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 hydrogen-producing 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.
Document Type: Research Article
Publication date: 2007-01-01
More about this publication?
- Proceedings of the Water Environment Federation is an archive of papers published in the proceedings of the annual Water Environment Federation® Technical Exhibition and Conference (WEFTEC® ) and specialty conferences held since the year 2000. These proceedings are not peer reviewed. A subscription to the Proceedings of the Water Environment Federation includes access to most papers presented at the annual WEF Technical Exhibition and Conference (WEFTEC) and other conferences held since 2000. Subscription access begins 12 months after the event and is valid for 12 months from month of purchase. A subscription to the Proceedings of the Water Environment Federation is included in Water Environment Federation (WEF) membership.
WEF Members: Sign in (right panel) with your IngentaConnect user name and password to receive complimentary access. Access begins 12 months after the conference or event
- Subscribe to this Title
- Membership Information
- About WEF Proceedings
- WEFTEC Conference Information
- Learn about the many other WEF member benefits and join today
- Ingenta Connect is not responsible for the content or availability of external websites