JP2008161187A - Primer set for detection of Saccharomyces yeasts and combinations thereof - Google Patents

Abstract

Provided is a primer set that can accurately, quickly and easily identify Saccharomyces yeast species.
A Saccharomyces pastorianus comprising a primer consisting of a polynucleotide comprising at least 10 bases that hybridizes to a polynucleotide consisting of a plurality of specific sequences or a polynucleotide represented by a complementary sequence of the base sequence, and / or It consists of a LAMP method primer set used for detection of Saccharomyces bayanus.
[Selection figure] None

Description

Field of Invention

  The present invention relates to a primer set for detecting Saccharomyces yeast and a combination thereof, and more particularly to a LAMP method primer set and a PCR method primer set for detecting Saccharomyces yeast and a combination thereof.

Saccharomyces yeasts are widely used for the production of alcoholic beverages such as beer, wine, sake, shochu and whiskey, as well as bread production. Saccharomyces cerevisiae (Saccharomyces cerevisiae) is top fermenting types of beer such as ale, wine, sake, brew such fruit wine such as cider, also shochu used distillery whiskey like. Saccharomyces bayanus is used to produce wine, sherry, or sparkling wine. The bottom fermenting yeast used for pilsner-type beer production is now classified as Saccharomyces pastorianus (Kurtzman, CP & Fell, JW The Yeasts, A Taxonomic Study, 4th edition, 1998, Elsevier Science BV , The Netherlands, Back, W .: Farbatlas und Handbuch der Geraenkebiologie, Teil I, 1994, Verlag Hans Carl, Nuernberg, Barnett, JA et al .: Yeasts, characteristics and identification, 3rd edition, 2000, Cambridge University Press, UK, Sake Yeast and Sake Study Group: "Sake Yeast Research" 2003, Shin Nippon Printing, Tokyo). Thus, in order to grasp whether the yeast used for production is an appropriate yeast, a technique for identifying the species of Saccharomyces yeast is important.

  However, if these yeasts remain in filtered liquor products or are mixed from the outside, they will ferment excessively and cause turbidity, producing a characteristic odor and transforming it into an exciting bitter taste. (Back, W .: Farbatlas und Handbuch der Geraenkebiologie, Teil I, 1994, Verlag Hans Carl, Nuernberg, European Brewery Convention: ANALYTICA-MICROBIOLOGICA-EBC, 2nd ed. 2005 Fachverlag Hans Carl, Nuernberg) .

  Saccharomyces yeasts may also be separated from soft drinks, especially fruit juice drinks. When these yeasts are mixed, carbon dioxide, ethanol, and unpleasant odors are produced from sugars such as glucose and saccharose, which greatly impairs product quality (Back, W .: Farbatlas und Handbuch der Geraenkebiologie, Teil II, 1999, Verlag Hans Carl, Nuernberg).

  In this way, when Saccharomyces yeast grows in a product, it has a great influence on the industry. Therefore, a technique for rapidly detecting and identifying these yeasts is important for quality control. In addition, if the Saccharomyces genus yeast separated from the product is the yeast used in the manufacturing process, it may be caused by leakage from the upstream of the manufacturing process, defective filtration process, etc. In such a case, there may be a cause such as poor filling machine cleaning or accumulation in the piping. Therefore, the technology of identifying yeast that has been separated from the product when it is contaminated is important to clarify the goals to be addressed.

However, Saccharomyces pastorianus, Saccharomyces cerevisiae, and Saccharomyces bayanus are taxonomically related and form a taxonomic group called Saccharomyces sensu stricto with several yeasts such as Saccharomyces paradoxus and Saccharomyces micatae. (Naumov, GI et al .: Int. J. Syst. Evol. Microbiol., 2000, vol. 50, 1931-1942). Furthermore, Saccharomyces pastorianus is considered to be a species formed by crossing Saccharomyces cerevisiae and Saccharomyces bayanus and has been confirmed to be a hybrid of both at the gene level and at the chromosome level (Kielland-Brandt, MC et al .: Genetics of brewing yeast. The Yeast, 2nd edn, vol. 6, pp223-254, Edited by Wheals, et al., Academic Press, New York, Ryu, S.-L. et al .: Yeast, 1996, vol. 12, 757, Tamai, Y. et al .: Yeast, 1998, vol. 14, 923-933, Tamai, Y. et al .: Yeast, 2000, vol. 16, 1335-1343). Morphological, physiological, and biochemical methods are used as traditional yeast identification methods. Especially, the ability to assimilate and ferment various sugars is often investigated, but the species belonging to Saccharomyces sensu stricto Are so phenotypically similar to each other that it is difficult to distinguish them by such traditional methods (Naumova, ES et al .: Antoni van Leeuwenhoek, 2003, vol. 83, 155-166). Molecular biological approaches to distinguish these strains include PCR fingerprinting, DNA / DNA recombination kinetics, karyotype analysis, mitochondrial DNA restriction enzyme cleavage analysis, rRNA gene nucleotide sequence analysis, rDNA restriction enzyme cleavage analysis, UP- Known are PCR, isozyme analysis, PCR-temperature gradient gel electrophoresis, real-time PCR, and the like.

  Up to now, a part of the FLO1 gene of Saccharomyces genus yeast has been amplified by PCR, or a part of rRNA gene has been amplified by PCR, and the yeast of Saccharomyces genus or other genera using RFLP, or for brewing A method for discriminating between yeast and non-brewing yeast has been developed (Japanese Patent Laid-Open No. 11-56366 (Patent Document 1)). Further, based on the knowledge that there are two types of spacer region sequences of 26S rRNA gene and 5S rRNA gene of bottom fermented brewer's yeast, specific PCR primer sets were developed for each of them (JP-A-2001-2001). No. 8684 (Patent Document 2)). Furthermore, a primer specific to an Lg-FLO1-like gene in which the N-terminal part of Lg-FLO1 and the gene of yeast chromosome IX were linked was developed for detection of bottom fermenting yeast (Japanese Patent Application Laid-Open No. 2002-233382). Publication (Patent Document 3)).

  However, since PCR and real-time PCR require advanced temperature control and fluorescence observation, expensive equipment is required. PCR requires electrophoresis, staining, photography, etc. after the reaction, and it takes time to determine the result after the gene amplification step. Furthermore, RAPD PCR, restriction enzyme treatment of amplification products, base sequence analysis, isozyme analysis, temperature gradient gel electrophoresis, etc. require more time and troublesome operations than ordinary PCR, and should be performed in daily microbial testing operations. Had a problem.

In addition, although a LAMP method primer set for detecting Saccharomyces pastorianus has been developed (WO2005 / 093059 (Patent Document 4)), there is still room for improvement in terms of detection accuracy.
JP-A-11-56366 JP 2001-8684 A JP 2002-233382 A WO2005 / 093059

Summary of the Invention

  The present inventors have recently reported the base sequence of FSY1 gene (GenBank Accession No. AJ250992; Goncalves, PM et al., J. Bacteriol., 2000, vol.19), which has been reported as a gene specific to bottom fermentation yeast. , 5628-5630), LAMP method primer set and PCR method primer set were designed to detect the species of Saccharomyces yeast. As a result, it was found that the prepared primer set cross-reacts not only with the bottom fermentation yeast but also with Saccharomyces bayanus.

  The present inventors also designed a LAMP method primer set and a PCR method primer set based on the nucleotide sequence of the promoter region of the CYS3 gene of Saccharomyces cerevisiae (GenBank Accession No. L05146) to detect the type of Saccharomyces yeast. Tried. As a result, it was found that the prepared primer set cross-reacts not only with Saccharomyces cerevisiae but also with Saccharomyces pastorianus.

  The present inventors further used a Saccharomyces genus yeast according to the type of bacteria by using a combination of a primer set prepared based on the FSY1 gene and a primer set prepared based on the CYS3 gene promoter region. Saccharomyces pastorianus, Saccharomyces cerevisiae, and Saccharomyces bayanus were found to be able to distinguish accurately.

According to a first aspect of the present invention, comprising a following polynucleotides, LAMP method primer set used for the detection of Saccharomyces pastorianus (Saccharomyces pastorianus) and / or Saccharomyces bayanus (Saccharomyces bayanus) are provided:
A polynucleotide of at least 10 bases that hybridizes to a polynucleotide represented by the base sequence of SEQ ID NO: 1 (FIP) or a polynucleotide represented by a complementary sequence of the base sequence;
A polynucleotide of at least 10 bases that hybridizes to the polynucleotide represented by the nucleotide sequence of SEQ ID NO: 2 (F3) or the polynucleotide represented by the complementary sequence of the nucleotide sequence;
A polynucleotide (BIP) represented by the nucleotide sequence of SEQ ID NO: 3 or a polynucleotide of at least 10 nucleotides that hybridizes to a polynucleotide represented by the complementary sequence of the nucleotide sequence; and represented by the nucleotide sequence of SEQ ID NO: 4. A polynucleotide having at least 10 bases that hybridizes to a polynucleotide (B3) or a polynucleotide represented by a complementary sequence of the base sequence.

According to the first aspect of the present invention, a polynucleotide having at least 10 bases that hybridizes to the polynucleotide represented by the base sequence of SEQ ID NO: 11 or the polynucleotide represented by the complementary sequence of the base sequence; Saccharomyces pastorianus comprising: a polynucleotide represented by the nucleotide sequence of SEQ ID NO: 12 or a polynucleotide of at least 10 nucleotides that hybridizes to a polynucleotide represented by a complementary sequence of the nucleotide sequence; Alternatively, a PCR primer set used for detection of Saccharomyces bayanus is provided.

According to a second aspect of the present invention, comprising a following polynucleotides, LAMP method primer set used for the detection of Saccharomyces pastorianus (Saccharomyces pastorianus) and / or Saccharomyces cerevisiae (Saccharomyces cerevisiae) are provided:
A polynucleotide of at least 10 bases that hybridizes to a polynucleotide represented by the base sequence of SEQ ID NO: 6 (FIP) or a polynucleotide represented by a complementary sequence of the base sequence;
A polynucleotide of at least 10 bases that hybridizes with the polynucleotide (F3) represented by the nucleotide sequence of SEQ ID NO: 7 or the polynucleotide represented by the complementary sequence of the nucleotide sequence;
A polynucleotide (BIP) represented by the base sequence of SEQ ID NO: 8 or a polynucleotide of at least 10 bases that hybridizes to a polynucleotide represented by the complementary sequence of the base sequence; and the base sequence of SEQ ID NO: 9 A polynucleotide having at least 10 bases that hybridizes to a polynucleotide (B3) or a polynucleotide represented by a complementary sequence of the base sequence.

According to the second aspect of the present invention, the polynucleotide represented by the nucleotide sequence of SEQ ID NO: 13 or a polynucleotide of at least 10 nucleotides that hybridizes to the polynucleotide represented by the complementary sequence of the nucleotide sequence; Saccharomyces pastorianus comprising: a polynucleotide represented by the nucleotide sequence of SEQ ID NO: 14 or a polynucleotide of at least 10 nucleotides that hybridizes to a polynucleotide represented by a complementary sequence of the nucleotide sequence; Alternatively, a PCR primer set used for detection of Saccharomyces cerevisiae is provided.

  According to the primer set of the present invention, Saccharomyces genus yeast can be accurately detected at the bacterial species level. Moreover, the primer set according to the present invention can be used for nucleic acid amplification reaction by the LAMP method, and the target bacterial species can be detected by the presence or absence of an amplification product. Therefore, according to the primer set of the present invention, Saccharomyces genus yeast can be identified accurately, rapidly and simply at the bacterial species level.

  Saccharomyces genus yeast is a causative bacterium that causes turbidity of various beverages such as alcoholic beverages and soft drinks, and the presence / absence of these bacterium can be an index for quality control of various beverages. Therefore, the primer set according to the present invention is useful for quality control of various beverages (particularly alcoholic beverages and soft drinks) and inspection of environmental samples.

Detailed description of the invention

Primer and primer set The LAMP method primer set according to the present invention comprises four types of primers, FIP, F3, BIP, and B3, and these primers correspond to six regions of the target nucleotide sequence. Specifically, with respect to the target base sequence, regions F3c, F2c, F1c, B1, B2, and B3 are defined in order from the 3 ′ end to the 5 ′ end, and four types of these six regions are defined. Primers, FIP, F3, BIP and B3. Here, regions complementary to the regions F3c, F2c, and F1c are F3, F2, and F1, respectively, and regions complementary to the regions B1, B2, and B3 are B1c, B2c, and B3c, respectively.

  FIP is a primer prepared so as to have an F2 region complementary to the F2c region of the target sequence 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 prepared so as to have an F3 region complementary to the F3c region of the target gene.

  BIP is a primer prepared so as to have a B2 region complementary to the B2c region of the target sequence 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 prepared so as 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 amplification product can be observed as one band after electrophoresis by treating the amplification product with the restriction enzyme after the nucleic acid amplification reaction by the LAMP method. In this case, if the target sequence has a restriction enzyme site, it is not necessary to artificially introduce the restriction enzyme site into the primer.

  In carrying out the LAMP method primer set according to the present invention, one or two kinds of loop primers (LF primer or LB primer) may be added in order to accelerate the nucleic acid amplification reaction. When loop primers are designed to anneal to the region between F1-F2 or the region between B1-B2 and used in addition to the LAMP method reaction system, these primers are not used in the nucleic acid amplification step. By binding to the nucleic acid, the nucleic acid reaction proceeds starting from all loop portions, and the nucleic acid amplification reaction is accelerated (see, for example, JP-A-2002-345499).

  Specifically, the LAMP method primer set of the first aspect is at least 10 that hybridizes to the polynucleotide (LB) represented by the nucleotide sequence of SEQ ID NO: 5 or the polynucleotide represented by the complementary sequence of the nucleotide sequence. A base polynucleotide may further be included as a loop primer.

  In the LAMP method primer set of the second aspect, the polynucleotide (LB) represented by the nucleotide sequence of SEQ ID NO: 10 or a polynucleotide of at least 10 nucleotides that hybridizes to the polynucleotide represented by the complementary sequence of the nucleotide sequence Further, it may be further included as a loop primer.

  In the present invention, not only the polynucleotides represented by the nucleotide sequences of SEQ ID NOs: 1 to 14 but also polynucleotides that hybridize to the polynucleotides represented by the complementary sequences of the nucleotide sequences of SEQ ID NOs: 1 to 14 (hereinafter referred to as “homologous”). Polynucleotide ") may also be used as a primer. Needless to say, polynucleotides complementary to these polynucleotides can also be used as primers.

  In the present specification, “hybridize” means that it hybridizes to a target polynucleotide and does not substantially hybridize to a polynucleotide other than the target polynucleotide. Hybridization can be performed under stringent conditions. Here, the “stringent conditions” can be determined depending on the Tm (° C.) of the duplex between the primer sequence and its complementary strand, the necessary salt concentration, etc. Setting stringent conditions is a technique well known to those skilled in the art (see, for example, J. Sambrook, EF Frisch, T. Maniatis; Molecular Cloning 2nd edition, Cold Spring Harbor Laboratory (1989), etc.). Stringent conditions include hybridization reactions in appropriate buffers normally used for hybridization at temperatures slightly below the Tm determined by the nucleotide sequence (eg, 0 to about 5 ° C. below the Tm). It is mentioned to carry out. Stringent conditions also include performing washing after the hybridization reaction with a high-concentration low-salt concentration solution. Examples of stringent conditions include 37 ° C. (for about 14 base oligonucleotides), 48 ° C. (for about 17 base oligonucleotides), 55 ° C. in 6 × SSC / 0.05% sodium pyrophosphate solution. Examples include washing conditions at 60 ° C. (for oligonucleotides of about 23 bases) (for oligonucleotides of about 20 bases).

  The nucleotide length of the homologous polynucleotide is at least 10 bases.

  In the LAMP method primer, the nucleotide length of homologous polynucleotides of FIP and BIP is preferably at least 30 bases (for example, 30 to 60 bases), more preferably at least 42 bases (for example, 42 to 47 bases). it can. In addition, the nucleotide length of F3, B3, LF, and LB homologous polynucleotides is preferably at least 12 bases (eg, 12-30 bases), more preferably at least 20 bases (eg, 20-28 bases). can do.

  In the PCR method primer, the nucleotide length of the homologous polynucleotide of the polynucleotide represented by the nucleotide sequence of SEQ ID NOS: 11 to 14 is preferably at least 15 bases (for example, 15 to 30 bases), more preferably at least 17 bases. (For example, 17 to 26 bases).

  The homologous polynucleotides can each be a polynucleotide comprising at least 10, preferably at least 15, more preferably at least 18, particularly preferably at least 20 nucleotides of the corresponding base sequence. .

  Examples of the homologous polynucleotides of the polynucleotides represented by the nucleotide sequences of SEQ ID NOs: 1 to 14 are as follows.

-FIP homologous polynucleotide represented by the nucleotide sequence of SEQ ID NO: 1: at least 38 (38-46) contiguous nucleotides of SEQ ID NO: 1, more preferably at least 42 (42-46) nucleotides (1 Or a plurality of mutations may be introduced) (nucleotide length may be up to 60 bases, preferably up to 53 bases, more preferably up to 47 bases).

F3 homologous polynucleotide represented by the base sequence of SEQ ID NO: 2: at least 19 (19-22) contiguous nucleotides of SEQ ID NO: 2, more preferably at least 20 (20-22) nucleotides (1 Or a plurality of mutations may be introduced) (nucleotide length can be up to 30 bases, preferably up to 28 bases, more preferably up to 25 bases).

-BIP homologous polynucleotide represented by the nucleotide sequence of SEQ ID NO: 3: at least 36 (36-42) contiguous nucleotides of SEQ ID NO: 3, more preferably at least 38 (38-42) nucleotides (1 Or a plurality of mutations may be introduced) (nucleotide length may be up to 60 bases, preferably up to 53 bases, more preferably up to 47 bases).

-B3 homologous polynucleotide represented by the nucleotide sequence of SEQ ID NO: 4: at least 19 (19-23) contiguous nucleotides of SEQ ID NO: 4, more preferably at least 20 (20-23) nucleotides (1 Or a plurality of mutations may be introduced) (nucleotide length can be up to 30 bases, preferably up to 28 bases, more preferably up to 25 bases).

A homologous polynucleotide of LB represented by the nucleotide sequence of SEQ ID NO: 5: at least 20 (20-28) consecutive nucleotides of SEQ ID NO: 5, more preferably at least 23 (23-28) nucleotides (1 Or a polynucleotide comprising several mutations (nucleotide length can be up to 30 bases).

-FIP homologous polynucleotide represented by the nucleotide sequence of SEQ ID NO: 6: at least 38 (38-47) contiguous nucleotides of SEQ ID NO: 6, more preferably at least 42 (42-47) nucleotides (1 Or a polynucleotide comprising several mutations) (nucleotide length can be up to 60 bases, preferably up to 53 bases).

F3 homologous polynucleotide represented by the nucleotide sequence of SEQ ID NO: 7: at least 18 (18-20) contiguous nucleotides of SEQ ID NO: 7, more preferably at least 19 (19-20) nucleotides (1 Or a plurality of mutations may be introduced) (nucleotide length can be up to 30 bases, preferably up to 28 bases, more preferably up to 25 bases).

-BIP homologous polynucleotide represented by the base sequence of SEQ ID NO: 8: at least 36 (36-42) contiguous nucleotides of SEQ ID NO: 8, more preferably at least 38 (38-42) nucleotides (1 Or a plurality of mutations may be introduced) (nucleotide length may be up to 60 bases, preferably up to 53 bases, more preferably up to 47 bases).

-B3 homologous polynucleotide represented by the nucleotide sequence of SEQ ID NO: 9: at least 18 (18-20) contiguous nucleotides of SEQ ID NO: 9, more preferably at least 19 (19-20) nucleotides (1 Or a plurality of mutations may be introduced) (nucleotide length can be up to 30 bases, preferably up to 28 bases, more preferably up to 25 bases).

A homologous polynucleotide of LB represented by the base sequence of SEQ ID NO: 10: at least 18 (18-20) contiguous nucleotides of SEQ ID NO: 10, more preferably at least 19 (19-20) nucleotides (1 Or a plurality of mutations may be introduced) (nucleotide length can be up to 30 bases, preferably up to 28 bases, more preferably up to 25 bases).

-Homologous polynucleotide of the polynucleotide represented by the nucleotide sequence of SEQ ID NO: 11: at least 15 (for example, 15 to 26) of SEQ ID NO: 11, more preferably at least 17 (for example, 17 to 26) ), Particularly preferably a polynucleotide comprising at least 21 (for example 21 to 26) nucleotides (one or several mutations may be introduced) (nucleotide length is at most 30 bases, preferably Can be up to 28 bases).

-Homologous polynucleotide of the polynucleotide represented by the nucleotide sequence of SEQ ID NO: 12: at least 15 (for example, 15 to 21) consecutive of SEQ ID NO: 12, more preferably at least 17 (for example, 17 to 21) ), Particularly preferably a polynucleotide comprising at least 18 (e.g. 18-21) nucleotides (in which one or several mutations may be introduced) (nucleotide length is at most 30 bases, preferably Can be up to 26 bases, more preferably up to 24 bases).

-Homologous polynucleotide of the polynucleotide represented by the base sequence of SEQ ID NO: 13: at least 15 (for example, 15-18) consecutive in SEQ ID NO: 13, more preferably at least 17 (for example, 17-18) ) Nucleotides (one or several mutations may be introduced) (nucleotide length is at most 30 bases, preferably at most 26 bases, more preferably at most 21 bases) Can do).

-Homologous polynucleotide of the polynucleotide represented by the base sequence of SEQ ID NO: 14: at least 15 (for example, 15 to 17) consecutive of SEQ ID NO: 14, more preferably at least 16 (for example, 16 to 17) ) Nucleotides (one or several mutations may be introduced) (nucleotide length is at most 30 bases, preferably at most 26 bases, more preferably at most 21 bases) Can do).

  Homologous polynucleotides can also be polynucleotides each having at least 90%, preferably at least 95% identity with the corresponding base sequence. The numerical value of identity can be calculated according to an algorithm well known in the art, and is the same using, for example, BLAST (http://www.ddbj.nig.ac.jp/search/blast-j.html). Sexual values can be calculated.

  The homologous polynucleotide further comprises a modified base sequence in which one or several mutations are introduced into the base sequences of SEQ ID NOs: 1 to 14, respectively, and is represented by a complementary sequence of the base sequences of SEQ ID NOs: 1-14. It can be a polynucleotide that hybridizes to a polynucleotide.

  Here, the “mutation” may be the same or different, and can be selected from substitution, deletion, insertion, and addition, and preferably “single base substitution” in which one base is replaced with another base. And “single base deletion” in which a certain base is deleted, “single base insertion” in which a certain base is inserted, and “single base addition” in which a certain base is added. Further, the number of mutations can be 1 to 6, 1, 2, 3, or 4, 1 or 2, or 1.

  In the present invention, “polynucleotide” is used to mean DNA, RNA, and PNA (peptide nucleic acid).

  The polynucleotide constituting the primer set according to the present invention is chemically synthesized according to a normal method such as the phosphite triester method (Hunkapiller, M. et al., Nature, 310, 105, 1984). Or the total DNA of the strain to be detected may be obtained, and a DNA fragment containing the target nucleotide sequence based on the nucleotide sequence disclosed in the present specification may be appropriately obtained by PCR or the like. Good.

  The LAMP method primer set according to the present invention may be used alone or in appropriate combination. By carrying out the combination of the primer sets according to the present invention, it is possible to more accurately distinguish and detect Saccharomyces pastorianus, Saccharomyces cerevisiae, and Saccharomyces bayanus.

  The primer set according to the first aspect of the present invention can detect Saccharomyces pastorianus and Saccharomyces bayanus. The primer set according to the second aspect of the present invention can detect Saccharomyces pastorianus and Saccharomyces cerevisiae. When it is desired to accurately detect Saccharomyces pastorianus, Saccharomyces bayanus, and Saccharomyces cerevisiae for each bacterial species, the primer set of the first aspect according to the present invention and the primer set of the second aspect according to the present invention are combined. Are preferably used. In this case, when an amplification reaction is observed in both the primer set of the first aspect and the primer set of the second aspect according to the present invention, it can be determined that Saccharomyces pastorianus is present in the sample. . When an amplification reaction is observed in the primer set of the first aspect according to the present invention and no amplification reaction is observed in the primer set of the second aspect according to the present invention, it is determined that Saccharomyces bayanus is present in the sample. be able to. When an amplification reaction is observed in the primer set of the second aspect according to the present invention and no amplification reaction is observed in the primer set of the first aspect according to the present invention, it is determined that Saccharomyces cerevisiae is present in the sample. be able to. When no amplification reaction was observed in any of the primer set of the first aspect according to the present invention and the primer set of the second aspect according to the present invention, Saccharomyces pastorianus, Saccharomyces bayanus, Saccharomyces cerevisiae were included in the sample. It can be determined that none of these exist.

  Saccharomyces pastorianus, Saccharomyces cerevisiae, and Saccharomyces bayanus can be detected by using specific primers for each, but in that case, one sample needs to be examined with three primer sets. However, if the primer set of the first aspect and the primer set of the second aspect according to the present invention are used in combination, three bacterial species can be identified by using two primer sets for one sample, The amount of reagent used per sample can be reduced.

  Further, the Saccharomyces pastorianus can be more accurately determined by combining the primer set according to the second aspect of the present invention with a primer set for detecting Saccharomyces pastorianus. The primers for Saccharomyces pastorianus may cross-react with Saccharomyces bayanus whose genomic structure is not uniform. The primer set of the second aspect according to the present invention reacts with the genomic sequence of Saccharomyces cerevisiae contained in Saccharomyces pastorianus, but Saccharomyces bayanus does not have the genomic sequence of this Saccharomyces cerevisiae. It does not react with the primer set of the embodiment. In this case, when an amplification reaction is observed in both the Saccharomyces pastorianus detection primer set and the primer set of the second aspect of the present invention, it can be determined that Saccharomyces pastorianus is present in the sample. When an amplification reaction occurs with the Saccharomyces pastorianus detection primer and no amplification reaction is observed with the primer set according to the second aspect of the present invention, it can be determined that Saccharomyces bayanus is present in the sample.

  The primer set according to the present invention can also be provided alone or in combination in the form of a kit. Therefore, according to the present invention, there is provided a Saccharomyces yeast detection kit comprising the primer set of the first aspect, the primer set of the second aspect, or a combination thereof.

  The kit according to the present invention including the LAMP method primer set includes reagents (for example, Bst DNA polymerase, reaction reagent mixture) and instruments (for example, reaction tubes) necessary for carrying out the nucleic acid amplification reaction by the LAMP method. Also good.

  The kit according to the present invention containing the PCR method primer set may contain reagents (for example, DNA polymerase, purified water) and instruments (for example, reaction tubes) necessary for carrying out the nucleic acid amplification reaction by the PCR method.

  When the nucleic acid amplification reaction is carried out using the primer set according to the present invention, Saccharomyces genus yeast that causes a reduction in the quality of alcoholic beverages and soft drinks can be accurately identified at the bacterial species level. Therefore, the primer set and kit according to the present invention are used for quality control of alcoholic beverages (for example, beer, sparkling wine, wine, fruit wine, sake) and / or soft drinks (for example, fruit juice beverages) and environmental samples (for example, raw water). ).

  Saccharomyces pastorianus, Saccharomyces cerevisiae, and Saccharomyces bayanus may be found in beer and happoshu production processes or end products as yeast species that cause quality degradation. Therefore, the primer set according to the present invention and the combination thereof can be preferably used for quality control of beer and sparkling wine.

  Saccharomyces cerevisiae and Saccharomyces bayanus may be found in the wine production process or in the final product as yeast species that cause quality degradation. Therefore, the primer set according to the present invention and combinations thereof can preferably be used for wine quality control.

  Saccharomyces cerevisiae and Saccharomyces bayanus may be found as yeast species that cause quality degradation in the production process or final product of soft drinks (especially fruit juice drinks). Therefore, the primer set and the combination thereof according to the present invention can be preferably used for quality control of soft drinks (particularly fruit juice drinks).

Nucleic acid amplification reaction by LAMP method The LAMP method primer set according to the present invention can be used as a primer for nucleic acid amplification reaction by LAMP method. The primer set according to the present invention can be used not only as a LAMP method but also as a primer for a nucleic acid amplification reaction obtained by improving the LAMP method. The principle of the LAMP method and a nucleic acid amplification method using the LAMP method are well known, and for carrying out a nucleic acid amplification reaction by the LAMP method, for example, WO00 / 28082 and Notomi T. et al., Nucleic Acids Research, 28 (12 ), e63 (2000).

  The nucleic acid amplification reaction by the LAMP method can be performed according to a commercially available LAMP method gene amplification reagent kit. For example, sample DNA, primer solution, and a commercially available LAMP method gene amplification reagent kit (for example, manufactured by Eiken Chemical Co., Ltd.) The reagent provided in the Loopamp DNA amplification kit) is mixed according to the instructions attached to the kit, kept at a constant temperature (60 to 65 ° C.), and allowed to react for a certain time (1 hour as a standard).

The nucleic acid amplification reaction by the LAMP method can be carried out 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 type DNA polymerase starting from its 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 a 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 is synthesized starting from the 3 ′ end of this BIP. In this process, the loop is peeled off and stretched. Further, 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 the 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 of (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.

  It will be apparent to those skilled in the art that the nucleic acid amplification reaction by the LAMP method can be carried out by appropriately modifying the above steps. The primer set according to the present invention can also be used in such a modified method.

The LAMP method primer set according to the present invention causes DNA strand synthesis at the same time as annealing at about 60 to about 65 ° C. (eg, 65 ° C.). By performing the reaction for about 1 hour by an annealing reaction and DNA strand synthesis, the nucleic acid can be amplified 10 ⁹ to 10 ¹⁰ times.

  When the LAMP method primer set according to the present invention is reacted with a sample nucleic acid under the conditions of a nucleic acid amplification reaction by the LAMP method, the target region in the strain to be detected is amplified. When such an amplification reaction occurs, the reaction solution becomes cloudy due to the influence of magnesium pyrophosphate formed as a by-product, and therefore the presence or absence of amplification can be visually determined based on this turbidity. The presence / absence of amplification may be measured optically using a turbidity measuring apparatus, or may be detected by detecting the presence / absence of a DNA fragment using agarose gel electrophoresis or the like.

  If nucleic acid amplification is observed, it means that the target base sequence is present, and represents positive (+) for the species that is the detection target of the primer set. On the contrary, when nucleic acid amplification is not observed, it means that the target base sequence is absent and represents a negative bacterial species (-) which is a detection target of the primer set.

Nucleic acid amplification reaction by PCR method The PCR method primer set according to the present invention can be used for identification of Saccharomyces yeasts using nucleic acid amplification methods such as PCR method, RT-PCR method, real-time PCR method, in situ PCR and the like. .

  If a nucleic acid amplification product is observed, it means that the target base sequence is present, and represents a positive (+) species of bacteria to be detected by the primer set. On the contrary, when nucleic acid amplification is not observed, it means that the target base sequence is absent and represents a negative bacterial species (-) which is a detection target of the primer set.

  In the PCR method, a nucleic acid amplification product can be detected according to a known method such as agarose gel electrophoresis. In the real-time PCR method, nucleic acid amplification products can be detected over time in an apparatus in which a thermal cycler and a spectrofluorometer are integrated using an intercalator or a fluorescently labeled probe.

Samples to be detected and their preparation Samples to be detected by the primer sets and kits according to the present invention include alcoholic beverages such as beer, sparkling liquor and wine; soft drinks such as cider, ramune and carbonated water; collected for raw materials Environmental samples such as water; semi-finished products collected from production processes such as alcoholic beverages and soft drinks.

  When these are used as samples in the LAMP method or PCR method, operations such as concentration, separation, and culture of bacteria present in the sample, separation of nucleic acids from cells, and concentration of nucleic acids may be performed as pretreatment. Good. Examples of the method for concentrating and separating the bacteria present in the sample include filtration and centrifugation, and can be selected as appropriate. In addition, the number of bacteria may be increased by further culturing the bacteria concentrated and separated from the sample. For the cultivation, an agar solid medium or a liquid medium suitable for the growth of the target yeast strain can be used, and a drug such as copper sulfate may be added to select the target yeast strain. In order to release nucleic acids from bacterial cells present in beverage samples and environmental samples, or cultured bacterial cells, for example, the bacterial cells are treated with a method using a commercially available kit or an alkaline solution at 100 ° C. It is possible to select a method for releasing nucleic acid from bacterial cells by heating. If it is necessary to further purify the nucleic acid, the nucleic acid may be purified by phenol / chloroform treatment, ethanol precipitation, centrifugation, etc., and finally redissolved in TE buffer or the like to be used as a template DNA for testing ( 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, Taiji Oshima et al: Protein Nucleic Acid Enzyme, vol. 35, 2523-2541, 1990).

  Detection of Saccharomyces yeasts using the primer set and kit according to the present invention can be carried out, for example, as follows.

  First, the Saccharomyces genus yeast considered to be present in the sample is enriched and cultured in an appropriate medium. Next, DNA is isolated from the colonies formed on the agar medium, and the LAMP method or PCR method using the primer set according to the present invention is performed on this DNA to amplify a specific gene region of Saccharomyces yeast. The presence of the gene amplification product indicates the presence of the bacterial species targeted by the primer set.

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

Example 1: Detection of Saccharomyces yeast using LAMP primer set (a) Genomic DNA extraction method The cells cultured on an agar plate medium were scraped and suspended in sterile distilled water. This suspension was centrifuged (15000 rpm, 5 minutes), and the supernatant was discarded. Sterile distilled water was added again to the precipitated cells, suspended, and centrifuged. The supernatant was discarded, and 100 μl of a solution of PrepMan Ultra (Applied Biosystems) was added to the obtained bacterial cells, and heated at 95 ° C. for 10 minutes. Thereafter, the mixture was centrifuged at 15000 rpm for 1 minute, and the supernatant was used as a genomic DNA solution. Alternatively, 100 μl of 0.1N NaOH solution was added to the washed cells and heated at 95 ° C. for 10 minutes. Thereafter, the mixture was neutralized with 1M Tris buffer (pH 7.0), and the supernatant was used as a genomic DNA solution.

(B) LAMP primer The following detection primers are chemically synthesized by Fujitsu System Solutions' eGenome Order (http://genome.e-mp.jp/index.html) or equivalent method, and TE buffer is used. The solution (pH 8.0) was dissolved to a concentration of 100 μM. These solutions were mixed and diluted so that FIP, BIP primer: 16 μM, F3, B3 primer: 2 μM, LF, LB primer: 8 μM.
[LAMP method primer set LGF1LB1]
FIP: CTGTCGTGCGAAGCAACGTCTGTGAAAGAAGAAGGCAATAAAATCG (SEQ ID NO: 1)
F3: ACGTCTGGAAAAGACTAAGAGA (SEQ ID NO: 2)
BIP: GTCCGTGTTCCACGTAACCGCCCAGTTAATTGACCCAGACCA (SEQ ID NO: 3)
B3: TGGACATGTAATACATGATGGCA (SEQ ID NO: 4)
LB: CGTGCTTTGGTATATTCTTGTATGATGA (SEQ ID NO: 5)
[LAMP method primer set SCC1LB1]
FIP: CCTCTTCCAGTTCTTGACTCTTTTCCTAAGATGAAAGTGCCGGGAGA (SEQ ID NO: 6)
F3: ACGAAGATGAGAAAGAGGCG (SEQ ID NO: 7)
BIP: TGACAGCAAGGAGAAGAGCACCCCTCGTTGTTTTCCTCCTCA (SEQ ID NO: 8)
B3: GTGCTGTATGCTCGTTTTCG (SEQ ID NO: 9)
LB: GAGCAAGGGGACGAAGGTGA (SEQ ID NO: 10)

(C) Preparation of LAMP amplification reaction solution A Loopamp DNA amplification kit manufactured by Eiken Chemical Co., Ltd. was used as a gene amplification reagent kit for the LAMP method. To the reaction tube, add genomic DNA solution: 2.5 μl, primer solution: 2.5 μl, double concentration buffer for reaction: 12.5 μl, Bst DNA polymerase: 1 μl, sterile water: 6.5 μl, total volume of 25 μl The reaction solution was prepared.

(D) LAMP reaction For the LAMP reaction, a real-time turbidimeter LA-200 manufactured by Teramex or LA-320C manufactured by Eiken Chemical Co., Ltd. was used. A reaction tube was set and reacted at a constant 65 ° C. (bonnet was 75 ° C.), and the turbidity change was measured every 6 seconds. Those in which turbidity increased were positive, and those in which no increase in turbidity was observed were negative.

(E) Evaluation of primers When LGF1LB1 was used, amplification was observed with a delay from Saccharomyces bayanus in addition to the bottom fermentation yeast. When SCC1LB1 was used, amplification was also observed from Saccharomyces cerevisiae and Saccharomyces pastorianus.

  Thus, LGF1LB1 and SCC1LB1 cannot be said to be specific primers for only one bacterial species, but when both are combined, SCC1LB1 is positive and LGF1LB1 is negative, and SCC1LB1 is negative and LGF1LB1 is delayed. When it becomes positive, it can be determined as Saccharomyces bayanus, and when both SCC1LB1 and LGF1LB1 are positive, it can be determined as Saccharomyces pastorianus.

Table 1: Evaluation of primers using various Saccharomyces yeast standard strains (numbers in squares: reaction time until amplification occurs,-: no amplification within 80 minutes of reaction)

Table 2: Evaluation of primers using various yeast standard strains and brewer's yeast (numbers in mass: reaction time until amplification occurs,-: no amplification within 80 minutes of reaction)

Example 2: Detection of Saccharomyces yeasts by PCR method primers (a) PCR method primers The LSY method primer set LGF1LB1 was designed based on the FSY primer set for PCR method, and the LAMP method primer set SCC1LB1 was designed. A CYS primer set for PCR was designed from the original base sequence, and evaluated with various Saccharomyces yeasts.

Ex Taq manufactured by Takara Bio Inc. was used for the PCR method. The base sequence of each primer used for PCR is as follows.
[FSY primer set (amplification product: about 350 bp)]
FSY3: GGTATATTCTTGTATGATGATTGGTC (SEQ ID NO: 11)
FSY5: GCATATGATCCAAAAGCCATG (SEQ ID NO: 12)
[CYS primer set (amplification product: about 420 bp)]
CYS3S3: GTCAAGAACTGGAAGAGG (SEQ ID NO: 13)
CYS3S4: GTCCTGGTAGCGATTGT (SEQ ID NO: 14)

  These reagents are mixed according to the instructions of Ex Taq manufactured by Takara Bio Inc. (total amount 50 μl), set in a thermal cycler, and a temperature program of 94 ° C. for 30 seconds, 50 ° C. for 30 seconds, 72 ° C. for 1 minute is performed 25 times. The PCR reaction was performed repeatedly.

  As a result, when FSY primer set is used, bands of expected molecular weight appear in Saccharomyces bayanus and Saccharomyces pastorianus, and when CYS primer set is used, bands of expected molecular weight appear in Saccharomyces cerevisiae and Saccharomyces pastorianus, respectively. The PCR method primer set was found to exhibit the same detection characteristics as the LAMP method primer set.

Table 3: Evaluation of primers using various Saccharomyces yeast standard strains (+: with amplification,-: without amplification)

Example 3 Evaluation of Primers in Patent Literature (a) Primers in Patent Literatures The following Saccharomyces detection primer set (SSC1LB1) and bottom fermentation yeast described in Tsuchiya et al. (WO 2005/093059) A primer solution for the LAMP method was prepared in the same manner as in Example 1 (b) using the detection primer set (SBFY1LF1LB1). The Saccharomyces genus primer set (SSC1LB1) targets the D2 region of the rRNA gene, and the bottom fermenting yeast detection primer set (SBFY1LF1LB1) targets the melibiase gene.
[Primer set for detecting the genus Saccharomyces (SSC1LB1)]
FIP: TGCGAGATTCCCCTACCCCAGACATGGTGTTTTGTGCC (SEQ ID NO: 15)
F3: AGACCGATAGCGAACAAGTA (SEQ ID NO: 16)
BIP: CTGTGGGAATACTGCCAGCTGGCCGTGTTTCAAGACGGGCGG (SEQ ID NO: 17)
B3: CTTGGTCCGTGTTTCAAGAC (SEQ ID NO: 18)
LB: CAAGGATGCTGGCATAATGGTT (SEQ ID NO: 19)
[Primer set for detection of bottom fermentation yeast (SBFY1LF1LB1)]
FIP: GCAATTGCTCACTGACGTCGCACACCCCTCAAATGGGTTGG (SEQ ID NO: 20)
F3: CCGAGTTACAATGGCCTTGG (SEQ ID NO: 21)
BIP: ACCGCTGACCGGATTTCTGAAAACTAGACCAGCAGTCATCCA (SEQ ID NO: 22)
B3: CCTTGTCTGCAACGAGTGT (SEQ ID NO: 23)
LF: GGCAAATGTGTTCCAATTGTC (SEQ ID NO: 24)
LB: GGACTAAAGGATTTGGGTTACAC (SEQ ID NO: 25)

  According to the description of Example 1 (c) and (d), various strains were detected by the LAMP method using the above primer set. The results were as shown in FIG. 1 and FIG.

(B) Evaluation of primers As a result, it was found that SSC1LB1 reacts with most of the Saccharomyces yeasts tested and cannot be distinguished among Saccharomyces yeasts. SBFY1LF1LB1 reacted with Saccharomyces bayanus in addition to the bottom fermentation yeast. SBFY1LF1LB1 is designed from the melibiase gene of bottom fermentation yeast, but the region used for the design was thought to have cross-reacted because a 96% homologous base sequence was also present in the genome of Saccharomyces bayanus. It was.

It is the figure which showed the reaction specificity to the detection object microbial species of the primer set for Saccharomyces genus detection (SSC1LB1). The strains used are as follows. Saccharomyces cerevisiae NBRC10217, Saccharomyces bayanus NBRC11022, Saccharomyces pastorianus NBRC11024, NBRC11023, NBRC10610, Saccharomyces cerevisiae Diaz Tati dregs DSM70487, Saccharomyces Paradokusasu NBRC10609, Saccharomyces Kariokanusu NBRC10947, Saccharomyces Mikatae NBRC1815, Saccharomyces Kudoriavuzevi NBRC1802, Saccharomyces Exigs NBRC1128, Saccharomyces cervusii NBRC1838, Saccharomyces unisporus NBRC0316, Saccharomyces direnensis NBRC0211, Saccharomyces kluyveri NBRC1685, Nega Genomic DNA additive-free. It is the figure which showed the reaction specificity to the detection object microbial species of the primer set for bottom fermentation yeast detection (SBFY1LF1LB1). The strains used are as follows. Saccharomyces cerevisiae NBRC10217, Saccharomyces bayanus NBRC11022, Saccharomyces pastorianus NBRC11024, NBRC11023, NBRC10610, Saccharomyces cerevisiae Diaz Tati dregs DSM70487, Saccharomyces Paradokusasu NBRC10609, Saccharomyces Kariokanusu NBRC10947, Saccharomyces Mikatae NBRC1815, Saccharomyces Kudoriavuzevi NBRC1802, Saccharomyces Exigs NBRC1128, Saccharomyces cervusii NBRC1838, Saccharomyces unisporus NBRC0316, Saccharomyces direnensis NBRC0211, Saccharomyces kluyveri NBRC1685, Nega Genomic DNA additive-free.

Claims (11)

Comprising the following polynucleotides, Saccharomyces pastorianus (Saccharomyces pastorianus) and / or LAMP method The primer set used to detect a Saccharomyces bayanus (Saccharomyces bayanus):
A polynucleotide of at least 10 bases that hybridizes to a polynucleotide represented by the base sequence of SEQ ID NO: 1 (FIP) or a polynucleotide represented by a complementary sequence of the base sequence;
A polynucleotide of at least 10 bases that hybridizes to the polynucleotide represented by the nucleotide sequence of SEQ ID NO: 2 (F3) or the polynucleotide represented by the complementary sequence of the nucleotide sequence;
A polynucleotide (BIP) represented by the nucleotide sequence of SEQ ID NO: 3 or a polynucleotide of at least 10 nucleotides that hybridizes to a polynucleotide represented by the complementary sequence of the nucleotide sequence; and represented by the nucleotide sequence of SEQ ID NO: 4. A polynucleotide having at least 10 bases that hybridizes to a polynucleotide (B3) or a polynucleotide represented by a complementary sequence of the base sequence.   The polynucleotide (LB) represented by the nucleotide sequence of SEQ ID NO: 5 or a polynucleotide of at least 10 nucleotides that hybridizes to the polynucleotide represented by the complementary sequence of the nucleotide sequence thereof. Primer set. Comprising the following polynucleotides, Saccharomyces pastorianus (Saccharomyces pastorianus) and / or Saccharomyces cerevisiae (Saccharomyces cerevisiae) LAMP method The primer set used to detect a:
A polynucleotide of at least 10 bases that hybridizes to a polynucleotide represented by the base sequence of SEQ ID NO: 6 (FIP) or a polynucleotide represented by a complementary sequence of the base sequence;
A polynucleotide of at least 10 bases that hybridizes with the polynucleotide (F3) represented by the nucleotide sequence of SEQ ID NO: 7 or the polynucleotide represented by the complementary sequence of the nucleotide sequence;
A polynucleotide (BIP) represented by the base sequence of SEQ ID NO: 8 or a polynucleotide of at least 10 bases that hybridizes to a polynucleotide represented by the complementary sequence of the base sequence; and the base sequence of SEQ ID NO: 9 A polynucleotide having at least 10 bases that hybridizes to a polynucleotide (B3) or a polynucleotide represented by a complementary sequence of the base sequence.   The polynucleotide (LB) represented by the nucleotide sequence of SEQ ID NO: 10 or a polynucleotide of at least 10 nucleotides that hybridizes to the polynucleotide represented by the complementary sequence of the nucleotide sequence thereof. Primer set. Comprising the following polynucleotides, Saccharomyces pastorianus (Saccharomyces pastorianus) and / or PCR methods primer set used for the detection of Saccharomyces bayanus (Saccharomyces bayanus):
A polynucleotide represented by the base sequence of SEQ ID NO: 11 or a polynucleotide of at least 10 bases that hybridizes to a polynucleotide represented by the complementary sequence of the base sequence; and a polynucleotide represented by the base sequence of SEQ ID NO: 12, A polynucleotide of at least 10 bases that hybridizes to a polynucleotide represented by a complementary sequence of the base sequence. Comprising the following polynucleotides, Saccharomyces pastorianus (Saccharomyces pastorianus) and / or Saccharomyces cerevisiae (Saccharomyces cerevisiae) PCR method primer set used for the detection of:
A polynucleotide represented by the base sequence of SEQ ID NO: 13 or a polynucleotide of at least 10 bases that hybridizes to a polynucleotide represented by the complementary sequence of the base sequence; and a polynucleotide represented by the base sequence of SEQ ID NO: 14, or A polynucleotide of at least 10 bases that hybridizes to a polynucleotide represented by a complementary sequence of the base sequence.   Polynucleotides comprising at least 10 bases that hybridize to a polynucleotide represented by a complementary sequence of the base sequences of SEQ ID NOs: 1 to 14 are each a polynucleotide comprising at least 10 consecutive nucleotides of the corresponding base sequence. The primer set according to any one of claims 1 to 6.   The polynucleotide of at least 10 bases that hybridizes to the polynucleotide represented by the complementary sequence of the base sequences of SEQ ID NOs: 1 to 14 is a polynucleotide having at least 90% identity with the corresponding base sequence, respectively. Item 7. The primer set according to any one of Items 1 to 6.   A modified nucleotide sequence in which at least 10 nucleotides hybridizing to a polynucleotide represented by a complementary sequence of the nucleotide sequences of SEQ ID NOs: 1 to 14 are modified in one or several nucleotides in the corresponding nucleotide sequences, respectively The primer set according to claim 1, which is a polynucleotide that hybridizes to a polynucleotide represented by a complementary sequence of a corresponding base sequence.   A kit for detecting Saccharomyces yeast, comprising a combination of the LAMP method primer set according to claim 1 or 2 and the LAMP method primer set according to claim 3 or 4.   A kit for detecting Saccharomyces yeast, comprising a combination of the PCR method primer set according to claim 5 and the PCR method primer set according to claim 6.

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