JP2007060953A - Method for analyzing bacterial flora - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To analyze a gene expression pattern as a whole bacterial flora to evaluate the inner and outer environments of the bacterial flora without specifying the kind and expression quantity of each microorganism existing in the environments. <P>SOLUTION: This method for analyzing the bacterial flora is characterized by analyzing the appearing patterns of genome DNAs originated from the bacterial flora in an analysis target specimen with a solid carrier on which probes containing base sequences related to genome DNA fragments obtained by fragmenting genome DNAs originated from two or more kinds of bacteria are immobilized, and analyzing the inner and/or outer states of the bacterial flora in the analysis target specimen on the basis of the expression patterns. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for analyzing bacterial flora. More specifically, the present invention relates to a method for analyzing the bacterial flora and environmental conditions surrounding the bacterial flora by analyzing the genomic DNA pattern of the bacterial flora contained in the analysis target sample.

  Many types of bacteria are resident in an environment such as soil, forming a resident microflora. Similarly, many kinds of bacteria are present in the intestines of animals including humans, and these are called intestinal flora. The general method for analyzing bacteria is by analyzing the properties of microbial colonies growing on an agar medium. Currently, the proportion of microorganisms that can be cultured is estimated to be about 1%, and most microbial species are difficult to cultivate. The actual condition of the bacterial flora is not clear. Therefore, in the analysis of the bacterial flora by the culture method, only specific microbial species that can be cultured were evaluated.

  In recent years, with the development of molecular biology, analytical methods using the sequence of ribosomal genes shared by living organisms have come to be used. One method for analyzing bacterial flora is denaturing gradient gel electrophoresis (DGGE method), in which ribosomal genes amplified by PCR are separated and analyzed using the difference in base composition of ribosomal genes of each microorganism. As another method, there is a T-RFLP method in which ribosome genes amplified by PCR are similarly separated using the recognition specificity of restriction enzymes. Since these methods eliminate the need for culturing microorganisms by using the PCR method, more microbial species information can be obtained compared to the analysis by the culturing method. However, the PCR method has a problem that the amplification efficiency is biased due to uncontrollable factors such as the degree of coincidence between the primer sequence used and the DNA sequence of the microorganism and the base composition of the microorganism DNA sequence.

  On the other hand, microarray and chip analysis methods that achieve analysis of many genes have been developed. In the analysis of bacterial flora, the use of chips (see Patent Document 1) in which ribosomal genes corresponding to various microorganisms are immobilized, and arrays in which genomic DNA or cDNA is immobilized (see Patent Document 2) are considered. ing. However, these methods have a problem that they can be applied only to microorganisms whose ribosomal genes and genomic DNA (or cDNA) have already been clarified.

  In addition, it has also been proposed to search for new environmental purification microorganisms and to examine optimum purification conditions using an array in which genes that are commonly expressed by known environmental purification microorganisms are analyzed and immobilized thereon (Patent Document 3). reference). However, this method is premised on accumulated genome information and analysis of known environmentally-cleaning microorganisms.

  Probing a cDNA sequence requires information on each gene sequence and isolation of DNA fragments having those gene sequences. In addition, the design of oligonucleotide probes requires ORF sequence information and EST sequence information, and more preferably genomic sequence information. Acquisition of these sequence information requires a large amount of cost. Is limited.

  On the other hand, in recent years, there has been a report on production of a DNA array using a random genomic library (see Non-Patent Documents 1 and 2). In these methods, DNA fragments to be immobilized on a substrate are prepared by PCR from the prepared genomic library, and a DNA array is prepared. These arrays are all intended for genetic analysis of specific microorganisms (using a single organism's genomic DNA as a material) and are targeted for analysis of "mixtures of multiple bacteria" is not.

JP2001-149073A JP 2000-253886 JP 2003-517843 A Zeigler et al., Mol. Microbiol. 48: 1089-1105, 2003 Parro et al., Proc. Natl. Acad. Sci. USA 100: 7883-7888

  The development of microarrays has enabled comprehensive analysis of microorganisms present in the environment. However, as described above, it is not realistic to individually analyze the types of microorganisms constituting the bacterial flora and the expression levels of specific microorganisms. An object of the present invention is to evaluate the environment surrounding the bacterial flora by analyzing the genomic DNA appearance pattern of the entire bacterial flora without specifying the type and abundance ratio of individual microorganisms present in the environment. There is.

  As means for solving the above problems, the inventors propose the following method.

  First, a genomic library is prepared based on DNA extracted from bacterial flora under a specific environmental condition, and a microarray to which nucleic acid probes derived from this library are immobilized is prepared. A labeled nucleic acid prepared from DNA extracted from the environment to be analyzed is hybridized to this array, and the genomic DNA appearance pattern is analyzed based on the signal intensity from each fixed probe on the array. By analyzing this appearance pattern for bacterial flora in various environments and creating a database, the internal and / or external state of the bacterial flora to be analyzed is evaluated.

  That is, the present invention is a method for analyzing a bacterial flora in a sample to be analyzed, and a probe comprising individual base sequences of genomic DNA fragments obtained by fragmenting genomic DNA derived from two or more kinds of bacteria. Is used to analyze the appearance pattern of genomic DNA derived from the bacterial flora in the sample to be analyzed, and from the appearance pattern, the inside and / or outside of the bacterial flora in the sample to be analyzed is analyzed. The present invention relates to a method for analyzing a bacterial flora characterized by analyzing a state.

In certain embodiments, the method comprises the following steps:
a) For a genomic DNA fragment obtained by fragmenting genomic DNA derived from two or more kinds of bacteria, a solid-phase support on which a nucleic acid probe containing each individual base sequence is immobilized is prepared,
b) hybridizing a labeled DNA prepared from genomic DNA derived from bacterial flora in the sample to be analyzed to the solid-phased carrier;
c) Analyzing the appearance pattern of genomic DNA derived from the bacterial flora in the sample to be analyzed based on the obtained signal intensity, and analyzing the internal and / or external state of the bacterial flora in the sample to be analyzed from the appearance pattern To do.

  In another embodiment, the method includes the appearance pattern of genomic DNA derived from bacterial flora in the sample to be analyzed obtained in step c) and the appearance of genomic DNA derived from bacterial flora under known conditions. It further includes a step of comparing and analyzing the pattern.

  This comparative analysis process may be performed using a database in which the appearance pattern of genomic DNA derived from the bacterial flora under known conditions and the information on the internal and / or external state of the bacterial flora are linked and stored. preferable.

  In the method of the present invention, the DNA fragment nucleic acid probe obtained by fragmenting genomic DNA is preferably 25 bp to 200 kb in base length.

  Moreover, as the labeled DNA to be prepared, labeled cDNA prepared using mRNA as a template can also be used.

  The form and shape of the solid-phase support used in the present invention are not particularly limited, and microarrays, chips, membrane filters, beads, or the like using glass, metal, silicon, synthetic resin, or the like as a substrate can be employed.

  The solid-phase support is obtained by homogenizing a genomic DNA fragment obtained by fragmenting genomic DNA derived from two or more kinds of bacteria, and then immobilizing probes containing the individual base sequences. Alternatively, it may be a solid-phase support on which a probe containing each individual base sequence is immobilized after homogenization of cDNA derived from RNA derived from two or more bacteria. .

  As will be described later, the method of the present invention can be used for the evaluation of the intestinal flora and the flora of a sewage treatment plant.

  According to the present invention, it is possible to analyze and evaluate the bacterial flora and the environment surrounding the bacterial flora without specifying in advance the type and abundance ratio of individual microorganisms present in the environment.

1. Method for producing bacterial flora genome array A method for producing a bacterial flora genome array according to the present invention will be described with reference to FIG. Extract and isolate the genomic DNA of the desired bacterial flora according to a standard method. The isolated genomic DNA is cleaved into fragments each having a length of about 25 bp to 200 kb, preferably about 500 bp to 2000 bp. The genomic DNA can be cleaved by any of chemical methods, enzymatic methods, and physical methods. A genomic DNA fragment is ligated to an appropriate vector according to a conventional method, and then cloned into Escherichia coli or the like to prepare a genomic DNA library. Each genomic DNA fragment in the genomic DNA library is PCR amplified using a vector primer and purified, and then immobilized on a suitable substrate (glass, metal, etc.) using a spotter or the like, or PCR amplification is performed. Immediately immobilize each genomic fragment linked to the vector used to construct the genomic DNA library such as plasmid, cosmid, fosmid, BAC on a suitable substrate (glass, metal, etc.) using a spotter or the like. .

  It should be noted that the DNA that is the basis of the genomic DNA library immobilized on the array may be a mixture of DNAs extracted from bacterial flora under a plurality of environmental conditions. Furthermore, in order to reduce the DNA ratio of each microorganism in library preparation, it is possible to perform a homogenization process in advance.

2. Bacterial flora analysis method using bacterial flora genome array With reference to FIG. 2 and FIG. 3, a bacterial flora analysis method using the bacterial flora genome array according to the present invention will be described.

(1) First, genomic DNA is extracted and isolated from a sample (bacteria flora) according to a conventional method. Using the isolated genomic DNA as a template, a labeled DNA (for example, Cy3 or Cy5) is prepared using a labeled random primer and a DNA synthase. After the labeled DNA is purified, it is hybridized with the bacterial flora array prepared in the previous section, and the resulting signal intensity (fluorescence intensity) pattern is read and analyzed with a scanner. This signal intensity may be a relative intensity obtained by competitive hybridization on a microarray after labeling a sample to be analyzed and an appropriate reference sample with different dyes using a two-fluorescence staining method. The signal intensity read by the scanner may be adjusted for error or normalized for each sample as necessary. Further, the reliability limit line may be specified to exclude data having low correlation. Note that the bacterial flora in the environment to be analyzed does not have to have the same configuration as the bacterial flora from which the genomic library that constitutes the array is based, and may be expected to be similar.

(2) According to the above procedure, the DNA appearance pattern of the bacterial flora under various environmental conditions is analyzed, and this DNA appearance pattern is linked (linked) with the characteristic information regarding the internal and / or external state of the bacterial flora. Store in a database ("bacteria flora array database").

(3) Genomic DNA is extracted and isolated from the analysis target sample (test bacterial flora) in the same manner as in (1), and the signal intensity pattern is analyzed using the bacterial flora array prepared in the previous section. By comparing this signal intensity pattern with the data stored in the bacterial flora array database and extracting similar signal intensity patterns and their characteristic information, the bacterial flora in the sample to be analyzed and the environmental conditions surrounding it Is estimated.

3. Use of Bacterial Flora Genome Array An outline of a method for analyzing human intestinal flora using the present invention and a method for analyzing flora such as a sewage treatment plant are described below.

(1) Method for analyzing intestinal bacterial flora A microflora microarray is prepared from human bacterial flora collected from stool or the like. For this purpose, it is desirable that the bacterial flora-derived DNA used for the production of the bacterial flora microarray is less biased by microbial species due to factors such as individual, age, and sex. Therefore, a microflora microarray is prepared from a mixture of DNA derived from a plurality of bacterial flora, and more preferably from a bacterial flora-derived DNA that uniformly includes the age and sex described above. The individual bacterial flora pattern is obtained by hybridizing the prepared bacterial microarray to the labeled nucleic acid synthesized from the individual bacterial flora-derived DNA as the specimen. The obtained bacterial flora pattern is made into a database together with biological information such as individual age, sex, and health condition, and the bacterial flora pattern characterizing the biological information is extracted. After the creation of the database, it becomes possible to infer the health condition of the subject by comparing the bacterial flora pattern obtained from the new specimen with the bacterial flora pattern of the database. Intestinal bacteria are an important tool for grasping not only the health condition of a subject but also the dietary state such as vegetarian and carnivorous, age, and race.

(2) Bacterial flora analysis method for sewage treatment plants, etc. Bacterial flora microarrays are prepared from the bacterial flora recovered from the sewage treatment plant. At this time, it is preferable to prepare a microflora microarray by mixing bacterial flora DNA derived from a bacterial flora recovered from a plant having an abnormality in the processing ability as well as a high flora having a high processing ability. The produced bacterial flora microarray is hybridized with a labeled nucleic acid synthesized from the bacterial flora-derived DNA collected from the plant to obtain a bacterial flora pattern. At this time, for each bacterial flora pattern, the database of the processing capacity of the plant from which they are based is simultaneously created. By comparing the bacterial flora pattern registered in this database with the bacterial flora pattern indicated by the bacterial flora to be verified, it is possible to grasp and manage the state of the bacterial flora present in the current plant.

Example 1: Preparation of bacterial flora-derived genomic DNA library and preparation of DNA array As the bacterial flora used in the test, the bacterial flora (post-accumulation flora) that showed ammonia-degrading ability in wastewater by 20-day enrichment culture and before accumulation Bacterial flora was used.

  DNA was extracted by the CTAB method from the post-accumulation bacterial flora recovered by centrifugation. The obtained bacterial flora-derived genomic DNA was physically cleaved using a DNA fragmentation apparatus Hydroshear (manufactured by GeneMachines), and then subjected to agarose gel electrophoresis to collect a cleaved DNA fragment of about 2,000 bp. The recovered genomic DNA fragment was smoothed using KOD DNA polymerase (Toyobo Co., Ltd.), which is a DNA synthase. Next, the blunted genomic DNA fragment was ligated to a pUC19 plasmid vector cleaved with the restriction enzyme HincII and introduced into a competent cell of E. coli XLI-Blue MRF ′ strain.

  From Escherichia coli colonies grown on an agar medium, plasmid DNA was collected from an arbitrary 1,920 colony by the alkali-SDS method, and after accumulation, a bacterial flora genomic DNA library was obtained as a plasmid 1,920 clone.

  Of these 1,920 clones, a partial base sequence analysis was performed on 365 clones using the M13 primer as a vector primer. BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) was used as the base sequence analysis reagent, and Applied Biosystems 3730xl DNA Analyzer (Applied Biosystems / HITACHI) was used as the base sequence analyzer. The GC content of the obtained base sequence was calculated for each analyzed plasmid, and the GC content was classified into 60% or more, 40% or more and less than 60%, and less than 40% (FIG. 4). The GC content in the genome sequence is an important indicator in the species classification of microorganisms, and the base sequence obtained in this test showed various GC contents as shown in FIG. Included multiple bacterial species, and it was confirmed that bacteria with a GC content of 60% or more are preferred in the constructed post-aggregation bacterial flora genomic library.

  The DNA array was prepared by spotting the recovered plasmid 1,920 clones on a slide glass with a spotter SP-BioIII (manufactured by Hitachi Soft) and then treating at 80 ° C. to fix the plasmid on the slide glass.

Example 2: DNA array hybridization method Pre-accumulation bacterial flora-derived genomic DNA and post-accumulation bacterial flora-derived genomic DNA were used as materials for producing labeled DNA. The labeled DNA was synthesized using 3 μg of bacterial flora genomic DNA as a template and a random 8-mer primer and Klenow fragment (Invitrogen) which is a DNA synthase.

  DNA array hybridization was performed by a competitive hybridization method in which Cy5 labeled DNA and Cy3 labeled DNA were given to one DNA array prepared in Example 1. Following hybridization, excess labeled DNA was removed by washing the DNA array, and the fluorescence signal intensity of each fixed spot on the DNA array was measured using ScanArray (Gsi Lumonics), which is a fluorescence signal measuring device. Next, MA-plots were calculated for each fixed spot, calculating log2 (Cy5 / Cy3) with the vertical axis as the intensity ratio and log10 (Cy5 × Cy3) with the horizontal axis as the signal intensity, and the results of competitive hybridization were evaluated.

Example 3: Results of control hybridization Cy5-labeled DNA and Cy3-labeled DNA were synthesized using the same post-aggregation bacterial flora-derived genomic DNA as the template used for genomic DNA library preparation, and competitive hybridization was performed. . The MA-plot of this result is shown in FIG.

Example 4: Comparison of bacterial flora genomic DNA before and after enrichment culture
Cy5-labeled DNA was synthesized using genomic DNA derived from the pre-accumulation bacterial flora, and Cy3-labeled DNA was synthesized using genomic DNA derived from the bacterial flora after accumulation. The MA-Plot obtained by competitive hybridization is shown in FIG.

  Compared with FIG. 5 which is the result of the control hybridization, it was observed in FIG. 6 that spots were dispersed in a cluster shape. The signal intensity on the horizontal axis represents the abundance ratio of each bacterium in the bacterial flora, and the signal intensity ratio on the vertical axis represents a change in the abundance ratio of each bacterium in the bacterial flora before and after accumulation. Therefore, the cluster-like dispersion pattern of MA-Plot as obtained in FIG. 6 shows bacterial flora transition, and if the difference of bacterial flora is large, a large dispersion pattern is shown. In this study, a clustered dispersion pattern was obtained by comparing the post-accumulation bacterial flora with high ammonia-degrading ability and the pre-accumulation flora with low resolution in wastewater. In other words, it is possible to evaluate the bacterial group transition that constitutes the bacterial flora by analyzing the MA-Plot pattern using this technique, and to further analyze the activity ability exhibited by the bacterial flora.

Example 5: Analysis of base sequence of specific spot A plasmid showing a bacterium whose abundance ratio was increased by accumulation was selected as a base sequence analysis target. The selected spot group is shown in FIG. The base sequences of these 270 clones were analyzed, and the GC content of each base sequence was calculated for each of the obtained plasmids. The GC content was classified into 60% or more, 40% or more and less than 60%, and less than 40% (FIG. 8).

  Unlike FIG. 4 shown in Example 1, a large number of plasmids have a nucleotide sequence with a GC content of 40% or more and less than 60%, and the GC content is not a bacterium with a GC content of 60% or more, which was prior to accumulation. It was found that 40% or more and less than 60% of bacteria were increased by enrichment culture. In this study, the transition of bacterial flora before and after enrichment culture was analyzed, and the activity of the bacterial flora was observed to increase ammonia degradability in wastewater. That is, it is presumed that bacteria having a GC content of 40% or more and less than 60% increased by enrichment culture are important for ammonia degradation.

  This result indicates that by selecting clusters obtained by MA-Plot analysis and analyzing the nucleotide sequences, it is possible to select bacterial groups important for bacterial flora activity and obtain gene sequence information. Therefore, it is possible to select a plasmid having a genomic DNA of a bacterial species that shows a characteristic transition even if the bacterial species composed by this analysis method is unknown. Genetic markers that can be used for identification can be obtained.

  According to the present invention, it is possible to analyze and evaluate the bacterial flora and the environment surrounding it without specifying the types and expression levels of individual microorganisms present in the environment. In other words, the present invention can be used in various fields such as health check by analysis of intestinal bacterial flora, bacterial flora analysis of sewage treatment plants, and detection of contaminating microorganisms in foods.

FIG. 1 shows an outline of a method for producing a bacterial flora genome array. FIG. 2 shows an outline of a method for analyzing a flora using a bacterial flora genome array. FIG. 3 shows an outline of a method for analyzing flora using a bacterial flora genome array using a database. FIG. 4 shows the GC content distribution of the genomic base sequence obtained by base sequence analysis of the post-accumulation bacterial flora genomic library. FIG. 5 shows MA-Plot of control hybridization. FIG. 6 shows MA-Plots obtained by comparing bacterial flora genomic DNA before and after enrichment culture. FIG. 7 shows a spot group (in a circle) selected as a base sequence analysis target. FIG. 8 shows the GC content distribution of genomic base sequences obtained by base sequence analysis of specific spots.

Claims (10)

A method for analyzing bacterial flora in a sample to be analyzed,
Genomic DNA fragments obtained by fragmenting genomic DNA derived from two or more bacteria, derived from the bacterial flora in the sample to be analyzed, using a solid-phased carrier on which probes containing the individual nucleotide sequences are immobilized Analyzing the appearance pattern of the genomic DNA to be analyzed, and analyzing the internal and / or external state of the bacterial flora in the sample to be analyzed from the appearance pattern. The method of claim 1 comprising the following steps:
a) For a genomic DNA fragment obtained by fragmenting genomic DNA derived from two or more kinds of bacteria, a solid-phase support on which a nucleic acid probe containing each individual base sequence is immobilized is prepared,
b) hybridizing a labeled DNA prepared from genomic DNA derived from bacterial flora in the sample to be analyzed to the solid-phased carrier;
c) Based on the obtained signal intensity, the appearance pattern of genomic DNA derived from the bacterial flora in the sample to be analyzed is analyzed, and the internal and / or external state of the bacterial flora in the sample to be analyzed is analyzed from the expression pattern. To do.   A step of comparing and analyzing the appearance pattern of the genomic DNA derived from the bacterial flora in the sample to be analyzed obtained in the step c) and the appearance pattern of the genomic DNA derived from the bacterial flora under a known condition; The method of claim 2.   The comparative analysis step is performed using a database in which the appearance pattern of genomic DNA derived from a bacterial flora under known conditions and information on the internal and / or external state of the bacterial flora are stored in cooperation with each other. The method of claim 3, wherein the method is characterized.   The method according to any one of claims 1 to 4, wherein a DNA fragment obtained by fragmenting genomic DNA has a base length of 25 bp to 200 kb.   The method according to any one of claims 1 to 5, wherein the labeled DNA is labeled cDNA prepared using mRNA as a template.   The method according to any one of claims 1 to 6, wherein the solid-phase support is a microarray, a chip, a membrane filter, or a bead.   The evaluation method of an intestinal microflora using the method of any one of Claims 1-7.   It is characterized by using a solid-phase support on which probes containing individual base sequences are immobilized after homogenizing the genomic DNA fragments obtained by fragmenting genomic DNA derived from two or more bacteria. The method for evaluating an intestinal bacterial flora according to claim 8. The method according to claim 8, wherein a homogenization treatment is performed on cDNA derived from RNA derived from two or more kinds of bacteria, and then a solid-phase support on which a probe including each base sequence is immobilized is used. The method for evaluating the intestinal bacterial flora as described.

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