Development of a rapid assay for determining the relative abundance of bacteria.
A sandwich hybridization assay for high-throughput, rapid, simple, and inexpensive quantification of specific microbial populations was evaluated. The assay is based on the hybridization of a target rRNA with differentially labeled capture and detector probes. Betaproteobacterial ammonia-oxidizing bacteria (AOB) were selected as the target group for the study, since they represent a phylogenetically coherent group of organisms that perform a well-defined geochemical function in natural and engineered environments. Reagent concentrations, probe combinations, and washing, blocking, and hybridization conditions were optimized to improve signal and reduce background. The detection limits for the optimized RNA assay were equivalent to approximately 10(3) to 10(4) and 10(4) to 10(5) bacterial cells, respectively, for E. coli rRNA and RNA extracted from activated sludge, by using probes targeting the majority of bacteria. Furthermore, the RNA assay had good specificity, permitted discrimination of rRNA sequences that differed by a 2-bp mismatch in the probe target region, and could distinguish the sizes of AOB populations in nitrifying and nonnitrifying wastewater treatment plants.
Rowan
Arlene K
AK
School of Civil Engineering and Geosciences, Cassie Building, University of Newcastle upon Tyne, Newcastle upon Tyne NE1 7RU, United Kingdom.
Davenport
Russell J
RJ
Snape
Jason R
JR
Fearnside
David
D
Barer
Michael R
MR
Curtis
Thomas P
TP
Head
Ian M
IM
D001419:Bacteria / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D005821:Genetic Techniques; D008163:Luminescent Measurements; D009693:Nucleic Acid Hybridization; D012329:RNA, Bacterial / Q000235:genetics; D012335:RNA, Ribosomal / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D015203:Reproducibility of Results; D012722:Sewage / Q000382:microbiology; D014865:Waste Disposal, Fluid
D012329:RNA, Bacterial; D012335:RNA, Ribosomal; D012336:RNA, Ribosomal, 16S; D012722:Sewage