Evaluation of quantitative polymerase chain reaction-based approaches for determining gene copy and gene transcript numbers in environmental samples.

Quantitative polymerase chain reaction (Q-PCR) amplification is widely applied for determining gene and transcript numbers within environmental samples. This research evaluated Q-PCR reproducibility via TaqMan assays quantifying 16S rRNA gene and transcript numbers in sediments, within and between replicate Q-PCR assays. Intra-assay variation in 16S rRNA gene numbers in replicate DNA samples was low (coefficients of variation; CV from 3.2 to 5.2%). However, variability increased using replicated standard curves within separate Q-PCR assays (CV from 11.2% to 26%), indicating absolute comparison of gene numbers between Q-PCR assays was less reliable. 16S rRNA transcript quantification was evaluated using standard curves of diluted RNA or cDNA (before, or following, reverse transcription). These standard curves were statistically different with cDNA-derived curves giving higher r(2) values and Q-PCR efficiencies. Template concentrations used in Q-PCR also affected 16S rRNA gene and transcript numbers. For DNA, 10(-3) dilutions yielded higher gene numbers than 10(-1) and 10(-2) dilutions. Conversely, RNA template dilution reduced numbers of transcripts detected. Finally, different nucleic acid isolation methods also resulted in gene and transcript number variability. This research demonstrates Q-PCR determination of absolute numbers of genes and transcripts using environmental nucleic acids should be treated cautiously.

[1]  R. Abramson,et al.  Detection of specific polymerase chain reaction product by utilizing the 5'----3' exonuclease activity of Thermus aquaticus DNA polymerase. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[2]  Darrell P. Chandler,et al.  Reverse Transcriptase (RT) Inhibition of PCR at Low Concentrations of Template and Its Implications for Quantitative RT-PCR , 1998, Applied and Environmental Microbiology.

[3]  R M Atlas,et al.  Polymerase chain reaction-gene probe detection of microorganisms by using filter-concentrated samples , 1991, Applied and environmental microbiology.

[4]  D. M. Ward,et al.  16S rRNA sequences reveal numerous uncultured microorganisms in a natural community , 1990, Nature.

[5]  S A Bustin,et al.  Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. , 2002, Journal of molecular endocrinology.

[6]  K. Scow,et al.  Detection and Quantification of Methyl tert-Butyl Ether-Degrading Strain PM1 by Real-Time TaqMan PCR , 2001, Applied and Environmental Microbiology.

[7]  J. Fry,et al.  DNase I treatment of Taq DNA polymerase for complete PCR decontamination. , 1992, BioTechniques.

[8]  G. Sayler,et al.  Power Analysis for Real-Time PCR Quantification of Genes in Activated Sludge and Analysis of the Variability Introduced by DNA Extraction , 2003, Applied and Environmental Microbiology.

[9]  A. Wright,et al.  Real-Time PCR Analysis of Vibrio vulnificus from Oysters , 2003, Applied and Environmental Microbiology.

[10]  Sébastien Flavier,et al.  Establishment of a Real-Time PCR-Based Approach for Accurate Quantification of Bacterial RNA Targets in Water, Using Salmonella as a Model Organism , 2004, Applied and Environmental Microbiology.

[11]  P. Lindgren,et al.  Quantification of Ammonia-Oxidizing Bacteria in Arable Soil by Real-Time PCR , 2001, Applied and Environmental Microbiology.

[12]  L. Jackson,et al.  Application of Real-Time PCR To Study Effects of Ammonium on Population Size of Ammonia-Oxidizing Bacteria in Soil , 2004, Applied and Environmental Microbiology.

[13]  E. Delong,et al.  Quantitative Analysis of Small-Subunit rRNA Genes in Mixed Microbial Populations via 5′-Nuclease Assays , 2000, Applied and Environmental Microbiology.

[14]  K. Horikoshi,et al.  Rapid Detection and Quantification of Members of the Archaeal Community by Quantitative PCR Using Fluorogenic Probes , 2000, Applied and Environmental Microbiology.

[15]  T. Schmidt,et al.  rRNA Operon Copy Number Reflects Ecological Strategies of Bacteria , 2000, Applied and Environmental Microbiology.

[16]  J. Hobman,et al.  Amplification of DNA from native populations of soil bacteria by using the polymerase chain reaction , 1992, Applied and environmental microbiology.

[17]  K. Scow,et al.  Naturally Occurring Bacteria Similar to the Methyl tert-Butyl Ether (MTBE)-Degrading Strain PM1 Are Present in MTBE-Contaminated Groundwater , 2003, Applied and Environmental Microbiology.

[18]  S. Giovannoni,et al.  Bias caused by template annealing in the amplification of mixtures of 16S rRNA genes by PCR , 1996, Applied and environmental microbiology.

[19]  N. Pace,et al.  Differential amplification of rRNA genes by polymerase chain reaction , 1992, Applied and environmental microbiology.

[20]  C. Schleper,et al.  Diversity and abundance of Crenarchaeota in terrestrial habitats studied by 16S RNA surveys and real time PCR. , 2003, Environmental microbiology.

[21]  K. Timmis,et al.  Detection and Diversity of Expressed Denitrification Genes in Estuarine Sediments after Reverse Transcription-PCR Amplification from mRNA , 2002, Applied and Environmental Microbiology.

[22]  R. Conrad,et al.  Quantitative Detection of Methanotrophs in Soil by Novel pmoA-Targeted Real-Time PCR Assays , 2003, Applied and Environmental Microbiology.

[23]  Darrell P. Chandler,et al.  Application of the 5′ Fluorogenic Exonuclease Assay (TaqMan) for Quantitative Ribosomal DNA and rRNA Analysis in Sediments , 2001, Applied and Environmental Microbiology.

[24]  C. Nakatsu,et al.  Detection and Enumeration of Aromatic Oxygenase Genes by Multiplex and Real-Time PCR , 2003, Applied and Environmental Microbiology.

[25]  Martin F. Polz,et al.  Bias in Template-to-Product Ratios in Multitemplate PCR , 1998, Applied and Environmental Microbiology.

[26]  A. Bej,et al.  Rapid Detection of Vibrio vulnificus in Shellfish and Gulf of Mexico Water by Real-Time PCR , 2004, Applied and Environmental Microbiology.

[27]  T. Lueders,et al.  Evaluation of PCR Amplification Bias by Terminal Restriction Fragment Length Polymorphism Analysis of Small-Subunit rRNA and mcrA Genes by Using Defined Template Mixtures of Methanogenic Pure Cultures and Soil DNA Extracts , 2003, Applied and Environmental Microbiology.

[28]  K. Livak,et al.  Real time quantitative PCR. , 1996, Genome research.

[29]  M. D. Perkins,et al.  Comparison of the ABI 7700 System (TaqMan) and Competitive PCR for Quantification of IS6110 DNA in Sputum during Treatment of Tuberculosis , 1998, Journal of Clinical Microbiology.

[30]  D. B. Nedwell,et al.  Rapid Extraction of DNA and rRNA from Sediments by a Novel Hydroxyapatite Spin-Column Method , 1996, Applied and environmental microbiology.

[31]  A. Holck,et al.  Application of 5′-Nuclease PCR for Quantitative Detection of Listeria monocytogenes in Pure Cultures, Water, Skim Milk, and Unpasteurized Whole Milk , 2000, Applied and Environmental Microbiology.

[32]  S. Bustin.  Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. , 2000, Journal of molecular endocrinology.

[33]  Willy Verstraete,et al.  Detection and quantification of the human-specific HF183 Bacteroides 16S rRNA genetic marker with real-time PCR for assessment of human faecal pollution in freshwater. , 2005, Environmental microbiology.

[34]  J. Paul,et al.  Real-Time PCR Quantification of rbcL (Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase) mRNA in Diatoms and Pelagophytes , 2002, Applied and Environmental Microbiology.

[35]  S. Giovannoni,et al.  Genetic diversity in Sargasso Sea bacterioplankton , 1990, Nature.

[36]  D. B. Nedwell,et al.  Denitrification in sediments of the River Colne estuary, England , 2000 .

[37]  R. Amann,et al.  Quantification of dissimilatory (bi)sulphite reductase gene expression in Desulfobacterium autotrophicum using real-time RT-PCR. , 2003, Environmental microbiology.

[38]  Sven Becker,et al.  Quantitative Tracing, by Taq Nuclease Assays, of a Synechococcus Ecotype in a Highly Diversified Natural Population , 2002, Applied and Environmental Microbiology.

[39]  Sven Becker,et al.  PCR Bias in Ecological Analysis: a Case Study for Quantitative Taq Nuclease Assays in Analyses of Microbial Communities , 2000, Applied and Environmental Microbiology.

[40]  Jizhong Zhou,et al.  Pseudomonas stutzeri Nitrite Reductase Gene Abundance in Environmental Samples Measured by Real-Time PCR , 2001, Applied and Environmental Microbiology.

[41]  S. Torriani,et al.  Development of Reverse Transcription (RT)-PCR and Real-Time RT-PCR Assays for Rapid Detection and Quantification of Viable Yeasts and Molds Contaminating Yogurts and Pasteurized Food Products , 2003, Applied and Environmental Microbiology.