JPWO2017043114A1 - Multi-item simultaneous detection method for isothermal amplification reaction products - Google Patents

Abstract

An object of the present invention is to provide a method for detecting multiple items at the same time quickly and easily by using chromatographic PAS for simultaneous detection of multiple items by the LAMP method. (A) a step of preparing a set of a first primer and a second primer for each target nucleic acid sequence, wherein a tag sequence is bound to the first primer, and when two or more target nucleic acid sequences are included, The tag sequence is different for each target nucleic acid sequence, and the step of binding the labeling substance binding substance to the second primer, (b) using one or a set of two or more first and second primers A step of performing isothermal amplification of nucleic acid by LAMP method in the same reaction solution; (c) preparing a development medium containing an amplification reaction solution containing the nucleic acid isothermal amplification product and a labeling substance that binds to the labeling substance binding substance; Nucleic acid chromatography on a carrier to hybridize one or more detection probes immobilized on the solid phase carrier with the nucleic acid isothermal amplification product and the nucleic acid isothermal increase A step of labeling a product with a labeling substance, and (d) a detection step of detecting the hybridized product generated in the step (c) with the labeling substance, wherein the set of the first primer and the second primer comprises The above problem is solved by a predetermined method.

Description

  The present invention relates to a multi-item simultaneous detection method for isothermal amplification reaction products. More specifically, the present invention relates to a method for simultaneously detecting a plurality of isothermal amplification reaction products present in the same reaction solution.

  The LAMP (Loop-Mediated Isolation amplification) method is a method of amplifying a target gene by an isothermal reaction (Patent Documents 1 and 2). Recently, the PCR method has been used for gene amplification. However, the PCR method takes time for the reaction and requires expensive equipment. On the other hand, the LAMP method can be amplified at a constant temperature in a short time and does not require expensive equipment. Further, since the LAMP method has a very high detection sensitivity compared with the PCR method, it is also possible to detect a trace amount sample. In the PCR method, an amplification reaction is performed with two types of primers for each detection target. On the other hand, the LAMP method uses 4-6 types of primers for one detection target in order to improve detection sensitivity and accuracy. Since the amplification reaction by the LAMP method requires a large number of primers for one detection target, as a result of including a large number of detection targets in the same reaction solution, it is not suitable for simultaneous detection of multiple items that will contain a large number of primers. Has been considered.

Conventionally, a turbidity detection method and an intercalator method are generally used as a method for detecting an amplification product by the LAMP method.
The turbidity detection method is a method of detecting white turbidity in which pyrophosphate, which is a byproduct of a nucleic acid amplification reaction, is precipitated with magnesium, as turbidity by the intensity of transmitted light.
The intercalator method is a method for detecting a DNA amplification reaction as an increase in fluorescence intensity by allowing a DNA intercalator such as SYBR (registered trademark) Green I to coexist in a nucleic acid amplification reaction solution. These methods detect one detection target and do not detect multiple items simultaneously.
Non-Patent Document 1 describes that detection was performed by an immunochromatography method in which a plurality of amplification products by RT-LAMP method were modified with different antigens, and antibodies against each antigen were developed and separated on chromatographic paper fixed on a solid phase. ing. However, for detection by immunochromatography, it is necessary to prepare an antibody against a labeling substance separately, the operation is complicated, and a simpler method is desired.

In the chromatographic PAS (PAS: Printed Array-Stripe) method, an amplification reaction is performed by a PCR method or the like using a primer labeled with one tag sequence for one target gene and a primer labeled with a labeling substance binding substance. After adding a tag sequence to the detection target and amplifying the detection target, a labeling substance such as a dye and a developing solution are added to the reaction product, and then a single-stranded DNA having a sequence complementary to the tag sequence (hereinafter, detection probe) Are developed on immobilized (PAS) chromatographic paper, so that a detection probe having a complementary strand of each immobilized tag sequence and a hybrid reaction between single-stranded DNAs in each tag sequence (STH: Single Tag) (Hybridization) occurs, and the detection target is detected as a band visualized by the labeling substance, for example. A capacity method (Patent Document 3). The chromatographic PAS method can be applied to simultaneous detection of multiple samples by labeling primers with different tags for each detection target in the PCR method.
However, as described above, the LAMP method requires a larger number of primers for a single detection target than the PCR method. Therefore, in order to detect a plurality of targets, a different tag sequence is bound to the primer for each detection target. Furthermore, for each detection target, when a labeling substance binding substance for detection is bound to a primer different from the primer to which the tag sequence is bound, the amplification reaction does not proceed with those primers, or the amplification reaction It was predicted that optimization would be difficult, and it was thought that it could not be applied.

In addition, in conventional nucleic acid amplification methods, for example, if the number of PCR method cycles exceeds 50, it is theoretically possible to amplify DNA at a level that can be detected from even one copy of template DNA, thus preventing contamination of DNA to reagents and samples. There is a basic problem that needs to be taken. In particular, when PCR is repeatedly performed for DNA diagnosis in a clinical laboratory or the like, contamination (carry over) due to PCR amplification products becomes a serious problem. Since the LAMP method is more sensitive than the PCR method, there is a strong demand for reducing the risk of contamination.
Regarding the PCR method, a method for preventing DNA carryover using uracil N glycosidase has been proposed (Non-patent Document 2). However, there is no example in which whether or not the same prevention method is effective in the combination of the LAMP method and the chromatographic PAS method.

Japanese Patent No. 3313358 JP 2002-345499 A Japanese Patent No. 5503302

Jung JH, Oh SJ, Kim YT, Kim SY, Kim WJ, Jung J, Seo TS. Combination of multiplex reverse-transcription loop-mediated isothermal amplification with an immunochromatographic strip for subtyping influenza A virus. Anal Chim Acta. 853: 541- 7, 2015 PMID: 25467501. Longo, M. C., Berninger, M. S. and Hartley, J. L. Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions. Gene. 1990 Sep 1; 93 (1): 125-8.

It is an object of the present invention to provide a method for detecting multiple items simultaneously and quickly by using a chromatographic PAS for simultaneous detection of multiple items by the LAMP method, which has been considered difficult in the past. It is another object of the present invention to provide a method capable of simultaneously detecting multiple items with high sensitivity even for a small amount of sample as compared with the STH chromatographic PAS method combining conventional PCR and DNA chromatography.
The present inventors have found that the labeling of the primer does not affect the amplification reaction of the LAMP method if the specific primer used in the LAMP method is labeled against the conventional prediction. In addition, the inventors have found that multi-item simultaneous detection by the LAMP method can be performed by combining the LAMP method and the chromatographic PAS method, and the present invention has been completed.
Furthermore, the present inventors, in the LAMP method, which is a highly sensitive detection method, in order to prevent contamination by the reaction product of the previous amplification reaction (contamination due to carryover) when repeating the amplification reaction, The inventors have found that a contamination prevention process using (UNG) is effective, and have completed the present invention.

That is, the present invention is as follows.
[1] A method for detecting a target nucleic acid sequence contained in one or more of the same specimen,
The following (a) to (d):
(A) preparing a set of a first primer and a second primer for each target nucleic acid sequence,
When a tag sequence is bound to the first primer and two or more target nucleic acid sequences are included, the tag sequence is different for each target nucleic acid sequence,
The step wherein a labeling substance-binding substance is bound to the second primer;
(B) a step of performing nucleic acid isothermal amplification by the LAMP method in the same reaction solution using a set of one or more first and second primers,
(C) preparing a development medium containing an amplification reaction solution containing the nucleic acid isothermal amplification product and a labeling substance that binds to the labeling substance binding substance, and performing nucleic acid chromatography on a solid phase carrier;
Including a step of hybridizing the one or more detection probes immobilized on the solid phase carrier and the nucleic acid isothermal amplification product, and a step of labeling the nucleic acid isothermal amplification product with a labeling substance;
(D) including a detection step of detecting the hybridized product generated in the step (c) with the labeling substance,
For the arbitrary sequences F3c, F2c, LF, F1c, B1, LBc, B2, and B3 arranged in the direction from the 3 ′ end to the 5 ′ end of the target nucleic acid sequence, the first and second primers are the following (i) to (iv) :
(I) FIP primers containing the same sequence as F1c on the 5 ′ side of sequences F2 and F2 complementary to F2c,
(Ii) a BIP primer comprising the same sequence as B2 and a sequence B1c complementary to B1 on the 5 ′ side of B2,
(Iii) A method arbitrarily selected from the group consisting of an LF primer having the same sequence as the LF region and (iv) an LB primer having a sequence complementary to the LBc region.
[2] The set of the first primer and the second primer, wherein the first primer is an FIP primer and the second primer is a BIP primer or the first primer is a BIP primer and the second primer is an FIP primer. the method of.
[3] The set of the first primer and the second primer of each target nucleic acid sequence is such that the first primer is an FIP primer and the second primer is a BIP primer or the first primer is a BIP primer and the second primer is an FIP primer, The method according to [1] or [2] above, wherein no primer other than the set of the first primer and the second primer of each target nucleic acid sequence is used in the nucleic acid isothermal amplification step.
[4] The labeling substance binding substance is one or more selected from the group consisting of an antibody in an antigen-antibody reaction and a hapten containing biotin, digoxigenin, and FITC.
The labeling substance has a site capable of binding to the labeling substance binding substance, and uses one or more kinds of labeling substances selected from the group consisting of fluorescence, radioactivity, enzyme, phosphorescence, chemiluminescence, and coloring. The method according to any one of [1] to [3], wherein
[5] The method according to any one of [1] to [4], wherein the nucleic acid isothermal amplification step is performed in the presence of dUTP, and the uracil DNA glycosylase (UNG) treatment is performed before the nucleic acid isothermal amplification step.
[6] A measuring reagent or reagent used for carrying out the detection method according to any one of [1] to [5] and comprising at least a set of a first primer and a second primer kit.
[7] The measuring reagent or reagent kit according to [6], further comprising one or more detection probes fixed on a solid phase carrier.

  Multi-item simultaneous detection by the LAMP method, which has been considered difficult in the past, can be realized by a simple method. In addition, it is possible to provide a multi-item simultaneous detection method with higher sensitivity compared to the STH chromatographic PAS method in which the conventional PCR method and DNA chromatography are combined. Further, in the multi-item simultaneous detection method using the LAMP method, contamination due to carry over can be prevented.

FIG. 2 illustrates the region setting on a target nucleic acid sequence for designing a primer used in the LAMP method. The primers used in the LAMP method are illustrated. The chromatographic development result of the amplification product by the LAMP method is shown. The result of having examined about the specificity of a detection primer is shown. The result of having examined about the combination of a labeled primer is shown. The examination result of the detection sensitivity of SAL and STA is shown. The examination result of the detection sensitivity by the multiplex of SAL and STA is shown. The result of the study on the position of the sign is shown. The influence on LAMP reaction by dUTP addition is shown. This shows the carryover reduction effect when UNG processing is performed. The chromatographic development result of the LAMP reaction product by UNG processing is shown. This shows the influence on the LAMP reaction when only the inner primer, only the loop primer, or only the outer primer is used.

The method of the present invention is a method for detecting a target nucleic acid sequence contained in one or more of the same specimen,
The following (a) to (d):
(A) preparing a set of a first primer and a second primer for each target nucleic acid sequence,
When a tag sequence is bound to the first primer and two or more target nucleic acid sequences are included, the tag sequence is different for each target nucleic acid sequence,
The step wherein a labeling substance-binding substance is bound to the second primer;
(B) a step of performing nucleic acid isothermal amplification by the LAMP method in the same reaction solution using a set of one or more first and second primers,
(C) preparing a development medium containing an amplification reaction solution containing the nucleic acid isothermal amplification product and a labeling substance that binds to the labeling substance binding substance, and performing nucleic acid chromatography on a solid phase carrier;
Including a step of hybridizing the one or more detection probes immobilized on the solid phase carrier and the nucleic acid isothermal amplification product, and a step of labeling the nucleic acid isothermal amplification product with a labeling substance;
(D) including a detection step of detecting the hybridized product generated in the step (c) with the labeling substance,
For the arbitrary sequences F3c, F2c, LF, F1c, B1, LBc, B2, and B3 arranged in the direction from the 3 ′ end to the 5 ′ end of the target nucleic acid sequence, the first and second primers are the following (i) to (iv) :
(I) FIP primers containing the same sequence as F1c on the 5 ′ side of sequences F2 and F2 complementary to F2c,
(Ii) a BIP primer comprising the same sequence as B2 and a sequence B1c complementary to B1 on the 5 ′ side of B2,
It is a method arbitrarily selected from the group consisting of (iii) an LF primer having the same sequence as the LF region and (iv) an LB primer having a sequence complementary to the LBc region.

As used herein, “specimen” refers to a specimen that may contain a target nucleic acid. The collection source of the specimen is not particularly limited, and refers to specimens derived from various living bodies such as plants and animals. Fractions containing nucleic acids from these various specimens can be obtained by those skilled in the art with reference to conventional techniques as appropriate.
In this specification, “nucleic acid” means a polymer of nucleotides, and the number of polymerizations is not particularly limited.
In the present specification, the “target nucleic acid” is any nucleic acid whose target is the presence and / or amount of the nucleic acid.

(Nucleic acid isothermal amplification process)
In the method of the present invention, nucleic acid amplification is performed by nucleic acid isothermal amplification using the LAMP method. In the LAMP (Loop-Mediated Isothermal amplification) method, 4 to 6 types of oligonucleotide primers, strand displacement type DNA synthase, nucleic acid monomer, etc. are mixed with a template nucleic acid chain containing the target nucleic acid sequence, and a constant temperature (around 65 ° C). ) In which the nucleic acid amplification reaction is performed.

The design of the LAMP method primer is performed by setting six regions of arbitrary sequences F3c, F2c, F1c, B1, B2, and B3 in the direction from the 3 ′ end to the 5 ′ end of the target nucleic acid sequence. The basic LAMP method uses 4 types of primers, 2 types of inner primer and 2 types of outer primer. The inner primer is a FIP primer containing the same sequence as F1c on the 5 ′ side of the sequences F2 and F2 complementary to F2c, and a BIP primer containing the same sequence as B2 and the sequence B1c complementary to B1 on the 5 ′ side of B2. Yes, the outer primer is an F3 primer containing a sequence complementary to F3c and a B3 primer containing the same sequence as B3.
Further, in order to improve the amplification efficiency, in addition to the four kinds of primers, two kinds of primers, that is, two kinds of loop primers may be used, and the amplification reaction may be carried out using a total of six kinds of primers. In this case, an arbitrary sequence LF between F2c and F1c of the target nucleic acid sequence and an arbitrary sequence LBc between B1 and B2 are set. The loop primer is an LF primer including the same sequence LF as LF and an LB primer including a sequence LB complementary to LBc (see FIGS. 1 and 2).
In the method of the present invention, a nucleic acid amplification reaction can be performed in the same manner as in the ordinary LAMP method except that the first primer and the second primer described below are used. The primer design and the amplification mechanism of amplification by the LAMP method are described in, for example, Japanese Patent No. 3313358 and Japanese Patent Application Laid-Open No. 2002-345499.

In the method of the present invention, a first primer to which a tag sequence is bound is used. When two or more target nucleic acid sequences are included, the tag sequence is different for each target nucleic acid sequence.
The tag sequence is a single-stranded nucleic acid sequence for allowing the amplification product to hybridize with the detection probe described later, and detects the target nucleic acid sequence. It is set to be capable of hybridizing to the detection sequence. Typically, it has a base sequence complementary to the detection sequence of the detection probe. The base length of the tag sequence preferably matches the base length of the detection sequence of the detection probe, preferably 20 bases or more and 50 bases or less, more preferably 20 bases or more and 25 bases or less.

In the present invention, a second primer to which a labeling substance binding substance is bound is used. Here, the labeling substance binding substance is a substance that can bind to the labeling substance, and will be described in detail later.
In this specification, the “labeling substance” is a substance that makes it possible to distinguish a substance or molecule to be detected from others. The labeling substance is not particularly limited, but typically, a labeling substance using fluorescence, radioactivity, enzyme (for example, peroxidase, alkaline phosphatase, etc.), phosphorescence, chemiluminescence, coloring and the like can be mentioned.

  The labeling substance is preferably a luminescent substance or a coloring substance that presents luminescence or coloring that can be detected visually from the viewpoint of performing detection quickly and easily. Such materials typically include various colorants such as various pigments and dyes. In addition to this, in addition to noble metals such as gold and silver, various metals or alloys such as copper, or organic compounds containing the metal (may be complex compounds) may be used. In addition, inorganic compounds such as mica, which are similar to the colorant, can be used.

  This kind of labeling substance typically includes various dyes, various pigments, luminol, isoluminol, acridinium compounds, olefins, enol ethers, enamines, aryl vinyl ethers, dioxene, aryl imidazoles, lucigenin, luciferin and eclion. A chemiluminescent substance is mentioned. Moreover, particles such as latex particles labeled with such a labeling substance are also included. Furthermore, colloids or sols including gold colloids or sols or silver colloids or sols can be mentioned. Furthermore, a metal particle, an inorganic particle, etc. are mentioned.

  As the labeling substance binding substance, a substance that can bind to the labeling substance using, for example, protein-protein interaction, low molecular compound-protein interaction, or the like can be selected. For example, antibodies in antigen-antibody reaction, biotin in avidin (streptavidin) -biotin system, digoxigenin in anti-digoxigenin (DIG) -digoxigenin (DIG) system, or haptens represented by FITC in anti-FITC-FITC system, etc. Is mentioned. In this case, the labeling substance finally used for detection is the other molecule or substance that interacts with the labeling substance binding substance (for example, an antigen, ie, streptavidin, anti-FITC, etc.) and the labeling substance binding substance. It is modified so as to be provided as a site for binding. When the amplification product includes a labeling substance binding substance, a label having a labeling substance binding substance of the amplification product and a site that binds to the labeling substance binding substance in the hybridization step, prior to or after this step. A complex with the substance is formed, and the amplification product is detected by the labeling substance.

The first primer and the second primer are (i) complementary to F2c for arbitrary sequences F3c, F2c, LF, F1c, B1, LBc, B2 and B3 arranged in the direction from the 3 ′ end to the 5 ′ end of the target nucleic acid sequence. FIP primer containing the same sequence as F1c on the 5 ′ side of the sequences F2 and F2, (ii) BIP primer containing the same sequence as B2 and the sequence B1c complementary to B1 on the 5 ′ side of B2, (iii) LF region And (iv) an LB primer having a sequence complementary to the LBc region.
That is, the first primer and the second primer are arbitrarily selected from two types of inner primers (FIP, BIP) and two types of loop primers (LF, LB). The combination of the types of the first primer and the second primer is not particularly limited.
In the method of the present invention, the set of the first primer and the second primer of each target nucleic acid sequence is such that the first primer is an FIP primer and the second primer is a BIP primer or the first primer is a BIP primer and the second primer is an FIP primer. In this case, the nucleic acid isothermal amplification step can be performed without using a primer other than the set of the first primer and the second primer of each target nucleic acid sequence.
The binding position of the tag sequence in the first primer and the binding position of the labeling substance binding substance in the second primer may be any of the 5 ′ end of the primer, the 3 ′ end of the primer, and the inside of the primer sequence.

(Nucleic acid chromatography)
In this specification, nucleic acid chromatography refers to a porous solid phase carrier capable of diffusing and moving a liquid (development medium) by a capillary phenomenon, and the nucleic acid is moved inside the solid phase carrier by the liquid to be solidified. This refers to chromatography in which a hybridized product is formed by specific base pairing with a detection probe immobilized on a phase carrier, and the nucleic acid is captured on a solid phase carrier.

The detection probe is a single-stranded nucleic acid sequence, and the length of the detection sequence is not particularly limited, but is preferably 20 bases or more and 50 bases or less. Within this range, hybridization efficiency can be ensured while ensuring the specificity of each detection sequence. For example, a design method and an example of such a detection sequence are disclosed in Japanese Patent No. 55503021.
Since the tag sequence bound to the first primer is a base sequence that is paired with the detection probe sequence, the base length of the tag sequence should be 20 to 50 bases, similar to the detection sequence. More preferably, it is 20 bases or more and 25 bases or less.

The detection probe is immobilized on a solid phase carrier. For example, the solid support may be plastic or glass, and the material is not particularly limited. Moreover, porous bodies, such as a cellulose, a nitrocellulose, and nylon, may be sufficient. This type of porous carrier is particularly suitable for hybridizing the detection probe immobilized on the solid phase carrier and the amplified fragment by affinity chromatography.
The solid phase carrier on which the detection probe is immobilized can be obtained commercially. For example, Chromato PAS (F4) (manufactured by TBA Co., Ltd.), Chromato PAS (F8) (manufactured by TBA Co., Ltd.), etc. Can be mentioned.

(Hybridization process)
The hybridization step is performed by preparing a development reaction medium containing an amplification reaction solution containing a nucleic acid isothermal amplification product and a labeling substance that binds to the labeling substance binding substance, performing nucleic acid chromatography, and fixing one or more immobilized on a solid phase carrier This is a step of hybridizing the detection probe and the nucleic acid isothermal amplification product. Furthermore, since the labeling substance is contained in the development medium, the labeling substance is labeled on the nucleic acid isothermal amplification product during the nucleic acid chromatography step.

  The development medium is a liquid that diffuses and moves the solid phase carrier of the chromatography main body by a capillary phenomenon, and is a medium for moving the by-product on the solid phase carrier. The development medium is an aqueous medium. The aqueous medium is not particularly limited, and examples thereof include water, an organic solvent compatible with water, or a mixture of water and one or more organic solvents. Organic solvents that are compatible with water are well known to those skilled in the art, and examples include lower alcohols having about 1 to 4 carbon atoms, esters such as DMSO, DMF, methyl acetate, and ethyl acetate, and acetone. The development medium is preferably mainly water.

  The development medium can contain a component for keeping the pH within a certain range. The buffer salt is usually in the range of 6.0 to 8.0, depending on the intended pH. More preferably, it is 7.0 or more and 8.0 or less. Components for obtaining such pH are, for example, acetic acid and sodium acetate (acetic acid buffer), citric acid and sodium citrate (citrate buffer), phosphoric acid and sodium phosphate (phosphate buffer), and the like. Furthermore, a phosphate buffered saline (PBS) etc. are mentioned. In addition, you may employ | adopt an amplification reaction liquid as a developing medium as it is. Further, the composition and concentration may be adjusted by adding an additional component such as a surfactant or an appropriate salt or a solvent to the amplification reaction solution, and the resultant may be used as a developing medium.

  The detection step is a step of detecting the final hybridized product based on the labeling substance. More specifically, this is a step of confirming the coloring and position of the region where the detection probe is immobilized. In order to detect the signal by the labeling substance, it is appropriately selected according to the type of the labeling substance. When a specific binding reaction or a color reaction with an enzyme is required, such an operation is appropriately performed.

(Contamination prevention)
In the method of the present invention, a nucleic acid isothermal amplification step (LAMP reaction) is performed in the presence of dUTP, and a uracil DNA glycosylase (UNG) treatment is performed before the nucleic acid isothermal amplification step (LAMP reaction), so that the preceding nucleic acid isothermal amplification is performed. “Carry over” contamination from the process can be prevented.
This method is a method for preventing contamination by modifying the amplified nucleic acid so as to be easily degraded.
First, the amplification reaction is performed using dUTP instead of dTTP as a nucleotide in the preceding LAMP reaction step so that the amplicon that is an amplification product of the previous LAMP reaction, which is a potential carry-over contamination source, contains uracil. Do. The preceding LAMP reaction is performed in the presence of dUTP, and in the reaction, part or all of dTTP is replaced with dUTP.
In the subsequent LAMP reaction, an amplification reaction is performed using dUTP, and uracil-N-DNA glycosylase is added to the reaction solution. Prior to the amplification reaction (eg 65 ° C., 30 minutes), incubation is carried out within the temperature range in which UNG is active (eg room temperature, 5 minutes). In the presence of uracil-containing contaminants from previous reactions, UNG cleaves uracil and leaves an abasic site. DNA having an abasic site is known to be unstable at high temperatures under high pH conditions, and such DNA is degraded when the amplification reaction starts. The UNG enzyme is inactivated at the temperature at which the amplification reaction is carried out, generating a new DNA amplicon containing uracil. Similarly, in the subsequent LAMP reaction, “carry over” contamination can be prevented by performing uracil-N-DNA glycosylase treatment.

  EXAMPLES Examples will be shown below for specifically explaining the present invention, but the present invention is not limited to the following examples.

Example 1
1. Objective To investigate whether LAMP reaction products could be detected by C-PAS.
2. material
<Reagents used in LAMP reaction>
・ 100μM forward inner primer (FIP)
・ 100μM Backward Inner Primer (BIP)
・ 100μM forward loop primer (LF) _tag sequence binding (5'end)
・ 100μM backward loop primer (LB) _biotin binding (5'end)
・ 100μM forward outer primer (F3)
・ 100μM Backward outer primer (B3)
・ Isothermal Master Mix (OptiGene)
・ Salmonella enterica (SAL) genomic DNA
Staphylococcus aureus (STA) genomic DNA
<Reagents used in STH chromatography PAS method> (manufactured by TBA Co., Ltd.)
・ Chromatographic PAS (F4)
-Chromatographic development buffer-Chromatographic development dye Method First, a primer set (FIP, BIP, LF, LB, F3 B3) was designed. Each primer set was designed using Primer Explorer (Eiken Chemical).

The sequences of primer sets (SAL detection primer sets) that can detect Salmonella enterica genomic DNA are as shown in the following table.

Table 1 Primer set for SAL detection

The sequences of primer sets (STA detection primer sets) that can detect Staphylococcus aureus genomic DNA are shown in the following table.

Table 2 STA detection primer set

After confirming the possibility of dimer formation, the designed primer has a tag sequence at the 5 ′ end of the LF primer and biotin at 5 ′ of the LB primer so that it can be detected by the STH chromatography PAS method. 'Attached to each end. Different tag sequences were used for SAL detection and STA detection.
Next, preparation of Probe Mixture was performed. Probe Mix is 0.4 μL of FIP, 0.4 μL of BIP, 0.4 μL of LF, 0.4 μL of LB, 0.05 μL of F3, 0.05 μL of B3, and 0.8 μL of sterile water per reaction. did. Next, a LAMP reaction solution was prepared. In the Tube Strip for LAMP reaction, 2 μL of the prepared Probe Mixture, 8 μL of the Islamic Master Mix, Temp. 2 μL of DNA was added and mixed. When the reaction was performed in a multiplex (when a plurality of nucleic acids to be detected were included in the same specimen), Probe Mix for SAL detection and Probe Mix for STA detection were mixed in a 1: 1 ratio. Genie II (OptiGene) was used for the LAMP isothermal reaction. The reaction temperature was 65 ° C. and the reaction was performed for 30 minutes.
The LAMP product after the reaction was diluted 1/10 with sterilized water. 1 μL of diluted product, 9 μL of sterilized water, 1 μL of chromatographic developing dye and 10 μL of chromatographic developing buffer were added and mixed. Chromatographic paper was immersed in the mixed reaction solution, and chromatographic development was performed at room temperature for 20 minutes.

4). Results The LAMP reaction was carried out using the LAMP primers LF and LB bound to the tag sequence and biotin, respectively. The chromatographic development result of the LAMP reaction product is shown in FIG. In FIG. 3, lane 1 contains only SAL genomic DNA in the sample, lane 2 contains only STA genomic DNA in the sample, and lane 3 contains SAL genomic DNA and STA genomic DNA in the sample. Lane 4 is negative control 1 (Escherichia coli), and lane 5 is negative control 2 (DW).
Specific bands could be detected for each of SAL genomic DNA and STA genomic DNA (FIG. 3, lane 1 and lane 2). In addition, when the LAMP reaction was performed in a multiplex, both SAL genomic DNA and STA genomic DNA could be detected (FIG. 3, lane 3). Neither SAL genomic DNA nor STA genomic DNA was detected in the negative control (FIG. 3, lane 4 and lane 5).
Furthermore, the result of having examined the specificity of the detection primer is shown in FIG. In FIG. 4, lanes 1 to 3 use primers for detecting SAL, the sample contains only SAL genomic DNA (lane 1), the sample contains only STA genomic DNA (lane 2), negative Control (lane 3). Lanes 1 to 3 use a STA detection primer, the sample contains only SAL genomic DNA (lane 4), the sample contains only STA genomic DNA (lane 5), and a negative control (lane 6).
When a LAMP reaction is performed using SAL and STA detection primers with a tag sequence and biotin bound to LF and LB, they are detected as bands only when SAL genomic DNA and STA genomic DNA are present, respectively. (FIG. 4, lane 1 and lane 5).
This revealed that the LAMP reaction was not inhibited even when a tag substance binding substance such as biotin or the like was bound to the LAMP primer. Further, the LAMP reaction product could be detected by the STH chromatography PAS method. Furthermore, it became clear that detection can be performed in a multiplex by mixing a plurality of primers. Moreover, it was shown that the reaction with the primers for detecting SAL and STA is specific.

(Example 2)
1. Objective Of the LAMP primers, which primer should be bound to the tag sequence or biotin was examined.
2. material
<Reagents used in LAMP reaction>
・ 100μM forward inner primer (FIP)
・ 100μM Backward Inner Primer (BIP)
・ 100μM forward inner primer (FIP) _tag sequence binding (5 'end)
・ 100μM Backward Inner Primer (BIP) _Biotin binding (5 'end)
・ 100μM forward loop primer (LF)
・ 100μM backward loop primer (LB)
・ 100μM forward loop primer (LF) _tag sequence binding (5'end)
・ 100μM backward loop primer (LB) _biotin binding (5'end)
・ 100μM forward outer primer (F3)
・ 100μM Backward outer primer (B3)
・ Isothermal Master Mix (OptiGene)
Staphylococcus aureus (STA) genomic DNA
<Reagents used in STH chromatography PAS method> (manufactured by TBA Co., Ltd.)
・ One chromatographic PAS (F4) ・ Chromatodevelopment buffer ・ Dye for chromatographic development

3. Method The primer sequence of the STA detection primer set used was the same as in Example 1. In accordance with the following table, a primer having a tag sequence or biotin bound to the 5 ′ end of each primer was prepared by a conventional method.
Table 3. Combinations of primers with tag sequences or biotin bound and detection results
Detectable: + Undetectable:-Preparation of Probe Mixture was performed as shown in "Primer combinations shown in the upper part of Table 3". In (i), a tag sequence or biotin is bound to the 5 ′ end portion of each of the FIP primer and BIP primer which are inner primers. In (ii), a tag sequence or biotin is bound to the inner primer FIP and the loop primer LB, respectively. In (iii), a tag sequence or biotin is bound to BIP of the inner primer and LF of the loop primer, respectively. Probe Mix is 0.4 μL of FIP, 0.4 μL of BIP, 0.4 μL of LF, 0.4 μL of LB, 0.05 μL of F3, 0.05 μL of B3, and 0.8 μL of sterile water per reaction. did. Next, a LAMP reaction solution was prepared. To the Tube Strip for LAMP reaction, 2 μL of the prepared Probe Mixture, 8 μL of Isotherm Master Mix, and 2 μL of Temp. DNA were added and mixed. Genie II (OptiGene) was used for the LAMP isothermal reaction. The reaction temperature was 65 ° C. and the reaction was performed for 30 minutes.
The LAMP product after the reaction was diluted 1/10 with sterilized water. 1 μL of diluted product, 9 μL of sterilized water, 1 μL of chromatographic developing dye and 10 μL of chromatographic developing buffer were added and mixed. Chromatographic paper was immersed in the mixed reaction solution, and chromatographic development was performed at room temperature for 20 minutes.

4). Results The results are shown in FIG. Among the LAMP primers, (i) when the set of the first primer and the second primer was the inner primer for both forward and backward (FIG. 5, lane 1), a specific band of STA genomic DNA was detected. Similarly, (ii) when the first primer and the second primer are a combination of a forward inner primer and a backward loop primer (FIG. 5, lane 3), or (iii) the first primer and the second primer set are forward. Even in the case of the combination of the inner primer and the backward loop primer (FIG. 5, lane 5), a specific band of STA genomic DNA was detected. In addition, lanes 2, 4 and 6 in FIG. 5 are negative controls (DW) when the combination of primers (i), (ii) and (iii) is used, respectively. Furthermore, since the set of the first primer and the second primer used in Example 1 is a loop primer for both forward and backward, it can be detected by binding a tag sequence or biotin in either the inner primer or the loop primer. I found out that In addition, although a result is not shown, it was not able to be detected when the outer primer which similarly made tag sequence or biotin couple | bond with 5 'terminal was used.

(Example 3)
1. The detection sensitivity of the target LAMP_C-PAS was confirmed.
2. material
<Reagents used in LAMP reaction>
・ 100μM forward inner primer (FIP)
・ 100μM Backward Inner Primer (BIP)
・ 100μM forward loop primer (LF) _tag sequence binding (5'end)
・ 100μM backward loop primer (LB) _biotin binding (5'end)
・ 100μM forward outer primer (F3)
・ 100μM Backward outer primer (B3)
・ Isothermal Master Mix (OptiGene)
Salmonella enterica (SAL) genomic DNA (10 ng, 1 ng, 100 pg, 10 pg, 1 pg, 100 fg, 10 fg and 1 fg)
Staphylococcus aureus (STA) genomic DNA (10 ng, 1 ng, 100 pg, 10 pg, 1 pg, 100 fg, 10 fg, 1 fg)

<Reagents used in STH chromatography PAS method> (manufactured by TBA Co., Ltd.)
・ One chromatographic PAS (F4) ・ Chromatodevelopment buffer ・ Dye for chromatographic development

3. Method The primer sequences of the primer sets for SAL detection and STA detection used were the same as in Example 1. The tag sequence was bound to the 5 ′ end of the LF primer and biotin was bound to the 5 ′ end of the LB primer by a conventional method. As in Example 1, different tag sequences were used for SAL detection and STA detection.
Dilution series were prepared so that SAL genomic DNA and STA genomic DNA were 10 ng, 1 ng, 100 pg, 10 pg, 1 pg, 100 fg, 10 fg, and 1 fg, respectively. When detecting SAL genomic DNA and STA genomic DNA in a mixed state, they were mixed at the same concentration.
Next, preparation of Probe Mixture was performed. Probe Mix is 0.4 μL of FIP, 0.4 μL of BIP, 0.4 μL of LF, 0.4 μL of LB, 0.05 μL of F3, 0.05 μL of B3, and 0.8 μL of sterile water per reaction. did. Next, a LAMP reaction solution was prepared. In the Tube Strip for LAMP reaction, 2 μL of the prepared Probe Mixture, 8 μL of the Islamic Master Mix, Temp. 2 μL of DNA was added and mixed. When the reaction was performed in a multiplex, SAL and Probe Mix for detecting STA were mixed at a ratio of 1: 1. Genie II (OptiGene) was used for the LAMP isothermal reaction. The reaction temperature was 65 ° C. and the reaction was performed for 30 minutes.
The LAMP product after the reaction was diluted 1/10 with sterilized water. 1 μL of diluted product, 9 μL of sterilized water, 1 μL of chromatographic developing dye and 10 μL of chromatographic developing buffer were added and mixed. Chromatographic paper was immersed in the mixed reaction solution, and chromatographic development was performed at room temperature for 20 minutes.

4). Results FIG. 6 shows the results of performing LAMP reaction after diluting SAL genomic DNA and STA genomic DNA and developing the LAMP reaction product by chromatography. In FIG. 6, lanes 1-4 contain 1 ng, 1 pg, 10 fg, 1 fg of SAL genomic DNA in order, and lanes 6-9 contain 1 ng, 1 pg, 10 fg, 1 fg of STA genomic DNA in order. Are negative controls.
Both SAL genomic DNA and STA genomic DNA could be detected up to 10 fg.
In addition, when SAL genomic DNA or STA genomic DNA was diluted and a LAMP reaction was performed and amplification products were detected by a conventional method using SYBRGreen, both of them could be detected up to 10 fg.
Further, FIG. 7 shows the results of mixing the SAL genomic DNA and the STA genomic DNA, diluting those contained in the same sample, performing the LAMP reaction, and chromatographically developing the LAMP reaction product. In FIG. 7, lanes 1 to 5 contain 1 ng, 1 pg, 100 fg, 10 fg, and 1 fg of SAL genomic DNA + STA genomic DNA in order, and lane 6 is a negative control. Both SAL genomic DNA and STA genomic DNA contained in the specimen were detectable up to 1 pg. In the case of 100 fg or less, only one of them was detected.

(Example 4)
1. Objective To study the effect of the position of binding within the primer sequence.
2. Material 100μM Forward Inner Primer (FIP) _Tag sequence binding (3 'end)
・ 100μM Backward Inner Primer (BIP) _Biotin binding (3 'end)
・ 100μM forward inner primer (FIP) _tag sequence binding (5 'and 3' ends)
・ 100μM Backward Inner Primer (BIP) _Biotin binding (5 'and 3' ends)
・ 100μM forward inner primer (FIP) _tag sequence binding (5 'end)
・ 100μM Backward Inner Primer (BIP) _Biotin binding (34 position T, inside B2)
・ 100μM forward inner primer (FIP) _tag sequence binding (5 'end)
・ 100μM Backward Inner Primer (BIP) _Biotin binding (10th T, inside B1c)
・ 100μM forward loop primer (LF) _tag sequence binding (3 'end)
・ 100μM backward loop primer (LB) _biotin binding (3 'end)
・ 100μM forward loop primer (LF) _tag sequence binding (5 'and 3' ends)
・ 100μM backward loop primer (LB) _biotin binding (5 'and 3' ends)
・ 100μM forward loop primer (LF) _tag sequence binding (5'end)
・ 100μM Backward Loop Primer (LB) _Biotin binding (10th position T, inside LB)
・ 100μM forward outer primer (F3) _tag sequence binding (5 'end)
・ 100μM Backward outer primer (B3) _Biotin binding (5 'end)
・ 100μM forward outer primer (F3) _tag sequence binding (3 'end)
・ 100μM Backward outer primer (B3) _Biotin binding (3 'end)
・ 100μM forward outer primer (F3) _Tag sequence binding (5 'and 3' ends)
・ 100μM Backward Outer Primer (B3) _Biotin binding (5 'and 3' ends)
・ 100μM forward outer primer (F3) _tag sequence binding (5 'end)
・ 100μM Backward Outer Primer (B3) _Biotin binding (11 position T, inside B3)
・ 100μM forward inner primer (FIP)
・ 100μM Backward Inner Primer (BIP)
・ 100μM forward loop primer (LF)
・ 100μM backward loop primer (LB)
・ 100μM forward outer primer (F3)
・ 100μM Backward outer primer (B3)
・ Isothermal Master Mix (OptiGene)
Staphylococcus aureus (STA) genomic DNA
<Reagents used in STH chromatography PAS method> (manufactured by TBA Co., Ltd.)
・ One chromatographic PAS (F4) ・ Chromatodevelopment buffer ・ Dye for chromatographic development

3. Method Among the primers necessary for the LAMP reaction, the position where biotin or a tag sequence is bound to the primer sequence was examined.
The primer sequence of the STA detection primer set used was the same as in Example 1. In the usual manner, primers were prepared by binding to both the 5 ′ end, 3 ′ end, 5 ′ end and 3 ′ end of the primer and the inside of the primer sequence. As in Example 1, different tag sequences were used for SAL detection and STA detection.

Next, preparation of Probe Mixture was performed. Probe Mix is 0.4 μL of FIP, 0.4 μL of BIP, 0.4 μL of LF, 0.4 μL of LB, 0.05 μL of F3, 0.05 μL of B3, and 0.8 μL of sterile water per reaction. did. Next, a LAMP reaction solution was prepared. In the Tube Strip for LAMP reaction, 2 μL of the prepared Probe Mixture, 8 μL of the Islamic Master Mix, Temp. 2 μL of DNA was added and mixed. When the reaction was performed in a multiplex, SAL and Probe Mix for detecting STA were mixed at a ratio of 1: 1. Genie II (OptiGene) was used for the LAMP isothermal reaction. The reaction temperature was 65 ° C. and the reaction was performed for 30 minutes.
The LAMP product after the reaction was diluted 1/10 with sterilized water. 1 μL of diluted product, 9 μL of sterilized water, 1 μL of chromatographic developing dye and 10 μL of chromatographic developing buffer were added and mixed. Chromatographic paper was immersed in the mixed reaction solution, and chromatographic development was performed at room temperature for 20 minutes.

4). Results The results are shown in FIG. Table 4 below summarizes the combinations and detection results of the primers to which the tag sequence or biotin is bound in FIG. Lane 1 is a tag sequence or biotin bound to the 5 ′ end of the outer primer, Lane 2 is a tag sequence or biotin bound to the 3 ′ end of the outer primer, and Lane 3 is a 5 ′ end of the outer primer. A tag sequence or biotin bound to both 3 ′ ends, Lane 4 is a tag sequence or biotin bound to the 3 ′ end of the inner primer, and Lane 5 is a 5 ′ end and 3 ′ end of the inner primer. A tag sequence or biotin bound to both, lane 6 is a tag sequence or biotin bound to the 3 ′ end of the loop primer, lane 7 is a tag to both the 5 ′ end and 3 ′ end of the loop primer Sequence or biotin conjugated, lane 8 has a tag sequence at the 5 'end of the inner primer and biochi in B1c of the inner primer Lane 9 is a tag sequence at the 5 ′ end of the inner primer, biotin is bound within B2 of the inner primer, lane 10 is a tag sequence at the 5 ′ end of the loop primer, and within the LB of the loop primer In the lane 11, a tag sequence is used at the 5 ′ end of the outer primer, and biotin is bonded to B3 of the outer primer.
A tag sequence or biotin bound to the 3 ′ end of the inner primer (lane 4), a tag sequence or biotin bound to both the 5 ′ end and 3 ′ end of the inner primer (lane 5), an inner primer A band was detected when a tag sequence was used at the 5 'end and biotin was bound inside the inner primer (lane 9). Similarly, a tag sequence or biotin bound to the 3 ′ end of the loop primer (lane 6), a tag sequence or biotin bound to both the 5 ′ end and the 3 ′ end of the loop primer (lane 7). A band was also detected when a tag sequence was used at the 5 ′ end of the loop primer and biotin was bound inside the loop primer (lane 8). On the other hand, a tag sequence or biotin bound to the 5 ′ end of the outer primer (lane 1), a tag sequence or biotin bound to the 3 ′ end of the outer primer (lane 2), and the 5 ′ end of the outer primer When a tag sequence or biotin is bound to both of the 3 ′ end and the 3 ′ end (lane 3), a tag sequence is bound to the 5 ′ end of the outer primer, and a biotin is bound to the inside of the outer primer (lane 11) No band was detected for any of the above.
It was.

Table 4 Combinations and detection results of the primer sequences bound to the tag sequence or biotin in FIG.


Detectable: + Undetectable:-

  Combined with the detection in Example 2 using a tag sequence or biotin bound to the 5 ′ end of the inner primer, or a tag primer or biotin bound to the 5 ′ end of the loop primer. Table 2 below summarizes the influence of the label position in the primer sequence on the detectability.

Table 5
Detectable: + Undetectable:-

(Example 5)
Contamination prevention measures Objective Examination of LAMP_C-PAS reaction system using Uracil-DNA Glycosylas (UNG) Materials ・ Isothermal Master Mix (OptiGene)
・ 2 × LAMP Master Mix (Nippon Gene Co., Ltd.)
・ Uracil-DNA Glycosylas (UNG) 1 Units / μL (Nippon Gene Co., Ltd.)
・ 100μM forward inner primer (FIP)
・ 100μM Backward Inner Primer (BIP)
・ 100μM forward loop primer (LF) _tag sequence binding (5'end)
・ 100μM backward loop primer (LB) _biotin binding (5'end)
・ 100μM forward outer primer (F3)
・ 100μM Backward outer primer (B3)
・ Staphylococcus aureus genomic DNA
<Reagents used in STH chromatography PAS method> (manufactured by TBA Co., Ltd.)
・ Chromatographic PAS (F8)
・ Chromatographic development buffer 10μL
・ Dye for chromatographic development 1μL

3. Method (1) In the LAMP reaction system, even when dUTP is added instead of dTTP, in order to confirm whether or not LAMP polymerase reacts, nucleotides using dNTPs are used so that dTTP: dUTP = 1: 1 Were compared with those obtained by substituting dUTP contained in dNTPs with dUTP and those obtained by substituting all dTTP contained in dNTPs with dUTP.
The primer sequence of the STA detection primer set used was the same as in Example 1. The tag sequence was bound to the 5 ′ end of the LF primer and biotin was bound to the 5 ′ end of the LB primer by a conventional method.
Probe Mix is 0.2 μL of FIP, 0.2 μL of BIP, 0.1 μL of LF, 0.1 μL of LB, 0.025 μL of F3, 0.025 μL of B3, and 1.85 μL of sterile water per reaction. did. Next, a LAMP reaction solution was prepared. In Tube Strip for LAMP reaction, 2.5 μL of prepared Probe Mixture, 12.5 μL of 2 × LAMP Master Mix (including Bst DNA polymerase), Temp. 2.5 μL of DNA and 6.5 μL of sterilized water were added and mixed.
For the LAMP isothermal reaction, LightCycler 96 (Roche Diagnostics Inc.) was used. The reaction temperature was 65 ° C. and the reaction was performed for 60 minutes.
(2) In order to prevent contamination due to carryover, Uracil-DNA Glycosylate (UNG) (Nippon Gene Co., Ltd.) was added to the LAMP reaction system to confirm the influence on the LAMP reaction. Probe Mix is 0.4 μL of FIP, 0.4 μL of BIP, 0.2 μL of LF, 0.2 μL of LB, 0.05 μL of F3, 0.05 μL of B3, and 1.2 μL of sterile water per reaction. did. Next, a LAMP reaction solution was prepared. In the Tube Strip for LAMP reaction, 2.5 μL of the prepared Probe Mixture, 12.5 μL of 2 × LAMP Master Mix (including Bst polymerase or Csa polymerase), 1.0 μL of UNG, Temp. 2.5 μL of DNA was added and mixed. For the LAMP isothermal reaction, LightCycler 96 (Roche Diagnostics Inc.) was used. The reaction temperature was 65 ° C. and the reaction was performed for 60 minutes. UNG treatment was performed by incubating at room temperature for 10 minutes prior to the LAMP isothermal reaction.
The LAMP product after the reaction was diluted 1/10 with sterilized water. 1 μL of diluted product, 9 μL of sterilized water, 1 μL of chromatographic developing dye and 10 μL of chromatographic developing buffer were added and mixed. Chromatographic paper was immersed in the mixed reaction solution, and chromatographic development was performed at room temperature for 20 minutes.

4). Results FIG. 9 shows the influence on the LAMP reaction when dTTP contained in dNTPs as a nucleotide is replaced with dUTP.
Similar to the normal LAMP reaction using dNTPs as nucleotides ((1) in FIG. 9), dTTP contained in dNTPs was replaced with dUTP so that dTTP: dUTP = 1: 1 ((2 in FIG. 9). )), The LAMP reaction proceeded without any problems even when all dTTP contained in dNTPs was replaced with dUTP ((3) in FIG. 9).
In addition, FIG. 10 and FIG. 11 show the results of examining the influence on carryover by the presence or absence of UNG processing. Fig. 10 shows whether a LAMP reaction using the LAMP reaction product as a template occurs with LAMP Master Mix using two enzymes, Bst DNA Polymerase or Csa DNA Polymerase, with and without UNG added. It is a thing. Even when LAMP Master Mix using any enzyme was used, the LAMP reaction did not proceed when the reaction was carried out by adding UNG.
In FIG. 11, lane 1 is a reaction obtained by adding UNG using STA genomic DNA as a template, lane 2 is a reaction obtained by adding UNG using STA genomic DNA and a STA LAMP amplification product as a template, lane 3 is an STA Reacted with LAMP amplified product as template and added UNG, Lane 4 reacted with STA genomic DNA as template and UNG not added, Lane 5 with STA genomic DNA and STA LAMP amplified product as template Lane 6 shows the reaction without addition of UNG using the STA LAMP amplification product as a template. In any case, in the system to which UNG was added, that is, the system in which UNG treatment was performed, no LAMP reaction was observed (lane 3 in FIGS. 10 and 11), and it was confirmed that no carryover occurred.

(Example 6)
1. Objective Examination when using only inner primer, loop primer or outer primer for LAMP reaction material
<Reagents used in LAMP reaction>
・ Isothermal Master Mix (manufactured by OptiGene)
・ 100μM forward inner primer (FIP) _ tag sequence binding (5 'end)
・ 100μM Backward Inner Primer (BIP) _Biotin binding (5 'end)
・ 100μM forward loop primer (LF) _tag sequence binding (5'end)
・ 100μM backward loop primer (LB) _biotin binding (5'end)
・ 100μM forward outer primer (F3) _tag sequence binding (5 'end)
・ 100μM Backward outer primer (B3) _Biotin binding (5 'end)
Staphylococcus aureus genomic DNA (STA)
<Reagents used in STH chromatography PAS method> (manufactured by TBA Co., Ltd.)
・ Chromatographic PAS (F8)
・ Chromatographic development buffer 10μL
・ Dye for chromatographic development 1μL

3. Method Whether only the inner primer, loop primer or outer primer was used, it was confirmed whether the LAMP reaction would proceed.
The primer sequence of the STA detection primer set used was the same as in Example 1. The position for binding the tag sequence or biotin was all at the 5 ′ end of the primer. In the preparation of Primer Mixture, 0.4 μL of FIP and 0.4 μL of BIP or 0.4 μL of LF and 0.4 μL of LB or 0.4 μL of F3 and 0.4 μL of B3 are added per reaction, and sterile water is added per reaction. To a total of 2.5 μL. Next, a LAMP reaction solution was prepared. In Tube Strip for LAMP reaction, 2.5 μL of prepared Primer Mixture, 8 μL of Isothermal Master Mix, Temp. 2 μL of DNA was added and mixed. When the reaction was performed in a multiplex, Salmonella enterica and Primer Mixture for Staphylococcus aureus were mixed 1: 1 respectively. Genie II (OptiGene) was used for the LAMP isothermal reaction. The reaction temperature was 65 ° C. and the reaction was performed for 30 minutes.
The LAMP product after the reaction was diluted 1/10 with sterilized water. 1 μL of diluted product, 9 μL of sterilized water, 1 μL of chromatographic developing dye and 10 μL of chromatographic developing buffer were added and mixed. Chromatographic paper was immersed in the mixed reaction solution, and chromatographic development was performed at room temperature for 20 minutes.

4). Results The results are shown in FIG. FIG. 12 shows STA genomic DNA (lane 1), negative control (lane 2) when only the inner primer is used, STA genomic DNA (lane 3), negative control (lane 4), and outer when only the loop primer is used. STA genomic DNA (lane 5) and negative control (lane 6) in the case of using only primers and primers are shown.
As a result of using any combination of inner primer (FIP and BIP), loop primer (LF and LB) and outer primer (F3 and B3) in the LAMP reaction, only the inner primer (FIP and BIP) is used (lane) It was detected as a band only in 1).

Claims (7)

A method for detecting a target nucleic acid sequence contained in one or more of the same specimen,
The following (a) to (d):
(A) preparing a set of a first primer and a second primer for each target nucleic acid sequence,
When a tag sequence is bound to the first primer and two or more target nucleic acid sequences are included, the tag sequence is different for each target nucleic acid sequence,
The step wherein a labeling substance-binding substance is bound to the second primer;
(B) a step of performing nucleic acid isothermal amplification by the LAMP method in the same reaction solution using a set of one or more first and second primers,
(C) preparing a development medium containing an amplification reaction solution containing the nucleic acid isothermal amplification product and a labeling substance that binds to the labeling substance binding substance, and performing nucleic acid chromatography on a solid phase carrier;
Including a step of hybridizing the one or more detection probes immobilized on the solid phase carrier and the nucleic acid isothermal amplification product, and a step of labeling the nucleic acid isothermal amplification product with a labeling substance;
(D) including a detection step of detecting the hybridized product generated in the step (c) with the labeling substance,

Arbitrary sequences F3c, F2c, LF, F1c, B1, LBc, B2, and B3 arranged in the direction from the 3 ′ end to the 5 ′ end of the target nucleic acid sequence The first and second primers are the following (i) to (iv) :
(I) FIP primers containing the same sequence as F1c on the 5 ′ side of sequences F2 and F2 complementary to F2c,
(Ii) a BIP primer comprising the same sequence as B2 and a sequence B1c complementary to B1 on the 5 ′ side of B2,
(Iii) an LF primer having the same sequence as the LF region, and (iv) an LB primer having a sequence complementary to the LBc region,
A method arbitrarily selected from the group consisting of:   The method according to claim 1, wherein the set of the first primer and the second primer is such that the first primer is an FIP primer and the second primer is a BIP primer or the first primer is a BIP primer and the second primer is an FIP primer.   The set of the first primer and the second primer of each target nucleic acid sequence is such that the first primer is an FIP primer and the second primer is a BIP primer or the first primer is a BIP primer and the second primer is an FIP primer, and the nucleic acid isothermal The method according to claim 1 or 2, wherein a primer other than the set of the first primer and the second primer of each target nucleic acid sequence is not used in the amplification step. The labeling substance binding substance is one or more selected from the group consisting of an antibody in an antigen-antibody reaction and a hapten containing biotin, digoxigenin and FITC,
The labeling substance has a site capable of binding to the labeling substance binding substance, and uses one or more kinds of labeling substances selected from the group consisting of fluorescence, radioactivity, enzyme, phosphorescence, chemiluminescence, and coloring. The method according to claim 1, wherein   The method according to any one of claims 1 to 4, wherein the nucleic acid isothermal amplification step is performed in the presence of dUTP, and the uracil DNA glycosylase (UNG) treatment is performed before the nucleic acid isothermal amplification step.   A measurement reagent or a reagent kit, which is used for carrying out the detection method according to any one of claims 1 to 5 and includes at least a set of a first primer and a second primer.   The measurement reagent or reagent kit according to claim 6, further comprising one or more detection probes immobilized on a solid phase carrier.