Improving PCR and qPCR detection of hydrogenase A (<Emphasis Type="Italic">hyd</Emphasis>A) associated with Clostridia in pure cultures and environmental sludges using bovine serum albumin |
| |
Authors: | Mei-Yun Wang Betty H Olson Jo-Shu Chang |
| |
Institution: | (1) Department of Chemical Engineering, National Cheng Kung University, no. 1 University Road, Tainan, 701, Taiwan;(2) Department of Civil and Environmental Engineering, University of California at Irvine, 1368 Social Ecology II, Irvine, CA 92697, USA |
| |
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 |
本文献已被 PubMed SpringerLink 等数据库收录! |
|