A solution hybridization assay for ribosomal RNA from bacteria using biotinylated DNA probes and enzyme-labeled antibody to DNA:RNA.
Rapid, convenient and non-isotopic nucleic-acid hybridization methods are needed for this technology to have practical use in clinical diagnostic tests. A method for hybridization of RNA with a DNA probe in solution followed by capture and measurement of the hybrid is described. DNA probes complementary to 23S rRNAs from Escherichia coli and Bacillus subtilis were labeled with a photoactivable biotin reagent. Hybridization of the biotinylated probes with rRNA was complete in less than 5 min. The resultant hybrids were allowed to bind simultaneously to succinylated avidin immobilized on latex and to beta-galactosidase-labeled Fab' fragments of a monoclonal antibody-specific for DNA:RNA. Finally, beta-galactosidase associated with the captured hybrids was measured colorimetrically. The hybridization method can detect less than 1000 bacteria per assay and has broad specificity to permit detection of the various genera of bacteria that infect the urinary tract.
Yehle
C O
CO
Ames Division, Miles Laboratories, Inc., Elkhart, IN 46515.
Patterson
W L
WL
Boguslawski
S J
SJ
Albarella
J P
JP
Yip
K F
KF
Carrico
R J
RJ
D000818:Animals; D001360:Avidin / Q000378:metabolism; D001431:Bacteriological Techniques; D001710:Biotin / Q000378:metabolism; D004247:DNA / Q000032:analysis; D004926:Escherichia coli / Q000032:analysis*; D007124:Immunoenzyme Techniques; D007962:Leukocytes / Q000032:analysis; D009693:Nucleic Acid Hybridization; D012329:RNA, Bacterial / Q000032:analysis* / Q000652:urine; D012335:RNA, Ribosomal / Q000032:analysis*; D012338:RNA, Ribosomal, 23S / Q000032:analysis*
D012329:RNA, Bacterial; D012335:RNA, Ribosomal; D012338:RNA, Ribosomal, 23S; D001360:Avidin; D001710:Biotin; D004247:DNA