JP5919631B2 - Mold inspection method - Google Patents

Description

  The present invention relates to a mold inspection method, and particularly to a mold inspection method for specifically detecting a plurality of types of molds simultaneously.

Conventionally, it has been important to check for the presence of fungi and other microorganisms at food production sites, clinical sites, and protected environments such as cultural properties, and to confirm their safety and prevent their propagation. ing.
The following three methods can be cited as confirmation of the presence of such mold.
First, as a first method, a sample is sampled from the environment, pre-cultured, and then cultured for about 20 days in an optimal medium for each bacterial species. The method of identifying and confirming the existence can be mentioned (refer patent document 1).

As a second method, after sampling and pre-culturing a sample from the environment, each bacterium is individually cultured, DNA is extracted from the cultured cells, and the target region is amplified by PCR (polymerase chain reaction) method. By analyzing the base sequence of the region, a method for identifying and confirming the presence of mold can be mentioned.
Furthermore, in recent years, as a new method for detecting mold, a probe comprising a base sequence that binds complementarily to the target region is prepared and immobilized on a DNA chip substrate, and a PCR amplification product is dropped onto the DNA chip. Therefore, a method has been developed in which a corresponding target region and a probe are hybridized to identify and confirm the presence of mold contained in a sample (see Patent Document 2).

JP 2007-195454 A JP 2008-35773 A

However, in the first method for morphological identification, an optimal medium and long-term culture is required for each bacterial species to express morphological characteristics, and further skill is required for identification. There was a problem that it was not suitable for inspection and simplification of inspection.
In addition, in the second method based on PCR and sequence analysis, since each cell type is cultured individually, a comparatively long test period of about 14 days is required, and it is necessary to analyze each cell type individually. For this reason, there has been a problem that it is not suitable when multi-sample processing is required.

  On the other hand, according to a new detection method using a DNA chip, it is theoretically possible to detect a plurality of types of molds collectively, and is expected as a quick and simple inspection method.

  On the other hand, molds are divided into dry mold (preferably dry), dry mold (can withstand dryness), and hygrophilic mold (preferably wet) based on the humidity suitable for growth. It was necessary to culture with a suitable different medium. Moreover, in the first and second methods that have been generally performed in the related art, since it was necessary to culture for each bacterial species, a plurality of molds were mixed and cultured. The concept of detecting each mold individually did not exist. For this reason, there has conventionally not been a technique for simultaneously cultivating a drought-resistant mold, a drought-resistant mold, and a hygrophilic mold so that each mold can be specifically detected.

  The present invention has been made in view of the above circumstances, and simultaneously cultivating a plurality of types of molds in the same medium without separating and culturing them, and mixing them together to extract genomic DNA all together, and using a DNA chip An object of the present invention is to provide a mold inspection method that allows each mold to be specifically detected.

In order to achieve the above object, the mold inspection method of the present invention cultivates a plurality of types of mold, mixes the plurality of types of cultured molds, collectively extracts genomic DNA, and uses a DNA chip. This is a method for simultaneously and specifically detecting each of a plurality of types of molds.
If the mold inspection method of the present invention is such a method, the cultured fungus species can be specifically detected even if the fungus is cultured in a mixed state without dividing the fungus separately for each fungus species. . That is, even when a plurality of types of cultured molds are mixed and genome DNA of each mold is extracted together, each mold can be detected by the DNA chip.

As a method for detecting the extracted genomic DNA using a DNA chip, a general method can be used.
Specifically, for example, a specific region of genomic DNA is amplified by a PCR method using a PCR reaction solution containing a primer set for amplifying a specific region of a mold to be detected. A probe selected in advance from the amplification region by this primer set is immobilized on a DNA chip. At this time, the primer set and the probe need to be prepared in advance for each specific region of the mold to be detected. Then, by dropping the amplification product obtained by the PCR method onto the DNA chip and detecting the amplification product bound to the probe, it is possible to specifically detect various molds contained in the mixture.

Further, in the mold inspection method of the present invention, at least two types of molds selected from the group consisting of at least a dry mold, a dry mold, and a wet mold are simultaneously cultured in a predetermined medium, It is also preferable to adopt a method for simultaneously and specifically detecting each of the cultured molds.
When the mold inspection method of the present invention is such a method, molds that are usually cultured individually and that require different humidity environments can be simultaneously cultured in a predetermined medium. Each mold can be specifically detected from these mixtures. For this reason, it is possible to carry out detection by culturing all together at the same time without considering the nature of the mold and the like, and it is possible to simplify the mold inspection.

In the mold inspection method of the present invention described above, a plurality of types of molds have a water activity value of less than 1.0, 0.90 or more, and a sugar concentration of 5 to 50%, more preferably 10%. It is preferable to set it to -40%. Specifically, glucose and sucrose are preferably used as the type of sugar.
According to such a solid medium having a water activity value and a sugar concentration, it is possible to suitably culture any of the dry mold, dry resistant mold, and wet mold, and simultaneously culture all molds at the same time, The detection can be performed.

Furthermore, in the mold inspection method of the present invention described above, a method of culturing a plurality of types of mold at a temperature of 25 ° C. ± 2 ° C. is also preferable.
If it is such a temperature range, it becomes possible to fully propagate any of dryness mold, dry-proof mold, and wetness mold.

In the mold inspection method of the present invention described above, a plurality of types of cultured molds are mixed in a container containing beads for physically crushing mold cell walls, and genomic DNA is extracted in a batch. It is also preferable to do.
If the mold inspection method of the present invention is such a method, genomic DNA can be extracted from a plurality of types of molds cultured at the same time.

Furthermore, in the mold inspection method of the present invention described above, the plurality of types of molds are preferably mold spores and mycelia floating or attached in the atmosphere.
If the mold inspection method of the present invention is such a method, molds in the environment can be collected and cultured, and these can be easily and simultaneously detected.

  According to the present invention, it is possible to simultaneously cultivate a plurality of types of molds in the same medium, mix them together, extract the genomic DNA at once, and specifically detect each mold using a DNA chip.

It is a figure which shows culture | cultivation evaluation of the culture | cultivation test by various culture medium composition. It is a figure which shows the diameter of a colony when a desiccating mold, a drought-resistant mold, and a hygroscopic mold are cultured with various culture media. It is a figure which shows the diameter of a colony when cultivating dryness mold, dry-proof mold, and hygroscopic mold at various temperatures. It is a figure which shows the photograph of a colony when dryness mold | fungi, dry-proof mold | fungi, and moisture-proof mold | fungi are cultured at various temperatures. It is a figure which shows the DNA chip analysis and sequence analysis result of the microbial species in the specimen 1-20. It is a figure which shows the DNA chip analysis and sequence | arrangement analysis result of the microbial species in the specimen 21-40. It is a figure which shows the DNA chip analysis and arrangement | sequence analysis result of a bacterial species in the specimen 45-60.

  Hereinafter, an embodiment of the mold inspection method of the present invention will be described in detail. The mold inspection method of the present embodiment cultivates a plurality of types of molds, mixes the plurality of types of cultured molds, collectively extracts genomic DNA, and uses a DNA chip to select a plurality of types of molds. As long as each of them is detected simultaneously and specifically, it is not limited to the specific contents of the following embodiments and examples.

The mold inspection method of the present embodiment may include the following steps.
(1) Collecting mold First, air is sampled at a food manufacturing site, clinical site, or a protected environment for cultural assets, using an air sampler. Next, the collected air is sprayed on a dedicated medium or the like made into a strip shape for an air sampler and cultured.

As the medium, as described later in Examples, it is preferable to use M40Y, MY10G medium, MY30G medium, or the like that can be cultivated in any of dryness, dryness resistance, and moisture resistance. Among these, use of M40Y medium is particularly preferable because molds having any of the above properties can be cultured with high efficiency.
Moreover, as culture conditions, it is preferable to let it stand for 2 to 7 days in a dark place at a temperature of 23 ° C to 27 ° C.
In general, the M40Y medium, the MY10G medium, and the MY30G medium are considered to be used for a dry mold, and have been considered not suitable for a wet mold culture.

  Next, various types of mold colonies generated in the medium are collected in a lump without individually separating them. The collected sample is placed in, for example, a vial containing φ0.5 mm zirconia beads, and the sample is frozen by immersing the vial in liquid nitrogen, and then the mold cells are crushed using a shaking device or the like. To do. The cells may be destroyed so that DNA can be extracted, and other methods may be used.

(2) DNA extraction As a method for extracting genomic DNA from a sample in which mold cells are destroyed, a general method such as a CTAB method (Cetyl trimethyl ammonium bromide) or a method using a DNA extraction apparatus can be used. .

(3) Amplification of ITS region by PCR method Next, a primer set capable of amplifying the ITS1 region of various mold rDNAs is added to the PCR reaction solution, and the genomic DNA of the mold in the sample is identified by the PCR method. Amplify the region. Specifically, those having the nucleotide sequences shown in SEQ ID NOs: 1 and 2 can be used as the forward primer and the reverse primer, respectively. Moreover, a general thermal cycler etc. can be used as a PCR apparatus.

  As the PCR reaction solution of this embodiment, for example, a PCR reaction 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 polymerase, etc.), labeling component (Cy5-dCTP, etc.), sample genomic DNA, buffer solution, and the rest It is possible to suitably use a PCR reaction solution containing water as a volume, and for example, Ampdirect (R) (Shimadzu Corporation) can be used as a buffer solution.

The PCR reaction conditions in the mold inspection method of the present embodiment are preferably as follows, 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

(4) Detection by DNA chip The DNA chip of this embodiment is not particularly limited as long as a probe selected from mold DNA to be detected is immobilized. For example, a spot type DNA chip or a synthetic type DNA chip can be used.
Specifically, a probe that binds to the amplification region amplified by the primer set included in the PCR reaction solution of the mold inspection method of this embodiment is synthesized in advance and immobilized on the substrate of the DNA chip. For example, as a probe for detecting Aspergillus vitricola, a probe having the base sequence shown in SEQ ID NO: 3 can be used. Moreover, as a probe for detecting Aspergillus penicillioides, a probe having the base sequence shown in SEQ ID NO: 4 can be used. Further, as a probe for detecting Eurotium sp., A probe having the base sequence shown in SEQ ID NO: 5 can be used.

Next, the PCR amplification product is dropped on the DNA chip, and the label of the PCR amplification product hybridized with the mold detection probe is detected. Specifically, for example, it can be performed as follows.
First, a predetermined buffer is mixed with the PCR amplification product and dropped onto the DNA chip.
Next, the DNA chip is allowed to stand at 45 ° C. for 1 hour, and then the PCR product that has not been hybridized with a predetermined buffer is washed away from the DNA chip.
Then, the label is detected by applying the DNA chip to the label detection device, and it is determined whether or not the detection target mold exists. In addition, as a label | marker detection apparatus, general things, such as a fluorescence scanning apparatus, can be used, for example. The label and its detection method are not limited to fluorescence, and other methods may be used.

As described above, according to the mold inspection method of the present embodiment, a plurality of molds are simultaneously cultured using a medium capable of cultivating any of dryness, dryness resistance, and moisture resistance. Can do. Then, the cultured molds are mixed, genomic DNA is extracted at once, and each of a plurality of types of molds can be detected simultaneously and specifically using a DNA chip.
Therefore, it is not necessary to separate and culture the collected mold, and it is possible to quickly and easily detect a plurality of types of mold.

  Hereinafter, the test result of mold detection by the mold inspection method of the present invention will be specifically described.

(Test 1: Culture test based on water activity values of various medium compositions)
Various media having various water activity values are prepared by adding sugar to PDA (Potato Dextrose Agar) medium and MY (Malt + Yeast) medium to produce dry mold, dry mold, and wet mold. Culture was performed and the results of each culture were evaluated.

1. Preparation method of medium (1) PDA medium PDA (manufactured by DIFCO) was added glucose (Wako Pure Chemical Industries, Ltd.) and / or sucrose (Wako Pure Chemical Industries, Ltd.) at various ratios, and 1 L It was suspended in ion-exchanged water, melted in an autoclave, and dispensed into a petri dish. Before dispensing the petri dish, chloramphenicol (Wako Pure Chemical Industries, Ltd.) was added to a final concentration of 50 ppm for the purpose of preventing bacterial growth. The same applies to the MY medium.

(2) MY medium To the following MY, at least one of sucrose (Wako Pure Chemical Industries, Ltd.), glucose, agar (Wako Pure Chemical Industries, Ltd.) and glycerin is added at various ratios, and 100 ml of ion exchange is performed. It was suspended in water, melted in an autoclave, and dispensed into a petri dish.
MY: Malt (Malt Extract, manufactured by DIFCO) + Yeast (Yeast Extract, manufactured by DIFCO)

2. Measurement of Water Activity Value of Medium Using a Rotronic water activity measuring device (manufactured by GSI Creos) for a predetermined amount of the medium actually used for culture, the water activity value was measured in a dedicated sealed container.

3. Cultivation Evaluation Using various media (Example 1-10 in FIG. 1, Reference Example 1-6) prepared according to the above production method, dry mold (Eurotium herbariorum), dry resistant mold (A. niger), and wet mold (Fusarium sp.) Was cultured at 25 ° C. in the dark for 72 hours, and the diameter of the obtained colonies was measured. When the diameter of the colony after the culture was 10 mm or more, it was rated as “◯” and less than 10 mm as “x”. The result is shown in FIG.
As shown in the figure, it can be seen that when the various culture media of Example 1-10 were used, all of the dry mold, the dry mold and the wet mold were sufficiently propagated. On the other hand, when the various culture media of Reference Example 1-6 are used, it can be seen that there are bacterial species that are not sufficiently propagated. For this reason, in order to culture a plurality of bacterial species simultaneously, it is preferable that the water activity value is less than 1.0, 0.90 or more, and the sugar concentration is in the range of 5% to 50%. Recognize.

(Test 2: Culture test using various media)
Various molds were cultured at 25 ° C. in the dark for 168 hours using the six types of culture media of Reference Examples 1, 2, 4-6 and Example 7 shown in FIG. It was measured.

  The following 14 types of test bacteria were used. The results are shown in FIG. Among these, the 1-4 test strains are desiccating molds, the 5-10 test strains are drought-resistant molds, and the 11-14 test strains are hygroscopic molds. . In addition, these test microbial species were collected and identified uniquely from the environment, and the numbers are given for convenience.

1. Aspergillus penicilloides (K-7-4)
2. Aspergillus restrictus (A.restrictus, I-2-1)
3. Eurotium herbariorum (I2-1)
4). Wallemia sebi (KSS-1127)
5. Aspergillus flavus (A.flavus, B-3-3)
6). Aspergillus fumigatus (A.fumigatus, KSS-1126)
7). Aspergillus niger (A.niger, A-1-1)
8). Aspergillus Versicolor (A.versicolor, i 3-1)
9. Penicillium glabrum (B-4-3)
10. Penicillium lugrosum (P.rugulosum, E-2-3)
11 Cladosporium.sphaerospermum (I-4-2)
12 Cladosporium (C.cladosporioides, A-2-1)
13. Fusarium sp. (B5-3-C)
14 Stachybotrys sp. (KSS-1125)

As shown in FIG. 2, according to the medium (M40Y) of Example 7, a plurality of types of mold colonies having different humidity suitable for growth are formed, and all the above-mentioned bacterial species 1-14 are sufficiently propagated. You can see that
Thus, using the medium of Example 7, it became clear that a plurality of molds having different properties with respect to the optimum humidity can be simultaneously cultured in the same medium.

(Test 3: Culture test at various temperatures)
Using the M40Y medium used in Example 7, culture was performed at various temperatures, and the diameter of the obtained colonies was measured. As the test bacteria species, 14 types including dryness mold, dry resistance mold, and wetness mold were used in the same manner as in Test 1, and cultured in the dark for 168 hours. The result is shown in FIG. Moreover, about 3, 8, and 13 microbial species, the photograph of a colony is shown in FIG.

  As shown in Examples 11 to 13 of FIG. 3, it can be seen that, when the culture temperature is in the range of 23 ° C. to 27 ° C., all of the dryness mold, the dry resistance mold, and the wetness mold are sufficiently propagated. . On the other hand, as shown in Reference Example 7-9, it can be seen that there are species that cannot grow when the culture temperature is in the range of 30 ° C to 35 ° C. For this reason, in order to culture several microbial species simultaneously, it turns out that it is suitable to make culture | cultivation temperature into the range of 25 degreeC +/- 2 degreeC.

(Test 4: DNA chip analysis test)
Various molds are cultured using the M40Y medium used in Example 7, the obtained colonies are mixed, genomic DNA is extracted at once, the ITS1 region is amplified by the PCR method, and the detection target mold becomes an amplification product. Whether it was contained or not was examined with a DNA chip. In addition, DNA sequence analysis was performed in order to verify the test results using the DNA chip. Specifically, it was performed as follows.

  First, as a sample using the M40Y medium used in Example 7, No. 1 was used. 1 to No. Sixty specimens up to 60 were prepared. Next, using an air sampler, air was collected from the general environment, and sprayed on each of the specimens and cultured. Culturing was performed in a dark place at 25 ° C. for 7 days.

Next, for each specimen, various types of mold colonies generated in the culture medium were individually collected in order to be used for DNA sequence analysis, and each sample was collected in a dark place at 25 ° C. for 7-10 days. Separate culture was performed.
In addition, for each specimen, various types of mold colonies generated in the culture medium were collected in a lump, placed in a vial containing φ0.5 mm zirconia beads, frozen in liquid nitrogen, and shaken. The device was used to disrupt mold cells.

Next, for each specimen, mold genomic DNA was extracted with a DNA extraction apparatus, and the ITS region of each mold was amplified by PCR.
Specifically, Ampdirect (R) (manufactured by Shimadzu Corporation) was used as a PCR reaction solution, and 20 μl of the following composition was prepared.
1. Ampdirect addition (G / Crich) 4.0 μl
2. Ampdirect (addition-4) 4.0 μl
3. dNTPmixture 1.0 μl
4). Cy5-dCTP 0.2 μl
5. ITS1 region amplification forward primer (10 μM, SEQ ID NO: 1, synthesized by Sigma-Aldrich) 1.0 μl
6). ITS1 region amplification reverse primer (10 μM, SEQ ID NO: 2, synthesized by Sigma-Aldrich) 1.0 μl
7). 1.0 μl of sample genomic DNA
8). NovaTaq polymerase 0.2 μl
9. Water (water until 20.0 μl total)

Using this PCR reaction solution, DNA was amplified by the nucleic acid amplification apparatus (TaKaRa PCR Thermal Cycler Dice (R) Gradient manufactured by Takara Bio Inc.) under the following conditions.
1.95 ° C 10 minutes 2.95 ° C 30 seconds 3.56 ° C 30 seconds 4.72 ° C 60 seconds (40 cycles of 2-4)
5.72 ° C 10 minutes

  As the DNA chip, Gene Silicon (R) (manufactured by Toyo Kohan Co., Ltd.) was used, to which a probe consisting of the base sequences shown in SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5 was immobilized. . These are respectively a probe for detecting Aspergillus vitricola, a probe for detecting Aspergillus penicillioides, and a probe for detecting Eurotium sp.

Next, the PCR amplification product was mixed with a buffer solution (3 × SSC citrate-saline + 0.3% SDS) and dropped onto the DNA chip.
The DNA chip was allowed to stand at 45 ° C. for 1 hour, and the PCR product that did not hybridize using the buffer solution was washed away from the microarray.
Subsequently, the fluorescence intensity of each probe was measured by applying the DNA chip to a labeling detection device (Genesis scanner dedicated to BIOS, manufactured by Toyo Kohan Co., Ltd.). The results are shown in FIGS.

Further, in order to analyze the DNA sequence of the bacterial species contained in each specimen, genomic DNA was extracted from the bacterial species obtained by separating and culturing for each colony as described above, and an amplification product was obtained by the PCR method.
At this time, a primer set consisting of the base sequences shown in SEQ ID NOs: 1 and 2 was used, and TAKARA ExTaq polymerase was used as the nucleic acid synthase. Moreover, TaKaRa PCR Thermal Cycler Dice (R) Gradient (manufactured by Takara Bio Inc.) was used as the nucleic acid amplification apparatus, and the PCR reaction was performed in the same manner as above.
The amplification product obtained by this PCR reaction and the primer set consisting of the nucleotide sequences shown in SEQ ID NOs: 6 and 7 were used as sequencing primers and consigned to Takara Bio Inc., and the sequence analysis of the ITS1 region was performed using a DNA sequencer. . As a result, as shown in FIGS. 5-7, a maximum of 4 bacterial species were confirmed from each specimen.

In “DNA chip analysis (probe fluorescence intensity)” of FIG. 5-7, a portion considered to be positive is surrounded by a thick frame. These indicate the same bacterial species in “bacterial species contained in the sample confirmed by ITS sequence analysis”.
It should be noted that Sample No. was found to contain drought-resistant mold (Penicillium sp.) And hygroscopic mold (Cladosporium sp.) By ITS sequence analysis. 3, the fluorescence intensity was measured with a label detection apparatus in the same manner as described above using a DNA chip on which a probe for detecting Penicillium sp and a probe for detecting Cladosporium sp was immobilized, and these bacterial species (Penicillium sp., Cladosporium sp.) Was detected.
Therefore, it was found that by the mold inspection method of the present invention, each mold can be specifically detected when a plurality of types of molds are simultaneously cultured in the same medium.

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, in the above embodiment, M40Y or the like is used as the medium, but the medium is not limited thereto, and the water activity value is less than 1.0, 0.90 or more, and the sugar concentration is 5% to 50%. It is possible to appropriately change such as using another solid medium of the solid medium.

  INDUSTRIAL APPLICABILITY The present invention can be suitably used for mold inspection at food manufacturing sites, clinical sites, cultural property protection environments, and the like.

Claims (3)

A mold inspection method for simultaneously and specifically detecting each of a plurality of types of molds cultured by collecting mold spores and mycelia floating in the atmosphere or adhering to the environment,
At least two types of molds selected from the group consisting of dry mold, dry resistant mold, and hygroscopic mold are simultaneously cultured in one solid medium without being separated and individually cultured;
The solid medium is a medium having a water activity value of less than 1.0, 0.90 or more, and a sugar concentration of 5% to 50%.
Collecting and mixing colonies in the fixed medium , extracting genomic DNA in a lump, and detecting each of the plurality of types of molds simultaneously and specifically using a DNA chip Method. The plural types of the mold, inspection method according to claim 1, wherein molds which comprises culturing at a temperature of 25 ° C. ± 2 ° C.. A plurality of types of molds described above culture was placed in a vessel containing beads to physically disrupt the cell walls of fungi are mixed, according to claim 1, wherein extracting the genomic DNA collectively Mold inspection method.