Quantification of Leishmania and Anaplasma in Diverse Samples

Total DNA was extracted from whole blood, skin, uterus, ovaries, placenta, liver, spleen and umbilical cord samples using the Genomic DNA Purification Kit (Gentra Systems, Minnesota, USA), while genomic DNA from amniotic fluid and bone marrow samples was extracted using QIAampDNA Micro Kit (Qiagen, GmbH, Hilden, Germany). For each sample, two qPCR reactions were individually performed for the detection and quantification of L. infantum and A. platys nucleic acids, using primers and probes targeting, respectively, the kinetoplast minicircle DNA (kDNA) and 16S rRNA gene, as described previously [18 (link), 23 (link)]. DNA extracted from lymph nodes and whole blood from L. infantum and A. platys-infected dogs were included as positive controls. Quantification of DNA of L. infantum and A. platys was performed using a 10-fold dilution series of standard DNA from promastigotes (log phase concentration, 1.7 × 10⁶ parasites/ml) of L. infantum (zymodeme MON-1) and from A. platys-infected blood with a concentration of 5.6 × 10⁵ infected platelets/100 μl. The detection limits of the qPCRs were assessed using serial dilutions from 1.7 × 10^-2 to 1.7 × 10^-7 parasites (L. infantum) and from 2.24 × 10² to 2.24 × 10^-6 infected platelets (A. platys) per reaction (2 μl of DNA template), respectively.