WO2003100400A1 - Procede de pre-test de microparticules, procede de detection de microparticules et instrument de detection de microparticules - Google Patents

Procede de pre-test de microparticules, procede de detection de microparticules et instrument de detection de microparticules Download PDF

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Publication number
WO2003100400A1
WO2003100400A1 PCT/JP2002/005110 JP0205110W WO03100400A1 WO 2003100400 A1 WO2003100400 A1 WO 2003100400A1 JP 0205110 W JP0205110 W JP 0205110W WO 03100400 A1 WO03100400 A1 WO 03100400A1
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WIPO (PCT)
Prior art keywords
procedure
fine particles
sample
detecting
solution
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PCT/JP2002/005110
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English (en)
Japanese (ja)
Inventor
Keisei Kimura
Masaaki Ogasawara
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Hitachi High-Technologies Corporation
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Application filed by Hitachi High-Technologies Corporation filed Critical Hitachi High-Technologies Corporation
Priority to PCT/JP2002/005110 priority Critical patent/WO2003100400A1/fr
Priority to JP2004507810A priority patent/JP3848960B2/ja
Publication of WO2003100400A1 publication Critical patent/WO2003100400A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence

Definitions

  • the present invention is used to check the safety and validity of work when handling samples containing harmful substances such as pathogenic bacteria or specific nucleic acids, etc., or to inspect the scattering state of samples in advance.
  • the present invention relates to a method for detecting fine particles and a detector device.
  • the present invention relates to a preliminary inspection method for fine particles in an environment in which a very small amount of a solution sample containing a nucleic acid or the like is operated.
  • microdroplets fine particles containing the gene may be generated. Such small droplets may drift in the work space and scatter in unexpected places.
  • Virus Gene concentration of critically ill patients are said to less than about 1 0 8 copies / m L.
  • rcopyj is the number of nucleic acids
  • L is the little (dm 3 ).
  • about 10 pL is sufficient for contamination with 1 copy of the hepatitis C virus gene. It is. That is, in genetic manipulation, the detection accuracy of such a very small amount of the gene solution is required.
  • viral gene concentrations should be accurately measured because physicians determine the optimal amount of therapeutic agent to administer according to the patient's viral gene concentration.
  • micro droplets are generated when a large number of samples are simultaneously performed in a liquid operation containing a virus gene, the micro droplets are mixed into other samples, making it impossible to measure the virus gene concentration accurately. For example, if a small sample containing a high concentration of a viral gene is mixed in a sample of an originally negative patient, it may be diagnosed as positive even without infection.
  • the present invention relates to providing a method and an apparatus for extremely easily and highly accurately inspecting the distribution of droplets generated by a liquid operation or a solid operation. Disclosure of the invention
  • the present invention relates to a preliminary inspection method including a simulation inspection procedure, a light emission procedure, and a detection procedure, which can confirm the state of generation of fine particles when a predetermined sample is processed according to a predetermined inspection procedure.
  • the simulated inspection procedure is a procedure in which a simulated sample containing a chemiluminescent catalyst is handled according to a predetermined inspection process.
  • the simulated sample has physical properties similar to the specified sample. Therefore, the scattering distribution of the simulated sample that has been scattered is a reproduction of the scattering state of the sample generated in the actual inspection process.
  • the luminescence procedure is a procedure in which the fine particles of the simulated sample scattered during the execution of the inspection step and the fine particle detection solution are reacted via the carrier.
  • the particle detection solution is a solution that reacts with the simulated sample and emits significant light.
  • the luminescence reaction lasts for a long time because the particle detection solution and the simulated sample come into contact via the carrier. For this reason, a remarkable luminescence is generated even in a very small amount of the simulated sample.
  • light emission occurs at a specific position on the carrier, which depends on the positional information of the fine particles.
  • the scattering distribution of the simulated sample is detected based on the light emission, and the scattering state of the sample generated in the actual inspection process is confirmed. As a result, the state of generation of fine particles when a predetermined sample is processed according to a predetermined inspection process can be inspected very easily and with high accuracy.
  • the present invention also relates to a method for detecting fine particles in which fine particles having a chemiluminescent catalyst are reacted with a fine particle detection solution via a carrier, and to a set of fine particle detection instruments.
  • the carrier is made of a material capable of holding the fine particle detection solution, and is almost uniformly moistened with the fine particle detection solution containing the substrate solution.
  • the chemiluminescent catalyst in the fine particles dissolves in the fine particle detection solution and diffuses while generating a luminescence reaction.
  • light emission starts around the particle position, and the light emission point expands around it. By observing this light emission, the position of the fine particles can be easily and accurately confirmed.
  • the terms used in the present invention have the following meanings.
  • Sample A solution or a solid containing nucleic acids, viruses, powders, crystals, etc., which is a substance that is actually subject to inspection and investigation.
  • detecting the state of scattering by performing actual work is based on the nuclei of pathogenic substances and specific nucleic acids. It is difficult to use actual samples for pre-tests because they can lead to acids.
  • Inspection step A combination of processing steps performed on a predetermined sample, for example, all or part of a group of steps for manipulating, analyzing, and inspecting a sample. Processing is a broad concept that includes:
  • One-reaction process such as biochemical reaction step (for example, the step of introducing the reagent of PCR)
  • Sample to be inspected means the sample to be processed in the specific inspection process.
  • Simulated sample A liquid sample or solid sample that simulates physical properties (viscosity, boiling point, specific gravity, particle size, particle size distribution, etc.) in place of the sample used for actual inspections and surveys.
  • physical properties viscosity, boiling point, specific gravity, particle size, particle size distribution, etc.
  • Approximate physical properties This means that at least one of the physical property items (viscosity, boiling point, specific gravity, particle size, particle size distribution, etc.) is similar. In particular, if the simulated sample and the viscosity of the sample are approximated, the scattering state of the sample caused by actual work can be reproduced.
  • the physical property items viscosity, boiling point, specific gravity, particle size, particle size distribution, etc.
  • Simulated experiment This is an operation, such as an inspection process or an investigation process, that is performed using a simulated sample in substantially the same procedure as the actual operation. However, it is not necessary to carry out all the inspection processes and investigation processes, etc., and in some cases, only some of the processes related to splashing may be carried out.
  • Fine particles For example, fine droplets consisting of a simulated sample scattered from the inspection process 02 05110
  • Liquid, solid, and mixtures thereof This includes the wet state during suspension and the dry state after fixation.
  • Substrate solution A chemical substance that emits significant light when catalyzed, for example, a mixture of luminol and an oxidizing agent.
  • the substrate solution reacts with high sensitivity to a trace amount of chemiluminescent catalyst and emits light.
  • the emission time profile is preferably one that reaches a maximum after 1 minute and emits light for a long time, considering the operation time of photon integration.
  • Chemiluminescent catalyst A chemical substance that reacts with a substrate solution and emits remarkable light regardless of whether it is dry or wet. Chemiluminescent catalysts are trace metals, trace enzymes, etc., for example, cobalt.
  • Carrier A substance capable of holding a substrate solution and supplying the substrate solution to a chemiluminescent catalyst, and having sufficient mechanical strength to be handled by an appropriate method. It is desirable to be colorless and transparent, but even if it is opaque, it is sufficient if chemiluminescence is sufficiently transmitted.
  • the filter material is a porous material (porous materia1) filter paper, agarose gel, porous silicon, or the like. However, a hydrophilic substance or the like can be used.
  • Sheet shape A shape in which the thickness of the fine particle detection region of the carrier is substantially constant. In other words, not only the general planar shape but also the central portion, which is the fine particle detection area, has a planar shape, and the peripheral portion includes a raised shape.
  • Nucleic acid processing device A device for processing a liquid, a solid, etc. containing a nucleic acid to achieve a predetermined purpose, for example, a genetic test device and a dispensing device.
  • FIG. 1 shows a use state of the sheet 1 and the transparent plate 8.
  • Fig. 2 shows how to check the scattering position and state of the fine particles.
  • FIG. 3 shows an image of the fine particles 2 of the chemiluminescent catalyst solution under the microscope 3.
  • FIG. 4 shows a photon integrated image by the image processing device 9.
  • FIG. 5 shows the position where the reagent was dispensed to the microplate.
  • FIG. 6 shows the results of contamination detection by actual liquid operation by the PCR method.
  • FIG. 7 shows the contamination detection result by actual liquid operation.
  • FIG. 8 shows a closed container.
  • Example 1 relates to the basic principle of particle detection, instruments used for particle detection, and reagents.
  • Example 2 relates to a method for detecting the scattering distribution of fine droplets (fine particles) generated by operating a nucleic acid processing apparatus with high sensitivity by preliminary experiments.
  • Embodiments 3 and 4 relate to a method of reducing scattering of fine droplets (fine particles) from a nucleic acid processing apparatus by performing preliminary experiments.
  • Example 5 relates to a container for storing an instrument used for detecting fine particles.
  • FIG. 1 is a schematic view of the detection of fine particles
  • FIG. 1 (a) is a perspective view thereof
  • FIG. 1 (b) is a cross-sectional view taken along AB in FIG. 1 (a).
  • the droplets (fine particles) 3 are present on the substrate 7 and consist of a chemiluminescent catalyst solution.
  • a sheet 1 (carrier) containing a particle detection solution and a transparent plate 8 are used to detect the droplets 3.
  • the sheet 1 (carrier) used for the droplet detection is a sheet-shaped porous body, and can infiltrate and hold the fine particle detection solution.
  • filter paper for example, filter paper, agarose gel and the like.
  • a filter paper having a square of 15 cm on a side and a thickness of 500 m is used.
  • the thickness of the sheet 1 may be large as long as the sheet 1 is colorless and transparent.
  • opaque materials such as filter paper, opaque materials such as agarose gel when thick, or colored, are as thin as possible. That is, the sheet 1 needs to transmit visible light to such an extent that chemiluminescence caused by the fine particles can be detected.
  • a filter paper having a thickness of about 500 m or less allows sufficient transmission of chemiluminescence. Further, the thickness of the sheet 1 needs to have a mechanical strength that does not break when the sheet 1 is placed on the fine droplets 3 or the like. It is preferable to use an elastic body having a low Young's modulus so as to cope with free bending.
  • a glass plate is used as the transparent plate 8.
  • the present invention is not limited to this. Any member may be used as long as it has a flat surface to which the sheet 1 is attached, transmits chemiluminescence, and has a certain mechanical strength.
  • a transparent resin such as an acrylic resin can be used.
  • the transparent plate 8 facilitates the work of infiltrating the sheet 1 with the substrate solution, and allows the sheet 1 to be accurately attached to the base 7. Further, drying of the substrate solution from the sheet 1 can be prevented. Further, when the surface of the transparent plate 8 is covered with a vinylidene chloride film or the like, the contamination of the transparent plate 8 by the substrate solution can be prevented.
  • the substrate 7 is a detection target for the presence or absence of fine particles, and is, for example, a desk on which a genetic diagnostic device is placed or a ventilation hole in a room where the genetic diagnostic device is located.
  • the droplet 3 to be detected is, for example, a small droplet of whole blood or plasma generated when a clinical sample is handled by a genetic diagnostic device, or a small droplet of an artificially generated Co 2 + solution. . It is assumed that the volume of the fine droplet 3 is l ⁇ L or less, especially 10 pL. The minimum volume of microdroplets that can be mixed with 1 copy of viral gene (contamination) is about 10 pL.
  • luminol luminescence which is a chemical luminescence
  • a mixed solution of luminol (chemiluminescent substrate) and hydrogen peroxide solution (oxidizing agent) is used as a substrate solution. Since luminol is used as the chemiluminescent substrate in this way, it is possible to detect minute droplets of whole blood, plasma, and serum. Therefore, it is possible to detect the scattered distribution of blood and the like before gene extraction, which has been scattered from the genetic diagnostic apparatus, without adding a special reagent to a clinical specimen such as blood. When micro droplets are artificially generated, use a solution containing cobalt ions (a divalent metal ion catalyst).
  • cobalt ions a divalent metal ion catalyst
  • the method for preparing the substrate solution is described. Specifically, 1.5 mM (X 1) luminol solution and 10 mM hydrogen peroxide solution are added in equal amounts and mixed using a pipette or the like. The substrate solution is prepared immediately immediately before the experiment. However, 1.5 mM (X 1) luminol solution and 1 O mM aqueous hydrogen peroxide are prepared immediately for each experiment. When stored in a cool place (4 ° C), it can be used for about one hour. The substrate solution stored in a cool, dark place (4 ° C) should be used after it has reached room temperature in a place.
  • boric acid (Boric Acid, manufacturer: Wako Pure Chemical Industries, serial number: 0 2 1 — 0 2 195) was added to 7 O mL of ultrapure water (H 2 ⁇ , manufactured by Millipore). Dissolve and add 1 M NaOH (Sodium Hydroxide, manufacturer, Wako Pure Chemical Industries) as appropriate until the solution has a pH of 3. Then, add ultrapure water appropriately until the total volume becomes 100 mL. However, since NaOH is deliquescent, the sample before preparation should be kept dry within a short time. Store the prepared solution in a location.
  • a method for preparing a 15 mM (X 20) luminol solution of a chemiluminescent substrate solution will be described.
  • the 20-fold solution is prepared because the luminol in the 1-fold solution cannot be weighed in a small amount. Specifically, 2.7 mg of luminol (manufactured by Wako Pure Chemical Industries, serial number: 123-025858) was added to 0.1 M boric acid. Dissolve in 1 mL of buffer. Luminol may be difficult to dissolve, so make sure it is completely dissolved. When used, further dilute to X1 solution with 0.1 M boric acid buffer. The luminol solution is prepared immediately. If it is stored in a location, it can be used for about two days. Luminol stored in a cool place (4 ° C) should be used after it has reached room temperature in the dark.
  • sheet 1 (15 cm square flat filter paper) is placed on the transparent plate 8 using metallically clean tweezers.
  • a substrate solution in this example, a mixture of a luminol solution as a chemiluminescent substrate and a hydrogen peroxide solution as an oxidizing agent
  • Infiltration is performed by uniformly dropping 5 mL of the substrate solution from above the sheet 1 fixed on the transparent plate 8.
  • the sheet 1 containing the substrate solution can be produced, and the sheet 1 containing the substrate solution can be fixed to the transparent plate 8. At this time, if the volume of the substrate solution is small or if the substrate solution is not dripped uniformly, irregularities are generated on the sheet 1 containing the substrate solution. Then, the sheet 1 containing the substrate solution is leveled with the tip of a metal-clean microchip or the like, and is closely adhered to the transparent plate 8 flat.
  • the sheet 1 is attached on the base 7 on which the fine droplets 3 are present.
  • the sheet 1 containing the substrate solution comes into contact with the fine droplets 3 of the chemiluminescent catalyst solution, the dried fine droplets 3 dissolve into the substrate solution.
  • the chemiluminescent catalyst reacts with the substrate solution, and a light emitting point 9 is generated on the sheet 1 at a position corresponding to the position of the droplet group.
  • the luminous point 9 expands as the fine droplets diffuse in the sheet 1.
  • the sheet 1 contains a large amount of the substrate solution, light emission can be maintained for a long time.
  • the sheet 1 uniformly contains the substrate solution, the light emission state (luminous intensity, etc.) of the light-emitting point 9 is hardly affected by the contact position between the sheet 1 and the fine droplets 3. Irrespective of the contact position of the droplet 3, a substantially uniform light emitting point 9 is generated.
  • the sheet 1 can be stuck loosely from above the base 7, the amount of movement of fine droplets at the time of detection is smaller than when, for example, a substrate solution is sprayed by spraying. Therefore, by observing the light emitting point 9 on the sheet 1, the scattering distribution of the minute droplet 3 can be accurately confirmed.
  • chemiluminescence from Sheet 1 can be sufficiently confirmed by the naked eye when the surroundings of Sheet 1 are darkened using a simple dark box, and the detection operation is very easy.
  • microdrops 3 of a chemiluminescent catalyst solution of about 10 pL are artificially generated on the base 7, and then the position of the microdroplets is detected by the microscope 10. Then, the position of the minute droplet is detected by the present droplet group detection method, and the result obtained by the microscope 10 is compared with the result obtained by the present method to evaluate.
  • microdroplets 3 of a chemiluminescent catalyst solution of about 10 pL on the substrate 7 will be described.
  • 110 In order to prevent contamination by micro droplets 3, 110
  • the microplate 10 is used to observe the base 7 on which the fine droplets 3 of the chemiluminescent catalyst solution are present, search for the fine droplets 3, and mark the droplet positions with a size that allows the naked eye to recognize the droplet positions.
  • the diameter of the fine droplet 3 of the chemiluminescent catalyst solution is recorded.
  • the capacity of the minute droplet 3 is calculated from the diameter measured by the microscope 10.
  • FIG. 2 shows a CCD camera 11 that detects the light-emitting point 9, a dark box 8 that shields the light-emitting reaction system (base 7, sheet 1, transparent plate 8), and data from the CCD camera 11
  • FIG. 4 is a schematic diagram of a method for detecting a light emitting point 9 present on a sheet 1 by a detection system including an image processing device 12 for processing and a computer 13 for controlling the image processing device 12.
  • a method of detecting the state of dispersion of the droplet group according to the present embodiment will be described with reference to FIG. The following operations are all performed in the dark box 14.
  • the minute droplets 3 on the base 7 are set in the measurement range of the CCD camera 11 with reference to the above-described marking.
  • CCD camera 11 has a field of view of about 16 cm x 16 cm.
  • photon integration is performed using the image processing device 12 so that the total photon amount from the light emitting point 9 can be measured.
  • the chemiluminescent substrate When the chemiluminescent substrate is brought into contact with the chemiluminescent catalyst, the light emission reaches its maximum after about 2 minutes and rapidly declines.
  • the chemiluminescent catalyst diffuses through the carrier and reacts with the chemiluminescent substrate one after another, it is possible to observe a very strong luminescent point that can be observed with the naked eye for a long time. As a result, it is possible to observe a very intense emission point that can be observed with the naked eye at the position of the microdroplet 3 of the chemiluminescent catalyst solution that rapidly declines. Also, the capacity of the fine droplet can be calculated from the detected total photon amount.
  • FIG. 3 shows an image of the microdroplets 3 of the chemiluminescent catalyst solution under a microscope 10.
  • FIG. 4 shows a photon integrated image obtained by the image processing apparatus 12. Comparing Figs. 3 and 4, it can be seen that there is a positional correlation between the fine droplet 3 (a, b, c) of the chemiluminescent catalyst solution and the luminescent point 9 (a, b, c). Is recognized.
  • the fine droplets 3 (a) of the chemiluminescent catalyst solution shown in FIG. 3 are estimated to correspond to about 1 OpL from a diameter of about 50 m, but in FIG. It is shown as a large emission point 9 (a) of about 1 mm or more. Therefore, it was shown that the distribution of fine droplets of about 10 pL can be detected with high sensitivity and with the naked eye.
  • the present invention is not limited to the steps disclosed in the present embodiment, and various embodiments exist.
  • a method is used in which a filter paper soaked with a fine particle detection solution is placed in an area where splash and scattering is expected, and then a splash is generated.
  • the present embodiment is a method for detecting the scattering distribution of fine particles generated from the nucleic acid processing apparatus with high sensitivity by performing a preliminary experiment.
  • a luminescent catalyst solution (Co 2 + solution), which is a simulated sample, is treated as a sample by a nucleic acid processing apparatus for which the state of scattering of fine particles is to be checked.
  • the scattering distribution of fine droplets (fine particles) of the chemiluminescent catalyst solution is detected using the fine particle detection method described in Example 1. That is, the sheet 1 soaked with the substrate solution is attached to a place where the scattering from the nucleic acid processing apparatus is expected, and the presence or absence of the light emitting point 9 is confirmed. As a result, it is possible to easily and accurately grasp the state of splash of about 10 pL, which is a cause of the contamination of 1 copy of the viral gene.
  • FIG. 5 is a schematic diagram showing the positional relationship between a well into which a simulation sample (ADNA solution or Co 2 + solution) was charged and a well in which contamination was detected.
  • FIG. 6 shows the results of detection by the PCR method.
  • FIG. 7 shows a detection result according to the present invention.
  • FIG. 5 (a) shows the position of the reagent dispensed into the microtiter plate.
  • the ⁇ DNA solution was placed as a positive model in the ⁇ in FIG. 5 (a).
  • FIG. 5 (b) is a cross-sectional view taken along line AB in FIG. 5 (a).
  • 200 L of ultrapure water was dispensed to the well adjacent to the A DNA well. Before and after suctioning and discharging ⁇ DNA, 50 L of this ultrapure water was used as a sample to evaluate contamination.
  • FIG. 6 shows the results of detection by the PCR method.
  • Fig. 6 shows the PCR product of the gel adjacent to the ⁇ DN After moving, it was stained with EtBr.
  • A-E indicates the well of a 96-well microtiter plate, and M indicates the molecular weight marker of the gene. ⁇ When the solution is sucked and discharged so that the droplet contacts the side wall of the well (Fig. 6
  • the chemiluminescent catalyst Co 2 + solution was placed as a positive model in the ⁇ part.
  • the C o 2 + and ⁇ a paraffin film on pre Ichito non Ueru solution, 2 0 times after sucking and discharging the C o 2 + solution, the paraffin film was mounted on the base 7 using metallically clean tweezers or the like.
  • the sheet 1 impregnated with the substrate solution fixed to the transparent plate 8 was brought into contact with the paraffin film to which the fine droplets of the Co 2 + solution had adhered. Twenty-five seconds after the paraffin film was brought into contact with the filter paper 4 fixed to the transparent plate 8, photon integration measurement was performed for one minute.
  • Figure 7 shows the detection results. In FIG.
  • a to E indicate the rows of a 96-well microplate, as in the PCR method, and numbers 1 to 4 indicate the same columns.
  • white triangles and white circles indicate the well positions of the 96-well micro tie plate.
  • the paraffin film adjacent to the relevant Co 2 + solution is used before performing the dispensing operation. No condensation mining was detected. However, after the dispensing operation in which the Co 2 + solution was sucked and discharged 20 times, many light emitting points 9 indicating contamination were observed. Specifically, there is a large luminescence in column 3 row D and row 4 row D adjacent to the Co 2 + solution, and there are a total of 5 luminescence points 9 including the surrounding area (number of experiments: 3 ).
  • the PCR method can detect only the nucleic acid amplification in the confinement in the well, that is, the confinement in the 3rd row D and 4th row D rows. .
  • this detection method can also detect contaminants other than pells. In other words, this detection method can clearly grasp the scattering distribution around the wells that cannot be detected by the PCR method, which is widely used as a highly sensitive gene detection method. Can be.
  • the present embodiment relates to a method for suppressing fine particles scattered from a nucleic acid processing apparatus by a preliminary experiment using the fine particle detection method of Example 1 or 2.
  • Example 1 or 2 the characteristic portions in comparison with Example 1 and Example 2 will be described.
  • experimental results may be obtained that are contrary to the expectations of workers.
  • a gene that is not originally amplified may be amplified, or conversely, a gene to be amplified may not be amplified at all. This is due to improper liquid or solid handling and contaminated workplaces. That is, amplification of genes that are not originally amplified is caused by unexpected gene contamination in the experimental system.
  • the fact that no amplification is performed is the result of contaminating digestive enzymes such as DNase and RNase or inhibitors of the amplification reaction in the experimental system.
  • the condominium mining is roughly divided into two. One is carryover between multiple samples, and the other is scattering of nucleic acid-containing microdroplets. Of these, carryover can often be resolved by cleaning and disposable equipment.
  • Embodiments 1 and 2 are performed in a workplace that handles genes, such as a room where a genetic diagnosis device is placed, the above problem is solved. I can decide. Hereinafter, the specific method will be described.
  • test procedures for handling samples such as genetic diagnosis methods and experimental methods, are specified, and simulated samples are prepared as substitutes for the samples.
  • the simulated sample particularly suitable for flying is a solution containing a chemiluminescent catalyst, and a simulated sample for the purpose is prepared by selecting the concentration, the solvent, and the like.
  • the simulated sample is preferably a liquid or solid sample simulating physical properties such as viscosity, boiling point, specific gravity, particle size, and particle size distribution.
  • “Approximation” has a broad meaning that the reproducibility of the flying situation can achieve a predetermined purpose.
  • a Co 2 + solution is sufficient for the investigation of the scattering state of a normal nucleic acid-containing sample.
  • an inert substance such as dextrose.
  • One that requires viscosity adjustment is, for example, a 6 M guanidine derivative that dissolves the viral outer membrane.
  • dispensing operation and liquid transfer operation are handled, for example, by Biomek2000.
  • a simulated sample is processed by Biomek2000 in a normal environment.
  • suction and discharge are repeated at the above flow rate at first.
  • the liquid transfer operation the solution once sucked is moved to another container by the X-axis and Y-axis rails. At this time, it is conceivable that droplets may be scattered during dispensing operation and movement.
  • the sheet 1 containing the substrate solution fixed to the transparent plate 8 described in Example 1 is brought into contact with a place where the scattering is expected.
  • the plate in the Biomek2000, the Biomek2000 body, and the Biomek2000 are open systems, attach sheet 1 around the workbench.
  • the sheet 1 is brought into contact with the part other than the plane without using the transparent plate 8.
  • work gloves are also contacted with sheet 1 containing the substrate solution.
  • the scattering distribution of the fine droplets 3 can be confirmed, it can be determined that the gene-containing solution has been scattered in the processing steps such as the liquid operation and the solid operation. In particular, when dealing with multiple samples simultaneously, the occurrence of contamination between samples can be predicted.
  • This embodiment is a method for ensuring the cleanliness of a facility in which a nucleic acid processing apparatus is arranged by using the particle detection method of the first or second embodiment.
  • the characteristic portions in comparison with Example 1 and Example 2 will be described.
  • Hazardous substances may be handled in various experimental systems. At that time, wearing safety gear etc. is required by the Ministry of Health, Labor and Welfare's safety standards, but workers may unintentionally contaminate the inside and outside of the workplace with harmful substances with protective gear etc. . That is, unconsciously touch the experimental equipment frequently used with the above harmful substances and its peripheral equipment, or the door knob of the work place, etc. while wearing protective gloves to which the harmful substances are likely to adhere.
  • the above problem is solved as follows. First, a simulated sample is used in an experimental system as a substitute for harmful substances, and workers perform liquid or solid operations as usual.
  • the simulated sample should be similar to the physical properties of the harmful substance (viscosity, boiling point, specific gravity, particle size, particle size distribution, etc.).
  • the sheet 1 containing the substrate solution fixed on the transparent plate 8 is brought into contact with the work place.
  • the sheet 1 containing the substrate solution is brought into contact with the portion other than the flat surface without using the transparent plate 8.
  • the substrate solution is contained around the inside and outside of the ventilation openings. Sheet 1 is brought into contact.
  • a light emitting point 9 is generated at the place where the simulation sample scattered. Then, by confirming the distribution of the light emitting points 9 (scattering distribution of the droplets 3), it can be determined that the distribution is a site contaminated by harmful substances. In particular, when the distribution of droplets is found outside the workplace, it is highly possible that the above harmful substances have leaked out of the workplace, and it is necessary to take immediate measures to prevent leakage.
  • the present embodiment is a sealed container for transporting and storing the sheet 1 described in Embodiments 1 to 4 in a ready-to-use state.
  • Fig. 8 (A) shows a perspective view during storage.
  • Fig. 8 (B) shows the line segment AB in Fig. 8 (A) during transportation.
  • FIG. FIG. 8 (C) shows a cross-sectional view of line segment AB in FIG. 8 (A) at the time of preparation.
  • the closed container 2 according to the fourth embodiment will be described with reference to FIGS. 8 (A) to 8 (C).
  • the substrate solution (a mixture of the chemiluminescent substrate 4 and the oxidizing agent 5) that is infiltrated into the sheet 1 reacts gradually upon contact even in the absence of a chemiluminescent catalyst (eg, cobalt ion). Therefore, the chemiluminescent substrate 4 and the oxidizing agent 5 need to be isolated until immediately before the detection of fine droplets.
  • a chemiluminescent catalyst eg, cobalt ion
  • the sealed container 2 is made of, for example, a material in which an aluminum foil is lined with a resin, has a space for separately accommodating the chemiluminescent substrate 4 and the oxidizing agent 5, and separates and stores them. .
  • the chemiluminescent substrate 4 and the oxidizing agent 5 are separated by a strong partition, and the chemiluminescent substrate 4 and the oxidizing agent 5 and the sheet 1 thereunder are separated by a brittle diaphragm. Save the person.
  • a force F is applied by pulling the center handle to both sides to break the fragile diaphragm, and the mixture of the chemiluminescent substrate 4 and the oxidizing agent 5 infiltrates the sheet 1. Since the cutout is formed on the side of the closed container 2 where the sheet 1 is stored, the operator can open the closed container 2 and take out the sheet 1 soaked with the substrate solution therefrom.
  • the sealed container 2 holds the sheet 1 separately from the chemiluminescent substrate 4 and the oxidizing agent 5, but is not limited to this, and the sheet 1 infiltrates the negative solution. It may be held as follows.
  • the sheet 1 does not need to be stored together with the chemiluminescent substrate 4 and the oxidizing agent 5.
  • the chemiluminescent substrate 4 and the oxidizing agent 5 can be separated and stored in a tubular sealed container 2 with a fragile diaphragm, and the sheet 1 can be separated and stored in another clean container.
  • the sheet 1 can be stored in such a state that microdroplets containing a chemiluminescent catalyst can be immediately detected.

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Abstract

La présente invention concerne un procédé de détection de microparticules et un détecteur à cet effet qu'on utilise lorsqu'on manipule des échantillons contenant des substances dangereuses (micro-organismes pathogènes, etc.) ou des acides nucléiques spécifiques afin de confirmer l'innocuité, le caractère raisonnable, etc. des procédures et des tests préliminaires de dispersion de cet échantillon. Dans un système expérimental, on effectue une expérience préliminaire en utilisant un échantillon de simulation possédant les mêmes propriétés ou des propriétés similaires à celles d'un échantillon, cet échantillon de simulation contenant un catalyseur chimioluminescent. Puis on lie une feuille 1 (un support) contenant une solution de substrat chimioluminescent à une partie dans laquelle on estime que des microparticules ont été dispersées. Dans le cas où ces microparticules sont présentes, le catalyseur chimioluminescent est dissout dans la solution de substrat chimique présente sur le support et il est dispersé dans ce support alors que la chimioluminescence apparaît. On peut facilement détecter très précisément les positions dispersées de ces microparticules en observant cette chimioluminescence. On examine ainsi préalablement la dispersion des microparticules à l'occasion de cette expérience préalable et l'innocuité, le caractère raisonnable, etc. des procédures du système expérimental sont confirmés.
PCT/JP2002/005110 2002-05-27 2002-05-27 Procede de pre-test de microparticules, procede de detection de microparticules et instrument de detection de microparticules WO2003100400A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
PCT/JP2002/005110 WO2003100400A1 (fr) 2002-05-27 2002-05-27 Procede de pre-test de microparticules, procede de detection de microparticules et instrument de detection de microparticules
JP2004507810A JP3848960B2 (ja) 2002-05-27 2002-05-27 微粒子の予備検査方法、微粒子検出方法、及び微粒子検出器具セット

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PCT/JP2002/005110 WO2003100400A1 (fr) 2002-05-27 2002-05-27 Procede de pre-test de microparticules, procede de detection de microparticules et instrument de detection de microparticules

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WO2003100400A1 true WO2003100400A1 (fr) 2003-12-04

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2010276468A (ja) * 2009-05-28 2010-12-09 Takenaka Komuten Co Ltd 粉末物質の飛散状態評価方法
CN113039425A (zh) * 2019-02-26 2021-06-25 松下知识产权经营株式会社 散射体测定方法及散射体测定装置

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JPH01289495A (ja) * 1988-05-13 1989-11-21 Konica Corp アルコール分析素子
JPH05302930A (ja) * 1992-04-24 1993-11-16 Canon Inc 分析装置及び分析方法
JPH07128315A (ja) * 1993-11-05 1995-05-19 Showa Denko Kk 化学発光検出用充填剤およびそれを用いる分析方法
JPH07209191A (ja) * 1994-01-25 1995-08-11 Toyobo Co Ltd 多孔性膜上の発光反応による物質の検出法およびその装置
JP2001264315A (ja) * 2000-03-15 2001-09-26 Arkray Inc 固体成分分離能を有する試験片

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Publication number Priority date Publication date Assignee Title
JPH01289495A (ja) * 1988-05-13 1989-11-21 Konica Corp アルコール分析素子
JPH05302930A (ja) * 1992-04-24 1993-11-16 Canon Inc 分析装置及び分析方法
JPH07128315A (ja) * 1993-11-05 1995-05-19 Showa Denko Kk 化学発光検出用充填剤およびそれを用いる分析方法
JPH07209191A (ja) * 1994-01-25 1995-08-11 Toyobo Co Ltd 多孔性膜上の発光反応による物質の検出法およびその装置
JP2001264315A (ja) * 2000-03-15 2001-09-26 Arkray Inc 固体成分分離能を有する試験片

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2010276468A (ja) * 2009-05-28 2010-12-09 Takenaka Komuten Co Ltd 粉末物質の飛散状態評価方法
CN113039425A (zh) * 2019-02-26 2021-06-25 松下知识产权经营株式会社 散射体测定方法及散射体测定装置

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