WO2013176136A1 - Mycoplasma- and acholeplasma-detecting composition - Google Patents

Mycoplasma- and acholeplasma-detecting composition Download PDF

Info

Publication number
WO2013176136A1
WO2013176136A1 PCT/JP2013/064101 JP2013064101W WO2013176136A1 WO 2013176136 A1 WO2013176136 A1 WO 2013176136A1 JP 2013064101 W JP2013064101 W JP 2013064101W WO 2013176136 A1 WO2013176136 A1 WO 2013176136A1
Authority
WO
WIPO (PCT)
Prior art keywords
probe
mycoplasma
seq
primer
dna
Prior art date
Application number
PCT/JP2013/064101
Other languages
French (fr)
Japanese (ja)
Inventor
早苗 田畑
上森　隆司
昌 磨谷
Original Assignee
タカラバイオ株式会社
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by タカラバイオ株式会社 filed Critical タカラバイオ株式会社
Publication of WO2013176136A1 publication Critical patent/WO2013176136A1/en

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • the present invention relates to a composition, a detection method, and a kit for detecting mycoplasma and acoleplasma.
  • Mycoplasma genus is a kind of eubacteria and widely inhabits the animal kingdom and the plant kingdom. Some of them are responsible for diseases such as primary atypical pneumonia in humans, respiratory mycoplasmosis in chickens, and mycoplasma pneumonia in pigs, and research on their diagnosis, treatment, and prevention has been actively conducted. Yes. Moreover, since mycoplasma does not have a cell wall and has a very small form among bacteria, it can pass through a filter for general filter sterilization. Therefore, when mycoplasma is present in the medium used for culturing cells, there is a problem that contamination (contamination) of mycoplasma cannot be removed even if the medium is sterilized by filtration.
  • Acholplasma is a kind of eubacteria and widely inhabits the animal kingdom and the plant kingdom. Acholplasma does not have a cell wall like mycoplasma, and has a very small form among bacteria, so it can pass through a filter for general filter sterilization. Therefore, when Acholplasma is present in the medium used for culturing cells, there is a problem that contamination (contamination) of Acholplasma cannot be removed even if the medium is sterilized by filtration.
  • Acholplasma laidlawii is known as an example of an Acoleplasma bacterium that causes contamination.
  • the influence of contamination by mycoplasma and acoleplasma in cell culture media includes growth inhibition of cultured cells, changes in metabolism and gene expression, and the like. When such an effect occurs, it is not possible to correctly evaluate an experiment using cultured cells. Therefore, when culturing animal cells, it is important to negate the presence (contamination) of mycoplasma and acholplasma in the medium by a highly sensitive method for detecting mycoplasma and acholplasma.
  • a direct culture method As a method for detecting mycoplasma, a direct culture method, a DNA fluorescent staining method, an ELISA method, a PCR method and the like are known. Of these, the PCR method is excellent in terms of sensitivity, rapidity, and simplicity, and is widely used.
  • a mycoplasma detection method using a combination of PCR and electrophoresis (endpoint PCR method) targeting a gene encoding rRNA is known (Patent Document 1).
  • the primer designed in this method compares only the base sequence between genes encoding rRNA of only 11 kinds of mycoplasma, and the primer is designed in a region conserved among the bacterial species, and detection The possible species are limited.
  • this method is highly sensitive, it is a method of detecting a band by electrophoresis after two-stage PCR, so that the operation is complicated and it takes time to detect.
  • Real-time PCR method is known as another existing technology. This method is a method for monitoring and analyzing the amount of PCR amplification in real time, and does not require electrophoresis and is excellent in rapidity and quantitativeness. A method for detecting mycoplasma by real-time PCR has also been studied, but this is also designed by comparing only 8 types of sequences, and the types of detectable species are limited (Patent Document 2).
  • An object of the present invention is to provide a composition, a detection method, and a kit for detecting mycoplasma and acoreplasma.
  • the present inventors have designed primers that amplify DNA of Mycoplasma bacteria and Acholplasma bacteria, but do not amplify DNA from other organisms, and are useful for detection of DNA of Mycoplasma bacteria and Acholplasma bacteria
  • the present invention was completed by designing a simple probe and conducting intensive studies.
  • the present invention [1] A composition for detecting mycoplasma and acholplasma, a primer comprising the base sequence represented by SEQ ID NO: 1 in the sequence listing, and a primer comprising the base sequence represented by SEQ ID NO: 2 in the sequence listing A composition comprising, [2] The composition according to [1], further comprising a primer having a base sequence represented by SEQ ID NO: 3 in the sequence listing.
  • composition according to [1] or [2], further comprising a probe comprising the base sequence represented by SEQ ID NO: 4 in the sequence listing, [4] The composition according to [3], wherein the probe is a DNA / RNA / DNA chimeric probe, [5] A method for detecting mycoplasma and acoleplasma, (A) a step of subjecting the specimen to a nucleic acid amplification reaction using a primer comprising a base sequence represented by SEQ ID NO: 1 in the sequence listing and a primer comprising a base sequence represented by SEQ ID NO: 2 in the sequence listing; and (b ) Detecting the amplification product obtained by step (a), [6] The method according to [5], wherein in step (a), a primer having the base sequence represented by SEQ ID NO: 3 is used.
  • step (b) The detection of the product obtained in step (a) in step (b) is performed using a probe consisting of the base sequence represented by SEQ ID NO: 4 in the sequence listing [5] or [6] the method of, [8] The method according to [7], wherein the probe is a DNA / RNA / DNA chimeric probe, [9]
  • a kit for detecting mycoplasma and acholplasma comprising: a primer comprising the base sequence represented by SEQ ID NO: 1 in the sequence listing; and a primer comprising the base sequence represented by SEQ ID NO: 2 in the sequence listing Including kit, [10] The kit according to [9], further comprising a primer having a base sequence represented by SEQ ID NO: 3 in the sequence listing.
  • the present invention provides a composition, a detection method, and a kit for detecting DNA derived from Mycoplasma bacteria and Acoleplasma bacteria.
  • FIG. 3 is a diagram showing the results of real-time PCR analysis in Example 1.
  • FIG. 3 is a diagram showing the results of real-time PCR analysis in Example 1. It is a figure which shows the result of real-time PCR in Example 2. It is a figure which shows the result of real-time PCR in Example 2.
  • the present invention provides a primer composed of the base sequence represented by SEQ ID NO: 1 in the sequence listing and a primer composed of the base sequence represented by SEQ ID NO: 2 in the sequence listing.
  • the primer of the present invention anneals to a gene encoding 16S rRNA derived from mycoplasma and a gene encoding 16S rRNA derived from Acholplasma, and is resident in the environment (for example, Bacillus subtilis, E. coli). And a gene encoding 16S rRNA derived from Pseudomonas (Pseudomonas sp.) And the like.
  • the present invention further provides a primer comprising the base sequence represented by SEQ ID NO: 3 in the sequence listing.
  • This primer is annealed to the same region as the primer consisting of the base sequence represented by SEQ ID NO: 1 on the gene encoding Mycoplasma 16S rRNA.
  • the present invention also provides a probe comprising the base sequence represented by SEQ ID NO: 4 in the sequence listing.
  • This probe is designed to anneal to a gene encoding 16S rRNA derived from mycoplasma and a gene encoding 16S rRNA derived from Acoreplasma and not to anneal to DNA derived from other species. Further, the probe is annealed to a DNA fragment amplified from the genomic DNA of Mycoplasma or Acoleplasma using the above-described primer set of the present invention and does not interact with the primer (using the probe as a template). It is designed so that primer extension reaction and formation of the ribonuclease H substrate by this probe and the primer do not occur).
  • the probe of the present invention is preferably modified at its 3 'end with, for example, a fluorescent dye, a quencher, a phosphate, or an amino group to prevent extension by DNA polymerase.
  • An example of the modification is a probe in which a fluorescent dye is added to the 5 'end and a quencher is added to the 3' end.
  • the fluorescent dye is not particularly limited, and examples thereof include 6-FAM (6-carbofluorescein), HEX (4, 7, 2 ′, 4 ′, 5 ′, 7′-hexachloro-6-carboxyfluorescein), ROX ( 6-carboxy-X-rhodamine) and TET (tetrafluorescein) (both manufactured by Life Technologies) can be preferably used.
  • the quenching substance is not particularly limited, and for example, Eclipse (manufactured by Epoch Biosciences) and DABCYL (4-dimethylaminoazobenzene-4'-sulfone) can be preferably used
  • This probe may be an oligonucleotide composed of DNA or a chimeric oligonucleotide composed of DNA / RNA / DNA. These probes are chemically synthesized, for example, by a known method. These probes may also contain nucleotide analogs and / or modified nucleotides.
  • the probe consisting of the base sequence represented by SEQ ID NO: 4 in the sequence listing of the present invention can be preferably a chimeric probe composed of DNA / RNA / DNA. More preferably, it is a probe in which the 9th base of the base sequence represented by SEQ ID NO: 4 in the sequence listing is changed to RNA, and this probe is represented by SEQ ID NO: 28 in the sequence listing.
  • the probe of the present invention is preferably a probe in which a fluorescent dye is added to one end and a quencher is added to the other end.
  • the present invention provides a kit and a composition for detecting mycoplasma and acoleplasma.
  • the kit and composition include a primer consisting of the base sequence represented by SEQ ID NO: 1 in the sequence listing, a primer consisting of the base sequence represented by SEQ ID NO: 2 in the sequence listing, and optionally, SEQ ID NO: 3 in the sequence listing.
  • the primer which consists of a base sequence represented is included.
  • the kit or composition further includes a probe having a base sequence represented by SEQ ID NO: 4 in the sequence listing.
  • the present invention provides a method for detecting mycoplasma and acholplasma using the primer of the present invention and / or the probe of the present invention.
  • a sample is subjected to a nucleic acid amplification reaction using a primer comprising a base sequence represented by SEQ ID NO: 1 in the sequence listing and a primer comprising a base sequence represented by SEQ ID NO: 2 in the sequence listing.
  • Specimens analyzed by the mycoplasma and acoreplasma detection method according to the present invention are not particularly limited, and examples thereof include a medium for culturing cells, cultured cells, and culture supernatants thereof. In the case of a specimen after culture, the culture supernatant is preferably subjected to analysis.
  • the method for preparing the sample is not particularly limited, but when using a culture supernatant as the sample, for example, the sample can be prepared by the following steps. Place the cell culture in a centrifuge and separate the cells and culture supernatant. Centrifugation conditions are well known to those skilled in the art. It is also possible to analyze the collected culture supernatant as it is as a specimen.
  • DNA is extracted and concentrated using a known DNA extraction method or a commercially available kit. There is no limitation in a commercially available kit, For example, NucleoSpin (trademark) Tissue XS (made by Machalai Nagel) is illustrated.
  • a specific region of the genomic DNA of mycoplasma and acoreplasma is amplified using the composition of the present invention to detect all mycoplasma in general and to detect bacteria belonging to the genus Acholplasma.
  • the mycoplasma is not particularly limited, for example, Mycoplasma hominis, Mycoplasma arginini, Mycoplasma fermentans, Mycoplasma hyorhinis, Mycoplasma orale, Mycoplasma pirum, Mycoplasma pneumoniae, Mycoplasma salivarium, include Mycoplasma bovis.
  • the Acholplasma is not particularly limited, and examples thereof include Acholplasma laidlawii, Acholplasma granularum, Acholplasma modicum, Acholplasma morum, and Acholplasma oculi.
  • Acholplasma laidlawii it is possible to specifically detect Mycoplasma bacteria and Acholplasma laidlawii.
  • the kit and composition of the present invention are useful for simple and rapid detection of various species of Mycoplasma bacteria and Acoplasma bacteria.
  • the above primers and probes can be chemically synthesized by known methods.
  • the nucleic acid amplification reaction in the method of the present invention is not particularly limited as long as it is a reaction in which DNA or RNA is used as a template to synthesize a complementary DNA thereto.
  • Polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT -Nucleic acid synthesis reactions well known in the art such as -PCR).
  • PCR is performed by a reaction comprising three steps: dissociation (denaturation) of double-stranded template DNA into single strands, annealing of primers to single-stranded template DNA, and synthesis of complementary strands from primers (extension).
  • dissociation denaturation
  • primers primers to single-stranded template DNA
  • synthesis of complementary strands from primers extension.
  • the Conditions for each step are appropriately set by those skilled in the art.
  • the method for detecting the amplification product obtained by the nucleic acid amplification reaction is not particularly limited as long as it is a method capable of detecting the amplification product, but a real-time PCR method is exemplified. Is done. Detection methods in the real-time PCR method are roughly classified into two types: a method using an intercalator and a method using a fluorescent probe. Examples of the method using a fluorescent probe include a cycling probe method, a TaqMan probe method, a molecular beacon probe method and the like.
  • the intercalator method is low in experimental cost and easy to construct a reaction system, but the detection specificity depends only on the primer sequence. on the other hand.
  • the fluorescent probe method requires an experimental cost, the sequences of both the primer and the probe contribute to the specificity of detection. Since the cycling probe method detects a combination of a chimeric probe composed of RNA and DNA and an endonuclease, detection with very high sequence specificity is possible even among fluorescent probe methods. Although not limiting the present invention, the cycling probe method is suitably used in the embodiment of the present invention in which a probe comprising the base sequence represented by SEQ ID NO: 4 in the sequence listing is used.
  • the intercalator method uses a substance (intercalator) that binds to double-stranded DNA synthesized by the PCR reaction and emits fluorescence when irradiated with excitation light. By detecting this fluorescence intensity, the amount of amplification product generated can be reduced. It is a detection method that can be monitored. Generally, SYBR (registered trademark) Green I is used as an intercalator. In the intercalator method, it is not necessary to design and synthesize a fluorescently labeled probe specific to the target.
  • the cycling probe method is a highly sensitive detection method using a combination of a chimeric probe composed of RNA and DNA and an endonuclease, and can efficiently detect specific sequences of gene fragments during and after amplification.
  • One probe is labeled with a fluorescent dye across the RNA portion, and the other is labeled with a substance (quencher) that quenches the fluorescence emitted by the fluorescent dye.
  • This probe does not emit strong fluorescence by quenching in an intact state, but emits strong fluorescence by forming a hybrid with a complementary sequence in the amplification product and then cleaving at the RNA portion by endonuclease. Become. By measuring the fluorescence intensity, the amount of amplification product can be monitored.
  • the TaqMan probe method uses an oligonucleotide probe (TaqMan probe) labeled with a fluorescent dye at one end and a quencher at the other end and the 5 ′ ⁇ 3 ′ exonuclease activity of DNA polymerase. is there.
  • the TaqMan probe anneals specifically to the template DNA in the annealing step, but since a quencher exists at the end of the probe, the generation of fluorescence is suppressed even when irradiated with excitation light.
  • the fluorescent dye is released from the probe, and the suppression by the quencher is released and the fluorescence is released. Is emitted. By detecting this fluorescence intensity, it is possible to monitor the amount of amplification product generated.
  • the molecular beacon probe method is a method in which one end is labeled with a fluorescent dye, the other end is labeled with a quenching substance, and an oligonucleotide (molecular beacon) having a stem-loop structure is used as a probe.
  • a molecular beacon in a single chain state, the stem region usually forms a double chain, and the fluorescent dyes at both ends are in close proximity to each other and are in a quenching state.
  • the fluorescent dye and the quenching substance are separated and a fluorescent signal is emitted. By detecting this fluorescence intensity, it is possible to monitor the amount of amplification product generated.
  • the reaction solution for detecting mycoplasma and acholplasma in the method of the present invention comprises a DNA polymerase, a reaction buffer, at least two kinds of primers of the present invention, and optionally a probe of the present invention and other components. It can be prepared by adding a nucleic acid derived from a more prepared specimen.
  • a composition containing a DNA polymerase, a reaction buffer, a primer of the present invention, and a probe of the present invention is one embodiment of the present invention.
  • a DNA polymerase can be used in the present invention as long as it has an activity of synthesizing DNA complementary to DNA or RNA as a template.
  • the DNA polymerase used in the present invention is particularly preferably a heat-resistant DNA-dependent DNA polymerase.
  • Examples of such a DNA polymerase include heat-resistant DNA polymerases derived from eubacteria typified by DNA polymerases derived from Thermus bacteria (such as Thermus aquaticus-derived DNA polymerases), and DNA polymerases derived from Pyrococcus archaea (Pyrococcus sp.-derived DNA polymerases). And the like, and thermococcus archaebacterium-derived DNA polymerases (Thermococcus kodakaraensis-derived DNA polymerase, etc.).
  • Two or more DNA polymerases may be used in combination as the DNA polymerase.
  • Examples of the two or more types of DNA polymerases include a combination of a DNA polymerase having 3 ' ⁇ 5' exonuclease activity and a DNA polymerase having substantially no 3 ' ⁇ 5' exonuclease activity.
  • a technique for performing PCR using a reaction solution containing two types of DNA polymerases is known as LA-PCR (Long and Accurate PCR).
  • an endonuclease that cleaves the probe in an amplification product-dependent manner is added to the reaction solution.
  • the endonuclease is not particularly limited as long as it is an enzyme that specifically cleaves the ribonucleotide portion of the probe of the present invention annealed to an amplification product derived from Mycoplasma bacteria or Acoleplasma bacteria, but Ribonuclease H (RNase H) Is exemplified.
  • the ribonuclease H is preferably a heat-resistant ribonuclease H, more preferably an archaea-derived heat-resistant ribonuclease H.
  • the reaction buffer means a compound or a mixture having an action of reducing the fluctuation of the hydrogen ion concentration (pH) of the reaction solution.
  • a mixed solution of a weak acid and its salt or a weak base and its salt is widely used as a reaction buffer for the purpose of pH control because it has a strong buffering action.
  • Various reaction buffers known in the biochemical field can be used in the present invention.
  • the reaction buffer is not particularly limited, and for example, bicine, tricine, hepes (HEPES), tris, and phosphate can be suitably used.
  • the final concentration of the reaction buffer is not particularly limited, but a range of 5 to 100 mM, preferably 20 to 50 mM can be suitably used.
  • the pH of the reaction solution in the method of the present invention is suitably set in the usual range in which the gene amplification reaction is carried out, for example, the pH at 25 ° C. is in the range of 8.0 to 9.5.
  • Example 1 Primer and Probe Design and Screening
  • Mycoplasma hominis Mycoplasma fermentans, which are targeted for gene regions encoding 16S rDNA and detected from the culture medium during animal cell culture, Mycoplasma hyorhinis, Mycoplasma oral, Mycoplasma pirum, Mycoplasma pneumoniae, etc. sp., Staphylococcus sp from the nucleotide sequence information. etc.
  • Mycoplasma hominis Mycoplasma fermentans, which are targeted for gene regions encoding 16S rDNA and detected from the culture medium during animal cell culture, Mycoplasma hyorhinis, Mycoplasma oral, Mycoplasma pirum, Mycoplasma pneumoniae, etc. sp., Staphylococcus sp from the nucleotide sequence information. etc.
  • the 5'-side primers were F12 (SEQ ID NO: 5), F233 (SEQ ID NO: 6), F259 (SEQ ID NO: 1), F344 (SEQ ID NO: 7), F362 (SEQ ID NO: 8), and F534 (SEQ ID NO: 9). It is a kind.
  • the 3 ′ primers were R605 (SEQ ID NO: 10), R750 (SEQ ID NO: 11), R827 (SEQ ID NO: 12), R850 (SEQ ID NO: 2), R1131 (SEQ ID NO: 13), R1155 (SEQ ID NO: 14), R1207 ( SEQ ID NO: 15), R1238 (SEQ ID NO: 16), R1352 (SEQ ID NO: 17), and R1440 (SEQ ID NO: 18).
  • Cycling probe In the amplification region with the designed primer, a cycling probe was designed that specifically detects typical mycoplasma and Acholplasma laidlawii detected from the culture medium during animal cell culture. Cycling probes are Probe 1 (SEQ ID NO: 19), Probe 2 (SEQ ID NO: 20), Probe 3 (SEQ ID NO: 21), Probe 4 (SEQ ID NO: 22), Probe 5 (SEQ ID NO: 23), Probe 6 (SEQ ID NO: 24). ), Probe 7 (SEQ ID NO: 25), Probe 8 (SEQ ID NO: 26), Probe 9 (SEQ ID NO: 27), Probe 10 (SEQ ID NO: 28), and Probe 11 (SEQ ID NO: 29). The design is performed according to the algorithm described in International Publication No.
  • Probe 4 and Probe 5 are positions shifted by 1 base from Probe 3
  • Probe 7 and Probe 8 are positions shifted by 1 base based on Probe 6
  • Probe 10 is Probe 9 shifted. The position is shifted by one base from the base, and Probe 11 is obtained by extending the chain length of Probe 9 by one base.
  • CyclePCR (registered trademark) Reaction Mix (2 ⁇ conc.) 12.5 ⁇ L, each 5 ⁇ M primer mix 1 ⁇ L, 5 ⁇ M cycling probe (ROX labeling) 1 ⁇ L, template 0.5 ⁇ L were mixed, and the total volume was adjusted to 25 ⁇ L with sterile water.
  • Thermal Cycler Dice registered trademark
  • Real Time System II Tekara Bio Inc. Code TP900
  • ⁇ represents a combination in which a good amplification signal was observed
  • ⁇ represents a combination in which an amplification signal was observed but low fluorescence intensity
  • x represents a combination in which no amplification signal was observed.
  • the primer combinations are F344 / R605, F362 / R605, F259 / R605, F344 / R827, F362 / R827, F344 / R850, F362 / R850, F259 / R850, and the cycling probe is Probe 6 or Probe 9 Was promising.
  • the same reaction as described above was performed using Probe 6 without adding a template. The results are shown in FIG.
  • ⁇ represents a combination in which a good amplification signal was observed
  • ⁇ represents a combination in which an amplification signal was observed but low fluorescence intensity
  • x represents a combination in which no amplification signal was observed.
  • Example 2 Examination using Mycoplasma Specimen (1) Examination of Primer Set Regarding the two combinations of primers F259 / R605 and F259 / R850 selected in Example 1, Acholplasma laidavii and Mycoplasma hominis, Mycoplasma Arginini, Mycoplasma fermentans, Mycoplasma hyorhinis, Mycoplasma orale, Mycoplasma pyrum, and Mycoplasma pneumoniae) were used as templates to compare the reactions. Probe 10 was used as the cycling probe. The reaction was performed with the following reaction composition using Cycle PCR (registered trademark) Reaction Mix (manufactured by Takara Bio Inc., code CY505).
  • Cycle PCR registered trademark
  • Reaction Mix manufactured by Takara Bio Inc., code CY505
  • FIG. 3A shows the measurement results for F259 / R605
  • FIG. 3B shows the measurement results for F259 / R850. It was confirmed that good detection can be performed with any combination of F259 / R605 and F259 / R850 primers.
  • FIG. 4 (a) is Probe 1
  • FIG. 4 (b) is Probe 3
  • FIG. 4 (c) is Probe 4
  • FIG. 4 (d) is Probe 5
  • FIG. 4 (e) is Probe 6
  • FIG. 4 (f) shows the measurement results for Probe 8
  • FIG. 4 (h) shows the measurement results for Probe 9
  • FIG. 4 (i) shows the measurement results for Probe 10, and FIG.
  • Probe 1 has a high fluorescence value, but has fewer types of mycoplasma that can be detected than other probes.
  • Probe 3, Probe 4, and Probe 5 resulted in low fluorescence values.
  • Probe 6, Probe 7, and Probe 8 were all good results.
  • Probe 9, Probe 10, and Probe 11 it was considered that Probe 10 had a higher fluorescence value than others and was favorable. Based on the above results, Probe 10 was selected.
  • Acholplasma laidlawiii, Mycoplasmm hominis, Mycoplasma arginini, Mycoplasma fermentans, Mycoplasma hyorhinis, Mycoplasma ore were amplified, and My was amplified.
  • Example 3 Examination of Primer Addition
  • amplification signals were not obtained when using Mycoplasma pyrum, Mycoplasma pneumoniae DNA. Therefore, a base sequence (SEQ ID NO: 30 to SEQ ID NO: 227) corresponding to each of primers F259 and R850 in Mycoplasma bacteria was compared, and a new additional primer F259P (SEQ ID NO: 3) was designed.
  • DNA prepared from Mycoplasma pyrum, Mycoplasma pneumoniae, and Mycoplasma oral was used as a template.
  • a forward primer and an R850 reverse primer in which F259 and F259P were mixed at a ratio of 1: 1 were used.
  • Probe 10 obtained with good results in Example 2 was used.
  • the reaction was performed using the following reaction composition using Cycleleave (registered trademark) Reaction Mix (manufactured by Takara Bio Inc., code CY505).
  • CyclePCR registered trademark
  • Reaction Mix (2 ⁇ conc.) 12.5 ⁇ L
  • 5 ⁇ M cycling probe ROX labeling
  • template 0.5 ⁇ L were mixed, and the total volume was adjusted to 25 ⁇ L with sterile water.
  • Thermal Cycler Dice registered trademark
  • Real Time System II manufactured by Takara Bio Inc., code TP900
  • the present invention is useful in a wide range of fields such as genetic engineering, biology, medicine and agriculture.
  • SEQ ID NO: 6 Oligonucleotide primer F233 to amplify the DNA fragment of 16S rRNA gene from Mycoplasma.
  • SEQ ID NO: 7 Oligonucleotide primer F344 to amplify the DNA fragment of 16S rRNA gene from Mycoplasma.
  • SEQ ID NO: 8 Oligonucleotide primer F362 to amplify the DNA fragment of 16S rRNA gene from Mycoplasma.
  • SEQ ID NO: 9 Oligonucleotide primer F534 to amplify the DNA fragment of 16S rRNA gene from Mycoplasma.
  • SEQ ID NO: 10 Oligonucleotide primer R605 to amplify the DNA fragment of 16S rRNA gene from Mycoplasma.
  • SEQ ID NO: 11 Oligonucleotide primer R750 to amplify the DNA fragment of 16S rRNA gene from Mycoplasma.
  • SEQ ID NO: 12 Oligonucleotide primer R827 to amplify the DNA fragment of 16S rRNA gene from Mycoplasma.
  • SEQ ID NO: 13 Oligonucleotide primer R1131 to amplify the DNA fragment of 16S rRNA gene from Mycoplasma.
  • SEQ ID NO: 14 Oligonucleotide primer R1155 to amplify the DNA fragment of 16S rRNA gene from Mycoplasma.
  • SEQ ID NO: 15 Oligonucleotide primer R1207 to amplify the DNA fragment of 16S rRNA gene from Mycoplasma.
  • SEQ ID NO: 16 Oligonucleotide primer R1238 to amplify the DNA fragment of 16S rRNA gene from Mycoplasma.
  • SEQ ID NO: 17 Oligonucleotide primer R1352 to amplify the DNA fragment of 16S rRNA gene from Mycoplasma.
  • SEQ ID NO: 18 Oligonucleotide primer R1440 to amplify the DNA fragment of 16S rRNA gene from Mycoplasma.
  • SEQ ID NO: 19 Oligonucleotide probe 1 to detect 16S rRNA gene from Mycoplasma.
  • Oligonucleotide 4 is ribonucleotide-other nucleotides are deoxyribonucleotide.
  • Oligonucleotide 4 is ribonucleotide-other nucleotides are deoxyribonucleotide.
  • Oligonucleotide 7 is ribonucleotide-other nucleotides are deoxyribonucleotide.
  • Oligonucleotide 6 is ribonucleotide-other nucleotides are deoxyribonucleotide.
  • SEQ ID NO: 23 Oligonucleotide probe 5 to detect 16S rRNA gene from Mycoplasma.
  • Oligonucleotide probe 5 is ribonucleotide-other nucleotides are deoxyribonucleotide.
  • SEQ ID NO: 24 Oligonucleotide probe 6 to detect 16S rRNA gene from Mycoplasma.
  • Oligonucleotide 9 is ribonucleotide-other nucleotides are deoxyribonucleotide.
  • SEQ ID NO: 25 Oligonucleotide probe 7 to detect 16S rRNA gene from Mycoplasma.
  • Oligonucleotide 8 is ribonucleotide-other nucleotides are deoxyribonucleotide.
  • SEQ ID NO: 26 Oligonucleotide probe 8 to detect 16S rRNA gene from Mycoplasma.
  • Oligonucleotide 7 is ribonucleotide-other nucleotides are deoxyribonucleotide.
  • SEQ ID NO: 27 Oligonucleotide probe 9 to detect 16S rRNA gene from Mycoplasma.
  • Oligonucleotide 10 is ribonucleotide-other nucleotides are deoxyribonucleotide.
  • SEQ ID NO: 28 Oligonucleotide probe 10 to detect 16S rRNA gene from Mycoplasma.
  • Oligonucleotide probe 11 to detect 16S rRNA gene from Mycoplasma Oligonucleotide probe 11 to detect 16S rRNA gene from Mycoplasma.
  • Oligonucleotide probe 11 to detect 16S rRNA gene from Mycoplasma Oligonucleotide probe 11 to detect 16S rRNA gene from Mycoplasma.
  • Oligonucleotide probe 11 to detect 16S rRNA gene from Mycoplasma Oligonucleotide probe 11 to detect 16S rRNA gene from Mycoplasma.
  • Oligonucleotide probe 11 to detect 16S rRNA gene from Mycoplasma Oligonucleotide probe 11 to detect 16S rRNA gene from Mycoplasma.
  • Oligonucleotide probe 11 to detect 16S rRNA gene from Mycoplasma Oligonucleotide probe 11 to detect 16S rRNA gene from Mycoplasma.

Abstract

Provided are: a composition that contains a primer for detecting Mycoplasma bacteria and Acholeplasma bacteria; a probe-containing composition; a method for detecting Mycoplasma bacteria and Acholeplasma bacteria; a kit that contains a primer for detecting Mycoplasma bacteria and Acholeplasma bacteria; and a probe-containing kit.

Description

マイコプラズマ及びアコレプラズマ検出用組成物Composition for detecting mycoplasma and acoleplasma
　本発明は、マイコプラズマ及びアコレプラズマを検出するための組成物、検出方法、及びキットに関する。 The present invention relates to a composition, a detection method, and a kit for detecting mycoplasma and acoleplasma.
　マイコプラズマ属細菌（Ｍｙｃｏｐｌａｓｍａ）は、真正細菌の一種であり、広く動物界、植物界で生息する。その一部は、例えば、ヒトの原発性異型肺炎、ニワトリの呼吸器マイコプラズマ症、ブタのマイコプラズマ肺炎等の疾病の原因となっており、その診断、治療、予防などの研究が活発に行われている。また、マイコプラズマは、細胞壁を有さず、細菌の中でも非常に小さな形態をしているため、一般的な濾過滅菌用のフィルターを通過し得る。そのため、細胞の培養に使用する培地中にマイコプラズマが存在していた場合、培地を濾過滅菌してもマイコプラズマのコンタミネーション（汚染）が除去できないことが問題となっている。 Mycoplasma genus (Mycoplasma) is a kind of eubacteria and widely inhabits the animal kingdom and the plant kingdom. Some of them are responsible for diseases such as primary atypical pneumonia in humans, respiratory mycoplasmosis in chickens, and mycoplasma pneumonia in pigs, and research on their diagnosis, treatment, and prevention has been actively conducted. Yes. Moreover, since mycoplasma does not have a cell wall and has a very small form among bacteria, it can pass through a filter for general filter sterilization. Therefore, when mycoplasma is present in the medium used for culturing cells, there is a problem that contamination (contamination) of mycoplasma cannot be removed even if the medium is sterilized by filtration.
　アコレプラズマ属細菌（Ａｃｈｏｌｅｐｌａｓｍａ）は、真正細菌の一種であり、広く動物界、植物界で生息する。アコレプラズマは、マイコプラズマと同じく細胞壁を有さず、細菌の中でも非常に小さな形態をしているため、一般的な濾過滅菌用のフィルターを通過し得る。そのため、細胞の培養に使用する培地中にアコレプラズマが存在していた場合、培地を濾過滅菌してもアコレプラズマのコンタミネーション（汚染）が除去できないことが問題となっている。コンタミネーションの原因となるアコレプラズマ属細菌の例として、Ａｃｈｏｌｅｐｌａｓｍａ　ｌａｉｄｌａｗｉｉが知られている。 Acholplasma is a kind of eubacteria and widely inhabits the animal kingdom and the plant kingdom. Acholplasma does not have a cell wall like mycoplasma, and has a very small form among bacteria, so it can pass through a filter for general filter sterilization. Therefore, when Acholplasma is present in the medium used for culturing cells, there is a problem that contamination (contamination) of Acholplasma cannot be removed even if the medium is sterilized by filtration. Acholplasma laidlawii is known as an example of an Acoleplasma bacterium that causes contamination.
　細胞培養用の培地のマイコプラズマ及びアコレプラズマによるコンタミネーションの影響としては、培養細胞の成長阻害、代謝や遺伝子発現の変動等が挙げられる。このような影響が発生すると、培養細胞を用いた実験について正しい評価を行うことができなくなる。そのため、動物細胞を培養する際には、培地中のマイコプラズマ及びアコレプラズマの存在（コンタミネーション）を、高感度なマイコプラズマ及びアコレプラズマの検出方法により否定することが重要である。 The influence of contamination by mycoplasma and acoleplasma in cell culture media includes growth inhibition of cultured cells, changes in metabolism and gene expression, and the like. When such an effect occurs, it is not possible to correctly evaluate an experiment using cultured cells. Therefore, when culturing animal cells, it is important to negate the presence (contamination) of mycoplasma and acholplasma in the medium by a highly sensitive method for detecting mycoplasma and acholplasma.
　マイコプラズマを検出する方法としては、直接培養方法、ＤＮＡ蛍光染色方法、ＥＬＩＳＡ法、及びＰＣＲ法等が知られている。このうちＰＣＲ法が感度、迅速性、簡便性の観点において優れており、広く利用されている。 As a method for detecting mycoplasma, a direct culture method, a DNA fluorescent staining method, an ELISA method, a PCR method and the like are known. Of these, the PCR method is excellent in terms of sensitivity, rapidity, and simplicity, and is widely used.
　既存技術の一例として、ｒＲＮＡをコードする遺伝子を標的としたＰＣＲと電気泳動との組み合わせ（エンドポイントＰＣＲ法）によるマイコプラズマの検出方法が知られている（特許文献１）。しかしながら、この方法において設計されたプライマーは、わずか１１種のマイコプラズマのｒＲＮＡをコードする遺伝子間の塩基配列のみを比較し、その菌種間で保存されている領域にプライマーを設計しており、検出可能な種が限定されている。また、この方法は、高感度ではあるが、２段階のＰＣＲの後に電気泳動によりバンドを検出する方法であるため、操作が煩雑であり検出に時間がかかる。 As an example of an existing technique, a mycoplasma detection method using a combination of PCR and electrophoresis (endpoint PCR method) targeting a gene encoding rRNA is known (Patent Document 1). However, the primer designed in this method compares only the base sequence between genes encoding rRNA of only 11 kinds of mycoplasma, and the primer is designed in a region conserved among the bacterial species, and detection The possible species are limited. In addition, although this method is highly sensitive, it is a method of detecting a band by electrophoresis after two-stage PCR, so that the operation is complicated and it takes time to detect.
　その他の既存技術として、リアルタイムＰＣＲ法が知られている。この方法はＰＣＲの増幅量をリアルタイムでモニターし解析する方法であり、電気泳動が不要で迅速性と定量性に優れている。リアルタイムＰＣＲによるマイコプラズマの検出方法についても検討されているが、これもわずか８種間の配列を比較して設計されているに過ぎず、検出可能な種が限定されている（特許文献２）。 Real-time PCR method is known as another existing technology. This method is a method for monitoring and analyzing the amount of PCR amplification in real time, and does not require electrophoresis and is excellent in rapidity and quantitativeness. A method for detecting mycoplasma by real-time PCR has also been studied, but this is also designed by comparing only 8 types of sequences, and the types of detectable species are limited (Patent Document 2).
　更に、マイコプラズマ属細菌及びアコレプラズマ属細菌の検出において、偽陽性（ｆａｌｓｅ　ｐｏｓｉｔｉｖｅ）とならないよう、哺乳類に広く存在している常在菌を検出しないプライマー、プローブ、及び反応系が必要であるが、既知の方法においてはこれが十分に検討されていない。 Furthermore, in detection of Mycoplasma bacteria and Acoleplasma bacteria, primers, probes, and reaction systems that do not detect resident bacteria widely present in mammals are necessary so as not to be false positives. This has not been fully studied in known methods.
特許第３０１６３９５号公報Japanese Patent No. 3016395 米国特許出願公開第２００５／０２５０１１２号US Patent Application Publication No. 2005/0250112
　マイコプラズマの検出について種々の方法が検討、開発されてきたが、現在においても様々な種のマイコプラズマ全般を迅速、簡便に、網羅的かつマイコプラズマ特異的に検出できる系は構築されていない。そのため、マイコプラズマ検出系のさらなる改善が望まれている。本発明の目的は、マイコプラズマ及びアコレプラズマを検出するための組成物、検出方法、及びキットを提供することにある。 Various methods have been studied and developed for the detection of mycoplasma, but even now, no system has been constructed that can detect various types of mycoplasma in general quickly, simply, comprehensively and specifically for mycoplasma. Therefore, further improvement of the mycoplasma detection system is desired. An object of the present invention is to provide a composition, a detection method, and a kit for detecting mycoplasma and acoreplasma.
　本発明者らは、マイコプラズマ属細菌及びアコレプラズマ属細菌のＤＮＡを増幅し、かつその他の生物由来のＤＮＡは増幅しないプライマーを設計するとともに、マイコプラズマ属細菌及びアコレプラズマ属細菌のＤＮＡの検出に有用なプローブを設計し、鋭意検討を行うことによって本発明を完成させるに至った。 The present inventors have designed primers that amplify DNA of Mycoplasma bacteria and Acholplasma bacteria, but do not amplify DNA from other organisms, and are useful for detection of DNA of Mycoplasma bacteria and Acholplasma bacteria The present invention was completed by designing a simple probe and conducting intensive studies.
　すなわち本発明は、
［１］マイコプラズマ及びアコレプラズマを検出するための組成物であって、配列表の配列番号１で表される塩基配列からなるプライマー、及び配列表の配列番号２で表される塩基配列からなるプライマーを含む組成物、
［２］更に、配列表の配列番号３で表される塩基配列からなるプライマーを含む、［１］記載の組成物、
［３］更に、配列表の配列番号４で表される塩基配列からなるプローブを含む、［１］又は［２］記載の組成物、
［４］プローブが、ＤＮＡ／ＲＮＡ／ＤＮＡキメラプローブである、［３］記載の組成物、
［５］マイコプラズマ及びアコレプラズマの検出方法であって、
（ａ）検体を配列表の配列番号１で表される塩基配列からなるプライマー、及び配列表の配列番号２で表される塩基配列からなるプライマーを用いた核酸増幅反応に供する工程、並びに（ｂ）工程（ａ）により得られた増幅産物を検出する工程、を含む方法、
［６］工程（ａ）において、更に配列番号３で表される塩基配列からなるプライマーを使用する、［５］記載の方法、
［７］工程（ｂ）における工程（ａ）により得られた産物の検出が、配列表の配列番号４で表される塩基配列からなるプローブを用いて行われる、［５］又は［６］記載の方法、
［８］プローブが、ＤＮＡ／ＲＮＡ／ＤＮＡキメラプローブである、［７］記載の方法、
［９］マイコプラズマ及びアコレプラズマを検出するためのキットであって、配列表の配列番号１で表される塩基配列からなるプライマー、及び配列表の配列番号２で表される塩基配列からなるプライマーを含むキット、
［１０］更に、配列表の配列番号３で表される塩基配列からなるプライマーを含む、［９］記載のキット、
［１１］更に、配列表の配列番号４で表される塩基配列からなるプローブを含む、［９］又は［１０］記載のキット、
［１２］プローブが、ＤＮＡ／ＲＮＡ／ＤＮＡキメラプローブである、［１１］記載のキット、
に関する。 That is, the present invention
[1] A composition for detecting mycoplasma and acholplasma, a primer comprising the base sequence represented by SEQ ID NO: 1 in the sequence listing, and a primer comprising the base sequence represented by SEQ ID NO: 2 in the sequence listing A composition comprising,
[2] The composition according to [1], further comprising a primer having a base sequence represented by SEQ ID NO: 3 in the sequence listing.
[3] The composition according to [1] or [2], further comprising a probe comprising the base sequence represented by SEQ ID NO: 4 in the sequence listing,
[4] The composition according to [3], wherein the probe is a DNA / RNA / DNA chimeric probe,
[5] A method for detecting mycoplasma and acoleplasma,
(A) a step of subjecting the specimen to a nucleic acid amplification reaction using a primer comprising a base sequence represented by SEQ ID NO: 1 in the sequence listing and a primer comprising a base sequence represented by SEQ ID NO: 2 in the sequence listing; and (b ) Detecting the amplification product obtained by step (a),
[6] The method according to [5], wherein in step (a), a primer having the base sequence represented by SEQ ID NO: 3 is used.
[7] The detection of the product obtained in step (a) in step (b) is performed using a probe consisting of the base sequence represented by SEQ ID NO: 4 in the sequence listing [5] or [6] the method of,
[8] The method according to [7], wherein the probe is a DNA / RNA / DNA chimeric probe,
[9] A kit for detecting mycoplasma and acholplasma, comprising: a primer comprising the base sequence represented by SEQ ID NO: 1 in the sequence listing; and a primer comprising the base sequence represented by SEQ ID NO: 2 in the sequence listing Including kit,
[10] The kit according to [9], further comprising a primer having a base sequence represented by SEQ ID NO: 3 in the sequence listing.
[11] The kit according to [9] or [10], further comprising a probe comprising the base sequence represented by SEQ ID NO: 4 in the sequence listing,
[12] The kit according to [11], wherein the probe is a DNA / RNA / DNA chimeric probe,
About.
　本発明により、マイコプラズマ属細菌及びアコレプラズマ属細菌由来のＤＮＡを検出するための組成物、検出方法、及びキットが提供される。 The present invention provides a composition, a detection method, and a kit for detecting DNA derived from Mycoplasma bacteria and Acoleplasma bacteria.
実施例１におけるリアルタイムＰＣＲの解析結果を示す図である。FIG. 3 is a diagram showing the results of real-time PCR analysis in Example 1. 実施例１におけるリアルタイムＰＣＲの解析結果を示す図である。FIG. 3 is a diagram showing the results of real-time PCR analysis in Example 1. 実施例２におけるリアルタイムＰＣＲの結果を示す図である。It is a figure which shows the result of real-time PCR in Example 2. 実施例２におけるリアルタイムＰＣＲの結果を示す図である。It is a figure which shows the result of real-time PCR in Example 2.
　本発明は、配列表の配列番号１で表される塩基配列からなるプライマー、及び配列表の配列番号２で表される塩基配列からなるプライマーを提供する。これら本発明のプライマーを用いることにより、様々な種（ｓｐｅｃｉｅｓ）のマイコプラズマ属細菌及びアコレプラズマ属細菌を簡便かつ迅速に検出することが可能である。 The present invention provides a primer composed of the base sequence represented by SEQ ID NO: 1 in the sequence listing and a primer composed of the base sequence represented by SEQ ID NO: 2 in the sequence listing. By using these primers of the present invention, it is possible to easily and rapidly detect various species of Mycoplasma bacteria and Acholplasma bacteria.
　本発明のプライマーはマイコプラズマ由来１６Ｓ　ｒＲＮＡをコードする遺伝子及びアコレプラズマ由来１６Ｓ　ｒＲＮＡをコードする遺伝子にアニールし、環境中に常在する微生物（例えば、枯草菌（Ｂａｃｉｌｌｕｓ　ｓｕｂｔｉｌｉｓ）、大腸菌（Ｅ．　ｃｏｌｉ）、シュードモナス（Ｐｓｅｕｄｏｍｏｎａｓ　ｓｐ．）等）由来の１６Ｓ　ｒＲＮＡをコードする遺伝子にはアニールしない配列となるよう設計されている。 The primer of the present invention anneals to a gene encoding 16S rRNA derived from mycoplasma and a gene encoding 16S rRNA derived from Acholplasma, and is resident in the environment (for example, Bacillus subtilis, E. coli). And a gene encoding 16S rRNA derived from Pseudomonas (Pseudomonas sp.) And the like.
　上記の、本発明のプライマーのセットに加え、本発明は更に配列表の配列番号３で表される塩基配列からなるプライマーを提供する。このプライマーは、マイコプラズマ　１６Ｓ　ｒＲＮＡをコードする遺伝子上で、上記の配列番号１で表される塩基配列からなるプライマーと同じ領域にアニーリングする。これら３本のプライマーのセットを用いたＰＣＲを行うことにより、更に広範囲の種のマイコプラズマ属細菌由来のＤＮＡを増幅することが可能である。 In addition to the primer set of the present invention described above, the present invention further provides a primer comprising the base sequence represented by SEQ ID NO: 3 in the sequence listing. This primer is annealed to the same region as the primer consisting of the base sequence represented by SEQ ID NO: 1 on the gene encoding Mycoplasma 16S rRNA. By performing PCR using these three primer sets, it is possible to amplify DNA from a wider range of species of Mycoplasma bacteria.
　また、本発明は、配列表の配列番号４で表される塩基配列からなるプローブを提供する。このプローブはマイコプラズマ由来１６Ｓ　ｒＲＮＡをコードする遺伝子及びアコレプラズマ由来１６Ｓ　ｒＲＮＡをコードする遺伝子にアニールし、他の生物種由来のＤＮＡにはアニールしない配列となるよう設計されている。更に、該プローブは、上記の本発明のプライマーセットを用いてマイコプラズマ又はアコレプラズマのゲノムＤＮＡより増幅されるＤＮＡ断片にアニールし、かつ前記プライマーと相互作用を生じないよう（該プローブを鋳型としたプライマー伸長反応や本プローブと前記プライマーとによるリボヌクレアーゼＨの基質の形成が起きないように）設計されている。本発明のプローブは、好適には、ＤＮＡポリメラーゼによる伸長を防ぐために、その３’末端が例えば蛍光色素、消光物質、リン酸、又はアミノ基により修飾されている。修飾の一例として、５’末端に蛍光色素を、３’末端に消光物質を付加したプローブが挙げられる。当該蛍光色素としては、特に限定は無いが、例えば、６－ＦＡＭ（６－ｃａｒｂｏｘｙｆｌｕｏｒｅｓｃｅｉｎ）、ＨＥＸ（４，７，２’，４’，５’，７’－ｈｅｘａｃｈｌｏｒｏ－６－ｃａｒｂｏｘｙｆｌｕｏｒｅｓｃｅｉｎ）、ＲＯＸ（６－ｃａｒｂｏｘｙ－Ｘ－ｒｈｏｄａｍｉｎｅ）、ＴＥＴ（ｔｅｔｒａｆｌｕｏｒｅｓｃｅｉｎ）（いずれもＬｉｆｅ　Ｔｅｃｈｎｏｌｏｇｉｅｓ社製）が好適に使用できる。当該消光物質としては、特に限定は無いが、例えば、Ｅｃｌｉｐｓｅ（Ｅｐｏｃｈ　Ｂｉｏｓｃｉｅｎｃｅｓ社製）、ＤＡＢＣＹＬ（４－ｄｉｍｅｔｈｙｌａｍｉｎｏａｚｏｂｅｎｚｅｎｅ－４’－ｓｕｌｆｏｎｅ）が好適に使用できる。 The present invention also provides a probe comprising the base sequence represented by SEQ ID NO: 4 in the sequence listing. This probe is designed to anneal to a gene encoding 16S rRNA derived from mycoplasma and a gene encoding 16S rRNA derived from Acoreplasma and not to anneal to DNA derived from other species. Further, the probe is annealed to a DNA fragment amplified from the genomic DNA of Mycoplasma or Acoleplasma using the above-described primer set of the present invention and does not interact with the primer (using the probe as a template). It is designed so that primer extension reaction and formation of the ribonuclease H substrate by this probe and the primer do not occur). The probe of the present invention is preferably modified at its 3 'end with, for example, a fluorescent dye, a quencher, a phosphate, or an amino group to prevent extension by DNA polymerase. An example of the modification is a probe in which a fluorescent dye is added to the 5 'end and a quencher is added to the 3' end. The fluorescent dye is not particularly limited, and examples thereof include 6-FAM (6-carbofluorescein), HEX (4, 7, 2 ′, 4 ′, 5 ′, 7′-hexachloro-6-carboxyfluorescein), ROX ( 6-carboxy-X-rhodamine) and TET (tetrafluorescein) (both manufactured by Life Technologies) can be preferably used. The quenching substance is not particularly limited, and for example, Eclipse (manufactured by Epoch Biosciences) and DABCYL (4-dimethylaminoazobenzene-4'-sulfone) can be preferably used.
　このプローブはＤＮＡで構成されるオリゴヌクレオチドでもよく、ＤＮＡ／ＲＮＡ／ＤＮＡで構成されるキメラオリゴヌクレオチドでもよい。これらプローブは、例えば公知の方法で化学的に合成される。又、これらプローブは、ヌクレオチドアナログ及び／又は修飾ヌクレオチドを含有していてもよい。 This probe may be an oligonucleotide composed of DNA or a chimeric oligonucleotide composed of DNA / RNA / DNA. These probes are chemically synthesized, for example, by a known method. These probes may also contain nucleotide analogs and / or modified nucleotides.
　本発明の配列表の配列番号４で表される塩基配列からなるプローブは、好ましくはＤＮＡ／ＲＮＡ／ＤＮＡで構成されるキメラプローブとすることができる。より好ましくは配列表の配列番号４で表される塩基配列の９番目に位置する塩基をＲＮＡに変更したプローブであり、当該プローブは、配列表の配列番号２８で表される。また、本発明のプローブは、好ましくは、一方の末端に蛍光色素を、もう一方の末端に消光物質が付加されたプローブである。 The probe consisting of the base sequence represented by SEQ ID NO: 4 in the sequence listing of the present invention can be preferably a chimeric probe composed of DNA / RNA / DNA. More preferably, it is a probe in which the 9th base of the base sequence represented by SEQ ID NO: 4 in the sequence listing is changed to RNA, and this probe is represented by SEQ ID NO: 28 in the sequence listing. The probe of the present invention is preferably a probe in which a fluorescent dye is added to one end and a quencher is added to the other end.
　本発明はマイコプラズマ及びアコレプラズマを検出するためのキット並びに組成物を提供する。前記のキット、組成物は、配列表の配列番号１で表される塩基配列からなるプライマー及び配列表の配列番号２で表される塩基配列からなるプライマー、更に任意に配列表の配列番号３で表される塩基配列からなるプライマーを含む。本発明の好適な態様においては、前記のキット、組成物は、更に配列表の配列番号４で表される塩基配列からなるプローブを含む。これら本発明の組成物は、核酸増幅反応によるマイコプラズマ属細菌及びアコレプラズマ属細菌の検出に有用である。 The present invention provides a kit and a composition for detecting mycoplasma and acoleplasma. The kit and composition include a primer consisting of the base sequence represented by SEQ ID NO: 1 in the sequence listing, a primer consisting of the base sequence represented by SEQ ID NO: 2 in the sequence listing, and optionally, SEQ ID NO: 3 in the sequence listing. The primer which consists of a base sequence represented is included. In a preferred embodiment of the present invention, the kit or composition further includes a probe having a base sequence represented by SEQ ID NO: 4 in the sequence listing. These compositions of the present invention are useful for detecting Mycoplasma bacteria and Acoleplasma bacteria by nucleic acid amplification reaction.
　本発明は前記の本発明のプライマー及び／又は本発明のプローブを使用するマイコプラズマ及びアコレプラズマの検出方法を提供する。本発明の方法は、（ａ）検体を配列表の配列番号１で表される塩基配列からなるプライマー、及び配列表の配列番号２で表される塩基配列からなるプライマーを用いた核酸増幅反応に供する工程、並びに（ｂ）工程（ａ）により得られた増幅産物を検出する工程を含む。 The present invention provides a method for detecting mycoplasma and acholplasma using the primer of the present invention and / or the probe of the present invention. In the method of the present invention, (a) a sample is subjected to a nucleic acid amplification reaction using a primer comprising a base sequence represented by SEQ ID NO: 1 in the sequence listing and a primer comprising a base sequence represented by SEQ ID NO: 2 in the sequence listing. And (b) a step of detecting the amplification product obtained in step (a).
　本発明によるマイコプラズマ及びアコレプラズマの検出方法にて解析される検体は、特に限定はないが、例えば細胞を培養するための培地、培養された細胞、及びその培養上清等がある。培養後の検体の場合、好ましくは、培養上清が解析に供される。 Specimens analyzed by the mycoplasma and acoreplasma detection method according to the present invention are not particularly limited, and examples thereof include a medium for culturing cells, cultured cells, and culture supernatants thereof. In the case of a specimen after culture, the culture supernatant is preferably subjected to analysis.
　この検体を準備する方法に特に限定はないが、検体として培養上清を用いる場合、例えば、下記工程によって検体を調製することができる。
　細胞培養液を遠心機にセットし、細胞と培養上清を分離する。遠心の条件は、当業者に周知である。回収した培養上清をそのまま検体として解析することも可能である。好ましくは、既知のＤＮＡ抽出方法又は市販のキット等を用いてＤＮＡを抽出・濃縮する。市販のキットに何ら限定はなく、例えば、ＮｕｃｌｅｏＳｐｉｎ（登録商標）　Ｔｉｓｓｕｅ　ＸＳ（マッハライ・ナーゲル社製）が例示される。 The method for preparing the sample is not particularly limited, but when using a culture supernatant as the sample, for example, the sample can be prepared by the following steps.
Place the cell culture in a centrifuge and separate the cells and culture supernatant. Centrifugation conditions are well known to those skilled in the art. It is also possible to analyze the collected culture supernatant as it is as a specimen. Preferably, DNA is extracted and concentrated using a known DNA extraction method or a commercially available kit. There is no limitation in a commercially available kit, For example, NucleoSpin (trademark) Tissue XS (made by Machalai Nagel) is illustrated.
　本発明によるマイコプラズマ及びアコレプラズマの検出方法においては、前記の本発明の組成物を用いてマイコプラズマ及びアコレプラズマのゲノムＤＮＡの特定領域を増幅して、マイコプラズマ全般を網羅的かつアコレプラズマ属細菌を検出することが可能である。当該マイコプラズマとしては、特に限定はないが、例えば、Ｍｙｃｏｐｌａｓｍａ　ｈｏｍｉｎｉｓ、Ｍｙｃｏｐｌａｓｍａ　ａｒｇｉｎｉｎｉ、Ｍｙｃｏｐｌａｓｍａ　ｆｅｒｍｅｎｔａｎｓ、Ｍｙｃｏｐｌａｓｍａ　ｈｙｏｒｈｉｎｉｓ、Ｍｙｃｏｐｌａｓｍａ　ｏｒａｌｅ、Ｍｙｃｏｐｌａｓｍａ　ｐｉｒｕｍ、Ｍｙｃｏｐｌａｓｍａ　ｐｎｅｕｍｏｎｉａｅ、Ｍｙｃｏｐｌａｓｍａ　ｓａｌｉｖａｒｉｕｍ、Ｍｙｃｏｐｌａｓｍａ　ｂｏｖｉｓが挙げられる。当該アコレプラズマとしては、特に限定はないが、例えば、Ａｃｈｏｌｅｐｌａｓｍａ　ｌａｉｄｌａｗｉｉ、Ａｃｈｏｌｅｐｌａｓｍａ　ｇｒａｎｕｌａｒｕｍ、Ａｃｈｏｌｅｐｌａｓｍａ　ｍｏｄｉcum、Ａｃｈｏｌｅｐｌａｓｍａ　ｍｏｒｕｍ、Ａｃｈｏｌｅｐｌａｓｍａ　ｏｃｕｌｉが挙げられる。例えば、マイコプラズマ属細菌及びＡｃｈｏｌｅｐｌａｓｍａ　ｌａｉｄｌａｗｉｉを特異的に検出することが可能である。また、本発明のキットや組成物は、様々な種のマイコプラズマ属細菌及びアコレプラズマ属細菌の簡便かつ迅速な検出に有用である。なお、上記のプライマー、プローブは公知の方法で化学的に合成できる。
　本発明の方法における核酸増幅反応としては、ＤＮＡ又はＲＮＡを鋳型としてこれに相補的なＤＮＡを合成する反応であれば特に限定はないが、ポリメラーゼ連鎖反応（ＰＣＲ）、逆転写ポリメラーゼ連鎖反応（ＲＴ－ＰＣＲ）等の当分野で周知の核酸合成反応が例示される。 In the method for detecting mycoplasma and acoreplasma according to the present invention, a specific region of the genomic DNA of mycoplasma and acoreplasma is amplified using the composition of the present invention to detect all mycoplasma in general and to detect bacteria belonging to the genus Acholplasma. Is possible. As the mycoplasma is not particularly limited, for example, Mycoplasma hominis, Mycoplasma arginini, Mycoplasma fermentans, Mycoplasma hyorhinis, Mycoplasma orale, Mycoplasma pirum, Mycoplasma pneumoniae, Mycoplasma salivarium, include Mycoplasma bovis. The Acholplasma is not particularly limited, and examples thereof include Acholplasma laidlawii, Acholplasma granularum, Acholplasma modicum, Acholplasma morum, and Acholplasma oculi. For example, it is possible to specifically detect Mycoplasma bacteria and Acholplasma laidlawii. In addition, the kit and composition of the present invention are useful for simple and rapid detection of various species of Mycoplasma bacteria and Acoplasma bacteria. The above primers and probes can be chemically synthesized by known methods.
The nucleic acid amplification reaction in the method of the present invention is not particularly limited as long as it is a reaction in which DNA or RNA is used as a template to synthesize a complementary DNA thereto. Polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT -Nucleic acid synthesis reactions well known in the art such as -PCR).
　ＰＣＲの温度サイクル条件としては一般的な条件が適用できる。例えば、二本鎖鋳型ＤＮＡの一本鎖への解離（変性）、一本鎖鋳型ＤＮＡへのプライマーのアニーリング、プライマーからの相補鎖合成（伸長）の３つのステップからなる反応によりＰＣＲが実施される。各ステップの条件は、当業者により適宜設定される。 General conditions can be applied as temperature cycle conditions for PCR. For example, PCR is performed by a reaction comprising three steps: dissociation (denaturation) of double-stranded template DNA into single strands, annealing of primers to single-stranded template DNA, and synthesis of complementary strands from primers (extension). The Conditions for each step are appropriately set by those skilled in the art.
　本発明のマイコプラズマ及びアコレプラズマの検出方法において、上記核酸増幅反応により得られた増幅産物を検出する方法としては、増幅産物を検出可能な方法であれば特に限定は無いが、リアルタイムＰＣＲ法が例示される。リアルタイムＰＣＲ法における検出方法は、インターカレーターを用いる方法と蛍光プローブを用いる方法の２種類に大別される。蛍光プローブを用いる方法としては、サイクリングプローブ法、ＴａｑＭａｎプローブ法、モレキュラービーコンプローブ法等が例示される。インターカレーター法は、実験コストが安く、反応系の構築が容易であるが、検出の特異性はプライマーの配列のみに依存する。一方。蛍光プローブ法は、実験コストはかかるが、プライマーおよびプローブ両者の配列が検出の特異性に寄与する。サイクリングプローブ法は、ＲＮＡとＤＮＡからなるキメラプローブとエンドヌクレアーゼを組み合わせて検出するため、蛍光プローブ法の中でも配列特異性の非常に高い検出が可能となる。本発明を限定するものではないが、配列表の配列番号４で表される塩基配列からなるプローブを使用する本発明の態様においては、サイクリングプローブ法が好適に使用される。 In the mycoplasma and acoreplasma detection method of the present invention, the method for detecting the amplification product obtained by the nucleic acid amplification reaction is not particularly limited as long as it is a method capable of detecting the amplification product, but a real-time PCR method is exemplified. Is done. Detection methods in the real-time PCR method are roughly classified into two types: a method using an intercalator and a method using a fluorescent probe. Examples of the method using a fluorescent probe include a cycling probe method, a TaqMan probe method, a molecular beacon probe method and the like. The intercalator method is low in experimental cost and easy to construct a reaction system, but the detection specificity depends only on the primer sequence. on the other hand. Although the fluorescent probe method requires an experimental cost, the sequences of both the primer and the probe contribute to the specificity of detection. Since the cycling probe method detects a combination of a chimeric probe composed of RNA and DNA and an endonuclease, detection with very high sequence specificity is possible even among fluorescent probe methods. Although not limiting the present invention, the cycling probe method is suitably used in the embodiment of the present invention in which a probe comprising the base sequence represented by SEQ ID NO: 4 in the sequence listing is used.
　インターカレーター法は、ＰＣＲ反応によって合成された二本鎖ＤＮＡに結合し、励起光の照射により蛍光を発する物質（インターカレーター）を用い、この蛍光強度を検出することにより、増幅産物の生成量をモニターすることが可能な検出法である。一般的にＳＹＢＲ（登録商標）　Ｇｒｅｅｎ　Ｉがインターカレーターとして使用される。インターカレーター法では、ターゲットに特異的な蛍光標識プローブを設計・合成する必要がない。 The intercalator method uses a substance (intercalator) that binds to double-stranded DNA synthesized by the PCR reaction and emits fluorescence when irradiated with excitation light. By detecting this fluorescence intensity, the amount of amplification product generated can be reduced. It is a detection method that can be monitored. Generally, SYBR (registered trademark) Green I is used as an intercalator. In the intercalator method, it is not necessary to design and synthesize a fluorescently labeled probe specific to the target.
　サイクリングプローブ法は、ＲＮＡとＤＮＡからなるキメラプローブとエンドヌクレアーゼの組み合わせによる高感度な検出法で、増幅中や増幅後の遺伝子断片の特定配列を効率良く検出することが可能である。プローブはＲＮＡ部分を挟んで一方が蛍光色素で、またもう一方がその蛍光色素の発する蛍光を消光する物質（クエンチャー）で標識されている。このプローブは、インタクトな状態ではクエンチングにより強い蛍光を発しないが、増幅産物中の相補的な配列とハイブリッドを形成した後にエンドヌクレアーゼによりＲＮＡ部分で切断されることにより、強い蛍光を発するようになる。この蛍光強度を測定することにより、増幅産物量をモニターすることが可能になる。 The cycling probe method is a highly sensitive detection method using a combination of a chimeric probe composed of RNA and DNA and an endonuclease, and can efficiently detect specific sequences of gene fragments during and after amplification. One probe is labeled with a fluorescent dye across the RNA portion, and the other is labeled with a substance (quencher) that quenches the fluorescence emitted by the fluorescent dye. This probe does not emit strong fluorescence by quenching in an intact state, but emits strong fluorescence by forming a hybrid with a complementary sequence in the amplification product and then cleaving at the RNA portion by endonuclease. Become. By measuring the fluorescence intensity, the amount of amplification product can be monitored.
　ＴａｑＭａｎプローブ法は、一方の末端が蛍光色素で、もう一方の末端が消光物質で標識されたオリゴヌクレオチドプローブ（ＴａｑＭａｎプローブ）とＤＮＡポリメラーゼのもつ５’→３’エキソヌクレアーゼ活性とを利用する方法である。ＴａｑＭａｎプローブは、アニーリングステップで鋳型ＤＮＡに特異的にアニールするが、プローブ末端に消光物質が存在するため、励起光を照射しても蛍光の発生は抑制される。伸長反応ステップのときに、ＤＮＡポリメラーゼのもつ５’→３’エキソヌクレアーゼ活性により、鋳型にアニールしたＴａｑＭａｎプローブが分解されると、蛍光色素がプローブから遊離し、消光物質による抑制が解除されて蛍光が発せられる。この蛍光強度を検出することにより、増幅産物の生成量をモニターすることが可能である。 The TaqMan probe method uses an oligonucleotide probe (TaqMan probe) labeled with a fluorescent dye at one end and a quencher at the other end and the 5 ′ → 3 ′ exonuclease activity of DNA polymerase. is there. The TaqMan probe anneals specifically to the template DNA in the annealing step, but since a quencher exists at the end of the probe, the generation of fluorescence is suppressed even when irradiated with excitation light. When the TaqMan probe annealed to the template is decomposed due to the 5 ′ → 3 ′ exonuclease activity of the DNA polymerase during the elongation reaction step, the fluorescent dye is released from the probe, and the suppression by the quencher is released and the fluorescence is released. Is emitted. By detecting this fluorescence intensity, it is possible to monitor the amount of amplification product generated.
　モレキュラービーコンプローブ法は、一方の末端が蛍光色素で、もう一方の末端が消光物質で標識され、かつステムループ構造を有するオリゴヌクレオチド（モレキュラービーコン）をプローブとして用いる方法である。モレキュラービーコンは通常、単鎖の状態では、ステム領域が二重鎖を形成し、両末端の蛍光色素と消光物質が近接し、消光状態となっている。一方、プローブ中のループ領域の塩基配列と相補的であるオリゴヌクレオチドとアニールしたときには、蛍光色素と消光物質が離れ、蛍光シグナルが発せられる。この蛍光強度を検出することにより、増幅産物の生成量をモニターすることが可能である。 The molecular beacon probe method is a method in which one end is labeled with a fluorescent dye, the other end is labeled with a quenching substance, and an oligonucleotide (molecular beacon) having a stem-loop structure is used as a probe. In a molecular beacon, in a single chain state, the stem region usually forms a double chain, and the fluorescent dyes at both ends are in close proximity to each other and are in a quenching state. On the other hand, when annealed with an oligonucleotide complementary to the base sequence of the loop region in the probe, the fluorescent dye and the quenching substance are separated and a fluorescent signal is emitted. By detecting this fluorescence intensity, it is possible to monitor the amount of amplification product generated.
　本発明の方法におけるマイコプラズマ及びアコレプラズマ検出のための反応液は、ＤＮＡポリメラーゼ、反応緩衝剤、少なくとも２種の本発明のプライマー、所望により本発明のプローブやその他の成分を組み合わせ、更に検体又は検体より調製した検体由来の核酸を加えて調製することができる。ＤＮＡポリメラーゼ、反応緩衝剤、本発明のプライマー、本発明のプローブを含有する組成物は、本発明の一態様である。 The reaction solution for detecting mycoplasma and acholplasma in the method of the present invention comprises a DNA polymerase, a reaction buffer, at least two kinds of primers of the present invention, and optionally a probe of the present invention and other components. It can be prepared by adding a nucleic acid derived from a more prepared specimen. A composition containing a DNA polymerase, a reaction buffer, a primer of the present invention, and a probe of the present invention is one embodiment of the present invention.
　ＤＮＡポリメラーゼは、ＤＮＡ又はＲＮＡを鋳型としてこれに相補的なＤＮＡを合成する活性を有するものであれば本発明に使用できる。本発明に使用されるＤＮＡポリメラーゼとしては、特に耐熱性のＤＮＡ依存性ＤＮＡポリメラーゼが好ましい。このようなＤＮＡポリメラーゼとしては、Ｔｈｅｒｍｕｓ属細菌由来ＤＮＡポリメラーゼ（Ｔｈｅｒｍｕｓ　ａｑｕａｔｉｃｕｓ由来ＤＮＡポリメラーゼ等）に代表される真正細菌由来の耐熱性ＤＮＡポリメラーゼ、及びＰｙｒｏｃｏｃｃｕｓ属古細菌由来ＤＮＡポリメラーゼ（Ｐｙｒｏｃｏｃｃｕｓ　ｓｐ．由来ＤＮＡポリメラーゼ等）やＴｈｅｒｍｏｃｏｃｃｕｓ属古細菌由来ＤＮＡポリメラーゼ（Ｔｈｅｒｍｏｃｏｃｃｕｓ　ｋｏｄａｋａｒａｅｎｓｉｓ由来ＤＮＡポリメラーゼ等）の古細菌由来の耐熱性ＤＮＡポリメラーゼが例示される。 A DNA polymerase can be used in the present invention as long as it has an activity of synthesizing DNA complementary to DNA or RNA as a template. The DNA polymerase used in the present invention is particularly preferably a heat-resistant DNA-dependent DNA polymerase. Examples of such a DNA polymerase include heat-resistant DNA polymerases derived from eubacteria typified by DNA polymerases derived from Thermus bacteria (such as Thermus aquaticus-derived DNA polymerases), and DNA polymerases derived from Pyrococcus archaea (Pyrococcus sp.-derived DNA polymerases). And the like, and thermococcus archaebacterium-derived DNA polymerases (Thermococcus kodakaraensis-derived DNA polymerase, etc.).
　ＤＮＡポリメラーゼとして２種以上のＤＮＡポリメラーゼを組み合わせて使用してもよい。２種類以上のＤＮＡポリメラーゼとしては、３’→５’エキソヌクレアーゼ活性を有するＤＮＡポリメラーゼと３’→５’エキソヌクレアーゼ活性を実質的に有さないＤＮＡポリメラーゼとの組み合わせが例示される。なお、このような２種類のＤＮＡポリメラーゼを含む反応液でＰＣＲを行う技術は、ＬＡ－ＰＣＲ（Ｌｏｎｇ　ａｎｄ　Ａｃｃｕｒａｔｅ　ＰＣＲ）として知られている。 Two or more DNA polymerases may be used in combination as the DNA polymerase. Examples of the two or more types of DNA polymerases include a combination of a DNA polymerase having 3 '→ 5' exonuclease activity and a DNA polymerase having substantially no 3 '→ 5' exonuclease activity. A technique for performing PCR using a reaction solution containing two types of DNA polymerases is known as LA-PCR (Long and Accurate PCR).
　前記のサイクリングプローブ法を本発明に使用する場合、反応液には増幅産物依存的にプローブを切断するエンドヌクレアーゼが添加される。当該エンドヌクレアーゼは、マイコプラズマ属細菌又はアコレプラズマ属細菌由来の増幅産物にアニーリングした本発明のプローブのリボヌクレオチド部分を特異的に切断する酵素であれば特に限定は無いが、リボヌクレアーゼＨ（ＲＮａｓｅ　Ｈ）が例示される。また、リボヌクレアーゼＨとしては、好ましくは耐熱性のリボヌクレアーゼＨが、より好ましくは古細菌由来の耐熱性のリボヌクレアーゼＨが例示される。 When the above-described cycling probe method is used in the present invention, an endonuclease that cleaves the probe in an amplification product-dependent manner is added to the reaction solution. The endonuclease is not particularly limited as long as it is an enzyme that specifically cleaves the ribonucleotide portion of the probe of the present invention annealed to an amplification product derived from Mycoplasma bacteria or Acoleplasma bacteria, but Ribonuclease H (RNase H) Is exemplified. The ribonuclease H is preferably a heat-resistant ribonuclease H, more preferably an archaea-derived heat-resistant ribonuclease H.
　本明細書において反応緩衝剤とは、反応液の水素イオン濃度（ｐＨ）の変動を和らげる作用を持つ化合物又は混合物のことをいう。一般に弱酸とその塩、あるいは弱塩基とその塩の混合溶液は、強い緩衝作用を持つため反応緩衝剤としてｐＨコントロールの目的で広く用いられている。本発明には生化学分野で公知の、各種の反応緩衝剤を使用することができる。当該反応緩衝剤としては、特に限定は無いが、例えば、ビシン（ｂｉｃｉｎｅ）、トリシン（ｔｒｉｃｉｎｅ）、ヘペス（ＨＥＰＥＳ）、トリス（ｔｒｉｓ）、リン酸塩が好適に使用できる。当該反応緩衝剤の最終濃度は、特に限定は無いが、５～１００ｍＭ、好ましくは２０～５０ｍＭの範囲が好適に使用できる。本発明の方法における反応液のｐＨは、遺伝子増幅反応が実施される通常の範囲、例えば２５℃におけるｐＨが８．０～９．５の範囲に設定されるのが適当である。 In the present specification, the reaction buffer means a compound or a mixture having an action of reducing the fluctuation of the hydrogen ion concentration (pH) of the reaction solution. In general, a mixed solution of a weak acid and its salt or a weak base and its salt is widely used as a reaction buffer for the purpose of pH control because it has a strong buffering action. Various reaction buffers known in the biochemical field can be used in the present invention. The reaction buffer is not particularly limited, and for example, bicine, tricine, hepes (HEPES), tris, and phosphate can be suitably used. The final concentration of the reaction buffer is not particularly limited, but a range of 5 to 100 mM, preferably 20 to 50 mM can be suitably used. The pH of the reaction solution in the method of the present invention is suitably set in the usual range in which the gene amplification reaction is carried out, for example, the pH at 25 ° C. is in the range of 8.0 to 9.5.
　以下に実施例を挙げて本発明を更に具体的に説明するが、本発明は以下の実施例のみに限定されるものではない。なお、以下の実施例におけるリアルタイムＰＣＲの反応装置としては、Ｔｈｅｒｍａｌ　Ｃｙｃｌｅｒ　Ｄｉｃｅ（登録商標）　Ｒｅａｌ　Ｔｉｍｅ　Ｓｙｓｔｅｍ　ＩＩ（タカラバイオ社製）を使用した。 Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to the following examples. As a reaction device for real-time PCR in the following examples, Thermal Cycler Dice (registered trademark) Real Time System II (manufactured by Takara Bio Inc.) was used.
実施例１　プライマー及びプローブの設計、スクリーニング
（１）プライマー設計
　１６Ｓ　ｒＤＮＡをコードする遺伝子領域を対象とし、動物細胞培養時に培養液から検出される代表的なマイコプラズマ（Ｍｙｃｏｐｌａｓｍａ　ｈｏｍｉｎｉｓ、Ｍｙｃｏｐｌａｓｍａ　ａｒｇｉｎｉｎｉ、Ｍｙｃｏｐｌａｓｍａ　ｆｅｒｍｅｎｔａｎｓ、Ｍｙｃｏｐｌａｓｍａ　ｈｙｏｒｈｉｎｉｓ、Ｍｙｃｏｐｌａｓｍａ　ｏｒａｌｅ、Ｍｙｃｏｐｌａｓｍａ　ｐｉｒｕｍ、及びＭｙｃｏｐｌａｓｍａ　ｐｎｅｕｍｏｎｉａｅ等）、Ａｃｈｏｌｅｐｌａｓｍａ　ｌａｉｄｌａｗｉｉ、代表的な常在菌（Ｂａｃｉｌｌｕｓ　ｓｕｂｔｉｌｉｓ、Ｅ．　ｃｏｌｉ、及びＰｓｅｕｄｏｍｏｎａｓ　ｓｐ．等）、マイコプラズマ以外に検出された常在菌（Ｓｐｈｉｎｇｏｍｏｎａｓ　ｓｐ．、Ｓｔａｐｈｙｌｏｃｏｃｃｕｓ　ｓｐ．等）の塩基配列情報より、マイコプラズマ及びＡｃｈｏｌｅｐｌａｓｍａ　ｌａｉｄｌａｗｉｉに特異的な領域で５’側プライマー及び３’側プライマーを設計した。５’側プライマーは、Ｆ１２（配列番号５）、Ｆ２３３（配列番号６）、Ｆ２５９（配列番号１）、Ｆ３４４（配列番号７）、Ｆ３６２（配列番号８）、及びＦ５３４（配列番号９）の６種類である。３’側プライマーは、Ｒ６０５（配列番号１０）、Ｒ７５０（配列番号１１）、Ｒ８２７（配列番号１２）、Ｒ８５０（配列番号２）、Ｒ１１３１（配列番号１３）、Ｒ１１５５（配列番号１４）、Ｒ１２０７（配列番号１５）、Ｒ１２３８（配列番号１６）、Ｒ１３５２（配列番号１７）、及びＲ１４４０（配列番号１８）、の１０種類である。 Example 1 Primer and Probe Design and Screening (1) Primer Design Representative mycoplasma (Mycoplasma hominis, Mycoplasma fermentans, which are targeted for gene regions encoding 16S rDNA and detected from the culture medium during animal cell culture, Mycoplasma hyorhinis, Mycoplasma oral, Mycoplasma pirum, Mycoplasma pneumoniae, etc. sp., Staphylococcus sp from the nucleotide sequence information. etc.), it was designed mycoplasma and Acholeplasma 5 'end primer and the 3' side primer specific regions laidlawii. The 5'-side primers were F12 (SEQ ID NO: 5), F233 (SEQ ID NO: 6), F259 (SEQ ID NO: 1), F344 (SEQ ID NO: 7), F362 (SEQ ID NO: 8), and F534 (SEQ ID NO: 9). It is a kind. The 3 ′ primers were R605 (SEQ ID NO: 10), R750 (SEQ ID NO: 11), R827 (SEQ ID NO: 12), R850 (SEQ ID NO: 2), R1131 (SEQ ID NO: 13), R1155 (SEQ ID NO: 14), R1207 ( SEQ ID NO: 15), R1238 (SEQ ID NO: 16), R1352 (SEQ ID NO: 17), and R1440 (SEQ ID NO: 18).
（２）プローブ設計
　設計したプライマーでの増幅領域において、動物細胞培養時に培養液から検出される代表的なマイコプラズマ及びＡｃｈｏｌｅｐｌａｓｍａ　ｌａｉｄｌａｗｉｉを特異的に検出するサイクリングプローブを設計した。サイクリングプローブは、Ｐｒｏｂｅ　１（配列番号１９）、Ｐｒｏｂｅ　２（配列番号２０）、Ｐｒｏｂｅ　３（配列番号２１）、Ｐｒｏｂｅ　４（配列番号２２）、Ｐｒｏｂｅ　５（配列番号２３）、Ｐｒｏｂｅ　６（配列番号２４）、Ｐｒｏｂｅ　７（配列番号２５）、Ｐｒｏｂｅ　８（配列番号２６）、Ｐｒｏｂｅ　９（配列番号２７）、Ｐｒｏｂｅ　１０（配列番号２８）、及びＰｒｏｂｅ　１１（配列番号２９）の１１種類である。
　設計は、国際公開公報第２０１２／０１４９８８号記載のアルゴリズムに従い設計を行い、マイコプラズマ全般とＡｃｈｏｌｅｐｌａｓｍａ　ｌａｉｄｌａｗｉｉとに共通で、常在菌は検出しない、また、上記（１）で設計したプライマーと相互作用しないものを採用した。なお、Ｐｒｏｂｅ　４及びＰｒｏｂｅ　５はＰｒｏｂｅ　３を基にして位置を１塩基ずつずらしたもの、Ｐｒｏｂｅ　７及びＰｒｏｂｅ　８はＰｒｏｂｅ　６を基にして位置を１塩基ずつずらしたもの、Ｐｒｏｂｅ　１０はＰｒｏｂｅ　９を基にして位置を１塩基ずらしたもの、並びにＰｒｏｂｅ　１１はＰｒｏｂｅ　９の鎖長を一塩基伸ばしたものである。 (2) Probe design In the amplification region with the designed primer, a cycling probe was designed that specifically detects typical mycoplasma and Acholplasma laidlawii detected from the culture medium during animal cell culture. Cycling probes are Probe 1 (SEQ ID NO: 19), Probe 2 (SEQ ID NO: 20), Probe 3 (SEQ ID NO: 21), Probe 4 (SEQ ID NO: 22), Probe 5 (SEQ ID NO: 23), Probe 6 (SEQ ID NO: 24). ), Probe 7 (SEQ ID NO: 25), Probe 8 (SEQ ID NO: 26), Probe 9 (SEQ ID NO: 27), Probe 10 (SEQ ID NO: 28), and Probe 11 (SEQ ID NO: 29).
The design is performed according to the algorithm described in International Publication No. 2012/014988, and is common to all mycoplasmas and Acholplasma laidlawii, and does not detect resident bacteria, and does not interact with the primer designed in (1) above. The thing was adopted. Probe 4 and Probe 5 are positions shifted by 1 base from Probe 3, Probe 7 and Probe 8 are positions shifted by 1 base based on Probe 6, and Probe 10 is Probe 9 shifted. The position is shifted by one base from the base, and Probe 11 is obtained by extending the chain length of Probe 9 by one base.
（３）スクリーニング
　前記（１）で設計したプライマーを組み合わせた６０通りのプライマーセット及びＰｒｏｂｅ　１、Ｐｒｏｂｅ　２、Ｐｒｏｂｅ　３、Ｐｒｏｂｅ　６、Ｐｒｏｂｅ　９の５種のプローブを使用して、Ａｃｈｏｌｅｐｌａｓｍａ　ｌａｉｄｌａｗｉｉの未精製ＤＮＡを鋳型として反応を行い、良好な増幅が確認されるプライマーセットをスクリーニングした。
　スクリーニングには、ＣｙｃｌｅａｖｅＰＣＲ（登録商標）　Ｒｅａｃｔｉｏｎ　Ｍｉｘ（タカラバイオ社製、コード　ＣＹ５０５）を使用し、以下の反応組成で行った。ＣｙｃｌｅａｖｅＰＣＲ（登録商標）　Ｒｅａｃｔｉｏｎ　Ｍｉｘ（２×ｃｏｎｃ．）　１２．５μＬ、各５μＭ　プライマーミックス　１μＬ、５μＭ　サイクリングプローブ（ＲＯＸ標識）　１μＬ、鋳型　０．５μＬを混合し、滅菌水にて全量を２５μＬにした。反応及び検出は、Ｔｈｅｒｍａｌ　Ｃｙｃｌｅｒ　Ｄｉｃｅ（登録商標）　Ｒｅａｌ　Ｔｉｍｅ　Ｓｙｓｔｅｍ　ＩＩ（タカラバイオ株式会社　コード　ＴＰ９００）を使用し、以下の条件で反応を行った。９５℃、１０秒（初期変性）の後、９５℃，５秒、５５℃，３０秒、７２℃，３０秒のサイクルを４５サイクル。検出は７２℃のステップで行った。
　結果を図１に示した。図中、○は良好な増幅シグナルが認められた組み合わせ、△は増幅シグナルは認められるが、蛍光強度の低い組み合わせ、×は増幅シグナルが認められない組み合わせ、をそれぞれ示す。
　この結果より、プライマーの組み合わせとしては、Ｆ３４４／Ｒ６０５、Ｆ３６２／Ｒ６０５、Ｆ２５９／Ｒ６０５、Ｆ３４４／Ｒ８２７、Ｆ３６２／Ｒ８２７、Ｆ３４４／Ｒ８５０、Ｆ３６２／Ｒ８５０、Ｆ２５９／Ｒ８５０、サイクリングプローブはＰｒｏｂｅ　６あるいはＰｒｏｂｅ　９が有望であった。
　次に、鋳型を加えずにＰｒｏｂｅ　６を使用して上記と同様の反応を行った。
　結果を図２に示した。図中、○は良好な増幅シグナルが認められた組み合わせ、△は増幅シグナルは認められるが、蛍光強度の低い組み合わせ、×は増幅シグナルが認められない組み合わせ、をそれぞれ示す。
　この結果より、Ｆ３４４／Ｒ６０５、Ｆ３６２／Ｒ６０５、Ｆ３４４／Ｒ８２７、Ｆ３６２／Ｒ８２７、Ｆ３４４／Ｒ８５０、Ｆ３６２／Ｒ８５０の組み合わせでは、鋳型を加えない場合においてもシグナルが得られるため、不具合が生じることが考えられた。
　以上の結果より、Ｆ２５９／Ｒ６０５、Ｆ２５９／Ｒ８５０の２組のプライマーの組み合わせを選出した。 (3) Screening Acholplasma laidlawii unpurified using 60 kinds of primer sets combining the primers designed in the above (1) and five probes of Probe 1, Probe 2, Probe 3, Probe 6, and Probe 9. Reaction was performed using DNA as a template, and a primer set that confirmed good amplification was screened.
For screening, CyclePCR (registered trademark) Reaction Mix (manufactured by Takara Bio Inc., code CY505) was used and the following reaction composition was used. CyclePCR (registered trademark) Reaction Mix (2 × conc.) 12.5 μL, each 5 μM primer mix 1 μL, 5 μM cycling probe (ROX labeling) 1 μL, template 0.5 μL were mixed, and the total volume was adjusted to 25 μL with sterile water. For the reaction and detection, Thermal Cycler Dice (registered trademark) Real Time System II (Takara Bio Inc. Code TP900) was used, and the reaction was performed under the following conditions. After 95 ° C for 10 seconds (initial modification), 45 cycles of 95 ° C for 5 seconds, 55 ° C for 30 seconds, 72 ° C for 30 seconds. Detection was performed in steps of 72 ° C.
The results are shown in FIG. In the figure, ◯ represents a combination in which a good amplification signal was observed, Δ represents a combination in which an amplification signal was observed but low fluorescence intensity, and x represents a combination in which no amplification signal was observed.
From these results, the primer combinations are F344 / R605, F362 / R605, F259 / R605, F344 / R827, F362 / R827, F344 / R850, F362 / R850, F259 / R850, and the cycling probe is Probe 6 or Probe 9 Was promising.
Next, the same reaction as described above was performed using Probe 6 without adding a template.
The results are shown in FIG. In the figure, ◯ represents a combination in which a good amplification signal was observed, Δ represents a combination in which an amplification signal was observed but low fluorescence intensity, and x represents a combination in which no amplification signal was observed.
From this result, it is considered that in the combination of F344 / R605, F362 / R605, F344 / R827, F362 / R827, F344 / R850, and F362 / R850, a signal is obtained even when a template is not added, so that a problem may occur. It was.
Based on the above results, two primer combinations F259 / R605 and F259 / R850 were selected.
実施例２　マイコプラズマ検体を使用した検討
（１）プライマーセットの検討
　前記実施例１にて選出した２組のプライマーの組み合わせＦ２５９／Ｒ６０５、Ｆ２５９／Ｒ８５０について、Ａｃｈｏｌｅｐｌａｓｍａ　ｌａｉｄｌａｗｉｉ、およびマイコプラズマ検体（Ｍｙｃｏｐｌａｓｍａ　ｈｏｍｉｎｉｓ、Ｍｙｃｏｐｌａｓｍａ　ａｒｇｉｎｉｎｉ、Ｍｙｃｏｐｌａｓｍａ　ｆｅｒｍｅｎｔａｎｓ、Ｍｙｃｏｐｌａｓｍａ　ｈｙｏｒｈｉｎｉｓ、Ｍｙｃｏｐｌａｓｍａ　ｏｒａｌｅ、Ｍｙｃｏｐｌａｓｍａ　ｐｉｒｕｍ、及びＭｙｃｏｐｌａｓｍａ　ｐｎｅｕｍｏｎｉａｅ）から抽出した各ＤＮＡを鋳型として、反応の比較を行った。サイクリングプローブはＰｒｏｂｅ　１０を使用した。反応はＣｙｃｌｅａｖｅＰＣＲ（登録商標）　Ｒｅａｃｔｉｏｎ　Ｍｉｘ（タカラバイオ社製、コード　ＣＹ５０５）を使用し、以下の反応組成で行った。ＣｙｃｌｅａｖｅＰＣＲ（登録商標）　Ｒｅａｃｔｉｏｎ　Ｍｉｘ（２×ｃｏｎｃ．）　１２．５μＬ、各５μＭ　プライマーミックス　１μL、５μＭ　サイクリングプローブ（ＲＯＸ標識）　１μＬ、鋳型　０．５μＬを混合し、滅菌水にて全量を２５μＬにした。反応及び検出は、Ｔｈｅｒｍａｌ　Ｃｙｃｌｅｒ　Ｄｉｃｅ（登録商標）　Ｒｅａｌ　Ｔｉｍｅ　Ｓｙｓｔｅｍ　ＩＩ　（タカラバイオ株式会社　コード　ＴＰ９００）を使用し、以下の条件で反応を行った。９５℃、１０秒（初期変性）の後、９５℃，５秒、５５℃，３０秒、７２℃，３０秒のサイクルを４５サイクル。検出は７２℃のステップで行った。
　結果を図３に示す。図３（ａ）はＦ２５９／Ｒ６０５、図３（ｂ）はＦ２５９／Ｒ８５０の測定結果である。
　Ｆ２５９／Ｒ６０５及びＦ２５９／Ｒ８５０いずれのプライマーの組み合わせにおいても、良好な検出が行えることが確認された。 Example 2 Examination using Mycoplasma Specimen (1) Examination of Primer Set Regarding the two combinations of primers F259 / R605 and F259 / R850 selected in Example 1, Acholplasma laidavii and Mycoplasma hominis, Mycoplasma Arginini, Mycoplasma fermentans, Mycoplasma hyorhinis, Mycoplasma orale, Mycoplasma pyrum, and Mycoplasma pneumoniae) were used as templates to compare the reactions. Probe 10 was used as the cycling probe. The reaction was performed with the following reaction composition using Cycle PCR (registered trademark) Reaction Mix (manufactured by Takara Bio Inc., code CY505). Cyclave PCR (registered trademark) Reaction Mix (2 × conc.) 12.5 μL, each 5 μM primer mix 1 μL, 5 μM cycling probe (ROX labeling) 1 μL, template 0.5 μL were mixed, and the total volume was adjusted to 25 μL with sterile water. For the reaction and detection, Thermal Cycler Dice (registered trademark) Real Time System II (Takara Bio Inc. Code TP900) was used, and the reaction was performed under the following conditions. After 95 ° C for 10 seconds (initial modification), 45 cycles of 95 ° C for 5 seconds, 55 ° C for 30 seconds, 72 ° C for 30 seconds. Detection was performed in steps of 72 ° C.
The results are shown in FIG. 3A shows the measurement results for F259 / R605, and FIG. 3B shows the measurement results for F259 / R850.
It was confirmed that good detection can be performed with any combination of F259 / R605 and F259 / R850 primers.
（２）サイクリングプローブの検討
　次に、同じくＡｃｈｏｌｅｐｌａｓｍａ　ｌａｉｄｌａｗｉｉおよびマイコプラズマ検体から抽出した各ＤＮＡを鋳型として、検出に用いるサイクリングプローブの比較を行った。反応組成及び反応条件は上記と同じで行った。サイクリングプローブはＰｒｏｂｅ　１、Ｐｒｏｂｅ　３、Ｐｒｏｂｅ　４、Ｐｒｏｂｅ　５、Ｐｒｏｂｅ　６、Ｐｒｏｂｅ　７、Ｐｒｏｂｅ　８、Ｐｒｏｂｅ　９、Ｐｒｏｂｅ　１０、Ｐｒｏｂｅ　１１を使用した。
　結果を図４に示す。図４（ａ）はＰｒｏｂｅ　１、図４（ｂ）はＰｒｏｂｅ　３、図４（ｃ）はＰｒｏｂｅ　４、図４（ｄ）はＰｒｏｂｅ　５、図４（ｅ）はＰｒｏｂｅ　６、図４（ｆ）はＰｒｏｂｅ　７、図４（ｇ）はＰｒｏｂｅ　８、図４（ｈ）はＰｒｏｂｅ　９、図４（ｉ）はＰｒｏｂｅ　１０、図４（ｊ）はＰｒｏｂｅ　１１の測定結果である。
　Ｐｒｏｂｅ　１は、高い蛍光値が得られているが、他のプローブに比べて検出できるマイコプラズマの種類が少ない結果であった。Ｐｒｏｂｅ　３、Ｐｒｏｂｅ　４、Ｐｒｏｂｅ　５は、蛍光値が低い結果であった。Ｐｒｏｂｅ　６、Ｐｒｏｂｅ　７、Ｐｒｏｂｅ　８はどれも良好な結果であった。Ｐｒｏｂｅ　９、Ｐｒｏｂｅ　１０、Ｐｒｏｂｅ　１１では、Ｐｒｏｂｅ　１０が他に比べて蛍光値が高く良好であると考えられた。
　以上の結果より、Ｐｒｏｂｅ　１０を選出した。
　また、Ａｃｈｏｌｅｐｌａｓｍａ　ｌａｉｄｌａｗｉｉ、Ｍｙｃｏｐｌａｓｍ　ｈｏｍｉｎｉｓ、Ｍｙｃｏｐｌａｓｍａ　ａｒｇｉｎｉｎｉ、Ｍｙｃｏｐｌａｓｍａ　ｆｅｒｍｅｎｔａｎｓ、Ｍｙｃｏｐｌａｓｍａ　ｈｙｏｒｈｉｎｉｓ、及びＭｙｃｏｐｌａｓｍａ　ｏｒａｌｅは増幅シグナルが得られたが、Ｍｙｃｏｐｌａｓｍａ　ｐｉｒｕｍおよびＭｙｃｏｐｌａｓｍａ　ｐｎｅｕｍｏｎｉａｅは増幅シグナルが得られなかった。 (2) Examination of cycling probe Next, the cycling probe used for detection was compared using each DNA extracted from Acholplasma laidlawii and Mycoplasma specimen as a template. The reaction composition and reaction conditions were the same as above. Probe 1, Probe 3, Probe 4, Probe 5, Probe 5, Probe 6, Probe 7, Probe 8, Probe 9, Probe 10, and Probe 11 were used as the cycling probe.
The results are shown in FIG. 4 (a) is Probe 1, FIG. 4 (b) is Probe 3, FIG. 4 (c) is Probe 4, FIG. 4 (d) is Probe 5, FIG. 4 (e) is Probe 6, and FIG. 4 (f). FIG. 4 (g) shows the measurement results for Probe 8, FIG. 4 (h) shows the measurement results for Probe 9, FIG. 4 (i) shows the measurement results for Probe 10, and FIG.
Probe 1 has a high fluorescence value, but has fewer types of mycoplasma that can be detected than other probes. Probe 3, Probe 4, and Probe 5 resulted in low fluorescence values. Probe 6, Probe 7, and Probe 8 were all good results. In Probe 9, Probe 10, and Probe 11, it was considered that Probe 10 had a higher fluorescence value than others and was favorable.
Based on the above results, Probe 10 was selected.
In addition, Acholplasma laidlawiii, Mycoplasmm hominis, Mycoplasma arginini, Mycoplasma fermentans, Mycoplasma hyorhinis, Mycoplasma ore were amplified, and My was amplified.
実施例３　プライマー追加の検討
　上記実施例１及び実施例２で使用したプライマーセット、サイクリングプローブでの反応において、Ｍｙｃｏｐｌａｓｍａ　ｐｉｒｕｍ、Ｍｙｃｏｐｌａｓｍａ　ｐｎｅｕｍｏｎｉａｅのＤＮＡを用いた場合では増幅シグナルが得られなかった。そのため、Ｍｙｃｏｐｌａｓｍａ属細菌におけるプライマーＦ２５９及びＲ８５０それぞれに相応する塩基配列（配列番号３０～配列番号２２７）を比較し、新たな追加プライマーＦ２５９Ｐ（配列番号３）を設計した。
　Ｍｙｃｏｐｌａｓｍａ　ｐｉｒｕｍ、Ｍｙｃｏｐｌａｓｍａ　ｐｎｅｕｍｏｎｉａｅ、及びＭｙｃｏｐｌａｓｍａ　ｏｒａｌｅより調製したＤＮＡを鋳型とした。プライマーは、Ｆ２５９とＦ２５９Ｐを１：１の比率で混合したフォワードプライマー及びＲ８５０リバースプライマーを使用した。プローブは、上記実施例２で良好な結果が得られたＰｒｏｂｅ　１０を使用した。
　反応はＣｙｃｌｅａｖｅＰＣＲ（登録商標）　Ｒｅａｃｔｉｏｎ　Ｍｉｘ（タカラバイオ株式会社製、コード　ＣＹ５０５）を使用し、以下の反応組成で行った。ＣｙｃｌｅａｖｅＰＣＲ（登録商標）　Ｒｅａｃｔｉｏｎ　Ｍｉｘ（２×ｃｏｎｃ．）　１２．５μＬ、各５μＭ　プライマーミックス　１μＬ、５μＭ　サイクリングプローブ（ＲＯＸ標識）　１μＬ、鋳型　０．５μＬを混合し、滅菌水にて全量を２５μＬにした。反応及び検出は、Ｔｈｅｒｍａｌ　Ｃｙｃｌｅｒ　Ｄｉｃｅ（登録商標）　Ｒｅａｌ　Ｔｉｍｅ　Ｓｙｓｔｅｍ　ＩＩ（タカラバイオ社製、コード　ＴＰ９００）を使用し、以下の条件で反応を行った。９５℃、１０秒（初期変性）の後、９５℃，５秒、５５℃，３０秒、７２℃，３０秒のサイクルを４５サイクル。検出は７２℃のステップで行った。
　その結果、Ｍｙｃｏｐｌａｓｍａ　ｐｉｒｕｍ、Ｍｙｃｏｐｌａｓｍａ　ｐｎｅｕｍｏｎｉａｅ、及びＭｙｃｏｐｌａｓｍａ　ｏｒａｌｅのいずれにおいても増幅シグナルが得られた。 Example 3 Examination of Primer Addition In the reaction with the primer set and cycling probe used in Examples 1 and 2 above, amplification signals were not obtained when using Mycoplasma pyrum, Mycoplasma pneumoniae DNA. Therefore, a base sequence (SEQ ID NO: 30 to SEQ ID NO: 227) corresponding to each of primers F259 and R850 in Mycoplasma bacteria was compared, and a new additional primer F259P (SEQ ID NO: 3) was designed.
DNA prepared from Mycoplasma pyrum, Mycoplasma pneumoniae, and Mycoplasma oral was used as a template. As the primer, a forward primer and an R850 reverse primer in which F259 and F259P were mixed at a ratio of 1: 1 were used. As the probe, Probe 10 obtained with good results in Example 2 was used.
The reaction was performed using the following reaction composition using Cycleleave (registered trademark) Reaction Mix (manufactured by Takara Bio Inc., code CY505). CyclePCR (registered trademark) Reaction Mix (2 × conc.) 12.5 μL, each 5 μM primer mix 1 μL, 5 μM cycling probe (ROX labeling) 1 μL, template 0.5 μL were mixed, and the total volume was adjusted to 25 μL with sterile water. For the reaction and detection, Thermal Cycler Dice (registered trademark) Real Time System II (manufactured by Takara Bio Inc., code TP900) was used, and the reaction was performed under the following conditions. After 95 ° C for 10 seconds (initial modification), 45 cycles of 95 ° C for 5 seconds, 55 ° C for 30 seconds, 72 ° C for 30 seconds. Detection was performed in steps of 72 ° C.
As a result, an amplified signal was obtained in any of Mycoplasma pyrum, Mycoplasma pneumoniae, and Mycoplasma oral.
　本発明は、遺伝子工学、生物学、医学、農業等の幅広い分野において有用である。 The present invention is useful in a wide range of fields such as genetic engineering, biology, medicine and agriculture.
SEQ ID NO:1 ; Oligonucleotide primer F259 to amplify the DNA fragment of 16S rRNA gene from Mycoplasma
SEQ ID NO:2 ; Oligonucleotide primer R850 to amplify the DNA fragment of 16S rRNA gene from Mycoplasma
SEQ ID NO:3 ; Oligonucleotide primer F259P to amplify the DNA fragment of 16S rRNA gene from Mycoplasma
SEQ ID NO:4 ; Oligonucleotide probe 10 to detect 16S rRNA gene from Mycoplasma.
SEQ ID NO:5 ; Oligonucleotide primer F12 to amplify the DNA fragment of 16S rRNA gene from Mycoplasma.
SEQ ID NO:6 ; Oligonucleotide primer F233 to amplify the DNA fragment of 16S rRNA gene from Mycoplasma.
SEQ ID NO:7 ; Oligonucleotide primer F344 to amplify the DNA fragment of 16S rRNA gene from Mycoplasma.
SEQ ID NO:8 ; Oligonucleotide primer F362 to amplify the DNA fragment of 16S rRNA gene from Mycoplasma.
SEQ ID NO:9 ; Oligonucleotide primer F534 to amplify the DNA fragment of 16S rRNA gene from Mycoplasma.
SEQ ID NO:10 ; Oligonucleotide primer R605 to amplify the DNA fragment of 16S rRNA gene from Mycoplasma.
SEQ ID NO:11 ; Oligonucleotide primer R750 to amplify the DNA fragment of 16S rRNA gene from Mycoplasma.
SEQ ID NO:12 ; Oligonucleotide primer R827 to amplify the DNA fragment of 16S rRNA gene from Mycoplasma.
SEQ ID NO:13 ; Oligonucleotide primer R1131 to amplify the DNA fragment of 16S rRNA gene from Mycoplasma.
SEQ ID NO:14 ; Oligonucleotide primer R1155 to amplify the DNA fragment of 16S rRNA gene from Mycoplasma.
SEQ ID NO:15 ; Oligonucleotide primer R1207 to amplify the DNA fragment of 16S rRNA gene from Mycoplasma.
SEQ ID NO:16; Oligonucleotide primer R1238 to amplify the DNA fragment of 16S rRNA gene from Mycoplasma.
SEQ ID NO:17 ; Oligonucleotide primer R1352 to amplify the DNA fragment of 16S rRNA gene from Mycoplasma.
SEQ ID NO:18 ; Oligonucleotide primer R1440 to amplify the DNA fragment of 16S rRNA gene from Mycoplasma.
SEQ ID NO:19 ; Oligonucleotide probe 1 to detect 16S rRNA gene from Mycoplasma. "Oligonucleotide 4 is ribonucleotide - other nucleotides are deoxyribonucleotide."
SEQ ID NO:20 ; Oligonucleotide probe 2 to detect 16S rRNA gene from Mycoplasma. "Oligonucleotide 4 is ribonucleotide - other nucleotides are deoxyribonucleotide."
SEQ ID NO:21 ; Oligonucleotide probe 3 to detect 16S rRNA gene from Mycoplasma."Oligonucleotide 7 is ribonucleotide - other nucleotides are deoxyribonucleotide."
SEQ ID NO:22 ; Oligonucleotide probe 4 to detect 16S rRNA gene from Mycoplasma."Oligonucleotide 6 is ribonucleotide - other nucleotides are deoxyribonucleotide."
SEQ ID NO:23 ; Oligonucleotide probe 5 to detect 16S rRNA gene from Mycoplasma. "Oligonucleotide 5 is ribonucleotide - other nucleotides are deoxyribonucleotide."
SEQ ID NO:24 ; Oligonucleotide probe 6 to detect 16S rRNA gene from Mycoplasma. "Oligonucleotide 9 is ribonucleotide - other nucleotides are deoxyribonucleotide."
SEQ ID NO:25 ; Oligonucleotide probe 7 to detect 16S rRNA gene from Mycoplasma. "Oligonucleotide 8 is ribonucleotide - other nucleotides are deoxyribonucleotide."
SEQ ID NO:26; Oligonucleotide probe 8 to detect 16S rRNA gene from Mycoplasma. "Oligonucleotide 7 is ribonucleotide - other nucleotides are deoxyribonucleotide."
SEQ ID NO:27 ; Oligonucleotide probe 9 to detect 16S rRNA gene from Mycoplasma. "Oligonucleotide 10 is ribonucleotide - other nucleotides are deoxyribonucleotide."
SEQ ID NO:28 ; Oligonucleotide probe 10 to detect 16S rRNA gene from  Mycoplasma. "Oligonucleotide 9 is ribonucleotide - other nucleotides are deoxyribonucleotide."
SEQ ID NO:29 ; Oligonucleotide probe 11 to detect 16S rRNA gene from Mycoplasma. "Oligonucleotide 10 is ribonucleotide - other nucleotides are deoxyribonucleotide." SEQ ID NO: 1; Oligonucleotide primer F259 to amplify the DNA fragment of 16S rRNA gene from Mycoplasma
SEQ ID NO: 2; Oligonucleotide primer R850 to amplify the DNA fragment of 16S rRNA gene from Mycoplasma
SEQ ID NO: 3; Oligonucleotide primer F259P to amplify the DNA fragment of 16S rRNA gene from Mycoplasma
SEQ ID NO: 4; Oligonucleotide probe 10 to detect 16S rRNA gene from Mycoplasma.
SEQ ID NO: 5; Oligonucleotide primer F12 to amplify the DNA fragment of 16S rRNA gene from Mycoplasma.
SEQ ID NO: 6; Oligonucleotide primer F233 to amplify the DNA fragment of 16S rRNA gene from Mycoplasma.
SEQ ID NO: 7; Oligonucleotide primer F344 to amplify the DNA fragment of 16S rRNA gene from Mycoplasma.
SEQ ID NO: 8; Oligonucleotide primer F362 to amplify the DNA fragment of 16S rRNA gene from Mycoplasma.
SEQ ID NO: 9; Oligonucleotide primer F534 to amplify the DNA fragment of 16S rRNA gene from Mycoplasma.
SEQ ID NO: 10; Oligonucleotide primer R605 to amplify the DNA fragment of 16S rRNA gene from Mycoplasma.
SEQ ID NO: 11; Oligonucleotide primer R750 to amplify the DNA fragment of 16S rRNA gene from Mycoplasma.
SEQ ID NO: 12; Oligonucleotide primer R827 to amplify the DNA fragment of 16S rRNA gene from Mycoplasma.
SEQ ID NO: 13; Oligonucleotide primer R1131 to amplify the DNA fragment of 16S rRNA gene from Mycoplasma.
SEQ ID NO: 14; Oligonucleotide primer R1155 to amplify the DNA fragment of 16S rRNA gene from Mycoplasma.
SEQ ID NO: 15; Oligonucleotide primer R1207 to amplify the DNA fragment of 16S rRNA gene from Mycoplasma.
SEQ ID NO: 16; Oligonucleotide primer R1238 to amplify the DNA fragment of 16S rRNA gene from Mycoplasma.
SEQ ID NO: 17; Oligonucleotide primer R1352 to amplify the DNA fragment of 16S rRNA gene from Mycoplasma.
SEQ ID NO: 18; Oligonucleotide primer R1440 to amplify the DNA fragment of 16S rRNA gene from Mycoplasma.
SEQ ID NO: 19; Oligonucleotide probe 1 to detect 16S rRNA gene from Mycoplasma. "Oligonucleotide 4 is ribonucleotide-other nucleotides are deoxyribonucleotide."
SEQ ID NO: 20; Oligonucleotide probe 2 to detect 16S rRNA gene from Mycoplasma. "Oligonucleotide 4 is ribonucleotide-other nucleotides are deoxyribonucleotide."
SEQ ID NO: 21; Oligonucleotide probe 3 to detect 16S rRNA gene from Mycoplasma. "Oligonucleotide 7 is ribonucleotide-other nucleotides are deoxyribonucleotide."
SEQ ID NO: 22; Oligonucleotide probe 4 to detect 16S rRNA gene from Mycoplasma. "Oligonucleotide 6 is ribonucleotide-other nucleotides are deoxyribonucleotide."
SEQ ID NO: 23; Oligonucleotide probe 5 to detect 16S rRNA gene from Mycoplasma. "Oligonucleotide 5 is ribonucleotide-other nucleotides are deoxyribonucleotide."
SEQ ID NO: 24; Oligonucleotide probe 6 to detect 16S rRNA gene from Mycoplasma. "Oligonucleotide 9 is ribonucleotide-other nucleotides are deoxyribonucleotide."
SEQ ID NO: 25; Oligonucleotide probe 7 to detect 16S rRNA gene from Mycoplasma. "Oligonucleotide 8 is ribonucleotide-other nucleotides are deoxyribonucleotide."
SEQ ID NO: 26; Oligonucleotide probe 8 to detect 16S rRNA gene from Mycoplasma. "Oligonucleotide 7 is ribonucleotide-other nucleotides are deoxyribonucleotide."
SEQ ID NO: 27; Oligonucleotide probe 9 to detect 16S rRNA gene from Mycoplasma. "Oligonucleotide 10 is ribonucleotide-other nucleotides are deoxyribonucleotide."
SEQ ID NO: 28; Oligonucleotide probe 10 to detect 16S rRNA gene from Mycoplasma. "Oligonucleotide 9 is ribonucleotide-other nucleotides are deoxyribonucleotide."
SEQ ID NO: 29; Oligonucleotide probe 11 to detect 16S rRNA gene from Mycoplasma. "Oligonucleotide 10 is ribonucleotide-other nucleotides are deoxyribonucleotide."

Claims (12)

  1. 　マイコプラズマ及びアコレプラズマを検出するための組成物であって、配列表の配列番号１で表される塩基配列からなるプライマー、及び配列表の配列番号２で表される塩基配列からなるプライマーを含む組成物。 A composition for detecting mycoplasma and acholplasma, comprising a primer comprising a base sequence represented by SEQ ID NO: 1 in the sequence listing, and a primer comprising a base sequence represented by SEQ ID NO: 2 in the sequence listing object.
  2. 　更に、配列表の配列番号３で表される塩基配列からなるプライマーを含む、請求項１記載の組成物。 Furthermore, the composition of Claim 1 containing the primer which consists of a base sequence represented by sequence number 3 of a sequence table.
  3. 　更に、配列表の配列番号４で表される塩基配列からなるプローブを含む、請求項１又は２記載の組成物。 Furthermore, the composition of Claim 1 or 2 containing the probe which consists of a base sequence represented by sequence number 4 of a sequence table.
  4. 　プローブが、ＤＮＡ／ＲＮＡ／ＤＮＡキメラプローブである、請求項３記載の組成物。 The composition according to claim 3, wherein the probe is a DNA / RNA / DNA chimeric probe.
  5. 　マイコプラズマ及びアコレプラズマの検出方法であって、
    （ａ）検体を配列表の配列番号１で表される塩基配列からなるプライマー、及び配列表の配列番号２で表される塩基配列からなるプライマーを用いた核酸増幅反応に供する工程、並びに
    （ｂ）工程（ａ）により得られた増幅産物を検出する工程、
    を含む方法。     A method for detecting mycoplasma and acoleplasma,
    (A) a step of subjecting the specimen to a nucleic acid amplification reaction using a primer comprising a base sequence represented by SEQ ID NO: 1 in the sequence listing and a primer comprising a base sequence represented by SEQ ID NO: 2 in the sequence listing; and (b ) Detecting the amplification product obtained in step (a),
    Including methods.
  6. 　工程（ａ）において、更に配列番号３で表される塩基配列からなるプライマーを使用する、請求項５記載の方法。 The method according to claim 5, wherein a primer comprising the base sequence represented by SEQ ID NO: 3 is further used in step (a).
  7. 　工程（ｂ）における工程（ａ）により得られた産物の検出が、配列表の配列番号４で表される塩基配列からなるプローブを用いて行われる、請求項５又は６記載の方法。 The method according to claim 5 or 6, wherein the detection of the product obtained in step (a) in step (b) is performed using a probe comprising the base sequence represented by SEQ ID NO: 4 in the sequence listing.
  8. 　プローブが、ＤＮＡ／ＲＮＡ／ＤＮＡキメラプローブである、請求項７記載の方法。 The method according to claim 7, wherein the probe is a DNA / RNA / DNA chimeric probe.
  9. 　マイコプラズマ及びアコレプラズマを検出するためのキットであって、配列表の配列番号１で表される塩基配列からなるプライマー、及び配列表の配列番号２で表される塩基配列からなるプライマーを含むキット。 A kit for detecting mycoplasma and acholplasma, comprising a primer comprising the base sequence represented by SEQ ID NO: 1 in the sequence listing and a primer comprising the base sequence represented by SEQ ID NO: 2 in the sequence listing.
  10. 　更に、配列表の配列番号３で表される塩基配列からなるプライマーを含む、請求項９記載のキット。 Furthermore, the kit of Claim 9 containing the primer which consists of a base sequence represented by sequence number 3 of a sequence table.
  11. 　更に、配列表の配列番号４で表される塩基配列からなるプローブを含む、請求項９又は１０記載のキット。 Furthermore, the kit of Claim 9 or 10 containing the probe which consists of a base sequence represented by sequence number 4 of a sequence table.
  12. 　プローブが、ＤＮＡ／ＲＮＡ／ＤＮＡキメラプローブである、請求項１１記載のキット。 The kit according to claim 11, wherein the probe is a DNA / RNA / DNA chimeric probe.
PCT/JP2013/064101 2012-05-23 2013-05-21 Mycoplasma- and acholeplasma-detecting composition WO2013176136A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2012117979 2012-05-23
JP2012-117979 2012-05-23

Publications (1)

Publication Number Publication Date
WO2013176136A1 true WO2013176136A1 (en) 2013-11-28

Family

ID=49623825

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2013/064101 WO2013176136A1 (en) 2012-05-23 2013-05-21 Mycoplasma- and acholeplasma-detecting composition

Country Status (1)

Country Link
WO (1) WO2013176136A1 (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3192878A4 (en) * 2014-09-10 2018-02-28 National University Corporation Tokyo Medical and Dental University Method for detecting mycoplasma
WO2021222144A1 (en) * 2020-04-27 2021-11-04 Cepheid Exponential base-3 and greater nucleic acid amplification with cycling probe

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007228977A (en) * 2001-06-12 2007-09-13 Takara Bio Inc Method of stabilizing reagent for amplifying or detecting nucleic acid and storage method
WO2012014988A1 (en) * 2010-07-29 2012-02-02 タカラバイオ株式会社 Method for manufacturing a probe containing rna for detecting a target base

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007228977A (en) * 2001-06-12 2007-09-13 Takara Bio Inc Method of stabilizing reagent for amplifying or detecting nucleic acid and storage method
WO2012014988A1 (en) * 2010-07-29 2012-02-02 タカラバイオ株式会社 Method for manufacturing a probe containing rna for detecting a target base

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
KOBAYASHI H. ET AL.: "In Vitro Amplificatin of the 16S rRNA Genes from Mycoplasma bovirhinis, Mycoplasma alkalescens and Mycoplasma bovigenitalium by PCR", J VET MED SCI, vol. 60, no. 12, 1998, pages 1299 - 1303 *
KOBAYASHI H. ET AL.: "Rapid Detection of Mycoplasma Contamination in Cell Cultures by Enzymatic Detection of Polymerase Chain Reaction (PCR) Products", J VET MED SCI, vol. 57, no. 4, 1995, pages 769 - 771 *
PEREDELTCHOUK M. ET AL.: "Detection of mycoplasma contamination in cell substrates using reverse transcription-PCR assays", J APPL MICROBIOL, vol. 110, no. 1, 2011, pages 54 - 60 *
WIRTH M. ET AL.: "Detection of mycoplasma contaminations by the polymerase chain reaction", CYTOTECHNOLOGY, vol. 16, no. 2, 1994, pages 67 - 77 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3192878A4 (en) * 2014-09-10 2018-02-28 National University Corporation Tokyo Medical and Dental University Method for detecting mycoplasma
US10640834B2 (en) 2014-09-10 2020-05-05 National University Corporation Tokyo Medical And Dental University Method for detecting mycoplasma
WO2021222144A1 (en) * 2020-04-27 2021-11-04 Cepheid Exponential base-3 and greater nucleic acid amplification with cycling probe

Similar Documents

Publication Publication Date Title
US9850545B2 (en) Multi-primer assay for Mycoplasma detection
JP5830461B2 (en) PCR primer set, PCR reaction solution, detection method for food poisoning bacteria
US20240018605A1 (en) Method and Kit of Detecting the Absence of Micro-Organisms
CN102242189A (en) Nucleic Acid Template preparation for real-time PCR
EP3192878B1 (en) Method for detecting mycoplasma
CN105121656A (en) PCR reaction mixtures and methods of using same
Castro et al. Ultrasensitive, direct detection of a specific DNA sequence of Bacillus anthracis in solution
US11479826B2 (en) Nucleic acids and methods for the detection of Enterobacter sakazakii (Cronobacter spp.)
US20180320227A1 (en) Method for quantifying target nucleic acid and kit therefor
KR20140071968A (en) Methods, systems, and compositions for detection of microbial dna by pcr
EP1772522A1 (en) Control of preservation by biomarkers
WO2013176136A1 (en) Mycoplasma- and acholeplasma-detecting composition
US20100297637A1 (en) Primer for amplification of rrna or bacterium belonging to the genus legionella, detection method, and detection kit
US20140087371A1 (en) Compositions and Methods for Detection of Clostridium Difficile
US7439022B2 (en) Nucleic acids for detection of Listeria
JP2009268413A (en) Primer and method for detecting bacillus bacterium using the primer
JP2012187067A (en) Method for quantifying bacterium of genus lactobacillus
US20230212697A1 (en) Isothermal real-time pcr method for determining presence of a pre-determined nucleic acid sequence of a bacterium of the mollicutes class in a sample
CN116536440A (en) Mycoplasma detection kit based on multiplex fluorescent probe method qPCR and application method thereof
US9738939B2 (en) Method for differentiating between living and dead cells
CN111183223A (en) Primer pair for mecA gene amplification, mecA gene detection kit and mecA gene detection method
Kubota Engineering a simple, rapid, inexpensive, nucleic acid based system for strain selective detection of the bacterial wilt pathogen Ralstonia solanacearum
GB2528647A (en) Detecting viable microorganisms

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 13794023

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 13794023

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: JP