Quantitative real-time PCR and fluorescence insitu hybridization approaches for enumerating Brevundimonas diminuta in drinking water

Brevundimonas diminuta is a small Gram-negative bacterium used for validation of membranes and filters used in the pharmaceutical and drinking water treatment industries. Current assays are time consuming, nonselective, and may be subject to interference by competing indigenous microorganisms. The focus of this study is to develop rapid and specific enumeration methodologies for B.diminuta. Quantitative real-time polymerase chain reaction (qPCR) and fluorescence insitu hybridization (FISH) assays were developed based on the gyrB (1,166bp) and rpoD (829bp) gene sequences of B.diminuta ATCC 19146. Species-specific primers and probes were designed, and a 100-200bp segment of each gene was targeted in the qPCR studies. For both the qPCR and FISH assays, an internal 25bp sequence was selected for use as a TaqMan probe (labeled with 6-FAM and a Black Hole Quencher). Probe specificity studies, conducted against Gram-negative and Gram-positive reference strains as well as environmental strains, revealed high specificity of the primer/probe pairs to B.diminuta. Sensitivities of the qPCR reactions using purified genomic DNA from B.diminuta were determined to be 0.89pg for rpoD and 8.9pg for gyrB. The feasibility of using whole-cell B.diminuta suspensions directly with the rpoD qPCR protocol was also evaluated. The greatest sensitivity observed for B.diminuta was 110 super(3) colony forming units (CFU) per mL when tryptic soy broth was used as the growth medium. When compared with direct microscopic enumeration using a 5' 6-FAM FISH probe, traditional plating methods showed significant underestimation of B.diminuta concentration (P=0.01) when this organism was cultivated in saline lactose broth. The results of this investigation demonstrate that qPCR and FISH are effective methods for rapid (