Bartonella DNA

Gonçalves-Oliveira, Jonathan, Rozental, Tatiana, Guterres, Alexandro, Teixeira, Bernardo Rodrigues, Andrade-Silva, Beatriz Elise, Socrates, Costa-Neto, Fraga da, Furtado, Marina Carvalho, Moratelli, Ricardo, S, Paulo, D’Andrea, ergio & Lemos, Elba Regina Sampaio, 2020, Investigation of Bartonella spp. in brazilian mammals with emphasis on rodents and bats from the Atlantic Forest, International Journal for Parasitology: Parasites and Wildlife 13, pp. 80-89 : 82

2.3. Laboratory methods for detection of Bartonella DNA

The DNA was extracted from samples of the liver and spleen using a commercial extraction kit (QIAamp DNA Mini Kit, Qiagen®), according to the manufacturer’ s instructions. Samples of the spleen are preferred for molecular testing (Kosoy et al., 2017), but when unavailable, liver samples were used. The quality of the extracted DNA was assessed in agarose gel. The DNA of Bartonella detected in wild rodents and bats in previous studies ( Ferreira et al., 2018; Rozental et al., 2017) was used as the positive control for the molecular assays, while nuclease-free water (UltraPure™ DNase/RNase-Free – Invitrogen) was used as the negative control. The DNA samples were submitted to conventional PCR for the gltA (731 bps - Rozental et al., 2017), ftsZ and groEL (present study) genes. The assay was run for each gene containing 0.5 μL of each primer (10 mM), 0.5 μL of 20 mM dNTP (Invitrogen™), 4.0 μL of 50 mM MgCl 2 (Applied Biosystems®), 2.5 μL of 10x PCR buffer (Applied Biosystems®), 0.2 μL of AmpliTaq Gold® DNA Polymerase (5U/μl, Applied Biosystems®), 13.8 μL of nuclease-free water (UltraPure™ DNase/RNase-Free – Invitrogen) and 3 μL of the sample DNA in a final volume of 25 μL. The cycle conditions and steps applied for each gene are listed in Table 1. For the sequencing reaction, the amplified products were purified using the illustra GFX PCR DNA and Gel Band Purification kit (GE Healthcare©) and were sequenced using the BigDye Terminator Cycle Sequencing Ready Reaction®v3.1kit (Thermo Fisher Scientific™, Waltham, MA, USA). Sequences were deposited in Genbank database (MN613434-MN613442).