Male adult Lu. longipalpis library from Cueva de Lapinha (Minas Gerais, Brasil)
In order to capture RNA viruses together with the rest of the associated microorganisms, total RNA was extracted from individual samples composed of 100 sandflies from the same gender, operation and location. Total RNA was amplified using a modified random PCR protocol (Bohlander et al., 1992). Briefly, the first cDNA strand was synthesised with a primer which includes a random octanucleotide portion followed by a known portion of 17 nucleotides (primer 1: 5’-GTT TCC CAG TAG GTC TCN NNN NNN N-3’). In the following step, cDNA was amplified using a mixture of the random primer and a second primer which targets the specific portion of the random primer (primer 2: 5’-CGC CGT TTC CCA GTA GGT CTC-3’). The amplified cDNA samples were pyrosequenced.