JP6291721B2 - Microbiological testing method - Google Patents

Description

  The present invention relates to an inspection method for confirming the presence or absence of microorganisms in the environment.

In recent years, it has been important to check the presence of microorganisms such as mold and bacteria in food production sites, clinical sites, and cultural property protection environments, to confirm the safety of the environment, and to prevent its propagation It has become.
For example, in the mold inspection, a sample is generally collected from the environment, pre-cultured, and then cultured for about 20 days in an optimal medium for each bacterial species, followed by morphological characteristics observation. A morphological observation method (culture method) for identifying molds has been performed (see Patent Document 1).

  However, this method has a problem that the inspection process becomes complicated because it is necessary to separate and culture for each mold species. In addition, since culture requires a long period of time, it is unsuitable for tests that require rapidity, such as indoor tests where people live and food tests. In addition, there is a problem that the identification cannot be performed unless the spore representing the morphological feature is formed, and the labor may be wasted.

  Recently, an identification method using DNA has been carried out for mold inspection. For example, after culturing a sample collected from the environment, DNA is extracted from the cultured cells, the target region is amplified by the PCR (polymerase chain reaction) method, and the amplification product is analyzed to remove mold in the sample. Identification has been done. Methods for analyzing amplification products include, for example, analysis of amplification product size by electrophoresis, and identification of molds present in a sample using a DNA chip on which a probe that binds complementary to the amplification product is immobilized. And the like have been proposed (see Patent Documents 2 and 3).

JP 2007-195454 A JP 2008-278848 A JP 2008-278861 A JP 2001-238700 A

However, according to such an identification method using DNA, it is possible to confirm the presence or absence of microorganisms in the environment, but it is not possible to confirm which microorganism is the dominant species in the environment. It was.
In addition, as described in Patent Document 1, according to the method of performing isolation culture for each bacterial species and identifying molds based on observation of morphological characteristics, each mold is counted by counting the number of identified molds. It is possible to confirm which mold is the dominant species. However, since this method requires a long time to confirm the dominant species, it is unsuitable for inspections that require a quick response such as indoor inspections and food inspections in which humans live.
Here, two or more samples are obtained from a population of organisms, DNA is extracted, a reaction solution containing DNA, a target gene-specific primer, and a control gene-specific primer is prepared and subjected to quantitative PCR. Measure the amount of each amplification product, determine the proportion of heterogeneous individuals in the population based on the amount of amplification product, and determine the reliability of the proportion by statistical processing. A measuring method has been proposed (see Patent Document 4).

However, the method for determining the proportion of heterogeneous individuals in a population based on the amount of amplification product is effective when the size of the amplification target region differs greatly among individuals, but the size of the amplification target region is large. If there is not enough difference, there is a problem that it cannot be used effectively.
For example, in the inspection of microorganisms using DNA, the same region in DNA is often used as an amplification target region for a plurality of types of microorganisms. At this time, depending on the microorganisms to be inspected, the sizes of the respective amplification target regions may not differ so much between different types. In such a case, even if the amount of the amplification product is analyzed by electrophoresis, for example, amplification products derived from a plurality of types of microorganisms are included in one band, so it is difficult to determine the existence ratio of each microorganism. Therefore, the dominant species of microorganisms in the environment could not be determined.

Therefore, the present inventors have intensively studied, and in examining microorganisms using DNA, even if the size of the amplification target region between the types of microorganisms is not so different, it is possible to easily determine the dominant species of microorganisms in the environment. Found a possible way.
The present invention has been made in view of the above circumstances, and it is possible to examine microorganisms that can be estimated almost appropriately and easily within a range that does not cause much problems if realistic use is considered for the dominant species of microorganisms in the environment. The purpose is to provide a method.

In order to achieve the above object, the method for examining microorganisms of the present invention comprises a culture step of collecting a sample from the environment, culturing the microorganism contained in the obtained sample in a medium, and cultivating the microorganism to produce a colony. When two or more are formed, the formed colonies are divided into two or more sets, and the nucleic acid extraction step for extracting the nucleic acid of the microorganisms included in the set for each set, and the region to be amplified using the extracted nucleic acids A nucleic acid amplification step for amplifying a nucleic acid fragment containing a nucleic acid, a microorganism detection step for detecting a microorganism contained in each set using the amplified nucleic acid fragment, and two or more microorganisms detected in the microorganism detection step For each detected microorganism, the ratio of the number of sets in which each microorganism was detected to the total number of sets, which is the total number of two or more sets, was calculated, and the species with the highest ratio was There as a method having the predominant species estimation step of predominant species estimated.

According to the present invention, the dominant species of microorganisms in the environment can be estimated almost appropriately and simply.

It is a figure which shows the base sequence of the primer set used in the test for confirming the effect of the microbe test | inspection method which concerns on embodiment of this invention. It is a figure which shows the base sequence of the probe used by the test for confirming the effect of the test method of the microorganisms concerning embodiment of this invention. It is a figure which shows the base sequence of the primer set for a sequence used in order to analyze the base sequence of an ITS area | region in the test for confirming the effect of the test | inspection method of the microorganisms concerning embodiment of this invention with a DNA sequencer. It is a figure which shows the test result (the number of chips used 5, 6) for confirming the effect of the microbe inspection method concerning the embodiment of the present invention. It is a figure which shows the test result (use chip number 4) for confirming the effect of the test method of the microorganisms which concern on embodiment of this invention. It is a figure which shows the test result (the number of used chips | tips 1, 2) for confirming the effect of the microbe inspection method which concerns on embodiment of this invention.

  Hereinafter, an embodiment of the microorganism testing method of the present invention will be described in detail. However, the present invention is not limited to the specific contents of this embodiment and the examples described later.

[Microbial inspection method]
In the microorganism testing method of the present embodiment, microorganisms included in a set are detected for each set, and when two or more types of microorganisms are detected, each microorganism for the total number of sets is detected for each detected microorganism. It is characterized by determining the dominant species of microorganisms in the environment by calculating the ratio of the number of aggregates.
Specifically, the method includes (a) a microorganism culture step, (b) a nucleic acid extraction step, (c) a nucleic acid amplification step, (d) a microorganism detection step, and (e) a dominant species determination step as follows. preferable.

(A) Microbial culture step This is a step of collecting a sample from the environment and culturing a microorganism contained in the obtained sample in a medium.
“In the environment” includes indoor and outdoor air, liquid, and other inspection objects in environmental inspection, food inspection, epidemiological environmental inspection, clinical test, livestock hygiene and the like. In addition, inspection objects such as food and drink and various instruments are also included.
The “microorganism” includes bacteria such as bacteria in addition to fungi such as mold and yeast, and the microorganism testing method of this embodiment can be applied as long as it is a microorganism that forms a colony. .

A method for collecting a sample from the environment is not particularly limited. For example, air in an environment to be inspected can be collected using an air sampler or the like.
The method for culturing microorganisms is not particularly limited, but the collected sample can be sprayed on a dedicated medium, and molds and the like contained in the sample can be cultured on an agar medium or the like. As a mold culture condition, it is preferable to stand at 25 ° C. in a dark place for 65 hours or more.

(B) Nucleic Acid Extraction Step In this step, when two or more colonies are formed by culturing microorganisms, the formed colonies are divided into two or more sets, and the nucleic acid of the microorganism is extracted for each set.
When a large number of colonies are formed, it is preferable to divide into a group of about 2 to 7 colonies. When only one colony is included in one set, the accuracy of determining the dominant species in the microorganism to be examined is 100%, but the number of samples is too large and the experiment becomes complicated. In addition, as the number of colonies in one set increases, the possibility that a single set includes a plurality of types of microorganisms increases. However, as the number of types amplified simultaneously by PCR increases, there is a risk that non-specific products are amplified. Therefore, the primer used for PCR does not function effectively, and the probe used for the DNA chip does not function effectively, or a non-specific product inhibits binding of the target product. From such a viewpoint, the number of colonies in one set is more preferably 4 or 5.

  In addition, when the number of aggregates is 1, the detected microorganisms can be determined as the dominant species, but when the sample includes a plurality of microorganisms, the detected microorganisms cannot be compared. The number is preferably 2 or more. Furthermore, it is more preferable to set the number of groups to 4 or more, because when a sample contains several types of microorganisms to be examined, their preferential order can be determined to some extent.

  The method for extracting a nucleic acid from a microorganism is not particularly limited, and can be performed as follows, for example. Collecting colonies of various types of microorganisms generated in the medium for each assembly, placing them in a vial containing φ0.5mm zirconia beads, freezing the samples by immersion in liquid nitrogen, and using a shaking device Crush the microbial cells. Then, nucleic acids can be extracted from the crushed cells of the obtained microorganism by a CTAB method (Cetyl trimethyl ammonium bromide) or a DNA extraction device.

(C) Nucleic acid amplification step In this step, a nucleic acid fragment containing a target region (amplification target region) is amplified using the extracted nucleic acid.
“Target region (amplification target region)” means a target region to be amplified by a PCR (polymerase chain reaction) method or the like.
“Nucleic acid fragment” means a part of DNA or the like that is amplified using a primer set by PCR or the like.

The method for amplifying the nucleic acid fragment 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 the microorganism testing method of the present embodiment, the target region of the microorganism 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, 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 present embodiment, DNA fragments can be amplified using the ITS region and / or β-tubulin gene in mold genomic DNA as a target region.
At this time, as a primer set for amplifying the ITS region, a primer set comprising the nucleotide sequences of SEQ ID NOs: 1 and 2 shown in FIG. 1 (SEQ ID NO: 1: forward primer, SEQ ID NO: 2: reverse primer) can be used. .
Further, as a primer set for amplifying the β-tubulin gene, a primer set consisting of the nucleotide sequences of SEQ ID NOs: 3 and 4 shown in FIG. 1 (SEQ ID NO: 3: forward primer, SEQ ID NO: 4: reverse primer) is used. it can.

(D) Microorganism detection step In this step, the amplified nucleic acid fragments are used to detect the microorganisms contained in the set for each set.
When examining microorganisms, it may be necessary to simultaneously amplify a plurality of types of microorganisms whose target region sizes are not significantly different, and to identify each microorganism based on the amplified product. For this reason, as a method for detecting microorganisms, a method for determining the type of microorganism by binding the amplification product to a corresponding probe using a DNA chip (carrier for microorganism testing) and detecting the fluorescent label of the amplification product Are preferably used. This is because according to this method, the sequence of the amplification product is recognized, whereby each microorganism is more accurately identified.

Specifically, a probe selected from a target region in the genomic DNA of a microorganism to be examined is immobilized in advance on a DNA chip. Each probe can hybridize only with the corresponding amplification product derived from the microorganism to be examined, thereby making it possible to specifically detect the microorganism to be examined.
For example, as shown in FIG. 2, when detecting Eurotium sp., A probe having the base sequence shown in SEQ ID NO: 5 can be suitably used as a probe selected from the ITS region. As the probe selected from the β tubulin gene, a probe having the base sequence shown in SEQ ID NO: 6 can be preferably used.

Further, when detecting Aspergillus penicillioides, a probe having the base sequence shown in SEQ ID NO: 7 can be preferably used as the probe selected from the ITS region, and selected from the β-tubulin gene. As the probe, a probe having the base sequence shown in SEQ ID NO: 8 can be preferably used.
In addition, when detecting Aspergillus versicolor, a probe having the base sequence shown in SEQ ID NO: 9 can be preferably used as the probe selected from the ITS region, and selected from the β tubulin gene. A probe having the base sequence shown in SEQ ID NO: 10 can be preferably used as the probe.

When detecting Penicillium sp., A probe having the base sequence shown in SEQ ID NO: 11 can be preferably used as the probe selected from the ITS region, and selected from the β tubulin gene. A probe having the base sequence shown in SEQ ID NO: 12 can be preferably used as the probe.
Moreover, when detecting Fusarium sp., A probe having the base sequence shown in SEQ ID NO: 13 can be suitably used as the probe selected from the ITS region.
In addition, when detecting Cladosporium sp., A probe having the base sequence shown in SEQ ID NO: 14 can be suitably used as the probe selected from the ITS region.

  For the genus Eurotium, Aspergillus penicillides, and Aspergillus bargecolor, both the ITS region and the probe selected from the β-tubulin gene are used, and the mold is only obtained when a positive reaction is obtained in both of them. It is preferable to determine that has been detected. This is because it is possible to prevent erroneous judgment based on a false positive reaction for these molds.

  For Penicillium spp., It is preferable to determine that the mold is present when a positive reaction is obtained in at least one of the probes selected from the ITS region or the β-tubulin gene. Penicillium spp. Include molds that can only be detected by one of the probes selected from the ITS region or the probe selected from the β-tubulin gene used to detect Penicillium spp. This is because the specificity of each probe is high, and it is possible to detect mold of Penicillium spp. Without causing a false positive reaction.

  For Fusarium and Cladosporium, it is preferable to determine that the mold is present when a positive reaction is obtained with a probe selected from only the ITS region. This is because probes selected from the ITS region of these molds have high specificity, and these molds can be detected without causing false positive reactions.

A DNA chip that can be used in the microorganism testing method of the present embodiment can be manufactured by an existing general method using a probe having the base sequences shown in SEQ ID NOs: 5 to 14.
For example, when an affixed type DNA chip is produced, the probe can be immobilized on a glass substrate by a DNA spotter and a spot corresponding to each probe can be formed. 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 amplification product obtained in the nucleic acid amplification step is dropped onto the DNA chip thus obtained, and the amplification product is hybridized to the probe immobilized on the DNA chip. Then, by detecting the label of the hybridized amplification product, it is possible to identify the microorganism to be examined that exists in the environment.
The detection of the label can be performed using a general label detection device such as a fluorescence scanning device, for example, by measuring the fluorescence intensity of the fluorescent label in the amplification product using BIOSHOT manufactured by Toyo Kohan Co., Ltd. be able to. The measurement result is preferably obtained as an S / N ratio value (Signal to Noise ratio, (median fluorescence intensity value−background value) ÷ background value). The label is not limited to fluorescence, and other labels can also be used.

(E) Dominant species determination step When two or more types of microorganisms are detected in the microorganism detection step, each of the detected microorganisms is detected with respect to the total number of sets, which is the total number of sets of two or more. This is a step of determining the dominant species of microorganisms in the environment by calculating the ratio of the number of aggregates.

  Specifically, for example, when the total number of sets is 3, fungus A and fungus B are detected from set 1, fungus A and fungus B are detected from set 2, and fungus A and fungus C are set from set 3. When is detected, the ratio of the number of sets detected for each fungus is calculated. At this time, the ratio of fungus A is 3/3, which is 100.0%, the ratio of fungus B is 2/3, which is 66.7%, and the ratio of fungus C is 1/3, which is 33.3%. As a result, the first dominant species is determined as A, the second dominant species is determined as B, and the third dominant species is determined as C.

At this time, if the number of colonies is 15, and all of them are separated and cultured, and identification is performed individually by morphological observation method, for example, 9 fungi A, 4 fungi B, and 2 fungi C are detected. Then, the respective existence ratios can be accurately calculated as 60.0% for fungus A, 26.7% for fungus B, and 13.3% for fungus C.
However, identification by this form observation method has a problem that enormous labor, time, and cost are required. The problem became enormous as the number of colonies increased.

By the way, when the number of colonies generated in the medium is small, or when the inspector has a high ability of identifying the form, the dominant species may be determined without performing the separation culture. In such a case, the dominant species can be determined without using the microorganism testing method of the present embodiment.
Further, according to the determination of the dominant species in the microorganism testing method of the present embodiment, the method of collecting colonies from the culture medium in which the microorganisms are cultured into each group may affect the determination result.

Furthermore, the microorganisms present in the environment may vary greatly depending on the environment. The probe of the dominant species in the tested environment is immobilized on the DNA chip used in the microorganism testing method of this embodiment. It may not be.
Therefore, the microorganism inspection method of the present embodiment is not a classification that can completely detect the dominant species of microorganisms in all environments. Even if many probes of microorganisms that are likely to be detected are immobilized on the DNA chip, there is a possibility that other microorganisms are dominant species depending on the environment.

However, considering the practical use of the microorganism testing method of the present embodiment, these are not so problematic.
That is, according to the microorganism testing method of the present embodiment, it is generally assumed that the microorganism is often used in a similar environment, and the dominant species can be determined almost appropriately in the microorganism to be tested. The ability to easily determine the dominant species in this way is very effective in view of the enormous effort, time, and cost of the conventional morphological observation method. In addition, the accuracy of determining the dominant species can be increased by increasing the number of sets.

Here, in the microorganism testing method of this embodiment, when all the microorganisms of each colony generated in the culture medium are microorganisms to be examined, if the total number of colonies is the same as the number of colonies, the dominant species is theoretically complete. (Except for exceptional cases such as cases where there is a failure to collect microorganisms at the time of sample collection from the environment, or cases where culture has failed).
However, when the number of colonies is large, it is desirable to reduce the number of aggregates from the viewpoint of labor, time, and cost reduction.
Therefore, as a result of confirming the effect of the microorganism testing method of the present embodiment, the following example shows that the dominant species can be confirmed if the number of sets is 2 or more, and in the sample if the number of sets is 4 or more. It was also shown that the approximate order of superiority of the dominant species can be confirmed when multiple organisms to be tested are included in

  Thus, according to the microorganism testing method of this embodiment, it is possible to quickly and easily confirm the dominant species of microorganisms in the environment.

  Hereinafter, the test performed in order to confirm the effectiveness of the microbe inspection method according to the embodiment of the present invention will be specifically described.

[Determination of dominant species by microbe inspection method of this embodiment]
(A) Microbial culture step First, wild mold was collected from the facility environment and used as detection target mold. Specifically, air in various facility environments was collected using an air sampler, and this was sprayed on each medium of Samples A to J and cultured. Culturing was performed in a dark place at 25 ° C. for 7 days. M40Y medium was used for each of the media A to J.

(B) Nucleic acid extraction step Next, for each sample, all the colonies are divided into groups (aggregates) of 5 colonies or less at the maximum. Each mold was collected in groups and divided into groups.
Specifically, as shown in FIG. 4, 29 colonies were generated in the sample A. For this reason, the number of groups in sample A was 5 (5 colonies) and 4 (4 colonies), a total of 6 groups (total number of groups was 6).
Moreover, as shown in the figure, 22 colonies were generated in the sample B. For this reason, the number of groups in sample B was set to 5 (total number of sets is 5) including 2 groups of 5 colonies and 3 groups of 4 colonies.
Further, as shown in the figure, 21 colonies were generated in the sample C. For this reason, the group in the sample C was set to 5 groups (3 in total), 3 groups of 5 colonies, 1 group of 4 colonies, and 1 group of 2 colonies.

Further, as shown in FIG. 5, 20 colonies were generated in the sample D. For this reason, the group in sample D was set to a total of four groups of four colonies (total number of sets is four).
Further, as shown in the figure, the sample E had 19 colonies. For this reason, the group in the sample E was set to 4 groups (3 groups of 5 colonies, 1 group of 4 colonies, total number of 4).
Further, as shown in the figure, 18 colonies were generated in the sample F. For this reason, the group in the sample F was set to 4 groups (total number of groups: 4) including 3 groups of 5 colonies and 1 group of 3 colonies.

Furthermore, as shown in FIG. 6, 9 colonies were generated in the sample G. For this reason, the group in the sample G was set to 2 groups (total number of groups: 2), one group of 5 colonies and one group of 4 colonies.
Moreover, as shown in the figure, the sample H had 6 colonies. For this reason, the group in the sample H was set to 2 groups (total number of sets: 2), one group of 5 colonies and one group of 1 colonies.
Moreover, as shown in the figure, Sample I had 3 colonies. For this reason, the group in sample I was set to one total of three colony groups (the total number of sets was 1).
Moreover, as shown in the figure, the sample J had 3 colonies. For this reason, the group in the sample J was set to one total of three colony groups (total number of sets is 1).

  Each group of colonies was placed in a vial containing Φ0.5 mm zirconia beads, immersed in liquid nitrogen to freeze the sample, and then the mold cells in the colonies were crushed using a shaking device. For each group, mold genomic DNA was extracted by a DNA extraction apparatus.

(C) Nucleic acid amplification step Subsequently, the ITS region of each mold and the β-tubulin gene were simultaneously amplified by PCR.
At this time, the forward primer (F primer) consisting of the base sequence of SEQ ID NO: 1 and the reverse primer (R primer) consisting of the base sequence of SEQ ID NO: 2 shown in FIG. 1 were used as the ITS region amplification primer set. Moreover, the forward primer which consists of a base sequence of sequence number 3 shown in the same figure and the reverse primer which consists of the base sequence of sequence number 4 shown in the figure were used as a primer set for β-tubulin gene amplification. In addition, all synthesized by Operon Technology Co., Ltd. were used.

Further, as a PCR reaction solution, Ampdirect (R) (manufactured by Shimadzu Corporation) was used for each of samples A to J, and 20 μl of the following composition was prepared.
1. Ampdirect (G / Crich) 4.0μl
2. Ampdirect (addition-4) 4.0μl
3. dNTPmix 1.0μl
4). Cy-5dCTP 0.2μl
5. ITS1-Fw primer (2.5μM) (SEQ ID NO: 1) 1.0μl
6). ITS1-Rv primer (2.5μM) (SEQ ID NO: 2) 1.0μl
7). BtF primer (10μM) (SEQ ID NO: 3) 1.0μl
8). BtR primer (10μM) (SEQ ID NO: 4) 1.0μl
9. Template DNA (each sample A to J) 1.0μl
10. NovaTaq HotStart DNA polymerase 0.2μl
11. Water (water until 20.0 μl total)

DNA was amplified by the nucleic acid amplification apparatus (TaKaRa PCR Thermal Cycler Dice (R) Gradient Takara Bio Inc.) using the above PCR reaction solutions 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

(D) Microorganism detection step Gene DNA (R) (manufactured by Toyo Kohan Co., Ltd.) was used as the DNA chip, and all the following probes shown in FIG. 2 were immobilized. In the following, (ITS) indicates a probe selected from the ITS region, and (β) indicates a probe selected from the β tubulin gene.
(1) For the genus Eurotium SEQ ID NO: 5 (ITS), SEQ ID NO: 6 (β)
(2) Aspergillus for Penicilliosides SEQ ID NO: 7 (ITS), SEQ ID NO: 8 (β)
(3) Aspergillus for barge color bacteria SEQ ID NO: 9 (ITS), SEQ ID NO: 10 (β)
(4) For Penicillium sp. SEQ ID NO: 11 (ITS), SEQ ID NO: 12 (β)
(5) For Fusarium spp. SEQ ID NO: 13 (ITS)
(6) For the genus Cladosporium SEQ ID NO: 14 (ITS)

Next, for each of samples A to J, the PCR amplification product was mixed with a buffer (3 × SSC citrate-saline + 0.3% SDS) and heated at 94 ° C. for 5 minutes. It was dripped in.
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 DNA chip.

Subsequently, the fluorescence intensity in each probe was measured by applying the DNA chip to a label detection device (GenePix4100A Molecular Devices), and the S / N ratio value was calculated.
Here, for (1) Eurotium, (2) Aspergillus penicillides, and (3) Aspergillus bargecolor, both S / B of the probe selected from the ITS region and the probe selected from the β-tubulin gene are used. A positive determination was made when the N ratio value was 3 or more.
In addition, (4) Penicillium is determined to be positive when the S / N ratio value of at least one of the probe selected from the ITS region and the probe selected from the β-tubulin gene is 3 or more. did.
Further, (5) Fusarium spp. And (6) Cladosporium spp. Were determined to be positive when the S / N ratio value of the probe selected from the ITS region was 3 or more.

(E) Predominant species determination step As a result, as shown in FIG. 4, in the group of sample A, Cladosporium spp. Was detected in 5 groups (5 chips showing positive), and Penicillium spp. Detected in groups (1 chip showing positive). The number of chips used is six.
Therefore, according to the dominant species determination method in the microorganism testing method of the present embodiment, the ratio of the number of cladosporium genus bacteria is 5 detected populations ÷ total population number 6 = 83.3%. The ratio of the number of Penicillium genus populations is 1 for the number of detected populations / 6 for the total number of populations = 16.7%.
Therefore, it is determined that the dominant species of sample A is the first genus Cladosporium and the second is the genus Penicillium.

Similarly, in the group in Sample B, Cladosporium spp. Were detected in 5 groups (5 chips showing positive), and Penicillium spp. Were detected in 2 groups (2 chips showing positive) ). The number of chips used is five.
Therefore, according to the dominant species determination method in the microorganism testing method of the present embodiment, the proportion of the number of cladosporum populations is 5 detected populations = 5 total populations = 100.0%. The ratio of the number of populations of the genus Penicillium is: the number of detected sets 2 ÷ the total number of sets 5 = 40.0%.
Therefore, it is determined that the dominant species of sample B is Cladosporium genus first and Penicillium genus second.

Similarly, in the group in sample C, Cladosporium spp. Was detected in 5 groups (5 chips showing positive), and Penicillium spp. Was detected in 1 group (1 chip showing positive) ). The number of chips used is five.
Therefore, according to the dominant species determination method in the microorganism testing method of the present embodiment, the proportion of the number of cladosporum populations is 5 detected populations = 5 total populations = 100.0%. The ratio of the number of Penicillium genus population is 1 for the number of detected populations ÷ 5 for the total number of populations = 20.0%.
Therefore, it is determined that the dominant species of sample C is the first genus Cladosporium and the second is the genus Penicillium.

Similarly, in the group in sample D, Cladosporium spp. Were detected in 4 groups (4 chips that showed positive), and Penicillium spp. Were detected in 2 groups (2 chips that showed positive). Eurotium bacteria were detected in 2 groups (2 chips showing positive). The number of chips used is four.
Therefore, according to the dominant species determination method in the microorganism testing method of the present embodiment, the ratio of the number of cladosporium genus bacteria is 4 detected populations = total population number 4 = 100.0%. The ratio of the number of populations of the genus Penicillium is the detected number of sets 2 ÷ total number of sets 4 = 50.0%, and the ratio of the number of populations of the genus Eurotium is 2 × the number of detected sets 4 = 50.0%.
Therefore, the dominant species of the sample D is determined to be the first genus Cladosporium and the second one is the genus Penicillium and the genus Eurotium.

Similarly, in the group in sample E, Cladosporium spp. Were detected in 4 groups (4 chips that showed positive), and Penicillium spp. Were detected in 2 groups (2 chips that showed positive). , Fusarium spp. Were detected in one group (1 chip showing positive). The number of chips used is four.
Therefore, according to the dominant species determination method in the microorganism testing method of the present embodiment, the ratio of the number of cladosporium genus bacteria is 4 detected populations = total population number 4 = 100.0%. The ratio of the number of populations of the genus Penicillium is the number of detected sets 2 ÷ total number of sets 4 = 50.0%, and the ratio of the number of populations of the genus Fusarium is 1 × the number of detected sets 4 = 25.0%.
Therefore, it is determined that the dominant species of sample E is the first genus Cladosporium, the second is the genus Penicillium, and the third is the genus Fusarium.

Similarly, in the group in sample F, Aspergillus virgin color bacteria were detected in 3 groups (3 chips showing positive), and Cladosporium bacteria were detected in 2 groups (2 chips showing positive). ), Aspergillus penicilliosides were detected in 2 groups (2 chips showing positive). The number of chips used is four.
Therefore, according to the dominant species determination method in the microorganism testing method of the present embodiment, the ratio of the number of Aspergillus bargecolor fungus populations is 3 detected populations = 4 total populations = 75.0%, The ratio of the number of cladosporium genus is 2 = the number of detected groups = 4 = 50.0%, and the percentage of the number of Aspergillus penicilliosides is 2 / total number of detected groups Number 4 = 50.0%.
Therefore, it is determined that the dominant species of sample F is Aspergillus bargecolor, the first is Cladosporium and Aspergillus penicillios.

Similarly, in the group in sample G, Cladosporium spp. Was detected in 2 groups (2 chips showing positive), and Penicillium spp. Was detected in 1 group (1 chip showing positive) ). Two chips were used.
Therefore, according to the dominant species determination method in the microorganism testing method of the present embodiment, the ratio of the number of cladsporium genus populations is: detected population number 2 ÷ total population number 2 = 100.0% The ratio of the number of populations of the genus Penicillium is 1 for the number of detected sets / total number of the sets 2 = 50.0%.
Therefore, it is determined that the dominant species of the sample G is the first genus Cladosporium and the second is the Penicillium genus.

Similarly, in the group in Sample H, Cladosporium spp. Were detected in 2 groups (two chips that showed positive). Two chips were used.
Therefore, according to the dominant species determination method in the microorganism testing method of the present embodiment, the ratio of the number of cladosporium genus populations is the number of detected populations 2 divided by the total number of populations 2 = 100.0%. .
Therefore, it is determined that the dominant species of sample H is the genus Cladosporium.

Similarly, in the group in Sample I, Cladosporium spp. Were detected in one group (one chip that showed positive). The number of chips used is one.
Therefore, according to the dominant species determination method in the microorganism testing method of the present embodiment, the ratio of the number of cladosporum bacteria is 1 / the number of detected aggregates = 1 = 100.0%. .
Therefore, it is determined that the dominant species of sample I is Cladosporium.

Similarly, in the group of sample J, Aspergillus penicilliosides was detected in one group (one chip showing positive), and Cladosporium was detected in one group (one chip showing positive) ). The number of chips used is one.
Therefore, according to the method for determining the dominant species in the microorganism testing method of the present embodiment, the ratio of the number of Aspergillus penicilliosides populations is 1 for the number of detected populations divided by 1 for the total population 1 = 100.0%. The ratio of the number of sporium genus is 1 = 100.0%.
Therefore, it is determined that the dominant species of sample J are Aspergillus penicilliosides and Cladosporium.

[Confirmation of dominant species by DNA sequence analysis]
For each of the samples A to J, a part of each type of various types of mold colonies generated in the medium was individually collected and separately cultured for 7 to 10 days in a dark place at 25 ° C. M40Y medium was used for each of the media A to J.
Then, the mold of each colony separated and cultured was subjected to DNA sequence analysis to confirm its bacterial species.

Specifically, a forward primer having a base sequence shown in SEQ ID NO: 15 and a reverse primer having a base sequence shown in SEQ ID NO: 16 are used as a primer set, TAKARA ExTaq polymerase is used as a nucleic acid synthesizing enzyme, and a nucleic acid amplification device is used. Using the TaKaRa PCR Thermal Cycler (R) Gradient (manufactured by Takara Bio Inc.), the ITS1 region in each mold genome was amplified in the same manner as the PCR conditions described above.
Each amplification product and the above primer set (primer set for sequencing) were outsourced to Takara Bio Inc., and DNA sequence analysis was performed with a DNA sequencer to confirm the type of mold corresponding to each amplification product. The results are shown in FIGS.

[Test results]
From the sample A, four types of microorganisms of Xylariales sp., Diaporthe sp., Pleosporales sp., And Stereum hirsutum were detected in addition to Cladosporium and Penicillium spp. The probes for detecting these four types of microorganisms are not immobilized on the DNA chip used in the microorganism testing method of this embodiment, and these are microorganisms that are not tested in this test.
The dominant species in sample A determined by the microorganism testing method of the present embodiment was Cladosporium genus first and Penicillium genus second.
On the other hand, as a result of DNA sequence analysis, among the colonies in sample A, the actual first dominant species was Xylariales sp.
However, this is a non-tested microorganism in this test, and the cladsporium and penicillium species that were tested were detected as the actual second and actual third dominant species, respectively. .
Therefore, it can be seen from this result that the dominant species and the rank order of the dominant species can be determined with high accuracy in a microorganism to be examined by a simple method.

From sample B, four types of microorganisms, Mycosphaerella crystallina, Xylariales sp., Artrinium sp., And Leptosphaerulina chartaru, were detected in addition to Cladosporium and Penicillium. The probes for detecting these four types of microorganisms are not immobilized on the DNA chip used in the microorganism testing method of this embodiment, and these are microorganisms that are not tested in this test.
The dominant species in the sample B determined by the microorganism testing method of the present embodiment was the first genus Cladosporium and the second genus Penicillium.
As a result of DNA sequence analysis, among the colonies in sample B, the actual first dominant species was Cladosporium, and the actual second dominant species was Penicillium.
Therefore, it can be seen from this result that the dominant species and the rank order of the dominant species can be determined with high accuracy in a microorganism to be examined by a simple method.

Sample C was analyzed by DNA sequence analysis in addition to Cladosporium and Penicillium, Arthrinium sp., Periconia macrospinosa, Toxicocladosporium irritans, Dothideomycete sp., Phoma sp., Pleosporales sp., And Unknown (unknown type) 7 types of microorganisms were detected. Probes for detecting these seven types of microorganisms are not immobilized on the DNA chip used in the microorganism testing method of this embodiment, and these are microorganisms that are not tested in this test.
As for the dominant species in the sample C determined by the microorganism testing method of the present embodiment, the first was the genus Cladosporium and the second was the genus Penicillium.
As a result of DNA sequence analysis, among the colonies in sample C, the actual first dominant species was Cladosporium, and the actual second dominant species was Penicillium.
Therefore, it can be seen from this result that the dominant species and the rank order of the dominant species can be determined with high accuracy in a microorganism to be examined by a simple method.

Sample D was analyzed by DNA sequence analysis, and in addition to Cladosporium spp, Penicillium spp, and Eurotium spp., Four types of Unknown (unknown types), Pleosporales sp., Diaporthe sp. Of microorganisms were detected. The probes for detecting these four types of microorganisms are not immobilized on the DNA chip used in the microorganism testing method of this embodiment, and these are microorganisms that are not tested in this test.
The dominant species in the sample D determined by the microorganism testing method of the present embodiment was Cladosporium genus in the first and Penicillium and Eurotium in the second.
As a result of DNA sequence analysis, among the colonies in sample D, the actual first dominant species is Cladosporium, the actual second dominant species is Penicillium, and the actual third The dominant species was Eurotium.
Therefore, it can be seen from this result that the dominant species can be determined with high accuracy in a microorganism to be examined by a simple method. It can also be seen that the order of superiority can be determined to some extent.

From the sample E, two types of microorganisms of Leptosphaeria sp. And Unknown (unknown type) were detected in addition to Cladosporium, Penicillium, and Fusarium by DNA sequence analysis. Probes for detecting these two types of microorganisms are not immobilized on the DNA chip used in the microorganism testing method of this embodiment, and these are microorganisms that are not tested in this test.
The dominant species in the sample E determined by the microorganism testing method of the present embodiment was Cladosporium genus first, Penicillium genus second, and Fusarium genus third.
As a result of DNA sequence analysis, among the colonies in sample E, the actual first dominant species is Cladosporium, the actual second dominant species is Penicillium, and the actual third The dominant species was Fusarium spp.
Therefore, it can be seen from this result that the dominant species and the rank order of the dominant species can be determined with high accuracy in a microorganism to be examined by a simple method.

In addition to Aspergillus bargecolor, Cladosporium, and Aspergillus Penicillides, sample F was analyzed by DNA sequence analysis, as well as Leptosphaerulina chartarum, Leptosphaeria sp., Sclerotinia sclerotiorum, Creosphaeria sassafrans, Fungal endophyte
Six types of microorganisms, sp. and Unknown (unknown types), were detected. Probes for detecting these six types of microorganisms are not immobilized on the DNA chip used in the microorganism testing method of the present embodiment, and these are microorganisms that are not tested in this test.
The dominant species in the sample F determined by the microorganism testing method of the present embodiment were Aspergillus bargecolor bacteria first, and Cladosporium sp. And Aspergillus penicilliosides.
As a result of DNA sequence analysis, among the colonies in sample F, the actual first dominant species is Aspergillus bargecolor, the actual second dominant species is Cladosporium, and the actual third The second dominant species was Aspergillus penicilliosides.
Therefore, it can be seen from this result that the dominant species can be determined with high accuracy in a microorganism to be examined by a simple method. It can also be seen that the order of superiority can be determined to some extent.

From sample G, Phaeosphaeriopsis sp. Was detected in addition to Cladosporium and Penicillium by DNA sequence analysis. The probe for detecting this microorganism is not immobilized on the DNA chip used in the microorganism testing method of the present embodiment, and this is a microorganism that is not subject to testing in this test.
As for the dominant species in the sample G determined by the microorganism testing method of the present embodiment, the first was Cladosporium and the second was Penicillium.
On the other hand, as a result of DNA sequence analysis, among the colonies in sample G, the actual first dominant species was Cladosporium sp., But the actual second dominant species was Phaeosphaeriopsis sp. The genus was the actual third dominant species.
However, Phaeosphaeriopsis sp. Is a non-tested microorganism in this test, and the test species Cladosporium and Penicillium were detected as the actual first and actual third dominant species, respectively. ing.
Therefore, it can be seen from this result that the dominant species and the rank order of the dominant species can be determined with high accuracy in a microorganism to be examined by a simple method.

In addition to Cladosporium sp., Pleosporales sp. Was detected from sample H by DNA sequence analysis. The probe for detecting this microorganism is not immobilized on the DNA chip used in the microorganism testing method of the present embodiment, and this is a microorganism that is not subject to testing in this test.
The dominant species in sample H determined by the microorganism testing method of the present embodiment was Cladosporium sp.
As a result of DNA sequence analysis, among the colonies in sample H, the actual first dominant species was Cladosporium, and the actual second dominant species was Pleosporales sp.
Therefore, it can be seen from this result that the dominant species can be determined with high accuracy in a microorganism to be examined by a simple method.

From Sample I, Alternaria alternata was detected in addition to Cladosporium spp. By DNA sequence analysis. Probes for detecting these microorganisms are not immobilized on the DNA chip used in the microorganism testing method of this embodiment, and these are microorganisms that are not tested in this test.
The dominant species in sample I determined by the microorganism testing method of the present embodiment was Cladosporium sp.
As a result of DNA sequence analysis, among the colonies in Sample I, the actual first dominant species was Cladosporium, and the actual second dominant species was Alternaria alternata.
Therefore, it can be seen from this result that the dominant species can be determined with high accuracy in a microorganism to be examined by a simple method.

From sample J, Aspergillus penicilliosides and Cladosporium were detected by DNA sequence analysis.
The dominant species in Sample J determined by the microorganism testing method of the present embodiment were Aspergillus penicilliosides and Cladosporium.
As a result of DNA sequence analysis, among the colonies in sample J, the actual first dominant species was Aspergillus penicillioides, and the actual second dominant species was Cladosporium.
Sample J uses only one chip, so if multiple microorganisms are present, the difference between these dominant species cannot be determined, but the actual dominant species is identified in the microorganism to be tested. I can do it.
As described above, according to the microorganism testing method of the present embodiment, the dominant species can be determined with high accuracy by a simple method.

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, components other than the ITS region amplification primer set and the β-tubulin gene amplification primer set in the PCR reaction solution of the above test can be appropriately changed. Further, instead of performing fluorescence detection using the DNA chip as described above, it is also possible to detect an amplification product hybridized to the probe using a DNA chip of another detection system such as a current detection system.

  The present invention quickly and easily detects the types of microorganisms and the dominant species in environmental inspections, food inspections, epidemiological environmental inspections, clinical tests, livestock hygiene, etc., when performing inspections to confirm the presence of microorganisms in the environment In some cases, it can be suitably used.

Claims (4)

A culture step of collecting a sample from the environment and culturing a microorganism contained in the obtained sample in a medium;
When two or more colonies are formed by culturing the microorganism, the nucleic acid extraction step of dividing the formed colonies into two or more sets and extracting the nucleic acid of the microorganisms included in the set for each set When,
A nucleic acid amplification step of amplifying a nucleic acid fragment containing an amplification target region using the extracted nucleic acid;
Using the amplified nucleic acid fragment, a microorganism detection step for detecting the microorganism contained in the assembly for each assembly;
When two or more types of microorganisms are detected in the microorganism detection step, the ratio of the number of sets in which each microorganism is detected to the total number of sets that is the total number of the two or more sets for each detected microorganism. And a dominant species estimation step of estimating the species having the highest ratio as the dominant species of the microorganism in the environment.   When four or more colonies are formed by culturing the microorganism, in the nucleic acid extraction step, the formed colonies are divided into four or more sets, and the nucleic acid of the microorganism included in the set for each set The method for inspecting microorganisms according to claim 1, wherein the microorganism is extracted.   3. The microorganism testing method according to claim 1, wherein the microorganism is mold, yeast, or bacteria.   In the microorganism detection step, the type of the microorganism included in the assembly for each assembly using a carrier on which a probe for detecting the microorganism that binds complementarily to the amplified nucleic acid fragment is immobilized. The method for examining microorganisms according to any one of claims 1 to 3, wherein:

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