Application of the 5' fluorogenic exonuclease assay (TaqMan) for quantitative ribosomal DNA and rRNA analysis in sediments.
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 < 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 (C(t)) 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.
Stults
J R
JR
Environmental Microbiology Group, Pacific Northwest National Laboratory, Richland, Washington 99352, USA.
Snoeyenbos-West
O
O
Methe
B
B
Lovley
D R
DR
Chandler
D P
DP
D004275:DNA, Ribosomal / Q000235:genetics / Q000302:isolation & purification*; D020564:Deltaproteobacteria / Q000235:genetics / Q000302:isolation & purification*; D005456:Fluorescent Dyes; D019015:Geologic Sediments / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics / Q000302:isolation & purification*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D019914:Taq Polymerase
D004275:DNA, Ribosomal; D005456:Fluorescent Dyes; D012336:RNA, Ribosomal, 16S; D019914:Taq Polymerase