JP2019103489A - Particle measurement method, specimen treating method, and particle imaging apparatus - Google Patents

Abstract

To provide particle measurement methods, specimen treating methods, and particle imaging apparatus capable of efficiently acquiring information effective for pathological diagnosis and the like.SOLUTION: The particle measurement method comprises a step S13 of labeling and amplifying in a particle a target nucleic acid within the particle; a step S14 of labeling the target polypeptide on the surface of the particle and/or another target nucleic acid different from the target nucleic acid; and a step S2 of measuring a labeled target nucleic acid, and a labeled target polypeptide and/or another labeled target nucleic acid.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a particle measurement method, an analyte processing method, and a particle imaging apparatus.

  As a method of detecting abnormal cells, there is an in situ PCR method of amplifying a target nucleic acid as a target nucleic acid. Non-Patent Document 1 discloses an experiment for detecting a specific nucleic acid in E. coli. As shown in FIG. 16, in this experiment, the operator fixes E. coli with formalin, and then protease treatment of E. coli, permeating reagents such as thermostable DNA polymerase and primers into E. coli, and target nucleic acid in E. coli Make it amplified. The operator confirms the presence of the target nucleic acid with a fluorescence microscope. Thus, in situ PCR can reliably detect a target nucleic acid by amplifying the target nucleic acid.

Kurokawa, K. et al., "Detection of enterohemorrhagic E. coli by in situ PCR method", Japanese Bacteriological Journal, 52 (2), 513-518, 1997, The Japanese Society of Bacteriology

  When there are abnormalities in cells, bacteria, etc. and the cause is nucleic acid, the method disclosed in Non-Patent Document 1 is effective. However, when the cause of the abnormality is not a nucleic acid, other causes include abnormalities of polypeptides such as proteins. Alternatively, abnormalities may exist in both the nucleic acid and the polypeptide.

  Also, for example, when there is a cause of abnormality in a plurality of nucleic acids contained in a cell, finding an abnormality using an effective detection method for each nucleic acid is important for performing appropriate pathological diagnosis. It is.

  The present invention provides a particle measurement method, a sample processing method, and a particle imaging apparatus capable of efficiently acquiring information effective in pathological diagnosis and the like.

  A first aspect of the present invention relates to a particle measurement method. The particle measurement method of the present embodiment comprises the steps of labeling and amplifying the target nucleic acid in the particle in the particle (S13), and the step of labeling the target polypeptide of the particle and / or another target nucleic acid different from the target nucleic acid (S13) Measuring S14), a labeled target nucleic acid, and a labeled target polypeptide and / or another labeled target nucleic acid (S2).

  "Particle" means cells, bacteria, etc. contained in a sample. The particles to be measured may be endoplasmic reticulum exosomes or the like. The above-mentioned "specimen" refers to blood directly collected from a person, tissue fragments such as skin, and the like. A "polypeptide" is a molecule in which amino acid residues are linked by peptide bonds, and includes, for example, relatively low molecular weight amino acid polymers and large molecular weight proteins.

  Also, "labeling and amplification of target nucleic acid" is not limited to the meaning of simply amplifying nucleic acid present in particles, ie, DNA or messenger RNA (mRNA). For example, non-coding RNAs (ncRNAs) such as microRNAs (miRNAs) and nucleic acids controlled thereby are also included in the inside of exosomes, which are a type of extracellular vesicles that are being actively studied in recent years . In amplification of a target nucleic acid, not only amplification of this miRNA but also nucleic acid transcribed from miRNA may be amplified. In the particle measurement method according to this aspect, all particles including the above-described nucleic acid are to be measured. Hereinafter, “labeling and amplification of target nucleic acid” will be described as having the same meaning.

  "Other target nucleic acid" means one of two target nucleic acids having genetic information different from each other. Moreover, in the base sequence of a nucleic acid, the case where one of two regions different from one another is meant is also included. The above target nucleic acid and other target nucleic acids may be double stranded or single stranded.

  According to the particle measurement method of this aspect, amplification or labeling of the target nucleic acid and labeling of the target polypeptide are simultaneously performed on one cell, or labeling of two target nucleic acids different from each other on one cell is performed. It is possible to do at the same time. In addition, in the case of the above configuration, information of one cell may be complementarily associated with each other regarding the presence or absence of the nucleic acid abnormality and the presence or absence of the polypeptide abnormality or the presence or absence of two different nucleic acids different from each other. Since it can, it is possible to obtain effective information in pathological diagnosis and the like.

  Moreover, in the particle measurement method according to the present embodiment, either of the labeling of the target nucleic acid and the amplification of the target nucleic acid may be performed first. Hereinafter, “labeling and amplification of target nucleic acid” is the same.

  In the particle measurement method according to the present embodiment, in the step (S14) of labeling the target polypeptide of the particle and / or another target nucleic acid different from the target nucleic acid (S14), the target polypeptide on the particle surface is labeled and labeled. In the step of measuring the nucleic acid and the labeled target polypeptide and / or the other target nucleic acid (S2), the labeled target nucleic acid and the labeled target polypeptide can be configured to be measured.

  According to the particle measurement method of the present embodiment, amplification and labeling of the target nucleic acid and labeling of the target polypeptide can be simultaneously performed on one cell. In addition, since information of both the presence or absence of nucleic acid abnormality and the presence or absence of abnormality of a polypeptide can be obtained for one cell, the reliability of evaluation for one cell is improved.

  In the particle measurement method according to this aspect, the steps of labeling and amplifying the target nucleic acid in the particle in the particle (S13), and the step of labeling another target nucleic acid different from the target polypeptide and / or target nucleic acid of the particle (S13) After performing S14), a step (S2) of measuring a labeled target nucleic acid and a labeled target polypeptide and / or another target nucleic acid may be performed.

  As a method of measuring a labeled target nucleic acid and a labeled target polypeptide or other target nucleic acid, for example, a flow cell irradiated with light from a light source is labeled with a measurement sample, that is, a labeled target nucleic acid There are a method of flowing a liquid containing the target polypeptide or other labeled target nucleic acid and imaging light generated from the liquid, a method of imaging with a fluorescence microscope, and the like. Such a measurement makes it possible to evaluate whether amplification of the target nucleic acid and labeling of the target polypeptide or amplification of the other target nucleic acid were performed on the same particle.

  In the particle measurement method according to the present embodiment, the step (S2) of measuring the labeled target nucleic acid and the labeled target polypeptide and / or the other target nucleic acid includes a step of imaging an image of the particle It can be configured.

  This allows the operator to visually confirm the presence or absence of amplification of the target nucleic acid and the presence or absence of amplification of the target polypeptide or other target nucleic acid.

  In the particle measurement method according to the present embodiment, the step (S2) of measuring the labeled target nucleic acid and the labeled target polypeptide and / or the other target nucleic acid comprises the labeled target nucleic acid and the labeled target nucleic acid The fluid containing the target polypeptide and / or the other target nucleic acid may be flowed to the flow cell (10) and configured to image light obtained by irradiating the fluid in the flow cell (10) with light.

  This makes it possible to determine, for a large number of particles, whether the cause of the abnormality is nucleic acid or polypeptide or both. Alternatively, it can be determined whether or not an abnormality is caused by different nucleic acids. Therefore, the reliability of the measurement result is improved.

  In the particle measurement method according to the present embodiment, the step (S3) of analyzing the imaged image after the step (S2) of measuring the labeled target nucleic acid and the labeled target polypeptide and / or the other target nucleic acid ) Can be done.

  Thus, based on the imaged image, it can be accurately evaluated whether the cause of the particle abnormality is a nucleic acid or a polypeptide or both of them. Alternatively, it can be accurately evaluated whether an abnormality is caused by mutually different nucleic acids.

  In the particle measurement method according to the present embodiment, the target nucleic acid is labeled with a first labeling substance (S13), and another target nucleic acid different from the target polypeptide and / or target nucleic acid is a second labeling substance different from the first labeling substance. It may be performed to label (S14).

  Thus, when the target nucleic acid and the target polypeptide or other target nucleic acid are respectively labeled, whether the target nucleic acid is amplified or not, whether the target polypeptide is labeled with a labeling substance, or the other target Whether or not the nucleic acid has been amplified can be appropriately determined.

  In the particle measurement method according to this aspect, the step of labeling and amplifying the target nucleic acid in the particle in the particle (S13) prepares a mixed solution containing the particle and a labeling substance that labels the target nucleic acid. In this case, the mixture can be prepared substantially free of the protease.

  When preparation of a mixture for use in amplification of a target nucleic acid is performed in a state in which the mixture contains a protease, the action of the protease causes the polypeptide on the surface of the particle to be degraded. The particles contained in such a mixture can not be labeled with the target polypeptide. On the other hand, when the preparation of the mixture is carried out substantially free of protease, the polypeptide on the surface of the particle is not completely degraded. Thereby, with the particle measurement method according to this aspect, the target polypeptide can be labeled. Thereby, it is possible to simultaneously obtain information based on the target polypeptide while obtaining information based on the target nucleic acid from one particle. Thus, the particles can be evaluated more accurately.

  In this case, the mixture is prepared to contain a polymerase, and can be prepared to further contain a primer. Thereby, reagents necessary for amplification of the target nucleic acid can be added to the mixture, and amplification of the target nucleic acid in the particle can proceed properly.

  In the particle measurement method according to this aspect, after the temperature of the liquid containing particles is set to 25 ° C. or more and 95 ° C. or less (S12), the step (S13) of labeling and amplifying the target nucleic acid in the particles in the particles may be performed.

  When the liquid containing the particles is adjusted to such a temperature range, the clearance of the polypeptide on the surface of the particle is expanded, but the polypeptide is not completely decomposed. Therefore, the particles remain in the liquid while maintaining the shape of the polypeptide on the surface. Then, as the gap widens, the reagent efficiently penetrates into the inside of the particle. Therefore, since the reagent required for amplification of the target nucleic acid is sufficiently present inside the particle, amplification of the target nucleic acid proceeds smoothly. Also, as described above, since the particles are not completely degraded, the same particles can be labeled with the target polypeptide.

  In the particle measurement method according to this aspect, after the temperature of the liquid containing particles is set to 25 ° C. or more and 75 ° C. or less (S12), a step (S13) may be performed to label and amplify the target nucleic acid in the particles in the particles.

  When the liquid containing particles is adjusted to such a temperature range, the particles remain in the liquid while maintaining the shape of the polypeptide on the surface, and the reagent penetrates the inside of the particles more efficiently. Thus, the amplification reaction of the target nucleic acid proceeds more smoothly. Also, as described above, since the polypeptide on the surface of the particle is not completely degraded, the same particle can be labeled with the target polypeptide.

  In the particle measurement method according to the present embodiment, the step of labeling and amplifying the target nucleic acid (S13) may be followed by the step of labeling the target polypeptide and / or another target nucleic acid different from the target nucleic acid (S14).

  Thus, after amplification of the target nucleic acid, the same particle is subjected to the step of labeling the target polypeptide or the step of labeling the other target nucleic acid, whereby the target nucleic acid and the target polypeptide or other target are obtained. Information on nucleic acids can be obtained at one time. Thus, the reliability of evaluation for one particle is increased.

  In the particle measurement method according to the present embodiment, the step of labeling the target polypeptide and / or another target nucleic acid different from the target nucleic acid (S14) comprises labeling the target polypeptide with a labeled antibody that specifically binds to the target polypeptide. It can be done as you

  Thereby, it can be appropriately determined whether the target polypeptide of interest is contained on the surface or inside of the particle.

  In the particle measurement method according to the present embodiment, the step of labeling the target polypeptide of the particle and / or another target nucleic acid different from the target nucleic acid (S14) comprises labeling the target polypeptide contained in the particle and labeling the target In the step of measuring the nucleic acid and the labeled target polypeptide and / or the other target nucleic acid (S2), the labeled target nucleic acid and the labeled target polypeptide can be configured to be measured.

  The “target polypeptide contained in the particle” means a target polypeptide located on the surface of the particle and a target polypeptide present inside the particle.

  According to the particle measurement method of the present embodiment, amplification and labeling of a target nucleic acid and labeling of a target polypeptide can be simultaneously performed on one cell. The cause of the disease may be a combination of multiple genetic abnormalities. Therefore, information on only one target nucleic acid can not make a sufficient diagnosis. With the configuration of this embodiment, the presence or absence of abnormality can be examined for the target nucleic acid and target polypeptide contained in one cell. Then, by correlating these pieces of information complementarily, it is possible to obtain information that is effective in pathological diagnosis and the like.

  In the particle measurement method according to this aspect, the step of labeling the target polypeptide of the particle and / or another target nucleic acid different from the target nucleic acid (S14) labels and labels the other target nucleic acid different from the target nucleic acid. In the step (S2) of measuring the target nucleic acid and the labeled target polypeptide and / or the other target nucleic acid, it may be configured to measure another target nucleic acid different from the labeled target nucleic acid.

  According to the particle measurement method of the present embodiment, labeling and amplification can be performed simultaneously on one target cell and two different target nucleic acids. Therefore, the presence or absence of abnormality of each target nucleic acid can be investigated. Then, by correlating these pieces of information complementarily, it is possible to obtain information that is effective in pathological diagnosis and the like.

  A second aspect of the present invention relates to a sample processing method. The sample processing method according to the present embodiment includes the steps of preparing a mixture containing a particle and a labeled probe that binds to a target nucleic acid in the particle and substantially containing no protease, and a step of amplifying the target nucleic acid in the particle (S13 And).

  In the sample processing method according to this aspect, the preparation of the mixed solution is performed substantially free of the protease. This means that the protease is not added to the mixture, or the protease is added to the mixture, but the effect of the protease is low. Usually, amplification of the target nucleic acid in the particle is performed after protease treatment, but in this embodiment, protease treatment is not necessary. Therefore, usually, when the mixed solution is treated with protease, treatment for inactivating the protease is also performed, but such treatment is also unnecessary. Therefore, the effort required for amplification of the target nucleic acid is saved, and amplification of the target nucleic acid proceeds efficiently. In addition, since the protease and the reagent for inactivating the protease are unnecessary, it contributes to cost reduction.

  Also, since the mixture is substantially free of protease, the polypeptide on the surface of the particle is not completely degraded. Thus, for example, after amplification of the target nucleic acid on the particle, the polypeptide on the surface of the particle is subsequently labeled to target the target polypeptide, and the presence or absence of abnormality in the target polypeptide can be examined. In this way, more tests can be performed on the same particle.

  In the sample processing method according to the present embodiment, the mixed solution is configured to further include a DNA polymerase, and may be prepared to further include a primer. Thereby, reagents necessary for amplification of the target nucleic acid can be added to the mixture, and amplification of the target nucleic acid in the particle can proceed properly.

  In the sample processing method according to this aspect, after the temperature of the liquid containing particles is adjusted to 25 ° C. or more and 95 ° C. or less (S12), a step of preparing a mixed liquid may be performed. Thereby, as in the first aspect, the amplification reaction of the target nucleic acid proceeds smoothly. In addition, since the protease reaction is not required for the amplification reaction of the target nucleic acid, the protease and the reagent for inactivating the protease become unnecessary, which contributes to cost reduction.

  In the sample processing method according to the present embodiment, after the temperature of the liquid containing particles is set to 25 ° C. or more and 75 ° C. or less (S12), the step of preparing the mixed liquid may be performed. Thus, as in the first aspect, the amplification reaction of the target nucleic acid proceeds more smoothly.

  In the sample processing method according to the present embodiment, the step (S2) of measuring the labeled target nucleic acid may be performed after the step (S13) of labeling and amplifying the target nucleic acid.

  As a method of measuring whether the amplification of the target nucleic acid has been properly performed, for example, a flow cell which is irradiated with light from a light source, ie, a liquid containing a labeled target nucleic acid and a labeled target polypeptide. And imaging the light generated from the liquid, and imaging with a fluorescence microscope. Such measurement makes it possible to evaluate whether amplification of the target nucleic acid has been performed.

  In the sample processing method according to this aspect, the step (S2) of measuring the labeled target nucleic acid can be configured to include a step of imaging an image of the particle.

  Thereby, the operator can visually confirm the presence or absence of amplification of the target nucleic acid.

  In the sample processing method according to this aspect, the step of imaging the image of particles is obtained by flowing a liquid containing a labeled target nucleic acid to the flow cell (10) and irradiating the liquid in the flow cell (10) with light. It may be configured to image the illumination light.

  Thereby, the presence or absence of amplification of a target nucleic acid can be confirmed about a lot of particles. Therefore, the reliability of the measurement result is improved.

  In the sample processing method according to this aspect, after the step (S2) of measuring the labeled target nucleic acid, the step of analyzing the imaged image may be performed.

  As described above, the cause of the particle abnormality can be accurately evaluated based on the captured image.

  A third aspect of the invention relates to a particle imaging device. In the particle imaging apparatus according to the present embodiment, the target nucleic acid in the particle is labeled and amplified in the particle, and a fluid containing particles labeled with the target polypeptide of the particle and / or another target nucleic acid different from the target nucleic acid is flowed A flow cell (10), a light source for irradiating light to the flow cell (10), and an imaging unit (154) for imaging light obtained from particles in the liquid by irradiating the light from the light source.

  Thereby, as in the first aspect, whether or not amplification of a target nucleic acid and amplification of a label of a target polypeptide or amplification of another target nucleic acid can be efficiently performed on the same particle.

  In the particle imaging device according to the present embodiment, the flow cell (10) may be configured to flow a liquid containing particles in which the target nucleic acid in the particles is labeled and amplified in the particles and the target polypeptide on the surface of the particles is labeled. .

  This makes it possible to determine, for a large number of particles, whether the cause of the abnormality is a nucleic acid or a polypeptide. Therefore, the reliability of the measurement result is improved.

  In the particle imaging device according to the present embodiment, the imaging unit is configured to label the fluorescence obtained from the first labeling substance that labels the target nucleic acid, and the second labeling substance that labels the target polypeptide and / or another target nucleic acid different from the target nucleic acid. And the fluorescence obtained from.

  As a result, for the same particle, the presence or absence of amplification of the target nucleic acid and the presence or absence of the label of the target polypeptide or the presence or absence of amplification of the other target nucleic acid can be clearly determined. Thus, for the particles to be measured, it can be determined whether the cause of the abnormality is nucleic acid or polypeptide or both. Alternatively, it can be determined whether the two nucleic acids are different from each other or one or the other. Therefore, the reliability of the measurement result is improved.

  In the particle imaging device according to this aspect, the liquid may be configured to be substantially free of the protease.

  This eliminates the need for treatment for inactivating the protease with respect to the labeling and amplification of the target nucleic acid and the labeling of the target polypeptide, which saves time and effort. In addition, since the protease and the reagent for inactivating the protease are unnecessary, it contributes to cost reduction.

  In the particle imaging device according to this aspect, the imaging unit (154) may be configured to be a CCD camera. If the imaging unit is a CCD (Charge-Coupled Device) camera, images of the labeled target nucleic acid and the labeled polypeptide can be clearly imaged.

  In the particle imaging device according to this aspect, the imaging unit (154) may be configured to be a TDI camera. If the imaging unit is a TDI (Time Delay Integration) camera, the fluorescence received at the light receiving surface of the imaging unit is integrated to generate a fluorescence image and a bright field image. This can enhance the quality of the fluorescent and bright field images of the cells.

  According to the present invention, a particle measurement method, a sample processing method, and a particle imaging apparatus can be obtained which can efficiently obtain information effective for pathological diagnosis and the like.

FIG. 1 is a flowchart illustrating the particle measurement method according to the embodiment. FIG. 2 is a schematic configuration view of a flow cytometer to which the particle measurement method according to the embodiment is applied. FIG. 3 is a view showing a display unit that displays a bright field image, a fluorescence image based on a target nucleic acid, a fluorescence image based on a target polypeptide, and a merged image. FIG. 4 is a diagram showing an example of a screen displaying an analysis result based on the measurement result of the measurement sample. FIGS. 5 (a) to 5 (d) are diagrams illustrating a particle measurement method according to a comparative example of the embodiment. FIG. 5 (a) is a schematic view illustrating amplification of a target nucleic acid. FIG. 5 (b) is a flowchart illustrating amplification of a target nucleic acid. FIG. 5 (c) is a schematic view illustrating labeling of a target polypeptide. FIG. 5 (d) is a flowchart illustrating labeling for a target polypeptide. Fig.6 (a) is a figure explaining the particle | grain measuring method which concerns on embodiment. FIG.6 (b) is the figure which showed the information obtained from one particle | grain by the particle | grain measuring method which concerns on embodiment. FIG. 7 is a graph showing the results of measurements 1 to 5 according to verification 1. FIG. 8 shows a bright field image, an image of a target nucleic acid, an image of a target polypeptide, and a mixed image of a target nucleic acid and a target polypeptide, obtained from measurement 1 according to Verification 1. FIG. 9 is a graph showing the results of measurements 4 and 6 to 8 according to verification 2. FIG. 10 shows a bright field image, an image of a target nucleic acid, an image of a target polypeptide, and a mixed image of a target nucleic acid and a target polypeptide obtained from measurement 4 according to Verification 5. FIG. 11 is a bright field image, an image of a target nucleic acid, an image of a target polypeptide, and a mixed image of a target nucleic acid and a target polypeptide, obtained from measurement 9 according to Verification 6. FIG. 12 is an image of the target nucleic acid of measurement 10 according to Verification 7. FIG. 13 (a) is a bright field image and a fluorescence image of the target nucleic acid of measurement 11 according to verification 8. FIG. 13 (b) is a scattergram before and after PCR of measurement 11 according to verification 8. FIG. 14 is a bright field image, an image of a target nucleic acid, an image of a target polypeptide, and a mixed image of a target nucleic acid and a target polypeptide, obtained from the measurement 12 according to Verification 9. FIG. 15 shows a bright field image, an image of an amplified target nucleic acid, an image of a target nucleic acid labeled by FISH, an image of a target nucleic acid labeled by FISH, and an amplified target nucleic acid and FISH obtained from measurement 13 according to verification 10 It is a mixed image with a target nucleic acid. FIG. 16 is a schematic view for explaining the related art.

Embodiment
The particle measurement method according to an embodiment of the present invention performs amplification of a target nucleic acid and binding of a target polypeptide and an antibody that labels the polypeptide to the same particle to be measured.

  Here, the “particles to be measured” refers to cells, bacteria, etc. which are extracted from the sample, have nucleic acid, and whose surface is covered with the polypeptide. The particles to be measured may be endoplasmic reticulum exosomes or the like. The above-mentioned "specimen" refers to blood directly collected from a person, tissue fragments such as skin, and the like. A "polypeptide" is a molecule in which amino acid residues are linked by peptide bonds, and includes, for example, relatively low molecular weight amino acid polymers and large molecular weight proteins. In the particle measuring method according to the embodiment, the particles to be measured are treated with a drug such as formalin, that is, the particles to be measured are not dispersed but dispersed in a liquid. .

  In the following, the operator prepares a sample to be subjected to measurement, and executes a particle measurement method using an apparatus for imaging fluorescence generated from the prepared sample. All steps of the particle measurement method may be performed automatically by the device.

1. Particle Measurement Method First, the particle measurement method according to the embodiment will be described based on FIG.

[Preparation of measurement sample]
In step S1, the operator prepares a sample to be subjected to measurement, ie, a measurement sample. The preparation of this measurement sample includes the four processes of steps S11 to S14. The details will be described below.

(1) Pretreatment As step S11, the operator collects a sample, that is, a tissue piece or the like from the subject. From this sample, particles to be measured, that is, cells and bacteria are extracted. The operator disperses the extracted particles in the liquid. Then, the operator mixes the buffer solution, the reagent, and the like with the liquid, and performs processing by centrifugation.

(2) Temperature Adjustment for Liquid After Pretreatment Next, in step S12, the operator adjusts the temperature of the liquid after pretreatment. This treatment is a very important treatment in the later binding of the target polypeptide to the labeled antibody. It is preferable that the temperature of the liquid after the pretreatment be adjusted in the range of 25 ° C. or more and 95 ° C. or less. More preferably, the temperature is in the range of 25 ° C. to 75 ° C. When the temperature of the liquid after pretreatment is adjusted to such a range, when the polymerase and the primer are added to the liquid, the efficiency of penetration of the reagent into the particles to be measured is increased, and amplification of the target nucleic acid is improved. Proceed smoothly.

(3) Amplification of Target Nucleic Acid Subsequently, in step S13, the operator adds a polymerase and a primer to the liquid after pretreatment to prepare a mixed solution in order to perform amplification of the target nucleic acid. In this process, the adjustment of the temperature performed earlier allows the polymerase and the primer to penetrate efficiently into the particles to be measured. The "polymerase" is, for example, a DNA polymerase.

  The operator then uses the PCR device to label and amplify the target nucleic acid. The PCR device performs temperature control on the measurement sample to amplify and label the target nucleic acid. Here, after the first labeling substance that labels the target nucleic acid is added to the mixture, amplification of the target nucleic acid may be performed, or after amplification of the target nucleic acid, the first labeling substance is added to the mixture. It is also good. The “first labeled substance” is a labeled substance for confirming whether or not amplification of the target nucleic acid has been performed, and is, for example, a labeled probe such as GAPDH_97_FAM. Moreover, in order to distinguish from the labeling substance in the labeling of the target polypeptide described below, it is referred to as "first labeling substance" for convenience of explanation.

(4) Binding Reaction Between Target Polypeptide and Labeled Antibody Next, in step S14, the operator carries out a binding reaction between the target polypeptide and the labeled antibody. As described above, since temperature control is performed on the pretreated liquid, the gap formed in the polypeptide is expanded while the shape of the polypeptide on the surface of the particle is maintained. For this reason, the target polypeptide can be labeled on the polypeptide on the surface of the particle. Therefore, according to the embodiment, both the labeling of the target nucleic acid and the labeling of the target polypeptide can be performed on the same particle.

  Whether or not the binding reaction between the target polypeptide and the labeled antibody has been performed is confirmed with a second labeled substance different from the first labeled substance of the target nucleic acid described above. The second labeled substance is, for example, a labeled probe such as Alexa Fluor 647 anti-human CD45 Antibody.

  Thus, preparation of the measurement sample is completed. Next, measurement is performed on the measurement sample. In the present embodiment, the case of performing measurement using an apparatus for imaging fluorescence generated from a measurement sample will be described.

[Measurement of measurement sample]
In step S2, the operator obtains an image of the labeled target nucleic acid and an image of the labeled target polypeptide using a device for imaging fluorescence generated from the measurement sample. The apparatus used in step S2 is a particle imaging apparatus, for example, a flow cytometer.

  FIG. 2 is a view schematically showing the configuration of a flow cytometer. The flow cytometer 10 includes an imaging unit 100, a processing unit 11, a storage unit 12, a display unit 13, and an input unit 14. The imaging unit 100 captures an image of the flow cell 110, the light sources 121 to 123, the focusing lenses 131 to 133, the mirror 141, the dichroic mirror 142, the focusing lens 151, the optical unit 152, the focusing lens 153, And a unit 154.

  As described above, in step S1, the prepared measurement sample is flowed to the flow path 111 of the flow cell 110 provided in the flow cytometer 10. The particles contained in the measurement sample are imaged by the imaging unit 100.

  The light sources 121 to 123 irradiate light to the measurement sample flowing through the flow cell 110. The light sources 121 to 123 are formed of, for example, a semiconductor laser or an argon laser. The lights emitted from the light sources 121 to 123 are laser lights of wavelengths λ11 to λ13 different from one another. The condenser lenses 131 to 133 condense the light emitted from the light sources 121 to 123, respectively. The mirror 141 reflects light of wavelength λ11. The dichroic mirror 142 transmits light of wavelength λ11 and reflects light of wavelength λ12. Thus, light of wavelengths λ11 to λ13 is applied to the measurement sample flowing through the flow path 111 of the flow cell 110.

  When the measurement sample is irradiated with light of wavelengths λ11 and λ12, fluorescence is generated from the fluorescent dye that is staining the particles. Specifically, the fluorescent dye that labels the target nucleic acid, ie, the first labeling substance, emits fluorescence of wavelength λ21 when it is irradiated with light of wavelength λ11. The fluorescent dye that labels the target polypeptide, ie, the second labeling substance, emits fluorescence of wavelength λ22 when it is irradiated with light of wavelength λ12. When the measurement sample is irradiated with light of wavelength λ13, this light passes through the cells. The light of wavelength λ13 transmitted through the cells is used to generate a bright field image. The wavelengths λ21, λ22, and λ13 are different from each other.

  The condenser lens 151 condenses the fluorescence of the wavelengths λ21 and λ22 generated from the measurement sample flowing in the flow path 111 of the flow cell 110 and the light of the wavelength λ13 transmitted through the measurement sample flowing in the flow path 111 of the flow cell 110. The optical unit 152 has a configuration in which three dichroic mirrors are combined. The three dichroic mirrors of the optical unit 152 reflect the fluorescence of the wavelengths λ21 and λ22 and the light of the wavelength λ13 at slightly different angles from each other and separate them on the light receiving surface of the imaging unit 154. The condensing lens 153 condenses the fluorescence of the wavelengths λ21 and λ22 and the light of the wavelength λ13.

  The imaging unit 154 is configured of, for example, a CCD (Charge-Coupled Device) camera or a CMOS (Complementary Metal-Oxide Semiconductor) camera. With a CCD camera, images of labeled target nucleic acid and labeled polypeptide can be clearly imaged. Further, the imaging unit 154 is preferably configured by a TDI (Time Delay Integration) camera. The TDI camera integrates fluorescence received at the light receiving surface to generate a fluorescence image and a bright field image. This can enhance the quality of the fluorescent and bright field images of the cells. The imaging unit 154 captures the fluorescence of the wavelengths λ21 and λ22 and the light of the wavelength λ13, and captures a fluorescence image corresponding to the fluorescence of the wavelengths λ21 and λ22 and a bright field image corresponding to the light of the wavelength λ13. Output as a signal.

  The processing unit 11 is configured of a CPU. The processing unit 11 is not limited to this, and may be configured by a CPU and a microcomputer. The processing unit 11 performs various processes based on the program stored in the storage unit 12. The processing unit 11 is connected to the imaging unit 100, the storage unit 12, the display unit 13, and the input unit 14, receives a signal from each unit, and controls each unit. The storage unit 12 is configured by a RAM, a ROM, a hard disk, and the like. The display unit 13 is configured of a liquid crystal display, a plasma display, a CRT (Cathode Ray Tube) display, or the like. The input unit 14 is configured of, for example, a mouse, a keyboard, and the like.

  The processing unit 11 stores the fluorescence image and the bright field image captured by the imaging unit 154 in the storage unit 12. When the operator inputs an instruction to display an image via the input unit 14, the processing unit 11 displays a measurement result screen 200 including a fluorescence image and a bright field image on the display unit 13.

  As shown in FIG. 3, the measurement result screen 200 displays a bright field image, a fluorescence image based on a target nucleic acid, a fluorescence image based on a target polypeptide, and a merged image (mixed image). The merge image is a fluorescence image in which the fluorescence image based on the target nucleic acid and the fluorescence image based on the target polypeptide are merged. Thus, the measurement result of the measurement sample measured by the flow cytometer 10 is displayed.

  In addition to the flow cytometer, the measurement of the measurement sample also includes, for example, acquisition of an image by a fluorescence microscope.

[analysis]
In step S3, the processing unit 11 analyzes predetermined items based on the image of the labeled target nucleic acid obtained in step S2 and the image of the labeled target polypeptide. For example, if the measurement sample is measured by the flow cytometer 10, the number of target nucleic acids or target polypeptides is counted from the measurement results displayed on the measurement result screen 200 shown in FIG. Assess whether it is normal or abnormal. Also, in terms of whether the target nucleic acid or target polypeptide appears clearly in the image, is the cause of the abnormality in the particle, that is, the cell or bacteria, is the nucleic acid or the polypeptide or both? Can also be evaluated.

[Display of analysis result]
In step S4, the processing unit 11 causes the display unit 13 to display an analysis result screen 201 as shown in FIG. 4 as a result of analysis in step S3. In FIG. 4, as an example of the display, a case where a measurement target is a white blood cell, a target nucleic acid is a GAPDH gene, and a target polypeptide is a human CDα antigen is shown. In the "Analysis Results" column, the presence or absence of abnormality of the nucleic acid and the polypeptide is displayed. FIG. 4 shows, for example, the case where the nucleic acid is abnormal and the polypeptide is normal. On the analysis result screen 201, the measurement result obtained in step S2 may be displayed. Also, scattergrams and histograms may be created.

  By displaying the analysis results in this manner, for example, a doctor or the like can use it to determine the treatment policy when examining abnormalities in nucleic acid and polypeptide in order to diagnose a disease.

2. Comparison of the particle measurement method according to the comparative example and the particle measurement method according to the embodiment In the particle measurement method according to the embodiment, amplification of the target nucleic acid and labeling of the target polypeptide are performed on the same particle, so The particle measurement method according to the embodiment is largely different from the conventional particle measurement method. Hereinafter, the conventional particle measurement method is used as a comparative example, and is compared with the particle measurement method according to the embodiment of the present invention. In the following, referring to FIGS. 5 (a) to 5 (d), a case is considered where amplification of nucleic acid is performed in order to investigate the cause of disease by applying the particle measurement method according to the comparative example.

[Particle Measuring Method According to Comparative Example]
FIGS. 5 (a) and 5 (b) are a schematic diagram and a flowchart showing the procedure of amplification of a target nucleic acid in a comparative example, respectively.

  In the comparative example, particles to be measured, that is, cells, bacteria and the like collected from a subject are treated with a drug such as formalin from the viewpoint of storage. Such a condition is a so-called "cell-fixed" condition, and can not be penetrated by a reagent containing DNA polymerase and a primer into the cell. Therefore, in the comparative example, as shown in FIG. 5 (a), when amplification of the target nucleic acid is performed, processing for degrading the polypeptide, ie, protease treatment is required.

  As shown in FIG. 5 (b), a protease treatment is performed in step S101. In the comparative example, as shown in FIG. 5A, since the polypeptide is degraded by the protease treatment, the reagent for amplifying the target nucleic acid can penetrate into the cell. Then, in step S102, amplification of the target nucleic acid is performed. Thereafter, the operator acquires an image of the target nucleic acid using, for example, a fluorescence microscope or the like.

  If amplification of the target nucleic acid in cells is not confirmed in the obtained image of the target nucleic acid, it can be understood that the cause of the disease is not in the nucleic acid. In this case, as a possible cause, there is an abnormality in the polypeptide.

  FIG.5 (c) and (d) are the schematic diagram and flowchart which respectively show the procedure of the label of the polypeptide in a comparative example.

  As shown in FIG. 5D, in step S103, the target polypeptide is labeled by performing a binding reaction between the target polypeptide and the labeled antibody. Here, in the cells used for amplification of the target nucleic acid, the polypeptide is degraded by protease treatment. Therefore, the binding reaction between the target polypeptide and the labeled antibody can not be performed on the cells used for amplification of the target nucleic acid. Therefore, the operator separately prepares a sample including other cells collected from the same subject, and then performs a binding reaction between the target polypeptide and the labeled antibody. Also in this case, the operator acquires an image of the target polypeptide using, for example, a fluorescence microscope or the like.

  Thus, in the comparative example, the operator is required to separately perform amplification of the target nucleic acid and a binding reaction between the target polypeptide and the labeled antibody, which is time-consuming and complicated. In addition, the above-described treatment is performed in a state where cells are mounted on a slide glass. Therefore, when amplification of target nucleic acid is desired for many particles, that is, cells, the operator is burdened with work.

  Furthermore, in the comparative example, the information found from one particle is either the presence or absence of an abnormal nucleic acid or the presence or absence of a target polypeptide. That is, only one piece of information can be obtained from one particle. For this reason, nucleic acid and polypeptide information can not be evaluated complementarily to one particle.

[Differences between the particle measurement method according to the comparative example and the particle measurement method according to the embodiment]
Next, differences between the particle measurement method according to the embodiment of the present invention and the particle measurement method according to the comparative example will be described.

  In embodiments, amplification of the target nucleic acid and labeling of the target polypeptide are performed on the same particle. The binding reaction between the target polypeptide and the labeled antibody in the embodiment can be performed because the polypeptide on the surface of the particle is not completely degraded.

  As a factor that enables the above, in the embodiment, the particles or cells to be measured are dispersed in a liquid. That is, in the embodiment, unlike the comparative example, the particles are not in a fixed state. For this reason, when a reagent containing a polymerase and a primer is allowed to penetrate into the particle, it is necessary to give a certain degree of impact to the polypeptide on the surface of the particle, but not as strong as in the comparative example.

  Therefore, in the particle measuring method according to the embodiment, the temperature of the liquid after the pretreatment is adjusted as shown in FIG. 6A in order to allow the reagent to permeate into the particles sufficiently. The temperature adjustment shown in FIG. 6A corresponds to the process of temperature adjustment (S12) on the liquid after the pretreatment in step S1 of FIG. Thereby, the polypeptide on the surface of the particle is not completely degraded, and the gap is expanded while maintaining its shape. The reagent containing the polymerase and the primer penetrates into the interior of the particle through the expanded gap. Thereby, amplification of the target nucleic acid is performed in the particle even in the state substantially free of the protease. The amplification of the target nucleic acid shown in FIG. 6 (a) corresponds to the process of labeling and amplification (S13) of the target nucleic acid in step S1 of FIG.

  In addition, in FIG. 6 (a), although a mode that the labeled probe which labels target nucleic acid is added before performing amplification of target nucleic acid is illustrated, you may carry out after amplification of target nucleic acid. .

  Here, "a state substantially containing no protease" means not only the case where no protease is added to the mixed solution, but the amount of the mixed solution is a very small amount although the protease is added. It also means the case where the effect of is not played.

  Therefore, after amplification of the target nucleic acid is performed on the same particle in step S1 of FIG. 1, the binding reaction between the target polypeptide and the labeled antibody is performed on the polypeptide on the surface of the non-degraded particle. It can be performed.

  Thus, in the particle measurement method according to the embodiment, amplification of a target nucleic acid and labeling of a target polypeptide can be performed on one particle, that is, one cell, bacteria, or the like. Therefore, amplification of the target nucleic acid and labeling of the target polypeptide can be efficiently performed.

[Effect by the particle measurement method according to the embodiment]
The particles of the embodiment, that is, cells, bacteria and the like are dispersed in a liquid. Thus, for example, as in the comparative example, when the particles are fixed with a drug such as formalin,
It is not necessary for the operator to carry out processing such as placing particles on a slide glass. Therefore, with the particle measurement method according to the embodiment, the operator can measure the target nucleic acid on a large number of cells at one time using a flow cytometer.

  In addition, it is not necessary to prepare cells for amplification of the target nucleic acid and labeling of the target polypeptide. This does not require, for example, multiple samples or scraps of diseased patients, leading to reduced stress on the patient.

  Also, in embodiments, the polymerase and primers are allowed to penetrate into the particle substantially free of the effects of the protease. For this reason, it is not necessary to carry out treatment to inactivate the protease. Thus, reagents can be saved.

  As described above, in the comparative example, the information obtained from one particle is the presence or absence of a nucleic acid abnormality or the presence or absence of a polypeptide abnormality. That is, only one piece of information can be obtained from one particle. On the other hand, in the particle measurement method according to the embodiment, as shown in FIG. 6 (b), one combination of four types of particles can be obtained from one particle depending on the combination of the presence or absence of nucleic acid abnormality and the presence or absence of polypeptide abnormality. Information matching any one is obtained. And, since information of both the nucleic acid and the polypeptide can be obtained at one time and in a large amount, more reliable information can be obtained. By complementarily relating these pieces of information, it is possible to accurately evaluate particles. As a result, it is possible to propose appropriate medications and treatment policies to the patient.

  In addition, since information of the target nucleic acid and information of the polypeptide on the surface of the particle can be obtained from one particle, it can be expected to be utilized for pathological diagnosis as a new biomarker. This is because, for example, when it is obtained from the information of nucleic acid that a nucleic acid is mutated and cancerous, PD-L1 (programmed cell death-1 ligand-1) is expressed on the surface of the cancered cell If it is possible to obtain the information, it is possible to select Nivolmal, which is an anti-PD-1 antibody drug, as a therapeutic drug for cancer.

  In addition, when it is obtained from the information of nucleic acid that a mutation occurs in the nucleic acid in the cell or exosome in the blood, the information on the surface antigen of the cell or exosome can be obtained to obtain the cell or exosome It is possible to identify which organ comes from. This helps to determine which organ the tumor has developed.

3. Verification of particle measurement method according to the embodiment
Next, the particle measurement method according to the embodiment of the present invention is used to verify the conditions for optimally amplifying the target nucleic acid and labeling the target polypeptide for the same particle, that is, cells, bacteria, etc. The authors performed verifications 1 to 8. First, the procedure of preparing the measurement sample to be subjected to verification 1 to 8 will be described.

[Preparation of measurement sample]
(A) Pretreatment White blood cells were used as particles to be measured. The target nucleic acid was GAPDH gene, and the target polypeptide was human CD45 antigen.
(A-1) White blood cells collected from a subject in an appropriate amount were dispersed in a liquid, and the total volume was adjusted to 200 μL. This was accommodated in a predetermined tube.
(A-2) 1 mL of hemolytic agent was added to the tube.
(A-3) ice-cold for 15 minutes.
(A-4) The tube was centrifuged at 400 rpm for 10 minutes in a 4 ° C. environment.
(A-5) The supernatant was removed.
(A-6) 400 μL of a hemolytic agent was added to a tube to wash the contents in the tube.
(A-7) The tube was centrifuged at 400 rpm for 10 minutes in a 4 ° C. environment.
(A-8) The supernatant was removed.
(A-9) 180 μL of PBS was put in a tube to suspend the contents.
(A-10) The contents were adjusted to a predetermined temperature and held for a predetermined time.
(A-11) The tube was centrifuged at 400 rpm for 10 minutes in a 4 ° C. environment.
(A-12) The supernatant was removed.

(B) Amplification of target nucleic acid (B-1) In order to amplify target nucleic acid, a reagent was added to a tube. The composition of the reagent was as follows.
-3 'end side primer: GAPDH_97_F
・ Primer on 5 'end side: GAPDH_97_R
-DNA polymerase: Taqman (registered trademark) Gene Expression Master Mix
· Labeled probe of target nucleic acid: GAPDH_97_FAM
-The concentration and amount of each composition contained in distilled water (B-2) reagent were as follows. In the following, "before use" means when in a product state or a storage state. "Final concentration" is the concentration when prepared as a reagent.
3 'end side primer: 0.5 μL was added to a tube. The concentration before the start of use was 10.0 μM, and the final concentration was 0.2 μM.
5 'end primer: 0.5 μL was added to the tube. The concentration before the start of use was 10.0 μM, and the final concentration was 0.2 μM.
Taqman® Gene Expression Master Mix: 2x concentrated as product but diluted 1x before use. To the tube was added 12.5 μL of 1 × diluted Taqman® Gene Expression Master Mix.
GAPDH_97_FAM: 0.5 μL was added to the tube. The concentration before the start of use was 10.0 μM, and the final concentration was 0.2 μM.
Distilled water: 11 μL was added to a tube.
The reagents were prepared as described above. The total volume was 25.0 μL.
(B-3) After adding the above-described reagent, the tube was set in a PCR device to carry out an amplification reaction of the target nucleic acid. The target nucleic acid amplification reaction is carried out at 50 ° C. for 2 minutes and then at 95 ° C. for 10 minutes, followed by two steps of 95 ° C. for 15 seconds as a first step and 60 seconds at 60 ° C. for a second step. 40 cycles were performed.

(C) Labeling of Target Polypeptide Following amplification of the target nucleic acid, a binding reaction between the target polypeptide and the labeled antibody was performed. The procedure is shown below.
(C-1) 75 μl of PBS was put in a tube to suspend a measurement sample.
(C-2) 5 μL of anti-CD45 antibody was added to a tube. In addition, Alexa Fluor 647 anti-human CD45 Antibody (BioLegend) was used as a labeled probe for labeling the binding reaction between the target polypeptide and the labeled antibody.
(C-3) The binding reaction between the target polypeptide and the labeled antibody was performed for 60 minutes under an environment of 4 ° C.
The following verification was performed using the measurement sample prepared by the above procedure.

[Verification 1: Relationship between efficiency of target nucleic acid amplification and temperature control of liquid after pretreatment]
In Verification 1, the efficiency of amplification of the target nucleic acid was measured, and it was verified whether amplification of the target nucleic acid was performed with the measurement sample prepared in the above procedure. In addition, the relationship with the contents, that is, the temperature control of the liquid after pretreatment was also verified. Specifically, in the procedure (A-10) of preparation of the measurement sample, it was verified whether or not there was a change in the efficiency of amplification of the target nucleic acid when the temperature was changed.

  In the procedure (A-10), the temperature of the contents is set to 1: 25 ° C., 2: 50 ° C., 3: 75 ° C., 4: 95 ° C., 5: 100 ° C. The temperature was 2 minutes. All conditions were the same except for the temperature condition.

  The verification results are shown in the graph of FIG. The Ct value shown in FIG. 7 is an index showing the efficiency of amplification of the target nucleic acid, and the lower the Ct value, the better the amplification efficiency of the target nucleic acid. As shown in the graph of FIG. 7, the Ct value was different for each temperature. Therefore, it was found that the temperature adjustment of the liquid after pretreatment affects the efficiency of amplification of the target nucleic acid. Among them, since the Ct value is the lowest in the case of measurement 4 (95 ° C.), the efficiency of amplification of the target nucleic acid is the best when the liquid after pretreatment is adjusted to 95 ° C. in procedure (A-10) I found that.

[Conclusion of Verification 1]
Even when the target polypeptide is labeled after amplification of the target nucleic acid on the measurement sample, amplification of the target nucleic acid is not affected.
Temperature during temperature control of the liquid after pretreatment affects the efficiency of target nucleic acid amplification.
When the temperature of the liquid after pretreatment is adjusted to 95 ° C. and the holding time is 2 minutes, the efficiency of amplification of the target nucleic acid is good.

[Verification 2: Target nucleic acid amplification and target polypeptide labeling]
In Verification 1, in the procedure (A-10), when the temperature of the liquid after pretreatment is adjusted to 95 ° C. and the holding time is 2 minutes (measurement 4), the efficiency of amplification of the target nucleic acid may be improved. I understood. However, verification 1 has not been verified as to whether labeling of the target polypeptide has been properly performed. Therefore, for each of the conditions of measurement 1 to 5 of verification 1, it was verified whether amplification of the target nucleic acid and labeling of the target polypeptide were both appropriately performed.

  In Verification 2, the ratio of the total number of cells in which amplification of the target nucleic acid was performed and the number of cells in which the target polypeptide was labeled was divided by total cells. That is, since this ratio is a ratio at which both amplification of the target nucleic acid and labeling of the target polypeptide were performed, it can be judged that the higher the value, the more efficiently the two reactions were performed. The results are shown in Table 1.

  The total number of cells (b) and the number of white blood cells (c) in Table 1 were measured by a general-purpose flow cytometer. (D) Amplification of target nucleic acid, and the number of cells labeled with the target polypeptide were calculated from the image captured by the flow cytometer. In the calculation of (d) in Table 1, the fluorescence intensity is higher than a predetermined threshold in the fluorescence image based on the target nucleic acid, and the fluorescence intensity is higher than the predetermined threshold in the fluorescence image based on the target polypeptide It was determined that the target nucleic acid was amplified and that the target polypeptide was labeled. Then, the target nucleic acid amplification and the number of cells determined to have labeled the target polypeptide were taken as the value of (d) in Table 1.

  In the verification 1, as shown in the graph of FIG. 7, the efficiency of amplification of the target nucleic acid was the best in the case of measurement 4. However, according to the results in Table 1, in the case of measurement 4, the percentage (a) of the target nucleic acid amplification and the target polypeptide labeled cells (e) was as low as 16.7%. On the other hand, the proportion of measurement 1 (e) was the highest 43.6%, and the proportions of measurement 2 and measurement 3 (e) were 28.2% and 22.5%, respectively. From this result, it was found that in the procedure (A-10), the proportion (e) decreases as the temperature of the liquid after the pretreatment is adjusted to a high temperature. In particular, the proportion (e) at 100 ° C. of Verification 5 was as low as 14.8%.

  From the results of Verification 2, the particle measurement method according to the embodiment is performed on the same cell when the temperature of the liquid after the pretreatment is in the range of 25 ° C. or more and 95 ° C. or less in the procedure (A-10). It has been found that both amplification of the target nucleic acid and labeling of the target polypeptide can be performed efficiently.

  Here, in the case of measurement 4, although the proportion (e) is as low as 16.7%, at least amplification of the target nucleic acid is well performed as shown in the graph of FIG. 7 which is the result of verification 1 The Therefore, in the case of measurement 4, it can be determined that both amplification of the target nucleic acid and labeling of the target polypeptide have been performed on the same cell.

  In the case of measurement 5, when the temperature of the liquid after pretreatment is adjusted to a high temperature as a cause of the low proportion (e), it affects the cells themselves and is contained in the sample before amplification of the target nucleic acid. It is possible that the number of cells that had been reduced has decreased and the number of cells to be measured has decreased. Therefore, when the temperature is raised to a high temperature in the procedure (A-10), good results are obtained regarding the state of efficient amplification of the target nucleic acid, the amplification of the target nucleic acid, and the proportion of cells labeled with the target polypeptide. It is thought that a large amount of cells are needed to

  Also, as described above, (d) amplification of the target nucleic acid in Table 1 and the number of cells labeled with the target polypeptide were calculated from the image captured by the flow cytometer. Such an image is specifically an image as shown in FIG. Each image in FIG. 8 is an image obtained by imaging the particles of measurement 1, and is a bright field image, an image of a target nucleic acid, an image of a target polypeptide in order from the left side. And the target polypeptide image. As described above, when the procedure of the measurement sample according to the embodiment is processed, the image of the target nucleic acid and the image of the target polypeptide imaged by the flow cytometer can be appropriately acquired.

[Conclusion of Verification 2]
When amplification of the target nucleic acid to the same cell and labeling of the target polypeptide are carried out with the temperature holding time being 2 minutes and the temperature in the range of 25 ° C. or more and 95 ° C. or less at the time of temperature control of the liquid after pretreatment. It works well.
-In particular, when it is in the range of 25 ° C. to 75 ° C., it is better.

[Verification 3: Efficiency of target nucleic acid amplification and time for temperature adjustment of liquid after pretreatment]
In Verification 1, when the temperature adjustment of the liquid after pretreatment was performed at 95 ° C., it was found that the efficiency of amplification of the target nucleic acid was the best. Here, in Verification 3, the relationship between the time for temperature adjustment of the liquid after pretreatment and the efficiency of amplification of the target nucleic acid was verified.

  In procedure (A-10), the temperature of the liquid after pretreatment was 95 ° C., and the holding time was set to a measurement of 6: 1 minutes and a measurement of 7:10 minutes. In addition, in the temperature adjustment of procedure (A-10), measurement 1 was performed by adjusting the temperature of the liquid after pretreatment to 25 ° C. In verification 2, amplification of the target nucleic acid and labeling of the target polypeptide were performed The proportion of cells was good. Therefore, the temperature of the liquid after pretreatment is adjusted to 25 ° C., and the case of holding time being 0 minutes is taken as measurement 8, and the measurement 8 is also time for performing temperature control of liquid after pretreatment and amplification of target nucleic acid The relationship between the efficiency of The Ct values of measurements 6-8 are shown in FIG.

  As a result, as shown in FIG. 9, the Ct value of measurement 6 was lower than the Ct value of measurement 4. Therefore, in procedure (A-10), when adjusting the temperature of the liquid after pre-processing to 95 degreeC, if holding time was made into 1 minute, it turned out that amplification of a target nucleic acid is performed efficiently.

  In addition, the efficiency of amplification of the target nucleic acid is worse than the measurements 6 and 7 because the Ct value is the highest in the measurement 8, but as shown in the graph of FIG. It was a Ct value. Therefore, under the condition of measurement 8, as in measurement 1, it was predicted that the proportion of cells in which amplification of the target nucleic acid and labeling of the target polypeptide were performed was high.

[Conclusion of Verification 3]
When the temperature of the liquid after pretreatment is adjusted to 95 ° C., the efficiency of amplification of the target nucleic acid is good if the holding time is 1 minute.

[Verification 4: Target nucleic acid amplification and target polypeptide labeling]
Next, as in the verification 2, conditions of measurement 6 to 8 were verified as to whether amplification of the target nucleic acid and labeling of the target polypeptide were performed. The total number of cells in which amplification of the target nucleic acid was performed and the number of cells in which the target polypeptide was labeled was divided by total cells to calculate the ratio. The results are shown in Table 2.

  From the results of Table 2, in the amplification of the target nucleic acid and the proportion of cells labeled with the target polypeptide (e), 45.5% of the measurement 8 was the highest. This agrees with the result predicted from the result of verification 3. In contrast, the proportions (e) of measurement 6 and measurement 7 were 14.5% and 16.1%. In Verification 4 as well as Verification 2, the ratio decreased as the temperature of the liquid after pretreatment was adjusted to a high temperature. This cause is considered to be the same as that of verification 2.

[Conclusion of Verification 4]
The amplification of the target nucleic acid and the labeling of the target polypeptide are carried out on the same cells, respectively, when the temperature of the liquid after pretreatment is adjusted to 95 ° C. and the holding time is 1 minute and 10 minutes.
· Target nucleic acid amplification and target polypeptide labeling are performed on the same cells also when the temperature of the liquid after pretreatment is adjusted to 25 ° C.

[Summary of Tests 1 to 4]
From the results of measurements 1 to 4 of verification 1 to 4, in the particle measurement method according to the embodiment, procedure (A-10), that is, temperature control of the liquid after pretreatment is adjusted to a range of 25 ° C. or more and 95 ° C. or less It was found that, in the case where the target nucleic acid was amplified, the target polypeptide was labeled efficiently. Moreover, it turned out that it is more efficiently performed when it is the range of 25 degreeC or more and 75 degrees C or less.

[Verification 5: Relationship between Labeling of Target Polypeptide and Adjustment of Temperature]
Verifications 1 to 4 show that the efficiency of amplification of the target nucleic acid is affected by the temperature of the measurement sample in the procedure (A-10). Next, in Verification 5, in the procedure (A-10), the temperature-adjusted pretreatment liquid is labeled with the target polypeptide instead of the target nucleic acid amplification, and the temperature adjustment is the target polypeptide. It was verified whether it would affect the sign of

  After adjusting the temperature in procedure (A-10) in the procedure 4 (when the temperature is adjusted at 95 ° C. for 2 minutes), the target polypeptide is labeled and an image of the target polypeptide is obtained by a flow cytometer. I got The image is shown in FIG.

  Each image in FIG. 10 is a bright field image, an image of a target nucleic acid, and an image of a target polypeptide in order from the left side, similarly to the image shown in FIG. It is the image which merged the image and the image of the target polypeptide. As shown in FIG. 10, measurement 4 shows that the target polypeptide is properly labeled with the labeled antibody. Thus, it was found that adjusting the temperature did not affect the labeling of the target polypeptide.

[Conclusion of Verification 5]
Adjustment of the temperature of the liquid after pretreatment does not affect the labeling of the target polypeptide.

[Verification 6: Order of target nucleic acid amplification and target polypeptide labeling]
In verifications 1 to 4, after amplification of the target nucleic acid was performed, labeling of the target polypeptide was performed. In Verification 6, the reverse, that is, when labeling of the target polypeptide was performed, amplification of the target nucleic acid was performed with respect to amplification of the target nucleic acid and accuracy of labeling of the target polypeptide.

  In measurement 4, after the procedure of preparation of the measurement sample (A-12), procedure (C), ie, labeling of the target polypeptide, was performed, followed by procedure (B), ie, amplification of the target nucleic acid. This measurement condition is referred to as measurement 9. Then, images of the target nucleic acid and the target polypeptide were obtained by a flow cytometer. The image is shown in FIG. Each image of FIG. 11 is a bright field image, an image of a target nucleic acid, and an image of a target polypeptide in order from the left side similarly to the images shown in FIG. 8 and FIG. The image of the amplification of and the image of the target polypeptide are merged.

  In the images other than the bright field image in FIG. 10, uneven coloring was observed, and the image had a blurred outline. In addition, the images other than the bright field image in FIG. 10, including the outline, were blurred as a whole and unclear. Therefore, it was shown that when target polypeptide is labeled with a labeled antibody and then target nucleic acid amplification is performed, both target nucleic acid amplification and target polypeptide labeling become difficult. Therefore, as in the embodiment, it has been shown that if amplification of the target nucleic acid is performed and then labeling of the target polypeptide is performed, amplification of the target nucleic acid and labeling of the target polypeptide can be accurately performed.

[Conclusion of verification 6]
-If the target polypeptide is labeled with a labeled antibody after amplification of the target nucleic acid, amplification of the target nucleic acid and labeling of the target polypeptide can be performed accurately.

[Verification 7: When the measurement target is exosome]
The above verifications 1 to 6 verified the measurement object as a white blood cell. Next, it was verified whether amplification of the target nucleic acid can be performed by the particle measurement method according to the embodiment, for example, bacteria etc. other than white blood cells.

  A measurement sample was prepared using exosome, which is an endoplasmic reticulum, as a measurement target. However, since the target polypeptide was not labeled, only the procedures (A) and (B) were performed. In the step of adjusting the temperature of procedure (A-10), the contents of the tube were adjusted to 25 ° C. and held for 2 minutes. This is taken as measurement 10.

  After amplification of the target nucleic acid for measurement 10, in order to confirm whether amplification of the target nucleic acid was performed, a superresolution image of exosomes was taken with a superresolution microscope. A super-resolution image of the imaged exosomes is shown in FIG. In imaging with a super resolution microscope, the fluorescent dye is switched to a state in which fluorescence is generated and a state in which fluorescence is not generated, and a plurality of fluorescence images are acquired. Then, based on the plurality of acquired fluorescence images, a high resolution image, that is, a super resolution image is acquired.

  As shown in FIG. 12, the circular outline was clearly imaged and the imaging of the target nucleic acid was confirmed. Therefore, it was found that amplification of the target nucleic acid was performed even when the measurement target was exosome.

[Conclusion of Verification 7]
The particle measurement method according to the embodiment can be applied to an exosome as a measurement target.

[Verification 8: When E. coli is to be measured]
Next, using E. coli as a measurement target, a measurement sample was prepared. However, since the target polypeptide was not labeled, only the procedures (A) and (B) were performed. In the step of adjusting the temperature of procedure (A-10), the contents of the tube were adjusted to 25 ° C. and held for 2 minutes. This is taken as measurement 11.

  After amplification of the target nucleic acid was performed on the measurement 11, in order to confirm whether amplification of the target nucleic acid was performed, an image of E. coli was imaged with a fluorescence microscope. The image of the imaged E. coli is shown in FIG. 13 (a).

  Moreover, the prepared measurement sample was measured using the flow cytometer. As a result, the area of the waveform signal of forward scattered light and the area of the waveform signal of side scattered light are acquired for each of the E. coli contained in the measurement sample, and the upper part of FIG. The scattergram shown has been created. In the scattergram shown in FIG. 13 (b), the region surrounded by a broken line is a region corresponding to the distribution of E. coli. Based on the E. coli contained in this region, as shown in the lower part of FIG. 13B, a histogram is created, in which the horizontal axis is the area of the fluorescent waveform signal and the vertical axis is the frequency. The left side of FIG. 13 (a) is a scattergram and a histogram created based on the measurement sample immediately before the processing for amplification of the target nucleic acid is performed, ie, between procedures (B-2) and (B-3). The right side of FIG. 13 (b) is prepared based on the measurement sample after amplification of the target nucleic acid, that is, the procedure (B-3) is performed.

  From the results of FIGS. 13A and 13B, it was shown that the target nucleic acid can be amplified by the particle measurement method according to the embodiment even when E. coli is the measurement target.

[Conclusion of verification 8]
The particle measurement method according to the embodiment can also be applied to E. coli as the measurement target.

  In addition, the exosome used in Verification 7 has received much attention in recent years, and research is being conducted as elucidation of the exosome leads to elucidation of the carcinogenic mechanism. It is expected that the particle measurement method according to the embodiment is applied to exosomes and can be useful as a biomarker for cancer.

[Verification 9: Labeling of target polypeptide in particles]
In the above verifications 1 to 6, the target polypeptide was present on the surface, that is, the outer surface of the particle to be measured, as shown in FIG. 6 (c). In Verification 9, it was verified whether the particle measurement method according to the embodiment can be applied even if the target polypeptide is contained inside the particle.

  In Verification 9, iPS cells were used as particles to be measured. The target nucleic acid was GAPDH gene and the target polypeptide was Oct4 protein. Labeling and amplification of the target nucleic acid were performed in the same manner as in the procedure of Verification 1 (A) and (B). The procedure for labeling a target polypeptide is shown below. In verification 9, in order to distinguish it from the procedure (C) of verification 1, it is referred to as procedure (D). Moreover, in the step of adjusting the temperature in procedure (A-10), the contents of the tube were adjusted to 25 ° C. and held for 2 minutes.

(D) Labeling of Target Polypeptide After amplification of the target nucleic acid, subsequently, a binding reaction between the target polypeptide contained inside the particles to be measured and the labeled antibody was carried out.
(D-1) 75 μl of PBS was put in a tube to suspend a measurement sample.
(D-2) 5 μL of anti-Oct4 antibody was added to a tube.
(D-3) The binding reaction between the target polypeptide and the labeled antibody was carried out at room temperature of 25 ° C. for 15 minutes.
The measurement sample was prepared by the above procedure. The above measurement condition in verification 9 is referred to as measurement 12.

  The measurement sample of measurement 12 was poured into a flow cytometer, and images of target nucleic acid and target polypeptide were imaged. Each image in FIG. 14 is an image obtained by imaging the measurement 12, and in order from the left, a bright field image, an image of a target nucleic acid, an image of a target polypeptide, and an image on the right side is an image of amplification of the target nucleic acid. And the image of the target polypeptide are merged. As described above, when the procedure of the measurement sample according to the embodiment is processed, the image of the target nucleic acid and the image of the target polypeptide imaged by the flow cytometer can be appropriately acquired.

[Conclusion of verification 9]
The particle measurement method according to the embodiment can also be applied to the labeling of a target polypeptide contained inside a cell.

[Verification 10: Labeling of Target Nucleic Acid by FISH Method]
In the above verifications 1 to 9, labeling of the target nucleic acid of the particles to be measured was performed by amplification of the target nucleic acid in the particles. In Verification 10, it was verified whether the fluorescence in situ hybridization method (FISH method) can be applied to the particle measurement method according to the embodiment.

  In Verification 10, after amplification and labeling of the target nucleic acid for the GAPDH gene, labeling of the PML gene was performed by the FISH method using the PML gene different from the GAPDH gene as the target nucleic acid. That is, it means that the label of the target polypeptide in step S14 in the flowchart of FIG. 1 is replaced with the label of the target nucleic acid by the FISH method. The amplification and labeling of the target nucleic acid for the GAPDH gene was performed in the same manner as in the procedure of Verification 1 (A) and (B). In verification 10, labeling of the target polypeptide was not performed, and procedure (E) was performed after procedure (B). The procedure (E) is shown below. In the step of adjusting the temperature in the procedure (A-10), the contents of the tube were adjusted to 25 ° C. and held for 2 minutes.

(E) FISH method In the procedure (B), after amplification on the GAPDH gene, the PML gene was subsequently labeled.
(E-1) A tube was added with 75 μL of Hybridization Solution A (Cytocell, HA 1000 L).
(E-2) 5 μL of PML Probe in Texas Red (Cytocell, MPH 6480) was added as a labeling probe to a tube.
(E-3) After adding the reagent above, the tube was heated at 95 ° C. for 5 minutes, and allowed to stand at 45 ° C. for 1 hour.
(E-4) The tube was centrifuged at 600 g for 3 minutes, and then the supernatant was removed.
(E-5) 100 μL of PBS was placed in a tube to suspend a measurement sample.

  The measurement sample was prepared by the above procedure. The above measurement condition in verification 10 is taken as measurement 13. The measurement sample of measurement 13 was flowed into a flow cytometer, and an image of the amplified target nucleic acid and the target nucleic acid to which the FISH method was applied was imaged. Each image in FIG. 15 is an image obtained by imaging the measurement 13, and is a bright-field image, an image of the amplified target nucleic acid, and an image of the target nucleic acid labeled by FISH in this order from the left side. Is an image obtained by merging the image of amplification of the target nucleic acid and the image of the target nucleic acid labeled by the FISH method. As described above, when the procedure of the measurement sample according to the embodiment is processed, an image of a target nucleic acid imaged by a flow cytometer and an image of another labeled target nucleic acid can be appropriately acquired.

[Conclusion of verification 10]
The particle measurement method according to the embodiment can apply the FISH method to labeling of a target nucleic acid.

  From the verification results of Verification 10, it was found that the particle measurement method according to the embodiment can detect an abnormality for a plurality of nucleic acids contained in one particle, that is, a cell, by nucleic acid amplification and FISH method.

  As described above, the FISH method is a very effective method for detecting gene copy number abnormalities. On the other hand, although amplification of nucleic acid can detect gene abnormality, it is unsuitable for detection of gene copy number abnormality. That is, mutations in genes that were difficult to detect by amplification of nucleic acids can be detected by FISH, and abnormalities in genes that are difficult to detect by FISH can be detected by amplification of nucleic acids. As described above, when nucleic acid amplification and FISH method are simultaneously used for one cell, each detection method detects an abnormality that each is good at, and as a result, compensates for each other's weaknesses, Anomalies can be detected. From these facts, a detection method that simultaneously performs amplification of nucleic acid and FISH method for one cell can detect gene abnormality more accurately, and can provide more reliable information with high detection clinical value. It is expected as a detection method.

  In addition, anti-HER2 therapy is given to breast cancer patients whose HER2 gene is amplified. However, about 20% of patients in which the HER2 gene is amplified are said to have a PIK3Ca gene mutation. Thus, there is a report that patients with HER2 gene amplification and PIK3Ca mutation have a complete remission rate reduced by half. Therefore, it is possible to determine whether anti-HER2 therapy works properly for breast cancer patients by measuring FISH for the HER2 gene and in situ PCR for the PIK3Ca gene. It can help determine the treatment plan.

  Thus, for a plurality of nucleic acids contained in one particle or cell, it is a very useful method in clinical diagnosis to detect an abnormality by nucleic acid amplification and FISH method.

  In Verification 10, the GAPDH gene corresponds to the "target nucleic acid" described in the claims, and the PML gene corresponds to the "other target nucleic acid" described in the claims.

  In verification 10, the target polypeptide was not labeled, but after amplification of the target nucleic acid and labeling of other target nucleic acids by FISH method, the measurement sample is prepared by the process of procedure (C) as in verification 1. You may Even when other target nucleic acids are labeled by the FISH method, as described with reference to FIG. 6, the polypeptide on the surface of the particle is not completely degraded, and the gap is widened while its shape is maintained. Thus, labeling of the target polypeptide can be performed. In addition, the labeling substance that labels each of the target nucleic acid, the other target nucleic acid, and the target polypeptide is respectively the “first labeling substance”, the “second labeling substance”, and the “second labeling substance” described in the claims. It corresponds to "3 labeled substances". Also, according to the verification result of verification 9, the target polypeptide may be contained inside the particle.

  In Verification 10, after target nucleic acid amplification was performed, the other target nucleic acids were labeled by the FISH method. Even if this order is reversed, each target nucleic acid may be labeled.

As described above, in the particle measurement method according to the embodiment, for each particle to be measured, amplification of a target nucleic acid, labeling of another target nucleic acid, and labeling of a target polypeptide are appropriately combined to detect abnormality of the particle. can do.
Furthermore, in the above verification, for particles to be measured, a combination of labeling and amplification of a target nucleic acid and a label of a target polypeptide, or combination of labeling and amplification of a target nucleic acid and labeling of another target nucleic acid In the particle measurement method according to the embodiment, it is possible to simultaneously perform labeling and amplification of a target nucleic acid, labeling of a target polypeptide, and labeling of other target nucleic acids to perform measurement. .

  One such example is the diagnosis of breast cancer. In the diagnosis of breast cancer, examination is made for abnormalities in the intracellular protein Ki67 and the above-mentioned HER2 gene and PIK3Ca gene. Conventionally, Ki67 was separately detected by immunostaining, the HER2 gene by FISH, and the PIK3Ca gene by in situ PCR. However, the particle measurement method according to the embodiment makes it possible to measure these proteins and genes simultaneously. By measuring these simultaneously, the working efficiency of measurement can be improved. Therefore, it is possible to present diagnosis results to breast cancer patients early. In addition, according to the method of the present invention, since it is possible to grasp the state of Ki67, HER2 gene, PIK3Ca gene for one cell, it is considered that there is a possibility of creating new clinical value from these relationships. Be

10 Flow Cytometer 110 Flow Cell 121 to 123 Light Source 154 Imaging Unit

Claims (31)

Labeling and amplifying target nucleic acid in the particle in the particle;
Labeling the target polypeptide of the particle and / or another target nucleic acid different from the target nucleic acid;
Measuring the labeled target nucleic acid and the labeled target polypeptide and / or the labeled other target nucleic acid. Labeling the target polypeptide on the surface of the particle in the step of labeling the target polypeptide of the particle and / or another target nucleic acid different from the target nucleic acid,
In the step of measuring the labeled target nucleic acid and the labeled target polypeptide and / or the other target nucleic acid, the labeled target nucleic acid and the labeled target polypeptide are measured The particle | grain measuring method of claim | item 1 item.   Labeling and amplifying the target nucleic acid in the particle in the particle, and labeling the target polypeptide of the particle and / or another target nucleic acid different from the target nucleic acid, followed by labeling The particle | grain measurement method of Claim 1 or 2 which performs the process of measuring the said target nucleic acid and the said labeled target polypeptide and / or said other target nucleic acid.   The method according to any one of claims 1 to 3, comprising the step of imaging an image of the particle in the step of measuring the labeled target nucleic acid and the labeled target polypeptide and / or the other target nucleic acid. The particle measurement method according to one item.   In the step of measuring the labeled target nucleic acid and the labeled target polypeptide and / or the other target nucleic acid, the labeled target nucleic acid and the labeled target polypeptide and / or the labeled target nucleic acid The particle measurement according to any one of claims 1 to 4, wherein a liquid containing another target nucleic acid is flowed to a flow cell, and light obtained by irradiating the liquid in the flow cell with light is imaged. Method.   The step of analyzing the imaged image is carried out after the step of measuring the labeled target nucleic acid and the labeled target polypeptide and / or the other target nucleic acid. The particle measurement method according to any one of the items.   The target nucleic acid is labeled with a first labeling substance, and the target polypeptide and / or another target nucleic acid different from the target nucleic acid is labeled with a second labeling substance different from the first labeling substance. The particle measurement method according to any one of the above. In the step of labeling and amplifying the target nucleic acid in the particle in the particle, a mixed solution containing the particle and a labeling substance for labeling the target nucleic acid is prepared;
The particle measurement method according to any one of claims 1 to 7, wherein the mixed solution is substantially free of a protease.   The particle measurement method according to claim 8, wherein the mixture further contains a polymerase.   The particle measurement method according to claim 8, wherein the liquid mixture further comprises a primer.   11. The method according to any one of claims 1 to 10, wherein the step of labeling and amplifying the target nucleic acid in the particle in the particle is performed after setting the temperature of the liquid containing the particle to 25 ° C to 95 ° C. Particle measurement method.   The method according to any one of claims 1 to 11, wherein the step of labeling and amplifying the target nucleic acid in the particle in the particle is performed after setting the temperature of the liquid containing the particle to 25 ° C to 75 ° C. Particle measurement method.   The particle according to any one of claims 1 to 12, wherein the step of labeling and amplifying the target nucleic acid is followed by the step of labeling the target polypeptide and / or another target nucleic acid different from the target nucleic acid. Measuring method.   The step of labeling the target polypeptide and / or another target nucleic acid different from the target nucleic acid, the target polypeptide is labeled with a labeled antibody that specifically binds to the target polypeptide. The particle | grain measuring method as described in any one. In the step of labeling the target polypeptide of the particle and / or another target nucleic acid different from the target nucleic acid, the target polypeptide contained in the particle is labeled;
In the step of measuring the labeled target nucleic acid and the labeled target polypeptide and / or the other target nucleic acid, the labeled target nucleic acid and the labeled target polypeptide are measured The particle | grain measuring method as described in any one of claim | item 1 thru | or 14. In the step of labeling the target polypeptide of the particle and / or another target nucleic acid different from the target nucleic acid, the other target nucleic acid different from the target nucleic acid is labeled;
In the step of measuring the labeled target nucleic acid and the labeled target polypeptide and / or the other target nucleic acid, another target nucleic acid different from the labeled target nucleic acid is measured. The particle measurement method according to any one of 1 to 15. Preparing a mixed solution substantially comprising a particle and a labeled probe which binds to a target nucleic acid in the particle and is substantially free of a protease;
Amplifying the target nucleic acid in the particle;
Sample treatment methods, including:   The sample processing method according to claim 17, wherein the mixed solution further contains a DNA polymerase.   The sample processing method according to claim 17, wherein the mixture further contains a primer.   The sample processing method according to any one of claims 17 to 19, wherein the step of preparing the mixed solution is performed after setting the temperature of the liquid containing the particles to 25 ° C to 95 ° C.   The sample processing method according to any one of claims 17 to 19, wherein the step of preparing the mixed solution is performed after setting the temperature of the liquid containing the particles to 25 ° C to 75 ° C.   The sample processing method according to any one of claims 17 to 21, wherein the step of measuring the labeled target nucleic acid is performed after the step of labeling and amplifying the target nucleic acid.   The method for treating an analyte according to claim 22, wherein the step of measuring the labeled target nucleic acid comprises the step of imaging an image of the particle.   24. The method according to claim 23, wherein in the step of imaging the image of the particles, a liquid containing the labeled target nucleic acid is flowed to a flow cell, and light obtained by irradiating the liquid in the flow cell is imaged. Sample processing method.   The sample processing method according to any one of claims 22 to 24, comprising the step of analyzing the imaged image after the step of measuring the labeled target nucleic acid. A flow cell for flowing a liquid containing the particle in which the target nucleic acid in the particle is labeled and amplified in the particle, and the target polypeptide of the particle and / or another target nucleic acid different from the target nucleic acid is labeled;
A light source for irradiating the flow cell with light;
An imaging unit configured to image light obtained from particles in the liquid by irradiating light from the light source.   27. The particle imaging device according to claim 26, wherein the flow cell flows a liquid including the particle in which a target nucleic acid in the particle is labeled and amplified in the particle, and the target polypeptide on the surface of the particle is labeled.   The imaging unit includes: fluorescence obtained from a first labeling substance that labels the target nucleic acid; fluorescence obtained from a second labeling substance that labels the target polypeptide and / or another target nucleic acid different from the target nucleic acid The particle imaging device according to claim 26 or 27, wherein   The particle imaging device according to any one of claims 26 to 28, wherein the liquid is substantially free of a protease.   The particle imaging device according to any one of claims 26 to 29, wherein the imaging unit is a CCD camera.   The particle imaging apparatus according to any one of claims 26 to 30, wherein the imaging unit is a TDI camera.