JP2009175074A - Apparatus and method for detecting amplified nucleic acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a compact and inexpensive apparatus for detecting an amplified nucleic acid, and to provide a simple and precise method for detecting the amplified nucleic acid by using the apparatus. <P>SOLUTION: The apparatus for detecting the amplified nucleic acid includes: a substrate which has a planar sample holding part on its surface for holding a nucleic acid solution and holds the nucleic acid solution and a coating liquid for coating the nucleic acid solution; a temperature adjusting means for adjusting a temperature of the substrate; a signal detecting means for detecting an optical signal in the nucleic acid solution; and a bubble detecting means for detecting the presence or absence of an air bubble in the coating liquid and the nucleic acid solution on the substrate. In the method for detecting the amplified nucleic acid, the nucleic acid solution is held by the planar sample holding part disposed on the surface of the substrate, and the nucleic acid solution is coated by the coating liquid, and the presence or absence of the air bubble in the coating liquid and the nucleic acid solution is detected, and if no air bubble is detected, a nucleic acid amplifying reaction is carried out at the sample holding part, and then an obtained amplification product is detected. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to an amplified nucleic acid detection apparatus and an amplified nucleic acid detection method using the apparatus.

The polymerase chain reaction (hereinafter abbreviated as PCR) is a reaction that amplifies a specific region in a nucleic acid molecule as much as 1 million times in a container. As a general method for detecting the amplified nucleic acid, the reaction solution after completion of PCR is subjected to agarose gel electrophoresis to separate each nucleic acid fragment and then stained, and the size of the separated nucleic acid fragment (molecular weight) ), A method for discriminating a target amplified nucleic acid, and a method for detecting a target amplified nucleic acid by subjecting the reaction solution to a dot hybridization method are widely used.
However, in the conventional method for detecting amplified nucleic acid using PCR, it is necessary to take out the nucleic acid amplification reaction mixture after the PCR from the reaction vessel once and process it. There is a problem that it involves a lot of work. In addition, amplified nucleic acid may be scattered in the environment and mixed with other samples used for detection. In this case, the mixed amplified nucleic acid becomes a template for PCR, and other samples can be accurately tested. There was a problem of disappearing. Another drawback of the PCR method is that the initial concentration of the target nucleic acid to be amplified in the sample is difficult to understand.

Therefore, a method for quantifying the amount of amplified nucleic acid while performing a PCR reaction has been proposed.
For example, a method for quantifying the amount of amplified nucleic acid while monitoring PCR using an apparatus having a temperature cycle block equipped with a vial containing a nucleic acid amplification reaction mixture has been proposed (see Patent Documents 1 to 3). ). In these methods, when double-stranded nucleic acid is present, the concentration of the nucleic acid is calculated by allowing the nucleic acid amplification reaction mixture to contain a fluorescent dye that emits fluorescence with an intensity proportional to the amount of the nucleic acid, and detecting the fluorescent signal. To do. The fluorescent signal is detected by irradiating the vial with an excitation beam that excites the fluorescent dye from the beam splitter to emit fluorescence from the fluorescent dye. The emitted light beam from the fluorescent dye is guided to the CCD detector by the beam splitter and detected.
In addition, a method of quantifying the amount of amplified nucleic acid while monitoring PCR using a device in which an optical fiber lead is connected to a PCR tube provided in a heating / cooling block has been proposed (Patent Document 4). reference). In this method, the optical fiber lead is used to irradiate the PCR tube with excitation light that excites the fluorescent dye, and the generated fluorescence is guided to the spectrofluorescence detection device using the same optical fiber lead for detection.
Special table 2003-524754 gazette JP 2005-526252 A JP 2005-274579 A Japanese Patent Laid-Open No. 10-201464

In the detection methods proposed in Patent Documents 1 to 4, a microtiter plate, a vial, or a sample tube is employed as a reaction container used for performing PCR and detecting amplified nucleic acids. In this case, the nucleic acid amplification reaction mixture is usually present below the sample filling portion in the reaction vessel.
In the detection methods proposed in Patent Documents 1 to 3, when detecting the amplified nucleic acid, it is necessary to accurately irradiate the nucleic acid amplification reaction mixture with the excitation light to focus. In this case, there are a plurality of reaction vessels, and in order to detect amplified nucleic acids in all of these reaction vessels without losing accuracy, it is necessary to focus on all of these nucleic acid amplification reaction mixtures. In this case, it is necessary to provide a lens above the reaction vessel and to provide a complicated and expensive telecentric optical system above the lens.
Further, the detection method proposed in Patent Document 4 requires an optical fiber for guiding excitation light and fluorescence.
Thus, the conventional detection device requires a special structure, and there is a problem that the device is large and expensive. Further, the detection method using such a detection apparatus has a problem that the operation is complicated. Thus, the actual situation is that it is difficult to detect a target amplified nucleic acid simply and at low cost without impairing accuracy.

  The present invention has been made in view of the above circumstances, and an object thereof is to provide a small and inexpensive amplified nucleic acid detection apparatus and a simple and highly accurate detection method of amplified nucleic acid using the apparatus.

To solve the above problem,
The invention according to claim 1 is an apparatus for detecting an amplified nucleic acid, wherein a planar sample holder for holding a nucleic acid solution is provided on the surface, and holds the nucleic acid solution and a coating solution for coating the nucleic acid solution. A substrate, temperature control means for adjusting the temperature of the substrate, signal detection means for detecting a light signal in the nucleic acid solution, and bubble detection for detecting the presence or absence of bubbles in the nucleic acid solution and coating liquid on the substrate Means for detecting an amplified nucleic acid.
In the invention according to claim 2, the substrate surface is provided with a first hydrophilic region that is a sample holding portion and a first water-repellent region that surrounds the periphery of the first hydrophilic region. The amplified nucleic acid detection device according to claim 1, wherein
According to a third aspect of the present invention, in the substrate, a second hydrophilic region that further surrounds the outer peripheral edge of the first water-repellent region, and a second repellent that surrounds the outer peripheral edge of the second hydrophilic region. The amplification nucleic acid detection device according to claim 2, wherein an aqueous region is provided.
According to a fourth aspect of the present invention, the image processing apparatus further includes an imaging unit that captures an image of the substrate, and the bubble detection unit detects the presence or absence of bubbles from the imaging data. The amplified nucleic acid detection device according to Item.
The invention according to claim 5 is the amplified nucleic acid detection apparatus according to any one of claims 1 to 4, wherein the imaging means and the signal detection means are integrated.
According to a sixth aspect of the invention, based on the detection result of the bubble detection means, a nucleic acid amplification reaction is performed when no bubble is detected, and a nucleic acid amplification reaction is not performed when a bubble is detected. The amplified nucleic acid detection apparatus according to claim 1, further comprising a reaction control unit that controls the reaction.

According to the seventh aspect of the present invention, a nucleic acid solution is held in a flat sample holder provided on a substrate surface, the nucleic acid solution is coated with a coating solution, and bubbles in the nucleic acid solution and the coating solution are detected. In the detection method of amplified nucleic acid, the presence / absence is detected and bubbles are not detected, a nucleic acid amplification reaction is performed in the sample holder, and the obtained amplification product is detected.
The invention according to claim 8 is the amplification nucleic acid according to claim 7, wherein the coating solution is a water-insoluble liquid having a specific gravity smaller than that of the nucleic acid solution and a boiling point of 100 ° C. or higher. It is a detection method.

  According to the present invention, amplified nucleic acid held on a substrate plane can be detected with high accuracy. And since the amplification nucleic acid hold | maintained on the board | substrate plane is detected, a telecentric optical system and an optical fiber become unnecessary for an apparatus. For example, an optical system of a microscope can be used, and light signals emitted from a plurality of nucleic acid amplification reaction mixtures can be detected simultaneously. Thus, since a special structure is not required, the detection apparatus can be reduced in size and can be manufactured at low cost. Furthermore, the detection method can be simplified.

Hereinafter, the present invention will be described in detail with reference to the drawings.
<Detection device>
The amplified nucleic acid detection apparatus according to the present invention has a planar sample holder for holding a nucleic acid solution provided on the surface thereof, a substrate holding the nucleic acid solution and a coating solution covering the nucleic acid solution, and a temperature of the substrate A temperature control means for adjusting the temperature, a signal detection means for detecting a light signal in the nucleic acid solution, and a bubble detection means for detecting the presence or absence of bubbles in the nucleic acid solution and the coating liquid on the substrate. It is a feature.
Here, the amplified nucleic acid is a nucleic acid amplified by applying a known method such as a PCR method, and the nucleic acid may be any of DNA, RNA, other oligonucleotides or polynucleotides.

  Examples of the nucleic acid in the solution include an amplified nucleic acid obtained by an amplification reaction, a nucleic acid that serves as a template during the amplification reaction, a nucleic acid that serves as a raw material for the amplified nucleic acid, and a primer. That is, examples of the nucleic acid solution include a reaction solution containing each component necessary for an amplification reaction such as an enzyme before the start of the amplification reaction, and a reaction solution during the amplification reaction. These reaction liquids are usually aqueous solutions.

  The optical signal is an optically detectable signal, and is emitted from a nucleic acid in the solution or a labeling substance separately contained in addition to the nucleic acid. An optical signal is usually generated as a result of excitation of a nucleic acid or a labeling substance in a solution by irradiation with excitation light or the like.

A preferable example of the labeling substance is a fluorescent substance.
As the fluorescent substance, any known substance can be used and is not particularly limited. Specifically, fluorescein, rhodamine (rhodamine green, TAMRA etc.), acriflavine, alexa (Alexa 647 etc.), cyber green (SYBR Green) etc. can be illustrated.
For example, SYBR GREEN I specifically intercalates with a nucleic acid forming a double helix structure, and as a result, absorbs blue light (wavelength 488 nm) and green light (wavelength 522 nm). It is known to emit fluorescence. Therefore, when a nucleic acid is amplified by subjecting the reaction solution containing Cyber Green I to an amplification reaction, Cyber Green I intercalates into the double helix of the amplified nucleic acid, and thus emits fluorescence when irradiated with excitation light. The fluorescence intensity of is proportional to the amount of amplified nucleic acid.

One kind of labeling substance may be used alone, or two or more kinds may be used in combination. When using 2 or more types together, the combination, ratio, etc. can be suitably selected according to the objective.
Further, the amount of the labeling substance to be introduced into the amplification product can be appropriately selected according to the purpose.

FIG. 1 is a schematic configuration diagram illustrating an amplified nucleic acid detection apparatus according to the present invention.
The detection apparatus exemplified here includes a substrate 2 and a temperature control unit 4 as a configuration mainly related to nucleic acid amplification, and includes a signal detection unit 13 and a bubble detection unit as a configuration mainly related to detection of amplified nucleic acid. As will be described later, here, the bubble detection means is integrated with the signal detection means 13. In addition, the illumination optical system 5, the light source 6, the excitation filter 7, the mirror 8, the objective lens 9, the absorption filter 10, and the like are mainly provided as a configuration mainly related to generation and transmission of optical signals. These may be used, for example, in a normal fluorescence detection apparatus.

Although the material of the board | substrate 2 is not specifically limited, A translucent material is preferable. The light-transmitting material refers to a material having high light transmittance and low autofluorescence, and specific examples thereof include glass and transparent resins. Of these, preferred examples include glass such as water plate glass, white plate glass, and half white glass.
The substrate 2 preferably has a smooth front and back surface. Here, the surface of the substrate refers to the surface of the substrate 2 on the side where the sample holding portion described later is provided, and the back surface of the substrate refers to the surface of the substrate 2 opposite to the surface.
The magnitude | size of the board | substrate 2 is not specifically limited, For example, the magnitude | size of the surface and a back surface can be suitably selected according to the objective. Further, the thickness of the substrate 2 can be appropriately selected according to the purpose, but it is preferably 0.9 to 1.1 mm from the viewpoint of easy handling in consideration of strength.

A sample holder for holding the nucleic acid solution is provided on the surface of the substrate. The sample holding part is also a part where a nucleic acid amplification reaction is performed in order to obtain an amplified nucleic acid to be detected.
The sample holder is flat and may take any form as long as it can hold the spotted solution, and the specific area on the substrate surface may be used as it is as the sample holder. However, it is preferable that the specimen can be stably held. In order to stably hold the sample, an example in which at least a part of the sample holding portion is hydrophilic is exemplified. As an example of such a substrate, a substrate holding a solution is illustrated in FIG. 2A is a perspective view of the substrate 2, and FIG. 2B is a cross-sectional view of the substrate 2 taken along line II-II in FIG.

  As shown in FIG. 2B, the surface 21 of the substrate is provided with a first hydrophilic region 211 and a first water repellent region 212 that surrounds the peripheral edge of the first hydrophilic region 211. . The nucleic acid solution 20 is stably held on the first hydrophilic region 211 which is a sample holding part. And by providing the 1st water-repellent area | region 212, since the movement of the solution 20 is suppressed, the solution 20 is hold | maintained more stably. The outer shape of the first hydrophilic region 211 is not particularly limited as long as it is planar, and may be selected according to the purpose. However, a substantially circular shape is preferable as shown in FIG. The first water repellent region 212 is also preferably planar. And the external shape is not specifically limited, What is necessary is just to select according to the objective, However, It is preferable that it is ring shape. By doing so, the nucleic acid solution 20 is held more stably.

  On the surface 21 of the substrate, as shown in FIG. 2, a second hydrophilic region 213 surrounding the outer peripheral edge of the first water-repellent region 212 and a second hydrophilic region 213 surrounding the outer peripheral edge of the second hydrophilic region 213. Two water-repellent regions 214 are preferably provided. By doing so, when the nucleic acid solution 20 is coated with the coating solution 25 in order to prevent evaporation of the held nucleic acid solution 20, the coating solution 25 can be stably held. The second hydrophilic region 213 and the second water repellent region 214 are preferably planar. The outer shape of the second hydrophilic region 213 is not particularly limited, but is preferably a ring shape. By doing in this way, the coating liquid 25 is hold | maintained more stably. Further, the outer shape of the second water repellent region 214 may be a ring shape or other shapes, and is not particularly limited. The first hydrophilic region 211, the first water repellent region 212, and the second hydrophilic region 213 are preferably provided concentrically.

The coating liquid 25 has a specific gravity smaller than that of the nucleic acid solution 20 for the purpose of suppressing evaporation of water that is the main solvent of the nucleic acid solution 20 and withstanding the heating of the nucleic acid solution 20 during nucleic acid amplification. A water-insoluble liquid having a boiling point of 100 ° C. or higher is preferred. Any material can be used as long as it has such physical properties, but various mineral oils can be exemplified as preferable commercial products. By directly coating the nucleic acid solution 20, a large amount of air layer does not exist in the vicinity of the nucleic acid solution 20 as in the case of using a microtiter plate or a sample tube. Does not evaporate and the concentration change of the nucleic acid solution 20 is suppressed, so that nucleic acid amplification can be performed stably.
Then, by providing the second hydrophilic region 213 and the second water repellent region 214 on the surface 21 of the substrate, the movement of the coating liquid 25 having the above physical properties is suppressed.

  The dimensions of the hydrophilic region and the water repellent region can be appropriately selected according to the purpose. For example, when holding a very small amount of nucleic acid solution 20 of about 0.2 to 3 μL, the diameter D1 of the first hydrophilic region 211 is preferably 0.5 mm to 6 mm. The width L1 and the width L2 of the second hydrophilic region 213 are preferably 0.2 mm to 3 mm. The dimension of the second water repellent region 214 can be arbitrarily selected in consideration of the size of the substrate 2 and the number of sample holders. For example, as shown in FIG. 2, the entire outer surface of the second hydrophilic region 213 may be a second water repellent region 214.

The substrate 2 having a hydrophilic region and a water repellent region can be produced by a known method. Specifically, a method of performing hydrophilic treatment and water repellent treatment on the substrate surface and a method of performing water repellent treatment on the hydrophilic substrate surface can be exemplified.
Examples of the hydrophilic treatment include those in which a hydrophilic functional group such as a hydroxyl group, an amino group or a carboxy group is introduced to the substrate surface. As the water repellent treatment, hydrophobic functional groups such as alkyl groups, alkenyl groups, alkoxy groups or alkenyloxy groups such as low polar functional groups, alkyl groups or alkoxy groups in which one or more hydrogen atoms are substituted with fluorine atoms. The thing which introduce | transduces a functional group into a substrate surface can be illustrated.
As such a substrate 2 having a hydrophilic region and a water-repellent region, a commercially available product may be used. For example, for the purpose of holding a very small amount of the nucleic acid solution 20, AmpliGrid (registered trademark) manufactured by Advaltyx Corporation Is preferred.

The substrate 2 is preferably covered with a cover 3 as shown in FIG. 1 in order to prevent foreign substances from entering the nucleic acid solution 20.
The material of the cover 3 is preferably a light-transmitting material so as not to hinder the excitation light irradiation to the substrate 2 and the detection of the optical signal. Specifically, a material having translucency similar to that of the substrate 2 can be exemplified.

As shown in FIG. 1, when the nucleic acid amplification 20 such as PCR is performed in the nucleic acid solution 20 held in the sample holding unit, the temperature adjusting means 4 is for mounting the substrate 2 and adjusting the temperature. The substrate 2 is usually mounted so that the substrate back surface 22 is in contact with the heat conducting portion of the temperature adjusting means 4.
The temperature control means 4 can be appropriately selected from known ones that can be heated and cooled. However, a commercially available product may be used from the viewpoint of quick and highly accurate temperature control, for example, for PCR. The thermal cycler is suitable.
The substrate 2 may be mounted in direct contact with the heat conducting portion of the temperature control means 4 or may be mounted in contact indirectly through an inclusion made of a heat conductive material.
Here, an example in which the substrate 2 is mounted on the temperature control means 4 is shown. However, for example, while supporting the substrate 2 with a support provided separately, the heat conduction portion of the temperature control means is directly or indirectly provided. The substrate 2 may be contacted.

  The material of the heat conduction part of the temperature control means 4 is preferably heat resistant and highly heat conductive, and examples thereof include metals such as aluminum, iron and copper, alloys such as stainless steel, and various heat conductive resins. Of these, metals and alloys are preferable, and aluminum is particularly preferable because of excellent stability and thermal conductivity.

  The temperature control means 4 is preferably electrically connected to a computer 15 as shown in FIG. In this way, the temperature, time, cycle number, etc. during nucleic acid amplification can be automatically controlled. Further, the progress or stop of the nucleic acid amplification reaction can be automatically controlled based on the detection result of the presence or absence of bubbles in the nucleic acid solution. Thereby, a huge number of samples can be processed quickly.

The arrangement position of the temperature control means 4 is not particularly limited as long as the detection of the light signal in the sample is not hindered.
The temperature adjusting means 4 can be produced in the same manner as a known heating / cooling device or the like.

The light source 6 generates a light signal by excitation by irradiation into a nucleic acid solution. For example, a halogen lamp or a xenon lamp can be used.
The illumination optical system 5 may be a known one and is not particularly limited.

  The excitation filter 7 transmits only light having a wavelength in a specific range necessary for excitation out of the irradiation light 60 from the light source 6 to be excitation light 61, and depends on the type of excitation target, for example, a labeling substance. Just choose. And in order to prevent the occurrence of ghost images resulting from reflected light and stray light, it is preferable to accurately attenuate light outside the wavelength range necessary for excitation, for example, when using Cyber Green as a fluorescent material, Those that transmit light in a wavelength band centered around 488 nm are preferable.

The mirror 8 reflects or transmits the excitation light 61 and guides it to the labeling substance.
The objective lens 9 enlarges and passes an optical signal generated by being excited by the excitation light 61.
The absorption filter 10 transmits only light having a wavelength within a specific range of the optical signal and serves as detection light, and may be selected according to the optical signal. In order to prevent generation of a ghost image caused by reflected light or stray light, it is preferable to attenuate light outside the target wavelength range with high accuracy. For example, when using Cyber Green as a fluorescent material, 522 nm. It is preferable to transmit light in a wavelength band centered around the vicinity.

  The signal detection means 13 may be any means as long as it can detect the intensity of the optical signal, and a known photodetector or the like can be used. Here, the detection light transmitted through the absorption filter 10 is detected from the light signals in the nucleic acid solution. Among these, those integrated with imaging means capable of imaging the substrate 2 are preferable, those capable of calculating the intensity of the optical signal from the imaging data of the substrate 2 are more preferable, and those equipped with a CCD camera can be exemplified.

  Further, the signal detection means 13 is preferably electrically connected to the computer 15. In this way, the amount of amplified nucleic acid can be automatically calculated based on the detection result of the optical signal. If the signal detection means 13 is integrated with the image pickup means, the number of times of image pickup at the time of optical signal detection, the image pickup timing, the exposure time, etc. can be automatically controlled. Thereby, a huge number of samples can be processed quickly, and desired information can be obtained efficiently and with high accuracy.

The bubble detection means is preferably one that can optically analyze an image of a nucleic acid solution or a coating solution, and preferably one that can detect the presence or absence of bubbles from imaging data of the substrate 2. In this case, it is preferable to acquire imaging data without using the absorption filter 10. Therefore, it is preferable that the absorption filter 10 is movable in the detection device. For example, the absorption filter 10 is fixed to a rotation shaft provided in the detection device, and is in a plane substantially parallel to the filter surface as the rotation shaft rotates. A turret type that can be moved in is preferable.
As shown in FIG. 1, when the image pickup means is integrated with the signal detection means 13, since the image data can be automatically analyzed by the computer 15, the presence or absence of bubbles can be automatically and efficiently determined.

  Here, an example in which the signal detection means, the imaging means, and the bubble detection means are provided integrally is shown, and the detection apparatus is miniaturized and streamlined by having such a configuration, but each of these means is individually provided. It may be provided. Also in this case, it is preferable that the signal detection means, the imaging means, and the bubble detection means are electrically connected to the computer 15.

  In the present invention, it is preferable to provide a dark box 14 as shown in FIG. By doing in this way, the excitation light of the wavelength of a specific range can be irradiated correctly, and an optical signal can be detected with high precision. Further, instead of using the dark box 14, the light signal may be detected in a dark room, or the light signal may be detected using a dark curtain.

  In the present invention, a dichroic mirror that transmits excitation light may be used instead of the excitation filter 7, and a dichroic mirror that transmits detection light may be used instead of the absorption filter 10.

As described above, the computer 15 can perform control and information processing of the detection device.
When bubbles are detected, imaging conditions and imaging data analysis conditions for the substrate 2 are set in advance, so that acquisition and analysis of imaging data and determination of the presence or absence of bubbles can be performed automatically.
When amplifying a nucleic acid, the progress of the amplification reaction is set in advance, so that the amplification reaction can be automatically advanced and stopped. In addition, the amplification reaction can be automatically performed by setting amplification conditions such as temperature, time, and cycle number during the amplification reaction in advance.
In detecting a light signal, the amount of amplified nucleic acid can be automatically calculated by setting signal data analysis conditions in advance. At this time, when the optical signal is detected by imaging the substrate 2, signal data is automatically acquired by setting detection conditions such as the number of times of imaging of the signal detection means 13, imaging timing, and exposure time in advance. Can be done.

  In addition, in the detection apparatus of this invention, it cannot be overemphasized that a part of said structure may be deleted or changed in the range which does not impair the effect of this invention.

  In the detection apparatus of the present invention, the amplified nucleic acid in the nucleic acid solution 20 held on the plane of the substrate 2 can be detected, so that a conventional telecentric optical system and optical fiber are unnecessary. For example, an optical system of a microscope can be used, and a plurality of optical signals on the substrate 2 can be detected simultaneously. Further, since the focus adjustment is performed on the contact surface between the substrate 2 and the nucleic acid solution 20, the focal position can be made constant without being affected by the amount of the solution, and there is no need to perform the focus adjustment for each nucleic acid solution 20. Therefore, the apparatus can be reduced in size and manufactured at low cost.

<Detection method>
In the method for detecting an amplified nucleic acid according to the present invention, a nucleic acid solution is held in a flat sample holder provided on a substrate surface, the nucleic acid solution is coated with a coating solution, and the nucleic acid solution and the coating solution The presence or absence of air bubbles is detected, and when no air bubbles are detected, a nucleic acid amplification reaction is performed in the sample holder, and the obtained amplification product is detected. It is preferable to use the detection device of the present invention.
In the present invention, the nucleic acid solution used for detecting the presence or absence of bubbles may be a reaction solution before the start of the amplification reaction or a reaction solution during the amplification reaction.
That is, the presence or absence of bubbles in the reaction solution and the coating solution before the start of the amplification reaction is detected, and it is confirmed that the bubbles do not exist before performing the nucleic acid amplification reaction. Alternatively, in the reaction solution and the coating solution in which bubbles did not exist before the start of the amplification reaction, bubbles may occur after the start of the amplification reaction for some reason. Therefore, in the reaction solution and the coating solution during the amplification reaction. If the presence or absence of bubbles is detected and the presence of bubbles is confirmed, the amplification reaction may be stopped.

  If bubbles exist in the nucleic acid solution, the bubbles may burst with the temperature change during the amplification reaction, and the nucleic acid solution may be scattered. In this case, the composition of the ruptured nucleic acid solution may be different from that before the rupture. Furthermore, if the sample holder is planar, the scattered liquid is likely to be mixed into other nucleic acid solutions that are simultaneously performing an amplification reaction. Then, the amplification reaction cannot be performed accurately, and the detection accuracy of the amplified nucleic acid is lowered. The same applies to the coating liquid. When the bubbles in the coating liquid burst, the coated nucleic acid solution may be scattered. However, in the present invention, when there are bubbles in either the nucleic acid solution or the coating liquid, the amplification reaction is not performed, so that the nucleic acid solution is prevented from being scattered and the scattered liquid is prevented from being mixed with high accuracy. Amplified nucleic acids can be detected. The presence / absence of bubbles in the coating solution may be detected in the same manner as in the nucleic acid solution.

In the detection method of the present invention, for example, as illustrated in FIG. 2, the nucleic acid solution is coated with a coating solution, preferably using a substrate provided with a hydrophilic region and a water-repellent region. By doing so, evaporation of the nucleic acid solution during the amplification reaction is suppressed, and nucleic acid amplification can be performed stably.
And while performing nucleic acid amplification reaction with the sample holding part of a detection apparatus, the presence or absence of the bubble in the obtained reaction liquid and coating liquid can be detected in real time, and amplified nucleic acid can be detected in real time.

The amount of the nucleic acid solution held in the sample holder may be set according to the purpose. However, since the solution is held on the plane of the sample holder, the amplified nucleic acid can be detected with high accuracy even if the amount is extremely small. For example, when a substrate as shown in FIG. 2 is used, the amount of the solution is preferably 0.1 to 5 μL, more preferably 0.2 to 3 μL, and 0.5 to 2 μL. It is particularly preferred.
The amount of the coating solution may be selected within a range that can cover the nucleic acid solution. For example, when the amount of the nucleic acid solution is within the above range, it is preferably 1 to 10 μL, and preferably 2 to 8 μL. Is more preferable, and 4 to 6 μL is particularly preferable.

  In the detection method of the present invention, the nucleic acid amplification reaction can be carried out by a known method except that the nucleic acid amplification reaction is carried out at the sample holder using the substrate. For example, since the substrate temperature can be adjusted in a specific temperature cycle, a nucleic acid amplification method such as a PCR method can be easily applied.

  The detection of the amplified nucleic acid can also be performed by a known method except that the substrate is used. For example, a light signal in the nucleic acid solution after the amplification reaction may be detected. Then, for example, by labeling the amplified nucleic acid using a labeling substance, preferably a fluorescent substance such as Cyber Green, the amount of the amplified nucleic acid is obtained by utilizing the fact that the intensity of the optical signal is proportional to the amount of the amplified product. Can be calculated.

When the temperature of the substrate is adjusted in a specific temperature cycle, it is preferable to detect the amplified nucleic acid once or twice or more for each temperature cycle. Thus, by detecting and quantifying the amplified nucleic acid in real time, the amount of the nucleic acid used as the template before the amplification reaction can be quantified quickly and accurately. The number of times to quantify the amplified nucleic acid may be selected according to the situation.
Similarly, the presence / absence of bubbles is preferably detected once or twice or more for each temperature cycle. Thus, by detecting in real time, an amplified nucleic acid can be detected with high accuracy.

Next, the detection method of the present invention when the detection apparatus shown in FIG. 1 is used will be specifically described.
Irradiation light 60 from the light source 6 is transmitted through the excitation filter 7 only to light in a specific range of wavelengths to become excitation light 61, passes through the illumination optical system 5, is reflected by the mirror 8, and is nucleic acid on the substrate 2 The solution 20 is irradiated.
The optical signal 200 generated by the irradiation of the excitation light 61 passes through the objective lens 9, and only the light having a specific range of wavelengths is transmitted by the absorption filter 10 to become detection light 201 to be detected. Then, the detection light 201 is detected by the signal detection means 13.

  In the detection method of the present invention, the amplified nucleic acid can be detected with high accuracy as described above. In addition, since the nucleic acid solution is held on a flat surface, a very small amount of amplified nucleic acid can be used for detection. Furthermore, since the nucleic acid solution held in a flat shape is prevented from being scattered due to the bursting of bubbles in the nucleic acid solution or the coating solution, it can be detected with high accuracy even with a very small amount of amplified nucleic acid. In addition, since the focus adjustment is performed on the contact surface between the substrate and the nucleic acid solution, the focus position can be made constant without being affected by the amount of the solution, and the detection operation can be simplified.

  Hereinafter, the present invention will be described in more detail with reference to specific examples. However, the present invention is not limited to the following examples.

[Example 1]
Using the detection apparatus shown in FIG. 1, RNA reverse transcription reaction and PCR were performed by the following method, and fluorescence amplification products were detected in real time.
(Detection device)
As the substrate for holding the nucleic acid solution, AmpliGrid AG480F (trade name, manufactured by Advalytix) having the configuration shown in FIG. 2 was used. In addition, AmpliSpeed (trade name, manufactured by Avalytic) was used as a thermal cycler, and connected to an in vivo living body observation system OV110 (trade name, manufactured by Olympus). This living body observation system includes a CCD camera and can image a substrate. The thermal cycler and the biological observation system were connected to a computer so that they could be controlled automatically.

(Nucleic acid solution)
As a sample for performing the amplification reaction, RNA solutions having five concentrations with the composition shown in Table 1 were prepared. In Table 1, 2 × SYBR Green Reaction Mix (manufactured by Invitrogen) includes Cyber Green I.

(Detection of presence of bubbles)
Using a pipette, 1 μl of each of the five RNA solutions was dispensed into the hydrophilic region of the substrate. Further, 5 μl of a sealing solution (manufactured by Advalix) was dispensed on top of these solutions, and the RNA solution was coated with the sealing solution as shown in FIG.
Next, this substrate was mounted on the detection device, and all RNA solutions and sealing solutions on the substrate were imaged at once. The acquired image is shown in FIG. FIG. 3 (a) is 16 ng / μl, (b) is 1.6 ng / μl, (c) is 160 pg / μl, (d) is 16 pg / μl, and (e) is 1.6 pg / μl. It is an image. As a result of analyzing the imaging data, no bubbles were detected in any of the five types of RNA solutions and sealing solutions.

(Nucleic acid amplification reaction)
Based on this detection result, the substrate was subsequently heated and cooled under the temperature conditions shown below, and reverse transcription reaction and real-time PCR were performed in the solution held on the substrate.
・ Reverse transcription reaction and PCR temperature conditions 55 ° C / 30 minutes → 94 ° C / 2 minutes → (94 ° C / 15 seconds → 55 ° C / 30 seconds → 72 ° C / 30 seconds) x 50 cycles → 25 ° C

(Real-time detection of light signal)
In each cycle at 72 ° C. during PCR, the substrate was irradiated with excitation light from above the substrate, and all the PCR reaction solutions on the substrate were imaged at once with a CCD camera. At this time, an amplification product was detected by setting the observation channel of the living body observation system as a GFP region and detecting the generated cyber green I fluorescence. The image acquired at this time is illustrated in FIG. 4A shows an image at one cycle, FIG. 4B shows an image at 20 cycles, and FIG. 4C shows an image at 30 cycles.

(Detection result)
From each acquired image, the fluorescence intensity of each PCR reaction solution was calculated using a kinetic computer.
At one cycle, all reaction solutions had the same fluorescence intensity. This indicates that the PCR product has not been amplified yet, and the fluorescent signal is derived from the DNA itself before the amplification reaction.
At 20 cycles, it was confirmed that the fluorescence intensity of the reaction solutions with RNA concentrations of 16 ng / μl, 1.6 ng / μl, and 160 pg / μl increased, and the fluorescence intensity was proportional to the concentration of the amplified product.
At 30 cycles, the fluorescence intensities of the reaction solutions having RNA concentrations of 16 ng / μl, 1.6 ng / μl, and 160 pg / μl were equal. On the other hand, in the reaction solutions with RNA concentrations of 16 pg / μl and 1.6 pg / μl, it was confirmed that the fluorescence intensity increased, and the fluorescence intensity was proportional to the concentration of the amplified RNA.
A graph in which the calculated fluorescence intensity is plotted against the number of cycles is shown in FIG. As is apparent from FIG. 5, it was confirmed that the rate at which the PCR product was amplified was proportional to the RNA concentration.

[Example 2]
As in Example 1, an RNA solution was prepared, the RNA solution and the sealing solution were imaged, and image analysis was performed. As a result, among the five RNA solutions, one bubble was found in the RNA concentration of 1.6 ng / μl. No air bubbles were detected in any of the remaining RNA solutions and sealing solutions. The image acquired at this time is shown in FIG. Since bubbles were detected in this way, PCR was temporarily stopped for all RNA solutions, the RNA solutions in which bubbles were detected were removed, RNA solutions with the same concentration were re-prepared, and dispensed onto the substrate. Coated with sealing solution.

  The present invention can be used for genome studies that require nucleic acid amplification reactions.

It is a schematic block diagram which illustrates the detection apparatus of the amplified nucleic acid which concerns on this invention. It is a figure which illustrates the board | substrate in this invention of the state holding the solution, (a) is a perspective view, (b) is sectional drawing in the II-II line of (a). It is an image of RNA solution before PCR and sealing solution in Example 1, (a) is 16 ng / μl, (b) is 1.6 ng / μl, (c) is 160 pg / μl, (d) is 16 pg / μl (E) is an image in the case of an RNA concentration of 1.6 pg / μl. It is an image of the PCR reaction liquid in Example 1, (a) is 1 cycle, (b) is 20 cycles, (c) is an image at 30 cycles. 3 is a graph showing a calculation result of fluorescence intensity in Example 1. 2 is an image of an RNA solution and a sealing solution in Example 2, wherein (a) is 16 ng / μl, (b) is 1.6 ng / μl, (c) is 160 pg / μl, (d) is 16 pg / μl, (e ) Is an image in the case of an RNA concentration of 1.6 pg / μl.

Explanation of symbols

DESCRIPTION OF SYMBOLS 2 ... Substrate, 20 ... Nucleic acid solution, 21 ... Substrate surface, 200 ... Optical signal, 201 ... Detection light, 211 ... First hydrophilic region, 212 ... First Water repellent region, 213 ... second hydrophilic region, 214 ... second water repellent region, 25 ... coating liquid, 4 ... temperature adjusting means, 60 ... irradiation light, 61 ... Excitation light, 13 ... detecting means, 15 ... computer

Claims (8)

An amplified nucleic acid detection device comprising:
A planar sample holder for holding a nucleic acid solution is provided on the surface, and a substrate for holding the nucleic acid solution and a coating solution for coating the nucleic acid solution;
Temperature control means for adjusting the temperature of the substrate;
A signal detection means for detecting a light signal in the nucleic acid solution;
Bubble detecting means for detecting the presence or absence of bubbles in the nucleic acid solution and the coating liquid on the substrate;
A device for detecting an amplified nucleic acid, comprising:   2. The substrate according to claim 1, wherein a first hydrophilic region as a sample holding portion and a first water repellent region surrounding the peripheral portion of the first hydrophilic region are provided on the substrate surface. Amplified nucleic acid detection device.   The substrate further includes a second hydrophilic region surrounding the outer peripheral edge of the first water-repellent region and a second water-repellent region surrounding the outer peripheral edge of the second hydrophilic region. The apparatus for detecting an amplified nucleic acid according to claim 2, wherein:   The amplification nucleic acid detection apparatus according to any one of claims 1 to 3, further comprising an imaging unit that captures an image of the substrate, wherein the bubble detection unit detects the presence or absence of bubbles from the imaging data. .   The apparatus for detecting an amplified nucleic acid according to any one of claims 1 to 4, wherein the imaging means and the signal detection means are integrated.   A reaction control means for performing a nucleic acid amplification reaction when a bubble is not detected based on a detection result of the bubble detection means, and for controlling the nucleic acid amplification reaction so as not to perform a nucleic acid amplification reaction when a bubble is detected; The amplified nucleic acid detection device according to any one of claims 1 to 5, further comprising:   A nucleic acid solution is held in a flat sample holder provided on the substrate surface, the nucleic acid solution is coated with a coating solution, and the presence or absence of bubbles in the nucleic acid solution and the coating solution is detected to detect bubbles. If not, a nucleic acid amplification reaction is performed in the sample holder, and the amplification product obtained is detected.   The method for detecting an amplified nucleic acid according to claim 7, wherein the coating liquid is a water-insoluble liquid having a specific gravity smaller than that of the nucleic acid solution and a boiling point of 100 ° C or higher.

Images