WO2016129249A1 - Method for detecting lactic acid bacteria, lactic acid bacteria detection kit, and lactic acid bacteria detection instrument - Google Patents

Method for detecting lactic acid bacteria, lactic acid bacteria detection kit, and lactic acid bacteria detection instrument Download PDF

Info

Publication number
WO2016129249A1
WO2016129249A1 PCT/JP2016/000546 JP2016000546W WO2016129249A1 WO 2016129249 A1 WO2016129249 A1 WO 2016129249A1 JP 2016000546 W JP2016000546 W JP 2016000546W WO 2016129249 A1 WO2016129249 A1 WO 2016129249A1
Authority
WO
WIPO (PCT)
Prior art keywords
lactobacillus
seq
lactic acid
acid bacteria
base sequence
Prior art date
Application number
PCT/JP2016/000546
Other languages
French (fr)
Japanese (ja)
Inventor
隆明 山崎
淳憲 一色
美穂 大河内
睦子 並木
京子 右田
宮下　隆
Original Assignee
東洋製罐グループホールディングス株式会社
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 東洋製罐グループホールディングス株式会社 filed Critical 東洋製罐グループホールディングス株式会社
Publication of WO2016129249A1 publication Critical patent/WO2016129249A1/en

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • 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

Definitions

  • the present invention relates to a technology for detecting lactic acid bacteria, and in particular, to a method for detecting lactic acid bacteria, a lactic acid bacteria detection kit, and a lactic acid bacteria detection instrument that simultaneously detect Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus buchnerii, and Lactobacillus fructivorans. .
  • a bacterial solution is prepared for each bacterial species, inoculated to several tens of types of carbon sources for each bacterial species, and grown on the basis of the color change of the carbon source after being grown for 2 days.
  • a separation culture step is required, and a certain number of bacteria is required.
  • a specific region of a gene possessed by a specific lactic acid bacterium is amplified by a PCR (Polymerase Chain Reaction) method or the like, and the amplified product is detected by an electrophoresis method or a DNA chip.
  • a method for determining whether or not a specific lactic acid bacterium is present in a food or drink has been proposed.
  • Lactobacillus buchneri and Lactobacillus plantarum can be detected by electrophoresis.
  • Lactobacillus brevis and Lactobacillus buchneri can be detected by electrophoresis and a DNA chip.
  • Lactobacillus fructivorans can be detected by electrophoresis or the like.
  • JP 2008-48652 A Japanese Patent No. 4791958 Japanese Patent No. 4446392
  • the present invention can detect Lactobacillus plantarum using the RNA polymerase gene (rpo) as a target gene, and also includes four types of Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus buchneri, and Lactobacillus fructivorans. It is an object to provide a method for detecting lactic acid bacteria, a kit for detecting lactic acid bacteria, and a lactic acid bacteria detection instrument capable of specifically detecting lactic acid bacteria at the same time.
  • rpo RNA polymerase gene
  • the method for detecting lactic acid bacteria of the present invention extracts DNA from cells contained in a sample, and targets the extracted DNA and the RNA polymerase gene of Lactobacillus plantarum.
  • a method for determining the presence or absence of Lactobacillus plantarum in the sample based on the amplification product obtained by performing an amplification reaction by PCR using the primer set consisting of the base sequence shown in SEQ ID NO: 1 and SEQ ID NO: 2 is there.
  • the lactic acid bacteria detection kit of the present invention comprises a primer set consisting of the nucleotide sequence shown in SEQ ID NO: 1 and SEQ ID NO: 2 targeting the RNA polymerase gene of Lactobacillus plantarum, Lactobacillus plantarum, Lactobacillus Contains a primer set consisting of the nucleotide sequence shown in SEQ ID NO: 3 and SEQ ID NO: 4 targeting the 16S rRNA gene of Lactobacillus brevis, Lactobacillus buchneri, and Lactobacillus fructivorans It is as composition to do.
  • the lactic acid bacteria detection instrument of the present invention comprises a probe consisting of SEQ ID NOs: 5 to 7 that complementarily bind to an RNA polymerase gene of Lactobacillus plantarum and a base sequence represented by at least one of these complementary sequences; A probe consisting of SEQ ID NOs: 8 and 9 that complementarily bind to the 16S rRNA gene of Lactobacillus brevis and a base sequence represented by at least one of these complementary sequences, and the 16S rRNA gene of Lactobacillus buchnerii that binds complementarily A probe consisting of a base sequence shown in at least one of SEQ ID NOs: 10 to 14 and their complementary sequences; and at least one of SEQ ID NO: 15 and its complementary sequence that binds complementarily to the 16S rRNA gene of Lactobacillus fructivorans Base sequence shown in either Made there a configuration in which the probe was immobilized.
  • Lactobacillus plantarum can be detected using RNA polymerase gene (rpo) as a target gene, and four types of Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus buchneri, and Lactobacillus fructivorans are used. It is possible to provide a lactic acid bacteria detection method, a lactic acid bacteria detection kit, and a lactic acid bacteria detection instrument that can specifically detect lactic acid bacteria simultaneously.
  • rpo RNA polymerase gene
  • the lactic acid bacteria detection method, lactic acid bacteria detection kit, and lactic acid bacteria detection instrument will be described in detail below.
  • Method for detecting lactic acid bacteria extracts DNA from cells contained in a sample, SEQ ID NO: 1 and SEQ ID NO: targeting the extracted DNA and the RNA polymerase gene of Lactobacillus plantarum An amplification reaction by PCR is performed using a primer set consisting of the base sequence shown in 2, and the presence or absence of Lactobacillus plantarum in the sample is determined based on the obtained amplification product.
  • the method for detecting lactic acid bacteria includes the extracted DNA, a primer set targeting the RNA polymerase gene of Lactobacillus plantarum, Lactobacillus plantarum, Lactobacillus brevis, Amplification reaction by PCR using Lactobacillus buchneri and Lactobacillus fructivorans 16S rRNA gene targeting primer sequences consisting of the nucleotide sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4 It is also preferable to determine the presence or absence of Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus buchneri, and Lactobacillus fructivorans in the sample based on the obtained amplification product. .
  • the method for detecting a lactic acid bacterium includes a probe comprising SEQ ID NOs: 5 to 7 that complementarily bind to an RNA polymerase gene of Lactobacillus plantarum and a base sequence represented by at least one of these complementary sequences And a probe comprising a base sequence represented by at least one of SEQ ID NOs: 8 and 9 and their complementary sequences, which complementarily bind to the 16S rRNA gene of Lactobacillus brevis, and complementary to the 16S rRNA gene of Lactobacillus bufuneri SEQ ID NOs: 10 to 14 and the nucleotide sequence represented by at least one of these complementary sequences, and SEQ ID NO: 15 and the complementary sequence thereof complementary to the 16S rRNA gene of Lactobacillus fructivorans
  • the DNA extraction method is not particularly limited, and can be performed, for example, as follows. First, 1 mL of the culture solution is collected and centrifuged at 5000 ⁇ g for 10 minutes. Next, the supernatant is discarded, and a 20 mg / mL lysozyme solution (20 mM Tris-HCl, pH 8.0 / 2 mM EDTA, 1.2% Triton X-100) is added to the resulting precipitate at 37 ° C. Lysis treatment is performed for 30 minutes. Furthermore, a DNA extract can be obtained by performing column purification. And this DNA extract can be used as a sample used in PCR.
  • a gene region (amplification target region) targeted for amplification is amplified by PCR.
  • a primer set consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 1 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 2 as a primer.
  • the Lactobacillus plantarum RNA polymerase gene (rpo) can be amplified.
  • a primer set consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 3 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 4 as a primer, Lactobacillus plantarum, Lactobacillus brevis , Lactobacillus buchnerii and Lactobacillus fructivorans 16S rRNA genes can be amplified.
  • a nucleic acid synthesis substrate for example, a nucleic acid synthesis substrate, a primer set, a nucleic acid synthase, a sample DNA, a buffer solution, and a solution containing water as the remaining components can be suitably used.
  • a label is added to the amplification product by PCR.
  • the labeling method is not particularly limited, but a fluorescent label can be preferably used.
  • an amplification product in which only the ends are labeled can be generated using a fluorescently labeled primer.
  • an amplification product containing a label therein can also be generated using a fluorescently labeled nucleic acid synthesis substrate.
  • Cy5 or Cy3 can be suitably used as the fluorescent labeling component.
  • a label it is also possible to use a label other than fluorescence, such as dicoxigenin, biotin, and a radioisotope.
  • a general thermal cycler etc. can be used as an apparatus which performs PCR reaction.
  • the reaction conditions for PCR can be performed, for example, as follows. (A) 94 ° C. 2 minutes, (b) 94 ° C. (DNA denaturation step) 30 seconds, (c) 60 ° C. (annealing step) 30 seconds, (d) 72 ° C. (DNA synthesis step) 60 seconds ((b) to (D) 35 cycles), (e) 72 ° C. 3 minutes
  • electrophoresis As a method for determining the presence or absence of lactic acid bacteria, for example, electrophoresis can be performed.
  • the electrophoresis can be performed by a general method such as agarose gel electrophoresis, acrylamide electrophoresis, or microchip electrophoresis.
  • electrophoresis the presence or absence of lactic acid bacteria is determined based on the size of the amplification product.
  • SEQ ID NOs: 5 to 7 that bind complementarily to the RNA polymerase gene (rpo) of L. plantarum and their complementary sequences
  • a probe comprising at least one of the nucleotide sequences shown in the above, a base sequence shown in at least one of SEQ ID NOs: 8 and 9 that binds complementarily to the 16S rRNA gene of L. brevis, and their complementary sequences
  • buchneri and a base sequence shown in at least one of these complementary sequences,
  • 16S rRNA gene (16S rRNA) of L. fructivorans It is preferable to use a DNA chip on which a probe consisting of a base sequence represented by at least one of SEQ ID NO: 15 and a complementary sequence thereof is immobilized.
  • These probes each have a specific sequence for each lactic acid bacterium, and can hybridize only with the amplification product of the corresponding gene region, so that each lactic acid bacterium to be tested can be specifically detected simultaneously. It is possible to do.
  • the DNA chip can be produced by an existing general method using the above probe.
  • the probe when an affixed type DNA chip is produced, the probe can be immobilized on a glass substrate by a DNA spotter and a spot corresponding to each probe can be formed.
  • a synthetic DNA chip when a synthetic DNA chip is produced, it can be produced by synthesizing a single-stranded oligo DNA having the above sequence on a glass substrate by a photolithography technique.
  • the substrate is not limited to glass, and a plastic substrate, a silicon wafer, or the like can also be used.
  • the shape of the substrate is not limited to a flat plate shape, and may be various three-dimensional shapes, and a substrate having a functional group introduced so that a chemical reaction can be performed on the surface can be used. .
  • the amplification product is dropped onto the DNA chip thus obtained, and the amplification product is hybridized to the probe immobilized on the DNA chip.
  • the kind of lactic acid bacteria to be examined can be specified by detecting the label of the hybridized amplification product.
  • the detection of the label can be performed using a general label detection device such as a fluorescence scanning device.
  • the fluorescence intensity of the fluorescent label in the amplification product is measured using BIOSHOT (R) manufactured by Toyo Kohan Co., Ltd. Can be done.
  • an S / N ratio value (Signal to Noise ratio, (median fluorescence intensity value ⁇ background value) ⁇ background value) as a measurement result. This is because it is possible to accurately determine whether the measurement result is positive or negative based on the S / N ratio value. Generally, when the S / N ratio value is 3 or more, it is determined to be positive. it can.
  • primers and probes in the present embodiment are not limited to the above base sequences, and those in which one or several bases are deleted, substituted or added in each base sequence can be used.
  • what consists of a nucleic acid fragment which can be hybridized on stringent conditions with respect to the nucleic acid fragment which consists of a base sequence complementary to each base sequence can also be used.
  • the stringent condition refers to a condition where a specific hybrid is formed and a non-specific hybrid is not formed.
  • a DNA having high homology (homology is 90% or more, preferably 95% or more) to the DNA comprising the sequences represented by SEQ ID NOs: 1 to 15 is determined from the sequences represented by SEQ ID NOs: 1 to 15.
  • the conditions for hybridizing with DNA consisting of a base sequence complementary to the DNA to be obtained can be mentioned. Usually, it means a case where hybridization occurs at a temperature about 5 ° C. to about 30 ° C., preferably about 10 ° C. to about 25 ° C. lower than the melting temperature (Tm) of the complete hybrid.
  • Tm melting temperature
  • the kit for detecting lactic acid bacteria of the present embodiment comprises a primer set consisting of the nucleotide sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2 targeting the RNA polymerase gene of Lactobacillus plantarum, Lactobacillus plantarum, Lactobacillus brevis And a primer set consisting of the nucleotide sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4 targeting the 16S rRNA gene of Lactobacillus buchneri and Lactobacillus fructivorans.
  • Such a lactic acid bacteria detection kit suitably amplifies the Lactobacillus plantarum RNA polymerase gene and the above-mentioned four types of lactic acid bacteria 16S rRNA genes by using them as a mixture of primers for preparing a PCR reaction solution. It is possible.
  • the lactic acid bacteria detection instrument of this embodiment comprises a probe comprising SEQ ID NOs: 5 to 7 that complementarily bind to an RNA polymerase gene of Lactobacillus plantarum, and a base sequence represented by at least one of these complementary sequences, It binds complementarily to the 16S rRNA gene of Lactobacillus bufuneri and a probe consisting of SEQ ID NOs: 8 and 9 that complementarily bind to the 16S rRNA gene of Bacillus brevis and at least one of these complementary sequences.
  • a probe comprising a base sequence represented by SEQ ID NOs: 10 to 14 and / or a complementary sequence thereof; and at least one of SEQ ID NO: 15 and a complementary sequence thereof which binds complementarily to the 16S rRNA gene of Lactobacillus fructivorans Consisting of the base sequence shown in And wherein the immobilized and lobes.
  • Such a lactic acid bacteria detection instrument is a Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus buchneri, by dropping an amplification product obtained by PCR using a PCR reaction solution to which the lactic acid bacteria detection kit is added. And Lactobacillus fructivorans can be specifically detected simultaneously.
  • the four types of lactic acid bacteria can be identified with excellent specificity, and false positive determination can be reduced. Moreover, it is excellent in detection sensitivity compared with the conventional method, and it is possible to suitably detect lactic acid bacteria even when a low concentration contaminated sample is used as a sample. Furthermore, according to the present embodiment, since the presence or absence of four types of lactic acid bacteria to be examined can be determined simultaneously with a single operation, the amount of reagents such as PCR can be reduced compared to the case of determining only one bacterial species. It is also possible to save work.
  • Test 1 Using the detection method of lactic acid bacteria and the lactic acid bacteria detection kit of this embodiment, a target gene of a specific lactic acid bacterium was amplified by the PCR method, and a verification test was performed to confirm whether lactic acid bacteria can be detected based on the amplification product. .
  • lactic acid bacteria a total of five samples were prepared for the following four types. These test strains were distributed from the National Institute for Product Evaluation.
  • Lactobacillus plantarum NBRC106468 (2) Lactobacillus plantarum NBRC15891 (3) Lactobacillus brevis NBRC107147 (4) Lactobacillus buchneri NBRC107764 (5) Lactobacillus fructivorans NBRC14747
  • Extraction of DNA from the cells contained in the sample was performed as follows. First, 1 mL of the culture solution was collected and centrifuged at 5000 ⁇ g for 10 minutes. Next, the supernatant is discarded, and a 20 mg / mL lysozyme solution (20 mM Tris-HCl, pH 8.0 / 2 mM EDTA, 1.2% Triton X-100) is added to the resulting precipitate at 37 ° C. Lysis treatment was performed for 30 minutes. Furthermore, a DNA extract was obtained by performing column purification using DNeasy Blood & Tissue Kit (manufactured by Qiagen). This DNA extract was used as a sample for PCR.
  • a primer set a primer set consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 1 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 2 was used.
  • the amplification target region is the RNA polymerase gene (rpo) of Lactobacillus plantarum, and the estimated amplification product length is 266 bp.
  • a primer set a primer set consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 3 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 4 was used.
  • the region to be amplified is the 16S rRNA gene (16S rRNA) of Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus buchnerii, and Lactobacillus fructivorans, and the estimated amplification product length is about 500 bp each.
  • a PCR reaction solution having the following composition was used. Primers were synthesized from Life Technology Japan. The others are manufactured by Takara Bio Inc. ⁇ Buffer 10 ⁇ Ex Taq buffer (20 mM Mg 2+ plus) 2.0 ⁇ l ⁇ Nucleic acid synthesis substrate dNTP Mixture (dATP, dCTP, dGTP, dTTP 2.5 mM each) 1.6 ⁇ l ⁇ 16SrRNA detection forward primer (10 ⁇ M) 0.5 ⁇ l ⁇ 16S rRNA detection reverse primer (10 ⁇ M) 0.5 ⁇ l -Rpo detection forward primer (10 ⁇ M) 0.2 ⁇ l ⁇ Rpo detection reverse primer (10 ⁇ M) 0.3 ⁇ l ⁇ Nucleic acid synthase EX Taq (5U / ⁇ l) 0.1 ⁇ l ⁇ Sample DNA 1.0 ⁇ l ⁇ Sterilized water 13.8 ⁇ l (Total 20 ⁇ l)
  • Electrophoresis was performed using MultiNA® (manufactured by Shimadzu Corporation). The result is shown in FIG. In the figure, for any of the test strains (1) to (5), a band was shown around 500 bp indicating the amplified product of the 16S rRNA gene (16S rRNA). For the test strains (1) and (2), a band was shown near 266 bp indicating the amplification product of the RNA polymerase gene (rpo).
  • Lactobacillus plantarum can be detected based on the amplified product of 16S rRNA gene and RNA polymerase gene. It was also confirmed that Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus buchneri, and Lactobacillus fructivorans could be detected based on the 16S rRNA gene amplification product.
  • Test 2 Using the lactic acid bacteria detection method, lactic acid bacteria detection kit, and lactic acid bacteria detection instrument of the present embodiment, a target gene of a specific lactic acid bacteria is amplified by the PCR method, and the amplified product is hybridized with a probe immobilized on the lactic acid bacteria detection instrument. Thus, a verification test was performed to confirm whether or not each of these lactic acid bacteria could be specifically detected simultaneously.
  • Test 1 a total of five samples were prepared for four types of lactic acid bacteria, and DNA was extracted.
  • the same primer set and PCR reaction solution as in Test 1 were used, and the gene was amplified by the PCR method in the same manner as in Test 1.
  • PCR a labeled amplification product was generated using a fluorescently labeled nucleic acid synthesis substrate.
  • a lactic acid bacterium detection instrument binds complementarily to the 16S rRNA gene of Lactobacillus brevis and a probe consisting of the nucleotide sequence shown in SEQ ID NOs: 5 to 7 that binds complementarily to the RNA polymerase gene of Lactobacillus plantarum.
  • a probe consisting of the base sequence shown in SEQ ID NOs: 8 and 9 a probe consisting of the base sequence shown in SEQ ID NOs: 10 to 14 that binds complementarily to the 16S rRNA gene of Lactobacillus buchnerii, and a 16S rRNA gene of Lactobacillus fructivorans
  • a DNA chip was prepared by immobilizing a probe consisting of the base sequence shown in SEQ ID NO: 15 that binds in a complementary manner.
  • a PCR amplification product was dropped onto the DNA chip, and the label of the amplification product hybridized with the probe was detected. Specifically, it was performed as follows. First, a PCR amplification product was mixed with a buffer solution (3 ⁇ SSC citrate-saline) added with 0.3% SDS (sodium dodecyl sulfate) and dropped onto a DNA chip. Next, the DNA chip was allowed to stand at 45 ° C. for 1 hour, and PCR products that were not hybridized using the buffer solution were washed away from the DNA chip.
  • a buffer solution 3 ⁇ SSC citrate-saline
  • SDS sodium dodecyl sulfate
  • the fluorescence intensity was measured by applying the DNA chip to a label detection device (BIOSHOT, manufactured by Toyo Kohan Co., Ltd.).
  • the fluorescence intensity was excited by laser light to cause the labeling component (Cy5) to emit light, and the amount of the light was replaced with an electric signal to quantify it to obtain the fluorescence intensity.
  • the S / N ratio value was calculated based on this fluorescence intensity. The result is shown in FIG. As shown in the figure, for any of the test strains (1) to (5), a positive reaction (S / N ratio value ⁇ 3) only when those lactic acid bacteria are detection target bacteria of each probe. It was confirmed that no false positive reaction occurred.
  • the PCR amplification product also includes an amplification product having a base sequence complementary to the amplification product that hybridizes with the probe having the base sequence shown in SEQ ID NOs: 5 to 15. Accordingly, a probe comprising a base sequence complementary to the base sequences shown in SEQ ID NOs: 5 to 15 can hybridize with an amplification product having such a complementary base sequence. Therefore, even when a probe having a base sequence complementary to the base sequences shown in SEQ ID NOs: 5 to 15 is immobilized on a DNA chip, the above four types of lactic acid bacteria can be specifically detected simultaneously. Is possible.
  • the present invention is not limited to the above embodiments and examples, and various modifications can be made within the scope of the present invention.
  • it is possible to appropriately change such as adding a probe other than the above to the lactic acid bacteria detection instrument according to the present embodiment and immobilizing it, or adding other components to the PCR reaction solution.
  • the present invention can be suitably used for specific detection of specific lactic acid bacteria simultaneously in food inspection, environmental inspection, and the like.

Abstract

In the present invention, four types of lactic acid bacteria (Lactobacillus plantarum, brevis, buchneri, and fructivorans) are each simultaneously detected specifically. An amplification reaction by PCR is conducted using DNA extracted from cells contained in a sample, a primer set shown by SEQ ID NO: 1 and SEQ ID NO: 2 that targets an RNA polymerase gene of Lactobacillus plantarum, and a primer set comprising base sequences shown by SEQ ID NO: 3 and SEQ ID NO: 4 that targets the 16SrRNA gene of Lactobacillus plantarum, brevis, buchneri, and fructivorans. The presence of Lactobacillus plantarum, brevis, buchneri, and fructivorans is determined simultaneously based on the amplified product obtained.

Description

乳酸菌の検出方法、乳酸菌検出キット、及び乳酸菌検出器具Lactic acid bacteria detection method, lactic acid bacteria detection kit, and lactic acid bacteria detection instrument
　本発明は、乳酸菌の検出技術に関し、特にラクトバチルス・プランタラム、ラクトバチルス・ブレビス、ラクトバチルス・ブフネリ、及びラクトバチルス・フルクチボランスを同時に検出する乳酸菌の検出方法、乳酸菌検出キット、及び乳酸菌検出器具に関する。 The present invention relates to a technology for detecting lactic acid bacteria, and in particular, to a method for detecting lactic acid bacteria, a lactic acid bacteria detection kit, and a lactic acid bacteria detection instrument that simultaneously detect Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus buchnerii, and Lactobacillus fructivorans. .
　従来から、酸性飲料や酸性食品に対して、特定の菌類が悪影響を及ぼすことが問題となっている。例えば、カビや酵母、特定の乳酸菌等が酸性飲料や酸性食品に混入すると、腐敗したり、風味を劣化させたりすることが知られている。一般に、カビや酵母と比べ乳酸菌は耐熱性が高く、低温殺菌で残存する事例があるため、酸性飲料や酸性食品における特定の乳酸菌の有無の検査は重要であった。 Conventionally, it has been a problem that certain fungi adversely affect acidic beverages and acidic foods. For example, it is known that when mold, yeast, specific lactic acid bacteria, or the like are mixed in an acidic beverage or an acidic food, it rots or degrades the flavor. In general, lactic acid bacteria have higher heat resistance than mold and yeast, and there are cases in which they remain after pasteurization, so it was important to check for the presence of specific lactic acid bacteria in acidic beverages and acidic foods.
　酸性飲料や酸性食品における特定の乳酸菌の有無の検査では、一般に、乳酸菌の菌種を判定するために、菌種毎に分離培養を行って、生理学的性質にもとづき１種類毎に判定が行われていた。
　具体的には、例えば、菌種毎に菌液を調製して、それぞれの菌種毎に数十種類の炭素源へ接種し、２日間生育させた後の炭素源の色調変化にもとづいて菌種の判定を行う方法がある。この方法では、分離培養工程が必要であり、また一定以上の菌数が必要であった。このため、特定の乳酸菌の有無の判定に最低１週間を要し、また作業員の手間も非常に掛かるものであった。このような判定に時間がかかることは、迅速性が求められる飲食品の検査においては問題であった。 In the inspection for the presence or absence of specific lactic acid bacteria in acidic beverages and acidic foods, in general, in order to determine the bacterial species of lactic acid bacteria, separate culture is performed for each bacterial species, and determination is performed for each type based on physiological properties. It was.
Specifically, for example, a bacterial solution is prepared for each bacterial species, inoculated to several tens of types of carbon sources for each bacterial species, and grown on the basis of the color change of the carbon source after being grown for 2 days. There is a method for determining the species. In this method, a separation culture step is required, and a certain number of bacteria is required. For this reason, it took at least one week to determine the presence or absence of a specific lactic acid bacterium, and it took much labor for the workers. It takes a long time to make such a determination, which has been a problem in the inspection of food and drink that requires quickness.
　そこで、このような問題を解消するために、特定の乳酸菌が有する遺伝子の一定領域をＰＣＲ（Polymerase　Chain　Reaction）法などによって増幅し、その増幅産物を電気泳動法やＤＮＡチップ等で検出することにより、飲食品に特定の乳酸菌が存在しているか否かの判定を行う手法が提案されている。
　具体的には、特許文献１に記載の手法によれば、ラクトバチルス・ブフネリ（Lactobacillus　buchneri）とラクトバチルス・プランタラム（Lactobacillus　plantarum）を電気泳動法によって検出できるとされている。また、特許文献２に記載の手法によれば、ラクトバチルス・ブレビス（Lactobacillus　brevis）とラクトバチルス・ブフネリを電気泳動法とＤＮＡチップによって検出できるとされている。さらに、特許文献３に記載の手法によれば、ラクトバチルス・フルクチボランス（Lactobacillus　fructivorans）を電気泳動法などによって検出できるとされている。 Therefore, in order to solve such problems, a specific region of a gene possessed by a specific lactic acid bacterium is amplified by a PCR (Polymerase Chain Reaction) method or the like, and the amplified product is detected by an electrophoresis method or a DNA chip. A method for determining whether or not a specific lactic acid bacterium is present in a food or drink has been proposed.
Specifically, according to the technique described in Patent Literature 1, it is said that Lactobacillus buchneri and Lactobacillus plantarum can be detected by electrophoresis. Further, according to the technique described in Patent Document 2, Lactobacillus brevis and Lactobacillus buchneri can be detected by electrophoresis and a DNA chip. Furthermore, according to the technique described in Patent Document 3, Lactobacillus fructivorans can be detected by electrophoresis or the like.
特開２００８－４８６５２号公報JP 2008-48652 A 特許第４７９１９５８号公報Japanese Patent No. 4791958 特許第４４６３５９２号公報Japanese Patent No. 4446392
　しかしながら、これら従来の手法では、ラクトバチルス・プランタラム、ラクトバチルス・ブレビス、ラクトバチルス・ブフネリ、及びラクトバチルス・フルクチボランスの４種類の乳酸菌を同時に検出することはできなかった。
　そこで、本発明者らは、特にラクトバチルス・プランタラムのＲＮＡポリメラーゼ遺伝子（ｒｐｏ）を標的遺伝子として選択することによって当該乳酸菌を高い精度で検出可能にすると共に、上記４種類の乳酸菌を同時にそれぞれ特異的に検出することに成功し、本発明を完成させた。このようにＲＮＡポリメラーゼ遺伝子（ｒｐｏ）を標的遺伝子として増幅して乳酸菌を検出することはこれまで見られず、また上記４種類の乳酸菌を同時にそれぞれ特異的に検出可能な本発明におけるプローブもこれまで見られなかった。 However, these conventional methods cannot simultaneously detect four types of lactic acid bacteria, Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus buchneri, and Lactobacillus fructivorans.
Accordingly, the present inventors have made it possible to detect the lactic acid bacteria with high accuracy by selecting the RNA polymerase gene (rpo) of Lactobacillus plantarum as a target gene, and at the same time specifically identify the four types of lactic acid bacteria. The present invention has been completed. Thus far, detection of lactic acid bacteria by amplifying the RNA polymerase gene (rpo) as a target gene has not been seen so far, and the probes in the present invention capable of specifically detecting each of the four types of lactic acid bacteria at the same time have also been found so far. I couldn't see it.
　すなわち、本発明は、ラクトバチルス・プランタラムをＲＮＡポリメラーゼ遺伝子（ｒｐｏ）を標的遺伝子として検出できると共に、ラクトバチルス・プランタラム、ラクトバチルス・ブレビス、ラクトバチルス・ブフネリ、及びラクトバチルス・フルクチボランスの４種類の乳酸菌を同時にそれぞれ特異的に検出可能な乳酸菌の検出方法、乳酸菌検出キット、及び乳酸菌検出器具の提供を目的とする。 That is, the present invention can detect Lactobacillus plantarum using the RNA polymerase gene (rpo) as a target gene, and also includes four types of Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus buchneri, and Lactobacillus fructivorans. It is an object to provide a method for detecting lactic acid bacteria, a kit for detecting lactic acid bacteria, and a lactic acid bacteria detection instrument capable of specifically detecting lactic acid bacteria at the same time.
　上記目的を達成するために、本発明の乳酸菌の検出方法は、試料に含まれる細胞からＤＮＡを抽出し、抽出されたＤＮＡと、ラクトバチルス・プランタラム（Lactobacillus　plantarum）のＲＮＡポリメラーゼ遺伝子を標的とする配列番号１及び配列番号２に示される塩基配列からなるプライマーセットとを用いてＰＣＲによる増幅反応を行い、得られた増幅産物にもとづき前記試料におけるラクトバチルス・プランタラムの有無を判定する方法としてある。 To achieve the above object, the method for detecting lactic acid bacteria of the present invention extracts DNA from cells contained in a sample, and targets the extracted DNA and the RNA polymerase gene of Lactobacillus plantarum. As a method for determining the presence or absence of Lactobacillus plantarum in the sample based on the amplification product obtained by performing an amplification reaction by PCR using the primer set consisting of the base sequence shown in SEQ ID NO: 1 and SEQ ID NO: 2 is there.
　また、本発明の乳酸菌検出キットは、ラクトバチルス・プランタラムのＲＮＡポリメラーゼ遺伝子を標的とする配列番号１及び配列番号２に示される塩基配列からなるプライマーセットと、ラクトバチルス・プランタラム、ラクトバチルス・ブレビス（Lactobacillus　brevis）、ラクトバチルス・ブフネリ（Lactobacillus　buchneri）、及びラクトバチルス・フルクチボランス（Lactobacillus　fructivorans）の16SrRNA遺伝子を標的とする配列番号３及び配列番号４に示される塩基配列からなるプライマーセットとを含有する構成としてある。 The lactic acid bacteria detection kit of the present invention comprises a primer set consisting of the nucleotide sequence shown in SEQ ID NO: 1 and SEQ ID NO: 2 targeting the RNA polymerase gene of Lactobacillus plantarum, Lactobacillus plantarum, Lactobacillus Contains a primer set consisting of the nucleotide sequence shown in SEQ ID NO: 3 and SEQ ID NO: 4 targeting the 16S rRNA gene of Lactobacillus brevis, Lactobacillus buchneri, and Lactobacillus fructivorans It is as composition to do.
　さらに、本発明の乳酸菌検出器具は、ラクトバチルス・プランタラムのＲＮＡポリメラーゼ遺伝子に相補的に結合する配列番号５～７及びこれらの相補的配列の少なくともいずれかに示される塩基配列からなるプローブと、ラクトバチルス・ブレビスの１６ＳｒＲＮＡ遺伝子に相補的に結合する配列番号８，９及びこれらの相補的配列の少なくともいずれかに示される塩基配列からなるプローブと、ラクトバチルス・ブフネリの１６ＳｒＲＮＡ遺伝子に相補的に結合する配列番号１０～１４及びこれらの相補的配列の少なくともいずれかに示される塩基配列からなるプローブと、ラクトバチルス・フルクチボランスの１６ＳｒＲＮＡ遺伝子に相補的に結合する配列番号１５及びこれの相補的配列の少なくともいずれかに示される塩基配列からなるプローブとを固定化した構成としてある。 Furthermore, the lactic acid bacteria detection instrument of the present invention comprises a probe consisting of SEQ ID NOs: 5 to 7 that complementarily bind to an RNA polymerase gene of Lactobacillus plantarum and a base sequence represented by at least one of these complementary sequences; A probe consisting of SEQ ID NOs: 8 and 9 that complementarily bind to the 16S rRNA gene of Lactobacillus brevis and a base sequence represented by at least one of these complementary sequences, and the 16S rRNA gene of Lactobacillus buchnerii that binds complementarily A probe consisting of a base sequence shown in at least one of SEQ ID NOs: 10 to 14 and their complementary sequences; and at least one of SEQ ID NO: 15 and its complementary sequence that binds complementarily to the 16S rRNA gene of Lactobacillus fructivorans Base sequence shown in either Made there a configuration in which the probe was immobilized.
　本発明によれば、ラクトバチルス・プランタラムをＲＮＡポリメラーゼ遺伝子（ｒｐｏ）を標的遺伝子として検出できると共に、ラクトバチルス・プランタラム、ラクトバチルス・ブレビス、ラクトバチルス・ブフネリ、及びラクトバチルス・フルクチボランスの４種類の乳酸菌を同時にそれぞれ特異的に検出可能な乳酸菌の検出方法、乳酸菌検出キット、及び乳酸菌検出器具の提供が可能となる。 According to the present invention, Lactobacillus plantarum can be detected using RNA polymerase gene (rpo) as a target gene, and four types of Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus buchneri, and Lactobacillus fructivorans are used. It is possible to provide a lactic acid bacteria detection method, a lactic acid bacteria detection kit, and a lactic acid bacteria detection instrument that can specifically detect lactic acid bacteria simultaneously.
本発明の実施形態に係る乳酸菌の検出方法、及び乳酸菌検出キットにおいて用いられるプライマーと、本発明の実施形態に係る乳酸菌の検出方法、及び乳酸菌検出器具において用いられるプローブの塩基配列を示す図である。It is a figure which shows the base sequence of the probe used in the detection method of lactic acid bacteria which concerns on embodiment of this invention, the primer used in a lactic acid bacteria detection kit, the detection method of lactic acid bacteria which concerns on embodiment of this invention, and a lactic acid bacteria detection instrument. . 本発明の実施形態に係る乳酸菌の検出方法、及び乳酸菌検出キットを用いてＰＣＲを行うことにより得られた増幅産物の電気泳動の結果を示す図である。It is a figure which shows the result of the electrophoresis of the amplification product obtained by performing PCR using the detection method of lactic acid bacteria which concerns on embodiment of this invention, and a lactic acid bacteria detection kit. 本発明の実施形態に係る乳酸菌の検出方法、乳酸菌検出キット、及び乳酸菌検出器具を用いた乳酸菌の検出結果（蛍光強度のＳ／Ｎ比値）を示す図である。It is a figure which shows the detection result (S / N ratio value of fluorescence intensity) of the lactic acid bacteria using the detection method of lactic acid bacteria which concerns on embodiment of this invention, a lactic acid bacteria detection kit, and a lactic acid bacteria detection instrument.
　以下、本発明の実施形態に係る乳酸菌の検出方法、乳酸菌検出キット、及び乳酸菌検出器具について、詳細に説明する。
［乳酸菌の検出方法］
　本実施形態の乳酸菌の検出方法は、試料に含まれる細胞からＤＮＡを抽出し、抽出されたＤＮＡと、ラクトバチルス・プランタラム（Lactobacillus　plantarum）のＲＮＡポリメラーゼ遺伝子を標的とする配列番号１及び配列番号２に示される塩基配列からなるプライマーセットとを用いてＰＣＲによる増幅反応を行い、得られた増幅産物にもとづき試料におけるラクトバチルス・プランタラムの有無を判定することを特徴とする。 The lactic acid bacteria detection method, lactic acid bacteria detection kit, and lactic acid bacteria detection instrument according to embodiments of the present invention will be described in detail below.
[Method for detecting lactic acid bacteria]
The method for detecting lactic acid bacteria of the present embodiment extracts DNA from cells contained in a sample, SEQ ID NO: 1 and SEQ ID NO: targeting the extracted DNA and the RNA polymerase gene of Lactobacillus plantarum An amplification reaction by PCR is performed using a primer set consisting of the base sequence shown in 2, and the presence or absence of Lactobacillus plantarum in the sample is determined based on the obtained amplification product.
　また、本実施形態の乳酸菌の検出方法を、抽出されたＤＮＡと、ラクトバチルス・プランタラムのＲＮＡポリメラーゼ遺伝子を標的とするプライマーセットと、ラクトバチルス・プランタラム、ラクトバチルス・ブレビス（Lactobacillus　brevis）、ラクトバチルス・ブフネリ（Lactobacillus　buchneri）、及びラクトバチルス・フルクチボランス（Lactobacillus　fructivorans）の１６ＳｒＲＮＡ遺伝子を標的とする配列番号３及び配列番号４に示される塩基配列からなるプライマーセットとを用いて、ＰＣＲによる増幅反応を行い、得られた増幅産物にもとづき試料におけるラクトバチルス・プランタラム、ラクトバチルス・ブレビス、ラクトバチルス・ブフネリ、及びラクトバチルス・フルクチボランスの有無を同時に判定するものとすることも好ましい。 In addition, the method for detecting lactic acid bacteria according to the present embodiment includes the extracted DNA, a primer set targeting the RNA polymerase gene of Lactobacillus plantarum, Lactobacillus plantarum, Lactobacillus brevis, Amplification reaction by PCR using Lactobacillus buchneri and Lactobacillus fructivorans 16S rRNA gene targeting primer sequences consisting of the nucleotide sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4 It is also preferable to determine the presence or absence of Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus buchneri, and Lactobacillus fructivorans in the sample based on the obtained amplification product. .
　さらに、本実施形態の乳酸菌の検出方法を、ラクトバチルス・プランタラムのＲＮＡポリメラーゼ遺伝子に相補的に結合する配列番号５～７及びこれらの相補的配列の少なくともいずれかに示される塩基配列からなるプローブと、ラクトバチルス・ブレビスの１６ＳｒＲＮＡ遺伝子に相補的に結合する配列番号８，９及びこれらの相補的配列の少なくともいずれかに示される塩基配列からなるプローブと、ラクトバチルス・ブフネリの１６ＳｒＲＮＡ遺伝子に相補的に結合する配列番号１０～１４及びこれらの相補的配列の少なくともいずれかに示される塩基配列からなるプローブと、ラクトバチルス・フルクチボランスの１６ＳｒＲＮＡ遺伝子に相補的に結合する配列番号１５及びこれの相補的配列の少なくともいずれかに示される塩基配列からなるプローブとを固定化した乳酸菌検出器具に、標識された増幅産物を接触させ、プローブと相補的に結合した増幅産物の標識を検出するものとすることも好ましい。 Furthermore, the method for detecting a lactic acid bacterium according to the present embodiment includes a probe comprising SEQ ID NOs: 5 to 7 that complementarily bind to an RNA polymerase gene of Lactobacillus plantarum and a base sequence represented by at least one of these complementary sequences And a probe comprising a base sequence represented by at least one of SEQ ID NOs: 8 and 9 and their complementary sequences, which complementarily bind to the 16S rRNA gene of Lactobacillus brevis, and complementary to the 16S rRNA gene of Lactobacillus bufuneri SEQ ID NOs: 10 to 14 and the nucleotide sequence represented by at least one of these complementary sequences, and SEQ ID NO: 15 and the complementary sequence thereof complementary to the 16S rRNA gene of Lactobacillus fructivorans A base represented by at least one of And a probe consisting column immobilized lactic acid bacteria detection instrument, contacting the labeled amplified products, it is also preferable to one that detects the labeled probe complementary bound amplification product.
（ＤＮＡの抽出）
　ＤＮＡの抽出方法は、特に限定されないが、例えば次のように行うことができる。
　まず、培養液を1ｍＬずつ回収し、５０００×ｇで、１０分間の遠心分離を行う。次に、上清を廃棄し、得られた沈殿に、２０ｍｇ／ｍＬ濃度のリゾチーム溶液（２０ｍＭ　Ｔｒｉｓ－ＨＣｌ，ｐＨ８．０／２ｍＭ　ＥＤＴＡ,１．２％ＴｒｉｔｏｎＸ－１００）を加えて、３７℃で３０分間溶菌処理を行う。さらに、カラム精製を行うことにより、ＤＮＡ抽出液を得ることができる。そして、このＤＮＡ抽出液をＰＣＲにおいて使用する試料として用いることができる。 (DNA extraction)
The DNA extraction method is not particularly limited, and can be performed, for example, as follows.
First, 1 mL of the culture solution is collected and centrifuged at 5000 × g for 10 minutes. Next, the supernatant is discarded, and a 20 mg / mL lysozyme solution (20 mM Tris-HCl, pH 8.0 / 2 mM EDTA, 1.2% Triton X-100) is added to the resulting precipitate at 37 ° C. Lysis treatment is performed for 30 minutes. Furthermore, a DNA extract can be obtained by performing column purification. And this DNA extract can be used as a sample used in PCR.
（ＰＣＲによる増幅反応）
　抽出したＤＮＡとプライマーを用いて、増幅の標的とする遺伝子領域（増幅対象領域）をＰＣＲ法により増幅させる。
　具体的には、図１に示すように、プライマーとして、配列番号１に示される塩基配列からなるフォワードプライマーと配列番号２に示される塩基配列からなるリバースプライマーとからなるプライマーセットを用いることによって、ラクトバチルス・プランタラムのＲＮＡポリメラーゼ遺伝子（ｒｐｏ）を増幅させることができる。
　また、プライマーとして、配列番号３に示される塩基配列からなるフォワードプライマーと配列番号４に示される塩基配列からなるリバースプライマーとからなるプライマーセットを用いることによって、ラクトバチルス・プランタラム、ラクトバチルス・ブレビス、ラクトバチルス・ブフネリ、及びラクトバチルス・フルクチボランスの１６ＳｒＲＮＡ遺伝子を増幅させることができる。 (Amplification reaction by PCR)
Using the extracted DNA and primers, a gene region (amplification target region) targeted for amplification is amplified by PCR.
Specifically, as shown in FIG. 1, by using a primer set consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 1 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 2 as a primer, The Lactobacillus plantarum RNA polymerase gene (rpo) can be amplified.
Further, by using a primer set consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 3 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 4 as a primer, Lactobacillus plantarum, Lactobacillus brevis , Lactobacillus buchnerii and Lactobacillus fructivorans 16S rRNA genes can be amplified.
　ＰＣＲ反応液としては、例えば、核酸合成基質、プライマーセット、核酸合成酵素、試料のＤＮＡ、緩衝液、及び残りの成分として水を含むものを好適に使用することができる。
　また、ＰＣＲによる増幅産物の有無をＤＮＡチップで特定する場合、ＰＣＲによって増幅産物に標識が付加される。標識の方法は特に限定されないが、蛍光標識を好適に用いることができる。 As the PCR reaction solution, for example, a nucleic acid synthesis substrate, a primer set, a nucleic acid synthase, a sample DNA, a buffer solution, and a solution containing water as the remaining components can be suitably used.
When the presence or absence of an amplification product by PCR is specified by a DNA chip, a label is added to the amplification product by PCR. The labeling method is not particularly limited, but a fluorescent label can be preferably used.
　ＰＣＲで蛍光標識を行う場合、蛍光標識されたプライマーを用いて、末端のみが標識された増幅産物を生成することができる。また、蛍光標識された核酸合成基質を用いて、内部に標識が含まれた増幅産物を生成することもできる。いずれの場合も蛍光標識成分として、Ｃｙ５やＣｙ３などを好適に用いることが可能である。
　さらに、標識として、ジコキシゲニン、ビオチン、放射性同位体などの蛍光以外のその他の方式のものを用いることも可能である。 When fluorescent labeling is performed by PCR, an amplification product in which only the ends are labeled can be generated using a fluorescently labeled primer. In addition, an amplification product containing a label therein can also be generated using a fluorescently labeled nucleic acid synthesis substrate. In any case, Cy5 or Cy3 can be suitably used as the fluorescent labeling component.
Furthermore, as a label, it is also possible to use a label other than fluorescence, such as dicoxigenin, biotin, and a radioisotope.
　また、ＰＣＲ反応を行う装置としては、一般的なサーマルサイクラーなどを用いることができる。
　ＰＣＲの反応条件としては、例えば以下の通りに行うことができる。
（ａ）９４℃　２分、（ｂ）９４℃（ＤＮＡ変性工程）　３０秒、（ｃ）６０℃（アニーリング工程）　３０秒、（ｄ）７２℃（ＤＮＡ合成工程）　６０秒（（ｂ）～（ｄ）を３５サイクル）、（ｅ）７２℃　３分 Moreover, a general thermal cycler etc. can be used as an apparatus which performs PCR reaction.
The reaction conditions for PCR can be performed, for example, as follows.
(A) 94 ° C. 2 minutes, (b) 94 ° C. (DNA denaturation step) 30 seconds, (c) 60 ° C. (annealing step) 30 seconds, (d) 72 ° C. (DNA synthesis step) 60 seconds ((b) to (D) 35 cycles), (e) 72 ° C. 3 minutes
（乳酸菌の有無の判定）
　乳酸菌の有無の判定方法としては、例えば電気泳動法により行うことができる。電気泳動法は、アガロースゲル電気泳動やアクリルアミド電気泳動、マイクロチップ電気泳動等の一般的な方法により行うことができる。電気泳動法では増幅産物の大きさにもとづいて、乳酸菌の有無の判定が行われる。 (Determination of the presence or absence of lactic acid bacteria)
As a method for determining the presence or absence of lactic acid bacteria, for example, electrophoresis can be performed. The electrophoresis can be performed by a general method such as agarose gel electrophoresis, acrylamide electrophoresis, or microchip electrophoresis. In electrophoresis, the presence or absence of lactic acid bacteria is determined based on the size of the amplification product.
　電気泳動法のように増幅産物の大きさにもとづき対象菌の有無を判定する場合、複数の種類の対象菌における標的とする遺伝子領域のサイズがあまり違わないときは、これらの対象菌を一つの系で同時に識別することは困難である。
　したがって、複数の種類の乳酸菌を一つの系で同時にそれぞれ特異的に識別するためには、ＤＮＡチップを用いることが好ましい。 When determining the presence or absence of target bacteria based on the size of the amplification product as in electrophoresis, if the size of the target gene region in multiple types of target bacteria is not so different, It is difficult to identify simultaneously in the system.
Therefore, it is preferable to use a DNA chip in order to specifically identify a plurality of types of lactic acid bacteria simultaneously in one system.
　具体的には、図１に示すように、プローブとして、ラクトバチルス・プランタラム（L.plantarum）のＲＮＡポリメラーゼ遺伝子（ｒｐｏ）に相補的に結合する配列番号５～７及びこれらの相補的配列の少なくともいずれかに示される塩基配列からなるプローブと、ラクトバチルス・ブレビス（L.brevis）の１６ＳｒＲＮＡ遺伝子に相補的に結合する配列番号８，９及びこれらの相補的配列の少なくともいずれかに示される塩基配列からなるプローブと、ラクトバチルス・ブフネリ（L.buchneri）の１６ＳｒＲＮＡ遺伝子に相補的に結合する配列番号１０～１４及びこれらの相補的配列の少なくともいずれかに示される塩基配列からなるプローブと、ラクトバチルス・フルクチボランス（L.fructivorans）の１６ＳｒＲＮＡ遺伝子（１６ＳｒＲＮＡ）に相補的に結合する配列番号１５及びこれの相補的配列の少なくともいずれかに示される塩基配列からなるプローブとを固定化したＤＮＡチップを用いることが好ましい。 Specifically, as shown in FIG. 1, as probes, SEQ ID NOs: 5 to 7 that bind complementarily to the RNA polymerase gene (rpo) of L. plantarum and their complementary sequences A probe comprising at least one of the nucleotide sequences shown in the above, a base sequence shown in at least one of SEQ ID NOs: 8 and 9 that binds complementarily to the 16S rRNA gene of L. brevis, and their complementary sequences A probe consisting of a sequence, a probe consisting of SEQ ID NOs: 10 to 14 that complementarily bind to the 16S rRNA gene of L. buchneri and a base sequence shown in at least one of these complementary sequences, Complementary to 16S rRNA gene (16S rRNA) of L. fructivorans It is preferable to use a DNA chip on which a probe consisting of a base sequence represented by at least one of SEQ ID NO: 15 and a complementary sequence thereof is immobilized.
　これらのプローブは、上記乳酸菌毎にそれぞれ特異的な配列を有しており、かつ対応する遺伝子領域の増幅産物とのみハイブリダイズできるため、検査対象の上記各乳酸菌を同時にそれぞれ特異的に検出可能にすることが可能となっている。 These probes each have a specific sequence for each lactic acid bacterium, and can hybridize only with the amplification product of the corresponding gene region, so that each lactic acid bacterium to be tested can be specifically detected simultaneously. It is possible to do.
　ＤＮＡチップは、上記のプローブを用いて、既存の一般的な方法で製造することができる。
　例えば、貼り付け型のＤＮＡチップを作成する場合は、ＤＮＡスポッターによりプローブをガラス基板上に固定化して、各プローブに対応するスポットを形成することにより作成することができる。また、合成型ＤＮＡチップを作成する場合は、光リソグラフィ技術により、ガラス基板上で上記配列を備えた一本鎖オリゴＤＮＡを合成することにより作成することができる。さらに、基板はガラス製に限定されず、プラスチック基板やシリコンウエハー等を用いることもできる。また、基板の形状は平板状のものに限定されず、様々な立体形状のものとすることもでき、その表面に化学反応が可能となるように官能基を導入したものなどを用いることもできる。 The DNA chip can be produced by an existing general method using the above probe.
For example, when an affixed type DNA chip is produced, the probe can be immobilized on a glass substrate by a DNA spotter and a spot corresponding to each probe can be formed. When a synthetic DNA chip is produced, it can be produced by synthesizing a single-stranded oligo DNA having the above sequence on a glass substrate by a photolithography technique. Furthermore, the substrate is not limited to glass, and a plastic substrate, a silicon wafer, or the like can also be used. Further, the shape of the substrate is not limited to a flat plate shape, and may be various three-dimensional shapes, and a substrate having a functional group introduced so that a chemical reaction can be performed on the surface can be used. .
　このようにして得られたＤＮＡチップに増幅産物を滴下して、増幅産物をＤＮＡチップに固定化されたプローブにハイブリダイズさせる。そして、ハイブリダイズした増幅産物の標識を検出することにより、検査対象の乳酸菌の種類を特定することができる。
　標識の検出は、蛍光スキャニング装置などの一般的な標識検出装置を用いて行うことができ、例えば東洋鋼鈑株式会社のＢＩＯＳＨＯＴ（Ｒ）を用いて、増幅産物における蛍光標識の蛍光強度を測定することにより行うことができる。 The amplification product is dropped onto the DNA chip thus obtained, and the amplification product is hybridized to the probe immobilized on the DNA chip. And the kind of lactic acid bacteria to be examined can be specified by detecting the label of the hybridized amplification product.
The detection of the label can be performed using a general label detection device such as a fluorescence scanning device. For example, the fluorescence intensity of the fluorescent label in the amplification product is measured using BIOSHOT (R) manufactured by Toyo Kohan Co., Ltd. Can be done.
　また、測定結果として、蛍光強度値の他、Ｓ／Ｎ比値（Signal　to　Noise　ratio，（メディアン蛍光強度値－バックグラウンド値）÷バックグラウンド値）を算出することも好ましい。Ｓ／Ｎ比値にもとづいて、測定結果が陽性であるか陰性であるかを精度高く判定することができるためであり、一般にＳ／Ｎ比値が３以上の場合、陽性と判定することができる。 In addition to the fluorescence intensity value, it is also preferable to calculate an S / N ratio value (Signal to Noise ratio, (median fluorescence intensity value−background value) ÷ background value) as a measurement result. This is because it is possible to accurately determine whether the measurement result is positive or negative based on the S / N ratio value. Generally, when the S / N ratio value is 3 or more, it is determined to be positive. it can.
　また、本実施形態におけるプライマー及びプローブは、上記の塩基配列に限定されるものではなく、それぞれの塩基配列において１又は数個の塩基が欠損、置換又は付加されたものを用いることができる。また、それぞれの塩基配列に対して相補的な塩基配列からなる核酸断片に対してストリンジェントな条件下でハイブリダイズできる核酸断片からなるものを用いることもできる。 In addition, the primers and probes in the present embodiment are not limited to the above base sequences, and those in which one or several bases are deleted, substituted or added in each base sequence can be used. Moreover, what consists of a nucleic acid fragment which can be hybridized on stringent conditions with respect to the nucleic acid fragment which consists of a base sequence complementary to each base sequence can also be used.
　なお、ストリンジェントな条件とは、特異的なハイブリッドが形成され、非特異的なハイブリッドが形成されない条件をいう。例えば、配列番号１～１５で表される配列からなるＤＮＡに対し高い相同性（相同性が９０％以上、好ましくは９５％以上）を有するＤＮＡが、配列番号１～１５で表される配列からなるＤＮＡと相補的な塩基配列からなるＤＮＡと、ハイブリダイズする条件が挙げられる。通常、完全ハイブリッドの溶解温度（Ｔｍ）より約５℃～約３０℃、好ましくは約１０℃～約２５℃低い温度でハイブリダイゼーションが起こる場合をいう。ストリンジェントな条件については、Ｊ．Ｓａｍｂｒｏｏｋら，Ｍｏｌｅｃｕｌａｒ　Ｃｌｏｎｉｎｇ，Ａ　Ｌａｂｏｒａｔｏｒｙ　Ｍａｎｎｕａｌ，Ｓｅｃｏｎｄ　Ｅｄｉｔｉｏｎ，Ｃｏｌｄ　Ｓｐｒｉｎｇ Ｈａｒｂｏｒ　Ｌａｂｏｒａｔｏｒｙ　Ｐｒｅｓｓ（１９８９）、特に11.45節「Ｃｏｎｄｉｔｉｏｎｓ　ｆｏｒ　Ｈｙｂｒｉｄｉｚａｔｉｏｎ　ｏｆ　Ｏｌｉｇｏｎｕｃｌｅｏｔｉｄｅ　Ｐｒｏｂｅｓ」に記載されている条件等を使用することができる。 The stringent condition refers to a condition where a specific hybrid is formed and a non-specific hybrid is not formed. For example, a DNA having high homology (homology is 90% or more, preferably 95% or more) to the DNA comprising the sequences represented by SEQ ID NOs: 1 to 15 is determined from the sequences represented by SEQ ID NOs: 1 to 15. The conditions for hybridizing with DNA consisting of a base sequence complementary to the DNA to be obtained can be mentioned. Usually, it means a case where hybridization occurs at a temperature about 5 ° C. to about 30 ° C., preferably about 10 ° C. to about 25 ° C. lower than the melting temperature (Tm) of the complete hybrid. For stringent conditions, see J.M. Sambrook et al., Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989), etc. can be used, especially in Section 11.45 “Conditions for HybridOboliprozation”.
［乳酸菌検出キット］
　本実施形態の乳酸菌検出キットは、ラクトバチルス・プランタラムのＲＮＡポリメラーゼ遺伝子を標的とする配列番号１及び配列番号２に示される塩基配列からなるプライマーセットと、ラクトバチルス・プランタラム、ラクトバチルス・ブレビス、ラクトバチルス・ブフネリ、及びラクトバチルス・フルクチボランスの１６ＳｒＲＮＡ遺伝子を標的とする配列番号３及び配列番号４に示される塩基配列からなるプライマーセットとを含有することを特徴とする。 [Lactic acid bacteria detection kit]
The kit for detecting lactic acid bacteria of the present embodiment comprises a primer set consisting of the nucleotide sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2 targeting the RNA polymerase gene of Lactobacillus plantarum, Lactobacillus plantarum, Lactobacillus brevis And a primer set consisting of the nucleotide sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4 targeting the 16S rRNA gene of Lactobacillus buchneri and Lactobacillus fructivorans.
　このような乳酸菌検出キットは、ＰＣＲ反応液を作成するためのプライマーの混合液として用いることにより、ラクトバチルス・プランタラムのＲＮＡポリメラーゼ遺伝子と、上記４種類の乳酸菌の１６ＳｒＲＮＡ遺伝子をそれぞれ好適に増幅させることが可能である。 Such a lactic acid bacteria detection kit suitably amplifies the Lactobacillus plantarum RNA polymerase gene and the above-mentioned four types of lactic acid bacteria 16S rRNA genes by using them as a mixture of primers for preparing a PCR reaction solution. It is possible.
［乳酸菌検出器具］
　本実施形態の乳酸菌検出器具は、ラクトバチルス・プランタラムのＲＮＡポリメラーゼ遺伝子に相補的に結合する配列番号５～７及びこれらの相補的配列の少なくともいずれかに示される塩基配列からなるプローブと、ラクトバチルス・ブレビスの１６ＳｒＲＮＡ遺伝子に相補的に結合する配列番号８，９及びこれらの相補的配列の少なくともいずれかに示される塩基配列からなるプローブと、ラクトバチルス・ブフネリの１６ＳｒＲＮＡ遺伝子に相補的に結合する配列番号１０～１４及びこれらの相補的配列の少なくともいずれかに示される塩基配列からなるプローブと、ラクトバチルス・フルクチボランスの１６ＳｒＲＮＡ遺伝子に相補的に結合する配列番号１５及びこれの相補的配列の少なくともいずれかに示される塩基配列からなるプローブとを固定化したことを特徴とする。 [Lactic acid bacteria detection instrument]
The lactic acid bacteria detection instrument of this embodiment comprises a probe comprising SEQ ID NOs: 5 to 7 that complementarily bind to an RNA polymerase gene of Lactobacillus plantarum, and a base sequence represented by at least one of these complementary sequences, It binds complementarily to the 16S rRNA gene of Lactobacillus bufuneri and a probe consisting of SEQ ID NOs: 8 and 9 that complementarily bind to the 16S rRNA gene of Bacillus brevis and at least one of these complementary sequences. A probe comprising a base sequence represented by SEQ ID NOs: 10 to 14 and / or a complementary sequence thereof; and at least one of SEQ ID NO: 15 and a complementary sequence thereof which binds complementarily to the 16S rRNA gene of Lactobacillus fructivorans Consisting of the base sequence shown in And wherein the immobilized and lobes.
　このような乳酸菌検出器具は、上記乳酸菌検出キットを加えたＰＣＲ反応液を用いてＰＣＲにより得られた増幅産物を滴下することにより、ラクトバチルス・プランタラム、ラクトバチルス・ブレビス、ラクトバチルス・ブフネリ、及びラクトバチルス・フルクチボランスを同時にそれぞれ特異的に検出することが可能である。 Such a lactic acid bacteria detection instrument is a Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus buchneri, by dropping an amplification product obtained by PCR using a PCR reaction solution to which the lactic acid bacteria detection kit is added. And Lactobacillus fructivorans can be specifically detected simultaneously.
　以上説明した通り、本実施形態の乳酸菌の検出方法、乳酸菌検出キット、及び乳酸菌検出器具によれば、上記４種類の乳酸菌を優れた特異性で識別でき、偽陽性判定を低減させることができる。
　また、従来法に比較して検出感度に優れ、低濃度の汚染サンプルを試料とする場合でも乳酸菌の検出を好適に行うことが可能である。
　さらに、本実施形態によれば、一回の操作で検査対象の４種類の乳酸菌の有無を同時に判定できるため、１菌種のみを判定する場合に比較して、ＰＣＲなどの試薬量を削減でき、作業を省力化することも可能である。 As described above, according to the detection method of lactic acid bacteria, the lactic acid bacteria detection kit, and the lactic acid bacteria detection instrument of the present embodiment, the four types of lactic acid bacteria can be identified with excellent specificity, and false positive determination can be reduced.
Moreover, it is excellent in detection sensitivity compared with the conventional method, and it is possible to suitably detect lactic acid bacteria even when a low concentration contaminated sample is used as a sample.
Furthermore, according to the present embodiment, since the presence or absence of four types of lactic acid bacteria to be examined can be determined simultaneously with a single operation, the amount of reagents such as PCR can be reduced compared to the case of determining only one bacterial species. It is also possible to save work.
（試験１）
　本実施形態の乳酸菌の検出方法、及び乳酸菌検出キットを用いて、特定の乳酸菌の標的遺伝子をＰＣＲ法により増幅し、その増幅産物にもとづき乳酸菌を検出できるかを確認するための検証試験を行った。
　乳酸菌としては、以下の４種類について、合計５つの試料を準備した。これらの供試菌株は、独立行政法人製品評価基盤機構から分譲されたものである。 (Test 1)
Using the detection method of lactic acid bacteria and the lactic acid bacteria detection kit of this embodiment, a target gene of a specific lactic acid bacterium was amplified by the PCR method, and a verification test was performed to confirm whether lactic acid bacteria can be detected based on the amplification product. .
As lactic acid bacteria, a total of five samples were prepared for the following four types. These test strains were distributed from the National Institute for Product Evaluation.
（１）ラクトバチルス・プランタラム（Lactobacillus　plantarum）NBRC106468
（２）ラクトバチルス・プランタラム（Lactobacillus　plantarum）NBRC15891
（３）ラクトバチルス・ブレビス（Lactobacillus　brevis）NBRC107147
（４）ラクトバチルス・ブフネリ（Lactobacillus　buchneri）NBRC107764
（５）ラクトバチルス・フルクチボランス（Lactobacillus　fructivorans）NBRC14747 (1) Lactobacillus plantarum NBRC106468
(2) Lactobacillus plantarum NBRC15891
(3) Lactobacillus brevis NBRC107147
(4) Lactobacillus buchneri NBRC107764
(5) Lactobacillus fructivorans NBRC14747
　試料に含まれる細胞からのＤＮＡの抽出は、次のように行った。
　まず、培養液を1ｍＬずつ回収し、５０００×ｇで、１０分間の遠心分離を行った。次に、上清を廃棄し、得られた沈殿に、２０ｍｇ／ｍＬ濃度のリゾチーム溶液（２０ｍＭ　Ｔｒｉｓ－ＨＣｌ，ｐＨ８．０／２ｍＭ ＥＤＴＡ,１．２％ＴｒｉｔｏｎＸ－１００）を加えて、３７℃で３０分間溶菌処理を行った。さらに、ＤＮｅａｓｙ　Ｂｌｏｏｄ＆Ｔｉｓｓｕｅ　Ｋｉｔ（株式会社キアゲン製）を用いて、カラム精製を行うことにより、ＤＮＡ抽出液を得た。このＤＮＡ抽出液をＰＣＲにおいて使用する試料とした。 Extraction of DNA from the cells contained in the sample was performed as follows.
First, 1 mL of the culture solution was collected and centrifuged at 5000 × g for 10 minutes. Next, the supernatant is discarded, and a 20 mg / mL lysozyme solution (20 mM Tris-HCl, pH 8.0 / 2 mM EDTA, 1.2% Triton X-100) is added to the resulting precipitate at 37 ° C. Lysis treatment was performed for 30 minutes. Furthermore, a DNA extract was obtained by performing column purification using DNeasy Blood & Tissue Kit (manufactured by Qiagen). This DNA extract was used as a sample for PCR.
　プライマーセットとして、配列番号１に示される塩基配列からなるフォワードプライマーと配列番号２に示される塩基配列からなるリバースプライマーとからなるプライマーセットを用いた。その増幅対象領域は、ラクトバチルス・プランタラムのＲＮＡポリメラーゼ遺伝子（ｒｐｏ）であり、推定増幅産物長は２６６ｂｐである。
　また、プライマーセットとして、配列番号３に示される塩基配列からなるフォワードプライマーと配列番号４に示される塩基配列からなるリバースプライマーとからなるプライマーセットを用いた。その増幅対象領域は、ラクトバチルス・プランタラム、ラクトバチルス・ブレビス、ラクトバチルス・ブフネリ、及びラクトバチルス・フルクチボランスの１６ＳｒＲＮＡ遺伝子（１６ＳｒＲＮＡ）であり、推定増幅産物長はそれぞれ約５００ｂｐである。 As a primer set, a primer set consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 1 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 2 was used. The amplification target region is the RNA polymerase gene (rpo) of Lactobacillus plantarum, and the estimated amplification product length is 266 bp.
As a primer set, a primer set consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 3 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 4 was used. The region to be amplified is the 16S rRNA gene (16S rRNA) of Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus buchnerii, and Lactobacillus fructivorans, and the estimated amplification product length is about 500 bp each.
　ＰＣＲ反応液としては、以下の組成のものを使用した。プライマーはライフテクノロジージャパン株式会社より合成した。それ以外は、タカラバイオ株式会社製である。
・緩衝液　10×Ex　Taq　buffer(20mM　Mg　2＋　plus)　2.0μl
・核酸合成基質　dNTP　Mixture(dATP、dCTP、dGTP、dTTP各2.5mM)　1.6μl
・16SrRNA検出用フォワードプライマー(10μM)　0.5μl
・16SrRNA検出用リバースプライマー　(10μM)　0.5μl
・rpo検出用フォワードプライマー(10μM)　0.2μl
・rpo検出用リバースプライマー　(10μM)　0.3μl
・核酸合成酵素　EX　Taq(5U/μl)　0.1μl
・試料のＤＮＡ　1.0μl
・滅菌水　13.8μl
（全量　20μl） A PCR reaction solution having the following composition was used. Primers were synthesized from Life Technology Japan. The others are manufactured by Takara Bio Inc.
・ Buffer 10 × Ex Taq buffer (20 mM Mg 2+ plus) 2.0 μl
・ Nucleic acid synthesis substrate dNTP Mixture (dATP, dCTP, dGTP, dTTP 2.5 mM each) 1.6 μl
・ 16SrRNA detection forward primer (10μM) 0.5μl
・ 16S rRNA detection reverse primer (10μM) 0.5μl
-Rpo detection forward primer (10 μM) 0.2 μl
・ Rpo detection reverse primer (10μM) 0.3μl
・ Nucleic acid synthase EX Taq (5U / μl) 0.1μl
・ Sample DNA 1.0μl
・ Sterilized water 13.8μl
(Total 20 μl)
　ＰＣＲ法による遺伝子の増幅には、ｅｐグラジエント（エッペンドルフ株式会社製）を使用した。また、ＰＣＲの反応条件は、実施形態で説明したものと同一の次の条件で行った。
（ａ）９４℃　２分
（ｂ）９４℃　３０秒（ＤＮＡ鎖の乖離工程）
（ｃ）６０℃　３０秒（アニーリング工程）
（ｄ）７２℃　６０秒（ＤＮＡ合成工程）
（ｅ）７２℃　３分
（ｂ）～（ｄ）を３５サイクル An ep gradient (manufactured by Eppendorf Co., Ltd.) was used for gene amplification by the PCR method. PCR reaction conditions were the same as those described in the embodiment.
(A) 94 ° C for 2 minutes (b) 94 ° C for 30 seconds (DNA strand dissociation step)
(C) 60 ° C. for 30 seconds (annealing process)
(D) 72 ° C. 60 seconds (DNA synthesis step)
(E) 72 ° C. 3 minutes (b) to (d) 35 cycles
　次に、ＰＣＲ法による増幅産物を、アガロースゲル電気泳動により増幅対象領域ごとに泳動させ、正しい増幅産物が得られているか否かを確認した。電気泳動は、ＭｕｌｔｉＮＡ（Ｒ）(株式会社島津製作所製)を用いて行った。その結果を図２に示す。
　同図において、供試菌株（１）～（５）のいずれについても、１６ＳｒＲＮＡ遺伝子（１６ＳｒＲＮＡ）の増幅産物を示す５００ｂｐ付近にバンドが示された。また、供試菌株（１），（２）については、ＲＮＡポリメラーゼ遺伝子（ｒｐｏ）の増幅産物を示す２６６ｂｐ付近にバンドが示された。 Next, the amplification product by PCR was migrated for each amplification target region by agarose gel electrophoresis, and it was confirmed whether or not the correct amplification product was obtained. Electrophoresis was performed using MultiNA® (manufactured by Shimadzu Corporation). The result is shown in FIG.
In the figure, for any of the test strains (1) to (5), a band was shown around 500 bp indicating the amplified product of the 16S rRNA gene (16S rRNA). For the test strains (1) and (2), a band was shown near 266 bp indicating the amplification product of the RNA polymerase gene (rpo).
　このことから、本実施形態の乳酸菌検出キットによれば、ラクトバチルス・プランタラムを１６ＳｒＲＮＡ遺伝子とＲＮＡポリメラーゼ遺伝子の増幅産物にもとづき検出できることが確認された。また、ラクトバチルス・プランタラム、ラクトバチルス・ブレビス、ラクトバチルス・ブフネリ、及びラクトバチルス・フルクチボランスを１６ＳｒＲＮＡ遺伝子の増幅産物にもとづきそれぞれ検出できることが確認された。 From this, it was confirmed that according to the lactic acid bacteria detection kit of this embodiment, Lactobacillus plantarum can be detected based on the amplified product of 16S rRNA gene and RNA polymerase gene. It was also confirmed that Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus buchneri, and Lactobacillus fructivorans could be detected based on the 16S rRNA gene amplification product.
（試験２）
　本実施形態の乳酸菌の検出方法、乳酸菌検出キット、及び乳酸菌検出器具を用いて、特定の乳酸菌の標的遺伝子をＰＣＲ法により増幅し、その増幅産物を乳酸菌検出器具に固定化されたプローブとハイブリダイズさせることによって、これらの乳酸菌を同時にそれぞれ特異的に検出できるかを確認するための検証試験を行った。 (Test 2)
Using the lactic acid bacteria detection method, lactic acid bacteria detection kit, and lactic acid bacteria detection instrument of the present embodiment, a target gene of a specific lactic acid bacteria is amplified by the PCR method, and the amplified product is hybridized with a probe immobilized on the lactic acid bacteria detection instrument. Thus, a verification test was performed to confirm whether or not each of these lactic acid bacteria could be specifically detected simultaneously.
　試験１と同様に、４種類の乳酸菌について合計５つの試料を準備し、ＤＮＡの抽出を行った。
　また、プライマーセット及びＰＣＲ反応液として試験１と同様のものを使用し、試験１と同様にＰＣＲ法による遺伝子の増幅を行った。ＰＣＲにおいては、蛍光標識された核酸合成基質を用いて、標識された増幅産物を生成した。 As in Test 1, a total of five samples were prepared for four types of lactic acid bacteria, and DNA was extracted.
In addition, the same primer set and PCR reaction solution as in Test 1 were used, and the gene was amplified by the PCR method in the same manner as in Test 1. In PCR, a labeled amplification product was generated using a fluorescently labeled nucleic acid synthesis substrate.
　さらに、乳酸菌検出器具として、ラクトバチルス・プランタラムのＲＮＡポリメラーゼ遺伝子に相補的に結合する配列番号５～７に示される塩基配列からなるプローブと、ラクトバチルス・ブレビスの１６ＳｒＲＮＡ遺伝子に相補的に結合する配列番号８，９に示される塩基配列からなるプローブと、ラクトバチルス・ブフネリの１６ＳｒＲＮＡ遺伝子に相補的に結合する配列番号１０～１４に示される塩基配列からなるプローブと、ラクトバチルス・フルクチボランスの１６ＳｒＲＮＡ遺伝子に相補的に結合する配列番号１５に示される塩基配列からなるプローブとを固定化したＤＮＡチップを作製した。 Furthermore, as a lactic acid bacterium detection instrument, it binds complementarily to the 16S rRNA gene of Lactobacillus brevis and a probe consisting of the nucleotide sequence shown in SEQ ID NOs: 5 to 7 that binds complementarily to the RNA polymerase gene of Lactobacillus plantarum. A probe consisting of the base sequence shown in SEQ ID NOs: 8 and 9, a probe consisting of the base sequence shown in SEQ ID NOs: 10 to 14 that binds complementarily to the 16S rRNA gene of Lactobacillus buchnerii, and a 16S rRNA gene of Lactobacillus fructivorans A DNA chip was prepared by immobilizing a probe consisting of the base sequence shown in SEQ ID NO: 15 that binds in a complementary manner.
　このＤＮＡチップにＰＣＲの増幅産物を滴下して、プローブにハイブリダイズした増幅産物の標識を検出した。具体的には、次のように行った。
　まず、ＰＣＲ増幅産物に、緩衝液（３×ＳＳＣ　クエン酸－生理食塩水）に０．３％ＳＤＳ（ドデシル硫酸ナトリウム）を添加したものを混合し、ＤＮＡチップに滴下した。
　次に、ＤＮＡチップを４５℃で１時間静置し、上記緩衝液を用いてハイブリダイズしなかったＰＣＲ産物をＤＮＡチップから洗い流した。 A PCR amplification product was dropped onto the DNA chip, and the label of the amplification product hybridized with the probe was detected. Specifically, it was performed as follows.
First, a PCR amplification product was mixed with a buffer solution (3 × SSC citrate-saline) added with 0.3% SDS (sodium dodecyl sulfate) and dropped onto a DNA chip.
Next, the DNA chip was allowed to stand at 45 ° C. for 1 hour, and PCR products that were not hybridized using the buffer solution were washed away from the DNA chip.
　そして、ＤＮＡチップを標識検出装置（ＢＩＯＳＨＯＴ，東洋鋼鈑株式会社製）にかけて蛍光強度を測定した。蛍光強度は、レーザー光で励起して標識成分（Ｃｙ５）を発光させ、その光量を電気信号に置換して数値化し、蛍光強度を得た。さらに、この蛍光強度にもとづきＳ／Ｎ比値を算出した。その結果を図３に示す。
　同図に示されるように、供試菌株（１）～（５）のいずれについても、それらの乳酸菌が、各プローブの検出対象菌である場合にのみ陽性反応（Ｓ／Ｎ比値≧３）が示されており、偽陽性反応が生じていないことが確認された。 Then, the fluorescence intensity was measured by applying the DNA chip to a label detection device (BIOSHOT, manufactured by Toyo Kohan Co., Ltd.). The fluorescence intensity was excited by laser light to cause the labeling component (Cy5) to emit light, and the amount of the light was replaced with an electric signal to quantify it to obtain the fluorescence intensity. Furthermore, the S / N ratio value was calculated based on this fluorescence intensity. The result is shown in FIG.
As shown in the figure, for any of the test strains (1) to (5), a positive reaction (S / N ratio value ≧ 3) only when those lactic acid bacteria are detection target bacteria of each probe. It was confirmed that no false positive reaction occurred.
　なお、ＰＣＲの増幅産物には、配列番号５～１５に示される塩基配列からなるプローブとハイブリダイズする増幅産物に対して相補的な塩基配列を有する増幅産物も含まれる。したがって、配列番号５～１５に示される塩基配列に対して相補的な塩基配列からなるプローブは、このような相補的な塩基配列を有する増幅産物とハイブリダイズすることができる。
　よって、配列番号５～１５に示される塩基配列に対して相補的な塩基配列からなるプローブをＤＮＡチップに固定化した場合にも、上記の４種類の乳酸菌を同時にそれぞれ特異的に検出することが可能である。 The PCR amplification product also includes an amplification product having a base sequence complementary to the amplification product that hybridizes with the probe having the base sequence shown in SEQ ID NOs: 5 to 15. Accordingly, a probe comprising a base sequence complementary to the base sequences shown in SEQ ID NOs: 5 to 15 can hybridize with an amplification product having such a complementary base sequence.
Therefore, even when a probe having a base sequence complementary to the base sequences shown in SEQ ID NOs: 5 to 15 is immobilized on a DNA chip, the above four types of lactic acid bacteria can be specifically detected simultaneously. Is possible.
　本発明は、以上の実施形態や実施例に限定されるものではなく、本発明の範囲内において、種々の変更実施が可能である。例えば、本実施形態に係る乳酸菌検出器具に上記以外のプローブをさらに追加して固定化したり、あるいはＰＣＲ反応液にその他の成分を含有させたりするなど適宜変更することが可能である。 The present invention is not limited to the above embodiments and examples, and various modifications can be made within the scope of the present invention. For example, it is possible to appropriately change such as adding a probe other than the above to the lactic acid bacteria detection instrument according to the present embodiment and immobilizing it, or adding other components to the PCR reaction solution.
　本発明は、食品検査、環境検査等において、特定の乳酸菌を同時にそれぞれ特異的に検出する場合に好適に利用することが可能である。 The present invention can be suitably used for specific detection of specific lactic acid bacteria simultaneously in food inspection, environmental inspection, and the like.
　この明細書に記載の文献及び本願のパリ優先の基礎となる日本出願明細書の内容を全てここに援用する。 All the contents of the documents described in this specification and the specification of the Japanese application that is the basis of Paris priority of this application are incorporated herein.

Claims (5)

  1. 　試料に含まれる細胞からＤＮＡを抽出し、
    　抽出されたＤＮＡと、ラクトバチルス・プランタラム（Lactobacillus　plantarum）のＲＮＡポリメラーゼ遺伝子を標的とする配列番号１及び配列番号２に示される塩基配列からなるプライマーセットとを用いてＰＣＲによる増幅反応を行い、
    　得られた増幅産物にもとづき前記試料におけるラクトバチルス・プランタラムの有無を判定する
    　ことを特徴とする乳酸菌の検出方法。     DNA is extracted from the cells contained in the sample,
    Using the extracted DNA and a primer set consisting of the nucleotide sequence shown in SEQ ID NO: 1 and SEQ ID NO: 2 targeting the RNA polymerase gene of Lactobacillus plantarum, an amplification reaction by PCR is performed,
    A method for detecting lactic acid bacteria, comprising determining the presence or absence of Lactobacillus plantarum in the sample based on the obtained amplification product.
  2. 　抽出された前記ＤＮＡと、ラクトバチルス・プランタラムのＲＮＡポリメラーゼ遺伝子を標的とする前記プライマーセットと、ラクトバチルス・プランタラム、ラクトバチルス・ブレビス（Lactobacillus　brevis）、ラクトバチルス・ブフネリ（Lactobacillus　buchneri）、及びラクトバチルス・フルクチボランス（Lactobacillus　fructivorans）の16SrRNA遺伝子を標的とする配列番号３及び配列番号４に示される塩基配列からなるプライマーセットと、を用いて、前記ＰＣＲによる増幅反応を行い、
    　得られた増幅産物にもとづき前記試料におけるラクトバチルス・プランタラム、ラクトバチルス・ブレビス、ラクトバチルス・ブフネリ、及びラクトバチルス・フルクチボランスの有無を同時に判定する
    　ことを特徴とする請求項１記載の乳酸菌の検出方法。     The extracted DNA, the primer set targeting the RNA polymerase gene of Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus brevis, Lactobacillus buchneri, and Using the primer set consisting of the nucleotide sequence shown in SEQ ID NO: 3 and SEQ ID NO: 4 targeting the 16S rRNA gene of Lactobacillus fructivorans (Lactobacillus fructivorans), an amplification reaction by the PCR is performed,
    The detection of lactic acid bacteria according to claim 1, wherein the presence or absence of Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus buchneri, and Lactobacillus fructivorans is simultaneously determined based on the obtained amplification product. Method.
  3. 　ラクトバチルス・プランタラムのＲＮＡポリメラーゼ遺伝子に相補的に結合する配列番号５～７及びこれらの相補的配列の少なくともいずれかに示される塩基配列からなるプローブと、
    　ラクトバチルス・ブレビスの１６ＳｒＲＮＡ遺伝子に相補的に結合する配列番号８，９及びこれらの相補的配列の少なくともいずれかに示される塩基配列からなるプローブと、
    　ラクトバチルス・ブフネリの１６ＳｒＲＮＡ遺伝子に相補的に結合する配列番号１０～１４及びこれらの相補的配列の少なくともいずれかに示される塩基配列からなるプローブと、
    　ラクトバチルス・フルクチボランスの１６ＳｒＲＮＡ遺伝子に相補的に結合する配列番号１５及びこれの相補的配列の少なくともいずれかに示される塩基配列からなるプローブと、を固定化した乳酸菌検出器具に、標識された前記増幅産物を接触させ、
    　前記プローブと相補的に結合した前記増幅産物の標識を検出する
    　ことを特徴とする請求項１又は２記載の乳酸菌の検出方法。     A probe comprising SEQ ID NOs: 5 to 7 that complementarily bind to an RNA polymerase gene of Lactobacillus plantarum and a base sequence represented by at least one of these complementary sequences;
    A probe consisting of SEQ ID NOs: 8 and 9 that bind complementarily to the 16S rRNA gene of Lactobacillus brevis and a base sequence represented by at least one of these complementary sequences;
    A probe comprising SEQ ID NOs: 10 to 14 that complementarily bind to the 16S rRNA gene of Lactobacillus buchnerii and a base sequence represented by at least one of these complementary sequences;
    The amplified amplification labeled with a lactic acid bacterium detection instrument having immobilized thereon a probe consisting of SEQ ID NO: 15 that binds complementarily to the 16S rRNA gene of Lactobacillus fructivorans and a base sequence represented by at least one of the complementary sequences thereof Contact the product,
    The method for detecting a lactic acid bacterium according to claim 1 or 2, wherein a label of the amplification product complementary to the probe is detected.
  4. 　ラクトバチルス・プランタラム（Lactobacillus　plantarum）のＲＮＡポリメラーゼ遺伝子を標的とする配列番号１及び配列番号２に示される塩基配列からなるプライマーセットと、
    　ラクトバチルス・プランタラム、ラクトバチルス・ブレビス（Lactobacillus　brevis）、ラクトバチルス・ブフネリ（Lactobacillus　buchneri）、及びラクトバチルス・フルクチボランス（Lactobacillus　fructivorans）の１６ＳｒＲＮＡ遺伝子を標的とする配列番号３及び配列番号４に示される塩基配列からなるプライマーセットと、を含有する
    　ことを特徴とする乳酸菌検出キット。     A primer set consisting of the base sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2 targeting the Lactobacillus plantarum RNA polymerase gene;
    As shown in SEQ ID NO: 3 and SEQ ID NO: 4, which target the 16S rRNA genes of Lactobacillus brevis, Lactobacillus buchneri, and Lactobacillus fructivorans. A lactic acid bacteria detection kit comprising a primer set consisting of a base sequence.
  5. 　ラクトバチルス・プランタラム（Lactobacillus　plantarum）のＲＮＡポリメラーゼ遺伝子に相補的に結合する配列番号５～７及びこれらの相補的配列の少なくともいずれかに示される塩基配列からなるプローブと、
    　ラクトバチルス・ブレビス（Lactobacillus　brevis）の１６ＳｒＲＮＡ遺伝子に相補的に結合する配列番号８，９及びこれらの相補的配列の少なくともいずれかに示される塩基配列からなるプローブと、
    　ラクトバチルス・ブフネリ（Lactobacillus　buchneri）の１６ＳｒＲＮＡ遺伝子に相補的に結合する配列番号１０～１４及びこれらの相補的配列の少なくともいずれかに示される塩基配列からなるプローブと、
    　ラクトバチルス・フルクチボランス（Lactobacillus　fructivorans）の１６ＳｒＲＮＡ遺伝子に相補的に結合する配列番号１５及びこれの相補的配列の少なくともいずれかに示される塩基配列からなるプローブと、を固定化した
    　ことを特徴とする乳酸菌検出器具。     A probe comprising SEQ ID NOs: 5 to 7 that complementarily bind to an RNA polymerase gene of Lactobacillus plantarum and a base sequence represented by at least one of these complementary sequences;
    A probe consisting of SEQ ID NOs: 8 and 9 that complementarily bind to the 16S rRNA gene of Lactobacillus brevis and a base sequence represented by at least one of these complementary sequences;
    A probe comprising SEQ ID NOs: 10 to 14 that complementarily bind to the 16S rRNA gene of Lactobacillus buchneri and a base sequence represented by at least one of these complementary sequences;
    A lactic acid bacterium characterized by immobilizing SEQ ID NO: 15 that complementarily binds to the 16S rRNA gene of Lactobacillus fructivorans and a base sequence represented by at least one of the complementary sequences thereof Detection instrument.
PCT/JP2016/000546 2015-02-10 2016-02-03 Method for detecting lactic acid bacteria, lactic acid bacteria detection kit, and lactic acid bacteria detection instrument WO2016129249A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2015024012A JP6530198B2 (en) 2015-02-10 2015-02-10 Lactic Acid Bacteria Detection Method, Lactic Acid Bacteria Detection Kit, and Lactic Acid Bacteria Detection Device
JP2015-024012 2015-02-10

Publications (1)

Publication Number Publication Date
WO2016129249A1 true WO2016129249A1 (en) 2016-08-18

Family

ID=56614538

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2016/000546 WO2016129249A1 (en) 2015-02-10 2016-02-03 Method for detecting lactic acid bacteria, lactic acid bacteria detection kit, and lactic acid bacteria detection instrument

Country Status (2)

Country Link
JP (1) JP6530198B2 (en)
WO (1) WO2016129249A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018168816A1 (en) * 2017-03-14 2018-09-20 株式会社ジーシー Dna chip for detecting dental caries bacteria

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH11178589A (en) * 1997-08-08 1999-07-06 Smithkline Beecham Corp New rpoa
JPH11206389A (en) * 1997-08-08 1999-08-03 Smithkline Beecham Corp New rpoa

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH11178589A (en) * 1997-08-08 1999-07-06 Smithkline Beecham Corp New rpoa
JPH11206389A (en) * 1997-08-08 1999-08-03 Smithkline Beecham Corp New rpoa

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DATABASE GenBank 27 June 2000 (2000-06-27), "Lactobacillus aviarius gene for 16S rRNA", Database accession no. AB001837 *
DATABASE GenBank 5 February 2014 (2014-02-05), "Lactobacillus plantarum subsp. argentoratensis rpoA gene for RNA polymerase alpha-subunit, partial cds, strain: NBRC 106468", Database accession no. AB907216 *
FERCHICHI,M. ET AL.: "A one-step reaction for the rapid identification of Lactobacillus mindensis, Lactobacillus panis, Lactobacillus paralimentarius, Lactobacillus pontis and Lactobacillus frumenti using oligonucleotide primers designed from the 16S-23S rRNA intergenic sequences.", J APPL MICROBIOL., vol. 104, no. 6, 2008, pages 1797 - 1807, ISSN: 1364-5072 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018168816A1 (en) * 2017-03-14 2018-09-20 株式会社ジーシー Dna chip for detecting dental caries bacteria
JP2018148855A (en) * 2017-03-14 2018-09-27 三菱ケミカル株式会社 Dna chip for detecting dental caries
US11732310B2 (en) 2017-03-14 2023-08-22 Gc Corporation DNA chip for detecting dental caries bacteria

Also Published As

Publication number Publication date
JP2016146764A (en) 2016-08-18
JP6530198B2 (en) 2019-06-12

Similar Documents

Publication Publication Date Title
US20220064715A1 (en) Polymerase Chain Reaction Primers and Probes for Mycobacterium Tuberculosis
JP5830461B2 (en) PCR primer set, PCR reaction solution, detection method for food poisoning bacteria
JP5851395B2 (en) Carrier for detecting food poisoning bacteria, and method for detecting food poisoning bacteria
CN102066573A (en) Amplicon rescue multiplex polymerase chain reaction for amplificaton of multiple targets
CA2692633A1 (en) Method for the simultaneous detection of multiple nucleic acid sequences in a sample
WO2015190107A1 (en) Method for detecting vibrio parahaemolyticus, and carrier for detecting vibrio parahaemolyticus
JP6623572B2 (en) Microorganism test method, microbial test kit, and microarray for microbial test
TW201202431A (en) Compositions and methods for the rapid detection of Legionella pneumophila
JP2017189135A (en) Carrier for detecting lactic acid bacterium and method for detecting lactic acid bacterium
JP2017136019A (en) Carrier for pathogenic escherichia coli detection, kit for pathogenic escherichia coli detection, and detection method of enteropathogenic escherichia coli
WO2016129249A1 (en) Method for detecting lactic acid bacteria, lactic acid bacteria detection kit, and lactic acid bacteria detection instrument
US20170081709A1 (en) Method for detecting escherichia coli and carrier for detecting escherichia coli
WO2021201091A1 (en) Primer set and probe for detecting klebsiella bacteria
US20120196765A1 (en) Method for detection or analysis of target sequence in genomic dna
WO2019221219A1 (en) Method for examining bacterium, microarray for examining bacterium, kit for examining bacterium, probe set for examining bacterium, and primer set for examining bacterium
GB2549799A (en) A multiplex assay for the sensitive and specific detection and differentiation of Clostridium difficile
JP6613627B2 (en) Nucleic acid amplification success / failure determination method and nucleic acid amplification test kit
JP6648559B2 (en) Bacteria test method, primer set, bacteria test carrier, and bacteria test kit
US20190284616A1 (en) Method for the detection of legionella
TWI531654B (en) Oligonucleotide probes, kit containing the same and method for pathotyping of h5 avian influenza viruses
JP5873356B2 (en) Method for detecting Moniliella spp.
JP6672760B2 (en) Carrier for detecting pathogenic E. coli, kit for detecting pathogenic E. coli, and method for detecting pathogenic E. coli
JP2019201626A (en) Method for examining bacterium, micro array for examining bacterium, kit for examining bacterium, probe set for examining bacterium, and primer set for examining bacterium
Masson et al. Molecular forensic approaches to the taxonomic assessment of bacteria in a commercial consortia
JP2006280273A (en) New polynucleotide, marker for detecting deep imported mycosis pathogen histoplasma bacteria(including histoplasma capsulatum var. capsulatum, histoplasma capsulatum var. farciminosum, histoplasma capsulatum var. duboisii and emmonsiella capsulata as bacterial name in sexual generation) using the same, probe, primer, detection method and kit

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: 16748896

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: 16748896

Country of ref document: EP

Kind code of ref document: A1