qPCR for Leishmania spp. Detection

Following a previously established protocol, targeting the conserved REPL repeats specific for L. donovani and L. infantum, qPCR was performed with extracted template DNA from whole blood and DBS (S1 Table) [2 (link),17 (link)]. In brief, to prepare a 20 μL reaction mix, 9 μL template DNA, 10 μL of TaqMan Gene Expression Master Mix (Applied Biosystems) and 1 μL pre-ordered Taqman primer-probe mix (Applied Biosystems) were combined. For amplification, Bio-Rad CFX96 iCycler system was utilized. The following were the amplification conditions: 10 min at 95°C, followed by 15 seconds at 95°C and 1 min at 60°C for 45 cycles. In each run, a standard curve was created with 10 ng—1 fg of parasite DNA isolated from in vitro cultured promastigotes (L. donovani MHOM/IN/80/DD8) corresponding to 10,000–0.1 parasites per reaction. One reaction with nuclease-free water was also included in each run as a negative control. A sample was considered as negative with a cycle threshold (Ct) > 40. Each sample was evaluated in duplicate, and an additional run was made in case of an inconclusive result.

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Ghosh P., Chowdhury R., Rahat M.A., Hossain F., Arpha N.E., Kristan M., Higgins M., El Wahed A.A., Goto Y., Islam M.M., Campino S., Cameron M., Duthie M.S., Haque R, & Mondal D. (2023). Dried Blood Spots (DBS): A suitable alternative to using whole blood samples for diagnostic testing of visceral leishmaniasis in the post-elimination era. PLOS Neglected Tropical Diseases, 17(10), e0011680.

Publication 2023

TaqMan Gene Expression Master Mix Taqman primer-probe mix CFX96 iCycler system

Blood Gene expression Parasites Primer Seconds

Corresponding Organization : International Centre for Diarrhoeal Disease Research

Other organizations : BRAC University, University of London, London School of Hygiene & Tropical Medicine, Leipzig University, The University of Tokyo, University of Dhaka

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Variable analysis

independent variables

Extracted template DNA from whole blood and DBS

dependent variables

Parasite DNA copy number (measured by qPCR)

control variables

Amplification conditions (10 min at 95°C, 15 seconds at 95°C and 1 min at 60°C for 45 cycles)
Taqman primer-probe mix
TaqMan Gene Expression Master Mix (Applied Biosystems)
Bio-Rad CFX96 iCycler system

controls

Positive control: Standard curve with 10 ng—1 fg of parasite DNA isolated from in vitro cultured promastigotes (L. donovani MHOM/IN/80/DD8) corresponding to 10,000–0.1 parasites per reaction
Negative control: One reaction with nuclease-free water

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