JP2009072168A - Method for distinguishing microorganisms - Google Patents

Abstract

【Task】
To provide a method for distinguishing microorganisms capable of distinguishing the hierarchy of a microflora to which microorganisms belong, based on the base sequence of the microorganism.
[Solution]
The method for discriminating microorganisms according to the present invention includes: (a) a first layer constituting the same layer having a partial base sequence that coincides with a first partial base sequence consisting of a predetermined number of bases of the first group constituting the same layer to which the microorganism belongs; The number of species in the second group other than the group (hereinafter referred to as “number of out-of-group matches”), and (b) a portion that matches the first partial base sequence in the first group constituting the same hierarchy. Based on the ratio of the number of species having a base sequence to the number of all species in the first group (hereinafter referred to as “in-group coincidence rate”) or only the number of out-of-group coincidence (a), the microorganism Can be discriminated, for example, species, genus, family, and the like.
[Selection figure] None

Description

  The present invention relates to a microorganism identification method for identifying a taxonomic hierarchy to which microorganisms belong. More specifically, the present invention relates to a microorganism identification method and classification method capable of determining the taxonomic hierarchy to which a microorganism belongs based on its base sequence, a probe used therefor, a microarray to which the probe is coupled, and the like. is there.

  Conventionally, organisms such as bacteria are generally cultivated by a culture method in a nutrient medium, morphological observation of formed colonies, Gram staining, oxygen demand, auxotrophy, biochemical examination, etc. Have been identified and classified based on However, this method requires a great deal of labor and cost because it is necessary to select a culture medium that is suitable for the microorganism to be discriminated for the culture, and in addition, when determining the taxonomic hierarchy, There is no objective judgment criterion such as morphological observation, and there are cases where it is based on subjective observation results, etc., so that objective discrimination is difficult, and there is a drawback that reliability may be lacking.

  Nowadays, it is now possible to analyze base sequences such as RNA and DNA from all microorganisms in the environment, and molecular biological techniques have become widely used for taxonomic analysis of microorganisms. Yes. As a phylogenetic index in a phylogenetic classification method based on the base sequence of RNA or DNA, a method using ribosomal RNA (rRNA) and its gene (rDNA) has been generally used (Non-patent Document 1). This is because ribosomal RNA genes are universally present in almost all microorganisms, have sufficient information to analogize past evolutionary (mutation) processes, and are appropriate differences that indicate species differences. And based on having enough diversity to serve as an indicator. Methods for using such rRNA and its gene (rDNA) for phylogenetic classification of microorganisms include determining the base sequences and comparing them, and using a specific sequence for a certain taxon as a DNA probe. Yes, this method is already in general use.

RDNA has different sizes depending on the organism, and is classified into, for example, 23S, 18S, 16S, 5S rDNA base sequences, etc. depending on the size, and a wide range using such small subunit rDNA base sequences. A tree of organisms can be created. For example, 18S in eukaryotes
For rDNA and prokaryotes, a phylogenetic method for all organisms using a 16S rDNA base sequence has been proposed (Non-patent Document 1). For this reason, 16S rDNA sequences with 10,000 or more bases have now been determined and registered and used in Japanese data banks and public gene banks such as GenBank.

  Small subunit rDNA base sequences, such as the abundant 16S rDNA base sequence accumulated so far, provide a specific sequence for a specific species or group of organisms by comparing them. be able to. By using such a specific sequence as, for example, a primer or a probe, it is possible to quickly and with high probability detect and identify a specific species or fungus group as a target.

  Information on specific probes consisting of such small DNA sequences can be obtained by distinguishing other groups from the group to which the microorganism belongs, or by selecting and classifying the desired microorganism group in the hierarchy of its species, genus, etc. Plays a decisive role in various applications.

  The first of many roles is, for example, investigating the characteristics of microorganisms in various environments, and this investigation is necessary to understand the material cycle and the role of microorganisms in various environments. It is useful in. In addition, the investigation of such microorganisms has already been carried out in various environments. For example, soil (Non-patent Document 2-12), heavy metal contaminated sediment (Non-patent Document 13), sludge (Non-patent Document 14), water (Non-patent documents 15 and 16), beach sand (non-patent document 17), Antarctic shelf deposits (non-patent document 18), marine nematode (non-patent document 19), and the like. Several of these studies show the importance of probes that can classify microbial groups at the genus level.

  Furthermore, in order to clarify the relationship between microorganisms in the human body and diseases and infectious diseases, investigations based on 16SrDNA are being conducted. As such investigations, for example, investigations on plaque (Non-Patent Documents 20 and 21), gastrointestinal mucosa (Non-Patent Document 22), excrement (Non-Patent Documents 23 and 24), vaginal fluid (Non-Patent Document 25) and the like have already been conducted. It has been implemented.

  The second role is the role of microbial DNA probe information in designing a microarray (for example, a DNA chip). Microarrays are now commonly used for gene expression analysis (Non-patent Document 25). DNA microarrays are used to detect the rough structure of the number and type of bacterial colonies and to detect microorganisms such as harmful bacteria.

  Thus, molecular methods based on nucleotide sequences are systematic and powerful. In addition, this molecular technique has the advantage of being able to evaluate a large number of uncultured microorganisms in a diverse environment.

The classification of microorganisms such as bacteria is a starting point for probe search, and the probe should have a high ability to distinguish a group classified by taxonomic hierarchy from another group. Numerous studies on molecular techniques have shown that 16S rDNA based studies are effective for microbial classification, and the data accumulated for 16S rDNA is registered as type strains (type cultures). Using these data, it is possible to classify most microorganisms. If the homology of a species belonging to one kind of bacteria with other bacteria can be specified, it is possible to select the bacterial species as a result. However, it is cumbersome to select only one group of higher taxonomic hierarchies: genus, family, order, class, phylum. In order to make such a choice possible, a new idea to overcome that difficulty is needed.
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  Therefore, as a result of intensive studies on a method for discriminating the taxonomic hierarchy to which microorganisms belong based on newly arrived ideas that systematically search for microorganism groups, the present inventors have found that the ribosomal gene of each microorganism includes The present invention was completed by finding that a common partial base sequence exists in a group, and by comparing the common partial base sequences, the taxonomic hierarchy of the microorganism can be determined and classified into the hierarchy to which it belongs. did.

  Therefore, the present invention discriminates microorganisms for a predetermined number of bases that match the partial base sequence of ribosomal DNA or RNA possessed by the microorganisms based on the tolerance of the number of out-of-group matches and the within-group match rate defined below. The main object is to provide a method for discriminating microorganisms comprising

  The present invention also provides a probe comprising a partial base sequence selected on the basis of the tolerance of the number of out-of-group matches alone or the combination of the number of out-of-group matches and the degree of in-group match, and microorganism discrimination / classification Another object is to provide a useful probe.

Furthermore, another object of the present invention is to provide a microarray in which the probe is bound by means such as immobilization.
Further, it can be understood that other objects and problems of the present invention will become clear from the description of the present specification.

In order to achieve the above object of the present invention, the present invention is a method for distinguishing microorganisms, wherein the taxonomic hierarchy of microorganisms is distinguished based on the base sequence of the microorganism,
a) In a second group other than the first group constituting the same layer having a partial base sequence that coincides with a first partial base sequence consisting of a predetermined number of bases of the first group constituting the same layer to which the microorganism belongs Tolerance of the number of seeds (hereinafter referred to as “the number of out-of-group matches”);
b) In the first group constituting the same layer, the ratio of the number of species having a partial base sequence that matches the first partial base sequence to the number of all species in the first group (hereinafter, The present invention provides a method for distinguishing microorganisms that identifies a hierarchy to which the microorganism belongs based only on a combination with a tolerance of “intra-group coincidence ratio” or a tolerance of the number of out-of-group matches. It should be noted that the number of out-of-group matches and the in-group match rate are set with tolerances that can be significantly determined in the taxonomic hierarchy to which the microorganism belongs.

  As a preferred embodiment of the present invention, there is provided a method for discriminating a microorganism in which the predetermined base number of the first partial sequence is set as the number of bases consisting of L from the i-th base of the base sequence of the microorganism belonging to the first group. Provided that i is an integer starting from 1 and corresponds to the number of bases constituting the base sequence, and L is not particularly limited as long as it is significant for taxonomic hierarchy discrimination of microorganisms. Generally, it is an integer of 10 to 50, preferably 10 to 40, more preferably 15 to 30).

  In a preferred embodiment of the present invention, the tolerance of the number of out-of-group matches is from 0 to 20, preferably from 0 to 10, more preferably from 0 to 5, particularly preferably. It should be zero. As a preferred embodiment of the present invention, the tolerance of the intra-group coincidence rate is 100% to 80%, preferably 100% to 90%, more preferably 100%.

  As a further preferred aspect, the present invention provides a method for distinguishing microorganisms that are ranked as shown in Table 1, for example, based on the tolerance of the number of out-of-group matches when the hierarchy is a species.

  According to a further preferred embodiment of the present invention, when the hierarchy is a genus, a family, an eye, a rope, or a gate, for example, in Table 2, based on the combination of the respective tolerances of the intra-group coincidence rate and the number of out-of-group coincidences. A method for distinguishing microorganisms comprising ranking as shown is provided.

  As a more preferred embodiment, the invention further ranks the ranked combinations of out-of-group matches and in-group matches as shown in, for example, Table 3, and the partial base sequence of the predetermined number of bases Provided is a microorganism discrimination method comprising attaching a symbol to the n-th base of a constituent base sequence according to the rank as shown in Table 3.

  As another embodiment, the present invention provides a probe in which the number of out-of-group matches and the in-group match rate are set with tolerances that can significantly distinguish the taxonomic hierarchy of microorganisms. In addition, the present invention provides a microbe discrimination / classification probe for discriminating the taxonomic hierarchy to which the microbe belongs and classifying into the hierarchy.

  Specifically, the present invention provides: a) the first layer constituting the same layer having a partial base sequence that matches the first partial base sequence consisting of a predetermined number of bases of the first group constituting the same layer to which the microorganism belongs. The number of species in the second group other than the first group, and b) the number of species having a partial base sequence that matches the first partial base sequence in the first group constituting the same hierarchy is the first. Providing probes consisting of partial base sequences that can identify and classify the taxonomic hierarchy to which the microorganism belongs based on the proportion of all species in the group and the probe for microorganism identification and classification To do.

  More specifically, in the probe according to the present invention and the microorganism discrimination / classification probe, the predetermined number of bases in the first partial sequence allows the number of bases that can significantly discriminate / classify the taxonomic hierarchy to which the microorganism belongs. The number of bases consisting of L from the i-th base of the base sequence of the microorganism (where i is an integer starting from 1 and corresponds to the number of bases constituting the base sequence, L is The number of bases that significantly discriminates and classifies the taxonomic hierarchy of microorganisms, and generally means an integer of 10 to 50, preferably 10 to 40, and more preferably 15 to 30).

  The present invention also includes (a) a probe or microorganism discrimination / classification probe ranked according to the tolerance of the number of out-of-group matches alone, (a) the tolerance of the number of out-of-group matches, and (b) Provided is a microarray in which probes ranked according to a combination with the tolerance of intra-group coincidence rate or probes for microorganism discrimination / classification are fixed. As a preferred embodiment of the present invention, the present invention provides a microarray in which probes that have been digging out or having a predetermined rank or higher than that rank or probes for microbial discrimination / classification are fixed.

Furthermore, as a preferred embodiment of the present invention, all groups or substantially all groups constituting the same hierarchy are selected so that the taxonomic hierarchy to which the microorganism to be determined belongs can be significantly distinguished and classified. Provided is a microarray in which the above-described probes or microorganism discrimination / classification probes are fixed.
As used herein, the term “substantially all groups” or a term related thereto consists of a necessary and sufficient number of all groups constituting the same hierarchy, which are necessary and sufficient for the purpose of the present invention. It means a group, which means a group consisting of numbers that can produce substantially the same effect as when all the groups are used.

  In addition, as a preferred embodiment of the present invention, all groups constituting substantially the same hierarchy or substantially all of the microorganisms can be classified and classified so that the taxonomic hierarchy to which the microorganism to be judged belongs can be more significantly distinguished and classified. In addition to the probe or microorganism identification / classification probe that covers a group, the probe or microorganism identification / classification that covers all groups or substantially all groups constituting a higher hierarchy of the genus to which the microorganism belongs A microarray is provided in which at least two types of probes or probes for microorganism discrimination / classification are fixed.

The present invention also provides a microarray production method for producing a microarray by fixing the probe or the microorganism discrimination / classification probe to the microarray by a conventional means such as solid phase, and using the produced microarray. A method of using a microarray for discriminating or classifying a taxonomic hierarchy to which microorganisms belong is provided.
Moreover, the present invention provides a microorganism screening method comprising screening microorganisms using the above-described microorganism discrimination method, probe and / or microarray.

  The microorganism discrimination method of the present invention has an effect that the taxonomic hierarchy to which each microorganism belongs can be distinguished and classified using a probe comprising a small base sequence of rDNA of each microorganism. In addition, if the microorganism discrimination method of the present invention is used, there is an effect that it is possible to comprehensively discriminate and search for all the bacterial species belonging to each bacterial group present in the sample.

The present invention also provides an arbitrary probe length and 16S
In addition to rDNA, there is an effect that a good probe can be searched for any gene other than 16S rDNA and its evaluation can be ranked.

  Furthermore, when the probe is mounted on the microarray, the present invention has an effect that all the bacterial species of the entire flora are covered and arranged on the microarray, and the entire bacterial flora can be distinguished and selected. In addition, the accuracy of discrimination of microorganisms can be increased by selecting a probe having a high tolerance for the above parameters. In addition, according to the present invention, the length of the prepared base sequence can be made almost constant, for example, about 550 bp, so that the accuracy of microbe discrimination can be made almost constant, and the base sequence can be compared with the probe. It is also possible to design a site for hybridization. In addition, there is an effect that the probes can be selected so that the proportions of the CG contents of the probes are uniform.

  In addition, the present invention can improve the accuracy of discrimination by simultaneously discriminating the genus and species to which the bacterial species belong. Further, by arranging the probes together so as to cover the genus and the species in the microarray, there is also an effect that the genus and the species can be simultaneously distinguished with high accuracy.

  According to the present invention, since the functions of bacteria are often almost the same at the genus level, it is possible to discriminate bacteria having only a certain function by searching by specifying the genus to which the microorganism belongs. It is. In addition, even when microorganisms cannot be searched at the species level with a probe that discriminates the species, it is likely that they have the same sequence at the genus level. This also has the effect of increasing the possibility that it can be discriminated.

  This invention discriminates the taxonomic hierarchy to which microorganisms belong, ie species, genus, family, order, class and phylum, and classifies the microorganism. The present invention relates to a novel method for distinguishing microorganisms that can be classified into a scientific hierarchy. In other words, the microorganism discriminating method according to the present invention is characterized in that the taxonomic hierarchy to which the microorganism belongs is discriminated / classified based on the partial base sequence common to the base sequences of the microorganism.

  In the method for discriminating microorganisms of the present invention, as a parameter for discriminating microorganisms, a partial sequence of rDNA of the microorganism is defined as follows: Coincidence Number of Outside Group (CNOG) and (Coincidence Rate Inside Group: CRIG).

  As used herein, the term “number of out-of-group matches” (CNOG) is a partial base sequence that matches a first partial base sequence consisting of a predetermined number of bases of the first group that constitutes the same hierarchy to which the microorganism to be measured belongs. Means the number of seeds in the second group other than the first group constituting the same hierarchy.

  More specifically, the “number of out-of-group coincidence” has, for example, a partial sequence identical to a partial base sequence consisting of a certain number of bases of 16S rDNA of a microorganism of a species belonging to a certain genus (genus) Means the number of other species belonging to the same genus or other genus. Similarly, the number of species belonging to the same family or other family having the same partial sequence as the rDNA partial base sequence of a microorganism of a certain species belonging to a family Means. The same applies to the upper hierarchy above the family.

  More specifically, for example, the same genus or genus having the same partial base sequence consisting of a certain number of bases of 16S rDNA of a species of microorganism belonging to a certain genus, This means that there are no other species belonging to other genera, that is, the number of out-of-group matches is “0”. In other words, it can be said that microorganisms having a common partial base sequence with an out-of-group match number of “0” belong to the same group in the same hierarchy with a very high probability. Therefore, it can be said that the common partial sequence having an out-of-group coincidence number of “0” can be used effectively for determining the hierarchy to which the microorganism, which is the object of the present invention, belongs.

  Similarly, the number of out-of-group coincidence of the partial base sequences of microorganisms belonging to a certain group is “1 to 5”, indicating that the bacterial species having the same partial base sequence as the partial base sequence of the microorganism is 1 in another group. It means that there is a high possibility that there are ~ 5 types. However, it is possible to determine that this possibility is unlikely to be a problem in determining the hierarchy of microorganisms.

  On the other hand, when the number of out-of-group matches is “11 to 20” or “more than 20”, 11 to 20 types of microorganisms belonging to another group having the same partial base sequence as the partial base sequence of the microorganism or 20 It means that there may be more than each species. Such a partial sequence having a large number of out-of-group matches can be judged to have low discrimination ability for the microorganism discrimination method that is the object of the present invention.

  Therefore, the tolerance of the number of out-of-group matches defined in the present invention is, for example, 0 to 20 out of group matches, preferably 0 to 15, more preferably 0 to 10, The number is preferably 0 to 5, particularly preferably 0. Further, depending on the object of the present invention, the lower limit may be increased or decreased. In some cases, the lower limit may be further subdivided or classified by a larger number.

On the other hand, the term “intra-group identity” (CRIG) as used herein is the number of species having a partial base sequence that matches the first partial base sequence in the first group constituting the same hierarchy. This means the ratio of the total number of all species in the first group.

  More specifically, the “in-group coincidence rate” means, for example, a bacterial species having the same partial sequence as a partial sequence consisting of a certain number of bases of rDNA of a species of microorganism belonging to a certain genus (genus) Means the ratio existing with respect to the number of all species belonging to the same genus. Further, when describing a family that is a higher hierarchy of a genus, it means a ratio in which a partial sequence of a certain kind of rDNA in a certain family is present with respect to other species in the family. The same applies to a higher hierarchy.

  More specifically, the fact that the in-group concordance rate of the partial sequence of rDNA of a certain group, for example, a genus species, is 100% means that all the bacterial species belonging to that group have such an identical part. It means having a base sequence. In other words, an intra-group identity rate of partial base sequences of microorganisms belonging to a certain group means 100% means that all of the bacterial species of the group have the same common partial sequence, This means that such microorganisms can be estimated with a very high probability that they belong to the same hierarchy. The same applies to other hierarchies.

  However, there is a possibility that a microorganism having the same partial sequence may also exist in another group belonging to the same hierarchy as the group of a certain hierarchy having a partial sequence with the intra-group coincidence rate of 100%. Therefore, in the present invention, in order to investigate such a possibility, it is possible to discriminate the hierarchy to which microorganisms belong with a certain degree of tolerance by combining the above-mentioned number of out-of-group matches with another in-group match rate. I have to.

  For example, when the intra-group coincidence rate 100% and the out-of-group coincidence number 0 are combined, it indicates that there is only one group having a common partial sequence with an intra-group coincidence rate of 100%. In this case, when the group is, for example, a genus, all the microorganisms having such a common partial sequence may be identified with a high tolerance of almost 100% that they belong to a single genus. it can. Further, when the intra-group match rate 100% and the out-of-group match number 1 are combined, there is a possibility that there are two groups having a common partial sequence with an intra-group match rate of 100%. Similarly, other combinations are the same. Therefore, in the present invention, it is preferable to combine the intra-group coincidence rate and the out-of-group coincidence number by determining how much tolerance is appropriate depending on the purpose. In other words, when strict discrimination of microorganisms is required, for example, when the intra-group match rate is 100% and the number of out-of-group matches is about 0 or 1, or when strict discrimination is not required, for example, the group It is also possible to combine the in-coincidence rate from less than 100% to 90% and the out-of-group coincidence number 0 or 1-10.

  Therefore, the tolerance of the intra-group coincidence rate defined in the present invention is, for example, that the intra-group coincidence rate is 100% to 80%, preferably 100% to 90%, and more preferably 100%. Further, depending on the object of the present invention, the lower limit may be increased or decreased. In some cases, the lower limit may be further subdivided or classified by a larger number.

  In this invention, in order to simplify the classification of the taxonomic hierarchy to which the microorganism belongs, the tolerance of the number of out-of-group coincidence alone or the combination of the tolerance of the within-group coincidence rate and the number of out-of-group coincidence, respectively. It is better to rank. The tolerance may be changed as appropriate according to the purpose, but in the classification of the taxonomic hierarchy of microorganisms in the present invention, for example, as shown in the following Table 1 and Table 2, ranking is performed with tolerance. be able to.

  Here, a partial base sequence of the base sequence of the microorganism used in the present invention will be described. In the present invention, from the predetermined base number (L) of the 16S rDNA base sequence of all the bacterial species whose bacterial species names are described in the Bergey's Manual of Systematic Bacteriology, 2nd Edition and registered as type cultures. For each partial sequence, the in-group coincidence rate and the number of out-of-group coincidences are respectively determined.

First, a type culture 16S rDNA library registered in a public organization such as GenBank is cleaved with two types of common primers (universal primers). Examples of common primers that can be used in the present invention include primers having the following sequences.
Primer 1: 5′-CCTACGGGAGGCAGCAG-3 ′ (SEQ ID NO: 2207)
Primer 2: 5'-CCGTCAATTCCTTTAAGTTT-3 '(SEQ ID NO: 2208)
Primer 3: 5'-CCGTCAATTCCTTTGAGTTT-3 '(SEQ ID NO: 2209)

  In this invention, for example, an about 550 bp sequence is extracted from an original 1600 bp 16S rDNA (3463 bases), and then bacteria having unclear bases such as symbols N, Y, and R are filtered from the extracted sequence. And 2206 species of species consisting only of A, C, G and T were extracted. 2206 species are classified into genus 697, family 186, order 75, class 31 and phylum 22. However, it should be noted that these numerical values may change depending on, for example, the discovery of unknown bacterial species or the identification of the base sequence of the type culture. In this case, the changed values are also the gist of the present invention. It should also be noted that it is obvious that it is included in

  A certain bacterial species (m-th species) extracted as described above (m = 1 to 2206. However, this number may change due to changes in type culture type, registered rDNA base sequence, etc. as described above. Or a group of fungi (hereinafter also referred to as “bacterial species / fungus group”), and from the i-th (i = 1 to n) of the base sequence (for example, L = about 10) ˜50) is selected, and whether or not this partial sequence is present in all remaining bacterial species and fungal groups is analyzed. The symbol “i = 1” means the first base number of the base sequence of each bacterial species / fungal group, and “i = n” means the nth base sequence of each bacterial species / fungal group. This means the base number of the number (that is, the last number of the base sequence). Further, “L = about 10-50” means that the number of bases is about 10-50. However, the symbol “L” is not particularly limited as long as it can be significantly used for discrimination of microorganisms, and the number can be arbitrarily determined. In the present invention, for example, about 10 ~ 50, preferably 12-40, more preferably 13-25.

  The i-th (i = 1) of the base sequence of a certain bacterial species and fungus group selected in this way, that is, L (for example, L = about 10-50 from the first base (i = 1) of the base sequence) ), The number of out-of-group matches is analyzed. In other words, how many bacterial species / fungal groups having the same partial sequence as the partial sequence (i = 1) are present in all the remaining bacterial species / fungal groups, and the number of the bacterial species / fungal groups by computer. To analyze. As a result, if the number is “0”, the number of out-of-group matches is defined as “0”. Similarly, if the number is more than 1, 2,..., 20 or more, the number of out-of-group matches is “1”, “2”,. Is defined as

  When the analysis for the partial sequence (i = 1) is completed, the second partial sequence (i = 2) of the base sequence is similarly analyzed for all remaining bacterial species and fungal groups. In this manner, the bases constituting the base sequence are sequentially analyzed, and the analysis for the final (i = n) base is completed. As a result, the out-of-group coincidence number is specified for all the bases constituting the base sequence of the (m = 1) strain / fungus group.

  As described above, when the analysis for all the bases of the (m = 1) bacterial species / fungal group is completed, next, the base sequence of another bacterial species (m = 2) or bacterial group is obtained. For all of the i-th (i = 1 to n) to L (for example, L = about 10 to 50) partial sequences, as in the case of the above-mentioned bacterial species (m = 1) or fungal group, Analyzes whether or not it exists in the bacterial species / bandit army. In this way, the number of out-of-group matches is determined for all partial sequences of the base sequence of the (m = 2) -th bacterial species / fungal group.

  When the analysis of the number of out-of-group matches for all the partial sequences of the base sequence of the (m = 2) th species is completed in this way, the base sequences of the bacterial species (m = 3 to 2206) or the bacterial group are subsequently obtained. In the same manner as described above, the i-th (i = 1 to n) to L (for example, L = about 10 to 50) partial sequences are analyzed. In this manner, the number of out-of-group matches is determined for all partial sequences of the base sequence of each bacterial species (m = 3 to 2206) or bacterial group.

Next, the number of out-of-group matches and their ranking for the upper layer will be described by taking a genus as an example.
First, the i-th base sequence of one bacterial species belonging to a certain genus (k = 1 to 697. The total number of genus in this invention may vary depending on the type of type culture, change of registered 16S rDNA, etc.) It is analyzed in the same manner as described above whether or not L (for example, L = about 10 to 50) partial sequences from i = 1 to n are present in other species of the genus. By this analysis, the number of bacterial species belonging to the same genus in which each partial sequence is present is calculated to calculate what percentage of the total number of all bacterial species in the same genus. Calculate the rate.

  In the same manner as described above, all bacterial species belonging to a genus (k = 2 to 697) other than the genus (k = 1) are analyzed, and the number of out-of-group matches and the within-group match rate of the genus are calculated. Further, in the same way, the number of out-of-group matches and the in-group match rate are analyzed for higher layers than the genus.

  In the present invention, it is preferable to rank according to Table 1 and Table 2 for the number of out-of-group matches and the within-group match rate of the bacterial species and fungus groups of each hierarchy analyzed as described above. Specifically, in the case of species, since there is no lower hierarchy, ranking is performed according to Table 1 for the number of out-of-group matches alone. In the case of a higher hierarchy than species, ranking is performed according to Table 2 based on the combination of the number of outside-group matches and the within-group match rate. It should be noted that the rank and tolerance can be changed according to the purpose, and should be appropriately determined.

  As an example of ranking as described above for the base sequence of the bacterial species, the base sequence of the bacterial species Holophaga foetida (SEQ ID NO: 1) is shown below for reference.

また、例えば、科の塩基配列について群外一致数と群内一致率との組合せにランク付けした例を下記に示す。

In addition, for example, the following is an example in which combinations of the number of matches outside the group and the match rate within the group are ranked for family base sequences.

  In this way, when the determination of the intra-group coincidence rate and the number of out-of-group coincidence and the ranking of all the partial sequences of all species, genera, families, eyes, classes, and gates are completed, according to Table 5 and Table 6. Symbolize all subsequences. This symboling for the partial sequences is performed by attaching a symbol corresponding to one leading base of each partial sequence. The results are shown in FIGS.

  For example, a case where L = 23 from the first base of Acidobacteriaceae, Holophaga, Holophaga foetida of SEQ ID NO: 1 (see 120 bases below) is described in more detail according to the above table.

  That is, in the above partial base sequence (note that the corresponding base sequences are described in symbols in the attached table. The same applies hereinafter), the first partial sequence (partial base sequence having 23 bases starting from the first base (probe) The symbol of the first base of the length: L = 23) is the same below) is “0”. This means that the partial sequence of 23 bases from the first base has a species, genus and family level rank of 0 as shown in Table 5, that is, the species, genus and family level. This means that the intra-group coincidence ratio at 80 is less than 80% or the number of out-of-group coincidence is larger than 20.

  Similarly, the symbol of the first base (that is, the second base) in the second partial sequence is also 0. Therefore, the rank of the species, genus and family level of this second partial sequence is 0. For example, since the 15th symbol of the base sequence is “u”, referring to Table 5, the rank at the species level is 3-1, the rank at the genus level is 7-1, the rank at the family level is 7 ~ 1. In other words, this means that the number of out-of-group matches at the species level is 6-20, the in-group match rate at the genus and family levels is 100% -80%, and the number of out-of-group matches is 0-20. I mean.

  Further, for example, since the 67th symbol of the base sequence is “A”, referring to Table 5, the rank at the species level is 5, the rank at the genus level is 9, and the rank at the family level is 9. That is, this means that the number of out-of-group matches at the species level is 0, the in-group match rate at the genus level and the family level is 100%, and the number of out-of-group matches is 0. In other words, all the bacterial species having a partial sequence consisting of 23 bases starting from the 67th symbol “A” in the above base sequence have a very high probability that they are bacterial species belonging to the same species, the same genus and the same family. It can be classified by. The same applies to other partial sequences.

  Even if the number of bases, that is, the probe length is, for example, 15 (L = 15), 19 (L = 19), 30 (L = 30), all the microorganisms are substantially the same. It is possible to determine the taxonomic hierarchy to which a species belongs. For example, when the number of bases is 15, 19, or 30, comparison and ranking of partial sequences of each base sequence is performed in the same manner as in the case where the number of bases described above is 23 (L = 23). The same processing is performed for determination. The other base numbers are substantially the same.

  In addition, as an example, for the same bacterial species Actinomycetaceae, Actinobaculum, Actinobaculum suis (SEQ ID NO: 3), ranking with 23 bases (L = 23) and 15 bases (L = 15) (notation of symbols) ) Are shown in Table 8-1 and Table 8-2.

  As described above, when the number of bases in the partial sequence is different, the identification of the intra-group match rate and the number of out-of-group matches for each base constituting the base sequence of the microorganism may be different, but even in this case, as described above Similar to the case of the probe length L = 23, it can be applied to the discrimination of microorganisms. That is, in this invention, when the number of bases in the partial base sequence, that is, the probe length is different, the number of target group matches (that is, the number of microorganism groups having a base sequence that matches the base sequence to be discriminated) is somewhat different. However, it is well suited for the purpose of discrimination of microorganisms within the probe length range defined above.

Below, in the case of probe length L = 23, L = 19, and L = 15, the target group coincidence number for 2206 bacterial species is as follows.
For example, in the case of probe length L = 23, when the tolerance rank of the out-of-group coincidence rank is rank 5, the target group coincidence number is 1799 bacterial species (82%), and rank 4 is 363 bacterial species (16%). Become. This means that 2162 bacterial species corresponding to 98% of the total 2206 bacterial species can be discriminated by setting the rank of tolerance for the number of out-of-group matches to ranks 5 and 4.

On the other hand, when the probe length L = 19, when the tolerance rank of the number of out-of-group matches is ranks 5 and 4, the target group hit numbers are 1770 (80%) and 386 (18%), respectively. When the probe length is set to L = 19, 2156 bacterial species corresponding to 98% of all 2206 bacterial species are discriminated by setting the rank of tolerance of the number of out-of-group matches to ranks 5 and 4. Will be able to.
Similarly, when the probe length is L = 15 and the rank of tolerance of the number of out-of-group matches is ranks 5 and 4, the target group hit numbers are 1732 (79%) and 406 (18%), respectively. When the probe length is set to L = 15, 2138 strains corresponding to 97% of all 2206 strains are discriminated by setting the rank of tolerance of the number of out-of-group matches to ranks 5 and 4. Will be able to.

On the other hand, in the case of probe length L = 23, for the genus (genus 697), family (family 186), eye (75th), class (31 class) and gate (22), out-of-group coincidence and intra-group coincidence The number of target group matches (rate) for each of ranks 9 and 8 based on the combination of the respective tolerances with the rate is as follows.
First, the number of target group matches (rate) for the genus is 574 genera (82%) at rank 9 and 81 genera (12%) at rank 8, and by setting ranks 9 and 8, 697 genera 94% can be discriminated.

Similarly, the number of coincidences (rates) for departments is 88 (47%) for rank 9 and 28 (15%) for rank 8, and 186 by setting ranks 9 and 8. 62% can be discriminated.
In addition, the number of objectives (rate) for eyes, ropes, and gates is 27 (36%) for rank 9 and 10 (13%) for rank 8, 49% for 75, 49 For rank 9, it is 12 classes (38%); for rank 8, it is 3 classes (10%); for class 31, 38%, and for gates it is 8 (36%) and rank 8 There are 2 gates (10%), and 46% of 22 gates can be discriminated.

  In this invention, the probe composed of the partial base sequence defined as described above can discriminate the hierarchy of the microbial flora to which the microorganism belongs based on the base sequence of the microorganism. The base sequence of the probe according to the present invention is selected based on the tolerance of the number of out-of-group matches alone or based on the combination of the number of out-of-group matches and the tolerance of each of the in-group matches. Can do.

  As described above, by using a probe selected based on the tolerance of the number of out-of-group matches alone, it is possible to determine a layer that is a seed among the layers to which the microorganism belongs. In addition, by using a probe selected based on the combination of the number of out-of-group matches and the in-group match rate, it is possible to discriminate hierarchies belonging to the genus, family, eye, class or gate among the hierarchies to which the microorganism belongs. can do.

  More specifically, by using the partial sequence defined above as a probe, it is possible to detect whether a nucleotide sequence complementary to the base sequence of the probe is present in a DNA or RNA sample. . Therefore, if the probe of the present invention is appropriately selected according to the purpose, a nucleotide sequence complementary to the base sequence of the probe present in a DNA or RNA sample can be detected. In other words, for example, if the objective is to investigate whether a species of a genus is present in a sample, a partial sequence common to all species in that genus is selected as a common probe. Strains having a nucleotide sequence complementary to the base sequence of the common probe present in can be detected. Taking the partial base sequence shown in Table 4 as an example, for example, by using a probe having a base sequence of 23 bases consecutive from the base having the 67th symbol “A”, all the probes belonging to the genus Holophaga It is possible to detect the bacterial species.

  As another method, a probe having a sequence complementary to the partial sequence may be artificially synthesized and used. In this case, the base sequence having the partial sequence hybridizes to the probe, so that it is possible to finally detect the bacterial species having the partial sequence.

  In this invention, in order to identify and detect the target probe, the probe is preferably labeled. As a probe labeling method, a method using an isotope or an immunomarker is generally used, but the labeling method can be performed by a conventional method commonly used in the technical field, and is not particularly limited. . The labeled probe can be denatured and separated into DNA single strands by means of heating or the like, and the target DNA or RNA can be hybridized.

  To detect the target probe, the target DNA or RNA may be immobilized on a membrane, or in situ. In this case, the DNA sequence and the RNA transcript can be observed by visualization by a conventional method commonly used in the technical field such as visualization techniques such as autoradiography and imaging.

  In addition, the probe according to the present invention can be immobilized on a surface of an inert carrier such as a glass slide or a chip as a high-density array such as a microarray, and target DNA can be hybridized with the probe. Any of these high-density arrays can be manufactured by techniques commonly used in the art. For example, as a method of creating a DNA chip, a method of synthesizing and purifying a DNA probe on a chip using a lithography technique used in the semiconductor industry, a method of forming a pre-synthesized DNA probe in a spot shape on a chip, A method of forming a DNA probe on a chip in a matrix array using an inkjet technique is known.

  The microarray prepared as described above has a partial base selected based on the tolerance of the number of matches outside the group alone or based on the combination of the tolerances of the number of matches outside the group and the percentage of matches within the group. For example, a probe comprising an array may be immobilized by conventional means.

  In the microarray according to the present invention, probes selected from all or substantially all groups constituting the same hierarchy are comprehensively immobilized on all or substantially all groups constituting the same hierarchy. It is good. In other words, for example, when searching for the species name of the microorganism to be discriminated, the microarray includes probes consisting of partial base sequences selected based on the tolerance of the number of out-of-group matches alone, All or substantially all the groups constituting a certain species are comprehensively immobilized. That is, in this invention, even if it is 2206 bacterial species extracted from the original 16SrDNA (3463 bases) of each bacterium or filtered, or less than that, the partial base sequence possessed by substantially all bacterial species. By using a microarray in which probes are arranged in an exhaustive manner, the species name of the microorganism to be discriminated can be specified.

On the other hand, in order to search for the genus species name of the microorganism to be discriminated using the microarray according to the present invention, the microarray includes the microorganisms based on the respective tolerances of the out-of-group coincidence number and the within-group coincidence rate. All or substantially all probes consisting of partial base sequences selected from all or substantially all genera (groups) constituting the genera (hierarchy) to which Is good.
In addition, in order to discriminate | determine a hierarchy higher than the genus to which the microorganisms of discrimination | determination belong, it can carry out by using the microarray which has arrange | positioned the probe similarly.

  In the present invention, in order to increase the discrimination accuracy of microorganisms, the microarray to be used includes, for example, the tolerance of the number of out-of-group matches or the combination of the degrees of tolerance of the number of out-of-group matches and the in-group match rate. Among the partial base sequences ranked based on the above, it is preferable to arrange a probe having a partial base sequence whose rank is higher than a predetermined rank.

  The microarray according to the present invention has an allowance of 0 to 20, preferably 0 to 10, more preferably 0 to 5, and particularly preferably 0. The ranked probe should be fixed.

  In the microarray according to the present invention, the number of out-of-group coincidence is 0 to 20, preferably 0 to 10, more preferably 0, and the intra-group coincidence rate is 100%. Probes ranked based on a combination of up to 80%, preferably 100% to 90%, more preferably 100% tolerance may be fixed.

  Furthermore, the microarray according to the present invention may be additionally coupled with probes that cover all or substantially all the layers different from the same layer. In addition, in the microarray, for example, when the same hierarchy belongs, it means that the upper hierarchy is a family that is the higher hierarchy of the genus. The same applies to other cases.

More specifically, for example, the object of the present invention is Holophaga
In the case of searching for bacterial species such as foetida, a rank (for example, rank 5) of probes representing each bacterial species, that is, the number of out-of-group matches with a high tolerance among the above-mentioned out-of-group matches as shown in Table 1. And 4) Probes consisting of the number of attached bases (i-th) to the number of bases (for example, L = 23) are preferably selected so as to cover all bacterial species and placed on the microarray. In addition, in this invention, although 2206 bacterial species are covered at present, the number of bacterial species changes due to an increase in type culture due to identification of unknown bacterial species in the future, and the current total number of bacterial species also changes. Even if it does, it is naturally included in the scope of the present invention. Of course, if the total number of bacterial species changes, the hierarchy to which the bacterial species belongs may also change.

  When the present invention is intended to detect a higher rank than the above bacterial species, that is, bacteria belonging to the genus, family, eye, class and / or gate, probes representing each hierarchy, that is, Table 1 and Among the combinations of the intra-group coincidence rate and the out-of-group coincidence as shown in any of 2 above, the number of bases (for example, the number of bases (for example, ranks 9, 8, and 7) assigned from the base (i-th) , L = 23) is preferably selected so as to cover all the layers and arranged on the microarray.

  Therefore, the present invention can provide a microarray in which probes are arranged so that only the species can be specified if the purpose is to search for only the species, and if the purpose is to simultaneously search for the species and the genus, It is also possible to provide a microarray in which probes are arranged so that species and genera can be identified simultaneously. Similarly, for the purpose of distinguishing microorganisms in a hierarchy higher than the genus, a microarray in which probes are arranged so that microorganisms belonging to the hierarchy can be specified can be provided.

  Currently, microarrays with tens of thousands of probes can be manufactured, so this invention also provides microarrays with all probes covering all levels arranged in the same array so that all levels can be searched simultaneously. can do.

  By using the above embodiment according to the present invention, not only known microorganisms but also unknown microorganisms can be screened. In particular, considering screening for unknown microorganisms, for example, if microorganisms belong to the same genus, they often have the same characteristic base sequence taxonomically, so we searched at the genus level. In this case, bacterial species belonging to the same genus and whose bacterial species name is unknown can be screened. If such an unknown bacterial species can be searched, the bacterial species can be further specified, and detailed information on the microorganism such as whether the bacterial species is a useful or harmful bacteria can be examined. it can.

Since the microorganism discrimination method according to the present invention can discriminate microorganisms at each hierarchical level, it is useful, for example, by searching at the genus level to determine whether or not bacteria having a specific function are present in the sample. It can also be used for screening unknown bacteria such as bacteria and harmful bacteria.
In addition, since the probe according to the present invention can discriminate microorganisms at each hierarchical level, microorganisms can be easily and quickly discriminated by, for example, being immobilized on a microarray such as a DNA chip. It is expected that it can be used widely in the fields of use and application.

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Claims (39)

A method for identifying microorganisms, wherein the hierarchy of the microflora to which microorganisms belong is determined based on the base sequence of the microorganisms,
a) In a second group other than the first group constituting the same layer having a partial base sequence that coincides with a first partial base sequence consisting of a predetermined number of bases of the first group constituting the same layer to which the microorganism belongs The number of seeds (hereinafter referred to as "the number of out-of-group matches"),
b) In the first group constituting the same layer, the ratio of the number of species having a partial base sequence that matches the first partial base sequence to the number of all species in the first group (hereinafter, "Intragroup match rate"),
A method for distinguishing microorganisms, characterized in that the hierarchy to which the microorganism belongs is determined based on the above.   A method of identifying a microorganism for determining a hierarchy of a microflora to which a microorganism belongs, based on a base sequence of the microorganism, and a first partial base sequence comprising a predetermined number of bases of a first group constituting the same hierarchy to which the microorganism belongs; Based on the number of species in the second group other than the first group constituting the same hierarchy having the same partial base sequence (hereinafter referred to as “number of out-of-group matches”), the hierarchy to which the microorganism belongs is determined. A method for discriminating microorganisms characterized by discriminating.   3. The method for distinguishing microorganisms according to claim 1, wherein the number of coincidences outside the group is 0 to 20.   4. The method for discriminating microorganisms according to claim 1, wherein the number of out-of-group matches is from 0 to 15.   5. The microorganism discrimination method according to claim 1, wherein the number of out-of-group matches is 0 to 10. 5.   6. The microorganism discrimination method according to any one of claims 1 to 5, wherein the number of out-of-group matches is zero.   2. The microorganism discrimination method according to claim 1, wherein the intra-group coincidence rate is 100% to 80%.   8. The microorganism discrimination method according to claim 1, wherein the intra-group coincidence rate is 100% to 90%.   9. The method for discriminating microorganisms according to claim 1, wherein the number of coincidence outside the group is 100%.   10. The microorganism identification method according to claim 1, wherein when the determination on the first partial sequence of the first group is completed, each group belonging to the second group is substantially reduced. Is a method for discriminating microorganisms characterized by discriminating similarly.   The method for distinguishing microorganisms according to any one of claims 1 to 10, wherein the predetermined number of bases in the first partial sequence is a number of bases that is significant for determining the hierarchy of microorganisms. .   The microorganism identification method according to any one of claims 1 to 11, wherein the predetermined number of bases of the first partial sequence is L from the i-th base of the base sequence of the microorganisms belonging to the first group. A method for distinguishing microorganisms characterized by the number of bases (where i is an integer starting from 1 and corresponds to the number of bases constituting the base sequence, and L means an integer of 10 to 50).   The method for discriminating microorganisms according to any one of claims 1 to 12, wherein when the hierarchy is a genus, a family, an eye, a rope, or a gate, a combination of the intra-group coincidence rate and the extra-group coincidence number A method for discriminating microorganisms, characterized by discriminating a hierarchy of microorganisms based on the method.   The method for discriminating microorganisms according to any one of claims 2 to 6 or any one of claims 10 to 12, wherein when the hierarchy is a species, the hierarchy of microorganisms is determined based on the number of out-of-group matches. A method for discriminating microorganisms characterized by discriminating.   14. The method for distinguishing microorganisms according to claim 1, wherein when the hierarchy is a genus, a family, an eye, a rope, or a gate, the intra-group coincidence rate and the extra-group coincidence number A method for discriminating microorganisms, characterized in that ranking is performed as shown in Table 1 based on combinations thereof.   15. The microorganism discrimination method according to any one of claims 2 to 6, wherein when the hierarchy is a species, ranking is performed as shown in Table 2 based on the number of out-of-group matches. A method for distinguishing a characteristic microorganism.   The combination of the ranked out-of-group coincidence according to claim 7 and the ranked within-group coincidence according to claim 8 in the microorganism discrimination method according to claim 15 is shown in Table 3. The method for distinguishing microorganisms is characterized in that the ranking is further performed, and a symbol as shown in Table 3 is attached to the nth base of the base sequence constituting the partial base sequence of the specified base number according to the rank .   The method for distinguishing microorganisms according to any one of claims 1 to 17, wherein the base sequence of the microorganisms is liposome DNA or RNA.   A method for classifying microorganisms, characterized in that the microorganisms are classified into respective levels to which the microorganisms belong by using the microorganism discrimination method according to any one of claims 1 to 18.   The probe used for the microorganisms discrimination method of any one of Claims 1 thru | or 18, or the microorganisms classification method of Claim 19.   A microorganism discrimination / classification probe used in the microorganism discrimination method according to any one of claims 1 to 18 or the microorganism classification method according to claim 19. A probe for discriminating the hierarchy of the microflora to which the microorganism belongs based on the base sequence of the microorganism,
a) In a second group other than the first group constituting the same layer having a partial base sequence that coincides with a first partial base sequence consisting of a predetermined number of bases of the first group constituting the same layer to which the microorganism belongs The number of seeds (hereinafter referred to as "the number of out-of-group matches"),
b) In the first group constituting the same layer, the ratio of the number of species having a partial base sequence that matches the first partial base sequence to the number of all species in the first group (hereinafter, The probe is selected based on the combination with the “in-group coincidence rate” or only the number of coincidence outside the group.   The probe according to any one of claims 20 to 22, wherein the probe is a probe selected based on a combination of the number of out-of-group matches and the in-group match rate, and belongs to the hierarchy. A probe capable of discriminating a hierarchy which is a family, an eye, a rope or a gate.   The probe according to any one of claims 20 to 23, wherein the probe is a probe selected based on the number of out-of-group matches, and a layer that is a seed among the layers can be determined. A probe characterized by being a probe.   The probe according to any one of claims 20 to 24, wherein the predetermined number of bases of the first partial sequence is a number of bases consisting of L from the i-th base of the base sequence of the microorganism belonging to the first group. A probe characterized in that i is an integer starting from 1 and corresponding to the number of bases constituting the base sequence, and L means an integer of 10 to 50. a) in a second group other than the first group constituting the same layer having a partial base sequence corresponding to the first partial base sequence consisting of a predetermined number of bases of the first group constituting the same layer to which the microorganism belongs The number of species (hereinafter referred to as “the number of out-of-group matches”);
b) In the first group constituting the same layer, the ratio of the number of species having a partial base sequence that matches the first partial base sequence to the number of all species in the first group (hereinafter, A probe selected based on only the combination number or the number of out-of-group matches, and selected from all or substantially all groups constituting the same hierarchy. Is a microarray in which all or substantially all of the groups constituting the same hierarchy are fixed comprehensively.   27. The microarray according to claim 26, wherein the probe is a probe ranked based on the number of out-of-group matches or a probe ranked based on a combination of the number of out-of-group matches and the in-group match rate. A microarray in which predetermined ranked probes are fixed.   28. The microarray according to claim 26 or 27, wherein probes ranked with an allowance of 0 to 20 out-of-group matches are fixed.   29. The microarray according to any one of claims 26 to 28, characterized in that probes ranked with a tolerance of 0 to 10 outside the group match are fixed. .   30. The microarray according to claim 26, wherein the number of out-of-group matches has a tolerance of 0 to 20, and the in-group match rate has a tolerance of 100% to 80%. A microarray in which probes ranked based on combinations are fixed.   28. The microarray according to claim 26 or 27, wherein the rank is based on a combination having a tolerance of 0 to 10 outside the group and a tolerance of 100% to 90% of the within-group coincidence rate. A microarray, wherein attached probes are fixed.   32. The microarray according to claim 31, wherein the number of out-of-group coincidence is 0, and probes ranked based on a combination in which the within-group coincidence rate is 100% are fixed. Microarray.   33. The microarray according to any one of claims 26 to 32, further comprising probes that cover all or substantially all of the same hierarchy and different hierarchy.   The microarray according to any one of claims 26 to 33, wherein the same hierarchy is a genus and the group is a species.   A method for producing a microarray, comprising producing the microarray by fixing the probe according to any one of claims 20 to 25 to the microarray by a conventional means such as immobilization.   35. A method of using a microarray, comprising distinguishing or classifying microorganisms using the microarray according to any one of claims 26 to 34.   A microorganism screening method, wherein the microorganism is screened using the microorganism discrimination method according to any one of claims 1 to 18.   A screening method for microorganisms, comprising screening microorganisms using the probe according to any one of claims 20 to 25. 35. A method for screening microorganisms, comprising screening microorganisms using the microarray according to any one of claims 26 to 34.

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