A new protocol for absolute quantification of haemosporidian parasites in raptors and comparison with current assays.

Parasites & Vectors
Xi HuangLu Dong

Abstract

Accurate quantification of infection intensity is essential to estimate infection patterns of avian haemosporidian parasites in order to understand the evolution of host-parasite associations. Traditional microscopy is cost-effective but requires high-quality blood smears and considerable experience, while the widely used semi-quantitative qPCR methods are mostly employed with ideal, laboratory-based golden samples and standard curves, which may limit the comparison of parasitemia from different laboratories. Here we present a digital droplet PCR (ddPCR) protocol for absolute quantification of avian haemosporidians in raptors. Novel primers were designed that target a conserved fragment of a rRNA region of the mitochondrial genome of the parasites. Sensitivity and repeatability were evaluated compared to qPCR and other assays. This ddPCR assay enables accurate quantification of haemosporidian parasites belonging to Plasmodium, Haemoproteus and Leucocytozoon with minimum infection quantities of 10-5 (i.e. one parasite copy in 105 host genomes) without the use of standard curves. Quantities assessed by ddPCR were more accurate than qPCR using the same primers through reduction of non-specific amplification in low-intensity sample...Continue Reading

References

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Datasets Mentioned

BETA
CM004177.1
AB250690.1
AB250415.1
LN835311.1
MT281522
MT281526

Methods Mentioned

BETA
light microscopy
PCR
Assay
electrophoresis

Software Mentioned

rptR
QuantaSoft
Geneious
Primer3

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