JP7006011B2 - Yeast detection oligonucleotide probes, yeast detection microarrays, and yeast detection kits - Google Patents

Description

The present invention relates to oligonucleotides that are effective in detecting and identifying fungi that are problematic in foods and the environment.

In recent years, in food manufacturing sites, clinical sites, agricultural sites, cultural property protection environments, etc., the presence or absence of fungi such as mold and yeast is inspected to confirm safety and soundness, and to prevent their reproduction. Is important.
In the examination of such fungi, an identification method using a gene is performed. That is, DNA is extracted from a fungus collected from the environment, the target region is amplified by a PCR (polymerase chain reaction) method or the like, and the amplified product is analyzed to identify the fungus in the environment. ing. As a method for analyzing the amplification product, a method of analyzing the size of the amplification product by electrophoresis and a method of using a microarray in which a probe that binds complementarily to the amplification product is arranged 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 developed a mold inspection technique in an indoor environment, and various oligonucleotide probes capable of detecting mold floating in the air or adhering to the environment are arranged on a substrate. I am creating a microarray.
By the way, yeast belonging to the same fungus as mold is collected from the environment together with mold by a collection method such as wiping, and the yeast gene is simultaneously amplified together with the mold gene in the DNA amplification step. However, while mold is detected by microarray, for yeast, the oligonucleotide probe that can detect it is not placed in the microarray, so that the amplification product of the yeast gene is actually obtained in the mold inspection step. Despite this, yeast was not detected.

Here, it is useful to make yeast detectable, because some yeasts cause problems such as deterioration of foods and candidiasis in humans depending on the type of yeast.
Examples of the technique 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 yeasts.

However, for many yeasts, oligonucleotide probes for detecting them have not been developed yet, and it has not been possible to detect them.
Therefore, the present inventors have diligently researched and organized the status of reports of harmfulness to a large number of yeasts, and developed an oligonucleotide probe (52 types of probes) capable of detecting 51 types of yeasts among them. I succeeded in doing so.

That is, the yeasts according to SEQ ID NOs: 1 to 12 are types that adversely affect foods, agriculture, etc., and oligonucleotide probes that can detect them (particularly those selected from the ITS region of yeast DNA) are selected. I haven't found it so far.
In addition, the yeasts according to SEQ ID NOs: 13 to 31 are detectable as yeasts that may cause a decay phenomenon in order to understand their distribution. No oligonucleotide probe capable of detecting these (particularly selected from the ITS region of yeast DNA) has been found so far.

Here, in the microarray for mold inspection that the inventors have already developed, it is possible to collectively detect molds of the same genus and subgenus. The reason is that closely related mold species have similar properties such as viable environment (temperature / humidity / water activity) and characteristics (heat resistance, psychrophilicity, toxin production), resulting in similar damage. Because. As for yeast, even if the harmfulness has not been reported at this time, there is a potential risk of causing similar damage if the properties are similar. Therefore, the present inventors have made it possible to detect yeasts according to SEQ ID NOs: 13 to 31 as such potentially dangerous yeasts.

In addition, the yeasts of SEQ ID NOs: 32 to 52 have been reported to be harmful in foods and medical settings, and there have already been reports on oligonucleotide probes capable of detecting them.
However, there has been no report on a unique oligonucleotide probe in which 51 types of yeast targeted by the present invention are arranged in one microarray and can be detected at the same time.

A probe is selected from a specific target region in the DNA of each of a plurality of yeasts by selecting a base sequence that has a base sequence that specifically binds only to each yeast and does not bind to other types of yeast. In theory, each probe can specifically detect these multiple yeasts. However, in reality, even the probe obtained in this way does not always function effectively. Therefore, in order to create each probe, it is necessary to confirm the effect of the probe by experiment and find a probe that functions effectively, which requires a lot of trial and error.

The present invention has been made in view of the above circumstances, and is an oligonucleotide probe for yeast detection, a microarray for yeast detection, and a kit for yeast detection capable of rapidly and accurately and specifically detecting a wide range of yeasts. 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, which comprises at least one of the following oligonucleotide probes (a) or (b).
(A) Each oligonucleotide probe consisting of the nucleotide sequences shown in SEQ ID NOs: 1 to 12 (b) Each oligonucleotide probe having 90% or more identity with respect to the nucleotide sequence of each oligonucleotide probe of (a) above.

Further, the yeast detection microarray of the present invention has a configuration in which at least the above oligonucleotide probe is arranged on a substrate.
In addition, the kit for detecting yeast of the present invention comprises at least a microarray in which the above-mentioned oligonucleotide probe is arranged on a substrate, and a primer and a sequence consisting of the base sequence shown in SEQ ID NO: 53 for amplifying the ITS region of DNA of yeast. It is configured to have 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 detect a wide range of yeasts rapidly, accurately and specifically.

It is a figure which shows the base sequence of the oligonucleotide probe (SEQ ID NO: 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 (SEQ ID NO: 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 (SEQ ID NO: 32 to 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 region of the target yeast to detect 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) individually detected in the test 1 about the oligonucleotide probe (SEQ ID NO: 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) individually detected in the test 1 about the oligonucleotide probe (SEQ ID NO: 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) individually detected in the test 1 about the oligonucleotide probe (SEQ ID NO: 32 to 52) for yeast detection which concerns on embodiment of this invention. It is a figure which shows the yeast mixed in the test 2 (samples 1 to 4), the strain number, and the 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 mixed in the test 2 (samples 5-8), the strain number, and the 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 mixed in the test 2 (samples 9-12), the strain number, and the corresponding sequence number 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) which was mixed and detected in the test 2 about the oligonucleotide probe (SEQ ID NO: 1-18) for yeast detection which concerns on embodiment of this invention. It is a figure which shows the fluorescence intensity (S / N ratio value) which was mixed and detected in the test 2 about the oligonucleotide probe (SEQ ID NO: 19-36) for yeast detection which concerns on embodiment of this invention. It is a figure which shows the fluorescence intensity (S / N ratio value) which was mixed and detected in the test 2 about the oligonucleotide probe (SEQ ID NO: 37-52) for yeast detection which concerns on embodiment of this invention.

Hereinafter, an embodiment of the oligonucleotide probe for yeast detection, the microarray for yeast detection, and the 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 embodiments and the examples described later.

[Oligonucleotide probe for yeast detection]
The oligonucleotide probe for yeast detection of the present embodiment is a nucleic acid fragment consisting of the base sequences specified by SEQ ID NOs: 1 to 52, respectively.
First, the yeasts according to SEQ ID NOs: 1 to 12 are a type that causes a deterioration phenomenon that adversely affects foods, agriculture, and the like.
Specifically, Kazachstania exigua detected by the probe of SEQ ID NO: 1 causes an offensive odor (ethanol odor), swelling (generation of gas due to putrefaction), and foreign matter generation (spots).
Hanseniaspora osmophila detected by the probe of SEQ ID NO: 2 has an offensive odor and is symbiotic with Drosophila sp., Therefore, yeast contamination of fruits and foods transmitted from the body surface of flies. There is a problem of being involved in.

In addition, Hanseniaspora occidentalis, Hanseniaspora valbyensis, Clavispora lusitaniae, and Lachansea thermotrelance, respectively, detected by the probes of SEQ ID NOs: 3 to 7. -Lorentinas (Zygotorulaspora florentinus) also has a problem of being involved in yeast contamination mediated by the fly body surface because it has a symbiotic relationship with the fruit fly (Drosophila sp.).

Debaryomyces carsonii detected by the probe of SEQ ID NO: 8 causes an offensive odor.
Saccharomycopsis capsularis detected by the probe of SEQ ID NO: 9 causes foreign body formation (black spots).
Torulaspora globosa and Torulaspora delbrueckii, which are detected by the probes of SEQ ID NOs: 10 and 11, respectively, are spoilage yeasts of 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 grain scab.

Therefore, the oligonucleotide probe for yeast detection of the present embodiment contains at least one of the following oligonucleotide probes (a) or (b), two or more of them, or all of them. (Hereinafter, it may be referred to as a probe of group A).
(A) Each oligonucleotide probe consisting of the nucleotide sequences shown in SEQ ID NOs: 1 to 12 (b) Each oligonucleotide probe having 90% or more identity with respect to the nucleotide sequence of each oligonucleotide probe of (a) above. According to the oligonucleotide probe for detecting yeast of the present embodiment, it is possible to detect yeast that causes a deterioration phenomenon that adversely affects food, agriculture, etc., which could not be detected so far.

In addition, the yeasts according to SEQ ID NOs: 13 to 31 are a type having a potential to cause a decay phenomenon.
Specifically, Barnettozyma pratensis detected by the probe of SEQ ID NO: 13, Candida berthetii detected by the probe of SEQ ID NO: 14, and Candida detected by the probe of SEQ ID NO: 15. Candida maltosa, Citeromyces matritensis detected by the probe of SEQ ID NO: 16, Issatchenkia occidentalis detected by the probe of SEQ ID NO: 17, detected by the probe of SEQ ID NO: 18. Kazachstania unispora, Kregervanrija fluxuum detected by the probe of SEQ ID NO: 19, Lindnera saturnus detected by the probe of SEQ ID NO: 20, Lindnera saturnus, SEQ ID NO: 21 Lodderomyces elongisporus detected by the probe and Nakaseomyces delphensis detected by the probe of SEQ ID NO: 22.

In addition, Naumovia castellii detected by the probe of SEQ ID NO: 23, Phaffomyces thermotolerans detected by the probe of SEQ ID NO: 24, and 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 tetrapisispora detected by the probe of SEQ ID NO: 28. • Fafi (Tetrapisispora phaffii), Vanderwaltozyma polyspora detected by the probe of SEQ ID NO: 29, Wickerhamomyces ciferrii detected by the probe of SEQ ID NO: 30, probe of SEQ ID NO: 31. Zygoascus hellenicus detected by.

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

In addition, the yeasts according to SEQ ID NOs: 32 to 38 have a large amount of offensive odor (ethanol odor), swelling (generation of gas due to putrefaction), foreign matter generation (spots), and putrefaction of preserved foods (high sugar / high salt). Wake up.
Specifically, Candida krusei, Zygosaccharomyces bailii, Zygosaccharomyces bisporus, and Zygosaccharomyces melis, which are detected by the probes of SEQ ID NOs: 32 to 35, respectively. mellis) can cause all of the above problems.
In addition, Saccharomyces cerevisiae detected by the probes of SEQ ID NOs: 36 and 37 causes an offensive odor (ethanol odor), swelling (generation of gas due to putrefaction), and foreign matter generation (spots), respectively.
In addition, Debaryomyces hansenii detected by the probe of SEQ ID NO: 38 causes stinks, foreign matter formation (spots), and spoilage of preserved foods (high sugar / high salt).

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

In addition, the yeasts of SEQ ID NOs: 43 to 48 cause offensive odors and foreign matter production.
Specifically, Rhodotorula glutinis, Rhodotorula minuta, and Rhodotorula mucilaginosa, which are detected by the probes of SEQ ID NOs: 43 to 45, respectively, have an offensive odor and foreign body formation (red). Spots). In addition, Brettanomyces custersianus, Brettanomyces naardenensis, and Dekkera bruxellensis, which are detected by the probes of SEQ ID NOs: 46 to 48, respectively, are beer spoilage. It is a yeast that causes offensive odors and foreign matter formation (turbidity).

Furthermore, the yeasts of SEQ ID NOs: 49-52 cause various problems.
Specifically, Yarrowia lipolytica detected by the probe of SEQ ID NO: 49 causes an offensive odor. Malassezia furfur, detected by the probe of SEQ ID NO: 50, is lipid-requiring and is present on 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 lipid-degrading activity and has the property of growing at low temperature.

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

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

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

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

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

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 to 35 and 37 to 52 were used. It is selected from the rDNA gene ITS region.

[Probe synthesis]
The oligonucleotide probes for yeast detection of the present embodiment are all 25 to 39 bases in length 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 the examples described later, those synthesized by a DNA synthesizer were used. The primers consisting of the base sequences shown in SEQ ID NOs: 53 to 56 used for PCR in the examples also had a length of 19 to 26 bases, and those synthesized by a DNA synthesizer were used.

Further, the probe consisting of the base sequences shown in SEQ ID NOs: 1 to 52 in the present embodiment is not limited to the sequence itself, and one or several bases are deleted, substituted or deleted in the base sequences shown in SEQ ID NOs: 1 to 52. An attached probe can be used. Further, a probe capable of hybridizing to a nucleic acid fragment consisting of a base sequence complementary to the base sequences shown in SEQ ID NOs: 1 to 52 under stringent conditions can also be used. Further, these probes and probes having a base sequence complementary to the probe consisting of the base sequences shown in SEQ ID NOs: 1 to 52 can also be used.

The stringent condition means a condition in which a specific hybrid is formed and a non-specific hybrid is not formed. For example, a DNA having high homology (similarity of 90% or more, preferably 95% or more) with respect to the DNA consisting of the base sequences shown in SEQ ID NOs: 1 to 52 comprises the base sequences shown in SEQ ID NOs: 1 to 52. Conditions for hybridizing with DNA having a base sequence complementary to DNA can be mentioned. Usually, it refers to the case where hybridization occurs at a temperature of about 5 ° C. to about 30 ° C., preferably about 10 ° C. to about 25 ° C. lower than the dissolution temperature (Tm) of the complete hybrid. For stringent conditions, see J.I. Sambrook et al., Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989), especially Section 11.45 "Conditions for Hybridization" can be described in Section 11.45 "Conditions for Hybrid".

[Microarray for yeast detection]
The yeast detection microarray of the present embodiment is characterized in that the above-mentioned yeast detection oligonucleotide probe is arranged on a substrate.
This yeast detection microarray can be produced by an existing general method using a probe having the base sequences shown in SEQ ID NOs: 1 to 52.
For example, when creating a paste-type DNA chip as this yeast detection microarray, the probe can be immobilized on a glass substrate with a DNA spotter to form a spot corresponding to each probe. can. Further, 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 optical lithography technology. Further, the substrate is not limited to glass, and a plastic substrate, a silicon wafer, or the like can be used. Further, the shape of the substrate is not limited to a flat plate shape, and various three-dimensional shapes can be used, and a substrate having a functional group introduced so as to enable a chemical reaction can be used. ..

In the yeast detection microarray of the present embodiment, all or part of the probe having the base sequence shown in SEQ ID NOs: 1 to 52 can be immobilized. Further, the probes can be fixed for each set of probes of the above-mentioned various groups.

[Yeast detection method]
The method for detecting yeast using the oligonucleotide probe for detecting yeast and the microarray for detecting yeast of the present embodiment can be, for example, the following method.
That is, it can include (1) yeast collection, (2) DNA extraction, (3) target region amplification, and (4) confirmation of amplification products.

(1) Collection of yeast First, yeast is collected from the environment. For example, the sample is collected by wiping the sample from the wall, floor, door, window, manufacturing equipment (instrument, kitchen knife, bat), tank, worker's finger, work clothes, etc. of a food factory. Next, the obtained sample is frozen in liquid nitrogen or the like to disrupt yeast cells.

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

(3) Amplification of target region Next, the target region in the extracted genomic DNA is amplified. That is, it amplifies a DNA fragment containing a target region in genomic DNA. The method for amplifying the target region is not particularly limited, but the PCR method can be preferably used. In the PCR method, the target region is amplified by using a PCR reaction solution containing a primer set for amplifying the target region. As the PCR device, a general thermal cycler or the like can be used.
In the present 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) ~ (D) for 40 cycles), (e) 72 ° C. for 10 minutes

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

In the yeast detection method using the yeast detection oligonucleotide probe and the yeast detection microarray of the present embodiment, DNA fragment amplification is performed by targeting the ITS region and / or β-tubulin gene of rDNA in the genomic DNA of yeast. Will be 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 preferably used. Further, 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 preferably used.

(4) Confirmation of Amplified Product Next, the amplified product is dropped onto the yeast detection microarray of the present embodiment, and the presence or absence of the amplified product is detected by detecting the label of the amplified product hybridized to the probe placed therein. confirm. This makes it possible to identify the yeast present in the environment to be inspected.
The label can be detected by using a general label detection device such as a fluorescent scanning device, for example, by measuring the fluorescence intensity of the amplified product using BIOSHOT (R) manufactured by Toyo Kohan Co., Ltd. Can be done. The measurement result is preferably obtained as an S / N ratio value (Signal to Noise ratio). This is because it is possible to accurately determine whether the measurement result is positive or negative based on the S / N ratio value, and generally, when the S / N ratio value is 3 or more, it is determined to be positive. Can be done. 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 may be used.

[Yeast detection kit]
In the yeast detection kit of the present embodiment, the above-mentioned 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 in the present embodiment. Includes a yeast detection microarray.
That is, the above-mentioned microarray in which the oligonucleotide probe for detecting yeast of the present embodiment is arranged on a substrate, the primer consisting of the base sequence shown in SEQ ID NO: 53 for amplifying the ITS region of DNA of yeast, and SEQ ID NO: 54. It is preferable to have a solution containing a primer set consisting of a primer consisting of the indicated base sequence.

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

According to the oligonucleotide probe for yeast detection, the microarray for yeast detection, and the kit for yeast detection of the present embodiment, a wide range of types of yeast can be appropriately used by using the above-mentioned 52 types of probes that can specifically identify yeast. Can be detected.
Therefore, it is possible to detect a wide range of yeasts quickly and with high accuracy in environmental inspections for confirming the existence of yeasts.

Hereinafter, the tests conducted to confirm the effects of the yeast detection oligonucleotide probe, the yeast detection microarray, and the yeast detection kit according to the embodiment of the present invention will be specifically described.

[Test 1]
As the yeast detection microarray, Gene Silicon (R) (manufactured by Toyo Kohan Co., Ltd.) was used, and the one in which the probes of SEQ ID NOs: 1 to 52 were arranged was used. Each probe used was synthesized by Sigma-Aldrich Japan GK. In addition, each probe was immobilized on a substrate by a microarray.

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

Candida exigua (NBRC 1128), Hansenia spora osmophylla (NBRC 10832), Hansenia spora occidentalis (NBRC 1819), Hansenia spora barbiensis (NBRC 0115), Clavispora lucitaniae (NBRC 10059), Lachansea thermotreans Zigotrraspora Florentinas (NBRC 1088), Debariyo Myces Calsoni (NBRC 0946), Saccharomycosis capsularis (NBRC 0672), Torraspora globosa (NBRC 1160), Torraspora delbricky (NBRC 0955), Elemosesium Ashbyi (NBRC) 0557), Burnett Zyma Platensis (NBRC 10696), Candida Berti (NBRC 10266), Candida Martosa (NBRC 1977), Citello Myces Matritensis (NBRC 0954), Isachenchia Occidentalis (NBRC 1904), Casaxtania Unispora (NBRC 0316) ), Kruger Van Ridgea Fruxhum (NBRC 0773), Lindnera Saturnus (NBRC 10697), Roderomyces erongisporus (NBRC 1676), Nakaseomyces Delphinsis (NBRC 10602), Naumobia Castelli (NBRC 1992), Performance Thermotreans (NBRC 10024), Pichia Farinosa (NBRC 1163), Pichia Fermentans (NBRC 1124), Saturni Spora Asharni (NBRC 10942), Tetrapsispora fafi (NBRC 1672), Van der Waldzaima Polyspora (NBRC 10782), Wicker Hamomises Siferii (NBRC 0793), Zigoascus Helenicas (NBRC 1575), Candida Krusei (NBRC 1395), Zigosaccaromyces baili (NBRC 1098), Zigosaccharomyces bisporus (NBRC 1131), Zigosaccharomyces mellis (NBRC 1615) ), Saccharomyces cerevisiae (NBRC 10217), Devalyomaises Hanseni (NBRC 0015), Candida parapsilosis (NBRC 1396), Candida Toro Picaris (NBRC 1400), Candida glabrata (NBRC 0622), Pichia Gilliel Mondi (NBRC 10279), Rhodotorula glutinis (NBRC 1125), Rhodotorula minuta (NBRC 0715), Rhodotorula mucilaginosa (NBRC 0909), Brettano Meisses Custercianus (NBRC 1585), Brettanomises nadenensis (NBRC 1588), Deckera burgselensis (NBRC 0677), Yarowia lipolytica (NBRC 1548), Malassezia furfur (NBRC 0656), Trichosporon kutanium (NBRC 0656) NBRC 1198), Cryptococcus Lorenty (NBRC 0609)

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

Here, as the reaction solution for PCR, a primer set consisting uniformly 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 DNA of yeast. , And a primer set consisting of 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 DNA of yeast was used. These primers were synthesized by Sigma-Aldrich Japan GK.

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

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

Next, a buffer solution (3 × SSC citric acid-physiological saline + 0.3% SDS) was mixed with the PCR amplification product, heated at 94 ° C. for 5 minutes, and dropped onto the above-mentioned yeast detection microarray. The microarray was allowed to stand at 45 ° C. for 1 hour to wash away unhybridized PCR products using the above buffer.
Then, using a label detection device (BioSHOT (R), manufactured by Toyo Kogyo Co., Ltd.), the fluorescence intensity (fluorescence intensity of the amplified product bound to the probe) in each probe arranged on the yeast detection microarray was measured. , The S / N ratio value ((median fluorescence intensity value-background value) ÷ background value) of each probe was acquired. The results are shown in FIGS. 5 to 7.

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

[Test 2]
A test was conducted to confirm whether the probe placed in the yeast detection microarray of the present embodiment functions effectively when multiplex PCR is performed to simultaneously amplify the target region in the DNA of a plurality of yeasts to be detected. rice field.

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

(Sample 1)
Hansenia spora osmophila (corresponding SEQ ID NO: 2), Performance Thermotolerance (corresponding SEQ ID NO: 24), Tetrapicis pola fafi (corresponding SEQ ID NO: 28), Zygosaccharomyces melis (corresponding SEQ ID NO: 35), Brettanomises nadenensis (corresponding) SEQ ID NO: 47)

(Sample 2)
Zigotrula spora Florentinas (corresponding SEQ ID NO: 7), Candida Berti (corresponding SEQ ID NO: 14), Roderomyces erongisporus (corresponding SEQ ID NO: 21), Pichia Farinosa (corresponding SEQ ID NO: 25), Devaliyo Myces Hanseni (corresponding SEQ ID NO: 38). )

(Sample 3)
Hansenia spora barbiensis (corresponding SEQ ID NO: 4), Isachenchia occidentalis (corresponding SEQ ID NO: 17), Nakaseomyces Delphinsis (corresponding SEQ ID NO: 22), Zygosaccharomyces baili (corresponding SEQ ID NO: 33), Candida parapsilosis (corresponding SEQ ID NO: 39) )

(Sample 4)
Lachansea Thermotolerance (corresponding SEQ ID NO: 6), Elemocesium Ashby (corresponding SEQ ID NO: 12), Burnett Zyma Platensis (corresponding SEQ ID NO: 13), Pichia fermentans (corresponding SEQ ID NO: 26), Brettanomises custercianus (Corresponding sequence number 46)

(Sample 5)
Kazakstania exigua (corresponding SEQ ID NO: 1), Naumovia castelli (corresponding SEQ ID NO: 23), Van der Waldzaima polyspora (corresponding SEQ ID NO: 29), Zygosaccharomyces bisporus (corresponding SEQ ID NO: 34), Deckera burgserensis (corresponding SEQ ID NO: 34). SEQ ID NO: 48)

(Sample 6)
Hansenia spora Occidentalis (corresponding SEQ ID NO: 3), Trulaspora globosa (corresponding SEQ ID NO: 10), Kazaxtania Unispora (corresponding SEQ ID NO: 18), Rodtorula glutinis (corresponding SEQ ID NO: 43), Cryptococcus lorenti (corresponding SEQ ID NO: 52)

(Sample 7)
Kravispora Lucitaniae (corresponding SEQ ID NO: 5), Candida Martosa (corresponding SEQ ID NO: 15), Kreger van Ridgea frucshum (corresponding SEQ ID NO: 19), Candida glabrata (corresponding SEQ ID NO: 41), Malassezia furfur (corresponding sequence) Number 50)

(Sample 8)
Saccharomyces capsularis (corresponding SEQ ID NO: 9), Wickerhamomices siferii (corresponding SEQ ID NO: 30), Saccharomyces cerevisiae (corresponding SEQ ID NOs 36, 37), Pichia Gilliermondi (corresponding SEQ ID NO: 42), Rodtorula・ Minuta (corresponding sequence number 44)

(Sample 9)
Trulaspora del Bricky (corresponding SEQ ID NO: 11), Lindnera saturnus (corresponding SEQ ID NO: 20), Candida krusei (corresponding SEQ ID NO: 32), Saccharomyces cerevisiae (corresponding SEQ ID NOs 36, 37), Trichosporon Kutaneum (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), Rodtorula mucilaginosa (corresponding SEQ ID NO: 45), Yarowia lipolytica (corresponding SEQ ID NO: 49).

(Sample 11)
Devaliyo Myces calconi (corresponding SEQ ID NO: 8), Elemocesium Ashby (corresponding SEQ ID NO: 12), Van der Waldzaima Polyspora (corresponding SEQ ID NO: 29), Zygosaccharomyces melis (corresponding SEQ ID NO: 35), Deckera burgserensis (corresponding SEQ ID NO: 35). SEQ ID NO: 48)

(Sample 12)
Kreger Van Ridgea Fruxhum (corresponding SEQ ID NO: 19), Saturnispora Asharni (corresponding SEQ ID NO: 27), Zigoascus Helenicas (corresponding SEQ ID NO: 31), Rodtorula minuta (corresponding SEQ ID NO: 44), Trichosporon Kutaneu (corresponding sequence) Number 51)

Culturing was carried out using PDA medium or M40Y medium in a dark place at 25 ° C. and allowed to stand for 7 days.
Then, the yeast colonies cultured for each sample are collectively collected, placed in a vial containing φ0.5 mm zirconia beads for each sample, immersed in liquid nitrogen to freeze the sample, and then shaken using a shaking device. Yeast cells were crushed.
Next, the yeast genomic DNA was extracted by a DNA extraction device, and the ITS region and / or β-tubulin gene of each yeast was simultaneously amplified for each sample by the PCR method. The primer set used in the PCR method is the same as in Test 1.

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

Using each of the above PCR reaction solutions, DNA is amplified by a nucleic acid amplification device (TaKaRa PCR Thermal Cycler Device (R) manufactured by Takara Bio Inc.) under the same conditions as in Test 1, and for yeast detection. The fluorescence intensity of each probe placed on 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. 11 to 13.

As shown in these figures, the S / N ratio values of the probes contained in each sample are all 3 or more, indicating positive. In addition, these probes were highly specific and did not show false positive reactions.
As described above, it was found that the oligonucleotide probe for yeast detection of the present embodiment functions effectively even when multiplex PCR is performed to simultaneously amplify the target region in the DNA of a plurality of yeasts to be detected.

It goes without saying that the present invention is not limited to the above embodiments and examples, and 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 for each type, and each probe may be arranged in a plurality of spots. Further, yeasts other than the yeast to be detected in the present embodiment and other probes for detecting mold may be arranged on the microarray together with the probes in the present embodiment, and can be appropriately changed.

INDUSTRIAL APPLICABILITY The present invention can be suitably used when a plurality of types of yeasts are specifically and multiplely detected in environmental inspections, food inspections, epidemiological environmental inspections, clinical trials, livestock hygiene, plant disease diagnosis, and the like. ..

Claims (5)

An oligonucleotide probe for detecting yeast, which comprises the base sequence shown in SEQ ID NO: 1. The oligonucleotide probe according to claim 1 is included, and the oligonucleotide probe is included.
A set of oligonucleotide probes for yeast detection, comprising at least one of the oligonucleotide probes consisting of the nucleotide sequences set forth in SEQ ID NOs : 2-52. A microarray for yeast detection, wherein the oligonucleotide probe according to claim 1 or 2 is arranged on a substrate. A microarray in which the oligonucleotide probe according to claim 1 or 2 is arranged on a substrate, and
It is characterized by having 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 yeast DNA. Yeast detection kit. A microarray in which the oligonucleotide probe according to claim 1 or 2 is arranged on a substrate, and
A primer set consisting of a primer consisting of the base sequence shown in SEQ ID NO: 53 for amplifying the ITS region of yeast DNA and a primer consisting of a primer consisting of the base sequence shown in SEQ ID NO: 54, and the β-tubulin gene of yeast DNA are amplified. A kit for detecting yeast, which comprises a primer set including a primer consisting of the base sequence shown in SEQ ID NO: 55 and a primer set consisting of a primer consisting of the base sequence shown in SEQ ID NO: 56.

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