Oligonucleotide probe for detecting Enterobacteriaceae by in situ hybridization.
OBJECTIVE
To develop oligonucleotide probes for visualizing bacteria belonging to Enterobacteriaceae.


RESULTS
24-mer oligonucleotide probe (probe D) was designed by comparison of 16S rDNA sequences of 35 species of Enterobacteriaceae, eight species of Vibrionaceae and six species of Pasteurellaceae. The sequence of the probe corresponding to the complementary sequence of a position 1251-1274 of Escherichia coli 16S rRNA was found to be a highly conserved region of 16S rDNA sequence in Enterobacteriaceae different from that of Vibrionaceae and Pasteurellaceae. The fluorescent dye-labelled probe was tested for the specificity by in situ hybridization and epifluorescence microscopy. Seventy-six out of 78 strains belonging to Enterobacteriaceae were visualized in an optimal hybridization condition. No bacterial strains belonging to Vibrionaceae (31 strains) and Gram-positive bacteria (three strains) were visualized.


CONCLUSIONS
In situ hybridization using probe D allows the detection of bacterial cells belonging to Enterobacteriaceae without false positive reaction.


CONCLUSIONS
In situ hybridization techniques using the probe D are potential tools for detecting Enterobacteriaceae in food and water samples.
Ootsubo
M
M
Research and Development Department, Hokkaido Industrial Technology Center, Japan.
Shimizu
T
T
Tanaka
R
R
Sawabe
T
T
Tajima
K
K
Yoshimizu
M
M
Ezura
Y
Y
Ezaki
T
T
Oyaizu
H
H
D004269:DNA, Bacterial / Q000032:analysis; D004275:DNA, Ribosomal / Q000032:analysis; D004755:Enterobacteriaceae / Q000235:genetics* / Q000302:isolation & purification*; D005516:Food Microbiology; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D015345:Oligonucleotide Probes / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000032:analysis; D012680:Sensitivity and Specificity; D014871:Water Microbiology; D014881:Water Supply / Q000592:standards*
D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D015345:Oligonucleotide Probes; D012336:RNA, Ribosomal, 16S