JP2008054632A - Primer for detecting turbid lactic acid bacteria in beer and detection method using the same - Google Patents

Abstract

The present invention provides a method for quickly and specifically detecting beer-turbid lactic acid bacteria resistant to beer hops, particularly in the inspection of alcoholic beverages such as beer and the production environment thereof.
[MEANS FOR SOLVING PROBLEMS] A LAMP primer set for detecting beer-turbid lactic acid bacteria, comprising four types of LAMP primer sets comprising base sequences FIP, BIP, F3 and B3 that can be annealed to a specific region of a turbid gene of beer-turbid lactic acid bacteria, and A method for detecting beer-turbid lactic acid bacteria using a LAMP primer set.
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Description

  The present invention relates to a primer set and method for detecting beer-turbid lactic acid bacteria quickly and accurately.

  More specifically, the present invention relates to a method and a primer set for detecting beer-spoilage lactic acid bacteria, including detecting amplification of a beer turbidity bacterium in beer-spoilage lactic acid bacteria by a loop-mediated isothermal amplification (LAMP) method.

Beer is a liquor that is not easily contaminated by microorganisms because isohumulones contained in hops have an antibacterial action. However, some lactic acid bacteria, such as Lactobacillus brevis and Pediococcus damnosus, have acquired hop resistance, and they grow and become turbid in beer while producing an off-flavor odor. Since it is known that there is a risk of damage, it is considered to be a very important bacterial species in terms of quality control (Non-patent Document 1).

  For this reason, it is important to quickly detect these cells from the beer production environment or in the product, but not all bacterial species belonging to these lactic acid bacteria have the property of causing turbidity in beer. Therefore, it is necessary to identify whether a strain such as Lactobacillus brevis is a strain having beer turbidity.

  As a detection method using antiserum, a detection method for Lactobacillus brevis having beer turbidity has been proposed (Patent Document 1). As a determination method based on the culture method, a method of determining by measuring turbidity during the culture period has been proposed, but the test requires a period of about one week (Patent Document 2). A method of discriminating the growth in beer from the mobility of polyacrylamide gel electrophoresis of D-lactate dehydrogenase of Lactobacillus brevis has also been proposed (Patent Document 3). However, this method takes time and requires caution in storage and handling, such as using acrylamide, which is highly suspected of carcinogenicity during gel preparation.

  As a method for determining hop-resistant beer lactic acid bacteria, a method of identifying a hop-resistant plasmid and performing hybridization using the plasmid as a probe has also been proposed (Patent Document 4). In addition, a method for labeling a restriction enzyme fragment of a plasmid related to hop resistance or a PCR cloning product to form a detection probe has been proposed (Patent Documents 5 and 6). However, these operations are extremely complicated and time consuming.

In the gene amplification method, a method for amplifying and detecting a part of the hop resistance gene present on the Lactobacillus brevis plasmid has also been proposed (Patent Document 7). In addition, a method for amplifying and detecting a part of a hop resistance gene present on a Lactobacillus lindneri plasmid has been proposed (Non-patent Document 2).

  In addition, a method for amplifying and detecting a part of the gene using a primer specific for the DNA gyrase subunit B gene has been proposed (Patent Document 8). In this method, a DNA fragment amplified by the PCR method is cleaved with a restriction enzyme, and the turbidity of beer is judged based on the obtained pattern. However, the bacterial detection method using the PCR method requires a special instrument for performing the reaction, and agarose gel electrophoresis, which is one of the determination methods, requires a lot of time and labor. In addition, since the determination based on the restriction enzyme cleavage pattern is complicated and takes time, there is a problem in conducting these tests in daily microorganism tests.

  A beer turbidity gene of beer turbid lactic acid bacteria is encoded on DNA, and its base sequence is available from, for example, GenBank and has a registration number of AB182366. In addition, identification of a beer turbidity gene and a method for amplifying the gene by PCR have already been reported (Patent Document 9, Non-Patent Document 1).

  In recent years, the LAMP reaction was developed by Eiken Chemical Co., Ltd. (Tochigi) as one of the new gene amplification methods. The LAMP method is an isothermal nucleic acid amplification method, has high specificity and amplification efficiency, and can be performed in about 1 hour from reaction to detection (Patent Document 10; Non-Patent Document 3).

Japanese Patent Laid-Open No. 10-104238 Japanese Unexamined Patent Publication No. 7-75596 Japanese Patent Laid-Open No. 11-56391 JP-A-9-260 Japanese Patent Laid-Open No. 10-14584 Japanese Patent Laid-Open No. 2001-86995 Japanese Patent Laid-Open No. 11-18780 JP 2003-250557 A Japanese Patent Laid-Open No. 2003-251 JP 2002-330796 A T. Fujii et al., J. Appl. Microbiol, 2005, 98 (5), 1209-20 K. Suzuki et al., Appl. Environ. Microbiol, 2005, 71 (9), 5089-97 T. Notomi et al., Nucleic Acids research, 2000, 28 (12), e63

  An object of the present invention is to provide a primer set capable of specifically detecting beer-turbid lactic acid bacteria by the LAMP method.

  Another object of the present invention is to provide a method for specifically detecting beer-turbid lactic acid bacteria using the above primer set.

  As a result of studies to solve the above problems, the present inventor has prepared an oligonucleotide primer that selectively hybridizes to a beer turbidity gene of beer-turbid lactic acid bacteria, and amplifies the oligonucleotide by using the LAMP method as a primer. Thus, it was found that beer-turbid lactic acid bacteria can be detected from a specimen in a specific, simple, rapid and sensitive manner, and the present invention has been completed.

That is, the present invention can be summarized as follows.
(1) The following LAMP primer set for detecting beer-turbid lactic acid bacteria.
(i) A set of four types of primers consisting of base sequences FIP, BIP, F3 and B3 shown in SEQ ID NOs: 1 to 4 that can be annealed to a specific region of a beer turbidity gene of beer turbid lactic acid bacteria.
(ii) A LAMP primer set further comprising a primer of the base sequence Loop-B represented by SEQ ID NO: 5 in addition to the four types of primers of (i) above.

(2) The following method for detecting beer-turbid lactic acid bacteria.
(iii) A method comprising detecting amplification of a beer turbidity gene of beer turbid lactic acid bacteria by the LAMP method.
(iv) In the method (iii), the LAMP method is performed using the LAMP primer set described in (i) or (ii).

Beer-turbid lactic acid bacteria are lactic acid bacteria that have acquired resistance to the antibacterial action of isohumulones contained in hops, and are bacterial species that grow and become turbid in beer and at the same time generate off-flavors. More specifically, Lactobacillus brevis (Lactobacillus brevis), Lactobacillus Rindoneri (Lactobacillus lindneri), Lactobacillus casei (Lactobacillus casei), Pediococcus Damunosasu (Pediococcus damnosus), Lactobacillus Parakorinoidesu (Lactobacillus paracollinoides ) Etc. are known.

  All of the primer sets for beer turbidity gene amplification of the present invention can specifically detect beer-turbid lactic acid bacteria by the LAMP method (Table 1 described later). As a result of testing for this bacterial strain, a strain having all the beer turbidity genes was positively detected by the primer set of the present invention. On the other hand, negative results were obtained for all the strains that did not have the beer turbidity genes evaluated.

  According to the present invention, beer-turbid lactic acid bacteria can be detected specifically, simply, rapidly and with high sensitivity.

  The base sequence of the beer clouding lactic acid bacterium was compared with other bacteria to identify a sequence specific to the beer clouding lactic acid bacterium, and the LAMP primer of the present invention was designed. The base sequence of this bacterial beer turbidity gene is available from, for example, GenBank, and has a registration number of AB182366. The beer turbidity gene has been analyzed in detail by Fujii et al. (Japanese Patent Laid-Open No. 2003-251, T. Fujii et al., J. Appl. Microbiol, 2005, 98 (5), 1209-20).

  The base sequences determined as regions that can be annealed to a specific region of the turbidity gene of beer-turbid lactic acid bacteria are the nucleotide sequences shown in SEQ ID NOs: 1 to 4 and 5, respectively.

  When the LAMP method is performed using a primer set of these sequences, beer-turbid lactic acid bacteria can be detected separately (ie, specifically) from other strains that do not have beer-turbidity.

  Therefore, the LAMP primer set for detecting beer cloudy lactic acid bacteria of the present invention uses a region where a base sequence different from other bacteria on the beer cloudy gene of beer clouded lactic acid bacteria is present, and 4 types of primers targeting this Primer set using (FIP, BIP, F3 and B3). Moreover, in order to accelerate the nucleic acid amplification reaction, one type (Loop-B) primer is added, and a total of five types of primers may be used.

  In the LAMP method, four primers (generally called FIP, BIP, F3, and B3) are set for six regions of the target gene, and a nucleic acid can be amplified isothermally using a strand displacement reaction. (JP 2002-330796 A; T. Notomi et al., Nucleic Acids research, 2000, 28 (12), e63).

  First, for the target gene, three regions, F3c, F2c, and F1c, from the 3 ′ end side, and regions B1, B2, and B3 toward the 5 ′ end side of the target gene are defined. Four types of primers are designed: FIP, F3, BIP and B3. Here, regions complementary to the F3c, F2c, and F1c regions are F3, F2, and F1, respectively, and regions complementary to the B1, B2, and B3 regions are B1c, B2c, and B3c, respectively.

  FIP is a primer designed to have an F2 region complementary to the F2c region of the target gene on the 3 ′ end side and the same sequence as the F1c region of the target gene on the 5 ′ end side. If necessary, a restriction enzyme site can be introduced between F1c and F2 of the FIP primer.

  F3 is a primer designed to have an F3 region complementary to the F3c region of the target gene.

  BIP is a primer designed to have a B2 region complementary to the B2c region of the target gene on the 3 ′ end side and the same sequence as the B1c region of the target gene on the 5 ′ end side. If necessary, a restriction enzyme site can be introduced between B1c and B2 of the BIP primer.

  B3 is a primer designed to have a B3 region complementary to the B3c region of the target gene.

  When the restriction enzyme site is contained in the FIP and BIP primers, the amplified product can be observed as one band after electrophoresis by treating the amplification product with the restriction enzyme after the reaction. In this case, if the target DNA has a restriction enzyme site, it is not necessary to artificially introduce the restriction enzyme site into the primer.

  In addition, when using Loop-B primers, these primers bind to loop portions that are not used in the nucleic acid amplification process, so that the nucleic acid reaction proceeds from all the loop portions, and the nucleic acid amplification reaction is accelerated. (Japanese Patent Laid-Open No. 2002-345499).

  The LAMP reaction can be performed in a one-step process until detection by keeping the sample gene, primer, strand-displacing DNA synthase, substrate and the like together at a constant temperature (about 60 to 65 ° C.).

  In this reaction, strand displacement type DNA synthase is used. This enzyme is inexpensive, unlike thermostable DNA polymerase in PCR, and can synthesize DNA while unfolding the double strand of template DNA. it can. Examples of enzymes are Bst DNA polymerase, Bca (exo-) DNA polymerase, Vent (Exo-) DNA polymerase and the like. For this reason, in the LAMP method, it is not necessary to thermally denature double-stranded DNA into a single strand in advance, unlike the PCR method.

For the LAMP reaction reagent, it is convenient to use a Loopamp DNA amplification reagent kit (excluding the primer set) commercially available from Eiken Chemical. Specifically, examples of the reaction solution are as follows. Buffer solution for double concentration: 40 mM Tris-HCl (pH 8.8), 20 mM KCl, 20 mM (NH ₄ ) ₂ SO ₄ , 16 mM MgSO ₄ , 0.2% Tween20, 1.6 M Betain, dATP with a final concentration of 2.8 mM, dCTP, dGTP, dTTP; DNA polymerase: Bst DNA Polymerase 8 units / μl; final concentration of each primer of the present invention, FIP, BIP: 40 μM, F3, B3: 5 μM, Loop-B: 20 μM.

  As the LAMP reaction solution, for example, 4.5 μl of sterilized distilled water, 12.5 μl of buffer solution for double concentration reaction, 1 μl of each primer of FIP, BIP, F3, B3, and Loop-B, 1 μl of DNA polymerase, sample solution (Template DNA) 2 μl is added to prepare a total volume of 25 μl of reaction solution.

  The reaction is performed through the following steps.

  (i) By the action of the strand displacement type DNA polymerase, a DNA strand complementary to the template DNA is synthesized starting from the 3 ′ end of the F2 region of FIP.

  (ii) The F3 primer anneals to the outside of the FIP, and DNA synthesis extends while peeling the DNA strand from the previously synthesized FIP by the action of the strand displacement DNA polymerase starting from the 3 ′ end.

  (iii) The DNA strand synthesized from the F3 primer and the template DNA are double-stranded.

  (iv) The DNA strand previously synthesized from FIP is peeled off by the DNA strand from the F3 primer to become single-stranded DNA. This DNA strand has complementary regions F1c and F1 on the 5 ′ end side. Then, self-annealing occurs and a loop is formed.

  (v) BIP anneals to the DNA strand that formed a loop in the process of (iv) above, and complementary DNA synthesis is performed starting from the 3 ′ end of this BIP. Furthermore, the B3 primer anneals to the outside of the BIP, and DNA synthesis extends while peeling the DNA strand from the previously synthesized BIP by the action of the strand displacement type DNA polymerase starting from its 3 ′ end.

  (vi) Double-stranded DNA is formed in the process of (v) above.

  (vii) Since the DNA strand synthesized from the BIP peeled off in the process (v) has a complementary sequence at both ends, it self-anneals and forms a loop to form a dumbbell-like structure.

  (viii) Starting from the DNA strand having the dumbbell structure, a desired DNA amplification cycle is performed through FIP and BIP annealing.

Each primer set of the present invention causes DNA strand synthesis at the same time as annealing at about 60 to 65 ° C. (for example, 65 ° C.). It is possible to amplify the nucleic acid 10 ⁹ to 10 ¹⁰ times by carrying out the reaction for about 1 hour by annealing reaction and DNA strand synthesis.

  When a specific DNA region of beer-turbid lactic acid bacteria is amplified, the reaction solution becomes cloudy due to the influence of magnesium pyrophosphate formed as a by-product, so the presence or absence of amplification can be determined visually based on this turbidity, or turbidity measurement Turbidity can also be measured optically using an apparatus. In addition, the presence or absence of DNA fragments can be confirmed and detected using agarose gel electrophoresis or the like.

  As a sample for identifying beer-turbid lactic acid bacteria by nucleic acid amplification, for example, alcoholic beverages such as beer and sparkling liquor, and as an environmental sample, for example, water or a wiped sample in a beer production environment may be used.

  When these specimens are used as samples for the LAMP method, operations such as concentration and separation of bacteria present in the specimen, separation of nucleic acids from bacterial cells, and concentration of nucleic acids can also be performed as pretreatment. As a method for concentrating and separating bacteria, filtration, centrifugation, and the like are known and can be appropriately selected. To release nucleic acids from bacterial cells present in food samples such as alcoholic beverages and environmental samples, for example, a method in which the bacterial cells are treated with Lysozyme, Proteinase K, etc., and nucleic acids are released from the bacterial cells by heating at 100 ° C. is there. In addition, depending on food samples, if further purification is required, nucleic acid is purified by, for example, phenol / chloroform or chloroform / isoamyl alcohol treatment, ethanol or isopropanol precipitation, centrifugation, etc., and finally redissolved in TE buffer or the like. May be used as a template DNA (BREWING MICROBIOLOGY, 1987, 127-140, European Brewery Convention: ANALYTICA-MICROBIOLOGICA-EBC, 2nd ed. 2005 Fachverlag Hans Carl, Nuernberg, Rolfs et al .: PCR-Clinical diagnostics and research, Springer-Verlag, Berlin, 1992, Oshima et al .: Protein Nucleic Acid Enzyme, 1990, 35, 2523-41).

  For example, beer cloudy lactic acid bacteria thought to be present in beer are enriched and cultured in an appropriate medium, DNA is separated from colonies formed on the agar medium, LAMP reaction is performed on the DNA using the above primers, and beer Amplifies specific gene region of cloudy lactic acid bacteria.

  Due to the influence of magnesium pyrophosphate formed as a by-product of the amplification reaction, the reaction solution becomes cloudy as described above, so the turbidity of the reaction solution can be visually checked or optical nucleic acid amplification using a turbidity measuring device can be used to determine whether nucleic acid is amplified Easy to check. If nucleic acid amplification is observed, it means that the target gene is present and as a result represents beer-turbid lactic acid bacteria positive (+). On the contrary, when nucleic acid amplification is not observed, it means that the target gene is absent, and as a result, beer-turbid lactic acid bacteria negative (-) is represented.

  As described above, according to the present invention, a primer set consisting of SEQ ID NOs: 1 to 4 for amplifying a specific gene region derived from a beer turbidity gene and a base sequence represented by SEQ ID NO: 5 that can be carried out as appropriate is provided. By using it as a LAMP primer set, beer-turbid lactic acid bacteria can be specifically detected. Here, “specifically” means that the detection reaction is negative for the other beer turbidity strains evaluated.

  The detection method of beer cloudy lactic acid bacteria by the LAMP method of the present invention can be used as a detection method that can quickly determine the presence or absence of beer clouded lactic acid bacteria present in test objects such as liquors such as beer and environmental samples, It can be used for detection of contamination of lactic acid bacteria causing turbidity in beer.

  EXAMPLES Hereinafter, although this invention is demonstrated concretely based on an Example, this invention is not limited to these Examples.

1. LAMP reaction
The concentration of each reagent used in the LAMP reaction is as follows. A LoopAMP DNA amplification reagent kit manufactured by Eiken Chemical Co., Ltd. was used as the LAMP reaction reagent. Double buffer solution: 40 mM Tris-HCl (pH 8.8), 20 mM KCl, 20 mM (NH ₄ ) ₂ SO ₄ , 16 mM MgSO ₄ , 0.2% Tween20, 1.6 M Betain, dATP with a final concentration of 2.8 mM, dCTP, dGTP, dTTP. DNA polymerase: Bst DNA Polymerase 8units / μl. Final concentration of each primer of the present invention, FIP, BIP: 40 μM, F3, B3: 5 μM, Loop-B: 20 μM. (Eiken Chemical Co., Ltd., instructions attached to DNA amplification reagent kit LMP201)

  The LAMP reaction solution was obtained by adding 4.5 μl of sterilized distilled water, 12.5 μl of double buffer solution, 1 μl of each of FIP, BIP, F3, B3, and Loop-B primers, and 1 μl of DNA polymerase from the above. A sample solution (template DNA) 2 μl was added to prepare a total volume of 25 μl of a reaction solution.

  For the LAMP reaction, a turbidity measuring device LA-200F manufactured by Teramex was used, an isothermal reaction at 65 ° C. was performed for 60 minutes, and then an enzyme deactivation treatment was performed at 80 ° C. for 2 minutes. The turbidity measuring device is capable of observing the white turbidity due to magnesium pyrophosphate, a by-product of the LAMP reaction, over time. Negative.

2. Primer design Compared with the turbidity gene of Lactobacillus brevis (GenBank accession number AB182366, SEQ ID NO: 6) and the base sequence of other lactic acid bacteria without beer turbidity, it is specific to beer-turbid lactic acid bacteria A LAMP primer was designed.

3. Results The primer sets according to SEQ ID NO: 1-4 (FIP, BIP, F3 and B3, respectively) and addable SEQ ID NO: 5 (Loop-B) are designed to amplify specific regions of the beer turbidity gene. As a result of actually carrying out the LAMP reaction using 20 strains of beer-turbid lactic acid bacteria shown in Table 1 and other strains, only the strains possessing the beer turbidity gene were found to have magnesium pyrophosphate as a by-product of the amplification reaction. Cloudiness was observed. Moreover, the crossing property with other strains was not seen. The results are shown in Table 1.

  The primer set for beer turbidity gene amplification was able to specifically detect beer turbid lactic acid bacteria and confirmed the amplification reaction within 1 hour.

  As a comparative example, detection by the PCR method (Japanese Patent Laid-Open No. 2003-251), which is a beer turbidity gene detection method, resulted in the same results as the LAMP method. This indicates that the test according to the invention gives the correct result.

  From this, it was judged that the primer set for amplifying beer turbidity genes was effective as a primer for specifically detecting beer turbid lactic acid bacteria.

  The detection method of beer cloudy lactic acid bacteria by the LAMP method of the present invention is a detection method that can quickly and specifically determine the presence or absence of beer clouded lactic acid bacteria present in test objects such as liquors such as beer and their production environment. Since it can be used, it is useful for detecting contamination of alcoholic beverages such as beer and beer-turbid lactic acid bacteria in its production environment.

Claims (4)

  A LAMP primer set for detecting beer-turbid lactic acid bacteria, which is a set of four types of primers consisting of base sequences FIP, BIP, F3 and B3 shown in SEQ ID NOs: 1 to 4 that can be annealed to specific regions of beer-turbid lactic acid bacteria .   The LAMP primer set according to claim 1, further comprising a primer of the base sequence Loop-B represented by SEQ ID NO: 5.   A method for detecting beer-turbid lactic acid bacteria, comprising detecting amplification of a beer-turbidity gene in beer-turbid lactic acid bacteria by the LAMP method.   The method according to claim 3, wherein the LAMP method is performed using the LAMP primer set according to claim 1 or 2.