JP2009225793A - Method for determining antigen type based on sugar chain antigen of microorganism - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a technique for simply determining an antigen type based on a sugar chain antigen such as a capsular antigen of a microorganism. <P>SOLUTION: A method for determining an antigen type based on a sugar chain antigen such as a capsular antigen of a microorganism includes preparing a probe set containing probes each targeting a plurality of glycosyltransferase genes associated with the antigen type, bringing these probes into contact with a sample nucleic acid obtained from a test specimen and detecting nucleic acid hybridization to the probes. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a technique for discriminating antigen types such as serotypes based on sugar chain antigens such as capsular antigens of microorganisms such as pathogenic microorganisms.

  Many species belonging to Gram-negative bacteria and positive bacteria have been identified as causative bacteria such as various infectious diseases. For example, Streptococcus pneumoniae accounts for more than 60% of causes of pneumonia, causing pneumonia in children and the elderly and often causing death. Pneumonia is still the world's leading cause of death today, and Streptococcus pneumoniae is one of the most important bacteria causing respiratory infections. Legionella is a cause of pneumonia in the city. It is present especially in public baths and cooling towers of air conditioning equipment, causing pneumonia in the surrounding elderly and often causing death. Legionella is an important fungus that causes respiratory infections. For this reason, detection of Legionella spp. Is listed as an inspection item for water quality standards in facilities equipped with public baths in many prefectures.

  Many pathogenic microorganisms such as pneumococci and staphylococci have a capsule (including a mucus layer whose boundary with the outer periphery is unclear) outside the cell wall. Most of the capsule is a polysaccharide, and the capsular polysaccharide is known as a heat-resistant sugar chain antigen (capsule antigen). Most of Gram-negative bacteria including Legionella have sugar chains (Lipopolysaccaride, LPS, Lipooligosaccaride, LOS) in the outer membrane. In addition, LPS is called endotoxin because it has a strong antigenic activity against animals including humans. In particular, when LPS or the like is released by destruction of microorganisms mixed in the bloodstream, an excessive immune reaction is induced, causing fever and shock symptoms.

  Thus, the sugar chain exposed to the outside of the cell is used for serological typing (serotype) of bacteria. Serotype refers to the type of bacteria corresponding to the antiserum when subdividing the bacterial species based on the difference in the structure of the antigen in the bacterial cell, and generally all the bacteria have. It is represented by a combination of antigens (bacterial antigen, capsule antigen, flagellar antigen, etc.). Therefore, even microorganisms that do not have a capsule and that contain polysaccharides and have a highly specific cell surface layer can be classified by sugar chain antigen (serotype). is there.

  The serotype is information necessary for testing and diagnosis as well as antiserum and vaccine production. For example, 90 types of serotypes with different antigenicity (capsular antigen type) are known for Streptococcus pneumoniae based on the diversity of the capsule. Since an antibody that recognizes the capsular antigen of Streptococcus pneumoniae is effective in protecting infection, vaccines that produce such an antibody in the human body have been developed. Currently, a 23-valent vaccine that is a mixture of 23 types of capsular polysaccharides corresponding to 23 types of capsular serotypes, and 7 types of capsular polysaccharides corresponding to the 7 main types of capsular serotypes. A 7-valent vaccine conjugated with a protein is provided.

  The effectiveness of these vaccines relies on containing capsular polysaccharides that cover the capsular serotypes of pneumococci that are then prevalent. However, pneumococci that are prevalent vary depending on the country and region, the type and age of the disease, and the time. Therefore, in the production of vaccines and the like, the capsular serotypes of pathogenic microorganisms that are likely to be infected must be continuously surveyed.

  Conventionally, as a method for identification of capsular antigens of such pathogenic microorganisms and discrimination of serotypes containing capsular antigens, there are methods using antibodies, for example, latex agglutination reaction and capsular swelling reaction. . In addition, a method for discriminating a sugar chain antigen type by multiplex PCR using a specific region of a gene specific for each sugar chain antigen type as a primer site is also disclosed (Non-patent Document 1). Furthermore, a method of amplifying the same target by PCR and performing hybridization using a DNA microarray is also disclosed (Non-patent Document 2).

  The Legionella genus is known to have 46 serotypes and 70 serotypes, but 6 serotypes classified as Legionella pneumophila are particularly highly pathogenic to humans. As for detection of Legionella, there are a method of detecting by culturing using an artificial medium, and a method of detecting a sugar chain antigen (urinary antigen) excreted in the urine of an infected person with an antibody for only serotype 1.

Dias et al., CA et al., J. Med. Microbaiol. 2007 Sep; 56: 1185-1188 Quan et., Al. J / Microbiol .. Methods, 68 (2007) 128-136

  However, the method using an antibody is not practically used for the following reasons. That is, in order to determine the capsular serotype of Streptococcus pneumoniae, it is necessary to prepare 90 kinds of sera, and there is a problem that the acquisition cost and labor are very high. Moreover, even if there are 90 types of serotypes, they may have common antigens and are difficult to determine due to cross-reactivity.

  In addition, when a specific region of a specific gene that is not directly related to the capsular antigen, such as 16S rDNA, is detected by capsular antigen type, it may become impossible to discriminate if a gene mutation occurs in the primer site. is there. In addition, a specific region of a gene specific for each serotype does not always exist for each capsule antigen type, and at present, only a limited capsule antigen type can be identified.

  As for Legionella, the method using urinary antigen is specialized only for serotype 1, and when it is matched by infection by other types, detection by culture is performed. Legionella is an artificial medium. Due to slow growth in the culture, detection by culture methods is generally difficult.

  From the above, to date, no realistic method has been established for easily distinguishing the capsular antigen type of microorganisms including pathogenic microorganisms.

  Accordingly, an object of the present invention is to provide a technique for easily discriminating an antigen type based on a capsular antigen of a microorganism.

  The present inventors paid attention to the diversity of polysaccharides found in serotypes based on sugar chain antigens such as capsules and LPS and glycosyltransferases involved in sugar chain antigen synthesis. And the knowledge that a sugar chain antigen type was distinguishable was obtained using this diversity. The present inventors have completed the present invention based on these findings. According to the present invention, the following means are provided.

According to the present invention, there is provided a method for discriminating an antigen type based on a sugar chain antigen of a microorganism, comprising preparing a probe set including probes each targeting a plurality of glycosyltransferase genes associated with the antigen type; Contacting the probe with a sample nucleic acid obtained from a subject;
And a step of detecting nucleic acid hybridization to the probe.

  In the discrimination method of the present invention, it is preferable that the plurality of glycosyltransferase genes are contained in a sugar chain antigen synthesis gene cluster. The probe set may further include a probe targeting a gene other than the glycosyltransferase gene contained in the sugar chain antigen synthesis gene cluster. Further, the microorganism is preferably one or more selected from pneumococci, Legionella, Salmonella, Escherichia coli, Shigella, and Vibrio. Furthermore, the microorganism preferably contains Streptococcus pneumoniae, and the glycosyltransferase gene is preferably a gene contained in the capsular polysaccharide synthesis gene cluster. It is also preferable that the microorganism contains Legionella. In the discrimination method of the present invention, the probe set preferably includes an antigen-specific probe that targets a region specific to the antigen type in the glycan antigen synthesis gene of the microorganism.

The microorganism preferably includes pneumococci, and the probe is preferably selected from the group consisting of the following sequences (a) and (b).
(A) a nucleotide sequence according to any one of SEQ ID NOs: 1 to 222 or a complementary sequence thereof (b) a base deletion, substitution or substitution with respect to any one of the sequences of SEQ ID NOs: 1 to 222 or their complementary sequences A sequence comprising a mutated sequence to which an addition has been made and having the function of the probe. The microorganism preferably comprises Legionella, and the probe is preferably selected from the group consisting of the following sequences (c) and (d): .
(C) the nucleotide sequence according to any one of SEQ ID NOS: 223 to 671 or a complementary sequence thereof (d) a nucleotide deletion, substitution or substitution with respect to any one of the sequences of SEQ ID NOS: 223 to 671 or their complementary sequences A sequence comprising an added mutant sequence and having the function of the probe In the discrimination method of the present invention, the probe set preparation step comprises a plurality of the probe sets corresponding to a plurality of the antigen types on a solid phase carrier. It may be a step of preparing an array to be held.

According to the present invention, a probe set comprising a solid phase carrier and a probe each targeting a plurality of glycosyltransferase genes that are held on the surface of the solid phase carrier and associated with an antigen type based on a sugar chain antigen of a microorganism, An array is provided. In the array of the present invention, the plurality of glycosyltransferase genes are preferably included in a sugar chain antigen synthesis gene cluster, and the microorganism is selected from pneumococci, Legionella, Salmonella, Escherichia coli, Shigella, and Vibrio. Preferably, the microorganisms include pneumococci, and preferably include Legionella. It is also preferable to hold a plurality of probe sets corresponding to a plurality of the antigen types. The probe set may include an antigen type-specific probe that targets a region specific to the antigen type in the sugar chain antigen synthesis gene of the microorganism. Furthermore, the probe set may further include a probe targeting a gene other than the glycosyltransferase gene contained in the sugar chain antigen synthesis gene cluster.
Further, the microorganism may include pneumococci, and the probe may be selected from the group consisting of the sequences (a) and (b). The microorganism may include Legionella, and the probe may be selected from the group consisting of the sequences (c) and (d).

  According to the present invention, a solid phase carrier and an antigen type-specific probe that is held on the surface of the solid phase carrier and targets a region specific to an antigen type based on a sugar chain antigen in a sugar chain antigen synthesis gene of a microorganism. An array is provided. Furthermore, a probe targeting a gene other than the isotransferase gene in the sugar chain antigen synthesis gene cluster may be provided. In the array of the present invention, the antigen type-specific probe selected from the group consisting of the sequences (a) and (b) can be provided so that two or more antigen types can be discriminated. In the array of the present invention, the probe selected from the group consisting of the sequences (c) and (d) can be provided so that two or more antigen types can be discriminated.

  According to the present invention, there is provided a method for preparing a probe used for discriminating an antigen type based on a sugar chain antigen of a microorganism, the bases obtained for a plurality of glycosyltransferase genes associated with the synthesis of the sugar chain antigen of the microorganism. There is also provided a production method comprising a step of determining base sequences of a plurality of probes targeting each of the plurality of glycosyltransferase genes based on sequence information. The production method of the present invention may further include a step of immobilizing the produced probes on the surface of the solid phase carrier.

  According to the present invention, there is provided a probe set for discriminating an antigen type based on a sugar chain antigen of a microorganism, and a plurality of glycosyltransferase genes included in the sugar chain synthesis gene cluster of the microorganism and associated with the antigen type A probe set is provided that includes probes that each target. The microorganism includes pneumococci, and the probe includes an antigen type-specific probe that targets a region specific to the antigen type selected from the group consisting of the sequences (a) and (b). Can do. The microorganism may include Legionella, and the probe may include a probe selected from the group consisting of the sequences (c) and (d).

It is a figure which shows the concept of this invention. It is a figure which shows the probe for Streptococcus pneumoniae of this invention. It is a figure which shows the probe for Streptococcus pneumoniae of this invention. It is a figure which shows the probe for Streptococcus pneumoniae of this invention. It is a figure which shows the probe for Streptococcus pneumoniae of this invention. It is a figure which shows the probe for Streptococcus pneumoniae of this invention. It is a figure which shows the probe for Streptococcus pneumoniae of this invention. It is a figure which shows the probe for Streptococcus pneumoniae of this invention. It is a figure which shows the probe for Streptococcus pneumoniae of this invention. It is a graph which shows the fluorescence intensity detected per serotype 1 in the Example. It is a graph which shows the fluorescence intensity detected about the serotype 12F in an Example. It is a graph which shows the fluorescence intensity detected about the serotype 14. FIG. It is a graph which shows the fluorescence intensity detected about the serotype 6B. It is a graph which shows the fluorescence intensity detected about the serotype 18B. It is a graph which shows the fluorescence intensity detected about serotype 18C. It is a graph which shows the fluorescence intensity detected about serotype 11C. It is a graph which shows the fluorescence intensity detected about serotype 11F. It is a figure which shows the probe for Legionella discrimination | determination of this invention. It is a figure which shows the probe for Legionella discrimination | determination of this invention. It is a figure which shows the probe for Legionella discrimination | determination of this invention. It is a figure which shows the probe for Legionella discrimination | determination of this invention. It is a figure which shows the probe for Legionella discrimination | determination of this invention. It is a figure which shows the probe for Legionella discrimination | determination of this invention. It is a figure which shows the probe for Legionella discrimination | determination of this invention. It is a figure which shows the probe for Legionella discrimination | determination of this invention. It is a figure which shows the probe for Legionella discrimination | determination of this invention. It is a figure which shows the probe for Legionella discrimination | determination of this invention. It is a figure which shows the probe for Legionella discrimination | determination of this invention. It is a figure which shows the probe for Legionella discrimination | determination of this invention. It is a figure which shows the probe for Legionella discrimination | determination of this invention. It is a figure which shows the probe for Legionella discrimination | determination of this invention. It is a figure which shows the probe for Legionella discrimination | determination of this invention. It is a graph which shows the fluorescence intensity detected about Legionella serotype 1 type (ST1) in the Example. The upper row shows the results for all probes, and the lower row shows the results for probes selected as effective for serotype discrimination. It is a graph which shows the fluorescence intensity detected about serotype 2 (ST2). The upper row shows the results for all probes, and the lower row shows the results for probes selected as effective for serotype discrimination. It is a graph which shows the fluorescence intensity detected about serotype 3 (ST3). The upper row shows the results for all probes, and the lower row shows the results for probes selected as effective for serotype discrimination. It is a graph which shows the fluorescence intensity detected about serotype 4 (ST4). The upper row shows the results for all probes, and the lower row shows the results for probes selected as effective for serotype discrimination. It is a graph which shows the fluorescence intensity detected about serotype 5 type (ST5). The upper row shows the results for all probes, and the lower row shows the results for probes selected as effective for serotype discrimination. It is a graph which shows the fluorescence intensity detected about serotype 6 (ST6). The upper row shows the results for all probes, and the lower row shows the results for probes selected as effective for serotype discrimination. It is a graph which shows the fluorescence intensity detected about colon_bacillus | E._coli. It is a graph which shows the fluorescence intensity detected about the Klebsiella pneumoniae. It is a graph which shows the fluorescence intensity detected about Pseudomonas aeruginosa.

  The present invention relates to an array for distinguishing antigen types (hereinafter simply referred to as capsule antigen types) based on sugar chain antigens such as capsular antigens of microorganisms and LPS, and a method for distinguishing sugar chain antigen types using the arrays It is related. The concept common to these embodiments of the present invention will be described with reference to FIG.

  As shown in FIG. 1, the difference in the sugar chain structure of a sugar chain antigen presented on the cell surface of a microorganism, that is, a polysaccharide such as a capsule polysaccharide or LPS on the cell surface, is expressed as a capsule in the microorganism. It can be related to the difference in a sugar chain antigen synthesis gene such as a polysaccharide synthesis gene. The sugar chain antigen is derived from a sugar chain structure in which sugars are sequentially bound in a certain order, and it is also known that such sugar chain structures are extremely diverse in each sugar chain antigen type. Such diversity of sugar chain structures can be related to the structure of various genes related to the synthesis of sugar chain antigens (including the presence or absence of various mutations in each gene). . Especially, it can link | relate with the structure and combination of the various glycosyltransferase genes which couple | bond together the structure sugar of a sugar_chain | carbohydrate antigen sequentially.

  For example, it is known that such a sugar chain antigen synthesis gene group exists as a Capsule Polysaccharide Synthesis gene cluster (CPS cluster) in Streptococcus pneumoniae (http: // www .sanger.ac.uk / Projects / S_pneumonie / CPS /). Moreover, it is known that such a sugar chain antigen synthesis gene group exists as a polysaccharide synthesis gene cluster in Legionella (Luneberg E et al., Mol. Microbiol. 2001 Mar; 28 (5): 1259-1271)

  In the present invention, a plurality of glycosyltransferase genes associated with the sugar chain antigen type of a microorganism are used to determine the sugar chain antigen type. That is, such a set of a plurality of glycosyltransferases is used as an element for discriminating a sugar chain antigen type. That is, by using a set of probes each targeting a plurality of glycosyltransferases and detecting hybridization with these probes, the microbial sugar chain antigen type can be more objectively and more directly compared to the conventional method. In addition, it can be more easily determined. In addition, since the determination is made based on the presence or absence of a predetermined plurality of glycosyltransferase genes, it can be expected to obtain higher discrimination accuracy than when the determination is made based on the presence or absence of a specific region of a specific gene. Furthermore, it can be expected that even when the sugar chain antigen of a microorganism changes due to acquisition of drug resistance, it can be easily detected. For this reason, it is possible to easily cope with a novel sugar chain antigen type after acquiring the drug resistance.

  Further, in the present invention, an antigen-specific probe that targets a region specific to the antigen type of a sugar chain antigen synthesis gene such as a capsular polysaccharide synthesis gene of a microorganism is used to discriminate the sugar chain antigen type. You can also. According to such a probe, since it is specific to a sugar chain antigen type, for example, it can be used by itself (single) for determining a sugar chain antigen type, as well as a plurality of glycosyltransferase genes associated with the sugar chain antigen type. The discrimination accuracy can be further increased by using the probe set with the target.

  Furthermore, in the present invention, a probe targeting a gene other than the glycosyltransferase gene belonging to the microorganism sugar chain antigen synthesis gene cluster can be used to discriminate the sugar chain antigen type. This is because such a gene and a combination thereof may have characteristics capable of distinguishing sugar chain antigen types.

  In addition, according to the present invention, since various kinds of probes are appropriately combined to form an array held on a solid support, a plurality of capsule antigen types can be discriminated at once. Furthermore, since discrimination is performed using probe hybridization, discrimination can be performed within a certain tolerance even if a genetic variation occurs in the site.

  Furthermore, according to the present invention, since it is possible to easily and comprehensively determine the sugar chain antigen type, for example, the capsular antigen type determination of pneumococcus, which is a pneumoniae, is routinely performed. Is also possible. As a result, a highly effective pneumonia-preventing vaccine capable of inducing antibody production against pneumococci with high possibility of infection can be produced. That is, the array and capsular antigen type discrimination method of the present invention can be used for the production of vaccines for preventing infectious diseases such as pneumonia.

  Furthermore, according to the present invention, since it is possible to easily and comprehensively discriminate sugar chain antigen types, for example, capsular antigen type discrimination of Legionella bacteria that cause pneumonia is routinely performed. It becomes possible. As a result, Legionella bacteria can be detected more easily than at present. Furthermore, Legionella bacteria other than serotype 1 that are difficult to detect at present can be detected. That is, it is a very significant detection method from the viewpoint of infection prevention and infection route identification.

  Hereinafter, various embodiments of the present invention will be described in detail.

(array)
The array of the present invention comprises a solid phase carrier, and a probe set comprising probes each targeting a plurality of glycosyltransferase genes that are held on the surface of the solid phase carrier and associated with an antigen type based on a capsular antigen of a microorganism. Can be provided.

  The array of the present invention is used for discriminating the sugar chain antigen type of microorganisms. The array of the present invention can target animals such as domestic animals and pets in addition to humans. In addition, examples of the subject include biological samples derived from these animals. For example, blood, spinal fluid, sputum, gastric fluid, vaginal secretions, bodily fluids such as mucus in the mouth, excrements such as urine and stool, and the like, which may contain microorganisms. In addition to food and beverages that are subject to infection and contamination, all media that can be attached to microorganisms, such as various appliances and indoor items, water in the environment such as water and hot spring water, etc. And other media that can cause bacterial contamination.

  In the present specification, the “sugar chain antigen” is an antigen based on a sugar chain (polysaccharide) present on the cell surface of a microorganism having a sugar chain antigen. The sugar chain antigen includes capsular membrane and endotoxin (lipopolysaccharide (LPS), lipo-oligosaccharide (LOS)). The capsule also includes a mucous envelope. Pneumococcus (gram positive) (pneumonia, otitis media, etc.), staphylococci (gram positive) (pneumonia, sepsis, meningitis, food poisoning, skin disease), hemolytic streptococci (gram positive) (pharynx) Inflammation, sepsis, fulminant infection, skin disease), oral streptococci (gram positive) (caries, endocarditis), gonorrhea (gram negative) (gonorrhea, pharyngitis), meningococcus (gram negative) ) (Meningitis), Klebsiella pneumoniae (typically Friedrendel bacilli, gram positive) (pneumonia), Bacillus spp (typically anthrax and cereus, gram positive) (pneumonia, sepsis, food poisoning), Clostridium perfringens (gram positive) (gas gangrene), Mycobacterium tuberculosis (gram positive) (tuberculosis), Campylobacter (gram negative) (enteritis), Helicobacter (typically H. pylori, gram negative) (gastric ulcer), influenza (Gram negative) (pneumonia, meningitis), Bordetella pertussis (gram negative) (pertussis, sore throat), Pseudomonas (typically Pseudomonas aeruginosa, Gram negative) (sepsis), E. coli (typically Enterohemorrhagic Escherichia coli O157, gram negative) (intestinal infection, ectopic infection, sepsis, pneumonia, sepsis), Salmonella (gram negative) (sepsis, intestinal infection), Vibrio spp. (Gram negative) (food poisoning, sepsis ) And the like. These are also pathogenic microorganisms, each of which is an infectious disease-causing fungus. The capsule is observed in both gram positive and gram negative bacteria. Examples of pathogenic microorganisms effective for the prevention and diagnosis of infectious diseases for which it is effective to specify capsular antigens include pneumococci, Haemophilus influenzae and Vibrio spp. For example, Streptococcus pneumoniae is classified into 90 antigen types (serotypes) according to the capsule antigen. LPS and LOS are basically possessed by all Gram-negative bacteria. Examples of pathogenic microorganisms that are effective for identifying LPS and LOS include Legionella, Escherichia coli, and Salmonella. There are 70 known antigenic types (serotypes) of Legionella.

  The array of the present invention is not only preferable for discriminating the sugar chain antigen type of such pathogenic microorganisms but also preferable for diagnosis of infectious diseases using these pathogenic microorganisms as causative bacteria.

(Solid phase carrier)
The solid phase carrier for holding the probe is not particularly limited. Any material capable of causing the intended hybridization may be used. The material and form of the solid support are not particularly limited. Examples of such a solid support include a flat plate such as a glass substrate, a plastic substrate, and a silicon substrate, a spherical body such as a bead, a three-dimensional structure having irregular or regular irregularities on the surface, a rod shape, a hollow tube, and the like. The linear body is mentioned. The surface of the solid phase carrier may be subjected to a treatment suitable for immobilizing the probe. For example, a functional group for probe adsorption or covalent bond may be introduced. The surface treatment of the solid phase carrier will be described later.

(Probe set)
The probe set used in the present invention includes a plurality of probes each targeting a plurality of glycosyltransferase genes associated with an antigen type (sugar chain antigen type) based on a sugar chain antigen of a microorganism. The probe is a polynucleotide, but is usually DNA. DNA may contain various derivatives such as natural nucleotides, functional groups for binding to solid phase carriers, labels, functional groups for detection, and functional groups for enhancing stability. Good. In some cases, a nucleotide chain as a linker that does not function as a probe is provided. Such linker sequences are distinct from the probe sequences.

(Probe targeting glycosyltransferase gene)
The plurality of glycosyltransferase genes associated with the sugar chain antigen type is preferably a plurality of glycosyltransferase genes associated with the sugar chain antigen type so as to be distinguishable. That is, it is preferable that the combination of these multiple glycosyltransferase genes is different from the other sugar chain antigen types to be distinguished. According to such a combination, the sugar chain antigen type can be discriminated only by this probe set. Considering the diversity of sugar chain antigens, that is, the diversity of glycosyltransferase genes, many sugar chain antigen types can be distinguished by a combination of a plurality of glycosyltransferase genes that can be associated with a sugar chain antigen type.

  A glycosyltransferase is an enzyme that forms a glycosidic bond and binds sugar. The glycosyltransferase is not particularly limited as long as it can be used for the capsule antigen type. Glycosyltransferases include glucose transferase, N-acetylfucosamine transferase, rhamnose transferase, sialyltransferase, fucose transferase, galactose transferase, N-acetylglucosamine transferase, N-acetylgalactosamine transferase, glucuronic acid Examples thereof include transferase and galacuronic transferase. In addition, even if the function is not yet known as a glycosyltransferase (protein), activity as a glycosyltransferase can be expected based on homology on the base sequence or amino acid sequence. When it can be used for discrimination from the capsule antigen type, it is handled as a glycosyltransferase in the present invention.

  Regarding glycosyltransferases, it is known that there are a plurality of glycosyltransferases that form the same glycosidic bond, and they may form a family. Such diversity of glycosyltransferases is thought to be related to the diversity, specificity and change of sugar chain antigens.

  The plurality of glycosyltransferase genes are preferably included in a sugar chain synthesis gene cluster including a cps cluster. This is because it can be identified as a glycosyltransferase gene related to the synthesis of a sugar chain antigen characterizing the sugar chain antigen type by being contained in this sugar chain synthesis gene cluster. Glycosynthetic gene clusters contain glycosyltransferase genes related to the synthesis of sugar chain antigens, and may contain other genes, but the base sequences of known glycosyltransferases, specific amino acid domains and motifs Glucosyltransferase gene is estimated by using various homology search programs (blast program, NCBI, http://www.ncbi.nlm.nih.gov/blast/Blast.cgi) be able to. A glycosyltransferase gene that has already been separated and identified as a protein in the glycosynthetic gene cluster can be handled as the glycosyltransferase gene of the present invention without estimating itself. The CPS cluster of Streptococcus pneumoniae is disclosed on the website of the Sanger Institute in the UK (http://www.sanger.ac.uk/Projects/S_pneumonie/CPS/) as already mentioned, Legionella, Escherichia coli, For Salmonella, etc., the website of Columbia University (http://genome3.cpmc.columbia.edu/~legion/g_info.html) and the University of Wisconsin website (http://www.genome.wisc.edu/ index.html), and can be confirmed in the genome sequence whose base sequence has already been published by the US Ventor Institute (http://msc.tigr.org/salmonella/index.shtml).

  There may be at least one type of probe per glycosyltransferase gene, but preferably two types, more preferably three types or more are prepared. By assigning a plurality of probes to one glycosyltransferase gene, the glycosyltransferase gene can be detected with higher accuracy, and as a result, the discrimination accuracy of the sugar chain antigen type can be improved.

  Whether multiple glycosyltransferase genes found from CPS clusters etc. can be used as multiple glycosyltransferase genes associated with the glycosylation antigen type It can be determined by comparing with the glycosyltransferase gene of. When a set of multiple glycosyltransferase genes is a plurality of glycosyltransferase genes associated with each other so that the glycan antigen type can be discriminated, a probe specific to each coding region of these glycosyltransferase genes can be designed. A probe set comprising a plurality of these probes can itself be used for discrimination of the sugar chain antigen type. For example, since the glycosyltransferase genes found in the CPS cluster of Streptococcus pneumoniae are rich in diversity, the capsular antigen type can be determined based only on the combination of a plurality of glycosyltransferase genes.

(Other probes)
The probe set can further include other probes. For example, when a gene other than a glycosyltransferase gene contained in a glycosynthetic gene cluster is useful for discriminating a sugar chain antigen type, by including a probe targeting the gene, Antigen type discrimination accuracy can be increased. In addition, by including such probes, it is possible to provide a set capable of discriminating the glycosyltransferase gene probe set even when it is difficult to discriminate the sugar chain antigen type. Proteins other than such glycosyltransferase genes that may be included in the CPS cluster include glycosynthetic enzymes (wzy3, manB) of Streptococcus pneumoniae and sugar-modifying enzymes in the glycosynthetic gene cluster of Legionella ( gip, neuC). In addition to the genes identified as genes other than glycosyltransferase genes, the other genes include genes of unknown function that have not been identified as glycosyltransferase genes at the time of selecting a probe.

  Such probes targeting other genes are particularly effective when the glycosyltransferase gene of the sugar chain antigen synthesis gene cluster has not been identified. For example, in Legionella, only some glycosyltransferase genes in the glycosynthetic gene cluster of bacteria classified as serotype 1 are known, and other serotype glycosynthetic gene clusters are known. So far, no glycosyltransferase genes have been identified.

  Under these circumstances, the present inventors have comprehensively applied to genes (including previously identified glycosyltransferase genes and other unidentified genes) included in the serotype 1 glycosylation gene cluster of Legionella bacteria. A plurality of probes were formed, and these probes were hybridized with sample nucleic acids prepared from glycan synthesis gene clusters derived from serotype 1 and other serotypes (serotypes 2 to 6). . Some of these probes react with sample nucleic acids of other serotypes and others do not react at all. In addition, hybridization reactivity (hybridization / non-hybridization) to a plurality of probes with respect to a serotype 1 glycosylation gene cluster is different in each serotype (type 2 to type 6). It was found that each serotype can be easily characterized by graphing the signal intensity distribution pattern or signal intensity based on the spectrum to form a spectrum. It was also found that a probe effective for discriminating each serotype can be found.

  From the above, it was found that even when a probe that is not specific for a sugar chain antigen was used, the sugar chain antigen type could be discriminated by the difference in hybridization reactivity with a plurality of probes. In addition, by using a comprehensive group of probes created for the glycan synthesis gene cluster of a specific glycan antigen type, the glycan antigen type can be changed even if the glycosyltransferase gene has not been identified. It has been found that it is possible to find probes or sets that are effective in discriminating.

(Probe design)
The base sequences of probes that specifically hybridize with the coding regions of a plurality of glycosyltransferase genes are based on the base sequence of the glycosyltransferase gene, such as OLIGO (manufactured by Takara Bio Inc., OLIGO is a trademark). It can be determined programmatically.

  Probes that target the glycosyltransferase gene are conserved between a certain range of sugar chain antigen types (for example, between serotypes, between subclasses, between serotypes and subclasses) to be distinguished within the coding region. It may be designed to target the coding region. These probes react specifically with the range of glycosyltransferase genes but do not react with other glycosyltransferase genes. By utilizing such a certain range of specificity, it is possible to distinguish a specific antigen type from other antigen types. In addition, a probe that targets a storage region can reduce the production cost of the probe. For this reason, since the array of this invention provided with the probe set containing such a probe can reduce probe cost, it can contribute to reduction of inspection cost.

  The probe that targets the glycosyltransferase gene may itself contain a sugar chain antigen type-specific probe. That is, when the coding region of a certain glycosyltransferase gene has a base sequence specific enough to distinguish the sugar chain antigen type from other sugar chain antigen types, the probe specific to the base sequence is As such, it is a probe that can discriminate the sugar chain antigen type. When the probe set includes such a sugar chain antigen type-specific probe, the sugar chain antigen type discrimination accuracy by the probe set can be further increased. Moreover, it is extremely useful when the probe set alone, that is, only a combination of a plurality of glycosyltransferase genes cannot be distinguished from other sugar chain antigen types.

  Such a sugar chain antigen type-specific probe may be contained only in one type in the probe set, but is preferably contained in two or more types. In addition, the sugar chain antigen type-specific probe may target one type of glycosyltransferase gene coding region, or two or more types each targeting two or more types of glycosyltransferase gene coding regions. May be.

  Further, the sugar chain antigen type specific probe may be at least one for each glycosyltransferase gene, but preferably two types, more preferably three types or more are prepared. By assigning multiple probes to one glycosyltransferase gene, the specificity of the glycosyl antigen type can be increased and the glycosyltransferase gene can be detected more accurately, resulting in improved accuracy of glycosyl antigen type discrimination. Can be made.

  The sugar chain antigen type-specific probe is designed so that the sugar chain antigen type can be specifically identified. That is, it has a specific base sequence that can be distinguished from other sugar chain antigen types by nucleic acid hybridization. Considering the discrimination accuracy, the probe is preferably one that can discriminate a single sugar chain antigen type, but may be one that can specifically discriminate two or more types of sugar chain antigen types. This is because even such a probe is useful for type discrimination.

  The glycan antigen type-specific probe targets a glycan antigen type-specific DNA region on the glycan antigen synthesis gene, but is preferably 30% or less identical to other glycan antigen types, more preferably Can target regions of less than 10% identity. Alternatively, a region having 42 or more, more preferably 54 or more base mismatches can be targeted.

  The sugar chain antigen type-specific probe is effective for many strains belonging to the sugar chain antigen type by determining the strain contained in the sugar chain antigen type. That is, the sugar chain antigen type is classified below the bacterial species, and is classified according to the sugar chain antigen, so if it is a sugar chain antigen type-specific probe in a certain strain, other strains of the same sugar chain antigen type It can also be said that it is sugar chain antigen type specific.

  The sugar chain antigen type-specific probe may be a probe specific to the coding region of a sugar chain antigen synthesis gene other than the glycosyltransferase gene. As described above, the glycosynthetic gene cluster includes a gene of a protein other than a glycosyltransferase gene depending on the microorganism, and a sugar chain antigen type-specific region is included in the coding region of such a gene. If you can, you can use it.

  Furthermore, as already described, the probe is based on the hybridization reactivity with the probe prepared comprehensively for the sugar chain antigen synthesis gene cluster of a specific sugar chain antigen type, and the specific sugar chain antigen type and others. It is also possible to select a probe effective for discriminating the sugar chain antigen type. Probes selected in this way include probes that target glycosyltransferase genes, probes that target other genes, and / or probes that target genes of unknown function.

  The length of the probe that can constitute the probe set described above is preferably 20 bases or more and 400 bases or less from the viewpoint of the specificity of glycosylation antigen type discrimination and hybridization. More preferably, it is about 45 to 75 bases.

  FIGS. 2 to 9 show 23 serotypes (capsular antigen types corresponding to 23-valent vaccines (sugar chain antigens) selected based on the nucleotide sequences of CPS clusters disclosed for 90 serotypes of Streptococcus pneumoniae. The capsular antigen type (sugar chain antigen type 9-specific probe base sequence (SEQ ID NO: 1-222)) suitable for discrimination of the type) is shown in the probe set of the present invention. In addition to a probe consisting of the above, a probe consisting of this complementary sequence can be used, and the base sequence shown in any of SEQ ID NOS: 1-222 or this complementary sequence can be used within the range that can retain the function as a probe. Probes comprising a mutated sequence from which a plurality of bases have been deleted, substituted or added are also included in the probe of the present invention.

  18 to 32, sugar chains suitable for discrimination of serotypes selected based on the base sequences of LPS synthetic gene clusters (glycan antigen synthetic gene clusters) disclosed for serotype 1 of Legionella bacteria. The base sequence (sequence number 223-671) of the probe which targets an antigen gene cluster is shown. In the probe set of the present invention, a probe selected from probes consisting of these complementary sequences in addition to the probes consisting of the base sequences shown in these can be used. In addition, from the nucleotide sequence shown in any one of SEQ ID NOS: 223 to 671 or a complementary sequence thereof, a mutant sequence in which one to a plurality of bases have been deleted, substituted or added within a range that can retain the function as a probe. These probes are also included in the probe of the present invention.

  The range in which the function of the probe is not impaired is a range in which specificity for discriminating a sugar chain antigen type such as a capsule antigen type of the probe is ensured. For example, it can be hybridized to the target nucleic acid under stringent hybridization conditions described in the examples.

  According to the present invention, various capsular antigen type-specific probes disclosed in FIGS. 2 to 9 and probe sets including two or more of these are provided. For example, a probe set for discriminating seven types of serotypes included in a 7-valent vaccine is also provided. These capsular antigen type-specific probes can also be used as a part of the probe set targeting the glycosyltransferase gene described above, but since capsular antigen type discrimination is possible by itself, Alternatively, it can be used as a set for capsular antigen type discrimination. According to the present invention, one or more of these antigen-specific probes described above can be used as a surface of a solid phase carrier so that a sugar chain antigen type including one or more capsule antigen types can be discriminated. An array comprising the above is also provided. Further, for example, a capsular antigen type-specific probe capable of discriminating seven serotypes of the 7-valent vaccine as described above can be provided, or 23 serotypes of the 23-valent vaccine can be discriminated. A capsular antigen type specific probe can also be provided. Even with only such a capsule antigen type-specific probe, the capsule antigen type can be distinguished very efficiently. In particular, various serotype-specific probes including a plurality of capsular antigen type-specific probes specific to one serotype are retained on the array, and various probes can be obtained. The serotype can also be discriminated based on the distribution of signals obtained by hybridization.

Table 1 shows an example of the determination of the sugar chain antigen type using the probes disclosed in FIGS. As shown in Table 1, serotypes 11F, 11A, 11B, 11C, and 11D, which are subclasses of serotype 11 of pneumococci, have common antigenicity, and the base sequences in the cps cluster are also common. For example, the first half of wchK is highly conserved among 11A, 11B, 11C, and 11D, and can be specifically detected by designing a probe (11A-9-1) that targets this part. . On the other hand, even within the same wchK gene, mutations were observed between serotypes in the latter half, and a probe (11A-9-2) targeting this serotype 11A or 11D was detected to detect only serotype 11A or 11D. It is possible to design. As glycosyltransferase variations, since wcyK and wcrL are conserved only in 11F, 11A, and 11D, probes that target the respective genes (11A-10-1, 11A-10-2, 11A for wcyK) -10-3 and wcrL can be distinguished from other serotypes (including 11B and 11C) by 11A-12-1, 11A-12-2, and 11A-12-3). That is, if only the first half (11A-9-1) of wchK reacts, it is 11B or 11C, and it does not react with the first half (11A-9-1, 11A-9-2) of wchK and the latter half (11A -9-3) can be determined as 11F if it reacts with wcyK and wcrL, and 11A or 11D when it reacts with all of wchK, wcyK and wcrL.

  According to the present invention, the probe disclosed in FIGS. 18 to 32 and a probe set including two or more of these are provided. This probe set includes probes targeting the glycosyltransferase genes of LPS synthesis gene cluster of serotype 1 of Legionella bacterium (probes with lpg775, lpg778, and lpg779), as well as the glycosyltransferase genes of the cluster Probes targeting other genes or genes with unknown functions (probes to which genes other than lpg775, lpg778, and lpg779 are attached) are included. According to the set of probes shown in FIGS. 18 to 32 or a probe selected from them, the sugar chain antigen type (serotype 1) of Legionella is based on the difference in hybridization reactivity when a plurality of probes are combined. Type-6) can be discriminated.

  More specifically, according to the probes selected from this probe set, Legionella serotype 1 can be distinguished from serotypes 2-6. Moreover, according to the probe selected from this probe set, Legionella can be distinguished from each of serotypes 2 to 6.

  In addition, according to the present invention, there is also provided an array comprising the probe set on the surface of a solid phase carrier so that one or more serotypes selected from Legionella serotypes 1 to 6 can be discriminated. Is done. Preferably, three or more serotypes can be discriminated, more preferably five or more, and even more preferably six. Moreover, it is preferable that the sugar chain antigen type | mold of Legionella bacteria which the array of this invention makes a discrimination | determination object is 1 type or 2 types or more selected from the serotypes 2-6 except a serotype 1. A mold may further be included.

The probes shown below are all selected from a group of probes that target the serotype 1 LPS synthetic gene cluster (sugar chain antigen synthetic gene cluster) shown in FIGS. It is a preferable probe for distinguishing each of type 1 to type 6. In combination with the probe corresponding to the serotype to be discriminated, the serotype can be discriminated by looking at the difference in hybridization reactivity with each probe. Probes useful for discrimination of serotype 1 include probes targeting the glycosyltransferase genes of the LPS synthetic gene cluster of Legionella serotype 1 (probes with lpg775, lpg778, and lpg779). Examples of probes useful for discrimination of other serotypes include probes corresponding to genes other than the identified glycosyltransferase genes.

  In the array of the present invention, the probe described above is held on a solid phase carrier. The technique for holding the probe on the surface of the solid support is not particularly limited. Various methods can be used to immobilize the probe. For example, a method using a combination of a maleimide group and a thiol (-SH) group (probe side) of a solid phase carrier, a method using a combination of an epoxy group and an amino group (probe side) on the solid phase carrier, and the like can be mentioned. In addition, for the introduction of various functional groups on the surface of the solid phase carrier, a surface treatment with various silane coupling agents can be used in addition to coating of a resin containing such functional groups. The functional group capable of reacting with the functional group previously introduced into the probe may be introduced. The introduction of the thiol group into the probe can be performed by using, for example, 5′-Thiol-Modifier C6 (Glen Research). The method for supplying the probe to the surface of the solid phase carrier is not particularly limited. A stamping method or an ink jet method may be used. The probe may be synthesized on a solid support.

  It is preferable that a probe for discriminating at least 5 sugar chain antigen types is provided on the solid phase carrier of the array of the present invention, more preferably 20 or more sugar chain antigen types are discriminated. A probe is provided. Moreover, it is preferable that the sugar chain serotypes in Streptococcus pneumoniae to be discriminated by the array of the present invention are part or all of the 23 sugar chain antigen types (serotypes) targeted by the 23-valent vaccine. Moreover, it is preferable that it is a part or all of seven types of sugar chain antigen types (serotypes) targeted by the 7-valent vaccine.

(Method for distinguishing microbial antigen types)
The method for discriminating the sugar chain antigen type of the microorganism of the present invention comprises the steps of preparing the probe set of the present invention, contacting the probe on the array with a sample nucleic acid, detecting nucleic acid hybridization to the probe, Can be provided. The array preparation step can be carried out by preparing the above-described array of the present invention, or obtaining a previously prepared array if the array of the present invention can be supplied.

(Contact process)
The contact step is a step of bringing the probe into contact with the sample nucleic acid obtained from the subject. In the contacting step, when the probe and the sample nucleic acid are brought into contact with each other, if there is a polynucleotide fragment that can hybridize with the probe in the sample nucleic acid, hybridization occurs. The conditions for bringing the probe into contact with the sample nucleic acid may be set to an appropriate temperature, salt concentration, and pH in consideration of the length of the probe and the like.

  In addition to the case where various subjects can be used as sample nucleic acids as they are, sample nucleic acids can be obtained from the subjects. The method for obtaining the sample nucleic acid is not particularly limited, and a known method can be adopted. In preparing the sample nucleic acid, DNA may be simply extracted, but preferably the DNA in the subject is amplified by a nucleic acid amplification method such as PCR. For example, a PCR product using a primer that uniformly amplifies the entire DNA of a microorganism to be discriminated may be used as a sample nucleic acid, or only a portion corresponding to a sugar chain antigen synthesis gene may be amplified using an appropriate primer. For example, it is preferable to amplify the cps cluster. For such a primer, an appropriate base sequence can be determined by obtaining a cps cluster or a base sequence in the vicinity thereof. The sample nucleic acid is preferably labeled with a fluorescent dye or the like.

(Detection process)
The detection step is a step of detecting nucleic acid hybridization to the probe of the sample nucleic acid. Hybridization of a probe and a DNA fragment in a sample nucleic acid can be performed by various known methods in addition to using various labels. As already described, the label may be attached to the probe or sample nucleic acid, or may be attached to the DNA fragment after hybridization. In the detection, a technique according to the detection method of the hybridized product such as the label used can be employed. When preparing a sample nucleic acid, a PCR product to which a fluorescent dye such as Cy3 or Cy5 is added as a sample nucleic acid, a scanner can be used at a wavelength corresponding to the excitation wavelength of these dyes.

  In the detection step, a signal expressed by a label or the like is detected based on the hybridization of the hybridized product, the hybridization is detected based on the presence or absence of the signal and its intensity, and the capsule antigen type is discriminated based on the detected signal. Can do.

  For example, when a hybridization signal can be detected for all probes in a probe set associated with a certain glycan antigen type, the subject has a glycan antigen-type microorganism associated with the probe set. A positive determination can be made. If a hybridization signal cannot be detected in some of the probes that can constitute these probe sets, a negative determination is made. In addition, when a certain probe can specifically identify one sugar chain antigen type, if the hybridization can be detected for this probe, the subject has said one sugar chain antigen type microorganism present. Can be positively determined. If hybridization cannot be detected for these probes, a negative determination can be made that the subject does not contain the one sugar chain antigen type microorganism.

(kit)
The kit of the present invention comprises the probe of the present invention described above (consisting of the nucleotide sequence of SEQ ID NOs: 1-222 and its complementary sequence, and a functionally equivalent base sequence), and a probe set comprising any of these probes Alternatively, an array in which these probes are held on the surface of a solid phase carrier and a reagent for detecting a reaction between the sample nucleic acid of the subject and the probe can be included. This kit can typically be used for capsular antigen type discrimination of pneumococci. This kit can also contain PCR primers for the preparation of the sample nucleic acid. When pneumococci are targeted for discrimination, the primer may be one that amplifies the entire DNA region of pneumococci or can selectively amplify the cps cluster. The kit of the present invention also comprises a probe of the present invention (comprising the base sequence of SEQ ID NOS: 223 to 671 and its complementary sequence and a functionally equivalent base sequence thereof) and a probe selected from these probes. A set or an array in which these probes are held on the surface of a solid phase carrier, and a reagent for detecting a reaction between a sample nucleic acid of a subject and the probe can be included. Typically, this kit can be used for distinguishing the sugar chain antigen type of Legionella. This kit also contains PCR primers that can selectively amplify the entire DNA region of Legionella or part thereof, or the whole or part of the glycan antigen synthesis gene cluster, as in the pneumococcal kit. be able to.

  The method for producing a probe used for discriminating an antigen type based on the capsular antigen of the present invention is based on the base sequence information obtained for a plurality of glycosyltransferase genes associated with the synthesis of a sugar chain antigen of a microorganism having a sugar chain antigen. And determining a base sequence of a plurality of probes targeting each of the plurality of glycosyltransferase genes based on the base sequence. The probes to be produced by the production method of the present invention are a plurality of probes targeting a plurality of glycosyltransferase genes associated with the sugar chain antigen type of microorganisms already described. According to this production method, a probe for easily discriminating a sugar chain antigen type can be easily obtained. Since the combination of glycosyltransferase genes, that is, the combination of probe sets has specificity, the specificity of the probe base sequence is not required, and the conserved region of the glycosyltransferase gene can also be targeted. Therefore, the probe can be provided at a low cost. Moreover, such a probe may be immobilized on a solid support to form an array.

  Various embodiments of the plurality of probes used in the above-described array of the present invention can be directly applied to the probe and the production thereof in this production method. Various aspects of the solid-phase carrier and the immobilization method in the above-described array of the present invention can be directly applied to the production of the array.

  Hereinafter, the present invention will be specifically described with reference to examples. However, the present invention is not limited to these examples.

In this example, genomic DNA was extracted from 55 serotypes of pneumococcal strains shown in Table 3 below, labeled to prepare a nucleic acid sample, and subjected to hybridization in a separately prepared array. Was detected.

(Genomic DNA extraction)
The pneumococcal strain was cultured in 5 mL of brain-heart infusion broth (0.3% yeast extract added) at 37 ° C. and 5% CO ₂ for 24 hours. 2 mL of this culture solution was centrifuged at 12,000 × g, 25 ° C. for 2 min, and the supernatant was discarded and the cells were collected. 450 μL of 50 mM EDTA and 12 μl of Lysozyme (100 mg / mL) were added and incubated at 37 ° C. for 1 hour. Thereafter, DNA purification was performed using Wizard genomic DNA purification kit (Promega) according to the attached protocol.

(Sample label)
The purified genomic DNA was adjusted to 21 μl (500 ng), and 20 μl of 2.5 × Random Primere Mix (Invitrogen) was added thereto. After incubating at 95 ° C. for 5 min, it was rapidly cooled on ice. 5 μl of 10 × dCTP mix, 3 μl of Cy3-dCTP or Cy5-dCTP (Invitrogen) were added, 1 μl of Exo-Klenow Fragment (Invitrogen) was added, and the mixture was incubated at 37 ° C. for 2 hours. The labeled sample was purified using a DNA purification kit (QIAGEN) and concentrated to 40 μl. Then, add 25 μl of 3 M sodium acetate and 1 μl of glycogen to 25 μl of the sample labeled with Cy3 or Cy5, and then add turbidity.Add 125 μl of ethanol and leave at -80 ° C for 30 min in the dark, then 12,000 × g, 4 ° C, Centrifugation was performed for 30 minutes, and the supernatant was discarded, and the pellet was collected. After air drying in the dark for 5 minutes, 140 μl of Hybridization buffer (25% formamide, 0.1% SDS, 6 × SSPE buffer) was added to suspend the pellet. The mixture was allowed to stand in the dark for 30 min, incubated at 75 ° C. for 8 min, and then incubated at 42 ° C. for 30 min.

(Production of array)
In addition to probes consisting of the nucleotide sequences of SEQ ID NOS: 1-222, positive control probes (26 types of probes targeting various housekeeping genes) and negative control probes (26 types of probes targeting genes of other microorganisms) ) Was used to produce an array. In this example, an inkjet type (piezoelectric drive type) microarray manufacturing apparatus was used. Specific production conditions were as follows.
(1) Probe synthesis: chain length 60mer or 59mer, no modification, cartridge purification (2) number of spots: 274
(3) Pattern: 2 rows of 13 rows x 11 columns (7 rows only for the 11th column), pitch 300 μm
(4) Solid phase carrier: 76.2 mm × 25.4 mm × 1 mm (active ester substrate)
(5) Supply of probe to solid phase carrier:
A predetermined probe solution was set in an inkjet microarray manufacturing apparatus and spotted on a solid phase carrier under the conditions (2) to (4).
(6) Immobilization The probe was immobilized on a solid support by the following operation.
a) Heat treatment at 80 ° C. for 1 hour
b) Soak for 15 minutes in (2 x SSC, 0.2% SDS) solution (room temperature)
c) Immerse in (2 x SSC, 0.2% SDS) solution for 5 minutes (95 ° C)
d) Shake about 10 times in sterile water e) Centrifuge dry

(Hybridization)
The prepared labeled sample nucleic acid was injected into the prepared DNA array and incubated at 42 ° C. for 72 hours for hybridization. In addition, a sample nucleic acid was separately prepared using half of each of the serotype 6B sample nucleic acid and the serotype 14 sample nucleic acid when hybridized in a single manner, and hybridization was performed. The composition of the hybridization solution was (25% formamide, 0.1% SDS, 6 × SSPE buffer).

(Fluorescence measurement)
After hybridization, the array was washed with Buffer A (1 × SSC / 0.1% SDS), Buffer B (0.05 × SSC), and Buffer C (95% ethanol). Array measurement was performed with a DNA Microarray Scanner G2565BA (Agilent). An example of the results (serotypes 1 and 12F) is shown in FIGS. In each figure, background data is also shown.

  As shown in FIGS. 10 and 11, when sample nucleic acids derived from each serotype of pneumococcal strain were used, the sample nucleic acids were hybridized with the corresponding serotype-specific probes and derived from the fluorescent dye used. Could be detected as fluorescence. Similarly, characteristic hybridization with serotype-specific probes could be detected for all other serotypes as well. From the above results, it was found that these probes are extremely effective for discrimination of serotype (capsular antigen type). Further, as is clear from FIGS. 10 and 11, the sample nucleic acid obtained from each serotype exhibits a characteristic fluorescence intensity distribution (spectrum) for the serotype-specific probe, thereby making each serotype clear and simple. It was found that can be distinguished.

  Further, as shown in FIGS. 12 and 13, when a mixed sample nucleic acid of serotype 6B sample nucleic acid and serotype 14 sample nucleic acid is used, each sample nucleic acid is hybridized with each serotype-specific probe. did. From the above, it was confirmed that these probes target a relatively close region of the cps cluster, but can hybridize with high specificity without crossing hybridization.

  The conventional methods of Non-Patent Documents 1 and 2 cannot distinguish serotypes 18A, 18B, 18C, and 18F. An example of this array is equipped with a probe that can identify serotype 18B or 18C. Specifically, the following 14 probes are designed to react specifically to serotypes 18B and 18C; 17F + 18C + 23F-8-1, 17F + 18C + 23F-8-2, 17F + 18C + 23F-8-3, 18C-9-1, 18C-9-2, 18C-10-1, 18C-10-2, 18C-10-3, 18C-11-1, 18C-11-2, 18C -11-3, 18C-12-1, 18C-13-2, 18C-13-3. When strains already known to be serotypes 18B and 18C by the swelling test were subjected to this method, as shown in FIGS. 14 and 15, each strain had its own serotype-specific probe. Hybridization was possible and it was possible to determine that it was serotype 18B or 18C.

  The conventional methods of Non-Patent Documents 1 and 2 cannot distinguish between serotypes 10C, 10F, 11A, 11D, and 11F. The probes mounted in the embodiments of this array are designed to distinguish them. Specifically, 11A-9-1 only for serotype 11C, 11A-9-3, 11A-10-1, 11A-10-2, 11A-10-3, 11A-12 for serotype 11F -7, 11A-12-2 and 11A-12-3 are designed to react specifically. Similarly, 11A, 11D, 10C, and 10F can be discriminated by the reacted probe. When strains already found to be 11C or 11F by the capsular swelling test were subjected to this method, as shown in FIGS. 16 and 17, each strain was hybridized with its serotype-specific probe. However, it could be determined as 11C or 11F, respectively.

In this example, genomic DNA was extracted from six serotypes of Legionella strains shown in Table 4 below, labeled to prepare a nucleic acid sample, and subjected to hybridization in a separately prepared array. I detected it. In addition, as a negative judgment material, genomic DNA of Escherichia coli, Klebsiella pneumophila, and Pseudomonas aeruginosa was subjected to the same treatment.

(Genomic DNA extraction)
Legionella strains were cultured in an artificial medium (yeast extract 1%, ACES 1%, α-ketoglutarate 1.5%, L-cysteine hydrochloride 0.04%, ferric pyrophosphate 0.025%) at 37 ° C for 72 hours. 2 ml of this culture solution was centrifuged at 12,000 × g, 25 ° C. for 2 minutes, and the supernatant was discarded and the cells were collected. 450 μl of 50 mM EDTA and 12 μl of Lysozyme (100 mg / mL) were added and incubated at 37 ° C. for 1 hour. Thereafter, DNA purification was performed using Wizard genomic DNA purification kit (Promega) according to the attached protocol.

(Sample label)
The purified genomic DNA was adjusted to 21 μl (500 ng), and 20 μl of 2.5 × Random Primere Mix (Invitrogen) was added thereto. After incubating at 95 ° C. for 5 minutes, it was rapidly cooled on ice. 5 μl of 10 × dCTP mix, 3 μl of Cy3-dCTP or Cy5-dCTP (Invitrogen) were added, 1 μl of Exo-Klenow Fragment (Invitrogen) was added, and the mixture was incubated at 37 ° C. for 2 hours. The labeled sample was purified using a DNA purification kit (QIAGEN) and concentrated to 40 μl. Then, add 25 μl of 3 M sodium acetate and 1 μl of glycogen to 25 μl of the sample labeled with Cy3 or Cy5, add turbidity, add 125 μl of ethanol, and leave at -80 ° C. for 30 minutes in the dark, then 12,000 × g, 4 ° C., Centrifugation was performed for 30 minutes, and the supernatant was discarded, and the pellet was collected. After air drying in the dark for 5 minutes, 140 μl of Hybridization buffer (25% formamide, 0.1% SDS, 6 × SSPE buffer) was added to suspend the pellet. The mixture was allowed to stand in the dark for 30 minutes and then incubated at 75 ° C. for 8 minutes, and then incubated at 42 ° C. for 30 minutes.

(Production of array)
In addition to the probes consisting of the base sequences of FIGS. 18 to 32 (SEQ ID NOS: 223 to 671), positive control probes (27 types of probes targeting various housekeeping genes) and negative control probes (genes of other microorganisms) An array was prepared using a total of 497 species (21 types of target probe total). In this example, an inkjet type (piezoelectric drive type) microarray manufacturing apparatus was used. Specific production conditions were as follows. Probes comprising the respective base sequences represented by SEQ ID NOs: 223 to 671 were prepared by targeting genes contained in the sugar chain antigen synthesis gene cluster containing the glycosyltransferase gene of serotype 1 Legionella.
(1) Probe synthesis: chain length 60mer or 59mer, no modification, cartridge purification (2) number of spots: 497
(3) Pattern: 4 rows of 13 rows x 11 columns, pitch 300 μm
(4) Solid phase carrier: 76.2 mm × 25.4 mm × 1 mm (active ester substrate)
(5) Supply of probe to solid phase carrier:
A predetermined probe solution was set in an inkjet microarray manufacturing apparatus and spotted on a solid phase carrier under the conditions (2) to (4).
(6) Immobilization The probe was immobilized on a solid support by the following operation.
a) Heat treatment at 80 ° C. for 1 hour
b) Soak for 15 minutes in (2 x SSC, 0.2% SDS) solution (room temperature)
c) Immerse in (2 x SSC, 0.2% SDS) solution for 5 minutes (95 ° C)
d) Shake about 10 times in sterile water e) Centrifuge dry

(Hybridization)
The prepared labeled sample nucleic acid was injected into the prepared DNA array and incubated at 42 ° C. for 72 hours for hybridization. In addition, a sample nucleic acid was separately prepared using half of each of the serotype 6B sample nucleic acid and the serotype 14 sample nucleic acid when hybridized in a single manner, and hybridization was performed. The composition of the hybridization solution was (25% formamide, 0.1% SDS, 6 × SSPE buffer).

(Fluorescence measurement)
After hybridization, the array was washed with Buffer A (1 × SSC / 0.1% SDS), Buffer B (0.05 × SSC), and Buffer C (95% ethanol). Array measurement was performed with a DNA Microarray Scanner G2565BA (Agilent). Fluorescence signal intensity distributions for serotypes 1 to 6 are shown in FIGS. In the upper part of each figure, the probes described in SEQ ID NOS: 223 to 671 are graphed by arranging the probes with smaller gene numbers on the left side (so that the larger sequence number is on the left side). ing. In addition, background data is also shown. The lower part of each figure shows a graph in which effective probes are selected and arranged so as to discriminate between serotypes 1 to 6. Moreover, the result about the microbe used as negative judgment material in FIGS. 39-41 is shown.

  As shown in FIGS. 33 to 38, sample nucleic acids derived from Legionella strains of each serotype have undergone a hybridization reaction specific to the probe group on the array and the serotype, and each serotype A unique fluorescence intensity distribution (spectrum) was obtained (see the upper graph of each figure). In addition, when the probe selected as effective for discrimination of each serotype shown in the lower part of each figure was graphed, a spectrum of fluorescence intensity peculiar to each serotype was obtained. Sample nucleic acids derived from serotype 1 strains had hybridization reactions with many probes, and fluorescent signals were obtained for many probes. Moreover, lpg0775_01, lpg0775_02, lpg0775_03, lpg0775_04, lpg0775_05, lpg0775_06, lpg0775_09, lpg0775_08, lpg0775_08, lpg0775_08, lpg0775_08, lpg0775_08, lpg0775_08, lpg0775_08, lpg0775_08, lpg0775_08, lpg0775_09, , lpg0778_01, lpg0778_02, lpg0778_03, lpg0778_04, lpg0778_05, lpg0778_06, lpg0778_07, lpg0778_08, lpg0778_09, lpg0778_10, lpg0778_11, lpg0779_01, lpg0779_02, lpg0779_03, lpg0779_04, lpg0779_05, lpg0779_06, lpg0779_07, lpg0779_08, lpg0779_09, lpg0779_10, lpg0779_11, probes lpg0779_12 and lpg0779_13 (It is described in order from the left side in the frame of GTasegenes in the lower part of FIG. 33 to FIG. 38.) specifically hybridizes with the sample nucleic acid derived from serotype 1 and other serotypes 2 to 6 And was distinguishable. That is, it is clearly distinguished from serotypes 2 to 6 by using one or more of these probes, preferably two or more probes targeting at least two glycosyltransferase genes. I knew it was possible.

In addition, the sample nucleic acid derived from the serotype 2 strain showed a spectrum of fluorescence intensity different from that of other serotypes, as shown in the upper part of FIG. Also, as shown in the lower part of FIG. 34, lpg0753_13, lpg0752_10, lpg0758_01, lpg0755_01, lpg0746_06, lpg0759_18, lpg0750_03, lpg0757_10, and lpg0757_06 (in the lower part of FIG. 33 to FIG. And lpg0779_09 (in the frame of GTgene) showed specific signals that were distinguishable from serotypes 1 and 3-6. That is, it was found that one or more selected from these probes is an effective probe for discriminating serotype 2.
As shown in the upper part of FIG. 35, the sample nucleic acid derived from the serotype 3 strain showed a spectrum of fluorescence intensity different from that of other serotypes. Also, as shown in the lower part of FIG. 35, lpg0746_14, lpg0750_07, lpg0758_08, lpg0746_08, lpg0753_03, lpg0748_17, lpg0751_04, lpg0759_05, lpg0756_07 and lpg0761_01 (in FIG. 33 to FIG. ) Showed a specific signal distinguishable from serotypes 1, 2, and 4-6. That is, it was found that one or more selected from these probes is an effective probe for discriminating serotype 3.
The sample nucleic acid derived from the serotype 4 strain showed a spectrum of fluorescence intensity different from that of other serotypes as shown in the upper part of FIG. 36, lpg0758_11, lpg0759_16, lpg0753_11, lpg0760_02, lpg0758_06, lpg0752_01, lpg0760_03, lpg0759_09, lpg0760_07, lpg0759_03 (in the bottom of FIG. 33 to FIG. ) Showed specific signals distinguishable from serotypes 1 to 3, 5 and 6. That is, it was found that one or more selected from these probes is an effective probe for discriminating serotype 4.
As shown in the upper part of FIG. 37, the sample nucleic acid derived from the serotype 5 strain showed a spectrum of fluorescence intensity different from that of other serotypes. As shown in the lower part of FIG. 37, lpg0780_09, lpg0755_09, lpg0759_17, lpg0760_11, lpg0757_07, lpg0747_01, lpg0752_06, lpg0746_17, lpg0780_08, lpg0759_02 (in the lower part of FIG. A specific signal distinguishable from serotypes 1 to 4 and 6. That is, it was found that one or more selected from these probes are effective probes for distinguishing serotype 5.
As shown in the upper part of FIG. 38, the sample nucleic acid derived from the serotype 6 strain showed a spectrum of fluorescence intensity different from that of other serotypes. 38, lpg0749_03, lpg0757_08, lpg0746_16, lpg0751_01, lpg0750_02, lpg0748_12, lpg0746_10, lpg0748_13, lpg0782_10, lpg0760_07 (in the bottom of FIG. A specific signal distinguishable from serotypes 1 to 4 and 6. That is, it was found that one or more selected from these probes is an effective probe for discriminating serotype 6.

  From the above, it was found that the probe set fixed to the array was a probe set capable of specifically detecting serotypes 1 to 6 respectively. It was also found that these serotypes can be clearly and easily distinguished by these probe sets. Furthermore, it was found that each serotype can be clearly and easily discriminated by a set of probes (see above) each having a characteristic hybridization reaction with each serotype selected from these probe sets. As described above, by using a probe that covers a specific serotype of a glycan antigen synthesis gene cluster, by evaluating the hybridization reactivity with a sample nucleic acid of another serotype glycan antigen synthesis gene cluster, It was found that a probe effective for discrimination of other serotypes can be selected. Such a probe is considered to target a gene other than a glycosyltransferase gene or a glycosyltransferase gene that has not yet been identified.

  Furthermore, as shown in FIGS. 39 to 41, when genomic DNA of Escherichia coli, Klebsiella pneumophila, and Pseudomonas aeruginosa is used, it is mounted as a negative control of this array. It hybridized with the probe and did not hybridize with the probe specific for Legionella. From the above, it was found that even if genomic DNA of different bacterial species is included, it can be clearly distinguished from Legionella.

  Sequence number 1-671: Probe

Claims (29)

A method for identifying an antigen type based on a sugar chain antigen of a microorganism,
Providing a probe set including probes each targeting a plurality of glycosyltransferase genes associated with the antigen type;
Contacting the probe with a sample nucleic acid obtained from a subject;
Detecting nucleic acid hybridization to the probe;
A discrimination method comprising:   The method according to claim 1, wherein the plurality of glycosyltransferase genes are included in a sugar chain antigen synthesis gene cluster.   The discrimination method according to claim 1 or 2, wherein the probe set further includes a probe targeting a gene other than a glycosyltransferase gene contained in the sugar chain antigen synthesis gene cluster.   The discrimination method according to any one of claims 1 to 3, wherein the microorganism is one or more selected from pneumococci, Legionella, Salmonella, Escherichia coli, Shigella, and Vibrio.   The method according to claim 1, wherein the microorganism includes pneumococci.   The determination method according to claim 1, wherein the microorganism includes Legionella bacteria. The probe set includes an antigen-specific probe that targets a region specific to the antigen type in the glycan antigen synthesis gene of the microorganism,
The determination method according to claim 1. The method according to claim 1, wherein the microorganism includes pneumococci, and the probe is selected from the group consisting of the following sequences (a) and (b).
(A) a nucleotide sequence according to any one of SEQ ID NOs: 1 to 222 or a complementary sequence thereof (b) a base deletion, substitution or substitution with respect to any one of the sequences of SEQ ID NOs: 1 to 222 or their complementary sequences A sequence comprising a mutant sequence to which addition has been made and having the function of the probe The method according to claim 1, wherein the microorganism includes Legionella, and the probe is selected from the group consisting of the following sequences (c) and (d).
(C) the nucleotide sequence according to any one of SEQ ID NOS: 223 to 671 or a complementary sequence thereof (d) a nucleotide deletion, substitution or substitution with respect to any one of the sequences of SEQ ID NOS: 223 to 671 or their complementary sequences A sequence comprising a mutant sequence to which addition has been made and having the function of the probe
  The discrimination method according to claim 1, wherein the probe set preparation step is a step of preparing an array holding a plurality of the probe sets corresponding to a plurality of the antigen types on a solid phase carrier. A solid support;
A probe set that includes probes that each target a plurality of glycosyltransferase genes that are held on the surface of the solid phase carrier and associated with an antigen type based on a sugar chain antigen of a microorganism;
An array.   The array according to claim 11, wherein the plurality of glycosyltransferase genes are included in a sugar chain antigen synthesis gene cluster.   The array according to claim 11 or 12, wherein the microorganism is one or more selected from pneumococci, Legionella, Salmonella, Escherichia coli, Shigella, and Vibrio.   The array according to any one of claims 11 to 13, wherein the microorganism comprises pneumococci.   The array according to any one of claims 11 to 14, wherein the microorganism comprises Legionella.   The array according to any one of claims 11 to 15, which holds a plurality of the probe sets corresponding to a plurality of the antigen types. The probe set includes an antigen type-specific probe that targets a region specific to the antigen type in the glycan antigen synthesis gene of the microorganism,
The array according to any of claims 11 to 16.   The array according to any one of claims 11 to 17, wherein the probe set further comprises a probe targeting a gene other than the glycosyltransferase gene contained in the sugar chain antigen synthesis gene cluster. The array according to any one of claims 11 to 18, wherein the microorganism comprises pneumococci, and the probe is selected from the group consisting of the following sequences (a) and (b).
(A) a nucleotide sequence according to any one of SEQ ID NOS: 1-222 or a complementary sequence thereof (b) a base deletion, substitution or substitution with respect to any one of the sequences of SEQ ID NOS: 1-222 or their complementary sequences A sequence comprising a mutant sequence to which addition has been made and having the function of the probe The array according to any one of claims 11 to 19, wherein the microorganism comprises Legionella, and the probe is selected from the group consisting of the following sequences (c) and (d).
(C) the nucleotide sequence according to any one of SEQ ID NOS: 223 to 671 or a complementary sequence thereof (d) a base deletion, substitution or substitution with respect to any one of the sequences of SEQ ID NOS: 223 to 671 or their complementary sequences A sequence comprising a mutant sequence to which addition has been made and having the function of the probe A solid support;
An antigen type-specific probe that is held on the surface of the solid phase carrier and targets a region specific to an antigen type based on a sugar chain antigen in a sugar chain antigen synthesis gene of a microorganism;
An array.   The array according to claim 21, further comprising a probe targeting a gene other than the glycosyltransferase gene in the sugar chain antigen synthesis gene cluster. The microorganism includes pneumococci,
The array according to claim 21 or 22, wherein the antigen-specific probe is selected from the group consisting of the following sequences (a) and (b) so that two or more types of antigens can be distinguished.
(A) a nucleotide sequence according to any one of SEQ ID NOS: 1-222 or a complementary sequence thereof (b) a base deletion, substitution or substitution with respect to any one of the sequences of SEQ ID NOS: 1-222 or their complementary sequences A sequence comprising a mutant sequence to which addition has been made and having the function of the probe A method for producing a probe used to discriminate an antigen type based on a sugar chain antigen of a microorganism,
Determining the base sequences of a plurality of probes targeting each of the plurality of glycosyltransferase genes based on the base sequence information obtained for a plurality of glycosyltransferase genes associated with the synthesis of the sugar chain antigen of the microorganism,
A manufacturing method comprising: A step of producing the plurality of probes based on the base sequence;
The manufacturing method of Claim 24 provided with these. A step of immobilizing the prepared probes on the surface of a solid phase carrier;
The manufacturing method of Claim 25 provided with these. A probe set for distinguishing an antigen type based on a sugar chain antigen of a microorganism,
A probe that is included in the glycan synthesis gene cluster of the microorganism and that targets each of a plurality of glycosyltransferase genes associated with the antigen type,
Probe set. 28. The probe set according to claim 27, wherein the microorganism includes the pneumococci, and the probe includes an antigen type-specific probe selected from the group consisting of the following sequences (a) and (b).
(A) a nucleotide sequence according to any one of SEQ ID NOS: 1-222 or a complementary sequence thereof (b) a base deletion, substitution or substitution with respect to any one of the sequences of SEQ ID NOS: 1-222 or their complementary sequences A sequence comprising a mutant sequence to which addition has been made and having the function of the probe The probe set according to claim 27 or 28, wherein the microorganism comprises Legionella, and the probe is selected from the group consisting of the following sequences (c) and (d).
(C) the nucleotide sequence according to any one of SEQ ID NOS: 223 to 671 or a complementary sequence thereof (d) a base deletion, substitution or substitution with respect to any one of the sequences of SEQ ID NOS: 223 to 671 or their complementary sequences A sequence comprising a mutant sequence to which addition has been made and having the function of the probe