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WO2005118804A1 - Méthode de détection spécifique de la bactérie appartenant au genre alicyclobacillus - Google Patents

Méthode de détection spécifique de la bactérie appartenant au genre alicyclobacillus Download PDF

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Publication number
WO2005118804A1
WO2005118804A1 PCT/JP2005/010259 JP2005010259W WO2005118804A1 WO 2005118804 A1 WO2005118804 A1 WO 2005118804A1 JP 2005010259 W JP2005010259 W JP 2005010259W WO 2005118804 A1 WO2005118804 A1 WO 2005118804A1
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Prior art keywords
oligonucleotide
seq
nucleotide sequence
sequence shown
primer
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Japanese (ja)
Inventor
Yoshihiko Tsuda
Fumihiro Arakawa
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San Ei Gen FFI Inc
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San Ei Gen FFI Inc
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Priority to JP2006514145A priority Critical patent/JPWO2005118804A1/ja
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    • 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

Definitions

  • the present invention relates to a method for specifically detecting Alicyclobacillus bacteria, particularly Alicyclobacillus acidoterrestris. Furthermore, the present invention relates to a primer for amplifying a DNA sequence specific to a bacterium belonging to the genus Alicyclobacillus, particularly Alicyclobacillus acidoterrestris, a probe for specifically labeling the DNA sequence, and a combination of the primer and the probe. . Background art
  • Alicyclobacillus bacteria are bacteria that have characteristics of growing well under acidic and high temperature conditions and forming spores. Alicyclobacillus bacteria are classified as Bacillus because of their spore formation.Proposed as a new genus independent of Bacillus in 1992, based on their unique properties and 16S rDNA gene sequence. Was done.
  • the peroxidase method is a method based on the ability of A. acidoterrestris to assimilate vanillic acid to produce guaial alcohol.
  • the determination is made by measuring the absorbance of tetraguai alcohol produced by treating guaia alcohol with hydrogen peroxide and peroxidase.
  • This method while working, However, there is a drawback that the determination result is likely to be false positive or false negative because it depends on the nature of the determination.
  • the temperature difference method is a method based on the difference in the optimal growth temperature between A. addoterrestris and other thermostable eosinophilic bacteria.
  • the test bacteria are cultured simultaneously at different temperatures, and the determination is made by observing the presence or absence of colony formation at each culture temperature.
  • this method has the disadvantage that it requires 3-4 days before results are obtained.
  • the force sequence method is conventionally used as a method for detecting the presence or absence of bacterial contamination.
  • the base sequence of the DNA (for example, 16S rDNA) of the test bacterium is determined, the obtained sequence is compared with a database, and the homologous bacteria are identified (for example, Non-Patent Document 2).
  • a method for identifying a bacterial species based on a nucleotide sequence can almost certainly identify a bacterium, but requires three to four days to obtain a result, and the operation is complicated. There is a problem that it is expensive and expensive.
  • Patent document 1 Japanese Patent Application Laid-Open No. 2004-201668
  • Non-patent document 1 Kei Goto-thermophilic eosinophilic spore-forming bacterium: Alicyclobadllus bacterium bacteriostatic fungus Vol.28 No.8 499-508 (2000)
  • Non-Patent Document 2 Tsugio Sasaki, Kenichi Tanamoto, 2001 Research Report on “Testing Methods in the Japanese Pharmacopoeia”, Rapid Identification of Microorganisms by Genetic Analysis, Pharmaceutical Research 33 (12) 7 63-769 (2002)
  • the present inventors have developed a novel method for specifically detecting bacteria of the genus Alicyclobadllus. Diligent research was done to radiate. As a result, by performing PCR using an oligonucleotide having a specific sequence as a primer, it is possible to amplify a DNA sequence specifically possessed by a bacterium belonging to the genus Alicyclobacillus. It was found that by using tide as a probe, the resulting amplified DNA product (PCR product) could be selectively labeled, thereby making Alicyclobacillus genus bacteria other than Escherichia coli, Bacillus subtilis, Staphylococcus aureus and Salmonella bacteria.
  • the present invention has the following aspects.
  • an oligonucleotide having a nucleotide sequence showing at least 90% identity to the nucleotide sequence shown in SEQ ID NO: 1 and a nucleotide sequence showing at least 90% identity to the nucleotide sequence shown in SEQ ID NO: 2 A primer set that also has an oligonucleotide power having the following characteristics, or an oligonucleotide having a nucleotide sequence showing at least 90% identity to the nucleotide sequence shown in SEQ ID NO: 3 and at least 90% identity to the nucleotide sequence shown in SEQ ID NO: 4 Performing a PCR using a primer set consisting of an oligonucleotide having the base sequence shown,
  • a method for specifically detecting Alicyclobacillus bacteria is provided.
  • a primer set comprising an oligonucleotide having the nucleotide sequence of SEQ ID NO: 1 and an oligonucleotide having the nucleotide sequence of SEQ ID NO: 2, Or PCR using an oligonucleotide having the nucleotide sequence shown in SEQ ID NO: 3 and an oligonucleotide having the nucleotide sequence shown in SEQ ID NO: 4 as a primer set,
  • Item 2 The method for specifically detecting Alicyclobadllus bacteria according to Item 1, which comprises:
  • Item 2 The method for specific detection of Alicyclobadllus bacteria according to Item 1, wherein the probe is immobilized on a solid phase.
  • Item 2 The method for specifically detecting Alicyclobadllus bacteria according to Item 1, which comprises:
  • Item 2 The method according to Item 1, wherein the bacterium belonging to the genus Alicyclobadllus has vanillin utilization.
  • Alicyclobacillus J3 ⁇ 4 Itoda fungus is Alicyclobacillus acidoterrestris, the method described in section 5 Item 7.
  • an oligonucleotide having a nucleotide sequence having at least 90% identity to the nucleotide sequence shown in SEQ ID NO: 1 and a nucleotide having at least 90% identity to the nucleotide sequence shown in SEQ ID NO: 2 A step of performing PCR using a primer set that also has the ability of an oligonucleotide having a sequence,
  • an oligonucleotide having a nucleotide sequence having at least 90% identity to the nucleotide sequence shown in SEQ ID NO: 3 and a nucleotide having at least 90% identity to the nucleotide sequence shown in SEQ ID NO: 4 A step of performing PCR using a primer set that also has the ability of an oligonucleotide having a sequence,
  • (2 ) a step of hybridizing the obtained PCR product with a probe consisting of an oligonucleotide having a nucleotide sequence having at least 90% identity to the nucleotide sequence shown in SEQ ID NO: 5 or 6, and
  • a method for specifically detecting Alicyclobacillus acidoterrestris comprising:
  • Test sample A step of performing PCR using a primer set consisting of an oligonucleotide having the nucleotide sequence shown in SEQ ID NO: 1 and an oligonucleotide having the nucleotide sequence shown in SEQ ID NO: 2,
  • Item 8 The method for specifically detecting Alicyclobacillus acidoterrestris according to Item 7, comprising:
  • Item 8 The method for specifically detecting Alicyclobacillus acidoterrestris according to Item 7, wherein the probe is immobilized on a solid phase.
  • Item 8 The method for specific detection of Alicyclobacillus acidoterrestris according to Item 7, wherein the method further comprises a step of immobilizing the product of ibridis on a solid phase before the detection step of (3 ′) or (3 ′′).
  • Item 8 The method for specifically detecting Alicyclobacillus acidoterrestris according to Item 7, wherein the test sample is a food or drink, a cosmetic, a hair care cosmetic, a dental care product, or a raw material thereof.
  • an oligonucleotide having at least 90% identity to the nucleotide sequence shown in SEQ ID NO: 1 and at least 90% identity to the nucleotide sequence shown in SEQ ID NO: 2 A step of performing PCR using a primer set that also has oligonucleotide power having a base sequence,
  • (2 "′) a step of hybridizing the obtained PCR product with a probe having a nucleotide sequence showing at least 90% identity to the nucleotide sequence shown in SEQ ID NO: 5, which also has an oligonucleotide power;
  • a method for specifically detecting a bacterium belonging to the genus Alicyclobacillus having vanillin assimilation comprising:
  • Item 13 The method for specific detection of a bacterium belonging to the genus Alicyclobacillus having vanillin assimilation according to Item 12, which comprises:
  • Item 13 The method for specific detection of a bacterium belonging to the genus Ali cyclobacillus having vanillin utilization, according to Item 12, wherein the probe is immobilized on a solid phase.
  • Item 15 The method for specifically detecting a bacterium belonging to the genus Alicyclobadllus capable of assimilating vanillin according to Item 12, which comprises a step of immobilizing the hybridized product on a solid phase before the detection step (3 "'). .
  • Item 13 The method for specifically detecting bacteria of the genus Alicyclobacillus having vanillin assimilation according to Item 12, wherein the test sample is food and drink, cosmetics, hair care cosmetics, dental care products, or raw materials thereof.
  • An oligonucleotide having a nucleotide sequence showing at least 90% identity to the nucleotide sequence shown in SEQ ID NO: 1 an oligonucleotide having a nucleotide sequence showing at least 90% identity to the nucleotide sequence shown in SEQ ID NO: 2, SEQ ID NO:
  • An oligonucleotide having the base sequence shown in SEQ ID NO: 1 an oligonucleotide having the base sequence shown in SEQ ID NO: 2, an oligonucleotide having the base sequence shown in SEQ ID NO: 3, and a base sequence shown in SEQ ID NO: 4
  • at least 90% identity to the nucleotide sequence shown in SEQ ID NO: 4 A primer set having a reverse primer consisting of an oligonucleotide having the nucleotide sequence of
  • a primer set for amplifying a DNA sequence specific to a bacterium belonging to the genus Alicyclobacillus according to item 19, which is either! Or (b) below:
  • a primer set comprising a forward primer having an oligonucleotide having the nucleotide sequence of SEQ ID NO: 3 and a reverse primer comprising an oligonucleotide having the nucleotide sequence of SEQ ID NO: 4;
  • Probes for marking DNA sequences specific to Alicyclobacillus bacteria either (c ') or (d') below:
  • a reagent kit for specifically detecting bacteria of the genus Alicyclobadllus comprising at least one probe selected from the group consisting of (c ′) and (d ′) described in 0.
  • An oligonucleotide having the base sequence shown in SEQ ID NO: 1, an oligonucleotide having the base sequence shown in SEQ ID NO: 2, an oligonucleotide having the base sequence shown in SEQ ID NO: 3, and a base sequence shown in SEQ ID NO: 4 Use of at least one oligonucleotide selected from the group consisting of oligonucleotides having as a primer in PCR for amplifying a DNA sequence specific to a bacterium belonging to the genus Alicyclobadllus.
  • primer sets (a) or (b) Use of any of the following primer sets (a) or (b) in PCR to amplify DNA sequences specific to Alicyclobacillus bacteria:
  • a primer set comprising a forward primer having an oligonucleotide having the nucleotide sequence of SEQ ID NO: 3 and a reverse primer comprising an oligonucleotide having the nucleotide sequence of SEQ ID NO: 4;
  • Oligonucleotide primer having the nucleotide sequence shown in SEQ ID NO: 1
  • a primer set having an oligonucleotide primer having the nucleotide sequence represented by SEQ ID NO: 1 and a reverse primer having an oligonucleotide primer having the nucleotide sequence represented by SEQ ID NO: 2, and a nucleotide sequence represented by SEQ ID NO: 6 Use of a probe consisting of an oligonucleotide having the following for specific detection of a bacterium belonging to the genus Alicyclobacillus capable of assimilating vanillin.
  • the present invention provides a method for specifically detecting a bacterium belonging to the genus Alicyclobacillus (hereinafter, also simply referred to as a bacterium of the genus Alicyclobacillus) in a test sample.
  • the method of the present invention basically comprises: (l) a step for amplifying a DNA sequence uniquely owned by a bacterium belonging to the genus Alicy clobacillus using PCR (Polymerase Chain Reaction) (PCR step); A step of hybridizing the DNA sequence (PCR product) unique to the genus Alicyclobacillus bacterium amplified by the PCR step with the probe (a hybridization step), and (3) a step obtained by the hybridization step. (Detection step) for detecting the hybridized product (PCR product-probe).
  • the PCR step is basically performed by repeatedly performing at least three steps of (I) denaturation, (II) annealing, and (III) extension.
  • a primer consisting of an oligonucleotide having a nucleotide sequence having at least 90% identity to the nucleotide sequence shown in SEQ ID NO: 1 and a primer comprising SEQ ID NO:
  • a primer consisting of an oligonucleotide having a nucleotide sequence showing at least 90% identity to the nucleotide sequence shown in 2 or an oligonucleotide having a nucleotide sequence showing at least 90% identity to the nucleotide sequence shown in SEQ ID NO: 3
  • an oligonucleotide having a nucleotide sequence showing at least 90% identity to the nucleotide sequence shown in SEQ ID NO: 4 A primer consisting of an oligonucleotide having the nucle
  • These primers may be oligonucleotides whose 5 'end is labeled.
  • a 5'-end labeled oligonucleotide is used as a primer, a PCR product can be obtained in a manner in which the 5 'end is labeled.
  • the label at the 5 'end is not limited, but includes, for example, digoxigenin (DIG), biotin, avidin, streptavidin, enzymes (such as horseradish peroxidase (HRP), alkaline phosphatase (AP)), and radioisotopes.
  • fluorescent dyes e.g., CyDye, Furuo receptacle in isothiocyanate Xia sulfonate (FITC), tetra methylol Honoré rhodamine isothiocyanate Xia sulfonate (TRIT C ) Etc.
  • FITC Furuo receptacle in isothiocyanate Xia sulfonate
  • T C tetra methylol Honoré rhodamine isothiocyanate Xia sulfonate
  • they are digoxigenin and biotin.
  • the most preferred primer set includes a primer set comprising a forward primer and a reverse primer, as shown in the following (A) or (B).
  • the forward primer and the reverse primer may be oligonucleotides each having a labeled 5 ′ end, as described above.
  • Each of these primers can be prepared by a conventional method (for example, the formamide method (Beaucage and Carruthers, Tetra. Letts. 22: 1859-1862, 1981)) or the triester method (Matteucci and Caruthers J. Amer. Chem. So, 103, 3185, 1981). )).
  • a conventional method for example, the formamide method (Beaucage and Carruthers, Tetra. Letts. 22: 1859-1862, 1981)
  • the triester method Miucci and Caruthers J. Amer. Chem. So, 103, 3185, 1981.
  • automatic It can also be synthesized using a synthesizer, or a synthetic product can be obtained from a contractor.
  • test sample to be subjected to PCR is not particularly limited as long as it can contain bacteria of the genus Alicyclobacillus.
  • “may include” does not need to actually include Alicyclobacillus genus bacteria as long as it may contain Alicyclobacillus genus bacteria.
  • test samples are not particularly limited, but are products whose commercial value and safety are impaired by containing Alicyclobacillus bacteria such as food and drink, cosmetics, hair care cosmetics, dental care products, and raw materials thereof. Particularly preferred are foods and drinks.
  • the powerful test sample Before the powerful test sample is subjected to the PCR step, it may be subjected to operations such as DNA extraction, lysis, incubation, stationary culture, shaking culture, concentration, dilution, and homogenization as necessary. May be performed.
  • (I) denaturation is a step of dissociating double-stranded DNA into single-stranded DNA. Although it is not limited as long as it is carried out, it can be usually carried out by treating at 92 to 96 ° C, preferably around 94 ° C for about 0.5 to 1 minute, preferably about 0.5 minute.
  • the subsequent (II) annealing is a reaction for forming a double strand by binding an oligonucleotide having a complementary base sequence to the single-stranded DNA dissociated by denaturation. Such annealing (II) is performed in the presence of the aforementioned primer set, most preferably the forward primer and the reverse primer of the primer set of the above (A) or (B).
  • the annealing step (II) can be carried out usually by treating at 50 to 68 ° C, preferably around 60 ° C for about 0.5 to 1 minute, preferably about 0.5 minute.
  • the primer is complementary to the single-stranded DNA. It binds to a basic base sequence site.
  • the subsequent (III) extension step is a step of extending the base sequence from the primer bound to the single-stranded DNA as a starting point to the single-stranded DNA in a ⁇ shape.
  • the brute force step is performed in the presence of DNA polymerase and four bases (doxynucleoside triphosphate (dNTP mixture): dATP, dCTP, dGTP and dTTP).
  • dNTP mixture doxynucleoside triphosphate
  • the (III) elongation step can be carried out usually by treating at 70 to 74 ° C, preferably around 72 ° C, for 0.5 to 2 minutes, preferably for about 1 minute.
  • the PCR step including the above steps (I) to (: III) is at least a type III DNA.
  • a reaction solution containing the test sample containing the obtained DNA, PCR buffer, primer set, dNTP mixture, and DNA polymerase raise and lower the temperature (for example, around 94 ° C ⁇ around 55 ° C ⁇ around 72 ° C ⁇ 94 (Around ° C), and so on, thus causing a chain reaction of DNA synthesis.
  • the DNA contained in the test sample is used as a template and the forward primer and the reverse primer are used. It becomes possible to amplify DNA having a specific base sequence sandwiched between them.
  • the number of PCR cycles is not particularly limited, but is usually 20 to 40, preferably 25 to 35.
  • DNA polymerase is a power capable of widely using a DNA polymerase generally used in PCR.
  • a commercially available thermostable polymerase such as Taq (manufactured by Promega), KOD (manufactured by TOYOBO) ), Pfo (manufactured by Promega), Vent (manufactured by NEB) and the like can be preferably used.
  • an oligonucleotide having a 5'-end labeled as described above may be used as a primer.
  • the dNTP mixture mixture of deoxynucleoside triphosphates used is dATP (5, -deoxyadenosine 5'-triphosphate), dCTP (5, -deoxycytidine 5'-triphosphate), dGTP ( DIG obtained by adding dexoxigenin (DIG) or biotin (Biotin) to a mixture of d-TPG (5, -deoxyguanosine 5'-triphosphate) and dTTP (5, -deoxythymidine 5'-triphosphate) -Those containing dUTP labeled with an arbitrary labeling agent, such as 11-dUTP (5, -deoxyperidine 5'-triphosphate) and Biotin-16-dUTP, can also be used.
  • an arbitrary labeling agent such as 11-dUTP (5, -deoxyperidine 5'
  • a dNTP mixture a mixture of at least one of dATP, dCTP, dGTP and dTTP labeled with any labeling agent such as a radioisotope (RI), a chemiluminescent agent, or a fluorescent dye may be used.
  • RI radioisotope
  • chemiluminescent agent a chemiluminescent agent
  • the PCR buffer can be appropriately prepared depending on the type of the DNA polymerase to be used and the like. For convenience, a commercially available product can also be used.
  • reaction temperature reaction temperature, reaction time, reaction cycle, etc.
  • Tm value of each primer and the like. Can be appropriately set and adjusted.
  • the temperature control of the PCR reaction is simply performed by using a commercially available thermal cycler (for example, iCy cler Thermal Cycler (manufactured by Bio-Rad Laboratory), TaKaRa PCR Thermal Cycler (manufactured by TaKa Ra) or the like.
  • a commercially available thermal cycler for example, iCy cler Thermal Cycler (manufactured by Bio-Rad Laboratory), TaKaRa PCR Thermal Cycler (manufactured by TaKa Ra) or the like.
  • the hybridization step is a step of hybridizing the DNA sequence (PCR product) amplified by the PCR step with an oligonucleotide probe (hybridization probe) that specifically recognizes the DNA sequence. It is.
  • the oligonucleotide used as a probe includes an oligonucleotide having a nucleotide sequence showing at least 90% identity to the nucleotide sequence shown in SEQ ID NO: 5, and an oligonucleotide having at least 90% nucleotide sequence shown in SEQ ID NO: 6.
  • Most preferred probes include those consisting of oligonucleotides shown in (C) or (D) below:
  • probes may be labeled with an appropriate labeling agent.
  • labeling agent digoxigenin (DIG), biotin, avidin, streptavidin, enzymes (horseradish peroxidase (HRP), alkaline phosphatase (AP), etc.), radioisotopes (AP), etc.
  • fluorescent dyes e.g., CyDye, Furuorese in isothiocyanate Xia sulfonate (FITC), tetra methylol Honoré rhodamine isothiocyanate Xia sulfonate (TRITC), etc.
  • fluorescent dyes e.g., CyDye, Furuorese in isothiocyanate Xia sulfonate (FITC), tetra methylol Honoré rhodamine isothiocyanate Xia sulfonate (TRITC), etc.
  • fluorescent dyes e.g., CyDye, Furuorese in isothiocyanate Xia sulfonate (FITC), tetra methylol Honoré rhodamine isothiocyanate Xia sulfonate (TRITC), etc.
  • fluorescent dyes e.g., CyDye, Furuores
  • the probe is preferably used in a specific combination with the aforementioned primer set.
  • any of the above probes may be any of the above-mentioned primer sets, most preferably the above-mentioned (A) or (B) primer set. And can be suitably used for hybridization with a PCR product obtained in a PCR step using
  • bacteria other than the genus Alicyclobacillus include Escherichia coli, Bacillus subtilis, Staphylococcus aureus, Salmonella and the like.
  • A1 icyclobacillus acidoterrestris can be named S.
  • A. acido terrestris has a property of assimilating vanillin and has a tendency to favor acidity under high temperature conditions.
  • acidoterrestris is a fungus that can generate odors based on its ability to utilize vanillin, so it is important to use fragrances such as cosmetics, hair care products, dental care products, and fragrances that are not limited to food and drink. Contamination of the A. acidoterrestris is also a problem in products.
  • an oligonucleotide having a nucleotide sequence having at least 90% identity to the nucleotide sequence shown in SEQ ID NO: 1 and an oligonucleotide having SEQ ID NO: 2 A primer set comprising an oligonucleotide having a nucleotide sequence showing at least 90% identity to the nucleotide sequence shown in SEQ ID NO: 1, more preferably having an oligonucleotide having the nucleotide sequence shown in SEQ ID NO: 1 and the nucleotide sequence shown in SEQ ID NO: 2
  • a probe consisting of an oligonucleotide having the nucleotide sequence shown in SEQ ID NO: 6 is more preferable. It is preferable to use a probe consisting of a peptide, most preferably a probe of the above (D), and an oligonucleotide having a nucleotide sequence showing at least 90% identity to the nucleotide sequence shown in SEQ ID NO: 3.
  • the primer set comprising an oligonucleotide having the above-mentioned (B) at least 90% of the nucleotide sequence represented by SEQ ID NO: 5 or 6
  • a primer set comprising an oligonucleotide having a nucleotide sequence showing at least 90% identity to the nucleotide sequence shown in SEQ ID NO: 2, more preferably having an oligonucleotide having the nucleotide sequence shown in SEQ ID NO: 1 and the nucleotide sequence shown in SEQ ID NO: 2 It shows at least 90% identity with the base sequence shown in SEQ ID NO: 5 to a PCR product obtained in a PCR step using a primer set that also has the ability to generate oligonucleotides, most preferably the
  • the detection step is a step of detecting a hybridized product (PCR product-probe) obtained by the above hybridization step.
  • a hybridized product PCR product-probe
  • the detection is performed using the PCR product (labeled PCR).
  • a labeled hybridized product (PCR product-labeled probe) is obtained by using the label of the product (probe) as an index and (2) using a labeled probe as a hybridization probe in the hybridization step. In this case, the label of the probe can be used as an index.
  • Jigokishi genin As the labeling agents used for the detection, Jigokishi genin (DIG), radioactive isotopes (32 P, 131 I, 35 S, 45 Ca, 3 H, 14 C , etc.), fluorescent dyes (e.g., Cy Dye It is preferable to use fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), or the like.
  • FITC fluorescein isothiocyanate
  • TRITC tetramethylrhodamine isothiocyanate
  • the detection of a strong labeling agent can be performed by a conventional method depending on the labeling agent to be used.
  • DIG digoxigenin
  • an anti-digoxigenin antibody obtained by labeling the labeled hybridized product with an enzyme such as alkaline phosphatase (AP) or peroxidase (POD) is used. And then react with the substrate of the enzyme (Nitroblue tetrazolium chloride and 5-blomo-4-chloro-3-indolyl phosphate in the case of AP: TMB or 2,2'-azinobis (3-E in the case of POD).
  • ABTS butylbenzothiazoline-6-sulfonic acid
  • the immobilization step of immobilizing the probe on an arbitrary solid phase or (3) prior to the detection step, It may also have a fixing step of fixing the hybridization product obtained in the hybridization step to an arbitrary solid phase.
  • the immobilization of the probe or the hybridized product on the solid phase is not limited, but is preferable.
  • the probe was prepared using a probe labeled with biotin, a hybridization product prepared using a PCR product labeled with biotin (biotin-labeled PCR product-probe), or a probe labeled with biotin.
  • B The reaction of an ibridiz product (PCR product-biotin-labeled probe) with a solid phase to which avidin (or streptavidin) has been previously bound to allow the binding of biotin to avidin (or streptavidin). Can be immobilized on a solid phase.
  • the PCR product to be hybridized to the probe is preferably labeled with a labeling agent suitably used for the above-described detection.
  • the side of the hybridized product that is not labeled with biotin ie, the PCR product is labeled with biotin, the probe is labeled with biotin, or the probe is labeled with biotin! R product
  • a labeling agent suitably used for the aforementioned detection.
  • PCR ELISA is a method for detecting the presence or absence of a product amplified by PCR by ELISA.
  • the PCR product and the probe are labeled in advance with different labeling agents.
  • the probe and the PCR product are hybridized to form a hybridized product, and the hybridized product is immobilized on a solid phase via a labeling agent of either the PCR product or the probe.
  • the immobilized substances immobilized on the solid phase are removed, and then the labeling agent of the hybridization product (another labeling agent not used for immobilization) is detected with an arbitrary detection substance (for example, an antibody).
  • an arbitrary detection substance for example, an antibody.
  • the solid phase used in the immobilization method is not particularly limited, and petri dishes, plates (including microplates), strips, beads, and the like used in the art can be widely used! Can be
  • the present invention also provides a primer (primer set) and a probe used in a method for specifically detecting the bacterium belonging to the genus Alicyclobacillus, particularly A. acidoterrestris.
  • primers (primer sets) and probes are as described above.
  • the present invention provides a reagent kit suitably used for easily carrying out a method for specifically detecting Alicyclobacillus bacteria, particularly A. acidoterrestris, of the present invention.
  • the reagent kit includes at least the above primer (primer set) and a probe as constituent components of the kit.
  • DNTP mixture which may contain NA polymerase, DIG-11-dUTP or Biotin-16-dUTP, sterile water, solid phase (microplate etc.), etc. may contain one kind or a combination of two or more kinds. .
  • PCR was performed using a set of forward primer and reverse primer of No. 1 to No. 4 shown in Table 1 above.
  • DIG digoxigenin
  • a commercially available kit PCR ELISA (DIG-Labeling) (Roche) equipped with a reagent containing dATP, dCTP, dGTP, dTTP and DIG-11-dUTP was used.
  • DIG-11-dUTP is incorporated into the amplification product, and a DIG-labeled amplification product is obtained.
  • a reaction solution for PCR was prepared to have the following composition.
  • Taq polymerase (5 Units) (TaKaRa) 0.05 ⁇ I
  • the DIG-labeled PCR product was denatured into a single strand, mixed with a probe whose 3 'end was labeled with biotin, and shaken at 50 ° C for 90 minutes.
  • the reaction solution was caloried onto a streptavidin solid-phased microplate (Roche) and shaken at 50 ° C for 90 minutes. Washing was performed 5 times with the washing solution included in the kit.
  • an anti-DIG antibody (Roche) labeled with peroxidase (POD) (Roche) was added, followed by shaking at 37 ° C for 30 minutes. After that, washing was performed 5 times with the washing solution included in the kit.
  • a DNA solution was prepared according to the same procedure as in Example 1 using four types of Itoda bacteria belonging to Alicyclobacillus Jill, A. acidoterrestris, A. acidocaldarius, A. hesperidensis and A. hesperidum.
  • PCR ELISA was performed in the same manner as in Example 1.
  • the primer and the probe the forward primer, reverse primer and probe shown in No. 1 to No. 4 in Table 1 were used.
  • No. 24 primer and probe A. acidoterrestris was detected specifically and distinguished from all other Alicyclobacillus bacteria. Therefore, the combination of No. 24 is useful as a combination for specifically detecting only A. acidoterrestris.
  • the yarn binding of No. 1 primer and probe is a low level of A. hesperidum and A. hesperidum, which have the ability to assimilate vanillin at a lower level as compared with A. acidoterrestris and can cause contamination.
  • A. hesperidensis a closely related species of A., is also detected. Therefore, the combination of No. 1 is useful for comprehensively detecting a kind of Alicyclobacillus bacterium which has vanillin assimilation properties and can cause off-flavor.
  • the present invention provides a rapid, simple, and low-cost method for specific detection of Alicyclobacillus bacteria. provide.
  • a primer for amplifying a DNA sequence specific to a bacterium of the genus Alicyclobacillus and a probe for specifically labeling the DNA sequence are used, a primer based on a DNA sequence specific to a bacterium of the genus Alicyclobacillus is used.
  • the bacterium can be specifically detected separately from bacteria of other genera. Therefore, according to the present invention, bacteria of the genus Alicyclobacillus can be detected without being affected by the activity of the bacteria.
  • the method of the present invention is a method utilizing PCR, even if the amount of Alicyclobacillus bacteria in the test sample is small, it can be detected, and the detection sensitivity is high.
  • bacteria of the genus Alicyclobacillus having vanillin assimilation may be comprehensively detected, or only Alicyclobacillus acidoterrestris may be specifically (or selectively) detected. can do.
  • the method of the present invention does not require time-consuming operations such as cultivation of bacteria or DNA sequencing, so that Alicyclobacillus bacteria can be detected quickly.
  • the method of the present invention does not require expensive reagents or equipment for sequencing, and can be performed at low cost.

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Abstract

L’intention est de fournir une méthode rapide, pratique et bon marché de détection d’une bactérie appartenant au genre Alicyclobacillus à la différence d’autres bactéries. Nommément, une méthode de détection d’une bactérie appartenant au genre Alicyclobacillus qui comprend : (1) l’étape où un échantillon test est soumis à une PCR au moyen d’un jeu de primer comprenant un oligonucléotide dont la séquence de base est représentée par SEQ ID NO:1 où la séquence de base d’un oligonucléotide est représentée par SEQ ID NO:2, ou un jeu de primer comprenant un oligonucléotide dont la séquence de base est représentée par SEQ ID NO:3 où la séquence de base d’un oligonucléotide est représentée par SEQ ID NO:4; (2) l’étape où le produit de la PCR ainsi obtenu est hybridée avec une sonde comprenant un oligonucléotide dont la séquence de base est représentée par SEQ ID NO:5 ou 6 ; et (3) l’étape de détection du produit de l’hybridation obtenu ci-dessus.
PCT/JP2005/010259 2004-06-03 2005-06-03 Méthode de détection spécifique de la bactérie appartenant au genre alicyclobacillus Ceased WO2005118804A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102854308A (zh) * 2012-09-20 2013-01-02 陕西师范大学 用于果汁中耐热菌双抗体夹心elisa检测试剂盒的制备方法及应用

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