A Sandwich Hybridization Assay Employing Enzyme Amplification for termination of Specific Ribosomal RNA from Unpurified Cell Lysates

Wicks, Ben; Cook, David B.; Barer, Michael R.; O'Donnell, Anthony G.; Self, Colin H.

doi:10.1006/abio.1998.2643

Cited by 12 publications

(11 citation statements)

References 26 publications

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“…In preparations of purified 16S-plus-23S rRNA from Escherichia coli MRE600 (Roche Diagnostics, United Kingdom), the 16S rRNA was estimated to account for 34% of the total RNA (31). All the usual precautions were utilized to prevent degradation of RNA (46).…”

Section: Methodsmentioning

confidence: 99%

“…Helper probes were included in certain assays to improve the accessibility of some target sites on the rRNA. The assay was extensively modified from the procedure developed by Wicks and coworkers (46). Two main modifications were made to adapt the assay for use with AOB and environmental samples.…”

Section: Methodsmentioning

confidence: 99%

“…Two main modifications were made to adapt the assay for use with AOB and environmental samples. Solid-phase capture of the probe-target hybrids on streptavidin-coated 96-well plates was employed in the original assay to capture target rRNA (45,46). In the modified assay, capture using suspensions of paramagnetic beads was used to improve the kinetics of the reaction and reduce hybridization time.…”

Section: Methodsmentioning

confidence: 99%

“…An alternative sensitive and specific method is a sandwich hybridization assay for the detection of rRNA, developed by Wicks and coworkers (46). The RNA assay is based on hybridization of a target rRNA with differentially labeled capture and detector probes.…”

mentioning

confidence: 99%

“…In the present study, the primary format of the RNA assay developed by Wicks et al was retained (46), but the assay was extensively modified, developed, and optimized for the specific detection and quantification of betaproteobacterial AOB. Autotrophic AOB are ideal candidates for such an approach, because they form a coherent phylogenetic group that performs a distinct metabolic and process function.…”

mentioning

confidence: 99%

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Development of a Rapid Assay for Determining the Relative Abundance of Bacteria

Rowan

Davenport

Snape

et al. 2005

Appl Environ Microbiol

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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.Quantification of specific bacterial populations is key to understanding the mechanisms that underlie many biologically mediated processes, including nitrification. The use of rRNAbased cultivation-independent techniques may be one potentially useful means to do this. Numerous rRNA-based techniques are now commonly employed to quantify microbial communities in environmental samples. These include fluorescence in situ hybridization (FISH) (see, e.g., references 11, 12, 17, 27, 37, and 40), quantitative dot blot hybridization (see, e.g., references 14 and 34), PCR-based methods (e.g., real time PCR [19] and competitive PCR [15,33]), and microarray technology (see, e.g., references 1, 18, and 38).Each of these methods has particular advantages and disadvantages. For example, FISH allows direct identification and quantification of microorganisms in environmental samples and, because of its ability to measure individuals accurately, represents the "gold standard." However, FISH has a low sample throughput and is thus too slow for routine analysis. In contrast, PCR-based methods allow high sample throughput, which may be required for statistically valid spatial and temporal analysis. However, PCR-based methods are prone to biases (typically introduced during nucleic acid extraction and amplification) that may make quantification of natural bacterial populations difficult (see, e.g., references 20, 39, 39a, and 43). In addition, relatively pure preparations of DNA are required to avoid problems of inhibition of DNA polymerases. Quantitative dot blots and microarrays, on the other hand, often require the use of radioactive isotopes or expensive equipment and consumables.An alternative sensitive and specific method is a sandwich hybridization assay for the detection of rRNA, developed by Wicks and coworkers (46). The RNA assay is based on hybridization of...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

Development of a Rapid Assay for Determining the Relative Abundance of Bacteria

Rowan

Davenport

Snape

et al. 2005

Appl Environ Microbiol

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Analytische Chemie 1999

Nießner¹,

Broekaert²,

Emons

et al. 2000

Nachr Chem

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Universal liposomes: preparation and usage for the detection of mRNA

et al. 2008

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Dye-encapsulating liposomes can serve as signaling reagents in biosensors and biochemical assays in place of enzymes or fluorophores. Detailed here is the use and preparation of streptavidin-coupled liposomes which offer a universal approach to biotinylated target detection. The universal approach provides two advantages, i.e. only one type of liposome is necessary despite varying target and probe sequences and the hybridization event can take place in the absence of potential steric hindrance occurring from liposomes directly conjugated to probes. One objective of this work was to optimize the one-step conjugation of SRB-encapsulating liposomes to streptavidin using EDC. Liposome, EDC, streptavidin concentrations, and reaction times were varied. The optimal coupling conditions were found to be an EDC:carboxylated lipid:streptavidin molar ratio of 600:120:1 and a reaction time of 15 min. The second goal was to utilize these liposomes in sandwich hybridization microtiter plate-based assays using biotinylated reported probes as biorecognition elements. The assay was optimized in terms of probe spacer length, probe concentration, liposome concentration, and streptavidin coverage. Subsequently, the optimized protocol was applied to the detection of DNA and RNA sequences. A detection limit of 1.7 pmol L(-1) and an assay range spanning four orders of magnitude (5 pmol L(-1)-50 nmol L(-1)) with a coefficient of variation

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A Sandwich Hybridization Assay Employing Enzyme Amplification for termination of Specific Ribosomal RNA from Unpurified Cell Lysates

Cited by 12 publications

References 26 publications

Development of a Rapid Assay for Determining the Relative Abundance of Bacteria

Development of a Rapid Assay for Determining the Relative Abundance of Bacteria

Analytische Chemie 1999

Universal liposomes: preparation and usage for the detection of mRNA

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