JP2014102227A - Inspection method and inspection device of microorganism or the like - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an inspection method and inspection device of a microorganism or the like capable of reducing background noise and measuring the number of a target microorganism (specific microorganism) to be examined.SOLUTION: A fluorescent-labeled antibody capable of binding with a specific microorganism and membrane permeable nucleic acid stain dye are added in a sample, and a specific microbial count is measured with a fluorescence cytometry method. Preferably, concentration by a magnetic bead and a fluorescence flow cytometry are used, the membrane permeable nucleic acid stain dyes and the fluorescent-labeled antibody are added in this order, and what emits both fluorescence is measured as a specific microorganism.

Description

  The present invention relates to a method and apparatus for inspecting microorganisms and the like, and more particularly, to a method and apparatus for inspecting microorganisms and the like suitable for inspecting specific microorganisms in an inspection object.

  In Patent Document 1, in order to quickly detect a target microorganism such as Escherichia coli O-157 in a sample without performing pre-culture, an antibody that specifically reacts with the target microorganism is bound to the magnetic bead, and the magnetic bead Is added to the sample so that only the target microorganism is adsorbed to the magnetic bead from the sample containing a large amount of contaminants, and the magnetic bead-microbe conjugate is reacted with an antibody labeled with a fluorescent dye, and the magnetic A method of detecting a bead-microbe-fluorescently labeled antibody complex by fluorescent flow cytometry has been proposed.

JP 2002-181823 A

  In Patent Document 1, in order to prevent contamination from entering during detection by flow cytometry, a fluorescently labeled antibody is added to a non-target microorganism (contamination) adsorbed non-specifically to magnetic beads. Rinse before. In Patent Document 1, a fluorescently labeled antibody is added to and reacted with a sample from which contaminants have been washed away, and then the sample is washed again.

  However, even if the sample is washed, impurities cannot be completely removed, and unreacted fluorescently labeled antibodies cannot be completely removed. For this reason, it is considered that there is a limit to the improvement of detection sensitivity. In Patent Document 1, measurement is possible until the number of bacteria is 1000 / mL in the sample, but as a sanitary standard of the Food Sanitation Law, for example, Staphylococcus aureus needs to be 100 / mL or less. It is.

  For this reason, it is desired to improve the detection sensitivity of specific microorganisms (target microorganisms to be examined) regardless of the cleaning performance of the sample.

  An object of the present invention is to provide an inspection method and an inspection apparatus for microorganisms and the like capable of measuring the number of specific microorganisms while reducing background noise.

  The present invention is characterized in that a fluorescently labeled antibody that binds to a specific microorganism and a membrane-permeable nucleic acid staining dye are added to a sample, and the number of specific microorganisms is measured by a fluorescence cytometry method.

  According to the present invention, it is possible to measure the number of specific microorganisms while reducing background noise.

  Problems, configurations, and effects other than those described above will be clarified by the following description of embodiments.

It is a flowchart which shows the test | inspection method of the specific microorganisms etc. which are one Example of this invention. It is a figure which shows the algorithm which discriminate | determines the specific live microorganisms in one Example of this invention. It is a figure which shows the influence of pH at the time of dyeing | staining with a membrane-permeable fluorescent dye. It is a figure which shows schematic structure of test | inspection apparatuses, such as microorganisms which are one Example of this invention. It is a figure which shows the example of a cross section containing the flow path for microorganisms detection in the microorganisms test | inspection cassette used for test | inspection apparatuses, such as microorganisms of the Example of this invention. It is a figure which shows the decomposition | disassembly structure example containing the flow path for microorganisms detection in the microorganisms test | inspection cassette used for test | inspection apparatuses, such as microorganisms of the Example of this invention. It is a figure explaining an example of the analysis process using test | inspection apparatuses, such as microorganisms, of the Example of this invention. It is a figure which shows an example of schematic structure of the microorganisms test | inspection cassette used for test | inspection apparatuses, such as microorganisms of the Example of this invention. It is a figure which shows the other example of schematic structure of the microorganisms test | inspection cassette used for test | inspection apparatuses, such as microorganisms of the Example of this invention.

  Embodiments of the present invention will be described below with reference to the drawings. In addition, the present invention can be similarly applied to the inspection of specific cells (cells to be detected) in addition to the inspection of specific microorganisms (microorganisms to be detected). Therefore, in the present specification and claims, “Specific microorganism or the like” means “specific microorganism and / or specific cell”. For the sake of simple explanation, “specific microorganisms” may be simply referred to as “specific microorganisms”.

  First, the background to the present invention will be described.

  In the present invention, a fluorescent labeled antibody that binds to an antigen of a specific microorganism is used to detect the specific microorganism using a fluorescent flow cytometry method. In the fluorescence flow cytometry method, it is necessary to reduce background noise in order to accurately measure the number of microorganisms. As described in Patent Document 1, it is possible to detect a specific microorganism by reducing background noise by combining a magnetic bead concentration method and washing a sample. However, there is a limit to improving detection sensitivity even if the sample is washed. That is, in Patent Document 1, there is a sample that can reduce impurities as much as possible by washing the sample as much as possible, but cannot be removed even by washing. Therefore, it may be difficult to achieve the measurement sensitivity required for detection of a specific microorganism only by combining the magnetic bead method and the fluorescence flow cytometry method.

  Therefore, the present invention detects specific microorganisms based on the new idea that even if contaminants other than specific microorganisms enter, it is sufficient to distinguish and detect specific microorganisms and contaminants at the time of detection. Is. Specifically, the present invention uses a membrane-permeable nucleic acid staining dye (a fluorescent dye whose amount of fluorescence is amplified by nucleic acid binding through membrane permeability) in addition to a fluorescently labeled antibody that binds to an antigen of a specific microorganism, In the detection by the fluorescence cytometry method, specific microorganisms and contaminants are distinguished and detected (details will be described with reference to FIG. 2).

  Thereby, it is possible to reduce background noise derived from microorganisms (contaminated microorganisms) other than the specific microorganisms and fluorescent dyes, and it is possible to more accurately measure the number of specific microorganisms.

  In principle, the present invention only needs to use a fluorescently labeled antibody that binds to a specific microorganism and a membrane-permeable nucleic acid staining dye. Concentration with magnetic beads is not essential. Ground noise can be further reduced. In principle, the present invention can also be applied to a fluorescence imaging cytometry method, but a fluorescence flow cytometry method is more preferable.

  In addition, specific microorganisms include live microorganisms and dead microorganisms, but in the present invention, nucleic acid staining dyes that are further membrane-impermeable nucleic acid dyes (fluorescent dyes that are membrane-impermeable and whose fluorescence amount is amplified by nucleic acid binding). It is possible to discriminate and count the number of specific live microorganisms. In the embodiments described below, a method for inspecting specific live microorganisms in which the number of specific live microorganisms is discriminated and counted will be mainly described.

(A) Background noise reduction method
FIG. 1 shows the procedure of a method for inspecting specific microorganisms or the like (addition procedure of fluorescent dye) according to an embodiment of the present invention. In this example, the nucleic acid of a specific microorganism is fluorescently stained using two types of nucleic acid staining dyes, and then an antibody labeled with the fluorescent dye is added and bound to the antigen of the specific microorganism, whereby only specific microorganisms can be selected. It is what I did. In the step of fluorescently staining the nucleic acid of the specific microorganism, the pH is preferably adjusted to 7.0 to 7.5.

  First, magnetic beads are added to a sample (specimen) prepared by suspending food in sterilized water, and the specific microorganisms are adsorbed onto the magnetic beads, whereby only the specific microorganisms are concentrated and separated. Since the concentration and separation of microorganisms using magnetic beads is a known technique as described in Patent Document 1, detailed description thereof is omitted.

  Next, two kinds of nucleic acid staining dyes are added, and the microorganisms in the specimen (specific microorganisms and microorganisms other than the specific microorganisms) are fluorescently stained. Specifically, a membrane-permeable nucleic acid staining dye and a membrane-impermeable nucleic acid staining dye are used. The membrane-permeable nucleic acid staining dye stains both live bacteria (live microorganisms) and dead bacteria (dead microorganisms). On the other hand, a membrane-impermeable nucleic acid staining dye stains only dead bacteria whose cell membrane is damaged. Thus, by using a membrane-permeable and membrane-impermeable nucleic acid staining dye, it is possible to distinguish between live and dead bacteria.

  Next, an antibody labeled with a fluorescent dye (hereinafter, fluorescent labeled antibody) is added. This antibody may be a monoclonal antibody or a polyclonal antibody as long as it specifically binds to the antigen of the target specific microorganism. Thereby, the fluorescence labeled antibody binds only to a specific microorganism. In addition, since the fluorescence labeled antibody is also a known technique, detailed description is omitted.

  Finally, the specimen is irradiated with excitation light to detect fluorescence of the specific microorganism that has been fluorescently stained.

  Next, an algorithm for discriminating and measuring a specific microorganism (specific live microorganism) by reducing background noise according to the inspection procedure of FIG. 1 will be described with reference to FIG.

  The target specific live microorganism is specifically bound by the fluorescently labeled antibody (A) and further contains a membrane-permeable nucleic acid staining dye (B), and the nucleic acid inside the cell membrane of the specific live microorganism is stained, and the specific live microorganism is stained. Microorganisms are dyed by a total of two types of staining dyes (the fluorescently labeled antibody (A) binds to a specific microorganism and is not directly stained by a staining dye as in nucleic acid staining. This will be described as “staining”.). The specific dead microorganisms further enter a membrane-impermeable nucleic acid staining dye (C) from the damaged part of the cell membrane, and the specific dead microorganisms are stained by a total of three types of staining dyes.

  On the other hand, contaminating microorganisms other than the specific living microorganisms mixed through the magnetic separation in the concentration and separation step are stained only by the membrane-permeable nucleic acid staining dye (B). Further, the contaminating dead microorganisms that have passed through the magnetic separation in the concentration and separation step are stained only by the membrane-permeable nucleic acid staining dye (B) and the membrane-impermeable nucleic acid staining dye (C). Therefore, it is possible to discriminate between specific living microorganisms and contaminating microorganisms (contaminated living microorganisms and contaminating dead microorganisms) depending on whether the fluorescently labeled antibody (A) is labeled.

  The specific live microorganism and the specific dead microorganism can be discriminated by the presence or absence of fluorescence derived from a membrane-impermeable nucleic acid staining dye (C).

  Each of the fluorescent dyes (A) to (C) has autofluorescence alone. For this reason, fluorescently labeled antibodies (A) that are not bound to specific microorganisms and nucleic acid staining dyes (B) and (C) that do not stain microorganisms become contaminants in the measurement of specific live microorganisms (specific dead microorganisms). Causes noise. In this example, the specific live microorganism simultaneously emits fluorescence from the fluorescently labeled antibody (A) and the membrane-permeable nucleic acid staining dye (B) (the specific dead microorganism further has a membrane-impermeable nucleic acid staining dye (C)). Since the fluorescence of the origin is emitted at the same time, it is possible to distinguish between the specific live microorganism (specific dead microorganism) and the fluorescent dyes (A) to (C).

  In this way, by combining fluorescent dyes with different staining mechanisms, that is, fluorescently labeled antibodies and membrane-permeable nucleic acid staining dyes (in combination with membrane-impermeable nucleic acid staining dyes as necessary), specific living microorganisms are specific. Can be detected.

  In addition, when using fluorescent dyes having different staining mechanisms, it is preferable to add a membrane-permeable nucleic acid staining dye first. The membrane-permeable nucleic acid staining dye permeates the cell membrane through the membrane protein of the cell membrane. When the fluorescently labeled antibody is added first, the fluorescently labeled antibody binds to a part of the antigen of the membrane protein of the cell membrane, so that the membrane-permeable nucleic acid staining dye does not easily permeate the cell membrane. Therefore, it is preferable to add a membrane-permeable nucleic acid staining dye before the fluorescently labeled antibody.

  FIG. 3 shows how the staining rate of the membrane-permeable nucleic acid staining dye changes depending on the pH. Fig. 3 shows E.aerogenes (Enterobacter aerogenes: a group of coliforms) added to a commercially available plastic bottle beverage and stained with cyanine blue fluorescent dye as a membrane-permeable nucleic acid dye. The dyeing rate is shown. In each condition, the experiment was performed twice at room temperature.

  When the pH was increased from the initial pH = 6.5 of a commercially available PET bottle beverage, the detection rate (= staining rate) became the best when the pH = 7.1 was neutral (the pH = generally called the neutral region = 6.8-7.2 are treated as neutral.) The staining rate is 62% on average at pH = 7.1, which is improved to nearly twice that at pH = 6.5, indicating that pH greatly affects the detection rate.

  In the results shown in FIG. 3, the detection rate is poor even at pH = 6.8, which is generally handled as neutral. From this, it can be said that the alkali side (pH = 7.0 to 7.2) is preferable even in the neutral region. Furthermore, the present inventors have confirmed that the staining rate is 50% or more even at pH = 7.6, as shown in FIG.

  That is, it can be said that the pH at the time of staining with a membrane-permeable nucleic acid staining dye is most preferably on the alkaline side of the neutral region (pH = 7.0 to 7.2), but pH = 7.0 to 7.6 at which the staining rate is 50% or more. It can be said that this is at least a practicable pH range. In addition, since vitamin C is normally added as an antioxidant to commercially available PET bottle beverages, the pH is close to slightly acidic. To adjust the pH, harmless sodium hydrogen carbonate or the like can be used as a food additive. The combination of the presence / absence of luminescence derived from the label of the fluorescently labeled antibody and the presence / absence of luminescence derived from the membrane-permeable nucleic acid staining dye by such pH adjustment (the presence / absence of luminescence derived from the membrane-impermeable nucleic acid staining dye is optionally included The number of specific viable microorganisms can be measured more accurately based on the combination.

  And according to the present Example, in the inspection method of the specific living microorganisms combining the magnetic beads and the fluorescence flow cytometry method, it becomes possible to reduce background noise derived from contaminating microorganisms and fluorescent dyes, and the number of specific living microorganisms Measurement can be performed more accurately.

(B) Example of overall configuration of microorganism testing apparatus
Below, the Example which applied this invention to the test | inspection method and test | inspection apparatus of microorganisms etc. by the fluorescence flow cytometry method using a microbe test | inspection cassette is described in detail. This embodiment is characterized in that a container for staining with a membrane-permeable nucleic acid staining dye is provided upstream of a container for staining a specific microorganism or the like with a fluorescently labeled antibody.

  In FIG. 4, the block diagram of the microorganisms testing apparatus 1 which concerns on one Example of this invention is shown. The microorganism testing apparatus 1 basically includes a microorganism testing cassette 10, a pressure supply device 14, an XY movable stage (holder) 125, a detection device 11, a system device 18, and an output device 19. ing.

  The microorganism testing cassette 10 includes a mechanism for holding a specimen and a reagent therein and performing a process necessary for microorganism measurement. The pressure supply device 14 controls the specimen and the conveyance in the microorganism testing cassette 10 via the chip connection tubes 1441 to 1444 connected to the microorganism testing cassette 10 in order to perform the steps necessary for the microorganism measurement. The XY movable stage (holder) 125 holds the microorganism testing cassette 10 and adjusts the position of the microorganism testing cassette 10. The detection device 11 irradiates microorganisms in the microorganism inspection cassette 10 with excitation light, and converts scattered light and fluorescence from the microorganisms into electrical signals. Details of these will be described later.

  The system device 18 connected to the microorganism testing device 1 executes signal processing on the output of the control signal to the pressure supply device 14 and the electric signal input from the detection device 11. The measurement result obtained by processing the electrical signal is displayed on the output device 19 connected to the system device 18.

  The pressure supply device 14 includes an air pump 141 with a regulator 1411. The air pump 141 and the vent holes 1591 to 1594 (FIG. 7) of the microorganism testing cassette 10 are connected by chip connecting tubes 1441 to 1444. Valves 1421 to 1424 are provided in the chip connection tubes 1441 to 1444, respectively. By opening and closing the valves 1421 to 1424, a gas having a predetermined pressure is supplied to the container of the microorganism testing cassette 10 or the container of the microorganism testing cassette 10 is opened to the atmosphere. By controlling this pressure, the transport of the specimen and the reagent in the microorganism testing cassette 10 is realized. The transportation of specimens and reagents in the microorganism testing cassette 10 by this pressure control is described in detail in Japanese Patent Application Laid-Open No. 2008-1557829.

  The microorganism testing cassette 10 basically includes a specimen container 151, a membrane permeation staining container 152, a labeled antibody staining container 155, a microorganism detection unit 17, a detection liquid disposal container 156, and a solution flow channel 1571 to 1574. (FIG. 7) and the flow path 1581-1584 (FIG. 7) for ventilation | gas_flowing. The sample container 151 holds the sample 1511. The membrane-permeable staining container 152 holds a membrane-permeable nucleic acid staining dye 1521 for staining microorganisms in the sample, and the sample and the membrane-permeable nucleic acid staining dye are mixed and reacted. The membrane permeation staining container 152 holds a membrane-impermeable nucleic acid staining dye for staining dead microorganisms in the specimen, if necessary. A permeable nucleic acid staining dye may be mixed and reacted. When using a membrane-impermeable nucleic acid staining dye, a membrane-impermeable staining container may be provided separately. The labeled antibody staining container 155 holds a fluorescently labeled antibody 1522 that binds only to a specific microorganism in the sample, and mixes and reacts the sample and the fluorescently labeled antibody. The microorganism detection unit 17 includes a microorganism detection channel 173 for irradiating the excitation light 113 from the excitation light sources 1111 and 1112 and observing the microorganism. In the detection liquid disposal container 156, the detection liquid that has passed through the microorganism detection flow path 173 is discarded. The solution flow channels 1571 to 1574 connect the sample container 151, the membrane permeation staining container 152, the labeled antibody staining container 155, and the microorganism detection flow channel 173, and the sample 1511 and the detection liquid flow. The ventilation channels 1581 to 1584 connect the pressure supply device 14 and each container in order to cause the specimen 1511 and the detection liquid to flow by atmospheric pressure.

  The detection device 11 includes excitation light sources 1111 and 1112, a scattered light detection unit, and fluorescence detection units 1201 to 1203. Among these, the scattered light detection unit includes the scattered light detector 123 for detecting the scattered light 124 from the microorganisms passing through the microorganism detection flow path 173 and the excitation light 113 from the excitation light source 112. It is comprised from the light-shielding plate 122 for preventing that it injects directly. On the other hand, the fluorescence detection unit condenses the fluorescence 121 from the microorganisms passing through the microorganism detection flow path 173 and reflects the excitation light 113 in the direction of the microorganism detection unit 17 while collimating the objective lens 114. The fluorescent light 121 reflects the transmitted dichroic mirror 112, condensing lenses 1181 and 1182 for condensing parallel light, a pinhole 119 used as a spatial filter for cutting stray light, and a partial wavelength of the fluorescent light 121. The dichroic mirrors 1151 and 1152 that transmit the remaining wavelengths, the bandpass filters 1171 to 1173 that pass the partial wavelengths of the fluorescence 121, and the photodetectors 1201 that detect the light that has passed through the bandpass filters 1171 to 1173 1203. Note that the irradiation unit and the detection unit are arranged so that their focal points overlap each other, and are configured so that the microorganism detection flow path 173 can be adjusted to the focal position during measurement.

  The detection device 11 irradiates the microorganism detection flow path 173 with the excitation light 113 output from the excitation light source 1112, and determines the amount of scattered light generated from the microorganism detection flow path 173 and the amount of fluorescence generated from the microorganism detection unit 17. By detecting, the relationship between the movable position of the XY stage 125 and each light quantity is acquired as a profile. Further, the detection device 11 controls the XY movable stage 125 based on the acquired profile, and adjusts the microorganism testing cassette 10 (specifically, the microorganism detection flow path 173) to a position suitable for detection. . That is, the position of the microorganism detection flow path where the amount of scattered light detected by the scattered light detection unit is maximized matches the optical axis of the excitation light, and the microorganism detection by which the fluorescence amount detected by the fluorescence detection unit is maximized The focus of the excitation light is made to coincide with the position of the flow path. This alignment is described in detail in Japanese Patent Application Laid-Open No. 2010-256278. For the alignment, another method, for example, a method described in Japanese Patent Application Laid-Open No. 2009-281753 may be used. In this case, the microorganism testing cassette is provided with a holding container such as an alignment reagent, a solution channel, a vent channel, and the like.

  In this embodiment, there are two excitation light sources, but the number of excitation light sources may be changed according to the type of fluorescent dye. In this embodiment, there are three fluorescence detection units, but the number of fluorescence detection units may be changed according to the type of fluorescent dye.

(C) Structure example of the microorganism detection part of the microorganism inspection cassette
The structure of the microorganism detecting unit 17 in the microorganism testing cassette 10 will be described with reference to FIGS. 5A and 5B. FIG. 5A shows a cross-sectional view of a joint portion between the main body 15 of the microorganism test cassette 10 and the microorganism detection unit 17. FIG. 5B shows an exploded perspective view of the microorganism detection unit 17.

In addition, the main body 15 and the microorganisms detection part 17 are each produced by a separate process, and each is joined. First, the manufacturing method of the microorganism detection part 17 is demonstrated. The microorganism detection unit 17 includes a cover member 171 and a flow path member 172, both of which are formed of a thin flat plate. A groove 1731 is formed in the flow path member 172, and through holes 1741 and 1751 are formed at both ends of the groove 1731. The cover member 171 and the flow path member 172 are bonded together so that the surface on which the groove 1731 is formed becomes the bonded surface. The microorganism detection part 17 is formed by this bonding. A microbe detection flow path 173 is configured by the groove 1731 of the flow path member 172 and the cover member 171. The through holes 1741 and 1751 of the flow path member 172 constitute a microorganism detection flow path inlet 174 and a microorganism detection flow path outlet 175.

  On the other hand, the flow path 1573 between the labeled antibody staining container and the microorganism detection flow path formed in the main body 15 changes the flow path direction at the lower end to form an opening on the surface of the main body 15. Similarly, the flow path 1574 between the microorganism detection flow path and the detection liquid disposal container changes the flow path direction at the upper end to form an opening on the surface of the main body 15. The opening of the labeled antibody staining container-microorganism detection flow path 1573 is connected to the microbe detection flow path inlet 174, and the opening of the microbe detection flow path-detection liquid disposal container flow path 1574 is for microbe detection. It is connected to the channel outlet 175.

  A detection window frame 161 is formed on the main body 15. The detection window frame portion 161 is a through hole or a through groove. The detection window frame 161 is formed between the opening of the flow path 1573 between the labeled antibody staining container and the microorganism detection flow path and the opening of the flow path 1574 between the flow path for microorganism detection and the detection liquid disposal container. The manufactured microorganism detection unit 17 is attached to the main body 15 as described above. As shown in FIG. 5A, the microorganism detection unit 17 is mounted such that the microorganism detection flow path 173 is disposed on the detection window frame 161 of the main body 15.

  In the case of the present embodiment, a detection window frame portion 161 that is a through hole or a through groove of the main body 15 is provided behind the microorganism detection flow path 173. Therefore, the excitation light 113 irradiates only the microorganism detection unit 17 and does not irradiate the main body 15. For this reason, the reflected light from the main body 15 and autofluorescence that cause an increase in background light are not generated. In order that the excitation light 113 that has passed through the microorganism detection flow path 173 is not irradiated to the main body 15, the cross-section of the through-hole constituting the detection window frame portion 161 increases along the radiation direction of the excitation light 113. Is preferred.

  The thickness of the cover member 171 is, for example, 0.01 μm to 1 mm. The thickness of the flow path member 172 is, for example, 0.01 μm to 1 mm. The cross-sectional shape of the microorganism detection flow path 173 is formed in, for example, a square, a rectangle, or a trapezoid. The larger the cross-sectional dimension of the microorganism detection channel 173 is, the smaller the pressure loss is. However, it is preferable that the microorganism is flowed one by one. One side of the cross section of the microorganism detection flow path 173 is preferably 1 μm to 1 mm, for example, and the length is preferably 0.01 mm to 10 mm, for example. The optical axis of the excitation light 113 applied to the microorganism detection channel 173 is perpendicular to the direction vector of the microorganism detection channel 173.

  The material which comprises the microorganisms detection part 17 is demonstrated. The microorganism testing cassette 10 is disposable. That is, after use, the microorganism detection unit 17 is discarded together with the main body 15. Therefore, the material used for the microorganism detection unit 17 must be inexpensive. The material used for the microorganism detection unit 17 needs to be excellent in optical characteristics so as to be suitable for fluorescence measurement. That is, it is desirable that the autofluorescence is low and the light transmittance, surface accuracy, refractive index and the like are excellent. In order not to inhibit detection of fluorescence from microorganisms, it is preferable that the amount of autofluorescence generated by the microorganism detection unit 17 itself be sufficiently smaller than the amount of fluorescence from microorganisms.

  If a curved surface, unevenness, or the like exists on the surface of the microorganism detection unit 17, the light amount of the excitation light 113 irradiated on the microorganism measurement flow path 173 varies due to light refraction or irregular reflection on the surface. For this reason, the amount of fluorescence detected also fluctuates, and the measurement accuracy decreases. Therefore, the surface of the microorganism detection unit 17 needs to have a desired flatness. The microorganism detecting unit 17 preferably has a flatness with an unevenness of 0.1 mm or less.

  Considering such conditions, the materials used for the microorganism detection unit 17 include glass, quartz, polymethacrylic acid methyl ester (PMMA), polydimethylsiloxane (PDMS), cycloolefin polymer (COP), polyethylene terephthalate, and polycarbonate. Etc. are considered. The microorganism detection unit 17 is formed of one or more kinds of substances selected from these substances.

  The cover member 171 is a simple flat plate, but the flow path member 172 is formed by forming a groove and a through hole in the flat plate. Therefore, the flow path member 172 is formed of a material that is easy to be finely processed and that has a low processing cost. Glass and quartz have excellent optical properties but are not easily processed finely. That is, processing costs increase when microfabrication is performed.

  Therefore, in the case of this embodiment, the cover member 171 is made of glass or quartz, and the flow path member 172 is made of polymethacrylic acid methyl ester, polydimethylsiloxane, cycloolefin polymer, polyethylene terephthalate, and polycarbonate. Preferably, the flow path member 172 is made of polydimethylsiloxane. In this case, the self-adhesiveness of polydimethylsiloxane is used for joining the cover member 171 and the flow path member 172.

  The amount of autofluorescence of the microorganism detection unit 17 depends not only on the material but also on the thickness dimension of the microorganism detection unit. In order to reduce the amount of autofluorescence, the thickness dimension of the microorganism detection unit may be reduced. The smaller the thickness of the microorganism detection unit 17, the smaller the amount of autofluorescence generated from the microorganism detection unit 17. However, if the thickness dimensions of the cover member 171 and the flow path member 172 are reduced, manufacturing becomes difficult and flatness deteriorates. In order to maintain the necessary flatness and suppress the amount of autofluorescence so as not to hinder the detection of fluorescence of microorganisms, the thickness dimensions of these parts must be limited to a predetermined range.

  The autofluorescence amounts of glass, quartz and polydimethylsiloxane are almost the same. Therefore, when the cover member 171 is made of glass or quartz, the thickness is preferably 0.05 mm or more and 1 mm or less, for example. When the flow path member 172 is manufactured from polydimethylsiloxane, the thickness is preferably 0.1 mm or more and 1 mm or less, for example.

  Further, the cover member 171 and the flow path member 172 may be made of cycloolefin polymer, polymethacrylic acid methyl ester, polyethylene terephthalate, or polycarbonate. In this case, the amount of autofluorescence per unit volume is increased about three times or more than when the cover member 171 is manufactured from glass or quartz and the flow path member 172 is manufactured from polydimethylsiloxane. Therefore, the thickness of the cover member 171 and the flow path member 172 is preferably 0.01 mm or more and 0.3 mm or less, for example.

(D) Measurement method (inspection method) example
Hereinafter, the Example in the case of measuring the number of specific living microorganisms (for example, colon_bacillus | E._coli O-157 etc.) in the sample derived from a foodstuff using the above-mentioned microbe inspection apparatus 1 is described. FIG. 6 is a flowchart showing the process of measuring the number of specific living microorganisms using the microorganism testing cassette 10. FIG. 7 shows a plan view of the microorganism testing cassette 10.

  As described above, the microorganism testing cassette 10 includes the testing container 151, the membrane permeation staining container 152, the labeled antibody staining container 155, the microorganism detection unit 17, the detection liquid disposal container 156, and the solution flow channels 1571 to 1574. And a flow path 1581 to 1584 for ventilation. Further, as shown in FIG. 7, a food residue removal unit 160 that is a filter for removing food residues contained in the sample is provided downstream of the sample container 151. In addition, vents 1591 to 1594 that connect the pressure supply device 14 and the respective flow channels 1581 to 1584 are provided.

  The flow path 1571 to 1574 for solution, the vents 1591 to 1594, and the flow paths 1581 to 1584 for ventilation are connected from the name of the container to be connected, the flow path 1571 between the specimen container and the membrane permeation staining container, the membrane permeation staining container and the labeled antibody staining container. Interchannel 1572, Labeled antibody staining container-microorganism detection flow path 1573, Microbe detection flow path-Detection liquid waste container flow path 1574, Sample container vent 1591, Membrane permeation staining container vent 1592, Label An antibody staining container vent 1593, a detection liquid disposal container vent 1594, a specimen container vent flow path 1581, a membrane permeation staining container vent flow path 1582, a labeled antibody staining container vent flow path 1583, and a detection liquid waste container vent flow path 1584. .

  The sample container 1511, the food residue removing unit 160, the membrane permeation staining container 152, the labeled antibody staining container 155, the microorganism detection flow path 173, and the detection liquid disposal container 156 are connected in series by a solution flow path 1571 to 1574. It is connected to.

  First, sterilized water with a mass ratio of 9 times is added to the food to be inspected, magnetic beads are added to the specimen prepared by the stomaching process, and the specific microorganisms are adsorbed on the magnetic beads, so that only the specific microorganisms are concentrated and separated. (S900). The concentration and separation of microorganisms using magnetic beads is a known technique as described in Patent Document 1 and will not be described in detail. There is also an automatic machine that automatically performs concentration using magnetic beads. In order to reduce the influence of food residues, it is preferable to concentrate and separate specific microorganisms using magnetic beads as a pretreatment step of the microorganism testing apparatus 1.

  Next, the specimen 1511 concentrated and separated with magnetic beads is injected into the specimen container 151 from the vent 1591 before the examination (S901).

  The volume of the sample container 151 is larger than the volume of the sample 1511. The volume of the membrane permeation staining container 152 is larger than the total volume of the specimen 1511 and the membrane permeable nucleic acid staining dye 1521. The volume of the labeled antibody staining container 155 is larger than the total volume of the specimen 1511, the membrane-permeable nucleic acid staining dye 1521, and the fluorescently labeled antibody 1522. Further, the highest point of the flow path 1571 between the specimen container and the membrane permeation staining container is formed to be higher than the water level of the specimen 1511 in the specimen container 151. Similarly, the highest point of the membrane permeation staining container-labeled antibody staining container flow path 1572 is formed so as to be higher than the water level of the mixed solution of the specimen 1511 and the membrane permeation nucleic acid staining dye 1521. Furthermore, the highest point of the flow path 1573 between the labeled antibody staining container and the microorganism detection flow path is formed to be higher than the water level of the detection liquid consisting of the specimen 1511, the membrane-permeable nucleic acid staining dye 1521, and the fluorescently labeled antibody 1522. The

  In this embodiment, the membrane-permeable staining container 152 also holds a membrane-impermeable nucleic acid staining dye so that it can be discriminated between dead and live bacteria. That is, in this embodiment, the membrane-permeable staining container 152 contains at least one membrane-impermeable nucleic acid staining dye (dead microorganism dye for staining dead microorganisms) and a membrane-permeable nucleic acid staining dye (total microorganisms). 1 or more mixed liquids are held. Examples of the dead microorganism dye include cyanine fluorescent dye for dead microorganism (concentration: 0.01 μM to 10 μM), PI (propidium iodide) (concentration: 0.1 mg / ml to 20 mg / ml), EB (ethidium). Iodide) (concentration: 0.1 mg / ml to 20 mg / ml), and for example, cyanine-based fluorescent dye for all microorganisms (concentration: 0.01 μM to 10 μM), LDS751 (concentration: 0) .1 mg / ml to 20 mg / ml), DAPI (4 ′, 6-diamidine-2′-phenylindole) (concentration: 0.1 mg / ml to 20 mg / ml), HOECHST33258 (concentration: 0.1 mg / ml to 20 mg) / ml), HOECHST 34580 (concentration: 0.1 mg / ml to 20 mg / ml) or the like. Here, a cyanine orange fluorescent dye for dead microorganisms is used as a dead microorganism dye, and LDS751 is used as a total microorganism dye.

  The fluorescently labeled antibody is obtained by labeling the above-mentioned all microbial dyes with respect to a monoclonal antibody or polyclonal antibody that specifically binds to an antigen of a target specific microorganism, and is not particularly limited. However, those different from the total microbial dyes held in the membrane permeation staining container are used. Here, a cyanine blue fluorescent dye for all microorganisms is used.

  DMSO (dimethyl sulfoxide), ethanols, water and the like are used as the dye solvent. The unit of concentration M means mol / L.

  As shown in FIG. 6, the measurement of the number of living microorganisms using the microorganism testing cassette 10 is started with the microorganism testing cassette 10 set in the microorganism testing apparatus (analyzer) 1 (S902). This measuring step includes an alignment step (S907) for aligning the microorganism testing cassette 10 and a pretreatment step for adding a membrane-permeable nucleic acid staining dye and a fluorescently labeled antibody to specific live microorganisms in the specimen for staining (see FIG. S903 to S906) and a measurement step (S908) for actually measuring the number of specific live microorganisms.

  Since the alignment step (S907) and the pretreatment steps (S903 to S906) are independent steps, they are performed in parallel, and the measurement step (S908) is performed when both steps are completed. Subsequently, the movement of each liquid in each step will be described.

  In the pretreatment step, first, the specimen 1511 is moved to the membrane permeation staining container 152 (S903). The pressure of the pressure supply device 14 is applied to the sample container 151 through the vent 1591. Thereby, the atmospheric pressure in the sample container 151 is increased. At the same time, the internal pressure of the membrane permeation dyeing container 152 is released to atmospheric pressure through the membrane permeation dyeing vent 1592. Due to the pressure difference, the specimen 1511 enters the membrane-permeable staining container 152 and is mixed with a membrane-permeable nucleic acid staining dye 1521 (in this embodiment, a mixed solution containing a membrane-impermeable nucleic acid staining dye). Bubbling is used for mixing (S904). Dead microorganisms in the specimen 1511 are stained with a dead microorganism dye (here, a cyanine orange fluorescent dye for dead microorganisms) and a whole microorganism dye (here, LDS751 is used), while the dead microorganisms in the specimen 1511. Viable microorganisms are stained with total microbial pigments only.

  When the sample 1511 flows through the food residue removing unit 160 to the membrane permeation staining container 152, the food residue in the sample 1511 is removed from the sample 1511 by the food residue removing unit 160.

  The water level of the mixture of the specimen 1511 and the membrane-permeable nucleic acid staining dye 1521 exceeds the highest point of the membrane-permeation staining container-labeled antibody staining container flow path 1572 connecting the membrane permeation staining container 152 and the labeled antibody staining container 155. Furthermore, the air contained in the membrane permeation dyeing container 152 is released to the outside through the membrane permeation dyeing container vent 1592. Since the pressure in the membrane permeation staining container 152 is equal to the atmospheric pressure, the mixed solution is not pushed out to the labeled antibody staining container 155, and the mixture can be held in the membrane permeation staining container 152 for a necessary time. The holding time is about 10 to 30 minutes.

  At this time, in order to prevent the mixed solution from flowing into the labeled antibody staining container 155, the pressure from the pressure supply device 14 is applied to each of the vents 1593 to 1594, and the labeled antibody staining is performed to a range lower than the atmospheric pressure of the specimen container 151. The air pressure in the container 155 and the detection liquid waste container 156 may be increased.

  Next, the mixed solution composed of the specimen 1511 and the membrane-permeable nucleic acid staining dye 1521 is moved to the labeled antibody staining container 155 (S905). The pressure of the pressure supply device 14 is applied to the membrane permeation dyeing container 152 through the vent 1592. Thereby, the atmospheric pressure in the membrane permeation staining container 152 is increased. At the same time, the internal pressure of the labeled antibody staining container 155 is released to atmospheric pressure through the vent 1593. Due to the pressure difference, the mixed solution enters the labeled antibody staining container 155 and is mixed with the fluorescently labeled antibody 1522. Bubbling is used for mixing (S906). Only specific microorganisms in the specimen 1511 are stained with a fluorescently labeled antibody (here, cyanine blue fluorescent dye for all microorganisms is used). The staining time is, for example, 5 to 20 minutes. During dyeing, it is desirable to keep the temperature of the microorganism testing cassette 10 constant to reduce the influence of dyeing due to temperature changes.

  In parallel with the above-described pretreatment process, alignment of the microorganism testing cassette 10 is executed (S907).

  When the above operation is completed, the detection liquid consisting of the specimen 1511, the membrane-permeable nucleic acid staining dye 1521, and the fluorescence-labeled antibody 1522 is moved to the microorganism detection flow path 173, and the specific live microorganism in the specimen is subjected to the fluorescence flow cytometry method. It is measured by. (S908). In the case of the inspection apparatus shown in FIG. 4, the excitation light 113 is irradiated from the direction perpendicular to the paper surface. For this reason, the fluorescence from the pigment | dye which dye | stained microorganisms and the scattered light by microorganisms arise. As described with reference to FIG. 2, the specific living microorganisms emit two colors of fluorescence, one of which is a fluorescently labeled antibody, and the other one of which is a dead microorganism, one of which is fluorescent. It emits fluorescence of two colors (labeled dye of labeled antibody) and one color of dead microorganism dye. The detection device 11 counts the two microorganisms that emit fluorescence of two colors, one of which is a fluorescent dye-labeled antibody, as a specific living microorganism, and the total microorganism dye (one is a fluorescent-labeled antibody-labeled dye). Those that emit fluorescence of two colors and one dead microorganism dye are counted as specific dead microorganisms. For this reason, it becomes possible to discriminate and count specific live microorganisms and specific dead microorganisms. In addition, since the amount of scattered light varies depending on the size of the microorganism, it is possible to determine the size of the microorganism.

  FIG. 8 shows a diagram of a microorganism testing cassette that holds an oxidizing agent and a staining solution in a container by a method different from that shown in FIG. The configurations of the internal containers and flow paths are the same as those in FIG. A glass tube 1521G held by a holder 1521H is disposed in the membrane permeation staining container 152, and a membrane permeable nucleic acid staining dye (in the case of measuring dead bacteria and viable bacteria for measurement) Liquid mixture with a natural nucleic acid staining dye) is held by capillary action in a glass tube. The assembly order is completed by holding the glass tube 1521G in the holder 1521H, filling the glass tube 1521G with a membrane-permeable nucleic acid staining dye, and inserting the holder 1521H into the membrane-permeable staining container 152 of the cassette. By arranging the upper end position of the glass tube 1521G below the water level of the sample liquid in the membrane permeation staining container 152, the glass tube is located below the water surface of the sample liquid when the sample liquid flows into the membrane permeation staining container 152. As a result, the capillary phenomenon in the glass tube is lost, and the membrane-permeable nucleic acid staining dye in the glass tube leaks into the specimen. Membrane-permeable nucleic acid staining dye that has oozed into the sample liquid is mixed by bubbling to stain the bacteria in the sample liquid. Thus, by arranging the glass tube below the water level of the sample liquid, the membrane-permeable nucleic acid staining dye in the glass tube can be easily exuded into the sample.

  Further, a glass tube 1522G held by a holder 1522H is arranged in the labeled antibody staining container 155, and the fluorescently labeled antibody is held in the glass tube 1522G by capillary action in the glass tube. The assembly order is completed by holding the glass tube 1522G in the holder 1522H, filling the glass tube 1522G with the fluorescently labeled antibody, and inserting the holder 1522H into the labeled antibody staining container 155 of the cassette. The upper end position of the glass tube 152G is placed below the water level of the specimen liquid (mixed liquid) in the labeled antibody staining container 155, so that the mixed liquid of the specimen liquid and the membrane-permeable nucleic acid staining dye is placed in the labeled antibody staining container 155. When flowing in, the glass tube is positioned below the water surface of the mixed solution, the capillary phenomenon in the glass tube is lost, and the fluorescently labeled antibody in the glass tube oozes out into the sample solution (mixed solution). The fluorescently labeled antibodies that have oozed into the mixed solution are mixed by bubbling and bind to specific microorganisms in the specimen. Thus, by arranging the glass tube below the water level of the mixed solution, the fluorescently labeled antibody in the glass tube can be easily leached into the mixed solution.

  Although the glass tube has an inner diameter of 0.6 mm and a length of 8 mm, the same effect can be obtained even when the inner diameter is 0.1 to 3 mm and the length is 1 to 30 mm.

  In this example, the membrane-permeable nucleic acid staining dye and the fluorescently labeled antibody are included in the microorganism testing cassette when the microorganism testing cassette is assembled, but the membrane-permeable nucleic acid staining dye and the fluorescently labeled antibody are included in the microorganism testing cassette. If there is a place to enclose, the membrane-permeable nucleic acid staining dye and fluorescently labeled antibody are not included when assembling the microorganism test cassette, and the membrane-permeable nucleic acid staining dye and fluorescently labeled antibody are manually or automatically The same effect as in the present embodiment can be obtained even if filling with.

  In this example, a membrane-permeable nucleic acid staining dye and a fluorescently labeled antibody are held in a glass tube, but the same effect can be obtained with a hydrophilic sponge structure instead of a glass tube. In particular, a glass sintered body or a melamine sponge is suitable.

  In the above-described embodiments, the fluorescence flow cytometry method is used as the fluorescence cytometry method, but the present invention can also be applied to the fluorescence imaging cytometry method. In the fluorescence imaging cytometry method, for example, a membrane-permeable nucleic acid staining dye (for membrane and impermeable nucleic acid staining dye in the case of measuring a dead cell and a living cell) is measured on a sample that has been concentrated and separated by magnetic beads. And a fluorescence-labeled antibody is added and held for a predetermined time (for example, 30 minutes) to prepare a detection liquid. Microorganisms are detected by holding the detection liquid in a filter and irradiating it with excitation light, acquiring images of the fluorescence emission of the microorganisms, and counting the light spots. Also in the fluorescence imaging cytometry method, the background noise derived from the fluorescent dye can be reduced and the number of specific living microorganisms can be measured more accurately.

  In addition, this invention is not limited to an above-described Example, Various modifications are included. For example, the above-described embodiments have been described in detail for easy understanding of the present invention, and are not necessarily limited to those having all the configurations described. Further, a part of the configuration of one embodiment can be replaced with the configuration of another embodiment, and the configuration of another embodiment can be added to the configuration of one embodiment. Moreover, it is possible to add, delete, and replace other configurations for a part of the configuration of each embodiment.

  For example, in the case of a microorganism testing cassette, if a specimen, a membrane-permeable nucleic acid staining dye and a fluorescently labeled antibody are mixed before injecting the specimen into the specimen container 151, the membrane-permeable staining container 152 or the labeled antibody staining container 155 is used. The associated solution flow path and the like can be deleted.

  DESCRIPTION OF SYMBOLS 1 ... Microbe test | inspection apparatus, 10 ... Microbe test | inspection cassette, 11 ... Detection apparatus, 14 ... Pressure supply apparatus, 17 ... Microbe detection part, 18 ... System apparatus, 19 ... Output device, 1111 ... Short wavelength excitation light source, 1112 ... Long wavelength Excitation light source, 1113 ... dichroic mirror for excitation light synthesis, 112 ... dichroic mirror, 113 ... excitation light, 114 ... objective lens, 119 ... pinhole, 120 ... photodetector, 121 ... fluorescence, 124 ... scattered light, 125 ... X- Y movable stage, 141 ... air pump, 1411 ... regulator, 151 ... specimen container, 1511 ... specimen, 152 ... membrane permeation staining container, 1521 ... membrane-permeable nucleic acid staining dye, 1521H ... holder, 1521G ... glass tube, 155 ... label Antibody staining container, 1522 ... fluorescently labeled antibody, 1522H ... holder, 1522G ... glass DESCRIPTION OF SYMBOLS 156 ... Detection liquid disposal container, 1571-1574 ... Solution flow path, 1581-1584 ... Ventilation flow path, 161 ... Detection window part, 173 ... Microorganism detection flow path, 1151 ... Short wavelength fluorescence separation dichroic mirror 1152 ... dichroic mirror for medium wavelength fluorescence separation, 1171 ... short wavelength band pass filter, 1172 ... medium wavelength band pass filter, 1173 ... long wavelength band pass filter, 1181, 1182 ... condensing lens, 1201 ... light detection for short wavelength 1202... Medium wavelength photodetector, 1203... Long wavelength photodetector.

Claims (10)

  A membrane-permeable nucleic acid staining dye and a fluorescently labeled antibody that binds to an antigen such as a target microorganism to be examined are added to the sample, and fluorescence and the fluorescence by the membrane-permeable nucleic acid staining dye are added using a fluorescence cytometry method. A method for inspecting microorganisms or the like, which comprises counting, as the target microorganisms or the like, those that emit both fluorescence from a labeled dye of a labeled antibody. In claim 1,
Further, a membrane-impermeable nucleic acid staining dye is added to the sample, and the target microorganism or the like emits two fluorescences, fluorescence by the membrane-permeable nucleic acid staining dye and fluorescence by the fluorescent dye-labeled dye. A method for inspecting microorganisms and the like characterized by counting as live microorganisms. In claim 1 or 2,
The sample is obtained by concentrating the target microorganism using magnetic beads, and the fluorescence cytometry method is a flow cytometry method. In claim 1,
A method for testing microorganisms or the like, characterized by adding the membrane-permeable nucleic acid staining dye before adding the fluorescently labeled antibody. In claim 2,
A method for testing microorganisms or the like, comprising adding the membrane-permeable nucleic acid staining dye and the membrane-impermeable nucleic acid staining dye before adding the fluorescently labeled antibody. In claim 3,
A method for testing microorganisms or the like, characterized by adding the membrane-permeable nucleic acid staining dye before adding the fluorescently labeled antibody. In claim 6,
A method for testing microorganisms or the like, wherein the pH is 7.0 to 7.6 when the membrane-permeable nucleic acid staining dye is added. A sample container for holding a sample liquid, a membrane permeation staining container for holding a membrane-permeable nucleic acid staining dye, a labeled antibody staining container for holding a fluorescently labeled antibody that binds to an antigen of a target microorganism, a microorganism detection flow path, The specimen container and the membrane permeation staining container, the membrane permeation staining container and the labeled antibody staining container, and a microorganism testing cassette comprising a flow path for connecting the labeled antibody staining container and the microorganism detection flow path,
A stage on which the microbial test cassette is mounted and moves the microbial test cassette;
Connected to the microorganism test cassette, transports the sample liquid from the sample container to the membrane permeation staining container, transports the mixed solution from the membrane permeation staining container to the labeled antibody staining container, and mixes from the labeled antibody staining container A pressure supply device for supplying a pressure for conveying the liquid as a detection liquid to the microorganism detection flow path;
A light source that irradiates the microorganism detection flow path with excitation light, and a detection device that includes a light detector that detects fluorescence from the detection liquid flowing through the microorganism detection flow path and converts the fluorescence into an electrical signal. Testing equipment for microorganisms. A microorganism testing cassette used in a testing apparatus such as a microorganism that detects microorganisms contained in the sample liquid as an electrical signal by irradiating excitation light to the sample liquid mixed with a fluorescent dye to detect fluorescence,
A sample container for holding the sample liquid;
A membrane-permeable staining container holding a membrane-permeable nucleic acid staining dye; and
A labeled antibody staining container holding a fluorescently labeled antibody that binds to an antigen of the target microorganism;
A microorganism-detecting channel irradiated with the excitation light;
The specimen container and the membrane permeation staining container, the membrane permeation staining container and the labeled antibody staining container, and the flow path connecting the labeled antibody staining container and the microorganism detection flow path, respectively. Microorganism test cassette. In claim 9,
A microorganism testing cassette, wherein the membrane-permeable nucleic acid staining dye and the fluorescently labeled antibody are held in glass tubes provided in the membrane-permeable staining container and the labeled antibody staining container, respectively.

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