Nucleic Acids Research, 2002, Vol. 30, No. 6 1292-1305
© 2002 Oxford University Press
Real-time PCR in virology
1Clinical Virology Research Unit, Sir Albert Sakzewski Virus Research Centre, Royal Children’s Hospital, Brisbane, Australia, 2Department of Paediatrics and 3Clinical Medical Virology Centre, University of Queensland, Queensland, Australia, 4Membrane Transport Laboratory, Division of Cancer and Cell Biology, Queensland Institute of Medical Research, Queensland, Australia and 5TIB MOLBIOL, Tempelhofer Weg 11–12, D-10829 Berlin, Germany
The use of the polymerase chain reaction (PCR) in molecular diagnostics has increased to the point where it is now accepted as the gold standard for detecting nucleic acids from a number of origins and it has become an essential tool in the research laboratory. Real-time PCR has engendered wider acceptance of the PCR due to its improved rapidity, sensitivity, reproducibility and the reduced risk of carry-over contamination. There are currently five main chemistries used for the detection of PCR product during real-time PCR. These are the DNA binding fluorophores, the 5' endonuclease, adjacent linear and hairpin oligoprobes and the self-fluorescing amplicons, which are described in detail. We also discuss factors that have restricted the development of multiplex real-time PCR as well as the role of real-time PCR in quantitating nucleic acids. Both amplification hardware and the fluorogenic detection chemistries have evolved rapidly as the understanding of real-time PCR has developed and this review aims to update the scientist on the current state of the art. We describe the background, advantages and limitations of real-time PCR and we review the literature as it applies to virus detection in the routine and research laboratory in order to focus on one of the many areas in which the application of real-time PCR has provided significant methodological benefits and improved patient outcomes. However, the technology discussed has been applied to other areas of microbiology as well as studies of gene expression and genetic disease.
* To whom correspondence should be addressed at: CVRU, SASVRC, Royal Children’s Hospital, Herston Road, Herston, Queensland 4029, Australia. Tel: +61 3636 8716; Fax: +61 3636 1401; Email: i.mackay{at}mailbox.uq.edu.au
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  R. van Doorn, M. Slawiak, M. Szemes, A. M. Dullemans, P. Bonants, G. A. Kowalchuk, and C. D. Schoen Robust Detection and Identification of Multiple Oomycetes and Fungi in Environmental Samples by Using a Novel Cleavable Padlock Probe-Based Ligation Detection Assay Appl. Envir. Microbiol., June 15, 2009; 75(12): 4185 - 4193. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Akiyama, H. Kimura, H. Tsukagoshi, K. Taira, K. Mizuta, M. Saitoh, M. Nagano, A. Sutoh, M. Noda, Y. Morita, et al. Development of an assay for the detection and quantification of the measles virus nucleoprotein (N) gene using real-time reverse transcriptase PCR J. Med. Microbiol., May 1, 2009; 58(5): 638 - 643. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. A. Bustin, V. Benes, J. A. Garson, J. Hellemans, J. Huggett, M. Kubista, R. Mueller, T. Nolan, M. W. Pfaffl, G. L. Shipley, et al. The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments Clin. Chem., April 1, 2009; 55(4): 611 - 622. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Balamurugan, K. D. Jayappa, M. Hosamani, V. Bhanuprakash, G. Venkatesan, and R. K. Singh Comparative efficacy of conventional and taqman polymerase chain reaction assays in the detection of capripoxviruses from clinical samples J Vet Diagn Invest, March 1, 2009; 21(2): 225 - 231. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. A. Rodriguez, I. L. Pepper, and C. P. Gerba Application of PCR-Based Methods To Assess the Infectivity of Enteric Viruses in Environmental Samples Appl. Envir. Microbiol., January 15, 2009; 75(2): 297 - 307. [Full Text] [PDF]
|
|
|
|
 |
|
 M. Hansen, C. Friis, S. Bowra, P. B. Holm, and E. Vincze A pathway-specific microarray analysis highlights the complex and co-ordinated transcriptional networks of the developing grain of field-grown barley J. Exp. Bot., January 1, 2009; 60(1): 153 - 167. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Vincent, Z. Gu, M. Morgenstern, C. Gibson, J. Pan, and R. T. Hayden Detection of Cytomegalovirus in Whole Blood Using Three Different Real-Time PCR Chemistries J. Mol. Diagn., January 1, 2009; 11(1): 54 - 59. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E.-S. Lee, D.-S. Son, S.-H. Kim, J. Lee, J. Jo, J. Han, H. Kim, H. J. Lee, H. Y. Choi, Y. Jung, et al. Prediction of Recurrence-Free Survival in Postoperative Non-Small Cell Lung Cancer Patients by Using an Integrated Model of Clinical Information and Gene Expression Clin. Cancer Res., November 15, 2008; 14(22): 7397 - 7404. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Kurth, J. Achenbach, L. Miller, I. M. Mackay, G. Pauli, and A. Nitsche Orthopoxvirus Detection in Environmental Specimens during Suspected Bioterror Attacks: Inhibitory Influences of Common Household Products Appl. Envir. Microbiol., January 1, 2008; 74(1): 32 - 37. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Navarro-Martinez, E. Navarro, M. J. Castano, and J. Solera Rapid Diagnosis of Human Brucellosis by Quantitative Real-Time PCR: a Case Report of Brucellar Spondylitis J. Clin. Microbiol., January 1, 2008; 46(1): 385 - 387. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. T. Hayden, K. M. Hokanson, S. B. Pounds, M. J Bankowski, S. W. Belzer, J. Carr, D. Diorio, M. S. Forman, Y. Joshi, D. Hillyard, et al. Multicenter Comparison of Different Real-Time PCR Assays for Quantitative Detection of Epstein-Barr Virus J. Clin. Microbiol., January 1, 2008; 46(1): 157 - 163. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Kramski, H. Meisel, B. Klempa, D. H. Kruger, G. Pauli, and A. Nitsche Detection and Typing of Human Pathogenic Hantaviruses by Real-Time Reverse Transcription-PCR and Pyrosequencing Clin. Chem., November 1, 2007; 53(11): 1899 - 1905. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Wada, N. Kubota, Y. Ito, H. Yagasaki, K. Kato, T. Yoshikawa, Y. Ono, H. Ando, Y. Fujimoto, T. Kiuchi, et al. Simultaneous Quantification of Epstein-Barr Virus, Cytomegalovirus, and Human Herpesvirus 6 DNA in Samples from Transplant Recipients by Multiplex Real-Time PCR Assay J. Clin. Microbiol., May 1, 2007; 45(5): 1426 - 1432. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J Bergqvist, J. Ohd, J Smeds, S Klaar, J Isola, H Nordgren, G. Elmberger, H Hellborg, J Bjohle, A-L Borg, et al. Quantitative real-time PCR analysis and microarray-based RNA expression of HER2 in relation to outcome Ann. Onc., May 1, 2007; 18(5): 845 - 850. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. M. Binstock Molecular Biology Techniques for Identifying Dermatophytes and Their Possible Use in Diagnosing Onychomycosis in Human Toenail: A Review J Am Podiatr Med Assoc, March 1, 2007; 97(2): 134 - 144. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. M. Parida, S. R. Santhosh, P. K. Dash, N. K. Tripathi, V. Lakshmi, N. Mamidi, A. Shrivastva, N. Gupta, P. Saxena, J. P. Babu, et al. Rapid and Real-Time Detection of Chikungunya Virus by Reverse Transcription Loop-Mediated Isothermal Amplification Assay J. Clin. Microbiol., February 1, 2007; 45(2): 351 - 357. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. S. Leland and C. C. Ginocchio Role of Cell Culture for Virus Detection in the Age of Technology Clin. Microbiol. Rev., January 1, 2007; 20(1): 49 - 78. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Mia Kim, C. L. Afonso, and D. L. Suarez Effect of probe-site mismatches on detection of virulent Newcastle disease viruses using a fusion-gene real-time reverse transcription polymerase chain reaction test J Vet Diagn Invest, November 1, 2006; 18(6): 519 - 528. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. M. Parida, S. R. Santhosh, P. K. Dash, N. K. Tripathi, P. Saxena, S. Ambuj, A. K. Sahni, P. V. Lakshmana Rao, and K. Morita Development and Evaluation of Reverse Transcription-Loop-Mediated Isothermal Amplification Assay for Rapid and Real-Time Detection of Japanese Encephalitis Virus J. Clin. Microbiol., November 1, 2006; 44(11): 4172 - 4178. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L.-J. Chien, T.-L. Liao, P.-Y. Shu, J.-H. Huang, D. J. Gubler, and G.-J. J. Chang Development of Real-Time Reverse Transcriptase PCR Assays To Detect and Serotype Dengue Viruses J. Clin. Microbiol., April 1, 2006; 44(4): 1295 - 1304. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Ronsin, A. Pillet, C. Bali, and G.-A. Denoyel Evaluation of the COBAS AmpliPrep-Total Nucleic Acid Isolation-COBAS TaqMan Hepatitis B Virus (HBV) Quantitative Test and Comparison to the VERSANT HBV DNA 3.0 Assay J. Clin. Microbiol., April 1, 2006; 44(4): 1390 - 1399. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Legoff, H. Bouhlal, G. Gresenguet, H. Weiss, N. Khonde, H. Hocini, N. Desire, A. Si-Mohamed, J. de Dieu Longo, C. Chemin, et al. Real-Time PCR Quantification of Genital Shedding of Herpes Simplex Virus (HSV) and Human Immunodeficiency Virus (HIV) in Women Coinfected with HSV and HIV J. Clin. Microbiol., February 1, 2006; 44(2): 423 - 432. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Nitsche, M. Buttner, S. Wilhelm, G. Pauli, and H. Meyer Real-Time PCR Detection of Parapoxvirus DNA, Clin. Chem., February 1, 2006; 52(2): 316 - 319. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. V. Nainan, G. Xia, G. Vaughan, and H. S. Margolis Diagnosis of Hepatitis A Virus Infection: a Molecular Approach Clin. Microbiol. Rev., January 1, 2006; 19(1): 63 - 79. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Hilscher, W. Vahrson, and D. P. Dittmer Faster quantitative real-time PCR protocols may lose sensitivity and show increased variability Nucleic Acids Res., November 27, 2005; 33(21): e182 - e182. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. A. Valasek and J. J. Repa The power of real-time PCR Advan Physiol Educ, September 1, 2005; 29(3): 151 - 159. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. K.-Y. Lai, L. Cook, E. M. Krantz, L. Corey, and K. R. Jerome Calibration Curves for Real-Time PCR Clin. Chem., July 1, 2005; 51(7): 1132 - 1136. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. S. Esworthy, L. Yang, P. H. Frankel, and F.-F. Chu Epithelium-Specific Glutathione Peroxidase, Gpx2, Is Involved in the Prevention of Intestinal Inflammation in Selenium-Deficient Mice J. Nutr., April 1, 2005; 135(4): 740 - 745. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. G. Rutledge Sigmoidal curve-fitting redefines quantitative real-time PCR with the prospective of developing automated high-throughput applications Nucleic Acids Res., December 15, 2004; 32(22): e178 - e178. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Stocher and J. Berg Internal Control DNA for PCR Assays Introduced into Lambda Phage Particles Exhibits Nuclease Resistance Clin. Chem., November 1, 2004; 50(11): 2163 - 2166. [Full Text] [PDF]
|
|
|
|
 |
|
 F. Patolsky, G. Zheng, O. Hayden, M. Lakadamyali, X. Zhuang, and C. M. Lieber Electrical detection of single viruses PNAS, September 28, 2004; 101(39): 14017 - 14022. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. L. Huff, M. P. Lynch, S. Nettikadan, J. C. Johnson, S. Vengasandra, and E. Henderson Label-Free Protein and Pathogen Detection Using the Atomic Force Microscope J Biomol Screen, September 1, 2004; 9(6): 491 - 497. [Abstract] [PDF]
|
|
|
|
|
|
K. H. Abd El Galil, M. A. El Sokkary, S. M. Kheira, A. M. Salazar, M. V. Yates, W. Chen, and A. Mulchandani Combined Immunomagnetic Separation-Molecular Beacon-Reverse Transcription-PCR Assay for Detection of Hepatitis A Virus from Environmental Samples Appl. Envir. Microbiol., July 1, 2004; 70(7): 4371 - 4374. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P.-Y. Shu and J.-H. Huang Current Advances in Dengue Diagnosis Clin. Vaccine Immunol., July 1, 2004; 11(4): 642 - 650. [Full Text]
|
|
|
|
|
|
G. Gallinella, F. Bonvicini, C. Filippone, S. Delbarba, E. Manaresi, M. Zerbini, and M. Musiani Calibrated Real-Time PCR for Evaluation of Parvovirus B19 Viral Load Clin. Chem., April 1, 2004; 50(4): 759 - 762. [Full Text] [PDF]
|
|
|
|
|
|
P. Wolffs, R. Knutsson, B. Norling, and P. Radstrom Rapid Quantification of Yersinia enterocolitica in Pork Samples by a Novel Sample Preparation Method, Flotation, Prior to Real-Time PCR J. Clin. Microbiol., March 1, 2004; 42(3): 1042 - 1047. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Nitsche, H. Ellerbrok, and G. Pauli Detection of Orthopoxvirus DNA by Real-Time PCR and Identification of Variola Virus DNA by Melting Analysis J. Clin. Microbiol., March 1, 2004; 42(3): 1207 - 1213. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. A. Sanchez, K. E. Pierce, J. E. Rice, and L. J. Wangh Linear-After-The-Exponential (LATE)-PCR: An advanced method of asymmetric PCR and its uses in quantitative real-time analysis PNAS, February 17, 2004; 101(7): 1933 - 1938. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. A. Kulesh, R. O. Baker, B. M. Loveless, D. Norwood, S. H. Zwiers, E. Mucker, C. Hartmann, R. Herrera, D. Miller, D. Christensen, et al. Smallpox and pan-Orthopox Virus Detection by Real-Time 3'-Minor Groove Binder TaqMan Assays on the Roche LightCycler and the Cepheid Smart Cycler Platforms J. Clin. Microbiol., February 1, 2004; 42(2): 601 - 609. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. S. Biel, A. Nitsche, A. Kurth, W. Siegert, M. Ozel, and H. R. Gelderblom Detection of Human Polyomaviruses in Urine from Bone Marrow Transplant Patients: Comparison of Electron Microscopy with PCR Clin. Chem., February 1, 2004; 50(2): 306 - 312. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Parida, G. Posadas, S. Inoue, F. Hasebe, and K. Morita Real-Time Reverse Transcription Loop-Mediated Isothermal Amplification for Rapid Detection of West Nile Virus J. Clin. Microbiol., January 1, 2004; 42(1): 257 - 263. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. G. Wise, D. L. Suarez, B. S. Seal, J. C. Pedersen, D. A. Senne, D. J. King, D. R. Kapczynski, and E. Spackman Development of a Real-Time Reverse-Transcription PCR for Detection of Newcastle Disease Virus RNA in Clinical Samples J. Clin. Microbiol., January 1, 2004; 42(1): 329 - 338. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Rondini, E. Mensah-Quainoo, H. Troll, T. Bodmer, and G. Pluschke Development and Application of Real-Time PCR Assay for Quantification of Mycobacterium ulcerans DNA J. Clin. Microbiol., September 1, 2003; 41(9): 4231 - 4237. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. B. Grace, C. B. McLeland, S. J. Gagliardi, J. M. Smith, W. E. Jackson III, and W. F. Blakely Development and Assessment of a Quantitative Reverse Transcription-PCR Assay for Simultaneous Measurement of Four Amplicons Clin. Chem., September 1, 2003; 49(9): 1467 - 1475. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. B. Kauffman, S. A. Jones, A. P. Dupuis II, K. A. Ngo, K. A. Bernard, and L. D. Kramer Virus Detection Protocols for West Nile Virus in Vertebrate and Mosquito Specimens J. Clin. Microbiol., August 1, 2003; 41(8): 3661 - 3667. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. K.-Y. Lai, L. Cook, S. Wendt, L. Corey, and K. R. Jerome Evaluation of Real-Time PCR versus PCR with Liquid-Phase Hybridization for Detection of Enterovirus RNA in Cerebrospinal Fluid J. Clin. Microbiol., July 1, 2003; 41(7): 3133 - 3141. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. P. Vaughn and K. S.J. Elenitoba-Johnson Hybridization-Induced Dequenching of Fluorescein-Labeled Oligonucleotides: A Novel Strategy for PCR Detection and Genotyping Am. J. Pathol., July 1, 2003; 163(1): 29 - 35. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. De Medici, L. Croci, E. Delibato, S. Di Pasquale, E. Filetici, and L. Toti Evaluation of DNA Extraction Methods for Use in Combination with SYBR Green I Real-Time PCR To Detect Salmonella enterica Serotype Enteritidis in Poultry Appl. Envir. Microbiol., June 1, 2003; 69(6): 3456 - 3461. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P.-Y. Shu, S.-F. Chang, Y.-C. Kuo, Y.-Y. Yueh, L.-J. Chien, C.-L. Sue, T.-H. Lin, and J.-H. Huang Development of Group- and Serotype-Specific One-Step SYBR Green I-Based Real-Time Reverse Transcription-PCR Assay for Dengue Virus J. Clin. Microbiol., June 1, 2003; 41(6): 2408 - 2416. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Francois, D. Pittet, M. Bento, B. Pepey, P. Vaudaux, D. Lew, and J. Schrenzel Rapid Detection of Methicillin-Resistant Staphylococcus aureus Directly from Sterile or Nonsterile Clinical Samples by a New Molecular Assay J. Clin. Microbiol., January 1, 2003; 41(1): 254 - 260. [Abstract] [Full Text] [PDF]
|
|