WO2013002185A1 - Array and method for detecting target polynucleotide - Google Patents

Abstract

[Problem] To provide a more practical method for detecting a target polynucleotide. [Solution] An array provided with a plurality of immobilization regions for an oligonucleotide probe preassociated with a target polynucleotide on solid-phase support is prepared; the oligonucleotide probe on the array and an analyte are hybridized; a macroscopically visible detection signal is applied to a double-stranded hybridized product obtained by hybridization; and the target polynucleotide is detected on the basis of the detection signal. Hybridization is carried out in 1 mL or less of liquid per array.

Description

Target polynucleotide detection method and array

The present specification relates to a method for detecting a target polynucleotide, an array therefor, and the like.
(Refer to related applications)
This application claims priority based on Japanese Patent Application No. 2011-143441, filed on June 28, 2011, the contents of which are incorporated herein by reference.

Conventionally, tests such as identifying a living body by targeting a specific nucleotide or base sequence in a gene of a biological sample have been performed. For example, a gene region required for each type of Mycobacterium tuberculosis is amplified by a PCR method, and the amplification product is hybridized with an oligonucleotide probe fixed on a membrane, thereby testing the strain type.

As this type of method, for example, there is a method using a membrane in which dozens of specific probes necessary for each type of bacteria to be detected are prepared and these probes are immobilized in a parallel line pattern. In this method, a PCR product prepared from a sample is supplied and hybridized using a blotter so as to be perpendicular to the line pattern of these probes. Kits for carrying out this method are also commercially available.

In this method, since one end of the PCR primer is labeled with biotin, the membrane after hybridization is incubated with peroxidase-labeled streptavidin. Thereby, the hybridized PCR product is labeled with peroxidase, and the chemiluminescence signal from the ECL reagent is exposed to the X-ray film. In this way, the type can be determined based on the sequence pattern of the specific probe expressed according to the type of bacteria contained in the biological sample.

In order to facilitate the detection of the target polynucleotide, two or more types of physiologically active substances are fixed on a test paper or the like, and the physiologically active substances are adjacent to each other so that a physiological complex reaction can be easily detected visually. It is also being studied (Patent Document 1). In addition, it has been attempted to easily identify the position of a nucleic acid probe bound to a target substance by spotting a fluorescently labeled reagent or the like around the spot where the nucleic acid probe is spotted (Patent Document 2). .

JP 2007-64927 A JP 2000-270896 A

However, in each of the conventional methods described above, it is difficult to perform a test for each sample or a test with a small number of samples. That is, the above-described typical commercial kit is a method of performing hybridization using a blotter, and therefore, several tens of kinds of probes are immobilized on a large (14 cm square) membrane, and a sample is also prepared at a time. Although several tens of samples can be hybridized, even when the number of samples was small, the same amount of reagent was used and the same treatment had to be performed. In addition, due to such circumstances, it is difficult to test each specimen.

Furthermore, in the above-mentioned commercial kit, the temperature must be kept at a specific constant temperature during the hybridization and washing steps. However, since an incubator that heats in the gas phase is used, it is difficult to strictly control the temperature, and it is nonspecific. It is easy to lead to misjudgment by combination. Furthermore, incubation in the gas phase took time until it reached a constant temperature due to the large membrane. In addition, in the detection process, since the primer is labeled, one signal is obtained for each PCR amplified fragment. To compensate for this low detection sensitivity, the chemiluminescent reagent and X-ray film are exposed to light. Adopting this leads to an increase in cost and working time.

Patent Document 1 discloses a test piece (60 to 300 mm ² ) having a relatively small area, but does not meet individual and prompt requests for each specimen. Furthermore, since Patent Document 2 requires a large amount of a fluorescent material such as Cy5 and a dedicated scanner, the cost increases.

Therefore, an object of the present specification is to provide a more practical method for detecting a target polynucleotide and an array therefor.

The present inventors provide a more practical method for detecting a target polynucleotide, that is, a detection method and an array therefor, which can reduce the number of specimens, and most preferably enable a test to be performed for each specimen. investigated. More specifically, a detection method and an array therefor have been studied that can realize speediness, simplicity, and low cost. As a result, it was found that the reaction system can be made compact and the detection can be visualized with the naked eye. According to the present invention, the following means are provided.

(1) A method for detecting a target polynucleotide, comprising:
Performing hybridization on the test sample and the oligonucleotide probe on the array comprising a plurality of immobilized regions of oligonucleotide probes pre-associated with the target polynucleotide on a solid phase carrier; and
Providing a double-stranded hybridization product obtained by the hybridization with a detection signal that can be visually recognized by the naked eye;
Detecting a target polynucleotide based on the detection signal;
With
The detection method, wherein the hybridization step is a step of performing hybridization in a solution of 1 ml or less for each array using the array.
(2) In the hybridization step, the size of the immobilization region is 0.2 mm ² or more and 150 mm ² or less, and the solid phase carrier has a plane area of 150 mm ² or less and an aspect ratio of 1.5 or more. The detection method according to (1), wherein hybridization is performed using the array that is a sheet-like body.
(3) In the hybridization step, hybridization is performed in a solution of 0.3 ml or less for each array, using the array in which the solid phase carrier is a sheet-like body having a plane area of 50 mm ² or less. The detection method according to (1) or (2), which is a step.
(4) The detection method according to any one of (1) to (3), wherein the aspect ratio is 20 or less.
(5) The detection method according to any one of (1) to (4), wherein the array is separately provided with a color development sample region based on the detection signal on the solid phase carrier.
(6) The detection method according to (5), wherein the color sample area is formed of pigment-based ink.
(7) The detection method according to (5) or (6), wherein the array includes two or more kinds of the color development sample regions having different degrees according to the color tone or shading of a detection signal.
(8) The array includes 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,
The detection method according to (7), wherein the detection step is a step of detecting the target polynucleotide using the upper limit and the lower limit as indices.
(9) The detection method according to any one of (1) to (8), wherein the solid phase carrier has liquid permeability or permeability.
(10) The detection method according to (9), wherein the solid phase carrier is selected from polyethersulfone, nitrocellulose, nylon, polyvinylidene fluoride, and filter paper.
(11) The detection method according to any one of (1) to (10), wherein the thickness of the solid phase carrier is 0.01 mm or more and 0.3 mm or less.
(12) The detection method according to any one of (1) to (11), wherein the array includes a handling portion at a predetermined part of an outer edge of the plurality of immobilization regions.
(13) The hybridization step and the signal imparting step are performed while maintaining the state in which the array is put in the same tube-shaped container in a predetermined direction. (13) Detection method.
(14) The detection method according to (13), wherein the detection step is performed while maintaining the state where the array is put into the container with a predetermined direction.
(15) The detection method according to (13) or (14), comprising a step of preparing a test sample by a gene amplification reaction in the container prior to the hybridization step.
(16) The detection step is a step of detecting a plurality of the target polynucleotides based on a combination of detection signals obtained from a predetermined plurality of the immobilized regions. The detection method described.
(17) The detection step is a step of detecting the combination in comparison with the coloring pattern of the immobilized region obtained on the array and the coloring pattern sample of the oligonucleotide probe corresponding to the combination. The detection method according to (16).
(18) The detection method according to any one of (1) to (17), wherein the number of the immobilized regions is 2 or more and 200 or less.
(19) The detection method according to any one of (1) to (18), wherein the oligonucleotide probe is a probe having a base sequence that is an orthonormal sequence.
(20) An array used for the detection method according to any one of (1) to (19),
A plurality of immobilization regions of oligonucleotide probes pre-associated with the target polynucleotide and a color sample region that presents color development by a detection signal that can be visually recognized by the naked eye are provided on a solid phase carrier.
The size of the immobilization region is 0.2 mm ² or more and 150 mm ² or less, and the solid phase carrier is a sheet-like body having a plane area of 150 mm ² or less and an aspect ratio of 1.5 or more and 20 or less. .
(21) An array sheet for detecting a target polynucleotide comprising:
Provided with a plurality of immobilized regions of oligonucleotide probes pre-associated with the target polynucleotide, a color sample region that presents color development by a detection signal visible to the naked eye, and a plurality of array regions on a solid phase carrier And
The size of the immobilization region is 0.2 mm ² or more and 150 mm ² or less, the array region has a plane area of 150 mm ² or less, and an aspect ratio of 1.5 or more and 20 or less,
The solid phase carrier is a sheet that is separable for each array region.

It is a figure which shows an example of the array and array sheet | seat of this invention. It is a figure which shows an example of the detection method of this invention.

This specification relates to a method for detecting a target polynucleotide, an array for the same, and the like. The detection method disclosed in the present specification can detect a target polynucleotide quickly and easily on a small scale. Reducing the scale of the reaction system can reduce the cost by reducing the amount of the reagent, and by controlling the scale of the array, the temperature can be controlled with high accuracy and speed. Furthermore, the detection by visible light can reduce the cost of the apparatus, and the observation of color development in real time contributes to the speed of inspection.

The array and sheet disclosed in the present specification are suitable for such a small-scale reaction, and particularly excellent in applicability to a tubular container of 0.5 ml or less.

In this specification, the target polynucleotide means a polymer of nucleotides, and the number thereof is not particularly limited. Therefore, the target polynucleotide includes an oligonucleotide in which several tens of nucleotides are linked. The target polynucleotide is a base that serves as a genetic index in organisms such as humans and non-human animals, such as constitution, genetic disease, onset of specific diseases such as cancer, disease diagnosis, treatment prognosis, drug and treatment selection, etc. Or the base sequence is included. 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 exemplified as base sequences possessed by the target polynucleotide.

Hereinafter, representative and non-limiting specific examples of the present invention will be described in detail with reference to the drawings. This detailed description is intended merely to present those skilled in the art with the details for practicing the preferred embodiments of the present invention and is not intended to limit the scope of the invention. In addition, the additional features and inventions disclosed below can be used separately or together with other features and inventions to provide further improved methods and arrays for detecting target polynucleotides.

Further, the combinations of features and steps disclosed in the following detailed description are not indispensable when practicing the present invention in the broadest sense, and are particularly only for explaining representative specific examples of the present invention. It is described. Moreover, various features of the representative embodiments described above and below, as well as those described in the independent and dependent claims, are described herein in providing additional and useful embodiments of the invention. They do not have to be combined in the specific examples given 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 disclosed in the present specification will be described.

(Target polynucleotide detection method)
The detection method disclosed in the present specification includes a hybridization step, a signal imparting step, and a target polynucleotide detection step.

(Hybridization process)
The hybridization step prepares an array comprising a plurality of immobilized regions of oligonucleotide probes pre-associated with the target polynucleotide on a solid phase carrier, and the oligonucleotide probe on the array and the test sample are It can be set as the process of performing the hybridization which implements hybridization in a 1 ml or less solution for every said array. Hereinafter, the array of the present invention used in this method will be described first.

(array)
The array disclosed herein comprises a plurality of immobilized regions of oligonucleotide probes pre-associated with a target polynucleotide on a solid support. As described above, this array has a form that allows hybridization in a liquid of 1 ml or less per solution. That is, the solid support of the said form (size and shape) is provided.

The form that allows hybridization in a solution of 1 ml or less in the array varies depending on the size of the hybridization container. In this specification, for example, a tapered or cylindrical tube container is intended as the container. It is preferable. 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 check the state of the internal array, and thus the detection signal. 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.

Also preferably, the rectangular shape has 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.

(Immobilization region of oligonucleotide probe)
The array comprises a plurality of immobilization regions of oligonucleotide probes pre-associated with a target polynucleotide on a solid support. The oligonucleotide probe is appropriately selected according to the object to be detected, the detection technique, or the target polynucleotide 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 oligonucleotide probes. In detecting a mutation, one or a plurality of oligonucleotide probe immobilization regions may correspond to one mutation.

As the oligonucleotide probe, a universal probe that is always a probe having a predetermined base sequence may be used regardless of the base sequence of the target polynucleotide. A universal probe is a base sequence designed by, for example, calculating a continuous match length, melting temperature prediction by the Nearest-Neighbor method, Hamming distance, and secondary structure prediction for a DNA sequence of a predetermined base length obtained from a random number. Can have an orthonormal array. An orthonormal 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. A base sequence that does not form a structure and does not inhibit hybridization with a complementary sequence, and that does not form a stable hybrid other than a complementary base sequence can be meant. A sequence included in one orthonormal 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 orthonormal sequence is amplified by PCR or the like, the amount of nucleic acid corresponding to the initial amount of the nucleic acid having the orthonormal sequence is quantitatively determined without being affected by the problem such as the above-described cross-hybridization. Can have the property of being amplified. The orthonormal sequences as described above are described in detail in H. Yoshida and A.Suyama, “Solution to 3-SAT by breadth first search”, DIMACS Vl.54, 9-20 (2000). An orthonormal array can be designed based on these documents.

When such a universal probe is used, a specific universal probe is associated with the target polynucleotide in advance. Then, the target polynucleotide is subjected to a hybridization reaction with a primer and a ligase reaction, and a ligase specific to the target polynucleotide having a base sequence identical or complementary to the base sequence of the associated universal probe. A product is obtained, and hybridization between the ligase product and the universal probe on the array is performed. The ligase product hybridizes with a pre-associated probe to produce a double stranded hybridized product. For detection of mutation such as SNP, a plurality of universal probes may be assigned to one target polynucleotide. This type of method is carried out with reference to Japanese Patent Application Laid-Open No. 2008-306941, Analytical Biochemistry, 364, 1, 2007, 78-85, as well as JP2009-232778, JP2009-24, etc. can do.

In one immobilization region, preferably one kind of oligonucleotide probe is immobilized. As for the method for immobilizing the oligonucleotide probe to the solid phase carrier, those skilled in the art can appropriately adopt various known methods as necessary.

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 areas is appropriately set according to the purpose of detection and the use 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.

It is preferable that the plurality of immobilization regions 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.

(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 degrees of color or shade 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.

Besides, the array can be equipped with information useful for operation 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.

(Solid phase carrier)
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 materials, for example, so-called porous membrane filters mainly composed of polymers such as polyethersulfone, nitrocellulose, nylon, and polyvinylidene fluoride, filter paper such as cellulose can be preferably used.

In the hybridization step, using such an array, hybridization is performed in a solution of 1 ml or less, preferably 0.5 ml or less, more preferably 0.3 ml or less per array. In the hybridization step, when the test sample contains an oligonucleotide having a target polynucleotide that forms a base pair with the probe, a double-stranded hybridization product is formed.

The hybridization step is preferably performed while the array is placed in a tubular container in a predetermined direction and maintained in that state. Specifically, it is preferable to carry out while maintaining a state in which the long side of the array is along the depth direction of the container in accordance with 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.

In the hybridization step, when a tube-like container is used, it can be applied to an existing temperature control device 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.

In addition, it is preferable to carry out a washing step for washing and removing excess test sample prior to the subsequent detection step. In the washing step, it is appropriate to remove the hybridization solution from the container in which the hybridization step has been performed and leave only the array, and then supply a solution suitable for washing under a predetermined temperature condition and contact for a suitable time. This can be done by repeating several 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 test sample to be subjected to the hybridization step is not particularly limited as long as it contains a target polynucleotide that may be a target of hybridization. As a test sample, known gene information to which probe hybridization is applied, a test sample in a detection, test, or diagnostic method using DNA can be used in this method.

Test samples include various biological samples (blood, urine, sputum, tissue, cells (including cultured animal cells, cultured plant cells, cultured microbial cells derived from various animals)) or DNA from such biological samples. Extracted DNA extracted samples, amplified samples by various gene amplification techniques from DNA and RNA of such biological samples are included. In consideration of the signal providing step described later, it is preferable that the oligonucleotide in the test sample is labeled in advance. A person skilled in the art can easily prepare such a test sample based on a well-known technique. For example, the following test sample preparation process may be performed.

(Test sample preparation process)
The method of the present invention may comprise a step of preparing a test sample by a gene amplification reaction using the same container as that used for the hybridization step prior to the hybridization step. By using the same container, the hybridization step can be performed by introducing the array into the container used for amplification. In addition, by doing this, the number of containers to be used can be reduced and the operation can be simplified.

As the gene amplification reaction, various known reactions can be used. Examples include PCR by various methods, LCR, SDA, ICAN, and the like. In addition, the polynucleotide in the test sample can be labeled by a method of incorporating dNTP linked with a predetermined label-binding substance. Examples of the label-binding substance include haptens (primary signal) such as digokigenin (DIG). In this case, the hapten-specific recognition antibody can be used, and a chromogenic substance (secondary signal) such as peroxidase imparted to the recognition antibody can be used as a detection signal.

It is preferable to supply a gene amplification reaction solution or a hybridization solution in the same container after the gene amplification reaction. Further, it is more preferable to carry out the denaturation step at a high temperature prior to the hybridization 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.

(Signal giving process)
The signal imparting step can be a step of imparting a detection signal visible to the naked eye to the double-stranded hybridized product obtained in the hybridization step. By giving a signal that can be visually recognized by the naked eye, it is possible to observe and determine the color development with the naked eye in real time without requiring a special detection device. In order to give a signal to the double-stranded hybridized product, for example, as already described, a primary signal such as biotin in addition to a hapten is previously given to a polynucleotide in a test sample. In this step, by giving a specific secondary signal to the primary signal, a detection signal that can be visually recognized by the naked eye can be given using the secondary signal. That is, an antigen-antibody reaction or a biotin-avidin reaction can be used as a detection system.

In order to obtain a detection signal that can be visually recognized using the secondary signal, the secondary signal is preferably an enzyme that produces a chromogenic product. By doing so, it is possible to detect the chromogenic product promptly and in real time using an enzyme reaction. In this case, the secondary signal can be a complex comprising an antibody that specifically binds to the hapten and such an enzyme.

The secondary signal does not necessarily include such an enzyme, and may simply be an antibody that binds to a primary signal such as a hapten. In this case, a complex of a secondary antibody and an enzyme that specifically binds to the antibody that is the secondary signal may be used as the tertiary signal. For detection by such multiple signals, a well-known technique in ELISA or biotin-avidin system can be used.

The signal imparting step is preferably performed in the same container used in the hybridization step. By doing so, the operation can be simplified and the speed can be improved. Further, by leaving the array in the same container, it is possible to easily maintain a certain directionality when the array is put into the container (in the hybridization step). For example, the labeling reaction can be carried out by introducing a solution containing a secondary signal into the same container in which the array after removal of the excess sample remains. Except for the case where the secondary signal can be visually recognized with the naked eye after washing after removing the excessive secondary signal, a color reaction for developing the secondary signal is further performed. For example, when an enzyme such as peroxidase is used as the secondary signal, after removing the excess peroxidase, a substrate is supplied to cause an enzyme reaction. If a tertiary signal is required, additional washing and reaction steps are performed.

(Detection process)
The detection step can be a step of detecting the target polynucleotide based on the detection signal imparted to the double-stranded hybridization product in the signal imparting step. As described above, since the detection signal is imparted to the double-stranded hybridization product, this is observed with the naked eye to detect the presence or absence of the target polynucleotide.

Detecting 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 liquid for signal provision is thrown in. In the signal application step, the same orientation as that used at the time of detection is detected when detecting the detection signal in the array by maintaining the orientation of the array in the same container as that used in the hybridization step. Therefore, the target polynucleotide can be easily detected and the accuracy is high.

When the array has a color sample area, it is possible to detect the color tone and the contrast in a quick and highly accurate manner by observing the color tone and the contrast. In addition, when the array includes at least two color development sample regions corresponding to the upper and lower limits of the color tone or shading of the detection signal, it can be a step of detecting the target polynucleotide using the upper limit and the lower limit 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 polynucleotides in the test 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 the type determination of causative bacteria of pneumonia, a combination of a plurality of immobilization regions corresponding to a plurality of target polynucleotides is 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.

Although the hybridization step and the signal imparting step are preferably performed in the same container, the present invention is not limited to this and may be performed in different containers. The test sample preparation process is the same. For example, an appropriate liquid may be prepared and arranged in different containers in advance for each process or for each finer step, and the array may be moved according to the reaction process.

(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 polynucleotide, and a plurality of array regions including a plurality of immobilized regions of oligonucleotide probes previously associated with the target polynucleotide are separable for each array region. An individual 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.

Hereinafter, the present invention will be specifically described by way of examples. However, the following examples illustrate the present invention and do not limit the scope of the present invention.

(Production of array)
A DNA probe solution consisting of a base sequence shown in the following table is made on a sheet made of polyethersulfone modified with an aldehyde group and having a size of 285 mm × 50 mm, a pore diameter of 0.5 μm, and a thickness of 0.15 μm. Spotting was performed using a GENSHOT (registered trademark) spotter using a discharge unit (inkjet method) described in Japanese Patent Application Laid-Open No. 2003-75305, and the spot was fixed by the following procedure.

(DNA probe solution)
Forty-four oligo DNAs synthesized and dissolved in Tris-EDTA buffer were mixed with SSC buffer and bromophenol blue to prepare a DNA probe solution having a DNA probe concentration of 2 to 60 μM.

 

(Procedure 1)
Before injecting the DNA probe solution into the liquid injection portion arranged in the discharge unit and spotting it on the sheet, an inspection spot was formed on the inspection sheet. For quality inspection, no spot is formed on the inspection spot, the spot is not distorted, the spot diameter is not deviated by more than 10% from the design value, or other than this spot called satellite is unnecessary The items of whether a spot was not generated or whether the spot position was shifted by more than one third of the spot diameter were implemented. Since the color of the sheet was white and the DNA probe solution was colored blue with bromophenol blue, the inspection was performed visually. Bromophenol blue is eluted and removed in hybridization, antibody-immune reaction with enzyme, and enzyme coloring pigmentation.

(Procedure 2)
When a defect is detected at the inspection spot, the inspection spot is performed again by adjusting the drive signal of the piezoelectric / electrostrictive element arranged in the ejection unit. The drive signal was adjusted by changing the voltage value, the rising time to the predetermined voltage, the keeping time of the predetermined voltage value, and the voltage falling time. If a defect is still detected, the DNA probe solution is extracted from the discharge unit by vacuum suction, the DNA probe is injected again into the liquid injection section, and an inspection spot is performed. This operation was repeated until no defective spot was detected.

(Procedure 3)
After confirming that no defective spots were detected, oligonucleotide probes were plotted on a sheet as shown in FIG. 1 by the following method. That is, for one type of DNA probe, the spot pitch is 0.05 mm in both the horizontal direction (X direction) and the vertical direction (Y direction), and nine spots are formed in both the horizontal direction and the vertical direction, and 0.5 mm in the horizontal direction. One immobilization area having a square shape of 0.5 mm in the vertical direction was formed. The corner of the immobilization region was R = 0.05 mm. Further, while the discharge unit moved on the sheet in the longitudinal direction (X direction) of the sheet, the sheet moved in the lateral direction (Y direction) of the sheet, and spotted without contact. 47 immobilization regions were formed and arranged from 47 types of DNA probe solutions in a 5 × 10 grid area with a square pitch of 0.6 mm in both the vertical and horizontal directions, and a plurality of immobilization regions were formed. One section (5.9 mm × 2.9 mm) was formed. Furthermore, 120 sections were formed on one sheet at a pitch of 13.5 mm in the vertical direction and a pitch of 6.8 mm in the horizontal direction.

(Procedure 4)
A plurality of pigment-based inks were mixed to obtain a solution to be used for the spot of the color development sample so that the color tone was the same as that of the detection signal. Further, the solution obtained previously was diluted 2 times, 3 times, or 4 times so that the color density of the detection signal was the same as that of the detection signal. These three kinds of solutions are spotted in the center of the upper part of each section by using the method of (Procedure 1) to (Procedure 3) to form a color sample area for each section on the sheet. did.

(Procedure 5)
In order to enable identification of each array, a spot of “A01... A12, B01... J12” is performed as an ID number using a pigment-based magenta ink at the top of the color sample area. It was.

(Procedure 6)
After the DNA probe solution, color sample, and ID number spot are completed,
The DNA probe solution was fixed, the color sample and the ID number were fixed.

(Procedure 7)
The inspection was performed using the same items as the inspection performed in (Procedure 1).

(Procedure 8)
About the sheet | seat in which the defect was not detected, 1 array was cut | disconnected with the roller cutter to 4 mm x 9 mm size (aspect ratio: 2.25), and 120 arrays were obtained from 1 sheet.

In this example, the application of this array to discriminate four types of Mycobacterium tuberculosis (BGC vaccine strain, Beijing family, LAM9, EAI2) was evaluated. A genome extraction sample was prepared according to a conventional method from specimens that were previously discriminated by these four types of fungi, and this sample was applied to the array prepared in Example 1 in the following steps. FIG. 2 shows an outline of the process.

(Gene amplification process)
Four types of genome extraction samples were taken in an Eppendorf tube, and using a DR sequence recognition primer, PCR was performed by a conventional method using DIG label NTP (PCR DIG labeling mix, manufactured by Roche) to obtain an amplification product solution.

(Modification process)
To each tube, 200 μl of 0.5% Tween 20, 1% BSA in PBS as a hybridization solution was added, mixed well, placed in a heat block heated to 95 ° C. for 5 minutes, then transferred to ice water and quenched. .

(Hybridization process)
Each of the arrays prepared in Example 1 was put into each tube so that the color sample area was on top, and inserted into a heat block controlled at 60 ° C., and hybridization was carried out for 15 minutes.

(Washing process)
The solution used in the hybridization step was once removed, 200 μl of hybridization solution was newly added to the array in the tube, and the hybridization solution was removed by heating in a heat block at 60 ° C. for 1 minute. Thereafter, the hybridization solution was changed in the same manner, and washing was repeated for 10 minutes and 1 minute, respectively.

(Signal giving process)
200 μl of a 1000-fold diluted solution of an anti-DIG antibody enzyme (peroxidase) -containing reagent (DIG-POD, manufactured by Roche) was supplied to the array in each tube, and the enzyme was bound by an antigen-antibody reaction.

(Washing process)
After removing the solution used in the signal imparting step, adding 250 μl of hybridization solution to the array in the tube and heating it in a stationary state for 1 minute in a heat block at 60 ° C. to remove the hybridization solution Further, in the same manner, the hybridization solution was changed, and washing was repeated for 10 minutes and 1 minute, respectively.

(Substrate reaction process)
200 μl of peroxidase substrate solution (manufactured by Vec. Lab, TMB kit) was supplied to the washed tube, and an enzyme reaction was performed at room temperature to produce a blue reaction product.

(Detection process)
When the color development pattern of the immobilization region was confirmed in the reaction solution in each tube, four types of Mycobacterium tuberculosis, BGC vaccine strain, Beijing family, LAM9, and EAI2 could be confirmed from each tube array. In confirming these four types, it is possible to easily and quickly determine whether the detection signal of each immobilization region can affirm the presence of the target polynucleotide by referring to the color development sample region. did it. Further, since the aspect ratio of the array was about 2, and the width was 4 mm × the length was 9 mm, each operation could be easily performed while maintaining the directionality. Furthermore, since the plurality of immobilization regions are square-shaped and aligned in 5 rows × 10 columns, and the aspect ratio is about 2, the detection signals of the plural immobilization regions are combined. Thus, it was easy to discriminate the patterns obtained, and the four types of Mycobacterium tuberculosis could be quickly determined.

SEQ ID NOs: 1-44: Probe

Claims (21)

1. A method for detecting a target polynucleotide, comprising:
   Performing hybridization on the test sample and the oligonucleotide probe on the array comprising a plurality of immobilized regions of oligonucleotide probes pre-associated with the target polynucleotide on a solid phase carrier; and
   Providing a double-stranded hybridization product obtained by the hybridization with a detection signal that can be visually recognized by the naked eye;
   Detecting a target polynucleotide based on a detection signal obtained from the immobilized region;
   With
   The detection method, wherein the hybridization step is a step of performing hybridization in a solution of 1 ml or less for each array using the array.
2. In the hybridization step, the immobilization region has a size of 0.2 mm ² or more and 150 mm ² or less, and the solid phase carrier has a planar area of 150 mm ² or less and an aspect ratio of 1.5 or more. The detection method according to claim 1, wherein hybridization is performed using the array.
3. The hybridization step is a step of performing hybridization in a solution of 0.3 ml or less for each array using the array in which the solid phase carrier is a sheet-like body having a plane area of 50 mm ² or less. The detection method according to claim 1 or 2.
4. The detection method according to any one of claims 1 to 3, wherein the aspect ratio is 20 or less.
5. The detection method according to any one of claims 1 to 4, wherein the array is separately provided with a color development sample region by the detection signal on the solid phase carrier.
6. The detection method according to claim 5, wherein the color sample area is formed of a pigment-based ink.
7. The detection method according to claim 5 or 6, wherein the array includes two or more kinds of the color sample regions having different degrees of color tone or shading of a detection signal.
8. The array includes 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,
   The detection method according to claim 7, wherein the detection step is a step of detecting the target polynucleotide using the upper limit and the lower limit as indices.
9. 9. The detection method according to claim 1, wherein the solid phase carrier has liquid permeability or permeability.
10. The detection method according to claim 9, wherein the solid phase carrier is selected from polyethersulfone, nitrocellulose, nylon, polyvinylidene fluoride, and filter paper.
11. The detection method according to any one of claims 1 to 10, wherein the thickness of the solid phase carrier is 0.01 mm or more and 0.3 mm or less.
12. The detection method according to any one of claims 1 to 11, wherein the array includes a handling portion at a predetermined part of an outer edge of the plurality of immobilization regions.
13. The detection method according to any one of claims 1 to 12, wherein the hybridization step and the signal imparting step are performed while maintaining the state in which the array is put in the same tube-like container with a predetermined direction.
14. Furthermore, the detection method according to claim 13, wherein the detection step is performed while maintaining the state where the array is put into the container with a predetermined direction.
15. The detection method according to claim 13 or 14, further comprising a step of preparing a test sample by a gene amplification reaction in the container prior to the hybridization step.
16. The detection method according to any one of claims 1 to 15, wherein the detection step is a step of detecting a plurality of the target polynucleotides based on a combination of detection signals obtained from a predetermined plurality of the immobilized regions.
17. The detection step is a step of detecting the combination in comparison with a coloring pattern of the immobilized region obtained on the array and a coloring pattern sample of the oligonucleotide probe corresponding to the combination. 16. The detection method according to 16.
18. The detection method according to any one of claims 1 to 17, wherein the number of the immobilized regions is 2 or more and 200 or less.
19. The detection method according to any one of claims 1 to 18, wherein the oligonucleotide probe is a probe having a base sequence which is a normal orthogonal sequence.
20. An array used in the detection method according to any one of claims 1 to 19,
    A plurality of immobilization regions of oligonucleotide probes pre-associated with the target polynucleotide and a color sample region that presents color development by a detection signal that can be visually recognized by the naked eye are provided on a solid phase carrier.
    The size of the immobilization region is 0.2 mm ² or more and 150 mm ² or less, and the solid phase carrier is a sheet-like body having a plane area of 150 mm ² or less and an aspect ratio of 1.5 or more and 20 or less. .
21. An array sheet for detecting a target polynucleotide comprising:
    Provided with a plurality of immobilized regions of oligonucleotide probes pre-associated with the target polynucleotide, a color sample region that presents color development by a detection signal visible to the naked eye, and a plurality of array regions on a solid phase carrier And
    The size of the immobilization region is 0.2 mm ² or more and 150 mm ² or less, the array region has a plane area of 150 mm ² or less, and an aspect ratio of 1.5 or more and 20 or less,
    The solid phase carrier is a sheet that is separable for each array region.

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