JP6581707B2 - Nucleic acid analyzer and nucleic acid analysis method - Google Patents

Description

  The present invention relates to an apparatus for performing nucleic acid analysis by fluorescence observation using a plurality of optical filters and a nucleic acid analysis method.

  The next-generation sequencer is measured using a flow cell in which a large number of minute reaction fields are fixed. By performing a chemical reaction on a micro reaction field fixed on the flow cell and performing fluorescence observation, it becomes possible to analyze the base sequence of the target DNA. A flow cell is a consumable product about the size of a slide glass on which a large number of micro reaction fields are fixed, and a flow path having an inlet and an outlet is constructed so that the reaction reagent can be replaced. A base extension reaction is carried out by pouring a reagent such as an enzyme necessary for the flow cell or a fluorescently labeled nucleotide. These reactions need to be controlled at an appropriate temperature as required. Since the flow cell is disposed in close contact with a heat block capable of controlling the temperature, an objective lens is disposed on the other side of the flow cell. The reaction field fixed to the flow cell is irradiated with excitation light through the objective lens, and fluorescence is detected. By capturing this fluorescence with a two-dimensional sensor such as a CCD camera or a CMOS camera, base information can be obtained as a fluorescence image.

The reaction field area of the flow cell is generally larger than the field of view that can be measured with an objective lens. The objective lens is fixed, and a fluorescent image is taken over the entire measurement region of the flow cell by driving the position of the flow cell with respect to the optical axis of the fixed objective lens. For example, when the reaction area of the flow cell is 35 mm × 4 mm, the area of the flow cell 140 mm ² can be acquired as a fluorescence image. Since the field of view of the objective lens is approximately 1 mm ² , the flow cell is fixed to the XY stage and driven by a certain distance, so that adjacent panels are sequentially observed with fluorescence.

  One of the important indexes in next-generation sequencers is throughput. Throughput is the total number of bases that can be analyzed per unit time, and technological development is progressing to increase this. There are two main strategies for improving throughput. One is to reduce the size of the reaction field fixed to the flow cell and to increase the base information that can be acquired per image taking by densely arranging them. Conventionally, the reaction field is fixed by random arrangement. Today, it is possible to pack more densely by miniaturizing the reaction field and arranging the pattern. On the other hand, there is a reduction in time for performing fluorescence observation. The next-generation sequencer uses four types of fluorescent dyes corresponding to each base. It is necessary to irradiate excitation light corresponding to each dye and to separate fluorescence. Conventionally, detection is generally performed using four types of filter sets corresponding to each dye. This method requires four types of excitation light, a dichroic mirror for separating excitation light and fluorescence, a filter for fluorescence and at least 12 types of optical elements, and a mechanism for switching each filter set. . With respect to the fluorescence observation time, for example, if the imaging time for one color is 200 ms and the number of panels to be measured is 140 panels, it takes about 2 minutes only for fluorescent imaging. In order to improve the throughput, it is necessary to further shorten these reading times. In Patent Document 1, by separating fluorescence with a plurality of dichroic mirrors and photographing with two two-dimensional sensors, it is possible to simultaneously read two color dyes in one photographing. By using this method, the mechanism for switching the filter set can be simplified, and the time required for fluorescence imaging can be reduced to about half. However, a change in spectral characteristics of a plurality of dichroic mirrors becomes a big problem. Since the spectral characteristic is changed by changing the angle of the incident light with respect to the dichroic mirror, there is a possibility that the target spectral characteristic cannot be obtained for a part of the region in the measurement visual field.

US2012 / 027305

  The separation of the fluorescence and the specific accuracy of the dye depends on the optical properties of the dichroic mirror. As the incident angle to the dichroic mirror changes, the optical characteristics change. For this reason, an area that cannot be analyzed in the measurement visual field is generated by the arrangement of each dichroic mirror. The present invention provides a configuration and analysis method of a nucleic acid analyzer capable of simultaneously observing a plurality of fluorescent dyes in the entire visual field, accurately identifying the dyes, and determining a nucleic acid sequence from the obtained fluorescent colors. .

In order to solve the above problems, a liquid feeding mechanism for feeding a reagent or a sample, a substrate having a flow path for feeding a liquid, a stage on which the substrate is installed, a temperature control mechanism for performing a base extension reaction, a fluorescence In the nucleic acid analyzer comprising a detection unit for detecting the fluorescence image and observing
The detection unit includes a light source, a plurality of optical filters that pass a predetermined wavelength range, an objective lens, a first dichroic mirror that reflects excitation light and passes fluorescence, and fluorescence emitted from the fluorescent dye. A second dichroic mirror that separates at different rates for each, and at least two two-dimensional sensors that detect the respective fluorescence after separation,
The substrate has a mark detected on an image by each of the two-dimensional sensors,
Image alignment of images acquired by each of the two-dimensional sensors is performed using a mark on the substrate detected on the image, and an area in the field of view is divided into a plurality of areas based on the mark, and for each divided area There are provided a nucleic acid analyzer and an analysis method characterized in that fluorescent dye analysis is performed using calculated values of separation ratio changes in spectral characteristics of the first and second dichroic mirrors.

  According to the present invention, the fluorescence separated by the dichroic mirror is photographed by two cameras, and the pigment is specified based on the luminance ratio of each point posted by each camera. When specifying the dye, the measurement field is divided into multiple areas and analysis is performed for each divided field, so that the dye can be accurately identified in the entire field of view, and the arrangement of the specified fluorescent color is determined. Finally, the base sequence can be determined.

  A plurality of fluorescent dyes can be detected simultaneously. In addition, since it becomes possible to accurately identify the dye throughout the entire measurement visual field, the apparatus throughput is improved. Problems, configurations, and effects other than those described above will become apparent from the following description of embodiments.

The figure which shows the structural example of the nucleic acid analyzer concerning an Example The figure which shows the structural example of the detection part of the nucleic acid analyzer concerning an Example. Diagram of light rays through the objective lens The figure which shows the example of a spectral characteristic change of the dichroic mirror concerning an Example The figure which shows the fluorescence wavelength and spectral example which concern on an Example The figure which shows the example of spectroscopy of the fluorescent dye concerning an Example The figure which shows the example of the field stop concerning an Example The figure which shows the example of the flow cell mark concerning an Example

  Hereinafter, embodiments of the present invention will be described with reference to the drawings. The embodiment of the present invention is not limited to the examples described later, and various modifications are possible within the scope of the technical idea.

  The outline of the nucleic acid analyzer 100 will be described with reference to FIG. The nucleic acid analyzer includes a reagent cooling storage box 102 that houses a reagent container 101 that holds a plurality of reagents, a reaction reagent feeding mechanism 103 that feeds a reagent held in the reagent container 101, and a flow path that combines DNA fragments. A flow cell 104, a temperature control mechanism 105 that promotes the reaction between the DNA fragment and the reagent in the flow cell, and a detection unit 106 that detects fluorescence incorporated into the DNA fragment. In the flow cell 104 adjusted in advance, each reaction solution is fed by the solution feeding mechanism 103. When necessary, the temperature control mechanism 105 heats and cools, thereby causing an extension reaction in the flow cell to emit fluorescence. The detection unit 106 detects fluorescence and determines the base sequence. Excess samples and reagents after the reaction are stored in the waste liquid tank 109.

  Various nucleic acid analyzes can be performed using the nucleic acid analyzer of the present invention. For example, DNA sequencing and hybridization can be performed. In particular, it is possible to perform DNA sequencing using the nucleic acid analyzer 100 according to the present invention. By feeding each reaction reagent into the flow cell, a base containing a fluorescent dye is bound to the DNA fragment immobilized on the flow cell. The reaction solution contains four kinds of fluorescently labeled bases and a polymerase. Each base is fluorescently labeled with FAM, Cy3, Texas Red, and Cy5, respectively. In the reaction solution, the salt concentration, the magnesium concentration and the pH are optimized so that the extension reaction can be carried out efficiently. The reaction solution contains a polymerase, and only one base of fluorescent nucleotide complementary to the DNA fragment is incorporated. The reason why the second base does not extend is that a substance that inhibits the extension of the second base dye is bound to the first base fluorescent dye. After one base has been incorporated, the floating fluorescent nucleotide is removed by washing.

  The detection unit 106 irradiates the fluorescent dye with excitation light to perform fluorescence observation. Thereafter, the fluorescent part is cut, an extension reaction is further performed, and the fluorescence observation is performed again. By repeating these steps, the sequences of the four fluorescent colors are determined, and finally the base sequence is determined. By this method, analysis of several tens to several hundreds of bp can be performed in one cycle, and data of several tens of Gbp can be analyzed in one run.

  The nucleic acid analyzer 100 of the present invention separates fluorescence with two dichroic mirrors and performs detection using two two-dimensional cameras.

  As shown in FIG. 2, the detection unit 106 includes an excitation light irradiation unit 201, an excitation fluorescence dichroic mirror 202, an objective lens 203, a fluorescence filter 204, a color separation dichroic mirror 205, tube lenses 206 and 207, and a two-dimensional image sensor. And 208 and 209. The excitation light irradiation unit 201 includes a white light source 220, a collimating lens 221, and a filter switching mechanism 222 that houses a plurality of optical filters. As the white light source, a light source capable of emitting high-intensity white light such as an Xe lamp or a white LED is used. Moreover, a cooling mechanism for cooling the light source is provided as necessary. The collimating lens 221 condenses light emitted from the light source and converts it into parallel light. A light guide is inserted between the light source and the collimating lens to make the light from the light source uniform. A liquid light guide or a light pipe can be used as the light guide. In this embodiment, a multi-array LED is used as a white light source, and emitted light is made uniform using a light pipe. The filter switching mechanism 222 accommodates a plurality of optical filters, and transmits light in a wavelength region used as excitation light from white light by switching to each filter. In this embodiment, two types of optical filters having a single band in a short wavelength region and an optical filter having a single band in a long wavelength region are accommodated, and a mechanism for switching to each filter is provided. For example, a motor, a solenoid, or the like can be used as the filter switching mechanism.

  In the excitation light irradiation unit 200, the light emitted from the white light source is converted into parallel light by the collimator lens, and then transmitted through the optical filter provided in the filter switching mechanism 222. Parallel light is emitted. The light emitted from the excitation light irradiation unit enters the excitation fluorescence dichroic mirror 202. The excitation fluorescence dichroic mirror is installed at an angle of 45 degrees with respect to the optical axis. It has bands in two wavelength ranges, reflects incident excitation light, and transmits fluorescence. The excitation light reflected by the excitation fluorescence dichroic mirror enters the objective lens 203. The objective lens 203 is attached to a Z stage 210 that is driven in the Z-axis direction. Focusing is performed on the measurement surface of the flow cell 104 by driving the Z stage. Fluorescence emitted from the fluorescent dye in the flow cell 104 passes through the objective lens again and passes through the excitation fluorescence dichroic mirror 202. The transmitted fluorescence is transmitted only through a specific band by the fluorescent filter 204. Thereafter, the fluorescence is incident on the dichroic mirror 205 for color separation. The color separation dichroic mirror 205 has gentle optical characteristics with respect to the fluorescence wavelength from the four dyes. Therefore, the fluorescence emitted from the fluorescent dye is transmitted or reflected by the dichroic mirror for color separation 205 at a different rate for each dye. The transmitted light and the reflected light are collected by the tube lenses 206 and 207, respectively, and formed on the two-dimensional cameras 208 and 209. By arranging the field flatteners 211 and 212 immediately in front of the two-dimensional cameras 208 and 209, it is possible to improve the optical performance. Due to the optical characteristics of the dichroic mirror for color separation 205, the fluorescence emitted from the bright spot in the measurement visual field is detected with different luminance. By obtaining the fluorescence intensity ratio of the bright spots of the fluorescence images photographed by each two-dimensional camera, it becomes possible to specify which of the four colors is the fluorescent dye.

  In this embodiment, as shown in FIG. 2, the arrangement of the excitation fluorescence dichroic mirror and the color separation dichroic mirror is arranged in the same direction with respect to the optical axis.

  The fluorescence that has passed through the objective lens 203 is not completely parallel light, but has an angle that depends on the position of the field of view that can be measured by the objective lens. For example, the measurable field of view of an objective lens with a magnification of FN26 and a magnification of 20 times is an area of Φ1.3 mm, but as shown in FIG. Rays 302 and 303 with an angle of The optical characteristics of the dichroic mirror change depending on the incident angle. An example is shown in FIG. Compared to characteristic 401 when incident at 45 degrees, light 303 incident at 49 degrees changes characteristic 402 in the short wavelength side transmission area, and light 302 incident at 41 degrees changes long wavelength side transmission area It changes to 403. If the two dichroic mirrors are arranged alternately, for example, a light beam incident on the excitation fluorescence dichroic mirror at 49 degrees enters the color separation dichroic mirror at 41 degrees. The characteristic change that occurs in each dichroic mirror changes the short wavelength and long wavelength in the opposite wavelength direction, so that all the fluorescence is transmitted or reflected in part of the measurement field. Therefore, the fluorescence cannot be separated at different ratios by the color separation dichroic mirror 205, and the pigment cannot be specified. The area that can be analyzed with one field of view is reduced.

  In this example, FAM, Cy3, Texas Red, and Cy5 are used as four-color fluorescent nucleotides. By using an optical filter with a peak near 500 nm, FAM and Cy3 are simultaneously excited and fluorescence observation is performed. In addition, by using an optical filter having a peak near 600 nm, both Texas Red and Cy5 are excited simultaneously and fluorescence observation is performed. As an example, FIG. 5 shows the spectral characteristics and fluorescence characteristics of each dichroic mirror in the case of FAM and Cy3. The FAM fluorescence transmitted through the excitation fluorescence separation dichroic mirror is separated into transmitted light 603 and reflected light 604 by the color separation dichroic mirror and detected. Thereafter, the fluorescence transmitted through the dichroic mirror for color separation is separated into transmitted light and reflected light by a dichroic mirror having gentle characteristics for each fluorescent dye as shown in FIG. The ratio of transmission and reflection in each color separation dye is 50:50 for FAM, 30:70 for Cy3, 70:30 for TxR, and 5:95 for Cy5. The separation ratio is arbitrary, but the dye can be specified with higher accuracy by avoiding the same ratio between the dyes.

  It is possible to put a field stop in front of the collimating lens of the excitation light irradiation unit. Since the field of view 304 to be measured in one imaging is a rectangle, when the excitation light is irradiated as it is, the region of the maximum field of view 701 of the objective lens shown in FIG. 7 is irradiated. By inserting the field stop, it is possible to irradiate only the region 702 of the measurement visual field. Irradiation to areas other than the measurement field causes fading of the fluorescent dye that is the object of measurement, so it is important to eliminate irradiation to areas other than the measurement field as much as possible. By inserting a field stop, it is possible to suppress fading of the fluorescent dye outside the measurement field. In order to securely block the excitation light to the outer periphery of the aperture, critical illumination or Koehler illumination is used as the collimating lens of the excitation light irradiation unit.

In this apparatus, the pigment is specified by calculating the ratio of the fluorescence detected by the two cameras. The detection method is as follows. First, the same bright spot is extracted from the fluorescence images acquired by the two cameras. Next, each ratio is calculated from the luminance information of each extracted point.
Thereafter, the fluorescent dye is specified based on the calculated luminance ratio. As shown in FIG. 7, the flow cell has a position detection mark 801 in each measurement field of view. FIG. 7 shows a cross mark or the like, but this shape and number may take any number. Can do. This mark is patterned so that it is always measured on each panel. On the image detected by the camera, the mark area is detected as a dark area without fluorescence. The position of the mark can be detected using pattern recognition or the like. In this embodiment, the same bright spot is extracted from the fluorescence image acquired by each camera based on the mark position coordinates. For each bright spot, the intensity ratio of the light detected on the transmission side or the reflection side is calculated. There are various methods for extracting the bright spot and calculating the brightness of each point, but it is possible to calculate the center coordinates and brightness of the bright spot by performing Gaussian fitting.

  Since the separation ratio for each dye changes depending on the characteristics of the filter and dichroic mirror, the separation ratio of each dye calculated from the design value of the dichroic mirror is compared with the ratio obtained from each bright spot. A point dye can be identified.

  The spectral characteristics of the dichroic mirror vary depending on the angle of light incident on the dichroic mirror. Like the light rays 301 to 303 shown in FIG. 3, the incident angle varies depending on the position of the visual field. If the entire field of view is separated at the same ratio, the analysis accuracy of the bright spot located at the edge of the field of view deteriorates. Therefore, it is possible to separate the entire field of view with high accuracy by dividing the field of view into several regions and separating the regions at different ratios. For dividing the field of view, for example, by dividing into 16 with reference to the mark in the field of view, it is possible to accurately identify the dye by using calculated values that take into account changes in the spectral characteristics of the dichroic mirror for each divided region It is.

DESCRIPTION OF SYMBOLS 100 Nucleic acid analyzer 101 Reagent container 102 Reagent storage 103 Liquid feeding mechanism 104 Flow cell 105 Temperature control mechanism 106 Detection part 107 XY stage 108 Liquid feeding syringe 109 Waste liquid part 201 Excitation light irradiation part 202 Excitation fluorescence dichroic mirror 203 Objective lens 204 Fluorescent filter 205 Color separation dichroic mirror 206, 207 Tube lens 208, 209 Two-dimensional camera 210 Z stage 212, 213 Field flattener 220 Light source 221 Collimator lens 222 Filter switching mechanism 301 Light rays 302, 303 from the center of the field of view From the edge of the field of view Light field 304 Measurement field 401 Spectral characteristic 402 for 45 degree incidence Spectral characteristic 403 for 49 degree incidence Spectral characteristic 501 for 41 degree incidence 501 Spectral characteristic 502 for excitation fluorescence dichroic mirror For color separation Spectral characteristics of dichroic mirror 503 FAM fluorescence characteristics 504 Cy3 fluorescence characteristics 601 FAM fluorescence separation 602 Figure 3 Cy3 fluorescence separation Figure 603 FAM fluorescence separation 603 Reflected by FAM fluorescence in the wavelength region 604 FAM Wavelength range 605 that forms an image on the side Wavelength region 606 that forms an image on the transmission side with fluorescence of Cy3 Wavelength region 701 that forms an image on the reflection side with fluorescence of Cy3 701 Maximum field of view 702 that can be imaged with an objective lens 801 Mark in the field of measurement

Claims (21)

A liquid feeding mechanism for feeding reagents and samples, a substrate having a flow path for feeding the liquid, a stage on which the substrate is installed, a temperature control mechanism for performing a base extension reaction, and detecting fluorescence and observing a fluorescent image In a nucleic acid analyzer comprising a detection unit that
The detection unit includes a light source, a plurality of optical filters that pass a predetermined wavelength range, an objective lens, a first dichroic mirror that reflects excitation light and passes fluorescence, and fluorescence emitted from the fluorescent dye. A second dichroic mirror that separates at different rates for each, and at least two two-dimensional sensors that detect the respective fluorescence after separation,
The substrate has a mark detected on an image by each of the two-dimensional sensors,
Image alignment of images acquired by each of the two-dimensional sensors is performed using a mark on the substrate detected on the image, and an area in the field of view is divided into a plurality of areas based on the mark, and for each divided area A nucleic acid analyzer characterized in that a fluorescent dye analysis is performed using a calculated value of a separation ratio change in spectral characteristics of the first and second dichroic mirrors. In claim 1,
The calculated value of the change in the separation ratio of the spectral characteristics of the first and second dichroic mirrors for each divided region in the field of view is the separation ratio of each dye calculated from the design value of each dichroic mirror, and each bright spot A nucleic acid analyzer characterized in that it is obtained by comparing the separation ratio of each dye obtained from the above. In claim 1,
The nucleic acid analyzer, wherein the first dichroic mirror and the second dichroic mirror are arranged to be inclined in the same direction with respect to the optical axis. In claim 1,
The plurality of optical filters include a first filter having a band in at least a short wavelength region and a second filter having a band in a long wavelength region,
Furthermore, the nucleic acid analyzer provided with the switching mechanism which switches these filters. In claim 4,
The nucleic acid analyzer according to claim 1, wherein the first filter is a filter having a peak in the vicinity of 500 nm and has a band capable of simultaneously exciting at least two kinds of short-wavelength fluorescent dyes. In claim 5,
The nucleic acid analyzer according to claim 1, wherein the first filter has a band between 480 nm and 520 nm, and can simultaneously excite the fluorescent dyes FAM and Cy3. In claim 4,
The nucleic acid analyzer characterized in that the second filter is a filter having a peak in the vicinity of 600 nm and has a band capable of simultaneously exciting at least two kinds of fluorescent dyes in a long wavelength region. In claim 7,
The nucleic acid analyzer according to claim 2, wherein the second filter has a band between 580 nm and 620 nm and can simultaneously excite the fluorescent dyes Texas Red and Cy5. In claim 1,
The nucleic acid analyzer according to the second dichroic mirror, wherein the fluorescent dye is separated into reflected light and transmitted light at a specific ratio for each fluorescent dye and detected by each of the two-dimensional sensors. In claim 9,
In the second dichroic mirror, a nucleic acid analyzer that separates reflected light and transmitted light into an arbitrary fluorescent dye having gentle edges near 550 nm and 630 nm and having a fluorescence peak near the edge. In claim 10,
The nucleic acid analyzer characterized in that the second dichroic mirror is capable of separating four types of fluorescent dyes of FAM, Cy3, Texas Red, and Cy5 at a specific ratio. In claim 4,
A nucleic acid analyzer that detects four different fluorescent dyes by switching optical filters. In claim 1,
A nucleic acid analyzer for photographing the same field of view with the same exposure time using the two-dimensional sensor. The fluorescence observation apparatus according to claim 1,
The nucleic acid analyzer is characterized in that the light source is a white LED or an Xe lamp. The fluorescence observation apparatus according to claim 1,
A nucleic acid analyzer characterized in that the type of fluorescent dye is specified by calculating the ratio of bright spots photographed simultaneously by the two-dimensional sensor. The fluorescence observation apparatus according to claim 1,
The nucleic acid analyzer, wherein the temperature control mechanism is installed on a stage and heats and cools the substrate. A liquid feeding mechanism for feeding reagents and samples, a substrate having a flow path for feeding the liquid, a stage on which the substrate is installed, a temperature control mechanism for performing a base extension reaction, and detecting fluorescence and observing a fluorescent image In a nucleic acid analysis method using a nucleic acid analyzer comprising a detection unit that performs
The detection unit includes a light source, a plurality of optical filters that pass a predetermined wavelength range, an objective lens, a first dichroic mirror that reflects excitation light and passes fluorescence, and fluorescence emitted from the fluorescent dye. A nucleic acid analysis method using a nucleic acid analyzer comprising a second dichroic mirror that separates at a different rate for each, and at least two two-dimensional sensors that detect respective fluorescence after separation,
The substrate has a mark detected on an image by each of the two-dimensional sensors,
Image alignment of images acquired by each of the two-dimensional sensors is performed using a mark on the substrate detected on the image, and an area in the field of view is divided into a plurality of areas based on the mark, and for each divided area A nucleic acid analysis method, wherein a fluorescent dye analysis is performed using a calculated value of a separation ratio change in spectral characteristics of the first and second dichroic mirrors, and a nucleic acid sequence is determined from the obtained fluorescent color. In claim 17,
The calculated value of the change in the separation ratio of the spectral characteristics of the first and second dichroic mirrors for each divided region in the field of view is the separation ratio of each dye calculated from the design value of each dichroic mirror, and each bright spot A method for nucleic acid analysis, characterized in that it is obtained by comparing the separation ratio of each dye obtained from the above. The nucleic acid analysis method according to claim 17,
The plurality of optical filters include a first filter having a band in a short wavelength region and a second filter having a band in a long wavelength region,
A nucleic acid analysis method characterized by switching between these filters. The nucleic acid analysis method according to claim 17,
The reaction solution used for nucleic acid base extension reaction includes a fluorescently labeled base and a polymerase for base extension, and the nucleic acid base extension reaction includes a step of cleaving the base fluorescent label after one base extension. A nucleic acid analysis method characterized by the above. The nucleic acid analysis method according to claim 20, comprising:
A nucleic acid analysis method comprising a step of obtaining a fluorescent color image of the extended base and cleaving the fluorescent label of the base after a single base extension reaction of the nucleic acid.

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