WO2015170362A1 - Procédé d'analyse de micro-organismes - Google Patents

Procédé d'analyse de micro-organismes Download PDF

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WO2015170362A1
WO2015170362A1 PCT/JP2014/002422 JP2014002422W WO2015170362A1 WO 2015170362 A1 WO2015170362 A1 WO 2015170362A1 JP 2014002422 W JP2014002422 W JP 2014002422W WO 2015170362 A1 WO2015170362 A1 WO 2015170362A1
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microorganism
microorganisms
sample
detected
nucleic acid
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PCT/JP2014/002422
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English (en)
Japanese (ja)
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淳憲 一色
真実 小梶
卓 田辺
充裕 吉田
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東洋製罐グループホールディングス株式会社
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Priority to PCT/JP2014/002422 priority Critical patent/WO2015170362A1/fr
Publication of WO2015170362A1 publication Critical patent/WO2015170362A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

Definitions

  • the present invention relates to an inspection method for confirming the presence or absence of microorganisms in the environment.
  • 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).
  • 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).
  • 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.
  • 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.
  • 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 detects microorganisms included in a set for each set. 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 an object of the present invention to provide a microorganism testing method that can determine the dominant species of microorganisms in the environment by calculating the ratio of the number of aggregates in which detection is detected.
  • 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.
  • 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 In addition, 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 sets of two or more, was calculated, whereby the superiority of microorganisms in the environment was calculated. There as a method having a predominant type determination step of determining the species.
  • 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
  • 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.
  • 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.
  • 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.
  • 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 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 them into groups 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.
  • 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.
  • the number of colonies in one set is more preferably 4 or 5.
  • 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.
  • the method for extracting nucleic acid from the 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 ⁇ 0.5mm zirconia beads, freezing the samples by immersion in liquid nitrogen, and using a shaking device Crush the microbial cells. Nucleic acids can be extracted from the microbial cell disruption obtained by the CTAB method (Cetyl trimethyl ammonium bromide) or a DNA extraction apparatus.
  • CTAB method Cetyl trimethyl ammonium bromide
  • 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.
  • 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.
  • the target region of the microorganism can be suitably amplified by performing PCR under the following reaction conditions, for example. (A) 95 ° C. for 10 minutes, (b) 95 ° C. (DNA denaturation step) for 30 seconds, (c) 56 ° C. (annealing step) for 30 seconds, (d) 72 ° C. (DNA synthesis step) for 60 seconds ((b) to (D) 40 cycles), (e) 72 ° C. 10 minutes
  • PCR reaction solution it is preferable to use, for example, one having the following composition. 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.
  • dNTPmixture dCTP, dATP, dTTP, dGTP
  • primer set primer set
  • nucleic acid synthase Nova Taq HotStart DNA polymerase, etc.
  • fluorescent labeling reagent Cy5-dCTP, etc.
  • sample genomic DNA sample genomic DNA
  • buffer solution a PCR reaction solution containing water as the remaining component
  • Ampdirect (R) manufactured by Shimadzu Corporation
  • R Ampdirect
  • DNA fragments can be amplified using the ITS region and / or ⁇ -tubulin gene in mold genomic DNA as a target region.
  • 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.
  • a primer set for amplifying the ⁇ -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.
  • the amplified nucleic acid fragments are used to detect the microorganisms contained in the set for each set.
  • 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.
  • 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.
  • a probe having the base sequence shown in SEQ ID NO: 5 can be suitably used as a probe selected from the ITS region.
  • a probe having the base sequence shown in SEQ ID NO: 6 can be preferably used as the probe selected from the ⁇ tubulin gene.
  • 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 ⁇ -tubulin gene.
  • a probe having the base sequence shown in SEQ ID NO: 8 can be preferably used.
  • 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 ⁇ tubulin gene.
  • a probe having the base sequence shown in SEQ ID NO: 10 can be preferably used as the probe.
  • 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 ⁇ tubulin gene.
  • a probe having the base sequence shown in SEQ ID NO: 12 can be preferably used as the probe.
  • a probe having the base sequence shown in SEQ ID NO: 13 can be suitably used as the probe selected from the ITS region.
  • a probe having the base sequence shown in SEQ ID NO: 14 can be suitably used as the probe selected from the ITS region.
  • both the ITS region and the probe selected from the ⁇ -tubulin gene were used, and the mold was only obtained when a positive reaction was 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.
  • 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 ⁇ -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 ⁇ -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.
  • the mold is present when a positive reaction is obtained with a probe selected only from 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 probes having the nucleotide sequences shown in SEQ ID NOs: 5 to 14.
  • the probe 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.
  • a synthetic DNA chip 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.
  • the substrate is not limited to glass, and a plastic substrate, a silicon wafer, or the like can also be used.
  • 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.
  • 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.
  • fungus A and fungus B are detected from set 1
  • fungus A and fungus B are detected from set 2
  • fungus A and fungus C are set from set 3.
  • the ratio of the number of sets detected for each fungus is calculated.
  • the ratio of fungus A is 3/3, which is 100.0%
  • the ratio of fungus B is 2/3, which is 66.7%
  • the ratio of fungus C is 1/3, which is 33.3%.
  • the first dominant species is determined as A
  • the second dominant species is determined as B
  • the third dominant species is determined as C.
  • the dominant species 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.
  • 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.
  • microorganism testing method of the present embodiment 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.
  • 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.
  • 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
  • microorganism testing method of the present embodiment it is possible to quickly and easily confirm the dominant species of microorganisms in the environment.
  • Each group of colonies was placed in a vial containing ⁇ 0.5 mm zirconia beads, immersed in liquid nitrogen to freeze the sample, and then mold cells in the colonies were crushed using a shaking device.
  • mold genomic DNA was extracted by a DNA extraction apparatus.
  • the ITS region of each mold and the ⁇ -tubulin gene were simultaneously amplified by PCR.
  • 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.
  • a primer set for amplifying ⁇ -tubulin gene a forward primer consisting of the base sequence of SEQ ID NO: 3 and a reverse primer consisting of the base sequence of SEQ ID NO: 4 shown in the same figure were used.
  • all synthesized by Operon Technology Co., Ltd. were used.
  • Ampdirect (R) (manufactured by Shimadzu Corporation) was used for each of samples A to J, and 20 ⁇ l of the following composition was prepared. 1. Ampdirect (G / Crich) 4.0 ⁇ l 2. Ampdirect (addition-4) 4.0 ⁇ l 3. dNTPmix 1.0 ⁇ l 4). Cy-5dCTP 0.2 ⁇ l 5. ITS1-Fw primer (2.5 ⁇ M) (SEQ ID NO: 1) 1.0 ⁇ l 6). ITS1-Rv primer (2.5 ⁇ M) (SEQ ID NO: 2) 1.0 ⁇ l 7). BtF primer (10 ⁇ M) (SEQ ID NO: 3) 1.0 ⁇ l 8). BtR primer (10 ⁇ M) (SEQ ID NO: 4) 1.0 ⁇ l 9. Template DNA (each sample A to J) 1.0 ⁇ l 10. NovaTaq HotStart DNA polymerase 0.2 ⁇ l 11. Water (water until 20.0 ⁇ l 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 for 10 minutes
  • b 95 ° C for 30 seconds
  • c 56 ° C for 30 seconds
  • d 72 ° C for 60 seconds
  • E 72 ° C 10 minutes
  • 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.
  • a buffer 3 ⁇ SSC citrate-saline + 0.3% SDS
  • 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.
  • a label detection device GenePix4100A Molecular Devices
  • both S / B of the probe selected from the ITS region and the probe selected from the ⁇ -tubulin gene are used.
  • a positive determination was made when the N ratio value was 3 or more.
  • 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 ⁇ -tubulin gene is 3 or more. did.
  • 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.
  • 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
  • a nucleic acid amplification device is used.
  • 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.
  • 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 B 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.
  • 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.
  • 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.
  • 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.
  • the actual first dominant species is Cladosporium
  • the actual second dominant species is Penicillium
  • 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.
  • 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 F was analyzed by DNA sequence analysis, as well as Leptosphaerulina chartarum, Leptosphaeria sp., Sclerotinia sclerotiorum, Creosphaeria sassafrans, Fungal endophyte sp. 6 types of microorganisms of unknown type) 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.
  • the actual first dominant species is Aspergillus bargecolor
  • the actual second dominant species is Cladosporium
  • 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.
  • 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.
  • 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.
  • the actual first dominant species was Cladosporium sp.
  • the actual second dominant species was Phaeosphaeriopsis sp.
  • the genus was the actual third dominant species.
  • 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.
  • 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.
  • the actual first dominant species was Cladosporium
  • 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.
  • sample J 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.
  • the actual first dominant species was Aspergillus penicillioides
  • 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.
  • 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.
  • components other than the ITS region amplification primer set and ⁇ -tubulin gene amplification primer set in the PCR reaction solution of the above test can be appropriately changed.
  • 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 or absence of microorganisms in the environment. In some cases, it can be suitably used.

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Abstract

L'invention a pour but de permettre la détection simple et rapide d'une espèce de micro-organisme dominante dans l'analyse sur la présence ou l'absence de micro-organismes dans un environnement. Pour atteindre ce but, l'invention porte sur un procédé d'analyse de micro-organismes, comprenant les étapes consistant : à prélever un échantillon dans un environnement et à effectuer, dans un milieu de culture, la culture de micro-organismes contenus dans l'échantillon ; lorsqu'au moins deux colonies sont formées par la culture des micro-organismes, à diviser les colonies formées en au moins deux masses et à extraire un acide nucléique d'un micro-organisme contenu dans chacune des masses ; à amplifier un fragment d'acide nucléique contenant un domaine d'intérêt à l'aide de l'acide nucléique extrait ; à détecter des micro-organismes contenus dans chacune des masses à l'aide du fragment d'acide nucléique amplifié ; lorsqu'au moins deux micro-organismes sont détectés dans l'étape de détection de micro-organismes susmentionnée, à calculer le rapport du nombre de masses dans lesquelles les micro-organismes sont détectés au nombre total de masses, qui est d'au moins deux, pour déterminer l'espèce de micro-organisme dominante dans l'environnement.
PCT/JP2014/002422 2014-05-07 2014-05-07 Procédé d'analyse de micro-organismes WO2015170362A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113430293A (zh) * 2021-06-18 2021-09-24 安徽农业大学 一种pcr检测猕猴桃软腐病病原菌拟茎点霉菌的特异性引物、方法及应用

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2012200213A (ja) * 2011-03-25 2012-10-22 Toyo Seikan Kaisha Ltd カビの検査方法

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2012200213A (ja) * 2011-03-25 2012-10-22 Toyo Seikan Kaisha Ltd カビの検査方法

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
EARTH KANKYO SERVICE KABUSHIKI KAISHA ET AL.: "Tanjikan·Tei-cost·Koseido de Biseibutsu Osen Jokyo o Ikkatsu Kensa", GEKKAN HACCP, vol. 19, no. 5, 2013, pages 40 - 43 *
EARTH KANKYO SERVICE KABUSHIKI KAISHA ET AL.: "Tanjikan·Tei-cost·Koseido de Biseibutsu Osen Jokyo o Ikkatsu Kensa", KEIRAN NIKU JOHO, vol. 43, no. 9, 2013, pages 78 - 81 *
MITSUHIRO YOSHIDA: "DNA Inspection Device for Fungi with DNA Chip", FOOD PROCESSING AND INGREDIENTS, vol. 47, no. 1, 2012, pages 38 - 40 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113430293A (zh) * 2021-06-18 2021-09-24 安徽农业大学 一种pcr检测猕猴桃软腐病病原菌拟茎点霉菌的特异性引物、方法及应用

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