Applied Microbiology and Biotechnology

, Volume 77, Issue 3, pp 645–656 | Cite as

Improving PCR and qPCR detection of hydrogenase A (hydA) associated with Clostridia in pure cultures and environmental sludges using bovine serum albumin

Applied Genetics and Molecular Biotechnology

Abstract

Detection of hydA genes of Clostridia spp. using degenerative and species specific primers for C. butyricum were optimized by the addition of bovine serum albumin (BSA) to polymerase chain reaction (PCR) and quantitative PCR (qPCR) reactions. BSA concentrations ranging from 100 to 400 ng/μl were examined using pure cultures and a variety of environmental samples as test targets. A BSA concentration of 100 ng/μl, which is lower than previously reported in the literature, was found to be most effective in improving the detection limit. The brightness of amplicons with 100 ng/μl BSA increased in ethidium bromide-treated gels, the minimum detection limit with BSA was at least one log greater, and cycle threshold (C T) values were lower than without BSA in qPCR indicating improved detection of target deoxyribonucleic acid for most samples tested. Although amplicon visualization was improved at BSA concentrations greater than or equal to 100 ng/μl, gene copy numbers detected by qPCR were less, CT values were increased, and T m values were altered. SYBR Green dissociation curves of qPCR products of DNA from pure culture or sludge samples showed that BSA at 100 ng/μl reduced the variability of peak areas and T m values.

Keywords

Bovine serum albumin Clostridia Hydrogenase PCR qPCR 

Notes

Acknowledgments

We thank EISG grant 06-54324A/05-01 and UC Pacific Rim Research Program grant 04-1299 for the funding to carry out this work. We thank Santa Margarita Water District wastewater treatment plant in Orange County for supplying several sets sludge samples. We also thank P. B. Gedalanga, Dr. Y. L. Tsai, W. Chu, and J. Y. Le for their assistance.

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Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  1. 1.Department of Chemical EngineeringNational Cheng Kung UniversityTainanTaiwan
  2. 2.Department of Civil and Environmental EngineeringUniversity of California at IrvineIrvineUSA

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