WO2017191715A1

WO2017191715A1 - Nucleic acid chromatography test instrument, nucleic acid chromatography test kit, and method of using nucleic acid chromatography test instrument

Info

Publication number: WO2017191715A1
Application number: PCT/JP2017/010554
Authority: WO
Inventors: 秀彦西塚; 充裕吉田; 恵美子小菅; 深澤　眞; 伊藤　好久
Original assignee: 東洋製罐グループホールディングス株式会社
Priority date: 2016-05-02
Filing date: 2017-03-16
Publication date: 2017-11-09
Also published as: JP2017200446A

Abstract

In accordance with the present invention, it is possible, when performing nucleic acid chromatography, to suppress contamination, and to reduce the amount of target-nucleic-acid-containing PCR reaction liquid used for detection so as to improve detection precision. Nucleic acid chromatography is performed using a nucleic acid chromatography test instrument that holds a gene testing strip 4 and uses the strip to detect target nucleic acids, the instrument comprising: an injector part 11 that is provided with a first injection mouth 111 into which a PCR tube 5 can be inserted, and a second injection mouth 112 into which a chromatography developer solution is injected; a developer part 12 that covers a detection region of the strip; a cutting-means-holding part 21 that supports a cutting means 3 for cutting open the tube; and a strip-holding part 22 for holding the strip.

Description

Nucleic acid chromatography inspection instrument, nucleic acid chromatography inspection kit, and method of using nucleic acid chromatography inspection instrument

The present invention relates to a nucleic acid chromatography technique, and in particular, a nucleic acid chromatography test instrument, a nucleic acid chromatography test kit, and a nucleic acid chromatography test instrument that detect a target nucleic acid using a strip for genetic testing on which a probe is immobilized. Regarding usage.

Conventionally, when performing nucleic acid chromatography, generally, a target nucleic acid is hybridized (complementarily bound) to a probe immobilized on a strip using a strip for genetic testing in which the probe is immobilized. The detection was performed.
Specifically, for example, genomic DNA is extracted from a sample to be tested, and a genetic test strip is immersed in a reaction solution containing an amplification product obtained by amplifying a target nucleic acid by a PCR (polymerase chain reaction) method or the like. Then, the target nucleic acid is detected by developing the amplification product on a strip and hybridizing with the probe, and visually confirming the labeling dye that binds to the amplification product hybridized with the probe.

At this time, the extracted genomic DNA was placed in a PCR tube together with a PCR reaction reagent and PCR was performed, and then a chromatographic developing solution containing a labeling dye was mixed in the PCR tube to color the amplification product. Then, as shown in FIG. 5, the gene test strip is immersed in the reaction solution in the PCR tube, the reaction solution is spread on the strip by capillary action, and the amplification product hybridized to the probe immobilized on the strip is used. It was done to check the colored lines shown.

However, such a conventional method has a problem that there is a risk of contamination because the amplification product is spread in an open form.
As a technique for suppressing such contamination, an analyte detection device described in Patent Document 1 can be exemplified. According to this device, the amplification product can be spread on a strip in a device configured to be a semi-closed system.
Specifically, by inserting a container containing a liquid sample into the cavity of the device, the lower part of the container is pierced so that the sample in the container is released to reach the strip and deployed. Such a device can be suitably used when the conventional nucleic acid chromatography protocol described above is performed.

Special table 2015-512250 gazette

By the way, in the method using such a device, since the amplification product by PCR or the like is colored and then developed on the strip, there is a problem that the coloring is unstable and the detection accuracy may not be sufficient. . For this reason, it is necessary to use a relatively large amount of PCR reaction solution, and there is a problem that a large amount of PCR reaction reagent is required.

As a method for solving such a problem, after the PCR reaction solution containing the amplification product is first developed on the strip, the chromatography development solution containing the labeling dye is later developed on the strip, and the amplification product hybridized with the probe. It is conceivable to use a coloration protocol for. According to such a protocol, the probe and the amplification product can be hybridized first, and then the labeling dye can be bound to the amplification product, so that the accuracy of hybridization can be further improved and stable coloring can be achieved. As a result, the detection accuracy can be improved.
However, the above-described conventional device is not suitable for the case where the PCR reaction solution and the chromatographic developing solution are separately developed, and it has been difficult to use in such a protocol.

Therefore, the present inventors have conducted intensive research and can perform nucleic acid chromatography in a semi-sealed system, and after developing the PCR reaction solution on the strip, the chromatography developing solution is separately developed on the strip. The present invention has been completed by developing a nucleic acid chromatography test instrument that can be used.
That is, the present invention can suppress contamination, reduce the amount of a PCR reaction solution containing a target nucleic acid used for detection, and improve the detection accuracy, a nucleic acid chromatography test instrument, a nucleic acid chromatography test kit, And it aims at providing the usage method of a nucleic acid chromatography test | inspection instrument.

In order to solve the above problems, a nucleic acid chromatography test instrument of the present invention is a nucleic acid chromatography test instrument that holds a strip for genetic testing and detects a target nucleic acid using the strip, and inserts a PCR tube. An injecting portion having a first inlet possible and a second inlet for injecting a chromatographic developing solution, a developing portion covering the detection region of the strip, and an incision supporting an incision means for incising the tube A means supporting portion and a strip holding portion for holding the strip are provided.

The nucleic acid chromatography test kit of the present invention comprises the above-described nucleic acid chromatography test instrument, at least a PCR reaction reagent containing a primer mix comprising one or more primer sets, at least a labeling dye and a buffer solution. And a chromatographic developing solution.

In addition, the method for using the nucleic acid chromatography test instrument of the present invention includes the step of inserting the tube in which the reaction liquid containing the target nucleic acid is sealed into the first inlet of the nucleic acid chromatography test instrument. A probe having a complementary base sequence immobilized on the strip, by incising the tube by means, allowing the reaction solution to permeate the strip from the tube, and developing the strip. There is a method in which after a predetermined time has elapsed, a chromatographic developing solution is injected from the second inlet, and the labeling dye contained in the developing solution is bound to the target nucleic acid hybridized to the probe. .

According to the present invention, in performing nucleic acid chromatography, contamination can be suppressed, the amount of PCR reaction solution containing the target nucleic acid used for detection can be reduced, and detection accuracy can be further improved.

It is a perspective view which shows the external appearance of the nucleic acid chromatography test | inspection instrument which concerns on embodiment of this invention. It is the top view and side view of the nucleic acid chromatography test | inspection instrument which concern on embodiment of this invention. It is the top view and side view of the bottom cover of the nucleic acid chromatography test | inspection instrument which concern on embodiment of this invention. It is a side view of the incision means of the nucleic acid chromatography test | inspection instrument which concerns on embodiment of this invention. It is a figure which shows using a PCR tube and a genetic test strip in the conventional nucleic acid chromatography method.

Hereinafter, preferred embodiments of a method for using a nucleic acid chromatography test instrument, a nucleic acid chromatography test kit, and a nucleic acid chromatography test instrument of the present invention will be described with reference to the drawings.
First, the nucleic acid chromatography protocol applied in the method for using the nucleic acid chromatography test instrument, the nucleic acid chromatography test kit, and the nucleic acid chromatography test instrument of the present embodiment will be described.
In the nucleic acid chromatography, the target nucleic acid moves (develops) in the genetic test strip in a certain direction, so that it is captured by the probe spotted on the strip, and at the spot where the probe is immobilized, By being concentrated, color development up to a level that can be visually observed by the labeling dye bound to the target nucleic acid is obtained, and the target nucleic acid is detected.

In the nucleic acid chromatography protocol applied in the present embodiment (hereinafter sometimes referred to as the present application protocol), first, the amplification process of the target nucleic acid is performed by the PCR method or the like. That is, the amplification product by the PCR method or the like consists of a target nucleic acid.
Specifically, a PCR reaction reagent containing a primer set for amplifying the target nucleic acid in the genomic DNA of the organism to be examined is injected into the PCR tube. As the primer set, a primer mix comprising one or more primer sets can be used, and multiplex PCR for simultaneously amplifying a plurality of target nucleic acids can also be performed.
The PCR reaction reagent includes a primer set, a nucleic acid synthesis substrate (for example, dNTPmixture (dCTP, dATP, dTTP, dGTP)), a nucleic acid synthase (for example, EX Taq HotStart DNA polymerase), a buffer, and the like. .
The PCR tube is injected with such a PCR reaction reagent, genomic DNA extracted from a biological sample, and a solution containing water as the remaining component, and this solution is used as a reaction solution used for PCR.

Genomic DNA can be extracted by a general method such as a method using a CTAB method (Cetyl trimethyl ammonium bromide) or a method using a DNA extraction apparatus.
Moreover, a general thermal cycler etc. can be used as a PCR apparatus. The PCR reaction can be performed, for example, under the following reaction conditions, but can be performed with appropriate changes, and the whole may be performed in about 15 to 30 minutes.
(A) 95 ° C. 2 minutes, (b) 95 ° C. (DNA denaturation step) 10 seconds, (c) 60 ° C. (annealing step) 20 seconds, (d) 72 ° C. (DNA synthesis step) 90 seconds ((b) to (D) 40 cycles), (e) 72 ° C. for 2 minutes

Next, a PCR reaction solution containing an amplification product (target nucleic acid) obtained by PCR is developed on a genetic test strip.
The genetic test strip is an elongated member on which a probe for capturing an amplification product is immobilized, and is formed so that the solution penetrates the entire member by capillary action and is easily developed. For this reason, cellulose such as cellulose membrane is preferably used as the strip member. Moreover, in order to give the strip strength and facilitate handling, a material having a resin adhered to one surface thereof can be suitably used.

In a genetic test strip, one or two or more probes are penetrated (impregnated) with a detection region fixed in a line in the short direction of the strip and a PCR reaction solution or a chromatographic development solution at a specific position of the strip. And a development start area which is a part where development starts. It should be noted that the deployment start area may be a very small part of the strip.

In the detection region, one or more regions (spots) to which the probe is immobilized are formed (spotted) in a line shape in the short direction of the strip. The number of lines on which the probes are immobilized is not particularly limited, and for example, a line on which 8 to 12 lines or the like are formed can be suitably used.
When, for example, a blue labeling dye is bound to the amplification product hybridized to the probe immobilized in such a line shape, the blue line can be visually detected on the detection region of the strip. Further, in order to make it possible to clearly confirm the position of the line on which the probe is immobilized, other color lines, for example, red lines, are preferably arranged as positioning markers in the detection region. The labeling dye is not particularly limited. For example, a labeling dye using an avidin / biotin system can be preferably used.

The development start region is a portion where the PCR reaction solution and the chromatographic development solution are first soaked. The development start area is continuously formed adjacent to the detection area, and the liquid soaked in the development start area develops to the detection area by capillary action.
It is also preferable to continuously form the water absorption promotion region adjacent to the detection region on the side opposite to the development start region so that the liquid can easily penetrate into the strip. The water absorption promotion region can be configured by, for example, laminating a member such as a water absorption pad or cellulose on the strip, and the water absorption of the water spread on the strip can be promoted by such a water absorption member. In addition, the strip can be made easier to handle by forming a region in which the thickness is increased by laminating members on the strip.

First, the PCR reaction solution is infiltrated into the development start region in such a genetic test strip. The PCR reaction solution is developed from the development start region to the detection region. When an amplification product having a base sequence complementary to the probe immobilized on the detection region is contained in the PCR reaction solution, the probe and the amplification product are hybridized.
Similarly, when multiple types of probes are individually immobilized on a strip in a line shape, amplification products that hybridize to each probe are obtained by multiplex PCR and included in the PCR reaction solution. The corresponding probe and the amplified product are hybridized.

Next, a chromatographic developing solution is impregnated into the development start region of the genetic test strip.
The chromatographic developing solution is desirably soaked after the PCR reaction solution has sufficiently developed to the detection region or the water absorption promoting region. This makes it possible to sufficiently soak the chromatography developing solution into the detection region. The chromatography developing solution is preferably soaked into the development start region after, for example, about 5 to 10 minutes have elapsed after the PCR reaction solution has been developed into the detection region.

The chromatographic developing solution contains at least a labeling dye (colored beads such as those produced by an avidin / biotin system) and a buffer solution. This labeling dye can bind to the amplification product. The developing solution is developed from the development start region to the detection region. The labeling dye contained in the developing solution binds to the amplified product hybridized with the probe, whereby a colored line appears at the position where the probe is immobilized.
Thus, the target nucleic acid can be detected by confirming the colored line on the strip.

According to such a protocol for nucleic acid chromatography, after a PCR reaction solution containing an amplification product is first developed on a strip, a chromatographic development solution containing a labeling dye is later developed on the strip and hybridized with the probe. The amplified product can be colored. For this reason, a colored line can be obtained stably and detection accuracy can be improved.
This is because in this application protocol, the probe and the amplification product are hybridized first, and then the labeling dye is bound to the amplification product. Therefore, the labeling dye is bound to the amplification product prior to the hybridization. This is considered to be because the steric hindrance of hybridization assumed in the case of the reaction is suppressed, and the accuracy of hybridization can be further improved.

Next, an outline of the nucleic acid chromatography test instrument of the present embodiment will be described with reference to FIG. FIG. 1 shows a perspective view of the nucleic acid chromatography test instrument of the present embodiment.
As shown in the figure, the nucleic acid chromatography test instrument of the present embodiment includes a first inlet 111 into which the PCR tube 5 can be inserted, and a second inlet 112 for injecting a chromatography developing solution. The injection part 11 provided, the development part 12 covering the detection region in the genetic test strip 4, the incision means support part 21 for supporting the incision means 3 for incising the PCR tube 5, and the strip for holding the genetic test strip 4 A holding part 22 is provided.
Further, the nucleic acid chromatography test instrument of this embodiment has a main body 1 having an injection part 11 and a developing part 12, a cutting means support part 21 and a strip holding part 22, and is fitted to the main body part 1. A configuration including the bottom lid 2 is also preferable.

Moreover, it is also preferable that the nucleic acid chromatography test instrument of the present embodiment is formed by integral molding instead of separately configuring the main body 1 and the bottom lid 2.
In this case, it is preferable to provide a slit into which the genetic test strip 4 can be inserted on any wall surface of the test instrument. The slit can be provided, for example, on any of the four side walls of the inspection instrument. Then, the genetic test strip 4 is inserted into the test instrument through the slit and disposed in the strip holding unit 22. FIG. 1 shows such a nucleic acid chromatography test instrument.

Thus, in the nucleic acid chromatography test instrument of this embodiment, the first inlet 111 into which the PCR tube 5 can be inserted and the second inlet 112 for injecting the chromatographic developing solution are separately provided. It is configured. For this reason, it is possible to suitably perform this application protocol in which the above-described PCR reaction solution is first developed on the strip, and then the chromatography development solution containing the labeling dye is later developed on the strip.

Further, the first injection port 111 and the second injection port 112 are formed in the longitudinal direction of the strip above the development start region in the genetic test strip 4. For this reason, the PCR tube 5 is inserted from the first inlet 111 and the PCR tube 5 is incised by the incision means 3, so that the reaction solution can be infiltrated into the strip from the PCR tube 5 and developed. By injecting a chromatographic developing solution from the second inlet 112 using a pipette or the like, the labeling dye is infiltrated into the strip and developed, and the amplified product that has been developed and hybridized to the probe A labeling dye can be attached. For this reason, it is possible to suitably perform the protocol applied to the present embodiment.

The arrangement order of the first inlet 111 and the second inlet 112 is not limited to that shown in FIG.
Further, by forming the first inlet 111 and the second inlet 112 side by side in the longitudinal direction of the genetic test strip 4 in this way, a linked nucleic acid chromatography test corresponding to a linked PCR tube described later is performed. It is also possible to make it suitable for constructing an instrument.

Moreover, in the nucleic acid chromatography test instrument of this embodiment, it is preferable that the detection region of the genetic test strip 4 can be easily confirmed visually. For this reason, it is preferable that at least one of the development part 12 covering the detection region in the genetic test strip 4 or the strip holding part 22 for holding the genetic test strip 4 is made of a transparent member. Of course, all of the injection part 11, the development part 12, the incision means support part 21, and the strip holding part 22 in the nucleic acid chromatography test instrument of the present embodiment can be made of a transparent member. As such a transparent member, for example, an acrylic resin such as polystyrene, polycarbonate, polyethylene terephthalate (PET), methyl methacrylate (MMA), or the like can be suitably used.

According to the nucleic acid chromatography test instrument of this embodiment, the nucleic acid chromatography method can be performed with the above-described application protocol using the general genetic test strip 4 and the PCR tube 5 with higher accuracy. It becomes possible to carry out.

Next, with reference to FIGS. 2 to 4, a nucleic acid chromatography test instrument in which the main body 1 and the bottom cover 2 are separately constructed will be described in detail. FIG. 2 is a plan view and a side view of the nucleic acid chromatography test instrument according to the present embodiment, FIG. 3 is a plan view and a side view of the bottom lid of the test instrument, and FIG. It is a side view of an incision means.

As shown in FIG. 2, the nucleic acid chromatography test instrument of the present embodiment includes a main body portion 1 having an injection portion 11 and a development portion 12 having a first injection port 111 and a second injection port 112, and a PCR tube. 5 has an incision means support portion 21 for supporting the incision means 3 for incising 5 and a strip holding portion 22 for holding the genetic test strip 4, and a bottom lid 2 fitted to the main body portion 1. .
Further, the incision means support portion 21 is provided so that at least a part of the incision means 3 is disposed in the first injection port 111.

The PCR tube 5 is filled with an amplification product (target nucleic acid) obtained by PCR. After performing PCR, the PCR tube 5 is pushed into the first inlet 111 without opening the lid.
At this time, the PCR tube 5 is cut, perforated, incised, or the like by the incision means 3, and a crack or an opening is generated at the bottom or side of the PCR tube 5. Then, the reaction solution in the PCR tube 5 flows into the genetic test strip 4. Thereby, the PCR reaction solution soaks into the genetic test strip 4 and develops.

As described above, the nucleic acid chromatography test instrument of the present embodiment can develop the reaction solution into a strip by opening a crack in the bottom or side surface of the PCR tube 5 without opening the lid. At this time, the inspection instrument inserts the PCR tube 5 into the first inlet 111, thereby closing the opening of the first inlet 111 and opening only the second inlet 112. It has become. For this reason, according to the nucleic acid chromatography test instrument of this embodiment, it is possible to reduce the risk of contamination.
In addition, the size of the opening of the second inlet 112 is preferably smaller than the size of the opening of the first inlet 111. As a result, the risk of contamination can be further reduced.

The shape of the first inlet 111 is not particularly limited, and can be formed, for example, in a substantially thin conical column or elliptical truncated cone, and the horizontal plane opening of the first inlet 111 is, for example, a perfect circle. Can be formed. Similarly, the shape of the second injection port 112 is not particularly limited, and can be formed, for example, in a substantially thin conical column having a narrow shape. The horizontal plane opening of the second injection port 112 is, for example, a perfect circle. It can be formed as follows.

In addition, the first inlet 111 can be formed so that the central axis of the first inlet 111 is inclined with respect to the vertical direction. For example, the central axis is inclined in the longitudinal direction of the genetic test strip 4 to the opposite side with respect to the detection region to form the first injection port 111, and the PCR tube 5 is obliquely inserted into the inclined first injection port 111. The incision position of the PCR tube 5 by the incision means 3 may be adjusted as appropriate.
Similarly, the second inlet 112 can be formed so that the central axis of the second inlet 112 is inclined with respect to the vertical direction.

Moreover, it is preferable to form the horizontal plane opening of the first inlet 111 so as to have a track shape (a shape similar to a track of an athletics), for example, two opposing linear sides are opposed to each other. It is preferable to use a semicircular shape (first shape). In addition, the track shape may include a shape (second shape) formed by two semi-circles that are convex on the inner side facing two opposite sides.

When the first inlet 111 is formed in this way, when the PCR tube 5 is pushed in and incised by the incision means 3, the PCR tube 5 becomes linear as the PCR tube 5 is pushed into the first inlet 111. Cracks that are compressed and incised by the two sides of the surface (in the case of the first shape) or by two semicircular surfaces convex inward (in the case of the second shape) As a result, the reaction solution can be easily opened out from the PCR tube 5.

It is also preferable that the first inlet 111 and the second inlet 112 have different shapes, sizes, heights, and the like. By making the first inlet 111 and the second inlet 112 different in this way, for example, the second inlet 112 is sized so that the PCR tube 5 cannot be inserted, so that the PCR tube 5 can be inserted incorrectly. It becomes possible to prevent.

In the nucleic acid chromatography test instrument of the present embodiment, an aluminum seal can be attached to the first inlet 111 and / or the second inlet 112 and peeled off during use.

As shown in FIG. 3, the strip holder 22 is preferably formed with a positioning column 221 composed of a plurality of protrusions for fixing the genetic test strip 4. The number thereof is not particularly limited, but it is preferable to provide a plurality of oppositely on the bottom lid 2 so that the genetic test strip 4 does not move in the nucleic acid chromatography test instrument of the present embodiment. In the example of the figure, six sets of twelve positioning columns 221 are formed.

The incision means 3 is not particularly limited as long as it can incise the PCR tube 5 in the first injection port 111. As the incision means 3, for example, a metal blade, a metal needle, a plastic blade, a plastic needle or the like can be used. Moreover, when forming with a plastic, it is also preferable to integrally mold with the incision means support part 21.

When a cutting tool such as a metal blade or a plastic blade is used as the incision means 3, for example, as shown in FIG. 4, an incision blade 31 for incising the PCR tube 5 inserted into the first inlet 111, and a gene It is preferable to have a strip contact portion 32 that contacts the test strip 4.
When the strip contact portion 32 is provided on the metal blade or the like in this way, the PCR reaction liquid flowing out from the cut PCR tube 5 can move to the genetic test strip 4 through the strip contact portion 32 and easily penetrates into the strip. Therefore, the development on the strip can be performed more reliably.

In the nucleic acid chromatography test instrument of this embodiment, the injection part 11 is formed in a substantially rectangular parallelepiped shape, and the development part 12 is formed thin and long enough to hold the genetic test strip 4 inside. By forming the injection part 11 and the development part 12 in this way, the volume of the inspection instrument can be reduced. However, the shapes of the injection part 11 and the development part 12 are not limited to these, and the injection part 11 can be formed in, for example, a substantially elliptical column shape, or the development part 12 can be formed thicker. .

Next, a test instrument having a configuration in which a plurality of nucleic acid chromatography test instruments described above are connected will be described.
The nucleic acid chromatography test instrument of this embodiment is arranged in a state where a plurality of the above-described nucleic acid chromatography test instruments are coupled in the short direction of the genetic test strip 4, and the same number as the nucleic acid chromatography test instrument is connected. Each tube of the connection type tube is connected to the first inlet 111 in the nucleic acid chromatography test instrument so as to be simultaneously insertable.

The nucleic acid chromatography test instrument of this embodiment is preferably formed by connecting, for example, eight nucleic acid chromatography test instruments described above in the short direction. By using such a connected nucleic acid chromatography test instrument, eight PCRs are simultaneously performed using a commonly used 8-series PCR tube, and target nucleic acid detection is simultaneously performed for each PCR reaction solution. Can be done.

As described above, according to the linked nucleic acid chromatography test instrument, it is possible to reduce the time and effort of processing when performing a plurality of tests, and it is possible to reduce the risk of sample mix-up. Moreover, according to such a plurality of processes, it is possible to contribute to cost reduction.
Needless to say, the number of nucleic acid chromatography test instruments to be connected is not limited to eight, and may be other numbers such as four or twelve.

As described above, according to the nucleic acid chromatography test instrument of the present embodiment, it is possible to reduce the risk of contamination due to scattering of the PCR reaction solution as compared with the conventional open-type nucleic acid chromatography method. Instability of detection accuracy due to liquid volatilization or the like can be eliminated.
Further, according to the nucleic acid chromatography test instrument of the present embodiment, the PCR reaction solution is developed on the strip and then the chromatography development solution is developed on the strip, as compared with the conventional semi-sealed device described above. It can be made suitable for this application protocol, and detection accuracy can be further improved.
Furthermore, by using a linked nucleic acid chromatography test instrument, it is possible to reduce the risk of processing troubles, sample mistaking, and the like, and further contribute to cost reduction.

Further, the nucleic acid chromatography test kit of the present embodiment includes the above-described nucleic acid chromatography test instrument, a PCR reaction reagent including a primer mix consisting of at least one or two or more primer sets, at least a labeling dye and a buffer. It is preferable to include a chromatography developing solution containing a liquid.

If the nucleic acid chromatography test kit according to the present embodiment is configured as described above, PCR can be easily executed, the PCR reaction solution can be developed on the strip, and then the chromatography development solution can be developed on the strip. For this reason, the amplification product hybridized with the probe can be colored, and the detection accuracy can be improved.

As a method for using the nucleic acid chromatography test instrument of this embodiment, the PCR tube 5 in which the reaction solution containing the target nucleic acid is sealed is inserted into the first inlet 111 of the nucleic acid chromatography test instrument described above. The PCR tube 5 is incised by the incision means 3, the reaction solution is permeated into the genetic test strip 4 from the PCR tube 5, and the target nucleic acid in the reaction solution is immobilized on the genetic test strip 4. Hybridize with a probe having a complementary base sequence, and after a lapse of a predetermined time, inject a chromatographic developing solution from the second inlet 112, and hybridize the labeling dye contained in the developing solution to the probe. It is preferable to use a method of binding to the target nucleic acid.

If the method of using the nucleic acid chromatography test instrument of this embodiment is configured as described above, the PCR reaction solution can be easily developed from the PCR tube to the strip first using the semi-enclosed nucleic acid chromatography test instrument. Can do. Then, this can be developed into a strip by injecting a chromatographic developing solution.
For this reason, after first hybridizing the amplification product to the probe on the strip, a labeling dye is bound to this amplification product, and the target nucleic acid is detected by visually recognizing the colored line indicated by the labeling dye. can do. Therefore, the accuracy of hybridization can be further improved, stable coloring is performed, and the detection accuracy can be improved.

While the present invention has been described with reference to the preferred embodiment, it is needless to say that the present invention is not limited to the above-described embodiment, and various modifications can be made within the scope of the present invention.
For example, a plurality of genetic test strips can be held in a single nucleic acid chromatography test instrument, and the test instrument can be appropriately changed to have a first inlet and a second inlet corresponding to each. Is possible.

The present invention can be suitably used for performing nucleic acid chromatography in order to suppress contamination and improve detection accuracy.
The contents of the documents described in this specification and the specification of the Japanese application that is the basis of Paris priority of the present application are all incorporated herein.

DESCRIPTION OF SYMBOLS 1 Main-body part 11 Injection | pouring part 111 1st injection | pouring port 112 2nd injection | pouring port 12 Expanding part 2 Bottom cover 21 Cutting means support part 22 Strip holding | maintenance part 221 Positioning column 3 Cutting means 31 Cutting blade 32 Strip contact part 4 Genetic test strip 5 PCR tubes

Claims

A nucleic acid chromatography test instrument for holding a strip for genetic testing and detecting a target nucleic acid by the strip,
An injection portion having a first injection port into which a PCR tube can be inserted and a second injection port for injecting a chromatographic developing solution, a development portion covering the detection region of the strip, and incising the tube A nucleic acid chromatography test instrument comprising an incision means support part for supporting the incision means, and a strip holding part for holding the strip.
A main body having the injection part and the development part;
The nucleic acid chromatography test instrument according to claim 1, further comprising a bottom cover that has the incision means support part and the strip holding part and fits with the main body part.
The nucleic acid chromatograph according to claim 1 or 2, wherein the first inlet and the second inlet are arranged in the longitudinal direction of the strip above the development start region of the strip. Graphy inspection instrument.
The nucleic acid chromatograph according to any one of claims 1 to 3, wherein the incision means support portion is provided so that at least a part of the incision means is disposed in the first inlet. Graphy inspection instrument.
5. The nucleic acid chromatography test instrument according to claim 1, wherein at least the development part and / or the strip holding part is made of a transparent member.
6. The cutting device according to claim 1, wherein the cutting means has a cutting blade for cutting the tube inserted into the first inlet, and a strip contact portion that contacts the strip. Nucleic acid chromatography instrument.
The nucleic acid chromatography test instrument according to any one of claims 1 to 6, wherein an opening having a horizontal cross section of the first injection port is formed in a track shape.
The nucleic acid chromatography test instrument according to any one of claims 1 to 7, wherein the strip holder has a positioning column composed of a plurality of protrusions for fixing the strip.
A plurality of nucleic acid chromatography test instruments according to any one of claims 1 to 8, wherein a plurality of connected tubes are arranged in the short direction of the strip and connected in the same number as the nucleic acid chromatography test instrument. Each of the nucleic acid chromatography test instruments is connected to the first inlet of the nucleic acid chromatography test instrument so as to be simultaneously insertable.
A nucleic acid chromatography test instrument according to any one of claims 1 to 9,
A reaction reagent for PCR containing at least a primer mix comprising one or more primer sets;
A kit for nucleic acid chromatography testing, comprising: a chromatography developing solution containing at least a labeling dye and a buffer solution.
10. The tube filled with a reaction solution containing a target nucleic acid is inserted into the first injection port of the nucleic acid chromatography test instrument according to claim 1, and the tube is opened by the cutting means. And
By infiltrating the reaction solution from the tube into the strip and spreading it, the target nucleic acid in the reaction solution is hybridized with a probe having a complementary base sequence immobilized on the strip,
After a lapse of a predetermined time, a chromatographic developing solution is injected from the second inlet, and a labeling dye contained in the developing solution is bound to a target nucleic acid hybridized with the probe. How to use a graphy inspection instrument.