Leishmania macropodum

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

2.6. Detecting and quantifying L. macropodum by real-time qPCR

Leishmania macropodum screening was performed with specific Taqman qPCR assays. Published primers rooME-F2 5′-AAACTTCCGGA ACCTGTCGT-3′, rooME-R2 5′-GTAGGCACCCGAAGAGACC-3′, and the Taqman probe LeishME 5′d FAM-CCGGCAAGATTTTGGGAGCG-BHQ-1 3′ were used to amplify L. macropodum ( Dougall et al., 2009) . PCR reactions were made in a 10 μL reaction of 1 × SsoAdvanced™ universal probe supermix, 6 mM MgCl 2 (Bio-Rad Laboratories, Australia), 0.3 μ M of primers, 0.05 μ M Taqman probe (Sigma-Aldrich, Australia) and 2 μL of DNA template extracted from either insect or FTAṜ card sample. PCR cycling conditions were as follows: 2 min at 95 ̊C followed by 35 cycles of 15 s at 95 ̊C and 40 s at 66 ̊C with the CFX96 Real Time System (C1000 Thermal Cycler; Bio-Rad Laboratories). Genomic L. macropodum DNA standards from cultivated promastigotes were included in every PCR run to quantify positive insect samples and FTAṜ cards. Standards were purified from cultured L. macropodum promastigotes and made up of serial dilutions 10 −1 – 10 − 7 in Tris-EDTA buffer (Sigma-Aldrich, Australia). Moreover, L. macropodum DNA standards were used to determine the qPCR assay's limit of detection. Parasite detection threshold was identified at ≥ 50 cultured parasites (data not shown here).