Inspection kit and method for designing inspection kit

Abstract

To provide an inspection kit that can enable a robust inspection of the amount of introduction of a reporter molecule precursor, and a method for designing an inspection kit.SOLUTION: An inspection kit for detecting a target nucleic acid sequence in a specimen comprises a lateral flow assay (LFA) test paper. The LFA test paper inspects a reaction solution obtained by adding the specimen to a reaction liquid including a Cas enzyme and a reporter molecule precursor and subjecting the reaction solution to the CRISPR-Cas reaction. The LFA test paper comprises a labeled part including a labeled antibody, a first detection line to which a capture molecule for capturing the reporter molecule precursor is fixed, and a second detection line to which a capture antibody for capturing the labeled antibody is fixed. The amount A of the labeled antibody and the mount S of the capture molecule on the LFA test paper satisfy the conditions of A≥10-12 mol and 1≤S / A.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] SUMMARY OF THE INVENTION The embodiments disclosed in this specification and drawings relate to test kits and methods for designing test kits. [Background technology]

[0002] A nucleic acid detection method (CRISPR-Cas LFA test) has been developed that combines the genome editing technology CRISPR-Cas with lateral flow assay (LFA), a testing method based on antigen-antibody reactions. In this CRISPR-Cas LFA test, for example, a sample containing DNA or RNA extracted from a subject is added to a reaction solution containing a Cas enzyme and a reporter molecule precursor, and subjected to a CRISPR-Cas reaction to generate a sample containing the reporter molecule precursor and / or reporter molecule. The resulting sample is then dropped onto an LFA test strip to test for the presence or absence (negative / positive) of the target nucleic acid in the sample. The reporter molecule precursor, for example, has a structure in which a first antigen and a second antigen are modified at both ends of a single-stranded DNA. The LFA test strip also has a labeled area where a labeled antibody for the first antigen is placed, a first detection line where a capture molecule for the second antigen is immobilized, and a second detection line where a capture antibody for the labeled antibody is placed. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Japanese Patent Publication No. 2023-11606 Summary of the Invention [Problem to be solved by the invention]

[0004] In conventional CRISPR-Cas LFA test kits, it was not uncommon for the second detection line to turn color despite negative conditions, resulting in a false positive. As described below, the inventors of the present invention discovered that one of the causes of this false positive is the tolerance to variations in the amount of reporter molecule precursor introduced.

[0005] The problem to be solved by the embodiments disclosed in this specification and the drawings is to enable robust testing against variations in the amount of reporter molecule precursor introduced. However, the problem to be solved by the embodiments disclosed in this specification and the drawings is not limited to the above problem. Problems corresponding to the effects of each configuration shown in the embodiments described below can also be positioned as other problems. [Means for solving the problem]

[0006] A test kit for detecting a target nucleic acid sequence in a sample according to an embodiment includes an LFA (lateral flow assay) test paper. The LFA test paper tests a reaction solution obtained by adding the sample to a reaction solution containing a Cas enzyme and a reporter molecule precursor and subjecting the reaction solution to a CRISPR-Cas reaction. The LFA test paper includes a labeling portion containing a labeled antibody, a first detection line on which a capture molecule that captures the reporter molecule precursor is immobilized, and a second detection line on which a capture antibody that captures the labeled antibody is immobilized. The amount A of the labeled antibody and the amount S of the capture molecule on the LFA test paper satisfy A≧10 -12 mol and 1≦S / A. [Brief explanation of the drawings]

[0007] [Figure 1] FIG. 1 is a diagram showing an example of a test kit according to an embodiment. [Figure 2] FIG. 1 is a diagram illustrating the CRISPR-Cas reaction process according to an embodiment. [Figure 3A] FIG. 2 is a diagram for explaining the flow (detection principle) of target nucleic acid detection under negative conditions according to an embodiment. [Figure 3B] FIG. 2 is a diagram for explaining the flow (detection principle) of target nucleic acid detection under negative conditions according to an embodiment. [Figure 4A] FIG. 2 is a diagram for explaining the flow (detection principle) of target nucleic acid detection under positive conditions according to an embodiment. [Figure 4B] FIG. 2 is a diagram for explaining the flow (detection principle) of target nucleic acid detection under positive conditions according to an embodiment. [Figure 5A] 1 is a graph showing the change in the amount of labeled antibody on each detection line relative to the amount Rp of introduced reporter molecule precursor under negative conditions for an LFA test paper according to the prior art. [Figure 5B] 1 is a graph showing the change in the amount of labeled antibody on each detection line relative to the amount Rp of introduced reporter molecule precursor under negative conditions for an LFA test paper according to the prior art. [Figure 6A] 10 is a graph showing the change in the amount of labeled antibody on each detection line relative to the amount Rp of introduced reporter molecule precursor under negative conditions for an LFA test paper according to an embodiment. [Figure 6B] 10 is a graph showing the change in the amount of labeled antibody on each detection line relative to the amount Rp of introduced reporter molecule precursor under negative conditions for an LFA test paper according to an embodiment. [Figure 7] FIG. 10 is a diagram showing the relationship between the tolerance range (Rp tolerance range) of the amount of reporter molecule precursor introduced Rp when the ratio S / A of the amount S of captured molecules on the first detection line to the amount A of labeled antibody on the first detection line according to the embodiment is changed. [Figure 8A] FIG. 10 is a diagram showing the change in color intensity with respect to the amount of reporter molecule precursor in the first detection line according to the embodiment. [Figure 8B] FIG. 10 is a diagram showing the change in color intensity relative to the amount of reporter molecule precursor in the second detection line according to the embodiment. [Figure 9A] 1 is a graph showing the relationship between the amount of labeled antibody and color intensity according to an embodiment. [Figure 9B] FIG. 9B is an enlarged view of a portion of the graph shown in FIG. 9A. [Figure 10A] 10 is a graph showing changes in specificity when the ratio S / A of the amount S of captured molecules on the first detection line to the amount A of labeled antibodies is changed according to the embodiment. [Figure 10B] 1 is a graph showing the change in specificity relative to the variation (standard deviation) in the amount Rp of reporter molecule precursor introduced according to an embodiment. [Figure 11] 1 is a graph showing the change in sensitivity relative to the variation (standard deviation) in the amount Rp of reporter molecule precursor introduced according to an embodiment. DETAILED DESCRIPTION OF THE INVENTION

[0008] Hereinafter, a test kit and a method for designing a test kit according to an embodiment will be described with reference to the drawings.

[0009] [Test kit composition] First, the configuration of a test kit according to an embodiment will be described. FIG. 1 is a diagram showing an example of a test kit 1 according to an embodiment. The test kit 1 performs a CRISPR-Cas LFA test. The test kit 1 detects a nucleic acid (hereinafter referred to as a "target nucleic acid") that is the target (subject) of detection. Examples of target nucleic acids include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The target nucleic acid may be single-stranded or double-stranded. The target nucleic acid is derived from, for example, a microorganism having a pathogen. Examples of microorganisms include viruses, bacteria, etc.

[0010] The test kit 1 includes, for example, a first reagent 10, a second reagent 12, and an LFA test paper 20 (device). The first reagent 10 includes a reporter molecule precursor. The reporter molecule precursor includes a nucleic acid (DNA or RNA) that serves as a substrate for the nonspecific nucleic acid cleavage activity of the CRISPR-Cas enzyme. Both ends of the nucleic acid in the reporter molecule precursor are modified with a molecule (antigen B) for binding to a labeled antibody and a molecule (antigen A) for binding to a capture molecule of the first detection line 23. Antigen B is, for example, 5-carboxyfluorescein (FAM). Antigen A is, for example, biotin. The nucleic acid sequence may be any sequence. The second reagent 12 includes a CRISPR-Cas enzyme. The CRISPR-Cas enzyme is a complex of a Cas protein (Cas12a, Cas12b, Cas12c, etc.) and RNA (crRNA). A sample is added to a reaction solution containing a first reagent 10 (a reporter molecule precursor) and a second reagent 12 (a CRISPR-Cas enzyme) to induce a CRISPR-Cas reaction. The CRISPR-Cas reaction solution after the CRISPR-Cas reaction is then tested using an LFA test paper 20.

[0011] The LFA test paper 20 is primarily made of a material used in conventional immunochromatographic tests. The primary material of the LFA test paper 20 is, for example, nitrocellulose. The LFA test paper 20 may be, for example, 60 mm long and 3 mm wide. The LFA test paper 20 includes, for example, a sample pad 21, a labeling section 22 (labeled antibody pad (conjugation pad)), a first detection line 23, a second detection line 24, and an absorbent pad 25. The labeling section 22, the first detection line 23, the second detection line 24, and the absorbent pad 25 are arranged in this order, with the sample pad 21, onto which the CRISPR-Cas reaction solution is dripped, being the most upstream.

[0012] The sample pad 21 controls the delivery of the dropped CRISPR-Cas reaction solution to the labeling unit 22. As in conventional immunochromatographic testing, the sample pad 21 uses, for example, a cellulose fiber filter.

[0013] The labeling unit 22 includes a labeled anti-antigen B antibody that binds to the antigen B of the reporter molecule precursor. The labeled anti-antigen B antibody includes an antibody that binds to the reporter molecule precursor or reporter molecule, and a labeled molecule that can exhibit color development on the LFA test paper. The labeled anti-antigen B antibody is, for example, an anti-FAM antibody labeled with gold nanoparticles. The labeling unit 22 allows the reporter molecule precursor or antigen B of the reporter molecule flowing from the sample pad 21 to form a complex with the labeled anti-antigen B antibody in the labeling unit 22 and flow downstream. The material of the labeling unit 22 can be, for example, glass fiber, which is used in conventional immunochromatographic tests.

[0014] The first detection line 23 is an area where a molecule that captures the reporter molecule precursor is immobilized. The first detection line 23 exhibits color when the captured reporter molecule precursor binds to a labeled antibody (anti-antigen B labeled antibody). The first detection line 23 is provided to determine a negative condition in a detection test for a target nucleic acid. An antigen A capturing molecule that binds to antigen A, a modifying molecule of the reporter molecule precursor, is immobilized on the first detection line 23. The antigen A capturing molecule is, for example, streptavidin. When antigen A of the reporter molecule precursor bound to the anti-antigen B labeled antibody binds to the antigen A capturing molecule, the anti-antigen B labeled antibody remains on the first detection line 23, and the first detection line 23 develops color.

[0015] The second detection line 24 is an area where molecules that bind to the labeled antibody (anti-antigen B labeled antibody) flowing from the first detection line 23 are immobilized. The second detection line 24 exhibits color due to the captured labeled antibody. The second detection line 24 is provided to determine a positive condition in a detection test for a target nucleic acid. An antibody of the labeled antibody (capture antibody), which is a molecule that binds to the anti-antigen B labeled antibody, is immobilized on the second detection line 24. The antibody of the labeled antibody is, for example, rabbit IgG. When the anti-antigen B labeled antibody bound to the reporter molecule (antigen B) binds to the antibody of the labeled antibody, the anti-antigen B labeled antibody remains on the second detection line 24, and the second detection line 24 develops color.

[0016] The water-absorbing pad 25 is provided downstream of the LFA test strip 20 and absorbs the flowing CRISPR-Cas reaction solution. For the material of this water-absorbing pad 25, for example, cellulose or the like used in conventional immunoassay tests is used.

[0017] [Testing method] Next, each step in the testing method using the test kit 1 will be described. This testing method includes a CRISPR-Cas reaction step and a target nucleic acid detection step (LFA step).

[0018] [CRISPR-Cas reaction step] First, the CRISPR-Cas reaction step will be described. FIG. 2 is a diagram for explaining the CRISPR-Cas reaction step according to the embodiment. For example, in this step, a sample is added to a reaction solution containing a CRISPR-Cas enzyme and a reporter molecule precursor to cause a CRISPR-Cas reaction. The CRISPR-Cas enzyme has the activity of recognizing a target nucleic acid (DNA or RNA) sequence by crRNA and non-specifically cleaving the surrounding nucleic acid (DNA or RNA). In the CRISPR-Cas reaction, when there is a nucleic acid having a target sequence in the sample, the CRISPR-Cas enzyme is activated, and the nucleic acid of the reporter molecule precursor is cleaved to convert it into a reporter molecule.One example of the CRISPR-Cas reaction conditions is to prepare 20 μL of the reaction solution in a PCR tube and react the PCR tube in a thermostat at 37° C. for 30 to 60 minutes.

[0019] When the sample contains DNA to be detected (hereinafter referred to as "target DNA"), that is, in the case of "positive", this target DNA specifically binds to the crRNA of the CRISPR-Cas enzyme. When the CRISPR-Cas enzyme is bound to the target DNA, it has cleavage activity against nucleic acids of any base sequence. Here, between the reporter molecule precursor and the CRISPR-Cas enzyme having cleavage activity, a cleavage reaction occurs at the ssDNA portion of the reporter molecule precursor. As a result, reporter molecules (antigen A reporter molecule, antigen B reporter molecule) in which the ssDNA portion of the reporter molecule precursor is cleaved are generated.

[0020] On the other hand, if the sample does not contain the target DNA to be detected (i.e., a "negative" result), the target DNA does not bind to the crRNA, and the CRISPR-Cas enzyme has no cleavage activity. In this case, no cleavage reaction occurs at the ssDNA portion of the reporter molecule precursor between the reporter molecule precursor and the CRISPR-Cas enzyme, which has no cleavage activity.

[0021] <Target nucleic acid detection process (LFA process)> Next, the target nucleic acid detection step (LFA step) will be described. In this step, the CRISPR-Cas reaction solution (hereinafter also referred to as "sample") after the CRISPR-Cas reaction is mixed with a buffer to adjust the pH, viscosity, etc. As an example, 100 μL of buffer is added to 10 μL of sample. For example, a Tris-Buffered Saline-based buffer (HybriDetect Assay Buffer, #MGCB, Milenia Biotec GmbH) is used as the buffer. Next, the sample mixed with the buffer is applied to the LFA test paper 20. For example, 10 μL of sample is dropped onto the sample pad 21 and allowed to flow through the LFA test paper 20 for 5 minutes, and the color development of each detection line is determined.

[0022] Alternatively, the test may be performed by placing a sample mixed with a buffer in a microtube, immersing the LFA test strip 20 in the sample, and then removing it after 5 minutes. The presence or absence (positive / negative) of the target nucleic acid sequence in the specimen can be determined based on the color development of each detection line on the LFA test strip 20 after the sample has been introduced. For example, if the first detection line 23 turns red and the second detection line 24 shows no color development, the result is determined to be negative; if the second detection line 24 shows color development regardless of whether the first detection line 23 turns red, the result is determined to be positive. Alternatively, a measuring device such as an immunochromatography reader may be used to determine whether the color development is above a threshold value, thereby determining whether the result is negative or positive.

[0023] 3A and 3B are diagrams illustrating the flow (detection principle) of target nucleic acid detection under negative conditions according to an embodiment, while FIGS. 4A and 4B are diagrams illustrating the flow (detection principle) of target nucleic acid detection under positive conditions according to an embodiment.

[0024] (Negative condition) As shown in Figure 3A, a sample of the CRISPR-Cas reaction solution is dropped onto the sample pad 21 of the LFA test strip 20. Under negative conditions, this sample contains a reporter molecule precursor but no reporter molecule. The sample dropped onto the sample pad 21 flows into the labeling section 22, where a complex (hereinafter referred to as the "first complex") is formed in which the anti-antigen B labeled antibody placed in the labeling section 22 binds to the antigen B of the reporter molecule precursor contained in the sample.

[0025] Next, as shown in FIG. 3B, the generated first complex permeates the LFA test paper 20 by capillary force and flows in the direction of arrow AR until it reaches the first detection line 23. The first complex (antigen A at one end of the reporter molecule precursor) that reaches the first detection line 23 binds to the antigen A capturing molecule immobilized on the first detection line 23. As the amount of first complex that binds to such antigen A capturing molecule increases, the detection line above the first detection line 23 changes color. On the other hand, the detection line above the second detection line 24 does not change color. By confirming that only the detection line above the first detection line 23 changes color, the examiner can confirm that the target nucleic acid was not detected and the test result is negative.

[0026] (Positive condition) As shown in FIG. 4A, a sample of the CRISPR-Cas reaction solution is dropped onto the sample pad 21 of the LFA test strip 20. Under positive conditions, the sample contains reporter molecules (antigen A reporter molecule, antigen B reporter molecule). Depending on the amount of reporter molecule precursor, the sample may contain unreacted reporter molecule precursor. The sample dropped onto the sample pad 21 flows into the labeling section 22, where a complex (hereinafter referred to as the "second complex") is formed in which the anti-antigen B labeled antibody placed in the labeling section 22 binds to the antigen B reporter molecule contained in the sample. If the sample contains a reporter molecule precursor, a first complex is also formed.

[0027] Next, as shown in FIG. 4B, the generated second complex and antigen A reporter molecule permeate the LFA test paper 20 by capillary force and flow in the direction of arrow AR until they reach the first detection line 23. The antigen A reporter molecule that reaches the first detection line 23 binds to the antigen A capturing molecule immobilized on the first detection line 23. On the other hand, the second complex does not bind to the antigen A capturing molecule. As a result, the anti-antigen B labeled antibody is not captured by the first detection line 23, and the detection line above the first detection line 23 does not develop a color. Note that if the sample contains a reporter molecule precursor, the first complex generated in the labeling section 22 will bind to the antigen A capturing molecule, causing the detection line above the first detection line 23 to develop a color.

[0028] Next, the second complex that has passed through the first detection line 23 continues to flow on the LFA test paper 20 in the direction of arrow AR and reaches the second detection line 24. The second complex (the anti-antigen B labeled antibody of the second complex) that has reached the second detection line 24 binds to the antibody of the labeled antibody immobilized on the second detection line 24. As the amount of the anti-antigen B labeled antibody that binds to the antibody of the labeled antibody increases, the detection line on the second detection line 24 changes color. By confirming the color change of the detection line on the second detection line 24, the examiner can confirm that the target nucleic acid has been detected in the sample and that the test result is positive.

[0029] <Test kit (LFA test strip) composition> To accurately perform a test based on the detection principle in the target nucleic acid detection process described above, it is necessary to limit the amount of reporter molecule precursor Rp introduced into the sample dropped onto the LFA test paper 20 to a tolerance range. This tolerance range is defined based on the amount A of labeled antibody on the LFA test paper 20 and the amount S of capture molecules on the first detection line. The present inventors discovered that in conventional LFA test papers, the ratio between the amount A of labeled antibody and the amount S of capture molecules was 1 > S / A, narrowing the tolerance range for the amount of reporter molecule precursor introduced into the sample dropped onto the LFA test paper. In contrast, the LFA test paper 20 according to the present embodiment is configured so that the ratio between the amount A of labeled antibody and the amount S of capture molecules is 1 ≦ S / A, thereby widening the tolerance range for the amount of reporter molecule precursor introduced. The following explains these differences in configuration.

[0030] (Conventional technology configuration) Figures 5A and 5B are graphs showing the change in the amount of labeled antibody at each detection line relative to the amount of reporter molecule precursor introduced, Rp, under negative conditions for a conventional LFA test strip. This graph is based on the mathematical model and experimental data described below. Figure 5B shows five states (states 1 to 5) on this graph.

[0031] The first state is a state in which the amount Rp of reporter molecule precursor introduced is small. In this first state, the amount of first complex produced in the labeling section 22 is small, and therefore the amount of labeled antibody (first complex) captured on the first detection line 23 is also small. As a result, the detection line on the first detection line 23 does not develop color. On the other hand, unreacted labeled antibody (anti-antigen B labeled antibody) flows over and is captured on the second detection line 24. As a result, the detection line on the second detection line 24 develops color.

[0032] In the second state, as the amount of reporter molecule precursor introduced Rp increases, the amount of the first complex produced in the labeling section 22 increases, and the amount of labeled antibody (first complex) captured on the first detection line 23 also increases. As a result, the color of the detection line on the first detection line 23 gradually becomes visible. Meanwhile, the amount of unreacted labeled antibody (anti-antigen B labeled antibody) that flows onto the second detection line 24 gradually decreases. As a result, the color of the detection line on the second detection line 24 gradually becomes invisible.

[0033] The third state is a state in which the amount of introduced reporter molecule precursor Rp is equal to the amount of captured molecules S on the first detection line. In this third state, the amount of labeled antibody (first complex) captured on the first detection line 23 is at its upper limit. In this state, the detection line on the first detection line 23 develops color. On the other hand, the amount of unreacted labeled antibody (anti-antigen B labeled antibody) that flows onto the second detection line 24 is at its lower limit. In this state, the detection line on the second detection line 24 does not develop color.

[0034] The fourth state is a state in which the amount Rp of introduced reporter molecule precursor is between the amount S of captured molecules on the first detection line and the amount A of labeled antibody on the LFA test paper 20. That is, in this fourth state, the amount Rp of introduced reporter molecule precursor is equal to or greater than the amount S of captured molecules and equal to or less than the amount A of labeled antibody. During this period, the number of reporter molecule precursors bound to the labeled antibody also increases, so there is no effect of competition, and the amount of labeled antibody (first complex) captured on the first detection line 23 maintains its upper peak. During this period, the detection line on the first detection line 23 develops color. Meanwhile, the amount of unreacted labeled antibody (anti-antigen B labeled antibody) flowing onto the second detection line 24 also maintains its lower peak. During this period, the detection line on the second detection line 24 does not develop color.

[0035] The fifth state is a state in which the amount of labeled antibody (first complex) captured on the first detection line 23 gradually decreases as the amount of introduced reporter molecule precursor Rp increases. When the amount of introduced reporter molecule precursor Rp becomes greater than the amount of labeled antibody A, competition occurs between the reporter molecule precursor that binds to the labeled antibody and the reporter molecule precursor that does not bind to the labeled antibody, and the amount of labeled antibody (first complex) captured on the first detection line 23 decreases due to the hook effect of the labeled antibody. As a result, the color of the detection line on the first detection line 23 gradually becomes undetectable. Meanwhile, the amount of labeled antibody (anti-antigen B labeled antibody) that flows onto the second detection line 24 gradually increases. As a result, the color of the detection line on the second detection line 24 gradually becomes visible.

[0036] Considering the five states of the LFA test paper according to the prior art, the tolerance for the amount of reporter molecule precursor introduced, Rp, can be defined as follows: If the amount of reporter molecule precursor introduced, Rp, is too small, the amount of labeled antibody on the first detection line 23 decreases (state 1); as the amount of reporter molecule precursor introduced, Rp, increases, the amount of labeled antibody on the first detection line 23 increases (state 2); if the amount of reporter molecule precursor introduced, Rp, is equal to or greater than the amount of capture molecule, S, but is equal to or less than the amount of labeled antibody, A, the number of reporter molecule precursors bound to the labeled antibody also increases, and no competitive effect is observed (states 3 and 4). Furthermore, if the amount of reporter molecule precursor introduced, Rp, exceeds the amount of labeled antibody, A, competition occurs between the reporter molecule precursors that bind to the labeled antibody and the reporter molecule precursors that do not bind to the labeled antibody, and the amount of labeled antibody (first complex) captured on the first detection line 23 decreases due to the hook effect of the labeled antibody (state 5). Therefore, the amount of labeled antibody on the first detection line 23 is greatest and the amount of labeled antibody on the second detection line 24 is least when the amount of introduced reporter molecule precursor Rp is within the range between the amount of capture molecule S on the first detection line and the amount of labeled antibody A. The range (the range between L1 and L2 shown in Figure 5A) in which the first detection line 23 develops color but the second detection line 24 does not, centered on the range from the amount of capture molecule S on the first detection line to the amount of labeled antibody A, is the allowable range of the amount of introduced reporter molecule precursor Rp. In other words, the design range of the amount of introduced reporter molecule precursor Rp is determined by the ratio (difference) between the amount of labeled antibody A and the amount of capture molecule S.

[0037] The width of the peak where the amount of labeled antibody in the first detection line 23 is the highest and the amount of labeled antibody in the second detection line 24 is the lowest determines the width to the base of the graph and affects the allowable range of the amount of reporter molecule precursor Rp introduced. In the configuration of the prior art shown in Figure 5A, when the amount of capture molecule S is 1.0 x 10 -12 mol, the amount of labeled antibody A is 2.0 × 10 -12 mol, and the tolerance of the amount of reporter molecule precursor introduced, Rp, is 2.4 × 10 -13 mol / LFA to 8 x 10 -12 mol / LFA and narrower.

[0038] (Design method of this embodiment) 6A and 6B are graphs showing the change in the amount of labeled antibody at each detection line relative to the amount of reporter molecule precursor introduced, Rp, under negative conditions for an LFA test strip according to an embodiment. This graph is based on the mathematical model and experimental data described below. Figure 6B shows three states (states 1A to 3A) on this graph.

[0039] State 1A is a state in which the amount of introduced reporter molecule precursor Rp is equal to the amount of labeled antibody A. In state 1A, the amount of labeled antibody (first complex) captured on the first detection line 23 is at its upper limit. In this state, the detection line on the first detection line 23 turns color. On the other hand, the amount of unreacted labeled antibody (anti-antigen B labeled antibody) that flows onto the second detection line 24 is at its lower limit. In this state, the detection line on the second detection line 24 does not turn color.

[0040] State 2A is a state in which the amount Rp of introduced reporter molecule precursor is between the amount A of labeled antibody and the amount S of captured molecules on the first detection line 23. That is, in state 2A, the amount Rp of introduced reporter molecule precursor is equal to or greater than the amount A of labeled antibody and less than the amount S of captured molecules. The hook effect does not occur until the amount Rp of introduced reporter molecule precursor becomes equal to or greater than the amount S of captured molecules. Therefore, the amount of labeled antibody (first complex) captured on the first detection line 23 maintains its upper peak and does not change. During this period, the detection line on the first detection line 23 develops color. Meanwhile, the amount of unreacted labeled antibody (anti-antigen B labeled antibody) that flows onto the second detection line 24 also maintains its lower peak. During this period, the detection line on the second detection line 24 does not develop color.

[0041] The third state (3A state) is a state where the introduced amount Rp of the reporter molecule precursor is not less than the amount S of the capture molecule. In this third state (3A state), as the introduced amount Rp of the reporter molecule precursor increases, the amount of the labeled antibody (the first complex) captured on the first detection line 23 gradually decreases. This is because when the introduced amount Rp of the reporter molecule precursor is greater than the amount S of the capture molecule, competition occurs on the first detection line 23 due to the hook effect, and the amount of the labeled antibody (the first complex) captured decreases. As a result, the color development of the detection line on the first detection line 23 gradually becomes unrecognizable. On the other hand, the amount of the labeled antibody (anti-antigen B labeled antibody) on the second detection line 24 gradually increases. As a result, the detection line on the second detection line 24 gradually becomes recognizable.

[0042] Considering the three states in the LFA test strip 20 according to the present embodiment as described above, the allowable range of the introduced amount Rp of the reporter molecule precursor can be defined based on the following theory. That is, under the condition that the amount A of the labeled antibody is greater than the amount S of the capture molecule (S < A) as in the configuration of the prior art, even if the amount S of the capture molecule is decreased, only the amount leaking at the peak increases, and actually, the allowable range of the introduced amount Rp of the reporter molecule precursor cannot be widened effectively. Similarly, even if the amount A of the labeled antibody is increased, the allowable range of the introduced amount Rp of the reporter molecule precursor cannot be widened. Under the condition that the amount A of the labeled antibody is greater than the amount S of the capture molecule (S < A), even if there is a difference between the amount S of the capture molecule and the amount A of the labeled antibody, the height of the upper limit (peak) of the amount of the labeled antibody captured on the first detection line 23 changes, so the allowable range of the introduced amount Rp of the reporter molecule precursor cannot be widened effectively.

[0043] On the other hand, under the condition that the amount S of the capture molecule is greater than the amount A of the labeled antibody (S > A), if the amount A of the labeled antibody is extremely decreased, a situation where the color development on the second detection line 24 cannot be seen during positive detection will occur. Therefore, in the present embodiment, the amount S of the capture molecule is not less than the amount A of the labeled antibody (S ≥ A), and the amount A of the labeled antibody is not less than a predetermined amount (A ≥ 10 -12Under the condition of becoming (mol), centering on the range from the amount A of this labeled antibody to the amount S of the capture molecule on the first detection line, the first detection line 23 develops color, and the range where the second detection line 24 does not develop color (the range between L1 and L3 shown in FIG. 6A) is defined as the allowable range of the introduction amount Rp of the reporter molecule precursor. Within this allowable range, the width of the above-mentioned state 2A (the state where the introduction amount Rp of the reporter molecule precursor is equal to or greater than the amount A of the labeled antibody and less than the amount S of the capture molecule) becomes the optimal allowable range. By designing so that the amount S of the capture molecule is equal to or greater than the amount A of the labeled antibody (S≧A), the width of the peak where the amount of the labeled antibody on the first detection line 23 reaches the upper limit extends more than before, and the allowable range can be widened.

[0044] FIG. 7 is a diagram showing the relationship of the allowable range (Rp allowable range) of the introduction amount Rp of the reporter molecule precursor when changing the ratio S / A of the amount S of the capture molecule to the amount A of the labeled antibody on the first detection line according to the embodiment. These values are calculated using the mathematical model described later. When the amount S of the capture molecule on the first detection line 23 is equal to or greater than the amount A of the labeled antibody (S≧A), it was confirmed that the width of the peak where the amount of the labeled antibody on the first detection line 23 reaches the upper limit extends more than before, and the allowable range widens. Thereby, a more robust inspection against fluctuations in the introduction amount Rp of the reporter molecule precursor becomes possible.

[0045] The test kit 1 is an example of a "test kit". The LFA test strip 20 is an example of an "LFA test strip". The test kit 1 detects the target nucleic acid sequence in a sample. The test kit 1 includes an LFA test strip 20 for testing the reaction solution obtained by adding a sample to a reaction solution containing a Cas enzyme and a reporter molecule precursor and subjecting it to a CRISPR-Cas reaction. The LFA test strip 20 includes a labeling part 22 containing a labeled antibody, a first detection line 23 on which a capture molecule for capturing the reporter molecule precursor is fixed, and a second detection line 24 on which a capture antibody for capturing the labeled antibody is fixed. The amount A of the labeled antibody and the amount S of the capture molecule on the LFA test strip 20 satisfy the conditions of A≧10 -12 mol and 1≦S / A. The introduction amount Rp of the reporter molecule precursor into the LFA test strip 20 satisfies the condition of A≦Rp<S.

[0046] Preferably, the amount A of labeled antibody and the amount S of capturing molecules may further satisfy the condition of 2≦S / A. More preferably, the amount A of labeled antibody and the amount S of capturing molecules may further satisfy the condition of 5≦S / A. Even more preferably, the amount A of labeled antibody and the amount S of capturing molecules may further satisfy the condition of 10≦S / A.

[0047] Preferably, the amount A of the labeled antibody and the amount S of the capture molecule are further such that A≧2×10 -12 More preferably, the amount A of the labeled antibody and the amount S of the capture molecule further satisfy the condition A ≥ 2 × 10 -12 More preferably, the amount A of the labeled antibody and the amount S of the capture molecule further satisfy the condition A ≥ 2 × 10 -12 More preferably, the amount A of the labeled antibody and the amount S of the capture molecule further satisfy the condition A ≥ 2 × 10 -12 mol and 10≦S / A may be satisfied.

[0048] <Mathematical model> The mathematical model used to determine the configuration of the test kit according to this embodiment is described below. This mathematical model is based on the following two settings. Setup 1: Complex formation All molecules meet the binding molecule immediately after mixing, giving each molecule introduced into the system an opportunity to bind (equilibrium is reached immediately, no deactivation occurs), and then form complexes with a binding rate that depends on the amount of molecule and the strength of affinity. Setting 2: Complex bonding (related to the hook effect) The binding rate of the target molecules is dependent on the amount of the molecule and the strength of the affinity. The binding rate between the target molecules does not change depending on whether or not another molecule is bound to them.

[0049] (initial conditions) The initial conditions of the mathematical model are as follows: CRISPR-Cas reaction process: Template amount [N0]≧0 Enzyme amount [E0]≧0 Reporter molecule precursor amount [Rp0]>0 LFA test strips: Labeled antibody amount [A0]>0 Capture molecular weight of reporter molecule precursor [S0]>0 Amount of captured antibody by labeled antibody [G0]>0

[0050] (1) Mathematical model (CRISPR-Cas reaction) (Binding of template DNA and Cas enzyme) The binding of template DNA to Cas enzyme is further assumed as follows: Namely, template DNA and Cas enzyme bind via the binding process described in Assumption 1 above. All Cas enzymes bound to template DNA become active enzymes. The amount of reporter molecule precursor cleaved per unit amount of active enzyme per unit time is defined as enzyme activity A. Enzyme activity A is a constant determined only by the type of enzyme and is not dependent on the amount of reporter molecule precursor or enzyme amount, etc.

[0051] The total amount of template DNA in the reaction system is [N0], and the total amount of Cas enzyme is [E0]. Here, the equilibrium dissociation constant of the binding of these two molecules is K NE Then, the amount of active enzyme [NE] can be calculated using the following formulas (1) to (5).

[0052]

number

number

[0053] Here, since [N]=[N0]-[NE] and [E]=[E0]-[NE], the above equation (2) can be rewritten as the following equation (3).

[0054]

number

[0055] K NE=K off / K on Using this, the above equation (3) can be rewritten as the following equation (4).

[0056]

number

[0057] From the solution of the quadratic equation in the above equation (4), the following equation (5) is obtained.

[0058]

number

[0059] (Cleavage of reporter molecule precursor by active enzyme) Let A be the enzyme activity, T be the reaction time, and [NE] be the amount of active enzyme. The amount of cleaved reporter molecule precursor is represented by AT[NE]. When [Rp0]>AT[NE], the following holds: [Rp] = [Rp0] - AT[NE] [R1]=AT[NE] [R2]=AT[NE]

[0060] Also, if [Rp0]≦AT[NE], all initial reporter molecule precursors are cleaved, resulting in: [Rp]=0 [R1]=[Rp0] [R2]=[Rp0]

[0061] For the cleavage reaction of the reporter molecule precursor in the CRISPR-Cas reaction, an enzyme reaction rate equation may be used, such as the total quasi-steady-state approximation (tQSSA), which is more accurate than the Michaelis-Menten equation.

[0062] (2) Mathematical model (LFA) (Binding of labeled antibody to reporter molecule precursor or first reporter molecule) All of the introduced CRISPR-Cas reaction products flow into the labeling section 22 (conjugation pad), and the labeled antibodies in the labeling section 22 are capable of binding to all of the reporter molecule precursors or the first reporter molecule (e.g., the antigen B reporter molecule). The amount of reporter molecule precursor is [Rp], the molecular weight of the first reporter is [R1], the amount of labeled antibody is [A], the total amount of labeled antibody-bound molecules is [RA], the amount of labeled antibody-bound reporter molecule precursor is [RpA], and the molecular weight of the labeled antibody-bound first reporter is [R1A].

[0063] In this reaction system, the total amount of the reporter molecule precursor and the first reporter molecule is [R0], and the total amount of the labeled antibody is [A0]. Here, the equilibrium dissociation constant of the binding of these two molecules is K RA Then, the total amount of labeled antibody-bound molecules [RA] is expressed by the following formula (6).

[0064]

number

[0065] The amount of reporter molecule precursor bound to the labeled antibody [RpA], the molecular weight of the first reporter bound to the labeled antibody [R1A], and the amount of reporter molecule precursor not bound to the labeled antibody [R] are expressed as follows using [RA]: [RpA]=[Rp][RA] / [Rp0] [R1A]=[R1][RA] / [Rp0] [R] = [Rp] - [RpA]

[0066] (coupling at first detection line) The binding at the first detection line 23 is assumed as follows: After mixing, all molecules flow to the first detection line 23. All captured molecules at the first detection line 23 have the ability to bind to a reporter molecule precursor or a second reporter molecule (e.g., an antigen A reporter molecule). Binding between the captured molecules and the reporter molecule precursor occurs regardless of whether a labeled antibody is bound. Molecules not captured at the first detection line 23 flow to the second detection line 24.

[0067] The unbound free capture molecule is [S], the total amount of molecules bound to the capture molecule is [RS], the amount of the labeled antibody-bound reporter molecule precursor bound to the capture molecule is [RpAS], the amount of the labeled antibody-unbound reporter molecule precursor bound to the capture molecule is [RpS], and the second reporter molecular weight bound to the capture molecule is [R2S].

[0068] The total amount of the second reporter molecule and the reporter molecule precursor in this reaction system is [Rp0], and the total amount of the capture molecule is [S0]. The equilibrium dissociation constant of the binding of these two molecules is K RS Then, the total amount of reporter bound to the capture molecule [RS] is expressed by the following equation (7).

[0069]

number

[0070] The amount of the reporter molecule precursor bound to the capture molecule by the labeled antibody [RpAS], the amount of the reporter molecule precursor not bound to the labeled antibody [RpS], and the second reporter molecular weight bound to the capture molecule [R2S] are expressed as follows: [RpAS] = [RpA][RS] / [Rp0] [RpS]=[R][RpS] / [Rp0] [R2S]=[R2][RS] / [Rp0] [S]=[S0]-[RS]

[0071] (coupling on the second detection line) The binding at the second detection line 24 is assumed as follows: All molecules that do not bind at the first detection line 23 flow to the second detection line 24. All capture antibodies on the second detection line 24 have the ability to bind to labeled antibodies. Binding between the capture antibody and labeled antibody occurs regardless of whether or not there is binding to the reporter molecule precursor or the first reporter molecule. Molecules that are not captured at the second detection line 24 flow to the absorbent pad 25.

[0072] The unbound free capture antibody is [G], and the total amount of AuNP antibody bound to the capture antibody is [AG]. The total amount of labeled antibody in this reaction system is [A t ] and the total amount of capture antibody is [G0]. The equilibrium dissociation constant of the binding of these two molecules is K AG Then, the total amount of labeled antibody bound to the capture antibody [AG] is expressed by the following formula (8).

[0073]

number

[0074] Here, the labeled antibody flowing from the first detection line 23 to the second detection line 24 is [A0]-[RpAS], so [At]=[A0]-[RpAS].

[0075] (3) Mathematical model (color intensity conversion) The color intensity of each detection line can be expressed from the concentration of the labeled antibody on each detection line using the 5PL model used in immunoassays such as ELISA. The 5PL model is expressed by the following equation (9):

[0076]

number

[0077] In the above equation (9), a is the theoretical response at zero concentration, b is the slope factor, c is the intermediate concentration (inflection point), d is the theoretical response at infinite concentration, and g is the asymmetry factor.

[0078] <Parameter setting for mathematical models> The parameters set in the mathematical model include the amount of labeled antibody, the captured molecular weight on the first detection line, and the parameters on each detection line of the color intensity conversion equation 5PL model. The implementation procedure was as follows: first, the color intensity of each detection line was measured when the amount of reporter molecule precursor was changed under negative conditions, and then the mathematical model equation was fitted using the measured values ​​and the data on the amount of reporter molecule precursor, and each of the above parameters was set.

[0079] 8A shows the change in color intensity relative to the amount of reporter molecule precursor in the first detection line 23 according to the embodiment. As a result of fitting a mathematical model equation to the reporter molecule precursor amount data shown in FIG. 8A, the amount of labeled antibody: 2×10 -12 mol, captured molecular weight: 1×10 -12 mol, 5PL model parameter: a=0.1,b=5.8×10 -1 ,c=4.3×10 -13 The following parameters were set: , d = 74, and g = 5.4. As shown in Figure 8A, the mathematical model equation in which these parameters were set well describes the relationship between the amount of reporter molecule precursor and the color intensity of the first detection line 23. The dissociation constant was assumed to be sufficiently smaller than the amount of the introduced substance.

[0080] 8B shows the change in color intensity relative to the amount of reporter molecule precursor in the second detection line 24 according to the embodiment. A mathematical model equation was fitted to the reporter molecule precursor amount data shown in FIG. 8B, resulting in a 5PL model with parameters: a = 5.26.1, b = 100, c = 1.9 × 10. -11 The parameters were set as follows: , d = 70, g = 1.67. As shown in Figure 8B, the mathematical model formula with these parameters set clearly expresses the relationship between the amount of reporter molecule precursor and the color intensity of the second detection line 24. The amount of capture antibody was 2 × 10, which is equal to the amount of labeled antibody. -12 The result was mol.

[0081] <Verification> Using the mathematical model with parameters set as described above, the following verification was performed. Specifically, by designing with A = 2×10 -12 mol, A ≦ Rp < S, 1 < S / A, it was verified whether it is possible to suppress a decrease in specificity with respect to variations in the introduction amount Rp of the reporter molecule precursor compared to the conventional design.

[0082] (Verification 1) Based on the measured values and the mathematical model, a graph showing the amount of labeled antibody and the color development intensity of each line was created. From the graph, a threshold value for determining the color development state of each line and the amount of labeled antibody at that time were determined. Also, from the graph, the amount of labeled antibody required for the second detection line to show color development when positive was obtained. FIG. 9A is a graph showing the relationship between the amount of labeled antibody and the color development intensity according to the embodiment. In this Verification 1, as the threshold value, the color development intensity at which the detection line is determined to be colored was set to "40", and the color development intensity at which the detection line is determined not to be colored was set to "5.2". Also, when the color development intensity is 40, the amount of labeled antibody for the first detection line 23 was determined to be 1.7×10 -15 mol, and the amount of labeled antibody for the second detection line 24 was determined to be 2.0×10 -12 mol. When the color development intensity is 5.2, the amount of labeled antibody for the second detection line 24 was determined to be 1.75×10 -12 mol.

[0083] FIG. 9B is an enlarged view of a part of the graph shown in FIG. 9A. The labeled antibody shows color development on the order of 10 -12 mol, and it was found that sufficient color development can be seen if it is above 2×10 -12 mol. Note that the amount of labeled antibody and the capture molecular weight shown in this embodiment indicate the amounts calculated as parameters by fitting the above mathematical model and the measured values.

[0084] (Verification 2) Random numbers were generated for the amount Rp of reporter molecule precursor introduced with a certain degree of variation, and the specificity was calculated when S / A was changed using a mathematical model and the threshold determined in Verification 1 above. The change in specificity with respect to the variation in the amount Rp of reporter molecule precursor introduced was compared between the conventional design (S / A<1) and the design of this embodiment (S / A≧1). The procedure involved generating random numbers for the amount Rp of reporter molecule precursor introduced with a certain degree of variation (log-normal distribution). These became the random numbers for samples under negative conditions. Next, the random numbers were substituted into the mathematical model to calculate the amount of labeled antibody on each detection line. When the amount of labeled antibody on each detection line was compared with the threshold, the number of samples determined to be negative was counted and the specificity was calculated.

[0085] 10A is a graph showing the change in specificity when the ratio S / A between the amount of capture molecules S and the amount A of labeled antibodies on the first detection line according to the embodiment is changed. FIG. 10A shows the results of calculating specificity by simulation using a mathematical model when the amount Rp of reporter molecule precursor introduced is varied. Specificity indicates the proportion of negative cases that were determined to be negative. As shown in FIG. 10A, it was confirmed that specificity increases when the ratio S / A between the amount of capture molecules S and the amount A of labeled antibodies is increased.

[0086] 10B is a graph showing the change in specificity with respect to the variation (standard deviation) in the amount Rp of reporter molecule precursor introduced according to the embodiment. FIG. 10B shows the results of calculating specificity by simulation using a mathematical model when the amount Rp of reporter molecule precursor introduced is varied. As shown in FIG. 10B, increasing the ratio S / A of the amount S of capture molecules to the amount A of labeled antibodies resulted in a small decrease in specificity, confirming high robustness of the test.

[0087] (Verification 3) We compared the change in sensitivity with variations in the amount of reporter molecule precursor introduced, Rp. Sensitivity refers to the percentage of positive cases that could be determined as positive. To do this, we generated random numbers (log-normal distribution) for the amount of reporter molecule precursor introduced, Rp, when 99.999% of the reporter molecule precursors with a certain degree of variation were converted to reporter molecules (when the cleavage rate of the reporter molecule precursor was set to 99.999%). This became the random number for the sample under the positive condition. Next, we substituted the random numbers into a mathematical model to calculate the amount of labeled antibody on each detection line, and when the amount of labeled antibody on each detection line was compared with the threshold, we counted the number of cases that were determined to be positive and calculated the sensitivity.

[0088] 11 is a graph showing the change in sensitivity with respect to the variation (standard deviation) in the amount Rp of reporter molecule precursor introduced according to the embodiment. Fig. 11 shows the results of calculating the sensitivity by simulation using a mathematical model when the amount Rp of reporter molecule precursor introduced is varied. As shown in Fig. 11, it was confirmed that the variation in the amount Rp of reporter molecule precursor introduced does not affect the sensitivity, and that the sensitivity does not change even when the ratio S / A of the amount S of the capture molecule to the amount A of the labeled antibody is changed.

[0089] The test of this embodiment may be performed while improving test sensitivity by combining CRISPR-Cas LFA with a nucleic acid amplification reaction. After amplifying the target nucleic acid sequence in the sample by a nucleic acid amplification reaction, the amplified product is subjected to a CRISPR-Cas reaction to perform the LFA test. The nucleic acid amplification reaction may be, for example, an isothermal nucleic acid amplification method such as PCR, LAMP, or RPA. As long as the target nucleic acid sequence can be amplified according to the test environment, there are no limitations on the method, including PCR, which requires temperature control, and LAMP, RPA, and other methods that do not require temperature control.

[0090] According to the embodiment described above, by widening the tolerance range for the amount of reporter molecule precursor introduced, it is possible to perform an inspection that is robust against fluctuations in the amount of reporter molecule precursor introduced.

[0091] Although several embodiments have been described, these embodiments are presented as examples and are not intended to limit the scope of the invention. These embodiments can be implemented in various other forms, and various omissions, substitutions, and modifications can be made without departing from the spirit of the invention. These embodiments and their modifications are included within the scope and spirit of the invention, as well as within the scope of the invention and its equivalents as defined in the claims. [Explanation of symbols]

[0092] 1...Test kit, 10...First reagent, 12...Second reagent, 20...LFA test paper, 21...Sample pad, 22...Label part, 23...First detection line, 24...Second detection line, 25...Water absorption pad

Claims

1. A test kit for detecting a target nucleic acid sequence in a sample, comprising: The sample is added to a reaction solution containing a Cas enzyme and a reporter molecule precursor, and the reaction solution is subjected to a CRISPR-Cas reaction. The sample is then subjected to a lateral flow assay (LFA) test paper for testing the resulting reaction solution. the LFA test paper comprises a labeled portion containing a labeled antibody, a first detection line to which a capture molecule that captures the reporter molecule precursor is immobilized, and a second detection line to which a capture antibody that captures the labeled antibody is immobilized; The amount A of the labeled antibody and the amount S of the capture molecule on the LFA test paper are A≧10 －１２ mol and satisfy the condition of 1≦S / A, Test kit.

2. the amount Rp of the reporter molecule precursor introduced into the LFA test paper satisfies the condition A≦Rp<S; The test kit of claim 1.

3. the amount A of the labeled antibody and the amount S of the capturing molecule further satisfy the condition of 2≦S / A; The test kit of claim 1.

4. the amount A of the labeled antibody and the amount S of the capturing molecule further satisfy the condition of 5≦S / A; The test kit of claim 1.

5. the amount A of the labeled antibody and the amount S of the capturing molecule further satisfy the condition of 10≦S / A; The test kit of claim 1.

6. The amount A of the labeled antibody and the amount S of the capture molecule are further such that A≧2×10 －１２ mol and satisfy the condition of 1≦S / A, The test kit of claim 1.

7. The amount A of the labeled antibody and the amount S of the capture molecule are further such that A≧2×10 －１２ mol and satisfy the condition of 2≦S / A, The test kit of claim 1.

8. The amount A of the labeled antibody and the amount S of the capture molecule are further such that A≧2×10 －１２ mol and satisfies the condition of 5≦S / A, The test kit of claim 1.

9. The amount A of the labeled antibody and the amount S of the capture molecule are further such that A≧2×10 －１２ mol and satisfies the condition of 10≦S / A, The test kit of claim 1.

10. The LFA test paper is configured to test a reaction solution obtained by subjecting an amplification product generated by amplifying a target nucleic acid sequence in the sample by a nucleic acid amplification reaction to a CRISPR-Cas reaction. The test kit according to any one of claims 1 to 9.

11. A method for designing a test kit for detecting a target nucleic acid sequence in a sample, comprising: The test kit includes an LFA (lateral flow assay) test paper for testing a reaction solution obtained by adding the sample to a reaction solution containing a Cas enzyme and a reporter molecule precursor and subjecting the reaction solution to a CRISPR-Cas reaction, the LFA test paper comprises a labeled portion containing a labeled antibody, a first detection line to which a capture molecule that captures the reporter molecule precursor is immobilized, and a second detection line to which a capture antibody that captures the labeled antibody is immobilized; The amount A of the labeled antibody and the amount S of the capture molecule on the LFA test paper are A≧10 －１２ The amount A of the labeled antibody and the amount S of the capture molecule are set so as to satisfy the conditions of 1 mol and 1≦S / A. How test kits are designed.

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