JP2008000045A - Method for detecting nucleic acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for easily detecting a nucleic acid at a low cost to enable the detection of a nucleic acid specimen of extremely small amount/dilute concentration without using a particular device. <P>SOLUTION: A specimen solution containing a nucleic acid is supplied to a specimen holding section on a substrate and the reaction and detection of the nucleic acid are carried out in the section. Concentration comprising a set of multiple specimen supplying steps and solvent evaporation steps is carried out in each specimen holding section and the detection of the product is carried out after proper amplification of the nucleic acid specimen. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for detecting a nucleic acid. More specifically, the present invention provides a method for detecting nucleic acid in a sample with high sensitivity using a nucleic acid, particularly a sample having a very small amount of nucleic acid in the sample such as a forensic sample.

  In general, the amount of nucleic acid contained in forensic samples and plasma is extremely small. Therefore, even if a normal nucleic acid detection reaction is performed on these samples, it is difficult to perform effective detection, analysis, diagnosis, and the like.

  By the way, in recent years, it has been shown that a large amount of DNA derived from cancer cells is present in plasma obtained by removing blood cell components from blood and serum obtained by removing coagulation components. Possibility is suggested (nonpatent literature 1). However, the amount of cancer cell-derived DNA (total DNA amount) in plasma is 2.8 μg per ml of plasma (non-patent document 2) for ovarian cancer, 211 ng per ml of plasma (non-patent document 3) for breast cancer, small In the case of cell lung cancer, it is about 39 ng per ml of plasma (Non-patent Document 4). Therefore, the abundance of a specific sequence (such as a cancer-related gene) that is desired to be detected is even smaller, and is present only at a concentration that is less than the detection limit in a normal detection method. Therefore, plasma cannot be used for effective analysis / diagnosis at present.

  Although the plasma DNA concentration of healthy people varies depending on the literature, it is generally lower than that of cancer patients, and is about several pg per μl of plasma (ten to several tens of ng per 10 ml of blood collected) (non-patent literature). 1). Therefore, the average plasma DNA concentration of healthy individuals is several tens to several hundred times lower than the plasma DNA concentration in cancer patients.

  In general, the amount of blood collected from a patient in clinical practice is about several ml to 10 ml. In order to ensure the absolute amount of nucleic acid to be detected, it is impossible to collect more blood than this because it is a burden on the patient. Therefore, when cancer screening tests are performed on a population including healthy individuals, nucleic acids can be detected with high sensitivity even at normal blood collection and normal DNA concentrations present in the plasma of healthy individuals. It is necessary to develop a nucleic acid detection method that contributes to effective analysis and diagnosis.

In the case of detecting a trace amount of DNA, a method of first performing a gene amplification step by PCR or the like and then detecting an amplified product is generally employed. That is,
(1) extracting a target nucleic acid to be detected from a sample;
(2) a step of transferring the extracted nucleic acid to be detected to another test system and amplifying it; and (3) a step of transferring the amplified nucleic acid to a detection system different from the amplification system and detecting it. It is considered that detection of nucleic acid at a very dilute concentration can be performed by carrying out the nucleic acid detection method constituted.

  However, in the case of amplifying nucleic acid, an enzyme reaction is usually used. In the enzyme reaction, when the nucleic acid to be analyzed is not contained in a certain concentration or more, the reaction efficiency becomes extremely low. There is. That is, there is an inherent problem that when a very small amount of nucleic acid sample is amplified by an enzymatic reaction, it is difficult to obtain a stable and reproducible result. Specifically, when the amount of the target nucleic acid after the nucleic acid extraction operation from the sample becomes a very small amount of about several pg to several tens of pg, normal reaction conditions, for example, the reaction volume is in the range of 10 μl to 100 μl. If it is within the range, it is difficult to amplify the nucleic acid beyond the detection limit amount in the detection system using a reaction system using an enzyme such as PCR. Therefore, even if it is a nucleic acid detection system which has the structure of said (1)-(3), the situation where nucleic acid detection itself cannot be performed will arise.

  Theoretically, this problem can be solved by concentrating the nucleic acid sample solution by means of ethanol precipitation of the diluted nucleic acid sample solution, or centrifugation to evaporate the solvent while heating the nucleic acid sample solution. be able to. However, when ethanol precipitation is used, the precipitation operation itself is complicated in clinical practice, and moreover, nucleic acid may be lost during precipitation or during pipetting when transferring a concentrated nucleic acid sample to the amplification process. Is quite expensive. In addition, when centrifuging while warming, a special device (speedback or the like) having a centrifuge / heating function is required in the clinical field, which is not realistic. Regardless of which method is used, the concentration step usually requires about 30 minutes to several hours. Furthermore, the nucleic acid sample may be contaminated by other nucleic acids during the above-described complicated operation, and this problem cannot be ignored particularly in the clinical laboratory where many kinds of nucleic acid samples are often processed in parallel. .

  Until now, several methods for detecting a very small amount of nucleic acid have been disclosed. For example, in Patent Document 1, a target nucleic acid and a single-stranded DNA having a sequence complementary to the nucleic acid are subjected to a hybridization reaction, and the target nucleic acid is concentrated and then detected. A method for detecting a nucleic acid contained only in a trace amount and in a dilute state with high sensitivity is disclosed. However, in this method, since it is essential that the nucleic acid sequence to be detected is known, it cannot be used when an unknown sequence or a polymorphism exists in the sequence. Furthermore, since a single-stranded DNA reagent is required in the concentration step, it is necessary to prepare a relatively large amount of relatively expensive single-stranded DNA reagents in order to analyze many types of sequences simultaneously. This method is not a suitable method in clinical practice.

  Patent Document 2 discloses a genetic testing container capable of performing from amplification to detection of nucleic acid in the same container. However, in this container, the portion where amplification is performed and the portion where detection is performed are not necessarily the same portion. Therefore, when the analysis object is a very small amount of nucleic acid or the like, it is necessary to move the amplified nucleic acid sample to the component to be detected after amplification. Therefore, the subject that the loss of a sample (nucleic acid) arises still remains.

Non-Patent Document 5 discloses the research content of high-sensitivity detection and quantification technology for free DNA. In this evaluation report, analysis of a sample having a serum DNA concentration of about 10 pg / ml has been achieved. However, no investigation has been made on plasma that is considered to have a lower DNA concentration.
Japanese Patent No. 3023368 Japanese Patent Laid-Open No. 2003-38161 LJ Herrera et al. (Clin. Chem) 2005, 51, 1, pp. 113-118. Gifford G. et al., Clin. Cancer Res., 2004, 10, 13, pp. 4420-4426. JM Silva et al., Ann. Surg. Oncol., 2002, Vol. 9, No. 1, pp.71-76. XQ Chen et al., Nature Med., 1996, Volume 2, Issue 9, pp.1033-1035. Technical Evaluation Committee, New Energy and Industrial Technology Development Organization, “Post-Fundamental Research Report on Highly Sensitive Genetic Testing System for Cancer Using Free DNA in Blood”, February 2003

  The present invention has been made in view of such a problem, and an object of the present invention is to provide a nucleic acid detection method capable of stably and reproducibly detecting even a very small amount of nucleic acid sample at a dilute concentration. In particular, it is an object of the present invention to provide a method capable of detecting a nucleic acid quickly, simply and at low cost even in a clinical site where many samples and many types of samples are handled at the same time.

In order to solve the above problems, the present invention has the following configuration.
(1) (a) supplying a nucleic acid-containing sample solution for analyzing a target nucleic acid to a sample holding section on a substrate;
(B) performing a reaction on the target nucleic acid in the nucleic acid-containing sample solution in the same compartment as the sample holding compartment; and (c) in the same compartment as the sample holding compartment in the step (b) Detecting a nucleic acid-containing reaction product;
A nucleic acid detection method comprising:

(2) After the step (a) and before the step (b),
(A-1) a step of appropriately evaporating at least a part of the solvent of the nucleic acid-containing sample solution in the sample holding compartment; and (a-2) a step consisting of the steps (a) and (a-1) Is performed once or a plurality of times (however, the evaporation in the step (a-1) is performed immediately after the first step (a) (a-1) or step (a-2)). Conducted in (a-1) at least once in any of the steps);
The nucleic acid detection method according to (1), which comprises

(3) The step of evaporating the solvent in the step (a-1)
Including a step of allowing the substrate to stand for a preset time at the same temperature as the temperature at which the step (a) is performed, and optionally exposing the substrate to dry air; or at least 5 ° C. than the temperature at which the step (a) is performed A step of heating the substrate to a higher temperature and appropriately exposing the substrate to dry air, including an operation of allowing the substrate to stand for a preset time as appropriate;
The nucleic acid detection method according to (2), comprising:

(4) The nucleic acid detection method according to (3), wherein the temperature higher than that in the step (a) is a temperature within a range of 25 to 95 ° C.

(5) The step (a)
(A-3) supplying a nucleic acid-containing sample solution to a substrate that has been previously kept at a constant temperature in the range of 25 to 95 ° C .; and
(A-4) Step of allowing the substrate to stand for a preset time as appropriate and exposing the substrate to dry air as appropriate;
The nucleic acid detection method according to (1), comprising:

(6) The step of reacting with the target nucleic acid in the nucleic acid-containing sample solution in the step (b),
(B ′) a step of performing any one reaction selected from the group consisting of an amplification reaction, a hybridization reaction, a reverse transcription reaction, a ligation reaction, and a cleavage reaction with a restriction enzyme,
(B-1) a step of supplying a reaction solution containing components essential for the reaction at a concentration suitable for the selected reaction into the same compartment as the sample holding compartment when supplied to the sample holding compartment And (b-2) holding the substrate at a temperature suitable for the selected reaction for a preset time, or each of the substrates in a plurality of temperature zones suitable for the selected reaction. A step of performing a cycle consisting of sequentially holding for a set time once or a plurality of times;
The method for detecting a nucleic acid according to (1), wherein

(7) The nucleic acid detection method according to (6), wherein the selected reaction is an amplification reaction by PCR.

(8) detecting the nucleic acid-containing reaction product in the step (c),
(C ′) a step of supplying a solution containing a nucleic acid detection reagent into the same compartment as the sample holding compartment and detecting a nucleic acid detection reaction product produced thereby, and the solution containing the nucleic acid detection reagent is The nucleic acid detection method according to (1), which contains the detection reagent at a concentration suitable for the detection reaction when it is supplied into the sample holding compartment.

(9) After the step (b) and before the step (c ′),
(B-3) evaporating at least a portion of the solvent of the solution in the sample holding compartment;
The nucleic acid detection method according to (8), comprising:

(10) detecting the nucleic acid detection reaction product in the step (c ′),
(C '') The nucleic acid detection method according to (8), which is a step of measuring spectroscopic characteristics of the nucleic acid detection reaction product.

(11) The nucleic acid detection method according to (10), wherein the spectroscopic characteristics relate to fluorescence intensity, emission intensity, color development, or absorbance.

(12) detecting the nucleic acid detection reaction product in the step (c ′),
(C ′ ″) The nucleic acid detection method according to (8), which is a step of measuring the amount of nucleic acid in the reaction product.

(13) The nucleic acid detection method according to (1), wherein the step (c) is to detect a label introduced into the reaction product of the target nucleic acid in the reaction of the step (b).

(14) supplying the nucleic acid-containing sample solution in the step (a),
The nucleic acid detection method according to (1), wherein the solution is dripped, a syringe piston pump method, or an ink jet method.

(15) The nucleic acid detection method according to (1), wherein the amount of the nucleic acid-containing sample solution supplied in the step (a) is a fixed amount within a range of 0.1 to 5 μl.

(16) The step of supplying the nucleic acid-containing sample solution in the step (a) is a step of supplying the predetermined amount of solution to the substrate a plurality of times before performing the step (b). Nucleic acid detection method.

(17) The nucleic acid-containing sample solution is a solution containing cells,
After the step (a) and before the step (b),
(A-5) adding a cell rupture solution to the sample holding section; and (a-6) holding the substrate at one temperature or a plurality of temperature zones for a preset time;
The nucleic acid detection method according to (1), comprising:

(18) The nucleic acid detection method according to (1), wherein the substrate has a plurality of sample holding sections.
(19) The nucleic acid detection method according to (18), wherein in the step (a), the same nucleic acid-containing sample solution is supplied to two or more sample holding sections.

(20) After the step (a) and before the step (b),
(A-1) a step of appropriately evaporating at least a part of the solvent of the nucleic acid-containing sample solution in the sample holding compartment; and (a-2) a step consisting of the steps (a) and (a-1) Is performed once or a plurality of times (however, the evaporation in the step (a-1) is performed immediately after the first step (a) (a-1) or step (a-2)). Conducted in (a-1) at least once in any of the steps);
The nucleic acid detection method according to (18), comprising:

(21) The step (a-2) is performed on at least one of the plurality of sample holding sections at a number different from the number of steps (a-2) performed in the other sample holding sections. ) Nucleic acid detection method according to the above.

(22) In all the steps (a-1), the evaporation operation is always performed, and the number of steps (a-1) to be performed for each of the plurality of sample holding sections is a set of natural numbers at regular intervals. (21) The nucleic acid detection method according to (21).

(23) (A) A nucleic acid-containing sample solution for analyzing the presence or absence of a target nucleic acid is supplied to at least two sample holding compartments on a substrate, and a standard nucleic acid-containing solution containing a standard nucleic acid in a known amount Supplying to at least two separate compartments on the same substrate having a sample holding compartment for supplying a nucleic acid-containing sample solution;
(B) The following sub-steps:
(B-1) evaporating at least part of the solvent of the nucleic acid-containing sample solution and at least part of the solvent of the standard nucleic acid-containing solution in the compartment; and (B-2) the step (A ) And (B-1) are further performed once or a plurality of times;
Comprising the step of concentrating nucleic acids in the solution,
The number of steps (B-1) performed on at least one of the series of compartments supplied with the nucleic acid-containing sample solution or standard nucleic acid-containing solution is a set of natural numbers at regular intervals;
(C) supplying a solution containing a nucleic acid detection reagent into each compartment after the concentration step (B), and detecting a nucleic acid detection reaction product produced thereby;
(D) The following sub-steps:
(D-1) A calibration curve between the amount of all standard nucleic acids supplied in the same compartment and the quantification result of the nucleic acid detection reaction product based on the quantification result in step (C) performed on the standard nucleic acid-containing solution. (D-2) calculating the amount of the target nucleic acid in the nucleic acid-containing sample solution based on the calibration curve created in the sub-step (D-1);
Performing a target nucleic acid quantification comprising:
A nucleic acid detection method comprising:

(24) After the step (B) and before the step (C),
(B-3) performing an amplification reaction using each of the target nucleic acid in the nucleic acid-containing sample solution and the standard nucleic acid in the standard nucleic acid-containing solution as a template in each of the compartments;
The nucleic acid detection method according to (23), comprising:

(25) The nucleic acid detection method according to any one of (1) to (24), wherein the substrate surface includes a hydrophobic region and a hydrophilic region, and the hydrophilic region is used as a sample holding section.

(26) The nucleic acid detection method according to (25), wherein the sample holding section is a depression having a substantially circular opening, and the diameter of the bottom of the depression is 0.5 mm to 6 mm.

(27) The nucleic acid detection method according to any one of (1) to (24), wherein the nucleic acid-containing sample solution is plasma.

(28) The nucleic acid concentration in the nucleic acid-containing sample solution is in the range of 5 pg / μl to 1 μg / μl, and the total amount of nucleic acids in the sample solution is in the range of 0.05 ng to 1 μg. ) To (24) The nucleic acid detection method according to any one of (24).

  By using the nucleic acid detection method of the present invention, it is possible to detect a very small amount of a nucleic acid sample that could not be detected by a normal method easily and at low cost without the need for a special apparatus. Can be used effectively for disease determination.

  In the present invention, it is possible to efficiently and accurately concentrate a nucleic acid sample. Therefore, by creating a series of nucleic acid concentrations of a nucleic acid sample by the method of the present invention, it is possible to effectively perform reaction and detection even for a sample whose concentration is unknown. Therefore, it is possible to omit a preliminary experiment for detecting the sample nucleic acid. For these reasons, the present invention is an extremely easy-to-use method that can be implemented without requiring complicated operations in clinical settings.

The nucleic acid detection method of the present invention comprises:
(A) supplying a nucleic acid-containing sample solution for analyzing a target nucleic acid to a sample holding section on a substrate;
(B) performing a reaction on the target nucleic acid in the nucleic acid-containing sample solution in the same compartment as the sample holding compartment; and (c) in the same compartment as the sample holding compartment in the step (b) Detecting a nucleic acid-containing reaction product;
Is included.

  That is, in the method of the present invention, the sample solution containing the target nucleic acid is supplied into the sample holding section formed on the substrate, and the reaction to the target nucleic acid is performed on the spot, and further in the place where the reaction is performed. Detection of the reaction product is performed.

  The “nucleic acid-containing sample” that can be used in the present invention is a biological sample including tissues, cells, bacteria, viruses, blood, plasma, serum, body fluid, urine, and the like. In the case where the biological sample is a tissue, cell, bacteria, virus-infected cell, blood, etc. and the target nucleic acid is not present in a released state, in the technical field to which the present invention belongs and its peripheral and related fields It is necessary to extract nucleic acids from tissues and cells by known methods. The operation for that can be performed before the implementation of the present invention, or can be performed by a known appropriate method immediately before the nucleic acid reaction. On the other hand, in the case of free nucleic acid in the target nucleic acid, for example, plasma, serum, body fluid, etc., the sample itself can be essentially subjected to the nucleic acid detection method of the present invention. It is. However, when it is known in advance that a substance that may inhibit the reaction to the nucleic acid, etc., performed in the method of the present invention is contained in the sample, such an inhibitory substance is included in the nucleic acid-containing sample. It is preferable to carry out a preliminary treatment such as removal of.

  The “target nucleic acid” in the present invention is a nucleic acid containing a gene sequence whose presence or mutation is desired to be detected / quantified due to a request in research, diagnosis, etc., and includes DNA and RNA.

  The “substrate” that can be used in the nucleic acid detection method of the present invention is a material, shape, suitable for holding a biological sample on the substrate, reacting the biological sample, and detecting the reaction product. If it is a thing of a dimension, it will not specifically limit. Specifically, a sample holding section suitable for holding, reaction, and detection of a nucleic acid-containing sample solution containing a target nucleic acid may be formed on a substrate such as glass.

  In the nucleic acid detection method of the present invention, after the nucleic acid-containing sample solution as described above is supplied to the sample holding compartment, the nucleic acid is reacted in the same compartment. In the next step (c), detection of a nucleic acid-containing reaction product that is predicted to be generated by the reaction is performed.

  Examples of the “nucleic acid-containing reaction product” in the present invention include reaction products in PCR, T7, NSABA and other amplification reactions, hybridization reactions, reverse transcription reactions, ligation reactions, and restriction enzyme reaction cleavage reactions. Can do.

  Examples of the detection step in the above step (c) of the nucleic acid detection method of the present invention include known methods in the technical field to which the present invention belongs and the surrounding and related fields.

  As described above, the present invention is characterized in that the sample supply step, the nucleic acid reaction step, and the nucleic acid-containing reaction product detection step are all performed in the same sample holding section on the substrate.

  In a normal method for detecting a small amount of nucleic acid, first, a reaction for amplifying a nucleic acid below the detection limit is performed in a container such as a tube, and then the amplified product is transferred to a system such as electrophoresis and then the detection is performed. To be done. On the other hand, in the method of the present invention, as described above, the reaction and detection can be performed in the same compartment. As a result, nucleic acid can be detected simply and effectively. In addition, in the conventional method, if an operation (such as changing the container) of moving a nucleic acid sample with a very small amount or dilute concentration is performed, the sample itself may be lost in the operation, or the sample may be contaminated due to an operation error. In the case of the present invention, such a situation can be prevented.

  Further, in the present invention, since reaction and detection can be performed in the same section, it is essentially possible to reduce the amount of reaction / detection instruments and consumables to be used. There are advantages.

  In the nucleic acid detection method of the present invention, since the sample reaction / detection is performed in the same compartment, it is a simpler system, and when configuring an apparatus for carrying out the nucleic acid detection method of the present invention, It is easy to automate.

In the nucleic acid detection method of the present invention, after the step (a) of supplying a sample and before the step (b) of performing a nucleic acid reaction,
(A-1) a step of appropriately evaporating at least a part of the solvent of the nucleic acid-containing sample solution in the sample holding compartment; and (a-2) a step consisting of the steps (a) and (a-1) Is further performed once or a plurality of times (however, the evaporation in the step (a-1) is performed immediately after the step (a) (a-1) or in the step (a-2) ( a-1) performed at least once in any of the steps);
A concentration step consisting of:

  In the step (a-1), at least a part of the solvent is appropriately evaporated from the nucleic acid-containing sample solution supplied in the immediately preceding step (a). However, when it is considered that the volume of the sample solution supplied in the step (a) is sufficiently smaller than the volume of the sample solution that can be held in each sample holding section of the substrate being used, for example, once in the section In the case where the volume of the sample solution supplied per unit is 80% or less with respect to the total volume of the compartment, the evaporation operation does not need to be performed every time the sample is supplied. Similarly, when it is desired to reduce the time and the number of operations required for carrying out the method of the present invention by reducing the number of evaporation operations in the step (a-1), the evaporation operations need to be performed every time the sample is supplied. Absent. However, it is necessary to perform the evaporation operation at least once during the execution of one set of all the steps of the method of the present invention so that the concentration of the nucleic acid-containing sample supplied into the sample holding compartment is performed. Therefore, the evaporation in the step (a-1) is performed either immediately after the first step (a) (a-1) or in the step (a-2) (a-1). At least once. In this case, from the viewpoint of maintaining the efficiency of the reaction in the next step (b), the step (a-1) performed immediately before the step (b), that is, the step (a-1) performed at the end of all the steps. It is preferable to perform the evaporation operation. Thus, when the evaporation operation is performed in the last step (a-1) of all the steps, the amount of solution present in the sample holding section with respect to the amount of solution supplied in the next reaction step (b) Therefore, it is possible to ensure efficient reaction progress in the step (b).

In the embodiment of the present invention in which the above sample solution is concentrated,
Evaporating the solvent of the step (a-1),
Including a step of allowing the substrate to stand for a preset time at the same temperature as the temperature at which the step (a) is performed, and optionally exposing the substrate to dry air; or at least 5 ° C. than the temperature at which the step (a) is performed A step of heating the substrate to a higher temperature and appropriately exposing the substrate to dry air, including an operation of allowing the substrate to stand for a preset time as appropriate;
Can be included. That is, one specific means for concentration is to leave the substrate supplied with the sample solution in the same environment without substantially changing the temperature in the system when the nucleic acid-containing sample solution is supplied. Then, as appropriate, dry air may be supplied to the substrate. This means can be employed when the humidity in the environment where the detection is performed is sufficiently low and the solvent tends to spontaneously evaporate. The additional operation of exposing to dry air is not particularly limited as long as the solution does not cause the solution in each sample holding compartment to move to another compartment or the solution splashes and scatters by this operation. The dehumidifying means can supply air having a lower humidity than the surroundings by blowing air.

  Another specific means for concentration is to bring the substrate up to a temperature that is at least 5 ° C higher than the temperature at which the sample solution was supplied to the sample holding compartment on the substrate, preferably to a temperature in the range of 25-95 ° C. Is heated after the sample is supplied, the substrate is allowed to stand for a preset time as appropriate, and the substrate is exposed to dry air as appropriate. Either the operation of leaving the substrate stationary for a certain period of time or the operation of exposing the substrate to dry air can be performed, or both can be performed together.

  The means for heating the substrate can be a means for directly heating the substrate or indirectly by heating air in an environment where the substrate is disposed. Further, when the difference between the temperature of the substrate that is finally reached by heating the substrate and the temperature at the time of supplying the sample in the step (a) is not sufficiently large from the viewpoint of the solvent evaporation rate, When the final temperature of the substrate is less than 50 ° C., it is preferable to leave the substrate in that state for a preset time after the heating operation. On the other hand, when the difference between the temperature of the substrate that is finally reached by heating the substrate and the temperature at the time of sample supply in step (a) is considered sufficiently large from the viewpoint of the solvent evaporation rate, specifically, When the final temperature of the substrate is 50 ° C or higher, it is not necessary to perform such a standing operation. Rather, the next operation (operation involving supply of the solution) is performed before the solution is completely dried. ) Is preferable. This is because, if the final temperature at which the substrate is reached by heating is too high, the nucleic acid-containing sample solution is completely dried in the sample holding section, and the sample is additionally supplied, and the steps (b) and (c) This is because even if the solution is supplied for the reaction in (2), the target nucleic acid may remain attached to the bottom of the sample holding section of the substrate and may not be involved in the reaction. In order to prevent such drying, it is desirable to provide a temperature control function that can adjust the temperature rise of the substrate and the temperature holding time at the final temperature, and preferably control the cooling of the substrate. However, since such a temperature control function causes complication of the method / system, the final temperature to be raised is preferably in the range of 37 to 70 ° C.

  There are two ways to heat the substrate, either directly heating the substrate or indirectly heating the substrate by heating the surrounding air, but from the viewpoint of easy means in the release system. Means for directly heating the substrate are preferred. More preferably, the device is a closed system.

In the nucleic acid detection method of the present invention, instead of concentrating the sample solution after supplying the sample solution to the substrate, the sample solution can be supplied and concentrated almost simultaneously. That is, in the nucleic acid detection method of the present invention including the above (a) to (c),
The step (a)
(A-3) supplying a nucleic acid-containing sample solution to a substrate that has been previously kept at a constant temperature in the range of 25 to 95 ° C .; and
(A-4) Step of allowing the substrate to stand for a preset time as appropriate and exposing the substrate to dry air as appropriate;
Can be included.

  Examples of means for keeping the substrate at a constant temperature include means for heating the substrate. Further, either one of the operation of allowing the substrate to stand for a certain period of time and the operation of exposing the substrate to dry air may be performed, or both may be performed together.

  In the embodiment using the means for heating the substrate for concentration of the sample solution as described above, when the concentration is sufficiently achieved, the next reaction step is performed without completely evaporating the solvent from the sample solution. In order to immediately perform the reaction in the liquid phase in (b), the substrate is heated in the vicinity of the temperature in step (b), or the temperature of the substrate is set immediately before step (b) in the reaction step (b). It is possible to provide a means for cooling to a suitable temperature. Such cooling means is when the substrate heating temperature is significantly higher than the sample solution supply temperature, and the natural solution may evaporate all the solution in the compartment after concentration. Is particularly effective.

In the nucleic acid detection method of the present invention, the step of reacting the target nucleic acid in the nucleic acid-containing sample solution in the step (b) includes (b ′) amplification reaction, hybridization reaction, reverse transcription reaction, ligation reaction, and restriction. The step may be any one reaction selected from the group consisting of enzymatic cleavage reactions, more preferably a PCR amplification reaction. And this step (b ')
(B-1) a step of supplying a reaction solution containing components essential for the reaction at a concentration suitable for the selected reaction into the same compartment as the sample holding compartment when supplied to the sample holding compartment And (b-2) holding the substrate at a temperature suitable for the selected reaction for a preset time, or each of the substrates in a plurality of temperature zones suitable for the selected reaction. A step of performing a cycle consisting of sequentially holding for a set time once or a plurality of times;
It is a process including.

  In the above step (b-1), a reaction solution suitable for any one reaction selected from the group consisting of an amplification reaction, a hybridization reaction, a reverse transcription reaction, a ligation reaction, and a cleavage reaction with a restriction enzyme (substrate or (Including enzyme etc.) is supplied to each compartment. Here, when the amount of the sample solution existing in each compartment before supply of the reaction solution is not negligible with respect to the supply amount of the reaction solution, the concentration of components in the reaction solution is increased by supplying the reaction solution into the compartment. Will change drastically. Therefore, when a reaction is considered that such concentration fluctuations may have a significant effect on the progress of the reaction, the amount of the reaction solution to be supplied should be taken into consideration before the reaction solution is supplied. It is preferable to adjust the component concentration.

  However, in the above-described aspect of the present invention in which concentration is performed, it is essentially possible to adjust the amount of solution in each compartment to a certain amount or less at the final stage of concentration. The adjustment of the component concentration of the reaction solution as described above is essentially unnecessary.

  In the next step (b-2), the substrate is set to a temperature suitable for the selected reaction, and the means for this can be the heating means used in the concentration step. If the selected reaction is an amplification reaction by PCR, the temperature of the substrate is sequentially changed to a temperature set suitable for thermal denaturation of template, annealing of single-stranded template and primer, and DNA extension reaction. The reaction can be allowed to proceed by holding at each temperature for a certain period of time.

  When the amplification reaction in this step (b ′) is PCR, it can be end point PCR or real time PCR. When using real-time PCR for the amplification reaction, PCR can be stopped when a signal is detected at a preset level during the PCR cycle, thus reducing the detection time. Is possible. In the method of the present invention, one of the features is that the reaction and the detection are performed in the same compartment. Therefore, when the real-time PCR is performed, the time for detection is significantly reduced.

In the nucleic acid detection method of the present invention including the steps (a) to (c), the step of detecting the nucleic acid-containing reaction product in the step (c) includes:
(C ′) a step of supplying a solution containing a nucleic acid detection reagent into the same compartment as the sample holding compartment and detecting a nucleic acid detection reaction product produced thereby, and the solution containing the nucleic acid detection reagent is When this is supplied into the sample holding compartment, the detection reagent can be contained at a concentration suitable for the detection reaction.

  The “nucleic acid detection reagent” in the method of the present invention is not particularly limited as long as it is used for high-sensitivity detection of nucleic acids known in the technical field to which the present invention belongs and its peripheral and related fields.

  The concentration of the nucleic acid detection reagent in the solution containing the nucleic acid detection reagent is not particularly limited as long as the reagent can effectively detect the nucleic acid. In addition, the volume of the sample solution in the sample holding section for supplying this is preferably sufficiently small relative to the volume of the solution containing the nucleic acid detection reagent, and more specifically, the volume of the solution containing the nucleic acid detection reagent. Is preferably 50% or less.

In order to satisfy such a condition, in the above-described method of the present invention, after the step (b) and before the step (c ′),
(B-3) It is preferable to include a step of evaporating at least a part of the solvent of the solution in the sample holding section. By providing this evaporation step, the nucleic acid detection reaction can be sufficiently and effectively advanced in step (c ′).

The step of detecting a nucleic acid detection reaction product in step (c ′) of the nucleic acid detection method of the present invention comprises:
(C ″) A step of measuring the spectroscopic characteristics of the nucleic acid detection reaction product. More specifically, the spectroscopic characteristics may relate to fluorescence intensity, emission intensity, color development, or absorbance. Therefore, in the nucleic acid detection method of the present invention, it is necessary to have a means capable of quantifying these spectroscopic characteristics, as long as it is known in the technical field to which the present invention belongs and its peripheral and related fields. There is no particular limitation. More specifically, it can be carried out by measuring the amount of nucleic acid, for example, using an intercalator that recognizes and binds quantitatively to the double strand of nucleic acid, such as PicoGreen (Molecular Probes, registered trademark). can do. Alternatively, instead of using such a reagent, labeling (for example, labeling with a fluorescent molecule) is performed in step (b), and then in step (c), FCS (fluorescence correlation analysis) or FIDA (fluorescence intensity distribution analysis) is performed. And the reaction product produced in step (b) is measured. As such a label, various fluorescent labels such as those known in the technical field to which the present invention belongs and its peripheral and related fields can be used.

  The step of supplying the nucleic acid-containing sample solution in the step (a) of the nucleic acid detection method of the present invention can be carried out, for example, by dropping the solution or performing a supply operation by an ink jet method. Other than these methods, the supply amount is accurate, the sample is not contaminated between the sample holding sections on the substrate, and the nucleic acid-containing sample solution can be brought into contact with the sample holding section ( For example, ordinary syringe piston pumps) can be employed.

  In supplying the nucleic acid-containing sample solution into the sample holding compartment, it is desirable that the amount of the solution is a certain amount within the range of 0.1 to 5 μl. If it is a certain amount within this range, it is possible to configure a system that simultaneously detects a large number of samples at once, and it is particularly easy to construct a system that uses a substrate as large as a slide glass. . Therefore, it becomes easy to introduce in clinical settings.

  In the system for supplying a certain amount of solution within the above range, the step of supplying the nucleic acid-containing sample solution in the step (a) may be performed by adding the fixed amount of solution before performing the step (b). It can be a step of supplying the substrate a plurality of times. Therefore, even if the size of the substrate is about the same as that of a slide glass, it is possible to supply the solution multiple times in the same sample holding compartment, and without overflowing the solution from the compartment. It is easy to increase the absolute amount of the target nucleic acid.

In the nucleic acid detection method of the present invention, the nucleic acid-containing sample solution can be a cell-containing solution. In this case, after the step (a) and before the step (b),
(A-5) adding a cell rupture solution to the sample holding section; and (a-6) holding the substrate at one temperature or a plurality of temperature zones for a preset time;
It can implement by including.

  The “cell” in the cell-containing solution used in this embodiment is preferably in a state where the cells are present in the liquid independently of each other. More preferably, the number of cells per unit volume is determined.

  In the step (a-5), the cells in the sample holding compartment are ruptured by the added cell rupture solution, and the nucleic acids in the cells are exposed. By subjecting this to the next step (a-6), a nucleic acid reaction is carried out.

  If the reaction in step (a-6) is an amplification reaction by PCR, after step (a-5), the nucleic acid is denatured, and then the PCR reagent is added to the sample holding compartment at an appropriate concentration to carry out the amplification reaction. be able to. In order to add the PCR reagent at an appropriate concentration, it is desirable to evaporate a part or all of the cell rupture solution.

  The “cell rupture solution” used in step (a-5) is a liquid that ruptures cells and releases nucleic acids from the cells into the liquid, and preferably does not inhibit the reaction in the subsequent step (b). Can be. Specific examples include hypotonic solutions and water (deionized water and distilled water) containing salts such as KCl. When, for example, water is added as a cell rupture solution into the sample holding compartment, the cells are ruptured due to a difference in osmotic pressure inside and outside the cell.

  The substrate used in the nucleic acid detection method of the present invention can be provided with a plurality of sample holding sections. By providing a plurality of sample holding sections on the same substrate, simultaneous detection of a plurality of types of samples can be performed. Also, with such a configuration, it is possible to perform reaction and detection after providing a concentration gradient for the same type of sample on the same substrate. By providing a concentration gradient in this way, even if the target nucleic acid concentration in the sample solution to be analyzed is not known in advance or is not accurately known in advance, the concentration gradient can be set wider. In this gradient, the target nucleic acid can be contained at a concentration suitable for the reaction and detection of the nucleic acid. As a result, it is possible to eliminate the burden of performing preliminary experiments or calculations for estimating the amount and concentration of the target nucleic acid accurately in advance before carrying out the method of the present invention.

Specifically, the concentration gradient of the target nucleic acid in the sample solution can be prepared by following the concentration step in the general method of the present invention described above. That is, after the step (a) and before the step (b),
(A-1) a step of appropriately evaporating at least a part of the solvent of the nucleic acid-containing sample solution in the sample holding compartment; and (a-2) a step consisting of the steps (a) and (a-1) Is performed once or a plurality of times (however, the evaporation in the step (a-1) is performed immediately after the first step (a) (a-1) or step (a-2)). Conducted in (a-1) at least once in any of the steps);
Can be carried out essentially by a method comprising Specifically, the step (a-2) is performed on at least one of the plurality of sample holding sections at a number different from the number of steps (a-2) performed in other sample holding sections. To create a concentration gradient. More preferably, the concentration gradient is created by performing an evaporation operation in all of the steps (a-1), and the number of steps (a-1) to be performed for each of the plurality of sample holding sections is constant. Is a set of natural numbers. Here, “a set of natural numbers at a constant interval” is, for example, {1, 2, 3,...}, {2, 4, 6, ...}, and {10, 100, 1000. Etc. Which set is used can be determined by those skilled in the art based on the predicted value of the target nucleic acid amount.

  In the above embodiment, the gradient of the amount of target nucleic acid supplied to each sample holding section was created by changing the number of times of performing step (a-2), but instead of such a method, Using the blank solution (buffer solution or pure water) used to prepare the sample solution containing nucleic acid, adjusting the number of times the blank solution is supplied without changing the number of times of supply in all compartments, It is also possible to create a gradient for the amount of standard nucleic acid supplied to the compartment.

The nucleic acid detection method of the present invention can be applied not only to detection of a target nucleic acid but also to quantification of the target nucleic acid. That is,
(A) A nucleic acid-containing sample solution for analyzing the presence or absence of a target nucleic acid is supplied to at least two sample holding compartments on a substrate, and a standard nucleic acid-containing solution containing a standard nucleic acid having a known sequence in a known amount, Supplying the nucleic acid-containing sample solution to at least two separate compartments on the same substrate having the sample holding compartment;
(B) The following sub-steps:
(B-1) evaporating at least part of the solvent of the nucleic acid-containing sample solution and at least part of the solvent of the standard nucleic acid-containing solution in the compartment; and (B-2) the step (A ) And (B-1) are further performed once or a plurality of times;
Comprising the step of concentrating nucleic acids in the solution,
The number of steps (B-1) performed on at least one of the series of compartments supplied with the nucleic acid-containing sample solution or standard nucleic acid-containing solution is a set of natural numbers at regular intervals;
(C) A step of supplying a solution containing a nucleic acid detection reagent into each of the compartments after the concentration step (B), and detecting a nucleic acid detection reaction product produced thereby or quantifying the nucleic acid detection reaction product And as appropriate
(D) The following sub-steps:
(D-1) A calibration curve between the amount of all standard nucleic acids supplied in the same compartment and the quantification result of the nucleic acid detection reaction product based on the quantification result in step (C) performed on the standard nucleic acid-containing solution. (D-2) calculating the amount of the target nucleic acid in the nucleic acid-containing sample solution based on the calibration curve created in the sub-step (D-1);
Performing a target nucleic acid quantification comprising:
With the configuration including the target nucleic acid, the target nucleic acid can be quantified.

  In the above step (A), not only a nucleic acid-containing sample solution but also a standard nucleic acid-containing solution containing a standard sequence in a known amount is prepared, and both the nucleic acid-containing sample solution and the standard nucleic acid-containing solution are placed in at least two compartments. Supply. It is preferable that the nucleic acid-containing sample solution and the standard nucleic acid-containing solution supplied to at least two compartments have different concentrations in each compartment, and more preferably a set of concentrations at a constant magnification.

  In order to create such a concentration gradient, specifically, the solution is concentrated by the step (B) including the sub-steps (B-1) and (B-2). That is, the concentration gradient can be created by changing the number of times the sub-step (B-2) is performed in each section. By setting the number of times as a natural number at a fixed interval, a concentration gradient such as {1, 2, 3,...} Or {2, 4, 6,.

  In addition, as described above, by varying the number of times of performing the sub-step (B-2), it is possible to create a gradient regarding the amount of target nucleic acid supplied to each sample holding section. In addition, the blank solution used to prepare the nucleic acid-containing sample solution is used, and in all the compartments, the standard nucleic acid solution or the nucleic acid-containing sample solution and the total number of blank solutions supplied are the same. It is also possible to create a gradient of the standard nucleic acid or nucleic acid-containing sample solution supplied to

  Next, the above step (C) is performed in order to detect the target nucleic acid in the nucleic acid-containing sample solution having a concentration gradient prepared as described above. In this step, a solution containing a nucleic acid detection reagent that can be used in the other embodiments of the present invention described above is supplied into the sample holding compartment, and the nucleic acid detection reaction product produced thereby is detected. This detection step makes it possible to determine whether the target nucleic acid is present in each section.

  The above step (D) is carried out in order to perform not only the detection of the nucleic acid detection reaction product but also its quantification. That is, based on the detection result of the standard nucleic acid-containing solution having a concentration gradient, based on the relationship between the amount of target nucleic acid supplied to each compartment and the measured value (for example, fluorescence intensity) in the detection of the step (C). First create a calibration curve. Next, based on this calibration curve and the measured value of the target nucleic acid in the sample nucleic acid-containing solution in the step (C), the amount of the target nucleic acid in the nucleic acid-containing sample solution in each sample holding section is calculated. Preferably, the standard nucleic acid used in the preparation of the calibration curve is used at a concentration where linearity exists between the abundance and the measured value in detection, and the amount of target nucleic acid in the concentration range where linearity is maintained. Perform the calculation.

In the above-described embodiment of the present invention, it is possible to amplify the target nucleic acid before detecting the target nucleic acid. That is, after the step (B) and before the step (C),
(B-3) performing an amplification reaction using each of the target nucleic acid in the nucleic acid-containing sample solution and the standard nucleic acid in the standard nucleic acid-containing solution as a template in each of the compartments;
Can be included. In this embodiment, it becomes possible to detect a target nucleic acid from a very small amount of a nucleic acid-containing sample solution that cannot be detected by concentration gradient creation of the nucleic acid-containing sample solution, that is, concentration. Specifically, by performing an amplification reaction using a standard nucleic acid as a template, preferably an amplification reaction by PCR, the target nucleic acid is amplified to a concentration far exceeding the target nucleic acid concentration achieved by the concentration of the above embodiment, and this is further performed. It becomes possible to detect.

  The surface of the substrate used in the present invention is preferably composed of a hydrophobic region and a hydrophilic region, and the hydrophilic region serves as a sample holding section. Since the nucleic acid-containing sample to be detected in the present invention is usually prepared and stored in an aqueous medium, the sample holding section formed on the substrate effectively holds the sample solution, and further the section. It is supplied to the hydrophilic region so that various reactions and detections (reactions) can be performed. In addition, the configuration in which the periphery of each hydrophilic region is enclosed by a hydrophobic region makes it easier to hold the sample solution supplied to the hydrophilic region in the region, and unintentional inter-compartment movement of the sample solution Can be suppressed.

For the formation of the hydrophilic region, the substrate surface is subjected to a hydrophilic treatment by various methods known in the technical field to which the present invention belongs and its peripheral and related fields, or the hydrophobic region is publicly known on the substrate surface that is originally hydrophilic. It is performed by forming by the method. Examples of groups introduced to the substrate surface by hydrophilic treatment include groups with high polarity such as —OH, —NH ₃ , —COOH, etc., while examples of groups introduced to the substrate surface by hydrophobic treatment include , -R, -OR (R is a hydrophobic group such as alkyl or alkenyl), -F or a group having low polarity such as an alkyl substituted with halogen or an alkoxy group.

  The shape / dimension of the sample holding section formed on the substrate is not particularly limited as long as it is suitable for holding / reaction / detection of an aqueous sample, but the nucleic acid detection method of the present invention is a very small amount of nucleic acid. Since it is suitably used for detection of a sample, for example, it is a depression having a substantially circular opening, and the diameter of the bottom of the depression can be 0.5 mm to 6 mm. As an example of the substrate having the sample holding section in such a form, the AmpliGrid (registered trademark) series manufactured by Advalytix AG can be cited.

  Since the nucleic acid detection method of the present invention is suitably used for detecting a very small amount of target nucleic acid, for example, plasma can be used as a nucleic acid-containing sample solution. The nucleic acid concentration detectable in the nucleic acid detection method of the present invention is in the range of 5 pg / μl to 1 μg / μl, and the total amount of nucleic acid in the sample solution is in the range of 0.05 ng to 1 μg. If there is, it can be detected.

  A substrate (Advalytics AmpliGrid (registered trademark)) having a total of 48 sample holding sections in an array of 12 rows × 4 columns and a Hamilton Micro Lab Star dispensing device manufactured by Hamilton were used. A concentration gradient consisting of four concentrations was prepared for the samples (nucleic acid 1 to nucleic acid 12). This substrate has minute hydrophilic regions with a diameter of approximately 1.6 mm formed at equal intervals on the surface, and a non-hydrophilic region is formed in a donut shape around each region.

  Dispensing was set at 1.0 μl per time. First, about 12 types of samples, 1.0 μl was dispensed into each of columns A to D of rows 1 to 12. Thereafter, the substrate was heated to 50 ° C. When about 2 minutes passed at this temperature, the sample solution supplied into each sample holding section was almost completely evaporated. After confirming the evaporation, the temperature of the substrate was lowered to room temperature, and then, for each of the 12 types of samples, 1.0 μl was dispensed into each of row B to column D in columns B to D. In the same manner as the first dispensing operation, water evaporation in each sample holding section was confirmed. Next, similarly, about 12 kinds of samples, 1.0 μl is dispensed into each of columns C and D in rows 1 to 12, and water is evaporated in the same manner. Finally, columns D in rows 1 to 12 are used. In each case, 1.0 μl was dispensed. By performing such an operation, a total of 1.0 μl, 2.0 μl, 3.0 μl, and 4.0 μl of the sample solution was supplied from column A to column D of row 1 to row 12, respectively.

Using the concentration gradient created in Example 1 above, nucleic acid amplification was performed by PCR in each sample holding compartment. For amplification, primers suitable for amplification of each nucleic acid and QIAGEN's QIAGEN Multiplex PCR kit were used, and 35 cycles of amplification reaction were performed.
Amplification was performed after adding a sample and a reagent solution for PCR reaction to each sample holding section on the substrate and then adding 5 μl of mineral oil manufactured by SIGMA.
The PCR product was analyzed by collecting 1 μl from the reaction solution below the oil in each sample holding compartment and applying it to a capillary sequencer.

(result)
All samples could be fragmented and analyzed using a capillary sequencer.

2 ml of plasma was prepared from 5 ml of blood collected from one healthy person. Using this QIAamp Midi Kit (QIAGEN), plasma DNA was prepared. Using this plasma DNA, four types of concentration gradients of 1 ×, 5 ×, 10 ×, and 20 × were prepared on one substrate in the same manner as in Example 1. Further, standard nucleic acids of known concentrations and DNase / RNase-free pure water were used as a positive control and a negative control, respectively, without a concentration gradient.
PCR was performed in the same manner as in Example 2.

(result)
Fragment analysis by a sequence synthesizer was possible at 10 × and 20 × out of 4 types of concentration gradients of 1 ×, 5 ×, 10 ×, and 20 ×. This is probably because the amount of PCR template was insufficient at 1 × and 5 ×.

Blood was obtained from one healthy person, and plasma preparation and amplification by PCR were performed on one substrate in the same manner as in Example 3.
In parallel with this experiment (after the above amplification reaction), a concentration gradient for the standard DNA attached to PicoGreen (registered trademark) manufactured by Molecular Probes was created on the (same) substrate.
The same amount of PicoGreen solution was added to each of the amplified product and the standard DNA, allowed to stand at room temperature for 5 minutes, and then the fluorescence was measured with a plate reader. A calibration curve was created from the relationship between the amount of standard DNA and the amount of fluorescence. Using this calibration curve, the amount of DNA was converted from the amount of fluorescence of the amplified product.

(result)
Of the four types of concentration gradients of 1 ×, 5 ×, 10 ×, and 20 ×, 5 ×, 10 ×, and 20 × can be quantitatively converted from the calibration curve. 1 × was not located within the linear range of the detection line (below the detection limit) and could not be quantified. This is probably because the amount of mold was insufficient.

Collect 5 ml of whole blood from healthy males and females in a blood collection tube with anticoagulant and centrifuge at 3000Xg 4 ° C for 15 minutes to enrich the lymphocytes located in the middle layer of the upper plasma component and lower blood cell component The containing buffy coat fraction was collected in a new centrifuge tube. After adding 5 ml of PBS solution to the collected buffy coat fraction and suspending lymphocytes in the solution, it was centrifuged again at 3000 × g at 4 ° C. for 15 minutes, the supernatant was removed, and 1 × 104 lymphocytes were added to the PBS solution again. The cell suspension solution was prepared by adding / ml.
Take 1 μl of the above cell suspension and dispense into one hydrophilic part on a slide glass (previously kept at 50 ° C.) with a hydrophilic part surrounded by a hydrophobic part. Evaporate to leave only lymphocyte cells attached.
After evaporating the solution and adding 3 μl of dH ₂ O to the lymphocyte cell-only compartment, the substrate is incubated at 95 ° C. for 5 minutes → 50 ° C. for 1 minute twice to destroy the lymphocyte cell membrane / nuclear membrane The solvent was evaporated at the same time as the lymphocyte DNA was exposed.
After 1 μl of PCR reaction solution was dispensed into lymphocyte DNA, 5 μl of sealing oil was dispensed, PCR was performed, and Y chromosome gene amplification was performed.
The presence or absence of DNA amplification was detected with Quant-iT PicoGreen dsDNA Reagent and Kits (Invitrogen, P7589).

(result)
Amplification was observed in healthy men but not in women.

The nucleic acid detection method of the present invention enables simple, efficient and accurate detection of a nucleic acid for a sample considered to have a very low nucleic acid concentration in the sample. Therefore, it can be effectively used in the determination of various diseases in the clinical field.

Claims (28)

(A) supplying a nucleic acid-containing sample solution for analyzing a target nucleic acid to a sample holding section on a substrate;
(B) performing a reaction on the target nucleic acid in the nucleic acid-containing sample solution in the same compartment as the sample holding compartment; and (c) in the same compartment as the sample holding compartment in the step (b) Detecting a nucleic acid-containing reaction product;
A nucleic acid detection method comprising: After the step (a) and before the step (b),
(A-1) a step of appropriately evaporating at least a part of the solvent of the nucleic acid-containing sample solution in the sample holding compartment; and (a-2) a step consisting of the steps (a) and (a-1) Is performed once or a plurality of times (however, the evaporation in the step (a-1) is performed immediately after the first step (a) (a-1) or step (a-2)). Conducted in (a-1) at least once in any of the steps);
The nucleic acid detection method according to claim 1, comprising: Evaporating the solvent of the step (a-1),
Including a step of allowing the substrate to stand for a preset time at the same temperature as the temperature at which the step (a) is performed, and optionally exposing the substrate to dry air; or at least 5 ° C. than the temperature at which the step (a) is performed A step of heating the substrate to a higher temperature and appropriately exposing the substrate to dry air, including an operation of allowing the substrate to stand for a preset time as appropriate;
The nucleic acid detection method of Claim 2 containing this. The method for detecting a nucleic acid according to claim 3, wherein the temperature higher than that in the step (a) is a temperature within a range of 25 to 95 ° C. The step (a)
(A-3) supplying a nucleic acid-containing sample solution to a substrate that has been previously kept at a constant temperature in the range of 25 to 95 ° C .; and
(A-4) Step of allowing the substrate to stand for a preset time as appropriate and exposing the substrate to dry air as appropriate;
The nucleic acid detection method according to claim 1, comprising: The step of reacting with the target nucleic acid in the nucleic acid-containing sample solution in the step (b),
(B ′) a step of performing any one reaction selected from the group consisting of an amplification reaction, a hybridization reaction, a reverse transcription reaction, a ligation reaction, and a cleavage reaction with a restriction enzyme,
(B-1) a step of supplying a reaction solution containing components essential for the reaction at a concentration suitable for the selected reaction into the same compartment as the sample holding compartment when supplied to the sample holding compartment And (b-2) holding the substrate at a temperature suitable for the selected reaction for a preset time, or each of the substrates in a plurality of temperature zones suitable for the selected reaction. A step of performing a cycle consisting of sequentially holding for a set time once or a plurality of times;
The method for detecting a nucleic acid according to claim 1, wherein the method comprises the steps of: The nucleic acid detection method according to claim 6, wherein the selected reaction is an amplification reaction by PCR. Detecting the nucleic acid-containing reaction product in the step (c),
(C ′) a step of supplying a solution containing a nucleic acid detection reagent into the same compartment as the sample holding compartment and detecting a nucleic acid detection reaction product produced thereby, and the solution containing the nucleic acid detection reagent is The nucleic acid detection method according to claim 1, wherein the detection reagent is contained at a concentration suitable for a detection reaction when the sample is supplied into the sample holding section. After the step (b) and before the step (c ′),
(B-3) evaporating at least a portion of the solvent of the solution in the sample holding compartment;
The method for detecting a nucleic acid according to claim 8, comprising: Detecting the nucleic acid detection reaction product in the step (c ′),
(C '') The nucleic acid detection method according to claim 8, which is a step of measuring spectroscopic characteristics of the nucleic acid detection reaction product. The nucleic acid detection method according to claim 10, wherein the spectroscopic characteristic relates to fluorescence intensity, emission intensity, color development, or absorbance. Detecting the nucleic acid detection reaction product in the step (c ′),
(C ′ ″) The nucleic acid detection method according to claim 8, which is a step of measuring the amount of nucleic acid in the reaction product. The nucleic acid detection method according to claim 1, wherein the step (c) is to detect a label introduced into the reaction product of the target nucleic acid in the reaction of the step (b). Supplying the nucleic acid-containing sample solution in the step (a),
The nucleic acid detection method according to claim 1, wherein the supply operation is performed by dropping the solution, a syringe piston pump system, or an ink jet system. The nucleic acid detection method according to claim 1, wherein the amount of the nucleic acid-containing sample solution supplied in the step (a) is a fixed amount within a range of 0.1 to 5 μl. The nucleic acid detection according to claim 15, wherein the step of supplying the nucleic acid-containing sample solution in the step (a) is a step of supplying the fixed amount of solution to the substrate a plurality of times before performing the step (b). Method. The nucleic acid-containing sample solution is a solution containing cells,
After the step (a) and before the step (b),
(A-5) adding a cell rupture solution to the sample holding section; and (a-6) holding the substrate at one temperature or a plurality of temperature zones for a preset time;
The nucleic acid detection method according to claim 1, comprising: The nucleic acid detection method according to claim 1, wherein the substrate has a plurality of sample holding sections. The nucleic acid detection method according to claim 18, wherein in the step (a), the same nucleic acid-containing sample solution is supplied to two or more sample holding sections. After the step (a) and before the step (b),
(A-1) a step of appropriately evaporating at least a part of the solvent of the nucleic acid-containing sample solution in the sample holding compartment; and (a-2) a step consisting of the steps (a) and (a-1) Is performed once or a plurality of times (however, the evaporation in the step (a-1) is performed immediately after the first step (a) (a-1) or step (a-2)). Conducted in (a-1) at least once in any of the steps);
The nucleic acid detection method of Claim 18 containing this. 21. The step (a-2) is performed on at least one of the plurality of sample holding sections at a number different from the number of steps (a-2) performed in another sample holding section. Nucleic acid detection method. In all of the steps (a-1), the operation of evaporating must be performed, and the number of steps (a-1) performed for each of the plurality of sample holding sections is a set of natural numbers at regular intervals. 22. The method for detecting a nucleic acid according to 21. (A) A nucleic acid-containing sample solution for analyzing the presence or absence of a target nucleic acid is supplied to at least two sample holding sections on the substrate, and a standard nucleic acid-containing solution containing a known amount of the standard nucleic acid is used as the nucleic acid-containing sample. Supplying separate, at least two compartments on the same substrate having a sample holding compartment for supplying a solution;
(B) The following sub-steps:
(B-1) evaporating at least part of the solvent of the nucleic acid-containing sample solution and at least part of the solvent of the standard nucleic acid-containing solution in the compartment; and (B-2) the step (A ) And (B-1) are further performed once or a plurality of times;
Comprising the step of concentrating nucleic acids in the solution,
The number of steps (B-1) performed on at least one of the series of compartments supplied with the nucleic acid-containing sample solution or standard nucleic acid-containing solution is a set of natural numbers at regular intervals;
(C) supplying a solution containing a nucleic acid detection reagent into each compartment after the concentration step (B), and detecting a nucleic acid detection reaction product produced thereby;
(D) The following sub-steps:
(D-1) A calibration curve between the amount of all standard nucleic acids supplied in the same compartment and the quantification result of the nucleic acid detection reaction product based on the quantification result in step (C) performed on the standard nucleic acid-containing solution. (D-2) calculating the amount of the target nucleic acid in the nucleic acid-containing sample solution based on the calibration curve created in the sub-step (D-1);
Performing a target nucleic acid quantification comprising:
A nucleic acid detection method comprising: After the step (B) and before the step (C),
(B-3) performing an amplification reaction using each of the target nucleic acid in the nucleic acid-containing sample solution and the standard nucleic acid in the standard nucleic acid-containing solution as a template in each of the compartments;
The nucleic acid detection method of Claim 23 containing this. The nucleic acid detection method according to any one of claims 1 to 24, wherein the substrate surface comprises a hydrophobic region and a hydrophilic region, and the hydrophilic region is used as a sample holding section. The nucleic acid detection method according to claim 25, wherein the sample holding section is a recess having a substantially circular opening, and the diameter of the bottom of the recess is 0.5 mm to 6 mm. The nucleic acid detection method according to any one of claims 1 to 24, wherein the nucleic acid-containing sample solution is plasma. 25. The nucleic acid concentration in the nucleic acid-containing sample solution is in the range of 5 pg / μl to 1 μg / μl, and the total amount of nucleic acid in the sample solution is in the range of 0.05 ng to 1 μg. The nucleic acid detection method according to any one of the above.