DNA was extracted from the peripheral blood, aqueous and vitreous humours using QIAamp DNA Blood Midi Kit (Qiagen, Valencia, California, USA) following the manufacturer’s instructions. After the DNA extraction, real-time PCR was performed using primers and probe designed for the B1 gene as described by Fekkar and collaborators.10 (link) Assays were performed using Taqman Universal Master Mix 2x (Applied Biosystems, USA) to a 25 μL final reaction volume containing 0.3 μM forward primer, 0.5 μM reverse primer, 0.15 μM flouorescent probe and 5 μL of DNA. PCR was performed on an Applied Biosystems 7500 Fast Real-Time PCR System. As a negative control, DNA was replaced with deionised water, and as positive control we used 4 μL of strain type I (RH).
For the PCR genotyping, a multilocus PCR–DNA was performed on genomic DNA at B1, SAG-1 and NTS2 loci as described previously.11 (link) PCR products were incubated for 15 min with ExoSAP-IT (USB Corp, Cleveland, Ohio, USA) prior to DNA sequencing. DNA sequencing was performed by the NIAID RML, Genomics Unit, Hamilton, Montana, USA.
For the PCR genotyping, a multilocus PCR–DNA was performed on genomic DNA at B1, SAG-1 and NTS2 loci as described previously.11 (link) PCR products were incubated for 15 min with ExoSAP-IT (USB Corp, Cleveland, Ohio, USA) prior to DNA sequencing. DNA sequencing was performed by the NIAID RML, Genomics Unit, Hamilton, Montana, USA.
