Quantitative PCR to detect, discriminate and quantify intracellular parasites in their host: an example from three microsporidians in Daphnia.
Reliable detection, discrimination and quantification of parasites are important for host-parasite studies and diagnostics. Microsporidial infections are problematic in this respect. Their discrimination and quantification using light microscopy is difficult because spores are the only light microscopically visible form of the parasite and they offer few distinct characters. We developed a quantitative PCR (qPCR) assay based on SYBR Green chemistry to quantify the microsporidia Glugoides intestinalis, Octosporea bayeri and Ordospora colligata in their host, the freshwater crustacean Daphnia magna. The assay allows the quantification of infection intensities in whole animals and is more than an order of magnitude more sensitive than light microscopy. Sampling and DNA extraction account for more than 90% of the residual variance in infection intensity data and this variance considerably impairs the resolution of qPCR. Where higher resolution is required, we propose using the ratio of parasite to host DNA as the measure of infection intensity. We show that this measure is robust and greatly improves resolution of qPCR. Additionally, this method can be applied to compare samples of unequal volume.
Unité Ecologie et Evolution, Département de Biologie, Université de Fribourg, chemin du Musée 10, CH-1700 Fribourg, Switzerland. dominik.refardt@gmail.com
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D000818:Animals; D004271:DNA, Fungal / Q000032:analysis*; D003621:Daphnia / Q000382:microbiology*; D016814:Microsporidia / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013045:Species Specificity; D013172:Spores, Fungal / Q000302:isolation & purification
D004271:DNA, Fungal