Combination of Competitive Quantitative PCR and Constant-Denaturant Capillary Electrophoresis for High-Resolution Detection and Enumeration of Microbial Cells

Eelin Lim — 2008-05-05T02:10:04-00:00

Appl. Environ. Microbiol., Vol. 67, No. 9. (1 September 2001), pp. 3897-3903.

A novel quantitative PCR (QPCR) approach, which combines competitive PCR with constant-denaturant capillary electrophoresis (CDCE), was adapted for enumerating microbial cells in environmental samples using the marine nanoflagellate Cafeteria roenbergensis as a model organism. Competitive PCR has been used successfully for quantification of DNA in environmental samples. However, this technique is labor intensive, and its accuracy is dependent on an internal competitor, which must possess the same amplification efficiency as the target yet can be easily discriminated from the target DNA. The use of CDCE circumvented these problems, as its high resolution permitted the use of an internal competitor which differed from the target DNA fragment by a single base and thus ensured that both sequences could be amplified with equal efficiency. The sensitivity of CDCE also enabled specific and precise detection of sequences over a broad range of concentrations. The combined competitive QPCR and CDCE approach accurately enumerated C. roenbergensis cells in eutrophic, coastal seawater at abundances ranging from approximately 10 to 104 cells ml[-]1. The QPCR cell estimates were confirmed by fluorescent in situ hybridization counts, but estimates of samples with <50 cells ml[-]1 by QPCR were less variable. This novel approach extends the usefulness of competitive QPCR by demonstrating its ability to reliably enumerate microorganisms at a range of environmentally relevant cell concentrations in complex aquatic samples. 10.1128/AEM.67.9.3897-3903.2001

Diversity and Dynamics of a North Atlantic Coastal Vibrio Community

Janelle Thompson — 2008-05-05T02:00:06-00:00

Appl. Environ. Microbiol., Vol. 70, No. 7. (1 July 2004), pp. 4103-4110.

Vibrios are ubiquitous marine bacteria that have long served as models for heterotrophic processes and have received renewed attention because of the discovery of increasing numbers of facultatively pathogenic strains. Because the occurrence of specific vibrios has frequently been linked to the temperature, salinity, and nutrient status of water, we hypothesized that seasonal changes in coastal water bodies lead to distinct vibrio communities and sought to characterize their level of differentiation. A novel technique was used to quantify shifts in 16S rRNA gene abundance in samples from Barnegat Bay, N.J., collected over a 15-month period. Quantitative PCR (QPCR) with primers specific for the genus Vibrio was combined with separation and quantification of amplicons by constant denaturant capillary electrophoresis (CDCE). Vibrio populations identified by QPCR-CDCE varied between summer and winter samples, suggesting distinct warm-water and year-round populations. Identification of the CDCE populations by cloning and sequencing of 16S rRNA genes from two summer and two winter samples confirmed this distinction. It further showed that CDCE populations corresponded in most cases to [~]98% rRNA similarity groups and suggested that the abundance of these follows temperature trends. Phylogenetic comparison yielded closely related cultured and often pathogenic representatives for most sequences, and the temperature ranges of these isolates confirmed the trends seen in the environmental samples. Overall, this suggests that temperature is a good predictor of the occurrence of closely related vibrios but that considerable microdiversity of unknown significance coexists within this trend. 10.1128/AEM.70.7.4103-4110.2004

CiteULike: vrich cdce

Combination of Competitive Quantitative PCR and Constant-Denaturant Capillary Electrophoresis for High-Resolution Detection and Enumeration of Microbial Cells

Diversity and Dynamics of a North Atlantic Coastal Vibrio Community