JP4347404B2 - Nucleic acid detection method - Google Patents

Description

  The present invention relates to a nucleic acid detection method and an apparatus therefor characterized by continuously performing a nucleic acid amplification reaction and analysis of amplification products under isothermal conditions on a microchip.

  As a technique for analyzing a minute amount of protein or nucleic acid, a microchip electrophoresis apparatus has been put to practical use (for example, Hitachi Microchip Electrophoresis Analysis System Cosmo Eye, Shimadzu Microchip Electrophoresis Apparatus MCE2010, etc.). In microchip electrophoresis, capillary electrophoresis is performed using a small chip in which a fine groove is formed on a plate substrate of several centimeters square and is bonded to another substrate to form a capillary. Detection of the nucleic acid after electrophoresis is usually performed by binding a fluorescent intercalator to the nucleic acid, irradiating it with fluorescence excitation light or laser light, and measuring the resulting fluorescence. Therefore, in microchip electrophoresis, a specific light source such as a fluorescence excitation light source or a laser light source and a detector are indispensable for detecting nucleic acid fragments.

  There is also known an integrated device that applies this microchip electrophoresis and continuously performs PCR amplification and electrophoresis analysis of a nucleic acid sample on the chip (see, for example, Patent Document 1 and Non-Patent Document 1). However, in PCR that amplifies nucleic acid by thermal cycling, it is difficult to selectively heat and cool only the amplification reaction site without affecting the adjacent electrophoresis site on the chip. Therefore, PCR on the chip has a problem in that heat stress due to repeated heating and cooling is applied to the chip, causing deformation and peeling of the bonded portion, thereby causing liquid leakage.

  On the other hand, the present inventors have succeeded in developing a LAMP method (Loop-mediated isothermal amplification), which is a new nucleic acid amplification method that does not require complicated temperature control that is indispensable in the PCR method (for example, Patent Document 2 and Non-patent document 2). In the LAMP method, amplification products having sequences complementary to each other at the upper end of the same strand are generated by using a specific primer and a strand displacement type DNA polymerase. The isothermal amplification reaction proceeds at a high amplification efficiency unprecedented by the extension reaction starting from the loop formed by annealing between the complementary sequences and the extension reaction by the primer that anneals to the loop.

Special table 2001-521622 International publication WO / 28082 pamphlet Woolly A.T. et.al., Analytical Chemistry 68 (23) 4081-4086 (1996) Notomi, T et al., Nucleic Acids Res. 28 (12): e63 (2000)

  An object of the present invention is to improve a conventional nucleic acid detection method by microchip analysis, and to provide a simple, reliable, and inexpensive method for performing nucleic acid amplification and analysis (especially electrophoresis analysis) on a microchip. .

  As a result of intensive studies to solve the above problems, the present inventors have thought that if the nucleic acid amplification process is performed under isothermal conditions, the entire chip can be heated without affecting the chip substrate. In addition, the inventors have found that nucleic acids can be detected visually by using an excess of intercalator than usual, and the present invention has been completed.

  That is, the present invention relates to a method for detecting a nucleic acid, which comprises performing a nucleic acid amplification reaction on a microchip under isothermal conditions, and subsequently analyzing the obtained amplification product on the microchip.

In certain embodiments, analysis of amplification products is performed using electrophoretic analysis.
In the above embodiment, the method of the present invention includes, for example, the following steps.
1) introducing a sample containing a target nucleic acid, a primer, a strand displacement polymerase and a substrate nucleotide into a sample well of a microchip;
2) a step of heating the microchip and performing a nucleic acid amplification reaction in the well under isothermal conditions; and 3) a step of subsequently electrophoresis of the amplification product on the microchip to separate and detect the target nucleic acid.

Furthermore, in another embodiment, the analysis of the amplification product may be a visual analysis in which an excess intercalator is added to the nucleic acid amplification product and the presence or absence of the target nucleic acid is visually detected by the resulting color change. Preferable examples of the intercalator include SYBR Green I.
In the method of the present invention, the nucleic acid amplification reaction under isothermal conditions is preferably performed using the LAMP method.

The present invention also provides a nucleic acid amplification and electrophoretic analysis integrated apparatus for carrying out the nucleic acid detection method of the present invention, comprising the following means.
1) Means for introducing a target nucleic acid, primer, strand displacement polymerase, and substrate nucleotide into the microchip;
2) Means for heating the chip to a constant temperature and performing a nucleic acid amplification reaction under isothermal conditions;
3) means for electrophoresis of the amplification product obtained by the amplification reaction on the chip; and
4) Means for detecting nucleic acids separated by electrophoresis.

  According to the present invention, the nucleic acid amplification reaction and analysis can be continuously performed on-chip without affecting the chip substrate. In the method of the present invention, the presence or absence of the target nucleic acid can be visually detected by adding an excess intercalator to the nucleic acid amplification product and observing the resulting color change. That is, the present invention enables simple, reliable, and inexpensive detection of trace nucleic acids by using the LAMP method with high amplification efficiency and microchip analysis suitable for the detection of trace nucleic acids.

Hereinafter, the present invention will be described in detail.
1. TECHNICAL FIELD The present invention provides a method for detecting a nucleic acid, which comprises performing a nucleic acid amplification reaction on a microchip under isothermal conditions, and subsequently analyzing the obtained amplification product on the microchip.
In the present invention, the analysis of the amplification product is preferably an electrophoretic analysis.

The method of the present invention specifically comprises the following steps:
1) introducing a sample containing a target nucleic acid, a primer, a strand displacement polymerase and a substrate nucleotide into a sample well of a microchip;
2) a step of heating the microchip and performing a nucleic acid amplification reaction in the well under isothermal conditions; and 3) a step of subsequently electrophoresis of the amplification product on the microchip to separate and detect the target nucleic acid.

1.1 Sample Introduction In the method of the present invention, first, a sample containing a target nucleic acid, a primer, a strand displacement polymerase, and a substrate nucleotide is introduced into a sample well of a microchip.
The “microchip” used in the method of the present invention is a microchip used for nucleic acid analysis, and preferably has a capillary between two substrates of several centimeters square, It means a microchip for electrophoresis for performing capillary electrophoresis by filling a gel or polymer. The substrate constituting the chip is not particularly limited, such as glass (quartz) or plastic. Since the quartz chip does not absorb in the ultraviolet region, it is suitable for measuring a wide region from the visible region to the ultraviolet region. However, in the present invention, when sealing wells described later, it is easier to bond using a plastic chip such as PMMA than a glass chip. Plastic chips are also excellent in that they are inexpensive. The microchip as described above may be manufactured based on a known method, but commercially available products (for example, manufactured by Hitachi or Hitachi Chemical Co., Ltd. or Shimadzu Corporation) can be suitably used.

  In the microchip, a sample introduction channel and a separation channel are usually provided so as to cross each other, and a sample well (reservoir) is provided at the end of the sample introduction channel. One end of the separation channel has an electrophoresis gel (polymer) or buffer well (reservoir), and the other end has a detector (see FIG. 1). In microchip electrophoresis of nucleic acids, the electrophoresis gel is first filled into a separation channel and a gel (polymer) or buffer well, and the sample is introduced into the sample well. When an appropriate voltage is applied to each reservoir, the sample in the sample well moves to the separation channel and is electrophoresed toward the other end.

  The order of introducing the target nucleic acid, primer, strand displacement polymerase, and substrate nucleotide into the sample well is not particularly limited, and may be introduced one by one in an appropriate order or all at the same time. If an amplification reaction solution containing the target nucleic acid, primer, strand displacement polymerase, and substrate nucleotide is prepared in advance and introduced into the well, the operation may be simple. The sample is introduced into the well according to the protocol attached to the product if it is a commercially available microchip.

  In the present invention, the “target nucleic acid” means a nucleic acid molecule to be detected. The “nucleic acid” may be natural or artificially synthesized and includes all of DNA, cDNA, RNA, mRNA, PNA and the like. It also includes both single-stranded nucleic acids and double-stranded nucleic acids. Further, even a nucleotide derivative partially modified or entirely composed of an artificial structure is included in the nucleic acid of the present invention as long as it can form a base pair bond. The term gene (nucleic acid carrying a specific function or information related to life) is also included in nucleic acid. The number of bases constituting the nucleic acid in the present invention is not limited.

  As the “primer” used in the method of the present invention, a primer suitable for the nucleic acid amplification reaction to be used is appropriately adjusted according to the sequence of the target nucleic acid. The primer design will be described in detail in the nucleic acid amplification method described later. As the “substrate nucleotide”, a commercially available dNTP or a modified product thereof may be used.

  Examples of the “strand displacement polymerase” used in the method of the present invention include Bst DNA polymerase, Bca (exo-) DNA polymerase, Klenow fragment of E. coli DNA polymerase I, Vent DNA polymerase, Vent (Exo-) DNA. Polymerase (Vent DNA polymerase minus exonuclease activity), DeepVent DNA polymerase, DeepVent (Exo-) DNA polymerase (DeepVent DNA polymerase minus exonuclease activity), Φ29 phage DNA polymerase, MS-2 phage DNA Polymerase, Z-Taq DNA polymerase (Takara Shuzo), KOD DNA polymerase (Toyobo), etc. may be used.

  If necessary, the well after sample introduction may be sealed with a seal. Sealing can be performed, for example, by attaching a seal such as a commercially available 96-hole PCR adhesive sheet or cellophane tape to the well. If the seal is transparent, it is preferable because the generation of bubbles due to heating can be confirmed. In particular, the 96-hole PCR adhesive sheet is excellent in that it can be easily peeled off, has adhesive strength, and can withstand heating.

1.2 Nucleic Acid Amplification Reaction under Isothermal Conditions Next, the microchip is heated, and a nucleic acid amplification reaction is performed in the wells under isothermal conditions. The method for heating the microchip is not particularly limited as long as the chip can be maintained at a constant temperature (about 65 ° C.) necessary for the nucleic acid amplification reaction. For example, the method of heating a chip | tip with a heat block is mentioned. In this case, the heat block may be applied to only one side of the chip. However, in order to heat the chip uniformly, it is better to apply heat to both the upper and lower surfaces.

  In the method of the present invention, since the nucleic acid amplification reaction is performed under isothermal conditions, the entire chip is heated to a constant temperature without affecting the chip substrate. Therefore, unlike the PCR, there is no fear of causing thermal stress due to repeated heating and cooling to the chip, causing deformation of the chip, peeling of the bonded portion, or liquid leakage.

  Examples of the nucleic acid amplification reaction used in the method of the present invention under isothermal conditions include the following SDA method and LAMP method. In particular, the LAMP method is suitable for detecting a small amount of nucleic acid because of its high amplification efficiency.

(1) SDA method
In the SDA method, when complementary strand synthesis is performed using a complementary primer on the 3 'side of a base sequence as a starting point, if a double-stranded region is present on the 5' side, the complementary strand is synthesized while replacing that strand. This is a method using a special DNA polymerase (strand displacement type DNA polymerase). In the SDA method, by inserting a restriction enzyme recognition sequence in advance into the annealed sequence as a primer, the temperature change step, which is essential in the PCR method, is omitted, and under isothermal conditions (around 65 ° C) Nucleic acid amplification can be realized. In other words, the nick produced by the restriction enzyme gives a 3′-OH group that is the starting point for complementary strand synthesis, and the complementary strand synthesized earlier is released as a single strand by performing strand displacement synthesis from there. Reused as a template for complementary strand synthesis.

(2) LAMP method
1) Overview of LAMP (Loop-mediated isothermal amplification) method The “LAMP method” is a nucleic acid amplification method developed by the present inventors. An inner primer pair or an outer primer pair and a loop primer pair are added to the inner primer pair. This is a method for rapidly and inexpensively amplifying DNA or RNA under isothermal conditions (around 65 ° C.) using two, four or six specific primers, a strand displacement polymerase and a substrate nucleotide. For an overview of the LAMP method, refer to: Notomi, T et al .: Nucleic Acids Res. 28 (12): e63 (2000), Patent: International Publication WO 00/28082, or Eiken Chemical Co., Ltd. website (http Please refer to http://www.loopamp.eiken.co.jp/).

  In the LAMP method, sequences complementary to each other are generated on the same strand of the amplified product, and these are annealed to form a hairpin-like loop, and an elongation reaction by the polymerase starting from the loop occurs. At the same time, a strand displacement type extension reaction occurs from the primer annealed in the loop, and the extension product is dissociated into single strands. Since the dissociated single strand also has a complementary sequence at the end, this reaction occurs repeatedly. Thus, in the LAMP method, since the extension reaction and the amplification reaction proceed simultaneously at a plurality of positions on the same strand of the amplified product, DNA amplification is achieved in an ultra-exponential and isothermal condition, and the tandem of the present invention. A probe nucleic acid having a structure can be efficiently synthesized.

2) Primer for LAMP method
In the LAMP method, specific primers called inner primer, outer primer and loop primer are used.
The inner primer is an essential primer for the LAMP method. When an arbitrary sequence X2c existing on the 3 ′ side and an arbitrary sequence X1c on the 5 ′ side are selected in each strand of the template DNA, the inner primer is complementary to the X2c. A primer containing the target sequence X2 and the same sequence X1c as X1c in this order from the 3 ′ side to the 5 ′ side (having the structure of X1c + X2). Functionally speaking, X2 on the inner primer is a part that specifically anneals to the template to give a starting point for complementary strand synthesis, and X1c is a complementary sequence for the amplification (extension) product to form a loop. give. This loop is the starting point for new complementary strand synthesis.

  The outer primer is a primer sequence that has a complementary sequence to an arbitrary sequence X3c on the outer side (3 'side of the template) of the inner primer, and can be annealed to it (one for each complementary to the double strand) ).

  In order to facilitate annealing of the primer to the template nucleic acid, the length of the above X1 (X1c), X2 (X2c), and X3 (X3c) is preferably 5 to 100 bases, more preferably 10 to 50 bases.

  The inner primer and outer primer are necessary for each of the double strands (F and R), and two types of inner primer (F1c + F2, R1c + R2) and outer primer (F3, R3) are designed.

  Each arbitrary sequence is preferably selected so that the amplification product obtained by the LAMP method preferentially causes intramolecular annealing rather than intermolecular annealing to form a terminal hairpin structure. For example, in order to preferentially cause intramolecular annealing, it is important to consider the distance between the F1c and F2c sequences and the distance between the R1 and R1c sequences when selecting an arbitrary sequence. . Specifically, it is preferable to select such that both sequences exist via a distance of 0 to 500 bases, preferably 0 to 100 bases, and most preferably 10 to 70 bases. Here, the numerical values respectively indicate the numbers of bases not including the F1c sequence and the F2c sequence itself, and the R1 sequence and the R2 sequence itself.

  The loop primer refers to a base sequence complementary to the sequence in the loop at the 3 'end when complementary sequences generated on the same strand of the amplified product by the LAMP method anneal to each other to form a loop. Two types of primers (one for each complementary to the double strand). The outer primer and loop primer are not essential for the LAMP method, but if they are present, the amplification (extension) reaction proceeds more efficiently.

3) Template nucleic acid for amplification
The template nucleic acid for amplification used in the LAMP method may be DNA or RNA, and can be prepared from a biological sample such as tissue or cell by a known method or a chemical synthesis method. In this case, the template polynucleotide is prepared so that sequences of appropriate length (referred to as double-sided sequences) exist on both sides of the region to be amplified (referred to as target region). Bilateral sequence means the sequence of the region from the 5 ′ end of the target region to the 5 ′ end of the polynucleotide chain, and the sequence of the region from the 3 ′ end of the target region to the 3 ′ end of the polynucleotide chain . The length of the both-side sequence is 10 to 1000 bases, preferably 30 to 500 bases in both the 5 ′ side and 3 ′ side regions of the target region.

4) Reaction conditions A series of reactions consist of a buffer that provides a suitable pH for the enzyme reaction, salts necessary for maintaining the catalytic activity of the enzyme and annealing, an enzyme protective agent, and, if necessary, a melting temperature (Tm ) Is preferably carried out in the presence of a regulator. As the buffering agent, a neutral to weakly alkaline buffering agent such as Tris-HCl is used. The pH may be adjusted according to the DNA polymerase used. As salts, for example, KCl, NaCl, (NH ₄ ) ₂ SO _4, or the like is appropriately added to maintain enzyme activity and adjust DNA melting temperature (Tm). As an enzyme protective agent, bovine serum albumin or saccharide is used. As the melting temperature (Tm) adjusting agent, betaine, proline, dimethyl sulfoxide, or formamide can be generally used.

5) LAMP reaction
In the reaction in the LAMP method, the following components (i), (ii), and (iii) are added to the template nucleic acid, and the inner primer forms a stable base pair bond with a complementary sequence on the template nucleic acid. It is possible to proceed by incubating at a temperature at which the strand displacement polymerase can maintain the enzyme activity. The incubation temperature is 50 to 75 ° C, preferably 55 to 70 ° C, and the incubation time is 1 minute to 10 hours, preferably 5 minutes to 4 hours.
(i) 2 types of inner primer, or 2 types of outer primer, or 2 types of loop primer
(ii) Strand displacement polymerase
(iii) Substrate nucleotide

1.3 Electrophoretic analysis After the amplification reaction, electrophoresis is continued on the microchip to separate and detect the target nucleic acid.
Separation and detection of the target nucleic acid by electrophoresis can be easily performed using a commercially available microchip electrophoresis analyzer (for example, Hitachi Microchip Electrophoresis Analysis System Cosmo Eye, Shimadzu Microchip Electrophoresis Apparatus MCE2010, etc.). In other words, the target nucleic acid is separated according to the applied voltage in the gel filled in the separation channel in the microchip.

  Nucleic acid detection is performed using light emission when fluorescence excitation light or laser light is applied to the nucleic acid from a light source built in the apparatus or absorption at a specific wavelength by the nucleic acid as an index. In general, a method is used in which electrophoresis is performed by binding a fluorescent intercalator to a nucleic acid, and the resulting fluorescence is irradiated with fluorescence excitation light or laser light. The detection may be performed at a specific time point or may be performed over time (real time). A commercially available apparatus is provided with an apparatus for analyzing the measurement value, and the obtained data can be immediately analyzed.

2. Visual analysis of amplification products by excess intercalator The present invention also allows visual confirmation of the presence or absence of a target nucleic acid without using a special light source or detection device by adding excess intercalator to the nucleic acid amplification product described above. A method for detecting a nucleic acid is provided.

  Here, an “intercalator” is a compound that can bind to double-stranded DNA, and in a narrow sense, refers to a compound that is inserted between the planes of nucleobase pairs. Including a compound (groove binder) capable of binding to a groove (main groove, sub groove) of DNA molecules is often referred to as an intercalator. Narrowly defined intercalators include ethidium bromide, acridine orange, TO-PRO-1, YO-PRO-1, methylene blue, actinomycin D, OliGreen, etc., and groove binders are SYBR Green I, SYBR Green II, Hoechst33258, Pico There are Green, DAPI, etc. In the present invention, the term “intercalator” means an intercalator in a broad sense including a groove binder.

  Usually, the fluorescence intercalator has background fluorescence, and when it is put in a large amount in the reaction solution, the background fluorescence also rises, so that a change in color tone cannot be confirmed visually. However, the present inventors have found that there is an intercalator that shows a subtle color change when bound to double-stranded DNA (for example, SYBR Green I: red brown → blue green). In the case of such an intercalator, it is possible to distinguish it from the background color tone, and when it is excessively added, the color tone change becomes strong and visual confirmation is possible. That is, it is possible to detect the binding of the intercalator to DNA without irradiating light from a special light source such as a UV lamp.

  In the present invention, “excess” means at least 10 times, preferably 100 times or more the amount used in normal detection. For example, in the case of SYBR Green I, if it is added at a concentration 100 times the normal usage, nucleic acid can be detected visually by a change in color tone (red brown → blue green) without using a UV lamp or the like. . However, since excess intercalator may inhibit the amplification reaction, the addition of excess intercalator must be performed after the amplification reaction.

3. Nucleic acid amplification and electrophoresis analysis integrated device The present invention also provides a nucleic acid amplification and electrophoresis analysis integrated device. The apparatus includes a means for introducing a target nucleic acid, a primer, a strand displacement polymerase, and a substrate nucleotide into a microchip, and a means for heating the chip to a constant temperature to perform a nucleic acid amplification reaction under isothermal conditions. And means for electrophoresis of the amplification product obtained by the amplification reaction on the chip, and means for detecting the nucleic acid separated by electrophoresis.

  Here, the means for introducing the target nucleic acid, primer, strand displacement polymerase, and substrate nucleotide into the microchip is the target nucleic acid, primer, strand displacement polymerase, and the sample well of the microchip held in the device. It is a means for automatically introducing a certain amount of substrate nucleotides simultaneously or sequentially.

  In addition, as a means for heating the chip to a constant temperature and performing a nucleic acid amplification reaction under an isothermal condition, for example, the chip is moved from above or below by a heat block provided in the apparatus, preferably both upper and lower surfaces. And a means for heating to a constant temperature suitable for the amplification reaction. This means may include means for sealing the well into which the sample has been introduced. The amplification reaction can be performed, for example, at a sample introduction port of a commercially available microchip electrophoresis apparatus, and can be directly applied to electrophoresis separation / detection described later.

Means for electrophoresis of amplification products obtained by the amplification reaction on the chip and means for detecting nucleic acids separated by electrophoresis are used in commercially available microchip electrophoresis apparatuses. Means similar to the means can be used.
The apparatus of the present invention may further include means for computer processing the obtained measurement data.

EXAMPLES Hereinafter, although this invention is demonstrated further in detail using an Example, this invention is not limited to these Examples.
(Example 1) Nucleic acid amplification and electrophoresis analysis using a microchip Experimental material
PMMA 3-lane microchip (COSMO eye i-chip: manufactured by HITACHI)
LAMP reaction solution (PSA and λ system kit accessories: manufactured by Eiken Chemical Co., Ltd.)
PCR96 hole seal (Thermowell; costar, Netherlands),
Gel (attached to Cosmo Eye iChip Kit),
Fluorescent dye EtBr (Cosmo eye i-chip kit attachment),
SYBR Green I (SG: Molecular Probes Inc.)

2. Test method (1) LAMP reaction Fill the separation channel of the chip with gel pressure. In addition, the gel is centrifuged in advance for about 10,000 rpm for 1 min to remove bubbles. A LAMP reaction solution (Table 1) containing PSA (prostate-specific antigen cDNA fragment; SEQ ID NO: 1) as a target (template) nucleic acid and four kinds of LAMP primers shown below was prepared. The sample well was filled with 10 μL of the LAMP reaction solution, and the other wells were filled with 10 μL of the above gel.
Primer for PSA LAMP:
FIP; 5'-TGTTCCTGATGCAGTGGGCAGCTTTAGTCTGCGGCGGTGTTCTG-3 '(SEQ ID NO: 2)
RIP; 5'-TGCTGGGTCGGCACAGCCTGAAGCTGACCTGAAATACCTGGCCTG-3 '(SEQ ID NO: 3)
F3; 5'-TGCTTGTGGCCTCTCGTG-3 '(SEQ ID NO: 4)
R3; 5'-GGGTGTGTGAAGCTGTG-3 '(SEQ ID NO: 5)
(FIP and RIP are Forward Inner Primer and Reverese Inner Primer, respectively, and F3 and R3 are Forward Outer Primer and Reverse Outer Primer, respectively.)

  After introducing the LAMP reaction solution, the wells were sealed using a PCR 96-hole seal. When sealing, it was performed while holding down so that air bubbles would not enter. Next, the chip was heated on an aluminum heat block at 65 ° C. for about 10 to 60 minutes. A heat block was also placed on the chip and heated in the same manner for the purpose of indirectly applying heat from above and preventing the seal from being lifted by bubbles (see FIG. 1). After the heating, the seal was peeled off, and a gel was added to the well as appropriate to remove bubbles in the lane.

(2) Confirmation of product
The chip after the LAMP reaction was subjected to electrophoresis using HITACHI Cosmo eye to confirm the presence of the product. The electrophoresis parameters were an introduction voltage of 300 V 60 sec and a separation electrophoresis voltage of 750 V (reverse electrophoresis voltage ^* 130 V) 80 sec. The measurement was first performed every 10 minutes after the start of the LAMP reaction, and then measured at shorter intervals. Furthermore, the LAMP amplification time was fixed (each reaction time was 30 minutes), and several times were measured in different reaction solutions to confirm the reproducibility of the measurement results.
* Reverse electrophoresis is a method in which a voltage is applied to the sample well and gel well during separation electrophoresis to place the sample on the cross site, and as a result, an electrophoretic diagram in which peaks repeatedly appear is obtained.

(3) Visible determination of the product SYBR Green I at a concentration of 10000 ×, 1000 ×, and 100 × was added to the product so as to be 10-fold diluted, respectively, and the final concentrations were 1000 ×, 100 ×, and 10 ×, respectively. It was made to become x. SYBR Green I is usually added at 1x in real-time PCR.
As a control, a product obtained by removing the template and the polymerase from the reaction solution was similarly introduced into the sample well and heated for detection.

3. Result (1) About heating time
From the results measured every 10 minutes after the start of the LAMP reaction (FIG. 2) and the results measured at shorter intervals (FIG. 3), it was found that the product was already obtained in about 10 minutes in the PSA system.
(2) Reproducibility As a result of repeated measurement, a peak of almost the same level was detected each time (FIG. 4). As a result, it was confirmed that a product of almost a fixed length was amplified to the same extent in the LAMP reaction.
(3) Visible determination of product FIG. 5 shows the LAMP product on the chip with about 1 μL of SYBR Green I added at 1000 × concentration (final concentration: 100 ×). FIG. 6 shows the result of adding 0.5 μL of SYBR Green I to 4.5 μL of the product obtained after LAMP on the chip. For ease of viewing, the photo was taken in a glass tube with an outer diameter of 1.5 mm.

  It can be seen that the control added to the LAMP product emits green fluorescence, whereas the control is not different from the case of SYBR Green I alone. Fluorescence is emitted immediately after mixing. When the back side is white, it is easy to identify the one with the final density of 1000 × because the change in control and color tone is large. When the back is dark, the fluorescence is most easily identified at the final density of 100 ×.

(Example 2) Multiplex measurement According to the method of Example 1, it was confirmed whether a plurality of samples could be simultaneously amplified and analyzed by the LAMP method.
1. Test method PSA (prostate-specific antigen cDNA fragment; SEQ ID NO: 1) and λ-DNA (Takara Shuzo; SEQ ID NO: 6) were used as target (template) nucleic acids and both were LAMP-amplified on the chip as in Example 1. Electrophoretic analysis was performed in the same manner. For the primer, the primer used in Example 1 was used for PSA, and the primer shown below was used for λ-DNA, and the reaction solution was prepared with the same composition as Example 1. Each reaction solution was introduced into a separate sample well, subjected to LAMP reaction at 65 ° C. for 30 minutes, and subjected to electrophoresis using Hitachi Cosmo Eye.

For comparison, only PSA primer and λ-DNA primer were added to the reaction solution, and LAMP amplification was performed in the same manner, followed by electrophoresis.
Primer for λ-DNA LAMP:
FIP; 5'-TCCCCTCAGAACATAACATAGTAATGCGGTAAGTCGCATAAAAACCATTC-3 '(SEQ ID NO: 7)
RIP; 5'-TGAAAATTCCCCTAATTCGATGAGGTCGGCGCATAGCTGATAACAAT-3 '(SEQ ID NO: 8)
F3; 5'-GCTTATCTTTCCCTTTATTTTTGC-3 '(SEQ ID NO: 9)
R3; 5'-GCTGATCGGCAAGGTGTTCT-3 '(SEQ ID NO: 10)
(FIP and RIP are Forward Inner Primer and Reverese Inner Primer, respectively, and F3 and R3 are Forward Outer Primer and Reverse Outer Primer, respectively.)

2. Results As shown in FIGS. 7 and 8, the graph obtained by performing LAMP amplification using two kinds of primers is close to a graph obtained by superimposing the graphs when LAMP amplification is performed by using each single primer. From this, it was confirmed that a plurality of samples can be analyzed simultaneously.

FIG. 1 is a diagram showing a structure of a microchip and an outline of microchip heating by a heat block. FIG. 2 is a graph showing the results of electrophoretic analysis of the LAMP product every 10 minutes after the start of the LAMP reaction. FIG. 3 is a graph showing the results of electrophoretic analysis of LAMP products at 0, 6, 9, and 10 minutes after the start of the LAMP reaction. FIG. 4 is a graph showing the reproducibility of the electrophoretic analysis results of the LAMP product (30 minutes after amplification). FIG. 5 is a photograph of the LAMP product when SYBR Green I at a concentration of 1000 × (final concentration: 100 ×) is added (the left side is the control and the right side is the LAMP product). Fig. 6 shows a photograph of SYBR Green I at a concentration of 1000x (final concentration: 100x) added to the LAMP product on the chip (taken in a glass tube with an outer diameter of 1.5 mm for ease of viewing. ). Figure 7 compares the results of multiplex measurements (PSA and λ-DNA) and single samples (either PSA or λ-DNA) for electrophoretic analysis of LAMP products on a chip. is there. From the top of the figure, only PSA is amplified, both PSA and λ-DNA are amplified, and only λ-DNA is amplified. FIG. 8 is a graph in which electrophoretic analysis of LAMP products on a chip is performed by superimposing results obtained by amplifying only PSA, amplifying both PSA and λ-DNA, and amplifying only λ-DNA.

SEQ ID NO: 2-description of artificial sequence: primer SEQ ID NO: 3-description of artificial sequence: primer SEQ ID NO: 4-description of artificial sequence: primer SEQ ID NO: 5-description of artificial sequence: primer SEQ ID NO: 7-description of artificial sequence: primer SEQ ID NO: 8-description of artificial sequence: primer SEQ ID NO: 9-description of artificial sequence: primer SEQ ID NO: 10-description of artificial sequence: primer

Claims (4)

After the nucleic acid amplification reaction, SYBR (registered trademark) Green I is added to the obtained amplification product so that the concentration is 10 to 1000 times the normal concentration, and visual analysis is performed to visually detect the presence or absence of the target nucleic acid based on the resulting color change. A method for detecting a nucleic acid, comprising: The method for detecting a nucleic acid according to claim 1 , wherein the nucleic acid amplification reaction is performed on a microchip under isothermal conditions. The method for detecting a nucleic acid according to claim 2 , wherein after the nucleic acid amplification reaction, the obtained amplification product is subsequently analyzed on a microchip. The method according to claim 2 or 3 , wherein the nucleic acid amplification reaction under isothermal conditions is performed using the LAMP method.

Images