Development of improved qPCR assay in the presence of bovine serum albumin for the hydA gene of Clostridium sp. from environmental samples
Hydrogen is considered to be an ideal energy source for the future because it can be efficiently converted to electricity via fuel cell technology and is also cleanly combustible without formation of carbon dioxide (1, 2). Biohydrogen is generated by hydrogen-producing bacterial genera using environmental wastewater as the substrate. Clostridium spp. are frequently found in hydrogenproducing bacterial consortia and are very effective in producing hydrogen from organic substrates, therefore we used Clostridium butyricum CGS5 (3), isolated from hydrogen-producing anaerobic
sewage sludge, to detect the hydA gene. Inhibition of PCR assays is a major problem when detecting molecular targets in microorganisms extracted from environmental samples. The inhibitors contain organic or inorganic compounds, and unknown materials. In addition, many of the reagents used to cultivate microorganisms or prepare PCR samples could inhibit PCR when present at contaminating levels (4-5). Various additives have been included in PCR to prevent inhibition effects, such as skim milk, bovine serum albumin (BSA), T4 gene 32 protein, and polyvinylpyrrolidone. Also, embedding DNA in agarose improves PCR amplification of templates contaminated with two powerful inhibitors (6), polysaccharides and humic acids. We optimized BSA concentration in the PCR assays of pure cultures, secondary sludges, and digester sludges to improve the detection of molecular targets.
BSA, qPCR, hydA, and Clostridium