Analysis method, method for classifying chromatin state, cancer-testing method, analysis system, testing system for cancer or small residual lesion, and analysis kit

Abstract

In actual clinical practice, there is a demand for a simple low-cost early-stage diagnosis technique or residual lesion-testing technique using a fluorescence detection method or the like. The purpose of the present technology is to provide a technique in which the use of a dye is reduced to a certain level or less, and which is capable of quickly detecting, at low cost, the expression level of a plurality of target nucleic acid molecules in a test object. As a result of intensive studies, the present inventors have found that by using a solution containing a plurality of nucleic acid molecules as a test object, classifying nucleic acid target sequences into at least two groups, and using fluorescent dyes assigned to the respective groups to analyze the result of detection of fluorescence of the fluorescent dyes, it is possible to suitably detect a plurality of target nucleic acid molecules in the test object at low cost without using NGS or dPCR.

Description

Analysis method, method for classifying chromatin state, cancer testing method, analysis system, cancer or minimal residual disease testing system, and analysis kit

[0001] The present invention relates to a method for analyzing nucleic acids such as cfDNA fragments, a cancer testing method, an analysis system, a cancer testing system, and an analysis kit. More specifically, the present invention relates to a technology that can detect the occurrence level of target nucleic acids in a test subject at low cost without using NGS or dPCR.

[0002] Conventionally, methods for detecting a target nucleic acid in a test subject or methods for comprehensively examining a base sequence have been known.

[0003] For example, Patent Document 1 listed below discloses a method for detecting RCA products by fluorescence detection, in which a solution containing nucleic acids is used as a test object and RCA is performed multiple times.

[0004] WO2022 / 117769 publication

[0005] For example, in the field of cancer treatment, there is a demand for simple and inexpensive early diagnosis techniques using fluorescence detection methods or residual lesion testing techniques.

[0006] Therefore, the present technology aims to provide a technology that can detect the occurrence levels of multiple target nucleic acids in a test subject quickly and at low cost while keeping the amount of dye used below a certain level.

[0007] As a result of extensive research, the inventors have found that by using a solution containing multiple nucleic acids as the test subject, classifying the nucleic acid target sequences into two or more groups, and using a fluorescent dye assigned to each group to analyze the fluorescence detection results of the fluorescent dye, multiple target nucleic acids as the test subject can be suitably detected at low cost without using NGS or dPCR.

[0008] Specifically, the present technology provides an analytical method including: a classification step of classifying multiple types of nucleic acid target sequences into groups fewer than the multiple types; an assignment step of assigning one or more fluorescent dyes to each group; a target molecule creation step of binding a detection probe containing a sequence complementary to the nucleic acid target sequence to a nucleic acid containing the nucleic acid target sequence among the multiple nucleic acids to create a target molecule; a fluorescent labeling step of hybridizing a fluorescent probe containing one or more fluorescent dyes to the target molecule to create a fluorescently labeled target molecule; a fluorescent detection step of detecting fluorescent information from the fluorescently labeled target molecule; and an analysis step of analyzing the fluorescent information detected in the fluorescent detection step. The detection probe may have a structure in which the sequence complementary to the nucleic acid target sequence is separated into two regions and located at both ends of the detection probe. In this case, after the target molecule creation step, the method may include a binding step in which the two regions of the detection probe are ligated to form a circular structure. Furthermore, the method may include a detection nucleic acid extension step, in which the detection nucleic acid is extended using the cyclic structure as a template, after the binding step. In this case, the detection nucleic acid may be synthesized by extending the 3'-end or 5'-end of the nucleic acid target sequence. In the analysis method of the present technology, it is preferable that the detection of the fluorescence of the nucleic acid is performed using a microparticle analysis device. In the analysis method of the present technology, the classification of the multiple types of nucleic acid target sequences in the classification step may be performed based on the analysis results of the base sequences contained in the multiple nucleic acids. In this case, the analysis results of the base sequences may be the analysis results of the occurrence levels of the multiple nucleic acids, and the analysis of the occurrence levels may be performed based on the degree of deviation from a reference occurrence level set as a reference. In this case, the base sequences may be sequence information comprehensively analyzed by NGS.In the analysis method of the present technology, the analysis of the fluorescence detection results in the analysis step may be performed based on the fluorescence detection pattern detected in the fluorescence detection step, and the fluorescence detection pattern may be corrected based on the fluorescence detection value of a reference fluorescent dye set as a reference. The nucleic acids in the solution containing multiple nucleic acids to be tested in the analysis method of the present technology may be DNA, and the DNA may be cfDNA. Next, the present technology provides a method for classifying the chromatin state of nucleic acids in a test subject derived from the test subject using the analysis method of the present technology. Furthermore, the present technology provides a cancer testing method for a test subject derived from the test subject using the analysis method of the present technology.

[0009] Furthermore, the present technology provides an analysis system that tests a solution containing multiple nucleic acids, and that has: a target molecule creation unit that binds a detection probe containing a sequence complementary to a nucleic acid target sequence in the nucleic acids to a nucleic acid among the multiple nucleic acids that has the nucleic acid target sequence to create a target molecule; a fluorescence detection unit that hybridizes a fluorescent probe having one or more fluorescent dyes to the target molecule and detects fluorescence information from the fluorescently labeled target molecule; and an analysis unit that analyzes the fluorescence detection results detected by the fluorescence detection unit, wherein the fluorescent probe classifies multiple types of nucleic acid target sequences into groups that are fewer than the multiple types, and has one or more fluorescent dyes assigned to each group. The present technology also provides a cancer or minimal residual disease testing system, which uses a solution containing multiple nucleic acids as a test subject, and includes: a target molecule preparation unit that binds detection probes containing sequences complementary to nucleic acid target sequences in the nucleic acids to nucleic acids among the multiple nucleic acids to create target molecules; a fluorescence detection unit that hybridizes fluorescent probes containing one or more fluorescent dyes to the target molecules and detects fluorescent information from the fluorescently labeled target molecules; an analysis unit that analyzes the fluorescence detection results detected by the fluorescence detection unit; and a judgment unit that determines the presence or absence of a disease in a test subject derived from the test subject based on the analysis results of the analysis unit, wherein the fluorescent probes classify the multiple types of nucleic acid target sequences into groups fewer than the multiple types, and each group has one or more fluorescent dyes assigned to it. Additionally, the present technology provides an analysis kit for a solution containing multiple nucleic acids, which classifies the multiple types of nucleic acid target sequences into groups fewer than the multiple types, and includes fluorescent probes containing one or more fluorescent dyes and detection probes having sequences complementary to the nucleic acid target sequences, each group having one or more fluorescent dyes assigned to it.

[0010] 1 shows an image of the closed and open chromatin states in unaffected and affected individuals, and an image of the detection level of cfDNA fragments by NGS. An example of a flow diagram of the analysis method of the present technology is shown. A modified example of the flow diagram of the analysis method of the present technology is shown. An example of a flow diagram for designing a detection probe used in the analysis method of the present technology is shown. A graph showing the amount of change in the pattern of the relative appearance level of mononucleosome units based on the detection results of cfDNA fragments for a group of unaffected individuals. An example of a classification panel for nucleic acid target sequences is shown. An image diagram showing an example of labeling of target molecules using a detection probe and a fluorescent probe. An image diagram showing a modified example of labeling of target molecules using a detection probe and a fluorescent probe. An image diagram of fluorescence detection patterns for unaffected and affected individuals is shown. An example of a flow diagram for testing for cancer or minimal residual disease using the analysis method of the present technology is shown. An image diagram for improving cfDNA appearance pattern class classification by associative learning is shown. A diagram schematically showing the overall configuration of a biological sample analyzer. A diagram schematically showing the overall configuration of a microscope system. A diagram showing an example of an imaging method. A diagram showing an example of an imaging method.

[0011] Preferred embodiments of the present technology will be described below. However, the embodiments shown below are examples of typical embodiments of the present technology, and the present technology is not limited to only the preferred embodiments below and can be freely modified within the scope of the present technology.

[0012] [Mechanism of cfDNA fragment generation] This technology tests a solution containing multiple nucleic acids, identifies a nucleic acid target sequence in the nucleic acids using a detection probe, fluorescently labels the detection probe with a fluorescent probe, detects the fluorescence, and analyzes the detection results.

[0013] The solution containing multiple nucleic acids that is the test target of the present technology is not particularly limited, but the target nucleic acids may be cell-free DNA (cfDNA) or circular tumor DNA (ctDNA) present in a biological fluid, or nucleic acids contained in extracellular vesicles (EVs) or circulating tumor cells (CTCs), and the biological fluid containing the cfDNA or the like may be the test target.

[0014] Generally, DNA in cells exists in a chromatin state, wrapped around proteins called histones. A structure in which one histone and DNA are wrapped around is called a nucleosome. In order for proteins to be synthesized from DNA in cells, the histones are unwound to form open chromatin, and the DNA in this open chromatin state is transcribed into RNA.

[0015] When cells are destroyed and DNA is released into the blood, it is decomposed in the liver and kidneys, and DNA in an open chromatin state is immediately decomposed. On the other hand, nucleosomes in an open chromatin state are decomposed into mononucleosomes and multiple nucleosome units, but the parts wrapped around histones are relatively resistant to decomposition and can be detected and analyzed.

[0016] For this reason, when these samples are analyzed using an NGS (next-generation sequencer), for example, the portion wrapped around histones (closed chromatin portion) is relatively resistant to degradation, resulting in a high level of cfDNA detection by NGS. On the other hand, the open chromatin portion is easily degraded, resulting in a very low probability of its presence and a low level of cfDNA detection by NGS. Therefore, by using NGS measurement data to analyze the cfDNA detection level at each position of the human genome reference sequence, the read depth record of the mononucleosome unit can be analyzed, and the closed / open chromatin state of the chromatin being examined can be classified according to any criteria.

[0017] Depending on the disease, closed and open chromatin states may differ from the original closed and open chromatin states. For example, it is assumed that healthy cells and cancer cells have different closed and open chromatin states due to differences in their characteristics. Similarly, immune cells that attack cancer cells also have chromatin states that differ from the healthy state due to interactions with cancer cells in the cancer microenvironment.

[0018] Based on this, by analyzing the abundance ratio of cfDNA sequence species present in biological fluids such as blood, it may be possible to analyze and classify, for example, cancer tissue, immune cell dynamics, and differences from non-affected individuals. Similarly, for diseases in which the chromatin state differs from the original closed or open state due to the disease, analyzing the abundance ratio of cfDNA sequence species may enable testing for the disease.

[0019] Figure 1 shows an image of the closed and open chromatin states in non-affected and affected individuals, as well as an image of the detection level of cfDNA fragments by NGS. In diseases such as cancer, non-affected and affected individuals may have different closed and open chromatin states. Therefore, as shown in Figure 1, it is expected that the cfDNA fragments detected by NGS will also differ between the two groups. In other words, if a cfDNA whose detection level changes depending on the presence or absence of a disease is identified, the cfDNA fragment can be used as a nucleic acid target sequence, and the presence or absence of the disease can be tested based on the detection level of the nucleic acid target sequence. Furthermore, rapid testing can be achieved by detecting the nucleic acid target sequence using fluorescent detection technology.

[0020] In the above, cfDNA fragments at each position of the human genome reference sequence are used as nucleic acid target sequences, and their detection levels are analyzed, so the cfDNA fragment pattern is not directly linked to cancer-specific gene mutations, etc. For this reason, it is preferable to derive the nucleic acid target sequence from the analysis of multiple sample data.

[0021] Although eight nucleosomes are shown in FIG. 1, the DNA in the human genome contains approximately 3.1 billion base pairs, and the number of nucleosomes is 10. ７ The number of nucleosome units is on the order of magnitude. Analysis of their occurrence frequency is a field of research called the fragmentome. Thus, the human genome contains a large number of nucleosome units. However, the number of fluorescent dyes that can detect fluorescence is finite.

[0022] Based on the above, the present technology provides a technology that can suitably analyze, by fluorescence detection, the detection level of a nucleic acid target sequence identified as a target cfDNA fragment corresponding to a large number of nucleosome units. The analytical method of the present technology will be described in more detail below.

[0023] [Analysis Method] The analysis method of the present technology reduces the dimension of multiple types of nucleic acid target sequences, classifies them into groups, and performs fluorescence detection on a group-by-group basis. More specifically, the analysis method of the present technology includes a classification step of classifying multiple types of nucleic acid target sequences into groups with a number smaller than the multiple types, an assignment step of assigning one or more fluorescent dyes to each group, a target molecule creation step of using a solution containing multiple nucleic acids as a test subject and binding a detection probe containing a sequence complementary to the nucleic acid target sequence to a nucleic acid containing the nucleic acid target sequence among the multiple nucleic acids to create a target molecule, a fluorescence labeling step of hybridizing a fluorescent probe containing the one or more fluorescent dyes to the target molecule to create a fluorescently labeled target molecule, a fluorescence detection step of detecting fluorescence information from the fluorescently labeled target molecule, and an analysis step of analyzing the fluorescence information detected in the fluorescence detection step.

[0024] The steps of the analytical method of the present technology may be performed consecutively at once, or may be performed separately for each step. For example, the classification step and allocation step may be performed in advance, and the subsequent steps may be performed at a later date at a different facility. Furthermore, the analytical method of the present technology does not need to be performed by the same person alone at the same facility. For example, each step may be performed by multiple people, and the multiple people may perform the analytical method of the present technology in collaboration at different facilities.

[0025] In the analytical method of the present technology, a solution containing multiple nucleic acids is tested. Here, the term "solution containing multiple nucleic acids" is not particularly limited, but the analytical method of the present technology can be suitably used to analyze even solutions containing nucleic acids containing a large number of nucleosome units. Therefore, for example, biological fluids can also be suitably analyzed. The biological fluid to be tested may be any biological fluid, such as blood plasma, tears, urine, or saliva. Furthermore, the nucleic acid contained in the solution to be tested in the analytical method of the present technology is expected to be, for example, DNA, particularly cfDNA, but is not limited thereto.

[0026] Fig. 2 shows an example of a flow diagram of the analytical method of the present technology. As shown in Fig. 2, the analytical method of the present technology proceeds in the order of a classification step S101, an assignment step S102, a target molecule preparation step S103, a fluorescent labeling step S104, a fluorescent detection step S105, and an analysis step S106. As shown in a modified example of the flow diagram in Fig. 3, the analytical method of the present technology may further include a binding step S107 and a detection nucleic acid extension step S108 after the fluorescent labeling step S104. Each step will be described in more detail.

[0027] In the analysis method of the present technology, the classification step classifies multiple types of nucleic acid target sequences into groups whose number is smaller than the multiple types, and the allocation step assigns one or more fluorescent dyes to each group. This allows the test subject to be suitably analyzed even if the number of nucleic acid target sequences contained in a test subject, such as a biological fluid, containing multiple nucleic acids is greater than the number of fluorescent dyes that can be detected by fluorescence detection.

[0028] Fig. 4 shows an example of a flow diagram for designing a detection probe to be used in the analytical method of the present technology. Note that, although the example shown in Fig. 4 shows an example of cancer as the disease to be tested, the disease to be tested in the analytical method of the present technology is not limited to cancer as long as the disease results in a closed / open chromatin state that is different from the original closed / open chromatin state.

[0029] First, as shown in <4A> in Figure 4, the biofluid of a control non-cancer subject (a person not affected by cancer) and the biofluid of a cancer patient are used as test subjects, and analysis is performed by NGS to obtain sequence information of nucleic acids such as cfDNA contained in the biofluids of the non-cancer subject and the cancer patient. As mentioned above, the nucleic acid target sequence set in this technology is not linked to a sequence specific to the target disease, and therefore it is preferable to collect sequence information from multiple sample data.

[0030] Here, the sequence information is preferably information on the base sequences of multiple nucleic acids contained in each biological fluid to be tested, and the base sequences are preferably sequence information comprehensively analyzed by NGS. Here, the larger the population of sequence information comprehensively analyzed by NGS, the more preferable it is from the perspective of improving the accuracy of analysis. In particular, it is preferable to use whole genome sequence information as the population. <4A> shows an example in which whole genome sequence information of both non-affected individuals and cancer patients is used.

[0031] By performing the data analysis procedure shown in <4B> using the nucleic acid sequence information obtained above, the base sequences contained in the multiple nucleic acids contained in the solution to be tested can be suitably analyzed. Specifically, the detection level of cfDNA at each position of the human genome reference sequence is analyzed, the read depth record of the mononucleosome unit is analyzed, and the fragment pattern to be tested is derived. Then, from the read depth records of non-affected and cancer patients, the relative intensity of the occurrence of mononucleosome units in cancer patients compared to non-affected patients is converted into a normalized score value, and these score patterns are compared. As a result, nucleosome sequences with large variations in occurrence can be identified. Based on the analysis results of the base sequences, the classification step of the analysis method of the present technology can suitably classify the multiple types of nucleic acid target sequences.

[0032] Figure 5 is a graph showing the change in the pattern of relative occurrence levels of mononucleosome units based on the detection results of cfDNA fragments in a group of unaffected individuals. In Figure 5, the X axis represents the human genome sequence of nucleosome units, and the Y axis represents the change in occurrence relative to unaffected individuals. The nucleosome units (N1 to N8) in this figure correspond to the nucleosome units (N1 to N8) in the schematic diagram of Figure 1.

[0033] As shown in Figure 1, the N3 nucleosome is in an open chromatin state in cancer patients but is closed in healthy, unaffected individuals, so the data shows a lower occurrence level compared to unaffected individuals. Therefore, in Figure 5, it can be seen that the occurrence level of nucleic acids associated with the N3 nucleosome is changed to (-) compared to unaffected individuals. On the other hand, the N6 nucleosome is in a closed chromatin state in cancer patients but is open chromatin in unaffected individuals, so it can be seen that the occurrence level of nucleic acids associated with the N6 nucleosome is changed to (+) compared to unaffected individuals.

[0034] By analyzing the change in the pattern of the relative expression level of mononucleosome units in a group of unaffected individuals on a whole nucleosome basis, the pattern of the relative expression level of mononucleosome units in unaffected individuals can be derived. Based on the obtained pattern of the relative expression level of mononucleosome units, mononucleosome units with a large change in relative expression level can be selected as nucleic acid target sequences.

[0035] The selection of the nucleic acid target sequence may be such that the mononucleosome unit changes in occurrence amount relative to that of a non-affected individual by a certain level of significance threshold or more. Specifically, a reference occurrence level may be set as a standard, and the mononucleosome unit may be selected as the nucleic acid target sequence based on the degree of deviation from the reference occurrence level.

[0036] In the analysis method of the present technology, the number of selected nucleic acid target sequences varies depending on the type of disease and the target site, and if the disease is cancer, also varies depending on the type of cancer, but is expected to be in the range of 100 to 100,000, for example. If the number of bases in the nucleic acid target sequence is approximately 160 bp, then the total nucleic acid target sequence can be approximately 160 bp × (100 to 100,000) bp.

[0037] In the analytical method of this technology, nucleic acid target sequences must be selected for each type of disease and target site, and if the disease is cancer, for each type of cancer. However, once these are selected, there is no need to select them again for each test.

[0038] The nucleic acid target sequences are classified (dimensionality reduced) into a number of groups fewer than the number of types of the nucleic acid target sequences. The number of groups is not particularly limited as long as it is within the range of types that can be detected by the fluorescence detection method used. For example, when a microparticle analyzer is used for fluorescence detection, the number of groups may be adjusted to within a range of 2 to 60, taking into account the number of fluorescent dye colors that can be analyzed by the current microparticle analyzer.

[0039] The dimensionality reduction method used in the present technology is not particularly limited, and any method can be used. For example, PCA (Principal Component Analysis), jNMF (Joint Nonnegative Matrix Factorization), etc. can be suitably used.

[0040] In the analytical method of this technology, the qualitative meaning of dimensionality reduction is to group nucleic acid target sequences that show the same amount of change and direction of change, and the minimum number of groups that can be classified is two: those showing negative changes and those showing positive changes. Meanwhile, the maximum number of groups that can be classified can be set within the range that can be detected by the fluorescence detection method used. For example, when a microparticle analysis device is used for fluorescence detection, as mentioned above, the number of groups is approximately 60, and depending on the device, it can be as many as 40 to 50.

[0041] In the analysis method of the present technology, it is possible to set criteria such as the amount of change and the direction of change within the range of the above-mentioned settable number of groups according to the purpose of analysis, etc., and to set any group.

[0042] In the classification step of the analysis method of the present technology, multiple types of nucleic acid target sequences are classified into groups with a number smaller than the multiple types, so that each group contains one or more nucleic acid target sequences, and some groups contain multiple nucleic acid target sequences. In the classification step, the nucleic acid target sequences are classified based on the group criteria set above.

[0043] Next, in the assignment step of the analysis method of the present technology, one or more fluorescent dyes are assigned to each group, and nucleic acid target sequences that meet the criteria set for each group are detected by the assigned one or more fluorescent dyes.

[0044] Figure 6 shows an example of a classification panel of nucleic acid target sequences, in which nucleic acid target sequences are classified based on set criteria. In the panel of Figure 6, N3, a nucleosome unit shown in Figure 5 that appears at a low level in unaffected individuals, is selected as nucleic acid target sequence #00001 and classified into Group 1. Nucleic acid target sequences #00020 and #00021, which show a similar pattern of appearance levels, are also classified into Group 1. As shown in Figure 6, fluorescent dye A is assigned to Group 1. Therefore, in fluorescent detection, fluorescent dye A tends to be detected in unaffected individuals, while fluorescent dye A tends to be difficult to detect in affected individuals.

[0045] Next, in the panel of Figure 6, N6, a nucleosome unit shown in Figure 5 that appears at a higher level in unaffected individuals, is selected as the nucleic acid target sequence for #00002 and classified into group 2. Nucleic acid target sequences #00025 and #00122, which show a similar pattern of appearance levels, are also classified into group 2. Fluorescent dye B is assigned to group 2. For this reason, while fluorescent dye B is detected in affected individuals in fluorescent detection, it tends to be difficult to detect in affected individuals.

[0046] For Group 3 and Group 4, the nucleic acid target sequences are also classified based on arbitrary criteria set as the criteria for these groups. Fluorescent dyes C and D are also assigned to these groups, respectively. Therefore, in fluorescence detection, the elements that serve as the criteria for the group can be determined based on whether or not these fluorescent dyes are detected.

[0047] Furthermore, in a classification panel of nucleic acid target sequences, a group may be provided as a measurement control. For example, in the panel of Figure 6, Group 5 classifies N8 and #00003, which are nucleic acid target sequences shown in Figure 5 and show no difference in nucleic acid expression levels between affected and unaffected individuals, as nucleic acid target sequences. Fluorescent dye E is assigned to Group 5. Fluorescent dye E can function as a positive control for measurement because it is detected in both affected and unaffected individuals.

[0048] Furthermore, gene mutation sequences that appear specifically in target diseases, such as tumor markers, may be classified into groups, and fluorescent dyes may be assigned to the groups. In this case, if the fluorescent dye is detected, it is determined that the subject may be suffering from the target disease. In this case, there are no particular limitations on the sequences that appear specifically in target diseases when classifying into groups, and any sequence can be classified as a nucleic acid target sequence according to the criteria of the set group. For example, if the sequence that appears specifically in target diseases is a tumor marker, any sequence, such as KRAS, BRAF, EGFR, SRSF2, ASXL1, AKT1, PIK3CA, or HER2, can be selected as the nucleic acid target sequence for the tumor marker.

[0049] When sequences that appear specifically in a target disease are classified as nucleic acid target sequences, the presence or absence of dyes related to these groups may enable the diagnosis of the disease, and therefore, these may be analyzed separately from the measurement results of other fragments. Alternatively, when dyes related to these groups are detected, the detection result data may be weighted in the analysis step described below.

[0050] As mentioned above, in the analytical method of the present technology, detection is performed on multiple nucleic acid target sequences, and multiple nucleic acid target sequences may be classified into each group. Therefore, it is necessary to take into consideration the sequence specificity between nucleic acid target sequences. In particular, if the (+) and (-) occurrence levels of nucleic acids in non-affected individuals are reversed, it may be desirable not to classify the nucleic acid target sequence into a group even if it meets the group classification criteria. (In this case, the nucleic acid target sequence is excluded from classification in the classification step.)

[0051] Next, a detection probe is designed based on the classification of the above-mentioned target nucleic acid sequences according to the procedure in <4B> of Figure 4. Here, the detection probe binds to a nucleic acid having the target nucleic acid sequence contained in the solution to be tested, and assists in fluorescent labeling of the target nucleic acid sequence.

[0052] The detection probe used in the analytical method of the present technology is designed for each nucleic acid target sequence, and has a sequence complementary to the nucleic acid target sequence and a sequence to which a fluorescent probe (described below) can hybridize or a sequence that serves as a template for a sequence to which a fluorescent probe can hybridize.

[0053] In the cleaning based on the probe design constraints in <4B>, sequences that may bind nonspecifically to nucleic acids are avoided, and a region of the base sequence that can bind specifically to the desired nucleic acid target sequence is identified and adopted as a sequence complementary to the nucleic acid target sequence of the detection probe. This cleaning process is performed for each nucleic acid target sequence, and a sequence complementary to the nucleic acid target sequence of each detection probe is determined.

[0054] The detection probe used in the analytical method of the present technology is designed by combining a sequence complementary to the nucleic acid target sequence determined above, as shown in <4C>, with a sequence that serves as a template for a sequence to which a fluorescent probe having an assigned fluorescent dye can hybridize according to the classification panel of nucleic acid target sequences determined by the classification and assignment steps described above, as shown in the example of Figure 6.

[0055] Furthermore, as mentioned above, in the analytical method of the present technology, since detection is performed for multiple nucleic acid target sequences and multiple nucleic acid target sequences may be classified into each group, it is desirable to compare the entire sequence of the designed detection probe with the sequences of all nucleic acids, such as cfDNA, that may be contained in the biological fluid to be tested, evaluate the degree of commonality of the base sequences (number of bases that differ), and select those with high specificity as nucleic acid target sequences. This process is expected to suppress an increase in the background (DC component) of the amount of fluorescence detected in the fluorescence detection step described below.

[0056] Next, the fluorescent probe used in the analytical method of the present technology has a sequence capable of hybridizing to a sequence formed from the detection probe, and one or more fluorescent dyes. By having one or more fluorescent dyes, the fluorescent probe can hybridize to a target molecule created in the target molecule creation step described below, and fluorescently label the target molecule.

[0057] The number of fluorescent dyes contained in the fluorescent probe used in the analysis method of the present technology is not particularly limited as long as it is one or more. Furthermore, depending on the purpose of fluorescence detection, the fluorescent probe may contain multiple fluorescent dyes.

[0058] The detection probes and fluorescent probes used in the analytical method of the present technology can be designed once and used to perform the analysis of the present technology without the need to redesign them for each test.

[0059] <Target Molecule Preparation Step and Fluorescent Labeling Step> In the analysis method of the present technology, in the target molecule preparation step, a solution containing multiple nucleic acids is used as a test object, and a detection probe containing a sequence complementary to the target nucleic acid sequence is bound to a nucleic acid containing the target nucleic acid sequence among the multiple nucleic acids to prepare a target molecule. In the subsequent fluorescent labeling step, a fluorescent probe containing one or more fluorescent dyes is hybridized to the target molecule to fluorescently label the target molecule.

[0060] Here, the target molecule created in the target molecule creation step is preferably capable of hybridizing with multiple fluorescent probes having fluorescent dyes, as this increases the fluorescence intensity and therefore improves the sensitivity of fluorescence detection. Therefore, for example, it is preferable to configure the detection probe to be capable of forming a cyclic structure, and perform RCA amplification using the cyclic structure of the detection probe as a template to elongate a detection nucleic acid having multiple sequences to which the fluorescent probes can hybridize. Since multiple fluorescent probes hybridize to the target molecule having the elongated detection nucleic acid, the fluorescence intensity can be increased.

[0061] In this way, by performing RCA amplification on a target molecule using the circular structure of the detection probe as a template, the detection nucleic acid is extended, and the target molecule can form microparticles called DNA nanoballs.

[0062] Here, if the shape of the DNA nanoballs can be adjusted to, for example, the order of several hundred nm, more specifically, 200 nm or more, 500 nm or more, the fluorescence of the DNA nanoballs associated with the detection nucleic acid identified by the fluorescent probe can be detected by a microparticle analyzer. Generally, detection using a FSC (Forward Scatter) or SSC (Side Scatter) / BSC (Back Scatter) detection system, which is the detection optical system installed in a microparticle analyzer, requires a short wavelength laser such as 405 nm and a high NA lens. However, since the DNA nanoballs formed in the analysis method of this technology are fluorescently labeled, they can also be detected by counting and brightness measurement triggered by fluorescence instead of scattering detection.

[0063] When the fluorescence is detected by a microparticle analyzer, the detection can be performed at a high throughput, which allows, for example, analyzing a solution such as a biological fluid of the test subject at a high throughput, and quickly testing the test subject for the presence or absence of a disease such as cancer that is derived from the test subject.

[0064] The method of the detection probe configured to form a circular structure is not particularly limited as long as it is capable of forming a circular structure, and examples include the padlock probe method, snail probe method, and Super RCA method. Furthermore, in the case of the padlock probe method, the detection nucleic acid extended by RCA amplification using the circular structure of the detection probe as a template is the 3'-end or 5'-end of the nucleic acid target sequence, so that the nucleic acid having the nucleic acid target sequence becomes the detection nucleic acid. Furthermore, in the case of the snail probe method, the 3'-end or 5'-end of a primer provided separately from the detection probe is extended by RCA amplification using the circular structure of the detection probe as a template to form the detection nucleic acid.

[0065] When the detection probe forms a circular structure by these methods, for example, the detection probe may be designed so that a sequence complementary to the nucleic acid target sequence is separated into two regions and arranged at both ends of the detection probe. When the detection probe used in the analysis method of the present technology has such a structure, in the target molecule preparation step, the sequences complementary to the two separated nucleic acid target sequences of the detection probe bind to the nucleic acid target sequence in the nucleic acid contained in the solution to be tested, and the detection probe becomes circular.

[0066] In this case, it is preferable to include a binding step in which, after the target molecule preparation step, two regions of the detection probe corresponding to sequences complementary to the nucleic acid target sequence are ligated to form a circular structure for the detection probe. By ligating the two regions of the detection probe, the detection probe becomes a circular structure, and RCA amplification using the circular structure as a template can be suitably performed. That is, after the binding step, a detection nucleic acid extension step can be performed in which the detection nucleic acid is extended by RCA amplification using the circular structure as a template. The resulting detection nucleic acid has multiple sequences to which fluorescent probes can hybridize, and therefore multiple fluorescent probes hybridize to target molecules having the detection nucleic acid, making it suitable for detection using a microparticle analyzer or the like, as described above. Note that, instead of performing RCA amplification, the target molecule can also be suitably detected using a microparticle analyzer or the like by forming a molecule or complex by specifically reacting or binding with the target molecule with a molecule comprising a dye that emits scattered light or fluorescence of an intensity that can be detected using a microparticle analyzer.

[0067] In other words, when a solution such as a biological fluid to be tested contains a nucleic acid having a nucleic acid target sequence, the detection probe binds to the nucleic acid target sequence, and a target molecule having an extended detection nucleic acid is formed through the aforementioned binding process and detection nucleic acid extension process, so that the fluorescent dye assigned to the classified group of the nucleic acid target sequence can be suitably detected using a microparticle analysis device, etc.

[0068] On the other hand, since the detection probe has a sequence complementary to the nucleic acid target sequence separated into two regions and both ends of the detection probe are not fixed, the detection probe will not form a loop unless a sequence complementary to the two separated nucleic acid target sequences of the detection probe binds to the nucleic acid target sequence portion of the nucleic acid contained in the solution to be tested. Unless the detection probe forms a loop, the detection nucleic acid will not be extended by RCA amplification. In other words, if the solution to be tested, such as a biological fluid, does not contain a nucleic acid having the nucleic acid target sequence, the detection probe cannot bind to the nucleic acid target sequence, and the detection nucleic acid will not be extended, so the fluorescent dye assigned to the classified group of the nucleic acid target sequence will not be detected.

[0069] In the detection probe used in the analytical method of the present technology, the sequence complementary to the nucleic acid target sequence is separated into two regions. If both ends of the detection probe are not fixed, the region of the sequence complementary to the nucleic acid target sequence becomes shorter in length when separated into two regions, which is detrimental to maintaining the specificity of each region. Therefore, in the classification process, probes with the same sequence or high similarity, such as those differing by only one base, may be classified into different groups. As a result, the nucleic acid target sequence may be erroneously labeled, which may increase noise in fluorescence detection.

[0070] In light of the above, when a detection probe has a structure in which a sequence complementary to a nucleic acid target sequence is separated into two regions and placed at both ends of the detection probe, careful consideration must be given to the selection and group classification of a common target probe or a nucleic acid target sequence that differs by one base at the 3' end of the nucleic acid target sequence. In particular, when the (+) and (-) of the occurrence levels of nucleic acids relative to non-affected individuals are reversed, it may be desirable not to classify the nucleic acid target sequence into a group even if it meets the group classification criteria, as is the case when the specificity of the nucleic acid target sequence is taken into consideration. (In this case, the nucleic acid target sequence is excluded from classification in the classification process.)

[0071] (1) Example of use of a detection probe using the padlock probe method The target molecule creation process, binding process, detection nucleic acid extension process, and fluorescent labeling process when using the padlock probe method as a detection probe configured to form a circular structure will be specifically described using Figure 7.

[0072] When the padlock probe system is used in the analytical method of the present technology, the detection probe has a configuration such as that shown in <7A>, where the detection probe 10 has a sequence 12-1 that serves as a template for a sequence to which the fluorescent probe can hybridize, and sequences 11-1 and 11-2 that are complementary to the nucleic acid target sequence separated into two regions are arranged at both ends.

[0073] <7B> shows a target molecule preparation step in which sequences 11-1 and 11-2 complementary to the nucleic acid target sequence provided in the detection probe 10 bind (hybridize) to a region of the nucleic acid target sequence 11 in cfDNA 13 containing the nucleic acid target sequence 11 among cfDNA present in a biological fluid or the like to be tested, thereby preparing a target molecule.

[0074] <7C> indicates a binding step. In this step, two regions of the detection probe 10, sequences 11-1 and 11-2, which are complementary to the nucleic acid target sequence, are ligated, and the detection probe 10 becomes a circular structure. In this step, the method for ligating the two regions of the detection probe 10 is not particularly limited, and any method can be used. For example, ligation may be performed using a DNA ligase or the like.

[0075] As described above, in the padlock probe method, the 3' or 5' end of the nucleic acid target sequence is extended to form the detection nucleic acid, and in the binding step <7C>, an exonuclease or the like is used to degrade the nucleic acid on the 3' or 5' end of the cfDNA down to the position where the detection probe is bound.

[0076] <7D> shows the detection nucleic acid extension step. In this step, the detection nucleic acid 14 is extended using the cyclic structure of the detection probe 10 as a template. In the padlock probe method shown in this example, the 3' or 5' end of the nucleic acid target sequence is extended to form the detection nucleic acid. The detection nucleic acid 14 formed in this step has multiple sequences 12 to which the fluorescent probe can hybridize, derived from sequence 12-1, which serves as a template for the sequence to which the fluorescent probe contained in the detection probe can hybridize. Note that the complementary strands of 11-1 and 11-2 in the extension portion are omitted in the figure.

[0077] <7E> denotes a fluorescent labeling step. In this step, fluorescent probes 15 having one or more fluorescent dyes are hybridized to a plurality of sequences 12 to which the fluorescent probes can hybridize, which are present in the detection nucleic acid 14 of the target molecule, to thereby fluorescently label the target molecule. Fluorescence information from the fluorescently labeled target molecule can be detected by a microparticle analyzer or the like in the fluorescence detection step described below.

[0078] (2) Example of use of snail probe type detection probe Next, the target molecule creation process, binding process, detection nucleic acid extension process, and fluorescent labeling process when using the snail probe type detection probe configured to form a circular structure will be explained using Figure 8.

[0079] In the analytical method of the present technology, the detection probe when using the snail probe system has the same structure as the detection probe when using the padlock probe system described above. Specifically, as shown in the example of <7A>, the detection probe has a sequence that serves as a template for the sequence to which the fluorescent probe can hybridize, and has sequences at both ends that are complementary to the nucleic acid target sequence separated into two regions. When using the snail probe system, in addition to the above configuration, the detection probe also has a region to which a primer for forming a detection nucleic acid can hybridize. As a result, when using the snail probe system, for example, the detection nucleic acid can be formed by RCA amplification using the 3' end of the primer as the starting point and the circular structure of the detection probe as a template. A detailed explanation is provided below using the figures.

[0080] <8A> shows a target molecule preparation step. In this step, a sequence complementary to the nucleic acid target sequence separated into two regions provided in the detection probe 10 binds (hybridizes) to the region of the nucleic acid target sequence 11 of the cfDNA 13 having the nucleic acid target sequence 11 among the cfDNA present in the biological fluid or the like to be tested, thereby preparing a target molecule. At this time, the primer 16 also hybridizes to the detection probe 10.

[0081] The next step, <8B>, is the binding step. In this step, two regions of the detection probe 10, which are sequences complementary to the nucleic acid target sequence, are ligated, resulting in the detection probe 10 forming a circular structure. In this step, the method for ligating the two regions of the detection probe 10 is not particularly limited, and any method that can be used in the padlock probe method described above can be suitably used. Note that in the snail probe method, there is no need to elongate a nucleic acid having a nucleic acid target sequence as a detection nucleic acid, and therefore there is no need to degrade the cfDNA nucleic acid to the position where the detection probe is bound using an exonuclease or the like.

[0082] <8C> shows the detection nucleic acid extension step. In this step, the detection nucleic acid 14 is extended using the circular structure of the detection probe 10 as a template. In the snail probe method shown in this example, for example, the 3' end of the primer 16 is extended by RCA amplification using the circular structure of the detection probe as a template to form the detection nucleic acid. The detection nucleic acid 14 formed in this step has multiple sequences 12 to which the fluorescent probe can hybridize, derived from the sequence that serves as a template for the sequence to which the fluorescent probe in the detection probe can hybridize.

[0083] <8D> shows a fluorescent labeling step. In this step, the fluorescent probe 15 having one or more fluorescent dyes is hybridized to a plurality of sequences 12 to which the fluorescent probe can hybridize, which are present in the detection nucleic acid 14 of the target molecule, to thereby fluorescently label the target molecule. Fluorescence information from the fluorescently labeled target molecule can be detected by a microparticle analyzer or the like in the fluorescence detection step described below.

[0084] <Fluorescence Detection Step and Analysis Step> In the analysis method of the present technology, the fluorescence detection step detects fluorescence information from fluorescently labeled target molecules, and the analysis step analyzes the fluorescence information detected in the fluorescence detection step.

[0085] Figure 9 shows an image of the fluorescence detection pattern of fluorescently labeled target molecules produced by subjecting biological fluids, etc., to be tested from non-cancer and cancer patients (cancer patients) to the aforementioned process. In the example shown in Figure 9, the nucleic acid target sequences are classified into five groups, and a fluorescent dye is assigned to each group. One of these groups is a control (in the example of Figure 9, dye E is assigned).

[0086] In the example of Figure 9, the disease of the affected individuals is cancer, and the cancer is of two types: cancer type A and cancer type B. <9A> shows the fluorescence detection pattern of a non-affected individual, <9B> shows the fluorescence detection pattern of a affected individual with cancer type A, and <9C> shows the fluorescence detection pattern of a affected individual with cancer type B.

[0087] In the example of Fig. 9, in the fluorescence detection process, fluorescence information from the test subject is detected by counting the number of DNA nanoballs, which are target molecules, for each dye using a microparticle analyzer. Based on the detected fluorescence information, the detection patterns of each dye can be compared and analyzed for non-affected individuals, patients with cancer type A, and patients with cancer type B, as shown in Fig. 9.

[0088] In the above analysis, the fluorescence detection pattern may be corrected based on the fluorescence detection value of a reference fluorescent dye. In the example of Figure 9, the detection value of the control dye E is used as the reference value, and the detection values ​​of each dye are corrected based on this reference value. While the example shown in Figure 9 illustrates the results using five dyes, the number of dyes used can be adjusted depending on the fluorescence detection method adopted and the performance of the fluorescence detection instrument. For example, when using a microparticle analyzer for fluorescence detection, as mentioned above, the number of fluorescent dyes that can be analyzed by current microparticle analyzers can be adjusted within a range of 2 to 60 groups. For example, a Sony microparticle analyzer (Flow Cytometer ID7000) has demonstrated the ability to analyze 44 colors using detectors with more than 100 channels.

[0089] Furthermore, even if the number of required clusters or groups exceeds the upper limit of the number of dyes that can be used, the required clusters or groups can be identified by labeling with a combination of multiple fluorescent dyes. For example, by labeling group N with fluorescent dyes A and B, it can be distinguished from groups labeled with fluorescent dyes A and B alone. This makes it possible to perform cluster analysis for more than the number of fluorescent dyes that can be used.

[0090] Furthermore, instead of combining multiple fluorescent dyes, groups can be distinguished by providing two or three brightness levels for the same fluorescent dye, which also enables cluster analysis for more than the number of fluorescent dyes that can be used.

[0091] Furthermore, in the explanation of this embodiment, a fluorescence detection method using a microparticle analyzer has been shown as a specific example of a fluorescence detection method for detecting fluorescence information from fluorescently labeled target molecules. However, any fluorescence detection method, such as counting using a microscope, can be used as a substitute as long as it is possible to count the fluorescence from target molecules that form DNA nanoballs, etc.

[0092] This section describes an example of the configuration of a biological sample analyzer that is a microparticle analyzer when a fluorescence detection method using a microparticle analyzer is used as the fluorescence detection method in the analysis method of the present technology. Below, we will explain the general functions of a biological sample analyzer that can detect fluorescence from various targets, including the test target of the present technology.

[0093] An example configuration of a biological sample analyzer according to the present disclosure is shown in Figure 12. The biological sample analyzer 6100 shown in Figure 12 includes a light irradiation unit 6101 that irradiates light onto a biological sample S flowing through a flow path C, a detection unit 6102 that detects light generated by irradiating the biological sample S with light, and an information processing unit 6103 that processes information related to the light detected by the detection unit. Examples of the biological sample analyzer 6100 include a flow cytometer and an imaging cytometer. The biological sample analyzer 6100 may also include a sorting unit 6104 that sorts specific biological particles P from within the biological sample. An example of a biological sample analyzer 6100 that includes the sorting unit is a cell sorter.

[0094] (Biological Sample) As described above, the biological sample S may be a liquid sample containing target molecules or a liquid sample containing biological particles. The biological particles may be, for example, cells or non-cellular biological particles. The cells may be living cells, and more specific examples include blood cells such as red blood cells and white blood cells, and reproductive cells such as sperm and fertilized eggs. The cells may be directly collected from a specimen such as whole blood, or may be cultured cells obtained after culturing. Examples of non-cellular biological particles include extracellular vesicles, particularly exosomes and microvesicles. The biological particles may be labeled with one or more labeling substances (e.g., dyes (especially fluorescent dyes) and fluorescent dye-labeled antibodies). Note that the biological sample analyzer of the present disclosure may analyze particles other than biological particles, or may analyze carriers containing beads or the like for calibration purposes. The carrier may also hold, for example, biological components (e.g., cells or cell-derived components, such as secretions) or the aforementioned target molecules (hereinafter collectively referred to as "biological components, etc."). Holding a biological component or the like on the carrier includes, for example, when the biological component or the like is captured on the carrier or when the biological component or the like is encapsulated in the carrier. Note that, as an alternative to RCA amplification, the target molecule may be a molecule or complex formed by a molecule having a fluorescent dye specifically reacting or binding with the target molecule. In this case, scattered light or fluorescence from the molecule or complex is detected and analyzed by the biological sample analyzer. The carrier may be, for example, a carrier used for secretion analysis. The carrier may also be an emulsion, in which case the biological particles may be collected while contained within the emulsion. In this case, the collected fractionated product may be an emulsion, and the dispersoid constituting the emulsion may be biological particles contained within the emulsion containing the particles to be collected. The dispersion medium constituting the emulsion may be appropriately selected by those skilled in the art, for example, depending on the type of emulsion particles. That is, the biological sample analyzer according to the present disclosure may be configured as a carrier fractionation device.In this way, the biological sample analyzer according to the present disclosure may be configured as a sorting device for particles other than biological particles (for example, carriers such as beads or emulsions).

[0095] (Flow Channel) The flow channel C is configured to allow the biological sample S to flow. In particular, the flow channel C can be configured to form a flow in which biological particles contained in the biological sample are aligned in a substantially straight line. The flow channel structure including the flow channel C may be designed to form a laminar flow. In particular, the flow channel structure is designed to form a laminar flow in which the flow of the biological sample (sample flow) is surrounded by the flow of sheath liquid. The design of the flow channel structure may be appropriately selected by those skilled in the art, and a known design may be adopted. The flow channel C may be formed in a flow channel structure such as a microchip (a chip having flow channels on the order of micrometers) or a flow cell. The width of the flow channel C may be 1 mm or less, particularly 10 μm or more and 1 mm or less. The flow channel C and the flow channel structure including it may be formed from a material such as plastic or glass.

[0096] The biological sample analyzer of the present disclosure is configured so that light from light irradiation unit 6101 is irradiated onto the biological sample flowing within flow path C, and particularly onto biological particles within the biological sample. The biological sample analyzer of the present disclosure may be configured so that the interrogation point of light on the biological sample is within the flow path structure in which flow path C is formed, or so that the interrogation point of light is outside the flow path structure. An example of the former is a configuration in which the light is irradiated onto flow path C within a microchip or flow cell. In the latter, the light may be irradiated onto biological particles after they have left the flow path structure (particularly its nozzle portion), and an example of this is a jet-in-air flow cytometer.

[0097] (Light Irradiation Unit) The light irradiation unit 6101 includes a light source unit that emits light and a light-guiding optical system that guides the light to an irradiation point. The light source unit includes one or more light sources. The type of light source is, for example, a laser light source or an LED. The wavelength of the light emitted from each light source may be any of ultraviolet light, visible light, and infrared light. The light-guiding optical system includes optical components such as a beam splitter group, a mirror group, or an optical fiber. The light-guiding optical system may also include a lens group for focusing light, such as an objective lens. There may be one or more irradiation points where the light intersects with the biological sample. The light irradiation unit 6101 may be configured to focus light irradiated from one or more different light sources onto one irradiation point.

[0098] (Detection Unit) The detection unit 6102 includes at least one photodetector that detects light generated by irradiating the bioparticles with light. The detected light is, for example, fluorescence or scattered light (e.g., one or more of forward scattered light, back scattered light, and side scattered light). Each photodetector includes one or more light-receiving elements, for example, a photodetector array. Each photodetector may include one or more PMTs (photomultiplier tubes) and / or photodiodes such as APDs and MPPCs as light-receiving elements. The photodetector includes, for example, a PMT array in which multiple PMTs are arranged in a one-dimensional direction. The detection unit 6102 may also include an imaging element such as a CCD or CMOS. The detection unit 6102 can acquire images of the bioparticles (e.g., bright-field images, dark-field images, and fluorescence images) using the imaging element.

[0099] The detection unit 6102 includes a detection optical system that allows light of a predetermined detection wavelength to reach a corresponding photodetector. The detection optical system includes a spectroscopic unit such as a prism or a diffraction grating, or a wavelength separation unit such as a dichroic mirror or an optical filter. The detection optical system is configured to, for example, disperse light generated by irradiating bioparticles with light, and detect the dispersed light using a plurality of photodetectors, the number of which is greater than the number of fluorescent dyes with which the bioparticles are labeled. A flow cytometer that includes such a detection optical system is called a spectral flow cytometer. The detection optical system is also configured to, for example, separate light corresponding to the fluorescent wavelength range of a specific fluorescent dye from the light generated by irradiating bioparticles with light, and detect the separated light using a corresponding photodetector.

[0100] The detection unit 6102 may also include a signal processing unit that converts the electrical signal obtained by the photodetector into a digital signal. The signal processing unit may include an A / D converter as a device that performs the conversion. The digital signal obtained by the conversion by the signal processing unit may be transmitted to the information processing unit 6103. The digital signal may be handled by the information processing unit 6103 as data related to light (hereinafter also referred to as "light data"). The light data may be light data including, for example, fluorescent light data. More specifically, the light data may be light intensity data, and the light intensity may be light intensity data of light including fluorescent light (which may include feature quantities such as area, height, and width).

[0101] (Information Processing Unit) The information processing unit 6103 includes, for example, a processing unit that processes various data (e.g., optical data) and a storage unit that stores various data. When the processing unit acquires optical data corresponding to a fluorescent dye from the detection unit 6102, the processing unit may perform fluorescence spillover correction (compensation processing) on ​​the light intensity data. Furthermore, in the case of a spectral flow cytometer, the processing unit performs fluorescence separation processing on the optical data to acquire light intensity data corresponding to the fluorescent dye. The fluorescence separation processing may be performed, for example, according to the unmixing method described in Japanese Patent Application Laid-Open No. 2011-232259. When the detection unit 6102 includes an image sensor, the processing unit may acquire morphological information of bioparticles based on images acquired by the image sensor. The storage unit may be configured to store the acquired optical data. The storage unit may further be configured to store spectral reference data used in the unmixing processing.

[0102] If the biological sample analyzer 6100 includes a fractionating unit 6104 (described below), the information processing unit 6103 can determine whether to fractionate bioparticles based on the optical data and / or morphological information. The information processing unit 6103 can then control the fractionating unit 6104 based on the result of this determination, allowing the fractionating unit 6104 to fractionate the bioparticles.

[0103] The information processing unit 6103 may be configured to output various data (e.g., optical data and images). For example, the information processing unit 6103 may output various data (e.g., two-dimensional plots, spectral plots, etc.) generated based on the optical data. The information processing unit 6103 may also be configured to accept input of various data, such as accepting gating processing on a plot by a user. The information processing unit 6103 may include an output unit (e.g., a display, etc.) or an input unit (e.g., a keyboard, etc.) for executing the output or input.

[0104] The information processing unit 6103 may be configured as a general-purpose computer, for example, as an information processing device including a CPU, RAM, and ROM. The information processing unit 6103 may be included in a housing that includes the light irradiation unit 6101 and the detection unit 6102, or may be located outside the housing. Furthermore, various processes or functions performed by the information processing unit 6103 may be realized by a server computer or a cloud connected via a network.

[0105] (Sorting unit) The sorting unit 6104 sorts the bioparticles according to the determination result by the information processing unit 6103. The sorting method may be a method of generating droplets containing bioparticles by vibration, applying an electric charge to the droplets to be sorted, and controlling the direction of travel of the droplets using electrodes. The sorting method may also be a method of controlling the direction of travel of the bioparticles within the flow channel structure to perform sorting. The flow channel structure is provided with, for example, a control mechanism using pressure (spray or suction) or electric charge. An example of such a flow channel structure is a chip (for example, the chip described in JP 2020-76736 A) having a flow channel structure in which a flow channel C branches downstream into a recovery flow channel and a waste flow channel, and specific bioparticles are recovered into the recovery flow channel.

[0106] An example of the configuration of a microscope when counting by a microscope is used as the fluorescence detection method used in the analysis method of the present technology will be described.

[0107] FIG. 13 shows an example configuration of a microscope system according to the present disclosure. The microscope system 5000 shown in FIG. 13 includes a microscope device 5100, a control unit 5110, and an information processing unit 5120. The microscope device 5100 includes a light irradiation unit 5101, an optical unit 5102, and a signal acquisition unit 5103. The microscope device 5100 may further include a sample mounting unit 5104 on which a biological sample S is placed. Note that the configuration of the microscope device is not limited to that shown in FIG. 13 . For example, the light irradiation unit 5101 may be located outside the microscope device 5100, and a light source not included in the microscope device 5100 may be used as the light irradiation unit 5101. Furthermore, the light irradiation unit 5101 may be arranged so that the sample mounting unit 5104 is sandwiched between the light irradiation unit 5101 and the optical unit 5102, and may be arranged on the side where the optical unit 5102 is located, for example. The microscope device 5100 may be configured to be able to perform one or more of bright-field observation, phase-contrast observation, differential interference observation, polarized light observation, fluorescence observation, and dark-field observation.

[0108] The microscope system 5000 may be configured as a so-called WSI (Whole Slide Imaging) system or a digital pathology imaging system and may be used for pathological diagnosis. The microscope system 5000 may also be configured as a fluorescence imaging system, particularly a multiplex fluorescence imaging system.

[0109] For example, the microscope system 5000 may be used to perform intraoperative pathological diagnosis or remote pathological diagnosis. In the intraoperative pathological diagnosis, the microscope device 5100 may acquire data of a biological sample S obtained from a subject of the surgery while the surgery is being performed, and transmit the data to an information processing unit 5120. In the remote pathological diagnosis, the microscope device 5100 may transmit data of the acquired biological sample S to an information processing device 5120 located in a location remote from the microscope device 5100 (such as a different room or building). In these diagnoses, the information processing device 5120 receives and outputs the data. A user of the information processing device 5120 may perform a pathological diagnosis based on the output data.

[0110] (Biological Samples) When performing an examination using the microscope system 5000, it is desirable to adjust the concentration of fluorescently labeled target nucleic acids so that they are dispersed on the slide, and then encapsulate them with a glass slide and cover glass. A mounting material used in creating pathology slides may be used to prevent fading of the fluorescence and fix the sample. In this case, the degree of dispersion is determined by the spatial resolution of the microscope system, and it is desirable to have a degree that allows particles to be separated and counted.

[0111] The biological sample may be one that has been subjected to a process such as staining or labeling. The process may be staining to reveal the morphology of the biological component or to reveal substances (such as surface antigens) contained in the biological component, and examples of such staining include hematoxylin-eosin (HE) staining and immunohistochemistry staining. The biological sample may be one that has been subjected to the process using one or more reagents, and the reagents may be a fluorescent dye, a color-developing reagent, a fluorescent protein, or a fluorescently labeled antibody.

[0112] The specimen may be prepared from a tissue sample for purposes such as pathological diagnosis or clinical testing. Furthermore, the specimen is not limited to being derived from the human body, but may also be derived from animals, plants, or other materials. The characteristics of the specimen vary depending on the type of tissue (e.g., organ or cell) used, the type of disease being treated, the subject's attributes (e.g., age, sex, blood type, or race), or the subject's lifestyle (e.g., diet, exercise, or smoking habits). The specimens may be managed with identifying information (e.g., a barcode or QR code (trademark)) attached to each specimen.

[0113] (Light Irradiation Unit) The light irradiation unit 5101 is a light source for illuminating the biological sample S and an optical unit that guides the light irradiated from the light source to the specimen. The light source can irradiate the biological sample with visible light, ultraviolet light, infrared light, or a combination thereof. The light source can be one or more of a halogen light source, a laser light source, an LED light source, a mercury light source, and a xenon light source. The type and / or wavelength of the light source for fluorescence observation may be multiple and can be selected appropriately by a person skilled in the art. The light irradiation unit can have a transmissive, reflective, or incident-light type (coaxial incident-light type or lateral-light type) configuration.

[0114] (Optical Unit) The optical unit 5102 is configured to guide light from the biological sample S to the signal acquisition unit 5103. The optical unit may be configured to enable the microscope device 5100 to observe or capture an image of the biological sample S. The optical unit 5102 may include an objective lens. The type of objective lens may be appropriately selected by a person skilled in the art depending on the observation method. The optical unit may also include a relay lens for relaying an image magnified by the objective lens to the signal acquisition unit. The optical unit may further include optical components other than the objective lens and the relay lens, such as an eyepiece, a phase plate, and a condenser lens. The optical unit 5102 may also include a wavelength separation unit configured to separate light having a predetermined wavelength from the light from the biological sample S. The wavelength separation unit may be configured to selectively allow light of a predetermined wavelength or wavelength range to reach the signal acquisition unit. The wavelength separation unit may include, for example, one or more of a filter that selectively transmits light, a polarizing plate, a prism (Wollaston prism), and a diffraction grating. The optical components included in the wavelength separation unit may be arranged, for example, on the optical path from the objective lens to the signal acquisition unit. The wavelength separation unit is provided in a microscope device when fluorescence observation is performed, particularly when the microscope device includes an excitation light irradiation unit. The wavelength separation unit may be configured to separate fluorescent light from each other or to separate white light from fluorescent light.

[0115] (Signal Acquisition Unit) The signal acquisition unit 5103 may be configured to receive light from the biological sample S and convert the light into an electrical signal, particularly a digital electrical signal. The signal acquisition unit may be configured to acquire data related to the biological sample S based on the electrical signal. The signal acquisition unit may be configured to acquire data on an image (particularly a still image, a time-lapse image, or a moving image) of the biological sample S, particularly data on an image enlarged by the optical unit. The signal acquisition unit includes one or more imaging elements, such as a CMOS or CCD, having a plurality of pixels arranged one-dimensionally or two-dimensionally. The signal acquisition unit may include an imaging element for acquiring low-resolution images and an imaging element for acquiring high-resolution images, or may include an imaging element for sensing (e.g., AF) and an imaging element for outputting images (e.g., observation). In addition to the plurality of pixels, the imaging element may include a signal processing unit (including one or more of a CPU, a DSP, and a memory) that performs signal processing using pixel signals from each pixel, and an output control unit that controls the output of image data generated from the pixel signals and processed data generated by the signal processing unit. The imaging element including the plurality of pixels, the signal processing unit, and the output control unit may preferably be configured as a single-chip semiconductor device. The microscope system 5000 may further include an event detection sensor. The event detection sensor may include a pixel that photoelectrically converts incident light and may be configured to detect an event when a luminance change of the pixel exceeds a predetermined threshold. The event detection sensor may be, in particular, an asynchronous type.

[0116] (Control Unit) The control unit 5110 controls imaging by the microscope device 5100. For imaging control, the control unit can adjust the positional relationship between the optical unit and the sample mounting unit by driving the movement of the optical unit 5102 and / or the sample mounting unit 5103. The control unit 5110 can move the optical unit and / or the sample mounting unit in a direction toward or away from each other (for example, in the optical axis direction of the objective lens). The control unit may also move the optical unit and / or the sample mounting unit in any direction in a plane perpendicular to the optical axis direction. For imaging control, the control unit may control the light irradiation unit 5101 and / or the signal acquisition unit 5103.

[0117] (Sample Mounting Unit) The sample mounting unit 5104 may be configured to fix the position of the biological sample on the sample mounting unit, and may be a so-called stage. The sample mounting unit 5104 may be configured to move the position of the biological sample in the optical axis direction of the objective lens and / or in a direction perpendicular to the optical axis direction.

[0118] (Information Processing Unit) The information processing unit 5120 may acquire data (such as imaging data) acquired by the microscope device 5100 from the microscope device 5100. The information processing unit may perform image processing on the imaging data. The image processing may include unmixing processing, particularly spectral unmixing processing. The unmixing processing may include processing for extracting data of light components of a predetermined wavelength or wavelength range from the imaging data to generate image data, or processing for removing data of light components of a predetermined wavelength or wavelength range from the imaging data. The image processing may also include autofluorescence separation processing for separating autofluorescence components and pigment components of tissue sections, or fluorescence separation processing for separating wavelengths between pigments with different fluorescence wavelengths. The autofluorescence separation processing may involve processing for removing autofluorescence components from image information of one of the multiple specimens, which are identical or have similar properties, using an autofluorescence signal extracted from the other specimen. The information processing unit 5120 may transmit data for imaging control to the control unit 5110, and the control unit 5110 may receive the data and control imaging by the microscope device 5100 in accordance with the data.

[0119] The information processing unit 5120 may be configured as an information processing device such as a general-purpose computer, and may include a CPU, RAM, and ROM. The information processing unit may be included in the housing of the microscope device 5100, or may be located outside the housing. Furthermore, various processes or functions performed by the information processing unit may be realized by a server computer or cloud connected via a network.

[0120] The method for imaging the biological sample S using the microscope device 5100 may be appropriately selected by those skilled in the art depending on the type of biological sample, the purpose of imaging, etc. Examples of the imaging method will be described below.

[0121] One example of an imaging method is as follows. The microscope apparatus may first identify an imaging target region. The imaging target region may be identified to cover the entire region where the biological sample is present, or may be identified to cover a target portion of the biological sample (a portion where a target tissue section, target cells, or target lesion is present). Next, the microscope apparatus divides the imaging target region into multiple divided regions of a predetermined size and sequentially images each divided region. This results in an image of each divided region being acquired. As shown in FIG. 14 , the microscope apparatus identifies an imaging target region R that covers the entire biological sample S. The microscope apparatus then divides the imaging target region R into 16 divided regions. The microscope apparatus then images divided region R1, and may then image any region included in the imaging target region R, such as a region adjacent to divided region R1. The divided regions are then imaged until no unimaged divided regions remain. Regions other than the imaging target region R may also be imaged based on the captured image information of the divided regions. After imaging a divided region, the positional relationship between the microscope device and the sample mounting unit is adjusted to image the next divided region. This adjustment may be performed by moving the microscope device, the sample mounting unit, or both. In this example, the imaging device that images each divided region may be a two-dimensional imaging element (area sensor) or a one-dimensional imaging element (line sensor). The signal acquisition unit may image each divided region via an optical unit. Furthermore, imaging of each divided region may be performed continuously while moving the microscope device and / or the sample mounting unit, or the movement of the microscope device and / or the sample mounting unit may be stopped when imaging each divided region. The imaging target region may be divided so that each divided region partially overlaps, or so that each divided region does not overlap. Each divided region may be imaged multiple times by changing imaging conditions such as focal length and / or exposure time. Furthermore, the information processing device may stitch adjacent divided regions together to generate image data for a larger region. By performing this stitching process over the entire imaging target area, it is possible to obtain an image of a wider area of ​​the imaging target area.Also, image data with a lower resolution can be generated from the images of the divided regions or from the images that have been subjected to stitching processing.

[0122] Another example of an imaging method is as follows. The microscope apparatus may first identify an imaging target area. The imaging target area may be identified to cover the entire area where the biological sample is present, or may be identified to cover a target portion of the biological sample (a portion where a target tissue slice or target cell is present). Next, the microscope apparatus scans a portion of the imaging target area (also referred to as a "divided scan area") in one direction (also referred to as the "scan direction") in a plane perpendicular to the optical axis to capture an image. After completing the scan of the divided scan area, the microscope apparatus then scans the adjacent divided scan area. These scanning operations are repeated until the entire imaging target area is captured. As shown in FIG. 15 , the microscope apparatus identifies the area of ​​the biological sample S where a tissue slice is present (gray portion) as the imaging target area Sa. Then, the microscope apparatus scans a divided scan area Rs of the imaging target area Sa in the Y-axis direction. After completing the scan of the divided scan area Rs, the microscope apparatus then scans the adjacent divided scan area in the X-axis direction. This operation is repeated until scanning of the entire imaging target area Sa is completed. The positional relationship between the microscope device and the sample mount unit is adjusted for scanning each divided scan area, and for imaging the next divided scan area after imaging a divided scan area. This adjustment may be performed by moving the microscope device, the sample mount unit, or both. In this example, the imaging device that images each divided scan area may be a one-dimensional imaging element (line sensor) or a two-dimensional imaging element (area sensor). The signal acquisition unit may image each divided scan area via a magnifying optical system. Furthermore, imaging of each divided scan area may be performed continuously while moving the microscope device and / or the sample mount unit. The imaging target area may be divided so that the divided scan areas partially overlap, or so that they do not overlap. Each divided scan area may be imaged multiple times by changing imaging conditions such as focal length and / or exposure time. Furthermore, the information processing device can stitch together a plurality of adjacent divided scan areas to generate image data of a larger area.By performing the stitching process across the entire imaging target area, it is possible to obtain images of a larger area of ​​the imaging target area. Also, it is possible to generate lower resolution image data from the images of the divided scan areas or the stitched images.

[0123] [Cancer or Minimal Residual Disease Testing Method] The present technology provides a cancer testing method for a subject derived from a test subject, such as a biological fluid, using the analytical method of the present technology. Figure 10 shows an example of a flow diagram for testing for cancer or minimal residual disease using the analytical method of the present technology. Here, "minimal residual disease" refers to small lesions or abnormalities that are easily overlooked by known simple testing techniques. Examples include cancer cells remaining after cancer treatment and metastatic lesions.

[0124] As described above, the analysis method of the present technology targets nucleic acids such as cfDNA (cell-free DNA), ctDNA (circular tumor DNA), nucleic acids contained in EVs (extracellular vesicles), and circulating tumor cells (CTCs), and can realize rapid testing for cancer or minimal residual disease using a fluorescence detection method or the like, with a biological fluid or the like containing the cfDNA or the like as the test subject.

[0125] The analytical method of the present technology can test any biological fluid that is relatively easily obtained, such as plasma in blood, tears, urine, or saliva. Specifically, cancer or minimal residual disease can be tested according to the flow shown in FIG. 10. For example, when blood is used as the biological fluid to be tested, the amount of blood collected is 4 to 10 mL. When collecting the blood, it is desirable to use a dedicated blood collection tube capable of maintaining cfDNA. (S201 in FIG. 10)

[0126] The collected blood is centrifuged in the first step S202 shown in Figure 10 to prepare a plasma sample. Then, cfDNA is extracted from the plasma as shown in S203. Suitable methods for extracting cfDNA include, for example, a spin column or magnetic beads (Qiagen, QIAamp Circulating Nucleic Acid Kit, etc.). At this stage, it is preferable to perform QC to determine whether the obtained sample contains a sufficient amount of cfDNA for analysis.

[0127] Thereafter, as shown in S204, adjustment for DNA detection is performed. In this process, pre-amplification and single-stranded DNA are performed. Next, as shown in S205, the solution containing the adjusted nucleic acid such as DNA is used as the test subject, and analysis by this technology is performed. As a result, the analysis result of the test subject is output.

[0128] Based on the analysis results of the analytical method of the present technology, for example, the chromatin state of the nucleic acid of a test subject derived from the test subject can be suitably classified. Therefore, for diseases in which the chromatin state differs from the original closed or open chromatin state due to the disease, by analyzing the abundance ratio of cfDNA sequence species, it is possible to suitably determine the presence or absence of the disease in the test subject derived from the test subject.

[0129] In particular, in the analysis method of the present technology, the target molecules created in the target molecule creation process are subjected to RCA amplification using the circular structure of the detection probe as a template, thereby extending the detection nucleic acid and forming DNA nanoballs large enough to be detected by a microparticle analyzer, thereby enabling high-throughput testing for cancer or minimal residual disease using a microparticle analyzer.

[0130] In this case, the method for testing for cancer or minimal residual disease in a test subject derived from a test subject specifically includes the following steps: a target molecule creation step in which a solution containing multiple nucleic acids is used as the test subject, and detection probes containing a sequence complementary to a nucleic acid target sequence, the sequence complementary to the nucleic acid target sequence being separated into two regions and arranged at both ends, are bound to a nucleic acid having the nucleic acid target sequence among the multiple nucleic acids to create a target molecule; a binding step in which the two regions of the detection probe are ligated to form the detection probe into a circular structure; a detection nucleic acid extension step in which the detection nucleic acid is extended using the circular structure as a template; a fluorescence detection step in which the fluorescence of the nucleic acid identified by the detection probe is detected by a microparticle analyzer through hybridization of the fluorescent probe to a sequence complementary to the dye-binding sequence; an analysis step in which the fluorescence detection results detected in the fluorescence detection step are analyzed; and a determination step in which the presence or absence of a disease in the test subject derived from the test subject is determined based on the analysis results of the analysis step.

[0131] [Analysis System and Cancer or Minimal Residual Disease Testing System] The present technology can also be implemented as an analysis system. Specifically, the analysis system of the present technology tests a solution containing multiple nucleic acids. The system includes a target molecule generation unit that binds a detection probe containing a sequence complementary to a nucleic acid target sequence in the nucleic acids to a nucleic acid having the target nucleic acid sequence among the multiple nucleic acids to create a target molecule; a fluorescence detection unit that hybridizes a fluorescent probe containing one or more fluorescent dyes to the target molecule and detects fluorescent information from the fluorescently labeled target molecule; and an analysis unit that analyzes the fluorescence detection results detected by the fluorescence detection unit. The fluorescent probes used in the analysis system of the present technology classify multiple types of nucleic acid target sequences into groups smaller than the multiple types, and each group has one or more fluorescent dyes assigned to it.

[0132] The present technology can also be implemented as a cancer or minimal residual disease testing system. Specifically, the cancer or minimal residual disease testing system of the present technology has as a test subject a solution containing multiple nucleic acids, and includes a target molecule preparation unit that binds a detection probe containing a sequence complementary to a nucleic acid target sequence in the nucleic acids to a nucleic acid having the nucleic acid target sequence among the multiple nucleic acids to create a target molecule, a fluorescence detection unit that hybridizes a fluorescent probe containing one or more fluorescent dyes to the target molecule and detects fluorescent information from the fluorescently labeled target molecule, an analysis unit that analyzes the fluorescence detection results detected by the fluorescence detection unit, and a judgment unit that determines the presence or absence of a disease in a test subject derived from the test subject based on the analysis results of the analysis unit. Furthermore, the fluorescent probes used in the cancer or minimal residual disease testing system of the present technology also classify multiple types of nucleic acid target sequences into groups smaller than the multiple types, and each group has one or more fluorescent dyes assigned to it.

[0133] The analysis system or cancer or minimal residual disease testing system of the present technology may be composed of a single device, or may be composed of multiple devices or optional components that can be separated from the device. Specifically, the target molecule preparation unit, fluorescence detection unit, analysis unit, and judgment unit that constitute the analysis system or cancer or minimal residual disease testing system of the present technology may be configured as an integrated unit, or each unit may be configured to be separable.

[0134] As shown in FIG. 11 , the analysis system or cancer or minimal residual disease testing system of the present technology may use hospitals or other testing locations as testing centers, and create a learning model based on data obtained by performing the analysis method or cancer or minimal residual disease testing of the present technology at multiple locations. In the analysis or testing system 20 shown in FIG. 11 , the analysis method of the present technology is performed using a microparticle analyzer 21 or the like provided at each location (locations X to Z in FIG. 11 ). Based on the results of the analysis, learning models accumulated on servers 22 at each location may be accumulated in a central cloud 23, and a system may be constructed that uses federated learning to improve cancer or minimal residual disease testing at all locations. In this case, federated learning based on the learning models accumulated at each location is expected to improve the accuracy of cancer or minimal residual disease testing over time as the learning models are accumulated.

[0135] In this case, machine learning may be used for the associative learning. Continuing this associative learning improves the accuracy of classification in the classification process of the present technology, and improves the classification of nucleic acid target sequences based on a learning model created based on accumulated cfDNA appearance patterns, thereby improving the accuracy of cancer or minimal residual disease testing. Examples of the machine learning that can be used include supervised learning using the k-nearest neighbor method, support vector machines, and decision trees.

[0136] The analysis system of the present technology or the cancer or minimal residual disease testing system may be combined with any system as needed, depending on the purpose of the analysis or testing, as long as the desired physical properties are not impaired.

[0137] [Analysis Kit] The present technology can also be implemented as an analysis kit. Specifically, an analysis kit for a solution containing multiple nucleic acids, which is the test subject of the present technology, classifies multiple types of nucleic acid target sequences into groups fewer than the multiple types, and includes multiple nucleic acids assigned to each group, each group including a fluorescent probe having one or more fluorescent dyes and a detection probe having a sequence complementary to the nucleic acid target sequence.

[0138] By using the analytical kit of the present technology, the analytical method of the present technology can be suitably realized, the biological fluid or the like that is the subject of the test can be suitably analyzed, and the presence or absence of a disease in the subject of the test that is derived from the subject of the test can be determined.

[0139] The present technology can be configured as follows: [1] An analytical method comprising: a classification step of classifying multiple types of nucleic acid target sequences into groups with a number smaller than the multiple types; an assignment step of assigning one or more fluorescent dyes to each group; a target molecule creation step of binding a detection probe containing a sequence complementary to the nucleic acid target sequence to a nucleic acid among the multiple nucleic acids to create a target molecule; a fluorescent labeling step of hybridizing a fluorescent probe containing one or more fluorescent dyes to the target molecule to create a fluorescently labeled target molecule; a fluorescent detection step of detecting fluorescent information from the fluorescently labeled target molecule; and an analysis step of analyzing the fluorescent information detected in the fluorescent detection step. [2] The analytical method according to [1], wherein the detection probe has a structure in which the sequence complementary to the nucleic acid target sequence is separated into two regions and arranged at both ends of the detection probe. [3] The analytical method according to [2], further comprising a binding step of ligating the two regions of the detection probe to form a circular structure. [4] The analytical method according to [3], which includes a detection nucleic acid extension step of extending the detection nucleic acid using the cyclic structure as a template. [5] The analytical method according to [4], in which the detection nucleic acid is synthesized by extending the 3'-end or 5'-end of the nucleic acid target sequence. [6] The analytical method according to any one of [1] to [5], in which the detection of the fluorescence of the nucleic acid is performed using a microparticle analyzer. [7] The analytical method according to any one of [1] to [6], in which the classification of the multiple types of nucleic acid target sequences in the classification step is performed based on the analysis results of base sequences contained in the multiple nucleic acids contained in the solution to be tested. [8] The analytical method according to [7], in which the analysis results of the base sequences are the analysis results of the occurrence levels of the multiple nucleic acids. [9] The analytical method according to [8], in which the analysis of the occurrence levels is performed based on the degree of deviation from a reference occurrence level set as a reference.

[10] The analytical method according to [8], in which the base sequences are sequence information comprehensively analyzed by NGS.

[11] The analysis method according to any one of [1] to

[10] , wherein the analysis of the fluorescence detection results in the analysis step is performed based on the detection pattern of the fluorescence detected in the fluorescence detection step.

[12] The analysis method according to

[11] , wherein a reference fluorescent dye is set as a reference and the fluorescence detection pattern is corrected based on the detection value of the fluorescence of the reference fluorescent dye.

[13] The analysis method according to any one of [1] to

[12] , wherein the nucleic acid is DNA.

[14] The analysis method according to

[13] , wherein the DNA is cfDNA.

[15] A method for classifying the chromatin state of nucleic acid in a test subject derived from the test subject, using the analysis method according to any one of [1] to

[14] .

[16] A method for testing for cancer or minimal residual disease in a test subject derived from the test subject, using the analysis method according to any one of [1] to

[16] .

[17] An analysis system comprising: a target molecule preparation unit that uses a solution containing multiple nucleic acids as a test subject, and that binds a detection probe containing a sequence complementary to a nucleic acid target sequence in the nucleic acids to a nucleic acid among the multiple nucleic acids that has the nucleic acid target sequence to prepare a target molecule; a fluorescence detection unit that hybridizes a fluorescent probe having one or more fluorescent dyes to the target molecule and detects fluorescence information from the fluorescently labeled target molecule; and an analysis unit that analyzes the fluorescence detection results detected by the fluorescence detection unit, wherein the fluorescent probe classifies multiple types of nucleic acid target sequences into groups that are smaller than the multiple types, and has one or more fluorescent dyes assigned to each group.

[18] A cancer or minimal residual disease testing system, wherein a solution containing multiple nucleic acids is used as a test subject, the system comprising: a target molecule preparation unit that binds a detection probe having a sequence complementary to a nucleic acid target sequence in the nucleic acids to a nucleic acid among the multiple nucleic acids that has the nucleic acid target sequence to create a target molecule; a fluorescence detection unit that hybridizes a fluorescent probe having one or more fluorescent dyes to the target molecule and detects fluorescent information from the fluorescently labeled target molecule; an analysis unit that analyzes the fluorescence detection results detected by the fluorescence detection unit; and a determination unit that determines the presence or absence of a disease in a test subject derived from the test subject based on the analysis results of the analysis unit, wherein the fluorescent probes classify multiple types of nucleic acid target sequences into groups fewer than the multiple types, and each group has one or more fluorescent dyes assigned to it.

[19] An analysis kit for a solution containing multiple nucleic acids, the system comprising: a fluorescent probe having one or more fluorescent dyes assigned to each group, and a detection probe having a sequence complementary to the nucleic acid target sequence.

[20] A method for testing for cancer or minimal residual disease in a test subject derived from the test subject, comprising: a target molecule creation step of binding a detection probe, which contains a sequence complementary to a nucleic acid target sequence and in which the sequence complementary to the nucleic acid target sequence is separated into two regions and arranged at both ends, to a nucleic acid among the plurality of nucleic acids having the nucleic acid target sequence to create a target molecule; a binding step of ligating the two regions of the detection probe to form a circular structure, a detection nucleic acid extension step of extending the detection nucleic acid using the circular structure as a template; a fluorescence detection step of detecting, with a microparticle analyzer, the fluorescence of the nucleic acid identified by the detection probe through hybridization of the fluorescent probe to a sequence complementary to the dye binding sequence; an analysis step of analyzing the fluorescence detection results detected in the fluorescence detection step; and a determination step of determining the presence or absence of a disease in the test subject derived from the test subject based on the analysis results of the analysis step.

[21] A testing system for cancer or minimal residual disease in a test subject derived from the test subject, comprising: a test subject is a solution containing multiple nucleic acids, and a detection probe containing a sequence complementary to a nucleic acid target sequence, the sequence complementary to the nucleic acid target sequence being separated into two regions and arranged at both ends, is bound to a nucleic acid having the nucleic acid target sequence among the multiple nucleic acids to create a target molecule; a target molecule creation unit that ligates the two regions of the detection probe to make the detection probe a circular structure and extends the detection nucleic acid using the circular structure as a template; a fluorescence detection unit that detects the fluorescence of the nucleic acid identified by the detection probe by hybridization of the fluorescent probe to a sequence complementary to the dye binding sequence using a microparticle analysis device; an analysis unit that analyzes the fluorescence detection results detected in the fluorescence detection step; and a judgment unit that judges whether or not the test subject has a disease derived from the test subject based on the analysis results of the analysis step.

[0140] 10 Detection probe 11 Nucleic acid target sequence 11-1, 11-2 Sequence complementary to nucleic acid target sequence 12 Sequence to which fluorescent probe can hybridize 12-1 Sequence serving as a template for sequence to which fluorescent probe can hybridize 13 Nucleic acid (cfDNA) 14 Detection nucleic acid 15 Fluorescent probe 16 Primer (detection nucleic acid extension primer) 20 Analysis or inspection system 21 Microparticle analyzer (FCM) 22 Server 23 Cloud

Claims

1. An analytical method comprising: a classification step of classifying a plurality of types of nucleic acid target sequences into groups the number of which is less than the plurality of types; an allocation step of assigning one or more fluorescent dyes to each of the groups; a target molecule creation step of testing a solution containing a plurality of nucleic acids and binding a detection probe containing a sequence complementary to the nucleic acid target sequence to a nucleic acid among the plurality of nucleic acids that has the nucleic acid target sequence to create a target molecule; a fluorescent labeling step of hybridizing a fluorescent probe containing the one or more fluorescent dyes to the target molecule to create a fluorescently labeled target molecule; a fluorescent detection step of detecting fluorescent information from the fluorescently labeled target molecule; and an analysis step of analyzing the fluorescent information detected in the fluorescent detection step.

2. The analytical method according to claim 1, wherein the detection probe has a structure in which a sequence complementary to the nucleic acid target sequence is separated into two regions and arranged at both ends of the detection probe.

3. The analytical method according to claim 2, further comprising a binding step of ligating the two regions of the detection probe to form a circular structure of the detection probe.

4. The analytical method according to claim 3, further comprising a step of extending the detection nucleic acid using the circular structure as a template.

5. The analytical method according to claim 4, wherein the detection of the fluorescence of the nucleic acid is carried out by a microparticle analyzer.

6. The analytical method according to claim 1, wherein the classification of the multiple types of nucleic acid target sequences in the classification step is performed based on the analysis results of the base sequences contained in the multiple nucleic acids.

7. The analytical method according to claim 6, wherein the analysis result of the base sequence is the analysis result of the occurrence levels of the plurality of nucleic acids.

8. The analysis method according to claim 7, wherein the analysis of the occurrence level is performed based on the degree of deviation from a reference occurrence level that is set as a reference.

9. The analytical method according to claim 7, wherein the base sequence is sequence information comprehensively analyzed by NGS.

10. The analytical method according to claim 1, wherein the analysis of the fluorescence detection results in the analysis step is performed based on the detection pattern of the fluorescence detected in the fluorescence detection step.

11. The analysis method according to claim 10, wherein the fluorescence detection pattern is corrected based on the fluorescence detection value of a reference fluorescent dye that is set as a reference.

12. The analytical method according to claim 1, wherein the nucleic acid is DNA.

13. The analytical method according to claim 12, wherein the DNA is cfDNA.

14. A method for classifying the chromatin state of a test subject from the cfDNA derived from the test subject, using the analytical method described in claim 13.

15. A method for testing a subject for cancer or minimal residual disease derived from the test subject, using the analytical method described in claim 13.

16. An analysis system having a solution containing multiple nucleic acids as a test subject, and comprising: a target molecule creation unit that binds a detection probe containing a sequence complementary to a nucleic acid target sequence in the nucleic acids to a nucleic acid among the multiple nucleic acids that has the nucleic acid target sequence to create a target molecule; a fluorescence detection unit that hybridizes a fluorescent probe having one or more fluorescent dyes to the target molecule and detects fluorescent information from the fluorescently labeled target molecule; and an analysis unit that analyzes the fluorescence detection results detected by the fluorescence detection unit, wherein the fluorescent probe classifies multiple types of nucleic acid target sequences into groups that are fewer than the multiple types, and has one or more fluorescent dyes assigned to each group.

17. A cancer or minimal residual disease testing system having a solution containing multiple nucleic acids as a test subject, comprising: a target molecule creation unit that binds a detection probe containing a sequence complementary to a nucleic acid target sequence in the nucleic acids to a nucleic acid among the multiple nucleic acids that has the nucleic acid target sequence to create a target molecule; a fluorescence detection unit that hybridizes a fluorescent probe having one or more fluorescent dyes to the target molecule and detects fluorescent information from the fluorescently labeled target molecule; an analysis unit that analyzes the fluorescence detection results detected by the fluorescence detection unit; and a judgment unit that judges the presence or absence of a disease in a test subject derived from the test subject based on the analysis results of the analysis unit, wherein the fluorescent probe classifies multiple types of nucleic acid target sequences into groups fewer than the multiple types, and has one or more fluorescent dyes assigned to each group.

18. A kit for analyzing a solution containing multiple nucleic acids, which classifies multiple types of nucleic acid target sequences into groups fewer than the multiple types, and includes a fluorescent probe having one or more fluorescent dyes assigned to each group, and a detection probe having a sequence complementary to the nucleic acid target sequence.

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