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Research Paper |
Institute of Dental Research, Westmead Centre For Oral Health, Westmead Hospital, PO Box 533, Wentworthville, NSW 2145, Australia1
Author for correspondence: Mangala A. Nadkarni. Tel: +61 2 9485 7826. Fax: +61 2 9485 7599. e-mail: mnadkarni{at}dental.wsahs.nsw.gov.au
The design and evaluation of a set of universal primers and probe for the amplification of 16S rDNA from the Domain Bacteria to estimate total bacterial load by real-time PCR is reported. Broad specificity of the universal detection system was confirmed by testing DNA isolated from 34 bacterial species encompassing most of the groups of bacteria outlined in Bergey’s Manual of Determinative Bacteriology. However, the nature of the chromosomal DNA used as a standard was critical. A DNA standard representing those bacteria most likely to predominate in a given habitat was important for a more accurate determination of total bacterial load due to variations in 16S rDNA copy number and the effect of generation time of the bacteria on this number, since rapid growth could result in multiple replication forks and hence, in effect, more than one copy of portions of the chromosome. The validity of applying these caveats to estimating bacterial load was confirmed by enumerating the number of bacteria in an artificial sample mixed in vitro and in clinical carious dentine samples. Taking these parameters into account, the number of anaerobic bacteria estimated by the universal probe and primers set in carious dentine was 40-fold greater than the total bacterial load detected by culture methods, demonstrating the utility of real-time PCR in the analysis of this environment.
Keywords: real-time PCR (TaqMan), detection of bacteria, universal probe, rDNA copy number, carious dentine
Abbreviations: ANGIS, Australian National Genomic Information Service; 6-FAM, 6-carboxyfluorescein; RTF, reduced transport fluid; TAMRA, 6-carboxy-tetramethylrhodamine; CT, threshold cycle; Tm, melting temperature of DNA; td, bacterial doubling time
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