JP2013005759A - Method for estimating intestinal bacterial flora of mouse - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for estimating intestinal bacterial flora of a mouse.SOLUTION: A bacterial strain and a phylotype existing in a sample is estimated by: amplifying DNA using an rRNA gene in the sample or a complementary DNA obtained by reverse transcription of the rRNA gene as a template; generating 5' and 3' terminal fragments by applying restriction enzyme treatment to the amplified DNA; separating the generated 5' and 3' terminal fragments by cataphoreses; measuring a number of base of the 5' and 3' terminal fragments; and using a database suitable for the estimation of the intestinal bacterial flora of a mouse based on the number of bases of the 5' and 3' terminal fragments originating from the sample measured by a T-RFLP method.

Description

  The present invention relates to a method for estimating intestinal flora and a database used in the method.

  The terminal-restriction fragment length polymorphism (T-RFLP) method obtains a gene from a bacterial flora in the target environment, amplifies the gene fragment by polymerase chain reaction (PCR) using the gene as a template, and the amplified This is a method of analyzing the bacterial flora in the environment by fragmenting the gene fragment with a restriction enzyme and then subjecting it to capillary electrophoresis to detect a gene fragment having a length specific to the bacterial species.

  According to the T-RFLP method, it is possible to comprehensively analyze a complex microbial flora, and it is possible to easily compare changes in the flora and flora patterns. In addition to this, if a gene fragment (peak) characteristic of the flora pattern and a bacterial species corresponding to the changed gene fragment (peak) can be estimated, more detailed data interpretation is possible. Software such as Microbial Community Analysis III (MiCA3) has been developed in order to estimate the bacterial species corresponding to a specific gene fragment in such a flora pattern. However, most of the databases contained in the software are databases constructed using the gene sequences registered in the Ribosomal Data Project (RDP) as they are, and the soil flora and soil A database constructed from gene sequences derived from bacterial species present in the human oral flora is also included.

  Microbial community structure analysis method using T-RFLP method is to analyze the community structure of environmental microorganisms using gene sequences registered in GenBank (US), EMBL (Europe), and DDBJ (Japan) as a database. A method is known (Patent Document 1). There is also a report analyzing the human intestinal flora using a database suitable for the human intestinal flora (Non-patent Document 1).

JP 2006-94803

Mitsuharu Matsumoto, Mitsuo Sakamoto, Hidenori Hayashi, and Yoshimi Benno, Journal of Microbiological Methods 61 (2005) 305-319

  However, when analyzing the bacterial flora in the intestinal environment of mice using an existing database such as MiCA3, a huge number of bacterial species in another environment (for example, soil, ocean, etc.) will be listed as candidates. The correct bacterial species cannot be estimated accurately. An object of the present invention is to perform estimation of a bacterial flora in an intestinal environment of a mouse simply, quickly, and with high accuracy.

  In view of the above circumstances, the inventor of the present application has focused on the database used for estimation, and as a result, has succeeded in independently constructing a database suitable for estimating the gut microbiota of mice. The present invention has been completed based on such findings.

Although this invention is not limited, the following estimation methods of (1)-(7) are provided.
(1) Amplifying DNA using a rRNA gene in a sample or a complementary DNA obtained by reverse transcription of the rRNA gene as a template,
Generate 5 'and 3' terminal fragments by treating the amplified DNA with restriction enzymes,
The generated 5 ′ and 3 ′ end fragments are separated by electrophoresis,
Measure the number of bases of the separated 5 ′ and 3 ′ terminal fragments,
Measured from a database containing information on the estimated number of bases of the 5 'and 3' terminal fragments and the bacterial species from which the 5 'and 3' terminal fragments are derived. Select 5 'and 3' terminal fragments corresponding to the number of bases given,
Selecting the bacterial species from which the selected 5 ′ and 3 ′ terminal fragments are derived from the database;
Inferring the selected strain to be present in the sample,
A method for estimating the gut microbiota of a mouse.

  (2) Restriction enzyme treatment of the amplified DNA is AccII, AfaI, AfIII, AluI, BamHI, BcuI, BfaI, Bme1580I, BseGI, Bsh1236I, Bsp106I, BspTI, BstUI, Bsu36I, BsuRI, CfoI, CviRI, DdeI, DpnI (MethA), DpnII, EcoRI, FneDII, HaeIII, HapII, HhaI, Hinp1I, HinfI, HpaII, Hpy1881, Hpy188III, HpyCH4IV, HpyCH4V, HpyAV, Hsp92II, MaeI, MaeII, MboI, Ms, MseI, Ms (1), using one or more selected from the group consisting of NdeII, NlaIII, PalI, RsaI, Sau3AI, Sau96I, ScrFI, StuI, TaqI, ThaI, Tru9I, TsoI, Tsp509I, and TthHB8I. The method described.

(3) The method according to (1) or (2), wherein the restriction enzyme treatment of the amplified DNA is performed using HhaI, MspI, HaeIII, and AluI.
(4) The database is as follows:
Lactobasillus sp. ASF360
Lactobacillus murinus
Uncultured bacterium clone LKMC043
Akkermansia muciniphila ATCC BAA-835
Parabacteroides goldsteinii
And a DNA amplified by using the rRNA gene derived from the bacterial species as a template, respectively, by treating them with restriction enzymes using HhaI, MspI, HaeIII, and AluI, respectively:
Lactobasillus sp. ASF360
HhaI 5 'end fragment 596 bp, 3' end fragment 407 bp,
MspI 5 'end fragment 181 bp, 3' end fragment 80 bp,
HaeIII 5 'end fragment 325 bp, 3' end fragment 74 bp,
AluI 5 'terminal fragment 63 bp, 3' terminal fragment 182 bp
Lactobacillus murinus
HhaI 5 'terminal fragment 256 bp, 3' terminal fragment 403 bp,
MspI 5 'terminal fragment 571 bp, 3' terminal fragment 76 bp,
HaeIII 5 'end fragment 247 bp, 3' end fragment 125 bp,
AluI 5 'end fragment 262 bp, 3' end fragment 178 bp,
Uncultured bacterium clone LKMC043
HhaI 5 'terminal fragment 100 bp, 3' terminal fragment 49 bp,
MspI 5 'end fragment 88 bp, 3' end fragment 35 bp,
HaeIII 5 'end fragment 458 bp, 3' end fragment 116 bp,
AluI 5 'terminal fragment 247 bp, 3' terminal fragment 170 bp,
Akkermansia muciniphila ATCC BAA-835
HhaI 5 'terminal fragment 98 bp, 3' terminal fragment 145 bp,
MspI 5 'terminal fragment 270 bp, 3' terminal fragment 130 bp,
HaeIII 5 'end fragment 219 bp, 3' end fragment 219 bp,
AluI 5 'terminal fragment 74 bp, 3' terminal fragment 94 bp,
Parabacteroides goldsteinii
HhaI 5 'terminal fragment 98 bp, 3' terminal fragment 135 bp,
MspI 5 ′ terminal fragment 93 bp, 3 ′ terminal fragment 120 bp,
HaeIII 5 'end fragment 915 bp, 3' end fragment 48 bp,
AluI 5 'terminal fragment 724 bp, 3' terminal fragment 170 bp
The estimated base number of the 5 ′ and 3 ′ terminal fragments of
Is contained as information, The method in any one of (1)-(3).

(5) The method according to any one of (1) to (4), wherein the sample is prepared from mouse feces.
(6) The method according to any one of (1) to (5), wherein the rRNA gene is a 16S rRNA gene.

(7) The method according to any one of (1) to (6), wherein complementary DNA obtained by reverse transcription of 16S rRNA gene is used as a template.
(8) The method according to any one of (1) to (7), wherein the electrophoresis is capillary electrophoresis.

  According to the present invention, it is possible to quickly and easily estimate the bacterial species and phylotype (phylotype, which cannot be cultured but has a known 16S rRNA gene sequence) in the mouse intestine.

  The present invention provides a method for estimating the bacterial species constituting the intestinal flora of mice using a database. The method involves amplifying DNA using the rRNA gene in the sample as a template, treating the amplified DNA with a restriction enzyme to generate 5 ′ and 3 ′ terminal fragments, and separating the 5 ′ and 3 ′ terminal fragments. The 5 'and 3' terminal fragments corresponding to the measured base number are selected from the database, the bacterial species from which the 5 'and 3' terminal fragments are derived is selected, and the bacterial species Is inferred as the species present in the sample.

  The database used in the present invention (sometimes referred to as PAD-MCM) is suitable for estimating the bacterial species and phylotype of the intestinal microflora of the mouse, and the bacterial species and phylotype present in the mouse intestine, and It contains the estimated lengths (also referred to as the number of bases in this specification) of the 5 ′ and 3 ′ terminal fragments derived from these as information. Such information may be obtained, for example, by extracting necessary information from existing databases such as GenBank, EMBL, and DDBJ, but from the viewpoint of accurately estimating the intestinal flora of mice, It is preferable to newly acquire information from a sample that well reflects the intestinal flora.

  In order to newly construct PAD-MCM, first, genes derived from enterobacteria are obtained from a sample that well reflects the intestinal flora of mice. The gene here includes a polynucleotide, specifically, polydeoxyribonucleotide or polyribonucleotide, preferably polyribonucleotide. Examples of polyribonucleotides include ribosome (r) RNA, messenger (m) RNA, transfer (t) RNA, and the like, and preferably an rRNA gene. Examples of the rRNA gene include 16S rRNA gene, 23S rRNA gene, and 5S rRNA gene present in prokaryotic cells, preferably 16S rRNA gene. Furthermore, a complementary DNA generated by reverse transcription of these rRNA genes may be used as a gene.

  DNA is amplified using the obtained gene as a template. PCR is used for DNA amplification. Various conditions such as the concentration of the template, primer, and other components in the PCR method, the reaction time, and the reaction temperature can be appropriately selected and set by those skilled in the art.

The amplified DNA is incorporated into an appropriate vector such as a plasmid and cloned by introducing it into a host such as Escherichia coli, and the base sequence of the DNA incorporated into each clone is determined.
It is judged whether the DNA whose base sequence has been determined is new or has been registered in an existing database such as GenBank, EMBL, and DDBJ. The determination can be made based on homology with DNA registered in an existing database. More specifically, if DNA having 98% or more homology with the DNA whose base sequence has been determined is not registered in the existing database, the DNA having the base sequence determined will be a new bacterial species. It can be judged that it originates. On the other hand, if DNA having 98% or more homology with the DNA whose base sequence is determined is registered in the existing database, the DNA whose base sequence is determined is derived from the existing sequence. Judgment can be made.

  Next, a phylogenetic tree is created based on the DNA base sequence, and the bacterial species and phylotype to be registered in the database of the present invention are selected. Here, homology search is performed between DNAs whose base sequences have been determined, and bacterial species and phylotypes derived from DNA having a high homology relationship are judged to be closely related species or closely related databases. Do not register with. Here, high homology means that the homology of the base sequence is 98% or more.

  Further, the number of bases of 5 ′ and 3 ′ terminal fragments (also referred to as restriction enzyme fragments or T-RFs in the present specification) derived from the bacterial species and phylotype selected above is estimated, and PAD-MCM Register as information. Since the number of bases of the 5 'and 3' end fragments differs depending on the bacterial species and phylotype, the bacterial species and phylotype of the mouse intestinal flora can be estimated based on the difference. Estimate the base sequence and the number of bases of the 5 'and 3' end fragments generated by applying a restriction enzyme whose recognition site is known to the DNA of each clone whose base sequence has been determined as described above. Is possible. Such estimation can be performed on a computer using NEBcutter V2.0, for example.

  As the restriction enzyme herein, any restriction enzyme known among those skilled in the art can be used. Specifically, AccII, AfaI, AfIII, AluI, BamHI, BcuI, BfaI, Bme1580I, BseGI, Bsh1236I , Bsp106I, BspTI, BstUI, Bsu36I, BsuRI, CfoI, CviRI, DdeI, DpnI (methA), DpnII, EcoRI, FneDII, HaeIII, HapII, HhaI, Hinp1I, HinfI, HpaII, Hpy1884, Hpy188III, HpyCHIV, Hpy188III, HpyCHIV Hsp92II, MaeI, MaeII, MboI, MIyI, MseI, MsII, MspI, MvnI, NdeII, NlaIII, PalI, RsaI, Sau3AI, Sau96I, ScrFI, StuI, TaqI, ThaI, Tru9I, TsoI, Tsp509I, and TthB. All of these restriction enzymes are commercially available. These restriction enzymes have the following recognition sites (Table 1).

  Although one type of restriction enzyme may be applied, it is preferable to use two or more types, preferably four or more types, from the viewpoint of improving the accuracy of estimation of the intestinal bacterial species and phylotype of mouse intestinal bacteria. For example, HhaI, MspI, HaeIII, and AluI are used.

One aspect of the PAD-MCM construction method will be described.
The stool of the mouse is washed with water, buffer solution, physiological saline or the like. The washed stool is suspended in a buffer containing crushing beads, a surfactant, and phenol and subjected to heat treatment. Cells are disrupted by homogenizing the suspension after heat treatment. The disrupted solution is subjected to centrifugation, and the supernatant is collected. A phenol / chloroform solution is added to the supernatant and mixed well, followed by centrifugation, and the supernatant is recovered. The gene contained in the supernatant is recovered by ethanol precipitation. Suspend the recovered gene in water to the desired concentration.

  Using the gene collected as described above as a template, the 16S rRNA gene is amplified by PCR using primers suitable for the amplification of the 16S rRNA gene. A person skilled in the art can appropriately set the PCR reaction temperature. DNA amplified by PCR is ligated to a plasmid vector, and the host is transformed by introducing the plasmid into the host.

  A plasmid is extracted from the colony of the transformant, and PCR is performed using this plasmid as a template gene. PCR primers can be appropriately selected according to the plasmid to be used, and PCR temperature conditions and PCR product purification conditions can also be set as appropriate.

  The base sequence of the PCR product purified as described above is analyzed by a method well known to those skilled in the art, and the sequence of almost the full length of the 16S rRNA gene is determined. A homology between the gene and a gene registered in an existing database is searched, and if it has 98% or more homology, it is determined that the gene is derived from an existing bacterial species and phylotype. On the other hand, if the homology is lower than 98%, it is determined that the gene is derived from a new bacterial species and phylotype that do not exist in the existing database.

  A phylogenetic tree is prepared based on the base sequence of the 16S rRNA gene determined as described above, and the bacterial species and phylotype used for the construction of PAD-MCM are selected. Here, a homology search is performed between the obtained 16S rRNA gene base sequences, and those having a homology of 98% or more are judged to be related species or related and are not registered in the PAD-MCM redundantly. Like that. In addition, a bacterial species having a 16S rRNA gene base sequence having 98% or more homology with an existing gene sequence is registered in the PAD-MCM as an existing species.

  Further, 5 ′ and 5 ′ produced by treating DNA amplified with the 16S rRNA gene derived from the above-mentioned bacterial species and phylotype registered in PAD-MCM with HhaI, MspI, HaeIII, and AluI, respectively. Estimate the number of bases of the 3 ′ end fragment using NEBcutter V2.0 or the like, and register it with PAD-MCM.

  By using the PAD-MCM constructed in this manner, it is possible to estimate the bacterial species and phylotype derived from the mouse intestine existing in the sample with high accuracy. Here, high accuracy means that 50% or more, preferably 60% or more, more preferably 70% or more of mouse enterobacteria obtained by the clone library method is estimated.

  Here, according to the clone library method, for example, the following 32 types of bacterial species and phylotypes derived from the mouse intestine can be obtained, and the base sequences of 16S rRNA genes derived from them are registered in DDBJ. (Table 2). That is, if PAD-MCM has information on the estimated base number of these bacterial species and phylotypes and the 5 ′ and 3 ′ terminal fragments derived from them, the bacterial species and phylotypes in the mouse intestine can be obtained. It becomes possible to guess.

  Of the 32 species and phylotypes, 56.3 percent of the five species and phylotypes were Uncultured bacterium clone LKMC043, Lactobasillus sp. ASF360, Lactobacillus murinus, Akkermansia muciniphila ATCC BAA-835, and Parabacteroides goldsteinii. Accounted for. That is, if the PAD-MCM has information on the estimated base number of these 5 species and phylotypes and the 5 ′ and 3 ′ terminal fragments derived from them, it can be used with sufficient accuracy. Can be guessed.

  As a specific example of the estimated base number of the 5 ′ and 3 ′ terminal fragments, DNA fragments amplified using the 16S rRNA gene in the above table as a template were treated with Msp I, Hha I, Alu I, and Hae III, respectively. The estimated base numbers of the 5 ′ and 3 ′ terminal fragments obtained are shown below (Table 3). Information on the estimated number of bases can be performed on a computer using NEBcutter V2.0 or the like.

  The sample as used herein refers to a sample used for estimating the bacterial species and phylotype in the mouse intestine by the method of the present invention, and containing a gene derived from the mouse intestinal flora. . In the preparation of the sample, mouse stool is preferably used. Feces may be dissolved or suspended in water, buffer solution, organic solvent or the like, and insoluble components may be removed from the sample by centrifugation or filtration. Furthermore, the gene in the sample may be concentrated or diluted and adjusted to an appropriate concentration by freeze drying or vacuum drying. The sample may be used as it is in subsequent steps without being purified to remove impurities, but may be purified as necessary.

The gene here refers to any polynucleotide that can be used to estimate the strain of mouse enteric bacteria, and includes polyribonucleotides and polydeoxynucleotides. Examples of polyribonucleotides include ribosome (r) RNA, messenger (m) RNA, transfer (t) RNA, and the like, and preferably an rRNA gene. Examples of the rRNA gene include 16S rRNA gene, 23S rRNA gene, and 5S rRNA gene present in prokaryotic cells, preferably 16S rRNA gene. In addition, complementary DNA generated by reverse transcription of these rRNA genes may be used as a gene. PCR is preferred as a method for amplifying DNA using the gene as a template. Those skilled in the art can appropriately set various conditions for PCR, for example, the concentration of the gene used as a template, the design of the primer, the obtaining method, the concentration of use, and the temperature conditions of denaturation, annealing, and extension reaction.

  Fragmentation of the amplified DNA is preferably performed using a restriction enzyme. The restriction enzymes used in the present invention are AccII, AfaI, AfIII, AluI, BamHI, BcuI, BfaI, Bme1580I, BseGI, Bsh1236I, Bsp106I, BspTI, BstUI, Bsu36I, BsuRI, CfoI, CviRI, DdeI, DpnI (methA), DpnII, EcoRI, FneDII, HaeIII, HapII, HhaI, Hinp1I, HinfI, HpaII, Hpy1881, Hpy188III, HpyCH4IV, HpyCH4V, HpyAV, Hsp92II, MaeI, MaeII, MboI, MIyI, MseI, MsII, MspI The same restriction enzyme selected from PalI, RsaI, Sau3AI, Sau96I, ScrFI, StuI, TaqI, ThaI, Tru9I, TsoI, Tsp509I, and TthHB8I and used in the construction of PAD-MCM is used.

  Since the 5 'and 3' terminal fragments obtained by the above restriction enzyme treatment are a mixture derived from multiple bacterial species, in order to determine the number of bases of individual 5 'and 3' terminal fragments, separate them. There is a need. As means for performing such separation, any means known to those skilled in the art can be used, and electrophoresis is preferable, and capillary electrophoresis is more preferable. The number of bases of the individual 5 'and 3' terminal fragments thus separated can be measured with a DNA sequencer. Based on the number of bases of the 5 'and 3' terminal fragments thus measured, the bacterial species and phylotype from which the 5 'and 3' terminal fragments are derived can be estimated from PAD-MCM.

In order to make the present invention easier to understand, specific examples will be given and described, but the scope of the present invention is not limited thereto.
[Test Example 1] Extraction of fecal DNA
Feces were collected from 6 ICR mice. Feces (100 mg) were washed twice with physiological saline (Phosphate buffered saline (PBS) (pH 7.4)) (1 ml) and stored frozen at −80 ° C. until used for DNA extraction. At the time of use, stool is thawed, suspended in ultrapure water (200 μl), 0.1 mm beads (300 mg), 100 mM Tris-HCl-50 mM EDTA buffer (pH 8.0) (350 μl), 10% SDS (50 μl) and phenol (500 μl) were added. The suspension was heat treated at 70 ° C. for 10 minutes, and then cells were disrupted by treatment with Micro Smash MS-100 (TOMY) at 4000 rpm for 60 seconds. The treatment solution was centrifuged at 13000 × g for 5 minutes at room temperature, and the supernatant (500 μl) was collected. A phenol / chloroform solution (500 μl) was added to the supernatant and mixed well, followed by centrifugation at 13000 × g for 5 minutes at room temperature to recover the supernatant (350 μl). By using Eta Precipitate Mate (Nippon Gene Co., Ltd.) under the conditions described in the instruction manual, the gene present in the supernatant was ethanol precipitated and collected. The collected gene was suspended in ultrapure water (350 μl). This was used as a sample in subsequent tests.

[Test Example 2] Preparation of 16S rRNA gene clone library (2-1) Amplification of 16S rRNA gene DNA was amplified using the 16S rRNA gene as a template from the sample prepared in Test Example 1 by PCR using the following primers. I let you.

27F primer: 5'-AGAGTTTGATCCTGGCTCAG-3 '(SEQ ID NO: 1)
1492R primer: 5'-GGTTACCTTGTTACGACTT-3 '(SEQ ID NO: 2)
Mix template sample (500 ng) (1 μl), 1.25 U TaKaRa Ex Taq, 10 × Ex Taq buffer (5 μl), dNTPmiture (4 μl) and the above primers (10 pmol each) into ultrapure water (37.75 μl) ) To adjust the total volume to 50 μl to prepare a PCR reaction solution. PCR was performed using a thermal cycler (RTC-200, MJ Research) set to the following temperature conditions.

反応終了後、GenElute（商標）PCR Clean−Up(SIGMA)を用い、製品説明書に記載された条件に従ってPCR産物を精製した。精製されたPCR産物をTA Cloning（登録商標）Kits（invitorogen）を用いて、pCR2.1プラスミドベクターにライゲーションし、INVαF’に形質転換した。PCR産物が挿入されたプラスミドを持つ形質転換体からランダムに96コロニーを選択し、プラスミドを抽出した。

After completion of the reaction, the PCR product was purified using GenElute ™ PCR Clean-Up (SIGMA) according to the conditions described in the product instructions. The purified PCR product was ligated to pCR2.1 plasmid vector using TA Cloning® Kits (invitorogen) and transformed into INVαF ′. Ninety-six colonies were randomly selected from the transformants having the plasmid inserted with the PCR product, and the plasmid was extracted.

  Plasmid extraction was performed by the direct boiling method shown below. A very small amount of the colony was scraped with a toothpick, suspended in TE buffer (100 μl), and boiled at 100 ° C. for 10 minutes. The suspension after boiling was subjected to centrifugation at 500 × g for 5 minutes, and the supernatant was recovered. PCR using the following primers was performed using the supernatant as a template gene.

M13F: 5′-GTAAAACGACGGCCAGT-3 ′ (SEQ ID NO: 3)
M13R: 5′-CAGGAAACAGCTATGAC-3 ′ (SEQ ID NO: 4)
PCR temperature conditions were the same as those described above except that the treatment of [98 ° C., 10 seconds → 50 ° C., 30 seconds → 72 ° C., 90 seconds] was set to 30 cycles. After the PCR was completed, the PCR product was purified using GenElute ™ PCR Clean-Up (SIGMA) as described above.

(2-2) Analysis of gene sequence The PCR product purified in (2-1) above was used as a template gene in the analysis of gene sequence. By using the following four primers, the nearly full-length sequence of the 16S rRNA gene was determined.

M13F (SEQ ID NO: 3);
M13R (SEQ ID NO: 4);
787F: 5′-ATTAGATACCCTGGTAGTCC-3 ′ (SEQ ID NO: 5);
920R: 5′-GTCAATTCCTTTGAGTTT-3 ′ (SEQ ID NO: 6).

  Using a BigDye (registered trademark) Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems), a sequencing reaction was performed according to the method of use described in the product manual. The nucleotide sequence was determined using 3130 Genetic Analyzer (Applied Biosystems).

  Perform a homology search of the PCR product whose base sequence is determined in the above and the genes registered in DDBJ, EMBL, and GenBank, determine a gene derived from an existing species having 98% or more homology, Those having a homology lower than 98% were judged to be derived from a new bacterial species. The homology search was performed using BLAST.

[Test Example 3] Construction of mouse intestinal flora database (PAD-MCM) (3-1) Registration of bacterial species A phylogenetic tree was created based on the sequence of the 16S rRNA gene determined in Test Example 2, and PAD -Species and phylotypes (phylotype: bacteria that cannot be cultured but the sequence of 16S rRNA gene is known) used for MCM construction were selected.

  Here, the homology search of 16S rRNA gene is performed using ClustalW, and phylotypes from which genes having homology of 98% or more are derived are considered to be related species or related, and PAD-MCM is duplicated. Don't register with

  In addition, the gene sequence of the 16S rRNA gene determined using the next-generation sequencer (FLX system) is subjected to a BLAST search, and a bacterial strain from which a gene having a homology of 98% or more with an existing gene is derived as a PAD -Registered with MCM (Table 4).

(3-2) Registration of 5 'and 3' terminal fragments DNA amplified by PCR using 27F primer and 1492R primer using 16S rRNA gene shown in Table 4 as a template, restriction enzymes HhaI, MspI, The number of bases of the 5 ′ terminal fragment and 3 ′ terminal fragment generated by treatment with HaeIII and AluI was estimated by NEBcutter V2.0 (New England Biolabs). The estimated number of bases of the terminal fragment was registered in the database (Table 5).

(3-3) Information registered in PAD-MCM In addition to the information in Table 4, the PAD-MCM constructed as described in (3-1) and (3-2) above contains enteric bacteria of mice. The information of Table 5 regarding the estimated base number of the restriction enzyme fragment of 16S rRNA gene acquired from 268 kinds of fungi derived from the flora is included. Of the 268 bacterial species, 236 are phylotypes and 31 are existing species. Specifically, 49 types belong to Clostridium cluster IV, 119 types belong to Clostridium cluster XIV, 63 types belong to other Clostridium genera, 1 type belongs to the genus Akkermansia, 7 The type belongs to the genus Lactobacillus, one type belongs to the segmented filamentous bacteria (SFB), 21 types belong to the genus Bacteroides, and 7 types belong to the other genera.

[Test Example 4] T-RFLP analysis (4-1) Primer fluorescent labeling The 5 ′ ends of the universal primer, 27F and 1492R were respectively 6′-carboxyfluorescein (6-FAM) and 2′-chloro- It labeled with 7'-phenyl-1,4'-dichloro-6-carboxyfluorescein (2'-chloro-7'-phenyl-1,4-dichloro-6-carboxyfluorescein (VIC)). Labeling was performed by methods well known to those skilled in the art. These labeled primers were used in the following PCR.

(4-2) PCR
PCR was performed using the sample prepared in Test Example 1 as a template. The PCR product was purified after performing PCR according to the method described in (2-1) above, except that the labeled primer prepared in (4-1) above was used.

(4-3) Restriction enzyme treatment of PCR product The PCR product (2 μl (50 ng)) purified in the above (4-2) was treated with 20 U HhaI, MspI, HaeIII, and AluI (Takara Bio Inc.), respectively. After mixing, the reaction volume was adjusted to 10 μl. The reaction solution was treated at 37 ° C. for 3 hours, 1 μl was mixed with GeneScanTM-1200LIZ (registered trademark) Size standard (Applied Biosystems) (0.5 μl), Hi-DiTMFormamide (9 μl), and treated at 95 ° C. for 3 minutes. , Cooled quickly. This was used as a sample for analysis of terminal-length fragments (T-RFs).

(4-4) Analysis of T-RFs By applying the analysis sample prepared in (4-3) above to 3130 Genetic Analyzer (Applied Biosystems), the peak of the terminal fragment was detected. The base number of the detected terminal fragment was calculated using Gene Mapper. In addition, the terminal fragment corresponding to the peak area whose ratio to the total peak area is less than 1% was excluded from the analysis target.

[Test Example 5] Presumption of intestinal flora using PAD-MCM Based on the number of bases of 5 'and 3' terminal fragments measured in Test Example 4 above, the bacterial species and phylotypes present in mouse feces are Presumed using the PAD-MCM constructed in Test Example 3. It has been reported that the number of base fragments in terminal fragments estimated by computer simulation is different from the measured values (Kitts, CL, 2001. Terminal Restriction Fragment Patterns: A Tool for Comparing Microbial Communities and Assessing. Curr. Issues Intest Microbiol. 2,17-25; Sakamoto, M., Takeuchi, Y., Umeda, M., Ishikawa, I., Benno, Y., 2003; Application of terminal RFLP analysis to characterize oral bacterial flora in saliva of healthy subjects and patients with periodontitis. J. Med. Microbiol. 52, 79-89).

  We have confirmed in preliminary experiments that terminal fragments having a base number of 100 to 1000 bp actually measured are estimated as 0 to 8 bp longer by computer simulation. Therefore, the terminal fragment having the actual number of bases of 300 bp was estimated as 300 to 308 bp by computer simulation.

  In addition, it was confirmed by preliminary tests that terminal fragments having a base number of less than 100 bp actually measured and 1000 bp or more actually measured were shifted in the range of 5-25 bp and 8-161 bp, respectively, depending on computer simulation. ing. Moreover, it has also been confirmed that the degree to which the estimated number of bases of such terminal fragments deviates from the actual measurement value varies greatly for each terminal fragment.

  From the above, terminal fragments having a base number of actually measured values of less than 100 bp and actually measured values of 1000 bp or more are excluded from the search target, and terminal fragments having actually measured values of 100 to 1000 bp are used as search targets. .

  The bacterial species and phylotype corresponding to the number of bases of the 3 'terminal fragment produced by Alu I treatment were estimated using PAD-MCM (Table 6).

  The result of estimating the bacterial species and phylotype using the existing database MiCA-DB is shown as a comparison. In the estimation using MiCA-DB, a large number of bacterial species are listed as candidates, and it became clear that it is difficult to narrow down bacterial species and phylotypes. In addition, it was also clarified that a number of bacterial species not related to the intestinal flora of mice such as Maribacter sp. W-8 and Flexithrix dorotheae BSs20185 (both derived from seawater) are listed as candidates. From these facts, it was confirmed that it was difficult to estimate the bacterial species of the mouse intestinal flora depending on the existing database. On the other hand, when the bacterial species and phylotype were estimated using PAD-MCM, it was shown that the bacterial species were effectively narrowed down.

  Next, the number of bases of 5 'and 3' terminal fragments generated by treatment of each of the four restriction enzymes HhaI, MspI, AluI, and HaeIII was compared with PAD-MCM (Table 7).

  As a result, the 20 species and phylotypes listed in Table 6 were estimated as shown in Table 8 (Table 8).

[Test Example 6] Comparison with bacterial species and phylotype obtained by the clone library method A clone library was prepared according to the method described in Test Example 2, and the obtained bacterial species and phylotype were used for PAD-MCM. It was compared with the guess result. (Table 9)

  Thirty-one bacterial species and phylotypes were detected by the clone library method. Nine of them: Uncultured bacterium clone LKMC043 (18.4%), Lactobasillus sp. ASF360 (17.2%), Lactobacillus murinus (8.0%), Akkermansia muciniphila ATCC BAA-835 (6.9%), Parabacteroides goldsteinii (5.7%), Bacteroides caccae (2.3%), Uncultured bacterium clone LKMC16 (2.3%), Uncultured bacterium clone LKMC162 (2.3%), and Bacteroides sp.SF519 (1.1%), which were estimated using PAD-MCM It was common. These accounted for 64.1% of the total clones. And the top 5 bacterial species and phylotypes that accounted for 56.3% of the total clones were detected in the method using PAD-MCM (Table 9).

  Since the bacterial species and phylotype obtained from the clone library were obtained from the feces of the mouse, all of them were present in the mouse individual. For this reason, it can be considered that the more accurate the speciation and phylotype estimated using PAD-MCM match with those obtained in the clone library, the higher the accuracy of the estimation. The above results indicate that by using PAD-MCM, it is possible to quickly and highly accurately estimate the dominant bacterial species and phylotype of the intestinal bacteria of mice.

  In addition, the five species and phylotypes shown in Table 9: Lactobasillus sp. ASF360, Lactobacillus murinus, Uncultured bacterium clone LKMC043, Akkermansia muciniphila ATCC BAA-835, and Parabacteroides goldsteinii account for more than half of the intestinal flora of mice It was shown that. That is, in the case of this research sample, among the information on the 268 species and phylotypes shown in Table 4, the information on the restriction enzyme terminal fragments for the above 5 species and phylotypes is included in the PAD-MCM. If so, it was shown that the bacterial species and phylotype of the mouse intestinal flora can be estimated with sufficiently high accuracy.

[Comparative Example 1] Presumption of bacterial species and phylotype using existing database Currently, in the estimation of bacterial species using the T-RFLP method, MiCA3 (Shyu, C., Soule, T., Bent, SJ, Foster, JA, Forney, LJ, 2007, MiCA: a web-based tool for the analysis of microbial communities based on terminal-restriction fragment length polymorphisms of 16S and 18S rRNA genes. Microbial Ecology 53, 562-570) Widely used.

This MiCA3 Virtual Digest (ISPaR) has the following conditions:
Selection data RDP (R10, U22) 606,188 Good Quality (> 1200) 16S rRNA gene;
Primers Eub-8f (5'-AGAGTTTGATCCTGGCTCAG-3 ') (same sequence as 27F primer), and 1492r (5'-GGTTACCTTGTTACGACTT-3') (same sequence as 1492R primer);
Restriction enzymes HhaI, MspI, HaeIII, and AluI
A Ribosome database project (RDP) database composed of 16S rRNA genes whose sequence of 1200 bp or more was determined was created and used as an existing database (MiCA-DB).

  Based on the number of bases of the 5 'and 3' end fragments measured in Test Example 4, the bacterial species and phylotype present in mouse feces were estimated using MiCA-DB. It was explained in Test Example 5 that the number of bases in the terminal fragment estimated by computer simulation was different from the actually measured value. Accordingly, terminal fragments having a base number of less than 100 bp and a measured value of 1000 bp or more were excluded from the search target, and a terminal fragment having a base number of 100 to 1000 bp was used as the search target.

  The estimation of the bacterial species and phylotype corresponding to the number of bases of the 3 'terminal fragment generated by Alu I treatment was performed using MiCA-DB (Table 6).

Claims (8)

Amplify DNA using the rRNA gene in the sample or complementary DNA obtained by reverse transcription of the rRNA gene as a template,
Generate 5 'and 3' terminal fragments by treating the amplified DNA with restriction enzymes,
The generated 5 ′ and 3 ′ end fragments are separated by electrophoresis,
Measure the number of bases of the separated 5 ′ and 3 ′ terminal fragments,
Measured from a database containing information on the estimated number of bases of the 5 'and 3' terminal fragments and the bacterial species from which the 5 'and 3' terminal fragments are derived. Select 5 'and 3' terminal fragments corresponding to the number of bases given,
Selecting the bacterial species from which the selected 5 ′ and 3 ′ terminal fragments are derived from the database;
Inferring the selected strain to be present in the sample,
A method for estimating the gut microbiota of a mouse.   Restriction enzyme treatment of the amplified DNA fragment was performed using AccII, AfaI, AfIII, AluI, BamHI, BcuI, BfaI, Bme1580I, BseGI, Bsh1236I, Bsp106I, BspTI, BstUI, Bsu36I, BsuRI, CfoI, CviRI, DdeI, DpnI (methA ), DpnII, EcoRI, FneDII, HaeIII, HapII, HhaI, Hinp1I, HinfI, HpaII, Hpy1881, Hpy188III, HpyCH4IV, HpyCH4V, HpyAV, Hsp92II, MaeI, MaeII, MboI, MIyI, MseI, MsII, MseI, MsII The method according to claim 1, wherein one or more selected from the group consisting of NlaIII, PalI, RsaI, Sau3AI, Sau96I, ScrFI, StuI, TaqI, ThaI, Tru9I, TsoI, Tsp509I, and TthHB8I and TthHB8I are used. The method described.   The method according to claim 1 or 2, wherein the restriction enzyme treatment of the amplified DNA fragment is performed using HhaI, MspI, HaeIII, and AluI. The database is the following:
Lactobasillus sp. ASF360
Lactobacillus murinus
Uncultured bacterium clone LKMC043
Akkermansia muciniphila ATCC BAA-835
Parabacteroides goldsteinii
And a DNA amplified by using the rRNA gene derived from the bacterial species as a template, respectively, by treating them with restriction enzymes using HhaI, MspI, HaeIII, and AluI, respectively:
Lactobasillus sp. ASF360
HhaI 5 'end fragment 596 bp, 3' end fragment 407 bp,
MspI 5 'end fragment 181 bp, 3' end fragment 80 bp,
HaeIII 5 'end fragment 325 bp, 3' end fragment 74 bp,
AluI 5 'terminal fragment 63 bp, 3' terminal fragment 182 bp,
Lactobacillus murinus
HhaI 5 'terminal fragment 256 bp, 3' terminal fragment 403 bp,
MspI 5 'terminal fragment 571 bp, 3' terminal fragment 76 bp,
HaeIII 5 'end fragment 247 bp, 3' end fragment 125 bp,
AluI 5 'end fragment 262 bp, 3' end fragment 178 bp,
Uncultured bacterium clone LKMC043
HhaI 5 'terminal fragment 100 bp, 3' terminal fragment 49 bp,
MspI 5 'end fragment 88 bp, 3' end fragment 35 bp,
HaeIII 5 'end fragment 458 bp, 3' end fragment 116 bp,
AluI 5 'terminal fragment 247 bp, 3' terminal fragment 170 bp,
Akkermansia muciniphila ATCC BAA-835
HhaI 5 'terminal fragment 98 bp, 3' terminal fragment 145 bp,
MspI 5 'terminal fragment 270 bp, 3' terminal fragment 130 bp,
HaeIII 5 'end fragment 219 bp, 3' end fragment 219 bp,
AluI 5 'terminal fragment 74 bp, 3' terminal fragment 94 bp,
Parabacteroides goldsteinii
HhaI 5 'terminal fragment 98 bp, 3' terminal fragment 135 bp,
MspI 5 ′ terminal fragment 93 bp, 3 ′ terminal fragment 120 bp,
HaeIII 5 'end fragment 915 bp, 3' end fragment 48 bp,
AluI 5 'terminal fragment 724 bp, 3' terminal fragment 170 bp,
The estimated base number of the 5 ′ and 3 ′ terminal fragments of
Is contained as information, The method of any one of Claims 1-3.   The method according to any one of claims 1 to 4, wherein the sample is prepared from mouse feces.   The method according to any one of claims 1 to 5, wherein the rRNA gene is a 16S rRNA gene.   The method according to any one of claims 1 to 6, wherein a complementary DNA obtained by reverse transcription of the 16S rRNA gene is used as a template.   The method according to any one of claims 1 to 7, wherein the electrophoresis is capillary electrophoresis.