JP2018143251A - Method for inspecting microorganism - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for inspecting microorganism in which the entire period of inspection including sampling can be shortened even more.SOLUTION: A method for inspecting microorganism in which existence of a plurality of types of microorganism in an environment is simultaneously determined has: a collection process of recovering microorganism floating in the air into liquid; a nucleic acid extraction process of extracting nucleic acid from microorganism in liquid; a nucleic acid amplification process of amplifying a part of extracted nucleic acid; detection process of specifying a type of microorganism in the case where solution containing amplified nucleic acid is dripped on a carrier on which nucleic acid of the plurality of types of microorganism is immobilized and nucleic acid which bonds with the amplified nucleic acid in a complementary manner is immobilized on the carrier, in which culture of microorganism is not performed after the collection process and before the nucleic acid extraction process.SELECTED DRAWING: None

Description

  The present invention relates to a microorganism inspection method, and more particularly to an inspection method for rapidly detecting a plurality of types of microorganisms simultaneously.

Conventionally, it has been important to check for the presence of mold and other microorganisms in food production sites, clinical sites, and protected environments such as cultural properties, to confirm their safety and to prevent their propagation. It has become.
As an inspection method for such microorganisms, particularly mold, the following three methods can be mentioned.

First, as a first method, a sample is sampled from the environment and pre-cultured (simultaneous culture of the collected bacteria), and then cultured for about 20 days in an optimal medium for each bacterial species, and then morphologically By observing the characteristics, a method for identifying a microorganism and confirming its presence or absence can be cited (see 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 a microorganism can be mentioned.
Furthermore, as a third method, a probe having a base sequence that binds complementarily to the target region is prepared and immobilized on the substrate of the DNA chip, and an amplification product by the PCR method is dropped on the DNA chip to obtain the target region and Examples include a method of hybridizing with a probe and identifying and confirming the presence of microorganisms contained in a sample (see Patent Document 2).

The first method for identifying microorganisms based on their form requires individual culturing after pre-culture and growth. At that time, in order to express the morphological characteristics of microorganisms, it is necessary to prepare an optimal medium for each bacterial species and perform long-term culture, so depending on the type of microorganism, a long test period of about 50 days is required. It was a thing. Moreover, since skill is required for identification of microorganisms, it was not suitable for quick inspection or simple inspection.
Even in the second method by sequence analysis, it is necessary to cultivate each bacterial species individually after pre-culture. For this reason, about 14 days were required as an inspection period. Moreover, since it is a method of analyzing for each bacterial species individually, it is not suitable for cases where it is necessary to examine a large number of specimens simultaneously.

On the other hand, in the third method using a DNA chip, microorganisms can be detected without individually culturing each bacterial species, and the test period can be shortened to about 9 days.
However, at the inspection site, there is a demand for further speeding up the inspection so as to know the result during the day when the sample is sampled from the environment.
However, the third method using a DNA chip also requires pre-culture, and this pre-culture requires about 7 days. Therefore, the third method cannot meet the demand.
Here, a method for rapid detection of micro-organisms has been proposed that makes it possible to carry out microorganism testing using a DNA chip without performing pre-culture (see Patent Document 3).

JP 2007-195454 A JP 2008-35773 A JP 2007-508811 A

However, this method for rapid detection of micro-organisms extracts RNA from a microorganism, but RNA cannot be obtained when the microorganism is in the form of spores. On the other hand, since it is not possible to selectively collect non-spore-form microorganisms, there is a problem that a recovery step for germinating spores becomes essential.
In addition, since microorganisms can be detected without amplifying RNA or DNA complementary thereto, it is necessary to collect a very large number of microorganism cells (in the examples, one million cells are collected). Use.Sampling takes a few days in a relatively dirty environment.), It was difficult to shorten the entire test period including sampling.
The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a microorganism testing method capable of further shortening the entire testing period including sampling.

  In order to achieve the above object, the microorganism testing method of the present invention is a microorganism testing method for simultaneously determining the presence or absence of a plurality of types of microorganisms in the environment, and collecting microorganisms floating in the air in a liquid A nucleic acid extraction step for extracting nucleic acid from microorganisms in a liquid, a nucleic acid amplification step for amplifying a part of the extracted nucleic acid, and a solution containing the amplified nucleic acid in advance, fixing nucleic acids of a plurality of types of microorganisms And a detection step for identifying the type of microorganism when a nucleic acid that is added dropwise to the carrier and complementarily binds to the amplified nucleic acid is immobilized on the carrier, and after the collection step, and Before the nucleic acid extraction step, the microorganism is not cultured.

  ADVANTAGE OF THE INVENTION According to this invention, it becomes possible to provide the microbe test | inspection method which can further shorten the period of the whole test | inspection including sampling.

It is a graph which shows the relationship between the air quantity collect | recovered with the air sampler in an Example, and the number of molds. It is a graph which shows the base sequence of the primer used in the Example. It is a graph which shows the base sequence of the probe used in the Example. It is a graph which shows the detection result (S / N ratio) by the DNA chip in an Example.

Hereinafter, preferred embodiments of the present invention will be described in detail.
The microorganism testing method of the embodiment of the present invention is a microorganism testing method for simultaneously determining the presence or absence of a plurality of types of microorganisms in the environment, a collection step of collecting the microorganisms floating in the air in a liquid, A nucleic acid extraction step for extracting nucleic acids from microorganisms therein, a nucleic acid amplification step for amplifying a part of the extracted nucleic acids, and a solution containing the amplified nucleic acids in a carrier on which nucleic acids of a plurality of types of microorganisms are immobilized in advance. And a detection step for identifying the type of microorganism when a nucleic acid that is added dropwise and binds complementarily to the amplified nucleic acid is immobilized on a carrier, and after the collection step and in the nucleic acid extraction step Before, the microorganism is not cultured.

The microorganism testing method of the present embodiment may include the following steps.
(1) Collection process In the collection process, microorganisms floating in the air are collected in a liquid. At this time, it is preferable to use a cyclone type air sampler as a sampling device for collecting microorganisms. Further, as the liquid, it is preferable to use a liquid containing a surfactant in order to increase the recovery efficiency of microorganisms.
By using a cyclone type air sampler, air is drawn into the liquid in the sampling container at a high speed and then discharged, and in the process, fine particles in the air adhere to the inner wall of the sampling container by centrifugal force. As a result, microorganisms in the air can be recovered in the liquid in the sampling container.

As the cyclone type air sampler, for example, Coriolis μ (liquid phase centrifugation method: liquid cyclone, manufactured by Bertin) can be suitably used.
When a cyclone type air sampler is used, it is preferable to perform air suction at a flow rate of 300 L / min for 1 to 10 minutes.

Thus, by using a cyclone type air sampler in the collection step, a relatively large number of microorganisms can be recovered in a liquid from a large amount of air in a short time. Therefore, even if the microorganism is not cultured (pre-culture) prior to the nucleic acid extraction step, the nucleic acid of the microorganism can be sufficiently amplified in the nucleic acid amplification step, and the microorganism can be detected in the detection step. .
In addition, since it is not necessary to perform pre-culture of microorganisms, it is possible to reduce the risk that the microorganisms cannot be detected because the microorganisms cannot grow properly due to the conditions of the medium.

In the microorganism testing method of this embodiment, “microorganism” includes fungi such as mold and yeast, bacteria such as food poisoning bacteria, and other minute organisms that cannot be seen without using a microscope.
In particular, according to the method for inspecting microorganisms of the present embodiment, spore or mycelium-type mold floating in the air, which is important in food inspection, epidemiological environmental inspection, environmental inspection, clinical test, livestock hygiene and the like, is suitable. Can be detected.
In addition, since it is not necessary to culture microorganisms, it is possible to specify the type of microorganisms not only from living microorganisms but also from the death of microorganisms such as dead bacteria.

(2) Nucleic acid extraction step In the nucleic acid extraction step, nucleic acids are extracted from microorganisms in the liquid obtained in the collection step. Thereby, genomic DNA can be obtained from the microorganism. Nucleic acid extraction can be performed by a normal method, and is not particularly limited.
For example, using a commercially available nucleic acid extraction kit (Water RNA / DNA Purification Kit-0.22 μm, manufactured by Norgen Biotek Corporation), the liquid is filtered through a filter to concentrate the microorganisms, and the cells are crushed and lysed. By purifying, nucleic acid can be suitably extracted. Further, nucleic acid may be extracted by a CTAB method (Cetyl trimethyl ammonium bromide) or a DNA extraction apparatus.

(3) Nucleic acid amplification step In the nucleic acid amplification step, a part of the nucleic acid extracted in the nucleic acid extraction step is amplified. As a result, the amplification target region in the genomic DNA serving as a sample can be amplified to a quantity necessary for detection.
As described above, according to the microorganism testing method of the present embodiment, the microorganisms floating in the air are collected in the liquid using the cyclone air sampler in the collection process, and the nucleic acids of the microorganisms are amplified in the nucleic acid amplification process. be able to.
For this reason, since it is possible to sufficiently amplify the nucleic acid of the microorganism in the nucleic acid amplification step without collecting the microorganism for a long time in the collection step, it is possible to appropriately detect the microorganism in the detection step. Yes.

The nucleic acid amplification method is not particularly limited, but for example, the PCR method can be suitably used.
It is preferable that the PCR reaction solution used in the PCR method has the following composition, for example. That is, a PCR reaction solution containing a nucleic acid synthesis substrate, a primer set, a nucleic acid synthase, a labeling component, a sample genomic DNA, a buffer solution, and water as the remaining volume can be preferably used.

  In the PCR method, an amplification product can be obtained by amplifying the amplification target region using a primer set for amplifying the amplification target region in the genomic DNA of the sample. Further, in the subsequent detection step, the amplification product is hybridized to a probe that can complementarily bind to a part of the amplification target region. And it becomes possible to identify the microorganisms which have the said genomic DNA by detecting the label component contained in the hybridized amplification product.

As a buffer solution, for example, Ampdirect (R) (Shimadzu Corporation) can be used.
A general thermal cycler or the like can be used as the PCR apparatus.
For example, the PCR reaction conditions are preferably as follows.
(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 Step In the detection step, a solution containing the amplification product obtained by the PCR method is dropped onto a carrier on which a plurality of types of microorganism probes are immobilized in advance. When a probe that binds complementarily to the amplification product is immobilized on this carrier, the amplification product and the probe hybridize. Then, by detecting the label component contained in the hybridized amplification product, the types of a plurality of types of microorganisms can be specifically identified simultaneously.
A DNA chip can be suitably used as such a carrier. The DNA chip is not particularly limited as long as it is prepared by previously synthesizing a probe selected from a region to be amplified by PCR in genomic DNA of a microorganism to be detected and immobilizing it on a substrate. For example, a spot type DNA chip or a synthetic type DNA chip can be used.

Specifically, the label component of the amplification product can be detected, for example, as follows.
First, a predetermined buffer solution is mixed with the amplification product and dropped onto the DNA chip, and the DNA chip is allowed to stand at 45 ° C. for 1 hour. Thereafter, the PCR product that has not hybridized is washed away from the DNA chip using a predetermined buffer.
Next, the label component is detected by applying the DNA chip to the label detection device, and it is determined whether or not the detection target microorganism exists. As the label detection device, for example, a general device such as a fluorescence scanning device can be used. The label and its detection method are not limited to those using fluorescence, and other methods may be used.

As described above, according to the microorganism testing method of the present embodiment, microorganisms floating in the air are collected in a liquid using a cyclone-type air sampler in the collection step, and the microorganism nucleic acid is collected in the nucleic acid amplification step. Can be amplified. For this reason, it is possible to obtain a sufficient amount of nucleic acid for detection without carrying out pre-culture of microorganisms, which was conventionally required before the nucleic acid extraction step, and to specifically detect a plurality of types of microorganisms simultaneously in the detection step. It is possible to detect.
In addition, since the nucleic acid of the microorganism can be sufficiently amplified in the nucleic acid amplification step without collecting the microorganism in the collection step for a long time, it is possible to appropriately detect the microorganism in the detection step.
For this reason, according to the microorganism testing method of the present embodiment, it is possible to further shorten the period of the entire test including sampling.

  In addition, according to the microorganism testing method of the present embodiment, since the microorganism can be detected without performing pre-culture of the microorganism, it is possible to identify the microorganism from the death of the microorganism floating in the air. . Even if microorganisms are dead, human inhalation can cause health hazards, and it is beneficial to be able to detect this more quickly.

Examples of the microorganism testing method of the present invention will be specifically described below.
In the collection process, in order to collect the microorganisms floating in the air in the liquid, a cyclone type air sampler (Coriolis μ, manufactured by Bertin) was used to suck in air at two points. Got one sample. The flow rate of the air sampler was fixed at 300 L / min.

First, air suction was performed three times at point A (toilet room), and samples 1-3 were obtained with collection times of 1 minute, 3 minutes, and 5 minutes, respectively. The collected air amounts of Sample 1-3 are 300L, 900L, and 1500L, respectively.
In addition, sample 4 was obtained by performing air suction once at the point B (the reception room) and setting the collection time to 10 minutes. The amount of recovered air of sample 4 is 3000L.
The amount of liquid for collecting microorganisms was 10 mL at the start of air collection for all samples. After the air recovery, the liquid volume decreased due to evaporation of the sample during the recovery, so the remaining liquid volume was smaller as the recovery time was longer, and the remaining liquid volume was present at 5.1 mL or more for all samples.

Each 0.1 mL was taken out from the liquid of sample 1-4, and applied to an M40Y medium sterilized in a petri dish having a diameter of 90 mm. This was performed for two samples for each sample and cultured at 25 ° C. for 7 days. This M40Y medium is a medium that can suitably cultivate any of dry, mildew, and wet molds, and is obtained by adding Malt extract 20 g, Yeast extract 5 g, Sucrose 400 g, and Agar 20 g to 1 L of distilled water. It is what was done.
Then, samples were taken out on the 4th and 7th days in each medium, and the number of colonies grown visually was counted, and the average was calculated for each sample. The result is shown in FIG.

  As shown in FIG. 1, the average number of molds in 0.1 mL of Sample 1-4 was 2, 9, 15, and 3 respectively. Accordingly, the number of molds in 5 mL of liquid is converted to 100, 450, 750, and 150, respectively. Considering the amount of recovered air, the number of molds in sample 1-3 at point A is greater than the number of molds in sample 4 at point B. The reason is considered that the air at the point A is more dirty than the air at the point B.

Next, in the nucleic acid extraction step, using a commercially available nucleic acid extraction kit (Water RNA / DNA Purification Kit-0.22 μm, manufactured by Norgen Biotek Corporation) Nucleic acids were extracted from molds.
At this time, 5 mL of the liquid of Sample 1-4 was filtered through a filter, and then the filter was separated and collected in a crushing tube containing glass beads, and then a lysis buffer was added, followed by crushing at 4800 rpm for 5 minutes. The mold collected in the liquid by this process was crushed with glass beads together with the filter, and a predetermined lysis treatment was performed. In the solution thus obtained, mold spores and mycelial cell walls recovered from the air into a liquid are both crushed and lysed.
And the solution obtained for every sample 1-4 was refine | purified, and the DNA solution was obtained.

Next, in the nucleic acid amplification step, a part of the DNA in the DNA solution for each sample 1-4 was amplified by the PCR method.
At this time, Aspergillus penicillioids, Cladosporium sp., And Wallemia sebi were selected as molds to be detected.
And as shown in FIG. 2, the forward primer shown to sequence number 1 and the reverse primer shown to sequence number 2 were used as a primer set for amplifying the ITS area | region in genomic DNA of these three types of mold. Moreover, the forward primer shown to sequence number 3 and the reverse primer shown to sequence number 4 were used as a primer set for amplifying the (beta) -tubulin area | region in the genomic DNA of Aspergillus penicillioides sp.

As a PCR reaction solution, Ampdirect (R) (manufactured by Shimadzu Corporation) was used, and 20 μl of the following composition was prepared for each sample.
1. Ampdirect addition (G / Crich) 4.0 μl
2. Ampdirect (addition-4) 4.0 μl
3. dNTPmixture 1.0 μl
4. 0.2 μl of 1.0 mM Cy5-dCTP
5. ITS1 region amplification forward primer (2.5 μM, SEQ ID NO: 1, synthesized by Sigma-Aldrich) 1.0 μl
6). ITS1 region amplification reverse primer (2.5 μM, SEQ ID NO: 2, synthesized by Sigma-Aldrich) 1.0 μl
7). β-tubulin region forward primer (10 μM, SEQ ID NO: 3, synthesized by Sigma-Aldrich) 1.0 μl
8). Reverse primer for amplification of β-tubulin region (10 μM, SEQ ID NO: 4, synthesized by Sigma Aldrich) 1.0 μl
9. Nova Taq Hot Start DNA polymerase 0.2 μl
10. 1.0 μl of sample genomic DNA
11. Water (water until 20.0 μl total)

Using this PCR reaction solution, DNA amplification was performed for each sample under the following conditions using a nucleic acid amplification apparatus (TaKaRa PCR Thermal Cycler Dice (R) Gradient manufactured by Takara Bio Inc.).
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

Next, in the detection step, a DNA chip on which a probe for detecting Aspergillus penicilloid spp., A probe for detecting Cladosporium spp., And a probe for detecting Waremia sp. Spp. Was used for each sample. Gene silicon (R) (manufactured by Toyo Kohan Co., Ltd.) was used for the DNA chip.
In addition, as shown in FIG. 3, a probe comprising the base sequences shown in SEQ ID NO: 5 and SEQ ID NO: 6 is used as a probe for detecting Aspergillus penicilliosides, and as a probe for detecting Cladosporium sp. The probe consisting of the base sequence shown in SEQ ID NO: 8 was used as the probe for detecting the seeds of Walemia spp.

  The probes consisting of the nucleotide sequences shown in SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 8 are hybridized with a part of the amplification target region of the primer set for amplifying the ITS region described above. The probe consisting of the base sequence shown in SEQ ID NO: 6 hybridizes with a part of the amplification target region of the primer set for amplifying the β-tubulin region described above.

A buffer solution (3 × SSC + 0.3% SDS) was mixed with the amplification product by the PCR method 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 DNA chip.

Next, the fluorescence intensity in each probe was measured by applying the DNA chip to a label detection apparatus (Genesis scanner dedicated to BIOSHOT manufactured by Toyo Kohan Co., Ltd.). The result is shown in FIG.
In the figure, each numerical value indicates an S / N ratio ((fluorescence intensity value−background value) / background value). If it is 3 or more, it can be determined that the corresponding mold exists. Moreover, about Aspergillus penicillioides inoculum, when both S / N ratio is 3 or more about both ITS area | region and (beta) -tubulin area | region, it can be judged that the said mold | fungi exist, and this produces a false positive reaction. This makes it possible to accurately prevent this and perform a more accurate inspection.

As shown in FIG. 4, at point A, the presence of Aspergillus penicilliosides inoculum can be confirmed by sample 1. In addition, the presence of Cladosporium spp. Can be confirmed by Sample 1-3, and the presence of Walemia spp.
Moreover, about the point B, the presence of Aspergillus penicillioides inoculum and Cladosporium genus bacteria can be confirmed by the sample 4.

As described above, according to the microorganism testing method of the present embodiment, it has been found that the microorganism can be detected without pre-culturing the microorganism. In addition, the time required for sampling is only about 10 minutes, and the time required for the entire inspection from the microbe collection process to the detection process can be greatly reduced.
As a result, according to the microorganism inspection method of the present embodiment, the inspection result can be obtained on the day when the sample is sampled from the environment. Conventionally, the inspection of microorganisms that took several days including sampling can be largely performed. It became clear that it could be speeded up. Moreover, it turned out that it can test | inspect from the said result also in the number of 750 or less, further 450 or less, 150 or less, and 100 or less in conversion mold number.

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, only three types of molds are used as targets, but the present invention can be applied regardless of the types of microorganisms due to its characteristics, and the same method can be applied to other types of molds. It can be appropriately changed, for example, used for detection of other microorganisms.

  INDUSTRIAL APPLICABILITY The present invention can be suitably used when simultaneously testing a plurality of types of molds in food inspection, epidemiological environmental inspection, environmental inspection, clinical test, livestock hygiene, and the like.

To achieve the above object, the inspection method of the microorganism of the present invention determines the presence or absence of a plurality of types of microorganisms in the environment at the same time, and to a method for inspecting microbes to identify the type of microorganisms which are dead microorganisms Are fungi in the form of spores or mycelia , collecting the microorganisms floating in the air into the liquid, obtaining the first liquid sample and the second liquid sample from the liquid, and before crushing Without culturing microorganisms, a disruption step of disrupting microorganism cells in at least a portion of the first liquid sample, a nucleic acid extraction step of extracting nucleic acids from the disrupted cells of microorganisms , and at least the extracted nucleic acids A nucleic acid amplification process for amplifying a part of the nucleic acid and a solution containing the amplified nucleic acid are dropped onto a carrier on which a plurality of types of microorganism nucleic acids are immobilized in advance. If it is immobilized on the body, a detection step of identifying the type of microorganisms, the medium was cultured a second liquid sample, counting the number of colonies of at least a portion of the microorganisms, in a first liquid sample The step of calculating the number of collected microorganisms, the step of determining that the type of microorganism can be specified when the calculated number of microorganisms is 100 or more, and the calculated number of microorganisms is 100. And when the type of microorganism is specified in the detection step, the microorganism includes a cause of death of the microorganism, and the step of detecting specifies the type of microorganism that is dead. .

Hereinafter, preferred embodiments of the present invention will be described in detail.
Method of inspecting microbes embodiment of the present invention determines the presence or absence of a plurality of types of microorganisms in the environment at the same time, and to a method for inspecting microbes to identify the type of microorganisms which are dead, microorganisms, spores or A mycelium-shaped mold that collects microorganisms floating in the air in a liquid, a step of obtaining a first liquid sample and a second liquid sample from the liquid, and culturing the microorganisms before crushing Without crushing, crushing step of crushing microbial cells in at least a part of the first liquid sample, nucleic acid extraction step of extracting nucleic acids from the crushed cells of the microorganism , and amplifying at least a part of the extracted nucleic acids The nucleic acid amplification step and a solution containing the amplified nucleic acid are dropped onto a carrier on which nucleic acids of a plurality of types of microorganisms are immobilized in advance, and the nucleic acid that complementarily binds to the amplified nucleic acid is immobilized on the carrier. If it has, a detection step of identifying the type of microorganisms, and the second liquid sample medium culture, and counting the number of colonies of at least a portion of the microorganisms, the microorganisms are recovered in the first liquid sample A step of calculating the number, a step of determining that the type of the microorganism can be specified when the calculated number of microorganisms is 100 or more, a calculated number of microorganisms of 100 or less, and When the type of microorganism is specified in the detection step, the microorganism includes a cause of death of the microorganism, and the step of detecting specifies the type of microorganism that is dead .

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, only three types of molds are used as targets, but the present invention can be applied regardless of the types of microorganisms due to its characteristics, and the same method can be applied to other types of molds. It can be appropriately changed, for example, used for detection of other microorganisms.
The invention disclosed in this specification can be configured as follows.
1. A method for testing microorganisms that simultaneously determines the presence or absence of multiple types of microorganisms in the environment,
A collection process for collecting microorganisms floating in the air in a liquid;
A nucleic acid extraction step of extracting nucleic acid from the microorganism in the liquid;
A nucleic acid amplification step for amplifying a part of the extracted nucleic acid;
When the solution containing the amplified nucleic acid is dropped onto a carrier on which nucleic acids of a plurality of types of microorganisms are immobilized in advance, and the nucleic acid that complementarily binds to the amplified nucleic acid is immobilized on the carrier And a detection step for identifying the type of the microorganism,
The microorganism is not cultured after the collection step and before the nucleic acid extraction step.
A method for inspecting microorganisms.
2. 2. The microorganism testing method according to item 1, wherein the microorganism is a spore or mycelium mold.
3. 3. The method for inspecting a microorganism according to the above item 1 or 2, wherein the microorganism includes a death of the microorganism, and the type of the microorganism having the death is specified in the detection step.
4). In the collection step, 300 to 3000 L of air is collected using an air sampler, and microorganisms floating in the collected air are collected in a liquid. The microbe inspection method described.

Claims (4)

A method for testing microorganisms that simultaneously determines the presence or absence of multiple types of microorganisms in the environment,
A collection process for collecting microorganisms floating in the air in a liquid;
A nucleic acid extraction step of extracting nucleic acid from the microorganism in the liquid;
A nucleic acid amplification step for amplifying a part of the extracted nucleic acid;
When the solution containing the amplified nucleic acid is dropped onto a carrier on which nucleic acids of a plurality of types of microorganisms are immobilized in advance, and the nucleic acid that complementarily binds to the amplified nucleic acid is immobilized on the carrier And a detection step for identifying the type of the microorganism,
The microorganism testing method, wherein the microorganism is not cultured after the collection step and before the nucleic acid extraction step.   2. The microorganism testing method according to claim 1, wherein the microorganism is a spore or mycelium mold.   The microorganism testing method according to claim 1 or 2, wherein the microorganism includes a death of the microorganism, and the type of the microorganism that is dead is specified in the detection step.   In the said collection | recovery process, 300-3000L of air is collect | recovered using an air sampler, The microorganisms which float in the collect | recovered air are collect | recovered in the liquid. Microbe inspection method.

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