Detection method of fusobacterium nucleatum and detection method of fusobacterium nucleatum subspecies

Abstract

A method for detecting fusobacterium nucleatum in a sample according to one aspect of the present invention comprises a step for performing a nucleic acid amplification reaction on DNA from the sample using a primer set having specificity for a citrate lyase beta subunit gene of fusobacterium nucleatum. According to the method, the fusobacterium nucleatum can be specifically detected. A method for detecting a fusobacterium nucleatum subsp. Nucleatum in a sample according to another aspect of the present invention comprises a step of performing a nucleic acid amplification reaction on DNA from the sample using a primer set having dicarboxylic acid / amino acid: cation co-transporter gene specificity of the fusobacterium nucleatum subsp. Nucleatum. According to the method, the fusobacterium nucleatum subsp. Nucleatum can be specifically detected.

Description

Technical Field

[0001] The invention relates to a detection method of Fusobacterium nucleatum and a detection method of Fusobacterium nucleatum subspecies nucleatum. Background Art

[0002] Fusobacterium nucleatum (F. nucleatum) is a bacterium that lives in the human mouth and colon. Fusobacterium nucleatum is an anaerobic bacterium that has attracted attention in recent years as a bacterium associated with various diseases such as colon cancer and periodontal disease. There are four subspecies of this bacterium: F. nucleatum subsp. animalis, F. nucleatum subsp. polymorphum, F. nucleatum subsp. nucleatum, and F. nucleatum subsp. vincentii.

[0003] As a method for detecting bacteria, there is a method for detecting 16S rRNA of bacteria. For example, in the example of Patent Document 1, a probe for 16S rRNA of Fusobacterium is used to detect Fusobacterium nucleatum. However, this method has a problem in terms of specificity. In the example of Patent Document 1, it is disclosed that the probe for 16S rRNA of Fusobacterium has cross-reactions with various bacteria including Campylobacter.

[0004] As a method for identifying Fusobacterium nucleatum in a sample at a finer classification than subspecies, that is, at the strain level, there is a method comprising the following steps: PCR is performed using a primer combination capable of amplifying DNA containing a CRISPR region, and the presence or absence of a CRISPR-Cas sequence containing the CRISPR region and the length of the sequence are detected (Patent Document 2). However, the detection of the presence or absence of a CRISPR-Cas sequence and the length of the sequence requires electrophoresis of the PCR product or base sequence determination, and this method has practical problems in terms of simplicity. In addition, this method also has practical problems in terms of detection sensitivity.

[0005] On the other hand, a method for broadly and specifically detecting the four subspecies or specific subspecies of Fusobacterium nucleatum rather than distinguishing them at the strain level is still unknown.

[0006] Prior art literature

[0007] Patent Literature

[0008] Patent Document 1: Japanese Patent No. 7040562

[0009] Patent Document 2: Japanese Patent No. 6945294 Summary of the invention

[0010] Problems to be solved by the invention

[0011] The present invention aims to specifically detect Fusobacterium nucleatum. Another object of the present invention is to specifically detect Fusobacterium nucleatum subsp. nucleatum.

[0012] Methods used to solve problems

[0013] In order to find species-specific and subspecies-specific sequences of Fusobacterium, it is necessary to analyze databases such as the National Center for Bioinformation (NCBI). However, it is very difficult to search for specific sequences in the entire 2000 kbp genome of Fusobacterium bacteria from about 50,000 sequence information, and there are no reports of such analysis. In such actual situations, no sequences common to species or subspecies of Fusobacterium were found except for the 16S rRNA gene and CRISPR.

[0014] In this regard, as described later, the present inventors conducted in-depth research, and as a result, they successfully analyzed a large amount of sequence information and found a gene with a species-specific sequence from the entire genome of Fusobacterium nucleatum. Furthermore, the present inventors found that by using a primer set for loop-mediated isothermal amplification (LAMP) reaction made based on the base sequence of the gene, Fusobacterium nucleatum can be specifically detected. In addition, as described later, the present inventors conducted in-depth research, and as a result, they found a gene with a subspecies-specific base sequence from the entire genome of Fusobacterium nucleatum subspecies nucleatum. Furthermore, they found that by using a primer set for LAMP reaction made based on the base sequence of the gene, Fusobacterium nucleatum subspecies nucleatum can be specifically detected. The present invention is a groundbreaking invention completed based on the insights of these present inventors.

[0015] That is, the present invention has the following aspects.

[0016] [1] A method for detecting Fusobacterium nucleatum in a sample, comprising the following steps:

[0017] A nucleic acid amplification reaction was performed on DNA from the sample using a primer set specific to the citrate lyase β subunit gene of Fusobacterium nucleatum.

[0018] [2] The method according to [1], wherein the citrate lyase β subunit gene is composed of a base sequence shown in any one of sequence numbers 1 to 4 or a base sequence having a sequence identity of 90% or more with the base sequence shown in any one of sequence numbers 1 to 4.

[0019] [3] The method according to [2], wherein the primer set is a region-specific primer set, and the region is:

[0020] In the base sequence shown in any one of SEQ ID NOs: 1 to 4, a region from bases 141 to 415, a region from bases 4 to 253, a region from bases 238 to 561, a region from bases 577 to 806, or a region from bases 32 to 636,

[0021] Alternatively, a region having a sequence identity of 90% or more with any of these regions.

[0022] [4] The method according to any one of [1] to [3], wherein the nucleic acid amplification reaction is a loop-mediated isothermal amplification reaction or a polymerase chain reaction.

[0023] [5] The method according to [4], wherein the nucleic acid amplification reaction is a loop-mediated isothermal amplification reaction,

[0024] The above primer sets are:

[0025] (a1) a primer set comprising an F3 primer consisting of the base sequence shown in SEQ ID NO: 5, a B3 primer consisting of the base sequence shown in SEQ ID NO: 6, a FIP primer consisting of the base sequence shown in SEQ ID NO: 7, and a BIP primer consisting of the base sequence shown in SEQ ID NO: 8;

[0026] (a2) a primer set comprising an F3 primer consisting of the base sequence shown in SEQ ID NO: 31, a B3 primer consisting of the base sequence shown in SEQ ID NO: 32, a FIP primer consisting of the base sequence shown in SEQ ID NO: 33, and a BIP primer consisting of the base sequence shown in SEQ ID NO: 34;

[0027] (a3) a primer set comprising an F3 primer consisting of the base sequence set forth in SEQ ID NO: 37, a B3 primer consisting of the base sequence set forth in SEQ ID NO: 38, a FIP primer consisting of the base sequence set forth in SEQ ID NO: 39, and a BIP primer consisting of the base sequence set forth in SEQ ID NO: 40; or

[0028] (a4) A primer set comprising an F3 primer consisting of the base sequence shown in sequence number 43, a B3 primer consisting of the base sequence shown in sequence number 44, a FIP primer consisting of the base sequence shown in sequence number 45, and a BIP primer consisting of the base sequence shown in sequence number 46.

[0029] [6] The method according to [5], wherein:

[0030] The primer set (a1) further comprises an LF primer consisting of the base sequence shown in SEQ ID NO: 9 and an LB primer consisting of the base sequence shown in SEQ ID NO: 10.

[0031] The primer set (a2) further comprises an LF primer consisting of the base sequence shown in SEQ ID NO: 35 and an LB primer consisting of the base sequence shown in SEQ ID NO: 36.

[0032] The primer set (a3) ​​further comprises an LF primer consisting of the base sequence shown in SEQ ID NO: 41 and an LB primer consisting of the base sequence shown in SEQ ID NO: 42.

[0033] The primer set (a4) described above further includes an LF primer consisting of the base sequence shown in SEQ ID NO:47 and an LB primer consisting of the base sequence shown in SEQ ID NO:48.

[0034] [7] The method according to [4], wherein the nucleic acid amplification reaction is a polymerase chain reaction, and the primer set is (a5) a primer set comprising a forward primer consisting of a base sequence shown in sequence number 67 and a reverse primer consisting of a base sequence shown in sequence number 68.

[0035] [8] The method according to any one of [1] to [7], wherein in the above step of performing the nucleic acid amplification reaction, a primer set specific for the dicarboxylic acid / amino acid:cation cotransporter gene of Fusobacterium nucleatum subspecies nucleatum is also used together with the above primer set specific for the citrate lyase β subunit gene of Fusobacterium nucleatum.

[0036] [9] A primer set for detecting Fusobacterium nucleatum, which is a primer set specific to the citrate lyase β subunit gene of Fusobacterium nucleatum.

[0037]

[10] A primer set according to [9], wherein the citrate lyase β subunit gene is composed of a base sequence shown in any one of sequence numbers 1 to 4 or a base sequence having a sequence identity of 90% or more with the base sequence shown in any one of sequence numbers 1 to 4.

[0038]

[11] The primer set according to

[10] , which is a region-specific primer set, wherein the region is:

[0039] In the base sequence shown in any one of SEQ ID NOs: 1 to 4, a region from bases 141 to 415, a region from bases 4 to 253, a region from bases 238 to 561, a region from bases 577 to 806, or a region from bases 32 to 636,

[0040] Alternatively, a region having a sequence identity of 90% or more with any of these regions.

[0041]

[12] The primer set according to any one of [9] to

[11] , which is used for a loop-mediated isothermal amplification reaction or for a polymerase chain reaction.

[0042]

[13] The primer set according to

[12] , which is used for loop-mediated isothermal amplification reaction, wherein the primer set is:

[0043] (a1) a primer set comprising an F3 primer consisting of the base sequence shown in SEQ ID NO: 5, a B3 primer consisting of the base sequence shown in SEQ ID NO: 6, a FIP primer consisting of the base sequence shown in SEQ ID NO: 7, and a BIP primer consisting of the base sequence shown in SEQ ID NO: 8;

[0044] (a2) a primer set comprising an F3 primer consisting of the base sequence shown in SEQ ID NO: 31, a B3 primer consisting of the base sequence shown in SEQ ID NO: 32, a FIP primer consisting of the base sequence shown in SEQ ID NO: 33, and a BIP primer consisting of the base sequence shown in SEQ ID NO: 34;

[0045] (a3) a primer set comprising an F3 primer consisting of the base sequence set forth in SEQ ID NO: 37, a B3 primer consisting of the base sequence set forth in SEQ ID NO: 38, a FIP primer consisting of the base sequence set forth in SEQ ID NO: 39, and a BIP primer consisting of the base sequence set forth in SEQ ID NO: 40; or

[0046] (a4) A primer set comprising an F3 primer consisting of the base sequence shown in sequence number 43, a B3 primer consisting of the base sequence shown in sequence number 44, a FIP primer consisting of the base sequence shown in sequence number 45, and a BIP primer consisting of the base sequence shown in sequence number 46.

[0047]

[14] The primer set according to

[13] , wherein:

[0048] The primer set (a1) further comprises an LF primer consisting of the base sequence shown in SEQ ID NO: 9 and an LB primer consisting of the base sequence shown in SEQ ID NO: 10.

[0049] The primer set (a2) further comprises an LF primer consisting of the base sequence shown in SEQ ID NO: 35 and an LB primer consisting of the base sequence shown in SEQ ID NO: 36.

[0050] The primer set (a3) ​​further comprises an LF primer consisting of the base sequence shown in SEQ ID NO: 41 and an LB primer consisting of the base sequence shown in SEQ ID NO: 42.

[0051] The primer set (a4) described above further includes an LF primer consisting of the base sequence shown in SEQ ID NO:47 and an LB primer consisting of the base sequence shown in SEQ ID NO:48.

[0052]

[15] The primer set according to

[12] , which is used for polymerase chain reaction, and the primer set is (a5) a primer set containing a forward primer composed of the base sequence shown in sequence number 67 and a reverse primer composed of the base sequence shown in sequence number 68.

[0053]

[16] A method for detecting Fusobacterium nucleatum subspecies nucleatum in a sample, comprising the following steps: performing a nucleic acid amplification reaction on DNA from the sample using a primer set specific to the dicarboxylic acid / amino acid:cation cotransporter gene of Fusobacterium nucleatum subspecies nucleatum.

[0054]

[17] The method according to

[16] , wherein the above-mentioned dicarboxylic acid / amino acid:cation co-transporter gene is composed of the base sequence shown in sequence number 11 or a base sequence having a sequence identity of more than 90% with the base sequence shown in sequence number 11.

[0055]

[18] The method according to

[17] , wherein the primer set is a region-specific primer set, and the region is:

[0056] In the base sequence shown in SEQ ID NO: 11, a region from bases 860 to 1121, a region from bases 196 to 449, a region from bases 580 to 869, or a region from bases 761 to 1038,

[0057] Alternatively, a region having a sequence identity of 90% or more with any of these regions.

[0058]

[19] The method according to any one of

[16] to

[18] , wherein the nucleic acid amplification reaction is a loop-mediated isothermal amplification reaction.

[0059]

[20] The method according to

[19] , wherein the primer set is:

[0060] (b1) a primer set comprising an F3 primer consisting of the base sequence shown in SEQ ID NO: 12, a B3 primer consisting of the base sequence shown in SEQ ID NO: 13, a FIP primer consisting of the base sequence shown in SEQ ID NO: 14, and a BIP primer consisting of the base sequence shown in SEQ ID NO: 15;

[0061] (b2) a primer set comprising an F3 primer consisting of the base sequence shown in SEQ ID NO: 49, a B3 primer consisting of the base sequence shown in SEQ ID NO: 50, a FIP primer consisting of the base sequence shown in SEQ ID NO: 51, and a BIP primer consisting of the base sequence shown in SEQ ID NO: 52;

[0062] (b3) a primer set comprising an F3 primer consisting of the base sequence set forth in SEQ ID NO: 55, a B3 primer consisting of the base sequence set forth in SEQ ID NO: 56, a FIP primer consisting of the base sequence set forth in SEQ ID NO: 57, and a BIP primer consisting of the base sequence set forth in SEQ ID NO: 58; or

[0063] (b4) A primer set comprising an F3 primer consisting of the base sequence shown in sequence number 61, a B3 primer consisting of the base sequence shown in sequence number 62, a FIP primer consisting of the base sequence shown in sequence number 63, and a BIP primer consisting of the base sequence shown in sequence number 64.

[0064]

[21] The method according to

[20] , wherein:

[0065] The primer set (b1) further comprises an LF primer consisting of the base sequence shown in SEQ ID NO: 16 and an LB primer consisting of the base sequence shown in SEQ ID NO: 17.

[0066] The primer set (b2) further comprises an LF primer consisting of the base sequence shown in SEQ ID NO: 53 and an LB primer consisting of the base sequence shown in SEQ ID NO: 54.

[0067] The primer set (b3) further comprises an LF primer consisting of the base sequence shown in SEQ ID NO: 59 and an LB primer consisting of the base sequence shown in SEQ ID NO: 60.

[0068] The primer set (b4) described above further includes an LF primer consisting of the base sequence shown in SEQ ID NO: 65 and an LB primer consisting of the base sequence shown in SEQ ID NO: 66.

[0069]

[22] A primer set for detecting Fusobacterium nucleatum subsp. nucleatum, which is a primer set specific to the dicarboxylic acid / amino acid:cation cotransporter gene of Fusobacterium nucleatum subsp. nucleatum.

[0070]

[23] A primer set according to

[22] , wherein the dicarboxylic acid / amino acid:cation co-transporter gene is composed of the base sequence shown in sequence number 11 or a base sequence having a sequence identity of more than 90% with the base sequence shown in sequence number 11.

[0071]

[24] The primer set according to

[23] , which is a region-specific primer set, wherein the region is:

[0072] In the base sequence shown in SEQ ID NO: 11, a region from bases 860 to 1121, a region from bases 196 to 449, a region from bases 580 to 869, or a region from bases 761 to 1038,

[0073] Alternatively, a region having a sequence identity of 90% or more with any of these regions.

[0074]

[25] The primer set according to any one of

[22] to

[24] , which is a primer set for loop-mediated isothermal amplification reaction.

[0075]

[26] The primer set according to

[25] , which is:

[0076] (b1) a primer set comprising an F3 primer consisting of the base sequence shown in SEQ ID NO: 12, a B3 primer consisting of the base sequence shown in SEQ ID NO: 13, a FIP primer consisting of the base sequence shown in SEQ ID NO: 14, and a BIP primer consisting of the base sequence shown in SEQ ID NO: 15;

[0077] (b2) a primer set comprising an F3 primer consisting of the base sequence shown in SEQ ID NO: 49, a B3 primer consisting of the base sequence shown in SEQ ID NO: 50, a FIP primer consisting of the base sequence shown in SEQ ID NO: 51, and a BIP primer consisting of the base sequence shown in SEQ ID NO: 52;

[0078] (b3) a primer set comprising an F3 primer consisting of the base sequence set forth in SEQ ID NO: 55, a B3 primer consisting of the base sequence set forth in SEQ ID NO: 56, a FIP primer consisting of the base sequence set forth in SEQ ID NO: 57, and a BIP primer consisting of the base sequence set forth in SEQ ID NO: 58; or

[0079] (b4) A primer set comprising an F3 primer consisting of the base sequence shown in sequence number 61, a B3 primer consisting of the base sequence shown in sequence number 62, a FIP primer consisting of the base sequence shown in sequence number 63, and a BIP primer consisting of the base sequence shown in sequence number 64.

[0080]

[27] The primer set according to

[26] , wherein:

[0081] The primer set (b1) further comprises an LF primer consisting of the base sequence shown in SEQ ID NO: 16 and an LB primer consisting of the base sequence shown in SEQ ID NO: 17.

[0082] The primer set (b2) further comprises an LF primer consisting of the base sequence shown in SEQ ID NO: 53 and an LB primer consisting of the base sequence shown in SEQ ID NO: 54.

[0083] The primer set (b3) further comprises an LF primer consisting of the base sequence shown in SEQ ID NO: 59 and an LB primer consisting of the base sequence shown in SEQ ID NO: 60.

[0084] The primer set (b4) described above further includes an LF primer consisting of the base sequence shown in SEQ ID NO: 65 and an LB primer consisting of the base sequence shown in SEQ ID NO: 66.

[0085] Effects of the Invention

[0086] According to the method of one aspect of the present invention, Fusobacterium nucleatum can be specifically detected. In addition, according to the method of another aspect of the present invention, Fusobacterium nucleatum subspecies can be specifically detected. The present invention is particularly useful in that it can specifically detect four subspecies of Fusobacterium nucleatum or Fusobacterium nucleatum subspecies contained in clinical samples such as saliva, feces, and tissues without culture. DETAILED DESCRIPTION

[0087] A method for detecting Fusobacterium nucleatum in a sample according to one aspect of the present invention includes a step of performing a nucleic acid amplification reaction on DNA from the sample using a primer set specific to the citrate lyase β subunit gene (CitE) of Fusobacterium nucleatum (hereinafter referred to as a nucleic acid amplification step).

[0088] The sample is not particularly limited, and may be, for example, a sample collected from a subject suspected of being infected with Fusobacterium nucleatum. The sample may be, for example, a biological sample such as nasal mucus, nasal aspirate, nasal swab, throat swab, vaginal swab, saliva, blood, plasma, serum, menstrual blood, urine, feces, tissue, or cells. The sample may be a substance obtained by separation, concentration, purification, or the like of the above biological samples.

[0089] The DNA from the sample may be DNA isolated from the sample, or may be DNA that is not isolated from the sample but dissolved in the sample. Therefore, the method for detecting Fusobacterium nucleatum in the sample of the present invention may include a step of extracting DNA from the sample and / or a step of isolating DNA from the sample before the nucleic acid amplification step. The extraction and isolation of DNA may be performed by known methods.

[0090] In the present specification, "citrate lyase β subunit gene" refers to a gene encoding the β subunit of citrate lyase. The β subunit of citrate lyase is sometimes referred to as "citrate lyase subunit β" (citrate lyase subunitbeta), "citrate (pro-3S)-lyase subunit β" (citrate (pro-3S)-lyase subunit beta), "citrate (pro-3S)-lyase, β subunit" (citrate (Pro-3S)-lyase, beta subunit), "citrate lyase beta chain" (citrate lyase beta chain) and other names. As long as these proteins function as the β subunit of citrate lyase, the genes encoding these proteins are also within the scope of the term "citrate lyase β subunit gene".

[0091] The citrate lyase β subunit gene of Fusobacterium nucleatum may be, for example, a gene consisting of a base sequence represented by any one of SEQ ID NOs. 1 to 4 or a base sequence having a sequence identity of 90% or more to the base sequence represented by any one of SEQ ID NOs. 1 to 4 respectively represent the base sequences of the citrate lyase β subunit gene of Fusobacterium nucleatum shown in the following table.

[0092]

[0093] From the viewpoint of more specifically detecting Fusobacterium nucleatum, the base sequence having a sequence identity of 90% or more with the base sequence represented by any one of SEQ ID NOs: 1 to 4 is preferably a base sequence having a sequence identity of 92% or more, 93% or more, or 95% or more with the base sequence represented by any one of SEQ ID NOs: 1 to 4.

[0094] In this specification, a primer set specific to a gene or a region in a gene refers to a combination of one or more forward primers and one or more reverse primers (also referred to as downstream primers) that anneal (i.e., specifically bind) to the base sequence or complementary base sequence of the gene or the region (i.e., the target gene or the target region). By using a primer set specific to the target gene or the target region for nucleic acid amplification reaction, a portion or the entire length of the target gene or the target region can be amplified.

[0095] The citrate lyase β subunit gene of Fusobacterium nucleatum has high sequence identity among subspecies, so a primer set specific to the citrate lyase β subunit gene of one subspecies is also specific to the citrate lyase β subunit genes of all other subspecies. That is, the primer set specific to the citrate lyase β subunit gene of Fusobacterium nucleatum is a primer set specific to the citrate lyase β subunit gene of all subspecies of Fusobacterium nucleatum. That is, it can be said that, for example, a primer set specific to the citrate lyase β subunit gene composed of the base sequence shown in any one of sequence numbers 1 to 4 is necessarily a primer set specific to all of the citrate lyase β subunit gene composed of the base sequence shown in sequence number 1, the citrate lyase β subunit gene composed of the base sequence shown in sequence number 2, the citrate lyase β subunit gene composed of the base sequence shown in sequence number 3, and the citrate lyase β subunit gene composed of the base sequence shown in sequence number 4. In addition, it can be said that such a primer set is also a primer set specific for the citrate lyase β subunit gene composed of a base sequence having a sequence identity of more than 90%, more than 92%, more than 93% or more than 95% with the base sequence shown in sequence number 1, the base sequence shown in sequence number 2, the base sequence shown in sequence number 3 and the base sequence shown in sequence number 4.

[0096] The primer set can be designed based on any region in the citrate lyase β subunit gene. For example, the primer set can be designed based on (i) the region of bases 141 to 415, (ii) the region of bases 4 to 253, (iii) the region of bases 238 to 561, (iv) the region of bases 577 to 806, or (v) the region of bases 32 to 636 in the citrate lyase β subunit gene. That is, the primer set specific to the citrate lyase β subunit gene may be, for example, a primer set specific to a region of the base sequence shown in any one of SEQ ID NOs: 1 to 4, (i) a region of bases 141 to 415, (ii) a region of bases 4 to 253, (iii) a region of bases 238 to 561, (iv) a region of bases 577 to 806, or (v) a region of bases 32 to 636. Alternatively, the primer set specific to the citrate lyase β subunit gene may be a primer set specific to a region corresponding to any one of the regions (i) to (v) in a base sequence having a sequence identity of 90% or more with the base sequence shown in any one of SEQ ID NOs: 1 to 4, that is, a primer set specific to a region having a sequence identity of 90% or more, 92% or more, 93% or more, or 95% or more with any one of the regions (i) to (v). As described above, the primer set specific to the region of bases 141 to 415 in the base sequence shown in any one of SEQ ID NOs. 1 to 4 can be said to be a primer set specific to the region of bases 141 to 415 in the base sequence shown in each of SEQ ID NOs. 1, 2, 3, and 4, and such a primer set can be said to be a primer set specific also to the citrate lyase β subunit gene consisting of regions having a sequence identity of 90% or more, 92% or more, 93% or more, or 95% or more with these regions. The same can be said of primer sets specific to other regions.

[0097] The primer set can be appropriately designed by a person skilled in the art according to the type of nucleic acid amplification reaction and based on the disclosure of known literature, etc. In addition, the number (type) of forward primers and reverse primers constituting the primer set can also be appropriately determined by a person skilled in the art according to the type of nucleic acid amplification reaction.

[0098] The nucleic acid amplification reaction can be, for example, a polymerase chain reaction (PCR), a loop-mediated isothermal amplification (LAMP) reaction, a transcription-mediated amplification (TMA) reaction, or a nucleic acid sequence-dependent amplification (NASBA) reaction.

[0099] When the nucleic acid amplification reaction is a LAMP reaction, the primer set contains F3 primer, FIP primer and optional LF primer as forward primers, and contains B3 primer, BIP primer and optional LB primer as reverse primers. Methods for designing these primers according to a certain target gene are well known.

[0100] Examples of the primer set for LAMP reaction include the following primer sets (a1) to (a4). The position of each base is the position in the base sequence shown in any one of SEQ ID NOs: 1 to 4.

[0101] (a1)

[0102] A primer set comprising:

[0103] F3 primers designed based on the region of bases 141 to 163 (region F3),

[0104] B3 primers designed based on the complementary region of the region from base 394 to base 415 (region B3),

[0105] The FIP primers at the 5' end are designed based on the complementary region of the region from base 268 to base 292 (region F1c) and the 3' end are designed based on the region from base 179 to base 205 (region F2), and

[0106] The BIP primer at the 5' end is designed based on the region of bases 301 to 325 (region B1c) and the BIP primer at the 3' end is designed based on the complementary region of the region of bases 367 to 386 (region B2), and

[0107] Optionally containing:

[0108] LF primers designed based on the complementary region of the region from base 226 to base 253, and

[0109] LB primers designed based on the region from base 336 to base 366.

[0110] (a2)

[0111] A primer set comprising:

[0112] F3 primers designed based on the region of bases 4 to 29 (region F3),

[0113] B3 primers designed based on the complementary region of the region from base 231 to base 253 (region B3),

[0114] The FIP primers at the 5' end are designed based on the complementary region of the region from base 106 to base 131 (region F1c) and the 3' end are designed based on the region from base 40 to base 63 (region F2), and

[0115] The BIP primer at the 5' end is designed based on the region of bases 133 to 163 (region B1c) and the BIP primer at the 3' end is designed based on the complementary region of the region of bases 200 to 223 (region B2), and

[0116] Optionally containing:

[0117] LF primers designed based on the complementary region of the region from base 64 to base 93, and

[0118] LB primers designed based on the region from base 166 to base 199.

[0119] (a3)

[0120] A primer set comprising:

[0121] F3 primers designed based on the region of bases 238 to 259 (region F3),

[0122] B3 primers designed based on the complementary region of the region from base 544 to base 561 (region B3),

[0123] The FIP primers at the 5' end are designed based on the complementary region of the region from base 428 to base 454 (region F1c), and the FIP primers at the 3' end are designed based on the region from base 269 to base 295 (region F2), and

[0124] The BIP primer at the 5' end is designed based on the region of bases 465 to 490 (region B1c) and the BIP primer at the 3' end is designed based on the complementary region of the region of bases 514 to 536 (region B2), and

[0125] Optionally containing:

[0126] LF primers designed based on the complementary region of the region from base 398 to base 427, and

[0127] LB primers designed based on the region from base 486 to base 513.

[0128] (a4)

[0129] A primer set comprising:

[0130] F3 primers designed based on the region of bases 577 to 599 (region F3),

[0131] B3 primers designed based on the complementary region of the region from base 784 to base 806 (region B3),

[0132] The FIP primers for designing the 5' end based on the complementary region of the region from base 652 to base 677 (region F1c) and designing the 3' end based on the region from base 601 to base 625 (region F2), and

[0133] The BIP primer at the 5' end is designed based on the region of bases 689 to 718 (region B1c) and the BIP primer at the 3' end is designed based on the complementary region of the region of bases 766 to 783 (region B2), and

[0134] Optionally containing:

[0135] LF primers designed based on the complementary region of the region from base 626 to base 658, and

[0136] LB primers designed based on the region from base 722 to base 756.

[0137] It should be noted that, in the present specification, "primers designed based on a certain region" refers to primers designed in a manner to anneal to the complementary sequence of the region, and usually have the same base sequence as the region or have a base sequence with a sequence identity of more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98% or more than 99% with the base sequence.

[0138] As a specific example of the primer set of (a1) above, there can be cited a primer set containing an F3 primer consisting of the base sequence shown in SEQ ID NO: 5, a B3 primer consisting of the base sequence shown in SEQ ID NO: 6, a FIP primer consisting of the base sequence shown in SEQ ID NO: 7, and a BIP primer consisting of the base sequence shown in SEQ ID NO: 8, and optionally containing an LF primer consisting of the base sequence shown in SEQ ID NO: 9 and an LB primer consisting of the base sequence shown in SEQ ID NO: 10. In addition, the F3, B3, FIP, BIP, LF, and LB primers can each be composed of a base sequence having a sequence identity of 90% or more with the base sequences shown in SEQ ID NOs: 5, 6, 7, 8, 9, and 10.

[0139] As a specific example of the primer set of (a2) above, there can be cited a primer set containing an F3 primer consisting of the base sequence shown in SEQ ID NO: 31, a B3 primer consisting of the base sequence shown in SEQ ID NO: 32, a FIP primer consisting of the base sequence shown in SEQ ID NO: 33, and a BIP primer consisting of the base sequence shown in SEQ ID NO: 34, and optionally containing an LF primer consisting of the base sequence shown in SEQ ID NO: 35 and an LB primer consisting of the base sequence shown in SEQ ID NO: 36. In addition, the F3, B3, FIP, BIP, LF, and LB primers can each be composed of a base sequence having a sequence identity of 90% or more with the base sequences shown in SEQ ID NOs: 31, 32, 33, 34, 35, and 36.

[0140] As a specific example of the primer set of (a3) ​​above, there can be cited a primer set containing an F3 primer consisting of the base sequence shown in SEQ ID NO: 37, a B3 primer consisting of the base sequence shown in SEQ ID NO: 38, a FIP primer consisting of the base sequence shown in SEQ ID NO: 39, and a BIP primer consisting of the base sequence shown in SEQ ID NO: 40, and optionally containing an LF primer consisting of the base sequence shown in SEQ ID NO: 41 and an LB primer consisting of the base sequence shown in SEQ ID NO: 42. In addition, the F3, B3, FIP, BIP, LF, and LB primers can each be composed of a base sequence having a sequence identity of 90% or more with the base sequences shown in SEQ ID NOs: 37, 38, 39, 40, 41, and 42.

[0141] As a specific example of the primer set of (a4) above, there can be cited a primer set containing an F3 primer consisting of the base sequence shown in SEQ ID NO: 43, a B3 primer consisting of the base sequence shown in SEQ ID NO: 44, a FIP primer consisting of the base sequence shown in SEQ ID NO: 45, and a BIP primer consisting of the base sequence shown in SEQ ID NO: 46, and optionally containing an LF primer consisting of the base sequence shown in SEQ ID NO: 47 and an LB primer consisting of the base sequence shown in SEQ ID NO: 48. In addition, the F3, B3, FIP, BIP, LF, and LB primers can each be composed of a base sequence having a sequence identity of 90% or more with the base sequences shown in SEQ ID NOs: 43, 44, 45, 46, 47, and 48.

[0142] The above sequence identities can all be 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0143] When the nucleic acid amplification reaction is PCR, examples of the primer set for PCR include the following primer set (a5). The position of each base is the position in the base sequence shown in any one of SEQ ID NOs: 1 to 4.

[0144] (a5)

[0145] A primer set comprising:

[0146] A forward primer designed based on the region from base 32 to base 59, and

[0147] The reverse primer was designed based on the complementary region of the region from base 613 to base 636.

[0148] As a specific example of the primer set of (a5) above, there can be cited a primer set comprising a forward primer consisting of the base sequence shown in SEQ ID NO: 67 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 68. In addition, the forward primer and the reverse primer can be composed of a base sequence having a sequence identity of 90% or more with the base sequences shown in SEQ ID NO: 67 and 68, respectively.

[0149] The above sequence identity may be 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0150] In the above nucleic acid amplification step, a primer set specific to the dicarboxylic acid / amino acid:cation cotransporter gene of Fusobacterium nucleatum subspecies nucleatum described later may be further used together with a primer set specific to the citrate lyase β subunit gene of Fusobacterium nucleatum. By using the primer set specific to the dicarboxylic acid / amino acid:cation cotransporter gene of Fusobacterium nucleatum subspecies nucleatum in combination, the detection sensitivity of Fusobacterium nucleatum subspecies nucleatum can be improved.

[0151] In the above-mentioned nucleic acid amplification step, known reagents for nucleic acid amplification reactions such as DNA polymerase, deoxyribonucleoside triphosphates (dNTP: dATP, dTTP, dCTP and dGTP) can be further used. Other known reagents for nucleic acid amplification reactions include, for example, buffers or salts that provide conditions suitable for enzyme reactions, and protective agents such as dithiothreitol that stabilize enzymes or DNA from samples.

[0152] The DNA polymerase can be appropriately selected depending on the type of nucleic acid amplification reaction, and can be, for example, Bst DNA polymerase (large fragment), Bca (exo-) DNA polymerase, Csa DNA polymerase, Klenow fragment of Escherichia coli DNA polymerase I, or Taq DNA polymerase.

[0153] In the above-mentioned nucleic acid amplification step, a labeled probe or fluorescent intercalator for detecting the amplification product of the nucleic acid amplification reaction can be further used. As the labeled probe, for example, a known fluorescent labeled probe such as a fluorescent quenching probe (QuenchingProbe: QProbe (registered trademark)) can be used. As the fluorescent intercalator, for example, a known fluorescent intercalator such as SYTO (trademark) 63 red fluorescent nucleic acid dye or SYBR (trademark) green I nucleic acid gel dye (both manufactured by Thermo Fisher Scientific) can be used.

[0154] The above-mentioned nucleic acid amplification step can be performed, for example, by incubating a reaction solution containing the DNA from the sample and the above-mentioned primer set. The reaction solution may also contain the above-mentioned known reagents for performing nucleic acid amplification reactions and the above-mentioned labeled probes or fluorescent intercalators. The temperature and time of incubation (i.e., the temperature and time of the reaction) can be appropriately set according to the type of nucleic acid amplification reaction, the type of DNA polymerase, and the melting temperature of the primers. For example, in the case of the LAMP reaction, the reaction temperature is not particularly limited as long as it is a temperature at which each primer can anneal with the citrate lyase β subunit gene, and for example, it can be below 65°C or below 60°C, and the reaction time can be, for example, below 90 minutes or below 60 minutes.

[0155] The concentration of each primer in the reaction solution can be appropriately adjusted according to the type of nucleic acid amplification reaction. For example, in the case of LAMP reaction, the concentration of the FIP primer and the concentration of the BIP primer in the reaction solution can be, for example, 0.8 to 2.4 μM, the concentration of the F3 primer and the concentration of the B3 primer can be, for example, 0.1 to 0.3 μM, and the concentration of the LF primer and the concentration of the LB primer can be, for example, 0.4 to 1.2 μM.

[0156] The method for detecting Fusobacterium nucleatum in a sample according to this aspect of the present invention may further include the step of detecting an amplification product of a nucleic acid amplification reaction. When the presence of an amplification product is detected, it can be determined that Fusobacterium nucleatum is present in the sample. On the other hand, when the presence of an amplification product is not detected, it can be determined that Fusobacterium nucleatum is not present in the sample. The detection of the amplification product may be performed after the nucleic acid amplification step, or may be performed in real time during the nucleic acid amplification step.

[0157] The method for detecting the amplification product is not particularly limited, and known techniques can be used. The amplification product can be detected, for example, by using the above-mentioned labeled probe or fluorescent intercalator, or by electrophoresis of the reaction solution. Alternatively, when the reaction solution contains magnesium ions, the amplification product can also be detected by measuring the turbidity of the reaction solution. Magnesium ions react with pyrophosphate ions as a by-product of nucleic acid synthesis to produce white magnesium pyrophosphate. From the perspective of more easily detecting Fusobacterium nucleatum in the sample, the detection of the amplification product is preferably performed by using a labeled probe or a fluorescent intercalator or by measuring the turbidity of the reaction solution.

[0158] According to the method for detecting Fusobacterium nucleatum in a sample of this aspect of the present invention, Fusobacterium nucleatum can be specifically detected by using a primer set specific to the citrate lyase β subunit gene that has high sequence consistency between subspecies of Fusobacterium nucleatum but low sequence consistency with other species of the genus Fusobacterium. In addition, the citrate lyase β subunit gene has high sequence consistency between subspecies of Fusobacterium nucleatum, so it is not necessary to prepare a primer set for each subspecies, and the four subspecies of Fusobacterium nucleatum can be detected using a single primer set specific to the citrate lyase β subunit gene. The existence of such a gene with a species-specific sequence is extremely rare, and it can be said that it would not have been discovered without the creative work and excellent information processing technology of the inventors, as well as the countless hours spent.

[0159] Specifically, usually when designing primers, the region of about hundreds of bp that can become the amplification region is explored with the homology and intersectionality that meet the target as an index. The inventors found out when exploring this region for the genome of Fusobacterium nucleatum that even the bacterial strain belonging to the same subspecies also has a variety of sequences. Therefore, in order to explore more comprehensively, it is necessary to refer to the public genome sequence of the American Type Culture Collection (ATCC) and multiple databases such as NCBI. However, it is difficult to find a region of hundreds of bp with high homology between Fusobacterium nucleatum subspecies and low homology with other species of Fusobacterium. Therefore, the inventors broke away from the conventional method and the genomic sequence of Fusobacterium was subdivided into about 20bp to explore the consensus sequence between Fusobacterium nucleatum subspecies. Then, by comprehensively analyzing the intersectionality of the consensus sequences obtained more than 30,000, the citrate lyase β subunit gene as a specific and rare region of only Fusobacterium nucleatum was found.

[0160] As described above, by using the above primer set specific to the citrate lyase β subunit gene of Fusobacterium nucleatum, Fusobacterium nucleatum can be specifically detected. Therefore, another aspect of the present invention can be said to be the above primer set specific to the citrate lyase β subunit gene of Fusobacterium nucleatum for detecting Fusobacterium nucleatum.

[0161] In addition, another aspect of the present invention is a kit for detecting Fusobacterium nucleatum, comprising the above primer set specific to the citrate lyase β subunit gene of Fusobacterium nucleatum. The kit may further comprise the above known reagents for nucleic acid amplification reaction such as DNA polymerase, dNTP, buffer, salt, protective agent, and the above labeled probe or fluorescent intercalator. In addition, the kit may further comprise a primer set specific to the dicarboxylic acid / amino acid: cation cotransporter gene of Fusobacterium nucleatum subspecies nucleatum described later.

[0162] According to one aspect of the present invention, a method for detecting Fusobacterium nucleatum subsp. nucleatum in a sample includes a step of performing a nucleic acid amplification reaction on DNA from the sample using a primer set specific to the dicarboxylic acid / amino acid:cation cotransporter (DAACS) gene of Fusobacterium nucleatum subsp. nucleatum (hereinafter referred to as the nucleic acid amplification step).

[0163] The sample is not particularly limited, and may be, for example, a sample collected from a subject suspected of being infected with Fusobacterium nucleatum subspecies nucleatum. Examples of the sample include the same samples as those exemplified in the method for detecting Fusobacterium nucleatum in a sample in the above aspect.

[0164] The details of the DNA from the sample are as described in the method for detecting Fusobacterium nucleatum in a sample in the above aspect.

[0165] In the present specification, "dicarboxylic acid / amino acid: cation cotransporter gene" refers to a gene encoding a dicarboxylic acid / amino acid: cation cotransporter. Dicarboxylic acid / amino acid: cation cotransporter is sometimes also referred to as "proton / sodium-aspartate symporter", "glutamate: protein symporter", "C4-dicarboxylate transporter" and other names. As long as these proteins function as dicarboxylic acid / amino acid: cation cotransporters (i.e., as long as they have dicarboxylic acid / amino acid: cation cotransporter activity), genes encoding these proteins are also included in the scope of the term "dicarboxylic acid / amino acid: cation cotransporter gene".

[0166] The DAACS gene of Fusobacterium nucleatum subsp. nucleatum may be, for example, a gene consisting of a base sequence as shown in SEQ ID NO: 11 or a base sequence having a sequence identity of 90% or more to the base sequence as shown in SEQ ID NO: 11. SEQ ID NO: 11 shows the base sequence of the DAACS gene of Fusobacterium nucleatum subsp. nucleatum strain ATCC25586 (Accession No.: CP028101). The base sequence having a sequence identity of 90% or more to the base sequence as shown in SEQ ID NO: 11 is preferably a base sequence having a sequence identity of 95% or more, 99% or more, or 99.5% or more to the base sequence as shown in SEQ ID NO: 11.

[0167] The primer set can be designed based on any region in the DAACS gene. For example, the primer set can be designed based on the region of (I) bases from position 860 to position 1121, (II) bases from position 196 to position 449, (III) bases from position 580 to position 869, or (IV) bases from position 761 to position 1038 in the DAACS gene. That is, the primer set specific to the DAACS gene can be, for example, a region-specific primer set, wherein the region is: in the base sequence shown in SEQ ID NO: 11, (I) the region of bases from position 860 to position 1121, (II) the region of bases from position 196 to position 449, (III) the region of bases from position 580 to position 869, or (IV) the region of bases from position 761 to position 1038. Alternatively, the DAACS gene-specific primer set can be a region-specific primer set corresponding to any one of the above-mentioned regions (I) to (IV) in a base sequence having a sequence identity of more than 90% with the base sequence shown in sequence number 11, that is, it can be a region-specific primer set having a sequence identity of more than 90%, more than 95%, more than 99% or more than 99.5% with any one of the above-mentioned regions (I) to (IV).

[0168] The primer set can be appropriately designed by a person skilled in the art according to the type of nucleic acid amplification reaction and based on the disclosure of known literature, etc. In addition, the number (type) of forward primers and reverse primers constituting the primer set can also be appropriately determined by a person skilled in the art according to the type of nucleic acid amplification reaction.

[0169] The nucleic acid amplification reaction may be, for example, PCR, LAMP reaction, TMA reaction or NASBA reaction.

[0170] When the nucleic acid amplification reaction is a LAMP reaction, the primer set contains an F3 primer, a FIP primer and an optional LF primer as a forward primer, and contains a B3 primer, a BIP primer and an optional LB primer as a reverse primer.

[0171] Examples of the primer set for LAMP reaction include the following primer sets (b1) to (b4). The position of each base is the position in the base sequence shown in SEQ ID NO:11.

[0172] (b1)

[0173] A primer set comprising:

[0174] F3 primers designed based on the region of bases 860 to 885 (region F3),

[0175] B3 primers designed based on the complementary region of the region from base 1098 to base 1121 (region B3),

[0176] The FIP primers for designing the 5' end based on the complementary region of the region from base 964 to base 993 (region F1c) and designing the 3' end based on the region from base 892 to base 914 (region F2), and

[0177] The BIP primer at the 5' end is designed based on the region of bases 997 to 1016 (region B1c), and the BIP primer at the 3' end is designed based on the complementary region of the region of bases 1070 to 1092 (region B2), and

[0178] Optionally containing:

[0179] LF primers designed based on the complementary region of the region from base 882 to base 925, and

[0180] LB primers designed based on the region from base 1032 to base 1064.

[0181] (b2)

[0182] A primer set comprising:

[0183] F3 primers designed based on the region of bases 196 to 220 (region F3),

[0184] B3 primers designed based on the complementary region of the region from base 420 to base 449 (region B3),

[0185] The FIP primers at the 5' end are designed based on the complementary region of the region from base 285 to base 307 (region F1c) and the 3' end are designed based on the region from base 230 to base 254 (region F2), and

[0186] The BIP primer at the 5' end is designed based on the region of bases 317 to 342 (region B1c), and the BIP primer at the 3' end is designed based on the complementary region of the region of bases 397 to 417 (region B2), and

[0187] Optionally containing:

[0188] LF primers designed based on the complementary region of the region from base 256 to base 284, and

[0189] LB primers designed based on the region from base 355 to base 380.

[0190] (b3)

[0191] A primer set comprising:

[0192] F3 primers designed based on the region of bases 580 to 605 (region F3),

[0193] B3 primers designed based on the complementary region of the region from base 844 to base 869 (region B3),

[0194] The FIP primers for designing the 5' end based on the complementary region of the region from base 688 to base 717 (region F1c) and designing the 3' end based on the region from base 619 to base 648 (region F2), and

[0195] The BIP primer at the 5' end is designed based on the region of bases 735 to 759 (region B1c) and the BIP primer at the 3' end is designed based on the complementary region of the region of bases 813 to 834 (region B2), and

[0196] Optionally containing:

[0197] LF primers designed based on the complementary region of the region from base 649 to base 684, and

[0198] LB primers designed based on the region from base 770 to base 792.

[0199] (b4)

[0200] A primer set comprising:

[0201] F3 primers designed based on the region of bases 761 to 780 (region F3),

[0202] B3 primers designed based on the complementary region of the region from base 1017 to base 1038 (region B3),

[0203] The FIP primers at the 5' end are designed based on the complementary region of the region from base 855 to base 884 (region F1c), and the FIP primers at the 3' end are designed based on the region from base 785 to base 810 (region F2), and

[0204] The BIP primer at the 5' end is designed based on the region of bases 894 to 921 (region B1c) and the BIP primer at the 3' end is designed based on the complementary region of the region of bases 975 to 996 (region B2), and

[0205] Optionally containing:

[0206] LF primers designed based on the complementary region of the region from base 823 to base 854, and

[0207] LB primers designed based on the region from base 932 to base 965.

[0208] As a specific example of the primer set of the above (b1), there can be cited a primer set containing an F3 primer consisting of the base sequence shown in SEQ ID NO: 12, a B3 primer consisting of the base sequence shown in SEQ ID NO: 13, a FIP primer consisting of the base sequence shown in SEQ ID NO: 14, and a BIP primer consisting of the base sequence shown in SEQ ID NO: 15, and optionally containing an LF primer consisting of the base sequence shown in SEQ ID NO: 16 and an LB primer consisting of the base sequence shown in SEQ ID NO: 17. In addition, the F3, B3, FIP, BIP, LF, and LB primers can be composed of base sequences having a sequence identity of 90% or more with the base sequences shown in SEQ ID NOs: 12, 13, 14, 15, 16, and 17, respectively.

[0209] As a specific example of the primer set of (b2) above, there can be cited a primer set containing an F3 primer consisting of the base sequence shown in SEQ ID NO: 49, a B3 primer consisting of the base sequence shown in SEQ ID NO: 50, a FIP primer consisting of the base sequence shown in SEQ ID NO: 51, and a BIP primer consisting of the base sequence shown in SEQ ID NO: 52, and optionally containing an LF primer consisting of the base sequence shown in SEQ ID NO: 53 and an LB primer consisting of the base sequence shown in SEQ ID NO: 54. In addition, the F3, B3, FIP, BIP, LF, and LB primers can be composed of base sequences having a sequence identity of 90% or more with the base sequences shown in SEQ ID NOs: 49, 50, 51, 52, 53, and 54, respectively.

[0210] As a specific example of the primer set of (b3) above, there can be cited a primer set containing an F3 primer consisting of the base sequence shown in SEQ ID NO: 55, a B3 primer consisting of the base sequence shown in SEQ ID NO: 56, a FIP primer consisting of the base sequence shown in SEQ ID NO: 57, and a BIP primer consisting of the base sequence shown in SEQ ID NO: 58, and optionally containing an LF primer consisting of the base sequence shown in SEQ ID NO: 59 and an LB primer consisting of the base sequence shown in SEQ ID NO: 60. In addition, the F3, B3, FIP, BIP, LF, and LB primers can each be composed of a base sequence having a sequence identity of 90% or more with the base sequences shown in SEQ ID NOs: 55, 56, 57, 58, 59, and 60.

[0211] As a specific example of the primer set of (b4) above, there can be cited a primer set containing an F3 primer consisting of the base sequence shown in SEQ ID NO: 61, a B3 primer consisting of the base sequence shown in SEQ ID NO: 62, a FIP primer consisting of the base sequence shown in SEQ ID NO: 63, and a BIP primer consisting of the base sequence shown in SEQ ID NO: 64, and optionally containing an LF primer consisting of the base sequence shown in SEQ ID NO: 65 and an LB primer consisting of the base sequence shown in SEQ ID NO: 66. In addition, the F3, B3, FIP, BIP, LF, and LB primers can each be composed of a base sequence having a sequence identity of 90% or more with the base sequences shown in SEQ ID NOs: 61, 62, 63, 64, 65, and 66.

[0212] The above sequence identities can all be 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0213] In the above-mentioned nucleic acid amplification step, a known reagent for detecting a nucleic acid amplification reaction and a labeled probe or fluorescent intercalator for detecting an amplification product of the nucleic acid amplification reaction may be further used. As the known reagent, labeled probe, and fluorescent intercalator for performing a nucleic acid amplification reaction, the same known reagent, labeled probe, and fluorescent intercalator as those exemplified in the method for detecting Fusobacterium nucleatum in a sample in the above aspect may be used.

[0214] The nucleic acid amplification step can be performed, for example, by incubating a reaction solution containing the DNA from the sample and the primer set. The reaction solution may also contain the known reagents for nucleic acid amplification reaction and the labeled probe or fluorescent intercalator. The incubation temperature and time (i.e., the reaction temperature and time) and the concentration of each primer in the reaction solution may be the same as in the method for detecting Fusobacterium nucleatum in the sample in the above aspect.

[0215] The method for detecting Fusobacterium nucleatum subspecies nucleatum in a sample according to this aspect of the present invention may further include the step of detecting an amplification product of a nucleic acid amplification reaction. When the presence of an amplification product is detected, it can be determined that Fusobacterium nucleatum subspecies nucleatum is present in the sample. On the other hand, when the presence of an amplification product is not detected, it can be determined that Fusobacterium nucleatum subspecies nucleatum is not present in the sample. Detection of the amplification product may be performed after the nucleic acid amplification step or in real time during the nucleic acid amplification step. Details of the method for detecting an amplification product are as described in the method for detecting Fusobacterium nucleatum in a sample according to the above aspect.

[0216] According to the method for detecting Fusobacterium nucleatum subspecies nucleatum in the sample of this aspect of the present invention, by using a primer set specific to the DAACS gene having high sequence consistency between strains of Fusobacterium nucleatum subspecies but low sequence consistency with other subspecies of Fusobacterium nucleatum and other species of the genus Fusobacterium, Fusobacterium nucleatum subspecies can be specifically detected. In addition, as described above, even strains belonging to the same Fusobacterium nucleatum subspecies have diverse sequences, but the DAACS gene has high sequence consistency between strains of Fusobacterium nucleatum subspecies nucleatum, so it is not necessary to prepare a primer set for each strain, and a single primer set specific to the DAACS gene can be used to detect various strains of Fusobacterium nucleatum subspecies nucleatum. Furthermore, according to the primer set specific to the DAACS gene, a higher detection sensitivity can be achieved compared to when using a primer set specific to other regions in the genome of Fusobacterium nucleatum subspecies nucleatum. The existence of such a gene with a subspecies-specific sequence is extremely rare, and it can be said that it would not have been discovered without the creative work of the inventor.

[0217] The present inventors first used the sequence comparison software "Mauve" (Darling, AC et al., Genome Res., 2004, 14: 1394-1403) to search for sequences specific to Fusobacterium nucleatum subspecies nucleatum. As a result, although a number of sequences specific to Fusobacterium nucleatum subspecies nucleatum were found, when preparing region-specific primer sets for them, only the DAACS gene was the primer set that provided sufficient detection sensitivity.

[0218] As described above, by using the above primer set specific to the DAACS gene of Fusobacterium nucleatum subsp. nucleatum, Fusobacterium nucleatum subsp. nucleatum can be specifically detected. Therefore, another aspect of the present invention can be said to be the above primer set specific to the DAACS gene of Fusobacterium nucleatum subsp. nucleatum for detecting Fusobacterium nucleatum subsp. nucleatum.

[0219] In addition, another aspect of the present invention is a kit for detecting Fusobacterium nucleatum subsp. nucleatum, comprising the above primer set specific to the DAACS gene of Fusobacterium nucleatum subsp. nucleatum. The kit may further contain the above known reagents for nucleic acid amplification reaction such as DNA polymerase, dNTP, buffer, salt, protective agent, and the like, and may further contain the above labeled probe or fluorescent intercalator.

[0220] Example

[0221] In the following, the template DNA is prepared as follows. The genomic DNA (gDNA) of the ATCC standard strain of each bacterial species was extracted using QIAamp (registered trademark) DNA Mini kit (manufactured by Qiagen Co., Ltd.). The genomic DNA of the GTC (Gifu type culture) strain of each bacterial species was extracted by heat extraction (95°C, 5 minutes). For Fusobacterium canis, instead of genomic DNA, an artificial gene (sequence number 18) containing a region with high homology to the citrate lyase β subunit gene having the base sequence shown in sequence numbers 1 to 4 in the genomic DNA of the FDAARGO_S999 strain (accession number: CP066022) was used. Each template DNA was appropriately diluted using a DNA diluent (5mM Tris hydrochloric acid buffer, pH8.0 containing 0.002mg / mL salmon testis-derived single-stranded DNA (manufactured by Sigma-Aldrich Co., Ltd.)).

[0222] Hereinafter, unless otherwise specified, a LAMP reaction solution was prepared by mixing 5 μL of a template DNA with 20 μL of a reaction mixture (Master Mix) having a composition shown in the following table.

[0223]

[0224] Hereinafter, LAMP reaction is carried out using a real-time PCR device LightCycler (registered trademark) 480 (manufactured by Roche) or CFX Maestro (manufactured by Bio-Rad) or a real-time turbidity measuring device Loopamp LA-320C (manufactured by Telemax Co., Ltd.). Unless otherwise specified, the detection of the amplified product is carried out by monitoring the fluorescence intensity of SYTO (trademark) 63 red fluorescent nucleic acid dyes. The time until the moving average of the fluorescence intensity reaches the threshold a certain number of times is measured, and the time when the threshold is exceeded for the first time is taken as the Tt value. As a no-template control (NTC), a reaction solution containing the above-mentioned DNA dilution is used instead of the template DNA.

[0225] <Example 1> Detection of Fusobacterium nucleatum

[0226] (1) Intersectionality

[0227] The reaction solution containing genomic DNA of various strains of Fusobacterium or an artificial gene from Fusobacterium felis (strain FDAARGO_S999) was subjected to LAMP reaction using the citrate lyase β subunit gene (CitE) specific primer set A1 shown in Table 3. The reaction was carried out at 60°C for 90 minutes using CFX Maestro. The Tt values ​​are shown in Table 4.

[0228]

[0229]

[0230] In the tables of this specification, "ND" means not detected. By using the citrate lyase β subunit gene-specific primer set A1 shown in Table 3, the four subspecies of Fusobacterium nucleatum can be specifically detected without cross-talk with other species of the genus Fusobacterium.

[0231] (2) Sensitivity

[0232] LAMP reaction was performed on a reaction solution containing 5 to 100 copies of genomic DNA of one of the four subspecies of Fusobacterium nucleatum (ATCC standard strain) using citrate lyase β subunit gene specific primer set A1 shown in Table 3. The reaction was performed at 60°C for 90 minutes using CFX Maestro. The Tt values ​​are shown in Table 5.

[0233]

[0234] (3) Combination of two primer sets

[0235] The LAMP reaction was performed on a reaction solution containing 10 to 100 copies of genomic DNA of one of the four subspecies of Fusobacterium nucleatum (ATCC standard strains) using the citrate lyase β subunit gene specific primer set A1 shown in Table 3 and the dicarboxylic acid / amino acid: cation cotransporter (DAACS) gene specific primer set B1 shown in Table 6. The reaction was performed at 60°C for 90 minutes using CFX Maestro. The Tt values ​​are shown in Table 7.

[0236]

[0237]

[0238] By using the citrate lyase β subunit gene-specific primer set A1 shown in Table 3 and the DAACS gene-specific primer set B1 shown in Table 6 in combination, the detection sensitivity of Fusobacterium nucleatum subspecies nucleatum was improved compared to when the citrate lyase β subunit gene-specific primer set A1 was used alone (Table 5). The detection sensitivity of subspecies other than Fusobacterium nucleatum subspecies nucleatum was at the same level as when the citrate lyase β subunit gene-specific primer set A1 was used alone.

[0239] <Example 2> Detection of Fusobacterium nucleatum subsp. nucleatum

[0240] (1) Intersectionality

[0241] The reaction solution containing genomic DNA of various strains of the genus Fusobacterium was subjected to LAMP reaction using the DAACS gene-specific primer set B1 shown in Table 6. The LAMP reaction was performed at 65° C. for 90 minutes using CFX Maestro. The Tt values ​​are shown in Table 8.

[0242]

[0243] By using the DAACS gene-specific primer set B1 shown in Table 6, it is possible to specifically detect Fusobacterium nucleatum subspecies nucleatum without overlapping with other subspecies and species.

[0244] (2) Sensitivity

[0245] LAMP reaction was performed on a reaction solution containing 5 to 80 copies of genomic DNA of Fusobacterium nucleatum subsp. nucleatum (ATCC25586 strain) using DAACS gene-specific primer set B1 shown in Table 6. The reaction was performed at 65°C for 90 minutes using LightCycler (registered trademark) 480. The Tt values ​​are shown in Table 9.

[0246]

[0247] (3) Comparison with other region-specific primer sets

[0248] Using the DAACS gene-specific primer set B1 shown in Table 6 or any one of the comparative primer sets 1 to 3 shown in Table 10, a LAMP reaction was performed on a reaction solution containing 100 or 1000 copies of the genomic DNA of Fusobacterium nucleatum subsp. nucleatum (ATCC25586 strain). Comparative primer sets 1 to 3 are primer sets specific to the "Cell Filamentation protein Fic" gene, which has the same high sequence identity as the DAACS gene between strains of Fusobacterium nucleatum subsp. nucleatum. The reaction was carried out at 65°C for 180 minutes using Loopamp LA-320C. However, SYTO (trademark) 63 red fluorescent nucleic acid dye was not added to the reaction solution, and the detection of the amplification product was carried out by monitoring the turbidity of the reaction solution. The time (Tt value) until the turbidity reached 0.1 was measured. The Tt value is shown in Table 11.

[0249]

[0250]

[0251] Comparative primer sets 1 to 3 specific for the filamentous protein Fic gene could not detect Fusobacterium nucleatum subsp. nucleatum, or detection required a longer time and had lower sensitivity than when the DAACS-specific primer set B1 shown in Table 6 was used.

[0252] <Example 3> Detection of Fusobacterium nucleatum

[0253] (1) Intersectionality

[0254] Using the CitE-specific primer sets A2 to A4 shown in Table 12, 1×10 5 The copied reaction solution was subjected to LAMP reaction. The reaction was carried out at 60°C for 140 minutes using CFX Maestro. The Tt values ​​are shown in Table 13.

[0255]

[0256]

[0257] By using the CitE-specific primer set A2 to A4, Fusobacterium nucleatum can be specifically detected without cross-talk with other species of the genus Fusobacterium.

[0258] (2) Sensitivity

[0259] Using CitE-specific primer sets A2 to A4, a LAMP reaction was performed on a reaction solution containing 22 to 100 copies of genomic DNA of one of the four subspecies of Fusobacterium nucleatum (ATCC standard strains). The reaction was performed at 60°C for 120 minutes using CFX Maestro. The Tt values ​​are shown in Table 14.

[0260]

[0261] <Example 4> Detection of Fusobacterium nucleatum subsp. nucleatum

[0262] (1) Intersectionality

[0263] Using the DAACS gene-specific primer sets B2 to B4 shown in Table 15, 1×10 genomic DNA of various strains of the genus Fusobacterium was detected. 5 The copied reaction solution was subjected to LAMP reaction. The LAMP reaction was carried out at 65°C for 90 minutes using CFX Maestro. The Tt values ​​are shown in Table 16.

[0264]

[0265]

[0266] By using the DAACS gene-specific primer sets B2 to B4, it is possible to specifically detect Fusobacterium nucleatum subspecies nucleatum without overlapping with other subspecies and species.

[0267] (2) Sensitivity

[0268] Using DAACS gene-specific primer sets B2 to B4, a LAMP reaction was performed on a reaction solution containing 22 to 1000 copies of genomic DNA of Fusobacterium nucleatum subsp. nucleatum (ATCC 25586 strain). The reaction was performed at 65°C for 90 minutes using a LightCycler (registered trademark) 480. The Tt values ​​are shown in Table 17.

[0269]

[0270] <Example 5> Detection of Fusobacterium nucleatum

[0271] (1) Intersectionality

[0272] Using the CitE-specific primer set A5 shown in Table 18, a PCR reaction was performed on a reaction solution containing genomic DNA of various strains of the genus Fusobacterium or an artificial gene derived from Fusobacterium felis and canis (FDAARGO_S999 strain).

[0273]

[0274] The PCR reaction solution was prepared by mixing 5 μL of the template DNA with 20 μL of the reaction mixture having the composition shown in the table below.

[0275]

[0276] The PCR reaction was performed using CFX Maestro (manufactured by Bio-Rad Co., Ltd.) (45 cycles of 2 minutes at 95°C, followed by 15 seconds at 95°C and 1 minute at 60°C). The detection of the amplified product was performed by monitoring the fluorescence intensity of SYBR (trademark) green nucleic acid gel dye. The time until the moving average of the fluorescence intensity reached the threshold a certain number of times was measured, and the time when the threshold was exceeded for the first time was taken as the Tt value. As NTC, a reaction solution containing the above-mentioned DNA dilution was used instead of the template DNA. The Tt values ​​are shown in Table 20.

[0277]

[0278] The results showed that by using the CitE-specific primer set A5, it was possible to specifically detect Fusobacterium nucleatum by PCR without cross-talk with F. felis and canis having the highest homology region with CitE of F. nucleatum.

Claims

1. A method for detecting Fusobacterium nucleatum in a sample, comprising the following steps: A nucleic acid amplification reaction was performed on DNA from the sample using a primer set specific to the citrate lyase β subunit gene of Fusobacterium nucleatum.

2. The method according to claim 1, wherein: The citrate lyase β subunit gene is composed of a base sequence represented by any one of SEQ ID NOs: 1 to 4, or a base sequence having a sequence identity of 90% or more to the base sequence represented by any one of SEQ ID NOs: 1 to 4.

3. The method according to claim 2, wherein: The primer set is a region-specific primer set, and the region is: In the base sequence shown in any one of SEQ ID NOs: 1 to 4, a region from bases 141 to 415, a region from bases 4 to 253, a region from bases 238 to 561, a region from bases 577 to 806, or a region from bases 32 to 636, Alternatively, a region having a sequence identity of 90% or more with any of these regions.

4. The method according to any one of claims 1 to 3, wherein: The nucleic acid amplification reaction is a loop-mediated isothermal amplification reaction or a polymerase chain reaction.

5. The method according to claim 4, wherein: The nucleic acid amplification reaction is a loop-mediated isothermal amplification reaction. The primer set is: (a1) a primer set comprising an F3 primer consisting of the base sequence shown in SEQ ID NO: 5, a B3 primer consisting of the base sequence shown in SEQ ID NO: 6, a FIP primer consisting of the base sequence shown in SEQ ID NO: 7, and a BIP primer consisting of the base sequence shown in SEQ ID NO: 8; (a2) a primer set comprising an F3 primer consisting of the base sequence shown in SEQ ID NO: 31, a B3 primer consisting of the base sequence shown in SEQ ID NO: 32, a FIP primer consisting of the base sequence shown in SEQ ID NO: 33, and a BIP primer consisting of the base sequence shown in SEQ ID NO: 34; (a3) a primer set comprising an F3 primer consisting of the base sequence set forth in SEQ ID NO: 37, a B3 primer consisting of the base sequence set forth in SEQ ID NO: 38, a FIP primer consisting of the base sequence set forth in SEQ ID NO: 39, and a BIP primer consisting of the base sequence set forth in SEQ ID NO: 40; or (a4) A primer set comprising an F3 primer consisting of the base sequence shown in sequence number 43, a B3 primer consisting of the base sequence shown in sequence number 44, a FIP primer consisting of the base sequence shown in sequence number 45, and a BIP primer consisting of the base sequence shown in sequence number 46.

6. The method according to claim 5, wherein: The primer set (a1) further comprises an LF primer consisting of the base sequence shown in SEQ ID NO: 9 and an LB primer consisting of the base sequence shown in SEQ ID NO:

10. The primer set (a2) further comprises an LF primer consisting of the base sequence shown in SEQ ID NO: 35 and an LB primer consisting of the base sequence shown in SEQ ID NO:

36. The primer set (a3) ​​further comprises an LF primer consisting of the base sequence shown in SEQ ID NO: 41 and an LB primer consisting of the base sequence shown in SEQ ID NO:

42. The primer set (a4) further includes an LF primer consisting of the base sequence shown in SEQ ID NO: 47 and an LB primer consisting of the base sequence shown in SEQ ID NO:

48.

7. The method according to claim 4, wherein: The nucleic acid amplification reaction is a polymerase chain reaction, The primer set is (a5) a primer set comprising a forward primer consisting of the base sequence shown in sequence number 67 and a reverse primer consisting of the base sequence shown in sequence number 68.

8. The method according to any one of claims 1 to 3, wherein: In the step of performing a nucleic acid amplification reaction, a primer set specific for the dicarboxylic acid / amino acid:cation cotransporter gene of Fusobacterium nucleatum subsp. nucleatum is used together with the primer set specific for the citrate lyase β subunit gene of Fusobacterium nucleatum.

9. A primer set for detecting Fusobacterium nucleatum, which is a primer set specific to the citrate lyase β subunit gene of Fusobacterium nucleatum.

10. The primer set according to claim 9, wherein: The citrate lyase β subunit gene is composed of a base sequence represented by any one of SEQ ID NOs: 1 to 4, or a base sequence having a sequence identity of 90% or more to the base sequence represented by any one of SEQ ID NOs: 1 to 4.

11. The primer set according to claim 10, which is a region-specific primer set, wherein the region is: In the base sequence shown in any one of SEQ ID NOs: 1 to 4, a region from bases 141 to 415, a region from bases 4 to 253, a region from bases 238 to 561, a region from bases 577 to 806, or a region from bases 32 to 636, Alternatively, a region having a sequence identity of 90% or more with any of these regions. 12 . The primer set according to claim 9 , which is used for loop-mediated isothermal amplification reaction or for polymerase chain reaction.

13. The primer set according to claim 12, which is used for loop-mediated isothermal amplification reaction, wherein the primer set is: (a1) a primer set comprising an F3 primer consisting of the base sequence shown in SEQ ID NO: 5, a B3 primer consisting of the base sequence shown in SEQ ID NO: 6, a FIP primer consisting of the base sequence shown in SEQ ID NO: 7, and a BIP primer consisting of the base sequence shown in SEQ ID NO: 8; (a2) a primer set comprising an F3 primer consisting of the base sequence shown in SEQ ID NO: 31, a B3 primer consisting of the base sequence shown in SEQ ID NO: 32, a FIP primer consisting of the base sequence shown in SEQ ID NO: 33, and a BIP primer consisting of the base sequence shown in SEQ ID NO: 34; (a3) a primer set comprising an F3 primer consisting of the base sequence set forth in SEQ ID NO: 37, a B3 primer consisting of the base sequence set forth in SEQ ID NO: 38, a FIP primer consisting of the base sequence set forth in SEQ ID NO: 39, and a BIP primer consisting of the base sequence set forth in SEQ ID NO: 40; or (a4) A primer set comprising an F3 primer consisting of the base sequence shown in sequence number 43, a B3 primer consisting of the base sequence shown in sequence number 44, a FIP primer consisting of the base sequence shown in sequence number 45, and a BIP primer consisting of the base sequence shown in sequence number 46.

14. The primer set according to claim 13, wherein: The primer set (a1) further comprises an LF primer consisting of the base sequence shown in SEQ ID NO: 9 and an LB primer consisting of the base sequence shown in SEQ ID NO:

10. The primer set (a2) further comprises an LF primer consisting of the base sequence shown in SEQ ID NO: 35 and an LB primer consisting of the base sequence shown in SEQ ID NO:

36. The primer set (a3) ​​further comprises an LF primer consisting of the base sequence shown in SEQ ID NO: 41 and an LB primer consisting of the base sequence shown in SEQ ID NO:

42. The primer set (a4) further includes an LF primer consisting of the base sequence shown in SEQ ID NO: 47 and an LB primer consisting of the base sequence shown in SEQ ID NO:

48.

15. The primer set according to claim 12, which is used for polymerase chain reaction, wherein the primer set is (a5) a primer set comprising a forward primer consisting of the base sequence shown in SEQ ID NO: 67 and a reverse primer consisting of the base sequence shown in SEQ ID NO:

68.

16. A method for detecting Fusobacterium nucleatum subspecies nucleatum in a sample, comprising the following steps: A nucleic acid amplification reaction was performed on DNA from the sample using a primer set specific to the dicarboxylic acid / amino acid:cation cotransporter gene of Fusobacterium nucleatum subsp. nucleatum.

17. The method according to claim 16, wherein: The dicarboxylic acid / amino acid:cation cotransporter gene is composed of the base sequence shown in sequence number 11 or a base sequence having a sequence identity of 90% or more with the base sequence shown in sequence number 11.

18. The method according to claim 17, wherein: The primer set is a region-specific primer set, and the region is: In the base sequence shown in SEQ ID NO: 11, a region from bases 860 to 1121, a region from bases 196 to 449, a region from bases 580 to 869, or a region from bases 761 to 1038, Alternatively, a region having a sequence identity of 90% or more with any of these regions.

19. The method according to any one of claims 16 to 18, wherein: The nucleic acid amplification reaction is a loop-mediated isothermal amplification reaction.

20. The method according to claim 19, wherein: The primer set is: (b1) a primer set comprising an F3 primer consisting of the base sequence shown in SEQ ID NO: 12, a B3 primer consisting of the base sequence shown in SEQ ID NO: 13, a FIP primer consisting of the base sequence shown in SEQ ID NO: 14, and a BIP primer consisting of the base sequence shown in SEQ ID NO: 15; (b2) a primer set comprising an F3 primer consisting of the base sequence shown in SEQ ID NO: 49, a B3 primer consisting of the base sequence shown in SEQ ID NO: 50, a FIP primer consisting of the base sequence shown in SEQ ID NO: 51, and a BIP primer consisting of the base sequence shown in SEQ ID NO: 52; (b3) a primer set comprising an F3 primer consisting of the base sequence shown in SEQ ID NO: 55, a B3 primer consisting of the base sequence shown in SEQ ID NO: 56, a FIP primer consisting of the base sequence shown in SEQ ID NO: 57, and a BIP primer consisting of the base sequence shown in SEQ ID NO: 58; or (b4) A primer set comprising an F3 primer consisting of the base sequence shown in sequence number 61, a B3 primer consisting of the base sequence shown in sequence number 62, a FIP primer consisting of the base sequence shown in sequence number 63, and a BIP primer consisting of the base sequence shown in sequence number 64.

21. The method according to claim 20, wherein: The primer set (b1) further comprises an LF primer consisting of the base sequence shown in SEQ ID NO: 16 and an LB primer consisting of the base sequence shown in SEQ ID NO:

17. The primer set (b2) further comprises an LF primer consisting of the base sequence shown in SEQ ID NO: 53 and an LB primer consisting of the base sequence shown in SEQ ID NO:

54. The primer set (b3) further comprises an LF primer consisting of the base sequence shown in SEQ ID NO: 59 and an LB primer consisting of the base sequence shown in SEQ ID NO:

60. The primer set (b4) further includes an LF primer consisting of the base sequence shown in SEQ ID NO: 65 and an LB primer consisting of the base sequence shown in SEQ ID NO:

66.

22. A primer set for detecting Fusobacterium nucleatum subspecies nucleatum, which is a primer set specific to a dicarboxylic acid / amino acid:cation cotransporter gene of Fusobacterium nucleatum subspecies nucleatum.

23. The primer set according to claim 22, wherein: The dicarboxylic acid / amino acid:cation cotransporter gene is composed of the base sequence shown in sequence number 11 or a base sequence having a sequence identity of 90% or more with the base sequence shown in sequence number 11.

24. The primer set according to claim 23, which is a region-specific primer set, wherein the region is: In the base sequence shown in SEQ ID NO: 11, a region from bases 860 to 1121, a region from bases 196 to 449, a region from bases 580 to 869, or a region from bases 761 to 1038, Alternatively, a region having a sequence identity of 90% or more with any of these regions. 25 . The primer set according to any one of claims 22 to 24 , which is a primer set for loop-mediated isothermal amplification reaction.

26. The primer set according to claim 25, which is: (b1) a primer set comprising an F3 primer consisting of the base sequence shown in SEQ ID NO: 12, a B3 primer consisting of the base sequence shown in SEQ ID NO: 13, a FIP primer consisting of the base sequence shown in SEQ ID NO: 14, and a BIP primer consisting of the base sequence shown in SEQ ID NO: 15; (b2) a primer set comprising an F3 primer consisting of the base sequence shown in SEQ ID NO: 49, a B3 primer consisting of the base sequence shown in SEQ ID NO: 50, a FIP primer consisting of the base sequence shown in SEQ ID NO: 51, and a BIP primer consisting of the base sequence shown in SEQ ID NO: 52; (b3) a primer set comprising an F3 primer consisting of the base sequence shown in SEQ ID NO: 55, a B3 primer consisting of the base sequence shown in SEQ ID NO: 56, a FIP primer consisting of the base sequence shown in SEQ ID NO: 57, and a BIP primer consisting of the base sequence shown in SEQ ID NO: 58; or (b4) A primer set comprising an F3 primer consisting of the base sequence shown in sequence number 61, a B3 primer consisting of the base sequence shown in sequence number 62, a FIP primer consisting of the base sequence shown in sequence number 63, and a BIP primer consisting of the base sequence shown in sequence number 64.

27. The primer set according to claim 26, wherein: The primer set (b1) further comprises an LF primer consisting of the base sequence shown in SEQ ID NO: 16 and an LB primer consisting of the base sequence shown in SEQ ID NO:

17. The primer set (b2) further comprises an LF primer consisting of the base sequence shown in SEQ ID NO: 53 and an LB primer consisting of the base sequence shown in SEQ ID NO:

54. The primer set (b3) further comprises an LF primer consisting of the base sequence shown in SEQ ID NO: 59 and an LB primer consisting of the base sequence shown in SEQ ID NO:

60. The primer set (b4) further includes an LF primer consisting of the base sequence shown in SEQ ID NO: 65 and an LB primer consisting of the base sequence shown in SEQ ID NO: 66.

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