A sandwich hybridization assay employing enzyme amplification for termination of specific ribosomal RNA from unpurified cell lysates.
We have employed the power of the cyclic NAD-based enzyme amplification system to the determination of 16S rRNA. This generally applicable system employs two oligonucleotide probes, one of which is captured on a microtiter well surface and the other labeled with alkaline phosphatase. The detection of very low levels of hybridization of the capture probe is then achieved by the means of the ultrasensitive enzyme-amplified assay system, resulting in a highly sensitive, convenient, and rapid technology which can be directly employed on unpurified samples. We have been able to demonstrate the detection of 20 amol (10(7) molecules) of pure rRNA, and specific signals from as few as 2000 bacterial cells have also been demonstrated. The total procedural time can be short-5 to 18 h-depending on the dynamic range and sensitivity required. RNA target in the range of 10(12)-10(8) molecules can be assayed within 5 h. Extending the substrate incubation time enables between 10(11) and 10(7) molecules to be determined within 18 h. The system has great potential use with respect to studying the distribution and physiological states of cellular organisms.
Wicks
B
B
Department of Microbiology, University of Newcastle upon Tyne, Tyne, NE2 4HH, United Kingdom.
Cook
D B
DB
Barer
M R
MR
O'Donnell
A G
AG
Self
C H
CH
D004798:Enzymes / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics; D009693:Nucleic Acid Hybridization / Q000379:methods*; D010802:Phylogeny; D012329:RNA, Bacterial / Q000032:analysis* / Q000145:classification; D012336:RNA, Ribosomal, 16S / Q000032:analysis*; D012680:Sensitivity and Specificity; D014733:Vibrio / Q000235:genetics
D004798:Enzymes; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S