JP2004121259A - Detection of pectinatus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for detecting a beer destructive fungus of Pectinatus, to provide a genetic sequence therefor, and to provide a method for detecting the fungus by using the sequence. <P>SOLUTION: The oligonucleotide comprises a sequence targeting a nucleotide cording for 16S ribosome RNA gene of Pectinatus and selected from specific sequences complementary to the nucleotide sequence or corresponding complementary sequences so as to selectively detect Pectinatus cerevisiiphillus belonging to the genus Pectinatus in a sample. <P>COPYRIGHT: (C)2004,JPO

Description

TECHNICAL FIELD The present invention relates to the detection of a beer harmful bacterium of the genus Pectinatus. More specifically, beer harmful bacteria, Pectinatus cerevisiiphilus (P. cerevisiiphillus), Pectinatus flisingensis (P. frisingensis), and Pectinatus
The present invention relates to a gene sequence for detecting each of the sp. DSM20764 bacteria and a method for detecting the same using the same.

  At present, in the microbiological test in beer production, in order to identify the bacterial species, the bacteria must be isolated through growth culture and separation culture, and it takes at least 7 days. Thereafter, the isolated bacteria are grown, and identification is performed by performing many property tests such as morphological observation, Gram staining, catalase test, and sugar utilization. These diverse tests are cumbersome, time consuming and expensive. In addition to these commonly used identification tests, DNA is extracted from the isolated bacteria, immobilized on a membrane or other support, and a hybridization test is performed using the DNA of the standard bacteria as a probe. Is performed to identify the bacterial species. However, this method also requires several days, and it is difficult to obtain sufficient detection sensitivity and selectivity.

As a more rapid bacterial detection method, recently, there is a method of detecting live bacteria by an ATP luminescence method using bioluminescence (see Non-Patent Document 1). Is not available at all. Also, some lactic acid bacteria harmful to beer, for example, Lactobacillus brevis, Lactobacillus casei,
Lactobacillus plantarum, Lactobacillus coryniformis and the like can be identified relatively early by using an antibody specific to lactic acid bacteria (see Patent Documents 1 and 2). However, the application of this method requires an operation until the bacteria are isolated, which requires days, and it is difficult to obtain sufficient detection sensitivity and selectivity.

Therefore, a quicker detection method has been studied, and recently, there is a method of extracting DNA of a lactic acid bacterium as a specimen and detecting the lactic acid bacterium using a PCR method in which an oligonucleotide complementary to this DNA functions as a primer. (See Patent Literature 3, Patent Literature 4, Non-Patent Literature 2).

On the other hand, in recent years, it has been confirmed that Pectinatus genus, which is an obligately anaerobic bacterium, is a bacterium having an adverse effect on product beer. For the detection of this bacterium, examination of a selective separation medium for detecting Pectinatus bacteria (see Non-Patent Document 3), identification of major antigenic determinants by immunoblot analysis (see Non-Patent Document 4), fluorescence immunoassay Has been attempted (see Non-Patent Document 5), but a method that is satisfactory in terms of detection sensitivity, detection time, and the like has not yet been established.
JP-A-6-46811 JP-A-6-311894 JP-A-5-15400 JP-A-6-141899 J. Am. Soc. Brew. Chem .: 52 (1) 19-23, 1994 J. Am. Soc. Brew. Chem .: 52 (3) 95-99, 1994 J. Am. Soc. Brew. Chem .: 52 (3) 115-119, 1994. FEMS. MICROBIOL. LETT .: 67 (3) 307-311, 1990 J. Am. Soc. Brew. Chem .: 51 (4) 158-163, 1993

Therefore, an object of the present invention is to develop a method that can more quickly and accurately detect a bacterium belonging to the genus Pectinatus and simultaneously identify it.

The present invention is characterized in that oligonucleotides specifically hybridizing with the 16S ribosomal gene of the genus Pectinatus are prepared, and these are used for gene amplification by functioning as primers, and each of the bacteria is selectively detected. It provides a method.

Oligonucleotides that specifically hybridize to Pectinatus cerevisiae are the following sequences 1 to 4,
5'-CAGGCGGATGACTAAGCG-3 '1
5'-TGGGATTCGAACTGGTCA-3'2
5'-CTCAAGATGACCAGTTCG-3 '3
5'-AATATGCATCTCTGCATACG-3 '4
Consisting of a sequence selected from or corresponding to a complementary sequence, ie
For the sequence of No. 1, 5'-CGCTTAGTCATCCGCCTG-3 '
For the sequence of 2, 5'-TGACCAGTTCGAATCCCA-3 '
For the sequence of No. 3, 5'-CGAACTGGTCATCTTGAG-3 '
For the sequence of No. 4, 5'-CGTATGCAGAGATGCATATT-3 '
It is characterized by being.

Oligonucleotides that specifically hybridize to Pectinus furysingensis are the following sequences 5 to 8,
5'-CAGGCGGAACATTAAGCG-3 '5
5'-ATGGGGTCCGAACTGAGG-3 '6
5'-CTCAAGAACCTCAGTTCG-3 '7
5'-AATATCCATCTCTGGATACG-3 '8
Consisting of a sequence selected from or corresponding to a complementary sequence, ie
For the sequence of 5, 5'-CGCTTAATGTTCCGCCTG-3 '
For the sequence of No. 6, 5'-CCTCAGTTCGGACCCCAT-3 '
For the sequence of No. 7, 5'-CGAACTGAGGTTCTTGAG-3 '
For the sequence of 8, 5′-CGTATCCAGAGATGGATATT-3 ′
It is characterized by being.

Further, an oligonucleotide that targets a nucleotide sequence encoding the 16S ribosomal RNA gene of Pectinus sp. DSM20764 and is complementary to the nucleotide sequence, wherein the oligonucleotide has the following sequences 9 to 10,

5'-TGGGGTCCGAACTGAATG-3 '9

5'-GCATCCATCTCTGAATGCG-3 '10
Consisting of a sequence selected from or corresponding to a complementary sequence, ie
For the sequence of No. 9, 5'-CATTCAGTTCGGACCCCA-3 '
For 10 sequences, 5'-CGCATTCAGAGATGGATGC-3 '
It is characterized by being.

The present invention also relates to a DNA encoding 16S ribosomal RNA of Pectinatus sp. DSM20764. And, the entire base sequence is shown in SEQ ID NO: 1 below. Pectinatus sp. DSM20764 is a German strain preservation agency.
DSM-Deutche Sammlung von
It is a fungus available from Mikroorganismen und Zellkulturen GmbH.

Techniques related to gene amplification are already known, and Polymerase Chain Reaction method (hereinafter abbreviated as PCR method) developed by Saiki et al .; Science
230, 1350, 1985). This method is a reaction that amplifies a specific gene sequence, and has rapid, high sensitivity, high specificity, and simplicity. Also, applications have been attempted for rapid detection of harmful bacteria in the food field. By performing the PCR method, even if only a small amount of the target sequence is present in the sample, the target nucleotide sequence sandwiched between the two primers is amplified by a factor of millions, A copy is produced. In addition, in order to perform the PCR method, it is necessary to release the nucleic acid component from the bacteria present in the sample.However, in PCR, if the target sequence is present in several molecules or more, the amplification reaction proceeds. The test can be sufficiently performed only by performing the simple pretreatment used. Therefore, the utility value is extremely high as compared with the conventional bacteria detection method.

In order to take advantage of these facts, the present invention provides that Pectinatus cerevisiae, Pectinatus flisingensis and Pectinatus
sp.DSM20764 by performing PCR using an oligonucleotide created to specifically hybridize with the 16S ribosomal gene of the bacterium as a primer, and detecting the sequence to be recognized, the pectinatus cerevisiaus, pectinatus Vlissingensis or Pectinatus
We developed a method to quickly and sensitively determine whether DSM20764 bacteria exist. The specimen may be beer or a semi-finished product during beer production, or a sample collected from an anaerobic environment such as sewage. Oligonucleotides used as primers may be chemically synthesized or natural, and any of them may be used.

The base length of the target sequence on the nucleotide sequence of the 16S ribosomal gene of Pectinatus cerevisiae defined by the two primers is about 70 base pairs when primers 1 and 3 are combined, and when the primers 2 and 4 are combined. It is about 390 base pairs and about 440 base pairs when primers 1 and 4 are combined. Since any combination of these primers is specific to Pectinatus cerevisiae, the combination of these primers can be detected. However, by using two or more pairs in parallel, more reliable identification can be performed.

The base length of the target sequence on the nucleotide sequence of the 16S ribosomal gene of Pectinatus flissingensis stipulated by the two primers was about 70 base pairs when primers 5 and 7 were combined, and primers 6 and 8 were combined. In this case, it is about 390 base pairs, and when primers 5 and 8 are combined, it is about 440 base pairs. Since any combination of these primers is specific to Pectinus furysingensis bacteria, this species can be detected. However, by using two or more pairs in parallel, more reliable identification can be performed.

Pectinatus defined by two primers 9 and 10
The base length of the target sequence on the nucleotide sequence of the 16S ribosomal RNA gene of sp. DSM20764 is about 390 base pairs. This combination of primers is
Since it is specific to sp. DSM20764, this strain can be detected.

It is sufficient that the DNA polymerase used in the PCR reaction has heat resistance at a temperature of 90 ° C. or higher. The temperature conditions in the PCR reaction are 90-98 ° C. in a heat denaturation reaction for converting double-stranded DNA into a single strand, 37-65 ° C. in an annealing reaction in which primers are hybridized to a template DNA, and a chain length reaction in which a DNA polymerase is allowed to act. The target sequence can be amplified by performing the above at 50 to 75 ° C., and performing this for one cycle for several tens of cycles. After the PCR reaction, the reaction product is separated by electrophoresis, and subjected to nucleic acid staining with ethidium bromide or the like.If the base length of the amplified nucleotide sequence is equal to the base length of the above-described target sequence, Pectinatus cerevisiaus is contained in the sample. Bacterium, Pectinatus furissingensis, or Pectinatus
sp. DSM20764 It can be determined that bacteria existed. Chromatography is also effective in detecting the amplified nucleotide sequence.

The present invention will be described with reference to examples.
(1) Method for detecting Pectinatus cerevisiae
Preparation of specimen As bacteria belonging to the genus Pectinatus, 10 strains of 3 species shown in Table 1 were used. In addition, in order to confirm the specificity of the brimer for Pectinatus cerevisiae, bacteria and yeasts that can be tested in addition to Pectinatus bacteria in a beer microbial test, and Escherichia coli that is commonly used in laboratories were used. After culturing them in an appropriate growth medium, the cells were collected by centrifugation. After that, DNA extraction from cells was carried out by
Nucleic acid I separation and purification
The procedure was performed according to p20-21 (edited by The Biochemical Society of Japan, Tokyo Kagaku Dojin), and 1 ml of each DNA solution was obtained.

Synthesis of primers The base sequence of the 16S ribosomal RNA gene of Pectinatus cerevisiae (KARL HEINZ SCHLEIFER, and HELGA SEIDEL-RUFER et.
al., Int. J. Syst. Bacteriol. 40 (1): 19-27, 1990), the following sequence groups 1 to 4 were selected, and oligonucleotides having the same sequences were chemically synthesized.
5'-CAGGCGGATGACTAAGCG-3 '1
5'-TGGGATTCGAACTGGTCA-3'2
5'-CTCAAGATGACCAGTTCG-3 '3
5'-AATATGCATCTCTGCATACG-3 '4

PCR
In a sterilized 0.5 ml tube, 1 μl of the sample solution, 78.5 μl of sterilized water, 10 × reaction buffer (manufactured by Toyobo Co., Ltd.)
10 × TAP) 10 μl, 25 mM aqueous magnesium chloride solution 6 μl, 20 mM dNTPs 1 μl, 100 μM primers 1 and 3 (or 2 and 4, or 1 and 4) each 1 μl, 5 U / μl Taq DNA polymerase (Toyobo TAP-101) 0.5 μl was added to prepare a total of 100 μl of the reaction solution. This is done under the following temperature conditions:
Thermal denaturation; 94 ° C
1 minute annealing; 54 ℃
1 minute chain length reaction; 74 ° C
One minute was defined as one cycle, and this was repeated 30 cycles to perform PCR. Before starting the cycle reaction,
1 minute, 74 ° C after completion of cycle reaction
Heat treatment was performed for 3 minutes. The temperature controller used was a program balance control system PC-800 manufactured by ASTEC.

detection
Using 10 μl of the reaction solution after the completion of PCR, electrophoresis was carried out on a 3.5% (w / v) agarose gel at a constant voltage of 100 V for 30 minutes. In addition to the reaction solution, φX174 / HincII was also electrophoresed as a molecular weight marker. After completion of the electrophoresis, the gel was stained in an ethidium bromide solution (about 0.5 μg / ml) for 15 minutes, and the gel was observed under ultraviolet irradiation and photographed. From the observation or photograph of the gel, the base length of the amplification product was determined from the relative mobility to the molecular weight marker.

Results The results of electrophoresis of the specimens of bacterial numbers 2 and 5 in Table 1 after the PCR reaction are shown in FIG.
Table 2 summarizes the results of the detection bands for all the bacteria tested.

In the table, the numbers indicate the base length (kilobase pairs) of the detection band, and (-) indicates that no band was detected.

From these results, when the oligonucleotides of the above-described sequences 1 to 4 were used as primers for PCR in the combinations shown in Table 2, any combination was detected as a band only in Pectinatus cerevisiae, Moreover, all the detected bands had the same number of bases as the target sequence. This indicates that each oligonucleotide of the present invention correctly recognizes the target sequence of the 16S ribosomal RNA gene of Pectinatus cerevisiae. And, since no band is detected in other strains, including the same genus Pectinatus furysingensis bacterium, the present invention shows that Pectinatus cerevisibillus can be detected in a species-specific manner. It was shown that identification can be performed simultaneously with detection.

(2) Method for detecting Pectinatus furissingensis bacteria
Preparation of specimen As bacteria belonging to the genus Pectinatus, 10 strains of 3 species shown in Table 1 were used in the same manner as described above. In addition, in order to confirm the specificity of the primers for Pectinatus furissingensis bacteria, bacteria and yeasts that can be tested in addition to Pectinatus bacteria in the beer microorganism test, and Escherichia coli that is commonly used in laboratories were used. After culturing them in an appropriate growth medium, the cells were collected by centrifugation. After that, DNA extraction from cells was carried out by
Nucleic acid I separation and purification
The procedure was performed according to p20-21 (edited by The Biochemical Society of Japan, Tokyo Kagaku Dojin), and 1 ml of each DNA solution was obtained.

Synthesis of primers The base sequence of the 16S ribosomal RNA gene of Pectinus furysingensis (KARL HEINZ SCHLEIFER, and HELGA SEIDEL-RUFER et
al, Int. J. Syst. Bacteriol. 40 (1): 19-27, 1990), the following sequence groups 5 to 8 were selected, and oligonucleotides having the same sequences were chemically synthesized.
5'-CAGGCGGAACATTAAGCG-3 '5
5'-ATGGGGTCCGAACTGAGG-3 '6
5'-CTCAAGAACCTCAGTTCG-3 '7
5'-AATATCCATCTCTGGATACG-3 '8

PCR
In a sterilized 0.5 ml tube, 1 μl of the sample solution, 78.5 μl of sterilized water, 10 × reaction buffer (manufactured by Toyobo Co., Ltd.)
10 × TAP) 10 μl, 25 mM aqueous magnesium chloride solution 6 μl, 20 mM dNTPs 1 μl, 100 μM primers 5 and 7 (or 6 and 8, or 5 and 8) each 1 μl, 5 U / μl Taq DNA polymerase (Toyobo TAP-101) 0.5 μl was added to prepare a total of 100 μl of the reaction solution. This is done under the following temperature conditions:
Thermal denaturation; 94 ° C
1 minute annealing; 54 ℃
1 minute chain length reaction; 74 ° C
One minute was defined as one cycle, and this was repeated 30 cycles to perform PCR. Before starting the cycle reaction,
1 minute, 74 ° C after completion of cycle reaction
Heat treatment was performed for 3 minutes. The temperature controller used was a program balance control system PC-800 manufactured by ASTEC.

detection
Using 10 μl of the reaction solution after the completion of PCR, electrophoresis was carried out on a 3.5% (w / v) agarose gel at a constant voltage of 100 V for 30 minutes. In addition to the reaction solution, φX174 / HincII was also electrophoresed as a molecular weight marker. After completion of the electrophoresis, the gel was stained in an ethidium bromide solution (about 0.5 μg / ml) for 15 minutes, and the gel was observed under ultraviolet irradiation and photographed. From the observation or photograph of the gel, the base length of the amplification product was determined from the relative mobility to the molecular weight marker.

Results The results of electrophoresis of the specimens of bacterial numbers 2 and 5 in Table 1 after the PCR reaction are shown in FIG.
Table 3 summarizes the results of the detection bands for all the bacteria tested.

In the table, the numbers indicate the base length (kilobase pairs) of the detection band, and (-) indicates that no band was detected.

From these results, when the oligonucleotides of the above-mentioned sequences 5 to 8 were used as primers for PCR in the combinations shown in Table 3, bands were detected only in Pectinatus furysingensis bacteria in any of the combinations. All the detected bands had the same number of bases as the target sequence. This indicated that each oligonucleotide of the present invention correctly recognized the target sequence of the 16S ribosomal RNA gene of Pectinus furysingensis. In addition, since no band is detected in other strains, including the same genus Pectinatus cerevisiae, the present invention shows that pectinatas furysingensis can be detected in a species-specific manner. It was shown that the bacteria can be detected and identified at the same time.

(3) Pectinatus
sp. DSM20764 Detection of bacteria
Preparation of specimensPectinatus bacteria belonging to the genus Pectinatus cerevisiiphilus (DSM20467), Pectinatus
frisingensis (DSM6306) and Pectinatus sp. DSM20764 were used. In order to confirm the specificity of the primer for Pectinatus sp. DSM20764, other obligate anaerobic bacteria shown in Table 4 were used. After culturing them in an appropriate growth medium, the cells were collected by centrifugation. After that, DNA extraction from cells was carried out by
Nucleic acid I was separated and purified according to p20-21 (edited by The Biochemical Society of Japan, Tokyo Kagaku Dojin) to obtain a DNA solution.

Pectinatas
Determination of base sequence of 16S ribosomal RNA gene of sp. DSM20764 bacterium Pectinatus prepared as described above
Using the DNA solution of sp. DSM20764 as a template,
Microbiological Methods 'Nucleic Acid Techniques in Bacterial Systematics'
PCR using a primer having an M13 primer sequence arranged 5 ′ to a universal primer common to many bacteria described in “16S / 23S rRNA Sequencing” p115 to 175 (J. Wiley & Sons Ltd., New York) Was done.

The amplified DNA fragment after PCR was separated and cut out by agarose gel electrophoresis, eluted from the gel using SUPRECTM-01 (trade name, manufactured by Takara Shuzo), and recovered by ethanol precipitation. Using the thus obtained DNA fragment as a template, a sequence reaction was performed. Sequencing primers are trade names
IRD41 Infared Dye Labeled M13
Forward primer (manufactured by Nisshinbo, sold by Aloka Co., Ltd.), and the reaction solution was SequiTherm (registered trademark) Long-Read (registered trademark) Cycle Sequencing Kit-LC (EPICENTRE
TECHNOLOGIES). The nucleotide sequence was determined using 4000L Long ReadIR (registered trademark) DNA Sequencing System (manufactured by LI-COR).
The obtained pectinatas
The 16S ribosomal RNA gene sequence of sp. DSM20764 is shown in SEQ ID NO: 1.

Synthesis of primers Pectinus of SEQ ID NO: 1
Oligonucleotides having the same sequence as the sequence from 630 to 647 on the 16S ribosomal RNA gene sequence of sp. DSM20764 and a sequence complementary to the sequence from 1004 to 1022 were chemically synthesized.
Pectinatus using primers
Detection and identification of sp. DSM20764 bacteria The DNA solution of each bacteria obtained in the preparation of the specimen was subjected to PCR using the synthesized primers. PCR is performed under the following temperature conditions:
Heat denaturation; 94 ° C for 30 seconds Annealing; 55 ° C for 30 seconds Chain length reaction; 72 ° C for 30 seconds as one cycle, and this cycle was repeated 35 cycles. After completion of PCR, the reaction solution was subjected to electrophoresis on an agarose gel at a constant voltage of 100 V for 30 minutes. In addition to the reaction solution, φX174 / HincII was also electrophoresed as a molecular weight marker. After completion of the electrophoresis, the gel was stained in an ethidium bromide solution of about 0.5 μg / ml for 20 minutes, and the gel was observed under ultraviolet irradiation and photographed. The base length of the amplification product was determined from the relative mobility with respect to the molecular weight marker from the observation or photograph of the gel.
As a result, as shown in FIG.
A band of about 390 bps was detected only in sp. DSM20764.

From these results, it was found that when the oligonucleotide was used as a PCR primer,
A band of the desired length was detected only in sp. DSM20764. This indicates that each oligonucleotide of the present invention
It was shown that the target base sequence on the 16S ribosomal RNA gene of sp. DSM20764 was correctly recognized. And Pectinatus cerevisiiphilus and Pectinatus of the same family
Since no band of the target length was detected even in closely related obligate anaerobic bacteria such as frisingensis, the present invention
It was shown that sp. DSM20764 bacteria can be detected in a species-specific manner, and that the bacteria can be detected and identified at the same time.

In the conventional method, it took at least about 10 days to identify the species of the genus Pectinatus, but by using the present invention, it is not always necessary to perform preculture, and it is also necessary to perform separate culture. Within 1 to 3 days, quickly and clearly, in the specimens Pectinatus cerevisiae, Pectinatus furysingensis, or Pectinatus
sp. DSM20764 can be detected and identified at the same time. In addition, the PCR method is easy to operate, including sample pretreatment, and uses inexpensive chemicals, instruments, and equipment. It becomes possible. Furthermore, recently, PCR peripheral devices have made great progress, and if they are used in the present invention, it will be possible to fully automate the test for Pectinatus bacteria.

FIG. 1 is a diagram showing the results of electrophoresis after PCR reaction of specimens of bacterial numbers 2 and 5 in Table 1 in combinations of primers 1 to 4. FIG. 2 is a view showing the results of electrophoresis after PCR reaction of specimens of bacterial numbers 2 and 5 in Table 1 in combinations of primers 5 to 8. FIG. 3 is a view showing the results of electrophoresis after PCR reaction of specimens of bacterial numbers 1 to 9 in Table 4 in the combination of primers 9 and 10.

Claims (7)

To selectively detect P. cerevisiiphillus belonging to the genus Pectinatus present in a sample, the nucleotide sequence encoding the 16S ribosomal RNA gene of the genus Pectinatus is targeted. An oligonucleotide comprising a sequence selected from the following sequences 1 to 4 which is complementary to, or a corresponding complementary sequence to:
5'-CAGGCGGATGACTAAGCG-3 '1
5'-TGGGATTCGAACTGGTCA-3'2
5'-CTCAAGATGACCAGTTCG-3 '3
5'-AATATGCATCTCTGCATACG-3 '4 To selectively detect P. frisingensis bacteria belonging to the genus Pectinatus present in a specimen, the nucleotide sequence encoding the 16S ribosomal RNA gene of the genus Pectinatus is targeted. An oligonucleotide comprising a sequence selected from the following sequences 5 to 8 that is complementary to a nucleotide sequence, or a corresponding complementary sequence.
5'-CAGGCGGAACATTAAGCG-3 '5
5'-ATGGGGTCCGAACTGAGG-3 '6
5'-CTCAAGAACCTCAGTTCG-3 '7
5'-AATATCCATCTCTGGATACG-3 '8 In order to selectively detect Pectinatus sp. DSM20764 present in the specimen, the following sequence 9 that is complementary to the nucleotide sequence encoding the 16S ribosomal RNA gene of Pectinatus sp. An oligonucleotide consisting of a sequence selected from 1010 to 10 or a corresponding complementary sequence.
5'-TGGGGTCCGAACTGAATG-3 '9
5'-GCATCCATCTCTGAATGCG-3 '10 An oligonucleotide comprising at least 10 consecutive bases among the oligonucleotides according to claim 1, 2 or 3. A method for amplifying a target nucleotide sequence, wherein the oligonucleotide according to claim 1, 2 or 3 functions as a primer for a chain length reaction. A method for causing at least one of the oligonucleotide sequences according to claim 1, 2 or 3 to function as a probe for detecting DNA. An oligonucleotide in which at least one of the oligonucleotide sequences according to claim 1, 2 or 3 is modified with a label.