JP7391321B2 - Oligonucleotide set and kit for determining the DNA type of P. acnes and method for determining the DNA type of P. acnes - Google Patents

Description

The present invention relates to an oligonucleotide set and kit for determining the DNA type of Cutibacterium acnes, which is a bacterial cause of acne vulgaris inflammation, and a method for determining the DNA type.

Cutibacterium acnes is a facultatively anaerobic Gram-positive bacillus of the phylum Actinobacteria, class Actinobacteria, order Actinomycetales, family Propionibacteriaceae. Until recently, the genus Cutibacterium was included in the genus Propionibacterium, but with the reclassification in 2016, it became the genus Cutibacterium (non-patent literature). 1). P. acnes is the most prevalent resident bacteria in sebum-producing areas such as the face, anterior chest, and back of humans, and is also known as the inflammatory cause of acne vulgaris (acne). When the infundibulum of the sebaceous hair follicle is blocked due to abnormal keratosis or overproduction of sebum, sebum accumulates within the hair follicle, and as a result, inflammation occurs due to the bacteria P. acnes that grows excessively within the hair follicle, resulting in an inflammatory condition. It is thought to cause acne.

Up to now, several methods have been reported for classifying P. acnes into types based on the depth of their involvement in acne. There is a classification (Non-Patent Document 2). In this method, an unspecified region of DNA extracted from P. acnes is amplified using predetermined primers, and electrophoresis is performed on an agarose gel. Depending on the band pattern of the expressed DNA, P. acnes is divided into four types of DNA-types. (D1, D2, D3 and D1/D3 intermediate type). Of these, D3 is most commonly detected in acne lesions, has high lipase activity, which is an inflammatory factor, and has a large amount of porphyrin, and is said to be most deeply involved in acne. On the other hand, D1 and D2 are less frequently detected from acne lesions and have low lipase activity, so their involvement in acne is thought to be low. As described above, there are different types of P. acnes bacteria, and identifying the type of P. acnes bacteria can be said to be a useful technique in the prevention and treatment of acne.

However, this DNA-type classification of P. acnes is a very labor-intensive and time-consuming typing method, and there is a major problem in that it can only be carried out by those who are familiar with DNA experiments. Furthermore, since the determination is made based on the DNA band pattern, there is a problem in that incorrect determinations are likely to occur if the amount of DNA amplified is insufficient.

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 that can more accurately, quickly, and simply determine the DNA type of Cutibacterium acnes.

The present inventors analyzed the gene sequences of strains of Cutibacterium acnes and found specific genes for each DNA type (D1 type, D2 type, and D3 type). As a result of further detailed examination of the base sequences of these genes, the present inventors identified regions in D1 type, D2 type, and D3 type that each have a specific sequence that can be distinguished from other DNA types. We have succeeded in establishing a set of oligonucleotides that can specifically and highly accurately identify the DNA type of P. acnes using a nucleic acid amplification method based on the base sequence of P. acnes, and have completed the present invention.

That is, the present invention includes the following inventions.
(1) An oligonucleotide set for determining the D1 type of Cutibacterium acnes using a nucleic acid amplification method, comprising an oligonucleotide that can hybridize to the base sequence of a partial region of the JCM18918_744 gene of Cutibacterium acnes or its complementary sequence, The partial region consists of two or more regions selected from the region from positions 76 to 93, the region from positions 110 to 227, the region from positions 241 to 331, and the region from positions 317 to 349 in the base 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 LAMP primer set consisting 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 the base sequence shown in SEQ ID NO: 4 in which one to several bases have been deleted, substituted, inserted, or added. F3 primer containing (a2) Oligonucleotide consisting of the base sequence shown in SEQ ID NO: 5, or consisting of a base sequence in which one to several bases are deleted, substituted, inserted, or added to the base sequence shown in SEQ ID NO: 5. B3 primer containing oligonucleotide (a3) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 6, or a base in which one to several bases have been deleted, substituted, inserted, or added to the base sequence shown in SEQ ID NO: 6. FIP primer containing an oligonucleotide consisting of the 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 (a5) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 8, or one to several bases deleted, substituted, or inserted in the base sequence shown in SEQ ID NO: 8. , 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 an oligonucleotide in which one to several bases are deleted from the base sequence shown in SEQ ID NO: 9, LB primer containing an oligonucleotide consisting of a substituted, inserted, or added base sequence (3) The oligonucleotide set includes a primer pair consisting of the following oligonucleotide (a7) and the oligonucleotide (a8), and the following A PCR primer and probe set consisting of a probe consisting of the oligonucleotide (a9), or a primer pair consisting of the following oligonucleotide (a10) and (a11), and the following oligonucleotide (a12) The oligonucleotide set according to (1), which is a set of PCR primers and probes composed of probes consisting 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 have been deleted, substituted, inserted, or added to 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 have been deleted, substituted, inserted, or added to 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 the base sequence shown in SEQ ID NO: 24 with one to several bases deleted, substituted, inserted, or added (a10) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 25, or an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 25 in which one to several bases have been deleted, substituted, inserted, or added (a11) Sequence An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 26, or an oligonucleotide consisting of a base sequence in which one to several bases are deleted, substituted, inserted, or added to the base sequence shown in SEQ ID NO: 26 (a12) SEQ ID NO: An oligonucleotide consisting of the base sequence shown in SEQ ID NO:27, or an oligonucleotide consisting of a base sequence in which one to several bases are deleted, substituted, inserted, or added to the base sequence shown in SEQ ID NO:27.

(4) An oligonucleotide set for determining the D2 type of Cutibacterium acnes using a nucleic acid amplification method, comprising an oligonucleotide that can hybridize to the base sequence of a partial region of the TIIST44_06360 gene of Cutibacterium acnes or its complementary sequence, The partial region consists of two or more regions selected from the region from positions 53 to 72, the region from positions 75 to 150, the region from positions 153 to 241, and the region from positions 244 to 261 in the base 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 LAMP primer set 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 have been deleted, substituted, inserted, or added to the base sequence shown in SEQ ID NO: 10. F3 primer containing (b2) Oligonucleotide consisting of the base sequence shown in SEQ ID NO: 11, or consisting of a base sequence in which one to several bases are deleted, substituted, inserted, or added to the base sequence shown in SEQ ID NO: 11 B3 primer containing oligonucleotide (b3) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 12, or a base in which one to several bases have been deleted, substituted, inserted, or added to the base sequence shown in SEQ ID NO: 12. FIP primer containing an oligonucleotide consisting of the 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 (b5) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 14, or one to several bases deleted, substituted, or inserted in the base sequence shown in SEQ ID NO: 14. , 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 an oligonucleotide in which one to several bases are deleted from the base sequence shown in SEQ ID NO: 15, LB primer containing an oligonucleotide consisting of a substituted, inserted, or added base sequence (6) The oligonucleotide set includes a primer pair consisting of the following oligonucleotide (b7) and the oligonucleotide (b8), and the following A PCR primer and probe set consisting 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 oligonucleotide of (b12) below. The oligonucleotide set according to (4), which is a set of PCR primers and probes composed of probes consisting 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 have been deleted, substituted, inserted, or added to the base sequence shown in SEQ ID NO: 28 ( 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 have been deleted, substituted, inserted, or added to 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 the base sequence shown in SEQ ID NO: 30 with one to several bases deleted, substituted, inserted, or added (b10) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 31, or an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 31 with one to several bases deleted, substituted, inserted, or added (b11) sequence An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 32, or an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 32 with one to several bases deleted, substituted, inserted, or added (b12) SEQ ID NO: An oligonucleotide consisting of the base sequence shown in SEQ ID NO:33, or an oligonucleotide consisting of a base sequence in which one to several bases are deleted, substituted, inserted, or added to the base sequence shown in SEQ ID NO:33.

(7) An oligonucleotide set for determining the D3 type of Cutibacterium acnes using a nucleic acid amplification method, comprising an oligonucleotide that can hybridize to the base sequence of a partial region of the FB33_0108 gene of Cutibacterium acnes or its complementary sequence, The partial region consists of two or more regions selected from the region from positions 219 to 236, the region from positions 298 to 412, the region from positions 450 to 521, and the region from positions 539 to 557 in the base 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 LAMP primer set 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 have been deleted, substituted, inserted, or added to the base sequence shown in SEQ ID NO: 16. Containing F3 primer (c2) Oligonucleotide consisting of the base sequence shown in SEQ ID NO: 17, or consisting of a base sequence in which one to several bases are deleted, substituted, inserted, or added to the base sequence shown in SEQ ID NO: 17. B3 primer containing oligonucleotide (c3) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 18, or a base in which one to several bases have been deleted, substituted, inserted, or added to the base sequence shown in SEQ ID NO: 18. FIP primer containing an oligonucleotide consisting of the 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 (c5) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 20, or one to several bases deleted, substituted, or inserted in the base sequence shown in SEQ ID NO: 20. , 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 an oligonucleotide in which one to several bases are deleted from the base sequence shown in SEQ ID NO: 21, LB primer containing an oligonucleotide consisting of a substituted, inserted, or added base sequence (9) The oligonucleotide set includes a primer pair consisting of the following oligonucleotide (c7) and the oligonucleotide (c8), and the following A PCR primer and probe set consisting of a probe consisting of the oligonucleotide (c9), or a primer pair consisting of the following oligonucleotide (c10) and (c11), and the following oligonucleotide (c12). The oligonucleotide set according to (7), which is a set of PCR primers and probes composed of probes consisting 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 have been deleted, substituted, inserted, or added to 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 have been deleted, substituted, inserted, or added to 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 the base sequence shown in SEQ ID NO: 36 with one to several bases deleted, substituted, inserted, or added (c10) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 37, or an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 37 in which one to several bases have been deleted, substituted, inserted, or added (c11) sequence An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 38, or an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 38 with one to several bases deleted, substituted, inserted, or added (c12) SEQ ID NO: An oligonucleotide consisting of the base sequence shown in SEQ ID NO:39, or an oligonucleotide consisting of a base sequence in which one to several bases are deleted, substituted, inserted, or added to 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 that differs depending on the DNA type is bound to the FIP primer, BIP primer, LF primer, or LB primer. .
(11) A kit for determining the DNA type of P. acnes, comprising one or more oligonucleotide sets 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) Performing an amplification reaction by a nucleic acid amplification method using DNA extracted from the sample as a template and at least one of the oligonucleotide sets described in any one of (1) to (3), and detecting the amplification product. A method for determining type D1 of acne bacteria, including the step of
(15) Performing an amplification reaction by a nucleic acid amplification method using DNA extracted from the sample as a template and at least one of the oligonucleotide sets described in any of (4) to (6), and detecting the amplification product. A method for determining type D2 of acne bacteria, including the step of
(16) Performing an amplification reaction by a nucleic acid amplification method using DNA extracted from the sample as a template and at least one of the oligonucleotide sets described in any of (7) to (9), and detecting the amplified product. A method for determining the D3 type of acne bacteria, including the step of
(17) Using DNA extracted from a specimen as a template, at least one oligonucleotide set according to any one of (1) to (3), and one oligonucleotide set according to any one of (4) to (6). a step of performing an amplification reaction by a nucleic acid amplification method using at least one set of the oligonucleotide set according to any one of (7) to (9), and a step of detecting the amplification product. Simultaneous DNA type determination method.
(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 specimen is a skin sample.

According to the present invention, an oligonucleotide set for determining the DNA type of Cutibacterium acnes is provided. By using this oligonucleotide set, the three DNA types (D1 type, D2 type, and D3 type) of Cutibacterium acnes contained in skin samples can be detected individually and simultaneously, resulting in false positives and false negatives. There is no occurrence and it can be determined accurately and easily.

The DNA in a sample was amplified by the LAMP method using the oligonucleotide set for determining the DNA type of Cutibacterium acnes of the present invention, and the results were detected using nucleic acid chromatography. 6425 DNA, primer used: JCM18918_744_LAMP), lane 2 (sample 2: DNA of Cutibacterium acnes JCM 6473, primer used: JCM18918_744_LAMP), lane 3 (sample 3: DNA of Cutibacterium acnes JCM 18918, primer used: JCM18918_744_LAMP), lane 4 (Sample 1: Cutibacterium acnes JCM 6425 DNA, used primer: TIIST44_06360_LAMP), Lane 5 (Sample 2: Cutibacterium acnes JCM 6473 DNA, used primer: TIIST44_06360_LAMP), Lane 6 (Sample 3: Cutibacterium acnes JCM 18918 DNA, 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 acnes JCM 18918 DNA, used primer: FB33_0108_LAMP), lane 10 (sample 4: Cutibacterium acnes JCM 6425, Cutibacterium acnes JCM 6473, and mixture of Cutibacterium acnes JCM 18918 DNA, used primers: JCM18918_744_LAMP, TIIST44_06360_L AMP, FB33_0108_LAMP mixed primer) , lane 11 (sample: distilled water, primers used: mixed primers of JCM18918_744_LAMP, TIIST44_06360_LAMP, FB33_0108_LAMP)].

The present invention will be explained in detail below.
1. Oligonucleotide set for determining the DNA type of Cutibacterium acnes The oligonucleotide set of the present invention is an oligonucleotide set for determining the DNA type of Cutibacterium acnes. This is an oligonucleotide set for individually or simultaneously identifying three DNA types (D1 type, D2 type, and D3 type) of Cutibacterium acnes by targeting partial regions and using nucleic acid amplification methods.

In the present invention, "DNA type" refers to DNA-type classification based on RAPD-PCR products using a predetermined primer (5'-AACGCGCAAC-3') (Journal of the Japanese Dermatological Association, vol.115, PP.2381- 2388, 2005), DNA-type 1 (D1), DNA-type 2 (D2), and DNA-type 3 (D3). In the D1 type, the electrophoretic band pattern of the above RAPD-PCR product is expressed at 3,000, 2,800, 1,500, 1,400, 800, 300bp, in the D2 type, the same pattern is expressed at 1,000, 350bp, and in the D3 type, the same pattern is expressed at 1,000, 350bp. , the same pattern is expressed at 3,000, 2,800, 1,300, 800, and 300bp. Among these, type D3 is isolated most frequently from acne vulgaris lesions and is characterized by higher lipase activity and higher coproporphyrin production than other DNAs. Lipase generates free fatty acids such as oleic acid by hydrolyzing triglycerides, which promotes the growth of P. acnes, and coproporphyrin, which is produced by P. acnes, causes a photosensitization reaction when exposed to ultraviolet light. This generates singlet oxygen, 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 D1 type-specific gene of P. acnes is the JCM18918_744 gene that encodes a phage-associated protein and consists of the base sequence shown in SEQ ID NO: 1. The D2 type-specific gene of P. acnes is the TIIST44_06360 gene that encodes a hypothetical protein and consists of the base sequence shown in SEQ ID NO:2. The D3 type-specific gene of P. acnes is the FB33_0108 gene that encodes glycosyl hydrolase family 20 and consists of the base sequence shown in SEQ ID NO: 3. Each gene of JCM18918_744, TIIST44_06360, and FB33_0108 has sequence identity of 90% or more, preferably 95% or more, more preferably 98% or more, and most preferably 99% or more to the base sequences shown in SEQ ID NOs: 1 to 3. It may be a gene that encodes a protein that has the same function as JCM18918_744, TIIST44_06360, and FB33_0108. Here, sequence identity can be determined, for example, by FASTA search or BLAST search.

The partial regions of the JCM18918_744 gene that are targets for D1 type determination of P. acnes include the region from positions 76 to 93, the region from positions 110 to 227, the region from positions 241 to 331, and the region from positions 317 to 317 in the base sequence of SEQ ID NO: 1. Ranked 349th to 349th. Therefore, the oligonucleotide set for determining the D1 type of P. acnes of the present invention includes oligonucleotides that can hybridize to the base sequences of these partial regions or their complementary sequences.

The partial regions of the TIIST44_06360 gene that are targeted for D2 type determination of P. acnes include the region from positions 53 to 72, the region from positions 75 to 150, the region from positions 153 to 241, and the region from positions 244 to 244 in the base sequence of SEQ ID NO: 2. Ranked from 1st to 261st. Therefore, the oligonucleotide set for determining the D2 type of P. acnes of the present invention includes oligonucleotides that can hybridize to the base sequences of these partial regions or their complementary sequences.

The partial regions of the FB33_0108 gene that are targets for D3 type determination of P. acnes include the region from positions 219 to 236, the region from positions 298 to 412, the region from positions 450 to 521, and the region from positions 539 to 539 in the base sequence of SEQ ID NO: 3. Ranked 557th to 557th. Therefore, the oligonucleotide set for determining the D3 type of P. acnes of the present invention includes oligonucleotides that can hybridize to the base sequences of these partial regions or their complementary sequences.

As used herein, "hybridize" means to hybridize to a partial region of the above-mentioned specific gene to be targeted, but not to substantially hybridize to a partial region of the above-mentioned specific gene to be targeted. It means that. Hybridization can be performed under stringent conditions. "Stringent conditions" refer to conditions under 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 determined as appropriate by referring to Specifically, stringent conditions can be set by changing the temperature during Southern hybridization and the salt concentration contained in the solution, and the temperature and salt concentration contained in the solution during the washing step of Southern hybridization. More specifically, stringent conditions include, 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 carried out at 1-7×SSC, 0.02-3% SDS, at a temperature of 40°C to 60°C. Further, a washing step may be performed after hybridization, and the washing step can be performed, for example, in 0.1 to 2×SSC, 0.1 to 0.3% SDS, and at a temperature of 50 to 65°C.

The oligonucleotide set for determining the DNA type of P. acnes of the present invention includes primers used in the LAMP method (hereinafter sometimes referred to as "LAMP primers"), primer pairs and probes used in the PCR method (hereinafter referred to as "PCR primers"). (sometimes referred to as "primer-probe").

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

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

In the LAMP method, in addition to the above four types of primers F3, FIP, BIP, and B3, primers called LF and LB (loop primers) can be used. The LF primer is designed to have a nucleotide sequence complementary to the nucleotide sequence between the F1 region and the F2 region. The LB primer is designed to have a nucleotide sequence complementary to the nucleotide sequence between the B1 region and the B2 region. By additionally using these primers, the number of starting points for nucleic acid synthesis in nucleic acid amplification by the LAMP method increases, making it possible to shorten reaction time and increase detection sensitivity.

For the F3, FIP, BIP, and B3 regions for designing the LAMP primers of the present invention, in the case of D1 type of P. acnes, the region from positions 76 to 93 and the region from positions 110 to 227 in the base sequence of SEQ ID NO: 1. , the areas 248th to 331st, and the areas 332nd to 349th correspond, respectively. In addition, in the case of D2 type of P. acnes, the region from positions 53 to 72, the region from positions 76 to 150, the region from positions 153 to 241, and the region from positions 244 to 261 in the base sequence of SEQ ID NO: 2. correspond to each other, and in the case of D3 type of P. acnes, the region from positions 219 to 236, the region from positions 298 to 408, the region from positions 450 to 521, and the region from positions 539 to 557 in the base sequence of SEQ ID NO: 3. The corresponding areas correspond to each other.

Using alignment software such as Clustal After creating an alignment file of base sequence information, use LAMP Designer (manufactured by OptiGene; http://www.optigene.co.uk/lamp-designer/) and Primer Explorer V4 based on the information in the alignment file. (Eiken Kagaku homepage) etc. can be used to design.

The LAMP primers of the present invention are desirably designed in regions with low sequence diversity (high conservation) for each type of DNA, but may be designed in regions containing a small number of genetic polymorphisms. The number of base substitutions that reflect genetic polymorphism is preferably 20% or less per primer, more preferably 15% or less. When using primers that include substitutions at genetic polymorphism sites, primers for each genetic polymorphism may be used, or mixed primers in which primers for each genetic polymorphism are mixed may be used. Mixed primers synthesized to have mixed bases depending on genetic polymorphism may be used.

In each of the above primers, "forward" refers to a primer that binds complementary to the sense strand of the target region and provides a starting point for synthesis, and "backward" refers to a primer that binds complementary to the sense strand of the target region and provides a starting point for synthesis. Primer designation that binds to and provides a starting point for synthesis. The base length of the oligonucleotide serving as the 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 of about 1 to 30 bases between F2 and F1c, and the BIP primer between B2 and B1c.

Furthermore, in the present invention, PCR primers and probes can also be designed based on the base sequences of the F3, B3, FIP, BIP, LF, and LB regions identified by the LAMP method. The forward primer is designed to have a base sequence in the F3 region that is complementary to the F3c region, the reverse primer is designed to have the same base sequence as the F1c region, and the probe is designed to have a base sequence in the F1c region that is complementary to the F3c region. Design to have a complementary base sequence 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 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 complementary base sequence to the base sequence between the B2 regions.

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

PCR probes can be designed using commercially available design software available 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 can be carried out based on the software included with the real-time PCR device, or the protocol attached to commercially available real-time PCR reagents such as TaqMan Universal PCR Master Mix (Life Technologies Japan). In general, the design criteria for probes are that the GC content should be within the range of 20 to 80%, that consecutive G or C bases of 4 or more bases should be avoided in the sequence, and that the Tm value should be lower than the Tm value of the corresponding primer pair. For example, the temperature should be set about 8 to 10 degrees Celsius higher.

The design criteria for specifically adopted LAMP primers and PCR primers are as follows.
(i) To enable specific annealing with the template DNA, the primer length is 15 to 30 bases for F3, B3, LF, and LB primers, and 30 bases for FIP, BIP primers. Must be in the range of base to 60 bases.
(ii) Avoid complementary sequences between primers to avoid forming dimers or tertiary structures.
(iii) Contains many mutations (genus-specific mutations) at the tip in the elongation direction.
(iv) The total free energy of each of the six bases at the 3' and 5' ends is 4 kcal/mol or less.
(v) Avoiding self-complementary sequences to prevent the formation of hairpin structures within the primer.
(vi) In order to ensure stable binding with template DNA, the GC content should be approximately 40 to 60%, and GC-rich or AT-rich should not be unevenly distributed in the primer.
(vii) There is high homology between the base sequence at the 3' end of the primer and the template DNA sequence.
(viii) The Tm value should 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 set for determining the DNA type of P. acnes of the present invention was established.
[LAMP primer set for determining D1 type of P. acnes]
(a1) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 4, or an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 4 in which one to several bases have been deleted, substituted, inserted, or added. F3 primer containing (a2) Oligonucleotide consisting of the base sequence shown in SEQ ID NO: 5, or consisting of a base sequence in which one to several bases are deleted, substituted, inserted, or added to the base sequence shown in SEQ ID NO: 5. B3 primer containing oligonucleotide (a3) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 6, or a base in which one to several bases have been deleted, substituted, inserted, or added to the base sequence shown in SEQ ID NO: 6. FIP primer containing an oligonucleotide consisting of the 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 (a5) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 8, or one to several bases deleted, substituted, or inserted in the base sequence shown in SEQ ID NO: 8. , 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 an oligonucleotide in which one to several bases are deleted from the base sequence shown in SEQ ID NO: 9, LB primer containing an oligonucleotide consisting of a substituted, inserted, or added base sequence

[PCR primer/probe set 1 for determining D1 type of P. acnes]
(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 have been deleted, substituted, inserted, or added to the base sequence shown in SEQ ID NO: 22. Forward primer containing (a8) Oligonucleotide consisting of the base sequence shown in SEQ ID NO: 23, or consisting of a base sequence in which one to several bases are deleted, substituted, inserted, or added to the base sequence shown in SEQ ID NO: 23 Reverse primer containing oligonucleotide (a9) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 24, or a base in which one to several bases have been deleted, substituted, inserted, or added to the base sequence shown in SEQ ID NO: 24. A probe containing an oligonucleotide consisting of a sequence

[PCR primer/probe set 2 for identifying D1 type of P. acnes]
(a10) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 25, or an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 25 in which one to several bases have been deleted, substituted, inserted, or added. Forward primer containing (a11) Oligonucleotide consisting of the base sequence shown in SEQ ID NO: 26, or consisting of a base sequence in which one to several bases are deleted, substituted, inserted, or added to the base sequence shown in SEQ ID NO: 26 Reverse primer containing oligonucleotide (a12) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 27, or a base in which one to several bases have been deleted, substituted, inserted, or added to the base sequence shown in SEQ ID NO: 27. A probe containing an oligonucleotide consisting of a sequence

[LAMP primer set for determining D2 type of P. acnes]
(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 have been deleted, substituted, inserted, or added to the base sequence shown in SEQ ID NO: 10. F3 primer containing (b2) Oligonucleotide consisting of the base sequence shown in SEQ ID NO: 11, or consisting of a base sequence in which one to several bases are deleted, substituted, inserted, or added to the base sequence shown in SEQ ID NO: 11 B3 primer containing oligonucleotide (b3) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 12, or a base in which one to several bases have been deleted, substituted, inserted, or added to the base sequence shown in SEQ ID NO: 12. FIP primer containing an oligonucleotide consisting of the 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 (b5) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 14, or one to several bases deleted, substituted, or inserted in the base sequence shown in SEQ ID NO: 14. , 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 an oligonucleotide in which one to several bases are deleted from the base sequence shown in SEQ ID NO: 15, LB primer containing an oligonucleotide consisting of a substituted, inserted, or added base sequence

[PCR primer/probe set 1 for determining D2 type of P. acnes]
(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 have been deleted, substituted, inserted, or added to the base sequence shown in SEQ ID NO: 28. Forward primer containing (b8) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 29, or a base sequence in which one to several bases have been deleted, substituted, inserted, or added to 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 have been deleted, substituted, inserted, or added to the base sequence shown in SEQ ID NO: 30. A probe containing an oligonucleotide consisting of a sequence

[PCR primer/probe set 2 for determining D2 type of P. acnes]
(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 have been deleted, substituted, inserted, or added to the base sequence shown in SEQ ID NO: 31. Forward primer containing (b11) 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 to the base sequence shown in SEQ ID NO: 32. Reverse primer containing oligonucleotide (b12) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 33, or a base in which one to several bases have been deleted, substituted, inserted, or added to the base sequence shown in SEQ ID NO: 33. A probe containing an oligonucleotide consisting of a sequence

[LAMP primer set for determining D3 type of P. acnes]
(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 have been deleted, substituted, inserted, or added to the base sequence shown in SEQ ID NO: 16. Containing F3 primer (c2) Oligonucleotide consisting of the base sequence shown in SEQ ID NO: 17, or consisting of a base sequence in which one to several bases are deleted, substituted, inserted, or added to the base sequence shown in SEQ ID NO: 17. B3 primer containing oligonucleotide (c3) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 18, or a base in which one to several bases have been deleted, substituted, inserted, or added to the base sequence shown in SEQ ID NO: 18. FIP primer containing an oligonucleotide consisting of the 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 (c5) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 20, or one to several bases deleted, substituted, or inserted in the base sequence shown in SEQ ID NO: 20. , 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 an oligonucleotide in which one to several bases are deleted from the base sequence shown in SEQ ID NO: 21, LB primer containing an oligonucleotide consisting of a substituted, inserted, or added base sequence

[PCR primer/probe set 1 for identifying D3 type of P. acnes]
(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 have been deleted, substituted, inserted, or added to the base sequence shown in SEQ ID NO: 34. Forward primer containing (c8) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 35, or a base sequence in which one to several bases have been deleted, substituted, inserted, or added to the base sequence shown in SEQ ID NO: 35. Reverse primer containing oligonucleotide (c9) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 36, or a base in which one to several bases have been deleted, substituted, inserted, or added to the base sequence shown in SEQ ID NO: 36. A probe containing an oligonucleotide consisting of a sequence

[PCR primer/probe set 2 for determining D3 type of P. acnes]
(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 have been deleted, substituted, inserted, or added to the base sequence shown in SEQ ID NO: 37. Forward primer containing (c11) 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 to the base sequence shown in SEQ ID NO: 38. Reverse primer containing oligonucleotide (c12) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 39, or a base in which one to several bases have been deleted, substituted, inserted, or added to the base sequence shown in SEQ ID NO: 39. A probe containing an oligonucleotide consisting of a sequence

In addition, in the oligonucleotide set for determining the D1 type of P. acnes, the oligonucleotide set for determining the D2 type of P. acnes, and the oligonucleotide set for discriminating the D3 type of P. acnes, "1 to several bases are missing". The range of ``1 to several'' in ``nucleotide sequences deleted, substituted, inserted, or added'' refers to sequences that hybridize to partial regions of genes specific to each of the above DNA types and that correspond to SEQ ID NOs: 4 to 39, respectively. For example, 1 to 5, preferably 1 to 4, more preferably 1 to 3, but not limited to, as long as they have substantially the same function as the oligonucleotide consisting of the base sequence shown as a primer or probe. Refers to an individual.

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

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

The LAMP primer set of the present invention makes it possible to detect amplification products by nucleic acid chromatography, so that it is possible to detect amplification products by nucleic acid chromatography. , a detection probe immobilized on a solid phase carrier described below and a hybridizable tag sequence are combined. In addition, a substance that can bind to the labeling substance through a specific binding pair (labeling substance binding substance) is attached to the primer that does not bind the tag sequence to the primer to visualize the hybridization between the tag sequence and the detection probe. let The specific binding pair may be any combination that recognizes and captures each other, and includes, for example, biotin-avidin, digoxigenin-anti-digoxigenin, FITC-anti-FITC antibody, and the like. In one embodiment of the invention, the labeling substance is included in a developing solution for carrying out a nucleic acid chromatography method.

The tag sequence is associated with the D1 type, D2 type, and D3 type of P. acnes, has a different sequence for each DNA type, and uses a base sequence that is 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 to 50 bases, more preferably 20 to 30 bases.

Labeling substances include fluorescent substances (fluorescence isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRIC), cyanine dyes (Cy3, Cy5, etc.), acetylaminofluorene (AFF), etc.), chemiluminescent substances (luminol, luminol Derivatives, luciferin, lucigenin, etc.), enzymes (alkaline phosphatase, peroxidase, β-galactosidase, β-glucosidase, etc.), etc., but from the viewpoint of quick and easy detection, display luminescence or color development that can be detected visually. It is preferable to use a luminescent substance or a color-forming substance. Alternatively, particles such as latex particles, metal particles, and inorganic particles labeled with such labeling substances may be used.

Further, in the PCR primer/probe set 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 P. acnes whose DNA type is to be determined. Labeled probes include TaqMan probes, Molecular Beacon probes, and cycling probes, with TaqMan probes being preferred. TaqMan probes are probes whose 5' end is modified with a fluorescent substance (reporter fluorescent dye) and whose 3' end is modified with a quencher (quencher fluorescent dye). A 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 substance (quencher fluorescent dye). Examples of reporter fluorescent dyes for the above TaqMan probes include 6-FAM (6-carboxyfluorescein), TET (6-carboxy-4, 7, 2',7'-tetrachlorofluorescein), and HEX (6-carboxy-2'). Examples of quencher fluorescent dyes include TAMRA (6-carboxytetramethylrhodamine), ROX (6-carboxy-X Rhodamine-based fluorescent dyes such as 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. These fluorescent dyes are known and included in commercially available real-time PCR kits, so they can be used.

2. Kit for determining the DNA type of Cutibacterium acnes 1. The oligonucleotide set can also be made into a kit. The kit of the present invention may contain at least one of the above-mentioned oligonucleotide sets, but it is preferable that the kit contains all the sets, and the kit may be selected depending on the type and number of DNA types of P. acnes to be determined, and the purpose. An appropriate set may be selected from among these oligonucleotide sets and used in combination. For example, by using all oligonucleotide sets, the DNA type of P. acnes can be simultaneously determined in one operation.

The kit of the present invention also includes reagents necessary for carrying out the nucleic acid amplification method, such as DNA extraction reagents, strand displacement DNA polymerases (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, four types of dNTPs (dATP, dCTP, dGTP and dTTP), labeling substances, sterile water, salts as cofactors (magnesium salts or manganese salts, etc.), protective agents to stabilize enzymes and templates, preparations (standard bacterial strains, etc.), instructions, etc. Good too.

The kit of the present invention also includes 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 on which a detection probe is immobilized and a developing solution.

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, depending on the tag sequence. . Within this range, hybridization efficiency can be ensured while ensuring the specificity of the tag sequence.

In this specification, the above-mentioned solid phase carrier on which a 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. Alternatively, it may be a porous material such as cellulose, nitrocellulose, or nylon. This type of porous body is particularly suitable for hybridizing a detection probe immobilized on a strip with an amplification product by affinity chromatography. Typical strips include printed array strips (PAS) used in the STH (Single-stranded Tag Hybridization) method, and these commercially available products can be used in the present invention. This strip is a test paper approximately 2 mm wide x 10 cm long, with nucleic acids printed as probes on the nitrocellulose membrane on the surface. The amplified nucleic acid can be detected by using a set of primers having a tag sequence that hybridizes complementary to this probe. On the strip, multiple detection probes corresponding to each DNA type are placed at appropriate positions, and position markers are placed so that each probe and the tag sequence bound to the probe can be easily identified. is preferred. Furthermore, a flow control line can be provided to visually confirm that the developing solution has been sufficiently applied.

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

It is not necessary to use all of the above oligonucleotide sets, depending on the type and number of DNA types of P. acnes to be discriminated, and the purpose of discrimination, and one or more sets of the above oligonucleotide sets may be used in appropriate combinations.

The specimen is not particularly limited as long as it is a biological sample or environmental sample that may contain P. acnes to be determined by DNA type, but a skin sample is preferred. The skin sample is typically collected from a human, but may also be a skin sample from an animal other than a human. The skin sample collected from a human is preferably a skin sample containing the stratum corneum of the human epidermis. The method for collecting skin samples is not particularly limited as long as it is a non-invasive method, but typically a method is used in which adhesive tape or adhesive is attached to the skin and then removed (tape stripping method). It will be done. The material (adhesive component) used for the adhesive tape and adhesive is not particularly limited.

For extraction of DNA from the specimen, any method known to those skilled in the art as a nucleic acid extraction method may be used, such as phenol extraction method, cetyltrimethylammonium bromide (CTAB) method, alkaline SDS method, etc. It will be done. Further, these methods may be modified as appropriate, and various DNA extraction kits sold by reagent manufacturers may be used. Depending on the type of sample, filtration with a membrane filter or homogenization may be performed. Alternatively, various DNA extraction kits such as DNeasy Plant mini Kit (manufactured by Qiagen Co., Ltd.) sold by reagent manufacturers may be used. The DNA extracted by these methods is preferably maintained in a state suitable for use as a PCR template, for example, preferably dissolved in an appropriate buffer and stored at low temperature. Further, after DNA extraction, purification treatments such as chloroform/isoamyl alcohol treatment, isopropanol precipitation, protein removal with phenol/chloroform, 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 in which a target region is amplified by repeating temperature changes, and the term PCR method also includes real-time PCR method.

Isothermal amplification methods are not particularly limited, and include loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), and chimeric primer isothermal nucleic acid amplification (Isothermal and Chimeric primer-initiated Amplification of Various isothermal amplification methods can be used, such as Nucleic acids (ICAN) method, Helicase-Dependent Amplification (HDA) method, and Nicking Enzyme Amplification Reaction (NEAR) method, but LAMP method is preferable.

The LAMP method uses 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 template nucleic acid strand containing a target nucleic acid sequence is mixed with a template nucleic acid strand containing a target nucleic acid sequence, a total of 6 types of primers (4 types of primers or 2 types of loop primers added to these, strand displacement DNA synthase, nucleic acid monomer, etc.), and isothermally This method performs a nucleic acid amplification reaction by incubating at a temperature of In the LAMP method, the 3' end of the nucleotide serving as a template is annealed to serve as the starting point for complementary strand synthesis, and by combining a primer that anneals to the loop formed at this time, complementary strand synthesis reaction is carried out at an isothermal temperature. This is a nucleic acid amplification method that has made it possible.

The amplification reaction using the LAMP method can be performed, for example, as follows. After mixing reagents (for example, the LAMP primer set of the present invention, Bst DNA polymerase, reaction buffer, dNTPs, and distilled water) and dispensing them into reaction tubes, a solution containing DNA extracted from a specimen is added. It is then incubated at a temperature suitable for enzyme activity, for example 60-70°C, preferably 65°C. The incubation time can be set to a time sufficient to complete DNA amplification by the LAMP method, for example, in the range of 30 to 120 minutes, preferably 60 to 90 minutes. Primer concentrations can be 0.4-0.025 μM for F3 and B3, 3.2-0.2 μM for FIP and BIP, and 1.6-0.1 μM for LF and LB.

There are no particular restrictions on the PCR method other than using the above-mentioned PCR primers, and it may be carried out according to conventional methods. Specifically, a cycle is repeated that includes denaturation of template DNA, annealing of the template to the primer, and extension reaction of the primer using a thermostable enzyme (a DNA polymerase such as Taq polymerase or Tth DNA polymerase from Thermus thermophilus). By doing so, a partial region of the gene specific to P. acnes to be identified is amplified. The composition of the PCR solution (template DNA amount, buffer type, primer concentration, DNA polymerase type and concentration, dNTP concentration, magnesium chloride concentration, etc.) and PCR reaction conditions (temperature cycle, number of cycles, etc.) can be 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 template DNA, 10× PCR reaction buffer, 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, For the diluted solution to a volume of 10 to 100 μl, 94 to 96°C 5 minutes x 1 cycle, (94 to 96°C 30 seconds, 52 to 58°C 30 seconds, 70 to 74°C 1 minute) x 30 cycles. , 70-74°C for 5 minutes x 1 cycle. This is just an example, and the composition of the PCR solution, reaction temperature and time can be appropriately set depending on the length and base composition of the oligonucleotide sequence serving as the primer. A series of these PCR operations can be performed using a commercially available PCR kit or PCR device according to the operating instructions.

Real-time PCR methods include the intercalator method, in which an intercalator such as SYBR Green I, which is a compound that emits fluorescence by binding to double-stranded DNA, is added to the PCR reaction system along with the primer pair, or the 5' end is added to the PCR reaction system using a compound called a reporter. There is a TaqMan method in which a fluorescent probe labeled with a quencher called a quencher at the 3' end (TaqMan ^™ probe) is added to the PCR reaction system, and both can be used in the present invention, but the TaqMan method is preferred. In the TaqMan method, the TaqMan ^TM probe specifically hybridizes to template DNA under the conditions used for the polymerase extension reaction in the PCR amplification reaction, and is degraded as the DNA strand is elongated, that is, the template DNA is amplified. By releasing the fluorescent substance, the amount of fluorescence in the PCR solution increases. This increase in the amount of fluorescence becomes an indicator of template DNA amplification, and the state of amplification in PCR can be easily detected in real time. Real-time PCR is carried out using commercially available real-time PCR kits and real-time PCR devices according to the operating instructions attached to them, based on conventional methods known to those skilled in the art, except for the use of the above oligonucleotide set. That's fine. As a 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 detect the amplification product obtained by the nucleic acid amplification method.

If the PCR method is used, the amplification product can be detected using conventional electrophoresis such as agarose gel electrophoresis or capillary electrophoresis, DNA hybridization method, intercalation method, quencher-mediated fluorescence detection method, etc. can. For example, in agarose gel electrophoresis, the DNA is stained with ethidium bromide, SYBR Green, etc., and the amplified product is detected as a single band, and the DNA type is determined based on the size of the band. Alternatively, the amplification product can be detected by performing the PCR using primers that have been labeled in advance with a labeling substance. As the labeling substance, fluorescent substances, radioisotopes, chemiluminescent substances, biotin, etc., which are well known in the technical field, can be used. In addition, in DNA hybridization, an amplified product is bound to an immobilized carrier such as a microarray, and the amplified product is confirmed by fluorescence or enzyme reaction. The immobilization 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 P. acnes to be determined is immobilized. As the support, for example, a nylon membrane, a nitrocellulose membrane, glass, a silicon chip, etc. can be used.

In the case of the isothermal amplification method (LAMP method), the amplification product can be detected using a method such as measuring the turbidity of the reaction solution, visually observing fluorescence, or using agarose gel electrophoresis. In the LAMP method, a large amount of the substrate is consumed during DNA synthesis, and the byproduct pyrophosphate reacts with the coexisting magnesium, resulting in white turbidity and white precipitate of magnesium pyrophosphate. Therefore, this white turbidity/white precipitate is measured by measuring the absorbance change at 650 nm using a measuring device that can optically observe the increase in turbidity of the reaction solution over time after the amplification reaction is completed or during the amplification reaction, such as a spectrophotometer. It is possible to confirm this by In addition, in the fluorescence visual method, after the amplification reaction is performed in the presence of a fluorescent intercalator that binds to double-stranded nucleic acids such as ethidium bromide, the amplified product can be confirmed by fluorescence in the presence of an ultraviolet lamp. It is.

As one embodiment of the present invention, detection of amplification products is preferably performed by nucleic acid chromatography. Nucleic acid chromatography is a method for detecting a target nucleic acid using chromatography, and this method involves hybridizing a chromatography solution (developing solution) containing a target nucleic acid to a specific region of the target nucleic acid. The method includes a step of forming a hybridization product between the target nucleic acid and the detection probe by developing it inside or on the surface of a solid phase carrier on which a detection probe is immobilized, and a step of detecting the hybridization product. Detection of the hybridization product is performed using a signal from a 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 solution is a liquid that diffuses and moves through the solid phase carrier of the chromatography body by capillary phenomenon, and is a medium for moving the multiplication product on the solid phase carrier. The developing solution may be one that allows the amplification product contained in the amplification reaction solution to be easily developed in the solid phase carrier, for example, water, an organic solvent that is compatible with water, or one or more types of developing solution with water. Examples include a mixture of organic solvents. Examples of the organic solvent include lower alcohols, DMSO, DMF, esters such as methyl acetate and ethyl acetate, and acetone. The developing solution contains buffer components for adjusting pH, such as acetate buffer, citrate buffer, and phosphate buffer. In addition, the amplification reaction solution can be used as it is as a developing solution, or the composition and concentration can be adjusted by adding additional components or solvents such as surfactants or appropriate salts to the amplification reaction solution. It may also be used as a developing solution.

The time for applying the developing solution to the solid support is not particularly limited, but is, for example, 5 minutes or more, preferably 10 minutes or more. The hybridization product is detected by confirming the coloring and position of the region on which the detection probe is immobilized. In order to detect a signal due to a labeling substance, an appropriate selection is made depending on the type of labeling substance.

EXAMPLES Hereinafter, the present invention will be explained in more detail using Examples, but the present invention is not limited to these Examples.

(Example 1) Construction of a LAMP primer set for determining the DNA type of C. acnes (1) Search for target genes From the public database NCBI (National Center for Biotechnology Information), C. acnes, its congener related bacteria, We obtained the whole genome sequences of 50 bacterial strains (Table 1-1) including other skin resident bacteria. Among these strains, the DNA types have already been determined, including D3 type Cutibacterium acnes JCM 6425 (ATCC 6919), D2 type Cutibacterium acnes JCM 6473 (ATCC 11828), and D3 type Cutibacterium acnes JCM. 18916, D1 type Cutibacterium acnes JCM 18918, and D2 type JCM 18920 (Table 1-2).

Comparative analysis of the obtained whole genome sequences of the above 50 bacterial strains using the PanOCT analysis tool (Nucleic Acids Res. 2012 Dec;40(22):e172) and a search for specific genes by DNA type revealed that D1 type The JCM18918_744 gene was selected as a gene specific to , the TIIST44_06360 gene was selected as a gene specific to the D2 type, and the FB33_0108 gene was selected as a gene specific to the D3 type (Table 2).

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

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

(Example 2) Performance evaluation of the LAMP primer set for determining the DNA type of P. acnes The performance of the LAMP primer set constructed in Example 1 for determining the DNA type of P. acnes was evaluated by the LAMP method and nucleic acid chromatography.
(1) Test Bacterial Strains The five strains of P. acnes obtained from the RIKEN BioResource Center Microbial Materials Development Office (JCM) shown in Table 6 were used as the bacterial strains obtained from the distribution organization. The culture method followed the method recommended by the distribution organization.

As the bacterial strains isolated from humans, 40 strains shown in Table 7 were used. The bacterial strain was cultured by the method described in the literature (J Dermatol, vol.40, PP.786-792, 2002.), and then cultured according to the method described in the literature (J Clin Microbiol, vol.40, PP.198-204, 2002.) It was identified as Cutibacterium acnes by the method described in . Furthermore, the DNA types of these isolated bacterial 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) Extraction of DNA 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 In order to detect amplification products by STH chromatography PAS method, each LAMP primer set for determining the DNA type of P. acnes constructed in Example 1 was The tag sequence was attached to the 5' end of the LF primer, and the biotin was attached to the 5' end of the LB primer using a method. Different tag sequences were 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. 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 to the LAMP reaction tube strip and mixed. A heat block was used for the LAMP isothermal reaction, and the reaction was carried out at 65°C for 30 minutes. After the LAMP product after the reaction was diluted 1/10 with sterilized water, 1 μL thereof was added to 20 μL of chromatography developer (1 μL of chromatography dye and 19 μL of chromatography buffer) and mixed. Chromatography paper was immersed in the mixture and chromatography was developed at room temperature for 20 minutes.

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

As shown in Tables 6 and 7, the LAMP primer set (JCM18918_744_LAMP) designed from the base sequence of the JCM18918_744 gene (SEQ ID NO: 1) is the D1 type of P. acnes, and the LAMP primer set (JCM18918_744_LAMP) designed from the base sequence of the TIIST44_06360 gene (SEQ ID NO: 2) It was confirmed that the primer set (TIIST44_06360_LAMP) can identify the D2 type of P. acnes, and the LAMP primer set (FB33_0108_LAMP) designed from the nucleotide sequence of the FB33_0108 gene (SEQ ID NO: 3) can specifically and highly accurately identify the D3 type of P. acnes. .

(Example 3) Construction and evaluation of simultaneous discrimination system for DNA type of P. acnes
DNA was extracted from Cutibacterium acnes JCM 6425, Cutibacterium acnes JCM 6473, and Cutibacterium acnes JCM 18918, respectively. Sample 1 containing only Cutibacterium acnes JCM 6425 DNA, Sample 2 containing only Cutibacterium acnes JCM 6473 DNA, Sample 3 containing only Cutibacterium acnes JCM 18918 DNA, Cutibacterium acnes JCM 6425 DNA, Cutibacterium acnes JCM 6473 DNA, Sample 4 containing all of the DNA of Cutibacterium acnes JCM 189186 was prepared.

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

The band patterns of nucleic acid chromatography for each sample are shown in Figure 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: DNA of Cutibacterium acnes JCM 18918, primer used: JCM18918_744_LAMP), lane 4 (sample 1: DNA of Cutibacterium acnes JCM 6425, primer used: TIIST44_06360_LAMP), lane 5 (sample 2: Cutibacterium acnes JCM 6473 DNA, 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, primers used: JCM18918_744_LAMP, TIIST44_06360_LAMP, mixed primers of FB33_0108_LAMP), lane 11 (sample: distilled water, primers used: JCM18918_744_LAMP, TIIST44_06360_LAMP, FB3) 3_0108_LAMP mixed primer)].

As shown in Figure 1, when the LAMP reaction was performed using JCM18918_744_LAMP (lane 3), sample 1 containing DNA of D1 type Cutibacterium acnes JCM 18918 was different from sample 1 containing DNA of D2 type Cutibacterium acnes JCM 6473. Sample 2 contains the DNA of D3 type Cutibacterium acnes JCM 6425 when the LAMP reaction was performed using TIIST44_06360_LAMP (Lane 5), and sample 3 containing the DNA of D3 type Cutibacterium acnes JCM 6425 performed the LAMP reaction using FB33_0108_LAMP (Lane 7). A specific band corresponding to the amplified product was detected. Furthermore, when LAMP reaction was performed in multiplex, bands corresponding to three DNA types were simultaneously detected (lane 10).

(Example 4) Construction and evaluation of a quantitative PCR primer/probe set for determining the DNA type of P. acnes (1) Construction of a quantitative PCR (qPCR) primer/probe set for determining the DNA type of P. acnes Specified in Example 1 We designed primers that can amplify partial nucleotide sequences of the JCM18918_744 gene, TIIST44_06360_ gene, and FB33_0108 gene of Cutibacterium acnes by PCR. Primer3 (https://primer3.org/) was used to design PCR primers.

Quantitative PCR primer and probe sets designed for JCM18918_744 gene, TIIST44_06360_ gene, and FB33_0108 gene are shown in Tables 8 to 10 below for DNA type determination of P. acnes. In the names, "F1", "F2", "R1", and "R2" indicate primers, and "P1" and "P2" indicate probes. The probe for determining D1 type (JCM18918_744) is modified with FAM at the 5' end and BHQ at the 3' end, and the probe for determining D2 type (TIIST44_06360) is modified with HEX at the 5' end and BHQ at the 3' end. However, the probe for determining the 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 determining the DNA type of P. acnes DNA of each test strain was extracted in the same manner as in Example 1 using the DNA extraction reagent "GenCheck" (Fasmac Co., Ltd.). The DNA type discrimination performance of the qPCR primer/probe set constructed in (1) was confirmed by quantitative PCR.

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

(3) Results The results are shown in Tables 11-1 to 11-3 (+: amplification product (fluorescence signal) detected, -: amplification product (fluorescence 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 a D1 type strain, and the qPCR primer/probe set designed from the base sequence of TIIST44_06360. It was confirmed that (TIIST44_06360_PCR_1, TIIST44_06360_PCR_2) can identify D2 type bacterial 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 specifically and highly accurately identify D3 type bacterial strains.

INDUSTRIAL APPLICABILITY The present invention can be used in the field of manufacturing reagents for testing P. acnes. By using the oligonucleotide set and method of the present invention, the DNA type of P. acnes, which causes acne and the like, can be accurately and quickly determined. Therefore, based on this result, it is possible to provide cosmetics and pharmaceuticals to prevent or improve acne in sample donors depending on the DNA type, to determine the therapeutic effect of the disease, and to provide advice on how to prevent or improve the disease. It will be possible to provide counseling and advice regarding care methods for patients.

Claims (14)

A kit for determining the DNA type of Cutibacterium acnes, which includes all of the following oligonucleotide sets (A) to (C).
(A) D1 type of Cutibacterium acnes (Primer: 5'-AACGCGCAAC-3' An oligonucleotide set for discriminating P. acnes DNA type characterized by the electrophoretic band pattern of the RAPD-PCR product using The partial region consists of two or more regions selected from the region from positions 76 to 93, the region from positions 110 to 227, the region from positions 241 to 331, and the region from positions 317 to 349 in the base sequence of SEQ ID NO: 1. The above oligonucleotide set , which is a region
(B) D2 type of Cutibacterium acnes (Primer: 5'-AACGCGCAAC-3' This is an oligonucleotide set for determining the DNA type of P. acnes characterized by the electrophoretic band pattern of the RAPD-PCR product expressed at 1,000,350 bp, the partial region being SEQ ID NO: 2. The above oligonucleotide set is two or more regions selected from the region from positions 53 to 72, the region from positions 75 to 150, the region from positions 153 to 241, and the region from positions 244 to 261 in the base sequence of
(C) D3 type of Cutibacterium acnes (Primer: 5'-AACGCGCAAC-3' An oligonucleotide set for discriminating the DNA type of P. acnes characterized by the electrophoretic band pattern of the RAPD-PCR product expressed at 3,000, 2,800, 1,300, 800, and 300 bp, The region is two or more regions selected from the region from positions 219 to 236, the region from positions 298 to 412, the region from positions 450 to 521, and the region from positions 539 to 557 in the base sequence of SEQ ID NO: 3. Yes, the above oligonucleotide set The kit according to claim 1, wherein the oligonucleotide set for determining the D1 type of P. acnes is a set of LAMP primers consisting of the following primers (a1) to (a6).
(a1) F3 primer containing an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 4 (a2) B3 primer containing an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 5 (a3) Consisting of the base sequence shown in SEQ ID NO: 6 FIP primer containing an oligonucleotide (a4) BIP primer containing an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 7 (a5) LF primer containing an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 8 (a6) SEQ ID NO: LB primer containing an oligonucleotide consisting of the base sequence shown in 9. The oligonucleotide set for determining D1 type of P. acnes is composed of a primer pair consisting of the following oligonucleotide (a7) and oligonucleotide (a8), and a probe consisting of the following oligonucleotide (a9). A set of PCR primers and probes, or a pair of primers consisting of the following oligonucleotides (a10) and (a11), and a probe consisting of the oligonucleotides (a12) below. The kit according to claim 1, which is a set.
(a7) Oligonucleotide consisting of the base sequence shown in SEQ ID NO: 22
(a8) Oligonucleotide consisting of the base sequence shown in SEQ ID NO: 23
(a9) Oligonucleotide consisting of the base sequence shown in SEQ ID NO: 24
(a10) Oligonucleotide consisting of the base sequence shown in SEQ ID NO: 25
(a11) Oligonucleotide consisting of the base sequence shown in SEQ ID NO: 26
(a12) Oligonucleotide consisting of the base sequence shown in SEQ ID NO: 27 The kit according to claim 1 , wherein the oligonucleotide set for determining the D2 type of P. acnes is a LAMP primer set consisting of the following primers (b1) to (b6).
(b1) F3 primer containing an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 10 (b2) B3 primer containing an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 11 (b3) Consisting of the base sequence shown in SEQ ID NO: 12 FIP primer containing an oligonucleotide (b4) BIP primer containing an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 13 (b5) LF primer containing an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 14 (b6) SEQ ID NO: LB primer containing an oligonucleotide consisting of the base sequence shown in 15 The oligonucleotide set for determining the D2 type of P. acnes is composed of a primer pair consisting of the following oligonucleotides (b7) and (b8), and a probe consisting of the following oligonucleotides (b9). A set of PCR primers and probes, or a pair of primers consisting of the following oligonucleotides (b10) and (b11), and a probe consisting of the oligonucleotides (b12) below. The kit according to claim 1 , which is a set.
(b7) Oligonucleotide consisting of the base sequence shown in SEQ ID NO: 28
(b8) Oligonucleotide consisting of the base sequence shown in SEQ ID NO: 29
(b9) Oligonucleotide consisting of the base sequence shown in SEQ ID NO: 30
(b10) Oligonucleotide consisting of the base sequence shown in SEQ ID NO: 31
(b11) Oligonucleotide consisting of the base sequence shown in SEQ ID NO: 32
(b12) Oligonucleotide consisting of the base sequence shown in SEQ ID NO: 33 The kit according to claim 1 , wherein the oligonucleotide set for determining the D3 type of P. acnes is a LAMP primer set consisting of the following primers (c1) to (c6).
(c1) F3 primer containing an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 16 (c2) B3 primer containing an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 17 (c3) Consisting of the base sequence shown in SEQ ID NO: 18 FIP primer containing an oligonucleotide (c4) BIP primer containing an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 19 (c5) LF primer containing an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 20 (c6) SEQ ID NO: LB primer containing an oligonucleotide consisting of the base sequence shown in 21 The oligonucleotide set for determining the D3 type of P. acnes is composed of a primer pair consisting of the following oligonucleotide (c7) and oligonucleotide (c8), and a probe consisting of the following oligonucleotide (c9). A set of PCR primers and probes, or a pair of primers consisting of the following oligonucleotides (c10) and (c11), and a probe consisting of the oligonucleotides (c12) below. The kit according to claim 1 , which is a set.
(c7) Oligonucleotide consisting of the base sequence shown in SEQ ID NO: 34
(c8) Oligonucleotide consisting of the base sequence shown in SEQ ID NO: 35
(c9) Oligonucleotide consisting of the base sequence shown in SEQ ID NO: 36
(c10) Oligonucleotide consisting of the base sequence shown in SEQ ID NO: 37
(c11) Oligonucleotide consisting of the base sequence shown in SEQ ID NO: 38
(c12) Oligonucleotide consisting of the base sequence shown in SEQ ID NO: 39 The kit according to any one of claims 2, 4 , and 6, wherein a different tag sequence for each DNA type is bound to the FIP primer, the BIP primer, the LF primer, or the LB primer. The kit according to claim 8 , further comprising a solid phase carrier on which a detection probe that hybridizes to the tag sequence is immobilized. The kit according to claim 9 , wherein the solid phase carrier is a strip for nucleic acid chromatography. A method for determining the DNA type of P. acnes, comprising the steps of performing an amplification reaction by a nucleic acid amplification method using the kit according to claim 1 using DNA extracted from a specimen as a template, and detecting the amplification product. A method for simultaneously determining the DNA type of P. acnes, comprising the steps of performing an amplification reaction by a nucleic acid amplification method using the kit according to claim 1 using DNA extracted from a specimen as a template, and detecting the amplification product. The discrimination method according to claim 11 or 12 , wherein the nucleic acid amplification method is an isothermal amplification method or a PCR method. The discrimination method according to any one of claims 11 to 13 , wherein the specimen is a skin sample.