Abstract
While infecting humans and other mammals, Leishmania spp. are obligate intracellular parasites. Therefore, for the purpose of therapeutic intervention and the study of infectivity, the relevant form of Leishmania spp. is the intracellular amastigote. Therefore, monitoring intracellular parasite load is an essential requirement in many fields of Leishmania research. Real-time quantitative PCR is a highly accurate technique for the detection and quantification of parasite burden in in vitro or in vivo infection experiments. The quantification of DNA for standard curves shows linearity over a 5 to 6-log concentration range indicating the high sensitivity of the method. Moreover, qPCR allows for the simultaneous quantification of host and parasite DNA in the same reaction, thereby allowing for an assessment of relative parasite load for basic research, but also for low- to medium-throughput compound screening. The method also allows to analyze late stages of in vitro infections where host cells and parasites have detached from surfaces and escape microscopy-based assays.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Weksberg R, Hughes S, Moldovan L, Bassett A, Chow E, Squire J (2005) A method for accurate detection of genomic micro-deletions using real-time quantitative PCR. BMC Genomics 6:180
Day E, Dear PH, McCaughan F (2013) Digital PCR strategies in the development and analysis of molecular biomarkers for personalized medicine. Methods 59(1):101–107. https://doi.org/10.1016/j.ymeth.2012.08.001
Nassirpour R, Mathur S, Gosink MM, Li Y, Shoieb AM, Wood J, O’Neil SP, Homer BL, Whiteley LO (2014) Identification of tubular injury microRNA biomarkers in urine: comparison of next-generation sequencing and qPCR-based profiling platforms. BMC Genomics 15:485. https://doi.org/10.1186/1471-2164-15-485
Kearns AM, Guiver M, James V, King J (2001) Development and evaluation of a real-time quantitative PCR for the detection of human cytomegalovirus. J Virol Methods 95(1–2):121–131
Nicolas L, Prina E, Lang T, Milon G (2002) Real-time PCR for detection and quantitation of leishmania in mouse tissues. J Clin Microbiol 40(5):1666–1669
Bifeld E, Tejera Nevado P, Bartsch J, Eick J, Clos J (2016) A versatile qPCR assay to quantify trypanosomatidic infections of host cells and tissues. Med Microbiol Immunol 205(5):449–458. https://doi.org/10.1007/s00430-016-0460-3
Chen C, Ridzon DA, Broomer AJ, Zhou Z, Lee DH, Nguyen JT, Barbisin M, Xu NL, Mahuvakar VR, Andersen MR, Lao KQ, Livak KJ, Guegler KJ (2005) Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res 33(20):e179. https://doi.org/10.1093/nar/gni178
VanGuilder HD, Vrana KE, Freeman WM (2008) Twenty-five years of quantitative PCR for gene expression analysis. Biotechniques 44(5):619–626. https://doi.org/10.2144/000112776
Morrison TB, Weis JJ, Wittwer CT (1998) Quantification of low-copy transcripts by continuous SYBR Green I monitoring during amplification. Biotechniques 24(6):954–958, 960, 962
Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29(9):e45
Prina E, Roux E, Mattei D, Milon G (2007) Leishmania DNA is rapidly degraded following parasite death: an analysis by microscopy and real-time PCR. Microbes Infect 9(11):1307–1315
Lachaud L, Marchergui-Hammami S, Chabbert E, Dereure J, Dedet JP, Bastien P (2002) Comparison of six PCR methods using peripheral blood for detection of canine visceral leishmaniasis. J Clin Microbiol 40(1):210–215
Mary C, Faraut F, Lascombe L, Dumon H (2004) Quantification of Leishmania infantum DNA by a real-time PCR assay with high sensitivity. J Clin Microbiol 42(11):5249–5255
Weirather JL, Jeronimo SM, Gautam S, Sundar S, Kang M, Kurtz MA, Haque R, Schriefer A, Talhari S, Carvalho EM, Donelson JE, Wilson ME (2011) Serial quantitative PCR assay for detection, species discrimination, and quantification of Leishmania spp. in human samples. J Clin Microbiol 49(11):3892–3904. https://doi.org/10.1128/JCM.r00764-11
Jara M, Berg M, Caljon G, de Muylder G, Cuypers B, Castillo D, Maes I, Orozco MDC, Vanaerschot M, Dujardin JC, Arevalo J (2017) Macromolecular biosynthetic parameters and metabolic profile in different life stages of Leishmania braziliensis: amastigotes as a functionally less active stage. PLoS One 12(7):e0180532. https://doi.org/10.1371/journal.pone.0180532
Acknowledgments
Thanks to Heidrun Von Thien for the advice and help with the establishment of the real-time qPCR assay.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Bifeld, E. (2019). Quantification of Intracellular Leishmania spp. Using Real-Time Quantitative PCR (qPCR). In: Clos, J. (eds) Leishmania. Methods in Molecular Biology, vol 1971. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9210-2_13
Download citation
DOI: https://doi.org/10.1007/978-1-4939-9210-2_13
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-9209-6
Online ISBN: 978-1-4939-9210-2
eBook Packages: Springer Protocols