JP2017158523A - Primer set, kit and method for detecting Listeria monocytogenes - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a detection method with high determination accuracy which makes it possible to avoid the formation of primer dimers in a method for detecting nucleic acid derived from Listeria monocytogenes including a nucleic acid amplification reaction and detection on a solid phase carrier.SOLUTION: A primer set is for target nucleic acids derived from Listeria monocytogenes which are amplified by a nucleic acid amplification reaction, with the amplification product being bound to a solid phase, and comprises: a first primer including a polynucleotide included at the 3' end of a base sequence homologous to a first base sequence in the target nucleic acid; a second primer including a polynucleotide included at the 3' end of a base sequence homologous to a complementary base sequence of a second base sequence downstream of the first base sequence in the target sequence; one of the first primer and the second primer further includes a tag which can bind to a labelling substance or includes a labelling site which is a labelling substance, and the other further includes a tag which is a binding site which can bind to the solid phase carrier.SELECTED DRAWING: None

Description

  The present invention relates to a primer set for amplifying and detecting a target nucleic acid derived from Listeria monocytogenes.

  The present invention also relates to a method for detecting Listeria monocytogenes and / or nucleic acid derived from Listeria monocytogenes.

  The present invention also relates to a kit for detecting Listeria monocytogenes and / or nucleic acid derived from Listeria monocytogenes.

  In order to prevent the occurrence of food poisoning and the spread of infection, it is important to quickly detect and identify the causative agent of food poisoning from food. In order to identify the causative bacteria of food poisoning, a culture test is generally used. However, since culture requires a number of days, there is a problem in terms of rapidity in preventing the occurrence of food poisoning and the spread of infection.

  In particular, Listeria monocytogenes, which is a harmful organism that is a zoonotic infectious animal and causes food poisoning, has a very slow growth rate, so it takes a long time to detect it in the culture test. There is a need to provide quick detection means.

  As a method for identifying a pathogenic bacterium instead of a culture method, a nucleic acid test method for amplifying and detecting a specific region on the genomic DNA of a target bacterium is being put into practical use. For example, in Non-Patent Document 1 and Non-Patent Document 2, each gene is amplified using each primer set designed in the iap gene, the hly gene, or the prf gene of Listeria monocytogenes, A nucleic acid test method for separation and detection by electrophoresis is disclosed.

  In Non-Patent Document 3 and Non-Patent Document 4, each gene is amplified by real-time PCR using each primer set designed in the hly gene or inl gene of Listeria monocytogenes, and nucleic acid amplification A nucleic acid test method (real-time PCR method) for monitoring and detecting the amount is disclosed.

  In the nucleic acid test method using electrophoresis, the amplified nucleic acid is separated by electrophoresis and detected by fluorescent dye and UV irradiation, but the operation is complicated and it takes time to confirm the result. There is still room for improvement because of the need for carcinogenic dyes.

  In addition, the nucleic acid test method using the real-time PCR method has improved convenience compared to the electrophoresis method in that it can be automatically monitored, but it requires a large-scale device, so that advanced analysis and work space are possible. There is a problem that is necessary.

  Nucleic acid chromatography has been developed as a simpler detection method. Nucleic acid chromatography is widely used because of its rapidity and simplicity. This detection method is roughly classified into two methods. The first method is disclosed in Patent Document 1. In Patent Document 1, a polymerase chain reaction (hereinafter referred to as “PCR”) is performed using a gene specific for Listeria monocytogenes as a target to obtain a target DNA as a double-stranded nucleic acid. On the other hand, a solid phase support on which a capture probe composed of a complementary oligonucleotide capable of hybridizing to a target DNA is bound and a labeling probe composed of a labeled complementary oligonucleotide capable of hybridizing to the target DNA is held Prepare. The target DNA is made into a single strand and brought into contact with a solid support, whereby the target DNA is captured, labeled and detected. This method is easy to operate because it does not require a large-scale apparatus used for separation of amplification products by electrophoresis or real-time PCR. However, this method of Patent Document 1 requires a step of denaturing the target DNA amplified by PCR from double-stranded to single-stranded, and the procedure is complicated, and reassociation to double-stranded nucleic acid is required. There is a problem that the sensitivity is lowered easily.

  As another method, there is a method disclosed in Patent Document 2. In Patent Document 2, a primer specific to a target nucleic acid is attached with a tag and a nucleic acid amplification reaction such as PCR is performed using a DNA containing the target nucleic acid as a template. The obtained amplification product is diffused and moved in the porous solid phase carrier by a capillary phenomenon, captured and bound to the label and the solid phase carrier via the tags attached to both ends of the amplification product, and the target nucleic acid is bound. Detect visually. This method, as described in Patent Document 1, can be easily detected without the need for heat denaturation, and is superior without worrying about a decrease in sensitivity due to reassociation from single-stranded nucleic acid to double-stranded. It can be said that. However, since this detection method relies on a tag derived from a primer attached to both ends of the amplification product to perform capture and binding to a label and a solid phase carrier, it is specific to the target nucleic acid. In some cases, an amplification product and a primer dimer formed by a non-specific reaction cannot be distinguished. Primer dimer formation is not a hindrance because it can be distinguished by the difference in target amplification product length in the conventional electrophoresis method and real-time PCR method, but it is derived from primers attached to both ends of the amplification product as in Patent Document 2. In the detection method that relies on the tag and captures and binds to the label and the solid phase carrier, there is a possibility that false positive determination is performed by detecting the formed primer dimer.

JP 2013-198482 A WO2012 / 070618

Annals of Biological Research, 2012, 3 (4): 1880-1884 Japanese Journal of Food Microbiology, 2000, 17 (2): 121-126, FOODBORNE PATHOGENS AND DISEASE, 2006, 3, Number 4, Biomed Environ Sciences, 2014, 27 (10): 770-778

  As described above, an amplification product of a target nucleic acid to which an immobilization tag derived from a primer and a detection tag or a labeling substance are added is generated by a nucleic acid amplification reaction, and the generated amplification product is placed on a solid phase carrier. In the nucleic acid chromatography method in which the primer dimer is formed in the nucleic acid amplification reaction, the primer dimer and the amplification product of the target nucleic acid cannot be distinguished at the time of detection and show false positives.

  Thus, the present invention avoids the formation of primer dimers in a method for detecting nucleic acids derived from Listeria monocytogenes, including nucleic acid amplification reactions and detection of nucleic acid amplification products on a solid phase carrier (for example, nucleic acid chromatography). It is an object of the present invention to provide a new detection means with high determination accuracy that makes it possible.

The present invention discloses the following invention as means for solving the above problems.
(1) A primer set for amplifying a target nucleic acid derived from Listeria monocytogenes by a nucleic acid amplification reaction and binding the amplified product to a solid phase carrier,
A first primer comprising a polynucleotide comprising a base sequence homologous to the first base sequence contained in the target nucleic acid at the 3 ′ end;
A second primer comprising a polynucleotide containing, at the 3 ′ end, a base sequence homologous to a complementary base sequence to a second base sequence located downstream of the first base sequence in the target nucleic acid,
One of the first primer and the second primer further includes a tag that can bind to the labeling substance or a labeling part that is a labeling substance, and the other further includes a binding part that is a tag that can bind to the solid phase carrier. set.
(2) The target nucleic acid is an iap gene, hly gene, lma gene, actA gene, inl gene, 16S rRNA gene, ssr gene, fla gene, plc gene, prf gene or mpl gene, (1) Primer set.
(3) The target nucleic acid is an iap gene,
The first primer is
(1a) a polynucleotide having a base sequence homologous to the base sequence of SEQ ID NO: 1 at the 3 'end, or a nucleotide number of 40 or less, or (1b) from the 3' end of the base sequence homologous to the base sequence of SEQ ID NO: 1. A polynucleotide having a base number of 40 or less, comprising a sequence of a portion consisting of 10 or more consecutive bases at the 3 ′ end;
The second primer
(2a) a polynucleotide having a base sequence homologous to the base sequence of SEQ ID NO: 2 at the 3 'end, or a polynucleotide having 40 bases or less, or (2b) from the 3' end of the base sequence homologous to the base sequence of SEQ ID NO: 2. The primer set according to (2), comprising a polynucleotide having a base number of 40 or less, comprising a sequence of a portion consisting of 10 or more consecutive bases at the 3 ′ end.
(4) The target nucleic acid is an hly gene,
The first primer is
(3a) a polynucleotide having a base sequence homologous to the nucleotide sequence of SEQ ID NO: 3 at the 3 'end, or a polynucleotide having 40 bases or less, or (3b) from the 3' end of the nucleotide sequence homologous to the nucleotide sequence of SEQ ID NO: 3 A polynucleotide having a base number of 40 or less, comprising a sequence of a portion consisting of 10 or more consecutive bases at the 3 ′ end;
The second primer
(4a) a polynucleotide having a base sequence homologous to the base sequence of SEQ ID NO: 4 at the 3 'end, or a polynucleotide having 40 bases or less, or (4b) from the 3' end of the base sequence homologous to the base sequence of SEQ ID NO: 4 The primer set according to (2), comprising a polynucleotide having a base number of 40 or less, comprising a sequence of a portion consisting of 10 or more consecutive bases at the 3 ′ end.
(5) The target nucleic acid is an hly gene,
The first primer is
(7a) a polynucleotide having a base number of 40 or less containing a base sequence homologous to the base sequence of SEQ ID NO: 7 at the 3 ′ end, or (7b) from the 3 ′ end of the base sequence homologous to the base sequence of SEQ ID NO: 7 A polynucleotide having a base number of 40 or less, comprising a sequence of a portion consisting of 10 or more consecutive bases at the 3 ′ end;
The second primer
(8a) a polynucleotide having a base number of 40 or less containing a base sequence homologous to the base sequence of SEQ ID NO: 8 at the 3 ′ end, or (8b) from the 3 ′ end of the base sequence homologous to the base sequence of SEQ ID NO: 8 The primer set according to (2), comprising a polynucleotide having a base number of 40 or less, comprising a sequence of a portion consisting of 10 or more consecutive bases at the 3 ′ end.
(6) The primer set according to any one of (1) to (5), wherein the labeling part is a tag containing a polynucleotide or a low molecular weight compound that can bind to the labeling substance.
(7) The primer set according to any one of (1) to (6), wherein the binding portion is a tag containing a polynucleotide that can bind to a solid phase carrier.
(8) A method for detecting a nucleic acid derived from Listeria monocytogenes and / or Listeria monocytogenes in a sample,
A nucleic acid amplification step of using a DNA obtained from a sample as a template and performing a nucleic acid amplification reaction using any one of the primer sets of (1) to (7);
A detection step of contacting the product of the nucleic acid amplification reaction with a solid phase carrier including at least a portion capable of binding to the binding portion, and detecting an amplification product in the portion of the solid phase carrier using the labeling portion as an index. Including methods.
(9) The labeling part is a tag that can bind to the labeling substance,
The method according to (8), further comprising a labeling step of binding a labeling substance to the tag of the labeling unit.
(10) The method according to (8) or (9), wherein the nucleic acid amplification step is a step of performing polymerase chain reaction (PCR) for 25 cycles or more.
(11) The nucleic acid amplification step is performed under the condition that the concentration at the start of each of the first primer and the second primer in the primer set of any one of (1) to (7) is 0.05 μM or more. The method according to any one of (8) to (10), which is a step of performing a reaction (PCR).
(12) A kit for detecting a nucleic acid derived from Listeria monocytogenes and / or Listeria monocytogenes,
A primer set of any one of (1) to (7);
A kit comprising a binding part and a solid phase carrier containing at least a part capable of binding.
(13) The kit according to (12), comprising a nucleic acid detection device comprising the solid phase carrier partially including a portion capable of binding to the binding portion, and a reaction system accepting portion for receiving a product of the nucleic acid amplification reaction. .
(14) The labeling part is a tag that can bind to the labeling substance,
The kit according to (12) or (13), further comprising a labeling substance that can bind to the tag of the labeling part.
(15) A reaction system for receiving a product (amplification product) of a nucleic acid amplification reaction, the solid phase carrier partially containing a binding part and a labeling substance holding part for holding the labeling substance The kit according to (14), comprising a nucleic acid detection device comprising a receiving part.

  According to the present invention, it is possible to detect Listeria monocytogenes in a sample with high accuracy.

FIG. 1 is a schematic diagram of a lateral flow type nucleic acid detection device 10. 1: solid phase carrier, 2: conjugate pad (labeling substance holding part), 3: sample pad (reaction system receiving part), 4: absorption pad, 5: base material, 6: tag capturing means of solid phase carrier 1 Including part. FIG. 2 is a photograph showing the results of a nucleic acid chromatography test of a negative control PCR reaction solution obtained in Example 1 Test 1 using each primer set. FIG. 3 is a photograph showing the results of analyzing a PCR reaction solution using the Mono primer set colored in the negative control test in Example 1 Test 1 by agarose electrophoresis. FIG. 4 shows the results of nucleic acid chromatography detection of PCR amplification products obtained in Example 1 Test 2 using Iap primers as a primer set and each concentration of Listeria monocytogenes genomic DNA as a template. It is a photograph. FIG. 5 shows the detection results of PCR amplification products obtained in Example 1 and Test 2 by PCR using the Hly primer as a primer set and each concentration of Listeria monocytogenes genomic DNA as a template. It is a photograph.

Hereinafter, the present invention will be described in detail.
1. Primer Set of the Present Invention The present invention provides a primer set for amplifying a target nucleic acid derived from Listeria monocytogenes by a nucleic acid amplification reaction and detecting the amplified product on a solid phase carrier.

  In the present invention, the terms nucleic acid and polynucleotide refer to DNA or RNA, more preferably DNA. The terms nucleic acid and polynucleotide are not particularly limited in the number of nucleotides, but also include oligonucleotides.

  In the present invention, the target nucleic acid refers to a nucleic acid whose whole or a part is to be amplified. The target nucleic acid may be any nucleic acid derived from Listeria monocytogenes, may be a nucleic acid naturally present in Listeria monocytogenes, or may be a nucleic acid artificially prepared from a naturally occurring nucleic acid. May be. Examples of the target nucleic acid include genomic DNA, mRNA transcribed from the genomic DNA, cDNA prepared from the mRNA, tRNA, rRNA and the like, and genomic DNA and cDNA are particularly preferable. Each term such as genomic DNA, mRNA, cDNA, tRNA, rRNA and the like also encompasses fragments thereof. The target nucleic acid can be single-stranded or double-stranded DNA, RNA, and / or a DNA / RNA hybrid. When a double-stranded target nucleic acid is used, it can be made into a single strand by normal denaturation treatment and used for a nucleic acid amplification reaction. When the target nucleic acid is double-stranded, the base sequence of the target nucleic acid refers to the base sequence contained in one of the strands.

  Specific examples of the target nucleic acid include Listeria monocytogenes iap gene, hly gene, lma gene, actA gene, inl gene, 16S rRNA gene, ssr gene, fla gene, plc gene, prf gene or mpl gene. As described above, each gene can be a nucleic acid such as genomic DNA, mRNA, cDNA or the like containing a base sequence corresponding to each gene, or a fragment consisting of a partial base sequence thereof.

  The iap gene is a gene encoding invasion associated protein (invasion-related protein) p60 having a sequence specific to Listeria monocytogenes, and has a base sequence shown in SEQ ID NO: 15 in genomic DNA.

  The hly gene is a gene encoding Listeriolsin O (Listeriolysin O) having a sequence specific to Listeria monocytogenes, and has a base sequence shown in SEQ ID NO: 16 in genomic DNA.

The lma gene is a gene having a sequence specific to Listeria monocytogenes, and has a base sequence shown in SEQ ID NO: 19 in genomic DNA.
The actA gene is a gene having a sequence specific to Listeria monocytogenes, and has a base sequence shown in SEQ ID NO: 20 in genomic DNA.

The inl gene is a gene having a sequence specific to Listeria monocytogenes, and has a base sequence shown in SEQ ID NO: 17 in genomic DNA.
The 16S rRNA gene is a gene having a sequence specific to Listeria monocytogenes, and has a base sequence shown in SEQ ID NO: 21 in genomic DNA.

The ssr gene is a gene having a sequence specific to Listeria monocytogenes, and has a base sequence shown in SEQ ID NO: 22 in genomic DNA.
The fla gene is a gene having a sequence specific to Listeria monocytogenes, and has a base sequence shown in SEQ ID NO: 23 in genomic DNA.

The plc gene is a gene having a sequence specific to Listeria monocytogenes, and has a base sequence shown in SEQ ID NO: 24 in genomic DNA.
The prf gene is a gene having a sequence specific to Listeria monocytogenes, and has a base sequence shown in SEQ ID NO: 25 in genomic DNA.
The mpl gene is a gene having a sequence specific to Listeria monocytogenes, and has a base sequence shown in SEQ ID NO: 18 in genomic DNA.

  The primer set of the present invention includes a first primer (functioning as a forward primer) and a second primer (functioning as a reverse primer), and one of the first primer and the second primer is a labeling substance. It further includes a labeling part that is a tag or a labeling substance that can be bound, and the other further includes a binding part that is a tag that can be bound to a solid phase carrier. Therefore, hereinafter, the features of the first primer, the second primer, the labeling part, and the binding part will be described in detail in order.

  In the present invention, when “base sequence Y homologous to base sequence X” or “base sequence X and base sequence Y are homologous”, when base sequences X and Y are the same, The nucleic acid amplification reaction is not limited to a nucleic acid containing a complementary sequence of the base sequence X (for example, a target nucleic acid or a complementary strand thereof) and a nucleic acid containing the base sequence Y (for example, the primer portion of the first or second primer). The base sequences X and Y may be partially different as long as the combination is capable of forming a hydrogen bond sufficient to hybridize under conditions to form a stable duplex. For example, a nucleic acid consisting of a complementary sequence of base sequence X and a nucleic acid consisting of base sequence Y have several mismatches such as 1 mismatch in 10 nucleotides, 1 mismatch in 20 nucleotides, or 1 mismatch in 30 nucleotides. May be present. Typically, when the base sequence X is “homologous” with the base sequence X, it means that the base sequences X and Y satisfy any of the following relationships.

  (A) The base sequence X and the base sequence Y are the same. When one of the base sequence X and the base sequence Y is a DNA base sequence and the other is an RNA base sequence, the thymine on one side and the uracil on the other side are regarded as the same base.

  (B) The base sequence Y is a base sequence in which one or several bases are deleted, substituted, added and / or inserted in the base sequence X.

(C) The base sequence Y is a base sequence having 70% or more identity with the base sequence X.
(D) A polynucleotide comprising a base sequence Y can hybridize with a polynucleotide comprising a base sequence complementary to SEQ ID NO: X under stringent conditions.

  In the above (B), “1 or several” preferably refers to 1 to 5, more preferably 1 to 4, more preferably 1 to 3, particularly preferably 1 or 2, most preferably One.

  In (C) above, the identity value indicates a value calculated by default using software (for example, FASTA, DANASYS, and BLAST) that calculates identity between a plurality of base sequences. The base sequence identity value is calculated by calculating the number of matching bases when aligning a pair of base sequences so that the degree of matching is maximized. Calculated as a percentage of the number. Here, when there is a gap, the total number of bases is the number of bases obtained by counting one gap as one base. For details of how identity is determined, see, for example, Altschul et al, Nuc. Acids. Res. 25, 3389-3402, 1977 and Altschul et al, J. Mol. Biol. 215, 403-410, 1990. .

  In the above (C), the identity is more preferably 80% or more, more preferably 90% or more, more preferably 95% or more, more preferably 96% or more, more preferably 97% or more, more preferably 98% or more. More preferably, the identity is 99% or more.

  In the above (D), the “stringent condition” means a condition in which a so-called specific hybrid is formed and a non-specific hybrid is not formed. For example, Green and Sambrook, Molecular Cloning, 4th Ed (2012) , Can be determined as appropriate with reference to Cold Spring Harbor Laboratory Press. Specifically, stringent conditions can be set according to the temperature at the time of Southern hybridization and the salt concentration contained in the solution, and the temperature at the time of the washing step of Southern hybridization and the salt concentration contained in the solution. More specifically, as stringent conditions, for example, in the hybridization step, the sodium concentration is 25 to 500 mM, preferably 25 to 300 mM, and the temperature is 40 to 68 ° C., preferably 40 to 65 ° C. More specifically, hybridization can be performed at 1 to 7 × SSC, 0.02 to 3% SDS, and a temperature of 40 ° C. to 60 ° C. In addition, a washing step may be performed after the hybridization, and the washing step can be performed, for example, at 0.1 to 2 × SSC, 0.1 to 0.3% SDS, and a temperature of 50 to 65 ° C.

1.1. 1st primer and 2nd primer The 1st primer contains the polynucleotide which contains the base sequence homologous to the 1st base sequence contained in a target nucleic acid at 3 'terminal as a primer part.
On the other hand, the second primer has a base sequence homologous to a complementary base sequence with respect to the second base sequence located downstream (that is, on the 3 ′ end side) of the target nucleic acid as the primer portion. Polynucleotides at the ends are included.

By using this primer set, it is possible to amplify a region from the base at the 5 ′ end of the first base sequence to the base at the 3 ′ end of the second base sequence in the target nucleic acid. The length of the region to be amplified is not particularly limited, but is usually 40 bases or more, or 100 bases or more, and the upper limit is not particularly limited, but is usually 1000 bases or less, 500 bases or less, or 400 bases or less. That's it.
“Homology” is as defined above.

  Each of the first base sequence and the second base sequence is not particularly limited in length, but may be, for example, 8 bases or more, 12 bases or more, or 15 bases or more, 40 bases or less, 30 bases or less, Or it can be 25 bases or less.

  In the present invention, the “primer portion” is a portion containing a polynucleotide that is annealed to a target nucleic acid or a complementary strand thereof in a nucleic acid amplification reaction and serves as a starting point for an extension reaction, and connects a labeling portion, a binding portion, and those described later. Does not contain groups.

  The polynucleotide constituting the primer portion of the first primer only needs to contain a base sequence homologous to the first base sequence at the 3 ′ end, and the 5 ′ end of the base sequence homologous to the first base sequence. Further, another base sequence may be added to the side. Although the total length of the polynucleotide constituting the primer portion of the first primer is not particularly limited, it can be 8 bases or more, 12 bases or more, or 15 bases or more, for example, 40 bases or less, 30 bases or less, or 25 bases It can be as follows.

  Similarly, the polynucleotide constituting the primer portion of the second primer only needs to contain a base sequence that is homologous to the complementary base sequence to the second base sequence at the 3 ′ end, and is complementary to the second base sequence. Another base sequence may be further added to the 5 ′ end side of the base sequence homologous to the base sequence. The total length of the polynucleotide constituting the primer portion of the second primer is not particularly limited, but can be, for example, 8 bases or more, 12 bases or more, or 15 bases or more, and 40 bases or less, 30 bases or less, or 25 bases It can be as follows.

  In the present invention, the polynucleotides constituting the respective primer portions of the first primer and the second primer can be produced by a known technique, and may be produced using a polynucleotide synthesizer or contracted. You may manufacture using a synthesis service.

  The first primer has a structure in which the polynucleotide and one of a labeling part or a binding part to be described later are connected and, if necessary, chemically linked via an appropriate spacer to be described later. In the first primer, the position where one of the labeling portion and the binding portion is linked to the polynucleotide is determined by annealing between the polynucleotide and the target nucleic acid or a nucleic acid complementary to the target nucleic acid, and extension in the nucleic acid amplification reaction. Although it will not specifically limit if it is a position which does not inhibit, Preferably, it is 5 'terminal of the said polynucleotide.

  Similarly, the second primer has a structure in which the polynucleotide and one of a labeling part or a binding part to be described later are connected and, if necessary, chemically linked via an appropriate spacer to be described later. In the second primer, the position at which one of the labeling portion and the binding portion is linked to the polynucleotide is determined by annealing between the polynucleotide and the target nucleic acid or a nucleic acid complementary to the target nucleic acid and extension in the nucleic acid amplification reaction. Although it will not specifically limit if it is a position which does not inhibit, Preferably, it is 5 'terminal of the said polynucleotide.

1.2. Preferred combination of primers 1
In the first preferred combination in the primer set of the present invention,
A first primer comprising the polynucleotide of (1a) or (1b), and
The second primer includes the polynucleotide (2a) or (2b).
This primer set is particularly suitable when the iap gene is used as a target nucleic acid.

  A nucleotide sequence homologous to the nucleotide sequence of SEQ ID NO: 1 in the polynucleotide (1a) is defined as nucleotide sequence 1a. This base sequence 1a satisfies the relationship (A), (B), (C) or (D) with the base sequence of SEQ ID NO: 1 (the base sequence of SEQ ID NO: 1 is base sequence X, and the base sequence 1a is Corresponding to base sequence Y).

  When the base sequence 1a satisfies the relationship of (B) with the base sequence of SEQ ID NO: 1, more preferably, the base sequence 1a is derived from one or both of the 5 ′ end and the 3 ′ end in the base sequence of SEQ ID NO: 1. A sequence in which one or several bases are deleted in total, particularly preferably a sequence in which one or several bases are deleted from only the 5 ′ end in the base sequence of SEQ ID NO: 1. In addition, when the base sequence 1a satisfies the relationship (B) with the base sequence of SEQ ID NO: 1, more preferably, the base sequence 1a is one or both of the 5 'end and the 3' end in the base sequence of SEQ ID NO: 1. In which a total of 1 or several bases are added, particularly preferably a sequence in which 1 or several bases are added only to the 5 ′ end in the base sequence of SEQ ID NO: 1, particularly preferably SEQ ID NO: 15. A partial base sequence of the base sequence, comprising the base sequence of SEQ ID NO: 1 and a total of one or several bases added to one or both of the 5 ′ end and the 3 ′ end of the base sequence of SEQ ID NO: 1. It is a partial base sequence.

  The polynucleotide (1a) may contain the base sequence 1a at the 3 'end, and another base sequence may be added to the 5' end of the base sequence 1a. The polynucleotide (1a) is a polynucleotide of 40 bases or less, preferably 35 bases or less, more preferably 30 bases or less, more preferably 25 bases or less, particularly preferably 22 bases or less. As a preferred embodiment of the polynucleotide of (1a), 40 bases or less, preferably 35 bases or less, more preferably 30 bases or less, more preferably, among the base sequence of the genomic DNA of the iap gene shown in SEQ ID NO: 15. Is a partial base sequence having 25 bases or less, particularly preferably 22 bases or less, and a partial base sequence containing the base sequence 1a at the 3 ′ end. More preferably, the polynucleotide of (1a) consists of the base sequence of the base sequence 1a, and most preferably the polynucleotide of the base sequence shown in SEQ ID NO: 1.

  The polynucleotide of (1b) is a 3 ′ end of a sequence consisting of 10 or more bases continuous from the 3 ′ end of a base sequence homologous to the base sequence of SEQ ID NO: 1 (ie, the above base sequence 1a). A polynucleotide having 40 or less bases. In the polynucleotide (1b), more preferably, the 3 'end of the base sequence 1a comprises a sequence consisting of 12 bases or more, more preferably 15 bases or more continuous from the 3' end.

  The polynucleotide (1b) only needs to include the partial sequence of the base sequence 1a at the 3 'end, and another base sequence may be further added to the 5' end of the partial sequence. The polynucleotide (1b) is a polynucleotide of 40 bases or less, preferably 35 bases or less, more preferably 30 bases or less, more preferably 25 bases or less, particularly preferably 22 bases or less. More preferably, the polynucleotide of (1b) comprises the partial base sequence of the base sequence 1a, and most preferably a partial base sequence comprising 10 bases or more consecutive from the 3 ′ end of the base sequence shown in SEQ ID NO: 1. A polynucleotide comprising:

  A nucleotide sequence homologous to the nucleotide sequence of SEQ ID NO: 2 in the polynucleotide (2a) is referred to as nucleotide sequence 2a. This base sequence 2a satisfies the relationship of (A), (B), (C) or (D) with the base sequence of SEQ ID NO: 2 (the base sequence of SEQ ID NO: 2 is base sequence X, and the base sequence 2a is Corresponding to base sequence Y).

  When the base sequence 2a satisfies the relationship of (B) above with the base sequence of SEQ ID NO: 2, more preferably, the base sequence 2a is derived from one or both of the 5 ′ end and the 3 ′ end in the base sequence of SEQ ID NO: 2. A sequence in which one or several bases have been deleted in total, particularly preferably a sequence in which one or several bases are deleted only from the 5 ′ end in the base sequence of SEQ ID NO: 2. In addition, when the base sequence 2a satisfies the relationship of (B) with the base sequence of SEQ ID NO: 2, more preferably, the base sequence 2a is one of the 5 ′ end and the 3 ′ end in the base sequence of SEQ ID NO: 2 or A sequence in which one or several bases are added to both in total, particularly preferably a sequence in which one or several bases are added only at the 5 ′ end in the base sequence of SEQ ID NO: 2, and particularly preferably SEQ ID NO: 15 A partial base sequence of a complementary base sequence to the base sequence of SEQ ID NO: 2, and a total of 1 or several nucleotide sequences added to one or both of the base sequence of SEQ ID NO: 2 and the 5 ′ end and 3 ′ end of the base sequence of SEQ ID NO: 2 It is the said partial base sequence which consists of a base.

  The polynucleotide (2a) may contain the base sequence 2a at the 3 'end, and another base sequence may be further added to the 5' end of the base sequence 2a. The polynucleotide (2a) is a polynucleotide of 40 bases or less, preferably 35 bases or less, more preferably 30 bases or less, more preferably 25 bases or less, particularly preferably 22 bases or less. As a preferred embodiment of the polynucleotide of (2a), 40 bases or less, preferably 35 bases or less, more preferably 30 bases or less, more preferably 25 bases or less of the base sequence complementary to the base sequence of SEQ ID NO: 15. Hereinafter, a partial base sequence having a base sequence of 22 bases or less and including the base sequence 2a at the 3 ′ end is particularly preferable. More preferably, the polynucleotide of (2a) consists of the nucleotide sequence of the nucleotide sequence 2a, most preferably the polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 2.

  The polynucleotide of (2b) is a 3 ′ end of a sequence consisting of 10 or more bases continuous from the 3 ′ end of the base sequence homologous to the base sequence of SEQ ID NO: 2 (that is, the base sequence 2a). A polynucleotide having 40 or less bases. In the polynucleotide (2b), more preferably, the 3 'end of the base sequence 2a comprises a sequence consisting of 12 or more bases, more preferably 15 or more bases continuous from the 3' end.

  The polynucleotide (2b) may contain the partial sequence of the base sequence 2a at the 3 'end, and another base sequence may be further added to the 5' end of the partial sequence. The polynucleotide (2b) is a polynucleotide of 40 bases or less, preferably 35 bases or less, more preferably 30 bases or less, more preferably 25 bases or less, particularly preferably 22 bases or less. More preferably, the polynucleotide of (2b) consists of the partial base sequence of the base sequence 2a, and most preferably a partial base sequence consisting of 10 bases or more continuous from the 3 ′ end of the base sequence shown in SEQ ID NO: 2. A polynucleotide comprising:

1.3. Primer combination 2
In the second preferred combination in the primer set of the present invention,
A first primer comprising the polynucleotide of (3a) or (3b), and
The second primer includes the polynucleotide (4a) or (4b).
This primer set is particularly suitable when the hly gene is used as a target nucleic acid.

  The nucleotide sequence homologous to the nucleotide sequence of SEQ ID NO: 3 in the polynucleotide (3a) is referred to as nucleotide sequence 3a. This base sequence 3a satisfies the relationship (A), (B), (C) or (D) with the base sequence of SEQ ID NO: 3 (the base sequence of SEQ ID NO: 3 is base sequence X, and the base sequence 3a is Corresponding to base sequence Y).

  When the base sequence 3a satisfies the relationship (B) with the base sequence of SEQ ID NO: 3, more preferably, the base sequence 3a is derived from one or both of the 5 ′ end and the 3 ′ end in the base sequence of SEQ ID NO: 3. A sequence in which one or several bases have been deleted in total, particularly preferably a sequence in which one or several bases are deleted only from the 5 ′ end in the base sequence of SEQ ID NO: 3. Further, when the base sequence 3a satisfies the relationship of (B) with the base sequence of SEQ ID NO: 3, more preferably, the base sequence 3a is one or both of the 5 ′ end and the 3 ′ end in the base sequence of SEQ ID NO: 3. In which a total of 1 or several bases are added, particularly preferably a sequence in which 1 or several bases are added only to the 5 ′ end in the base sequence of SEQ ID NO: 3, particularly preferably SEQ ID NO: 16. A partial base sequence of the base sequence, comprising the base sequence of SEQ ID NO: 3 and a total of one or several bases added to one or both of the 5 ′ end and the 3 ′ end of the base sequence of SEQ ID NO: 3 It is a partial base sequence.

  The polynucleotide (3a) may contain the base sequence 3a at the 3 'end, and another base sequence may be further added to the 5' end of the base sequence 3a. The polynucleotide (3a) is a polynucleotide of 40 bases or less, preferably 35 bases or less, more preferably 30 bases or less, more preferably 25 bases or less, particularly preferably 22 bases or less. As a preferred embodiment of the polynucleotide of (3a), 40 bases or less, preferably 35 bases or less, more preferably 30 bases or less, more preferably, among the base sequence of the genomic DNA of the hly gene shown in SEQ ID NO: 16. Is a partial base sequence of 25 bases or less, particularly preferably 22 bases or less, and includes a partial base sequence containing the base sequence 3a at the 3 ′ end. More preferably, the polynucleotide of (3a) is a polynucleotide consisting of the base sequence 3a, and most preferably the polynucleotide consisting of the base sequence shown in SEQ ID NO: 3.

  The polynucleotide of (3b) is a 3 'end of a sequence consisting of 10 or more bases continuous from the 3' end of a base sequence homologous to the base sequence of SEQ ID NO: 3 (ie, the above base sequence 3a). A polynucleotide having 40 or less bases. In the polynucleotide (3b), more preferably, the 3 'end of the base sequence 3a comprises a sequence consisting of 12 or more bases, more preferably 15 or more bases continuous from the 3' end.

  The polynucleotide (3b) only needs to contain the partial sequence of the base sequence 3a at the 3 'end, and another base sequence may be further added to the 5' end of the partial sequence. The polynucleotide (3b) is a polynucleotide of 40 bases or less, preferably 35 bases or less, more preferably 30 bases or less, more preferably 25 bases or less, particularly preferably 22 bases or less. More preferably, the polynucleotide of (3b) consists of the partial base sequence of the base sequence 3a, and most preferably a partial base sequence consisting of 10 bases or more consecutive from the 3 ′ end of the base sequence shown in SEQ ID NO: 3. A polynucleotide comprising:

  A nucleotide sequence that is homologous to the nucleotide sequence of SEQ ID NO: 4 in the polynucleotide (4a) is referred to as nucleotide sequence 4a. This base sequence 4a satisfies the relationship of (A), (B), (C) or (D) with the base sequence of SEQ ID NO: 4 (the base sequence of SEQ ID NO: 4 is base sequence X, and the base sequence 4a is Corresponding to base sequence Y).

  When the base sequence 4a satisfies the relationship of (B) above with the base sequence of SEQ ID NO: 4, more preferably, the base sequence 4a is derived from one or both of the 5 ′ end and the 3 ′ end in the base sequence of SEQ ID NO: 4. A sequence in which one or several bases are deleted in total, particularly preferably a sequence in which one or several bases are deleted only from the 5 ′ end in the base sequence of SEQ ID NO: 4. In addition, when the base sequence 4a satisfies the relationship (B) with the base sequence of SEQ ID NO: 4, more preferably, the base sequence 4a is one of the 5 ′ end and the 3 ′ end in the base sequence of SEQ ID NO: 4 or A sequence in which one or several bases are added to both in total, particularly preferably a sequence in which one or several bases are added only to the 5 ′ end in the base sequence of SEQ ID NO: 4, and particularly preferably SEQ ID NO: 16 A partial base sequence of a complementary base sequence to the base sequence of SEQ ID NO: 4, and a total of 1 or several nucleotide sequences added to one or both of the base sequence of SEQ ID NO: 4 and the 5 ′ end and 3 ′ end of the base sequence of SEQ ID NO: 4 It is the said partial base sequence which consists of a base.

  The polynucleotide (4a) may contain the base sequence 4a at the 3 'end, and another base sequence may be added to the 5' end of the base sequence 4a. The polynucleotide (4a) is a polynucleotide of 40 bases or less, preferably 35 bases or less, more preferably 30 bases or less, more preferably 25 bases or less, particularly preferably 22 bases or less. As a preferred embodiment of the polynucleotide of (4a), 40 bases or less, preferably 35 bases or less, more preferably 30 bases or less, more preferably 25 bases in the complementary base sequence to the base sequence of SEQ ID NO: 16 Hereinafter, a partial base sequence having a base sequence of 22 bases or less and including the base sequence 4a at the 3 ′ end is particularly preferable. More preferably, the polynucleotide (4a) comprises a base sequence of the base sequence 4a, and most preferably a polynucleotide comprising the base sequence shown in SEQ ID NO: 4.

  The polynucleotide of (4b) is a 3 ′ end of a sequence consisting of 10 or more bases consecutive from the 3 ′ end of a base sequence homologous to the base sequence of SEQ ID NO: 4 (ie, the above base sequence 4a). A polynucleotide having 40 or less bases. In the polynucleotide (4b), more preferably, the 3 'end of the base sequence 4a comprises a sequence consisting of 12 bases or more, more preferably 15 bases or more continuous from the 3' end.

  The polynucleotide (4b) only needs to contain the partial sequence of the base sequence 4a at the 3 'end, and another base sequence may be further added to the 5' end of the partial sequence. The polynucleotide (4b) is a polynucleotide of 40 bases or less, preferably 35 bases or less, more preferably 30 bases or less, more preferably 25 bases or less, particularly preferably 22 bases or less. More preferably, the polynucleotide of (4b) comprises the partial base sequence of the base sequence 4a, and most preferably a partial base sequence comprising 10 or more bases continuous from the 3 ′ end of the base sequence shown in SEQ ID NO: 4. A polynucleotide comprising:

1.4. Preferred primer combinations 3
In the third preferred combination in the primer set of the present invention,
A first primer comprising the polynucleotide of (7a) or (7b), and
The second primer includes the polynucleotide (8a) or (8b).
This primer set is particularly suitable when the hly gene is used as a target nucleic acid.

  A nucleotide sequence homologous to the nucleotide sequence of SEQ ID NO: 7 in the polynucleotide (7a) is referred to as nucleotide sequence 7a. This base sequence 7a satisfies the relationship (A), (B), (C) or (D) with the base sequence of SEQ ID NO: 7 (the base sequence of SEQ ID NO: 7 is base sequence X, and the base sequence 7a is Corresponding to base sequence Y).

  When the base sequence 7a satisfies the relationship of (B) above with the base sequence of SEQ ID NO: 7, more preferably, the base sequence 7a is derived from one or both of the 5 ′ end and the 3 ′ end in the base sequence of SEQ ID NO: 7. A sequence in which one or several bases have been deleted in total, particularly preferably a sequence in which one or several bases are deleted from only the 5 ′ end in the base sequence of SEQ ID NO: 7. Further, when the base sequence 7a satisfies the relationship (B) with the base sequence of SEQ ID NO: 7, more preferably, the base sequence 7a is one or both of the 5 ′ end and the 3 ′ end in the base sequence of SEQ ID NO: 7. In which a total of 1 or several bases are added, particularly preferably a sequence in which 1 or several bases are added only at the 5 ′ end in the base sequence of SEQ ID NO: 7, particularly preferably A partial base sequence of the base sequence comprising the base sequence of SEQ ID NO: 7 and a total of one or several bases added to one or both of the 5 ′ end and the 3 ′ end of the base sequence of SEQ ID NO: 7 It is a partial base sequence.

  The polynucleotide (7a) only needs to contain the base sequence 7a at the 3 'end, and another base sequence may be further added to the 5' end of the base sequence 7a. The polynucleotide (7a) is a polynucleotide of 40 bases or less, preferably 35 bases or less, more preferably 30 bases or less, more preferably 25 bases or less, particularly preferably 22 bases or less. As a preferred embodiment of the polynucleotide (7a), the base sequence of the genomic DNA of the hly gene shown in SEQ ID NO: 16 is 40 bases or less, preferably 35 bases or less, more preferably 30 bases or less, more preferably Is a partial base sequence having 25 bases or less, particularly preferably 22 bases or less, and a partial base sequence containing the base sequence 7a at the 3 ′ end. More preferably, the polynucleotide (7a) comprises a nucleotide sequence of the nucleotide sequence 7a, most preferably a polynucleotide comprising the nucleotide sequence shown in SEQ ID NO: 7.

  The polynucleotide (7b) is a 3'-terminal sequence consisting of 10 or more bases continuous from the 3'-end of a base sequence homologous to the base sequence of SEQ ID NO: 7 (ie, the above-mentioned base sequence 7a). A polynucleotide having 40 or less bases. In the polynucleotide (7b), more preferably, the 3 'end of the base sequence 7a comprises a sequence consisting of 12 bases or more, more preferably 15 bases or more continuous from the 3' end.

  The polynucleotide (7b) only needs to contain the partial sequence of the base sequence 7a at the 3 'end, and another base sequence may be further added to the 5' end of the partial sequence. The polynucleotide (7b) is a polynucleotide of 40 bases or less, preferably 35 bases or less, more preferably 30 bases or less, more preferably 25 bases or less, particularly preferably 22 bases or less. More preferably, the polynucleotide of (7b) comprises the partial base sequence of the base sequence 7a, and most preferably a partial base sequence comprising 10 or more bases continuous from the 3 ′ end of the base sequence shown in SEQ ID NO: 7. A polynucleotide comprising:

  In the polynucleotide (8a), a base sequence homologous to the base sequence of SEQ ID NO: 8 is defined as a base sequence 8a. This base sequence 8a satisfies the relationship of (A), (B), (C) or (D) with the base sequence of SEQ ID NO: 8 (the base sequence of SEQ ID NO: 8 is base sequence X, and the base sequence 8a is Corresponding to base sequence Y).

  When the base sequence 8a satisfies the relationship (B) above with the base sequence of SEQ ID NO: 8, more preferably, the base sequence 8a is from one or both of the 5 ′ end and the 3 ′ end in the base sequence of SEQ ID NO: 8. A sequence in which one or several bases have been deleted in total, particularly preferably a sequence in which one or several bases are deleted only from the 5 ′ end in the base sequence of SEQ ID NO: 8. Further, when the base sequence 8a satisfies the relationship of (B) with the base sequence of SEQ ID NO: 8, more preferably, the base sequence 8a is one of the 5 'end and the 3' end in the base sequence of SEQ ID NO: 8 or A sequence in which one or several bases are added to both in total, particularly preferably a sequence in which one or several bases are added only to the 5 ′ end in the base sequence of SEQ ID NO: 8, particularly preferably SEQ ID NO: 16 A partial base sequence of a complementary base sequence to the base sequence of SEQ ID NO: 8, a total of 1 or several nucleotide sequences added to one or both of the base sequence of SEQ ID NO: 8 and the 5 ′ end and 3 ′ end of the base sequence of SEQ ID NO: 8 It is the said partial base sequence which consists of a base.

  The polynucleotide (8a) may contain the base sequence 8a at the 3 'end, and another base sequence may be further added to the 5' end of the base sequence 8a. The polynucleotide (8a) is a polynucleotide of 40 bases or less, preferably 35 bases or less, more preferably 30 bases or less, more preferably 25 bases or less, particularly preferably 22 bases or less. As a preferred embodiment of the polynucleotide of (8a), 40 nucleotides or less, preferably 35 nucleotides or less, more preferably 30 nucleotides or less, more preferably 25 nucleotides or less of the complementary nucleotide sequence to the nucleotide sequence of SEQ ID NO: 16. Hereinafter, a partial base sequence having a base sequence of 22 bases or less and including the base sequence 8a at the 3 ′ end is particularly preferable. More preferably, the polynucleotide (8a) comprises a nucleotide sequence of the nucleotide sequence 8a, and most preferably a polynucleotide comprising the nucleotide sequence shown in SEQ ID NO: 8.

  The polynucleotide (8b) is a 3'-terminal sequence consisting of 10 or more bases consecutive from the 3'-end of the base sequence homologous to the base sequence of SEQ ID NO: 8 (ie, the above-mentioned base sequence 8a). A polynucleotide having 40 or less bases. In the polynucleotide (8b), more preferably, the base sequence 8a includes a sequence of a portion consisting of 12 bases or more, preferably 15 bases or more continuous from the 3 'end, at the 3' end.

  The polynucleotide (8b) may contain the partial sequence of the base sequence 8a at the 3 'end, and another base sequence may be further added to the 5' end of the partial sequence. The polynucleotide (8b) is a polynucleotide of 40 bases or less, preferably 35 bases or less, more preferably 30 bases or less, more preferably 25 bases or less, particularly preferably 22 bases or less. More preferably, the polynucleotide of (8b) comprises the partial base sequence of the base sequence 8a, and most preferably a partial base sequence comprising 10 or more bases continuous from the 3 ′ end of the base sequence shown in SEQ ID NO: 8. A polynucleotide comprising:

1.5. The labeling part is either a tag or a labeling substance that can bind to the labeling substance, preferably a tag that can bind to the labeling substance. In this specification, a tag that can bind to a labeling substance may be referred to as a labeling tag. When the labeling part is a tag for labeling, the tag contained at one end of the amplification product can be labeled by bringing the amplification product of the nucleic acid amplification reaction into contact with the labeling substance. When the labeling part is a labeling substance, an amplification product containing the labeling substance at one end can be obtained as an amplification product of the nucleic acid amplification reaction.

  The labeling substance is not particularly limited as long as it enables detection of the amplification product, but preferably enables visual detection of the amplification product. Examples of such a labeling substance include colored particles, pigments, enzymes (peroxidase, alkaline phosphatase, luciferase, etc.), and preferably colored particles. Examples of the “colored particles” include, but are not limited to, metal (eg, gold, silver, copper, platinum, etc.) particles, metal rods, colored latex particles, silica nanoparticles including a pigment, and the like. The size of the labeling substance is not limited as long as it does not prevent the amplification product from being captured on the solid phase carrier described later. The labeling substance preferably has a good color development upon detection, and can be appropriately selected so as to have a size smaller than the pore diameter of various porous members of a solid phase carrier and a nucleic acid detection device described later. For example, the size of the labeling substance can be about 500 nm or less, preferably about 0.1 nm to 250 nm, more preferably about 1 nm to 100 nm. Fluorescent dyes (fluorescein, cyanine, etc.) can be used as the dye, but in that case, it is preferable to detect by irradiating the excitation wavelength light of each fluorescent dye.

  The labeling tag that can be used as the labeling part is not particularly limited as long as it can bind to the labeling substance, and may be appropriately selected depending on the structure of the labeling substance. For example, nucleic acid (DNA, RNA, polynucleotide, etc. ), Proteins, peptides, or other compounds (eg, low molecular weight compounds such as biotin, fluorescein isothiocyanate (FITC), digoxigenin (DIG)), or combinations thereof can be utilized. One preferred form of the tag for labeling includes a polynucleotide or consists of a polynucleotide. The polynucleotide that can be contained in the labeling tag is not particularly limited as long as it does not substantially interfere with the nucleic acid amplification reaction by the primer set of the present invention, but the number of bases is, for example, 5 to 50, preferably 10 to 35. More preferably, a polynucleotide comprising (or consisting of) the base sequence shown in SEQ ID NO: 26 or a partial base sequence thereof or a complementary base sequence thereof, or the base sequence shown in SEQ ID NO: 27 or a part thereof A polynucleotide containing (or consisting of) a base sequence or a complementary base sequence thereof can be used. Another preferred form of the labeling tag is a low molecular weight compound such as biotin, FITC, DIG or the like.

  When the labeling unit is the labeling tag described above, the labeling substance and the tag for labeling may be directly coupled or indirectly coupled, and the coupling means uses the labeling substance to be used and the labeling tag. A suitable one can be appropriately selected according to the combination with the tag. For example, when the labeling tag contains a polynucleotide, the labeling substance is bound to a polynucleotide capable of hybridizing to the polynucleotide (for example, a polynucleotide containing a sequence complementary to the base sequence of the polynucleotide), By hybridizing the polynucleotide, the labeling substance and the labeling tag can be indirectly bound. The binding between the labeling substance and the polynucleotide may be performed via a peptide, protein, nucleic acid, or the like, or may be performed via an appropriate functional group. The hybridization conditions are not particularly limited as long as hybridization occurs. For example, the reaction is performed at 20 ° C. to 40 ° C. in a buffer solution (pH 6 to 7) containing 10 mM to 50 mM phosphate. Can be performed. In order to increase the hybridization efficiency, the buffer may further contain a salt such as sodium chloride.

  When the labeling tag is a low molecular weight compound, it is linked to a binding substance such as a protein that specifically binds to the tag (for example, avidin that binds to biotin, a protein that binds to FITC), an antibody (for example, an anti-DIG antibody), or an aptamer. The labeling substance can be labeled. In this case, the labeling tag and the binding substance can be bound using various neutral buffers.

  The labeling portion (tag for labeling or labeling substance) and the polynucleotide contained in the first primer or the polynucleotide contained in the second primer can be bound by any means, directly or Can be indirectly bound. However, when at least a portion of the labeling portion connected to the polynucleotide is composed of a nucleic acid, the progress of the DNA polymerase reaction should be suppressed or stopped so that the portion is not double-stranded with the polynucleotide by the nucleic acid amplification reaction. The polynucleotide and the labeling part are bound to each other through a spacer capable of. Such a “spacer” is not particularly limited as long as it can suppress or stop the progress of the DNA polymerase reaction and prevents the labeling portion from becoming double-stranded. For example, it has a strong hairpin structure or pseudoknot structure. Nucleic acid sequence, L-type nucleic acid, peptide nucleic acid (PNA), cross-linked nucleic acid (Bridged Nucleic Acid (BNA) or Locked Nucleic Acid (LNA)), fluorescein, Cy3, Cy5, two containing an azobenzene structure represented by the following formula I Examples thereof include, but are not limited to, a valent group, an aliphatic chain (an alkylene chain or a polyoxyalkylene chain), a divalent group including an inverted sequence structure such as a 5′-5 ′ bond and a 3′-3 ′ bond. .

  When two polynucleotide molecules are connected via a divalent group represented by Formula I, the phosphate group at one 3 ′ end of the divalent group is the number of nucleotides at the 5 ′ end of the one polynucleotide molecule. This refers to a phosphate group, and the oxygen atom at the other 5 'end forms a phosphate ester bond with the phosphate group of the nucleotide at the 3' end of the other polynucleotide molecule.

Examples of the fatty chain spacer include a spacer represented by the following formula (II).
5′-O—C _m H _2m —O-3 ′ Formula (II)
(In the formula, 5 ′ represents an oxygen atom of a 5′-side phosphodiester bond, 3 ′ represents an oxygen atom of a 3′-side phosphodiester bond, and m represents an integer of 1 to 40. H may be substituted with a substituent.)

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

Moreover, as another spacer, the spacer represented by the following formula | equation (III) is mentioned.
5 ′-(OC _n H _2n ) _L —O-3 ′ Formula (III)
(In the formula, 5 ′ represents an oxygen atom of a phosphodiester bond on the 5 ′ side, 3 ′ represents an oxygen atom of a phosphodiester bond on the 3 ′ side, and n represents an integer of 2 or more and 4 or less. , L is an integer of 1 or more, and (n + 1) × L represents an integer of 40 or less, and H may be substituted with a substituent.

  In the formula (III), (n + 1) × L is preferably 2 or more and 36 or less, and more preferably 3 or more and 16 or less. A mode similar to the substituent in formula (II) is applied to the substituent of H in formula (III).

Examples of other fatty chain spacers include the following divalent groups.

  When two polynucleotide molecules are connected via these divalent groups, the phosphate group at one end of each divalent group is phosphated to the nucleotide at the 3 ′ end or 5 ′ end of one polynucleotide molecule. The oxygen atom at the other end forms a phosphate ester bond with the phosphate group of the nucleotide at the 5 'end or 3' end of the other polynucleotide molecule.

1.6. The binding portion binding portion is a tag that can be bound to a solid phase carrier described later. In the present specification, a tag capable of binding to a solid phase carrier may be referred to as an immobilization tag.
The immobilization tag that can be used as the binding portion is not particularly limited as long as it can be bound to the solid phase carrier, and may be appropriately selected depending on the structure of the solid phase carrier. For example, nucleic acid (DNA, RNA, Polynucleotides, etc.), proteins, peptides, or other compounds (eg, low molecular weight compounds), or combinations thereof can be utilized. The immobilization tag preferably contains a polynucleotide or consists of a polynucleotide. The polynucleotide that can be contained in the immobilization tag is not particularly limited as long as it does not substantially interfere with the nucleic acid amplification reaction by the primer set of the present invention, but the number of bases is, for example, 5 to 50, preferably 10 to 10. 35, more preferably a polynucleotide comprising (or consisting of) the base sequence shown in SEQ ID NO: 26 or a partial base sequence thereof or a complementary base sequence thereof, or the base sequence shown in SEQ ID NO: 27 or A polynucleotide containing (or consisting of) a partial base sequence or a complementary base sequence thereof can be used.

  The solid phase carrier is not particularly limited, but may be made of a resin, metal, polysaccharide, mineral or the like, and may be in the form of a membrane, film, nonwoven fabric, plate, gel or the like. Preferably, the solid phase carrier has a porous structure so that the amplification product and the labeling substance in the solution can be developed. Examples of the solid phase carrier usable in the present invention include filter paper, nitrocellulose membrane, polyethersulfone membrane, nylon membrane, various dried gels (silica gel, agarose gel, dextran gel, gelatin gel), silicon, glass, Examples include plastics. As the size and form of the solid phase carrier, those suitable for various operations and detection can be appropriately selected.

  The solid phase carrier only needs to be configured such that at least a part of the solid phase carrier can be bound to the immobilization tag, and more preferably, only a part of the solid phase carrier can be bound to the immobilization tag. Yes. By configuring so that only a specific part of the solid phase carrier can be bound to the immobilization tag, the amplification product captured on the solid phase carrier is detected only in the above part, and therefore positive or negative. Discrimination can be facilitated.

  The binding between the solid phase carrier and the immobilization tag may be direct coupling or indirect coupling, and the coupling means is appropriately determined depending on the combination of the solid phase carrier and the immobilization tag to be used. A suitable one can be selected. For example, when the immobilization tag contains a polynucleotide, a polynucleotide capable of hybridizing to the polynucleotide (eg, a polynucleotide containing a sequence complementary to the base sequence of the polynucleotide) is immobilized on a solid phase carrier. By using the tag capturing means and hybridizing both polynucleotides, the solid phase carrier and the immobilization tag can be indirectly bound. Immobilization of the polynucleotide to the solid phase carrier may be performed via a peptide, protein, nucleic acid or the like, or may be performed via an appropriate functional group. Hybridization can be performed under the conditions described above for the binding between the labeling tag and the labeling substance. When immobilizing a polynucleotide on a solid phase carrier, the trapped amplification product is detected only on a specific part by immobilizing it on a specific part. Can be easily.

  The binding part (immobilization tag) and the polynucleotide contained in the first primer or the polynucleotide contained in the second primer can be bound by any means, directly or indirectly. Can be combined. However, if at least the portion of the immobilization tag that is connected to the polynucleotide is composed of a nucleic acid, the progress of the DNA polymerase reaction is suppressed or stopped so that the portion is not double-stranded with the polynucleotide by the nucleic acid amplification reaction. The polynucleotide and the immobilization tag are bound to each other through a spacer that can be used. Specific examples of the spacer provided between the binding portion and the polynucleotide are the same as those described above regarding the spacer provided between the labeling portion and the polynucleotide.

2. Detection Method of the Present Invention The present invention provides a method for detecting Listeria monocytogenes and / or nucleic acid derived from Listeria monocytogenes in a sample using the above primer set.
The sample to be detected is not particularly limited, but typically, a sample containing a part of a living body (organ, tissue, cell, etc.) such as foods and drinks, animal plants, feces, microorganisms, viruses and the like can be mentioned.

The DNA obtained from the sample is used as a template for the nucleic acid amplification reaction. The DNA may be in a form extracted from a sample and purified, or in a form extracted from a sample and roughly purified. Alternatively, if the sample contains DNA at a relatively high concentration, such as a part of a cell or tissue, the sample itself can be directly included in the nucleic acid amplification reaction. Further, cDNA prepared from a sample can also be used as “DNA obtained from the sample”.
Hereinafter, the nucleic acid amplification process, the detection process, and the labeling process will be described.

2.1. Nucleic Acid Amplification Step The nucleic acid amplification step is a step of performing a nucleic acid amplification reaction using DNA obtained from a sample as a template and using the primer set described above.
The nucleic acid amplification reaction can be performed according to a general PCR method. That is, when PCR is performed on the template DNA containing the target nucleic acid using the primer set of the present invention, it can be performed under PCR conditions that amplify a desired region.

  The DNA polymerase used for PCR is not particularly limited as long as it is a heat-resistant DNA polymerase. In the present invention, a commercially available DNA polymerase can be used, and for example, TaKaRa Ex Taq (registered trademark) can be preferably used. The temperature, time, buffer composition, etc. can be appropriately selected depending on the DNA polymerase used, the concentration of each primer, and the like.

  The hot start PCR method is useful because it can be expected to suppress the formation of primer dimers. Hot start PCR method can be performed using commercially available hot start PCR reagents such as TaKaRa Ex Taq (registered trademark) Hot Start Version (Takara Bio). Further, TaKaRa Taq HS PCR Kit, UNG plus (registered trademark) can be used, and by using this kit, an effect of suppressing carryover contamination of the nucleic acid amplification product can be expected.

  The number of cycles in the PCR method is preferably 25 cycles or more, more preferably 30 cycles or more. The upper limit of the number of cycles is not particularly limited, but is usually 60 cycles or less, preferably 50 cycles or less. In general, the greater the number of cycles, the higher the amplification factor, and at the same time, primer dimers are more easily generated in the reaction system. However, when the primer set of the present invention is used, even if the number of cycles in the PCR method is 25 cycles or more, the generation of primer dimers is small, and a very small amount of target nucleic acid in the sample can be sufficiently amplified, and the sensitivity is high. Detection is possible.

  The primer concentration at the start of the reaction system in the PCR method is not particularly limited and can be appropriately adjusted according to the amount of DNA serving as a template. A preferable primer concentration at the start of the reaction in the reaction system includes concentrations of 0.05 μM or more and 1.0 μM or less for the first primer and the second primer, respectively. If it is less than 0.05 μM, the detection sensitivity may be lowered, and the reference detection sensitivity may be lowered. Further, the upper limit of the primer concentration is not particularly limited, but is preferably 1.0 μM.

  When the target DNA is contained in the template DNA in the nucleic acid amplification step, the double-stranded target DNA in which the labeling portion is linked to one end and the binding portion is linked to the other end is reacted as an amplification product. Formed in the system.

2.2. In the detection step, the product of the nucleic acid amplification reaction in the nucleic acid amplification step is brought into contact with a solid phase carrier including at least a portion capable of binding to the binding portion, and the portion of the solid phase carrier is used with the labeling portion as an index. This is a step of detecting an amplification product at.

  When the labeling part is the above-described labeling tag, a labeling step of binding the labeling substance to the labeling tag may be further performed. When the labeling part is the above-mentioned labeling substance, the labeling step is unnecessary. In the detection step, “using the labeling portion as an index” means that when the labeling portion is a tag for labeling, detection of an amplification product using the labeling substance bound through the labeling step as an index. In some cases, it means detecting an amplification product using the labeling substance as an index.

The product of the nucleic acid amplification reaction refers to a reaction solution of a nucleic acid amplification reaction that may contain an amplification product, a sample obtained by further increasing the concentration of the amplification product from the reaction solution, and the like.
The solid phase carrier is as described in detail in “1.6.

  According to the combination of the solid phase carrier and the binding part, the contact of the product with the binding part of the solid phase carrier can be performed when the amplification product is contained in the product. May be performed under conditions appropriately adjusted so that the binding part of the amplification product is bound to (for example, the hybridization conditions described in the above-mentioned “1.6. Binding part” or the buffer conditions of about pH 5-9).

  The amplification product can be detected by detecting the labeling substance bound to the amplification product captured on the solid phase carrier, preferably by visual detection. When the amplification product is present, the labeling substance of the amplification product captured and bound to the solid phase carrier is detected. The presence / absence of the detection can be used as an index to determine the presence / absence of an amplification product (target nucleic acid) in the reaction system of the nucleic acid amplification reaction, as well as for L. monocytogenes and / or L. monocytogenes in the sample. The presence or absence of nucleic acids derived from can be determined.

2.3. Labeling step The labeling step is a step performed when the labeling part contained in the primer set of the present invention is the above-described labeling tag, and the product of the nucleic acid amplification reaction and the labeling substance are brought into contact with each other to form a labeling tag. This is done by binding to a labeling substance. The labeling step may be performed before the product of the nucleic acid amplification reaction is contacted with the solid phase carrier, may be performed after the contact, or may be performed simultaneously with the contact.

  According to the combination of the labeling substance and the tag for labeling, the labeling substance and the tag for labeling of the amplification product are bound when the labeling substance and the product are in contact with each other. The conditions may be adjusted as appropriate (for example, the hybridization conditions described in “1.6. Binding part” or the buffer conditions of about pH 5-9).

3. Kit of the present invention The present invention is also a kit for detecting Listeria monocytogenes and / or nucleic acid derived from Listeria monocytogenes, the primer set of the present invention and a portion capable of binding to the binding portion. And a solid phase carrier containing at least a part thereof. The kit can be used in the detection method of the present invention.

When the labeling part in the primer set of the present invention is a labeling tag, the kit of the present invention can further contain a labeling substance.
The solid phase carrier and the labeling substance can be in the form of a nucleic acid detection device to be described later.
The kit of the present invention can further contain a PCR buffer, dNTPs, DNA polymerase, a nucleic acid chromatography developing solution, and the like.

4). Nucleic acid detection device The above-described detection step and labeling step can be performed using a nucleic acid detection device utilizing nucleic acid chromatography. By using this nucleic acid detection device, the presence or absence of amplification products (target nucleic acids) in the reaction system of the nucleic acid amplification reaction can be detected and discriminated without requiring a special device, and the results can be obtained easily and quickly. Can be obtained.

  As the nucleic acid detection device, a known nucleic acid detection device (WO2012 / 070618) used for detecting a nucleic acid amplification product labeled by a nucleic acid chromatography method can be used.

  A schematic diagram of an embodiment of a nucleic acid detection device that can be used in the present invention is shown in FIG. 1, but the nucleic acid detection device is not limited to this embodiment. In the following description, the reference numerals given to the respective members correspond to the reference numerals shown in FIG.

  The nucleic acid detection device 10 in FIG. 1 includes a sample pad 3 that is a reaction system receiving unit for receiving a reaction system of a nucleic acid amplification reaction, a conjugate pad 2 that holds a labeling substance, and an amplification product on a substrate 5. The porous solid phase carrier 1 including a portion 6 that can be coupled to the coupling portion contained therein and the absorbent pad 4 are arranged so as to contact each other in this order. The portion 6 of the solid phase carrier 1 is a portion where a means (capturing means) for capturing an amplification product (for example, the above-described oligonucleotide or the like) is locally arranged and immobilized. The sample pad 3, the conjugate pad 2, the solid phase carrier 1 and the absorption pad 4 can be constituted by a member having a porous structure that can be used as the solid phase carrier. These may be comprised from the same member, and may be comprised from a different member. The base material 5 may support various members disposed thereon, and may be any material that facilitates the operation of the nucleic acid detection device. For example, a material made of resin, metal, mineral, or the like can be used. . When the labeling substance is mixed in the developing solution, or when the labeling part is a labeling substance, the conjugate pad 2 can be omitted.

  The reaction system of the nucleic acid amplification reaction obtained by the nucleic acid amplification step is added to the sample pad 3. The reaction system may be added as it is, or may be added together with an appropriate developing solution (for example, phosphate buffer, Tris buffer, Good buffer, SSC buffer). The developing solution can further contain a surfactant, a salt, a protein, a nucleic acid and the like, if necessary. The reaction system added to the sample pad 3 develops by a capillary phenomenon from upstream to downstream in the direction indicated by the arrow in FIG.

  In another embodiment, the nucleic acid detection device is placed in a container (for example, a PCR tube, an Eppendorf tube, a 96-well plate, etc.) holding the product of the nucleic acid amplification reaction and / or the developing solution, and the sample pad 3 is amplified by nucleic acid. It can also be developed by a method of immersing in a reaction product and / or a developing solution. In that case, the width of the sample pad 3 is preferably 2.0 to 10.0 mm so that the sample pad 3 is placed in a container holding the product of the nucleic acid amplification reaction and / or the developing solution. More preferably, it is 5.0 mm.

  In an embodiment in which the labeling part is a labeling tag, the amplification product in the reaction system comes into contact with the labeling substance when passing through the conjugate pad 2 holding the labeling substance, and the labeling substance is passed through the labeling tag. Labeled.

  Next, when the amplification product in the reaction system passes through the solid phase carrier 1, it contacts the capture means fixed to the portion 6, and is captured and bound to the solid phase carrier 1 via the immobilization tag.

  When the target nucleic acid is present in the sample, the labeling substance bound to the amplification product captured and bound to the portion 6 of the solid phase carrier 1 including the capturing means is detected in the portion 6. If the labeling substance can be visually confirmed, the portion 6 is colored due to the labeling substance. The presence or absence of the target nucleic acid in the sample can be determined using the presence or absence of detection (coloration) of the labeling substance as an index.

(Example)
The present invention will be specifically described based on the following experimental results, but the present invention is not limited thereto.

[Example 1]
<Material>
(I) Design of candidate primers for detecting genes characteristic of Listeria The following seven types of primer sets (forward primers (forward primers)) are used as candidate primer sets for amplifying genes characteristic of L. monocytogenes. F) and reverse primer (R) pair) were designed.

The Iap primer set was designed to amplify the region of Nos. 486 to 616 of the iap gene shown in SEQ ID NO: 15.
The Hly primer set was designed to amplify the region from 1044 to 1277 of the hly gene shown in SEQ ID NO: 16.

The Hly1 primer set was designed to amplify the region from 831 to 1286 of the hly gene shown in SEQ ID NO: 16.
The Hly2 primer set was designed to amplify the region from 652 to 891 of the hly gene shown in SEQ ID NO: 16.

The Mono primer set was designed to amplify the region from 736 to 1088 of the iap gene shown in SEQ ID NO: 15.
The Inl primer set was designed to amplify the region of 1211-1370 of the inlA gene shown in SEQ ID NO: 17.

  The CD primer set was designed to amplify the region of Nos. 25 to 136 of the mpl gene shown in SEQ ID NO: 18. The forward primer (SEQ ID NO: 13) of the CD primer set is −25 to − in the upstream region of the mpl gene shown in SEQ ID NO: 18 (region between the hly gene and the mpl gene) in the genomic DNA of Listeria monocytogenes. Designed at position 6.

Through the divalent group represented by the above formula I containing an azobenzene structure that inhibits the polymerase reaction at the 5 ′ end of the DNA (shown in Table 1) constituting the primer portion of the forward primer of each primer set, DNA comprising the following forward primer tag sequences was ligated. At this time, the 5 ′ end of the divalent group containing the structure of azobenzene represented by the above formula I and the phosphate group of the nucleotide at the 3 ′ end of the DNA comprising the forward primer tag sequence form a phosphate ester bond, The phosphate group at the 3 ′ end of the divalent group and the 5′-position hydroxyl group of deoxyribose of the nucleotide at the 5 ′ end of the primer portion of each forward primer form a phosphate ester bond.
5′-GACAACGGAGACAGAGCCCAA-3 ′ (SEQ ID NO: 26)

Through the divalent group represented by the above formula I containing an azobenzene structure that inhibits the polymerase reaction at the 5 ′ end of the DNA (shown in Table 1) constituting the primer part of the reverse primer of each primer set, DNA consisting of the following reverse primer tag sequences was ligated. At this time, the 5 ′ end of the divalent group containing the structure of azobenzene represented by the above formula I and the phosphate group of the nucleotide at the 3 ′ end of the DNA consisting of the reverse primer tag sequence form a phosphate ester bond, The phosphate group at the 3 ′ end of the divalent group and the hydroxyl group at the 5 ′ position of the deoxyribose of the nucleotide at the 5 ′ end of the primer portion of each reverse primer form a phosphate ester bond.
5′-ATGCTCACCTATGCCCCAGTG-3 ′ (SEQ ID NO: 27)

The entire sequence of the forward primer of the Iap primer set including the tag sequence and the linker sequence is shown in SEQ ID NO: 28, and the entire sequence of the reverse primer is shown in SEQ ID NO: 29.
The entire sequence of the forward primer of the Hly primer set including the tag sequence and the linker sequence is shown in SEQ ID NO: 30, and the entire sequence of the reverse primer is shown in SEQ ID NO: 31.

The whole sequence of the forward primer of the Hly1 primer set including the tag sequence and the linker sequence is shown in SEQ ID NO: 32, and the whole sequence of the reverse primer is shown in SEQ ID NO: 33.
The entire sequence of the forward primer of the Hly2 primer set including the tag sequence and the linker sequence is shown in SEQ ID NO: 34, and the entire sequence of the reverse primer is shown in SEQ ID NO: 35.

The entire sequence of the forward primer of the Mono primer set including the tag sequence and the linker sequence is shown in SEQ ID NO: 36, and the entire sequence of the reverse primer is shown in SEQ ID NO: 37.
The whole sequence of the forward primer of the Inl primer set including the tag sequence and the linker sequence is shown in SEQ ID NO: 38, and the whole sequence of the reverse primer is shown in SEQ ID NO: 39.

The entire sequence of the forward primer of the CD primer set including the tag sequence and the linker sequence is shown in SEQ ID NO: 40, and the entire sequence of the reverse primer is shown in SEQ ID NO: 41.
The synthesis of each primer including a tag sequence consisting of the base sequence shown in SEQ ID NOs: 28 to 41 and a linker sequence was purchased through a custom synthesis at Tsukuba Oligo Service Co., Ltd.

(II) Preparation of oligonucleotide-bound gold colloid Gold Colloid (40 nm, 9.0 × 10 ¹⁰ (number of particles / ml), manufactured by British Biocell International) and a thiol group-containing oligonucleotide represented by SEQ ID NO: 42 below Mix and incubate at 50 ° C. for 16 hours. Centrifugation was performed at 6000 rpm for 15 minutes, the supernatant was removed, 0.05 M sodium chloride, 5 mM phosphate buffer (pH 7) was added and mixed, and then incubated again at 50 ° C. for 40 hours.

  After incubation, centrifugation (6000 rpm, 15 minutes) was performed, the supernatant was removed, and 5 mM phosphate buffer (pH 7) was added. This buffer replacement was performed again.

The prepared gold colloid solution was uniformly added to a glass fiber pad, and then dried with a vacuum dryer to obtain a conjugate pad.
(Thiol group-containing oligonucleotide): 5′-TTGGCTCTGTCTCCGTTGTC-SH-3 ′ (SEQ ID NO: 42)

The thiol group-containing oligonucleotide represented by SEQ ID NO: 42 comprises a phosphate group at the 3 ′ position of a cytosine nucleotide at the 3 ′ end, and a hydroxyl group of a compound represented by HO— (CH ₂ ) _m —SH (m is 6). Are bonded by a phosphate ester bond.

(III) Preparation of membrane with immobilized tag capturing means As a tag capturing means, a solution containing the oligonucleotide probe represented by SEQ ID NO: 43 below was applied to a nitrocellulose membrane (Hi-Flow 180) manufactured by Merck Millipore, and a dispenser. It was applied to a line having a width of 1 mm perpendicular to the developing direction. Thereafter, the membrane was dried at 40 ° C. for 30 minutes to obtain a membrane equipped with tag capturing means.
(Oligonucleotide probe): 5′-CACTGGGGCATACGGTAGCAT-3 ′ (SEQ ID NO: 43)

(IV) Production of Lateral Flow Type Nucleic Acid Detection Device The produced lateral flow type nucleic acid detection device was produced based on the detection device shown in the schematic diagram of FIG.
That is, a polypropylene backing sheet (Lohmann) was used as the substrate 5, the conjugate pad prepared in (II) above as the conjugate pad 2, and the tag capturing means prepared in (III) above as the capturing means in the portion 6. As shown in FIG. 1, a membrane (solid phase carrier) 1, a glass fiber sample pad as the sample pad 3, and a cellulose absorbent pad as the absorption pad 4 are bonded to each other as shown in FIG. 10 was produced.

<Test 1: Confirmation test for non-specific detection>
The template DNA derived from Listeria monocytogenes was subjected to PCR amplification using each of the above primer sets, and it was confirmed whether or not false positives due to non-specific coloring of the primer dimer occurred.

  A Listeria monocytogenes DNA control (manufactured by Vircell) diluted with sterilized water to 50 pg / μL was used in this test as a template DNA solution derived from Listeria monocytogenes.

  Using TaKaRa Ex Taq (registered trademark) Hot Start Version (Takara Bio) as a PCR reagent, a total amount of 25 μL of a PCR reaction solution having the composition shown in the following table was prepared.

As a negative control, a PCR reaction solution having the same composition was prepared except that 1 μL of sterilized distilled water was used instead of the template DNA solution.
The 5 μM forward primer solution and the 5 μM reverse primer solution are prepared by dissolving a predetermined primer in sterile distilled water so as to have a concentration of 5 μM.

  PCR was performed at 95 ° C. for 2 minutes, followed by 40 cycles of 95 ° C. for 20 seconds−60 ° C. for 30 seconds−72 ° C. for 30 seconds, and cooling to 4 ° C. after the completion of the 40th cycle. went.

  The reaction solution after PCR under the above conditions was applied to the lateral flow nucleic acid detection device 10 to try to detect an amplification product. Specifically, 5 μL of the reaction solution after PCR is added to the sample pad 3 on the device 10, and further, 80 μL of a developing solution (citrate buffer mixed with a surfactant) is added to thereby prepare the reaction solution. Expanded. After 10 minutes at room temperature, the presence or absence of coloring of the portion 6 including the tag capturing means fixed in a line on the membrane 1 was visually confirmed.

  As a result of the above test, when any of the seven primer sets shown in Table 1 was used, the red color derived from the colloidal gold in the portion 6 including the tag capturing means was observed in the test section where the template DNA was PCR amplified. It could be confirmed. However, when the Hly1 primer set, the Mono primer set, and the Inl primer set were used, coloring of part 6 was observed even in a negative control test in which no template DNA was added to the reaction system. On the other hand, when the Iap primer set, the Hly primer set, the Hly2 primer set, and the CD primer set were used, no coloring of the portion 6 in the negative control test was observed. The results of the negative control test are shown in Table 3 and FIG.

  In order to investigate the cause of coloration in the negative control test using the Hly1 primer set, Mono primer set, and Inl primer set, the PCR reaction solution was analyzed by agarose gel electrophoresis. As shown in FIG. Since the amplification of a short region was seen unlike the amplification length of, formation of primer dimers was observed, and these were detected on the nucleic acid chromatographic chip, and were found to be the cause of false positive determination. In FIG. 3, the result of agarose gel electrophoresis of the PCR reaction product of the negative control test using the Mono primer set is shown on the left, and the result of agarose gel electrophoresis of the standard sample containing the primer and primer dimer is shown on the right. Show. Although not shown, similar results were obtained when the Hly1 primer set and the Inl primer set were used.

  From the above results, by using the Iap primer set, the Hly primer set, the Hly2 primer set, and the CD primer set, the Listeria monocytogenes genomic DNA can be specifically amplified, and the amplification product is derived from the DNA. It was confirmed that no false positives were generated by the primer dimer.

<Test 2: Confirmation test for high sensitivity detection>
From the four primer sets (Iap primer set, Hly primer set, Hly2 primer set, and CD primer set) that were confirmed not to cause non-specific coloring (false positive) in Test 1 above, Listeria monocytogenes In order to select a primer set capable of detecting genomic DNA with high sensitivity, the following test was performed.

In the PCR reaction solution composition shown in Table 2, the same conditions as in Test 1 except that 1 μL of the template DNA solution adjusted to each concentration of 50 pg / μL, 5 pg / μL, or 0.5 pg / μL was used as the template DNA solution. Detection was performed by PCR amplification and nucleic acid chromatography according to the procedure. As a negative control test (0 pg / test), a similar test was performed using sterilized distilled water instead of the template DNA solution.
The following table shows the observation results of the presence or absence of coloring of the portion 6 including the tag capturing means of the device 10.

  FIG. 4 shows a photograph of the solid phase carrier 1 including the portion 6 in the device 10 in the detection test using the Iap primer set. FIG. 5 shows a photograph of the solid phase carrier 1 including the portion 6 in the device 10 in a detection test using the Hly primer set. The “Listeria detection line” in FIGS. The “internal standard line” is a control detection unit that is formed in a part other than the part 6 of the solid phase carrier 1 and detects the amplification and / or labeling reaction product of a control nucleic acid (internal standard substance) added in advance as a control. It is. The “internal standard line” is used to evaluate whether the amplification reaction and / or labeling reaction has progressed normally. By detecting the signal in the “internal standard line”, reagents such as enzymes function normally. It is used to evaluate the reliability of the test results by evaluating that the test was performed properly and without any problems.

  From the above results, it was confirmed that the Iap primer set and the Hly primer set can detect a small amount of genomic DNA of 0.5 pg / test. That is, it was found that the Iap primer set and the Hly primer set are primer sets that can detect Listeria monocytogenes with high sensitivity and specificity using a nucleic acid chromatography method.

[Example 2]
<Material>
(I) Design of candidate primers for detecting genes characteristic of Listeria Seven types of primer sets shown in Table 1 of Example 1 were used.
A biotin-containing labeling tag represented by the following formula IV was linked to the 5 ′ end of the primer portion of the forward primer of each primer set. Specifically, a monovalent phosphate group including a biotin structure represented by the following formula IV, a 5′-position hydroxyl group of deoxyribose of the 5′-end nucleotide of each forward primer primer portion, The monovalent group represented by Formula IV was linked to the 5 ′ end of the primer portion by forming a phosphate ester bond.

  The 5 'end of DNA constituting the primer part of the reverse primer of each primer set (shown in Table 1) contains an inverted sequence structure that is a 5'-5' bond that inhibits the polymerase reaction, and has the following formula V The DNA consisting of the reverse primer tag sequence shown in SEQ ID NO: 27 described in Example 1 (I) was ligated through the divalent group represented by At this time, the 3 ′ end of the DNA consisting of the reverse primer tag sequence is connected to the 5 ′ end of the divalent group containing the inverted sequence structure represented by the following formula V, and the 3 ′ of the divalent group The ends were linked so that the 5 ′ end of the DNA (shown in Table 1) constituting the primer portion of each reverse primer was connected. At this time, the 3 ′ position on the deoxyribose of the nucleotide at the 3 ′ end of the DNA consisting of the 5 ′ end phosphate group of the divalent group represented by the following formula V and the reverse primer tag sequence of SEQ ID NO: 27 Form a phosphate ester bond, and the 3 ′ end of the divalent group and the phosphate group at the 5 ′ position on the deoxyribose of the 5 ′ end nucleotide of the primer part of each reverse primer are phosphoric acid. An ester bond is formed.

The whole sequence of the forward primer of the Iap primer set including the tag sequence and the linker sequence is shown in SEQ ID NO: 44, and the whole sequence of the reverse primer is shown in SEQ ID NO: 45.
The entire sequence of the forward primer of the Hly primer set including the tag sequence and the linker sequence is shown in SEQ ID NO: 46, and the entire sequence of the reverse primer is shown in SEQ ID NO: 47.

The entire sequence of the forward primer of the Hly1 primer set including the tag sequence and the linker sequence is shown in SEQ ID NO: 48, and the entire sequence of the reverse primer is shown in SEQ ID NO: 49.
The whole sequence of the forward primer of the Hly2 primer set including the tag sequence and the linker sequence is shown in SEQ ID NO: 50, and the whole sequence of the reverse primer is shown in SEQ ID NO: 51.

The entire sequence of the forward primer of the Mono primer set including the tag sequence and the linker sequence is shown in SEQ ID NO: 52, and the entire sequence of the reverse primer is shown in SEQ ID NO: 53.
The entire sequence of the forward primer of the Inl primer set including the tag sequence and the linker sequence is shown in SEQ ID NO: 54, and the entire sequence of the reverse primer is shown in SEQ ID NO: 55.

The entire sequence of the forward primer of the CD primer set including the tag sequence and the linker sequence is shown in SEQ ID NO: 56, and the entire sequence of the reverse primer is shown in SEQ ID NO: 57.
The synthesis of each primer including a tag sequence consisting of the base sequence shown in SEQ ID NOs: 44 to 57 and a linker sequence was purchased through a custom synthesis at Tsukuba Oligo Service Co., Ltd.

(II) Preparation of streptavidin-bonded latex Carboxyl group-containing polystyrene latex (blue) (solid content 10% (w / w), manufactured by Bangs) and streptavidin (manufactured by Wako Pure Chemical Industries) require water-soluble carbodiimide After mixing in an added amount of MES buffer, the mixture was blocked with monoethanolamine. The reaction solution was centrifuged, the supernatant was removed, and the resulting precipitate was washed with water. After washing, the suspension was resuspended in a HEPES buffer containing a surfactant to prepare streptavidin-binding latex (blue). After this latex solution was uniformly added to a glass fiber pad, it was dried with a hot air dryer to obtain a conjugation pad.

(III) Fabrication of membrane with tag capture means immobilized A membrane having tag capture probes arranged in the same manner as described in Example 1 (III) was fabricated.

(IV) Production of Lateral Flow Type Nucleic Acid Detection Device A nucleic acid detection device was produced in the same manner as in (IV) of Example 1 except that the conjugate pad produced in (II) above was used as conjugate pad 2. .

<Test 1>
PCR amplification was performed using each primer set prepared in the above (I), and it was confirmed in the same manner as in Example 1 whether or not a false positive due to non-specific coloring of the primer dimer occurred.
As the template DNA solution, 50 pg / μL of Listeria monocytogenes DNA prepared in Example 1 was used.

  Using Takara Taq HS (registered trademark), UNG plus PCR kit (Takara Bio) as a PCR reagent, a total amount of 25 μL of a PCR reaction solution having the composition shown in the following table was prepared.

  As a negative control, a PCR reaction solution having the same composition was prepared except that 1 μL of sterilized distilled water was used instead of the template DNA solution.

The 5 μM forward primer solution and the 5 μM reverse primer solution are prepared by dissolving a predetermined primer in sterile distilled water so as to have a concentration of 5 μM.
UNG refers to uracil-N-glycosylase.

  PCR was heated at 25 ° C. for 10 minutes, heated at 95 ° C. for 2 minutes, and then carried out for 40 cycles of 98 ° C. for 10 seconds−60 ° C. for 30 seconds−72 ° C. for 30 seconds. It was performed under the condition of cooling to ° C.

  The reaction solution after PCR under the above conditions was applied to a nucleic acid detection device to try to detect an amplification product. Specifically, after adding and mixing 80 μl of a development buffer (citrate buffer mixed with a surfactant) into a tube containing a PCR reaction solution, the nucleic acid detection device prepared in (IV) above is sampled in the reaction tube. Insertion and deployment started from the pad 3 side. After 10 minutes at room temperature, the presence or absence of coloring of the portion 6 including the tag capturing means fixed in a line on the membrane 1 was visually confirmed.

  As a result of the above test, when any of the seven primer sets shown in Table 1 was used, in the test section where the template DNA was PCR-amplified, the blue coloration by the polystyrene latex (blue) of the portion 6 including the tag capturing means Was confirmed. However, when the Hly1 primer set, the Mono primer set, and the Inl primer set were used, coloring of part 6 was observed even in a negative control test in which no template DNA was added to the reaction system. On the other hand, when the Iap primer set, the Hly primer set, the Hly2 primer set, and the CD primer set were used, no coloring of the portion 6 in the negative control test was observed.

<Test 2: Confirmation test for high sensitivity detection>
From the four primer sets (Iap primer set, Hly primer set, Hly2 primer set, and CD primer set) that were confirmed not to cause non-specific coloring (false positive) in Test 1 above, Listeria monocytogenes In order to select a primer set capable of detecting genomic DNA with high sensitivity, the following test was performed.

In the PCR reaction solution composition shown in Table 5, the same conditions as in Test 1 except that 1 μL of the template DNA solution adjusted to each concentration of 50 pg / μL, 5 pg / μL, or 0.5 pg / μL was used as the template DNA solution. Detection was performed by PCR amplification and nucleic acid chromatography according to the procedure. As a negative control test (0 pg / test), a similar test was performed using sterilized distilled water instead of the template DNA solution.
The observation results of the presence or absence of coloring of the portion 6 including the tag capturing means of the device are shown in the following table.

  From the above results, even in the Iap primer set and the Hly primer set using biotin for the tag for labeling and the oligonucleotide tag with the inverted structure as a spacer in the binding part, a small amount of genome of 0.5 pg / test It was confirmed that DNA could be detected. It was also confirmed that 50 pg / test genomic DNA could be detected when the Hly2 primer set and the CD primer set were used. That is, even when a tag containing a low molecular weight compound is used on one side, the Iap primer set, Hly primer set, Hly2 primer set and CD primer set are highly sensitive and specific using the nucleic acid chromatography method. It was found to be a detectable primer set.

  The primer set, method and kit of the present invention are useful for accurate and simple detection of Listeria monocytogenes which is a causative agent of food poisoning.

SEQ ID NO: 1: Iap forward primer SEQ ID NO: 2: Iap reverse primer SEQ ID NO: 3: Hly forward primer SEQ ID NO: 4: Hly reverse primer SEQ ID NO: 5: Hly1 forward primer SEQ ID NO: 6: Hly1 reverse primer SEQ ID NO: 7: Hly2 forward primer sequence Number 8: Hly2 reverse primer SEQ ID NO: 9: Mono forward primer SEQ ID NO: 10: Mono reverse primer SEQ ID NO: 11: Inl forward primer SEQ ID NO: 12: Inl reverse primer SEQ ID NO: 13: CD forward primer SEQ ID NO: 14: CD reverse primer SEQ ID NO: 15: iap gene SEQ ID NO: 16: hly gene SEQ ID NO: 17: inlA gene SEQ ID NO: 18: mpl gene SEQ ID NO: 19: lma inheritance SEQ ID NO: 20: actA gene SEQ ID NO: 21: 16S rRNA gene SEQ ID NO: 22: ssr gene SEQ ID NO: 23: fla gene SEQ ID NO: 24: plc gene SEQ ID NO: 25: prf gene SEQ ID NO: 26: tag sequence SEQ ID NO: 27: tag sequence sequence Number 28: Iap forward primer / Adenine nucleotide at position 20 and adenine nucleotide at position 21 are linked via a divalent group of formula I.
SEQ ID NO: 29: Iap reverse primer / thymidine nucleotide at position 20 and thymidine nucleotide at position 21 are linked via a divalent group of formula I.
SEQ ID NO: 30: Hly forward primer / Adenine nucleotide at position 20 and guanine nucleotide at position 21 are linked via a divalent group of formula I.
SEQ ID NO: 31: Hly reverse primer / thymidine nucleotide at position 20 and cytosine nucleotide at position 21 are linked via a divalent group of formula I.
SEQ ID NO: 32: Hly1 forward primer / Adenine nucleotide at position 20 and guanine nucleotide at position 21 are linked via a divalent group of formula I.
SEQ ID NO: 33: Hly1 reverse primer / thymidine nucleotide at position 20 and guanine nucleotide at position 21 are linked via a divalent group of formula I.
SEQ ID NO: 34: Hly2 forward primer / Adenine nucleotide at position 20 and adenine nucleotide at position 21 are linked via a divalent group of formula I.
SEQ ID NO: 35: Hly2 reverse primer / thymidine nucleotide at position 20 and cytosine nucleotide at position 21 are linked via a divalent group of formula I.
SEQ ID NO: 36: Mono forward primer / Adenine nucleotide at position 20 and cytosine nucleotide at position 21 are linked via a divalent group of formula I.
SEQ ID NO: 37: Mono reverse primer / thymidine nucleotide at position 20 and guanine nucleotide at position 21 are linked via a divalent group of formula I.
SEQ ID NO: 38: Inl forward primer / Adenine nucleotide at position 20 and thymidine nucleotide at position 21 are linked via a divalent group of formula I.
SEQ ID NO: 39: Inl reverse primer / thymidine nucleotide at position 20 and guanine nucleotide at position 21 are linked via a divalent group of formula I.
SEQ ID NO: 40: CD forward primer / Adenine nucleotide at position 20 and thymidine nucleotide at position 21 are linked via a divalent group of formula I.
SEQ ID NO: 41: CD reverse primer / thymidine nucleotide at position 20 and thymidine nucleotide at position 21 are linked via a divalent group of formula I.
SEQ ID NO: 42: A thiol group is introduced at the probe / 3 ′ end.
SEQ ID NO: 43: Probe SEQ ID NO: 44: Iap forward primer / biotin-containing group of formula IV is attached to the 5 ′ end.
SEQ ID NO: 45: Iap reverse primer / thymidine nucleotide at position 20 and thymidine nucleotide at position 21 are linked via a divalent group of formula V
SEQ ID NO: 46: Hly forward primer / biotin-containing group of formula IV attached to 5 ′ end
SEQ ID NO: 47: Hly reverse primer / thymidine nucleotide at position 20 and cytosine nucleotide at position 21 are linked via a divalent group of formula V.
SEQ ID NO: 48: Hly1 forward primer / biotin-containing group of formula IV is attached to the 5 ′ end.
SEQ ID NO: 49: Hly1 reverse primer / thymidine nucleotide at position 20 and guanine nucleotide at position 21 are linked via a divalent group of formula V.
SEQ ID NO: 50: Hly2 forward primer / biotin-containing group of formula IV is attached to the 5 ′ end.
SEQ ID NO: 51: Hly2 reverse primer / thymidine nucleotide at position 20 and cytosine nucleotide at position 21 are linked via a divalent group of formula V.
SEQ ID NO: 52: Mono forward primer / Biotin-containing group of formula IV attached to 5 ′ end
SEQ ID NO: 53: Mono reverse primer / thymidine nucleotide at position 20 and guanine nucleotide at position 21 are linked via a divalent group of formula V
SEQ ID NO: 54: Inl forward primer / biotin-containing group of formula IV is attached to the 5 ′ end.
SEQ ID NO: 55: Inl reverse primer / thymidine nucleotide at position 20 and guanine nucleotide at position 21 are linked via a divalent group of formula V.
SEQ ID NO: 56: CD forward primer / biotin-containing group of formula IV attached to the 5 ′ end
SEQ ID NO: 57: CD reverse primer / thymidine nucleotide at position 20 and thymidine nucleotide at position 21 are linked via a divalent group of formula V

Claims (15)

A primer set for amplifying a target nucleic acid derived from Listeria monocytogenes by a nucleic acid amplification reaction and binding the amplified product to a solid phase carrier,
A first primer comprising a polynucleotide comprising a base sequence homologous to the first base sequence contained in the target nucleic acid at the 3 ′ end;
A second primer comprising a polynucleotide containing, at the 3 ′ end, a base sequence homologous to a complementary base sequence to a second base sequence located downstream of the first base sequence in the target nucleic acid,
One of the first primer and the second primer further includes a tag that can bind to the labeling substance or a labeling part that is a labeling substance, and the other further includes a binding part that is a tag that can bind to the solid phase carrier. set.   The target nucleic acid according to claim 1, wherein the target nucleic acid is an iap gene, hly gene, lma gene, actA gene, inl gene, 16S rRNA gene, ssr gene, fla gene, plc gene, prf gene or mpl gene. Primer set. The target nucleic acid is the iap gene;
The first primer is
(1a) a polynucleotide having a base sequence homologous to the base sequence of SEQ ID NO: 1 at the 3 'end, or a nucleotide number of 40 or less, or (1b) from the 3' end of the base sequence homologous to the base sequence of SEQ ID NO: 1. A polynucleotide having a base number of 40 or less, comprising a sequence of a portion consisting of 10 or more consecutive bases at the 3 ′ end;
The second primer
(2a) a polynucleotide having a base sequence homologous to the base sequence of SEQ ID NO: 2 at the 3 'end, or a polynucleotide having 40 bases or less, or (2b) from the 3' end of the base sequence homologous to the base sequence of SEQ ID NO: 2. The primer set according to claim 2, comprising a polynucleotide having a base number of 40 or less, comprising a sequence of a portion consisting of 10 or more consecutive bases at the 3 'end. The target nucleic acid is the hly gene,
The first primer is
(3a) a polynucleotide having a base sequence homologous to the nucleotide sequence of SEQ ID NO: 3 at the 3 'end, or a polynucleotide having 40 bases or less, or (3b) from the 3' end of the nucleotide sequence homologous to the nucleotide sequence of SEQ ID NO: 3 A polynucleotide having a base number of 40 or less, comprising a sequence of a portion consisting of 10 or more consecutive bases at the 3 ′ end;
The second primer
(4a) a polynucleotide having a base sequence homologous to the base sequence of SEQ ID NO: 4 at the 3 'end, or a polynucleotide having 40 bases or less, or (4b) from the 3' end of the base sequence homologous to the base sequence of SEQ ID NO: 4 The primer set according to claim 2, comprising a polynucleotide having a base number of 40 or less, comprising a sequence of a portion consisting of 10 or more consecutive bases at the 3 'end. The target nucleic acid is the hly gene,
The first primer is
(7a) a polynucleotide having a base number of 40 or less containing a base sequence homologous to the base sequence of SEQ ID NO: 7 at the 3 ′ end, or (7b) from the 3 ′ end of the base sequence homologous to the base sequence of SEQ ID NO: 7 A polynucleotide having a base number of 40 or less, comprising a sequence of a portion consisting of 10 or more consecutive bases at the 3 ′ end;
The second primer
(8a) a polynucleotide having a base number of 40 or less containing a base sequence homologous to the base sequence of SEQ ID NO: 8 at the 3 ′ end, or (8b) from the 3 ′ end of the base sequence homologous to the base sequence of SEQ ID NO: 8 The primer set according to claim 2, comprising a polynucleotide having a base number of 40 or less, comprising a sequence of a portion consisting of 10 or more consecutive bases at the 3 'end.   The primer set according to any one of claims 1 to 5, wherein the labeling part is a tag containing a polynucleotide or a low molecular weight compound that can bind to the labeling substance.   The primer set according to any one of claims 1 to 6, wherein the binding part is a tag containing a polynucleotide capable of binding to a solid phase carrier. A method for detecting a nucleic acid derived from Listeria monocytogenes and / or Listeria monocytogenes in a sample, comprising:
A nucleic acid amplification step of performing a nucleic acid amplification reaction using the DNA obtained from the sample as a template and using the primer set according to any one of claims 1 to 7;
A detection step of contacting the product of the nucleic acid amplification reaction with a solid phase carrier including at least a portion capable of binding to the binding portion, and detecting an amplification product in the portion of the solid phase carrier using the labeling portion as an index. Including methods. The labeling part is a tag that can bind to the labeling substance,
The method according to claim 8, further comprising a labeling step of binding a labeling substance to the tag of the labeling unit.   The method according to claim 8 or 9, wherein the nucleic acid amplification step is a step of performing polymerase chain reaction (PCR) for 25 cycles or more.   The nucleic acid amplification step is performed under the condition that the concentration of each of the first primer and the second primer in the primer set according to any one of claims 1 to 7 is 0.05 μM or more under the condition that the concentration is 0.05 μM or more. The method according to any one of claims 8 to 10, which is a step of performing (PCR). A kit for detecting a nucleic acid derived from Listeria monocytogenes and / or Listeria monocytogenes comprising:
The primer set according to any one of claims 1 to 7,
A kit comprising a binding part and a solid phase carrier containing at least a part capable of binding.   The kit according to claim 12, comprising a nucleic acid detection device comprising the solid phase carrier partly including a binding part and a part capable of binding to a binding part, and a reaction system accepting part for receiving a product of a nucleic acid amplification reaction. The labeling part is a tag that can bind to the labeling substance,
The kit according to claim 12 or 13, further comprising a labeling substance that can bind to the tag of the labeling part.   Nucleic acid detection comprising the solid phase carrier partially including a binding part and a part capable of binding, a labeling substance holding part for holding the labeling substance, and a reaction system receiving part for receiving a product of a nucleic acid amplification reaction 15. A kit according to claim 14, comprising a device.

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