How Many Microorganisms Are Present? Quantitative Reverse Transcription PCR (qRT-PCR)

  • Andy Price
  • Laura Acuña Álvarez
  • Corinne Whitby
  • Jan Larsen
Conference paper

Abstract

Quantitative reverse transcription PCR (qRT-PCR) is a variation of conventional quantitative or real-time PCR, whereby mRNA is first converted into the complementary DNA (cDNA) by reverse transcription, the cDNA is then subsequently quantified by qPCR. The use of mRNA as the initial template allows the quantification of gene transcripts, rather than gene copy numbers. mRNA is only produced by actively metabolising cells and is produced by its corresponding gene to provide a ‘blueprint’ in order for a cell to manufacture a specific protein. Conventional qPCR detects not only DNA present in actively metabolising cells but also inactive and dead cells. qRT-PCR has the advantage that only actively metabolising cells are detected, hence provides a more reliable measure of microbial activity in oilfield samples. When qRT-PCR is combined with primers and probes for specific genes, the activity of microbial processes important in the oilfield, such as sulphate reduction, methanogenesis and nitrate reduction can be monitored.

Keywords

Sulphate Reduction dsrAB Gene Molecular Beacon Probe dsrA Gene Sulphite Reductase 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Notes

Acknowledgements

Laboratory experiments and field monitoring were sponsored by DUC Partners (A.P. Møller-Mærsk, Shell and Chevron).

References

  1. Amann R, Ludwig W, Schleifer KH (1995) Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol Rev 59:143–169Google Scholar
  2. Chin KJ, Esteve-Nunez A, Leang C, Lovley DK (2004) Direct correlation between rates of anaerobic respiration and levels of mRNA for key respiratory genes in Geobacter sulfurreducens. Appl Environ Microbiol 70:5183–5189CrossRefGoogle Scholar
  3. Chin KJ, Sharma ML, Russell LA, O’Neill KR, Lovely DR (2008) Quantifying expression of a dissimilatory (bi) sulfite reductase gene in petroleum-contaminated marine harbour sediments. Microbial Ecol 55:489–499CrossRefGoogle Scholar
  4. Dalsgaard T, Bak F (1994) Nitrate reduction in a sulphate-reducing bacterium, Desulfovibrio desulfuricans, isolated from rice paddy soil: sulfide inhibition, kinetics and regulation. Appl Environ Microbiol 60:291–297Google Scholar
  5. Davies M, Scott PJB (2006) Oilfield water technology. NACE – National Association of Corrosion Engineers, Houston, p 249Google Scholar
  6. Fauque G, Ollivier B (2004) Anaerobes: the sulphate-reducing bacteria as an example of metabolic diversity. In: Bull AT (ed) Microbial diversity and bioprospecting. ASM Press, Washington, DC, pp 169–176Google Scholar
  7. Krekeler D, Cypionka H (1994) The preferred electron-acceptor of Desulfovibrio salixigens. Arch Microbiol 161:271–277Google Scholar
  8. McKew BA, Coulon F, Yakimov MM, Denaro R, Genovese M, Smith CJ, Osborn AM, Timmis KN, McGenity TJ (2007) Determining the identity and roles of oil-metabolizing marine bacteria from the Thames estuary. Environ Microbiol 9:165–176CrossRefGoogle Scholar
  9. Mitchell GJ, Jones JG, Cole JA (1986) Distribution and regulation of nitrate and nitrite reduction by Desulfovibrio and Desulfotomaculum species. Arch Microbiol 144:35–40CrossRefGoogle Scholar
  10. Moura JJG, Brondino CD, Trincao J, Romao MJ (2004) Mo and W bis-MGD enzymes: nitrate reductases and formate dehydrogenases. J Biol Inorg Chem 9:791–799CrossRefGoogle Scholar
  11. Plugge CM, Balk M, Stams AJM (2002) Desulfotomaculum themobenzoicum subsp. thermosyntrophicum subsp. nov., a thermophilic syntrophic, propionate-oxidizing, spore-forming bacterium. Intern J Syst Evol Microbiol 52:391–399Google Scholar
  12. Roszak DB, Grimes DJ, Colwell RR (1984) Viable but non-recoverable stage of Salmonella enteritidus in aquatic systems. Can J Microbiol 30:131–139CrossRefGoogle Scholar
  13. Turpin PE, Maycroft KA, Rowlands CL, Wellington EMH (1993) Viable but non-culturable Salmonellas in soil. J Appl Bacteriol 74:421–427CrossRefGoogle Scholar
  14. Xu HS, Roberts N, Singleton FL, Attwell RW, Grimes DJ, Colwell RR (1982) Survival and viability of non-culturable Escherichia coli and Vibrio cholerae in the estuarine environment. Microbial Ecol 8:313–323CrossRefGoogle Scholar

Copyright information

© Springer Netherlands 2010

Authors and Affiliations

  • Andy Price
    • 1
  • Laura Acuña Álvarez
    • 2
  • Corinne Whitby
    • 3
  • Jan Larsen
    • 4
  1. 1.Oil Plus LtdBerkshireUK
  2. 2.Centro de Innovación y transferencia de Tecnologia de Tecnología (CITT), Universidad de Santiago de Compostela (USC), Edificio EmprendiaSantiago de compostelaSpain
  3. 3.Department of Biological SciencesUniversity of EssexColchesterUK
  4. 4.Maersk Oil and Gas ASCopenhagenDenmark

Personalised recommendations