Species-specific oligonucleotides for bifidobacteria and a method of detection using the same

Oligonucleotides which specifically hybridize to rRNAs of Bifidobacterium infantis, Bifidobacterium bifidum, Bifidobacterium adolescentis, Bifidobacterium breve, and Bifidobacterium longum represented by the sequences of formulae (I) to (V), respectively, or sequences complementary thereto: EQU UAUCGGGGAGCAAGCGUGA (I)SEQ ID NO: 1 EQU UCGGAUCGGAGCCUGC (II)SEQ ID NO: 4 EQU CGCUUUUGACUGGGAGC (III)SEQ ID NO: 7 EQU UGUGUUGAGUGUACCUU (IV)SEQ ID NO: 10 EQU UAUCGGGGAGCAAGCGAGA (V)SEQ ID NO: 13 and a method of species-specifically detecting species of the genus Bifidobacterium of human origin comprising using these oligonucleotides as a labeled probe.

FIELD OF THE INVENTION 
This invention relates to oligonucleotides having species specificity for 
various Bifidobacterium species, particularly those of human origin, and a 
species-specific method of detection using the same. 
BACKGROUND OF THE INVENTION 
Identification of species belonging to the genus Bifidobacterium has mainly 
been performed on the basis of phenotypic characteristics, such as 
carbohydrate fermentation patterns, gas-liquid chromatographic analyses of 
fatty acids, and cellular morphology. Recently, identification based on 
DNA-DNA homology has also been used (see Benno, Y., BISEIBUTSU, Vol. 6, 
pp. 1-12 (1990). 
Identification based on phenotypic characteristics requires a high degree 
of skill and takes a period of time before an identification can be made. 
The DNA-DNA homology approach, which requires extraction of DNA from 
microbial cells, is disadvantageous in that bifidobacteria are 
gram-positive bacteria which require more complicated procedures for 
bacteriolysis than those needed with gram-negative bacteria. In addition, 
the values for DNA-DNA homology between closely related species with no 
distinct difference are high. 
Hence, there has been keen demand for the development of a rapid, simple, 
and accurate method for detecting Bifidobacterium species, especially 
those found in humans, i.e., Bifidobacterium infantis, B. bifidum, B. 
adolescentis, B. breve, and B. longum. 
SUMMARY OF THE INVENTION 
In light of the demand for a better detection method, the present inventors 
have conducted extensive investigations. Studies were performed on 
detecting bifidobacteria using a labeled oligonucleotide having species 
specificity for a Bifidobacterium species as a probe and observing 
hybridization of the probe to the bacterium. This study was focused on the 
use of short oligonucleotides comprising about 20 bases, which would 
perform in situ hybridization without requiring extraction of nucleic 
acids, thereby making it feasible to detect even a 1-nucleotide 
difference. 
Species-specific portions of 16S rRNAs were chosen as the target site for 
assays because the 16S rRNA sequences have recently been accepted as a 
highly reliable indication of phylogenetic classification and also because 
16S rRNAs have a much higher copy number per cell than chromosomal DNA and 
are expected to exhibit increased detection sensitivity. 
Standard strains of various bifidobacteria were subjected to enzymatic 
bacteriolysis, and crude high-molecular-weight RNAs were extracted by a 
conventional method. The 16S rRNA portion of the crude high-molecular 
weight RNA was partially sequenced according to a Sanger's [dideoxy chain 
termination] method using reverse transcriptase. After comparing the thus 
determined base sequences, it was found that the oligonucleotides having 
sequences represented by formulae (I) to (V) (SEQ ID NO:1, SEQ ID NO:4, 
SEQ ID NO:7, SEQ ID NO:10 and SEQ ID NO:13, respectively) shown below are 
specific to Bifidobacterium infantis, B. bifidum, B. adolescentis, B. 
breve, and B. longum, respectively, and are useful as primers for 
detecting bacteria in a polymerase chain reaction (PCR) method and that 
the oligonucleotides, when labeled, serve as a probe for 
species-specifically detecting bifidobacteria rapidly, simply, and 
accurately. The present invention has been completed based on these 
findings. 
The present invention relates to (1) an oligonucleotide which specifically 
hybridizes to Bifidobacterium infantis rRNA which has a sequence 
represented by formula (I) (SEQ ID NO:1) or a sequence complementary 
thereto: 
EQU UAUCGGGGAGCAAGCGUGA (I) 
(2) an oligonucleotide which specifically hybridizes to Bifidobacterium 
bifidum rRNA which has a sequence represented by formula (II) (SEQ ID 
NO:4) or a sequence complementary thereto: 
EQU UCGGAUCGGAGCCUGC (II) 
(3) an oligonucleotide which specifically hybridizes to Bifidobacterium 
adolescentis rRNA which has a sequence represented by formula (III) (SEQ 
ID NO:7) or a sequence complementary thereto: 
EQU CGCUUUUGACUGGGAGC (III) 
(4) an oligonucleotide which specifically hybridizes to Bifidobacterium 
breve rRNA which has a sequence represented by formula (IV) (SEQ ID NO:10) 
or a sequence complementary thereto: 
EQU UGUGUUGAGUGUACCUU (IV) 
(5) an oligonucleotide which specifically hybridizes to Bifidobacterium 
longum rRNA which has a sequence represented by formula (V) (SEQ ID NO:13) 
or a sequence complementary thereto: 
EQU UAUCGGGGAGCAAGCGAGA (V) 
and (6) a species-specific method for detecting various species of the 
genus Bifidobacterium found in humans comprising using these 
oligonucleotides as a labeled probe.