JPH11127898A - Primer for aerofaciens bacterium - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new primer comprising a primer or a probe for Eubacterium aerofaciens having a specific base sequence or a sequence complementary to the specific base sequence and useful for identification and detection or the like of species of bacterium which is predominant in intestine. SOLUTION: This new primer or probe for Eubacterium aerofaciens has a sequence represented by formula I or II or a sequence complementary to the base sequence and is capable of rapidly, simply and precisely identifying Eubacterium aerofaciens at a low cost and used also for analysis or the like of intestinal flora and grasps conditions of digestive tract or the like from the result and enables prevention, treatment or the like of various diseases. The primer is obtained by comparing a base sequence of plural bacterial strains of Eubacterium aerociens with a base sequence of 16Sr RNA gene of Eubacterium lentum which is the closest related species and designing the structure and artificially synthesizing these sequences by DNA synthesizer according to the base sequence.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a DN useful for the identification of Eubacterium aerofaciens , a dominant bacterium in the intestine of humans and animals.
The present invention relates to an A primer and a probe, and a method for identifying and detecting an Aerofaciens strain using the same.

[0002]

2. Description of the Related Art Various bacterial groups are resident in the intestinal tract of humans and animals, and form a complicated intestinal flora. These are known to be in a stable state as long as the host is healthy. Eubacterium is one of the most predominant bacteria that form the intestinal flora. Thus, bacteria of the genus Eubacterium are often isolated from soil and mud, but are mainly isolated from the intestinal tract and clinical material of humans and animals.

[0003] Of the bacteria belonging to the genus Eubacterium, Aerofasciens, which is the most prevalent in the intestine, accounts for about 80% of the bacteria belonging to the genus Eubacterium in the intestine and about 10% of the human intestinal flora. Occupy. It is known that the ratio of the number of bacteria in the intestine of Aerofaciens varies depending on the dietary component. For example, it has been reported that aerofacience was isolated at a high rate of 25.1% from the intestines of rural Japanese, but as low as 5.2% for urban North Americans. In addition, it has been reported that when dietary fiber (brown rice diet) is supplied to humans, the composition ratio of aerofasciens is significantly reduced, and differences in the number of bacteria and appearance frequency of aerofasciens are caused by differences in living environment and individuals. Is suggested. Furthermore, it has been found that the trends of Eubacterium bacteria are similar to those of Bifidobacterium bacteria, which are useful bacteria in the intestine.

[0004] From these facts, it is considered that the trend of aerofacience in the intestinal tract of humans and animals is closely related to the health condition of living bodies. For this reason,
It is desired to conduct further research on Eubacterium bacteria and accumulate knowledge such as physiological effects. For this reason, it is important to quickly and accurately detect aerofacience from feces and the like, and to grasp the health condition of the individual from the trend.

However, at present, Aerofaciens is a gram-positive, non-spore-forming bacillus belonging to the genus Propionibacterium, the genus Bifidobacterium and the lactobacillus (L.
Actobacillus) is a bacterium that is only classified as a bacterium other than the genus Bacteria and has many unknown parts such as physiological activities. Therefore, its detection and identification is performed by examining the colony shape, microscopic observation, and glycolytic properties,
The identification method based on such a phenotypic trait requires complicated operations and requires the skill of the tester.

[0006] In recent years, determinations based on DNA-DNA homology have also been made (TAKAYUKI
EZAKI, YASUHIRO HASHIMOTO, AND EIKO YABUUCHI. IJS
B, 1989, vol. 39, p224-229). However, this identification method also requires a long time, and is not preferable for rapid bacterial identification.

[0007]

As described above, it takes a long time to identify and analyze Aerofaciens from differences in fermentation properties and DNA-DNA homology, and the operation is complicated. was there. Accordingly, an object of the present invention is to provide a method capable of quickly and easily identifying and detecting aerofasciens in feces and intestinal bacterial flora.

[0008]

Means for Solving the Problems In view of this situation, the present inventors have conducted intensive studies, and found a DNA primer having a base sequence specific to Aerofaciliens.
By using this, DN-derived bacteria extracted from a specimen can be used without performing complicated operations such as confirming the glycolytic properties of the bacteria.
The present inventors have found that the PCR reaction of A enables rapid and simple identification and detection of Aerofaciens, and completed the present invention.

That is, the present invention provides a primer or probe for E. aerofaciens having the nucleotide sequence of SEQ ID NO: 1 or 2 or a sequence complementary to the nucleotide sequence.

[0010] The present invention also provides a method for identifying or detecting Eubacterium aerofaciens, which comprises using the above-mentioned primer for Eubacterium aerofaciens.

Further, the present invention provides (1) a step of extracting DNA in a specimen, (2) a step of performing a PCR reaction using the above-mentioned primers for E. aerofaciens, and (3) a step (2). And a method for detecting E. bacteria aerofaciens, which comprises a step of detecting a DNA fragment amplified by E. coli.

[0012]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Eubacterium is an anaerobic, non-flagellar, non-motile, non-spore, non-branched, gram-positive bacillus having a paired or short-chain sequence of bacteria. It is a fungus genus named and proposed. In addition, metabolites are distinguished from other Gram-positive bacilli such as Propionibacterium, Bifidobacterium, and Lactobacillus. Among them, as a glycolytic property, glucose, mannose, galactose, fructose, maltose and produce acid from lactose, arabinose, xylose, meletitose, soluble starch,
A group of bacteria that do not produce acid from glycogen, mannitol, sorbit, inosit, and erythrit is E. bacteria aerofaciens.

In the above-mentioned classification, in preparing the primers for Aerofasciens, it was first decided to clarify the genetic classification. That is, the classification was performed using a highly reliable 16S rRNA gene as a target as an index for phylogenetic classification. Here, as a comparison target, the present inventors newly performed sequencing of the 16S rRNA gene, and specifically, the Aerofaciliens strain was specifically a reference strain, Eubacterium aerofaciens JCM6479 strain and a reference strain. The strains are JCM7790 strain and JCM7791 strain. These sequences will be registered in the NIG database DDBJ.

The present inventor compares and examines the nucleotide sequence obtained by performing the sequencing with the sequence obtained from a database (DDBJ, Genbank, etc.) and examines the NJ method (Saitou, N., and M. Ne).
i. 1987. The neighbor-Joining method: a new metho
d for reconstracthiong phylogenetic trees. Mol. Bi
ol. Evol. 4: 406-425). As a result, it was considered appropriate that Aerofaciens belong to the genus Coriobacterium instead of the genus Eubacteria. However, in this specification, it is described as belonging to the genus Eubacterium for convenience.

[0015] On the other hand, the preparation of the Aerofacience-specific primer was carried out as follows. First, a 16S rRNA gene was used as a target, and DNA was used instead of RNA because identification and analysis required means such as PCR.

Primers were designed by comparing the gene sequences of the above three strains and the closest related species, Eubacterium lentum. At that time, a portion where the number of bases was about 20 and the GC content was about 50% was searched. As a result, three primers were designed for each of the forward and reverse primers (Table 1). Then, when the specificity was examined using AERO-F and AERO-R, a result of amplifying not only aerofascience but also rentum was obtained. Therefore, AER
A-F1 and AERO-R1, AERO-F2 and AERO
AERO-F1 and AE
Good results were obtained in combination with RO-R1.

AERO-F1 and AE thus obtained
The length of the RO-R1 oligonucleotide is 21 b. p
And shown in SEQ ID NOs: 1 and 2, respectively. These are PC
It is the most suitable length for the operation such as R reaction, but it is necessary to use several to several tens of b. You may use what added the base sequence of p. At this time, the primer whose base sequence has been increased or decreased may amplify other kinds of bacteria. Therefore, it is necessary not to use such a primer or to modify PCR conditions.

At this time, the conditions of the PCR reaction were also examined. That is, the annealing temperature is 58,
The temperature was changed to 60 and 63 ° C., and the number of cycles was also examined with two types of 25 and 30 cycles. As a result, the most preferable conditions were 63 ° C. and 25 cycles.

The above primer is artificially synthesized by a DNA synthesizer according to its base sequence. The species specificity was determined by the DN of 11 reference strains and 9 reference strains of Eubacterium bacteria and 3 isolates of Aerofaciens.
The presence or absence of amplification when the primer was reacted with A was confirmed as an index. As a result, there was no problem with the specificity of the primers for Aerofaciens. In addition, when four bacterial strains of other genera were examined, no amplification was observed. Furthermore, from about 300 fresh isolates of gram-positive bacilli, E.
When 178 strains identified as Aerofaciens were reacted with the primers of the present invention, bands were formed with all the strains. In addition, the strain which was not identified as Aerofasciens in the sugar fermentation test did not react with the primer of the present invention.

The primer of the present invention is a DNA oligonucleotide specific to Eubacterium aerofaciens , and can be used as a primer or a probe for identifying and analyzing aerofaciens. . As a primer, it is possible to use in combination with a known universal primer or the like. However, some combinations cannot perform species-specific detection.
Alternatively, it is preferable to combine with an oligonucleotide having the nucleotide sequence described in 2.

It should be noted that species-specific sequences other than the sequences shown in SEQ ID NOs: 1 and 2 have been found by the new sequencing. However, identification using these sequences or other known primers often causes a reaction with a strain belonging to another species or does not react with a strain of a target species, which is not preferable in use. Even if such primers are used, it is considered that species-specific reactivity can be achieved by setting conditions during the PCR reaction, but such conditions have not been found at present.

Since the primers of the present invention have such species specificities, they can be used to rapidly identify Aerofaciliens in feces and tissue sections. In addition, Aerofasciens was transformed into EG (Eggerth-Gagnon) medium (Eiken)
By simply extracting DNA directly from the formed colonies or by extracting DNA from the cultured cells and examining the reactivity with the primers, simple identification of the bacterial species can be performed.

Further, by using the primer of the present invention, it is also possible to directly identify from the DNA of Aerofasciens. For example, DNA is extracted from a pellet obtained by centrifuging feces or the like by a benzyl chloride method or the like,
This is used as a template DNA, and an amplification reaction is carried out with the primer of the present invention, whereby an Aerofasciens-specific DN
A sequence (PCR product) can be obtained. When the DNA thus obtained is subjected to electrophoresis, Aerofascient can be identified from the presence or absence of the band.

As a method for extracting DNA, the Marmur method, which is a conventional method, the enzyme method, which is a modification thereof, and the benzyl chloride method, which is a simple method, are preferable. Although these methods are somewhat complicated, enzymatic methods can extract DNA from a wide variety of bacterial species in good yield. For DNA extracted from purely cultured bacteria and the like, a phenol method or the like can be suitably used in addition to the above-described method.

Usually, when primers are used in the PCR method or the like, it is necessary to use two types of primers as one set. For example, if the primers according to SEQ ID NOs: 1 and 2 are used, an amplification reaction occurs between both primers only in the DNA of E. aerofaciens, among other types of bacteria, and this is identified. Can be. In addition, the above primer and a known universal primer may be used in combination, but in such a case, it is necessary that both of them be a combination of a leading strand and a lagging strand. In addition, when performing PCR, if the amount of template DNA is serially diluted and the same analysis is performed, it is possible to quantify Aerofasciens. Also,
Since the primer of the present invention has an Aerofacience-specific sequence, it can be used alone as a probe.

The use of the primers of the present invention enables analysis of the intestinal flora of humans and animals. At present, it is impossible to detect bacteria other than Aerofaciliens, but if the distribution and number of bacteria of Aerofaciliens are known, the number of bacteria such as Bifidobacterium can be predicted. Various information such as status can be obtained. Furthermore, when used in combination with other diverse primers of bacteria, such as primers for Bifidobacterium genus bacteria, which the applicant of the present invention previously filed (Japanese Patent Application No. 9-219567), the overall image of the bacterial flora can be obtained. It is also possible to grasp.

[0027]

EFFECT OF THE INVENTION By using the primer of the present invention, eubacterium aerofaciens can be identified quickly, simply, at low cost and with high accuracy. Also,
When used in combination with other species-specific primers, etc., analysis of the intestinal flora can be performed.
Furthermore, since the state of the digestive tract and the like can be grasped from the results of the analysis, prevention and treatment of various diseases and the like become easy.

[0028]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.

Example 1 Design and synthesis of primers:
Based on the 16S rRNA gene sequence of eubacterium rentum at a phylogenetically adjacent position and the above 16S rRNA gene sequence decoded by the present inventors, an aerofaciens-specific primer was designed. . At that time, a portion where the number of bases was about 20 and the GC content was about 50% was searched. As a result, three primers were designed for each of the forward and reverse primers (Table 1). The positions of these primers in the numbering of Escherichia coli were 439 to 486 for AERO-F, 436 to 466 for AERO-F1, 694 to 713 for AERO-F2,
ERO-R: 1055-1031, AERO-R1: 1
060-1039, AERO-F2 is 1001-982
Met. According to the base sequence thus designed, a primer was synthesized using a DNA synthesizer, and the specificity was confirmed. As a result, the primers shown in Table 1 (AERO-F1 and AERO
-R1) yielded favorable results.

[0030]

[Table 1]

Example 2 Investigation of the reactivity between the primer and the bacterium belonging to the genus Eubacterium: In order to confirm whether the primer of the present invention actually has the specificity of Aerofasciens, each primer of the bacterium belonging to the genus Eubacterium was used. The reactivity of the reference strain, reference strain and isolate of the strain with the primers was examined. (1) Pure culture of strains and isolation of colonies As shown in Table 2, nine strains of 14 strains of Eubacterium belonging to the genus Eubacterium and 5 strains of intestinal bacteria belonging to other genera were subjected to EG medium (Eiken).
Anaerobically for two nights. Cells 15 thus obtained
Colonies of each type were scraped and suspended in sterile water using a 0.5 ml Eppendorf tube. This is 10
After boiled at 0 ° C. for 5 minutes and denatured by heat, a PCR reaction was performed using the primers of the present invention.

(2) PCR reaction The total volume was set to 100 μl, and 10 mM Tris-HCl (pH
8.3), 50 mM KCl, 1.5 mM MgCl ₂ and 200 μM dNTP mixture, 0.25 pM primer (AERO-F1 and AERO-R1), 2.5 U Taq D, respectively.
A reaction solution containing NA polymerase (manufactured by Takara) and template DNA, and a DNA thermal cycler (Perkin-Elmerce)
tus, Norwalk, conn.) after 94 minutes at 94 ° C.
The PCR reaction was performed at 25 ° C. for 25 cycles at 30 ° C. for 1 minute 30 seconds at 63 ° C.

(3) Investigation of species specificity of primers The PCR product obtained in (2) was subjected to electrophoresis, and the ability of the primers to bind to DNA of each strain was confirmed based on the presence or absence of a band. Was determined. 1% Agarose Type
II (manufactured by Sigma)
After electrophoresis for 5 minutes and staining with ethidium bromide, bands were observed under a UV lamp. As a result, the primer formed a band specifically for aerofacience (Table 2).

[0034]

[Table 2]

T indicates Type (reference strain). The results of PCR are the results when AERO-F1 and AERP-R1 were used. A107, A235 and A243 are isolates.

Example 3 Identification of Aerofaciens from Isolates Using Primers: Approximately 300 gram-positive bacilli were selected from human fecal isolates by a property test. For these, a conventional property test and identification with the primer of the present invention were performed to confirm the specificity of the primer. (1) Pure culture of strains and isolation of colonies Approximately 300 fresh isolates identified as genus Eubacterium from property tests were anaerobically cultured in EG medium for 2 days, and colonies suspended in sterilized water and heat-denatured Was used as a sample. (2) PCR reaction Using the DNA obtained in (1) as a template, a PCR reaction was performed under the same conditions as in Example 2. (3) Examination of bacterial species specificity of primers A sugar degradation test was performed on about 300 strains obtained by anaerobically culturing the isolates in an EG medium.
Eight strains were identified as Eubacterium aerofaciens. On the other hand, when a PCR reaction was performed on 300 strains using the primers of the present invention, the same 17
Amplification was observed only in 8 strains of DNA. This 178
From among the three strains, A107 strain, A235 strain, and A243 strain (described in Table 2) were sequenced. As a result, E. coli Aerofasciens JC was determined.
Like the M6479 strain, the JCM7790 strain, and the JCM7791 strain, it was confirmed to have the designed primer sequence. Further, JCM6479 for the A107 strain
Strain, JCM7790 strain and JCM7791 strain and DNA-
DNA homology was performed to confirm that they were the same species.

[0037]

[Sequence list]

SEQ ID NO: 1 Sequence length: 21 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: DNA Origin Organism: Eubacteria aerofaciens (Euba)
cterium aerofaciens) Strain name: JCM 6479 Sequence CTTTCAGCAG GGAAGAGTCA A 21

SEQ ID NO: 2 Sequence length: 21 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: DNA origin Organism: Eubacteria aerofaciens (Euba)
cterium aerofaciens) Strain name: JCM 6479 sequence AGCCATGCAC CACCTGTATG G 21

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code FI C12R 1:01)

Claims (4)

[Claims] 1. A primer or probe for Eubacterium aerofaciens having the nucleotide sequence of SEQ ID NO: 1 or 2 or a sequence complementary to the nucleotide sequence. 2. A method for identifying Eubacterium aerofaciens, comprising using the primer according to claim 1. 3. A method for detecting Eubacterium aerofaciens, comprising using the primer according to claim 1. 4. A step of (1) extracting DNA in a sample,
(2) a step of performing a PCR reaction using the primer according to claim 1, and (3) DN amplified in step (2).
A method for detecting Eubacterium aerofaciens, comprising a step of detecting the A fragment.