WO2011122034A1 - 核酸クロマトグラフ法を利用した肺炎原因菌の検出方法 - Google Patents
核酸クロマトグラフ法を利用した肺炎原因菌の検出方法 Download PDFInfo
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Definitions
- the present invention relates to a detection method and detection kit for pneumonia-causing bacteria, and more particularly, Streptococcus pneumoniae, Haemophilus influenzae, Mycoplasma pneumoniae, Chlamydophilia pneumonia, Legionella pneumophila, Klebsiella pneumoniae, Pseudomonas aeruginosa, Moraxella cataur (MR)
- the present invention relates to a detection method and a detection kit for pneumonia-causing bacteria that detect Staphylococcus aureus.
- Pneumonia currently ranks fourth in Japanese mortality rates by cause of death, is often a complication of basic diseases such as cancer, and is known as a disease with a very large number of affected people.
- culture tests that have been conducted as exploratory tests for microorganisms that cause pneumonia take at least several days, and when a drug susceptibility test is performed on the cultured causative bacteria, it can take up to a week. Therefore, it is said that the examination method does not sufficiently contribute to treatment selection.
- ICU emergency life unit
- the causative bacteria that cause pneumonia account for nearly 50% of the bacterial species with high frequency of occurrence, and it is said that there are about 20 to 30 main causative bacteria including viruses. Some of them cannot be cultured by a normal technique, and the causative bacteria may be difficult to determine by culture. In particular, in pneumonia, where antibiotics need to be appropriately selected and treated depending on the bacterial species and amount, it is possible to detect multiple types of pneumoniae at the same time and to quantitatively analyze the detected signals. Very important. In addition, although the optimal therapeutic drug varies depending on the type of causative bacteria, it is actually unavoidable to start treatment before determining the causative bacteria in terms of medical ethics. In order to solve these problems, development of a method capable of detecting specific bacteria quickly and quantitatively from a plurality of bacterial species has been awaited.
- the lytA gene encoding Streptococcus pneumoniae autolytic enzyme (LytA), the gene encoding 16S rRNA of Haemophilus influenza, the gene encoding 16S rRNA of Streptococcus pneumoniae, Mycoplasma pneumoniae )
- Primers derived from each of the 16S rRNA-encoding genes or a mip gene encoding a gene encoding 16S rRNA of Legionella pneumophila and a protein MIP that is a pathogenic factor of Legionella pneumophila
- a method for simultaneously detecting four types of respiratory infection-causing bacteria using a primer set to which a primer derived from is added see, for example, Patent Document 1).
- a pertussis bacteria containing a first oligonucleotide and a second oligonucleotide using a nucleic acid separated from a sample containing bacteria as a template a primer set that specifically amplifies a target sequence present in P.
- a nucleic acid primer set capable of simultaneously amplifying 5 or more types of respiratory disease-causing virus target sequences selected from oligonucleotides containing 10 or more consecutive base fragments, and 10 or more consecutive bases
- the step of obtaining cDNA is performed using, for example, reverse transcriptase, and reverse transcriptase reaction using reverse transcriptase is performed by RT-PCR. It is described that it may be used and that the amplification step can be performed by PCR ( In example, see Patent Document 4).
- a method for specifically amplifying genes of genogroup I (GI) and genogroup II (GII), which are gene groups of noroviruses roughly classified in a trace amount A step of obtaining a complementary single-stranded nucleic acid by the NASBA method capable of amplifying a nucleic acid from the extracted RNA at a predetermined temperature, and an RT-LAMP method capable of amplifying the nucleic acid from the amplified product by the NASBA method at a predetermined temperature
- a simple and highly sensitive detection method for norovirus which further comprises a step of amplifying a nucleic acid (see, for example, Patent Document 5).
- the present inventors are primer sets used for detecting a plurality of types of pneumonia-causing bacteria.
- multiplex PCR, real-time PCR, RT-PCR and the like are used for pneumococcus, influenza, From a primer set that enables simultaneous detection of Mycoplasma pneumoniae and Chlamydophila pneumoniae (see, for example, Patent Document 6), and a DNA sequence corresponding to a specific sequence of a target RNA from a fungus-specific RNA strand in 16S rRNA
- a method for detecting and quantifying target RNA comprising preparing a liquid-phase universal primer having an RNA polymerase promoter sequence added to the 5 ′ end of the tag sequence, Staphylococcus spp., Streptococcus spp.
- Two or more kinds selected from bacteria at least one first primer set having a tag sequence and a base sequence that selectively anneals to a target nucleic acid on a DnaJ gene held by the pathogenic microorganism, and the tag
- a method for detecting pathogenic microorganisms (for example, see Patent Document 8) is proposed.
- a method for specifically detecting or quantifying a target nucleic acid in a sample which is a single strand using a primer not bound to a hapten or peptide from a target nucleic acid arbitrarily extracted from a sample.
- Amplifying as a nucleic acid detecting the amplified product by hybridizing with a first oligonucleotide probe complementary to the amplified product bound to the membrane and a complementary second oligonucleotide probe labeled with a colored polymer carrier
- a method for detecting or quantifying a nucleic acid including a step and a step of evaluating the detected image by visual judgment has already been developed.
- NASBA amplification using total RNA extracted from a cultured strain of methicillin-resistant Staphylococcus aureus (MRSA) as a template A method has been established for detecting amplification products using nucleic acid chromatostrips. For example, see Patent Document 9).
- the object of the present invention is to provide Streptococcus pneumoniae, Haemophilus influenza, Mycoplasma pneumoniae, Chlamydophila pneumoniae, Legionella pneumophila, Klebsiella pneumoniae, Pseudomonas aeruginosa, Moraxella catarrhalis, methicillin resistance -Aureus (MRSA), Staphylococcus aureus (these individual bacteria are hereinafter referred to as “detection pneumoniae” or simply “pneumoniae”, sometimes collectively referred to as "10 types of detection pneumoniae”). It is to provide a rapid and accurate detection method and a detection kit used therefor.
- the present inventors examined a method for further improving the detection accuracy of the causative agent of pneumonia for practical use in clinical practice, and found that the DnaJ gene has a gene polymorphism about 10 times the 16S rRNA sequence. Based on the DnaJ region and other gene regions specific to various pneumococci, primer pairs for each target region of 10 detection target pneumoniae contained in the DnaJ gene of 10 types of pneumoniae Designing a set, using this primer pair set, amplify the gene product by NASBA method, qualitatively quantitate the target nucleic acid that is the amplification product, and found that multiple types of pneumoniae causative bacteria can be detected with high accuracy It was.
- the present invention relates to (1) Streptococcus pneumoniae, Hemophilus influenza, Mycoplasma pneumoniae, Chlamydophila pneumoniae, Legionella pneumophila, Klebsiella pneumoniae, Pseudomonas aeruginosa, Moraxella catarrhalis, methicillin resistant staphylococcus MRSA), a method for detecting pneumonia causative organisms to detect at least three types of pneumoniae selected from Staphylococcus aureus, 1) a target specific to each pneumoniae arbitrarily extracted from a sample Amplifying as a single-stranded nucleic acid from a nucleic acid using a primer (a); 2) a step of preparing at least three types of probe pairs different for each pneumococci selected from nucleotide sequences complementary to the amplification product ( 3) A step of preparing a first probe-bound labeled polymer carrier by binding at least three kinds of first probes of each pneumococci to the labeled polymer carrier
- Reverse primer consisting of RNA polymerase promoter sequence added to the terminal side; forward primer consisting of the nucleotide sequence shown in SEQ ID NO: 22, nucleotide sequence shown in SEQ ID NO: 32, and RNA polymerase promoter added to the 5 ′ terminal side thereof Reverse primer consisting of a sequence; a forward primer consisting of a nucleotide sequence shown in SEQ ID NO: 23, a reverse primer consisting of a nucleotide sequence shown in SEQ ID NO: 33 and an RNA polymerase promoter sequence added to the 5 ′ end side thereof; SEQ ID NO: 24 A forward primer comprising the nucleotide sequence shown in FIG.
- RNA polymerase promoter sequence added to the 5 ′ end thereof; a forward comprising a nucleotide sequence represented by SEQ ID No. 28
- a reverse primer comprising a nucleotide primer represented by SEQ ID NO: 38 and an RNA polymerase promoter sequence added to the 5 ′ end thereof; a forward primer comprising a nucleotide sequence represented by SEQ ID NO: 29; and represented by SEQ ID NO: 39
- the primer comprises at least three different primer pairs selected from pneumoniae selected from a reverse primer comprising an RNA polymerase promoter sequence added to The at least three types of pneumoniae that comprise at least three types of pneumonia
- the present invention also relates to (4) Streptococcus pneumoniae, Haemophilus influenza, Mycoplasma pneumoniae, Chlamydophila pneumoniae, Legionella pneumophila, Klebsiella pneumoniae, Pseudomonas aeruginosa, Moraxella catarrhalis, methicillin resistant staphylococcus MRSA), a kit for detecting pneumonia causing causative organisms for detection of at least three types of pneumoniae selected from Staphylococcus aureus, wherein a target nucleic acid specific for each pneumoniae arbitrarily extracted from a sample
- a forward primer comprising the nucleotide sequence shown in SEQ ID NO: 21 that can be amplified, the nucleotide sequence shown in SEQ ID NO: 31, and an RNA polymerase added to the 5 ′ end side thereof Reverse primer consisting of a promoter sequence; a forward primer consisting of a nucleotide sequence shown in SEQ ID NO: 22, a reverse primer consisting of a
- a first probe-bound labeled polymer carrier in which at least three kinds of first probes of pneumoniae different for each pneumoniae are bound to a labeled polymer carrier; 2) at least three types paired with the first probe
- the second probe of each of the pneumoniae comprises a second probe-carrying development support that is solid-phased at a predetermined position that can be identified for each pneumoniae.
- a kit, and (6) at least three kinds of first probes of each pneumococci are composed of at least three kinds of DNAs selected from the nucleotide sequences shown in SEQ ID NOs: 1 to 10, and at least paired with the first probes
- the present invention makes it possible to quickly and accurately identify pneumonia-causing bacteria by a simple method when diagnosing pneumonia patients.
- RNA amplification product by the NASBA method using a multiplex primer was detected about Legionella pneumophila, Pseudomonas aeruginosa, Klebsiella pneumoniae, MRSA, Haemophilus influenza.
- FIG. 3 is a diagram showing that a target RNA amplification product was detected.
- Moraxella catarrhalis it is a figure which shows that RNA amplified by NASBA was confirmed by real-time PCR that it was the target NASBA product of interest.
- One specific example of the nucleic acid chromatography method of the present invention is shown as a schematic diagram. The figure which examined the non-specific binding between each probe for pneumoniae is shown.
- RNA amplification product was detected by the nucleic acid chromatography when RNA amplification was separately performed by NASBA method. It is a figure which shows the verification result of the presence or absence of nonspecific reaction with object microbes other than the target microbe of each probe.
- RNA amplification product was detected by the nucleic acid chromatography when RNA amplification was performed by NASBA method using a multiplex primer. It is the figure which used for the test piece for nucleic acid chromatograph measurement of this invention the 5 types of pneumonia causative bacteria.
- a step of amplifying as a single-stranded nucleic acid using a primer from a target nucleic acid specific to each pneumoniae arbitrarily extracted from a sample (a); 2 ) Step (b) of preparing at least three kinds of probe pairs different from each pneumoniae selected from nucleotide sequences shown in SEQ ID NOs: 1 to 20 complementary to the amplification product; 3) shown in SEQ ID NOs: 1 to 10
- Step (d) of preparing a development support carrying two probes (d); 5 The amplification
- Detecting step (e); 6) step (f) for evaluating by determining a detection image; and at least three types of detection target pneumoniae selected from ten types of detection target pneumoniae as detection targets Although it is not particularly limited as long as it is a method, it is preferable to detect at least 5 types of pneumoniae to be detected, that is, 5 to 10 types.
- a forward primer comprising the nucleotide sequence shown in SEQ ID NO: 21 capable of amplifying a target nucleic acid specific to each pneumoniae arbitrarily extracted from a sample
- a reverse primer comprising a nucleotide sequence represented by SEQ ID NO: 31 and an RNA polymerase promoter sequence added to the 5 ′ end thereof
- a forward primer comprising a nucleotide sequence represented by SEQ ID NO: 22
- a nucleotide represented by SEQ ID NO: 32 A reverse primer comprising a sequence and an RNA polymerase promoter sequence added to the 5 ′ end thereof
- a forward primer comprising a nucleotide sequence represented by SEQ ID NO: 23; a nucleotide sequence represented by SEQ ID NO: 33 and an addition to the 5 ′ end thereof
- RNA Reverse primer consisting of a remerase promoter sequence
- a forward primer consisting of a nucleotide sequence shown in SEQ ID NO: 21 capable of amplifying
- a reverse primer consisting of the nucleotide sequence shown in SEQ ID No. 35 and the RNA polymerase promoter sequence added to the 5 ′ end thereof; consisting of the nucleotide sequence shown in SEQ ID No. 26 A reverse primer comprising a forward primer and a nucleotide sequence shown in SEQ ID NO: 36 and an RNA polymerase promoter sequence added to the 5 ′ end thereof; SEQ ID NO: 2 A forward primer consisting of the nucleotide sequence shown in SEQ ID NO: 37, a reverse primer consisting of the nucleotide sequence shown in SEQ ID NO: 37 and an RNA polymerase promoter sequence added to the 5 ′ end thereof; a forward primer consisting of the nucleotide sequence shown in SEQ ID NO: 28 A reverse primer comprising a nucleotide sequence represented by SEQ ID NO: 38 and an RNA polymerase promoter sequence added to the 5 ′ end thereof; a forward primer comprising a nucleotide sequence represented by SEQ ID
- Streptococcus pneumoniae Haemophilus influenzae, Mycoplasma pneumoniae, Legionella pneumophila, and Chlamydophila pneumoniae (hereinafter referred to as “Pneumoniae for detection of outpatients”) when the above-mentioned three types of pneumoniae are detected.
- Streptococcus pneumoniae such as Streptococcus pneumoniae, Haemophilus influenza and Mycoplasma pneumoniae, Streptococcus pneumoniae, Haemophilus influenza and Legionella pneumophila, Streptococcus pneumoniae, Haemophilus influenzae, and Chlamydophila pneumoniae Pseudomonas aeruginosa often detected in hospitalized patients due to hospital combinations or hospital infections , Klebsiella pneumoniae, Staphylococcus aureus, MRSA, Moraxella catarrhalis (hereinafter also referred to as “detected pneumoniae for inpatients”), Pseudomonas aeruginosa, Klebsiella pneumoniae and Staphylococcus aureus, and Klebsiella aureus Pneumonie, Staphylococcus aureus and MRSA, Pseudomonas aeruginosa, MRSA and Moraxella catarrhalis, Staphylococcus
- Streptococcus pneumoniae When there are four types of pneumoniae to be detected, Streptococcus pneumoniae, Haemophilus influenza, Mycoplasma pneumoniae and Legionella pneumophila, Streptococcus pneumoniae, Haemophilus influenzae, Mycoplasma pneumoniae among the detection target pneumoniae for outpatients , Streptococcus pneumoniae, Streptococcus pneumoniae, Hemophilus influenza, Legionella pneumophila and Chlamydophila pneumoniae, Streptococcus pneumoniae, Mycoplasma pneumoniae, Legionella pneumophila and Chlamydophila pneumoniae, Hemophile, Emoplasma Legionella pneumophila and Among the four types of combinations such as Midophila pneumoniae and pneumoniae to be detected for hospitalized patients, Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus and MRSA, Pseudomonas aeruginosa, Klebsiella
- MRA Streptococcus pneumoniae
- Haemophilus influenza Mycoplasma pneumoniae, Legionella pneumophila, MRSA and Moraxella catarrhalis
- Streptococcus pneumoniae Haemophilus influenza, Mycoplasma pneumoniae, Chlamydophila pneumoniae, MRSA and Moraxella catarrhalis
- Streptococcus pneumoniae Hemophilus influenza, Mycoplasma pneumoniae, MRSA, Staphylococcus aureus and Moraxella catarrhalis
- Streptococcus pneumoniae Influenza, Mycoplasma pneumoniae, cash register Nera pneumophila, Chlamydophila pneumoniae, Pseudomonas aeruginosa, Streptococcus pneumoniae, Hemophilus influenza, Mycoplasma pneumoniae, Legionella pneumophila, Pseudomonas aeruginosa, MRSA, Streptococcus pneumoniae, Hemoni
- detection pneumoniae for outpatients MRSA and Staphylococcus aureus
- detection target pneumoniae for outpatients MRSA and Moraxella catarrhalis
- Streptococcus pneumoniae hemophilus influenza , Mycoplasma pneumoniae, Legionella pneumophila, MRSA, Staphylococcus aureus and Moraxella catarrhalis
- Streptococcus pneumoniae Hemophilus influenza, Mycoplasma pneumoniae, Chlamyphila pneumoniae, MRSA, Staphylococcus aureus Streptococcus pneumoniae, Haemophilus influenza, Mycoplasma pneumoniae, Pseudo 120 streets consisting of eggplant aeruginosa, MRSA, Staphylococcus aureus and Moraxella catarrhalis
- Streptococcus pneumoniae Haemophilus influenza, Pseudomonas aeruginosa, Klebsiella pneumoniae, MRSA, Sta
- MRA Staphylococcus aureus and Moraxella catarrhalis
- Pneumoniae to be detected for outpatients Pseudomonas aeruginosa, MRSA and Staphylococcus Aureus
- Pneumoniae to be detected for outpatients Pseudomonas aeruginosa, MRSA and Moraxella catarrhalis
- Streptococcus pneumoniae Hemophilus influenza, Mycoplasma pneumoniae, Legionella pneumophila, Pseudomonas aeruginosa, MRSA, Staphylococcus aureus Moraxella catarrhalis
- Streptococcus pneumoniae Hemophilus influenza, Mycoplasma pneumoniae, Kramidov La pneumoniae, Pseudomonas aeruginosa, MRSA
- MRA Staphylococcus aureus
- Moraxella catarrhalis Klebsiella pneumoniae, Streptococcus pneumoniae, Hemophilus influenza, Mycoplasma pneumoniae, Legionella ⁇ Pneumophila, Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus, MRSA, Moraxella catarrhalis, Streptococcus pneumoniae, Haemophilus influenza, Mycoplasma pneumoniae, Legionella pneumophila, Pseudomonas aergiosa aergiosa
- lungs including Coccus aureus, Moraxella catarrhalis, etc. It can be exemplified a combination of fungi.
- a target nucleic acid specific to each of the detection target pneumoniae arbitrarily extracted from a sample that may contain the detection target pneumoniae, the target nucleic acid
- the target nucleic acid There is no particular limitation as long as it is a method of amplifying as a single-stranded nucleic acid using a primer capable of amplifying.
- Specific target nucleic acids for each of the above pneumococci include Streptococcus pneumoniae lytA, Haemophilus influenza dnaJ, Mycoplasma pneumoniae dnaJ1, Chlamydophila pneumoniae dnaJ, Staphylococcus aureus spaA, Examples include nucleic acids in regions contained in MRSA mecA, Legionella pneumophila dnaJ, Moraxella catarrhalis dnaJ, Pseudomonas aeruginosa dnaJ, and Klebsiella pneumoniae dnaJ.
- the primer pairs used in the above step (a) or (a ′) are primer pairs that can amplify pneumoniae-specific target nucleic acids in 10 types of pneumoniae to be detected. It is a primer pair including a target sequence that is different for each detection target pneumococci selected from the nucleotide sequences shown.
- a combination of primer pairs consisting of (chimeric) primers is used. Also, instead of the above sequences, 5′-AGCGTATGAAATCCCTACTACTAT-3 ′ (SEQ ID NO: 54) and 5′-CAAAGATATCGCTGGAAGTCG-3 ′ (SEQ ID NO: 56) for Klebsiella pneumoniae, and 5′-GCGAGGTGGTCGTCTGCATGCATGCATGCATG A combination of a primer pair consisting of a forward primer and a reverse (chimeric) primer containing the target nucleotide sequence shown in (SEQ ID NO: 55) and 5′-GATGTGCAAGGTGGTGGGGA-3 ′ (SEQ ID NO: 57) can also be used.
- a primer having the nucleotide sequence shown in SEQ ID NO: 21 to 30, 54 or 55 is used as the forward primer.
- a tag sequence can be added to the 5'-end side of each of 10 types of target sequences that differ depending on the pneumoniae to be detected.
- An example of such a tag sequence is 5'-TAGCAGGATCCCTCTAAG-3 '(SEQ ID NO: 41).
- the reverse primer comprises a nucleotide sequence shown in SEQ ID NOs: 31 to 40, 56 or 57, and an RNA polymerase promoter sequence added to the 5 ′ end of each of 10 types of target sequences that differ depending on the pneumoniae to be detected. Use primers.
- RNA polymerase promoter sequence examples include a T7 RNA polymerase promoter sequence, a T3 RNA polymerase promoter sequence, an SP6 RNA polymerase promoter sequence, and the like. Among them, the T7 RNA polymerase promoter sequence is preferable in terms of high RNA amplification efficiency. .
- T7 RNA polymerase promoter sequences include 5'-AATTCTAATACGACTCACTATAGGGAG-3 '(SEQ ID NO: 42) and 5'-CTAATACGACTCACTATAGGGAG-3' (SEQ ID NO: 43).
- any primer that can amplify target pneumoniae-specific target nucleic acids can be used as a primer in the present invention.
- the forward primer and reverse primer can be synthesized by a conventional method using a DNA synthesizer or the like.
- test samples that may contain the nucleic acid of the pneumoniae fungus to be detected in the above step (a) or (b ′) include saliva, sputum, peripheral blood, bronchoalveolar lavage, nasal lavage, and gargle from patients with pneumonia. Liquids, nasopharyngeal swabs, microorganisms contained in them, and cell lysates of cultures of such microorganisms.
- RNA extraction method using guanidine thiocyanate or a nucleic acid extraction method using EDTA-SDS-phenol-ethanol
- Extragen II manufactured by Tosoh Corporation
- Mora-Extract Advanced Microorganism Research
- the extracted RNA or DNA can be appropriately cleaved to prepare RNA or DNA containing one or a plurality of target RNAs or target DNA 5'-side sequences and 3'-side target-specific sequences.
- RNA amplification method such as RNA amplification / detection by concerted reaction (Transcription Reverse Transcription Concerted Reaction, TRC method) can be used, but the NASBA method, particularly the NASBA method described in International Publication No. 2009/057330 is advantageous. Can be used.
- This NASBA method is a method of amplifying a target RNA in a sample via RNA polymerase or reverse transcriptase.
- a step of preparing a solid phase DNA (+) primer by immobilizing the 5 ′ end of the chimeric primer shown in SEQ ID NOs: 1 to 10 containing a DNA sequence corresponding to the 5 ′ target-specific sequence on the substrate surface;
- B A step of preparing a liquid phase cDNA ( ⁇ ) primer shown in SEQ ID NOs: 11 to 20, in which a T7 RNA polymerase promoter sequence is added to the 5 ′ end side of a primer containing a cDNA sequence complementary to the 3 ′ side sequence of the target RNA.
- (D) the liquid phase cDNA prepared in step (B) ( -) The primer and the sample RNA strand prepared in step (C) are contacted in the liquid phase, the liquid phase cDNA (-) primer and the sample RNA are hybridized, and then the DNA (-) strand by reverse transcriptase A step of preparing a cDNA strand-RNA strand complex by extending the RNA; and (E) the RNA strand in the DNA strand-RNA strand complex is specifically added to the cDNA strand-RNA strand complex prepared in step (D).
- Phase DNA (+) primer in liquid phase Touch hybridize the single-stranded DNA (-) and the solid-phase DNA (+) primer, and then extend the DNA (+) strand with an enzyme having DNA-dependent DNA polymerase activity to (G) RNA polymerase is allowed to act on the double-stranded DNA prepared in step (F), and the single-stranded RNA ( ⁇ ) is converted using the RNA polymerase promoter sequence derived from the DNA ( ⁇ ) strand.
- a method comprising the step of amplifying is disclosed.
- the NASBA method the PCR method, the strand displacement amplification method (SDA method), the ligase chain reaction method (in order to amplify the target nucleic acid used in the above step (a) or (b ′) ( LCR method) can be used.
- a forward primer a primer selected from the nucleotide sequences shown in SEQ ID NOs: 21 to 30, 54 or 55 is used, and a primer including the nucleotide sequence shown in SEQ ID NOs: 31 to 40, 56 or 57 is used as a reverse primer.
- the probe pair used in the step (b) is different for each pneumococci selected from the nucleotide sequence complementary to the amplification product, for example, for each pneumoniae selected from the nucleotide sequences shown in SEQ ID NOs: 1 to 20.
- they are different probe pairs and are shown as DNA sequences for convenience in the sequence listing, they may be probe pairs composed of DNA nucleotide sequences or probe pairs composed of RNA nucleotide sequences.
- a probe pair consisting of a nucleotide sequence of DNA is preferable because of its excellent stability as a probe.
- the probe pairs that differ depending on the pneumoniae to be detected include Streptococcus pneumoniae 5′-ACGCACGAGTATTGCACGAATAACC-3 ′ (SEQ ID NO: 1) and 5′-TGCCGAAAACCGCTTGATACAGGGAGT-3 ′ (SEQ ID NO: 11), and hemophilus influenza.
- 5′-AACTTGTCCGCATTGCCACGGTTCT-3 ′ (SEQ ID NO: 44) and 5′-TGTGATAGGCTGTGGTGGCTCTGGGGG-3 ′ (SEQ ID NO: 49) of Chlamyphila pneumoniae (5′-TCAAGGGATTGAATCCTGATGCATGAATCGATGAATCGATGAATCG 45) and 5'-ATCTTGTGAAACCCTTTCTGGTCAA-3 '(SEQ ID NO: 50), Legionella pneumophila 5'-AAGAAGCAGCTATAGGAAAAAGAGT-3' (SEQ ID NO: 46) and 5'-GGGGCGCTGATTTGCAATTTAATGT-3 5'-TCGAACAGGGGCGGCATGGGC GCGG-3 '(SEQ ID NO: 47) and 5'-CAGAAGCGTGCGGCCTACGATCAGGT-3' (SEQ ID NO: 52), Pseudomonas aeruginosa 5'-TCCAGGTTCACCGGGGTTTC
- the first probe is paired with at least three kinds of probes paired with the second probe, for example, a second probe selected from the nucleotide sequences shown in SEQ ID NOs: 1 to 10.
- a second probe selected from the nucleotide sequences shown in SEQ ID NOs: 1 to 10.
- examples include at least three types, preferably at least five types of first probes of pneumoniae.
- a first probe-bound labeled polymer in which five types of first probes of pneumoniae are bound to a labeled polymer carrier. Two sets of carriers can be used simultaneously.
- DNA having the nucleotide sequence shown in SEQ ID NOs: 1 to 10 having an additional group introduced at the 5 ′ or 3 ′ end is introduced into the additional group.
- the additional group include an amino group, a carboxyl group, a hydroxyl group, and a thiol group.
- an amino group is preferable.
- a first probe in which one first probe is bound to one labeled polymer carrier as in the prior art. Since there is no need for an operation of mixing a plurality of probe-bound labeled polymer carriers, the detection operation is simplified, and the labeled polymer carrier concentration described below, such as labeled latex, can be set to an optimum concentration. This is preferable.
- FIG. 13 One specific example of the present invention is shown in FIG. 13 as a schematic diagram.
- Examples of the polymer carrier in the labeled polymer carrier include carboxymethyl cellulose (CMC), hydrophilic resins such as polyacrylate having a carboxyl group, acrylic latex, polyester latex, polystyrene latex, polyurethane latex, and polyacetic acid.
- Examples of the latex include vinyl latex, SBR resin, NBR resin, polyamide latex, and carboxy-modified polystyrene latex.
- carboxyl group-containing polystyrene latex solid content 4% (w / W)) (Duke Scientific) or carboxyl group-containing polystyrene latex (solid content 10% (w / w)) (Bangs).
- Specific methods for preparing the first probe-bound labeled polymer carrier include, for example, at least three types selected from nucleotide sequences represented by SEQ ID NOs: 1 to 10, respectively.
- the particle size of the polymer carrier can be appropriately selected, but is preferably selected from particle sizes smaller than the membrane pore size. For example, the size of particles having a diameter of 500 nm or less can be selected.
- a polymer carrier exhibiting a color distinguishable from the color of the development support may be used, and a polymer carrier colored with a pigment or the like may be used.
- a polymer carrier can also be used.
- the second probe in the step (d) at least three kinds of probes paired with the first probe, for example, at least paired with the first probe selected from the nucleotide sequences shown in SEQ ID NOs: 11 to 20 are used.
- Examples of the method for preparing the support carrying and carrying the second probe include a method in which the second probe is immobilized on a predetermined position on the development support for each pneumoniae.
- a method in which a probe having the nucleotide sequence shown in SEQ ID NOs: 11 to 20 having an additional group introduced at the 3 ′ end is immobilized on a development support.
- Examples of the addition group include an amino group, a carboxyl group, a hydroxyl group, and a thiol group.
- an amino group is preferable. Regardless of the presence or absence, it is possible to produce the second probe using a known method such as a chemical synthesis method.
- Examples of the development support include nylon membrane derivatives such as nylon membranes and carboxyl group-modified nylon membranes, cellulose membrane derivatives such as cellulose membranes and nitrocellulose membranes, and the nucleotide sequences shown in SEQ ID NOs: 11 to 20 above.
- nylon membrane derivatives such as nylon membranes and carboxyl group-modified nylon membranes
- cellulose membrane derivatives such as cellulose membranes and nitrocellulose membranes
- the nucleotide sequences shown in SEQ ID NOs: 11 to 20 above In the case where an amino group is introduced, at least three kinds, preferably at least five kinds, selected from the nucleotide sequences shown in SEQ ID NOs: 11 to 20 by a reaction that forms a covalent bond via an amino group and a carboxyalkyl group. Since the second probe of each pneumoniae can be easily solid-phased at a predetermined position that can be identified on the development support, a carboxyl group-modified nylon membrane is preferable.
- a carboxyl group-modified nylon membrane is treated with water-soluble carbodiimide, activated by washing with deionized water, and the activated carboxyl group-modified nylon membrane.
- the nucleotide having the second probe sequence is immobilized on a predetermined position appropriately allocated so as to be distinguishable for each pneumoniae, air-dried for 15 minutes, and then the nucleotide having the second probe sequence is immobilized.
- An example is a method in which the carboxyl group-modified nylon membrane is treated with a Tris base buffer to eliminate active groups, and the membrane on which nucleotides are immobilized is washed with deionized water.
- the form of immobilizing the nucleotide having the second probe sequence is not particularly limited, and may be a line shape or a circular spot shape.
- a method of detecting the amplification product in the step (e) a method of detecting by hybridizing the amplified single-stranded nucleic acid to the second probe carried on the development support and the labeled polymer carrier-bound first probe. If it is not particularly limited, it is preferable to hold a first probe-bound labeled polymer carrier in which a plurality of first probes are bound to a labeled polymer carrier in advance in a holding part, and as such a holding part, ADVANTEC made by ADVANTEC An absorbent pad can be preferably exemplified.
- Such a holding part composed of an absorption pad or the like holding the first probe-bound labeled polymer carrier is sequentially connected to the other end of the second probe-carrying development support so that it can be advantageously used in the detection method of the present invention. It can be used as a test piece for nucleic acid chromatography.
- Examples of the method for producing the holding part include a method in which a labeled polymer carrier to which a plurality of nucleotides having the first probe sequence are bonded is applied to the holding part and dried.
- step (e) as a method of detecting the amplification product in the step (a) by hybridizing to the second probe supported on the development support and the first probe bound to the labeled polymer carrier, for example, By immersing the nucleic acid chromatograph test piece in a solution containing an amplification product, the first probe-bound labeled polymer carrier is leached from the holding part to the development support, and the first probe on the development support
- the second probe of each detection target pneumococci forming a pair reaches a predetermined position where the solid phase is immobilized, the amplification product can be captured by sandwich hybridization at the predetermined position. Even when a plurality of types of target single-stranded nucleic acids are present in the amplification product, they are sequentially captured by each second probe at a predetermined position.
- step (f) the labeled polymer carrier to which the nucleotide having the first probe sequence is bound has reached the position where the nucleotide having the second probe sequence has been immobilized on the development support.
- nucleotides consisting of the sequences shown in SEQ ID NOs: 44 to 48 can be used, and the sequences shown in SEQ ID NOs: 11 to 20
- nucleotides consisting of the sequences shown in SEQ ID NOs: 49 to 53 can be used.
- Examples of the method for qualitatively and quantitatively determining the target nucleic acid in the step (c ′) include electrophoresis, hybridization method, and sequencing method.
- Examples of the electrophoresis method include a method of analyzing the molecular weight of the amplified product by a molecular sieving effect using an agarose gel, and an analysis system of Agilent Technology Co., Ltd. using capillary electrophoresis.
- Examples of the hybridization method include a method of monitoring and analyzing the production process of the amplification product in real time using a reagent for real time monitoring, such as real time PCR. Examples of the real-time monitoring reagent include TaqMan (registered trademark: Applied Biosystems) probe.
- the hybridization method can also include a Northern blot method.
- Examples of the sequencing method include a dideoxy method in which the synthesis of DNA polymerase is stopped in a base-specific manner using dideoxynucleotides.
- the detection kit for pneumoniae causing bacteria of the present invention can be used in the detection method for the pneumoniae causing bacteria of the present invention, and a target nucleic acid specific to each pneumoniae arbitrarily extracted from a sample can be amplified.
- a forward primer comprising a nucleotide sequence represented by SEQ ID NO: 23; and SEQ ID NO: 33 Nucleus shown in A reverse primer comprising a tide sequence and an RNA polymerase promoter sequence added to the 5 ′ end thereof; a forward primer comprising a nucleo
- Such a kit includes the above-described primer pair, various reagents used in the RNA amplification method in the step (b ′), and the target nucleic acid in the step (c ′).
- Various reagents used for qualitative / quantitative determination may be provided.
- an amplification set that amplifies as a single-stranded nucleic acid using a primer from a target nucleic acid specific to each pneumoniae arbitrarily extracted from a sample; a nucleotide sequence complementary to the amplification product, eg, SEQ ID NOs: 1 to 10 Or a first probe-bound labeled polymer carrier in which at least three kinds of first probes of pneumococcus selected from the nucleotide sequences shown in 44 to 48 are bound to the labeled polymer carrier; A second probe made of, for example, a second probe of at least three types of pneumococci selected from the nucleotide sequences shown in SEQ ID NOs: 11 to 20 or 49 to 53, at a predetermined position identifiable for each pneumoniae
- a kit including a second probe-carrying development support dedicated to each pneumococcus or shared by a plurality of pneumococci, and the amplification set and the nucleic acid chroma A kit and a graph for
- PCR was performed using EX Taq Hot start (manufactured by TaKaRa bio) as an instrument, using TaKaRaPCR Thermal CyclerGP as a reagent kit in accordance with the protocol described in the following. 95 ° C .; 3 min, (95 ° C .; 10 sec, 65 ° C .; 10 sec, 72 ° C .; 10 sec) ⁇ 40 cycles.
- the obtained PCR product was analyzed by electrophoresis using MultiNA. The result of electrophoresis is shown in FIG. Since the PCR product obtained in any strain was amplified, it was found that the newly designed primer pair was useful for detecting Moraxella catarrhalis.
- RNA as a template was prepared by DNA cloning using a plasmid vector.
- the target gene region was cloned to produce a recombinant.
- each DNA fragment of the target gene dnaJ of Mycoplasma pneumoniae or Chlamyphila pneumoniae was inserted into a plasmid vector, and then E. coli serving as a host was transformed. After extracting the plasmid from E.
- RNA target gene by NASBA method As a forward primer, a primer in which TAGCAGGATCCCTCTAG (SEQ ID NO: 41) is added to the 5 ′ end side of each of 10 types of target sequences for each detection target pneumoniae selected from the nucleotide sequences shown in SEQ ID NOs: 21 to 40, and reverse is used.
- a promoter in which CTAATACGACTCACTATAGGGGAG (SEQ ID NO: 43) was added as a T7 RNA polymerase promoter sequence on the 5 ′ end side of each of 10 types of target sequences that differ for each pneumoniae to be detected was used.
- RNA extracted from the seven standard strains of the above Streptococcus pneumoniae, Legionella pneumophila, Klebsiella pneumoniae, Moraxella catarrhalis, Staphylococcus aureus, Mycoplasma pneumoniae, and Chlamydophila pneumoniae for each bacterial species Data obtained by performing NASBA method to amplify RNA of a target gene is shown.
- RNA extraction was carried out using MORA-EXTRACT (manufactured by Advanced Microorganism Research) according to the attached protocol, and NASBA method was carried out using the NASBA Amplification kit (manufactured by Kainos) according to the attached protocol.
- RNA samples used as templates for Streptococcus pneumoniae, Mycoplasma pneumoniae, Chlamydophila pneumoniae, Legionella pneumophila, Klebsiella pneumoniae, Moraxella catarrhalis, Staphylococcus aureus are 1, 1/10, 1/100, 1 / 1000-fold dilutions were used for each, using Bio Analyzer; Agilent RNA Pico 6000.
- Bio Analyzer Agilent RNA Pico 6000.
- an enzyme-free reaction solution was prepared and carried out in the same process. The results are shown in FIGS. It was confirmed that RNA was amplified by the NASBA method for all target detection-target pneumonia-causing species.
- FIG. 10 shows the results of studies on multiplexing using the present primer pairs for Legionella pneumophila, Pseudomonas aeruginosa, Chlamydophila pneumoniae, MRSA, and Haemophilus influenza.
- FIG. 11 shows the multiplex examination results using this primer pair for Staphylococcus aureus, Streptococcus pneumoniae, Moraxella catarrhalis, Mycoplasma pneumoniae, and Chlamydophila pneumoniae. It was confirmed that RNAs of all target detection target pneumonia causative species were amplified by the NASBA method.
- RNA amplified by NASBA It is necessary to determine whether the RNA amplified by NASBA has the target sequence of interest. Perform real-time PCR targeting DNA synthesized as a by-product of NASBA reaction, measure the Ct value of the control of NASBA product and NASBA unreacted sample, and indirectly confirm the target NASBA product by comparing this difference did. Using SYBR premixEX Taq Hot start (TaKaRa bio), PCR was performed under the conditions of 95 ° C .; 3 min, (95 ° C .; 10 sec, 65 ° C .; 10 sec, 72 ° C .; 10 sec) ⁇ 40 cycles. It was. In all bacterial species, RNA amplified by NASBA indirectly confirmed the target NASBA product of interest. An example of Moraxella catarrhalis is shown in FIG.
- sequence information analysis software DNASISSpro registered trademark (Hitachi Software Co., Ltd.) was used for analysis, and oligonucleotide probes having the sequences shown in SEQ ID NOs: 1 to 20 were determined.
- HEPES [4- (2-Hydroxyethyl) -1-piperazinyl] ethanesulfonic acid
- a nonionic surfactant in the resulting precipitate manufactured by Saikyo Kasei Co., Ltd.
- a labeled polymer carrier having a plurality of nucleotides having the first probe sequence bound thereto was prepared and stored at 4 ° C. until use.
- One kind of the above 3′-terminal amino group-introduced oligonucleotide (second probe) was bound to the activated carboxyl group-modified nylon membrane and air-dried for 15 minutes. After treating the air-dried membrane with a Tris base buffer to remove the remaining active groups, the membrane is washed with deionized water and air-dried again, so that the oligonucleotide having the second probe sequence can be Thus, a development support that was solid-phased in a line at a predetermined position that was distinguishable from each other was produced. Since there are 3 or more types of pneumoniae to be detected, the above treatment was performed at least 3 times for each pneumoniae.
- the reference line is a line detected for the purpose. Since this line is detected for the purpose of confirming the flow of the labeled polymer carrier, it is preferable to detect the reference line even when there is a nucleic acid of the pneumonia causing bacteria to be detected.
- a test piece for nucleic acid chromatography used in the present invention was prepared as follows.
- a labeled polymer carrier to which a plurality of nucleotides having the first probe sequence of each of the aforementioned pneumococci was bonded was dissolved in a buffer solution, applied to a development pad (manufactured by ADVANTEC), and then dried to form a holding part.
- a holding portion was connected to one end of the development support at a portion that did not overlap the development support. Further, an absorption pad (manufactured by ADVANTEC) was connected to the other end of the development support to obtain a test piece for nucleic acid chromatography measurement.
- the first probe-bound labeled polymer carrier to which one probe is bound is used, and the second probe-carrying development support is 5 sheets each carrying 5 types of second probes of detection target pneumoniae for outpatients.
- a second probe-carrying deployment support (dedicated) and five second probe-carrying deployment supports (dedicated) each carrying 5 types of second probes of pneumoniae to be detected for hospitalized patients were used. The results are shown in FIG. In any strain used in the experiment, a red colored line is observed at the reference position, and in the absence of the amplification product, non-specific binding is present between the labeled polymer carrier-bound first probe and the second probe. It was confirmed that it did not occur.
- NASBA product by nucleic acid chromatography
- Legionella pneumophila, Pseudomonas aeruginosa, Klebsiella pneumoniae, MRSA, Haemophilus influenza, Staphylococcus aureus, Streptococcus pneumoniae, Moraxella catarrhalis, Mycoplasma pneumoniae, Chlamydophila pneumoniae NASBA products were detected and examined by nucleic acid chromatography. Test specimens for measurement were prepared individually for each detection target pneumococci using the probes shown in Table 2 according to the above method. The result is shown in FIG. All NASBA products were detected on nucleic acid chromatostrips to which probes for each pneumococcus were bound.
- NASBA product amplified with multiplex primer by nucleic acid chromatography Multiple primer pairs using Legionella pneumophila, Pseudomonas aeruginosa, Klebsiella pneumoniae, MRSA, Haemophilus influenza, Staphylococcus aureus, Streptococcus pneumoniae, Moraxella catarrhalis, Mycoplasma pneumoniae, Chlamydophila pneumoniae NASBA products amplified with the primers were detected by nucleic acid chromatography. The result is shown in FIG. All NASBA products amplified using multiplex primers were detected by nucleic acid chromatography.
- Legionella pneumophila is cultured at 35 ° C. using BCYE ⁇ medium
- Chlamydia pneumoniae is cultured using HEp-2 cells
- Mycoplasma pneumoniae is cultured at 37 ° C. using PPLO medium
- Haemophilus influenzae is The culture was performed at 37 ° C. using a chocolate medium
- Streptococcus pneumoniae was cultured at 37 ° C. using a sheep blood medium.
- Nucleic acids were extracted from each sample from 100 ⁇ L of each bacterial suspension using EXTRAGENII reagent (manufactured by Tosoh Corporation), and the nucleic acids of the above five types of bacteria were dissolved with 50 ⁇ L of RNase free water.
- Each target of 10 bacterial species different from the probe pair set of the present invention using 2.5 ⁇ L of nucleic acid solution using NASBA Amplification Kit (manufactured by Kainos) and using NASBA Amplification Kit (manufactured by Kainos) with totalRNA as a template NASBA amplification reaction was performed using a primer pair specific to each bacterial species that hybridizes to a sequence in the region.
- the primer concentration for each bacterium was prepared so that the final concentration was 0.2 ⁇ M.
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Abstract
Description
これまでに、モラクセラ・カタラーリスのゲノム情報が解読されておらず、類縁属種のdnaJ遺伝子配列を基に設計を進めていたが、本菌に特異的なプライマーを設計することは困難であった。一方近年、本菌のゲノム配列情報のみが明らかとなったため, この配列を基にORF抽出によってdnaJ遺伝子のアノテーションをアライメントソフトDNASISpro(Hitachi Software社製)により実施し、この配列から新たなプライマーの設計を試みた。8株のモラクセラ・カタラーリス菌株を用いて新規プライマーの検証を実施した、試薬キットとしてTaKaRaPCR Thermal CyclerGPを記載のプロトコルに準じて用い、機器としてEX Taq Hot start(TaKaRa bio社製)を用いてPCRを95℃;3min、(95℃;10sec、65℃;10sec、72℃;10sec)×40cycles の条件で実施した。得られたPCR産物は、MultiNAを用いて電気泳動により解析された。電気泳動の結果を図1に示す。いずれの菌株においても得られたPCR産物は増幅していたことから、新たに設計したプライマー対がモラクセラ・カタラーリスの検出に有用であることがわかった。
(鋳型となるRNAの作製)
マイコプラズマ・ニューモニエ及びクラミドフィラ・ニューモニエは培養が困難であるため、プラスミドベクターを利用したDNAクローニングによって、鋳型となるRNAを作製した。標的遺伝子領域をクローニングし組換え体を作製した。まず、プラスミドベクターに、マイコプラズマ・ニューモニエ又はクラミドフィラ・ニューモニエの標的遺伝子dnaJの各DNA断片を挿入後、宿主となる大腸菌をそれぞれ形質転換した。大腸菌からプラスミドを抽出後PCRで標的遺伝子のDNA増幅をPCRで行い、得られたPCR産物からRiboMAX(登録商標)T7Expressシステム(Promega社製)を用いてプロトコールにしたがってRNAを増幅し、得られたRNAをNASBA法の鋳型とした。マイコプラズマ・ニューモニエ及びクラミドフィラ・ニューモニエ以外の検出対象肺炎原因菌のRNAは常法により調製した。
フォワードプライマーとしては、配列番号21~40に示されるヌクレオチド配列から選ばれる検出対象肺炎菌ごとに10種類の各標的配列の5’末端側にTAGCAGGATCCCTCTAAG(配列番号41)を付加したプライマーを用い、リバースプライマーとしては、検出対象肺炎菌ごとに異なる10種類の各標的配列の5’末端側にT7RNAポリメラーゼのプロモーター配列として、CTAATACGACTCACTATAGGGAG(配列番号43)が付加されたプロモーターを用いた。上記ストレプトコッカス・ニューモニエ、レジオネラ・ニューモフィラ、クレブシエラ・ニューモニエ、モラクセラ・カタラーリス、スタフィロコッカス・アウレウス、マイコプラズマ・ニューモニエ、クラミドフィラ・ニューモニエの7種の標準菌株から抽出したRNAを鋳型とし、1菌種ごとにNASBA法を実施して標的遺伝子のRNAを増幅したデータを示す。RNA抽出は、MORA-EXTRACT(Advanced Microorganism Research社製)を用いて添付のプロトコールにしたがって実施し、NASBA法は、NASBA Amplificationキット(カイノス製)を用いて添付のプロトコールにしたがって実施した。ストレプトコッカス・ニューモニエ、マイコプラズマ・ニューモニエ、クラミドフィラ・ニューモニエ、レジオネラ・ニューモフィラ、クレブシエラ・ニューモニエ、モラクセラ・カタラーリス、スタフィロコッカス・アウレウスの各鋳型としたRNA試料は、1、1/10、1/100、1/1000倍に希釈したものをそれぞれ用い、Bio Analyzer ; Agilent RNA Pico 6000を用いて行った。コントロールとして、酵素無添加の反応液を調製し、同様の工程にて実施した。結果を図2~8に示す。すべての標的検出対象肺炎原因菌種について、NASBA法によりRNAが増幅したことを確認した。
本件プライマー10対のマルチプレックス化を実施した際に、各プライマーが標的菌以外の対象菌と非特異反応を起こすか否かを検証した。結果を図9に示す。本件プライマー10対のすべてのプライマーにおいて非特異反応は認められなかった.
レジオネラ・ニューモフィラ、シュードモナス・アエルギノーサ、クラミドフィラ・ニューモニエ、MRSA、ヘモフィルス・インフルエンザについて、本件プライマー対を用いたマルチプレックス化の検討結果を図10に示す。また同様に、スタフィロコッカス・アウレウス、ストレプトコッカス・ニューモニエ、モラクセラ・カタラーリス、マイコプラズマ・ニューモニエ、クラミドフィラ・ニューモニエについて、本件プライマー対を用いたマルチプレックス化の検討結果を図11に示す。すべての標的検出対象肺炎原因菌種のRNAがNASBA法により増幅されることが確認された。
NASBAによって増幅したRNAが目的の標的配列を有するか否かを判断する必要がある。NASBA反応の副産物として合成されるDNAを標的としたリアルタイムPCRを実施し、NASBA産物とNASBA未反応サンプルのコントロールのCt値を測定し、この差を比較することによって標的NASBA産物を間接的に確認した。SYBR premixEX Taq Hot start(TaKaRa bio社製)を用いて、プロトコルに準じ、PCRは、95℃;3min、(95℃;10sec、65℃;10 sec、72℃;10sec)×40cyclesの条件で行った。全菌種において、NASBAによって増幅したRNAが目的の標的NASBA産物を間接的に確認された。モラクセラ・カタラーリスの例を図12に示す。
検出対象となる各検出対象肺炎菌の以下の表1に示される遺伝子領域の核酸の標的配列に対し相補的配列を持つ1対のアミノ基含有オリゴヌクレオチドプローブの作製を試みた。
オリゴヌクレオチドプローブとカルボキシル基含有ポリスチレンラテックスとを結合させるために、5’末端にアミノ基を導入した5’末端アミノ基導入オリゴヌクレオチドを合成した。上記で選択された配列からなる20種類の各オリゴヌクレオチドの5’末端アミノ基導入オリゴヌクレオチドを各肺炎菌の第一のプローブ配列として作製した。検出対象とされる、少なくとも3種類、好ましくは少なくとも5種類の各肺炎菌の第1のプローブ配列を有するヌクレオチドと、カルボキシル基含有ポリスチレンラテックス(Bangs社製)とを、水溶性カルボジイミドを50mMのMES(2-Morpholinoethanesulfonic acid、monohydrate)(同仁化学研究所社製)緩衝液中で混合し、ラテックス中のカルボキシル基と複数の第1プローブのアミノ基とを結合させた後、モノエタノールアミン(和光純薬工業社製)を添加し、さらに反応させた。上記反応液を遠心分離後上清除去し、得られた沈殿に非イオン性の界面活性剤を含むHEPES(2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonicacid)(埼京化成社製)緩衝液で洗浄及び再懸濁して、第1のプローブ配列を有するヌクレオチドが複数結合している標識高分子担体が調製され、使用まで4℃にて保存された。
上記で選択された第2プローブ配列からなるオリゴヌクレオチドと、カルボキシル基修飾ナイロンメンブレン(ポール社製)とを結合させるために、選択された第2プローブ配列の3’末端にアミノ基を導入した3’末端アミノ基導入オリゴヌクレオチド(第2プローブ)をDNA合成機により作製した。上記メンブレンを水溶性カルボジイミドにより処理し、脱イオン水で洗浄し活性化させた。かかる活性化されたカルボキシル基修飾ナイロンメンブレンに上記3’末端アミノ基導入オリゴヌクレオチド(第2プローブ)の1種類を結合させ、15分間風乾した。風乾されたメンブレンをTrisベース緩衝液で処理し、残存する活性基を除去した後、メンブレンを脱イオン水で洗浄し、再び風乾することで、第2プローブ配列を有するオリゴヌクレオチドが、肺炎菌ごとに識別可能な所定の位置にライン状に固相化された展開支持体が作製された。検出対象の肺炎菌が3種以上のため、上記処理を肺炎菌ごとに少なくとも3回行った。
肺炎原因菌の検出位置にラインが検出されなかった場合、検出される核酸が無いことがその要因である点を明らかとするために、クロマトストリップ上を標識高分子担体がフローできたことを証明する目的で検出されるラインがリファレンスラインである。なお、このラインは標識高分子担体のフローの確認を行う目的で検出されるため、検出される肺炎原因菌の核酸がある場合もリファレンスラインが検出されるようにすることが好ましい。
本発明に用いられる核酸クロマトグラフ用試験片を以下のように作製した。上記各肺炎菌の第1プローブ配列を有するヌクレオチドが複数結合している標識高分子担体を緩衝液に溶解して展開パッド(ADVANTEC社製)に塗布後、乾燥させて保持部とした。上記展開支持体の一端に、上記展開支持体とは重なり合わない部分で保持部を連結した。さらに、該展開支持体の他端に吸収パッド(ADVANTEC社製)を連結し、核酸クロマトグラフ測定用試験片とした。
外来患者の肺炎菌特定に特に有効とされる5種類の肺炎原因菌についての外来用プローブセットと、入院患者の肺炎菌特定に特に有効とされる5種類の肺炎原因菌についての入院用プローブセットについて、各肺炎菌用の標識高分子担体結合第1プローブと第2プローブ(専用)との間において直接的にハイブリダイズして特異的結合が生じないかどうかを増幅産物の非存在下でチェックした。第1プローブ結合標識高分子担体として、外来患者用検出対象肺炎菌の5種類の第1プローブが結合された第1プローブ結合標識高分子担体と、入院患者用検出対象肺炎菌の5種類の第1プローブが結合された第1プローブ結合標識高分子担体とを用い、第2プローブ担持展開支持体としては、5種類の外来患者用検出対象肺炎菌の第2プローブがそれぞれ担持された5枚の第2プローブ担持展開支持体(専用)と、5種類の入院患者用検出対象肺炎菌の第2プローブがそれぞれ担持された5枚の第2プローブ担持展開支持体(専用)とを用いた。結果を図14に示す。実験に供したいずれの菌株においても、リファレンスの位置に赤色の着色線が観察され、増幅産物の非存在下において、標識高分子担体結合第1プローブと第2プローブとの間に非特異結合が生じないことが確認された。
レジオネラ・ニューモフィラ、シュードモナス・アエルギノーサ、クレブシエラ・ニューモニエ、MRSA、ヘモフィルス・インフルエンザ、スタフィロコッカス・アウレウス、ストレプトコッカス・ニューモニエ、モラクセラ・カタラーリス、マイコプラズマ・ニューモニエ、クラミドフィラ・ニューモニエについて、各菌に対するプライマーで増幅されたNASBA産物を核酸クロマトグラフィーで検出検討した。測定用試験片は、表2に示されるプローブを用いて、上記の方法に準じて各検出対象肺炎菌毎に個別に作製して行った。その結果を図15に示す。いずれのNASBA産物も、各肺炎菌専用プローブが結合されている核酸クロマトストリップで検出された。
各プローブが標的菌以外の対象菌のNASBA産物と非特異反応を起こすか否かを検証した。結果を図16に示す。本件プローブのすべてにおいて非特異反応は認められなかった。
レジオネラ・ニューモフィラ、シュードモナス・アエルギノーサ、クレブシエラ・ニューモニエ、MRSA、ヘモフィルス・インフルエンザ、スタフィロコッカス・アウレウス、ストレプトコッカス・ニューモニエ、モラクセラ・カタラーリス、マイコプラズマ・ニューモニエ、クラミドフィラ・ニューモニエについて、本件プライマー対を用いたマルチプレックスプライマーで増幅されたNASBA産物を核酸クロマトグラフィーで検出した。その結果を図17に示す。マルチプレックスプライマーを用いて増幅されたNASBA産物は、いずれも核酸クロマトグラフィーで検出された。
レジオネラ・ニューモフィラは、BCYEα培地を用い35℃で培養し、クラミドフィラ・ニューモニエは、HEp-2細胞を用いて培養し、マイコプラズマ・ニューモニエは、PPLO培地を用い37℃で培養し、ヘモフィルス・インフルエンザは、チョコレート培地を用い37℃で培養し、ストレプトコッカス・ニューモニエは、羊血液培地を用い37℃で培養した。培養後の各菌懸濁液100μLからEXTRAGENII試薬(東ソー社製)を用いて各試料より核酸を抽出し、50μLのRNase free waterで上記5種類の菌の核酸を溶解した。2.5μLの核酸溶液をNASBA Amplificationキット(カイノス社製)を用いてtotalRNAを鋳型としてNASBA Amplificationキット(カイノス社製)を使用し、本発明のプローブ対のセットとは異なる10菌種の各ターゲット領域内の配列にハイブリダイズする各菌種固有のプライマー対を用いて、NASBA増幅反応を行った。この際の各菌専用プライマー濃度は、最終濃度が0.2μMになるように調製した。増幅反応後各菌の溶液は、増幅終了後のNASBA増幅産物を前処理する事なく1本にまとめ、直ちに核酸クロマトストリップを用いて核酸クロマトグラフ法による肺炎菌の検出を試みた。NASBA産物をフローさせなかった場合は、フローが行われた事を示すリファレンスラインのみ検出された(図18A参照)。上記肺炎原因菌5菌種のNASBA産物をフローさせた場合リファレンスラインとは別に各々の検出位置にラインが検出された(図18B参照)。
Claims (6)
- ストレプトコッカス・ニューモニエ、ヘモフィルス・インフルエンザ、マイコプラズマ・ニューモニエ、クラミドフィラ・ニューモニエ、レジオネラ・ニューモフィラ、クレブシエラ・ニューモニエ、シュードモナス・アエルギノーサ、モラクセラ・カタラーリス、メチシリン耐性スタフィロコッカス・アウレウス(MRSA)、スタフィロコッカス・アウレウスから選ばれる少なくとも3種類の肺炎菌類を検出対象とする肺炎原因菌の検出方法であって、以下の工程(a)~工程(f)を備えたことを特徴とする検出方法。
1)試料中より任意に抽出された各肺炎菌に特異的な標的核酸からプライマーを用いて1本鎖核酸として増幅する工程(a);
2)増幅産物と相補的なヌクレオチド配列から選ばれる、肺炎菌ごとに異なる少なくとも3種類のプローブ対を調製する工程(b);
3)標識高分子担体に少なくとも3種類の各肺炎菌の第1のプローブを結合させ、第1プローブ結合標識高分子担体を調製する工程(c);
4)第1のプローブと対をなす少なくとも3種類の各肺炎菌の第2のプローブを、肺炎菌ごとに識別可能な所定の位置に固相化した第2プローブ担持展開支持体を調製する工程(d);
5)前記増幅産物を、展開支持体に担持された第2プローブ及び標識高分子担体に結合した第1プローブにハイブリダイズさせて検出する工程(e);
6)検出像を判定することにより評価する工程(f); - プライマーが、配列番号21に示されるヌクレオチド配列からなるフォワードプライマーと、配列番号31に示されるヌクレオチド配列及びその5’末端側に付加されたRNAポリメラーゼプロモーター配列からなるリバースプライマー;配列番号22に示されるヌクレオチド配列からなるフォワードプライマーと、配列番号32に示されるヌクレオチド配列及びその5’末端側に付加されたRNAポリメラーゼプロモーター配列からなるリバースプライマー;配列番号23に示されるヌクレオチド配列からなるフォワードプライマーと、配列番号33に示されるヌクレオチド配列及びその5’末端側に付加されたRNAポリメラーゼプロモーター配列からなるリバースプライマー;配列番号24に示されるヌクレオチド配列からなるフォワードプライマーと、配列番号34に示されるヌクレオチド配列及びその5’末端側に付加されたRNAポリメラーゼプロモーター配列からなるリバースプライマー;配列番号25に示されるヌクレオチド配列からなるフォワードプライマーと、配列番号35に示されるヌクレオチド配列及びその5’末端側に付加されたRNAポリメラーゼプロモーター配列からなるリバースプライマー;配列番号26に示されるヌクレオチド配列からなるフォワードプライマーと、配列番号36に示されるヌクレオチド配列及びその5’末端側に付加されたRNAポリメラーゼプロモーター配列からなるリバースプライマー;配列番号27に示されるヌクレオチド配列からなるフォワードプライマーと、配列番号37に示されるヌクレオチド配列及びその5’末端側に付加されたRNAポリメラーゼプロモーター配列からなるリバースプライマー;配列番号28に示されるヌクレオチド配列からなるフォワードプライマーと、配列番号38に示されるヌクレオチド配列及びその5’末端側に付加されたRNAポリメラーゼプロモーター配列からなるリバースプライマー;配列番号29に示されるヌクレオチド配列からなるフォワードプライマーと、配列番号39に示されるヌクレオチド配列及びその5’末端側に付加されたRNAポリメラーゼプロモーター配列からなるリバースプライマー;配列番号30に示されるヌクレオチド配列からなるフォワードプライマーと、配列番号40に示されるヌクレオチド配列及びその5’末端側に付加されたRNAポリメラーゼプロモーター配列からなるリバースプライマー;から選ばれる、肺炎菌ごとに異なる少なくとも3種類のプライマー対であることを特徴とする請求項1記載の検出方法。
- 少なくとも3種類の各肺炎菌の第1のプローブが、配列番号1~10に示されるヌクレオチド配列から選ばれる少なくとも3種類のDNAからなり、第1のプローブと対をなす少なくとも3種類の各肺炎菌の第2のプローブが、配列番号11~20に示されるヌクレオチド配列から選ばれる少なくとも3種類のDNAであることを特徴とする請求項1又は2記載の検出方法。
- ストレプトコッカス・ニューモニエ、ヘモフィルス・インフルエンザ、マイコプラズマ・ニューモニエ、クラミドフィラ・ニューモニエ、レジオネラ・ニューモフィラ、クレブシエラ・ニューモニエ、シュードモナス・アエルギノーサ、モラクセラ・カタラーリス、メチシリン耐性スタフィロコッカス・アウレウス(MRSA)、スタフィロコッカス・アウレウスから選ばれる少なくとも3種類の肺炎菌類を検出対象とする肺炎原因菌の検出キットであって、試料中より任意に抽出された各肺炎菌に特異的な標的核酸を増幅することができる配列番号21に示されるヌクレオチド配列からなるフォワードプライマーと、配列番号31に示されるヌクレオチド配列及びその5’末端側に付加されたRNAポリメラーゼプロモーター配列からなるリバースプライマー;配列番号22に示されるヌクレオチド配列からなるフォワードプライマーと、配列番号32に示されるヌクレオチド配列及びその5’末端側に付加されたRNAポリメラーゼプロモーター配列からなるリバースプライマー;配列番号23に示されるヌクレオチド配列からなるフォワードプライマーと、配列番号33に示されるヌクレオチド配列及びその5’末端側に付加されたRNAポリメラーゼプロモーター配列からなるリバースプライマー;配列番号24に示されるヌクレオチド配列からなるフォワードプライマーと、配列番号34に示されるヌクレオチド配列及びその5’末端側に付加されたRNAポリメラーゼプロモーター配列からなるリバースプライマー;配列番号25に示されるヌクレオチド配列からなるフォワードプライマーと、配列番号35に示されるヌクレオチド配列及びその5’末端側に付加されたRNAポリメラーゼプロモーター配列からなるリバースプライマー;配列番号26に示されるヌクレオチド配列からなるフォワードプライマーと、配列番号36に示されるヌクレオチド配列及びその5’末端側に付加されたRNAポリメラーゼプロモーター配列からなるリバースプライマー;配列番号27に示されるヌクレオチド配列からなるフォワードプライマーと、配列番号37に示されるヌクレオチド配列及びその5’末端側に付加されたRNAポリメラーゼプロモーター配列からなるリバースプライマー;配列番号28に示されるヌクレオチド配列からなるフォワードプライマーと、配列番号38に示されるヌクレオチド配列及びその5’末端側に付加されたRNAポリメラーゼプロモーター配列からなるリバースプライマー;配列番号29に示されるヌクレオチド配列からなるフォワードプライマーと、配列番号39に示されるヌクレオチド配列及びその5’末端側に付加されたRNAポリメラーゼプロモーター配列からなるリバースプライマー;配列番号30に示されるヌクレオチド配列からなるフォワードプライマーと、配列番号40に示されるヌクレオチド配列及びその5’末端側に付加されたRNAポリメラーゼプロモーター配列からなるリバースプライマー;から選ばれる、肺炎菌ごとに異なる少なくとも3種類のプライマー対を備えたことを特徴とするキット。
- さらに、1)増幅産物と相補的なヌクレオチド配列から選ばれる、肺炎菌ごとに異なる少なくとも3種類の各肺炎菌の第1のプローブが標識高分子担体に結合された第1プローブ結合標識高分子担体;
2)第1のプローブと対をなす少なくとも3種類の各肺炎菌の第2のプローブが、肺炎菌ごとに識別可能な所定の位置に固相化された第2プローブ担持展開支持体;
を備えたことを特徴とする請求項4記載のキット。 - 少なくとも3種類の各肺炎菌の第1のプローブが、配列番号1~10に示されるヌクレオチド配列から選ばれる少なくとも3種類のDNAからなり、第1のプローブと対をなす少なくとも3種類の各肺炎菌の第2のプローブが、配列番号11~20に示されるヌクレオチド配列から選ばれる少なくとも3種類のDNAであることを特徴とする請求項5記載のキット。
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JP2013198482A (ja) * | 2012-02-22 | 2013-10-03 | Nippon Meat Packers Inc | 核酸の検出方法 |
JP2014082977A (ja) * | 2012-10-23 | 2014-05-12 | Kainosu:Kk | 食品中からのセレウリド産生セレウス菌の検出方法、これに用いられるプライマーおよびプローブ |
JP2015500027A (ja) * | 2011-12-09 | 2015-01-05 | ザ セクレタリー オブ ステート フォー ヘルスThe Secretary Of State For Health | 呼吸器感染症アッセイ |
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Cited By (10)
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JP2015500027A (ja) * | 2011-12-09 | 2015-01-05 | ザ セクレタリー オブ ステート フォー ヘルスThe Secretary Of State For Health | 呼吸器感染症アッセイ |
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JP2013198482A (ja) * | 2012-02-22 | 2013-10-03 | Nippon Meat Packers Inc | 核酸の検出方法 |
JP2014082977A (ja) * | 2012-10-23 | 2014-05-12 | Kainosu:Kk | 食品中からのセレウリド産生セレウス菌の検出方法、これに用いられるプライマーおよびプローブ |
US10640834B2 (en) | 2014-09-10 | 2020-05-05 | National University Corporation Tokyo Medical And Dental University | Method for detecting mycoplasma |
WO2018069185A1 (en) | 2016-10-10 | 2018-04-19 | Total Research & Technology Feluy | Improved expandable vinyl aromatic polymers |
WO2018069186A1 (en) | 2016-10-10 | 2018-04-19 | Total Research & Technology Feluy | Improved expandable vinyl aromatic polymers |
WO2018069178A1 (en) | 2016-10-10 | 2018-04-19 | Total Research & Technology Feluy | Improved expandable vinyl aromatic polymers |
US11834563B2 (en) | 2016-10-10 | 2023-12-05 | Totalenergies Onetech Belgium | Expandable vinyl aromatic polymers |
WO2021043552A1 (en) | 2019-09-04 | 2021-03-11 | Total Research & Technology Feluy | Expandable vinyl aromatic polymers with improved flame retardancy |
Also Published As
Publication number | Publication date |
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CN102822352B (zh) | 2015-07-29 |
US20130023443A1 (en) | 2013-01-24 |
EP2557178B1 (en) | 2014-11-26 |
CN102822352A (zh) | 2012-12-12 |
JPWO2011122034A1 (ja) | 2013-07-04 |
JP5565781B2 (ja) | 2014-08-06 |
EP2557178A4 (en) | 2013-11-13 |
EP2557178A1 (en) | 2013-02-13 |
US9347944B2 (en) | 2016-05-24 |
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