Abstract
Sensitive TaqMan® real-time polymerase chain reaction (PCR)-based methods have been developed recently for the detection and quantitation of feline leukemia virus (FeLV) proviral DNA in infected cats. In this chapter, we outline the design and implementation of a TaqMan® real-time PCR assay to quantify total FeLV proviral and viral RNA loads in infected cats. The assay is designed to amplify all three FeLV subtypes (A-C), but not FeLV-related endogenous retroviral sequences. The system is tested and optimized using proviral DNA or viral RNA from cells infected with reference strains. The sequence used to produce the standard DNA and RNA is amplified, subcloned into a vector, and sequenced. cRNA is synthesized from the linearized plasmid DNA. Standard DNA and RNA are quantified, diluted and used to determine efficiency, sensitivity, linear amplification range, and precision of the quantitative TaqMan® real-time PCR assays.
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
Hofmann-Lehmann, R., Tandon, R., Boretti, F. S., et al. (2006) Vaccine 24, 1087–1094.
Holland, P. M., Abramson, R. D., Watson, R., and Gelfand, D. H. (1991) Proc. Natl Acad. Sci. 88, 7276–7280.
Lyamichev, V., Brow, M. A., and Dahlberg, J. E. (1993) Science 260, 778–783.
Förster, V. T. (1948) Ann. Phys. 2, 55–75.
Hofmann-Lehmann, R., Huder, J. B., Gruber, S., Boretti, F., Sigrist, B., and Lutz, H. (2001) J. Gen. Virol. 82, 1589–1596.
Tandon, R., Cattori, V., Gomes-Keller, M. A., et al. (2005) J. Virol. Methods 130, 124–132.
Torres, A. N., Mathiason, C. K., and Hoover, E. A. (2005) Virology 332, 272–283.
Gibson, U. E., Heid, C. A., and Williams, P. M. (1996) Genome Res. 6, 995–1001.
Heid, C. A., Stevens, J., Livak, K. J., and Williams, P. M. (1996) Genome Res. 6, 986–994.
Livak, K. J., Flood, S. J., Marmaro, J., Giusti, W., and Deetz, K. (1995) PCR Meth. Appl. 4, 357–362.
Lutz, H., Pedersen, N. C., Durbin, R., and Theilen, G. H. (1983) J. Immunol. Methods 56, 209–220.
Hardy, W. D. Jr., Geering, G., Old, L. J., de Harven, E., Brodey, R. S., and McDonough, S. K. (1970) Bibl. Haematol. 36, 343–354.
Jarrett, O., Golder, M. C., and Weijer, K. (1982) Vet. Rec. 110, 325–328.
Rohn, J. L. and Overbaugh, J. (1995) Virology 206, 661–665.
Klein, D., Janda, P., Steinborn, R., Muller, M., Salmons, B., and Gunzburg, W. H. (1999) Electrophoresis 20, 291–299.
Lockey, C., Otto, E., and Long, Z. (1998) Biotechniques 24, 744–746.
Hofmann-Lehmann, R., Swenerton, R. K., Liska, V., et al. (2000) AIDS Res. Hum. Retroviruses 16, 1247–1257.
Hofmann-Lehmann, R., Williams, A. L., Swenerton, R. K., et al. (2002) AIDS Res. Hum. Retroviruses 18, 627–639.
McDonagh, S. (2003) Equipping and Establishing a PCR Laboratory. In PCR Protocols (Bartlett, J. M. S. and Stirling, D., eds.), Vol. 226, Humana Press, Totowa, NJ.
O’Connell, J. (2002) The basics of RT-PCR-some practical considerations. In RT-PCR Protocols (O’Connell, J., ed.), Vol. 193, Humana Press, Totowa, NJ.
Schenborn, E. T. and Mierendorf, R. C. (1985) Nucleic Acids Res. 13, 6223–6236.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2008 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Cattori, V., Hofmann-Lehmann, R. (2008). Absolute Quantitation of Feline Leukemia Virus Proviral DNA and Viral RNA Loads by TaqMan® Real-time PCR and RT-PCR. In: Marx, A., Seitz, O. (eds) Molecular Beacons: Signalling Nucleic Acid Probes, Methods, and Protocols. Methods in Molecular Biology, vol 429. Humana Press. https://doi.org/10.1007/978-1-60327-040-3_6
Download citation
DOI: https://doi.org/10.1007/978-1-60327-040-3_6
Publisher Name: Humana Press
Print ISBN: 978-1-58829-700-6
Online ISBN: 978-1-60327-040-3
eBook Packages: Springer Protocols