, Volume 77, Issue 3, pp 645-656
Date: 02 Oct 2007

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

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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.