Luminex detection of fecal indicators in river samples, marine recreational water, and beach sand

Abstract

Research to understand and remediate coastal pollution is moving toward a multitiered approach in which traditional enumeration of fecal indicators is accompanied by molecular analysis of a variety of targets. Technology that rapidly detects multiple microbial contaminants would benefit from such an approach. The Luminex ® 100™ system is a suspension array that assays multiple analytes rapidly in a single well of a microtiter plate. The ability of the system to simultaneously detect multiple fecal indicating bacteria in environmental samples was tested. Primer/probe sets were designed to simultaneously detect the following fecal indicators: the Bacteroides fragilis group, Enterococcus spp., Escherichia coli & Shigella spp., B. distasonis, and Ent. faecalis. Specificity and sensitivity of the Luminex probes was tested against laboratory cultures. In addition, sequencing, culture plate testing, and specificity testing with environmental isolates were steps taken to validate the function of the assay with environmental samples. Luminex response to cultures and to environmental samples was consistent with sequencing results, suggesting that the technology has the potential to simultaneously detect multiple targets for coastal water quality applications, particularly as progress is made to efficiently extract DNA from water and sediment matrices.

Enterococci in the Environment

“…and pathogenic bacteria such as Shigella spp. in a multiplex format (21). In brief, the extracted DNA is marked with probes, and a detection system determines the overall concentration of the target microbes.…”

“…Experiments using natural waters found that the methodology worked best on river water samples. The targets were not as concentrated in beach water and sand; therefore, group-specific primers were developed to optimize the technique for these natural waters (21). Initial results indicated that the system could detect Enterococcus spp., but quantities were often quite different from those obtained by culture methods (21).…”

Temporal and spatial distributions of marine Synechococcus in the Southern California Bight assessed by hybridization to bead-arrays

“…Luminex beadarrays have been developed for detecting microbial pathogens from clinical (e.g. Bandyopadhyay et al 2007) and environmental (Baums et al 2007) samples. Applications of this technology have also been used to determine bacteria and phytoplankton abundances from the marine environment (Ellison & Burton 2005, Scorzetti et al 2009, Mayali et al 2010.…”

Geo-Chip analysis reveals reduced functional diversity of the bacterial community at a dumping site for dredged Elbe sediment

“…Even if the functional inventory (DNA level) of the bacterial communities at the dumping site is significantly lower, the transcription of for instance pollutant specific genes might be enhanced instead. Environmental pollution, resulting from organic, heavy metal antibiotic contamination, was reported to influence gene regulation in previous studies (Baums et al, 2007;Busenlehner et al, 2003;Diaz and Prieto, 2000). While organic pollutants might be used as an alternative energy source (Gibson et al, 1988;Kanaly and Harayama, 2000), heavy metal contamination results in the up-regulation of transporter genes (Hu et al, 2005) or a chemical transformation of the substances, as in the case of mercury contamination (Nascimento and Chartone-Souza, 2003).…”