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WO2009104641A1 - Procédé de détection et de quantification de nématodes dans le sol et instrument permettant de consolider un échantillon de sol destiné à être utilisé dans ledit procédé - Google Patents

Procédé de détection et de quantification de nématodes dans le sol et instrument permettant de consolider un échantillon de sol destiné à être utilisé dans ledit procédé Download PDF

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Publication number
WO2009104641A1
WO2009104641A1 PCT/JP2009/052794 JP2009052794W WO2009104641A1 WO 2009104641 A1 WO2009104641 A1 WO 2009104641A1 JP 2009052794 W JP2009052794 W JP 2009052794W WO 2009104641 A1 WO2009104641 A1 WO 2009104641A1
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Prior art keywords
soil
nucleic acid
nematodes
sample
soil sample
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English (en)
Japanese (ja)
Inventor
豊田剛己
大島忠男
佐藤恵利華
後藤圭太
大石正行
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Tokyo University of Agriculture and Technology NUC
University of Tokyo NUC
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Tokyo University of Agriculture and Technology NUC
University of Tokyo NUC
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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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43536—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms
    • C07K14/4354—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms from nematodes
    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae

Definitions

  • the present invention relates to a method for extracting nucleic acids from nematodes in soil, and a soil diagnostic method for detecting and quantifying plant parasitic nematodes in soil that are involved in crop diseases. More specifically, a method of extracting a nucleic acid with high efficiency by destroying a nematode in a soil sample by compacting the soil sample, and a line in the soil by detecting or quantifying the extracted nucleic acid.
  • the present invention relates to a method for detecting and quantifying insects quickly and accurately.
  • the present invention also relates to a compacting device having a mechanism capable of manually applying a strong pressure to a soil sample.
  • Autoactive nematodes include predatory nematodes that prey on protozoa and other nematodes, and corrosive nematodes that decompose organic matter such as fallen leaves, which are beneficial for crops. Is.
  • plant parasitic nematodes are harmful to crops because they infest the roots of crops and die crops or reduce commercial value.
  • root-knot nematodes, cyst nematodes, and neptune nematodes are called three major parasitic nematodes and cause serious damage.
  • soil fumigants In order to control plant parasitic nematodes, soil fumigants etc. are used, but soil fumigants can not selectively control only plant parasitic nematodes, so by using soil fumigants, There is a problem that it also eliminates autoactive nematodes and other soil microorganisms that are beneficial to crops. Therefore, it is preferable to minimize the use of soil fumigants.
  • a fumigant In order to reduce the use of soil fumigants, to prevent the use of unnecessary soil fumigants, avoid preventive use as much as possible, and determine how many nematodes parasitic on the cultivated crops live in the soil. A fumigant must be used after detection or quantification.
  • the type of nematode is identified by observing the difference in the shape of the mouth or tail of the nematode or mouth needle by observation with an optical microscope, and the number of nematodes is counted.
  • this method not only requires specialized knowledge and skill, but also requires a lot of labor and is not easy to perform.
  • Patent Document 1 As a method for easily detecting or quantifying nematodes in soil, the present inventors previously isolated nematodes from soil, amplified nucleic acids of the nematodes by PCR method or real-time PCR method, and detected or quantified. A method for quantification was developed (Patent Document 1, Non-Patent Document 1, and Non-Patent Document 2).
  • nucleic acids when nucleic acids are extracted directly from soil, nucleic acids are hardly obtained or extraction efficiency is very poor.
  • nematodes are separated from soil using the Bellman method or the two-layer centrifugal suspension method. There was a need to do.
  • the Bellman method is a separation method using the property that nematodes move from soil to water by their own activity. Specifically, (1) place a Japanese paper filter on the pan and put a soil sample, (2) fill the glass funnel with water, and (3) place the net so that the bottom of the pan is immersed in the water of the glass funnel. Place the dish in a glass funnel and (4) leave the nematode for 3 days at a temperature at which the nematode is active, and move the nematode from the soil into the water to separate the nematode from the soil. .
  • Non-patent Document 3 The Bellman method is the most widely used nematode separation method, but it requires 2 to 3 days for separation as described above, and the separation rate is low, which affects the motility of nematodes. This has a serious drawback that the separation rate changes under the influence of physiological conditions and soil conditions of nematodes (Non-patent Document 3).
  • the double-layer centrifugal suspension method is a method for separating nematodes and soil particles by the difference in density.
  • the soil particles with relatively high density are precipitated to form a soil particle layer, and the nematodes with relatively low density are separated near the interface between the water layer and the specific gravity liquid layer, and (4) the water layer and the specific gravity liquid layer.
  • the nematode is separated by passing through the mesh.
  • the two-layer centrifugal suspension method is a method having a higher separation rate than the Bellman method, but requires a complicated operation with the centrifugal device as described above.
  • cyst nematodes that survive in the form of eggs with a small amount of soil that can be tested tend to settle due to the adhesion of soil particles, so the separation rate is low, and in soils with high organic matter and clay content, the nematode separation rate decreases. It has various drawbacks such as poor separation of large nematodes (Non-patent Document 3).
  • the present invention provides a method for detecting or quantifying nematodes in soil easily and accurately by extracting nucleic acids from nematodes in soil in a short time and with high efficiency. With the goal.
  • the present inventors have conducted extensive studies, and surprisingly, when the soil sample is consolidated, the pores in which nematodes can inhabit are reduced and nematodes are destroyed. It was found that the nematode nucleic acid can be extracted from the soil in a short time and with high efficiency by omitting the step of separating nematodes by the double-layer centrifugal suspension method. In addition, when soil samples are consolidated, nematode nucleic acids can be extracted with high efficiency, so that nematodes in soil can be accurately detected or quantified without being greatly affected by differences in soil type or worm motility. As a result, the inventors have found out that the present invention can be achieved.
  • the present invention provides the following methods (I) to (XII) for detecting and quantifying nematodes in soil, a compacting device used in the method, and a kit including the compacting device.
  • a method for extracting nucleic acid from nematodes in soil comprising the following steps: (I) a step of compacting a soil sample collected from soil; (B) A step of extracting nucleic acid from the soil sample consolidated by the step (a).
  • a method for detecting or quantifying nematodes in soil comprising the following steps: (I) a step of compacting a soil sample collected from soil; (B) a step of extracting nucleic acid from the soil sample consolidated by the step (a); (C) A step of detecting or quantifying the nucleic acid extracted in the step (b).
  • step (b) the soil sample is consolidated until the volume of pores having a diameter of 15 ⁇ m or more in the soil sample is reduced by 30 to 100%, as described in (I) or (II) above the method of.
  • step (IV) In the step (I), the soil sample is consolidated until the dry density of the soil sample becomes 0.85 to 2.7 g / cm 3. The method described.
  • step (V) The method according to any one of (I) to (IV) above, wherein in the step (b), a nucleic acid is extracted using a surfactant and / or bead beating.
  • the nematode is a plant parasitic nematode
  • the nucleic acid in the step (c) is a nucleic acid having a base sequence specific to the plant parasitic nematode
  • plant parasitic nematode and the nucleic acid having a base sequence specific to the plant parasitic nematode are any of the following four combinations: : Potato cyst nematode / nucleic acid having a base sequence of 100 to 200 residues out of the base sequence set forth in SEQ ID NO: 1; Sweet potato root nematode / Nucleic acid having a base sequence of 100 to 200 residues out of the base sequence set forth in SEQ ID NO: 2; A nucleic acid having a base sequence of 100 to 200 residues among the base sequences set forth in soybean cyst nematode / SEQ ID NO: 3.
  • step (IX) The method according to any one of (II) to (VIII) above, which further comprises the following step (d): (D) Quantifying nematodes in a soil sample by comparing the amount of nucleic acid quantified in the above step (c) with the amount of nucleic acid quantified in advance using a sample with a known amount of nematode. Process.
  • the compression mechanism includes a compression portion that can be inserted into the sample container, and a control mechanism that controls a depth of insertion of the compression portion into the sample container, and the compression portion enters the sample container.
  • a support base (3) on which the sample container (1) can be installed a support base (3) on which the sample container (1) can be installed, a main body (4) connected to the support base (3), an upper part of the main body (4), A fastener (5) slidable in the vertical direction by screwing, and a pressing portion (2) attached to the tip of the fastener (5), and sliding the fastener (5) downward.
  • a compaction device according to claim 9 characterized in that when it is moved, the compression part (2) fits into the sample container (1) and can be compacted by applying pressure to the soil sample.
  • the main body (4) has a substantially inverted U shape, and one end of the connecting portion of the support base (3) and the main body (4) is connected to the pivot portion (6).
  • the other end of the connecting part of the support base (3) and the main body (4) is fixed by a jaw part (7) and a rotatable hook (8).
  • (XIV) A kit for extracting nucleic acids from nematodes in soil, comprising the compacting device according to any one of (X) to (XIII) and a reagent for extracting nucleic acids in soil samples.
  • (XV) Detecting nematodes in soil comprising the consolidation device according to any one of (X) to (XIII), a reagent for extracting nucleic acid in a soil sample, and a reagent for detecting or quantifying nucleic acid Kit to quantify.
  • the method for extracting nucleic acids from nematodes in soil according to the present invention reduces the pores in which nematodes can inhabit and destroys nematodes by compacting the soil, so that nematode nucleic acids are efficiently extracted from soil. There is an effect that it can be extracted.
  • nucleic acids can be extracted with high efficiency by compacting a soil sample. Therefore, nematodes from soil such as the Bellman method and the two-layer centrifugal suspension method can be extracted. It does not require a separation step and enables detection or quantification in a short time.
  • nematodes can be detected or quantified more accurately.
  • the soil sample when the soil sample is consolidated until the number of pores having a diameter of 15 ⁇ m or more in the soil sample is reduced by 30 to 100%, or the dry density of the soil sample is 0.85 to 2.7 g / cm 3 .
  • the nematode When the soil sample is consolidated until it becomes, the nematode is more reliably destroyed and the nucleic acid extraction efficiency is increased to 1.2 to 7 times or more (see Example 2). Since it is possible to extract with efficiency near 100% (see Example 5), nematodes can be detected or quantified more accurately.
  • step (c) of the method of the present invention when a nucleic acid having a sequence specific to a plant parasitic nematode is detected or quantified, it is parasitic on a specific plant parasitic nematode, for example, a crop to be cultivated. It is possible to accurately and easily detect or quantify only the nematodes that do so, making it possible to accurately determine the suitability of the use of soil fumigants, reducing the number of wasteful use of pesticides, and reducing the microbial environment of the soil. A pesticide-reducing farming method that can keep as much as possible can be implemented.
  • FIG. 3 is a diagram showing pF-water curves of soils consolidated at different degrees. It is the figure which showed the relationship between the dry density at the time of compacting soil, and a nematode reduction amount. It is a figure which shows the result of having compared the extraction efficiency of a nucleic acid.
  • the method for extracting nucleic acids from nematodes in soil of the present invention includes the following steps (a) and (b).
  • the soil sample collected from the soil is preferably a soil sample simply sampled from cultivated land or the like, but is a soil sample obtained by adding physical or chemical treatment such as cryopreservation to the sampled one. Also good.
  • consolidation means increasing the density of soil by applying pressure. Any method may be used for the compaction as long as it reduces the pores in the soil where nematodes can inhabit. However, a soil sample is packed in a closed space such as a container, and pressure is applied. The method of further narrowing the space filled with soil samples is preferable.
  • the degree of consolidation may be such that the volume of voids having a diameter of 15 ⁇ m or more inhabiting nematodes is reduced by 30% or more, preferably 60% or more, more preferably 90% to 100%.
  • the volume of voids having a diameter of 15 ⁇ m or more can be measured by the method described in Example 3.
  • the pressure required to perform such consolidation varies depending on the soil sample.
  • Another index for the degree of compaction of the soil sample includes the dry density of the soil sample.
  • the soil sample is preferably compacted until the dry density of the soil sample is 0.85 to 2.7 g / cm 3 . More preferably, the soil sample is compacted until the dry density of the soil sample becomes 0.95 to 1.2 g / cm 3 .
  • the dry density measurement method is based on the method described in “Soil Environmental Analysis Method” (Japan Soil Fertilizer Society, 1997).
  • the volume of the soil sample can be mentioned, and it is preferable to compact the soil sample until the volume of the soil sample is reduced by 50 to 80%. More preferably, compaction is performed until the volume of the soil sample is reduced by 60% or more.
  • a method for extracting nucleic acid from a compacted soil sample a method for extracting nucleic acid from microorganisms in soil or an improved method thereof can be used.
  • a method called direct extraction method (Direct Lysis Method) for extracting nucleic acids of microorganisms directly from soil is preferable.
  • the nucleic acid is DNA or RNA.
  • the direct extraction method is based on the method originally developed by Ogram et al. (Ogram et al., 1987, J.Microbiol.Methods, vol.7, pp.57-66). It consists of two steps: I) destruction of cells in soil and (II) separation and purification of nucleic acids.
  • steps (1) There are three types of steps (1): (1) chemical treatment, (2) physical treatment, and (3) enzymatic treatment.
  • a chemical treatment surfactants such as SDS and LDS, phenol / chloroform, benzyl chloride, guanidine thioocyanate (GTC) and the like can be used. Of these, use of a surfactant, particularly SDS, is preferable because of good extraction efficiency.
  • GTC guanidine thioocyanate
  • use of a surfactant, particularly SDS is preferable because of good extraction efficiency.
  • As physical treatment bead beating (bead-beating) that breaks cells by shaking vigorously with small glass beads or zirconia beads, heating, freezing / thawing, etc. can be used. When used, it is preferable because cells of various forms can be destroyed.
  • enzymatic treatment lysozyme, proteinase K, achromopeptidase, pronase and the like are used, and it is particularly preferable to use
  • a chelating agent such as EDTA or Chelex100, sodium phosphate buffer, or a high concentration of 1 M or more A salt solution or the like can be added.
  • RNA can be extracted while preventing degradation of RNA by using guanidine thiocyanate, sarkosyl, mercaptoethanol or the like (YU-Li et al., 1991, Applied and Environmental Microbiology). , vol.57, pp.765-768)
  • the step (I) since the nematode is destroyed by the process of compacting the soil sample, the step (I) is not absolutely necessary, but the synergistic effect combined with the step (I)
  • nucleic acid can be extracted with high efficiency. For example, by combining the step of compacting a soil sample and the step (I), it is possible to extract nucleic acid with 1.2 to 7 times higher efficiency than the case of only the step (I). (See Examples 2 and 5).
  • step (II) since only nucleic acid must be extracted from a soil sample containing various contaminants, multiple stages of purification are often required.
  • humic substances in the soil can be removed by adding denatured proteins and polysaccharides, cetyltrimethylammonium bromide (CTAB) and polyvinylpolypyrrolidone (PVPP) that form a complex with cell residues.
  • CAB cetyltrimethylammonium bromide
  • PVPP polyvinylpolypyrrolidone
  • the nucleic acid can be purified by deproteinization by organic solvent extraction and the like, and concentration of the nucleic acid by alcohol precipitation or the like.
  • Deproteinization can be performed by salting out using a saturated salt solution in addition to extraction with an organic solvent such as phenol, phenol-chloroform, chloroform-isoamyl alcohol and the like.
  • alcohol precipitation with ethanol or isopropanol or precipitation with polyethylene glycol (PEG) can be used.
  • nucleic acid purified as described above agarose electrophoresis, CsCl density gradient centrifugation, gel filtration chromatography, or the like can be used.
  • nucleic acid can be purified by using a commercially available DNA purification kit or column.
  • a nucleic acid can also be extracted from a compacted soil sample using a commercially available soil extraction kit.
  • a soil extraction kit for example, ISOIL for Beads Beating (Nippon Gene), Fast DNA spin kit for soil (registered trademark, Qbio gene, USA), UltraClean Soil DNA kit (registered trademark, Mobio, USA) and the like are commercially available.
  • a kit for extracting RNA Bio101 FastRNA Pro Soil-Direct Kit (registered trademark, Qbiogene, USA) is commercially available.
  • Detecting or quantifying nucleic acids extracted by the above method makes it possible to detect or quantify nematodes in soil.
  • the present invention provides a method for detecting or quantifying nematodes in soil, comprising the following steps (a) to (c).
  • (I) a step of compacting a soil sample collected from soil; (B) a step of extracting nucleic acid from the soil sample consolidated by the step (a); (C) A step of detecting or quantifying the nucleic acid extracted in the step (b).
  • the extracted nucleic acid may be detected or quantified as it is, but since the amount of nematode nucleic acid is very small, the extracted nucleic acid may be amplified for detection or quantification. preferable.
  • the method for amplifying the nucleic acid is not particularly limited, and PCR method, LAMP method, ICAN method and the like can be used.
  • nucleic acid When nucleic acid is detected or quantified, if a nucleic acid having a base sequence specific to a specific plant parasitic nematode is detected or quantified, whether the specific plant parasitic nematode exists in the soil. It is possible to detect and quantify whether or not it exists. In order to detect or quantify a nucleic acid having such a base sequence, for example, it is specifically amplified using a primer complementary to the nucleic acid having the base sequence and / or complementary to the nucleic acid having the base sequence. Can be detected or quantified by hybridizing a typical probe.
  • the method for detecting or quantifying the extracted nucleic acid or the nucleic acid obtained by amplifying the nucleic acid is not limited thereto, but a method of hybridizing with a complementary nucleic acid labeled with fluorescence, chemiluminescence, enzyme or radioactive signal, Examples include real-time PCR, a method for separation by electrophoresis, a method for binding with a labeled antibody, a method for hybridizing with a labeled peptide nucleic acid, a method using an electrochemical sensor, and a method using a microarray / gene chip. Among these, it is preferable to quantify by real-time PCR.
  • Real-time PCR uses a nucleic acid to be quantified as a template, and causes a fluorescent substance to act when performing a double-stranded DNA synthesis reaction using two PCR primers (primer sets) called F and R primers.
  • F and R primers two PCR primers (primer sets)
  • F and R primers two PCR primers
  • This is a PCR method in which a fluorescent signal is generated when the synthesis reaction occurs.
  • the amount of PCR product generated can be monitored in real time while continuing the PCR reaction, and the template can be quantified based on the amplification curve.
  • Such a real-time PCR method can be performed using various commercially available instruments and systems.
  • Applied Biosystems 7900HT Fast Applied Biosystems
  • LightCycler registered trademark, Roche Diagnostics
  • iCycler iQ registered trademark
  • Bio-Rad Laboratories
  • a specific nematode for example, potato cyst nematode, sweet potato nematode, soybean cyst nematode, or northern nematode nematode can be detected or quantified. That is, in order to detect or quantify potato cyst nematodes, in the step (c) of the present invention, a nucleic acid having a base sequence of 100 to 200 residues of the base sequence set forth in SEQ ID NO: 1 is detected or quantified. For example, it can be detected or quantified by specifically amplifying the nucleic acid using a primer having the base sequences described in SEQ ID NOs: 5 and 6.
  • a nucleic acid having a base sequence of 100 to 200 residues of the base sequence described in SEQ ID NO: 2 may be detected or quantified.
  • it can be detected or quantified by specifically amplifying the nucleic acid using a primer having the nucleotide sequence set forth in SEQ ID NOs: 7 and 8.
  • RKNf GCTGGTGTCTAAGTGTTGCTGATAC (SEQ ID NO: 7)
  • RKNr (5 ' ⁇ 3'): GAGCCTAGTGATCCACCGATAAG (SEQ ID NO: 8) Since the above-mentioned primer pair can also detect Java root-knot nematodes and arenaria root-knot nematodes, it can also be used as a primer for detecting root-knot nematodes.
  • a nucleic acid having a base sequence of 100 to 200 residues of the base sequence set forth in SEQ ID NO: 3 is detected or quantified.
  • it can be detected or quantified by specifically amplifying the nucleic acid using a primer having the base sequence described in SEQ ID NOs: 9 and 10.
  • a nucleic acid having a base sequence of 100 to 200 residues out of the base sequence set forth in SEQ ID NO: 4 is detected or quantified.
  • it can be detected or quantified by specifically amplifying the nucleic acid using primers having the nucleotide sequences set forth in SEQ ID NOs: 11 and 12.
  • NEGf (5 ' ⁇ 3'): ATTCCGTCCGTGGTTGCTATG (SEQ ID NO: 11)
  • NEGr (5 ' ⁇ 3'): GCCGAGTGATCCACCGATAAG (SEQ ID NO: 12)
  • Nematodes can be quantified.
  • examples of the sample having a known amount of nematodes include, for example, a soil sample in which the amount of nematodes is known by counting nematodes in soil, or a plant parasitic line separated from soil and selected by type. Nucleic acids having sequences specific to the worm itself or plant parasitic nematodes can be used. Quantification of a sample with a known amount of nematode may be performed separately from the soil sample for which the nematode is to be quantified, or may be added to the soil sample for which the nematode is to be quantified to serve as an internal standard.
  • the method for quantifying nematodes in soil according to the present invention can extract nucleic acids with high efficiency by compacting a soil sample, and in some cases can extract with nearly 100% efficiency. Therefore, even if a sample with a known amount of nematode is quantified separately from the soil for which the nematode is to be quantified without using it as an internal standard, the error due to the difference in extraction efficiency due to the type of soil is small, Accurate quantification is possible.
  • the present invention also provides an apparatus suitable for compacting a soil sample, which is used for extracting nucleic acid with high efficiency from nematodes contained in the soil sample as described above.
  • the compacting device of the present invention includes a sample container for storing a soil sample, and a compression mechanism for compacting the soil sample by applying a desired pressure to the soil sample in the sample container.
  • the compression mechanism is not particularly limited as long as it is a configuration capable of compacting the soil sample as described above, and may have any configuration.
  • the compression mechanism may include a compression portion that can be inserted into the sample container and a control mechanism that controls the depth of insertion of the compression portion into the sample container.
  • the compacting device can adjust the pressure applied to the soil sample by the depth of insertion of the compression portion into the sample container. That is, a desired pressure can be applied to the soil sample accommodated in the sample container by freely moving the pressing portion and the sample container in the direction of contacting and separating.
  • One of the preferred embodiments of the consolidation device of the present invention is a device as shown in FIG. That is, the compacting device is positioned in the vertical direction by screwing the support base (3) on which the sample container (1) can be installed, the main body (4) connected to the support base (3), and the upper part of the main body (4).
  • the compaction device shown in FIG. 1 when the fastener (5) is moved downward, the compression portion (2) is fitted into the sample container (1), and the soil sample can be pressed and compacted. .
  • FIG. 2 shows a state in which the fastener (5) attached to the main body (4) is moved up and down by screwing in the compacting device of the present invention.
  • the consolidation device of the present invention may be manufactured using any material, but iron is preferably used as a material from the viewpoint of stability.
  • This compacting device can manually apply a high pressure to the soil sample, and is compact and easy to carry, so that the method of the present invention can be easily carried out. Furthermore, in this compaction instrument, the compression part (2) can be positioned at a desired depth in the sample container (1) by manually rotating the fastener (5) to a desired degree. Thereby, according to this compaction instrument, the soil sample accommodated in the sample container (1) can be compacted (consolidated) with a desired pressure.
  • the compaction device has a main body (4) having a substantially inverted U shape, and a pivot portion (6) that rotatably connects one end of the approximately inverted U shape to the support base (3). It is preferable to have a hook (8) for fixing the other end of the inverted U shape to the support base (3).
  • the hook (8) is rotated so as to be detached from the jaw (7) of the support base (3), and the main body (4) is rotated around the pivot (6) as a fulcrum. 1) can be easily removed. Further, the compacting device can easily install the sample container (1) by the reverse operation.
  • the sample container (1) can be a cylindrical metal container.
  • a sample container (1) is pressurized in the state which accommodated the soil sample in the inside, it is produced so that it may have intensity
  • the compacting device is configured to apply a desired pressure to the soil sample by moving the fastening tool (5) attached to the main body (4) up and down by screwing.
  • the compacting device according to the present invention is not limited to this configuration.
  • the compaction instrument according to the present invention may be configured, for example, to apply a desired pressure to the soil sample by fixing the compression portion (2) and moving the support base (3) in the vertical direction.
  • the mechanism for moving the compression portion (2) and the support base (3) in the vertical direction is not limited to the above-described movement method, and any mechanism such as a hydraulic method or a pressurization method using a vacuum may be applied. good.
  • the present invention also provides a kit for extracting nucleic acids from nematodes in soil, comprising the above-described compaction device and a reagent for extracting nucleic acids in soil samples.
  • the reagent for extracting nucleic acid in the soil sample is not limited to these, but is a cell lysis reagent including a surfactant, beads and / or lysozyme, phenol, chloroform, alcohol, PEG, CTAB. And / or a nucleic acid purification reagent containing PVPP, and a purification column.
  • the present invention also provides a kit for detecting or quantifying nematodes in soil, comprising the above-described compaction device, a reagent for extracting nucleic acid in a soil sample, and a reagent for detecting or quantifying nucleic acid.
  • the reagent for detecting or quantifying the nucleic acid is not limited to these, but labeled with polymerase, reverse transcriptase, reaction buffer, dNTP mix, primer, fluorescent dye, radioisotope, or the like. Examples include probes and hybridization buffers.
  • Example 1 Extraction of nematode nucleic acids in soil
  • Consolidation of soil samples Using the compaction apparatus shown in FIG. 1, about 30 g of soil (humic black soil) was filled into a 100 cm 3 cylindrical sample container, and compacted under the following three conditions. Next, using a spatula or a flat-blade screwdriver, the soil was taken out from the cylindrical sample container and loosened sufficiently to prepare the following three types of samples.
  • the nematode suspension obtained by the above stirring was put in a tube with a sterilized lid containing 0.75 g of zirconia beads ( ⁇ 0.1 mm) and 0.25 g of glass beads ( ⁇ 0.5 mm).
  • Chloroform was added again to the aqueous layer, and after centrifugation, 1200 ⁇ L of the aqueous layer was transferred to a new eppen (at 15000 g for 20 minutes).
  • Example 2 Quantification by real-time PCR (1) The PC on which Smart Cycler (registered trademark) software Version 2.0 was installed and the Smart Cycler (registered trademark) II System were connected with a USB cable.
  • Potato cyst nematode PCN280f (5 ' ⁇ 3'): GCGTCGTTGAGCGGTTGTT PCN280r (5 ' ⁇ 3'): CCACGGACGTAGCACACAAG Root-knot nematode RKNf (5 ' ⁇ 3'): GCTGGTGTCTAAGTGTTGCTGATAC RKNr (5 ' ⁇ 3'): GAGCCTAGTGATCCACCGATAAG Soybean cyst nematode forward (5 ' ⁇ 3'): CTAGCGTTGGCACCACCAA reverse (5 ' ⁇ 3'): AATGTTGGGCAGCGTCCACA Kitanegusa nematode NEGf (5 ' ⁇ 3'): ATTCCGTCCGTGGTTGCTATG NEGr (5 ' ⁇ 3'): GCCGAGTGATCCACCGATAAG (5) 5 ⁇ L of 100-fold diluted template DNA was placed in each tube and
  • the Ct value was 27.1 ⁇ 0.17 in the consolidated sample, whereas the Ct value was 30.0 ⁇ 0.26 in the sample that was not consolidated, which was significantly faster by 2.9 times. It was. This means that the extraction efficiency of cyst nematode-derived DNA is increased by about 7.6 times due to consolidation.
  • the amount of pores can be easily determined by a pressure plate method (for example, DIK-3404 wide area soil pF measuring instrument manufactured by Daikai Rika Kogyo Co., Ltd.).
  • the portion with a moisture content lower than pF2.3 is shown by a single curve, which indicates that the pore volume of 15 ⁇ m or less is not affected by consolidation.
  • Example 4 Using the apparatus of FIG. 1, the soil can be compacted to any dry density.
  • the height of the black soil used in this experiment is 5 cm.
  • the dry density at this time corresponds to 0.5 g / cm 3 . If this is consolidated sequentially and the height is 2.5 cm, it becomes 1.0 g / cm 3 . That is, it is possible to adjust the soil to an arbitrary dry density by adjusting the height according to the degree of consolidation while keeping the soil to be used constant.
  • Fig. 6 is a graph plotting the amount of nematode reduction when the black soil is consolidated to each dry density.
  • Negussa nematode was bisected, one was added to the soil and the other was not added to the soil and completely destroyed in solution.
  • the soil to which Negusa nematode was added was consolidated, DNA was extracted from it, and the northern nematode nematode was quantified by real-time PCR.
  • the result is shown in FIG.
  • the value obtained by quantifying the nematode nematode using a solution that was completely destroyed in the control solution was taken as 100
  • when the soil was consolidated it became the same 100 as the solution, but the soil was not consolidated
  • the amount of Negusale nematode extracted from the soil using ordinary methods was quantified and was less than 20. In other words, it is understood that the efficiency increased by a factor of 4 due to compaction, and that all the northern root nematodes added to the soil by consolidation could be recovered and detected.
  • the method for extracting nucleic acids from nematodes in soil, the method for detecting and quantifying nematodes in soil, the compacting device used in the method and the kit including the compacting device according to the present invention are: It is useful in the field of

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Abstract

La présente invention concerne un procédé selon lequel des acides nucléiques sont extraits à partir de nématodes dans le sol sur une courte période de temps avec un rendement élevé, les nématodes dans le sol pouvant ainsi être détectés ou quantifiés de façon pratique et précise. La présente invention concerne un procédé d'extraction d'acides nucléiques à partir de nématodes dans le sol caractérisé en ce qu'il comprend les étapes (a) et (b) suivantes ; et un procédé de détection ou de quantification de nématodes dans le sol caractérisé en ce qu'il comprend les étapes (a), (b) et (c) suivantes : (a) une étape de consolidation d'un échantillon de sol prélevé dans le sol ; (b) une étape d'extraction des acides nucléiques à partir de l'échantillon de sol qui a été consolidé dans l'étape (a) ; et (c) une étape de détection ou de quantification des acides nucléiques qui ont été extraits dans l'étape (b).
PCT/JP2009/052794 2008-02-20 2009-02-18 Procédé de détection et de quantification de nématodes dans le sol et instrument permettant de consolider un échantillon de sol destiné à être utilisé dans ledit procédé Ceased WO2009104641A1 (fr)

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EP2486155A4 (fr) * 2009-10-09 2013-08-14 Monsanto Technology Llc Méthodes de quantification d'organismes cibles et création de plants de coton résistants au nématode réniforme
CN113960285A (zh) * 2021-09-27 2022-01-21 中国热带农业科学院热带作物品种资源研究所 一种便携式土壤病原线虫综合监测系统

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JP5831731B2 (ja) * 2010-08-04 2015-12-09 有限会社ネマテンケン 土壌線虫の分離方法
JP2018143184A (ja) * 2017-03-07 2018-09-20 東ソー株式会社 微生物ｒｎａの検出方法
JP7745916B1 (ja) * 2024-07-25 2025-09-30 国立研究開発法人農業・食品産業技術総合研究機構 ダイズシストセンチュウの検出定量方法

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2486155A4 (fr) * 2009-10-09 2013-08-14 Monsanto Technology Llc Méthodes de quantification d'organismes cibles et création de plants de coton résistants au nématode réniforme
US8686219B2 (en) 2009-10-09 2014-04-01 Monsanto Technology Llc Methods of quantifying target organisms and creating reniform resistant cotton plants
CN113960285A (zh) * 2021-09-27 2022-01-21 中国热带农业科学院热带作物品种资源研究所 一种便携式土壤病原线虫综合监测系统
CN113960285B (zh) * 2021-09-27 2023-11-17 中国热带农业科学院热带作物品种资源研究所 一种便携式土壤病原线虫综合监测系统

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