JP2017163971A - Oligonucleotide for detecting staphylococcus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To solve the problem associated with the conventional art in which: Staphylococcus schweitzeri is sometimes erroneously detected as Staphylococcus aureus, and therefore, there is a need for a method that differentiates S.schweitzeri from S.aureus for quick detection.SOLUTION: An oligonucleotide has a base sequence consisting of at least 10 continuous bases in the genome sequence of Staphylococcus schweitzeri, and also has a mismatch of one base or more when the base sequence is contrasted with the genome sequence of Staphylococcus aureus.SELECTED DRAWING: Figure 2

Description

The present invention relates to an oligonucleotide for detecting staphylococci. More specifically, the present invention relates to a method for detecting Staphylococcus schweitzeri contained in a sample using an oligonucleotide probe, and a kit therefor.

The staphylococci are gram-positive cocci belonging to the genus Staphylococcus (hereinafter, the genus name may be abbreviated as S.). In particular, S. aureus is known to exhibit drug resistance by having a drug resistance gene such as methicillin resistance gene (mecA) as well as pathogenicity to humans (Patent Document 1). ). Therefore, for the purpose of selection of therapeutic agents, prevention of spread of infection, prevention of food poisoning, etc. There is a need for a method that can quickly detect and classify aureus from other staphylococci such as S. epidermidis, and as such a method, use the difference in gene sequences possessed by each microorganism. Many methods have been proposed that can be detected by detecting a part of the base sequence (for example, Patent Document 1, Patent Document 2, Patent Document 3, and Patent Document 4).

[S. About schweitzeri]
By the way, in recent years, S.A. As a result of whole genome analysis, some strains classified as aureus were phylogenetically identified as different species (Non-patent Document 1). For example, one of the newly classified strains is S. cerevisiae. There is schweitzeri. S. schweitzeri has been isolated from animals other than humans (Non-patent Document 3), and details of infectivity and pathogenicity to humans are not known. However, in the prior art, S.I. schweitzeri is S. Since there is a risk of erroneous determination as aureus, it is extremely important in clinical diagnosis to accurately detect and identify this bacterial species.

S. schweitzeri is a S. As well as aureus, it does not only show a positive coagulase test, but also an identification sensitivity test commonly used in clinical settings (for example, Vitek (registered trademark) 2, bioMerieux) and 16S rRNA commonly used for bacterial identification Even if the gene sequence is used, the S. identified as aureus (Non-patent Document 3).

S. Aureus is known as a representative causative bacterium of nosocomial infections. aureus and S.M. It is very important to distinguish schweitzeri.

By performing whole genome analysis using next-generation sequencing, etc. schweitzeri and S. It was possible to identify aureus (Non-patent Document 1). However, these methods have problems such as high cost, complicated operation, and long test time.

JP2013-048620A Special table 2013-535951 Special table 2001-518283 JP2015-073457

International Journal of Systemic and Evolutionary Microbiology (2015), 65, 15-22. JOURNAL OF CLINICAL MICROBIOLOGY, July 1992, p. 1654-1660 JOURNAL OF CLINICAL MICROBIOLOGY, November 2014, p. 4036-4038 Nucleic Acids Research, 2006, Vol. 34, no. 8 e60

The present invention has been made against the background of such prior art problems. That is, the object of the present invention is S.I. A distinction from S. aureus The object is to provide a method for detecting schweitzeri and a kit therefor.

As a result of intensive studies, the present inventors have conducted a simple method for using S. cerevisiae by using an oligonucleotide probe containing a specific nucleic acid sequence. A distinction from S. aureus The inventors have found that schweitzeri can be detected and have reached the present invention. That is, the outline of the present invention is as follows.

An oligonucleotide characterized by the following (X) and (Y):
(X) It has a base sequence consisting of at least 10 consecutive bases in the genome sequence of Staphylococcus schweitzeri.
(Y) When the base sequence described in (X) is compared with the genomic sequence of Staphylococcus aureus, it has a mismatch of 1 base or more.
[Section 2]
The oligonucleotide according to Item 1, characterized by the following (X2) instead of (X):
(X2) It has a base sequence consisting of at least 10 consecutive bases in the base sequence represented by SEQ ID NO: 1 or SEQ ID NO: 57.
[Section 3]
Item 3. The oligonucleotide according to Item 2, comprising the base sequence represented by the following (1A) or (2A).
(1A) A base sequence composed of at least 10 consecutive bases in the base sequence represented by SEQ ID NO: 1, wherein the sequence is a group represented by the following (I) among the base sequences represented by SEQ ID NO: 1 A base sequence comprising at least one base.
(2A) A base sequence complementary to the base sequence of (1A).
(I) 15, 32, 35, 49, 52, 62, 72, 78, 84, 85, 88, 94, 95, 99, 102, 105, 111, 141, 156 of the nucleotide sequence represented by SEQ ID NO: 1. 158, 162, 164, 166, 167, 168, 171, 176, 177, 180, 187, 188, 193, 197, 199, 204, 205, 208, 209, 210, 219, 221, 228, 229, 230, 232, 234, 235, 240, 242, 243, 258, 273, 279, 300, 315, 316, 334, 336, 343, 351, 354, 360, 363, 378, 381, 384, 390, 396, 405, 429, 435, 450, 453, 470, 492, 498, 510, 549, 552, 555, 564, 570, 5 2,600,633,634,636,637,651,656,657 and the group consisting of 663 th.
[Claim 4]
The group represented by (I) is 84, 85, 240, 242, 243, 258, 273, 279, 300, 315, 316, 351, 354, 360, 363, 378, 381, 384, 390, 450, 453, 470, 492, 498, 549, 564, 570, 582, 636, and the oligonucleotide of claim | item 3 which is a group which consists of 637th.
[Section 5]
Item 5. The oligonucleotide according to Item 3, comprising the base sequence represented by the following (1B) or (2B).
(1B) A base sequence consisting of at least 10 consecutive bases in the base sequence represented by SEQ ID NO: 1, wherein the sequence is at least one of the group represented by (II) below.
(2B) A base sequence complementary to the base sequence of (1B).
(II) SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 25 SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38 SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, No. 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, the group consisting of SEQ ID NO: 55 and SEQ ID NO: 59.
[Claim 6]
The group represented by (II) is a group consisting of SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 59. Item 6. The oligonucleotide according to Item 5.
[Claim 7]
Item 7. The oligonucleotide according to any one of Items 1 to 6, which is labeled with one or more labeling substances.
[Section 8]
A method for detecting staphylococcus schweitzeri comprising the following steps (1) to (3):
(1) A first step of amplifying a nucleic acid fragment that can contain the base sequence represented by (1A) or (2A) of Item 3 or the base sequence represented by (1B) or (2B) of Item 5.
(2) A compound obtained by hybridizing the amplification product obtainable in the first step with the oligonucleotide capable of hybridizing to the amplification product obtainable in the first step among the oligonucleotides according to any one of Items 2 to 7. Second step to form the body.
(3) A third step of detecting a complex that can be obtained in the second step.
[Claim 9]
Item 9. The method according to Item 8, wherein the step (3) comprises melting curve analysis.
[Section 10]
A detection kit for Staphylococcus schweitzeri comprising the following (P) and (Q).
(P) An oligonucleotide primer set that can amplify a nucleic acid fragment that can contain the base sequence represented by (1A) or (2A) in Item 3 or the base sequence represented by (1B) or (2B) in Item 5.
(Q) The oligonucleotide according to any one of Items 2 to 7.

In accordance with the present invention, S.M. A distinction from S. aureus schweitzeri can be detected. That is, by using the method or kit described in the present invention, S. schweitzeri is It can now be detected without being misjudged as aureus. Use of the method and the kit can greatly contribute to the selection of therapeutic agents for staphylococcal infection, prevention of spread of infection, prevention of food poisoning, and the like.

S. schweitzeri, S.M. It is the figure which aligned a part of genomic DNA of aureus. It is a figure which shows the result of Example 1. “S. schweitzeri” indicates the result of melting curve analysis using the synthetic oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 1 as a template. “S. aureus” indicates the result when the synthetic oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 56 is used as a template, and “water” indicates the result when purified water is used instead of the synthetic oligonucleotide. It is a figure which shows the result of Example 2. “Sample 1”, “Sample 2”, and “Sample 3” indicate the results obtained by amplifying nucleic acid for each sample and performing melting curve analysis. “Water” indicates the result when purified water is used instead of each sample. It is a figure which shows the result (sequence obtained by the sequence analysis of the sample 1) of the reference example 1. S. It showed high homology with a part of genomic DNA of schweitzeri. It is a figure which shows the result (sequence obtained by the sequence analysis of the sample 2) of the reference example 1. S. It showed high homology with a part of the genomic DNA of aureus. It is a figure which shows the result of Example 3. It is a figure which shows the result of the comparative example 1. S. schweitzeri, S.M. It is the figure which aligned a part of genomic DNA of aureus.

[Oligonucleotide]
[1]
One embodiment of the present invention is an oligonucleotide characterized by the following (X) and (Y).
(X) It has a base sequence consisting of at least 10 consecutive bases in the genome sequence of Staphylococcus schweitzeri.
(Y) When the base sequence described in (X) is compared with the genomic sequence of Staphylococcus aureus, it has a mismatch of 1 base or more.

The use of the oligonucleotide of the present invention is not particularly limited. For example, S.I. It can be used as a probe in a method for detecting schweitzeri, whereby S. schweitzeri. schweitzeri to S. It can be detected separately from aureus.

S. The genomic sequence of schweitzeri is known from Non-Patent Document 1. Regarding the feature (X), the oligonucleotide of the present invention is not particularly limited as long as it has a base sequence composed of at least 10 consecutive bases in the genome sequence. Preferably, it may have a base sequence composed of at least 10 consecutive bases in the base sequence described in SEQ ID NO: 1 or SEQ ID NO: 57 described later.

S. The genome sequence of aureus is known from Non-Patent Document 1. Regarding the feature (Y), the oligonucleotide of the present invention is not particularly limited as long as it has a mismatch of 1 base or more when compared with the genomic sequence.

The number of mismatches when comparing base sequences can be determined by alignment analysis. Alignment analysis is generally performed in this field, and in this specification, Clustal Omega (clustered omega), which is a multiple alignment program provided by The European Bioinformatics Institute (EMBL-EBI), is used. In this program, S.M. schweitzeri genomic sequence and S. cerevisiae. The number and position of mismatches are specified by aligning the aureus genome sequences.

In the comparison, there is no particular limitation on which part of the genome sequence is compared. For example, S.M. What is necessary is just to select from the nuc gene known as a gene specific to aureus, the femA gene that can be distinguished from other staphylococci using the difference in the base sequence, the tuf gene, and the like. Oligonucleotides that can be compared with these genes include, for example, S. cerevisiae. When used as a detection probe for schweitzeri, S. Not only can it be distinguished from Aureus, but also microorganisms other than the genus Staphylococcus (e.g., Enterococcus, Streptococcus, Escherichia, Klebsiella, Serratia, Pseudocoterm, Haechobacter, Haetobacter, Haetobacter, Haetobacter , Citrobacter genus and the like), and is preferable in that it has a specificity that can be distinguished from the above. In particular, the nuc gene and the femA gene are S. phyllococcus among the genus Staphylococcus. schweitzeri and S. Other microorganisms of the genus Staphylococcus other than Aureus (for example, S. epidermidis, S. capitis, S. caprae, S. carnosus, S. chromogenes, S. cohni, S. condienti, S. condenti, S. felis, S. fleurettii, S. gallinarum, S. haemolyticus, S. hominis, S. hyicus, S. intermedius, S. kloosii, S. lentus, S. ludunensis, S. lutraus, S. lutraus, S. lutraus, S. lutraus, S. lutraus, S. lutraus, S. pasteuri, S. piscifermentans, S. sac and specificities that can be distinguished from halolyticus, S. saprophyticus, S. schleiferi, S. sciuri, S. simulans, S. succinus, S. vitrinus, S. warneri, S. xylosus, etc.). preferable.

[1-1]
The base sequence shown in SEQ ID NO: 1 is S. cerevisiae. It is the nucleotide sequence of the nuc gene of schweitzeri. The nuc gene is It has been conventionally used as an aureus identification marker (Non-patent Document 2). However, in recent years, even when a nucleic acid amplification test targeting the nuc gene is performed using the primers described in Non-Patent Document 2, S.P. It was revealed that schweitzeri cannot be detected (Non-Patent Document 1, Non-Patent Document 3).

However, the inventors have described S.I. schweitzeri and S. By using an oligonucleotide probe containing a specific base sequence of the nuc gene by alignment analysis of the Aureus nuc gene sequence, S. aureus It was found that schweitzeri can be detected. Further, the method is described in S.H. A distinction from S. aureus schweitzeri could be detected.

S. schweitzeri 3 strain, S. FIG. 1 shows the result of alignment analysis of a part of the nucleotide sequence of the nuc gene of Aureus 3 strain (corresponding to the 412th to 591th in SEQ ID NO: 1).

As a result of alignment analysis, S.I. In the nuc gene of schweitzeri, the nucleotide sequence is S. cerevisiae. It became clear that there is a part different from aureus. Such a position is 15, 32, 35, 49, 52, 62, 72, 78, 84, 85, 88, 94, 95, 99, 102, 105, 111, 141 in the base sequence of SEQ ID NO: 1. 156, 158, 162, 164, 166, 167, 168, 171, 176, 177, 180, 187, 188, 193, 197, 199, 204, 205, 208, 209, 210, 219, 221, 228, 229 230, 232, 234, 235, 240, 242, 243, 258, 273, 279, 300, 315, 316, 334, 336, 343, 351, 354, 360, 363, 378, 381, 384, 390, 396 , 405, 429, 435, 450, 453, 470, 492, 498, 510, 549, 552, 555, 56 , Corresponding to the 570,582,600,633,634,636,637,651,656,657 and 663 th. Since these portions have no difference between strains in each bacterial species, the oligonucleotide containing the base sequence is S. cerevisiae. A distinction from S. aureus It is preferable as an oligonucleotide probe for detecting schweitzeri.

[1-2]
The base sequence represented by SEQ ID NO: 57 It is the base sequence of the femA gene of schweitzeri. The femA gene is also S. cerevisiae. It has been conventionally used as an aureus identification marker (Patent Document 2, Patent Document 3).

S. schweitzeri 3 strain, S. FIG. 8 shows the result of alignment analysis of a part of the base sequence of the femA gene of Aureus 3 strain (corresponding to the 421st to 600th in SEQ ID NO: 57).

As a result of alignment analysis, S.I. In the schweitzeri femA gene, the nucleotide sequence is S. cerevisiae. It became clear that there is a part different from aureus. Such a position corresponds to the 546th, 939th, 984th and 993th positions in the base sequence of SEQ ID NO: 57. Since these portions have no difference between strains in each bacterial species, the oligonucleotide containing the base sequence is S. cerevisiae. A distinction from S. aureus It is preferable as an oligonucleotide probe for detecting schweitzeri.

[2]
Specific examples of the oligonucleotide include an oligonucleotide having a base sequence represented by the following (1A) or (2A).
(1A) A base sequence composed of at least 10 consecutive bases in the base sequence represented by SEQ ID NO: 1, wherein the sequence is a group represented by the following (I) among the base sequences represented by SEQ ID NO: 1 A base sequence comprising at least one base.
(2A) A base sequence complementary to the base sequence of (1A).
(I) 15, 32, 35, 49, 52, 62, 72, 78, 84, 85, 88, 94, 95, 99, 102, 105, 111, 141, 156 of the nucleotide sequence represented by SEQ ID NO: 1. 158, 162, 164, 166, 167, 168, 171, 176, 177, 180, 187, 188, 193, 197, 199, 204, 205, 208, 209, 210, 219, 221, 228, 229, 230, 232, 234, 235, 240, 242, 243, 258, 273, 279, 300, 315, 316, 334, 336, 343, 351, 354, 360, 363, 378, 381, 384, 390, 396, 405, 429, 435, 450, 453, 470, 492, 498, 510, 549, 552, 555, 564, 570, 5 2,600,633,634,636,637,651,656,657 and the group consisting of 663 th.

As said oligonucleotide, you may use the oligonucleotide which consists of a base sequence shown by the following (3A) or (4A).
(3A) A base sequence consisting of at least 10 consecutive bases in the base sequence represented by SEQ ID NO: 57, wherein the sequence is a group represented by the following (III) among the base sequences represented by SEQ ID NO: 57 A base sequence comprising at least one base.
(4A) A base sequence complementary to the base sequence of (3A).
(III) A group consisting of the 546th, 939th, 984th and 993th nucleotide sequences of SEQ ID NO: 57.

The oligonucleotide of the present invention has a base sequence consisting of at least 10 consecutive bases including at least one base among the above-identified positions in the base sequence represented by SEQ ID NO: 1 or SEQ ID NO: 57.

In the present specification, the number of bases of “at least 10 consecutive bases” is not particularly limited.
Assuming use as an oligonucleotide probe, the number of bases in the region that hybridizes to the target nucleic acid varies depending on the test method, but the preferred upper limit is 50 bases, more preferably 35 bases, even more preferably. Is 30 bases. On the other hand, the minimum with the preferable number of bases is 10 bases, More preferably, it is 15 bases. Long oligonucleotide probes may not provide sufficient specificity. On the other hand, a short oligonucleotide probe may reduce the hybridization efficiency.

In this specification, the number of “at least one” is not particularly limited. There is preferably a single nucleotide polymorphism of several bases with respect to aureus. The melting temperature (Melting Temperature, Tm) of the oligonucleotide changes depending on the number of single nucleotide polymorphisms contained in the oligonucleotide. Tm refers to the temperature at which the proportion of the oligonucleotide forming a double strand with its complementary strand is equal to the proportion of the single strand without forming a double strand. When the oligonucleotide contains a single nucleotide polymorphism with respect to the target nucleic acid (for example, S. aureus genome), the Tm value is generally small because the interaction with the target nucleic acid is weakened. Therefore, since the Tm value becomes smaller as the number of single nucleotide polymorphisms of the oligonucleotide relative to the target nucleic acid is larger than when there is no single nucleotide polymorphism, the target nucleic acid (for example, S. schweitzeri genome or S. aureus genome) ) By measuring the single nucleotide polymorphism of the oligonucleotide for S. schweitzeri and S. aureus can be clearly distinguished. However, when the number of single nucleotide polymorphisms is too large, the hybridization efficiency of the oligonucleotide is lowered. Therefore, the number of preferable single nucleotide polymorphisms is 1 to 4 places. More preferably, there are 1 to 3 locations, and even more preferably 1 to 2 locations.

In particular, the nucleotide sequence represented by SEQ ID NO: 1 is 84, 85, 240, 242, 243, 258, 273, 279, 300, 315, 316, 351, 354, 360, 363, 378, 381, 384, 390, 450. , 453, 470, 492, 498, 549, 564, 570, 582, 636, and 637, the base sequences before and after that match without any difference between strains, and therefore include single nucleotide polymorphisms depending on the purpose. However, it is more preferable because it is easy to design an arbitrary oligonucleotide.

[3]
Specific examples of the oligonucleotide having the base sequence represented by (1A) or (2A) include the following oligonucleotides having the base sequence represented by (1B) or (2B).
(1B) A base sequence consisting of at least 10 consecutive bases in the base sequence represented by SEQ ID NO: 1, wherein the sequence is at least one of the group represented by (II) below.
(2B) A base sequence complementary to the base sequence of (1B).
(II) SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 25 SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38 SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, No. 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, the group consisting of SEQ ID NO: 55 and SEQ ID NO: 59.

As the oligonucleotide consisting of the base sequence represented by the above (3A) or (4A), specifically, the oligonucleotide comprising the base sequence represented by the following (3B) or (4B) may be used.
(3B) A base sequence comprising at least 10 consecutive bases in the base sequence represented by SEQ ID NO: 57, wherein the sequence is at least one of the group represented by (IV) below.
(4B) A base sequence complementary to the base sequence of (3B).
(IV) A group consisting of SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, and SEQ ID NO: 70.

The oligonucleotides described above are S. cerevisiae contained in the sample. In the method for detecting schweitzeri, it can be used as a probe. A distinction from S. aureus schweitzeri can be detected.

The present inventors have found by alignment analysis that the oligonucleotides do not differ between strains in each bacterial species. Thus, the oligonucleotide is S. cerevisiae. A distinction from S. aureus It is preferable as an oligonucleotide probe for detecting schweitzeri.

Among the groups represented by (II) above, in particular, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 41 and SEQ ID NO: 59 The base sequence is S.P. There is no mismatch with schweitzeri and S. S. aureus contains a mismatch of 1 to 2 bases. schweitzeri and S. It is more preferable as a nucleotide probe for distinguishing Aureus.

Of the group represented by (IV), the base sequences represented by SEQ ID NO: 66, SEQ ID NO: 68, and SEQ ID NO: 70 are S. There is no mismatch with schweitzeri and S. S. aureus contains a mismatch of 1 to 2 bases. schweitzeri and S. It is more preferable as a nucleotide probe for distinguishing Aureus.

Also, the difference in Tm values for single nucleotide polymorphisms in melting curve analysis is generally small. For example, when there is one mismatch, there is a report that the difference in Tm value of the oligonucleotide probe is 0.5 to 3 ° C. (Non-Patent Document 4). Therefore, in order to increase the difference in Tm value, an oligonucleotide probe containing a nucleotide analog such as LNA (Locked Nucleic Acid) is used (Non-patent Document 4). However, since oligonucleotide probes containing LNA have problems such as difficulty in design and high price, another method has been required to increase the difference in Tm values.

However, as a result of continual research, the present inventors have used S.P. in melting curve analysis by using the oligonucleotide described in the claims as an oligonucleotide probe. schweitzeri and S. It was found that the difference in Tm value of aureus is larger than expected. Even if the oligonucleotide does not contain a nucleotide analog such as LNA, the difference in Tm value is large. schweitzeri and S. Since aureus can be clearly distinguished, erroneous determination can be easily prevented.

For example, the base sequences shown in SEQ ID NO: 40 and SEQ ID NO: 58 are S.P. schweitzeri, S.M. Part of the aureus nuc gene sequence. As a result of conducting a melting curve analysis using a fluorescent oligonucleotide probe (SEQ ID NO: 59) that binds to these regions, S. schweitzeri and S. aureus could be isolated (Example 1).

More surprisingly, S.M. schweitzeri and S. The temperature difference of the melting curve peak in Aureus was about 10 ° C. (FIG. 2). The fluorescent oligonucleotide probe represented by SEQ ID NO: 59 There is no mismatch with schweitzeri and S. There are two mismatches with aureus. Even in the case of two mismatches, there is a difference in Tm value of about 10 ° C. schweitzeri and S. aureus can be clearly distinguished.

On the other hand, a part of the nucleotide sequence of the nuc gene is targeted. A technique for detecting Aureus has already been published (for example, Patent Document 4). When targeting schweitzeri, there is a diversity of genome sequences depending on the strain. It is not easy to detect schweitzeri without strain differences. In the base sequence shown in the document, S. Since there were many mismatches with respect to schweitzeri, appropriate measurement results could not be obtained by melting curve analysis (Comparative Example 1) (FIG. 7).

[4]
The oligonucleotide of the present invention is preferably labeled with one or more labeling substances. Such oligonucleotides can be used, for example, as oligonucleotide probes.

Nucleic acid testing methods using oligonucleotide probes have been practiced and have already been established in the art (for example, Patent Document 4).

One or more labeling substances can be added to the oligonucleotide probe depending on the purpose. Examples of labeling substances include fluorescent substances, chemiluminescent substances, radioactive substances, biotin, alkaline phosphatase, digoxigenin, and peroxidase. In the present invention, except for the matters described in the claims, there are no particular limitations, and publicly known according to the test method An oligonucleotide probe labeled with a labeling substance can be used. Moreover, a linker or the like may be added for labeling the oligonucleotide probe.

For example, fluorescent oligonucleotide probes labeled with a fluorescent substance include TaqMan (registered trademark) probes, molecular beacons, cycling probes, QProbe (registered trademark), Eprobe (registered trademark), etc. It is not limited.

The base constituting the oligonucleotide probe may include nucleotide analogs such as inosine, peptide nucleic acid (PNA), and LNA. By using a nucleotide analog, an improvement in specificity can be expected.

[5]
[S. Method for detecting schweitzeri]
Another embodiment of the present invention is an S.D. comprising the following steps (1) to (3). This is a method for detecting schweitzeri.
(1) A first step of amplifying a nucleic acid fragment that may contain the base sequence represented by (1A) or (2A) or the base sequence represented by (1B) or (2B).
(2) Secondly, the amplification product obtainable in the first step and the oligonucleotide capable of hybridizing to the amplification product obtainable in the first step among the oligonucleotides of the present invention are hybridized to form a complex. Process.
(3) A third step of detecting a complex that can be obtained in the second step.
Here, in the above (1), “the nucleic acid fragment that can contain the base sequence represented by. This is because, in the method for detecting schweitzeri, it is not usually assumed that the nucleic acid fragment is necessarily included in the sample to be detected.

[First step (nucleic acid amplification step)]
The nucleic acid amplification method is not particularly limited except the matters described in the claims in the present invention, and a known method can be used. For example, PCR, real-time PCR, digital PCR, quantitative PCR (qPCR), LA-PCR (Long and Accurate PCR), competitive PCR including ABC-PCR, In situ PCR, RNA-primed PCR, multiplex PCR, shuttle PCR, Examples include PCR / GC-calmp method, Immuno PCR, reverse transcription polymerase chain reaction, NASBA method, TMA method, TRC method, SDA method, and LAMP method. The conditions for the nucleic acid amplification reaction in each method are not particularly limited, and can be performed under each condition.

As the nucleic acid amplification enzyme, DNA polymerase, RNA polymerase, reverse transcriptase and the like can be used depending on the nucleic acid amplification method. Further, as the nucleic acid amplification enzyme, not only a naturally derived enzyme but also a recombinant enzyme having an artificially altered amino acid sequence may be used. For example, when PCR is used in the nucleic acid amplification method, examples of DNA polymerase that can be used include Taq polymerase, Tth polymerase, KOD polymerase, Pfu polymerase, and the like, but are not limited to these in the present invention.

The composition of the nucleic acid amplification reaction requires several reaction components depending on the method. As an example, in a nucleic acid amplification method using PCR, a measurement sample, a DNA polymerase, an oligonucleotide primer, deoxyribonucleoside triphosphates (dNTPs), a reaction buffer, and a magnesium salt are generally required. Depending on the purpose, UNG (Uracil-N-Glycosylas), BSA, DMSO, betaine, glycerol, saccharides, amino acids, peptides, amines, potassium salts and the like may be included as additives, but the present invention is not limited thereto. Not.

An oligonucleotide primer is an oligonucleotide used as a starting point for nucleic acid amplification in a nucleic acid amplification reaction, and is commonly used in the art (for example, Patent Document 4 and Non-Patent Document 2). In addition, multiple types of oligonucleotide primers are used depending on the nucleic acid amplification method. The oligonucleotide primer is characterized in that it contains a base sequence complementary to the target nucleic acid, and the number of bases in the region that hybridizes to the target nucleic acid varies depending on the nucleic acid amplification method, but the preferred upper limit is 50. It is a base, more preferably 35 bases, still more preferably 30 bases. On the other hand, the minimum with the preferable number of bases is 10 bases, More preferably, it is 15 bases. Long oligonucleotide primers may increase non-specific products and decrease annealing efficiency, and short oligonucleotide primers may not provide sufficient specificity.

In general, it is important for highly specific oligonucleotide primers that the 3 'end is complementary to the target sequence, in other words, the 5' end is complementary if the 3 'end is complementary. It is said that it works as an effective primer. Therefore, the oligonucleotide primer used in the present invention is also preferably designed so that the 3 'end has a sequence complementary to the genomic sequence.

For example, in general PCR, at least two oligonucleotide primers are used. These oligonucleotide primers are preferably designed so as to be able to hybridize to the complementary strand of the target nucleic acid, respectively, and the amplification region preferably has a region where the oligonucleotide probe can hybridize. Preferable examples include combinations of oligonucleotide primers represented by SEQ ID NO: 60 and SEQ ID NO: 61.

One or more labeling substances can be added to the oligonucleotide primer depending on the purpose. Examples of the labeling substance include a fluorescent substance, a chemiluminescent substance, a radioactive substance, biotin, alkaline phosphatase, digoxigenin, and peroxidase. However, the present invention is not particularly limited except for the matters described in the claims, and depends on the nucleic acid amplification method. An oligonucleotide primer labeled with a known labeling substance can be used. Moreover, a linker or the like may be added for labeling the oligonucleotide primer.

The bases constituting the oligonucleotide primer may include not only mixed bases but also nucleotide analogs such as inosine, peptide nucleic acid (PNA), and LNA. By using a nucleotide analog or the like, an improvement in specificity can be expected.

[Second step and third step (step of detecting amplification product)]
The detection method of the amplification product is not particularly limited except the matters described in the claims in the present invention, and a known method can be used. Specific methods include melting curve analysis, DNA probe method, nucleic acid hybridization, Northern blotting, etc., but the present invention is not limited to these. Among these, S.D. schweitzeri and S. Since aureus can be detected separately, melting curve analysis is preferred as a method for detecting amplification products.

The melting curve analysis is an analysis method for measuring the Tm value by measuring and monitoring the fluorescence intensity at each temperature while changing the temperature of the reaction solution from a low temperature to a high temperature. The obtained fluorescence intensity is first-order differentiated by temperature, whereby the melting temperature (Tm value) specific to the oligonucleotide probe to be used can be determined. Since the Tm value is unique to the base sequence, the melting curve analysis can be used as a method for measuring the base sequence. Therefore, as an example, melting curve analysis is also applied to SNP analysis and the like.

For example, when melting curve analysis is used as a method for detecting an amplification product, a fluorescent oligonucleotide probe may be added to the composition of the nucleic acid amplification reaction.

As an example, when performing a melting curve analysis using QProbe (registered trademark), the concentration of KOD polymerase is 0.001 to 1.0 U / μL, more preferably 0.01 to 0.1 U / μL, and the primer concentration is 0.01 to 10 μM, more preferably 0.1 μM to 8 μM, dNTPs concentration is 0.01 to 2 mM, more preferably 0.1 to 0.5 mM, and pH of the reaction buffer is 2 to 13, more preferably Is 4-10, the magnesium salt concentration is 0.1-10 mM, more preferably 1.0-5 mM, and the QProbe concentration is 0.01-2 μM, more preferably 0.1-0.8 μM. . PCR and melting curve analysis can be performed under known conditions. A plurality of types of QProbe may be added depending on the measurement.

[About sample]
The measurement sample that can be used in the present invention is not limited to biological samples and foods, but can be prepared nucleic acids and the like, but is not limited thereto.

The biological sample is not particularly limited, but blood, blood culture medium, pus, spinal fluid, pleural effusion, throat wiping liquid, nasal cavity wiping liquid, sputum, tissue section, skin, sputum, feces, isolated culture colony, catheter washing liquid, etc. Is mentioned. When a biological sample is used as a measurement sample, pretreatment such as dilution or suspension may be performed according to each biological sample.

Examples of the food include, but are not limited to, water, alcoholic beverages, soft drinks, processed foods, vegetables, livestock products, marine products, eggs, dairy products, raw meat, raw fish, side dishes, and the like. When food is used as a measurement sample, not only a part or all of the food can be used, but also the food surface can be used. Depending on the sample, pretreatment such as fine crushing or suspending in purified water may be performed.

Moreover, the material which wiped off the cooking utensil and the doorknob, or the washing | cleaning liquid which wash | cleaned them can also be used as a sample.

The prepared nucleic acid refers to DNA or RNA extracted from each sample, artificially synthesized nucleic acid, or the like, but is not limited thereto. For nucleic acid extraction, a kit sold by each manufacturer may be used.

The sample collection method, the preparation method, and the like are not particularly limited, and known methods can be used depending on the type and purpose of the sample.

[About kit for carrying out the method]
In another embodiment of the present invention, S. cerevisiae contained in a sample is obtained using an oligonucleotide probe. A kit for detecting schweitzeri can be mentioned.
As such, S., including the following (P) and (Q): An example is a detection kit of schweitzeri.
(P) An oligonucleotide primer set that can amplify a nucleic acid fragment that can contain the base sequence represented by (1A) or (2A) in Item 3 or the base sequence represented by (1B) or (2B) in Item 5.
(Q) The oligonucleotide according to any one of Items 2 to 7.
The primer applicable to the kit of the present invention and the oligonucleotide of the present invention are as described above.

The composition contained in the kit for carrying out the method of the present invention is not particularly limited except for the matters described in the claims, and reagents necessary for nucleic acid amplification / nucleic acid detection can be appropriately used. As the nucleic acid amplification / detection method, the above-mentioned various methods can be used, and these methods have already been established in the art.

EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to the examples.

Example 1: Melting curve analysis using fluorescent probe (1) Method Melting curve analysis using a fluorescent oligonucleotide probe (QProbe (registered trademark)) represented by SEQ ID NO: 59 was performed. Specifically, melting curve analysis was performed using synthetic oligonucleotides having the nucleotide sequences represented by SEQ ID NO: 1 and SEQ ID NO: 56 as templates. Gene Cube (registered trademark) Test Basic (Toyobo Co., Ltd.) was used as a reagent. Using the attached primer / probe dilution, probe, template DNA and the like were mixed as follows.
Fluorescent oligonucleotide probe 0.3 μM
Template synthesis oligonucleotide 1 μM
Melting curve analysis was performed using GENECUBE (registered trademark) (Toyobo Co., Ltd.) as a measuring device.
(2) Results The measurement results are shown in FIG. It was revealed that the Tm value was different depending on the template synthesis oligonucleotide used for the measurement. The base sequences represented by SEQ ID NO: 1 and SEQ ID NO: 56 are S.P. schweitzeri, S.M. aureus nuc gene, which contains the nucleotide sequences of SEQ ID NO: 40 and SEQ ID NO: 58, respectively. The fluorescent oligonucleotide probe used was a probe that binds to these regions, and the Tm value in the melting curve analysis differed depending on the single nucleotide polymorphism. Therefore, by comparing Tm values, S.I. schweitzeri and S. It was shown that each aureus can be classified.

Example 2: Measurement of biological sample by melting curve analysis (1) Method Three specimens of biological sample (blood culture solution) were measured by melting curve analysis. Nucleic acid amplification and subsequent melting curve analysis were performed using the primers shown by SEQ ID NO: 60, SEQ ID NO: 61, and the fluorescent oligonucleotide probe shown by SEQ ID NO: 59 (QProbe (registered trademark)). GeneCube (registered trademark) Test Basic (Toyobo) was used as a reagent, and GENECUBE (registered trademark) (Toyobo) was used as a measuring instrument. In addition, amplification reaction conditions and melting curve analysis conditions were performed under the following conditions, and the reaction solution preparation and the like were in accordance with the instruction manual of GeneCube (registered trademark) Test Basic. As a measurement sample, a blood culture fluid sample determined to be S. aureus or S. epidermidis by an identification sensitivity test was used.

Amplification reaction conditions 94 ° C 30 seconds 98 ° C 1 second -60 ° C 3 seconds-63 ° C 5 seconds (50 cycles)

Melting curve analysis conditions 94 ° C 30 seconds 39 ° C 30 seconds 40-75 ° C 0.09 ° C / second

(2) Results The measurement results are shown in FIG. Specimen 1 and Specimen 2 are specimens determined to be S. aureus by the identification sensitivity test, and specimen 3 is a specimen determined to be S. epidermidis by the identification sensitivity test. It was. When sequence analysis was performed on Sample 1 and Sample 2 by the method of Reference Example 1, the sequence obtained in Sample 1 was S. pylori. While showing high homology with schweitzeri, the sequence obtained in specimen 2 was S. schweitzeri. High homology with aureus. Therefore, S. cerevisiae contained in a biological sample is analyzed by melting curve analysis. schweitzeri can be detected and S. cerevisiae can be detected. It was shown that it can be distinguished from aureus. In addition, a strain erroneously determined as S. aureus by the identification sensitivity test is S. aureus. It was correctly classified into schweitzeri. Moreover, it was shown that the primer and the probe do not detect Staphylococcus epidermidis.

Reference Example 1: Sequence Analysis (1) Method Sequence analysis was performed on two strains of Sample 1 and Sample 2 used in Example 2. Specifically, nucleic acid amplification was performed using the primers represented by SEQ ID NO: 60 and SEQ ID NO: 61, and then the obtained nucleic acid amplification product was subjected to sequence analysis.
(A) The nucleic acid amplification reaction solution was prepared as follows according to the instruction manual of Blend Taq (registered trademark) -Plus- (Toyobo Co., Ltd.). The measurement sample was added in an amount corresponding to 1/40 volume of the reaction solution.
PCR Buffer for Blend Taq 1x
Blend Taq (registered trademark) -Plus- 0.025 U / μL
Primer 0.2μM each
dNTPs 0.2 mM
The nucleic acid amplification reaction was performed under the following conditions.
94 ° C, 1 minute -55 ° C, 30 seconds -72 ° C, 1 minute 30 seconds (37 cycles)
72 ° C., 3 minutes 30 seconds (b) The sequence analysis reagent used was BigDye (registered trademark) Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems), and the preparation of the reaction solution was in accordance with the instruction manual of the reagent . As a measuring instrument, 3730 DNA Analyzer (Applied Biosystems) was used.
(2) Results The sequence analysis results of Sample 1 and Sample 2 are shown in FIGS. 4 and 5, respectively. When the homology search of the base sequence shown in FIG. 4 (Sample 1) was performed, it showed high homology with a part of the base sequence shown in SEQ ID NO: 1. The nucleotide sequence represented by SEQ ID NO: 1 is S. cerevisiae. Since this is a schweitzeri nuc gene, this strain is S. cerevisiae. Classified as schweitzeri. On the other hand, when the homology search of the base sequence shown in FIG. 5 (specimen 2) was performed, it showed high homology with a part of the base sequence shown in SEQ ID NO: 56. The base sequence represented by SEQ ID NO: 56 is S.P. This strain is a S. aureus nuc gene and is therefore S. aureus. Classified as aureus. From the above results, the sample 1 is S.P. schweitzeri, specimen 2 is S. schweitzeri. It was aureu. Thus, as in Example 2, S. schweitzeri can be detected and S. cerevisiae can be detected. It was shown that it can be distinguished from aureus.

Example 3 and Comparative Example 1: Comparison with Prior Art (1) Method Using a primer represented by SEQ ID NO: 60, SEQ ID NO: 61, and a fluorescent oligonucleotide probe (QProbe (registered trademark)) represented by SEQ ID NO: 59, Nucleic acid amplification followed by melting curve analysis was performed (Example 3). Similarly, nucleic acid amplification and subsequent melting curve analysis were performed using the primers and fluorescent nucleotide probes described in Patent Document 4 (SEQ ID NO: 62, primer shown by SEQ ID NO: 63, probe shown by SEQ ID NO: 64). (Comparative Example 1). GeneCube (registered trademark) Test Basic (Toyobo) was used as a reagent, and GENECUBE (registered trademark) (Toyobo) was used as a measuring instrument. The amplification reaction conditions, the melting curve analysis conditions, the preparation of the reaction solution, and the like were in accordance with the instruction manual for Gene Cube (registered trademark) Test Basic as in Example 2. The measurement sample is S.I. aureus or S. Schweitzeri colonies were used.
(2) Results The measurement results are shown in FIGS. FIG. 6 shows the measurement results (Example 3) according to the present invention, and FIG. 7 shows the measurement results (Comparative Example 1) by the method described in Patent Document 4. In FIG. schweitzeri and S. In contrast to the fact that the Tm values of the peaks of the aureus were different, each could be separated, whereas in FIG. Since the Tm value of the schweitzeri peak was too low, an appropriate measurement result could not be obtained. In order to investigate the cause of the low Tm value of the peak, the samples used were sequenced and compared with the oligonucleotide probes used in each test. The number of mismatches to schweitzeri (3) was larger than the number of mismatches (2) of the oligonucleotide probe used in Example 3.
Therefore, the present invention is difficult to achieve with S.P. schweitzeri and S. It has been shown that aureus measurement has become possible.

By using the method or kit according to the invention, S. A distinction from S. aureus schweitzeri can be detected. Use of the method or kit described in the present invention is particularly useful for selection of therapeutic agents for staphylococcal infection, prevention of spread of infection, prevention of food poisoning, etc. The present invention is not only used for research purposes but also for clinical tests and environmental tests. It can also contribute greatly to food inspections.

Claims (10)

An oligonucleotide characterized by the following (X) and (Y):
(X) It has a base sequence consisting of at least 10 consecutive bases in the genome sequence of Staphylococcus schweitzeri.
(Y) When the base sequence described in (X) is compared with the genomic sequence of Staphylococcus aureus, it has a mismatch of 1 base or more. The oligonucleotide according to claim 1, which has the following (X2) instead of (X).
(X2) It has a base sequence consisting of at least 10 consecutive bases in the base sequence represented by SEQ ID NO: 1 or SEQ ID NO: 57. The oligonucleotide according to claim 2, comprising the base sequence represented by the following (1A) or (2A).
(1A) A base sequence composed of at least 10 consecutive bases in the base sequence represented by SEQ ID NO: 1, wherein the sequence is a group represented by the following (I) among the base sequences represented by SEQ ID NO: 1 A base sequence comprising at least one base.
(2A) A base sequence complementary to the base sequence of (1A).
(I) 15, 32, 35, 49, 52, 62, 72, 78, 84, 85, 88, 94, 95, 99, 102, 105, 111, 141, 156 of the nucleotide sequence represented by SEQ ID NO: 1. 158, 162, 164, 166, 167, 168, 171, 176, 177, 180, 187, 188, 193, 197, 199, 204, 205, 208, 209, 210, 219, 221, 228, 229, 230, 232, 234, 235, 240, 242, 243, 258, 273, 279, 300, 315, 316, 334, 336, 343, 351, 354, 360, 363, 378, 381, 384, 390, 396, 405, 429, 435, 450, 453, 470, 492, 498, 510, 549, 552, 555, 564, 570, 5 2,600,633,634,636,637,651,656,657 and the group consisting of 663 th. The group represented by (I) is 84, 85, 240, 242, 243, 258, 273, 279, 300, 315, 316, 351, 354, 360, 363, 378, 381, 384, 390, 450, The oligonucleotide according to claim 3, which is a group consisting of 453, 470, 492, 498, 549, 564, 570, 582, 636 and 637. The oligonucleotide according to claim 3, comprising the base sequence represented by the following (1B) or (2B).
(1B) A base sequence consisting of at least 10 consecutive bases in the base sequence represented by SEQ ID NO: 1, wherein the sequence is at least one of the group represented by (II) below.
(2B) A base sequence complementary to the base sequence of (1B).
(II) SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 25 SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38 SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, No. 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, the group consisting of SEQ ID NO: 55 and SEQ ID NO: 59. The group represented by (II) is a group consisting of SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 59. The oligonucleotide according to claim 5. The oligonucleotide according to any one of claims 1 to 6, which is labeled with one or more labeling substances. A method for detecting staphylococcus schweitzeri comprising the following steps (1) to (3):
(1) A first step of amplifying a nucleic acid fragment that can comprise the base sequence represented by (1A) or (2A) of claim 3 or the base sequence represented by (1B) or (2B) of claim 5.
(2) Hybridizing the amplification product obtainable in the first step with the oligonucleotide capable of hybridizing to the amplification product obtainable in the first step among the oligonucleotides according to any one of claims 2 to 7. And a second step of forming a complex.
(3) A third step of detecting a complex that can be obtained in the second step. The method according to claim 8, wherein the step (3) includes melting curve analysis. A detection kit for Staphylococcus schweitzeri comprising the following (P) and (Q).
(P) An oligonucleotide primer set capable of amplifying the nucleic acid fragment that can comprise the base sequence represented by (1A) or (2A) of claim 3 or the base sequence represented by (1B) or (2B) of claim 5 .
(Q) The oligonucleotide according to any one of claims 2 to 7.