WO2016129249A1 - Procédé pour détecter des bactéries d'acide lactique, kit de détection de bactéries d'acide lactique et instrument de détection de bactéries d'acide lactique - Google Patents

Procédé pour détecter des bactéries d'acide lactique, kit de détection de bactéries d'acide lactique et instrument de détection de bactéries d'acide lactique Download PDF

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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
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lactobacillus
seq
lactic acid
acid bacteria
base sequence
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Japanese (ja)
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隆明 山崎
淳憲 一色
美穂 大河内
睦子 並木
京子 右田
宮下　隆
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東洋製罐グループホールディングス株式会社
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    • 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

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  • 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.

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Abstract

Dans la présente invention, quatre types de bactéries d'acide lactique (Lactobacillus plantarum, brevis, buchneri et fructivorans) sont chacun détectés simultanément et spécifiquement. Une réaction d'amplification par PCR est effectuée à l'aide d'ADN extrait de cellules contenues dans un échantillon, d'un ensemble d'amorces représenté par les séquences SEQ ID NO : 1 et SEQ ID NO : 2, ciblant un gène d'ARN polymérase de Lactobacillus plantarum, et d'un ensemble d'amorces comprenant les séquences de bases représentées par les séquences SEQ ID NO : 3 et SEQ ID NO : 4, ciblant le gène de l'ARNr 16S de Lactobacillus plantarum, brevis, buchneri, et fructivorans. La présence de Lactobacillus plantarum, brevis, buchneri et fructivorans est déterminée simultanément sur base du produit amplifié obtenu.
PCT/JP2016/000546 2015-02-10 2016-02-03 Procédé pour détecter des bactéries d'acide lactique, kit de détection de bactéries d'acide lactique et instrument de détection de bactéries d'acide lactique WO2016129249A1 (fr)

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