Design and use of group-specific primers and probes for real-time quantitative PCR

Abstract

Real-time quantitative polymerase chain reaction (qPCR) has gained popularity as a technique to detect and quantify a specific group of target microorganisms from various environmental samples including soil, water, sediments, and sludge. Although qPCR is a very useful technique for nucleic acid quantification, accurately quantifying the target microbial group strongly depends on the quality of the primer and probe used. Many aspects of conducting qPCR assays have become increasingly routine and automated; however, one of the most important aspects, designing and selecting primer and probe sets, is often a somewhat arcane process. In many cases, failed or non-specific amplification can be attributed to improperly designed primer-probe sets. This paper is intended to provide guidelines and general principles for designing group-specific primers and probes for qPCR assays. We demonstrate the effectiveness of these guidelines by reviewing the use of qPCR to study anaerobic processes and biologic nutrient removal processes. qPCR assays using group-specific primers and probes designed with this method, have been used to successfully quantify 16S ribosomal Ribonucleic Acid (16S rRNA) gene copy numbers from target methanogenic and ammonia- oxidizing bacteria in various laboratory- and full-scale biologic processes. Researchers with a good command of primer and probe design can use qPCR as a valuable tool to study biodiversity and to develop more efficient control strategies for biologic processes.

Keywords

absolute quantification / design guideline / primer / probe / real-time quantitative polymerase chain reaction (qPCR)

References

[1] Koops H, Pommerening-Roser A. The lithoautotrophic ammonia-oxidizing bacteria. In: Brenner D J, Krieg N R, Staley J T, Garrity G M, eds. Bergey’s Manual of Systematic Bacteriology. New York: Springer, 2005, 141-147

[2] Pommerening-Roser A, Rath G, Koops H. Phylogenetic diversity within the genus Nitrosomonas. Systematic and Applied Microbiology, 1996, 19: 344-351

[3] Radl V, Pritsch K, Munch J C, Schloter M. Structural and functional diversity of microbial communities from a lake sediment contaminated with trenbolone, an endocrine-disrupting chemical. Environmental Pollution, 2005, 137(2): 345-353

[4] Muller A KWestergaard KChristensen S, Sorensen S J. The effect of long-term mercury pollution on the soil microbial community. FEMS Microbiology Ecology, 2001, 36(1): 11-19

[5] Wong M T, Mino T, Seviour R J, Onuki M, Liu W T. In situ identification and characterization of the microbial community structure of full-scale enhanced biological phosphorous removal plants in Japan. Water Research, 2005, 39(13): 2901-2914

[6] McMahon K D, Stroot P G, Mackie R I, Raskin L. Anaerobic codigestion of municipal solid waste and biosolids under various mixing conditions—II: Microbial population dynamics. Water Research, 2001, 35(7): 1817-1827

[7] Rowan A K, Snape J R, Fearnside D, Barer M R, Curtis T P, Head I M. Composition and diversity of ammonia-oxidising bacterial communities in wastewater treatment reactors of different design treating identical wastewater. FEMS Microbiology Ecology, 2003, 43(2): 195-206

[8] Schmeisser C, Steele H, Streit W R. Metagenomics, biotechnology with non-culturable microbes. Applied Microbiology and Biotechnology, 2007, 75(5): 955-962

[9] Nicolaisen M H, Ramsing N B. Denaturing gradient gel electrophoresis (DGGE) approaches to study the diversity of ammonia-oxidizing bacteria. Journal of Microbiological Methods, 2002, 50(2): 189-203

[10] Lee C, Kim J, Hwang K, O’Flaherty V, Hwang S. Quantitative analysis of methanogenic community dynamics in three anaerobic batch digesters treating different wastewaters. Water Research, 2009, 43(1): 157-165

[11] Yu Y, Kim J, Hwang S. Use of real-time PCR for group-specific quantification of aceticlastic methanogens in anaerobic processes: population dynamics and community structures. Biotechnology and Bioengineering, 2006, 93(3): 424-433

[12] Ahn J H, Kim J, Lim J, Hwang S. Biokinetic evaluation and modeling of continuous thiocyanate biodegradation by Klebsiella sp. Biotechnology Progress, 2004, 20(4): 1069-1075

[13] Beller H R, Kane S R, Legler T C, Alvarez P J J. A real-time polymerase chain reaction method for monitoring anaerobic, hydrocarbon-degrading bacteria based on a catabolic gene. Environmental Science & Technology, 2002, 36(18): 3977-3984

[14] Zhang T, Fang H H P. Applications of real-time polymerase chain reaction for quantification of microorganisms in environmental samples. Applied Microbiology and Biotechnology, 2006, 70(3): 281-289

[15] Khot P D, Fredricks D N. PCR-based diagnosis of human fungal infections. Expert Review of Anti-Infective Therapy, 2009, 7(10): 1201-1221

[16] Song M, Shin S G, Hwang S. Methanogenic population dynamics assessed by real-time quantitative PCR in sludge granule in upflow anaerobic sludge blanket treating swine wastewater. Bioresource Technology, 2010, 101(1 Suppl 1): S23-S28

[17] Klein D. Quantification using real-time PCR technology: applications and limitations. Trends in Molecular Medicine, 2002, 8(6): 257-260

[18] Schena L, Nigro F, Ippolito A, Gallitelli D. Real-time quantitative PCR: a new technology to detect and study phytopathogenic and antagonistic fungi. European Journal of Plant Pathology, 2004, 110(9): 893-908

[19] Suzuki M T, Taylor L T, DeLong E F. Quantitative analysis of small-subunit rRNA genes in mixed microbial populations via 5′-nuclease assays. Applied and Environmental Microbiology, 2000, 66(11): 4605-4614

[20] Takai K, Horikoshi K. Rapid detection and quantification of members of the archaeal community by quantitative PCR using fluorogenic probes. Applied and Environmental Microbiology, 2000, 66(11): 5066-5072

[21] Hori T, Haruta S, Ueno Y, Ishii M, Igarashi Y. Direct comparison of single-strand conformation polymorphism (SSCP) and denaturing gradient gel electrophoresis (DGGE) to characterize a microbial community on the basis of 16S rRNA gene fragments. Journal of Microbiological Methods, 2006, 66(1): 165-169

[22] Wong M L, Medrano J F. Real-time PCR for mRNA quantitation. BioTechniques, 2005, 39(1): 75-85

[23] Smith C J, Osborn A M. Advantages and limitations of quantitative PCR (Q-PCR)-based approaches in microbial ecology. FEMS Microbiology Ecology, 2009, 67(1): 6-20

[24] Giulietti A, Overbergh L, Valckx D, Decallonne B, Bouillon R, Mathieu C. An overview of real-time quantitative PCR: applications to quantify cytokine gene expression. Methods (San Diego, Calif.), 2001, 25(4): 386-401

[25] Cook K L, Whitehead T R, Spence C, Cotta M A. Evaluation of the sulfate-reducing bacterial population associated with stored swine slurry. Anaerobe, 2008, 14(3): 172-180

[26] Harms G, Layton A C, Dionisi H M, Gregory I R, Garrett V M, Hawkins S A, Robinson K G, Sayler G S. Real-time PCR quantification of nitrifying bacteria in a municipal wastewater treatment plant. Environmental Science & Technology, 2003, 37(2): 343-351

[27] Hermansson A, Lindgren P E. Quantification of ammonia-oxidizing bacteria in arable soil by real-time PCR. Applied and Environmental Microbiology, 2001, 67(2): 972-976

[28] Layton A C, Dionisi H, Kuo H W, Robinson K G, Garrett V M, Meyers A, Sayler G S. Emergence of competitive dominant ammonia-oxidizing bacterial populations in a full-scale industrial wastewater treatment plant. Applied and Environmental Microbiology, 2005, 71(2): 1105-1108

[29] Lim J, Do H, Shin S G, Hwang S. Primer and probe sets for group-specific quantification of the genera Nitrosomonas and Nitrosospira using real-time PCR. Biotechnology and Bioengineering, 2008, 99(6): 1374-1383

[30] Yu Y, Lee C, Kim J, Hwang S. Group-specific primer and probe sets to detect methanogenic communities using quantitative real-time polymerase chain reaction. Biotechnology and Bioengineering, 2005, 89(6): 670-679

[31] Houghton S G, Cockerill F R 3rd. Real-time PCR: overview and applications. Surgery, 2006, 139(1): 1-5

[32] Hermansson A, Backman J S K, Svensson B H, Lindgren P. Quantification of ammonia-oxidising bacteria in limed and non-limed acidic coniferous forest soil using real-time PCR. Soil Biology & Biochemistry, 2004, 36(12): 1935-1941

[33] Sekido T, Bodelier P L E, Shoji T, Suwa Y, Laanbroek H J. Limitations of the use of group-specific primers in real-time PCR as appear from quantitative analyses of closely related ammonia-oxidising species. Water Research, 2008, 42(4-5): 1093-1101

[34] Ashelford K E, Weightman A J, Fry J C. PRIMROSE: a computer program for generating and estimating the phylogenetic range of 16S rRNA oligonucleotide probes and primers in conjunction with the RDP-II database. Nucleic Acids Research, 2002, 30(15): 3481-3489

[35] Marchesi J R. Primer design for PCR amplification of environmental DNA targets. In: Rochelle P A, ed. Environmental Molecular Microbiology: Protocols and Applications. Norfolk, England: Horizon Scientific Press, 2001, 43-54

[36] Giulietti A, Overbergh L, Valckx D, Decallonne B, Bouillon R, Mathieu C. An overview of real-time quantitative PCR: applications to quantify cytokine gene expression. Methods (San Diego, Calif.), 2001, 25(4): 386-401

[37] Polz M F, Cavanaugh C M. Bias in template-to-product ratios in multitemplate PCR. Applied and Environmental Microbiology, 1998, 64(10): 3724-3730

[38] Hall S J, Hugenholtz P, Siyambalapitiya N, Keller J, Blackall L L. The development and use of real-time PCR for the quantification of nitrifiers in activated sludge. Water Science and Technology, 2002, 46(1-2): 267-272

[39] Rochelle P A, De Leon R, Stewart M H, Wolfe R L. Comparison of primers and optimization of PCR conditions for detection of Cryptosporidium parvum and Giardia lamblia in water. Applied and Environmental Microbiology, 1997, 63(1): 106-114

[40] Labrenz M, Brettar I, Christen R, Flavier S, Bötel J, Höfle M G. Development and application of a real-time PCR approach for quantification of uncultured bacteria in the central Baltic Sea. Applied and Environmental Microbiology, 2004, 70(8): 4971-4979

[41] Zinder S H. Physiological ecology of methanogens. In: Ferry J G, ed. Methanogenesis. New York: Chapman & Hall, 1993, 128-206

[42] Banning N, Brock F, Fry J C, Parkes R J, Hornibrook E R C, Weightman A J. Investigation of the methanogen population structure and activity in a brackish lake sediment. Environmental Microbiology, 2005, 7(7): 947-960

[43] Garrity G M, Holt J G. Phylum AII. Euryarchaeota phy. nov. In: Boone D R, Castenholz R W, eds. Bergey’s manual of systematic bacteriology. New York: Springer-Verlag, 2001, 211-355

[44] Karakashev D, Batstone D J, Angelidaki I. Influence of environmental conditions on methanogenic compositions in anaerobic biogas reactors. Applied and Environmental Microbiology, 2005, 71(1): 331-338

[45] Raskin L, Stromley J M, Rittmann B E, Stahl D A. Group-specific 16S rRNA hybridization probes to describe natural communities of methanogens. Applied and Environmental Microbiology, 1994, 60(4): 1232-1240

[46] Smith K S, Ingram-Smith C. Methanosaeta, the forgotten methanogen? Trends in Microbiology, 2007, 15(4): 150-155

[47] Raskin L, Zheng D, Griffin M E, Stroot P G, Misra P. Characterization of microbial communities in anaerobic bioreactors using molecular probes. Antonie van Leeuwenhoek, 1995, 68(4): 297-308

[48] Yu Y, Lee C, Hwang S. Analysis of community structures in anaerobic processes using a quantitative real-time PCR method. Water Science and Technology, 2005, 52(1-2): 85-91

[49] Tatara M, Makiuchi T, Ueno Y, Goto M, Sode K. Methanogenesis from acetate and propionate by thermophilic down-flow anaerobic packed-bed reactor. Bioresource Technology, 2008, 99(11): 4786-4795

[50] Kobayashi T, Li Y Y, Harada H. Analysis of microbial community structure and diversity in the thermophilic anaerobic digestion of waste activated sludge. Water Science and Technology, 2008, 57(8): 1199-1205

[51] Kobayashi T, Yasuda D, Li Y Y, Kubota K, Harada H, Yu H Q. Characterization of start-up performance and archaeal community shifts during anaerobic self-degradation of waste-activated sludge. Bioresource Technology, 2009, 100(21): 4981-4988

[52] Nettmann E, Bergmann I, Mundt K, Linke B, Klocke M. Archaea diversity within a commercial biogas plant utilizing herbal biomass determined by 16S rDNA and mcrA analysis. Journal of Applied Microbiology, 2008, 105(6): 1835-1850

[53] Nettmann E, Bergmann I, Pramschüfer S, Mundt K, Plogsties V, Herrmann C, Klocke M. Polyphasic analyses of methanogenic archaeal communities in agricultural biogas plants. Applied and Environmental Microbiology, 2010, 76(8): 2540-2548

[54] Shin S G, Lee C, Hwang K, Ahn J H, Hwang S. Use of order-specific primers to investigate the methanogenic diversity in acetate enrichment system. Journal of Industrial Microbiology and Biotechnology, 2008, 35(11): 1345-1352

[55] Lee C, Kim J, Shin S G, O’Flaherty V, Hwang S. Quantitative and qualitative transitions of methanogen community structure during the batch anaerobic digestion of cheese-processing wastewater. Applied Microbiology and Biotechnology, 2010, 87(5): 1963-1973

[56] O’Reilly J, Lee C, Collins G, Chinalia F, Mahony T, O’Flaherty V. Quantitative and qualitative analysis of methanogenic communities in mesophilically and psychrophilically cultivated anaerobic granular biofilims. Water Research, 2009, 43(14): 3365-3374

[57] Saengkerdsub S, Anderson R C, Wilkinson H H, Kim W K, Nisbet D J, Ricke S C. Identification and quantification of methanogenic Archaea in adult chicken ceca. Applied and Environmental Microbiology, 2007, 73(1): 353-356

[58] Saengkerdsub S, Herrera P, Woodward C L, Anderson R C, Nisbet D J, Ricke S C. Detection of methane and quantification of methanogenic archaea in faeces from young broiler chickens using real-time PCR. Letters in Applied Microbiology, 2007, 45(6): 629-634

[59] Denman S E, Tomkins N W, McSweeney C S. Quantitation and diversity analysis of ruminal methanogenic populations in response to the antimethanogenic compound bromochloromethane. FEMS Microbiology Ecology, 2007, 62(3): 313-322

[60] Knapp C W, Dolfing J, Ehlert P A I, Graham D W. Evidence of increasing antibiotic resistance gene abundances in archived soils since 1940. Environmental Science & Technology, 2010, 44(2): 580-587

[61] Kim H S, Choung Y K, Ahn S, Oh H S. Enhancing nitrogen removal of piggery wastewater by membrane bioreactor combined with nitrification reactor. Desalination, 2008, 223(1-3): 194-204

[62] Lay-Son M, Drakides C. New approach to optimize operational conditions for the biological treatment of a high-strength thiocyanate and ammonium waste: pH as key factor. Water Research, 2008, 42(3): 774-780

[63] Neufeld R, Greenfield J, Rieder B. Temperature, cyanide and phenolic nitrification inhibition. Water Research, 1986, 20(5): 633-642

[64] Yuan X J, Gao D W. Effect of dissolved oxygen on nitrogen removal and process control in aerobic granular sludge reactor. Journal of Hazardous Materials, 2010, 178(1-3): 1041-1045

[65] Giotta L, Agostiano A, Italiano F, Milano F, Trotta M. Heavy metal ion influence on the photosynthetic growth of Rhodobacter sphaeroides. Chemosphere, 2006, 62(9): 1490-1499

[66] Koops H P, Moller U C. The lithotrophic ammonia-oxidizing bacteria. In: Balow A, Truper H G, Dworkin M, Harder W, Schleifer K H, eds. The Prokaryotes. New York: Springer Verlag, 1992, 2625-2637

[67] Head I M, Hiorns W D, Embley T M, McCarthy A J, Saunders J R. The phylogeny of autotrophic ammonia-oxidizing bacteria as determined by analysis of 16S ribosomal RNA gene sequences. Journal of General Microbiology, 1993, 139(6): 1147-1153

[68] Bothe H, Jost G, Schloter M, Ward B B, Witzel K. Molecular analysis of ammonia oxidation and denitrification in natural environments. FEMS Microbiology Reviews, 2000, 24(5): 673-690

[69] Teske A, Alm E, Regan J M, Toze S, Rittmann B E, Stahl D A. Evolutionary relationships among ammonia- and nitrite-oxidizing bacteria. Journal of Bacteriology, 1994, 176(21): 6623-6630

[70] Prosser J I, Embley T M. Cultivation-based and molecular approaches to characterisation of terrestrial and aquatic nitrifiers. Antonie van Leeuwenhoek, 2002, 81(1-4): 165-179

[71] Kowalchuk G A, Stephen J R. Ammonia-oxidizing bacteria: a model for molecular microbial ecology. Annual Review of Microbiology, 2001, 55(1): 485-529

[72] Bano N, Hollibaugh J T. Diversity and distribution of DNA sequences with affinity to ammonia-oxidizing bacteria of the β subdivision of the class Proteobacteria in the Arctic Ocean. Applied and Environmental Microbiology, 2000, 66(5): 1960-1969

[73] Kowalchuk G A, Stephen J R, De Boer W, Prosser J I, Embley T M, Woldendorp J W. Analysis of ammonia-oxidizing bacteria of the β subdivision of the class Proteobacteria in coastal sand dunes by denaturing gradient gel electrophoresis and sequencing of PCR-amplified 16S ribosomal DNA fragments. Applied and Environmental Microbiology, 1997, 63(4): 1489-1497

[74] Purkhold U, Pommerening-Röser A, Juretschko S, Schmid M C, Koops H P, Wagner M. Phylogeny of all recognized species of ammonia oxidizers based on comparative 16S rRNA and amoA sequence analysis: implications for molecular diversity surveys. Applied and Environmental Microbiology, 2000, 66(12): 5368-5382

[75] Stephen J R, McCaig A E, Smith Z, Prosser J I, Embley T M. Molecular diversity of soil and marine 16S rRNA gene sequences related to β-subgroup ammonia-oxidizing bacteria. Applied and Environmental Microbiology, 1996, 62(11): 4147-4154

[76] Lim J, Lee S, Hwang S. Use of quantitative real-time PCR to monitor population dynamics of ammonia-oxidizing bacteria in batch process. Journal of Industrial Microbiology and Biotechnology, 2008, 35(11): 1339-1344

[77] Limpiyakorn T, Shinohara Y, Kurisu F, Yagi O. Communities of ammonia-oxidizing bacteria in activated sludge of various sewage treatment plants in Tokyo. FEMS Microbiology Ecology, 2005, 54(2): 205-217

[78] Schramm A. In situ analysis of structure and activity of the nitrifying community in biofilms, aggregates, and sediments. Geomicrobiology Journal, 2003, 20(4): 313-333

[79] Do H, Lim J, Shin S G, Wu Y J, Ahn J H, Hwang S. Simultaneous effect of temperature, cyanide and ammonia-oxidizing bacteria concentrations on ammonia oxidation. Journal of Industrial Microbiology & Biotechnology, 2008, 35(11): 1331-1338

[80] Forney L J, Zhou X, Brown C J. Molecular microbial ecology: land of the one-eyed king. Current Opinion in Microbiology, 2004, 7(3): 210-220

[81] Nocker A, Camper A K. Selective removal of DNA from dead cells of mixed bacterial communities by use of ethidium monoazide. Applied and Environmental Microbiology, 2006, 72(3): 1997-2004

[82] Bergmann I, Mundt K, Sontag M, Baumstark I, Nettmann E, Klocke M. Influence of DNA isolation on Q-PCR-based quantification of methanogenic Archaea in biogas fermenters. Systematic and Applied Microbiology, 2010, 33(2): 78-84

[83] Steinberg L M, Regan J M. mcrA-targeted real-time quantitative PCR method to examine methanogen communities. Applied and Environmental Microbiology, 2009, 75(13): 4435-4442

[84] Wells G F, Park H D, Yeung C H, Eggleston B, Francis C A, Criddle C S. Ammonia-oxidizing communities in a highly aerated full-scale activated sludge bioreactor: betaproteobacterial dynamics and low relative abundance of Crenarchaea. Environmental Microbiology, 2009, 11(9): 2310-2328

[85] Lozada M, Figuerola E L M, Itria R F, Erijman L. Replicability of dominant bacterial populations after long-term surfactant-enrichment in lab-scale activated sludge. Environmental Microbiology, 2006, 8(4): 625-638

[86] Lee C, Kim J, Shin S G, Hwang S. Monitoring bacterial and archaeal community shifts in a mesophilic anaerobic batch reactor treating a high-strength organic wastewater. FEMS Microbiology Ecology, 2008, 65(3): 544-554

[87] Shigematsu T, Era S, Mizuno Y, Ninomiya K, Kamegawa Y, Morimura S, Kida K. Microbial community of a mesophilic propionate-degrading methanogenic consortium in chemostat cultivation analyzed based on 16S rRNA and acetate kinase genes. Applied Microbiology and Biotechnology, 2006, 72(2): 401-415

[88] Zhang H, Banaszak J E, Parameswaran P, Alder J, Krajmalnik-Brown R, Rittmann B E. Focused-Pulsed sludge pre-treatment increases the bacterial diversity and relative abundance of acetoclastic methanogens in a full-scale anaerobic digester. Water Research, 2009, 43(18): 4517-4526

Acknowledgements

This work was supported in part by the Korea Ministry of Environment as ‘Human Resource Development Project for Energy from Waste & Recycling’ project. This work was also supported by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD) and by New & Renewable Energy of the Korea Institute of Energy Technology Evaluation and Planning (KETEP) grant (No. 20101T100100366). The authors would like to thank Youngseob Yu (Daewoo E&C) for Fig. 2 and useful discussion.

RIGHTS & PERMISSIONS

2014 Higher Education Press and Springer-Verlag Berlin Heidelberg