Quantification of 16S rRNAs in Complex Bacterial Communities by Multiple Competitive Reverse Transcription-PCR in Temperature Gradient Gel Electrophoresis Fingerprints

Abstract

A novel approach was developed to quantify rRNA sequences in complex bacterial communities. The main bacterial 16S rRNAs in Drentse A grassland soils (The Netherlands) were amplified by reverse transcription (RT)-PCR with bacterium-specific primers and were separated by temperature gradient gel electrophoresis (TGGE). The primer pair used (primers U968-GC and L1401) was found to amplify with the same efficiency 16S rRNAs from bacterial cultures containing different taxa and cloned 16S ribosomal DNA amplicons from uncultured soil bacteria. The sequence-specific efficiency of amplification was determined by monitoring the amplification kinetics by kinetic PCR. The primer-specific amplification efficiency was assessed by competitive PCR and RT-PCR, and identical input amounts of different 16S rRNAs resulted in identical amplicon yields. The sequence-specific detection system used for competitive amplifications was TGGE, which also has been found to be suitable for simultaneous quantification of more than one sequence. We demonstrate that this approach can be applied to TGGE fingerprints of soil bacteria to estimate the ratios of the bacterial 16S rRNAs.

Effect of fermentable carbohydrates on piglet faecal bacterial communities as revealed by denaturing gradient gel electrophoresis analysis of 16S ribosomal DNA

“…DGGE visualised only the dominant bacterial fraction in pig faeces, while a cloning approach randomly selected 16S rDNA amplicons. It has been shown previously that all ribotypes matching clearly visible ¢ngerprint bands only represent approximately one-half of all of the rRNA extracted from the soil [40]. This suggests that a large number of less dominant bacteria do not form detectable DGGE bands, although some of them can be selected using a cloning approach [17].…”

Molecular analysis of microbial communities identified in different developmental stages of Ixodes scapularis ticks from Westchester and Dutchess Counties, New York

“…in our tick samples, a pathogen that we (data not shown) and others (Benson et al, 2004) have been able to detect by PCR. The DNA band corresponding to Rickettsia was variable in intensity in the sampled groups of ticks, suggesting differences in bacterial concentration, differences that might in the future be quantified by reverse transcription polymerase chain reaction (RT-PCR) of 16S rRNAs, much as has been done with soil bacteria (Felske et al, 1998).…”

Advances in molecular ecology: tracking trophic links through predator–prey food‐webs

“…DNA profiling techniques, especially DGGE/TGGE, pioneered through studies of microbial diversity in environmental samples, have achieved this aim in examining bacterial diversity (e.g. Felske, Akkermans & De Vos 1998;Sheppard et al 2005b). Generalist predators eat multiple prey species, and it is not always practical to analyse predator responses to prey diversity using the species-specific primer approach.…”