Leishmania macropodum, DNA

Panahi, Elina, Shivas, Martin, Hall-Mendelin, Sonja, Kurucz, Nina, Rudd, Penny A., Araujo, Rachel De, Skinner, Eloise B., Melville, Lorna & Herrero, Lara J., 2020, Utilising a novel surveillance system to investigate species of Forcipomyia (Lasiohelea) (Diptera: Ceratopogonidae) as the suspected vectors of Leishmania macropodum (Kinetoplastida: Trypanosomatidae) in the Darwin region of Australia, International Journal for Parasitology: Parasites and Wildlife 12, pp. 192-198 : 194

2.3. Stability of L. macropodum DNA on FTA Ṝ cards

To ensure that honey-coated FTAṜ cards could be used to preserve and identify Leishmania DNA , a pilot experiment was designed to investigate the cards’ ability to detect cultured L. macropodum promastigotes over a 10-week time course. FTAṜ cards were inoculated with a parasite load between 0 to 10 6 parasites/card in 10-fold increments in triplicates and stability of DNA was tested at five time points (week 0, 2, 5, 8, and 10). Furthermore, cards had either been coated with honey or without (plain, control group) in order to ensure the use of honey had no effect on parasite detection.

For parasite DNA elution from FTAṜ cards, the cards were cut into strips and added to a 5 mL tube containing 1 mL molecular grade water kept on ice. To release the DNA from the matrix of the FTAṜ cards, the tubes were vortexed every 5 min for 10 s for a total of 20 min. The strips and suspension were separated with a syringe, and strips were discarded ( Hall-Mendelin et al., 2010). The suspension, containing the DNA, was aliquoted and 200 μL (1/5 of the card) was used for DNA extraction using DNeasyṜ Blood & Tissue Kit (Qiagen) following the manufacturer's protocol for purification of total DNA from cells. DNA was eluted in 200 μL AE buffer.