JP2007289176A - Probe, probe-immobilized carrier and method for testing - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a nucleic acid probe which is a probe for detecting a DNA of Peptoniphilus asaccharolyticus that is an infectious disease pathogenic bacterium, collectively detecting bacterial strains of the same species irrespective of the strains and differentially detecting the bacterial strains from other bacterial species for the purpose of classifying pathogenic bacteria by the species. <P>SOLUTION: The probe of a specific base sequence is used for detecting the DNA of the infectious disease pathogenic bacterium Peptoniphilus asaccharolyticus. A method for detecting the DNA of the Peptoniphilus asaccharolyticus comprises using the probe. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a probe for detecting DNA of Peptoniphilus asaccharolyticus, which is an infectious disease-causing bacterium useful for detection and identification of causative bacteria of diseases caused by infection. The present invention also relates to a probe-immobilized carrier in which the probe is immobilized on a carrier, a DNA testing method using these, and a kit for DNA testing.

  Reagents and methods for detecting a causative bacterium of an infectious disease in a specimen more quickly and reliably have been proposed. For example, Patent Document 1 discloses an oligonucleotide consisting of a specific base sequence that can be used as a probe and primer for detecting candidiasis and aspergillosis-causing fungi, and a method for detecting a target bacterium using the oligonucleotide. . Moreover, the same patent document discloses a primer set for amplifying a plurality of target bacteria in common by PCR. These primers are used to amplify a plurality of target fungal nucleic acid fragments in a sample by PCR. By detecting the presence or absence of a sequence portion specific to each bacterium in the nucleic acid fragments amplified by these primers, the species of the fungus in the sample is identified by detecting the hybridization using a probe specific to each bacterium. be able to.

On the other hand, as a method capable of simultaneously detecting oligonucleotides composed of a plurality of different base sequences, there is a method using a probe array in which probes having sequences complementary to each of the base sequences are separated on a solid phase. Known (Patent Document 2).
JP-A-8-89254 JP 2004-3131181 A

However, it is not easy to set up a probe that specifically detects DNA of infectious disease-causing bacteria contained in a sample. That is, there is a possibility that not only infectious disease-causing bacteria DNA to be detected but also other infectious disease-causing bacteria DNA exists in the sample. It is never easy to set up a probe that can specifically detect infectious disease-causing bacteria DNA that is the detection target while minimizing the influence (cross-contamination) due to the presence of infectious disease-causing bacteria DNA that is not the detection target. There is no. Under such circumstances, the inventors of the present invention have the following infectious disease-causing bacteria DNA while suppressing cross-contamination to a lower level from a sample in which a plurality of bacterial DNAs exist. The purpose of this study was to obtain a probe that can accurately detect. As a result, a probe capable of accurately detecting the infectious disease-causing bacteria DNA has been obtained.
[Bacteria name]
Peptoniphilus asaccharolyticus
Then, the 1st objective of this invention aims at providing the probe which can identify the DNA of object microbe correctly.

  Another object of the present invention is to provide a probe-immobilizing carrier capable of accurately identifying a target fungus from a specimen in which various fungi can exist simultaneously.

  Furthermore, another object of the present invention is to provide a DNA test method for infectious disease-causing bacteria capable of more rapidly and more accurately detecting various bacteria in a specimen, and therefore The purpose is to provide a kit.

A probe for detecting DNA of Peptoniphilus asaccharolyticus which is an infectious disease-causing bacterium according to the present invention comprises the following base sequence (1) or (2): It is a probe.
(1) GAGTACGTGCGCAAGCATGAAACT (SEQ ID NO: 55) or its complementary sequence,
(2) A probe comprising a mutant sequence in which a base is deleted, substituted or added to the sequence of SEQ ID NO: 55 or a complementary sequence thereof within a range that can retain the function as the probe.

A probe set for detecting DNA of Peptoniphilus asaccharolyticus, which is an infectious disease-causing bacterium according to the present invention, comprises at least two of the probes (A) to (D) below. It is a probe set characterized by including.
(A) a probe comprising a base sequence represented by GAGTACGTGCGCAAGCATGAAACT (SEQ ID NO: 55),
(B) a probe comprising a complementary sequence of the nucleotide sequence of SEQ ID NO: 55,
(C) a probe consisting of a mutant sequence in which a base is deleted, substituted or added to the base sequence of SEQ ID NO: 55 within the range that can retain the function as the probe;
(D) A probe comprising a mutated sequence in which a base is deleted, substituted or added to the complementary sequence of the base sequence of SEQ ID NO: 55 within a range that can retain the function as the probe.

  In the probe fixing carrier of the present invention, at least one of the probes (A) to (D) is fixed on a solid phase carrier, and when there are a plurality of probes to be fixed, the probes are arranged separately from each other. A probe-immobilizing carrier.

The method for detecting Peptoniphylus asaccharolyticus DNA in a sample using the probe-immobilized carrier of the present invention is as follows:
(I) a step of reacting the probe-immobilized carrier according to the present invention with a specimen;
(Ii) detecting the presence or absence of reaction between the probe on the probe-immobilized carrier and the nucleic acid in the specimen, or the reaction intensity;
A method for detecting peptoniphyllus Asaccharolyticus DNA, comprising:

  The kit for detecting an infectious disease-causing fungus of the present invention comprises at least one of the probes (A) to (D) above and a reagent for detecting the reaction between the probe and the target nucleic acid. A kit for detecting Peptoniphylla asaccharolyticus characterized by

According to the present invention, when the specimen is infected with the causative bacterium shown above, even if the specimen is complexly infected at the same time as the bacterium other than the bacteria shown above, Can be identified more rapidly and accurately. Especially, Anaerococcus with high possibility of cross-contamination
Peptoniphilus is distinguished from prevotii (anaerococcus prebotti) with high accuracy.
Asaccharolyticus can be detected. Furthermore, it becomes possible to detect Peptoniphilus asaccharolyticus with high accuracy and distinction from other infectious disease-causing bacteria.

  The present inventors have obtained almost all bacteria (shown in (1) to (62) of Table 1) known to date as sepsis-causing fungi from each depository institution and obtained 16S in all fungi. The DNA sequence of the rRNA gene region was identified.

  A detailed analysis of the probe sequence in Peptoniphilus asaccharolyticus was performed while comparing all of the identified sequences. As a result, the present inventors have found a probe that can identify the Peptoniphilus asaccharolyticus bacterium of the present invention.

The present invention provides a probe set comprising an oligonucleotide probe (hereinafter simply referred to as a probe) for identifying an infectious disease-causing bacterium and a combination of two or more thereof. By using this probe or probe set, the following bacteria that cause inflammation due to infection can be detected.
[Bacteria name]
Peptoniphilus asaccharolyticus (Peptoniphilus asaccharolyticus)
That is, the probe of the present invention can detect the DNA sequence of the 16S rRNA gene region in the above-mentioned fungal DNA, and includes those consisting of the following base sequences.
(A) a probe comprising a base sequence represented by GAGTACGTGCGCAAGCATGAAACT (SEQ ID NO: 55),
(B) a probe comprising a complementary sequence of the nucleotide sequence of SEQ ID NO: 55,
(C) a probe consisting of a mutant sequence in which a base is deleted, substituted or added to the base sequence of SEQ ID NO: 55 within the range that can retain the function as the probe;
(D) A probe comprising a mutated sequence in which a base is deleted, substituted or added to the complementary sequence of the base sequence of SEQ ID NO: 55 within a range that can retain the function as the probe.

  A probe set can be constructed using two or more of these probes.

  The above mutant sequence has a mutation within a range that does not impair the function as a probe, that is, within a range where the target nucleic acid sequence to be detected can be hybridized and detected. Especially, it is preferable to have the variation | mutation in the range which can hybridize to the target nucleic acid sequence as a detection object on stringent conditions. Preferred conditions for hybridization that define the range of mutation include the conditions in Examples described later. The target to be detected here is contained in the sample to be hybridized, and may be the base sequence itself unique to the infectious disease-causing bacteria, or a complementary sequence of this unique base sequence. Also good. Furthermore, examples of such mutations include mutant sequences in which deletion, substitution or addition of one to several bases is performed within a range that retains the probe function.

  The functions of these probes are largely dependent on the specificity of the probe sequence for the target nucleic acid sequence to be examined. The specificity of the probe sequence can be evaluated from the degree of base coincidence with the target nucleic acid sequence and the melting temperature between the target nucleic acid sequence and the probe sequence. Further, when the probe constitutes a probe set, the variation from the melting temperature of other probe sequences is also a condition that affects the performance.

  In designing these probe sequences, a region having high specificity for the bacterial species is selected so that even if different strains are the same species, there is no variation. In addition, a region having a mismatched portion of 3 or more bases with a sequence of a bacterial species other than the bacterial species is selected. In addition, the difference in melting temperature between the probe sequence and the bacterial species sequence and the melting temperature between the probe sequence and the bacterial species other than the bacterial species are designed to be 10 ° C. or higher. Furthermore, the base is deleted or added to a highly specific region so that the melting temperatures of the probes immobilized on the same carrier fall within a certain range.

  According to the experiments by the inventors of the present application, it has been found that when 80% or more of contiguous sequences are conserved among probe sequences, the attenuation of hybridization intensity is small. From this, it can be said that the function of the probe is not impaired as long as it is a mutant sequence in which 80% or more of the base sequence is conserved among the probe sequences disclosed in the present application.

  Since these probe sequences are specific only to the DNA sequence coding for the 16S rRNA gene of the fungus, sufficient hybridization sensitivity is expected for the sequence even when stringent conditions are set. Is done. In addition, these probe sequences are designed so as to form a stable hybrid in a hybridization reaction with a target sample and obtain good results even when immobilized on a carrier.

  Furthermore, the probe fixing carrier (for example, DNA chip) on which the probe for detecting infectious disease causing bacteria according to the present invention is fixed can be obtained by supplying the probe to a predetermined position of the carrier and fixing it. Various methods can be used for supplying the probe to the carrier. For example, a method in which the probe can be fixed to the carrier by chemical bonding (covalent bonding or the like) and a liquid containing the probe is applied to a predetermined position of the carrier by an ink jet method can be suitably used. As a result, the probe is less likely to be peeled off from the carrier, and an additional effect that sensitivity is improved is obtained. That is, when a DNA chip is prepared by a stamping method called a Stanford method that has been generally used, there is a drawback that the applied DNA may be easily peeled off. As a method for producing a DNA chip, there is a method in which probes are arranged by DNA synthesis on a carrier surface (for example, a DNA chip manufactured by Affymetrix). In this method of synthesizing probes on the carrier, it is difficult to uniformly control the amount of synthesis for each probe sequence, so the amount of probe immobilization differs greatly for each probe immobilization region (spot). Is likely to occur. If there is a variation in the fixed amount of each probe, there is a case where an accurate evaluation for a detection result using the probe cannot be performed. From such a viewpoint, it is preferable that the probe carrier according to the present invention is prepared by using the above-described ink jet method. According to the above-described ink jet method, there is an advantage that a probe carrier can be efficiently provided with a high sensitivity and high accuracy, since the probe is stably fixed to the carrier and hardly peeled off.

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

  Samples to be examined using a probe carrier (for example, a DNA chip) in which the probe of the present invention is fixed to a carrier include those derived from animals such as humans and livestock. For example, all substances that are considered to contain bacteria such as blood, cerebrospinal fluid, sputum, gastric fluid, vaginal secretions, bodily fluids such as oral mucus, urine and feces such as feces are to be examined. In addition, all media that may cause contamination by bacteria, such as food poisoning, contaminated food, drinking water and hot water in the environment, or filters such as air purifiers are subject to inspection. Can be mentioned. In addition, animals and plants such as quarantine at the time of import / export are also subject to specimens.

When the sample as described above can be used as it is for the reaction with the DNA chip, it is reacted with the DNA chip as a specimen and the result is analyzed. In addition, when the specimen cannot be directly reacted with the DNA chip, the sample obtained by performing processing on the specimen according to necessity such as extraction and purification of the target substance is reacted with the DNA chip as the specimen. For example, if the target nucleic acid is contained, an extract that is assumed to contain the target nucleic acid is prepared from the sample, and further subjected to treatment such as washing and dilution as necessary to obtain a sample solution, which is reacted with the DNA chip. You may let them. Furthermore, when a target nucleic acid is contained in a specimen by performing various amplification processes including a PCR amplification process, the sample may be amplified and reacted with a DNA chip. Such amplified nucleic acid samples include the following.
(A) Amplified specimen prepared using PCR reaction primers designed for DNA detection of the 16S rRNA gene region.
(B) A sample prepared by further performing a PCR reaction or the like based on the PCR amplification product.
(C) Sample prepared by an amplification method other than PCR.
(D) Samples labeled by various labeling methods for visualization.

  In addition, the carrier used for the preparation of the probe-immobilized carrier such as a DNA chip is not particularly limited as long as it satisfies the characteristics capable of performing the intended solid-liquid phase reaction. For example, a planar substrate such as a glass substrate, a plastic substrate, or a silicon wafer, a three-dimensional structure with unevenness, a spherical shape such as a bead, a rod shape, a string shape, a thread shape, or the like can be used as a carrier. Further, the surface of the carrier may be treated so that the probe can be immobilized. In particular, those in which a functional group is introduced so that a chemical reaction can be performed on the surface are preferable in terms of reproducibility because the probe is stably bound in the course of the hybridization reaction.

Various methods can be used to immobilize the probe. An example is a method using a combination of a maleimide group and a thiol (—SH) group. In this method, a thiol (—SH) group is bonded to the end of the probe, and the support (solid phase) surface is treated so as to have a maleimide group. As a result, the thiol group of the probe supplied to the carrier surface reacts with the maleimide group on the carrier surface to form a covalent bond and immobilize the probe.
To introduce a maleimide group, first, an aminosilane coupling agent is reacted with a glass substrate, and then a maleimide group is introduced by a reaction between the amino group and an EMCS reagent (N- (6-Maleimidocaproyloxy) succinimide: Dojin). A method to do is available. Introduction of a thiol group into DNA can be performed by using 5′-Thiol-Modifier C6 (manufactured by Glen Research) on an automatic DNA synthesizer.

  Examples of combinations of functional groups used for immobilization include combinations of epoxy groups (on the solid phase) and amino groups (ends of nucleic acid probes) in addition to the combinations of thiol groups and maleimide groups described above. . Further, surface treatment with various silane coupling agents is also effective, and a probe into which a functional group capable of reacting with a functional group introduced with the silane coupling agent is used. Furthermore, a method of coating a resin having a functional group can also be used.

Detection of infectious disease-causing bacteria DNA using the probe-immobilized carrier according to the present invention can be performed by a DNA detection method having at least the following steps (i) and (ii).
(I) A step of reacting a probe-immobilized carrier on which such a probe of the present invention is immobilized with a specimen.
(Ii) A step of detecting the presence or absence of reaction between the probe on the probe-immobilized carrier and the nucleic acid in the specimen, or the reaction intensity thereof.

Furthermore, it can also be set as the detection method which has the process of at least the following processes (A)-(C).
(A) A step of reacting a probe-immobilized carrier on which such a probe of the present invention is immobilized with a specimen.
(B) A step of detecting the presence or absence of a reaction between the probe on the probe-immobilized carrier and the nucleic acid in the specimen.
(C) When the reaction between the probe and the nucleic acid in the sample is detected, the probe that has reacted with the nucleic acid in the sample is identified, and the infectious disease-causing bacteria DNA contained in the sample is identified based on the base sequence of the probe Process.

  The probe to be immobilized on the probe-immobilizing carrier is at least one of the above-mentioned (A) to (D), and the carrier is used for detecting other types of probes (species other than Peptoniphilus asaccharolyticus depending on the purpose of inspection). Probe) may be fixed. In this case, by using a probe that can detect bacterial species other than Peptoniphilus asaccharolyticus without causing cross-contamination as other probes, it is possible to simultaneously detect a plurality of bacterial species with high accuracy. As other probes, the base sequences of SEQ ID NOs: 35 to 54 and SEQ ID NOs: 56 to 67 shown in Table 9, and their complementary sequences, and probes for detecting the DNA of the detection target bacteria in each probe in these base sequences As long as the above function can be maintained, at least one probe selected from the group consisting of a mutant sequence in which a base is deleted, substituted, or added can be preferably used. The probe may be used by being fixed at a position separated from the solid phase carrier Peptoniphilus asaccharolyticus bacterium DNA detection probe.

Further, as described above, in the case where the DNA sequence of the 16S rRNA gene region of the infectious disease-causing fungus contained in the specimen is amplified by PCR and used as a sample to be reacted with the probe carrier, infectious disease initiation A primer set for detecting bacteria can be used. As this primer set, one containing at least one selected from the oligonucleotides listed in (1) to (21) below and at least one selected from the oligonucleotides listed in (22) to (28) is preferable. It is. More preferably, those containing all kinds of oligonucleotides listed in (1) to (28) are suitable.
(1) An oligonucleotide having a base sequence of 5 ′ gcggcgtgcctaatacatgcaag 3 ′ (SEQ ID NO: 1).
(2) An oligonucleotide having a base sequence of 5 ′ gcggcaggcctaacacatgcaag 3 ′ (SEQ ID NO: 2).
(3) An oligonucleotide having a base sequence of 5 ′ gcggcaggcttaacacatgcaag 3 ′ (SEQ ID NO: 3).
(4) An oligonucleotide having a base sequence of 5 ′ gcggtaggcctaacacatgcaag 3 ′ (SEQ ID NO: 4).
(5) An oligonucleotide having a base sequence of 5 ′ gcggcgtgcttaacacatgcaag 3 ′ (SEQ ID NO: 5).
(6) An oligonucleotide having a base sequence of 5 ′ gcgggatgccttacacatgcaag 3 ′ (SEQ ID NO: 6).
(7) An oligonucleotide having a base sequence of 5 ′ gcggcatgccttacacatgcaag 3 ′ (SEQ ID NO: 7).
(8) An oligonucleotide having a base sequence of 5 ′ gcggcatgcttaacacatgcaag 3 ′ (SEQ ID NO: 8).
(9) An oligonucleotide having a base sequence of 5 ′ gcggcgtgcttaatacatgcaag 3 ′ (SEQ ID NO: 9).
(10) An oligonucleotide having a base sequence of 5 ′ gcggcaggcctaatacatgcaag 3 ′ (SEQ ID NO: 10).
(11) An oligonucleotide having a base sequence of 5 ′ gcgggatgctttacacatgcaag 3 ′ (SEQ ID NO: 11).
(12) An oligonucleotide having a base sequence of 5 ′ gcggcgtgcctaacacatgcaag 3 ′ (SEQ ID NO: 12).
(13) An oligonucleotide having a base sequence of 5 ′ gcggcgtgcataacacatgcaag 3 ′ (SEQ ID NO: 13).
(14) An oligonucleotide having a base sequence of 5 ′ gcggcatgcctaacacatgcaag 3 ′ (SEQ ID NO: 14).
(15) An oligonucleotide having a base sequence of 5 ′ gcggcgcgcctaacacatgcaag 3 ′ (SEQ ID NO: 15).
(16) An oligonucleotide having a base sequence of 5 ′ gcggcgcgcttaacacatgcaag 3 ′ (SEQ ID NO: 16).
(17) An oligonucleotide having a base sequence of 5 ′ gcgtcatgcctaacacatgcaag 3 ′ (SEQ ID NO: 17).
(18) An oligonucleotide having a base sequence of 5 ′ gcgataggcttaacacatgcaag 3 ′ (SEQ ID NO: 18).
(19) An oligonucleotide having a base sequence of 5 ′ gcgacaggcttaacacatgcaag 3 ′ (SEQ ID NO: 19).
(20) An oligonucleotide having a base sequence of 5 ′ gctacaggcttaacacatgcaag 3 ′ (SEQ ID NO: 20).
(21) An oligonucleotide having a base sequence of 5 ′ acagaatgcttaacacatgcaag 3 ′ (SEQ ID NO: 21).
(22) An oligonucleotide having a base sequence of 5 ′ atccagccgcaccttccgatac 3 ′ (SEQ ID NO: 22).
(23) An oligonucleotide having a base sequence of 5 ′ atccaaccgcaggttcccctac 3 ′ (SEQ ID NO: 23).
(24) An oligonucleotide having a base sequence of 5 ′ atccagccgcaggttcccctac 3 ′ (SEQ ID NO: 24).
(25) An oligonucleotide having a base sequence of 5 ′ atccagccgcaccttccggtac 3 ′ (SEQ ID NO: 25).
(26) An oligonucleotide having a base sequence of 5 ′ atccagcgccaggttcccctag 3 ′ (SEQ ID NO: 26).
(27) An oligonucleotide having a base sequence of 5 ′ atccagccgcaggttctcctac 3 ′ (SEQ ID NO: 27).
(28) An oligonucleotide having a base sequence of 5 ′ atccagccgcacgttcccgtac 3 ′ (SEQ ID NO: 28).

  Among them, primer sets configured to be able to amplify DNA of 16S rRNA region of Peptoniphilus asaccharolyticus bacteria include at least one of the oligonucleotides listed in (15) and (16) below and (22) and ( Those containing at least one of the oligonucleotides listed in 25) are preferred.

Specifically, the 16S rRNA region DNA sequence of Peptoniphilus asaccharolyticus (JCM8143) shown in SEQ ID NO: 68 is compared with the above-mentioned SEQ ID NOs: (1) to (28). As a result, it can be confirmed that the above primer sequences are suitable.
(15) An oligonucleotide having a base sequence of 5 ′ gcggcgcgcctaacacatgcaag 3 ′ (SEQ ID NO: 15).
(16) An oligonucleotide having a base sequence of 5 ′ gcggcgcgcttaacacatgcaag 3 ′ (SEQ ID NO: 16).
(22) An oligonucleotide having a base sequence of 5 ′ atccagccgcaccttccgatac 3 ′ (SEQ ID NO: 22).
(25) An oligonucleotide having a base sequence of 5 ′ atccagccgcaccttccggtac 3 ′ (SEQ ID NO: 25).

  For the purpose of detecting Peptoniphilus asaccharolyticus DNA, at least one of the oligonucleotides listed in (15) and (16) above and at least one of the oligonucleotides listed in (22) and (25) It only has to have.

  A kit for detecting infectious disease-causing bacteria can be constructed using at least the probe described above and a reagent for detecting the reaction between the nucleic acid in the specimen and the probe. The probe in this kit is preferably provided as the probe solid carrier described above. The detection reagent includes a label for detecting the reaction and a primer for amplification as a pretreatment. When the detection reagent contains a primer, the primer is preferably composed of a primer for amplifying DNA of 16S rRNA region of Peptoniphilus asaccharolyticus. Furthermore, a primer for amplifying the DNA of the region of an infectious disease-causing fungus other than Peptoniphilus asaccharolyticus as a detection reagent can be included together with the primer for amplifying the region DNA of Peptoniphilus asaccharolyticus. As a kit for detecting at least the DNA of Peptoniphilus asaccharolyticus, a configuration having the DNA detection probe of Peptoniphilus asaccharolyticus according to the present invention and a primer for amplifying DNA of the 16S rRNA region of Peptoniphilus asaccharolyticus as a detection reagent is suitable. . When the kit has a probe-fixing carrier on which a probe for detecting infectious disease-causing bacteria other than Peptoniphilus asaccharolyticus is immobilized together with a DNA detection probe for Peptoniphilus asaccharolyticus, as a detection reagent, infection other than Peptoniphilus asaccharolyticus It is also preferable to include a primer for amplifying the DNA of the region of the pathogenic fungus.

  Hereinafter, the present invention will be described in more detail with reference to examples using probes for detecting infectious disease pathogens for detecting Peptoniphilus asaccharolyticus bacteria.

Example 1
In the following examples, detection of microorganisms using the 2 Step PCR method will be described.

[1. Preparation of probe DNA]
The nucleic acid sequences shown in Table 2 were designed as the base sequence of the probe for detecting Peptoniphilus asaccharolyticus. Specifically, these nucleotide sequences were selected from the genome part coding for 16S rRNA gene of Peptoniphilus asaccharolyticus. These probe base sequence groups are designed so as to be highly specific to the bacterium and to be expected to have sufficient hybridization sensitivity without variation among the probe base sequences. As the probe, in addition to the probe having the base sequence shown in Table 2, a probe having a base length of about 20 to 30 including each base sequence can be used. In this case, portions other than the base sequence defined in Table 2 need to have a base sequence that does not affect the detection accuracy.

In the probe having the base sequence shown in Table 2, a thiol group was introduced into the 5 ′ end of the nucleic acid as a functional group for immobilization on the DNA chip after synthesis according to a conventional method. After introduction of the functional group, it was purified and lyophilized. The lyophilized probe for internal standard was stored in a refrigerator at -30 ° C.

[2. Preparation of PCR primers]
[2-1. Preparation of PCR primers for sample amplification]
The nucleic acid sequences shown in Table 3 below were designed as PCR primers for amplification of DNA (target DNA) of the 16s rRNA gene region for detection of pathogenic bacteria. Specifically, a primer set that specifically amplifies the genomic portion coding for 16s rRNA, that is, designed to align specific melting temperatures as much as possible at both ends of the approximately 1500 base-length 16s rRNA coding region. Primers were used. In addition, a plurality of types of primers are designed so that a plurality of different bacterial species listed in (1) to (62) of Table 1, mutant strains, and a plurality of 16s rRNA gene region DNAs present on the genome can be amplified simultaneously. Yes. Needless to say, the primer set need not be limited to the primer sets listed in Table 3 below as long as the primer set can amplify almost the entire length in common with the DNA of the 16s rRNA gene region of the causative fungus.

  The primers shown in Table 3 were purified by high performance liquid chromatography (HPLC) after synthesis. Then, 21 kinds of Forward primer and 7 kinds of Reverse primer were mixed, and each primer concentration was dissolved in TE buffer so that the final concentration would be 10 pmol / μl.

[2-2. Preparation of PCR primers for labeling]
Similarly to the above-described sample amplification primers, oligonucleotides having the sequences shown in Table 4 below were used as labeling primers.

The primers shown in Table 4 were labeled using Cy3 as the fluorescent dye. After the synthesis, the product was purified by high performance liquid chromatography (HPLC), 6 kinds of labeled primers were mixed, and each labeled primer concentration was dissolved in TE buffer so that the final concentration would be 10 pmol / μl.

[3. Extraction of Peptoniphilus asaccharolyticus bacteria Genome DNA (model specimen)]
[3-1. Microbial culture & Genome DNA extraction]
First, Peptoniphilus asaccharolyticus (JCM 8143) was cultured according to a standard method. Genome DNA was extracted and purified from this microorganism culture solution using a nucleic acid purification kit (FastPrep FP100A / FastDNA Kit: manufactured by Funakoshi Co., Ltd.).

[3-2. Inspection of recovered Genome DNA]
Genome DNA of the collected microorganism (Peptoniphilus asaccharolyticus) was subjected to agarose electrophoresis and absorbance measurement at 260/280 nm according to a conventional method, and the quality (contamination amount of low-molecular nucleic acid, degree of degradation) and the recovery amount were tested. In this example, about 10 μg of Genome DNA was recovered, and Genome DNA degradation and rRNA contamination were not observed. The recovered Genome DNA was dissolved in TE buffer so as to have a final concentration of 50 ng / μl and used in the following examples.

[4. Creation of DNA chip]
[4-1. Glass substrate cleaning]
A synthetic quartz glass substrate (size: 25 mm x 75 mm x 1 mm, manufactured by Iiyama Special Glass Co., Ltd.) was placed in a heat-resistant and alkali-resistant rack and immersed in a cleaning solution for ultrasonic cleaning adjusted to a predetermined concentration. After soaking in the cleaning solution overnight, ultrasonic cleaning was performed for 20 minutes. Subsequently, the substrate was taken out, rinsed lightly with pure water, and then ultrasonically cleaned in ultrapure water for 20 minutes. Next, the substrate was immersed in a 1N sodium hydroxide aqueous solution heated to 80 ° C. for 10 minutes. Pure water cleaning and ultrapure water cleaning were performed again to prepare a quartz glass substrate for a DNA chip.

[4-2. surface treatment]
A silane coupling agent KBM-603 (manufactured by Shin-Etsu Silicone Co., Ltd.) was dissolved in pure water to a concentration of 1% by weight (wt) and stirred at room temperature for 2 hours. Subsequently, the previously cleaned glass substrate was immersed in an aqueous silane coupling agent solution and allowed to stand at room temperature for 20 minutes. The glass substrate was pulled up and the surface was lightly washed with pure water, and then nitrogen gas was blown onto both sides of the substrate to dry it. Next, the dried substrate was baked in an oven heated to 120 ° C. for 1 hour to complete the coupling agent treatment, and amino groups were introduced onto the substrate surface. Next, an EMCS solution in which N-maleimidocaproyloxy succinimide (hereinafter abbreviated as EMCS) is dissolved in a 1: 1 mixed (volume ratio) solvent of dimethyl sulfoxide and ethanol to a final concentration of 0.3 mg / ml. Prepared. EMCS is N-maleimidocaproyloxysuccinimide (N- (6-Maleimidocaproyloxy) succinimido) manufactured by Dojindo Laboratories.

  The glass substrate after baking was allowed to cool and immersed in the prepared EMCS solution for 2 hours at room temperature. By this treatment, the amino group introduced on the surface by the silane coupling agent and the succinimide group of EMCS reacted to introduce a maleimide group on the glass substrate surface. The glass substrate pulled up from the EMCS solution was washed with the above-mentioned mixed solvent in which EMCS was dissolved, further washed with ethanol, and then dried in a nitrogen gas atmosphere.

[4-3. Probe DNA]
[1. The probe for detecting microorganisms prepared in [Preparation of probe DNA] was dissolved in pure water, dispensed to a final concentration (at the time of ink dissolution) of 10 μM, and then freeze-dried to remove moisture.

[4-4. DNA ejection by BJ printer and bonding to substrate]
An aqueous solution containing glycerin 7.5 wt%, thiodiglycol 7.5 wt%, urea 7.5 wt%, and acetylenol EH (manufactured by Kawaken Fine Chemical Co., Ltd.) 1.0 wt% was prepared. Subsequently, each of the previously prepared one type of probe (Table 2) was dissolved in the above mixed solvent so as to have a specified concentration. The obtained DNA solution was filled in an ink tank for an ink jet printer (trade name: BJF-850 manufactured by Canon Inc.) and mounted on a print head.

  The ink jet printer used here is modified so that printing on a flat plate is possible. The ink jet printer can spot a DNA solution of about 5 picoliters at a pitch of about 120 μm by inputting a print pattern according to a predetermined file creation method. Subsequently, using this modified inkjet printer, a printing operation was performed on one glass substrate to produce an array. After confirming that printing was performed reliably, it was left in a humidified chamber for 30 minutes to react the maleimide group on the glass substrate surface with the thiol group at the end of the nucleic acid probe.

[4-5. Washing]
After the reaction for 30 minutes, the DNA solution remaining on the surface was washed away with 10 mM phosphate buffer (pH 7.0) containing 100 mM NaCl to obtain a DNA chip having single-stranded DNA immobilized on the glass substrate surface.

[5. Sample amplification and labeling]
[5-1. Sample amplification: 1st PCR]
Table 5 below shows amplification (1st PCR) and labeling (2nd PCR) reactions of microbial DNA serving as a specimen.

The reaction solution having the above composition was subjected to an amplification reaction using a commercially available thermal cycler according to the protocol shown in FIG. After completion of the reaction, the product was purified using a purification column (QIAGEN QIAquick PCR Purification Kit), and the amplification product was quantified.

[5-2. Labeling reaction: 2nd PCR]
Amplification reaction of the reaction liquid having the composition shown in Table 6 was performed using a commercially available thermal cycler according to the protocol shown in FIG.

After completion of the reaction, the sample was purified using a purification column (QIAGEN QIAquick PCR Purification Kit) to prepare a labeled sample.

[6. Hybridization]
[4. DNA chip created in [Creation of DNA chip] and [5. Detection reaction was carried out using the labeled specimen prepared in [Amplification and labeling of specimen].

[6-1. DNA chip blocking]
BSA (bovine serum albumin Fraction V: manufactured by Sigma) was dissolved in 100 mM NaCl / 10 mM Phosphate Buffer to a concentration of 1 wt%. Next, [4. The DNA chip prepared in [Preparation of DNA chip] was soaked at room temperature for 2 hours for blocking. After completion of blocking, the plate was washed with the following washing solution, rinsed with pure water, and then drained with a spin dry apparatus.
Cleaning solution:
0.1wt% SDS 2 × SSC solution containing (sodium dodecyl sulfate) (NaCl 300mM, Sodium Citrate ( trisodium citrate dihydrate, C 6 H 5 Na 3 · 2H 2 O) 30mM, pH7.0)
[6-2. Hybridization]
The drained DNA chip was set in a hybridization apparatus (Genomic Solutions Inc. Hybridization Station), and a hybridization reaction was performed with the following hybridization solution and conditions.

[6-3. Hybridization solution]
6xSSPE / 10% Formamide / Target (2nd PCR Products total amount) / 0.05wt% SDS
_{(6xSSPE: NaCl 900mM, NaH 2} PO 4 · H 2 O 50mM, EDTA 6mM, pH 7.4)
[6-4. Hybridization conditions]
65 ℃ 3min → 55 ℃ 4hr → Wash 2xSSC / 0.1% SDS at 50 ℃ → Wash 2xSSC at 20 ℃ → (Rinse with H ₂ O: Manual) → Spin dry
[7. Detection of microorganism Genome (fluorescence measurement)]
The DNA chip after completion of the hybridization reaction was subjected to fluorescence measurement using a fluorescence detection apparatus for DNA chip (Axon, GenePix 4000B). As a result, Peptoniphilus asaccharolyticus bacteria could be detected with a sufficient signal with good reproducibility.
Table 7 below shows the fluorescence measurement results of Peptoniphilus asaccharolyticus bacteria.

[8. Hybridization with other species]
In order to prove that the probe shown in Table 2 specifically forms a hybrid with only Peptoniphilus asaccharolyticus bacteria, the results of hybridization reaction with Anaerococcus prevotii (JCM 6490) are shown in Table 8 below.

[9. result]
As described above, according to the above example, a DNA chip on which a probe capable of specifically detecting only Peptoniphilus asaccharolyticus bacteria was immobilized could be produced. Furthermore, it became possible to identify infectious disease-causing bacteria using this DNA chip, and solved the problem of DNA probes derived from microorganisms. That is, oligonucleotide probes can be chemically synthesized in large quantities, and purification and concentration control are possible. In addition, for the purpose of classification by microorganism species, it was possible to provide a probe that could detect all species of the same species at once, and could distinguish and detect other species.

  Further, in addition to Anaerococcus prevotii in the same manner as described above, hybridization reactions were also performed on nucleic acids extracted from the bacteria shown in Table 1 (1) to (62). These results were also confirmed to be substantially non-reactive like Anaerococcus prevotii except for Peptoniphilus asaccharolyticus.

  The bacteria shown in (1) to (62) of Table 1 are considered to be septic infectious bacteria and satisfy almost all of the infectious bacteria previously detected in human blood. Therefore, by extracting the nucleic acid of the infectious disease-causing bacteria in the blood using the primer of this embodiment and reacting this with the probe of the present invention, it is possible to identify Peptoniphilus asaccharolyticus bacteria almost certainly. .

  Moreover, according to the said Example, by detecting the DNA sequence of 16S rRNA gene region of infectious disease-causing bacteria DNA without excess and deficiency, the presence of the infectious disease-causing bacteria is determined efficiently and with high accuracy. be able to.

(Example 2) Preparation of DNA chip capable of simultaneously determining various types of fungi [1. In the same manner as in [Preparation of probe DNA], probes having base sequences shown in Table 9 below were prepared.

  Similar to the probe specific to Peptoniphilus asaccharolyticus bacterium in Example 1, these probes are probes that can specifically detect only the specific bacterial species shown in the left column of the table.

  These probes are designed with the same Tm value with the target, reactivity with the non-target sequence, and the like so that the nucleic acid of each target bacterial species can be specifically detected under the same reaction conditions.

  A probe solution was prepared for each probe in the same manner as in [4-3] of Example 1. Thereafter, using the ink jet printer used in [4-4], each probe solution is discharged onto the same substrate, and a plurality of DNA chips in which spots of each probe are spaced apart at a pitch interval of 120 μm are provided. Created.

  One DNA chip was used for a hybridization reaction with a nucleic acid extracted from Peptoniphilus asaccharolyticus bacteria in the same manner as in [6] of Example 1. As a result, the spot of the probe that specifically detects Peptoniphilus asaccharolyticus bacteria showed almost the same fluorescence brightness as in Example 1. Further, the remaining spots of other probes showed very low fluorescence luminance.

  Furthermore, using other prepared DNA chips, hybridization reactions were similarly carried out for bacteria other than Peptoniphilus asaccharolyticus described in Table 9. As a result, the spot of Peptoniphilus asaccharolyticus showed very low fluorescence brightness, and the spot of the probe of the target strain showed very high brightness. Therefore, it was confirmed that the DNA chip prepared in this example can simultaneously determine 11 types of bacteria listed in Table 9 in addition to Peptoniphilus asaccharolyticus bacteria.

(Example 3)
Using the DNA chip prepared in Example 2, detection was attempted when a plurality of bacterial species were present in the sample.

  A culture solution in which both Peptoniphilus asaccharolyticus bacteria and Clostridium difficile bacteria were cultured was prepared, treated in the same manner as in Example 1, and reacted with a DNA chip.

  As a result, the spots of the probes having only SEQ ID NOS: 43, 44, 45, and 55 showed high fluorescence luminance, and it was confirmed at the same time that these bacteria were present together.

It is a figure explaining 1st PCR protocol. It is a figure explaining 2nd PCR protocol.

Claims (10)

Peptoniphilus, an infection-causing fungus
A probe for detecting DNA of asaccharolyticus, which comprises the following base sequence (1) or (2):
(1) GAGTACGTGCGCAAGCATGAAACT (SEQ ID NO: 55) or its complementary sequence,
(2) A probe comprising a mutant sequence in which a base is deleted, substituted or added to the sequence of SEQ ID NO: 55 or a complementary sequence thereof within a range that can retain the function as the probe. Peptoniphilus, an infection-causing fungus
A probe set for detecting DNA of asaccharolyticus, comprising two or more of the probes (A) to (D) below.
(A) a probe comprising a base sequence represented by GAGTACGTGCGCAAGCATGAAACT (SEQ ID NO: 55),
(B) a probe comprising a complementary sequence of the nucleotide sequence of SEQ ID NO: 55,
(C) A probe comprising a mutated sequence in which a base is deleted, substituted or added to the base sequence of SEQ ID NO: 55 within a range capable of retaining the function as the probe.
(D) A probe comprising a mutated sequence in which a base is deleted, substituted or added to the complementary sequence of the base sequence of SEQ ID NO: 55 within a range that can retain the function as the probe.   Each of the plurality of probes constituting the probe according to claim 1 or the probe set according to claim 2 is fixed on a solid phase carrier, and when there are a plurality of fixed probes, the probes are separated from each other. Probe fixing carrier arranged.   The base sequences of SEQ ID NOs: 35 to 54 and SEQ ID NOs: 56 to 67 and their complementary sequences are held at positions separated from the probes, and further, these base sequences retain functions as probes for detecting the DNA of each detection target bacterium. The probe-immobilized carrier according to claim 3, wherein at least one probe selected from the group consisting of a mutant sequence in which base deletion, substitution, or addition has been made is fixed within a possible range.   A peptoniphyll ascer comprising the probes according to claim 1 or the probes constituting the probe set according to claim 2 and a reagent for detecting a reaction between the probes and the target nucleic acid. Kit for detection of Loritacus DNA. The reagent includes a primer for amplifying Peptoniphylus asaccharolyticus DNA,
The kit according to claim 5, wherein the primer comprises at least one of the following oligonucleotides (15) and (16) and at least one of the following oligonucleotides (22) and (25).
(15) An oligonucleotide having a base sequence of 5 ′ gcggcgcgcctaacacatgcaag 3 ′ (SEQ ID NO: 15).
(16) An oligonucleotide having a base sequence of 5 ′ gcggcgcgcttaacacatgcaag 3 ′ (SEQ ID NO: 16).
(22) An oligonucleotide having a base sequence of 5 ′ atccagccgcaccttccgatac 3 ′ (SEQ ID NO: 22).
(25) An oligonucleotide having a base sequence of 5 ′ atccagccgcaccttccggtac 3 ′ (SEQ ID NO: 25).   The kit according to claim 5 or 6, wherein the probe is provided in a state of being immobilized on a solid phase carrier. A DNA detection kit comprising the probe fixing carrier according to claim 4 and a reagent for detecting a reaction between the probe and a target nucleic acid,
The reagent includes a primer for amplifying infectious disease-causing bacteria DNA,
DNA comprising at least one oligonucleotide selected from the following (1) to (21) and at least one oligonucleotide selected from the following (22) to (28) as the primer: Detection kit.
(1) An oligonucleotide having a base sequence of 5 ′ gcggcgtgcctaatacatgcaag 3 ′ (SEQ ID NO: 1).
(2) An oligonucleotide having a base sequence of 5 ′ gcggcaggcctaacacatgcaag 3 ′ (SEQ ID NO: 2).
(3) An oligonucleotide having a base sequence of 5 ′ gcggcaggcttaacacatgcaag 3 ′ (SEQ ID NO: 3).
(4) An oligonucleotide having a base sequence of 5 ′ gcggtaggcctaacacatgcaag 3 ′ (SEQ ID NO: 4).
(5) An oligonucleotide having a base sequence of 5 ′ gcggcgtgcttaacacatgcaag 3 ′ (SEQ ID NO: 5).
(6) An oligonucleotide having a base sequence of 5 ′ gcgggatgccttacacatgcaag 3 ′ (SEQ ID NO: 6).
(7) An oligonucleotide having a base sequence of 5 ′ gcggcatgccttacacatgcaag 3 ′ (SEQ ID NO: 7).
(8) An oligonucleotide having a base sequence of 5 ′ gcggcatgcttaacacatgcaag 3 ′ (SEQ ID NO: 8).
(9) An oligonucleotide having a base sequence of 5 ′ gcggcgtgcttaatacatgcaag 3 ′ (SEQ ID NO: 9).
(10) An oligonucleotide having a base sequence of 5 ′ gcggcaggcctaatacatgcaag 3 ′ (SEQ ID NO: 10).
(11) An oligonucleotide having a base sequence of 5 ′ gcgggatgctttacacatgcaag 3 ′ (SEQ ID NO: 11).
(12) An oligonucleotide having a base sequence of 5 ′ gcggcgtgcctaacacatgcaag 3 ′ (SEQ ID NO: 12).
(13) An oligonucleotide having a base sequence of 5 ′ gcggcgtgcataacacatgcaag 3 ′ (SEQ ID NO: 13).
(14) An oligonucleotide having a base sequence of 5 ′ gcggcatgcctaacacatgcaag 3 ′ (SEQ ID NO: 14).
(15) An oligonucleotide having a base sequence of 5 ′ gcggcgcgcctaacacatgcaag 3 ′ (SEQ ID NO: 15).
(16) An oligonucleotide having a base sequence of 5 ′ gcggcgcgcttaacacatgcaag 3 ′ (SEQ ID NO: 16).
(17) An oligonucleotide having a base sequence of 5 ′ gcgtcatgcctaacacatgcaag 3 ′ (SEQ ID NO: 17).
(18) An oligonucleotide having a base sequence of 5 ′ gcgataggcttaacacatgcaag 3 ′ (SEQ ID NO: 18).
(19) An oligonucleotide having a base sequence of 5 ′ gcgacaggcttaacacatgcaag 3 ′ (SEQ ID NO: 19).
(20) An oligonucleotide having a base sequence of 5 ′ gctacaggcttaacacatgcaag 3 ′ (SEQ ID NO: 20).
(21) An oligonucleotide having a base sequence of 5 ′ acagaatgcttaacacatgcaag 3 ′ (SEQ ID NO: 21).
(22) An oligonucleotide having a base sequence of 5 ′ atccagccgcaccttccgatac 3 ′ (SEQ ID NO: 22).
(23) An oligonucleotide having a base sequence of 5 ′ atccaaccgcaggttcccctac 3 ′ (SEQ ID NO: 23).
(24) An oligonucleotide having a base sequence of 5 ′ atccagccgcaggttcccctac 3 ′ (SEQ ID NO: 24).
(25) An oligonucleotide having a base sequence of 5 ′ atccagccgcaccttccggtac 3 ′ (SEQ ID NO: 25).
(26) An oligonucleotide having a base sequence of 5 ′ atccagcgccaggttcccctag 3 ′ (SEQ ID NO: 26).
(27) An oligonucleotide having a base sequence of 5 ′ atccagccgcaggttctcctac 3 ′ (SEQ ID NO: 27).
(28) An oligonucleotide having a base sequence of 5 ′ atccagccgcacgttcccgtac 3 ′ (SEQ ID NO: 28). In a method for detecting Peptoniphylus asaccharolyticus DNA in a sample using a probe-immobilized carrier,
(I) reacting the probe-immobilized carrier according to claim 3 with a specimen;
(Ii) detecting the presence or absence of the reaction between the probe on the probe-immobilized carrier and the nucleic acid in the specimen, or the reaction intensity thereof;
A method for detecting peptidophilus asaccharolyticus DNA, comprising: PCR amplification of the target nucleic acid in the sample is performed using at least one of the following oligonucleotides (15) and (16) and at least one of the following oligonucleotides (22) and (25) as primers: The detection method according to claim 9, further comprising a step.
(15) An oligonucleotide having a base sequence of 5 ′ gcggcgcgcctaacacatgcaag 3 ′ (SEQ ID NO: 15).
(16) An oligonucleotide having a base sequence of 5 ′ gcggcgcgcttaacacatgcaag 3 ′ (SEQ ID NO: 16).
(22) An oligonucleotide having a base sequence of 5 ′ atccagccgcaccttccgatac 3 ′ (SEQ ID NO: 22).
(25) An oligonucleotide having a base sequence of 5 ′ atccagccgcaccttccggtac 3 ′ (SEQ ID NO: 25).

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