JP2021078393A - Oligonucleotide set for determining dna type of cutibacterium acnes, kit, and determination method of dna type of cutibacterium acnes - Google Patents

Abstract

To establish a test system that can further accurately, quickly, and easily identify the DNA type of Cutibacterium acnes.SOLUTION: There are provided: an oligonucleotide set for determining the DNA type of Cutibacterium acnes by a nucleic acid amplification method that targets a partial region of each gene of JCM18918_744 gene specific for D1 type, TIIST44_06360 gene specific for D2 type, and FB33_0108 gene specific for D3 type, of Cutibacterium acnes; a kit; and a determination method of the DNA type of Cutibacterium acnes, which comprises the steps of performing an amplification reaction by a nucleic acid amplification method using the oligonucleotide set, and detecting an amplified product.SELECTED DRAWING: Figure 1

Description

The present invention relates to an oligonucleotide set for discriminating the DNA type of P. acnes, which is an inflammation-causing bacterium of acne vulgaris (acne), a kit, and a method for discriminating the DNA type.

P. acnes (Cutibacterium acnes) is a facultative anaerobic gram-positive bacillus of the phylum Actinobacteria, Actinomycetales, and Propionibacteriaceae. The genus Cutibacterium was included in the genus Propionibacterium until recent years, but with the reclassification in 2016, it became the genus Cutibacterium (non-patent literature). 1). Acne is the most predominant indigenous bacterium in sebum-producing sites such as the human face, precordium and back, and is also known as an inflammation-causing bacterium for acne vulgaris. When the funnel of a sebaceous hair follicle is occluded due to abnormal keratinization or overproduction of sebum, sebum accumulates in the hair follicle, and as a result, P. acnes that overgrow in the hair follicle causes inflammation and is inflammatory. It is believed that acne will occur.

Several methods of typing acne bacteria based on their deep involvement in acne have been reported so far, and one of them is the DNA-type by Randomly Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR). There is a classification (Non-Patent Document 2). In this method, when DNA extracted from Acne bacteria is amplified in an unspecified region using a predetermined primer and electrophoresed on an agarose gel, Acne bacteria have four types of DNA-types depending on the band pattern of the expressed DNA. It is classified into (D1, D2, D3 and D1 / D3 intermediate type). Of these, D3 is most often detected in acne lesions, has high lipase activity, which is an inflammatory factor, and has a large amount of porphyrin, so it is said to be most deeply involved in acne. On the other hand, D1 and D2 are considered to be less likely to be involved in acne because they are rarely detected in acne lesions and have low lipase activity. Thus, there are types of acne bacteria, and identifying the type of acne bacteria can be said to be a useful technique in the prevention and treatment of acne.

However, this DNA-type classification of Acne bacteria has a big problem that it is a very laborious and time-consuming typing method and can be performed only by a person who is familiar with DNA experiments. Further, since the determination is made based on the band pattern of DNA, there is a problem that an erroneous determination is likely to occur when the amplification amount of DNA is not sufficient.

Int J Syst Evol Microbiol, vol.67, PP.4422-4432, 2016. Journal of the Japanese Dermatological Association, vol.115, PP.2381-2388, 2005.

Therefore, an object of the present invention is to establish a test system capable of more accurately, quickly, and easily discriminating the DNA type of P. acnes.

The present inventors analyzed the gene sequences of the strains of P. acnes and found specific genes for each DNA type (D1 type, D2 type, D3 type). As a result of examining the base sequences of these genes in more detail, the present inventors have identified regions having specific sequences that can be distinguished from other DNA types in each of D1 type, D2 type, and D3 type, and the regions concerned. We have succeeded in establishing a set of oligonucletides capable of discriminating the DNA type of Acne bacteria with specificity and high accuracy by the nucleic acid amplification method based on the nucleotide sequence of the above, and have completed the present invention.

That is, the present invention includes the following inventions.
(1) An oligonucleotide set for discriminating the D1 type of P. acnes by a nucleic acid amplification method, which comprises an oligonucleotide capable of hybridizing to the nucleotide sequence of a partial region of the JCM18918_744 gene of P. acnes or its complementary sequence. Two or more of the partial regions selected from the 76-93 position region, the 110-227 position region, the 241-331 position region, and the 317-349 position region in the nucleotide sequence of SEQ ID NO: 1. The above oligonucleotide set, which is a region.
(2) The oligonucleotide set according to (1), wherein the oligonucleotide set is a set of LAMP primers composed of the following primers (a1) to (a6).
(A1) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 4 or an oligonucleotide consisting of a base sequence in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 4. F3 Primer (a2) Containing an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 5, or a base sequence in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 5. B3 primer containing an oligonucleotide (a3) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 6, or a base in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 6. FIP primer containing an oligonucleotide consisting of a sequence (a4) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 7 or one to several bases deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 7. BIP primer containing the nucleotide sequence of the nucleotide sequence (a5) The oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 8 or the nucleotide sequence shown in SEQ ID NO: 8 is deleted, substituted, or inserted with one to several bases. Or, an LF primer containing an oligonucleotide consisting of the added base sequence (a6) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 9 or a base sequence shown in SEQ ID NO: 9 in which one to several bases are deleted. LB primer containing an oligonucleotide consisting of a substituted, inserted, or added base sequence (3) The above-mentioned oligonucleotide set consists of a primer pair consisting of the following oligonucleotides (a7) and (a8), and the following. A set of PCR primers and probes composed of a probe consisting of the oligonucleotide of (a9), or a primer pair consisting of the oligonucleotide of (a10) below and the oligonucleotide of (a11), and the oligo of (a12) below. The oligonucleotide set according to (1), which is a set of PCR primers and probes composed of a probe composed of nucleotides.
(A7) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 22 or an oligonucleotide consisting of a base sequence in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 22 ( a8) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 23, or an oligonucleotide consisting of a base sequence in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 23 (a9). ) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 24, or an oligonucleotide consisting of a base sequence in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 24 (a10). An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 25, or an oligonucleotide (a11) sequence consisting of a base sequence in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 25. An oligonucleotide consisting of the base sequence shown in No. 26, or an oligonucleotide consisting of a base sequence in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 26 (a12) SEQ ID NO: An oligonucleotide consisting of the base sequence shown in 27, or an oligonucleotide consisting of a base sequence in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 27.

(4) An oligonucleotide set for discriminating the D2 type of P. acnes by a nucleic acid amplification method, which comprises an oligonucleotide capable of hybridizing to the nucleotide sequence of a partial region of the TIIST44_06360 gene of P. acnes or its complementary sequence. Two or more of the partial regions selected from the 53-72 region, the 75-150 region, the 153-241 region, and the 244-261 region in the nucleotide sequence of SEQ ID NO: 2. The above oligonucleotide set, which is a region.
(5) The oligonucleotide set according to (4), wherein the oligonucleotide set is a set of LAMP primers composed of the following primers (b1) to (b6).
(B1) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 10 or an oligonucleotide consisting of a base sequence in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 10. F3 Primer (b2) Consists of an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 11 or a base sequence in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 11. B3 primer containing an oligonucleotide (b3) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 12, or a base in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 12. FIP primer containing an oligonucleotide consisting of a sequence (b4) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 13 or one to several bases deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 13. BIP primer containing the nucleotide sequence of the nucleotide sequence (b5) The oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 14 or the nucleotide sequence shown in SEQ ID NO: 14 lacks, substitutes, or inserts one to several bases. Or an LF primer containing an oligonucleotide consisting of the added base sequence (b6) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 15 or a base sequence shown in SEQ ID NO: 15 lacking one to several bases. LB primer containing an oligonucleotide consisting of a substituted, inserted, or added base sequence (6) The above-mentioned oligonucleotide set consists of a primer pair consisting of the following oligonucleotides (b7) and (b8), and the following. A set of PCR primers and probes composed of a probe consisting of the oligonucleotide of (b9), or a primer pair consisting of the oligonucleotide of (b10) and the oligonucleotide of (b11) below, and the oligo of (b12) below. The oligonucleotide set according to (4), which is a set of PCR primers and probes composed of a probe composed of nucleotides.
(B7) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 28, or an oligonucleotide consisting of a base sequence in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 28 (b7) b8) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 29, or an oligonucleotide consisting of a base sequence in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 29 (b9). ) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 30, or an oligonucleotide consisting of a base sequence in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 30 (b10). An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 31 or an oligonucleotide (b11) sequence consisting of a base sequence in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 31. An oligonucleotide consisting of the base sequence shown in No. 32, or an oligonucleotide consisting of a base sequence in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 32 (b12) SEQ ID NO: An oligonucleotide consisting of the base sequence shown in 33, or an oligonucleotide consisting of a base sequence in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 33.

(7) An oligonucleotide set for discriminating the D3 type of P. acnes by a nucleic acid amplification method, which comprises an oligonucleotide capable of hybridizing to the nucleotide sequence of a partial region of the FB33_0108 gene of P. acnes or its complementary sequence. Two or more of the partial regions selected from the 219th to 236th region, the 298th to 412th region, the 450th to 521st region, and the 539th to 557th region in the nucleotide sequence of SEQ ID NO: 3. The above oligonucleotide set, which is a region.
(8) The oligonucleotide set according to (7), wherein the oligonucleotide set is a set of LAMP primers composed of the following primers (c1) to (c6).
(C1) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 16 or an oligonucleotide consisting of a base sequence in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 16. F3 primer containing F3 primer (c2) Consists of an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 17, or a base sequence in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 17. B3 primer containing an oligonucleotide (c3) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 18, or a base in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 18. FIP primer containing an oligonucleotide consisting of a sequence (c4) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 19 or one to several bases deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 19. BIP primer containing the nucleotide sequence of the nucleotide sequence (c5) The oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 20 or the nucleotide sequence shown in SEQ ID NO: 20 with one to several bases deleted, substituted, or inserted. Or, an LF primer containing an oligonucleotide consisting of the added base sequence (c6) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 21 or a base sequence shown in SEQ ID NO: 21 in which one to several bases are deleted. LB primer containing an oligonucleotide consisting of a substituted, inserted, or added base sequence (9) The above-mentioned oligonucleotide set consists of a primer pair consisting of the following oligonucleotides (c7) and (c8), and the following A set of PCR primers and probes composed of a probe consisting of the oligonucleotide of (c9), or a primer pair consisting of the oligonucleotide of (c10) and the oligonucleotide of (c11) below, and the oligo of (c12) below. The oligonucleotide set according to (7), which is a set of PCR primers and probes composed of a probe composed of nucleotides.
(C7) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 34, or an oligonucleotide consisting of a base sequence in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 34 ( c8) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 35, or an oligonucleotide consisting of a base sequence in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 35 (c9). ) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 36, or an oligonucleotide consisting of a base sequence in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 36 (c10) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 37, or an oligonucleotide (c11) sequence consisting of a base sequence in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 37. An oligonucleotide consisting of the base sequence shown in No. 38, or an oligonucleotide consisting of a base sequence in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 38 (c12) SEQ ID NO: An oligonucleotide consisting of the base sequence shown in 39, or an oligonucleotide consisting of a base sequence in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 39.

(10) The oligonucleotide set according to any one of (2), (5), and (8), wherein a tag sequence different for each DNA type is bound to the FIP primer or BIP primer, or the LF primer or LB primer. ..
(11) A kit for determining the DNA type of acne bacteria, which comprises at least one set of oligonucleotides according to any one of (1) to (10).
(12) The kit according to (11), further comprising a solid-phase carrier on which a detection probe that hybridizes to the tag sequence is immobilized.
(13) The kit according to (12), wherein the solid phase carrier is a strip for nucleic acid chromatography.

(14) A step of performing an amplification reaction by a nucleic acid amplification method using at least one set of oligonucleotides according to any one of (1) to (3) using DNA extracted from a sample as a template, and detection of an amplification product. A method for discriminating the D1 type of acne bacteria, including the step of performing.
(15) A step of performing an amplification reaction by a nucleic acid amplification method using at least one set of oligonucleotides according to any one of (4) to (6) using DNA extracted from a sample as a template, and detection of an amplification product. A method for discriminating the D2 type of acne bacteria, including the step of performing.
(16) A step of performing an amplification reaction by a nucleic acid amplification method using at least one set of oligonucleotides according to any one of (7) to (9) using DNA extracted from a sample as a template, and detection of an amplification product. A method for discriminating the D3 type of acne bacteria, including the step of performing.
(17) Using the DNA extracted from the sample as a template, at least one set of the oligonucleotide set according to any one of (1) to (3), and the oligonucleotide set according to any one of (4) to (6). Acne bacteria, including a step of performing an amplification reaction by a nucleic acid amplification method using at least one set, at least one set of oligonucleotide sets according to any one of (7) to (9), and a step of detecting an amplification product. Simultaneous discrimination method of DNA type.
(18) The discrimination method according to any one of (14) to (17), wherein the nucleic acid amplification method is an isothermal amplification method or a PCR method.
(19) The discrimination method according to any one of (14) to (18), wherein the sample is a skin sample.

According to the present invention, there is provided a set of oligonucleotides for discriminating the DNA type of P. acnes. By using the oligonucleotide set, the three DNA types (D1 type, D2 type, D3 type) of P. acnes contained in the skin sample can be individually and simultaneously false-positive or false-negative. It does not occur and can be identified accurately and easily.

Using the oligonucleotide set for determining the DNA type of P. acnes of the present invention, the DNA in the sample is amplified by the LAMP method, and the result of detection by nucleic acid chromatography is shown [Lane 1 (Sample 1: Cutibacterium acnes JCM). 6425 DNA, Primer used: JCM18918_744_LAMP), Lane 2 (Sample 2: Cutibacterium acnes JCM 6473 DNA, Primer used: JCM18918_744_LAMP), Lane 3 (Sample 3: Cutibacterium acnes JCM 18918 DNA, Primer used: JCM18918_744_LAMP), Lane 4 (Sample 1: DNA of Cutibacterium acnes JCM 6425, primer used: TIIST44_06360_LAMP), Lane 5 (Sample 2: DNA of Cutibacterium acnes JCM 6473, primer used: TIIST44_06360_LAMP), Lane 6 (Sample 3: DNA of Cutibacterium acnes JCM 18918, used Primer: TIIST44_06360_LAMP), Lane 7 (Sample 1: DNA of Cutibacterium acnes JCM 6425, Primer used: FB33_0108_LAMP), Lane 8 (Sample 2: DNA of Cutibacterium acnes JCM 6473, Primer used: FB33_0108_LAMP), Lane 9 (Sample 3: Cutibacterium) DNA of acnes JCM 18918, primer used: FB33_0108_LAMP), lane 10 (sample 4: mixture of DNA of Cutibacterium acnes JCM 6425, Cutibacterium acnes JCM 6473, and Cutibacterium acnes JCM 18918, primer used: JCM18918_744_LAMP, TIIST44_06360_LAMP, FB33_0108_LAMP , Lane 11 (Sample: distilled water, primer used: JCM18918_744_LAMP, TIIST44_06360_LAMP, FB33_0108_LAMP mixed primer)].

Hereinafter, the present invention will be described in detail.
1. 1. An oligonucleotide set for determining the DNA type of P. acnes (Cutibacterium acnes) The oligonucleotide set of the present invention is a specific gene possessed by P. acnes (Cutibacterium acnes), which is an inflammation-causing bacterium of acne vulgaris (acne). It is a set of oligonucleotides for discriminating three DNA types (D1 type, D2 type, D3 type) of P. acnes individually or simultaneously by targeting a partial region by a nucleic acid amplification method.

In the present invention, the "DNA type" is a DNA-type classification based on RAPD-PCR products using a predetermined primer (5'-AACGCGCAAC-3') (Journal of the Japanese Society of Dermatology, vol.115, PP.2381- It refers to DNA-type 1 (D1), DNA-type 2 (D2), and DNA-type 3 (D3) classified according to 2388, 2005). In the D1 type, the electrophoresis band pattern of the above RAPD-PCR product is expressed at 3,000, 2,800, 1,500, 1,400, 800, 300 bp, in the D2 type, the same pattern is expressed in 1,000, 350 bp, and in the D3 type. , The same pattern is expressed at 3,000, 2,800, 1,300, 800, 300 bp. Of these, the D3 type is characterized by being the most isolated from acne vulgaris lesions, having higher lipase activity than other DNAs, and producing a large amount of coproporphyrin. In lipase, free fatty acids such as oleic acid produced by hydrolyzing triglyceride promote the growth of P. acnes, and coproporphyrin produced by P. acnes undergoes a photosensitization reaction when exposed to ultraviolet rays. Generates singlet oxygen, which is a type of active oxygen, which irritates the skin. Therefore, it is strongly suggested that the D3 type is related to acne vulgaris and its severity.

The gene specific to the D1 type of Acne is the JCM18918_744 gene, which encodes a phage-associated protein, and consists of the nucleotide sequence shown in SEQ ID NO: 1. The gene specific to the D2 type of Acne is the TIIST44_06360 gene, which encodes a hypothetical protein, and consists of the nucleotide sequence shown in SEQ ID NO: 2. The gene specific to the D3 type of Acne is the FB33_0108 gene encoding glycosyl hydrolase family 20, and consists of the nucleotide sequence shown in SEQ ID NO: 3. The genes of JCM18918_744, TIIST44_06360, and FB33_0108 have the same sequence of 90% or more, preferably 95% or more, more preferably 98% or more, and most preferably 99% or more of the nucleotide sequences shown in SEQ ID NOs: 1 to 3, respectively. It may be a gene encoding a protein having sex and having a function equivalent to that of JCM18918_744, TIIST44_06360, and FB33_0108. Here, the sequence identity can be determined by, for example, FASTA search or BLAST search.

The partial regions of the JCM18918_744 gene, which is the target of D1 type discrimination of Acne bacteria, are the regions at positions 76 to 93, 110 to 227, 241 to 331, and 317 in the nucleotide sequence of SEQ ID NO: 1. The rank is from 349th. Therefore, the D1 type discrimination oligonucleotide set of Acne bacterium of the present invention contains an oligonucleotide capable of hybridizing to the base sequence of these partial regions or its complementary sequence.

The partial regions of the TIIST44_06360 gene, which are the targets of D2 type discrimination of Acne bacteria, are the regions at positions 53 to 72, 75 to 150, 153 to 241 and 244 in the nucleotide sequence of SEQ ID NO: 2. The rank is from 261st. Therefore, the D2 type discrimination oligonucleotide set of Acne bacteria of the present invention contains oligonucleotides capable of hybridizing to the nucleotide sequences of these partial regions or their complementary sequences.

The partial regions of the FB33_0108 gene, which is the target of D3 type discrimination of Acne, are the regions 219 to 236, 298 to 412, 450 to 521, and 539 in the nucleotide sequence of SEQ ID NO: 3. The rank is from 557th to 557th. Therefore, the oligonucleotide set for discriminating the D3 type of Acne bacterium of the present invention contains an oligonucleotide capable of hybridizing to the base sequence of these partial regions or its complementary sequence.

As used herein, the term "hybridize" means that it hybridizes to a partial region of the specific gene to be targeted and does not substantially hybridize to a partial region of the specific gene to be targeted. It means that. Hybridization can be performed under stringent conditions. "Stringent conditions" mean conditions in which so-called specific hybrids are formed and non-specific hybrids are not formed, for example, Green and Sambrook, Molecular Cloning, 4th Ed (2012), Cold Spring Harbor Laboratory Press. Can be appropriately determined with reference to. Specifically, stringent conditions can be set by the temperature at the time of Southern hybridization and the salt concentration contained in the solution, and the temperature at the time of the washing step of Southern hybridization and the salt concentration contained in the solution. More specifically, as stringent conditions, for example, in the hybridization step, the sodium concentration is 25 to 500 mM, preferably 25 to 300 mM, and the temperature is 40 to 68 ° C, preferably 40 to 65 ° C. More specifically, hybridization can be performed at 1-7 × SSC, 0.02-3% SDS, and a temperature of 40 ° C-60 ° C. Further, a washing step may be performed after hybridization, and the washing step can be performed, for example, at 0.1 to 2 × SSC, 0.1 to 0.3% SDS, and a temperature of 50 to 65 ° C.

The oligonucleotide set for discriminating the DNA type of Acne bacteria of the present invention includes a primer used in the LAMP method (hereinafter, may be referred to as "LAMP primer"), a primer pair and a probe used in the PCR method (hereinafter, "PCR"). A set of "primers and probes") is included.

In the LAMP method, the regions named F3c, F2c, and F1c in order from the 3'end side of one strand of the double-stranded template nucleic acid and the regions named B3, B2, and B1 in order from the 5'end side of the same strand, respectively. Based on the nucleotide sequences of these regions, four types of primers called F3, FIP, BIP, and B3 are designed.

The F3 primer (also called a forward primer) is designed to have the base sequence of the F3 region, which is a base sequence complementary to the F3c region. The FIP primer (also called the forward inner primer) has the base sequence of the F2 region, which is a complementary base sequence to the F2c region, on the 3'end side and the same base sequence as the base sequence of the F1c region on the 5'end side. Design to. The BIP primer (also called the backward inner primer) is designed to have the base sequence of the B2 region on the 3'end side and the base sequence of the B1c region, which is a complementary base sequence to the B1 region, on the 5'end side. To do. The B3 primer (also called the backward primer) is designed to have the base sequence of the B3 region.

In the LAMP method, in addition to the above-mentioned four types of primers of F3, FIP, BIP, and B3, primers (loop primers) called LF and LB can be used. The LF primer is designed to have a base sequence complementary to the base sequence between the F1 region and the F2 region. The LB primer is designed to have a base sequence complementary to the base sequence between the B1 region and the B2 region. By additionally using these primers, the starting point of nucleic acid synthesis in nucleic acid amplification by the LAMP method is increased, and the reaction time can be shortened and the detection sensitivity can be increased.

As the regions of F3, FIP, BIP, and B3 for which the LAMP primer of the present invention is designed, in the case of D1 type of Acne bacteria, the region of positions 76 to 93 and the region of positions 110 to 227 in the nucleotide sequence of SEQ ID NO: 1 , 248th to 331st regions, and 332nd to 349th regions correspond to each other. In the case of the D2 type of Acne, the region of positions 53 to 72, the region of positions 76 to 150, the region of positions 153 to 241 and the region of positions 244 to 261 in the nucleotide sequence of SEQ ID NO: 2 In the case of the D3 type of Acne, the region at positions 219 to 236, the region at positions 298 to 408, the region at positions 450 to 521, and the region at positions 539 to 557 in the nucleotide sequence of SEQ ID NO: 3 The corresponding areas correspond to each other.

For each primer of F3, B3, FIP, BIP, LF, and LB, using alignment software such as Clustal X, a partial region of a gene specific to the D1 type, D2 type, and D3 type of the acne bacterium to be discriminated, respectively. After creating the alignment file of the base sequence information of, LAMP Designer (manufactured by OptiGene; http://www.optigene.co.uk/lamp-designer/), Primer Explorer V4 based on the information in the alignment file. It can be designed using (Eiken Chemical website).

The LAMP primer of the present invention is preferably designed in a region where the sequence diversity is small (highly conserved) for each type of DNA, but it may be designed in a region containing a small number of gene polymorphisms. The number of base substitutions reflecting the gene polymorphism is preferably 20% or less, more preferably 15% or less per primer. When using a primer containing a substitution at a gene polymorphism site, a primer for each gene polymorphism may be used, or a mixed primer in which a primer for each gene polymorphism is mixed may be used, and the gene polymorphism site may be used. A mixed primer synthesized so as to be a mixed base according to the gene polymorphism may be used.

In each of the above primers, "forward" is a primer display that complementarily binds to the sense strand of the target region and provides a starting point for synthesis, and "backward" is complementary to the antisense strand of the target region. It is a primer display that binds to and provides a starting point for synthesis. The base length of the oligonucleotide used as a primer is not limited, but is usually 15 to 50 bases, preferably 20 to 40 bases. The FIP primer may contain an arbitrary base sequence having about 1 to 30 bases between F2 and F1c, and the BIP primer may contain between B2 and B1c.

Further, in the present invention, a PCR primer probe can also be designed based on the base sequence of the above-mentioned F3, B3, FIP, BIP, LF, and LB regions specified by the LAMP method. The forward primer is designed to have the base sequence of the F3 region, which is a base sequence complementary to the F3c region, the reverse primer is designed to have the same base sequence as the base sequence of the F1c region, and the probe is F1. Design to have a base sequence complementary to the base sequence between the region and the F2 region. Alternatively, the forward primer is designed to have the base sequence of the B3 region, the reverse primer is designed to have the base sequence of the B1c region which is a base sequence complementary to the B1 region, and the probe is designed to have the base sequence of the B1 region. It can also be designed to have a base sequence complementary to the base sequence between the B2 regions.

PCR primers are designed in consideration of oligonucleotide length, GC content, Tm value, complementarity between oligonucleotides, secondary structure within the oligonucleotide, and the like. Primer design can also be done using design software on the website, such as Primer3 (https://primer3.org/).

In addition, PCR probe design can be done on the market with design software on the website, such as GENETYX (https://www.genetyx.co.jp/) and ABI Prism 7900HT Real-time PCR System (Life Technologies Japan Co., Ltd.). It may be performed based on the protocol attached to a commercially available real-time PCR reagent such as TaqMan Universal PCR Master Mix (Life Technologies Japan), which is the software attached to the real-time PCR device of. Probe design criteria generally include a GC content in the range of 20-80%, avoiding contiguous G or C of 4 or more bases in the sequence, and the Tm value of the corresponding primer pair. It is also possible to set the temperature as high as 8 to 10 ° C.

The design criteria for the specifically adopted LAMP and PCR primers are as follows.
(i) Primer lengths are 15-30 bases for F3, B3, LF, and LB primers and 30 for FIP, BIP primers to allow specific annealing to the template DNA. Must be in the range of bases to 60 bases.
(ii) Avoid complementary sequences between primers so as not to form dimers or conformations.
(iii) Many mutations (genus-specific mutations) are contained at the tip in the elongation direction.
(iv) The total free energy of each of the 6 bases at the 3'end and 5'end is 4 kcal / mol or less.
(v) Avoid self-complementary sequences to prevent the formation of hairpin structures within the primer.
(vi) To ensure stable binding to the template DNA, the GC content should be approximately 40-60% so that GC-rich or AT-rich is not unevenly distributed in the primers.
(vii) High homology between the base sequence at the 3'end of the primer and the template DNA sequence.
(viii) The Tm value shall be around 60 ° C for F3c, F2c, B2, and B3, and around 65 ° C for LF, F1c, B1, and LBc.

Based on the above criteria, the following oligonucleotide sets for discriminating the DNA type of the acne bacterium of the present invention were established.
[LAMP primer set for distinguishing D1 type of acne bacteria]
(A1) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 4 or an oligonucleotide consisting of a base sequence in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 4. F3 Primer (a2) Containing an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 5, or a base sequence in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 5. B3 primer containing an oligonucleotide (a3) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 6, or a base in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 6. FIP primer containing an oligonucleotide consisting of a sequence (a4) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 7 or one to several bases deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 7. BIP primer containing the nucleotide sequence of the nucleotide sequence (a5) The oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 8 or the nucleotide sequence shown in SEQ ID NO: 8 is deleted, substituted, or inserted with one to several bases. Or, an LF primer containing an oligonucleotide consisting of the added base sequence (a6) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 9 or a base sequence shown in SEQ ID NO: 9 in which one to several bases are deleted. LB primer containing an oligonucleotide consisting of a substituted, inserted, or added base sequence

[PCR primer / probe set 1 for distinguishing D1 type of Acne bacteria]
(A7) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 22 or an oligonucleotide consisting of a base sequence in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 22. Forward Primer (a8) Consists of an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 23, or a base sequence in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 23. Reverse primer containing oligonucleotide (a9) An oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 24, or a nucleotide in which one to several bases are deleted, substituted, inserted, or added in the nucleotide sequence shown in SEQ ID NO: 24. Probe containing an oligonucleotide consisting of a sequence

[PCR primer / probe set 2 for distinguishing D1 type of Acne bacteria]
(A10) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 25, or an oligonucleotide consisting of a base sequence in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 25. Forward Primer (a11) Consists of an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 26, or a base sequence in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 26. Reverse primer containing oligonucleotide (a12) An oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 27, or a nucleotide in which one to several bases are deleted, substituted, inserted, or added in the nucleotide sequence shown in SEQ ID NO: 27. Probe containing an oligonucleotide consisting of a sequence

[LAMP primer set for distinguishing D2 type of acne bacteria]
(B1) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 10 or an oligonucleotide consisting of a base sequence in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 10. F3 Primer (b2) Consists of an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 11 or a base sequence in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 11. B3 primer containing an oligonucleotide (b3) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 12, or a base in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 12. FIP primer containing an oligonucleotide consisting of a sequence (b4) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 13 or one to several bases deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 13. BIP primer containing the nucleotide sequence of the nucleotide sequence (b5) The oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 14 or the nucleotide sequence shown in SEQ ID NO: 14 lacks, substitutes, or inserts one to several bases. Or an LF primer containing an oligonucleotide consisting of the added base sequence (b6) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 15 or a base sequence shown in SEQ ID NO: 15 lacking one to several bases. LB primer containing an oligonucleotide consisting of a substituted, inserted, or added base sequence

[PCR primer / probe set 1 for distinguishing D2 type of acne bacteria]
(B7) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 28, or an oligonucleotide consisting of a base sequence in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 28. Forward Primer (b8) Consists of an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 29, or a base sequence in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 29. Reverse primer containing oligonucleotide (b9) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 30, or a base in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 30. Probe containing an oligonucleotide consisting of a sequence

[PCR primer / probe set 2 for distinguishing D2 type of acne bacteria]
(B10) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 31 or an oligonucleotide consisting of a base sequence in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 31. Forward Primer (b11) Consists of an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 32, or a base sequence in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 32. Reverse primer containing oligonucleotide (b12) An oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 33, or a nucleotide in which one to several bases are deleted, substituted, inserted, or added in the nucleotide sequence shown in SEQ ID NO: 33. Probe containing an oligonucleotide consisting of a sequence

[LAMP primer set for distinguishing D3 type of acne bacteria]
(C1) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 16 or an oligonucleotide consisting of a base sequence in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 16. F3 primer containing F3 primer (c2) Consists of an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 17, or a base sequence in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 17. B3 primer containing an oligonucleotide (c3) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 18, or a base in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 18. FIP primer containing an oligonucleotide consisting of a sequence (c4) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 19 or one to several bases deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 19. BIP primer containing the nucleotide sequence of the nucleotide sequence (c5) The oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 20 or the nucleotide sequence shown in SEQ ID NO: 20 with one to several bases deleted, substituted, or inserted. Or, an LF primer containing an oligonucleotide consisting of the added base sequence (c6) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 21 or a base sequence shown in SEQ ID NO: 21 in which one to several bases are deleted. LB primer containing an oligonucleotide consisting of a substituted, inserted, or added base sequence

[PCR primer / probe set 1 for distinguishing D3 type of acne bacteria]
(C7) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 34, or an oligonucleotide consisting of a base sequence in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 34. Forward Primer (c8) Consists of an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 35, or a base sequence in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 35. Reverse primer containing oligonucleotide (c9) An oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 36, or a nucleotide in which one to several bases are deleted, substituted, inserted, or added in the nucleotide sequence shown in SEQ ID NO: 36. Probe containing an oligonucleotide consisting of a sequence

[PCR primer / probe set 2 for distinguishing D3 type of acne bacteria]
(C10) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 37, or an oligonucleotide consisting of a base sequence in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 37. Forward Primer (c11) Consists of an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 38, or a base sequence in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 38. Reverse primer containing oligonucleotide (c12) An oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 39, or a nucleotide in which one to several bases are deleted, substituted, inserted, or added in the nucleotide sequence shown in SEQ ID NO: 39. Probe containing an oligonucleotide consisting of a sequence

In addition, in the above-mentioned oligonucleotide set for discriminating D1 type of Acne bacteria, oligonucleotide set for discriminating D2 type of Acne bacteria, and oligonucleotide set for discriminating D3 type of Acne bacteria, "1 to several bases are missing". The range of "1 to several" in the "lost, substituted, inserted, or added base sequence" hybridizes to the partial region of the gene specific to each of the above DNA types, and is represented by SEQ ID NOs: 4-39, respectively. As long as it has substantially the same function as an oligonucleotide having the nucleotide sequence shown and a primer or probe, for example, 1 to 5, preferably 1 to 4, more preferably 1 to 3 is used. Refers to an individual.

Alternatively, another example of the mutant oligonucleotide may be an oligonucleotide consisting of a base sequence containing at least 15 consecutive bases in the base sequences shown in SEQ ID NOs: 4 to 39, respectively, and bases other than the continuous at least 15 bases. There is no particular limitation on the type and the site of the continuous mutant oligonucleotide of at least 15 bases (3'end side, 5'end side).

The above-mentioned oligonucleotide is a DNA automatic synthesizer usually used by a method known in the art as a method for synthesizing an oligonucleotide, for example, a phosphotriethyl method, a phosphodiester method, or the like (for example, Model 394 manufactured by Applied Biosystems). It is possible to synthesize using.

The LAMP primer set of the present invention can be used on either the FIP primer or the BIP primer, or on either the LF primer or the LB primer, so that the amplification product can be detected by the nucleic acid chromatography method. , A tag sequence capable of hybridizing with a detection probe immobilized on a solid phase carrier described later is bound. In addition, a substance that can be bound via a specific binding pair to a labeling substance for visualizing hybridization between the tag sequence and the detection probe (labeling substance binding substance) is bound to the primer that does not bind the tag sequence. Let me. The specific binding pair may be a combination that recognizes and captures each other, and examples thereof include biotin-avidin, digoxigenin-anti-digoxigenin, and FITC-anti-FITC antibody. In one embodiment of the invention, the labeling substance is contained in a developing solution that carries out a nucleic acid chromatography method.

The tag sequence is associated with the D1 type, D2 type, and D3 type of Acne bacteria, and is a sequence different for each DNA type, and uses a base sequence complementary to the base sequence of the detection probe. The base length of the tag sequence preferably matches the base length of the base sequence of the detection probe, preferably 20 bases to 50 bases, and more preferably 20 bases to 30 bases.

Labeling substances include fluorescent substances (fluorescein isothiocyanate (FITC), tetramethyllodamine isothiocyanate (TRIC), cyanine pigments (Cy3, Cy5, etc.), acetylaminofluorene (AFF), etc.), chemiluminescent substances (luminol, luminol, etc.). Derivatives, luciferin, luciferin, etc.), enzymes (alkaline phosphatase, peroxidase, β-galactosidase, β-glucosidase, etc.), etc., but from the viewpoint of quick and easy detection, chemiluminescence or color development that can be visually detected is presented. It is preferably a luminescent substance or a coloring substance. Further, particles such as latex particles, metal particles, and inorganic particles labeled with such a labeling substance may be used.

Further, in the set of PCR primers and probes of the present invention, the probe may be labeled with a labeling substance for detecting a fluorescent signal derived from a target amplification product of Acne bacteria to be discriminated against the DNA type. Labeled probes include TaqMan probe, Molecular Beacon probe, and cycling probe, with TaqMan probe being preferred. The TaqMan probe is a probe in which the 5'end is modified with a fluorescent substance (reporter fluorescent dye) and the 3'end is modified with a quencher (quencher fluorescent dye). The Molecular Beacon probe is a probe that can have a stem-loop structure in which the 5'end is modified with a fluorescent substance (reporter fluorescent dye) and the 3'end is modified with a quencher (quencher fluorescent dye). A cycling probe is a probe consisting of RNA and DNA in which the 5'end of an oligonucleotide is modified with a fluorescent substance (reporter fluorescent dye) and the 3'end is modified with a quencher (quencher fluorescent dye). Examples of the reporter fluorescent dyes of the above TaqMan probe are 6-FAM (6-carboxyfluorescein), TET (6-carboxy-4, 7, 2', 7'-tetrachlorofluorescein), HEX (6-carboxy-2'). Fluorescein-based fluorescent dyes such as, 4', 7', 4,7-hexachlorofluorescein) can be mentioned, and examples of quencher fluorescent dyes include TAMRA (6-carboxytetramethylrhodamine) and ROX (6-carboxy-X). -Rhodamine fluorescent dyes such as (-Rhodamine), BHQ-1 ([(4- (2-nitro-4-methyl-phenyl) -azo) -yl-((2-methoxy-5-methyl-phenyl) -azo) ]-Aniline), BHQ-2 ([(4- (1-nitro-phenyl) -azo) -yl-((2,5-dimethoxy-phenyl) -azo)]-aniline), Dabcyl (4-[[[ 4- (Dimethylamino) -phenyl] -azo] -benzoic acid), Eclipse (4-[[2-chloro-4-nitro-phenyl] -azo] -aniline) and the like can be mentioned. These fluorescent dyes are known and can be used because they are included in a commercially available real-time PCR kit.

2. Kit for determining the DNA type of P. acnes (Cutibacterium acnes) 1. Oligonucleotide sets can also be kitted. The kit of the present invention may contain at least one set of the above oligonucleotides, but preferably includes the entire set, depending on the type, number, and purpose of the DNA type of the acne bacterium to be discriminated. An appropriate set may be appropriately selected from the oligonucleotide sets of the above and used in combination. For example, by using the entire set of oligonucleotides, it can be used to simultaneously determine the DNA type of acne bacteria in a single operation.

In addition, the kit of the present invention includes reagents necessary for performing a nucleic acid amplification method, for example, a reagent for DNA extraction, a strand-substituted DNA polymerase (for example, Bst DNA polymerase, Bca (Exo-) DNA polymerase, Escherichia coli DNA polymerase). Klenow Fragment of I, Vent DNA polymerase, Vent (Exo-) DNA polymerase, Z-Taq DNA polymerase, KOD DNA polymerase, etc.), PCR buffer, 4 types of dNTPs (dATP, dCTP, which are substrates for nucleic acid synthesis, etc.) Includes dGTP and dTTP), labeling substances, sterile water, salts as cofactors (magnesium or manganese salts, etc.), protective agents that stabilize enzymes and templates, standards (standard strains, etc.), instructions, etc. May be good.

The kit of the present invention also contains reagents necessary for detecting the amplification product amplified by the nucleic acid amplification method. For example, when the amplification product is detected by nucleic acid chromatography, it is preferable to include a solid-phase carrier or a developing solution on which a detection probe is immobilized.

The detection probe is a single-stranded nucleic acid sequence, and the length of the sequence is not particularly limited, but is preferably 20 to 50 bases, more preferably 20 to 30 bases, corresponding to the above-mentioned tag sequence. .. Within this range, hybridization efficiency can be ensured while ensuring the specificity of the tag sequence.

In the present specification, the above-mentioned solid-phase carrier on which the detection probe is immobilized is referred to as a strip. Conventionally known strips can be used. The material of the base material of the strip is not particularly limited, and may be plastic or glass. Further, it may be a porous body such as cellulose, nitrocellulose, or nylon. This type of porous material is particularly suitable for hybridizing the detection probe immobilized on the strip with the amplification product by affinity chromatography. A typical strip includes a test piece for visual determination (PAS: Printed Array-Strip) used in the STH (Single-stranded Tag Hybridization) method, and these commercially available products can be used in the present invention. This strip is an inspection test paper having a width of about 2 mm and a length of about 10 cm, and is printed with nucleic acid as a probe on a nitrocellulose membrane on the surface. Amplified nucleic acid can be detected by using a primer having a tag sequence that hybridizes complementaryly with this probe as a set. Multiple detection probes corresponding to each DNA type are placed at appropriate positions on the strip, and position markers are placed so that each probe and the tag sequence bound to the probe can be easily identified. Is preferable. It is also possible to arrange a flow control line that can visually confirm that the developing liquid has been sufficiently applied.

3. 3. Method for discriminating the DNA type of P. acnes (Cutibacterium acnes) The method for discriminating the DNA type of P. acnes (Cutibacterium acnes) of the present invention uses a DNA extracted from a sample as a template and a nucleic acid amplification method using the above oligonucleotide set. It includes a step of performing an amplification reaction and a step of detecting an amplification product.

It is not necessary to use all of the oligonucleotide sets depending on the type and number of DNA types of the acne bacteria to be discriminated and the purpose of discrimination, and one set or a plurality of sets of the oligonucleotide sets may be used as appropriate.

The sample is not particularly limited as long as it is a biological sample or an environmental sample that may contain acne bacteria for DNA type discrimination, but a skin sample is preferable. The skin sample is typically taken from a human, but may be a skin sample of a non-human animal. As the skin sample collected from humans, a skin sample containing the human epidermal stratum corneum is preferable. The method for collecting the skin sample is not particularly limited as long as it is a non-invasive method, but typically, a method of attaching an adhesive tape or adhesive to the skin and then peeling it off (tape stripping method) is used. Be done. The material (adhesive component) used for the adhesive tape or adhesive is not particularly limited.

For the extraction of DNA from the above sample, for example, any method known to those skilled in the art may be used as a nucleic acid extraction method, and examples thereof include a phenol extraction method, a cetyltrimethylammonium bromide (CTAB) method, and an alkaline SDS method. Be done. In addition, these methods may be appropriately modified, and various DNA extraction kits sold by reagent manufacturers may be used. Depending on the type of sample, filtration or homogenization with a membrane filter is performed. Further, various DNA extraction kits such as DN easy Plant mini Kit (manufactured by Qiagen Co., Ltd.) sold by the reagent manufacturer may be used. The DNA extracted by these methods is preferably retained in a state suitable for use as a PCR template, for example, dissolved in an appropriate buffer solution and stored at a low temperature. Further, after the DNA is extracted, purification treatments such as chloroform / isoamyl alcohol treatment, isopropanol precipitation, phenol / chloroform deproteinization, and ethanol precipitation may be performed.

As the nucleic acid amplification method, various known methods such as PCR method and isothermal amplification method can be used. In the present invention, the PCR method refers to a nucleic acid amplification method that amplifies a target region by repeating temperature changes, and the term PCR method also includes a real-time PCR method.

The isothermal amplification method is not particularly limited, and is a loop-mediated isothermal amplification (LAMP) method, a strand displacement amplification (SDA) method, and an isothermal and chimeric primer-initiated amplification of. Various isothermal amplification methods such as Nucleic acids; ICAN), Helicase-Dependent Amplification (HDA), and Nicking Enzyme Amplification Reaction (NEAR) can be used, but the LAMP method is used. preferable.

The LAMP method is a total of two types of inner primers and two types of outer primers designed based on arbitrary sequences F3c, F2c, F1c, B1, B2 and B3 set from the 3'end to the 5'end of the target nucleic acid sequence. A total of 6 types of primers, including 4 types of primers or 2 types of loop primers, a strand-substituted DNA synthase, a nucleic acid monomer, and other reagents and a template nucleic acid chain containing the target nucleic acid sequence are mixed and isothermally (approximately 65). It is a method of carrying out a nucleic acid amplification reaction by keeping warm at (° C.). In the LAMP method, the 3'end of itself is annealed to the nucleotide as a template to serve as the starting point for complementary strand synthesis, and the complementary strand synthesis reaction at an isothermal temperature is carried out by combining a primer that anneals to the loop formed at this time. This is a nucleic acid amplification method that has been made possible.

The amplification reaction by the LAMP method can be carried out, for example, as follows. Reagents (eg, LAMP primer set of the present invention, Bst DNA polymerase, reaction buffer, dNTPs, distilled water) are mixed, dispensed into a reaction tube, and then a solution containing DNA extracted from the sample is added. The incubation is then performed at a temperature suitable for enzyme activity, such as 60-70 ° C, preferably 65 ° C. The incubation time can be set to a time sufficient to complete the DNA amplification by the LAMP method, for example, 30 to 120 minutes, preferably 60 to 90 minutes. The primer concentration can be 0.4 to 0.025 μM for F3 and B3, 3.2 to 0.2 μM for FIP and BIP, and 1.6 to 0.1 μM for LF and LB.

The PCR method is not particularly limited except that the above-mentioned PCR primers are used, and may be carried out according to a conventional method. Specifically, the cycle is repeated including denaturation of the template DNA, annealing of the primer to the template, and extension of the primer using a thermostable enzyme (DNA polymerase such as Taq polymerase or Tth DNA polymerase derived from Thermophilus). This amplifies the partial region of the gene specific to the acne bacterium to be discriminated. The composition of the PCR solution (amount of template DNA, type of buffer solution, primer concentration, type and concentration of DNA polymerase, dNTP concentration, magnesium chloride concentration, etc.) and PCR reaction conditions (temperature cycle, number of cycles, etc.) are determined by those skilled in the art. If so, it can be selected and set appropriately.

For example, after mixing 0.1 to 100 ng of DNA as a template, buffer solution for 10 × PCR reaction, 0.25 to 1 μM each of primers, 0.25 to 2.5 U of DNA polymerase (Taq polymerase, Tth DNA polymerase, etc.), and 250 μM each of dNTP, the whole Diluted to a liquid volume of 10 to 100 μl, 94 to 96 ° C for 5 minutes x 1 cycle, (94 to 96 ° C for 30 seconds, 52 to 58 ° C for 30 seconds, 70 to 74 ° C for 1 minute) x 30 cycles , 70-74 ° C 5 minutes x 1 cycle. This is only an example, and the composition of the PCR solution, the reaction temperature and the time can be appropriately set according to the length of the oligonucleotide sequence as a primer, the base composition and the like. A series of these PCR operations can be performed by using a commercially available PCR kit or PCR device and following the operation manual.

The real-time PCR method is an intercalator method in which an intercalator such as SYBR Green I, which is a compound that fluoresces by binding to double-stranded DNA together with a primer pair, is added to the PCR reaction system, or the 5'end is called a reporter. ^{There is a TaqMan method in which a probe (TaqMan TM} probe) labeled with a quencher at the 3'end of a fluorescent substance is added to the PCR reaction system, and both can be used in the present invention, but the TaqMan method is preferable. In the TaqMan method, the TaqMan ^TM probe specifically hybridizes to the template DNA under the conditions used for the polymerase extension reaction in the amplification reaction by PCR, and is degraded as the DNA strand is extended, that is, the template DNA is amplified. By liberating the fluorescent substance, the amount of fluorescence in the PCR solution increases. This increase in the amount of fluorescence serves as an index for amplification of the template DNA, and the state of amplification in PCR can be easily detected in real time. The real-time PCR method is performed by using a commercially available real-time PCR kit or a real-time PCR device based on a usual method known to those skilled in the art except using the above-mentioned oligonucleotide set, and following the operating instructions attached to them. Just do it. As the real-time PCR device, for example, ABI Prism 7900HT Real-time PCR System or the like is used.

Any known detection method can be applied to the detection of the amplification product obtained by the nucleic acid amplification method.

In the case of the PCR method, the amplification product can be detected by using conventional electrophoresis such as agarose gel electrophoresis or capillary electrophoresis, DNA hybridization method, intercalation method, quencher-mediated fluorescence detection method, or the like. it can. For example, in agarose gel electrophoresis, staining is performed with ethidium bromide, SYBR Green solution, etc., and the amplified product is detected as a single band, and the DNA type is determined based on its size. It is also possible to detect the amplification product by performing the PCR using a primer labeled with a labeling substance in advance. As the labeling substance, a fluorescent substance, a radioisotope, a chemical luminescent substance, biotin and the like, which are well known in the art, can be used. Further, in DNA hybridization, the amplification product is bound to an immobilized carrier such as a microarray, and the amplification product is confirmed by fluorescence, enzymatic reaction, or the like. The immobilized carrier for detection is a support on which an oligonucleotide having a sequence capable of hybridizing to a partial region of a gene specific to the DNA type of the acne bacterium to be discriminated is immobilized. As the support, for example, a nylon film, a nitrocellulose film, glass, a silicon chip, or the like can be used.

In the case of the isothermal amplification method (LAMP method), the amplification product can be detected by using a method of measuring the turbidity of the reaction solution, a method of visual fluorescence, a method of agarose gel electrophoresis, or the like. In the LAMP method, a large amount of substrate is consumed by DNA synthesis, and it reacts with magnesium in which pyrophosphate, which is a by-product, coexists, causing cloudiness and whitening of magnesium pyrophosphate. Therefore, the change in absorbance at 650 nm is measured using a measuring device that can optically observe the increase in turbidity of the reaction solution after the completion of the amplification reaction or during the amplification reaction, for example, a spectrophotometer. It is possible to confirm by. In addition, in the fluorescent visual method, after performing an amplification reaction in the presence of a fluorescent intercalator that binds to a double-stranded nucleic acid such as ethidium bromide, the amplified product can be confirmed by fluorescence coloring in the presence of an ultraviolet lamp. Is.

As one embodiment of the present invention, it is preferable that the amplification product is detected by nucleic acid chromatography. Nucleic acid chromatography is a method for detecting a target nucleic acid using chromatography, and this method hybridizes a chromatography solution (developing solution) containing the target nucleic acid to a specific region of the target nucleic acid. This includes a step of developing a solid phase carrier on which the detection probe is immobilized to form a hybrid product of the target nucleic acid and the detection probe, and a step of detecting the hybrid product. Detection of the hybridization product is carried out by utilizing the signal of the labeling substance imparted to the target nucleic acid prior to development (for example, during nucleic acid amplification) or during development. The form of nucleic acid chromatography is not particularly limited, and may be a so-called lateral flow type intended for development in a substantially horizontal state, or a so-called dipstick type intended for development in a substantially vertical direction.

The developing liquid is a liquid that diffuses and moves the solid phase carrier of the chromatography body by a capillary phenomenon, and is a medium for transferring the augmented by-products on the solid phase carrier. The developing solution may be any as long as the amplification product contained in the amplification reaction solution can be easily developed in the solid phase carrier. For example, water, an organic solvent compatible with water, or one or more kinds of water. A mixed solution of the organic solvent of the above can be mentioned. Examples of the organic solvent include lower alcohols, DMSO, DMF, esters such as methyl acetate and ethyl acetate, and acetone. The developing solution contains a buffer component for adjusting pH, for example, an acetate buffer solution, a citrate buffer solution, and a phosphate buffer solution. Further, the amplification reaction solution can be used as it is as the developing solution, and the composition and concentration can be adjusted by adding an additional component or solvent such as a surfactant or an appropriate salt to the amplification reaction solution. May be used as a developing solution.

The time for applying the developing solution to the solid phase carrier is not particularly limited, but is, for example, 5 minutes or more, preferably 10 minutes or more. Detection of hybridized products confirms the coloration and location of the region where the detection probe is immobilized. In order to detect the signal due to the labeling substance, it is appropriately selected according to the type of the labeling substance.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.

(Example 1) Construction of LAMP primer set for discriminating the DNA type of P. acnes (1) Search for target genes From the public database NCBI (National Center for Biotechnology Information), P. acnes (Cutibacterium acnes) and its related bacteria, The entire genome sequences of 50 strains including other skin flora (Table 1-1) were obtained. Among these strains, D3 type Cutibacterium acnes JCM 6425 (ATCC 6919), D2 type Cutibacterium acnes JCM 6473 (ATCC 11828), and D3 type Cutibacterium acnes JCM whose DNA types have already been clarified. Includes 18916, D1 type Cutibacterium acnes JCM 18918, and D2 type JCM 18920 (Table 1-2).

The whole genome sequences obtained from the above 50 strains were comparatively analyzed using the PanOCT analysis tool (Nucleic Acids Res. 2012 Dec; 40 (22): e172), and specific genes were searched for by DNA type. The JCM18918_744 gene was selected as a gene specific to D2, the TIIST44_06360 gene was selected as a gene specific to D2 type, and the FB33_0108 gene was selected as a gene specific to D3 type (Table 2).

(2) Construction of LAMP primer set for discriminating the DNA type of Acne bacteria The nucleotide sequence of the JCM18918_744 gene (SEQ ID NO: 1) specific to the D1 type selected in (1), and the nucleotide sequence of the TIIST44_06360 gene specific to the D2 type. The sequence (SEQ ID NO: 2) and the nucleotide sequence of the FB33_0108 gene specific to the D3 type (SEQ ID NO: 3) were examined in more detail, and the nucleotide sequence specific to each DNA type was identified. Next, we designed a primer that can amplify the specified base sequence by the LAMP method. For the design of the LAMP primer, the known LAMP method primer design support software LAMP Designer (manufactured by OptiGene); http://www.optigene.co.uk/lamp-designer/ was used.

The LAMP primers designed for D1 type, D2 type, and D3 type of Acne bacteria are shown in Tables 3 to 5 below.

(Example 2) Performance evaluation of the LAMP primer set for discriminating the DNA type of Acne bacteria The DNA type discrimination performance of the Acne bacteria of the LAMP primer set constructed in Example 1 was evaluated by the LAMP method and nucleic acid chromatography.
(1) Test strains As the strains obtained from the distribution organization, 5 strains of Acne strains obtained from the Japan Collection of Microorganisms (JCM), RIKEN BioResource Center, shown in Table 6 were used. The culture method was based on the method recommended by the condominium organization.

As the strains isolated from humans, 40 strains shown in Table 7 were used. Strains are cultivated by the method described in the literature (J Dermatol, vol.40, PP.786-792, 2002.) and then in the literature (J Clin Microbiol, vol.40, PP.198-204, 2002.). It was identified as Cutibacterium acnes by the method described in. In addition, the DNA types of these isolated strains were identified in advance by the method described in the literature (Journal of the Japanese Dermatological Association, vol.115, PP.2381-2388, 2005.).

(2) DNA extraction The DNA of each test strain was extracted using the DNA extraction reagent "GenCheck (Fasmac Co., Ltd.)".

(3) Gene amplification by LAMP method and detection by nucleic acid chromatography Since the amplification product is detected by the STH chromato PAS method, each LAMP primer set for discriminating the DNA type of Acne bacteria constructed in Example 1 is always used. By the method, the tag sequence was attached to the 5'end of the LF primer, and biotin was attached to the 5'end of the LB primer. A different tag sequence was used for each type.

Next, Primer Mixture was prepared. Distilled water was added to 0.012 μL of F3 primer and B3 primer, 0.096 μL of FIP primer and BIP primer, and 0.048 μL of LF primer and LB primer to make a total of 3 μL.

Next, a LAMP reaction solution was prepared. To the Tube Strip for LAMP reaction, 3 μL of the prepared Primer Mixture, 6 μL of Isothermal Master Mix (OptiGene), and 3 μL of the DNA extracted in (2) were added and mixed. A heat block was used for the LAMP isothermal reaction, and the reaction was carried out at 65 ° C. for 30 minutes. The LAMP product after the reaction was diluted 1/10 with sterilized water, and 1 μL thereof was added to 20 μL of the chromatographic developing solution (1 μL of the chromatographic developing dye and 19 μL of the chromatographic developing buffer) and mixed. The chromatographic paper was immersed in the mixed solution and chromatographically developed at room temperature for 20 minutes.

(4) Results The results are shown in Tables 6 and 7 (+: Amplified product (band) detected,-: Amplified product (band) not detected).

As shown in Tables 6 and 7, the LAMP primer set (JCM18918_744_LAMP) designed from the nucleotide sequence of the JCM18918_744 gene (SEQ ID NO: 1) is the LAMP designed from the nucleotide sequence of the TIIST44_06360 gene (SEQ ID NO: 2) of the Acne bacterium D1 type. It was confirmed that the primer set (TIIST44_06360_LAMP) can discriminate the D2 type of Acne bacteria, and the LAMP primer set (FB33_0108_LAMP) designed from the nucleotide sequence of the FB33_0108 gene (SEQ ID NO: 3) can discriminate the D3 type of Acne bacteria specifically and with high accuracy. ..

(Example 3) Construction and evaluation of a simultaneous discrimination system for the DNA type of acne bacteria
DNA was extracted from Cutibacterium acnes JCM 6425, Cutibacterium acnes JCM 6473, and Cutibacterium acnes JCM 18918, respectively. Specimen containing only the DNA of Cutibacterium acnes JCM 6425, Specimen containing only the DNA of Cutibacterium acnes JCM 6473 2, Specimen containing only the DNA of Cutibacterium acnes JCM 18918, DNA of Cutibacterium acnes JCM 6425, DNA of Cutibacterium acnes JCM 6473, Sample 4 containing all of the DNA of Cutibacterium acnes JCM 189186 was prepared.

As a LAMP reaction solution for sample 4, a LAMP reaction solution was prepared by mixing Primer Mixtures of each DNA type primer set (JCM18918_744_LAMP, TIIST44_06360_LAMP, FB33_0108_LAMP) at a ratio of 1: 1: 1. The LAMP reaction was carried out in the same manner as in Example 2, and the amplified product was detected by nucleic acid chromatography.

The band pattern of nucleic acid chromatography of each sample is shown in FIG. 1 [Lane 1 (Sample 1: DNA of Cutibacterium acnes JCM 6425, primer used: JCM18918_744_LAMP), Lane 2 (Sample 2: DNA of Cutibacterium acnes JCM 6473, primer used: JCM18918_744_LAMP), Lane 3 (Sample 3: Cutibacterium acnes JCM 18918 DNA, Primer used: JCM18918_744_LAMP), Lane 4 (Sample 1: Cutibacterium acnes JCM 6425 DNA, Primer used: TIIST44_06360_LAMP), Lane 5 (Sample 2: Cutibacterium acnes JCM) DNA of 6473, primer used: TIIST44_06360_LAMP), lane 6 (sample 3: DNA of Cutibacterium acnes JCM 18918, primer used: TIIST44_06360_LAMP), lane 7 (sample 1: DNA of Cutibacterium acnes JCM 6425, primer used: FB33_0108_LAMP), lane 8 (Sample 2: DNA of Cutibacterium acnes JCM 6473, primer used: FB33_0108_LAMP), Lane 9 (Sample 3: DNA of Cutibacterium acnes JCM 18918, primer used: FB33_0108_LAMP), Lane 10 (Sample 4: Cutibacterium acnes JCM 6425, Cutibacterium acnes JCM) 6473 and Cutibacterium acnes JCM 18918 DNA mixture, Primer used: JCM18918_744_LAMP, TIIST44_06360_LAMP, FB33_0108_LAMP mixed primer), Lane 11 (Sample: Distilled water, Primer used: JCM18918_744_LAMP, TIIST44_06360_LAMP, FB33_0108_LAMP.

As shown in FIG. 1, the sample 1 containing the DNA of D1 type Cutibacterium acnes JCM 18918 is a sample containing the DNA of D2 type Cutibacterium acnes JCM 6473 when the LAMP reaction is performed using JCM18918_744_LAMP (lane 3). In case 2, the LAMP reaction was carried out using TIIST44_06360_LAMP (lane 5), and the sample 3 containing the DNA of D3 type Cutibacterium acnes JCM 6425 was subjected to the LAMP reaction using FB33_0108_LAMP (lane 7). A specific band corresponding to the amplification product of was detected. Moreover, when the LAMP reaction was carried out in the multiplex, bands corresponding to the three DNA types were detected at the same time (lane 10).

(Example 4) Construction and evaluation of a quantitative PCR primer / probe set for discriminating the DNA type of Acne bacteria (1) Construction of a quantitative PCR (qPCR) primer / probe set for discriminating the DNA type of Acne bacteria Specified in Example 1 We designed a primer that can amplify the base sequence of a partial region of the base sequence of the JCM18918_744 gene, TIIST44_06360_ gene, and FB33_0108 gene of the acne bacterium (Cutibacterium acnes) by PCR. Primer3 (https://primer3.org/) was used to design the PCR primer.

Tables 8 to 10 below show quantitative PCR primer / probe sets designed for the JCM18918_744 gene, TIIST44_06360_ gene, and FB33_0108 gene for DNA type determination of Acne bacteria. In the name, those described as "F1", "F2", "R1", and "R2" indicate primers, and those described as "P1" and "P2" indicate probes. The probe for distinguishing D1 type (JCM18918_744) has the 5'end modified with FAM and the 3'end with BHQ, and the probe for D2 type (TIIST44_06360) has the 5'end modified with HEX and the 3'end with BHQ. However, the probe for distinguishing D3 type (FB33_0108) was modified with Texas RED at the 5'end and BHQ at the 3'end.

(2) Performance evaluation of qPCR primer / probe set for discriminating the DNA type of Acne bacteria The DNA of each test strain was extracted using the DNA extraction reagent "GenCheck (Fasmac Co., Ltd.)" in the same manner as in Example 1. The DNA type discrimination performance of the qPCR primer / probe set constructed in (1) was confirmed by a quantitative PCR method.

Quantitative (real-time) PCR was performed using StepOne Plus (Thermo Fisher) / Light Cycler LC 96 System (Roche). The composition of the PCR reaction solution was 2 μl of the template DNA sample, 1 μl of the forward primer (10 μM), 1 μl of the reverse primer (10 μM), 1 μl of the probe (2.5 μM), 10 μl of Takara premix EX Taq, and 5 μl of distilled water, and the total volume was 20 μl. The PCR reaction consists of 45 cycles of 2-step PCR, which consists of pretreatment at 42 ° C for 5 minutes and 95 ° C for 10 minutes, heat denaturation at 95 ° C for 30 seconds, annealing treatment at 60 ° C for 60 seconds, and extension reaction. went.

(3) Results The results are shown in Tables 11-1 to 11-3 (+: Amplified product (fluorescent signal) detected,-: Amplified product (fluorescent signal) not detected).

As shown in Tables 11-1 to 11-3, the qPCR primer probe set (JCM18918_744_PCR_1, JCM18918_744_PCR_2) designed from the base sequence of the JCM18918_744 gene is the qPCR primer probe set designed from the base sequence of TIIST44_06360, a D1 type strain. It was confirmed that (TIIST44_06360_PCR_1, TIIST44_06360_PCR_2) can discriminate D2 type strains, and the qPCR primer probe set (FB33_0108_PCR_1, FB33_0108_PCR_2) designed from the base sequence of the FB33_0108 gene can discriminate D3 type strains specifically and with high accuracy.

The present invention can be used in the field of manufacturing reagents for testing acne bacteria. By using the oligonucleotide set and method of the present invention, the DNA type of acne bacteria that cause acne and the like can be accurately and quickly determined. Therefore, based on this result, providing cosmetics and pharmaceuticals for preventing or improving acne of the sample provider according to the DNA type, determining the therapeutic effect of the disease, preventing or improving the disease. It will be possible to provide counseling and advice on how to care for the patient.

Claims (19)

A set of oligonucleotides for discriminating D1 type of P. acnes by nucleic acid amplification method, which comprises an oligonucleotide capable of hybridizing to the nucleotide sequence of a partial region of the JCM18918_744 gene of P. acnes (Cutibacterium acnes) or its complementary sequence. Is two or more regions selected from the 76-93 position region, the 110-227 position region, the 241-331 position region, and the 317-349 position region in the nucleotide sequence of SEQ ID NO: 1. , The above oligonucleotide set. The oligonucleotide set according to claim 1, wherein the oligonucleotide set is a set of LAMP primers composed of the following primers (a1) to (a6).
(A1) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 4 or an oligonucleotide consisting of a base sequence in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 4. F3 Primer (a2) Containing an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 5, or a base sequence in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 5. B3 primer containing an oligonucleotide (a3) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 6, or a base in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 6. FIP primer containing an oligonucleotide consisting of a sequence (a4) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 7 or one to several bases deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 7. BIP primer containing the nucleotide sequence of the nucleotide sequence (a5) The oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 8 or the nucleotide sequence shown in SEQ ID NO: 8 is deleted, substituted, or inserted with one to several bases. Or, an LF primer containing an oligonucleotide consisting of the added base sequence (a6) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 9 or a base sequence shown in SEQ ID NO: 9 in which one to several bases are deleted. LB primer containing an oligonucleotide consisting of a substituted, inserted, or added base sequence The oligonucleotide set is a primer pair consisting of the following oligonucleotide (a7) and the oligonucleotide (a8), and a set of PCR primers and probes composed of the probe consisting of the following oligonucleotide (a9). 1. A set of PCR primers and probes composed of a primer pair consisting of the following oligonucleotide (a10) and an oligonucleotide (a11), and a probe consisting of the following oligonucleotide (a12). The oligonucleotide set described.
(A7) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 22 or an oligonucleotide consisting of a base sequence in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 22 ( a8) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 23, or an oligonucleotide consisting of a base sequence in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 23 (a9). ) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 24, or an oligonucleotide consisting of a base sequence in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 24 (a10). An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 25, or an oligonucleotide (a11) sequence consisting of a base sequence in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 25. An oligonucleotide consisting of the base sequence shown in No. 26, or an oligonucleotide consisting of a base sequence in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 26 (a12) SEQ ID NO: An oligonucleotide consisting of the base sequence shown in 27, or an oligonucleotide consisting of a base sequence in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 27. An oligonucleotide set for discriminating the D2 type of P. acnes by a nucleic acid amplification method, which comprises an oligonucleotide capable of hybridizing to the nucleotide sequence of a partial region of the TIIST44_06360 gene of P. acnes (Cutibacterium acnes) or a complementary sequence thereof. Is two or more regions selected from the 53-72 region, the 75-150 region, the 153-241 region, and the 244-261 region in the nucleotide sequence of SEQ ID NO: 2. , The above oligonucleotide set. The oligonucleotide set according to claim 4, wherein the oligonucleotide set is a set of LAMP primers composed of the following primers (b1) to (b6).
(B1) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 10 or an oligonucleotide consisting of a base sequence in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 10. F3 Primer (b2) Consists of an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 11 or a base sequence in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 11. B3 primer containing an oligonucleotide (b3) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 12, or a base in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 12. FIP primer containing an oligonucleotide consisting of a sequence (b4) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 13 or one to several bases deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 13. BIP primer containing the nucleotide sequence of the nucleotide sequence (b5) The oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 14 or the nucleotide sequence shown in SEQ ID NO: 14 lacks, substitutes, or inserts one to several bases. Or an LF primer containing an oligonucleotide consisting of the added base sequence (b6) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 15 or a base sequence shown in SEQ ID NO: 15 lacking one to several bases. LB primer containing an oligonucleotide consisting of a substituted, inserted, or added base sequence The oligonucleotide set is a primer pair consisting of the following oligonucleotide (b7) and (b8) oligonucleotide, and a set of PCR primers and probes composed of the following probe consisting of the oligonucleotide (b9). 4. A set of PCR primers and probes composed of the following (b10) oligonucleotide and (b11) oligonucleotide pair, and the following (b12) oligonucleotide probe. The oligonucleotide set described.
(B7) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 28, or an oligonucleotide consisting of a base sequence in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 28 (b7) b8) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 29, or an oligonucleotide consisting of a base sequence in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 29 (b9). ) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 30, or an oligonucleotide consisting of a base sequence in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 30 (b10). An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 31 or an oligonucleotide (b11) sequence consisting of a base sequence in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 31. An oligonucleotide consisting of the base sequence shown in No. 32, or an oligonucleotide consisting of a base sequence in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 32 (b12) SEQ ID NO: An oligonucleotide consisting of the base sequence shown in 33, or an oligonucleotide consisting of a base sequence in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 33. An oligonucleotide set for discriminating the D3 type of P. acnes by a nucleic acid amplification method, which comprises an oligonucleotide capable of hybridizing to a nucleotide sequence of a partial region of the FB33_0108 gene of P. acnes (Cutibacterium acnes) or a complementary sequence thereof. Is two or more regions selected from the region at positions 219 to 236, the region at positions 298 to 412, the region at positions 450 to 521, and the region at positions 539 to 557 in the nucleotide sequence of SEQ ID NO: 3. , The above oligonucleotide set. The oligonucleotide set according to claim 7, wherein the oligonucleotide set is a set of LAMP primers composed of the following primers (c1) to (c6).
(C1) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 16 or an oligonucleotide consisting of a base sequence in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 16. F3 primer containing F3 primer (c2) Consists of an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 17, or a base sequence in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 17. B3 primer containing an oligonucleotide (c3) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 18, or a base in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 18. FIP primer containing an oligonucleotide consisting of a sequence (c4) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 19 or one to several bases deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 19. BIP primer containing the nucleotide sequence of the nucleotide sequence (c5) The oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 20 or the nucleotide sequence shown in SEQ ID NO: 20 with one to several bases deleted, substituted, or inserted. Or, an LF primer containing an oligonucleotide consisting of the added base sequence (c6) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 21 or a base sequence shown in SEQ ID NO: 21 in which one to several bases are deleted. LB primer containing an oligonucleotide consisting of a substituted, inserted, or added base sequence The oligonucleotide set is a set of PCR primers and probes composed of a primer pair consisting of the following oligonucleotides (c7) and (c8) oligonucleotides, and a probe consisting of the following oligonucleotides (c9). 7. A set of PCR primers and probes composed of the following (c10) oligonucleotide and (c11) oligonucleotide pair, and the following (c12) oligonucleotide probe. The oligonucleotide set described.
(C7) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 34, or an oligonucleotide consisting of a base sequence in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 34 ( c8) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 35, or an oligonucleotide consisting of a base sequence in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 35 (c9). ) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 36, or an oligonucleotide consisting of a base sequence in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 36 (c10) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 37, or an oligonucleotide (c11) sequence consisting of a base sequence in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 37. An oligonucleotide consisting of the base sequence shown in No. 38, or an oligonucleotide consisting of a base sequence in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 38 (c12) SEQ ID NO: An oligonucleotide consisting of the base sequence shown in 39, or an oligonucleotide consisting of a base sequence in which one to several bases are deleted, substituted, inserted, or added in the base sequence shown in SEQ ID NO: 39. The oligonucleotide set according to any one of claims 2, 5 and 8, wherein a tag sequence different for each DNA type is bound to the FIP primer or BIP primer, or the LF primer or LB primer. A kit for determining the DNA type of acne bacteria, which comprises at least one set of oligonucleotides according to any one of claims 1 to 10. The kit according to claim 11, further comprising a solid-phase carrier on which a detection probe that hybridizes to the tag sequence is immobilized. The kit according to claim 12, wherein the solid phase carrier is a strip for nucleic acid chromatography. A step of performing an amplification reaction by a nucleic acid amplification method using at least one set of oligonucleotides according to any one of claims 1 to 3 using DNA extracted from a sample as a template, and a step of detecting an amplification product are performed. Method for determining D1 type of Acne bacteria, including. A step of performing an amplification reaction by a nucleic acid amplification method using at least one set of oligonucleotides according to any one of claims 4 to 6 using DNA extracted from a sample as a template, and a step of detecting an amplification product are performed. Method for determining D2 type of acne bacteria, including. A step of performing an amplification reaction by a nucleic acid amplification method using at least one set of oligonucleotides according to any one of claims 7 to 9 using DNA extracted from a sample as a template, and a step of detecting an amplification product are performed. Method for determining D3 type of acne bacteria, including. Using the DNA extracted from the sample as a template, at least one set of the oligonucleotide set according to any one of claims 1 to 3 and at least one set of the oligonucleotide set according to any one of claims 4 to 6. , And a step of performing an amplification reaction by a nucleic acid amplification method using at least one set of oligonucleotides according to any one of claims 7 to 9, and a step of detecting an amplification product, a DNA type of Acne bacteria. Simultaneous discrimination method. The discrimination method according to any one of claims 14 to 17, wherein the nucleic acid amplification method is an isothermal amplification method or a PCR method. The discrimination method according to any one of claims 14 to 18, wherein the sample is a skin sample.

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