) 72 ° C. (DNA synthesis step) 60 seconds ((b) to (D) 40 cycles), (e) 72 ° C. 10 minutes

  As a reaction solution for PCR, for example, a solution having the following composition is preferably used. That is, a nucleic acid synthesis substrate (dNTPmixture (dCTP, dATP, dTTP, dGTP)), primer set, nucleic acid synthase (Nova Taq HotStart DNA polymerase, etc.), fluorescent labeling reagent (Cy5-dCTP, etc.), sample genomic DNA, buffer solution , And a PCR reaction solution containing water as the remaining component can be preferably used. For example, Ampdirect (R) (manufactured by Shimadzu Corporation) can be used as the buffer solution.

In the yeast detection method using the yeast probe for oligonucleotide detection and the microarray for yeast detection according to this embodiment, DNA fragments are amplified using the ITS region of rDNA and / or the β tubulin gene in the genomic DNA of yeast as a target region. Done.
As a primer set for amplifying the ITS region, a primer set consisting of the nucleotide sequences of SEQ ID NOs: 53 and 54 (SEQ ID NO: 53: forward primer, SEQ ID NO: 54: reverse primer) can be suitably used. Moreover, as a primer set for amplifying the β tubulin gene, a primer set consisting of the nucleotide sequences of SEQ ID NOs: 55 and 56 (SEQ ID NO: 55: forward primer, SEQ ID NO: 56: reverse primer) can be suitably used.

(4) Confirmation of amplification product Next, the amplification product is dropped on the yeast detection microarray of this embodiment, and the presence or absence of the amplification product is detected by detecting the label of the amplification product hybridized to the probe arranged on this. Check. Thereby, the yeast which exists in the environment of test object can be specified.
The detection of the label can be performed by using a general label detection device such as a fluorescence scanning device, for example, by measuring the fluorescence intensity of the amplification product using BIOSHOT (R) of Toyo Kohan Co., Ltd. Can do. The measurement result is preferably obtained as an S / N ratio (Signal to Noise ratio) value. This is because it is possible to accurately determine whether the measurement result is positive or negative based on the S / N ratio value. Generally, when the S / N ratio value is 3 or more, it is determined to be positive. Can do. The S / N ratio value described in the present specification is calculated by (median fluorescence intensity value−background value) ÷ background value. The label is not limited to fluorescence, and other labels can also be used.

[Yeast detection kit]
In the yeast detection kit of this embodiment, the above primer set for amplifying a nucleic acid fragment containing a target region in yeast DNA and a probe for confirming the presence or absence of an amplification product are arranged. Yeast detection microarray.
That is, a microarray in which the oligonucleotide probe for yeast detection of this embodiment described above is arranged on a substrate, a primer comprising the base sequence shown in SEQ ID NO: 53 for amplifying the ITS region of yeast DNA, and SEQ ID NO: 54 It is preferable to have a solution containing a primer set consisting of a primer having the base sequence shown.

  Further, a microarray in which the oligonucleotide probe for yeast detection of this embodiment described above is arranged on a substrate, a primer comprising the base sequence shown in SEQ ID NO: 53 for amplifying the ITS region of yeast DNA, and SEQ ID NO: 54 A primer set consisting of a primer consisting of the nucleotide sequence shown, and a primer consisting of the nucleotide sequence shown in SEQ ID NO: 55 and a primer consisting of the nucleotide sequence shown in SEQ ID NO: 56 for amplifying the β-tubulin gene of yeast DNA It is also preferable to have a solution containing the primer set.

According to the oligonucleotide probe for yeast detection, the microarray for yeast detection, and the kit for yeast detection of this embodiment, a wide variety of yeasts can be appropriately selected by using the above 52 types of probes that can specifically identify yeast. Can be detected.
Therefore, it is possible to quickly and accurately detect a wide variety of yeasts in an environmental test or the like for confirming the presence or absence of yeast.

  Hereinafter, the test performed in order to confirm the effect of the oligonucleotide probe for yeast detection which concerns on embodiment of this invention, the microarray for yeast detection, and the kit for yeast detection is demonstrated concretely.

[Test 1]
As the microarray for yeast detection, Gene Silicon (R) (manufactured by Toyo Kohan Co., Ltd.) was used, and the probes with SEQ ID NOS: 1 to 52 were arranged. Each probe was synthesized by Sigma Aldrich Japan LLC. In addition, each probe was immobilized on the substrate by microarrayer.

  As the yeast to be detected, a strain obtained from the NITE Biological Resource Center, Biotechnology Headquarters, National Institute of Technology and Evaluation, Product Evaluation Technology was used. Specifically, as shown in FIGS. 5 to 7, the following 51 strains of yeast were used.

  Kazakstania Excigua (NBRC 1128), Hansenia Spora Ozmofila (NBRC 10832), Hansenia Spora Occidentalis (NBRC 1819), Hansenia Spora Barbiensis (NBRC 0115), Clavispora Lucitaniae (NBRC 10059), La Chanlancea RB Gigotorraspola Florentinas (NBRC 1088), Debaryomyces Carsoni (NBRC 0946), Saccharomycopsis Capsularis (NBRC 0672), Torlaspola Globosa (NBRC 1160), Torraspola Del Bricky (NBRC 0955), Eremosecium Ashby (NBRC 0557), Barnet Zaima Platensis (NBRC 10696), Candida Berti (NBRC 10266), Candida Martosa (NBRC 1977), Citellomyces Matritensis (NBRC 0954), Isachenchia Occiden Talis (NBRC 1904), Kazaxstania Unispora (NBRC 0316), Kregor Van Ridge Fulksum (NBRC 0773), Lindnera Saturnus (NBRC 10697), Roderomyces elongisporus (NBRC 1676), Nakaseomyces delfensis (NBRC 10602), Naumovia Castelli (NBRC 1992), Paffymyces thermotolerance (NBRC 10024), Pichia Farinosa (NBRC 1163), Pichia Fermentans (NBRC 1124), Saturnispora Asharni (NBRC 10942), Tetrapysis Pora Faffy (NBRC 1672) , Vanderwaldzaima Polyspora (NBRC 10782), Wicker Hamomises ciferii (NBRC 0793), Gigoascus Helenikas (NBRC 1575), Candida Crusei (NBRC 1395), Gigosaccharomyces baili (NBRC 1098), Gigosaccharomyces・ Bisporus (NBRC 1131), Gigosaccharomyces meris (NBRC 1615), Saccharomyces cerevisiae (NBRC 10217), Debaryomyces Hanseni (NBRC 0015), Candida parapsilosis (NBRC 1396), Candida tropicalis (NBRC 1400), Candida・ Grabrata (NBRC 0622), Pichia Guillermondi (NBRC 10279), Rhodotorula Glutinis (NBRC 1125), Rhodotorula Minutta (NBRC 0715), Rhodotorula Musiraginosa (NBRC 0909), Brettanomyces Custellianus (NBRC 1585) ), Brettanomyces nadenensis (NBRC 1588), Deckera Burgerensis (NBRC 0677), Yarrowia Lipolitica (NBRC 1548), Malassezia fullfur (NBRC 0656), Trichosporon kutanium (NBRC 1198), Cryptococcus lauren Tee (NBR C 0609)

These yeasts were individually cultured for each strain. Culturing was carried out using a PDA medium or an M40Y medium and allowed to stand for 7 days in the dark at 25 ° C.
Furthermore, the cultured yeast colonies were collected, individually placed in vials containing φ0.5 mm zirconia beads, frozen in samples by immersion in liquid nitrogen, and then the yeast cells were disrupted using a shaking device.
Subsequently, yeast genomic DNA was extracted with a DNA extraction apparatus, and the ITS region and / or β-tubulin gene of each yeast was amplified for each strain by PCR.

  Here, as the PCR reaction solution, a primer set consisting of a primer consisting of the base sequence shown in SEQ ID NO: 53 and a primer consisting of the base sequence shown in SEQ ID NO: 54 for amplifying the ITS region of the yeast DNA is uniformly used. And a primer set consisting of a primer consisting of the base sequence shown in SEQ ID NO: 55 and a primer consisting of the base sequence shown in SEQ ID NO: 56 for amplifying the β-tubulin gene of yeast DNA was used. These primers were synthesized by Sigma Aldrich Japan LLC.

As a reaction solution for PCR, Ampdirect (R) (manufactured by Shimadzu Corporation) was used, and 20 μl of the following composition was prepared for each strain.
1. Ampdirect (G / Crich) 4.0μl
2. Ampdirect (addition-4) 4.0μl
3. dNTPmix 1.0μl
4). Cy-5dCTP 0.2μl
5. ITS region forward primer (2.5μM) 1.0μl
6). ITS region reverse primer (2.5μM) 1.0μl
7). β-tubulin gene forward primer (10μM) 1.0μl
8). β-tubulin gene reverse primer (10μM) 1.0μl
9. NovaTaq HotStart DNA polymerase 0.2μl
10. Template DNA (100pg / μl) 1.0μl
11. Water (water until 20.0 μl total)

Using each of the PCR reaction solutions, DNA was amplified using the nucleic acid amplification apparatus (TaKaRa PCR Thermal Cycler Dice (R) Gradient manufactured by Takara Bio Inc.) under the following conditions.
(A) 95 ° C. 10 minutes (b) 95 ° C. 30 seconds (c) 56 ° C. 30 seconds (d) 72 ° C. 60 seconds (40 cycles of (b) to (d))
(E) 72 ° C 10 minutes

Next, a buffer solution (3 × SSC citric acid-saline + 0.3% SDS) was mixed with the PCR amplification product, heated at 94 ° C. for 5 minutes, and dropped onto the yeast detection microarray. The microarray was allowed to stand at 45 ° C. for 1 hour, and the PCR product that did not hybridize was washed away using the above buffer.
Then, using a label detection device (BIOSHOT (R), manufactured by Toyo Kohan Co., Ltd.), the fluorescence intensity (fluorescence intensity of the amplification product bound to the probe) in each probe arranged in the yeast detection microarray is measured. The S / N ratio value ((median fluorescence intensity value−background value) ÷ background value) for each probe was obtained. The results are shown in FIGS.

As shown in these figures, the S / N ratio values of the probes each having the base sequence shown in SEQ ID NOs: 1 to 52 are all 3 or more, indicating a positive result.
Thus, it was found that these probes function effectively for detecting each target yeast.

[Test 2]
Tests were conducted to confirm that the probes placed in the yeast detection microarray of this embodiment function effectively when multiplex PCR is performed to simultaneously amplify target regions in the DNA of a plurality of yeasts to be detected. It was.

The same yeast array as in Test 1 was used. Moreover, the same strain as test 1 was used for the yeast to be detected.
In this test, as shown in FIGS. 8 to 10, five kinds of these yeasts were randomly combined to prepare the following 12 groups of samples, and the yeasts were cultured for each sample. Therefore, some yeasts are included in a plurality of samples.

(Sample 1)
Hansenia spora osmophila (corresponding SEQ ID NO: 2), Phatomyces thermotolerance (corresponding SEQ ID NO: 24), Tetrapysispora faphy (corresponding SEQ ID NO: 28), Gigosaccharomyces meris (corresponding SEQ ID NO: 35), Brettanomyces nadenensis (corresponding SEQ ID NO: 47)

(Sample 2)
Gigotorraspola Florentinas (Corresponding SEQ ID NO: 7), Candida Berti (Corresponding SEQ ID NO: 14), Roderomyces elongisporus (Corresponding SEQ ID NO: 21), Pichia Farinosa (Corresponding SEQ ID NO: 25), Debaryomyces Hanseni (Corresponding SEQ ID NO: 38) )

(Sample 3)
Hansenia spora balbiensis (corresponding sequence number 4), Isachenchia occidentalis (corresponding sequence number 17), Nakaseomyces delfensis (corresponding sequence number 22), Digosaccharomyces baili (corresponding sequence number 33), Candida parapsilosis (corresponding sequence number 39) )

(Sample 4)
Lachanthea thermotolerance (corresponding SEQ ID NO: 6), Eremocesium ashbee (corresponding SEQ ID NO: 12), Barnet Zyma Platensis (corresponding SEQ ID NO: 13), Pichia Fermentans (corresponding SEQ ID NO: 26), Brettanomyces custercianus (Corresponding sequence number 46)

(Sample 5)
Kazaxstania Exigua (corresponding SEQ ID NO: 1), Naumovia Castelli (corresponding SEQ ID NO: 23), Vanderwald Zyma Polyspora (corresponding SEQ ID NO: 29), Gigosaccharomyces bisporus (corresponding SEQ ID NO: 34), Deckella Burgcerensis (corresponding SEQ ID NO: 48)

(Sample 6)
Hansenia spora occidentalis (corresponding sequence number 3), Torlas pora globusa (corresponding sequence number 10), Kazakstania unispora (corresponding sequence number 18), Rhodotorula glutinis (corresponding sequence number 43), cryptococcus lorenti (corresponding sequence number 52)

(Sample 7)
Clavispora lucitaniae (corresponding SEQ ID NO: 5), Candida maltosa (corresponding SEQ ID NO: 15), Krega Van Ridge Frucsum (corresponding SEQ ID NO: 19), Candida glabrata (corresponding SEQ ID NO: 41), Malassezia full full (corresponding array Number 50)

(Sample 8)
Saccharomycopsis capsularis (corresponding SEQ ID NO: 9), Wicker Hammyces ciferii (corresponding SEQ ID NO: 30), Saccharomyces cerevisiae (corresponding SEQ ID NO: 36, 37), Pichia guilier mondi (corresponding SEQ ID NO: 42), Rhodotorra Minuta (corresponding sequence number 44)

(Sample 9)
Torlaspola del Bricky (corresponding SEQ ID NO: 11), Lindnera Saturnus (corresponding SEQ ID NO: 20), Candida Crusei (corresponding SEQ ID NO: 32), Saccharomyces cerevisiae (corresponding SEQ ID NO: 36, 37), Trichosporon tatanium (corresponding SEQ ID NO: 51) )

(Sample 10)
Citeromyces matritensis (corresponding SEQ ID NO: 16), Saturnispora asharni (corresponding SEQ ID NO: 27), Candida tropicalis (corresponding SEQ ID NO: 40), Rhodotorula mucilainosa (corresponding SEQ ID NO: 45), Yarrowia lipolytica (corresponding SEQ ID NO: 49)

(Sample 11)
Debaryomyces Karsoni (corresponding SEQ ID NO: 8), Eremocesium Ashby (corresponding SEQ ID NO: 12), Vanderwald Zyma Polyspora (corresponding SEQ ID NO: 29), Gigosaccharomyces meris (corresponding SEQ ID NO: 35), Deckella Burgcerensis (corresponding SEQ ID NO: 48)

(Sample 12)
Kregor Van Ridge Fulsum (corresponding SEQ ID NO: 19), Saturnispora Asharni (corresponding SEQ ID NO: 27), Digoascus Helenikas (corresponding SEQ ID NO: 31), Rhodotorula minuta (corresponding SEQ ID NO: 44), Trichosporon kutanium (corresponding array) Number 51)

Culturing was carried out using a PDA medium or an M40Y medium and allowed to stand for 7 days in the dark at 25 ° C.
Then, collect the colonies of yeast cultured for each sample in a lump, put each sample in a vial containing φ0.5 mm zirconia beads, immerse in liquid nitrogen and freeze the sample, then use a shaking device, Yeast cells were disrupted.
Subsequently, yeast genomic DNA was extracted with a DNA extraction apparatus, and the ITS region and / or β-tubulin gene of each yeast was simultaneously amplified for each sample by PCR. The primer set used in the PCR method is the same as in Test 1.

As a reaction solution for PCR, Ampdirect (R) (manufactured by Shimadzu Corporation) was used, and 20 μl of the following composition was prepared for each sample.
1. Ampdirect (G / Crich) 4.0μl
2. Ampdirect (addition-4) 4.0μl
3. dNTPmix 1.0μl
4). Cy-5dCTP 0.2μl
5. ITS region forward primer (2.5μM) 1.0μl
6). ITS region reverse primer (2.5μM) 1.0μl
7). β-tubulin gene forward primer (10μM) 1.0μl
8). β-tubulin gene reverse primer (10μM) 1.0μl
9. NovaTaq HotStart DNA polymerase 0.2μl
10. Template DNA (50pg / μl each, total 250pg / μl) 1.0μl
11. Water (water until 20.0 μl total)

  Using each of the above PCR reaction solutions, a nucleic acid amplification apparatus (TaKaRa PCR Thermal Cycler Dice (R) Gradient manufactured by Takara Bio Inc.) is used to amplify DNA under the same conditions as in Test 1, and for yeast detection The fluorescence intensity of each probe arranged in the microarray (the fluorescence intensity of the amplification product bound to the probe) was measured, and the S / N ratio value of each probe was obtained. The results are shown in FIGS.

As shown in these figures, the S / N ratio values of the probes contained in each sample are all 3 or more, indicating a positive result. Moreover, these probes had high specificity and no false positive reaction was observed.
As described above, it was found that the oligonucleotide probe for yeast detection of the present embodiment functions effectively even when multiplex PCR for simultaneously amplifying target regions in a plurality of yeast DNAs to be detected is performed.

The present invention is not limited to the above-described embodiments and examples, and it goes without saying that various modifications can be made within the scope of the present invention.
For example, the probes arranged in the yeast detection microarray of the present embodiment are not limited to one spot per type, and a plurality of each probe may be arranged. In addition, yeasts other than the detection target yeast of this embodiment and other probes for detecting mold may be arranged in the microarray together with the probes in this embodiment, and can be changed as appropriate.

  INDUSTRIAL APPLICABILITY The present invention can be suitably used for specific and multiplex detection of multiple types of yeast in environmental inspection, food inspection, epidemiological environmental inspection, clinical test, livestock hygiene, plant disease diagnosis, and the like. .

Claims (10)

The oligonucleotide probe for a yeast detection containing at least any one of the following oligonucleotide probes of (a) or (b).
(A) Each oligonucleotide probe consisting of the base sequence shown in SEQ ID NO: 1 to 12 (b) Each oligonucleotide probe having 90% or more identity to the base sequence of each oligonucleotide probe of (a) above A set of oligonucleotide probes for yeast detection comprising the oligonucleotide probe according to claim 1 and at least one of the following oligonucleotide probes (c) or (d):
(C) Each oligonucleotide probe consisting of the nucleotide sequence shown in SEQ ID NOs: 13 to 31 (d) Each oligonucleotide probe having 90% or more identity to the nucleotide sequence of each oligonucleotide probe of (c) above A set of oligonucleotide probes for yeast detection comprising the oligonucleotide probe according to claim 1 or 2 and at least one of the following oligonucleotide probes (e) or (f):
(E) Each oligonucleotide probe consisting of the base sequences shown in SEQ ID NOs: 32-38 (f) Each oligonucleotide probe having 90% or more identity to the base sequence of each oligonucleotide probe of (e) above The set of the oligonucleotide probe for yeast detection containing the oligonucleotide probe in any one of Claims 1-3 and at least any one of the following oligonucleotide probes of (g) or (h).
(G) Each oligonucleotide probe consisting of the base sequences shown in SEQ ID NOs: 39 to 42 (h) Each oligonucleotide probe having 90% or more identity to the base sequence of each oligonucleotide probe of (g) above A set of oligonucleotide probes for yeast detection comprising the oligonucleotide probe according to any one of claims 1 to 4 and at least one of the following oligonucleotide probes (i) or (j):
(I) Each oligonucleotide probe comprising the nucleotide sequence shown in SEQ ID NOs: 43 to 48 (j) Each oligonucleotide probe having 90% or more identity to the nucleotide sequence of each oligonucleotide probe of (i) above A set of oligonucleotide probes for yeast detection comprising the oligonucleotide probe according to any one of claims 1 to 5 and at least one of the following oligonucleotide probes (k) or (l):
(K) Each oligonucleotide probe comprising the nucleotide sequence shown in SEQ ID NOs: 49 to 52 (l) Each oligonucleotide probe having 90% or more identity to the nucleotide sequence of each oligonucleotide probe of (k) above A set of oligonucleotide probes for detecting yeast, comprising the oligonucleotide probe according to claim 1 or 2 and at least one of the following oligonucleotide probes (m) or (n):
(M) Each oligonucleotide probe comprising the nucleotide sequence shown in SEQ ID NOs: 32-52 (n) Each oligonucleotide probe having 90% or more identity to the nucleotide sequence of each oligonucleotide probe of (m) above   A microarray for yeast detection, wherein the oligonucleotide probe according to any one of claims 1 to 7 is arranged on a substrate. A microarray in which the oligonucleotide probes according to at least one of claims 1 to 7 are arranged on a substrate;
And a solution containing a primer set consisting of a primer consisting of the base sequence shown in SEQ ID NO: 53 and a primer consisting of the base sequence shown in SEQ ID NO: 54 for amplifying the ITS region of the yeast DNA. Yeast detection kit. A microarray in which the oligonucleotide probes according to at least one of claims 1 to 7 are arranged on a substrate;
A primer set consisting of a primer consisting of the base sequence shown in SEQ ID NO: 53 and a primer consisting of the base sequence shown in SEQ ID NO: 54 for amplifying the ITS region of yeast DNA, and a β-tubulin gene of yeast DNA And a solution containing a primer set consisting of a primer consisting of the base sequence shown in SEQ ID NO: 55 and a primer consisting of the base sequence shown in SEQ ID NO: 56.

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