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Leishmania pp 249-263 | Cite as

Quantification of Intracellular Leishmania spp. Using Real-Time Quantitative PCR (qPCR)

  • Eugenia Bifeld
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1971)

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.

Key words

Leishmania Real-time PCR Parasite burden Leishmania actin 

Notes

Acknowledgments

Thanks to Heidrun Von Thien for the advice and help with the establishment of the real-time qPCR assay.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Eugenia Bifeld
    • 1
    • 2
  1. 1.Bernhard Nocht Institute for Tropical MedicineHamburgGermany
  2. 2.altona Diagnostics GmbHHamburgGermany

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