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Applications of real-time polymerase chain reaction for quantification of microorganisms in environmental samples

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Abstract

Due to the advanced development of fluorogenic chemistry, quantitative real-time polymerase chain reaction (qRT-PCR) has become an emerging technique for the detection and quantification of microorganisms in the environment. Compared with the conventional hybridization- and PCR-based techniques, qRT-PCR not only has better sensitivity and reproducibility, but it is also quicker to perform and has a minimum risk of amplicon carryover contamination. This article reviews the principle of this emerging technique, its detection reagents, target DNAs, quantification procedures, and affecting factors. The applications of qRT-PCR for the quantification of microorganisms in the environment are also summarized.

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References

  • Abell GCJ, Bowman JP (2005) Colonization and community dynamics of class Flavobacteria on diatom detritus in experimental mesocosms based on Southern Ocean seawater. FEMS Microbiol Ecol 53:379–391

    CAS  PubMed  Google Scholar 

  • Bassler HA, Flood SJA, Livak KJ, Marmaro J, Knorr R, Batt CA (1995) Use of a fluorogenic probe in a PCR-based assay for the detection of Listeria monocytogenes. Appl Environ Microbiol 61:3724–3728

    CAS  PubMed  PubMed Central  Google Scholar 

  • Belanger SD, Boissinot M, Clairoux N, Picard FJ, Bergeron MG (2003) Rapid detection of Clostridium difficile in feces by real-time PCR. J Clin Microbiol 41:730–734

    CAS  PubMed  PubMed Central  Google Scholar 

  • Beller HR, Kane SR, Legler TC, Alvarez PJJ (2002) A real-time polymerase chain reaction method for monitoring anaerobic, hydrocarbon-degrading bacteria based on a catabolic gene. Environ Sci Technol 36:3977–3984

    CAS  PubMed  Google Scholar 

  • Bengtsson M, Karlsson HJ, Westman G, Kubista M (2003) A new minor groove binding asymmetric cyanine reporter dye for real-time PCR. Nucleic Acids Res 31:e45

    PubMed  PubMed Central  Google Scholar 

  • Benveniste O, Vaslin B, Villinger F, Grand RL, Ansari AA, Dormont D (1996) Cytokine mRNA levels in unmanipulated (ex vivo) and in vitro stimulated monkey PBMCs using a semi-quantitative RT-PCR and high sensitivity fluorescence-based detection strategy. Cytokine 8:32–41

    CAS  PubMed  Google Scholar 

  • Bischoff C, Lüthy J, Altwegg M, Baggi F (2005) Rapid detection of diarrheagenic E. coli by real-time PCR. J Microbiol Methods 61:335–341

    CAS  PubMed  Google Scholar 

  • Briones A, Raskin L (2003) Diversity and dynamics of microbial communities in engineered environments and their implications for process stability. Curr Opin Biotechnol 14:270–276

    CAS  PubMed  Google Scholar 

  • Chen S, Yee A, Griffiths M, Larkin C, Yamashiro CT, Behari R, Paszko-Kolva C, Grandis SAD (1997) The evaluation of a fluorogenic polymerase chain reaction assay for the detection of Salmonella species in food commodities. Int J Food Microbiol 35:239–250

    PubMed  Google Scholar 

  • DeFrancesco L (2003) Real-time PCR takes center stage. Anal Chem 75(7):175A–179A

    Google Scholar 

  • Devereux R, Kane MD, Winfrey J, Stahl DA (1992) Genus- and group-specific hybridization probes for determinative and environmental studies of sulfate-reducing bacteria. Syst Appl Microbiol 15:601–609

    CAS  Google Scholar 

  • Devers M, Soulas G, Martin-Laurent F (2004) Real-time PCR reverse transcription PCR analysis of expression of atrazine catabolism genes in two bacterial strains isolated from soil. J Microbiol Methods 56:3–15

    CAS  PubMed  Google Scholar 

  • Dionisi HM, Layton AC, Harms G, Gregory IR, Robinson KG, Sayler GS (2002) Quantification of Nitrosomonas oligotropha-like ammonia-oxidizing bacteria and Nitrospira spp from full-scale wastewater treatment plants by competitive PCR. Appl Environ Microbiol 68:245–253

    CAS  PubMed  PubMed Central  Google Scholar 

  • Dionisi HM, Harms G, Layton AC, Gregory IR, Parker J, Hawkins SA, Robinson KG, Sayler GS (2003) Power analysis for real-time PCR quantification of genes in activated sludge and analysis of the variability introduced by DNA extraction. Appl Environ Microbiol 69:6597–6604

    CAS  PubMed  PubMed Central  Google Scholar 

  • Donia MD, Pana A (2005) Use of armored RNA as a standard to construct a calibration curve for real-time RT-PCR. J Virol Methods 126:157–163

    CAS  PubMed  Google Scholar 

  • Foulds IV, Granacki A, Xiao C, Krull UJ, Castle A, Horgen PA (2002) Quantification of microcystin-producing cyanobacteria and E. coli in water by 5′ nuclease PCR. J Appl Microbiol 93:825–834

    CAS  PubMed  Google Scholar 

  • Gibson UE, Heid CA, Williams PM (1996) A novel method for real time quantitative RT-PCR. Genome Res 6:995–1001

    CAS  PubMed  Google Scholar 

  • Ginzinger DG (2002) Gene quantification using real-time quantitative PCR: an emerging technology hits the mainstream. Exp Hematol 30:503–512

    CAS  PubMed  Google Scholar 

  • Glazer AN, Mathies RA (1997) Energy-transfer fluorescent reagents for DNA analyses. Curr Opin Biotechnol 8:94–102

    CAS  PubMed  Google Scholar 

  • Gruntzig V, Nold SC, Zhou J, Tiedje JM (2001) Pseudomonas stutzeri nitrite reductase gene abundance in environmental samples measured by real-time PCR. Appl Environ Microbiol 67:760–768

    CAS  PubMed  PubMed Central  Google Scholar 

  • Hales BA, Edwards C, Ritchie DA, Hall G, Pickup RW, Saunders JR (1996) Isolation and identification of methanogen-specific DNA from blanket bog peat by PCR amplification and sequence analysis. Appl Environ Microbiol 62:668–675

    CAS  PubMed  PubMed Central  Google Scholar 

  • Hall SJ, Hugenholtz P, Siyambalapitiya N, Keller J, Blackall LL (2002) The development and use of real-time PCR for the quantification of nitrifiers in activated sludge. Water Sci Technol 46:267–272

    CAS  PubMed  Google Scholar 

  • Harms G, Layton AC, Dionisi HM, Gregory IM, Garrett VM, Hawkins SA, Robinson KG, Sayler GS (2003) Real-time PCR quantification of nitrifying bacteria in a municipal wastewater treatment plant. Environ Sci Technol 37:343–351

    CAS  Google Scholar 

  • Haugland RA, Vesper SJ, Wymer LJ (1999) Quantitative measurement of Stachybotrys chartarum conidia using real time detection of PCR products with TaqMan fluorogenic probe system. Mol Cell Probes 13:329–340

    CAS  PubMed  Google Scholar 

  • Heid CA, Stevens J, Livak KJ, Williams PM (1996) Real time quantitative PCR. Genome Res 6:986–994

    CAS  Google Scholar 

  • Hermansson A, Lindgren PE (2001) Quantification of ammonia oxidizing bacteria in arable soil by real-time PCR. Appl Environ Microbiol 67:972–976

    CAS  PubMed  PubMed Central  Google Scholar 

  • Hernández M, Estev Te, Prat S, Pla M (2004) Development of real-time PCR systems based on SYBR Green I, Amplifluor and TaqMan technologies for specific quantitative detection of the transgenic maize event GA21. J Cereal Sci 39:99–107

    Google Scholar 

  • Higuchi R, Fockler C, Dollinger G, Watson R (1993) Kinetic PCR analysis: real-time monitoring of DNA amplification reactions. Biotechnology (N Y) 11:1026–1030

    CAS  Google Scholar 

  • Holland PM, Abramson RD, Watson R, Gelfand DH (1991) Detection of specific polymerase chain reaction product by utilizing the 5′-3′ exonuclease activity of Thermus aquaticus DNA polymerase. Proc National Acad Sci U S A 88:7276–7280

    CAS  Google Scholar 

  • Hristova KR, Lutenegger CM, Scow KM (2001) Detection and quantification of MTBE-degrading strain PM1 by real-time TaqMan PCR. Appl Environ Microbiol 67:5154–5160

    CAS  PubMed  PubMed Central  Google Scholar 

  • Jordan FL, Cantera JJL, Fenn ME, Stein LY (2005) Autotrophic ammonia-oxidizing bacteria contribute minimally to nitrification in a nitrogen-impacted forested ecosystem. Appl Environ Microbiol 71:197–206

    CAS  PubMed  PubMed Central  Google Scholar 

  • Kikuchi T, Iwasaki K, Nishihara H, Takamura Y, Yagi O (2002) Quantitative and rapid detection of the trichloroethylene-degrading bacterium Methylocyctis sp. M in groundwater by real-time PCR. Appl Microbiol Biotechnol 59:731–736

    CAS  PubMed  Google Scholar 

  • Klein D (2002) Quantification using real-time PCR technology: applications and limitations. Trends Mol Med 8:257–260

    CAS  PubMed  Google Scholar 

  • Kolb S, Knief C, Stubner S, Conrad R (2003) Quantitative detection of methanotrophs in soil by novel pmoA-targeted real-time PCR assays. Appl Environ Microbiol 69:2423–2429

    CAS  PubMed  PubMed Central  Google Scholar 

  • Leutenegger CM, Pusterla N, Mislin CN, Weber R, Lutz H (1999) Molecular evidence of ticks coinfected with Borrelia burgdorferi sensu lato and the human granulocytic ehrlichiosis agent in Switzerland. J Clin Microbiol 37:3390–3391

    CAS  PubMed  PubMed Central  Google Scholar 

  • Limpiyakorna T, Shinoharab Y, Kurisub F, Yagib O (2005) Communities of ammonia-oxidizing bacteria in activated sludge of various sewage treatment plants in Tokyo. FEMS Microbiol Ecol 54:205–217

    Google Scholar 

  • Lopez-Gutierrez JC, Henry S, Hallet S, Martin-Laurent F, Catroux G, Philippot L (2004) Quantification of a novel group of nitrate-reducing bacteria in the environment by real-time PCR. J Microbiol Methods 57:399–407

    CAS  PubMed  Google Scholar 

  • Lyon WJ (2001) TaqMan PCR for detection of Vibrio cholerae O1, O139, non-O1, and non-O139 in pure cultures, raw oysters, and synthetic seawater. Appl Environ Microbiol 67:4685–4693

    CAS  PubMed  PubMed Central  Google Scholar 

  • Manz W, Eisenbrecher M, Neu TR, Szewzyk U (1998) Abundance and spatial organization of gram-negative sulfate-reducing bacteria in activated sludge investigated by in situ probing with specific 16S rRNA targeted oligonucleotides. FEMS Microbiol Ecol 25:43–61

    CAS  Google Scholar 

  • Maria PO, McDonald IR, Groleau D, Murrell JC, Miguez CB (2002) Detection of methanotrophs with highly divergent pmoA genes from Arctic soils. FEMS Microbiol Lett 209:313–319

    Google Scholar 

  • Mhlanga MM, Malmberg L (2001) Using molecular beacons to detect single-nucleotide polymorphisms with real-time PCR. Methods 25:463–471

    CAS  PubMed  Google Scholar 

  • Morrison TM, Weiss JJ, Wittwer CT (1998) Quantification of low-copy transcripts by continuous SYBR green I monitoring during amplification. Biotechniques 24:954–962

    CAS  PubMed  Google Scholar 

  • Muyzer G, de Waal EC, Uitterlinden AG (1993) Profiling of complex microbial population by DGGE analysis of polymerase chain reaction amplified genes encoding for 16S rRNA. Appl Environ Microbiol 62:2676–2680

    Google Scholar 

  • Nadkarni MA, Martin FE, Jacques NA, Hunter N (2002) Determination of bacterial load by real-time PCR using a broad-range (universal) probe and primers set. Microbiol 148:257–266

    CAS  Google Scholar 

  • Nogva HK, Rudi K, Naterstad K, Holck A, LilleHaug D (2000) Application of 5′-nuclease PCR for quantitative detection of Listeria monocytogenes in pure cultures, water, skim milk, and pasteurized whole milk. Appl Environ Microbiol 66:4266–4271

    CAS  PubMed  PubMed Central  Google Scholar 

  • Nonneman D, Zimba PA (2002) PCR-based test to assess the potential for microcystin occurrence and channel catfish production ponds. J Phycol 38:230–233

    Google Scholar 

  • Oberst RD, Hays MP, Bohra LK, Phebus RK, Yamashiro CT, Paszko-Kolva C, Flood SJA, Sargeant JM, Gillespie JR (1998) PCR-based DNA amplification and presumptive detection of Escherichia coli O157:H7 with an internal fluorogenic probe and the 5′ nuclease TaqMan assay. Appl Environ Microbiol 64:3389–3396

    CAS  PubMed  PubMed Central  Google Scholar 

  • Okabe S, Satoh H, Watanabe Y (1999) In situ analysis of nitrifying biofilms as determined by the in situ hybridisation and the use of microeletrodes. Appl Environ Microbiol 65:3182–3191

    CAS  PubMed  PubMed Central  Google Scholar 

  • Okano Y, Hristova KR, Leutenegger CM, Jackson LE, Denison RF, Gebreyesus B, Lebauer D, Scow KM (2004) Application of real-time PCR to study effects of ammonium on population size of ammonia-oxidizing bacteria in soil. Appl Environ Microbiol 70:1008–1016

    CAS  PubMed  PubMed Central  Google Scholar 

  • Panicker G, Myers ML, Bej AK (2004) Rapid detection of Vibrio vulnificus in shellfish and Gulf of Mexico water by real-time PCR. Appl Environ Microbiol 70:498–507

    CAS  PubMed  PubMed Central  Google Scholar 

  • Ponchel F, Toomes C, Bransfield K, Leong FT, Douglas SH, Field SL, Bell SM, Combaret V, Puisieux A, Mighell AJ, Robinson PA, Inglehearn CF, Isaacs JD, Markham AF (2003) Real-time PCR based on SYBR Green I fluorescence: an alternative to the TaqMan assay for a relative quantification of gene rearrangements, gene amplifications and micro gene deletions. BMC Biotechnol 13:18

    Google Scholar 

  • Purkhold U, Pommerening-Roser A, Juretschko S, Schmid MC, Koops HP, Wagner M (2000) Phylogeny of all recognized species of ammonia oxidizers based on comparative 16S rRNA and amoA sequence analysis: implications for molecular diversity surveys. Appl Environ Microbiol 66:5368–5382

    CAS  PubMed  PubMed Central  Google Scholar 

  • Qiu XY, Hurt RA, Wu LY, Chen CH, Tiedje JM, Zhou JZ (2004) Detection and quantification of copper-denitrifying bacteria by quantitative competitive PCR. J Microbiol Methods 59:199–210

    CAS  PubMed  Google Scholar 

  • Raskin L, Amann RI, Poulsen LK, Rittmann BE, Stahl DA (1995) Use of ribosomal RNA-based molecular probes for characterization of complex microbial communities in anaerobic biofilms. Water Sci Technol 31:261–272

    CAS  Google Scholar 

  • Reina EI, Kurokawa K, Fujioka A, Sharma A, Mayer BJ, Matsuda M (2005) A FRET-based probe for epidermal growth factor receptor bound non-covalently to a pair of synthetic amphipathic helixes. Exp Cell Res 307:142–152

    Google Scholar 

  • Rhee SK, Liu XD, Wu LY, Chong SC, Wan XF, Zhou JZ (2004) Detection of genes involved in biodegradation and biotransformation in microbial communities by using 50-mer oligonucleotide microarrays. Appl Environ Microbiol 70:4303–4317

    CAS  PubMed  PubMed Central  Google Scholar 

  • Rinta-Kanto JM, Ouellette AJA, Boyer GL, Twiss MR, Bridgeman TB, Wilhelm SW (2005) Quantification of toxic Microcystis spp during the 2003 and 2004 blooms in western lake Erie using quantitative real-time PCR. Environ Sci Technol 39:4198–4205

    CAS  PubMed  Google Scholar 

  • Sawayama S, Tsukahara K, Yagishita T (2006) Phylogenetic description of immobilized methanogenic community using real-time PCR in a fixed-bed anaerobic digester. Bioresour Technol 97:69–76

    CAS  PubMed  Google Scholar 

  • Scheid D, Stubner S (2001) Structure and diversity of gram-negative sulfate-reducing bacteria on rice roots. FEMS Microbiol Ecol 36:175–183

    CAS  PubMed  Google Scholar 

  • Schneegurt MA, Kulpa CF (1998) The application of molecular techniques in environmental biotechnology for monitoring microbial systems. Biotechnol Appl Biochem 27:73–79

    CAS  Google Scholar 

  • Sharkey FH, Banat IM, Marchant R (2004) Detection and quantification of gene expression in environmental bacteriology. Appl Environ Microbiol 70:3795–3806

    CAS  PubMed  PubMed Central  Google Scholar 

  • Skovhus TL, Ramsing NB, Holmstrom C, Kjelleberg S, Dahllof I (2004) Real-time quantitative PCR for assessment of abundance of Pseudoalteromonas species in marine samples. Appl Environ Microbiol 70:2373–2382

    CAS  PubMed  PubMed Central  Google Scholar 

  • Sonia H, Baudoin E, López-Gutiérrez JC, Martin-Laurent F, Brauman A, Philippot L (2004) Quantification of denitrifying bacteria in soils by nirK gene targeted real-time PCR. J Microbiol Methods 59:327–335

    Google Scholar 

  • Stubner S (2002) Enumeration of 16S rDNA of Desulfotomaculum lineage 1 in rice field soil by real-time PCR with SybrGreen detection. J Microbiol Methods 50:155–164

    CAS  PubMed  Google Scholar 

  • Stubner S (2004) Quantification of gram-negative sulphate-reducing bacteria in rice field soil by 16S rRNA gene-targeted real-time PCR. J Microbiol Methods 57:219–230

    CAS  PubMed  Google Scholar 

  • Stubner S, Meuser K (2000) Detection of Desulfotomaculum in an Italian rice paddy soil by 16S ribosomal nucleic acid analyses. FEMS Microbiol Ecol 34:73–80

    CAS  PubMed  Google Scholar 

  • Suzuki MT, Taylor LT, DeLong EF (2000) Quantitative analysis of small-subunit rRNA genes in mixed microbial populations via 5′-nuclease assays. Appl Environ Microbiol 66:4605–4614

    CAS  PubMed  PubMed Central  Google Scholar 

  • Takai K, Horikoshi K (2000) Rapid detection and quantification of members of the archaeal community quantitative PCR using fluorogenic probes. Appl Environ Microbiol 66:5066–5072

    CAS  PubMed  PubMed Central  Google Scholar 

  • Tang Y, Shigematsu T, Morimura IS, Kida K (2004) The effects of micro-aeration on the phylogenetic diversity of microorganisms in a thermophilic anaerobic municipal solid-waste digester. Water Res 38:2537–2550

    CAS  PubMed  Google Scholar 

  • Taveau M, Stockholm D, Spencer M, Richard I (2002) Quantification of splice variants using molecular beacon or scorpion primers. Anal Biochem 305:227–235

    CAS  PubMed  Google Scholar 

  • Tyagi S, Kramer FR (1996) Molecular beacons: probes that fluoresce upon hybridization. Nat Biotechnol 14:303–308

    CAS  PubMed  Google Scholar 

  • van der Velden VH, Hochhaus A, Cazzaniga G, Szczepanski T, Gabert J, van Dongen JJ (2003) Detection of minimal residual disease in hematologic malignancies by real-time quantitative PCR: principles, approaches, and laboratory aspects. Leukemia 17:1013–1034

    PubMed  Google Scholar 

  • Wagner M, Loy A (2002) Bacterial community composition and function in sewage treatment systems. Curr Opin Biotechnol 13:218–227

    CAS  PubMed  Google Scholar 

  • Wagner M, Roger AJ, Flax JL, Brusseau GA, Stahl DA (1998) Phylogeny of dissimilatory sulfite reductases supports an early origin of sulfate respiration. J Bacteriol 180:2975–2982

    CAS  PubMed  PubMed Central  Google Scholar 

  • Wilson PE, Kazadi W, Kamwendo DD, Mwapasa V, Purfield A, Meshnick SR (2005) Prevalence of pfcrt mutations in Congolese and Malawian Plasmodium falciparum isolates as determined by a new Taqman assay. Acta Trop 93:97–106

    CAS  PubMed  Google Scholar 

  • Woese CR, Kandler O, Wheelis ML (1990) Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya. Proc National Acad Sci U S A 87:4576–4579

    CAS  Google Scholar 

  • Wu LY, Thompson DK, Li GS, Hurt RA, Tiedje JM, Zhou JZ (2001) Development and evaluation of functional gene arrays for detection of selected genes in the environment. Appl Environ Microbiol 67:5780–5790

    CAS  PubMed  PubMed Central  Google Scholar 

  • Zhang T, Fang HHP (2001) Phylogenetic diversity of a SRB-rich marine biofilm. Appl Microbiol Biotechnol 57:437–440

    CAS  PubMed  Google Scholar 

  • Zhang T, Fang HHP (2005) 16S rDNA clone library screening of environmental samples using melting curve analysis. J Chin Inst Chem Eng 28:1153–1155

    CAS  Google Scholar 

  • Zhang T, Fang HHP, Ko BCB (2003a) Methane producing bacteria in the corrosion biofilm on mild steel. Appl Microbiol Biotechnol 63:101–106

    CAS  PubMed  Google Scholar 

  • Zhang Y, Zhang D, Li W, Chen J, Peng Y, Cao W (2003b) A novel real-time quantitative PCR method using attached universal template probe. Nucleic Acids Res 31:e123

    PubMed  PubMed Central  Google Scholar 

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Acknowledgement

The authors wish to thank the Hong Kong Research Grants Council for the financial support of this study (HKU 7106/04E).

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  1. Environmental Biotechnology Laboratory, Department of Civil Engineering, The University of Hong Kong, Pokfulam Road, Hong Kong, China

    Tong Zhang & Herbert H. P. Fang

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Zhang, T., Fang, H.H. Applications of real-time polymerase chain reaction for quantification of microorganisms in environmental samples. Appl Microbiol Biotechnol 70, 281–289 (2006). https://doi.org/10.1007/s00253-006-0333-6

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