JP6037445B2 - Fluorescence measuring apparatus and fluorescence measuring method - Google Patents

Description

  The present invention relates to a fluorescence measuring apparatus and a fluorescence measuring method using the same and particularly suitable for PCR.

  The PCR (Polymerase Chain Reaction) method is known as a method for proliferating a target DNA region in a short time from a very small amount of DNA. The principle uses two known sequences on double-stranded DNA to design two primer DNAs that face each other in the synthesis direction. When the DNA in the region sandwiched between these primers is synthesized, it becomes a template in the next cycle. By repeating such a reaction, the DNA strand can be amplified geometrically.

By using such a PCR method, virus diagnosis in serum can be performed. For example, in testing for HBV (hepatitis B virus), the concentration of how much HBV is present in the serum can be measured. In this case, the sensitivity at a virus copy number of 10 ² to 10 ⁶ in 1 ml of serum is particularly important. In order to measure this, a method is used in which a fluorescent primer is attached to the DNA of HBV virus, and the increase or decrease in fluorescence is measured by amplifying the DNA by the PCR method.

  First, the virus is lysed and extracted from the serum, and the HVB DNA is taken out therefrom. Next, specific DNA in HBV is amplified. For this purpose, primers suitable for HBV-DNA are designed to amplify HBV-DNA. In particular, when the concentration of HBV-DNA is low, it cannot be measured unless it is increased. At this time, a specific fluorescence is imparted to the primer, and this fluorescence is “proportionally increased or attenuated” with an increase in HBV-DNA, whereby the concentration of HBV-DNA can be measured. The increase in HBV-DNA depends on the PCR method, but theoretically doubles at one time. When an appropriate primer is designed for HBV-DNA, any type of subtypes A to H can detect HBV-DNA because it is amplified or attenuated after about 40 PCRs.

For example, performing the HBV-DNA 40 rounds of PCR, the initial becomes ^{2 40} times that, which is substantially equal to ^{10 12} times (a trillion times). That is, performing PCR 40 times increases the original HBV-DNA by a trillion times. Therefore, if PCR is performed 40 times, even if it is a diluted nucleic acid of 100 copies / ml, the copy number becomes 100 × 10 ¹² / ml. Therefore, fluorescence increase / decrease is detected by applying fluorescence to the primer. It becomes possible. For example, as the fluorescence imparted to the primer, N, N′-bis-3-aminopropyl-2,7-diamino-1,8-naphthyridine so-called DANP or the like can be used.

  In the three-step method, the PCR apparatus applies a primer to single-stranded DNA (= nucleic acid) at 53 to 56 ° C., amplifies it at 60 to 70 ° C., and at 95 ° C. Decompose into main chains and repeat this. In the two-step method, primer application and amplification are performed at the same temperature (60 to 65 ° C.), so that it can be performed in a shorter time than the three-step method.

  As a real-time PCR method using such a fluorescent label, there are an interaction method, a hybridization method, and a hairpin primer PCR method. It is known that the hybridization method has a lower probability of non-specific amplification than the intercalation method, and the amplification efficiency of the target gene chain is higher.

  FIG. 6 is a conceptual diagram of the hairpin primer PCR method. In this method, as shown in FIG. 6A, a hairpin primer 103 in which a DNA sequence forming a hairpin structure is added to the end of the primer is used. A cytosine bulge region to which a small molecule specifically binds is introduced into the hairpin region. Prior to PCR, a low molecular weight compound that emits fluorescence when bound to cytosine bulge is bound.

  As shown in FIG. 6B, when the synthesis of the DNA strand by the polymerase 107 proceeds and the hairpin structure of the hairpin primer 103 is opened by PCR, the cytosine bulge structure disappears. For this reason, the fluorescent dye 105 is liberated. Therefore, the fluorescence intensity decreases. That is, the progress of PCR can be grasped by detecting the decrease in the fluorescence intensity of the reaction system.

  FIG. 7 is a schematic view showing the vicinity of a well for measuring fluorescence by such real-time PCR. First, the reaction solution 113 including the various components described above is placed in the well 109. In this state, light is irradiated from above by the irradiation / light receiving unit 111 (in the direction of arrow E in the figure). The irradiation / light receiving unit 111 is, for example, an optical fiber, and is connected to a light source and a fluorescence measuring device (not shown).

  From the reaction solution 113 irradiated with light, fluorescence corresponding to the progress of PCR is generated (disappears). A part of the fluorescence generated from the reaction solution 113 is received by the irradiation / light receiving unit 111, and the fluorescence is measured.

  In Patent Document 1, excitation light is guided to an optical fiber from a lamp 14 that generates excitation light via an optical demultiplexer 5, and in the microchannel structure 2 via a movable mirror 32 and optical lenses 22 and 24. The fluorescent material 7 disposed in the vicinity of the flow path 3 receives excitation light to generate fluorescence, and the fluorescent light generated by the fluorescent material is reflected by the movable mirror 32 and is passed through the optical demultiplexer 5 to the light detection unit 8. A fluorescence measuring apparatus having an optical switch for switching an optical path between a light source and a light detection unit and a light emitter is disclosed as a measuring apparatus for guiding and measuring fluorescence intensity. It is described that the movable mirror 32 is disposed directly above the optical path forming portion of the base member 11.

  In Patent Document 2, excitation light emitted from an excitation light source passes through a dichroic mirror, and is obliquely reflected by a mirror disposed obliquely upward onto a spot of a fluorescently labeled array existing on an ultrathin substrate. From above, it passes through the mirror 116 arranged at an angle θ. The emitted fluorescence is collected by a lens 127, passed through a filter 128, and from there through a computer controlled 132 CCD camera aperture 126 is disclosed.

  In Patent Document 3, the sample plate 10 is configured to be in close contact with the heating / cooling block 3 disposed below the sample plate 10, and excitation light is transmitted from the diagonally upper side of the sample plate 10 to the beam splitter 6 and the beam direction. The sample is passed through the change mirror 7 and the Fresnel lens 8 to irradiate the sample plate 10, and the fluorescence generated from the sample plate 10 is passed through the beam direction change mirror 7, the beam splitter 6, the radiation filter 11, and the lens 6. Fluorescence measurement to be incident on a CCD camera 13 provided obliquely above is described.

  Various apparatuses for performing such fluorescence measurement have been developed. For example, Patent Document 1 irradiates excitation light from an optical fiber toward the fluorescent material of the microchannel structure and emits fluorescence from the fluorescent material. This is a fluorescence measuring apparatus having an optical switch that switches and uses a movable mirror on a base member when reflecting toward a fiber. In this case, the optical switch is disposed on the side or top of the microchannel structure.

  In Patent Document 2, the excitation light emitted from the excitation light source is irradiated from an obliquely upper side to the spot of the fluorescently labeled array on the ultrathin substrate through an optical system such as a dichroic mirror, and conversely The fluorescence emitted from the spot passes through a lens and a filter, and from there, passes through an opening of a CCD camera disposed on an ultrathin substrate.

  In Patent Document 3, the sample plate is configured so as to be in close contact with a heating / cooling block disposed on the lower side of the sample plate, and excitation light is transmitted obliquely from above the sample plate through various optical components. Is a fluorescence measurement device that causes fluorescence generated from the sample plate to enter a CCD camera provided obliquely above the sample plate.

  As described above, in any patent document, excitation light is irradiated from above the sample plate or microarray, and fluorescence generated from the well or array on the sample plate is received above the sample plate or array. is there. This is because a heating / cooling block, or a heating / cooling component such as a Peltier element or a fan, for example, is usually disposed below the sample plate, regardless of whether or not shown.

  FIG. 8 is a schematic view of a conventional fluorescence measuring apparatus generally used. A plurality of wells 109 arranged in a well holding part 119 made of, for example, an aluminum block are used in the upper part of the fluorescence measuring instrument. A Peltier heating unit 115 is provided below the well holding unit 119. The temperature of the reaction solution in the well 109 can be raised by the Peltier heating unit 115. Further, a cooling unit 117 is provided below the Peltier heating unit 115. The cooling unit 117 has a built-in cooling fan, for example, and can lower the temperature of the reaction solution in the well 109 via the Peltier heating unit 115.

JP 2003-247893 A JP 2005-535905 A Special table 2003-514223 gazette

  However, in the conventional measurement method, as shown in FIG. 8, if a hole is formed in the Peltier element of the Peltier heating unit 115, the Peltier element is damaged, and it is difficult to provide a hole in the Peltier heating unit. Further, since the fan rotates in the cooling unit 117, the well 109 cannot be inserted up to the cooling unit 117. For this reason, a light source and a measuring instrument could not be arranged below the well 109.

  As described above, in the conventional measurement method, since the laser beam is irradiated from the upper part of the well and the fluorescence is measured from the upper part of the well, it is necessary to take a large space in the upper part of the apparatus, and the measurement unit is large. In addition, in a measuring apparatus using an optical fiber, since the fluorescence intensity taken into the optical fiber is insufficient, in many cases, it is necessary to use a lens system for condensing light. Since the fluorescence spreads evenly over the entire space above the well, there is a large amount of fluorescence that cannot be collected in the lens, and there is a problem that the fluorescence intensity decreases.

  In addition, in the case of a measuring apparatus in which various optical components are arranged above a well, the number of components is large, the structure is complicated, and fluorescence cannot be directly captured from the well, so that fluorescence is captured in the optical system. However, there is a problem that the fluorescence intensity is lowered.

  The present invention has been made in view of such problems, and an object of the present invention is to provide a fluorescence measuring apparatus and the like that have a compact and simple structure and at the same time have high fluorescence collection efficiency and a short measurement time.

  In order to achieve the above-mentioned object, the first invention is a fluorescence measuring apparatus, which is provided separately from a plurality of wells for holding a reaction solution, a lid provided on the top of the well, and the well. The temperature of the reaction solution can be adjusted by contacting the holding unit as a heat receiving block that holds the wells in parallel, a plurality of waveguides directly connected to the wells below the well, and the holding unit. A temperature controller, a light source that irradiates the reaction solution in the well via the waveguide with excitation light, and a fluorescence measuring instrument that measures fluorescence generated in the well, and the holding unit includes Provides a through-hole so as to expose and hold the bottom surface of the well, and allows excitation light to enter the waveguide from the lower side of the waveguide by the light source, and from the bottom surface of the well through the waveguide. Reaction solution in well Irradiating the excitation light to the well, the fluorescence generated in the well is made incident on the same waveguide as the waveguide in which the excitation light is incident from the bottom of the well, and the fluorescence is passed through the waveguide, A fluorescence measuring apparatus having a structure that can be measured by the fluorescence measuring instrument below the waveguide.

  Here, the holding portion can hold the well and the waveguide in parallel by inserting an end portion of the waveguide into a through hole provided in the holding portion.

  The temperature controller includes a Peltier element and a fan, and is provided in contact with a side portion of the holding portion. Furthermore, the diameter of the upper part of the waveguide is substantially the same as the size of the bottom surface of the well, and the waveguide is pressed against the bottom surface of the well, so that the waveguide is in close contact with the bottom surface of the well. .

  By adopting such a structure, it is possible to reduce light leakage and diffusion of light from the well bottom surface when fluorescent light from the well bottom surface is sent to the waveguides disposed immediately below the well bottom surface corresponding to each well position. . If necessary, a matching oil that is transparent and has excellent light transmittance can be applied to the top surface of the waveguide very thinly, but the matching oil is used because the bottom of the well and the waveguide are in close contact. It is not necessary.

  The waveguide may have substantially the same diameter in the upper and lower sides, and the waveguide has a tapered shape so that the diameter is slightly reduced in the lower part, or on the end surface opposite to the end surface connected to the well. The lens may be processed. Since the waveguide has a tapered shape, it becomes easy to capture fluorescence into the fluorescence measuring device by making it coincide with the aperture diameter of the fluorescence measuring device. Further, by processing the lens on the end surface opposite to the end surface connected to the well, the focal point can be made substantially coincident with the fluorescence capturing portion of the fluorescence measuring instrument, and the fluorescence extraction efficiency on the emission side of the waveguide is improved. be able to.

  A reflective film is preferably formed on the lid. A reflective film may be formed on the outer peripheral surface of the waveguide. By forming a reflective film on at least one of the outer peripheral surface of the waveguide and the lid, light leakage from the lid of the well and the outer peripheral surface of the waveguide can be prevented. In addition, by providing a rubber pedestal inside the lid and pressing the lid against the well, the lid can be brought into close contact with the well, and leakage of the reaction solution can be reliably prevented.

  The fluorescence measuring apparatus is characterized in that the light source and the fluorescence measuring instrument are movable in accordance with the arrangement of the well and the waveguide. By moving the fluorescence measuring instrument, it is possible to measure a large number of wells. The fluorescence measurement device is used for fluorescence measurement for PCR using the temperature adjustment function of the temperature controller.

  According to the first invention, the holding part for holding the well is joined to the temperature controller, and the holding part serves as a heat receiving block. Therefore, separation by thermal denaturation, binding of a primer to single-stranded DNA, Amplification by DNA synthesis and measurement of fluorescence can be performed continuously. In addition, since the fluorescence measuring instrument can move below a plurality of wells and waveguides arranged in parallel, a single fluorescence measuring instrument can measure fluorescence of a plurality of wells.

  In addition, by providing a through hole so that the bottom surface of the well is exposed and held at the bottom of the holding unit that is a heat receiving block, the waveguide can be directly connected to the well through the through hole. Since the excitation light is irradiated through the waveguide and the generated fluorescence is detected below, the apparatus is compact. When exchanging the well, the light source of excitation light and the fluorescence measuring device do not interfere with the exchanging operation. Since one measurement can be performed in a relatively short time, the fluorescence measurement of a plurality of wells can be performed in a relatively short time by moving the light source and the measuring device at a high speed.

  In addition, since the well is provided with a lid, the reaction solution can be prevented from evaporating and scattering at the time of temperature rise for DNA amplification. It is also necessary to heat the well lid. This is because if the well lid is not heated, a temperature difference occurs between the well and the well lid when the well is heated, such as during PCR, and the reaction in the well may not proceed uniformly. Here, it is desirable to use a heat resistant resin for the lid of the well, and Teflon (registered trademark) having excellent heat resistance can be used. It is necessary to have a structure that prevents the lid from being removed even when the pressure increases with the temperature history during PCR heating.

  For example, as a mechanism for this, one end of an arm provided perpendicular to the rotation axis is fixed to the rotation axis, a lid is fixed to the other end of the arm opposite to the rotation axis, and the rotation of the arm to which this lid is attached is performed. By rotating the shaft 180 ° by driving the motor, a mechanism that opens and closes by reversing and forming a fitting unevenness on the side surface of the outer peripheral part of the lid, and a lid fixing frame that fits this A mechanism for installing and attaching the lid by press fitting, and further using a hollow metal tube, reducing the pressure inside the tube to vacuum or negative pressure, moving the lid directly above the well and covering the well with the lid, A mechanism for pressing the lid with the tip of the metal tube is conceivable.

  By doing in this way, a well and a lid | cover can be fixed firmly and the leakage of the reaction solution during a measurement can be prevented. Here, in the method using an arm and a motor, leakage of the reaction solution can be more reliably prevented by applying the driving force of the motor as a pressing force even after the cover is put on.

  Further, the device for covering the lid is provided with a rubber pedestal, and the well is pressed through the pedestal so that the pressing force against the lid can be made uniform and the adhesion between the well and the lid can be improved.

  Further, since the waveguide is pressed against the bottom surface of the well, leakage due to light reflection and diffusion at the boundary between the waveguide and the well can be suppressed.

  In addition, by providing a reflective film on the lid, it is possible to suppress the fluorescence generated in the reaction solution from leaking to the outside through the lid or being absorbed by the lid. Further, by forming a reflective film on the outer peripheral surface of the waveguide, it is possible to prevent light from leaking in the middle of the waveguide.

  In the present invention, since at least one excitation light source is sufficient, even if a reflection film is not provided on the outer periphery of the waveguide, it is not affected by light leakage between adjacent waveguides when measuring fluorescence intensity.

  If the outer diameter of the waveguide is substantially constant in the vertical direction, light leakage can be suppressed and manufacturing is easy. Further, if the tapered shape is formed so that the diameter of the lower portion of the waveguide is reduced, the volume of the well can be increased and the measurement unit can be reduced.

  A second invention uses the fluorescence measuring apparatus according to the first invention, injects a reaction solution into the well, and seals the upper part of the well with the lid, and the waveguide is connected to the bottom surface of the well. In this fluorescence measurement method, the waveguide is directly connected to the bottom surface of the well by being pressed and adhered, and the fluorescence generated in the well is measured.

  The light source and the fluorescence measuring instrument can be moved and measured in accordance with the arrangement of the well and the waveguide. In addition, after the fluorescence generated in the well is measured by the fluorescence measuring device, a new measurement can be repeated by replacing only the well.

  Furthermore, by adjusting the temperature of the reaction solution with the temperature controller, separation by thermal denaturation of DNA in the reaction solution, binding of a primer to single-stranded DNA, and amplification by DNA synthesis are performed, Is repeated a plurality of times, and the fluorescence generated in the well can be measured by the fluorescence measuring instrument while the light source and the fluorescence measuring instrument are relatively moved below the waveguide.

  According to the second invention, it is possible to measure the fluorescence of a large number of wells in a short time by moving the fluorescence measuring instrument using a compact measuring apparatus. In addition, since the measurement can be newly repeated simply by exchanging the wells, real-time fluorescence measurement using fluorescence measurement can be performed, and a measurement method applicable to real-time PCR can be provided.

  According to the present invention, it is possible to provide a fluorescence measuring apparatus that is compact and has a short measurement time.

1 is an exploded perspective view showing a fluorescence measuring device 1. FIG. 1 is a cross-sectional view of a fluorescence measuring device 1. The figure which shows operation | movement of the fluorescence measuring device 23 with respect to the fluorescence measuring apparatus 1. FIG. The figure which shows the relationship between a fluorescence residual rate and DNA copy number. Sectional drawing of the fluorescence measuring device 1a. The conceptual diagram which shows the process of PCR using a hairpin primer. The conceptual diagram which shows the conventional fluorescence measurement. The conceptual diagram which shows the conventional fluorescence measuring device.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings. FIG. 1 is an exploded perspective view showing the fluorescence measuring apparatus 1. The fluorescence measuring apparatus 1 mainly includes a temperature controller 3, a well holding unit 5, a well 7, a waveguide 11, a fluorescence measuring instrument, a light source, and the like. For simplicity, the fluorescence measuring instrument, the light source, and the like are not shown in FIG. 1, and details will be described later.

  The well holding unit 5 is provided with a plurality of holes 13. The well 7 is inserted into each hole 13. In the illustrated example, the plurality of wells 7 are integrally formed, but the wells 7 may be divided individually. In addition, the number of wells 7 is not limited to the illustrated example, and usually a plurality of wells 7 are often arranged vertically and horizontally. The well 7 is made of, for example, resin, and polystyrene, polycarbonate, acrylic resin, or the like can be used. Other resins than the above can be used as long as they have heat resistance and light transmittance.

  Each well 7 is covered with a lid 9. The lid 9 prevents the reaction solution in the well 7 from being evaporated and scattered by heat. Further, the lid 9 can prevent viruses and the like from diffusing into the room. In the illustrated example, the plurality of lids 9 are integrally formed. However, like the wells 7, the lids 9 may be divided individually. Although details will be described later, it is desirable that a reflective film such as metal is formed on the inner surface of the lid 9. The reflective film prevents light in the well 7 from leaking from the upper end of the well 7. As the reflective film, for example, a material such as silicon oxide, titanium oxide, or niobium oxide may be formed by vapor deposition, or a reflective film component may be applied.

  The well holding part 5 is joined to the side of the temperature controller 3 so as to be in contact with the temperature controller, and can be used as a heat receiving block when the temperature is adjusted. As the temperature controller 3, the temperature is adjusted by a Peltier heater and a cooler (cooling fan), but a heater block, a heater such as resistance heating, or the like can be used. By adjusting the temperature with the temperature controller 3, the temperature of the well 7 and the reaction solution in the well 7 can be adjusted via the well holding part 5 which is a heat receiving block. Therefore, the well holding part 5 is preferably made of a metal having a good thermal conductivity and a high rigidity. At this time, in order to further shorten the temperature adjustment time, a simple heating device can be provided above the lid 9 as described above. By doing in this way, since it can heat from two directions with the temperature controller provided in the side part of the well 7 and the heating device provided above the lid, it becomes possible to adjust the temperature with high accuracy in a shorter time.

  The waveguides 11 are inserted into the respective holes 13 of the well holding part 5 from below. The waveguide 11 has a substantially cylindrical shape. The waveguide 11 is pressed upward from below. That is, the waveguide 11 is used by being pressed against the bottom surface of the well 7. The entire waveguide 11 is made of a substantially uniform material, and glass can be used as the material. As the glass material, silica-based glass with few impurities is preferable. Furthermore, in order to improve the reflection characteristics inside the waveguide 11, glass doped with fluorine at the outer periphery of the silica glass or glass doped with germanium at the center of the silica glass can be used. Thus, by doping the silica-based glass with impurities so that the refractive index of the central portion of the glass is higher than that of the outer peripheral portion, leakage of excitation light and fluorescence from the waveguide 11 can be reduced, and fluorescence measurement is performed. Sensitivity will be further improved. Here, as the material of the waveguide 11, glass is preferable in consideration of heat resistance, but a resin such as a polycarbonate resin, a polyimide resin, and an acrylic resin can also be used. Note that a reflective film similar to the lid 9 may be formed on the outer peripheral surface of the waveguide 11. By doing so, it is possible to prevent light from leaking from the waveguide 11 to the outside.

  Next, the function and detailed structure of the fluorescence measuring apparatus 1 will be described. FIG. 2 is a cross-sectional view of the well 7 and the waveguide 11 of the fluorescence measuring apparatus 1. A reaction solution such as DNA (nucleic acid) and a reagent is placed in the well 7. The reaction solution put into the well 7 is, for example, about 50 to 60 μl. A lid 9 is placed over the well 7 to seal the well 7.

  The well 7 has a tapered shape such that the diameter of the well 7 is reduced. In addition, the taper shape of the outer surface of the well 7 may be about 5 ° to 10 ° in consideration of the insertion property into the hole 13. As illustrated, the internal shape of the well 7 may be an inverted conical shape, or may be configured with substantially the same diameter in the depth direction. In the example shown in the figure, the shape of each part is simplified, but the well 7 has a depth of about 15 mm into which the reaction solution can be placed, an upper inner diameter of about 6 mm, and a lower inner diameter. Is about 1.0 to 3.5 mm.

  Here, normally, when the resin receives the excitation light, the resin emits light having a slightly longer wavelength than the excitation light. In particular, autofluorescence is what emits strong fluorescence in the visible range. Such autofluorescence has a wavelength in the wavelength band of 400 nm to 700 nm, and decreases as the autofluorescence becomes longer. Therefore, considering the effect of autofluorescence of the well 7 and considering the heat conduction from the metal well holding part 5, the resin thickness on the bottom surface of the well 7 needs to be thin. Therefore, the thickness of the bottom surface and side surface of the well 7 is desirably about 0.5 mm to 1.5 mm, for example. Recently, cycloolefins having low light transmission and autofluorescence have been proposed, and these materials can also be used as the material for the well 7. In addition, autofluorescence due to the resin can be removed as a background by measuring the intensity of autofluorescence in advance.

  Further, the upper surface and inner surface of the well 7 may be subjected to a hydrophilic treatment. By performing the hydrophilic treatment, wettability between the well 7 and the reaction solution can be enhanced. Therefore, it becomes easy to put the reaction solution into the well 7. As such a hydrophilic treatment, for example, a hydrophilic polymer or the like may be applied to the surface of the well 7, or there is a method of modifying the surface of the resin. As surface modification methods, surface modification methods such as UV irradiation, corona discharge, plasma treatment, and primer treatment are known, and these methods can be used.

  A portion of the upper portion of the hole 13 where the well 7 is inserted has a shape corresponding to the outer shape of the well 7. That is, the hole 13 has a tapered shape in which the upper part is expanded in diameter. When the well 7 is inserted into the hole 13, the well 7 comes into close contact with the well holding portion 5 and is fitted to each other. For example, as shown in the drawing, a minute convex portion 19 is formed on the outer peripheral surface of the well 7, and a concave portion 21 having a shape corresponding to the convex portion 19 is provided on the inner surface of the hole 13. By doing so, the well 7 does not float above the hole 13.

  The waveguide 11 is inserted from below the hole 13. A flange 15 is attached to a part of the waveguide 11 by adhesion or the like. An elastic member 17 is disposed below the flange 15 and pushes up the flange 15 (arrow A in the figure). That is, the waveguide 11 is pushed upward. Both the bottom surface of the well 7 and the top surface of the waveguide 11 are formed smoothly. Therefore, when the waveguide 11 is pushed upward, the upper surface of the waveguide 11 is pressed against the bottom surface of the well 7 and closely contacts. By doing in this way, it can prevent that an air layer is formed in the boundary part of the well 7 and the waveguide 11. FIG. Note that the method is not limited as long as the waveguide 11 can be pressed against the well 7, and the method is not limited to the illustrated example.

  In such a state, the temperature controller 3 changes the temperature of the reaction solution in the well 7 in a predetermined temperature cycle. For example, the temperature is sequentially controlled to a temperature (55 ° C. to 95 ° C.) at which DNA (nucleic acid) is amplified. Is done. As described above, DNA (nucleic acid) can be amplified. When amplification is completed a predetermined number of times, the light source 25 emits light having a predetermined wavelength. The light source 25 can irradiate a laser, for example. The laser is not particularly limited, but is, for example, 405 nm ± 5 nm and a 50 mW laser.

  The light source 25 is provided in a direction substantially perpendicular to the waveguide 11. A filter 27 is provided in the vicinity of the intersection on the extension line of the light source 25 and the waveguide 11. The filter 27 transmits only light having a specific wavelength. The filter 27 is disposed at an angle of approximately 45 ° with respect to the irradiation direction from the light source 25. Since the transmission wavelength of the filter 27 is different from the wavelength of the excitation light emitted from the light source 25, the light emitted from the light source 25 is reflected by the filter 27 and introduced into the waveguide 11 (arrow B in the figure). ). A lens for condensing light may be disposed on the optical path as appropriate.

  The excitation light introduced into the waveguide 11 propagates in the waveguide 11 in the direction of the well 7, is introduced into the well 7, and further irradiates the reaction solution inside the well 7. Since the well 7 and the waveguide 11 are in close contact with each other, reflection of excitation light at the boundary can be suppressed.

  When the reaction solution in the well 7 is irradiated with excitation light, as described above, fluorescence having a predetermined wavelength is generated with an intensity corresponding to the amplification of DNA. Here, the fluorescence wavelength is usually shifted to the long wavelength side by about 20 to 50 nm with respect to the wavelength of the excitation light. The fluorescence generated in the well 7 is again introduced into the same waveguide 11 as the waveguide through which the excitation light is guided, and propagates downward in the waveguide.

  Here, the wavelength of the fluorescence generated in the well 7 (for example, 380 to 460 nm) coincides with the transmission wavelength of the filter 27. Therefore, since the wavelength of the fluorescence emitted from the lower end of the waveguide 11 is longer than that of the excitation light, it can be measured by the fluorescence measuring instrument 23 that is transmitted through the filter 27 and disposed below the filter 27. Thus, by arranging the filter 27 that reflects the excitation light and transmits the fluorescence at an angle of 45 ° with respect to the optical path, the light source 25 and the fluorescence measuring instrument 23 are arranged in the vertical direction, and the fluorescence intensity is increased. Can be measured.

  FIG. 3 is a diagram showing a method for measuring the fluorescence of the plurality of wells 7 in this way. First, one well 7 is irradiated with light from a light source 25 (not shown), and fluorescence is measured by the fluorescence measuring instrument 23. Next, the fluorescence measuring instrument 23, the light source 25, and the filter 27 are moved together under the adjacent waveguide 11 (arrow D in the figure), and the fluorescence measurement is performed in the same manner. That is, DNA amplification or the like in the well 7 is performed simultaneously, but fluorescence measurement is performed for each well 7.

  Note that the fluorescence measuring instrument 23 or the like may be stopped below each waveguide 11 or may be moved continuously. The moving speed of the fluorescence measuring instrument 23 etc. is 30-80 mm / sec. It is. As described above, since the fluorescence is measured by moving the set of fluorescence measuring instruments 23 and the like, the number of fluorescence measuring instruments 23 and the like corresponding to the number of the wells 7 is not necessary. For this reason, the fluorescence measuring apparatus 1 becomes compact. Moreover, by measuring the fluorescence of the plurality of wells 7 at the same time, the fluorescence leaks into the adjacent waveguides 11 and does not affect the measurement accuracy of the fluorescence. Moreover, since the fluorescence measurement of one well 7 is completed in an extremely short time, the fluorescence measurement of a plurality of wells 7 can be performed in a short time.

  Next, the relationship between the fluorescence intensity and the number (copy number) of HBV-DNA will be described. FIG. 4 is a graph showing the relationship between the fluorescence residual rate and the total number of copies of HBV-DNA in the reaction solution. The dummy reaction solution containing no virus was irradiated with light and the fluorescence intensity measured was taken as 100%. On the other hand, HBV-DNA copy number amplification was performed, and fluorescence was measured at each copy number.

As described above, since fluorescence disappears when HBV-DNA is amplified, the residual fluorescence rate decreases. Therefore, the total copy number of HBV-DNA in the reaction solution can be estimated by measuring the fluorescence residual ratio with respect to the dummy measurement value. For example, although illustration is omitted, as a result of performing the same measurement a plurality of times, in consideration of measurement variation, when the residual fluorescence rate is 90%, it is about 30 to 110 copies, and when the residual fluorescence rate is 80%, it is 200. When the fluorescence remaining rate was 55%, it was 1 million to 2.5 million copies, and when the fluorescence remaining rate was 45%, it was about 10 ⁸ copies. Note that when the fluorescence residual ratio is reduced to a certain level, it does not decrease any more and becomes substantially constant. Therefore, when the fluorescence residual ratio is 45% to 90%, it can be determined that a predetermined amount of virus is present.

  As described above, according to the present embodiment, it is possible to irradiate light from below the well 7 and measure fluorescence below, and further perform fluorescence measurement while the set of fluorescence measuring instruments 23 and the like move. Thereby, the fluorescence measuring apparatus 1 which is compact and can be measured in a short time can be obtained. In addition, since the well 7 and the waveguide 11 are separated without being integrally formed, the structure of each member is simple, and furthermore, since the well 7 and the waveguide 11 are integrally connected, there is little light leakage. . If only the well 7 is exchanged, the measurement can be repeated.

  In addition, excitation light and fluorescence can be received below the apparatus. For this reason, the fluorescence measurement part and sample arrangement | positioning part by a fluorescence measuring device can be isolate | separated up and down. Therefore, the fluorescence measurement unit does not hinder the replacement work of the well 7, and rapid measurement is possible. Furthermore, since the fluorescence measurement unit and the sample placement unit are spatially separated, the measurement apparatus is not contaminated by the processing liquid in the well 7.

  In addition, since the well 7 is covered with the lid 9, the reaction solution is not evaporated and scattered. At this time, since a reflective film is formed on the lid 9, it is possible to prevent fluorescence from leaking from the lid 9. Similarly, by providing a reflective film on the outer peripheral surface of the waveguide 11, it is possible to prevent light from leaking from the waveguide 11.

  Further, since the waveguide 11 is pressed against the well 7, it is possible to suppress the excitation light and fluorescence from being reflected or diffused at the boundary portion and leaking. Further, by pressing the lid 9 of the well 7 from above or pressing the waveguide 11 from below, the sealing performance of the well 7 can be further enhanced. Furthermore, by heating the well 7 from above the lid 9, the temperature of the well 7 can be kept uniform, and the reaction in the well 7 can be stabilized.

  Next, another embodiment will be described. FIG. 5 is a schematic diagram showing a fluorescence measuring apparatus 1a according to the second embodiment. In the following description, components having the same functions as those of the fluorescence measuring apparatus 1 are denoted by the same reference numerals as those in FIGS.

  The fluorescence measuring device 1a has substantially the same configuration as that of the fluorescence measuring device 1, but differs in that the waveguide 11 is not inserted into the hole 13 of the well holding portion 5. That is, only the well 7 is inserted into the hole 13 of the well holding part 5. The waveguide 11 is supported by a guide 29. The guide 29 is preferably a member that has, for example, heat insulation and reflects light on the inner surface.

  Thus, even if the waveguide 11 is supported by the guide 29, the same effect as that of the fluorescence measuring apparatus 1 described above can be obtained.

  As mentioned above, although embodiment of this invention was described referring an accompanying drawing, the technical scope of this invention is not influenced by embodiment mentioned above. It is obvious for those skilled in the art that various modifications or modifications can be conceived within the scope of the technical idea described in the claims, and these are naturally within the technical scope of the present invention. It is understood that it belongs.

  For example, in the above-described example, the application to the hairpin primer PCR method has been described, but the present invention is not limited to this. It can be applied to other PCR methods. Moreover, although the waveguide 11 was comprised by the substantially constant diameter over the full length, it is good also as a taper shape which diameter reduces gradually as it goes below. By doing in this way, the volume of the well 7 can be enlarged and a measurement part can be made small. A similar effect can be obtained by processing the lens at the lower end of the waveguide.

DESCRIPTION OF SYMBOLS 1, 1a ......... Fluorescence measuring apparatus 3 ......... Temperature adjuster 5 ......... Well holding part 7 ......... Well 9 ......... Cover 11 ......... Waveguide 13 ......... Hole 15 ......... Flange 17 ......... Elastic member 19 ... ... Convex part 21 ... ... Concave part 23 ... ... Fluorescence measuring instrument 25 ... ... Light source 27 ... ... Filter 29 ... ... Guide 103 ... ... Hairpin primer 105 ... ... Fluorescent dye 107... Polymerase 109... Well 111... Irradiation / light receiving unit 113... Reaction solution 115... Peltier heater 117.

Claims (13)

A fluorescence measuring device comprising:
A plurality of wells for holding the reaction solution;
A lid provided on top of the well;
A holding unit as a heat receiving block provided separately from the well and holding the wells in parallel;
A plurality of waveguides directly connected to each of the wells below the wells and a temperature controller capable of adjusting the temperature of the reaction solution in contact with the holding unit;
A light source that irradiates the reaction solution in the well with excitation light through the waveguide;
A fluorescence measuring instrument for measuring fluorescence generated in the well,
The holding portion is provided with a through hole so as to expose and hold the bottom surface of the well,
Excitation light is incident on the waveguide by the light source from below the waveguide,
Irradiating the reaction solution in the well with excitation light from the bottom of the well through the waveguide,
Fluorescence generated in the well is incident on the same waveguide as the waveguide in which the excitation light is incident from the bottom of the well,
A fluorescence measuring apparatus having a structure in which the fluorescence can be measured by the fluorescence measuring device below the waveguide through the waveguide.   The holding unit inserts an end of the waveguide into a through hole provided in the holding unit, and holds both the well and the waveguide in parallel in the holding unit. The fluorescence measuring apparatus according to 1.   The fluorescence measuring apparatus according to claim 1, wherein the temperature controller includes a Peltier element and a fan, and is provided in contact with a side portion of the holding unit.   The diameter of the upper part of the said waveguide is substantially the same as the bottom face of the said well, The said waveguide is pressed and adhered to the bottom face of the said well, The Claim 1 characterized by the above-mentioned. Fluorescence measuring device.   The waveguide has substantially the same diameter in the upper and lower directions, or has a tapered shape so that the diameter of the waveguide is reduced, or a lens is processed on an end surface opposite to the end surface connected to the well. The fluorescence measuring device according to any one of claims 1 to 4.   6. The fluorescence measuring apparatus according to claim 1, wherein a reflective film is formed on at least one of an outer peripheral surface of the waveguide and the lid.   A rubber pedestal is provided inside the lid, and the lid is brought into close contact with the well by pressing the pedestal against the well, thereby preventing leakage of a reaction solution. Item 7. The fluorescence measurement device according to any one of Items 6.   The fluorescence measuring apparatus according to claim 1, wherein the light source and the fluorescence measuring instrument are movable in accordance with the arrangement of the well and the waveguide.   9. The fluorescence measuring apparatus according to claim 1, wherein the fluorescence measuring apparatus is used for fluorescence measurement for PCR using a temperature control function of the temperature controller.   A reaction solution is injected into the well using the fluorescence measuring device according to claim 1, and the waveguide is connected to the bottom surface of the well in a state where the top of the well is sealed with the lid. A fluorescence measuring method comprising: measuring the fluorescence generated in the well by directly connecting the waveguide to a bottom surface of the well by pressing and closely contacting the waveguide.   The fluorescence measurement method according to claim 10, wherein the light source and the fluorescence measuring instrument are moved and measured in accordance with the arrangement of the well and the waveguide.   The fluorescence measurement method according to claim 11, wherein after the fluorescence generated in the well is measured by the fluorescence measuring instrument, a new measurement is repeated by exchanging only the well. By adjusting the temperature of the reaction solution with the temperature controller, separation by thermal denaturation of DNA in the reaction solution, primer binding to single-stranded DNA and amplification by DNA synthesis are performed,
The fluorescence generated in the well is measured by the fluorescence measuring device while the light source and the fluorescence measuring device are relatively moved below the waveguide after the above process is repeated a plurality of times. Item 11. The fluorescence measurement method according to Item 10.

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