A versatile qPCR assay to quantify trypanosomatidic infections of host cells and tissues.
Eugenia BifeldPaloma Tejera NevadoJanika BartschJulia EickJoachim ClosPublished in: Medical microbiology and immunology (2016)
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.
Keyphrases
- trypanosoma cruzi
- drug induced
- plasmodium falciparum
- cell cycle arrest
- cell death
- liver injury
- induced apoptosis
- toxoplasma gondii
- single cell
- circulating tumor
- cell therapy
- cell free
- adverse drug
- single molecule
- life cycle
- genome wide
- gene expression
- oxidative stress
- copy number
- pi k akt
- high throughput
- endoplasmic reticulum stress
- signaling pathway
- real time pcr
- stem cells
- mass spectrometry
- newly diagnosed
- dna methylation
- cell proliferation
- bone marrow
- mesenchymal stem cells
- transcription factor
- wild type
- circulating tumor cells