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Applied and Environmental Microbiology, June 2001, p. 2781-2789, Vol. 67, No. 6
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.6.2781-2789.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Application of the 5' Fluorogenic Exonuclease Assay (TaqMan) for Quantitative Ribosomal DNA and rRNA Analysis in Sediments

Jennie R. Stults,1 Oona Snoeyenbos-West,2 Barbara Methe,2 Derek R. Lovley,2 and Darrell P. Chandler1,*

Environmental Microbiology Group, Pacific Northwest National Laboratory, Richland, Washington 99352,1 and Department of Microbiology, University of Massachusetts, Amherst, Massachusetts 010032

Received 13 November 2000/Accepted 4 April 2001

In this study, we report on the development of quantitative PCR and reverse transcriptase PCR assays for the 16S rRNA of Geobacter spp. and identify key issues related to fluorogenic reporter systems for nucleic acid analyses of sediments. The lower detection limit of each assay was 5 to 50 fg of genomic DNA or <= 2 pg of 16S rRNA. TaqMan PCR spectral traces from uncontaminated, amended aquifer sediments were significantly lower (P < 0.0002) than traces for the external standard curve. We also observed a similar, significant decrease in mean quencher emissions for undiluted extracts relative to those for diluted extracts (P < 0.0001). If PCR enumerations were based solely upon the undiluted sample eluant, the TaqMan assay generated an inaccurate result even though the threshold cycle (Ct) measurements were precise and reproducible in the sediment extracts. Assay accuracy was significantly improved by employing a system of replicate dilutions and replicate analyses for both DNA and rRNA quantitation. Our results clearly demonstrate that fluorescence quenching and autofluorescence can significantly affect TaqMan PCR enumeration accuracy, with subsequent implications for the design and implementation of TaqMan PCR to sediments and related environmental samples.

* Corresponding author. Mailing address: 900 Battelle Blvd., Mail Stop P7-50, Richland, WA 99352. Phone: (509) 376-8644. Fax: (509) 376-1321. E-mail: dp.chandler{at}pnl.gov.

Applied and Environmental Microbiology, June 2001, p. 2781-2789, Vol. 67, No. 6
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.6.2781-2789.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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