Leishmania screening
We tested each extracted pool of sandflies for the presence ofLeishmania species with real-time quantitative PCR (qPCR) using
primer pairs kDNA1 and L. braziliensis kDNA3 as described by
Weirather et al. (2011). The kDNA1 primers amplify DNA for the speciesL. amazonensis , L. chagasi , L. donovani , L.
infantum , L. major , L. mexicana , and L. tropica ,
while the kDNA3 primers primarily amplify DNA for L. braziliensis(Weirather et al., 2011). PCR reactions were carried out in a volume of
10 ul using 5 ul PowerTrack SYBR Green Master Mix (final concentration
of 1x), 0.05 ul of the forward primer (final concentration of 500 nM),
0.05 ul of the reverse primer (final concentration of 500 nM), 2.9 ul of
water, and 2 ul of the DNA template. The PCR cycling was as follows
(Weirather et al., 2011): 95°C for 10 min; 40 cycles of 95°C for 15 sec;
and 60°C for 1 min. We carried out a total of 2,260 reactions (1,130
reactions using the kDNA1 primers and 1,130 reactions using the L.
braziliensis kDNA3 primers) to screen for the presence ofLeishmania species.