Abstract
In spite of the impressive technical refinement of the PCR technology, new-generation real-time PCR assays still suffer from two major limitations: the impossibility to control both for PCR artifacts (with the important caveat of false-negative results) and for the efficiency of nucleic acid recovery during the preliminary extraction phase of DNA from the biological sample.
The calibrator technology developed at the Unit of Human Virology overcomes both of these limitations, leading to a substantially higher degree of accuracy and reproducibility in the quantification, which is especially useful for the measurement of pathogen loads in sequential samples and for the reliable detection of low-copy pathogens.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Heid CA, Stevens J, Livak KJ et al (1996) Real-time quantitative PCR. Genome Res 6:986–994
Kalinina O, Lebedeva I, Brown J et al (1997) Nanoliter scale PCR with TaqMan detection. Nucleic Acids Res 25:1999–2004
Whitcombe D, Brownie J, Gillard HL et al (1998) A homogeneous fluorescence assay for PCR amplicons: its application to real-time, single-tube genotyping. Clin Chem 44: 918–923
Solinas A, Brown LJ, McKeen C et al (2001) Duplex Scorpion primers in SNP analysis and FRET applications. Nucleic Acids Res 29:E96
Gelsthorpe AR, Wells RS, Lowe AP et al (1999) High-throughput class I HLA genotyping using fluorescence resonance energy transfer (FRET) probes and sequence-specific primer-polymerase chain reaction (SSP-PCR). Tissue Antigens 54:603–614
Chen W, Martinez G, Mulchandani A (2000) Molecular beacons: a real-time polymerase chain reaction assay for detecting Salmonella. Anal Biochem 280:166–172
Chen X, Livak KJ, Kwok PY (1998) A homogeneous, ligase-mediated DNA diagnostic test. Genome Res 8:549–556
Carters R, Ferguson J, Gaut R et al (2008) Design and use of scorpions fluorescent signaling molecules. Methods Mol Biol 429:99–115
Ortiz E, Estrada G, Lizardi PM (1998) PNA molecular beacons for rapid detection of PCR amplicons. Mol Cell Probes 12:219–226
Broccolo F, Locatelli G, Sarmati L et al (2002) Calibrated real-time PCR assay for quantitation of human herpesvirus 8 DNA in biological fluids. J Clin Microbiol 40:4652–4658
Broccolo F, Scarpellini P, Locatelli G et al (2003) Rapid diagnosis of mycobacterial infections and quantitation of Mycobacterium tuberculosis load using two real-time calibrated PCR assays. J Clin Microbiol 41:4565–4572
Flamand L, Gravel A, Boutolleau D et al (2008) Multicenter comparison of PCR assays for detection of human herpesvirus 6 DNA in serum. J Clin Microbiol 46:2700–2706
Gatto F, Cassina G, Broccolo F et al (2011) A multiplex calibrated real-time PCR assay for quantitation of DNA of EBV-1 and 2. J Virol Methods 178:98–105
Cassina G, Russo D, De Battista D et al (2013) Calibrated real-time polymerase chain reaction for specific quantitation of HHV-6A and HHV-6B in clinical samples. J Virol Methods 189:172–179
Tedeschi R, Enbom M, Bidoli E et al (2001) Viral load of human herpesvirus 8 in peripheral blood of human immunodeficiency virus infected patients with Kaposi’s sarcoma. J Clin Microbiol 39:4269–4273
Malnati MS, Broccolo F, Nozza S et al (2002) Retrospective analysis of HHV-8 viremia and cellular viral load in HIV-seropositive patients receiving interleukin 2 in combination with antiretroviral therapy. Blood 100:1575–1578
Dagna L, Broccolo F, Paties CT et al (2005) A relapsing inflammatory syndrome and active human herpesvirus 8 infection. N Engl J Med 353:156–163
Broccolo F, Drago F, Careddu AM et al (2005) Additional evidence that pityriasis rosea is associated with reactivation of HHV-6 and-7. J Invest Dermatol 124:1234–1240
Drago F, Broccolo F, Zaccaria E et al (2008) Pregnancy outcome in patients with pityriasis rosea. J Am Acad Dermatol 58:S78–S83
Broccolo F, Drago F, Paolino S et al (2009) Reactivation of human herpesvirus 6 (HHV-6) infection in patients with connective tissue disorders. J Clin Virol 46:43–46
Broccolo F, Lusso P, Malnati M (2013) Calibration technologies for correct determination of Epstein-Barr virus, human herpesvirus 6 (HHV-6), and HHV-8 antiviral drug susceptibilities by use of real-time PCR-based assays. J Clin Microbiol 51:2013
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media New York
About this protocol
Cite this protocol
Russo, D., Malnati, M.S. (2014). Absolute Quantification of Viral DNA: The Quest for Perfection. In: Biassoni, R., Raso, A. (eds) Quantitative Real-Time PCR. Methods in Molecular Biology, vol 1160. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0733-5_7
Download citation
DOI: https://doi.org/10.1007/978-1-4939-0733-5_7
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-0732-8
Online ISBN: 978-1-4939-0733-5
eBook Packages: Springer Protocols