A versatile qPCR assay to quantify trypanosomatidic infections of host cells and tissues.
The majority of PCR-based detection systems for Leishmania spp. and Trypanosoma cruzi aim at high sensitivity and specificity, rather than an accurate parasite load quantification required for experimental infections in basic research and drug development. Here, we describe the use of a dual-labelled probe qPCR to detect and quantify intracellular Old World Leishmania spp. and T. cruzi amastigotes after in vitro and in vivo infection experiments. We show that quantification of parasite actin gene DNA relative to the host cell actin gene DNA accurately reflects the parasite load relative to the host cells and that qPCR quantification is highly sensible to drug-induced cell death. Furthermore, qPCR allows to determine parasite loads even after host cell detachment and/or rupture, important when comparing untreated versus drug-treated samples. The method is also suitable for the quantification of parasites from infected mouse tissue, making it suitable for drug testing and mutant phenotype analysis.
Bernhard Nocht Institute for Tropical Medicine, Bernhard Nocht St 74, 20359, Hamburg, Germany.
Eugenia
E
Bifeld
Bernhard Nocht Institute for Tropical Medicine, Bernhard Nocht St 74, 20359, Hamburg, Germany.
Paloma
P
Tejera Nevado
Bernhard Nocht Institute for Tropical Medicine, Bernhard Nocht St 74, 20359, Hamburg, Germany.|Beuth Hochschule für Technik, Berlin, Germany.
Janika
J
Bartsch
Bernhard Nocht Institute for Tropical Medicine, Bernhard Nocht St 74, 20359, Hamburg, Germany.
Julia
J
Eick
Bernhard Nocht Institute for Tropical Medicine, Bernhard Nocht St 74, 20359, Hamburg, Germany. clos@bnitm.de.
Joachim
http://orcid.org/0000-0003-4377-8173
J
Clos
D000199:Actins / Q000235:genetics; D000818:Animals; D005260:Female; D006801:Humans; D007891:Leishmania / Q000235:genetics / Q000302:isolation & purification*; D008810:Mice, Inbred C57BL; D059208:Parasite Load / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D014345:Trypanosoma / Q000235:genetics / Q000302:isolation & purification*
D000199:Actins
Leishmania; Parasite load; RT-PCR; Trypanosoma cruzi