JP6001374B2 - Target nucleic acid detection method and kit used therefor - Google Patents

Description

  The present specification relates to a method for detecting a target nucleic acid and a kit used therefor.

  As a method for detecting various nucleic acid-derived target nucleic acids, an amplification product obtained by amplifying the target nucleic acid in advance is hybridized with a detection probe immobilized in advance on a solid phase carrier, and the hybridized product is subjected to hybridization. The method of giving a signal is widely used.

  For example, a highly versatile test method for tuberculosis drug susceptibility has been disclosed in clinical settings (Non-Patent Document 1). In this method, DNA extracted from bacteria is detected visually using an array. In general, this type of method requires about five steps in order to easily detect the hybridized product visually. For example, an amplification product labeled with biotin is obtained, hybridization between the amplification product and a detection probe is performed, and then the hybridized amplification product is labeled with peroxidase, and further a color reaction is performed.

  Further, in this type of method, not only the color development step, but also the preparation step of the amplification product from the target nucleic acid leading to the color development step, the hybridization step, etc. include a plurality of substeps.

Journal of Clinical Microorganism Rapid Diagnosis Research Society, 14: 45-50

  However, in the conventional method, in order to visually determine the hybridization product, a signal imparting step including multiple steps is performed. For this reason, not only the complexity of operation and the working time have increased, but also the possibility of operation mistakes and human error have increased, and there has been a risk that the detection accuracy will decrease.

  Further, in the conventional method, due to the complexity of the process, it is difficult to perform a single specimen or a small number of specimens individually and quickly. On the other hand, when a large number of samples are processed at once, a dedicated device is required, and there is a problem that the cost is increased if there is no more than a certain number of samples.

  Furthermore, normally, in the hybridization process and the washing process, the process temperature must be maintained at a specific constant temperature. However, in an incubator that is heated in a generally used gas phase, it is difficult to strictly control the temperature. There was a problem that it was easy to lead to a misjudgment due to specific binding.

  The present specification provides a more practical method for detecting a target nucleic acid and a kit used therefor.

  The present inventors examined a more practical method for detecting a target nucleic acid, that is, providing a detection method and a kit used therefor that can realize simplicity, rapidity, and low cost. As a result, it has been found that the reaction system can be made compact and the detection of the hybridized product can be simplified at the same time. The present specification provides the following means based on these findings.

  The present specification provides a method for detecting a target nucleic acid. This detection method comprises a tag sequence that hybridizes with a detection probe that is associated in advance with the target nucleic acid, an amplification product derived from the target nucleic acid, a labeling substance that can be visually confirmed, and a part of the amplification product. A labeling probe having a labeling sequence to hybridize and an array having the detection probe on a solid phase carrier can be used. The detection method also includes a first hybridization in which the amplification product and the detection probe in the array are brought into contact with each other in a hybridizable manner, and a first hybridization in which the labeling probe and the amplification product are brought into contact with each other in a hybridizable manner. A hybridization step of performing the second hybridization. Furthermore, a step of detecting a hybridization product by the first hybridization and the second hybridization with the labeling substance can be provided. In this detection method, the labeling substance can be a particle including a coloring substance. The immobilization region may be 2 or more and 200 or less. The detection probe may be a probe having a base sequence that is a normal orthogonal sequence.

  In addition, the amplification product can be provided at the 5 'protruding end of each chain that pairs the tag sequence with a tag sequence for labeling that hybridizes with the labeling sequence. In this embodiment, the hybridization step can be a step of simultaneously performing the first hybridization and the second hybridization on the same array.

The hybridization step of the present detection method may be a step of performing the first and second hybridization using the array in a liquid of 1 ml or less for each array. In this aspect, in the hybridization step, the size of the immobilization region of the detection probe is 0.2 mm ² or more and 150 mm ² or less, the solid phase carrier has a plane area of 150 mm ² or less, and an aspect ratio. It can be set as the process of implementing hybridization using the said array which is 1.5 or more sheet-like bodies. In this embodiment, the hybridization step is performed by using the array in which the solid-phase carrier is a sheet-like body having a plane area of 50 mm ² or less in a liquid of 0.3 ml or less for each array. It can be a step of performing hybridization. Furthermore, in this aspect, the aspect ratio may be 20 or less.

  In this detection method, the array may further include a color development sample region by the labeling substance on the solid phase carrier. The color sample area may be formed of pigment-based ink. Furthermore, the solid phase carrier may have liquid permeability or permeability, and in this case, the material of the solid phase carrier may be selected from polyethersulfone, nitrocellulose, nylon, polyvinylidene fluoride and cellulose. Good. The thickness of the solid phase carrier may be 0.01 mm or more and 0.3 mm or less.

  In the present detection method, the array may include a handling portion at a predetermined part of an outer edge of the plurality of immobilization regions. In addition, the hybridization step can be performed while maintaining the state where the array is put in the same tube-like container with a predetermined direction. Furthermore, prior to the hybridization step, a step of preparing a test sample by a nucleic acid amplification reaction in the container can be provided.

  The present specification provides a kit for detecting a target nucleic acid. This kit is provided with a tag sequence that hybridizes with a detection probe pre-associated with a target nucleic acid, and is visually recognized with one or more primer sets that amplify an amplification product derived from the target nucleic acid by a gene amplification method. One or more labeling probes comprising a possible labeling substance and a labeling sequence that hybridizes with a part of the amplification product, and an array comprising the detection probe on a solid phase carrier .

It is a figure which shows an example of the flow until a target nucleic acid is detected. It is a figure which shows an example of acquisition of the amplification product provided with the tag sequence | arrangement hybridized to a probe for a detection, and the label | marker tag sequence | arrangement hybridized to a label | marker probe at a 5 'overhanging end, respectively. It is a figure which shows an example of the final hybridization product in this detection method. It is a figure which shows the amplification result of the genomic DNA in an Example. It is a figure which shows the detection result obtained in the Example of this invention. It is a figure which shows the detection result obtained in the Example of this invention. It is a figure which shows the detection method (upper stage) of the target nucleic acid in an Example, and the detection method (lower stage) of the conventional target nucleic acid.

  The present specification discloses a method for detecting a target nucleic acid, a kit therefor, and the like. In the detection method disclosed in the present specification, the target nucleic acid can be detected quickly and easily by performing the first hybridization and the second hybridization. Furthermore, this detection method uses an amplification product that protrudes to the 5 ′ end side of each strand that couples the tag sequence and the labeling tag sequence that can hybridize with the labeling sequence, thereby denaturing the amplification product. Thus, the first and second hybridizations can be carried out without going through a single-stranded process. Furthermore, the first and second hybridizations can be performed simultaneously on the same array.

  The amplification product of this embodiment can be obtained, for example, by amplifying a target nucleic acid with a first primer set and a second primer set as shown in FIG. The first primer includes a first arbitrary base sequence such as a tag sequence complementary to a detection probe previously associated with the target nucleic acid, and a first identification sequence for identifying the first base sequence in the target nucleic acid. Including a linking site capable of suppressing or stopping the DNA polymerase reaction between the first arbitrary base sequence and the first recognition sequence, and the second primer includes a tag sequence for labeling, A second identification sequence for identifying a second base sequence in the target nucleic acid, and having a linking site capable of suppressing or stopping the DNA polymerase reaction between the tag sequence for labeling and the second identification sequence Yes.

  The ligation site suppresses or stops the DNA polymerase reaction. That is, the linking site cannot be a template for a DNA extension reaction by DNA polymerase because it does not contain a natural base or the like. Therefore, as shown in FIG. 2, when the DNA single strand amplified by the first primer becomes a template strand and further amplified by the second primer, the DNA extension reaction from the second primer is In addition, it is suppressed or stopped on the 3 ′ side from the site that matches the linking site.

  Similarly to the first primer, the second primer becomes a template strand when the single strand of DNA amplified by the second primer is further amplified by the first primer. The DNA elongation reaction is suppressed or stopped on the 3 ′ side from the site that pairs with the ligation site. For this reason, as a result, the amplified fragment (DNA double-stranded fragment) obtained by the amplification step is provided as a single strand with a tag sequence protruding at one end and an arbitrary base sequence protruding at the other end. It is inferred that it is provided as a single strand and has a double-stranded portion by base pairing.

  As described above, amplification using a DNA polymerase for a sample containing a target nucleic acid using a primer set in which the first arbitrary base sequence is a tag sequence complementary to a detection probe previously associated with the target nucleic acid It is also supported that the target nucleic acid can be detected with extremely high sensitivity and speed when the amplified fragment obtained by carrying out the step is hybridized with the detection probe without being denatured as it is. As shown in FIG. 1, the obtained DNA double-stranded fragment forms a double-stranded portion in the first base sequence and the second base sequence in the target nucleic acid, and the tag sequence is single-stranded at the end. Therefore, it is considered that this single strand is efficiently hybridized with the probe. Sensitivity improves as hybridization efficiency increases.

By using such an amplification product, the detection method of the present invention can realize at least one of the following effects.
(1) Efficiency improvement (acceleration) of hybridization
(2) Efficient labeling (hybridization with a labeling probe) (3) Enhancement of detection sensitivity (4) Simplification (acceleration) of the process-Especially, omission of the denaturation process with a double strand as a single strand by

  An oligonucleotide derivative having a base sequence including such a linking site is itself useful as a nucleic acid amplification agent such as a primer. Moreover, the nucleic acid amplification method using such a primer, the obtained DNA double-stranded fragment and the hybridization composition containing the fragment can also exhibit at least one effect corresponding to the form of each.

  Furthermore, the present detection method can be combined with the reduction in the scale of the solid phase array to reduce the cost by reducing the amount of the reagent, and also enables accurate and rapid temperature control. Furthermore, the detection by visible light can reduce the cost of the apparatus and can observe the color development in real time, which is suitable for the speed of inspection.

  In this specification, the target nucleic acid means a polymer of nucleotides, and the number thereof is not particularly limited. Accordingly, target nucleic acids include oligonucleotides in which several tens of nucleotides are linked. The target nucleic acid is, for example, a base serving as a genetic index in a living organism such as a human or non-human animal, such as constitution, genetic disease, onset of a specific disease such as cancer, disease diagnosis, treatment prognosis, drug or treatment selection Contains the base sequence. Typically, polymorphisms such as SNP and congenital or acquired mutations can be mentioned. In addition, base sequences derived from microorganisms such as pathogenic bacteria and viruses are also included as base sequences possessed by the target nucleic acid.

  In the present specification, samples that can contain a target nucleic acid include various biological samples (blood, urine, sputum, tissue, cells (including cultured animal cells, cultured plant cells, and cultured microbial cells derived from various animals). Or a DNA extraction sample obtained by extracting DNA from such a biological sample.

  Hereinafter, representative and non-limiting specific examples of the disclosure of the present specification will be described in detail with reference to the drawings. This detailed description is intended merely to provide those skilled in the art with details for practicing the preferred embodiments of the present disclosure and is not intended to limit the scope of the present disclosure. Absent. Further, the additional features and disclosure disclosed below can be used separately from or together with other features and inventions to provide improved methods for detecting target nucleic acids and the like.

  In addition, the combination of features and steps disclosed in the following detailed description are not essential in carrying out the disclosure of the present specification in the broadest sense, and are particularly representative specific examples of the disclosure of the present specification. It is described only for the purpose of explaining. Furthermore, the various features of the exemplary embodiments described above and below, as well as those described in the independent and dependent claims, provide additional and useful embodiments of the disclosure herein. They do not have to be combined according to the specific examples described herein or in the order listed.

  All features described in this specification and / or claims, apart from the configuration of the features described in the examples and / or claims, are individually disclosed as limitations on the original disclosure and claimed specific matters. And are intended to be disclosed independently of each other. Further, all numerical ranges and group or group descriptions are intended to disclose intermediate configurations thereof as a limitation to the original disclosure and claimed subject matter.

  Hereinafter, embodiments of the present invention will be described.

(Target nucleic acid detection method)
The method for detecting a target nucleic acid disclosed in the present specification comprises a tag sequence that hybridizes with a detection probe that is previously associated with the target nucleic acid, an amplification product derived from the target nucleic acid, and a labeling substance that can be visually recognized A labeling probe comprising a labeling sequence that hybridizes with a part of the amplification product and an array comprising the detection probe on a solid phase carrier can be prepared and used. This detection method is applied to various known hybridization techniques. For example, hybridization (hereinafter referred to as “in-liquid type high hybridization”) is performed by immersing the entire array in a hybridization medium containing labeled probes and amplification products, in which an array of dot-like immobilization regions arranged in a matrix or the like on a flat solid support. Also referred to as hybridization). In addition, a hybrid medium that is also a mobile phase is supplied to a part of an array in which immobilization regions of various forms such as a stream (band) are arranged on a flat solid support, and a predetermined orientation with respect to the array The method is also applied to hybridization in affinity chromatography (hereinafter also referred to as development type hybridization) performed by developing a mobile phase in (1). Hereinafter, the array, the amplification product, and the labeling probe will be described in this order.

(array)
The array includes a plurality of immobilization regions for detection probes previously associated with target nucleic acids on a solid phase carrier. The shape and size of the array are not particularly limited, and can be various known shapes and sizes. The array is configured according to various known hybridizations. When the array is applied to submerged hybridization, the array has a unique form capable of submerged hybridization of 1 ml or less per array. In other words, the present array preferably includes a solid phase carrier having such a unique form (size and shape). In addition, when applied to development-type hybridization, at least the end of the array is immersed in a hybridization solution supplied to a microtube such as an Eppendorf tube (trademark), which is widely used for testing and research. It is preferable to have a possible size (width direction) and shape. Preferably, it is a long body provided with a part that can be accommodated from the vicinity of the bottom of this type of tube to the upper end. The array may be prepared in any way and may be obtained commercially, but the present detection method may comprise an array preparation step for preparing the array.

  The form that enables development-type hybridization using 1 ml or less submerged hybridization or 1 ml or less mobile phase to the array varies depending on the size of the hybridization container. In the present invention, for example, It is preferred that a tapered or slotted tubular container is contemplated. That is, it is preferable to implement this detection method using such a container. An example of the tapered container is typically an Eppendorf tube (trade name), and an example of a cylindrical container is typically a general test tube. The tube-like container preferably has a volume capable of being filled with, for example, 1 ml or less, 0.5 ml or less, and 0.3 ml or less hybridization solution. Such tubular containers each have an inner diameter of 7-9 mm, typically 8 mm, a depth of 37-39 mm, typically 38 mm, an inner diameter of 5-7 mm, typically 6 mm, a depth of 29-31 mm, typically Specifically, those having a size of 30 mm and an inner diameter of 4 to 6 mm, typically 5 mm, a depth of 19 to 21 mm, and typically 20 mm can be mentioned.

  The container is also preferably transparent. This is because it is convenient to visually recognize the state of the internal array and thus the labeling substance with the naked eye. Moreover, the container may be provided with a detachable lid to open and close its opening (usually the upper part). By providing such a lid, it is possible to prevent evaporation of a small amount of liquid and the like and to quickly and easily control the temperature.

More specifically, the form of the array preferably has a plane area of 150 mm ² or less. When the area is less than this plane area, hybridization is possible in the immobilization region even in a solution of 1 ml or less. More preferably, it is 100 mm ² or less, and further preferably 50 mm ² or less. When it is 50 mm ² or less, it is also effective for hybridization in a solution of 0.3 ml or less.

  Further, it is preferably a rectangular shape having an aspect ratio of 1.5 or more. Such a square shape is easy to fit in a tubular container and is easy to handle. Moreover, the form at the time of injection | throwing-in is hold | maintained in a container, without reversing in a tube-shaped container, and the predetermined fixed directionality is ensured. Furthermore, when the shape is rectangular, the directionality of the array can be easily determined visually, which is convenient for the hybridization step, the signal application step, and the detection step. More preferably, the aspect ratio is 20 or less. This is because when the aspect ratio exceeds 20, it is difficult to grasp the detection pattern of the fixed region. When the aspect ratio is 20 or less, it is easy to visually grasp the signals in the plurality of fixed regions as a pattern. Can be improved. The aspect ratio is preferably 15 or less, more preferably 10 or less, still more preferably 5 or less, and even more preferably 3 or less.

  The array is preferably in the form of a sheet. When it is in the form of a sheet, it is easy to handle and is advantageous for storage as inspection data. Furthermore, when it is in the form of a sheet, it is easy to impart a certain degree of flexibility, which can advantageously contribute to the filling property, storage property, and reactivity in a small-scale container. The thickness of the array is preferably 0.01 mm or more and 0.3 mm or less as the thickness of the solid phase carrier.

(Detection probe immobilization area)
The array holds detection probes previously associated with target nucleic acids on a solid support. In order to facilitate detection, the detection probe is usually held so as to form an immobilization region having a predetermined shape. The detection probe is appropriately selected according to the object to be detected, the detection technique, or the target nucleic acid detection system. For example, in order to determine the species (type) of an infectious agent such as an infectious disease, detection is performed by hybridization with a plurality of detection probes. In detecting a mutation, one or a plurality of detection probe immobilization regions may correspond to one mutation.

  Each of the detection probes has a detection sequence that is a unique base sequence for probing. Such a detection sequence can be set independently of the sequence characteristic of the target nucleic acid, that is, the target sequence. By setting the detection sequence independent of the target sequence, the detection sequence of the detection probe can suppress or avoid non-specific binding between a plurality of detection probes, and is suitable for hybridization at a suitable temperature and time. Can be set in consideration of the hybridization conditions. In addition, the same detection probe can always be used regardless of the type of target nucleic acid.

  The length of the detection sequence is not particularly limited, but is preferably 20 bases or more and 50 bases or less. This is because within this range, hybridization efficiency can be ensured while ensuring the specificity of each detection sequence. For example, such a base length detection sequence is a 46 base length sequence obtained by combining two base sequences each having a base length of 23 bases each selected from SEQ ID NOs: 1 to 100 and a complementary sequence thereof, or the combined base sequence. Can be obtained by appropriately adding or deleting a base. More preferably, it is 20 bases or more and 25 bases or less. For example, such a base length detection sequence can be obtained by appropriately adding or deleting bases to the 23 base length sequences of SEQ ID NOS: 1 to 100 and their complementary sequences or these base sequences. it can. Since the tag sequence in the first primer is a base sequence that is paired with the detection sequence, the base length of the tag sequence is preferably 20 bases or more and 50 bases or less, like the detection sequence. Preferably, it is 20 bases or more and 25 bases or less.

  As a detection sequence of such a detection probe, for example, the base sequence described in SEQ ID NO: 1 to SEQ ID NO: 100 or a base sequence complementary to this base sequence can be used. These base sequences all have the same base length (23 base length), and have a melting temperature (Tm) of 40 ° C. or higher and 80 ° C. or lower, preferably 50 ° C. or higher and 70 ° C. or lower, and are homogeneous in hybridization under the same conditions. A hybrid result can be obtained. In addition, as above-mentioned, 2 types selected from these base sequence groups can also be combined. Furthermore, bases can be added, deleted, substituted, etc. within such a range that the specificity is not lost. The detection sequence for the detection probe to be used at the same time is selected from either the base sequence (group) represented by SEQ ID NOs: 1 to 100 or a base sequence (group) complementary thereto. Is preferred.

  The detection sequence of the detection probe can be appropriately selected from such candidate base sequences or their complementary sequences, and is selected from the base sequences shown in the following table or their complementary sequences. Only a probe set consisting of only one or two or more probes each having one or two or more base sequences as a detection sequence, or only a probe having all the following base sequences or their complementary sequences as detection sequences It is preferable to use a probe set consisting of By selecting such a base sequence as a detection sequence, it is possible to perform hybridization in a short time and realize further rapid hybridization.

  The detection sequence in such a detection probe is also referred to as an orthonormalized sequence, for example, a continuous match length for a DNA sequence of a predetermined base length obtained from a random number, melting temperature prediction by Nearest-Neighbor method, Hamming distance, Designed by performing secondary structure prediction calculations. The orthonormalized sequence is a base sequence of nucleic acid having a uniform melting temperature, that is, a sequence designed so that the melting temperature is within a certain range, and the nucleic acid itself is intramolecular. It means a base sequence that does not form a stable hybrid other than a base sequence that is structured in the above and does not inhibit hybridization with a complementary sequence. A sequence included in one orthonormalized sequence group hardly reacts between sequences other than the desired combination and within a self-sequence, or does not generate a reaction. Further, when the orthonormalized sequence is amplified in PCR, the amount of nucleic acid corresponding to the initial amount of the nucleic acid having the orthonormalized sequence is quantitatively affected without being affected by the problems such as the above-mentioned cross-hybridization. Has the property of being amplified. The orthonormalized sequence as described above is described in detail in H. Yoshida and A. Suyama, “Solution to 3-SAT by breadth first search”, DIMACS Vl. 54, 9-20 (2000) and Japanese Patent Application No. 2003-108126. Have been described. Orthonormalized sequences can be designed using the methods described in these references.

  In one immobilization region, one kind of oligonucleotide probe is preferably immobilized. The 3 'end of the detection probe may be bound to a carrier, or the 5' end may be bound. It may be covalent or non-covalent. As for the method for immobilizing the oligonucleotide probe to the solid phase carrier, those skilled in the art can appropriately employ various known methods as necessary. The 3 'end of the detection probe may be bound to a carrier, or the 5' end may be bound. It may be covalent or non-covalent. The detection probe can be immobilized on the surface of the carrier by various conventionally known methods. For example, the carrier is supplied so as to draw a predetermined plane form by a method of discharging a micro droplet of a solution containing a detection probe. And the probe for a detection is fix | immobilized by drying by heating etc. as needed. Furthermore, for example, in order to immobilize the detection probe on the solid phase carrier, an amino group or the like may be added to the detection probe, or a protein such as albumin is linked to increase the adhesion to the carrier. You can also. Further, the sticking property can be enhanced by various types of radiation such as heat treatment and UV irradiation.

The size of each immobilization region is 0.2 mm ² or more and 150 mm ² or less. When the thickness is less than 0.2 mm ² , the visual visibility is excessively lowered, and when it exceeds 150 mm ² , there is a problem in the size of the array.

  The shape of the immobilization region is not particularly limited, and may be various known forms applied in the array, such as a circular shape and a rectangular shape. In view of the packing density and visibility of the immobilization region, a rectangular shape is preferable, and a square shape is more preferable.

  The number of immobilization regions is appropriately set according to the detection intention and the application of the detection method. Considering the size of the array and the like, it is not particularly limited, but it is preferably 2 or more and 200 or less. This is because if the number exceeds 200, it may be difficult to determine the size of the array and to visually detect and judge. When visual signal detection in one or a plurality of immobilization regions is taken into consideration, it is preferably 150 or less, more preferably 100 or less. Further, the number of immobilization regions is preferably 20 or more, more preferably 40 or more. When it is 20 or more, for example, it is highly versatile in various diagnoses.

  The plurality of immobilization regions preferably form one compartment on the solid support. It is preferable that the section has an alignment form by a combination of a vertical row and a horizontal row according to the shape of the array. Typically, these compartments are square.

  The detection probe is immobilized on a solid phase carrier in a predetermined pattern according to the form of hybridization described later. In liquid type hybridization, typically, a pattern is formed in which dots corresponding to individual detection probes are arranged. In the development type hybridization, typically, a stream (band) corresponding to each detection probe is a pattern arranged at one or more development positions along the development direction.

(Color sample area)
The array can be separately provided with a color sample region by a detection signal in addition to a plurality of immobilization regions on a solid phase carrier. By providing such a color sample area, the detection accuracy and accuracy can be ensured even if the array is reduced in scale or by visual signal discrimination. The color development sample region is a color development sample of a signal applied in a signal application step described later, and is provided in advance with a color tone, lightness, and saturation corresponding to the signal. That is, it is given so that it can be maintained through the detection method. The color sample area is provided by ink, and pigment ink is preferably used in consideration of fixability to a solid phase carrier and reactivity. Such inks are typically applied to a solid phase carrier in the same manner as when an oligonucleotide probe is applied to a solid phase carrier to form an immobilized region (for example, an inkjet method using a piezo element). Yes. The color development sample region is preferably provided on the solid phase carrier in the same size and shape as the immobilization region because of the requirement for comparative observation. Furthermore, it is preferable that the color development sample region is formed adjacent to the immobilization region so that it can be easily compared with the signal in the immobilization region.

  The color development sample region is preferably composed of two or more types of regions having different levels of color tone or shading of the detection signal. With such a configuration, visual signal detection can be performed more easily and reliably. Preferably, at least two color sample regions corresponding to the upper limit and the lower limit of the color tone or shading of the detection signal are provided. By providing such a color sample area, signal detection can be determined using the above upper and lower limits as indices, and easier and more reliable detection is possible regardless of the skill level of the tester and reaction errors.

(Handling part)
The array can be provided with a handling site outside the compartment consisting of a plurality of immobilization regions. By having a handling site, handling can be ensured even in a small array, the array can be handled in a certain direction, experimental accuracy or detection accuracy can be improved, and signal detection can be facilitated. . As the handling site, typically, a margin region in which an immobilization region or a color sample region on a solid phase carrier is not formed can be applied. In addition, it is convenient to handle the array by providing a handling part on a part of the array, for example, one end (especially one short edge) of the square sheet, and the array can be easily maintained in a certain direction. Various operations can be performed. The handling site can be provided with specimen identification information for individually identifying the array, and such specimen identification information may be previously given to ink or the like. The specimen identification information may be numbers, symbols, characters or figures, or a combination thereof.

  In addition, the array can comprise information useful for manipulation and identification. For example, direction identification information that determines the directionality of the array at the time of operation or detection can be given. The margin area (handling part), color sample area, and specimen identification information described above can also be used as direction identification information.

  The solid phase carrier constituting the array is not particularly limited, but it is preferable to have liquid permeability or permeability in consideration of downsizing, handling on a small scale, reactivity, visibility of detection signal, and the like. . As such a material, for example, a so-called porous membrane filter mainly composed of a polymer such as polyethersulfone, nitrocellulose, nylon, polyvinylidene fluoride, or cellulose such as filter paper can be preferably used. This kind of porous material is also suitable for developmental hybridization.

(Amplification product)
An amplification product is a product derived from a target nucleic acid in a sample and obtained by a nucleic acid amplification reaction. Various known reactions can be used as the nucleic acid amplification reaction. For example, in addition to PCR by various methods, LCR, SDA, ICAN and the like can be mentioned. The amplification product may be prepared in any way, but the present detection method may include an amplification step for preparing the amplification product.

  The amplification product is provided with a tag sequence that hybridizes with the detection probe. The amplification product usually has a full duplex form. In the case of this type of amplification product, it is provided from the primer for obtaining the amplification product at the 5 'end or 3' end of either strand. For example, if an oligonucleotide strand having a tag sequence or a complementary sequence thereto is provided in advance on the 5 ′ end side of one primer of a primer set for obtaining an amplification product, 5 ′ of one strand of the amplification product is provided. A tag sequence is provided at the terminal or 3 ′ terminal.

  The amplification product has a labeling tag sequence that can hybridize with a labeling sequence of a labeled probe described later. Similarly to the tag sequence, the tag sequence for labeling is provided at the 5 'end or 3' end of either strand derived from a primer for obtaining an amplification product. However, in the case of a full duplex amplification product, it is preferable to provide a tag sequence and a labeling tag sequence at each end of one of the single strands. The labeling tag sequence can be amplified, for example, by providing an oligonucleotide chain that becomes a labeling tag sequence or a complementary sequence thereof at the 5 ′ end side of one primer of a primer set for obtaining an amplification product. A tag sequence for labeling is provided at the 5 ′ end or 3 ′ end of one strand of the product. As will be described later, the amplification product in such a complete double-stranded form is converted into a single strand and then subjected to the first and second hybridizations.

  In this detection method, an amplification product of another aspect can also be used. In other words, an amplification product having a tag sequence at the 5 'end and having a single-stranded portion protruding from the double-stranded site (5' protruding end) can also be used. By providing the tag sequence at the 5 ′ protruding end, the tag sequence can be hybridized with the detection probe even in the amplification product after the amplification reaction including the double-stranded site in the paired state. Therefore, the amplification product can be subjected to the first hybridization without undergoing thermal denaturation as described later.

  In addition, the amplification product of this embodiment may be an amplification product having a labeling tag sequence at the 5 'end and a single-stranded portion protruding from the double-stranded site (5' protruding end). By providing the tag sequence for labeling at the 5 ′ protruding end, the tag sequence for labeling can be hybridized with the labeling probe even in the amplification product after the amplification reaction including the paired double-stranded sites. . Therefore, the amplification product can be subjected to the second hybridization without undergoing thermal denaturation as described later.

  Preferably, the amplification product of this embodiment has a tag sequence at the 5 'protruding end of one strand and a tagging tag sequence at the 5' protruding end of the other strand. The amplification product of such an embodiment can be subjected to the first and second hybridizations without thermal denaturation.

  The amplification product of this aspect may be subjected to nucleic acid amplification using, for example, the following first primer and second primer. Here, the acquisition of an amplification product having a tag sequence at the 5 ′ protruding end of one strand and a tagging tag sequence at the 5 ′ protruding end of the other strand will be described. For the amplification product having only the tag sequence or the tag sequence for labeling, one of the paired primers may be a primer shown below and the other may be a normal primer.

(First primer)
The first primer includes a tag sequence complementary to a detection probe previously associated with the target nucleic acid and a first identification sequence for identifying the first base sequence in the target nucleic acid. The lengths and the like of these base sequences are not particularly limited, and are appropriately determined according to the contents of the target sequence of the target nucleic acid.

(First identification sequence)
The first identification sequence is a sequence for amplifying the target nucleic acid by nucleic acid amplification, and can specifically hybridize with the first base sequence constituting a part of the target sequence in the target nucleic acid. The first identification sequence is set complementarily to the extent that it can hybridize with the first base sequence with high selectivity. Preferably, it is set to be completely complementary (specific).

(Tag array)
The tag sequence is a sequence for allowing the amplified fragment to hybridize with the detection probe and detects the target nucleic acid. Therefore, the tag sequence hybridizes to the detection sequence of the detection probe for each target nucleic acid. It is set to be able to soy. Typically, the base sequence is complementary to the detection sequence. Therefore, one target nucleic acid is associated with one detection probe. As already explained, the base length of the tag sequence preferably matches the base length of the detection sequence of the detection probe, preferably 20 bases to 50 bases, more preferably 20 bases to 25 bases. It is as follows.

  The first base sequence and the second base sequence in the target nucleic acid may have any configuration with respect to the target nucleic acid. For example, when detecting a mutation on DNA, only one of the base sequences may contain a mutation site of one or more bases, or both may contain a mutation site. . The first primer has such a tag sequence and a first identification sequence, has a natural base constituting such a base sequence or an artificial base homologous thereto, and a base pair with a natural nucleic acid. It has a skeleton that can be combined. Typically an oligonucleotide or a derivative thereof.

(Linked part)
A part of the primer having the tag sequence and the other part of the primer having the first identification sequence are not directly linked and have a linking site between them. The ligation site is a site capable of suppressing or stopping the DNA polymerase reaction when included in the template strand. In the DNA polymerase reaction, it is said that if there is no nucleic acid (or base) as a template, the DNA strand will not be extended any further. For this reason, the linking site of the present invention has a structure that cannot serve as a template during DNA elongation by DNA polymerase. That is, this linking site does not include a natural base or a derivative of a natural base (such as a natural base) that pairs with a natural base.

  The ligation site cannot be a template for a DNA extension reaction by DNA polymerase because it does not contain a natural base or the like. Therefore, as shown in FIG. 1, when the DNA single strand amplified by the first primer becomes a template strand and further amplified by the second primer, the DNA extension reaction from the second primer is In addition, it is suppressed or stopped on the 3 ′ side from the site that matches the linking site. Therefore, as a result, the amplified fragment (DNA double-stranded fragment) obtained by the amplification step is provided with a first arbitrary base sequence protruding from one end as a single strand and double-paired by base pairing. A chain portion is provided.

  This linking site may be a chain linking group containing a single chain structure having 2 to 40 elements adjacent to the nucleotide via a phosphodiester bond. This is because if the number of elements is 1 or less, the DNA polymerase reaction is likely to be incompletely inhibited or stopped, and if the number of elements exceeds 40, the solubility of nucleotides may be reduced. Considering the effect of suppressing or stopping the DNA polymerase reaction, the chain linking group element is preferably 2 or more and 36 or less, more preferably 3 or more and 16 or less.

  This linking site includes a single bond to facilitate rotation at the linking site, and the single bond includes a carbon-carbon single bond, a carbon-oxygen single bond, a carbon-nitrogen single bond, and an S-S single bond. Examples include bonding. It is preferable that this connection site is mainly composed of such a single bond. In addition, this linking site may partially contain an aromatic ring or cycloalkane as long as it contains a single bond.

  The linking site preferably includes an alkylene chain or a polyoxyalkylene chain having 2 to 40 elements and optionally substituted. Such a chain-like connection structure is structurally simple and can be easily introduced as a connection site.

As such a connection part, the connection part represented by the following formula | equation (1) is mentioned, for example.
5′-O—C _m H _2m —O-3 ′ Formula (1)
(In the formula, 5 ′ represents an oxygen atom of a phosphodiester bond on the 5 ′ side, 3 ′ represents a phosphate atom of a phosphodiester bond on the 3 ′ side, and m represents an integer of 2 to 40. Represents.)

  In the formula (1), m is preferably 2 or more and 36 or less, and more preferably 3 or more and 16 or less. Typical examples of the substituent of H in formula (1) include an alkyl group, an alkoxy group, and a hydroxyl group. It is preferable that carbon number of an alkyl group and an alkoxy group is 1-8, More preferably, it is 1-4. Moreover, when it has two or more substituents, the substituents may be the same or different. Furthermore, it is also preferable that it does not have a substituent.

Moreover, as another connection part, the connection part represented by the following formula | equation (2) is mentioned.
_{5 '- (OC n H 2n} ) l -v3' formula (2)
(In the formula, 5 ′ represents an oxygen atom of a phosphodiester bond on the 5 ′ side, 3 ′ represents a phosphate atom of a phosphodiester bond on the 3 ′ side, and n represents an integer of 2 or more and 4 or less. And l is an integer of 2 or more, and (n + 1) × l represents an integer of 40 or less.)

  In the formula (2), (n + 1) × l is preferably 2 or more and 36 or less, and more preferably 3 or more and 16 or less. The same aspect as the substituent in Formula (1) is applied to the substituent of H in Formula (2).

  Examples of the linking site include the following chain sites.

  Furthermore, as this connection site | part, the following chain | strand-shaped site | parts are mentioned, for example.

  The first primer has a first identification sequence and a tag sequence, and has a natural base constituting such a base sequence or an artificial base homologous thereto, and allows base pairing with a natural nucleic acid. As a main component. Typically an oligonucleotide or a derivative thereof.

  The first primer preferably has a tag sequence, a linking site, and a first identification sequence in that order from the 5 'side. With such a configuration, when the DNA strand amplified by such a primer is amplified as a template strand, it is extended 5 ′ from the ligation site derived from the first primer in the template strand, that is, extended by DNA polymerase. The extension reaction is stopped or suppressed on the 3 ′ side further in the DNA strand to be processed. As a result, the 5 'end of the nucleotide base adjacent to the 3 ′ side of the ligation site derived from the first primer in the template strand or the base in the vicinity thereof is the 5 ′ end, and the tag sequence in the first primer An amplified fragment having no complementary strand is obtained.

  It should be noted that a sequence unrelated to the tag sequence or the first identification sequence can also be included in the vicinity of the linking site, that is, on the 3 ′ side and 5 ′ side of the linking site. When the first primer becomes a template strand, the presence of a ligation site can reduce or avoid the influence of unintended DNA extension reaction progress or termination on the tag sequence or the first identification sequence in the extended strand. Because.

(Second primer)
As shown in FIG. 2, the second primer includes a second identification sequence that identifies the second base sequence in the target nucleic acid. The lengths and the like of these base sequences are not particularly limited, and are appropriately determined according to the contents of the target sequence of the target nucleic acid.

(Second identification sequence)
The second identification sequence is a sequence for amplifying the target nucleic acid together with the first primer by nucleic acid amplification, and specifically with the second base sequence constituting the other part of the target sequence in the target nucleic acid. Can hybridize. The second identification sequence is set complementarily to the extent that it can hybridize with the second base sequence with high selectivity. Preferably, it is set to be completely complementary (specific).

(Tag array for labeling)
As shown in FIG. 2, the tag sequence for labeling may be configured to be capable of binding the labeling substance. That is, a labeled probe having a predetermined base sequence and having a labeling substance and a base sequence for identifying a labeling sequence may be capable of binding. Such a labeled probe is supplied to the amplification product on the solid phase in the hybridization step and detection step described later, and can be labeled.

  The second primer has a natural base constituting the base sequence of the second identification sequence or an artificial base homologous thereto, and a skeleton that allows base pairing with the natural nucleic acid. Typically an oligonucleotide or a derivative thereof.

(Linked part)
When the labeling tag sequence is provided, the labeling tag sequence and the second identification sequence may be directly linked, but preferably have a linking site between them. In particular, as shown in FIG. 2, it is preferable when the labeling substance binding region has a base sequence that interacts with and binds to the labeling probe. The linking site is as described in the first primer.

  The second primer preferably has a labeling tag sequence, a linking site, and a second identification sequence in that order from the 5 'side. By adopting such a configuration, when the DNA strand amplified by the second primer becomes a template strand and is amplified by the first primer, it is 5% more than the linking site derived from the second primer in the template strand. The extension reaction is stopped or suppressed on the 'side, that is, the 3 ′ side ahead in the new DNA strand that is extended by the DNA polymerase. As a result, the labeling tag in the second primer having the 5 'end as the base pairing with the nucleotide base adjacent to or adjacent to the 3' side of the linking site derived from the second primer in the template strand A DNA amplified fragment that does not have a complementary strand of the sequence (its base sequence) will be obtained.

  It should be noted that a sequence unrelated to the labeling tag sequence and the second identification sequence can also be included in the vicinity of the linking site, that is, on the 3 ′ side and 5 ′ side of the linking site. When the second primer becomes a template strand, due to the presence of the ligation site, the influence of unintended DNA extension reaction progress or termination on the labeling tag sequence or the second identification sequence in the extended strand is reduced or This is because it can be avoided.

  Such primers can be synthesized according to ordinary oligonucleotide synthesis methods. For example, the linking site can be synthesized using a phosphoramidite reagent having an alkylene chain. Such a reagent itself is known and can be obtained from, for example, GlenResearch. For example, the following reagents can be mentioned. In the following formula, DMT represents a typical dimethoxytrityl group as a hydroxyl protecting group, but may be other known hydroxyl protecting groups. In the following formula, PA represents a phosphoramidite group.

  Nucleic acid amplification is performed using these primers. As described above, various known methods can be applied to the nucleic acid amplification method, but typically, various PCRs such as PCR and multiplex PCR are used. A person skilled in the art can appropriately set the solution composition, temperature control, and the like in carrying out the nucleic acid amplification step.

Hereinafter, nucleic acid amplification using such a first primer and a second primer as a primer set will be described with reference to FIG.
As already described, for example, a tag sequence, a linking site, and a first identification sequence in this order from the 5 ′ side, and a tag sequence for labeling, a linking site, and a second from the 5 ′ side. When PCR is performed on a sample that may contain a target nucleic acid using a second primer having these in the order of the identification sequences, as shown in (a) to (c) of FIG. A template strand containing the primer derived from the first primer and the second primer is formed by the DNA extension reaction of the DNA polymerase.

  Then, the DNA extension reaction is again performed by the DNA polymerase using the second primer and the first primer different from the primers from which the template strands are derived. At this time, as shown in FIG. 2 (d), the DNA elongation reaction of the DNA polymerase with respect to the template strand starting from the second primer and containing the first primer is carried out from the ligation site derived from the first primer in the template strand. On the 5 ′ side, that is, on the extended strand, on the 3 ′ side of the ligation site, DNA elongation is suppressed or stopped.

  In addition, as shown in FIG. 2 (d), the DNA polymerase DNA extension reaction to the template strand starting from the first primer and containing the second primer is performed 5 times from the ligation site derived from the second primer in the template strand. On the 'side, that is, on the extended strand, 3' from the ligation site, DNA elongation is suppressed or stopped.

  As a result, as shown in FIG. 2 (e), the resulting amplified fragment comprises a single-stranded tag sequence protruding from the 5 ′ end and a tag sequence for labeling, and includes a first identification sequence and a second tag sequence. In the identification sequence, it becomes a DNA double-stranded fragment having a double strand. That is, in the heavy chain fragment of this DNA, the tag sequence protrudes into a single strand on the 5 ′ side of one DNA strand, and the labeling tag sequence protrudes on the 5 ′ side of the other DNA strand. Yes.

(Labeling probe)
The labeling probe can include a labeling sequence that hybridizes with a part of the amplification product (labeling tag sequence) and a labeling substance. The labeling sequence has complementarity to such an extent that it can specifically hybridize with the tagging tag sequence, and is preferably completely complementary. The length of the base sequence of the labeling sequence is not particularly limited, and is appropriately determined according to the content of the target sequence of the target nucleic acid, its Tm related to hybridization, and the like.

  The labeling substance can be visually confirmed. That is, it is a substance that itself can directly generate a signal that is visible without the need for other components. Such materials typically include various colorants such as various pigments and dyes. In addition to this, in addition to noble metals such as gold and silver, various metals or alloys such as copper, or organic compounds containing the metal (may be complex compounds) may be used. In addition, inorganic compounds such as mica, which are similar to the colorant, can be used.

  The labeling substances typically include chemiluminescent substances including various dyes, luminol, isoluminol, acridinium compounds, olefins, enol ethers, enamines, aryl vinyl ethers, dioxene, aryl imidazoles, lucigenin, luciferin and eclion. . Moreover, particles such as latex particles labeled with such a labeling substance can be mentioned. Furthermore, colloids or sols including gold colloids or sols or silver colloids or sols can be mentioned.

  The average particle diameter of the particles constituting a part of the labeling substance is usually 20 nm or more and 20 μm or less, typically 40 nm to 10 μm, preferably 0.1 to 10 μm, particularly preferably 0.1 to 5 μm, More preferably, it has an average particle diameter of 0.15 to 2 μm. Further, depending on the pore diameter of the solid phase carrier 110, it is preferably 500 nm or less, more preferably 0.1 nm or more and 100 nm or less, and further preferably 1 nm or more and 50 nm or less. Preferred particles are particles that can be suspended in an aqueous solution and consist of a water-insoluble polymeric material, preferably a substituted polyethylene. Particularly preferred are latex particles such as polyethylene, acrylic acid polymer, methacrylic acid polymer, acrylonitrile polymer, acrylonitrile-butadiene-styrene, polyvinyl acetate-acrylate, polyvinylpyrrolidone or vinyl chloride-acrylate. Latex particles having active groups such as carboxyl, amino or aldehyde groups on their surface.

  Such a labeling substance and particles labeled with the labeling substance are commercially available, and the production of the labeling substance and the method of labeling the label on the particle are also known, and those skilled in the art can obtain them appropriately using known techniques. be able to. Furthermore, the binding between such a labeling substance or particles labeled with the labeling substance and an oligonucleotide such as DNA is also possible via a functional group such as an amino group, which is well known in the art.

(Hybridization process)
In the present method, the first hybridization in which the amplification product and the detection probe are brought into contact with each other in a hybridizable manner and the second hybridization in which the labeling probe and the amplification product are brought into contact with each other in a hybridizable manner in the array. A hybridization step to be performed can be provided. By performing the first hybridization, a hybridization product between the detection probe and the amplification product is obtained by hybridization between the tag sequence and the detection probe. In addition, by performing the second hybridization, a hybridized product of the amplification product and the labeling probe can be obtained by hybridization of the labeling sequence and the labeling tag sequence. As a result of these first and second hybridizations, as shown in FIG. 3, a hybridized product of an amplification product, a detection probe and a labeling probe is obtained on the solid phase. Since the labeling substance is held in the immobilization region of the detection probe associated with the target nucleic acid in advance, the presence of the hybridized product is visualized visually. Since the labeling substance is accumulated or concentrated in the region where the detection probe is immobilized, compared with the region where the detection probe is not immobilized, visual recognition is easy.

  The order of the first and second hybridizations is not particularly limited. The first hybridization may be performed first, followed by the second hybridization, or vice versa. Further, the first hybridization and the second hybridization may be performed simultaneously. In any case, the final hybridized product is retained in the immobilization region of the detection probe associated with the target nucleic acid in advance.

  When the amplification product is a complete duplex, it is preferable to carry out a denaturation step for making the duplex into a single strand prior to the first hybridization and / or the second hybridization. This is because it is difficult to hybridize with a detection probe or a labeling probe unless it is single-stranded. In the denaturation step, a temperature or the like is appropriately set based on Tm corresponding to the sequence of the amplification product. Usually, it is rapidly cooled after being heated to about 90 ° C to 95 ° C. In this way, a single strand (having a tag sequence and a label tag sequence) derived from the amplification product can be hybridized with a detection probe or a label probe on the array. Therefore, this kind of amplification product can be first subjected to a denaturation step, and then the first hybridization and the second hybridization can be performed in any order or simultaneously.

  When the amplification product has a tag sequence at one 5 ′ protruding end of the duplex and a labeling tag sequence at the other 5 ′ protruding end, the first high-end product is not subjected to the denaturation step as it is. Hybridization and second hybridization can be performed in any order or simultaneously.

  Hereinafter, an example of a hybridization process in which the first hybridization and the second hybridization are performed in a solution of 1 ml or less on the array will be described with reference to FIG. 1 as appropriate.

  The following hybridization steps are performed sequentially or simultaneously in a tube-shaped container in a solution of 1 ml or less, preferably 0.5 ml or less, more preferably 0.3 ml or less per array. This is an example.

  First, when carrying out the first hybridization, the array in which the detection probe is fixed and the amplification product form a state existing in an appropriate hybridization medium in a container, and the temperature is set in advance. And hybridization at time. The array is loaded into the container in a predetermined direction. Hybridization is preferably performed while maintaining the state in which the long side of the array is along the depth direction of the container according to the internal form of the tubular container. By using the array of rectangular sheets having the aspect ratio described above, it can be stably held in various forms of tubular containers of about 1 to 0.3 ml that are commercially available.

  When a tube-like container is used for hybridization, it can be applied to an existing temperature control apparatus for PCR. By performing such temperature control, temperature control can be performed accurately and quickly. The conditions for the hybridization step are not particularly limited. A normal hybridization medium can be used. Moreover, it can set to moderate temperature.

  When the second hybridization is performed subsequent to the first hybridization, a labeling probe used for the second hybridization is added to the container after the first hybridization, and a preset temperature and temperature are set. Perform hybridization with time. As described above, the first hybridization and the second hybridization can be performed in the reverse order or simultaneously. That is, the hybridization step can be performed all at once in the presence of the amplification product, the array (detection probe) and the labeling probe.

  Thus, it is preferable to implement 1st hybridization and 2nd hybridization in the same container irrespective of the order. By doing so, the operation can be simplified and the speed can be improved. In addition, by keeping the array in the same container, it is possible to easily maintain a certain direction when the array is put into the container (during the hybridization step), and the first hybridization and the second hybridization can be performed. It can be carried out under more stable and favorable conditions.

  1A shows a step of performing the second hybridization after the first hybridization, FIG. 1B shows a step of performing the first hybridization after the second hybridization, FIG. (C) shows the process of simultaneously performing the first and second hybridizations.

  In addition, as shown in FIG. 1 (d), it is preferable that the nucleic acid amplification reaction for obtaining an amplification product is also performed prior to the hybridization step in the same container in which the hybridization step is performed. After carrying out the amplification reaction, the labeling probe and / or the array can be supplied to the amplification reaction solution in the container, and a medium suitable for hybridization can also be applied.

  In addition, when a denaturation step of the amplification product is necessary prior to the hybridization step, the denaturation step may be performed in the same container, and then the hybridization step may be subsequently performed in the same container. By doing so, it is possible to simplify the operation, further speeding up the operation, speeding up the temperature control and increasing the accuracy.

  In addition, you may implement the washing | cleaning process which wash | cleans and removes an excess test sample after a hybridization process. For example, in the washing step, the hybridization solution is removed from the container in which the hybridization step has been performed and only the array remains, and a solution suitable for washing is supplied at a predetermined temperature condition and contacted for an appropriate time. Can be carried out by repeating a suitable number of times. By carrying out such a cleaning process in the same container, the operation can be simplified, the number of containers can be reduced, and temperature control can be performed quickly and accurately.

  The hybridization step illustrated in FIG. 2 is in-liquid type hybridization, but is not limited to this form, and can be applied to development-type hybridization.

(Detection process)
The detection step is a step of detecting the final hybridized product based on the labeling substance. More specifically, it is a step of confirming the coloring and position of the immobilization region where the detection probe is immobilized. In this detection method, since the labeling substance that can be visually confirmed is used, the presence or absence of the target nucleic acid can be confirmed easily and quickly.

  The detection may be performed in a container filled with the array, or may be performed outside the container after the array is taken out of the container. Moreover, you may carry out in the state in which the hybridization medium was accommodated. In this detection method, after the hybridization step, the detection step can be carried out without any special signal imparting step, and coloring can be detected while maintaining the same orientation of the array as used in the hybridization step. For this reason, there is no misjudgment based on directional mistakes, and the accuracy is high.

  When the array has a color sample area, the color of the detection probe-immobilized area is observed by contrasting with the color tone and shading of the sample area, thereby enabling rapid and highly accurate detection. Further, when the array is provided with at least two color development sample regions corresponding to the upper and lower limits of the assumed color tone or shading, it is possible to detect the target nucleic acid using the upper and lower limits as an index. . In this way, highly accurate detection is possible without depending on skill level or reaction error.

  The detection step may be a step of detecting a combination of a plurality of target nucleic acids in a sample according to the detection target and application. For example, in the case of detecting a plurality of sequences of a plurality of genes as in pneumonia causative bacteria typing, combinations of a plurality of immobilized regions corresponding to a plurality of target nucleic acids are detected. In this case, such a combination may be detected by comparing the coloring pattern of the immobilization region obtained on the array with the coloring pattern sample of the oligonucleotide probe corresponding to the combination. Such a color pattern sample is prepared in advance. For example, by preparing for each pattern to be detected, it is possible to quickly and easily determine with high accuracy.

(Array and array sheet)
The present invention also provides an array for use in the method of the present invention. The array of the various aspects described above is also included in the present invention. Furthermore, according to the present invention, an array sheet including a plurality of such arrays as an array region is also provided. That is, an array sheet for detecting a target nucleic acid, which includes a plurality of array regions including a plurality of immobilized regions of detection probes previously associated with the target nucleic acid so as to be separable for each array region An array sheet is provided. According to this sheet, the array used in the method of the present invention can be easily manufactured and supplied. In order to be separable for each array region, for example, when the above-described preferred solid support is used, cutting can be performed with a commonly used cutter or scissors. Alternatively, the array region can be easily separated by manual operation by providing a fragile portion that can be cut between the array regions.

  A kit for detecting a target nucleic acid disclosed in the present specification comprises a tag sequence that hybridizes with a detection probe previously associated with a target nucleic acid, and amplifies an amplification product derived from the target nucleic acid by a gene amplification method 1 Alternatively, one or more labeling probes comprising two or more primer sets, a labeling substance that can be visually recognized, and a labeling sequence that hybridizes with a part of the amplification product, and the detection probe as a solid phase carrier And an array provided thereon. According to this kit, the target nucleic acid can be determined easily and rapidly.

  EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples. However, the following examples illustrate the present invention and do not limit the scope of the present invention.

In the following examples, the target nucleic acid was detected by the following procedure according to the detection method of the present invention. Hereinafter, it demonstrates according to these order.
(1) Preparation of DNA microarray (2) Preparation and amplification of target nucleic acid and primer (3) Hybridization (4) Detection using scanner

(1) Preparation of DNA microarray GENESHT (Nippon Gaishi Co., Ltd.) uses an aqueous solution prepared by dissolving a synthetic oligo DNA (manufactured by Nippon Genetic Laboratory Co., Ltd.) having a 3 ′ end modified with an amino group on a plastic plate. Spotted with a spotter (registered trademark). As synthetic oligo DNA sequences used, the following 33 types capable of high-speed hybridization were selected from SEQ ID NOs: 1 to 100.

  After the spot, baking was performed at 80 ° C. for 1 hour. Furthermore, the synthetic oligo DNA was immobilized by the procedure described below. That is, after washing with 2 × SSC / 0.2% SDS for 15 minutes, washing with 2 × SSC / 0.2% SDS at 95 ° C. for 5 minutes, and then washing with sterilized water (shaking up and down 10 times) 3 Repeated times. Then, it dehydrated by centrifugation (1000 rpm x 3 minutes).

(2) Amplification of target nucleic acid Genomic DNA used for amplification is derived from a human, and primers P1-1 to P1 shown in the following table specific to six target nucleic acids ((1) to (6)) in the human genome -6 (manufactured by Nippon Genetic Institute), P2-1 to P2-6 (manufactured by Nippon Genetic Institute) and P3-1 to P3-6 (manufactured by Nippon Genetic Institute) were prepared. Each series has the following configuration (displayed as 5 ′ to 3 ′). The propylene group portion of the P3-based primer was synthesized according to an ordinary oligonucleotide synthesis method using Spacer Phophoamidite C3, a phosphoramidite reagent of GlenResearch, shown in the following formula.

P1-based primers: F, R: P2-based primers including a base sequence for specific target nucleic acids (1) to (6) in human DNA:
F: Binding sequence (tag sequence) of labeled probe + base sequence for each target nucleic acid of P1 system
R: Tag sequence consisting of the same base sequence as the base sequence of the synthetic oligonucleotide probe + base sequence for each target nucleic acid of P1 system (in the case of using P2 system primer, complement of the base sequence complementary to this tag sequence) Since the strand is also amplified, the complementary strand hybridizes with the probe, and the amplified fragment can be detected.)
P3 primer:
F: Binding sequence of labeled probe + linkage site X (propylene chain) + base sequence for each target nucleic acid of P1 system
R: Tag sequence consisting of a base sequence complementary to the base sequence of the synthetic oligonucleotide probe + linkage site X (propylene chain) + base sequence for each target nucleic acid of the P1 system

  Next, genomic DNA was amplified using these primers as follows. A multiplex PCR master mix manufactured by QIAGEN was used as a sample amplification reagent. As a thermal cycler, Applied Biosystems GeneAmp PCR System 9700 was used.

First, the following reagents were prepared for each sample.
(Reagent preparation)
dH ₂ O 4.0 μl
2 × multiplex PCR master mix 5.0μl
Primer mix (500 nM each) 0.5 μl
Genomic DNA (50 ng / μl) 0.5 μl
Total 10.0 μl

  Next, the amplification reagent is transferred to the thermal cycle plate, and the thermal cycle reaction (after 95 minutes at 95 ° C .; 30 seconds at 95 ° C., 1 second at 80 ° C., 40 minutes for 6 minutes at 64 ° C., then lowered to 10 ° C.) ) The amplified sample was purified with QIAGEN's MinElute PCR Purification Kit and then confirmed to be amplified with the intended length by agarose electrophoresis. The results are shown in FIG. The upper part of FIG. 4 shows the result of electrophoresis, and the lower part shows the amount of amplification calculated from the fluorescence intensity.

(3) Hybridization In order to hybridize the amplified sample obtained in (2) with the detection probe immobilized on the microarray, the following Hybri control and Hybri solution were prepared, and a reagent for hybridization was prepared therefrom. PrimerMix contains a labeling probe (fluorescently modified oligonucleotide that binds to the 5 ′ side of F of P2 and P3 primers) (25 μM). In addition, Alexa555-rD1_100 used for hybrid control used what labeled the 5 'end of the sequence complementary to the sequence corresponding to D1_100 with Alexa555.

(Hybri control)
Alexa555-rD1_100 (100 nM) 10 μl
TE (pH 8.0) 390 μl
400 μl total

(Hybri solution)
20 x SSC 2.0ml
10% SDS 0.8ml
100% Formamide 12.0ml
100 mM EDTA 0.8 ml
milliQ 24.4ml
Total 40.0ml

(Reagent for hybridization)
Hybri control 1.5μl
Primer Mix 1.0μl
Hybri solution 9.0μl
Subtotal 10.5μl
Amplified sample 3.0 μl
18.0 μl total

  Apply 9 μl of the prepared hybridization reagent (labeled sample solution) to the spot area of the microarray without heating for denaturation, etc., and use a comfort / plus thermoblock slide (Eppendorf) to prevent drying. Then, the hybridization reaction was performed by allowing to stand at 37 ° C. for 30 minutes.

(Washing)
After hybridization, the microarray substrate after the hybridization reaction is immersed in a glass staining vat filled with a cleaning solution having the following composition, shaken up and down for 5 minutes, and transferred to a glass staining vat containing sterilized water, The mixture was shaken up and down for 1 minute and centrifuged at 2000 rpm for 1 minute to remove water remaining on the surface of the microarray substrate.
(Composition of cleaning solution)
milliQ 188.0ml
20 x SSC 10.0ml
10% SDS 2.0ml
Total 200.0ml

(4) Detection using a scanner
Using an Appleied Precision ArrayWoRx, the exposure time was appropriately adjusted to obtain a fluorescence image. The results for the plastic substrate are shown in FIGS.

  First, as shown in the upper part of FIG. 4, it was found that the intended target nucleic acid in the genomic DNA can be amplified regardless of the presence or absence of the tag sequence. In addition, as shown in the lower table of FIG. 4, it was found that the amplification amount did not change greatly even when the tag sequence was directly linked to the identification sequence or via a linking site containing a propylene group. .

  In addition, as shown in FIGS. 5 and 6, when using the P2 primer (tag sequence + identification sequence) and the P3 primer (tag sequence + linking site + identification sequence), the P3 primer is clearly As a result of hybridization with the DNA fragment obtained by amplification using the DNA, roughly constant strong fluorescence could be observed regardless of individual tag sequences. On the other hand, in the hybridization results using the P2 primer, almost no fluorescence was observed regardless of the tag sequence.

  Furthermore, in the hybridization results obtained by diluting the sample concentration 10 times, fluorescence could still be observed when the P3 primer was used. The same result as in the plastic substrate described above was confirmed for the glass substrate.

  From the above, it was found that the use of the P3-based primer improves the detection sensitivity by at least 10 times. In the above example, the amplification sample was applied to the array without performing the denaturation step, and as shown in FIG. 4, the synthesis amount of the amplification sample was almost the same as that by the P2-based primer. From the above, it can be seen that a double-stranded fragment with high efficiency and good label efficiency was obtained by using the P3 primer.

  In this example, in (1) preparation of DNA microarray of Example 1, a glass substrate (geneslide manufactured by Toyo Steel Co., Ltd.) was used as a substrate instead of a plastic substrate, and the base sequences of detection probes are shown in the following table. 33 types were selected, and (3) in hybridization, except that a reagent having the following composition was used as a hybridization reagent, (1) Preparation of DNA microarray of Example 1, (2) Preparation of target nucleic acid and primer The target nucleic acid was detected in the same manner as in amplification, (3) hybridization, and (4) detection using a scanner. The results are the same as in Example 1 when the P3 primer (tag sequence + linking site + identification sequence) was used compared to the P2 primer (tag sequence + identification sequence) without performing the denaturation step. Obviously, it was possible to obtain a hybridization signal that was 10 times or more intense.

(Composition of hybridizing reagent)
Hybri control 1.5μl
Primer Mix 3.5μl
Hybri solution 9.0μl
Subtotal 14.0 μl
Amplified sample 4.0 μl
18.0 μl total

Hereinafter, examples embodying the disclosure of this specification will be described.
(1) Preparation of Membrane Type DNA Microarray A detection probe solution consisting of a base sequence shown in the following table on a Hi-Flow Plus membrane sheet (60 mm x 600 mm) manufactured by Merck Millipore is described in JP-A-2003-75305. The sample was spotted in a matrix using a GENISHOT (registered trademark) spotter using NGK Corporation using a discharge unit (inkjet method). All the spots were sufficiently within the width when the sheet was cut to a width of 3.5 mm so as to fit in the 0.2 ml tube. As the detection sequences of the detection probes used, arbitrary 16 types out of 100 types (SEQ ID NO: 1 to SEQ ID NO: 100) of D1_1 to D1_100 described in Supplementary Table 1 of the literature (Analytical Biochemistry 364 (2007) 78-85) were used. A selected sequence was used in which the 3 ′ end of the sequence was modified with an amino group.

It may be possible to fix the synthetic oligo DNA by performing baking or UV irradiation at 80 ° C. for 1 hour after the spot, but this time, as a treatment after the spot, a UV irradiation apparatus (XL-1500 UV Crosslinker) manufactured by Spectroline. Was used to carry out immobilization by irradiating with ultraviolet light of about 200 to 500 mJ / cm ² .

(2) Amplification of target nucleic acid Genomic DNA used for amplification is derived from humans and is specific to four target nucleic acids ((1) to (4)) in the human genome. P3-4 (manufactured by Nippon Genetic Institute) was prepared. Each primer set had the following configuration (indicated as 5 ′ to 3 ′). The propylene group portion of the P3-based primer was synthesized according to an ordinary oligonucleotide synthesis method using Spacer Phophoamidite C3, a phosphoramidite reagent of GlenResearch, shown in the following formula.

P3 primer:
F: tag sequence for labeling + linkage site X (propylene chain) + base sequence for each target nucleic acid
R: tag sequence + linkage site X (propylene chain) + base sequence for each target nucleic acid

  Next, genomic DNA was amplified using these primers as follows. A multiplex PCR master mix manufactured by QIAGEN was used as a sample amplification reagent. As a thermal cycler, Applied Biosystems GeneAmp PCR System 9700 was used.

(Reagent preparation)
dH ₂ O 4.0 μl
2 × multiplex PCR master mix 5.0μl
Primer mix (500 nM each) 0.5 μl
Genomic DNA (50 ng / μl) 0.5 μl
Total 10.0 μl

  Next, the amplification reagent is transferred to the thermal cycle plate, and the thermal cycle reaction (after 95 minutes at 95 ° C .; 30 seconds at 95 ° C., 1 second at 80 ° C., 40 minutes for 6 minutes at 64 ° C., then lowered to 10 ° C.) ) The amplified sample was purified with QIAGEN's MinElute PCR Purification Kit and then confirmed to be amplified with the intended length by agarose electrophoresis.

(3) Detection Using Membrane Type DNA Microarray and Color Latex Using the sample amplified in (2), the reaction to membrane type DNA microarray (in-liquid hybridization) and its detection procedure were as follows (Fig. 7). The hybridization solution applied to the array had the following composition. A latex liquid manufactured by AMR Co., Ltd. was used. The latex solution is a polystyrene latex bead containing a blue colorant and DNA of a sequence complementary to the ED-1 sequence immobilized (synthesis of a sequence complementary to the 5 'amino group-modified ED-1). DNA and a functional group on the latex surface having a carboxyl group formed a covalent bond) were prepared with PBS so as to have an arbitrary concentration.

The hybridization solution applied to the array had the following composition.
(Hybridization solution composition)
Hybrid Solution ^* 140.0μl
(0.5% Tween 20-1% BSA-PBS)
Latex solution ^* 50.0 μl
Sample 10.0 μl
total 200.0 μl

  The DNA microarray prepared in (1) is cut to a size that can fit into a 0.2 ml tube, set in the tube, and 200 μl of each of the prepared hybridized samples is added without heating for denaturation, etc., and the heat block temperature Reaction (shaking) was performed at 37 ° C. for 1 hour to carry out submerged hybridization. After completion of the reaction, the hybridization solution was discharged, and the DNA microarray was air-dried.

(4) Detection using membrane-type DNA microarray and color reaction The reaction (membrane-type hybridization) to the membrane-type DNA microarray using the sample amplified in (2) and its detection procedure were as follows (Fig. 7). The hybridization solution applied to the array had the following composition.

(Hybridized sample composition)
Hybri Solution ^* 200.0μl
(0.5% Tween20-1% BSA-PBS)
Biotin-labeled oligo DNA complementary to the tag sequence for labeling the F-side primer (25 μM)
4.0 μl
Sample 4.0 μl
Total 208.0 μl

  The DNA microarray prepared in (1) is cut into a size that can be accommodated in a 0.2 ml tube, set in the tube, and 208 μl of the prepared hybridized sample is added without heating for denaturation or the like, and the heat block temperature 37 Reaction was carried out at 0 ° C. for 1 hour to carry out submerged hybridization.

  After completion of the hybridization reaction, the membrane type DNA microarray was transferred to a 0.2 ml tube containing a washing solution (0.1% Tween 20-1 mM EDTA-TBS) and washed in a 37 ° C. heat block (37 ° C. × 1 min, 37 ° C. × 10 min, 37 ° C. × 1 min).

  The washed membrane-type DNA microarray was transferred to a 0.2 ml tube containing a mixture of biotin-HRP and streptavidin and reacted at room temperature for 20 minutes.

  After completion of the reaction, the membrane type DNA microarray was transferred to a 0.2 ml tube containing a washing solution (0.1% Tween 20-1 mM EDTA-TBS), and washing operations (room temperature × 1 min, room temperature × 10 min, room temperature × 1 min) were performed.

The washed membrane-type DNA microarray was subjected to a color reaction for about 5 minutes at room temperature using Vector Laboratories TMB Peroxidase Substrate Kit, 3, 3 ', 5, 5'-tetramethylbenzidine. Thereafter, the color developing solution was discharged and washed, and then the DNA microarray was air-dried.
Washing was performed.

(5) Detection The presence or absence of color development after drying of the array after the reactions of (3) and (4) was visually confirmed. In any case, the target nucleic acid could be detected by using the P3 primer. Further, as shown in the upper part of FIG. 7, when the color latex (3) was used, the target nucleic acid could be detected only by the hybridization step after the amplification reaction. On the other hand, as shown in the lower part of FIG. 7, in the case of the color development reaction of (4), a multi-stage color development process is required after the amplification reaction, the work is complicated, and the results cannot be obtained quickly. I understood it. From the above results, as shown in (3), by using a labeling substance that directly shows a visible signal, sufficient detection can be achieved even if the number of steps to obtain the result is reduced and the work time is shortened. Was found to be possible.

SEQ ID NOs: 1 to 136: probe

Claims (9)

A method for detecting a target nucleic acid, comprising:
An amplification product derived from the target nucleic acid, comprising a tag sequence that hybridizes with a detection probe pre-associated with the target nucleic acid;
A labeling probe comprising a particle comprising a coloring substance that can be visually confirmed, and a labeling sequence that hybridizes with a part of the amplification product;
An array comprising the detection probe on a solid phase carrier having an aspect ratio of 1.5 or more ;
Use
The first hybridization for bringing the amplification product and the detection probe in the array into contact with each other so as to be capable of hybridizing, and the second hybridization for bringing the labeling probe and the amplification product into contact with each other so as to be capable of hybridizing are performed. A hybridization step,
Detecting a hybridization product of the first hybridization and the second hybridization with the labeling substance;
With
The amplification product is provided at the 5 ′ protruding end of each strand pairing the tag sequence with a tag sequence for labeling that hybridizes to the labeling sequence ,
The hybridization step is performed by immersing the array in a tubular container containing a liquid containing a labeling probe that is 1 ml or less for each array, and the long side of the array is in the depth direction of the tubular container. Maintaining the substantially vertical state along the first and second hybridization,
The detection method , wherein the solid phase carrier has a color sample region formed of pigment-based ink and has liquid permeability or permeability . The hybridization step is a step of performing hybridization in a solution of 0.3 ml or less for each array using an array in which the solid phase carrier is a sheet-like body having a plane area of 50 mm ² or less. Item 2. The detection method according to Item 1. The detection method according to claim 1 or 2 , comprising a step of preparing a test sample by a gene amplification reaction in the tubular container prior to the hybridization step. Said aspect ratio is 20 or less, the detection method according to any one of claims 1-3. The solid support, polyethersulfone, nitrocellulose, nylon, is selected from polyvinylidene fluoride and cellulose, the detection method according to any one of claims 1-4. The solid support thickness is 0.01mm or more 0.3mm or less, the detection method according to any one of claims 1-5. Said array, said comprising a plurality of handling site to a predetermined portion of the outer edge of the fixing area, the detection method according to any one of claims 1-6. The detection method according to claim 7 , wherein the number of immobilization regions is 2 or more and 200 or less. The detection probe is a probe having a nucleotide sequence which is orthonormal sequence detecting method according to any one of claims 1-8.