JP4001699B2 - Primer for Enterococcus bacteria - Google Patents

Description

【００３９】
【実施例２】
エンテロコッカス属細菌用プライマーの特異性の確認
本発明のプライマーが、実際に特異性を有しているかを確認するため、近縁種の株とプライマーとの反応性を検討した。
【００４０】
（１）菌株の純粋培養及びＤＮＡの抽出
表２に示す、２４菌種の細菌を血液寒天培地（ベクトンデッキンソン社製）にて好気的に一晩純粋培養した。こうして得た菌体各々から、塩化ベンジル法(Zhu, H. et al.,(1993) Nucleic Acids Research, 21,5279-5280)により、ＤＮＡを抽出した。
【００４１】
すなわち、血液寒天培地を用いた一夜培養菌液１．５ｍｌを作成し、遠心した後、菌体にＥｘｔｒａｃｔｉｏｎ buffer（１００ｍM Tris,４０ｍM EDTA ｐH9.0）２５０μｌ、ベンジルクロライド１５０μｌ，１０％SDS５０μｌを加え、５０℃で３０分間激しく振盪した。３M酢酸ナトリウム１５０μｌを加えて氷中で１５分間静置した後、１５０００ｒｐｍ、１０分間の遠心で得られた上清をイソプロパノールで沈殿させ７０％エタノールで洗浄した。沈殿を５０μｌのＴＥ ｂｕｆｆｅｒ（１０ｍＭ Ｔｒｉｓ−ＨＣｌ （ｐＨ８．０）／１ｍＭ ＥＤＴＡ）に溶かして濃度を測定し、１０μｇ／ｍｌの濃度に調製して鋳型ＤＮＡ溶液とした。
【００４２】
（２）ＰＣＲ反応
総量を２５μｌとし、１０ｍＭ Ｔｒｉｓ−ＨＣｌ （ｐＨ８．３）、５０ｍＭＫＣｌ、１．５ｍｌ ＭｇＣｌ２、２００μＭ ｄＮＴＰ ｍｉｘｔｕｒｅに、０．８μＭプライマー（Ｅｎｔ−１，２）、１．０Ｕ Ｔａｑ ＤＮＡ ｐｏｌｙｍｅｒａｓｅ（タカラ社製）、１０ｎｇ 鋳型ＤＮＡを含む反応液で、ＤＮＡサーマルサイクラーＰＪ４８０（タカラ社製）により、９４℃、２０秒、５0℃、２０秒、７２℃、３０秒を３０回のＰＣＲ反応を行った。
【００４３】
（３）プライマーの特異性の検討
ＰＣＲ反応で得られたＰＣＲ産物を電気泳動し、バンドの有無によりプライマーの各菌株のＤＮＡとの結合能を確認することにより、プライマーの特異性を判定した。１．５％アガロース(Ｍｏlｅｃｕｌａｒ Ｂｉｏｌｏｇｙ Ｃｅｒｔｉｆｉｅｄ Ａｇａｒｏｓｅ；バイオラッド社製)で、Ｍｕｐｉｄ−２（コスモ・バイオ）により１００Ｖ、２５分電気泳動し、Ｅｔｈｉｄｉｕｍ Ｂｒｏｍｉｄｅ（０．５ｍｇ／ｍｌ）で染色後、ＵＶランプ下でバンドを観察した。その結果は表２に示すとおりであり、プライマーはエンテロコッカス属細菌特異的にバンドを形成した。
【００４４】
【表２】

【００４５】
【実施例３】
嫌気性連鎖球菌用プライマーの特異性の確認
本発明のプライマーが、実際に特異性を有しているかを確認するため、実施例２と同様に近縁種の株とプライマーとの反応性を検討した。
【００４６】
（１）菌株の純粋培養及びＤＮＡの抽出
表３に示す、２５菌種細菌をＢｒｕｃｅｌｌａ ＨＫ 寒天培地（極東社製）にて嫌気性的に一晩純粋培養した。こうして得た菌体各々から、実施例１と同様に塩化ベンジル法(Zhu, H. et al.,(1993) Nucleic Acids Research, 21, 5279-5280)により、ＤＮＡを抽出した。
【００４７】
（２）ＰＣＲ反応
総量を２５μｌとし、１０ｍＭ Ｔｒｉｓ−ＨＣｌ （ｐＨ８．３）、５０ｍＭＫＣｌ、１．５ｍｌ ＭｇＣｌ２、２００μＭ ｄＮＴＰ ｍｉｘｔｕｒｅに、各々０．８μＭプライマー、１．０Ｕ Ｔａｑ ＤＮＡ ｐｏｌｙｍｅｒａｓｅ（タカラ社製）、１０ｎｇ 鋳型ＤＮＡを含む反応液で、ＤＮＡサーマルサイクラーＰＪ４８０（タカラ社製）により、９４℃、２０秒、５0℃、２０秒、７２℃、３０秒を３０回のＰＣＲ反応を行った。
【００４８】
（３）プライマーの特異性の検討
ＰＣＲ反応で得られたＰＣＲ産物を電気泳動し、バンドの有無によりプライマーの各菌株のＤＮＡとの結合能を確認することにより、プライマーの特異性を判定した。１．５％アガロース(Ｍｏｒｅｃｕｌａｒ Ｂｉｏｌｏｇｙ Ｃｅｒｔｉｆｉｅｄ Ａｇａｒｏｓｅ；バイオラッド社製)で、Ｍｕｐｉｄ−２（コスモ・バイオ）により１００Ｖ、２５分電気泳動し、Ｅｔｈｉｄｉｕｍ Ｂｒｏｍｉｄｅ（０．５ｍｇ／ｍｌ）で染色後、ＵＶランプ下でバンドを観察した。その結果は表３に示すとおりであり、プライマーは対象となる各菌特異的にバンドを形成した。
【００４９】
【表３】

【配列表】

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a DNA primer useful for identification of anaerobic streptococci and Enterococcus bacteria, and a detection / identification / analysis method using the same.
[0002]
[Prior art]
Anaerobic streptococci such as Peptostreptococcus bacteria and Sarcinia bacteria are frequently detected in inflammatory sites such as the oral cavity and lungs of humans, and some inhabit in the intestines. Other anaerobic streptococci include atopobium bacteria, gemera bacteria, beironella bacteria, acidaminococcus bacteria, and megasfera bacteria. Moreover, Enterococcus bacteria are bacteria that mainly live in the intestinal tract.
[0003]
It is very useful to identify and analyze these bacteria, for example, to grasp the health state of an individual by grasping the trend of the bacteria in the intestinal flora, and to conduct pathological studies in the intestinal tract. Further, if the bacteria involved in the onset can be identified at the lesion site, it is possible to quickly cope with the selection of antibiotics. At present, as a means for identification, a selection method using a combination of various selective media and microscopic observation are mainly performed.
[0004]
In order to identify and analyze the fungus, it is necessary to dilute the feces of the target individual with a diluent under anaerobic conditions, spread it on a medium, and perform anaerobic culture. However, the culture takes several days to several weeks, and operations such as counting the number of colonies are complicated.
[0005]
In recent years, determination based on DNA-DNA homology has also been performed. (Collins, MD, et al., INTERNATIONAL JOURNAL of SYSTEMATIC BACTERIOLOGY, 39, 105-108 (1989)). However, this identification method also takes a long time, and is not preferable for rapid bacterial species identification.
[0006]
In order to solve the problem of the above detection method, the present applicants examined a method for identifying a bacterium using a probe or primer specific to a certain type of intestinal bacterium. For example, Japanese Patent Laid-Open No. 11-28090 Discloses a probe specific to Bacteroides group bacteria, and JP-A-11-123093 discloses a primer specific to Bifidobacterium. According to this method, bacteria can be identified and detected easily and rapidly. However, since there are various other bacteria in the human intestine and lesions, the above-mentioned peptostreptococcus bacteria and the like There is a demand for a set of primers and probes that can cover other bacteria.
[0007]
[Problems to be solved by the invention]
However, designing primers that can be used to identify enterobacteria, etc., requires considerable effort, such as gene sequencing and alignment. Even if primers are synthesized with a specially designed sequence, specificity can be improved. It may not be obtained. For this reason, the kind of primer does not increase easily, and it is still insufficient to grasp the trend of bacteria in the intestines and lesions.
[0008]
Therefore, an object of the present invention is to synthesize a primer that can specifically identify various bacteria detected in the human intestine and lesion site, and to use this primer to quickly and easily identify and analyze the bacteria. It is to provide.
[0009]
[Means for Solving the Problems]
In view of such a current situation, the present invention has been intensively researched, found a DNA primer having a base sequence specific to various anaerobic streptococci and Enterococcus bacteria, and using this, without culturing bacteria, The present invention was completed by finding that the above-mentioned bacteria can be identified and analyzed quickly and easily by PCR reaction of bacteria-derived DNA extracted from a specimen.
[0011]
That is, the present invention is to provide Enterococcus ShokuHoso bacteria specific flop Lima over pairs having a sequence complementary to the combination or combinations of these nucleotide sequence of the nucleotide sequence set forth in SEQ ID NO: 1 and 9.
[0012]
Furthermore, the present invention is to provide a method for detecting Enterococcus bacteria, characterized by using the above primer pair.
[0013]
Furthermore, the present invention provides (1) extracting DNA in a specimen, (2) process and (3) performing a PCR reaction using the primers pair step (2) detecting the amplified DNA fragments by The present invention provides a method for detecting Enterococcus bacteria comprising the step of:
[0014]
DETAILED DESCRIPTION OF THE INVENTION
The primer specific to the anaerobic streptococci or Enterococcus bacterium of the present invention has a sequence specific to bacteria frequently detected in the human intestinal tract or lesion site. More specifically, Enterococcus faecalis (Enterococcus faecalis), Enterococcus faecium (Enterococcus faecium), Enterococcus Durance (Enterococcus durans), Enterococcus cut Seri Philadelphia bus (Enterococcus casserifilavus), Enterococcus Garinaramu (Enterococcus gallinarum), Enterococcus Sekoramu (Enterococcus cecorum), Enterococcus Koranbi (Enterococcus columbae) or a specific enteritidis in 8 species of Enterococcus Sarufuroisu (Enterococcus sulfureus) Genus bacteria primer, Peptostreptococcus (Peptostreptococcus) Anaerobiusu (anaerobius) groups in the genus, Asaka Lori Genetics (asaccharolyticus) group, primers specific to each Pureboti (prevotii) group or Magnus (magnus) group, Sarusaina & Bentorikuri (Sarcina ventriculi) or Sarusaina maxima Sarusaina bacteria for primers specific to (Sarcina maxima), species-specific primers Beironera-Parubura (Veillonella parvula), the a Sid amino Lactococcus · fermentans (Acidaminococcus fermentans) It can be exemplified specific primers.
[0015]
Here, the four groups in the genus Peptostreptococcus refer to genetically similar populations that are subdivided into four lines from the sequence of 16S ribosome and possess common sequences in each group.
[0016]
In preparing primers specific to the above-mentioned anaerobic streptococci or Enterococcus bacteria, the target is a highly reliable 16S rRNA gene as a phylogenetic index, and identification / analysis is performed by means such as PCR. DNA was used instead of RNA.
[0017]
The primer was obtained by comparing and examining a base sequence obtained by sequencing by the present inventor and a base sequence obtained from a database (DDBJ, Genbank, etc.). Here, the bacterial species newly sequenced by the present inventor for 16S rRNA gene are, specifically, Enterococcus dulans (E. durans) ATCC 19432, Peptostreptococcus anaerobius (Ps. Anaerobius) GIFU7882, Ps. Asacarolytics GIFU 7656, Ps. Asacarolytics GIFU7715, Ps. Asacarolytics GIFU7717, Ps. Asacarolytics GIFU7752, Ps. Asacarolytics GIFU8124, Ps. Prevoti GIFU 7658, Pept. Streptococcus baginalis (Ps. GIFU 7732, Pept. Str. icus) GIFU 8586 and Pept. Streptococcus heliotrin redugens GIFU 9910. These sequences will be registered in the NIG database DDBJ.
[0018]
When designing the primer, the sequences of the bacteria to be identified and the closely related bacteria were aligned. As a result, in Enterococcus bacteria, the Escherichia coli numbering was around 170 and 560, and in the Anaerobius group, Asacarolytics group, Prevoti group and Magnus group, around 310 and 660, -Specificity was recognized near 170 and 660 in Maxima, and around 140 and 660 in Beironella parvula and Acidaminococcus fermentans, respectively, and this part was used as a target. Further, the length of the oligonucleotide varies depending on the primer, and 19-21b. p. These are the most suitable lengths for operation, but in use, in each 16S rRNA gene, several to ten or more b. You may use what increased or decreased the base sequence of p.
[0019]
Among the primers obtained in this manner, those having the nucleotide sequences of SEQ ID NOS: 1 and 9 are DNA oligonucleotides specific for Enterococcus bacteria, and primers for identifying and analyzing bacterial species Can be used as a probe. As a primer, it can be used in combination with a known universal primer for lactic acid bacteria or the like, but some combinations cannot perform species-specific detection, so it is desirable to combine both.
[0020]
Moreover, what has a base sequence of sequence number 2 and 10 is an oligonucleotide specific to an Anaerobius group bacterium. For this reason, it is preferable to use both in combination as a primer.
[0021]
Moreover, what has a base sequence of sequence number 3 and 11 is an oligonucleotide specific to Asa calolytics group bacteria (Ps. Asa calolytics and Ps. Indolicus). For this reason, it is preferable to use both in combination as a primer.
[0022]
Moreover, what has a base sequence of sequence number 4 and 12 is an oligonucleotide specific to Prevoti group bacteria (Ps. Prevoti, Ps. Tetradius, and Ps. Vaginalis). For this reason, it is preferable to use both in combination as a primer.
[0023]
Those having the nucleotide sequences of SEQ ID NOs: 5 and 13 are oligonucleotides specific to Magnus group bacteria (Ps. Magnus and Ps. Micros). For this reason, it is preferable to use both in combination as a primer.
[0024]
Moreover, what has a base sequence of sequence number 6 and 14 is an oligonucleotide specific to Sarcina bentrikuri and Sarcina maxima. For this reason, it is preferable to use both in combination as a primer.
[0025]
Moreover, what has a base sequence of sequence number 7 and 15 is an oligonucleotide specific to Beironella parvula. For this reason, it is preferable to use both in combination as a primer.
[0026]
Furthermore, those having the base sequences shown in SEQ ID NOs: 8 and 16 are oligonucleotides specific for acid aminococcus fermentans. For this reason, it is preferable to use both in combination as a primer.
[0027]
The oligonucleotide primer designed as described above is artificially synthesized by a DNA synthesizer according to the base sequence. The specificity was confirmed using the band-forming ability of the primers for the closely related DNAs shown in the following examples as an index. As a result, there was no problem in the specificity of all the above-mentioned bacterial species.
[0028]
On the other hand, by performing a new sequence this time, species-specific sequences other than the sequences shown in SEQ ID NOs: 1 to 16 have been found. However, identification with these sequences and other known primers is not preferable in use because it may react with a strain belonging to another species or may not react with a strain of a target species. Even if such a primer is used, it is considered that species-specific reactivity can be achieved by setting conditions during the PCR reaction, but at present no such condition has been found.
[0029]
Since the primer of the present invention has specificity to various anaerobic streptococci or Enterococcus bacteria as described above, feces, enterobacteria, and lesions can be rapidly identified using these primers. In addition, bacterial cells directly or cultured from a colony formed on a selective medium of each bacterium (for example, in the case of Enterococcus bacteria, a BCP-added colony count agar medium (Eiken Chemical Co., Ltd.) that is a selective medium for lactic acid bacteria). DNA can be extracted from the bacterium and the reactivity with each primer can be examined to easily identify the bacteria.
[0030]
In order to specifically detect the target bacteria from stool without culturing, for example, first, DNA is extracted from a pellet obtained by centrifuging about 1 ml of stool sample by benzyl chloride method or the like. This is used as template DNA. By combining the template DNA with the primer of the present invention and performing an amplification reaction, a DNA sequence (PCR product) specific to the bacterium can be obtained. When the DNA thus obtained is electrophoresed, bacteria can be specifically detected from the presence or absence of bands and the primers used.
[0031]
In addition, in order to identify bacteria without complicated operations such as DNA-DNA homology tests and biological and biochemical property tests, for example, DNA is first extracted by a boiling method or the like, and this is used as a template. Let it be DNA. By combining the template DNA with the primer of the present invention and performing an amplification reaction, a DNA sequence (PCR product) specific to the bacterium can be obtained. When the DNA thus obtained is electrophoresed, bacteria can be specifically detected from the presence or absence of bands and the primers used.
[0032]
As a method for extracting DNA, the above-described boiling method, the conventional Marauur method, an enzyme method which is a modified method thereof, and the benzyl chloride method which is a simple method are preferable. Although these methods are somewhat complicated, DNA can be extracted with a high yield from a wide variety of bacterial species in the enzymatic method.
[0033]
As described above, if a DNA extracted from a colony or a stool sample is used as a template and a bacterium is identified using the primer of the present invention, it is difficult to discriminate strains and species that were below the detection limit by the conventional method. It was also possible to easily detect the strains. In addition, when PCR is performed, if the amount of template DNA is serially diluted and PCR is performed, the target bacteria can be quantified.
[0034]
The primer of the present invention can be used alone as a probe. These can also be used in combination with other known universal primers and oligonucleotides.
[0035]
Moreover, if the primer of this invention is used, intestinal microflora, such as a human and an animal, can also be analyzed. That is, when used in combination with the above-mentioned primers for Bifidobacterium and other various bacteria, it is possible to grasp the whole image of the bacterial flora.
[0036]
【The invention's effect】
By using the primer of the present invention, anaerobic streptococci or Enterococcus bacteria can be identified quickly, simply, at low cost and with high accuracy without culturing the bacteria. Moreover, by using in combination with primers specific to other bacteria, analysis of intestinal bacterial flora can be performed. Furthermore, since the state of the diseased part such as bacteria and digestive tract can be grasped from the results of identification and analysis, prevention and treatment of various diseases and the like are facilitated.
[0037]
[Example 1]
Primer Design and Synthesis Primers were designed based on the bacterial 16S rRNA gene sequence obtained from databases such as DDBJ and Genbank and the 16S rRNA gene sequence decoded by the present inventors. Enterococcus bacteria are around 170 and 560 in the Escherichia coli numbering, Anaerobius, Asacarolytics, Prevoti and Magnus are around 310 and 660, and Sarsina Bentricli and Sarsina Maxima Specificity was observed in the vicinity of the 170 region and the 660 region, and in the Beironella parvula and Acidaminococcus fermentans, in the vicinity of the 140 region and the 660 region, respectively, and this portion was used as a target. Primers were synthesized using a DNA synthesizer according to the designed base sequence (Table 1).
[0038]
[Table 1]

[0039]
[Example 2]
Confirmation of Specificity of Primer for Enterococcus Bacteria In order to confirm whether the primer of the present invention actually has specificity, the reactivity between closely related strains and the primer was examined.
[0040]
(1) Pure culture of bacterial strain and extraction of DNA The bacteria of 24 species shown in Table 2 were aerobically pure cultured overnight on a blood agar medium (Becton Dickinson). From each of the cells thus obtained, DNA was extracted by the benzyl chloride method (Zhu, H. et al., (1993) Nucleic Acids Research, 21,5279-5280).
[0041]
That is, 1.5 ml of an overnight culture using a blood agar medium was prepared, centrifuged, and then 250 μl of Extraction buffer (100 mM Tris, 40 mM EDTA pH 9.0), 150 μl of benzyl chloride, 50 μl of 10% SDS were added to the cells, Shake vigorously at 50 ° C. for 30 minutes. After adding 150 μl of 3M sodium acetate and allowing to stand in ice for 15 minutes, the supernatant obtained by centrifugation at 15000 rpm for 10 minutes was precipitated with isopropanol and washed with 70% ethanol. The precipitate was dissolved in 50 μl of TE buffer (10 mM Tris-HCl (pH 8.0) / 1 mM EDTA), the concentration was measured, and the concentration was adjusted to 10 μg / ml to obtain a template DNA solution.
[0042]
(2) The total PCR reaction volume was 25 μl, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 ml MgCl 2, 200 μM dNTP mix, 0.8 μM primer (Ent-1, 2), 1.0 U Taq DNA polymerase (Manufactured by Takara Co., Ltd.) In a reaction solution containing 10 ng template DNA, PCR reaction was performed 30 times at 94 ° C., 20 seconds, 50 ° C., 20 seconds, 72 ° C., 30 seconds using DNA thermal cycler PJ480 (manufactured by Takara) went.
[0043]
(3) Examination of primer specificity The PCR product obtained by the PCR reaction was electrophoresed, and the primer specificity was determined by confirming the binding ability of the primer to the DNA of each strain by the presence or absence of a band. Electrophoresis with 1.5% agarose (Molecular Biology Certified Agarose; manufactured by Bio-Rad) with Mupid-2 (Cosmo Bio) for 25 minutes, staining with Ethidium Bromide (0.5 mg / ml), UV lamp The band was observed below. The results are shown in Table 2, and the primer formed a band specifically for Enterococcus bacteria.
[0044]
[Table 2]

[0045]
[Example 3]
Confirmation of specificity of anaerobic streptococcal primers In order to confirm whether or not the primer of the present invention actually has specificity, the reactivity of closely related strains and primers was examined in the same manner as in Example 2. did.
[0046]
(1) Pure culture of bacterial strain and extraction of DNA The 25 bacterial strains shown in Table 3 were anaerobically pure cultured overnight on Brucella HK agar medium (manufactured by Kyokuto). From each of the cells thus obtained, DNA was extracted by the benzyl chloride method (Zhu, H. et al., (1993) Nucleic Acids Research, 21, 5279-5280) in the same manner as in Example 1.
[0047]
(2) The total amount of PCR reaction was 25 μl, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 ml MgCl 2, 200 μM dNTP mix, 0.8 μM primer, 1.0 U Taq DNA polymerase (manufactured by Takara), PCR reaction was carried out 30 times at 94 ° C., 20 seconds, 50 ° C., 20 seconds, 72 ° C., 30 seconds using a DNA thermal cycler PJ480 (manufactured by Takara) with a reaction solution containing 10 ng template DNA.
[0048]
(3) Examination of primer specificity The PCR product obtained by the PCR reaction was electrophoresed, and the primer specificity was determined by confirming the binding ability of the primer to the DNA of each strain by the presence or absence of a band. Electrophoresis with 1.5% agarose (Molecular Biology Certified Agarose; manufactured by Bio-Rad) with Mupid-2 (Cosmo Bio) for 25 minutes, staining with Ethidium Bromide (0.5 mg / ml), UV lamp The band was observed below. The results are shown in Table 3, and the primer formed a band specifically for each target bacterium.
[0049]
[Table 3]

[Sequence Listing]

Claims (3)

SEQ ID NO: 1 and 9 Enterococcus ShokuHoso bacteria specific flop Lima over pairs having a sequence complementary to the combination or combinations of these nucleotide sequence of the nucleotide sequence set forth in. Detection method of Enterococcus bacteria, characterized by using the primers pairs according to claim 1, wherein. (1) extracting the DNA in a specimen process, the step of detecting a DNA fragment amplified by (2) process and (3) performing PCR reaction using primers pairs according to claim 1, wherein step (2) A method for detecting Enterococcus bacteria, comprising: