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

作者：Xi Huang, Di Huang, Yuge Liang, Linlin Zhang, Guocheng Yang, Boye Liu, Yangyang Peng, Wenhong Deng, Lü Dong · 发表于：Parasites & Vectors · 年份：2020 · DOI：10.1186/s13071-020-04195-y · 被引用次数：34 · 研究领域：Bird parasitology and diseases、Coccidia and coccidiosis research、Vector-borne infectious diseases

Abstract Background 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. Methods 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. Results 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 10 5 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 samples. The ddPCR technique was more consistent among technical duplicates and reactions, especially when infection intensities were low, and this technique demonstrated equal sensitivity with high correspondence ( R 2 ...