JP6872222B2 - How to determine the user of an object - Google Patents

Description

The present invention relates to a method for determining a user (including an owner) of an object based on a composition ratio of a strain genotype of an indigenous skin flora.

In a criminal investigation, the user is identified from the remains, etc. by DNA test, chemical test, handwriting test, fingerprint test, etc., and the ability of police dogs to sort out odors. Of these, fingerprint identification, which is a commonly performed method, focuses on the characteristic that fingerprints are all different for each person and finger and are invariant for the rest of their lives. Nevertheless, the rate of erroneous recognition of the same fingerprint (erroneous acceptance rate) is about 1 / 100,000 in actual measurement. In addition, fingerprint authentication is used as a method for personal identification in many fields such as ATMs.

By the way, as many as 1 trillion bacteria (indigenous skin bacteria) inhabit human skin. In recent years, it has become clear that the composition of indigenous skin flora changes little over time and varies greatly among individuals (Non-Patent Document 1). Therefore, it is considered that an individual can be identified by the composition of the indigenous skin flora as in the above fingerprint test.

Previous studies have revealed that the bacterial flora of a computer keyboard and the bacterial flora of the user's finger are similar, suggesting that the indigenous skin flora may identify the user. (Non-Patent Document 1). In the previous study, the authors wiped the computer keyboard and its user's fingertips with a swab, amplified the gene encoding 16S rRNA in the collected bacterial sample using a universal primer, and based on the sequence, the bacterial flora. By identifying the composition of the bacterial flora (composition ratio of the types of bacteria contained in the bacterial flora), it was revealed that the composition of the bacterial flora of the keyboard and its user's fingertips is similar. Such teaching suggests the possibility of determining the user of the keyboard based on the similarity in composition of the bacterial flora, but since the amount of bacterial DNA that can be collected from the skin is very small, it is diverse. In the daily environment where various bacteria live, the influence of contamination becomes large, and highly accurate analysis is impossible.

Fierer, N., Lauber, C.L., et al. Forensic identification using skin bacterial communities. Proc.Natl.Acad.Sci.U.S.A., 107, 6477-6481 (2010)

An object of the present invention is to provide a method for determining a user of an object by analyzing the composition ratio of a strain genotype of a skin flora on the surface of the object and the skin of a candidate.

As a result of intensive research to solve the above problems, the present inventors have found that the composition ratio of the acne strain genotype collected from the surface of the object and the acne strain genotype collected from the skin of the candidate. The composition ratio is determined, the composition ratio obtained from the object and the candidate is compared by machine learning, and the similarity of the composition ratio acquired from the object and the candidate is calculated. We have found that it is possible to determine whether or not the candidate is a user of the object, and have completed the present invention.

That is, the present invention is as follows.
(1) A method for determining a user of an object, which comprises the following steps (A) to (D).
(A) A step of amplifying the genes of one or more types of bacteria collected from the surface of the object and the genes of the one or more types of bacteria collected from the skin of a candidate;
(B) The nucleotide sequence of the amplification product obtained in the step (A) was determined, and based on the polymorphism of the nucleotide sequence, the strain gene of the one or more kinds of bacteria collected from the surface of the object. A step of determining the composition ratio M of the type and the composition ratio N of the strain genotype of the one or more kinds of bacteria collected from the skin of the candidate;
(C) A step of comparing the constituent ratios M and N to determine the degree of similarity between the constituent ratios M and N;
(D) A step of determining whether the candidate is a user of the object based on the similarity;
(2) One or more kinds of bacteria are bacteria selected from Propionibacterium spp., Staphylococcus spp., Ralstonia spp., Corinebacterium spp. And Streptococcus spp. (1). The method described in.
(3) The method according to (1) or (2) above, wherein one or more types of bacteria are Propionibacterium spp.
(4) The method according to any one of (1) to (3) above, wherein one or two or more kinds of bacteria are P. acnes.
(5) The gene of one or more kinds of bacteria is the genotype of 16S rRNA of P. acnes, and the genotype of the strain of one or more kinds of bacteria is the genotype of 16S rRNA of P. acnes. The method according to any one of (4).
(6) The genotype of 16S rRNA of Acne bacteria is the genotype of the region corresponding to the region of positions 950 to 1335 in the gene of 16S rRNA of KPA171202, which is the reference strain of Acne bacteria, according to the above (5). Method.
(7) The genotype of 16S rRNA of Acne bacteria is 976th, 981st, 996th, 1012th, 1034th, 1041st, 1044th, 1045th, of the 16S rRNA gene of KPA171202, which is the reference strain of Acne. 1047th, 1049th, 1050th, 1052nd, 1054th, 1056th, 1057th, 1059th, 1060th, 1064th, 1065th, 1075th, 1076th, 1077th, 1079th, 1081st, 1089th 1090th, 1095th, 1097th, 1099th, 1100th, 1104th, 1107th, 1108th, 1110th, 1111th, 1112th, 1113th, 1121st, 1122th, 1123th, 1125th, 1127th 1130th, 1142th, 1144th, 1145th, 1147th, 1151st, 1154th, 1164th, 1169th, 1175th, 1176th, 1177th, 1186th, 1187th, 1189th, 1199th, From the parts corresponding to 1206, 1210, 1213, 1215, 1219, 1222, 1226, 1227, 1229, 1232, 1236, 1242, 1243, 1244 and 1248, respectively. The method according to (5) or (6) above, which is a genotype based on a gene polymorphism of one or more sites selected from the group.
(8) The step according to any one of (5) to (7) above, wherein the step of amplifying the 16S rRNA gene of Acne bacterium is carried out by using the nested PCR method including the following steps (a) and (b). the method of.
(A) A step of performing the first-stage PCR using the oligonucleotides of SEQ ID NO: 1 and SEQ ID NO: 2 as primers;
(B) A step of performing a second-stage PCR using the oligonucleotides of SEQ ID NO: 3 and SEQ ID NO: 4 as primers;
(9) The method according to any one of (1) to (8) above, wherein the nucleotide sequence is determined using a massively parallel sequencer.
(10) The method according to any one of (1) to (9) above, wherein the comparison is performed for the top 90 to the top 110 of the strain genotypes of one or more types of bacteria.
(11) The method according to any one of (1) to (10) above, wherein the comparison is performed by machine learning.
(12) The method according to (11) above, wherein the machine learning is supervised machine learning.
(13) The method according to (12) above, wherein the supervised machine learning is supervised machine learning using a random forest algorithm.
(14) A region corresponding to the region of positions 950 to 1335 (preferably positions 968 to 1314) of the 16S rRNA gene (GenBank accession number NR_074675.1: SEQ ID NO: 109) of KPA171202, which is a reference strain of Acne bacteria. A kit for determining the user of an object based on the composition ratio of the strain genotype of the 16S rRNA gene of Acne, which contains a primer set capable of amplifying a polynucleotide.
(15) The primer set includes a primer consisting of the nucleotide sequence shown in SEQ ID NO: 1 and a primer set for first-stage PCR consisting of the nucleotide sequence shown in SEQ ID NO: 2.
A primer consisting of the nucleotide sequence shown in SEQ ID NO: 3 and a primer set for second-stage PCR consisting of the nucleotide sequence shown in SEQ ID NO: 4;
The kit according to (14) above.

According to the method of the present invention, the user of the object is determined with higher accuracy than in the case of analyzing the bacterial species composition of the indigenous skin flora (the composition ratio of the types of bacteria contained in the skin flora). You can do things like that.

The alignment of the regions corresponding to the regions at positions 968 to 1027 of the 16S rRNA gene of KPA171202 (GenBank accession number NR_074675.1: SEQ ID NO: 109) is shown for Acne bacteria MT1 to MT50. The nucleotide with a white background represents a nucleotide (polymorphism) different from the corresponding nucleotide of MT1 (KPA171202). The alignment of the regions corresponding to the regions at positions 968 to 1027 of the 16S rRNA gene of KPA171202 (GenBank accession number NR_074675.1: SEQ ID NO: 109) is shown for Acne bacteria MT51 to MT100. The nucleotide with a white background represents a nucleotide (polymorphism) different from the corresponding nucleotide of MT1 (KPA171202). The alignment of the regions corresponding to the regions 1028 to 1087 of the 16S rRNA gene of KPA171202 (GenBank accession number NR_074675.1: SEQ ID NO: 109) is shown for Acne bacteria MT1 to MT50. The nucleotide with a white background represents a nucleotide (polymorphism) different from the corresponding nucleotide of MT1 (KPA171202). The alignment of the regions corresponding to the regions 1028 to 1087 of the 16S rRNA gene of KPA171202 (GenBank accession number NR_074675.1: SEQ ID NO: 109) is shown for Acne bacteria MT51 to MT100. The nucleotide with a white background represents a nucleotide (polymorphism) different from the corresponding nucleotide of MT1 (KPA171202). The alignment of the regions corresponding to the regions at positions 1088 to 1147 of the 16S rRNA gene of KPA171202 (GenBank accession number NR_074675.1: SEQ ID NO: 109) is shown for Acne bacteria MT1 to MT50. The nucleotide with a white background represents a nucleotide (polymorphism) different from the corresponding nucleotide of MT1 (KPA171202). The alignment of the regions corresponding to the regions at positions 1088 to 1147 of the 16S rRNA gene of KPA171202 (GenBank accession number NR_074675.1: SEQ ID NO: 109) is shown for Acne bacteria MT51 to MT100. The nucleotide with a white background represents a nucleotide (polymorphism) different from the corresponding nucleotide of MT1 (KPA171202). The alignment of the regions corresponding to the regions 1148 to 1207 of the 16S rRNA gene of KPA171202 (GenBank accession number NR_074675.1: SEQ ID NO: 109) is shown for Acne bacteria MT1 to MT50. The nucleotide with a white background represents a nucleotide (polymorphism) different from the corresponding nucleotide of MT1 (KPA171202). The alignment of the regions corresponding to the regions 1148 to 1207 of the 16S rRNA gene of KPA171202 (GenBank accession number NR_074675.1: SEQ ID NO: 109) is shown for Acne bacteria MT51 to MT100. The nucleotide with a white background represents a nucleotide (polymorphism) different from the corresponding nucleotide of MT1 (KPA171202). The alignment of the regions corresponding to the 1208 to 1267 positions of the 16S rRNA gene of KPA171202 (GenBank accession number NR_074675.1: SEQ ID NO: 109) is shown for Acne bacteria MT1 to MT50. The nucleotide with a white background represents a nucleotide (polymorphism) different from the corresponding nucleotide of MT1 (KPA171202). The alignment of the regions corresponding to the 1208 to 1267 positions of the 16S rRNA gene of KPA171202 (GenBank accession number NR_074675.1: SEQ ID NO: 109) is shown for Acne bacteria MT51 to MT100. The nucleotide with a white background represents a nucleotide (polymorphism) different from the corresponding nucleotide of MT1 (KPA171202). The alignment of the regions corresponding to the regions 1268 to 1314 of the 16S rRNA gene of KPA171202 (GenBank accession number NR_074675.1: SEQ ID NO: 109) is shown for Acne bacteria MT1 to MT50. The nucleotide with a white background represents a nucleotide (polymorphism) different from the corresponding nucleotide of MT1 (KPA171202). The alignment of the regions corresponding to the regions 1268 to 1314 of the 16S rRNA gene of KPA171202 (GenBank accession number NR_074675.1: SEQ ID NO: 109) is shown for Acne bacteria MT51 to MT100. The nucleotide with a white background represents a nucleotide (polymorphism) different from the corresponding nucleotide of MT1 (KPA171202). It is a figure which shows the electrophoretic photograph of each PCR product obtained in Example 3. It is a figure which showed the composition ratio of OTU which the most similar species of Example 7 is acne fungus, divided into MT1 to MT36. In Example 7, it is the figure which showed the composition ratio paying attention to MT1-10. It is a figure which shows the result of the principal coordinate analysis (PCoA) in Example 8. FIG. 5 left panel: It is a figure which shows the result of having performed the principal coordinate analysis about the bacterial flora composition. FIG. 5 right panel: It is a figure which shows the result of having performed the principal coordinate analysis about the acne strain genotype composition. In addition, S1 to S10 each represent 10 subjects (subjects). It is a figure which shows the phylogenetic tree created by calculating the Euclidean distance from the result of the principal coordinate analysis of Example 8. FIG. 6 left panel: is a diagram showing a phylogenetic tree created for bacterial flora composition. FIG. 6 right panel: is a diagram showing a phylogenetic tree prepared for the acne strain genotype composition. In addition, S1 to S10 each represent 10 subjects (subjects). The results of calculating the similarity (Pearson's correlation coefficient) of the acne strain genotype composition or the bacterial flora composition between both hands of three subjects (S1, S3, S5), that is, six kinds of hands are shown. It is a figure. The upper right part of the dotted line connecting the upper left to the lower right represents the correlation coefficient r of the P. acnes genotype composition, and the lower left part of the dotted line represents the correlation coefficient r of the bacterial flora composition. When r> 0.99, it is shown in dark gray, when 0.80 <r <0.99, it is shown in medium gray, and when r <0.80, it is shown in light gray. S1 RH represents the right hand of S1, S1 LH represents the left hand of S1, S3 RH represents the right hand of S3, S3 LH represents the left hand of S3, S5 RH represents the right hand of S5, and S5 LH represents the right hand of S5. Represents the left hand. For example, the correlation coefficient r of the acne genotype composition of S1 RH and S1 LH is shown as 0.9989 on the panel just above S1 LH, just to the right of the indication S1 RH, and S1 RH and S5 LH. The correlation coefficient r of the acne genotype composition of S1 RH is shown as 0.3459 on the panel at the intersection of the right column from the S1 RH display and the upward column from the S5 LH display. The correlation coefficient r of the bacterial flora composition of S1 RH and S5 LH is shown as 0.7431 on the panel at the intersection of the downward column from the S1 RH display and the left column from the S5 LH display. .. It is a figure which shows the result of having performed the judgment test of the user of the object using the random forest which is one of the machine learning algorithms in Example 11. FIG. 8 upper left panel: is a diagram showing the results of a determination test using the acne strain genotype composition. The numerical value on the vertical axis represents the correct answer rate (%), and the numerical value on the horizontal axis represents the number of types of acne strains used in the analysis of the judgment. FIG. 8 lower left panel: It is a figure which shows the result of the judgment test using the bacterial flora composition. The numerical value on the vertical axis represents the correct answer rate (%), and the numerical value on the horizontal axis represents the number of types of bacteria used in the analysis of the judgment. FIG. 8 right panel: It is a figure which shows the result of the judgment test using both the acne strain genotype composition and the bacterial flora composition. The numerical value on the vertical axis represents the correct answer rate (%), and the numerical value on the horizontal axis represents the number of types of bacteria having the bacterial flora composition used. In Example 12, it is a figure which shows the result of having analyzed the time-dependent change of the acne strain genotype composition and the bacterial flora composition in a subject. FIG. 9 upper left panel: is a diagram showing the results of PC1 of the main coordinate analysis performed on the bacterial flora composition of the first sample and the second sample. FIG. 9 upper right panel: is a diagram showing the results of PC1 of the main coordinate analysis performed on the acne strain genotype composition of the first sample and the second sample. FIG. 9 lower left panel: It is a figure which shows the similarity index (Yue-Clayton theta similarity Index) of the 1st sample and the 2nd sample. P. acnes represents the similarity when using the P. acnes strain genotype composition, and Microbiome represents the similarity when using the bacterial flora composition. FIG. 9 lower right panel: It is a figure showing the result of performing random forest using the data of the first sampling as the teacher data and the data of the second sampling as the test data. P. acnes represents the correct answer rate (%) when the acne strain genotype composition is used, and Microbiome represents the correct answer rate (%) when the bacterial flora composition is used.

The method for determining the user of the object of the present invention is as follows: (A) the gene of one or two or more types of bacteria collected from the surface of the object, and the one or more types of bacteria collected from the skin of a candidate. (B) The nucleotide sequence of the amplification product obtained in the step (A) was determined, and the above 1 was collected from the surface of the object based on the polytype of the nucleotide sequence. Alternatively, a step of determining the composition ratio M of the strain genotypes of two or more types of bacteria and the composition ratio N of the strain genotypes of the one or more types of bacteria collected from the skin of the candidate; (C). The step of comparing the composition ratios M and N to determine the similarity between the composition ratios M and N; and (D) whether the candidate is a user of the object based on the similarity. This is a method including the step of determining.

In the present specification, the "user of an object" includes a person who has experience of skin contact with the object, and preferably, bacteria existing on the surface of the skin are attached to the surface of the object. Includes those who have. The user of the object also includes the owner of the object. Such owners may or may not have legal ownership of the object.

In the present invention, the one or more types of bacteria are not particularly limited as long as they are bacterial species that can be collected from the surface of the skin, and include so-called indigenous bacteria on the skin. Specific examples thereof include propionibacterium. Examples thereof include bacteria of the genus Umm, bacteria of the genus Staphylococcus, bacteria of the genus Ralstonia, bacteria of the genus Corynebacterium, and bacteria of the genus Streptococcus. In addition, among the above-mentioned bacteria, Propionibacterium genus bacteria, particularly Propionibacterium acnes, are more abundant among the indigenous skin bacteria, and therefore are less susceptible to contamination. Since it is possible to make a judgment with high accuracy, it can be preferably mentioned.

The object in the present invention is not particularly limited as long as it contains at least a solid portion, but from the viewpoint of determining the user of the object, an object that may come into contact with the skin of the candidate is preferable and specific. Includes computer keyboards, computer mice, smartphones, tablets, bags, pens, pencils, chopsticks, mobile phones, doorknobs, keys, remote controls, watches, regular pockets, and more.

The one or more types of bacteria used in the determination method of the present invention may be one type of bacteria or two or more types of bacteria, but from the viewpoint of ease of work and analysis, they may be used. It is preferably one type of bacterium (a bacterium of the same genus and the same type). As one such bacterium, the above-mentioned P. acnes Propionibacterium acnes can be preferably used.

In the above step (A), the method of collecting one or more kinds of bacteria from the surface of the object is not particularly limited as long as the method can collect the bacteria from the surface of the object, and is, for example, a porous material containing a solvent. A method of recovering bacteria by rubbing the surface of an object with a material and immersing the porous material in a separately prepared solvent can be preferably mentioned. Examples of such a solvent include a buffer solution such as TE10 buffer (1 to 100 mM (preferably 10 mM) Tris, 1 to 100 mM (preferably 10 mM) EDTA) and water, and among them, the buffer solution is preferably mentioned. Yes, and above all, TE10 buffer can be mentioned more preferably. Examples of the above-mentioned porous material include absorbent cotton, cloth, sponge, paper and the like. Among them, absorbent cotton can be preferably mentioned, and among them, a cotton swab can be more preferably mentioned. From the viewpoint of avoiding contamination, these porous materials are preferably sterilized porous materials.

In the above step (A), when one or more kinds of bacteria (preferably P. acnes) are collected from the skin of the candidate, one or more kinds of bacteria (preferably P. acnes) are collected from the surface of the object. ) And / or before or after the time when the skin of the user of the object comes into contact with the object, but from the viewpoint of enjoying the significance of the present invention more. When to collect one or more bacteria (preferably P. acnes) from the candidate's skin, when to collect one or more bacteria (preferably P. acnes) from the surface of the object, and / Or, the time between the time when the skin of the user of the object comes into contact with the object is preferably 1 month or more, more preferably 2 months or more, still more preferably 3 months or more, and more preferably 4 months. It is preferably more than 2 months, more preferably 5 months or more, and examples of the upper limit of such a period include 24 months or less, 18 months or less, 12 months or less, 10 months or less, and the like. If the above period is longer than a certain period, the significance of the present invention can be enjoyed more than the composition of the strain genotype of one or more kinds of bacteria (preferably P. acnes) used in the determination method of the present invention. This is because the ratio changes less with the passage of time than the composition of two or more types of bacterial flora (composition ratio of bacterial types) used in the conventional determination method.

In the present specification, the "gene" amplified in the above step (A) includes a polynucleotide encoding a gene (preferably DNA) and / or its complementary polynucleotide (preferably DNA). In addition to a full-length gene, a gene includes a partial region of the gene (hereinafter referred to as "gene region") for convenience. The type of gene to be amplified in the above step (A) is not particularly limited as long as it is a gene containing a polymorphism, but a gene containing a large amount of polymorphism is preferable, and a 16S rRNA gene is particularly preferable. As the above-mentioned "gene containing a large amount of polymorphism (including a gene region)", a gene containing two or more types of polymorphism variations, preferably five or more types, more preferably 10 types or more, and further preferably 15 or more types ( (Including gene region). The upper limit of the variation of the polymorphism is not particularly limited, and examples thereof include 300 types, 200 types, and 100 types.

When the bacterium used in the determination method of the present invention is Acne, the 16S rRNA gene is preferably mentioned as the gene to be amplified in the above step (A), and among them, the 16S rRNA gene of KPA171202, which is the acne reference strain. The region containing the region corresponding to the region at positions 950 to 1335 of (SEQ ID NO: 109) is more preferably mentioned, and the region containing the region corresponding to the region at positions 968 to 1314 of the 16S rRNA gene of KPA171202 is particularly preferable. Can be mentioned. Specific mutations in the region corresponding to the region of the 16S rRNA gene of KPA171202 at positions 950 to 1335 (preferably at positions 968 to 1314) include positions 976, 981 and 996 of the 16S rRNA gene of KPA171202. 1012th, 1034th, 1041st, 1044th, 1045th, 1047th, 1049th, 1050th, 1052nd, 1054th, 1056th, 1057th, 1059th, 1060th, 1064th, 1065th, 1075th 1076th, 1077th, 1079th, 1081st, 1089th, 1090th, 1095th, 1097th, 1099th, 1100th, 1104th, 1107th, 1108th, 1110th, 1111th, 1112th, 1113, 1121, 1122, 1123, 1125, 1127, 1130, 1142, 1144, 1145, 1147, 1151, 1154, 1164, 1169, 1175, 1176 , 1177th, 1186th, 1187th, 1189th, 1199th, 1206th, 1210th, 1213th, 1215th, 1219th, 1222th, 1226th, 1227th, 1229th, 1232th, 1236th, 1242 One-base substitutions in nucleotides at one or more sites selected from the group consisting of sites corresponding to positions, 1243, 1244 and 1248, respectively. It should be noted that which region of the 16S rRNA gene of Acne bacteria other than KPA171202 corresponds to the region of the 16S rRNA gene of KPA171202 at positions 950 to 1335 (preferably 968 to 1314) is CLUSTALW. It can be specified by creating an alignment with sequence analysis software such as.

When there is one type of bacterium that amplifies a gene in the above step (A), the gene or gene region may be one type or two or more types, but it is easy to work and analyze. From the viewpoint, one type is preferable. When there are two or more types of bacteria that amplify the gene in the above step (A), the amplified gene or gene region may be the same regardless of the bacterial species, or may differ depending on the bacterial species. It may be different depending on the bacterial species, but from the viewpoint of ease of work and analysis, the gene or gene region to be amplified may be the same regardless of the bacterial species or may differ depending on the bacterial species. Preferably, the gene or gene region to be amplified is the same regardless of the bacterial species.

In the above step (A), the amplification of the bacterial gene may be carried out by any known method. For example, a cotton swab rubbed against the surface of the object or the surface of the candidate's skin is placed in a TE buffer. A polymerase chain reaction (PCR) that is stirred and the buffer is used as a template can be mentioned. The primers used for PCR can be arbitrarily designed based on the nucleotide sequences at both ends of the gene region to be amplified, and PCR may be performed using a set of primer pairs, but non-specific amplification may be performed. In order to reduce the amount and enhance the final judgment system, it is preferable to carry out by the nested PCR method using two or more sets of primer pairs. Further, the amplified gene may be extracted from the excised band by agarose gel electrophoresis, and a known PCR product purification method (GE Healthcare's "NAP Columns", "MicroSpin Columns", Beckman Coulter). It may be purified using "Agencourt AMPure XP" manufactured by the company.

In the above step (A), the region including the region corresponding to the region at positions 950 to 1335 (preferably at positions 968 to 1314) of the 16S rRNA gene of KPA171202, which is the reference strain of Acne bacteria, was subjected to the above-mentioned nested PCR method. As a suitable method for amplification, (a) an oligonucleotide primer consisting of the nucleotide sequence shown in SEQ ID NO: 1 and an oligonucleotide primer consisting of the nucleotide sequence shown in SEQ ID NO: 2 are used to perform the first-stage PCR. Steps; (b) Performing a second-step PCR using an oligonucleotide primer consisting of the nucleotide sequence shown in SEQ ID NO: 3 and an oligonucleotide primer consisting of the nucleotide sequence shown in SEQ ID NO: 4; The PCR method can be mentioned. By using such a nested PCR method, the region corresponding to the region of the 16S rRNA gene of Acne bacterium KPA171202 at positions 950 to 1335 (preferably at positions 968 to 1314) can be amplified particularly efficiently. Therefore, the kit containing the following first primer set and second primer set can be suitably used as a kit for determining the user of the object based on the composition ratio of the strain genotype of Acne. it can.
A first primer set consisting of an oligonucleotide primer consisting of the nucleotide sequence shown in SEQ ID NO: 1 and an oligonucleotide primer consisting of the nucleotide sequence shown in SEQ ID NO: 2;
A second primer set consisting of an oligonucleotide primer consisting of the nucleotide sequence shown in SEQ ID NO: 3 and an oligonucleotide primer consisting of the nucleotide sequence shown in SEQ ID NO: 4;

In the above step (B), the nucleotide sequence of the amplification product obtained in the step (A) may be determined by any known method, but the sequencing using a massively parallel sequencer is extremely large. It is more preferable in that the nucleotide sequence can be determined at once. Examples of the massively parallel sequencer include MiSeq, HiSeq2000, Genome Analyzer IIx (GAIIx) manufactured by Illumina, and Genome Sequencer-FLX (GS-FLX) manufactured by Roche. Massively parallel sequencers have devices with 100 million to several billion sequences (reads) that can be determined at one time, and can quickly and easily determine the nucleotide sequences of an extremely large number of amplification products.

In step (B) above, the strain genotype of one or more bacteria is determined based on the polymorphism of the nucleotide sequence of the gene. The polymorphism of the nucleotide sequence means that there are a plurality of base types at specific positions of the nucleotide, and examples thereof include base substitution, insertion of one or more bases, and deletion. Such a polymorphism can be identified by analyzing the sequence difference between the nucleotide sequences of the plurality of types of amplification products determined in the step (B) with sequence analysis software such as CLUSTALW.

In the experiments of the examples described later, the present inventors identified 100 types of genotypes of regions corresponding to the regions at positions 968 to 1314 in the 16S rRNA gene of KPA171202, which is a reference strain of Acne bacteria. The 100 genotypes (MT1 to MT100) of this gene region are shown as nucleotide sequences of SEQ ID NOs: 9 to 108, respectively. The alignment of the nucleotide sequences of the above gene regions of MT1 to MT100 is shown in FIGS. 1-1 to 1-12. The genotype of MT1 (SEQ ID NO: 9) is positioned as the wild-type genotype of this gene region.

In addition, the composition ratio M of the strain genotypes of the one or more types of bacteria collected from the surface of the object, and the composition ratio N of the strain genotypes of the one or more types of bacteria collected from the skin of the candidate. In order from the determined nucleotide sequence having the highest abundance ratio, the top 10 to the top 1000, preferably the top 10 to the top 500, more preferably the top 10 to the top 300, and even more preferably the top 20. It is possible to determine the number of pieces to the top 60 pieces, particularly preferably the top 30 pieces to the top 50 pieces (more preferably the top 30 pieces). Further, when the composition ratios of the strain genotypes of two or more types of bacteria are used, the composition ratios M and N are determined individually for each bacterial species even if they are collectively determined for the entire two or more types of bacteria. You may. However, from the viewpoint of ease of work and analysis, it is preferable to use the composition ratio of the genotype of one type of bacterium (bacteria of the same genus and the same type). P. acnes is preferably mentioned as one such bacterium.

The step (C): a step of comparing the constituent ratios M and N to determine the similarity between the constituent ratios M and N, and the step (D): the candidate is based on the similarity. As a step of determining whether or not the object is a user, any known method can be used, and machine learning and data mining can be exemplified. Among them, machine learning, more preferably supervised machine learning. , More preferably, supervised machine learning using a random forest algorithm and supervised machine learning using a support vector machine are applied. For such machine learning and data mining, it is preferable to use multivariate analysis such as principal component analysis (PCA) and principal component analysis (PCA) in combination. The similarity is preferably calculated as a numerical value such as the Euclidean distance. A person skilled in the art can determine how similar the candidate is to be the user of the object, depending on the situation. For example, of the composition ratios M and N, When the degree of similarity is converted into the Euclidean distance, if the Euclidean distance is 0.1 or less, preferably 0.05 or less, it can be determined that the candidate is a user of the object. The Euclidean distance can be calculated using, for example, the software "R".

As described above, the determination method of the present invention is a method of determining the user of an object by utilizing the similarity of the composition ratio of the bacterial strain genotype, but at the time of the determination, the bacterial flora composition is determined. It is preferable to use the degree of similarity together from the viewpoint of making a more accurate determination. When the similarity of bacterial flora composition is used together, for example,
In addition to including the above steps (A) to (D),
(I) A step of amplifying one or more bacterial genes collected from the surface of an object and one or more bacterial genes collected from a candidate's skin, respectively;
(J) Based on the nucleotide sequence of the amplification product obtained in the step (I), the composition ratio V of the one or more types of bacteria collected from the surface of the object, and the skin of the candidate. Step of determining the composition ratio W of each of the above-mentioned one or more types of bacteria collected from
(K) A step of comparing the constituent ratios V and W to determine the degree of similarity between the constituent ratios V and W;
Including
The above step (D)
(D') A step of determining whether the candidate is a user of the object based on the similarity determined in the step C and the similarity determined in the step K;
There is a method of determining the user of the object.

The step (D') is a step of determining whether the candidate is a user of the object based on the similarity determined in the step C and the similarity determined in the step K. The degree of similarity is not particularly limited as long as it is, but for example, the average value of the similarity of the composition ratio of the strain genotype determined in the above step C and the similarity of the bacterial composition determined in the above step K is used as the similarity. , A method of determining whether the candidate is a user of the target person is preferably mentioned. As a method for determining the bacterial flora composition of a certain sample, the method described in Non-Patent Document 2 can be used.

Aspects of the present invention also include a kit for determining the user of an object based on the composition ratio of the strain genotype of the 16S rRNA gene of Acne. Such a kit is a region corresponding to the region of positions 950 to 1335 (preferably positions 968 to 1314) of the 16S rRNA gene (GenBank accession number NR_074675.1: SEQ ID NO: 109) of KPA171202, which is a reference strain of Acne bacteria. It is not particularly limited as long as it contains a primer set capable of amplifying the polynucleotide of. The kit is also a kit for detecting polymorphisms in the region corresponding to the region of the 16S rRNA gene of Acne bacterium KPA171202 at positions 950 to 1335 (preferably at positions 968 to 1314).

The nucleotide sequences of the forward primer and the reverse primer in the above primer set can be designed by those skilled in the art based on the nucleotide sequence shown in SEQ ID NO: 109. For example, the nucleotide sequence of the primer can be selected from the nucleotide sequences capable of hybridizing to the nucleotide sequence outside the region corresponding to the region at positions 950 to 1335 (preferably at positions 968 to 1314). The number of nucleotides in the primer is preferably 17 to 25. It is preferable that the Tm value of the primer is approximately the same for the forward primer and the reverse primer and is set to about 55 to 65 ° C. It is preferable to select a primer pair having less complementarity between the primers so that the primers do not anneal to each other. In particular, in order to prevent a decrease in amplification efficiency due to the formation of primer dimers, it is preferable to design the 3'ends of each primer so that three or more nucleotides are not continuous and complementary. In addition, it is usually preferable not to contain a self-complementary sequence of 4 or more nucleotides in order to avoid the formation of secondary structure in the primer. It is preferable that the GC content is around 40 to 60% so as not to be partially GC-rich or AT-rich. Further, it is preferable that the 3'end of the primer and the template DNA are stably bound to each other so that the 3'side of the primer is not AT-rich or GC-rich.

A suitable primer capable of amplifying the polynucleotide in the region containing the region corresponding to the region at positions 968 to 1314 of the 16S rRNA gene (GenBank accession number NR_074675.1: SEQ ID NO: 109) of KPA171202, which is a reference strain of Acne bacteria. As a set, a primer set for the first stage PCR (a primer consisting of the nucleotide sequence shown in SEQ ID NO: 1 and a primer consisting of the nucleotide sequence shown in SEQ ID NO: 2) and a primer set for the second stage PCR. Examples thereof include a primer set containing (a primer consisting of the nucleotide sequence shown in SEQ ID NO: 3 and a primer consisting of the nucleotide sequence shown in SEQ ID NO: 4).

In the aspect of the present invention, it is determined whether the subject is one of X candidates (where X represents any integer of 1 or more), which comprises the following steps (E) to (H). A method of determination is also included. (E) A step of amplifying genes of one or more types of bacteria collected from the skin of the subject;
(F) The nucleotide sequence of the amplification product obtained in the step (E) was determined, and based on the polymorphism of the nucleotide sequence, the strain genotype of the one or more bacteria collected from the skin of the subject. Step of determining the composition ratio P of
(G) Constituent ratios of strain genotypes of the one or more bacteria collected from the skin of the X candidates Q _{1 to} Q _x (where X represents any integer of 1 or more). , by comparing the composition ratio P, the step of determining the similarity of the composition ratio P and the constituent ratio Q ₁ to Q _x;
(H) A step of determining whether the subject is one of the X candidates based on the similarity;

The composition ratios Q _{1 to} Q _x in the candidate (where X represents any integer of 1 or more) may be determined before or after the composition ratio P in the subject. may be determined, preferably to be determined before determining the composition ratio P in a subject, the composition ratio Q ₂ to Q x _(provided that in advance a plurality of candidates, X is one or more represents an arbitrary integer.) to determine, it is more preferable to such a database of its component ratio Q ₂ to Q _x.

The X candidates may be one or two or more, but preferably three or more, more preferably 10 or more, and even more preferably 50 or more. The upper limit of X people is not particularly limited, and examples thereof include 1000 people and 500 people.

As a method in each step of the method of determining whether the subject is one of the X candidates, the same method as the above-mentioned "user determination method" can be used.

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

1. 1. Sample collection and DNA extraction In examining whether it is possible to identify the user based on the composition ratio of the strain genotype of P. acnes, 3 people at 6 locations (5 left and right fingertips, left and right keyboard, PC pad) , Smartphone screen) was the subject of research. A sterile cotton swab dipped in TE10 buffer (10 mM Tris, 10 mM EDTA) was rubbed against each site for 30 seconds, and a sample dipped in 500 μL TE10 buffer was used as a sample and stored frozen at -80 ° C. DNA extraction was performed from these samples according to the following procedure.
[1] 25 μL of lysozyme (300 mg / mL) was added to the sample, and the sample was incubated overnight at 37 ° C.
[2] Add 15 μL of achromopeptidase (20,000 units / mL), incubate at 37 ° C. for 8 hours, then add 50 μL of 10% SDS and 15 μL of proteinase K (25 mg / mL) and overnight at 55 ° C. Incubated. Then, 600 μL of a phenol / chloroform / isoamyl alcohol mixed solution was added, and the mixture was stirred with a Micro Shaker for 3 minutes, and centrifuged at 17,800 × g at 20 ° C. for 10 minutes.
[3] Add 600 μL of the phenol / chloroform / isoamyl alcohol mixture to the supernatant again, stir at 1,500 rpm for 3 minutes using a Micro Shaker, and centrifuge at 17,800 × g at 20 ° C. for 10 minutes. Was done.
[4] The aqueous layer was recovered, 60 μL of 3M sodium acetate buffer (pH 5.2) was added, 1.2 mL of 100% ethanol was added, and the mixture was cooled at −80 ° C. for 1 hour.
[5] After centrifugation at 17,800 × g and 4 ° C. for 10 minutes, the supernatant was removed and 500 μL of 70% ethanol was added.
[6] After centrifugation at 17,800 × g at 4 ° C. for 5 minutes, the supernatant was removed, 50 μL of 1 × TE buffer was added, and the mixture was stirred with a Micro Shaker for 5 minutes to dissolve the DNA. ..

2. PCR for amplification of DNA fragments of Acne bacteria and purification of PCR products Acne bacteria 16S rRNA-specific primers designed by the present inventors (414F: 5'-GGGTTGTAAACCGCTTTCGCCT-3'(SEQ ID NO: 1), 1445R: 5 '-GTTGTGGGGGAGCCGTCGAA-3' (SEQ ID NO: 2)) was used to amplify the 16S rRNA gene of the DNA extract obtained in Example 1. The PCR conditions at that time are as follows.
Initial denaturation reaction: Repeat 3 steps at 94 ° C for 5 minutes 96 ° C for 10 seconds, 64 ° C for 15 seconds, 68 ° C for 1 minute 30 times Final extension reaction: 2 minutes at 68 ° C Polymerase is Tks Gflex DNA Polymerase (Takara Bio Inc.) Made) was used. Then, 36 μL of Agencourt AMPure XP reagent (manufactured by Beckman Coulter) was added to 20 μL of the PCR product, and purification was performed according to the attached manual.

3. 3. PCR for the purpose of amplifying the strain discrimination region, purification and concentration determination of PCR products Primers for strain discrimination designed by the present inventors (950F: 5'-AGAACCTTACCTGGGTTTGA-3'(SEQ ID NO: 3), 1334R: 5'- The 16S rRNA gene was amplified using GATCTGCGATTACTAGCGAC-3'(SEQ ID NO: 4). The PCR products obtained in Example 2 were diluted 300-fold and used as templates. The PCR conditions are as follows.
Initial denaturation reaction: Repeat 3 steps at 94 ° C for 2 minutes 96 ° C for 10 seconds, 64 ° C for 15 seconds, 68 ° C for 30 seconds 20 times Final extension reaction: 2 minutes at 68 ° C Polymerase is Tks Gflex DNA Polymerase (Takara Bio Inc.) Made) was used. Then, 36 μL of Agencourt AMPure XP reagent (manufactured by Beckman Coulter) was added to 20 μL of the PCR product, and purification was performed according to the attached manual. After purification, electrophoresis was performed on a 1.5% agarose gel for 15 minutes, stained with Gel Red (manufactured by Wako Pure Chemical Industries, Ltd.), and electrophoretic photographs were taken. The result is shown in FIG. As can be seen from FIG. 2, it was shown that the band of interest was amplified by nested PCR. Moreover, when the sequence result of the PCR product was confirmed, 99% of the amplified products were DNA fragments of Acne bacteria. From this, it was shown that the DNA fragment of interest can be amplified specifically for acne bacteria by nested PCR using the above primers. In the obtained PCR product solution, the concentration of double-stranded DNA was quantified using Picogreen (manufactured by Thermo Fisher Scientific Co., Ltd.). Based on these results, the volume molar concentration of the PCR product was calculated. Each sample was diluted so that the template used in the PCR of Example 4 had a final concentration of 4 nM.

4. PCR for universal primer sequence addition, purification and concentration determination of PCR products In order to add the Index sequence used in the massively parallel sequencer MiSeq (manufactured by Illumina) to the DNA fragment, the universal primer sequence (27F_mod, 338R) is added in advance. There is a need. Therefore, a primer in which the sequences of 27F_mod and 338R were added to the strain discrimination primer (27_950F: 5'-AGRGTTTGATYMTGGCTCAGAGAACCTTACCTGGGTTTGA-3'(SEQ ID NO: 5), 338_1334R: 5'-TGCTGCCTCCCGTAGGAGTGATCTGCGATTACTAGCGAC-3'(SEQ ID NO: 6)) And PCR was performed. The PCR conditions are as follows.
Initial denaturation reaction: Repeat 3 steps at 94 ° C for 2 minutes 96 ° C for 10 seconds, 64 ° C for 15 seconds, 68 ° C for 30 seconds 5 times Final extension reaction: 2 minutes at 68 ° C Polymerase is Tks Gflex DNA Polymerase (Takara Bio Inc.) Made) was used. Then, 90 μL of Agencourt AMPure XP reagent (manufactured by Beckman Coulter) was added to 50 μL of the PCR product, and purification was performed according to the attached manual. After purification, concentration quantification was performed using Bioanalyzer 2100 (manufactured by Agilent Technologies). Based on these results, the amount of template to be used in the PCR of Example 5 was determined, and dilution was performed so that the final concentration was 1 nM.

5. Index PCR and concentration determination Index PCR was performed to add the adapter sequence and Index sequence required for sequencing with the massively parallel sequencer MiSeq (manufactured by Illumina). Primers used were Index F: 5'-AATGATACGGCGACCACCGAGATCTACAC-NNNNNNNN-TATGGTAATTGTAGRGTTTGATYMTGGCTCAG-3' (SEQ ID NO: 7) and Index R: 5'-CAAGCAGAAGACGGCATACGAGAT-NNNNNNNN-AGTCAGTCAGCCTGCTGCCTCCCGTAGGAGT-3'. The base sequence described by N in the above is an Index sequence, and a different sequence was used for each sample. The PCR conditions are as follows.
Initial denaturation reaction: 98 ° C for 1 minute 98 ° C for 10 seconds, 55 ° C for 15 seconds, 68 ° C for 30 seconds Repeat 3 steps 8 times Final extension reaction: 68 ° C for 3 minutes Polymerase is Tks Gflex DNA Polymerase (Takara Bio Inc.) Made) was used. Then, 90 μL of Agencourt AMPure XP reagent (manufactured by Beckman Coulter) was added to 50 μL of the PCR product, and purification was performed according to the attached manual. Concentration quantification of Index PCR products was performed using qPCR, Picogreen (manufactured by Thermo Fisher Scientific Co., Ltd.), and Bioanalyzer 2100 (manufactured by Agilent Technologies). Based on these results, each sample was diluted to 4 nM.

6. Sequencing with MiSeq Samples diluted to 4 nM were mixed and sequenced with MiSeq. The sequence was performed according to the protocol indicated by Illumina.

7. Data analysis First, the base sequence sequenced at the pair end was assembled by FLASH (Version 1.2.11). Nucleotide sequences with an average Q-value of 25 or less were excluded from the analysis. Then, the primer sequences of 950F and 1334R were removed from the sequenced base sequence. The obtained data was analyzed using Qime (Version 1.8.0). After that, the number of occurrences of completely matching base sequences was counted for each sample, and the duplicate base sequences were removed (Operational Taxonomic Units (OTU) with 100% homology as a threshold value were created). A homology search was performed using the Ribosomal Database Project (RDP) as a database, and OTU, whose most similar species was Acne, was used for the subsequent analysis. For OTUs for which the bacterial species could not be identified by RDP, homology search was performed using the Nucleotide collection as a database using NCBI BLAST, and OTUs whose most similar species was Acne bacteria were used for subsequent analysis. Using. OTUs, the species with the highest similarity being Acne, were arranged in descending order of the number of reads, and the strain numbers were assigned (the strain with the highest abundance was MT1 and MT1 (SEQ ID NO: 9) to MT100 in descending order of abundance. (SEQ ID NO: 108) was numbered.) The nucleotide sequence alignments of the MT1 to MT100 gene regions (regions corresponding to the 968 to 1314 positions of the 16S rRNA gene of KPA171202 (SEQ ID NO: 109)) are shown in FIGS. 1-1 to 1-12. .. A random forest algorithm was used to examine whether individual discrimination was possible. Random forest analysis is performed by software that implements a package called "random forest" for random forest analysis in software "R" (Version 3.1.2), and possession data (keyboard left and right, PC pad) as teacher data. , MT1 to MT100 composition ratio calculated collectively for the smartphone screen), and user data (composition ratio of MT1 to MT100 calculated collectively for five left and right fingertips) was used as test data.

FIG. 3 is a diagram showing the composition ratio of OTU in which the most similar species is acne, divided into MT1 to MT36. Further, FIG. 4 is a diagram showing the composition ratios focusing on MTs 1 to 10. From all the figures, it was found that the composition ratio of the strain genotype of the object and its user was similar, and it was shown that the composition ratio could be used for determining the user of the object. The analysis results by Random Forest are shown below.
Type of random forest: classification
Number of trees: 5000
No. of variables tried at each split: 5
OOB estimate of error rate: 0%
Confusion matrix:
S1 S2 S3 class.error
S1 2 0 0 0
S2 0 2 0 0
S3 0 0 2 0

From this analysis result, it was possible to determine the users of all three tested subjects based on the strain genotype of Acne bacteria collected from the objects (keyboard left and right, PC pad, smartphone screen). That is, the method for determining the user of the object in the present invention has a correct answer rate of 100% (error rate of 0%), indicating that such a determination method has excellent accuracy. Although the composition ratios of MT1 to MT100 were used in this analysis, the result of 100% correct answer rate was also obtained in the analysis using the composition ratios of MT1 to MT20.

In Non-Patent Document 1 (Proc.Natl.Acad.Sci.USA, 107, 6477-6481 (2010)), 16S rRNA of a bacterial sample collected by wiping the keyboard of a computer and the fingertips of the user with a swab. The gene encoding the above was amplified using a universal primer, and the composition of the bacterial flora (composition ratio of the types of bacteria contained in the bacterial flora) was identified based on the sequence. It is disclosed that the compositions of the above were similar. Using this finding, the present inventors conducted a method for determining the user of an object based on the composition of the bacterial flora as a comparative experiment. This method does not focus on the difference in genotype between "strains of a certain bacterial species" as in the determination method of the present invention, but focuses on the difference in genotype between "bacterial species". It is clearly different from the method of.

When the above-mentioned comparative experiment (an experiment using a method of determining the user of the object based on the bacterial flora composition), which is a known prior art, was performed on the object of Example 1 and the subjects (3 persons). Of the three objects, the user of one object was erroneously determined. That is, the correct answer rate in the comparative experiment was 66.7% (error rate 33.3%). From this, it was shown that the above-mentioned determination method of the present invention has excellent accuracy.

8. Principal Coordinate Analysis (PCoA) for bacterial flora composition (composition ratio of types of bacteria contained in bacterial flora) and 10 principal coordinate analysis (S1 to S10) for P. acnes genotype composition For each of the (subjects), sampling (sampling) was performed at 6 locations (5 fingertips of the left hand, 5 fingertips of the right hand, the left side of the keyboard, the right side of the keyboard, the PC pad, and the smartphone screen). The genotype composition of Acne was measured and analyzed by the method described in Examples 1 to 7 above. However, for the analysis, a known principal coordinate analysis was used. The results are shown in the right panel of FIG.

In addition, as a comparative experiment, sampling at the above-mentioned 6 locations was performed on the above-mentioned 10 subjects according to the method of Non-Patent Document 1 (Proc.Natl.Acad.Sci.USA, 107, 6477-6481 (2010)). , And the bacterial flora composition was measured and analyzed. However, for the analysis, a known principal coordinate analysis was used. The results are shown in the left panel of FIG. Samples with the same mark (for example, "○") indicate that they are samples of the same subject.

In the principal coordinate analysis, plots are made so that highly correlated samples are arranged close to each other. In the analysis results of the bacterial flora composition, the positions of the six plots of the same type of mark (for example, "○") were relatively widely dispersed, that is, the positions of each plot of the six samples of a specific subject. Was relatively large and varied (left panel in FIG. 5). On the other hand, in the analysis of the acne genotype composition, the positions of the six plots of the same type of marks (for example, "○") are closer to each other than in the case of the bacterial flora composition analysis, that is, a specific one. The plot positions of the six samples of the subjects were relatively close (right panel in FIG. 5). From the results of FIG. 5, it is shown that the P. acnes genotype composition analysis is more sensitive to individual identification than the bacterial flora composition analysis, and is very excellent for identifying the user of an object. Was done.

9. Phylogenetic tree of bacterial flora composition and P. acnes genotype composition For each measurement result of Example 8 above, the Euclidean distance was calculated using software "R" to prepare a phylogenetic tree. The result is shown in FIG. The left side of FIG. 6 shows the phylogenetic tree obtained by measuring and analyzing the bacterial flora composition, and the right side of FIG. 6 shows the phylogenetic tree prepared by measuring and analyzing the P. acnes genotype composition.

In these phylogenetic trees, similar samples were plotted so that they were closely arranged. The length of a branch connecting a sample in a phylogenetic tree represents the Euclidean distance between the samples. In the phylogenetic tree, samples with the same mark indicate that they are samples of the same subject. As can be seen from FIG. 6, in the phylogenetic tree of bacterial flora composition, the positions of the six plots of the same type of mark (for example, “○”) were relatively widely dispersed, that is, six locations of a specific subject. The plot positions of the sample were relatively large and varied (left panel in FIG. 6). In contrast, in the P. acnes genotype composition phylogenetic tree, the positions of the six plots of homologous marks (eg, "○") are closer together than in the bacterial flora composition analysis, i.e. The plot positions of the six samples of the subjects were relatively close (right panel in FIG. 6). From the results of FIG. 6, it is shown that the P. acnes genotype composition analysis is more sensitive to individual identification than the bacterial flora composition analysis, and is very excellent for identifying the user of an object. Was done.

10. Similarity in bacterial flora composition and P. acnes genotype composition Of the 10 subjects in Examples 8 and 9, 3 (S1, S3, S5) subjects' hands (S1 RH (S1's right hand), The composition of S1 LH (left hand of S1), S3 RH (right hand of S3), S3 LH (left hand of S3), S5 RH (right hand of S5), S5 LH (left hand of S5)) is picked up and 6 kinds of hands. Correlation values were calculated for the composition of. The result is shown in FIG. Pearson's correlation coefficient was used as the correlation coefficient. The upper right part of the dotted line connecting the upper left to the lower right represents the correlation coefficient of P. acnes genotype composition, and the lower left part of the dotted line represents the correlation coefficient of bacterial flora composition.

As can be seen from the results of FIG. 7, in the correlation value of the bacterial flora composition, the correlation coefficient of S1 RH and S1 LH was high, and the correlation coefficient of S5 RH and S5 LH was also high to some extent, but S3 RH and S3 The correlation coefficient of LH was relatively low, and the correlation coefficient between S3 RH and S1 LH was higher. On the other hand, in the correlation coefficient of the acne genotype composition, the correlation coefficient of the samples (S1 RH and S1 LH, S3 RH and S3 LH, S5 RH and S5 LH) between the same subjects is very high (r>. 0.99) On the other hand, it was shown that the correlation coefficient of the samples between different subjects was low. From the results of FIG. 7, from the results of FIG. 7, the P. acnes genotype composition analysis is more sensitive to individual identification than the bacterial flora composition analysis, and is very useful for identifying the user of an object. It was shown to be excellent.

11. Judgment test of users of objects using random forest By the same method as in Examples 1 to 7 above, 10 subjects (S1 to S10) were subjected to objects (left side of keyboard, right side of keyboard, PC). A test was conducted to determine the user of the pad (pad, smartphone screen).

In the analysis method using the acne genotype composition, 10 types and 30 types of acne strains are selected in order to investigate how many types of acne strains are used for analysis and the correct answer rate differs. , 50 types, 100 types, 300 types, 500 types, and 1000 types were changed for analysis. These results are shown in the upper left panel of FIG.

In addition, even in the analysis method using the bacterial flora composition, in order to investigate how many types of bacteria are used for analysis from the one having the larger abundance, the correct answer rate differs, and the types are 50 types, 100 types, or 130 types. The analysis was performed by changing to types, 150 types, 300 types, 500 types, and 700 types. These results are shown in the lower left panel of FIG.

As can be seen from the results of the upper left panel and the lower left panel of FIG. 8, in the judgment using the bacterial flora composition, the correct answer rate was 53.15% even at the highest rate (lower left panel of FIG. 8), whereas the acne bacteria. In the judgment using the genotype composition, the correct answer rate is 79.49% at the highest and exceeds 60% at the lowest. From this, it was shown that the determination using the P. acnes genotype composition is more accurate than the determination using the conventional bacterial flora composition.

As can be seen from the results in the upper left panel of FIG. 8, in the determination using the acne genotype composition, 30 acne strains from the most abundant acne strains were used for the analysis. The correct answer rate was the highest, followed by the correct answer rate when 50 types of acne strains were used. From this, it was found that in the determination method of the present invention using the bacterial strain genotype composition, the type of bacterial strain used for analysis is preferably 20 to 60 types, more preferably 30 to 50 types.

In addition, the present inventors used the method with the highest correct answer rate in the judgment using the acne genotype composition (the method using 30 acne strains from the one with the highest abundance among various acne strains for analysis). ) And the determination method using the bacterial flora composition (composition of 130 types, 150 types or 700 types of bacteria) were tried (Mix). These methods are methods for analyzing both the acne strain genotype composition and the bacterial flora composition at the time of determination. The results of these combined methods are shown in the right panel of FIG. As can be seen from the results on the right panel of FIG. 8, the correct answer rate was further improved. Among them, the method in which the acne strain genotype composition and the 130 kinds of bacterial flora composition were combined had the highest correct answer rate, which was 95.7%. From these results, it was shown that the use of the bacterial flora composition in addition to the bacterial strain genotype composition enables more accurate determination.

12. Changes in the genotype composition and bacterial flora composition of the Acne strain in the subject over time Whether or not there is a change in the genotype composition and bacterial flora composition of the Acne strain in the subject over time, that is, by the passage of a certain period of time, the subject The following experiments were conducted to investigate whether the genotype composition and bacterial flora composition of the Acne strain were changed.

Sampling (sampling) of 6 locations (5 fingertips of the left hand, 5 fingertips of the right hand, left side of the keyboard, right side of the keyboard, PC pad, smartphone screen) was taken for each of the 3 subjects (S1 to S3). went. Sampling was performed twice, and the second sampling was performed 5 months after the first sampling. The acne genotype composition of the obtained sample was measured and analyzed by the method described in Examples 1 to 7 above. However, for the analysis, a known principal coordinate analysis was used. Further, regarding the sampling of the determination method (Non-Patent Document 1) using the bacterial flora composition, which is a comparative experiment, the second sampling was performed 5 months after the first sampling.

The results of PC1 (Weighted) of the main coordinate analysis (PCAA) performed on the acne strain genotype composition are shown in the upper left panel of FIG. 9, and the results of PC1 (Weighted) of the main coordinate analysis performed on the bacterial flora composition are shown in FIG. Shown in the upper right panel. The sample for the first sampling is indicated by a black circle, and the sample for the second sampling is indicated by a white circle. The similarity between the result of the first sampling and the result of the second sampling was evaluated by the Yue-Clayton theta similarity Index. In addition, two groups, the P. acnes genotype composition group (P. acnes) and the bacterial flora composition group (Microbiome), were compared by the Mann-Whitney U test. The results are shown in the lower left panel of FIG. As can be seen from the results in the lower left panel of FIG. 9, in the determination method using the acne strain genotype composition, the result of the first sampling and the result of the second sampling are compared with the determination method using the bacterial flora composition. The similarity of the results was significantly (p <0.01) higher.

Further, the result of performing random forest using the data of the first sampling as the teacher data and the data of the second sampling as the test data is shown in the lower right panel of FIG. As can be seen from the lower right panel of FIG. 9, the correct answer rate was about 40% in the judgment method using the bacterial flora composition, whereas the correct answer rate was 100% in the judgment method using the acne strain genotype composition. It was.

From the results of the four panels in FIG. 9, it was shown that the acne strain genotype composition had less change over time and was less affected by the sampling time than the bacterial flora composition. Therefore, the user can be identified with relatively high accuracy even if the object has been touched for a relatively long period of time.

According to the method of the present invention, the user of the object is determined with higher accuracy than in the case of analyzing the bacterial species composition of the skin flora (the composition ratio of the types of bacteria contained in the skin flora). It is highly industrially useful in fields such as criminal investigations where it is necessary to determine the user of the object. In addition, by registering the composition ratio of the bacterial strain genotype collected from the skin of a subject in advance and comparing it with the composition ratio in the subject, it can be used as the identity verification of the subject. It is conceivable that

Claims (14)

1 or 2 or more kinds of bacterial genes taken from the surface of the (A) Target material, and the gene of the one or more bacteria collected from candidates of the skin, amplifying each step;
(B) The nucleotide sequence of the amplification product obtained in the step (A) was determined, and based on the polymorphism of the nucleotide sequence, the strain gene of the one or more kinds of bacteria collected from the surface of the object. A step of determining the composition ratio M of the type and the composition ratio N of the strain genotype of the one or more kinds of bacteria collected from the skin of the candidate;
(C) by comparing the component ratios M and N, step determines the similarity of the composition ratio M and N; and,
(D) A step of determining whether the candidate is a user of the object based on the similarity;
Including
The one or more kinds of bacteria are bacteria selected from Propionibacterium spp., Staphylococcus spp., Ralstonia spp., Corinebacterium spp. And Streptococcus spp.
A method of determining the user of an object. The method according to claim 1, wherein one or more of the bacteria are Propionibacterium spp. The method according to claim 1, wherein one or more of the bacteria are P. acnes. The method according to claim 3 , wherein the gene of one or more types of bacteria is the gene of 16S rRNA of P. acnes, and the genotype of the strain of one or more types of bacteria is the genotype of 16S rRNA of P. acnes. .. The method according to claim 4 , wherein the genotype of 16S rRNA of Acne is the genotype of the region corresponding to positions 950 to 1335 in the gene of 16S rRNA of KPA171202, which is a reference strain of Acne. The 16S rRNA genotype of Acne bacteria is 976th, 981st, 996th, 1012th, 1034th, 1041st, 1044th, 1045th, 1047th, etc. 1049th, 1050th, 1052nd, 1054th, 1056th, 1057th, 1059th, 1060th, 1064th, 1065th, 1075th, 1076th, 1077th, 1079th, 1081st, 1089th, 1090th 1095th, 1097th, 1099th, 1100th, 1104th, 1107th, 1108th, 1110th, 1111th, 1112th, 1113th, 1121st, 1122th, 1123th, 1125th, 1127th, 1130th 1142, 1144, 1145, 1147, 1151, 1154, 1164, 1169, 1175, 1176, 1177, 1186, 1187, 1189, 1199, 1206, From a group consisting of parts corresponding to 1210, 1213, 1215, 1219, 1222, 1226, 1227, 1229, 1232, 1236, 1242, 1243, 1244 and 1248, respectively. The method according to claim 4 or 5 , which is a genotype based on a gene polymorphism of one or more sites to be selected. The method according to any one of claims 4 to 6 , wherein the step of amplifying the 16S rRNA gene of Acne bacterium is carried out by using the nested PCR method including the following steps (a) and (b).
(A) A step of performing the first-stage PCR using the oligonucleotides of SEQ ID NO: 1 and SEQ ID NO: 2 as primers;
(B) A step of performing a second-stage PCR using the oligonucleotides of SEQ ID NO: 3 and SEQ ID NO: 4 as primers; The method according to any one of claims 1 to 7 , wherein the nucleotide sequence is determined using a massively parallel sequencer. The method according to any one of claims 1 to 8 , wherein the comparison is made for the top 90 to 110 of the strain genotypes of one or more bacteria. Comparison is performed by machine learning method according to any one of claims 1-9. The method of claim 10 , wherein the machine learning is supervised machine learning. The method of claim 11 , wherein the supervised machine learning is supervised machine learning using a random forest algorithm. Acne containing a primer set capable of amplifying the polynucleotide in the region corresponding to the region at positions 950 to 1335 of the 16S rRNA gene (GenBank accession number NR_074675.1: SEQ ID NO: 109) of KPA171202, which is a reference strain of Acne bacteria. A kit for determining the user of an object based on the composition ratio of the strain genotype of the 16S rRNA gene of the bacterium. The primer set is a primer consisting of the nucleotide sequence shown in SEQ ID NO: 1 and a primer set for first-stage PCR consisting of the nucleotide sequence shown in SEQ ID NO: 2.
A primer consisting of the nucleotide sequence shown in SEQ ID NO: 3 and a primer set for second-stage PCR consisting of the nucleotide sequence shown in SEQ ID NO: 4;
13. The kit according to claim 13.

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